Describe the basic procedure of an autopsy
• External examination
o Rigor mortis
o Liver mortis (color change)
o Algor mortis (temperature change)
o Assess injuries, identifying features, evidence of medical intervention
- Internal examination and evisceration
- Neuropathologic exam
- Microscopic exam
- Ancillary testing (case-by-case)
List the manners of death
Definition: Based on circumstances surrounding death (vs. COD, an injury or disease resulting in death)
- Suicide
- Homicide
- Accident
- Undetermined
- Natural
Recognize deaths that fall outside of a physician’s jurisdiction
Coroner case
oCoroners and medial examiners certify deaths with non-natural MOD oReferred when death is/of:
- <24 hrs hospital admission
- Under anesthetic
- Violent death (even after hospital stay)
- After in-hospital accident
- Prisoners
- Infant sudden death
- Unidentified person
Medical case
o Physicians can certify deaths with natural MOD
Distinguish between mechanism of death and cause of death
•MOD: circumstances surrounding death
•COD: injury or disease producing physiologic derangement in body leading to death
o Etiologically specific
o If non-natural, the MOD is then also non-natural (coroner assumes jurisdiction)
• OSC (other significant conditions): contributed to death, but distinct from major COD
Define Hyaline change
describes a glassy pink appearance, usually from protein accumulation (ex. Amyloids, Russell bodies)
define anthracosis
black discoloration of lung tissue and draining lymph nodes from inhaled and phagocytosed carbon
Define caspase
cysteine proteases, exist as pro-enzymes and activate apoptosis cascade when cleaved
List the types of intracellular accumulations and give examples of each, including morphologic descriptions.
•Lipids:
oSteatosis: accumulation of TAGs
- Common in liver
- Round, clear spaces in cytoplasm
oAtherosclerosis: accumulation of cholesterol in smooth muscle cells
- Intracellular: look foamy
- Extracellular: crystallizes, looks like shards
•Proteins
oAppear pink, many different shapes possible
oAmyloidosis: if proteins fold abnormally and deposit
- Usually extracellular
- Appear glassy, pink, “hyaline”
- Non-branching fibrils, bound by congo red stain
•Hyaline Change
oDescribes a glassy pink appearance, usually from protein accumulation (ex. Amyloids, Russell bodies in plasma cells)
•Glycogen
oClear cytoplasmic vacuoles, can appear in nucleus in liver cells
•Pigments
oCarbon: black (anthracosis)
oLipofuscin: brown-yellow, granular, cytoplasmic; from incomplete breakdown of subcellular components
oMelanin: brown-black, from melanocytes
oHemosiderin: golden yellow-brown, granular, from iron bound to ferritin
oBilirubin: yellow, from hemoglobin
Explain the difference between apoptotic and necrotic cell death.
•Apoptotic
o Physiologic or pathologic
o No inflammation
o Membranes intact
o Cell shrinkage
•Necrotic
o Pathologic
o Inflammation
o Membranes disrupted
o Cell swelling
Describe the extrinsic and intrinsic pathways of apoptosis, and know the names of the major components involved in each.
•Initiation phase can be intrinsic or extrinsic
•Intrinsic pathway (mitochondrial)
o Normally, balance between pro- and anti-apoptotic proteins (Bcl family)
o Signal: irradiation, lack of survival signals
o Cause pro-apoptotic proteins to dominate
o Forms channel in mitochondrial membrane
o Release of contents (cytochrome c)
o Cytochrome c activates caspases, which activate cascade
•Extrinsic pathway (death receptor)
o T lymphocytes express FasL molecule on membrane
o FasL binds Fas receptor on cell (a death receptor)
o Causes caspase cleavage and activation of cascade •
COMMON: Execution phase
o DNA fragmentation, endonuclease activation. cytoskeleton breakdown
o Membrane blebs into Apoptotic Body
o Phagocytosed
Explain the role of ATP depletion in the major mechanisms of cell damage
o Na+/K+ pump fails, leads to cell swelling
o Switch to anaerobic metabolism, leads to lactic acid accumulation and decreased pH
o Ca2+ pump fails, leads to influx of Ca2+
o Damaged/degradation of protein synthesis apparatus
• Ribosomes detach from RER
o Abnormal protein folding
Describe the morphologic changes associated with apoptosis
o Cell shrinkage, darkly stained cytoplasm
o Nuclear pyknois
o Cytoplasmic blebs
o Apoptotic bodies rapidly phagocytosed
Describe the morphologic changes associated with reversible necrotic cell death
oElectron microscope
- Membrane blebbing, loss of microvilli
- Mitochondrial swelling
- ER dilation
oLight microscope
- Cell swelling, clear vacuoles
- Fatty change
Describe the morphologic changes associated with irreversible necrotic cell death
oElectron microscope
- Membranc discontinuity
- Greater mitochondrial swelling
- Myelin figures (whorled phospholipids from membranes)
oLight microscope
- Cytoplasmic eosinophilia (dark pink cytoplasm)
- Dystrophic calcification (fatty acid calcification)
- Nuclear changes (karyolysis, pyknosis, karyorrhexis, loss of nucleus)
Types of Necrosis
- Coagulative necrosis— often with ischemia, dead tissue architecture maintained
- Liquefactive necrosis— often with bacterial and fungal infections, hypoxic damage in CNS; dead cells completely digested leaving only liquid
- Fat necrosis— often in pancreatitis; FA’s combine with calcium, makes chalky white areas
- Caseous necrosis— often in tuberculosis infection; tissue is “cheesy” granular material
Pyknosis
chromatin condensation (darkening) and breaking up into fragments
Karyorrhexis
chromatin fragmentation
Karyolysis
fading of chromatin
Types of calcification
•Dystrophic calcification—calcium deposition in dying tissues; can occur with normal Ca2+ levels; appears dark blue-purple (basophilic) & variable shape, both intra- and extra-cellular. Also in areas of atherosclerosis.
•Metastatic calcification—when high serum Ca2+, occurs in otherwise normal (not damaged/dead) tissue. Appears same as dystrophic under light microscope.
Define inflammation
- Reaction to injurious agent that limits damage and promotes repair
- See rubor/redness, tumor/swelling, calor/heat, and dolor/pain
- Mediated by vessels and blood cells
- Tightly regulated chain of molecular and cellular events
- Acute or chronic by duration of response and types of cells involved
Describe the difference between innate and acquired immunity
•Innate
o Non-specific defense mechanisms
o Barriers (skin, pH) and inflammation
•Acquired
o Response to specific injurious agent
o Mediated by antibodies
o “immune” system
Neutrophils/ Polymorphonuclear leukocyte (PMN)
o Most numerous cells in peripheral blood (50-70%)
o Segmented nucleus with 3-5 lobes
o Pseudopods—moves to site of injury
o Granules (lysosomes)
o Motile phagocyte
o Primary cell responder in acute inflammation
Lymphocytes
o Single nucleus, scant cytoplasm
o Includes: B cells, T cells, NK cells
o Produce antibodies, cytokines, or toxic granules to eliminate foreign invaders
o Primary cell in chronic inflammation
Monocytes (circulating) /Macrophages (when in tissues)
o Produced in bone marrow, move to tissues after few days in circulation
o Horseshoe nucleus, granules
o Motile phagocyte
o Recognizes foreign material when opsonized
o Presents antigens via Class II MHC receptors
o Helps transition between innate to acquired immunity
Eosinophils
o Bi- or tri-lobed nucleus
o Cytoplasm filled with pink granules
• Contain numerous mediators of inflammation: Histamine, proteolytic enzymes and major basic protein
o Involved in inflammatory reactions to allergens and parasites
Basophils/Mast Cells
o Bilobed nucleus (but often obscured)
o Cytoplasm filled with blue/basophilic granules: Histamine, proteoglycans, proteolytic enzymes, lipid mediators of inflammation
o Analogous cells in tissues = “mast cells”
Platelets
o Anuclear fragments of megakaryocyte cytoplasm
o Contain RNA, mitochondria, canalicular system and granules of platelet activating factor (stimulates inflammation)
o Primary “cells” in blood clot formation
List the classes of chemical mediators of inflammation.
•Plasma derived (in precursor forms)
o Factor XII (Hageman factor)
o Complement proteins
o Kinins
o Clotting proteins
•Cell derived (in organelles or produced when stimulated)
o Vasoactive amines (histamine, serotonin)
o Arachidonic acid metabolites (prostaglandins, leukotrienes, lipoxins)
o Cytokines and chemokines
o Nitric oxide
o Lysosomal contents
o Oxygen-derived free radicals
Define acute and chronic inflammation.
•Acute
o Immediate response but short duration
o Changes in blood vessels so WBCs move into tissues where they neutralize injurious agent
o Important cells: granulocytes (primarily neutrophil)
•Chronic
o Prolonged onset, prolonged duration
o Usually follows acute inflammation, but may begin insidiously and asymptomatically
o Important cells: mononuclear (lymphocytes, macrophages, plasma cells)
Describe the processes involved in tissue homeostasis
- Maintain normal cell population:
- Rate of cell proliferation: regenerates lost tissue mass
- Rate of cell death by apoptosis: eliminates damaged cells
- Rate of cellular differentiation: regulates cell population size
Define regeneration and distinguish it from repair
• Regeneration restores damaged or lost cells/tissues to their original state
o Via cell proliferation from remaining cells or stem cells
o Requires ECM scaffold
o Ex. Hematopoietic system, skin and GI epithelium
• Repair involves a combination of regeneration and scar formation by collagen deposition
o Requires cell proliferation from parenchyma or stem cells
o ECM is usually damaged so new ECM is deposited
o Ex. Healing of a burn
Describe the components of the cell cycle
• Cycle regulated by cyclin and cdk (cyclin dependent kinases) dimer
• Phosphorylate Rb (retinoblastoma protein) which releases inhibition
o Allows transcription to occur (S phase)
• Interphase
o G1: cells prepare for DNA synthesis, but not yet committed to replication
• Checkpoint (G1/S): checks for DNA integrity
o S: DNA synthesis, cell now committed to replicate or die
• Checkpoint (S/G2): checks for damaged or unduplicated DNA
o G2: cell prepares to enter mitosis
• Mitosis
o Prophase, metaphase, anaphase, telophase
• Cell types
o Labile cells: continually dividing
• Epidermis, GI tract epithelium
o Quiescent cells: stable cells; low level of replication, in G0 stage
• Have potential to reenter cell cycle
• Hapatocytes
o Permanent cells: have left the cell cycle
• Neurons, cardiac myocytes
Define and explain the various types of stem cells
• Population of cells that have not terminally differentiated and can regenerate lost cells
o Self renewal (one progeny remains a stem cell after cell division)
o Asymmetric replication (other progeny differentiates)
• Types
o Embryonic
• Derived from inner cell mass of blastocyst
• Each cell is pluripotent
• Have self renewal capacity
• Potential use to treat diseases with loss of critical cell type (ex. MI)
• Challenges
• Ethical issues
• Transplant rejection
• Tumor formation (teratomas)
• Persistence of underlying disease
• Making differentiated cells function normally
o Induced pluripotent stem cells (iPS)
o Adult
• Some tissues have a reservoir of self-renewing cells
• Hair follicle, intestinal crypts, skin
• Restricted in their differentiation potential
• Restricted niches (location)
• But can transdifferentiate in vitro
• Function to regenerate cells lost by normal wear and tear
• Response post-injury
• Increase # dividing stem cells
• Increase # replications of cells
• Decrease cell-cycle time for each division
o Cancer stem cell
List the functions of the extracellular matrix.
o Provides turgor by sequestering water, or rigidity by sequestering minerals
o Reservoir for secreted growth factors
o Framework for cells to adhere, migrate, and proliferate
o Mediates cell-cell interactions
o Site of remodeling during wound healing
List the components of the extracellular matrix
Fibrous structural proteins
Collagen
Elastin and fibrillin
Cell adhesion proteins
- Secreted: fibronectin and laminin
- Cell-surface bound: integrins and cadherins
- Function as receptors, allow cells to interact with each other and ECM
Proteoglycans and hyaluronic acid
Distinguish between the interstitial matrix and basement membranes.
• Two types of ECM
• BOTH: made of fibrous structural proteins, adhesive glycoproteins, proteoglycans and hyaluronic acid
• Interstitial matrix: space between cells
o Fibrillar and nonfibrillar collagen, elastin, fibronectin, proteoglycans, hyaluronate, others.
• Basement Membrane: structure that separates epithelial cells from mesenchymal cells
o Type IV collagen, laminin, heparin sulfate, proteoglycans, other glycoproteins
Describe the function of each discussed collagen types
o Type I: most common form; in bone, tendons, mature scars
o Type II: articular and hyaline cartilage
o Type III: embryonic collagen, first collagen deposited in wound healing and is eventually replaced by Type I
o Type IV: NON-fibrillar; in basement membranes
Describe the formation of collagen
• Triple helix of 3 polypeptide chains
o Modified
• Hydroxylation on proline and lysine residues
• Glycosylation on lysine residues
o Secreted
o Procollagen is cleaved into active form
o Cross-linked into fibrils
• Via oxidation of lysine and hydroxylysine residues
• Requires Vitamin C
• Provides tensile strength
Elastic Fibers in wound healing
o Structure: elastin core surrounded by fibrillin
o Function: allows recoil after stretching
o Located: arteries, skin, uterus, lung
o Marfan Syndrome: from inherited defects in fibillin
Fibronectin in wound healing
o A secreted cell adhesion protein
o Location
• Plasma: stabilizes early clot (fibrin plug)
• Cellular: secreted from fibroblasts, macrophages, endothelial cells
o Function: binds numerous molecules of ECM and integrin
• Adds structural integrity to clot
• Chemotactic for many cells
• Substrate for cellular adhesion (RGD domain)
• Substrate for other ECM protein attachment and assembly
o First ECM deposited during wound healing
Laminin in wound healing
o A secreted cell adhesion protein
o Located primarily in basement membrane
o Forms tight network with Type IV collagen to maintain BM integrity
o Functions: similar to fibronectin
• Substrate for ECM protein binding (heparin, collagen)
• Substrate for cell adhesion
Integrin in wound healing
o A cell surface-bound cell adhesion protein
o Transmembrane receptors
o Functions:
• Facilitate cell-cell interactions
• Facilitates cell interaction with ECM
• Binds collagen, fibronectin, laminin
• Links cell surface to cytoskeleton
• Conformation changes in cytoskeleton can initiate signal transduction → cell proliferation, apoptosis, differentiation
Cadherin in wound healing
o A calcium-dependent cell surface-bound cell adhesion protein
o Functions:
• Facilitate cell-cell interactions between similar cells types
• Facilitate formation of cell junctions (zonula adherens, desmosomes)
• Linked to actin cytoskeleton via catenins to regulate cell motility, proliferation, and differentiation
Proteoglycans in wound healing
o Structure: repeating disaccharide polymers bound to protein core
• Chondroitin sulfate, heparin sulfate, dermatan sulfate
o Function: forms ECM scaffold for structure and permeability
Hyaluronic Acid in wound healing
o Structure: huge molecule of long repeating polysaccharides WITHOUT protein core
• Can bind lots of water
• Forms viscous, hydrated gel
o Function: allows ECM to resist compressive forces
o Deposited early in wound healing
• Facilitates cell migration and proliferation
Explain the two mechanisms by which angiogenesis can occur
**• From pre-existing vessels** o Nitric Oxide and VEGF cause vasodilation and increased permeability o Metalloproteinases degrade vessel’s BM and plasmingogen activator disrupts cell-cell contact between endothelial cells o Endothelial cells migrate toward angiogenic activator o Endothelial cells proliferate o Endothelial cells mature and from capillary tubes o Periendothelial (pericytes and smooth muscle cells) cells are recruited to form mature vessels • **From endothelial precursor cells derived from the bone marrow** o Hemangioblast cells can differentiate into hematopoietic cells and vascular cells o Recruited into injured tissues to initiate angiogenesis o Unknown mechanism
List the phases in wound healing
- hemostasis phase
- inflammatory
- proliferative
- maturation
Describe the Hemostasis Phase in wound healing
o Function: stops the bleeding and facilitates inflammation
o Platelets activated by exposed collagen
• Aggregate
• Secrete cytokines to activate clotting and complement cascades (serotonin, bradykinin, prostaglandins, prostacyclins, thromboxane, histamine)
• Release thromboxame: transient vasoconstriction and platelet aggregation (10 minutes long)
• Factors like histamine then cause vasodilation and increased permeability for edema (entrance of water) and inflammatory cells to enter
o Plasma fibronectin/fibrin forms plug at injury site
• Early structural support
Describe the Inflammatory phase in wound healing
o Function: Stop infection, clear debris, induce repair
o Early phase:
• Neutrophils appear within hours
• Attracted by chemokines (fibronectin, PDGF, TGF-b, C5b, TNF)
• Phagocytose debris and pathogens
• Secrete proteases to break down wound site
• Macrophages become predominant cell after 2 days
• Secrete growth factors, cytokines, proteases
• Phagocytose debris and pathogens
o Late phase
• Macrophage secretion peaks on days 3-4
• Secreted factors attract and activate multiple cell types needed for the proliferative phase
• Neutrophil/macrophage numbers begin to decline
• Fibroblasts become predominant cell
• Early wound ECM: fibrin, fibronectin, hyaluronic acid, adhesion glycoproteins
• Serves as anchor for cell adhesion and collagen deposition
List the steps in the proliferative phase
- Angiogenesis
- Formation of granulation tissue
- Fibroplasia (collagen deposition)
- Parenchymal cell migration/proliferation (epithelialization)
- Wound contraction
Describe the angiogenesis of the proliferative phase
o Purpose: to restore perfusion to wound
o VEGF
• Increased vascular permeability
• Endothelial cell migration
• Promotes angiogenesis
o Fibrin/Fibronectin deposition in ECM facilitates endothelial cell migration
• Proteases degrade existing ECM for endothelial migration
o New thin walled vessels in wound site (granulation tissue)
• Most vessels regress via apoptosis when no longer needed
Describe granulation tissue formation in the proliferative phase
o Purpose: fills defect left by injury prior to collagen deposition (fibrosis)
o 2-5 days after injury
o Composed of highly vascular loose fibrous tissue
• Scattered inflammatory cells and proliferating fibroblasts
o ECM composed of
• Fibronectin (adherence)
• Type III collagen
• Hyaluronic acid (binds water)
o Growth factors
• PDGF and TGF-beta: facilitate fibroblast migration and proliferation
Describe fibroplasia in the proliferative phase
o Purpose: influx and proliferation of fibroblasts with subsequent collagen deposition
o Fibroblasts attracted and activated by TGF-b, PDGF, EGF, FGF, and cytokines
• Secreted from platelets, inflammatory cells, endothelial cells
o TGF-b is a critical mediator of fibrogenesis
• Increases fibroblast proliferation and migration
• Increases collagen and fibronectin synthesis
• Decreases ECM degradation by metalloproteinases
• Chemotactic for monocytes
o Ends 2-4 weeks after injury
• When collagen degradation > deposition
Describe epithelialization in the proliferative phase
o Purpose: restores injured epithelium
o 2-3 days after injury
o Basal keratinocytes (in skin wounds)
• Proliferate from edges of wound and migrate until contact inhibition
• Migrate over granulation tissue, but under scab
• Proliferation and migration stimulated by EGF, KGF, FGF
• Migration facilitated by interaction with fibronectin/fibrin in provisional ECM of granulation tissue
• Secrete:
• Plasminogen activator
o Activates plasmin to degrade fibrin to dissolve scab
• Matrix metalloproteases
o Dissolve damaged ECM and old BM
• Growth factors and new BM
o Includes laminin and Type IV collagen
Describe wound contraction in the proliferative phase
o Pulls edges of wound together to reduce wound surface area by 40-80%
o Mediated by myofibroblasts
• TGF-b induces differentiation of fibroblasts to myofibroblasts
• Myofibroblasts contain actin filaments linked to ECM
• Facilitate contraction
• As contract, fibroblasts lay down new collagen
Describe the maturation phase
o Remodeling of ECM with mature scar to increase tensile strength
o Process can last over 1 year
o Collagen production = degradation
o Matrix metalloproteases degrade collagen and ECM
o Replace Type III collagen with Type I
• Collagen is organized, cross-linked, and aligned along tension lines
• Final wound strength = 80% of normal
o Replace hyaluronic acid with proteoglycans
• Heparin sulfate, chondroitin sulfate
o Decreased number of blood vessels, fibroblasts
o Mature scar: dense Type I collagen, elastic fibers, proteoglycans, scattered fibroblasts
Describe the processes of liver regeneration and liver fibrosis following injury
• Regeneration
o Following a partial hepatectomy
o Compensatory growth: hepatocytes re-enter cell cycle and replicate once or twice
o Return to quiescent state
o Nonparenchymal cells also replicate (Kupffer cells, endothelial cells, stellate cells)
o Mediated by cytokines: TNF, IL-6; and growth factors: HGF, TGF-a
o No fibrosis occurs
• Liver fibrosis
o With more chronic forms of injury
• Chronic viral hepatitis, alcohol abuse
o Repeated cell injury, inflammation, repair → fibrosis
o Can result in cirrhosis
Define each type of skin wound: abrasion, laceration, incision, avulsion, amputation, and puncture
- Abrasion: superficial injury from contact between skin and parallel rough surface; scrapes off epidermis
- Laceration: tears in tissues with jagged margins
- Incision: division of soft parts made with a knife
- Avulsion: tissue torn loose and left hanging by flaps
- Amputation: portion of body is completely detached
- Puncture: narrow object penetrates body’s tissues; entrance wound is often small so don’t know the harm done to underlying tissues and vessels
Describe the various types of surgical wound healing
• Primary Closure
o Wound edges are closely connected by suture, staples, tape, etc.
• Delayed primary intention/closure
o Wound is initially left open
o Closed when granulation tissue is sufficiently clean and vital
• Secondary intention/ closure
o When wound is contaminated or tissue has questionable vitality
o Tissues left open to scar from bottom up
o Prevents morbidity/complications and mortality
o In secondary intention (vs primary)
• Larger tissue defects
• Larger inflammatory response
• Larger amounts of granulation tissue formed
• Greater amount of wound contraction and collagen cross-linking
List the local factors that can disrupt (enhance or retard) normal wound healing
o Wound type, size, location
o Vascular supply
• Poor blood supply = slower healing
• Ischemic wounds do not heal
o Oxygen supply
• Increases O2 promotes healing
o Infection
• Delays or prevents healing
• Wounds may be edematous, red, tender, drain purulent material, febrile
o Necrosis
• Lowers pH of area, causes inflammation, prevents healing
o Foreign material
• Impair healing, increased likelihood of bacterial infection
o Movement
• Pulls wounds apart
• Increases glucocorticoid circulation, inhibiting repair
o Radiation
• Prevents cells from dividing, injures epithelial cells
List the systemic factors that can disrupt (enhance or retard) normal wound healing.
o Circulatory compromise
o Nutritional status
• Malnutrition: decreased protein synthesis
• Zinc deficiency: needed for metalloprotease function
• Vitamin C deficiency: collagen synthesis (scurvy)
• Vitamin A deficiency: increased requirements in wound healing, aids in epithelialization
• Vitamin B1 (thiamine) and B2 (riboflavin) deficiencies
o Diabetes mellitus: impaired circulation
o Obesity: unclear reasons, maybe from poor circulation in fatty tissues
o Hormones: glucocorticoids impair (inhibit inflammation and collagen synthesis)
o Chemotherapy: prevents cells from dividing
o Others: age, genetic disorders, malignancy, uremia
Explain the complications of wound healing
• Deficient scar formation
o Can lead to dehiscence (rupture) or ulceration (can occur due to poor vascularization during healing)
• Excessive formation of repair components
o Hypertrophic scars: excessive amounts of collagen in wound leading to raised scar
o Keloids: scar grows beyond original borders and doesn’t regress; can recur after removal
o Excessive granulation tissue: can prevent re-epithelialization
o Excessive fibroblast proliferation: creates a mass lesion called a desmoid or aggressive fibromatosis
• Formation of contractures
o If excessive: results in disfigurement and loss of function (permanent muscle or joint fixation)
o Especially after burns
Explain the role of mitochondrial damage in the major mechanisms of cell damage
o From increased Ca2+, ROS, or oxygen deprivation o Creates mitochondrial permeability transition pore (channel in membrane, so destroys membrane potential) o Contents (cytochrome c) leak into cytoplasm, trigger apoptosis
Explain the role of loss of Calcium homeostasis or defects in membrane permeability in the mechanism of cell damage
o High concentration of Ca2+
o Opens mitochondrial permeability transition pore
o Activates enzymes
o Activates caspases → apoptosis
Explain the role of oxidative stress from free radicals in the mechanism of cell damage
o Sources:
• Mitochondrial oxidative phosphorylation
• Radiant energy
• Leukocytes in inflammatory response
• Metals (Fe, Cu)
• Nitric oxide
o Results:
• Lipid peroxidation in membranes
• Chain reaction
• Oxidative modification of proteins
• Lesions in DNA
o Removed by:
• Antioxidants
• Transport proteins for reactive metals
• Enzymes (superoxide dismutase, catalase, glutathione peroxidase)
Explain the role of oxidative stress from free radicals in the mechanism of cell damage
o Oxidative damage
o Decreased phospholipid synthesis from low ATP
o Increased phospholipid breakdown from Ca2+ activation of phospholipases
• Breakdown products also cause detergent action
o Damage to cytoskeleton scaffolding
o Results
• Loss of membrane potentials, osmotic balance
• Leakage of cell and lysosome contents
• Damage to DNA and proteins
Neutrophil
Polymorphonuclear leukocyte
Lymphocyte
Monocyte (circulating)
Macrophage (in tissues)
Eosinophil
Basophil
Mast Cells (in tissues)
Describe the vascular changes that occur with inflammation
• Changes in vascular flow and caliber
o Dilation of capillary bed’s pre-arterioles and post-venules
o Results in stasis (expansion of capillary bed) so sluggish blood flow
• Increased vascular permeability:
o Endothelial gaps in venules
• From vasoactive mediators (histamine, bradykinin, substance P) and cytokines (IL-1, TNF, IFN-y) causing cell constriction
o Endothelial cell injury
• Toxins, burns, chemicals → immediate sustained response, or delayed prolonged leakage
• Leukocytes
o Increased transcytosis
• VEGF induces leakage of fluid and cells via “vesiculovacuolar organelle”
o Leakage from new blood vessels
• Because leaky until maturity
• Endothelial cells are more sensitive to vasoactive mediators
End Result: edema, erythema
List the steps involved in leukocyte recruitment and activation
- Stasis
- Margination
- Adherence
- Transmigration/Diapedesis
- Migration
- Chemotaxis
- Leukocyte activation
- Phagocytosis
- Release of Leukocyte products
Leukocyte chemotaxis in inflammation
WBCs move along chemical gradient
o Agents:
• Exogenous agents: bacterial products
• Endogenous agents: complement products (C5a), lipoxygenase pathway products (LTB4), cytokines (IL-8)
o Agents bind G-protein coupled receptors on WBC membrane
o Activation → stimulates polymerization of actin filaments and extension of filopodia at leading edge of WBC
Leukocyte Phagoccytosis
• Recognition and attachment
o Macrophage mannose receptor recognizes mannose and fucose residues from glycoproteins and glycolipids on microbial cell walls
o Enhanced recognition and attachment if foreign object is opsonized by IgG or C3b or plasma lectins
• Engulfment
o Cytoplasm wraps around foreign material
o Forms phagosome
o Fuses with lysosome
o Lysosomal enzymes mix with phagosome contents
• Killing and degradation
o Oxygen-dependent mechanisms
• Reactive Oxygen Intermediates (ROI)
• While oxidizing NADPH, reduce oxygen to superoxide anion → hydrogen peroxide → hydroxyl radical
• Myeloperoxidase converts hydrogen peroxide → hypochlorite
• Hypochlorite destroys microbes by halogenation or lipid peroxidation
o Oxygen-Independent mechanisms
• Substances in leukocyte granules: lysozyme, lactoferrin, major basic protein, defensins, and other enzymes
o After organism is killed, pH in phagolysosome decreases
• Acid hydrolases degrade dead microbes
Describe the factors involved in Leukocyte margination, adherence, and diapedesis
• E-selectin
o Expressed on cell surface of endothelial cells when cytokines present
o Bind to Sialyl-Lewis X–modified glycoprotein on leukocytes
o Weak binding, but slows down WBCs = margination
• ICAM-1 and VCAM-1
o Types of integrin ligands
o Expressed on cell surface of endothelial cells when cytokines present
o Bind to integrins on leukocytes (induced to high-affinity state from proteoglycans stimulated by chemokines)
o Strong bond, fixes leukocyte to endothelium = adherence
• PECAM-1 (CD31)
o An integrin ligand on endothelial cell surface
o Mediates diapedesis
List four powerful chemoattractants for leukocytes
- Bacterial products (LFB)
- Complement products (C5a)
- Products of lipoxygenase pathway (LTB4)
- Cytokines (IL-8)
Describe the functional effects of leukocyte activation
- Modulation of leukocyte adhesion molecules
- Phagocytosis
- Degranulation of phagocytes and platelets
- Secretion of cytokines (to amplify and regulate inflammation)
- Production of arachidonic acid metabolites
- Aggregation of platelets
Compare emigration, chemotaxis, and phagocytosis in terms of function and sequence in acute inflammation
- Emigration: to extravascular space
- Chemotaxis: leukocyte movement along chemical gradient
- Phagocytosis: elimination of injurious agent by WBCs
List the chemical products involved in killing during phagocytosis
o Oxygen-dependent mechanisms
• Reactive Oxygen Intermediates (ROI)
• While oxidizing NADPH, reduce oxygen to superoxide anion → hydrogen peroxide → hydroxyl radical
• Myeloperoxidase converts hydrogen peroxide → hypochlorite
• Hypochlorite destroys microbes by halogenation or lipid peroxidation
o Oxygen-Independent mechanisms
• Substances in leukocyte granules: lysozyme, lactoferrin, major basic protein, defensins, and other enzymes
o After organism is killed, pH in phagolysosome decreases
• Acid hydrolases degrade dead microbes
Describe the causes and manifestations of each of the four diseases related to deranged leukocyte function.
1. Defects in Leukocyte adhesion
• genetic deficiency in leukocyte adhesion molecues (LAD types 1 & 2)
• susceptible to recurrent bacterial infections, impaired wound healing
2. Defects in phagolysosome function
• Chediak-Higashi syndrome
o Autosomal recessive
o Susceptible to infection due to neutropenia, defective granulation, defects in oxygen-dependent microbicidal activity
o Causes delayed microbial killing, albinism, nerve defects, bleeding disorders
• Chronic Granulomatous disease
o Defects in genes for NADPH oxidase
o Deficient oxygen-dependent microbicidal activity
o Recurrent infections
3. Bone marrow suppression
• Common cause of decreased leukocyte activity
• Primary to bone marrow diseases (myelodysplasia, leukemia)
• Secondary to tumor metastases, iatrogenic bone marrow depletion (radiation therapy, chemotherapy) or acute or chronic diseases (burns, sepsis, diabetes, malnutrition, anemia)
4. Aberrant release of leukocyte products
• Underlies many inflammatory diseases: arthritis, asthma, atherosclerosis
• Causes include:
o Regurgitation during feeding—phagolysosome opens to outside
o Frustrated phagocytosis—leukocyte attaches to immune complexes that are fixed on tissues so can’t be digested
o Cytotoxic release—leukocyte ingests substance that damages its own membrane, so becomes incontinent
o Exocytosis—lysosomal granules actively secreted to outside
List the types of acute inflammation
- serous
- fibrinous
- suppurative/purulent
- pseudomembranous
- ulcerative
- gangrene
Serous inflammation
Thin, watery fluid (transudate) at site of injury
Fibrinous inflammation
o Clear fluid AND fibrinogen escape vessel
o Fibringogen polymerizes → tan-pink, stringy fibrin coat
o Characteristic inflammation of linings of body cavities (pleural, pericardial, peritoneal)
Suppurative/Purulent inflammation
o Lots of pus made from neutrophils, necrotic cells, and edema fluid
o Response characteristic of “pyogenic” bacteria (staphylococcus aureus, Neisseria meningitides)
o Ex. Acute appendicitis, abscess, empyma (hollow viscus filled with pus)
Pseudomembranous inflammation
o Characteristic of colonic overgrowth of C. difficle or fungi
• Secondary to broad-spectrum antibiotic use or immunosuppression
o Inflammatory cells, necrotic epithelium, fibrin, and mucus form a thick film over mucosa
Ulcerative Inflammation
o Destruction of an epithelial lining due to ischemic damage or infection
o Ex. Bed sore, diabetic foot ulcer, amebiasis, H. pylori, Herpes
Gangrene inflammation
o Tissue necrosis secondary to interruption of the blood supply by trauma, infection, or thrombosis
o Necrotizing bacterial infection can be superimposed
o Types:
• Dry—without superinfection
• Wet—leading to liquefactive necrosis
• Gas—due to superinfection with gas-forming organisms (clostridium perfringens)
• Necrotizing fasciitis—in deep tissues
• Fournier’s gangrene of the scrotum
Describe the relationship between acute and chronic inflammation. Give examples of each.
• Injurious agent in acute inflammation persists
• Progression to chronic inflammation and acquired immune system is activated
o Via antigen presenting macrophages
• Examples of chronic inflammation:
o Autoimmune diseases
o Persistent infections: Tuberculosis, syphilis, diabetic foot ulcers, gastric or duodenal ulcers, hepatitis C virus infection
o Some malignancies
o Prolonged exposure to toxic agents (particle dusts, reactive lipids)
Define and describe the morphologic/histologic types of chronic inflammation, and give examples of each
• Non-specific chronic inflammation
o Mononuclear cell infiltration: Macrophages, lymphocytes, plasma cells
o Tissue destruction
o Fibrosis
• Granulomatous inflammation
o Granuloma formation
• Collection of activated (plump) macrophages
• Surrounded by collar of lymphocytes
• Can have central necrosis (caseation)
• Can have giant cells (lots nuclei)
o Limited differential diagnosis:
• Infectious: tuberculosis, leprosy, brucellosis, cat-scratch disease, fungi
• Foreign/insoluble objects
• Sarcoidosis: don’t know cause
o Granuloma contains injurious agent so not spread elsewhere
List the systemic effects of inflammation and the responsible cytokines
• Fever
o Pyrogens (bacterial products, IL-1, TNF) → prostaglandin synthesis in hypothalamus → reset temperature
**• Acute phase proteins: **made in liver in response to IL-6 from macrophages
o C-reactive protein: marker for acute MI
o Fibrinogen: makes erythrocytes sticky and form stacks (basis of ESR test)
o Serum amyloid protein: brings HDL to macrophages for metabolism; shows elevated macrophage activity when elevated
• Leukocytosis (elevated WBC)
o Accelerated release of WBC from bone marrow
o Increased immature forms of WBC in circulating blood (“left shift”)
o Mediated by IL-1, TNF-a from macrophages
• Leukopenia: in some viral infections, malnourishment, chronic debilitation
• Acute Phase Reaction: increased pulse, BP, sweating, rigors, chills, anorexia, somnolence, malaise
• Sepsis (TNF, IL-1)
o Tachycardia, fever or hypothermia, hyperventilation, leukocytosis or leukopenia
Discuss the causes and symptoms of sepsis
• From overwhelming bacterial infection or bacterial toxins in blood
• Mediated by TNF, IL-1
• Tachycardia
• Fever or hypothermia
• Hyperventilation
• Leukocytosis or leukopenia
• Disseminated intravascular coagulation (DIC)
o LPS and TNF induce tissue factor expression
o Induces coagulation
• Hypoglycemia: liver can’t maintain glucose levels
• Cardiovascular failure (decreased perfusion pressure)
Compare and contrast resolution and scar
• Resolution
o Inflammation mediators have a short half live
o Released in rapid bursts
o Released only as stimulus persists
o Produce their own stop signals or anti-inflammatory cytokines and lipoxins
o If persists→ chronic inflammation
• Scar formation
o Removal of injurious agent
o Damaged tissue is replaced with fibrous tissue
• When extensive injury
• When involves cells with limited regenerative capacity
Describe the role played by the kinin system in inflammation
• Factor XII/Hageman Factor activated by exposed collagen, BM, activated platelets
• It then activates precursor prekallikrein to kallikrein
• Converts high molecular weight kininogen (HMWK) to Bradykinin
o Effects: pain, increased vascular permeability
o Rapidly cleared by kininases (Ex. ACE in lungs)
Describe the role played by vasoactive amines in inflammation
• Histamine
o Mast cells, basophils, platelets
o Dilation of arterioles, increased venule permeability
o Principle mediator of immediate transient response
• Serotonin
o Platelets and enterochromaffin cells
o Released when platelets aggregate
o Causes increased vessel permeability
Describe the role played by the complement system in inflammation
• Produced by liver, circulate in plasma in inactive configuration
• Activation cascade
• Activated by:
o Classical pathway
• Antigen-antibody complexes bind to complement protein C1
• Initiates cascade
o Alternative pathway
• Direct activation of complement protein C3 from microbial surface molecules or Factor XII
o Lectin Pathway
• C1 activated by serum lectin bound to mannose on bacteria surface
• Common part in pathways: C3 cleavage and activation
• Effector complement proteins:
o C5a: chemotactic agent, attracts leukocytes, activates arachidonic acid metabolism
o C3a and C5a: anaphylatoxins (release and cause vascular dilation)
o C3b: an opsonin, targets bacteria for phagocytosis
o Membrane Attack Complex (MAC): from aggregation of proteins C5-C9, create holes in microbe membrane to lyse
• Regulation
o Complement control proteins acting on convertases
o Inhibitors of complement in serum, also protect self-cells
o C1 inhibitor: circulating serine protease that irreversibly binds to activated C1
Describe the role played by arachidonic acid metabolites in inflammation
- Made from cell membrane in response to external stimuli
- Bind to G-protein couples receptors
• Mediate inflammatory processes:
o Vasodilation/constriction
o Platelet aggregation
o Leukocyte adhesion and chemotaxis
• Eicosanoid production pathway:
o Cyclooxygenase pathway → prostaglandins and thromboxane
o Lipoxygenase pathway → leukotrienes and lipoxins
• Blocked or inhibited by drugs: steroids, aspirin, NSAIDS, COX-2 inhibitors
Describe the role played by the clotting system in inflammation
• Initiated by Factor XII (kinin system): activated by collagen, BM, activated platelets
• Produces Thrombin
o Links clotting cascade to inflammation
• Binds G-protein coupled receptors
• Activates platelets, endothelial cells, smooth muscle cells
• Causes:
• Mobilization of selectins
• Change in endothelial cell shape
• Chemokine production
• Induction of arachidonic acid pathway
• Production of PAF and NO
o Regulation
• Enzymes inhibited by antithrombins, proteins C and S, tissue factor pathway inhibitor (TFPI)
o Fibrinolytic System
• Antagonizes clotting cascade
• Tissue plasminogen activator cleaves plasminogen → plasmin
• Plasmin cleaves fibrin → fibrin split products
• Plasmin cleaves C3 → C3a (increased vascular permeability)
• Plasmin can activate Factor XII
Describe the role played by cytokines in inflammation
- Protein that modulates function of other cell types
- In inflammation: TNF-1, IL-1
State how the complement, coagulation and kinin systems interact
• Kallikrein directly activates C5 to C5a
• Plasmin can directly activate C3
• Complement cascade can activate C5 and C3
• Factor XII activates clotting cascade, kinin system, fibrinolytic system, complement system
• Factor XII and Kallikrein are autocatalytic amplifiers
o Can activate each other
• Bradykinin, C3a, C5a, and thrombin are all inflammation mediators
Describe the effect of steroids, NSAIDs, aspirin and other anti-inflammatory drugs on arachidonic acid metabolism
• Steroids
o Inhibit phospholipases
o Arachidonic acid not available to make eicosanoids
• NSAIDs, acetaminophen, aspirin, COX inhibitors
o Inhibit cyclooxygenase pathway
o Blocks prostaglandin, prostacyclin, and thromboxane synthesis
Discuss the role of free radicals in inflammation.
• Produced by leukocytes when microbes, chemokines or immune complexes
• Products of NADPH oxidative system:
o Superoxide anion, hydrogen peroxide, hydroxyl radical
o Reactive nitrogen intermediates when combined with NO
• Effects
o Destroy phagocytosed microbes
o Extracellular release potentiates inflammatory response
• Increased chemokines, cytokines, endothelial leukocyte adhesion
o Damaging if extracellular release
• Injure endothelial cells, parenchymal cells, RBCs
• Inactivate antiproteases in extracellular tissues
o Counteracted by antioxidants
• Ceruloplasmin, transferrin, superoxide dismutase, glutathione peroxidase, catalase
Platelet Activating Factor (PAF)
• Derived from phospholipids
• Produced by platelets, basophils/mast cells, neutrophils, monocytes/macrophages, endothelial cells
• Specific G-protein coupled receptor on effector cells
• Effects
o Platelet aggregation
o Vasodilation (more potent than histamine)
o Constriction of non-vascular smooth muscles
o Increased leukocyte adhesion
o Chemotaxis
o Degranulation
o Oxidative burst
o Potentiation of arachidonic acid metabolism
****elicits most of cardinal features of inflammation *
Nitric Oxide
• Gaseous signaling molecule
• 3 different nitric oxide synthetase enzymes:
1)phagocytes
• produces NO as free radical
• causes injury to bacteria and parasites
2) endothelial cells
• NO produced causes vasodilation by smooth muscle relaxation
3) Brain
• Produced when increased cytosolic calcium or cytokine stimulation of macrophages
• Effects
o Paracrine inflammation inhibitor
• Reduces platelet aggregation
• Reduces leukocyte recruitment
o Vasodilator
o Microbicidal (via reactive nitrogen intermediates)
• Short half life
• Involved in diseases
o Atherosclerosis, hypertension, diabetes
Substance P
- Pro-inflammatory neuropepetide
- Released in response to psychological stress
- Linked to systemic stress response mediated by CRF
- Activate mast cells and leukocytes
- Involved in some poorly understood inflammatory conditions: Asthma, eczema, migraine, fibromyalgia, rosacea, psoriasis
Anaphylaxis
• Type I hypersensitivity allergic reaction
• Cross-linking of IgE receptors on mast cells
• Degranulation of mast cells
• Histamine release
o Vasodilation → rash, redness, edema
o Bronchoconstriction → respiratory distress
• Medical emergency
o Administer epinephrine (bronchodilation, regulate heart beat)
Complement deficiency
• C3 deficiency
o Increases susceptibility to infection
• Defective MAC formation
o Unable to clear infection by Neisseria
Paroxysmal nocturnal hemoglobinuria
- Complement Inhibitor deficiency
- Mutation in gene controlling complement activation
- Recurrent complement-mediated intravascular hemolysis
- Chronic hemolytic anemia
Alpha-1 antitrypsin deficiency
• Alpha-1 antitrypsin is an antiprotease
o Major inhibitor of neutrophil elastase and other proteases
• Sustained action of elastases cause digestion of extracellular tissues
• Autosomal recessive
o Mutation in protease inhibitor gene
• Pathologic effects
o Panacinar pulmonary emphysema
o Cholestatic hepatitis leading to cirrhosis
o Variable expression throughout life
Discuss how chemical mediators of inflammation might be developed for therapy.
Give examples of their current therapeutic use
• Disrupt inflammatory processes in disease
o Allergy, autoimmune, transplant, malignancy
• Block histamine in allergic diseases
o Block release (cromolyn sodium)
o Block histamine receptor (loratadine)
• Block arachidonic acid metabolism
o Steroids →autoimmune diseases, transplants, allergy
o Aspirin and NSAIDs →arthritis, etc.
o COX-2 inhibitors (Vioxx, Celebrex, Bextra) → arthritis
• Block effect of TNF
o Autoimmune diseases → Crohn’s, rheumatoid arthritis
• Block effect of IL-1
o Clinical trials to treat rheumatoid arthritis