What are the five pathological processes? Development of cell injury Key concepts: 1. What is cell injury 2. Reversible vs. irreversible cell injury 3. Characteristics of Apoptosis 4. Characteristics of Necrosis 5. Central role of membrane function/dysfunction 6. Importance of free radicals in damaging membranes 7. Morphology of cell injury - apoptosis and non apoptosis cell injury 8. Morphology of necrosis 9. Fate of necrotic tissue 10. Diagnostic detection of tissue injury 2 most important requirement in cell is Membrane damage: How free radicals are damaging the cell membrane Energy depletion What is cell injury? Normal cells adapt to changes could lead to 3 responses Cellular responses to the injury includes Causes of cell injury ● Hypoxia ● Physical agents ● Chemical agents ● Genetic factors ● Infectious agent ● Inflammation Hypoxia may develop with a lesion anywhere from the nostrils down to the mitochondrial enzyme thus may involve: 1. 2. 3. Vocab that relate with normal cells adapt to changes hypertrophy hyperplasia Atrophy Metaplasia Another response included epidermal adaptations Includes (2 reason) 1. 2. Cell injury occurs once the cell’s adaptive capacity is surpassed which lead to 1. 2. What determines the extent of cell injury 6 answer 2 basic mechanism of action that agents of disease to injury the cells are: ● Membrane damage ● Energy depletion Membrane damage - direct damage 1. 2. 3. 4. Disease agents that can directly cause damage to the cell membrane: radiation, complement, vit E or selenium deficiency Introduction to immunology, inflammation and repair Inflammation & repair : Structural perspective Immunology: functional perspective Try to understand the innate/adaptive graph ! categories of blood stem cell (slide 9) Time of each phase 1. Peracute (within few hours) 2. Acute (4-6 hours to 3-5 days) 3. Subacute (5-14 days) 4. Chronic (>7-14 days) Stimulus that tend to encourage certain cell types - Extracellular bacteria (Neutrophils) - Intracellular bacteria & virus, neoplasia(tumor) (MONONUCLEAR CELLS lymphocytes, plasma cells, macrophages) - Parasites (eosinphills) Fungi and mycobacteria/ TB (macrophages - granulomatous inflammation) Inflammation and repair Includes - Vascular changes - Chemical mediators release from damaged cell (communication between cells) - Cellular response (inflammatory cells) - Chemical mediators released from inflammatory cells It respond to - Cellular danger signals - Exposure of connective tissue to plasma - Not just infectious agents Wound healing Goal is the regeneration of the tissue, either by ● Regeneration (repair by same type of cell, ie. liver) ● Connective tissue deposition 1. 2. 3. 4. Haemorrhage coagulation Inflammation Proliferation Maturation (remodelling) Haemostasis ● Immediate ● Vasospasm ● Platelet aggregation ● Release of vasoconstrictive substances ● Deposition of fibrin, clot formation Inflammation ● Variable duration ● Neutrophils first ● Macrophages later ● Phagocytosis ● Involves ○ Growth factors, chemotactic factors, degradative enzymes Chronic inflammation and repair The macrophage/histiocyte - Phagocytosis and intracellular killing - Antigen processing and presentation - Transition to the proliferative and remodelling phases Proliferative phase - Begins after 4 days and last up to 3 to 4 weeks or longer - Angiogenesis and fibroplasia = granulation tissue Synthesis of extracellular matrix proteins and collagen deposition Re-epithelialization Restoration of structure and function Remodelling - After about 3-4 weeks - Contraction (cause of scar) - Remodeling of granulation tissue by immature connective tissue and then mature connective tissue → scar formation - Improve of wound strength (absence of hair follicles) Times could be different under clean surgery condition And takes longer to heal when there is open/contaminated wound/deficit Steps In angiogenesis 1. Breakdown of ECM and basement membrane around parent vessel 2. Migration of immature endothelial wound 3. Maturation of tubes and formation of lumina 4. Establishment of gap junctions and cellular adhesion molecules between endothelial cells 5. Pericytes and smooth muscle cells surround the newly formed vessel Fibroplasia Migration and proliferation of fibroblasts involves growth factors and cytokines Platelets, inflammatory cells, activated epithelium and endothelium can all secrete growth factors and cytokines Proliferating fibroblasts will often align themselves parallels with lines of tension stress Collagen synthesis begins in 3-5 days and continues for at least several weeks Re-epithelialization - Epithelial cells proliferate almost immediately - Re-epirh in 3-5 days for 1st intension healing - New basement membrane need to be deposited first - Migrating epithelium pushed under the scab Contraction - Myofibroblasts are specialised fibroblast with contractile activity - contain actin fibres Systemic factors affecting wound healing - Nutrition - Metabolic status - Circulatory status - Hormones - Age, anaemia, genetic disorders, malignant disease, obesity, fever, etc Local factors - infection, mechanical factor, foreign bodies, (size, location and type of wound) Peracute inflammation and the fluid phase of the innate response Acute inflammation has a fluid component and a cellular component - Fluid component - Dilutes stimulus - Localises stimulus (stop it from spreading to the body open a route for cells to come in) - Cellular component - Delivers leucocytes to the localised site of injury Hyperaemia: 1. Arteriolar dilation 2. Additional parts of the capillary bed opened Resulting in 1. Increase flow volume 2. Congestion of veins Leads to redness, swelling, heat Vascular permeability 1. Increase permeability of capillaries and post-capillary venules allows exudation of fibrin 2. Emigration of leucocytes(WBC) BIGGER PROTEIN MOVE INTO THE SITE Fluid phase - Dilution of the pathogen - Swelling - Separation of connective tissue elements allow movement of cells and nutrients - Fibrinogen allowed into tissue - Polymerises to form fibrin clot to localise the pathogen Excess interstitial fluid drains to lymophartics which drain to lymph nodes, and then to the systemic circulation Adaptive: transport of antigens to the cells of the adaptive response Innate: triggering of acute phase proteins and other inflammatory mediators that cause systemic signs and local inflammation 3 main triggers ● Dying or distressed cells >alarmins ○ Heat shock proteins ○ Received by sentinel cells ● Pathogen associated molecular patterns ○ Triggers sentinel cells ○ Conserved across many pathogen ● Exposure of connective tissued to plasma ○ Releases cascade-triggering factors Sentinel cells ● ● Classic cells of the innate response ○ Macrophages and dendritic cells ○ Mast cells and some granulocytes ○ Epithelial cells )respiratory, intestinal and urinary) On sentinel cells ○ Alarmins released from damaged cells attach to receptors ○ PAMPS attach to toll like receptors ■ Possible source of ‘memory’ - many different TLRs, variably expressed ○ On mast cells, antigens attactch to IgE on their surface Mast cells ● Sit on skin and mucosal surface ● Degranulate (release histamine) in response to ○ Physical or chemical insult ○ Activated complement proteins ○ Use of IgE (produced by adaptive response) as a surface receptor by mast cells illustrates a feedback from the adaptive response to the innate response Sentinel cells are stimulated and produce - Pro - inflammatory cytokines - Chemokines Interconnected cascades ● Coagulation cascade ● Kinin cascades ● Complement ○ Classical and non-classical ● Cascades fuelled by acute phase proteins Plasma protein is always present in the blood vessel (synthesized in the liver) and can increase or decrease by 25% during inflammation Different active modes: 1. C reactive protein (binds bacteria and fungi and activated complement) 2. Complement factors(precursors for the complement cascade) 3. Fibrinogen (the precursor of fibrin) 4. Haptoglobin (limits free iron) In combination with inflammatory cytokines - Affect heart rate, blood pressure and temp regulation Remember the diagram !!!! B1 B lymphocytes ● Sentinel cells in tissues - have PAMP receptor ● Activated by PAMPS ○ Non specific ○ Divide and transform to plasma cells ● Produce IgM - very effective for ○ ● Opsonization (process where opsonins make an invading pathogen more susceptible to phagocytosis) ○ Agglutination (a clump of bacteria or red cells when held together by antibodies) ○ Fixation of complement Normally too few to be visible Complement cascade ● C5am C3A stimulate release of histamine ○ ↑ vascular permeability ● C3b opsonized antigens ○ Facilitated phagocytosis ● C5a is a chemotaxin ○ Attract a range of inflammatory cells ● C5-9 form the membrane attack complex ○ Opens a hole in the cell/pathogen membrane ○ Kill Why use cascade? Very powerful, pro-inflammatory products - Effective, but potentially damaging Each substance has a short half life - Thus many potential places to stop the reaction Acute to chronic Innate to adaptive - antibody mediated - No memory Pre- dates the adaptive response But ● Growing appreciation that it plays a critical role in directing adaptive response Front line therefore major component of the acute response Continues into chronic active inflammation Margination, adhesion, migration ● Complement and chemokines from sentinel cells ○ Stimulation of endothelial cells ■ Endothelial cells express adhesion molecules to “grab” passing leukocytes ○ Vascular permeability and chemotaxins ■ Attract leukocytes through the vessel walls into tissue Acute(<3-5 d) - locally ● Background ○ Continued role of kinins, prostaglandins (pro- inflammatory mediators, chemotactic factors, vasodilators/ permeabilizes) ○ Oedema and exudation ● ● Frontline troops ○ Rapid chemotaxis and exudation of neutrophils ○ Some chemotaxis and activity of NK and Tc cells, depending upon stimulus (viruses/intracellular) Garbage collectors and generals ○ Phagocytosis by local macrophages and dendritic cells ○ Links to the adaptive response Innate: neutrophils (bacteria) ● Bone marrow ● Segmented neutrophils are released into circulation ● Immature forms are released in times of high demand ● 1 day in circulation ● 1-2 days in tissue ● Once in the tissues they do not re-enter the blood Packed full of lysosomes, containing enzymes, proteases Phagocytic cells - Form phagosomes by engulfing foreign material by pseudopodesis, which then merge with lysosomes intracellularly to form phagolysosomes Good at finding bacteria and killing them Most commonly involved in a inflammatory process in large numbers when bacteria are an important part of the pathogens of the lesion Suppurative exudates ● Composed predominantly of neutrophils and necrotic cells ● Abscess (swelling of suppuration) ● Often bacterial in aetiology Slide 16! Important for practical ! check a bit Innate: macrophage ● Two sources ○ Macrophages in tissues and lymph nodes ○ Circulating monocytes ● Specialist phagocytes ● Long tissue life ● Bridge between innate and adaptive immunity by presenting antigens to lymphocytes Chronic: adaptive antibody - mediated response ● More focus ● More actions ● Memory First response - IgM Very effective for - Opsonization - Agglutination - Fixation of complement Produced first then replaced after class- switching Important to understand IgM, IgG, IgA Outcome of inappropriate IgM or IgG production? ● Opsonization and phagocytosis → cell destruction ● ● Activation of complement (inflammation and necrosis) Blockade of receptors ○ Neuro (paralysis) ○ Hormonal (endocrine dysfunction) Depends on where IgG and IgM bind ● Type 2 hypersensitivity ○ Cytotoxic type ○ Opsonization and complement fixation by antibody attachment ● Type 3 hypersensitivity ○ Immune complex type ○ Deposition of antigen antibody complexes in bystander tissues (and subsequent fixation of complement) Stil type 3 ● Antigens and antibodies in circulation ● Form complexes and deposit ● Ag-ab complexes result in complement fixation ● → pro- inflammatory effects ● → endothelial damage ● → coagulation cascade, fibrinoid necrosis, neutrophils, thrombosis, infarcts Acute to chronic Innate to adaptive- cell mediated The adaptive response effector mechanisms - cell mediated immunity (intracellular pathogens and neoplasia) Surface defences ● Structural ○ Epithelial cells, cilia, stratum corneum ● Antimicrobial peptides ○ Defensins ● Commensal / normal flora ● Local environment ● Non-compatibility of adhesion molecules Natural killer cells ● Type of lymphocyte ● ● ● ● ● 15% of circulating Lc Activated by PAMPs ○ non - specific ○ Have no memory Attracted and activated by cytokines released by sentinel cells Kill neoplastic and infected cells Direct further activation of cells Another important diagram slide 13 Naive cells ● Requires activation of dendritic cells ○ PAMPS ○ Tissue damage ○ Alarmin ● And Th1 cells ○ Activated dendritic cells ○ Activated NK cells ● Antigen presence isn't enough - in absence of tissue damage antigen exposure leads to deletion of the Lc Primary Tc cell activation (Th1 response) ● Interaction of antigen with ○ MHCll (on dendritic cell) + CD4 Th1 cells ○ MHC1 (on dendritic cell) + CD8 Tc cells ● Results in clonal expansion ○ Some become effector and some become memory cells How MHC1 presents peptides to keep NK cells away and Tc cell only target abnormal cell Strong inhibition, reduced inhibition, strong activation Type 4 delayed type hypersensitivity - chlamydia Antibody reduces cell to cell transmission Apoptosis of infected cells essential for elimination SO Th1/Th17 responses essential for elimination BUT ● th1/th17 induced damage (type 4 hypersensitivity) ● Th2 ↑ ○ ○ Inhibits Th1 and allows persistence Promotes fibrosis Neoplasia Anti-neoplastic responses ● NK cells ○ NK receptors ○ Antibody guided ● CD8+ Tc cells ○ Need Th1 ● Tumour necrosis factor ○ Needs Th1 Th1 produced MHC2 inhibits MP/B lymphocytes that produced IL2 /IFNg which inhibits Tc which then triggers apoptosis in abnormal cell Immunology, inflammation and repair Host defence and response (surface defense mechanisms) ● Physical barriers, antimicrobial proteins, clearance mechanism BAD: HYPERKERATOSIS, HYPERPLASIA ● Loss of structure/impaired function ○ Secondary infection ○ Loss of fluid and nutrients ○ Discomfort Modulation Balanced responded needed ● Intracellular vs extracellular pathogens ● Active inflammation vs repair and immunity ● Modulated by mutual inhibition (Th1 vs Th2) ● Determined by ○ The pathogen ■ Interactions with sentinel cells that direct subsequent responses ○ Host ■ Life history - past exposures ■ Current state - stress, hormones ○ Genetics ■ Inbreeding and host - pathogen co-evolution Tuberculous granuloma: complex composition ● Granulomatous inflammation = sheets of macrophages ● NOT granulation tissue (migration of fibroblasts) ● Classic lesion ● Basis is in Th1 ○ Tc cells ● Components of Th2 ○ Fibrosis ○ Giant cells (multinucleated macrophages) ● Th2 contains, Th1 eliminates Roles of macrophages ● Determined by ○ Cell-pathogen interactions ○ Feedback from the adaptive response ● Phagocytosis ● Directing the adaptive and innate responses ● Directed by adaptive responses via feedback Immunomodulation ● Th2 promoted by ● ○ Cortisone (stress response) ○ Progesterone ○ Hypoproteinaemia (poor nutrition) Th1 promoted by decreasing day length in some spp ○ Protection against respiratory viruses in winter ■ Viral survival ■ Sheltering indoors Regulatory T cells ● CD4+ so interact with MHC 2 ● Created during thymic selection ○ Strong reaction to self - apoptosis ○ weak / no reaction to sef - Th cells ○ Medium reaction to self - Treg cells ● Treg cells, when activated, produce IL10 ○ Wide range of immunosuppressive activities ○ Decreased both sizes but tend to skew towards Th2 Treg cell failure - Can due to : - Genetic susceptibility - Environmental allergen load - Lack concurrent parasitic disease - Cutaneous allergen exposure - Which lead to Hypersensitivities of both Th1 and Th2 Treg stimulation - immunosuppressive viruses ● Feline and bovine leukaemia viruses ● Conserved protein bind to receptor on Treg cell and activated it ● This activated T reg cell → increased IL10 ● Results in immunosuppression Th17 pathways ● Particularly promote mucosal inflammation ● Th17 lymphocytes ○ CD4+ Th cells ○ Produce IL17 ● NK CELLS ○ Produce IL17 ● IL17 ○ Stimulated proinflammatory pathways ■ Stimulated Tc cells, inhibits Treg cells ■ Stimulates epithelial cells to secrete chemotaxins ■ Attract and active neutrophils Tolerance Sources of antigens for hypersensitivity ● Self antigens (auto-immune) ○ Normal self antigens ○ Modified self antigens ○ Exposed cryptic antigens ● Non-self antigens（hypersensitivity) ○ ○ From pathogens/allergens From transplants/transfusion What does it need? ● A mechanisms so lymphocytes can ○ Target affected cells and only affected cells ■ (Tc cells via TCR and MHC1, supported by Th1 cells) ○ Produce antibodies only to things we want directly responded to ■ (B cells via BCR, supported by Th2 cells) ● ● Lots of different specificities to respond to lots of different pathogens ○ Not self(thymic selection) ○ Not benign substances (peripheral selection - need for tissue damage and activated dendritic cells) Enough effector cells to do the jo quickly Lymphocytes are antigen specific ● T cells have t cell receptors ● B cells have B cell receptors ● These are surface bound immunoglobulin ○ Like antibodies, but membrane bound ● Conformation of the antigen binding site determine specificity The t cell receptor ● On Th cell, Tc cells and Treg cells ○ So important for both Th1 and Th2 pathway ● In the foetus, TCRs are generated at random to produce million of different T cells ○ Some will be to pathogens ○ Some will be to self ○ ~ to environmental molecules Thymic selection for self-tolerance ● Presentation of antigens to immature lymphocytes in the thymus leads to apoptosis ● APC = antigen presenting cell ● Requires MHC2 so poor MHC diversity / certain types impairs thymic selection Dendritic cells ● Similar to macrophages ● In places they can contact Ag ○ mucosa/skin ● ● ● ○ Lymph nodes Phagocytose Ag Present it to Lc ○ Professional APC Direct the response ○ Cytokines MHC The major histocompatibility complex ● MHC1 ○ Present on all cells ○ Samples peptides from the cell cytoplasm ○ Presents it to CD8+ cells (Tc cells) ● MHC2 ○ Present only on professional antigen presenting cells (dendritic cells, macrophages, B cells) ○ Some epithelial cells if stimulated by inflammation ○ Picks up phagocytosed antigens from the phagosome ○ Presents them to CD4+ cells (Th cells) ○ Essential for primary activation of the adaptive response Intracellular vs extracellular Diagrams of both T cell and B cell tolerance through peripheral selection Failure/ bypassing of tolerance leading to hypersensitivity To normal self antigens ● Breakdown / bypassing of thymic selection ○ Normal imperfection ○ Genetic predisposition ○ Cryptic antigens not available to induce deletion ○ Change to self proteins to appear non-self ● Inappropriate activation ○ Persistent inflammation (cytokine environment) ○ Receptor cross linking by superantigens ○ Molecular mimicry Failure of tolerance ● Most self - reactive cells are deleted in the thymus during development ● Most of those that escaped are deleted peripherally, by exposure to Ag in absence of co-stimulatory signals ● But there are always some remain(very few) can be genetically predisposed ○ By MHC type/function To self antigens(auto-immunity) Exposure to hidden antigens ● Spermatozoa are not present or accessible to the immune system during thymic selection ● Normally a barrier “immunologically privileged’ ● ● Exposed to the immune system following trauma immune - mediated male infertility Modification of self antigens into nonself Ag ● Proteins structure modified by a substance ○ toxins/drugs ○ Mutation ● Attachment of foreign substance to a protein makes it appear to be foreign Molecular mimicry ● Some pathogens have antigens almost identical to our own ● Somatic hypermutation can fine tune B cells to self ● Presence in large quantities,in association with co-stimulatory factors (inflammatory lesion) ● Increase chance of activating left over self Lc Somatic hypermutation!!! Persistent inflammatory environment ● The activation requires ○ Presentation on MHC2 ○ Co-stimulation ● Inflammatory environment ○ Encourages MHC2 expression ○ Encourages presentation of self Ah on many cell types ○ Provides co-stimulatory factors ○ Activates bystander remnant(剩余） Receptor cross linking ● Superantigens can cause non-specific activation and clonal expansion ● Crosslinking of B cell receptors ● Non specific ○ Increases chance that a remnant self B lymphocyte will be activated and expanded
