PHYSIOLOGY OF BLOOD FUNCTIONS OF BLOOD HOMEOSTATIC FUNCTION buffering thermoregulation (transport of heat) TRANSPORT OF SUBSTANCES (blood gases, nutrients, metabolites, vitamins, electrolytes...) HUMORAL CONTROL OF ORGANISM (hormones) DEFENCE OF ORGANISM (immune functions) BLOOD CLOTTING BASIC CHARACTERISTICS 'Suspension character »6 - 8% total body mass ■55% - fluid phase (plasma) 45% - formed phase (blood cells and platelets) White üioud cells Platelets Red blood cell «Adam Serum: from plasma during blood clotting - after consumption of fibrinogen BONE MARROW Size (1600-3000 grams), activity. Red bone marrow, yellow bone marrow. Pluripotent stem cells. Unipotent (determined) stem cells - differentiated cells. Extra-medullar haematopoiesis - liver, lien - CHILDREN. Medullar haematopoiesis - ADULTS. Bone marrow examination - punction. Bone marrow diseases. Bone marrow transplantation. (a} The bone marrow, hidden within the bones of the skeleton, is easily overlooked as a tissue, although collectively it ps nearly the size and weight of the liver] (b) Marrow is a highly vascular tissue, filled with blood sinuses, widened regions lined wfth epithelium. Mature blood cells squeeze through the endothelium to reach the circulation. Fragments of megakaryocyte break off to become plateEets. The stroma is composed of fibroblast-tike reticular cells, collagenous fibers, and extracellular matrix. Silverthorn, D. U. Human Physiology - an Integrated Approach. 6th. edition. Pearson Education, Inc. 2012. Section of yellow bone marrow. This bone marrow is yellow in its fresh state because of the abundance of yellowish adipocytes present. The hemopoietic (*) tissue is comparatively less abundant than in red bone marrow. The adipocytes, or fat cells, (Ad) appear as large circular clear spaces in this field. A megakaryocyte (M) and venous sinuses (S) are also labelled. Source: http://audilab.bmed.mcgill.ca/HA/ht ml/blood 7 E.html This bone marrow is referred to as red marrow because it contains few adipocytes, or fat cells, among an abundance of hemopoietic cells. It is difficult to identify the individual precursors of red and white blood cells because they are closely packed and condensed during the fixation of the tissue (*). The following elements are identified: a megakaryocyte (M), which is a very large polyploid cell responsible for the production of blood platelets one adipocyte (Ad) two blood sinuses (S). The walls of these vessels are the sites where newly formed erythrocytes and leukocytes pass from the connective tissue into the blood circulation. Epi physeal-Sí^ř-. irssr.«^ lins Articular surface Trabecular: bone Artery — Central sinus- Cortical — bone Marrow cavity- Epi physis Metaphysis Diaphysis Metaphysis Epiphysis Figure 11 Bone marrow anatomy. Haematopoietic stem cells {HSCs) reside jiiamly ivitluii turtle man'ov.'during adulth< n rd Bone marrow is a complex organ, containing many different haematopoietic and non-haematopoietic cell types, that is surrounded by a shell of vascularized and innervated bone, a, Minute projections of hone (trabeculae) are found throughout the metaphysis such that many cells in this region are closetothe bonesurface. b,The interface of hone and bone marrow is known as the endosteum, which is covered by bone-lining cells that include hone-forming osteoblasts and houe-resorbing osteoclasts. Arteries carry oxygen, nutrients and growth factors into the bone marrow, before feeding into sinusoids, which coalesce as a central sinus to form the venous circulation. Sinusoids are specialized venules that form a reticular network of fenestrated vessels that allow cells to pass in and out of circulation. There is a particularly rich supply of arterioles, as well as sinusoids, near the endosteum. c, Three-dimensional reconstructed photomicrograph from the bone marrow towards the endosteal surface (blue) from 50 um below the surface, revealing the rich network of vessels (red) (image courtesy of C. Lin, J. Spencer and J. Wu). Sma Her arteriolar vessels (white arrows) become larger sinusoidal vessels. The field of view is 350 um * 350 um. d, A cross-sectional view of blood vessels that run along the endosteal surface (EV) and that trans ition (white arrow) into sinusoids (S) that then course towards the central sinus (adapted with permission from ref. 31). e, The bone marrow is cellularly complex with CD 150+CD48~CD4L "lineage" HSCs (arrow) residing in close contact not only with vascular and perivascular cells (*, sinusoid lumens) but also megakaryocytes (large yellow cells) and other haematopoietic cells (image adapted with permission from ref. 125). In the enlargement on the right, CD 150 is shown in red and CD48, CD41 and lineage are shown in green. Morrison SJ, Scadden DT: The bone marrow niche for haematopoietic stem cells. Nature 2014, 505(7483):327-334. Proerythroblast Myeloblast Hernocytoblast 1 Lymphoblast Monoblast l Polychromatic erythroblast 1 Progranulocyte Lymphocyte Monocyte Erythrocytes Basophil Eosinophil Neutrophi L Granulocytes Acjranulocytes Leukocytes Megakaryoblast Megakaryocyte y Thrombocytes ff Ä Source: Wikimedia Commons s*ir-'tw^*i Ervirirocyics GranulrscysEa ri.ii-.s- Tnlli ndndrlhc^allc Fif I. Hierarchical organiiaiion of the hjemsLopotcric system. Hu lorsi-tcjitt hjematopoictic stem ccU (LT-H5C) resides at the apex of the literatehicjl rLifniatopoiclic SYHtcm jnd tan undergo idf-rencwal or sequential muLtilirieaEC different JatLujj In produce all the specij]-ixed blood cells in the body. The LT-HSC riiat dirfcientiates inro the short-term haematopoietic stem cell fST-HSCh which yields one at' '.in- types a\ multipoteut progenitors fMPP): the common myeloid progenitor (CMP) or the lymphoid multipotenf progenitor HLMfl3). Downstream, these progenitor celts gradually become more restricted in their potential to differentiate into oetls i>t other linearis. Eventually, the committed progenitors grdmitocyto-macjophasje progenitor (tiMP), mc^akflrycKytc^rytirrocyte progenitor (MEP( and common Lymphoid progenitor (CLP), can only give rise to one Lineage and produce mature blood cells. Fig 2. Haematopoietic stem cell niches in the bone marrow. Hac-matopokLk stem cells (HSCs) reside in specialiifid supportive iruc-fOQnvtrorjments or niches within the bone marrow. Quiescent or slow-cycling Jong-term haematopoietic stem cells fLT-HS("s> localize dose to the bone j bone manuH interface, a site known as the endosteal 01 osteoblastic iiUk-. Jn contract, fjst-cycjinr^ sborr-term-HSCs (ST-H5Cs) may be found in dose proximity to sinusoidal endothelial ceUs and ncri-l cells. This site, which is known as the perivascular niche, supports the proliferation and differentiation of HSCs. Endorteiid-Jteli Bene raw space ■stromal ce^l OiriublairL kT f lt-hsc i [ Bone matrix Ho MSH, Medcalf RL, Livesey SA, Traianedes K: The dynamics of adult haematopoiesis in the bone and bone marrow environment. Br J Haematol 2015, 170(4).472-486. Regulatory function of megakaryocytes (MKs)! control of bone marrow homeostasis mesenchyal stem cells (MSCs) = an important regulator of MKs function via production of cytokines and soluble factors role of MKs in modulating the replication and differentiation of osteoclasts and osteoblasts = regulation of bone formation and matrix reorganization MKs represent an important reservoir of bioactive hemopoietic and angiogenic factors MKs can directly regulate hemopoietic stem cells (HSCs) and next hemopoietic cells (mainly via IL-6) OSTEOCLASTS OSTEOBLASTS O PROLIFERATION £ř PROLIFERATION íl DIFFERENTIATION JJ DIFFERENTIATION OPG.TGF-ft. IL-10, TGF-r.VEGF, IL-13, GM-C5F POCF.SMPi, tc-50 koa ptcrteln [?> CELL-CELL CONTACTS f :. !- ! ANGIOGEŇEŠIS HSCs « ) ADIPOCYTES TSP3.I JM* PLASM A CELLS STEADY-STATE BONE MARROW OSTEOCLASTS OSTEOBLASTS MEGAKARYOCYTES /? mm M: •JTGF-fJl '.! PDGF /YSINUSOIDS' bone marrow fibros MEGAKARYOCYTES V ECM SYNTHESIS SDF-1 BONE MARROW POSTINJ Malara A, Abbonante V, Di Buduo CA, Tozzi L, Currao Mks participate in angiogenesis m, Balduini A: The secret life of a megakaryocyte: emerging roles in bone marrow homeostasis control. Cellular and Molecular Life Sciences 2015, 72(8):1517-1536. Sympathetic nerve Bone Mobilization Mesenchymal stromal cell osteolineage progenitors Figure 3 | Haematopoietic stem cells (HSCs) and restricted haematopoietic progenitors occupy distinct niches in the ooiie marrow. HSCs are found mainly adjacent to sinusoids throughout the bone marrow27™'31,33, where endothelial cells and mesenchymal stromal cells promote HSC maintenance by producing SCF84, CXCL12 (refs 17,33,62) and probably other factors. Similar cells may also promote HSC maintenance around other types of blood vessels, such as arterioles. The mesenchymal stromal cells can be identified based on their expression ofLepr-Cre64, Prxl -Cre6J, Cxcll2-G¥Pli or Nes-GFP transgenes63 in mice and similar cells are likely to be identified by CD 146 expression in humans54. Perivascular stromal cells, which probably include Cxcll 2-abundant reticular (CAR) cells33, are fated to form bone in vivo, express Ms-1 -Cre and overlap with CD45/Terl 19"PDGFRa*Sca-1* stromal cells that are highly enriched for mesenchymal stromal cells in culture6*. It is likely that other cells also contribute to this niche, these probably include cells near bone surfaces in trabecular-rich areas. Other cell types that regulate HSC niches include sympathetic nerves91,9!, non-myelinating Schwann cells (which are also Nes4)96, macrophages95 and osteoclasts97. The extracellular matrix120'121 and calcium56 also regulate HSCs. Osteoblasts do not directly promote HSC maintenance but do promote the maintenance and perhaps the differentiation of certain lymphoid progenitors by secreting CXCL12 and probably other factors'317,iM0. Early lymphoid restricted progenitors thus reside in an endosteal niche that is spatially and cellularly distinct from HSCs. O GCSF mobilizes HSCs by causing the release of proteolytic enzymes like elaslase, cathepsin G, MMP-2, and MMP-9 from neutrophils... H ...which inactivate SDF-1 by cleaving itsNH2-leiminal signal sequence. Homing Osteoblastic niche FIGURE 3 Simplistic model for hematopoietic stem cell mobilization and homing GCSF, granulocyte colony-stimulating factor; HSC, hematopoietic stem ceil; HPC, hematopoietic progenitor cell; SDF, stromal cell-derived factor; ECM, extracellular matrix. A_ E I Presentation of ECM-tethered SDF-1 induces transendothelial migraLion and homing to the HSC niche much like a hapmtactic molecular guidance system. Kopp HG, Avecilla ST, Hooper AT, Rafii S: The bone marrow vascular niche: Home of HSC differentiation and mobilization. Physiology 2005, 20:349-356. Morrison SJ, Scadden DT: The bone marrow niche for haematopoietic stem cells. Nature 2014, 505(7483):327-334. bone marrow contains endothelial cell precursors BONE MARROW-DERIVED ENDOTHELIAL CELLS H5 Herna i ig i oblast (Stem Cell) Fig, L Some possible differentiation pathways for endothelial cells ATP 3-Phosphoglycerate -* Rapoport-Luberin shunt 1 Phosphatase 2-Phosphoglycerate PEP L-ADP f^ATP Pyruvate NADH ^11 NAD* *\ Lactate Conducted vasodilation Highi oxygen demand Normal oxygan demand Figure 1. Cascade of events initiated by the entrance of erythrocytes into a tissue region (dashed oval) in which oxygen demand exceeds oxygen supply. |For clarity, a single erythrocyte (RBC) is enlarged along with the associated vascular cells to show the events that occur following the entrance of an erythrocyte into the region of tissue with high oxygen demand.] When oxygen supply does not meet oxygen demand, tissue oxygen tension (P03) decreases. This decrease in tissue PO? causes the hemoglobin oxygen content of the erythrocytes that perfuse the tissue region to decrease proportionally. This decrease in oxygen contenl initiates a series of events resulting in the release of ATP from the erythrocyte. The ATP then diffuses to the endothelium (Endc) where it binds to purinergic (P^) receptors resulting in the production of vasoactive mediators, either within the endothelium or the smooth muscle (SMC), which initiate vasodilation. This vasodilation is conducted (dashed arrow) in a retrograde fashion increasing flow and thus oxygen supply to the tissue region in need. Erythrocyte Adenylyl cyclase PKA CFiR ATP Reduced 02 content of hemoglobin Increased O2 demand in skeletal muscle Blood vessel endothelium TRENDS fl Endocrinology S Meiaholisrn Figure 2. Proposed pathway for regulated ATP release from erythrocytes in response to passage of these cells through areas of increased oxygen demand in skeletal muscle. The increase in oxygen demand leads to oxygen release from hemoglobin within the erythrocyte. Consequently, hemoglobin oxygen content decreases resulting in activation of the heterotrimeric G protein, Gi, leading to ATP release. ATP released from the erythrocyte can bind to purinergic receptors [P^) on the vascular endothelium resulting in the release of vasodilators and, ultimately, an increase in blood flow (oxygen delivery). Abbreviations: Gi and Gs= heterotrimeric G proteins - i = inhibitory, s = stimulatory; ATP = adenosine 5'-triphosphate; cAMP = 3'5'-cyclic adenosine monophosphate; Hb = hemoglobin; PKA= protein kinase A; CFTR= cystic fibrosis transmembrane conductance regulator; ? = an as yet unidentified mechanism; Pzy = Pzy purinergic receptor; ± = stimulation. Sprague RS, Stephenson AH, Ellsworth ML: Red not dead: signaling in and from erythrocytes. TRENDS in Endocrinology and Metabolism 2007, 18(9):350-355. MORPHOLOGICAL VARIATIONS OF ERYTHROCYTES Poikilocytes - drop-like erythrocytes Schizocytes - fragmented erythrocytes Spherocytes - volume normal, diameter smaller, thickness bigger Eliptocytes - ecliptic shape Leptocytes - thin, centrally concentrated haemoglobin (thalasemia, after splenectomy) Akantocytes - prickly prominences FRAGILITY OF ERYTHROCYTES Haemolysis - destruction of red blood cell membrane. Types of haemolysis: a) physical b) chemical c) osmotic d) biological (toxic) e) immunological Spherocytosis - disorders of protein net responsible for shape and elasticity of erythrocyte membrane - actin, ankyrin, spectrin. Disorders of glucose-6-phosphate-dehydrogenase. Erythrocytes life span: 120 days, role of lien (double circulation), splenectomy. Reticulocytes. Fig. 2. Peripheral blood smears in hereditary spherocytosis. {A) Typical hereditary spherocytosis. Characteristic spherocytes lacking central pallor are seen. (B) Severe, recessively inherited spherocytosis. Numerous small, dense spherocytes and bizarre erythrocyte morphology with anisocytosis and poikilocytosis associated with severe hemolysis are seen. Table 1 Classification of hereditary spherocytosis Carrier Mild Spherocytosis Moderate Spherocytosis Severe Spherocytosis3 Hemoglobin (g/dL) Normal 11-15 8-12 6-8 Reticulocytes (%) <3 3-6 >6 >10 Bilirubin (mg/dL) 0-1 1-2 >2 >2 Spectrin content (% of normal) 100 80-100 50-80 40-60 Peripheral smear Normal Mild spherocytosis Spherocytosis Spherocytosis Osmotic fragility fresh blood Normal Normal or slightly increased Distinctly increased Distinctly increased Incubated blood Slightly increased Distinctly increased Distinctly increased Distinctly increased a Values in untransfused patients. From Eber SW, Armbrust R, Schroter W. Variable clinical severity of hereditary spherocytosis: relation to erythrocytic spectrin concentration, osmotic fragility, and autoriemolysis. J Pediatr 1990;117:409-16. Gallagher PG: Abnormalities of the Erythrocyte Membrane. Pediatric Clinics of North America 2013, 60(6):1349-+. ERYTHROCYTE SEDIMENTATION Sedimentation rate indirectly corresponds to suspension stability of blood. Method of Fahreus-Westergren (FW). Physiological values: men - women Units: mm/10min, 1 hr, 2 hrs, 24 hrs Physiological causes of increased sedimentation. Pathological causes of increased sedimentation. THE BLOOD COUNT This table lists the norma! ranges of values. -58% plasma volume 100% MALES FEMALES Hematocrit Hematocrit is the percentage of total blood volume that is occupied by packed (centrifuged) red blood cells. 40-54% 37-^17% Hemoglobin (g Hb/dL* whole blood) The hemoglobin value reflects the oxygen-carrying capacity of red blood cells. ("1 deciliter (dl_) = 100 mL) 14-17 12-16 Red cell count (cel!s/uL) A machine counts erythrocytes as they stream through a beam of light. 4.5-6.5 x 103 3.9-5.6 x 103 Total white count (cells/uL) A total white cell count includes all types of leukocytes but does not distinguish between them. 4-11 x 103 4-11 x 103 Differential white cell count The differential white cell count presents estimates of the relative proportions of the five types of leukocytes in a thin blood smear stained with biological dyes. Neutrophils 50-70% 50-70% Eosinophils 1-4% 1-4% Basophils <1% <1% Lymphocytes 20-10% 20-40% Monocytes 2-8% 2-B% Platelets (per uL) Platelet count is suggestive of the blood's ability tD clot. ! 150-450 x 103 15D-450 x 103 ■ Fig. 16.3 Silverthorn, D. U. Human Physiology - an Integrated Approach. 6th. edition. Pearson Education, Inc. 2012. I able t actors causmg false crianges m trythrocyte ^edi mentation Kate Fitters ca using False increases Fatten causing: raise decreases Increased fibrinogen, globulin, cholesterol levels Gcheria Nigh room temperature Coagulation of Lhe blood sample Macrocytic anemia Increase In bile sails Menstruation Increase In plwspholiplcb Pregnancy Making the sedimentation simple wait mo re than two hours Tilting Of lying dewn of the ESR lube Increase In adrenal steroids Drugs:Dextrine, meUryldopa, ntethyserigicfe, pertJdllamine,procainamide, HypoJibfinofjenemia terjphylline, trifluoperida-le, vilampn A Hyperglycemia Hyperalbuminentla Leukocytosis Microcytic anemia. Drugs: ACTH, cortisone, ethambutolr quinine, salicylates (Adapted! I'm in A TexLbflof Natural Medicine, PiiittfnO and Murray, 1992) Table 3. Factors affecting Erythrocyte Sedimentation Rate (E5R) Increased E5R Decreased F. SR ht'jle Heavy Me til Poisoning Congestive heart failure Gal jVasculai Disease Polycythemia! Carcinomas SkkJe Cell Anemia Cell ot tissue Injury Göut afthri Iii Infections Inflammatory dtsord-ef s Leukemia Myocardial Infarction rJeohrrfe Syphilis (Ada pied from A Texlbouk uJ Natural Medici ne, Piiiurno and Murray, HAEMOGLOBIN Red pigment transporting oxygen. Protein, 64 450, 4 subunits. Hem - derivative of porphyrine containing iron, conjugated with polypeptides (globin). Embryonic haemoglobin: Gower I a Gower II (t2s2, a2s2), Portland Fetal haemoglobin: Hb F, (32y2, weaker binding of 2,3 DPG Adult haemoglobin: Hb A, a2(32 (141/146) Forms of haemoglobin: oxyhaemoglobin - 02 carbaminohaemoglobin - C02 methaemoglobin - Fe3+ in hem carboxyhaemoglobin - CO Gestation (months) Age (months) FIGURE 32-8 Development of human hemoglobin chains. HEMOGLOBIN (o) Hemoglobin and iron Iron {Fe) fngested from the diet. t Fe absorbed by active transport. f Transferrin protefn transports Fe in plasma. - Fe •transferrin - Intestine Plasma Uf — Bile-*-1_ Liver (Liver stores excess Fe as ferritin. Liver metabolizes bilirubin and excretes it in bile. Bone Marrow Bilirubin metabolites in feces Bone marrow uses Fe to make hemoglobin (Hb( as part of HBC synthesis ■ Fe- ■Heme- Hb- RBC synthesis Spleen Spfeen destroys w aid RBCs and converts Hb to bilirubin. Hb i SiFirubin RBCs live about 120 days in the blood. 0 Bilirubin and metabolites are excreted in urine and feces. Kidney Bilirubin metabolites in urine Silverthorn, D. U. Human Physiology -an Integrated Approach. 6th. edition. Pearson Education, Inc. 2012. 4 02 + Hbj • BPG 4 » HbR - (02)4 + H® + BPC : BPG stabilizes T form Tform Rform Abnormalities of haemoglobin production •haemoglobinopathy (abnormal structure of chains) •thalasemia (lower production of normal chains) •Sickle cell anaemia (Hb J) Synthesis and destruction of haemoglobin Hem: glycin a succinyl-CoA Globin: AMK Hem - globin: biliverdin, bilirubin (lumirubin - photo-therapy), bil TABLE 32-3 Partial amino acid composition of normal human |'i chain, and some hemoglobins with abnormal [3 chains.3 Hemoglobin Positions on Polypeptide Cha n of Hemoglobin 123 67 26 63 67 121 146 A (normal) Val-His-Leu Glu-Glu Glu His Val Glu His S (sickle cell) Val C ^San Jose Gly E Lys ^Saskatoon Tyr ^Milwaukee Glu ^Arabia Lys aOther hemoglobins have abnormal a chains. Abnormal hemoglobins that are very similar eiectrophoretically but differ slightly in composition are indicated by the same letter and a subscript indicating the geographic location where they were first discovered; hence, M5as|(atoon and MMi|waiJkee. Clinical aspects - Glycosylated haemoglobin (HbA^ • formed by hemoglobin's exposure to high plasma levels of glucose • non-enzymatic glycolysation (glycation)- sugar bonding to a protein • normal level HbA1- 5%; a buildup of HbA1- increased glucose concentration • the HbA1 level is proportional to average blood glucose concentration over previous weeks; in individuals with poorly controlled diabetes, increases in the quantities of these glycated hemoglobins are noted (patients monitoring) Sugar-CHO + NH2 — CH2—Protein Sugar—CH =N -CH2 —Protein Schiff base Amadori reaction Sugar—CH2—NH— CH2—Protein Glycosylated protein ERYTHROPOETIN Glycoprotein, 39 000, a2-globulin. Recombinant erythropoetin. Small amount in plasma, urine, lymph, foetal blood. Inactivation: liver Origin: kidneys (85-90%) - endothelial cells of peri-tubular capillaries in kidney core, liver (10-15%) Stimulation of release: tissue hypoxia of any origin, alkalosis, cobalt salts, androgens, catecholamines (ß-receptors) Effects: Erythropoetin responsive cell - differentiation into erythroid line: increase of synthesis of nucleic acids, increase of iron absorption in erythroid cells, stimulation of cells release from bone marrow into circulation Acclimation - adaptation to high altitude Osteoblasts - next cellular source of erythropoietin HIF signaling in cells of the osteoblastic lineage regulate EPO expression in bone under physiologic and pathophysiologic conditions. In addition to regulating erythropoiesis, EPO has also been implicated in the regulation of bone formation and repair. Wu C, Giaccia AJ, Rankin EB: Osteoblasts: a Novel Source of Erythropoietin. Current Osteoporosis Reports 2014, 12(4):428-432. Source Model Phenolype Reference Osteoblast (OSX- Re model my Increased trabecular bone volume associated with increased angiogenesis and Rankin et al. VHL) (moose) erythrupoiesis. EPO (4500: 6.000 Remodeling Increased bone volume in neonatal and adult mice associated with increased osteoblasts and Shiozawa et al U/Kg) (mouse) erythropoiesis. EPO {300 U/K.g) Remodeling Modest decrease in bone volume. Singbrant etal. (mouse) EPO (5000 U/Kg) Repair (mouse) Increased torsinal stiffness, callus density, and mineralised bone. HoJstetn et al. EPO (40 tig} Repair (mouse) Increased cartilaginous callus formation and bona healing associated with increased Wan et al. angiogenesis. EPO (1000 Ul Repair (mouse) Increased LlMT-i induced kmc rctvncratmn in a cranial detect model associated ^vnh Sun cc al. enhanced angiogenesis. EPO (500 IU) Repair (mouse) Increased bone v ulumc m Lin bndgaiy calvaria] delect model. Nair et al. EPO (500 IE/Kg) Repair (mouse) Increased bone volume and repair in an femoral segmental defect model associated with Holstetn et al. increased angiogenesis. EPO(500U/K.g) Repair (mouse) increased callus formation in a closed femoral fracture model. Garcia et al. EPO (250 IU/K&I Repair (rabbit) Increased bone fusion in a posLerulalerttl spinal fusion model associated with enhanced Rolling et ai. angiogenesis. (2011} EPO(900 IU) Repair (porcine) Modest increase in bone formation in a caKariat defect model. Rolfing cl al. (2013) EPO (000 IU) Repair (porcine) Increase in bone formation when combined tvith bone marrow concentrate in a Betseh etal, osteochondral defect model. EPO erythropoietin Myoblast proliteranbri Moused Rat * Human * ^-* 4" Myoblast dirrer^riafkwi and fusion Mo'JSe ■'' Rat * ^_Human *_ f Protgctkw -l,..!-!- i !|i....!. Mouse r Feat* EPO l).it?Hi-i Healthy if ^ Mimle 4 e«erwrstiofi and funtlaon *t Anaipgenesi-i ^ Hypertrophy Ť Angingenesii. ^ Oxidative capacity At vitro studies Rodent stud i pí FIGURE Z I Effects oř EPO in ikeferal mu.sc!«..', actuated fry EPO. X, nan activated by EPO. f, conrradictorv results. Iff wijq EPO treatment increases -mause, hut nnl ra: or human rryublasl proliferation. E50 treatment oiecreasea diiterentiaiion arid fusion at mnuaa, but '-a: fat or human myoblasls. EPO treatment protects against apootosji in mause but not in rat myoblasts. In rodem s, EPO treatment increases muscle regeneration ana anginoenesis follůvving injury In humans, EPO trearment increases slelet6l muscJa hypertrophy and engioganesis in diseased concfiiions (ctironic renal failure and Friedreich ataxia, respectively), but has fin affect in fiealthy muscle. In boTn mcenr and human stucfias: EPO has been shown to increase or have no effect on muscle c*joative capacity. Male that it is prasemly urrknoATi if the affects of EPO treatment csjserved in rodent and human skeletal muscle are direct or indirect Lamon S, Russell AP: The role and regulation of erythropoietin (EPO) and its receptor in skeletal muscle: how much do we really know? Frontiers in Physiology 2013, 4. EPO and brain No.J'On Table 1. functions of Epe Function Endothelial calls FIGURE '. Expressen pattern of Epc/EpoR in the brain Whereas Epo expression iE restricted to astrocytes and neurians, EpaR is expressed an the surface of endothelial cells, microglia, astrocytes, oligodendrocytes, and neurons. Epe ts thought to act in an autocrine as well as paracrine mann Er. Description Neuroprotection Infusion of soluble Epofi into the brain of gerbils, subjet'.ed to a mild form of iit-iemia, cajsed neu- ronal death in the h ippOCampus. Neurotrophic factor Regeneration of septal cholinergic neurons in aduk rats, which had undergone fimbria-fornix transections. Promotion of the survival end differentiation of dopaminergic precursor neurons in vitro. Neurogenesis Anti-inflammation AngiOgenesis Hypoxia-induced Epo production acts directly on neuronal stem Cells in the tOrehrain. Indirectly by inducing BDNF expression. Reduced production of inflammatory mediators leading to: Cerebral ischemia: smaller infarcts. Multipfe sclerosis: protection. Optic neuritis: improved survival of retinal ganglion cetls. Mitogenic action ore Human umbilical vein. Adrenal capillary endothelial cells. Brain Capillary endothelial cells. Angiogenic action on: Rat aortic rings. Mouse endometrium. Chick embryo thorioallantonic membrane. Vascular permeability vitro: BBS protection against VEGF-induted increase in vascular permeability Refs. 95 107 107 99 113 112 2, 96 ■: 4 121 19 123 90 75 BDNF, brain-derived neurotrophic factor; BBB, blood-brain barrier; VEGF, vascular endothelial growth factor. Rabie T, Marti HH: Brain Protection by Erythropoietin: A Manifold Task. Physiology 2008, 23(5):263-274. tHuEPO Oxygen tension Production ol endogenous EPO Figur« 1. How high levels of circulating recombinant EPO may result in suppression of endogenous EPO synthesis secondary to a decrease In in tra renal oxygen corny mption. by Intrinsic renal effects (1) EPO ses raabsofption of sodium and fluid in the proximal lubufe. thereby direnly reducing the major owytiHCDiisuminy process in the kidney; {2) increase in euiJ-prmimai tubular deiweiy to she macula densa decreases renin release and subsequent angiotensin Ik and aldosiercme-dependent reabsorption tfi more distal nephron segments; {3; decreased proximal lubuEar reabsorpticm activates the tubdoglümerulaf feedback mechanism producing a Fall m GFR and- reduction of the filtered Foad; (4) the resulting increase in renal oxygen partial pressure in vie environment a( interstitial Fibrobiast-like cells down-regulates the hypoxia-inducible factof-2'dependent praductton of endogenous EPO. Lundby C, Olsen NV: Effects of recombinant human erythropoietin in normal humans. Journal of Physiology-London 2011, 589(6):1265-1271. ERYTHROPOESIS Substances affecting erythropoesis Need of copper Ceruloplasmin - binding protein (a2-globulin) with ferroxidase activity. Oxidation of Fe2+ to Fe3+ is necessary for binding of iron to transferrin. Need of cobalt Part of vitamin B12 molecule. Vitamin Bl2 (cyancobalamin) Produced by bacteria in GIT. Source: liver, kidneys, meet, milk products... Resorption: necessity of s.c. intrinsic factor secreted by parietal cells of gastric fundus and body. Bound to transcobalamins in blood. Stored in liver, pancreas, kidneys, brain, myocardium. Function: synthesis of nucleic acids, co-factor in conversion of ribonucleotids to deoxyribonucleotids, production of metabolic active forms of folic acid NECESSARY FOR NORMAL DIVISION AND MATURATION OF RED BLOOD CELL LINE ELEMENTS. Symptoms of anaemia after years only!!! Pernicious anaemia. Folic acid (pteroylglutamic) Produced by higher plants and micro-organisms. Source: green vegetables, yeast, liver, kidneys... Function: part of co-enzymes during synthesis of DNA, participation in cell division and differentiation Deficiency: deficient nutrition, treatment with cytostatics (methotrexate) Symptoms of anaemia already after couple of months!!! Macrocyte hyperchromic anaemia. Other vitamins Vitamin B6 (pyridoxine) - metabolism of amino acids, synthesis of hem Vitamin B2 (riboflavin) - part of flavoprotein enzymes - reductases of erythrocytes (normal function and survival of erythrocytes). Normocyte anaemia with lower reticulocytes count. Vitamin C (ascorbic acid) - non-specific function in erythropoesis. Hormonal influences Androgens, estrogens, hormones of thyroid gland, glucocorticoids, growth hormone. ANAEMIA Disorder, in which basic and characteristic feature is lower amount of haemoglobin. Usually also haematocrit and red blood cell count in 1 litre of blood are below physiological value. CLASSIFICATION OF ANEMIAS MORPHOLOGICAL CLASSIFICATION Evaluation of erythrocyte volume and concentration of haemoglobin in erythrocytes 1. Normocyte anaemia 2. Microcyte a. 3. Macrocyte 1. Normochromic anaemia 2. Hypochromic a. PATHOPHYSIOLOGICAL CLASSIFICATION Anaemias caused by inefficient blood production Sideropenic anaemias - lack of iron Megaloblastic a. - lack of vitamin B12 or folic acid Anaemias caused by suppression of blood production Anaemias in chronic diseases and symptomatic anaemias Thalasemia Anaemias caused by increased losses Haemolytic a - caused by increased destruction of erythrocytes Chronic posthaemorhagic anemia Acute posthaemorhagic anaemia ANTIGENS AND ANTIBODIES OF RED BLOOD CELLS 1) History of blood transfusions. 2) Posttransfusion reactions: aglutination, haemolysis (immediate or delayed), life-threatening complications (jaundice, damage of kidneys, anuria, death - in case of full blood or RBCs administration, in case of plasma - dilution of aglutinins!!! Autoimmune diseases. Paternity tests, event, transplantology. 3) Antigens of blood cells: a) 30 antigen systems (ABO, Rh, MNSs, Lutheran, Kell, Kidd, Lewis, Diego, P, Duffy...) b) hundreds of other - „weak" - antigens (important for paternity testing, organ transplantations) 4) Aglutinogen: antigen of plasmatic membrane of cells - complex oligosaccharide - erytrocytes, salivary glands, pancreas, liver, kidney, lungs, testes - saliva, sperm, amnionic fluid, milk, urine 5) Aglutinin: antibody against aglutinogen, y-globulin (IgM -ABO system, IgG - Rh system), produced in the same way as other antibodies - after births almost zero concentration in blood - production of aglutinins begins 2-8 months after birth: stimulation by antigens similar to aglutinogens - in food, in GIT bacteria - maximal concentration of antibodies is reached in 8-10 years, decreases gradually with age Blood group systems ISBTNS™ I System name ; System symbol ; Epitope or carrier, notes * Chromosome ; 001 ABO ABO Carbohydrate (N-Acetylgalactosamine, galactose). A. B and H antigens mainly elicit IgM antibody reactions, although anti-H is very rare, see the Hh antigen system (Bombay pbenotype ISBT#18). 9q34.2 002 MNS MNS GPA/ GPB (grycophorins A and B) Main antigens M. N. S. s. 4q31.21 003 P P Glycotipid. Three antigens: Pi. 1'. and Pk 22q13.2 004 Rh RH Protein. C. c. D. E. e antigens (there is no "d" antigen: lowercase "d" indicates the absence of D). 1p36.11 005 Lutheran LU Protein (member of the Immunoglobulin superfamiiy). Set of 21 antigens 19q13.32 006 Kell KEL Glycoprotein. Kj can cause hemolytic disease of the newborn (anti-Kelt), which can he severe. 7q34 007 Lewis LE Carhohydrate (Tucose residue) Main antigens Lea and Leb— associated with tissue ABH antigen secretion. 19p13.3 008 Duffy FY Protein (chemokine receptor). Main antigens Fya and Fyb. Individuals lacking Duffy antigens altogether are immune to malaria caused by Plasmodium vtvax and Plasmodium knowlesi. 1q23.2 009 Ktdd JK Protein (urea transporter) Main antigens Jka and Jkb. 1Bq12.3 010 Diego Dl Glycoprotein (band 3. AE 1. or anion exchange). Positive blood is found only among East Asians and Native Americans. 17021.31 011 Yt YT Protein (AChE: acetylcholinesterase). 7g22.1 012 XG XG Glycoprotein. Xp22.33 013 Scianna SC Glycoprotein. 1p34.2 014 Dombrock DO Glycoprotein (fixed to cell membrane by GPI, or glycosyl-phosphatidyl-inositol). 12p12.3 015 Co Bon CO Aquaporin 1. Main antigens Co(a) and Co(b). 7p14.3 D16 Landsteiner-Wiener LW Protein (member ol the immunoglobulin superfamiiy). 19p13.2 017 Clrido CH C4A C4B (complement fractions). 6p21.3 018 Hh H Carbohydrate (fucose residue) 19q13.33 019 XK XK Glycoprotein. Xp21.1 020 Gerbtcb GE GPC / GPD (Grycophorins C and D). 2qt4.3 021 Cromer CROM Glycoprotein (DAF or CD55, regulates complement tractions C3 and C5. attached to the membrane by GPI). 1q32.2 022 Knops KN Glycoprotein (CR1 or CD35, immune complex receptor). 1q32.2 023 Indian IN Glycoprotein (CD44 adhesion function?). ffp13 024 OK OK Glycoprotein (CD147). 19p13.3 025 Raph RAPH Transmembrane glycoprotein. f1p15.5 026 JMH JMH Protein (fixed to cell membrane by GPI). Also known as Semaphore 7A or GDI 08. 15q24.1 027 li 1 Branched (1) i unbranched (i) polysaccharide. 6p24.2 028 Globoside GLOB Glycolipid. Antigen P. 3q26.1 029 GIL GIL Aquaporin 3. 9p13.3 030 Rh-associated glycoprotein RHAg Rh-associated glycoprotein. 6p21-qter 031 Forssman FORS Globoside alpha-1.3-N-acetylgalactosaminyltransterase 1 (GBGT1) 9q 34.13 032 Längere is^ LAN ABC BE. Porphyrin transporter 2q36 033 Junior^ JR ABCG2. Multi-drug transporter protein 4q22 034 Vel Vel Human red cell antigens 1p36.32 035 CD59 CD59 11p13 A-B-O SYSTEM Genotype Blood group Aglutinogen Aglutinin 00 0 (H) anti-A a anti-B OA or AA A A anti-B OB or BB B B anti-A AB AB A and B - Described by Landsteiner in 1901, 1930 - awarded by Nobel Price. Janský-1906. Frequency of blood groups in ABO system: O 47% (38%) A 41% (42%) B 9% (14%) AB 3% (6,5%) Subgroups in A a B blood groups. Aj (1 million copies of antigen on 1 ery), A2 (250 thousands copies). Heredity: both A and B is inherited dominantly, according to Mendel's law. Rh SYSTEM Monkey Maccacus rhesus. 40th of the 20th century, Wiener a Landsteiner. Frequency: 85% - Rh+, 15% - Rh". Antigens D, C, E, d, c, e. Present only on erythrocytes. D - the „strongest" antigen: Rh - positive, Rh - negative (produces anti-D aglutinin after contact with D-erythrocytes). Aglutinins production: only after the contact with D-erythrocytes (transfusion, foetal erythroblastosis). High concentration of anti-D antibodies lasts for many years!!! HAEMOLYTIC JAUNDICE OF NEWBORNS Rh-negative mother x Rh-positive foetus. First pregnancy - immunisation of mother during delivery (or interruption or miscarriage!!!). Next pregnancy - anti-D aglutinins (IgG) cross foetoplacental barrier. Foetus damage: approx. in 17% of next pregnancies Haemolysis of foetal erythrocytes - haemolyti disease of newborn (erythroblastosis fetalis): •anaemia •jaundice •oedemas - event, hydrops fetalis •CNS damage (icterus) -bile acids enter CNS (no haematoencephalic barrier!) •deaths of foetus in utero Prevention of foetal damage: 1) administration of small doses of anti-D antibodies to mother during pregnancy 2) administration of one dose of anti-D antibodies during postpartum period Success of therapy: up 90%. Newborn (RhD +) 1st Pregnancy Mother (RhD -) Fetus (RhD+) Fetal-maternal blood transfer during labor Coombs test of mother for anti-D Abs RhJD) tg therapy to mother to prevent sensitization to RhD Mother (RhD -) (sensitized to RhD antigen) m Next Pregnancy (RhD+ fetus) Father is DAi or D/D increased bilirubin, CNS damage fkemicterus), death Mild anemia, jaundice Severe Mifd case Repeat encounter with fetal RhD antigen Fetal or Newborn Hemolytic Anemia MaternaUgG anti-D crosses placenta IgG anti-D attaches to fetal RBCs & marks them for destruction \7 Rapid production of IgG anti-D by mother