Cellular Pathology

Question 1

ER

Answer 1

Rough - ribosomes, new proteins synthesised here (assembled in golgi)
Smooth - no ribosomes, used for steroid and lipoprotein synthesis, modification of hydrophobic compounds into water soluble molecules

3 sites for breakdown - lysosomes, proteasomes, peroxisomes

Question 2

Mitochondria

Answer 2

ATP through oxidative phosphorylation, sensors of cell damage and regulate programmed cell death
Last 10 days

Question 3

Cell cycle

Answer 3

Interphase - cell growth not division (far longer than mitosis)
Checkpoints between interphase phases - monitor integrity before replication occurs
G1 - longest, cell growth
S - DNA synthesis
G2 - prepare for mitosis

Cyclins drive cell cycle, paired with CDK (enzyme)
CDK inhibitors modulate complex activity enforcing checkpoints

Question 4

Apoptosis

Answer 4

Tightly regulated cell death
NO LEAKAGE OF CONTENTS AND NO INFLAMMATORY REACTION

Pathological examples
DNA damage during radiation, chemo or hypoxia - p53
Protein misfolding - Targeted for proteolysis in proteasomes
- Accumulate in ER - decreased synthesis, increased degradation, increased chaperones (which control proper folding)
- If this doesn’t work - activate caspases —> apoptosis
- Feature in neurodegenerative disease
Infections - Cytotoxic T lymphocyte apoptosis —> secrete performing —> entry of protease called granzymes into host cell (cleaves proteins and activates caspases within cell)
Pathological atrophy - dysregulated or excessive

Mechanisms:
Cysteine proteases that cleave proteins
- Cleaved ones are marker for cells undergoing apoptosis

Initiation (intrinsic mitochondrial, extrinsic death receptor) and execution phase (caspases activation leads to final phase of apoptosis)

Mitochondrial - BCL2 - BAX and BAK (increased membrane permeability) - there are also anti-apoptosis proteins that prevent leakage of cytochrome C and other death inducing proteins from mitochondria into cytosol
Sensors - BH3 only proteins - sense cellular stress and damage

SENSE PROTEINS —> ACTIVATE PRO or ANTI-APOPTOTIC proteins —> activation or no activation

Death receptor pathway:
Engagement of plasma membrane death receptors - TNF (TNFR!) and a related FAS ligand - expressed on T cells that recognise self-antigens

EXECUTION
Sequential activation of executioner caspases —> endonuclease activation or breakdown of cytoskeleton

Question 5

Necrosis

Answer 5

These cells do not maintain membrane integrity —> ALWAYS ACUTE INFLAMMTION and always pathological
Lysosomes from dying cells and from leucocytes cause digestion

Is ischaemia, toxins, trauma and certain infections cause this

Show increased eosinophilia

Karyolysis, pyknosis, karyorrhexis

Types:
Coagulative - infarct except brain
Liquefaction - death within CNS, focal bacterial - liquid viscous mass (creamy yellow)
Gangrenous - Limb that has lost its blood supply and has undergone necrosis
Caseous necrosis - Tubercolsis, granule a
Fibrinogen - vasculitis syndromes, immune reactions involving blood vessels
Fat necrosis - Acute pancreatitis - fat saponification

There are broadly six types of tissue necrosis: coagulative (not coagulopathic), fibrinoid, fat, caseous, liquefactive, and gangrenous. Coagulative and liquefactive are distinct pathological processes and the remaining four types are descriptions of necrosis in specific circumstances.

In coagulative necrosis, tissue architecture is preserved at least for several days, whereas in liquefactive necrosis, the cells are digested early, resulting in a thick liquid mass, often called “pus”.

Fat necrosis is specifically necrosis of fat tissue, and classically occurs in severe pancreatitis, where enzymes digest the cells of the peritoneum. Released triglycerides combine with calcium in a process called “saponification”.

Fibrinoid necrosis is immune-mediated and common in autoimmune reactions involving blood vessels. It results from deposition of antigen–antibody complexes in vessel walls.

Caseous necrosis is seen in TB and other inflammatory conditions. It is characterised by foci of friable, cheese-like material surrounded by inflammatory cells, known as “granulomas”.

Gangrenous necrosis is used to describe cell death associated with ischaemia, typically of the lower limbs.

Necrosis, or cell death, occurs when an injury to the cell becomes irreversible and the cell cannot recover from it. Its features include:

• Increased cell size (swelling)
• Nuclear changes observed (pyknosis -> karyorrhexis -> karyolysis)
• Plasma membrane disrupted
• Cellular contents leak from the cell
• Associated inflammation present

Question 6

Differences between necrosis and apoptosis

Answer 6

Cell size - enlarged vs reduced
Nucleus - pyknosis vs fragmentation
Membrane - Disrupted vs intact/altered structure
Cellular contents - Enzymatic digestion vs intact
Inflammation - Frequent vs No
Physiologic or pathological - usually pathological vs physiologic

Question 7

Cell injury and cell death

Answer 7

Cell injury - decreased ATP and cellular swelling (potassium efflux)

Causes - hypoxia, chemical agents, infectious agents, immunologic reactions/genetic derangement/nutritional imbalances

Biochemical mechanisms of cell injury -
Depletion of ATP —> increased cellular swelling AND lactic acid —> acidosis —> failure of calcium pump AND protein misfolding
Mitochondrial damage —> mitochondrial membrane pore —> failure of oxidative phosphorylation
Influx of calcium and loss of calcium homeostasis —> phosphipases/proteases/endonucleases/ATPases OR mitochondrial permeability
Accumulation of oxygen derived free radicals (ischaemia repercussion) - these are produced normally but too much - Lipid peroxidation, oxidative modifications of proteins, lesions in DNA
Defects in membrane permeability - ROS, decreased phospholipid synthesis or increased breakdown of these, cytoskeleton abnormalities ALL OF THESE lead to —> plasma or mitochondrial membrane damage OR injury to lysosomal membranes
Damage to DNA and proteins

Question 8

Ischaemia-repercussion injury

Answer 8

Oxidative stress, increased intercellular calcium, inflammation, activation of complement

Question 9

Metastatic calcification

Answer 9

Metastatic calcification is deposition of calcium in normal tissues due to elevated serum calcium levels (hypercalcemia). There are multiple causes of hypercalcemia including:

• Increased serum levels of parathyroid hormone (e.g. due to parathyroid adenoma)
• Bone destruction
• Renal failure
• Vitamin D intoxication
• Certain malignancies (e.g. lung cancer)
• Medications (e.g. thiazide diuretics)

Question 10

Dystrophic calcification

Answer 10

Dystrophic calcification refers to calcium deposition in abnormal (usually dying) tissues in the context of normal serum calcium levels. By contrast, metastatic calcification occurs in normal tissues when serum calcium levels are elevated, such as in hyperparathyroidism or malignancy. All of the above result in serum hypercalcaemia.

Question 11

Irreversibility

Answer 11

Progress to irreversibility is characterised by mitochondrial swelling, calcium influx, and leakage of cellular enzymes.

Irreversible cellular injury is characterised by profound disruption of cell membrane function and failure of mitochondrial energy generation, even after removal of the injurious stimulus. Cell membrane blebbing or blunting, and endoplasmic reticulum dilatation, are both associated with reversible cell injury. The point of no return for cellular injury is still not well defined.

Question 12

Apoptosis microscopy

Answer 12

Cellular bleb

Leucocytes infiltration

Question 13

Telomere

Answer 13

Telomeres are short repetitive nucleotide sequences present at the end of the chromosome that protect the ends and ensure their complete replication. Shortening of the telomeres can lead to cell loss and eventually cellular aging. The centromere is the region of the chromosome that separates the p-arm from the q-arm. The promotor, terminator, and exons are parts of a gene.

Question 14

Calcium accumulation in cells

Answer 14

Calcium levels are tightly controlled within the cell, much lower than extracellular concentrations. Most intracellular calcium is found in the endoplasmic reticulum and mitochondria.

Calcium accumulation in the cell results in cell injury by several mechanisms. Mitochondria become more permeable and fail to generate ATP; intracellular enzymes are activated, which break down ATP, DNA and cellular proteins; and apoptosis is directly induced.

DIRECT, enzymes OR mitochondrial permeability

Question 15

Cellular Aging

Answer 15

Cellular aging occurs due to a reduction in the functions of the cell, eventually leading to cell death. There are various mechanisms of cellular aging, such as accumulation of damaged DNA, shortening of telomeres, and abnormal protein homeostasis. Certain environmental factors can cause decreased growth factor signaling, which leads to increased DNA repair and increased protein homeostasis to counteract the cellular aging process.

Question 16

Intrinsic apoptosis pathway

Answer 16

BAX and BAK
These are pro-apoptotic mediators, activated when sensors of stress or cellular damage are activated. They trigger insertion of proteins that increase mitochondrial permeability, causing cytochrome C release. This triggers caspase 9 activation, which is an initiator caspase that can then activate the executioner caspases 3 and 6.

Question 17

Cell injury mechanisms

Answer 17

There are several mechanisms of cell injury, such as:

• Depleted ATP levels, which leads to reduced activity of the ATPase pumps and anaerobic glycolysis
• Damage to mitochondria, leading to impaired oxidative phosphorylation
• Calcium influx which activates a number of enzymes
• Reactive oxygen species (ROS) accumulation which injures the cell by lipid peroxidation
• DNA damage and the presence of misfolded proteins leading to apoptosis

Question 18

SNP’s

Answer 18

SNPs are defined as variants at single nucleotide positions.

SNPs can occur across the genome, within exons, introns, intergenic regions and coding regions. They are the most common type of genetic variation among people, occurring roughly once in every 1000 nucleotides. Each individual has an average of 4-5 million SNPs in their genome.

SNPs can occur across the genome but only about 1% of SNPs occur in coding regions. Since coding regions comprise about 1.5% of the genome, this percentage shows that SNPs happen randomly occurring to probability.

SNPs in noncoding regions such as promoters, silencers, enhancers and introns can alter regulatory elements in the genome, thereby altering gene expression. Each SNP represents a difference in a single nucleotide, for example replacing a thymine base with a cytosine base. This difference then leads to the production of a transcript that will behave differently than that coded by the original gene.

The effect of most SNPs on disease susceptibility is weak. When SNPs occur within a gene or in a regulatory region near a gene, they may play a more direct role in disease by affecting a gene’s function, however, since the chance of a SNP occurring in a coding or regulatory region are low, they often do not have any deleterious effect.

Question 19

Micro-RNA’s

Answer 19

miRNAs function primarily to modulate translation of RNA by posttranscriptional silencing of gene expression. This is often accomplished physically by binding to mRNA, thus preventing the ribomsome from binding and translation from taking place. This is a highly conserved mechanism of gene regulation present in all eukayotes (plants and animals). It is not present in bacteria, though they have a primitive version of the same general machinery.

Question 20

Gene splicing

Answer 20

The first transcript during the transcription process, pre-mRNA, includes UTRs, exons and introns. As the spliceosome reads along the pre-mRNA segment, it cuts the transcript at specific points signalled by the intron, thereby removing the introns from the RNA strand. Exons are then linked together by RNA ligase to produce the open reading frame, which is used to produce proteins.

Question 21

Gangrenous

Answer 21

Gangrenous necrosis is a condition in which most commonly the lower limb loses its blood supply, causing tissue death. Some risk factors for gangrenous necrosis include diabetes mellitus, smoking, peripheral arterial disease, and Raynaud’s phenomenon. There are two major types of gangrenous necrosis: dry and wet gangrene. Dry gangrene is a form of coagulative necrosis without superimposed infection, whereas wet gangrene is a form of liquefactive necrosis with superimposed infection.

Question 22

Free Radicals

Answer 22

Proteasomal degradation
ZBreaks in DNA
Lipid membrane breakdown
Enzyme site damage

Reactive oxygen species (e.g. superoxide, hydroxyl radical, hydrogen peroxide) are produced in small amounts in all cells and in large amounts in neutrophils and macrophages, to help in the degradation of microorganisms. They cause cellular injury by various mechanisms such as lipid peroxidation, DNA damage, and protein misfolding. Antioxidants such as vitamins A, C and E prevent the formation of and promote the degradation of reactive oxygen species.

Question 23

Non Coding DNA

Answer 23

About 98.5% of the human genome does not encode proteins, however, more than 85% of non-coding DNA is ultimately transcribed to produce RNAs such as miRNA that are used to regulate gene expression and protein syntheses. In the past non-coding DNA was thought to be ‘junk DNA’; DNA that had outlived its evolutionary purpose. However, recent research has shown that non-coding DNA is often essential to epigenetic regulation and gene expression.

Question 24

Cytotoxic T cells

Answer 24

Cytotoxic T cells have multiple mechanisms by which they may kill damaged or infected cells. Lymphocytes express both Fas and the Fas ligand (FasL) on their surface. Binding of Fas to FasL can induce cellular apoptosis and is thought to play a role in removing lymphocytes that recognise (and target) self-antigens.

Cytotoxic T cells secrete perforins, which insert onto the target cell membrane and allow entry of granzyme enzymes, which may trigger apoptosis through the cleavage of precursor caspases.

Bcl-2 factors are anti-inflammatory and anti-apoptotic proteins, involved in promoting cell survival. Their dysregulation can lead to development of cancers.

Question 25

Stains

Answer 25

Iron is visualized as either purple or blue deposits on histopathology using the Prussian blue stain. Sudan black is used to identify fat deposits in tissues. Gram stain and Giemsa stain are mostly used to identify bacteria and parasites (such as malaria). Silver nitrate can be used to identify fungi such as Pneumocystis jiroveci.