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Comprehensive Notes on 6 Pathology Topics

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1. Epigenetics

• Robbins Pathologic Basis of Disease: "Epigenetics — heritable changes in gene expression that are not caused by variations in DNA sequence."

Mechanisms of Epigenetic Regulation

A. Histone Modifications

• Euchromatin - loosely wound, transcriptionally active
• Heterochromatin - densely packed, transcriptionally silent

B. DNA Methylation

• Addition of a methyl group to cytosine residues, predominantly at CpG dinucleotides (CpG islands)
• High methylation at gene regulatory regions → transcriptional silencing
• Regulated by DNA methyltransferases (DNMTs), demethylating enzymes, and methyl-CpG binding proteins
• Promoter hypermethylation silences tumor suppressor genes in many cancers

C. MicroRNAs (miRNAs) and Non-coding RNAs

• miRNAs: ~22 nucleotide single-stranded RNAs that do NOT encode proteins
• Processed from primary transcripts (pri-miRNA) → pre-miRNA → mature miRNA by the enzyme Dicer
• Associate with RISC (RNA-Induced Silencing Complex)
• Base pairing with target mRNA → mRNA cleavage OR translational repression (post-transcriptional silencing)
• ~6,000 miRNA genes in the human genome; each miRNA can regulate multiple protein-coding genes
• lncRNAs (long non-coding RNAs, >200 nt): regulate chromatin structure, transcription, and post-transcriptional processing

Disease Relevance

• Cancer: Promoter hypermethylation silences tumor suppressors; histone deacetylation compacts DNA around oncosuppressors. HDAC inhibitors and DNA methylation inhibitors are clinically used
• Monozygotic twins: Epigenetics explains phenotypic divergence despite identical DNA sequences
• Imprinting: Differential parental methylation (e.g., Prader-Willi, Angelman syndromes)
• X-inactivation (lyonization): One X chromosome silenced via epigenetic mechanisms in females
• Transgenerational inheritance: Some epigenetic marks can be transmitted across generations

Source: Robbins Pathologic Basis of Disease, Chapter 1 (Histone Organization, p.20)

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2. Retrogressive Changes

A. Atrophy

B. Degenerations

1. Cellular Swelling (Hydropic/Vacuolar Degeneration)

• Earliest and most common reaction to injury
• Failure of Na+/K+ ATPase pump → Na+ and water enter cell → cytoplasmic vacuolation
• Grossly: organ pale, enlarged, heavy
• Microscopically: cytoplasmic clearing, small vacuoles, may progress to "ballooning degeneration"

2. Fatty Change (Steatosis)

• Accumulation of lipid droplets in parenchymal cells (especially hepatocytes and cardiac myocytes)
• Causes: alcoholism, malnutrition, diabetes, toxins (CCl4), obesity, anoxia
• Mechanisms: excess fatty acid delivery, defective oxidation, decreased apoprotein synthesis, impaired lipoprotein export
• Liver: enlarged, yellow, greasy ("foie gras" appearance); microscopically - small vacuoles coalescing to large vacuoles displacing nucleus ("signet ring" in severe cases)
• Heart (tigroid/tabby heart): alternating bands of pale (fatty) and normal myocardium

3. Mucoid (Myxomatous) Degeneration

• Accumulation of mucopolysaccharides (glycosaminoglycans) in connective tissue
• Example: myxoid degeneration of heart valves (mitral valve prolapse), myxoma

4. Hyaline Change (Hyalinization)

• Non-specific term for any homogeneous, glassy, eosinophilic intracellular or extracellular material on H&E stain
• Intracellular hyaline: Mallory-Denk bodies (alcoholic liver disease), Russell bodies (plasma cells), Lewy bodies (Parkinson's), Councilman bodies (viral hepatitis)
• Extracellular hyaline: hyalinized collagen, amyloid deposits, hyaline arteriosclerosis

5. Amyloid Degeneration

• Extracellular deposit of abnormal fibrillar proteins with a beta-pleated sheet configuration
• Congo red stain → apple-green birefringence under polarized light
• Types: AA (reactive/secondary), AL (myeloma-related), Abeta (Alzheimer's), transthyretin (TTR), etc.

6. Calcification

• Occurs in dead or dying tissue
• Serum calcium normal
• Examples: necrotic foci (TB), atherosclerotic plaques, damaged heart valves, tumors (psammoma bodies in papillary thyroid/ovarian carcinoma/meningioma/mesothelioma)

• Occurs in normal tissue due to hypercalcemia
• Causes: hyperparathyroidism, vitamin D toxicity, milk-alkali syndrome, multiple myeloma, widespread bone metastases
• Sites: kidneys (nephrocalcinosis), lungs, gastric mucosa, blood vessels, cornea

7. Pigmentary Changes

• Lipofuscin (wear-and-tear pigment): yellowish-brown, perinuclear, lysosomal residue; seen in heart and liver in aging and atrophy ("brown atrophy")
• Melanin accumulation/depletion

C. Necrosis

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3. Stress Proteins (Heat Shock Proteins) in Cell Injury

Definition and Discovery

Classification of HSPs (7 Major Categories)

Induction Mechanisms

1. Under normal conditions, HSF-1 is bound to HSP90 in an inactive monomer form
2. Stress → accumulation of misfolded/unfolded proteins → HSP90 sequestered to misfolded proteins
3. Free HSF-1 trimerizes → translocates to nucleus → binds Heat Shock Elements (HSEs) in promoters → transcribes HSP genes
4. After stress resolution, newly synthesized HSPs bind and inactivate HSF-1 (negative feedback)

The Three Major Stress Response Systems (Harrison's)

1. Heat Shock Response (HSR): Cytoplasm/nucleus; regulated by HSF-1; induced by proteotoxic, thermal, oxidative stress
2. Unfolded Protein Response (UPR) in ER (UPRER): Induced by ER stress → sensors IRE1, PERK, ATF6 → reduce protein synthesis + increase folding capacity; prolonged/severe UPR triggers apoptosis
3. Mitochondrial UPR (UPRmt): Regulated by ATF5 in mammals; responds to mitochondrial stress; upregulates mitochondrial chaperones and metabolic genes

Functions of Stress Proteins in Cell Injury

1. Molecular chaperones - HSP70/HSP90: Recognize unfolded proteins; facilitate proper folding or direct them to degradation pathways; prevent pathological protein aggregation
2. Proteolytic function - Some HSPs tag irreversibly damaged proteins; ubiquitin marks proteins for 26S proteasome degradation (UPS pathway)
3. DNA and RNA repair - Protect genetic material from stress-induced damage
4. Metabolic enzyme restoration - Re-establish metabolic pathways after heat stress
5. Regulatory proteins - Cell cycle control, transcriptional regulation
6. Cytoskeletal support - Small HSPs (HSP27) stabilize actin filaments; prevent cytoskeletal disruption
7. Transport, detoxification, membrane modulation - 7th category

Autophagy-Lysosomal Pathway (ALP)

CHIP (Carboxyl Terminus of Hsc70 Interacting Protein)

• Client refolding (via HSP70/HSP90 complex)
• Proteasome-mediated degradation (via ubiquitination)

Cytoprotective vs. Pathological Roles

• Cytoprotective: Preconditioning (brief stress makes cells more tolerant of subsequent lethal stress; "hormesis")
• Pathological: HSP dysregulation in aging → accumulation of misfolded proteins → neurodegeneration (Alzheimer's: Abeta; Parkinson's: alpha-synuclein; Huntington's: polyQ aggregates)
• Cancer: HSPs overexpressed in tumors → promote survival, resist therapy → targets for anti-cancer drugs (e.g., HSP90 inhibitors like geldanamycin)
• Inflammation: HSPs released extracellularly act as DAMPs (damage-associated molecular patterns) → activate innate immunity

Sources: Harrison's Principles of Internal Medicine 22E, Chapter 504; Miller's Anesthesia 10e, Chapter 29

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4. Hemoprotein-Derived Pigments

A. Hemoglobin Breakdown Pathway

Hemoglobin (Fe²⁺-porphyrin)
        ↓ (RBC destruction by macrophages in spleen/liver/bone marrow)
Globin (recycled as amino acids) + Heme
        ↓ (Heme oxygenase)
Biliverdin (green) + CO + Fe³⁺
        ↓ (Biliverdin reductase)
Bilirubin (yellow-orange, unconjugated/indirect)
        ↓ (Hepatocyte uptake and conjugation with glucuronate)
Conjugated bilirubin (direct bilirubin, water-soluble)
        ↓ (Secreted into bile)
Urobilinogen (in gut, by bacterial action) → Urobilin (urine - yellow)
                                          → Stercobilin (feces - brown)

B. Hemosiderin

• Storage form of iron within cells
• Derived from ferritin aggregates; golden-brown granular pigment
• Composed of aggregated ferritin molecules + denatured protein + lipid
• Prussian blue reaction (Perls' stain): positive (blue-green)
• Normally found in small amounts in macrophages of spleen, liver, bone marrow

• Local: hemorrhage → macrophages ("heart failure cells" in pulmonary congestion contain hemosiderin from extravasated RBCs)
• Systemic: hemolytic anemia, multiple transfusions

• Primary (hereditary): HFE gene mutation → excessive intestinal iron absorption
• Secondary: repeated transfusions, ineffective erythropoiesis
• Deposits in liver (cirrhosis), pancreas ("bronze diabetes"), heart (restrictive cardiomyopathy), joints, skin (bronze pigmentation), gonads (hypogonadism)
• "Cirrhosis + Diabetes + Skin bronzing" = classic triad

C. Bilirubin and Jaundice

• Lipid-soluble, albumin-bound in blood
• Cannot be excreted in urine (absent in urine = acholuric jaundice)
• Toxic to brain (kernicterus in neonates)

• Water-soluble; excreted in urine when elevated (tea-colored/dark urine)
• Conjugated with 2 molecules of glucuronic acid by UDP-glucuronosyltransferase in hepatocytes

D. Hematoidin

• Chemically identical to bilirubin; formed within tissues during hematoma resolution
• Formed at sites of old hemorrhage in relatively avascular areas (no macrophages)
• Iron-FREE pigment (unlike hemosiderin)
• Appears as orange-yellow rhomboid crystals extracellularly
• Negative Prussian blue stain

E. Malarial Pigment (Hemozoin)

• Brown-black, crystalline, insoluble complex formed by Plasmodium species
• Parasite digests hemoglobin in RBCs; heme is polymerized to hemozoin (biocrystallization as a detoxification mechanism)
• Accumulates in macrophages, spleen, liver, brain (in cerebral malaria)
• Negative iron stain; birefringent under polarized light

F. Porphyrins

• Precursors in heme synthesis pathway
• Accumulate in porphyrias (enzyme deficiencies in heme synthesis)
• Photosensitivity (porphyrins absorb light → reactive oxygen species → skin damage)
• Uroporphyrin (type I) in urine → port-wine colored urine in acute porphyrias

Summary Table

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5. Role of Autopsy in Diagnosis of Disease

Definition

Types of Autopsy

1. Medicolegal (Forensic) Autopsy
   • Ordered by legal authority (coroner/medical examiner)
   • Purpose: determine cause of death in unnatural, violent, suspicious, sudden, or unexplained deaths
   • Does not require family consent
2. Clinical (Hospital/Pathological) Autopsy
   • Performed with family consent
   • Purpose: correlate clinical findings with pathological findings; assess accuracy of clinical diagnosis and treatment
   • Has declined in frequency but remains educationally and scientifically valuable
3. Partial Autopsy
   • Restricted examination (e.g., cranial only, abdominal only) at family request

Diagnostic Roles of Autopsy

A. Establishing Cause of Death

• Definitive determination when cause is unclear clinically
• Identification of lethal lesions (massive MI, pulmonary embolism, ruptured aortic aneurysm, subarachnoid hemorrhage)

B. Quality Assurance and Clinical Audit

• Studies consistently show 20-30% discordance between clinical and autopsy diagnoses
• Major discrepancies: unsuspected MI, pulmonary embolism, aortic dissection, sepsis, malignancy
• Essential for hospital quality improvement programs
• Identifies errors in clinical management

C. Diagnosis of Infectious Disease

• Identifies unsuspected infections (tuberculosis, fungal infections, HIV-related opportunistic infections)
• Important during epidemics and pandemics (COVID-19 autopsy studies revealed diffuse alveolar damage, microvascular thrombi)
• Allows microbiological sampling (culture, PCR on fixed tissue)

D. Diagnosis of Genetic/Hereditary Conditions

• Confirms storage diseases (Gaucher's, Niemann-Pick), metabolic disorders
• SIDS (Sudden Infant Death Syndrome) investigation
• Enables genetic counseling for families when inherited disease is confirmed

E. Cancer Diagnosis and Staging

• Confirms primary site of unknown primary malignancy
• Documents metastatic spread; assists staging accuracy
• Identifies treatment-related changes

F. Research and Education

• Major source of tissue for medical research
• Training platform for pathology residents and medical students
• Understanding of natural history of disease

G. Forensic/Medicolegal Significance

• Establishes manner of death (natural, accident, homicide, suicide, undetermined)
• Documents injuries (blunt, sharp, gunshot wound trajectory)
• Collects toxicological samples (blood, vitreous humor, urine, liver)
• Evidence in legal proceedings
• Detection of iatrogenic complications

H. Public Health

• Disease surveillance; identification of new/emerging diseases
• Occupational disease identification (asbestosis, silicosis, mesothelioma)
• Epidemiological data collection

Components of an Autopsy

1. External examination: Body habitus, skin, injuries, surgical scars, decomposition
2. Internal examination (in situ): Systematic opening of body cavities
3. Organ dissection: Systematic examination, weighing, and sectioning of organs
4. Histopathology: Microscopic examination of sampled tissues
5. Special investigations: Microbiology, toxicology, virology, molecular studies, electron microscopy
6. Documentation: Photographs, diagrams, written report

Limitations and Declining Rates

• Autopsy rates have declined from ~50% (mid-20th century) to <10% in many countries
• Causes of decline: clinical confidence in imaging/labs, family reluctance, cost, religious/cultural objections, medicolegal concerns
• Professional bodies (CAP, Royal College of Pathologists) advocate for maintaining autopsy rates

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6. The Aging Process

Definition

Theories of Aging

A. Molecular/Genetic Theories

• Telomeres = repetitive DNA sequences (TTAGGG)n at chromosomal ends that protect chromosomes from degradation and end-to-end fusion
• Each mitotic division → 50-100 bp loss (due to inability of DNA polymerase to replicate extreme 5' end - "end replication problem")
• After ~50-70 divisions (Hayflick limit), telomeres become critically short → trigger senescence signals (p53/Rb pathways) or apoptosis
• Telomerase (reverse transcriptase that adds telomere repeats) is active in: stem cells, germ cells, most cancers (immortalization); absent in most somatic cells
• Werner syndrome (premature aging) and dyskeratosis congenita: defective telomere maintenance

• Reactive oxygen species (ROS), UV radiation, ionizing radiation, chemical mutagens
• DNA double-strand breaks accumulate; repair mechanisms become less efficient
• Critical genes (tumor suppressors, metabolic genes) are inactivated
• Progeria (Hutchinson-Gilford): LMNA mutation → nuclear lamina instability → impaired DNA repair → accelerated aging

• Systematic changes in DNA methylation patterns with aging ("epigenetic clock"; Horvath clock)
• Altered expression of longevity genes (sirtuins, FOXO, mTOR pathway)
• Caloric restriction → activates SIRT1 (deacetylase) → longevity in multiple organisms

• Harman (1956): ROS produced as byproducts of mitochondrial oxidative phosphorylation damage lipids, proteins, and DNA
• Mitochondrial DNA (mtDNA) especially vulnerable (no histones; close to ROS source)
• Accumulation of mitochondrial mutations → reduced energy production → cellular dysfunction
• "Vicious cycle": mitochondrial damage → more ROS → more damage

B. Cellular Theories

• Senescent cells are metabolically active but permanently arrested (G1 phase)
• Cannot be stimulated to divide by growth factors
• Express p16INK4a, p21, p53; β-galactosidase at pH 6.0 (senescence marker)
• Develop SASP (Senescence-Associated Secretory Phenotype): secrete pro-inflammatory cytokines (IL-6, IL-8, MMP), promoting inflammation and tissue dysfunction
• Initially protective (tumor suppressor mechanism; prevents propagation of damaged cells)
• With age, accumulation of senescent cells in tissues → "inflammaging"

• Reduced number and regenerative capacity of adult stem cells
• Impaired tissue homeostasis and repair
• Hematopoietic stem cell dysfunction → immunosenescence

• Decreased efficiency of HSPs, UPS, and autophagy with aging
• Accumulation of misfolded and aggregated proteins
• Contributes to neurodegeneration (Alzheimer's, Parkinson's)

C. Organismal Theories

Hallmarks of Aging (Lopez-Otin et al., 2013/2023)

1. Genomic instability
2. Telomere attrition
3. Epigenetic alterations
4. Loss of proteostasis
5. Deregulated nutrient-sensing (mTOR, AMPK, IGF-1 signaling)
6. Mitochondrial dysfunction
7. Cellular senescence
8. Stem cell exhaustion
9. Altered intercellular communication (inflammaging)
10. (2023 update adds): Disabled macroautophagy, chronic inflammation, dysbiosis

Cellular Changes in Aging

• Accumulation of lipofuscin (aging pigment) in heart, liver, neurons - "brown atrophy"
• Increased vacuolization of cells
• Nuclear irregularities (pyknosis, increased inclusions)
• Mitochondrial pleomorphism and reduced number
• Reduced ribosomal density

Systemic Effects of Aging

Longevity Pathways (Therapeutic Targets)

• mTOR inhibition (rapamycin): extends lifespan in multiple organisms
• Caloric restriction / CR mimetics (metformin, resveratrol)
• Senolytics (drugs that clear senescent cells): dasatinib + quercetin; navitoclax
• NAD+ boosters (NMN, NR): activate sirtuins
• Telomerase activation: experimental

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