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HAEMATOPOIESIS Dr.Michael Bennett March 2007. HAEMATOPOIESIS RED CELLS ERYTHROPOIESIS GRANULOCYTES MYELOPOIESIS MONOCYTES PLATELETS THROMBOPOIESIS.

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Presentation on theme: "HAEMATOPOIESIS Dr.Michael Bennett March 2007. HAEMATOPOIESIS RED CELLS ERYTHROPOIESIS GRANULOCYTES MYELOPOIESIS MONOCYTES PLATELETS THROMBOPOIESIS."— Presentation transcript:

1 HAEMATOPOIESIS Dr.Michael Bennett March 2007

2 HAEMATOPOIESIS RED CELLS ERYTHROPOIESIS GRANULOCYTES MYELOPOIESIS MONOCYTES PLATELETS THROMBOPOIESIS

3 HAEMATOPOIESIS 1.Examination of the blood 2.Examination of the bone marrow 3.Stem cells 4.Conditions for haematopoiesis a.Bone marrow stroma b.Adhesion molecules c.Haemopoietic growth factors 5.Intracellular molecular mechanisms a.The cell cycle b. Apoptosis c. Signal transduction

4 HAEMATOPOIESIS 1.Examination of the blood 2.Examination of the bone marrow 3.Stem cells 4.Conditions for haematopoiesis a.Bone marrow stroma b.Adhesion molecules c.Haemopoietic growth factors 5.Intracellular molecular mechanisms a.The cell cycle b. Apoptosis c. Signal transduction

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9 CAUSES RED CELL ABNORMALITIES normal Liver disease, alcoholism, Oval in megaloblastosis macrocyte Iron deficiency,liver disease, haemoglobinopathies, post splenectomy Target cell Liver disease, alcoholismstomatocyte Iron deficiencyPencil cell Liver disease, post splenectomyechinocyte Liver disease, abetalipo-proteinaemia, renal failure acanthocyte

10 CAUSES RED CELL ABNORMALITIES Hereditary spherocytosis, autoimmune haemolytic anaemia, septicaemia microspherocyte DIC, HUS, TTP,cardiac valvesfragments Hereditary elliptocytosiselliptocyte myelofibrosis tear drops Oxidant damage eg G6PD deficiencybasket cell Sickle cell diseaseSickle cell Iron deficiency, thalassemiamicrocyte

11 MEAN LIFE SPAN Red cells 120 days Platelets 8-10 days Neutrophils 2-4 days Monocytes months Lymphocytes months

12 SITE OF HAEMOPOIESIS YOLK SAC up to 6 weeks FOETAL LIVER up to 6-7 months and SPLEEN BONE MARROW from 6-7 months

13 BONE MARROW Haematopoietic (Red Marrow) all bones at birth adults – axial skeleton and proximal long bones Fatty marrow (Yellow Marrow) begins to replace red marrow from 5-7 years

14 HAEMATOPOIESIS 1.Examination of the blood 2.Examination of the bone marrow 3.Stem cells 4.Conditions for haematopoiesis a.Bone marrow stroma b.Adhesion molecules c.Haemopoietic growth factors 5.Intracellular molecular mechanisms a. The cell cycle b. Apoptosis c. Signal transduction

15 BONE MARROW Haematopoietic (Red Marrow) all bones at birth adults – axial skeleton and proximal long bones Fatty marrow (Yellow Marrow) begins to replace red marrow from 5-7 years

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18 MARROW ASPIRATE MARROW TREPHINE

19 CELLS IN THE BONE MARROW 1. MYELOID PRECURSORS for neutrophils, eosinophils, basophils and monocytes 2. ERYTHROID PRECURSORS 3. MEGAKARYOCYTES 4. LYMPHOCYTES 5. PLASMA CELLS 6. RETICULOENDOTHELIAL CELLS 7. FAT CELLS 8. OSTEOBLASTS 9. OSTEOCLASTS

20 Bone Marrow Differential %

21 ERYTHROBLAST

22 NORMOBLASTIC ERYTHROPOIESIS proerythroblast basophilic erythroblast polychromatic erythroblast pyknotic erythroblast

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25 myeloblast promyelocyte myelocyte metamyelocyte stab polymorph immature nucleolus no granulation primary granulation secondary granulation no nucleolus indentation of nucleus MYELOPOIESIS

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32 EOSINOPHIL MYELOCYTE EOSINOPHIL

33 BASOPHIL MYELOCYTE BASOPHIL

34 Cirulating neutrophils Marginating neutrophils Stem cells Progenitor cells Myeloblasts promyelocytes myelocytes Metamyelocytes Bands Segmented neutrophils Mitotic pool Post-mitotic pool bone marrowblood 6-10 days6-10 hrs Myelopoiesis

35 MEGAKARYOCYTES PLATELETS

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40 MONOCYTES PROMONOCYTES MONOBLASTS

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43 monocyte migration sites

44 LYMPHOBLASTS LYMPHOCYTES

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46 PERIPHERAL BLOOD LYMPHOCYTES A. T CELLS (65-80%) CD2 POS CD4 helper - majority in PB CD8 suppressor/cytotoxic - majority in BM B. B CELLS (5-15%) CD 19, 20, 22 POS endogenous Ig molecules on surface membrane C. Natural Killer Cells kill target cells without MHC restriction

47 LYMPHOCYTE PRODUCTION Although marrow is a major site of “virgin” lymphocyte production, most circulating cells are produced in lymphoid tissue such as lymph nodes, spleen, thymus and lymphoid tissue of the GI and respiratory tracts. Lymphoblasts are difficult to differentiate from other blast cells.

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50 HAEMATOPOIESIS 1.Examination of the blood 2.Examination of the bone marrow 3.Stem cells 4.Conditions for haematopoiesis a.Bone marrow stroma b.Adhesion molecules c.Haemopoietic growth factors 5.Intracellular molecular mechanisms a. The cell cycle b. Apoptosis c. Signal transduction

51 PLURIPOTENT STEM CELLS LINEAGE COMMITTED PROGENITOR CELLS RECOGNIZABLE MARROW PRECURSORS

52 hemangioblast Hematopoietic stem cell endothelial cells MEP CMP GMP MKPEP RBCPlts CLP gransmonos GPMonoP HSC PROGENITOR CELLS

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54 ERYTHROID BURST

55 MARROW CULTURE : GE AND EOSINOPHIL COLONY

56 CD38- CD34+ HAEMOPOIETIC STEM CELL 1 in 20x10 6 Nucleated cells in marrow Capacity for Renewal Differentiation After 20 divisions 10 6 mature cells are formed Found also in peripheral blood

57 STEM CELL PLASTICITY Stem cells may be totipotent and generate all tissues of the body

58 HAEMATOPOIESIS 1.Examination of the blood 2.Examination of the bone marrow 3.Stem cells 4.Conditions for haematopoiesis a.Bone marrow stroma b.Adhesion molecules c.Haemopoietic growth factors 5.Intracellular molecular mechanisms a. The cell cycle b. Apoptosis c. Signal transduction

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60 STROMAL CELLS 1.MACROPHAGES 2.FAT CELLS 3.ENDOTHELIAL CELLS 4.FIBROBLASTS

61 EXTRACELLULAR MATRIX 1.FIBRONECTIN 2.HAEMONECTIN 3.LAMININ 4.COLLAGEN 5.PROTEOGLYCANS eg heparan, chondroitin

62 ADHESION MOLECULES Glycoprotein molecules that mediate attachment of haemopoietic cells, leucocytes and platelets to the extracellular matrix, endothelium and each other.

63 ADHESION MOLECULES These molecules on leucocytes are termed receptors and they bind to ligands on surfaces of target cells

64 EXPRESSION OF ADHESION MOLECULES MAY BE MODIFIED QUANTITATIVELY OR FUNCTIONALLY BY INTRA OR EXTRACELLULAR FACTORS eg upregulation by IL1,TNF, INF- γ, viral infection

65 IMPORTANCE OF ADHESION MOLECULES 1.INFLAMMATORY AND IMMUNE RESPONSES 2.PLATELET AND LEUCOCYTE VESSEL WALL INTERACTIONS 3.ON TUMOUR CELLS – MODE OF SPREAD AND TISSUE LOCALISATION

66 ADHESION MOLECULES 1.Immunoglobulin superfamily – Antigen receptors, T-cell receptors, growth factor receptors 2.Selectins – attach leucocytes and platelets to vessels walls 3.Integrins – mediate cell adhesion during migration and signals for growth and development 4.Cadherins 5.Syndecams 6.ADAMs

67 HAEMOPOIETIC GROWTH FACTORS 1.ERYTHROPOIETIN6. THROMBOPOIETIN 2.G-CSF 7. INTERLEUKINS 3.GM-CSF 8. TNF 4.M-CSF9. Flt LIGAND 5.SCF 10. INF   

68 HAEMOPOIETIC GROWTH FACTORS general properties 1.Glycoproteins 2.Act locally where produced or systemically 3.Act at low concentrations 4.Specific receptors on target cells 5.Produced by many cell types 6.Synergistic effects 7.Action of one factor may stimulate production of another growth factor or receptor 8. May effect more than one lineage 9. Multiple actions - self renewal of stem cells, differentiation of lineage-committed progenitor cells, maturation, functional activation, prevention of apoptosis

69 GROWTH FACTORS IN NORMAL HAEMATOPOIESIS IL-5 SCF GM-CSF

70 ERYTHROPOIETIN Source : Kidney and liver 34 kd glycoprotein Lineage specific Level regulated by a simple feedback mechanism in response to oxygen tension Uses: 1. Anaemia of chronic renal failure 2. AIDS HIV infection 3. Cancer patients receiving chemotherapy 4. MDS 5. Autologous blood transfusion

71 Peritubular Interstitial cells of outer cortex Peritubular Interstitial cells of outer cortex

72 G-CSF SOURCE : stromal cells endothelial cells monocytes and macrophages ACTIVITY : Induces proliferation and maturation of neutrophil progenitors Mobilises myeloid stem cells into PB Activates neutrophil function and prolongs their life span CLINICAL 1. Post chemotherapy APPLICATIONS 2. After BMT 3. collection of stem cells from PB 4. Kostman’s syndrome Causes bone pain

73 THROMBOPOIETIN Originally cloned as the ligand for the c-mpl receptor

74 THROMBOPOIETIN ACTIVITY lineage dominant factor megakaryocyte progenitor cell proliferation, maturation and platelet production. Affects also erythropoiesis and pluripotent stem cell proliferation

75 LEVELS OF THROMBOPOIETIN (TPO) INVERSELY RELATED TO PLATELET COUNTS

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77 THROMBOPOIETIN APPICATIONS not yet established a single dose may increase platelets from day 4, peaking even after day 8 half life 20-30 hours

78 HAEMATOPOIESIS 1.Examination of the blood 2.Examination of the bone marrow 3.Stem cells 4.Conditions for haematopoiesis a.Bone marrow stroma b.Adhesion molecules c.Haemopoietic growth factors 5.Intracellular molecular mechanisms a. The cell cycle b. Apoptosis c. Signal transduction

79 The Cell Cycle During development from stem to fully differentiated, cells in the body alternately divide (mitosis) and "appear" to be resting (interphase). This sequence of activities exhibited by cells is called the cell cycle. Interphase, which appears to the eye to be a resting stage between cell divisions, is actually a period of diverse activities. Those interphase activities are indispensible in making the next mitosis possible.

80 Interphase Lasts at least 12 to 24 hours in mammalian tissue. During this period, the cell is constantly synthesizing RNA, producing protein and growing in size. It can be divided into 4 steps: Gap 0 (G0), Gap 1 (G1), S (synthesis) phase, Gap 2 (G2).

81 Gap 0 (G0) There are times when a cell will leave the cycle and quit dividing. This may be a temporary resting period or more permanent. An example of the latter is a cell that has reached an end stage of development and will no longer divide.

82 Gap 1 (G1) Cells increase in size in Gap 1, produce RNA and synthesize protein. An important cell cycle control mechanism activated during this period (G1 Checkpoint) ensures that everything is ready for DNA synthesis.

83 S Phase To produce two similar daughter cells, the complete DNA instructions in the cell must be duplicated. DNA replication occurs during this S (synthesis) phase.

84 Gap 2 (G2) During the gap between DNA synthesis and mitosis, the cell will continue to grow and produce new proteins. At the end of this gap is another control checkpoint (G2 Checkpoint) to determine if the cell can now proceed to enter M (mitosis) and divide.

85 Mitosis or M Phase Orderly division into two similar daughter cells. Mitosis is much shorter than interphase, lasting one to two hours. As in both G1 and G2, there is a Checkpoint in the middle of mitosis (Metaphase Checkpoint) that ensures the cell is ready to complete cell division.

86 G1 & G2 CHECKPOINTS CONTROLLED BY 1.Cyclin dependent protein kinases (Cdk) that phosphorylate downstream protein targets 2. Cyclins which regulate Cdks

87 THE CELL CYCLE

88 APOPTOSIS Programmed cell death

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90 APOPTOSIS CASPASES Cysteinyl Aspartate-Specific Proteases Many caspase isoforms promote apoptosis activated by two main pathways: 1.the death receptor pathway 2. the mitochondrial pathway

91 DEATH RECEPTOR PATHWAY EXTRINSIC Fas (CD-95) and TNF receptor are membrane proteins exposed at the surface of cells Binding of FasL and TNF results in the formation of a death inducing signalling complex (DISC) which contains Fas-associated death domain protein

92 MITOCHONDRIAL PATHWAY INTRINSIC Mitochondria release cytochrome C cytochrome C binds to Apaf-1 aggregate to form apoptosomes Activate caspase-9

93 BAX Promotes apoptosis causes holes in the mitochondrial membrane and cytochrome C to leak out p53 induces the transcription of BAX after DNA damage

94 CD95L CD95 Death Domain Procaspase Active Caspase Cell death

95 Bcl-2 Anti-apoptotic stabilise mitochrondrial membrane Increased expression of the Bcl-2 gene may lead to neoplasia eg in follicular lymphoma t(14;18)

96 IMPORTANCE OF APOPTOSIS Normal modeling and embryonal development Removing calls with DNA damage, viral infection, cancer cells Cellular immune function

97 LACK OF APOPTOSIS IN CANCER Bcl-2 over expression in follicular lymphoma p53 mutation or lack leads to a poor prognosis in leukemia

98 SIGNAL TRANSDUCTION

99 CELL SIGNALING cells respond to external stimuli and respond by : 1.Changes in metabolism 2.Changes in electrical charge across plasma membrane 3.Changes in gene expression

100 SIGNAL (Ligand) RECEPTOR SIGNAL TRANSDUCTION NUCLEUS GENE EXPRESSION

101 3 major pathways in signal transduction 1.JAK/STAT 2.MAP kinase (mitogen activated protein) 3.PI3 kinase (phosphatidylinositol 3) All involve phosphorylation of tyrosine residues

102 JAK kinase = Just Another Kinase

103 JANUS god of gates and pathways

104 Type I cytokine receptors eg for Epo, G-CSF, GM-CSF,TPO, IL lack a cytoplasmic tyrosine kinase domain Use the JAK-STAT pathway to initiate signalling

105 Signal Transducers and Activators of Transcription

106 STRUCTURE OF JAK2 Four-point-one Ezrin Radixin Moesin domain

107 Epo R exists in a homodimeric state in the absence of ligand with the cytoplasmic domains 73 Å apart JAK bound to receptor by FERM domain to box 1 in a conformation that JH2 inhibits JH1 upon ligand binding – conformational shift bringing the cytoplasmic domains 39 Å apart The tethered JAK2 also undergoes a conformational change which results in a loss of apposition of JH2 to JH1 allowing its kinase activity.

108 The juxtaposition of the 2 tethered JAK2 kinases also allows cross activation and initiation of signal transduction by phosphorylation of : 1.tyrosine residues within the receptor 2.Molecules that promote cell survival and proliferation eg STATs,PI3K, Mapks. 3.Molecules that limit cell signaling eg SHP1, SHIP1 phosphatases

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110 JAK2 (V617F) Somatic Does not occur in normal population Polycythemia Vera 90-95% Essential Thrombocythemia 50-70% Idiopathic Myelofibrosis 40-50% Rare in other hematological conditions

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112 THE END


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