Blood and Immune

• the simplest and most successful form of life on earth

• consists of DNA/RNA genome, lipid and protein coat

• viruses cannot replicate themselves so must invade cells to multiply

• billions of different viruses — infects all living things from bacteria to humans

• intracellular pathogens

  • they need the cellular machinery (ribosomes and protein synthesis) so they need a way of getting inside a cell

outline viruses

• bacteria are prokaryotes — no nucleus or organelles but a well-organised soup

• 10x the size of a virus

• replicate rapidly by themselves and evolve

• quickly by mutation and/or gene swapping

• extracellular pathogens (mostly)

  • they grow outside cells, defence is primarily mediated by innate mechanisms and phagocytosis

outline bacteria

• plant-like multicellular eukaryotic organisms

outline fungi and yeasts

• complex multicellular organism too large to be eaten by phagocytes

outline parasites

• anatomical and physiological barriers

  • intact skin

  • ciliary clearance

  • low stomach pH

  • lysozome in tears and saliva

• innate immunity

  • cellular:

    • neutral killer cells

    • neutrophils

    • neutral killer T cells

    • eosinophils

    • macrophages

    • mast cells

    • dendritic cells

  • humoral:

    • complement

    • mannose binding lectin

    • antimicrobial peptides

    • LPS binding protein

    • C-reactive protein

• adaptive immunity:

  • cellular:

    • T cells

    • B cells

  • humoral:

    • antibodies

how does our body protect against pathogens

distribute oxygenated blood to tissues

what is the role of the heart in the circulatory system

• arteries:

  • thick, muscular, elastic

  • carry blood away from heart

  • maintain back pressure for even flow

• veins:

  • thinner walled

  • carry blood back to heart

  • have valves to prevent backflow

• large vessels near heart = high volume/low flow; capillaries in tissue = low volume/high flow

describe the different blood vessels and their roles

• 120 = systolic (LV contracting); 80 = diastolic (heart at rest)

• hypotension (too low) → blood can’t reach capillaries → fatigue, fainting

• hypertension (too high) → capillary damage, abnormal clotting, stroke risk

outline blood pressure (120/80)

• oxyhaemoglobin (bright red) → lungs to tissue via arteries

• carbaminohaemoglobin (dark red) → tissue back to lungs via veins

• exchange happens at lung alveoli (vast surface area)

outline the haemoglobin cycle in the transport of oxygen and CO₂ transport

• O₂ binds/releases based on partial pressure (pO₂):

Location	pO₂ (mmHg)	pCO₂ (mmHg)
Air	160	0.3
Lung alveoli	100	35
Arterial blood	80–100	40
Venous blood	20–40	50

• O₂ associates in the lungs (high pO₂), dissociates in tissue (low pO₂) — CO₂ is the reverse

• cyanide and carbon monoxide can displace O₂ from haem (cherry red/pink appearance)

outline how haem and partial pressure causes O₂ to bind/release

Component	Examples & Notes
Cells	Erythroid (O₂ transport), Myeloid (innate immunity), Lymphoid (adaptive immunity), Platelets (clotting)
Proteins	Albumin (~50% of blood protein; pH/osmolarity balance), Haemoglobin (O₂/CO₂ transport), Fibrinogen (coagulation), Immunoglobulins (antibodies), Complement (innate defence)
Lipids	Carried in lipoproteins — HDL (good), LDL (bad), VLDL
Electrolytes	HCO₃⁻, Na⁺, Cl⁻, Ca²⁺, Mg²⁺, K⁺
Other	Vitamins, hormones, glucose

outline the major components of blood

• plasma (55%) — top layer; liquid fraction; still contains fibrinogen (needs anticoagulant, e.g. heparin)

• buffy coat — thin middle layer; white blood cells + platelets

• red blood cells (45%) — bottom layer

• serum = what remains after clotting (no fibrinogen)

what does blood separate into when centrifuged

• all cells originate from CD34+ haematopoietic stem cells (HSC) in thebone marrow (1 in 10,000 white cells)

• CD = “cluster of differentiation” — cell surface markers used to identify cell types via monoclonal antibodies (mAbs)

outline what CD34+ is

Cell Type	Function	Count/mL
Erythrocytes (RBCs)	O₂ transport	~5–6 million
Leukocytes (WBCs)	Immune defence	~10,000
Platelets	Coagulation & repair	~400,000

outline the function and abundance of each blood cell type

Innate immunity (fast, non-specific):

• neutrophil — most abundant, phagocytic

• eosinophil and basophil — secretory granules; fight parasitic infection

• monocyte → becomes Macrophage (phagocytic) or Dendritic cell

Adaptive immunity (slow, specific):

• B cells → become Plasma cells (produce antibodies); marker: CD19+

• T cells (CD3+) → CD4+ (helper) or CD8+ (cytotoxic); marker: CD3+

• NK cells

outline the different leukocyte types

• AIDS → very low CD4+ + T cell count

• Neutropenia → low myeloid count (signals infectionor cancer)

what are the clinical uses of immunophenotyping

• Core concept: a proteolytic cascade — each factor activates the next (zymogen → active protease), with amplification at each step

• Two activation pathways:

Pathway	Trigger	Key Factors
Intrinsic (Contact)	Contact with activating surface (e.g. glass, prosthetic valve)	XII, XI, IX, VIII
Extrinsic (Tissue Damage)	Cut, bruise, infection; platelet aggregation	Tissue Factor, V, VII,

• Both converge on Factor X → Xa, then:
  Factor Xa → Prothrombin → Thrombin → Fibrinogen → Fibrin → CLOT

  • calcium is essential at multiple steps — remove it and blood won’t clot

  • plasminogen → plasmin disolves the fibrin clot (thrombolysis)

  • TPA (tissue plasminogen activator) used clinically for stroke, MI, DVT, PE — must be given early

• Anticoagulants:

  • Herparin — inhibits thrombin

  • Hirudin — from leeches; also targets thrombin

• Key clinical points:

  • haemophilia — genetic defect in a clotting factor; most common is X-linked VIII Factor deficiency

  • many blood-feeding parasites produce anticoagulants targeting the thrombin step

Outline the the coagulation cascade

Purpose: First-line innate defence — rapidly coats and destroys pathogens

9 major proteins (C1-C9) attach to bacteria in a proteolytic cascade

3 Activation Pathways (all converge on C3):

Pathway	Trigger
Classical	Antibodies (IgM/IgG) binding to microbe → C1
Lectin	Complement recognises unique sugars on bacteria
Alternative	Direct C3 activation on bacterial surface (most important — self-amplifying loop)

Key steps and Terms:

• C3 — most abundant complement protein in blood; activated C3b binds covalent to bacterial surface (opsonisation)

• Convertases — stable enzyme complexes formed on bacteria surface; irreversibly bound; amplify cascade

• Anaphylatoxins (C3a, C4a, C5a) — small fragments released during cascade; powerful chemoattractants for neutrophils

• MAC (Membrane Attack Complex) — terminal pore formed by C5b-C9; lyses some bacteria

Clincial Points:

• complement deficiency → susceptibility to chronic infections

• many microbes produce virulence factors that inhibit complement

Outline the Complement System

There are three interconnected layers of defense against pathogens:

Layer	Examples
Anatomical & Physiological Barriers	Intact skin, ciliary clearance in lungs, low stomach pH, lysozyme in tears/saliva
Innate Immunity	Complement, neutrophils, macrophages, NK cells, PRRs, antimicrobial peptides
Adaptive Immunity	T cells, B cells, antibodies

Outline the layers of defense

Feature	Innate	Adaptive
Speed	Immediate (minutes)	Slow (days–weeks)
Specificity	Broad (recognises patterns)	Highly specific (recognises antigens)
Memory	No memory	Forms memory
Age	Ancient — found in all living things	Appeared ~300 million years ago

• key point: innate immunity provides the first-line response, it also acts as the alarm switch tht activates the adaptive response

Comapre Innate immunity to Adaptive immunity

Pathogen	Location	Defence Strategy
Viruses	Intracellular	Mainly adaptive (cellular immunity + antibodies)
Bacteria	Mostly extracellular	Innate + adaptive (complement, phagocytosis, antibodies)
Fungi/Yeast	Extracellular	Innate + adaptive
Parasites	Multicellular, extracellular	Innate + adaptive (eosinophils important)

outline the different types of pathogens and the respetive immune strategies that are used to combat them

How neutrophils travel from blood vessels to the site of infection

1. Activation

   • chemoskines released by opsonisation/tissue injury activate nearby capillary endothelial cells

2. Tethering

   • neutrophil loosely attaches to the capillary wall — mediated by selectins (on endothelial cells) binding sialyl Lewis X (sLex) (on neutrophil surface)

3. Adhesion

   • strong binding between neutrophil integrins and ICAM-1 on the endothelium

   • neutrophil flattens and immobilises

4. Diapadesis

   • neutrophil squeezes between endothelial cells into the interstitial space (tissue)

5. Chemotaxis

   • neutrophil follows a chemokine gradient (e.g. C5a) to migrate toward infection site

The entire process takes only minutes from initial infection

Outline Neutrophil extravasation

• chemoattractants (e.g. C5a) radiate outward from bacteria

• neutrophil senses gradient at its leading edge

• moves via actin polymerisation at leading edge and depolymerisation at trailing edge

outline how neutrophils find bacteria

• bacteria coated in C3b via complement (opsonisation)

• neutrophil complement receptors (CR1, CR2, CR3, CR4) bind C3b

• CR1 is the main receptor

• cross-linking of surface CRs → triggers phagocytosis

• C5a receptor also activates the neutrophil further

outline how phagocytosis is triggered by the complement receptor

• antibodies (IgM or IgG) bind bacteria surface antigens → Fc region exposed

• neutrophil Fc receptors (FcR) bind the multivalent Fc regions

• triggers phagocytosis

outline how phagocytosis is triggered by Fc receptors

1. Capture — neutrophil adheres to bacterium

2. Invagination — membrane engulfs bacterium, forming a phagosome

3. Phagosome/lysosome fusion → phagolysosome

4. Killing — phagolysosome acidifies; superoxides and digestive enzymes destroy the bacterium

5. Exocytosis — waste expelled from cell

Outline the general steps of phagocytosis

PAMPs — what PRRs recognise

• PAMPs = Pathogen-Associated Molecular Patterns

• molecules unique to microbes (not found on human cells)

• structural complex (e.g. LPS) and evolutionary stable — don’t change much

• PRRs bridge innate and adaptive immunity — they tell the immunity system if pathogen is present and what type it is

outline Pattern Recognition Receptors (PRRs)

• most well-known class of PRR

• rich in leucine repeats (coiled-spring shape)

• bind many different PAMPs; often work together

• activation → signalling cascade via NFkB (nuclear factor) → strong innate immune response

outline Toll-Like Receptors (TLRs)

LPS (Lipopolysaccharide):

• a membrane component of all gram-negative bacteria

• extremely complex molecule; only a small part (lipid A) is recognised by TLR4

• acts as a pyrogen — tiny amounts in the bloodstream cause rapid fever

• must be removed from any injectable pharmaceutical products (common contaminant)

Clinical significance:

• gram-negative bacterial infection → LPS release → massive TLR4 activation → septic shock

• septic shock = life-threatening, systemic inflammatory response

outline TLR4 and LPS

Feature	Gram-Positive	Gram-Negative
Cell wall	Thick peptidoglycan layer	Thin peptidoglycan + outer membrane containing LPS
Gram stain	Purple	Pink/red
LPS present?	No	Yes
Septic shock risk	Lower	Higher (due to LPS)

compare gram negative bacteria to gram positive bacteria

• protective immunity that develops after exposure to infection or vaccination

• unlike innate immunity, it adapts over time — responds faster and more effectively with each encounter

• can provide lifelong protection (e.g. measles)

• relies on billions of naive B and T lymphocytes, each with a unique antigen specificity, generated randomly before birth

• diversity come from random gene rearrangement in BCR and TCR loci — the only region in your genome that does this

what is adaptive immunity

• transposition is a gene changing location within a genome

• two essential elements of transposition:

  • transpoease (recombinase) — enzyme that cuts and repositions DNA; in B/T cells these are RAG1 and RAG2 (only active in B and T lymphocytes)

  • recognition signal sequences (RSS) — short conserved sequences (7 or 9bp) at the end of each segment, recognised by RAG1/2 which then cuts and rejoins segments that can be millions of base pairs apart

outline transposition

The Ig domain (building block):

• ~110 amino acids in length; ~12.5kD

• structural shape: β-barrel fold — two anti-parallel β-pleated sheets (like two cupped hands)

• held together by a central covalent disulphide bond

• three unconstrained loops join the sheets — these loops can vary greatly in amino acid sequence without disrupting the overall structure → this is where diversity arises

Overall Antibody structure

• made of 2 protein chain types: Heavy (H) chains and Light (L) chains, both built from repeated Ig domains

• assembly: L—S-S—H—S-S—H—S-S—L (connected by disulphide bonds)

• shape: Y-shaped

Region	Location	Function
Variable domain (Fab)	Tips of the two arms	Antigen binding; where all diversity occurs
Effector region (Fc)	Stem (CH2 + CH3)	Binds Fc receptors and complement C1; defines antibody class and function
Hinge region	Junction of arms and stem	Flexibility

describe the immunoglobulin (Ig) structure

• each antibody has two identical antigen binding sites (one per arm)

• each site is made up of 6 loops total — 3 from the L-chain + 3 from the H-chain

• these loops are the hypervariable regions = complementarity determining regions (CDRs): CDR1, CDR2, CDR3

• CDR3 (the VDJ join loop) is the most variable and contributes most to antibody diversity

outline the antigen binding site

Class	Gene	Form	Key Functions
IgG	γ	Monomer	Most abundant in blood; neutralises toxins; activates complement; crosses placenta; long-lived; undergoes affinity maturation
IgA	α	Dimer	Found in mucosal secretions (breast milk, gut, tears, genitourinary tract); passive gut immunity to neonates
IgM	μ	Pentamer (blood) / Monomer (BCR)	Default Ig of naïve B cells; 10 antigen binding sites; high avidity; excellent at activating complement (classical pathway) via 5 Fc regions
IgE	ε	Monomer	Least abundant; defence against parasites; causes atopic allergies via high-affinity FcεR on mast cells
IgD	δ	Monomer	BCR form; role in B cell differentiation

outline the five antibody classes

• the strength of binding between a single antibody binding site and its antigen — sum of attractive forces exceeding repulsive forces

• example: high affinity IgG after maturation

what is affinity

• combined binding strength from multiple simultaneous weak contacts — orders of magnitude stronger than single affinity

• example: IgM pentameter with 10 binding sites — like Velcro

• key point: avidity allows naive, low-affinity antibodies (like IgM) to still effectively bind pathogen surfaces before affinity maturation has occurred

what is avidity

• the Ig H-chain gene locus is divided into four clusters:

  Segment	Name	Number of variants
  V	Variable	~100
  D	Diversity	6
  J	Junctional	27
  C	Constant	Defines antibody class (μ, α, γ, ε, δ)

what are the 4 gene segments (V-D-J-C)