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Sample records for interleukin-3

  1. RECOMBINANT HUMAN INTERLEUKIN-3 IN CLINICAL ONCOLOGY

    NARCIS (Netherlands)

    DEVRIES, EGE; VANGAMEREN, MM; WILLEMSE, PHB

    Interleukin 3 (IL-3) is a multipotent hematopoietic growth factor which became available as a recombinant (rh) growth factor for use in the clinic a few years ago. In dose-finding studies, this hematopoietic growth factor has been evaluated without and after standard chemotherapy. Stimulatory

  2. Interleukin-3 prevents neuronal death induced by amyloid peptide

    Directory of Open Access Journals (Sweden)

    Otth Carola

    2007-10-01

    Full Text Available Abstract Background Interleukin-3 (IL-3 is an important glycoprotein involved in regulating biological responses such as cell proliferation, survival and differentiation. Its effects are mediated via interaction with cell surface receptors. Several studies have demonstrated the expression of IL-3 in neurons and astrocytes of the hippocampus and cortices in normal mouse brain, suggesting a physiological role of IL-3 in the central nervous system. Although there is evidence indicating that IL-3 is expressed in some neuronal populations, its physiological role in these cells is poorly known. Results In this study, we demonstrated the expression of IL-3 receptor in cortical neurons, and analyzed its influence on amyloid β (Aβ-treated cells. In these cells, IL-3 can activate at least three classical signalling pathways, Jak/STAT, Ras/MAP kinase and the PI 3-kinase. Viability assays indicated that IL-3 might play a neuroprotective role in cells treated with Aβ fibrils. It is of interest to note that our results suggest that cell survival induced by IL-3 required PI 3-kinase and Jak/STAT pathway activation, but not MAP kinase. In addition, IL-3 induced an increase of the anti-apoptotic protein Bcl-2. Conclusion Altogether these data strongly suggest that IL-3 neuroprotects neuronal cells against neurodegenerative agents like Aβ.

  3. Cloning and sequencing of Indian Water buffalo (Bubalus bubalis) interleukin-3 cDNA

    KAUST Repository

    Sugumar, Thennarasu

    2011-12-12

    Full-length cDNA (435 bp) of the interleukin-3(IL-3) gene of the Indian water buffalo was amplified by reverse transcriptase-polymerase chain reaction and sequenced. This sequence had 96% nucleotide identity and 92% amino acid identity with bovine IL-3. There are 10 amino acid substitutions in buffalo compared with that of bovine. The amino acid sequence of buffalo IL-3 also showed very high identity with that of other ruminants, indicating functional cross-reactivity. Structural homology modelling of buffalo IL-3 protein with human IL-3 showed the presence of five helical structures.

  4. Effects of interleukin-3 on hemopoietic function after combined radiation-burn injury in mice

    International Nuclear Information System (INIS)

    Luo Chengji; Guo Chaohua; Zhou Yanhong; Liu Xin

    1996-01-01

    Destruction of hemopoietic function is one of the most basic pathologic changes in combined radiation-burn injury. Interleukin-3 exhibits a broad-spectrum stimulating effect on proliferation and differentiation for hemopoietic cells. This study was aimed to investigate the effects of interleukin-3 on hemopoietic function after combined radiation-burn injury in mice. The results show that preventing and additionally treating radiation injury or the combined radiation-burn injury with IL-3 have better effects on the stimulation of hematopoiesis in vivo than only preventing or treating with IL-3. This favourable stimulation of hemopoiesis was not only expressed in granulocyte-macrophage progenitors (CFU-GM) or erythroid progenitors (CFU-E, BFU-E), but also in bone marrow stromal elements (CFU-F). Stimulating effect on hemopoiesis was not shown in animals with burn injury in which hemopoietic failures did not appear. The results indicate that excellent prospects are expected by using IL-3 in treating radiation injury or combined radiation-burn injury

  5. Synergism between erythropoietin and interleukin-3 in the induction of hematopoietic stem cell proliferation and erythroid burst colony formation

    NARCIS (Netherlands)

    Migliaccio, G.; Migliaccio, A.R.; Visser, J.W.M

    1988-01-01

    The influence of recombinant erythropoietin (Ep) and interleukin-3 (IL-3) on the proliferation and differentiation of murine hematopoietic stem and progenitor cells was investigated in serum-deprived cultures. The differentiation of progenitor cells, purified by collecting blast cell colonies from

  6. Dual Mechanism of Interleukin-3 Receptor Blockade by an Anti-Cancer Antibody

    Directory of Open Access Journals (Sweden)

    Sophie E. Broughton

    2014-07-01

    Full Text Available Interleukin-3 (IL-3 is an activated T cell product that bridges innate and adaptive immunity and contributes to several immunopathologies. Here, we report the crystal structure of the IL-3 receptor α chain (IL3Rα in complex with the anti-leukemia antibody CSL362 that reveals the N-terminal domain (NTD, a domain also present in the granulocyte-macrophage colony-stimulating factor (GM-CSF, IL-5, and IL-13 receptors, adopting unique “open” and classical “closed” conformations. Although extensive mutational analyses of the NTD epitope of CSL362 show minor overlap with the IL-3 binding site, CSL362 only inhibits IL-3 binding to the closed conformation, indicating alternative mechanisms for blocking IL-3 signaling. Significantly, whereas “open-like” IL3Rα mutants can simultaneously bind IL-3 and CSL362, CSL362 still prevents the assembly of a higher-order IL-3 receptor-signaling complex. The discovery of open forms of cytokine receptors provides the framework for development of potent antibodies that can achieve a “double hit” cytokine receptor blockade.

  7. Physiology of natural killer cells. In vivo regulation of progenitors by interleukin 3

    International Nuclear Information System (INIS)

    Kalland, T.

    1987-01-01

    Adoptive transfer of bone marrow cells to syngeneic lethally irradiated C57BL/6 mice was used to study the maturation of natural killer (NK) cells from their progenitors. The NK progenitor cell was found to be asialomonoganglioside-negative, (aGM1-) Thy-1-, NK-1-, Ly-1-, Ly-2-, and L3T4-. The NK cells emerging from the bone marrow grafts were aGM1+, NK-1+, Thy-1+/-, Ly-1-, Ly-2-, and L3T4- and to have a target specter similar to that of NK cells isolated from the spleen of normal mice. The regulatory role of interleukin 2 (IL-2) and interleukin 3 (IL-3) for the maturation of NK cells was examined by exposure of the bone marrow cells to the lymphokines in vitro before bone marrow grafting or by treatment of bone marrow-grafted mice with lymphokines through s.c. implanted miniosmotic pumps. IL-3 antagonized the IL-2-induced maturation of NK cells in vitro and strongly inhibited the generation of NK cells after adoptive transfer of bone marrow cells in vivo. The suppressive effect of IL-3 was evident throughout the treatment period (8 or 16 days) but was apparently reversible because NK activity returned to control levels within 8 days after cessation of treatment. The inhibition of cytotoxic activity was accompanied by a reduced appearance of cells with the NK phenotypic markers aGM1 or NK-1, indicating that not only the cytotoxic activity of NK cells but also their actual formation was inhibited. Concomitantly, a moderate increase in cells expressing the T cell marker L3T4 and an increased proliferative response to the T cell mitogen concanavalin A was observed. A direct estimate of the effect of IL-3 on the frequency of NK cell progenitors was obtained by limiting dilution analysis of bone marrow cells at day 8 after bone marrow transplantation

  8. Interleukin-3 enhances the migration of human mesenchymal stem cells by regulating expression of CXCR4.

    Science.gov (United States)

    Barhanpurkar-Naik, Amruta; Mhaske, Suhas T; Pote, Satish T; Singh, Kanupriya; Wani, Mohan R

    2017-07-14

    Mesenchymal stem cells (MSCs) represent an important source for cell therapy in regenerative medicine. MSCs have shown promising results for repair of damaged tissues in various degenerative diseases in animal models and also in human clinical trials. However, little is known about the factors that could enhance the migration and tissue-specific engraftment of exogenously infused MSCs for successful regenerative cell therapy. Previously, we have reported that interleukin-3 (IL-3) prevents bone and cartilage damage in animal models of rheumatoid arthritis and osteoarthritis. Also, IL-3 promotes the differentiation of human MSCs into functional osteoblasts and increases their in-vivo bone regenerative potential in immunocompromised mice. However, the role of IL-3 in migration of MSCs is not yet known. In the present study, we investigated the role of IL-3 in migration of human MSCs under both in-vitro and in-vivo conditions. MSCs isolated from human bone marrow, adipose and gingival tissues were used for in-vitro cell migration, motility and wound healing assays in the presence or absence of IL-3. The effect of IL-3 preconditioning on expression of chemokine receptors and integrins was examined by flow cytometry and real-time PCR. The in-vivo migration of IL-3-preconditioned MSCs was investigated using a subcutaneous matrigel-releasing stromal cell-derived factor-1 alpha (SDF-1α) model in immunocompromised mice. We observed that human MSCs isolated from all three sources express IL-3 receptor-α (IL-3Rα) both at gene and protein levels. IL-3 significantly enhances in-vitro migration, motility and wound healing abilities of MSCs. Moreover, IL-3 preconditioning upregulates expression of chemokine (C-X-C motif) receptor 4 (CXCR4) on MSCs, which leads to increased migration of cells towards SDF-1α. Furthermore, CXCR4 antagonist AMD3100 decreases the migration of IL-3-treated MSCs towards SDF-1α. Importantly, IL-3 also induces in-vivo migration of MSCs towards

  9. Cloning and expression of recombinant equine interleukin-3 and its effect on sulfidoleukotriene and cytokine production by equine peripheral blood leukocytes

    Czech Academy of Sciences Publication Activity Database

    Janda, Jozef; Lehmann, M.; Luttmann, W.; Marti, E.

    2015-01-01

    Roč. 163, 3-4 (2015), s. 202-209 ISSN 0165-2427 R&D Projects: GA MŠk ED2.1.00/03.0124 Institutional support: RVO:67985904 Keywords : interleukin-3 * horse * sulphidoleukotriene release * cytokines Subject RIV: EC - Immunology Impact factor: 1.664, year: 2015

  10. Novel antibody-cytokine fusion proteins featuring granulocyte-colony stimulating factor, interleukin-3 and interleukin-4 as payloads.

    Science.gov (United States)

    Schmid, Anja Sophie; Tintor, Diana; Neri, Dario

    2018-04-10

    Neutrophils can strongly influence disease activity in cancer and in chronic inflammation. Here, we report for the first time the construction and characterization of antibody-fusion proteins featuring granulocyte-colony stimulating factor and interleukin-3 as payloads capable of enhancing neutrophil activity and a novel antibody-interleukin-4 fusion protein with neutrophil inhibitory potential. We used the F8 antibody specific to the alternatively-spliced extra domain A (EDA) of fibronectin as a targeting agent, since the cognate antigen is strongly upregulated in diseases characterized by angiogenesis. The fusion proteins GCSF-F8, F8-IL3 and F8-IL4-F8, were cloned, expressed, and their targeting ability assessed, exhibiting preferential tumor uptake with tumor:blood ratios at 24 h after injection of 3.3, 18.2 and 27.3, respectively. In F9 tumor bearing-mice GCSF-F8 and F8-IL3 did not provide a therapeutic benefit, while F8-IL4-F8 showed a potent tumor growth retardation. In the collagen-induced model of arthritis, GCSF-F8 and F8-IL3 induced a worsening of the disease, while F8-IL4-F8 slowed arthritis progression but, surprisingly, exhibited substantial toxicity when used in combination with dexamethasone. Collectively, the results indicate that the novel fusion proteins could be expressed and efficiently delivered to the site of disease. However, they were not superior to other antibody-cytokine fusions previously described by our laboratory. Copyright © 2018 Elsevier B.V. All rights reserved.

  11. 3T3 fibroblasts induce cloned interleukin 3-dependent mouse mast cells to resemble connective tissue mast cells in granular constituency

    International Nuclear Information System (INIS)

    Dayton, E.T.; Pharr, P.; Ogawa, M.; Serafin, W.E.; Austen, K.F.; Levi-Schaffer, F.; Stevens, R.L.

    1988-01-01

    As assessed by ultrastructure, histochemical staining, and T-cell dependency, in vitro-differentiated interleukin 3-dependent mouse mast cells are comparable to the mast cells that reside in the gastrointestinal mucosa but not in the skin or the serosal cavity of the mouse. The authors now demonstrate that when cloned interleukin 3-dependent mast cells are cocultured with mouse skin-derived 3T3 fibroblasts in the presence of WEHI-3 conditioned medium for 28 days, the mast cells acquire the ability to stain with safranin, increase their histamine content ∼ 50-fold and their carboxypeptidase. A content ∼ 100-fold, and augment ∼ their biosynthesis of proteoglycans bearing 35 S-labeled haparin relative to 35 S-labeled chondroitin sulfate glycosaminoglycans. Thus, fibroblasts induce interleukin 3-dependent mouse mast cells to change phenotype from mucosal-like to connective tissue-like, indicating that the biochemical and functional characteristics of this mast cell type are strongly influenced by the connective tissue microenvironment

  12. Phosphatidylinositol 3-kinase is essential for kit ligand-mediated survival, whereas interleukin-3 and flt3 ligand induce expression of antiapoptotic Bcl-2 family genes

    DEFF Research Database (Denmark)

    Karlsson, Richard; Engström, Maria; Jönsson, Maria

    2003-01-01

    Cytokines such as interleukin 3 (IL-3), kit ligand (KL), and flt3 ligand (FL) promote survival of hematopoietic stem cells and myeloid progenitor cells. In many cell types, members of the Bcl-2 gene family are major regulators of survival, but the mediating mechanisms are not fully understood....... Using two myeloid progenitor cell lines, FDCP-mix and FDC-P1, as well as primary mouse bone marrow progenitors, we demonstrate that KL-mediated survival is dependent on the activation of phosphatidylinositol-3 (PI-3) kinase. The inhibitor LY294002 was able to completely abolish survival mediated by KL...

  13. Skin CD4+ Memory T Cells Play an Essential Role in Acquired Anti-Tick Immunity through Interleukin-3-Mediated Basophil Recruitment to Tick-Feeding Sites

    Directory of Open Access Journals (Sweden)

    Takuya Ohta

    2017-10-01

    Full Text Available Ticks, blood-sucking arthropods, serve as vectors for transmission of infectious diseases including Lyme borreliosis. After tick infestation, several animal species can develop resistance to subsequent infestations, reducing the risk of transmission. In a mouse model, basophils reportedly infiltrate tick-feeding sites during the second but not first infestation and play a crucial role in the expression of acquired tick resistance. However, the mechanism underlying basophil recruitment to the second tick-feeding site remains ill-defined. Here, we investigated cells and their products responsible for the basophil recruitment. Little or no basophil infiltration was detected in T-cell-deficient mice, and adoptive transfer of CD4+ but not CD8+ T cells reconstituted it. Il3 gene expression was highly upregulated at the second tick-feeding site, and adoptive transfer of interleukin-3 (IL-3-sufficient but not IL-3-deficient CD4+ T cells conferred the basophil infiltration on T-cell-deficient mice, indicating that the CD4+ T-cell-derived IL-3 is essential for the basophil recruitment. Notably, IL-3+ resident CD4+ memory T cells were detected even before the second infestation in previously uninfested skin distant from the first tick-feeding site. Taken together, IL-3 produced locally by skin CD4+ memory T cells appears to play a crucial role in basophil recruitment to the second tick-feeding site.

  14. Skin CD4+Memory T Cells Play an Essential Role in Acquired Anti-Tick Immunity through Interleukin-3-Mediated Basophil Recruitment to Tick-Feeding Sites.

    Science.gov (United States)

    Ohta, Takuya; Yoshikawa, Soichiro; Tabakawa, Yuya; Yamaji, Kayoko; Ishiwata, Kenji; Shitara, Hiroshi; Taya, Choji; Oh-Hora, Masatsugu; Kawano, Yohei; Miyake, Kensuke; Yamanishi, Yoshinori; Yonekawa, Hiromichi; Watanabe, Naohiro; Kanuka, Hirotaka; Karasuyama, Hajime

    2017-01-01

    Ticks, blood-sucking arthropods, serve as vectors for transmission of infectious diseases including Lyme borreliosis. After tick infestation, several animal species can develop resistance to subsequent infestations, reducing the risk of transmission. In a mouse model, basophils reportedly infiltrate tick-feeding sites during the second but not first infestation and play a crucial role in the expression of acquired tick resistance. However, the mechanism underlying basophil recruitment to the second tick-feeding site remains ill-defined. Here, we investigated cells and their products responsible for the basophil recruitment. Little or no basophil infiltration was detected in T-cell-deficient mice, and adoptive transfer of CD4 + but not CD8 + T cells reconstituted it. Il3 gene expression was highly upregulated at the second tick-feeding site, and adoptive transfer of interleukin-3 (IL-3)-sufficient but not IL-3-deficient CD4 + T cells conferred the basophil infiltration on T-cell-deficient mice, indicating that the CD4 + T-cell-derived IL-3 is essential for the basophil recruitment. Notably, IL-3 + resident CD4 + memory T cells were detected even before the second infestation in previously uninfested skin distant from the first tick-feeding site. Taken together, IL-3 produced locally by skin CD4 + memory T cells appears to play a crucial role in basophil recruitment to the second tick-feeding site.

  15. Enhancement of the grafting efficiency of transplanted marrow cells by preincubation with interleukin-3 and granulocyte-macrophage colony-stimulating factor

    International Nuclear Information System (INIS)

    Tavassoli, M.; Konno, M.; Shiota, Y.; Omoto, E.; Minguell, J.J.; Zanjani, E.D.

    1991-01-01

    To improve the grafting efficiency of transplanted murine hematopoietic progenitors, we briefly preincubated mouse bone marrow cells with interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF) ex vivo before their transplantation into irradiated recipients. This treatment was translated into an increase in the seeding efficiency of colony-forming unit-spleen (CFU-S) and CFU-GM after transplantation. Not only was the concentration of CFU-S in the tibia increased 2 and 24 hours after transplantation, but the total cell number and CFU-S and CFU-GM concentrations were persistently higher in IL-3- and GM-CSF-treated groups 1 to 3 weeks after transplantation. In addition, the survival of animals as a function of transplanted cell number was persistently higher in IL-3- and GM-CSF-treated groups compared with controls. The data indicate that the pretreatment of marrow cells with IL-3 and GM-CSF before transplantation increases the seeding efficiency of hematopoietic stem cells and probably other progenitor cells after transplantation. This increased efficiency may be mediated by upward modulation of homing receptors. Therefore, ex vivo preincubation of donor marrow cells with IL-3 and GM-CSF may be a useful tactic in bone marrow transplantation

  16. Enhancement of the grafting efficiency of transplanted marrow cells by preincubation with interleukin-3 and granulocyte-macrophage colony-stimulating factor

    Energy Technology Data Exchange (ETDEWEB)

    Tavassoli, M.; Konno, M.; Shiota, Y.; Omoto, E.; Minguell, J.J.; Zanjani, E.D.

    1991-04-01

    To improve the grafting efficiency of transplanted murine hematopoietic progenitors, we briefly preincubated mouse bone marrow cells with interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF) ex vivo before their transplantation into irradiated recipients. This treatment was translated into an increase in the seeding efficiency of colony-forming unit-spleen (CFU-S) and CFU-GM after transplantation. Not only was the concentration of CFU-S in the tibia increased 2 and 24 hours after transplantation, but the total cell number and CFU-S and CFU-GM concentrations were persistently higher in IL-3- and GM-CSF-treated groups 1 to 3 weeks after transplantation. In addition, the survival of animals as a function of transplanted cell number was persistently higher in IL-3- and GM-CSF-treated groups compared with controls. The data indicate that the pretreatment of marrow cells with IL-3 and GM-CSF before transplantation increases the seeding efficiency of hematopoietic stem cells and probably other progenitor cells after transplantation. This increased efficiency may be mediated by upward modulation of homing receptors. Therefore, ex vivo preincubation of donor marrow cells with IL-3 and GM-CSF may be a useful tactic in bone marrow transplantation.

  17. Interleukin 3 gene is located on human chromosome 5 and is deleted in myeloid leukemias with a deletion of 5q

    International Nuclear Information System (INIS)

    Le Beau, M.M.; Epstein, N.D.; O'Brien, S.J.; Nienhuis, A.W.; Yang, Y.C.; Clark, S.C.; Rowley, J.D.

    1987-01-01

    The gene IL-3 encodes interleukin 3, a hematopoietic colony-stimulating factor (CSF) that is capable of supporting the proliferation of a broad range of hematopoietic cell types. By using somatic cell hybrids and in situ chromosomal hybridization, the authors localized this gene to human chromosome 5 at bands q23-31, a chromosomal region that is frequently deleted [del(5q)] in patients with myeloid disorders. By in situ hybridization, IL-3 was found to be deleted in the 5q-chromosome of one patient with refractory anemia who had a del(5)(q15q33.3), of three patients with refractory anemia (two patients) or acute nonlymphocytic leukemia (ANLL) de novo who had a similar distal breakpoint [del(5)(q13q33.3)], and of a fifth patient, with therapy-related ANLL, who had a similar distal breakpoint in band q33[del(5)(q14q33.3)]. Southern blot analysis of somatic cell hybrids retaining the normal or the deleted chromosome 5 from two patients with the refractory anemia 5q- syndrome indicated that IL-3 sequences were absent from the hybrids retaining the deleted chromosome 5 but not from hybrids that had a cytologically normal chromosome 5. Thus, a small segment of chromosome 5 contains IL-3, GM-CSF, CSF-1, and FMS. The findings and earlier results indicating that GM-CSF, CSF-1, and FMS were deleted in the 5q- chromosome, suggest that loss of IL-3 or of other CSF genes may play an important role in the pathogenesis of hematologic disorders associated with a del(5q)

  18. Interleukin-3 plays dual roles in osteoclastogenesis by promoting the development of osteoclast progenitors but inhibiting the osteoclastogenic process

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Huixian [Department of Hematology, Guangzhou First People’s Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510180 (China); Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Shi, Zhenqi [Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Qiao, Ping [Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Department of Pharmacology, Norman Bethune Medical College, Jilin University, Changchun, Jilin 130021 (China); Li, Hui [Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); McCoy, Erin M. [Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Mao, Ping [Department of Hematology, Guangzhou First People’s Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510180 (China); Xu, Hui [Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Feng, Xu [Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Wang, Shunqing, E-mail: shqwang_cn@yahoo.com [Department of Hematology, Guangzhou First People’s Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510180 (China)

    2013-11-01

    Highlights: •IL-3 treatment of bone marrow cells generates a population of hematopoietic cells. •IL-3-dependent hematopoietic cells are capable of differentiating into osteoclasts. •Osteoclasts derived from IL-3-dependent hematopoietic cells are functional. •IL-3 promotes the development of osteoclast progenitors. •IL-3 inhibits the osteoclastogenic process. -- Abstract: Interleukin (IL)-3, a multilineage hematopoietic growth factor, is implicated in the regulation of osteoclastogenesis. However, the role of IL-3 in osteoclastogenesis remains controversial; whereas early studies showed that IL-3 stimulates osteoclastogenesis, recent investigations demonstrated that IL-3 inhibits osteoclast formation. The objective of this work is to further address the role of IL-3 in osteoclastogenesis. We found that IL-3 treatment of bone marrow cells generated a population of cells capable of differentiating into osteoclasts in tissue culture dishes in response to the stimulation of the monocyte/macrophage-colony stimulating factor (M-CSF) and the receptor activator of nuclear factor kappa B ligand (RANKL). The IL-3-dependent hematopoietic cells were able to further proliferate and differentiate in response to M-CSF stimulation and the resulting cells were also capable of forming osteoclasts with M-CSF and RANKL treatment. Interestingly, IL-3 inhibits M-CSF-/RANKL-induced differentiation of the IL-3-dependent hematopoietic cells into osteoclasts. The flow cytometry analysis indicates that while IL-3 treatment of bone marrow cells slightly affected the percentage of osteoclast precursors in the surviving populations, it considerably increased the percentage of osteoclast precursors in the populations after subsequent M-CSF treatment. Moreover, osteoclasts derived from IL-3-dependent hematopoietic cells were fully functional. Thus, we conclude that IL-3 plays dual roles in osteoclastogenesis by promoting the development of osteoclast progenitors but inhibiting the osteoclastogenic process. These findings provide a better understanding of the role of IL-3 in osteoclastogenesis.

  19. Astrocyte-targeted expression of interleukin-3 and interferon-alpha causes region-specific changes in metallothionein expression in the brain

    DEFF Research Database (Denmark)

    Giralt, M; Carrasco, J; Penkowa, M

    2001-01-01

    Transgenic mice expressing IL-3 and IFN-alpha under the regulatory control of the GFAP gene promoter (GFAP-IL3 and GFAP-IFNalpha mice) exhibit a cytokine-specific, late-onset chronic-progressive neurological disorder which resemble many of the features of human diseases such as multiple sclerosis...... was confirmed by immunohistochemistry. MT-III immunoreactivity was present in cells that were mainly round or amoeboid monocytes/macrophages and in astrocytes. MT-I+II induction was more generalized in the GFAP-IFNalpha (GIFN12 and GIFN39 lines) mice, with significant increases in the cerebellum, thalamus...

  20. Important role of IL-3 during intiation of collagen induced arthritis

    Czech Academy of Sciences Publication Activity Database

    Brühl, E.; Cihak, J.; Niedermeier, M.; Denzel, A.; Gomez, M.R.; Talke, Y.; Goebel, N.; Plachý, Jiří; Stangassinger, M.; Mack, M.

    2009-01-01

    Roč. 60, č. 5 (2009), s. 1352-1361 ISSN 0004-3591 Institutional research plan: CEZ:AV0Z50520514 Keywords : experimental arthritis * interleukin-3 * basophils Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 7.332, year: 2009

  1. Genetically Modified T-cell Immunotherapy in Treating Patients With Relapsed/Refractory Acute Myeloid Leukemia and Persistent/Recurrent Blastic Plasmacytoid Dendritic Cell Neoplasm

    Science.gov (United States)

    2018-03-02

    Adult Acute Myeloid Leukemia in Remission; Acute Biphenotypic Leukemia; Early Relapse of Acute Myeloid Leukemia; Late Relapse of Acute Myeloid Leukemia; Recurrent Adult Acute Myeloid Leukemia; Secondary Acute Myeloid Leukemia; Blastic Plasmacytoid Dendritic Cell Neoplasm; Acute Myeloid Leukemia; Adult Acute Lymphoblastic Leukemia; Interleukin-3 Receptor Subunit Alpha Positive; Minimal Residual Disease; Refractory Acute Myeloid Leukemia; Untreated Adult Acute Myeloid Leukemia

  2. Research and Development Strategies for the Current and Future Medical Treatment of Radiation Casualties

    Science.gov (United States)

    2014-09-01

    currently FDA-approved for the treatment of urea cycle disorders, and it can be administered orally and is stored at room temperature.102 Phenylbutyrate can...in Figure 5. No DRF was calculated from the filgrastim NHP study. However, MacVittie, Farese, and Jackson performed a study in canines where they...Treatment of Canine Cyclic Hematopoiesis with Recombinant Human Granulocyte-Macrophage Colony-Macrophage Stimulating Factor (GM-CSF), Interleukin-3, and

  3. Pretreatment with rh-GMCSF, but not rh-IL3, enhances PAF-induced eosinophil accumulation in guinea-pig airways.

    OpenAIRE

    Sanjar, S.; Smith, D.; Kings, M. A.; Morley, J.

    1990-01-01

    Intraperitoneal injections of recombinant human granulocyte-macrophage colony stimulating factor (rh-GMCSF, 50 micrograms/kg-1 daily) or interleukin-3 (rh-IL3, 50 micrograms kg-1 daily) for two days, induced an increase in the percentage of bone marrow and pulmonary airway eosinophils in the guinea-pig. In addition, rh-IL3-treated animals exhibited an increase (21%) in blood neutrophils. Exposure of guinea-pigs to an aerosol of platelet activating factor (PAF) gives rise to a selective pulmon...

  4. Spontaneous and cytokine induced basophil adhesion evaluated by microtiter assay

    DEFF Research Database (Denmark)

    Quan, Sha; Poulsen, Lars K; Reimert, Claus Michael

    2002-01-01

    We have developed a microtiter assay for evaluating basophil spontaneous adhesion to extracellular matrix (ECM) proteins exemplified by fibronectin and cytokine induced basophil adhesion to bovine serum albumin (BSA). The percentage of basophils adhering to either ECM or BSA was quantified...... by the histamine content of the adhering basophils. The spontaneous adhesion to fibronectin was higher than to laminin and collagen type I. Both spontaneous adhesion to fibronectin and interleukin-3 (IL-3), interleukin-5 (IL-5), granulocyte/macrophage colony stimulating factor (GM-CSF) induced adhesion to BSA...

  5. Comparison of the levels of inositol metabolites in transformed haemopoietic cells and their normal counterparts.

    OpenAIRE

    Bunce, C M; French, P J; Allen, P; Mountford, J C; Moor, B; Greaves, M F; Michell, R H; Brown, G

    1993-01-01

    We have compared the levels of inositol metabolites in three pairs of normal and transformed cells which have been matched with respect to their cell lineage, differentiation and proliferation status: (i) normal human myeloid blast cells and the human promyelocytic leukaemic cell line, HL60; (ii) human umbilical-cord T-helper cells and C8166 cells, a HTLV-1-transformed T-helper cell line; and (iii) an interleukin 3-dependent long-term culture of murine pro-B-cells (BAF3) and BAF3 cells transf...

  6. Effect of the industrial nanoparticles TiO2, SiO2 and ZnO on cell viability and gene expression in red bone marrow of Mus musculus

    OpenAIRE

    Zarria-Romero, Jacquelyne; Laboratorio de Reproducción y Biología del Desarrollo. Facultad de Ciencias Biológicas. Universidad Nacional Mayor de San Marcos. Lima, Perú. Biólogo, magíster en Biología Molecular; Osorio, Ana; Laboratorio de Nanotecnología e Innovación Tecnológica. Facultad de Química e Ingeniería Química, Universidad Nacional Mayor de San Marcos. Lima, Perú. ingeniero químico, magíster en Ciencias Químicas; Pino, José; Laboratorio de Reproducción y Biología del Desarrollo. Facultad de Ciencias Biológicas. Universidad Nacional Mayor de San Marcos. Lima, Perú. biólogo, bachiller en Ciencias Biológicas; Shiga, Betty; Laboratorio de Reproducción y Biología del Desarrollo. Facultad de Ciencias Biológicas. Universidad Nacional Mayor de San Marcos. Lima, Perú. biólogo, bachiller en Ciencias Biológicas; Vivas-Ruiz, Dan; Laboratorio de Biología Molecular. Facultad de Ciencias Biológicas. Universidad Nacional Mayor de San Marcos. Lima, Perú. biólogo, doctor en Ciencias Biológicas

    2017-01-01

    Objectives. To evaluate the effect of ZnO, TiO2 and SiO2 nanoparticles on cell viability and expression of the interleukin 7, interleukin 3, and granulocyte-macrophage colony stimulating factor (GM-CSF) genes in Mus musculus. Materials and methods. Red bone marrow was extracted from five Balb/c mice for the analysis of cell viability using the MTT test. The mice were divided into two groups of five each: one group was inoculated intraperitoneally with 0.5, 1.0, 2.5, 5.0, and 10 mg/kg of ZnO a...

  7. IRS-1: essential for insulin- and IL-4-stimulated mitogenesis in hematopoietic cells.

    Science.gov (United States)

    Wang, L M; Myers, M G; Sun, X J; Aaronson, S A; White, M; Pierce, J H

    1993-09-17

    Although several interleukin-3 (IL-3)-dependent cell lines proliferate in response to IL-4 or insulin, the 32D line does not. Insulin and IL-4 sensitivity was restored to 32D cells by expression of IRS-1, the principal substrate of the insulin receptor. Although 32D cells possessed receptors for both factors, they lacked the IRS-1--related protein, 4PS, which becomes phosphorylated by tyrosine in insulin- or IL-4--responsive lines after stimulation. These results indicate that factors that bind unrelated receptors can use similar mitogenic signaling pathways in hematopoietic cells and that 4PS and IRS-1 are functionally similar proteins that are essential for insulin- and IL-4--induced proliferation.

  8. FLT3 ligand preserves the uncommitted CD34+CD38- progenitor cells during cytokine prestimulation for retroviral transduction

    DEFF Research Database (Denmark)

    Nielsen, S D; Husemoen, L L; Sørensen, T U

    2000-01-01

    Before stem cell gene therapy can be considered for clinical applications, problems regarding cytokine prestimulation remain to be solved. In this study, a retroviral vector carrying the genes for the enhanced version of green fluorescent protein (EGFP) and neomycin resistance (neo(r)) was used...... (SCF), FLT3 ligand, interleukin-3 (IL-3), IL-6, and IL-7 prior to transduction. Expression of the two genes was assessed by flow cytometry and determination of neomycin-resistant colonies in a selective colony-forming unit (CFU) assay, respectively. The neomycin resistance gene was expressed...... in a higher percentage of cells than the EGFP gene, but there seemed to be a positive correlation between expression of the two genes. The effect of cytokine prestimulation was therefore monitored using EGFP as marker for transduction. When SCF was compared to SCF in combination with more potent cytokines...

  9. Cytokine receptor signaling activates an IKK-dependent phosphorylation of PUMA to prevent cell death

    Science.gov (United States)

    Sandow, J J; Jabbour, A M; Condina, M R; Daunt, C P; Stomski, F C; Green, B D; Riffkin, C D; Hoffmann, P; Guthridge, M A; Silke, J; Lopez, A F; Ekert, P G

    2012-01-01

    P53-upregulated modifier of apoptosis (PUMA), a pro-apoptotic member of the Bcl-2 family, is transcriptionally activated by p53 and is a key effector of p53-dependent apoptosis. We show that PUMA protein is subject to rapid post-translational regulation by phosphorylation at a conserved residue, serine 10, following serum or interleukin-3 (IL-3) stimulation. Serine 10 is not within the Bcl-2 homology (BH3) domain, and PUMA phosphorylated at serine 10 retained the ability to co-immunoprecipitate with antiapoptotic Bcl-2 family members. However, phosphorylated PUMA was targeted for proteasomal degradation indicating that it is less stable than unphosphorylated PUMA. Importantly, we identified IKK1/IKK2/Nemo as the kinase complex that interacts with and phosphorylates PUMA, thereby also demonstrating that IL-3 activates NFκB signaling. The identification and characterization of this novel survival pathway has important implications for IL-3 signaling and hematopoietic cell development. PMID:21997190

  10. Cell proliferation and differentiation in chemical leukemogenesis

    Science.gov (United States)

    Irons, R. D.; Stillman, W. S.; Clarkson, T. W. (Principal Investigator)

    1993-01-01

    In tissues such as bone marrow with normally high rates of cell division, proliferation is tightly coordinated with cell differentiation. Survival, proliferation and differentiation of early hematopoietic progenitor cells depend on the growth factors, interleukin 3 (IL-3) and/or granulocyte-macrophage colony stimulating factor (GM-CSF) and their synergism with other cytokines. We provide evidence that a characteristic shared by a diverse group of compounds with demonstrated leukemogenic potential is the ability to act synergistically with GM-CSF. This results in an increase in recruitment of a resting population of hematopoietic progenitor cells normally unresponsive to the cytokine and a twofold increase in the size of the proliferating cell population normally regarded to be at risk of transformation in leukemogenesis. These findings support the possibility that transient alterations in hematopoietic progenitor cell differentiation may be an important factor in the early stages of development of leukemia secondary to chemical or drug exposure.

  11. Tissue localization and fate in mice of injected multipotential colony-stimulating factor

    International Nuclear Information System (INIS)

    Metcalf, D.; Nicola, N.A.

    1988-01-01

    The hemopoietic regulator multipotential colony-stimulating factor [Multi-CSF (interleukin 3)] has proliferative effects on a wide range of hemopoietic cells in vitro and in vivo. Native or recombination Multi-CSF injected intravenoulsy into adult mice had an initial half-life of 3-5 min and a second phase of 50 min. Clear labeling of hemopoietic cells was observed in the bone marrow and spleen of mice injected intravenously with recombinant 125 I-labeled Multi-CSF showing that injected Multi-CSF can obtain access to such cells in situ. A high proportion of injected 125 I-labeled Multi-CSF of both types became localized in the liver and in the kidney (in cells of the Bowman's capsule and proximal renal tubules). The kidney appeared to be an active site of degradation of Multi-CSF with the early appearance of low molecular weight labeled material in the urine

  12. Drug Target Optimization in Chronic Myeloid Leukemia Using Innovative Computational Platform

    Science.gov (United States)

    Chuang, Ryan; Hall, Benjamin A.; Benque, David; Cook, Byron; Ishtiaq, Samin; Piterman, Nir; Taylor, Alex; Vardi, Moshe; Koschmieder, Steffen; Gottgens, Berthold; Fisher, Jasmin

    2015-02-01

    Chronic Myeloid Leukemia (CML) represents a paradigm for the wider cancer field. Despite the fact that tyrosine kinase inhibitors have established targeted molecular therapy in CML, patients often face the risk of developing drug resistance, caused by mutations and/or activation of alternative cellular pathways. To optimize drug development, one needs to systematically test all possible combinations of drug targets within the genetic network that regulates the disease. The BioModelAnalyzer (BMA) is a user-friendly computational tool that allows us to do exactly that. We used BMA to build a CML network-model composed of 54 nodes linked by 104 interactions that encapsulates experimental data collected from 160 publications. While previous studies were limited by their focus on a single pathway or cellular process, our executable model allowed us to probe dynamic interactions between multiple pathways and cellular outcomes, suggest new combinatorial therapeutic targets, and highlight previously unexplored sensitivities to Interleukin-3.

  13. TEL/ETV6 induces apoptosis in 32D cells through p53-dependent pathways

    International Nuclear Information System (INIS)

    Yamagata, Tetsuya; Maki, Kazuhiro; Waga, Kazuo; Mitani, Kinuko

    2006-01-01

    TEL is an ETS family transcription factor that is critical for maintaining hematopoietic stem cells in adult bone marrow. To investigate the roles of TEL in myeloid proliferation and differentiation, we introduced TEL cDNA into mouse myeloid 32Dcl3 cells. Overexpression of TEL repressed interleukin-3-dependent proliferation through blocking cell cycle progression. Also, the presence of TEL triggered apoptosis through the mitochondrial intrinsic pathway on exposure to granulocyte colony-stimulating factor. We found an increase in p53 protein and its DNA binding in the TEL-overexpressing cells. Forced expression of TEL stimulated transcription via the p53-responsive element and increased the expression of cellular target genes for p53 such as cell cycle regulator p21 and apoptosis inducer Puma. Consistently, induction of apoptosis was delayed by pifithrin-α treatment and completely blocked by increased expression of Bcl-2 in the TEL-overexpressing cells. These data collectively suggest that TEL exerts a tumor suppressive function through augmenting the p53 pathway and facilitates normal development of myelopoiesis

  14. Subcloning, expression, purification, and characterization of recombinant human leptin-binding domain.

    Science.gov (United States)

    Sandowski, Yael; Raver, Nina; Gussakovsky, Eugene E; Shochat, Suzan; Dym, Orly; Livnah, Oded; Rubinstein, Menachem; Krishna, Radha; Gertler, Arieh

    2002-11-29

    A subdomain of the human leptin receptor encoding part of the extracellular domain (amino acids 428 to 635) was subcloned, expressed in a prokaryotic host, and purified to homogeneity, as evidenced by SDS-PAGE, with over 95% monomeric protein. The purified leptin-binding domain (LBD) exhibited the predicted beta structure, was capable of binding human, ovine, and chicken leptins, and formed a stable 1:1 complex with all mammalian leptins. The binding kinetics, assayed by surface plasmon resonance methodology, showed respective k(on) and k(off) values (mean +/- S.E.) of 1.20 +/- 0.23 x 10(-5) mol(-1) s(-1) and 1.85 +/- 0.30 x 10(-3) s(-1) and a K(d) value of 1.54 x 10(-8) m. Similar results were achieved with conventional binding experiments. LBD blocked leptin-induced, but not interleukin-3-induced, proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor. The modeled LBD structure and the known three-dimensional structure of human leptin were used to construct a model of 1:1 LBD.human leptin complex. Two main residues, Phe-500, located in loop L3, and Tyr-441, located in L1, are suggested to contribute to leptin binding.

  15. Nerve growth factor interactions with mast cells.

    Science.gov (United States)

    Kritas, S K; Caraffa, A; Antinolfi, P; Saggini, A; Pantalone, A; Rosati, M; Tei, M; Speziali, A; Saggini, R; Pandolfi, F; Cerulli, G; Conti, P

    2014-01-01

    Neuropeptides are involved in neurogenic inflammation where there is vasodilation and plasma protein extravasion in response to this stimulus. Nerve growth factor (NGF), identified by Rita Levi Montalcini, is a neurotrophin family compound which is important for survival of nociceptive neurons during their development. Therefore, NGF is an important neuropeptide which mediates the development and functions of the central and peripheral nervous system. It also exerts its proinflammatory action, not only on mast cells but also in B and T cells, neutrophils and eosinophils. Human mast cells can be activated by neuropeptides to release potent mediators of inflammation, and they are found throughout the body, especially near blood vessels, epithelial tissue and nerves. Mast cells generate and release NGF after degranulation and they are involved in iperalgesia, neuroimmune interactions and tissue inflammation. NGF is also a potent degranulation factor for mast cells in vitro and in vivo, promoting differentiation and maturation of these cells and their precursor, acting as a co-factor with interleukin-3. In conclusion, these studies are focused on cross-talk between neuropeptide NGF and inflammatory mast cells.

  16. Successful hematopoietic reconstitution with transplantation of erythrocyte-depleted allogeneic human umbilical cord blood cells in a child with leukemia.

    Science.gov (United States)

    Pahwa, R N; Fleischer, A; Than, S; Good, R A

    1994-05-10

    Cord blood, a potent source of hematopoietic stem cells, has been shown to successfully reconstitute hematopoiesis following allogeneic transplantation in a variety of disorders. A major drawback of cord blood has been the risk of transfusion reactions in ABO blood group incompatibility and drastic reduction in the stem cell pool if the cord blood is manipulated to remove red cells prior to cryopreservation or after thawing. This report describes an erythrocyte depletion method employing 3% gelatin-induced erythrocyte sedimentation for the selective removal of red cells from cord blood. The red cell-depleted fraction was shown to be enriched in progenitor cells and in cells secreting hematopoietic cytokines interleukin 3, granulocyte/macrophage colony-stimulating factor, and interleukin 6; a major source for cytokines was from cord T cells. This preparative technique was employed to separate out red cells from cord blood of an infant delivered by cesarean section who had an 8-year-old sibling with leukemia. Histocompatibility testing of cord cells revealed complete matching with the patient. A cord cell transplant of cryopreserved and thawed cells consisting of 4 x 10(7) nucleated cells per kg was administered to the patient following myeloablative chemotherapy. The patient's quick hematologic recovery and 9-month disease-free period to date suggest that 3% gelatin separation of erythrocytes is a simple method that can be successfully used for transplanting cord cells for malignant/nonmalignant diseases.

  17. Protection of zidovudine-induced toxicity against murine erythroid progenitor cells by vitamin E.

    Science.gov (United States)

    Gogu, S R; Lertora, J J; George, W J; Hyslop, N E; Agrawal, K C

    1991-08-01

    The ability of vitamin E (alpha-tocopherol) to stimulate erythroid progenitor cells was investigated in an attempt to identify ways to ameliorate zidovudine (azidothymidine, AZT)-induced anemia. In vitro, alpha-tocopherol acid succinate (ATS), upon incubation with murine bone marrow cells at concentrations of up to 4 micrograms/ml, caused a dose-dependent increase in erythroid colony-forming unit (CFU-E)-derived colonies. This increase was equivalent to the effect demonstrated by 50 mU of recombinant human erythropoietin (rhEpo) or 200 U of recombinant interleukin 3 (rIL-3). For in vivo studies, anemia was produced in CD-1 male mice by administering AZT in drinking water (1.5 mg/ml). Treatment with vitamin E (50 mg/kg body weight) or Epo (0.4 U per mouse) was initiated 24 h later and continued for five consecutive days. Seventh day bone marrow cells from femurs were assayed for CFU-E-derived colonies. Both vitamin E and Epo significantly increased the number of CFU-E-derived colonies by 75% and 86% of control, respectively, indicating that these agents were approximately similar in protecting the bone marrow from AZT-induced toxicity.

  18. The Oncoprotein E2A-Pbx1a Collaborates with Hoxa9 To Acutely Transform Primary Bone Marrow Cells

    Science.gov (United States)

    Thorsteinsdottir, Unnur; Krosl, Jana; Kroon, Evert; Haman, André; Hoang, Trang; Sauvageau, Guy

    1999-01-01

    A recurrent translocation between chromosome 1 (Pbx1) and 19 (E2A) leading to the expression of the E2A-Pbx1 fusion oncoprotein occurs in ∼5 to 10% of acute leukemias in humans. It has been proposed that some of the oncogenic potential of E2A-Pbx1 could be mediated through heterocomplex formation with Hox proteins, which are also involved in human and mouse leukemias. To directly test this possibility, mouse bone marrow cells were engineered by retroviral gene transfer to overexpress E2A-Pbx1a together with Hoxa9. The results obtained demonstrated a strong synergistic interaction between E2A-Pbx1a and Hoxa9 in inducing growth factor-independent proliferation of transduced bone marrow cells in vitro and leukemic growth in vivo in only 39 ± 2 days. The leukemic blasts which coexpress E2A-Pbx1a and Hoxa9 showed little differentiation and produced cytokines such as interleukin-3, granulocyte colony-stimulating factor, and Steel. Together, these studies demonstrate that the Hoxa9 and E2A-Pbx1a gene products collaborate to produce a highly aggressive acute leukemic disease. PMID:10454582

  19. Microarray analysis of gene expression in disk abalone Haliotis discus discus after bacterial challenge.

    Science.gov (United States)

    De Zoysa, Mahanama; Nikapitiya, Chamilani; Oh, Chulhong; Lee, Youngdeuk; Whang, Ilson; Lee, Jae-Seong; Choi, Cheol Young; Lee, Jehee

    2011-02-01

    In this study, we investigated the gene expression profiling of disk abalone, Haliotis discus discus challenged by a mixture of three pathogenic bacteria Vibrio alginolyticus, Vibrio parahemolyticus, and Listeria monocytogenes using a cDNA microarray. Upon bacteria challenge, 68 (1.6%) and 112 (2.7%) gene transcripts changed their expression levels ≥2 or ≤2 -fold in gills and digestive tract, respectively. There were 46 tissue-specific transcripts that up-regulated specifically in the digestive tract. In contrast, only 13 transcripts showed gill-specific up-regulation. Quantitative real-time PCR was performed to verify microarray data and results revealed that candidate genes namely Krüppell-like factor (KLF), lachesin, muscle lim protein, thioredoxin-2 (TRx-2), nuclear factor interleukin 3 (NFIL-3) and abalone protein 38 were up-regulated. Also, our results further indicated that bacteria challenge may activate the transcription factors or their activators (Krüppell-like factor, inhibitor of NF-κB or Ik-B), inflammatory cytokines (IL-3 regulated protein, allograft inflammatory factor), other cytokines (IFN-44-like protein, SOCS-2), antioxidant enzymes (glutathione-S-transferase, thioredoxin-2 and thioredoxin peroxidase), and apoptosis-related proteins (TNF-α, archeron) in abalone. The identification of immune and stress response genes and their expression profiles in this microarray will permit detailed investigation of the stress and immune responses of abalone genes. Copyright © 2010 Elsevier Ltd. All rights reserved.

  20. Introduction of mismatches in a random shRNA-encoding library improves potency for phenotypic selection.

    Directory of Open Access Journals (Sweden)

    Yongping Wang

    Full Text Available RNA interference (RNAi is a mechanism for interfering with gene expression through the action of small, non-coding RNAs. We previously constructed a short-hairpin-loop RNA (shRNA encoding library that is random at the nucleotide level [1]. In this library, the stems of the hairpin are completely complementary. To improve the potency of initial hits, and therefore signal-to-noise ratios in library screening, as well as to simplify hit-sequence retrieval by PCR, we constructed a second-generation library in which we introduced random mismatches between the two halves of the stem of each hairpin, on a random template background. In a screen for shRNAs that protect an interleukin-3 (IL3 dependent cell line from IL3 withdrawal, our second-generation library yielded hit sequences with significantly higher potencies than those from the first-generation library in the same screen. Our method of random mutagenesis was effective for a random template and is likely suitable, therefore, for any DNA template of interest. The improved potency of our second-generation library expands the range of possible unbiased screens for small-RNA therapeutics and biologic tools.

  1. The protective effect of Royal Jelly against the hemopoiesis dysfunction in X-irradiated mice

    Energy Technology Data Exchange (ETDEWEB)

    Emori, Yutaka; Oka, Hideki; Ohya, Osamu; Tamaki, Hajime; Hayashi, Yoshiro [Zeria Pharmaceutical Co., Ltd., Konan, Saitama (Japan). Central Research Laboratories; Nomoto, Kikuo

    1998-02-01

    The protective effect of Royal Jelly (RJ) against the hemopoietic dysfunction in whole body X-irradiated C57BL/6 mice was investigated. When RJ (1.0 g/kg, po or 0.5 g/kg, ip) was administered every day beginning two weeks before X-irradiation (10 Gy), a significant increase in the number of leukocytes and erythrocytes was observed in mice treated with RJ, as compared with X-irradiated control. In addition, the number of colony forming units in culture (CFU-C) of bone marrow cells or splenocytes was significantly increased in mice treated with RJ. Therefore, when granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) in peripheral blood was measured by ELISA kit, a significant increase in the amount of GM-CSF and IL-3 was observed. These results suggest that the protective effect of RJ against hemopoietic dysfunction could be expressed through an increase in the number of hemopoietic stem cells by the induction of hemopoietic factor such as GM-CSF and IL-3. (author)

  2. Enhanced normal short-term human myelopoiesis in mice engineered to express human-specific myeloid growth factors.

    Science.gov (United States)

    Miller, Paul H; Cheung, Alice M S; Beer, Philip A; Knapp, David J H F; Dhillon, Kiran; Rabu, Gabrielle; Rostamirad, Shabnam; Humphries, R Keith; Eaves, Connie J

    2013-01-31

    Better methods to characterize normal human hematopoietic cells with short-term repopulating activity cells (STRCs) are needed to facilitate improving recovery rates in transplanted patients.We now show that 5-fold more human myeloid cells are produced in sublethally irradiated NOD/SCID-IL-2Receptor-γchain-null (NSG) mice engineered to constitutively produce human interleukin-3, granulocyte-macrophage colony-stimulating factor and Steel factor (NSG-3GS mice) than in regular NSG mice 3 weeks after an intravenous injection of CD34 human cord blood cells. Importantly, the NSG-3GS mice also show a concomitant and matched increase in circulating mature human neutrophils. Imaging NSG-3GS recipients of lenti-luciferase-transduced cells showed that human cells being produced 3 weeks posttransplant were heterogeneously distributed, validating the blood as a more representative measure of transplanted STRC activity. Limiting dilution transplants further demonstrated that the early increase in human granulopoiesis in NSG-3GS mice reflects an expanded output of differentiated cells per STRC rather than an increase in STRC detection. NSG-3GS mice support enhanced clonal outputs from human short-term repopulating cells (STRCs) without affecting their engrafting efficiency. Increased human STRC clone sizes enable their more precise and efficient measurement by peripheral blood monitoring.

  3. Blastic plasmacytoid dendritic cell neoplasm: challenges and future prospects

    Directory of Open Access Journals (Sweden)

    Trottier AM

    2017-12-01

    Full Text Available Amy M Trottier, Sonia Cerquozzi, Carolyn J Owen Division of Hematology and Hematological Malignancies, University of Calgary, Foothills Medical Centre, Calgary, AB, Canada Abstract: Blastic plasmacytoid dendritic cell neoplasm (BPDCN is a rare CD4+ CD56+ myeloid malignancy that is challenging to diagnose and treat. BPDCN typically presents with nonspecific cutaneous lesions with or without extra-cutaneous manifestations before progressing to leukemia. Currently, there is no standard of care for the treatment of BPDCN and various approaches have been used including acute myeloid leukemia, acute lymphoblastic leukemia, and lymphoma-based regimens with or without stem cell transplantation. Despite these treatment approaches, the prognosis of BPDCN remains poor and there is a lack of prospective data upon which to base treatment decisions. Recent work examining the mutational landscape and gene expression profiles of BPDCN has identified a number of potential therapeutic targets. One such target is CD123, the α subunit of the human interleukin-3 receptor, which is the subject of intervention studies using the novel agent SL-401. Other investigational therapies include UCART123, T-cell immunotherapy, and venetoclax. Prospective trials are needed to determine the best treatment for this uncommon and aggressive neoplasm. Keywords: BPDCN, myeloid, neoplasm, cutaneous, dendritic cell

  4. Autologous cell therapy as a new approach to treatment of radiation-induced bone marrow aplasia: preliminary study in a baboon model

    Energy Technology Data Exchange (ETDEWEB)

    Herodin, F.; Drouet, M. [Radiohematology Unit, Centre de Recherches du Service de Sante des Armees, La Tronche CEDEX (France)

    2002-07-01

    The sparing of viable hematopoietic stem and progenitor cells located in underexposed bone marrow territories associated with the relative radioresistance of certain stem cell populations is the rationale for autologous cell therapy consisting of ex vivo expansion of residual cells after collection postirradiation. The feasibility of this treatment mainly depends on time constraints and hematopoietic cell threshold. We showed in this study that in the absence of early-acting mobilizing agent administration, subliminar amounts of CD34{sup +} cells can be collected (1 x 10{sup 6} CD34{sup +} cells/100 mL bone marrow or for 1 L apheresis) from 6-Gy {gamma} globally irradiated baboons. Residual CD34{sup +} cells were successfully expanded in serum-free medium in the presence of antiapoptotic cytokine combination (stem cell factor + FLT-3 ligand + thrombopoietin + interleukin 3, 50 ng/mL each, i.e., 4F): K{sub CD34{sup +}} = x2.8 and x13.7 (n=2). Moreover, we demonstrated the short-term neutrophil engraftment potential of a low-size mixed expanded graft (1.5 x 10{sup 6} final CD34{sup +}cells/kg) issued from the coculture of unirradiated (20%) and 2.5-Gy in vitro irradiated (80%) CD34{sup +} cells on an allogeneic stromal cell layer in the presence of 4F. Further preclinical research needs to be performed to clearly establish this therapeutic approach that could be optimized by the early administration of antiapoptotic cytokines. (author)

  5. Identification of chondroitin sulfate E proteoglycans and heparin proteoglycans in the secretory granules of human lung mast cells

    Energy Technology Data Exchange (ETDEWEB)

    Stevens, R.L.; Austen, K.F. (Brigham and Women' s Hospital, Boston, MA (USA)); Fox, C.C.; Lichtenstein, L.M. (Johns Hopkins School of Medicine, Baltimore, MD (USA))

    1988-04-01

    The predominant subclasses of mast cells in both the rat and the mouse can be distinguished from one another by their preferential synthesis of {sup 35}S-labeled proteoglycans that contain either heparin or oversulfated chondroitin sulfate glycosaminoglycans. Although ({sup 35}S)heparin proteoglycans have been isolated from human lung mast cells of 40-70% purity and from a skin biopsy specimen of a patient with urticaria pigmentosa, no highly sulfated chondroitin sulfate proteoglycan has been isolated from any enriched or highly purified population of human mast cells. The authors demonstrate that human lung mast cells of 96% purity incorporate ({sup 35}S)sulfate into separate heparin and chondroitin sulfate proteoglycans in an {approx}2:1 ratio. As assessed by HPLC of the chondroitinase ABC digests, the chondroitin ({sup 35}S)sulfate proteoglycans isolated from these human lung mast cells contain the same unusual chondroitin sulfate E disaccharide that is present in proteoglycans produced by interleukin 3-dependent mucosal-like mouse mast cells. Both the chondroitin ({sup 35}S)sulfate E proteoglycans and the ({sup 35}S)heparin proteoglycans were exocytosed from the ({sup 35}S)sulfate-labeled cells via perturbation of the IgE receptor, indicating that both types of {sup 35}S-labeled proteoglycans reside in the secretory granules of these human lung mast cells.

  6. The production of lymphokines by primary alloreactive T-cell clones: a co-ordinate analysis of 233 clones in seven lymphokine assays.

    Science.gov (United States)

    Sanderson, C J; Strath, M; Warren, D J; O'Garra, A; Kirkwood, T B

    1985-01-01

    A total of 233 primary alloreactive T-cell clones have been tested for the production of interleukin-2 (IL-2), interleukin-3 (IL-3), immune(gamma) interferon (IFN) and granulocyte-macrophage colony-stimulating factor (CSF-2), B-cell growth factor I and II (BCGFI, BCGFII), and eosinophil differentiation factor (EDF). EDF was assayed by means of the eosinophil differentiation assay (EDA). Two principal correlations were observed: IL-3 was shown to be the major lymphokine detected in the bone marrow proliferation assay (BMPA) used to detect CSF-2, and there was a high correlation between the EDA and BCGFII. Subsequent work has suggested that this latter correlation is because a single factor is responsible for both activities. Apart from these two exceptions, and low level correlations probably due to the fact that different assays detect more than one lymphokine, there was no evidence for co-ordinate expression of lymphokines. There was a large variation in amounts of individual lymphokines produced. More clones produced multiple lymphokines than would be expected from independent control. Taken together, this pattern of regulation is consistent with the hypothesis that antigen stimulation of T cells results in the activation of all the lymphokine genes, but the amount of each produced is determined by secondary controlling mechanisms. PMID:3935571

  7. Inhibited interferon production after space flight

    Science.gov (United States)

    Sonnenfeld, G.; Gould, C. L.; Williams, J.; Mandel, A. D.

    1988-01-01

    Several studies have been performed in our laboratories indicating that interferon production may be impaired in rodents after space flight. Using an antiorthostatic suspension model that simulates some of the effects of microgravity seen during space flight, we have shown that interferon-alpha/beta production was inhibited. The inhibition was not due solely to the stress of suspension. The inhibited interferon production was transient, as suspended animals returned to normal caging recovered the ability to produce interferon. Antiorthostatic suspension of mice also resulted in a loss of resistance to infection with the diabetogenic strain of encephalomyocarditis virus, which correlated with the drop in interferon production. In rats flown in US Space Shuttle mission SL-3, interferon-gamma production was inhibited severely when spleen cells were challenged with concanavalin-A upon return to earth. In contrast, interleukin-3 production by these cells was normal. These results suggest that immune responses may be altered after antiorthostatic modeling or space flight, and the resistance to viral infections may be especially affected.

  8. Redirecting Specificity of T cells Using the Sleeping Beauty System to Express Chimeric Antigen Receptors by Mix-and-Matching of VL and VH Domains Targeting CD123+ Tumors.

    Directory of Open Access Journals (Sweden)

    Radhika Thokala

    Full Text Available Adoptive immunotherapy infusing T cells with engineered specificity for CD19 expressed on B- cell malignancies is generating enthusiasm to extend this approach to other hematological malignancies, such as acute myelogenous leukemia (AML. CD123, or interleukin 3 receptor alpha, is overexpressed on most AML and some lymphoid malignancies, such as acute lymphocytic leukemia (ALL, and has been an effective target for T cells expressing chimeric antigen receptors (CARs. The prototypical CAR encodes a VH and VL from one monoclonal antibody (mAb, coupled to a transmembrane domain and one or more cytoplasmic signaling domains. Previous studies showed that treatment of an experimental AML model with CD123-specific CAR T cells was therapeutic, but at the cost of impaired myelopoiesis, highlighting the need for systems to define the antigen threshold for CAR recognition. Here, we show that CARs can be engineered using VH and VL chains derived from different CD123-specific mAbs to generate a panel of CAR+ T cells. While all CARs exhibited specificity to CD123, one VH and VL combination had reduced lysis of normal hematopoietic stem cells. This CAR's in vivo anti-tumor activity was similar whether signaling occurred via chimeric CD28 or CD137, prolonging survival in both AML and ALL models. Co-expression of inducible caspase 9 eliminated CAR+ T cells. These data help support the use of CD123-specific CARs for treatment of CD123+ hematologic malignancies.

  9. Dexamethasone Effects in the Strongyloides venezuelensis Infection in A Murine Model

    Science.gov (United States)

    Machado, Eleuza R.; Carlos, Daniela; Sorgi, Carlos A.; Ramos, Simone G.; Souza, Daniela I.; Soares, Edson G.; Costa-Cruz, Julia M.; Ueta, Marlene T.; Aronoff, David M.; Faccioli, Lúcia H.

    2011-01-01

    The aim of this study was to investigate the immunomodulatory effects of glucocorticoids on the immune response to Strongyloides venezuelensis in mice. Balb/c mice were infected with S. venezuelensis and treated with Dexamethasone (Dexa) or vehicle. Dexa treatment increased circulating blood neutrophil numbers and inhibited eosinophil and mononuclear cell accumulation in the blood, bronchoalveolar, and peritoneal fluid compared with control animals. Moreover, Dexa decreased tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-3 (IL-3), IL-4, IL-5, IL-10, and IL-12 production in the lungs and circulating immunoglobulin G1 (IgG1), IgG2a, and IgE antibody levels while increasing the overall parasite burden in the feces and intestine. Dexa treatment enhanced the fertility of female nematodes relative to untreated and infected mice. In summary, the alterations in the immune response induced by Dexa resulted in a blunted, aberrant immune response associated with increased parasite burden. This phenomenon is similar to that observed in S. stercoralis-infected humans who are taking immunosuppressive or antiinflammatory drugs, including corticosteroids. PMID:21633034

  10. A dual-color fluorescence-based platform to identify selective inhibitors of Akt signaling.

    Directory of Open Access Journals (Sweden)

    Aranzazú Rosado

    Full Text Available BACKGROUND: Inhibition of Akt signaling is considered one of the most promising therapeutic strategies for many cancers. However, rational target-orientated approaches to cell based drug screens for anti-cancer agents have historically been compromised by the notorious absence of suitable control cells. METHODOLOGY/PRINCIPAL FINDINGS: In order to address this fundamental problem, we have developed BaFiso, a live-cell screening platform to identify specific inhibitors of this pathway. BaFiso relies on the co-culture of isogenic cell lines that have been engineered to sustain interleukin-3 independent survival of the parental Ba/F3 cells, and that are individually tagged with different fluorescent proteins. Whilst in the first of these two lines cell survival in the absence of IL-3 is dependent on the expression of activated Akt, the cells expressing constitutively-activated Stat5 signaling display IL-3 independent growth and survival in an Akt-independent manner. Small molecules can then be screened in these lines to identify inhibitors that rescue IL-3 dependence. CONCLUSIONS/SIGNIFICANCE: BaFiso measures differential cell survival using multiparametric live cell imaging and permits selective inhibitors of Akt signaling to be identified. BaFiso is a platform technology suitable for the identification of small molecule inhibitors of IL-3 mediated survival signaling.

  11. Exploring the use of expanded erythroid cells for autologous transfusion for anemia of prematurity.

    Science.gov (United States)

    Khodabux, Chantal M; van Hensbergen, Yvette; Slot, Manon C; Bakker-Verweij, Margreet; Giordano, Piero C; Brand, Anneke

    2013-12-01

    Autologous cord blood (CB) red blood cells (RBCs) can partly substitute transfusion needs in premature infants suffering from anemia. To explore whether expanded CB cells could provide additional autologous cells suitable for transfusion, we set up a simple one-step protocol to expand premature CB cells. CB buffy coat cells and isolated CD34-positive (CD34(pos) ) cells from premature and full-term CB and adult blood were tested with several combinations of growth factors while omitting xenogeneic proteins from the culture medium. Cell differentiation was analyzed serially during 21 days using flow cytometry, progenitor assays, and high-performance liquid chromatography. Expanded CB buffy coat cells resulted in a threefold higher number of erythroblasts than the isolated CD34(pos) cells. However, the RBCs contaminating the buffy coat remained present during the culture with uncertain quality. Premature and full-term CB CD34(pos) cells had similar fold expansion capacity and erythroid differentiation. With the use of interleukin-3, stem cell factor, and erythropoietin, the fold increases of all CD34(pos) cell sources were similar: CB 3942 ± 1554, adult peripheral mobilized blood 4702 ± 1826, and bone marrow (BM) 4143 ± 1908. The proportion of CD235a expression indicating erythroblast presence on Day 21 was slightly higher in the adult CD34(pos) cell sources: peripheral blood stem cells (96.7 ± 0.8%) and BM (98.9 ± 0.5%) compared to CB (87.7 ± 2.7%; p = 0.002). We were not able to induce further erythroid maturation in vitro. This explorative study showed that fairly pure autologous erythroid-expanded cell populations could be obtained by a simple culture method, which should be optimized. Future challenges comprise obtaining ex vivo enucleation of RBCs with the use of a minimal manipulating approach, which can add up to autologous RBCs derived from CB in the treatment of anemia of prematurity. © 2013 American Association of Blood Banks.

  12. Transcription factor RBP-J-mediated signalling regulates basophil immunoregulatory function in mouse asthma model.

    Science.gov (United States)

    Qu, Shuo-Yao; He, Ya-Long; Zhang, Jian; Wu, Chang-Gui

    2017-09-01

    Basophils (BA) play an important role in the promotion of aberrant T helper type 2 (Th2) immune responses in asthma. It is not only the effective cell, but also modulates the initiation of Th2 immune responses. We earlier demonstrated that Notch signalling regulates the biological function of BAin vitro. However, whether this pathway plays the same role in vivo is not clear. The purpose of the present study was to investigate the effect of Notch signalling on BA function in the regulation of allergic airway inflammation in a murine model of asthma. Bone marrow BA were prepared by bone marrow cell culture in the presence of recombinant interleukin-3 (rIL-3; 300 pg/ml) for 7 days, followed by isolation of the CD49b + microbeads. The recombination signal binding protein J (RBP-J -/- ) BA were co-cultured with T cells, and the supernatant and the T-cell subtypes were examined. The results indicated disruption of the capacity of BA for antigen presentation alongside an up-regulation of the immunoregulatory function. This was possibly due to the low expression of OX40L in the RBP-J -/- BA. Basophils were adoptively transferred to ovalbumin-sensitized recipient mice, to establish an asthma model. Lung pathology, cytokine profiles of brobchoalveolar fluid, airway hyperactivity and the absolute number of Th1/Th2 cells in lungs were determined. Overall, our results indicate that the RBP-J-mediated Notch signalling is critical for BA-dependent immunoregulation. Deficiency of RBP-J influences the immunoregulatory functions of BA, which include activation of T cells and their differentiation into T helper cell subtypes. The Notch signalling pathway is a potential therapeutic target for BA-based immunotherapy against asthma. © 2017 John Wiley & Sons Ltd.

  13. Strategy to confirm the presence of anti-erythropoietin neutralizing antibodies in human serum.

    Science.gov (United States)

    Sanchez, Sergio; Barger, Troy; Zhou, Lei; Hale, Michael; Mytych, Daniel; Gupta, Shalini; Swanson, Steven J; Civoli, Francesca

    2011-07-15

    Functional cell-based assays are the preferred method to test for the presence of anti-rHuEPO neutralizing antibodies (NAbs). However, due to the unpredictable nature of test serum matrix effects on cell-based assays, confirmatory assays are essential for verifying NAb positive results observed during the course of sample testing. The cell-based assay used for the detection of NAbs described by Wei et al. [1] used 32D-EPOR cells, a murine myeloid cell line transfected with the human EPO receptor (EPOR). The 32D-EPOR cell line responded to either rHuEPO or murine interleukin 3 (mIL-3) with proliferation. NAbs were expected to only inhibit rHuEPO-induced cell proliferation and not mIL-3 induced proliferation. Due to reliance on proliferation, the results from this cell-based assay can be confounded by the presence of non-antibody inhibitory serum factors. This paper describes a strategy for confirming that the inhibition of rHuEPO-induced proliferation in a cell-based assay is only attributable to NAbs. The strategy of antibody depletion uses a resin mixture composed of Protein G Sepharose and Protein L Sepharose (Protein G/L resin) to significantly reduce the concentration of immunoglobulins of IgG, IgM and IgA isotypes from human serum prior to testing in the cell-based assay. If the reduction in immunoglobulins in a serum sample corresponds to a reduction in inhibition of EPO-induced proliferation, it would infer that EPO neutralizing activity is antibody-mediated and not due to non-antibody inhibitory serum factors. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. c-Kit-Positive Adipose Tissue-Derived Mesenchymal Stem Cells Promote the Growth and Angiogenesis of Breast Cancer

    Directory of Open Access Journals (Sweden)

    Wenjie Li

    2017-01-01

    Full Text Available Background. Adipose tissue-derived mesenchymal stem cells (ASCs improve the regenerative ability and retention of fat grafts for breast reconstruction in cancer patients following mastectomy. However, ASCs have also been shown to promote breast cancer cell growth and metastasis. For the safety of ASC application, we aimed to identify specific markers for the subpopulation of ASCs that enhance the growth of breast cancer. Methods. ASCs and bone marrow-derived vascular endothelial progenitor cells (EPCs were isolated from Balb/c mice. c-Kit-positive (c-Kit+ or c-Kit-negative (c-Kit- ASCs were cocultured with 4T1 breast cancer cells. Orthotropic murine models of 4T1, EPCs + 4T1, and c-Kit+/-ASCs + 4T1/EPCs were established in Balb/c mice. Results. In coculture, c-Kit+ ASCs enhanced the viability and proliferation of 4T1 cells and stimulated c-Kit expression and interleukin-3 (IL-3 release. In mouse models, c-Kit+ASCs + 4T1/EPCs coinjection increased the tumor volume and vessel formation. Moreover, IL-3, stromal cell-derived factor-1, and vascular endothelial growth factor A in the c-Kit+ASCs + 4T1/EPCs coinjection group were higher than those in the 4T1, EPCs + 4T1, and c-Kit-ASCs + 4T1/EPCs groups. Conclusions. c-Kit+ ASCs may promote breast cancer growth and angiogenesis by a synergistic effect of c-Kit and IL-3. Our findings suggest that c-Kit+ subpopulations of ASCs should be eliminated in fat grafts for breast reconstruction of cancer patients following mastectomy.

  15. The leukemogenic transcription factor E2a-Pbx1 induces expression of the putative N-myc and p53 target gene NDRG1 in Ba/F3 cells.

    Science.gov (United States)

    Rutherford, M N; Bayly, G R; Matthews, B P; Okuda, T; Dinjens, W M; Kondoh, H; LeBrun, D P

    2001-03-01

    The chimeric transcription factor E2a-Pbx1 is expressed as a result of the 1;19 chromosomal translocation in some 5% of cases of pediatric acute lymphoblastic leukemia. We investigated the biological and transcriptional consequences of forced expression of E2a-Pbx1 in the interleukin-3 (IL-3) dependent, bone marrow-derived cell line Ba/F3. We show that forced expression of E2a-Pbx1 induces apoptosis in Ba/F3 cells without apparent effects on cell cycle progression. This pro-apoptotic effect is enhanced on cytokine deprivation. Furthermore, using cDNA representational difference analysis (RDA), we show that these cellular effects are associated with marked induction of the gene NDRG1, which was previously identified as a target of transcriptional repression by N-myc and induction by the tumor suppressor protein p53. We identify a portion of the NDRG1 promoter capable of mediating transcriptional induction by E2a-Pbx1 and show that NDRG1 is also induced on simple IL-3 deprivation of BaF3 cells. Although we show that E2a-Pbx1 induction of NDRG1 is not impaired as a result of targeting p53 using HPV E6, and therefore does not appear to be p53-dependent, our results overall are consistent with the notion that induction of NDRG1 by E2a-Pbx1 may represent part of an apoptotic or cytostatic cellular response to oncogene activation.

  16. Hematopoietic stem cell cytokines and fibroblast growth factor-2 stimulate human endothelial cell-pericyte tube co-assembly in 3D fibrin matrices under serum-free defined conditions.

    Directory of Open Access Journals (Sweden)

    Annie O Smith

    Full Text Available We describe a novel 3D fibrin matrix model using recombinant hematopoietic stem cell cytokines under serum-free defined conditions which promotes the assembly of human endothelial cell (EC tubes with co-associated pericytes. Individual ECs and pericytes are randomly mixed together and EC tubes form that is accompanied by pericyte recruitment to the EC tube abluminal surface over a 3-5 day period. These morphogenic processes are stimulated by a combination of the hematopoietic stem cell cytokines, stem cell factor, interleukin-3, stromal derived factor-1α, and Flt-3 ligand which are added in conjunction with fibroblast growth factor (FGF-2 into the fibrin matrix. In contrast, this tube morphogenic response does not occur under serum-free defined conditions when VEGF and FGF-2 are added together in the fibrin matrices. We recently demonstrated that VEGF and FGF-2 are able to prime EC tube morphogenic responses (i.e. added overnight prior to the morphogenic assay to hematopoietic stem cell cytokines in collagen matrices and, interestingly, they also prime EC tube morphogenesis in 3D fibrin matrices. EC-pericyte interactions in 3D fibrin matrices leads to marked vascular basement membrane assembly as demonstrated using immunofluorescence and transmission electron microscopy. Furthermore, we show that hematopoietic stem cell cytokines and pericytes stimulate EC sprouting in fibrin matrices in a manner dependent on the α5β1 integrin. This novel co-culture system, under serum-free defined conditions, allows for a molecular analysis of EC tube assembly, pericyte recruitment and maturation events in a critical ECM environment (i.e. fibrin matrices that regulates angiogenic events in postnatal life.

  17. Bone marrow-derived cultured mast cells and peritoneal mast cells as targets of a growth activity secreted by BALB/3T3 fibroblasts

    International Nuclear Information System (INIS)

    Jozaki, K.; Kuriu, A.; Hirota, S.; Onoue, H.; Ebi, Y.; Adachi, S.; Ma, J.Y.; Tarui, S.; Kitamura, Y.

    1991-01-01

    When fibroblast cell lines were cultured in contact with bone marrow-derived cultured mast cells (CMC), both NIH/3T3 and BALB/3T3 cell lines supported the proliferation of CMC. In contrast, when contact between fibroblasts and CMC was prohibited by Biopore membranes or soft agar, only BALB/3T3 fibroblasts supported CMC proliferation, suggesting that BALB/3T3 but not NIH/3T3 cells secreted a significant amount of a mast cell growth activity. Moreover, the BALB/3T3-derived growth activity induced the incorporation of [3H]thymidine by CMC and the clonal growth of peritoneal mast cells in methylcellulose. The mast cell growth activity appeared to be different from interleukin 3 (IL-3) and interleukin 4 (IL-4), because mRNAs for these interleukins were not detectable in BALB/3T3 fibroblasts. Although mast cells are genetically deficient in tissues of W/Wv mice, CMC did develop when bone marrow cells of W/Wv mice were cultured with pokeweed mitogen-stimulated spleen cell-conditioned medium. Because BALB/3T3 fibroblast-conditioned medium (BALB-FCM) did not induce the incorporation of [3H]thymidine by W/Wv CMC, the growth activity in BALB-FCM appeared to be a ligand for the receptor encoded by the W (c-kit) locus. Because CMC and peritoneal mast cells are obtained as homogeneous suspensions rather easily, these cells may be potentially useful as targets for the fibroblast-derived mast cell growth activity

  18. Macrophage colony-stimulating factor, CSF-1, and its proto-oncogene-encoded receptor

    International Nuclear Information System (INIS)

    Sherr, C.J.; Rettenmier, C.W.; Roussel, M.F.

    1988-01-01

    The macrophage colony-stimulating factor, CSF-1, or M-CSF, is one of a family of hematopoietic growth factors that stimulates the proliferation of monocytes, macrophages, and their committed bone marrow progenitors. Unlike pluripotent hemopoietins such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3 or multi-CSF), which affect the growth of myeloid cells of several different hematopoietic lineages, CSF-1 acts only on cells of the mononuclear phagocyte series to stimulate their growth and enhance their survival. Retroviral transduction of the feline c-fms gene in the Susan McDonough and Hardy Zuckerman-5 (HZ-5) strains of feline sarcoma virus (FeSV) led to genetic alterations that endowed the recombined viral oncogene (v-fms) with the ability to transform cells in culture morphologically and to induce firbrosarcomas and hematopoietic neoplasms in susceptible animals. The v-fms oncogene product differs from the normal CSF-1 receptor in certain of its cardinal biochemical properties, most notably in exhibiting constitutively high basal levels of tyrosine kinase activity in the absence of its ligand. Comparative studies of the c-fms and v-fms genes coupled with analyses of engineered mutants and receptor chimeras have begun to pinpoint pertinent genetic alterations in the normal receptor gene that unmask its latent oncogenic potential. In addition, the availability of biologically active c-fms, v-fms, and CSF-1 cDNAs has allowed these genes to be mobilized and expressed in naive cells, thereby facilitating assays for receptor coupling with downstream components of the mitogenic pathway in diverse cell types

  19. Eosinophilia in a patient with cyclical vomiting: a case report

    Directory of Open Access Journals (Sweden)

    Fitzgerald S Matthew

    2004-05-01

    Full Text Available Abstract Background Eosinophilic gastritis is related to eosinophilic gastroenteritis, varying only in regards to the extent of disease and small bowel involvement. Common symptoms reported are similar to our patient's including: abdominal pain, epigastric pain, anorexia, bloating, weight loss, diarrhea, ankle edema, dysphagia, melaena and postprandial nausea and vomiting. Microscopic features of eosinophilic infiltration usually occur in the lamina propria or submucosa with perivascular aggregates. The disease is likely mediated by eosinophils activated by various cytokines and chemokines. Therapy centers around the use of immunosuppressive agents and dietary therapy if food allergy is a factor. Case presentation The patient is a 31 year old Caucasian female with a past medical history significant for ulcerative colitis. She presented with recurrent bouts of vomiting, abdominal pain and chest discomfort of 11 months duration. The bouts of vomiting had been reoccurring every 7–10 days, with each episode lasting for 1–3 days. This was associated with extreme weakness and cachexia. Gastric biopsies revealed intense eosinophilic infiltration. The patient responded to glucocorticoids and azathioprine. The differential diagnosis and molecular pathogenesis of eosinophilic gastritis as well as the molecular effects of glucocorticoids in eosinophilic disorders are discussed. Conclusions The patient responded to a combination of glucocorticosteroids and azathioprine with decreased eosinophilia and symptoms. It is likely that eosinophil-active cytokines such as interleukin-3 (IL-3, granulocyte macrophage colony stimulating factor (GM-CSF and IL-5 play pivotal roles in this disease. Chemokines such as eotaxin may be involved in eosinophil recruitment. These mediators are downregulated or inhibited by the use of immunosuppressive medications.

  20. Neutrophil-induced transmigration of tumour cells treated with tumour-conditioned medium is facilitated by granulocyte-macrophage colony-stimulating factor.

    LENUS (Irish Health Repository)

    Wu, Q D

    2012-02-03

    OBJECTIVE: To investigate the effect of different cytokines that are present in tumour-conditioned medium on human neutrophil (PMN)-induced tumour cell transmigration. DESIGN: Laboratory study. SETTING: University hospital, Ireland. MATERIAL: Isolated human PMN and cultured human breast tumour cell line, MDA-MB-231. Interventions: Human PMN treated with either tumour-conditioned medium or different media neutralised with monoclonal antibodies (MoAb), and MDA-MB-231 cells were plated on macrovascular and microvascular endothelial monolayers in collagen-coated transwells to assess migration of tumour cells. MAIN OUTCOME MEASURES: Cytokines present in tumour-conditioned medium, PMN cytocidal function and receptor expression, and tumour cell transmigration. RESULTS: tumour-conditioned medium contained high concentrations of granulocyte-macrophage colony-stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), and interleukin 8 (IL-8), but not granulocyte colony-stimulating factor (G-CSF) and interleukin 3 (IL-3). Anti-GM-CSF MoAb significantly reduced PMN-induced transmigration of tumour cells treated with tumour-conditioned medium (p < 0.05), whereas anti-VEGF and anti-IL-8 MoAbs did not affect their migration. In addition, anti-GM-CSF MoAb, but not anti-VEGF or anti-IL-8 MoAb, reduced PMN CD11b and CD18 overexpression induced by tumour-conditioned medium (p < 0.05). CONCLUSION: These results indicate that the GM-CSF that is present in tumour-conditioned medium may be involved, at least in part, in alterations in PMN function mediated by the medium and subsequently PMN-induced transmigration of tumour cells.

  1. Effects of combined radiation-burn injury on the serum level of IL-3 in mice

    International Nuclear Information System (INIS)

    He Pei; Ding Zhenhua; Zheng Li; Yang Jun; Luo Chenji

    2001-01-01

    Objective: To explore the relationship between endogenous interleukin-3 and hematopoietic failure caused by combined radiation-burn injury. Methods: Mice were randomized into normal (N), irradiation (R), burn (B) and combined irradiation-burn injury (C) groups. Serum level of IL-3 was detected by ELISA using the Endogenous Mouse IL-3 ELISA Kit at different times after injury. Results: Compared with group N the serum level of IL-3 was significantly decreased in group R, especially on the 3rd and the 5th day (below the detecting sensitivity). It recovered slowly from the 15th day and did not elevate to the normal value on the 28th day. In group B. IL-3 was less than normal value on the 3rd and 5th day, then it increased rapidly and reached the peak (210% of that of the group N) on the 15 th day. In group C, the serum level of IL-3 was higher than that of group R but lower than that of group B. The changes of the peripheral white blood cell in each group were parallel with the changes of IL-3. The serum of burnt mice collected from the 1 st to 11 day showed inhibitory effect on CFU-F, stromal cell adhering rate of bone marrow stromal cells (BMSC), whereas serum harvested from the 13 th to 15 th days showed stimulation effect. Conclusion: The serum of burnt mice collected at different times showing different actions on BMSC, and abnormal WBC caused by combined radiation-burn injury might be related with the changes in endogenous IL-3. The results of this experiment indicates: inhibition of endogenous IL-3 is one of the causes of hematopoietic failure induced by combined radiation-burn injury in mice

  2. Effect of ionizing radiation on hematopoietic stem cells and progenitor cells: Role of apoptosis and potential therapeutic significance of anti-apoptotic treatments; Effet des rayonnements ionisants sur les cellules souches et progeniteurs hematopoietiques : place de l'apoptose et interet therapeutique potentiel des traitements antiapoptotiques

    Energy Technology Data Exchange (ETDEWEB)

    Drouet, M.; Mourcin, F.; Grenier, N.; Mayol, J.F.; Leroux, V. [Unite de Radiohematologie experimentale, Centre de Recherches du Service de Sante des Armees, La Tronche CEDEX (France); Sotto, J.J. [Inst. Albert Bonniot, La Tronche (France); Herodin, F. [Unite de Radiohematologie experimentale, Centre de Recherches du Service de Sante des Armees, La Tronche CEDEX (France)

    2002-07-01

    Bone marrow aplasia observed following ionizing radiation exposure (Total Body Irradiation; gamma dose range: 2-10 Gy) is a result, in particular, of the radiation-induced (RI) apoptosis in hematopoietic stem and progenitor cells (HSPC). We have previously shown in a baboon model of mobilized peripheral blood CD34{sup +} cell irradiation in vitro that RI apoptosis in HSPC was an early event, mostly occurring within the first 24 hours, which involves the CD95 Fas pathway. Apoptosis may be significantly reduced with a combination of 4 cytokines (4F): Stem Cell Factor (SCF), FLT-3 Ligand (FL), thrombopoietin (TPO), and interleukin-3 (IL-3), each at 50 ng{center_dot}mL{sup -1} (15% survival versus <3% untreated cells, 24 h post-irradiation at 2.5 Gy). In this study we show that addition of TNF-{alpha}(800 IU/ml) induces an increase in 4F efficacy in terms of cell survival 24 h after incubation (26% survival after 24 h irradiation exposure at 2.5 Gy) and amplification (k) of CD34{sup +} cells after 6 days in a serum free culture medium (SFM) (k{sub CD34{sup +}} = 4.3 and 6.3 respectively for 4F and successive 4F + TNF-{alpha}/4F treatments). In addition, the 4F combination allows culture on pre-established allogenic irradiated stromal cells in vitro at 4 Gy (k{sub CD34{sup +}} = 4.5). Overall this study suggests (i) the potential therapeutic interest for an early administration of anti-apoptotic cytokines with or without hematopoiesis inhibitors (emergency cytokine therapy) and (ii) the feasibility in the accidentally irradiated individual, of autologous cell therapy based on ex vivo expansion in order to perform autograft of residual HSPC collected after the accident. (author)

  3. Basophil markers for identification and activation in the indirect basophil activation test by flow cytometry for diagnosis of autoimmune urticaria.

    Science.gov (United States)

    Kim, Zehwan; Choi, Bong Seok; Kim, Jong Kun; Won, Dong Il

    2016-01-01

    The indirect basophil activation test using flow cytometry is a promising tool for autoimmune urticaria diagnosis. We aimed to identify better donor basophils (from atopic vs. non-atopic donors and interleukin-3 primed vs. unprimed basophils) and improve basophil identification and activation markers (eotaxin CC chemokine receptor-3 [CCR3] vs. CD123 and CD63 vs. CD203c). Donor basophils were obtained from non-atopic and atopic group O donors. Positive control sera were artificially prepared to simulate autoimmune urticaria patients' sera. Patient sera were obtained from nine children with chronic urticaria. Assay sensitivity was compared among each variation by using positive control sera (n=21), applying cutoff values defined from negative control sera (n=20). For basophil identification, a combination of CCR3 and CD123 markers revealed a higher correlation with automated complete blood count (r=0.530) compared with that observed using CD123 (r=0.498) or CCR3 alone (r=0.195). Three activation markers on the atopic donor basophils attained 100% assay sensitivity: CD203c on unprimed basophils, CD63+CD203+ or CD63 alone on primed basophils; however, these markers on the non-atopic donor basophils attained lower assay sensitivity. For basophil identification markers, a combination of CD123 and CCR3 is recommended, while CD123 alone may be used as an alternative. Donor basophils should be obtained from an atopic donor. For basophil activation markers, either CD203c alone on unprimed basophils or CD203c and CD63 on primed basophils are recommended, while CD63 alone on primed basophils may be used as an alternative.

  4. Genetically engineered mesenchymal stromal cells produce IL-3 and TPO to further improve human scaffold-based xenograft models.

    Science.gov (United States)

    Carretta, Marco; de Boer, Bauke; Jaques, Jenny; Antonelli, Antonella; Horton, Sarah J; Yuan, Huipin; de Bruijn, Joost D; Groen, Richard W J; Vellenga, Edo; Schuringa, Jan Jacob

    2017-07-01

    Recently, NOD-SCID IL2Rγ -/- (NSG) mice were implanted with human mesenchymal stromal cells (MSCs) in the presence of ceramic scaffolds or Matrigel to mimic the human bone marrow (BM) microenvironment. This approach allowed the engraftment of leukemic samples that failed to engraft in NSG mice without humanized niches and resulted in a better preservation of leukemic stem cell self-renewal properties. To further improve our humanized niche scaffold model, we genetically engineered human MSCs to secrete human interleukin-3 (IL-3) and thrombopoietin (TPO). In vitro, these IL-3- and TPO-producing MSCs were superior in expanding human cord blood (CB) CD34 + hematopoietic stem/progenitor cells. MLL-AF9-transduced CB CD34 + cells could be transformed efficiently along myeloid or lymphoid lineages on IL-3- and TPO-producing MSCs. In vivo, these genetically engineered MSCs maintained their ability to differentiate into bone, adipocytes, and other stromal components. Upon transplantation of MLL-AF9-transduced CB CD34 + cells, acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) developed in engineered scaffolds, in which a significantly higher percentage of myeloid clones was observed in the mouse compartments compared with previous models. Engraftment of primary AML, B-cell ALL, and biphenotypic acute leukemia (BAL) patient samples was also evaluated, and all patient samples could engraft efficiently; the myeloid compartment of the BAL samples was better preserved in the human cytokine scaffold model. In conclusion, we show that we can genetically engineer the ectopic human BM microenvironment in a humanized scaffold xenograft model. This approach will be useful for functional study of the importance of niche factors in normal and malignant human hematopoiesis. Copyright © 2017 ISEH - International Society for Experimental Hematology. All rights reserved.

  5. In vitro validation of bioluminescent monitoring of disease progression and therapeutic response in leukaemia model animals

    International Nuclear Information System (INIS)

    Inoue, Yusuke; Okubo, Toshiyuki; Tojo, Arinobu; Sekine, Rieko; Soda, Yasushi; Kobayashi, Seiichiro; Nomura, Akiko; Izawa, Kiyoko; Kitamura, Toshio; Ohtomo, Kuni

    2006-01-01

    The application of in vivo bioluminescence imaging to non-invasive, quantitative monitoring of tumour models relies on a positive correlation between the intensity of bioluminescence and the tumour burden. We conducted cell culture studies to investigate the relationship between bioluminescent signal intensity and viable cell numbers in murine leukaemia model cells. Interleukin-3 (IL-3)-dependent murine pro-B cell line Ba/F3 was transduced with firefly luciferase to generate cells expressing luciferase stably under the control of a retroviral long terminal repeat. The luciferase-expressing cells were transduced with p190 BCR-ABL to give factor-independent proliferation. The cells were cultured under various conditions, and bioluminescent signal intensity was compared with viable cell numbers and the cell cycle stage. The Ba/F3 cells showed autonomous growth as well as stable luciferase expression following transduction with both luciferase and p190 BCR-ABL, and in vivo bioluminescence imaging permitted external detection of these cells implanted into mice. The bioluminescence intensities tended to reflect cell proliferation and responses to imatinib in cell culture studies. However, the luminescence per viable cell was influenced by the IL-3 concentration in factor-dependent cells and by the stage of proliferation and imatinib concentration in factor-independent cells, thereby impairing the proportionality between viable cell number and bioluminescent signal intensity. Luminescence per cell tended to vary in association with the fraction of proliferating cells. Although in vivo bioluminescence imaging would allow non-invasive monitoring of leukaemia model animals, environmental factors and therapeutic interventions may cause some discrepancies between tumour burden and bioluminescence intensity. (orig.)

  6. In vitro validation of bioluminescent monitoring of disease progression and therapeutic response in leukaemia model animals

    Energy Technology Data Exchange (ETDEWEB)

    Inoue, Yusuke; Okubo, Toshiyuki [University of Tokyo, Department of Radiology, Institute of Medical Science, Tokyo (Japan); Tojo, Arinobu; Sekine, Rieko; Soda, Yasushi; Kobayashi, Seiichiro; Nomura, Akiko; Izawa, Kiyoko [University of Tokyo, Division of Molecular Therapy, Advanced Clinical Research Centre, Tokyo (Japan); Kitamura, Toshio [University of Tokyo, Division of Cellular Therapy, Advanced Clinical Research Centre, Tokyo (Japan); Ohtomo, Kuni [University of Tokyo, Department of Radiology, Graduate School of Medicine, Tokyo (Japan)

    2006-05-15

    The application of in vivo bioluminescence imaging to non-invasive, quantitative monitoring of tumour models relies on a positive correlation between the intensity of bioluminescence and the tumour burden. We conducted cell culture studies to investigate the relationship between bioluminescent signal intensity and viable cell numbers in murine leukaemia model cells. Interleukin-3 (IL-3)-dependent murine pro-B cell line Ba/F3 was transduced with firefly luciferase to generate cells expressing luciferase stably under the control of a retroviral long terminal repeat. The luciferase-expressing cells were transduced with p190 BCR-ABL to give factor-independent proliferation. The cells were cultured under various conditions, and bioluminescent signal intensity was compared with viable cell numbers and the cell cycle stage. The Ba/F3 cells showed autonomous growth as well as stable luciferase expression following transduction with both luciferase and p190 BCR-ABL, and in vivo bioluminescence imaging permitted external detection of these cells implanted into mice. The bioluminescence intensities tended to reflect cell proliferation and responses to imatinib in cell culture studies. However, the luminescence per viable cell was influenced by the IL-3 concentration in factor-dependent cells and by the stage of proliferation and imatinib concentration in factor-independent cells, thereby impairing the proportionality between viable cell number and bioluminescent signal intensity. Luminescence per cell tended to vary in association with the fraction of proliferating cells. Although in vivo bioluminescence imaging would allow non-invasive monitoring of leukaemia model animals, environmental factors and therapeutic interventions may cause some discrepancies between tumour burden and bioluminescence intensity. (orig.)

  7. NFIL3 is a negative regulator of hepatic gluconeogenesis.

    Science.gov (United States)

    Kang, Geon; Han, Hye-Sook; Koo, Seung-Hoi

    2017-12-01

    Nuclear factor interleukin-3 regulated (NFIL3) has been known as an important transcriptional regulator of the development and the differentiation of immune cells. Although expression of NFIL3 is regulated by nutritional cues in the liver, the role of NFIL3 in the glucose metabolism has not been extensively studied. Thus, we wanted to explore the potential role of NFIL3 in the control of hepatic glucose metabolism. Mouse primary hepatocytes were cultured to perform western blot analysis, Q-PCR and chromatin immunoprecipitation assay. 293T cells were cultured to perform luciferase assay. Male C57BL/6 mice (fed a normal chow diet or high fat diet for 27weeks) as well as ob/ob mice were used for experiments with adenoviral delivery. We observed that NFIL3 reduced glucose production in hepatocytes by reducing expression of gluconeogenic gene transcription. The repression by NFIL3 required its basic leucine zipper DNA binding domain, and it competed with CREB onto the binding of cAMP response element in the gluconeogenic promoters. The protein levels of hepatic NFIL3 were decreased in the mouse models of genetic- and diet-induced obesity and insulin resistance, and ectopic expression of NFIL3 in the livers of insulin resistant mice ameliorated hyperglycemia and glucose intolerance, with concomitant reduction in expression of hepatic gluconeogenic genes. Finally, we witnessed that knockdown of NFIL3 in the livers of normal chow-fed mice promoted elevations in the glucose levels and expression of hepatic gluconeogenic genes. In this study, we showed that NFIL3 functions as an important regulator of glucose homeostasis in the liver by limiting CREB-mediated hepatic gluconeogenesis. Thus, enhancement of hepatic NFIL3 activity in insulin resistant state could be potentially beneficial in relieving glycemic symptoms in the metabolic diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. The effects of X-irradiation on ex vivo expansion of cryopreserved human hematopoietic stem/progenitor cells

    International Nuclear Information System (INIS)

    Hayashi, Naoki; Takahashi, Kenji; Kashiwakura, Ikuo

    2010-01-01

    In our previous study (Life Sciences 84: 598, 2009), we demonstrated that placental/umbilical cord blood-derived mesenchymal stem cell-like stromal cells have the effect to support the regeneration of freshly prepared X-irradiated hematopoietic stem/progenitor cells (HSPCs). Generally, HSPCs are supplied from companies, institutions, and cell banks that cryopreserve them for clinical and experimental use. In this study, the influence of cryopreservation on the responses of HSPCs to irradiation and co-culture with stromal cells is assessed. After cryopreservation with the optimal procedure, 2 Gy-irradiated HSPCs were cultured with or without stromal cells supplemented with combination of interleukin-3, stem cell factor, and thrombopoietin. The population of relatively immature CD34 + /CD38 - cells in cryopreserved cells was significantly higher than in fresh cells prior to cryopreservation; furthermore, the hematopoietic progenitor populations of CD34 + /CD45RA + cells and CD34 + /CD117 + cells in cryopreserved cells were significantly lower than that in fresh cells. However, the rate of expansion in the cryopreserved HSPCs was lower than in the fresh HSPCs. In the culture of cryopreserved cells irradiated with 2 Gy, the growth rates of CD34 + cells, CD34 + /CD38 - cells, and hematopoietic progenitors were greater than growth rates of their counter parts in the culture of fresh cells. Surprisingly, the effect to support the hematopoiesis in co-culture with stromal cells was never observed in the X-irradiated HSPCs after cryopreservation. The present results demonstrated that cryopreserving process increased the rate of immature and radio-resistant HSPCs but decreased the effects to support the hematopoiesis by stromal cells, thus suggesting that cryopreservation changes the character of HSPCs. (author)

  9. Further phenotypic characterization of the primitive lineage− CD34+CD38−CD90+CD45RA− hematopoietic stem cell/progenitor cell sub-population isolated from cord blood, mobilized peripheral blood and patients with chronic myelogenous leukemia

    International Nuclear Information System (INIS)

    Wisniewski, D; Affer, M; Willshire, J; Clarkson, B

    2011-01-01

    The most primitive hematopoietic stem cell (HSC)/progenitor cell (PC) population reported to date is characterized as being Lin−CD34+CD38−CD90+CD45R. We have a long-standing interest in comparing the characteristics of hematopoietic progenitor cell populations enriched from normal subjects and patients with chronic myelogenous leukemia (CML). In order to investigate further purification of HSCs and for potential targetable differences between the very primitive normal and CML stem/PCs, we have phenotypically compared the normal and CML Lin−CD34+CD38−CD90+CD45RA− HSC/PC populations. The additional antigens analyzed were HLA-DR, the receptor tyrosine kinases c-kit and Tie2, the interleukin-3 cytokine receptor, CD33 and the activation antigen CD69, the latter of which was recently reported to be selectively elevated in cell lines expressing the Bcr-Abl tyrosine kinase. Notably, we found a strikingly low percentage of cells from the HSC/PC sub-population isolated from CML patients that were found to express the c-kit receptor (<1%) compared with the percentages of HSC/PCs expressing the c-kitR isolated from umbilical cord blood (50%) and mobilized peripheral blood (10%). Surprisingly, Tie2 receptor expression within the HSC/PC subset was extremely low from both normal and CML samples. Using in vivo transplantation studies, we provide evidence that HLA-DR, c-kitR, Tie2 and IL-3R may not be suitable markers for further partitioning of HSCs from the Lin−CD34+CD38−CD90+CD45RA− sub-population

  10. Plasma based markers of [11C] PiB-PET brain amyloid burden.

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    Steven John Kiddle

    Full Text Available Changes in brain amyloid burden have been shown to relate to Alzheimer's disease pathology, and are believed to precede the development of cognitive decline. There is thus a need for inexpensive and non-invasive screening methods that are able to accurately estimate brain amyloid burden as a marker of Alzheimer's disease. One potential method would involve using demographic information and measurements on plasma samples to establish biomarkers of brain amyloid burden; in this study data from the Alzheimer's Disease Neuroimaging Initiative was used to explore this possibility. Sixteen of the analytes on the Rules Based Medicine Human Discovery Multi-Analyte Profile 1.0 panel were found to associate with [(11C]-PiB PET measurements. Some of these markers of brain amyloid burden were also found to associate with other AD related phenotypes. Thirteen of these markers of brain amyloid burden--c-peptide, fibrinogen, alpha-1-antitrypsin, pancreatic polypeptide, complement C3, vitronectin, cortisol, AXL receptor kinase, interleukin-3, interleukin-13, matrix metalloproteinase-9 total, apolipoprotein E and immunoglobulin E--were used along with co-variates in multiple linear regression, and were shown by cross-validation to explain >30% of the variance of brain amyloid burden. When a threshold was used to classify subjects as PiB positive, the regression model was found to predict actual PiB positive individuals with a sensitivity of 0.918 and a specificity of 0.545. The number of APOE [Symbol: see text] 4 alleles and plasma apolipoprotein E level were found to contribute most to this model, and the relationship between these variables and brain amyloid burden was explored.

  11. Antisense myb inhibition of purified erythroid progenitors in development and differentiation is linked to cycling activity and expression of DNA polymerase alpha

    International Nuclear Information System (INIS)

    Valtieri, M.; Venturelli, D.; Care, A.; Fossati, C.; Pelosi, E.; Labbaye, C.; Mattia, G.; Gewirtz, A.M.; Calabretta, B.; Peschle, C.

    1991-01-01

    These studies aimed to determine the expression and functional role of c-myb in erythroid progenitors with different cycling activities. In the first series of experiments the erythroid burst-forming unit (BFU-E) and colony-forming unit (CFU-E) populations from adult peripheral blood (PB), bone marrow (BM), and embryonic-fetal liver (FL) were treated with either c-myb antisense oligomers or 3H-thymidine (3H-TdR). A direct correlation was always observed between the inhibitory effect of anti-myb oligomers and the level of cycling activity. Thus, the inhibitory effect of antisense c-myb on the number of BFU-E colonies was 28.3% +/- 15.8% in PB, 53.4% +/- 9.3% in BM, and 68.2% +/- 24.5% in FL. Both adult and embryonic CFU-E were markedly inhibited. Using purified PB progenitors, we observed a similar pattern, although with slightly lower inhibitory effects. In the 3H-TdR suicide assay the killing index of BFU-E was 8.9% +/- 4.2% in PB, 29.4% +/- 6.5% in BM, and 40.1% +/- 9.6% in FL. The values for adult and embryonic CFU-E were 55.7% +/- 7.9% and 60.98% +/- 6.6%, respectively. We then investigated the kinetics of c-myb mRNA level during the erythroid differentiation of purified adult PB and FL BFU-E, as evaluated in liquid-phase culture by reverse transcription-polymerase chain reaction. Adult erythroid precursors showed a gradual increase of c-myb mRNA from day 4 through day 8 of culture and a sharp decrease at later times, whereas the expression of c-myb mRNA and protein in differentiation embryonic precursors peaked 2 days earlier. In both cases, c-myb mRNA level peaked at the CFU-E stage of differentiation. Finally, highly purified adult PB BFU-E were stimulated into cycling by a 3-day treatment with interleukin-3 in liquid phase: both the sensitivity to c-myb antisense oligomers and the 3H-TdR suicide index showed a gradual, strictly parallel increase

  12. IL-3R-alpha blockade inhibits tumor endothelial cell-derived extracellular vesicle (EV)-mediated vessel formation by targeting the β-catenin pathway.

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    Lombardo, Giusy; Gili, Maddalena; Grange, Cristina; Cavallari, Claudia; Dentelli, Patrizia; Togliatto, Gabriele; Taverna, Daniela; Camussi, Giovanni; Brizzi, Maria Felice

    2018-03-01

    The proangiogenic cytokine Interleukin-3 (IL-3) is released by inflammatory cells in breast and ovarian cancer tissue microenvironments and also acts as an autocrine factor for human breast and kidney tumor-derived endothelial cells (TECs). We have previously shown that IL-3-treated endothelial cells (ECs) release extracellular vesicles (EVs), which serve as a paracrine mechanism for neighboring ECs, by transferring active molecules. The impact of an anti-IL-3R-alpha blocking antibody on the proangiogenic effect of EVs released from TECs (anti-IL-3R-EVs) has therefore been investigated in this study. We have found that anti-IL-3R-EV treatment prevented neovessel formation and, more importantly, also induced the regression of in vivo TEC-derived neovessels. Two miRs that target the canonical wingless (Wnt)/β-catenin pathway, at different levels, were found to be differentially regulated when comparing the miR-cargo of naive TEC-derived EVs (EVs) and anti-IL-3R-EVs. miR-214-3p, which directly targets β-catenin, was found to be upregulated, whereas miR-24-3p, which targets adenomatous polyposis coli (APC) and glycogen synthase kinase-3β (GSK3β), was found to be downregulated. In fact, upon their transfer into the cell, low β-catenin content and high levels of the two members of the "β-catenin destruction complex" were detected. Moreover, c-myc downregulation was found in TECs treated with anti-IL-3R-EVs, pre-miR-214-3p-EVs and antago-miR-24-3p-EVs, which is consistent with network analyses of miR-214-3p and miR-24-3p gene targeting. Finally, in vivo studies have demonstrated the impaired growth of vessels in pre-miR-214-3p-EV- and antago-miR-24-3p-EV-treated animals. These effects became much more evident when combo treatment was applied. The results of the present study identify the canonical Wnt/β-catenin pathway as a relevant mechanism of TEC-derived EV proangiogenic action. Furthermore, we herein provide evidence that IL-3R blockade may yield some

  13. Short-term effects of early-acting and multilineage hematopoietic growth factors on the repair and proliferation of irradiated pure cord blood (CB) CD34 hematopoietic progenitor cells

    International Nuclear Information System (INIS)

    Ziegler, Benedikt L.; Sandor, Peter S.; Plappert, Ulla; Thoma, Stefan; Mueller, Robert; Bock, Thomas; Thomas, Christian A.; Nothdurft, Wilhelm; Fliedner, Theodor M.

    1998-01-01

    Purpose: Hematopoietic growth factor(s) (GF) may exert positive effects in vitro or in vivo on the survival of hematopoietic stem and progenitor cells after accidental or therapeutic total body irradiation. Methods and Materials: We studied the clonogenic survival and DNA repair of irradiated (0.36, 0.73, and 1.46 Gy) CD34 + cord blood (CB) cells after short-term incubation (24 h) with GFs. CD34 + cells were stimulated with basic fibroblast growth factor (bFGF), stem cell factor/c-kit ligand (SCF), interleukin-3 (IL-3), IL-6, leukemia inhibitory factor (LIF), and granulocyte-monocyte colony stimulating factor (GM-CSF) alone or in combination in short-term serum-free liquid suspension cultures (LSC) immediately after irradiation and then assayed for clonogenic progenitors. DNA repair was evaluated by analysis of DNA strand breaks using the comet assay. Survival of CFU-GM, BFU-E, and CFU-Mix was determined and dose-response curves were fitted to the data. Results: The radiobiological parameters (D 0 and n) showed significant GF(s) effects. Combination of IL-3 with IL-6, SCF or GM-CSF resulted in best survival for CFU-GM BFU-E and CFU-Mix, respectively. Combinations of three or more GFs did not increase the survival of clonogenic CD34 + cells compared to optimal two-factor combinations. The D 0 values for CFU-GM, BFU-E, and CFU-Mix ranged between 0.56-1.15, 0.41-2.24, and 0.56-1.29 Gy, respectively. As for controls, the curves remained strictly exponential, i.e., all survival curves were strictly exponential without any shoulder (extrapolation numbers n = 1 for all tested GF(s). DNA repair capacity of CD34 + cells determined by comet assay, was measured before, immediately after irradiation, as well as 30 and 120 min after irradiation at 1 Gy. Notably, after irradiation the 2-h repair of cytokine-stimulated and unstimulated CD34 + cells was similar. Conclusion: Our data indicate that increased survival of irradiated CB CD34 + cells after short-term GF treatment is

  14. [Biological characteristics of mesenchymal stem cell and hematopoietic stem cell in the co-culture system].

    Science.gov (United States)

    Wei, Wei; Xu, Chao; Ye, Zhi-Yong; Huang, Xiao-Jun; Yuan, Jia-En; Ma, Tian-Bao; Lin, Han-Biao; Chen, Xiu-Qiong

    2016-10-25

    The aim of the present study was to obtain the qualified hematopoietic stem/progenitor cells (HSC/HPC) and human umbilical cord-mesenchymal stem cells (MSC) in vitro in the co-culture system. Cord blood mononuclear cells were separated from umbilical cord blood by Ficoll lymphocyte separation medium, and then CD34 + HSC was collected by MACS immunomagnetic beads. The selected CD34 + HSC/HPC and MSC were transferred into culture flask. IMDM culture medium with 15% AB-type cord plasma supplemented with interleukin-3 (IL-3), IL-6, thrombopoietin (TPO), stem cell factor (SCF) and FMS-like tyrosine kinase 3 ligand (Flt-3L) factors were used as the co-culture system for the amplification of HSC/HPC and MSC. The cellular growth status and proliferation on day 6 and 10 after co-culture were observed by using inverted microscope. The percentage of positive expression of CD34 in HSC/HPC, as well as the percentages of positive expressions of CD105, CD90, CD73, CD45, CD34 and HLA-DR in the 4 th generation MSC, was tested by flow cytometry. Semisolid colony culture was used to test the HSC/HPC colony forming ability. The osteogenic, chondrogenesis and adipogenic ability of the 4 th generation MSC were assessed. The karyotype analysis of MSC was conducted by colchicines. The results demonstrated that the HSC/HPC of co-culture group showed higher ability of amplification, CFU-GM and higher CD34 + percentage compared with the control group. The co-cultured MSC maintained the ability to differentiate into bone cells, fat cells and chondrocytes. And the karyotype stability of MSC remained normal. These results reveal that the appropriate co-culture system for MSC and HSC is developed, and via this co-culture system we could gain both two kinds of these cells. The MSCs under the co-culture system maintain the biological characteristics. The CFU-GM ability, cell counting and the flow cytometry results of HSC/HPC under the co-culture system are conform to the criterion, showing that

  15. Effects of cyclosporin A and FK506 on Fc epsilon receptor type I-initiated increases in cytokine mRNA in mouse bone marrow-derived progenitor mast cells: resistance to FK506 is associated with a deficiency in FK506-binding protein FKBP12.

    Science.gov (United States)

    Kaye, R E; Fruman, D A; Bierer, B E; Albers, M W; Zydowsky, L D; Ho, S I; Jin, Y J; Castells, M C; Schreiber, S L; Walsh, C T

    1992-09-15

    The inhibitory effects of cyclosporin A (CsA) and FK506 on Fc epsilon receptor type I-initiated increases in cytokine mRNA and the expression of their intracellular binding proteins were studied in interleukin 3 (IL-3)-dependent, mouse bone marrow-derived mast cells (BMMCs). In BMMCs sensitized with IgE anti-trinitrophenyl, CsA inhibited trinitrophenylated bovine serum albumin-induced increases in mRNA for IL-1 beta, tumor necrosis factor alpha (TNF-alpha), and IL-6 in a dose-related manner (IC50 values of 4, 65, and 130 nM, respectively). FK506 did not inhibit hapten-specific increases of mRNA for TNF-alpha or IL-6, and for IL-1 beta the IC50 was greater than 50-fold higher than that of CsA. Neither agent inhibited exocytosis of the endogenous secretory granule mediators beta-hexosaminidase and histamine at the IC50 values for inhibition of increases in cytokine mRNA. BMMCs expressed cyclophilin, and CsA inhibited the phosphatase activity of cellular calcineurin with an IC50 of approximately 8 nM. That CsA inhibited IL-1 beta mRNA accumulation in IgE-activated BMMCs with an IC50 similar to that for inhibition of calcineurin activity, whereas the IC50 values were approximately 20-fold higher for the inhibition of TNF-alpha and IL-6 mRNA, suggests that the induction of TNF-alpha and IL-6 is less dependent upon calcineurin activity than is the induction of IL-1 beta. BMMCs were deficient in the 12-kDa FK506-binding protein FKBP12, but not FKBP13, as assessed by RNA and protein blot analyses. FK506 did not inhibit calcineurin phosphatase activity in BMMCs, even at drug concentrations of 1000 nM. The resistance of BMMCs to inhibition of Fc epsilon receptor type I-mediated increases in cytokine mRNA by FK506 is most likely due to their deficiency of FKBP12 and the related inability to inhibit the activity of calcineurin.

  16. Expression profiling of skeletal muscle following acute and chronic β2-adrenergic stimulation: implications for hypertrophy, metabolism and circadian rhythm

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    Lynch Gordon S

    2009-09-01

    administration also appeared to influence some genes associated with the peripheral regulation of circadian rhythm (including nuclear factor interleukin 3 regulated, D site albumin promoter binding protein, and cryptochrome 2. Conclusion This is the first study to utilize gene expression profiling to examine global gene expression in response to acute β2-AR agonist treatment of skeletal muscle. In summary, systemic administration of a β2-AR agonist had a profound effect on global gene expression in skeletal muscle. In terms of hypertrophy, β2-AR agonist treatment altered the expression of several genes associated with myostatin signaling, a previously unreported effect of β-AR signaling in skeletal muscle. This study also demonstrates a β2-AR agonist regulation of circadian rhythm genes, indicating crosstalk between β-AR signaling and circadian cycling in skeletal muscle. Gene expression alterations discovered in this study provides insight into many of the underlying changes in gene expression that mediate β-AR induced skeletal muscle hypertrophy and altered metabolism.

  17. Niveles de citoquinas megacariocitopoyéticas en pacientes con trombocitemia esencial y su relación con características clínicas y bioquímicas Megakaryopoietic cytokine levels in patients with essential thrombocythemia and their relationship with clinical and biochemical features

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    Rosana F. Marta

    2006-12-01

    high platelet count and megakaryocytic hyperplasia. In the present work we evaluated plasmatic levels of cytokines involved in megakaryocytic development in a group of patients with ET that were not on treatment, as well as thrombopoietin (TPO levels before and during anagrelide treatment. The assays were carried out using ELISA techniques. Among the cytokines mainly involved in proliferation of megakaryocytic progenitors, interleukin 3 (IL-3 levels were found increased in patients compared to normal controls (p=0.0383. Granulocyte-macrophage colony stimulating factor and stem cell factor levels were normal. Interleukin 6, as well as interleukin 11 and erythropoietin (EPO, cytokines mainly related to megakaryocytic maturation, were normal. Plasma TPO levels before treatment were within the normal range and increased during treatment but the difference was not statistically significant. Patients who displayed spontaneous platelet aggregation had higher plasma TPO levels compared to those who did not (p=0.049. We did not find any relationship between cytokine levels and clinical or laboratory parameters. The high IL-3 levels seen in some patients with ET could contribute to megakaryocytic proliferation. The simultaneous occurrence of higher TPO levels and elevated platelet count could be a predisposing factor for the development of spontaneous platelet aggregation in ET patients.