1. Apoptosis and Intracellular Accumulations
Dr Bikramajit Singh Saroya
Windsor University SOM

2. Apoptosis Programmed cell death Suicide program
Cells destined to die activate activate enzymes that degrade the cells' own nuclear DNA and nuclear and cytoplasmic proteins
Intact plasma membrane
Apoptosis eliminates cells that are genetically altered or injured beyond repair and does so without eliciting a severe host reaction, thereby keeping the extent of tissue damage to a minimum

4. Physiologic Apoptosis
Programmed destruction of cells during embryogenesis
Involution of hormone dependent tissues upon hormone withdrawal Ex: Endometrial cell breakdown during menstrual cycle, Ovarian follicular atresia in menopause
Cell loss in proliferating cell populations to maintain homeostasis Ex: Immature lymphocytes in the bone marrow and thymus that fail to express useful antigen receptors
Elimination of potentially harmful self-reactive lymphocytes
Elimination of cells that have served their useful purpose

5. Apoptosis in Pathologic Conditions
DNA damage - radiation, cytotoxic anticancer drugs, extremes of temperature, and even hypoxia
Accumulation of misfolded proteins
Cell injury in certain infections - especially viral
Pathologic atrophy in parenchymal organs after duct obstruction

6. Mechanism of Apoptosis
The Mitochondrial (Intrinsic) Pathway of Apoptosis
The Death Receptor (Extrinsic) Pathway of Apoptosis
Activation and Function of Caspases
Clearance of Apoptotic Cells

8. Mechanism of Apoptosis
The two pathways of apoptosis differ in their induction and regulation, and both culminate in the activation of caspases.
In the mitochondrial pathway, proteins of the BCL2 family, which regulate mitochondrial permeability, become imbalanced and leakage of various substances from mitochondria leads to caspase activation.
In death receptor pathway, signals from plasma membrane receptors lead to the assembly of adaptor proteins into a “death-including signaling complex,” which activates caspases, and the end result is the same.

11. Anti-apoptotic genes (BCL-2 gene) and Pro-apoptotic genes (BAX, BAK genes)
BCL-2 proteins
maintain mitochondrial membrane integrity and prevent leakage of mitochondrial proteins that can trigger apoptosis (e.g., cytochrome c)
BAX and BAK - proapoptotic genes
activated by damage to DNA, misfolded proteins, viral infections, and other injurious events
produces protein products that form channels in the mitochondrial membrane that cause leakage of cytochrome c into the cytosol

12. Intrinsic(Mitochondrial) Pathway
A: Survival signals induce anti apoptotic proteins like BCL-2 that maintain integrity of mitochondrial membranes, prevent leakage of mitochondrial proteins
B: Loss of survival signals, DNA damage, other insults - activate the pro-apoptotic proteins BAX and BAK, which form channels in the mitochondrial membrane causing leakage of cytochrome c and other proteins leading to caspase activation and apoptosis

13. Extrinsic (Death Receptor) Pathway
Responsible for elimination of self-reactive lymphocytes and damage by cytotoxic T lymphocytes
Initiated by engagement of plasma membrane death receptors
Death receptors - members of the TNF receptor family
FAAD- Fas-associated death domain; FasL - Fas ligand)

14. Biochemical Features of Apoptosis
Activation of caspases
Caspases : Initiator and Executioner
Initiator caspases – caspase 8 and 9
Executioner caspases – caspase 3 and 6
DNA and Protein Breakdown
Membrane alteration to promote recognition by phagocytes :
Movement of some phospholipids (notably phosphatidylserine) from the inner leaflet to the outer leaflet of the membrane,
Binding of a protein called annexin V
Annexin V staining is commonly used to identify apoptotic cells

15. Morphologic Changes
Cell shrinkage: dense cytoplasm, tightly packed organelles
Chromatin condensation and DNA fragmentation (most characteristic feature of apoptosis)
Cytoplasmic blebs and apoptotic bodies
Phagocytosis of apoptotic cell bodies by macrophages
Intact plasma membranes

16. Apoptosis of an epidermal cell in an immune reaction
Apoptosis of an epidermal cell in an immune reaction. The cell is reduced in size and contains brightly eosinophilic cytoplasm and a condensed nucleus.

20. Disorders of Apoptosis
Defective apoptosis and increased cell survival.
Cancer
Autoimmune disorders
Increased apoptosis and excessive cell death.
Neurodegenerative diseases,
Death of virus-infected cells

21. Autophagy Cell eats its own contents
Survival mechanism in times of nutrient deprivation.
Autophagolysosome = autophagic vacuole + lysosome
Mechanism of cell loss in : degenerative disorders of CNS.

22. Intracellular Accumulations
2 main pathways of abnormal intracellular accumulations
Inadequate removal of normal substance (defects in packaging and transport) FATTY LIVER
Failure to degrade a metabolite (inherited enzyme deficiencies) STORAGE DISORDERS
Based on duration - Transient/ permanent
Based on Location - Cytoplasm/ nucleus

23. Mechanism of Intracellular accumulation
A normal endogenous substance is produced at a normal or increased rate, but the rate of metabolism is inadequate to remove it ex: Fatty change in the liver
Defects in protein folding and transport and an inability to degrade the abnormal protein efficiently ex: Accumulation of mutated alpha 1- antitrypsin in liver cells.
Inherited defects of enzymes required for metabolism of a particular substance ex: Lysosomal storage disorders
Exogenous substance ex: Carbon particles, Silica

24. Abnormal metabolism, as in fatty change in the liver
Mutations causing alterations in protein folding and transport, so that defective molecules accumulate intracellularly

25. (3) Deficiency of critical enzymes for breaking down certain compounds, causes substrates to accumulate in lysosomes (lysosomal storage diseases)
(4) An inability to degrade phagocytosed particles, as in carbon pigment accumulation

26. Intracellular Accumulations
Fatty Change (Steatosis) - mainly in the liver
Cholesterol and Cholesteryl Esters - Atherosclerosis
Proteins - Mallory hyaline, Russell bodies, Neurofibrillary tangles
Glycogen
Pigments
Carbon (Anthracosis)
Lipofuscin (Brown atrophy)
Melanin
Hemosiderin

27. Steatosis ( Fatty change)
Abnormal accumulation of triglycerides within parenchymal cells
Most common cause of fatty change in the liver in developed nations – alcohol abuse & non alcoholic fatty liver disease(NAFLD/NASH) is associated with malnutrition, obesity, diabetes.
Seen in liver and the heart commonly
Clear vacuoles within parenchymal cells
Stains: Sudan IV / Oil red O – Orange red color

28. Steatosis Morphology Gross – enlarged bright yellow soft greasy
Histological features :
Small vacuoles in the cytoplasm around the nucleus
Vacuoles coalesce
Clear spaces with Nucleus displaced to periphery
Fatty cysts – enclosed fat globules coalesce after rupture of contiguous cells

30. Cholesterol and Cholesterol Esters
Atherosclerosis: In atherosclerotic plaques, smooth muscle cells and macrophages are filled with lipid vacuoles, made up of cholesterol and cholesterol esters
Xanthomas: Clusters of foamy cells in sub-epithelial connective tissue of skin and in tendons, producing masses
Cholesterolosis: focal accumulations of cholesterol-laden macrophages in the lamina propria of the gallbladder
Niemann-Pick disease, type C: mutations affecting an enzyme involved in cholesterol trafficking resulting in cholesterol accumulation in multiple organs

31. A: Achilles tendon xanthoma - slightly yellow nodular lesions at the distal end of the Achilles.
B: Xanthelasma - Yellow, raised lesions on the lower left eyelid
C: Palmar xanthomas - yellow macules on the palm accentuated in the creases
D: Eruptive xanthomas - numerous small yellow papular lesions distributed over the buttocks

32. Proteins
Reabsorption droplets in proximal renal tubules – in renal diseases associated with proteinuria
Normal secreted proteins produced in excessive amounts – Russell bodies(abnormal immunoglobulins)
Defective intracellular transport and secretion of critical proteins - α1-antitrypsin deficiency
Accumulation of cytoskeletal proteins – Mallory Hyaline(keratin intermediate filaments), Nuerofibrillary tangles(Alzeimer’s)
Aggregation of abnormal proteins - Amyloidosis

33. Bone marrow core biopsy showing numerous plasma cells, some with eccentric nuclei and round cherry red inclusions (Russell bodies)

34. Mallory's hyaline or "alcoholic" hyaline – globular red hyaline material within hepatocytes. The globules are aggregates of intermediate filaments in the cytoplasm resulting from hepatocyte injury

35. Neurofibrillary "tangle" of Alzheimer's disease

36. Hyaline Change Descriptive histologic term
An alteration within cells or in the extracellular space that gives a homogeneous, glassy, pink appearance in routine histologic sections stained with hematoxylin and eosin
In long-standing hypertension and diabetes mellitus, the walls of arterioles, especially in the kidney, show hyaline change

37. Accumulation of Glycogen
Readily available energy source stored in cytoplasm of healthy cells
Excessive intracellular deposits seen in patients with genetic disorders collectively referred to as the “glycogen storage diseases”, or “glycogenoses”
Rose-to-violet color with PAS stain

38. Pigments Exogenous Pigments Endogenous Pigments
Anthracosis: carbon (coal dust) in lungs and lymph nodes
Tattooing
Endogenous Pigments
Lipofuscin ( wear and tear pigment, Brown Atrophy)
Melanin ( Melanoma, Addison Disease)
Homogentisic acid, a black pigment accumulates in patients with alkaptonuria (Oochronosis)
Hemosiderin ( Hemosiderosis, Heart failure cells)
Bilirubin (Jaundice, kernicterus

40. Pathologic calcification
Abnormal tissue deposition of calcium salts, smaller amounts of iron, magnesium & other mineral salts
2 types: Dystrophic calcification
Metastatic calcification

41. Dystrophic calcification
Normal serum levels of calcium
Normal calcium metabolism
Deposition of calcium in dying tissues
Encountered in areas of necrosis
Seen in aging or damaged heart valves
Atheromas of advanced atherosclerosis
Dystrophic calcification of the aortic valve. View looking down onto the unopened aortic valve in a heart with calcific aortic stenosis. It is markedly narrowed (stenosis). The semilunar cusps are thickened and fibrotic, and behind each cusp are irregular masses of piled-up dystrophic calcification

42. Dystrophic calcification
Pathogenesis : Calcium enters the necrotic cells and binds to phosphate producing calcium phosphate, which is basophilic in the hematoxylin-eosin stain
Gross : Fine, white granules or clumps, felt as gritty stones
Histological appearance: basophilic amorphous, granular, sometimes clumped
Calcification in areas of caseous necrosis in tuberculosis
Calcified atherosclerotic plaques
Calcification of aortic valves in elderly
Calcification in chronic pancreatitis
Periventricular calcification in congenital cytomegalovirus infection
Psammoma bodies (Papillary cancer of thyroid, Meningioma)

43. Metastatic Calcification
In normal tissues when there is HYPERCALCEMIA
Causes of hypercalcemia:
Hyperparathyroidism
Destruction of bone tissue
Vitamin D–related disorders
Renal failure
Aluminum intoxication
Milk-alkali syndrome.

44. Metastatic Calcification
Principally affects the interstitial tissues of the
gastric mucosa
kidneys,
lungs,
systemic arteries,
and pulmonary veins
Noncrystalline amorphous deposits or as hydroxyapatite crystals