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These are lecture summaries from the haematology part of the subject. ...

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Lecture 2- Cells of the blood and immune system 

Production of cells means you have a way to transport things and defend yourself

Blood components  Blood is a liquid tissue that consists of  Cellular components  Red blood cells (erythrocytes)  Platelets (thrombocytes)  5 types of white blood cells (leukocytes)  Plasma (liquid that cells are suspended in) Functions of blood  Transport of gases, food molecules, ions, wastes, hormones and heat through the body  Defence of the body against infections, injury and cancer Blood examination  Hematocrit- the portion of red cell that settle to the bottom of the tube once it’s been centrifuged  Normally the hematocrit is 45%  Other cell components (e.g. leukocytes, platelets) sit on top of this and it’s called the buffy coat  Remainder is plasma (approx. half)  Can also examine blood by smearing it on a glass slide and staining it with a dye to aid in visualization  Blood biochemistry- a blood sample can also be analysed to detect other components such as hormones, proteins and metabolites Blood components Plasma  Consists of water, ions (e.g. Na+, Ca2+, HCO3-), organic molecules (e.g. glucose, amino acids, lipids, urea), proteins, trace elements, vitamins, gases (e.g. CO2, O2) Plasma proteins  Albumin  54% of plasma proteins  acts as a carrier for steroids, fatty acids, cholesterols, which are then transported throughout the body  contributes to plasma osmotic pressure, which is why your body can stand trauma through blood loss. The albumin keeps enough liquid in the blood for it to keep functioning 1

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Globulins  38% of plasma proteins  3 types- Alpha, beta and gamma  Alpha transport bilirubin and steroids  Beta transport ion and copper  Gama are antibodies in the immune system Fibrinogen  7% of plasma proteins  Play an important role in the clotting process (it is converted to fibrin Other proteins that are transported in the plasma such as hormones (e.g. insulin, growth hormone, leptin, EPO) and cell signaling molecules (e.g. cytokines, chemokines)

Cell components Red blood cells (erythrocytes)  Most numerous cell type in the blood  Highly specialized cells  Do not have a nucleus, nor other organelles  Essentially just a sack filled with hemoglobin  Major role in gas transport- O2 from lungs to tissues, CO2 from tissues  Terminally differentiated cells- they cannot divide  120-day life span- then ingested by leukocytes in the liver and spleen at a rate of 3 million per second  Most of the iron in the hemoglobin is recycled  Some of the haeme portion of the molecule is degraded into bile pigments and excreted by the liver Platelets  Cell fragments from megakaryocytes  Key role in blood clotting  Not as many RBC  Blood loss is stopped by solidification of the blood; a process known as coagulation or clotting  A blood cloth consists of a plug of platelets in a network of insoluble fibrin molecules White blood cells (leukocytes)  These cells have nuclei but altered morphology  Key role is in defending the body from insults such as infection and injury  There are five cell types divided into two groups  Granulocytes- neutrophils, eosinophils, basophils  Agranulocytes- monocytes/macrophages, lymphocytes 2

Neutrophils  Most abundant WBC- 50-70%  Key role is destruction of microbes (e.g. bacteria, fungus)  Numbers increase is response to infection  Possess neutral staining granules which contain a range of antimicrobial enzymes  Have a lifespan in the blood of 6-10 hours so they need to be replenished a high rate Eosinophils  Low levels in the blood (1-4%)  Increase in numbers in certain diseases, especially infections by parasitic worms  Are cytotoxic- they release the contents of their eosinophilic granules to kill invading organisms Basophils  Rarest WBC (less than 1% on blood)  Increase in numbers during infection and inflammation  Leave the blood and accumulate at the site of infection or inflammation  Discharge the contents of their basophilic granules Monocytes/Macrophages  2-8% of WBC’s  Monocytes leave the blood and develop into specialized cells, including macrophages  Macrophages are large phagocytic cells that engulf foreign material and dead/dying cells Lymphocytes  Second most common WBC (20-40%)  Several kinds of lymphocytes each with a different function  B lymphocytes (B cells) are the most common and are responsible for making antibodies  T lymphocytes (T cells) include three types  Helper T cells (Th) enhance the production of antibodies and recruit macrophages and neutrophils  Cytotoxic T cells (Tc) kill virus infected and tumour cells  Regulatory T cells (Treg) control immune responses

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Organs and tissues of the blood and immune system AGM/Foetal liver  The first blood and immune precursors originate in the AGM  These then travel to the foetal liver and the thymus to generate the first mature blood cells Bone marrow  Major source of blood cell production in adults  Found mostly in the pelvis, ribs and long bones  Blood cell development occurs in the active red marrow  At birth there is 100% red marrow, which is replaced by inactive yellow marrow (around 50%) which is composed of adipose tissue  Composed of a network of epithelial cell lined sinusoids interspersed with islands of heamatopoietic cells  Supported by connective tissue elements that form the bone marrow stroma (fibroblasts, adipose tissue and endothelial cells Lymphatic system  A series of canals that are filled with fluid from the interstitial space  Filter through at least one lymph node before it re-enters the circulation  Organs of the lymphatic system: Tonsils, thymus, lymph nodes, lymphatic vessels, liver, spleen, payer’s patch on small intestine, appendix and bone marrow 4

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Lymph nodes  Encapsulated kidney shaped organs  Distributed along lymphatic vessels in clusters  Contain lymphocytes Spleen  Blood cell development (both as a fetus and an adult during infection  Immunity- contains 25% of lymphoid mass of the body  Macrophages remove of old/damaged RCBs, bacteria Thymus  Key site of T cell maturation and differentiation

Lecture 3- Blood and immune cell development Haematopoiesis  Blood cells are constantly being replaced- 1011 a day  Blood cell development is an ongoing process throughout life  Occurs in several phases and locations during the life cycle  Represents a classical stem cell driven process Stem cells  Stem cells are unique in their ability to undergo asymmetric division to yield daughter cells that are able to either  Self-renew to produce more stem cells  Differentiate along specific developmental lineages  In the transition from stem cell to fully differentiated (mature) cell the ability to self-renew and proliferate changes dramatically

Hematopoietic stem cells (HSC’s)  Adult HSC’s that are in the bone marrow are very rare (only 1 in 10,000 bone marrow cells)  They are intimately mixed up with connective tissue and stromal cells (niche) 5

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Characterized by specific surface proteins (e.g. CD34) Are generally in a quiescent state Initially pluripotent, the HSC first becomes committed to ever more specific lineages This is followed by rounds of replication and further commitment to generate all mature blood cell lineages Complex control to balance  Self-renewal  Lineage commitment  Proliferation  Differentiation

Hematopoiesis  HSC’s both self-renew as well as yield daughter cells that are multipotent

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Multipotent progenitor become committed to specific lineages via either the common lymphoid progenitor (CLP) or the common myeloid progenitor (CMP)

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CMPs become further committed to the granulocyte/macrophage progenitor (GMP) or the megakaryocyte/erythrocyte progenitor (MEP) lineage CLP’s become further committed to the T cell and B cell lineages

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Further rounds of lineage commitment give rise to other cell types  MEP- megakaryocytes/platelets and erythrocytes  CMP- granulocytes (neutrophils, basophils and eosinophils) and monocytes/macrophages  Lymphoid cells- T cells/subsets, B cells/plasma cells and NK cells

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Along each lineage  There is a massive increase in cell number  Peak of proliferation is at progenitor stage  Cells differentiate into mature cells that lose the capacity to proliferate

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Hematopoiesis during the life cycle  Occurs in a number of distinct waves  These occur at different anatomical sites and exhibit unique developmental programs Primitive hematopoiesis  Very early in embryonic development  Occurs in so-called blood islands across the yolk sac  Restricted cell outputs  Anterior- primitive macrophages  Posterior- primitive RBC’s and neutrophils Definitive hematopoiesis  First HSC’s develop in the AGM and then move to the fetal liver and spleen, and then to the bone marrow and thymus  Full hematopoietic program Emergency hematopoiesis  Occurs in response to major insults such as infection, trauma or anemia  HSC’s are mobilized into the blood and can differentiate there  Liver and spleen can recommence ‘extramedullary’ hematopoiesis Clonality in hematopoiesis  Hematopoiesis is a stem cell mediated process  However, at any one time only a subset of HSC’s contributes to blood cell production  Therefore, each blood cell is derived from a limited number of parent stem cells 8

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The process is best described as clonal- strictly oligoclonal The Clonality concept continues with the various progenitor cells

Diseases of hematopoiesis Disorders of deficiency

Disorders of excess

Lecture 4- Factors controlling blood/immune cell development and function Control of haematopoiesis  

Complex control to balance self-renewal, lineage commitment, proliferation, differentiation and survival Two levels of control  Intrinsic- internal factors 9

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Extrinsic- external factors

Intrinsic control  Because there is a constant need for new blood and immune cells, there is a basal level of production of all cell lineages  For each progenitor cell this is at least partially stochastic (by chance) following pre-determined blue-prints  This is largely mediated by transcription factors Transcription factors  Proteins that control the transcription of specific target genes  Bind to specific sequences usually upstream of a gene  Can activate or repress transcription  Often work with other transcription factors  Often called master regulators  Target genes may include other transcription factors which increase specialization  Mediates a self-propagating wave of differentiation, reflecting lineage commitment Hematopoietic transcription factors  SCL- HSC’s/MPs only (not lineage specific)  GATA2- only CMP’s  IKAROS- CLP and some lymphoid cells  GATA1- MEP’s/erythroid lineage  SPI1- GMP’s and some lymphoid cells  PAX5- B cells  C/EBPα- GMP’s  These mediate a transcriptional program that mediates the cellular changes that define each cell type  GATA1-globin genes  C/EBPα- myeloperoxidase  IKAROS- recombination genes  Provide the blue print that makes each specific cell  Can positively or negatively regulate transcription  They are often antagonistic- lineage switches whereby one increases and the other will decrease

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Extrinsic control  In bone marrow HSC’s are exposed to support cells (endosteal niche) and the circulation (vascular niche)  This represents the physical machinery that controls quiescence, differentiation, proliferation, survival, homing and mobilization  The HSC’s receive signals in the form of direct cell-cell interactions, as well as a variety of soluble factors including oxygen, cytokines, growth factors and chemokines Cell-cell interactions  Principally via interactions between stromal cells and HSC’s  Mediated by cell surface molecules such as adhesion molecules (e.g. integrin’s and cadherin’s) and receptor-ligand interactions (e.g. Tie/Ang, Notch-Jagged)  Send signals into the HSC’s that control key events such as quiescence or activation and subsequent lineage commitment and differentiation Oxygen  If there is less than 1% oxygen in the HSC niche, then quiescence will be favored  1-4% of oxygen in the sinusoids then self-renewal, expansion and differentiation will be favored  these lower levels of oxygen contrast with the 10% oxygen in peripheral blood  Acts via the hypoxia inducible factor (HIF) which is induced by low oxygen levels

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Cytokines, growth factors and chemokines  Are secreted proteins and used for cell-cell communications  They are often produced/modulated in response to act via external/environmental stimuli such as infection and hypoxia  Act via specific cell-surface receptors  Many are involved in haematopoiesis or blood/immune functions  Action is tightly regulated  Provide a mechanism to respond to environmental insults  There is a tight negative feedback control that extinguishes the signal to maintain homeostasis Hematopoietic cytokine and growth factors  20+ cytokines and growth factors that affect hematopoietic cell proliferation and differentiation including  Interleukins (IL’s)  Colony-stimulating factors (CSF’s)  Erythropoietin (EPO)  Thrombopoietin (TPO)  Stem cell factor (SCF)  Each target specific hematopoietic populations which is determined by expression of the corresponding receptor  Receptor binding activates specific intracellular pathways, which may be shared between different receptors  The key difference between receptors is that growth factor receptors have intrinsic tyrosine kinase activity but cytokine receptors do not  Act as a timer switch to send specific instructions to the cellI  In terms of haematopoiesis they  Guide cells down specific pathways by influencing lineage choice, enhancing proliferation and differentiation  Impact on cell function  Are not master regulators Cytokine receptor signaling  Cytokine receptors act via associated tyrosine kinases including the Janus kinase (JAK) family  These activate the signal transducer and activators of transcription (STAT) transcription factors  STAT’s bind to specific promoter sequences to regulate genes involved in the key pathway affecting survival, differentiation, proliferation and negative feedback (suppressor of cytokine signaling or SOCS)

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Chemokines  This class of proteins acts via a different type of receptor- the G-protein coupled 7 transmembrane receptor  Utilize alternative signaling pathways to alter gene expression  Key role is in controlling the migration of blood and immune cells by chemotaxis and retention

Lecture 5- Erythrocytes and their function Erythrocyte properties       

6 Most numerous cell in the body ~5 x 10 /ul x ~5 litres blood = 2.5 x 13 10 cells Highly specialised for gas transport Biconcave disc- large surface area Flexible- able to squeeze through small blood vessels Lipid bilayer membrane that has integral membrane proteins for strength and flexibility No nucleus, mitochondria or ribosomes It has enzymes for glycolysis (main energy source is glucose) and pentose phosphate pathway

Oxygen transport- Hemoglobin  Erythrocytes are packed with hemoglobin  Hemoglobin is composed of i. Globin  a tetrameric protein complex  a pair of α chains and a pair of β chains 13

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Haem  iron containing prosthetic group  that gives erythrocytes their red colour.  There is one haem per globin chain (4 in total)  Consists of a protoporphyrin ring that is complexed with an iron molecule  Each can bind one molecule of oxygen (4 oxygen per Hb) Key role for Hb is to load and unload oxygen Affinity of Hb for oxygen is impacted on by several factors such as temperature and pH During development the specific Hb expressed changes Foetal hemoglobin (HbF: α2Y2) is the major oxygen transport protein during the last seven months of development HbF differs most from adult haemoglobin (HbA: α2β2) in that it is able to bind oxygen with greater affinity so that it can access oxygen from the mother’s bloodstream

Carbon dioxide transport  About one half is directly bound to hemoglobin (at a site different from the one that binds oxygen)  The rest is converted by an enzyme (carbonic anhydrase) into  Bicarbonate ions (HCO3-) that diffuse back out into the plasma  Hydrogen ions (H+) that bind to hemoglobin Erythrocyte production  Erythrocytes are derived from specific precursors in the bone marrow  During this process (erythropoiesis)  They manufacture hemoglobin until it accounts for 90% of the dry weight of the cell  The nucleus is squeezed out of the cell (enucleation) and ingested by macrophages

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Transcription factors  Erythropoiesis is driven by members of the GATA and EKLF families of zinc finger transcription factors Cytokines and growth factors  Early phase- stem cell to committed erythroid precursor (stem cell factor (SCF) 14

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 Late phase- erythroid precursor to mature red blood cell (EPO) Erythropoietin  EPO is produced by the kidney in response to low oxygen levels  Stimulates erythrocyte precursor cell to proliferate and mature into RBC’s and release into the circulation  EPO acts via several pathways  Key role is to protect against cell death (it is anti-apoptotic)  JAK2/STAT5 increases the anti-apoptotic gene BcL-XL  PI3K/AKT1 inhibits the pro-apoptotic genes Bad, Caspase 9

Erythrocyte destruction  Normal life span is 120 days  Destruction due to wear and tear  Shearing forces  Repeated RBC deformation  Oxidative damage  Osmotic forces  Reduction in normal enzyme activity  Altered protein expression  Old RBC’s are destroyed by specialized macrophages in the spleen and liver  Some degradation produced (iron and amino acids) and recycled  The haem molecule is converted to bilirubin and transported to the liver  It is excreted into the bile, converted to urobilirubin and reabsorbed  The urobilirubin is then excreted by the kidneys Nutritional requirements for erythrocyte production  Three essential dietary components: Iron, Folate, B12  Iron  Meat, liver, vegetables, eggs and dairy  Body stores- 3000mg-4000mg  Daily needs of 0.5-1mg for men, 1-2mg for women and 4-6mg for pregnant woman  Absorption in the small intestine- DMT1 (in) and ferroportin (out)  Transport in the blood- bound to transferrin  Delivery to cells- transferrin receptor (in) and ferroportin (out)  Stroage- ferritin  Excretion- no pathway of iron excretion because of toxicity  Downregulation occurs at the level of absorption (DMT/ferroportin)  It is downregulated when there is iron overload or infection/inflammation  It is upregulated when there is erythropoietic demand, decrease iron stores, anemia or hypoxia  Haem protein is involved in oxygen transport (hemoglobin and myoglobin) 15

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Folate  Meats, dairy, eggs, green leafy vegetables, fruits, yeast, mushrooms  Only need a small amount per day- 400 micrograms for adults, 500-600 micrograms for lactating women and 50-200 micrograms for kids  Body stores of 5-20mg (half of it is in the liver)  Absportion in the jejunum- in monoglutamate form  Transport in the blood- 1/3 free and 2/3 non-specific protein binding  Delivery into the cells- via folate receptors, and it becomes polyglutamated (active and trapped)  Modifications- converted to dihydrofolate (DHF) in the liver  Demethylated to THF participates in purine synthesis (DNA replication) and amino acid synthesis (protein production) Vitamin B12  Legumes, fish, meat, eggs, milk, poultry  Small amount needed each day- 1.5-2.6 micrograms a day  Body stores of 2-5mg (1mg in liver)  Obligate losses of 1.3 micrograms per day- takes 3-4 years to deplete stores  Absorption in the ile...