VM605 General Pathology

need to add lectures 15+

Describe the process of primary hemostasis including receptor-ligand interactions which allow for platelet adhesion, activation, and aggregation

Step 1: Vascular injury

Step 2: Gp-1b on platelets binds vWF expressed on subendothelial collagen

Step 3: after adhesion platelets become activated, conformational changes in membrane Gp-2b and Gp-3a receptor increases affinity for fibrinogen, phosphatidylserine translocates to cell surface to allow for Ca2+ binding, secretion of granule contents promotes platelet aggregation (ADP, which activates nearby platelets; Thromboxane A2, which induces aggregation)

Step 4: once bound to fibrinogen a bridge between adjacent platelets form, leading to aggregation, initially reversible but becomes permanent once thrombin formed through coagulation cascade

Outline the coagulation cascade and know which factors are involved in the extrinsic, intrinsic, and common pathways

Factors are named 1-13

1 = fibrin (fibrinogen to fibrin), fibrin is crosslinked with Factor 13 to stabilize the clot

2 = prothrombin to thrombin (when activated)

3 = tissue factor

4 = calcium (Ca2+)

Extrinsic Pathway:

7-->7a via 3 and Ca2+

7a helps activate 10 to 10a (then starts the common pathway)

Intrinsic Pathway:

12-->12a via collagen

12a turns 11-->11a

11a (and 7a can also activate 9) turns 9-->9a

9a, with Ca2+ and 8a, helps activate 10 to 10a (to start the common pathway)

Common Pathway:

10-->10a (via 7a, 9a, Ca2+, 8a)

10a -->helps convert 2 -->2a (along with Ca2+ and 5a) (prothrombin to thrombin)

2a then helps convert 1-->1a (fibrinogen to fibrin)

2a also activates 13-->13a which crosslinks with fibrin to stabilize clot

Factor 8 is activated independently and forms a complex with vWF

Recall Vitamin K-dependent coagulation factors

2, 7, 9, 10

Outline the basic process of fibrinolysis

plasminogen (via t-PA, Factor 12a, and Kallikrein) is turned to plasmin

t-PA = tissue plasminogen activator

plasmin helps turn fibrin into FDPs (which can be measured clinically)

fibrinolysis is a tightly regulated process which should allow for controlled clot formation and clot breakdown depending on the situation

Total Process: vasoconstriction --> primary hemostasis --> secondary hemostasis --> fibrinolysis

Realize the purpose of the major components (heart,vessels,interstitium)

blood, pump (heart), distribution/exchange network (arteries/veins/capillaries, lymphatic vessels)

all vessels lined by endothelium - healthy endothelium is antithrombotic, damaged endothelium can become prothrombotic

Review the mechanism of fluid homeostasis, including concentration and pressure gradients

interendothelial pores is generally passive for substances

O2, CO2 and non-charged fatty acids move freely but depend on pressure and concentration gradient

charged particles may require pumps/receptors/ATP to get across the plasma membrane

osmotic pressure: primarily albumin

hydrostatic pressure: what is measured with blood pressure measurements

Understand the mechanisms of edema formation and be familiar with the four different types

edema forms when there is an imbalance in fluid distribution between intravascular and interstitial compartments

four mechanisms: increased microvascular permeability, increased intravascular hydrostatic pressure (cardiac function), decreased intravascular oncotic pressure (albumin), decreased lymphatic drainage (physical obstruction or dilation of lymphatic vessels)

Recognize the gross changes and clinical presentation of local versus systemic edema

edema looks like clear slightly yellow watery fluid, can fill an organ or a cavity and can cause dysfunction of the organ

Know the diseases that can cause edema and the associated mechanisms

Increased vascular permeability: inflammation is a common cause (mediators cause vasodilation and increased vascular permeability (histamine and Substance P), fluid accumulation is an exudate (high protein, high cells))

Increased intravascular hydrostatic pressure: increased blood volume either from an active or passive increase in blood flow (heart failure)

Decreased oncotic (osmotic) pressure: hypoproteinemia, more commonly hypoalbuminemia, clinical signs are effusions and peripheral edema

causes of hypoalbuminemia: loss (hemorrhage, GI loss, renal loss) or decreased production by the liver (generally severe liver disease/failure)

Decreased lymphatic drainage: mass or dilation of lymphatics resulting in obstructed or impaired flow of lymphatic fluid, lymphatic vessels drain excess fluid from interstitial space, mass will often result in local edema, dilation of lymphatics can cause ascites or effucions in other locations

Realize the difference between hyperemia and congestion

Hyperemia: avtive increase in blood flow, tissue will appear more red due to the increased blood flow

physiologic causes: exercise, GI trace after a meal, blushing

pathologic causes: inflammation, neovascularization

Congestion: passive accumulation of blood, can be local or generalized

hypostatic congestion: pooling of blood in dependent regions shortly before death

liver mortis (post-mortem lividity): gravity dependent pooling of blood in tissues after death

Know the definition and mechanism of thrombus formation (Virchow triad)

thrombus = blood clot inside a blood vessel that disrupts blood flow to a tissue often resulting in ischemic necrosis and damage or death of the tissue

it is an aggregate of platelets/fibrin/other components of blood formed on a vascular wall

Virchow triad: if you have endothelial injury/abnormal blood flow/hypercoagulability then can lead to thrombosis

How does endothelial injury contribute to thrombus formation

endothelial injury leads to abnormal blood flow which leads to thrombosis, it also leads to a state of hypercoagulability which leads to thrombosis

endothelial injury --> exposure of collagen --> release of TF and vWF leads to platelet activation and accumulation

endothelial activation secondary to inflammation or injury can shift to prothrombotic gene expression by the endothelial cells

procoagulant: thrombomodulin downregulation

antifibrinolytic: secretion of plasminogen activator inhibitors (PAI)

Recognize the differences between venous and arterial thrombi

arterial thrombi: usually form secondary to endothelial damage, usually pale and consit of platelets and fibrin

venous thrombi: more likely to form in areas of stasis or congestion, often red due to accumulation of erythrocytes

Review the mechanism of embolus formation and the types of emboli

mural thrombus: thrombi attached to the endocardium (the entire cardiac ventricle or atrium)

vegetative thrombi: septic thrombi on the heart valves that have a cauliflower appearance

Know the different types of shock and their mechanism

Mechanism: hypotension --> impaired tissue perfusion --> cellular hypoxia resulting in a shift to anaerobic metabolism, cellular degeneration, cell death

Caused by loss of blood volume, decreased cardiac output, inappropriate vascular resistance

Types: cardiogenic, hypovolemic, blood maldistribution

Cardiogenic: failure of the heart to adequately pump blood (decreased cardiac output)

Hypovolemic: decreased circulating blood volume from blood loss (hemorrhage) or fluid loss (dehydration)

Blood maldistribution: decreased peripheral vascular resistance and pooling of blood in peripheral tissues, neural- or cytokine-induced vasodilation (anaphylactic shock/neurogenic shock/septic shock)

Be able to recognize the gross and clinical signs of shock

Stages:

nonprogressive: compensatory responses to decreased pressure

progressive: disease too severe to be controlled by compensatory mechanisms, blood pools and tissues are hypoperfused resulting in progressive cell injury (systemic acidosis, inefficient production of ATP, increased production of lactic acid)

irreversible: fall in peripheral resistance, multiple organ dysfunction

Clinical signs: hypotension, tachycardia, decreased urine output, hypothermia, can progress to DIC

Gross changes: edema, petechial hemorrhages, microthrombi, pooling of blood

Learning Objective 1: Understand the basic features of mitochondrial disease and possible manifestations

mutations occur and spread slowly since they divide like bacteria, some cells carry mostly mutant mitochondria

mitochondrial encephalopathies: genetic disease that affects tissues with high metabolic rates (brain and muscle), variable patterns of inheritance (inherited from mother), delayed progressive course, neurologic signs, bilateral symmetry with white matter necrosis

mitochondrial myopathies: "parking lot" paracrystalline arrays in mitochondria, no cristae (uncertain significance), exercise intolerance

mitochondrial neoplasms: oncocytoma - tumor cells filled with compact masses of mitochondria

Understand the basic features of autophagy

cellular housekeeping: inhibits apoptosis, autophagosome formation, SNARE docking proteins

natural/protective process

relevant in cancers (some have high rate of autophagy, fermentation of glucose) and anti-viral responses

lipofuscin: brown pigment from stressed or long-lived cells from the junk of the cells

Understand and be able to articulate the biochemical changes that underlie the morphologic manifestations of apoptosis, including the role of caspases, calcium, and mitochondria

Cell death with: cytoplasmic shrinkage, pyknosis, karyorhexis, membrane blebbing, apoptotic bodies/vessels

Intrinsic and Extrinsic Apoptosis: perturbation of intrinsic or extrinsic environment

Intrinsic: mitochondrial pathway = development of mitochondrial outer membrane permeabilization (MOMP), Caspase 9 activation --> Caspase 3 activation (leads to cell domolition)

caused by: ER stress, ROS, mitotic stress, DNA damage (radiation, heat), withdrawal of trophic stimulus/survival factor

MOMP controlled by: BAX and BAK+, BCL-XL-; releases apoptogenic factors and leads to cessation of mitochondrial function (ATP production)

SMAC binds apoptosis inhibitors (IAPs)

Extrinsic: death receptor pathway = membrane receptor-dependent, Caspase 8 activation --> Caspase 3 activation, ligation leads to death-inducing signaling complex (DISC) (which leads to activation of Caspases 8 and 3)

caused by: ligand attachment to surface death receptors (FAS and TRAILR)

TNFalpha (from macrophages) can bind FAS --> apoptosis

T cells have FAS ligant --> apoptosis

Know the ways in which apoptosis can be triggered in a cell

embryology (involution), atrophy (loss of stimulus), neoplasia (genetically unstable cells), cell-meidated immune disease (hepatitis), toxins/hyperthermia/radiation/drugs, genomic injury, cytotoxic T cells induce apoptosis, underlying triggers overlap with those that lead to necrosis

Be able to identify, describe, and compare/contrast the sequelae to necrosis and apoptosis

Sequelae to necrosis: DAMPs (damage associated molecular patterns) are released from dying/injured cells with spread into the tissue and bind to receptors on leukocytes to alert them; neutrophils commonly respond with inconsiderate enzyme use and generally die in the tissue which mediates further inflammation leading to scarring; scar tissue - the organ/animal survives but parenchyma is lost and does not return (primary function continues but if too much then can lose function)

examples of DAMPs: extracellular chromatin/DNA, extracellular ATP/adenosine/uric acid, partially-degraded membrane fragments (phospholipids), heat shock proteins

Sequelae to apoptosis: no inflammatory response (no neutrophil attractants but macrophages and adjacent cells come in), very little release of intracellular enzymes into tissue, atrophy is a result (cell deletion leads to shrinkage of tissue), tissue can recover since the stroma is intact (no fibrosis) and parenchymal cells are healthy

Compare/Contrast:

Necrosis: occurs when cell loses control of its biochemical processes, mitochondrion is the key organelle, occurs when the damage is so severe repairs cannot be made before mitochondrial collapse occurs

Apoptosis: cell is in complete control of process, mitochondrion is also key organelle but it regulates the process so it is orderly, "preferred" method for disposing of injured or obsolescent cells especially if DNA is damaged

Recognize the morphologic features of apoptosis and be able to distinguish them from the morphologic features of necrosis

cells round up, fragment, and are then ingested by neighbors (heterophagy)

tightly regulated, controlled by the cell itself

cell gragments are self-contained (intact membranes and organelles)

no inflammation

cytoplasmic changes: cell shrinkage and condensation --> rounded with clear halo, also see hyperbasophilia or hypereosinophilia

nuclear changes: chromatin caps/crescents and nuclear membrane stays intact

in late stages, cell fragments into membrane-bound bodies

Differs from necrosis by:

in necrosis: leakage of contents from cell, formation of myelin figures, and amorphous densities in mitochondria; neutrophils are the phagocytic cell

in apoptosis: no leakage, get cellular fragmentation and apoptotic bodies, phagocytes come in to clean up the cell

Understand the two basic mechanisms of lethal cell injury and the underlying pathogeneses

1. Interference with energy supply:

common causes: hypoxia (decreased O2), ischemia (no blood flow), infarction (tissue death due to ischemia), anoxia (no O2 in tissues)

myocardium/proximal renal tubules/neurons need constant ATP - these are very susceptible to hypoxia/ischemia

for other cell types, hypoxia is not the most common cause of necrosis

2. Damage to cell membrane:

common causes: many; irradiation, toxins (chemical/bacterial), metabolic products, depleted antioxidants, immune-mediated reactions, free radicals (superoxide is most common, hydroxyl radical is most damaging)

liver and kidney are especially susceptible

Understand and be able to outline the biochemical/ultrastructural progression of lethal cell injury

Mitochondrial damage: decreases ATP (which has multiple downstream effects) and increases ROS (which leads to damage to lipids/proteins/DNA)

Entry of Ca2+: increased mitochondrial permeability and activation of multiple cellular enzymes

Membrane damage: the plasma membrane sees loss of cellular components; the lysosomal membrane sees enzymatic digestion of cellular components

Understand the concept of reperfusion injury

reperfusion injury: return of blood flow to damaged but living cells

much of the injury to cells occurs in this stage - damaged mitochondria end up with free electrons, producing radicals

inefficient ATP production

Ca/water flood into cells --> pumps don't work well --> increased swelling

Understand the concepts of free radicals and the mechanisms by which they cause cell injury and cell death

leukocytes produce them to kill bacteria

hemorrhage releases iron, which catalyzes free radical reactions

injury releases arachidonic acids from cell membranes --> produces free radicals

ischemia from reperfusion injury leads to free radical damage

sunlight --> free radical production

x-rays produce free radicals

drugs/toxins/pollutants

Lethal injury comes from the initiation of chain reactions (initiation, propagation, termination)

Initiation: oxygen gets into the cell membrane and breaks the double bond (cell membrane lipids are bent/broken/cross-linked due to radical attack)

Propagation: PUFA radicals with oxygen lead to further break-up of bonds

Termination: a polymerized fatty acid is produced = a molecular lesion

radicals induce the MPT (mitochondrial permeability transition)

Understand and be able to describe the protective mechanisms and pathways the cell has against free radical damage

Detoxification: how the body deals with free radicals

Antioxidant scavengers: Vitamin E (lipid-soluble, eats radicals in the membranes), Vitamin C (eats up radicals, regenerates Vitamin E); freezes radicals

SOD (superoxide dismutase) gets rid of O2- in the cytosol and mitochondria

Catalase and Peroxidase: remove hydrogen peroxide

Glutathione peroxidase: detoxifies H2O2, requires Selenium

tissues with high oxygen consumption (like muscle and liver) are prone to damage without adequate selenium

Recognize the morphologic changes reflective of irreversible cell injury leading to necrosis - both cytoplasmic and nuclear

cells swell (oncosis) and eventually burst

internal structure falls apart

often groups of cells

sequelae: inflammation --> scarring --> loss of function

cytoplasm: pallor in early stages, loff of cell detail, can see some fragmentation and hypereosinophilia

nucleus: pyknosis, karyorhexis, karyolysis

pyknosis: condensation of the chromatin and the nuclear membrane

karyorhexis: fragmentation of chromatin and nuclear membrane

karyolysis: dissolution of chromatin and nuclear membrane

Recognize the gross and histological abnormalities associated with necrosis and recognize the three major morphologic manifestations of necrosis - coagulation necrosis, liquefactive necrosis, and caseous necrosis

1. Coagulation Necrosis: tissue structure maintained, all cells are dead, inflammation minor; seen with infarctions (blood supply is cut off)

initially: pallor from blood loss

subsequently: reddening from hemorrhage and inflammation

cell outlines are preserved but details are lacking (no or lacking a nucleus), seen as cell shadows

2. Liquefactive Necrosis: tissues are liquid - structure is lost, inflammation is prominent, many neutrophils; an example is in classic foal sepsis

can see refractile patterns on ultrasound

can also see some petichia and ecchymosis

3. Caseous Necrosis: tissue develops a cheesy semi-solid quality - loss of structure, inflammation is prominent, many macrophages; an example is in Mycobacteria infections

tissues are pale yellow and pasty

a special term for infections where many macrophages are present

may see multi-nucleated giant cells (hallmark of granulous response)

Recognize the three major morphologic manifestations of gangrene - dry, wet, and gas

Gangrene = coagulative necrosis with other modifiers/changes

Dry gangrene (mummification) = coagulative + dessication, better one to get

Wet gangrene = coagulative + moisture & saprophytic bacteria

Gas gangrene = coagulative + saprophytic anaerobic bacteria, worst of all

Fat necrosis: coagulative necrosis of adipose, fatty acids combine with Ca/Na/K in dead tissue --> for soaps (saponification)

Intracellular accumulations

one of the manifestations of metabolic derangements in cells (accumulation of abnormal amounts of substances)

two categories of substances that accumulate

normal: normal cell constituents (water, lipids, proteins, carbohydrates)

abnormal:

exogenous: mineral, products of infectious agents/pathologic processes elsewhere

endogenous: product of abnormal synthesis or metabolism

may accumulate in cytoplasm (typically) or nucleus; may accumulate transiently or permanently

possible consequences of accumulation: harmless (incidental) to varying degrees of injury (toxic)

if the overload can be stopped/controlled then it is reversible

Many processes result in abnormal intracellular accumulations that can often be categorized into four types of abnormalities

1. Abnormal metabolism: a normal endogenous substance is produced at a normal or increased rate but rate fo metabolism is inadequate to remove it leading to accumulation

ex. hepatic lipidosis, renal tubular proteinosis, steroid hepatopathy (glycogen accumulation)

2. Defect in protein folding/transport: an abnormal endogenous substance (abnormal usually from a genetic defect; abnormality --> improper folding/transport/degradation --> accumulation

ex. mutated alpha-1-antitrypsin in liver, degenerative CNS diseases (Alzheimer's)

3. Lack of an enzyme: a normal endogenous substance accumulates because of defects in enzymes required to metabolize the substance (defects typically inherited); substance will generally accumulate in lysosomes

ex. lysosomal storage diseases

4. Ingestion of indigestible materials: an abnormal exogenous substance is deposited and accumulates because the cell doesn't have the enzymatic machinery needed to break it down NOR the ability to transport the substance to another site

ex. pneumoconiosis (accumulation of inhaled junk in macrophages of the lungs)

Accumulations of Lipids:

lipidosis/steatosis/fatty change: accumulation of triglycerides and other lipid metabolites (fats and cholesterols) within parenchymal cells (hepatocytes), most commonly seen in the liver (hepatic lipidosis) - the main organ involved in lipid metabolism

may see in other organs as well (heart/kidneys/skeletal muscle) typically as a result of altered liver function

fatty change is lipidosis often related to altered lipid metabolism --> fatty vacuolation WITHIN cell cytoplasm

fatty infiltration refers to infiltration of mature adipocytes into non-adipose tissue --> NORMAL lipid metabolism; in some instances of tissue atrophy the cells that are lost may be replaced by adipocytes

Accumulation of Proteins:

ex. renal tubular proteinosis

Mott Cells and Russell Bodies: accumulation of protein within the cytoplasm of plasma cells

globules are called Russell bodies, described as a manifestation of cellular indigestion of the ER

seen in some chronic inflammatory diseases like plasma cell stomatitis in cats/plasma cell pododermatitis in cats/inflammatory bowel disease

Accumulation of Glycogen:

most reserves found in the liver and skeletal muscle

excessive accumulation of glycogen can occur in different pathologic processes (generally abnormal glucose or glycogen metabolism)

ex. glucocorticoid (steroid) hepatopathy

Hepatic Lipid vs Glycogen Accumulation:

Lipid: abnormal lipid/fat metabolism, clear punctate vacuoles (microvesicular or macrovesicular), possibly displaced nucleus, stains with Oil Red O positive and is PAS negative

Glycogen: abnormal glucose and glycogen metabolism, small fuzzy to foamy vacuoles, no displaced nucleus, stains with Oil Red O negative and is PAS positive

Lysosomal Storage Diseases:

a group of disorders characterized by accumulation of material within lysosomes due to absence of specific enzymes required for breakdown of the material (material can be a variety and combination of proteins/carbohydrates/lipids)

most of these diseases are genetic/inherited (some can be induced by toxins targeting the specific enzyme)

long-lived post-mitotic cells tend to be the most affected (neurons and muscle)

clinically will see young animals with progressive neuromuscular impairment

ex. alpha-mannosidosis in cats = deficiency of alpha-mannosidase which blocks catabolism of mannose/N-acetylglucosamine oligosacchardies leading to accumulation of material within lysosomes of neurons and other cells which contributes to neurologic disease

ex. globoid cell leukodystrophy = deficiency in galactocerebrosidase (component of myelin) which leads to material accumulation in oligodendrocytes/Schwann cells/macrophages which leads to destruction of myelin-producting cells and myelin that is produced is wrong; macrophages (Gitter cells) come in to clean up but they also accumulate material

Inclusions:

non-infectious inclusions: rhomboid crystalline protein inclusions (crystalloids or "brick" inclusions) = incidental finding, significance unknown, large/eosinophilic/rhomboidal (can be so large they distort the nucleus or cell); lead inclusion bodies = in renal tubular epithelial cells (in lead toxicity/poisoning), best highlighted by acid-fast staining, inclusion represents lead + protein

viral inclusion bodies: may be in nucleus, cytoplasm, or both; presence or absence depends on timeline of infection (more often seen early in infection); generally DNA viruses are intranuclear inclusions and RNA viruses are intracytoplasmic inclusions

understand significance of iron in context of erythrocytes

hemaglobin is present in RBCs (carries oxygen), hemaglobin contains four iron atoms (Fe2+), oxygen binds to Fe2+

know common causes fo iron deficiency

1. nutritional deficiency (not ingesting enough)

2. true deficiency (increased loss - chronic hemorrhage)

3. functional deficiency (sequestration and decreased absorption (with inflammation))

understand the basics of iron metabolism, including transport, storage, and alterations in inflammatory disease

Iron Metabolism: most (50-70%) exists in erythrocyte Hgb, iron absorption is tightly regulated, occurs in proximal duodenum, majority of iron used comes from recycled Hgb iron, stores are regulated to avoid overload/deficit, body preserves iron present in erythrocytes using the mononuclear phagocyte system (macrophages phagocytose senescent RBCs so that hemoglobin is split into heme and globin, the heme releases Fe2+ and is degraded, the free iron can now be stored or exported), ferroportin also found in macrophages allowing export of Fe3+

Transport: ingestion as either Fe2+ or Fe3+ --> changed into Fe2+ if needed --> transporter moves Fe2+ into enterocyte --> Fe2+ moved out of enterocyte and into plasma by ferroportin --> Fe2+ converted back to Fe3+ --> Fe3+ binds to apotransferrin to form transferrin --> transferring moves iron around body to necessary tissues --> binds with transferrin receptor at target --> entire complex moved into cell via endosome --> Fe3+ dissociates from transferrin due to low pH of endosome --> Fe3+ reduced to Fe2+ --> transporter protein in endosome membrane moves Fe2+ into cytosol for heme synthesis or storage

Storage:

Ferritin: water-soluble, mobile, iron-protein complex; mainly found in developing RBCs/macrophages/hepatocytes/enterocytes; synthesis increases in inflammation and when more Fe is present

Hemosiderin: poorly soluble, less mobile, more stable; major storage form of iron; predominantly in macrophages of liver/spleen/bone marrow

Alterations: Hepcidin = small peptide produced by the liver, negative regulator of iron movement, prevents iron absorption, prevents iron movement out of storage

binds ferroportin (internalizes in the cell) --> results in decreased iron export from macrophages and hepatocytes --> intracellular iron increases --> decreased iron absorption from intestinal lumen and decreased iron uptake by erythroid precursors

suspected that this mechanism of iron sequestration developed to prevent bacterial proliferation during infection, this also contributes to the anemia of chronic/inflammatory diseases, functional iron deficiency ("pseudo-iron deficiency")

Decreased hepcidin production = increased absorption of intestinal iron, normal enterocyte iron export into plasma, normal release of stored iron from tissue macrophages and hepatocytes

Increased hepcidin production = prevention of iron absorption by enterocytes, decreased iron export from macrophages and enterocytes

be familiar with methods of assessing body iron (serum iron,serum ferritin)

iron testing with non-regenerative anemia, difficult to assess total body iron, must interpret results in light of clinical picture, a panel of tests has the greatest clinical utility - analytes affected by inflammation/organ function/etc. --> results may be confusing and difficult to interpret

Serum Iron Concentration: measures amount of iron bound to transferrin, only assesses circulating iron, increases seen in inappropriate iron injections/potentially excess dietary iron/hemolysis, decreases seen in true/absolute iron deficiency (chronic external blood loss and decreased dietary iron or ability to absorb iron from the GI tract) and increased iron storage (chronic inflammation leading to functional iron deficiency)

Serum Ferritin Concentration: ferritin in serum is secreted into blood by cells (primarily macrophages), correlates with tissue iron stores in domestic animals (can be used to separate a true iron deficiency anemia from anemia of chronic disease), species-specific immunoassay required to measure; increases seen in diseases that cause increased iron storage and increased ferritin production (inflammatory disease - ferritin is a positive acute phase protein), decreases seen in true iron deficiency, if animal also has a concurrent inflammatory disease then some ferritin may be normal or even elevated

Reversible injury

injured cell can regain homeostasis and return to morphologically and functionally normal state

seeing purely reversible cell injury alone is not common, see a spectrum of lesions from normal to necrosis (with degeneration between)

Cell swelling

one consistent lesion of injury

grossly (if many cells affected): pallor, organ swelling (organomegaly), decreased specific gravity (increased water)

histopathology: increased cell size with rounding, pale finely vacuolated to granular appearance, nuclei are not displaced and may be swollen

Hypertrophy

organomegaly, increased workload on cell, increased size from increased numbers and sizes of organelles, no difference in water amount

Degeneration

organomegaly, cell injury and damage, increased size from increased water in cells (organ swelling), increased water amount, concurrent necrosis or other findings associated w/ cell injury/damage

Major controller of cell volume

Na/K ATPase pump

susceptible players: physical barrier function of membranes, ATP requires oxygen, membrane proteins/enzymes maintaining ion concentrations and membrane functions

Mechanisms

damage to cellular membranes, injury to enzymes regulating ion channels on membranes, failure of ATP production

Potential causes

mechanical injury (trauma), hypoxia, toxicity, free radicals, infectious (viral and bacterial), immune-mediated

Injured cells cannot control

water/electrolytes cannot maintain other cell functions

if only a few cells affected, you may not see clinical signs (except for regions of the heart/brain)

most of the cells affected will show clinical signs

this change is reversible, however if injury is severe enough or not removed promptly enough then can lead to cell death

Reversible Disorders

atrophy, involution, hypertrophy, hyperplasia

Increase

hyperplasia (cell NUMBER) and hypertrophy (cell SIZE); tissue is bigger

hypertrophy: rare to see solely, histologic architecture is normal but cells are bigger, cytoplasm swells due to increased numbers and size of organelles, cell is doing more work so needs more machinery, subtle change; 2 major notes - increased gene expression (more relaxed chromatin pattern) and increased SER (more organelle to detox the drug), both expand the cytoplasm and makes the cell bigger, nuleus looks paler than normal on histo

pathologic: hypertrophic cardiomyopathy or genetic mutations resulting in overdevelopment of muscle (double-muscling)

physiologic: cardiac/skeletal muscle hypertrophy from exercising, hypertrophy of uterine wall during pregnancy, compensatory renal hypertrophy after unilateral nephrectomy, drug metabolism in the liver

hyperplasia: increase in cell number increases mitotic divisions --> chronic hyperplasia leads to increased times genetic info is replicated --> more opportunities for mutations (neoplasia), increase is in response to increased and/or ongoing stimulus or to accomodate tissue loss

hypertrophy may also be seen concurrently

pathologic: typically chronic irritation or hormonal stimulation

physiologic: often related to sexual development (development of secondary sex characteristics at puberty - mammary, uterus, testes, prostate, endometrial hyperplasia during estrus and diestrus, mammary gland development during pregnancy) and in response to tissue loss (compensatory, hepatic regeneration, epithelial regeneration)

Decrease

atrophy and involution; tissue is smaller AFTER normal growth has been reached

atrophy: cause by adverse environment, lack of/decreased nutrition/blood flow/stimulatory factors/workload or use/innervation/endocrine stimulation, pressure atrophy (a combination)

involution: physiologic (normal) process, job is done and tissue is no longer needed, examples are thymus, uterus, mammary tissue

Reversible/Irreversible Disorders: metaplasia, dysplasia

Metaplasia: one cell type is replaced by another cell type, less-differentiated reserve (stem) cells differentiate along a different line (basal cells), cause is chronic irritation or damage, within the same germ line (usually epithelial is specialized to less specialized, less commonly mesenchymal which is connective tissue to bone)

not neoplastic but may be pre-neoplastic; is reversible to a point, but constant turnover can turn neoplastic

Dysplasia: abnormal organization and cell maturation, disarray +/- cellular atypia

two major forms:

1. developmental/primary: congenital conditions, generally referring to an organ/tissue, an issue from birth

2. acquired/secondary: viral infection, chronic irritation/damage, carries the potential to progress to neoplasia (pre-neoplastic), similar to metaplasia

Homeostasis

normal cell is confined to a fairly narrow range of fucntion and structure by its state of metabolism/differentiation/specialization, constraints of neighboring cells, and availability of metabolic substrates

when the cell is able to handle physiologic demands to maintain a steady state

Cell Stress & Injury

the cause/stimulus, what is causing the harm

intrinsic etiologies: primary = genetic mutations, secondary = influences that may modify expression of disease (age, gender, coat color, genus, familial, breed, idiosyncratic reactions)

extrinsic etiologies: inanimate forces = weather/radiation/trauma/toxins/chemicals/drugs, animate agents = bacteria/viruses/parasites/protozoa/prions

things that do NOT cause injury or stress: oxygen deficiency, physical agents, infectious agents, nutritional deficiencies/imbalances, genetic derangements, workload imbalances, chemicals/drugs/toxins, immunologic dysfunction, aging

Mechanisms

things that occur in cells secondary to the cause of damage/stimuli, can occur simultaneously, lot of cross-talk/intertwining of these

1. decreased ATP: major causes = decreased oxygen/nutrients, mitochondrial damage, specific targeting of ATP-generating mechanisms by toxins; consequences = ER swelling, cellular swelling, loss of microvilli, blebs, clumping of nuclear chromatin, lipid deposition

2. mitochondrial damage: major causes = increased cytosolic Ca, reactive oxygen species, oxygen deprivation, mutations in mitochondrial genes, consequences = necrosis, apoptosis

3. entry of Ca2+ = big role in overall cell damage, major causes = intracytoplasmic Ca levels increase from either extracellular sources due to increased membrane permeablity or release from mitochondria and ER, consequences = decreased ATP and activation of numerous enzymes --> membrane/nuclear damage --> further depletion of ATP

4. increased ROS (reactive oxygen species): free radicals are chemicals that have an unpaired electron in outer orbit so they are unstable, can disrupt other molecules and make those free radicals also (autocatalytic reaction); ROS is a free radical derived from oxygen, major causes = normal metabolic/mitochondrial respiration, absorption of radiant energy, inflammation, enzymatic metabolism of exogenous chemicals/drugs, transition metals, nitric oxide, consequences = damage to lipids (membranes), proteins (enzymes, ribosomes, structural proteins, etc), carbohydrates (membrane proteins), nucleic acids (DNA, RNA)

5. membrane damage: major causes = ROS's (lipid peroxidation), decreased phospholipid synthesis, increased phospholipid breakdown, cytoskeletal abnormalities, consequences = mitochondrial damage, lysosomal injury, plasma membrane leakage

6. protein misfolding/DNA damage: major causes = several, consequences = irreparable damage --> apoptosis, broken down/recycled, trigger apoptosis

Lesions: structural (morphologic) alterations in cells or tissues; visual representations of tissue injury and response

macroscopic: gross pathology, see with plain eye

microscopic: histopathology, need a microscope

ultrastructure: electron microscopy, need special microscope

Strong terms: colors (can further clarify with bright/dull/dark/muted), shapes/structures, surfaces, margins, texture

Objective measurements: percentages (with context), three planes of measurement (lengthxwidthxheight), distance from anatomical landmarks, amount of fluid, weight, descriptive terms in relation to commonly known items, any measurements (preference is for metric units)

Feel/Smell: soft/firm/gritty/hard/gelatinous/semi-dry; friable/elastic/fixed in place/freely moveable/pitting/crepitus/emphysematous; rancid butter/alcohol/sweet/sour/rotten/chemical-like/metallic

Anatomical locations: carpus/tarsus/antebrachium/brachium/bony pertuberances/ventral/dorsal/rostral/caudal/medial/lateral/superficial/deep/

Common/standard terms: abrasion = grazes/scratches, contusion = bruises, laceration = tears, incised wounds = stabs

Morphological Diagnosis: descriptive in nature and focused on one lesion or one set of related lesions

specific cause (etiology) usually not provided, can develop a list of differential etiologies

based heavily on medical terminology

sentence structure: Organ: [severity], [duration], [distribution], [lesion] +/- "with..." [additional features]

severity, duration, and distribution are all modifiers; optional and not always applicable

Pathology: the study of disease

structural and functional changes in cells/tissues/organs and relating these changes to specific causes

how these changes manifest and contribute to disease

Health --> Insult --> Change --> Disease

the connection between normal anatomy/physiology with clinical medicine

Etiology: the cause

Intrinsic etiology (from within the body):

Primary: genetic mutation

Secondary: influences that may modify expression of disease (age/gender/coat color/genus/familial/breed/idiosyncratic reactions)

Extrinsic etiology (from outside the body):

Inanimate forces: weather/radiation/trauma/toxins/chemicals/drugs

Aminate forces: bacteria/viruses/parasites/protozoa/prions

Pathogenesis: how the disease develops

a running flow chart: etiology --> sequence of events --> lesion

pathogenesis statement: follows the above to form a script of what happened

Lesions: structural (morphological) alterations in cells or tissues

macroscopic (gross pathology) - can see with eye alone

microscopic (histopathology) - need microscope

ultrastructure (electron microscopy) - need special microscope

visual representation of tissue injury and response

pathognomonic lesions - a lesion, when taken in context of the clinical picture, that is diagnostic for a particular process; incredibly rare

Types:

neoplasia, bacteria, neuromuscular junctional disease/some toxins/arrhythmias

not every lesion is significant

consider statistical significance versus clinical significance

incidental findings - ask yourself: is this really what is causing the clinical problem/disease/death? And does it make sense?

Lesions secondary to euthanasia:

barbiturate salts

splenomegaly (barbiturate euthanasia or anesthesia)

cranial hemorrhage (captive bolt)

Postmortem changes:

algor mortis: postmortem cooling

rigor mortis: rigidity (lack of can be telling, will eventually fade to laxity)

liver mortis: blood pooling (can tell if a body was moved)

staining: bile imbibition, hemoglobin imbibition, pseudomelanosis, melanosis, gas distension (emphysema, prolapses, gastric rupture)

Differentiating between antemortem and postmortem:

is there a tissue reaction (inflammation, fibrosis, edema, hemorrhage)

does it make sense in context of case

what is post-mortem interval

Morphological change --> functional change --> disease manifestation (clinical signs and symptoms)

Clinical Signs vs. Symptoms:

Signs: a manifestation that the physician perceives, points to something that can be quite specific

Objective - can be measured

temperature, blood pressure, respiratory rate, heart rate, rash/pustules/erythema, diarrhea, vomiting

Symptoms: a manifestation of disease apparent to the patient reported by owner, can be misleading, can be quite vague/non-specific, NEVER to be taken alone as a basis for diagnosis

Subjective - usually not measureable

back pain, fatigue, headache, anxiety, nausea

Diagnosis: an interpretation of the nature of the disease taking into context the lesions and clinical signs

Few different types:

morphologic diagnosis (gross and histopathology) - descriptive in nature and focused on one lesion or one set of related lesions, specific cause usually not provided so sets the stage for developing a list of differential etiologies, based heavily on medical terminology

script: organ: [severity], [duration], [distribution], [lesion] +/- "with..." [additional features]

etiologic diagnosis - less descriptive and does not always identify a specific lesion, focused on the cause/etiology of the lesion in affected organ/tissue

example: coronaviral vasculitis (meaning FIP)

disease or condition - a bit of a summation of everything you are seeing/observing, no descriptive component

example: feline infectious peritonitis

Prognosis: an estimate of future outcome

combines case history/lesions/clinical signs/diagnosis together with established literature and/or prior experience

expressed in terms of optimism: excellent --> good --> fair --> guarded --> poor/grave

Important Cell Structures:

Nucleus: the brain, two-layer membrane studded by nuclear pore complexes for selective passage of proteins and RNAs in and out, contains chromatin (heterochromatin is inactive and euchromatin is active), nuclear proteins, RNA, and nucleolus

Nucleolus: site where rRNA is transcribed and ribosomal subunits are assembled, rRNA used to determine bacterial and fungal identification (very conserved area), larger nucleolus = more protein synthesis

Cytoplasm: the substance where everything lives inside the cell

Ribosomes: synthesize proteins from mRNA (translation), attached to ribosomes are proteins destined for secretion, membrane, ER, Golgi, or lysosomes

Rough Endoplasmic Reticulum: processes/sorts proteins destined for secretion/plasma membrane/ER/Golgi/lysosomes, further processing is required for protein to become functional, has several modifications

Smooth Endoplasmic Reticulum: lipid metabolism and membrane lipid synthesis, major production site of molecules composed of lipids (steroid hormones derived from cholesterol), major role in metabolizing lipid-soluble compounds (home of cytochrome p450 in hepatocytes, inactivate numerous drugs by converting them to water-soluble compounds that are eliminated in urine)

Golgi apparatus: the post office, receives proteins from ER, processes/sorts them for transport to eventual destinations (lysosomes, plasma membrane, secretion)

Plasma cells: secrete antibodies, also called Mott cells

Lysosomes: waste/recycling center, membrane-bound, contains array of enzymes, degrades material from endocytosis, degrades obsolete cell components (autophagy)

Mitochondria: power plant, generates ATP from breakdown of carbohydrates and fatty acids, in the Citric Acid cycle and oxidative phosphorylation

Structural components

Cytoskeleton: either intermediate filaments, microtubules, microfilaments

Plasma membrane: fluid mosaic

Membrane Proteins:

Transport: passive diffusion, facilitated passive transport, active transport, endocytosis

Cell-to-cell: tight junctions, desmosomes, adherens junctions, hemidesmosomes, gap junctions

4 major groups of cell-adhesion molecules (CAMs): selectins, integrins, immunoglobulin superfamily (IgSF), cadherins

Cell signaling

cell-cell contact: gap junctions, juxtacrine (membrane-bound ligands)

secreted signaling molecules: autocrine, paracrine, endocrine, synaptic/neural

signaling molecules: gases to complex proteins, when interacting with receptor on target cell = ligand

Receptors for cell communication:

ligand-gated ion channels

G protein-coupled receptors (7 transmembrane receptors)

enzyme-linked receptors

cytokine/chemokine receptors

Pattern recognition receptors (PRRs):

toll-like receptors (TLRs)

nucleotide-binding oligomerization domain-like receptors (NLRs)

C-type lectin receptors (CLRs)

RIG-1 like receptors (RLRs)

Cluster Differentiation (CD) markers

DNA Packing and Organization:

DNA = negatively charged, attracted to positive charge of histone proteins and stains blue with Hematoxylin stain

Nucleosome = DNA strand wrapped around 8 histone proteins

modification of these histone proteins can allow or shut off transcription/replication

Transcription:

Generation of RNA sequence (by RNA polymerase 2)

also includes messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA)

Gene = sequence of DNA ready to develop mRNA that encodes a protein

Regulated via transcription factors, enhancers, repressors, and epigenetics

epigenetics act on top of DNA but not directly to it (can include histone modifications)

Cell Proliferation - The Cell Cycle

Goal: ensure appropriate DNA replication and integrity

Regulation: through interplay of multiple proteins (cyclins, cyclin-dependent kinases (CDKs), CDK-inhibitors (many of the p-proteins), p53, Rb)

Checkpoints act as "spellcheck" to ensure integrity after replication (S phase)

if everything is good, proceeds to mitosis (M phase)

DNA Repair

Mechanisms in place to repair DNA damage

many small alterations are fixed with high rate of fidelity (accuracy), NER is used most commonly

larger alterations are more challenging to fix (mechanisms are not as consistent and may allow for mistakes (mutations)

High fidelity = high accuracy and low fidelity = low/less accuracy

Provide a basic definition of inflammation

inflammation = reaction of vascularized living tissue to local injury (needs to be living), reactions are designed to eliminate the injurious stimulus and repair associated tissue damage

a complex dynamic process

the process involves many mediators, cells, and components of the blood vascular system

an evoked defensive response (does not happen spontaneously or proactively), a stereotypical phenomenon (limited number and type of inflammatory responses, different stimuli can elicit the same response - can vary in intensity/magnitude/duration)

depends on multiple factors - innate immune response, immunologic status of host, type of agent involved

Know the five cardinal signs of inflammation

Heat, Redness, Swelling, Pain, Loss of Function

Understand how inflammation can help to differentiate antemortem necrosis from postmortem necrosis

occurs in living animals, cannot continue or begin after death

inflammation changes can differentiate antemortem necrosis from postmortem autolysis

Describe the beneficial aspects of inflammatory responses as a component of host defense

Remove injurious stimulus

killing/sequestration: microbes, necrotic tissue, neoplastic cells, etc.

degrading foreign materials

dilution/inactivation of biologic and chemical toxins

Tissue repair aspect

increasing local or systemic temperature to inhibit microbial replication

providing wound healing factors

restricting movement of joints or appendages to allow for healing and repair

Know the five main phases of the inflammatory response

1. Recognition of Inflammatory Stimulus

2. Acute Vascular Response

3. Acute Cellular Response

4. Chronic Cellular Response

5. Resolution

Recognize the endogenous and exogenous stimuli that can induce an inflammatory response

Endogenous substances: autoimmune reactions (antibodies directed against self-antigens, may be primary or secondary to infectious disease or neoplasia, examples are IMHA or systemic lupus erythematosus), intracellular signals released from injured or dying cells

Exogenous substances: microbes (viruses/bacteria/protozoa/etc), foreign bodies (suture material/plant material/etc), mechanical action (traumatic injury), physical actions (thermal or freezing injury), chemical substances (caustic agents/venoms/etc)

Explain the difference between PAMPs and DAMPs, provide examples of each, and describe the effects of binding to PRRs

PAMP (pathogen-associated molecular pattern): microbial ligands, recognized as "non-self" by host cells (macrophages, leukocytes, mucosal epithelium), binding triggers release of inflammatory mediators, unique to microbes

bind PRRs on or within host cells --> binding results in downstream induction of inflammatory mediators

example is LPS or double-stranded RNA

DAMP (damage-associated molecular pattern): endogenous molecules released from damaged or dying cells, presence extracellularly alerts nearby cells to the presence of tissue injury

bind PRRs for downstream induction of inflammatory mediators

extracellular presence of some molecules signals for damaged cell membranes (HMGB1, monosodium urate, galectins, S100s)

release of intracellular enzymes causes breakdown of extracellular components which can also be recognized as DAMPs (hyaluronic acid, heparan sulfate, collagen-derived peptide)

circulating antibodies may recognize intracellular antigens to activate the complement system

PAMPs are exogenous molecules that differentiate "self" from "non-self"

DAMPs are endogenous molecules that differentiate "healthy self" from "damaged self"

BOTH bind PRRs to induce production and/or release of inflammatory mediators

Know how mast cells are activated during initial tissue injury and describe the initial effects of mast cell degranulation

already present in tissue, often close to site of injury (situated around vessels and close to peripheral nerves), contain preformed vasoactive mediators within granules (histamine and serotonin)

receptors: PRRs (TLRs), FcE (binds IgE), complement receptors, others

functions: degranulate in response to receptor binding/physical trauma/temperature extremes/etc., release histamine and serotonin (which lead to vasodilation and increased vascular permeability)

Recognize other cells involved in the initial recognition of the inflammatory stimulus

Resident tissue macrophages - recognize inflammatory stimuli and initiate inflammatory response via binding of PRRs, already present in tissue so do not need to be recruited from distant sites

Epithelial cells - may secrete cytokines when injured or recognize inflammatory stimuli via PRRs (especially mucosal epithelium)

Platelets - initiators of inflammation, active role in inflammation and coagulation, aggregate and accumulate at sites of endothelial injury, activated by collagen to release inflammatory mediators (vasoactive amines (serotonin, histamine), complement activators, platelet-activating factor, coagulation factors)

Other vasoactive mediators: these become active later in the inflammatory response, need to be produced

Bradykinin: released from damaged vascular endothelium, causes vasodilation

Prostaglandins and leukotrienes: produced by many cell types in response to PRR activation, cause vasodilation and increased vascular permeability

Platelet-Activating Factor (PAF): produced by many cell types in response to PRR activation, causes increased vascular permeability and smooth muscle contraction

Understand the mechanisms underlying vascular permeability changes in inflammatory responses

occurs concurrently with the acute cellular response

biphasic - initial arteriolar dilation and increased vascular permeability occur almost immediately (histamine, kinins, etc)

second wave of vascular permeability changes occurs following de novo synthesis of inflammatory mediators (IL-1, TNF-alpha, kinins, etc)

early phase: histamine-dependent

later phase: cytokine-dependent (IL-1, TNF-alpha, kinins)

arteriolar dilation drives increased blood flow, venule dilation causes blood stasis --> net effect: increased capillary hydrostatis pressure --> leakage of fluid and plasma proteins (if pressure alone) & is compounded by increased vascular permeability (cells also leave vessels)

histamine MOA = retraction of endothelial cells

Recognize the clinical manifestations of the acute vascular response

tissue redness (hyperemia) = vasodilation & blood stasis

tissue swelling (edema) = leakage of plasma proteins (increased vascular permeability) & +/- leakage of macromolecules and leukocytes (endothelial gaps and endothelial injury)

fluid which leaks from the vasculature during inflammatory responses is usually rich in both cells and proteins

cell types may reflect the cause and duration of inflammation, characteristics change with time (reflect the duration of the response)

analyzing the cellular makeup of an inflammatory lesion is an important tool in the diagnosis of many infectious diseases and other pathologic processes

Make inferences about underlying inflammatory stimuli based on the cellular composition of inflammatory lesions

Neutrophils: often predominant if the response is acute and/or bacterial

Neutrophils and Macrophages: lesions which are subacute to chronic (relative number of macrophages tends to increase with chronicity)

Eosinophils: parasitic disease and acute hypersensitivity reactions

Lymphocytes and Plasma cells: antigenic stimulation or delayed-type hypersensitivity (Type 4 hypersensitivity)

Visually recognize the morphologic characteristics of the cell types involved in the acute inflammatory response (neutrophils, eosinophils, basophils, monocytes, macrophages, NK cells)

Polymorphonuclear cells (aka Granulocytes): contain distinctly lobulated/segmented nuclei (ALL have segmented nucleus and cytoplasmic granules that determine effector function), terminally differentiated (do not divide, short half-life), found circulating in blood and in tissues (normal concentrations vary by species)

neutrophils: nonstaining to pale pink granules; key effector cell in the acute inflammatory response, first cell to enter an area of injury from the bloodstream (move rapidly by amoeboid motion), intensely phagocytic, granules are lysosomes which contain powerful degradative enzymes (for things they ingest), unstable and short-lived in circulation (more persistent in tissues)

eosinophils: bright red or orange granules

basophils: dark blue to purple

Know the general sequence in which different leukocytes enter inflammatory lesions

Mast cells: already present in tissues

Neutrophils: 20 minutes to hours, keep moving in until they are no longer needed

Macrophages: at least 24 hours to days, separate chronic and acute injury

Lymphocytes: days, exclusive to chronic responses

Fibroblasts: days to weeks, major mediator of tissue repair

Eosinophils: hours to days, has very specific signals to activate (IL-5)

Recognize morphologic and functional differences between neutrophils and heterophils across veterinary species

Heterophils:

Birds/Reptiles: elongated red granules, not as antibacterial as mammalian neutrophils (lack myeloperoxidase, both morphologic and functional difference)

Rabbits/Guinea pigs/Elephants/etc: bright red granules, no difference in function compared to neutrophils, different on morphology (heterophil appearance, neutrophil function)

Describe the three phases of neutrophil movement into inflammatory lesions (adherence, migration, chemotaxis) and identify the mediators and receptors involved in each step

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1. Adherence:

   1. Stasis: blood flow through affected tissues is slowed (stasis)

   2. Margination: neutrophils move towards endothelial surface as blood stasis occurs, endothelium expresses selectins (P-selectin, E-selectin) in response to histamine/thrombin/PAF/etc, preformed in endothelial granules (Weibel-Palade bodies) (needs to be induced)

   3. Rolling: neutrophil ligands specifically interact with endothelial selectins (Sialyl Lewis X-modified glycoproteins & L-selectin (variable by species)), continued binding and unbinding slows neutrophil to an eventual stop along endothelial surface

   4. Pavementing: stable adhesion, irreversible process, flattening of neutrophil on endothelial surface, induced by IL-1 and INF-alpha from adjacent tissue, mediated by beta-2 integrins (CD11 & Cd18) on neutrophils and ICAM-1 and VCAM-1 on endothelial cells

2. Migration: neutrophils exit the vessel by diapedesis, PECAM-1 in endothelial cell junctions and on leukocytes binds to itself (homotypic binding), leukocytes squeeze through endothelial cell junctions and occurs at post-capillary venules and capillaries

3. Chemotaxis: migration down a cencentration gradient (chemotaxis), bind receptors on neutrophil membrane, change membrane fluidity to allow for formation of pseudopodia, net movement towards inciting agent

Explain neutrophil effector functions within inflammatory lesions (phagocytosis, mechanisms of microbial killing, degradation, and secretory responses)

Phagocytosis: ingestion of foreign material/debris/injurious agents; small particles (up to 0.5 micrometers - bacteria/foreign bodies/senescent cells/debris), receptor-mediated (complement via CR1 and CR3 for opsonization and Fc receptors), after internalization phagocytized particle is contained in a phagolysosome

Microbial killing:

Respiratory burst: essential for effective killing of bacterial and fungal agents, dramatic increase in O2 consumption, NADPH oxidase is phagocytic vacuole converts O2 to O2- (superoxide anion) which is highly cytotoxic, O2- reacts with H+ to form H2O2; some organisms can produce catalase to inactivate H2O2 (Staphylococcus spp.)

Fenton Reaction: generation of hydroxyl radical (most potent free radical), requires iron (lactoferrin in neutrophil granules is the source of iron), animals deficient in lactoferrin have impaired microbial killing, is paired with respiratory burst

Myeloperoxidase: present in neutrophil granules, converts H2O2 and Cl- ions to oxidizing agents (hypochlorous acid), contributes to liquefaction and necrosis (pus), absent in avian/reptilian heterophils (these species tend to rely on non-oxidative mechanisms of microbial killing

Neutrophil Extracellular Traps: released upon neutrophil cell death, DNA backbone embedded with antimicrobial peptides and granule contents, physically entrap bacteria and can be microbicidal

Degradation: of ingested microbes and necrotic tissue components; two major types of neutrophil granules - specific granules (fuse with phagolysosome first, granule contents function to acidify phagolysosomal compartment) and azurophilic granules (fuse with phagolysosome slightly later), similar to oxidative burst these degradative enzymes can cause collateral tissue damage if released from phagolysosome

specific granules: lysozyme, lactoferrin

azurophilic granules: myeloperoxidase, lysozyme

Secretory responses: neutrophils secrete products which further stimulate or enhance the inflammatory response (proinflammatory cytokines, chemokines (IL-8), eicosanoids, kinins, mast cell activators, others)

Understand the major functions and stimulatory cytokines of other cell types involved in the acute cellular response (eosinophils, NK cells, macrophages)

Eosinophils: parasite-mediated inflammatory reactions, immune-mediated reactions, found in all tissues (most abundant in GI tract), enter tissue during transition from acute to chronic inflammation, weakly phagocytic and bactericidal, selective response to chemotactic stimuli, large brightly eosinophilic cytoplasmic granules

major granule contents: major basic protein (MBP) (parasiticidal - creates holes in nematode cuticle, mast cells ingest MBP to limit collateral tissue damage), eosinophil cationic protein (similar to MBP), eosinophil peroxidase

other granule contents: degradative enzymes (arylsulfatase B (neutralizes heparin), histaminase (inactivates histamine), phospholipase D (inactivates PAF))

mast cells keep eosinophils in check

IL-5: produced by eosinophils/mast cells/Th2 lymphocytes, in eosinophils it stimulates eosinophil proliferation, promotes differentiation/activation/maintenance of eosinophils, primes eosinophils for effects of other cytokines

NK cells: involved in lysis of tumor cells and virus-infected cells without previous encounter, enter sites of tissue inflammation hours to days after initiation of lesion, IL-21 is the major cytokine driving NK cell function and differentiation

granules contain perforin (destroys target cells by creating pores in their membranes), visible as discrete magenta cytoplasmic granules on cytology or blood smear, also expressed on CD8+ cytotoxic T cells

Macrophages: monocytes develop in bone marrow and circulate in blood stream and enter inflammatory lesions 12-48 hours after initiation of lesion, in tissue they rapidly differentiate into macrophages

Know the difference between M1 and M2 macrophages

If driven by cytokines/antigens/other inflammatory stimuli then monocytes differentiate into M1 macrophages

sets the stage for development of chronic inflammation, accumulate in sites of persistent antigen/persistent microbes/foreign material/repeated injury

innate functions: phagocytosis and cytokine release

adaptive functions: capable of presenting antigen and regulating T-lymphocyte activity

M2 macrophages (aka fixed tissue macrophages) - reside in organs/connective tissue long-term and enter tissue sites as blood monocytes under physiologic conditions (rather than inflammatory conditions), perform housekeeping functions (removing cells and debris associated with tissue remodeling)

ex. Kupffer cells in the liver, alveolar macrophages in the lung, microglial cells in the brain, osteoclasts in bone

Recognize whether inflammatory mediators are preformed, plasma-derived, or newly synthesized and how this affects when mediators are active during inflammatory responses

Preformed: released immediately upon cellular activation (ex. histamine), see in the initial spike

Plasma-derived: constantly circulating in precursor forms, require cleavage to active forms (rapid) (ex. acute phase proteins, kinins, complement)

Synthesized: produced after inflammatory cell becomes activated or injured, require more time for release (ex. cytokines, prostaglandins, adhesion molecules), see in the delayed/sustained spike

Understand how histamine and serotonin induce the initial stages of vascular permeability changes

Histamine: primarily released from mast cells and basophils, rapidly enhances vascular permeability (acute vascular effects are immediate and transient), variety of stimuli induce histamine release (IgE, C3a and C5a, temperature extremes, substance P, etc.)

other effects: vasodilation, neural/vagal reflexes, bronchial constriction, release of PGF2alpha, pain and itching, tachycardia, eosinophil chemotaxis

Serotonin: released from activated platelets in mammals (exposure to subendothelial collagen, thrombin, ADP, etc), released from mast cells in rodents, similar actions to histamine

Know the effects of bradykinin on the microvasculature

major product of kinin pathway

slow-acting vasodilator --> increases capillary permeability, produces sustained redness/heat/pain

Recall the major products of the complement cascade and explain how each product contributes to the inflammatory process

generation of multiple active molecules with important inflammatory and immune effects

C3a and C5a = anaphylatoxins (induce histamine release from mast cells to increase vascular permeability)

C5a = also chemoattractant (brings granulocytes and monocytes to site of inflammation)

C3b = opsonin (coats microbes/apoptotic cells/etc. for enhanced phagocytosis, binds CR1/CR3 receptors on neutrophils

C5b = initiates formation of the MAC complex (acts as a tube inserted into lipid bilayer of microbe on cell causing bacterial or cell lysis)

Define "acute phase protein" and differentiate the major acute phase proteins for common domestic animal species

APPs = plasma proteins synthesized in the liver whose concentrations change by 25% or more during inflammation (positive APPs increase in concentration, negative APPs decrease in concentration), mainly induced by IL-6, can be measured on a blood sample, useful in both identification and monitoring of inflammatory responses

positive APPs = CRP, serum amyloid A, fibrinogen

nefative APPs = albumin, transferrin

Dog = C-reactive protein

Cat = serum amyloid A

Horse = fibrinogen

Cow = fibrinogen

Recognize the opposing actions of arachidonic acid metabolites (eicosanoids) and how this allows for regulation of inflammatory responses

eicosanoids = prostaglandins, leukotrienes, lipoxins; produced by cyclooxygenase and lipoxygenase pathways

COX-1 = constitutive (physiologic)

COX-2 = induced (inflammation)

Prostaglandin E series: vasodilation, mediate pain, fever (PGE2), inflammatory tachycardia

Prostaglandin I series (prostacyclin): vasodilation, bronchodilation, inhibits platelet activation

Thromboxane A2: produced by platelets, vasoconstriction, facilitates platelet aggregation

Leukotrienes: pro-inflammatory

LTB4: produced by neutrophils, chemotactic for neutrophils, promotes respiratory burst

LTC4/LTD4/LTE4: produced by mast cells/endothelial cells/ platelets/macrophages, increase capillary permeability, chemotactic for eosinophils, induce airway smooth muscle contraction

Lipoxins: anti-inflammatory; formed by platelets late in inflammatory response, anti-inflammatory properties (reduce neutrophil recruitment), drive the resolution phase of inflammation (clean up apoptotic neutrophils, promote tissue repair)

Know the major functions of key interleukins

IL-1: fever, T cell and macrophage activation, pro-inflammatory

IL-6: acute phase protein production, pro-inflammatory

IL-5: chemotaxis for eosinophils

IL-8: chemotaxis for neutrophils

IL-10: suppresses macrophage function, anti-inflammatory

Identify the major stimulators of acute phase protein production and fever

TNF-alpha (along with IL-6) stimulates acute phase protein production

TNF-alpha (along with IL-1) promotes neutrophil pavementing and stimulates IL-1 secretion to induce fever