Immune Response — Guyton and Hall Textbook of Medical Physiology

1. Innate vs. Acquired (Adaptive) Immunity

• Innate immunity — non-specific, present from birth; includes skin barriers, phagocytic cells (neutrophils, macrophages), complement, and natural killer (NK) cells.
• Acquired (adaptive) immunity — develops after exposure to a specific invader; highly specific and forms the basis of immunological memory and vaccination.

2. Two Types of Acquired Immunity

3. Antigens

• Antigens are the chemical substances (usually proteins or large polysaccharides, MW ≥ 8,000 Da) that initiate acquired immunity.
• They bear repeating surface groups called epitopes that interact with antigen-specific receptors on lymphocytes.
• Almost any large foreign molecule can be antigenic.

4. Lymphocytes — Central to Acquired Immunity

• Lymph nodes (most abundantly)
• Spleen, thymus, bone marrow
• Submucosal areas of the GI tract (GALT)
• Tonsils and adenoids (intercept upper respiratory antigens)

Origin of B and T Lymphocytes

• Both arise from pluripotent hematopoietic stem cells in the bone marrow.
• T lymphocytes are preprocessed in the thymus (hence "T") → gain antigen specificity and self-tolerance.
• B lymphocytes are preprocessed in the bone marrow (and fetal liver) → gain the ability to form antibodies.
• After preprocessing, millions of B and T cells with different antigen specificities seed the lymphoid tissues of the body.

5. Humoral Immunity — The B-Lymphocyte System

Clonal Selection and Activation

1. The matching B cell proliferates rapidly → clonal expansion
2. Most daughter cells differentiate into plasma cells, which secrete large quantities of antibody
3. Some become memory B cells for a faster secondary response

Antibodies (Immunoglobulins)

• Two heavy chains + two light chains (connected by disulfide bonds)
• Two Fab regions (antigen-binding)
• One Fc region (activates complement or binds phagocyte receptors)

Mechanisms by Which Antibodies Attack Antigens

1. Agglutination — cross-links particulate antigens into clumps
2. Precipitation — cross-links soluble antigens until they become insoluble
3. Neutralization — covers the toxic or infective sites of antigens
4. Lysis — direct rupture of bacterial/cellular membranes (via complement)
5. Opsonization — coating pathogens with antibody (and C3b) to enhance phagocytosis
6. Activation of complement — triggering the entire complement cascade

Primary vs. Secondary Response

• Primary: Latent period of several days before antibodies appear; peak at ~1–2 weeks; mainly IgM
• Secondary (anamnestic): Starts within hours; far higher antibody titers; mainly IgG; prolonged persistence — basis of immunological memory and vaccination

6. Cell-Mediated Immunity — The T-Lymphocyte System

T-Cell Activation

Major T-Cell Subsets

How T Cells Kill

• Direct cell lysis — CTLs attach to target cell via TCR-MHC interaction and release perforin/granzymes → apoptosis
• Lymphokines — soluble signals secreted by T-helper cells that amplify the immune response:
  • Interleukins (IL-2 and others) — stimulate T and B cell proliferation
  • Macrophage migration inhibition factor — keeps macrophages at infection site
  • Macrophage-activating factor — boosts phagocytic power of macrophages
  • Lymphotoxin (TNF-β) — directly kills cells
  • Chemotactic factor — attracts macrophages
  • Interferon — antiviral protection

7. Tolerance — Preventing Autoimmunity

• Central tolerance in the thymus (T cells): T cells that react strongly to self-antigens presented by thymic cells undergo clonal deletion (apoptosis) during development.
• Central tolerance in bone marrow (B cells): Self-reactive B cells are also clonally deleted.
• Peripheral tolerance: Anergy and Treg cells suppress any self-reactive lymphocytes that escape central deletion.

8. Immunization

• Active immunization (vaccination): Injecting antigens (killed organisms, attenuated organisms, or toxoids) to generate primary immunity and memory; protects against subsequent real infection.
• Passive immunization: Injecting pre-formed antibodies (immunoglobulins) → immediate but transient protection (no memory cells formed). Example: tetanus immunoglobulin after wound exposure.

9. Allergies and Hypersensitivity (Type I — Anaphylactic)

• Antigen bridges two IgE molecules → mast cell degranulation
• Release of histamine, leukotrienes (slow-reacting substance of anaphylaxis), bradykinin, prostaglandins
• Clinical manifestations:
  • Anaphylaxis — systemic vasodilation, circulatory shock
  • Urticaria (hives) — localized skin histamine release
  • Hay fever — nasal mucosal swelling and hypersecretion
  • Asthma — bronchiolar smooth muscle spasm (mainly from leukotrienes)

10. Sex Differences in Immunity

• Females generally mount stronger innate and adaptive immune responses than males (influenced by sex chromosomes and estrogen/testosterone)
• Greater interferon production, T-cell expansion, and antibody responses in females
• This confers better vaccine responses but also explains why ~80% of autoimmune disease patients are female (e.g., SLE affects women ~10× more than men)

11. Immune Reactions in Transplantation

• Transplanted tissue expresses foreign MHC (HLA) antigens
• Host T cells recognize these → rejection (primarily cell-mediated)
• Prevention strategies: HLA matching, immunosuppressive drugs (corticosteroids, cyclosporine, tacrolimus, azathioprine), sometimes thymic irradiation

Routes of Drug Administration

Overview

• Rate and extent of absorption (bioavailability, F)
• Onset and duration of drug action
• Whether first-pass metabolism occurs
• Suitability for the drug's physicochemical properties

1. Enteral Routes (via GI Tract)

Oral (PO)

• Most common, safest, most convenient, most economical
• Absorption governed by: surface area, blood flow, drug solubility, ionization state, GI motility
• Most absorption occurs in the upper small intestine (large surface area ~200 m²) via passive diffusion — even if the drug is ionized there, the surface area advantage dominates over the stomach
• Disadvantages:
  • Erratic/incomplete bioavailability (0.05 < F < 1)
  • Destruction by gastric acid or digestive enzymes
  • First-pass effect — drug absorbed from GI tract passes through the liver before reaching systemic circulation; extensive hepatic/intestinal metabolism can drastically reduce bioavailability (e.g., morphine, nitroglycerin, lidocaine)
  • Requires patient cooperation
  • Drug interactions with food, other drugs, gut microbiome (>1,000 species)
  • Emesis from GI irritation

• Recumbent right-side position speeds emptying
• Caloric content, food volume, osmolality slow emptying
• Estrogen slows emptying in premenopausal women

• Slow, uniform absorption over 8+ hours
• Benefits: reduced dosing frequency, better compliance, reduced peak-trough fluctuations
• Most suitable for drugs with short t½ (<4 h)

Sublingual (SL)

• Absorbed through oral mucosa directly into systemic venous drainage, bypassing first-pass metabolism
• Rapid onset (e.g., nitroglycerin — onset ~2 min)
• Ideal for drugs with high first-pass extraction orally
• Small surface area limits use to highly potent, lipid-soluble drugs

Rectal (PR)

• ~50% of rectal venous drainage bypasses portal circulation → partial avoidance of first-pass effect
• Useful when oral route unavailable (unconscious patient, vomiting, pediatrics)
• Absorption often irregular and incomplete
• Can cause rectal irritation
• Examples: diazepam rectal gel (seizures), suppository antiemetics, analgesics

2. Parenteral Routes (bypassing GI tract)

Intravenous (IV)

• F = 1 (100% bioavailability by definition)
• Immediate onset — useful in emergencies
• Allows precise titration of dose
• Can deliver large volumes, irritating substances (when diluted), and high-molecular-weight proteins/peptides (e.g., biologics, insulin)
• Disadvantages:
  • Highest risk of adverse effects (no recall once injected)
  • Must inject slowly in most cases
  • Not suitable for oily solutions or poorly soluble substances
  • Risk of infection, phlebitis, air embolism

Intramuscular (IM)

• F: 0.75–1
• Prompt absorption from aqueous solutions; slow/sustained from repository (depot) preparations (e.g., haloperidol decanoate, medroxyprogesterone)
• Can handle moderate volumes; suitable for oily vehicles and some irritating substances
• Appropriate for self-administration (e.g., insulin, epinephrine autoinjector)
• Disadvantages:
  • Pain or necrosis from irritating substances
  • Contraindicated during anticoagulant therapy (risk of hematoma)
  • Can elevate creatine kinase (confounds cardiac enzyme interpretation)

Subcutaneous (SC/SQ)

• F: 0.75–1
• Prompt from aqueous solutions; slow/sustained from implants or repository preparations
• Suitable for poorly soluble suspensions (e.g., insulin suspensions) and slow-release implants (e.g., etonogestrel contraceptive implant)
• Not suitable for large volumes
• Less risk of vascular injury than IM

Intra-arterial

• Delivers drug to specific tissue/organ with high local concentration before systemic dilution
• Used in cancer chemotherapy (e.g., hepatic artery infusion) and diagnostic contrast injection
• Requires great caution — inadvertent injection can cause severe ischemia

Intrathecal / Epidural

• Direct delivery to the cerebrospinal fluid (CSF) or epidural space, bypassing the blood-brain barrier (BBB)
• Used for: spinal anesthesia, intrathecal chemotherapy (e.g., methotrexate), opioid analgesia, baclofen pump
• Allows lower doses with reduced systemic effects

Intraosseous (IO)

• Emergency vascular access into bone marrow cavity when IV access unavailable
• Comparable drug kinetics to IV; used in pediatric resuscitation and adult emergencies

3. Topical / Local Routes

Transdermal

• Systemic absorption through skin; bypasses first-pass metabolism
• Rate-limited by skin permeability
• Suitable for lipid-soluble drugs with low required doses
• Provides sustained, steady plasma levels (e.g., fentanyl patch, nicotine patch, nitroglycerin patch, estradiol patch, scopolamine)
• Patches: controlled by membrane or drug-in-adhesive systems

Inhalation

• Rapid absorption via large alveolar surface area (~70 m²) and rich blood supply
• Near-immediate systemic effect (e.g., volatile anesthetics, inhaled bronchodilators)
• Local pulmonary delivery with inhaled corticosteroids (ICS) reduces systemic side effects
• Requires proper inhaler technique; spacer devices improve lung deposition and reduce oropharyngeal deposition
• Adverse effects of ICS: hoarseness, oropharyngeal candidiasis → rinse mouth after use

Topical (Skin/Mucous Membranes)

• Applied locally for local effect (e.g., dermatological creams, eye drops, nasal sprays)
• Minimal systemic absorption desired; however, systemic toxicity possible with overuse (e.g., topical corticosteroids)

Nasal (Intranasal)

• Can be used for local effects (decongestants, corticosteroids) or systemic delivery (e.g., intranasal desmopressin, fentanyl, sumatriptan)
• Avoids first-pass metabolism
• Rapid absorption via nasal mucosa

Ocular, Otic, Vaginal, Urethral

• Primarily local; systemic absorption may occur (e.g., timolol eye drops → bradycardia)

4. Summary Comparison Table

Spectrophotometer

Definition

Basic Optical Principle

• A fraction is absorbed by the solute
• The remainder is transmitted as Iₛ

T = Iₛ / I₀

A = −log T

Beer-Lambert Law

A = a · b · c

• Beer's law holds only over a linear range — calibrators must not exceed this range
• The law is valid at a single, fixed wavelength; polychromatic light causes deviation
• Best measurement at λ_max (wavelength of maximum absorption) for greatest sensitivity

Components of a Spectrophotometer

1. Radiant Energy Source (Light Source)

• Continuum sources (emit radiation continuously over a range):
  • Tungsten/tungsten-halogen lamp — visible region (380–780 nm); halogen gas prevents filament vaporization, giving brighter/whiter light
  • Deuterium lamp — UV region (~190–380 nm); ~100 V applied excites atoms to emit UV photons
  • Xenon arc lamp — covers both UV and visible; high-pressure discharge; pulsed output
• Line sources (emit discrete wavelengths):
  • Mercury/sodium vapor lamps — sharp lines in UV/visible
  • Hollow cathode lamp — used in atomic absorption spectroscopy; metal-specific
  • Laser — extremely narrow bandwidth, high intensity; used in high-resolution spectroscopy, nephelometry, MALDI-MS

2. Wavelength Selector (Monochromator)

• Filters (simple photometers):
  • Absorption filters — broad bandpass; transmit a range of wavelengths
  • Interference filters — narrow bandpass (~2–10 nm); preferred for greater specificity
• Prisms — disperse light by refraction; less commonly used
• Diffraction gratings — disperse light by diffraction; most widely used in modern spectrophotometers; provide uniform dispersion across the spectrum
• Holographic gratings — laser-etched, superior to ruled gratings; minimal stray light

3. Sample Holder (Cuvette)

• Holds the solution to be measured
• Must be optically clear at the wavelength used:
  • Glass — suitable for visible light
  • Quartz/fused silica — required for UV measurements
• Path length is standardized at 1 cm for most clinical work

4. Photodetector

• Photomultiplier tube (PMT) — most sensitive and widely used:
  • Contains a photocathode + 10–15 dynodes in a vacuum tube
  • Photons hit photocathode → emit electrons → cascade amplification at dynodes → 10⁵ to 10⁷ electrons per photon
  • ~1,500 V applied; extremely rapid response; sensitive to stray light (must be shielded)
  • Dark current = residual current in the absence of light (noise)
• Photodiodes — solid-state (silicon, GaAs, etc.); detect 250–1,100 nm; more durable
• Photodiode array (PDA) — multiple diodes detecting different wavelengths simultaneously; allows full-spectrum capture in one measurement (1–2 nm resolution per diode)
• Charge-coupled device (CCD) and charge-injection device (CID) — two-dimensional arrays; read entire wavelength range at once; CCD reads row-by-row destructively; CID reads each pixel independently and non-destructively

5. Signal Processor

• Amplifies, rectifies, filters, and mathematically transforms the electrical signal
• Performs operations: log conversion (T → A), integration, differentiation

6. Readout Device

• Digital meters, LED displays, LCD screens, X-Y recorders, computer interfaces

Single-Beam vs. Double-Beam Configuration

Spectrophotometry Nomenclature (Tietz Table)

Types of Photometric Instruments (Spectrum of Complexity)

Quality Assurance in Spectrophotometry

1. Wavelength accuracy — verified using didymium glass (broad peak ~600 nm) or holmium oxide (sharp peak at 360 nm)
2. Photometric (absorbance) accuracy — measured using glass filters or solutions of known absorbance
3. Linearity — ability to yield a linear A vs. concentration relationship (Beer's law range); evaluated by slope and intercept
4. Stray light — any light reaching the detector that does not originate from the monochromator; assessed using cutoff filters; causes positive error at high absorbances

Sources of Error / Deviations from Beer's Law