DOCSLIB.ORG

And Insulin-Like Growth Factor-I (IGF-I) in Regulating Human Erythropoiesis

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
And Insulin-Like Growth Factor-I (IGF-I) in Regulating Human Erythropoiesis Leukemia (1998) 12, 371–381 1998 Stockton Press All rights reserved 0887-6924/98 $12.00 The role of insulin (INS) and insulin-like growth factor-I (IGF-I) in regulating human erythropoiesis. Studies in vitro under serum-free conditions – comparison to other cytokines and growth factors J Ratajczak, Q Zhang, E Pertusini, BS Wojczyk, MA Wasik and MZ Ratajczak Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA The role of insulin (INS), and insulin-like growth factor-I (IGF- has been difficult to assess. The fact that EpO alone fails to I) in the regulation of human erythropoiesis is not completely stimulate BFU-E in serum-free conditions, but does do in understood. To address this issue we employed several comp- lementary strategies including: serum free cloning of CD34؉ serum containing cultures indicates that serum contains some cells, RT-PCR, FACS analysis, and mRNA perturbation with oli- crucial growth factors necessary for the BFU-E development. godeoxynucleotides (ODN). In a serum-free culture model, both In previous studies from our laboratory, we examined the ؉ INS and IGF-I enhanced survival of CD34 cells, but neither of role of IGF-I12 and KL9,11,13 in the regulation of early human these growth factors stimulated their proliferation. The influ- erythropoiesis. Both of these growth factors are considered to ence of INS and IGF-I on erythroid colony development was be crucial for the BFU-E growth.3,6,8,14 Unexpectedly, that dependent on a combination of growth factors used for stimul- + ating BFU-E growth. When BFU-E growth was optimally stimu- addition of IGF-I to cultures of the purified human CD34 cells lated with erythropoietin (EpO) ؉ kit ligand (KL) the large did not enhance formation of the hematopoietic colonies.12 erythroid colonies developed normally even in the absence of Similarly, downregulation of IGF-I R using antisense oligo- INS or IGF-I. However, the addition of both of these growth fac- deoxynucleotides was without any apparent effect on the tors slightly enhanced colony size. On the other hand, if eruthroid colonies were stimulated suboptimally with EpO + IL- growth of normal human hematopoietic progenitors. In con- 3 only, INS or IGF-I increased the number of small erythroid trast, antisense oligodeoxynucleotides against c-Kit R totally bursts by ෂ30%. Both INS and IGF-I activated signal transduc- inhibited the BFU-E colony formation, if CD34+ cells were co- tion in maturing human erythropoietic cells as determined by stimulated with EpO + KL alone.9,11,13 We concluded there- phosphorylation of the insulin receptor substrate-2 (IRS-2) pro- fore, that KL, but not IGF-I, is a critical regulator of human tein. We also found by RT-PCR that mRNA coding for INS-R is expressed in FACS sorted CD34؉, c-kit-R؉ marrow cells, and erythroid colony formation. We concluded also, that EpO is 11 in cells isolated from BFU-E and CFU-GM colonies. Expression not able to support BFU-E colony growth if KL is not present of INS-R protein on these cells was subsequently confirmed by in the cultures. This last observation was recently confirmed cytofluorometry. In contrast, the receptor for insulin-like 1–3 .؉ in several elegant studies by the other investigators growth factor-I (IGF-IR) was not detected on CD34 cells, and Nevertheless, since our previous studies were performed in was first easily detectable on more differentiated cells derived 8 from day 6 BFU-E and CFU-GM colonies. We conclude that INS the presence of serum, which is known to contain INS, the and IGF-I may be survival factors for human CD34؉ cells, but effect of IGF-I on colony formation was uncertain. As it is well are not required during early erythropoiesis. In contrast, both known, INS, IGF-I and IGF-II show a high degree of the mol- growth factors may play some role at the final stages of ecular homology, and crossreact with their corresponding erythroid maturation. receptors. In addition, INS R and IGF-I R are structurally Keywords: erythropoiesis; antisense oligonucleotides; insulin; insulin-like growth factors; insulin receptor; insulin-like growth factor related heterodimers, which possess intrinsic tyrosine kinase I receptor activity, and after interaction with IRS-1 and IRS-2 ‘docking proteins’ activate appropriate signal transduction proteins. In contrast, the third receptor of this family, IGF-II R, is a single Introduction highly glycosylated transmembrane peptide, which does not possess tyrosine kinase activity. The ability of IGF-II R to The formation of red blood cells is regulated by various cyto- recruit signal transduction proteins is not well documented in kines and growth factors. Of these, erythropoietin (EpO) is the literature.15 absolutely required for survival and differentiation of the To learn more about the role of the insulin growth factor erythropoietic progenitors.1–3 Other growth factors, and cyto- family in regulating human erythropoiesis, we employed a kines such as insulin (INS), insulin-like growth factors (IGF-I, well-defined serum-free cloning system to examine the direct IGF-II), basic fibroblast growth factor (FGF-2), hepatocyte effect of INS and IGF-I on survival and cloning efficiency of growth factors (HGF), prolactin (PRL), interleukins (IL-3, IL- the CD34+ cells. We also studied the development of BFU-E 4, IL-6, IL-9, IL-11), granulocyte–monocyte stimulatory factor colonies compared to the effect of other cytokines and growth (GM-CSF), and kit ligand (KL), have been reported to co-stimu- factors which have been reported to enhance erythropo- late with EpO the growth of the erythroid colonies.4–11 Since iesis.4,6,16–19 Finally, we analyzed by FACS and RT-PCR the several of these factors including KL, INS and IGF-I, are usu- expression of INS-R and IGF-IR in the early progenitor cells ally present in the serum used to supplement cell culture and their more differentiated, cultured derivatives. Our results media, contribution of the individual factors to erythropoiesis suggest that neither INS nor IGF-I are critical for human in experiments performed in serum supplemented conditions erythropoiesis. These factors seem to inhibit apoptosis in the human early hematopoietic cells but do not appear to affect the initial proliferation and differentiation of these cells. INS Correspondence: MZ Ratajczak, Rm 515 Stellar-Chance Laboratories, and IGF-I may, however contribute more effectively to the University of Pennsylvania School of Medicine, 422 Curie Blvd, Phila- final maturation of erythroid colonies. delphia, PA 19104, USA; Fax 215 573 2078 Received 23 May 1997; accepted 14 November 1997 Insulin and human hematopoiesis MZ Ratajczak et al 372 Materials and methods growth factor, 20 ng/ml), G-CSF (10 ng/ml), GM-CSF (5 ng/ml), IL-3 (20 U/ml) and KL (100 ng/ml). Recombinant Oligodeoxynucleotides (ODN) human growth factors were used in all experiments (R&D, Minneapolis, MN, USA). Colonies were counted with an Unmodified 18-base ODN were synthesized on an Applied inverted microscope on day 11 (CFU-GM), on day 14 (BFU- Biosystems (Foster City, CA, USA) 380B DNA synthesizer by E), and on day 15 (CFU-Mix). means of ␤-cyanoethyl phosphoroamidite chemistry as In some experiments cells were cultured in serum-free con- described. ODN were purified by ethanol precipitation and ditions with or without exogenously added insulin (20 ␮g/ml) multiple washes in 70% ethanol, lyophilized to dryness, and for 7 days before growth factors were added to stimulate re-dissolved in culture medium at a concentration of 1 mg/ml growth of CFU-Mix, BFU-E and CFU-GM colonies. (0.167 mmol/l). ODN sequences utilized were based on the published human IGF-I R sequence,20 and were targeted to codons −21−26 of the pro-receptor signal peptide: antisense Cell selection and mRNA isolation ODN; 5′-TCC TCC GGA GCC AGA CTT-3′, sense ODN: 5′- AAG TCT GGC TCC GGA GGA-3′ and scrambled ODN; 5′- A-T-MNC were enriched for CD34+ cells by immunoaffinity TGA GAC TCC TTA CCG CCG-3′ as described.21 selection with murine monoclonal antibody HPCA-1 (Becton Dickinson, San Jose, CA, USA) and magnetic beads according to the manufacturer’s protocol (Dynal, Oslo, Norway). The c- Cells kit R+ subset of CD34+ cells was isolated by FACS. In brief, 2 × 107 human A-T-MNC were suspended in PBS sup- Light-density marrow mononuclear cells (MNC) were plemented with 5% bovine calf serum (Hyclone, Logan, UT, obtained from 11 consenting healthy donors and depleted USA) (BCS) and labeled for 30 min at 4°C with anti-c-kit R of adherent cells and T lymphocytes (A-T-MNC) as monoclonal antibodies (SR-1) (1:1000) (generously provided described.10–12,22 by V Broudy, University of Washington, Seattle, WA, USA). Cells were washed three times in ice-cold PBS supplemented with 5% BCS and then incubated with phycoerythrin (PE)-con- Cell lines jugated goat anti-mouse monoclonal antibody (Sigma) (1:100) for 30 min at 4°C. Then the cells were washed again and incu- Chinese-hamster ovary (CHO) cell line transfected with bated with anti-CD34 monoclonal antibody conjugated with human INS-R was made available to us by P Rothenberg from fluorescein FITC anti-HPCA-2 (Becton Dickinson) (20 ␮l/106 the University of Pennsylvania. HTLV-1 positive T cell lines: cells) for 30 min at 4°C. After incubation, cells were washed HUT-102B and C91PL were obtained from G Trinchieri from three times in ice-cold PBS supplemented with 5% BCS and Wistar Institute, Philadelphia, and M Kamoun from the Uni- then subjected to fluorescence-activated cell sorting using a versity of Pennsylvania, respectively.
Load more

Recommended publications
  • ARTICLES Fibroblast Growth Factors 1, 2, 17, and 19 Are The
    0031-3998/07/6103-0267 PEDIATRIC RESEARCH Vol. 61, No. 3, 2007 Copyright © 2007 International Pediatric Research Foundation, Inc. Printed in U.S.A. ARTICLES Fibroblast Growth Factors 1, 2, 17, and 19 Are the Predominant FGF Ligands Expressed in Human Fetal Growth Plate Cartilage PAVEL KREJCI, DEBORAH KRAKOW, PERTCHOUI B. MEKIKIAN, AND WILLIAM R. WILCOX Medical Genetics Institute [P.K., D.K., P.B.M., W.R.W.], Cedars-Sinai Medical Center, Los Angeles, California 90048; Department of Obstetrics and Gynecology [D.K.] and Department of Pediatrics [W.R.W.], UCLA School of Medicine, Los Angeles, California 90095 ABSTRACT: Fibroblast growth factors (FGF) regulate bone growth, (G380R) or TD (K650E) mutations (4–6). When expressed at but their expression in human cartilage is unclear. Here, we deter- physiologic levels, FGFR3-G380R required, like its wild-type mined the expression of entire FGF family in human fetal growth counterpart, ligand for activation (7). Similarly, in vitro cul- plate cartilage. Using reverse transcriptase PCR, the transcripts for tivated human TD chondrocytes as well as chondrocytes FGF1, 2, 5, 8–14, 16–19, and 21 were found. However, only FGF1, isolated from Fgfr3-K644M mice had an identical time course 2, 17, and 19 were detectable at the protein level. By immunohisto- of Fgfr3 activation compared with wild-type chondrocytes and chemistry, FGF17 and 19 were uniformly expressed within the showed no receptor activation in the absence of ligand (8,9). growth plate. In contrast, FGF1 was found only in proliferating and hypertrophic chondrocytes whereas FGF2 localized predominantly to Despite the importance of the FGF ligand for activation of the resting and proliferating cartilage.
    [Show full text]
  • Insights Into the Cellular Mechanisms of Erythropoietin-Thrombopoietin Synergy
    Papayannopoulou et al.: Epo and Tpo Synergy Experimental Hematology 24:660-669 (19961 661 @ 1996 International Society for Experimental Hematology Rapid Communication ulation with fluorescence microscopy. Purified subsets were grown in plasma clot and methylcellulose clonal cultures and in suspension cultures using the combinations of cytokines Insights into the cellular mechanisms cadaveric bone marrow cells obtained from Northwest described in the text. Single cells from the different subsets Center, Puget Sound Blood Bank (Seattle, WA), were were. also deposited (by FACS) on 96-well plates containing of erythropoietin-thrombopoietin synergy washed, and incubated overnight in IMDM with 10% medmm and cytokines. Clonal growth from single-cell wells calf serum on tissue culture plates to remove adherent were double-labeled with antiglycophorin A-PE and anti­ Thalia Papayannopoulou, Martha Brice, Denise Farrer, Kenneth Kaushansky From the nonadherent cells, CD34+ cells were isolated CD41- FITC between days 10 and 19. direct immunoadherence on anti-CD34 monoclonal anti­ University of Washington, Department of Medicine, Seattle, WA (mAb)-coated plates, as previously described [15]. Purity Immunocytochemistry Offprint requests to: Thalia Papayannopoulou, MD, DrSci, University of Washington, isolated CD34+ cells ranged from 80 to 96% by this For immunocytochemistry, either plasma clot or cytospin cell Division of Hematology, Box 357710, Seattle, WA 98195-7710 od. Peripheral blood CD34 + cells from granulocyte preparations were used. These were fixed at days 6-7 and (Received 24 January 1996; revised 14 February 1996; accepted 16 February 1996) ulating factor (G-CSF)-mobilized normal donors 12-13 with pH 6.5 Histochoice (Amresco, Solon, OH) and provided by Dr.
    [Show full text]
  • The Molecular Mechanisms That Control Thrombopoiesis
    The molecular mechanisms that control thrombopoiesis Kenneth Kaushansky J Clin Invest. 2005;115(12):3339-3347. https://doi.org/10.1172/JCI26674. Review Series Our understanding of thrombopoiesis — the formation of blood platelets — has improved greatly in the last decade, with the cloning and characterization of thrombopoietin, the primary regulator of this process. Thrombopoietin affects nearly all aspects of platelet production, from self-renewal and expansion of HSCs, through stimulation of the proliferation of megakaryocyte progenitor cells, to support of the maturation of these cells into platelet-producing cells. The molecular and cellular mechanisms through which thrombopoietin affects platelet production provide new insights into the interplay between intrinsic and extrinsic influences on hematopoiesis and highlight new opportunities to translate basic biology into clinical advances. Find the latest version: https://jci.me/26674/pdf Review series The molecular mechanisms that control thrombopoiesis Kenneth Kaushansky Department of Medicine, Division of Hematology/Oncology, University of California, San Diego, San Diego, California, USA. Our understanding of thrombopoiesis — the formation of blood platelets — has improved greatly in the last decade, with the cloning and characterization of thrombopoietin, the primary regulator of this process. Thrombopoietin affects nearly all aspects of platelet production, from self-renewal and expansion of HSCs, through stimulation of the proliferation of megakaryocyte progenitor cells, to support of the maturation of these cells into platelet-pro- ducing cells. The molecular and cellular mechanisms through which thrombopoietin affects platelet production provide new insights into the interplay between intrinsic and extrinsic influences on hematopoiesis and highlight new opportunities to translate basic biology into clinical advances.
    [Show full text]
  • The Thrombopoietin Receptor : Revisiting the Master Regulator of Platelet Production
    This is a repository copy of The thrombopoietin receptor : revisiting the master regulator of platelet production. White Rose Research Online URL for this paper: https://eprints.whiterose.ac.uk/175234/ Version: Published Version Article: Hitchcock, Ian S orcid.org/0000-0001-7170-6703, Hafer, Maximillian, Sangkhae, Veena et al. (1 more author) (2021) The thrombopoietin receptor : revisiting the master regulator of platelet production. Platelets. pp. 1-9. ISSN 0953-7104 https://doi.org/10.1080/09537104.2021.1925102 Reuse This article is distributed under the terms of the Creative Commons Attribution (CC BY) licence. This licence allows you to distribute, remix, tweak, and build upon the work, even commercially, as long as you credit the authors for the original work. More information and the full terms of the licence here: https://creativecommons.org/licenses/ Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing [email protected] including the URL of the record and the reason for the withdrawal request. [email protected] https://eprints.whiterose.ac.uk/ Platelets ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/iplt20 The thrombopoietin receptor: revisiting the master regulator of platelet production Ian S. Hitchcock, Maximillian Hafer, Veena Sangkhae & Julie A. Tucker To cite this article: Ian S. Hitchcock, Maximillian Hafer, Veena Sangkhae & Julie A. Tucker (2021): The thrombopoietin receptor: revisiting the master regulator of platelet production, Platelets, DOI: 10.1080/09537104.2021.1925102 To link to this article: https://doi.org/10.1080/09537104.2021.1925102 © 2021 The Author(s).
    [Show full text]
  • Factor-The Controlling Growth Factor-At a Sufficiently Low Value, While the Cell Population Has Ceased to Grow and Is in a Sort
    708 GENETICS: NO VICK A ND SZILARD PROC. N. A. S. EXPERIMENTS WITH THE CHEMOSTAT ON SPONTANEOUS MUTATIONS OF BACTERIA By AARON NOVICK AND LEO SZILARD INSTITUTRE OF RADIOBIOLOGY AND BIOPHYSICS, UNIVERSITY OF CHICAGO Communicated by H. J. Muller, October 18, 1950 Introduction.-All bacteria require for growth the presence of certain inorganic chemical components in the nutrient, such as potassium, phos- phorus, sulphur, etc., and with a few exceptions all bacteria require an energy-yielding carbon source, such as, for instance, glucose or lactate, etc. In addition to these elements or simple compounds, certain bacteria require more complex compounds, for instance an amino acid, which they are not capable of synthesizing. For the purposes of this presentation, any of the chemical compounds which a given strain of bacteria requires for its growth will be called a "growth factor." In general, the growth rate of a bacterial strain may be within very wide limits independent of the concentration of a given growth factor; but since at zero concentration the growth rate is zero, there must of necessity exist, at sufficiently low concentrations of the growth factor, a region in which the growth rate falls with falling concentration of the growth factor. It therefore should be possible to maintain a bacterial population over an indefinite period of time growing at a rate considerably lower than normal simply by maintaining the concentration of one growth factor-the controlling growth factor-at a sufficiently low value, while the concentrations of all other growth factors may at the same time be main- tained at high values.
    [Show full text]
  • Erythropoietin Prevents Haloperidol Treatment-Induced Neuronal Apoptosis Through Regulation of BDNF
    Neuropsychopharmacology (2008) 33, 1942–1951 & 2008 Nature Publishing Group All rights reserved 0893-133X/08 $30.00 www.neuropsychopharmacology.org Erythropoietin Prevents Haloperidol Treatment-Induced Neuronal Apoptosis through Regulation of BDNF ,1,2 3 1,2 4 Anilkumar Pillai* , Krishnan M Dhandapani , Bindu A Pillai , Alvin V Terry Jr and 1,2 Sahebarao P Mahadik 1 2 Department of Psychiatry and Health Behavior, Medical College of Georgia, Augusta, GA, USA; Medical Research Service Line, Veterans Affairs 3 4 Medical Center, Augusta, GA, USA; Department of Neurosurgery, Medical College of Georgia, Augusta, GA, USA; Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, GA, USA Functional alterations in the neurotrophin, brain-derived neurotrophic factor (BDNF) have recently been implicated in the pathophysiology of schizophrenia. Furthermore, animal studies have indicated that several antipsychotic drugs have time-dependent (and differential) effects on BDNF levels in the brain. For example, our previous studies in rats indicated that chronic treatment with the conventional antipsychotic, haloperidol, was associated with decreases in BDNF (and other neurotrophins) in the brain as well as deficits in cognitive function (an especially important consideration for the therapeutics of schizophrenia). Additional studies indicate that haloperidol has other deleterious effects on the brain (eg increased apoptosis). Despite such limitations, haloperidol remains one of the more commonly prescribed antipsychotic agents worldwide due to its efficacy for the positive symptoms of schizophrenia and its low cost. Interestingly, the hematopoietic hormone, erythropoietin, in its recombinant human form rhEPO has been reported to increase the expression of BDNF in neuronal tissues and to have neuroprotective effects.
    [Show full text]
  • Phase II Study of Sorafenib Plus 5-Azacitidine for the Initial Therapy of Patients with Acute Myeloid Leukemia and High Risk
    2014-0076 March 9, 2015 Page 1 Protocol Page Phase II Study of Sorafenib Plus 5-Azacitidine for the Initial Therapy of Patients with Acute Myeloid Leukemia and High Risk Myelodysplastic Syndrome with FLT3-ITD Mutation 2014-0076 Core Protocol Information Short Title Sorafenib Plus 5-Azacitidine initial therapy of patients with AML and high risk MS with FLT3-ITD Mutation Study Chair: Farhad Ravandi-Kashani Additional Contact: Andrea L. Booker Mary Ann Richie Leukemia Protocol Review Group Department: Leukemia Phone: 713-792-7305 Unit: 428 Full Title: Phase II Study of Sorafenib Plus 5-Azacitidine for the Initial Therapy of Patients with Acute Myeloid Leukemia and High Risk Myelodysplastic Syndrome with FLT3-ITD Mutation Protocol Type: Standard Protocol Protocol Phase: Phase II Version Status: Terminated 11/27/2018 Version: 12 Submitted by: Andrea L. Booker--2/23/2017 1:07:00 PM OPR Action: Accepted by: Julie Arevalo -- 3/3/2017 2:39:37 PM Which Committee will review this protocol? The Clinical Research Committee - (CRC) 2014-0076 March 9, 2015 Page 2 Protocol Body Sorafenib plus 5-Azacitidine Initial Therapy – 2014-0076 March 05, 2015 1 Phase II Study Of Sorafenib Plus 5-Azacitidine For The Initial Therapy Of Patients With Acute Myeloid Leukemia And High Risk Myelodysplastic Syndrome With FLT3-ITD Mutation Short Title: Sorafenib Plus 5-Azacitidine initial therapy of patients with AML and high risk MS with FLT3-ITD mutation PI: Farhad Ravandi, MD Professor of Medicine, Department of Leukemia University of Texas – MD Anderson Cancer Center 1 Sorafenib plus 5-Azacitidine Initial Therapy – 2014-0076 March 05, 2015 2 Contents 1.0 Objectives ........................................................................................................................................
    [Show full text]
  • Updated November 2019 KIT Mutation the KIT Gene Is Associated With
    Updated November 2019 KIT Mutation The KIT gene is associated with autosomal dominant piebaldism, gastrointestinal stromal tumors (GISTs), and familial mastocytosis.1-3 The type of mutation identified in the KIT gene determines the symptoms/cancer risks that the individual may have. Pathogenic loss-of-function (LOF) mutations in the KIT gene are generally only associated with Piebaldism. However, pathogenic gain of function mutations in the KIT gene are associated with systemic mastocytosis and gastrointestinal stromal tumors.4,5 Piebaldism: This is a rare condition characterized by the patchy absence of melanocytes in certain areas of the skin and hair. Melanocytes produce the pigment melanin, which contributes to hair, eye, and skin color. The absence of melanocytes leads to patches of skin and hair that are lighter than normal. These unpigmented areas are typically present at birth and do not increase in size or number.6-8 Gastrointestinal stromal tumors (GIST): GISTs are tumors that occur in the gastrointestinal tract, most commonly in the stomach or small intestine. Small tumors may cause no signs or symptoms. However, some people with GISTs may experience pain or swelling in the abdomen, nausea, vomiting, loss of appetite, or weight loss. These tumors can be cancerous (malignant) or noncancerous (benign). Mastocytosis: This is a blood disorder that occurs when white blood cells called mast cells accumulate in one or more tissues (most commonly in the bone marrow). Mast cells normally trigger inflammation during an allergic reaction. When an environmental trigger activates mast cells, they release proteins that signal an immune response. In systemic mastocytosis, excess mast cells mean more proteins are being released in the tissues where the cells accumulate, leading to an increased immune response.
    [Show full text]
  • B-Cell Growth Factor
    Proc. Natl Acad. Sci. USA Vol. 79, pp. 7455-7459, December 1982 Immunology B-cell growth factor: Distinction from T-cell growth factor and B-cell maturation factor (B lymphocytes/T-cell hybridomas) TOMAS LEANDERSON*, ERIK LUNDGRENt, ERIK RUUTH*, HAKAN BORGt, HAKAN PERSSONt, AND ANTONIO COUTINHOt tLaboratory for Cell and Tissue Culture Research, *Institute of Pathology, and +Department of Immunology, UmeA University, S-901 87 UmeA, Sweden Communicated by Niels K. Jerne, July 12, 1982 ABSTRACT A T-cell hybridoma was derived by somatic cell We have initiated these studies by systematically screening hybridization between concanavalin A-activated BALB/c spleen hybridoma activities in different assays that measure either cells and the AKR thymoma BW 5147. Media conditioned by hy- growth or maturation ofall B lymphocytes, regardless ofclonal bridoma cells, even at high dilutions (1:1,000) support the growth specificity. We chose to study activated rather than resting B oflipopolysaccharide-stimulated B-cell blasts but not that ofT-cell cells because of the-overwhelming evidence that initiation of growth factor (TCGF)-reactive T-cells. This activity, herein des- cooperative B-cell responses requires direct cellular interaction ignated B-cell growth factor (BCGF), has a Mr of =20,000 and it (13, 14). In this report we describe a hybridoma clone secreting can readily be separated from TCGF (Mr "30,000) by gel filtra- a factor that supports growth but not maturation of lipopoly- tion. BCGF is constitutively produced by the hybridoma cells, it saccharide (LPS)-activated B-cell blasts and is devoid of is removed from conditioned media by incubation with target cells T-cell at +4°C, and it is equally effective on B-cell blasts carrying dif- growthfactor(TCGF) andT-cellreplacingfactor(TRF) activities.
    [Show full text]
  • Lab Dept: Hematology Test Name: ERYTHROPOIETIN
    Lab Dept: Hematology Test Name: ERYTHROPOIETIN General Information Lab Order Codes: EPOS Synonyms: Erythropoietin (EPO), Serum CPT Codes: 82668 - Erythropoietin Test Includes: Erythropoietin level reported in mIU/mL. Logistics Test Indications: This test is mainly used for the differential diagnosis of primary and secondary polycythemia and to determine the cause of anemia. In the diagnosis of primary polycythemia (polycythemia rubra vera) due to an uncontrolled increase in the number of erythrocytes carrying high concentrations of oxygen, the EPO level is suppressed. The test is also useful for diagnosis of appropriate secondary polycythemia caused by high-altitude living, pulmonary disease, and tobacco use, which increase EPO levels. In patients with inappropriate secondary polycythemia caused by renal tumors and extrarenal tumors, the EPO level is also increased. Patients with anemia of bone marrow failure, iron deficiency, or thalassemia also have increased EPO levels Lab Testing Sections: Hematology - Sendouts Referred to: Mayo Medical Laboratories (MML Test: EPO) Phone Numbers: MIN Lab: 612-813-6280 STP Lab: 651-220-6550 Test Availability: Daily, 24 hours Turnaround Time: 2 - 4 days, test set up Monday - Saturday Special Instructions: N/A Specimen Specimen Type: Blood Container: SST (Gold, marble or red) tube Draw Volume: 1.8 mL (Minumum: 1.5 mL) blood Processed Volume: 0.6 mL (Minimum: 0.5 mL) serum Collection: Routine blood collection Special Processing: Lab Staff: Centrifuge specimen, aliquot into a screw-capped plastic vial. Store and ship at refrigerated temperatures. Forward promptly. Patient Preparation: None Sample Rejection: Mislabeled or unlabeled specimen; gross hemolysis Interpretive Reference Range: 2.6 – 18.5 mIU/mL Interpretation: In the appropriate clinical setting (eg, confirmed elevation of hemoglobin >18.5 gm/dL, persistent leukocytosis, persistent thrombocystosis, unusual thrombosis, splenomegaly, and erythromegaly), polycythemia vera is unlikely when EPO levels are elevated and polycythemia vera is likely when EPO levels are suppressed.
    [Show full text]
  • Erythropoietin Using ALZET Osmotic Pumps
    ALZET® Bibliography References on the Administration of Erythropoietin Using ALZET Osmotic Pumps Q8440: S. Dey, et al. Sex-specific brain erythropoietin regulation of mouse metabolism and hypothalamic inflammation. JCI Insight 2020;5(5): Agents: Erythropoietin, recombinant human Vehicle: Saline; Route: CSF/CNS (intracerebral); IV; Species: Mice; Pump: 2006; Duration: 14 days; ALZET Comments: Dose (3000 U/kg); Controls received mp w/ vehicle; animal info (Tg21 mice); recombinant human Erythropoietin aka recombinant human EPO; ALZET brain infusion kit 3 used; Brain coordinates (midline, 1.00 mm; antero- posterior, 0.34 mm; dorsoventral, 2.30 mm); dental cement used;replacement therapy (Erythropoietin); Q8045: E. K. Kim, et al. Local Subcutaneous Injection of Erythropoietin Might Improve Fat Graft Survival, Whereas Continuous Infusion Using an Osmotic Pump Device Was Harmful by Provoking an Overwhelming Foreign Body Reaction in a Nude Mouse Model. Archives of Aesthetic Plastic Surgery 2018;24(3):128-133 Agents: Erythropoietin Vehicle: Saline; Route: SC; Species: Mice; Pump: 1007D; Duration: 1 week; ALZET Comments: Dose (1,000 IU of EPO); animal info (36 weeks old, CD-1, Male, 20-25 g); EPO aka hemangiogenic and antiapoptotic factor ; dependence; Q4880: E. H. Sanchez-Mendoza, et al. Implantation of Miniosmotic Pumps and Delivery of Tract Tracers to Study Brain Reorganization in Pathophysiological Conditions. Journal of Visualized Experiments 2016;107(1-9 ALZET Comments: Erythropoietin, recombinant human; CSF/CNS; Mice; 30 days; Controls received
    [Show full text]
  • Inactivation of Erythropoietin Receptor Function by Point Mutations in a Region Having Homology with Other Cytokine Receptors OSAMU MIURA,'T JOHN L
    MOLECULAR AND CELLULAR BIOLOGY, Mar. 1993, p. 1788-1795 Vol. 13, No. 3 0270-7306/93/031788-08$02.00/0 Copyright © 1993, American Society for Microbiology Inactivation of Erythropoietin Receptor Function by Point Mutations in a Region Having Homology with Other Cytokine Receptors OSAMU MIURA,'t JOHN L. CLEVELAND,' AND JAMES N. IHLEl,2* Department ofBiochemistry, St. Jude Children's Research Hospital, Memphis, Tennessee 31051,1 and Department ofBiochemistry, University of Tennessee, Memphis, Tennessee 3816332 Received 13 July 1992/Returned for modification 12 August 1992/Accepted 21 December 1992 The cytoplasmic domain of the erythropoietin receptor (EpoR) contains a region, proximal to the transmembrane domain, that is essential for function and has homology with other members of the cytokine receptor family. To explore the functional significance of this region and to identify critical residues, we introduced several amino acid substitutions and examined their effects on erythropoietin-induced mitogenesis, tyrosine phosphorylation, and expression of immediate-early (c-fos, c-myc, and egr-1) and early (ornithine decarboxylase and T-cell receptor 'y) genes in interleukin-3-dependent cell lines. Amino acid substitution of W-282, which is strictly conserved at the middle portion of the homology region, completely abolished all the functions of the EpoR. Point mutation at L-306 or E-307, both of which are in a conserved LEVL motif, drastically impaired the function of the receptor in all assays. Other point mutations, introduced into less conserved amino acid residues, did not significantly impair the function of the receptor. These results demonstrate that conserved amino acid residues in this domain of the EpoR are required for mitogenesis, stimulation of tyrosine phosphorylation, and induction of immediate-early and early genes.
    [Show full text]