Adaptation, Injury and Death

 Cells constantly alter their functional state to maintain homeostasis  But: 1) Excessive physiologic stresses; or 2) Adverse pathological stimuli (injury) result in: 1) Adaptation (Hypertrophy/plasia, atrophy, metaplasia) 2) Reversible injury 3) Irreversible injury and cell death (necrosis, apoptosis)

› hypoxia › toxins › metabolic › infective › ischaemia › physical agents

1) Hydropic change 2) Fatty change 3) Cellular inclusions  may be indicative of exposure to previous insults

 Cells cannot maintain ionic and fluid homeostasis  Due to loss of activity in plasma membrane energy- dependent ion pumps  Cells become vacuolated and pale

Above: liver showing hydropic degeneration (H)Above: liver showing hydropic degeneration (H) Right: renal tubules showing hydropic degeneration (H)Right: renal tubules showing hydropic degeneration (H)

 Cytoplasmic lipid (triglyceride) vacuoles  Principally encountered in cells involved in or dependent on fat metabolism Eg. Hepatocytes (in response to ethanol) Myocardial cells Renal tubular epithelial cells

 Chronic sublethal damage  Damaged organelles are removed by autophagy  Damaged proteins are removed by ubiquitin- mediated proteolysis  Residual materials may remain as inclusion bodies – stigmata of damage  Eg. Pigments (Hemosiderin, Lipofuscin) Cholesterol

 If cellular damage is severe or maintained  cell death  Damaged cells die by three main mechanisms: 1) Coagulative necrosis (uncommon) - Some stimuli to cells instantly coagulate proteins - Cells die immediately - Stimuli: extreme heat, extreme pH change 2) Necrosis (very common) 3) Pathological apoptosis (very common)

 Relate closely to aetiology › Coagulative  Ischaemia › Liquefactive (most common)  Brain pathology › Haemorrhagic  complete venous obstruction (volvulus) › Caseous  “cheese” TB › Gummatous  ‘’rubbery” Syphilis › Fibrinoid  reserved for vascular structures Eg. Hypertension, vasculitis

 Cell death by activation of internal “suicide” program  Minimal disruption of surrounding tissue, therefore, minimal inflammation  Causes: -Surface receptor activation -Surface membrane damage -Damage to mitochondrial membranes -DNA damage

 Cells which are damaged can show changes of sublethal injury which is potentially recoverable › Hydropic change › Fatty change › Cellular inclusions may be indicative of exposure to previous insults

 Damaged cells die by three main mechanisms › Coagulative necrosis (uncommon) › necrosis (very common) › pathological apoptosis (very common)

Loss of function Total loss of activity Partial loss of activity Gain of function Increased activity Novel activity

Allele Heterogeneity: Different mutations on same gene but at different locations Locus heterogeneity: Mutations on different genes altogether

If there is a hotspot, then mutation detection can be undertaken in a targeted way If there are mulitple sites of mutation, it will be necessary to scan for mutation

 Post PCR analysis : A. Size analysis to detect expansion mutations B. MLPA C. PCR to detect common point mutations: ARMS D. Analysis by heteroduplex E. Sequencing

 Using electrophoresis  Used for expansion mutations ( triplet repeats)  Allows “anticipation” in successive generations with increasing number of repeats  Polymorphic markers can be used instead B. Absence/Presence of a product   Multiplex PCR uses several primers to amplify several regions  Absence of a PCR product can be used to identify deletion of exons / genes /sequence

 Technique used to detect an abnormal number of chromosomes, gene deletions, gene duplications, and gene expansions  Eg. Downs Syndrome, DMD, Fragile X syndrome  Multiple probe sizes with different gene targets allow 40 or more gene exons to be tested for abnormalities at the same time

Useful when there are very few mutations since primers are “mutation” specific If the mutation is not there, the primer will not bind and there will be no PCR product Several mutations can be detected in same assay

Can be used to detect imprinted or epigenetically silenced alleles DNA is modified by bisulphite reaction which converts non-methylated cytosines to thymine Methylation specific primers can then be used to test for the presence or absence of a PCR product

In cases of allelic heterogeneity, a lot of regions may need to be tested Testing each one by sequencing would be expensive A number of strategies will allow the PCR product to be tested for sequence change before definitive sequencing E.g. Heteroduplex formation, single strand conformation polymorphism, protein truncation test)