Cellular Pathology and its Morphology

Cellular Pathology

§  Pathology includes study of etiology (what initiates a process), pathogenesis (mechanism), morphology (how it’s recognized) and functional consequences (how it produces disease).

§  The three fundamental processes of cellular pathology are cell injury, cell death and cellular adaptation.  In response to stressors, cells may adapt or die/be injured. 

§  The etiology of cell injury may be extrinsic or intrinsic, including hypoxia, physical/chemical agents, drugs, infection, immunologic reactions, genetic, or nutritional problems.

§  Hypoxia is the reduction or absence of normal oxygen supply.  It may be a result of ischemia, which is the reduction or absence of blood supply.  They most often go together, but ischemia alone can be damaging from a lack of trophic substances or accumulation of toxins, and hypoxia alone can still be damaging (ex: anemia, pulmonary disease, cyanide poisoning).

§  Infarction is the process in which a portion of a tissue dies as a result of ischemia. An infarct is the end result of this process.  They may be white or red (still some blood supply to the dead tissue), but both result from the same underlying mechanism.

§  The mechanisms by which ischemia causes cell death are inter-dependent and synergistic.  Toxins often influence one of these mechanisms.  They include: 

o   Decreases in ATP – oxidative phosphorylation shuts down first in ischemia.  Pumps stop running and cells/organelles swell and have blebs, glycolysis lowers pH and chromatin clumps, protein synthesis decreases and lipids accumulate since proteins aren’t available to export lipids as lipoproteins.  These early changes are reversible. 

o   Increased intracellular Ca, which is critical to homeostasis.  Many enzymes only function within a very narrow range of [Ca].  Ischemia inactivates calcium pumps, resulting in calcium activation of enzymes and increased membrane breakdown along with decreased membrane synthesis. 

o   Membrane damage (considered the point of no return)

o   Reactive oxygen species – endogenous or exogenous free radicals.  Reperfusion of an ischemic area can add to ischemic damage, likely by sudden calcium influx or exposure to free radicals from an influx of inflammatory cells.  We have some level of antioxidant defense from vitamins C and E, glutathione peroxidase, catalase and superoxide dismutase.  But oxidative stress can result from imbalance between ROS and antioxidants.  Oxidative stress is associated with aging, diabetes, atherosclerosis, Alzheimer, etc.

§  Cell injury becomes irreversible most quickly in neurons, followed by myocardium and hepatocytes, and most slowly in skeletal muscle. 

§  Reversible cell injury is characterized by cell swelling, vacuolar degeneration, and lipid deposition (especially in the myocardium and liver).

§  Irreversible injury:  necrosis – the morphological changes in the nucleus and cytoplasm occurring after death in a living tissue, regardless of the cause of injury.  By the time necrosis is observed, the cell is dead.  This need not occur at the tissue level.

o   Features of necrosis include:  eosinophilia due to loss of RNA/ribosomes and denatured protein, nuclear changes (small-pyknosis, broken apart-karyorrhexis, or dissolved-karyolysis), INFLAMMATION.  The inflammation is the big distinguishing feature, and it usually occurs about 8 hours after cell death.  Thus, it won’t occur if the cell dies and organism also dies at the same time. 

§  Types of Necrosis:

o   Coagulative:  Results from ischemic cell death, and is seen in most tissues except for the brain.  In this type of necrosis, the tissue architecture is retained.  You typically see ‘tombstones’ of hyper-eosinophilic cells.  It typically resolves by scar formation.

o   Liquefactive:  Characterized by complete hydrolysis of dead cells, resulting in a loss of tissue architecture and usually resulting in a cyst or cavity.  This is the usual response to infarction in the brain. 

o   Abscess:  Liquefactive necrosis resulting from localized bacterial/fungal/parasitic infections.  It usually has lots of neutrophils within the abscess (pus=dead neutrophils and debris) which are the source of hydrolytic enzymes.  It often requires surgical drainage, since vasculature is commonly damaged and antibiotics won’t be delivered effectively.

o   Caseous (cheese-like): Characteristic of TB and fungal infections.  It has a center of cheese-like necrosis surrounded by a rim of inflammatory cells (all together called a granuloma).

o   Fat:  Common in the pancreas (from release of pancreatic enzymes) and breast (from minor trauma).  Membrane lipids are digested into FFAs, and combine with calcium (saponification) to form chalky white deposits.  May mimic a carcinoma clinically.

§  Cellular adaptations include hyperplasis, hypertrophy, atrophy, and metaplasia. 

§  Hyperplasia is an increase in the number of cells in a tissue/organ.  In hyperplasia, you see dividing, mitotic cells.  Hyperplasia may be physiologic, such as in lactating breasts.  But is can also be pathologic, as in endometrial hyperplasia with unopposed estrogen stimulation that results in prolonged cycles with menorrhagia (heavy bleeding).  Another example of hyperplasia is in BPH. 

§  Hypertrophy is an increase in individual cell mass, and is usually reversible.  In benign prostatic hyperplasia, bladder muscle hypertrophies as a results of needing to push the urine out with more force.  Other examples of pathologic hypertrophy may be hypertrophy of the heart in someone with aortic valve stenosis or chronic hypertension.  Hypertrophy may results from greater workload, as above, but also from increased levels of hormones (anabolic or in pregnancy, etc).  Genetic mutation in the myostatin gene has been shown to cause muscle hypertrophy in animals. 

§  Atrophy is cellular shrinkage due to a loss of substance, and may result from disuse, denervation (polio), ischemia, starvation (protein-calorie malnutrition, or marasmus), or absence of endocrine stimulation.  Ex:  menopause can result in atrophy of cells making up endometrial glands.  Ex:  cachexia, a wasting associated with cancer, AIDS and other chronic inflammatory diseases.

o   Cellular atrophy may culminate or be accompanied by progressive cell loss, and if enough occurs an organ or tissue may shrink.  In these cases, atrophy describes both cell shrinkage and cell loss. 

§  Metaplasia is the reversible replacement of one differentiated cell type by another differentiated cell type.  It can be considered an adaptive substitution by cells that can better withstand an adverse environment.  In smokers, you see squamous metaplasia where respiratory epithelium is replaced by stratified squamous cells.  Stem cells at the basement layer differentiate into squamous cells.  Metaplastic epithelium may undergo neoplastic progression to dysplasia (not yet cancer, but cells lose normal architecture on the way there), and ultimately to neoplasia (cancer w/ clonal cells having a genetic mutation).  Not all metplasias become cancers, but if the stimulus persists, they may. 

§  Cells may accumulate endogenous or exogenous substances (lipids, protein, glycogen, carbs, minerals, pigments, etc). 

o   Pigment accumulations:  anthracosis (accumulation of dark carbon pigment in city dwellers) or lipofuscin (wear and tear pigment where you have lots of cell turnover) are examples of benign pigment accumulations.

o   Intracellular lipid accumulation:  Fatty change (steatosis) is potentially reversible.  It most commonly occurs in the liver, and is caused most commonly by obesity or alcoholic liver disease.  The liver is typically enlarged and rather than red, it appears yellow/white. 

§  Cell death is not always pathologic; apoptosis is normal in embryogenesis, immune cell differentiation, menstruation, etc.  Apoptosis may be pathologic too, resulting from DNA damage (w/ p53 as a principal mediator), viral infection or CD8 T cell mediated injury. 

o   Apoptosis is mediated by caspases, cysteine protesases that require activation.  Bcl-2 is an anti-apoptotic protein, and its family contains both pro and anti apoptotic factors.  P53 stops cell division in response to DNA damage in order to facilitate recovery, and if recover fails it initiates apoptosis. 

o   Morphologically, apoptosis is characterized by nuclei breaking into apoptotic bodies.  This occurs in single cells with apoptosis, whereas necrosis tends to occur in large regions of an organ.  In apoptosis, DNA is systematically fragmented and shows as a ladder on a gel, whereas necrotic cell death breaks it down at random, resulting in a smear. 

o   Basically, apoptosis is usually physiologic, occurs in single cells, fragments DNA between nucleosomes, and produces apoptotic bodies WITHOUT inflammation.  Necrosis is usually pathologic, occurs in groups of cells, randomly fragments DNA and shows swelling, degeneration, and inflammation. 

o   Inhibition of apoptosis facilitates tumorigenesis.  HPV blocks p53 and apoptosis, and can result in squamous cell carcinoma of the cervix.  Constitutive activation of Bcl-2 blocks apoptosis and facilitates follicular lymphomas.  

Why Is Apoptosis Necessary?

Why Is Apoptosis Necessary?

Here are some points to keep in mind.

Normally, billions of cells die every day by apoptosis. If they did not, disaster would follow. The embryo could not “chisel” itself into the proper shape; the developing nervous system would be distorted by swarms of unwanted neurons; the maturing immune system would rapidly be overcome by autoimmune lymphocytes; the epidermis and other surface epithelia would become absurdly thick; and in the adult, the lack of a mechanism to counterbalance mitosis would lead to organ deformities incompatible with life.

Apoptosis is planned to perform as Nature's surgical knife: it acts swiftly (at least twice as fast as oncosis) and quietly (it does not trigger inflammation, as oncosis does).

Why There Is No Inflammation In Apoptosis?

Granulocytes are rarely seen around apoptotic cells. This in stark contrast with the response to ischemic necrosis: crowds of granulocytes and other inflammatory cells pour into the area. The “inflammatory silence” of apoptosis makes sense: if apoptosis is to carry out its discrete function of eliminating single unwanted cells, without damage to the tissue structure as a whole, the neutrophils must be kept out of the picture. When activated, these cells are known to secrete bactericidal molecules in such large amounts that collateral damage to bystander cells, as in Inflammation, is bound to occur. Actually, it is not quite correct to say that apoptosis does not induce inflammation: macrophages do appear on the scene and phagocytize the apoptotic cells; and macrophages are almost the prototype of inflammatory cells.

It is a general rule of inflammation that when macrophages are activated, they secrete chemical messages to recruit other types of inflammatory cells; we rationalize this as a call for help. But macrophages phagocytizing apoptotic cells do not call for help. Quite to the contrary: they secrete anti-inflammatory substances such as prostaglandin E₂. It seems obvious that the overall plan for the apoptotic cells is to die in incognito.

Microscopic features of apoptosis

As usual, details vary, but the following description is representative; the whole performance lasts about an hour, sometimes less

• The cell shrinks, becomes denser (dark by electron microscopy), rounds up and detaches itself from its neighbors. The loss of volume is the first change; it may be as high as 60 percent for eosinophils; it may have the purpose of reducing the labor of phagocytosis. Both shrinkage and density may reflect (a) loss of fluid: the smooth endoplasmic reticulum swells into vesicles that fuse with the cell membrane, dumping out fluid, while the Na/KATP pump extracts cations from the cell; and/or (b) protein cross-linking by transglutaminases, which may also explain the increased density.
• The chromatin becomes very dense, and separates into deeply stained, homogeneous, often semilunar or sickleshaped masses plastered against the nuclear membrane. At least half a dozen factors are known to induce this chromatin condensation, including a factor called acinus and the caspases. We may wonder why the cell takes the trouble of shredding its DNA: the purpose may be to prevent it from being misused.
• Budding: Seen in a time-lapse movie the cell performs a striking (death dance): it sends out and pulls back short processes, which contain dense cytoplasm and often a piece of the nucleus.
• Blebbing: Small blebs may develop, but this is not typical of apoptosis, whereas it is constant and extensive in oncosis. Blebs, blister-like structures with a watery content, are biologically different phenomenon from budding. There has been some confusion between budding and blebbing, because scanning electron micrographs show balloon-like “protrusions” that are difficult to interpret. Another confusing factor is that intermediate forms may also occur.
• The “buds” break off, to become apoptotic bodies.
• What is left of the cell body is taken up by neighboring cells or extruded (in epithelia); or it is phagocytized by macrophages.
• The corpses of apoptotic cells, if shed into a space such as a glandular lumen, may undergo what was called “secondary necrosis”. They can also be picked up by macrophages or by neighboring cells. These former neighbors are induced to cannibalize the remains because they express a number of “eat me signals”, perhaps as many as 10; the most important are a vitronectin receptor, annexin 5, and molecules of phosphatidylserine, normally inserted into the inner leaflet of the cell membrane, but flipped out on the apoptotic cells. Remarkably absent around the corpses are the neutrophils.

Difference between Necrosis and apoptosis

Cell Injury I & II

A.  Definitions.  Be able to recognize examples of these conditions and vignettes and in pathologic images from text, lecture outlines, and case studies.  Several of these conditions are discussed in greater detail in later pages of the text.

Necrosis:  irreversible cell injury, sequence of morphological changes that follow cell death in living tissue.  Occurs after a loss of blood supply or after an exposure to toxins.  Characterized by cell swelling, protein denaturation, and organelle breakdown.  Leads to tissue dysfunction.  Inflammatory response initiated.
Apoptosis:  programmed cell death, dead cells removed without much disruption to surrounding tissues.  Where unwanted cells (ex: embryogenesis) or pathological cells (ex: irreparable mutations) are eliminated.  Inflammatory response is not initiated.  Cells shrink in size.
Hypertrophy:  increase in the size of the cells due to increased workload (ex: myocardial cells increase in size if have excessive HTN or aortic stenosis because of the increased work load)
Atrophy:  decrease in cell size without change in # of cells (ex: casted broken leg---don’t use will see atrophy, remember use it or lose it)
Infarction:  irreversible injury after complete or prolonged occlusion, cells are dead (ex: myocardial infarction aka “heart attack”), irreversible
Cachexia:  weight loss after periods of prolonged starvation (ex: the setting of malignant tumors)
Hyperplasia:  increase in the number of cells, can be physiologic or pathologic, associated with hypertrophy at times (exception:  renal epithelial cells can undergo hypertrophy but not hyperplasia).   Physiological hyperplasia: a)  hormonally induced (ex: during breast development and during pregnancy have breast and uterine development) or b)  compensatory hyperplasia (occurs when tissue is removed or diseased ex: partial liver resection: cells will proliferate to fill in space where liver tissue once resided).  Hyperplasia is also important in wound healing.  Pathological hyperplasia:  mainly due to excessive hormonal stimulation or growth factor stimulation (ex: warts caused by the papillovirus, tumors)

Aplasia: incomplete or underdeveloped cells/organs

Hypoplasia: underdeveloped cells/organs

Metaplasia:  reversible change in which one adult cell type (epith or mesenchymal) is replaced by another adult cell type

Hypoxia:  oxygen deficiency which interferes with aerobic oxidative respiration, common cause of cell injury/death

Hypoxemia: low oxygenation of blood

Anorexia: self induced starvation, results in cachexia, anemia may result leading to hypoxic tissues and hypoxemia
Ischemia: loss of blood supply due to impeded arterial flow (due to occlusion) or decreased venous drainage

Phagocytosis:  engulfment of bacteria via invagination of membrane of pathogen to make phagosome which later fuses with lysosome to make phagolysosome.  With this fusion, the pathogen is ingested via heterophagy or autophagy.

Autophagy:  intracellular organelles (not outside sources) and portions of cytosol are sequestered from the cytoplasm in the autophagic vacuole form from the ribosome-free parts of the RER (prominent n cells undergoing atrophy).  The vacuole fuses with a primary lysosome to form a autophagolysosome.  This will completely digest proteins, carbs, but some lipids remain undigested.

Heterophagy:  materials from the external environment are taken up through a process generically called endocytosis; uptake of larger particular matter is phagocytosis; and uptake of smaller macromolecules is denoted pinocytosis.  Mainly limited to phagocytotic cells: PMNs and macrophages

B. (pg 4-8)  Understand causes and biochemical mechanisms of cell injury.  Be able to recognize specific examples and what happens at the subcellular level

Causes:                                  Examples:                                     Subcellular events:
1. O2 deprivation                  anemia, CO poisoning           *  ↓ Oxidative phosph = ↓ ATP
Leads to mito and cell swelling increase intracell Ca++ which activates endonucleases, proteases, ATPases via activation of phospholipases

2. Chemical agents   glucose, salt, high partial      * leads to free radical formation
                                              pressures of O2, asbestos,        causing lipid peroxidation
                                              drugs, insecticides

3. Infectious agents  tapeworm, bacteria, viruses,    *viruses take over nuclear
                                              fungi, protozoa                          activity making its own DNA

4. Immunologic rxts   incidental or intended,             *increase mast cell degranul.
   anaphylaxis, autoimmune dz     immune complexes deposited
          in blood vessel walls etc

5. Genetic defects          Down’s syndrome, sickle cell         *cells misshaped in sickle cell

6. Nutritional imbalances     protein insufficiency, vitamin     *decreased cell function
 deficiencies, DM, atherosclerosis

7. Physical agents          trauma, extreme temps, radiation,  *decreased cell function
                       electric shock, sudden changes in
                       atmospheric pressure

8. Aging          repeated injury                 *decreased cell function


**NOTE: there subcellular events are difficult to find**




C.  Understand the sequence of events in ischemic injury in the mitochondria

Ischemia
                                ↓
Decreased Oxidative       Phosporylation
                                ↓
    Decreased ATP

                                                              ↓
Decreased Na Pump
     
      ↓

Increased influx of calcium, water, and, Na, efflux of K

      ↓

Cellular swelling, loss of microvilli, blebs, ER swelling     Increased Glycolysis

      ↓                       ↓

Decreased       Decreased
Glycogen         pH

                               ↓


                        Clumping of
                        Nuclear
                        Chormatin Other Effects

          ↓

Detachment of ribosomes, etc.

          ↓

Decreased protein synthesis

          ↓

Lipid deposition
 



D.   Understand how reperfusion of ischemic tissue can lead to further tissue damage

The restoration of blood flow to ischemic but otherwise viable tissues results in exacerbated and accelerated injury.  As a result, tissues sustain the loss of cells in addition to those that are irreversibly damaged at the end of the ischemic episode.  This is call ischemia/reperfusion injury that contributes to tissue damage in myocardial and cerebral infarctions, but is also amendable to therapeutic intervention.

Reperfusion to ischemic tissues may cause further damage by the following means:

1. Restoration of blood flow bathes compromised cells in high concentrations of Ca++ when the cells themselves cannot regulate themselves.  With high intracellular Ca++, this activates proteases (decreased cytoskeletal and disruption of membrane), endonucleases (nuclear chromatin damage), phospholipases (decrease phospholipids in membrane), and ATPases (decreased ATP).  This all culminates into the loss of cell integrity.

2. Increase in blood flow will also recruit inflammatory cells which release high levels of oxygen derived reactive species and damage cell membrane and mitochondrial membrane permeability.

3. Damaged mitochondria yield incomplete oxygen production and thus increase the production of free radicals.  Cells also have compromised antioxidant effects.

E. (pg 9-10)  Know what free radicals are, how they are formed, how they injure cells, and examples of diseases in which free radicals are involved in pathogenesis

Free radical damage underlies chemical and radiation injury, toxicity from oxygen and other gases, cellular aging, microbial killing by phagocytic cells, inflammatory cell damage, tumor destruction by macrophages, and other injurious processes.  Free radical generation is also a part of respiration and other normal functions like microbial defense.  (Superoxide breaks down water into O2 and hydrogen peroxide)

Free radicals:  chemical species with a single unpaired electron in the outer orbital.  These states are unstable and react readily.  When made inside cells they attack and degrade nucleic acids and membrane molecules.  They initiate autocatalytic reactions: molecules that react with free radicals are converted into free radicals (chain reaction).

Free radicals generated by:  1. redox reactions:  that occur during normal processes like respiration (reduce 02 by adding 4 electrons to generate water) intermediates are formed such as hydrogen peroxide, superoxide radicals (also produced by intracellular oxidases), and OH∙.  Fenton reaction forms free radicals and is catalyzed by transition metals (Cu and Fe) who donate or accept free electrons. The reduction step of the Fenton reaction is catalyzed by superoxide ion.

NO can act as a free radical.  Absorbing UV light, X-Rays  (radiant energy) can hydrolyze water into hydroxyl (OH∙) and hydrogen (H∙) free radicals.  Metabolizing exogenous chemical by enzymes can produce free radicals.

Injury:
Lipid peroxidation of membranes: double bonds in membrane lipids are vulnerable to attack by O2 deprived free radicals.  This reaction yields peroxides which are unable and can lead to chain autocatalysis.

DNA fragmentation:  free radicals react with the thymidine in the nuclear/mitochondrial DNA produce single strand breaks.  This DNA damage has been seen in cell killing and malignant transformation of cells

Cross-linking of proteins:  Cross-linking promoted by free radicals result in enhanced rates of degradation or loss of enzymatic activity.



F. (pg 11)  Describe and understand 2 examples of how chemical agents injure cells.

Some chemicals act directly by combining with a critical molecular component or cellular component or cellular organelle.  Ex: mercuric chloride poisoning, mercury binds to the sulfhydryl groups of various cell membrane proteins, causing inhibition of ATPase –dependent transport and increased membrane permeability. (How antineoplastic agents and antibiotics work by cell damage).  The greatest damage is sustained by the cells that use, absorb, excrete, or concentrate the compounds.

Many other chemicals are not intrinsically biologically active but must be converted to reactive toxic metabolites, which will then act on target cells.   This usually happens by P-450 oxidases in the SER of the liver and other organs.   The metabolites may bind covelantly to proteins and lipids in the membrane and damage/injuring cells, however free radicals injure the cell the greatest.  For instance CCl4  used in the dry cleaning industry can be converted to free radical CCl3 ∙ in the liver causing phospholipids peroxidation breaking down the ER.  Will see less export of lipids because they cannot make apoprotein (a protein) that hooks triglycerides to be secreted by liver.  These patients get fatty liver from CCl4  poisening. Eventually the mitochondria decrease ATP production and the mitochondria start to swell with subsequent calcium influx and cell death.

G. (pg 14)  Define and understand processes of phagocytosis, endocytosis, and pinocytosis

Phagocytosis:  Uptake of large matter via engulfment by the cell.

Endocytosis: Uptake of materials from the external environment.

Pinocytosis:  Uptake of soluable smaller macromolecules

With the above the endocytosed vacuoles and their contents fuse with a lysosomes creating phaogolysomes and degrade by heterophagy (pinocytosis, endocytosis, or phagocytosis) or autophagy (intracellular organelles/cytosol are put into an autophagic vacuole and form from ribosome free parts of the RER.  The autophagic vacuole then fuses with lysosome creating a autophagolysosome---this is to remove damages organelles or to remodel—seen in cells undergoing atrophy.)  Lysosomes have enzymes that degrade proteins, carbohydrates, while some lipid remain undigested.  When undigested debris occurs (lipofuscin pigments, tattoo ink, etc) lysosomes store these in residual bodies

Lysosomal storage disorders caused by deficiencies in enzymes thus, will see an abnormal accumulation of intermediate metabolites all over the body (especially neurons).

H. (pg 15)  Understand the role of lipofuscin and its role in the aging process

Lipofuscin pigment granules represent indigestible material from intracellular lipid peroxidation.  Also known as the “wear and tear” pigment.  It is an insoluable brownish-yellow granular intracellular material that accumulates tissues (heart, brain, and liver) as a function of age or atrophy.  It is a complex of lipid and protein that results from free radical catalyzed perioxidation of lipids of subcellular membranes.  It is NOT injurious to cells but is a marker of past free radical injury.  Aka brown atrophy when you can see the pigment on gross specimen examination.

I. (pg 16-17)  Understand the pathophysiology and causes of hepatic steatosis, recognize examples in lab case studies

Hepatic steatosis:  aka fatty change which is an abnormal accumulation of triglycerides within the parenchymal cells.  Indication of reversible injury, but can be found adjacent to necrosed cells.  Most often seen in the liver since it is the major organ involved in fat metabolism but also seen in kidney, heart, and skeletal muscle.  Causes include: toxins, protein malnutrition, diabetes, obesity, and anorexia, but the MOST COMMON cause is alcoholism.
Excess accumulation of trigyclerides may result in defects at any step from fatty acid entry to the lipoprotein exit.  (last paragraph pg 16 tell about normal fat metabolism pathway)   Hepatotoxins (alcohol etc) alter mitochondrial and SER function; CCl4 and protein malnutrition decrease the synthesis of apoproteins; anorexia inhibits fatty acid oxidation; and starvation increases fatty acid mobilization from peripheral stores.
Mild fatty change: no effect on cellular function
Moderate fatty change: transiently impaired cellular function, reversible unless some vital intracellular process is impaired
Severe fatty change: fatty change may precede cell death but cells can also die without undergoing fatty change.

J. (pg 20)  Define hemosiderin and hemosiderosis and contrast with hemochromatosis

Hemosiderin:  a yellow to brown hemoglobin derived granular pigment that accumulates in tissues when there is an excess of iron.  Iron is normally stored within cells in association with the protein apoferritin forming micelles.  Small amounts are normal in mononuclear phagocytes of bone marrow, liver, and spleen where excessive RBC breakdown occurs.  However, usually if present they are pathologic.   Excess of local iron and hemosiderin results from hemorrhage like with bruising.  After lysis of RBCs in area of bruise, the RBS are phagocytosed by macrophages, hemoglobin is catabolized by lysosomes and the heme iron accumulates in hemosiderin.
Hemosiderosis:  Deposition of hemosiderin in orgasm in tissues due to overload of iron.  Found at first in mononuclear phagocytes of liver, marrow, spleen, and lymph nodes.  These deposits then progressively accumulate the liver, pancreas, heart, and endocrine organs and then the organs become “bronzed” with this pigment.  This occurs when 1) increased absorption of iron  2)  impaired ultilization of iron  3) hemolytic anemias  4) transfusions.   Most of the time the accumulation does not damage the parenchymal cells or impair organ function.  However excessive accumulation results in hemochromatosis.
Hemochromatosis:  Excessive accumulations of iron (hemosiderin) that results in tissue injury including liver fibrosis, heart failure, and diabetes.

K. (pg 21-22)  Define, understand the pathologic process, and recognize diseases associated with metastatic versus dystrophic calcification

Dystrophic calcification:  deposition of calcium salts (along with small amounts of iron, magnesium, and other minerals) in dead or dying tissues.  It occurs in the absence of calcium metabolic derangements (ie: with NORMAL calcium levels).  Comes with any necrotic areas (in atheromas seen in advance atherosclerosis where the initima is damaged in large arteries and see accumulation of lipids).  May be seen with organ dysfunction or cell injury.  It is a cause of aortic stenosis in older people when heart valves are damage causing cuspal calcification.

Morphology:  calcium salts occur as fine white granules or clumps and feel gritty.  On histo slides, will see intracellular and/or extracellular basophilic deposits.  In time, heterotrophic bone may develop in those areas.

Pathogenesis:  Iniation (nucleation) and propagation (intra or extracellular processes) start with end product forming calcium phosphate.  Initiation in extracellular sites occurs in membrane bound vesicles (in bone they occur in matrix vesicle and in pathologic calcification they derive from degenerating cells where the calcium is concentrated because calcium has affinity for membrane phospholipids and phosphatases accumulate as a result of membrane bound phosphatases.  Initiation in intracellular calcification occurs in the mitochondria of dead/dying cells that cannot regulate intracellular calcium anymore. Propagation then occurs and is dependent on the concentration of calcium and phosphate in the extracellular spaces, the presence of mineral inhibitors, and degree of collagenization.  Collagen enhances the rate of crystal growth but osteopontin (protein that binds calcium) is also involved.

Metastatic calcification: deposition of calcium salts (along with small amounts of iron, magnesium, and other minerals) in normal tissue.  It almost ALWAYS reflects some derangement in calcium metabolism (ie: hypercalcemia).  Thus, high calcium levels exacerbate metastatic calcification.

4 causes of hypercalcemia:  1) increased PTH due to parathyroid tumors or other malignant tumors  2)  destruction of bone due to accelerated bone turnover (Paget’s dz), immobilization, or tumors (increased bone breakdown associated with leukemias, bone cancer, or multiple myeloma)  3) vitamin D disorders like Vit D intoxication and sarcoidosis where macrophages activate vitamin D precursor  4)  renal failure which you retain phosphate which leads to secondary hyperparathyroidism

Morphology:  Mainly occurs interstitial tissues of the vasculature, kidneys, lungs, and gastric mucosa.  Granules look like dystrophic granules, but do NOT generally cause clinical dysfunction.  However, extensive calcifications in lungs and kidneys may lead to damage.

L. (pg 22-24 Fig 1.19)  Provide examples and understand the processes of reversible versus irreversible cell injury

Within limits, the cell can compensate for disturbance to ATP production, cell membrane, protein synthesis and DNA and can return the cell to normalcy.  However, after repeated and persistent injury causes cells to surpass the threshold and lead to irreversible cell injury.  Irreversible injury causes problems with oxidative phosphorylation and eventually deplete the ATP supply.  Membrane damage is a critical step in the development of lethal injury and increased intracellular calcium can turn on enzymes which can chew up proteins, cell membrane components, and DNA.

Irreversible cell injury hallmarks: 1) cannot reverse mitochondrial dysfunction after original injury is resolved (ie: restoration of blood flow)  2)  profound membrane function problems (key of irreversible cell injury) due to loss of phospholipids, cytoskeletal abnormalities, toxic oxygen radicals, and lipid breakdown.

Usually functional changes occur before morphological changes.  Reversible injury ultrastructural changes:  plasma membrane problems (blebbing, distortion of microvilli, loosening of intracellular attachments), mitochondrial changes (swelling and dense opacities of mitochondria), dilation of the ER (with ribosome detachment), and nuclear alterations.    Reversible injury starts with cell swelling (hydropic changes) and will see fatty change (from hypoxic/toxic/metabolite injury).    Irreversible cell injury: (necrosis)  morphologic changes that  follow cell death in living tissue.   Get mitochondrial and cell swelling.  DNA degeneration with pyknosis, karryohexis, karyolysis.   Dead cells increase in eosinophilia (pink dye) because of increase eosin binding.

M. (pg 25)  Understand definitions of karyolysis, karyorrhexis, pyknosis

Pyknosis:  nuclear shrinkage and increased basophilia (blue staining)
Karyorrhexis:  pyknotic nuclease fragments
Karylysis:  basophilia of the chromatin may fade secondary to DNAse activity

N. (pg 25)  Compare and contrast liquefactive, caseation, coagulative, fat, and gangrenous necrosis.   Understand what is happening at the cellular level in each of these processes, which organs tend to exhibit each of them, and recognize examples of diseases in which they play an important role

Liquefactive Necrosis:  enzymatic digestion is the primary pattern, due to bacteria or fungal infections.  WBCs accumulate.  Hypoxic death of cells within the CNS also exhibit this type of necrosis.  This necrosis completely digests dead cells.

Caseous Necrosis:  most often due to TB, it is a cheesy white gross appearance of the central necrotic area. Granular debris is enclosed in a ring of inflammation.  Cannot tell what type of tissue you are looking at (unlike coagulative).

Coagulative Necrosis:  denaturation is the primary pattern recognized, basic outer structure of the cells is preserved (ie: can tell what cell is dead) for days.  Classic characteristic of hypoxic death of cells in all tissues EXCEPT the brain.  See increasing acidosis and the injury causes denaturation of structural and enzyme proteins blocking cellular proteolysis.  Ex: myocardial infarction.

Fat Necrosis:  focal areas of fat destruction due to pancreatic injury secondary to the release of pancreatic enzymes.  Causes: pancreatitis, pancreas trauma.  Fat is liquefied by pancreatic enzymes.  This releases fatty acids which combine with calcium to produce chalky white aresas (fat supponification).  On histo slides, only shadowy outlines of necrotic fat cells may be seen with calcium deposits and surrounding inflammatory reaction occurring.

Gangrenous Necrosis: refers to ischemic coagulative necrosis (usually of a limb).  When there is a infection with it is called wet gangrene.

Most of the time, necrotic cells are phagocytized.  If not, these cells attract calcium salt and other minerals leading to dystrophic calcification.

O. (pg 26)  Understand the mechanisms and various causes of apoptosis at the subcellular level (Fig. 1.26)
Apoptosis: programmed cell death, cell suicide
Examples of apoptosis at work: destruction of cells in embryogenesis, hormone dependent involution (ie endometrial thickening during menstruation, pathologic atrophy in prostate after castration, decrease in breast size/lactation with weaning, rapid turnover cells (intestinal epithelium), deletion of autoreactive T-cells in thymus)  If failed to undergo apoptosis, would have tumors or autoimmune dz.  Usually involves single or clusters of cells that have eosinophillic cytoplasm. CELLS SHRINK and form buds with cytosol or organelles called apoptotic bodies.  NO INFLAMMATORY RESPONSE OCCURS.

APOPTOSIS:
Stimulus:  physiologic and pathologic factors
Histo appearance:  single cells, chromatin condensation, apoptotic bodies
DNA breakdown: internucleosomal
Mechanisms: gene activation, endonucleases, proteases
Tissue reaction: NO inflammation, phagocytosis of apoptotic bodies

Mechanisms of apoptosis:
Signaling: due to lack of growth factor, intrinsic programmed event, release of granzymes from cytotoxic T cells, radiated cells, etc.  TNF receptor shares the death domain that when oligimerized leads to activation of caspases and cascade leading to cell death
Control and Integration:  proteins that connect the original death signals to the final execution program  (not sure how indepth he is wanting us to go thru---review pg 27)

P. (pg 28-30)  Understand and provide the examples of events that occur in cell aging

 As we age, oxidative phosphorylation, synthesis of proteins, nutritional uptake, and the ability to repair chromosomal damage is reduced.  Morphological changes include: irregular nuclei, vacuolated mitochondria, diminished ER, and distorted Golgi, increased accumulation of lipofuscin, abnormally folded proteins and AGES (advanced glycosylated end products occur.  Involves intrinsic clock of aging, and extrinsic wear and tear. Intrinsic clock: fibroblast stop dividing after 50 doublings in adults (65 in neonates and 35 in patients with progeria who age prematurely)

Incomplete replication of chromosome ends (telomere shortening) and clock genes are apart of the explanations behind intrinsic clock.  

Cellular aging mechanisms involve both programmed cell events and the consequences of progressive environmental injury.  Programmed aging assumes a predetermined sequence of events including repression and derepression of specific genetic programs leading ultimately to senescence.

What are the Causes of Cellular Injury?

CELLULAR PATHOLOGY

Etiology = what initiates a process, the cause of a disease.
Pathogenesis = what is its mechanism = pathophysiology
Morphology = how it is recognized
Functional Consequences = how it produces disease
Homeostasis = maintenance of a stable internal environment despite environmental variations.

An example of this syllogism:  hemmorhagic interventricular septum
Etiology: Occluded coronary artery, ischemia of myocardium
Pathogenesis: Free radical damage, necrosis, and weakening/rupture of septum
Morphology: Signs of MI (gross and microscopic) and hole in septum
Func conseq: CV collapse and death

Priniciples of Injury:
• Cells normally exist in a steady state
• Adaptation: achieve a new steady state, survival in the presence of the stimulus is now possible
• Examples – hypertrophy of cardiac mm cells b/c of incr work load; due to incr myofilaments and mitochondria
• Cells can reach a point where they can no longer adapt  injury
• Can be reveresible or irreversible (leads to cell death)

•The system that undergoes the primary damage depends on the injurious agent
• Damage in one system leads to damage in other systems
• Inhibition of aerobic respiration  depletion of ATP  no ptn synthesis
• Morphological changes develop more slowly than the biochemical derangements
• Microscopic evidence of cell death may not be apparent for 10-12 hrs
• Reaction to an injurious stimulus depends on its duration and severity
• Results of cell injury depend on the type of cell and its enviornment – neurons are more sensitive than skeletal mm cells

Causes of Cell Injury:

• Hypoxia – most common cause of cell death
• Both MI’s and strokes are ultimately due to hypoxia
• Results from:
1. Loss of blood supply due to blood loss, blockage of vessels, etc
2. Loss of osygen-carrying capacity due to anemia, CO poisoning
3. Intracellular blockage of oxidative phosphorylation by toxins (ex – cyanide)
• Physical Agents:
1. Direct mechanical trauma
2. Extremes of temperature
3. Radiation
4. Electrical shock
5. Changes in atm pressure

• Chemical agents
• Includes toxins, manufactured products, natural products, and therapeutic drugs
• Toxicity may depend on concentration

• Biological Agents – viruses, bacteria, parasites

• Immunologic Reactions
• Example – anaphylaxis, autoimmune diseases

• Genetic Derangements
• Point mutation of DNA – sickle cell anemia
• Inborn errors of metabolism
• Extra chromosomes – Down’s syndrome

• Nutritional Imbalances
• Deficiences and excesses of vitamins, minerals, ptn, kcal, etc

Cell injury and necrosis:
• Cellular response depends on type, duration, and severity
• Consequences of cell injury depend on the type, state, and adaptability of the injured cell
• Four intracellular systems are especially vulnerable:
1. maintenance of integrity of cell mbr
2. aerobic respiration
3. ptn synthesis
4. preservation of the intregrity of the genetic apparatus of the cell

General Biochemical Mechanisms:
• Particularly vulnerable are glycolysis, TCA cycle, and oxidative phosphorylation
• ATP depletion
• Tissues with greater glycolytic ability have an advantage when ATP levels are falling
• ATP depletion and decr ATP synthesis are common consequences of ischemic and toxic injury

• Oxygen/oxygen-derived free radicals
• Have production of reduced reactive oxygen forms, free radicals as by-products
• Cells have defense systems
• Oxidative stress: imbalance b/t free-radical generating and radical-scavenging systems

• Intracellular calcium and loss of calcium homeostatsis
• Ischemia and toxins cause early incr in cytosolic Ca concentration  activates enzymes
• Enzymes that are activated are:
• Phospholipases – damages cell mbr
• Proteases
• ATPases
• Endonucleases
• Defects in membrane permeability
• Irreversible mitochondrial damage
• Cells are dependent on mitochondria for ATP
• Can be damaged by:
• Incr of cytosolic Ca
• Oxidatve stress
• Breakdown on phospholipids t/ phospholipase A2 and sphingomyelin pathways
• Lipid breakdown products – ceramide and free fatty acids

• Mitochondrial Permeability Transition – in inner mitochondrial membrane; high-conductance channel
• Reversible in early stages
• Becomes permanent if stimuli persist  cell death
• Mitochondrial damage is associated with leakage of cytochrome c into the cytoplasm
• Can trigger apoptotic death p’ways in the cytosol

Hypoxic/Ischemic Injury:

• Hypoxia – glycolytic energy production can continue
• Ischemia – compromises the delivery of substrates for glycolysis, so anaerobic energy production stops
• Ischemia tends to injure tissues faster than hypoxia
• Ischemia  reversible cell injury  point of no return  irreversible cell injury
• When blood flow is restored: injury is often exacerbated  tissues lose more cells b/c of ischemia/reperfusion injury
• Let us summarize the drama of ischemic cell death? The drama varies from one tissue to another and with the type of injury, but for ischemic cell death of liver cells we can suggest the following, drawn from many sources. The reader should try to see it as a movie:

 Mitochondrial ATP production stops.
 The ATP-driven membrane ionic pumps run down.
 Sodium and water seep into the cell.
 The cell swells, and the plasma membrane is stretched.
 Glycolysis enables the cell to limp on for a while.
 The cell initiates a heat-shock (stress) response, which will probably not help if the ischemia persists.
 The pH drops.
 Calcium enters the cell.
 Calcium activates phospholipases, causing the cell membranes to lose phospholipid and producing lysophosphatides and fatty acids, both of which cause more membrane damage, initiating a vicious circle.
 Calcium activates proteases, damaging cytoskeletal structures; blebbing develops.
 Calcium activates ATPase, causing more loss of ATP.
 Calcium activates endonucleases, the nuclear chromatin forms clumps, some seeps out. Uric acid is released.
 Protein denaturation starts (calcium may be involved).
 All cell membranes are damaged.
 The ER and other organelles swell.
 The final blow is probably related to the massive inflow of calcium.

Reversible Cell Injury:
• First point of attack is the aerobic respiration – oxidative phosphorylation by the mitochondria  decr synthesis of ATP
• Depletion of ATP has many effects:


1. Decreased activity of Na/K pump in cell mbr • Na incr inside cell, K diffuses outside cell
• Influx of Ca into cell
• Net gain of solute  osmotic cell swelling and dilation of the ER
• Incr intracellular osmotic load b/c of accumulation of catabolites and net gain of solute

2. Switch to anaerobic glycolysis • Decr in glycogen stores, buildup of lactic acid and phosphates   decr pH of cell

3. Detachment of ribosomes • Detachment of ribosomes from ER and dissociation of polysomes  decr in ptn synthesis



• If ATP depletion gets worse:
• Cytoskeleton disintegrates  loss of ultrastructural features (ex – microvilli) and formation of blebs
• Myelin figures can be seen – Myelin figures are stacks of phospholipid bilayers in the shape of spheres, cylinders, and spirals that develop wherever cells are destroyed; derived from plasma and organellar membranes
• Mitochondria are swollen
• ER remains dilated
• Early chromatin clumping
• If oxygen is restored, all these changes are reversible

Irreversible Cell Injury:
• Hallmark of irreversibility:
1. Inability to reverse mitochonrial dysfunction
2. Profound problems in membrane fx – central factor in pathogenesis of irreversible cell injury
3. Nuclear changes are the light microscopist's hallmark for irreversible injury. PYKNOSIS is a shriveling and darkening of the nucleus attributed to very low pH. (RULE: If the nucleus is smaller and darker than a resting lymphocyte's, or is small and dark and shows no euchromatin-heterochromatin textures, that cell is very dead.) Other sure signs of cell death include KARYORRHEXIS, or fragmentation of the shriveled nucleus (into "NUCLEAR DUST"), and KARYOLYSIS, which simply means that nothing of the nucleus is visible any longer, except perhaps a purple haze.




Mitochondrial dysfunction • Incr of Ca and ATP depletion  mitochondria takes up more Ca  activates
    mitochondrial phospholipases accumulation of free fatty acids
• Causes changes such as mitochondrial permeablilty transtion
• Also causes changes in outer membrane
• Severe swelling of mitochondria
Loss of membrane phospholipids • Ca enters cells  activates phospholipases  phospholipid breakdown  incr in lipid breakdown products
• Extensive damge to plasma membranes; problems with permeabilility
• Massive influx of calcium into the cell occurs
• Continued loss of ptns, enzymes, conenzymes, and ribonucleic acids from the
     hyperpermeable membranes
Cytoskeletal problems • Activation of proteases by Ca  causes damage to cytoskeleton
• Can have detachemnt of cytoskeleton from mbr  mbr suceptible to stretching and tearing
Lipid breakdown products • Include free fatty acids, acyl carnitine, and lysophospholipids
• Act as detergents on membrane
• Insert into lipid bilayer
• Exchange with mebrane phospholipids
• Cause problems with permeability and retention of ions
Loss of intracellular amino acids • Glycine and other aa’s protect hypoxic cells from irreversible mbr damage in vitro
• Glycine helps ATP-depleted cells to resist the lethal effects of high Ca
• Loss of such aa’s that occurs in hypoxia predisposed to mbr injury
Swelling of lysosomes
• Injury to lysosomal membranes occurs  leakage of enzymes into the cytoplasm  activation of acid hydrolases
• Lysosomes contain RNAases, DNAases, proteases, phosphatases, glucosidases, and cathespins
• Have enzymatic degradation of cell components
Large, flocculent, amorphous densities develop in the mitochondrial matrix

• Early fall in pH is followed by a shift to neutral or alkaline pH
• Acidosis protects against lethal injury in many ischemia models and reperfusion
• Probably as a result in the inhibitory effect of low pH on enzymatic reactions

After cell death:
• Cell components are degraded
• Widespread leakage of cellular enzymes into x-cell space
• Dead cells become replaced by large masses composed of phospholipids in the form of myelin figures  phagocytosed or degraded into fatty acids
• Calcification of fatty acids can occur

Reperfusion:
• Cells may die after reperfusion, by both necrosis and apoptosis
• May be b/c cells are structurally intact, but are biochemically compromised and lose integrity during reperfusion
• New damaging processes are set in motion during reperfusion

Reactive oxygen species
• Incr in reactive oxygen species during reperfusion
• From parenchyma and endothelial cells; also infiltrating leukocytes
• Cellular anti-oxidant defenses may have damaged during ischemia, and therefore be more damaged during reperfusion

Mitochondrial permeablility transition • Can be further promoted by reactive oxygen species

Additional inflammation • Caused by cytokines and incr expression of adhesion molecules by hypoxic cells
• Recruits PMN’s to reperfused tissues  inflammation  injury


Free-Radical Induced Cell Injury:

4) Free Radicals = have single unpaired electron in outer orbit, thus extra –ve charge
Reactive Oxygen Species (ROS) = Superoxide (O2-), Hydrogen Peroxide H2O2, Hydroxyl ion OH-
Reactive Nitrogen Species = Peroxynitrite

How, in a living tissue, can any atom or molecule find itself limited to a single electron in an outer orbit? This accident can occur in several ways.
• Energy supplied by the environment can split the covalent bond between two atoms in such a way that one electron remains attached to either side (homolytic fission). This is what happens when tissues are irradiated: water molecules are split into free radicals (radiolysis) which account for much of the tissue damage. The energy of sunlight does something similar to the skin (photolysis).
• Even without the application of external energy, susceptible atoms can capture an electron; for instance, an electron that strays off the electron transport chain in a mitochondrion. Normally, the electron-transfer reactions are carried out by enzymes that are firmly embedded and properly ordered in lipid membranes, so that the production of free radicals is minimized; but this order can be broken. Under normal conditions, about 1 percent of the electrons that pass along the transport chain stray away and react with molecular oxygen; fortunately, the resulting free radical is captured by defensive free-radical scavengers and damage is avoided. The rate of this electron escape is directly proportional to the partial pressure of oxygen. In a person breathing 100 percent O2 the mitochondria of the pulmonary alveolar cells may produce five times the usual amount of free radicals, too much for the scavenger mechanisms to absorb.
• Several oxidative enzymes can produce free radicals, in some cases because the substrate diffuses away from the enzyme surface before it is wholly oxidized or reduced to an even electron number.


Sources of Free Radicals


• May be initiated w/in cell by:
• Absorption of radiant energy – UV light, x-rays
• Enzymatic metabolism of exogenous chemicals or drugs – carbon tetrachloride
• Reduction-oxidation rxns
• Can produce superoxide anion radical (O2-), hydrogen peroxide (H2O2), and hydroxyl ions (OH-)
• Rapid bursts of superoxide production occur in activated PMN’s  - uses NADPH oxidase
• Some intracellular oxidases generate superoxide radicals
• Transition metals can donate or accept free electrons
• Reduction by Fe can be enhanced by superoxide
• Sources of iron and superoxide are required for maximal oxidative damage
• Nitric Oxide can act as a free radical; can be converted to more reactive forms

•Effects of reactive species:
• Lipid peroxidation of membranes
• Double bonds in unsaturated fatty acids in mbr lipids are attacked  yields peroxides
• Autocatalytic chain propagates  extensive damage
                                                     ROS + Lipids = Lipid Peroxidases + Lipids = More Lipid Peroxidases
• Termination can occur if free radical is captured by a scavenger in the cell mbr (Vit E)
• Oxidative modification of ptns
• Free radicals promote oxidation of amino acid side chains and ptn backbone  ptn fragmentation
• Lesion in DNA
• Free radicals react with thymine to produce single-stranded breaks in DNA

• Cell defenses:
• Free radicals are unstable and decay spontaneously
• Antioxidants – block initiation or inactivate free radicals; terminate radical damage (Vitamin E, Vitamin C,  Retinoids, etc)
• Minimize levels of iron and copper by binding ions to ptns (transferrin, ferritin, etc)
             • Enzymes can act as free-radical-scavenging systems (Detoxifying Enzymes)
            • Superoxide dimutases – both in mitochondria and cytosol; 2 O2- + 2 H+  O2 + H2O2
• Catalase – in peroxisomes; 2 H2O2  2 H2O + O2
• Glutathione peroxidase – both in mitochondria and cytosol; H2O2 + 2 GSH  2 H2O + GSSG

Chemical Injury
• Chemicals can act directly by binding to critical molecular comonent or cellular organelle
• Mercuric chloride poisoning
• Cyanide – poisons mitochondrial cytocrome oxidase
• Antineoplastic drugs

• Must be converted into reactive toxic metabolites
• Conversion is done via a P450 mixed function oxidase in the SER of the liver and other organs
• Metabolites can cause damage by direct covalent bonding
• Can form free radicals  lipid preoxidation

Carbon Tetrachloride Induced Liver Injury:
• CCl4 used in dry cleaning
• Toxic effect due to conversion by P450 in the SER: CCl4   CCl3 + Cl-
• CCl3 is a highly reactive toxic free radical  lipid peroxidation  propagation along microsomal membrane
• In the hepatocyte, this can lead to attach of PUFA’s  forms an organic radical
• Organic radical can meet with O2 to form an organic lipid peroxide radical
• Rapid breakdown of structure and fx of the ER

• Can form malondialdehyde from lipid peroxide
• Can form lipid-lipid, lipid-ptn, or ptn-ptn crosslinks

• Free radicals can attach ptns  disulfide crosslinking, ptn-ptn crosslinking, and ptn scission

• Net result is damage to the integrity of the membranes
• Have dissociation of polyribosomes from the ER and dissociation of the polyribosomes  decr ptn synthesis
• Inhibits secretion of TG b/c no apoproteins
• Hepatocyte accumulates large droplets of TG in its cytosol

• Have damage to mitochondria  decr ATP synthesis
• Also have damage to plasma mbr  changes in ionic homeostatsis  cell death

Acetominophen:
• Detoxified in the liver via sulfation and glucuronidation
• Small amount are converted via P450 to highly toxic metabolite  detoxified by interactions w/ GSH
• Large doses of drug ingested  GSH depleted  covalent binding of toxic metabolites  incr drug toxicity  liver cell necrosis
• Hepatotoxicity correlates with lipid peroxidation, can be reduced by administration of antibiotics

Apoptosis:
• Occurs in:
• Tissue remodeling during development
• Negative selection of B and T cells
• In response to loss of hormonal stimulation – postmenopausal, etc
• In cancer cells, spontaneously and in response to radiation or chemo
• In viral infections
• During atrophy of tissues
• Death of neutrophils during an acute inflammatory response
• Death induced by Tc cells
• Cell deletion in proliferating cell populations – intestinal crypts
• Pathologic atrophy after duct obstruction – pancreas, parotid, etc
• In response to injurious stimuli

• Morphology:
• Cell shrinkage; surface convolutions
• Chromatin condensation
• Formation of cytoplasmic blebls and apoptotic bodies
• Phagocytosis of apoptotic cells by adjacent paraenchymal cells and macrophages

• Biochemical Features
• Ptn cleavage – involves the activation of a family of cysteine proteases named caspases
• Caspases cleave nuclear scaffold and cytoskeletal ptns and triggers endonucleases
• Extensive ptn cross-linking by transglutaminase activation
• Breakdown of DNA into large pieces  interneucleosomal cleavage into smaller pieces by Ca and Mg endonucleases
• Express phosphatidylserine and/or thrombospondin in outer layers of cell mbr for recognition by macrophages

• Initiators of Apoptosis:
• Variety of signals
• Lack of growth factor or hormone
• Positive ligand-receptor interaction
• Specific injurious agents

• Signaling Pathways
• Transmembrane signals – can be negative or positive
  • FAS ligand on cytotoxic T cells – can bind to FAS receptors on target cells and trigger apoptosis
• Important in clonal selection; may play a role in killing virus-infected cells
• Ligand binding of plasma mbr receptors of the TNF superfamily

• Intracellular perturbation
• Ionizing radiation  DNA damage  apoptosis
• p53 protein
• Altered in cancers, loss of fx is important for malignant transformation
• c-myc protein
• Fx’s in cell cycle
• Overexpressed  apoptosis only in absence of an exogenous growth factor

• Certain viral proteins and bacterial toxins
• gp120 protein
• Can induce apoptosis of CD4+ T cells; imp in HIV infection

• Modulators of Apoptosis
• Specific ptns that connect death signals to execution
• bcl-2 protein – in outer mitochondrial membrane, ER, and nuclear mbr
• Altered in malignant tumors, plays a role in preventing apotosis

• bcl-x, bax, bak, and other related molecules
• Regulate apoptosis t/ dimerization reactions with other bcl-2 family members

• APAF-1
• Binds to cytochrome c and promotes apoptosis and activiting caspase 3
• Apoptotic signals results in mitochondrial permeability transitions  mitoch swelling
• Also cause incr permeability of outer mitoch mbr  release cytochrome c into cytosol
• Cytochrome c release precedes the morphologic changes of apoptosis

• Effectors of Apoptosis
• Caspases play a key role – cleave a variety of substrates
• Caspase 3  activates a cytoplasmic endonuclease  cleaves DNA b/t nucleosomes
• Gives rise to DNA ladder seen on gels of DNA from apoptotic cells
• Present new cell surface molecules – i.e. thrombospondin (facilitates phagocytosis)
• Transglutaminase activity  cross-linkage of cytoplasmic ptns
• Induced during apoptosis, may lead to incr in eosinophilia of cytoplasm

Induction of SER:
• Protracted use of barbituates  hypertrophy of the SER of hepatocytes
• Enzyme modification of barbituates uses the P-450 mixed function oxidase system
• P-450 mixed oxidase system:
• Incr solubility of a variety of compounds
• Facilatate their secretion
• Many compounds renedered more injurious by the P450 system; forms many reactive oxygen species

Cell Injury and its Adaptation part 2

Cell Injury II

1. Know the definitions of necrosis, apoptosis, autolysis, heterolysis, atrophy, hypertrophy, hyperplasia, and metaplasia.
a. Necrosis: a sequence of morphological changes that follow irreversible injury and cell death in a tissue
b. Apoptosis: programmed cell death
c. Autolysis: occurs when the source of the hydrolytic enzymes is from the dead cells themselves
d. Heterolysis: occurs when the enzymes originate from inflammatory cells that invade the necrotic focus
e. Atrophy: decrease in cell size
f. Hypertrophy: increase in cell size
g. Hyperplasia: increase in mitosis resulting in a greater number of cells
h. Metaplasia: chang in the state of differentiation of a cell in response to a stress, most commonly an irritation

2. Know the 2 biochemical components of necrosis and understand which prevails in different forms of necrosis.
a. Enzymatic digestion: produces liquefactive necrosis
b. Denaturation of proteins: produces coagulation necrosis

3. Understand the morphology, etiology and common tissue sites of coagulation, liquefactive, caseous, gangrenous, and fat necrosis.
a. Coagulation necrosis: the most common form of necrosis and occurs in a number of solid organs.
i. Hypoxic death of cells in all tissues except the brain will result in coagulation necrosis.
ii. The structural outline of elements of the tissue is preserved for days.
iii. Necrotic tissue contains eosinophilic ghosts of structures or cells that are devoid of nuclear detail.
iv. The necrotic area may have an inflammatory infiltrate.
b. Liquefactive necrosis: occurs in any tissue where there is a focus of bacterial or fungal infection (abscess).
c. Gangrenous necrosis: normally involves the distal region of the limb (usually the leg)
i. Loss of blood supply initiates a coagulative necrosis with a superimposed bacterial infection (wet gangrene)
d. Caseous necrosis: a form of coagulation necrosis at the focus of a tuberculous infection.
i. The necrotic area is white and cheesy and devoid of structure but surrounded by a ring of granulomatous inflammation.
ii. Once resolved, this too leaves cavities at the site.
e. Fat necrosis: may be seen in focal areas of the peritoneal cavity and caused by release of pancreatic enzymes into the peritoneum.
i. The pancreatic lipases attack the fat associated with the mesenteries and omentum, hydrolyzing triglyceride esters to release fatty acids.
ii. The fatty acids combine with calcium to form chalky white areas (fat saponification).
iii. Traumatic fat necrosis is also seen in the breast.

4. Know the differences between dystrophic and metastatic calcification.
a. Dystrophic calcification: occurs in areas of necrosis despite normal circulating levels of calcium
i. Crystal formation begins as calcium combines with phosphate to form a hydroxyapatite-like deposit in the dead cells or mitochondria of the necrotic tissues.
ii. In some cases, these sites may initiate heterotopic bone formation
iii. This can also contribute to further organ dysfunction.
b. Metastatic calcification: occurs in normal tissues
i. Abnormally high levels of circulating calcium (hypercalcemia) due to endocrine dysfunction or a tumor causing bone destruction are the cause of this sort of calcium deposition.

5. Understand the difference between cell death by necrosis and apoptosis.  When do you see each and how do they differ in morphology?
a. Necrotic cell death always follows injury.
i. Whole tissues may be affected
b. Apoptotic cell death is a well-planned and highly-regulated process that may follow injury but also occurs in normal tissues.
i. Apoptotic cells are often isolated and seen in smaller numbers
ii. Not accompanied or followed by inflammation
iii. Occurrences:
1. During embryogenesis (limb bud development)
2. Hormone-regulated cell changes involve apoptosis.  (shedding of the endometrium)
3. Cell deletion in tumors or normal proliferating populations of cells.
4. Selective removal of cells with DNA damage.
iv. Stimulation, signaling and activation:
1. Extrinsic signals act as stimuli, including specific growth factors, alterations in hormone levels, and specific receptor-ligand interactions (TNF, FAS)
2. Injurious agents may also trigger signals, resulting in a cascade of enzymatic reactions involving caspases leading to DNA degradation and cell and nuclear fragmentation.
3. Generally programmed intrinsic signals may also result in activation of the execution caspases.
v. Regulation:
1. Apoptosis is controlled by the BCL-2 family of proteins, involving the mitochondrion and regulation of the permeability transition pore (PTP).
2. Bcl-2 and others inhibit apoptotic cell death by stabilizing the pore complex to prevent leakage of cytochrome c and also stabilizes Apaf-1 protein so that it is not available to interact with procaspase 9.
3. Bax and others promote apoptosis by binding Bcl-2 and modulating this anti-apoptotic effect.
vi. Morphology:
1. Nucleus exhibits distinctive condensation of chromatin, followed by formation of nuclear fragments.
2. Plasma membrane is intact and packaged budding cytoplasmic fragments containing organelles and nuclear fragments into discrete apoptotic bodies are quickly phagocytosed by macrophages and other cells without inflammation.
3. If DNA is isolated from apoptotic cells, necrotic cells, and viable cells:
a. Control DNA runs as a single band.
b. DNA from necrotic cells will travel as a streaked band due to general degradation.
c. DNA from apoptotic cells forms distinct subunits or fragments that “ladder” along the band of the gel.

6. Know why substances may accumulate in cells.  What are some of the defects responsible for accumulation of endogenous substances?
a. Accumulation of substances may be an adaptation to stress or these substances may be normal constituents of the cell (endogenous substances) or abnormal substances (exogenous).
b. Accumulations of normal endogenous materials may occur when that substance is produced by the cell at normal or greater than normal rates where the removal is inadequate to prevent accumulation.
i. Ex: fatty liver and protein accumulations in proximal tubule cells
c. Both normal and abnormal endogenous substances may accumulate if the cell has defects in the metabolic pathway necessary to process the substance as in lysosomal storage diseases.
d. Exogenous substances accumulate in cells because there is simply no biological mechanism for their removal.
i. Ex: carbon in the lung and associated lymph nodes (anthracosis leading to emphysema) and tattoos
e. Endogenous accumulations:
i. Fat: accumulation of triglycerides due to blockage of steps in triglyceride metabolism.  Lipids accumulate in small vesicles surrounding the nucleus and then coalesce to form large intracellular lipid vacuoles that displace the hepatocyte nucleus and may cause rupture of the cell. (hepatocytes in fatty liver)
ii. Proteins:
1. When greater than normal amounts of protein escape in the glomerular filtrate in glomerulonephritis, the proximal tubule cells fill with eosinophilic droplets.
2. A plasma cell neoplasm (multiple myeloma) produces excess amounts of immunoglobulins and may form inclusion bodies with the RER called Russell bodies.
iii. Glycogen: may accumulate in diabetic individuals due to altered glucose metabolism.
1. Defect in glucose-6-phosphate results in accumulation of glycogen in the liver and hepatomegaly.
2. Defect in lysosomal glucosidase results in glycogen stores in the cardiac muscle cells and cardiomegaly.
iv. Cholesterol: Following endothelial injury, macrophages and smooth muscle cells filled with lipid (foam cells) may accumulate in developing atherosclerotic plaques.
1. When the lipid containing cells die, the extracellular lipids form the cholesterol core of the atheroma.
v. Pigments:
1. Bilirubin: When concentrations of bile in the blood exceed 2.0 mg/dL, jaundice occurs.  Dubin-Johnson disease involves a defect in the hepatic excretion of bilirubin.
2. Hemosiderin: hemoglobin-derived, iron-containing pigment.
a. Local excesses founding focal hemorrhage in a bruise or in the congested lungs of heart failure patients.
b. Systemic excesses in multiple organ systems due to massive doses of dietary iron or impaired iron usage causing hemosiderosis snd extreme iron overload found in hemochromatosis.
3. Lipofuscin: “wear and tear” pigment that accumulates with age or atrophy
4. Melanin: synthesized by oxidation of tyrosine in melanosomes of the melanocyte

7. Know the causes of atrophy, hypertrophy, hyperplasia, and metaplasia.  How may they contribute to organ size?
a. Atrophy: decrease in cell size leading to a reduction in organ size if enough cells atrophy.
i. Cause: decreased work load, decreased blood supply to an organ, alteration in nutritional status, change in hormonal stimulation, or aging in general
b. Hypertrophy: increase in cell size leading to an increase in organ size if enough cells hypertrophy.
i. Cause: increased physical or mechanical demand or hormonal stimulation
c. Hyperplasia: due to increase in the rate of mitosis resulting in a greater number of cells.  Can contribute to increased organ size.
i. Cause: growth factor stimulation (compensatory) as in hyperplasia of one kidney following unilateral nephrectomy, change in hormonal stimulation as in prostatic hyperplasia with advancing age as estrogens increase and testosterone decreases
ii. Hyperplasia may be a precursor for neoplasia.
d. Metaplasia: change in the state of differentiation of a cell in response to stress, usually irritation.
i. Cause: In response to potentially damaging irritant, growth factors signal to reserve stem cells to produce a population of cells better suited to handle the stress.
1. Respiratory epithelium that is chronically irritated may respond by metaplastic change to stratified squamous.
2. Esophageal reflux disease leads to replacement of squamous epithelium in areas of the lower esophagus by a columnar epithelial cell type with characteristics of the stomach or intestinal mucosa (Barrett’s esophagus).