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BI2219 Medical Biochemistry 29/09 - Blood Composition Haemopoesis - how blood cells are developed

Pluripotent stem cell - can develop into any blood cell, capable of self-renewal Blood needs the right quantity of each cell Imbalance in B/T lymphocytes leads to flooding of system - reducing levels of other cells Dysplastic syndrome - bone marrow fails to produce particular cell types - leading to lack of function The cells must also die at the right time Red blood cell 120 days Neutrophil 24 hours Lymphocytes can survive decades Cytokines - proteins produced in small quantities, promote development along the pathway, regulating haemopoesis Erythropoietin increases number of red blood cells, often illegally taken by athletes, in medicine used to treat symptoms of kidney dialysis to upregulate RBC production IL - interleukin ‘between white cells’ Sites of blood production: Prenatal - 0-1.5 months yolk sac 3-7 months 60% liver 3-7 months 20% spleen 4 months onwards bone marrow starts to take over Postnatal - tibia and femur production cease in 20s Vertebrae and pelvis and sternum remain highest contributors 1

Rib production decreases more rapidly with age Bone marrow - stromal matrix - stem cell embedded in matrix on bed of macrophages, fibroblasts and fat cells Protected by surrounding ECM, held in place by adhesion molecules and acted upon by growth factors Pleiotropic - has more than one effect, some cytokines are pleiotropic Multipotential cell - slightly more mature than pluripotent, pathways are restricted Committed progenitor cells - even more mature Haemopoietic growth factors: Act on stromal cells - IL-1, TNF Act on pluripotent stem cells - stem cell factor Act on early multipotential cells - IL-3, IL-4, IL-6, GM-CSF Act on committed progenitor cells - G-CSF, M-CSF, IL-5, erythropoietin, thrombopoietin CFU-GM - colony forming unit - forms granulocyte or monocyte PSC - pluripotent stem cell CSF - colony stimulating factor Growth factor acts on receptor - communicated through membrane to activate second messengers within cell cytoplasm, these activate transcription factors in the nucleus to cause cell proliferation/differentiation/activation The transitioning cells feel the effects of different growth factors at the same time

Cells can be produced that do not reach endpoint - not functional, can affect immune system This can happen on mass, eg leukemia - due to block in differentiation pathway (mainly genetic)

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NK cell - natural killer cell - a lymphocyte non-T, non-B Monocyte becomes macrophage that controls inflammation Monocyte circulates in blood - moves into connective tissue and develops into macrophage responsible for engulfing and killing bacteria Granulocytes - contain granules in cytoplasm - significant in function Polymorphonuclear cells - have different shaped nuclei Macrophage - phagocytosis and activation of bactericidal mechanisms and antigen presentation Dendritic cell - antigen uptake in peripheral sites, antigen presentation in lymph nodes Neutrophil - phagocytosis and activation of bactericidal mechanisms Eosinophil - killing of antibody-coated parasites, very granular Basophil - function largely unknown, appears to have a role in inflammation Mast cell - release of granules containing histamine and other active agents, role in allergy and sensitivity Phagocytosis does not necessarily kill the cell, eg leprosy and TB bacteria live inside cell, historically difficult to treat 3

Myeloid lineage: Erythrocytes - diameter 7.2μm, haemoglobin 2.2mmol/l, life span 120 days Platelets - diameter 3μm, dense granules containing calcium ions, thromboxane, serotonin and ADP, functions for haemostasis, life span 6-8 days Neutrophil - diameter 12-15μM, 2-5 lobed nucleus, cytoplasm contains acidophilic granules which contain lysozyme, myeloperoxidase, cationic proteins, lactoferrin, lifespan 3-4 days Monitors the blood for infection, after phagocytosis the cell dies itself Monocyte - diameter 16-20μM, has irregular nucleus and irregular cytoplasm, lifespan varies from daysmonths, tissue form is termed ‘macrophage’ Eosinophil - diameter 12-17μM, bilobed nucleus, granules contain myeloperoxidase and acid phosphatase, lifespan 8-12 days Basophil - diameter 10-14μM, lifespan 7-10 days, coarse granules containing histamine, kallikrein, myeloperoxidase, esterases, proteases, mucopolysaccharide Kallikrein - involved in haemostasis and blood clotting Primary lymphoid organs - thymus, bone marrow Secondary lymphoid organs - adenoid, tonsil, lymph node, kidney, spleen, Peyer’s patch in small intestine, large intestine, appendix, lymphatics in limbs T-cells reside in thymus B-cells in spleen, lymph nodes, lymphoid tissue Lymphocytes after production in bone marrow move to secondary lymphoid organ to finalise maturation Lymphocyte - diameter 10μM, 45% of white cell population, large nucleus dominating cytoplasm, circular cell shape, critical role in humoral and cell mediated immunity 20% B, 80% T 30/09 - Components of the Immune System The immune response: Recognition - foreign invading agents Communication - to the rest of the defence system Regulation - knowing when to slow/speed up, control Disposal Balanced immune system = optimal effectiveness Immune over-reaction → internal threats from autoimmune problems eg Hashimoto’s thyroiditis, rheumatoid arthritis, inflammatory bowel disease, type 1 diabetes → external threats from allergic reactions including food sensitivities, allergies, eczema, asthma, sinusitis Immune under-reaction → infection or cancer, hepatitis, HIV, shingles, TB Cancer - failure to kill malignant cells Shingles - derived from chicken pox virus, lays dormant until immune under-reaction 4

Worms are the most common infection, followed by malaria Respiratory infections are the biggest killer followed by diarrhoea Functions of immune system - detect presence of infection, contain and eliminate infection, regulate the immune system to prevent self harm, remember the infection to be able to make a fast response on second exposure It protects against four classes of pathogens: Extracellular bacteria, fungi, parasites eg Tetanus: Clostridium tetani Intracellular bacteria, parasites eg Leprosy: mycobacterium leprae Intracellular pathogens hide from the immune system Viruses eg Chicken pox: Varicella Zoster Parasitic worms eg Schistosomiasis Innate immune response - immediate, initiated within seconds, targets groups of pathogens, has no memory - can’t tell the difference between infections - only vague groups, response is always the same ‘fixed’, is able to trigger adaptive immune response Adaptive immune response - gradual response generated over 3-4 days, targets specific pathogens, has memory - more sophisticated, highly developed in mammals Able to recognise specific epitopes, can distinguish self from nonself, able to trigger innate immune response Exterior defences important in innate response - first stop to try to prevent infection from entering Anatomical barriers - skin (mechanical and acidic), mucous membranes entrap organisms Physiological barriers - temperature, pH acidity in stomach, lysozyme, chemicals including complement Commensals - normal flora in gut and on skin ‘healthy bacteria’ compete with foreign bacteria Phagocytic barriers Inflammatory barriers - causes tissue damage and leakage of antibacterial serum proteins and phagocytic cells Phagocytes, macrophages, neutrophils Complement proteins link together to form hole in bacterial membrane - bacteria loses control over its contents, form thousands of channels Innate immunity - mature after 9 months of gestation - premature babies are very vulnerable Adaptive - acquired during life progression, becomes better able to fight infection, slower because it takes longer to develop cells to fight infection, once it recognises a pathogen it mounts a large, specific response Macrophages monitor tissues Neutrophils monitor blood Phagocytosis - bacteria engulfed by macrophage enters lysosomal pathway

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Inflammation - increased blood flow and swelling allows blood contents to flow into tissues including cytokines, complement proteins, leukocytes and immunoglobulins - localise to site of infection Immunoglobulins - antibodies, part of adaptive system Humoral system - production of antibody molecules: B lymphocytes - plasma cells, memory cells, antibodies Cell-mediated response occurs during viral infection - T lymphocytes, T helper cells, cytotoxic T cells B lymphocyte engulfs and degrades bacteria via immunoglobulin recognition and endocytic pathway, remnants are then displayed on the outside of cell T helper cell receptor recognises the bacteria molecule being displayed by the B cell - results in cytokine production released from T cell → immune response occurs, stimulates cell proliferation - forms clones of original cell - same structure recognising same infection B cells differentiate and secrete antibodies with same receptor as initial B cell immunoglobulin Surface-bound = immunoglobulin Secreted form = antibody (both are the same molecule) Antibodies bind to bacterial cells with same molecular structure → they are recognised by phagocytes → engulfed and destroyed Immunoglobulins can activate complement better than the bacteria alone and guard susceptible sites such as epithelial surfaces, foetus and mast cell activation Viral cell - any cell infected by a virus Cytotoxic T-cell has T cell receptor (structure varies) Infected host cell has mechanism to trigger displaying of virus on cell surface (only able to do this if cell has a nucleus and is able to produce protein, eg not RBC) Cytotoxic T-cell recognises and binds - cytokines are stimulated T-cell replicates and releases chemicals to cause cell death of the infected cells ‘suicide signals’ HIV resides in T cells and macrophages - need to kill immune cells to eliminate infection SCID - severe combined immunodeficiency - overwhelmed by infection, cured by bone marrow transplant 30/09 - The Adaptive Response - Antigen Recognition Immunoglobulins - 11% of plasma proteins, 5 classes IgM, IgG, IgA, IgE, IgD, bind to surface molecules of pathogen, bind to proteins in plasma or on cell surfaces - can block pathogen binding sites for entry, can be recognised by phagocyte receptor and activate complement Epitope - on pathogen where immunoglobulin binds to 6

Antigen - anything with capability to generate antibodies - pathogens, allergens, toxins released by bacteria Antibodies are secreted by plasma cells

Antigen binds to NH3+ end (Fab) Phagocyte binds to COO- end (non-variant) (Fc) VL - variable light VH - variable heavy Heavy-heavy are identical in structure Light-light are identical in structure CL - constant light CH1, CH2, CH3 - constant heavy At hinge - able to move in/out depending on size of antigen to bind to CHO - carbohydrate loops 214 amino acids in light chain 446 amino acids in heavy chain Heavy chain denotes class of antibody, specificity varies within classes Papain digestion splits antigen binding region from phagocyte binding region Pepsin digestion fragments Fc region Mercaptoethanol reduction splits heavy chains from light chains Particular regions of Hv and Lv with hypervariability - higher binding capabilities at 30, 50, 100 residue marks, they are at the tip of the arm of the antibody - these regions bind CDRs - complementarity determining region Epitope - molecular pattern/structure that the variable region can bind to 7

Noncovalent forces: Electrostatic - attraction between opposite charges Hydrogen bonds - hydrogen shared between electronegative atoms (N,O) Van der Waals - fluctuations in electron clouds around molecules oppositely polarized neighbouring atoms Hydrophobic Affinity - strength of binding between a single antibody-antigen bond Avidity - overall strength of binding between antibody and antigen TBC 02/10 - The Innate Response - Phagocytosis and Microbial Killing Uptake of material into cells - removal of debris and dead cells, host defence - destruction of invading pathogens Macrophages engulf and digest bacteria to which they bind, they also release cytokines to affect other cells and can act as a cytokine factory secreting anti-inflammatory cytokines such as IL-10 Neutrophils are guided rapidly to the site of infection and leave the bloodstream via adhesion and extravasation after the vessel has become leaky Phagosome - engulfed bacteria Phagolysosome - lysosome fused with phagosome to destroy bacteria Getting bacteria into cells: Enhanced attachment - attachment of microbes to phagocytes via Ab, C3b and C4b complement and proteins eg mannose-binding lectin Is more specific and efficient than unenhanced Unenhanced attachment - innate recognition of pathogen-associated molecular patterns eg peptidoglycan, lipopolysaccharides, etc from bacterial cell walls. Pseudopods form to engulf bacteria Killing bacteria via degradation by proteases and reactive oxygen species: Oxygen independent pathway - lysosomes Lysosomes contain hydrolytic enzymes necessary for intracellular digestion - glycosidases, proteases, sulfatases, deoxyribonucleases, ribonucleases and lipases pH 4.8 maintained by proton pumps and chloride ion channels Enzymes are pH sensitive and don’t function well in the cytosol - protective Uncontrolled release of lysosomal contents into the cytoplasm is also a component of necrotic cell death Bacteria can interfere with this pathway to cause cell death Oxygen dependent - either myeloperoxidase dependent or independent: Myeloperoxidase independent: Reactive oxygen species cause damage - free radicals (molecules with unpaired electrons on an otherwise open shell configuration), they are very reactive, eg oxygen ions, peroxides, hydroxyl If two reactive oxygen species meet they can contribute their unpaired electrons to form a covalent bond 8

Metabolic burst - response of phagocytes to particles → enhanced uptake of oxygen leads to production of H2O2 and eventually superoxide anions (O2-) and hydroxyl radicals (OH-) H2O2, O2- and OH- are all bactericidal (An NADH dependent system) Respiratory burst is NADPH oxidase dependent, toxic compounds produced: O2-, H2O2, OHIf no NADPH oxidase is present it is fatal - largely unable to fight infection Myeloperoxidase dependent: Activated macrophages/neutrophils make bleach and can use it to generate hydroxyl radicals Myeloperoxidase catalyses the reaction: H2O2 + Cl- → HOCl + OHHOCl is the active constituent in bleach HOCl reactions can form highly reactive hydroxyl radicals HOCl + O2- → OH- + Cl- + O2 HOCl + Fe2+ → OH- + Cl- + Fe Respiratory burst is not NADPH dependent, toxic compounds produced: OCl- and singlet oxygen 1O2 NADPH oxidase complex - made of several proteins, susceptible to point mutations, there can also be mutations in promoters but these don’t tend to be fatal Chronic granulomatous disease - fatal in childhood, production of hydrogen peroxide by phagocytes does not occur due to lesion in NADP dependent oxidase → catalase negative bacteria are not killed, the absence of the oxygen dependent killing mechanism is not fatal itself but seriously compromises the primary defence system Oxidants are very toxic - good for killing bacteria, but bad if released into tissue, they are involved in inflammatory disease processes eg rheumatoid arthritis Detoxification reactions - to counteract stress caused by H2O2 Reactions leading to H2O2 production are minimised by sequestering metal ions into proteins, eg ferritin, transferrin protect the cell against oxidative damage Enzymes rapidly dismutate H2O2 to water eg superoxide dismutase and catalase 2H2O2 → H2O + O2 Removing the extra electron After detoxification the contents are released out of the cell and metabolised as nutrients 06/10 - The Complement System Complements action of lysis when coupled with antibody Two components are needed for bacterial inactivation - a heat-stable immune component (antibody) and a heat-labile non-immune component (complement) 9

Complement system comprised of many proteins that react with each other and other compounds to opsonise (make molecules easily phagocytosed), kill cells and induce inflammation Complement activation results in an enzyme cascade that amplifies the response and requires tight regulation Cell lysis - can be antibody dependent or independent Inflammation caused via - vasoactive, chemoattractant (attracts chemicals to target area), phagocyte activator Complement also clears immune complexes - clearing up after immune attack, including viral neutralisation Complement precursors - proenzymes (protein is inactive, must be cleaved to become active) continuously circulate in inactive state, mostly come from hepatocytes, some from macrophages and epithelial cells Membrane attack complex - many pores in cell surface, causes major leakage and cell lysis

Complement proteins numbered C1-C9, numbered in order of discovery not order of action Triggered by antigen:antbody complexes, or pathogenic foreign substances, or mannose binding lectin

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C3 convertase is an enzyme that activates C3 cleaving it to C3a and C3b - both have different roles a is small, b is big C3 is the inactive form C1 binds to Ag-Ab complex, activated C1 cleaves C4 and C2 and these are then reassembled to form C3 convertase Then C3 is cleaved which acts on C5 Then there is sequential binding of C6-C9 C1 is able to activate itself on the surface of certain bacteria Binding of two of the C1q heads to the surface of a bacteria activate C1r to cleave and activate C1s Classic pathway: Fab of IgG or IgM bind to epitopes on the Ag → C1q, C1r and C1s assemble on the Fc portion of the Ab C1 cleaves C4 to C4a and C4b C4b binds to prpteins and carbohydrates on the Ag surface C2 binds to C4b and cleaves C2 into C2a and C2b C2a remains bound and C2b is released 11

C4b2a = C3 convertase - cleaves hundreds of molecules of C3 into C3a and C3b Much C3b binds to adjacent proteins and carbohydrates on the Ag = opsonisation, C3a can stimulate inflammatory response Some C3b binds C4b2a C4b2a3b = a C5 convertase that can cleave C5 to C5a and C5b C5b binds the surface of the target cell and subsequently C6, C7, C8 and several C9 monomers = MAC The MAC puts pores in the lipid bilauer - cell lysis Alternative pathway - innate immunity, spontaneous cleavage of C3 based on multiple initiators, requires factors B and D, formation of C3 convertase requires properdin This C3 less stable than classic pathway Stronger response - more likely to stay together and be able to act C3b mediated either from classical pathway or water hydrolysis Water hydrolises C3 to C3i C3b or C3i binds cell wall and other surface components of microbes Factor B combines with C3b → C3bB Factor D splits bound factor B → Bb and Ba Properdin binds Bb → C3bBbP = C3 convertase, alternative pathway is now active Alternative pathway does not require antibodies to begin MB lectin pathway - innate immunity, MB lectin protein bound to microbial cell wall components, follow up binding of serine proteases (cleave proteins to activate), this complex cleaves C4 and C2 much like activated C1 Receptors of innate immunity recognise molecular patterns that are common to many pathogens (pattern recognition receptors) eg mannose - with correct spacing to indicate pathogen so not to self harm Mannan-binding lectin similar to C1 but binds to mannose then activates C4 and C2 MB-lectin pathway is important in newborn children without much antibody MASP1 and MASP2 (equivalent to C1r and C1s) bind to mannose binding protein (MBP) Forms an enzyme similar to C1 - cleaves C4 and C2 to form C4b2a (C3 convertase which cleaves C3) C3a and C5a float off and induce inflammation - recruit cells to site of infection, enable cells to move out f blood vessels into tissues C3b - increases opsonization Cell lysis - membrane attack complex formed by C5b-C9 Complement receptors - CR* CR1 - found on macrophages, neutrophils, act to bind immune complexes and induce phagocytosis CR3/4 found on macrophages and neutrophils, also bind immune complexes and enhance phagocytosis Results of complement activation not always same - varying levels of inflammation/phagocytosis/MAC due to locat...