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Sample records for adult murine hematopoiesis

  1. Histone acetyltransferase activity of MOF is required for adult but not early fetal hematopoiesis in mice.

    PubMed

    Valerio, Daria G; Xu, Haiming; Eisold, Meghan E; Woolthuis, Carolien M; Pandita, Tej K; Armstrong, Scott A

    2017-01-05

    K(lysine) acetyltransferase 8 (KAT8, also known as MOF) mediates the acetylation of histone H4 at lysine 16 (H4K16ac) and is crucial for murine embryogenesis. Lysine acetyltransferases have been shown to regulate various stages of normal hematopoiesis. However, the function of MOF in hematopoietic stem cell (HSC) development has not yet been elucidated. We set out to study the role of MOF in general hematopoiesis by using a Vav1-cre-induced conditional murine Mof knockout system and found that MOF is critical for hematopoietic cell maintenance and HSC engraftment capacity in adult hematopoiesis. Rescue experiments with a MOF histone acetyltransferase domain mutant illustrated the requirement for MOF acetyltransferase activity in the clonogenic capacity of HSCs and progenitors. In stark contrast, fetal steady-state hematopoiesis at embryonic day (E) 14.5 was not affected by homozygous Mof deletion despite dramatic loss of global H4K16ac. Hematopoietic defects start manifesting in late gestation at E17.5. The discovery that MOF and its H4K16ac activity are required for adult but not early and midgestational hematopoiesis supports the notion that multiple chromatin regulators may be crucial for hematopoiesis at varying stages of development. MOF is therefore a developmental-stage-specific chromatin regulator found to be essential for adult but not early fetal hematopoiesis. © 2017 by The American Society of Hematology.

  2. Kras is required for adult hematopoiesis

    PubMed Central

    Damnernsawad, Alisa; Kong, Guangyao; Wen, Zhi; Liu, Yangang; Rajagopalan, Adhithi; You, Xiaona; Wang, Jinyong; Zhou, Yun; Ranheim, Erik A.; Luo, Hongbo R.; Chang, Qiang; Zhang, Jing

    2017-01-01

    Previous studies indicate that Kras is dispensable for fetal liver hematopoiesis, but its rolein adult hematopoiesis remains unclear. Here, we generated a Kras conditional knockout allele to address this question. Deletion of Kras in adult bone marrow is mediated by Vav-Cre or inducible Mx1-Cre. We find that loss of Kras leads to greatly reduced TPO signaling in hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs), while SCF-evoked ERK1/2 activation is not affected. The compromised TPO signaling is associated with reduced long term- and intermediate-term HSC compartments and a bias towards myeloid differentiation in MPPs. Although GM-CSF-evoked ERK1/2 activation is only moderately decreased in Kras−/− myeloid progenitors, it is blunted in neutrophils and neutrophil survival is significantly reduced in vitro. At 9–12 months old, Kras conditional knockout mice develop profound hematopoietic defects, including splenomegaly, an expanded neutrophil compartment, and reduced B cell number. In a serial transplantation assay, the reconstitution potential of Kras−/− bone marrow cells is greatly compromised, which is attributable to defects in the self-renewal of Kras−/− HSCs and defects in differentiated hematopietic cells. Our results demonstrate that Kras is a major regulator of TPO and GM-CSF signaling in specific populations of hematopoietic cells and its function is required for adult hematopoiesis. PMID:26972179

  3. Hematopoiesis

    PubMed Central

    Jagannathan-Bogdan, Madhumita; Zon, Leonard I.

    2013-01-01

    Hematopoiesis – the process by which blood cells are formed – has been studied intensely for over a century using a variety of model systems. There is conservation of the overall hematopoietic process between vertebrates, although some differences do exist. Over the last decade, the zebrafish has come to the forefront as a new model in hematopoiesis research, as it allows the use of large-scale genetics, chemical screens and transgenics. This comparative approach to understanding hematopoiesis has led to fundamental knowledge about the process and to the development of new therapies for disease. Here, we provide a broad overview of vertebrate hematopoiesis. We also highlight the benefits of using zebrafish as a model. PMID:23715539

  4. Loss of Runx1 perturbs adult hematopoiesis and is associated with a myeloproliferative phenotype

    PubMed Central

    Growney, Joseph D.; Shigematsu, Hirokazu; Li, Zhe; Lee, Benjamin H.; Adelsperger, Jennifer; Rowan, Rebecca; Curley, David P.; Kutok, Jeffery L.; Akashi, Koichi; Williams, Ifor R.; Speck, Nancy A.; Gilliland, D. Gary

    2005-01-01

    Homozygous loss of function of Runx1 (Runt-related transcription factor 1 gene) during murine development results in an embryonic lethal phenotype characterized by a complete lack of definitive hematopoiesis. In light of recent reports of disparate requirements for hematopoietic transcription factors during development as opposed to adult hematopoiesis, we used a conditional gene-targeting strategy to effect the loss of Runx1 function in adult mice. In contrast with the critical role of Runx1 during development, Runx1 was not essential for hematopoiesis in the adult hematopoietic compartment, though a number of significant hematopoietic abnormalities were observed. Runx1 excision had lineage-specific effects on B- and T-cell maturation and pronounced inhibition of common lymphocyte progenitor production. Runx1 excision also resulted in inefficient platelet production. Of note, Runx1-deficient mice developed a mild myeloproliferative phenotype characterized by an increase in peripheral blood neutrophils, an increase in myeloid progenitor populations, and extramedullary hematopoiesis composed of maturing myeloid and erythroid elements. These findings indicate that Runx1 deficiency has markedly different consequences during development compared with adult hematopoiesis, and they provide insight into the phenotypic manifestations of Runx1 deficiency in hematopoietic malignancies. PMID:15784726

  5. Stable long-term blood formation by stem cells in murine steady-state hematopoiesis.

    PubMed

    Zavidij, Oksana; Ball, Claudia R; Herbst, Friederike; Oppel, Felix; Fessler, Sylvia; Schmidt, Manfred; von Kalle, Christof; Glimm, Hanno

    2012-09-01

    Hematopoietic stem cells (HSCs) generate all mature blood cells during the whole lifespan of an individual. However, the clonal contribution of individual HSC and progenitor cells in steady-state hematopoiesis is poorly understood. To investigate the activity of HSCs under steady-state conditions, murine HSC and progenitor cells were genetically marked in vivo by integrating lentiviral vectors (LVs) encoding green fluorescent protein (GFP). Hematopoietic contribution of individual marked clones was monitored by determination of lentiviral integration sites using highly sensitive linear amplification-mediated-polymerase chain reaction. A remarkably stable small proportion of hematopoietic cells expressed GFP in LV-injected animals for up to 24 months, indicating stable marking of murine steady-state hematopoiesis. Analysis of the lentiviral integration sites revealed that multiple hematopoietic clones with both myeloid and lymphoid differentiation potential contributed to long-term hematopoiesis. In contrast to intrafemoral vector injection, intravenous administration of LV preferentially targeted short-lived progenitor cells. Myelosuppressive treatment of mice prior to LV-injection did not affect the marking efficiency. Our study represents the first continuous analysis of clonal behavior of genetically marked hematopoietic cells in an unmanipulated system, providing evidence that multiple clones are simultaneously active in murine steady-state hematopoiesis.

  6. From Embryo to Adult: Hematopoiesis along the Drosophila Life Cycle.

    PubMed

    Ramond, Elodie; Meister, Marie; Lemaitre, Bruno

    2015-05-26

    Studies on Drosophila hematopoiesis have thus far focused on the embryonic and larval origin of hemocytes, the fly blood cells. In this issue of Developmental Cell, Ghosh et al. (2015) identify adult hematopoietic hubs containing progenitors that can differentiate into different blood cell types.

  7. Functional significance of mononuclear phagocyte populations generated through adult hematopoiesis

    PubMed Central

    Gutknecht, Michael F.; Bouton, Amy H.

    2014-01-01

    Tissue homeostasis requires a complete repertoire of functional macrophages in peripheral tissues. Recent evidence indicates that many resident tissue macrophages are seeded during embryonic development and persist through adulthood as a consequence of localized proliferation. Mononuclear phagocytes are also produced during adult hematopoiesis; these cells are then recruited to sites throughout the body, where they function in tissue repair and remodeling, resolution of inflammation, maintenance of homeostasis, and disease progression. The focus of this review is on mononuclear phagocytes that comprise the nonresident monocyte/macrophage populations in the body. Key features of monocyte differentiation are presented, focusing primarily on the developmental hierarchy that is established through this process, the markers used to identify discrete cell populations, and novel, functional attributes of these cells. These features are then explored in the context of the tumor microenvironment, where mononuclear phagocytes exhibit extensive plasticity in phenotype and function. PMID:25225678

  8. Adult somatic progenitor cells and hematopoiesis in oysters.

    PubMed

    Jemaà, Mohamed; Morin, Nathalie; Cavelier, Patricia; Cau, Julien; Strub, Jean Marc; Delsert, Claude

    2014-09-01

    Long-lived animals show a non-observable age-related decline in immune defense, which is provided by blood cells that derive from self-renewing stem cells. The oldest living animals are bivalves. Yet, the origin of hemocytes, the cells involved in innate immunity, is unknown in bivalves and current knowledge about mollusk adult somatic stem cells is scarce. Here we identify a population of adult somatic precursor cells and show their differentiation into hemocytes. Oyster gill contains an as yet unreported irregularly folded structure (IFS) with stem-like cells bathing into the hemolymph. BrdU labeling revealed that the stem-like cells in the gill epithelium and in the nearby hemolymph replicate DNA. Proliferation of this cell population was further evidenced by phosphorylated-histone H3 mitotic staining. Finally, these small cells, most abundant in the IFS epithelium, were found to be positive for the stemness marker Sox2. We provide evidence for hematopoiesis by showing that co-expression of Sox2 and Cu/Zn superoxide dismutase, a hemocyte-specific enzyme, does not occur in the gill epithelial cells but rather in the underlying tissues and vessels. We further confirm the hematopoietic features of these cells by the detection of Filamin, a protein specific for a sub-population of hemocytes, in large BrdU-labeled cells bathing into gill vessels. Altogether, our data show that progenitor cells differentiate into hemocytes in the gill, which suggests that hematopoiesis occurs in oyster gills. © 2014. Published by The Company of Biologists Ltd.

  9. AML1/Runx1 as a versatile regulator of hematopoiesis: regulation of its function and a role in adult hematopoiesis.

    PubMed

    Kurokawa, Mineo

    2006-08-01

    AML1/Runx1, originally identified as a gene located at the breakpoint of the t(8;21) translocation, encodes a transcription factor that is widely expressed in multiple hematopoietic lineages and that regulates the expression of a variety of hematopoietic genes. Numerous studies have shown that AML1 is a critical regulator of hematopoietic development. In addition, AML1 is a frequent target for chromosomal translocation in human leukemia. The activity of AML1 can be modulated by various types of posttranslational modification, including phosphorylation and acetylation. Phosphorylation by extracellular signal-regulated kinase (ERK) is one of the mechanisms that dictate whether AML1 acts as either a transcriptional repressor or an activator of gene expression. Recently, a physiological role for AML1 in adult hematopoiesis was revealed by conditional gene targeting in mice. Remarkably, adult hematopoietic progenitors are maintained even in the absence of AML1, in stark contrast to the total disruption of definitive hematopoiesis during embryogenesis. AML1 is, however, critical for megakaryopoiesis and plays an important role in T-cell and B-cell development in adult mice. Recent analyses engineered to recreate hematopoiesis in vitro revealed that the transcriptional activity of AML1 is closely related with the potential of AML1 to generate hematopoietic cells and support thymocyte development.

  10. The Corepressor Tle4 Is a Novel Regulator of Murine Hematopoiesis and Bone Development

    PubMed Central

    Chen, Xi; Wang, Jianfeng; Ding, Dacheng; Yamin, Rae’e; Sweetser, David A.

    2014-01-01

    Hematopoiesis is a complex process that relies on various cell types, signaling pathways, transcription factors and a specific niche. The integration of these various components is of critical importance to normal blood development, as deregulation of these may lead to bone marrow failure or malignancy. Tle4, a transcriptional corepressor, acts as a tumor suppressor gene in a subset of acute myeloid leukemia, yet little is known about its function in normal and malignant hematopoiesis or in mammalian development. We report here that Tle4 knockout mice are runted and die at around four weeks with defects in bone development and BM aplasia. By two weeks of age, Tle4 knockout mice exhibit leukocytopenia, B cell lymphopenia, and significant reductions in hematopoietic stem and progenitor cells. Tle4 deficient hematopoietic stem cells are intrinsically defective in B lymphopoiesis and exhaust upon stress, such as serial transplantation. In the absence of Tle4 there is a profound decrease in bone mineralization. In addition, Tle4 knockout stromal cells are defective at maintaining wild-type hematopoietic stem cell function in vitro. In summary, we illustrate a novel and essential role for Tle4 in the extrinsic and intrinsic regulation of hematopoiesis and in bone development. PMID:25153823

  11. Arginine methyltransferase PRMT5 is essential for sustaining normal adult hematopoiesis

    PubMed Central

    Liu, Fan; Cheng, Guoyan; Hamard, Pierre-Jacques; Greenblatt, Sarah; Wang, Lan; Man, Na; Perna, Fabiana; Xu, Haiming; Tadi, Madhavi; Luciani, Luisa; Nimer, Stephen D.

    2015-01-01

    Epigenetic regulators play critical roles in normal hematopoiesis, and the activity of these enzymes is frequently altered in hematopoietic cancers. The major type II protein arginine methyltransferase PRMT5 catalyzes the formation of symmetric dimethyl arginine and has been implicated in various cellular processes, including pluripotency and tumorigenesis. Here, we generated Prmt5 conditional KO mice to evaluate the contribution of PRMT5 to adult hematopoiesis. Loss of PRMT5 triggered an initial but transient expansion of hematopoietic stem cells (HSCs); however, Prmt5 deletion resulted in a concurrent loss of hematopoietic progenitor cells (HPCs), leading to fatal BM aplasia. PRMT5-specific effects on hematopoiesis were cell intrinsic and depended on PRMT5 methyltransferase activity. We found that PRMT5-deficient hematopoietic stem and progenitor cells exhibited severely impaired cytokine signaling as well as upregulation of p53 and expression of its downstream targets. Together, our results demonstrate that PRMT5 plays distinct roles in the behavior of HSCs compared with HPCs and is essential for the maintenance of adult hematopoietic cells. PMID:26258414

  12. Modulation of TGF-β signaling by endoglin in murine hemangioblast development and primitive hematopoiesis.

    PubMed

    Zhang, Liying; Magli, Alessandro; Catanese, Jacquelyn; Xu, Zhaohui; Kyba, Michael; Perlingeiro, Rita C R

    2011-07-07

    Endoglin (Eng), an accessory receptor for the transforming growth factor β (TGF-β) superfamily, is required for proper hemangioblast and primitive hematopoietic development. However the mechanism by which endoglin functions at this early developmental stage is currently unknown. Transcriptional analyses of differentiating eng(-/-) and eng(+/+) ES cells revealed that lack of endoglin leads to profound reductions in the levels of key hematopoietic regulators, including Scl, Lmo2, and Gata2. We also detected lower levels of phosphorylated Smad1 (pSmad1), a downstream target signaling molecule associated with the TGF-β pathway. Using doxycycline-inducible ES cell lines, we interrogated the TGF-β signaling pathway by expressing activated forms of ALK-1 and ALK-5, type I receptors for TGF-β. Our results indicate that ALK-1 signaling promotes hemangioblast development and hematopoiesis, as evidenced by colony assays, gene expression and FACS analyses, whereas signaling by ALK-5 leads to the opposite effect, inhibition of hemangioblast and hematopoietic development. In Eng(-/-) ES cells, ALK-1 rescued both the defective hemangioblast development, and primitive erythropoiesis, indicating that ALK-1 signaling can compensate for the absence of endoglin. We propose that endoglin regulates primitive hematopoiesis by modulating the activity of the Smad1/5 signaling pathway in early stages of development.

  13. Role of Vitamin A/Retinoic Acid in Regulation of Embryonic and Adult Hematopoiesis

    PubMed Central

    Cañete, Ana; Cano, Elena; Muñoz-Chápuli, Ramón; Carmona, Rita

    2017-01-01

    Vitamin A is an essential micronutrient throughout life. Its physiologically active metabolite retinoic acid (RA), acting through nuclear retinoic acid receptors (RARs), is a potent regulator of patterning during embryonic development, as well as being necessary for adult tissue homeostasis. Vitamin A deficiency during pregnancy increases risk of maternal night blindness and anemia and may be a cause of congenital malformations. Childhood Vitamin A deficiency can cause xerophthalmia, lower resistance to infection and increased risk of mortality. RA signaling appears to be essential for expression of genes involved in developmental hematopoiesis, regulating the endothelial/blood cells balance in the yolk sac, promoting the hemogenic program in the aorta-gonad-mesonephros area and stimulating eryrthropoiesis in fetal liver by activating the expression of erythropoietin. In adults, RA signaling regulates differentiation of granulocytes and enhances erythropoiesis. Vitamin A may facilitate iron absorption and metabolism to prevent anemia and plays a key role in mucosal immune responses, modulating the function of regulatory T cells. Furthermore, defective RA/RARα signaling is involved in the pathogenesis of acute promyelocytic leukemia due to a failure in differentiation of promyelocytes. This review focuses on the different roles played by vitamin A/RA signaling in physiological and pathological mouse hematopoiesis duddurring both, embryonic and adult life, and the consequences of vitamin A deficiency for the blood system. PMID:28230720

  14. RCAD/Ufl1, a Ufm1 E3 ligase, is essential for hematopoietic stem cell function and murine hematopoiesis

    PubMed Central

    Zhang, M; Zhu, X; Zhang, Y; Cai, Y; Chen, J; Sivaprakasam, S; Gurav, A; Pi, W; Makala, L; Wu, J; Pace, B; Tuan-Lo, D; Ganapathy, V; Singh, N; Li, H

    2015-01-01

    The Ufm1 conjugation system is a novel ubiquitin-like modification system, consisting of Ufm1, Uba5 (E1), Ufc1 (E2) and poorly characterized E3 ligase(s). RCAD/Ufl1 (also known as KIAA0776, NLBP and Maxer) was reported to function as a Ufm1 E3 ligase in ufmylation (Ufm1-mediated conjugation) of DDRGK1 and ASC1 proteins. It has also been implicated in estrogen receptor signaling, unfolded protein response (UPR) and neurodegeneration, yet its physiological function remains completely unknown. In this study, we report that RCAD/Ufl1 is essential for embryonic development, hematopoietic stem cell (HSC) survival and erythroid differentiation. Both germ-line and somatic deletion of RCAD/Ufl1 impaired hematopoietic development, resulting in severe anemia, cytopenia and ultimately animal death. Depletion of RCAD/Ufl1 caused elevated endoplasmic reticulum stress and evoked UPR in bone marrow cells. In addition, loss of RCAD/Ufl1 blocked autophagic degradation, increased mitochondrial mass and reactive oxygen species, and led to DNA damage response, p53 activation and enhanced cell death of HSCs. Collectively, our study provides the first genetic evidence for the indispensable role of RCAD/Ufl1 in murine hematopoiesis and development. The finding of RCAD/Ufl1 as a key regulator of cellular stress response sheds a light into the role of a novel protein network including RCAD/Ufl1 and its associated proteins in regulating cellular homeostasis. PMID:25952549

  15. Fetal hyperglycemia and a high-fat diet contribute to aberrant glucose tolerance and hematopoiesis in adult rats.

    PubMed

    Blue, Emily K; Ballman, Kimberly; Boyle, Frances; Oh, Eunjin; Kono, Tatsuyoshi; Quinney, Sara K; Thurmond, Debbie C; Evans-Molina, Carmella; Haneline, Laura S

    2015-02-01

    Children exposed to gestational diabetes mellitus (GDM) during pregnancy are at increased risk of obesity, diabetes, and hypertension. Our goal was to identify metabolic and hematopoietic alterations after intrauterine exposure to maternal hyperglycemia that may contribute to the pathogenesis of chronic morbidities. Streptozotocin treatment induced maternal hyperglycemia during the last third of gestation in rat dams. Offspring of control mothers (OCM) and diabetic mothers (ODM) were evaluated for weight, glucose tolerance, insulin tolerance, and hematopoiesis defects. The effects of aging were examined in normal and high-fat diet (HFD)-fed young (8-wk-old) and aged (11-mo-old) OCM and ODM rats. Young adult ODM males on a normal diet, but not females, displayed improved glucose tolerance due to increased insulin levels. Aged ODM males and females gained more weight than OCM on a HFD and had worse glucose tolerance. Aged ODM males fed a HFD were also neutrophilic. Increases in bone marrow cellularity and myeloid progenitors preceded neutrophilia in ODM males fed a HFD. When combined with other risk factors like HFD and aging, changes in glucose metabolism and hematopoiesis may contribute to the increased risk of obesity, type 2 diabetes, and hypertension observed in children of GDM mothers.

  16. Crustacean hematopoiesis.

    PubMed

    Söderhäll, Irene

    2016-05-01

    Crustacean hemocytes are important mediators of immune reactions, and the regulation of hemocyte homeostasis is of utmost importance for the health of these animals. This review discusses the current knowledge on the lineages, synthesis and differentiation of hemocytes in crustaceans. Hematopoietic tissues, their origins, and the regulation of hematopoiesis during molting, seasonal variation and infection are discussed. Furthermore, studies concerning the molecular regulation of hemocyte formation in crustaceans are also described, and the different lineages and their molecular markers are discussed and compared with several insect species. Signaling pathways and the regulation of hematopoiesis by transcription factors are typically conserved among these arthropods, whereas cytokines and growth factors are more variable and species specific. However, considering the great diversity among the crustaceans, one should be cautious in drawing general conclusions from studies of only a few species.

  17. SIRT1 deficiency compromises mouse embryonic stem cell hematopoietic differentiation, and embryonic and adult hematopoiesis in the mouse

    PubMed Central

    Ou, Xuan; Chae, Hee-Don; Wang, Rui-Hong; Shelley, William C.; Cooper, Scott; Taylor, Tammi; Kim, Young-June; Deng, Chu-Xia; Yoder, Mervin C.

    2011-01-01

    SIRT1 is a founding member of a sirtuin family of 7 proteins and histone deacetylases. It is involved in cellular resistance to stress, metabolism, differentiation, aging, and tumor suppression. SIRT1−/− mice demonstrate embryonic and postnatal development defects. We examined hematopoietic and endothelial cell differentiation of SIRT1−/− mouse embryonic stem cells (ESCs) in vitro, and hematopoietic progenitors in SIRT1+/++/−, and −/− mice. SIRT1−/− ESCs formed fewer mature blast cell colonies. Replated SIRT1−/− blast colony-forming cells demonstrated defective hematopoietic potential. Endothelial cell production was unaltered, but there were defects in formation of a primitive vascular network from SIRT1−/−-derived embryoid bodies. Development of primitive and definitive progenitors derived from SIRT1−/− ESCs were also delayed and/or defective. Differentiation delay/defects were associated with delayed capacity to switch off Oct4, Nanog and Fgf5 expression, decreased β-H1 globin, β-major globin, and Scl gene expression, and reduced activation of Erk1/2. Ectopic expression of SIRT1 rescued SIRT1−/− ESC differentiation deficiencies. SIRT1−/− yolk sacs manifested fewer primitive erythroid precursors. SIRT1−/− and SIRT1+/− adult marrow had decreased numbers and cycling of hematopoietic progenitors, effects more apparent at 5%, than at 20%, oxygen tension, and these progenitors survived less well in vitro under conditions of delayed growth factor addition. This suggests a role for SIRT1 in ESC differentiation and mouse hematopoiesis. PMID:20966168

  18. Wnts are dispensable for differentiation and self-renewal of adult murine hematopoietic stem cells

    PubMed Central

    Kabiri, Zahra; Numata, Akihiko; Kawasaki, Akira; Tenen, Daniel G.

    2015-01-01

    Wnt signaling controls early embryonic hematopoiesis and dysregulated β-catenin is implicated in leukemia. However, the role of Wnts and their source in adult hematopoiesis is still unclear, and is clinically important as upstream Wnt inhibitors enter clinical trials. We blocked Wnt secretion in hematopoietic lineages by targeting Porcn, a membrane-bound O-acyltransferase that is indispensable for the activity and secretion of all vertebrate Wnts. Surprisingly, deletion of Porcn in Rosa-CreERT2/PorcnDel, MX1-Cre/PorcnDel, and Vav-Cre/PorcnDel mice had no effects on proliferation, differentiation, or self-renewal of adult hematopoietic stem cells. Targeting Wnt secretion in the bone marrow niche by treatment with a PORCN inhibitor, C59, similarly had no effect on hematopoiesis. These results exclude a role for hematopoietic PORCN-dependent Wnts in adult hematopoiesis. Clinical use of upstream Wnt inhibitors is not likely to be limited by effects on hematopoiesis. PMID:26089398

  19. Cyclic Hematopoiesis: animal models

    SciTech Connect

    Jones, J.B.; Lange, R.D.

    1983-08-01

    The four existing animal models of cyclic hematopoiesis are briefly described. The unusual erythropoietin (Ep) responses of the W/Wv mouse, the Sl/Sld mouse, and cyclic hematopoietic dog are reviewed. The facts reviewed indicate that the bone marrow itself is capable of influencing regulatory events of hematopoiesis.

  20. Role of Polycomb RYBP in Maintaining the B-1-to-B-2 B-Cell Lineage Switch in Adult Hematopoiesis

    PubMed Central

    Pavón, Leticia; Starowicz, Katarzyna; Pérez, Claudia; Bravo, Mónica; Ikawa, Tomokatsu; Koseki, Haruhiko

    2015-01-01

    Polycomb chromatin modifiers regulate hematopoietic pluripotent stem and progenitor cell self-renewal and expansion. Polycomb complex redundancy and biochemical heterogeneity complicate the unraveling of the functional contributions of distinct components. We have studied the hematopoietic activity of RYBP, a direct interactor and proposed modulator of RING1A/RING1B-dependent histone H2A monoubiquitylation (H2AUb). Using a mouse model to conditionally inactivate Rybp in adult hematopoiesis, we have found that RYBP deletion results in a reversion of B-1-to-B-2 B-cell progenitor ratios, i.e., of the innate (predominantly fetal) to acquired (mostly adult) immunity precursors. Increased numbers of B-1 progenitors correlated with a loss of pre-proB cells, the B-2 progenitors. RYBP-deficient stem and progenitor cell populations (LKS) and isolated common lymphoid progenitors (CLP) gave rise to increased numbers of B-1 progenitors in vitro. Rybp inactivation, however, did not result in changes of global H2AUb and did not interact genetically with Ring1A or Ring1B deletions. These results show that a sustained regulation of the B-1-to-B-2 switch is needed throughout adult life and that RYBP plays an important role in keeping B-2 dominance, most likely independently of its Polycomb affiliation. PMID:26711264

  1. Tachykinins and hematopoiesis.

    PubMed

    Liu, Katherine; Castillo, Marianne D; Murthy, Raghav G; Patel, Nitixa; Rameshwar, Pranela

    2007-10-01

    Originally discovered in the 1930s, tachykinins have been a subject of renewed interest. Antagonists to the tachykinin receptors have shown potential in the treatment of a variety of maladies including neurodegenerative disorders, heart disease, pain perception and malignancies. Tachykinins have been the subject of intense studies due to their impact on hematopoiesis that has significant effects on endothelial tissue and vascular conditions. Hematopoiesis relies on a relatively small subset of bone marrow-resident hematopoietic stem cells. This review discusses the network developed by cytokines and the tachykinins to regulate hematopoiesis. An understanding of tachykinin effect on normal hematopoietic functions and their involvement in hematological disorders could lead to new treatments for bone marrow disorders such as fibrosis, leukemia and anemia.

  2. From transplantation to transgenics: mouse models of developmental hematopoiesis.

    PubMed

    Schmitt, Christopher E; Lizama, Carlos O; Zovein, Ann C

    2014-08-01

    The mouse is integral to our understanding of hematopoietic biology. Serving as a mammalian model system, the mouse has allowed for the discovery of self-renewing multipotent stem cells, provided functional assays to establish hematopoietic stem cell identity and function, and has become a tool for understanding the differentiation capacity of early hematopoietic progenitors. The advent of genetic technology has strengthened the use of mouse models for identifying critical pathways in hematopoiesis. Full genetic knockout models, tissue-specific gene deletion, and genetic overexpression models create a system for the dissection and identification of critical cellular and genetic processes underlying hematopoiesis. However, the murine model has also introduced perplexity in understanding developmental hematopoiesis. Requisite in utero development paired with circulation has historically made defining sites of origin and expansion in the murine hematopoietic system challenging. However, the genetic accessibility of the mouse as a mammalian system has identified key regulators of hematopoietic development. Technological advances continue to generate extremely powerful tools that when translated to the murine system provide refined in vivo spatial and temporal control of genetic deletion or overexpression. Future advancements may add the ability of reversible genetic manipulation. In this review, we describe the major contributions of the murine model to our understanding of hematopoiesis. Copyright © 2014 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  3. Inhibition of Thrombopoietin/Mpl Signaling in Adult Hematopoiesis Identifies New Candidates for Hematopoietic Stem Cell Maintenance

    PubMed Central

    Schwarzer, Adrian; Kamp, Christel; Brugman, Martijn H.; Breuer, Daniel C.; Büsche, Guntram; Baum, Christopher; Modlich, Ute

    2015-01-01

    Thrombopoietin (Thpo) signals via its receptor Mpl and regulates megakaryopoiesis, hematopoietic stem cell (HSC) maintenance and post-transplant expansion. Mpl expression is tightly controlled and deregulation of Thpo/Mpl-signaling is linked to hematological disorders. Here, we constructed an intracellular-truncated, signaling-deficient Mpl protein which is presented on the cell surface (dnMpl). The transplantation of bone marrow cells retrovirally transduced to express dnMpl into wildtype mice induced thrombocytopenia, and a progressive loss of HSC. The aplastic BM allowed the engraftment of a second BM transplant without further conditioning. Functional analysis of the truncated Mpl in vitro and in vivo demonstrated no internalization after Thpo binding and the inhibition of Thpo/Mpl-signaling in wildtype cells due to dominant-negative (dn) effects by receptor competition with wildtype Mpl for Thpo binding. Intracellular inhibition of Mpl could be excluded as the major mechanism by the use of a constitutive-dimerized dnMpl. To further elucidate the molecular changes induced by Thpo/Mpl-inhibition on the HSC-enriched cell population in the BM, we performed gene expression analysis of Lin-Sca1+cKit+ (LSK) cells isolated from mice transplanted with dnMpl transduced BM cells. The gene expression profile supported the exhaustion of HSC due to increased cell cycle progression and identified new and known downstream effectors of Thpo/Mpl-signaling in HSC (namely TIE2, ESAM1 and EPCR detected on the HSC-enriched LSK cell population). We further compared gene expression profiles in LSK cells of dnMpl mice with human CD34+ cells of aplastic anemia patients and identified similar deregulations of important stemness genes in both cell populations. In summary, we established a novel way of Thpo/Mpl inhibition in the adult mouse and performed in depth analysis of the phenotype including gene expression profiling. PMID:26147434

  4. Lithium and hematopoiesis.

    PubMed Central

    Barr, R. D.; Galbraith, P. R.

    1983-01-01

    Some of lithium's effects on blood cell formation suggest that the element may be of value in treating hematologic disorders. Lithium enhances granulopoiesis and thereby induces neutrophilia. Two possible mechanisms of action are suggested: a direct action on the pluripotent stem cells, or an inhibition of the suppressor cells (thymus-dependent lymphocytes) that limit hematopoiesis. Lithium also inhibits erythropoiesis. Although most studies use concentrations at or above pharmacologic levels there is evidence that lithium plays a role in normal cell metabolism. PMID:6336655

  5. Insect immunology and hematopoiesis.

    PubMed

    Hillyer, Julián F

    2016-05-01

    Insects combat infection by mounting powerful immune responses that are mediated by hemocytes, the fat body, the midgut, the salivary glands and other tissues. Foreign organisms that have entered the body of an insect are recognized by the immune system when pathogen-associated molecular patterns bind host-derived pattern recognition receptors. This, in turn, activates immune signaling pathways that amplify the immune response, induce the production of factors with antimicrobial activity, and activate effector pathways. Among the immune signaling pathways are the Toll, Imd, Jak/Stat, JNK, and insulin pathways. Activation of these and other pathways leads to pathogen killing via phagocytosis, melanization, cellular encapsulation, nodulation, lysis, RNAi-mediated virus destruction, autophagy and apoptosis. This review details these and other aspects of immunity in insects, and discusses how the immune and circulatory systems have co-adapted to combat infection, how hemocyte replication and differentiation takes place (hematopoiesis), how an infection prepares an insect for a subsequent infection (immune priming), how environmental factors such as temperature and the age of the insect impact the immune response, and how social immunity protects entire groups. Finally, this review highlights some underexplored areas in the field of insect immunobiology.

  6. Differential expression of murine adult hemoglobins in early ontogeny

    SciTech Connect

    Wawrzyniak, C.J.; Lewis, S.E.; Popp, R.A.

    1985-01-01

    A hemoglobin mutation is described that permits study of the expression of the two adult ..beta..-globin genes throughout fetal and postnatal development. Mice with a mutation at the Hbb/sup s/, ..beta..-globin locus, were used to study the relative levels of ..beta..-s2major and ..beta..-sminor globins specified by the mutant Hbb/sup s2/ haplotype during development. At 11.5 days of gestation ..beta..-sminor comprised over 80% and ..beta..-s2major under 20% of the adult beta-globin. The relative level of ..beta..-sminor decreased through fetal development; at birth ..beta..-sminor represented 33.7% of the ..beta..-globin. The adult values of 71.0% ..beta..-s2major and 29.0% ..beta..-sminor globin are expressed in mice six days after birth. Because the two ..beta..-globin genes are expressed in mice of the Hbb/sup 2s/ haplotype, both the ..beta..-smajor and ..beta..-sminor genes must be expressed in mice of the Hbb/sup s/ haplotype. Expression of the ..beta..-sminor gene is elevated to 35.6% in Hbb/sup s2/ mice that have been bled repeatedly. Thus, the 5' ..beta..-s2major and 3' ..beta..-sminor genes of the Hbb/sup s2/ haplotype and, presumably the 5' ..beta..-smajor and 3' ..beta..-sminor genes of the Hbb/sup s/ haplotype, are regulated independently and are homologous to the 5' ..beta..-dmajor and 3' ..beta..-dminor genes of the Hbb/sup d/ haplotype. Mice of the Hbb/sup s2/ haplotype are better than mice of the Hbb/sup d/ haplotytpe for studying the mechanisms of hemoglobin switching because the Hbb/sup s2/ each of the three embryonic and two adult hemoglobins can be separated by electrophoresis. 17 refs., 3 figs.

  7. The megakaryocyte lineage originates from hemangioblast precursors and is an integral component both of primitive and of definitive hematopoiesis

    PubMed Central

    Tober, Joanna; Koniski, Anne; McGrath, Kathleen E.; Vemishetti, Radhika; Emerson, Rachael; de Mesy-Bentley, Karen K. L.; Waugh, Richard; Palis, James

    2007-01-01

    In the adult, platelets are derived from unipotential megakaryocyte colony-forming cells (Meg-CFCs) that arise from bipotential megakaryocyte/erythroid progenitors (MEPs). To better define the developmental origin of the megakaryocyte lineage, several aspects of megakaryopoiesis, including progenitors, maturing megakaryocytes, and circulating platelets, were examined in the murine embryo. We found that a majority of hemangioblast precursors during early gastrulation contains megakaryocyte potential. Combining progenitor assays with immunohistochemical analysis, we identified 2 waves of MEPs in the yolk sac associated with the primitive and definitive erythroid lineages. Primitive MEPs emerge at E7.25 along with megakaryocyte and primitive erythroid progenitors, indicating that primitive hematopoiesis is bilineage in nature. Subsequently, definitive MEPs expand in the yolk sac with Meg-CFCs and definitive erythroid progenitors. The first GP1bβ-positive cells in the conceptus were identified in the yolk sac at E9.5, while large, highly reticulated platelets were detected in the embryonic bloodstream beginning at E10.5. At this time, the number of megakaryocyte progenitors begins to decline in the yolk sac and expand in the fetal liver. We conclude that the megakaryocyte lineage initially originates from hemangioblast precursors during early gastrulation and is closely associated both with primitive and with definitive erythroid lineages in the yolk sac prior to the transition of hematopoiesis to intraembryonic sites. PMID:17062726

  8. Developmental hematopoiesis: ontogeny, genetic programming and conservation.

    PubMed

    Ciau-Uitz, Aldo; Monteiro, Rui; Kirmizitas, Arif; Patient, Roger

    2014-08-01

    Hematopoietic stem cells (HSCs) sustain blood production throughout life and are of pivotal importance in regenerative medicine. Although HSC generation from pluripotent stem cells would resolve their shortage for clinical applications, this has not yet been achieved mainly because of the poor mechanistic understanding of their programming. Bone marrow HSCs are first created during embryogenesis in the dorsal aorta (DA) of the midgestation conceptus, from where they migrate to the fetal liver and, eventually, the bone marrow. It is currently accepted that HSCs emerge from specialized endothelium, the hemogenic endothelium, localized in the ventral wall of the DA through an evolutionarily conserved process called the endothelial-to-hematopoietic transition. However, the endothelial-to-hematopoietic transition represents one of the last steps in HSC creation, and an understanding of earlier events in the specification of their progenitors is required if we are to create them from naïve pluripotent cells. Because of their ready availability and external development, zebrafish and Xenopus embryos have enormously facilitated our understanding of the early developmental processes leading to the programming of HSCs from nascent lateral plate mesoderm to hemogenic endothelium in the DA. The amenity of the Xenopus model to lineage tracing experiments has also contributed to the establishment of the distinct origins of embryonic (yolk sac) and adult (HSC) hematopoiesis, whereas the transparency of the zebrafish has allowed in vivo imaging of developing blood cells, particularly during and after the emergence of HSCs in the DA. Here, we discuss the key contributions of these model organisms to our understanding of developmental hematopoiesis. Copyright © 2014 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  9. Crustacean hematopoiesis and the astakine cytokines.

    PubMed

    Lin, Xionghui; Söderhäll, Irene

    2011-06-16

    Major contributions to research in hematopoiesis in invertebrate animals have come from studies in the fruit fly, Drosophila melanogaster, and the freshwater crayfish, Pacifastacus leniusculus. These animals lack oxygen-carrying erythrocytes and blood cells of the lymphoid lineage, which participate in adaptive immune defense, thus making them suitable model animals to study the regulation of blood cells of the innate immune system. This review presents an overview of crustacean blood cell formation, the role of these cells in innate immunity, and how their synthesis is regulated by the astakine cytokines. Astakines are among the first invertebrate cytokines shown to be involved in hematopoiesis, and they can stimulate the proliferation, differentiation, and survival of hematopoietic tissue cells. The astakines and their vertebrate homologues, prokineticins, share similar functions in hematopoiesis; thus, studies of astakine-induced hematopoiesis in crustaceans may not only advance our understanding of the regulation of invertebrate hematopoiesis but may also provide new evolutionary perspectives about this process.

  10. Long noncoding RNAs in hematopoiesis

    PubMed Central

    Zhang, Xu; Hu, Wenqian

    2016-01-01

    Mammalian development is under tight control to ensure precise gene expression. Recent studies reveal a new layer of regulation of gene expression mediated by long noncoding RNAs. These transcripts are longer than 200nt that do not have functional protein coding capacity. Interestingly, many of these long noncoding RNAs are expressed with high specificity in different types of cells, tissues, and developmental stages in mammals, suggesting that they may have functional roles in diverse biological processes. Here, we summarize recent findings of long noncoding RNAs in hematopoiesis, which is one of the best-characterized mammalian cell differentiation processes. Then we provide our own perspectives on future studies of long noncoding RNAs in this field. PMID:27508063

  11. Stat5-deficient hematopoiesis is permissive for Myc-induced B-cell leukemogenesis.

    PubMed

    Wang, Zhengqi; Medrzycki, Magdalena; Bunting, Silvia T; Bunting, Kevin D

    2015-10-06

    Despite being an attractive molecular target for both lymphoid and myeloid leukemias characterized by activated tyrosine kinases, the molecular and physiological consequences of reduced signal transducer and activator of transcription-5 (Stat5) during leukemogenesis are not well known. Stat5 is a critical regulator of mouse hematopoietic stem cell (HSC) self-renewal and is essential for normal lymphocyte development. We report that pan-hematopoietic deletion in viable adult Vav1-Cre conditional knockout mice as well as Stat5ab(null/null) fetal liver transplant chimeras generated HSCs with reduced expression of quiescence regulating genes (Tie2, Mpl, Slamf1, Spi1, Cited2) and increased expression of B-cell development genes (Satb1, Dntt, Btla, Flk2). Using a classical murine B-cell acute lymphoblastic leukemia (B-ALL) model, we demonstrate that these HSCs were also poised to produce a burst of B-cell precursors upon expression of Bcl-2 combined with oncogenic Myc. This strong selective advantage for leukemic transformation in the background of Stat5 deficient hematopoiesis was permissive for faster initiation of Myc-induced transformation to B-ALL. However, once established, the B-ALL progression in secondary transplant recipients was Stat5-independent. Overall, these studies suggest that Stat5 can play multiple important roles that not only preserve the HSC compartment but can limit accumulation of potential pre-leukemic lymphoid populations.

  12. Partial Characterization of the Sox2+ Cell Population in an Adult Murine Model of Digit Amputation

    PubMed Central

    Agrawal, Vineet; Siu, Bernard F.; Chao, Hsu; Hirschi, Karen K.; Raborn, Eric; Johnson, Scott A.; Tottey, Stephen; Hurley, Katherine B.; Medberry, Chris J.

    2012-01-01

    Tissue regeneration in response to injury in adult mammals is generally limited to select tissues. Nonmammalian species such as newts and axolotls undergo regeneration of complex tissues such as limbs and digits via recruitment and accumulation of local and circulating multipotent progenitors preprogrammed to recapitulate the missing tissue. Directed recruitment and activation of progenitor cells at a site of injury in adult mammals may alter the default wound-healing response from scar tissue toward regeneration. Bioactive molecules derived from proteolytic degradation of extracellular matrix (ECM) proteins have been shown to recruit a variety of progenitor cells in vitro and in vivo to the site of injury. The present study further characterized the population of cells accumulating at the site of injury after treatment with ECM degradation products in a well-established model of murine digit amputation. After a mid-second phalanx digit amputation in 6–8-week-old adult mice, treatment with ECM degradation products resulted in the accumulation of a heterogeneous population of cells, a subset of which expressed the transcription factor Sox2, a marker of pluripotent and adult progenitor cells. Sox2+ cells were localized lateral to the amputated P2 bone and coexpressed progenitor cell markers CD90 and Sca1. Transgenic Sox2 eGFP/+ and bone marrow chimeric mice showed that the bone marrow and blood circulation did not contribute to the Sox2+ cell population. The present study showed that, in addition to circulating progenitor cells, resident tissue-derived cells also populate at the site of injury after treatment with ECM degradation products. Although future work is necessary to determine the contribution of Sox2+ cells to functional tissue at the site of injury, recruitment and/or activation of local tissue-derived cells may be a viable approach to tissue engineering of more complex tissues in adult mammals. PMID:22530556

  13. Assessment of DNA synthesis in Islet-1{sup +} cells in the adult murine heart

    SciTech Connect

    Weinberger, Florian Mehrkens, Dennis Starbatty, Jutta Nicol, Philipp Eschenhagen, Thomas

    2015-01-02

    Highlights: • Islet-1 was expressed in the adult heart. • Islet-1-positive cells did not proliferate in the adult heart. • Sinoatrial node cells did not proliferate in the adult heart. - Abstract: Rationale: Islet-1 positive (Islet-1{sup +}) cardiac progenitor cells give rise to the right ventricle, atria and outflow tract during murine cardiac development. In the adult heart Islet-1 expression is limited to parasympathetic neurons, few cardiomyocytes, smooth muscle cells, within the proximal aorta and pulmonary artery and sinoatrial node cells. Its role in these cells is unknown. Here we tested the hypothesis that Islet-1{sup +} cells retain proliferative activity and may therefore play a role in regenerating specialized regions in the heart. Methods and results: DNA synthesis was analyzed by the incorporation of tritiated thymidine ({sup 3}H-thymidine) in Isl-1-nLacZ mice, a transgenic model with an insertion of a nuclear beta-galactosidase in the Islet-1 locus. Mice received daily injections of {sup 3}H-thymidine for 5 days. DNA synthesis was visualized throughout the heart by dipping autoradiography of cryosections. Colocalization of an nLacZ-signal and silver grains would indicate DNA synthesis in Islet-1{sup +} cells. Whereas Islet{sup −} non-myocyte nuclei were regularly marked by accumulation of silver grains, colocalization with nLacZ-signals was not detected in >25,000 cells analyzed. Conclusions: Islet-1{sup +} cells are quiescent in the adult heart, suggesting that, under normal conditions, even pacemaking cells do not proliferate at higher rates than normal cardiac myocytes.

  14. Label-Retaining Cells in the Adult Murine Salivary Glands Possess Characteristics of Adult Progenitor Cells

    PubMed Central

    Chibly, Alejandro M.; Querin, Lauren; Harris, Zoey; Limesand, Kirsten H.

    2014-01-01

    Radiotherapy is the primary treatment for patients with head and neck cancer, which account for roughly 500,000 annual cases worldwide. Dysfunction of the salivary glands and associated conditions like xerostomia and dysphagia are often developed by these patients, greatly diminishing their life quality. Current preventative and palliative care fail to deliver an improvement in the quality of life, thus accentuating the need for regenerative therapies. In this study, a model of label retaining cells (LRCs) in murine salivary glands was developed, in which LRCs demonstrated proliferative potential and possessed markers of putative salivary progenitors. Mice were labeled with 5-Ethynyl-2′-deoxyuridine (EdU) at postnatal day 10 and chased for 8 weeks. Tissue sections from salivary glands obtained at the end of chase demonstrated co-localization between LRCs and the salivary progenitor markers keratin 5 and keratin 14, as well as kit mRNA, indicating that LRCs encompass a heterogeneous population of salivary progenitors. Proliferative potential of LRCs was demonstrated by a sphere assay, in which LRCs were found in primary and secondary spheres and they co-localized with the proliferation marker Ki67 throughout sphere formation. Surprisingly, LRCs were shown to be radio-resistant and evade apoptosis following radiation treatment. The clinical significance of these findings lie in the potential of this model to study the mechanisms that prevent salivary progenitors from maintaining homeostasis upon exposure to radiation, which will in turn facilitate the development of regenerative therapies for salivary gland dysfunction. PMID:25238060

  15. Identification and enrichment of colony-forming cells from the adult murine pituitary

    SciTech Connect

    Lepore, D.A.; Roeszler, K.; Wagner, J.; Ross, S.A.; Bauer, K.; Thomas, P.Q. , E-Mail: paul.thomas@mcri.edu.au

    2005-08-01

    Stem and progenitor cells have been identified in many adult tissues including bone marrow, the central nervous system, and skin. While there is direct evidence to indicate the activity of a progenitor cell population in the pituitary gland, this putative subpopulation has not yet been identified. Herein we describe the isolation and characterization of a novel clonogenic cell type in the adult murine pituitary, which we have termed Pituitary Colony-Forming Cells (PCFCs). PCFCs constitute 0.2% of pituitary cells, and generate heterogeneous colonies from single cells. PCFCs exhibit variable proliferative potential, and may exceed 11 population doublings in 14 days. Enrichment of PCFCs to 61.5-fold with 100% recovery can be obtained through the active uptake of the fluorescent dipeptide, {beta}-Ala-Lys-N{epsilon}-AMCA. PCFCs are mostly contained within the large, agranular subpopulation of AMCA{sup +} cells, and constitute 28% of this fraction, corresponding to 140.5-fold enrichment. Interestingly, the AMCA{sup +} population contains rare cells that are GH{sup +} or PRL{sup +}. GH{sup +} cells were also identified in PCFC single cell colonies, suggesting that PCFCs have the potential to differentiate into GH{sup +} cells. Together, these data show that the pituitary contains a rare clonogenic population which may correspond to the somatotrope/lactotrope progenitors suggested by previous experiments.

  16. Alternatively activated macrophages determine repair of the infarcted adult murine heart

    PubMed Central

    Shiraishi, Manabu; Shintani, Yasunori; Shintani, Yusuke; Ishida, Hidekazu; Saba, Rie; Yamaguchi, Atsushi; Adachi, Hideo; Yashiro, Kenta

    2016-01-01

    Alternatively activated (also known as M2) macrophages are involved in the repair of various types of organs. However, the contribution of M2 macrophages to cardiac repair after myocardial infarction (MI) remains to be fully characterized. Here, we identified CD206+F4/80+CD11b+ M2-like macrophages in the murine heart and demonstrated that this cell population predominantly increases in the infarct area and exhibits strengthened reparative abilities after MI. We evaluated mice lacking the kinase TRIB1 (Trib1–/–), which exhibit a selective depletion of M2 macrophages after MI. Compared with control animals, Trib1–/– mice had a catastrophic prognosis, with frequent cardiac rupture, as the result of markedly reduced collagen fibril formation in the infarct area due to impaired fibroblast activation. The decreased tissue repair observed in Trib1–/– mice was entirely rescued by an external supply of M2-like macrophages. Furthermore, IL-1α and osteopontin were suggested to be mediators of M2-like macrophage–induced fibroblast activation. In addition, IL-4 administration achieved a targeted increase in the number of M2-like macrophages and enhanced the post-MI prognosis of WT mice, corresponding with amplified fibroblast activation and formation of more supportive fibrous tissues in the infarcts. Together, these data demonstrate that M2-like macrophages critically determine the repair of infarcted adult murine heart by regulating fibroblast activation and suggest that IL-4 is a potential biological drug for treating MI. PMID:27140396

  17. Ginger Stimulates Hematopoiesis via Bmp Pathway in Zebrafish

    PubMed Central

    Ferri-Lagneau, Karine F.; Moshal, Karni S.; Grimes, Matthew; Zahora, Braden; Lv, Lishuang; Sang, Shengmin; Leung, TinChung

    2012-01-01

    effects in adults. Our results will provide the basis for future research into the effect of ginger during mammalian hematopoiesis to develop novel erythropoiesis promoting agents. PMID:22761764

  18. Cannabinoid receptor 2 and its agonists mediate hematopoiesis and hematopoietic stem and progenitor cell mobilization

    PubMed Central

    Jiang, Shuxian; Alberich-Jorda, Meritxell; Zagozdzon, Radoslaw; Parmar, Kalindi; Fu, Yigong; Mauch, Peter; Banu, Naheed; Makriyannis, Alexandros; Tenen, Daniel G.; Avraham, Shalom; Groopman, Jerome E.

    2011-01-01

    Endocannabinoids are arachidonic acid derivatives and part of a novel bioactive lipid signaling system, along with their G-coupled cannabinoid receptors (CB1 and CB2) and the enzymes involved in their biosynthesis and degradation. However, their roles in hematopoiesis and hematopoietic stem and progenitor cell (HSPC) functions are not well characterized. Here, we show that bone marrow stromal cells express endocannabinoids (anandamide and 2-arachidonylglycerol), whereas CB2 receptors are expressed in human and murine HSPCs. On ligand stimulation with CB2 agonists, CB2 receptors induced chemotaxis, migration, and enhanced colony formation of bone marrow cells, which were mediated via ERK, PI3-kinase, and Gαi-Rac1 pathways. In vivo, the CB2 agonist AM1241 induced mobilization of murine HSPCs with short- and long-term repopulating abilities. In addition, granulocyte colony-stimulating factor -induced mobilization of HSPCs was significantly decreased by specific CB2 antagonists and was impaired in Cnr2−/− cannabinoid type 2 receptor knockout mice. Taken together, these results demonstrate that the endocannabinoid system is involved in hematopoiesis and that CB2/CB2 agonist axis mediates repopulation of hematopoiesis and mobilization of HSPCs. Thus, CB2 agonists may be therapeutically applied in clinical conditions, such as bone marrow transplantation. PMID:21063029

  19. The histone demethylase UTX regulates stem cell migration and hematopoiesis.

    PubMed

    Thieme, Sebastian; Gyárfás, Tobias; Richter, Cornelia; Özhan, Günes; Fu, Jun; Alexopoulou, Dimitra; Muders, Michael H; Michalk, Irene; Jakob, Christiane; Dahl, Andreas; Klink, Barbara; Bandola, Joanna; Bachmann, Michael; Schröck, Evelin; Buchholz, Frank; Stewart, A Francis; Weidinger, Gilbert; Anastassiadis, Konstantinos; Brenner, Sebastian

    2013-03-28

    Regulated migration of hematopoietic stem cells is fundamental for hematopoiesis. The molecular mechanisms underlying stem cell trafficking are poorly defined. Based on a short hairpin RNA library and stromal cell-derived factor-1 (SDF-1) migration screening assay, we identified the histone 3 lysine 27 demethylase UTX (Kdm6a) as a novel regulator for hematopoietic cell migration. Using hematopoietic stem and progenitor cells from our conditional UTX knockout (KO) mice, we were able to confirm the regulatory function of UTX on cell migration. Moreover, adult female conditional UTX KO mice displayed myelodysplasia and splenic erythropoiesis, whereas UTX KO males showed no phenotype. During development, all UTX KO female and a portion of UTX KO male embryos developed a cardiac defect, cranioschisis, and died in utero. Therefore, UTY, the male homolog of UTX, can compensate for UTX in adults and partially during development. Additionally, we found that UTX knockdown in zebrafish significantly impairs SDF-1/CXCR4-dependent migration of primordial germ cells. Our data suggest that UTX is a critical regulator for stem cell migration and hematopoiesis.

  20. Posterior mediastinal extramedullary hematopoiesis secondary to hypoxia

    PubMed Central

    Solazzo, A; D’Auria, V; Moccia, LG; Vatrella, A; Bocchino, M; Rea, G

    2016-01-01

    Two mediastinal masses were incidentally detected at high resolution computed tomography (HRCT) of a 72 year-old male patient, former smoker, affected by chronic obstructive pulmonary disease with worsening dyspnea and 2-year medical history of polycythemia secondary to hypoxia. Integration with a multidetector computed tomography (MDCT) scan after administration of intravenous injection contrast medium showed slightly inhomogeneous increase of enhancement of masses, suggesting in the first case potential malignancy. Diagnosis of extramedullary hematopoiesis was achieved by fine needle aspiration citology (FNAC). Extramedullary hematopoiesis must be considered in differential diagnosis in patients with medical history of polycythemia and severe hypoxia. PMID:27326388

  1. Posterior mediastinal extramedullary hematopoiesis secondary to hypoxia.

    PubMed

    Solazzo, A; D'Auria, V; Moccia, L G; Vatrella, A; Bocchino, M; Rea, G

    2016-05-01

    Two mediastinal masses were incidentally detected at high resolution computed tomography (HRCT) of a 72 year-old male patient, former smoker, affected by chronic obstructive pulmonary disease with worsening dyspnea and 2-year medical history of polycythemia secondary to hypoxia. Integration with a multidetector computed tomography (MDCT) scan after administration of intravenous injection contrast medium showed slightly inhomogeneous increase of enhancement of masses, suggesting in the first case potential malignancy. Diagnosis of extramedullary hematopoiesis was achieved by fine needle aspiration citology (FNAC). Extramedullary hematopoiesis must be considered in differential diagnosis in patients with medical history of polycythemia and severe hypoxia.

  2. The Proteome of Native Adult Müller Glial Cells From Murine Retina*

    PubMed Central

    Hauser, Alexandra; Lepper, Marlen Franziska; Mayo, Rebecca

    2016-01-01

    To date, the proteomic profiling of Müller cells, the dominant macroglia of the retina, has been hampered because of the absence of suitable enrichment methods. We established a novel protocol to isolate native, intact Müller cells from adult murine retinae at excellent purity which retain in situ morphology and are well suited for proteomic analyses. Two different strategies of sample preparation - an in StageTips (iST) and a subcellular fractionation approach including cell surface protein profiling were used for quantitative liquid chromatography-mass spectrometry (LC-MSMS) comparing Müller cell-enriched to depleted neuronal fractions. Pathway enrichment analyses on both data sets enabled us to identify Müller cell-specific functions which included focal adhesion kinase signaling, signal transduction mediated by calcium as second messenger, transmembrane neurotransmitter transport and antioxidant activity. Pathways associated with RNA processing, cellular respiration and phototransduction were enriched in the neuronal subpopulation. Proteomic results were validated for selected Müller cell genes by quantitative real time PCR, confirming the high expression levels of numerous members of the angiogenic and anti-inflammatory annexins and antioxidant enzymes (e.g. paraoxonase 2, peroxiredoxin 1, 4 and 6). Finally, the significant enrichment of antioxidant proteins in Müller cells was confirmed by measurements on vital retinal cells using the oxidative stress indicator CM-H2DCFDA. In contrast to photoreceptors or bipolar cells, Müller cells were most efficiently protected against H2O2-induced reactive oxygen species formation, which is in line with the protein repertoire identified in the proteomic profiling. Our novel approach to isolate intact glial cells from adult retina in combination with proteomic profiling enabled the identification of novel Müller glia specific proteins, which were validated as markers and for their functional impact in glial

  3. The role of Smad signaling in hematopoiesis and translational hematology.

    PubMed

    Blank, U; Karlsson, S

    2011-09-01

    Hematopoietic stem cells (HSCs) reside in the bone marrow (BM) of adult individuals and function to produce and regenerate the entire blood and immune system over the course of an individual's lifetime. Historically, HSCs are among the most thoroughly characterized tissue-specific stem cells. Despite this, the regulation of fate options, such as self-renewal and differentiation, has remained elusive, partly because of the expansive plethora of factors and signaling cues that govern HSC behavior in vivo. In the BM, HSCs are housed in specialized niches that dovetail the behavior of HSCs with the need of the organism. The Smad-signaling pathway, which operates downstream of the transforming growth factor-β (TGF-β) superfamily of ligands, regulates a diverse set of biological processes, including proliferation, differentiation and apoptosis, in many different organ systems. Much of the function of Smad signaling in hematopoiesis has remained nebulous due to early embryonic lethality of most knockout mouse models. However, recently new data have been uncovered, suggesting that the Smad-signaling circuitry is intimately linked to HSC regulation. In this review, we bring the Smad-signaling pathway into focus, chronicling key concepts and recent advances with respect to TGF-β-superfamily signaling in normal and leukemic hematopoiesis.

  4. Hematopoiesis In The Equine Fetal Liver Suggests Immune Preparedness

    PubMed Central

    Battista, JM; Tallmadge, RL; Stokol, T; Felippe, MJB

    2014-01-01

    We investigated how the equine fetus prepares its pre-immune humoral repertoire for an imminent exposure to pathogens in the neonatal period, particularly how the primary hematopoietic organs are equipped to support B cell hematopoiesis and immunoglobulin (Ig) diversity. We demonstrated that the liver and the bone marrow at approximately 100 days of gestation (DG) are active sites of hematopoiesis based on the expression of signature mRNA (c-KIT, CD34, IL7R, CXCL12, IRF8, PU.1, PAX5, NOTCH1, GATA1, CEBPA) and protein markers (CD34, CD19, IgM, CD3, CD4, CD5, CD8, CD11b, CD172A) of hematopoietic development and leukocyte differentiation molecules, respectively. To verify Ig diversity achieved during the production of B cells, V(D)J segments were sequenced in primary lymphoid organs of the equine fetus and adult horse, revealing that similar heavy chain VDJ segments and CDR3 lengths were most frequently used independent of life stage. In contrast, different lambda light chain segments were predominant in equine fetal compared to adult stage and, surprisingly, the fetus had less restricted use of variable gene segments to construct the lambda chain. Fetal Igs also contained elements of sequence diversity, albeit to a smaller degree than that of the adult horse. Our data suggest that the B cells produced in the liver and bone marrow of the equine fetus generate a wide repertoire of pre-immune Igs for protection, and the more diverse use of different lambda variable gene segments in fetal life may provide the neonate an opportunity to respond to a wider range of antigens at birth. PMID:25179685

  5. Hematopoiesis Primer Modeling Combined Injury

    DTIC Science & Technology

    2012-05-01

    2010 Feb; 125( Suppl 2):S3-23. Dainiak N, J.K. Waselenko. Biology and clinical features of radiation injury in adults [Internet]. UpToDate ® 2004...Available from: http://www.sassit.co.za/Journals/General%20complications/ UpToDate %C2%AE%20%27Biolog y%20and%20clinical%20features%20of%20radiation

  6. Stochasticity and determinism in models of hematopoiesis.

    PubMed

    Kimmel, Marek

    2014-01-01

    This chapter represents a novel view of modeling in hematopoiesis, synthesizing both deterministic and stochastic approaches. Whereas the stochastic models work in situations where chance dominates, for example when the number of cells is small, or under random mutations, the deterministic models are more important for large-scale, normal hematopoiesis. New types of models are on the horizon. These models attempt to account for distributed environments such as hematopoietic niches and their impact on dynamics. Mixed effects of such structures and chance events are largely unknown and constitute both a challenge and promise for modeling. Our discussion is presented under the separate headings of deterministic and stochastic modeling; however, the connections between both are frequently mentioned. Four case studies are included to elucidate important examples. We also include a primer of deterministic and stochastic dynamics for the reader's use.

  7. What do we know about the participation of hematopoietic stem cells in hematopoiesis?

    PubMed Central

    Drize, Nina; Petinati, Nataliya

    2015-01-01

    The demonstrated presence in adult tissues of cells with sustained tissue regenerative potential has given rise to the concept of tissue stem cells. Assays to detect and measure such cells indicate that they have enormous proliferative potential and usually an ability to produce all or many of the mature cell types that define the specialized functionality of the tissue. In the hematopoietic system, one or only a few cells can restore lifelong hematopoiesis of the whole organism. To what extent is the maintenance of hematopoietic stem cells required during normal hematopoiesis? How does the constant maintenance of hematopoiesis occur and what is the behavior of the hematopoietic stem cells in the normal organism? How many of the hematopoietic stem cells are created during the development of the organism? How many hematopoietic stem cells are generating more mature progeny at any given moment? What happens to the population of hematopoietic stem cells in aging? This review will attempt to describe the results of recent research which contradict some of the ideas established over the past 30 years about how hematopoiesis is regulated. PMID:27081472

  8. Expression of Pitx2 in stromal cells is required for normal hematopoiesis.

    PubMed

    Kieusseian, Aurélie; Chagraoui, Jalila; Kerdudo, Cécile; Mangeot, Philippe-Emmanuel; Gage, Philip J; Navarro, Nicole; Izac, Brigitte; Uzan, Georges; Forget, Bernard G; Dubart-Kupperschmitt, Anne

    2006-01-15

    Although the expression of Pitx2, a bicoid family homeodomain transcription factor, is highly regulated during hematopoiesis, its function during this process was not documented; we thus studied hematopoiesis in Pitx2-null mice. We found that Pitx2(-/-) embryos display hypoplastic livers with reduced numbers of hematopoietic cells, but these cells had normal hematopoietic potential, as evidenced by colony-forming assays, immature progenitor cell assays, and long-term repopulation assays. Because the microenvironment is also crucial to the development of normal hematopoiesis, we established Pitx2(-/-) and Pitx2(+/+) stromas from fetal liver and studied their hematopoietic supportive capacity. We showed that the frequency of cobblestone area-forming cells was 4-fold decreased when using Pitx2(-/-) stromal cells compared with Pitx2(+/+) stromal cells, whatever the Pitx2 genotype of hematopoietic cells tested in this assay. This defect was rescued by expression of Pitx2 into Pitx2(-/-) fetal liver stromal cells, demonstrating a major and direct role of Pitx2 in the hematopoietic supportive capacity of fetal liver stroma. Finally, we showed a reduced capacity of MS5 stromal cells expressing Pitx2 RNAi to support human hematopoiesis. Altogether these data showed that Pitx2 has major functions in the hematopoietic supportive capacity of fetal liver and adult bone marrow stromal cells.

  9. Expression of Pitx2 in stromal cells is required for normal hematopoiesis

    PubMed Central

    Kieusseian, Aurélie; Chagraoui, Jalila; Kerdudo, Cécile; Mangeot, Philippe-Emmanuel; Gage, Philip J.; Navarro, Nicole; Izac, Brigitte; Uzan, Georges; Forget, Bernard G.; Dubart-Kupperschmitt, Anne

    2006-01-01

    Although the expression of Pitx2, a bicoid family homeodomain transcription factor, is highly regulated during hematopoiesis, its function during this process was not documented; we thus studied hematopoiesis in Pitx2-null mice. We found that Pitx2–/– embryos display hypoplastic livers with reduced numbers of hematopoietic cells, but these cells had normal hematopoietic potential, as evidenced by colony-forming assays, immature progenitor cell assays, and long-term repopulation assays. Because the microenvironment is also crucial to the development of normal hematopoiesis, we established Pitx2–/– and Pitx2+/+ stromas from fetal liver and studied their hematopoietic supportive capacity. We showed that the frequency of cobblestone area-forming cells was 4-fold decreased when using Pitx2–/– stromal cells compared with Pitx2+/+ stromal cells, whatever the Pitx2 genotype of hematopoietic cells tested in this assay. This defect was rescued by expression of Pitx2 into Pitx2–/– fetal liver stromal cells, demonstrating a major and direct role of Pitx2 in the hematopoietic supportive capacity of fetal liver stroma. Finally, we showed a reduced capacity of MS5 stromal cells expressing Pitx2 RNAi to support human hematopoiesis. Altogether these data showed that Pitx2 has major functions in the hematopoietic supportive capacity of fetal liver and adult bone marrow stromal cells. PMID:16195330

  10. FANCA safeguards interphase and mitosis during hematopoiesis in vivo

    PubMed Central

    Abdul-Sater, Zahi; Cerabona, Donna; Sierra Potchanant, Elizabeth; Sun, Zejin; Enzor, Rikki; He, Ying; Robertson, Kent; Goebel, W. Scott; Nalepa, Grzegorz

    2015-01-01

    Fanconi anemia (FA/BRCA) signaling network controls multiple genome-housekeeping checkpoints, from interphase DNA repair to mitosis. The in vivo role of abnormal cell division in FA remains unknown. Here, we quantified the origins of genomic instability in FA patients and mice in vivo and ex vivo. We found that both mitotic errors and interphase DNA damage significantly contribute to genomic instability during FA-deficient hematopoiesis and in non-hematopoietic human and murine FA primary cells. Super-resolution microscopy coupled with functional assays revealed that FANCA shuttles to the pericentriolar material (PCM) to regulate spindle assembly at mitotic entry. Loss of FA signaling rendered cells hypersensitive to spindle chemotherapeutics and allowed escape from the chemotherapy-induced spindle assembly checkpoint. In support of these findings, direct comparison of DNA cross-linking and antimitotic chemotherapeutics in primary FANCA−/− cells revealed genomic instability originating through divergent cell cycle checkpoint aberrations. Our data indicate that the FA/BRCA signaling functions as an in vivo gatekeeper of genomic integrity throughout interphase and mitosis, which may have implications for future targeted therapies in FA and FA-deficient cancers. PMID:26366677

  11. Impaired Hematopoiesis and Disrupted Monocyte/Macrophage Homeostasis in Mucopolysaccharidosis Type I Mice.

    PubMed

    Viana, Gustavo Monteiro; Buri, Marcus Vinícius; Paredes-Gamero, Edgar Julian; Martins, Ana Maria; D'Almeida, Vânia

    2016-03-01

    Mucopolysaccharidosis type I (MPS I) is a rare autosomal recessive disease caused by alpha-L-iduronidase deficiency in which heparan and dermatan sulfate degradation is compromised. Besides primary lysosomal glycosaminoglycan accumulation, further changes in cellular functions have also been described in several murine MPS models. Herein, we evaluated alterations in hematopoiesis and its implications on the production of mature progeny in a MPS I murine model. Despite the significant increase in hematopoietic stem cells, a reduction in common myeloid progenitors and granulocyte-macrophage progenitor cells was observed in Idua -/- mice bone marrow. Furthermore, no alterations in number, viability nor activation of cell death mechanisms were observed in Idua -/- mice mature macrophages but they presented higher sensitivity to apoptotic induction after staurosporine treatment. In addition, changes in Ca(2+) signaling and a reduction in phagocytosis ability were also found. In summary, our results revealed significant intracellular changes in mature Idua -/- macrophages related to alterations in Idua -/- mice hematopoiesis, revealing a disruption in cell homeostasis. These results provide new insights into physiopathology of MPS I. © 2015 Wiley Periodicals, Inc.

  12. Autosomal dominant cyclic hematopoiesis: Genetics, phenotype, and natural history

    SciTech Connect

    Palmer, S.E.; Stephens, K.; Dale, D.C.

    1994-09-01

    Autosomal dominant cyclic hematopoiesis (ADCH; cyclic neutropenia) is a rare disorder manifested by transient neutropenia that recurs every three weeks. To facilitate mapping the ADCH gene by genetic linkage analysis, we studied 9 ADCH families with 42 affected individuals. Pedigrees revealed AD inheritance with no evidence for decreased penetrance. Similar intra- and interfamilial variable expression was observed, with no evidence to support heterogeneity. At least 3 families displayed apparent new mutations. Many adults developed chronic neutropenia, while offspring always cycled during childhood. Children displayed recurrent oral ulcers, gingivitis, lymphadenopathy, fever, and skin and other infections with additional symptoms. Interestingly, there were no cases of neonatal infection. Some children required multiple hospitalizations for treatment. Four males under age 18 died of Clostridium sepsis following necrotizing enterocolitis; all had affected mothers. No other deaths due to ADCH were found; most had improvement of symptoms and infections as adults. Adults experienced increased tooth loss prior to age 30 (16 out of 27 adults, with 9 edentulous). No increase in myelodysplasia, malignancy, or congenital anomalies was observed. Recombinant G-CSF treatment resulted in dramatic improvement of symptoms and infections. The results suggest that ADCH is not a benign disorder, especially in childhood, and abdominal pain requires immediate evaluation. Diagnosis of ADCH requires serial blood counts in the proband and at least one CBC in relatives to exclude similar disorders. Genetic counseling requires specific histories as well as CBCs of each family member at risk to determine status regardless of symptom history, especially to assess apparent new mutations.

  13. Spatial and temporal expression of FoxO transcription factors in the developing and adult murine brain.

    PubMed

    Hoekman, Marco F M; Jacobs, Frank M J; Smidt, Marten P; Burbach, J Peter H

    2006-01-01

    In order to obtain leads to molecular mechanisms of signal transduction pathways and controlled gene expression in neuronal development we have screened the adult mouse brain for expressed forkhead transcription factors using a degenerate RT-PCR approach. Here, we focus on three FoxO genes found to be expressed in the brain: FoxO1, FoxO3 and FoxO6. The FoxO subfamily of forkhead transcription family is emerging as a central keypoint in an array of cellular functions, such as metabolism, differentiation and transformation. In situ hybridization experiments on adult and embryonic mouse brain showed differential expression patterns for three FoxO members. FoxO1 was strongly expressed in the striatum and neuronal subsets of the hippocampus (dentate gyrus and the ventral/posterior part of the CA regions), whereas FoxO3 was more diffusely expressed throughout the brain including all hippocampal areas, cortex and cerebellum. FoxO6 expression was eminent in various parts of the adult mouse brain, including the entire hippocampus, the amygdalohippocampal area and the shell of the nucleus accumbens. Remarkably, all three FoxO transcription factors were expressed relatively late in the developing murine brain, starting between E12.5 and E14. In summary, the presented data show FoxO factors to be expressed in the adult and developing mouse brain, in a spatially end temporally restricted manner.

  14. Pulmonary Extramedullary Hematopoiesis Involving the Pulmonary Artery

    PubMed Central

    Monga, Varun; Silverman, Margarida

    2015-01-01

    Extramedullary hematopoiesis (EMH) occurs as a complication of hematologic disorders such as myelofibrosis, sickle cell anemia and thalassemia. The extramedullary tissue usually involves liver, spleen and lymph nodes, less frequently the chest. We present a recent case of a man with myeloproliferative neoplasm who developed pulmonary hemorrhage secondary to EMH in the lung and pulmonary artery. Radiation therapy was considered the best approach, but it didn’t work and the patient died a week after radiation therapy was completed. We also review herein the present literature. PMID:25852851

  15. Understanding leukemic hematopoiesis as a complex adaptive system

    PubMed Central

    Thomas, Xavier

    2015-01-01

    Normal and abnormal hematopoiesis is working as a complex adaptive system. From this perspective, the development and the behavior of hematopoietic cell lineages appear as a balance between normal and abnormal hematopoiesis in the setting of a functioning or malfunctioning microenvironment under the control of the immune system and the influence of hereditary and environmental events. PMID:26516407

  16. Understanding leukemic hematopoiesis as a complex adaptive system.

    PubMed

    Thomas, Xavier

    2015-10-26

    Normal and abnormal hematopoiesis is working as a complex adaptive system. From this perspective, the development and the behavior of hematopoietic cell lineages appear as a balance between normal and abnormal hematopoiesis in the setting of a functioning or malfunctioning microenvironment under the control of the immune system and the influence of hereditary and environmental events.

  17. Hematopoiesis on cellulose ester membranes (CEM). X. Effects of in vitro irradiation of stromal cells prior to application on CEM

    SciTech Connect

    Knospe, W.H.; Husseini, S.G.

    1986-11-01

    Cellulose ester membranes (CEM) were coated with stromal cells from murine bone or bone marrow irradiated in vitro with 1000, 2000, or 4000 rad and then implanted i.p. in CAF1 mice for periods of six and 12 months. CEM coated with stromal cells from bone showed excellent regeneration of bone and hematopoiesis after 1000 rad in vitro irradiation. After 2000 rad, hematopoietic and bone regeneration was reduced by about 50%, and after 4000 rad it was completely absent in CEM coated with stromal cells from bone. CEM coated with stromal cells from bone marrow showed no regeneration of hematopoiesis or bone after 1000, 2000, and 4000 rad in vitro irradiation and residence i.p. for six and 12 months. These results indicate that regeneration of the hematopoietic microenvironment is dependent upon living stromal cells. A difference in radiation sensitivity is demonstrated between stromal cells from bone and from bone marrow.

  18. Perturbed hematopoiesis in mice lacking ATMIN

    PubMed Central

    Anjos-Afonso, Fernando; Loizou, Joanna I.; Bradburn, Amy; Kanu, Nnennaya; Purewal, Sukhveer; Da Costa, Clive; Behrens, Axel

    2016-01-01

    The ataxia telangiectasia mutated (ATM)-interacting protein ATMIN mediates noncanonical ATM signaling in response to oxidative and replicative stress conditions. Like ATM, ATMIN can function as a tumor suppressor in the hematopoietic system: deletion of Atmin under the control of CD19-Cre results in B-cell lymphomas in aging mice. ATM signaling is essential for lymphopoiesis and hematopoietic stem cell (HSC) function; however, little is known about the role of ATMIN in hematopoiesis. We thus sought to investigate whether the absence of ATMIN would affect primitive hematopoietic cells in an ATM-dependent or -independent manner. Apart from its role in B-cell development, we show that ATMIN has an ATM-independent function in the common myeloid progenitors (CMPs) by deletion of Atmin in the entire hematopoietic system using Vav-Cre. Despite the lack of lymphoma formation, ATMIN-deficient mice developed chronic leukopenia as a result of high levels of apoptosis in B cells and CMPs and induced a compensatory mechanism in which HSCs displayed enhanced cycling. Consequently, ATMIN-deficient HSCs showed impaired regeneration ability with the induction of the DNA oxidative stress response, especially when aged. ATMIN, therefore, has multiple roles in different cell types, and its absence results in perturbed hematopoiesis, especially during stress conditions and aging. PMID:27581360

  19. Mutant U2AF1 Expression Alters Hematopoiesis and Pre-mRNA Splicing In Vivo

    PubMed Central

    Shirai, Cara Lunn; Ley, James N.; White, Brian S.; Kim, Sanghyun; Tibbitts, Justin; Shao, Jin; Ndonwi, Matthew; Wadugu, Brian; Duncavage, Eric J.; Okeyo-Owuor, Theresa; Liu, Tuoen; Griffith, Malachi; McGrath, Sean; Magrini, Vincent; Fulton, Robert S.; Fronick, Catrina; O’Laughlin, Michelle; Graubert, Timothy A.; Walter, Matthew J.

    2015-01-01

    SUMMARY Heterozygous somatic mutations in the spliceosome gene U2AF1 occur in ~11% of patients with myelodysplastic syndromes (MDS), the most common adult myeloid malignancy. It is unclear how these mutations contribute to disease. We examined in vivo hematopoietic consequences of the most common U2AF1 mutation using a doxycycline-inducible transgenic mouse model. Mice expressing mutant U2AF1(S34F) display altered hematopoiesis and changes in pre-mRNA splicing in hematopoietic progenitor cells by whole transcriptome analysis (RNA-seq). Integration with human RNA-seq datasets determined that common mutant U2AF1-induced splicing alterations are enriched in RNA processing genes, ribosomal genes, and recurrently-mutated MDS and acute myeloid leukemia-associated genes. These findings support the hypothesis that mutant U2AF1 alters downstream gene isoform expression, thereby contributing to abnormal hematopoiesis in MDS patients. PMID:25965570

  20. Hypocellularity in the Murine Model for Down Syndrome Ts65Dn Is Not Affected by Adult Neurogenesis.

    PubMed

    López-Hidalgo, Rosa; Ballestín, Raul; Vega, Jessica; Blasco-Ibáñez, José M; Crespo, Carlos; Gilabert-Juan, Javier; Nácher, Juan; Varea, Emilio

    2016-01-01

    Down syndrome (DS) is caused by the presence of an extra copy of the chromosome 21 and it is the most common aneuploidy producing intellectual disability. Neural mechanisms underlying this alteration may include defects in the formation of neuronal networks, information processing and brain plasticity. The murine model for DS, Ts65Dn, presents reduced adult neurogenesis. This reduction has been suggested to underlie the hypocellularity of the hippocampus as well as the deficit in olfactory learning in the Ts65Dn mice. Similar alterations have also been observed in individuals with DS. To determine whether the impairment in adult neurogenesis is, in fact, responsible for the hypocellularity in the hippocampus and physiology of the olfactory bulb, we have analyzed cell proliferation and neuronal maturation in the two major adult neurogenic niches in the Ts656Dn mice: the subgranular zone (SGZ) of the hippocampus and the subventricular zone (SVZ). Additionally, we carried out a study to determine the survival rate and phenotypic fate of newly generated cells in both regions, injecting 5'BrdU and sacrificing the mice 21 days later, and analyzing the number and phenotype of the remaining 5'BrdU-positive cells. We observed a reduction in the number of proliferating (Ki67 positive) cells and immature (doublecortin positive) neurons in the subgranular and SVZ of Ts65Dn mice, but we did not observe changes in the number of surviving cells or in their phenotype. These data correlated with a lower number of apoptotic cells (cleaved caspase 3 positive) in Ts65Dn. We conclude that although adult Ts65Dn mice have a lower number of proliferating cells, it is compensated by a lower level of cell death. This higher survival rate in Ts65Dn produces a final number of mature cells similar to controls. Therefore, the reduction of adult neurogenesis cannot be held responsible for the neuronal hypocellularity in the hippocampus or for the olfactory learning deficit of Ts65Dn mice.

  1. Expression and localization of laminin 5, laminin 10, type IV collagen, and amelotin in adult murine gingiva.

    PubMed

    Sawada, Takashi; Yamazaki, Takaki; Shibayama, Kazuko; Kumazawa, Kaido; Yamaguchi, Yoko; Ohshima, Mitsuhiro

    2014-06-01

    The biochemical composition of the internal and external basal laminae in the junctional epithelium differs significantly, and the precise cellular origin of their respective molecules remains to be determined. In the present study, the expression and localization of three basement membrane-specific molecules-laminin 5 (γ2 chain), type IV collagen (α1 chain), and laminin 10 (α5 chain)-and one tooth-specific molecule, amelotin, was analyzed in adult murine gingiva by using in situ hybridization and immunohistochemistry. The results showed that the outermost cells in junctional epithelium facing the tooth enamel strongly expressed laminin 5 mRNA, supporting the immunohistochemical staining data. This suggests that laminin 5 is actively synthesized in junctional epithelial cells and that the products are incorporated into the internal basal lamina to maintain firm epithelial adhesion to the tooth enamel throughout life. Conversely, no amelotin mRNA signals were detected in the junctional epithelial cells, suggesting that the molecules localized on the internal basal lamina are mainly derived from maturation-stage ameloblasts. Weak and sporadic expression of type IV collagen in addition to laminin 10 in the gingiva indicates that these molecules undergo turnover less frequently in adult animals.

  2. Epigenetics of hematopoiesis and hematological malignancies

    PubMed Central

    Hu, Deqing; Shilatifard, Ali

    2016-01-01

    Hematological malignancies comprise a diverse set of lymphoid and myeloid neoplasms in which normal hematopoiesis has gone awry and together account for ∼10% of all new cancer cases diagnosed in the United States in 2016. Recent intensive genomic sequencing of hematopoietic malignancies has identified recurrent mutations in genes that encode regulators of chromatin structure and function, highlighting the central role that aberrant epigenetic regulation plays in the pathogenesis of these neoplasms. Deciphering the molecular mechanisms for how alterations in epigenetic modifiers, specifically histone and DNA methylases and demethylases, drive hematopoietic cancer could provide new avenues for developing novel targeted epigenetic therapies for treating hematological malignancies. Just as past studies of blood cancers led to pioneering discoveries relevant to other cancers, determining the contribution of epigenetic modifiers in hematologic cancers could also have a broader impact on our understanding of the pathogenesis of solid tumors in which these factors are mutated. PMID:27798847

  3. Congenital Lobar Emphysema with Pulmonary Extramedullary Hematopoiesis.

    PubMed

    Suryawanshi, Kishor; Nikumbh, Dhiraj; Singhavi, Sudhir; Damle, Rajshri; Dravid, Nandkumar

    2017-01-01

    Congenital lobar emphysema is a one of the rare variety of congenital abnormality of the lung causing respiratory distress in newborns and infants. Herein, we report a case of congenital lobar emphysema in a 3-week-old female admitted to the pediatric intensive care unit with severe respiratory distress. CT scan revealed a hyperinflated, hyperlucent left upper lobe, collapse of the ipsilateral lung and marked mediastinal shift to the right. The patient underwent emergency left upper lobectomy with improvement of her condition in the postoperative period. Histopathological findings confirmed the clinical diagnosis of congenital lobar emphysema with an unusual finding of pulmonary extramedullary hematopoiesis. Congenital lobar emphysema often presents a diagnostic and therapeutic dilemma and therefore requires a high index of suspicion in neonates presenting with respiratory distress to avoid morbidity and mortality.

  4. Myc Roles in Hematopoiesis and Leukemia

    PubMed Central

    Delgado, M. Dolores; León, Javier

    2010-01-01

    Hematopoiesis is a process capable of generating millions of cells every second, as distributed in many cell types. The process is regulated by a number of transcription factors that regulate the differentiation along the distinct lineages and dictate the genetic program that defines each mature phenotype. Myc was first discovered as the oncogene of avian leukemogenic retroviruses; it was later found translocated in human lymphoma. From then on, evidence accumulated showing that c-Myc is one of the transcription factors playing a major role in hematopoiesis. The study of genetically modified mice with overexpression or deletion of Myc has shown that c-Myc is required for the correct balance between self-renewal and differentiation of hematopoietic stem cells (HSCs). Enforced Myc expression in mice leads to reduced HSC pools owing to loss of self-renewal activity at the expense of increased proliferation of progenitor cells and differentiation. c-Myc deficiency consistently results in the accumulation of HSCs. Other models with conditional Myc deletion have demonstrated that different lineages of hematopoietic cells differ in their requirement for c-Myc to regulate their proliferation and differentiation. When transgenic mice overexpress c-Myc or N-Myc in mature cells from the lymphoid or myeloid lineages, the result is lymphoma or leukemia. In agreement, enforced expression of c-Myc blocks the differentiation in several leukemia-derived cell lines capable of differentiating in culture. Not surprising, MYC deregulation is recurrently found in many types of human lymphoma and leukemia. Whereas MYC is deregulated by translocation in Burkitt lymphoma and, less frequently, other types of lymphoma, MYC is frequently overexpressed in acute lymphoblastic and myeloid leukemia, through mechanisms unrelated to chromosomal translocation, and is often associated with disease progression. PMID:21779460

  5. Erythro-Myeloid Progenitors: “definitive” hematopoiesis in the conceptus prior to the emergence of hematopoietic stem cells

    PubMed Central

    Frame, Jenna M.; McGrath, Kathleen E.; Palis, James

    2013-01-01

    Erythro-myeloid progenitors (EMP) serve as a major source of hematopoiesis in the developing conceptus prior to the formation of a permanent blood system. In this review, we summarize the current knowledge regarding the emergence, fate, and potential of this hematopoietic stem cell (HSC)-independent wave of hematopoietic progenitors, focusing on the murine embryo as a model system. A better understanding of the temporal and spatial control of hematopoietic emergence in the embryo will ultimately improve our ability to derive hematopoietic stem and progenitor cells from embryonic stem cells and induced pluripotent stem cells to serve therapeutic purposes. PMID:24095199

  6. Regulation of haematopoietic stem cell proliferation by stimulatory factors produced by murine fetal and adult liver.

    PubMed Central

    Dawood, K A; Briscoe, C V; Thomas, D B; Riches, A C

    1990-01-01

    Haematopoietic stem cells in murine fetal liver are in a proliferative state unlike those in normal bone marrow which are quiescent. A regulatory activity is produced by cells in the fetal liver which will switch quiescent normal bone marrow haematopoietic stem cells into cell cycle in vitro. This regulator from Day 15 fetal liver cells is produced by adherent cells and by cells fractionated on a Percoll gradient in the 1.064 and 1.076 g per cm3 density bands but not in the 1.123 g per cm3 band. Colony-stimulating factor cannot be detected in the supernatants containing the stem cell regulatory activity. The stimulator can be detected in supernatants produced from cell suspensions of liver cells at Day 15 and Day 17 of gestation and 24 hours and 72 hours after birth. However by 1 week after birth the production of the stimulator decreases and is undetectable 3 and 10 weeks after birth. The total numbers of haematopoietic stem cells (CFU-S) in fetal liver decrease from Day 15 of gestation and only small numbers are present 1 week after birth. Thus the decline in the production of haematopoietic stem cell proliferation stimulator correlates with the decrease in haematopoietic stem cell numbers in the liver through gestation and after birth. PMID:2323992

  7. The controversial role of the Hedgehog pathway in normal and malignant hematopoiesis

    PubMed Central

    Mar, BG; Amakye, D; Aifantis, I; Buonamici, S

    2015-01-01

    Hedgehog (Hh) is a developmental signaling pathway in which Hh ligands bind Patched (Ptch), which relieves its inhibition of Smoothened (Smo), allowing the Gli family of transcription factors to translocate to the nucleus and activate Hh target genes. The role of Hh signaling in hematopoiesis is controversial and ill defined. Although some groups observed self-renewal defects with decreased replating and reduced efficiency of secondary murine transplants, other groups reported no hematopoietic phenotypes, which may be related to the timing of Hh abrogation. In malignant hematopoiesis, most attention has been focused on the role of Hh signaling in chronic myeloid leukemia (CML), considered by many to be a stem cell disorder that bears the constitutively active BCR-ABL tyrosine kinase. Despite the elimination of most leukemia cells through BCR-ABL inhibition, most patients remain PCR positive, suggesting that the putative CML stem cell may be resistant to kinase antagonism. Groups are now exploring the Hh pathway as an alternate pathway supporting CML stem cell survival. Knockdown or inhibition of Smo abrogates or delays the appearance of CML in several in vitro and in vivo models. These data have lead to clinical trials using BCR-ABL kinase and novel Smo inhibitors in combination. PMID:21660044

  8. Ultrafiltered pig leukocyte extract (IMUNOR) decreases nitric oxide formation and hematopoiesis-stimulating cytokine production in lipopolysaccharide-stimulated RAW 264.7 macrophages.

    PubMed

    Hofer, Michal; Vacek, Antonín; Lojek, Antonín; Holá, Jirina; Streitová, Denisa

    2007-10-01

    A low-molecular-weight (<12 kDa) ultrafiltered pig leukocyte extract, IMUNOR, was tested in experiments in vitro on non-stimulated and lipopolysaccharide (LPS)-stimulated murine RAW 264.7 macrophages in order to assess modulation of nitric oxide (NO) production (measured indirectly as the concentration of nitrites), hematopoiesis-stimulating activity of the supernatant of the macrophage cells (ascertained by counting cell colonies growing from progenitor cells for granulocytes and macrophages (GM-CFC) in vitro), and the release of hematopoiesis-stimulating cytokines. No hematopoiesis-stimulating activity and cytokine or NO production were found in the supernatant of non-stimulated macrophages. It was found that IMUNOR does not influence this status. Supernatant of LPS-stimulated macrophages was characterized by hematopoiesis-stimulating activity, as well as by the presence of nitrites, interleukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF). A key role in the hematopoiesis-stimulating activity of the supernatant of LPS-stimulated macrophages could be ascribed to G-CSF since the formation of the colonies could be abrogated nearly completely by monoclonal antibodies against G-CSF. IMUNOR was found to suppress all the mentioned manifestations of the LPS-activated macrophages. When considering these results together with those from our previous in vivo study revealing stimulatory effects of IMUNOR on radiation-suppressed hematopoiesis, a hypothesis may be formulated which postulates a homeostatic role of IMUNOR, consisting in stimulation of impaired immune and hematopoietic systems but also in cutting back the production of proinflammatory mediators in cases of overstimulation which threats with undesirable consequences.

  9. Hypocellularity in the Murine Model for Down Syndrome Ts65Dn Is Not Affected by Adult Neurogenesis

    PubMed Central

    López-Hidalgo, Rosa; Ballestín, Raul; Vega, Jessica; Blasco-Ibáñez, José M.; Crespo, Carlos; Gilabert-Juan, Javier; Nácher, Juan; Varea, Emilio

    2016-01-01

    Down syndrome (DS) is caused by the presence of an extra copy of the chromosome 21 and it is the most common aneuploidy producing intellectual disability. Neural mechanisms underlying this alteration may include defects in the formation of neuronal networks, information processing and brain plasticity. The murine model for DS, Ts65Dn, presents reduced adult neurogenesis. This reduction has been suggested to underlie the hypocellularity of the hippocampus as well as the deficit in olfactory learning in the Ts65Dn mice. Similar alterations have also been observed in individuals with DS. To determine whether the impairment in adult neurogenesis is, in fact, responsible for the hypocellularity in the hippocampus and physiology of the olfactory bulb, we have analyzed cell proliferation and neuronal maturation in the two major adult neurogenic niches in the Ts656Dn mice: the subgranular zone (SGZ) of the hippocampus and the subventricular zone (SVZ). Additionally, we carried out a study to determine the survival rate and phenotypic fate of newly generated cells in both regions, injecting 5′BrdU and sacrificing the mice 21 days later, and analyzing the number and phenotype of the remaining 5′BrdU-positive cells. We observed a reduction in the number of proliferating (Ki67 positive) cells and immature (doublecortin positive) neurons in the subgranular and SVZ of Ts65Dn mice, but we did not observe changes in the number of surviving cells or in their phenotype. These data correlated with a lower number of apoptotic cells (cleaved caspase 3 positive) in Ts65Dn. We conclude that although adult Ts65Dn mice have a lower number of proliferating cells, it is compensated by a lower level of cell death. This higher survival rate in Ts65Dn produces a final number of mature cells similar to controls. Therefore, the reduction of adult neurogenesis cannot be held responsible for the neuronal hypocellularity in the hippocampus or for the olfactory learning deficit of Ts65Dn mice

  10. Modeling notch signaling in normal and neoplastic hematopoiesis: global gene expression profiling in response to activated notch expression.

    PubMed

    Ganapati, Uma; Tan, Hongying Tina; Lynch, Maureen; Dolezal, Milana; de Vos, Sven; Gasson, Judith C

    2007-08-01

    In normal hematopoiesis, proliferation is tightly linked to differentiation in ways that involve cell-cell interaction with stromal elements in the bone marrow stem cell niche. Numerous in vitro and in vivo studies strongly support a role for Notch signaling in the regulation of stem cell renewal and hematopoiesis. Not surprisingly, mutations in the Notch gene have been linked to a number of types of malignancies. To better define the function of Notch in both normal and neoplastic hematopoiesis, a tetracycline-inducible system regulating expression of a ligand-independent, constitutively active form of Notch1 was introduced into murine E14Tg2a embryonic stem cells. During coculture, OP9 stromal cells induce the embryonic stem cells to differentiate first to hemangioblasts and subsequently to hematopoietic stem cells. Our studies indicate that activation of Notch signaling in flk+ hemangioblasts dramatically reduces their survival and proliferative capacity and lowers the levels of hematopoietic stem cell markers CD34 and c-Kit and the myeloid marker CD11b. Global gene expression profiling of day 8 hematopoietic progenitors in the absence and presence of activated Notch yield candidate genes required for normal hematopoietic differentiation, as well as putative downstream targets of oncogenic forms of Notch including the noncanonical Wnts Wnt4 and 5A. Disclosure of potential conflicts of interest is found at the end of this article.

  11. Strain-dependent protective effect of adult thymectomy on murine infection by Mycobacterium lepraemurium.

    PubMed Central

    Bach, M A; Hoffenbach, A

    1987-01-01

    C57BL/6, DBA/2, BALB/c and CBA mice were thymectomized as adults, or sham-thymectomized, and infected subcutaneously with 10(6) MLM. The number of MLM in the spleen and in the inoculated footpad was measured after 1 year of infection as well as the DTH reactions and the IgM and IgG antibody levels to MLM. Non-thymectomized mice exhibited a broad spectrum of resistance to MLM infection and of T cell mediated immunity grading from the highly resistant C57BL/6 strain to the highly susceptible CBA strain. In between, DBA/2 was found more resistant than BALB/c mice. Adult thymectomy reduced by 100 times the MLM number in the spleen of infected DBA/2 mice, without affecting that measured in the inoculated footpad, and significantly decreased DTH reaction in the same strain. No effect of adult thymectomy was observed in any other strain, except for an increase of anti MLM antibodies in BALB/c mice. These results may suggest that the medium-resistant DBA/2 strain develops after MLM infection suppressor T cells which favour MLM dissemination and are sensitive to adult. PMID:2958188

  12. Zebrafish kidney stromal cell lines support multilineage hematopoiesis

    PubMed Central

    Stachura, David L.; Reyes, Jason R.; Bartunek, Petr; Paw, Barry H.; Zon, Leonard I.

    2009-01-01

    Studies of zebrafish hematopoiesis have been largely performed using mutagenesis approaches and retrospective analyses based upon gene expression patterns in whole embryos. We previously developed transplantation assays to test the repopulation potentials of candidate hematopoietic progenitor cells. We have been impaired, however, in determining cellular differentiation potentials by a lack of short-term functional assays. To enable more precise analyses of hematopoietic progenitor cells, we have created zebrafish kidney stromal (ZKS) cell lines. Culture of adult whole kidney marrow with ZKS cells results in the maintenance and expansion of hematopoietic precursor cells. Hematopoietic growth is dependent upon ZKS cells, and we show that ZKS cells express many growth factors and ligands previously demonstrated to be important in maintaining mammalian hematopoietic cells. In the absence of exogenous growth factors, ZKS cells maintain early hematopoietic precursors and support differentiation of lymphoid and myeloid cells. With the addition of zebrafish erythropoietin, ZKS cells also support the differentiation of erythroid precursors. These conditions have enabled the ability to ascertain more precisely the points at which hematopoietic mutants are defective. The development of robust in vitro assays now provide the means to track defined, functional outcomes for prospectively isolated blood cell subsets in the zebrafish. PMID:19433857

  13. Ablating hedgehog signaling in tenocytes during development impairs biomechanics and matrix organization of the adult murine patellar tendon enthesis.

    PubMed

    Breidenbach, Andrew P; Aschbacher-Smith, Lindsey; Lu, Yinhui; Dyment, Nathaniel A; Liu, Chia-Feng; Liu, Han; Wylie, Chris; Rao, Marepalli; Shearn, Jason T; Rowe, David W; Kadler, Karl E; Jiang, Rulang; Butler, David L

    2015-08-01

    Restoring the native structure of the tendon enthesis, where collagen fibers of the midsubstance are integrated within a fibrocartilaginous structure, is problematic following injury. As current surgical methods fail to restore this region adequately, engineers, biologists, and clinicians are working to understand how this structure forms as a prerequisite to improving repair outcomes. We recently reported on the role of Indian hedgehog (Ihh), a novel enthesis marker, in regulating early postnatal enthesis formation. Here, we investigate how inactivating the Hh pathway in tendon cells affects adult (12-week) murine patellar tendon (PT) enthesis mechanics, fibrocartilage morphology, and collagen fiber organization. We show that ablating Hh signaling resulted in greater than 100% increased failure insertion strain (0.10 v. 0.05 mm/mm, p<0.01) as well as sub-failure biomechanical deficiencies. Although collagen fiber orientation appears overtly normal in the midsubstance, ablating Hh signaling reduces mineralized fibrocartilage by 32%, leading to less collagen embedded within mineralized tissue. Ablating Hh signaling also caused collagen fibers to coalesce at the insertion, which may explain in part the increased strains. These results indicate that Ihh signaling plays a critical role in the mineralization process of fibrocartilaginous entheses and may be a novel therapeutic to promote tendon-to-bone healing. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  14. Interleukin-1beta causes pulmonary inflammation, emphysema, and airway remodeling in the adult murine lung.

    PubMed

    Lappalainen, Urpo; Whitsett, Jeffrey A; Wert, Susan E; Tichelaar, Jay W; Bry, Kristina

    2005-04-01

    The production of the inflammatory cytokine interleukin (IL)-1 is increased in lungs of patients with chronic obstructive pulmonary disease (COPD) or asthma. To characterize the in vivo actions of IL-1 in the lung, transgenic mice were generated in which human IL-1beta was expressed in the lung epithelium with a doxycycline-inducible system controlled by the rat Clara cell secretory protein (CCSP) promoter. Induction of IL-1beta expression in the lungs of adult mice caused pulmonary inflammation characterized by neutrophil and macrophage infiltrates. IL-1beta caused distal airspace enlargement, consistent with emphysema. IL-1beta caused disruption of elastin fibers in alveolar septa and fibrosis in airway walls and in the pleura. IL-1beta increased the thickness of conducting airways, enhanced mucin production, and caused lymphocytic aggregates in the airways. Decreased immunostaining for the winged helix transcription factor FOXA2 was associated with goblet cell hyperplasia in IL-1beta-expressing mice. The production of the neutrophil attractant CXC chemokines KC (CXCL1) and MIP-2 (CXCL2), and of matrix metalloproteases MMP-9 and MMP-12, was increased by IL-1beta. Chronic production of IL-1beta in respiratory epithelial cells of adult mice causes lung inflammation, enlargement of distal airspaces, mucus metaplasia, and airway fibrosis in the adult mouse.

  15. Lifestyle effects on hematopoiesis and atherosclerosis.

    PubMed

    Nahrendorf, Matthias; Swirski, Filip K

    2015-02-27

    Diet, exercise, stress, and sleep are receiving attention as environmental modifiers of chronic inflammatory diseases, including atherosclerosis, the culprit condition of myocardial infarction and stroke. Accumulating data indicate that psychosocial stress and a high-fat, high-cholesterol diet aggravate cardiovascular disease, whereas regular physical activity and healthy sleeping habits help prevent it. Here, we raise the possibility that inflammation-associated leukocyte production plays a causal role in lifestyle effects on atherosclerosis progression. Specifically, we explore whether and how potent real-life disease modifiers influence hematopoiesis' molecular and cellular machinery. Lifestyle, we hypothesize, may rearrange hematopoietic topography, diverting production from the bone marrow to the periphery, thus propagating a quantitative and qualitative drift of the macrophage supply chain. These changes may involve progenitor-extrinsic and intrinsic communication nodes that connect organ systems along neuroimmune and immunometabolic axes, ultimately leading to an altered number and phenotype of lesional macrophages. We propose that, in conjunction with improved public health policy, future therapeutics could aim to modulate the quantitative and qualitative output, as well as the location, of the hematopoietic tree to decrease the risk of atherosclerosis complications. © 2015 American Heart Association, Inc.

  16. Differential vascular permeability along the forebrain ventricular neurogenic niche in the adult murine brain.

    PubMed

    Colín-Castelán, Dannia; Ramírez-Santos, Jesús; Gutiérrez-Ospina, Gabriel

    2016-02-01

    Adult neurogenesis is influenced by blood-borne factors. In this context, greater or lesser vascular permeability along neurogenic niches would expose differentially neural stem cells (NSCs), transit amplifying cells (TACs), and neuroblasts to such factors. Here we evaluate endothelial cell morphology and vascular permeability along the forebrain neurogenic niche in the adult brain. Our results confirm that the subventricular zone (SVZ) contains highly permeable, discontinuous blood vessels, some of which allow the extravasation of molecules larger than those previously reported. In contrast, the rostral migratory stream (RMS) and the olfactory bulb core (OBc) display mostly impermeable, continuous blood vessels. These results imply that NSCs, TACs, and neuroblasts located within the SVZ are exposed more readily to blood-borne molecules, including those with very high molecular weights, than those positioned along the RMS and the OBc, subregions in which every stage of neurogenesis also takes place. These observations suggest that the existence of specialized vascular niches is not a precondition for neurogenesis to occur; specialized vascular beds might be essential for keeping high rates of proliferation and/or differential differentiation of neural precursors located at distinct domains. © 2015 Wiley Periodicals, Inc.

  17. Hematopoiesis on cellulose ester membranes. XI. Induction of new bone and a hematopoietic microenvironment by matrix factors secreted by marrow stromal cells.

    PubMed

    Knospe, W H; Husseini, S G; Fried, W

    1989-07-01

    Cellulose ester membranes (CEM) were coated with stromal cells from bone marrow (BM) or bone and implanted intraperitoneally (IP) in CAF1 mice for intervals of 1 to 6 months. Previous studies indicated that matrix factors [glycoproteins (GPs), proteoglycans (PGs), and glycosaminoglycans (GAGs)] were secreted by the regenerating stromal cells and adsorbed by the CEM. After 1 to 6 months, the CEMs were removed, scraped free of adherent cells, and irradiated in vitro with 40 Gy. The scraped and irradiated CEMs were then reimplanted IP or subcutaneously (SC) for periods of 1 to 6 months in secondary syngeneic murine hosts. They were then removed for histologic study. CEMs reimplanted in SC sites developed bone and hematopoiesis as early as 1 month after implantation. Maximum hematopoiesis and bone formation was observed after 3 months. CEMs coated during the initial implantation with bone-derived stromal cells contained more bone and hematopoietic cells than did CEMs coated with marrow-derived stromal cells after SC implementation. Neither the CEMs coated with bone stromal cells nor those coated with marrow stromal cells developed new bone or trilineal hematopoiesis after being implanted IP. A few CEMs contained small foci of granulopoiesis only. We conclude that noncellular matrix substances deposited on CEMs by bone, and to a lesser degree by marrow cells, can induce prestromal cells in the SC tissues to produce a microenvironment suitable for trilineal hematopoiesis.

  18. Genetics, phenotype, and natural history of autosomal dominant cyclic hematopoiesis

    SciTech Connect

    Palmer, S.E. |; Dale, D.C.

    1996-12-30

    Cyclic hematopoiesis (CH, or cyclic neutropenia) is a rare disease manifested by transient severe neutropenia that recurs approximately every 21 days. The hematologic profile of families with the autosomal dominant form (ADCH) has not been well characterized, and it is unknown if the phenotype is distinct from the more common sporadic congenital or acquired forms of CH. We studied nine ADCH families whose children displayed typical CH blood patterns. Pedigrees confirmed dominant inheritance without evidence of heterogeneity or decreased penetrance; three pedigrees suggested new mutations. Families were Caucasian with exception of one with a Cherokee Native American founder. A wide spectrum of symptom severity, ranging from asymptomatic to life-threatening illness, was observed within families. The phenotype changed with age. Children displayed typical neutrophil cycles with symptoms of mucosal ulceration, lymphadenopathy, and infections. Adults often had fewer and milder symptoms, sometimes accompanied by mild chronic neutropenia without distinct cycles. While CH is commonly described as {open_quotes}benign{close_quotes}, four children in three of the nine families died of Clostridium or E. coli colitis, documenting the need for urgent evaluation of abdominal pain. Misdiagnosis with other neutropenias was common but can be avoided by serial blood counts in index cases. Genetic counseling requires specific histories and complete blood counts in relatives at risk to assess status regardless of symptoms, especially to determine individuals with new mutations. We propose diagnostic criteria for ADCH in affected children and adults. Recombinant human granulocyte colony-stimulating factor treatment resulted in dramatic improvement of neutropenia and morbidity. The differential diagnosis from other forms of familial neutropenia is reviewed. 45 refs., 4 figs., 1 tab.

  19. Wnt Signaling Regulates Airway Epithelial Stem Cells in Adult Murine Submucosal Glands.

    PubMed

    Lynch, Thomas J; Anderson, Preston J; Xie, Weiliang; Crooke, Adrianne K; Liu, Xiaoming; Tyler, Scott R; Luo, Meihui; Kusner, David M; Zhang, Yulong; Neff, Traci; Burnette, Daniel C; Walters, Katherine S; Goodheart, Michael J; Parekh, Kalpaj R; Engelhardt, John F

    2016-06-24

    Wnt signaling is required for lineage commitment of glandular stem cells (SCs) during tracheal submucosal gland (SMG) morphogenesis from the surface airway epithelium (SAE). Whether similar Wnt-dependent processes coordinate SC expansion in adult SMGs following airway injury remains unknown. We found that two Wnt-reporters in mice (BAT-gal and TCF/Lef:H2B-GFP) are coexpressed in actively cycling SCs of primordial glandular placodes and in a small subset of adult SMG progenitor cells that enter the cell cycle 24 hours following airway injury. At homeostasis, these Wnt reporters showed nonoverlapping cellular patterns of expression in the SAE and SMGs. Following tracheal injury, proliferation was accompanied by dynamic changes in Wnt-reporter activity and the analysis of 56 Wnt-related signaling genes revealed unique temporal changes in expression within proximal (gland-containing) and distal (gland-free) portions of the trachea. Wnt stimulation in vivo and in vitro promoted epithelial proliferation in both SMGs and the SAE. Interestingly, slowly cycling nucleotide label-retaining cells (LRCs) of SMGs were spatially positioned near clusters of BAT-gal positive serous tubules. Isolation and culture of tet-inducible H2B-GFP LRCs demonstrated that SMG LRCs were more proliferative than SAE LRCs and culture expanded SMG-derived progenitor cells outcompeted SAE-derived progenitors in regeneration of tracheal xenograft epithelium using a clonal analysis competition assay. SMG-derived progenitors were also multipotent for cell types in the SAE and formed gland-like structures in xenografts. These studies demonstrate the importance of Wnt signals in modulating SC phenotypes within tracheal niches and provide new insight into phenotypic differences of SMG and SAE SCs. Stem Cells 2016.

  20. Pharmacological analysis of epithelial chloride secretion mechanisms in adult murine airways.

    PubMed

    Gianotti, Ambra; Ferrera, Loretta; Philp, Amber R; Caci, Emanuela; Zegarra-Moran, Olga; Galietta, Luis J V; Flores, Carlos A

    2016-06-15

    Defective epithelial chloride secretion occurs in humans with cystic fibrosis (CF), a genetic defect due to loss of function of CFTR, a cAMP-activated chloride channel. In the airways, absence of an active CFTR causes a severe lung disease. In mice, genetic ablation of CFTR function does not result in similar lung pathology. This may be due to the expression of an alternative chloride channel which is activated by calcium. The most probable protein performing this function is TMEM16A, a calcium-activated chloride channel (CaCC). Our aim was to assess the relative contribution of CFTR and TMEM16A to chloride secretion in adult mouse trachea. For this purpose we tested pharmacological inhibitors of chloride channels in normal and CF mice. The amplitude of the cAMP-activated current was similar in both types of animals and was not affected by a selective CFTR inhibitor. In contrast, a CaCC inhibitor (CaCCinh-A01) strongly blocked the cAMP-activated current as well as the calcium-activated chloride secretion triggered by apical UTP. Although control experiments revealed that CaCCinh-A01 also shows inhibitory activity on CFTR, our results indicate that transepithelial chloride secretion in adult mouse trachea is independent of CFTR and that another channel, possibly TMEM16A, performs both cAMP- and calcium-activated chloride transport. The prevalent function of a non-CFTR channel may explain the absence of a defect in chloride transport in CF mice.

  1. Embryonic hematopoiesis in vertebrate somites gives rise to definitive hematopoietic stem cells

    PubMed Central

    Qiu, Juhui; Fan, Xiaoying; Wang, Yixia; Jin, Hongbin; Song, Yixiao; Han, Yang; Huang, Shenghong; Meng, Yaping; Tang, Fuchou; Meng, Anming

    2016-01-01

    Hematopoietic stem cells (HSCs) replenish all types of blood cells. It is debating whether HSCs in adults solely originate from the aorta-gonad-mesonephros (AGM) region, more specifically, the dorsal aorta, during embryogenesis. Here, we report that somite hematopoiesis, a previously unwitnessed hematopoiesis, can generate definitive HSCs (dHSCs) in zebrafish. By transgenic lineage tracing, we found that a subset of cells within the forming somites emigrate ventromedially and mix with lateral plate mesoderm-derived primitive hematopoietic cells before the blood circulation starts. These somite-derived hematopoietic precursors and stem cells (sHPSCs) subsequently enter the circulation and colonize the kidney of larvae and adults. RNA-seq analysis reveals that sHPSCs express hematopoietic genes with sustained expression of many muscle/skeletal genes. Embryonic sHPSCs transplanted into wild-type embryos expand during growth and survive for life time with differentiation into various hematopoietic lineages, indicating self-renewal and multipotency features. Therefore, the embryonic origin of dHSCs in adults is not restricted to the AGM. PMID:27252540

  2. Proteomics identifies differentially expressed proteins in neonatal murine thymus compared with adults

    PubMed Central

    2012-01-01

    Background The thymus is an immune organ essential for life and plays a crucial role in the development of T cells. It undergoes a fetal to adult developmental maturation process occurring in mouse during the postnatal months. The molecular modifications underlying these ontogenic changes are essentially unknown. Here we used a differential proteomic-based technique (2D-Difference Gel Electrophoresis) coupled with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry to search for key proteins in the postnatal development of the thymus. Eight different BALB/c mice were used in the study: four mice aged of 1 day (neonatal) and four mice aged of 60 days (adult). Protein samples derived from thymus were labeled and run in 2D-PAGE (Two-Dimensional Polyacrylamide Gel Electrophoresis). One whole-thymus tissue from each mouse was run on gels and each gel containing a pooled sample of the eight mice was run in parallel. The pooled sample was set as the internal pool, containing equal amount of each protein extract used in the experiment. Gels were matched and compared with Difference In-gel Analysis software. Differential spots were picked, in-gel digested and peptide mass fingerprints were obtained. Results Among the differentially regulated proteins in neonatal thymus group, 111 proteins were identified by mass spectrometry, of which 95 proteins were up-regulated and 16 proteins were down-regulated. The identified proteins belong to several functional categories, including cell proliferation, cycle and apoptosis, transcription regulation, signal transduction, nucleotide processing, proteolysis and translation, protein folding, metabolism, oxidoreduction, cytoskeleton, immune response, and embryonic development. The major interaction networks comprised of cellular function and maintenance, cellular assembly and organization, and metabolism were also identified by STRING analysis. Conclusions The demonstrated molecular changes are

  3. Long-Term Retinal PEDF Overexpression Prevents Neovascularization in a Murine Adult Model of Retinopathy

    PubMed Central

    Ribera, Albert; Bosch, Assumpcio; Ramos, David; Ruberte, Jesus; Bosch, Fatima

    2012-01-01

    Neovascularization associated with diabetic retinopathy (DR) and other ocular disorders is a leading cause of visual impairment and adult-onset blindness. Currently available treatments are merely palliative and offer temporary solutions. Here, we tested the efficacy of antiangiogenic gene transfer in an animal model that mimics the chronic progression of human DR. Adeno-associated viral (AAV) vectors of serotype 2 coding for antiangiogenic Pigment Epithelium Derived Factor (PEDF) were injected in the vitreous of a 1.5 month-old transgenic model of retinopathy that develops progressive neovascularization. A single intravitreal injection led to long-term production of PEDF and to a striking inhibition of intravitreal neovascularization, normalization of retinal capillary density, and prevention of retinal detachment. This was parallel to a reduction in the intraocular levels of Vascular Endothelial Growth Factor (VEGF). Normalization of VEGF was consistent with a downregulation of downstream effectors of angiogenesis, such as the activity of Matrix Metalloproteinases (MMP) 2 and 9 and the content of Connective Tissue Growth Factor (CTGF). These results demonstrate long-term efficacy of AAV-mediated PEDF overexpression in counteracting retinal neovascularization in a relevant animal model, and provides evidence towards the use of this strategy to treat angiogenesis in DR and other chronic proliferative retinal disorders. PMID:22911805

  4. Continuous cell supply from Krt7-expressing hematopoietic stem cells during native hematopoiesis revealed by targeted in vivo gene transfer method

    PubMed Central

    Tajima, Yoko; Ito, Keiichi; Umino, Ayumi; Wilkinson, Adam C.; Nakauchi, Hiromitsu; Yamazaki, Satoshi

    2017-01-01

    The nature of hematopoietic stem cells under normal hematopoiesis remained largely unknown due to the limited assays available to monitor their behavior in situ. Here, we develop a new mouse model to transfer genes specifically into the primitive hematopoietic stem cell compartment through the utilization of a modified Rcas/TVA system. We succeeded in transferring a GFP reporter gene into adult hematopoietic stem cells in vivo, which are predominantly quiescent, by generating pseudotyped-lentivirus. Furthermore, we demonstrate the utility of this system to study neonatal hematopoiesis, a developmental stage that has been difficult to analyze to date. Using the system developed in this study, we observed continuous multi-lineage hematopoietic cell supply in peripheral blood from Krt7-positive hematopoietic stem cells during unperturbed homeostatic condition. This powerful experimental system could provide a new standard tool to analyze hematopoiesis under physiological condition without transplantation. PMID:28098173

  5. Leptin in the regulation of immunity, inflammation, and hematopoiesis.

    PubMed

    Fantuzzi, G; Faggioni, R

    2000-10-01

    Leptin, the product of the ob gene, is a pleiotropic molecule that regulates food intake as well as metabolic and endocrine functions. Leptin also plays a regulatory role in immunity, inflammation, and hematopoiesis. Alterations in immune and inflammatory responses are present in leptin- or leptin-receptor-deficient animals, as well as during starvation and malnutrition, two conditions characterized by low levels of circulating leptin. Both leptin and its receptor share structural and functional similarities with the interleukin-6 family of cytokines. Leptin exerts proliferative and antiapoptotic activities in a variety of cell types, including T lymphocytes, leukemia cells, and hematopoietic progenitors. Leptin also affects cytokine production, the activation of monocytes/macrophages, wound healing, angiogenesis, and hematopoiesis. Moreover, leptin production is acutely increased during infection and inflammation. This review focuses on the role of leptin in the modulation of the innate immune response, inflammation, and hematopoiesis.

  6. The Cytokine Flt3-Ligand in Normal and Malignant Hematopoiesis

    PubMed Central

    Tsapogas, Panagiotis; Mooney, Ciaran James; Brown, Geoffrey; Rolink, Antonius

    2017-01-01

    The cytokine Fms-like tyrosine kinase 3 ligand (FL) is an important regulator of hematopoiesis. Its receptor, Flt3, is expressed on myeloid, lymphoid and dendritic cell progenitors and is considered an important growth and differentiation factor for several hematopoietic lineages. Activating mutations of Flt3 are frequently found in acute myeloid leukemia (AML) patients and associated with a poor clinical prognosis. In the present review we provide an overview of our current knowledge on the role of FL in the generation of blood cell lineages. We examine recent studies on Flt3 expression by hematopoietic stem cells and its potential instructive action at early stages of hematopoiesis. In addition, we review current findings on the role of mutated FLT3 in leukemia and the development of FLT3 inhibitors for therapeutic use to treat AML. The importance of mouse models in elucidating the role of Flt3-ligand in normal and malignant hematopoiesis is discussed. PMID:28538663

  7. Cis-element mutated in GATA2-dependent immunodeficiency governs hematopoiesis and vascular integrity

    PubMed Central

    Johnson, Kirby D.; Hsu, Amy P.; Ryu, Myung-Jeom; Wang, Jinyong; Gao, Xin; Boyer, Meghan E.; Liu, Yangang; Lee, Youngsook; Calvo, Katherine R.; Keles, Sunduz; Zhang, Jing; Holland, Steven M.; Bresnick, Emery H.

    2012-01-01

    Haploinsufficiency for GATA2 causes human immunodeficiency syndromes characterized by mycobacterial infection, myelodysplasia, lymphedema, or aplastic anemia that progress to myeloid leukemia. GATA2 encodes a master regulator of hematopoiesis that is also linked to endothelial biology. Though the disease-causing mutations commonly occur in the GATA-2 DNA binding domain, we identified a patient with mycobacterial infection and myelodysplasia who had an uncharacterized heterozygous deletion in a GATA2 cis-element consisting of an E-box and a GATA motif. Targeted deletion of the equivalent murine element to yield homozygous mutant mice revealed embryonic lethality later than occurred with global Gata2 knockout, hematopoietic stem/progenitor cell depletion, and impaired vascular integrity. Heterozygous mutant mice were viable, but embryos exhibited deficits in definitive, but not primitive, hematopoietic stem/progenitor activity and reduced expression of Gata2 and its target genes. Mechanistic analysis revealed disruption of the endothelial cell transcriptome and loss of vascular integrity. Thus, the composite element disrupted in a human immunodeficiency is essential for establishment of the murine hematopoietic stem/progenitor cell compartment in the fetal liver and for essential vascular processes. PMID:22996659

  8. Regulation of hematopoiesis by activators and inhibitors of Wnt signaling from the niche.

    PubMed

    Schreck, Christina; Bock, Franziska; Grziwok, Sandra; Oostendorp, Robert A J; Istvánffy, Rouzanna

    2014-03-01

    Hematopoietic stem cells (HSCs) are a rare population of somatic stem cells that have the ability to regenerate the entire mature blood system in a hierarchical way for the duration of an adult life. Adult HSCs reside in the bone marrow niche. Different niche cell types and molecules regulate the balance of HSC dormancy and activation as well as HSC behavior in both normal and malignant hematopoiesis. Here, we describe the interplay of HSCs and their niche, in particular the involvement of the Wnt signaling pathway. Although the prevailing notion has been that malignant transformation of HSCs is the main cause of leukemia, evidence is mounting that disruption of niche regulation by transformed hematopoietic cells, which may overexpress Wnt signaling or intrinsic stromal defects in gene expression, is at least a collaborative factor in leukemogenesis. Thus, insights into the normal and altered functions of niche components will help to obtain a better understanding of normal and malignant hematopoiesis and how environmental factors affect these processes. © 2014 New York Academy of Sciences.

  9. Correlating Histone Modification Patterns with Gene Expression Data During Hematopoiesis

    PubMed Central

    Hu, Gangqing; Zhao, Keji

    2014-01-01

    Hematopoietic stem cells (HSC) in mammals are an ideal system to study differentiation. While transcription factors (TFs) control the differentiation of HSCs to distinctive terminal blood cells, accumulating evidence suggests that chromatin structure and modifications constitute another critical layer of gene regulation. Recent genome-wide studies based on next-generation sequencing reveal that histone modifications are linked to gene expression and contribute to hematopoiesis. Here, we briefl y review the bioinformatics aspects for ChIP-Seq and RNA-Seq data analysis with applications to the epigenetic studies of hematopoiesis and provide a practical guide to several basic data analysis methods. PMID:24743998

  10. Communication of bone cells with hematopoiesis, immunity and energy metabolism

    PubMed Central

    Asada, Noboru; Sato, Mari; Katayama, Yoshio

    2015-01-01

    The bone contains the bone marrow. The functional communication between bone cells and hematopoiesis has been extensively studied in the past decade or so. Osteolineage cells and their modulators, such as the sympathetic nervous system, macrophages and osteoclasts, form a complex unit to maintain the homeostasis of hematopoiesis, called the ‘microenvironment'. Recently, bone-embedded osteocytes, the sensors of gravity and mechanical stress, have joined the microenvironment, and they are demonstrated to contribute to whole body homeostasis through the control of immunity and energy metabolism. The inter-organ communication orchestrated by the bone is summarized in this article. PMID:26512322

  11. Intracranial extramedullary hematopoiesis. CT and bone marrow scan findings

    SciTech Connect

    Urman, M.; O'Sullivan, R.A.; Nugent, R.A.; Lentle, B.C. )

    1991-06-01

    This case concerns a patient with intracranial extramedullary hematopoiesis (EH) suspected on a CT scan and subsequently confirmed with In-111 chloride and Tc-99m SC bone marrow scans. The bone marrow scans also provided additional information by demonstrating other sites of EH in the paravertebral tissues and bone marrow expansion into the distal extremities.

  12. Glucose-Dependent Insulinotropic Polypeptide Receptor Deficiency Leads to Impaired Bone Marrow Hematopoiesis.

    PubMed

    Mantelmacher, Fernanda Dana; Fishman, Sigal; Cohen, Keren; Pasmanik Chor, Metsada; Yamada, Yuichiro; Zvibel, Isabel; Varol, Chen

    2017-04-15

    The bone marrow (BM) contains controlled specialized microenvironments, or niches, that regulate the quiescence, proliferation, and differentiation of hematopoietic stem and progenitor cells (HSPC). The glucose-dependent insulinotropic polypeptide (GIP) is a gut-derived incretin hormone that mediates postprandial insulin secretion and has anabolic effects on adipose tissue. Previous studies demonstrated altered bone microarchitecture in mice deficient for GIP receptor (Gipr(-/-) ), as well as the expression of high-affinity GIP receptor by distinct cells constructing the BM HSPC niche. Nevertheless, the involvement of GIP in the process of BM hematopoiesis remains elusive. In this article, we show significantly reduced representation and proliferation of HSPC and myeloid progenitors in the BM of Gipr(-/-) mice. This was further manifested by reduced levels of BM and circulating differentiated immune cells in young and old adult mice. Moreover, GIP signaling was required for the establishment of supportive BM HSPC niches during HSPC repopulation in radioablated BM chimera mice. Finally, molecular profiling of various factors involved in retention, survival, and expansion of HSPC revealed significantly lower expression of the Notch-receptor ligands Jagged 1 and Jagged 2 in osteoblast-enriched bone extracts from Gipr(-/-) mice, which are important for HSPC expansion. In addition, there was increased expression of CXCL12, a factor important for HSPC retention and quiescence, in whole-BM extracts from Gipr(-/-) mice. Collectively, our data suggest that the metabolic hormone GIP plays an important role in BM hematopoiesis.

  13. Fetal hyperglycemia and a high fat diet contribute to aberrant glucose tolerance and hematopoiesis in adulthood

    PubMed Central

    Blue, Emily K.; Ballman, Kimberly; Boyle, Frances; Oh, Eunjin; Kono, Tatsuyoshi; Quinney, Sara K.; Thurmond, Debbie C.; Evans-Molina, Carmella; Haneline, Laura S.

    2014-01-01

    Background Children exposed to gestational diabetes mellitus (GDM) during pregnancy are at increased risk of obesity, diabetes, and hypertension. Our goal was to identify metabolic and hematopoietic alterations after intrauterine exposure to maternal hyperglycemia that may contribute to the pathogenesis of chronic morbidities. Methods Streptozotocin treatment induced maternal hyperglycemia during the last third of gestation in rat dams. Offspring of control mothers (OCM) and diabetic mothers (ODM) were evaluated for weight, glucose tolerance, insulin tolerance, and hematopoiesis defects. The effects of aging were examined in normal and high fat diet (HFD)-fed young (8-week-old) and aged (11-month-old) OCM and ODM rats. Results Young adult ODM males on a normal diet, but not females, displayed improved glucose tolerance due to increased insulin levels. Aged ODM males and females gained more weight than OCM on a HFD and had worse glucose tolerance. Aged ODM males fed a HFD were also neutrophilic. Increases in bone marrow cellularity and myeloid progenitors preceded neutrophilia in ODM males fed a HFD. Conclusion When combined with other risk factors like HFD and aging, changes in glucose metabolism and hematopoiesis may contribute to the increased risk of obesity, type 2 diabetes, and hypertension observed in children of GDM mothers. PMID:25412163

  14. Haemopedia: An Expression Atlas of Murine Hematopoietic Cells.

    PubMed

    de Graaf, Carolyn A; Choi, Jarny; Baldwin, Tracey M; Bolden, Jessica E; Fairfax, Kirsten A; Robinson, Aaron J; Biben, Christine; Morgan, Clare; Ramsay, Kerry; Ng, Ashley P; Kauppi, Maria; Kruse, Elizabeth A; Sargeant, Tobias J; Seidenman, Nick; D'Amico, Angela; D'Ombrain, Marthe C; Lucas, Erin C; Koernig, Sandra; Baz Morelli, Adriana; Wilson, Michael J; Dower, Steven K; Williams, Brenda; Heazlewood, Shen Y; Hu, Yifang; Nilsson, Susan K; Wu, Li; Smyth, Gordon K; Alexander, Warren S; Hilton, Douglas J

    2016-09-13

    Hematopoiesis is a multistage process involving the differentiation of stem and progenitor cells into distinct mature cell lineages. Here we present Haemopedia, an atlas of murine gene-expression data containing 54 hematopoietic cell types, covering all the mature lineages in hematopoiesis. We include rare cell populations such as eosinophils, mast cells, basophils, and megakaryocytes, and a broad collection of progenitor and stem cells. We show that lineage branching and maturation during hematopoiesis can be reconstructed using the expression patterns of small sets of genes. We also have identified genes with enriched expression in each of the mature blood cell lineages, many of which show conserved lineage-enriched expression in human hematopoiesis. We have created an online web portal called Haemosphere to make analyses of Haemopedia and other blood cell transcriptional datasets easier. This resource provides simple tools to interrogate gene-expression-based relationships between hematopoietic cell types and genes of interest. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  15. Long noncoding RNAs during normal and malignant hematopoiesis

    PubMed Central

    Alvarez-Dominguez, Juan R.; Hu, Wenqian; Gromatzky, Austin A.

    2014-01-01

    Long noncoding RNAs (lncRNAs) are increasingly recognized to contribute to cellular development via diverse mechanisms during both health and disease. Here, we highlight recent progress on the study of lncRNAs that function in the development of blood cells. We emphasize lncRNAs that regulate blood cell fates through epigenetic control of gene expression, an emerging theme among functional lncRNAs. Many of these noncoding genes and their targets become dysregulated during malignant hematopoiesis, directly implicating lncRNAs in blood cancers such as leukemia. In a few cases, dysregulation of an lncRNA alone leads to malignant hematopoiesis in a mouse model. Thus, lncRNAs may be not only useful as markers for the diagnosis and prognosis of cancers of the blood, but also as potential targets for novel therapies. PMID:24609766

  16. Leukemic blast cells and controversies in models of hematopoiesis.

    PubMed

    Gluzman, D F; Sklyarenko, L M; Zavelevich, M P; Koval, S V; Ivanovskaya, T S

    2015-03-01

    Classical and up-to-date models of hematopoietic lineage determination are briefly reviewed with the focus on myeloid-based models challenging the existence of the common progenitor for T cells, B cells and NK cells. The analysis of immunophenotype of leukemic blast cells seems to be a promising approach for interpreting some controversies in the schemes of normal hematopoiesis. The literature data as well as our own findings in the patients with various types of acute leukemias are in favor of the concept postulating that common myeloid-lymphoid progenitors giving rise to T and B cell branches retain the myeloid potential. The similarity of some immunophenotypic features of blast cells in pro-B acute lymphoblastic leukemia and acute monoblastic leukemia is consistent with monocyte origin postulated in the studies of normal hematopoiesis. Study of acute leukemias may be the challenging area of research allowing for new insight into the origin of hematopoietic cell lineages.

  17. Cell state-specific metabolic dependency in hematopoiesis and leukemogenesis

    PubMed Central

    Wang, Ying-Hua; Israelsen, William J.; Lee, Dongjun; Yu, Vionnie W.C.; Jeanson, Nathaniel T.; Clish, Clary B; Cantley, Lewis C.; Heiden, Matthew G. Vander; Scadden, David T.

    2014-01-01

    SUMMARY The balance between oxidative and non-oxidative glucose metabolism is essential for a number of pathophysiological processes. By deleting enzymes that affect aerobic glycolysis with different potencies, we examine how modulating glucose metabolism specifically affects hematopoietic and leukemic cell populations. We find that deficiency in the M2 pyruvate kinase isoform (PKM2) reduces levels of metabolic intermediates important for biosynthesis and impairs progenitor function without perturbing hematopoietic stem cells (HSC), whereas lactate dehydrogenase-A (LDHA) deletion significantly inhibits the function of both HSC and progenitors during hematopoiesis. In contrast, leukemia initiation by transforming alleles putatively affecting either HSC or progenitors is inhibited in the absence of either PKM2 or LDHA, indicating that the cell state-specific responses to metabolic manipulation in hematopoiesis do not apply to the setting of leukemia. This finding suggests that fine-tuning the level of glycolysis may be therapeutically explored for treating leukemia while preserving HSC function. PMID:25215489

  18. Gut microbiota promote hematopoiesis to control bacterial infection.

    PubMed

    Khosravi, Arya; Yáñez, Alberto; Price, Jeremy G; Chow, Andrew; Merad, Miriam; Goodridge, Helen S; Mazmanian, Sarkis K

    2014-03-12

    The commensal microbiota impacts specific immune cell populations and their functions at peripheral sites, such as gut mucosal tissues. However, it remains unknown whether gut microbiota control immunity through regulation of hematopoiesis at primary immune sites. We reveal that germ-free mice display reduced proportions and differentiation potential of specific myeloid cell progenitors of both yolk sac and bone marrow origin. Homeostatic innate immune defects may lead to impaired early responses to pathogens. Indeed, following systemic infection with Listeria monocytogenes, germ-free and oral-antibiotic-treated mice display increased pathogen burden and acute death. Recolonization of germ-free mice with a complex microbiota restores defects in myelopoiesis and resistance to Listeria. These findings reveal that gut bacteria direct innate immune cell development via promoting hematopoiesis, contributing to our appreciation of the deep evolutionary connection between mammals and their microbiota.

  19. Densely interconnected transcriptional circuits control cell states in human hematopoiesis

    PubMed Central

    Novershtern, Noa; Subramanian, Aravind; Lawton, Lee N.; Mak, Raymond H.; Haining, W. Nicholas; McConkey, Marie E.; Habib, Naomi; Yosef, Nir; Chang, Cindy Y.; Shay, Tal; Frampton, Garrett M.; Drake, Adam C. B.; Leskov, Ilya; Nilsson, Bjorn; Preffer, Fred; Dombkowski, David; Evans, John W.; Liefeld, Ted; Smutko, John S.; Chen, Jianzhu; Friedman, Nir; Young, Richard A.; Golub, Todd R.; Regev, Aviv; Ebert, Benjamin L.

    2011-01-01

    While many individual transcription factors are known to regulate hematopoietic differentiation, major aspects of the global architecture of hematopoiesis remain unknown. Here, we profiled gene expression in 38 distinct purified populations of human hematopoietic cells and used probabilistic models of gene expression and analysis of cis-elements in gene promoters to decipher the general organization of their regulatory circuitry. We identified modules of highly co-expressed genes, some of which are restricted to a single lineage, but most are expressed at variable levels across multiple lineages. We found densely interconnected cis-regulatory circuits and a large number of transcription factors that are differentially expressed across hematopoietic states. These findings suggest a more complex regulatory system for hematopoiesis than previously assumed. PMID:21241896

  20. Long noncoding RNAs during normal and malignant hematopoiesis.

    PubMed

    Alvarez-Dominguez, Juan R; Hu, Wenqian; Gromatzky, Austin A; Lodish, Harvey F

    2014-01-01

    Long noncoding RNAs (lncRNAs) are increasingly recognized to contribute to cellular development via diverse mechanisms during both health and disease. Here, we highlight recent progress on the study of lncRNAs that function in the development of blood cells. We emphasize lncRNAs that regulate blood cell fates through epigenetic control of gene expression, an emerging theme among functional lncRNAs. Many of these noncoding genes and their targets become dysregulated during malignant hematopoiesis, directly implicating lncRNAs in blood cancers such as leukemia. In a few cases, dysregulation of an lncRNA alone leads to malignant hematopoiesis in a mouse model. Thus, lncRNAs may be not only useful as markers for the diagnosis and prognosis of cancers of the blood, but also as potential targets for novel therapies.

  1. Suppression of hepatic hematopoiesis with radioactive gold (/sup 198/Au)

    SciTech Connect

    Turner, A.R.; Gummerman, L.W.; Boggs, D.R.

    1985-04-01

    A patient with idiopathic myelofibrosis of some 20 yr duration developed esophageal varices and ascites. No explanation for increased portal pressure other than hepatic hematopoiesis was found. Consequently, a trial of cobalt irradiation to the liver was undertaken with definite but transient decrease in ascites. Subsequently, two courses of radioactive colloidal gold were given, again with definite but transient beneficial effects on the degree of ascites. This latter benefit occurred without suppression of marrow function.

  2. CircRNAs in hematopoiesis and hematological malignancies

    PubMed Central

    Bonizzato, A; Gaffo, E; te Kronnie, G; Bortoluzzi, S

    2016-01-01

    Cell states in hematopoiesis are controlled by master regulators and by complex circuits of a growing family of RNA species impacting cell phenotype maintenance and plasticity. Circular RNAs (circRNAs) are rapidly gaining the status of particularly stable transcriptome members with distinctive qualities. RNA-seq identified thousands of circRNAs with developmental stage- and tissue-specific expression corroborating earlier suggestions that circular isoforms are a natural feature of the cell expression program. CircRNAs are abundantly expressed also in the hematopoietic compartment. There are a number of studies on circRNAs in blood cells, a specific overview is however lacking. In this review we first present current insight in circRNA biogenesis discussing the relevance for hematopoiesis of the highly interleaved processes of splicing and circRNA biogenesis. Regarding molecular functions circRNAs modulate host gene expression, but also compete for binding of microRNAs, RNA-binding proteins or translation initiation and participate in regulatory circuits. We examine circRNA expression in the hematopoietic compartment and in hematologic malignancies and review the recent breakthrough study that identified pathogenic circRNAs derived from leukemia fusion genes. CircRNA high and regulated expression in blood cell types indicate that further studies are warranted to inform the position of these regulators in normal and malignant hematopoiesis. PMID:27740630

  3. [Blueberry muffin baby: the pathogenesis of cutaneous extramedullary hematopoiesis].

    PubMed

    Hödl, S; Auböck, L; Reiterer, F; Soyer, H P; Müller, W D

    2001-11-01

    Two neonates exhibited the clinical picture of the "blueberry muffin baby" at delivery. The integument manifested petechiae and purpuric magenta-colored macules, papules, and plaques, as well as blueberry-colored ecchymoses. These findings led to the diagnosis of a connatal cytomegalovirus infection and fetal erythroblastosis, respectively. The hemorrhagic-purpuric looking skin lesions reflected extramedullary hematopoiesis with ultrastructural study disclosing evidence of both erythro- and granulopoietic lineage. For the first time, we were able to demonstrate that complexes of red cells in various stages of maturation can occur in the skin, similarly to the erythroblastic islands of the bone marrow. In the pathogenesis of extramedullary hematopoiesis, mechanisms underlying the reconstitution of blood cells must be considered. These may reactivate hematopoiesis in organs where it previously occurred in embryonic and fetal life. Possible causative factors may be great compensatory demand, deficient replacement, or loss or dysfunction of corpuscular blood elements. This would explain the occurrence of this disease entity in conjunction with etiologically completely heterogeneous systemic diseases.

  4. Single cell analysis of normal and leukemic hematopoiesis.

    PubMed

    Povinelli, Benjamin J; Rodriguez-Meira, Alba; Mead, Adam J

    2017-09-07

    The hematopoietic system is well established as a paradigm for the study of cellular hierarchies, their disruption in disease and therapeutic use in regenerative medicine. Traditional approaches to study hematopoiesis involve purification of cell populations based on a small number of surface markers. However, such population-based analysis obscures underlying heterogeneity contained within any phenotypically defined cell population. This heterogeneity can only be resolved through single cell analysis. Recent advances in single cell techniques allow analysis of the genome, transcriptome, epigenome and proteome in single cells at an unprecedented scale. The application of these new single cell methods to investigate the hematopoietic system has led to paradigm shifts in our understanding of cellular heterogeneity in hematopoiesis and how this is disrupted in disease. In this review, we summarize how single cell techniques have been applied to the analysis of hematopoietic stem/progenitor cells in normal and malignant hematopoiesis, with a particular focus on recent advances in single-cell genomics, including how these might be utilized for clinical application. Copyright © 2017. Published by Elsevier Ltd.

  5. Intrathoracic extramedullary hematopoiesis: appearance on /sup 99m/Tc sulfur colloid marrow scan

    SciTech Connect

    Bronn, L.J.; Paquelet, J.R.; Tetalman, M.R.

    1980-06-01

    Imaging of the bone marrow by radionuclide scanning was performed using colloids, which are phagocytized by the reticuloendothelial cells of the marrow, or radioiron, which is incorporated into reticulocytes. The use of the former radiopharmaceutical is based on the assumption, generally valid except in aplastic states or after irradiation, that the distribution of hematopoietic and reticuloendothelial tissue in the marrow is similar. Regardless of the method used, active adult marrow is normally distributed only in the axial skeleton and proximal humeri and femurs. Marrow imaging has been used in the evaluation of myeloproliferative disorders, leukemia, lymphoma, aplastic states, malignancy metastatic to marrow, and hemolytic anemia. We report a case of thalassemia major in which the diagnosis of intrathoracic extramedullary hematopoiesis was confirmed with the /sup 99m/Tc sulfur colloid bone marrow scan.

  6. A regulatory role for Fc gamma receptors (CD16 and CD32) in hematopoiesis.

    PubMed

    de Andres, B; Hagen, M; Sandor, M; Verbeek, S; Rokhlin, O; Lynch, R G

    1999-05-03

    Progenitor cells of the T- and B-lineages in mice express (CD32) and Fc gamma RIII (CD16) but as the developing lymphocytes begin to express clonal antigen receptors, CD16 and CD32 are downregulated in T-cells, and CD16 is downregulated in B-cells. Considering that counter-receptors for Fc gamma R occur on thymic and bone marrow stromal cells, the possibility exists that Fc gamma R might participate in some aspect of T- and B-lineage development prior to the stage of antigen receptor expression. Previous studies provided evidence that Fc gamma R can influence murine T-lineage development. In the present studies we found that anti-Fc gamma RII/III mAb accelerated B-lineage development in bone marrow cultures from normal mice, but not in cultures from CD16-/- or CD32-/- mice. Similar results were observed when FACS-purified B-progenitor cells were co-cultured with BMS2, a bone marrow stromal cell line. Fresh bone marrow from CD32-/- mice contained about two-fold more B-lineage cells compared to bone marrow from normal or CD16-/- mice. These studies indicate that the Fc gamma R on B-lineage progenitor cells can influence their further development and add to a growing body of evidence that implicates Fc gamma R as regulatory elements in hematopoiesis.

  7. Use of partially phosphorothioated "antisense" oligodeoxynucleotides for sequence-dependent modulation of hematopoiesis in culture.

    PubMed

    Ehrlich, G; Patinkin, D; Ginzberg, D; Zakut, H; Eckstein, F; Soreq, H

    1994-01-01

    To distinguish between sequence-dependent effects and non-specific cytotoxicity of phosphorothioate antisense oligonucleotides (AS-oligos), we introduced AS-oligos blocking expression of 2Hs, the Homo sapiens cell division controller cdc2 kinase, its hematopoietically expressed homolog CHED, and the acetylcholine-hydrolyzing enzyme butyrylcholinesterase (BCHE) into primary murine bone marrow (BM) culture. Antisense oligonucleotides were fully phosphorothioated (Ts) or prepared with three phosphorothioate groups at their 3' termini (S3). Each of these oligos could cause reductions in colony counts either as a result of its sequence-dependent biological capacity or due to sequence-independent cytotoxicity. The Ts and S3 forms of the matching sense oligo, S-BCHE, served for comparison. The S3 forms of AS-2Hs, AS-BCHE, and S-BCHE caused more limited drops in colony counts than their Ts counterparts, reflecting lower cytotoxicity. When incubated with electroblotted BM proteins, Ts but not S3 oligos intensively labeled two protein bands. Moreover, 5'-end 32P-labeled (Ts) S-BCHE labeled nuclear proteins in situ in small, mitotic cells, suggesting correlation between oligo-protein interactions and the sequence-independent cytotoxicity of Ts AS-oligos. Extension of the apparently nontoxic AS-CHED by two adenosine residues at the 3' end, creating a potential for intramolecular hydrogen bond formation, resulted in increased toxicity. These findings recommend the use of nonlooped, partially phosphorothioated oligos for the modulation of hematopoiesis.

  8. Histone demethylase KDM2B regulates lineage commitment in normal and malignant hematopoiesis

    PubMed Central

    Andricovich, Jaclyn; Kai, Yan; Peng, Weiqun; Foudi, Adlen; Tzatsos, Alexandros

    2016-01-01

    The development of the hematopoietic system is a dynamic process that is controlled by the interplay between transcriptional and epigenetic networks to determine cellular identity. These networks are critical for lineage specification and are frequently dysregulated in leukemias. Here, we identified histone demethylase KDM2B as a critical regulator of definitive hematopoiesis and lineage commitment of murine hematopoietic stem and progenitor cells (HSPCs). RNA sequencing of Kdm2b-null HSPCs and genome-wide ChIP studies in human leukemias revealed that KDM2B cooperates with polycomb and trithorax complexes to regulate differentiation, lineage choice, cytokine signaling, and cell cycle. Furthermore, we demonstrated that KDM2B exhibits a dichotomous role in hematopoietic malignancies. Specifically, we determined that KDM2B maintains lymphoid leukemias, but restrains RAS-driven myeloid transformation. Our study reveals that KDM2B is an important mediator of hematopoietic cell development and has opposing roles in tumor progression that are dependent on cellular context. PMID:26808549

  9. Fell Pony syndrome: characterization of developmental hematopoiesis failure and associated gene expression profiles.

    PubMed

    Tallmadge, Rebecca L; Stokol, Tracy; Gould-Earley, Mary Jean; Earley, Ed; Secor, Erica J; Matychak, Mary Beth; Felippe, M Julia B

    2012-07-01

    Fell Pony syndrome (FPS) is a fatal immunodeficiency that occurs in foals of the Fell Pony breed. Affected foals present with severe anemia, B cell lymphopenia, and opportunistic infections. Our objective was to conduct a prospective study of potential FPS-affected Fell Pony foals to establish clinical, immunological, and molecular parameters at birth and in the first few weeks of life. Complete blood counts, peripheral blood lymphocyte phenotyping, and serum immunoglobulin concentrations were determined for 3 FPS-affected foals, 49 unaffected foals, and 6 adult horses. In addition, cytology of bone marrow aspirates was performed sequentially in a subset of foals. At birth, the FPS-affected foals were not noticeably ill and had hematocrit and circulating B cell counts comparable to those of unaffected foals; however, over 6 weeks, values for both parameters steadily declined. A bone marrow aspirate from a 3-week-old FPS-affected foal revealed erythroid hyperplasia and concurrent erythroid and myeloid dysplasia, which progressed to a severe erythroid hypoplasia at 5 weeks of life. Immunohistochemical staining confirmed the paucity of B cells in primary and secondary lymphoid tissues. The mRNA expression of genes involved in B cell development, signaling, and maturation was investigated using qualitative and quantitative reverse transcriptase PCR (RT-PCR). Several genes, including CREB1, EP300, MYB, PAX5, and SPI1/PU.1, were sequenced from FPS-affected and unaffected foals. Our study presents evidence of fetal erythrocyte and B cell hematopoiesis with rapid postnatal development of anemia and B lymphopenia in FPS-affected foals. The transition between fetal/neonatal and adult-like hematopoiesis may be an important aspect of the pathogenesis of FPS.

  10. Expansion of Human and Murine Hematopoietic Stem and Progenitor Cells Ex Vivo without Genetic Modification Using MYC and Bcl-2 Fusion Proteins

    PubMed Central

    Bird, Gregory A.; Polsky, Avital; Estes, Patricia; Hanlon, Teri; Hamilton, Haley; Morton, John J.; Gutman, Jonathan; Jimeno, Antonio

    2014-01-01

    The long-term repopulating hematopoietic stem cell (HSC) population can self-renew in vivo, support hematopoiesis for the lifetime of the individual, and is of critical importance in the context of bone marrow stem cell transplantation. The mechanisms that regulate the expansion of HSCs in vivo and in vitro remain unclear to date. Since the current set of surface markers only allow for the identification of a population of cells that is highly enriched for HSC activity, we will refer to the population of cells we expand as Hematopoietic Stem and Progenitor cells (HSPCs). We describe here a novel approach to expand a cytokine-dependent Hematopoietic Stem and Progenitor Cell (HSPC) population ex vivo by culturing primary adult human or murine HSPCs with fusion proteins including the protein transduction domain of the HIV-1 transactivation protein (Tat) and either MYC or Bcl-2. HSPCs obtained from either mouse bone marrow, human cord blood, human G-CSF mobilized peripheral blood, or human bone marrow were expanded an average of 87 fold, 16.6 fold, 13.6 fold, or 10 fold, respectively. The expanded cell populations were able to give rise to different types of colonies in methylcellulose assays in vitro, as well as mature hematopoietic populations in vivo upon transplantation into irradiated mice. Importantly, for both the human and murine case, the ex vivo expanded cells also gave rise to a self-renewing cell population in vivo, following initial transplantation, that was able to support hematopoiesis upon serial transplantation. Our results show that a self-renewing cell population, capable of reconstituting the hematopoietic compartment, expanded ex vivo in the presence of Tat-MYC and Tat-Bcl-2 suggesting that this may be an attractive approach to expand human HSPCs ex vivo for clinical use. PMID:25170611

  11. Expansion of human and murine hematopoietic stem and progenitor cells ex vivo without genetic modification using MYC and Bcl-2 fusion proteins.

    PubMed

    Bird, Gregory A; Polsky, Avital; Estes, Patricia; Hanlon, Teri; Hamilton, Haley; Morton, John J; Gutman, Jonathan; Jimeno, Antonio; Turner, Brian C; Refaeli, Yosef

    2014-01-01

    The long-term repopulating hematopoietic stem cell (HSC) population can self-renew in vivo, support hematopoiesis for the lifetime of the individual, and is of critical importance in the context of bone marrow stem cell transplantation. The mechanisms that regulate the expansion of HSCs in vivo and in vitro remain unclear to date. Since the current set of surface markers only allow for the identification of a population of cells that is highly enriched for HSC activity, we will refer to the population of cells we expand as Hematopoietic Stem and Progenitor cells (HSPCs). We describe here a novel approach to expand a cytokine-dependent Hematopoietic Stem and Progenitor Cell (HSPC) population ex vivo by culturing primary adult human or murine HSPCs with fusion proteins including the protein transduction domain of the HIV-1 transactivation protein (Tat) and either MYC or Bcl-2. HSPCs obtained from either mouse bone marrow, human cord blood, human G-CSF mobilized peripheral blood, or human bone marrow were expanded an average of 87 fold, 16.6 fold, 13.6 fold, or 10 fold, respectively. The expanded cell populations were able to give rise to different types of colonies in methylcellulose assays in vitro, as well as mature hematopoietic populations in vivo upon transplantation into irradiated mice. Importantly, for both the human and murine case, the ex vivo expanded cells also gave rise to a self-renewing cell population in vivo, following initial transplantation, that was able to support hematopoiesis upon serial transplantation. Our results show that a self-renewing cell population, capable of reconstituting the hematopoietic compartment, expanded ex vivo in the presence of Tat-MYC and Tat-Bcl-2 suggesting that this may be an attractive approach to expand human HSPCs ex vivo for clinical use.

  12. Very Small Embryonic-Like Stem Cells: A Potential Developmental Link Between Germinal Lineage and Hematopoiesis in Humans.

    PubMed

    Virant-Klun, Irma

    2016-01-15

    It has been suggested that hematopoietic stem/progenitor cells (HSPCs) could become specified from a population of migrating primordial germ cells (PGCs), precursors of gametes, during embryogenesis. Some recent experimental data demonstrated that the cell population that is usually considered to be PGCs, moving toward the gonadal ridges of an embryo, contains a subset of cells coexpressing several germ cell and hematopoietic markers and possessing hematopoietic activity. Experimental data showed that bone morphogenetic protein 4 (BMP4) generates PGCs from mouse bone marrow-derived pluripotent stem cells. Interestingly, functional reproductive hormone receptors have been identified in HSPCs, thus indicating their potential role in reproductive function. Several reports have demonstrated fertility restoration and germ cell generation after bone marrow transplantation in both animal models and humans. A potential link between HSPCs and germinal lineage might be represented by very small embryonic-like stem cells (VSELs), which have been found in adult human bone marrow, peripheral blood, and umbilical cord blood, express a specific pattern of pluripotency, germinal lineage, and hematopoiesis, and are proposed to persist in adult tissues and organs from the embryonic period of life. Stem cell populations, similar to VSELs, expressing several genes related to pluripotency and germinal lineage, especially to PGCs, have been discovered in adult human reproductive organs, ovaries and testicles, and were related to primitive germ cell-like cell development in vitro, thus supporting the idea of VSELs as a potential link between germinal lineage and hematopoiesis.

  13. Spleen hypoplasia leads to abnormal stress hematopoiesis in mice with loss of Pbx homeoproteins in splenic mesenchyme.

    PubMed

    Zewdu, Rediet; Risolino, Maurizio; Barbulescu, Alexandru; Ramalingam, Pradeep; Butler, Jason M; Selleri, Licia

    2016-07-01

    The spleen plays critical roles in immunity and also provides a permissive microenvironment for hematopoiesis. Previous studies have reported that the TALE-class homeodomain transcription factor Pbx1 is essential in hematopoietic stem and progenitor cells (HSPCs) for stem cell maintenance and progenitor expansion. However, the role of Pbx1 in the hematopoietic niche has not been investigated. Here we explored the effects that genetic perturbation of the splenic mesenchymal niche has on hematopoiesis upon loss of members of the Pbx family of homeoproteins. Splenic mesenchyme-specific inactivation of Pbx1 (SKO) on a Pbx2- or Pbx3-deficient genetic background (DKO) resulted in abnormal development of the spleen, which is dysmorphic and severely hypoplastic. This phenotype, in turn, affected the number of HSPCs in the fetal and adult spleen at steady state, as well as markedly impairing the kinetics of hematopoietic regeneration in adult mice after sub-lethal and lethal myelosuppressive irradiation. Spleens of mice with compound Pyx deficiency 8 days following sublethal irradiation displayed significant downregulation of multiple cytokine-encoding genes, including KitL/SCF, Cxcl12/SDF-1, IL-3, IL-4, GM-CSF/Csf2 IL-10, and Igf-1, compared with controls. KitL/SCF and Cxcl12/SDF-1 were recently shown to play key roles in the splenic niche in response to various haematopoietic stresses such as myeloablation, blood loss, or pregnancy. Our results demonstrate that, in addition to their intrinsic roles in HSPCs, non-cell autonomous functions of Pbx factors within the splenic niche contribute to the regulation of hematopoiesis, at least in part via the control of KitL/SCF and Cxcl12/SDF-1. Furthermore, our study establishes that abnormal spleen development and hypoplasia have deleterious effects on the efficiency of hematopoietic recovery after bone marrow injury. © 2016 Anatomical Society.

  14. The effect of the zeolite clinoptilolite on serum chemistry and hematopoiesis in mice.

    PubMed

    Martin-Kleiner, I; Flegar-Mestric, Z; Zadro, R; Breljak, D; Stanovic Janda, S; Stojkovic, R; Marusic, M; Radacic, M; Boranic, M

    2001-07-01

    Zeolites are natural or synthetic crystalline alumosilicates with ion exchanging properties. Supplied in fodder, they promote biomass production and animal health. Our aim was to assess the effects of the natural zeolite, clinoptilolite, on hematopoiesis, serum electrolytes and essential biochemical indicators of kidney and liver function in mice. Two preparations differing in particle size were tested: a powderized form obtained by countercurrent mechanical treatment of the clinoptilolite (MTCp) and normally ground clinoptilolite (NGCp). Young adult mice were supplied with food containing 12.5, 25 or 50% clinoptilolite powder. Control animals received the same food ration without the clinoptilolite. After 10, 20, 30 and 40 days, six animals from each group were exsanguinated to obtain blood for hematological and serum for biochemical measurements as well as to collect femoral bone marrow for determination of hematopoietic activity. Clinoptilolite ingestion was well tolerated, as judged by comparable body masses of treated and control animals. A 20% increase of the potassium level was detected in mice receiving the zeolite-rich diet, without other changes in serum chemistry. Erythrocyte, hemoglobin and platelet levels in peripheral blood were not materially affected. NGCp caused leukocytosis, with concomitant decline of the GM-CFU content in the bone marrow, which was attributed to intestinal irritation by rough zeolite particles. The mechanically treated clinoptilolite preparation caused similar, albeit less pronounced, changes. In a limited experiment, mice having transplanted mammary carcinoma in the terminal stage showed increased potassium and decreased sodium and chloride levels, severe anemia and leukocytosis, decreased bone marrow cellularity and diminished content of hematopoietic progenitor cells in the marrow. The clinoptilolite preparations ameliorated the sodium and chloride decline, whereas the effects on hematopoiesis were erratic.

  15. Polymorphisms of the murine mitochondrial ND4, CYTB and COX3 genes impact hematopoiesis during aging

    PubMed Central

    Timmer, Katrin; Sekora, Anett; Knübel, Gudrun; Escobar, Hugo Murua; Fuellen, Georg; Ibrahim, Saleh M.; Tiedge, Markus; Baltrusch, Simone; Jaster, Robert; Köhling, Rüdiger; Junghanss, Christian

    2016-01-01

    During aging, mitochondrial DNA (mtDNA) can accumulate mutations leading to increasing levels of reactive oxygen species (ROS). Increased ROS were described to activate formerly quiescent hematopoietic stem cells (HSC). Mutations in mtDNA were shown to enhance the risk for myelodysplastic syndrome and leukemia. However, the complex relationship between mtDNA variations, ROS and aging of the hematopoietic system is not fully understood. Herein, three mouse strains with mtDNA polymorphisms in genes of respiratory chain complexes I (ND4), III (CYTB) and IV (COX3) were compared to a reference strain during aging. Analysis focused on ROS and ATP levels, bone marrow composition and blood counts. Additionally, hematopoietic restoration capacity following cytotoxic stress was tested. Mice with polymorphisms in ND4 and CYTB gene had significantly decreasing ROS levels in bone marrow cells during aging, without effecting ATP levels. In addition, the frequency of stem and progenitor cells increased during aging but the amount of lymphocytes in the peripheral blood decreased during aging. In summary, the presence of mtDNA polymorphisms affecting the respiratory chain complexes I, III and IV was associated with altered ROS levels as well as changes in BM and peripheral blood composition during aging. PMID:27626489

  16. MicroRNA-155 and Its Role in Malignant Hematopoiesis

    PubMed Central

    Ranganath, Prajnya

    2015-01-01

    MicroRNA-155 (miR-155) is a multifunctional molecule involved in both normal and malignant hematopoiesis. It has been found to be involved in the pathogenesis of many different hematological malignancies with either an oncogenic or a tumor-repressor effect, depending on the nature of the cell and the type of malignancy. In particular, it has been strongly implicated in the causation of diffuse large B-cell lymphomas. This review focuses on the molecular interactions of miR-155, its oncogenic mechanisms, and its potential as an effective therapeutic target for the associated malignancies. PMID:26523117

  17. Megaloblastic hematopoiesis in a 20 year old pregnant female

    PubMed Central

    Trivette, Evan T.; Hoedebecke, Kyle; Berry-Cabán, Cristóbal S.; Jacobs, Brandy R.

    2013-01-01

    Summary Background: Nitrous oxide can cause disordered blood cell proliferation and lead to pancytopenia and altered immune function. Case Report: A young pregnant female patient presented after binge nitrous oxide abuse with altered mental status and abnormal vital signs. From her initial assessment she was noted to have pancytopenia and was found to have megaloblastic, hyper-cellular changes in a subsequent bone marrow biopsy. This presentation was determined to be secondary to toxic effects after heavy use of nitrous oxide. Conclusions: Nitrous oxide exposure, including use as an inhalant, over 12 hours can lead to bone marrow abnormalities such as megaloblastic hematopoiesis. PMID:23569553

  18. Murine T-box transcription factor Tbx20 acts as a repressor during heart development, and is essential for adult heart integrity, function and adaptation.

    PubMed

    Stennard, Fiona A; Costa, Mauro W; Lai, Donna; Biben, Christine; Furtado, Milena B; Solloway, Mark J; McCulley, David J; Leimena, Christiana; Preis, Jost I; Dunwoodie, Sally L; Elliott, David E; Prall, Owen W J; Black, Brian L; Fatkin, Diane; Harvey, Richard P

    2005-05-01

    The genetic hierarchies guiding lineage specification and morphogenesis of the mammalian embryonic heart are poorly understood. We now show by gene targeting that murine T-box transcription factor Tbx20 plays a central role in these pathways, and has important activities in both cardiac development and adult function. Loss of Tbx20 results in death of embryos at mid-gestation with grossly abnormal heart morphogenesis. Underlying these disturbances was a severely compromised cardiac transcriptional program, defects in the molecular pre-pattern, reduced expansion of cardiac progenitors and a block to chamber differentiation. Notably, Tbx20-null embryos showed ectopic activation of Tbx2 across the whole heart myogenic field. Tbx2 encodes a transcriptional repressor normally expressed in non-chamber myocardium, and in the atrioventricular canal it has been proposed to inhibit chamber-specific gene expression through competition with positive factor Tbx5. Our data demonstrate a repressive activity for Tbx20 and place it upstream of Tbx2 in the cardiac genetic program. Thus, hierarchical, repressive interactions between Tbx20 and other T-box genes and factors underlie the primary lineage split into chamber and non-chamber myocardium in the forming heart, an early event upon which all subsequent morphogenesis depends. Additional roles for Tbx20 in adult heart integrity and contractile function were revealed by in-vivo cardiac functional analysis of Tbx20 heterozygous mutant mice. These data suggest that mutations in human cardiac transcription factor genes, possibly including TBX20, underlie both congenital heart disease and adult cardiomyopathies.

  19. The changing balance between osteoblastogenesis and adipogenesis in aging and its impact on hematopoiesis.

    PubMed

    Bethel, Monique; Chitteti, Brahmananda R; Srour, Edward F; Kacena, Melissa A

    2013-06-01

    Osteoblasts (OBs) and adipocytes (APs) share a common mesenchymal ancestor. It is now clear that mesenchymal stem cell (MSC) maturation along the OB lineage comes at the expense of adipogenesis and vice versa. During aging, this balance increasingly favors the formation of APs. Hematopoiesis also slowly declines during the aging process. The role of OB lineage cells in hematopoiesis has been studied, but less is known about how APs regulate hematopoiesis. A few studies have demonstrated a negative relationship between APs and hematopoiesis; however, there is also evidence that brown adipose tissue (BAT) may promote hematopoiesis. This review will examine the current knowledge of how adipogenesis and osteogenesis change with aging and the implications of this changing environment on hematopoeisis.

  20. The Changing Balance Between Osteoblastogenesis and Adipogenesis in Aging and its Impact on Hematopoiesis

    PubMed Central

    Bethel, Monique; Chitteti, Brahmananda R.; Srour, Edward F.

    2013-01-01

    Osteoblasts (OBs) and adipocytes (APs) share a common mesenchymal ancestor. It is now clear that mesenchymal stem cell (MSC) maturation along the OB lineage comes at the expense of adipogenesis and vice versa. During aging, this balance increasingly favors the formation of APs. Hematopoiesis also slowly declines during the aging process. The role of OB lineage cells in hematopoiesis has been studied, but less is known about how APs regulate hematopoiesis. A few studies have demonstrated a negative relationship between APs and hematopoiesis; however, there is also evidence that brown adipose tissue (BAT) may promote hematopoiesis. This review will examine the current knowledge of how adipogenesis and osteogenesis change with aging and the implications of this changing environment on hematopoeisis. PMID:23423562

  1. The NFKB Inducing Kinase Modulates Hematopoiesis During Stress.

    PubMed

    González-Murillo, África; Fernández, Lucía; Baena, Sara; Melen, Gustavo J; Sánchez, Rebeca; Sánchez-Valdepeñas, Carmen; Segovia, José C; Liou, Hsiou-Chi; Schmid, Roland; Madero, Luís; Fresno, Manuel; Ramírez, Manuel

    2015-09-01

    The genetic programs that maintain hematopoiesis during steady state in physiologic conditions are different from those activated during stress. Here, we show that hematopoietic stem cells (HSCs) with deficiencies in components of the alternative NFκB pathway (the NFκB inducing kinase, NIK, and the downstream molecule NFκB2) had a defect in response to stressors such as supraphysiological doses of cytokines, chemotherapy, and hematopoietic transplantation. NIK-deficient mice had peripheral blood and bone marrow leukocyte numbers within normal ranges (except for the already reported defects in B-cell maturation); however, HSCs showed significantly slower expansion capacity in in vitro cultures compared to wild-type HSCs. This was due to a delayed cell cycle and increased apoptosis. In vivo experiments showed that NIK-deficient HSCs did not recover at the same pace as controls when challenged with myeloablative chemotherapy. Finally, NIK-deficient HSCs showed a significantly decreased competitive repopulation capacity in vivo. Using HSCs from mice deficient in one of two downstream targets of NIK, that is, either NFκB2 or c-Rel, only NFκB2 deficiency recapitulated the defects detected with NIK-deficient HSCs. Our results underscore the role of NIK and the alternative NFκB pathway for the recovery of normal levels of hematopoiesis after stress. © 2015 AlphaMed Press.

  2. The Hippo pathway regulates hematopoiesis in Drosophila melanogaster.

    PubMed

    Milton, Claire C; Grusche, Felix A; Degoutin, Joffrey L; Yu, Eefang; Dai, Qi; Lai, Eric C; Harvey, Kieran F

    2014-11-17

    The Salvador-Warts-Hippo (Hippo) pathway is an evolutionarily conserved regulator of organ growth and cell fate. It performs these functions in epithelial and neural tissues of both insects and mammals, as well as in mammalian organs such as the liver and heart. Despite rapid advances in Hippo pathway research, a definitive role for this pathway in hematopoiesis has remained enigmatic. The hematopoietic compartments of Drosophila melanogaster and mammals possess several conserved features. D. melanogaster possess three types of hematopoietic cells that most closely resemble mammalian myeloid cells: plasmatocytes (macrophage-like cells), crystal cells (involved in wound healing), and lamellocytes (which encapsulate parasites). The proteins that control differentiation of these cells also control important blood lineage decisions in mammals. Here, we define the Hippo pathway as a key mediator of hematopoiesis by showing that it controls differentiation and proliferation of the two major types of D. melanogaster blood cells, plasmatocytes and crystal cells. In animals lacking the downstream Hippo pathway kinase Warts, lymph gland cells overproliferated, differentiated prematurely, and often adopted a mixed lineage fate. The Hippo pathway regulated crystal cell numbers by both cell-autonomous and non-cell-autonomous mechanisms. Yorkie and its partner transcription factor Scalloped were found to regulate transcription of the Runx family transcription factor Lozenge, which is a key regulator of crystal cell fate. Further, Yorkie or Scalloped hyperactivation induced ectopic crystal cells in a non-cell-autonomous and Notch-pathway-dependent fashion.

  3. Effects of deuteration on hematopoiesis in the mouse

    SciTech Connect

    Adams, W.H.; Adams, D.G.

    1988-02-01

    Mice ingesting 30 to 50% D/sub 2/O (heavy water, deuterium oxide) developed a dose-dependent depression of formed peripheral blood elements in 4 to 9 days. The principal mechanism of anemia and thrombocytopenia is impaired hematopoiesis. Despite pancytopenia in the peripheral blood, bone marrow cellularity and morphology remained normal. Upon replacement of D/sub 2/O with tap water, platelet and neutrophil concentrations returned to normal within 48 to 72 hr. In contrast, blood lymphocyte concentrations remained low for several weeks. B-lymphocytes may be more affected by deuteration than other lymphocyte subsets. In vivo reticuloendothelial cell function, as assessed by /sup 51/Cr-labeled sheep erythrocyte clearance, was unaffected by D/sub 2/O. Although a dose-dependent decrease in fluid intake occurred during deuteration, hematocytopenia was not a consequence of dehydration. In view of the known kinetics of D/sub 2/O in biological systems, the rapid response of myeloid elements to deuteration must be due primarily to the solvent (nonmetabolic) isotope effect. Prolonged deuteration has proven toxic when included in regimens for treatment of neoplasia, including leukemia, in animal models. The present study shows that modulation of hematopoiesis by D/sub 2/O is possible without invoking the toxicities associated with prolonged deuteration.

  4. Cloning and antisense oligodeoxynucleotide inhibition of a human homolog of cdc2 required in hematopoiesis.

    PubMed

    Lapidot-Lifson, Y; Patinkin, D; Prody, C A; Ehrlich, G; Seidman, S; Ben-Aziz, R; Benseler, F; Eckstein, F; Zakut, H; Soreq, H

    1992-01-15

    Mechanisms triggering the commitment of pluripotent bone marrow stem cells to differentiated lineages such as mononuclear macrophages or multinucleated megakaryocytes are still unknown, although several lines of evidence suggested correlation between cholinergic signaling and hematopoietic differentiation. We now present cloning of a cDNA coding for CHED (cholinesterase-related cell division controller), a human homolog of the Schizosaccharomyces pombe cell division cycle 2 (cdc2)-like kinases, universal controllers of the mitotic cell cycle. Library screening, RNA blot hybridization, and direct PCR amplification of cDNA reverse-transcribed from cellular mRNA revealed that CHED mRNA is expressed in multiple tissues, including bone marrow. The CHED protein includes the consensus ATP binding and phosphorylation domains characteristic of kinases, displays 34-42% identically aligned amino acid residues with other cdc2-related kinases, and is considerably longer at its amino and carboxyl termini. An antisense oligodeoxynucleotide designed to interrupt CHED's expression (AS-CHED) significantly reduced the ratio between CHED mRNA and actin mRNA within 1 hr of its addition to cultures, a reduction that persisted for 4 days. AS-CHED treatment selectively inhibited megakaryocyte development in murine bone marrow cultures but did not prevent other hematopoietic pathways, as evidenced by increasing numbers of mononuclear cells. An oligodeoxynucleotide blocking production of the acetylcholine-hydrolyzing enzyme, butyrylcholinesterase, displayed a similar inhibition of megakaryocytopoiesis. In contrast, an oligodeoxynucleotide blocking production of the human 2Hs cdc2 homolog interfered with production of the human 2Hs cdc2 homolog interfered with cellular proliferation without altering the cell-type composition of these cultures. Therefore, these findings strengthen the link between cholinergic signaling and cell division control in hematopoiesis and implicate both CHED and

  5. Cloning and antisense oligodeoxynucleotide inhibition of a human homolog of cdc2 required in hematopoiesis.

    PubMed Central

    Lapidot-Lifson, Y; Patinkin, D; Prody, C A; Ehrlich, G; Seidman, S; Ben-Aziz, R; Benseler, F; Eckstein, F; Zakut, H; Soreq, H

    1992-01-01

    Mechanisms triggering the commitment of pluripotent bone marrow stem cells to differentiated lineages such as mononuclear macrophages or multinucleated megakaryocytes are still unknown, although several lines of evidence suggested correlation between cholinergic signaling and hematopoietic differentiation. We now present cloning of a cDNA coding for CHED (cholinesterase-related cell division controller), a human homolog of the Schizosaccharomyces pombe cell division cycle 2 (cdc2)-like kinases, universal controllers of the mitotic cell cycle. Library screening, RNA blot hybridization, and direct PCR amplification of cDNA reverse-transcribed from cellular mRNA revealed that CHED mRNA is expressed in multiple tissues, including bone marrow. The CHED protein includes the consensus ATP binding and phosphorylation domains characteristic of kinases, displays 34-42% identically aligned amino acid residues with other cdc2-related kinases, and is considerably longer at its amino and carboxyl termini. An antisense oligodeoxynucleotide designed to interrupt CHED's expression (AS-CHED) significantly reduced the ratio between CHED mRNA and actin mRNA within 1 hr of its addition to cultures, a reduction that persisted for 4 days. AS-CHED treatment selectively inhibited megakaryocyte development in murine bone marrow cultures but did not prevent other hematopoietic pathways, as evidenced by increasing numbers of mononuclear cells. An oligodeoxynucleotide blocking production of the acetylcholine-hydrolyzing enzyme, butyrylcholinesterase, displayed a similar inhibition of megakaryocytopoiesis. In contrast, an oligodeoxynucleotide blocking production of the human 2Hs cdc2 homolog interfered with production of the human 2Hs cdc2 homolog interfered with cellular proliferation without altering the cell-type composition of these cultures. Therefore, these findings strengthen the link between cholinergic signaling and cell division control in hematopoiesis and implicate both CHED and

  6. Human T-cell leukemia virus type 1 tax oncoprotein suppression of multilineage hematopoiesis of CD34+ cells in vitro.

    PubMed

    Tripp, Adam; Liu, Yingxian; Sieburg, Michelle; Montalbano, Joanne; Wrzesinski, Stephen; Feuer, Gerold

    2003-11-01

    Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 are highly related viruses that differ in disease manifestation. HTLV-1 is the etiologic agent of adult T-cell leukemia and lymphoma, an aggressive clonal malignancy of human CD4-bearing T lymphocytes. Infection with HTLV-2 has not been conclusively linked to lymphoproliferative disorders. We previously showed that human hematopoietic progenitor (CD34(+)) cells can be infected by HTLV-1 and that proviral sequences were maintained after differentiation of infected CD34(+) cells in vitro and in vivo. To investigate the role of the Tax oncoprotein of HTLV on hematopoiesis, bicistronic lentiviral vectors were constructed encoding the HTLV-1 or HTLV-2 tax genes (Tax1 and Tax2, respectively) and the green fluorescent protein marker gene. Human hematopoietic progenitor (CD34(+)) cells were infected with lentivirus vectors, and transduced cells were cultured in a semisolid medium permissive for the development of erythroid, myeloid, and primitive progenitor colonies. Tax1-transduced CD34(+) cells displayed a two- to fivefold reduction in the total number of hematopoietic clonogenic colonies that arose in vitro, in contrast to Tax2-transduced cells, which showed no perturbation of hematopoiesis. The ratio of colony types that developed from Tax1-transduced CD34(+) cells remained unaffected, suggesting that Tax1 inhibited the maturation of relatively early, uncommitted hematopoietic stem cells. Since previous reports have linked Tax1 expression with initiation of apoptosis, lentiviral vector-mediated transduction of Tax1 or Tax2 was investigated in CEM and Jurkat T-cell lines. Ectopic expression of either Tax1 or Tax2 failed to induce apoptosis in T-cell lines. These data demonstrate that Tax1 expression perturbs development and maturation of pluripotent hematopoietic progenitor cells, an activity that is not displayed by Tax2, and that the suppression of hematopoiesis is not attributable to induction of apoptosis. Since

  7. The Dtk receptor tyrosine kinase, which binds protein S, is expressed during hematopoiesis.

    PubMed

    Crosier, P S; Freeman, S A; Orlic, D; Bodine, D M; Crosier, K E

    1996-02-01

    Dtk (Tyro 3/Sky/Rse/Brt/Tif) belongs to a recently recognized subfamily of receptor tyrosine kinases that also includes Ufo (Axl/Ark) and Mer (Eyk). Ligands for Dtk and Ufo have been identified as protein S and the related molecule Gas6, respectively. This study examined expression of Dtk during ontogeny of the hematopoietic system and compared the pattern of expression with that of Ufo. Both receptors were abundantly expressed in differentiating embryonic stem cells, yolk sac blood islands, para-aortic splanchnopleural mesoderm, fractionated AA4+ fetal liver cells, and fetal thymus from day 14 until birth. Although Ufo was expressed at moderate levels in adult bone marrow, expression of Dtk in this tissue was barely detectable. In adult bone marrow subpopulations fractionated using counterflow centrifugal elutriation, immunomagnetic bead selection for lineage-depletion and FACS sorting for c-kit expression, very low levels of Dtk and/or Ufo were detected in some cell fractions. These results suggest that Dtk and Ufo are likely to be involved in the regulation of hematopoiesis, particularly during the embryonic stages of blood cell development.

  8. Murine Typhus

    PubMed Central

    Dzul-Rosado, Karla R; Zavala Velázquez, Jorge Ernesto; Zavala-Castro, Jorge

    2012-01-01

    Rickettsia typhi: is an intracellular bacteria who causes murine typhus. His importance is reflected in the high frequency founding specific antibodies against Rickettsia typhi in several worldwide seroepidemiological studies, the seroprevalence ranging between 3-36%. Natural reservoirs of R. typhi are rats (some species belonging the Rattus Genus) and fleas (Xenopsylla cheopis) are his vector. This infection is associated with overcrowding, pollution and poor hygiene. Typically presents fever, headache, rash on trunk and extremities, in some cases may occur organ-specific complications, affecting liver, kidney, lung or brain. Initially the disease is very similar to other diseases, is very common to confuse the murine typhus with Dengue fever, therefore, ignorance of the disease is a factor related to complications or non-specific treatments for the resolution of this infection. This paper presents the most relevant information to consider about the rickettsiosis caused by Rickettsia typhi. PMID:24893060

  9. Roles of p53 and p27 Kip1 in the regulation of neurogenesis in the murine adult subventricular zone

    PubMed Central

    Gil-Perotin, Sara; Haines, Jeffery D.; Kaur, Jasbir; Marin-Husstege, Mireya; Spinetta, Michael J.; Kim, Kwi-Hye; Duran-Moreno, Maria; Schallert, Timothy; Zindy, Frederique; Roussel, Martine F.; Garcia-Verdugo, Jose M.; Casaccia, Patrizia

    2011-01-01

    The tumor suppressor protein p53 (Trp53) and the cell cycle inhibitor p27 Kip1 (Cdknb1) have both been implicated in regulating proliferation of adult subventricular zone (aSVZ) cells. We previously reported that genetic ablation of Trp53 (Trp53 −/−) or Cdknb1 (p27 Kip1−/−) increased proliferation of cells in the aSVZ, but differentially affected the number of adult born neuroblasts. We therefore hypothesized that these molecules might play non-redundant roles. To test this hypothesis we generated mice lacking both genes (Trp53 −/−;p27 Kip1−/−) and analysed the consequences on aSVZ cells and adult neuroblasts. Proliferation and self-renewal of cultured aSVZ cells were increased in the double mutants compared with control, but the mice did not develop spontaneous brain tumors. In contrast, the number of adult-born neuroblasts in the double mutants was similar to wild-type animals and suggested a complementation of the p27 Kip1−/− phenotype due to loss of Trp53. Cellular differences detected in the aSVZ correlated with cellular changes in the olfactory bulb and behavioral data on novel odor recognition. The exploration time for new odors was reduced in p27 Kip1−/− mice, increased in Trp53 −/− mice and normalized in the double Trp53−/−;p27 Kip1−/− mutants. At the molecular level, Trp53 −/− aSVZ cells were characterized by higher levels of NeuroD and Math3 and by the ability to generate neurons more readily. In contrast, p27 Kip1−/− cells generated fewer neurons, due to enhanced proteasomal degradation of pro-neural transcription factors. Together, these results suggest that p27 Kip1 and p53 function non-redundantly to modulate proliferation and self-renewal of aSVZ cells and antagonistically in regulating adult neurogenesis. PMID:21899604

  10. SRSF2 Is Essential for Hematopoiesis, and Its Myelodysplastic Syndrome-Related Mutations Dysregulate Alternative Pre-mRNA Splicing

    PubMed Central

    Komeno, Yukiko; Huang, Yi-Jou; Qiu, Jinsong; Lin, Leo; Xu, YiJun; Zhou, Yu; Chen, Liang; Monterroza, Dora D.; Li, Hairi; DeKelver, Russell C.; Yan, Ming

    2015-01-01

    Myelodysplastic syndromes (MDS) are a group of neoplasms characterized by ineffective myeloid hematopoiesis and various risks for leukemia. SRSF2, a member of the serine/arginine-rich (SR) family of splicing factors, is one of the mutation targets associated with poor survival in patients suffering from myelodysplastic syndromes. Here we report the biological function of SRSF2 in hematopoiesis by using conditional knockout mouse models. Ablation of SRSF2 in the hematopoietic lineage caused embryonic lethality, and Srsf2-deficient fetal liver cells showed significantly enhanced apoptosis and decreased levels of hematopoietic stem/progenitor cells. Induced ablation of SRSF2 in adult Mx1-Cre Srsf2flox/flox mice upon poly(I):poly(C) injection demonstrated a significant decrease in lineage− Sca+ c-Kit+ cells in bone marrow. To reveal the functional impact of myelodysplastic syndromes-associated mutations in SRSF2, we analyzed splicing responses on the MSD-L cell line and found that the missense mutation of proline 95 to histidine (P95H) and a P95-to-R102 in-frame 8-amino-acid deletion caused significant changes in alternative splicing. The affected genes were enriched in cancer development and apoptosis. These findings suggest that intact SRSF2 is essential for the functional integrity of the hematopoietic system and that its mutations likely contribute to development of myelodysplastic syndromes. PMID:26124281

  11. Maintenance of the functional integrity of mouse hematopoiesis by EED and promotion of leukemogenesis by EED haploinsufficiency

    PubMed Central

    Ikeda, Kenichiro; Ueda, Takeshi; Yamasaki, Norimasa; Nakata, Yuichiro; Sera, Yasuyuki; Nagamachi, Akiko; Miyama, Takahiko; Kobayashi, Hiroshi; Takubo, Keiyo; Kanai, Akinori; Oda, Hideaki; Wolff, Linda; Honda, Zen-ichiro; Ichinohe, Tatsuo; Matsubara, Akio; Suda, Toshio; Inaba, Toshiya; Honda, Hiroaki

    2016-01-01

    Polycomb repressive complex 2 (PRC2) participates in transcriptional repression through methylation of histone H3K27. The WD-repeat protein embryonic ectoderm development (EED) is a non-catalytic but an essential component of PRC2 and its mutations were identified in hematopoietic malignancies. To clarify the role(s) of EED in adult hematopoiesis and leukemogenesis, we generated Eed conditional knockout (EedΔ/Δ) mice. EedΔ/Δ mice died in a short period with rapid decrease of hematopoietic cells. Hematopoietic stem/progenitor cells (HSPCs) were markedly decreased with impaired bone marrow (BM) repopulation ability. Cell cycle analysis of HSPCs demonstrated increased S-phase fraction coupled with suppressed G0/G1 entry. Genes encoding cell adhesion molecules are significantly enriched in EedΔ/Δ HSPCs, and consistently, EedΔ/Δ HSPCs exhibited increased attachment to a major extracellular matrix component, fibronectin. Thus, EED deficiency increases proliferation on one side but promotes quiescence possibly by enhanced adhesion to the hematopoietic niche on the other, and these conflicting events would lead to abnormal differentiation and functional defect of EedΔ/Δ HSPCs. In addition, Eed haploinsufficiency induced hematopoietic dysplasia, and Eed heterozygous mice were susceptible to malignant transformation and developed leukemia in cooperation with Evi1 overexpression. Our results demonstrated differentiation stage-specific and dose-dependent roles of EED in normal hematopoiesis and leukemogenesis. PMID:27432459

  12. The human microbiome in hematopoiesis and hematologic disorders

    PubMed Central

    Manzo, Veronica E.

    2015-01-01

    Humans are now understood to be in complex symbiosis with a diverse ecosystem of microbial organisms, including bacteria, viruses, and fungi. Efforts to characterize the role of these microorganisms, commonly referred as the microbiota, in human health have sought to answer the fundamental questions of what organisms are present, how are they functioning to interact with human cells, and by what mechanism are these interactions occurring. In this review, we describe recent efforts to describe the microbiota in healthy and diseased individuals, summarize the role of various molecular technologies (ranging from 16S ribosomal RNA to shotgun metagenomic sequencing) in enumerating the community structure of the microbiota, and explore known interactions between the microbiota and humans, with a focus on the microbiota’s role in hematopoiesis and hematologic diseases. PMID:26012569

  13. Long non-coding RNAs in normal and malignant hematopoiesis

    PubMed Central

    Nobili, Lucia; Lionetti, Marta; Neri, Antonino

    2016-01-01

    Long non-coding RNAs (lncRNAs) are defined as ncRNAs of more than 200 nt in length. They are involved in a large spectrum of biological processes, such as maintenance of genome integrity, genomic imprinting, cell differentiation, and development by means of mechanisms that remain to be fully elucidated. Besides their role in normal cellular physiology, accumulating evidence has linked lncRNA expression and functions to cancer development and progression. In this review, we summarize and discuss what is known about their expression and roles in hematopoiesis with a particular focus on their cell-type specificity, functional interactions, and involvement in the pathobiology of hematological malignancies. PMID:27177333

  14. Molecular mechanisms underlying lineage bias in aging hematopoiesis.

    PubMed

    Elias, Harold K; Bryder, David; Park, Christopher Y

    2017-01-01

    Although hematopoietic stem cells (HSCs) have traditionally been thought to possess the ability to give rise to all the mature cell types in the hematopoietic system, this conception of hematopoiesis was based on evaluation of hematopoietic output from large numbers of HSCs using transplantation models.  More recent studies evaluating HSCs at the clonal or near-clonal level, both in transplantation studies and during in situ hematopoiesis, have established that individual HSCs can exhibit lineage bias, giving rise to myeloid-biased, lymphoid-biased, or more balanced differentiation, with the proportion of myeloid-biased HSCs increasing with age.  This age-associated shift in lineage potential is associated with decreased cellular immunity and increased incidence of diseases with prominent inflammatory components including atherosclerosis, autoimmunity, neurodegenerative disease, and carcinogenesis. Understanding the molecular mechanisms that regulate this shift in linage bias therefore represents an important area of investigation in numerous human diseases.  In this review, we summarize our current understanding of the cell-intrinsic (autonomous) and cell-extrinsic factors that regulate HSC lineage fate bias during aging.  In addition, we have attempted to bring attention to important caveats and unanswered questions related to the issue of HSC lineage bias to encourage explorations of these important lines of inquiry. Ultimately, we expect a comprehensive understanding of HSC lineage bias during aging to have important implications for human health, since strategies to alter lineage bias in old HSCs not only has the potential to restore immune function in the elderly, but also to reduce the incidence of inflammation-associated diseases, many for which there is a current unmet need for novel and more effective treatments. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Induction of murine tumors in adult mice by a combination of either avian sarcoma virus or human adenovirus and syngeneic mouse embryo cells.

    PubMed

    Takeuchi, M; Nitta, K

    1983-01-01

    Primary murine Rous sarcoma was produced in adult mice of seven strains, C57BL/6, DBA/2, BALB/c, C3H/He, CBAJ, AKR, and DDD, by s.c. inoculation of a mixture of 5 X 10(6) chicken tumor cells containing Schmidt-Ruppin Rous sarcoma virus and 9- to 12-day-old mouse embryo cells (MEC) (2 X 10(6) ) of the syngeneic strain. The sarcoma developed at the site of injection in almost all mice tested, but there were some differences in the latent period and the survival time among mouse strains. When the number of cells inoculated was reduced to 5 X 10(4) for chicken tumor cells induced by the Schmidt-Ruppin strain of Rous sarcoma virus (SR-CTC) and 2 X 10(4) for MEC, no tumor was produced in C3H/He mice. These tumors had strain specificity and the Schmidt-Ruppin strain of Rous sarcoma virus genome in masked form. The tumor at the site of injection originated in the embryo cells injected along with SR-CTC. This was confirmed by CBAT6/T6 marker chromosome analysis of the tumor cells of CBA mice induced with SR-CTC plus CBAT6/T6 MEC and also confirmed by transplantation of a C57BL/6 X C3H/He F1 tumor which had been induced with SR-CTC plus C3H/He or C57BL/6 MEC. Tumor induction in adult mouse by a mixture of virus and syngeneic 9- to 14-day-old embryo cells was tested for human adenovirus serotype 12 (Ad12) and simian virus 40. Primary Ad12 tumor was also induced in adult CBA, C3H/He, and DDD mice by 4 X 10(5 to 6) 50% tissue culture infective dose of Ad12 with 5 X 10(6) syngeneic embryo cells. This tumor contained Ad12 T-antigen-positive particles in cells. But in the case of simian virus 40, the tumor did not appear for about 300 days of observation.

  16. Practical Murine Hematopathology: A Comparative Review and Implications for Research

    PubMed Central

    O'Connell, Karyn E; Mikkola, Amy M; Stepanek, Aaron M; Vernet, Andyna; Hall, Christopher D; Sun, Chia C; Yildirim, Eda; Staropoli, John F; Lee, Jeannie T; Brown, Diane E

    2015-01-01

    Hematologic parameters are important markers of disease in human and veterinary medicine. Biomedical research has benefited from mouse models that recapitulate such disease, thus expanding knowledge of pathogenetic mechanisms and investigative therapies that translate across species. Mice in health have many notable hematologic differences from humans and other veterinary species, including smaller erythrocytes, higher percentage of circulating reticulocytes or polychromasia, lower peripheral blood neutrophil and higher peripheral blood and bone marrow lymphocyte percentages, variable leukocyte morphologies, physiologic splenic hematopoiesis and iron storage, and more numerous and shorter-lived erythrocytes and platelets. For accurate and complete hematologic analyses of disease and response to investigative therapeutic interventions, these differences and the unique features of murine hematopathology must be understood. Here we review murine hematology and hematopathology for practical application to translational investigation. PMID:25926395

  17. A comparison of murine T-cell-depleted adult bone marrow and full-term fetal blood cells in hematopoietic engraftment and immune reconstitution.

    PubMed

    Chen, Benny J; Cui, Xiuyu; Sempowski, Gregory D; Gooding, Maria E; Liu, Congxiao; Haynes, Barton F; Chao, Nelson J

    2002-01-01

    Umbilical cord blood has been increasingly used as a source of hematopoietic stem cells. A major area of concern for the use of cord blood transplantation is the delay in myeloid and lymphoid recovery. To directly compare myeloid and lymphoid recovery using an animal model of bone marrow and cord blood as sources of stem cells, hematopoietic engraftment and immune recovery were studied following infusion of T-cell-depleted adult bone marrow or full-term fetal blood cells, as a model of cord blood in a murine allogeneic transplantation model (C57BL/6 [H-2(b)] --> BALB/c [H-2(d)]). Allogeneic full-term fetal blood has poorer radioprotective capacity but greater long-term engraftment potential on a cell-to-cell basis compared with T-cell-depleted bone marrow. Allogeneic full-term fetal blood recipients had decreased absolute numbers of T, B, and dendritic cells compared with bone marrow recipients. Splenic T cells in allogeneic full-term fetal blood recipients proliferated poorly, were unable to generate cytotoxic effectors against third-party alloantigens in vitro, and failed to generate alloantigen-specific cytotoxic antibodies in vivo. In addition, reconstituting T cells in fetal blood recipients had decreased mouse T-cell receptor delta single-joint excision circles compared with bone marrow recipients. At a per-cell level, B cells from fetal blood recipients did not proliferate as well as those found in bone marrow recipients. These results suggest that full-term fetal blood can engraft allogeneic hosts across the major histocompatibility barrier with slower hematopoietic engraftment and impaired immune reconstitution.

  18. Impact of oxygen concentration on adult murine pre-antral follicle development in vitro and the corresponding metabolic profile.

    PubMed

    Gook, Debra A; Edgar, D H; Lewis, K; Sheedy, J R; Gardner, D K

    2014-01-01

    Oxygen concentration during in vitro culture has a significant effect on the physiology of embryos, altering metabolic profile and developmental outcome. Although atmospheric oxygen has been used routinely for the culture of ovarian follicles, oxygen concentration may also be critical for follicle growth but the optimal concentration has not been determined. In this study, mechanically isolated primary and secondary follicles (80-140 µm diameter) from adult mouse ovaries were cultured in serum-free conditions for 8 days in either 5 or 20% oxygen to determine growth (follicular diameter), morphology and viability. For each oxygen concentration, half of the medium was replaced on Days 2, 4 and 6 or on Day 4 only. In the latter group, metabolic analysis of spent follicular culture media was performed by (1)H-NMR. The proportion of viable, growing follicles was significantly (P < 0.0001) higher in 5% than in 20% oxygen (59% versus 8%). Reducing the frequency of medium replacement during culture in 5% oxygen resulted in significantly (P < 0.001) more viable follicles (79 versus 46%). In 20% oxygen, poor follicular viability was observed irrespective of the frequency of medium replacement (8 and 10% respectively). Metabolic profiles showed marked differences in amino acid and carbohydrate utilization with respect to both oxygen concentration and between Days 4 and 8 of development. Metabolites which significantly discriminated between oxygen concentration at both time points were glucose consumption, lactate utilization, alanine, alanyl-glutamine, leucine and proline. In conclusion, the poor in vitro follicular development previously observed in minimal culture conditions may reflect the use of 20% oxygen. Frequent medium replenishment is not necessary and does not overcome the detrimental effect of high oxygen on follicle viability. Further optimization of culture conditions would benefit from metabolic analyses and the use of 5% oxygen should be tested further for

  19. Extensive double humanization of both liver and hematopoiesis in FRGN mice.

    PubMed

    Wilson, Elizabeth M; Bial, J; Tarlow, Branden; Bial, G; Jensen, B; Greiner, D L; Brehm, M A; Grompe, M

    2014-11-01

    Preclinical research in animals often fails to adequately predict the outcomes observed in human patients. Chimeric animals bearing individual human tissues have been developed to provide improved models of human-specific cellular processes. Mice transplanted with human hematopoietic stem cells can be used to study human immune responses, infections of blood cells and processes of hematopoiesis. Animals with humanized livers are useful for modeling hepatotropic infections as well as drug metabolism and hepatotoxicity. However, many pathophysiologic processes involve both the liver and the hematolymphoid system. Examples include hepatitis C/HIV co-infection, immune mediated liver diseases, liver injuries with inflammation such as steatohepatitis and alcoholic liver disease. We developed a robust protocol enabling the concurrent double-humanization of mice with mature hepatocytes and human blood. Immune-deficient, fumarylacetoacetate hydrolase (Fah(-/-)), Rag2(-/-) and Il2rg(-/-) deficient animals on the NOD-strain background (FRGN) were simultaneously co-transplanted with adult human hepatocytes and hematopoietic stem cells after busulfan and Ad:uPA pre-conditioning. Four months after transplantation the average human liver repopulation exceeded 80% and hematopoietic chimerism also was high (40-80% in bone marrow). Importantly, human macrophages (Kupffer cells) were present in the chimeric livers. Double-chimeric FRGN mice will serve as a new model for disease processes that involve interactions between hepatocytes and hematolymphoid cells.

  20. [The investigation of hematopoietic capacity of HPP-CFC derived from murine embryonic stem cells in vitro and in vivo].

    PubMed

    Liu, Bing; Hou, Chun-Mei; Wu, Ying; Zhang, Shuang-Xi; Mao, Ning

    2003-05-01

    The hematopoietic system of the mouse arises from extraembryonic mesoderm that migrate through primitive streak to the presumptive yolk sac at day 7.0 of gestation. However, the mechanisms regulating mesoderm commitment to hematopoietic lineages remain poorly understood. Previous studies demonstrated that the development kinetics and growth factor responsiveness of hematopoietic precursors derived from embryonic stem cells (ES cells) is similar to that found in the yolk sac, indicating that the onset of hematopoiesis within the embryoid bodies (EBs) parallels that found in the embryo. Furthermore, in vitro differentiation of ES cells to hematopoietic cells is valuable for establishment of therapeutic clone against a variety of hematological disorders. Despite the identification of multipotential hematopoietic progenitors in EBs, a subset of more primitive progenitors, identical to the high proliferative potential colony-forming cells (HPP-CFC) derived from human and murine hematopoietic tissues, have not been clearly identified regarding particular their replating potential in vitro. HPP-CFC is among the most primitive hematopoietic multipotent precursors cultured in vitro. In this study, our aim was to investigate the in vitro and in vivo hematopoietic capacity of HPP-CFC within the day 12 EBs, rather than the expansion of more committed progenitors. In this study the HPP-CFC could be detected within EBs differentiated for 5 to 14 days of murine ES cells, but the development dynamics of the HPP-CFC differed greatly among distinct serum lots. Qualitatively HPP-CFC is capable of forming secondary colonies. As to our expectation the ES cells-derived HPP-CFC demonstrated similar regeneration capacity to those from yolk sac, giving rise to secondary granulocyte, erythrocyte, macrophage and mast cells, however largely differed from the counterparts of adult bone marrow. In addition, by RT-PCR ES cells-derived HPP-CFC were found to express transcription factors

  1. Database setup for preclinical studies of gene-modified hematopoiesis.

    PubMed

    Balcik, Brenden; Grassman, Elke; Reeves, Lilith

    2009-01-01

    Murine safety studies are routinely used for gathering preclinical safety and efficacy data and, for Phase I studies, Good Laboratory Practice (GLP) compliance is not mandated. However, extensive amounts of data must be gathered and analyzed. An inter-relational database is an effective tool for storing, sorting, and reviewing data.

  2. ROS-mediated iron overload injures the hematopoiesis of bone marrow by damaging hematopoietic stem/progenitor cells in mice

    PubMed Central

    Chai, Xiao; Li, Deguan; Cao, Xiaoli; Zhang, Yuchen; Mu, Juan; Lu, Wenyi; Xiao, Xia; Li, Chengcheng; Meng, Juanxia; Chen, Jie; Li, Qing; Wang, Jishi; Meng, Aimin; Zhao, Mingfeng

    2015-01-01

    Iron overload, caused by hereditary hemochromatosis or repeated blood transfusions in some diseases, such as beta thalassemia, bone marrow failure and myelodysplastic syndrome, can significantly induce injured bone marrow (BM) function as well as parenchyma organ dysfunctions. However, the effect of iron overload and its mechanism remain elusive. In this study, we investigated the effects of iron overload on the hematopoietic stem and progenitor cells (HSPCs) from a mouse model. Our results showed that iron overload markedly decreased the ratio and clonogenic function of murine HSPCs by the elevation of reactive oxygen species (ROS). This finding is supported by the results of NAC or DFX treatment, which reduced ROS level by inhibiting NOX4 and p38MAPK and improved the long-term and multi-lineage engrafment of iron overload HSCs after transplantation. Therefore, all of these data demonstrate that iron overload injures the hematopoiesis of BM by enhancing ROS through NOX4 and p38MAPK. This will be helpful for the treatment of iron overload in patients with hematopoietic dysfunction. PMID:25970748

  3. Gene expression profiling to define the cell intrinsic role of the SKI proto-oncogene in hematopoiesis and myeloid neoplasms.

    PubMed

    Chalk, Alistair M; Liddicoat, Brian J J; Walkley, Carl R; Singbrant, Sofie

    2014-12-01

    The proto-oncogene SKI is highly expressed in human myeloid leukemia and also in murine hematopoietic stem cells. However, its operative relevance in these cells remains elusive. We have over-expressed SKI to define its intrinsic role in hematopoiesis and myeloid neoplasms, which resulted in a robust competitive advantage upon transplantation, a complete dominance of the stem and progenitor compartments, and a marked enhancement of myeloid differentiation at the expense of other lineages. Accordingly, enforced expression of SKI induced gene signatures associated with hematopoietic stem cells and myeloid differentiation. Here we provide detailed experimental methods and analysis for the gene expression profiling described in our recently published study of Singbrant et al. (2014) in Haematologica. Our data sets (available at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE39457) provide a resource for exploring the underlying molecular mechanisms of the involvement of the proto-oncogene SKI in hematopoietic stem cell function and development of myeloid neoplasms.

  4. Retroviral vector integration in post-transplant hematopoiesis in mice conditioned with either submyeloablative or ablative irradiation.

    PubMed

    Sadat, M A; Dirscherl, S; Sastry, L; Dantzer, J; Pech, N; Griffin, S; Hawkins, T; Zhao, Y; Barese, C N; Cross, S; Orazi, A; An, C; Goebel, W S; Yoder, M C; Li, X; Grez, M; Cornetta, K; Mooney, S D; Dinauer, M C

    2009-12-01

    X-linked chronic granulomatous disease (X-CGD) is an inherited immunodeficiency with absent phagocyte NADPH-oxidase activity caused by defects in the gene-encoding gp91(phox). Here, we evaluated strategies for less intensive conditioning for gene therapy of genetic blood disorders without selective advantage for gene correction, such as might be used in a human X-CGD protocol. We compared submyeloablative with ablative irradiation as conditioning in murine X-CGD, examining engraftment, oxidase activity and vector integration in mice transplanted with marrow transduced with a gamma-retroviral vector for gp91(phox) expression. The frequency of oxidase-positive neutrophils in the donor population was unexpectedly higher in many 300 cGy-conditioned mice compared with lethally irradiated recipients, as was the fraction of vector-marked donor secondary CFU-S12. Vector integration sites in marrow, spleen and secondary CFU-S12 DNA from primary recipients were enriched for cancer-associated genes, including Evi1, and integrations in or near cancer-associated genes were more frequent in marrow and secondary CFU-S12 from 300 cGy-conditioned mice compared with fully ablated mice. These findings support the concept that vector integration can confer a selection bias, and suggest that the intensity of the conditioning regimen may further influence the effects of vector integration on clonal selection in post-transplant engraftment and hematopoiesis.

  5. A multiscale model of the bone marrow and hematopoiesis

    PubMed Central

    Silva, Ariosto S; Anderson, Alexander R.A.

    2013-01-01

    The bone marrow is necessary for renewal of all hematopoietic cells and critical for maintenance of a wide range of physiologic functions. Multiple human diseases result from bone marrow dysfunction. It is also the site in which “liquid” tumors, including leukemia and multiple myeloma, develop as well as a frequent site of metastases. Understanding the complex cellular and microenvironmental interactions that govern normal bone marrow function as well as diseases and cancers of the bone marrow would be a valuable medical advance. Our goal is the development of a spatially-explicit in silico model of the bone marrow to understand both its normal function and the evolutionary dynamics that govern the emergence of bone marrow malignancy. Here we introduce a multiscale computational model of the bone marrow that incorporates three distinct spatial scales, cell, hematopoietic subunit, whole marrow. Implemented as a fixed lattice 3D cellular automaton, it reproduces the spatial characteristics of the normal bone marrow and is validated against data from the daily production of mature blood cells and response of hematopoiesis after irradiation. The major mechanisms modeled in this work are: (1) replication, specialization and migration of hematopoietic cells, (2) optimized spatial configuration of sinuses and hematopoietic compartments and, (3) intravasation of mature hematopoietic cells into sinuses. Our results, using parameter estimates from literature, recapitulates normal bone marrow function and suggest an explanation for the fractal-like structure of trabeculae and sinuses in the marrow, which would be an optimization of the hematopoietic function in order to maximize the number of mature blood cells produced daily within the volumetric restrictions of the marrow. PMID:21631151

  6. Dietary Supplementation with Probiotics Improves Hematopoiesis in Malnourished Mice

    PubMed Central

    Salva, Susana; Merino, María Cecilia; Agüero, Graciela; Gruppi, Adriana; Alvarez, Susana

    2012-01-01

    Background Lactobacillus rhamnosus CRL1505 (Lr) administered during the repletion of immunocompromised-malnourished mice improves the resistance against intestinal and respiratory infections. This effect is associated with an increase in the number and functionality of immune cells, indicating that Lr could have some influence on myeloid and lymphoid cell production and maturation. Objective This study analyzed the extent of the damage caused by malnutrition on myeloid and lymphoid cell development in the spleen and bone marrow (BM). We also evaluated the impact of immunobiotics on the recovery of hematopoiesis affected in malnourished mice. Methods Protein malnourished mice were fed on a balanced conventional diet for 7 or 14 consecutive d with or without supplemental Lr or fermented goat's milk (FGM). Malnourished mice and well-nourished mice were used as controls. Histological and flow cytometry studies were carried out in BM and spleen to study myeloid and lymphoid cells. Results Malnutrition induced quantitative alterations in spleen B and T cells; however, no alteration was observed in the ability of splenic B cells to produce immunoglobulins after challenge with LPS or CpG. The analysis of BM B cell subsets based on B220, CD24, IgM and IgD expression showed that malnutrition affected B cell development. In addition, BM myeloid cells decreased in malnourished mice. On the contrary, protein deprivation increased BM T cell number. These alterations were reverted with Lr or FGM repletion treatments since normal numbers of BM myeloid, T and B cells were observed in these groups. Conclusions Protein malnutrition significantly alters B cell development in BM. The treatment of malnourished mice with L. rhamnosus CRL1505 was able to induce a recovery of B cells that would explain its ability to increase immunity against infections. This work highlights the possibility of using immunobiotics to accelerate the recovery of lymphopoyesis in immunocompromised

  7. Drosophila hematopoiesis under normal conditions and in response to immune stress.

    PubMed

    Letourneau, Manon; Lapraz, Francois; Sharma, Anurag; Vanzo, Nathalie; Waltzer, Lucas; Crozatier, Michèle

    2016-11-01

    The emergence of hematopoietic progenitors and their differentiation into various highly specialized blood cell types constitute a finely tuned process. Unveiling the genetic cascades that control blood cell progenitor fate and understanding how they are modulated in response to environmental changes are two major challenges in the field of hematopoiesis. In the last 20 years, many studies have established important functional analogies between blood cell development in vertebrates and in the fruit fly, Drosophila melanogaster. Thereby, Drosophila has emerged as a powerful genetic model for studying mechanisms that control hematopoiesis during normal development or in pathological situations. Moreover, recent advances in Drosophila have highlighted how intricate cell communication networks and microenvironmental cues regulate blood cell homeostasis. They have also revealed the striking plasticity of Drosophila mature blood cells and the presence of different sites of hematopoiesis in the larva. This review provides an overview of Drosophila hematopoiesis during development and summarizes our current knowledge on the molecular processes controlling larval hematopoiesis, both under normal conditions and in response to an immune challenge, such as wasp parasitism. © 2016 Federation of European Biochemical Societies.

  8. Inflamm-Aging of Hematopoiesis, Hematopoietic Stem Cells, and the Bone Marrow Microenvironment

    PubMed Central

    Kovtonyuk, Larisa V.; Fritsch, Kristin; Feng, Xiaomin; Manz, Markus G.; Takizawa, Hitoshi

    2016-01-01

    All hematopoietic and immune cells are continuously generated by hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) through highly organized process of stepwise lineage commitment. In the steady state, HSCs are mostly quiescent, while HPCs are actively proliferating and contributing to daily hematopoiesis. In response to hematopoietic challenges, e.g., life-threatening blood loss, infection, and inflammation, HSCs can be activated to proliferate and engage in blood formation. The HSC activation induced by hematopoietic demand is mediated by direct or indirect sensing mechanisms involving pattern recognition receptors or cytokine/chemokine receptors. In contrast to the hematopoietic challenges with obvious clinical symptoms, how the aging process, which involves low-grade chronic inflammation, impacts hematopoiesis remains undefined. Herein, we summarize recent findings pertaining to functional alternations of hematopoiesis, HSCs, and the bone marrow (BM) microenvironment during the processes of aging and inflammation and highlight some common cellular and molecular changes during the processes that influence hematopoiesis and its cells of origin, HSCs and HPCs, as well as the BM microenvironment. We also discuss how age-dependent alterations of the immune system lead to subclinical inflammatory states and how inflammatory signaling might be involved in hematopoietic aging. Our aim is to present evidence supporting the concept of “Inflamm-Aging,” or inflammation-associated aging of hematopoiesis. PMID:27895645

  9. Emergence of Clonal Hematopoiesis in the Majority of Patients with Acquired Aplastic Anemia

    PubMed Central

    Babushok, Daria V.; Perdigones, Nieves; Perin, Juan C.; Olson, Timothy S.; Ye, Wenda; Roth, Jacquelyn J.; Lind, Curt; Cattier, Carine; Li, Yimei; Hartung, Helge; Paessler, Michele E.; Frank, Dale M.; Xie, Hongbo M.; Cross, Shanna; Cockroft, Joshua D.; Podsakoff, Gregory M.; Monos, Dimitrios; Biegel, Jaclyn A.; Mason, Philip J.; Bessler, Monica

    2015-01-01

    Acquired aplastic anemia (aAA) is a non-malignant disease caused by autoimmune destruction of early hematopoietic cells. Clonal hematopoiesis is a late complication, seen in 20–25% of older patients. We hypothesized that clonal hematopoiesis in aAA is a more general phenomenon, which can arise early in disease even in younger patients. To evaluate clonal hematopoiesis in aAA, we used comparative whole exome sequencing of paired bone marrow and skin in 22 patients. We found somatic mutations in sixteen patients (72.7%) with a median disease duration of 1 year; twelve (66.7%) were patients with pediatriconset aAA. Fifty-eight mutations in 51 unique genes were primarily in pathways of immunity and transcriptional regulation. Most frequently mutated was PIGA, with 7 mutations. Only two mutations were in genes recurrently-mutated in MDS. Two patients had oligoclonal loss of HLA alleles, linking immune escape to clone emergence. Two patients had activating mutations in key signaling pathways (STAT5B(p.N642H), CAMK2G(p.T306M)). Our results suggest that clonal hematopoiesis in aAA is common, with two mechanisms emerging― immune escape and increased proliferation. Our findings expand conceptual understanding of this non-neoplastic blood disorder. Future prospective studies of clonal hematopoiesis in aAA will be critical for understanding outcomes, and for designing personalized treatment strategies. PMID:25800665

  10. Fight or flight: regulation of emergency hematopoiesis by pyroptosis and necroptosis.

    PubMed

    Croker, Ben A; Silke, John; Gerlic, Motti

    2015-07-01

    A feature of the innate immune response that is conserved across kingdoms is the induction of cell death. In this review, we discuss the direct and indirect effects of increased inflammatory cell death, including pyroptosis - a caspase-1-dependent cell death - and necroptosis - a receptor-interacting protein kinase 3/mixed lineage kinase domain-like protein-dependent, caspase-independent cell death - on emergency hematopoiesis. Activation of nonapoptotic cell death pathways during infection can trigger release of cytokines and/or damage-associated molecular patterns such as interleukin (IL)-1α, IL-1β, IL-18, IL-33, high-mobility group protein B1, and mitochondrial DNA to promote emergency hematopoiesis. During systemic infection, pyroptosis and necroptosis can directly kill hematopoietic stem and progenitor cells, which results in impaired hematopoiesis, cytopenia, and immunosuppression. Although originally described as discrete entities, there now appear to be more intimate connections between the nonapoptotic and death receptor signaling pathways. The choice to undergo pyroptotic and necroptotic cell death constitutes a rapid response system serving to eliminate infected cells, including hematopoietic stem and progenitor cells. This system has the potential to be detrimental to emergency hematopoiesis during severe infection. We discuss the potential of pharmacological intervention for the pyroptosis and necroptosis pathways that may be beneficial during periods of infection and emergency hematopoiesis.

  11. Aminolevulinate synthase 2 mediates erythrocyte differentiation by regulating larval globin expression during Xenopus primary hematopoiesis.

    PubMed

    Ogawa-Otomo, Asako; Kurisaki, Akira; Ito, Yuzuru

    2015-01-02

    Hemoglobin synthesis by erythrocytes continues throughout a vertebrate's lifetime. The mechanism of mammalian heme synthesis has been studied for many years; aminolevulinate synthase 2 (ALAS2), a heme synthetase, is associated with X-linked dominant protoporphyria in humans. Amphibian and mammalian blood cells differ, but little is known about amphibian embryonic hemoglobin synthesis. We investigated the function of the Xenopus alas2 gene (Xalas2) in primitive amphibian erythrocytes and found that it is first expressed in primitive erythroid cells before hemoglobin alpha 3 subunit (hba3) during primary hematopoiesis and in the posterior ventral blood islands at the tailbud stage. Xalas2 is not expressed during secondary hematopoiesis in the dorsal lateral plate. Hemoglobin was barely detectable by o-dianisidine staining and hba3 transcript levels decreased in Xalas2-knockdown embryos. These results suggest that Xalas2 might be able to synthesize hemoglobin during hematopoiesis and mediate erythrocyte differentiation by regulating hba3 expression in Xenopus laevis.

  12. Spliceosomal gene mutations in myelodysplasia: molecular links to clonal abnormalities of hematopoiesis

    PubMed Central

    Inoue, Daichi; Bradley, Robert K.; Abdel-Wahab, Omar

    2016-01-01

    Genomic analyses of the myeloid malignancies and clonal disorders of hematopoiesis that may give rise to these disorders have identified that mutations in genes encoding core spliceosomal proteins and accessory regulatory splicing factors are among the most common targets of somatic mutations. These spliceosomal mutations often occur in a mutually exclusive manner with one another and, in aggregate, account for the most frequent class of mutations in patients with myelodysplastic syndromes (MDSs) in particular. Although substantial progress has been made in understanding the effects of several of these mutations on splicing and splice site recognition, functional connections linking the mechanistic changes in splicing induced by these mutations to the phenotypic consequences of clonal and aberrant hematopoiesis are not yet well defined. This review describes our current understanding of the mechanistic and biological effects of spliceosomal gene mutations in MDSs as well as the regulation of splicing throughout normal hematopoiesis. PMID:27151974

  13. Clonal Hematopoiesis Associated With Adverse Outcomes After Autologous Stem-Cell Transplantation for Lymphoma.

    PubMed

    Gibson, Christopher J; Lindsley, R Coleman; Tchekmedyian, Vatche; Mar, Brenton G; Shi, Jiantao; Jaiswal, Siddhartha; Bosworth, Alysia; Francisco, Liton; He, Jianbo; Bansal, Anita; Morgan, Elizabeth A; Lacasce, Ann S; Freedman, Arnold S; Fisher, David C; Jacobsen, Eric; Armand, Philippe; Alyea, Edwin P; Koreth, John; Ho, Vincent; Soiffer, Robert J; Antin, Joseph H; Ritz, Jerome; Nikiforow, Sarah; Forman, Stephen J; Michor, Franziska; Neuberg, Donna; Bhatia, Ravi; Bhatia, Smita; Ebert, Benjamin L

    2017-01-09

    Purpose Clonal hematopoiesis of indeterminate potential (CHIP) is an age-related condition characterized by somatic mutations in the blood of otherwise healthy adults. We hypothesized that in patients undergoing autologous stem-cell transplantation (ASCT) for lymphoma, CHIP at the time of ASCT would be associated with an increased risk of myelodysplastic syndrome and acute myeloid leukemia, collectively termed therapy-related myeloid neoplasm (TMN), and other adverse outcomes. Methods We performed whole-exome sequencing on pre- and post-ASCT samples from 12 patients who developed TMN after autologous transplantation for Hodgkin lymphoma or non-Hodgkin lymphoma and targeted sequencing on cryopreserved aliquots of autologous stem-cell products from 401 patients who underwent ASCT for non-Hodgkin lymphoma between 2003 and 2010. We assessed the effect of CHIP at the time of ASCT on subsequent outcomes, including TMN, cause-specific mortality, and overall survival. Results For six of 12 patients in the exome sequencing cohort, mutations found in the TMN specimen were also detectable in the pre-ASCT specimen. In the targeted sequencing cohort, 120 patients (29.9%) had CHIP at the time of ASCT, which was associated with an increased rate of TMN (10-year cumulative incidence, 14.1% v 4.3% for those with and without CHIP, respectively; P = .002). Patients with CHIP had significantly inferior overall survival compared with those without CHIP (10-year overall survival, 30.4% v 60.9%, respectively; P < .001), including increased risk of death from TMN and cardiovascular disease. Conclusion In patients undergoing ASCT for lymphoma, CHIP at the time of transplantation is associated with inferior survival and increased risk of TMN.

  14. Spinal osteoblastic meningioma with hematopoiesis: radiologic-pathologic correlation and review of the literature.

    PubMed

    Cochran, Elizabeth J; Schlauderaff, Abraham; Rand, Scott D; Eckardt, Gerald W; Kurpad, Shekar

    2016-10-01

    Spinal meningiomas associated with bone formation and hematopoiesis are rare tumors with only 3 prior case reports in the literature. We describe a case report of a woman who presented with back pain and an isolated event of urinary incontinence. A calcified spinal canal mass at T8 was identified on computed tomographic and magnetic resonance imaging. A gross total resection of the tumor was performed and pathologic examination showed a meningioma, World Health Organization grade 1, containing bone and bone marrow elements. A review of previously reported cases and a discussion of possible mechanisms of bone and hematopoiesis development in meningioma are presented. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Engineering vascularized bone: osteogenic and proangiogenic potential of murine periosteal cells.

    PubMed

    van Gastel, Nick; Torrekens, Sophie; Roberts, Scott J; Moermans, Karen; Schrooten, Jan; Carmeliet, Peter; Luttun, Aernout; Luyten, Frank P; Carmeliet, Geert

    2012-11-01

    One of the key challenges in bone tissue engineering is the timely formation of blood vessels that promote the survival of the implanted cells in the construct. Fracture healing largely depends on the presence of an intact periosteum but it is still unknown whether periosteum-derived cells (PDC) are critical for bone repair only by promoting bone formation or also by inducing neovascularization. We first established a protocol to specifically isolate murine PDC (mPDC) from long bones of adult mice. Mesenchymal stem cells were abundantly present in this cell population as more than 50% of the mPDC expressed mesenchymal markers (CD73, CD90, CD105, and stem cell antigen-1) and the cells exhibited trilineage differentiation potential (chondrogenic, osteogenic, and adipogenic). When transplanted on a collagen-calcium phosphate scaffold in vivo, mPDC attracted numerous blood vessels and formed mature bone which comprises a hematopoiesis-supportive stroma. We explored the proangiogenic properties of mPDC using in vitro culture systems and showed that mPDC promote the survival and proliferation of endothelial cells through the production of vascular endothelial growth factor. Coimplantation with endothelial cells demonstrated that mPDC can enhance vasculogenesis by adapting a pericyte-like phenotype, in addition to their ability to stimulate blood vessel ingrowth from the host. In conclusion, these findings demonstrate that periosteal cells contribute to fracture repair, not only through their strong osteogenic potential but also through their proangiogenic features and thus provide an ideal cell source for bone regeneration therapies. Copyright © 2012 AlphaMed Press.

  16. Inhibitory effects of homeodomain-interacting protein kinase 2 on the aorta-gonad-mapharsen hematopoiesis

    SciTech Connect

    Ohtsu, Naoki; Nobuhisa, Ikuo; Mochita, Miyuki; Taga, Tetsuya . E-mail: taga@kaiju.medic.kumamoto-u.ac.jp

    2007-01-01

    Definitive hematopoiesis starts in the aorta-gonad-mesonephros (AGM) region of the mouse embryo. Our previous studies revealed that STAT3, a gp130 downstream transcription factor, is required for AGM hematopoiesis and that homeodomain-interacting protein kinase 2 (HIPK2) phosphorylates serine-727 of STAT3. HIPK2 is a serine/threonine kinase known to be involved in transcriptional repression and apoptosis. In the present study, we examined the role of HIPK2 in hematopoiesis in mouse embryo. HIPK2 transcripts were found in fetal hematopoietic tissues such as the mouse AGM region and fetal liver. In cultured AGM cells, HIPK2 protein was detected in adherent cells. Functional analyses of HIPK2 were carried out by introducing wild-type and mutant HIPK2 constructs into AGM cultures. Production of CD45{sup +} hematopoietic cells was suppressed by forced expression of HIPK2 in AGM cultures. This suppression required the kinase domain and nuclear localization signals of HIPK2, but the kinase activity was dispensable. HIPK2-overexpressing AGM-derived nonadherent cells did not form cobblestone-like colonies in cultures with stromal cells. Furthermore, overexpression of HIPK2 in AGM cultures impeded the expansion of CD45{sup low}c-Kit{sup +} cells, which exhibit the immature hematopoietic progenitor phenotype. These data indicate that HIPK2 plays a negative regulatory role in AGM hematopoiesis in the mouse embryo.

  17. Sympathetic neural-immune interactions regulate hematopoiesis, thermoregulation and inflammation in mammals.

    PubMed

    Madden, Kelley S

    2017-01-01

    This review will highlight recently discovered mechanisms underlying sympathetic nervous system (SNS) regulation of the immune system in hematopoiesis, thermogenesis, and inflammation. This work in mammals illuminates potential mechanisms by which the nervous and immune systems may interact in invertebrate and early vertebrate species and allow diverse organisms to thrive under varying and extreme conditions and ultimately improve survival.

  18. Versatility of stem and progenitor cells and the instructive actions of cytokines on hematopoiesis.

    PubMed

    Brown, Geoffrey; Mooney, Ciaran James; Alberti-Servera, Llucia; Muenchow, Lilly von; Toellner, Kai-Michael; Ceredig, Rhodri; Rolink, Antonius

    2015-01-01

    For many years, developing hematopoietic cells have been strictly compartmentalized into a rare population of multi-potent self-renewing hematopoietic stem cells (HSC), multi-potent hematopoietic progenitor cells (MPP) that are undergoing commitment to particular lineage fates, and recognizable precursor cells that mature towards functional blood and immune cells. A single route to each end-cell type is prescribed in the "classical" model for the architecture of hematopoiesis. Recent findings have led to the viewpoint that HSCs and MPPs are more versatile than previously thought. Underlying this are multiple routes to a particular fate and cells having clandestine fate options even when they have progressed some way along a pathway. The primary role of cytokines during hematopoiesis has long been seen to be regulation of the survival and proliferation of developing hematopoietic cells. Some cytokines now clearly have instructive actions on cell-fate decisions. All this leads to a new way of viewing hematopoiesis whereby versatile HSC and MPP are directed towards lineage outcomes via cytokine regulated cell-fate decisions. This means greater flexibility to the shaping of hematopoiesis.

  19. Identification of novel mRNA transcripts of the nm23-M1 gene that are modulated during mouse embryo development and are differently expressed in adult murine tissues.

    PubMed

    Gervasi, F; Capozza, F; Bruno, T; Fanciulli, M; Lombardi, D

    1998-12-01

    The nm23-M1, a putative metastasis-suppressor gene, and its homologs are involved in development and differentiation. We have shown previously that in vitro neuronal cell proliferation and differentiation can be modulated by nm23-M1 expression levels. In the present study, by the yeast two-hybrid system, we have shown that, at the onset of mouse tissue differentiation, the Nm23-M1 protein forms either homodimers, or heterodimers with Nm23-M2. Furthermore, we have isolated two cDNA variants of the nm23-M1 gene in the 3'-untranslated region (UTR). The two variants related to novel mRNA transcripts that are modulated in mouse embryo and are differently expressed in adult murine tissues.

  20. A Non-Canonical Function of Zebrafish Telomerase Reverse Transcriptase Is Required for Developmental Hematopoiesis

    PubMed Central

    Koshimizu, Eriko; Hanai, Jun-ichi; Raftopoulou, Christina; Murphey, Ryan D.; Bayliss, Peter E.; Imai, Yoichi; Burns, Caroline Erter; Masutomi, Kenkichi; Gagos, Sarantis; Zon, Leonard I.; Roberts, Thomas M.; Kishi, Shuji

    2008-01-01

    Although it is clear that telomerase expression is crucial for the maintenance of telomere homeostasis, there is increasing evidence that the TERT protein can have physiological roles that are independent of this central function. To further examine the role of telomerase during vertebrate development, the zebrafish telomerase reverse transcriptase (zTERT) was functionally characterized. Upon zTERT knockdown, zebrafish embryos show reduced telomerase activity and are viable, but develop pancytopenia resulting from aberrant hematopoiesis. The blood cell counts in TERT-depleted zebrafish embryos are markedly decreased and hematopoietic cell differentiation is impaired, whereas other somatic lineages remain morphologically unaffected. Although both primitive and definitive hematopoiesis is disrupted by zTERT knockdown, the telomere lengths are not significantly altered throughout early development. Induced p53 deficiency, as well as overexpression of the anti-apoptotic proteins Bcl-2 and E1B-19K, significantly relieves the decreased blood cells numbers caused by zTERT knockdown, but not the impaired blood cell differentiation. Surprisingly, only the reverse transcriptase motifs of zTERT are crucial, but the telomerase RNA-binding domain of zTERT is not required, for rescuing complete hematopoiesis. This is therefore the first demonstration of a non-canonical catalytic activity of TERT, which is different from “authentic” telomerase activity, is required for during vertebrate hematopoiesis. On the other hand, zTERT deficiency induced a defect in hematopoiesis through a potent and specific effect on the gene expression of key regulators in the absence of telomere dysfunction. These results suggest that TERT non-canonically functions in hematopoietic cell differentiation and survival in vertebrates, independently of its role in telomere homeostasis. The data also provide insights into a non-canonical pathway by which TERT functions to modulate specification of

  1. The histone H2A deubiquitinase Usp16 regulates hematopoiesis and hematopoietic stem cell function

    PubMed Central

    Gu, Yue; Jones, Amanda E.; Yang, Wei; Liu, Shanrun; Dai, Qian; Liu, Yudong; Swindle, C. Scott; Zhou, Dewang; Zhang, Zhuo; Ryan, Thomas M.; Townes, Tim M.; Klug, Christopher A.; Chen, Dongquan; Wang, Hengbin

    2016-01-01

    Epigenetic mechanisms play important regulatory roles in hematopoiesis and hematopoietic stem cell (HSC) function. Subunits of polycomb repressive complex 1 (PRC1), the major histone H2A ubiquitin ligase, are critical for both normal and pathological hematopoiesis; however, it is unclear which of the several counteracting H2A deubiquitinases functions along with PRC1 to control H2A ubiquitination (ubH2A) level and regulates hematopoiesis in vivo. Here we investigated the function of Usp16 in mouse hematopoiesis. Conditional deletion of Usp16 in bone marrow resulted in a significant increase of global ubH2A level and lethality. Usp16 deletion did not change HSC number but was associated with a dramatic reduction of mature and progenitor cell populations, revealing a role in governing HSC lineage commitment. ChIP- and RNA-sequencing studies in HSC and progenitor cells revealed that Usp16 bound to many important hematopoietic regulators and that Usp16 deletion altered the expression of genes in transcription/chromosome organization, immune response, hematopoietic/lymphoid organ development, and myeloid/leukocyte differentiation. The altered gene expression was partly rescued by knockdown of PRC1 subunits, suggesting that Usp16 and PRC1 counterbalance each other to regulate cellular ubH2A level and gene expression in the hematopoietic system. We further discovered that knocking down Cdkn1a (p21cip1), a Usp16 target and regulated gene, rescued the altered cell cycle profile and differentiation defect of Usp16-deleted HSCs. Collectively, these studies identified Usp16 as one of the histone H2A deubiquitinases, which coordinates with the H2A ubiquitin ligase PRC1 to regulate hematopoiesis, and revealed cell cycle regulation by Usp16 as key for HSC differentiation. PMID:26699484

  2. Active Hematopoietic Hubs in Drosophila Adults Generate Hemocytes and Contribute to Immune Response

    PubMed Central

    Ghosh, Saikat; Singh, Arashdeep; Mandal, Sudip; Mandal, Lolitika

    2015-01-01

    Summary Blood cell development in Drosophila shares significant similarities with vertebrate. The conservation ranges from biphasic mode of hematopoiesis to signaling molecules crucial for progenitor cell formation, maintenance, and differentiation. Primitive hematopoiesis in Drosophila ensues in embryonic head mesoderm, whereas definitive hematopoiesis happens in larval hematopoietic organ, the lymph gland. This organ, with the onset of pupation, ruptures to release hemocytes into circulation. It is believed that the adult lacks a hematopoietic organ and survives on the contribution of both embryonic and larval hematopoiesis. However, our studies revealed a surge of blood cell development in the dorsal abdominal hemocyte clusters of adult fly. These active hematopoietic hubs are capable of blood cell specification and can respond to bacterial challenges. The presence of progenitors and differentiated hemocytes embedded in a functional network of Laminin A and Pericardin within this hematopoietic hub projects it as a simple version of the vertebrate bone marrow. PMID:25959225

  3. Low-dose busulphan conditioning and neonatal stem cell transplantation preserves vision and restores hematopoiesis in severe murine osteopetrosis.

    PubMed

    Askmyr, Maria; Holmberg, Johan; Flores, Carmen; Ehinger, Mats; Hjalt, Tord; Richter, Johan

    2009-02-01

    Infantile malignant osteopetrosis is a fatal disease caused by lack of functional osteoclasts. In most of patients, TCIRG1, encoding a subunit of a proton pump essential for bone resorption, is mutated. Osteopetrosis leads to bone marrow failure and blindness due to optic nerve compression. Oc/oc mice have a deletion in Tcirg1 and die around 3 to 4 weeks, but can be rescued by neonatal stem cell transplantation (SCT) after irradiation conditioning. However, as irradiation of neonatal mice results in retinal degeneration, we wanted to investigate whether conditioning with busulphan prior to SCT can lead to preservation of vision and reversal of osteopetrosis in the oc/oc mouse model. Pregnant dams were conditioned with busulphan and their litters transplanted with 1 x 10(6) normal lineage-depleted bone marrow cells intravenously or intraperitoneally. Mice were followed in terms of survival and engraftment level, as well as with peripheral blood lineage analysis, bone and eye histopathology and a visual-tracking drum test to assess vision. Busulphan at 15 mg/kg was toxic to oc/oc mice. However, six of seven oc/oc mice conditioned with busulphan 7.5 mg/kg survived past the normal lifespan with 10% engraftment, correction of the skeletal phenotype, and normalization of peripheral blood lineages. Busulphan, in contrast to irradiation, did not have adverse effects on the retina as determined by histopathology, and 8 weeks after transplantation control and oc/oc mice retained their vision. Low-dose busulphan conditioning and neonatal SCT leads to prolonged survival of oc/oc mice, reverses osteopetrosis and prevents blindness even at low engraftment levels.

  4. Reliable Genetic Labeling of Adult-Born Dentate Granule Cells Using Ascl1CreERT2 and GlastCreERT2 Murine Lines

    PubMed Central

    Yang, Sung M.; Alvarez, Diego D.

    2015-01-01

    Newly generated dentate granule cells (GCs) are relevant for input discrimination in the adult hippocampus. Yet, their precise contribution to information processing remains unclear. To address this question, it is essential to develop approaches to precisely label entire cohorts of adult-born GCs. In this work, we used genetically modified mice to allow conditional expression of tdTomato (Tom) in adult-born GCs and characterized their development and functional integration. Ascl1CreERT2;CAGfloxStopTom and GlastCreERT2;CAGfloxStopTom mice resulted in indelible expression of Tom in adult neural stem cells and their lineage upon tamoxifen induction. Whole-cell recordings were performed to measure intrinsic excitability, firing behavior, and afferent excitatory connectivity. Developing GCs were also staged by the expression of early and late neuronal markers. The slow development of adult-born GCs characterized here is consistent with previous reports using retroviral approaches that have revealed that a mature phenotype is typically achieved after 6–8 weeks. Our findings demonstrate that Ascl1CreERT2 and GlastCreERT2 mouse lines enable simple and reliable labeling of adult-born GC lineages within restricted time windows. Therefore, these mice greatly facilitate tagging new neurons and manipulating their activity, required for understanding adult neurogenesis in the context of network remodeling, learning, and behavior. SIGNIFICANCE STATEMENT Our study shows that Ascl1CreERT2 and GlastCreERT2 mice lines can be used to label large cohorts of adult-born dentate granule cells with excellent time resolution. Neurons labeled in this manner display developmental and functional profiles that are in full agreement with previous findings using thymidine analogs and retroviral labeling, thus providing an alternative approach to tackle fundamental questions on circuit remodeling. Because of the massive neuronal targeting and the simplicity of this method, genetic labeling will

  5. Case report and literature review: cardiac tamponade as a complication of pericardial extramedullary hematopoiesis.

    PubMed

    Mahadevan, Navin R; Morgan, Elizabeth A; Mitchell, Richard N

    2016-01-01

    Pericardial effusion can cause cardiac tamponade physiology with resultant cardiogenic shock and death. Myelofibrosis, the replacement of marrow cavity by fibrous connective tissue, is a secondary complication of a group of disorders known as myeloproliferative neoplasms, which are clonal processes characterized by abnormal proliferative growth of one or more hematopoietic lineages. One consequence of myelofibrosis is the development of hematopoiesis at other anatomic sites, most commonly the spleen and liver, a phenomenon known as extramedullary hematopoiesis (EMH). Herein we report a case of a man who died from pericardial tamponade due to a subacute pericardial effusion secondary to EMH in the pericardium in the setting of myelofibrosis. This case highlights an unusual etiology for pericardial effusion and tamponade that should be considered in cases of myelofibrosis and stimulates a discussion regarding the mechanisms and anatomic distribution of EMH.

  6. In vivo Mapping of Notch Pathway Activity in Normal and Stress Hematopoiesis

    PubMed Central

    Gao, Jie; Tikhonova, Anastasia; Loizou, Evangelia; Manet, Jan; van Handel, Ben; Ibrahim, Sherif; Greve, Jeffrey; Mikkola, Hanna; Artavanis-Tsakonas, Spyros; Aifantis, Iannis

    2013-01-01

    SUMMARY Accumulating evidence suggests that Notch signaling is active at multiple points during hematopoiesis. Until recently, the majority of such studies focused on Notch signaling in lymphocyte differentiation and knowledge of individual Notch receptor roles has been limited due to a paucity of genetic tools available. In this manuscript we generate and describe animal models to identify and fate-map stem and progenitor cells expressing each Notch receptor, delineate Notch pathway activation, and perform in vivo gain and loss of function studies dissecting Notch signaling in early hematopoiesis. These models provide comprehensive genetic maps of lineage-specific Notch receptor expression and activation in hematopoietic stem and progenitor cells. Moreover, they establish a previously unknown role for Notch signaling in the commitment of blood progenitors towards the erythrocytic lineage and link Notch signaling to optimal organismal response to stress erythropoiesis. PMID:23791481

  7. In vivo mapping of notch pathway activity in normal and stress hematopoiesis.

    PubMed

    Oh, Philmo; Lobry, Camille; Gao, Jie; Tikhonova, Anastasia; Loizou, Evangelia; Manet, Jan; van Handel, Ben; Ibrahim, Sherif; Greve, Jeffrey; Mikkola, Hanna; Artavanis-Tsakonas, Spyros; Aifantis, Iannis

    2013-08-01

    Accumulating evidence suggests that Notch signaling is active at multiple points during hematopoiesis. Until recently, the majority of such studies focused on Notch signaling in lymphocyte differentiation and knowledge of individual Notch receptor roles has been limited due to a paucity of genetic tools available. In this manuscript we generate and describe animal models to identify and fate-map stem and progenitor cells expressing each Notch receptor, delineate Notch pathway activation, and perform in vivo gain- and loss-of-function studies dissecting Notch signaling in early hematopoiesis. These models provide comprehensive genetic maps of lineage-specific Notch receptor expression and activation in hematopoietic stem and progenitor cells. Moreover, they establish a previously unknown role for Notch signaling in the commitment of blood progenitors toward the erythrocytic lineage and link Notch signaling to optimal organismal response to stress erythropoiesis. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. Clinical management of the homozygous α-thalassemia with unusual mandibular manifestation of hematopoiesis.

    PubMed

    Ruiz-Roca, J A; Oñate-Sánchez, R E; Urrutia-Rodríguez, I; Martínez-Izquierdo, A; Mengual-Pujante, D; Rodríguez-Lozano, F J

    2017-02-01

    Alpha (α)-thalassemias are the most common genetic disorder of hemoglobin (Hb) synthesis, affecting up to 5% of the world's population. These congenital hemolytic anemias induce extramedullary hematopoiesis, including the liver, spleen, sinuses, and the diploic spaces of the skull. Oral health problems in patients with thalassemias are mostly related to a varied degree of facial deformities, malocclusions, and/or dental arch dimensions. We present a case with a 49-year-old man, diagnosed with homozygous α thalassemia that came to the Faculty of Dentistry at the University of Murcia for a dental treatment. It was observed that the patient had an unusual mandibular manifestation of hematopoiesis. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  9. Cocaine exposure impairs multineage hematopoiesis of human hematopoietic progenitor cells mediated by the sigma-1 receptor

    PubMed Central

    Nixon, Christopher C.; Schwartz, Brandon H.; Dixit, Dhaval; Zack, Jerome A.; Vatakis, Dimitrios N.

    2015-01-01

    Prenatal exposure to cocaine is a significant source of fetal and neonatal developmental defects. While cocaine associated neurological and cardiac pathologies are well-documented, it is apparent that cocaine use has far more diverse physiological effects. It is known that in some cell types, the sigma-1 receptor mediates many of cocaine's cellular effects. Here we present a novel and concise investigation into the mechanism that underlies cocaine associated hematopoietic pathology. Indeed, this is the first examination of the effects of cocaine on hematopoiesis. We show that cocaine impairs multilineage hematopoiesis from human progenitors from multiple donors and tissue types. We go on to present the first demonstration of the expression of the sigma-1 receptor in human CD34 + human hematopoietic stem/progenitor cells. Furthermore, we demonstrate that these cocaine-induced hematopoietic defects can be reversed through sigma-1 receptor blockade. PMID:25728014

  10. Sympathoadrenergic modulation of hematopoiesis: a review of available evidence and of therapeutic perspectives

    PubMed Central

    Cosentino, Marco; Marino, Franca; Maestroni, Georges J. M.

    2015-01-01

    Innervation of the bone marrow (BM) has been described more than one century ago, however the first in vivo evidence that sympathoadrenergic fibers have a role in hematopoiesis dates back to less than 25 years ago. Evidence has since increased showing that adrenergic nerves in the BM release noradrenaline and possibly also dopamine, which act on adrenoceptors and dopaminergic receptors (DR) expressed on hematopoietic cells and affect cell survival, proliferation, migration and engraftment ability. Remarkably, dysregulation of adrenergic fibers to the BM is associated with hematopoietic disturbances and myeloproliferative disease. Several adrenergic and dopaminergic agents are already in clinical use for non-hematological indications and with a usually favorable risk-benefit profile, and are therefore potential candidates for non-conventional modulation of hematopoiesis. PMID:26300737

  11. Effect of 2450 MHz microwave radiation on hematopoiesis of preganant mice

    SciTech Connect

    Galvin, M.J.; MacNichols, G.L.; McRee, D.I.

    1984-11-01

    In this study, the influence of 2450 MHz CW microwave radiation on hematopoiesis in pregnant mice was examined. Dams (mice CD-1 strain) were irradiated during Days 1-6 or 6-15 of pregnancy. The animals were irradiated for a total of 8 hr per day at an average power density of 30 mW/cm/sup 2/. Peripheral blood and bone marrow samples were obtained on Day 18 of pregnancy. The total leukocyte and differential leukocyte counts of peripheral blood samples were not affected by either exposure regimen. In addition, no effects were noted in either the erythroid or myeloid mitotic indices of bone marrow samples. Exposure of pregnant mice to microwave radiation under the conditions of these experiments had no effects on the investigated aspects of hematopoiesis.

  12. Myelopathy due to Spinal Extramedullary Hematopoiesis in a Patient with Polycythemia Vera

    PubMed Central

    Ito, Shuhei; Hosogane, Naobumi; Nagoshi, Narihito; Yagi, Mitsuru; Iwanami, Akio; Watanabe, Kota; Tsuji, Takashi; Nakamura, Masaya; Matsumoto, Morio; Ishii, Ken

    2017-01-01

    Extramedullary hematopoiesis (EMH) occasionally occurs in patients exhibiting hematological disorders with decreased hematopoietic efficacy. EMH is rarely observed in the spinal epidural space and patients are usually asymptomatic. In particular, in the patients with polycythemia vera, spinal cord compression due to EMH is extremely rare. We report a case of polycythemia vera, in which operative therapy proved to be an effective treatment for myelopathy caused by spinal EMH. PMID:28133558

  13. Feedback signals in myelodysplastic syndromes: increased self-renewal of the malignant clone suppresses normal hematopoiesis.

    PubMed

    Walenda, Thomas; Stiehl, Thomas; Braun, Hanna; Fröbel, Julia; Ho, Anthony D; Schroeder, Thomas; Goecke, Tamme W; Rath, Björn; Germing, Ulrich; Marciniak-Czochra, Anna; Wagner, Wolfgang

    2014-04-01

    Myelodysplastic syndromes (MDS) are triggered by an aberrant hematopoietic stem cell (HSC). It is, however, unclear how this clone interferes with physiologic blood formation. In this study, we followed the hypothesis that the MDS clone impinges on feedback signals for self-renewal and differentiation and thereby suppresses normal hematopoiesis. Based on the theory that the MDS clone affects feedback signals for self-renewal and differentiation and hence suppresses normal hematopoiesis, we have developed a mathematical model to simulate different modifications in MDS-initiating cells and systemic feedback signals during disease development. These simulations revealed that the disease initiating cells must have higher self-renewal rates than normal HSCs to outcompete normal hematopoiesis. We assumed that self-renewal is the default pathway of stem and progenitor cells which is down-regulated by an increasing number of primitive cells in the bone marrow niche--including the premature MDS cells. Furthermore, the proliferative signal is up-regulated by cytopenia. Overall, our model is compatible with clinically observed MDS development, even though a single mutation scenario is unlikely for real disease progression which is usually associated with complex clonal hierarchy. For experimental validation of systemic feedback signals, we analyzed the impact of MDS patient derived serum on hematopoietic progenitor cells in vitro: in fact, MDS serum slightly increased proliferation, whereas maintenance of primitive phenotype was reduced. However, MDS serum did not significantly affect colony forming unit (CFU) frequencies indicating that regulation of self-renewal may involve local signals from the niche. Taken together, we suggest that initial mutations in MDS particularly favor aberrant high self-renewal rates. Accumulation of primitive MDS cells in the bone marrow then interferes with feedback signals for normal hematopoiesis--which then results in cytopenia.

  14. Absence of yolk sac hematopoiesis from mice with a targeted disruption of the scl gene.

    PubMed Central

    Robb, L; Lyons, I; Li, R; Hartley, L; Köntgen, F; Harvey, R P; Metcalf, D; Begley, C G

    1995-01-01

    The scl gene encodes a basic-helix-loop-helix transcription factor which was identified through its involvement in chromosomal translocations in T-cell leukemia. To elucidate its physiological role, scl was targeted in embryonic stem cells. Mice heterozygous for the scl null mutation were intercrossed and their offspring were genotyped. Homozygous mutant (scl-/-) pups were not detected in newborn litters, and analysis at earlier time points demonstrated that scl-/- embryos were dying around embryonic day 9.5. The scl-/- embryos were pale, edematous, and markedly growth retarded after embryonic day 8.75. Histological studies showed complete absence of recognizable hematopoiesis in the yolk sac of these embryos. Early organogenesis appeared to be otherwise normal. Culture of yolk sac cells of wild-type, heterozygous, and homozygous littermates confirmed the absence of hematopoietic cells in scl-/- yolk sacs. Reverse transcription PCR was used to examine the transcripts of several genes implicated in early hematopoiesis. Transcripts of GATA-1 and PU.1 transcription factors were absent from RNA from scl-/- yolk sacs and embryos. These results implicate scl as a crucial regulator of early hematopoiesis. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7624372

  15. Fish Oil–Rich Diet Promotes Hematopoiesis and Alters Hematopoietic Niche

    PubMed Central

    Li, Xiao-ping; Cheng, Lu; Han, Mu-tian; Zhang, Miao-miao; Shao, Qi-xiang; Xu, Hua-xi

    2015-01-01

    The self-renewal and differentiation of hematopoietic stem cells (HSCs) in bone marrow are essential to replenish all blood cell types, but how this process is influenced by diet remains largely unclear. Here we show that a diet rich in fish oils promotes self-renewal of HSCs and extramedullary hematopoiesis. Chronic intake of a fish oil–rich diet increases the abundance of HSCs, alters the hematopoietic microenvironment, and, intriguingly, induces the expression of matrix metalloproteinase 12 (MMP12) in the bone marrow. Pointing to a direct effect of fish oil on MMP12 expression, omega-3 polyunsaturated fatty acids induce the expression of MMP12 in a dose-dependent manner in bone marrow cells. Importantly, down-regulation of MMP12 activity using an MMP12-specific inhibitor attenuates diet-induced myelopoiesis in both bone marrow and spleen. Thus, a fish oil–rich diet promotes hematopoiesis in the bone marrow and spleen, in part via the activity of MMP12. Taken together, these data provide new insights into diet-mediated regulation of hematopoiesis. PMID:26061726

  16. The Hox cofactors Meis1 and Pbx act upstream of gata1 to regulate primitive hematopoiesis.

    PubMed

    Pillay, Laura M; Forrester, A Michael; Erickson, Timothy; Berman, Jason N; Waskiewicz, Andrew Jan

    2010-04-15

    During vertebrate development, the initial wave of hematopoiesis produces cells that help to shape the developing circulatory system and oxygenate the early embryo. The differentiation of primitive erythroid and myeloid cells occurs within a short transitory period, and is subject to precise molecular regulation by a hierarchical cascade of transcription factors. The TALE-class homeodomain transcription factors Meis and Pbx function to regulate embryonic hematopoiesis, but it is not known where Meis and Pbx proteins participate in the hematopoietic transcription factor cascade. To address these questions, we have ablated Meis1 and Pbx proteins in zebrafish, and characterized their molecular effects on known markers of primitive hematopoiesis. Embryos lacking Meis1 and Pbx exhibit a severe reduction in the expression of gata1, the earliest marker of erythroid cell fate, and fail to produce visible circulating blood cells. Concomitant with a loss of gata1, Meis1- and Pbx-depleted embryos exhibit downregulated embryonic hemoglobin (hbae3) expression, and possess increased numbers of pu.1-positive myeloid cells. gata1-overexpression rescues hbae3 expression in Pbx-depleted; meis1-morphant embryos, placing Pbx and Meis1 upstream of gata1 in the erythropoietic transcription factor hierarchy. Our study conclusively demonstrates that Meis1 and Pbx act to specify the erythropoietic cell lineage and inhibit myelopoiesis. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  17. Impact of fluorescent silicon nanoparticles on circulating hemolymph and hematopoiesis in an invertebrate model organism.

    PubMed

    Xing, Rui; Li, Kai-Le; Zhou, Yan-Feng; Su, Yuan-Yuan; Yan, Si-Qi; Zhang, Kai-Long; Wu, Si-Cong; Sima, Yang-Hu; Zhang, Ke-Qin; He, Yao; Xu, Shi-Qing

    2016-09-01

    Silicon nanoparticles (SiNPs) have attractive potential applications in biological and medical fields, and yet their impact on animals is still controversial, and there have been no reports of their effects on hematopoiesis. In this study, the effects of SiNPs on hemocytes and hematopoiesis were investigated by administering SiNPs via a vascular injection into an invertebrate model, the silkworm. Our results show that the ability of SiNPs to enter different types of circulating hemocytes and their impact on those hemocytes differed significantly. Rapid accumulation of SiNPs was observed in granulocytes, oenocytoids, and spherulocytes, which have immune functions in the circulating hemolymph, whereas SiNPs did not easily enter prohemocytes, which can differentiate into granulocytes, oenocytoids, and spherulocytes and replenish them. The SiNPs that entered the hemocytes initiated autophagy and apoptosis via the lysosomal/mitochondrial pathway. High-dose SiNPs weakly stimulated lysosomal activity in hematopoietic organs, but did not lead to a significant increase in reactive oxygen species or severe autophagy or apoptosis in the organ tissues. We suggest that the damage caused by high-dose SiNPs to hematopoiesis is self-healing, because few SiNPs entered the hematopoietic stem cells in the circulating hemolymph, so the damage to the hematopoietic tissues was limited. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Each cell counts: Hematopoiesis and immunity research in the era of single cell genomics.

    PubMed

    Jaitin, Diego Adhemar; Keren-Shaul, Hadas; Elefant, Naama; Amit, Ido

    2015-02-01

    Hematopoiesis and immunity are mediated through complex interactions between multiple cell types and states. This complexity is currently addressed following a reductionist approach of characterizing cell types by a small number of cell surface molecular features and gross functions. While the introduction of global transcriptional profiling technologies enabled a more comprehensive view, heterogeneity within sampled populations remained unaddressed, obscuring the true picture of hematopoiesis and immune system function. A critical mass of technological advances in molecular biology and genomics has enabled genome-wide measurements of single cells - the fundamental unit of immunity. These new advances are expected to boost detection of less frequent cell types and fuzzy intermediate cell states, greatly expanding the resolution of current available classifications. This new era of single-cell genomics in immunology research holds great promise for further understanding of the mechanisms and circuits regulating hematopoiesis and immunity in both health and disease. In the near future, the accuracy of single-cell genomics will ultimately enable precise diagnostics and treatment of multiple hematopoietic and immune related diseases.

  19. The impact of chronic intermittent hypoxia on hematopoiesis and the bone marrow microenvironment.

    PubMed

    Alvarez-Martins, Inês; Remédio, Leonor; Matias, Inês; Diogo, Lucília N; Monteiro, Emília C; Dias, Sérgio

    2016-05-01

    Obstructive sleep apnea (OSA) is a highly prevalent sleep-related breathing disorder which is associated with patient morbidity and an elevated risk of developing hypertension and cardiovascular diseases. There is ample evidence for the involvement of bone marrow (BM) cells in the pathophysiology of cardiovascular diseases but a connection between OSA and modulation of the BM microenvironment had not been established. Here, we studied how chronic intermittent hypoxia (CIH) affected hematopoiesis and the BM microenvironment, in a rat model of OSA. We show that CIH followed by normoxia increases the bone marrow hypoxic area, increases the number of multipotent hematopoietic progenitors (CFU assay), promotes erythropoiesis, and increases monocyte counts. In the BM microenvironment of CIH-subjected animals, the number of VE-cadherin-expressing blood vessels, particularly sinusoids, increased, accompanied by increased smooth muscle cell coverage, while vWF-positive vessels decreased. Molecularly, we investigated the expression of endothelial cell-derived genes (angiocrine factors) that could explain the cellular phenotypes. Accordingly, we observed an increase in colony-stimulating factor 1, vascular endothelium growth factor, delta-like 4, and angiopoietin-1 expression. Our data shows that CIH induces vascular remodeling in the BM microenvironment, which modulates hematopoiesis, increasing erythropoiesis, and circulating monocytes. Our study reveals for the first time the effect of CIH in hematopoiesis and suggests that hematopoietic changes may occur in OSA patients.

  20. Morphologic and GATA1 sequencing analysis of hematopoiesis in fetuses with trisomy 21.

    PubMed

    Hoeller, Sylvia; Bihl, Michel P; Tzankov, Alexandar; Chaffard, Rosemarie; Hirschmann, Petra; Miny, Peter; Kühne, Thomas; Bruder, Elisabeth

    2014-05-01

    Trisomy 21 alters fetal liver hematopoiesis and, in combination with somatic globin transcription factor 1 (GATA1) mutations, leads to development of transient myeloproliferative disease in newborns. However, little is known about the morphological hematopoietic changes caused by trisomy 21 in the fetus, and to date, the exact onset of GATA1 mutations remains uncertain. Therefore, we analyzed fetal liver hematopoiesis from second trimester pregnancies in trisomy 21 and screened for GATA1 mutations. We examined 57 formalin-fixed and paraffin-embedded fetal liver specimens (49 harboring trisomy 21 and 8 controls) by immunohistochemistry for CD34, CD61, factor VIII, and glycophorin A. GATA1 exon 2 was sequenced in fetal livers and corresponding nonhematologic tissue. Cell counts of megakaryocytes (P = .022), megakaryocytic precursors (P = .021), and erythroid precursors were higher in trisomy 21 cases. CD34-positive hematopoietic blasts showed no statistically significant differences. No mutation was detected by GATA1 exon 2 sequencing in fetal livers from 12 to 25 weeks of gestation. Our results suggest that GATA1 exon 2 mutations occur late in trisomy 21 fetal hematopoiesis. However, trisomy 21 alone provides a proliferative stimulus of fetal megakaryopoiesis and erythropoiesis. CD34-positive precursor cells are not increased in trisomy 21 fetal livers.

  1. [Chronic myelogenous leukemia initially presenting with multiple subcutaneous tumors due to extramedullary hematopoiesis].

    PubMed

    Kurosawa, Shuhei; Doki, Noriko; Kaito, Satoshi; Sakaguchi, Masahiro; Harada, Kaito; Hino, Yutaro; Yamamoto, Keita; Ikegawa, Shuntaro; Yosioka, Kosuke; Watanabe, Daisuke; Senoo, Yasushi; Watakabe, Kyoko; Hagino, Takeshi; Igarashi, Aiko; Najima, Yuho; Kobayashi, Takeshi; Kakihana, Kazuhiko; Miyawaki, Shuichi; Sakamaki, Hisashi; Ohashi, Kazuteru

    2016-04-01

    A 53-year-old woman was admitted with right upper-extremity pain and multiple subcutaneous masses. Bone marrow aspirate showed hypercellular marrow with increased myeloid components at all stages of maturation. Cytogenetic analysis of the bone marrow revealed 100% Philadelphia chromosome positivity along with BCR/ABL gene rearrangement, as demonstrated by polymerase chain reaction (PCR). A diagnosis of chronic phase of chronic myeloid leukemia (CML) was therefore made. Biopsy of one of the subcutaneous masses showed proliferation of granulocytes in various stages of differentiation. There were also erythroid cells and megakaryocytes, without p53 and CD34-positive blasts. These results suggested that the subcutaneous masses had developed from extramedullary hematopoiesis, not blastomas. The patient was administered dasatinib (DA) 140 mg, combined with radiation therapy for pain and peripheral neuropathy from the right axial extramedullary tumor. The patient showed complete hematological remission and the subcutaneous masses had disappeared 1 month after starting administration of DA. Because the patient did not achieve a cytogenetic response, the tyrosine kinase inhibitor nilotinib was administered. She will undergo allogeneic stem cell transplantation in the near future. Extramedullary hematopoiesis in the early stages of CML is uncommon. Our case emphasizes the need to elucidate the pathogenesis of extramedullary hematopoiesis in the early stages of CML.

  2. Role of the clathrin adaptor PICALM in normal hematopoiesis and polycythemia vera pathophysiology.

    PubMed

    Ishikawa, Yuichi; Maeda, Manami; Pasham, Mithun; Aguet, Francois; Tacheva-Grigorova, Silvia K; Masuda, Takeshi; Yi, Hai; Lee, Sung-Uk; Xu, Jian; Teruya-Feldstein, Julie; Ericsson, Maria; Mullally, Ann; Heuser, John; Kirchhausen, Tom; Maeda, Takahiro

    2015-04-01

    Clathrin-dependent endocytosis is an essential cellular process shared by all cell types. Despite this, precisely how endocytosis is regulated in a cell-type-specific manner and how this key pathway functions physiologically or pathophysiologically remain largely unknown. PICALM, which encodes the clathrin adaptor protein PICALM, was originally identified as a component of the CALM/AF10 leukemia oncogene. Here we show, by employing a series of conditional Picalm knockout mice, that PICALM critically regulates transferrin uptake in erythroid cells by functioning as a cell-type-specific regulator of transferrin receptor endocytosis. While transferrin receptor is essential for the development of all hematopoietic lineages, Picalm was dispensable for myeloid and B-lymphoid development. Furthermore, global Picalm inactivation in adult mice did not cause gross defects in mouse fitness, except for anemia and a coat color change. Freeze-etch electron microscopy of primary erythroblasts and live-cell imaging of murine embryonic fibroblasts revealed that Picalm function is required for efficient clathrin coat maturation. We showed that the PICALM PIP2 binding domain is necessary for transferrin receptor endocytosis in erythroblasts and absolutely essential for erythroid development from mouse hematopoietic stem/progenitor cells in an erythroid culture system. We further showed that Picalm deletion entirely abrogated the disease phenotype in a Jak2(V617F) knock-in murine model of polycythemia vera. Our findings provide new insights into the regulation of cell-type-specific transferrin receptor endocytosis in vivo. They also suggest a new strategy to block cellular uptake of transferrin-bound iron, with therapeutic potential for disorders characterized by inappropriate red blood cell production, such as polycythemia vera.

  3. Role of the clathrin adaptor PICALM in normal hematopoiesis and polycythemia vera pathophysiology

    PubMed Central

    Ishikawa, Yuichi; Maeda, Manami; Pasham, Mithun; Aguet, Francois; Tacheva-Grigorova, Silvia K.; Masuda, Takeshi; Yi, Hai; Lee, Sung-Uk; Xu, Jian; Teruya-Feldstein, Julie; Ericsson, Maria; Mullally, Ann; Heuser, John; Kirchhausen, Tom; Maeda, Takahiro

    2015-01-01

    Clathrin-dependent endocytosis is an essential cellular process shared by all cell types. Despite this, precisely how endocytosis is regulated in a cell-type-specific manner and how this key pathway functions physiologically or pathophysiologically remain largely unknown. PICALM, which encodes the clathrin adaptor protein PICALM, was originally identified as a component of the CALM/AF10 leukemia oncogene. Here we show, by employing a series of conditional Picalm knockout mice, that PICALM critically regulates transferrin uptake in erythroid cells by functioning as a cell-type-specific regulator of transferrin receptor endocytosis. While transferrin receptor is essential for the development of all hematopoietic lineages, Picalm was dispensable for myeloid and B-lymphoid development. Furthermore, global Picalm inactivation in adult mice did not cause gross defects in mouse fitness, except for anemia and a coat color change. Freeze-etch electron microscopy of primary erythroblasts and live-cell imaging of murine embryonic fibroblasts revealed that Picalm function is required for efficient clathrin coat maturation. We showed that the PICALM PIP2 binding domain is necessary for transferrin receptor endocytosis in erythroblasts and absolutely essential for erythroid development from mouse hematopoietic stem/progenitor cells in an erythroid culture system. We further showed that Picalm deletion entirely abrogated the disease phenotype in a Jak2V617F knock-in murine model of polycythemia vera. Our findings provide new insights into the regulation of cell-type-specific transferrin receptor endocytosis in vivo. They also suggest a new strategy to block cellular uptake of transferrin-bound iron, with therapeutic potential for disorders characterized by inappropriate red blood cell production, such as polycythemia vera. PMID:25552701

  4. Enhancement of Umbilical Cord Blood Cell Hematopoiesis by Maitake Beta-Glucan Is Mediated by Granulocyte Colony-Stimulating Factor Production▿

    PubMed Central

    Lin, Hong; Cheung, Sandy W. Y.; Nesin, Mirjana; Cassileth, Barrie R.; Cunningham-Rundles, Susanna

    2007-01-01

    Maitake beta-glucan (MBG) is an extract from the fruit body of the Grifola frondosa mushroom that is being widely used to treat cancer in Asia. We have previously reported that MBG enhances mouse bone marrow cell (BMC) hematopoiesis in vitro and protects BMC from doxorubicin (DOX) toxicity. In the current study, we investigated the ability of MBG to enhance hematopoiesis and to reduce the toxic effects of DOX on fresh human umbilical cord blood (CB) cells. MBG treatment significantly enhanced the colony formation unit (CFU) response of granulocytes-macrophages (CFU-GM response) over the whole dose range of 12.5 to 100 μg/ml (P < 0.05). The addition of MBG to DOX-treated CB cells significantly protected granulocyte-macrophage colony formation from the toxicity of DOX, which otherwise produced strong hematopoietic repression. MBG also partially replaced recombinant human granulocyte colony-stimulating factor (rhG-CSF), as shown by a significant augmentation of the CFU-GM response in the absence of rhG-CSF. We found that MBG induces granulocyte colony-stimulating factor (G-CSF) production in CB CD33+ monocytes, as detected by intracellular cytokine flow cytometric assessment. In contrast, we found that adult peripheral blood monocytes did not produce a significant G-CSF response to MBG, whereas both adult and CB monocytes produced G-CSF in response to lipopolysaccharide. These studies provide the first evidence that MBG induces hematopoietic stem cell proliferation and differentiation of CFU-GM in umbilical CB cells and acts directly to induce G-CSF. PMID:17093103

  5. Reliable Genetic Labeling of Adult-Born Dentate Granule Cells Using Ascl1 CreERT2 and Glast CreERT2 Murine Lines.

    PubMed

    Yang, Sung M; Alvarez, Diego D; Schinder, Alejandro F

    2015-11-18

    Newly generated dentate granule cells (GCs) are relevant for input discrimination in the adult hippocampus. Yet, their precise contribution to information processing remains unclear. To address this question, it is essential to develop approaches to precisely label entire cohorts of adult-born GCs. In this work, we used genetically modified mice to allow conditional expression of tdTomato (Tom) in adult-born GCs and characterized their development and functional integration. Ascl1(CreERT2);CAG(floxStopTom) and Glast(CreERT2);CAG(floxStopTom) mice resulted in indelible expression of Tom in adult neural stem cells and their lineage upon tamoxifen induction. Whole-cell recordings were performed to measure intrinsic excitability, firing behavior, and afferent excitatory connectivity. Developing GCs were also staged by the expression of early and late neuronal markers. The slow development of adult-born GCs characterized here is consistent with previous reports using retroviral approaches that have revealed that a mature phenotype is typically achieved after 6-8 weeks. Our findings demonstrate that Ascl1(CreERT2) and Glast(CreERT2) mouse lines enable simple and reliable labeling of adult-born GC lineages within restricted time windows. Therefore, these mice greatly facilitate tagging new neurons and manipulating their activity, required for understanding adult neurogenesis in the context of network remodeling, learning, and behavior. Our study shows that Ascl1(CreERT2) and Glast(CreERT2) mice lines can be used to label large cohorts of adult-born dentate granule cells with excellent time resolution. Neurons labeled in this manner display developmental and functional profiles that are in full agreement with previous findings using thymidine analogs and retroviral labeling, thus providing an alternative approach to tackle fundamental questions on circuit remodeling. Because of the massive neuronal targeting and the simplicity of this method, genetic labeling will

  6. A method for high purity intestinal epithelial cell culture from adult human and murine tissues for the investigation of innate immune function

    PubMed Central

    Graves, Christina L.; Harden, Scott W.; LaPato, Melissa; Nelson, Michael; Amador, Byron; Sorenson, Heather; Frazier, Charles J.; Wallet, Shannon M.

    2015-01-01

    Intestinal epithelial cells (IECs) serve as an important physiologic barrier between environmental antigens and the host intestinal immune system. Thus, IECs serve as a first line of defense and may act as sentinel cells during inflammatory insults. Despite recent renewed interest in IEC contributions to host immune function, the study of primary IEC has been hindered by lack of a robust culture technique, particularly for small intestinal and adult tissues. Here, a novel adaptation for culture of primary IEC is described for human duodenal organ donor tissue as well as duodenum and colon of adult mice. These epithelial cell cultures display characteristic phenotypes and are of high purity. In addition, the innate immune function of human primary IEC, specifically with regard to Toll-like receptor (TLR) expression and microbial ligand responsiveness, is contrasted with a commonly used intestinal epithelial cell line (HT-29). Specifically, TLR expression at the mRNA level and production of cytokine (IFNγ and TNFα) in response to TLR agonist stimulation is assessed. Differential expression of TLRs as well as innate immune responses to ligand stimulation is observed in human-derived cultures compared to that of HT-29. Thus, use of this adapted method to culture primary epithelial cells from adult human donors and from adult mice will allow for more appropriate studies of IECs as innate immune effectors. PMID:25193428

  7. A method for high purity intestinal epithelial cell culture from adult human and murine tissues for the investigation of innate immune function.

    PubMed

    Graves, Christina L; Harden, Scott W; LaPato, Melissa; Nelson, Michael; Amador, Byron; Sorenson, Heather; Frazier, Charles J; Wallet, Shannon M

    2014-12-01

    Intestinal epithelial cells (IECs) serve as an important physiologic barrier between environmental antigens and the host intestinal immune system. Thus, IECs serve as a first line of defense and may act as sentinel cells during inflammatory insults. Despite recent renewed interest in IEC contributions to host immune function, the study of primary IEC has been hindered by lack of a robust culture technique, particularly for small intestinal and adult tissues. Here, a novel adaptation for culture of primary IEC is described for human duodenal organ donor tissue as well as duodenum and colon of adult mice. These epithelial cell cultures display characteristic phenotypes and are of high purity. In addition, the innate immune function of human primary IEC, specifically with regard to Toll-like receptor (TLR) expression and microbial ligand responsiveness, is contrasted with a commonly used intestinal epithelial cell line (HT-29). Specifically, TLR expression at the mRNA level and production of cytokine (IFNγ and TNFα) in response to TLR agonist stimulation is assessed. Differential expression of TLRs as well as innate immune responses to ligand stimulation is observed in human-derived cultures compared to that of HT-29. Thus, use of this adapted method to culture primary epithelial cells from adult human donors and from adult mice will allow for more appropriate studies of IECs as innate immune effectors.

  8. Symbiont-induced odorant binding proteins mediate insect host hematopoiesis

    PubMed Central

    Benoit, Joshua B; Vigneron, Aurélien; Broderick, Nichole A; Wu, Yineng; Sun, Jennifer S; Carlson, John R; Aksoy, Serap; Weiss, Brian L

    2017-01-01

    Symbiotic bacteria assist in maintaining homeostasis of the animal immune system. However, the molecular mechanisms that underlie symbiont-mediated host immunity are largely unknown. Tsetse flies (Glossina spp.) house maternally transmitted symbionts that regulate the development and function of their host’s immune system. Herein we demonstrate that the obligate mutualist, Wigglesworthia, up-regulates expression of odorant binding protein six in the gut of intrauterine tsetse larvae. This process is necessary and sufficient to induce systemic expression of the hematopoietic RUNX transcription factor lozenge and the subsequent production of crystal cells, which actuate the melanotic immune response in adult tsetse. Larval Drosophila’s indigenous microbiota, which is acquired from the environment, regulates an orthologous hematopoietic pathway in their host. These findings provide insight into the molecular mechanisms that underlie enteric symbiont-stimulated systemic immune system development, and indicate that these processes are evolutionarily conserved despite the divergent nature of host-symbiont interactions in these model systems. DOI: http://dx.doi.org/10.7554/eLife.19535.001 PMID:28079523

  9. Human and Murine Hematopoietic Stem Cell Aging Is Associated with Functional Impairments and Intrinsic Megakaryocytic/Erythroid Bias

    PubMed Central

    Rundberg Nilsson, Alexandra; Soneji, Shamit; Adolfsson, Sofia; Bryder, David; Pronk, Cornelis Jan

    2016-01-01

    Aging within the human hematopoietic system associates with various deficiencies and disease states, including anemia, myeloid neoplasms and reduced adaptive immune responses. Similar phenotypes are observed in mice and have been linked to alterations arising at the hematopoietic stem cell (HSC) level. Such an association is, however, less established in human hematopoiesis and prompted us here to detail characteristics of the most primitive human hematopoietic compartments throughout ontogeny. In addition, we also attempted to interrogate similarities between aging human and murine hematopoiesis. Coupled to the transition from human cord blood (CB) to young and aged bone marrow (BM), we observed a gradual increase in frequency of candidate HSCs. This was accompanied by functional impairments, including decreased lymphoid output and reduced proliferative potential. Downstream of human HSCs, we observed decreasing levels of common lymphoid progenitors (CLPs), and increasing frequencies of megakaryocyte/erythrocyte progenitors (MEPs) with age, which could be linked to changes in lineage-affiliated gene expression patterns in aged human HSCs. These findings were paralleled in mice. Therefore, our data support the notion that age-related changes also in human hematopoiesis involve the HSC pool, with a prominent skewing towards the megakaryocytic/erythroid lineages, and suggests conserved mechanisms underlying aging of the blood cell system. PMID:27368054

  10. Single episode of mild murine malaria induces neuroinflammation, alters microglial profile, impairs adult neurogenesis, and causes deficits in social and anxiety-like behavior.

    PubMed

    Guha, Suman K; Tillu, Rucha; Sood, Ankit; Patgaonkar, Mandar; Nanavaty, Ishira N; Sengupta, Arjun; Sharma, Shobhona; Vaidya, Vidita A; Pathak, Sulabha

    2014-11-01

    Cerebral malaria is associated with cerebrovascular damage and neurological sequelae. However, the neurological consequences of uncomplicated malaria, the most prevalent form of the disease, remain uninvestigated. Here, using a mild malaria model, we show that a single Plasmodium chabaudi adami infection in adult mice induces neuroinflammation, neurogenic, and behavioral changes in the absence of a blood-brain barrier breach. Using cytokine arrays we show that the infection induces differential serum and brain cytokine profiles, both at peak parasitemia and 15days post-parasite clearance. At the peak of infection, along with the serum, the brain also exhibited a definitive pro-inflammatory cytokine profile, and gene expression analysis revealed that pro-inflammatory cytokines were also produced locally in the hippocampus, an adult neurogenic niche. Hippocampal microglia numbers were enhanced, and we noted a shift to an activated profile at this time point, accompanied by a striking redistribution of the microglia to the subgranular zone adjacent to hippocampal neuronal progenitors. In the hippocampus, a distinct decline in progenitor turnover and survival was observed at peak parasitemia, accompanied by a shift from neuronal to glial fate specification. Studies in transgenic Nestin-GFP reporter mice demonstrated a decline in the Nestin-GFP(+)/GFAP(+) quiescent neural stem cell pool at peak parasitemia. Although these cellular changes reverted to normal 15days post-parasite clearance, specific brain cytokines continued to exhibit dysregulation. Behavioral analysis revealed selective deficits in social and anxiety-like behaviors, with no change observed in locomotor, cognitive, and depression-like behaviors, with a return to baseline at recovery. Collectively, these findings indicate that even a single episode of mild malaria results in alterations of the brain cytokine profile, causes specific behavioral dysfunction, is accompanied by hippocampal microglial

  11. Elk3 deficiency causes transient impairment in post-natal retinal vascular development and formation of tortuous arteries in adult murine retinae.

    PubMed

    Weinl, Christine; Wasylyk, Christine; Garcia Garrido, Marina; Sothilingam, Vithiyanjali; Beck, Susanne C; Riehle, Heidemarie; Stritt, Christine; Roux, Michel J; Seeliger, Mathias W; Wasylyk, Bohdan; Nordheim, Alfred

    2014-01-01

    Serum Response Factor (SRF) fulfills essential roles in post-natal retinal angiogenesis and adult neovascularization. These functions have been attributed to the recruitment by SRF of the cofactors Myocardin-Related Transcription Factors MRTF-A and -B, but not the Ternary Complex Factors (TCFs) Elk1 and Elk4. The role of the third TCF, Elk3, remained unknown. We generated a new Elk3 knockout mouse line and showed that Elk3 had specific, non-redundant functions in the retinal vasculature. In Elk3(-/-) mice, post-natal retinal angiogenesis was transiently delayed until P8, after which it proceeded normally. Interestingly, tortuous arteries developed in Elk3(-/-) mice from the age of four weeks, and persisted into late adulthood. Tortuous vessels have been observed in human pathologies, e.g. in ROP and FEVR. These human disorders were linked to altered activities of vascular endothelial growth factor (VEGF) in the affected eyes. However, in Elk3(-/-) mice, we did not observe any changes in VEGF or several other potential confounding factors, including mural cell coverage and blood pressure. Instead, concurrent with the post-natal transient delay of radial outgrowth and the formation of adult tortuous arteries, Elk3-dependent effects on the expression of Angiopoietin/Tie-signalling components were observed. Moreover, in vitro microvessel sprouting and microtube formation from P10 and adult aortic ring explants were reduced. Collectively, these results indicate that Elk3 has distinct roles in maintaining retinal artery integrity. The Elk3 knockout mouse is presented as a new animal model to study retinal artery tortuousity in mice and human patients.

  12. Hematopoietic stem cell development: Using the zebrafish to identify extrinsic and intrinsic mechanisms regulating hematopoiesis.

    PubMed

    Frame, J M; Lim, S-E; North, T E

    2017-01-01

    Hematopoietic stem cells (HSCs) reside at the apex of the hematopoietic hierarchy, giving rise to each of the blood lineages found throughout the lifetime of the organism. Since the genetic programs regulating HSC development are highly conserved between vertebrate species, experimental studies in zebrafish have not only complemented observations reported in mammals but have also yielded important discoveries that continue to influence our understanding of HSC biology and homeostasis. Here, we summarize findings that have established zebrafish as an important conserved model for the study of hematopoiesis, and describe methods that can be utilized for future investigations of zebrafish HSC biology. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Identification of novel targets of CSL-dependent Notch signaling in hematopoiesis.

    PubMed

    Hamidi, Habib; Gustafason, Derek; Pellegrini, Matteo; Gasson, Judith

    2011-01-01

    Somatic activating mutations in the Notch1 receptor result in the overexpression of activated Notch1, which can be tumorigenic. The goal of this study is to understand the molecular mechanisms underlying the phenotypic changes caused by the overexpression of ligand independent Notch 1 by using a tetracycline inducible promoter in an in vitro embryonic stem (ES) cells/OP9 stromal cells coculture system, recapitulating normal hematopoiesis. First, an in silico analysis of the promoters of Notch regulated genes (previously determined by microarray analysis) revealed that the motifs recognized by regulatory proteins known to mediate hematopoiesis were overrepresented. Notch 1 does not bind DNA but instead binds the CSL transcription factor to regulate gene expression. The in silico analysis also showed that there were putative CSL binding sites observed in the promoters of 28 out of 148 genes. A custom ChIP-chip array was used to assess the occupancy of CSL in the promoter regions of the Notch1 regulated genes in vivo and showed that 61 genes were bound by activated Notch responsive CSL. Then, comprehensive mapping of the CSL binding sites genome-wide using ChIP-seq analysis revealed that over 10,000 genes were bound within 10 kb of the TSS (transcription start site). The majority of the targets discovered by ChIP-seq belong to pathways that have been shown by others to crosstalk with Notch signaling. Finally, 83 miRNAs were significantly differentially expressed by greater than 1.5-fold during the course of in vitro hematopoiesis. Thirty one miRNA were up-regulated and fifty two were down-regulated. Overexpression of Notch1 altered this pattern of expression of microRNA: six miRNAs were up-regulated and four were down regulated as a result of activated Notch1 overexpression during the course of hematopoiesis. Time course analysis of hematopoietic development revealed that cells with Notch 1 overexpression mimic miRNA expression of cells in a less mature stage, which

  14. Bloody nipple discharge in 2 infants with interesting cytologic findings of extramedullary hematopoiesis and hemophagocytosis.

    PubMed

    Pampal, Arzu; Gokoz, Aytac; Sipahi, Tansu; Dogan, Handan; Ergur, Ayca Torel

    2012-04-01

    Bloody nipple discharge in the infantile period is an uncommon finding. Despite its stressful course to the parents, it is generally a benign condition with a spontaneous resolution. The approach to bloody nipple discharge in the infantile period is well documented in the literature even though the number of these cases is limited. We report 2 infants with unilateral bloody nipple discharge. Their physical examination, laboratory, and ultrasound findings were normal but the cytologic examinations of the discharge revealed signs of extramedullary hematopoiesis and hemophagocytosis. These extraordinary findings made us brainstorm on the probable ongoing processes in the infantile breast tissue.

  15. Structurally specific heparan sulfates support primitive human hematopoiesis by formation of a multimolecular stem cell niche.

    PubMed

    Gupta, P; Oegema, T R; Brazil, J J; Dudek, A Z; Slungaard, A; Verfaillie, C M

    1998-12-15

    Stem cell localization, conservation, and differentiation is believed to occur in niches in the marrow stromal microenvironment. Our recent observation that long-term in vitro human hematopoiesis requires a stromal heparan sulfate proteoglycan (HSPG) led us to hypothesize that such HSPG may orchestrate the formation of the stem cell niche. We compared the structure and function of HS from M2-10B4, a hematopoiesis-supportive cell line, with HS from a nonsupportive cell line, FHS-173-We. Long-term culture-initiating cell (LTC-IC) maintenance was enhanced by PG from supportive cells but not by PG from nonsupportive cells (P <.005). The supportive HS were significantly larger and more highly sulfated than the nonsupportive HS. Specifically, supportive HS contained higher 6-O-sulfation on the glucosamine residues. In agreement with these observations, purified 6-O-sulfated heparin and highly 6-O-sulfated bovine kidney HS similarly maintained LTC-IC. In contrast, completely desulfated heparin, N-sulfated heparin, and unmodified heparin did not support LTC-IC maintenance. Moreover, the supportive HS promoted LTC-IC maintenance but not differentiation of CD34(+)/HLA-DR- cells into colony-forming cells (CFCs) and mature blood cells. The supportive HS but not the nonsupportive HS bound both cytokines and matrix components critical for hematopoiesis, including interleukin-3 (IL-3), macrophage inflammatory protein-1 (MIP-1), and thrombospondin (TSP). Significantly more CD34(+) cells adhered directly to immobilized O-sulfated heparin than to N-sulfated or desulfated heparin. Thus, hematopoiesis-supportive stromal HSPG possessing large, highly 6-O-sulfated HS mediate the juxtaposition of hematopoietic progenitors with stromal cells, specific growth-promoting (IL-3) and growth-inhibitory (MIP-1 and platelet factor 4 [PF4]) cytokines, and extracellular matrix (ECM) proteins such as TSP. We conclude that the structural specificity of stromal HSPG that determines the selective

  16. In utero transplantation of adult bone marrow decreases perinatal lethality and rescues the bone phenotype in the knockin murine model for classical, dominant osteogenesis imperfecta

    PubMed Central

    Panaroni, Cristina; Gioia, Roberta; Lupi, Anna; Besio, Roberta; Goldstein, Steven A.; Kreider, Jaclynn; Leikin, Sergey; Vera, Juan Carlos; Mertz, Edward L.; Perilli, Egon; Baruffaldi, Fabio; Villa, Isabella; Farina, Aurora; Casasco, Marco; Cetta, Giuseppe; Rossi, Antonio; Frattini, Annalisa; Marini, Joan C.; Vezzoni, Paolo

    2009-01-01

    Autosomal dominant osteogenesis imperfecta (OI) caused by glycine substitutions in type I collagen is a paradigmatic disorder for stem cell therapy. Bone marrow transplantation in OI children has produced a low engraftment rate, but surprisingly encouraging symptomatic improvements. In utero transplantation (IUT) may hold even more promise. However, systematic studies of both methods have so far been limited to a recessive mouse model. In this study, we evaluated intrauterine transplantation of adult bone marrow into heterozygous BrtlIV mice. Brtl is a knockin mouse with a classical glycine substitution in type I collagen [α1(I)-Gly349Cys], dominant trait transmission, and a phenotype resembling moderately severe and lethal OI. Adult bone marrow donor cells from enhanced green fluorescent protein (eGFP) transgenic mice engrafted in hematopoietic and nonhematopoietic tissues differentiated to trabecular and cortical bone cells and synthesized up to 20% of all type I collagen in the host bone. The transplantation eliminated the perinatal lethality of heterozygous BrtlIV mice. At 2 months of age, femora of treated Brtl mice had significant improvement in geometric parameters (P < .05) versus untreated Brtl mice, and their mechanical properties attained wild-type values. Our results suggest that the engrafted cells form bone with higher efficiency than the endogenous cells, supporting IUT as a promising approach for the treatment of genetic bone diseases. PMID:19414862

  17. Murine adult neural progenitor cells alter their proliferative behavior and gene expression after the activation of Toll-like-receptor 3

    PubMed Central

    Melnik, A.; Tauber, S.; Dumrese, C.; Ullrich, O.; Wolf, S. A.

    2012-01-01

    Viral infections during pregnancy significantly increase the risk for psychological pathologies like schizophrenia in the offspring. One of the main morphological hallmarks of schizophrenia is a reduced size of the hippocampus. Since new neurons are produced in this particular brain compartment throughout life, it might be possible that low neurogenesis levels triggered by a maternal viral infection contribute to developmental deficits of the hippocampus. We injected polyinosinic:polycytidylic acid (Poly I:C) in pregnant C57Bl/6 mice to stimulate an anti-viral response through TLR3 and examined gene expressions in the neuronal progenitor cells (NPCs) of the offspring at different ages. Additionally, we treated adult NPC lines with Poly I:C to investigate its direct effect. We could show for the first time that TLR3 and its downstream effector molecule IRF3 are expressed in adult NPCs. Poly I:C treatment in vitro and in vivo led to the regulation of proliferation and genes involved in antiviral response, migration, and survival. These findings indicate that NPCs of the fetus are able to react towards an in utero immune response, and thus, changes in the neuronal stem cell pool can contribute to the development of neurological diseases like schizophrenia. PMID:24688771

  18. EphB2 and EphB3 play an important role in the lymphoid seeding of murine adult thymus.

    PubMed

    Alfaro, David; García-Ceca, Javier; Farias-de-Oliveira, Desio A; Terra-Granado, Eugenia; Montero-Herradón, Sara; Cotta-de-Almeida, Vinicius; Savino, Wilson; Zapata, Agustín

    2015-12-01

    Adult thymuses lacking either ephrin type B receptor 2 (EphB2) or EphB3, or expressing a truncated form of EphB2, the forward signal-deficient EphB2LacZ, have low numbers of early thymic progenitors (ETPs) and are colonized in vivo by reduced numbers of injected bone marrow (BM) lineage-negative (Lin(-)) cells. Hematopoietic progenitors from these EphB mutants showed decreased capacities to colonize wild type (WT) thymuses compared with WT precursors, with EphB2(-/-) cells exhibiting the greatest reduction. WT BM Lin(-) cells also showed decreased colonizing capacity into mutant thymuses. The reduction was also more severe in EphB2(-/-) host thymuses, with a less severe phenotype in the EphB2LacZ thymus. These results suggest a major function for forward signaling through EphB2 and, to a lesser extent, EphB3, in either colonizing progenitor cells or thymic stromal cells, for in vivo adult thymus recruitment. Furthermore, the altered expression of the molecules involved in thymic colonization that occurs in the mutant thymus correlates with the observed colonizing capacities of different mutant mice. Reduced production of CCL21 and CCL25 occurred in the thymus of the 3 EphB-deficient mice, but their expression, similar to that of P-selectin, on blood vessels, the method of entry of progenitor cells into the vascular thymus, only showed a significant reduction in EphB2(-/-) and EphB3(-/-) thymuses. Decreased migration into the EphB2(-/-) thymuses correlated also with reduced expression of both ephrinB1 and ephrinB2, without changes in the EphB2LacZ thymuses. In the EphB3(-/-) thymuses, only ephrinB1 expression appeared significantly diminished, confirming the relevance of forward signals mediated by the EphB2-ephrinB1 pair in cell recruitment into the adult thymus. © Society for Leukocyte Biology.

  19. Identification of a new adtrp1-tfpi regulatory axis for the specification of primitive myelopoiesis and definitive hematopoiesis.

    PubMed

    Wang, Li; Wang, Xiaojing; Wang, Longfei; Yousaf, Muhammad; Li, Jia; Zuo, Mengxia; Yang, Zhongcheng; Gou, Dongzhi; Bao, Binghao; Li, Lei; Xiang, Ning; Jia, Haibo; Xu, Chengqi; Chen, Qiuyun; Wang, Qing Kenneth

    2017-09-06

    A genomic variant in the human ADTRP [androgen-dependent tissue factor (TF) pathway inhibitor (TFPI) regulating protein] gene increases the risk of coronary artery disease, the leading cause of death worldwide. TFPI is the TF pathway inhibitor that is involved in coagulation. Here, we report that adtrp and tfpi form a regulatory axis that specifies primitive myelopoiesis and definitive hematopoiesis, but not primitive erythropoiesis or vasculogenesis. In zebrafish, there are 2 paralogues for adtrp (i.e., adtrp1 and adtrp2). Knockdown of adtrp1 expression inhibits the specification of hemangioblasts as shown by decreased expression of the hemangioblast markers, etsrp, fli1a, and scl; blocks primitive hematopoiesis as shown by decreased expression of pu.1, mpo, and l-plastin; and disrupts the specification of hematopoietic stem cells (definitive hematopoiesis) as shown by decreased expression of runx1 and c-myb However, adtrp1 knockdown does not affect erythropoiesis during primitive hematopoiesis (no effect on gata1 or h-bae1) or vasculogenesis (no effect on kdrl, ephb2a, notch3, dab2, or flt4). Knockdown of adtrp2 expression does not have apparent effects on all markers tested. Knockdown of adtrp1 reduced the expression of tfpi, and hematopoietic defects in adtrp1 morphants were rescued by tfpi overexpression. These data suggest that the regulation of tfpi expression is one potential mechanism by which adtrp1 regulates primitive myelopoiesis and definitive hematopoiesis.-Wang, L., Wang, X., Wang, L., Yousaf, M., Li, J., Zuo, M., Yang, Z., Gou, D., Bao, B., Li, L., Xiang, N., Jia, H., Xu, C., Chen, Q., Wang, Q. K. Identification of a new adtrp1-tfpi regulatory axis for the specification of primitive myelopoiesis and definitive hematopoiesis. © FASEB.

  20. Conditional overexpression of receptors for advanced glycation end-products in the adult murine lung causes airspace enlargement and induces inflammation.

    PubMed

    Stogsdill, Megan P; Stogsdill, Jeffrey A; Bodine, B Garrett; Fredrickson, Ali C; Sefcik, Tayler L; Wood, Tyler T; Kasteler, Stephen D; Reynolds, Paul R

    2013-07-01

    Receptors for advanced glycation end-products (RAGE) are multiligand surface receptors detected abundantly in pulmonary tissue. Our previous work revealed increased RAGE expression in cells and lungs exposed to tobacco smoke and RAGE-mediated cytokine expression via proinflammatory mechanisms involving NF-κB. RAGE expression is elevated in various pathological states, including chronic obstructive pulmonary disease; however, precise contributions of RAGE to the progression of emphysema and pulmonary inflammation in the adult lung are unknown. In the current study, we generated a RAGE transgenic (RAGE TG) mouse and conditionally induced adult alveolar epithelium to overexpress RAGE. RAGE was induced after the period of alveologenesis, from weaning (20 d of age) until animals were killed at 50, 80, and 110 days (representing 30, 60, and 90 d of RAGE overexpression). Hematoxylin and eosin staining and mean chord length revealed incremental dilation of alveolar spaces as RAGE overexpression persisted. TUNEL staining and electron microscopy confirmed increased apoptosis and blebbing of alveolar epithelium in lungs from RAGE TG mice when compared with control mice. Immunohistochemistry for matrix metalloproteinase 9 revealed an overall increase in matrix metalloproteinase 9, which correlated with decreased elastin expression in RAGE TG mice. Furthermore, RAGE TG mice manifested significant inflammation measured by elevated bronchoalveolar lavage protein, leukocyte infiltration, and secreted cytokines. These data support the concept that innovative transgenic mice that overexpress RAGE may model pulmonary inflammation and alveolar destabilization independent of tobacco smoke and validate RAGE signaling as a target pathway in the prevention or attenuation of smoke-related inflammatory lung diseases.

  1. Conventional murine gene targeting.

    PubMed

    Zimmermann, Albert G; Sun, Yue

    2013-01-01

    Murine gene knockout models engineered over the last two decades have continued to demonstrate their potential as invaluable tools in understanding the role of gene function in the context of normal human development and disease. The more recent elucidation of the human and mouse genomes through sequencing has opened up the capability to elucidate the function of every human gene. State-of-the-art mouse model generation allows, through a multitude of experimental steps requiring careful standardization, gene function to be reliably and predictably ablated in a live model system. The application of these standardized methodologies to directly target gene function through murine gene knockout has to date provided comprehensive and verifiable genetic models that have contributed tremendously to our understanding of the cellular and molecular pathways underlying normal and disease states in humans. The ensuing chapter provides an overview of the latest steps and procedures required to ablate gene function in a murine model.

  2. Jak1 Integrates Cytokine Sensing to Regulate Hematopoietic Stem Cell Function and Stress Hematopoiesis.

    PubMed

    Kleppe, Maria; Spitzer, Matthew H; Li, Sheng; Hill, Corinne E; Dong, Lauren; Papalexi, Efthymia; De Groote, Sofie; Bowman, Robert L; Keller, Matthew; Koppikar, Priya; Rapaport, Franck T; Teruya-Feldstein, Julie; Gandara, Jorge; Mason, Christopher E; Nolan, Garry P; Levine, Ross L

    2017-10-05

    JAK1 is a critical effector of pro-inflammatory cytokine signaling and plays important roles in immune function, while abnormal JAK1 activity has been linked to immunological and neoplastic diseases. Specific functions of JAK1 in the context of hematopoiesis, and specifically within hematopoietic stem cells (HSCs), have not clearly been delineated. Here, we show that conditional Jak1 loss in HSCs reduces their self-renewal and markedly alters lymphoid/myeloid differentiation in vivo. Jak1-deficient HSCs exhibit decreased competitiveness in vivo and are unable to rescue hematopoiesis in the setting of myelosuppression. They exhibit increased quiescence, an inability to enter the cell cycle in response to hematopoietic stress, and a marked reduction in cytokine sensing, including in response to type I interferons and IL-3. Moreover, Jak1 loss is not fully rescued by expression of a constitutively active Jak2 allele. Together, these data highlight an essential role for Jak1 in HSC homeostasis and stress responses. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Multiple allogeneic progenitors in combination function as a unit to support early transient hematopoiesis in transplantation

    PubMed Central

    Takahashi, Satoshi; Lai, Chen-Yi; Nojima, Masanori; Yamamoto, Ryo; Takeuchi, Yasuo; Higashihara, Masaaki; Nakauchi, Hiromitsu

    2016-01-01

    Cord blood (CB) is a valuable donor source in hematopoietic cell transplantation. However, the initial time to engraftment in CB transplantation (CBT) is often delayed because of low graft cell numbers. This limits the use of CB. To overcome this cell dose barrier, we modeled an insufficient dose CBT setting in lethally irradiated mice and then added hematopoietic stem/progenitor cells (HSCs/HPCs; HSPCs) derived from four mouse allogeneic strains. The mixture of HSPCs rescued recipients and significantly accelerated hematopoietic recovery. Including T cells from one strain favored single-donor chimerism through graft versus graft reactions, with early hematopoietic recovery unaffected. Furthermore, using clinically relevant procedures, we successfully isolated a mixture of CD34+ cells from multiple frozen CB units at one time regardless of HLA-type disparities. These CD34+ cells in combination proved transplantable into immunodeficient mice. This work provides proof of concept that when circumstances require support of hematopoiesis, combined multiple units of allogeneic HSPCs are capable of early hematopoietic reconstitution while allowing single-donor hematopoiesis by a principal graft. PMID:27503070

  4. Arrested Hematopoiesis and Vascular Relaxation Defects in Mice with a Mutation in Dhfr

    PubMed Central

    Thoms, Julie A. I.; Knezevic, Kathy; Liu, Jia Jenny; Glaros, Elias N.; Thai, Thuan; Qiao, Qiao; Campbell, Heather; Packham, Deborah; Huang, Yizhou; Papathanasiou, Peter; Tunningley, Robert; Whittle, Belinda; Yeung, Amanda W. S.; Chandrakanthan, Vashe; Hesson, Luke; Chen, Vivien; Wong, Jason W. H.; Purton, Louise E.; Ward, Robyn L.

    2016-01-01

    Dihydrofolate reductase (DHFR) is a critical enzyme in the folate metabolism pathway and also plays a role in regulating nitric oxide (NO) signaling in endothelial cells. Although both coding and noncoding mutations with phenotypic effects have been identified in the human DHFR gene, no mouse model is currently available to study the consequences of perturbing DHFR in vivo. In order to identify genes involved in definitive hematopoiesis, we performed a forward genetic screen and produced a mouse line, here referred to as Orana, with a point mutation in the Dhfr locus leading to a Thr136Ala substitution in the DHFR protein. Homozygote Orana mice initiate definitive hematopoiesis, but expansion of progenitors in the fetal liver is compromised, and the animals die between embryonic day 13.5 (E13.5) and E14.5. Heterozygote Orana mice survive to adulthood but have tissue-specific alterations in folate abundance and distribution, perturbed stress erythropoiesis, and impaired endothelium-dependent relaxation of the aorta consistent with the role of DHFR in regulating NO signaling. Orana mice provide insight into the dual roles of DHFR and are a useful model for investigating the role of environmental and dietary factors in the context of vascular defects caused by altered NO signaling. PMID:26830229

  5. DEK oncogene expression during normal hematopoiesis and in Acute Myeloid Leukemia (AML).

    PubMed

    Logan, Gemma E; Mor-Vaknin, Nirit; Braunschweig, Till; Jost, Edgar; Schmidt, Pia Verena; Markovitz, David M; Mills, Ken I; Kappes, Ferdinand; Percy, Melanie J

    2015-01-01

    DEK is important in regulating cellular processes including proliferation, differentiation and maintenance of stem cell phenotype. The translocation t(6;9) in Acute Myeloid Leukemia (AML), which fuses DEK with NUP214, confers a poor prognosis and a higher risk of relapse. The over-expression of DEK in AML has been reported, but different studies have shown diminished levels in pediatric and promyelocytic leukemias. This study has characterized DEK expression, in silico, using a large multi-center cohort of leukemic and normal control cases. Overall, DEK was under-expressed in AML compared to normal bone marrow (NBM). Studying specific subtypes of AML confirmed either no significant change or a significant reduction in DEK expression compared to NBM. Importantly, the similarity of DEK expression between AML and NBM was confirmed using immunohistochemistry analysis of tissue mircorarrays. In addition, stratification of AML patients based on median DEK expression levels indicated that DEK showed no effect on the overall survival of patients. DEK expression during normal hematopoiesis did reveal a relationship with specific cell types implicating a distinct function during myeloid differentiation. Whilst DEK may play a potential role in hematopoiesis, it remains to be established whether it is important for leukemagenesis, except when involved in the t(6;9) translocation. Copyright © 2014. Published by Elsevier Inc.

  6. Stimulation of canine hematopoiesis by recombinant human granulocyte-macrophage colony-stimulating factor.

    PubMed

    Schuening, F G; Storb, R; Goehle, S; Nash, R; Graham, T C; Appelbaum, F R; Hackman, R; Sandmaier, B M; Urdal, D L

    1989-09-01

    The effect of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on canine hematopoiesis was evaluated. rhGM-CSF stimulated granulocyte-macrophage colony formation of canine marrow depleted of accessory cells up to tenfold. Stimulation of colony formation was abrogated by anti-rhGM-CSF antiserum or heat inactivation. rhGM-CSF also stimulated in vivo canine hematopoiesis both when given as continuous i.v. infusion and as intermittent s.c. injections. Neutrophil, monocyte, and lymphocyte counts were increased three- to eightfold above controls, whereas values for eosinophils, reticulocytes, and hematocrits were not changed. Bone marrow histology after 2 weeks of treatment with rhGM-CSF showed hypercellularity with myeloid hyperplasia and left-shifted granulocytopoiesis. After discontinuation of rhGM-CSF, peripheral leukocyte counts returned to control level within 3-7 days. Platelet counts decreased rapidly after starting rhGM-CSF, to 5000-15,000 platelets/mm3, and increased within 24 h after stopping rhGM-CSF treatment, whereas marrow histology after 2 weeks of rhGM-CSF application showed the normal number and morphology of megakaryocytes.

  7. Hydrogen-rich saline protects spermatogenesis and hematopoiesis in irradiated BALB/c mice

    PubMed Central

    Chuai, Yunhai; Shen, Jianliang; Qian, Liren; Wang, Yicun; Huang, Yuecheng; Gao, Fu; Cui, Jianguo; Ni, Jin; Zhao, Luqian; Liu, Shulin; Sun, Xuejun; Li, Bailong; Cai, Jianming

    2012-01-01

    Summary Background Recent studies show that molecular hydrogen (dihydrogen, H2) has potential as an effective and safe radioprotective agent through reducing oxidative stress. The aim of this study was to investigate whether H2 is able to protect spermatogenesis and hematopoiesis from radiation-induced injuries. Material/Methods H2 was dissolved in physiological saline using an apparatus produced by our department. 60Co-gamma rays in the irradiation centre were used for irradiation. Spermatid head counts and histological analysis were used to evaluate spermatogenesis. Endogenous hematopoietic spleen colony formation (endoCFUs), bone marrow nucleated cells (BMNC) and peripheral blood (PB) leukocytes were used to evaluate hemopoiesis. Results This study demonstrates that treating mice with H2 before ionizing radiation (IR) can increase the spermatid head count and protect seminiferous epithelium from IR. This study also demonstrates that H2 could significantly increase the number of endoCFUs, BMNC and PB leukocyte. Conclusions This study suggests that hydrogen-rich saline could partially protect spermatogenesis and hematopoiesis in irradiated mice. PMID:22367121

  8. TopBP1 Governs Hematopoietic Stem/Progenitor Cells Survival in Zebrafish Definitive Hematopoiesis

    PubMed Central

    Gao, Lei; Li, Dantong; Ma, Ke; Zhang, Wenjuan; Xu, Tao; Fu, Cong; Jing, Changbin; Jia, Xiaoe; Wu, Shuang; Sun, Xin; Dong, Mei; Deng, Min; Chen, Yi; Zhu, Wenge; Peng, Jinrong; Wan, Fengyi; Zhou, Yi; Zon, Leonard I.; Pan, Weijun

    2015-01-01

    In vertebrate definitive hematopoiesis, nascent hematopoietic stem/progenitor cells (HSPCs) migrate to and reside in proliferative hematopoietic microenvironment for transitory expansion. In this process, well-established DNA damage response pathways are vital to resolve the replication stress, which is deleterious for genome stability and cell survival. However, the detailed mechanism on the response and repair of the replication stress-induced DNA damage during hematopoietic progenitor expansion remains elusive. Here we report that a novel zebrafish mutantcas003 with nonsense mutation in topbp1 gene encoding topoisomerase II β binding protein 1 (TopBP1) exhibits severe definitive hematopoiesis failure. Homozygous topbp1cas003 mutants manifest reduced number of HSPCs during definitive hematopoietic cell expansion, without affecting the formation and migration of HSPCs. Moreover, HSPCs in the caudal hematopoietic tissue (an equivalent of the fetal liver in mammals) in topbp1cas003 mutant embryos are more sensitive to hydroxyurea (HU) treatment. Mechanistically, subcellular mislocalization of TopBP1cas003 protein results in ATR/Chk1 activation failure and DNA damage accumulation in HSPCs, and eventually induces the p53-dependent apoptosis of HSPCs. Collectively, this study demonstrates a novel and vital role of TopBP1 in the maintenance of HSPCs genome integrity and survival during hematopoietic progenitor expansion. PMID:26131719

  9. Hematopoiesis toxicity induced by 4-methylumbelliferon determined in an invertebrate model organism.

    PubMed

    Fang, Yan; He, Yue; Wang, Hua; Yan, Siqi; Xing, Rui; Yin, Weimin; Sima, Yanghu; Xu, Shiqing

    2016-01-01

    Umbelliferone has potential value as it has an inhibitory effect on tumor cells; however, its impact on an animal's circulatory system and hematopoietic function has not been reported. In this study, 4-methylumbelliferon (4-MU), an umbelliferone derivative, was used as a model drug, and its potential toxicity on hemocytes and hematopoietic organs (HOs) was investigated using an invertebrate animal model, the silkworm, Bombyx mori. The results showed that the level of reactive oxygen species in HOs increased when larvae (third day of the fifth instar) were orally exposed to 4 mM 4-MU for 8 min, followed by the induction of improved antioxidative metabolism of coenzymes in hemolymph. Exposure to 4-MU also significantly upregulated the expression levels of several genes in the hemolymph and fat body (a detoxification tissue similar to the liver in mammals) including antimicrobial peptide gene cecropinA and moricin, and a phagocytosis-related gene, tetraspanin E, suggesting an increased antioxidant level and antimicrobial ability of the circulatory system. However, the percentage of dead hemocytes increased and hematopoiesis significantly decreased in HOs, indicating the toxic effect of 4-MU on hemocytes and hematopoiesis, despite it inducing enhanced antioxidant and antimicrobial activity in the circulatory system.

  10. A Quantitative Proteomic Analysis of Hemogenic Endothelium Reveals Differential Regulation of Hematopoiesis by SOX17

    PubMed Central

    Clarke, Raedun L.; Robitaille, Aaron M.; Moon, Randall T.; Keller, Gordon

    2015-01-01

    Summary The in vitro derivation of hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) is complicated by the existence of multiple overlapping embryonic blood cell programs called primitive, erythromyeloid progenitor (EMP), and definitive. As HSCs are only generated during the definitive stage of hematopoiesis, deciphering the regulatory pathways that control the emergence of this program and identifying markers that distinguish it from the other programs are essential. To identify definitive specific pathways and marker sets, we used label-free proteomics to determine the proteome of embryo-derived and mouse embryonic stem cell-derived VE-CADHERIN+CD45− definitive hematopoietic progenitors. With this approach, we identified Stat1 as a marker that distinguishes the definitive erythroid lineage from the primitive- and EMP-derived lineages. Additionally, we provide evidence that the generation of the Stat1+ definitive lineage is dependent on Sox17. These findings establish an approach for monitoring the emergence of definitive hematopoiesis in the PSC differentiation cultures. PMID:26267830

  11. The Rothmund-Thomson syndrome helicase RECQL4 is essential for hematopoiesis

    PubMed Central

    Smeets, Monique F.; DeLuca, Elisabetta; Wall, Meaghan; Quach, Julie M.; Chalk, Alistair M.; Deans, Andrew J.; Heierhorst, Jörg; Purton, Louise E.; Izon, David J.; Walkley, Carl R.

    2014-01-01

    Mutations within the gene encoding the DNA helicase RECQL4 underlie the autosomal recessive cancer-predisposition disorder Rothmund-Thomson syndrome, though it is unclear how these mutations lead to disease. Here, we demonstrated that somatic deletion of Recql4 causes a rapid bone marrow failure in mice that involves cells from across the myeloid, lymphoid, and, most profoundly, erythroid lineages. Apoptosis was markedly elevated in multipotent progenitors lacking RECQL4 compared with WT cells. While the stem cell compartment was relatively spared in RECQL4-deficent mice, HSCs from these animals were not transplantable and even selected against. The requirement for RECQL4 was intrinsic in hematopoietic cells, and loss of RECQL4 in these cells was associated with increased replicative DNA damage and failed cell-cycle progression. Concurrent deletion of p53, which rescues loss of function in animals lacking the related helicase BLM, did not rescue BM phenotypes in RECQL4-deficient animals. In contrast, hematopoietic defects in cells from Recql4Δ/Δ mice were fully rescued by a RECQL4 variant without RecQ helicase activity, demonstrating that RECQL4 maintains hematopoiesis independently of helicase activity. Together, our data indicate that RECQL4 participates in DNA replication rather than genome stability and identify RECQL4 as a regulator of hematopoiesis with a nonredundant role compared with other RecQ helicases. PMID:24960165

  12. Perturbed hematopoiesis in the Tc1 mouse model of Down syndrome.

    PubMed

    Alford, Kate A; Slender, Amy; Vanes, Lesley; Li, Zhe; Fisher, Elizabeth M C; Nizetic, Dean; Orkin, Stuart H; Roberts, Irene; Tybulewicz, Victor L J

    2010-04-08

    Trisomy of human chromosome 21 (Hsa21) results in Down syndrome (DS), a disorder that affects many aspects of physiology, including hematopoiesis. DS children have greatly increased rates of acute lymphoblastic leukemia and acute megakaryoblastic leukemia (AMKL); DS newborns present with transient myeloproliferative disorder (TMD), a preleukemic form of AMKL. TMD and DS-AMKL almost always carry an acquired mutation in GATA1 resulting in exclusive synthesis of a truncated protein (GATA1s), suggesting that both trisomy 21 and GATA1 mutations are required for leukemogenesis. To gain further understanding of how Hsa21 contributes to hematopoietic abnormalities, we examined the Tc1 mouse model of DS, which carries an almost complete freely segregating copy of Hsa21, and is the most complete model of DS available. We show that although Tc1 mice do not develop leukemia, they have macrocytic anemia and increased extramedullary hematopoiesis. Introduction of GATA1s into Tc1 mice resulted in a synergistic increase in megakaryopoiesis, but did not result in leukemia or a TMD-like phenotype, demonstrating that GATA1s and trisomy of approximately 80% of Hsa21 perturb megakaryopoiesis but are insufficient to induce leukemia.

  13. TC1(C8orf4) regulates hematopoietic stem/progenitor cells and hematopoiesis.

    PubMed

    Jung, Yusun; Kim, Minsung; Soh, Hyunsu; Lee, Soyoung; Kim, Jungtae; Park, Surim; Song, Kyuyoung; Lee, Inchul

    2014-01-01

    Hematopoiesis is a complex process requiring multiple regulators for hematopoietic stem/progenitor cells (HSPC) and differentiation to multi-lineage blood cells. TC1(C8orf4) is implicated in cancers, hematological malignancies and inflammatory activation. Here, we report that Tc1 regulates hematopoiesis in mice. Myeloid and lymphoid cells are increased markedly in peripheral blood of Tc1-deleted mice compared to wild type controls. Red blood cells are small-sized but increased in number. The bone marrow of Tc1-/- mice is normocellular histologically. However, Lin-Sca-1+c-Kit+ (LSK) cells are expanded in Tc1-/- mice compared to wild type controls. The expanded population mostly consists of CD150-CD48+ cells, suggesting the expansion of lineage-restricted hematopoietic progenitor cells. Colony forming units (CFU) are increased in Tc1-/- mice bone marrow cells compared to controls. In wild type mice bone marrow, Tc1 is expressed in a limited population of HSPC but not in differentiated cells. Major myeloid transcriptional regulators such as Pu.1 and Cebpα are not up-regulated in Tc1-/- mice bone marrow. Our findings indicate that TC1 is a novel hematopoietic regulator. The mechanisms of TC1-dependent HSPC regulation and lineage determination are unknown.

  14. [Effect of zinc-intensified milk powder on hematopoiesis in mice].

    PubMed

    Wang, G; Li, S; Yuan, Y

    1999-12-01

    To observe the effect of Zn-intensified milk powder and zinc on hematopoiesis in mice. Kunming mice were randomly divided into four groups (n = 9 each) (control group; milk powder group; equal zinc group; high zinc group) and fed on experimental diet for 24 weeks. 1. In high zinc group: the mice hemoglobin (Hb) declined (P < 0.05) after 12 wk of supplementation and were significantly lower (P < 0.01) than those in control group at 16 wk. At 24 wk, the mice showed lower Hb, red blood cell(RBC), hematocrit (HCT), and mean corpuscular volume (MCV) compared with control group (P < 0.05). Bone marrow aspirates revealed erythroid hyperplasia. Zinc concentrations in serum, liver and kidney were higher (P < 0.01) and copper was lower than that of control group(P < 0.01). 2. At 16 wk, no change occurred in milk powder group and equal zinc group. At 24 wk, parameters changed in milk powder group similar to those in high zinc group, and in equal zinc group liver and kidney copper was lower than that in control group (P < 0.05). Ingestion of over-dosed zinc could induce anemia. Generally, intake of Zn-intensified milk powder is safe, but long-term intake of the milk powder may interfere with hematopoiesis.

  15. Effective treatment of a murine model of adult T-cell leukemia using 211At-7G7/B6 and its combination with unmodified anti-Tac (daclizumab) directed toward CD25

    PubMed Central

    Zhang, Zhuo; Zhang, Meili; Garmestani, Kayhan; Talanov, Vladimir S.; Plascjak, Paul S.; Beck, Barbara; Goldman, Carolyn; Brechbiel, Martin W.; Waldmann, Thomas A.

    2006-01-01

    Adult T-cell leukemia (ATL) consists of an overabundance of T cells, which express CD25. Therapeutic efficacy of astatine-211 (211At)–labeled murine monoclonal antibody 7G7/B6 alone and in combination with daclizumab was evaluated in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice given injections of MET-1 human T-cell leukemia cells. Daclizumab and 7G7/B6 are directed toward different epitopes of CD25. Either a single dose of 12 μCi (0.444 MBq) 211At-7G7/B6 per mouse given intravenously or receptor-saturating doses of daclizumab given at 100 μg weekly for 4 weeks intravenously inhibited tumor growth as monitored by serum levels of human β-2 microglobulin (β2μ) and by prolonged survival of leukemia-bearing mice compared with the control groups (P < .001). The combination of 2 agents enhanced the antitumor effect when compared with groups treated with 12 μCi (0.444 MBq) of 211At-7G7/B6 (P < .05) or daclizumab alone (P < .05). The median survival duration of the PBS group was 62.6 days and 61.5 days in the radiolabeled nonspecific antibody 211At-11F11–treated group. In contrast, 91% of mice in the combination group survived through day 94. These results that demonstrate a significantly improved therapeutic efficacy by combining 211At-7G7/B6 with daclizumab support a clinical trial of this regimen in patients with ATL. PMID:16569769

  16. Sense and antisense transcripts of the developmentally regulated murine hsp70.2 gene are expressed in distinct and only partially overlapping areas in the adult brain

    NASA Technical Reports Server (NTRS)

    Murashov, A. K.; Wolgemuth, D. J.

    1996-01-01

    We have examined the spatial pattern of expression of a member of the hsp70 gene family, hsp70.2, in the mouse central nervous system. Surprisingly, RNA blot analysis and in situ hybridization revealed abundant expression of an 'antisense' hsp70.2 transcript in several areas of adult mouse brain. Two different transcripts recognized by sense and antisense riboprobes for the hsp70.2 gene were expressed in distinct and only partially overlapping neuronal populations. RNA blot analysis revealed low levels of the 2.7 kb transcript of hsp70.2 in several areas of the brain, with highest signal in the hippocampus. Abundant expression of a slightly larger (approximately 2.8 kb) 'antisense' transcript was detected in several brain regions, notably in the brainstem, cerebellum, mesencephalic tectum, thalamus, cortex, and hippocampus. In situ hybridization revealed that the sense and antisense transcripts were both predominantly neuronal and localized to the same cell types in the granular layer of the cerebellum, trapezoid nucleus of the superior olivary complex, locus coeruleus and hippocampus. The hsp70.2 antisense transcripts were particularly abundant in the frontal cortex, dentate gyrus, subthalamic nucleus, zona incerta, superior and inferior colliculi, central gray, brainstem, and cerebellar Purkinje cells. Our findings have revealed a distinct cellular and spatial localization of both sense and antisense transcripts, demonstrating a new level of complexity in the function of the heat shock genes.

  17. Sense and antisense transcripts of the developmentally regulated murine hsp70.2 gene are expressed in distinct and only partially overlapping areas in the adult brain

    NASA Technical Reports Server (NTRS)

    Murashov, A. K.; Wolgemuth, D. J.

    1996-01-01

    We have examined the spatial pattern of expression of a member of the hsp70 gene family, hsp70.2, in the mouse central nervous system. Surprisingly, RNA blot analysis and in situ hybridization revealed abundant expression of an 'antisense' hsp70.2 transcript in several areas of adult mouse brain. Two different transcripts recognized by sense and antisense riboprobes for the hsp70.2 gene were expressed in distinct and only partially overlapping neuronal populations. RNA blot analysis revealed low levels of the 2.7 kb transcript of hsp70.2 in several areas of the brain, with highest signal in the hippocampus. Abundant expression of a slightly larger (approximately 2.8 kb) 'antisense' transcript was detected in several brain regions, notably in the brainstem, cerebellum, mesencephalic tectum, thalamus, cortex, and hippocampus. In situ hybridization revealed that the sense and antisense transcripts were both predominantly neuronal and localized to the same cell types in the granular layer of the cerebellum, trapezoid nucleus of the superior olivary complex, locus coeruleus and hippocampus. The hsp70.2 antisense transcripts were particularly abundant in the frontal cortex, dentate gyrus, subthalamic nucleus, zona incerta, superior and inferior colliculi, central gray, brainstem, and cerebellar Purkinje cells. Our findings have revealed a distinct cellular and spatial localization of both sense and antisense transcripts, demonstrating a new level of complexity in the function of the heat shock genes.

  18. Brain-Derived Neurotrophic Factor Induces Cell Survival and the Migration of Murine Adult Hippocampal Precursor Cells During Differentiation In Vitro.

    PubMed

    Ortiz-López, Leonardo; Vega-Rivera, Nelly Maritza; Babu, Harish; Ramírez-Rodríguez, Gerardo Bernabé

    2017-01-01

    The generation of new neurons during adulthood involves local precursor cell migration and terminal differentiation in the dentate gyrus. These events are influenced by the hippocampal microenvironment. Brain-derived neurotrophic factor (BDNF) is relevant for hippocampal neuronal development and behavior. Interestingly, studies that have been performed in controlled in vitro systems that involve isolated precursor cells that were derived from the dentate gyrus (AHPCs) have shown that BDNF induces the activation of the TrkB receptor and, consequentially, might activate signaling pathways that favor survival and neuronal differentiation. Based on the fact that the cellular events of AHPCs that are induced by single factors can be studied in this controlled in vitro system, we investigated the ability of BDNF and the involvement of protein kinase C (PKC), as one of the TrkB-downstream activated signaling proteins, in the regulation of migration, here reflected by motility, of AHPCs. Precursor cells were cultured following a concentration-response curve (1-640 ng/ml) for 24 or 96 h. We found that BDNF favored cell survival without altering the viability under culture proliferative conditions of the AHPCs. Concomitantly, glial- and neuronal-differentiated precursor cells increased as a consequence of survival promoted by BDNF. Additionally, pharmacological approaches showed that BDNF (40 ng/ml)-induced migration of AHPCs was blocked with the compounds K252a and GF109203x, which prevent the activation of TrkB and PKC, respectively. The results indicate that in the in vitro migration of differentiated AHPCs it is involved the BDNF and TrkB cascade. Our results provide additional information about the mechanism by which BDNF impacts adult neurogenesis in the hippocampus.

  19. Hematopoietic Stem/Progenitor Cells Express Several Functional Sex Hormone Receptors—Novel Evidence for a Potential Developmental Link Between Hematopoiesis and Primordial Germ Cells

    PubMed Central

    Mierzejewska, Katarzyna; Borkowska, Sylwia; Suszynska, Ewa; Suszynska, Malwina; Poniewierska-Baran, Agata; Maj, Magda; Pedziwiatr, Daniel; Adamiak, Mateusz; Abdel-Latif, Ahmed; Kakar, Sham S.; Ratajczak, Janina; Kucia, Magda

    2015-01-01

    Evidence has accumulated that hematopoietic stem progenitor cells (HSPCs) share several markers with the germline, a connection supported by reports that prolactin, androgens, and estrogens stimulate hematopoiesis. To address this issue more directly, we tested the expression of receptors for pituitary-derived hormones, such as follicle-stimulating hormone (FSH) and luteinizing hormone (LH), on purified murine bone marrow (BM) cells enriched for HSPCs and tested the functionality of these receptors in ex vivo signal transduction studies and in vitro clonogenic assays. We also tested whether administration of pituitary- and gonad-derived sex hormones (SexHs) increases incorporation of bromodeoxyuridine (BrdU) into HSPCs and expansion of hematopoietic clonogenic progenitors in mice and promotes recovery of blood counts in sublethally irradiated animals. We report for the first time that HSPCs express functional FSH and LH receptors and that both proliferate in vivo and in vitro in response to stimulation by pituitary SexHs. Furthermore, based on our observations that at least some of CD45− very small embryonic-like stem cells (VSELs) may become specified into CD45+ HSPCs, we also evaluated the expression of pituitary and gonadal SexHs receptors on these cells and tested whether these quiescent cells may expand in vivo in response to SexHs administration. We found that VSELs express SexHs receptors and respond in vivo to SexHs stimulation, as evidenced by BrdU accumulation. Since at least some VSELs share several markers characteristic of migrating primordial germ cells and can be specified into HSPCs, this observation sheds new light on the BM stem cell hierarchy. PMID:25607657

  20. Lentiviral-mediated RNAi inhibition of Sbds in murine hematopoietic progenitors impairs their hematopoietic potential

    PubMed Central

    Rawls, Amy S.; Gregory, Alyssa D.; Woloszynek, Jill R.; Liu, Fulu

    2007-01-01

    Shwachman-Diamond syndrome (SDS) is a rare multisystem disorder characterized by exocrine pancreatic insufficiency, multilineage hematopoietic dysfunction, and metaphyseal chondrodysplasia. Bone marrow dysfunction is present in nearly all patients with SDS, with neutropenia being the most common abnormality. The majority of patients with SDS have mutations in the Shwachman Bodian Diamond syndrome (SBDS) gene. We have developed a strategy to examine the consequences of lentiviral-mediated RNA interference (RNAi) of Sbds on hematopoiesis. Here, we report that both Sbds RNA and protein expression can be efficiently inhibited in primary murine hematopoietic cells using lentiviral-mediated RNAi. Inhibition of Sbds results in a defect in granulocytic differentiation in vitro and impairs myeloid progenitor generation in vivo. In addition, short-term hematopoietic engraftment was impaired, which is due in part to reduced homing of hematopoietic progenitors to the bone marrow. Finally, we show that inhibition of Sbds is associated with a decrease in circulating B lymphocytes, despite evidence of normal B lymphopoiesis. These data provide the first evidence that loss of Sbds is sufficient to induce abnormalities in hematopoiesis. PMID:17638857

  1. Ectopic extramedullary hematopoiesis: evaluation and treatment of a rare and benign paraspinal/epidural tumor.

    PubMed

    Mattei, Tobias A; Higgins, Michael; Joseph, Flynn; Mendel, Ehud

    2013-03-01

    Ectopic extramedullary hematopoiesis (EMH), defined as the formation of blood cells outside the bone marrow, usually occurs in a scenario of chronic anemia when, even after conversion of the bony yellow marrow to red marrow, the body is still unable to meet the demand for red blood cells. Ectopic extramedullary hematopoiesis most commonly occurs in the liver and spleen but may, in fact, occur almost anywhere in the body. Although previous reports have documented EMH presenting as paraspinal masses, such lesions have almost always been associated with a predisposing hematological disorder such as hemolytic anemia, myelofibrosis or myelodysplastic syndromes, thalassemia, polycythemia vera, leukemia, or lymphoma. The authors of this report describe the first reported instance of EMH in a patient presenting with a symptomatic epidural and paraspinal cervical lesion arising from the posterior spinal elements and no known predisposing hematological disease. Initial radiographs revealed a bony lesion arising posteriorly from the C2-3 laminae and spinous processes. Subsequent imaging suggested the diagnosis, which was confirmed by CT-guided biopsy, peripheral blood smears, and bone marrow aspirate. Despite epidural compression and slight displacement of the cervical cord and thecal sac, the patient's symptoms were limited to pain and diminished cervical range of motion. Therefore, surgery was deferred in favor of nonsurgical therapy. Several alternative modalities for the treatment of EMH have been suggested in the literature, including cytotoxic agents and radiotherapy. The authors opted for an approach utilizing directed low-dose radiotherapy of a total of 25 Gy divided in 2.5-Gy fractions. At the 3-month follow-up, the patient continued to be asymptomatic, and MRI demonstrated a significant reduction in the dimensions of the lesion. Extramedullary hematopoiesis with spinal cord compression in the absence of a preexisting hematological disorder has not been described in

  2. Developmentally regulated promoter-switch transcriptionally controls Runx1 function during embryonic hematopoiesis

    PubMed Central

    Pozner, Amir; Lotem, Joseph; Xiao, Cuiying; Goldenberg, Dalia; Brenner, Ori; Negreanu, Varda; Levanon, Ditsa; Groner, Yoram

    2007-01-01

    Background Alternative promoters usage is an important paradigm in transcriptional control of mammalian gene expression. However, despite the growing interest in alternative promoters and their role in genome diversification, very little is known about how and on what occasions those promoters are differentially regulated. Runx1 transcription factor is a key regulator of early hematopoiesis and a frequent target of chromosomal translocations in acute leukemias. Mice deficient in Runx1 lack definitive hematopoiesis and die in mid-gestation. Expression of Runx1 is regulated by two functionally distinct promoters designated P1 and P2. Differential usage of these two promoters creates diversity in distribution and protein-coding potential of the mRNA transcripts. While the alternative usage of P1 and P2 likely plays an important role in Runx1 biology, very little is known about the function of the P1/P2 switch in mediating tissue and stage specific expression of Runx1 during development. Results We employed mice bearing a hypomorphic Runx1 allele, with a largely diminished P2 activity, to investigate the biological role of alternative P1/P2 usage. Mice homozygous for the hypomorphic allele developed to term, but died within a few days after birth. During embryogenesis the P1/P2 activity is spatially and temporally modulated. P2 activity is required in early hematopoiesis and when attenuated, development of liver hematopoietic progenitor cells (HPC) was impaired. Early thymus development and thymopoiesis were also abrogated as reflected by thymic hypocellularity and loss of corticomedullary demarcation. Differentiation of CD4/CD8 thymocytes was impaired and their apoptosis was enhanced due to altered expression of T-cell receptors. Conclusion The data delineate the activity of P1 and P2 in embryogenesis and describe previously unknown functions of Runx1. The findings show unequivocally that the role of P1/P2 during development is non redundant and underscore the

  3. Single-Cell Network Analysis Identifies DDIT3 as a Nodal Lineage Regulator in Hematopoiesis.

    PubMed

    Pina, Cristina; Teles, José; Fugazza, Cristina; May, Gillian; Wang, Dapeng; Guo, Yanping; Soneji, Shamit; Brown, John; Edén, Patrik; Ohlsson, Mattias; Peterson, Carsten; Enver, Tariq

    2015-06-16

    We explore cell heterogeneity during spontaneous and transcription-factor-driven commitment for network inference in hematopoiesis. Since individual genes display discrete OFF states or a distribution of ON levels, we compute and combine pairwise gene associations from binary and continuous components of gene expression in single cells. Ddit3 emerges as a regulatory node with positive linkage to erythroid regulators and negative association with myeloid determinants. Ddit3 loss impairs erythroid colony output from multipotent cells, while forcing Ddit3 in granulo-monocytic progenitors (GMPs) enhances self-renewal and impedes differentiation. Network analysis of Ddit3-transduced GMPs reveals uncoupling of myeloid networks and strengthening of erythroid linkages. RNA sequencing suggests that Ddit3 acts through development or stabilization of a precursor upstream of GMPs with inherent Meg-E potential. The enrichment of Gata2 target genes in Ddit3-dependent transcriptional responses suggests that Ddit3 functions in an erythroid transcriptional network nucleated by Gata2.

  4. Mitotic History Reveals Distinct Stem Cell Populations and Their Contributions to Hematopoiesis.

    PubMed

    Säwén, Petter; Lang, Stefan; Mandal, Pankaj; Rossi, Derrick J; Soneji, Shamit; Bryder, David

    2016-03-29

    Homeostasis of short-lived blood cells is dependent on rapid proliferation of immature precursors. Using a conditional histone 2B-mCherry-labeling mouse model, we characterize hematopoietic stem cell (HSC) and progenitor proliferation dynamics in steady state and following several types of induced stress. HSC proliferation following HSC transplantation into lethally irradiated mice is fundamentally different not only from native hematopoiesis but also from other stress contexts. Whereas transplantation promoted sustained, long-term proliferation of HSCs, both cytokine-induced mobilization and acute depletion of selected blood cell lineages elicited very limited recruitment of HSCs to the proliferative pool. By coupling mCherry-based analysis of proliferation history with multiplex gene expression analyses on single cells, we have found that HSCs can be stratified into four distinct subtypes. These subtypes have distinct molecular signatures and differ significantly in their reconstitution potentials, showcasing the power of tracking proliferation history when resolving functional heterogeneity of HSCs.

  5. Effect of vitamin A supplementation on hematopoiesis in children with anemia.

    PubMed

    Garg, Arun; Abrol, Pankaj; Tewari, Ad; Sen, Rajiv; Lal, Harbans

    2005-01-01

    Fifty children (1-4 years age) presenting with microcytic hypochromic anemia (hemoglobin less than 10g/dl) were studied in two groups of 25 each. Group I was supplemented with iron (ferrous sulphate 6 mg/kg/d) while group II in addition to iron was also supplemented with vitamin A (5000 IU/d). Hemoglobin concentration was found to be significantly increased after 4 weeks of iron supplementation. Rise in hemoglobin was comparatively more in-group II, as compared to group I, after 8 and 12 weeks. Serum iron was significantly higher after 4 weeks in both the groups. Packed cell volume (PCV) and retinol levels increased significantly in-group II only. The data suggests that supplementation of vitamin A improves hematopoiesis.

  6. Postnatal Hematopoiesis and Gut Microbiota in NOD Mice Deviate from C57BL/6 Mice

    PubMed Central

    Damlund, Dina Silke Malling; Metzdorff, Stine Broeng; Hasselby, Jane Preuss; Wiese, Maria; Lundsager, Mia; Buschard, Karsten Stig; Hansen, Axel Kornerup; Frøkiær, Hanne

    2016-01-01

    Neonatal studies in different mouse strains reveal that early life colonization affects the development of adaptive immunity in mice. The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes, but neonatal studies of NOD mice are lacking. We hypothesized that NOD mice deviate from another much used mouse strain, C57BL/6, with respect to postnatal microbiota and/or hematopoiesis and compared this in newborn mice of dams housed under the same conditions. A distinct bacteria profile rich in staphylococci was found at postnatal days (PND) 1–4 in NOD mice. Furthermore, a distinct splenic cell profile high in a granulocytic phenotype was evident in the neonatal NOD mice whereas neonatal C57BL/6 mice showed a profile rich in monocytes. Neonatal expression of Reg3g and Muc2 in the gut was deviating in NOD mice and coincided with fewer bacteria attaching to the Mucosal surface in NOD compared to C57BL/6 mice. PMID:26783537

  7. Concise review: Exploring miRNAs--toward a better understanding of hematopoiesis.

    PubMed

    Hong, Seok-Ho; Kim, Kye-Seong; Oh, Il-Hoan

    2015-01-01

    Hematopoiesis is governed by a multidimensional regulatory network involving both intrinsic and extrinsic factors that control self-renewal and differentiation of hematopoietic stem cells (HSCs) through the coordination of influences that affect cell fate. Increasing evidence indicates that microRNAs (miRNAs), short noncoding RNAs of approximately 22 nucleotides, play a central role in orchestrating these regulatory mechanisms to modulate the multiple entities of hematopoietic function in a cell-type specific manner, including self-renewal, lineage commitment, and survival of HSCs as well as their microenvironmental crosstalk. Here, we summarize the current understanding regarding the regulatory effects of miRNA on hematopoietic cells, thus enlightening their role in fine-tuning HSC function and hematopoietic homeostasis. © 2014 AlphaMed Press.

  8. Spinal Cord Compression Secondary to Extramedullary Hematopoiesis: Case Report and Review of the Literature

    PubMed Central

    Wang, Arthur; Carberry, Nathan; Solli, Elena; Gillick, John; Islam, Humayun; Hillard, Virany

    2016-01-01

    Extramedullary hematopoiesis (EMH) is a rare cause of spinal cord compression (SCC). EMH represents the growth of blood cells outside of the bone marrow and occurs in a variety of hematologic illnesses, including various types of anemia and myeloproliferative disorders. Although EMH usually occurs in the liver, spleen, and lymph nodes, it may also occur within the spinal canal. When this occurs, the mass effect can compress the spinal cord, potentially leading to the development of neurological deficits. We present a case of SCC secondary to EMH. This report illustrates the importance of considering EMH in the differential diagnosis of SCC, even in the absence of signs of its most common etiologies. PMID:27462228

  9. The genetics of myelodysplastic syndrome: from clonal hematopoiesis to secondary leukemia

    PubMed Central

    Sperling, Adam S.; Gibson, Christopher J.; Ebert, Benjamin L.

    2017-01-01

    Myelodysplastic syndrome (MDS) is a clonal disease that arises from the expansion of mutated hematopoietic stem cells. In a spectrum of myeloid disorders ranging from clonal hematopoiesis of indeterminate potential (CHIP) to secondary acute myeloid leukemia (sAML), MDS is distinguished by the presence of peripheral blood cytopenias, dysplastic hematopoietic differentiation, and the absence of features that define acute leukemia. Over 50 recurrently mutated genes are involved in the pathogenesis of MDS, including genes that encode proteins involved in pre-mRNA splicing, epigenetic regulation, and transcription. In this review we discuss the molecular processes that lead to CHIP and further clonal evolution to MDS and sAML. We also highlight the ways in which these insights are shaping the clinical management of MDS, including classification schemata, prognostic scoring systems, and therapeutic approaches. PMID:27834397

  10. [Research Progress on Muscle-derived Stem Cells Capable of Hematopoiesis].

    PubMed

    Chen, Yu-Fang; Wang, Yuan-Yuan; Wang, Juan-Juan; Gao, Xiao-Ning; Wang, Xiao-Ling; Zhao, Shu-Wu; Wang, Tao; Dou, Hao-Ying

    2015-10-01

    Muscle-derived stem cells (MDSC) are defined as myogenic stem cells endowed with their ability to self-renew and differentiate into multiple cell types of their derivative tissue, and are proved to be over 10 times more efficient in hematopoiesis than hematopoietic stem cells (HSC). Although the mechanism which MDSC differentiate into blood cells is still unclear, MDSC were considered to replace HSC to treat the patients suffering from bone marrow diseases such as aplastic anemia and tumor. MDSC are different from HSC in a variety aspects like biological characteristics, protein expression and cell proliferation. On the other hand, MDSC contain multiple distinct stem cell populations. Among these, there is only a small part with the ability to repopulate hematopoietic cells, and it is still uncertain whether their origin is same as HSC. This review summarizes the difference between MDSC and HSC, the ability of MDSC to repopulate hematopoietic cells, and the prospect of MDSCs' transplantation.

  11. The emerging role of MIR-146A in the control of hematopoiesis, immune function and cancer

    PubMed Central

    2012-01-01

    MicroRNA (miRs) represent a class of small non-coding regulatory RNAs playing a major role in the control of gene expression by repressing protein synthesis at the post-transcriptional level. Studies carried out during the last years have shown that some miRNAs plays a key role in the control of normal and malignant hgematopoiesis. In this review we focus on recent progress in analyzing the functional role of miR-146a in the control of normal and malignant hematopoiesis. On the other hand, this miRNA has shown to impact in the control of innate immune responses. Finally, many recent studies indicate a deregulation of miR-146 in many solid tumors and gene knockout studies indicate a role for this miRNA as a tumor suppressor. PMID:22453030

  12. Oncomir miR-125b regulates hematopoiesis by targeting the gene Lin28A.

    PubMed

    Chaudhuri, Aadel A; So, Alex Yick-Lun; Mehta, Arnav; Minisandram, Aarathi; Sinha, Nikita; Jonsson, Vanessa D; Rao, Dinesh S; O'Connell, Ryan M; Baltimore, David

    2012-03-13

    MicroRNA-125b (miR-125b) is up-regulated in patients with leukemia. Overexpression of miR-125b alone in mice causes a very aggressive, transplantable myeloid leukemia. Before leukemia, these mice do not display elevation of white blood cells in the spleen or bone marrow; rather, the hematopoietic compartment shows lineage-skewing, with myeloid cell numbers dramatically increased and B-cell numbers severely diminished. miR-125b exerts this effect by up-regulating the number of common myeloid progenitors while inhibiting development of pre-B cells. We applied a miR-125b sponge loss of function system in vivo to show that miR-125b physiologically regulates hematopoietic development. Investigating the mechanism by which miR-125b regulates hematopoiesis, we found that, among a panel of candidate targets, the mRNA for Lin28A, an induced pluripotent stem cell gene, was most repressed by miR-125b in mouse hematopoietic stem and progenitor cells. Overexpressing Lin28A in the mouse hematopoietic system mimicked the phenotype observed on inhibiting miR-125b function, leading to a decrease in hematopoietic output. Relevant to the miR-125b overexpression phenotype, we also found that knockdown of Lin28A led to hematopoietic lineage-skewing, with increased myeloid and decreased B-cell numbers. Thus, the miR-125b target Lin28A is an important regulator of hematopoiesis and a primary target of miR-125b in the hematopoietic system.

  13. Oncomir miR-125b regulates hematopoiesis by targeting the gene Lin28A

    PubMed Central

    Chaudhuri, Aadel A.; So, Alex Yick-Lun; Mehta, Arnav; Minisandram, Aarathi; Sinha, Nikita; Jonsson, Vanessa D.; Rao, Dinesh S.; O'Connell, Ryan M.; Baltimore, David

    2012-01-01

    MicroRNA-125b (miR-125b) is up-regulated in patients with leukemia. Overexpression of miR-125b alone in mice causes a very aggressive, transplantable myeloid leukemia. Before leukemia, these mice do not display elevation of white blood cells in the spleen or bone marrow; rather, the hematopoietic compartment shows lineage-skewing, with myeloid cell numbers dramatically increased and B-cell numbers severely diminished. miR-125b exerts this effect by up-regulating the number of common myeloid progenitors while inhibiting development of pre-B cells. We applied a miR-125b sponge loss of function system in vivo to show that miR-125b physiologically regulates hematopoietic development. Investigating the mechanism by which miR-125b regulates hematopoiesis, we found that, among a panel of candidate targets, the mRNA for Lin28A, an induced pluripotent stem cell gene, was most repressed by miR-125b in mouse hematopoietic stem and progenitor cells. Overexpressing Lin28A in the mouse hematopoietic system mimicked the phenotype observed on inhibiting miR-125b function, leading to a decrease in hematopoietic output. Relevant to the miR-125b overexpression phenotype, we also found that knockdown of Lin28A led to hematopoietic lineage-skewing, with increased myeloid and decreased B-cell numbers. Thus, the miR-125b target Lin28A is an important regulator of hematopoiesis and a primary target of miR-125b in the hematopoietic system. PMID:22366319

  14. Expression of the erythropoietin receptor by germline-derived cells - further support for a potential developmental link between the germline and hematopoiesis

    PubMed Central

    2014-01-01

    Background Expressing several markers of migrating primordial germ cells (PGCs), the rare population of quiescent, bone marrow (BM)-residing very small embryonic-like stem cells (VSELs) can be specified like PGCs into hematopoietic stem/progenitor cells (HSPCs). These two properties of VSELs support the possibility of a developmental origin of HSPCs from migrating PGCs. Methods To address a potential link between VSELs and germ line cells we analyzed by RT-PCR and FACS expression of erythropoietin receptor (EpoR) on murine bone marrow- and human umbilical cord blood-derived VSELs, murine and human teratocarcinoma cell lines and human ovarian cancer cells. A proper gating strategy and immunostaining excluded from FACS analysis potential contamination by erythroblasts. Furthermore, the transwell chemotaxis assays as well as adhesion and signaling studies were performed to demonstrate functionality of erythropoietin - EpoR axes on these cells. Results We report here that murine and human VSELs as well as murine and human teratocarcinoma cell lines and ovarian cancer cell lines share a functional EpoR. Conclusions Our data provide more evidence of a potential developmental link between germline cells, VSELs, and HSCs and sheds more light on the developmental hierarchy of the stem cell compartment in adult tissues. PMID:24982693

  15. The transcriptional regulation of the Colony-Stimulating Factor 1 Receptor (csf1r) gene during hematopoiesis.

    PubMed

    Bonifer, Constanze; Hume, David A

    2008-01-01

    The colony-stimulating-factor 1 receptor (CSF-1 R) is a tyrosine kinase receptor that is absolutely required for macrophage differentiation and thus occupies a central role in hematopoiesis. Mice deficient for the csf1r gene show multiple defects in macrophage development, reproduction and tissue remodeling. Moreover, deregulation of this gene is a hallmark of many tumors. This includes repression of expression in acute myeloid leukemia and aberrant activation in certain solid tumors, such as breast cancer. Expression of this gene therefore needs to be tightly controlled. This review summarizes experiments providing a detailed picture of how transcription of csf1r gene expression is regulated. Aside from the direct relevance to hematopoiesis, studies of csf1r transcriptional regulation provide a model for understanding the molecular mechanisms that control mammalian cell fate.

  16. Cocaine exposure impairs multilineage hematopoiesis of human hematopoietic progenitor cells mediated by the sigma-1 receptor [corrected].

    PubMed

    Nixon, Christopher C; Schwartz, Brandon H; Dixit, Dhaval; Zack, Jerome A; Vatakis, Dimitrios N

    2015-03-02

    Prenatal exposure to cocaine is a significant source of fetal and neonatal developmental defects. While cocaine associated neurological and cardiac pathologies are well-documented, it is apparent that cocaine use has far more diverse physiological effects. It is known that in some cell types, the sigma-1 receptor mediates many of cocaine's cellular effects. Here we present a novel and concise investigation into the mechanism that underlies cocaine associated hematopoietic pathology. Indeed, this is the first examination of the effects of cocaine on hematopoiesis. We show that cocaine impairs multilineage hematopoiesis from human progenitors from multiple donors and tissue types. We go on to present the first demonstration of the expression of the sigma-1 receptor in human CD34 + human hematopoietic stem/progenitor cells. Furthermore, we demonstrate that these cocaine-induced hematopoietic defects can be reversed through sigma-1 receptor blockade.

  17. Parallels between immune driven-hematopoiesis and T cell activation: 3 signals that relay inflammatory stress to the bone marrow

    SciTech Connect

    Libregts, Sten F.W.M.; Nolte, Martijn A.

    2014-12-10

    Quiescence, self-renewal, lineage commitment and differentiation of hematopoietic stem cells (HSCs) towards fully mature blood cells are a complex process that involves both intrinsic and extrinsic signals. During steady-state conditions, most hematopoietic signals are provided by various resident cells inside the bone marrow (BM), which establish the HSC micro-environment. However, upon infection, the hematopoietic process is also affected by pathogens and activated immune cells, which illustrates an effective feedback mechanism to hematopoietic stem and progenitor cells (HSPCs) via immune-mediated signals. Here, we review the impact of pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), costimulatory molecules and pro-inflammatory cytokines on the quiescence, proliferation and differentiation of HSCs and more committed progenitors. As modulation of HSPC function via these immune-mediated signals holds an interesting parallel with the “three-signal-model” described for the activation and differentiation of naïve T-cells, we propose a novel “three-signal” concept for immune-driven hematopoiesis. In this model, the recognition of PAMPs and DAMPs will activate HSCs and induce proliferation, while costimulatory molecules and pro-inflammatory cytokines confer a second and third signal, respectively, which further regulate expansion, lineage commitment and differentiation of HSPCs. We review the impact of inflammatory stress on hematopoiesis along these three signals and we discuss whether they act independently from each other or that concurrence of these signals is important for an adequate response of HSPCs upon infection. - Highlights: • Inflammation and infection have a direct impact on hematopoiesis in the bone marrow. • We draw a striking parallel between immune-driven hematopoiesis and T cell activation. • We review how PAMPs and DAMPs, costimulation and cytokines influence HSPC function.

  18. Modulation of hematopoiesis via alpha 1-adrenergic receptors on bone marrow cells.

    PubMed

    Maestroni, G J; Conti, A

    1994-03-01

    We have recently demonstrated that adrenergic agents can affect hematopoiesis after syngeneic bone marrow transplantation in mice. In particular, chemical sympathectomy by 6-hydroxydopamine (6-OHDA) and/or administration of the alpha 1-adrenergic antagonist prazosin were shown to increase the concentration of blood granulocytes, platelets, and bone marrow colony-forming units-granulocyte/macrophage (CFU-GM), and to induce a granulocytic hyperplasia of the spleen. Here we show that prazosin can also enhance myelopoiesis and platelet formation in normal mice. Furthermore, noradrenaline and the alpha 1-adrenergic agonist methoxamine could directly inhibit the in vitro growth of GM-CFU. The effect of noradrenaline was counteracted by prazosin and by other alpha-adrenergic antagonists such as phentolamine and yohimbine, in the following order of potency: prazosin > phentolamine > yohimbine. In line with these results, we were able to demonstrate that 3H-prazosin binds specifically to both bone marrow cell membranes and intact bone marrow cells. Scatchard analysis of the binding to intact cells revealed the presence of two binding sites. A kd of 0.98 +/- 0.32 nM and a B max of 5 +/- 2.9 fM/2 x 10(6) cells characterized the higher affinity site, while the lower affinity site displayed a kd of 55.9 +/- 8.2 nM and a B max of 44 +/- 7.7 fM/mg protein. These saturation studies, together with competition experiments to evaluate the ability of various adrenergic compounds to displace 3H-prazosin binding, classified the higher affinity site as an alpha 1-adrenergic receptor. The remaining low affinity binding site remains to be characterized. Furthermore, separation of bone marrow cells by counterflow centrifugal elutriation (CCE) showed that the high-affinity binding is due to a lymphoid/stem cell fraction with no blasts and no GM-CFU progenitors. The low-affinity site was apparent on the rotor-off fraction, which was enriched with GM-CFU progenitor cells. These findings

  19. Impact of Viral Infections on Hematopoiesis: From Beneficial to Detrimental Effects on Bone Marrow Output

    PubMed Central

    Pascutti, Maria Fernanda; Erkelens, Martje N.; Nolte, Martijn A.

    2016-01-01

    The ability of the bone marrow (BM) to generate copious amounts of blood cells required on a daily basis depends on a highly orchestrated process of proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs). This process can be rapidly adapted under stress conditions, such as infections, to meet the specific cellular needs of the immune response and the ensuing physiological changes. This requires a tight regulation in order to prevent either hematopoietic failure or transformation. Although adaptation to bacterial infections or systemic inflammation has been studied and reviewed in depth, specific alterations of hematopoiesis to viral infections have received less attention so far. Viruses constantly pose a significant health risk and demand an adequate, balanced response from our immune system, which also affects the BM. In fact, both the virus itself and the ensuing immune response can have a tremendous impact on the hematopoietic process. On one hand, this can be beneficial: it helps to boost the cellular response of the body to resolve the viral infection. But on the other hand, when the virus and the resulting antiviral response persist, the inflammatory feedback to the hematopoietic system will become chronic, which can be detrimental for a balanced BM output. Chronic viral infections frequently have clinical manifestations at the level of blood cell formation, and we summarize which viruses can lead to BM pathologies, like aplastic anemia, pancytopenia, hemophagocytic lymphohistiocytosis, lymphoproliferative disorders, and malignancies. Regarding the underlying mechanisms, we address specific effects of acute and chronic viral infections on blood cell production. As such, we distinguish four different levels in which this can occur: (1) direct viral infection of HSPCs, (2) viral recognition by HSPCs, (3) indirect effects on HSPCs by inflammatory mediators, and (4) the role of the BM microenvironment on hematopoiesis upon virus

  20. Germ line variants predispose to both JAK2 V617F clonal hematopoiesis and myeloproliferative neoplasms

    PubMed Central

    Hinds, David A.; Barnholt, Kimberly E.; Mesa, Ruben A.; Kiefer, Amy K.; Do, Chuong B.; Eriksson, Nicholas; Mountain, Joanna L.; Francke, Uta; Tung, Joyce Y.; Nguyen, Huong (Marie); Zhang, Haiyu; Gojenola, Linda; Zehnder, James L.

    2016-01-01

    We conducted a genome-wide association study (GWAS) to identify novel predisposition alleles associated with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) and JAK2 V617F clonal hematopoiesis in the general population. We recruited a web-based cohort of 726 individuals with polycythemia vera, essential thrombocythemia, and myelofibrosis and 252 637 population controls unselected for hematologic phenotypes. Using a single-nucleotide polymorphism (SNP) array platform with custom probes for the JAK2 V617F mutation (V617F), we identified 497 individuals (0.2%) among the population controls who were V617F carriers. We performed a combined GWAS of the MPN cases plus V617F carriers in the control population (n = 1223) vs the remaining controls who were noncarriers for V617F (n = 252 140). For these MPN cases plus V617F carriers, we replicated the germ line JAK2 46/1 haplotype (rs59384377: odds ratio [OR] = 2.4, P = 6.6 × 10−89), previously associated with V617F-positive MPN. We also identified genome-wide significant associations in the TERT gene (rs7705526: OR = 1.8, P = 1.1 × 10−32), in SH2B3 (rs7310615: OR = 1.4, P = 3.1 × 10−14), and upstream of TET2 (rs1548483: OR = 2.0, P = 2.0 × 10−9). These associations were confirmed in a separate replication cohort of 446 V617F carriers vs 169 021 noncarriers. In a joint analysis of the combined GWAS and replication results, we identified additional genome-wide significant predisposition alleles associated with CHEK2, ATM, PINT, and GFI1B. All SNP ORs were similar for MPN patients and controls who were V617F carriers. These data indicate that the same germ line variants endow individuals with a predisposition not only to MPN, but also to JAK2 V617F clonal hematopoiesis, a more common phenomenon that may foreshadow the development of an overt neoplasm. PMID:27365426

  1. Expression profile analysis of aorta-gonad-mesonephros region-derived stromal cells reveals genes that regulate hematopoiesis

    SciTech Connect

    Nagao, Kenji; Ohta, Takayuki; Hinohara, Atsushi; Tahara, Tomoyuki; Hagiwara, Tetsuya; Maeda, Yoshitake; Yoneya, Takashi; Sohma, Yoshiaki; Heike, Toshio; Nakahata, Tatsutoshi; Inagaki, Yoshimasa Nishikawa, Mitsuo

    2008-12-05

    The aorta-gonad-mesonephros (AGM) region is involved in the generation and maintenance of the first definitive hematopoietic stem cells (HSCs). A mouse AGM-derived cell line, AGM-S3, was shown to support the development of HSCs. To elucidate the molecular mechanisms regulating early hematopoiesis, we obtained subclones from AGM-S3, one of which was hematopoiesis supportive (S3-A9) and the other one of which was non-supportive (S3-A7), and we analyzed their gene expression profiles by gene chip analysis. In the present study, we found that Glypican-1 (GPC1) was highly expressed in the supportive subclone AGM-S3-A9. Over-expression of GPC1 in non-supportive cells led to the proliferation of progenitor cells in human cord blood when cocultured with the transfected-stromal cells. Thus, GPC1 may have an important role in the establishment of a microenvironment that supports early events in hematopoiesis.

  2. Beyond Hox: the role of ParaHox genes in normal and malignant hematopoiesis.

    PubMed

    Rawat, Vijay P S; Humphries, R Keith; Buske, Christian

    2012-07-19

    During the past decade it was recognized that homeobox gene families such as the clustered Hox genes play pivotal roles both in normal and malignant hematopoiesis. More recently, similar roles have also become apparent for members of the ParaHox gene cluster, evolutionarily closely related to the Hox gene cluster. This is in particular found for the caudal-type homeobox genes (Cdx) genes, known to act as upstream regulators of Hox genes. The CDX gene family member CDX2 belongs to the most frequent aberrantly expressed proto-oncogenes in human acute leukemias and is highly leukemogenic in experimental models. Correlative studies indicate that CDX2 functions as master regulator of perturbed HOX gene expression in human acute myeloid leukemia, locating this ParaHox gene at a central position for initiating and maintaining HOX gene dysregulation as a driving leukemogenic force. There are still few data about potential upstream regulators initiating aberrant CDX2 expression in human leukemias or about critical downstream targets of CDX2 in leukemic cells. Characterizing this network will hopefully open the way to therapeutic approaches that target deregulated ParaHox genes in human leukemia.

  3. Hematopoiesis from human embryonic stem cells: overcoming the immune barrier in stem cell therapies.

    PubMed

    Priddle, Helen; Jones, D Rhodri E; Burridge, Paul W; Patient, Roger

    2006-04-01

    The multipotency and proliferative capacity of human embryonic stem cells (hESCs) make them a promising source of stem cells for transplant therapies and of vital importance given the shortage in organ donation. Recent studies suggest some immune privilege associated with hESC-derived tissues. However, the adaptability of the immune system makes it unlikely that fully differentiated tissues will permanently evade immune rejection. One promising solution is to induce a state of immune tolerance to a hESC line using tolerogenic hematopoietic cells derived from it. This could provide acceptance of other differentiated tissues from the same line. However, this approach will require efficient multilineage hematopoiesis from hESCs. This review proposes that more efficient differentiation of hESCs to the tolerogenic cell types required is most likely to occur through applying knowledge gained of the ontogeny of complex regulatory signals used by the embryo for definitive hematopoietic development in vivo. Stepwise formation of mesoderm, induction of definitive hematopoietic stem cells, and the application of factors key to their self-renewal may improve in vitro production both quantitatively and qualitatively.

  4. Traveling waves in a coupled reaction-diffusion and difference model of hematopoiesis

    NASA Astrophysics Data System (ADS)

    Adimy, M.; Chekroun, A.; Kazmierczak, B.

    2017-04-01

    The formation and development of blood cells is a very complex process, called hematopoiesis. This process involves a small population of cells called hematopoietic stem cells (HSCs). The HSCs are undifferentiated cells, located in the bone marrow before they become mature blood cells and enter the blood stream. They have a unique ability to produce either similar cells (self-renewal), or cells engaged in one of different lineages of blood cells: red blood cells, white cells and platelets (differentiation). The HSCs can be either in a proliferating or in a quiescent phase. In this paper, we distinguish between dividing cells that enter directly to the quiescent phase and dividing cells that return to the proliferating phase to divide again. We propose a mathematical model describing the dynamics of HSC population, taking into account their spatial distribution. The resulting model is a coupled reaction-diffusion equation and difference equation with delay. We study the existence of monotone traveling wave fronts and the asymptotic speed of spread.

  5. Differential use of SCL/TAL-1 DNA-binding domain in developmental hematopoiesis.

    PubMed

    Kassouf, Mira T; Chagraoui, Hedia; Vyas, Paresh; Porcher, Catherine

    2008-08-15

    Dissecting the molecular mechanisms used by developmental regulators is essential to understand tissue specification/differentiation. SCL/TAL-1 is a basic helix-loop-helix transcription factor absolutely critical for hematopoietic stem/progenitor cell specification and lineage maturation. Using in vitro and forced expression experimental systems, we previously suggested that SCL might have DNA-binding-independent functions. Here, to assess the requirements for SCL DNA-binding activity in vivo, we examined hematopoietic development in mice carrying a germline DNA-binding mutation. Remarkably, in contrast to complete absence of hematopoiesis and early lethality in scl-null embryos, specification of hematopoietic cells occurred in homozygous mutant embryos, indicating that direct DNA binding is dispensable for this process. Lethality was forestalled to later in development, although some mice survived to adulthood. Anemia was documented throughout development and in adulthood. Cellular and molecular studies showed requirements for SCL direct DNA binding in red cell maturation and indicated that scl expression is positively autoregulated in terminally differentiating erythroid cells. Thus, different mechanisms of SCL's action predominate depending on the developmental/cellular context: indirect DNA binding activities and/or sequestration of other nuclear regulators are sufficient in specification processes, whereas direct DNA binding functions with transcriptional autoregulation are critically required in terminal maturation processes.

  6. The protumorigenic potential of FTY720 by promoting extramedullary hematopoiesis and MDSC accumulation.

    PubMed

    Li, Y; Zhou, T; Wang, Y; Ning, C; Lv, Z; Han, G; Morris, J C; Taylor, E N; Wang, R; Xiao, H; Hou, C; Ma, Y; Shen, B; Feng, J; Guo, R; Li, Y; Chen, G

    2017-02-20

    FTY720 (also called fingolimod) is recognized as an immunosuppressant and has been approved by the Food and Drug Administration to treat refractory multiple sclerosis. However, long-term administration of FTY720 potentially increases the risk for cancer in recipients. The underlying mechanisms remain poorly understood. Herein, we provided evidence that FTY720 administration potentiated tumor growth. Mechanistically, FTY720 enhanced extramedullary hematopoiesis and massive accumulation of myeloid-derived suppressor cells (MDSCs), which actively suppressed antitumor immune responses. Granulocyte-macrophage colony-stimulating factor (GM-CSF), mainly produced by MDSCs, was identified as a key factor to mediate these effects of FTY720 in tumor microenvironment. Furthermore, we showed that FTY720 triggers MDSCs to release GM-CSF via S1P receptor 3 (S1pr3) through Rho kinase and extracellular signal-regulated kinase-dependent pathway. Thus, our findings provide mechanistic explanation for the protumorigenic potentials of FTY720 and suggest that targeting S1pr3 simultaneously may be beneficial for the patients receiving FTY720 treatment.Oncogene advance online publication, 20 February 2017; doi:10.1038/onc.2017.2.

  7. Modeling normal and malignant human hematopoiesis in vivo through newborn NSG xenotransplantation.

    PubMed

    Ishikawa, Fumihiko

    2013-12-01

    Various strains of immune-compromised mice have been developed to investigate human normal and malignant stem cells in vivo. NOD/SCID mice harboring complete null mutation of Il2rg (NSG mice) lack T cells, B cells, and NK cells, and support high levels of engraftment by human cord blood hematopoietic stem cells (CB HSCs) and acute myeloid leukemia stem cells (AML LSCs). In addition to achieving high levels of human hematopoietic cell engraftment, use of newborn NSG mice as recipients has enabled the investigation into how human CB HSCs generate mature immune subsets in vivo. Moreover, through establishing an in vivo model of human primary AML by xenotransplantation of human LSCs into newborn NSG mice, functional properties of human AML such as cell cycle, location, and self-renewal capacity can be examined in vivo. Newborn NSG xenogeneic transplantation model may facilitate the understanding of human normal and malignant hematopoiesis and contribute to the development of novel therapies against hematologic diseases.

  8. The role of variant histone H2AV in Drosophila melanogaster larval hematopoiesis.

    PubMed

    Grigorian, Melina; DeBruhl, Heather; Lipsick, Joseph S

    2017-04-15

    Replication-independent histone variants can replace the canonical replication-dependent histones. Vertebrates have multiple H2A variant histones, including H2AZ and H2AX that are present in most eukaryotes. H2AZ regulates transcriptional activation as well as the maintenance of gene silencing, while H2AX is important in DNA damage repair. The fruit fly Drosophila melanogaster has only one histone H2A variant (H2AV), which is a chimera of H2AZ and H2AX. In this study we found that lack of H2AV led to the formation of black melanotic masses in Drosophila third instar larvae. The formation of these masses was found in conjunction with a loss of the majority of the primary lymph gland lobes. Interestingly, the cells of the posterior signaling center were preserved in these mutants. Reduction of H2AV levels by RNAi knockdown caused a milder phenotype that preserved the lymph gland structure but that included precocious differentiation of the prohemocytes located within the medullary zone and the secondary lobes of the lymph gland. Mutant rescue experiments suggest that the H2AZ-like rather than the H2AX-like function of H2AV is primarily required for normal hematopoiesis. © 2017. Published by The Company of Biologists Ltd.

  9. Single-cell transcriptional analysis of normal, aberrant, and malignant hematopoiesis in zebrafish

    PubMed Central

    Garcia, Elaine G.; Lobbardi, Riadh; Jain, Esha; Tang, Qin; Moore, John C.; Cortes, Mauricio; Molodtsov, Aleksey; Kasheta, Melissa; Luo, Christina C.; Garcia, Amaris J.; Mylvaganam, Ravi; Yoder, Jeffrey A.; Blackburn, Jessica S.; Sadreyev, Ruslan I.; Ceol, Craig J.; North, Trista E.

    2016-01-01

    Hematopoiesis culminates in the production of functionally heterogeneous blood cell types. In zebrafish, the lack of cell surface antibodies has compelled researchers to use fluorescent transgenic reporter lines to label specific blood cell fractions. However, these approaches are limited by the availability of transgenic lines and fluorescent protein combinations that can be distinguished. Here, we have transcriptionally profiled single hematopoietic cells from zebrafish to define erythroid, myeloid, B, and T cell lineages. We also used our approach to identify hematopoietic stem and progenitor cells and a novel NK-lysin 4+ cell type, representing a putative cytotoxic T/NK cell. Our platform also quantified hematopoietic defects in rag2E450fs mutant fish and showed that these fish have reduced T cells with a subsequent expansion of NK-lysin 4+ cells and myeloid cells. These data suggest compensatory regulation of the innate immune system in rag2E450fs mutant zebrafish. Finally, analysis of Myc-induced T cell acute lymphoblastic leukemia showed that cells are arrested at the CD4+/CD8+ cortical thymocyte stage and that a subset of leukemia cells inappropriately reexpress stem cell genes, including bmi1 and cmyb. In total, our experiments provide new tools and biological insights into single-cell heterogeneity found in zebrafish blood and leukemia. PMID:27139488

  10. PDGF/VEGF-Related Receptor Affects Transglutaminase Activity to Control Cell Migration During Crustacean Hematopoiesis.

    PubMed

    Junkunlo, Kingkamon; Söderhäll, Kenneth; Noonin, Chadanat; Söderhäll, Irene

    2017-09-14

    The platelet-derived growth factor (PDGF) receptor, a tyrosine kinase (TK) receptor whose ligand is PDGF, is crucial in the transduction of extracellular signals into cells and mediates numerous processes, such as cell proliferation, differentiation, survival, and migration. We demonstrate the important roles of a receptor TK related to the PDGF/VEGF family protein (PVR) in controlling hematopoietic progenitor cell migration by affecting extracellular transglutaminase (TGase) activity. Pl_PVR1, GenBank accession No. KY444650, is highly expressed in hemocytes and the hematopoietic tissue (HPT). Sunitinib malate was used to block the PVF/PVR downstream pathway in HPT cell culture. The addition of Sunitinib also caused the HPT cells to increase in size and begin spreading. An increase in extracellular TGase activity on the HPT cell membrane was observed in a dose-dependent manner after treatment with Sunitinib malate. The presence of crude Ast1 provided a combinatorial beneficial effect that enhanced the number of spreading cells after inhibition of the Pl_PVR downstream signaling cascade. In addition, an increased immunoreactivity for β-tubulin and elongation of β-tubulin filaments were found in Pl_PVR signaling-inhibited cells. The potential roles of PVF/PVR signaling in controlling progenitor cell activity during hematopoiesis in crayfish were investigated and discussed.

  11. Adenosine A(3) receptor agonist acts as a homeostatic regulator of bone marrow hematopoiesis.

    PubMed

    Hofer, Michal; Pospísil, Milan; Znojil, Vladimír; Holá, Jirina; Vacek, Antonín; Streitová, Denisa

    2007-07-01

    The present study was performed to define the optimum conditions of the stimulatory action of the adenosine A(3) receptor agonist, N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA), on bone marrow hematopoiesis in mice. Effects of 2-day treatment with IB-MECA given at single doses of 200nmol/kg twice daily were investigated in normal mice and in mice whose femoral bone marrow cells were either depleted or regenerating after pretreatment with the cytotoxic drug 5-fluorouracil. Morphological criteria were used to determine the proliferation state of the granulocytic and erythroid cell systems. Significant negative correlation between the control proliferation state and the increase of cell proliferation after IB-MECA treatment irrespective of the cell lineage investigated was found. The results suggest the homeostatic character of the induced stimulatory effects and the need to respect the functional state of the target tissue when investigating effects of adenosine receptor agonists under in vivo conditions.

  12. Embryonic Hematopoietic Progenitor Cells Reside in Muscle before Bone Marrow Hematopoiesis.

    PubMed

    Tanaka, Yuka; Inoue-Yokoo, Tomoko; Kulkeaw, Kasem; Yanagi-Mizuochi, Chiyo; Shirasawa, Senji; Nakanishi, Yoichi; Sugiyama, Daisuke

    2015-01-01

    In mice, hematopoietic cells home to bone marrow from fetal liver prenatally. To elucidate mechanisms underlying homing, we performed immunohistochemistry with the hematopoietic cell marker c-Kit, and observed c-Kit(+) cells localized inside muscle surrounding bone after 14.5 days post coitum. Flow cytometric analysis showed that CD45(+) c-Kit(+) hematopoietic cells were more abundant in muscle than in bone marrow between 14.5 and 17.5 days post coitum, peaking at 16.5 days post coitum. CD45(+) c-Kit(+) cells in muscle at 16.5 days post coitum exhibited higher expression of Gata2, among several hematopoietic genes, than did fetal liver or bone marrow cells. Colony formation assays revealed that muscle hematopoietic cells possess hematopoietic progenitor activity. Furthermore, exo utero transplantation revealed that fetal liver hematopoietic progenitor cells home to muscle and then to BM. Our findings demonstrate that hematopoietic progenitor cell homing occurs earlier than previously reported and that hematopoietic progenitor cells reside in muscle tissue before bone marrow hematopoiesis occurs during mouse embryogenesis.

  13. Dose-dependent role of the cohesin complex in normal and malignant hematopoiesis

    PubMed Central

    Viny, Aaron D.; Ott, Christopher J.; Spitzer, Barbara; Rivas, Martin; Meydan, Cem; Papalexi, Efthymia; Yelin, Dana; Shank, Kaitlyn; Reyes, Jaime; Chiu, April; Romin, Yevgeniy; Boyko, Vitaly; Thota, Swapna; Maciejewski, Jaroslaw P.; Melnick, Ari

    2015-01-01

    Cohesin complex members have recently been identified as putative tumor suppressors in hematologic and epithelial malignancies. The cohesin complex guides chromosome segregation; however, cohesin mutant leukemias do not show genomic instability. We hypothesized that reduced cohesin function alters chromatin structure and disrupts cis-regulatory architecture of hematopoietic progenitors. We investigated the consequences of Smc3 deletion in normal and malignant hematopoiesis. Biallelic Smc3 loss induced bone marrow aplasia with premature sister chromatid separation and revealed an absolute requirement for cohesin in hematopoietic stem cell (HSC) function. In contrast, Smc3 haploinsufficiency increased self-renewal in vitro and in vivo, including competitive transplantation. Smc3 haploinsufficiency reduced coordinated transcriptional output, including reduced expression of transcription factors and other genes associated with lineage commitment. Smc3 haploinsufficiency cooperated with Flt3-ITD to induce acute leukemia in vivo, with potentiated Stat5 signaling and altered nucleolar topology. These data establish a dose dependency for cohesin in regulating chromatin structure and HSC function. PMID:26438361

  14. Effects of combined sunitinib and extracranial stereotactic radiotherapy on bone marrow hematopoiesis

    PubMed Central

    Kao, Johnny; Timmins, Jonathan; Ozao-Choy, Junko; Packer, Stuart

    2016-01-01

    There is considerable interest in deploying stereotactic body radiotherapy in combination with immune therapy for patients with extracranial oligometastases. In addition to angiogenesis inhibition, sunitinib appears to mediate antitumor immunity through effects on circulating monocytic cells. The current study investigated the effects of combined sunitinib and stereotactic radiotherapy on hematopoiesis. As part of a phase I/II clinical trial utilizing concurrent sunitinib (25–50 mg on days 1–28) and image-guided radiation therapy (40–50 Gy in 10 fractions starting on days 8–19) for patients with metastatic cancer, the complete blood count, platelet count and automatic differential were performed pretreatment and on days 8 and 19. On average, sunitinib monotherapy for 7 days resulted in a 33% decrease in monocytes and an 18% decrease in neutrophils (P<0.01 for all). Compared to sunitinib alone, combined sunitinib and radiation resulted in a further decrease in neutrophils, lymphocytes and platelets (P<0.05). Following sunitinib and radiation treatment, a greater than average decrease in monocytes (≥200/µl) was associated with a significant increase in progression-free and overall survival times. This exploratory study provides further evidence that monocytes represent a potential biomarker in patients with solid tumors treated with sunitinib. PMID:27602153

  15. Metformin improves defective hematopoiesis and delays tumor formation in Fanconi anemia mice.

    PubMed

    Zhang, Qing-Shuo; Tang, Weiliang; Deater, Matthew; Phan, Ngoc; Marcogliese, Andrea N; Li, Hui; Al-Dhalimy, Muhsen; Major, Angela; Olson, Susan; Monnat, Raymond J; Grompe, Markus

    2016-12-15

    Fanconi anemia (FA) is an inherited bone marrow failure disorder associated with a high incidence of leukemia and solid tumors. Bone marrow transplantation is currently the only curative therapy for the hematopoietic complications of this disorder. However, long-term morbidity and mortality remain very high, and new therapeutics are badly needed. Here we show that the widely used diabetes drug metformin improves hematopoiesis and delays tumor formation in Fancd2(-/-) mice. Metformin is the first compound reported to improve both of these FA phenotypes. Importantly, the beneficial effects are specific to FA mice and are not seen in the wild-type controls. In this preclinical model of FA, metformin outperformed the current standard of care, oxymetholone, by improving peripheral blood counts in Fancd2(-/-) mice significantly faster. Metformin increased the size of the hematopoietic stem cell compartment and enhanced quiescence in hematopoietic stem and progenitor cells. In tumor-prone Fancd2(-/-)Trp53(+/-) mice, metformin delayed the onset of tumors and significantly extended the tumor-free survival time. In addition, we found that metformin and the structurally related compound aminoguanidine reduced DNA damage and ameliorated spontaneous chromosome breakage and radials in human FA patient-derived cells. Our results also indicate that aldehyde detoxification might be one of the mechanisms by which metformin reduces DNA damage in FA cells. © 2016 by The American Society of Hematology.

  16. Leptin in chronic kidney disease: a link between hematopoiesis, bone metabolism, and nutrition.

    PubMed

    Zhang, Jingjing; Wang, Ningning

    2014-06-01

    Anemia, dyslipidemia, malnutrition, together with mineral and bone disorders are common complications in patients with chronic kidney disease (CKD). All are associated with increased risk of mortality. Leptin is a small peptide hormone that is mainly but not exclusively produced in adipose tissue. It is also secreted by normal human osteoblasts, subchondral osteoblasts, placental syncytiotrophoblasts, and the gastric epithelium. Leptin binds to its receptors in the hypothalamus to regulate bone metabolism and food intake. Leptin also has several other important metabolic effects on peripheral tissues, including the liver, skeletal muscle, and bone marrow. Leptin is cleared principally by the kidney. Not surprisingly, serum leptin appears to increase concurrently with declines in the glomerular filtration rate in patients with CKD. A growing body of evidence suggests that leptin might be closely related to hematopoiesis, nutrition, and bone metabolism in CKD patients. Results are conflicting regarding leptin in patients with CKD, in whom both beneficial and detrimental effects on uremia outcome are found. This review elucidates the discovery of leptin and its receptors, changes in serum or plasma leptin levels, the functions of leptin, relationships between leptin and the complications mentioned above, and pharmaceutical interventions in serum leptin levels in patients with CKD.

  17. Parp-2 is required to maintain hematopoiesis following sublethal γ-irradiation in mice.

    PubMed

    Farrés, Jordi; Martín-Caballero, Juan; Martínez, Carlos; Lozano, Juan J; Llacuna, Laura; Ampurdanés, Coral; Ruiz-Herguido, Cristina; Dantzer, Françoise; Schreiber, Valérie; Villunger, Andreas; Bigas, Anna; Yélamos, José

    2013-07-04

    Hematopoietic stem cells self-renew for life to guarantee the continuous supply of all blood cell lineages. Here we show that Poly(ADP-ribose) polymerase-2 (Parp-2) plays an essential role in hematopoietic stem/progenitor cells (HSPC) survival under steady-state conditions and in response to stress. Increased levels of cell death were observed in HSPC from untreated Parp-2-/- mice, but this deficit was compensated by increased rates of self-renewal, associated with impaired reconstitution of hematopoiesis upon serial bone marrow transplantation. Cell death after γ-irradiation correlated with an impaired capacity to repair DNA damage in the absence of Parp-2. Upon exposure to sublethal doses of γ-irradiation, Parp-2-/- mice exhibited bone marrow failure that correlated with reduced long-term repopulation potential of irradiated Parp-2-/- HSPC under competitive conditions. In line with a protective role of Parp-2 against irradiation-induced apoptosis, loss of p53 or the pro-apoptotic BH3-only protein Puma restored survival of irradiated Parp-2-/- mice, whereas loss of Noxa had no such effect. Our results show that Parp-2 plays essential roles in the surveillance of genome integrity of HSPC by orchestrating DNA repair and restraining p53-induced and Puma-mediated apoptosis. The data may affect the design of drugs targeting Parp proteins and the improvement of radiotherapy-based therapeutic strategies.

  18. Impaired hematopoiesis and delayed thrombopoietic recovery following sublethal irradiation in SRC‑3 knockout mice.

    PubMed

    Jin, J; Wang, Y; Wang, J; Xu, Y; Chen, S L; Wang, J P; Su, Y P

    2014-05-01

    The objective of the present study was to investigate the role of the steroid receptor coactivator-3 (SRC-3) in hematopoiesis of mouse bone marrow (BM) following total body irradiation (TBI). SRC-3-/‑ mice and wild-type (WT) mice were exposed to 4.5 Gy γ rays. Immunoblotting analysis revealed that the SRC-3 protein (p160) levels in normal BM-nucleated cells in WT were higher than in SRC-3-/‑ mice. Furthermore, peripheral blood cell counts, BM cellularity and colony-forming unit (CFU) assays were performed following irradiation. The results showed that peripheral blood cells were significantly lower in number and recovered less rapidly in irradiated SRC-3-/‑ mice as compared with control animals. BM-nucleated cell and CFU counts were significantly decreased in SRC-3-/‑ mice on the 7th and 14th day. Of note, the recovery of platelet (PLT) and megakaryocytic lineage were more depressed than the granulocytic and erythroid lineage in SRC-3-/‑ mice. In conclusion, the present study demonstrated that the hematopoietic ability in SRC-3 knockout mice is severely impaired following a sublethal dose of irradiation.

  19. CD137 ligand reverse signaling skews hematopoiesis towards myelopoiesis during aging.

    PubMed

    Tang, Qianqiao; Koh, Liang Kai; Jiang, Dongsheng; Schwarz, Herbert

    2013-09-01

    CD137 is a costimulatory molecule expressed on activated T cells. Its ligand, CD137L, is expressed on the surface of hematopoietic progenitor cells, and upon binding to CD137 induces reverse signaling into hematopoietic progenitor cells promoting their activation, proliferation and myeloid differentiation. Since aging is associated with an increasing number of myeloid cells we investigated the role of CD137 and CD137L on myelopoiesis during aging. Comparing 3 and 12 months old WT, CD137‐/‐ and CD137L‐/‐ mice we found significantly more granulocytes and monocytes in the bone marrow of older WT mice, while this age‐dependent increase was absent in CD137‐/‐ and CD137L‐/‐ mice. Instead, the bone marrow of 12 months old CD137‐/‐ and CD137L‐/‐ mice was characterized by an accumulation of hematopoietic progenitor cells, suggesting that the differentiation of hematopoietic progenitor cells became arrested in the absence of CD137L signaling. CD137L signaling is initiated by activated CD137‐expressing, CD4+ T cells. These data identify a novel molecular mechanisms underlying immune aging by demonstrating that CD137‐expressing CD4+ T cells in the bone marrow engage CD137L on hematopoietic progenitor cells, and that this CD137L signaling biases hematopoiesis towards myelopoiesis during aging.

  20. Gut microbiota metabolism of dietary fiber influences allergic airway disease and hematopoiesis.

    PubMed

    Trompette, Aurélien; Gollwitzer, Eva S; Yadava, Koshika; Sichelstiel, Anke K; Sprenger, Norbert; Ngom-Bru, Catherine; Blanchard, Carine; Junt, Tobias; Nicod, Laurent P; Harris, Nicola L; Marsland, Benjamin J

    2014-02-01

    Metabolites from intestinal microbiota are key determinants of host-microbe mutualism and, consequently, the health or disease of the intestinal tract. However, whether such host-microbe crosstalk influences inflammation in peripheral tissues, such as the lung, is poorly understood. We found that dietary fermentable fiber content changed the composition of the gut and lung microbiota, in particular by altering the ratio of Firmicutes to Bacteroidetes. The gut microbiota metabolized the fiber, consequently increasing the concentration of circulating short-chain fatty acids (SCFAs). Mice fed a high-fiber diet had increased circulating levels of SCFAs and were protected against allergic inflammation in the lung, whereas a low-fiber diet decreased levels of SCFAs and increased allergic airway disease. Treatment of mice with the SCFA propionate led to alterations in bone marrow hematopoiesis that were characterized by enhanced generation of macrophage and dendritic cell (DC) precursors and subsequent seeding of the lungs by DCs with high phagocytic capacity but an impaired ability to promote T helper type 2 (TH2) cell effector function. The effects of propionate on allergic inflammation were dependent on G protein-coupled receptor 41 (GPR41, also called free fatty acid receptor 3 or FFAR3), but not GPR43 (also called free fatty acid receptor 2 or FFAR2). Our results show that dietary fermentable fiber and SCFAs can shape the immunological environment in the lung and influence the severity of allergic inflammation.

  1. Commensal bacterial–derived signals regulate basophil hematopoiesis and allergic inflammation

    PubMed Central

    Hill, David A.; Siracusa, Mark C.; Abt, Michael C.; Kim, Brian S.; Kobuley, Dmytro; Kubo, Masato; Kambayashi, Taku; LaRosa, David F.; Renner, Ellen D.; Orange, Jordan S.; Bushman, Frederic D.; Artis, David

    2012-01-01

    Commensal bacteria that colonize mammalian barrier surfaces are reported to influence T helper type 2 (TH2) cytokine–dependent inflammation and susceptibility to allergic disease, although the mechanisms that underlie these observations are poorly understood. In this report, we identify that deliberate alteration of commensal bacterial populations via oral antibiotic treatment resulted in elevated serum immunoglobulin E (IgE) levels, increased steady–state circulating basophil populations, and exaggerated basophil–mediated TH2 cell responses and allergic inflammation. Elevated serum IgE levels correlated with increased circulating basophil populations in mice and subjects with hyperimmunoglobulinemia E syndrome. Furthermore, B cell–intrinsic expression of MyD88 was required to limit serum IgE levels and circulating basophil populations in mice. Commensal–derived signals were found to influence basophil development by limiting proliferation of bone marrow–resident precursor populations. Collectively, these results identify a previously unrecognized pathway through which commensal–derived signals influence basophil hematopoiesis and susceptibility to TH2 cytokine–dependent inflammation and allergic disease. PMID:22447074

  2. Administration of Interleukin-6 Stimulates Multilineage Hematopoiesis and Accelerates Recovery from Radiation-Induced Hematopoietic Depression

    DTIC Science & Technology

    1991-02-01

    Htirano T, Kishimoto T, Nakahata T. Asano S: In vitro hernatopoietic growth factors. J1 Natl Cancer Inst 81: t370. 1989 expansion of the murine...a.i phas S. %’ink A. Billiau A. VanSnick 1: Identification of the nutohlbyrcmiatntrekn.Cllmuolt12. human Zh-kd protein, interferon beta ,, as a B... beta . B-cell %timulatory factor type 2 shares identity T. Takaku F. Akivama Y: In vivo effects of recombinant human with rnsanc~tc-derived

  3. In vitro myelotoxicity of propanil and 3,4-dichloroaniline on murine and human CFU-E/BFU-E progenitors.

    PubMed

    Malerba, I; Castoldi, A F; Parent-Massin, D; Gribaldo, L

    2002-10-01

    Because of the wide use of pesticides for domestic and industrial purposes, the evaluation of their potential effects is of major concern for public health. The myelotoxicity of the herbicide propanil (3,4-dichloroproprioanilide) and its metabolite 3,4-dichloroaniline (DCA) is well documented in mice, but evidence that pesticides may severely compromise hematopoiesis in humans is lacking. In this study, an interspecies comparison of in vitro toxicity of these two compounds on murine and human burst- and colony-forming unit-erythrocyte (BFU-E, CFU-E) and colony-forming unit-granulocyte/macrophage (CFU-GM) progenitors, has been carried out. Murine bone marrow progenitors and human cord blood cells were exposed to propanil or DCA in doses ranging from 10 micro M to 1000 micro M, and the toxic effect was detected by a clonogenic assay with continuous exposure to the compounds. The results on murine cells indicate that the erythrocytic lineage is the most sensitive target for propanil and DCA. On the other hand, human progenitors seem to be less sensitive to the toxic effects of both compounds than murine progenitors at the same concentrations (IC(50) values are 305.2 +/- 22.6 micro M [total erythroid colonies] and >500 micro M [CFU-GM] for propanil). Propanil was significantly more toxic to human erythroid progenitors than to human CFU-GM progenitors, as was found for the murine cells, emphasizing the role of the heme pathway as the target for propanil. These data confirm the evidence that the compounds investigated interfere with erythroid colony formation at different stages of the differentiation pathway and have different effects according to the dose.

  4. MDS-associated somatic mutations and clonal hematopoiesis are common in idiopathic cytopenias of undetermined significance.

    PubMed

    Kwok, Brian; Hall, Jeff M; Witte, John S; Xu, Yin; Reddy, Prashanti; Lin, Keming; Flamholz, Rachel; Dabbas, Bashar; Yung, Aine; Al-Hafidh, Jenan; Balmert, Emily; Vaupel, Christine; El Hader, Carlos; McGinniss, Matthew J; Nahas, Shareef A; Kines, Julie; Bejar, Rafael

    2015-11-19

    Establishing a diagnosis in patients suspected of having a myelodysplastic syndrome (MDS) can be challenging and could be informed by the identification of somatic mutations. We performed a prospective study to examine the frequency and types of mutations encountered in 144 patients with unexplained cytopenias. Based on bone marrow findings, 17% were diagnosed with MDS, 15% with idiopathic cytopenias of undetermined significance (ICUS) and some evidence of dysplasia, and 69% with ICUS and no dysplasia. Bone marrow DNA was sequenced for mutations in 22 frequently mutated myeloid malignancy genes. Somatic mutations were identified in 71% of MDS patients, 62% of patients with ICUS and some dysplasia, and 20% of ICUS patients and no dysplasia. In total, 35% of ICUS patients carried a somatic mutation or chromosomal abnormality indicative of clonal hematopoiesis. We validated these results in a cohort of 91 lower-risk MDS and 249 ICUS cases identified over a 6-month interval. Mutations were found in 79% of those with MDS, in 45% of those with ICUS with dysplasia, and in 17% of those with ICUS without dysplasia. The spectrum of mutated genes was similar with the exception of SF3B1 which was rarely mutated in patients without dysplasia. Variant allele fractions were comparable between clonal ICUS (CCUS) and MDS as were mean age and blood counts. We demonstrate that CCUS is a more frequent diagnosis than MDS in cytopenic patients. Clinical and mutational features are similar in these groups and may have diagnostic utility once outcomes in CCUS patients are better understood.

  5. Conserved hemopoietic transcription factor Cg-SCL delineates hematopoiesis of Pacific oyster Crassostrea gigas.

    PubMed

    Song, Xiaorui; Wang, Hao; Chen, Hao; Sun, Mingzhe; Liang, Zhongxiu; Wang, Lingling; Song, Linsheng

    2016-04-01

    Hemocytes are the effective immunocytes in bivalves, which have been reported to be derived from stem-like cells in gill epithelium of oyster. In the present work, a conserved haematopoietic transcription factor Tal-1/Scl (Stem Cell Leukemia) was identified in Pacific oyster (Cg-SCL), and it was evolutionarily close to the orthologs in deuterostomes. Cg-SCL was highly distributed in the hemocytes as well as gill and mantle. The hemocyte specific genes Integrin, EcSOD and haematopoietic transcription factors GATA3, C-Myb, c-kit, were down-regulated when Cg-SCL was interfered by dsRNA. During the larval developmental stages, the mRNA transcripts of Cg-SCL gradually increased after fertilization and peaked at early trochophore larvae stage (10 hpf, hours post fertilization), then sharply decreased in late trochophore larvae stage (15 hpf) before resuming in umbo larvae (120 hpf). Whole-mount immunofluorescence assay further revealed that the immunoreactivity of Cg-SCL appeared in blastula larvae with two approximate symmetric spots, and this expression pattern lasted in gastrula larvae. By trochophore, the immunoreactivity formed a ring around the dorsal region and then separated into two remarkable spots at the dorsal side in D-veliger larvae. After bacterial challenge, the mRNA expression levels of Cg-SCL were significantly up-regulated in the D-veliger and umbo larvae, indicating the available hematopoietic regulation in oyster larvae. These results demonstrated that Cg-SCL could be used as haematopoietic specific marker to trace potential developmental events of hematopoiesis during ontogenesis of oyster, which occurred early in blastula stage and maintained until D-veliger larvae.

  6. Extramedullary hematopoiesis is associated with lower cardiac iron loading in chronically transfused thalassemia patients.

    PubMed

    Ricchi, Paolo; Meloni, Antonella; Spasiano, Anna; Neri, Maria Giovanna; Gamberini, Maria Rita; Cuccia, Liana; Caruso, Vincenzo; Gerardi, Calogera; D'Ascola, Domenico Giuseppe; Rosso, Rosamaria; Campisi, Saveria; Rizzo, Michele; Terrazzino, Fabrizia; Vangosa, Alessandra Briatico; Chiodi, Elisabetta; Missere, Massimiliano; Mangione, Maurizio; Positano, Vincenzo; Pepe, Alessia

    2015-11-01

    The aim of this study was to evaluate, in a large cohort of chronically transfused patients, whether the presence of extramedullary hematopoiesis (EMH) accounts for the typical patterns of cardiac iron distribution and/or cardiac function parameters. We retrospectively selected 1,266 thalassemia major patients who had undergone regular transfusions (611 men and 655 women; mean age: 31.3 ± 8.9 years, range: 4.2-66.6 years) and were consecutively enrolled within the Myocardial Iron Overload in Thalassemia network. The presence of EMH was evaluated based on steady-state free precession sequences; cardiac and liver iron overloads were quantified using a multiecho T2* approach; cardiac function parameters and pulmonary diameter were quantified using the steady-state free precession sequences; and myocardial fibrosis was evaluated using the late gadolinium enhancement technique. EMH was detected in 167 (13.2%) patients. The EMH+ patients had significantly lower cardiac iron overload than that of the EMH- patients (P = 0.003). The patterns of cardiac iron distribution were significantly different in the EMH+ and EMH- patients (P < 0.0001), with a higher prevalence of patients with no myocardial iron overload and heterogeneous myocardial iron overload and no significant global heart iron in the EMH+ group EMH+ patients had a significantly higher left ventricle mass index (P = 0.001) and a significantly higher pulmonary artery diameter (P = 0.002). In conclusion, in regularly transfused thalassemia patients, EMH was common and was associated with a thalassemia intermedia-like pattern of cardiac iron deposition despite regular transfusion therapy.

  7. Hyaluronan Expressed by the Hematopoietic Microenvironment Is Required for Bone Marrow Hematopoiesis*

    PubMed Central

    Goncharova, Valentina; Serobyan, Naira; Iizuka, Shinji; Schraufstatter, Ingrid; de Ridder, Audrey; Povaliy, Tatiana; Wacker, Valentina; Itano, Naoki; Kimata, Koji; Orlovskaja, Irina A.; Yamaguchi, Yu; Khaldoyanidi, Sophia

    2012-01-01

    The contribution of hyaluronan (HA) to the regulatory network of the hematopoietic microenvironment was studied using knock-out mice of three hyaluronan synthase genes (Has1, Has2, and Has3). The number of hematopoietic progenitors was decreased in bone marrow and increased in extramedullary sites of Prx1-Cre;Has2flox/flox;Has1−/−;Has3−/− triple knock-out (tKO) mice as compared with wild type (WT) and Has1−/−;Has3−/− double knock-out (dKO) mice. In line with this observation, decreased hematopoietic activity was observed in long term bone marrow cultures (LTBMC) from tKO mice, whereas the formation of the adherent layer and generation of hematopoietic cells in WT and dKO cultures was not different. 4-Methylumbelliferone (4MU) was used to pharmacologically inhibit the production of HA in LTBMC. Treatment with 4MU inhibited HA synthesis, decreased expression of HAS2 and HAS3, and eliminated hematopoiesis in LTBMC, and this effect was alleviated by the addition of exogenous HA. Exogenous HA also augmented the cell motility in LTBMC, which correlated with the HA-stimulated production of chemokines and growth factors. Conditioned media from HA-induced LTBMC enhanced the chemotaxis of hematopoietic stem/progenitor cells (HSPC) in response to SDF-1. Exposure of endothelial cells to 4MU decreased their ability to support HSPC rolling and adhesion. In addition, migration of transplanted HSPC into the marrow of 4MU-pretreated mice was lower than in untreated mice. Collectively, the results suggest that HA depletion reduces the ability of the microenvironment to support HSPC, and confirm a role for HA as a necessary regulatory element in the structure of the hematopoietic microenvironment. PMID:22654110

  8. Effects of Dosimetrically Guided I-131 Therapy on Hematopoiesis in Patients With Differentiated Thyroid Cancer

    PubMed Central

    Bikas, Athanasios; Schneider, Mark; Desale, Sameer; Atkins, Frank; Mete, Mihriye; Burman, Kenneth D.; Wartofsky, Leonard

    2016-01-01

    Objective: The objective of the study was to evaluate the effects of dosimetrically guided I-131 prescribed activities on hematopoiesis reflected by changes in complete blood counts (CBCs). Design: This was a retrospective analysis. Setting: The study was conducted at an academic center. Patients: A total of 152 patients with differentiated thyroid cancer who had 185 dosimetrically guided I-131 treatments. Interventions: There were no interventions. Main Outcome Measures: Repeated-measure ANOVA was used for the analysis of the differences in the averages of CBCs that were documented at baseline and 1, 6, 12, 24–36, and 48–60 months after I-131 treatment. Results: All parameters decreased to their respective nadir at 1 month and then gradually returned toward baseline values. White blood cells (WBCs) and platelets (PLTs) were the most significantly affected cells. At 1 month, the decrease was 29.6% (P < .0001) for WBCs and 25% (P < .0001) for PLTs, whereas at 12 months, the decrease was 15.5% (P < .0001) and 13% (P < .0001), respectively. Lymphocytes appeared to be more susceptible to I-131 than neutrophils (ANCs). The decreases were small in absolute numbers for red blood cells, hematocrit and hemoglobin not surpassing 10%. Multivariate analysis demonstrated that the ratio of administered prescribed activity-to-maximum tolerated activity was associated with the decreases in WBCs (P = .0038), ANCs (P = .0063), and red blood cells (P = .029), with borderline significance for PLTs (P = .057) and hemoglobin (P = .057). Conclusions: Dosimetrically guided I-131 resulted in statistically significant decreases in CBC parameters, which were more prominent in WBCs and PLTs. Lymphocytes were more severely affected than ANCs, whereas all parameters reached a nadir at 1 month and then gradually returned toward baseline values over the 5-year follow-up of our study. PMID:26900639

  9. MDS-associated somatic mutations and clonal hematopoiesis are common in idiopathic cytopenias of undetermined significance

    PubMed Central

    Kwok, Brian; Hall, Jeff M.; Witte, John S.; Xu, Yin; Reddy, Prashanti; Lin, Keming; Flamholz, Rachel; Dabbas, Bashar; Yung, Aine; Al-Hafidh, Jenan; Balmert, Emily; Vaupel, Christine; El Hader, Carlos; McGinniss, Matthew J.; Nahas, Shareef A.; Kines, Julie

    2015-01-01

    Establishing a diagnosis in patients suspected of having a myelodysplastic syndrome (MDS) can be challenging and could be informed by the identification of somatic mutations. We performed a prospective study to examine the frequency and types of mutations encountered in 144 patients with unexplained cytopenias. Based on bone marrow findings, 17% were diagnosed with MDS, 15% with idiopathic cytopenias of undetermined significance (ICUS) and some evidence of dysplasia, and 69% with ICUS and no dysplasia. Bone marrow DNA was sequenced for mutations in 22 frequently mutated myeloid malignancy genes. Somatic mutations were identified in 71% of MDS patients, 62% of patients with ICUS and some dysplasia, and 20% of ICUS patients and no dysplasia. In total, 35% of ICUS patients carried a somatic mutation or chromosomal abnormality indicative of clonal hematopoiesis. We validated these results in a cohort of 91 lower-risk MDS and 249 ICUS cases identified over a 6-month interval. Mutations were found in 79% of those with MDS, in 45% of those with ICUS with dysplasia, and in 17% of those with ICUS without dysplasia. The spectrum of mutated genes was similar with the exception of SF3B1 which was rarely mutated in patients without dysplasia. Variant allele fractions were comparable between clonal ICUS (CCUS) and MDS as were mean age and blood counts. We demonstrate that CCUS is a more frequent diagnosis than MDS in cytopenic patients. Clinical and mutational features are similar in these groups and may have diagnostic utility once outcomes in CCUS patients are better understood. PMID:26429975

  10. Artesunate and artelinic acid: association of embryotoxicity, reticulocytopenia, and delayed stimulation of hematopoiesis in pregnant rats.

    PubMed

    Clark, Robert L; Brannen, Kimberly C; Sanders, James E; Hoberman, Alan M

    2011-02-01

    The artemisinin antimalarials cause embryo death and malformations in animals by killing embryonic erythroblasts. Groups of pregnant rats (N = 4) were administered 35 and 48 µmol/kg artesunate and 17.2, 28.7, 48, 96, and 191 µmol/kg artelinic acid as a single oral dose on gestational day (GD) 12. Litters were examined on GD21. The ED(50) for embryo death with artelinic acid (23.4 µmol/kg) was just slightly lower than that for decreased reticulocyte count at 24 hr postdose (33.5 µmol/kg) and both had similarly steep dose responses (maximal effects of total litter loss and ∼60% decreases in reticulocyte count at 48 µmol/kg). Results with artesunate were similar. The correlation coefficient between embryo death and decreased reticulocyte count was 0.82 (p<0.01). The close relationship between embryotoxicity and reticulocytopenia is suggestive of a common mechanism-artemisinin-induced mitochondrial damage leading to cell death. At 9 days postdose, treatment with artesunate and artelinic acid also caused increases in counts of reticulocytes, lymphocytes, basophils, and monocytes (up to 3.7 ×, 1.7 ×, 4.7 ×, and 1.7 × control, respectively). This stimulation of hematopoiesis may have been mediated by the direct oxidative conversion of artesunate or artelinic acid to the artemisininyl hydroperoxide within the bone marrow cells or by an indirect increase in reactive oxygen species. The high correlation between embryotoxicity and reticulocytopenia further supports the assertion that therapeutic dosage regimens of artemisinins that cause decreases in reticulocyte count in pregnant women during the putative critical period (approximately postconception wk 3 to 9) are at risk of also causing adverse effects on the embryo.

  11. Long-term human hematopoiesis in the SCID-hu mouse

    PubMed Central

    1990-01-01

    Coimplantation of small fragments of human fetal thymus and fetal liver into immunodeficient SCID mice resulted in the formation of a unique structure (Thy/Liv). Thereafter, the SCID-hu mice showed reproducible and long-term reconstitution of human hematopoietic activity. For periods lasting 5-11 mo after transplantation, active T lymphopoiesis was observed inside the grafts and cells that were negative for T cell markers were found to have colony-forming units for granulocyte/macrophage (CFU-GM) and erythroid burst-forming unit (BFU- E) activity in the methylcellulose colony assay. In addition, structures similar to normal human bone marrow were observed inside the Thy/Liv grafts, consisting of blast cells, mature and immature forms of myelomonocytic cells, and megakaryocytes. These data indicate long-term maintenance, in vivo, of human progenitor cells for the T lymphoid, myelomonocytic, erythroid, and megakaryocytic lineages. The role of the implanted fetal liver fragments was analyzed using HLA-mismatched Thy/Liv implants. The HLA type of the liver donor was found on T cells and macrophages in the graft. In addition, cells grown in the methylcellulose colony assay and cells in a bone marrow-like structure, the "thymic isle," expressed the HLA type of the liver donor. Thus, the Thy/Liv implants provided a microenvironment in which to follow human hematopoietic progenitor cells for multiple lineages. The formation of the Thy/Liv structures also results in a continuous source of human T cells in the peripheral circulation of the SCID-hu mouse. Though present for 5-11 mo, these cells did not engage in a xenograft (graft- versus-host) reaction. This animal model, the first in which multilineage human hematopoietic activity is maintained for long periods of time, should be useful for the analysis of human hematopoiesis in vivo. PMID:2212942

  12. Lymph node extramedullary hematopoiesis in breast cancer patients receiving neoadjuvant therapy: a potential diagnostic pitfall.

    PubMed

    Prieto-Granada, Carlos; Setia, Namrata; Otis, Christopher N

    2013-06-01

    Extramedullary hematopoiesis (EMH) develops as a compensatory mechanism associated with hematologic processes but it may occur in association with chemotherapy. Three cases of EMH arising in axillary lymph nodes following neoadjuvant therapy for breast carcinoma are reported herein. Three women ranging in age from 41 to 47 years presented with unilateral breast masses measuring 0.6 to 4.0 cm in greatest dimension and were diagnosed with infiltrating ductal carcinoma, grade III by core needle biopsies. Two of the tumors were triple negative and one was estrogen receptor positive. All patients subsequently received neoadjuvant therapy followed by lumpectomies. No residual carcinoma was identified in postchemotherapy breast resection specimens. One patient underwent a sentinel lymph node procedure, the second patient an axillary lymph node dissection, and the third patient had a core biopsy of an enlarged axillary lymph node. The patient that underwent axillary lymph node dissection had metastatic carcinoma in one of her lymph nodes. Foci of EMH consisting of myeloid, erythroid, and megakaryocytic precursors were present within the nodal parenchyma and/or subcapsular sinuses of axillary lymph nodes of all three cases. Megakaryocytes were immunoreactive with factor VIII, erythroid elements with Glycophorin and myeloid precursors with myeloperoxidase. With increasing use of neoadjuvant therapy for breast carcinoma, EMH within lymph nodes is more likely to be encountered. Hematopoietic precursors present in lymph nodes may potentially be misdiagnosed as metastatic tumor cells, particularly as lobular carcinoma or metaplastic carcinoma. Therefore, caution should be exercised when evaluating axillary lymph nodes in the clinical setting of neoadjuvant therapy for breast carcinoma.

  13. [Regulation of erythropoiesis in patients with suppressed hematopoiesis during mountain climatic treatment].

    PubMed

    Ismailova, A Z; Makeshova, A B; Eralieva, M O; Levina, A A; Makukova, Iu I; Raimzhanov, A R

    2010-01-01

    to estimate the regulation of erythropoiesis and the coagulation system in patients with suppressed hematopoiesis in a mountain hospital (3200 m above sea level). The investigation included 12 patients with aplastic anemia (AA) and 10 with idiopathic thrombocytopenic purpura (ITP). Blood was received at a Bishkek hospital, then on days 20 and 40 of stay in the mountains. The authors studied erythropoietin (EPO) by enzyme immunoassay (Protein Contour kit, Russia), serum ferritin (SF) by immunoradioassay (Immunotech kit, Czech Republic), hypoxia-inducible factor-1alpha (HIF-1alpha), homocysteine (HC), hepcidin, endothelin (ET), and thrombomodulin (TM) by sandwich enzyme immunoassay, by applying monospecific antisera and monoclonal antibodies against relevant antigens (IDG Int Inc, USA). On staying in the mountains, there was a gradual increase in the content of hemoglobin in patients with AA and ITP. On day 40, in keeping with higher hemoglobin (Hb) levels, both groups showed a decrease in HIF-1alpha concentrations to the normal values (from 8.2 to 4.5 pg/ml). Due to the anemic syndrome, baseline EPO was increased by 5-7 times in the patients from both groups. On days 20-40, the content of EPO showed a 1.3-2.5-fold increase. In AA, HC was almost 3 times greater than the normal values; in ITP, it was 1.5-fold increased. On day 20 and during the patients'stay in the mountains, the level of HC remained in the normal range in both groups. Hypoxic hypoxia positively affects a number of hematological parameters, by normalizing erythropoiesis (Hb, EPO, and HIF-1alpha), iron metabolism (SF), and the coagulation system (HC, ET, and TM).

  14. Effects of the murine skull in optoacoustic brain microscopy.

    PubMed

    Kneipp, Moritz; Turner, Jake; Estrada, Héctor; Rebling, Johannes; Shoham, Shy; Razansky, Daniel

    2016-01-01

    Despite the great promise behind the recent introduction of optoacoustic technology into the arsenal of small-animal neuroimaging methods, a variety of acoustic and light-related effects introduced by adult murine skull severely compromise the performance of optoacoustics in transcranial imaging. As a result, high-resolution noninvasive optoacoustic microscopy studies are still limited to a thin layer of pial microvasculature, which can be effectively resolved by tight focusing of the excitation light. We examined a range of distortions introduced by an adult murine skull in transcranial optoacoustic imaging under both acoustically- and optically-determined resolution scenarios. It is shown that strong low-pass filtering characteristics of the skull may significantly deteriorate the achievable spatial resolution in deep brain imaging where no light focusing is possible. While only brain vasculature with a diameter larger than 60 µm was effectively resolved via transcranial measurements with acoustic resolution, significant improvements are seen through cranial windows and thinned skull experiments.

  15. Histological Analyses Demonstrate the Temporary Contribution of Yolk Sac, Liver, and Bone Marrow to Hematopoiesis during Chicken Development

    PubMed Central

    Guedes, Priscila Tavares; de Oliveira, Barbara Cristina Euzébio Pereira Dias; Manso, Pedro Paulo de Abreu; Caputo, Luzia Fátima Gonçalves; Pelajo-Machado, Marcelo

    2014-01-01

    The use of avian animal models has contributed to the understanding of many aspects of the ontogeny of the hematopoietic system in vertebrates. However, specific events that occur in the model itself are still unclear. There is a lack of consensus, among previous studies, about which is the intermediate site responsible for expansion and differentiation of hematopoietic cells, and the liver's contribution to the development of this system. Here we aimed to evaluate the presence of hematopoiesis in the yolk sac and liver in chickens, from the stages of intra-aortic clusters in the aorta-genital ridges-mesonephros (AGM) region until hatching, and how it relates to the establishment of the bone marrow. Gallus gallus domesticus L. embryos and their respective yolk sacs at embryonic day 3 (E3) and up to E21 were collected and processed according to standard histological techniques for paraffin embedding. The slides were stained with hematoxylin-eosin, Lennert's Giemsa, and Sirius Red at pH 10.2, and investigated by light microscopy. This study demonstrated that the yolk sac was a unique hematopoietic site between E4 and E12. Hematopoiesis occurred in the yolk sac and bone marrow between E13 and E20. The liver showed granulocytic differentiation in the connective tissue of portal spaces at E15 and onwards. The yolk sac showed expansion of erythrocytic and granulocytic lineages from E6 to E19, and E7 to E20, respectively. The results suggest that the yolk sac is the major intermediate erythropoietic and granulopoietic site where expansion and differentiation occur during chicken development. The hepatic hematopoiesis is restricted to the portal spaces and represented by the granulocytic lineage. PMID:24621665

  16. Correlation between nicotine-induced inhibition of hematopoiesis and decreased CD44 expression on bone marrow stromal cells.

    PubMed

    Khaldoyanidi, S; Sikora, L; Orlovskaya, I; Matrosova, V; Kozlov, V; Sriramarao, P

    2001-07-15

    This study demonstrates that in vivo exposure to cigarette smoke (CS) and in vitro treatment of long-term bone marrow cultures (LTBMCs) with nicotine, a major constituent of CS, result in inhibition of hematopoiesis. Nicotine treatment significantly delayed the onset of hematopoietic foci and reduced their size. Furthermore, the number of long-term culture-initiating cells (LTC-ICs) within an adherent layer of LTBMCs was significantly reduced in cultures treated with nicotine. Although the production of nonadherent mature cells and their progenitors in nicotine-treated LTBMCs was inhibited, this treatment failed to influence the proliferation of committed hematopoietic progenitors when added into methylcellulose cultures. Bone marrow stromal cells are an integral component of the hematopoietic microenvironment and play a critical role in the regulation of hematopoietic stem cell proliferation and self-renewal. Exposure to nicotine decreased CD44 surface expression on primary bone marrow-derived fibroblastlike stromal cells and MS-5 stromal cell line, but not on hematopoietic cells. In addition, mainstream CS altered the trafficking of hematopoietic stem/progenitor cells (HSPC) in vivo. Exposure of mice to CS resulted in the inhibition of HSPC homing into bone marrow. Nicotine and cotinine treatment resulted in reduction of CD44 surface expression on lung microvascular endothelial cell line (LEISVO) and bone marrow-derived (STR-12) endothelial cell line. Nicotine treatment increased E-selectin expression on LEISVO cells, but not on STR-12 cells. These findings demonstrate that nicotine can modulate hematopoiesis by affecting the functions of the hematopoiesis-supportive stromal microenvironment, resulting in the inhibition of bone marrow seeding by LTC-ICs and interfering with stem cell homing by targeting microvascular endothelial cells.

  17. Oral feeding with polyunsaturated fatty acids fosters hematopoiesis and thrombopoiesis in healthy and bone marrow-transplanted mice.

    PubMed

    Limbkar, Kedar; Dhenge, Ankita; Jadhav, Dipesh D; Thulasiram, Hirekodathakallu V; Kale, Vaijayanti; Limaye, Lalita

    2017-09-01

    Hematopoietic stem cells play the vital role of maintaining appropriate levels of cells in blood. Therefore, regulation of their fate is essential for their effective therapeutic use. Here we report the role of polyunsaturated fatty acids (PUFAs) in regulating hematopoiesis which has not been explored well so far. Mice were fed daily for 10 days with n-6/n-3 PUFAs, viz. linoleic acid (LA), arachidonic acid (AA), alpha-linolenic acid and docosahexanoic acid (DHA) in four separate test groups with phosphate-buffered saline fed mice as control set. The bone marrow cells of PUFA-fed mice showed a significantly higher hematopoiesis as assessed using side population, Lin-Sca-1(+)ckit+, colony-forming unit (CFU), long-term culture, CFU-spleen assay and engraftment potential as compared to the control set. Thrombopoiesis was also stimulated in PUFA-fed mice. A combination of DHA and AA was found to be more effective than when either was fed individually. Higher incorporation of PUFAs as well as products of their metabolism was observed in the bone marrow cells of PUFA-fed mice. A stimulation of the Wnt, CXCR4 and Notch1 pathways was observed in PUFA-fed mice. The clinical relevance of this study was evident when bone marrow-transplanted recipient mice, which were fed with PUFAs, showed higher engraftment of donor cells, suggesting that the bone marrow microenvironment may also be stimulated by feeding with PUFAs. These data indicate that oral administration of PUFAs in mice stimulates hematopoiesis and thrombopoiesis and could serve as a valuable supplemental therapy in situations of hematopoietic failure. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Posttraumatic Chondrocyte Apoptosis in the Murine Xiphoid.

    PubMed

    Davis, Christopher G; Eisner, Eric; McGlynn, Margaret; Shelton, John M; Richardson, James; Borrelli, Joseph; Chen, Christopher C T

    2013-10-01

    To demonstrate posttraumatic chondrocyte apoptosis in the murine xiphoid after a crush-type injury and to ultimately determine the pathway (i.e., intrinsic or extrinsic) by which chondrocytes undergo apoptosis in response to mechanical injury. The xiphoids of adult female wild-type mice were injured with the use of a modified Kelly clamp. Postinjury xiphoid cartilage was analyzed via 3 well-described independent means of assessing apoptosis in chondrocytes: hematoxylin and eosin staining, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, and activated caspase-3 staining. Injured specimens contained many chondrocytes with evidence of apoptosis, which is characterized by cell shrinkage, chromatin condensation, nuclear fragmentation, and the liberation of apoptotic bodies. There was a statistically significant increase in the number of chondrocytes undergoing apoptosis in the injured specimens as compared with the uninjured specimens. Chondrocytes can be stimulated to undergo apoptosis as a result of mechanical injury. These experiments involving predominantly cartilaginous murine xiphoid in vivo establish a baseline for future investigations that employ the genetic and therapeutic modulation of chondrocyte apoptosis in response to mechanical injury.

  19. Extramedullary hematopoiesis presenting as a compressive cord and cerebral lesion in a patient without a significant hematologic disorder: a case report

    PubMed Central

    2010-01-01

    Introduction Intracranial or spinal compressive lesions due to extramedullary hematopoiesis have been reported in the medical literature. Most of the reported cases are extradural lesions or, on rare occasions, foci within another neoplasm such as hemangioblastoma, meningioma or pilocytic astrocytoma. Often these cases occur in patients with an underlying hematological disorder such as acute myelogenic leukemia, myelofibrosis, or other myelodysplastic syndromes. Such lesions have also been reported in thalassemia major. Case presentation We report the case of a 43-year-old Iranian woman in whom extramedullary hematopoiesis presented as a compressive cord lesion and then later as an intracranial lesion. Conclusions To the best of our knowledge, we document the first reported case of sacral, lumbar, thoracic and cranial involvement in the same patient with extramedullary hematopoiesis, which seems both rare and remarkable. PMID:20939863

  20. Extramedullary hematopoiesis presenting as a compressive cord and cerebral lesion in a patient without a significant hematologic disorder: a case report.

    PubMed

    Seddighi, Amir Saied; Seddighi, Afsoun

    2010-10-12

    Intracranial or spinal compressive lesions due to extramedullary hematopoiesis have been reported in the medical literature. Most of the reported cases are extradural lesions or, on rare occasions, foci within another neoplasm such as hemangioblastoma, meningioma or pilocytic astrocytoma. Often these cases occur in patients with an underlying hematological disorder such as acute myelogenic leukemia, myelofibrosis, or other myelodysplastic syndromes. Such lesions have also been reported in thalassemia major. We report the case of a 43-year-old Iranian woman in whom extramedullary hematopoiesis presented as a compressive cord lesion and then later as an intracranial lesion. To the best of our knowledge, we document the first reported case of sacral, lumbar, thoracic and cranial involvement in the same patient with extramedullary hematopoiesis, which seems both rare and remarkable.

  1. Targeted disruption of the murine fps/fes proto-oncogene reveals that Fps/Fes kinase activity is dispensable for hematopoiesis.

    PubMed

    Senis, Y; Zirngibl, R; McVeigh, J; Haman, A; Hoang, T; Greer, P A

    1999-11-01

    The fps/fes proto-oncogene encodes a cytoplasmic protein-tyrosine kinase that is functionally implicated in the survival and terminal differentiation of myeloid progenitors and in signaling from several members of the cytokine receptor superfamily. To gain further insight into the physiological function of fps/fes, we targeted the mouse locus with a kinase-inactivating missense mutation. Mutant Fps/Fes protein was expressed at normal levels in these mice, but it lacked detectable kinase activity. Homozygous mutant animals were viable and fertile, and they showed no obvious defects. Flow cytometry analysis of bone marrow showed no statistically significant differences in the levels of myeloid, erythroid, or B-cell precursors. Subtle abnormalities observed in mutant mice included slightly elevated total leukocyte counts and splenomegaly. In bone marrow hematopoietic progenitor cell colony-forming assays, mutant mice gave slightly elevated numbers and variable sizes of CFU-granulocyte macrophage in response to interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Tyrosine phosphorylation of Stat3 and Stat5A in bone marrow-derived macrophages was dramatically reduced in response to GM-CSF but not to IL-3 or IL-6. This suggests a distinct nonredundant role for Fps/Fes in signaling from the GM-CSF receptor that does not extend to the closely related IL-3 receptor. Lipopolysaccharide-induced Erk1/2 activation was also reduced in mutant macrophages. These subtle molecular phenotypes suggest a possible nonredundant role for Fps/Fes in myelopoiesis and immune responses.

  2. Spatial and temporal expression of c-Kit in the development of the murine submandibular gland.

    PubMed

    Wang, Xuejiu; Qi, Senrong; Wang, Jinsong; Xia, Dengsheng; Qin, Lizheng; Zheng, Zongmei; Wang, Liping; Zhang, Chunmei; Jin, Luyuan; Ding, Gang; Wang, Songlin; Fan, Zhipeng

    2014-08-01

    The c-Kit pathway is important in the development of many mammalian cells and organs and is indispensable for the development of hematopoiesis, melanocytes, and primordial germ cells. Loss-of-function mutations in c-Kit lead to perinatal death in mouse embryos. Previously, c-Kit has been used as one of salivary epithelial stem or progenitor cell markers in mouse, its specific temporo-spatial expression pattern and function in developing murine submandibular gland (SMG) is still unclear. Here we used quantitative real-time PCR, in situ hybridization, and immunohistochemistry analysis to detect c-Kit expression during the development of the murine SMG. We found that c-Kit was expressed in the epithelia of developing SMGs from embryonic day 11.5 (E11.5; initial bud stage) to postnatal day 90 (P90; when the SMG is completely mature). c-Kit expression in the end bud epithelium increased during prenatal development and then gradually decreased after birth until its expression was undetectable in mature acini at P30. Moreover, c-Kit was expressed in the SMG primordial cord at the initial bud, pseudoglandular, canacular, and terminal end bud stages. c-Kit was also expressed in the presumptive ductal cells adjacent to the developing acini. By the late terminal end bud stage on P14, c-Kit expression could not be detected in ductal cells. However, c-Kit expression was detected in ductal cells at P30, and its expression had increased dramatically at P90. Taken together, these findings describe the spatial and temporal expression pattern of c-Kit in the developing murine SMG and suggest that c-Kit may play roles in epithelial histo-morphogenesis and in ductal progenitor cell homeostasis in the SMG.

  3. Monoclonal antibodies reacting with murine teratocarcinoma cells.

    PubMed Central

    Goodfellow, P N; Levinson, J R; Williams, V E; McDevitt, H O

    1979-01-01

    Monoclonal antibodies were produced in vitro by fusing mouse myeloma cells with spleen cells from a rat immunized with the C3H mouse teratocarcinoma C86-S1. After the fusion two clones were chosen for further analysis. The first clone, 3C4-10, produced an antibody recognizing an antigen with a distribution restricted to teratocarcinoma cell lines, an endoderm cell line, and a neuroblastoma. The second clone, 4A1-9, produced an antibody that reacted with all cultured murine cells tested and adult brain. Neither antibody reacted with preimplantation embryos. The 3C4-10 antibody recognized an antigen associated with proteins. The apparent molecular weight of the 3C4-10 antigen was greater than 100,000. PMID:284353

  4. Extramedullary hematopoiesis generates Ly-6C(high) monocytes that infiltrate atherosclerotic lesions.

    PubMed

    Robbins, Clinton S; Chudnovskiy, Aleksey; Rauch, Philipp J; Figueiredo, Jose-Luiz; Iwamoto, Yoshiko; Gorbatov, Rostic; Etzrodt, Martin; Weber, Georg F; Ueno, Takuya; van Rooijen, Nico; Mulligan-Kehoe, Mary Jo; Libby, Peter; Nahrendorf, Matthias; Pittet, Mikael J; Weissleder, Ralph; Swirski, Filip K

    2012-01-17

    Atherosclerotic lesions are believed to grow via the recruitment of bone marrow-derived monocytes. Among the known murine monocyte subsets, Ly-6C(high) monocytes are inflammatory, accumulate in lesions preferentially, and differentiate. Here, we hypothesized that the bone marrow outsources the production of Ly-6C(high) monocytes during atherosclerosis. Using murine models of atherosclerosis and fate-mapping approaches, we show that hematopoietic stem and progenitor cells progressively relocate from the bone marrow to the splenic red pulp, where they encounter granulocyte macrophage colony-stimulating factor and interleukin-3, clonally expand, and differentiate to Ly-6C(high) monocytes. Monocytes born in such extramedullary niches intravasate, circulate, and accumulate abundantly in atheromata. On lesional infiltration, Ly-6C(high) monocytes secrete inflammatory cytokines, reactive oxygen species, and proteases. Eventually, they ingest lipids and become foam cells. Our findings indicate that extramedullary sites supplement the hematopoietic function of the bone marrow by producing circulating inflammatory cells that infiltrate atherosclerotic lesions. © 2011 American Heart Association, Inc.

  5. ZIP8 Zinc Transporter: Indispensable Role for Both Multiple-Organ Organogenesis and Hematopoiesis In Utero

    PubMed Central

    Gálvez-Peralta, Marina; He, Lei; Jorge-Nebert, Lucia F.; Wang, Bin; Miller, Marian L.; Eppert, Bryan L.; Afton, Scott; Nebert, Daniel W.

    2012-01-01

    Previously this laboratory characterized Slc39a8-encoded ZIP8 as a Zn2+/(HCO3–)2 symporter; yet, the overall physiological importance of ZIP8 at the whole-organism level remains unclear. Herein we describe the phenotype of the hypomorphic Slc39a8(neo/neo) mouse which has retained the neomycin-resistance gene in intron 3, hence causing significantly decreased ZIP8 mRNA and protein levels in embryo, fetus, placenta, yolk sac, and several tissues of neonates. The Slc39a8(neo) allele is associated with diminished zinc and iron uptake in mouse fetal fibroblast and liver-derived cultures; consequently, Slc39a8(neo/neo) newborns exhibit diminished zinc and iron levels in several tissues. Slc39a8(neo/neo) homozygotes from gestational day(GD)-11.5 onward are pale, growth-stunted, and die between GD18.5 and 48 h postnatally. Defects include: severely hypoplastic spleen; hypoplasia of liver, kidney, lung, and lower limbs. Histologically, Slc39a8(neo/neo) neonates show decreased numbers of hematopoietic islands in yolk sac and liver. Low hemoglobin, hematocrit, red cell count, serum iron, and total iron-binding capacity confirmed severe anemia. Flow cytometry of fetal liver cells revealed the erythroid series strikingly affected in the hypomorph. Zinc-dependent 5-aminolevulinic acid dehydratase, required for heme synthesis, was not different between Slc39a8(+/+) and Slc39a8(neo/neo) offspring. To demonstrate further that the mouse phenotype is due to ZIP8 deficiency, we bred Slc39a8(+/neo) with BAC-transgenic BTZIP8-3 line (carrying three extra copies of the Slc39a8 allele); this cross generated viable Slc39a8(neo/neo)_BTZIP8-3(+/+) pups showing none of the above-mentioned congenital defects–proving Slc39a8(neo/neo) causes the described phenotype. Our study demonstrates that ZIP8-mediated zinc transport plays an unappreciated critical role during in utero and neonatal growth, organ morphogenesis, and hematopoiesis. PMID:22563477

  6. ZIP8 zinc transporter: indispensable role for both multiple-organ organogenesis and hematopoiesis in utero.

    PubMed

    Gálvez-Peralta, Marina; He, Lei; Jorge-Nebert, Lucia F; Wang, Bin; Miller, Marian L; Eppert, Bryan L; Afton, Scott; Nebert, Daniel W

    2012-01-01

    Previously this laboratory characterized Slc39a8-encoded ZIP8 as a Zn(2+)/(HCO(3)(-))(2) symporter; yet, the overall physiological importance of ZIP8 at the whole-organism level remains unclear. Herein we describe the phenotype of the hypomorphic Slc39a8(neo/neo) mouse which has retained the neomycin-resistance gene in intron 3, hence causing significantly decreased ZIP8 mRNA and protein levels in embryo, fetus, placenta, yolk sac, and several tissues of neonates. The Slc39a8(neo) allele is associated with diminished zinc and iron uptake in mouse fetal fibroblast and liver-derived cultures; consequently, Slc39a8(neo/neo) newborns exhibit diminished zinc and iron levels in several tissues. Slc39a8(neo/neo) homozygotes from gestational day(GD)-11.5 onward are pale, growth-stunted, and die between GD18.5 and 48 h postnatally. Defects include: severely hypoplastic spleen; hypoplasia of liver, kidney, lung, and lower limbs. Histologically, Slc39a8(neo/neo) neonates show decreased numbers of hematopoietic islands in yolk sac and liver. Low hemoglobin, hematocrit, red cell count, serum iron, and total iron-binding capacity confirmed severe anemia. Flow cytometry of fetal liver cells revealed the erythroid series strikingly affected in the hypomorph. Zinc-dependent 5-aminolevulinic acid dehydratase, required for heme synthesis, was not different between Slc39a8(+/+) and Slc39a8(neo/neo) offspring. To demonstrate further that the mouse phenotype is due to ZIP8 deficiency, we bred Slc39a8(+/neo) with BAC-transgenic BTZIP8-3 line (carrying three extra copies of the Slc39a8 allele); this cross generated viable Slc39a8(neo/neo)_BTZIP8-3(+/+) pups showing none of the above-mentioned congenital defects-proving Slc39a8(neo/neo) causes the described phenotype. Our study demonstrates that ZIP8-mediated zinc transport plays an unappreciated critical role during in utero and neonatal growth, organ morphogenesis, and hematopoiesis.

  7. Zebrafish hoxd4a Acts Upstream of meis1.1 to Direct Vasculogenesis, Angiogenesis and Hematopoiesis

    PubMed Central

    Amali, Aseervatham Anusha; Sie, Lawrence; Winkler, Christoph; Featherstone, Mark

    2013-01-01

    Mice lacking the 4th-group paralog Hoxd4 display malformations of the anterior vertebral column, but are viable and fertile. Here, we report that zebrafish embryos having decreased function of the orthologous hoxd4a gene manifest striking perturbations in vasculogenesis, angiogenesis and primitive and definitive hematopoiesis. These defects are preceded by reduced expression of the hemangioblast markers scl1, lmo2 and fli1 within the posterior lateral plate mesoderm (PLM) at 13 hours post fertilization (hpf). Epistasis analysis revealed that hoxd4a acts upstream of meis1.1 but downstream of cdx4 as early as the shield stage in ventral-most mesoderm fated to give rise to hemangioblasts, leading us to propose that loss of hoxd4a function disrupts hemangioblast specification. These findings place hoxd4a high in a genetic hierarchy directing hemangioblast formation downstream of cdx1/cdx4 and upstream of meis1.1. An additional consequence of impaired hoxd4a and meis1.1 expression is the deregulation of multiple Hox genes implicated in vasculogenesis and hematopoiesis which may further contribute to the defects described here. Our results add to evidence implicating key roles for Hox genes in their initial phase of expression early in gastrulation. PMID:23554940

  8. Platelet factor 4 promotes adhesion of hematopoietic progenitor cells and binds IL-8: novel mechanisms for modulation of hematopoiesis.

    PubMed

    Dudek, Arkadiusz Z; Nesmelova, Irina; Mayo, Kevin; Verfaillie, Catherine M; Pitchford, Simon; Slungaard, Arne

    2003-06-15

    Platelet factor 4 (PF4) is an abundant platelet alpha-granule C-X-C chemokine that has weak chemotactic potency but strongly inhibits hematopoiesis through an unknown mechanism. We find that PF4 binds to human CD34+ hematopoietic progenitor cells (HPCs) with a median effective concentration of 1 microg/mL but not after exposure to chondroitinase ABC. PF4 enhances adhesion of HPCs to intact stroma. Committed progenitors also adhere avidly to immobilized PF4. This adhesion is time-dependent, requires metabolic activity, causes cytoskeletal rearrangement, and induces cell-cycle inhibition. Using extracellular acidification rate to indicate transmembrane signaling, we find that interleukin-8 (IL-8), but not PF4, activates CD34+ progenitors, and PF4 blocks IL-8-mediated activation. Surface plasmon resonance analysis shows that PF4 binds IL-8 with high (dissociation constant [Kd] = 42 nM) affinity. Nuclear magnetic resonance analysis of IL-8 and PF4 in solution confirms this interaction. We conclude that PF4 has the capacity to influence hematopoiesis through mechanisms not mediated by a classical high-affinity, 7-transmembrane domain chemokine receptor. Instead, PF4 may modulate the hematopoietic milieu both directly, by promoting progenitor adhesion and quiescence through interaction with an HPC chondroitin sulfate-containing moiety, and indirectly, by binding to or interfering with signaling caused by other, hematopoietically active chemokines, such as IL-8.

  9. Mice bearing a targeted interruption of the homeobox gene HOXA9 have defects in myeloid, erythroid, and lymphoid hematopoiesis.

    PubMed

    Lawrence, H J; Helgason, C D; Sauvageau, G; Fong, S; Izon, D J; Humphries, R K; Largman, C

    1997-03-15

    Several homeobox genes of the HOXA and HOXB clusters are expressed in primitive blood cells, suggesting a role for HOX genes in normal hematopoiesis. The HOXA9 gene is expressed in CD34+ marrow cells and in developing lymphocytes. We examined blood-forming organs of mice homozygous for an interrupted HOXA9 allele to determine if loss of HOX gene function is deleterious to hematopoiesis. HOXA9-/- mice have approximately 30% to 40% reductions in total leukocytes and lymphocytes (P < .001) and a blunted granulocytic response to granulocyte colony-stimulating factor (G-CSF). Homozygous mice have significantly smaller spleens and thymuses. Myeloid/erythroid and pre-B progenitors in the marrow are significantly reduced, but no significant decreases are noted in mixed colonies, day 12 colony-forming units-spleen (CFU-S), or long-term culture-initiating cells (LTC-IC), suggesting little or no perturbation in earlier progenitors. Heterozygous animals display no hematopoietic defects. The abnormalities in leukocyte production are transplantable, indicating that the defect resides in the hematopoietic cells. These studies demonstrate a physiologic role for a HOX gene in blood cell differentiation, with the greatest apparent influence of HOXA9 at the level of the committed progenitor.

  10. Murine typhus in travelers returning from Indonesia.

    PubMed Central

    Parola, P.; Vogelaers, D.; Roure, C.; Janbon, F.; Raoult, D.

    1998-01-01

    We report the first three documented cases of murine typhus imported into Europe from Indonesia, discuss clues for the diagnosis of the disease, and urge that murine fever be considered in the diagnosis of febrile disease in travelers. PMID:9866749

  11. Murine typhus in travelers returning from Indonesia.

    PubMed

    Parola, P; Vogelaers, D; Roure, C; Janbon, F; Raoult, D

    1998-01-01

    We report the first three documented cases of murine typhus imported into Europe from Indonesia, discuss clues for the diagnosis of the disease, and urge that murine fever be considered in the diagnosis of febrile disease in travelers.

  12. Tissue-Resident Macrophages in Pancreatic Ductal Adenocarcinoma Originate from Embryonic Hematopoiesis and Promote Tumor Progression.

    PubMed

    Zhu, Yu; Herndon, John M; Sojka, Dorothy K; Kim, Ki-Wook; Knolhoff, Brett L; Zuo, Chong; Cullinan, Darren R; Luo, Jingqin; Bearden, Audrey R; Lavine, Kory J; Yokoyama, Wayne M; Hawkins, William G; Fields, Ryan C; Randolph, Gwendalyn J; DeNardo, David G

    2017-08-15

    Tumor-associated macrophages (TAMs) are essential components of the cancer microenvironment and play critical roles in the regulation of tumor progression. Optimal therapeutic intervention requires in-depth understanding of the sources that sustain macrophages in malignant tissues. In this study, we investigated the ontogeny of TAMs in murine pancreatic ductal adenocarcinoma (PDAC) models. We identified both inflammatory monocytes and tissue-resident macrophages as sources of TAMs. Unexpectedly, significant portions of pancreas-resident macrophages originated from embryonic development and expanded through in situ proliferation during tumor progression. Whereas monocyte-derived TAMs played more potent roles in antigen presentation, embryonically derived TAMs exhibited a pro-fibrotic transcriptional profile, indicative of their role in producing and remodeling molecules in the extracellular matrix. Collectively, these findings uncover the heterogeneity of TAM origin and functions and could provide therapeutic insight for PDAC treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. The role of G-CSF and IL-6 in the granulopoiesis-stimulating activity of murine blood serum induced by perorally administered ultrafiltered pig leukocyte extract, IMUNOR.

    PubMed

    Vacek, Antonín; Hofer, Michal; Holá, Jirina; Weiterová, Lenka; Streitová, Denisa; Svoboda, Jaroslav

    2007-05-01

    IMUNOR, a low-molecular weight (< 12 kD) ultrafiltered pig leukocyte extract, has been previously found to have significant stimulatory effects on murine hematopoiesis supressed by ionizing radiation or cytotoxic drugs. This communication shows data on the mechanisms of these effects. Using ELISA assay, significantly increased levels of granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6) were observed. On the contrary, no detectable levels of granulocyte-macrophage colony-stimulating factor (GM-CFC) and interleukin-3 (IL-3) have been found in blood serum of IMUNOR-treated mice. Incubation of the serum from IMUNOR-treated mice with antibodies against G-CSF caused abrogation of the ability of the sera to stimulate in vitro growth of colonies originating from granulocyte-macrophage progenitor cells (GM-CFC). In contrast, incubation of the serum with antibodies against IL-6 did not change its colony-stimulating activity. It may be inferred from these findings that G-CSF is probably the main cytokine responsible for the granulopoiesis-stimulating effects of IMUNOR. When the serum from IMUNOR-treated mice with G-CSF inactivated by anti-G-CSF antibodies (but with elevated IL-6) was added to cultures of bone marrow cells together with a suboptimum concentration of IL-3, a significant increase in the numbers of GM-CFC colonies was found. Moreover, conjoint inactivation of G-CSF and IL-6 significantly decreased the numbers of GM-CFC colonies in comparison with those observed when only G-CSF was inactivated. This observation strongly suggests that though IMUNOR-induced IL-6 is not able to induce the growth of GM-CFC colonies alone, it is able to potentiate the hematopoiesis-stimulating effect of IL-3. These findings represent a new knowledge concerning the hematopoiesis-stimulating action of IMUNOR, a promising immunomodulatory agent.

  14. Activation of adenosine A(3) receptors supports hematopoiesis-stimulating effects of granulocyte colony-stimulating factor in sublethally irradiated mice.

    PubMed

    Hofer, Michal; Pospísil, Milan; Sefc, Ludek; Dusek, Ladislav; Vacek, Antonín; Holá, Jirina; Hoferová, Zuzana; Streitová, Denisa

    2010-08-01

    Research areas of 'post-exposure treatment' and 'cytokines and growth factors' have top priority among studies aimed at radiological nuclear threat countermeasures. The experiments were aimed at testing the ability of N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA), an adenosine A(3) receptor agonist, to modulate hematopoiesis in sublethally irradiated mice, when administered alone or in a combination with granulocyte colony-stimulating factor (G-CSF) in a two-day post-irradiation treatment regimen. A complete analysis of hematopoiesis including determination of numbers of bone marrow hematopoietic progenitor and precursor cells, as well as of numbers of peripheral blood cells, was performed. The outcomes of the treatment were assessed at days 3 to 22 after irradiation. IB-MECA alone has been found to induce a significant elevation of numbers of bone marrow granulocyte-macrophage progenitor cells (GM-CFC) and peripheral blood neutrophils. IB-MECA given concomitantly with G-CSF increased significantly bone marrow GM-CFC and erythroid progenitor cells (BFU-E) in comparison with the controls and with animals administered each of the drugs alone. The findings suggest the ability of IB-MECA to stimulate hematopoiesis and to support the hematopoiesis-stimulating effects of G-CSF in sublethally irradiated mice.

  15. Antimicrobial proteins of murine macrophages.

    PubMed Central

    Hiemstra, P S; Eisenhauer, P B; Harwig, S S; van den Barselaar, M T; van Furth, R; Lehrer, R I

    1993-01-01

    Three murine microbicidal proteins (MUMPs) were purified from cells of the murine macrophage cell line RAW264.7 that had been activated by gamma interferon. Similar proteins were also present in nonactivated RAW264.7 cells, in cells of the murine macrophage cell line J774A.1, and in resident and activated murine peritoneal macrophages. MUMP-1, MUMP-2, and MUMP-3 killed Salmonella typhimurium, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Mycobacterium fortuitum, and Cryptococcus neoformans in vitro. MUMP-1 resembled an H1 histone but was unusual because its N-terminal residue (serine) was not N acetylated. Although MUMP-2 was N terminally blocked, its high lysine/arginine ratio and its reactivity with an antibody to H1 histones suggested that it also belonged to the H1 histone family. MUMP-3 was identical to histone H2B in 30 of 30 amino-terminal residues. Although the antimicrobial properties of histones have been recognized for decades, this is the first evidence that such proteins may endow the lysosomal apparatus of macrophages with nonoxidative antimicrobial potential. Other MUMPs, including some with a more restricted antimicrobial spectrum and one that appeared to be induced in RAW264.7 cells after gamma interferon stimulation, were noted but remain to be characterized. Images PMID:8514411

  16. Conversion of danger signals into cytokine signals by hematopoietic stem and progenitor cells for regulation of stress-induced hematopoiesis

    PubMed Central

    O'Connell, Ryan M.; Mehta, Arnav; DiLoreto, Race; Heath, James R.; Baltimore, David

    2014-01-01

    SUMMARY During an infection, the body increases the output of mature immune cells to fight off the pathogen. Despite convincing evidence that hematopoietic stem and progenitor cells (HSPCs) can sense pathogens directly, how this contributes to hematopoietic cell output remains unknown. Here we have combined mouse models with a single cell proteomics platform to show that in response to toll-like receptor stimulation, short-term HSCs and multipotent progenitor cells produce copious amount of diverse cytokines through the NF-κB signaling. Interestingly, the cytokine production ability of HSPCs trumps mature immune cells in both magnitude and breadth. Among cytokines produced by HSPCs, IL-6 is a particularly important regulator of myeloid differentiation and HSPC proliferation in a paracrine manner and in mediating rapid myeloid cell recovery during neutropenia. This study has uncovered a novel property of HSPCs that enables them to convert danger signals into versatile cytokine signals for regulation of stress hematopoiesis. PMID:24561084

  17. [Bone metabolism and cardiovascular function update. Inter-communication between bone marrow hematopoiesis and skeletal/vascular network].

    PubMed

    Katayama, Yoshio

    2014-07-01

    The hematopoiesis takes place in the bone marrow. Because bone marrow is the "marrow" of the bone, bone marrow does not exist without bone. The specialized microenvironment for hematopoietic stem cells (HSCs) to be appropriately functional is called "niche" . In the recent ten years since the bone-forming osteoblast was identified as a HSC niche, the entire mesenchymal lineage cells from mesenchymal stem cells to end-terminal osteocytes have been recognized as niche cells or niche-modulators. Among these, mesenchymal stem/progenitor cells are located at perivascular area. The very recent study showed the difference between arteriolar and sinusoidal niches. It is likely that the vascular network and the bone tissue are connected by the mesenchymal lineage cells as a complex of bone forming system, and HSCs utilize this complex as a series of niche.

  18. Inferring Cell Differentiation Processes Based on Phylogenetic Analysis of Genome-Wide Epigenetic Information: Hematopoiesis as a Model Case

    PubMed Central

    Koyanagi, Kanako O.

    2015-01-01

    How cells divide and differentiate is a fundamental question in organismal development; however, the discovery of differentiation processes in various cell types is laborious and sometimes impossible. Phylogenetic analysis is typically used to reconstruct evolutionary processes based on inherent characters. It could also be used to reconstruct developmental processes based on the developmental changes that occur during cell proliferation and differentiation. In this study, DNA methylation information from differentiated hematopoietic cells was used to perform phylogenetic analyses. The results were assessed for their validity in inferring hierarchical differentiation processes of hematopoietic cells and DNA methylation processes of differentiating progenitor cells. Overall, phylogenetic analyses based on DNA methylation information facilitated inferences regarding hematopoiesis. PMID:25638259

  19. Acute arrest of hematopoiesis induced by infection with Staphylococcus epidermidis following total knee arthroplasty: A case report and literature review.

    PubMed

    Bi, Lintao; Li, Jun; Lu, Zhenxia; Shao, Hui; Wang, Ying

    2016-03-01

    Infection is one of the most severe complications of total knee prosthesis implantation. The present study reported the case of a 74-year-old female that developed a Staphylococcus epidermidis infection following a cemented total knee arthroplasty. A routine blood test revealed neutropenia and anemia, while S. epidermidis was detected in the peripheral blood and bone marrow. In the present case, S. epidermidis infection led to acute arrest of hematopoiesis (AAH), also known as aplastic crisis, which is the temporary cessation of red cell production. The development of AAH secondary to S. epidermidis infection is rare and, to the best of our knowledge, this is the first case reported in the literature. The present study increased our knowledge of this rare disease and its characteristics, which will enable physicians to be aware of the development of AAH as a rare complication of S. epidermidis infection.

  20. ZFP521 regulates murine hematopoietic stem cell function and facilitates MLL-AF9 leukemogenesis in mouse and human cells.

    PubMed

    Garrison, Brian S; Rybak, Adrian P; Beerman, Isabel; Heesters, Balthasar; Mercier, Francois E; Scadden, David T; Bryder, David; Baron, Roland; Rossi, Derrick J

    2017-08-03

    The concept that tumor-initiating cells can co-opt the self-renewal program of endogenous stem cells as a means of enforcing their unlimited proliferative potential is widely accepted, yet identification of specific factors that regulate self-renewal of normal and cancer stem cells remains limited. Using a comparative transcriptomic approach, we identify ZNF521/Zfp521 as a conserved hematopoietic stem cell (HSC)-enriched transcription factor in human and murine hematopoiesis whose function in HSC biology remains elusive. Competitive serial transplantation assays using Zfp521-deficient mice revealed that ZFP521 regulates HSC self-renewal and differentiation. In contrast, ectopic expression of ZFP521 in HSCs led to a robust maintenance of progenitor activity in vitro. Transcriptional analysis of human acute myeloid leukemia (AML) patient samples revealed that ZNF521 is highly and specifically upregulated in AMLs with MLL translocations. Using an MLL-AF9 murine leukemia model and serial transplantation studies, we show that ZFP521 is not required for leukemogenesis, although its absence leads to a significant delay in leukemia onset. Furthermore, knockdown of ZNF521 reduced proliferation in human leukemia cell lines possessing MLL-AF9 translocations. Taken together, these results identify ZNF521/ZFP521 as a critical regulator of HSC function, which facilitates MLL-AF9-mediated leukemic disease in mice.

  1. Blood morphology and the levels of selected cytokines related to hematopoiesis in occupational short-term exposure to lead.

    PubMed

    Dobrakowski, Michał; Boroń, Marta; Czuba, Zenon P; Birkner, Ewa; Chwalba, Artur; Hudziec, Edyta; Kasperczyk, Sławomir

    2016-08-15

    The aim of the study was to investigate the influence of a short-term exposure to lead on the blood morphology and the levels of selected cytokines related to hematopoiesis in occupationally exposed workers. The study population included 37 males occupationally exposed to lead for 36 to 44days. Their blood lead level raised from 10.7±7.67μg/dl at baseline to the level of 49.1±14.1μg/dl at the end of the study. The level of hemoglobin and values of MCH and MCHC were decreased due to a short-term exposure to lead by 2%, 2%, and 1%, respectively. The counts of WBC, LYM, and MXD increased significantly by 5%, 7%, and 35%. Similarly, the count of PLT increased by 7%, while PDW, MPV, and P-LCR decreased by 6%, 3%, and 9%, respectively. The levels of IL-7, G-CSF, HGF, PDGF AB/BB, SCF, and PECAM-1, decreased significantly by 30%, 33%, 8%, 30%, 25%, and 20%, respectively. A short-term occupational exposure to lead results in a decreased hemoglobin level and increased counts of WBC and PLT. Changes in counts and proportions of different types of leukocytes and decreased values of PLT indices, such as PDW, MPV, and P-LCR, due to the subacute lead-exposure may be associated with lead-induced decreased levels of cytokines related to hematopoiesis, including SCF, G-CSF, IL-7, and PDGF. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. The adult murine heart has a sparse, phagocytically active macrophage population that expands through monocyte recruitment and adopts an ‘M2’ phenotype in response to Th2 immunologic challenge

    PubMed Central

    Mylonas, Katie J.; Jenkins, Stephen J.; Castellan, Raphael F.P.; Ruckerl, Dominik; McGregor, Kieran; Phythian-Adams, Alexander T.; Hewitson, James P.; Campbell, Sharon M.; MacDonald, Andrew S.; Allen, Judith E.; Gray, Gillian A.

    2015-01-01

    Tissue resident macrophages have vital homeostatic roles in many tissues but their roles are less well defined in the heart. The present study aimed to identify the density, polarisation status and distribution of macrophages in the healthy murine heart and to investigate their ability to respond to immune challenge. Histological analysis of hearts from CSF-1 receptor (csf1-GFP; MacGreen) and CX3CR1 (Cx3cr1GFP/+) reporter mice revealed a sparse population of GFP positive macrophages that were evenly distributed throughout the left and right ventricular free walls and septum. F4/80+CD11b+ cardiac macrophages, sorted from myocardial homogenates, were able to phagocytose fluorescent beads in vitro and expressed markers typical of both ‘M1’ (IL-1β, TNF and CCR2) and ‘M2’ activation (Ym1, Arg 1, RELMα and IL-10), suggesting no specific polarisation in healthy myocardium. Exposure to Th2 challenge by infection of mice with helminth parasites Schistosoma mansoni, or Heligmosomoides polygyrus, resulted in an increase in cardiac macrophage density, adoption of a stellate morphology and increased expression of Ym1, RELMα and CD206 (mannose receptor), indicative of ‘M2’ polarisation. This was dependent on recruitment of Ly6ChighCCR2+ monocytes and was accompanied by an increase in collagen content. In conclusion, in the healthy heart resident macrophages are relatively sparse and have a phagocytic role. Following Th2 challenge this population expands due to monocyte recruitment and adopts an ‘M2’ phenotype associated with increased tissue fibrosis. PMID:25700973

  3. The adult murine heart has a sparse, phagocytically active macrophage population that expands through monocyte recruitment and adopts an 'M2' phenotype in response to Th2 immunologic challenge.

    PubMed

    Mylonas, Katie J; Jenkins, Stephen J; Castellan, Raphael F P; Ruckerl, Dominik; McGregor, Kieran; Phythian-Adams, Alexander T; Hewitson, James P; Campbell, Sharon M; MacDonald, Andrew S; Allen, Judith E; Gray, Gillian A

    2015-07-01

    Tissue resident macrophages have vital homeostatic roles in many tissues but their roles are less well defined in the heart. The present study aimed to identify the density, polarisation status and distribution of macrophages in the healthy murine heart and to investigate their ability to respond to immune challenge. Histological analysis of hearts from CSF-1 receptor (csf1-GFP; MacGreen) and CX3CR1 (Cx3cr1(GFP/+)) reporter mice revealed a sparse population of GFP positive macrophages that were evenly distributed throughout the left and right ventricular free walls and septum. F4/80+CD11b+ cardiac macrophages, sorted from myocardial homogenates, were able to phagocytose fluorescent beads in vitro and expressed markers typical of both 'M1' (IL-1β, TNF and CCR2) and 'M2' activation (Ym1, Arg 1, RELMα and IL-10), suggesting no specific polarisation in healthy myocardium. Exposure to Th2 challenge by infection of mice with helminth parasites Schistosoma mansoni, or Heligmosomoides polygyrus, resulted in an increase in cardiac macrophage density, adoption of a stellate morphology and increased expression of Ym1, RELMα and CD206 (mannose receptor), indicative of 'M2' polarisation. This was dependent on recruitment of Ly6ChighCCR2+ monocytes and was accompanied by an increase in collagen content. In conclusion, in the healthy heart resident macrophages are relatively sparse and have a phagocytic role. Following Th2 challenge this population expands due to monocyte recruitment and adopts an 'M2' phenotype associated with increased tissue fibrosis.

  4. Quantitative trait gene Slit2 positively regulates murine hematopoietic stem cell numbers

    PubMed Central

    Waterstrat, Amanda; Rector, Kyle; Geiger, Hartmut; Liang, Ying

    2016-01-01

    Hematopoietic stem cells (HSC) demonstrate natural variation in number and function. The genetic factors responsible for the variations (or quantitative traits) are largely unknown. We previously identified a gene whose differential expression underlies the natural variation of HSC numbers in C57BL/6 (B6) and DBA/2 (D2) mice. We now report the finding of another gene, Slit2, on chromosome 5 that also accounts for variation in HSC number. In reciprocal chromosome 5 congenic mice, introgressed D2 alleles increased HSC numbers, whereas B6 alleles had the opposite effect. Using gene array and quantitative polymerase chain reaction, we identified Slit2 as a quantitative trait gene whose expression was positively correlated with the number of HSCs. Ectopic expression of Slit2 not only increased the number of the long-term colony forming HSCs, but also enhanced their repopulation capacity upon transplantation. Therefore, Slit2 is a novel quantitative trait gene and a positive regulator of the number and function of murine HSCs. This finding suggests that Slit2 may be a potential therapeutic target for the effective in vitro and in vivo expansion of HSCs without compromising normal hematopoiesis. PMID:27503415

  5. Epigenetic chromatin states uniquely define the developmental plasticity of murine hematopoietic stem cells.

    PubMed

    Weishaupt, Holger; Sigvardsson, Mikael; Attema, Joanne L

    2010-01-14

    Heritable epigenetic signatures are proposed to serve as an important regulatory mechanism in lineage fate determination. To investigate this, we profiled chromatin modifications in murine hematopoietic stem cells, lineage-restricted progenitors, and CD4(+) T cells using modified genome-scale mini-chromatin immunoprecipitation technology. We show that genes involved in mature hematopoietic cell function associate with distinct chromatin states in stem and progenitor cells, before their activation or silencing upon cellular maturation. Many lineage-restricted promoters are associated with bivalent histone methylation and highly combinatorial histone modification patterns, which may determine their selective priming of gene expression during lineage commitment. These bivalent chromatin states are conserved in mammalian evolution, with a particular overrepresentation of promoters encoding key regulators of hematopoiesis. After differentiation into progenitors and T cells, activating histone modifications persist at transcriptionally repressed promoters, suggesting that these transcriptional programs might be reactivated after lineage restriction. Collectively, our data reveal the epigenetic framework that underlies the cell fate options of hematopoietic stem cells.

  6. The proteasome inhibitor bortezomib induces testicular toxicity by upregulation of oxidative stress, AMP-activated protein kinase (AMPK) activation and deregulation of germ cell development in adult murine testis

    SciTech Connect

    Li, Wei; Fu, Jianfang; Zhang, Shun; Zhao, Jie; Xie, Nianlin; Cai, Guoqing

    2015-06-01

    Understanding how chemotherapeutic agents mediate testicular toxicity is crucial in light of compelling evidence that male infertility, one of the severe late side effects of intensive cancer treatment, occurs more often than they are expected to. Previous study demonstrated that bortezomib (BTZ), a 26S proteasome inhibitor used to treat refractory multiple myeloma (MM), exerts deleterious impacts on spermatogenesis in pubertal mice via unknown mechanisms. Here, we showed that intermittent treatment with BTZ resulted in fertility impairment in adult mice, evidenced by testicular atrophy, desquamation of immature germ cells and reduced caudal sperm storage. These deleterious effects may originate from the elevated apoptosis in distinct germ cells during the acute phase and the subsequent disruption of Sertoli–germ cell anchoring junctions (AJs) during the late recovery. Mechanistically, balance between AMP-activated protein kinase (AMPK) activation and Akt/ERK pathway appeared to be indispensable for AJ integrity during the late testicular recovery. Of particular interest, the upregulated testicular apoptosis and the following disturbance of Sertoli–germ cell interaction may both stem from the excessive oxidative stress elicited by BTZ exposure. We also provided the in vitro evidence that AMPK-dependent mechanisms counteract follicle-stimulating hormone (FSH) proliferative effects in BTZ-exposed Sertoli cells. Collectively, BTZ appeared to efficiently prevent germ cells from normal development via multiple mechanisms in adult mice. Employment of antioxidants and/or AMPK inhibitor may represent an attractive strategy of fertility preservation in male MM patients exposed to conventional BTZ therapy and warrants further investigation. - Highlights: • Intermittent treatment with BTZ caused fertility impairment in adult mice. • BTZ treatment elicited apoptosis during early phase of testicular recovery. • Up-regulation of oxidative stress by BTZ treatment

  7. Ultrasound backscatter microscopy image-guided intraventricular gene delivery at murine embryonic age 9.5 and 10.5 produces distinct transgene expression patterns at the adult stage.

    PubMed

    Jang, Jiwon; Ahn, Jyhyun; Lee, Nayeon; Kim, Seong-Tae; Kweon, Dae-Hyuk; Cho, Jae Youl; Park, Kye Won; Kim, Sunyoung; Yoon, Keejung

    2013-01-01

    In utero injection of a retroviral vector into the embryonic telencephalon aided by ultrasound backscatter microscopy permits introduction of a gene of interest at an early stage of development. In this study, we compared the tissue distribution of gene expression in adult mice injected with retroviral vectors at different embryonic ages in utero. Following ultrasound image-guided gene delivery (UIGD) into the embryonic telencephalon, adult mice were subjected to whole-body luciferase imaging and immunohistochemical analysis at 6 weeks and 1 year postinjection. Luciferase activity was observed in a wide range of tissues in animals injected at embryonic age 9.5 (E9.5), whereas animals injected at E10.5 showed brain-localized reporter gene expression. These results suggest that mouse embryonic brain creates a closed and impermeable structure around E10. Therefore, by injecting a transgene before or after E10, transgene expression can be manipulated to be local or systemic. Our results also provide information that widens the applicability of UIGD beyond neuroscience studies.

  8. EphA2 Is a Therapy Target in EphA2-Positive Leukemias but Is Not Essential for Normal Hematopoiesis or Leukemia.

    PubMed

    Charmsaz, Sara; Beckett, Kirrilee; Smith, Fiona M; Bruedigam, Claudia; Moore, Andrew S; Al-Ejeh, Fares; Lane, Steven W; Boyd, Andrew W

    2015-01-01

    Members of the Eph family of receptor tyrosine kinases and their membrane bound ephrin ligands have been shown to play critical roles in many developmental processes and more recently have been implicated in both normal and pathological processes in post-embryonic tissues. In particular, expression studies of Eph receptors and limited functional studies have demonstrated a role for the Eph/ephrin system in hematopoiesis and leukemogenesis. In particular, EphA2 was reported on hematopoietic stem cells and stromal cells. There are also reports of EphA2 expression in many different types of malignancies including leukemia, however there is a lack of knowledge in understanding the role of EphA2 in hematopoiesis and leukemogenesis. We explored the role of EphA2 in hematopoiesis by analyzing wild type and EphA2 knockout mice. Mature, differentiated cells, progenitors and hematopoietic stem cells derived from knockout and control mice were analyzed and no significant abnormality was detected. These studies showed that EphA2 does not have an obligatory role in normal hematopoiesis. Comparative studies using EphA2-negative MLL-AF9 leukemias derived from EphA2-knockout animals showed that there was no detectable functional role for EphA2 in the initiation or progression of the leukemic process. However, expression of EphA2 in leukemias initiated by MLL-AF9 suggested that this protein might be a possible therapy target in this type of leukemia. We showed that treatment with EphA2 monoclonal antibody IF7 alone had no effect on tumorigenicity and latency of the MLL-AF9 leukemias, while targeting of EphA2 using EphA2 monoclonal antibody with a radioactive payload significantly impaired the leukemic process. Altogether, these results identify EphA2 as a potential radio-therapeutic target in leukemias with MLL translocation.

  9. EphA2 Is a Therapy Target in EphA2-Positive Leukemias but Is Not Essential for Normal Hematopoiesis or Leukemia

    PubMed Central

    Charmsaz, Sara; Beckett, Kirrilee; Smith, Fiona M.; Bruedigam, Claudia; Moore, Andrew S.; Al-Ejeh, Fares; Lane, Steven W.; Boyd, Andrew W.

    2015-01-01

    Members of the Eph family of receptor tyrosine kinases and their membrane bound ephrin ligands have been shown to play critical roles in many developmental processes and more recently have been implicated in both normal and pathological processes in post-embryonic tissues. In particular, expression studies of Eph receptors and limited functional studies have demonstrated a role for the Eph/ephrin system in hematopoiesis and leukemogenesis. In particular, EphA2 was reported on hematopoietic stem cells and stromal cells. There are also reports of EphA2 expression in many different types of malignancies including leukemia, however there is a lack of knowledge in understanding the role of EphA2 in hematopoiesis and leukemogenesis. We explored the role of EphA2 in hematopoiesis by analyzing wild type and EphA2 knockout mice. Mature, differentiated cells, progenitors and hematopoietic stem cells derived from knockout and control mice were analyzed and no significant abnormality was detected. These studies showed that EphA2 does not have an obligatory role in normal hematopoiesis. Comparative studies using EphA2-negative MLL-AF9 leukemias derived from EphA2-knockout animals showed that there was no detectable functional role for EphA2 in the initiation or progression of the leukemic process. However, expression of EphA2 in leukemias initiated by MLL-AF9 suggested that this protein might be a possible therapy target in this type of leukemia. We showed that treatment with EphA2 monoclonal antibody IF7 alone had no effect on tumorigenicity and latency of the MLL-AF9 leukemias, while targeting of EphA2 using EphA2 monoclonal antibody with a radioactive payload significantly impaired the leukemic process. Altogether, these results identify EphA2 as a potential radio-therapeutic target in leukemias with MLL translocation. PMID:26083390

  10. Integrated analysis of miRNA and mRNA during differentiation of human CD34+ cells delineates the regulatory roles of microRNA in hematopoiesis.

    PubMed

    Raghavachari, Nalini; Liu, Poching; Barb, Jennifer J; Yang, Yanqin; Wang, Richard; Nguyen, Quang Tri; Munson, Peter J

    2014-01-01

    In the process of human hematopoiesis, precise regulation of the expression of lineage-specific gene products is critical for multiple cell-fate decisions that govern cell differentiation, proliferation, and self-renewal. Given the important role of microRNAs (miRNAs) in development and differentiation, we examined the global expression of miRNA in CD34(+) cells during lineage specific hematopoiesis and found 49 miRNAs to be differentially expressed, with functional roles in cellular growth and proliferation, and apoptosis. miR-18a was upregulated during erythropoiesis and downregulated during megakaryopoiesis. miR-145 was upregulated during granulopoiesis and down regulated during erythropoiesis. Megakaryopoitic differentiation resulted in significant alteration in the expression of many miRNAs that are believed to play critical roles in the regulation of B and T cell differentiation. Target prediction analyses on three different miRNA databases indicated that TargetScan outperformed microCosm and miRDB in identifying potential miRNA targets associated with hematopoietic differentiation process. An integrated analysis of the observed miRNAs and messenger RNAs (mRNAs) resulted in 87 highly correlated miRNA-mRNA pairs that have major functional roles in cellular growth and proliferation, hematopoietic system development, and Wnt/B-catenin and Flt 3 signaling pathways. We believe that this study will enhance our understanding on the regulatory roles of miRNA in hematopoiesis by providing a library of mRNA-miRNA networks.

  11. Quantitative trait mapping reveals a regulatory axis involving peroxisome proliferator-activated receptors, PRDM16, transforming growth factor-β2 and FLT3 in hematopoiesis

    PubMed Central

    Avagyan, Serine; Aguilo, Francesca; Kamezaki, Kenjiro

    2011-01-01

    Hematopoiesis is the process whereby BM HSCs renew to maintain their number or to differentiate into committed progenitors to generate all blood cells. One approach to gain mechanistic insight into this complex process is the investigation of quantitative genetic variation in hematopoietic function among inbred mouse strains. We previously showed that TGF-β2 is a genetically determined positive regulator of hematopoiesis. In the presence of unknown nonprotein serum factors TGF-β2, but not TGF-β1 or -β3, enhances progenitor proliferation in vitro, an effect that is subject to mouse strain-dependent variation mapping to a locus on chr.4, Tb2r1. TGF-β2–deficient mice show hematopoietic defects, demonstrating the physiologic role of this cytokine. Here, we show that TGF-β2 specifically and predominantly cell autonomously enhances signaling by FLT3 in vitro and in vivo. A coding polymorphism in Prdm16 (PR-domain-containing 16) underlies Tb2r1 and differentially regulates transcriptional activity of peroxisome proliferator-activated receptor-γ (PPARγ), identifying lipid PPAR ligands as the serum factors required for regulation of FLT3 signaling by TGF-β2. We furthermore show that PPARγ agonists play a FLT3-dependent role in stress responses of progenitor cells. These observations identify a novel regulatory axis that includes PPARs, Prdm16, and TGF-β2 in hematopoiesis. PMID:21967974

  12. Quantitative trait mapping reveals a regulatory axis involving peroxisome proliferator-activated receptors, PRDM16, transforming growth factor-β2 and FLT3 in hematopoiesis.

    PubMed

    Avagyan, Serine; Aguilo, Francesca; Kamezaki, Kenjiro; Snoeck, Hans-Willem

    2011-12-01

    Hematopoiesis is the process whereby BM HSCs renew to maintain their number or to differentiate into committed progenitors to generate all blood cells. One approach to gain mechanistic insight into this complex process is the investigation of quantitative genetic variation in hematopoietic function among inbred mouse strains. We previously showed that TGF-β2 is a genetically determined positive regulator of hematopoiesis. In the presence of unknown nonprotein serum factors TGF-β2, but not TGF-β1 or -β3, enhances progenitor proliferation in vitro, an effect that is subject to mouse strain-dependent variation mapping to a locus on chr.4, Tb2r1. TGF-β2-deficient mice show hematopoietic defects, demonstrating the physiologic role of this cytokine. Here, we show that TGF-β2 specifically and predominantly cell autonomously enhances signaling by FLT3 in vitro and in vivo. A coding polymorphism in Prdm16 (PR-domain-containing 16) underlies Tb2r1 and differentially regulates transcriptional activity of peroxisome proliferator-activated receptor-γ (PPARγ), identifying lipid PPAR ligands as the serum factors required for regulation of FLT3 signaling by TGF-β2. We furthermore show that PPARγ agonists play a FLT3-dependent role in stress responses of progenitor cells. These observations identify a novel regulatory axis that includes PPARs, Prdm16, and TGF-β2 in hematopoiesis.

  13. Role of the thrombopoietin (TPO)/Mpl system: c-Mpl-like molecule/TPO signaling enhances early hematopoiesis in Xenopus laevis.

    PubMed

    Kakeda, Minoru; Kyuno, Jun-ichi; Kato, Takashi; Nishikawa, Mitsuo; Asashima, Makoto

    2002-02-01

    Multiple organs are induced in the primitive embryonic ectoderm excised from blastula stage Xenopus laevis embryos, under the strict control of mesoderm inducing factors. This in vitro system is useful for exploring the mechanisms of development. In this study, the function of thrombopoietin (TPO)/c-Mpl signaling in the development of hematopoietic cells was investigated. An optimal hematopoietic cell induction system was established to evaluate the influence of growth factors on hematopoiesis. It was found that exogenous TPO enhanced hematopoiesis in explants induced by activin and bone morphogenetic protein (BMP)-4 and increased the number of both erythrocytes and leukocytes in a dose-dependent manner. Addition of anti-c-Mpl antibody completely inhibited the expansion of hematopoietic cells stimulated by TPO, and the antibody specifically recognized blood-like cells. These results demonstrate that TPO acts on hematopoietic progenitors induced in explants and the c-Mpl-like molecule in Xenopus mediates the cellular function of TPO. We also found that forced expression of TPO in embryos promoted hematopoiesis in the ventral blood island and the dorsal-- lateral plate mesoderm. These results suggest that hematopoietic stem and progenitor cells are regulated by TPO/c-Mpl signaling from when they appear in their ontogeny. They also suggest that TPO/c-Mpl signaling play a crucial role in the formation of hematopoietic cells in Xenopus.

  14. Protein kinase R as mediator of the effects of interferon (IFN) gamma and tumor necrosis factor (TNF) alpha on normal and dysplastic hematopoiesis.

    PubMed

    Sharma, Bhumika; Altman, Jessica K; Goussetis, Dennis J; Verma, Amit K; Platanias, Leonidas C

    2011-08-05

    IFNγ and TNFα are potent inhibitors of hematopoiesis and have been implicated in the pathophysiology of bone marrow failure and myelodysplastic syndromes (MDS). We examined the role of protein kinase R (PKR) in the generation of the inhibitory effects of these myelosuppressive cytokines on hematopoiesis. Our data demonstrate that PKR is rapidly phosphorylated/activated in response to engagement of IFNγ or TNFα receptors in normal human hematopoietic progenitors. Such engagement of PKR is important for the suppressive effects of these cytokines on normal hematopoiesis. Pharmacological targeting of PKR using a specific inhibitor or siRNA-mediated PKR knockdown results in partial reversal of the suppressive effects of IFNγ and TNFα on normal human CD34+-derived myeloid (colony-forming unit-granulocyte-monocytic) and erythroid (burst-forming unit-erythroid) progenitors. Importantly, inhibition of PKR activity or expression increases hematopoietic colony formation from human MDS progenitors, suggesting that drugs that target PKR may provide a novel approach for the treatment of MDS and marrow failure syndromes. Altogether, our data establish that beyond its key role in the induction of IFN-antiviral responses, PKR plays important roles in signaling for IFNγ and other myelosuppressive cytokine receptors as a common mediator of signals for hematopoietic suppression.

  15. Murine Typhus, Reunion, France, 2011–2013

    PubMed Central

    Camuset, Guillaume; Socolovschi, Cristina; Moiton, Marie-Pierre; Kuli, Barbara; Foucher, Aurélie; Poubeau, Patrice; Borgherini, Gianandrea; Wartel, Guillaume; Audin, Héla; Raoult, Didier; Filleul, Laurent; Parola, Philippe; Pagès, Fréderic

    2015-01-01

    Murine typhus case was initially identified in Reunion, France, in 2012 in a tourist. Our investigation confirmed 8 autochthonous cases that occurred during January 2011–January 2013 in Reunion. Murine typhus should be considered in local patients and in travelers returning from Reunion who have fevers of unknown origin. PMID:25625653

  16. Pleiotropic roles of Notch signaling in normal, malignant, and developmental hematopoiesis in the human

    PubMed Central

    Kushwah, Rahul; Guezguez, Borhane; Lee, Jung Bok; Hopkins, Claudia I; Bhatia, Mickie

    2014-01-01

    The Notch signaling pathway is evolutionarily conserved across species and plays an important role in regulating cell differentiation, proliferation, and survival. It has been implicated in several different hematopoietic processes including early hematopoietic development as well as adult hematological malignancies in humans. This review focuses on recent developments in understanding the role of Notch signaling in the human hematopoietic system with an emphasis on hematopoietic initiation from human pluripotent stem cells and regulation within the bone marrow. Based on recent insights, we summarize potential strategies for treatment of human hematological malignancies toward the concept of targeting Notch signaling for fate regulation. PMID:25252682

  17. IL-10 regulates murine lupus.

    PubMed

    Yin, Zhinan; Bahtiyar, Gul; Zhang, Na; Liu, Lanzhen; Zhu, Ping; Robert, Marie E; McNiff, Jennifer; Madaio, Michael P; Craft, Joe

    2002-08-15

    MRL/MpJ-Tnfrsf6(lpr) (MRL/MpJ-Fas(lpr); MRL-Fas(lpr)) mice develop a spontaneous lupus syndrome closely resembling human systemic lupus erythematosus. To define the role of IL-10 in the regulation of murine lupus, IL-10 gene-deficient (IL-10(-/-)) MRL-Fas(lpr) (MRL-Fas(lpr) IL-10(-/-)) mice were generated and their disease phenotype was compared with littermates with one or two copies of an intact IL-10 locus (MRL-Fas(lpr) IL-10(+/-) and MRL-Fas(lpr) IL-10(+/+) mice, respectively). MRL-Fas(lpr) IL-10(-/-) mice developed severe lupus, with earlier appearance of skin lesions, increased lymphadenopathy, more severe glomerulonephritis, and higher mortality than their IL-10-intact littermate controls. The increased severity of lupus in MRL-Fas(lpr) IL-10(-/-) mice was closely associated with enhanced IFN-gamma production by both CD4(+) and CD8(+) cells and increased serum concentration of IgG2a anti-dsDNA autoantibodies. The protective effect of IL-10 in this lupus model was further supported by the observation that administration of rIL-10 reduced IgG2a anti-dsDNA autoantibody production in wild-type MRL-Fas(lpr) animals. In summary, our results provide evidence that IL-10 can down-modulate murine lupus through inhibition of pathogenic Th1 cytokine responses. Modulation of the level of IL-10 may be of potential therapeutic benefit for human lupus.

  18. Kit ligand cytoplasmic domain is essential for basolateral sorting in vivo and has roles in spermatogenesis and hematopoiesis

    PubMed Central

    Deshpande, Shayu; Agosti, Valter; Manova, Katia; Moore, Malcolm A.S.; Hardy, Matthew P.

    2009-01-01

    Juxtamembrane signaling via the membrane growth factor KitL is critical for Kit mediated functions. KitL has a conserved cytoplasmic domain, and has been shown to possess a monomeric leucine dependent basolateral targeting signal. To investigate the consequences in vivo of impaired basolateral KitL targeting in polarized epithelial cells, we have mutated this critical leucine to alanine using a knock-in strategy. KitLL263A/L263A mutant mice are pigmented normally and steady-state hematopoiesis is unaffected although peritoneal and skin mast cell numbers are significantly increased. KitL localization is affected in the Sertoli cells of the KitLL263A/L263A testis and testis size is reduced in these mice due to aberrant spermatogonial proliferation. Further, the effect of the KitL L263A mutation on the testicular phenotype is dosage dependent. The tubules of hemizygous KitLL263A/Sl mice completely lack germ cells in contrast to the weaker testicular phenotype of KitLL263A/L263A mice. The onset of the testis phenotype coincides with the formation of tight junctions between Sertoli cells during postnatal development. Thus, the altered sorting of KitL is dispensable for hematopoietic and melanogenic lineages, yet is crucial in the testicular environment, where the basal membranes of adjacent polarized Sertoli cells form a niche for the proliferating spermatogonia. PMID:19874813

  19. Conversion of danger signals into cytokine signals by hematopoietic stem and progenitor cells for regulation of stress-induced hematopoiesis.

    PubMed

    Zhao, Jimmy L; Ma, Chao; O'Connell, Ryan M; Mehta, Arnav; DiLoreto, Race; Heath, James R; Baltimore, David

    2014-04-03

    During an infection, the body increases the output of mature immune cells in order to fight off the pathogen. Despite convincing evidence that hematopoietic stem and progenitor cells (HSPCs) can sense pathogens directly, how this contributes to hematopoietic cell output remains unknown. Here, we have combined mouse models with a single-cell proteomics platform to show that, in response to Toll-like receptor stimulation, short-term HSCs and multipotent progenitor cells produce copious amounts of diverse cytokines through nuclear factor κB (NF-κB) signaling. Interestingly, the cytokine production ability of HSPCs trumps mature immune cells in both magnitude and breadth. Among cytokines produced by HSPCs, IL-6 is a particularly important regulator of myeloid differentiation and HSPC proliferation in a paracrine manner and in mediating rapid myeloid cell recovery during neutropenia. This study has uncovered an important property of HSPCs that enables them to convert danger signals into versatile cytokine signals for the regulation of stress hematopoiesis.

  20. Far Upstream Element Binding Protein Plays a Crucial Role in Embryonic Development, Hematopoiesis, and Stabilizing Myc Expression Levels

    PubMed Central

    Zhou, Weixin; Chung, Yang Jo; Parrilla Castellar, Edgardo R.; Zheng, Ying; Chung, Hye-Jung; Bandle, Russell; Liu, Juhong; Tessarollo, Lino; Batchelor, Eric; Aplan, Peter D.; Levens, David

    2017-01-01

    The transcription factor far upstream element binding protein (FBP) binds and activates the MYC promoter when far upstream element is via TFIIH helicase activity early in the transcription cycle. The fundamental biology and pathology of FBP are complex. In some tumors FBP seems pro-oncogenic, whereas in others it is a tumor suppressor. We generated an FBP knockout (Fubp1−/−) mouse to study FBP deficiency. FBP is embryo lethal from embryonic day 10.5 to birth. A spectrum of pathology is associated with FBP loss; besides cerebral hyperplasia and pulmonary hypoplasia, pale livers, hypoplastic spleen, thymus, and bone marrow, cardiac hypertrophy, placental distress, and small size were all indicative of anemia. Immunophenotyping of hematopoietic cells in wild-type versus knockout livers revealed irregular trilineage anemia, with deficits in colony formation. Despite normal numbers of hematopoietic stem cells, transplantation of Fubp1−/− hematopoietic stem cells into irradiated mice entirely failed to reconstitute hematopoiesis. In competitive transplantation assays against wild-type donor bone marrow, Fubp1−/− hematopoietic stem cells functioned only sporadically at a low level. Although cultures of wild-type mouse embryo fibroblasts set Myc levels precisely, Myc levels of mouse varied wildly between fibroblasts harvested from different Fubp1−/− embryos, suggesting that FBP contributes to Myc set point fixation. FBP helps to hold multiple physiologic processes to close tolerances, at least in part by constraining Myc expression. PMID:26774856

  1. Analysis of hematopoiesis in mice irradiated with 500 mGy of X rays at different stages of development

    SciTech Connect

    Grande, T.; Bueren, J.A.

    1995-09-01

    We have investigated whether a relatively low dose of 500 mGy of X rays given as a single acute irradiation at different stages of pre-and postnatal development induces significant changes in the content of femoral hematopoietic progenitores during a 1-year period after irradiation. Data obtained show that, in the case of 4-day-old embryos as well as in 2-day, 8-day and 12-week-old mice, this dose is below the threshold capable of inducing a long-term impairment of hematopoiesis in the mouse. Nevertheless, in mice irradiated at the 13th or the 17th day postconception, a hematopoietic dysfunction consisting of a significant reduction in the proportion of femoral granulocyte-macrophage colony-forming units (CFU-GM) was manifested 1 year after irradiation. Our study confirms that, for most stages of development in the mouse, a single acute X irradiation of 500 mGy is below the threshold dose capable of inducing deterministic effects in the mouse hematopoietic system, although it reveals the induction of a significant impairment in the CFU-GM population when irradiation is given at the late stages of embryonic development. 24 refs., 4 figs.

  2. Co-transplantation of Hematopoietic Stem Cells and Cxcr4 Gene-Transduced Mesenchymal Stem Cells Promotes Hematopoiesis.

    PubMed

    Chen, Wei; Li, Miao; Su, Guizhen; Zang, Yu; Yan, Zhiling; Cheng, Hai; Pan, Bin; Cao, Jiang; Wu, Qingyun; Zhao, Kai; Zhu, Feng; Zeng, Lingyu; Li, Zhenyu; Xu, Kailin

    2015-04-01

    Mesenchymal stem cells (MSCs) are a promising candidate for cellular therapies. Co-transplantation of MSCs and hematopoietic stem cells (HSCs) promotes successful engraftment and improves hematopoietic recovery. In this study, the effects of co-transplantation of HSCs and mouse bone marrow (BM)-derived MSCs overexpressing CXCR4 (CXCR4-MSC) on CXCR4-MSC homing capacity and the reconstitution potential in lethally irradiated mice were evaluated. Recovery of donor-derived peripheral blood leukocytes and platelets was accelerated when CXCR4-MSCs were co-transplanted with BM cells. The frequency of c-kit(+)Sca(+)Lin(-) HSCs was higher in recipient BM following co-transplantation of CXCR4-MSCs compared with the EGFP-MSC control and the BMT only groups. Surprisingly, the rate of early engraftment of donor-derived BM cells in recipients co-transplanted with CXCR4-MSCs was slightly lower than in the absence of MSCs on day 7. Moreover, co-transplantation of CXCR4-MSCs regulated the balance of T helper cells subsets. Hematopoietic tissue reconstitution was evaluated by histopathological analysis of BM and spleen. Co-transplantation of CXCR4-MSCs was shown to promote the recovery of hematopoietic organs. These findings indicate that co-transplantation of CXCR4-MSCs promotes the early phase of hematopoietic recovery and sustained hematopoiesis.

  3. Hemorrhage, Impaired Hematopoiesis, and Lethality in Mouse Embryos Carrying a Targeted Disruption of the Fli1 Transcription Factor†

    PubMed Central

    Spyropoulos, Demetri D.; Pharr, Pamela N.; Lavenburg, Kim R.; Jackers, Pascale; Papas, Takis S.; Ogawa, Makio; Watson, Dennis K.

    2000-01-01

    The Ets family of transcription factors have been suggested to function as key regulators of hematopoeisis. Here we describe aberrant hematopoeisis and hemorrhaging in mouse embryos homozygous for a targeted disruption in the Ets family member, Fli1. Mutant embryos are found to hemorrhage from the dorsal aorta to the lumen of the neural tube and ventricles of the brain (hematorrhachis) on embryonic day 11.0 (E11.0) and are dead by E12.5. Histological examinations and in situ hybridization reveal disorganization of columnar epithelium and the presence of hematomas within the neuroepithelium and disruption of the basement membrane lying between this and mesenchymal tissues, both of which express Fli1 at the time of hemorrhaging. Livers from mutant embryos contain few pronormoblasts and basophilic normoblasts and have drastically reduced numbers of colony forming cells. These defects occur with complete penetrance of phenotype regardless of the genetic background (inbred B6, hybrid 129/B6, or outbred CD1) or the targeted embryonic stem cell line used for the generation of knockout lines. Taken together, these results provide in vivo evidence for the role of Fli1 in the regulation of hematopoiesis and hemostasis. PMID:10891501

  4. Extramedullary hematopoiesis associated with organizing peritoneal hemorrhage: a report of 5 cases in patients presenting with primary gynecologic disorders.

    PubMed

    Ardakani, Nima Mesbah; Kumarasinghe, Marian Priyanthi; Spagnolo, Dominic V; Stewart, Colin J R

    2014-05-01

    Extramedullary hematopoiesis (EMH) usually occurs in patients with severe anemia or myelofibrosis, and involvement of the serous cavities is uncommon. A total of 5 cases of peritoneal EMH are presented in patients presenting with primary gynecologic pathology including endometrial adenosarcoma (n=2), ovarian leiomyosarcoma, and ovarian endometrioid adenocarcinoma (each n=1), all of which were associated with peritoneal metastases; the remaining patient had a hemorrhagic benign ovarian cyst. All cases were associated with organizing peritoneal hemorrhage, and EMH was localized to the reactive granulation tissue. EMH was not identified within the tumor tissue in the 4 neoplastic cases. Erythroid precursors were present in all cases and granulocytic precursors and megakaryocytes were identified in two and three cases, respectively. There was no evidence of EMH in the corresponding peritoneal fluid cytology preparations examined in 4 cases. None of the patients had a significant hematological abnormality at the time of presentation or during a mean follow-up period of 35 mo (range, 2-66 mo). The mechanism of peritoneal EMH in these cases is uncertain but most likely related to tissue hemorrhage and repair as described in other sites such as dura, myocardium, and synovium. Pathologists should be aware that EMH may involve the peritoneum to avoid misinterpretation of the findings, particularly in small biopsy or cytology samples.

  5. Extramedullary hematopoiesis in a case of benign mixed mammary tumor in a female dog: cytological and histopathological assessment

    PubMed Central

    2010-01-01

    Backgroud Extramedullary hematopoiesis (EMH) is defined as the presence of hematopoietic stem cells such as erythroid and myeloid lineage plus megakaryocytes in extramedullary sites like liver, spleen and lymph nodes and is usually associated with either bone marrow or hematological disorders. Mammary EMH is a rare condition either in human and veterinary medicine and can be associated with benign mixed mammary tumors, similarly to that described in this case. Case presentation Hematopoietic stem cells were found in a benign mixed mammary tumor of a 7-year-old female mongrel dog that presents a nodule in the left inguinal mammary gland. The patient did not have any hematological abnormalities. Cytological evaluation demonstrated two distinct cell populations, composed of either epithelial or mesenchymal cells, sometimes associated with a fibrillar acidophilic matrix, apart from megakaryocytes, osteoclasts, metarubricytes, prorubricytes, rubricytes, rubriblasts, promyelocytes, myeloblasts. Histological examination confirmed the presence of an active hematopoietic bone marrow within the bone tissue of a benign mammary mixed tumor. Conclusions EMH is a rare condition described in veterinary medicine that can be associated with mammary mixed tumors. It's detection can be associated with several neoplastic and non-neoplastic mammary lesions, i.e. osteosarcomas, mixed tumors and bone metaplasia. PMID:20846427

  6. Expression of the fetal hematopoiesis regulator FEV indicates leukemias of prenatal origin.

    PubMed

    Liu, T-H; Tang, Y-J; Huang, Y; Wang, L; Guo, X-L; Mi, J-Q; Liu, L-G; Zhu, H; Zhang, Y; Chen, L; Liu, X; Zhang, L-H; Ye, Q-J; Li, B-S; Tang, J-Y; Ford, A; Enver, T; Liu, F; Chen, G-Q; Hong, D-L

    2017-05-01

    The origin of cancers is associated with etiology as well as therapeutics. Several studies reveal that malignancies in children can originate in utero. However, a diagnostic approach to distinguish between cancers initiated pre- or postnatally is absent. Here we identified a transcriptional factor FEV (fifth Ewing variant) that was expressed in fetal hematopoietic cells and became silent after birth. We characterized that FEV was essential for the self-renewal of hematopoietic stem cells (HSCs). We next found that FEV was expressed in most infant leukemia samples, but seldom in adult samples, in accord with the known prenatal origins of the former. We further determined the majority of pediatric acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML) were FEV positive. Moreover, FEV knockdown markedly impaired the leukemia-propagating ability of leukemic stem cells. We therefore identified FEV is unique to fetal HSCs and stably expressed in leukemic cells of prenatal origin. It may also provide a tractable therapeutic target.

  7. Plaque assay for murine norovirus.

    PubMed

    Gonzalez-Hernandez, Mariam B; Bragazzi Cunha, Juliana; Wobus, Christiane E

    2012-08-22

    Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques. Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50). This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration. However, its limit of detection is higher compared to a plaque assay. In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of

  8. The aryl hydrocarbon receptor: Regulation of hematopoiesis and involvement in the progression of blood diseases

    PubMed Central

    Casado, Fanny L.; Singh, Kameshwar P.; Gasiewicz, Thomas A.

    2010-01-01

    The aryl hydrocarbon receptor (AhR) is a basic helix-loop-helix protein that belongs to the superfamily of environment-sensing PAS (Per-ARNT-Sim) proteins. A large number of ligands have been described to bind AhR and promote its nuclear translocation. In the nucleus, the AhR and its dimerization partner the AhR nuclear translocase (ARNT), also known as HIF1β, form a DNA-binding complex that acts as a transcriptional regulator. Animal and human data suggest that, beyond its mediating responses to xenobiotic and/or unknown endogenous ligands, the AhR has a role, although as yet undefined, in the regulation of cell cycle and inflammation. The AhR also appears to regulate the hematopoietic and immune systems during development and adult life in a cell-specific manner. While accidental exposure to xenobiotic AhR ligands has been associated with leukemia in humans, the specific mechanisms of AhR involvement are still not completely understood. However, recent data are consistent with a functional role of the AhR in the maintenance of hematopoietic stem and/or progenitor cells (HSCs/HPCs). Studies highlighting AhR-regulation of HSCs/HPCs provide a rational framework to understand their biology, a role of the AhR in hematopoietic diseases, and a means to develop interventions for these diseases. PMID:20171126

  9. MicroRNAs in Control of Stem Cells in Normal and Malignant Hematopoiesis

    PubMed Central

    Roden, Christine; Lu, Jun

    2016-01-01

    Studies on hematopoietic stem cells (HSCs) and leukemia stem cells (LSCs) have helped to establish the paradigms of normal and cancer stem cell concepts. For both HSCs and LSCs, specific gene expression programs endowed by their epigenome functionally distinguish them from their differentiated progenies. MicroRNAs (miRNAs), as a class of small non-coding RNAs, act to control post-transcriptional gene expression. Research in the past decade has yielded exciting findings elucidating the roles of miRNAs in control of multiple facets of HSC and LSC biology. Here we review recent progresses on the functions of miRNAs in HSC emergence during development, HSC switch from a fetal/neonatal program to an adult program, HSC self-renewal and quiescence, HSC aging, HSC niche, and malignant stem cells. While multiple different miRNAs regulate a diverse array of targets, two common themes emerge in HSC and LSC biology: miRNA mediated regulation of epigenetic machinery and cell signaling pathways. In addition, we propose that miRNAs themselves behave like epigenetic regulators, as they possess key biochemical and biological properties that can provide both stability and alterability to the epigenetic program. Overall, the studies of miRNAs in stem cells in the hematologic contexts not only provide key understandings to post-transcriptional gene regulation mechanisms in HSCs and LSCs, but also will lend key insights for other stem cell fields. PMID:27547713

  10. Tacrolimus prevents murine cerebral malaria.

    PubMed

    Bao, Lam Quoc; Nhi, Dang My; Huy, Nguyen Tien; Hamano, Shinjiro; Hirayama, Kenji

    2017-02-01

    Tacrolimus and mycophenolate mofetil are immunosuppressants frequently used in human organ transplantation. Tacrolimus is also reported to inhibit Plasmodium falciparum growth in vitro. Here, we report that tacrolimus prevented the death from cerebral malaria of Plasmodium berghei ANKA-infected C57BL/6J mice, but not their death from malaria due to the high parasitaemia and severe anaemia. The mycophenolate mofetil-treated mice showed higher mortality from cerebral malaria and succumbed to malaria earlier than tacrolimus-treated littermates. Tacrolimus attenuated the infiltration of mononuclear cells including pathogenic CD8(+) T cells into the brain. It appears to prevent murine cerebral malaria through the inhibition of cerebral infiltration of CD8(+) T cells. © 2016 John Wiley & Sons Ltd.

  11. Murine models of ulcerative colitis.

    PubMed

    Flynn, Christopher; Levine, Joel; Rosenberg, Daniel W

    2003-06-01

    Ulcerative colitis (UC) is an inflammatory bowel disease of unknown etiology limited to the large intestine. The disease is prevalent in industrial societies and is associated with specific ethnic populations. A number of murine models, each focused on distinct aspects of the disease process, were developed over the past 20 years to further our understanding of the pathogenesis of UC. These models have been and remain our best resource for the study of the disorder as a result of their homology to human UC and the ease in which they can be manipulated and examined. This review examines and distills what has been leamed from these models and how this information is related back to human UC.

  12. Murine model of TB meningitis.

    PubMed

    Gupta, Umesh Datta; Abbas, Ali; Kashyap, Raj Pal Singh; Gupta, Pushpa

    2016-12-01

    Central nervous system (CNS) infections caused by Mycobacterium tuberculosis (MTB) are the most severe forms of extrapulmonary TB (EPTB) due to high levels of mortality and neurological morbidity. Limited studies are available on CNS-TB animal-model development, despite the steady rise in cerebral-TB cases in India over the past decade. This study describes the development of a murine model of CNS-TB using a clinical strain (C3) isolated from the cerebrospinal fluid (CSF) of CNS-TB patients. Groups of mice were infected intravenously with an MTB C3 strain isolated from the CSF of CNS-TB patients in order to mimic the dynamics of actual infection. Brain and lung tissue were evaluated for bacterial burden, as well as histopathology and surrogate markers of TB infection at 30- and 50-days post-infection. Mice infected intravenously with MTB C3 strains showed progressive development of CNS disease, with high bacillary burden in the lungs during the initial stage (30days), which eventually disseminated to the brain at a later stage (50days). All C3-infected mice showed elevated levels of mycobacterial antigens and antibodies, as well as increased T cell adenosine deaminase activity in brain homogenates, which explicitly correlated with mycobacterial load in the brain and chronic brain pathology. High mortality rates (60%) were associated with mice infected with the C3 strain as compared to those of controls. Our findings demonstrated the design of a novel murine model of CNS-TB using a C3 strain and that replicated events of EPTB dissemination. This model will promote efforts to understand the pathogenesis CNS-TB infection for development of improved therapeutic interventions in the future. Copyright © 2016.

  13. Polysaccharides from the root of Angelica sinensis promotes hematopoiesis and thrombopoiesis through the PI3K/AKT pathway

    PubMed Central

    2010-01-01

    Background Dozens of Traditional Chinese Medicine (TCM) formulas have been used for promotion of "blood production" for centuries, and we are interested in developing novel thrombopoietic medicines from these TCMs. Our previous studies have demonstrated the hematopoietic effects of DangGui BuXue Tong (DBT), a formula composed of Radix Angelicae Sinensis and Radix Astragali in animal and cellular models. As a step further to identify and characterize the active chemical components of DBT, we tested the hematopoietic and particularly, thrombopoietic effects of polysaccharide-enriched fractions from the root of Radix Angelicae Sinensis (APS) in this study. Methods A myelosuppression mouse model was treated with APS (10 mg/kg/day). Peripheral blood cells from APS, thrombopoietin and vehicle-treated samples were then counted at different time-points. Using the colony-forming unit (CFU) assays, we determined the effects of APS on the proliferation and differentiation of hematopoietic stem/progenitor cells and megakaryocytic lineages. Using a megakaryocytic cell line M-07e as model, we analyzed the cellular apoptosis progression with and without APS treatment by Annexin V, Mitochondrial Membrane Potential and Caspase 3 assays. Last, the anti-apoptotic effect of APS on cells treated with Ly294002, a Phosphatidylinositol 3-Kinse inhibitor (PI3K) was also tested. Results In animal models, APS significantly enhanced not only the recovery of platelets, other blood cells and their progenitor cells, but also the formation of Colony Forming Unit (CFU). In M-07e cells, we observed the anti-apoptotic effect of APS. Treatment by Ly294002 alone increased the percentage of cells undergoing apoptosis. However, addition of APS to Ly294002-treated cells significantly reduced the percentage of cells undergoing apoptosis. Conclusions APS promotes hematopoiesis and thrombopoiesis in the mouse model. This effect likely resulted from the anti-apoptosis activity of APS and is likely to involve

  14. The midregion, nuclear localization sequence, and C terminus of PTHrP regulate skeletal development, hematopoiesis, and survival in mice

    PubMed Central

    Toribio, Ramiro E.; Brown, Holly A.; Novince, Chad M.; Marlow, Brandlyn; Hernon, Krista; Lanigan, Lisa G.; Hildreth, Blake E.; Werbeck, Jillian L.; Shu, Sherry T.; Lorch, Gwendolen; Carlton, Michelle; Foley, John; Boyaka, Prosper; McCauley, Laurie K.; Rosol, Thomas J.

    2010-01-01

    The functions of parathyroid hormone-related protein (PTHrP) on morphogenesis, cell proliferation, apoptosis, and calcium homeostasis have been attributed to its N terminus. Evidence suggests that many of these effects are not mediated by the N terminus but by the midregion, a nuclear localization sequence (NLS), and C terminus of the protein. A knock-in mouse lacking the midregion, NLS, and C terminus of PTHrP (PthrpΔ/Δ) was developed. PthrpΔ/Δ mice had craniofacial dysplasia, chondrodysplasia, and kyphosis, with most mice dying by d 5 of age. In bone, there were fewer chondrocytes and osteoblasts per area, bone mass was decreased, and the marrow was less cellular, with erythroid hypoplasia. Cellular proliferation was impaired, and apoptosis was increased. Runx2, Ocn, Sox9, Crtl1, β-catenin, Runx1, ephrin B2, cyclin D1, and Gata1 were underexpressed while P16/Ink4a, P21, GSK-3β, Il-6, Ffg3, and Ihh were overexpressed. Mammary gland development was aberrant, and energy metabolism was deregulated. These results establish that the midregion, NLS, and C terminus of PTHrP are crucial for the commitment of osteogenic and hematopoietic precursors to their lineages, and for survival, and many of the effects of PTHrP on development are not mediated by its N terminus. The down-regulation of Runx1, Runx2, and Sox9 indicates that PTHrP is a modulator of transcriptional activation during stem cell commitment. Toribio, R. E., Brown, H. A., Novince, C. M., Marlow, B. Hernon, K., Lanigan, L. G., Hildreth III, B. E., Werbeck, J. L., Shu, S. T., Lorch, G., Carlton, M., Foley, J., Boyaka, P., McCauley, L. K., Rosol, T. J. The midregion, nuclear localization sequence, and C terminus of PTHrP regulate skeletal development, hematopoiesis, and survival in mice. PMID:20145205

  15. Knockdown of a Zebrafish Aryl Hydrocarbon Receptor Repressor (AHRRa) Affects Expression of Genes Related to Photoreceptor Development and Hematopoiesis

    PubMed Central

    Aluru, Neelakanteswar; Jenny, Matthew J.; Hahn, Mark E.

    2014-01-01

    The aryl hydrocarbon receptor repressor (AHRR) is a transcriptional repressor of aryl hydrocarbon receptor (AHR) and hypoxia-inducible factor (HIF) and is regulated by an AHR-dependent mechanism. Zebrafish (Danio rerio) possess two AHRR paralogs; AHRRa regulates constitutive AHR signaling during development, whereas AHRRb regulates polyaromatic hydrocarbon-induced gene expression. However, little is known about the endogenous roles and targets of AHRRs. The objective of this study was to elucidate the role of AHRRs during zebrafish development using a loss-of-function approach followed by gene expression analysis. Zebrafish embryos were microinjected with morpholino oligonucleotides against AHRRa or AHRRb to knockdown AHRR protein expression. At 72 h postfertilization (hpf), microarray analysis revealed that the expression of 279 and 116 genes was altered by knockdown of AHRRa and AHRRb, respectively. In AHRRa-morphant embryos, 97 genes were up-regulated and 182 genes were down-regulated. Among the down-regulated genes were several related to photoreceptor function, including cone-specific genes such as several opsins (opn1sw1, opn1sw2, opn1mw1, and opn1lw2), phosphodiesterases (pde6H and pde6C), retinol binding protein (rbp4l), phosducin, and arrestins. Down-regulation was confirmed by RT-PCR and with samples from an independent experiment. The four genes tested (opn1sw1, pde6H, pde6C, and arr3b) were not inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin. AHRRa knockdown also caused up-regulation of embryonic hemoglobin (hbbe3), suggesting a role for AHRR in regulating hematopoiesis. Knockdown of AHRRb caused up-regulation of 31 genes and down-regulation of 85 genes, without enrichment for any specific biological process. Overall, these results suggest that AHRRs may have important roles in development, in addition to their roles in regulating xenobiotic signaling. PMID:24675095

  16. Optimized flow cytometry isolation of murine spermatocytes

    PubMed Central

    Gaysinskaya, Valeriya; Soh, Ina Y.; van der Heijden, Godfried W.; Bortvin, Alex

    2014-01-01

    Meiotic prophase I (MPI), is an initial stage of meiosis characterized by intricate homologous chromosome interactions, synapsis and DNA recombination. These processes depend on the complex, but poorly understood early MPI events of homologous chromosome search, alignment and pairing. Detailed molecular investigation of these early events requires isolation of individual MPI substages. Enrichment for Pachytene (P) and Diplotene (D) substages of late MPI was previously accomplished using flow cytometry. However, separation of early MPI spermatocytes, specifically, of Leptotene (L) and Zygotene (Z) substages, has been a challenge due to these cells’ similar characteristics. In this report, we describe an optimized Hoechst-33342 (Hoechst)-based flow cytometry approach for isolating individual MPI populations from adult murine testis. We get significant enrichment for individual L and Z spermatocytes, previously inseparable from each other, and optimize the isolation of other MPI substages. Our flow cytometry approach is a combination of three optimized strategies. The first is optimization of testis dissociation protocol that yields more consistent and reproducible testicular single cell suspension. The second involves optimization of flow cytometric gating protocol where a critical addition to the standard protocol for cell discrimination based on Hoechst fluorescence, involves a back-gating technique based on light scattering parameters. This step specifies selection of individual MPI substages. The third, is an addition of DNA content restriction to the gating protocol to minimize contamination from non-meiotic cells. Finally, we confirm significant enrichment of high-purity Preleptotene (PreL), L, Z, P and D MPI spermatocytes using stage-specific marker distribution. The technique will facilitate understanding of the molecular events underlying meiotic prophase I. PMID:24664803

  17. Contrasting spatial distribution and risk factors for past infection with scrub typhus and murine typhus in Vientiane City, Lao PDR.

    PubMed

    Vallée, Julie; Thaojaikong, Thaksinaporn; Moore, Catrin E; Phetsouvanh, Rattanaphone; Richards, Allen L; Souris, Marc; Fournet, Florence; Salem, Gérard; Gonzalez, Jean-Paul J; Newton, Paul N

    2010-12-07

    The aetiological diagnostic of fevers in Laos remains difficult due to limited laboratory diagnostic facilities. However, it has recently become apparent that both scrub and murine typhus are common causes of previous undiagnosed fever. Epidemiological data suggests that scrub typhus would be more common in rural areas and murine typhus in urban areas, but there is very little recent information on factors involved in scrub and murine typhus transmission, especially where they are sympatric - as is the case in Vientiane, the capital of the Lao PDR. We therefore determined the frequency of IgG seropositivity against scrub typhus (Orientia tsutsugamushi) and murine typhus (Rickettsia typhi), as indices of prior exposure to these pathogens, in randomly selected adults in urban and peri-urban Vientiane City (n = 2,002, ≥35 years). Anti-scrub and murine typhus IgG were detected by ELISA assays using filter paper elutes. We validated the accuracy of ELISA of these elutes against ELISA using serum samples. The overall prevalence of scrub and murine typhus IgG antibodies was 20.3% and 20.6%, respectively. Scrub typhus seropositivity was significantly higher among adults living in the periphery (28.4%) than in the central zone (13.1%) of Vientiane. In contrast, seroprevalence of murine typhus IgG antibodies was significantly higher in the central zone (30.8%) as compared to the periphery (14.4%). In multivariate analysis, adults with a longer residence in Vientiane were at significant greater risk of past infection with murine typhus and at lower risk for scrub typhus. Those with no education, living on low incomes, living on plots of land with poor sanitary conditions, living in large households, and farmers were at higher risk of scrub typhus and those living in neighborhoods with high building density and close to markets were at greater risk for murine typhus and at lower risk of scrub typhus past infection. This study underscores the intense circulation of both scrub

  18. Contrasting Spatial Distribution and Risk Factors for Past Infection with Scrub Typhus and Murine Typhus in Vientiane City, Lao PDR

    PubMed Central

    Vallée, Julie; Thaojaikong, Thaksinaporn; Moore, Catrin E.; Phetsouvanh, Rattanaphone; Richards, Allen L.; Souris, Marc; Fournet, Florence; Salem, Gérard; Gonzalez, Jean-Paul J.; Newton, Paul N.

    2010-01-01

    Background The aetiological diagnostic of fevers in Laos remains difficult due to limited laboratory diagnostic facilities. However, it has recently become apparent that both scrub and murine typhus are common causes of previous undiagnosed fever. Epidemiological data suggests that scrub typhus would be more common in rural areas and murine typhus in urban areas, but there is very little recent information on factors involved in scrub and murine typhus transmission, especially where they are sympatric - as is the case in Vientiane, the capital of the Lao PDR. Methodology and Principal Findings We therefore determined the frequency of IgG seropositivity against scrub typhus (Orientia tsutsugamushi) and murine typhus (Rickettsia typhi), as indices of prior exposure to these pathogens, in randomly selected adults in urban and peri-urban Vientiane City (n = 2,002, ≥35 years). Anti-scrub and murine typhus IgG were detected by ELISA assays using filter paper elutes. We validated the accuracy of ELISA of these elutes against ELISA using serum samples. The overall prevalence of scrub and murine typhus IgG antibodies was 20.3% and 20.6%, respectively. Scrub typhus seropositivity was significantly higher among adults living in the periphery (28.4%) than in the central zone (13.1%) of Vientiane. In contrast, seroprevalence of murine typhus IgG antibodies was significantly higher in the central zone (30.8%) as compared to the periphery (14.4%). In multivariate analysis, adults with a longer residence in Vientiane were at significant greater risk of past infection with murine typhus and at lower risk for scrub typhus. Those with no education, living on low incomes, living on plots of land with poor sanitary conditions, living in large households, and farmers were at higher risk of scrub typhus and those living in neighborhoods with high building density and close to markets were at greater risk for murine typhus and at lower risk of scrub typhus past infection

  19. Normal hematopoiesis and lack of β-catenin activation in osteoblasts of patients and mice harboring Lrp5 gain-of-function mutations.

    PubMed

    Galán-Díez, Marta; Isa, Adiba; Ponzetti, Marco; Nielsen, Morten Frost; Kassem, Moustapha; Kousteni, Stavroula

    2016-03-01

    Osteoblasts are emerging regulators of myeloid malignancies since genetic alterations in them, such as constitutive activation of β-catenin, instigate their appearance. The LDL receptor-related protein 5 (LRP5), initially proposed to be a co-receptor for Wnt proteins, in fact favors bone formation by suppressing gut-serotonin synthesis. This function of Lrp5 occurring in the gut is independent of β-catenin activation in osteoblasts. However, it is unknown whether Lrp5 can act directly in osteoblast to influence other functions that require β-catenin signaling, particularly, the deregulation of hematopoiesis and leukemogenic properties of β-catenin activation in osteoblasts, that lead to development of acute myeloid leukemia (AML). Using mice with gain-of-function (GOF) Lrp5 alleles (Lrp5(A214V)) that recapitulate the human high bone mass (HBM) phenotype, as well as patients with the T253I HBM Lrp5 mutation, we show here that Lrp5 GOF mutations in both humans and mice do not activate β-catenin signaling in osteoblasts. Consistent with a lack of β-catenin activation in their osteoblasts, Lrp5(A214V) mice have normal trilinear hematopoiesis. In contrast to leukemic mice with constitutive activation of β-catenin in osteoblasts (Ctnnb1(CAosb)), accumulation of early myeloid progenitors, a characteristic of AML, myeloid-blasts in blood, and segmented neutrophils or dysplastic megakaryocytes in the bone marrow, are not observed in Lrp5(A214V) mice. Likewise, peripheral blood count analysis in HBM patients showed normal hematopoiesis, normal percentage of myeloid cells, and lack of anemia. We conclude that Lrp5 GOF mutations do not activate β-catenin signaling in osteoblasts. As a result, myeloid lineage differentiation is normal in HBM patients and mice. This article is part of a Special Issue entitled: Tumor Microenvironment Regulation of Cancer Cell Survival, Metastasis, Inflammation, and Immune Surveillance edited by Peter Ruvolo and Gregg L. Semenza.

  20. Normal hematopoiesis and lack of β-catenin activation in osteoblasts of patients and mice harboring Lrp5 gain-of-function mutations☆, ☆☆

    PubMed Central

    Galán-Díez, Marta; Isa, Adiba; Ponzetti, Marco; Nielsen, Morten Frost; Kassem, Moustapha; Kousteni, Stavroula

    2016-01-01

    Osteoblasts are emerging regulators of myeloid malignancies since genetic alterations in them, such as constitutive activation of β-catenin, instigate their appearance. The LDL receptor-related protein 5 (LRP5), initially proposed to be a co-receptor for Wnt proteins, in fact favors bone formation by suppressing gut-serotonin synthesis. This function of Lrp5 occurring in the gut is independent of β-catenin activation in osteoblasts. However, it is unknown whether Lrp5 can act directly in osteoblast to influence other functions that require β-catenin signaling, particularly, the deregulation of hematopoiesis and leukemogenic properties of β-catenin activation in osteoblasts, that lead to development of acute myeloid leukemia (AML). Using mice with gain-of-function (GOF) Lrp5 alleles (Lrp5A214V) that recapitulate the human high bone mass (HBM) phenotype, as well as patients with the T253I HBM Lrp5 mutation, we show here that Lrp5 GOF mutations in both humans and mice do not activate β-catenin signaling in osteoblasts. Consistent with a lack of β-catenin activation in their osteoblasts, Lrp5A214V mice have normal trilinear hematopoiesis. In contrast to leukemic mice with constitutive activation of β-catenin in osteoblasts (Ctnnb1CAosb), accumulation of early myeloid progenitors, a characteristic of AML, myeloid-blasts in blood, and segmented neutrophils or dysplastic megakaryocytes in the bone marrow, are not observed in Lrp5A214V mice. Likewise, peripheral blood count analysis in HBM patients showed normal hematopoiesis, normal percentage of myeloid cells, and lack of anemia. We conclude that Lrp5 GOF mutations do not activate β-catenin signaling in osteoblasts. As a result, myeloid lineage differentiation is normal in HBM patients and mice. This article is part of a Special Issue entitled: Tumor Microenvironment Regulation of Cancer Cell Survival, Metastasis, Inflammation, and Immune Surveillance edited by Peter Ruvolo and Gregg L. Semenza. PMID:26681532

  1. IFN-γ-mediated hematopoietic cell destruction in murine models of immune-mediated bone marrow failure

    PubMed Central

    Feng, Xingmin; Desierto, Marie J.; Keyvanfar, Keyvan; Young, Neal S.

    2015-01-01

    Interferon gamma (IFN-γ) has been reported to have both negative and positive activity on hematopoietic cells, adding complexity to the interpretation of its pleiotropic functions. We examined the effects of IFN-γ on murine hematopoietic stem cells (HSCs) and progenitors in vitro and in vivo by using mouse models. IFN-γ treatment expanded bone marrow (BM) c-Kit+Sca1+Lin– (KSL) cell number but reduced BM KLCD150+ and KLCD150+CD48– cells. IFN-γ-expanded KSL cells engrafted poorly when tested by competitive repopulation in vivo. KSL, KLCD150+, and KLCD150+CD48– cells from IFN-γ-treated animals all showed significant upregulation in Fas expression. When cocultured with activated T cells in vitro, KSL and KLCD150+ cells from IFN-γ-treated donors showed increased apoptosis relative to those from untreated animals, and infusion of activated CD8 T cells into IFN-γ-injected animals in vivo led to partial elimination of KSL cells. Exposure of BM cells or KSL cells to IFN-γ increased expression of Fas, caspases, and related proapoptotic genes and decreased expression of Ets-1 and other hematopoietic genes. In mouse models of BM failure, mice genetically deficient in IFN-γ receptor expression showed attenuation of immune-mediated marrow destruction, whereas effector lymphocytes from IFN-γ-deficient donors were much less potent in initiating BM damage. We conclude that the activity of IFN-γ on murine hematopoiesis is context dependent. IFN-γ-augmented apoptotic gene expression facilitates destruction of HSCs and progenitors in the presence of activated cytotoxic T cells, as occurs in human BM failure. PMID:26491068

  2. IFN-γ-mediated hematopoietic cell destruction in murine models of immune-mediated bone marrow failure.

    PubMed

    Chen, Jichun; Feng, Xingmin; Desierto, Marie J; Keyvanfar, Keyvan; Young, Neal S

    2015-12-10

    Interferon gamma (IFN-γ) has been reported to have both negative and positive activity on hematopoietic cells, adding complexity to the interpretation of its pleiotropic functions. We examined the effects of IFN-γ on murine hematopoietic stem cells (HSCs) and progenitors in vitro and in vivo by using mouse models. IFN-γ treatment expanded bone marrow (BM) c-Kit(+)Sca1(+)Lin(-) (KSL) cell number but reduced BM KLCD150(+) and KLCD150(+)CD48(-) cells. IFN-γ-expanded KSL cells engrafted poorly when tested by competitive repopulation in vivo. KSL, KLCD150(+), and KLCD150(+)CD48(-) cells from IFN-γ-treated animals all showed significant upregulation in Fas expression. When cocultured with activated T cells in vitro, KSL and KLCD150(+) cells from IFN-γ-treated donors showed increased apoptosis relative to those from untreated animals, and infusion of activated CD8 T cells into IFN-γ-injected animals in vivo led to partial elimination of KSL cells. Exposure of BM cells or KSL cells to IFN-γ increased expression of Fas, caspases, and related proapoptotic genes and decreased expression of Ets-1 and other hematopoietic genes. In mouse models of BM failure, mice genetically deficient in IFN-γ receptor expression showed attenuation of immune-mediated marrow destruction, whereas effector lymphocytes from IFN-γ-deficient donors were much less potent in initiating BM damage. We conclude that the activity of IFN-γ on murine hematopoiesis is context dependent. IFN-γ-augmented apoptotic gene expression facilitates destruction of HSCs and progenitors in the presence of activated cytotoxic T cells, as occurs in human BM failure.

  3. The target cell of transformation is distinct from the leukemia stem cell in murine CALM/AF10 leukemia models.

    PubMed

    Dutta, S; Krause, A; Vosberg, S; Herold, T; Ksienzyk, B; Quintanilla-Martinez, L; Tizazu, B; Chopra, M; Graf, A; Krebs, S; Blum, H; Greif, P A; Vetter, A; Metzeler, K; Rothenberg-Thurley, M; Schneider, M R; Dahlhoff, M; Spiekermann, K; Zimber-Strobl, U; Wolf, E; Bohlander, S K

    2016-05-01

    The CALM/AF10 fusion gene is found in various hematological malignancies including acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia and malignant lymphoma. We have previously identified the leukemia stem cell (LSC) in a CALM/AF10-driven murine bone marrow transplant AML model as B220+ lymphoid cells with B-cell characteristics. To identify the target cell for leukemic transformation or 'cell of origin of leukemia' (COL) in non-disturbed steady-state hematopoiesis, we inserted the CALM/AF10 fusion gene preceded by a loxP-flanked transcriptional stop cassette into the Rosa26 locus. Vav-Cre-induced panhematopoietic expression of the CALM/AF10 fusion gene led to acute leukemia with a median latency of 12 months. Mice expressing CALM/AF10 in the B-lymphoid compartment using Mb1-Cre or CD19-Cre inducer lines did not develop leukemia. Leukemias had a predominantly myeloid phenotype but showed coexpression of the B-cell marker B220, and had clonal B-cell receptor rearrangements. Using whole-exome sequencing, we identified an average of two to three additional mutations per leukemia, including activating mutations in known oncogenes such as FLT3 and PTPN11. Our results show that the COL for CALM/AF10 leukemia is a stem or early progenitor cell and not a cell of B-cell lineage with a phenotype similar to that of the LSC in CALM/AF10+ leukemia.

  4. CD45 phosphatase is crucial for human and murine acute myeloid leukemia maintenance through its localization in lipid rafts

    PubMed Central

    Saint-Paul, Laetitia; Nguyen, Chi-Hung; Buffière, Anne; de Barros, Jean-Paul Pais; Hammann, Arlette; Landras-Guetta, Corinne; Filomenko, Rodolphe; Chrétien, Marie-Lorraine; Johnson, Pauline; Bastie, Jean-Noël; Delva, Laurent; Quéré, Ronan

    2016-01-01

    CD45 is a pan-leukocyte protein with tyrosine phosphatase activity involved in the regulation of signal transduction in hematopoiesis. Exploiting CD45 KO mice and lentiviral shRNA, we prove the crucial role that CD45 plays in acute myeloid leukemia (AML) development and maintenance. We discovered that CD45 does not colocalize with lipid rafts on murine and human non-transformed hematopoietic cells. Using a mouse model, we proved that CD45 positioning within lipid rafts is modified during their oncogenic transformation to AML. CD45 colocalized with lipid rafts on AML cells, which contributes to elevated GM-CSF signal intensity involved in proliferation of leukemic cells. We furthermore proved that the GM-CSF/Lyn/Stat3 pathway that contributes to growth of leukemic cells could be profoundly affected, by using a new plasma membrane disrupting agent, which rapidly delocalized CD45 away from lipid rafts. We provide evidence that this mechanism is also effective on human primary AML samples and xenograft transplantation. In conclusion, this study highlights the emerging evidence of the involvement of lipid rafts in oncogenic development of AML and the targeting of CD45 positioning among lipid rafts as a new strategy in the treatment of AML. PMID:27579617

  5. CD45 phosphatase is crucial for human and murine acute myeloid leukemia maintenance through its localization in lipid rafts.

    PubMed

    Saint-Paul, Laetitia; Nguyen, Chi-Hung; Buffière, Anne; Pais de Barros, Jean-Paul; Hammann, Arlette; Landras-Guetta, Corinne; Filomenko, Rodolphe; Chrétien, Marie-Lorraine; Johnson, Pauline; Bastie, Jean-Noël; Delva, Laurent; Quéré, Ronan

    2016-10-04

    CD45 is a pan-leukocyte protein with tyrosine phosphatase activity involved in the regulation of signal transduction in hematopoiesis. Exploiting CD45 KO mice and lentiviral shRNA, we prove the crucial role that CD45 plays in acute myeloid leukemia (AML) development and maintenance. We discovered that CD45 does not colocalize with lipid rafts on murine and human non-transformed hematopoietic cells. Using a mouse model, we proved that CD45 positioning within lipid rafts is modified during their oncogenic transformation to AML. CD45 colocalized with lipid rafts on AML cells, which contributes to elevated GM-CSF signal intensity involved in proliferation of leukemic cells. We furthermore proved that the GM-CSF/Lyn/Stat3 pathway that contributes to growth of leukemic cells could be profoundly affected, by using a new plasma membrane disrupting agent, which rapidly delocalized CD45 away from lipid rafts. We provide evidence that this mechanism is also effective on human primary AML samples and xenograft transplantation. In conclusion, this study highlights the emerging evidence of the involvement of lipid rafts in oncogenic development of AML and the targeting of CD45 positioning among lipid rafts as a new strategy in the treatment of AML.

  6. DNA methylation analysis of murine hematopoietic side population cells during aging.

    PubMed

    Taiwo, Oluwatosin; Wilson, Gareth A; Emmett, Warren; Morris, Tiffany; Bonnet, Dominique; Schuster, Eugene; Adejumo, Tomas; Beck, Stephan; Pearce, Daniel J

    2013-10-01

    Stem cells have been found in most tissues/organs. These somatic stem cells produce replacements for lost and damaged cells, and it is not completely understood how this regenerative capacity becomes diminished during aging. To study the possible involvement of epigenetic changes in somatic stem cell aging, we used murine hematopoiesis as a model system. Hematopoietic stem cells (HSCs) were enriched for via Hoechst exclusion activity (SP-HSC) from young, medium-aged and old mice and subjected to comprehensive, global methylome (MeDIP-seq) analysis. With age, we observed a global loss of DNA methylation of approximately 5%, but an increase in methylation at some CpG islands. Just over 100 significant (FDR<0.2) aging-specific differentially methylated regions (aDMRs) were identified, which are surprisingly few considering the profound age-based changes that occur in HSC biology. Interestingly, the polycomb repressive complex -2 (PCRC2) target genes Kiss1r, Nav2 and Hsf4 were hypermethylated with age. The promoter for the Sdpr gene was determined to be progressively hypomethylated with age. This occurred concurrently with an increase in gene expression with age. To explore this relationship further, we cultured isolated SP-HSC in the presence of 5-aza-deoxycytdine and demonstrated a negative correlation between Sdpr promoter methylation and gene expression. We report that DNA methylation patterns are well preserved during hematopoietic stem cell aging, confirm that PCRC2 targets are increasingly methylated with age, and suggest that SDPR expression changes with age in HSCs may be regulated via age-based alterations in DNA methylation.

  7. Tumor necrosis factor receptors support murine hematopoietic progenitor function in the early stages of engraftment.

    PubMed

    Pearl-Yafe, Michal; Mizrahi, Keren; Stein, Jerry; Yolcu, Esma S; Kaplan, Ofer; Shirwan, Haval; Yaniv, Isaac; Askenasy, Nadir

    2010-07-01

    Tumor necrosis factor (TNF) family receptors/ligands are important participants in hematopoietic homeostasis, in particular as essential negative expansion regulators of differentiated clones. As a prominent injury cytokine, TNF-alpha has been traditionally considered to suppress donor hematopoietic stem and progenitor cell function after transplantation. We monitored the involvement of TNF receptors (TNF-R) 1 and 2 in murine hematopoietic cell engraftment and their inter-relationship with Fas. Transplantation of lineage-negative (lin(-)) bone marrow cells (BMC) from TNF receptor-deficient mice into wild-type recipients showed defective early engraftment and loss of durable hematopoietic contribution upon recovery of host hematopoiesis. Consistently, cells deficient in TNF receptors had reduced competitive capacity as compared to wild-type progenitors. The TNF receptors were acutely upregulated in bone marrow (BM)-homed donor cells (wild-type) early after transplantation, being expressed in 60%-75% of the donor cells after 6 days. Both TNF receptors were detected in fast cycling, early differentiating progenitors, and were ubiquitously expressed in the most primitive progenitors with long-term reconstituting potential (lin(-)c-kit(+) stem cell antigen (SCA)-1(+)). BM-homed donor cells were insensitive to apoptosis induced by TNF-alpha and Fas-ligand and their combination, despite reciprocal inductive cross talk between the TNF and Fas receptors. The engraftment supporting effect of TNF-alpha is attributed to stimulation of progenitors through TNF-R1, which involves activation of the caspase cascade. This stimulatory effect was not observed for TNF-R2, and this receptor did not assume redundant stimulatory function in TNFR1-deficient cells. It is concluded that TNF-alpha plays a tropic role early after transplantation, which is essential to successful progenitor engraftment.

  8. The aryl hydrocarbon receptor nuclear translocator is an essential regulator of murine hematopoietic stem cell viability.

    PubMed

    Krock, Bryan L; Eisinger-Mathason, Tzipora S; Giannoukos, Dionysios N; Shay, Jessica E; Gohil, Mercy; Lee, David S; Nakazawa, Michael S; Sesen, Julie; Skuli, Nicolas; Simon, M Celeste

    2015-05-21

    Hypoxia-inducible factors (HIFs) are master regulators of the transcriptional response to low oxygen and play essential roles in embryonic development, tissue homeostasis, and disease. Recent studies have demonstrated that hematopoietic stem cells (HSCs) within the bone marrow localize to a hypoxic niche and that HIF-1α promotes HSC adaptation to stress. Because the related factor HIF-2α is also expressed in HSCs, the combined role of HIF-1α and HIF-2α in HSC maintenance is unclear. To this end, we have conditionally deleted the HIF-α dimerization partner, the aryl hydrocarbon receptor nuclear translocator (ARNT) in the hematopoietic system to ablate activity of both HIF-1α and HIF-2α and assessed the functional consequence of ARNT deficiency on fetal liver and adult hematopoiesis. We determined that ARNT is essential for adult and fetal HSC viability and homeostasis. Importantly, conditional knockout of both Hif-1α and Hif-2α phenocopied key aspects of these HSC phenotypes, demonstrating that the impact of Arnt deletion is primarily HIF dependent. ARNT-deficient long-term HSCs underwent apoptosis, potentially because of reduced B-cell lymphoma 2 (BCL-2) and vascular endothelial growth factor A (VEGF-A) expression. Our results suggest that HIF activity may regulate HSC homeostasis through these prosurvival factors.

  9. The aryl hydrocarbon receptor nuclear translocator is an essential regulator of murine hematopoietic stem cell viability

    PubMed Central

    Krock, Bryan L.; Eisinger-Mathason, Tzipora S.; Giannoukos, Dionysios N.; Shay, Jessica E.; Gohil, Mercy; Lee, David S.; Nakazawa, Michael S.; Sesen, Julie; Skuli, Nicolas

    2015-01-01

    Hypoxia-inducible factors (HIFs) are master regulators of the transcriptional response to low oxygen and play essential roles in embryonic development, tissue homeostasis, and disease. Recent studies have demonstrated that hematopoietic stem cells (HSCs) within the bone marrow localize to a hypoxic niche and that HIF-1α promotes HSC adaptation to stress. Because the related factor HIF-2α is also expressed in HSCs, the combined role of HIF-1α and HIF-2α in HSC maintenance is unclear. To this end, we have conditionally deleted the HIF-α dimerization partner, the aryl hydrocarbon receptor nuclear translocator (ARNT) in the hematopoietic system to ablate activity of both HIF-1α and HIF-2α and assessed the functional consequence of ARNT deficiency on fetal liver and adult hematopoiesis. We determined that ARNT is essential for adult and fetal HSC viability and homeostasis. Importantly, conditional knockout of both Hif-1α and Hif-2α phenocopied key aspects of these HSC phenotypes, demonstrating that the impact of Arnt deletion is primarily HIF dependent. ARNT-deficient long-term HSCs underwent apoptosis, potentially because of reduced B-cell lymphoma 2 (BCL-2) and vascular endothelial growth factor A (VEGF-A) expression. Our results suggest that HIF activity may regulate HSC homeostasis through these prosurvival factors. PMID:25855602

  10. Isolation, cultivation, and characterization of adult murine prostate stem cells

    PubMed Central

    Lukacs, Rita U.; Goldstein, Andrew S.; Lawson, Devon A.; Cheng, Donghui; Witte, Owen N.

    2010-01-01

    ABSTRACT/SUMMARY The successful isolation and cultivation of prostate stem cells will allow us to study their unique biological properties and their application in therapeutic approaches. Here we provide step-by-step procedures on the basis of previous work in our laboratory for: the harvesting of primary prostate cells from adolescent male mice by a modified enzymatic procedure; the isolation of an enriched population of prostate stem cells through cell sorting; the cultivation of prostate stem cells in vitro; and characterization of these cells and their stem-like activity, including in vivo tubule regeneration. Normally it will take approximately 8 hours to harvest prostate cells, isolate the stem cell enriched population, and set up the in vitro sphere assay. It will take up to 8 weeks to analyze the unique properties of the stem cells, including their regenerative capacity in vivo. PMID:20360765

  11. Isolation, cultivation and characterization of adult murine prostate stem cells.

    PubMed

    Lukacs, Rita U; Goldstein, Andrew S; Lawson, Devon A; Cheng, Donghui; Witte, Owen N

    2010-04-01

    The successful isolation and cultivation of prostate stem cells will allow us to study their unique biological properties and their application in therapeutic approaches. Here we describe step-by-step procedures on the basis of previous work in our laboratory for the harvesting of primary prostate cells from adolescent male mice by a modified enzymatic procedure; the isolation of an enriched population of prostate stem cells through cell sorting; and the cultivation of prostate stem cells in vitro and characterization of these cells and their stem-like activity, including in vivo tubule regeneration. Normally, it will take approximately 8 h to harvest prostate cells, isolate the stem cell-enriched population and set up the in vitro sphere assay. It will take up to 8 weeks to analyze the unique properties of the stem cells, including their regenerative capacity in vivo.

  12. Long-term clinical and molecular follow-up of large animals receiving retrovirally transduced stem and progenitor cells: no progression to clonal hematopoiesis or leukemia.

    PubMed

    Kiem, Hans-Peter; Sellers, Stephanie; Thomasson, Bobbie; Morris, Julia C; Tisdale, John F; Horn, Peter A; Hematti, Peiman; Adler, Rima; Kuramoto, Ken; Calmels, Boris; Bonifacino, Aylin; Hu, Jiong; von Kalle, Christof; Schmidt, Manfred; Sorrentino, Brian; Nienhuis, Arthur; Blau, C Anthony; Andrews, Robert G; Donahue, Robert E; Dunbar, Cynthia E

    2004-03-01

    There has been significant progress toward clinically relevant levels of retroviral gene transfer into hematopoietic stem cells (HSC), and the therapeutic potential of HSC-based gene transfer has been convincingly demonstrated in children with severe combined immunodeficiency syndrome (SCID). However, the subsequent development of leukemia in two children with X-linked SCID who were apparently cured after transplantation of retrovirally corrected CD34+ cells has raised concerns regarding the safety of gene therapy approaches utilizing integrating vectors. Nonhuman primates and dogs represent the best available models for gene transfer safety and efficacy and are particularly valuable for evaluation of long-term effects. We have followed 42 rhesus macaques, 23 baboons, and 17 dogs with significant levels of gene transfer for a median of 3.5 years (range 1-7) after infusion of CD34+ cells transduced with retroviral vectors expressing marker or drug-resistance genes. None developed abnormal hematopoiesis or leukemia. Integration site analysis confirmed stable, polyclonal retrovirally marked hematopoiesis, without progression toward mono- or oligoclonality over time. These results suggest that retroviral integrations using replication-incompetent vectors, at copy numbers achieved using standard protocols, are unlikely to result in leukemogenesis and that patient- or transgene-specific factors most likely contributed to the occurrence of leukemia in the X-SCID gene therapy trial.

  13. Isolation, Purification, and Culture of Primary Murine Sensory Neurons

    PubMed Central

    Katzenell, Sarah; Cabrera, Jorge R.; North, Brian J.; Leib, David A.

    2017-01-01

    Cultured primary neurons have been of extraordinary value for the study of neuronal anatomy, cell biology, and physiology. While use of neuronal cell lines has ease and utility, there are often caveats that arise due to their mitotic nature. This methods article presents detailed methodology for the preparation, purification, and culture of adult murine sensory neurons for the study of herpes simplex virus lytic and latent infections. While virology is the application for our laboratory, these cultures also have broad utility for neurobiologists and cell biologists. While these primary cultures have been highly informative, the methodology is challenging to many investigators. Through publication of this highly detailed protocol, it is our hope that the use of this culture system can spread in the field to allow more rapid progress in furthering our understanding of neurotropic virus infection. PMID:28808974

  14. Murine Norovirus: Propagation, Quantification and Genetic Manipulation

    PubMed Central

    Hwang, Seungmin; Alhatlani, Bader; Arias, Armando; Caddy, Sarah L; Christodoulou, Constantina; Cunha, Juliana; Emmott, Ed; Gonzalez-Hernandez, Marta; Kolawole, Abimbola; Lu, Jia; Rippinger, Christine; Sorgeloos, Frédéric; Thorne, Lucy; Vashist, Surender; Goodfellow, Ian

    2014-01-01

    Murine norovirus (MNV) is a positive-sense, plus-stranded RNA virus in the Caliciviridae family. It is the most common pathogen in biomedical research colonies. MNV is also related to the human noroviruses, which cause the majority of non-bacterial gastroenteritis worldwide. Like the human noroviruses, MNV is an enteric virus that replicates in the intestine and is transmitted by the fecal-oral route. MNV replicates in murine macrophages and dendritic cells in cells in culture and in the murine host. This virus is often used to study mechanisms in norovirus biology, because the human noroviruses are refractory to growth in cell culture. MNV combines the availability of a cell culture and reverse genetics system with the ability to study infection in the native host. Herein, we describe a panel of techniques that are commonly used to study MNV biology. PMID:24789596

  15. Isolation and Differentiation of Murine Macrophages.

    PubMed

    Rios, Francisco J; Touyz, Rhian M; Montezano, Augusto C

    2017-01-01

    Macrophages play a major role in inflammation, wound healing, and tissue repair. Infiltrated monocytes differentiate into different macrophage subtypes with protective or pathogenic activities in vascular lesions. In the heart and vascular tissues, pathological activation promotes cardiovascular inflammation and remodeling and there is increasing evidence that macrophages play important mechanisms in this environment. Primary murine macrophages can be obtained from: bone marrow by different treatments (granulocyte-macrophage colony-stimulating factor-GM-CSF, macrophage colony-stimulating factor-M-CSF or supernatant of murine fibroblast L929), peritoneal cavity (resident or thioglycolate elicit macrophages), from the lung (alveolar macrophages) or from adipose tissue. In this chapter we describe some protocols to obtain primary murine macrophages and how to identify a pure macrophage population or activation phenotypes using different markers.

  16. Regulation of Hematopoiesis

    PubMed Central

    Desforges, Jane F.

    1984-01-01

    These discussions are selected from the weekly staff conferences in the Department of Medicine, University of California, San Francisco. Taken from transcriptions, they are prepared by Drs Homer A. Boushey, Associate Professor of Medicine, and David G. Warnock, Associate Professor of Medicine, under the direction of Dr Lloyd H. Smith, Jr, Professor of Medicine and Chairman of the Department of Medicine. Requests for reprints should be sent to the Department of Medicine, University of California, San Francisco, School of Medicine, San Francisco, CA 94143. PMID:6382801

  17. Cell signaling directing the formation and function of hemogenic endothelium during murine embryogenesis

    USDA-ARS?s Scientific Manuscript database

    During developmental hematopoiesis, multilineage hematopoietic progenitors are thought to derive from a subset of vascular endothelium. Herein, we define the phenotype of such hemogenic endothelial cells and demonstrate, on a clonal level, that they exhibit multilineage hematopoietic potential. Furt...

  18. Commonly dysregulated genes in murine APL cells

    PubMed Central

    Yuan, Wenlin; Payton, Jacqueline E.; Holt, Matthew S.; Link, Daniel C.; Watson, Mark A.; DiPersio, John F.; Ley, Timothy J.

    2007-01-01

    To identify genes that are commonly dysregulated in a murine model of acute promyelocytic leukemia (APL), we first defined gene expression patterns during normal murine myeloid development; serial gene expression profiling studies were performed with primary murine hematopoietic progenitors that were induced to undergo myeloid maturation in vitro with G-CSF. Many genes were reproducibly expressed in restricted developmental “windows,” suggesting a structured hierarchy of expression that is relevant for the induction of developmental fates and/or differentiated cell functions. We compared the normal myeloid developmental transcriptome with that of APL cells derived from mice expressing PML-RARα under control of the murine cathepsin G locus. While many promyelocyte-specific genes were highly expressed in all APL samples, 116 genes were reproducibly dysregulated in many independent APL samples, including Fos, Jun, Egr1, Tnf, and Vcam1. However, this set of commonly dysregulated genes was expressed normally in preleukemic, early myeloid cells from the same mouse model, suggesting that dysregulation occurs as a “downstream” event during disease progression. These studies suggest that the genetic events that lead to APL progression may converge on common pathways that are important for leukemia pathogenesis. PMID:17008535

  19. Murine typhus in child, Yucatan, Mexico.

    PubMed

    Zavala-Castro, Jorge E; Zavala-Velázquez, Jorge E; Sulú Uicab, Justo Eduardo

    2009-06-01

    A case of murine typhus in Yucatan was diagnosed in a child with nonspecific signs and symptoms. The finding of Rickettsia typhi increases the number of Rickettsia species identified in Yucatan and shows that studies are needed to determine the prevalence and incidence of rickettsioses in Mexico.

  20. Murine erythroleukemia cell line GM979 contains factors that can activate silent chromosomal human. gamma. -globin genes

    SciTech Connect

    Zitnik, G.; Hines, P.; Stamatoyannopoulos, G.; Papayannopoulou, T. )

    1991-03-15

    The authors introduced a normal chromosome 11 into GM979 murine erythroleukemia cells by fusing them with Epstein-Barr virus-transformed lymphocytes from a normal individual. In contrast to precious data obtained with other murine erythroleukemia cells, they detected activation of human chromosomal {gamma}-globin genes in GM979 cells. GM979, unlike previously used murine erythroleukemia cell lines, expresses murine embryonic globin in addition to adult globin. While all the hybrids expressed {gamma}- and {beta}-globin, they displayed a wide range of {gamma}-globin expression in relation to that of {beta}-globin. No correlation, however, was found in quantitative expression between murine embryonic globin and human {gamma}-globin in these hybrids, suggesting that the two globins are regulated independently, at least in this cell line. These data indicate that {gamma}-globin genes from normal, nonerythroid chromosomes are not irreversibly silenced, and they can be activated by a positive trans factor(s) present in GM979 cells.

  1. Cutting edge: murine UL16-binding protein-like transcript 1: a newly described transcript encoding a high-affinity ligand for murine NKG2D.

    PubMed

    Carayannopoulos, Leonidas N; Naidenko, Olga V; Fremont, Daved H; Yokoyama, Wayne M

    2002-10-15

    Murine NKG2D is known to recognize H60 and five RAE1 variants. The human homologue recognizes both inducible MHC class I chain-related gene and constitutive (UL16-binding protein (ULBP)) ligands. Widely expressed, the latter are thought to mark transformed or infected cells for destruction by NK cells in the context of down-regulated cell surface class I (i.e., the "missing self"-response). Unlike MIC and ULBP however, mRNA for the murine ligands appears only in very limited contexts in the mature animal. In this study, we describe a NKG2D ligand termed "murine ULBP-like transcript 1 (MULT1) whose mRNA appears to be widely expressed in adult parenchyma. This molecule possesses MHC class I-like alpha1 and alpha2 domains as well as a large cytoplasmic domain. Recombinant MULT1 binds NKG2D with relatively high affinity (K(D) approximately 6 nM) and low k(off) (approximately 0.006s(-1)). Expression of MULT1 by normally resistant RMA cells results in their susceptibility to lysis by C57BL/6 splenocytes.

  2. A novel murine homeobox gene isolated by a tissue specific PCR cloning strategy.

    PubMed Central

    Kern, M J; Witte, D P; Valerius, M T; Aronow, B J; Potter, S S

    1992-01-01

    We have identified a novel homeobox gene, designated K-2, using a reverse transcription PCR cloning strategy. Sequence analysis reveals that the homeobox of K-2 is 77.6% homologous at the nucleotide level and 97% identical at the amino acid sequence level to another murine gene, S8. Homeodomain sequence comparisons indicate that K-2 and S8 represent a distinct subclass of paired type homeobox genes. Northern blot analysis of RNA from murine embryos and adult tissues identified multiple transcripts that are expressed in a developmentally specific and tissue restricted manner. Alternate splicing of K-2 at the 3-coding region leads to the inclusion of a chain terminating sequence. In addition, the developmental expression pattern of this gene at day 12 of gestation was determined by in situ hybridization. Expression was observed in diverse mesenchymal cells in craniofacial, pericardial, primitive dermal, prevertebral, and genital structures. Images PMID:1383943

  3. Developmentally-Dynamic Murine Brain Proteomes and Phosphoproteomes Revealed by Quantitative Proteomics

    PubMed Central

    Doubleday, Peter F.; Ballif, Bryan A.

    2014-01-01

    Developmental processes are governed by a diverse suite of signaling pathways employing reversible phosphorylation. Recent advances in large-scale phosphoproteomic methodologies have made possible the identification and quantification of hundreds to thousands of phosphorylation sites from primary tissues. Towards a global characterization of proteomic changes across brain development, we present the results of a large-scale quantitative mass spectrometry study comparing embryonic, newborn and adult murine brain. Using anti-phosphotyrosine immuno-affinity chromatography and strong cation exchange (SCX) chromatography, coupled to immobilized metal affinity chromatography (IMAC), we identified and quantified over 1,750 phosphorylation sites and over 1,300 proteins between three developmental states. Bioinformatic analyses highlight functions associated with the identified proteins and phosphoproteins and their enrichment at distinct developmental stages. These results serve as a primary reference resource and reveal dynamic developmental profiles of proteins and phosphoproteins from the developing murine brain. PMID:25177544

  4. Deep Sequencing of the Murine Olfactory Receptor Neuron Transcriptome

    PubMed Central

    Kanageswaran, Ninthujah; Demond, Marilen; Nagel, Maximilian; Schreiner, Benjamin S. P.; Baumgart, Sabrina; Scholz, Paul; Altmüller, Janine; Becker, Christian; Doerner, Julia F.; Conrad, Heike; Oberland, Sonja; Wetzel, Christian H.; Neuhaus, Eva M.; Hatt, Hanns; Gisselmann, Günter

    2015-01-01

    The ability of animals to sense and differentiate among thousands of odorants relies on a large set of olfactory receptors (OR) and a multitude of accessory proteins within the olfactory epithelium (OE). ORs and related signaling mechanisms have been the subject of intensive studies over the past years, but our knowledge regarding olfactory processing remains limited. The recent development of next generation sequencing (NGS) techniques encouraged us to assess the transcriptome of the murine OE. We analyzed RNA from OEs of female and male adult mice and from fluorescence-activated cell sorting (FACS)-sorted olfactory receptor neurons (ORNs) obtained from transgenic OMP-GFP mice. The Illumina RNA-Seq protocol was utilized to generate up to 86 million reads per transcriptome. In OE samples, nearly all OR and trace amine-associated receptor (TAAR) genes involved in the perception of volatile amines were detectably expressed. Other genes known to participate in olfactory signaling pathways were among the 200 genes with the highest expression levels in the OE. To identify OE-specific genes, we compared olfactory neuron expression profiles with RNA-Seq transcriptome data from different murine tissues. By analyzing different transcript classes, we detected the expression of non-olfactory GPCRs in ORNs and established an expression ranking for GPCRs detected in the OE. We also identified other previously undescribed membrane proteins as potential new players in olfaction. The quantitative and comprehensive transcriptome data provide a virtually complete catalogue of genes expressed in the OE and present a useful tool to uncover candidate genes involved in, for example, olfactory signaling, OR trafficking and recycling, and proliferation. PMID:25590618

  5. Neonatal CD71+ erythroid cells do not modify murine sepsis mortality

    PubMed Central

    Wynn, James L.; Scumpia, Philip O.; Stocks, Blair T.; Romano-Keeler, Joann; Alrifai, Mhd Wael; Liu, Jin-Hua; Kim, Annette S.; Alford, Catherine E.; Matta, Pranathi; Weitkamp, Jörn-Hendrik; Moore, Daniel J.

    2015-01-01

    Sepsis is a major cause of neonatal mortality and morbidity worldwide. A recent report suggested murine neonatal host defense against infection could be compromised by immunosuppressive CD71+ erythroid splenocytes. We examined the impact of CD71+ erythroid splenocytes on murine neonatal mortality to endotoxin challenge or polymicrobial sepsis and characterized circulating CD71+ erythroid (CD235a+) cells in human neonates. Adoptive transfer or antibody-mediated reduction of neonatal CD71+ erythroid splenocytes did not alter murine neonatal survival to endotoxin challenge or polymicrobial sepsis challenge. Ex vivo immunosuppression of stimulated adult CD11b+ cells was not limited to neonatal splenocytes as it also occurred with adult and neonatal bone marrow. Animals treated with anti-CD71 antibody showed reduced splenic bacterial load following bacterial challenge compared to isotype-treated mice. However, adoptive transfer of enriched CD71+ erythroid splenocytes to CD71+-reduced animals did not reduce bacterial clearance. Human CD71+CD235a+ cells were common among cord blood mononuclear cells and were shown to be reticulocytes. In summary, a lack of effect on murine survival to polymicrobial sepsis following adoptive transfer or diminution of CD71+ erythroid splenocytes under these experimental conditions suggests the impact of these cells on neonatal infection risk and progression may be limited. An unanticipated immune priming effect of anti-CD71 antibody treatment was likely responsible for the reported enhanced bacterial clearance, rather than a reduction of immunosuppressive CD71+ erythroid splenocytes. In humans, the well-described rapid decrease in circulating reticulocytes after birth suggests they may have a limited role in reducing inflammation secondary to microbial colonization. PMID:26101326

  6. Advances in Murine Models of Diabetic Nephropathy

    PubMed Central

    Kong, Li-li; Wu, Hao; Cui, Wen-peng; Zhou, Wen-hua; Luo, Ping; Sun, Jing; Yuan, Hang; Miao, Li-ning

    2013-01-01

    Diabetic nephropathy (DN) is one of the microvascular complications of both type 1 and type 2 diabetes, which is also associated with a poor life expectancy of diabetic patients. However, the pathogenesis of DN is still unclear. Thus, it is of great use to establish appropriate animal models of DN for doing research on pathogenesis and developing novel therapeutic strategies. Although a large number of murine models of DN including artificially induced, spontaneous, and genetically engineered (knockout and transgenic) animal models have been developed, none of them develops renal changes sufficiently reflecting those seen in humans. Here we review the identified murine models of DN from the aspects of genetic background, type of diabetes, method of induction, gene deficiency, animal age and gender, kidney histopathology, and phenotypic alterations in the hope of enhancing our comprehension of genetic susceptibility and molecular mechanisms responsible for this disease and providing new clues as to how to choose appropriate animal models of DN. PMID:23844375

  7. Murine Toxicity of Agrobacterium tumefaciens1

    PubMed Central

    Hamilton, Pat B.; Huisingh, Donald

    1968-01-01

    Eleven strains of the crown gall organism, Agrobacterium tumefaciens, tested by intraperitoneal injection into mice, were lethal within 48 hr. Five other species had some lethal strains. The lethal effect of A. tumefaciens appeared to be the result of a toxic rather than an infectious process, since histopathological anomalies were not found in mice injected with live cultures and since heat-killed cultures were lethal. The murine toxin disappeared when A. tumefaciens was grown at 36 C and reappeared when the organism was subsequently incubated below 30 C. The murine toxin itself was not inactivated by exposure to 100 C for 30 min. The toxin was associated with the cells and was not excreted into the medium. Centrifugal fractionation revealed that the toxin was associated with the smaller cells in 3-day stationary-phase cultures. These data suggested a possible relationship between toxin production and the production of the agents responsible for the initiation of plant tumors. PMID:5643064

  8. Murine typhus: an unrecognized suburban vectorborne disease.

    PubMed

    Civen, Rachel; Ngo, Van

    2008-03-15

    Murine typhus, an acute febrile illness caused by Rickettsia typhi, is distributed worldwide. Mainly transmitted by the fleas of rodents, it is associated with cities and ports where urban rats (Rattus rattus and Rattus norvegicus) are abundant. In the United States, cases are concentrated in suburban areas of Texas and California. Contrary to the classic rat-flea-rat cycle, the most important reservoirs of infection in these areas are opossums and cats. The cat flea, Ctenocephalides felis, has been identified as the principal vector. In Texas, murine typhus cases occur in spring and summer, whereas, in California, cases have been documented in summer and fall. Most patients present with fever, and many have rash and headache. Serologic testing with the indirect immunofluorescence assay is the preferred diagnostic method. Doxycycline is the antibiotic of choice and has been shown to shorten the course of illness.

  9. Enhanced Cultivation Of Stimulated Murine B Cells

    NASA Technical Reports Server (NTRS)

    Sammons, David W.

    1994-01-01

    Method of in vitro cultivation of large numbers of stimulated murine B lymphocytes. Cells electrofused with other cells to produce hybridomas and monoclonal antibodies. Offers several advantages: polyclonally stimulated B-cell blasts cultivated for as long as 14 days, hybridomas created throughout culture period, yield of hybridomas increases during cultivation, and possible to expand polyclonally in vitro number of B cells specific for antigenic determinants first recognized in vivo.

  10. Stage-specific roles for CXCR4 signaling in murine hematopoietic stem/progenitor cells in the process of bone marrow repopulation.

    PubMed

    Lai, Chen-Yi; Yamazaki, Satoshi; Okabe, Motohito; Suzuki, Sachie; Maeyama, Yoshihiro; Iimura, Yasuaki; Onodera, Masafumi; Kakuta, Shigeru; Iwakura, Yoichiro; Nojima, Masanori; Otsu, Makoto; Nakauchi, Hiromitsu

    2014-07-01

    Hematopoietic cell transplantation has proven beneficial for various intractable diseases, but it remains unclear how hematopoietic stem/progenitor cells (HSPCs) home to the bone marrow (BM) microenvironment, initiate hematopoietic reconstitution, and maintain life-long hematopoiesis. The use of newly elucidated molecular determinants for overall HSPC engraftment should benefit patients. Here, we report that modification of C-X-C chemokine receptor type 4 (Cxcr4) signaling in murine HSPCs does not significantly affect initial homing/lodging events, but leads to alteration in subsequent BM repopulation kinetics, with observations confirmed by both gain- and loss-of-function approaches. By using C-terminal truncated Cxcr4 as a gain-of-function effector, we demonstrated that signal augmentation likely led to favorable in vivo repopulation of primitive cell populations in BM. These improved features were correlated with enhanced seeding efficiencies in stromal cell cocultures and altered ligand-mediated phosphorylation kinetics of extracellular signal-regulated kinases observed in Cxcr4 signal-augmented HSPCs in vitro. Unexpectedly, however, sustained signal enhancement even with wild-type Cxcr4 overexpression resulted in impaired peripheral blood (PB) reconstitution, most likely by preventing release of donor hematopoietic cells from the marrow environment. We thus conclude that timely regulation of Cxcr4/CXCR4 signaling is key in providing donor HSPCs with enhanced repopulation potential following transplantation, whilst preserving the ability to release HSPC progeny into PB for improved transplantation outcomes.

  11. Depletion of Jak2V617F myeloproliferative neoplasm-propagating stem cells by interferon-α in a murine model of polycythemia vera

    PubMed Central

    Bruedigam, Claudia; Poveromo, Luke; Heidel, Florian H.; Purdon, Amy; Vu, Therese; Austin, Rebecca; Heckl, Dirk; Breyfogle, Lawrence J.; Kuhn, Catherine Paine; Kalaitzidis, Demetrios; Armstrong, Scott A.; Williams, David A.; Hill, Geoff R.; Ebert, Benjamin L.

    2013-01-01

    Interferon-α (IFNα) is an effective treatment of patients with myeloproliferative neoplasms (MPNs). In addition to inducing hematological responses in most MPN patients, IFNα reduces the JAK2V617F allelic burden and can render the JAK2V617F mutant clone undetectable in some patients. The precise mechanism underlying these responses is incompletely understood and whether the molecular responses that are seen occur due to the effects of IFNα on JAK2V617F mutant stem cells is debated. Using a murine model of Jak2V617F MPN, we investigated the effects of IFNα on Jak2V617F MPN-propagating stem cells in vivo. We report that IFNα treatment induces hematological responses in the model and causes depletion of Jak2V617F MPN-propagating cells over time, impairing disease transplantation. We demonstrate that IFNα treatment induces cell cycle activation of Jak2V617F mutant long-term hematopoietic stem cells and promotes a predetermined erythroid-lineage differentiation program. These findings provide insights into the differential effects of IFNα on Jak2V617F mutant and normal hematopoiesis and suggest that IFNα achieves molecular remissions in MPN patients through its effects on MPN stem cells. Furthermore, these results support combinatorial therapeutic approaches in MPN by concurrently depleting dormant JAK2V617F MPN-propagating stem cells with IFNα and targeting the proliferating downstream progeny with JAK2 inhibitors or cytotoxic chemotherapy. PMID:23487027

  12. Tissue-specific in vivo transcription start sites of the human and murine cystic fibrosis genes.

    PubMed

    White, N L; Higgins, C F; Trezise, A E

    1998-03-01

    The in vivo transcription start sites of the human cystic fibrosis transmembrane conductance regulator gene ( CFTR ) and its murine homologue ( Cftr ) have been mapped in a range of tissues using the technique of 5' rapid amplification of cDNA ends (5' RACE). These are the first in vivo transcription start sites for CFTR or Cftr to be reported. Distinct, tissue-specific patterns of CFTR start site usage were identified in both mouse and human. In particular, striking variation in the position of the murine Cftr transcription start site was seen along the length of the intestinal tract; different start sites being utilized in ileum and in duodenum. In humans, distinct transcription start sites are utilized in adult and foetal lungs. In addition, a novel 5'-untranslated exon of murine Cftr , denoted exon -1, was identified and shown to be expressed exclusively in mouse testis. Expression of exon -1-containing Cftr transcripts was shown by mRNA in situ hybridization to be confined to the germ cells and to be regulated during spermatogenesis.

  13. Differential Secreted Proteome Approach in Murine Model for Candidate Biomarker Discovery in Colon Cancer

    PubMed Central

    Rangiah, Kannan; Tippornwong, Montri; Sangar, Vineet; Austin, David; Tétreault, Marie-Pier; Rustgi, Anil K.; Blair, Ian A.; Yu, Kenneth H.

    2009-01-01

    The complexity and heterogeneity of the plasma proteome have presented significant challenges in the identification of protein changes associated with tumor development. We used cell culture as a model system and identified differentially expressed, secreted proteins which may constitute serological biomarkers. A stable isotope labeling by amino acids in cell culture (SILAC) approach was used to label the entire secreted proteomes of the CT26 murine colon cancer cell line and normal young adult mouse colon (YAMC) cell line, thereby creating a stable isotope labeled proteome (SILAP) standard. This SILAP standard was added to unlabeled murine CT26 colon cancer cell or normal murine YAMC colon epithelial cell secreted proteome samples. A multidimensional approach combining isoelectric focusing (IEF), strong cation exchange (SCX) followed by reversed phase liquid chromatography was used for extensive protein and peptide separation. A total of 614 and 929 proteins were identified from the YAMC and CT26 cell lines, with 418 proteins common to both cell lines. Twenty highly abundant differentially expressed proteins from these groups were selected for liquid chromatography-multiple reaction monitoring/mass spectrometry (LC-MRM/MS) analysis in sera. Differential secretion into the serum was observed for several proteins when Apcmin mice were compared with control mice. These findings were then confirmed by Western blot analysis. PMID:19769411

  14. Potent Inhibition of Junín Virus Infection by Interferon in Murine Cells

    PubMed Central

    Huang, Cheng; Walker, Aida G.; Grant, Ashley M.; Kolokoltsova, Olga A.; Yun, Nadezhda E.; Seregin, Alexey V.; Paessler, Slobodan

    2014-01-01

    The new world arenavirus Junín virus (JUNV) is the causative agent of Argentine hemorrhagic fever, a lethal human infectious disease. Adult laboratory mice are generally resistant to peripheral infection by JUNV. The mechanism underlying the mouse resistance to JUNV infection is largely unknown. We have reported that interferon receptor knockout mice succumb to JUNV infection, indicating the critical role of interferon in restricting JUNV infection in mice. Here we report that the pathogenic and vaccine strains of JUNV were highly sensitive to interferon in murine primary cells. Treatment with low concentrations of interferon abrogated viral NP protein expression in murine cells. The replication of both JUNVs was enhanced in IRF3/IRF7 deficient cells. In addition, the vaccine strain of JUNV displayed impaired growth in primary murine cells. Our data suggested a direct and potent role of host interferon response in restricting JUNV replication in mice. The defect in viral growth for vaccine JUNV might also partially explain its attenuation in mice. PMID:24901990

  15. IL-22 exacerbates weight loss in a murine model of chronic pulmonary Pseudomonas aeruginosa infection.

    PubMed

    Bayes, Hannah K; Ritchie, Neil D; Ward, Christopher; Corris, Paul A; Brodlie, Malcolm; Evans, Thomas J

    2016-11-01

    Interleukin (IL)-22 is a critical mediator of mucosal immunity and tissue regeneration, protecting against a number of respiratory pathogens. Whether IL-22 confers protection against chronic Pseudomonas aeruginosa (PA) infection in cystic fibrosis (CF) is unknown. Explanted CF lungs were examined for IL-22 production and immune-localization. A murine model of persistent pulmonary PA infection was used to examine production of IL-22 following infective challenge. The role of IL-22 was examined using IL-22 knockout (KO) animals. IL-22 is produced within the adult CF lung and localizes to the airway epithelium. IL-22 is produced by murine pulmonary lymph node cells following lung infection. The absence of IL-22 resulted in no significant difference in acute mortality, bacterial burden, chronic infection rates, histological changes or neutrophilic inflammation in the chronic PA infection model. However, IL-22 KO animals lost less weight following infection. IL-22 is produced in the CF lung and in response to PA infection yet is dispensable in protection against chronic pulmonary P. aeruginosa infection in a murine model. However, we identified a novel role for the cytokine in promoting infection-related weight-loss, a significant prognostic factor in the CF population. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  16. Potent inhibition of Junín virus infection by interferon in murine cells.

    PubMed

    Huang, Cheng; Walker, Aida G; Grant, Ashley M; Kolokoltsova, Olga A; Yun, Nadezhda E; Seregin, Alexey V; Paessler, Slobodan

    2014-06-01

    The new world arenavirus Junín virus (JUNV) is the causative agent of Argentine hemorrhagic fever, a lethal human infectious disease. Adult laboratory mice are generally resistant to peripheral infection by JUNV. The mechanism underlying the mouse resistance to JUNV infection is largely unknown. We have reported that interferon receptor knockout mice succumb to JUNV infection, indicating the critical role of interferon in restricting JUNV infection in mice. Here we report that the pathogenic and vaccine strains of JUNV were highly sensitive to interferon in murine primary cells. Treatment with low concentrations of interferon abrogated viral NP protein expression in murine cells. The replication of both JUNVs was enhanced in IRF3/IRF7 deficient cells. In addition, the vaccine strain of JUNV displayed impaired growth in primary murine cells. Our data suggested a direct and potent role of host interferon response in restricting JUNV replication in mice. The defect in viral growth for vaccine JUNV might also partially explain its attenuation in mice.

  17. Differential requirement for irf8 in formation of embryonic and adult macrophages in zebrafish

    DOE PAGES

    Shiau, Celia E.; Kaufman, Zoe; Meireles, Ana M.; ...

    2015-01-23

    Interferon regulatory factor 8 (Irf8) is critical for mammalian macrophage development and innate immunity, but its role in teleost myelopoiesis remains incompletely understood. Specifically, genetic tools to analyze the role of irf8 in zebrafish macrophage development at larval and adult stages are lacking. In this study, we generated irf8 null mutants in zebrafish using TALEN-mediated targeting. Our analysis defines different requirements for irf8 at different stages. irf8 is required for formation of all macrophages during primitive and transient definitive hematopoiesis, but not during adult-phase definitive hematopoiesis starting at 5-6 days postfertilization. At early stages, irf8 mutants have excess neutrophils andmore » excess cell death in pu.1-expressing myeloid cells. Macrophage fates were recovered in irf8 mutants after wildtype irf8 expression in neutrophil and macrophage lineages, suggesting that irf8 regulates macrophage specification and survival. In juvenile irf8 mutant fish, mature macrophages are present, but at numbers significantly reduced compared to wildtype, indicating an ongoing requirement for irf8 after embryogenesis. As development progresses, tissue macrophages become apparent in zebrafish irf8 mutants, with the possible exception of microglia. Our study defines distinct requirement for irf8 in myelopoiesis before and after transition to the adult hematopoietic system.« less

  18. Differential Requirement for irf8 in Formation of Embryonic and Adult Macrophages in Zebrafish

    PubMed Central

    Shiau, Celia E.; Kaufman, Zoe; Meireles, Ana M.; Talbot, William S.

    2015-01-01

    Interferon regulatory factor 8 (Irf8) is critical for mammalian macrophage development and innate immunity, but its role in teleost myelopoiesis remains incompletely understood. In particular, genetic tools to analyze the role of Irf8 in zebrafish macrophage development at larval and adult stages are lacking. We generated irf8 null mutants in zebrafish using TALEN-mediated targeting. Our analysis defines different requirements for irf8 at different stages. irf8 is required for formation of all macrophages during primitive and transient definitive hematopoiesis, but not during adult-phase definitive hematopoiesis starting at 5-6 days postfertilization. At early stages, irf8 mutants have excess neutrophils and excess cell death in pu.1-expressing myeloid cells. Macrophage fates were recovered in irf8 mutants after wildtype irf8 expression in neutrophil and macrophage lineages, suggesting that irf8 regulates macrophage specification and survival. In juvenile irf8 mutant fish, mature macrophages are present, but at numbers significantly reduced compared to wildtype, indicating an ongoing requirement for irf8 after embryogenesis. As development progresses, tissue macrophages become apparent in zebrafish irf8 mutants, with the possible exception of microglia. Our study defines distinct requirement for irf8 in myelopoiesis before and after transition to the adult hematopoietic system. PMID:25615614

  19. Adult Learning, Adult Teaching.

    ERIC Educational Resources Information Center

    Daines, John; And Others

    This book is designed to provide specific and practical suggestions for teachers and tutors of adult students. It is divided into two major parts. Part 1, which is devoted to adult learning, includes information on the characteristics of adult learners, their expectations and motivation, and issues related to equal opportunities and access. Part 2…

  20. Dietary supplementation with apple juice decreases endogenous amyloid-beta levels in murine brain.

    PubMed

    Chan, Amy; Shea, Thomas B

    2009-01-01

    Folate deficiency has been associated with age-related neurodegeneration. We demonstrate herein that dietary deficiency in folate and vitamin E, coupled pro-oxidant stress induced by dietary iron, increased amyloid-beta (Abeta) levels in normal adult mice. This increase was potentiated by apolipoprotein E (ApoE) deficiency as shown by treatment of transgenic mice homozygously lacking murine ApoE. Dietary supplementation with apple juice concentrate in drinking water alleviated the increase in Abeta for both mouse genotypes. These findings provide further evidence linking nutritional and genetic risk factors for age-related neurodegeneration, and underscore that dietary supplementation may be useful to augment therapeutic approaches.

  1. B cells modulate systemic responses to Pneumocystis murina lung infection and protect on-demand hematopoiesis via T cell-independent innate mechanisms when type I interferon signaling is absent.

    PubMed

    Hoyt, Teri R; Dobrinen, Erin; Kochetkova, Irina; Meissner, Nicole

    2015-02-01

    HIV infection results in a complex immunodeficiency due to loss of CD4(+) T cells, impaired type I interferon (IFN) responses, and B cell dysfunctions causing susceptibility to opportunistic infections such as Pneumocystis murina pneumonia and unexplained comorbidities, including bone marrow dysfunctions. Type I IFNs and B cells critically contribute to immunity to Pneumocystis lung infection. We recently also identified B cells as supporters of on-demand hematopoiesis following Pneumocystis infection that would otherwise be hampered due to systemic immune effects initiated in the context of a defective type I IFN system. While studying the role of type I IFNs in immunity to Pneumocystis infection, we discovered that mice lacking both lymphocytes and type I IFN receptor (IFrag(-/-)) developed progressive bone marrow failure following infection, while lymphocyte-competent type I IFN receptor-deficient mice (IFNAR(-/-)) showed transient bone marrow depression and extramedullary hematopoiesis. Lymphocyte reconstitution of lymphocyte-deficient IFrag(-/-) mice pointed to B cells as a key player in bone marrow protection. Here we define how B cells protect on-demand hematopoiesis following Pneumocystis lung infection in our model. We demonstrate that adoptive transfer of B cells into IFrag(-/-) mice protects early hematopoietic progenitor activity during systemic responses to Pneumocystis infection, thus promoting replenishment of depleted bone marrow cells. This activity is independent of CD4(+) T cell help and B cell receptor specificity and does not require B cell migration to bone marrow. Furthermore, we show that B cells protect on-demand hematopoiesis in part by induction of interleukin-10 (IL-10)- and IL-27-mediated mechanisms. Thus, our data demonstrate an important immune modulatory role of B cells during Pneumocystis lung infection that complement the modulatory role of type I IFNs to prevent systemic complications.

  2. The hematopoiesis in gill and its role in the immune response of Pacific oyster Crassostrea gigas against secondary challenge with Vibrio splendidus.

    PubMed

    Li, Yiqun; Song, Xiaorui; Wang, Weilin; Wang, Lingling; Yi, Qilin; Jiang, Shuai; Jia, Zhihao; Du, Xinyu; Qiu, Limei; Song, Linsheng

    2017-06-01

    Increasing evidences have demonstrated that the invertebrate gill is a predominant tissue participating in the immune response during pathogen challenge. In the present study, the hematopoiesis and immune activities in gill of Pacific oyster Crassostrea gigas were investigated. Stem-like cells with big nuclei and thin cytoplasm were found in the tubules of gill filaments, where DNA synthesis is active and hemocytes production are exuberant. The oysters primarily stimulated by formaldehyde-killed Vibrio splendidus exhibited stronger immune responses and enhanced cell regeneration in gill when they encountered the secondary challenge of live V. splendidus. After the secondary stimulation with V. splendidus, the expression levels of CgClec-4 and CgIFN in the gill of oysters pre-stimulated with formaldehyde-killed V. splendidus were significantly higher (p < 0.05) than that in the oysters pre-stimulated with filter-sterilized (0.22 μm pore size) sea water, while the expression level of CgIL-17 was significantly decreased (p < 0.05). Meanwhile, the protein expression level of hematopoietic transcription factor CgGATA3 and immune-related protein CgEcSOD in gill increased apparently after the secondary challenge with V. splendidus. ROS production was also enhanced (p < 0.05) at 6 h and 24 h after the secondary challenge. The phagocytic rate in gill of oysters pre-stimulated with formaldehyde-killed V. splendidus was significantly increased (p < 0.05) at 6 h after the secondary challenge with live V. splendidus, showing faster response than that pre-stimulated with filter-sterilized sea water. These results collectively showed that the immune parameters in gill were apparently enhanced after secondary challenge with live V. splendidus, indicating that hematopoiesis might participate in immune priming in Pacific oyster C. gigas.

  3. [Long-term ability for hematopoiesis of CD34(+)CD59(+) cells from patients with paroxysmal nocturnal hemoglobinuria: experiment with mice].

    PubMed

    Sun, Wan-Ling; Han, Bing; Wang, Xuan; Zhong, Yu-Ping; Zhuang, Jun-Ling; Shen, Ti; Wu, Yong-Ji

    2007-11-13

    To obtain the evidence of long-term hematopoiesis by normal clone stem cells from patients with paroxysmal nocturnal hemoglobinuria (PNH). 10 ml fresh bone marrow was collected from 2 PNH patients. Normal bone marrow was obtained from the ribs resected during operation of 2 patients without hematopathy. CD34(+)CD59(+) cells were isolated, purified, and expended. 12 total body irradiated SCID mice were divided into 2 equal group to undergo transplantation of the expanded CD34(+)CD59(+) cells from PNH patients (experiment group 1) or normal patients (control group 1). Four mice underwent transfusion of cell-free medium as blank control group 1. 30 and 60 days after the transplantation peripheral blood samples were collected and 90 days after the transplantation heart blood samples were collected. Spleens were taken out to prepare suspension of splenocytes and bacterium-free bone marrow cell suspension was prepared from the bilateral femurs. 90 days after the primary transplantation another 16 mice underwent total body irradiation and then divided into 2 equal groups to undergo transplantation of the bone marrow cells of the mice of the experiment group 1 and control group 1 (experiment group 2 and control group 2). Another 4 mice were transfused with cell-free medium fluid (blank control group 2). The levels of blood cells of all groups were calculated 30, 60, and 90 days after the primary and 30 days after the secondary transplantation. The cell percentages of peripheral blood, spleen, and bone marrow after the primary transplantation and 30 days after the secondary transplantation were detected by flow cytometry. PCR was used to detect whether the sex determining region Y existed in the female SCID mice after transplantation. Ninety days after primary transplantation, the peripheral blood cell levels of both experiment group 1 and control group 1 recovered to normal. Expression of CD45 was detected in the mice without significant difference between the 2 groups

  4. Epidemiological, clinical and laboratory features of murine typhus in central Tunisia.

    PubMed

    Aouam, A; Toumi, A; Ben Brahim, H; Loussaief, C; Jelliti, B; Ben Romdhane, F; Ben Yahia, S; Khairallah, M; Chakroun, M

    2015-04-01

    Murine typhus is an endemic zoonosis. It is difficult to diagnose because of its non-specific clinical manifestations. Our objective was to describe the epidemiological, clinical, laboratory, and treatment features of murine typhus. We conducted a retrospective study of 73 adult patients hospitalized for murine typhus from 2006 to 2011. The diagnosis was confirmed by a single titer of IgM≥128 or by seroconversion to typhus group antigen identified by indirect fluorescent assay. The mean age of patients was 33.1 years (range, 13-68 years). Thirty-eight patients (52%) lived in rural or suburban areas; neither fleabites nor exposure to rats were reported. The most common clinical symptoms were: fever, headache, and myalgia. A maculopapular and non-confluent rash was observed in 47 patients (64.4%). No inoculation eschar was observed in any patient. Eight patients presented with interstitial pneumonia and two with lymphocytic meningitis. The diagnosis was confirmed by indirect fluorescence assay in every case. A single titer of IgM ≥ 128 was found in 62 (84.9%) cases. The other 11 cases were diagnosed by seroconversion. All patients were given antibiotics. Tetracyclines were prescribed in 57 cases (78%). The two patients presenting with meningitis were treated with fluoroquinolone. The outcome was favorable for all patients and no relapse was observed. The features of murine typhus are non-specific. The definitive diagnosis is based on serologic testing by indirect fluorescent assay. Cyclins were the most prescribed antibiotics. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  5. Zebrafish scube1 (Signal Peptide-CUB (Complement Protein C1r/C1s, Uegf, and Bmp1)-EGF (Epidermal Growth Factor) Domain-containing Protein 1) Is Involved in Primitive Hematopoiesis*

    PubMed Central

    Tsao, Ku-Chi; Tu, Cheng-Fen; Lee, Shyh-Jye; Yang, Ruey-Bing

    2013-01-01

    scube1 (signal peptide-CUB (complement protein C1r/C1s, Uegf, and Bmp1)-EGF domain-containing protein 1), the founding member of a novel secreted and cell surface SCUBE protein family, is expressed predominantly in various developing tissues in mice. However, its function in primitive hematopoiesis remains unknown. In this study, we identified and characterized zebrafish scube1 and analyzed its function by injecting antisense morpholino-oligonucleotide into embryos. Whole-mount in situ hybridization revealed that zebrafish scube1 mRNA is maternally expressed and widely distributed during early embryonic development. Knockdown of scube1 by morpholino-oligonucleotide down-regulated the expression of marker genes associated with early primitive hematopoietic precursors (scl) and erythroid (gata1 and hbbe1), as well as early (pu.1) and late (mpo and l-plastin) myelomonocytic lineages. However, the expression of an early endothelial marker fli1a and vascular morphogenesis appeared normal in scube1 morphants. Overexpression of bone morphogenetic protein (bmp) rescued the expression of scl in the posterior lateral mesoderm during early primitive hematopoiesis in scube1 morphants. Biochemical and molecular analysis revealed that Scube1 could be a BMP co-receptor to augment BMP signaling. Our results suggest that scube1 is critical for and functions at the top of the regulatory hierarchy of primitive hematopoiesis by modulating BMP activity during zebrafish embryogenesis. PMID:23271740

  6. Generation of mesenchymal stem cell lines from murine bone marrow.

    PubMed

    Sreejit, P; Dilip, K B; Verma, R S

    2012-10-01

    Mesenchymal stem cells (MSC), because of their multipotency and ease of purification and amplification, are an ideal stem cell source for cell therapies. Bone-marrow-derived stem cells (BMSC) can be used to develop MSC-like immortalized cell lines with large proliferation and differentiation potentialities. Their immortalized status prevents the maintenance of MSC function and characters; this can be negated by modifying the isolation and maintenance protocol. Adult murine BMSC were isolated and maintained in media without additional growth factors together with passage-dependent reseeding following trypsinization. Cells maintained over 25 passages were considered as putative cell lines and characterized. The phenotypic and genotypic characteristics and multilineage differentiation potential of the cells were assessed by morphological, phenotypic, and molecular assays at various passages. The putative BMSC cell lines showed the characteristics of MSC and were able to maintain these characteristics, even after immortalization. The phenotypic data demonstrated difference among two cell lines; this was further validated by the difference in their multilineage differentiation potential following specific induction. More importantly, no changes were observed in the genotypic level in comparison with control cells, even after more than 50 passages. Our protocol thus advances the isolation and maintenance of BMSC and the development of putative BMSC cell lines that maintain characteristics of MSC, including multilineage differentiation potential, after more than 40 passages.

  7. Novel transient outward K+ current of mature murine hippocampal neurones.

    PubMed

    Li, X Y; McArdle, J J

    1997-06-01

    Hippocampal neurones were freshly isolated from the brain of adult mice and voltage-dependent K+ currents were recorded with whole-cell patch-clamp technique. Three components of transient K+ current (IA) were isolated when analyzing data with exponential functions or treating neurones with a variety of voltage protocols and pharmacologic agents. Subtraction of the delayed rectifier current (IK) from the K+ currents elicited after prepulses to -120 mV of varying duration revealed fast (IAf) and slow (IAs) components with decay time constants of 45 +/- 8 and 612 +/- 140 ms, respectively; the corresponding time constants for the removal of inactivation were 12.3 and 189.6 ms. both tetraethylammonium and dendrotoxin selectively inhibited IAs. 4-Aminopyridine (4-AP) specifically blocked IAf and 40% of IAs with different affinities. Therefore, the properties of a 4-AP-resistant (IAsR) and 4-AP-sensitive (IAsS) component of IAs were compared. These data suggest that three distinct subtypes of K+ currents contribute to the IA of mature murine hippocampal neurones.

  8. Murine Asb-17 expression during mouse testis development and spermatogenesis.

    PubMed

    Kim, Kye-Seong; Kim, Myung-Sun; Kim, Soo-Kyung; Baek, Kwang-Hyun

    2004-05-01

    In this study we isolated a murine mAsb-17 from mouse testis by RT-PCR using primers designed based on the sequences from the GenBank database. The sequence analysis showed that mAsb-17 encodes a 295 amino acid polypeptide with a molecular weight of approximately 34 kDa containing two ankyrin repeats and one SOCS box. The amino acid sequence of mASB-17 showed 87.5%, 98.3% and 92.9% identity with that of human, rat and dog, respectively. Interestingly, northern blot analysis showed that mAsb-17 was expressed only in the testis. The expression analysis by RT-PCR for mAsb-17 in mouse indicates that mAsb-17 is expressed from the fourth week after birth to adult, with the highest expression in round spermatids. Both northern blot and RT-PCR analyses suggest that mASB-17 may play essential roles in testis development and spermatogenesis.

  9. Neonatal umbilical cord blood transplantation halts skeletal disease progression in the murine model of MPS-I.

    PubMed

    Azario, Isabella; Pievani, Alice; Del Priore, Federica; Antolini, Laura; Santi, Ludovica; Corsi, Alessandro; Cardinale, Lucia; Sawamoto, Kazuki; Kubaski, Francyne; Gentner, Bernhard; Bernardo, Maria Ester; Valsecchi, Maria Grazia; Riminucci, Mara; Tomatsu, Shunji; Aiuti, Alessandro; Biondi, Andrea; Serafini, Marta

    2017-08-25

    Umbilical cord blood (UCB) is a promising source of stem cells to use in early haematopoietic stem cell transplantation (HSCT) approaches for several genetic diseases that can be diagnosed at birth. Mucopolysaccharidosis type I (MPS-I) is a progressive multi-system disorder caused by deficiency of lysosomal enzyme α-L-iduronidase, and patients treated with allogeneic HSCT at the onset have improved outcome, suggesting to administer such therapy as early as possible. Given that the best characterized MPS-I murine model is an immunocompetent mouse, we here developed a transplantation system based on murine UCB. With the final aim of testing the therapeutic efficacy of UCB in MPS-I mice transplanted at birth, we first defined the features of murine UCB cells and demonstrated that they are capable of multi-lineage haematopoietic repopulation of myeloablated adult mice similarly to bone marrow cells. We then assessed the effectiveness of murine UCB cells transplantation in busulfan-conditioned newborn MPS-I mice. Twenty weeks after treatment, iduronidase activity was increased in visceral organs of MPS-I animals, glycosaminoglycans storage was reduced, and skeletal phenotype was ameliorated. This study explores a potential therapy for MPS-I at a very early stage in life and represents a novel model to test UCB-based transplantation approaches for various diseases.

  10. Murine Toxicity of Cochliobolus carbonum1

    PubMed Central

    Hamilton, Pat B.; Nelson, R. R.; Harris, B. S. H.

    1968-01-01

    Seventeen wild-type strains of the phytopathogenic fungus Cochliobolus carbonum, tested by intraperitoneal injection into mice, were lethal within 48 hr. The lethal effect appeared to be a toxic rather than an infectious process, because death occurred within 3 hr after injection of two of the isolates and heat-killed cultures were lethal. Assays of ascospore progeny from two crosses involving three isolates indicated that the toxic metabolites were under genetic control and quantitative regulation. Studies of the toxicological, cultural, and chemical characteristics of these three strains indicated that more than one murine toxin was present. PMID:16349821

  11. Irradiation Design for an Experimental Murine Model

    SciTech Connect

    Ballesteros-Zebadua, P.; Moreno-Jimenez, S.; Suarez-Campos, J. E.; Celis, M. A.; Larraga-Gutierrez, J. M.; Garcia-Garduno, O. A.; Rubio-Osornio, M. C.; Custodio-Ramirez, V.; Paz, C.

    2010-12-07

    In radiotherapy and stereotactic radiosurgery, small animal experimental models are frequently used, since there are still a lot of unsolved questions about the biological and biochemical effects of ionizing radiation. This work presents a method for small-animal brain radiotherapy compatible with a dedicated 6MV Linac. This rodent model is focused on the research of the inflammatory effects produced by ionizing radiation in the brain. In this work comparisons between Pencil Beam and Monte Carlo techniques, were used in order to evaluate accuracy of the calculated dose using a commercial planning system. Challenges in this murine model are discussed.

  12. Characterization of Murine Gammaherpesvirus 68 Glycoprotein B

    PubMed Central

    Lopes, Filipa B.; Colaco, Susanna; May, Janet S.; Stevenson, Philip G.

    2004-01-01

    Murine gammaherpesvirus 68 (MHV-68) glycoprotein B (gB) was identified in purified virions by immunoblotting, immunoprecipitation, and immunoelectron microscopy. It was synthesized as a 120-kDa precursor in infected cells and cleaved into 65-kDa and 55-kDa disulfide-linked subunits close to the time of virion release. The N-linked glycans on the cleaved, virion gB remained partially endoglycosidase H sensitive. The processing of MHV-68 gB therefore appears similar to that of Kaposi's sarcoma-associated herpesvirus gB and human cytomegalovirus gB. PMID:15542690

  13. Irradiation Design for an Experimental Murine Model

    NASA Astrophysics Data System (ADS)

    Ballesteros-Zebadúa, P.; Lárraga-Gutierrez, J. M.; García-Garduño, O. A.; Rubio-Osornio, M. C.; Custodio-Ramírez, V.; Moreno-Jimenez, S.; Suarez-Campos, J. E.; Paz, C.; Celis, M. A.

    2010-12-01

    In radiotherapy and stereotactic radiosurgery, small animal experimental models are frequently used, since there are still a lot of unsolved questions about the biological and biochemical effects of ionizing radiation. This work presents a method for small-animal brain radiotherapy compatible with a dedicated 6MV Linac. This rodent model is focused on the research of the inflammatory effects produced by ionizing radiation in the brain. In this work comparisons between Pencil Beam and Monte Carlo techniques, were used in order to evaluate accuracy of the calculated dose using a commercial planning system. Challenges in this murine model are discussed.

  14. Transplantation sites for human and murine islets.

    PubMed

    Stokes, Rebecca A; Cheng, Kim; Lalwani, Amit; Swarbrick, Michael M; Thomas, Helen E; Loudovaris, Thomas; Kay, Tom W; Hawthorne, Wayne J; O'Connell, Philip J; Gunton, Jenny E

    2017-07-22

    Beta cell replacement is a potential cure for type 1 diabetes. In humans, islet transplants are currently infused into the liver via the portal vein, although this site has disadvantages. Here, we investigated alternative transplantation sites for human and murine islets in recipient mice, comparing the portal vein with quadriceps muscle and kidney, liver and spleen capsules. Murine islets were isolated from C57BL6/J mice and transplanted into syngeneic recipients. Human islets were isolated and transplanted into either severe combined immunodeficiency (SCID) or recombination-activating gene 1 (RAG-1) immunodeficient recipient mice. All recipient mice were 8-12 weeks of age and had been rendered diabetic (defined as blood glucose concentrations ≥20 mmol/l on two consecutive days before transplantation) by alloxan tetrahydrate treatment. Islets were transplanted into five different sites (portal vein, quadriceps muscle, kidney, liver and spleen capsules). Blood glucose concentrations were monitored twice weekly until mice were killed. Dose-response studies were also performed to determine the minimum number of islets required to cure diabetes ('cure' is defined for this study as random fed blood glucose of <15 mmol/l). For transplantation of murine islets into the different sites, the kidney yielded 100% success, followed by muscle (70%), portal vein (60%), spleen capsule (29%) and liver capsule (0%). For human islets, transplantation into the kidney cured diabetes in 75-80% of recipient mice. Transplantation into muscle and portal vein had intermediate success (both 29% at 2000 islet equivalents), while transplantation into liver and spleen capsule failed (0%). With increased islet mass, success rates for muscle grafts improved to 52-56%. For both human and murine islets, equivalent or superior glucose lowering results were obtained for transplantation into skeletal muscle, compared with the portal vein. Unfortunately, kidney grafts are not feasible in human

  15. Efficacy of posaconazole in murine experimental sporotrichosis.

    PubMed

    Fernández-Silva, Fabiola; Capilla, Javier; Mayayo, Emilio; Guarro, Josep

    2012-05-01

    We developed a murine model of systemic sporotrichosis by using three strains of each of the two commonest species causing sporotrichosis, i.e., Sporothrix schenckii sensu stricto and Sporothrix brasiliensis, in order to evaluate the efficacy of posaconazole (PSC). The drug was administered at a dose of 2.5 or 5 mg/kg of body weight twice a day by gavage, and one group was treated with amphotericin B (AMB) as a control treatment. Posaconazole, especially at 5 mg/kg, showed good efficacy against all the strains tested, regardless of their MICs, as measured by prolonged survival, tissue burden reduction, and histopathology.

  16. Efficacy of Posaconazole in Murine Experimental Sporotrichosis

    PubMed Central

    Fernández-Silva, Fabiola; Capilla, Javier; Mayayo, Emilio

    2012-01-01

    We developed a murine model of systemic sporotrichosis by using three strains of each of the two commonest species causing sporotrichosis, i.e., Sporothrix schenckii sensu stricto and Sporothrix brasiliensis, in order to evaluate the efficacy of posaconazole (PSC). The drug was administered at a dose of 2.5 or 5 mg/kg of body weight twice a day by gavage, and one group was treated with amphotericin B (AMB) as a control treatment. Posaconazole, especially at 5 mg/kg, showed good efficacy against all the strains tested, regardless of their MICs, as measured by prolonged survival, tissue burden reduction, and histopathology. PMID:22330929

  17. Factors affecting responses to murine oncogenic viral infections.

    PubMed Central

    Harvey, J. J.; Rager-Zisman, B.; Wheelock, E. F.; Nevin, P. A.

    1980-01-01

    Silica specifically kills macrophages in vitro, and in vivo has been used as a method of determining the possible immunological or other roles of macrophages in a number of viral infections. In experiments reported here, injection of 30 or 50 mg silica i.p. increased the severity of the oncogenic effects of the murine sarcoma virus (MSV) and Friend virus (FV) in BALB/c mice. Unlike Herpes simplex and Coxsackie B-3 infections, however, passive transfer of adult macrophages to suckling mice did not protect the latter against MSV. In mice injected with silica, histological evidence of the compensatory proliferation of macrophages suggests that precursors of these cells may act as target cells for the virus and that this may override any immunosuppressive response effected by the silica. In addition, there was a considerable enhancing effect on the erythroproliferative response to both MSV and FV by injection of saline 5 h before the virus, and indeed to FV after only a simple abdominal needle puncture. We attributed this to the lymphopenic immunodepressive effects of stress, and our data may explain previously published findings of augmented oncogenic responses in mice after "normal" serum injections. Newborn BALB/c (FV-1b) mice were susceptible to N-tropic FV, but developed resistance by 29 days of age. Antithymocyte serum (ATS) but not silica injections or adult thymectomy ablated this resistance. C57BL (FV-2r) mice were completely resistant to FV; however, those receiving FV and ATS developed late-onset leukaemia histologically characteristic of that produced by the helper component of the FV complex. Images Fig. PMID:6248095

  18. An Unusual Cutaneous Manifestation in a Patient with Murine Typhus

    PubMed Central

    Blanton, Lucas S.; Lea, Alfred S.; Kelly, Brent C.; Walker, David H.

    2015-01-01

    Murine typhus is a flea-borne febrile illness caused by Rickettsia typhi. Although often accompanied by rash, an inoculation lesion has not been observed as it is with many tick- and mite-transmitted rickettsioses. We describe a patient with murine typhus and an unusual cutaneous manifestation at the site of rickettsial inoculation. PMID:26416115

  19. Reemergence of murine typhus in Galveston, Texas, USA, 2013.

    PubMed

    Blanton, Lucas S; Vohra, Rahat F; Bouyer, Donald H; Walker, David H

    2015-03-01

    Twelve patients with murine typhus were identified in Galveston, Texas, USA, in 2013. An isolate from 1 patient was confirmed to be Rickettsia typhi. Reemergence of murine typhus in Galveston emphasizes the importance of vector control and awareness of this disease by physicians and public health officials.

  20. Reemergence of Murine Typhus in Galveston, Texas, USA, 2013

    PubMed Central

    Vohra, Rahat F.; Bouyer, Donald H.; Walker, David H.

    2015-01-01

    Twelve patients with murine typhus were identified in Galveston, Texas, USA, in 2013. An isolate from 1 patient was confirmed to be Rickettsia typhi. Reemergence of murine typhus in Galveston emphasizes the importance of vector control and awareness of this disease by physicians and public health officials. PMID:25695758

  1. Whole-Exome Sequencing Identifies Loci Associated with Blood Cell Traits and Reveals a Role for Alternative GFI1B Splice Variants in Human Hematopoiesis.

    PubMed

    Polfus, Linda M; Khajuria, Rajiv K; Schick, Ursula M; Pankratz, Nathan; Pazoki, Raha; Brody, Jennifer A; Chen, Ming-Huei; Auer, Paul L; Floyd, James S; Huang, Jie; Lange, Leslie; van Rooij, Frank J A; Gibbs, Richard A; Metcalf, Ginger; Muzny, Donna; Veeraraghavan, Narayanan; Walter, Klaudia; Chen, Lu; Yanek, Lisa; Becker, Lewis C; Peloso, Gina M; Wakabayashi, Aoi; Kals, Mart; Metspalu, Andres; Esko, Tõnu; Fox, Keolu; Wallace, Robert; Franceshini, Nora; Matijevic, Nena; Rice, Kenneth M; Bartz, Traci M; Lyytikäinen, Leo-Pekka; Kähönen, Mika; Lehtimäki, Terho; Raitakari, Olli T; Li-Gao, Ruifang; Mook-Kanamori, Dennis O; Lettre, Guillaume; van Duijn, Cornelia M; Franco, Oscar H; Rich, Stephen S; Rivadeneira, Fernando; Hofman, Albert; Uitterlinden, André G; Wilson, James G; Psaty, Bruce M; Soranzo, Nicole; Dehghan, Abbas; Boerwinkle, Eric; Zhang, Xiaoling; Johnson, Andrew D; O'Donnell, Christopher J; Johnsen, Jill M; Reiner, Alexander P; Ganesh, Santhi K; Sankaran, Vijay G

    2016-08-04

    Circulating blood cell counts and indices are important indicators of hematopoietic function and a number of clinical parameters, such as blood oxygen-carrying capacity, inflammation, and hemostasis. By performing whole-exome sequence association analyses of hematologic quantitative traits in 15,459 community-dwelling individuals, followed by in silico replication in up to 52,024 independent samples, we identified two previously undescribed coding variants associated with lower platelet count: a common missense variant in CPS1 (rs1047891, MAF = 0.33, discovery + replication p = 6.38 × 10(-10)) and a rare synonymous variant in GFI1B (rs150813342, MAF = 0.009, discovery + replication p = 1.79 × 10(-27)). By performing CRISPR/Cas9 genome editing in hematopoietic cell lines and follow-up targeted knockdown experiments in primary human hematopoietic stem and progenitor cells, we demonstrate an alternative splicing mechanism by which the GFI1B rs150813342 variant suppresses formation of a GFI1B isoform that preferentially promotes megakaryocyte differentiation and platelet production. These results demonstrate how unbiased studies of natural variation in blood cell traits can provide insight into the regulation of human hematopoiesis.

  2. Mesenchymal stromal cells (MSC) from JAK2+ myeloproliferative neoplasms differ from normal MSC and contribute to the maintenance of neoplastic hematopoiesis.

    PubMed

    Ramos, Teresa L; Sánchez-Abarca, Luis Ignacio; Rosón-Burgo, Beatriz; Redondo, Alba; Rico, Ana; Preciado, Silvia; Ortega, Rebeca; Rodríguez, Concepción; Muntión, Sandra; Hernández-Hernández, Ángel; De Las Rivas, Javier; González, Marcos; González Porras, José Ramón; Del Cañizo, Consuelo; Sánchez-Guijo, Fermín

    2017-01-01

    There is evidence of continuous bidirectional cross-talk between malignant cells and bone marrow-derived mesenchymal stromal cells (BM-MSC), which favors the emergence and progression of myeloproliferative neoplastic (MPN) diseases. In the current work we have compared the function and gene expression profile of BM-MSC from healthy donors (HD-MSC) and patients with MPN (JAK2V617F), showing no differences in the morphology, proliferation and differentiation capacity between both groups. However, BM-MSC from MPN expressed higher mean fluorescence intensity (MIF) of CD73, CD44 and CD90, whereas CD105 was lower when compared to controls. Gene expression profile of BM-MSC showed a total of 169 genes that were differentially expressed in BM-MSC from MPN patients compared to HD-MSC. In addition, we studied the ability of BM-MSC to support the growth and survival of hematopoietic stem/progenitor cells (HSPC), showing a significant increase in the number of CFU-GM colonies when MPN-HSPC were co-cultured with MPN-MSC. Furthermore, MPN-MSC showed alteration in the expression of genes associated to the maintenance of hematopoiesis, with an overexpression of SPP1 and NF-kB, and a downregulation of ANGPT1 and THPO. Our results suggest that BM-MSC from JAK2+ patients differ from their normal counterparts and favor the maintenance of malignant clonal hematopoietic cells.

  3. Mesenchymal stromal cells (MSC) from JAK2+ myeloproliferative neoplasms differ from normal MSC and contribute to the maintenance of neoplastic hematopoiesis

    PubMed Central

    Sánchez-Abarca, Luis Ignacio; Rosón-Burgo, Beatriz; Redondo, Alba; Rico, Ana; Preciado, Silvia; Ortega, Rebeca; Rodríguez, Concepción; Muntión, Sandra; Hernández-Hernández, Ángel; De Las Rivas, Javier; González, Marcos; González Porras, José Ramón; del Cañizo, Consuelo; Sánchez-Guijo, Fermín

    2017-01-01

    There is evidence of continuous bidirectional cross-talk between malignant cells and bone marrow-derived mesenchymal stromal cells (BM-MSC), which favors the emergence and progression of myeloproliferative neoplastic (MPN) diseases. In the current work we have compared the function and gene expression profile of BM-MSC from healthy donors (HD-MSC) and patients with MPN (JAK2V617F), showing no differences in the morphology, proliferation and differentiation capacity between both groups. However, BM-MSC from MPN expressed higher mean fluorescence intensity (MIF) of CD73, CD44 and CD90, whereas CD105 was lower when compared to controls. Gene expression profile of BM-MSC showed a total of 169 genes that were differentially expressed in BM-MSC from MPN patients compared to HD-MSC. In addition, we studied the ability of BM-MSC to support the growth and survival of hematopoietic stem/progenitor cells (HSPC), showing a significant increase in the number of CFU-GM colonies when MPN-HSPC were co-cultured with MPN-MSC. Furthermore, MPN-MSC showed alteration in the expression of genes associated to the maintenance of hematopoiesis, with an overexpression of SPP1 and NF-kB, and a downregulation of ANGPT1 and THPO. Our results suggest that BM-MSC from JAK2+ patients differ from their normal counterparts and favor the maintenance of malignant clonal hematopoietic cells. PMID:28796790

  4. Saponins from Sanguisorba officinalis Improve Hematopoiesis by Promoting Survival through FAK and Erk1/2 Activation and Modulating Cytokine Production in Bone Marrow

    PubMed Central

    Chen, Xin; Li, Bogang; Gao, Yue; Ji, Jianxin; Wu, Zhongliu; Chen, Shuang

    2017-01-01

    Radix Sanguisorbae, the root of Sanguisorba officinalis L. is used as traditional Chinese medicine. In recent decades, it has been reported to be clinically effective against myelosuppression induced by chemotherapy and/ or radiotherapy. However, the underlining mechanism has not been well studied. In this work, we evaluated the hematopoietic effect of total saponins from S. officinalis L. on myelosuppressive mice induced by cyclophosphamide and by60Co-γ-irradiation and confirmed the therapeutic effect. Then, we found total saponins and their characteristic constituents Ziyuglycoside I and Ziyuglycoside II can inhibit apoptosis of TF-1 cells caused by cytokine deprivation, and promote survival of mouse bone marrow nuclear cells through focal adhesion kinase (FAK) and extracellular signal-regulated kinase 1/2 (Erk1/2) activation in vitro. In addition, they can down-regulate macrophage inflammatory protein 2 (MIP-2), platelet factor 4 (PF4) and P-selectin secretion, which are reported to be suppressive to hematopoiesis, both in vitro and in vivo. These results suggest that promotion of survival through FAK and Erk1/2 activation and inhibition of suppressive cytokines in the bone marrow is likely to be the pharmacological mechanism underlying the hematopoietic effect of saponins from S. officinalis L. PMID:28360858

  5. Loss of Tifab, a del(5q) MDS gene, alters hematopoiesis through derepression of Toll-like receptor–TRAF6 signaling

    PubMed Central

    Varney, Melinda E.; Niederkorn, Madeline; Konno, Hiroyasu; Matsumura, Takayuki; Gohda, Jin; Yoshida, Nobuaki; Akiyama, Taishin; Christie, Susanne; Fang, Jing; Miller, David; Jerez, Andres; Karsan, Aly; Maciejewski, Jaroslaw P.; Meetei, Ruhikanta A.; Inoue, Jun-ichiro

    2015-01-01

    TRAF-interacting protein with forkhead-associated domain B (TIFAB) is a haploinsufficient gene in del(5q) myelodysplastic syndrome (MDS). Deletion of Tifab results in progressive bone marrow (BM) and blood defects, including skewed hematopoietic stem/progenitor cell (HSPC) proportions and altered myeloid differentiation. A subset of mice transplanted with Tifab knockout (KO) HSPCs develop a BM failure with neutrophil dysplasia and cytopenia. In competitive transplants, Tifab KO HSPCs are out-competed by wild-type (WT) cells, suggesting a cell-intrinsic defect. Gene expression analysis of Tifab KO HSPCs identified dysregulation of immune-related signatures, and hypersensitivity to TLR4 stimulation. TIFAB forms a complex with TRAF6, a mediator of immune signaling, and reduces TRAF6 protein stability by a lysosome-dependent mechanism. In contrast, TIFAB loss increases TRAF6 protein and the dynamic range of TLR4 signaling, contributing to ineffective hematopoiesis. Moreover, combined deletion of TIFAB and miR-146a, two genes associated with del(5q) MDS/AML, results in a cooperative increase in TRAF6 expression and hematopoietic dysfunction. Re-expression of TIFAB in del(5q) MDS/AML cells results in attenuated TLR4 signaling and reduced viability. These findings underscore the importance of efficient regulation of innate immune/TRAF6 signaling within HSPCs by TIFAB, and its cooperation with miR-146a as it relates to the pathogenesis of hematopoietic malignancies, such as del(5q) MDS/AML. PMID:26458771

  6. Structural features of hematopoiesis-specific RhoH/ARHH gene: high diversity of 5'-UTR in different hematopoietic lineages suggests a complex post-transcriptional regulation.

    PubMed

    Lahousse, Sébastien; Smorowski, Anne-Lise; Denis, Claude; Lantoine, Danièle; Kerckaert, Jean-Pierre; Galiègue-Zouitina, Sylvie

    2004-12-08

    The hematopoiesis-specific RhoH gene is thought to be deregulated in B-cell non-Hodgkin's lymphoma (B-NHL), by either a chromosomal translocation or mutations, which affect its 5' regulatory region. The encoded Rho protein, always GTP-bound in vivo, was hypothesized to behave as a Rac antagonist. Extensive expression analysis allowed the detection of RhoH transcripts in all hematopoietic lineages (lymphoid, erythroid, myeloid), with a high level in lymphoid cells. To initiate investigations on the molecular mechanisms that regulate RhoH gene expression, Race-PCR and primer extension were conducted in the B-cell line Raji, which allowed (i) the establishment of RhoH complex intron/exon organization and (ii) the detection of several transcription initiation sites. In addition, a high 5' end heterogeneity of RhoH mRNAs was observed, due to alternative splicing of some 5' exons and to the use of these different transcription start sites. RT-PCR analysis led to the identification of this 5' end heterogeneity in different hematopoietic lineages. Discrepancies were particularly observed between B and T cells, due to an alternative splicing of one 5' exon (1b), which might be an important element in RhoH gene regulation. Such specific features have never been described for any Rho family member gene. They provide a molecular basis to study complex mechanisms involved in the control of RhoH expression.

  7. The Gottingen minipig is a model of the hematopoietic acute radiation syndrome: G-CSF stimulates hematopoiesis and enhances survival from lethal total-body gamma-irradiation

    PubMed Central

    Moroni, Maria; Ngudiankama, Barbara F.; Christensen, Christine; Olsen, Cara H.; Owens, Rossitsa; Lombardini, Eric D.; Holt, Rebecca K.; Whitnall, Mark H.

    2013-01-01

    Purpose We are characterizing the Gottingen minipig as an additional large animal model for advanced drug testing for the Acute Radiation Syndrome (ARS), to enhance discovery and development of novel radiation countermeasures. Among the advantages provided by this model, the similarities to human hematological parameters and dynamics of cell loss/recovery following irradiation provide a convenient means to compare efficacy of drugs known to affect bone marrow cellularity and hematopoiesis. Methods and Materials Male Gottingen minipigs, 4–5 months old and weighing 9–11 kg were used for this study. We tested the standard off-label treatment for ARS, rhG-CSF (Neupogen®, 10 μg/kg/day for 17 days), at the estimated LD70/30 total-body gamma-irradiation (TBI) radiation dose for the hematopoietic syndrome, starting 24 hours after irradiation. Results Results indicate G-CSF enhanced survival, stimulated recovery from neutropenia, and induced mobilization of hematopoietic progenitor cells. In addition, administration of G-CSF resulted in maturation of monocytes/macrophages. Conclusion These results support continuing efforts toward validation of the minipig as a large animal model for advanced testing of radiation countermeasures and characterization of the pathophysiology of ARS, and suggest that the efficacy of G-CSF in improving survival after total body irradiation may involve mechanisms other than increasing numbers of circulating granulocytes. PMID:23845847

  8. Effects of the bifunctional sulfoxide MMS350, a radiation mitigator, on hematopoiesis in long-term bone marrow cultures and on radioresistance of marrow stromal cell lines.

    PubMed

    Shinde, Ashwin; Epperly, Michael W; Cao, Shaonan; Franicola, Darcy; Shields, Donna; Wang, Hong; Wipf, Peter; Sprachman, Melissa M; Greenberger, Joel S

    2014-01-01

    The ionizing irradiation mitigator MMS350 prolongs survival of mice treated with total-body irradiation and prevents radiation-induced pulmonary fibrosis when added to drinking water at day 100 after thoracic irradiation. The effects of MMS350 on hematopoiesis in long-term bone marrow culture and on the radiobiology of derived bone marrow stromal cell lines were tested. Long-term bone marrow cultures were established from C57BL/6NTac mice and maintained in a high-humidity incubator, with 7% CO2 and the addition of 100 μM MMS350 at the weekly media change. Over 10 weeks in culture, MMS350 had no significant effect on maintenance of hematopoietic stem cell production, or on nonadherent cells or colony-forming units of hematopoietic progenitor cells. Stromal cell lines derived from non MMS350-treated long-term cultures or control stromal cells treated with MMS350 were radioresistant in the clonogenic survival curve assay. MMS350 is a non-toxic, highly water-soluble radiation mitigator that exhibits radioprotective effects on bone marrow stromal cells.

  9. Novel Therapeutic Approach to Improve Hematopoiesis in low risk MDS by Targeting MDSCs with The Fc-engineered CD33 Antibody BI 836858

    PubMed Central

    Eksioglu, Erika A.; Chen, Xianghong; Heider, Karl-Heinz; Rueter, Bjoern; McGraw, Kathy L.; Basiorka, Ashley A.; Wei, Max; Burnette, Alexis; Cheng, Pinyang; Lancet, Jeffrey; Komrokji, Rami; Djeu, Julie; List, Alan; Wei, Sheng

    2017-01-01

    We recently reported that the accumulation of myeloid-derived suppressor cells (MDSC), defined as CD33+HLA-DR−Lin−, plays a direct role in the pathogenesis of myelodysplastic syndrome (MDS). In particular, CD33 is strongly expressed in MDSC isolated from patients with MDS where it plays an important role in MDSC-mediated hematopoietic suppressive function through its activation by S100A9. Therefore, we tested whether blocking this interaction with a fully human, Fc-engineered monoclonal antibody against CD33 (BI 836858) suppresses CD33-mediated signal transduction and improves the bone marrow microenvironment in MDS. We observed that BI 836858 can reduce MDSC by antibody-dependent cellular cytotoxicity (ADCC), which correlated with increases in granule mobilization and cell death. BI 836858 can also block CD33 downstream signaling preventing immune-suppressive cytokine secretion, which correlates with a significant increase in the formation of CFU-GM and BFU-E colonies. Activation of the CD33 pathway can cause reactive oxygen species (ROS)-induced genomic instability but BI 836858 reduced both ROS and the levels of double strand breaks and adducts (measured by comet assay and γH2AX). This work provides the ground for the development of a novel group of therapies for MDS aimed at MDSC and their disease-promoting properties with the goal of improving hematopoiesis in patients. PMID:28096534

  10. Expression of p21(Cip1/Waf1/Sdi1) and p27(Kip1) cyclin-dependent kinase inhibitors during human hematopoiesis.

    PubMed

    Taniguchi, T; Endo, H; Chikatsu, N; Uchimaru, K; Asano, S; Fujita, T; Nakahata, T; Motokura, T

    1999-06-15

    Expression of p21 and p27 cyclin-dependent kinase inhibitors is associated with induced differentiation and cell-cycle arrest in some hematopoietic cell lines. However, it is not clear how these inhibitors are expressed during normal hematopoiesis. We examined various human hematopoietic colonies derived from cord blood CD34(+) cells, bone marrow, and peripheral blood cells using a quantitative reverse transcription-polymerase chain reaction assay, immunochemistry, and/or Western blot analysis. p21 mRNA was expressed increasingly over time in all of the colonies examined (granulocytes, macrophages, megakaryocytes, and erythroblasts), whereas p27 mRNA levels remained low, except for erythroid bursts. Erythroid bursts expressed both p21 and p27 mRNAs with differentiation but expressed neither protein, whereas both proteins were expressed in megakaryocytes and peripheral blood monocytes. In bone marrow, p21 was immunostained almost exclusively in a subset of megakaryocytes and p27 protein was present in megakaryocytes, plasma cells, and endothelial cells. In megakaryocytes, reciprocal expression of p27 to Ki-67 was evident and an inverse relationship between p21 and Ki-67 positivities was also present, albeit less obvious. These observations suggest that a complex lineage-specific regulation is involved in p21 and p27 expression and that these inhibitors are involved in cell-cycle exit in megakaryocytes.

  11. Loss of c-Kit and bone marrow failure upon conditional removal of the GATA-2 C-terminal zinc finger domain in adult mice.

    PubMed

    Li, Haiyan S; Jin, Jin; Liang, Xiaoxuan; Matatall, Katie A; Ma, Ying; Zhang, Huiyuan; Ullrich, Stephen E; King, Katherine Y; Sun, Shao-Cong; Watowich, Stephanie S

    2016-09-01

    Heterozygous mutations in the transcriptional regulator GATA-2 associate with multilineage immunodeficiency, myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML). The majority of these mutations localize in the zinc finger (ZnF) domains, which mediate GATA-2 DNA binding. Deregulated hematopoiesis with GATA-2 mutation frequently develops in adulthood, yet GATA-2 function in the bone marrow remains unresolved. To investigate this, we conditionally deleted the GATA-2 C-terminal ZnF (C-ZnF) coding sequences in adult mice. Upon Gata2 C-ZnF deletion, we observed rapid peripheral cytopenia, bone marrow failure, and decreased c-Kit expression on hematopoietic progenitors. Transplant studies indicated GATA-2 has a cell-autonomous role in bone marrow hematopoiesis. Moreover, myeloid lineage populations were particularly sensitive to Gata2 hemizygosity, while molecular assays indicated GATA-2 regulates c-Kit expression in multilineage progenitor cells. Enforced c-Kit expression in Gata2 C-ZnF-deficient hematopoietic progenitors enhanced myeloid colony activity, suggesting GATA-2 sustains myelopoiesis via a cell intrinsic role involving maintenance of c-Kit expression. Our results provide insight into mechanisms regulating hematopoiesis in bone marrow and may contribute to a better understanding of immunodeficiency and bone marrow failure associated with GATA-2 mutation.

  12. The future of murine sepsis and trauma research models

    PubMed Central

    Efron, Philip A.; Mohr, Alicia M.; Moore, Frederick A.; Moldawer, Lyle L.

    2015-01-01

    Recent comparisons of the murine and human transcriptome in health and disease have called into question the appropriateness of the use of murine models for human sepsis and trauma research. More specifically, researchers have debated the suitability of mouse models of severe inflammation that is intended for eventual translation to human patients. This mini-review outlines this recent research, as well as specifically defines the arguments for and against murine models of sepsis and trauma research based on these transcriptional studies. In addition, we review newer advancements in murine models of infection and injury and define what we envision as an evolving but viable future for murine studies of sepsis and trauma. PMID:26034205

  13. Bone marrow mononuclears from murine tibia after spaceflight on biosatellite

    NASA Astrophysics Data System (ADS)

    Andreeva, Elena; Roe, Maria; Buravkova, Ludmila; Andrianova, Irina; Goncharova, Elena; Gornostaeva, Alexandra

    Elucidation of the space flight effects on the adult stem and progenitor cells is an important goal in space biology and medicine. A unique opportunity for this is provided by project "BION -M1". The purpose of this study was to evaluate the effects of a 30-day flight on biosatellite "BION - M1" and the subsequent 7-day recovery on the quantity, viability, immunophenotype of mononuclears from murine tibia bone marrow. Also the in vitro characterization of functional capacity of multipotent mesenchymal stromal cells (MSCs) was scheduled. Under the project, the S57black/6 mice were divided into groups: spaceflight/vivarium control, recovery after spaceflight/ vivarium control to recovery. Bone marrow mononuclears were isolated from the tibia and immunophenotyped using antibodies against CD45, CD34, CD90 on a flow cytometer Epics XL (Beckman Coulter). A part of the each pool was frozen for subsequent estimation of hematopoietic colony-forming units (CFU), the rest was used for the evaluation of fibroblast CFU (CFUf) number, MSC proliferative activity and osteogenic potency. The cell number in the flight group was significantly lower than in the vivarium control group. There were no differences in this parameter between flight and control groups after 7 days of recovery. The mononuclears viability was more than 95 percent in all examined groups. Flow cytometric analysis showed no differences in the bone marrow cell immunophenotype (CD45, CD34, CD90.1 (Thy1)), but the flight animals had more large-sized CD45+mononuclears, than the control groups of mice. There was no difference in the CFUf number between groups. After 7 days in vitro the MSC number in flight group was twice higher than in vivarium group, after 10 days - 4 times higher. These data may indicate a higher proliferative activity of MSCs after spaceflight. MSCs showed the same and high alkaline phosphatase activity, both in flight and in the control groups, suggesting no effect of spaceflight factors on early

  14. Pathogenesis and immunity in murine salmonellosis.

    PubMed Central

    Hsu, H S

    1989-01-01

    Salmonella is traditionally described as a facultative intracellular parasite, and host macrophages are regarded as the primary effector cells in both native and acquired immunity in mouse typhoid. This concept has not been unanimously accepted in the literature. Based on cell culture experiments and electron microscopic examinations of infected tissues, we observed that virulent Salmonella typhimurium is killed within polymorphs and macrophages of guinea pigs and mice. In a systemic disease, the organism propagates primarily in the extracellular locations of sinusoids and tissue lesions and within hepatocytes. Hence, it is more likely to be an extracellular pathogen and its virulence is directly related to its antiphagocytic property. The conspicuous absence of macrophages in the primary lesions of murine salmonellosis disputes the likelihood of their significant role in native resistance to the disease. Acquired cellular immunity is expressed as an enhanced antibacterial activity of macrophages facilitated by cytophilic antibodies rather than as an altered antibacterial action of immune macrophages. It is proposed that acquired immunity in murine salmonellosis is a synergistic manifestation of the innate capacity of polymorphs and macrophages to destroy ingested salmonellae, the activated antibacterial functions of macrophages mediated by cytophilic antibodies, the opsonic and agglutinating actions of antiserum, and the accelerated inflammation associated with delayed hypersensitivity to bacterial antigens. Unlike live attenuated vaccines, nonviable vaccines offer a significant, though not a solid, protection against subsequent challenges. Images PMID:2687679

  15. Murine models of breast cancer bone metastasis

    PubMed Central

    Wright, Laura E; Ottewell, Penelope D; Rucci, Nadia; Peyruchaud, Olivier; Pagnotti, Gabriel M; Chiechi, Antonella; Buijs, Jeroen T; Sterling, Julie A

    2016-01-01

    Bone metastases cause significant morbidity and mortality in late-stage breast cancer patients and are currently considered incurable. Investigators rely on translational models to better understand the pathogenesis of skeletal complications of malignancy in order to identify therapeutic targets that may ultimately prevent and treat solid tumor metastasis to bone. Many experimental models of breast cancer bone metastases are in use today, each with its own caveats. In this methods review, we characterize the bone phenotype of commonly utilized human- and murine-derived breast cell lines that elicit osteoblastic and/or osteolytic destruction of bone in mice and report methods for optimizing tumor-take in murine models of bone metastasis. We then provide protocols for four of the most common xenograft and syngeneic inoculation routes for modeling breast cancer metastasis to the skeleton in mice, including the intra-cardiac, intra-arterial, orthotopic and intra-tibial methods of tumor cell injection. Recommendations for in vivo and ex vivo assessment of tumor progression and bone destruction are provided, followed by discussion of the strengths and limitations of the available tools and translational models that aid investigators in the study of breast cancer metastasis to bone. PMID:27867497

  16. Effect of zidovudine on preimplantation murine embryos.

    PubMed Central

    Toltzis, P; Mourton, T; Magnuson, T

    1993-01-01

    It previously has been demonstrated that zidovudine (AZT) is lethal to early murine embryos. The effect of the drug on pre- and postimplantation embryos was examined to delineate the timing of this toxicity and to investigate its possible mechanisms. Embryos exposed in the whole mouse during preblastocyst development were unable to proceed beyond the blastocyst stage. Similarly, when two-cell embryos harvested from unexposed females were exposed to low-concentration (1 microM) AZT in vitro over 24 h, development beyond the blastocyst stage was inhibited. In contrast, drug exposure during in vitro blastocyst and postblastocyst development resulted in little or no morphologic toxicity. Further investigation revealed that preblastocyst AZT exposure resulted in the development of blastocysts with significantly lower cell numbers than control embryos. While embryonic exposure to AZT at the blastocyst and postblastocyst stages also resulted in retarded cell division, the effects were milder than those recorded after preblastocyst exposure. These data demonstrate that the critical period of AZT toxicity toward murine embryos is between ovulation and implantation and indicate that AZT directly suppresses cell division in the preimplantation embryo. PMID:8215271

  17. Benzaldehyde suppresses murine allergic asthma and rhinitis.

    PubMed

    Jang, Tae Young; Park, Chang-Shin; Kim, Kyu-Sung; Heo, Min-Jeong; Kim, Young Hyo

    2014-10-01

    To evaluate the antiallergic effects of oral benzaldehyde in a murine model of allergic asthma and rhinitis, we divided 20 female BALB/c mice aged 8-10 weeks into nonallergic (intraperitoneally sensitized and intranasally challenged to normal saline), allergic (intraperitoneally sensitized and intranasally challenged to ovalbumin), and 200- and 400-mg/kg benzaldehyde (allergic but treated) groups. The number of nose-scratching events in 10 min, levels of total and ovalbumin-specific IgE in serum, differential counts of inflammatory cells in bronchoalveolar lavage (BAL) fluid, titers of Th2 cytokines (IL-4, IL-5, IL-13) in BAL fluid, histopathologic findings of lung and nasal tissues, and expressions of proteins involved in apoptosis (Bcl-2, Bax, caspase-3), inflammation (COX-2), antioxidation (extracellular SOD, HO-1), and hypoxia (HIF-1α, VEGF) in lung tissue were evaluated. The treated mice had significantly fewer nose-scratching events, less inflammatory cell infiltration in lung and nasal tissues, and lower HIF-1α and VEGF expressions in lung tissue than the allergic group. The number of eosinophils and neutrophils and Th2 cytokine titers in BAL fluid significantly decreased after the treatment (P<0.05). These results imply that oral benzaldehyde exerts antiallergic effects in murine allergic asthma and rhinitis, possibly through inhibition of HIF-1α and VEGF. Copyright © 2014. Published by Elsevier B.V.

  18. Glucosylceramides stimulate mitogenesis in aged murine epidermis.

    PubMed

    Marchell, N L; Uchida, Y; Brown, B E; Elias, P M; Holleran, W M

    1998-04-01

    Glucosylceramides (GlcCer) and ceramides (Cer) appear to have opposite effects on epidermal growth and differentiation. Whereas Cer inhibit mitosis and induce terminal differentiation and apoptosis in cultured keratinocytes, GlcCer is mitogenic in young murine epidermis. Using a recently described murine model of chronologic senescence we explored whether GlcCer is mitogenic in aged epidermis. Epidermal GlcCer content increases following topical applications of either conduritol-B epoxide (CBE), an inhibitor of GlcCer hydrolysis, or exogenous GlcCer in a penetration-enhancing vehicle. During chronologic aging in the hairless mouse, baseline epidermal DNA synthesis rates remain normal until 18 mo, but decline significantly at 24 mo. Topical CBE stimulates a 1.5- to 1.9-fold increase in epidermal DNA synthesis in all age groups (i.e., 1-2, 18, and 24 mo). Although the CBE induced increase in [3H]thymidine incorporation in 24 mo old animals is significant (p < 0.01), it is not sufficient to reach the absolute levels reached in similarly treated, younger mouse epidermis. Moreover, topical GlcCer induced mitogenesis is both dose dependent and hexose specific in young (1-2 mo old) animals, and remains effective in aged (< or = 24 mo old) animals. Furthermore, the CBE induced increase in DNA synthesis in aged epidermis is sufficient to produce epidermal hyperplasia. Finally, although an increased GlcCer:Cer ratio can alter stratum corneum barrier function and membrane structure, neither stratum corneum function nor extracellular membrane structure change under these experimental conditions, and therefore the mitogenic effects of increased epidermal GlcCer cannot be attributed to effects on the stratum corneum. These results show that: (i) elevations in endogenous GlcCer are mitogenic for aged as well as young murine epidermis; (ii) topical GlcCer is also mitogenic when delivered in an enhancing vehicle; and (iii) despite the putative importance of epidermal DNA synthesis

  19. Differential requirement for irf8 in formation of embryonic and adult macrophages in zebrafish

    SciTech Connect

    Shiau, Celia E.; Kaufman, Zoe; Meireles, Ana M.; Talbot, William S.

    2015-01-23

    Interferon regulatory factor 8 (Irf8) is critical for mammalian macrophage development and innate immunity, but its role in teleost myelopoiesis remains incompletely understood. Specifically, genetic tools to analyze the role of irf8 in zebrafish macrophage development at larval and adult stages are lacking. In this study, we generated irf8 null mutants in zebrafish using TALEN-mediated targeting. Our analysis defines different requirements for irf8 at different stages. irf8 is required for formation of all macrophages during primitive and transient definitive hematopoiesis, but not during adult-phase definitive hematopoiesis starting at 5-6 days postfertilization. At early stages, irf8 mutants have excess neutrophils and excess cell death in pu.1-expressing myeloid cells. Macrophage fates were recovered in irf8 mutants after wildtype irf8 expression in neutrophil and macrophage lineages, suggesting that irf8 regulates macrophage specification and survival. In juvenile irf8 mutant fish, mature macrophages are present, but at numbers significantly reduced compared to wildtype, indicating an ongoing requirement for irf8 after embryogenesis. As development progresses, tissue macrophages become apparent in zebrafish irf8 mutants, with the possible exception of microglia. Our study defines distinct requirement for irf8 in myelopoiesis before and after transition to the adult hematopoietic system.

  20. Dissection of the adult zebrafish kidney.

    PubMed

    Gerlach, Gary F; Schrader, Lauran N; Wingert, Rebecca A

    2011-08-29

    Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans. The zebrafish has become a powerful animal model for the study of vertebrate cell biology. There has been extensive documentation and analysis of embryonic development in the zebrafish. Only recently have scientists sought to document adult anatomy and surgical dissection techniques, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two

  1. Dissection of the Adult Zebrafish Kidney

    PubMed Central

    Wingert, Rebecca A.

    2011-01-01

    Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the

  2. Peptide nucleic acids targeting β-globin mRNAs selectively inhibit hemoglobin production in murine erythroleukemia cells

    PubMed Central

    MONTAGNER, GIULIA; GEMMO, CHIARA; FABBRI, ENRICA; MANICARDI, ALEX; ACCARDO, IGEA; BIANCHI, NICOLETTA; FINOTTI, ALESSIA; BREVEGLIERI, GIULIA; SALVATORI, FRANCESCA; BORGATTI, MONICA; LAMPRONTI, ILARIA; BRESCIANI, ALBERTO; ALTAMURA, SERGIO; CORRADINI, ROBERTO; GAMBARI, ROBERTO

    2015-01-01

    In the treatment of hemoglobinopathies, amending altered hemoglobins and/or globins produced in excess is an important part of therapeutic strategies and the selective inhibition of globin production may be clinically beneficial. Therefore the development of drug-based methods for the selective inhibition of globin accumulation is required. In this study, we employed peptide nucleic acids (PNAs) to alter globin gene expression. The main conclusion of the present study was that PNAs designed to target adult murine β-globin mRNA inhibit hemoglobin accumulation and erythroid differentiation of murine erythroleukemia (MEL) cells with high efficiency and fair selectivity. No major effects were observed on cell proliferation. Our study supports the concept that PNAs may be used to target mRNAs that, similar to globin mRNAs, are expressed at very high levels in differentiating erythroid cells. Our data suggest that PNAs inhibit the excess production of globins involved in the pathophysiology of hemoglobinopathies. PMID:25405921

  3. Glucocorticoid receptors in murine erythroleukaemic cells

    SciTech Connect

    Hammond, K.D.; Torrance, J.M.; DiDomenico, M.

    1987-01-01

    Glucocorticoid receptors in murine erythroleukaemic cells were studied in relation to hexamethylene bisacetamide (HMBA) induced differentiation. Specific binding of dexamethasone was measured. A single class of saturable, high affinity binding sites was demonstrated in intact cells; with cell homogenates or fractions binding was low and could not be reliably quantified. Receptor binding in whole cell suspensions was lower in cells which had been treated with HMBA (36.5 +/- 8.2 pmol/g protein) than in untreated controls (87.9 +/- 23.6 pmol/g protein); dissociation constants were similar in treated (2.7 nM) and untreated cells (2.5 nM). Dexamethasone, hydrocortisone, corticosterone and progesterone competed with tritium-labelled dexamethasone for receptor binding sites; cortisone, deoxycorticosterone and oestradiol had little effect.

  4. Hematopoiesis-restricted minor histocompatibility antigens HA-1- or HA-2-specific T cells can induce complete remissions of relapsed leukemia

    PubMed Central

    Marijt, W. A. Erik; Heemskerk, Mirjam H. M.; Kloosterboer, Freke M.; Goulmy, Els; Kester, Michel G. D.; van der Hoorn, Menno A. W. G.; van Luxemburg-Heys, Simone A. P.; Hoogeboom, Manja; Mutis, Tuna; Drijfhout, Jan Wouter; van Rood, Jon J.; Willemze, Roel; Falkenburg, J. H. Frederik

    2003-01-01

    Donor lymphocyte infusion (DLI) into patients with a relapse of their leukemia or multiple myeloma after allogeneic stem cell transplantation (alloSCT) has been shown to be a successful treatment approach. The hematopoiesis-restricted minor histocompatibility antigens (mHAgs) HA-1 or HA-2 expressed on malignant cells of the recipient may serve as target antigens for alloreactive donor T cells. Recently we treated three mHAg HA-1- and/or HA-2-positive patients with a relapse of their disease after alloSCT with DLI from their mHAg HA-1- and/or HA-2-negative donors. Using HLA-A2/HA-1 and HA-2 peptide tetrameric complexes we showed the emergence of HA-1- and HA-2-specific CD8+ T cells in the blood of the recipients 5–7 weeks after DLI. The appearance of these tetramer-positive cells was followed immediately by a complete remission of the disease and restoration of 100% donor chimerism in each of the patients. Furthermore, cloned tetramer-positive T cells isolated during the clinical response specifically recognized HA-1 and HA-2 expressing malignant progenitor cells of the recipient and inhibited the growth of leukemic precursor cells in vitro. Thus, HA-1- and HA-2-specific cytotoxic T lymphocytes emerging in the blood of patients after DLI demonstrate graft-versus-leukemia or myeloma reactivity resulting in a durable remission. This finding implies that in vitro generated HA-1- and HA-2-specific cytotoxic T lymphocytes could be used as adoptive immunotherapy to treat hematological malignances relapsing after alloSCT. PMID:12601144

  5. Hemorrhage Exacerbates Radiation Effects on Survival, Leukocytopenia, Thrombopenia, Erythropenia, Bone Marrow Cell Depletion and Hematopoiesis, and Inflammation-Associated microRNAs Expression in Kidney

    PubMed Central

    Kiang, Juliann G.; Smith, Joan T.; Anderson, Marsha N.; Swift, Joshua M.; Christensen, Christine L.; Gupta, Paridhi; Balakathiresan, Nagaraja; Maheshwari, Radha K.

    2015-01-01

    Exposure to high-dose radiation results in detrimental effects on survival. The effects of combined trauma, such as radiation in combination with hemorrhage, the typical injury of victims exposed to a radiation blast, on survival and hematopoietic effects have yet to be understood. The purpose of this study was to evaluate the effects of radiation injury (RI) combined with hemorrhage (i.e., combined injury, CI) on survival and hematopoietic effects, and to investigate whether hemorrhage (Hemo) enhanced RI-induced mortality and hematopoietic syndrome. Male CD2F1 mice (10 weeks old) were given one single exposure of γ- radiation (60Co) at various doses (0.6 Gy/min). Within 2 hr after RI, animals under anesthesia were bled 0% (Sham) or 20% (Hemo) of total blood volume via the submandibular vein. In these mice, Hemo reduced the LD50/30 for 30-day survival from 9.1 Gy (RI) to 8.75 Gy (CI) with a DMF of 1.046. RI resulted in leukocytopenia, thrombopenia, erythropenia, and bone marrow cell depletion, but decreased the caspase-3 activation response. RI increased IL-1β, IL-6, IL-17A, and TNF-α concentrations in serum, bone marrow, ileum, spleen, and kidney. Some of these adverse alterations were magnified by CI. Erythropoietin production was increased in kidney and blood more after CI than RI. Furthermore, CI altered the global miRNAs expression in kidney and the ingenuity pathway analysis showed that miRNAs viz., let-7e, miR-30e and miR-29b that were associated with hematopoiesis and inflammation. This study provides preliminary evidence that non-lethal Hemo exacerbates RI-induced mortality and cell losses associated with high-dose γ-radiation. We identified some of the initial changes occurring due to CI which may have facilitated in worsening the injury and hampering the recovery of animals ultimately resulting in higher mortality. PMID:26422254

  6. Hemorrhage Exacerbates Radiation Effects on Survival, Leukocytopenia, Thrombopenia, Erythropenia, Bone Marrow Cell Depletion and Hematopoiesis, and Inflammation-Associated microRNAs Expression in Kidney.

    PubMed

    Kiang, Juliann G; Smith, Joan T; Anderson, Marsha N; Swift, Joshua M; Christensen, Christine L; Gupta, Paridhi; Balakathiresan, Nagaraja; Maheshwari, Radha K

    2015-01-01

    Exposure to high-dose radiation results in detrimental effects on survival. The effects of combined trauma, such as radiation in combination with hemorrhage, the typical injury of victims exposed to a radiation blast, on survival and hematopoietic effects have yet to be understood. The purpose of this study was to evaluate the effects of radiation injury (RI) combined with hemorrhage (i.e., combined injury, CI) on survival and hematopoietic effects, and to investigate whether hemorrhage (Hemo) enhanced RI-induced mortality and hematopoietic syndrome. Male CD2F1 mice (10 weeks old) were given one single exposure of γ- radiation (60Co) at various doses (0.6 Gy/min). Within 2 hr after RI, animals under anesthesia were bled 0% (Sham) or 20% (Hemo) of total blood volume via the submandibular vein. In these mice, Hemo reduced the LD50/30 for 30-day survival from 9.1 Gy (RI) to 8.75 Gy (CI) with a DMF of 1.046. RI resulted in leukocytopenia, thrombopenia, erythropenia, and bone marrow cell depletion, but decreased the caspase-3 activation response. RI increased IL-1β, IL-6, IL-17A, and TNF-α concentrations in serum, bone marrow, ileum, spleen, and kidney. Some of these adverse alterations were magnified by CI. Erythropoietin production was increased in kidney and blood more after CI than RI. Furthermore, CI altered the global miRNAs expression in kidney and the ingenuity pathway analysis showed that miRNAs viz., let-7e, miR-30e and miR-29b that were associated with hematopoiesis and inflammation. This study provides preliminary evidence that non-lethal Hemo exacerbates RI-induced mortality and cell losses associated with high-dose γ-radiation. We identified some of the initial changes occurring due to CI which may have facilitated in worsening the injury and hampering the recovery of animals ultimately resulting in higher mortality.

  7. Hypoxia pathway and hypoxia-mediated extensive extramedullary hematopoiesis are involved in ursolic acid's anti-metastatic effect in 4T1 tumor bearing mice

    PubMed Central

    Gao, Jian-Li; Shui, Yan-Mei; Jiang, Wei; Huang, En-Yi; Shou, Qi-Yang; Ji, Xin; He, Bai-Cheng; Lv, Gui-Yuan; He, Tong-Chuan

    2016-01-01

    Hypoxic in the tumor mass is leading to the myeloproliferative-like disease (leukemoid reaction) and anemia of body, which characterized by strong extensive extramedullary hematopoiesis (EMH) in spleen. As the key transcription factor of hypoxia, hypoxia-inducible factor-1 (HIF-1) activates the expression of genes essential for EMH processes including enhanced blood cell production and angiogenesis. We found ursolic acid (UA), a natural pentacyclic triterpenoid carboxylic acid, inhibited growth of breast cancer both in vivo and in vitro. The suppression was mediated through the inhibition of multiple cell pathways linked to inflammation, proliferation, angiogenesis, and metastasis. UA also suppressed the leukemoid reaction and the EMH phenomenon of the tumor bearing mice without any significant suppression on body weight (i.p. by 20 mg/kg for 28 days). This is associated with the significant decrease in white blood cells (WBC), platelets (PLT) and spleen weight. During this process, we also detected the down-regulation of cell proliferative genes (PCNA, and β-catenin), and metastatic genes (VEGF, and HIF-1α), as well as the depression of nuclear protein intensity of HIF-1α. Furthermore, the expression of E2F1, p53 and MDM2 genes were increased in UA group when the VEGF and HIF-1α was over-expressed. Cancer cells were sensitive to UA treating after the silencing of HIF-1α and the response of Hypoxic pathway reporter to UA was suppressed when HIF-1α was over expressed. Overall, our results from experimental and predictive studies suggest that the anticancer activity of UA may be at least in part caused by suppressing the cancer hypoxia and hypoxia-mediated EMH. PMID:27708244

  8. dUbc9 negatively regulates the Toll-NF-kappa B pathways in larval hematopoiesis and drosomycin activation in Drosophila.

    PubMed

    Chiu, Hsiling; Ring, Brian C; Sorrentino, Richard Paul; Kalamarz, Marta; Garza, Dan; Govind, Shubha

    2005-12-01

    Highly conserved during evolution, the enzyme Ubc9 activates the small ubiquitin-like modifier (SUMO) prior to its covalent ligation to target proteins. We have used mutations in the Drosophila Ubc9 (dUbc9) gene to understand Ubc9 functions in vivo. Loss-of-function mutations in dUbc9 cause strong mitotic defects in larval hematopoietic tissues, an increase in the number of hematopoietic precursors in the lymph gland and of mature blood cells in circulation, and an increase in the proportion of cyclin-B-positive cells. Some blood cells are polyploid and multinucleate, exhibiting signs of genomic instability. We also observe an overabundance of highly differentiated blood cells (lamellocytes), normally not found in healthy larvae. Lamellocytes in mutants are either free in circulation or recruited to form tumorous masses. Hematopoietic defects of dUbc9 mutants are strongly suppressed in the absence of the Rel/NF-kappaB-family transcription factors Dorsal and Dif or in the presence of a non-signaling allele of Cactus, the IkappaB protein in Drosophila. In the larval fat body, dUbc9 negatively regulates the expression of the antifungal peptide gene drosomycin, which is constitutively expressed in dUbc9 mutants in the absence of immune challenge. dUbc9-mediated drosomycin expression requires Dorsal and Dif. Together, our results support a role for dUbc9 in the negative regulation of the Drosophila NF-kappaB signaling pathways in larval hematopoiesis and humoral immunity.

  9. Dynamic shifts in occupancy by TAL1 are guided by GATA factors and drive large-scale reprogramming of gene expression during hematopoiesis

    PubMed Central

    Wu, Weisheng; Morrissey, Christapher S.; Keller, Cheryl A.; Mishra, Tejaswini; Pimkin, Maxim; Blobel, Gerd A.; Weiss, Mitchell J.

    2014-01-01

    We used mouse ENCODE data along with complementary data from other laboratories to study the dynamics of occupancy and the role in gene regulation of the transcription factor TAL1, a critical regulator of hematopoiesis, at multiple stages of hematopoietic differentiation. We combined ChIP-seq and RNA-seq data in six mouse cell types representing a progression from multilineage precursors to differentiated erythroblasts and megakaryocytes. We found that sites of occupancy shift dramatically during commitment to the erythroid lineage, vary further during terminal maturation, and are strongly associated with changes in gene expression. In multilineage progenitors, the likely target genes are enriched for hematopoietic growth and functions associated with the mature cells of specific daughter lineages (such as megakaryocytes). In contrast, target genes in erythroblasts are specifically enriched for red cell functions. Furthermore, shifts in TAL1 occupancy during erythroid differentiation are associated with gene repression (dissociation) and induction (co-occupancy with GATA1). Based on both enrichment for transcription factor binding site motifs and co-occupancy determined by ChIP-seq, recruitment by GATA transcription factors appears to be a stronger determinant of TAL1 binding to chromatin than the canonical E-box binding site motif. Studies of additional proteins lead to the model that TAL1 regulates expression after being directed to a distinct subset of genomic binding sites in each cell type via its association with different complexes containing master regulators such as GATA2, ERG, and RUNX1 in multilineage cells and the lineage-specific master regulator GATA1 in erythroblasts. PMID:25319994

  10. Prevention of murine norovirus infection in neonatal mice by fostering.

    PubMed

    Compton, Susan R

    2008-05-01

    Murine norovirus (MNV) causes subclinical chronic infections in adult immunocompetent mice and is endemic in many mouse colonies. The susceptibility of neonatal mice to MNV infection was investigated. Intestinal homogenates from Swiss Webster (SW) mice inoculated orally with MNV-L on postpartum days (ppd) 1 to 3 were negative for MNV by RT-PCR at postinoculation days (pid) 3 and 7. In contrast, 69% of intestinal homogenates prepared on pid 3 and 7 from mice inoculated orally at ppd 5 to 8 were MNV-positive by RT-PCR. Because only mice 10 d of age or older were infected by contact with infected dams, a study was performed to determine whether fostering of neonatal mice from MNV-infected to MNV-naïve dams could be effective at preventing infection of neonatal mice. Four litters each of 1-, 2-, 4-, or 6 d-old mice from MNV-L- infected dams were transferred to naïve dams with similar-aged litters and vice versa. On ppd 21, feces from all MNV-infected dams and litters transferred to them were MNV-positive. In contrast on ppd 21, feces from all MNV-naïve dams and litters transferred to them were MNV-negative. Fostering of 2-d-old mice from 5 of 5 MNV-C-, 5 of 6 MNV-D-, and 7 of 8 MNV-G- infected dams onto MNV-naïve dams prevented MNV infection of the foster mice. In the 2 litters where MNV was detected, dams were infected within 7 d of transfer, suggesting that the neonatal mice had served as fomites. In summary, fostering was effective at preventing MNV infection in 33 of 35 litters of neonatal mice. Precautions to prevent transmission of virus on the surface of neonatal mice to foster dams could increase the efficiency of the fostering process.

  11. Thymopoietic and Bone Marrow Response to Murine Pneumocystis Pneumonia▿

    PubMed Central

    Shi, Xin; Zhang, Ping; Sempowski, Gregory D.; Shellito, Judd E.

    2011-01-01

    CD4+ T cells play a key role in host defense against Pneumocystis infection. To define the role of naïve CD4+ T cell production through the thymopoietic response in host defense against Pneumocystis infection, Pneumocystis murina infection in the lung was induced in adult male C57BL/6 mice with and without prior thymectomy. Pneumocystis infection caused a significant increase in the number of CCR9+ multipotent progenitor (MPP) cells in the bone marrow and peripheral circulation, an increase in populations of earliest thymic progenitors (ETPs) and double negative (DN) thymocytes in the thymus, and recruitment of naïve and total CD4+ T cells into the alveolar space. The level of murine signal joint T cell receptor excision circles (msjTRECs) in spleen CD4+ cells was increased at 5 weeks post-Pneumocystis infection. In thymectomized mice, the numbers of naïve, central memory, and total CD4+ T cells in all tissues examined were markedly reduced following Pneumocystis infection. This deficiency of naïve and central memory CD4+ T cells was associated with delayed pulmonary clearance of Pneumocystis. Extracts of Pneumocystis resulted in an increase in the number of CCR9+ MPPs in the cultured bone marrow cells. Stimulation of cultured bone marrow cells with ligands to Toll-like receptor 2 ([TLR-2] zymosan) and TLR-9 (ODN M362) each caused a similar increase in CCR9+ MPP cells via activation of the Jun N-terminal protein kinase (JNK) pathway. These results demonstrate that enhanced production of naïve CD4+ T lymphocytes through the thymopoietic response and enhanced delivery of lymphopoietic precursors from the bone marrow play an important role in host defense against Pneumocystis infection. PMID:21343353

  12. Prevention of Murine Norovirus Infection in Neonatal Mice by Fostering

    PubMed Central

    Compton, Susan R

    2008-01-01

    Murine norovirus (MNV) causes subclinical chronic infections in adult immunocompetent mice and is endemic in many mouse colonies. The susceptibility of neonatal mice to MNV infection was investigated. Intestinal homogenates from Swiss Webster (SW) mice inoculated orally with MNV-L on postpartum days (ppd) 1 to 3 were negative for MNV by RT-PCR at postinoculation days (pid) 3 and 7. In contrast, 69% of intestinal homogenates prepared on pid 3 and 7 from mice inoculated orally at ppd 5 to 8 were MNV-positive by RT-PCR. Because only mice 10 d of age or older were infected by contact with infected dams, a study was performed to determine whether fostering of neonatal mice from MNV-infected to MNV-naïve dams could be effective at preventing infection of neonatal mice. Four litters each of 1-, 2-, 4-, or 6 d-old mice from MNV-L–infected dams were transferred to naïve dams with similar-aged litters and vice versa. On ppd 21, feces from all MNV-infected dams and litters transferred to them were MNV-positive. In contrast on ppd 21, feces from all MNV-naïve dams and litters transferred to them were MNV-negative. Fostering of 2-d-old mice from 5 of 5 MNV-C–, 5 of 6 MNV-D–, and 7 of 8 MNV-G–infected dams onto MNV-naïve dams prevented MNV infection of the foster mice. In the 2 litters where MNV was detected, dams were infected within 7 d of transfer, suggesting that the neonatal mice had served as fomites. In summary, fostering was effective at preventing MNV infection in 33 of 35 litters of neonatal mice. Precautions to prevent transmission of virus on the surface of neonatal mice to foster dams could increase the efficiency of the fostering process. PMID:18459709

  13. A comparison of treatment of canine cyclic hematopoiesis with recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF interleukin-3, and canine G-CSF.

    PubMed

    Hammond, W P; Boone, T C; Donahue, R E; Souza, L M; Dale, D C

    1990-08-01

    Cyclic hematopoiesis in gray collie dogs is a stem cell disease in which abnormal regulation of cell production in the bone marrow causes cyclic fluctuations of blood cell counts. In vitro studies demonstrated that recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and granulocyte colony stimulating factor (G-CSF) all stimulated increases in colony formation by canine bone marrow progenitor cells. Based on these results, gray collie dogs were then treated with recombinant human (rh) GM-CSF, IL-3, or G-CSF subcutaneously to test the hypothesis that pharmacologic doses of one of these hematopoietic growth factors could alter cyclic production of cells. When recombinant canine G-CSF became available, it was tested over a range of doses. In vivo rhIL-3 had no effect on the recurrent neutropenia but was associated with eosinophilia, rhGM-CSF caused neutrophilia and eosinophilia but cycling of hematopoiesis persisted. However, rhG-CSF caused neutrophilia, prevented the recurrent neutropenia and, in the two animals not developing antibodies to rhG-CSF, obliterated periodic fluctuation of monocyte, eosinophil, reticulocyte, and platelet counts. Recombinant canine G-CSF increased the nadir neutrophil counts and amplitude of fluctuations at low doses (1 micrograms/kg/d) and eliminated all cycling of cell counts at high doses (5 and 10 micrograms/kg/d). These data suggest significant differences in the actions of these growth factors and imply a critical role for G-CSF in the homeostatic regulation of hematopoiesis.

  14. Cultivation and characterization of three strains of murine rotavirus.

    PubMed Central

    Greenberg, H B; Vo, P T; Jones, R

    1986-01-01

    Three distinct strains of murine rotavirus were adapted to growth in cell culture. These strains are genetically related but not identical; they are serotypically heterogeneous. The cultivatable strains were substantially more infectious (approximately 10(6)-fold) for suckling mice than heterologous simian rotaviruses were. Homologous murine rotavirus strains spread from inoculated to uninoculated litter mates and caused diarrhea, while heterologous rotaviruses did not spread and cause illness. Images PMID:3003390

  15. Regeneration of thyroid follicles from primordial cells in a murine thyroidectomized model.

    PubMed

    Lee, Junguee; Yi, Shinae; Chang, Joon Young; Kang, Yea Eun; Kim, Hyun Jung; Park, Ki Cheol; Yang, Keum-Jin; Sul, Hae Joung; Kim, Jong Ok; Yi, Hyon-Seung; Zhu, Xuguang; Cheng, Sheue-Yann; Shong, Minho

    2017-04-01

    The functional unit of the thyroid gland, the thyroid follicle, dynamically responds to various stimuli to maintain thyroid hormone homeostasis. However, thyroid follicles in the adult human thyroid gland have a very limited regenerative capacity following partial resection of the thyroid gland. To gain insight into follicle regeneration in the adult thyroid gland, we observed the regeneration processes of murine thyroid follicles after partial resection of the lower third of the thyroid gland in 10-week-old male C57BL/6 mice. Based on sequential observation of the partially resected thyroid lobe, we found primitive follicles forming in the area corresponding to the central zone of the intact lateral thyroid lobe. The primitive thyroid follicles were multiciliated and had coarsely vacuolated cytoplasm and large vesicular nuclei. Consistently, these primitive follicular cells did not express the differentiation markers paired box gene-8 and thyroid transcription factor-1 (clone SPT24), but were positive for forkhead box protein A2 and leucine-rich repeat-containing G-protein-coupled receptor 4/GPR48. Follicles newly generated from the primitive follicles had clear or vacuolar cytoplasm with dense, darkly stained nuclei. At day 21 after partial thyroidectomy, the tall cuboidal follicular epithelial cells had clear or vacuolar cytoplasm, and the intraluminal colloid displayed pale staining. Smaller activated follicles were found in the central zone of the lateral lobe, whereas larger mature follicles were located in the peripheral zone. Based on these observations, we propose that the follicle regeneration process in the partially resected adult murine thyroid gland associated with the appearance of primitive follicular cells may be a platform for the budding of differentiated follicles in mice.

  16. Characterization of msim, a murine homologue of the Drosophila sim transcription factor

    SciTech Connect

    Moffett, P.; Reece, M.; Pelletier, J.

    1996-07-01

    Mutations in the Drosophila single-minded (sim) gene result in loss of precursor cells that give rise to midline cells of the embryonic central nervous system. During the course of an exon-trapping strategy aimed at identifying transcripts that contribute to the etiology and pathophysiology of Down syndrome, we identified a human exon from the Down syndrome, we identified a human exon from the Down syndrome critical region showing significantly homology to the Drosophila sim gene. Using a cross-hybridization approach, we have isolated a murine homolog of Drosophila sim gene, which we designated msim. Nucleotide and predicted amino acid sequence analyses of msim cDNA clones indicate the this gene encodes a member of the basic-helix-loop-helix class of transcription factors. The murine and Drosophila proteins share 88% residues within the basic-helix-loop helix domain, with an overall homology of 92%. In addition, the N-terminal domain of MSIM contains two PAS dimerization motifs also featured in the Drosophila sim gene product, as well as a small number of other transcription factors. Northern blot analysis of adult murine tissues revealed that the msim gene produces a single mRNA species of {approximately}4 kb expressed in a small number of tissues, with the highest levels in the kidneys and lower levels present in skeletal muscle, lung, testis, brain, and heart. In situ hybridization experiments demonstrate that msim is also expressed in early fetal development in the central nervous system and in cartilage primordia. The characteristics of the msim gene are consistent with its putative function as a transcriptional regulator. 51 refs., 6 figs., 1 tab.

  17. A Neonatal Murine Model of MRSA Pneumonia

    PubMed Central

    Shrestha, Bishwas; Siefker, David; Patel, Vivek S.; Yadav, Nikki; Jaligama, Sridhar; Cormier, Stephania A.

    2017-01-01

    Pneumonia due to methicillin-resistant Staphylococcus aureus (MRSA) is a significant cause of morbidity and mortality in infants particularly following lower respiratory tract viral infections such as Respiratory Syncytial Virus (RSV). However, the mechanisms by which co-infection of infants by MRSA and RSV cause increased lung pathology are unknown. Because the infant immune system is qualitatively and quantitatively different from adults we developed a model of infant MRSA pneumonia which will allow us to investigate the effects of RSV co-infection on disease severity. We infected neonatal and adult mice with increasing doses of MRSA and demonstrate that neonatal mice have delayed kinetics in clearing the bacteria in comparison to adult mice. There were differences in recruitment of immune cells into the lung following infection. Adult mice exhibited an increase in neutrophil recruitment that coincided with reduced bacterial titers followed by an increase in macrophages. Neonatal mice, however, exhibited an early increase in neutrophils that did not persist despite continued presence of the bacteria. Unlike the adult mice, neonatal mice failed to exhibit an increase in macrophages. Neonates exhibited a decrease in phagocytosis of MRSA suggesting that the decrease in clearance was partially due to deficient phagocytosis of the bacteria. Both neonates and adults responded with an increase in pro-inflammatory cytokines following infection. However, in contrast to the adult mice, neonates did not express constitutive levels of the anti-microbial peptide Reg3γ in the lung. Infection of neonates did not stimulate expression of the co-stimulatory molecule CD86 by dendritic cells and neonates exhibited a diminished T cell response compared to adult mice. Overall, we have developed a neonatal model of MRSA pneumonia that displays a similar delay in bacterial clearance as is observed in the neonatal intensive care unit and will be useful for performing co

  18. Exposure to the environmental endocrine disruptor TCDD and human reproductive dysfunction: Translating lessons from murine models.

    PubMed

    Bruner-Tran, Kaylon L; Gnecco, Juan; Ding, Tianbing; Glore, Dana R; Pensabene, Virginia; Osteen, Kevin G

    2017-03-01

    Humans and other animals are exposed to a wide array of man-made toxicants, many of which act as endocrine disruptors that exhibit differential effects across the lifespan. In humans, while the impact of adult exposure is known for some compounds, the potential consequences of developmental exposure to endocrine disrupting chemicals (EDCs) is more difficult to ascertain. Animal studies have revealed that exposure to EDCs prior to puberty can lead to adult reproductive disease and dysfunction. Specifically, in adult female mice with an early life exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), we demonstrated a transgenerational occurrence of several reproductive diseases that have been linked to endometriosis in women. Herein, we review the evidence for TCDD-associated development of adult reproductive disease as well as known epigenetic alterations associated with TCDD and/or endometriosis. We will also introduce new "Organ-on-Chip" models which, combined with our established murine model, are expected to further enhance our ability to examine alterations in gene-environment interactions that lead to heritable disease. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Contrasting roles of leukemia inhibitory factor in murine bone development and remodeling involve region-specific changes in vascularization.

    PubMed

    Poulton, Ingrid J; McGregor, Narelle E; Pompolo, Sueli; Walker, Emma C; Sims, Natalie A

    2012-03-01

    We describe here distinct functions of leukemia inhibitory factor (LIF) in bone development/growth and adult skeletal homeostasis. In the growth plate and developing neonate bones, LIF deficiency enhanced vascular endothelial growth factor (VEGF) levels, enlarged blood vessel formation, and increased the formation of "giant" osteoclasts/chondroclasts that rapidly destroyed the mineralized regions of the growth plate and developing neonatal bone. Below this region, osteoblasts formed large quantities of woven bone. In contrast, in adult bone undergoing remodeling osteoclast formation was unaffected by LIF deficiency, whereas osteoblast formation and function were both significantly impaired, resulting in osteopenia. Consistent with LIF promoting osteoblast commitment, enhanced marrow adipocyte formation was also observed in adult LIF null mice, and adipocytic differentiation of murine stromal cells was delayed by LIF treatment. LIF, therefore, controls vascular size and osteoclast differentiation during the transition of cartilage to bone, whereas an anatomically separate LIF-dependent pathway regulates osteoblast and adipocyte commitment in bone remodeling.

  20. Preclinical testing of the safety and tolerability of LV-mediated above normal alpha-L-iduronidase expression in murine and human hematopoietic cells using toxicology and biodistribution GLP studies.

    PubMed

    Visigalli, Ilaria; Delai, Stefania; Ferro, Francesca; Cecere, Francesca; Vezzoli, Michela; Sanvito, Francesca; Chanut, Franck; Benedicenti, Fabrizio; Spinozzi, Giulio; Wynn, Rob; Calabria, Andrea; Naldini, Luigi; Montini, Eugenio; Cristofori, Patrizia; Biffi, Alessandra

    2016-07-18

    In order to support the clinical application of hematopoietic stem cell (HSC) gene therapy for Mucopolysaccharidosis I (MPS I), we conducted biosafety studies to assess the toxicity and tumorigenic potential, as well as the biodistribution of HSCs and progenitor cells (HSPCs) transduced with lentiviral vectors (LV) encoding the cDNA of the alpha-iduronidase (IDUA) gene, which is mutated in MPS I patients. To this goal, toxicology and biodistribution studies were conducted employing Good Laboratory Practice (GLP) study practices. Vector integration sites studies were applied in order to predict adverse consequences of vector gene transfer and obtain HSC-related information. Overall, the results obtained in these studies provided robust evidence to support the safety and tolerability of high-efficiency LV-mediated gene transfer and above normal IDUA enzyme expression in both murine and human HSPCs and their in vivo progeny. Taken together these investigations provide essential safety data to support clinical testing of HSC gene therapy in MPS I patients. These studies have also underlined criticisms associated to the use of currently available models and highlighted the value of surrogate markers of tumorigenicity that may be further explored in the future. Notably, biological evidence supporting the efficacy of gene therapy on MPS I disease was also generated and the clonal contribution of LV-transduced HSPCs to hematopoiesis along serial transplantation was quantified in a minimum of 200-300 clones, with the different level of repopulating cells in primary recipients being reflected in the secondary.

  1. Prioritization and Association Analysis of Murine-Derived Candidate Genes in Anxiety-Spectrum Disorders

    PubMed Central

    Hettema, John M.; Webb, Bradley T.; Guo, An-Yuan; Zhao, Zhongming; Maher, Brion S.; Chen, Xiangning; An, Seon-Sook; Sun, Cuie; Aggen, Steven H.; Kendler, Kenneth S.; Kuo, Po-Hsiu; Otowa, Takeshi; Flint, Jonathan; van den Oord, Edwin J.

    2011-01-01

    Background Anxiety disorders are common psychiatric conditions that are highly comorbid with each other and related phenotypes such as depression, likely due to a shared genetic basis. Fear-related behaviors in mice have long been investigated as potential models of anxiety disorders, making integration of information from both murine and human genetic data a powerful strategy for identifying potential susceptibility genes for these conditions. Methods We combined genome-wide association analysis of fear-related behaviours with strain distribution pattern analysis in heterogeneous stock mice to identify a preliminary list of 52 novel candidate genes. We ranked these according to three complementary sources of prior anxiety-related genetic data: (1) extant linkage and knock-out studies in mice, (2) a meta-analysis of human linkage scans, and (3) a preliminary human genomewide association study. We genotyped tagging SNPs covering the nine top-ranked regions in a two-stage association study of 1316 subjects from the Virginia Adult Twin Study of Psychiatric and Substance Use Disorders chosen for high or low genetic loading for anxiety-spectrum phenotypes (anxiety disorders, neuroticism, and major depression). Results Multiple SNPs in the PPARGC1A gene demonstrated association in both stages that survived gene-wise correction for multiple testing. Conclusions Integration of genetic data across human and murine studies suggests PPARGC1A as a potential susceptibility gene for anxiety-related disorders. PMID:21871609

  2. Establishment of a murine epidermal cell line suitable for in vitro and in vivo skin modelling

    PubMed Central

    2011-01-01

    Background Skin diseases are a major health problem. Some of the most severe conditions involve genetic disorders, including cancer. Several of these human diseases have been modelled in genetically modified mice, thus becoming a highly valuable preclinical tool for the treatment of these pathologies. However, development of three-dimensional models of skin using keratinocytes from normal and/or genetically modified mice has been hindered by the difficulty to subculture murine epidermal keratinocytes. Methods We have generated a murine epidermal cell line by serially passaging keratinocytes isolated from the back skin of adult mice. We have termed this cell line COCA. Cell culture is done in fully defined media and does not require feeder cells or any other coating methods. Results COCA retained its capacity to differentiate and stratify in response to increased calcium concentration in the cell culture medium for more than 75 passages. These cells, including late passage, can form epidermis-like structures in three-dimensional in vitro models with a well-preserved pattern of proliferation and differentiation. Furthermore, these cells form epidermis in grafting assays in vivo, and do not develop tumorigenic ability. Conclusions We propose that COCA constitutes a good experimental system for in vitro and in vivo skin modelling. Also, cell lines from genetically modified mice of interest in skin biology could be established using the method we have developed. COCA keratinocytes would be a suitable control, within a similar background, when studying the biological implications of these alterations. PMID:21510892

  3. Hhex is Required at Multiple Stages of Adult Hematopoietic Stem and Progenitor Cell Differentiation

    PubMed Central

    Goodings, Charnise; Smith, Elizabeth; Mathias, Elizabeth; Elliott, Natalina; Cleveland, Susan M.; Tripathi, Rati M.; Layer, Justin H.; Chen, Xi; Guo, Yan; Shyr, Yu; Hamid, Rizwan; Du, Yang; Davé, Utpal P.

    2015-01-01

    Hhex encodes a homeodomain transcription factor that is widely expressed in hematopoietic stem and progenitor cell populations. Its enforced expression induces T-cell leukemia and we have implicated it as an important oncogene in early T-cell precursor leukemias where it is immediately downstream of an LMO2-associated protein complex. Conventional Hhex knockouts cause embryonic lethality precluding analysis of adult hematopoiesis. Thus, we induced highly efficient conditional knockout (cKO) using vav-Cre transgenic mice. Hhex cKO mice were viable and born at normal litter sizes. At steady state, we observed a defect in B-cell development that we localized to the earliest B-cell precursor, the pro-B-cell stage. Most remarkably, bone marrow transplantation using Hhex cKO donor cells revealed a more profound defect in all hematopoietic lineages. In contrast, sublethal irradiation resulted in normal myeloid cell repopulation of the bone marrow but markedly impaired repopulation of T- and B-cell compartments. We noted that Hhex cKO stem and progenitor cell populations were skewed in their distribution and showed enhanced proliferation compared to WT cells. Our results implicate Hhex in the maintenance of LT-HSCs and in lineage allocation from multipotent progenitors especially in stress hematopoiesis. PMID:25968920

  4. Genetic predisposition to myelodysplastic syndrome and acute myeloid leukemia in children and young adults

    PubMed Central

    Babushok, Daria V.; Bessler, Monica; Olson, Timothy S.

    2016-01-01

    Myelodysplastic syndrome (MDS) is a clonal blood disorder characterized by ineffective hematopoiesis, cytopenias, dysplasia and an increased risk of acute myeloid leukemia (AML). With the growing availability of clinical genetic testing, there is an increasing appreciation that a number of genetic predisposition syndromes may underlie apparent de novo presentations of MDS/AML, particularly in children and young adults. Recent findings of clonal hematopoiesis in acquired aplastic anemia add another facet to our understanding of the mechanisms of MDS/AML predisposition. As more predisposition syndromes are recognized, it is becoming increasingly important for hematologists and oncologists to have familiarity with the common as well as emerging syndromes, and to have a systematic approach to diagnosis and screening of at risk patient populations. Here, we provide a practical algorithm for approaching a patient with a suspected MDS/AML predisposition, and provide an in-depth review of the established and emerging familial MDS/AML syndromes caused by mutations in the ANKRD26, CEBPA, DDX41, ETV6, GATA2, RUNX1, SRP72 genes. Finally, we discuss recent data on the role of somatic mutations in malignant transformation in acquired aplastic anemia, and review the practical aspects of MDS/AML management in patients and families with predisposition syndromes. PMID:26693794

  5. Characterization of ozone disinfection of murine norovirus.

    PubMed

    Lim, Mi Young; Kim, Ju-Mi; Lee, Jung Eun; Ko, GwangPyo

    2010-02-01

    Despite the importance of human noroviruses (NoVs) in public health, little information concerning the effectiveness of ozone against NoVs is available. We determined the efficacy of ozone disinfection using murine norovirus (MNV) as a surrogate of human NoV. MNV in ozone demand-free buffer was exposed to a predetermined dose of ozone at two different pHs and temperatures. The virus remaining in the solution was analyzed by plaque assay, real-time TaqMan reverse transcriptase PCR (RT-PCR) (short template), and long-template conventional RT-PCR. Under all conditions, more than 99% of the MNV was inactivated by ozone at 1 mg/liter within 2 min. Both RT-PCR assays significantly underestimated the inactivation of MNV, compared with that measured by plaque assay. Our results indicate that NoV may be more resistant to ozone than has been previously reported. Nevertheless, proper ozone disinfection practices can be used to easily control its transmission in water.

  6. Regulation of Murine Natural Killer Cell Development

    PubMed Central

    Goh, Wilford; Huntington, Nicholas D.

    2017-01-01

    Natural killer (NK) cells are effector lymphocytes of the innate immune system that are known for their ability to kill transformed and virus-infected cells. NK cells originate from hematopoietic stem cells in the bone marrow, and studies on mouse models have revealed that NK cell development is a complex, yet tightly regulated process, which is dependent on both intrinsic and extrinsic factors. The development of NK cells can be broadly categorized into two phases: lineage commitment and maturation. Efforts to better define the developmental framework of NK cells have led to the identification of several murine NK progenitor populations and mature NK cell subsets, each defined by a varied set of cell surface markers. Nevertheless, the relationship between some of these NK cell subsets remains to be determined. The classical approach to studying both NK cell development and function is to identify the transcription factors involved and elucidate the mechanistic action of each transcription factor. In this regard, recent studies have provided further insight into the mechanisms by which transcription factors, such as ID2, FOXO1, Kruppel-like factor 2, and GATA-binding protein 3 regulate various aspects of NK cell biology. It is also becoming evident that the biology of NK cells is not only transcriptionally regulated but also determined by epigenetic alterations and posttranscriptional regulation of gene expression by microRNAs. This review summarizes recent progress made in NK development, focusing primarily on transcriptional regulators and their mechanistic actions. PMID:28261203

  7. Murine cytomegalovirus infection of cultured mouse embryos.

    PubMed Central

    Tsutsui, Y.; Naruse, I.

    1987-01-01

    Isolated mouse whole embryos of 7.5 days' gestation were infected with murine cytomegalovirus (MCMV) and cultured in pure rat serum. Although the MCMV infection had little effect on the survival and development of the embryos during 3 days of cultivation, immunohistochemical analysis of their serial sections using monoclonal antibody showed MCMV-infected cells in various portions of the embryos. This monoclonal antibody, when tested with the use of infected cultured mouse fibroblasts, reacted with nuclear antigen within 2 hours after infection and also reacted with nuclear inclusions in the late phase of infection. The viral antigen-positive cells detected by the monoclonal antibody were present in almost all of the ectoplacental cone and the yolk sac and in about 82% of the embryos. In the embryos, antigen-positive cells were frequently observed in the epithelium of the digestive tracts, endothelial cells of the blood vessels, and the mesodermal cells. In some of the embryos, viral antigen-positive cells were clearly observed in a small percentage of the blood cells. These findings indicate that blood cells, in addition to cell migration during embryogenesis, may play an important role in transmission of infectious virus into the embryos. Mouse whole embryo culture infected with MCMV can provide a model for the study of cellular tropism related to congenital infection by cytomegalovirus. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:3034066

  8. ESCRT Requirements for Murine Leukemia Virus Release.

    PubMed

    Bartusch, Christina; Prange, Reinhild

    2016-04-18

    The Murine Leukemia Virus (MLV) is a gammaretrovirus that hijack host components of the endosomal sorting complex required for transport (ESCRT) for budding. To determine the minimal requirements for ESCRT factors in MLV viral and viral-like particles (VLP) release, an siRNA knockdown screen of ESCRT(-associated) proteins was performed in MLV-producing human cells. We found that MLV VLPs and virions primarily engage the ESCRT-I factor Tsg101 and marginally the ESCRT-associated adaptors Nedd4-1 and Alix to enter the ESCRT pathway. Conversely, the inactivation of ESCRT-II had no impact on VLP and virion egress. By analyzing the effects of individual ESCRT-III knockdowns, VLP and virion release was profoundly inhibited in CHMP2A- and CHMP4B-knockdown cells. In contrast, neither the CHMP2B and CHMP4A isoforms nor CHMP3, CHMP5, and CHMP6 were found to be essential. In case of CHMP1, we unexpectedly observed that the CHMP1A isoform was specifically required for virus budding, but dispensable for VLP release. Hence, MLV utilizes only a subset of ESCRT factors, and viral and viral-like particles differ in ESCRT-III factor requirements.

  9. Metabolic syndrome components in murine models

    PubMed Central

    Lawson, Heather A.; Cheverud, James M.

    2010-01-01

    1. Abstract Animal models have enriched understanding of the physiological basis of metabolic disorders and advanced identification of genetic risk factors underlying the metabolic syndrome (MetS). Murine models are especially appropriate for this type of research, and are an excellent resource not only for identifying candidate genomic regions, but also for illuminating the possible molecular mechanisms or pathways affected in individual components of MetS. In this review, we briefly discuss findings from mouse models of metabolic disorders, particularly in light of issues raised by the recent flood of human genome-wide association studies (GWAS) results. We describe how mouse models are revealing that genotype interacts with environment in important ways, indicating that the underlying genetics of MetS is highly context dependant. Further we show that epistasis, imprinting and maternal effects each contribute to the genetic architecture underlying variation in metabolic traits, and mouse models provide an opportunity to dissect these aspects of the genetic architecture that are difficult if not impossible to ascertain in humans. Finally we discuss how knowledge gained from mouse models can be used in conjunction with comparative genomic methods and bioinformatic resources to inform human MetS research. PMID:20088816

  10. A murine model of Nijmegen breakage syndrome.

    PubMed

    Williams, Bret R; Mirzoeva, Olga K; Morgan, William F; Lin, Junyu; Dunnick, Wesley; Petrini, John H J

    2002-04-16

    Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by microcephaly, immunodeficiency, and predisposition to hematopoietic malignancy. The clinical and cellular phenotypes of NBS substantially overlap those of ataxia-telangiectasia (A-T). NBS is caused by mutation of the NBS1 gene, which encodes a member of the Mre11 complex, a trimeric protein complex also containing Mre11 and Rad50. Several lines of evidence indicate that the ataxia-telangiectasia mutated (ATM) kinase and the Mre11 complex functionally interact. Both NBS and A-T cells exhibit ionizing radiation (IR) sensitivity and defects in the intra S phase checkpoint, resulting in radioresistant DNA synthesis (RDS)-the failure to suppress DNA replication origin firing after IR exposure. NBS1 is phosphorylated by ATM in response to IR, and this event is required for activation of the intra S phase checkpoint (the RDS checkpoint). We derived a murine model of NBS, the Nbs1(DeltaB/DeltaB) mouse. Nbs1(DeltaB/DeltaB) cells are phenotypically identical to those established from NBS patients. The Nbs1(DeltaB) allele was synthetically lethal with ATM deficiency. We propose that the ATM-Mre11 complex DNA damage response pathway is essential and that ATM or the Mre11 complex serves as a nexus to additional components of the pathway.

  11. Implantable Micropump Technologies for Murine Intracochlear Infusions

    PubMed Central

    Johnson, D. G.; Waldron, M. J.; Frisina, R. D.; Borkholder, D. A.

    2011-01-01

    Due to the very small size of the mouse inner ear, 600 nL volume, developing effective, controlled infusion systems is quite challenging. Key technologies have been created to minimize both size and power for an implantable pump for murine intracochlear infusions. A method for coupling fine capillary tubing to microfluidic channels is presented which provides low volume, biocompatible interconnects withstanding pressures as high as 827 kPa (120 psi) and consuming less than 20 nL of volume exiting in-plane with the pump. Surface micromachined resistive bridges integrated into the flow channel for anemometry based flow rate measurement have been optimized for low power operation in the ultra-low flow rate regime. A process for creation of deformable diaphragms over pump chambers with simultaneous coating of the microfluidic channels has been developed allowing integration of a biocompatible fluid flow path. These advances represent enabling capabilities for a drug delivery system suitable for space constrained applications such as subcutaneous implantation in mice. PMID:21096713

  12. Cellular Localization of Latent Murine Cytomegalovirus

    PubMed Central

    Koffron, Alan J.; Hummel, Mary; Patterson, Bruce K.; Yan, Shixian; Kaufman, Dixon B.; Fryer, Jonathan P.; Stuart, Frank P.; Abecassis, Michael I.

    1998-01-01

    Herpesviruses typically establish latent infection in their hosts. The cell(s) responsible for harboring latent virus, in most cases, is not known. Using immunofluorescence and PCR-in situ hybridization (PISH), a technique which combines the sensitivity of PCR with the localization and specificity of in situ hybridization, we provide the first direct evidence that endothelial cells are a major site of murine cytomegalovirus (MCMV) DNA in latently infected animals. These findings are consistent with existing knowledge of the biological behavior of CMV, in particular the transmission of latent CMV by solid organ and bone marrow transplantation, in both human and animal models. In addition, we have localized MCMV DNA in the lung alveolar macrophage and in bone marrow cells. Our findings confirm that bone marrow-derived hematopoietic cells are a site of CMV latency and further suggest that bone marrow may be a reservoir of infected progeny capable of migrating into the circulation and establishing latency in various tissues. These findings provide clearly needed insight into the site of latent infection which is central to an understanding of the mechanisms of reactivation. PMID:9420204

  13. Nuclear Nonhistone Proteins in Murine Melanoma Cells

    PubMed Central

    Wikswo, Muriel A.; Mcguire, Joseph S.; Shansky, Janet E.; Boshes, Roger A.

    1976-01-01

    Nuclear nonhistone proteins (NHP's) have been implicated as regulatory agents involved in controlling genetic expression. Utilizing murine melanoma cells, we describe a method for isolating and fractionating NHP's which greatly increases the yield of these proteins as well as the level of resolution required for detecting small differences in particular NHP's. Mouse melanoma cells were grown in medium labeled with [3H]leucine. Following 48 hr of incubation, the cells were harvested and nuclei isolated. The NHP's were extracted from the nuclei in a series of steps which yielded four major fractions: NHP1, NHP2, NHP3, NHP4. This method solubilized 80-90% of the protein from the nuclear homogenate. The NHP fractions were then separated on DEAE-cellulose columns in a series of salt steps increasing in concentration from 0.05 to 0.50 M NaCl, followed by steps of 2 M NaCl and 4 and 7 M guanidine-hydrochloride. The 40 NHP fractions eluted from these columns were further separated on polyacrylamide-SDS gels and ranged in molecular weight from 9000 to 110,000 daltons. Differences were observed in the electrophoretic pattern of each of these 40 fractions. The high resolution of these fractionation procedures greatly enhances the possibility of observing small changes in proteins which may play a role in gene regulation. ImagesFIG. 2FIG. 5 PMID:997593

  14. Tumor gangliosides accelerate murine tumor angiogenesis.

    PubMed

    Liu, Yihui; Wondimu, Assefa; Yan, Su; Bobb, Daniel; Ladisch, Stephan

    2014-07-01

    Tumor cells shed gangliosides and populate their microenvironment with these biologically active membrane glycosphingolipids. In vitro, ganglioside enrichment amplifies receptor tyrosine kinase signaling and activation of vascular endothelial cells. However, a long-standing question is whether in the actual microenvironment of a neoplasm, in vivo, tumor cell ganglioside shedding stimulates angiogenesis. Here we tested the hypothesis that tumor gangliosides have a critical proangiogenic role in vivo using novel murine tumor cells, GM3synthase/GM2synthase double knockout (DKO) cells, genetically completely incapable of ganglioside synthesis and impaired in tumor growth versus wild-type (WT) ganglioside-rich cells. We studied angiogenesis during tumor formation by these ganglioside-depleted cells, quantifying vessel formation, angiogenic factor production/release, and consequences of reconstitution with purified WT gangliosides. DKO cells formed virtually avascular tumors, much smaller than ganglioside-rich WT tumors and displaying a striking paucity of blood vessels, despite levels of VEGF and other angiogenic factors that were similar to those of WT cells. Transient enrichment of the ganglioside milieu of the DKO cell inoculum by adding purified WT gangliosides partially restored angiogenesis and tumor growth. We conclude that tumor gangliosides trigger robust angiogenesis important for tumor growth. Our findings suggest strategies to eliminate their synthesis and shedding by tumor cells should be pursued.

  15. Tumor gangliosides accelerate murine tumor angiogenesis

    PubMed Central

    Liu, Yihui; Wondimu, Assefa; Yan, Su; Bob, Daniel; Ladisch, Stephan

    2013-01-01

    Tumor cells shed gangliosides and populate their microenvironment with these biologically active membrane glycosphingolipids. In vitro, ganglioside enrichment amplifies receptor tyrosine kinase signaling and activation of vascular endothelial cells. However, a long-standing question is whether in the actual microenvironment of a neoplasm, in vivo, tumor cell ganglioside shedding stimulates angiogenesis. Here we tested the hypothesis that tumor gangliosides have a critical proangiogenic role in vivo using novel murine tumor cells (DKO) genetically completely incapable of ganglioside synthesis and impaired in tumor growth vs. wild-type (WT) ganglioside-rich cells. We studied angiogenesis during tumor formation by these ganglioside-depleted cells, quantifying vessel formation, angiogenic factor production/release, and consequences of reconstitution with purified WT gangliosides. DKO cells formed virtually avascular tumors, much smaller than ganglioside-rich WT tumors and displaying a striking paucity of blood vessels, despite levels of VEGF and other angiogenic factors that were similar to those of WT cells. Transient enrichment of the ganglioside milieu of the DKO cell inoculum by adding purified WT gangliosides partially restored angiogenesis and tumor growth. We conclude that tumor gangliosides trigger robust angiogenesis important for tumor growth. Our findings suggest strategies to eliminate their synthesis and shedding by tumor cells should be pursued. PMID:24165965

  16. Murine Ileocolic Bowel Resection with Primary Anastomosis

    PubMed Central

    Perry, Troy; Borowiec, Anna; Dicken, Bryan; Fedorak, Richard; Madsen, Karen

    2014-01-01

    Intestinal resections are frequently required for treatment of diseases involving the gastrointestinal tract, with Crohn’s disease and colon cancer being two common examples. Despite the frequency of these procedures, a significant knowledge gap remains in describing the inherent effects of intestinal resection on host physiology and disease pathophysiology. This article provides detailed instructions for an ileocolic resection with primary end-to-end anastomosis in mice, as well as essential aspects of peri-operative care to maximize post-operative success. When followed closely, this procedure yields a 95% long-term survival rate, no failure to thrive, and minimizes post-operative complications of bowel obstruction and anastomotic leak. The technical challenges of performing the procedure in mice are a barrier to its wide spread use in research. The skills described in this article can be acquired without previous surgical experience. Once mastered, the murine ileocolic resection procedure will provide a reproducible tool for studying the effects of intestinal resection in models of human disease. PMID:25406841

  17. Eliminating Murine Norovirus by Cross-Fostering

    PubMed Central

    Buxbaum, Laurence U.; DeRitis, Pierina C.; Chu, Niansheng; Conti, Pierre A.

    2011-01-01

    Murine norovirus (MNV) is a newly discovered and extremely prevalent pathogen of laboratory mouse colonies. MNV causes severe disease in some immunocompromised mouse strains and can cause persistent infections even in immunocompetent mice. Despite the fact that immunocompetent mice are generally asymptomatic, the possibility that MNV infection might alter immune responses makes its eradication a potentially useful goal for many facilities. Initial attempts by others to use a strategy of testing and culling were unsuccessful, whereas complete depopulation and facility decontamination was successful. However, these measures may be impractical, and finding less drastic approaches seemed prudent. Based on a report that cross-fostering of pups from MNV-positive mothers to MNV-negative ones could be successful in experimental MNV infection, we undertook a comprehensive fostering program using Swiss Webster mothers, careful sanitary measures, and fecal PCR testing to eradicate the virus from a mouse colony recently infected with MNV. We successfully decontaminated 17 of 18 (94%) litters and managed to prevent spread when a new MNV-infected mouse strain entered quarantine at our facility. These results suggest that cross-fostering, when performed in a setting of excellent sanitary procedures, may be practical for the large number of mouse facilities in which MNV is endemic. PMID:21838978

  18. ESCRT Requirements for Murine Leukemia Virus Release

    PubMed Central

    Bartusch, Christina; Prange, Reinhild

    2016-01-01

    The Murine Leukemia Virus (MLV) is a gammaretrovirus that hijack host components of the endosomal sorting complex required for transport (ESCRT) for budding. To determine the minimal requirements for ESCRT factors in MLV viral and viral-like particles (VLP) release, an siRNA knockdown screen of ESCRT(-associated) proteins was performed in MLV-producing human cells. We found that MLV VLPs and virions primarily engage the ESCRT-I factor Tsg101 and marginally the ESCRT-associated adaptors Nedd4-1 and Alix to enter the ESCRT pathway. Conversely, the inactivation of ESCRT-II had no impact on VLP and virion egress. By analyzing the effects of individual ESCRT-III knockdowns, VLP and virion release was profoundly inhibited in CHMP2A- and CHMP4B-knockdown cells. In contrast, neither the CHMP2B and CHMP4A isoforms nor CHMP3, CHMP5, and CHMP6 were found to be essential. In case of CHMP1, we unexpectedly observed that the CHMP1A isoform was specifically required for virus budding, but dispensable for VLP release. Hence, MLV utilizes only a subset of ESCRT factors, and viral and viral-like particles differ in ESCRT-III factor requirements. PMID:27096867

  19. Implantable micropump technologies for murine intracochlear infusions.

    PubMed

    Johnson, D G; Waldron, M J; Frisina, R D; Borkholder, D A

    2010-01-01

    Due to the very small size of the mouse inner ear, 600 nL volume, developing effective, controlled infusion systems is quite challenging. Key technologies have been created to minimize both size and power for an implantable pump for murine intracochlear infusions. A method for coupling fine capillary tubing to microfluidic channels is presented which provides low volume, biocompatible interconnects withstanding pressures as high as 827 kPa (120 psi) and consuming less than 20 nL of volume exiting in-plane with the pump. Surface micromachined resistive bridges integrated into the flow channel for anemometry based flow rate measurement have been optimized for low power operation in the ultra-low flow rate regime. A process for creation of deformable diaphragms over pump chambers with simultaneous coating of the microfluidic channels has been developed allowing integration of a biocompatible fluid flow path. These advances represent enabling capabilities for a drug delivery system suitable for space constrained applications such as subcutaneous implantation in mice.

  20. Quantitative Trait Loci for Murine Growth

    PubMed Central

    Cheverud, J. M.; Routman, E. J.; Duarte, FAM.; van-Swinderen, B.; Cothran, K.; Perel, C.

    1996-01-01

    Body size is an archetypal quantitative trait with variation due to the segregation of many gene loci, each of relatively minor effect, and the environment. We examine the effects of quantitative trait loci (QTLs) on age-specific body weights and growth in the F(2) intercross of the LG/J and SM/J strains of inbred mice. Weekly weights (1-10 wk) and 75 microsatellite genotypes were obtained for 535 mice. Interval mapping was used to locate and measure the genotypic effects of QTLs on body weight and growth. QTL effects were detected on 16 of the 19 autosomes with several chromosomes carrying more than one QTL. The number of QTLs for age-specific weights varied from seven at 1 week to 17 at 10 wk. The QTLs were each of relatively minor, subequal effect. QTLs affecting early and late growth were generally distinct, mapping to different chromosomal locations indicating separate genetic and physiological systems for early and later murine growth. PMID:8846907

  1. Isolation in a single step of a highly enriched murine hematopoietic stem cell population with competitive long-term repopulating ability

    SciTech Connect

    Szilvassy, S.J.; Lansdorp, P.M.; Humphries, R.K.; Eaves, A.C.; Eaves, C.J. )

    1989-08-15

    A simple procedure is described for the quantitation and enrichment of murine hematopoietic cells with the capacity for long-term repopulation of lymphoid and myeloid tissues in lethally irradiated mice. To ensure detection of the most primitive marrow cells with this potential, we used a competitive assay in which female recipients were injected with male test cells and 1 to 2 x 10(5) compromised female marrow cells with normal short-term repopulating ability, but whose long-term repopulating ability had been reduced by serial transplantation. Primitive hematopoietic cells were purified by flow cytometry and sorting based on their forward and orthogonal light-scattering properties, and Thy-1 and H-2K antigen expression. Enrichment profiles for normal marrow, and marrow of mice injected with 5-fluorouracil (5-FU) four days previously, were established for each of these parameters using an in vitro assay for high proliferative potential, pluripotent colony-forming cells. When all four parameters were gated simultaneously, these clonogenic cells were enriched 100-fold. Both day 9 and day 12 CFU-S were copurified; however, the purity (23%) and enrichment (75-fold) of day 12 CFU-S in the sorted population was greater with 5-FU-treated cells. Five hundred of the sorted 5-FU marrow cells consistently repopulated recipient lymphoid and myeloid tissues (greater than 50% male, 1 to 3 months post-transplant) when co-injected with 1 to 2 x 10(5) compromised female marrow cells, and approximately 100 were sufficient to achieve the same result in 50% of recipients under the same conditions. This relatively simple purification and assay strategy should facilitate further analysis of the heterogeneity and regulation of stem cells that maintain hematopoiesis in vivo.

  2. The possible role of liver kinase B1 in hydroquinone-induced toxicity of murine fetal liver and bone marrow hematopoietic stem cells.

    PubMed

    Li, Zhen; Wang, Chunhong; Zhu, Jie; Bai, YuE; Wang, Wei; Zhou, Yanfeng; Zhang, Shaozun; Liu, Xiangxiang; Zhou, Sheng; Huang, Wenting; Bi, Yongyi; Wang, Hong

    2016-07-01

    Epidemiological studies suggest that the increasing incidence of childhood leukemia may be due to maternal exposure to benzene, which is a known human carcinogen; however, the mechanisms involved remain unknown. Liver Kinase B1 (LKB1) acts as a regulator of cellular energy metabolism and functions to regulate hematopoietic stem cell (HSC) homeostasis. We hypothesize that LKB1 contributes to the deregulation of fetal or bone hematopoiesis caused by the benzene metabolite hydroquinone (HQ). To evaluate this hypothesis, we compared the effects of HQ on murine fetal liver hematopoietic stem cells (FL-HSCs) and bone marrow hematopoietic stem cells (BM-HSCs). FL-HSCs and BM-HSCs were isolated and enriched by a magnetic cell sorting system and exposed to various concentrations of HQ (0, 1.25, 2.5, 5, 10, 20, and 40 μM) for 24 h. We found that the inhibition of differentiation and growth, as well as the apoptosis rate of FL-HSCs, induced by HQ were consistent with the changes in BM-HSCs. Furthermore, G1 cell cycle arrest was observed in BM-HSCs and FL-HSCs in response to HQ. Importantly, FL-HSCs were more sensitive than BM-HSCs after exposure to HQ. The highest induction of LKB1 and adenosine monophosphate-activated protein kinase (AMPK) was observed with a much lower concentration of HQ in FL-HSCs than in BM-HSCs. LKB1 may play a critical role in apoptosis and cell cycle arrest of HQ-treated HSCs. This research has developed innovative ideas concerning benzene-induced hematopoietic toxicity or embryotoxicity, which can provide a new experimental evidence for preventing childhood leukemia. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 830-841, 2016.

  3. Optogenetic Control of Heart Rhythm by Selective Stimulation of Cardiomyocytes Derived from Pnmt+ Cells in Murine Heart

    PubMed Central

    Wang, Yanwen; Lin, Wee Khang; Crawford, William; Ni, Haibo; Bolton, Emma L.; Khan, Huma; Shanks, Julia; Bub, Gil; Wang, Xin; Paterson, David J.; Zhang, Henggui; Galione, Antony; Ebert, Steven N.; Terrar, Derek A.; Lei, Ming

    2017-01-01

    In the present study, channelrhodopsin 2 (ChR2) was specifically introduced into murine cells expressing the Phenylethanolamine n-methyltransferase (Pnmt) gene, which encodes for the enzyme responsible for conversion of noradrenaline to adrenaline. The new murine model enabled the identification of a distinctive class of Pnmt-expressing neuroendocrine cells and their descendants (i.e. Pnmt+ cell derived cells) within the heart. Here, we show that Pnmt+ cells predominantly localized to the left side of the adult heart. Remarkably, many of the Pnmt+ cells in the left atrium and ventricle appeared to be working cardiomyocytes based on their morphological appearance and functional properties. These Pnmt+ cell derived cardiomyocytes (PdCMs) are similar to conventional myocytes in morphological, electrical and contractile properties. By stimulating PdCMs selectively with blue light, we were able to control cardiac rhythm in the whole heart, isolated tissue preparations and single cardiomyocytes. Our new murine model effectively demonstrates functional dissection of cardiomyocyte subpopulations using optogenetics, and opens new frontiers of exploration into their physiological roles in normal heart function as well as their potential application for selective cardiac repair and regeneration strategies. PMID:28084430

  4. Murine and Human Tissue-Engineered Esophagus Form from Sufficient Stem/Progenitor Cells and Do Not Require Microdesigned Biomaterials

    PubMed Central

    Spurrier, Ryan Gregory; Speer, Allison L.; Hou, Xiaogang; El-Nachef, Wael N.

    2015-01-01

    Purpose: Tissue-engineered esophagus (TEE) may serve as a therapeutic replacement for absent foregut. Most prior esophagus studies have favored microdesigned biomaterials and yielded epithelial growth alone. None have generated human TEE with mesenchymal components. We hypothesized that sufficient progenitor cells might only require basic support for successful generation of murine and human TEE. Materials and Methods: Esophageal organoid units (EOUs) were isolated from murine or human esophagi and implanted on a polyglycolic acid/poly-l-lactic acid collagen-coated scaffold in adult allogeneic or immune-deficient mice. Alternatively, EOU were cultured for 10 days in vitro prior to implantation. Results: TEE recapitulated all key components of native esophagus with an epithelium and subjacent muscularis. Differentiated suprabasal and proliferative basal layers of esophageal epithelium, muscle, and nerve were identified. Lineage tracing demonstrated that multiple EOU could contribute to the epithelium and mesenchyme of a single TEE. Cultured murine EOU grew as an expanding sphere of proliferative basal cells on a neuromuscular network that demonstrated spontaneous peristalsis in culture. Subsequently, cultured EOU generated TEE. Conclusions: TEE forms after transplantation of mouse and human organ-specific stem/progenitor cells in vivo on a relatively simple biodegradable scaffold. This is a first step toward future human therapies. PMID:25298083

  5. Primary Murine Small Intestinal Epithelial Cells, Maintained in Long-Term Culture, Are Susceptible to Rotavirus Infection

    PubMed Central

    Macartney, Kristine K.; Baumgart, Daniel C.; Carding, Simon R.; Brubaker, Jeffery O.; Offit, Paul A.

    2000-01-01

    We describe a method for long-term culture of primary small intestinal epithelial cells (IEC) from suckling mice. IEC were digested from intestinal fragments as small intact units of epithelium (organoids) by using collagenase and dispase. IEC proliferated from organoids on a basement-membrane-coated culture surface and remained viable for 3 weeks. Cultured IEC had the morphologic and functional characteristics of immature enterocytes, notably sustained expression of cytokeratin and alkaline phosphatase. Few mesenchymal cells were present in the IEC cultures. IEC were also cultured from adult BALB/c mice and expressed major histocompatibility complex (MHC) class II antigens for at least 48 h in vitro. Primary IEC supported the growth of rhesus rotavirus (RRV) to a greater extent than a murine small intestinal cell line, m-ICcl2. Cell-culture-adapted murine rotavirus strain EDIM infected primary IEC and m-ICcl2 cells to a lesser extent than RRV. Wild-type EDIM did not infect either cell type. Long-term culture of primary murine small intestinal epithelial cells provides a method to study (i) virus-cell interactions, (ii) the capacity of IEC to act as antigen-presenting cells using a wide variety of MHC haplotypes, and (iii) IEC biology. PMID:10823867

  6. A rapid murine coma and behavior scale for quantitative assessment of murine cerebral malaria.

    PubMed

    Carroll, Ryan W; Wainwright, Mark S; Kim, Kwang-Youn; Kidambi, Trilokesh; Gómez, Noé D; Taylor, Terrie; Haldar, Kasturi

    2010-10-01

    Cerebral malaria (CM) is a neurological syndrome that includes coma and seizures following malaria parasite infection. The pathophysiology is not fully understood and cannot be accounted for by infection alone: patients still succumb to CM, even if the underlying parasite infection has resolved. To that effect, there is no known adjuvant therapy for CM. Current murine CM (MCM) models do not allow for rapid clinical identification of affected animals following infection. An animal model that more closely mimics the clinical features of human CM would be helpful in elucidating potential mechanisms of disease pathogenesis and evaluating new adjuvant therapies. A quantitative, rapid murine coma and behavior scale (RMCBS) comprised of 10 parameters was developed to assess MCM manifested in C57BL/6 mice infected with Plasmodium berghei ANKA (PbA). Using this method a single mouse can be completely assessed within 3 minutes. The RMCBS enables the operator to follow the evolution of the clinical syndrome, validated here by correlations with intracerebral hemorrhages. It provides a tool by which subjects can be identified as symptomatic prior to the initiation of trial treatment. Since the RMCBS enables an operator to rapidly follow the course of disease, la