IIH

Revision Tutorial

BIMS / BirminghamMES Pre-clinical

Revision Series
[December] 2017
Jessica McGinty
Year 4 MBChB
DISCLAIMER

The BIMS Pre-Clinical Revision Series is developed and run entirely by senior
MBChB students who voluntarily give up their time to teach.

Teaching is to the best of tutors’ knowledge fully-factually correct, but please bear
in mind that neither the tutor(s), nor BIMS accepts any responsibility for any
inaccuracies in the material taught.

This teaching session is in no way affiliated with or endorsed by the medical

school.

[ 2
 Outline of lecture
Immunology Haematology:
- Barriers - Haematopoiesis
- Innate Immunity – cells involved, complement - Red Cells and anaemia
- Adaptive Immunity – B cells, T cells, Antibodies - Clotting
Pathogens and specific immune responses
- Prions, Fungi, Bacteria, Viruses, Parasites Anatomy:
- Lymphatics system
Clinical application of immunology: - Spleen
- Vaccination - Thymus
- Allergy - MALT/Tonsils
- Immunodeficiency
- Tolerance and Autoimmune diseases
 Barriers
Types of protection: mechanical, chemical and microbiological, can be compromised
Skin rrier Cuts, sun burn, cannula, skin diseases
D efects in Ba such as eczema
• Mechanical protection: tight junctions, longitudinal flow of air
• Chemical protection: fatty acids, antibacterial peptides
• Microbiological: normal flora – help inhibit invasion
Respiratory:
Respiratory tract: in B a rrier
D efects • Cystic fibrosis:
• Mechanical: tight junctions, cilia to aid mucus movement Defects in mucus – can’t be cleared
• Chemical: antibacterial peptides: mucin  Recurrent infections
- mucin prevents bacterial adherence, traps droplets, protects epithelial • Kartagener’s ciliary dysfunction:
cells. Inability to move bacteria away from
- Mucocilliary escalator moves mucus to main bronchi and trachea, removal lower airways
by coughing • Recurrent infections:
scarring, dilation, distortion e.g.
GI tract:
• Mechanical: tight junctions, longitudinal flow of fluid, peristalsis Defects in Barrier Bronchiestasis
• Chemical: low pH (sterilise), enzymes (e.g. pepsin), antibacterial GI Inflammation
peptides
• Microbiological: Normal gut flora

Also secretion and drainage: r

Defects in Barrie • Vesicouteric valve failure 
• E.g. lysozymes in tears are bacteriocidal
• Constant drainage of urine helps prevent infections of urinary tract recurrent infection
Features of the Innate Immune System
- No memory
- Triggered within seconds – immediate immunity

- Pre-programmed response to pathogens

- Recognises non-self through preformed non-specific receptors
- The general patterns recognised as called PAMPs: Pathogen Associated Molecular Patterns
- Recognised using PPRs: Pattern Recognition Receptors – can be found on the surface of or
intracellular of innate immune cells (mast cells, dentritic cells etc..)
- A major group of PPRs is TLRs: Toll-like receptors
- 1 TLR can recognise several different PAMPS.
E.g. of Surface TLRs and pathogen product
TLR2 – peptidoglycan,
TLR4 – Lipopolysaccharide (LPS), parasitic phospholipids

E.g. of Intracellular TLRs and pathogen product

TLR3 – viral double stranded DNA

Most signalling through MyD88 and NFKappaB

 Proinflammatory cytokines e.g. IL-6
Innate immune cell

Genetic defects in pathways  recurrent pyogenic infections

 Cells of the Innate Immune System Other important components are:
- Complement
- Mast cells - Cytokines
- Dendritic cells
- Phagocytes: Macrophages (tissue), monocytes (blood), Neutrophils (bacteria, blood  tissue)
- Natural Killer Cells
- Granulocytes: Neutrophils, Basophils, Eosinophils
Mast Cells
• Found in bodies tissue, surrounding blood vessels, nerves, skin and mucosal
surface
• Sense pathogens, dying cells and tissue damage
• Activates mast cells to degranulate, release pre-formed mediators e.g. histamine
• leads to inflammation: vasodilation (increases blood flow) and increase vascular
permeability bringing immune cells to the site of infection
• Leads to signs of inflammation: redness, heat, swelling and pain
Dendritic Cells
• Found in body tissues, especially abundant at the body surface
• Function is to activate naïve T cells  link to adaptive immune system
• By picking up material (e.g. pathogens) (using PAMP/PPRS) from their
surroundings by pinocytosis and present this to T cells
• Have long cytoplasmic branching extensions  large surface area
 Macrophages
• Found in bodies tissue, in serous cavities e.g. the peritoneal cavity
• Fixed beds of macrophages in the liver (kupffer cells) and spleen
• Effectively the dust-bin
• Phagocytose (engulf) cells undergoing apoptosis and pathogens (recognised by PAMPs/PPPs)
• Dying neutrophils are engulfed by macrophages
• Activation of PPRs also induces release of acute phase cytokines (e.g. TNF-α, IL-1β and IL-6)
and upregulates acute phase proteins e.g. C-reactive protein (CRP)
• Acute phase cytokines lead to systemic features such as pyrexia, malaise, anorexia
Monocytes
• Found in the blood
• Macrophages are derived from monocytes
• Move from blood into tissues to help phagocytose dying cells (e.g. neutrophils) with the
correct cytokine signals

Neutrophils
• Found in the blood
• Move from blood into tissue with the correct cytokine signals (e.g. IL-8)
• Kill extracellular bacteria and fungi through phagocytosis and NETs
• Granules contain perforin, myeloperoxidase, nitrates and bactericidal proteins
e.g. lactoferrin. Superoxide anions can be generated too
Neutrophil Function
1. Infected tissue releases cytokines to attract neutrophils to area (eg IL-8)
2. Released systematically in the blood and form a gradient leading back to the site of injury
 Leukocyte adhesion cascade
- Reversible binding of neutrophils to vascular endothelium using selectins, neutrophils roll along endothelium
- Activated integrins bind neutrophils to endothelium. These are ICAMS (intracellular adhesion molecules)
- Extravasation: The neutrophil moves into the tissue, between endothelial cells
3. Once inside the tissue, move directly straight towards the infection, releasing proteases to digest extracellular matrix

Once neutrophil reaches bacteria:

• Bacteria opsonised with C3b and Ig
• Recognised by CR1 and Fc receptors on neutrophil respectively
• After binding, pathogen is engulfed in a phagosome
• Lysosomal granules containing antimicrobial mediators (e.g. hydrolytic enzymes,
lactoferrin, myeloperoxidase) which fuse with phagosomes
• Neutrophils can also produce reactive oxygen species (E.g. superoxide anions O2-,
hydrogen peroxide H2O2, nitric oxide NO) through the membrane bound NADPH oxidase
complex, result in transient increase in O2 called respiratory burst
• Neutrophils continue to kill after they die by producing NETs – fibrous bands with cellular contents than capture bacteria
Macrophages will phagocytose dying neutrophils. Monocytes will move into tissue from blood to phagocytose dying
neutrophils too.
 Basophils
• Found in the blood
• Effectively are the mast cells of the blood
• Primary defence to anything toxic
• Involved in allergy and atopy
• Release histamine (and produce IL-4, IL-3 – IgE switching)

Eosinophils
• Found in the blood, move into tissues with correct cytokines (e.g. IL-5)
• Fight parasitic infection (helminth infection)
• Not phagocytic, instead releases preformed toxic mediators onto surface of helminth
• Associated with allergy and atopy

Natural Killer cells

• They kill cells!
• No antigen-specific receptors, therefore part of the innate immune system. Although function
relies on components related to the adaptive immune system
• Recognise mammalian cells via MHC molecules
• If cell has downregulated MHC (because it is infected by virus or cancer), NK cell will kill the target
cell by inducing apoptosis
• Also if an infected cell is coated with antibodies, NK cells will induce apoptosis of infected cells via
interaction with the FcεRI
• Kill by releasing perforin and granzyme
The Complement System
• Consists of a number of soluble proteins or pro-enzymes
• In the blood and tissue fluids
• Cascade: proteins are activated in a sequence

Three pathways to trigger complement:

- The alternative pathway
- The lectin binding pathway All converge with the formation of
- The classical pathway C3 CONVERTASE

1. The alternative Pathway

- Unknown pathogen, no specific antibodies
- Activated by endotoxin and bacterial cell walls, relies on high concentrations of soluble C3
- Spontaneous C3 hydrolyses to produce insoluble C3b.
- C3b binds to microbial membrane. Multiple bound C3b forms C3 convertase (to convert more C3
etc..) with interactions with other factors
2. The Lectin Binding Pathway
- Pro-inflammatory cytokines released from macrophages – e.g. IL-1β and IL-6
- Act on the liver to produce acute phase proteins, which recognise constituents of
bacterial cell walls e.g. Mannose Binding Lectin (MBL) and C-reactive protein (CRP).

3.The Classical Pathway

- Activated by antigen-antibody complexes (AKA immune complexes)
- Immune complexes activate other molecules (e.g. C1) which in turn activate other
molecules and eventually form C3 convertase
 C3

C3 Convertase

Opsonises pathogens (allows bacteria

Activate mast cell C3a C3b to be identified for phagocytosis)
degranulation, as - C3b binds to CR1 receptor found on
express C3a receptor neutrophils and macrophages
C5 - Also iC3b can binds to CR3/4
C5 Convertase receptor

Attracts neutrophils to
the site of complement C5a C5b Activates the terminal lytic pathway,
activation components C5b  9 bind in
succession.

Forms a transmembrane pore called

Control of Complement: the membrane attack complex (MAC)
- Factor H or I: cleaves C3b to an inactive form: iC3b. This allows water into the cell, resulting
- C1 esterase inhibitor: C1 esterase involved in producing C1. C1 is in swelling and ultimately destruction
the first step in the classical pathway. Inhibitor reduces formation of the cell
of C1 and thus less activation of complement
C1 Esterase
Key Features of Adaptive Immune System
• Recognises non-self specifically
• Each cell expresses a specific unique receptor
• Cells involved are lymphocytes: B cell and T cells
• Takes 4-6 weeks as there are insufficient number of lymphocytes specific to the micro-
organism
• Lymphocytes multiply exponentially
• Memory – adaptive immune system can rapidly upregulate on re-exposure to pathogen and
act as fast as the innate immune system

OVERALL: Enhances the actions of the innate immune system

 T cell Receptor
• Composed of 2 non-identical polypeptide chains
• Which are linked by a disulphide bond
• Either Alpha-beta OR gamma-delta (majority express alpha-beta)
• Have a constant and a variable region . Upper variable region is the
location of the antigen recognition site
• TCR has short cytoplasmic tail, has accessory signalling molecules: CD3
complexes
• T cell will have either a CD4 and CD8 molecule

TCRs only recognise:

- Short peptide sequence from a whole antigen
- Peptide = amino acids (protein)(only recognise proteins!)
- Peptides only recognised when presented on MHC molecules (aka
HLA molecules). Two types: MHC I and MHC II
- Therefore another cell needs to cleave the antigen into peptides and
present this using an MHC molecule.
B Cell Receptor
• The B cell receptor will recognise the surface of an antigen (detect ‘whole’ antigen)
• Specifically will recognise an epitome of an antigen
Antigen = anything that will provoke an immune response
Epitome = a specific molecular structure of the antigen that R binds to
• Short cytoplasmic tail, accessory molecule CD19
• Soluble B cell receptor is antibody (aka immunoglobulin)

Antibody structure
• Structure: 2 identical heavy chains and 2 identical light chains
• Connections via disulphide bonds
• Has 3 regions connected at the flexible hinge region:
- 2 Fragment antigen binding regions (Fab)
- 1 Fragment crystallisable (Fc) region
• Antigen bind to Fab – has a variable region, allows specificity
• Fc has constant domain – available for effect ligands following formation
of antigen-antibody complex. Fc binds to Fc receptor on effector cells.
Antibody structure (continued)
The constant domain has a conserved sequence for a particular type of heavy or light chain
- The heavy chain can be: IgM, IgD, IgG, IgA, or IgE
- The light chain can be: kappa or lambda
The type of heavy and long chain determine the function of the antibody, examples:
- IgM and IgD what immature B cells express on surface of cell
- IgG memory cells
- IgE for mast cells
 Generating lymphocyte receptor diversity
i.e. how does each cell produce a different receptor?
• Variable domains of the heavy and light chains are highly variable in amino
acid sequence – determines antigen-binding specificity
• Infact, it is only the amino acid sequence occurring in the hypervariable
region that determines specificity

Many different genetic sequences can be generated due to

somatic recombination
• There are many genetic segments which encode part of the variable region
of BCR/TCR and there are many ways these can be joined together.
• There are three segments of genes: variable domain (v), diversity (D) and
joining (J)

Heavy Chain (for BCR) and Beta chain (for TCR) Light Chain (for BCR) and alpha chain (for TCR):
• First D and J genes undergo rearrangement Only have V and J segments of the variable
• Rearrangement of V genes region
• V genes joined to DJ genes
• Enzyme that cleaves DNA: RAG1 and RAG2 (Recombinase activating genes)
• Joining by Tdt (terminal deoxynucleotidyl transferase)
 DNA:
V – V – V – V – V – V – V – V – V – V – V – V – D – D – D – D – D – D –D – J – J – J – J

First recombine DJ genes:

D D
D
D J RAG
V–V–V–V–V–V–V–V–V–V–V–V–D–D J–J–J

V–V–V–V–V–V–V–V–V–V–V–V–D–D J–J
Tdt

V–V–V–V–V–V–V–V–V–V–V–V–D–D–J –J

Then recombine V genes and join to DJ genes:

V
V V
D RAG Then converted to mRNA
V–V–V–V–V–V D–J –J Once the mRNA for the variable and
constant segments are formed, these
V–V–V–V–V–V D–J –J are spliced together and translated
Tdt
Whole process happening in developing
V–V–V–V–V–V–D–J –J
B and T cells (bone marrow)
 T cells and Function
T cells express:
(TCR Binds to MHC 2) (TCR Binds to MHC I)

CD4 or CD8
Treg cells Tc CD8+
Regulatory T cells:
Cytotoxic T cells
Suppress autoreactive
Th Cells: Kill infected cells by
lymphocytes, part of
T Helper cells - inducing apoptosis. Cells
peripheral tolerance,
Help another cell do may be infected by virus,
(CD4, CD25, CTLA-4, FoxP3)
something intracellular bacteria, live
vaccine or abnormal
change in cell (cancer)

Tfh Cells:
Th1 Cells: Th2 Cells: Th17 Cells:
Follicular helper cells,
Help infected macrophages to Help B cells in TD antibody Associated with
provide necessary signals
increase killing ability – e.g. response. Involved in class autoimmune/inflammatory
for class switching and
with intracellular bacteria switching – especially IgE, produce conditions e.g. psoriasis, MS. Produce
rescue signals for B cells in
germinal centres. Express IL-4 and IL-13 – ALLERGY and anti- IL-17. Protection against fungi,
CXCR5 to migrate to b zone helminth. also can activate bacteria, by increasing NP recruitment
of lymph node macrophages and barrier function of epithelium
Before any cells can work need to be activated: T cell
priming
When T cells leave thymus, they are immature (not come across antigen) and no effector function at
this stage. They need to be activated:
1. Antigen breaks barrier and enters tissue
2. Immature Dendritic cell (found in tissues) detects antigen through PAMPs/PPRs
3. Activates dendritic cell – phagocytoses and internalises antigen
4. Dendritic cell migrates through lymphatics to the draining lymph node and remains in the T Zone

T-zone
Before any cells can work need to be activated: T cell
priming
When T cells leave thymus, they are immature (not come across antigen) and no effector function at
this stage. They need to be activated:
1. Antigen breaks barrier and enters tissue
2. Immature Dendritic cell (found in tissues) detects antigen through PAMPs/PPRs
3. Activates dendritic cell – phagocytoses and internalises antigen
4. Dendritic cell migrates through lymphatics to the draining lymph node and remains in the T Zone
5. Antigen peptide is presented on the dendritic cell surface using MHC molecule
6. Circulating naïve T cell (has not encountered antigen) encounters dendritic cell in T zone. TCR of
T cell binds to MHC/Antigen peptide complex of dendritic cell
Co-stimulatory molecules include:
- CD80/86 on DC to CD28 on T cell
- CD40 on DC to CD40 Ligand on T cell
- Cytokines
(Type of Cytokines released determine which effector cell
the T cell differentiates into. Not limited to DC. )

e.g. Primed Th2 cell  help to B cells

Primed Th1 cell  help to macrophages
Which Cytokines for which T cell?
Cytokines released during T cell Priming will determine which cell naïve T cell becomes:
• IL-12, IFNγ  Th1 Cell. Th1 cells will produce IFNγ and TNFα to help killing ability of macrophages
• IL-18  CD8
• IL-10  Treg
• IL-4  Th2  secretes IL-4, IL-5 IL-13
• IL-17  Th17
 T cells and Function
T cells express:
(TCR Binds to MHC 2) (TCR Binds to MHC I)

CD4 or CD8
Treg cells Tc CD8+
Regulatory T cells:
Cytotoxic T cells
Suppress autoreactive
Th Cells: Kill infected cells by
lymphocytes, part of
T Helper cells - inducing apoptosis. Cells
peripheral tolerance,
Help another cell do may be infected by virus,
(CD4, CD25, CTLA-4, FoxP3)
something intracellular bacteria, live
vaccine or abnormal
change in cell (cancer)

Tfh Cells:
Th1 Cells: Th2 Cells: Th17 Cells:
Follicular helper cells,
Help infected macrophages to Help B cells in TD antibody Associated with
provide necessary signals
increase killing ability – e.g. response. Involved in class autoimmune/inflammatory
for class switching and
with intracellular bacteria switching – especially IgE, produce conditions e.g. psoriasis, MS. Produce
rescue signals for B cells in
(Help B cells in TD antibody IL-4 and IL-13 – ALLERGY and anti- IL-17. Protection against fungi,
germinal centres. Express
response) helminth. also can activate bacteria, by increasing NP recruitment
CXCR5 to migrate to b zone
of lymph node macrophages and barrier function of epithelium
Cytotoxic T cells (CD8+)
• Kill infected cells by inducing apoptosis
• Cells may be infected with a virus, intracellular bacteria, live vaccine or cancer
• CD8 T cell recognise infected cells using MHC I
• Infected cells present peptides of endogenous proteins (i.e. derived from within the cell) on MHC I
• All nucleated cells have MHC I as they can be infected by viruses etc..
• Not found on red blood cells (no nucleus) nor neurones

TCR with CD8 co-receptor of T cell binds to MHC I peptide complex of infected cell. Triggers apoptosis
• Another signal is from Fas ligand of T cell binds to Fas on infected cell  also induce apoptosis
• CD8 Tc release granules containing perforin and granzyme
HOWEVER…
• Some sneaky viruses can interfere with this process, reducing the amount of MHC I on cell surface
• Therefore cytotoxic CD8 T cells cannot induce apoptosis as they do not recognise which cells are
infected

BUT
• Natural Killer cells are inhibited when the bind to MHC molecules
• Therefore a decline in MHC on infected cells leads to natural killer cells being activated
• Kill infected cell with cytotoxic products: perforin and granzyme
CD4+ Cells
• CD4 cells recognise peptides on MHC II molecules
• MHC II molecules present peptides of exogenous proteins, i.e. produced outside the cell, which
have been brought into the cell (by pinocytosis) and cleaved within the cell e.g. soluble antigen,
extracellular bacteria, immune complexes, non-live vaccines
• MHC II molecules are found on antigen-presenting cells, found on the cells that need T cell
help: B cells, macrophages, and also monocytes
• MHC II is also found on dendritic cells, which are involved in priming T cells

One of the main functions of CD4 Th cells is to help antibody production so…
 B- cells: produce antibodies Where Tfh, Th2 (and Th1)
have their functions in
Main antibody responses helping antibody
production

Natural Thymus Independent Thymus dependent

Antibodies response response
(i.e. without T cells) (i.e. with T cells)
High affinity, class-switched
antibodies, memory

Type 1 Type 2
Extra-follicular Follicular 
produces
germinal centres
Features of T Independent Antibody
Response
• No T cell help
• Antibodies against non-protein molecules
• Involves BCR signal and innate/toll signal
• Strong primary response (fast response on first expose to antigen)
• No Memory
• This is extra follicular (i.e. does not occur in the follicles of lymph nodes)

Two Types:
Type 1:
- Present from second half of foetal life and throughout life time
- Against lipopolysaccharide (and other components)

Type 2:
- Only present several months after birth, only fully developed after 5 years
- Recognises polysaccharide capsule around bacteria – i.e. encapsulated bacteria
- E.g. streptococcus pneumonia, Haemophilus Influenzae B, Niesseria meningitides
- These diseases are likely to cause serious infection in children under age of 5 years – septicaemia and
bacterial meningitis  vaccination
- Population of B cells in splenic marginal zone implicated for these responses
Features of T Dependent Antibody Response
• Involves T cells
• Recognise protein/peptide antigens (have to as T cells can only recognise peptides)
• Slower primary response than T independent reaction – takes time for T cells primed and B and T cells to meet etc..
• Memory and long-term protection – secondary response is much greater
• Produces antibodies that are higher affinity than TI antibody response

Memory cells produced:

• Do not secrete antibodies themselves
• But can be rapidly reactivated
• Secondary memory antibody responses are faster
• High affinity and high specificity
Process:
1. BCR of immature B cell binds to “whole” antigen that has been found in the lymph/node
2. B cell internalises it, processes it and produces part of its peptide on MHC II molecule
This is signal 1  Makes B cell highly efficient at interacting with primed T cell
3. The TCR of a primed Th2 T cell (CD4+) recognises the same antigen and binds to the MHC II peptide complex
Also co-stimulatory molecules:
- CD40 (B cell) and CD40 Ligand (T cell) – this is signal 2
- Cytokines released
NB: This occurs in the T zone of lymph nodes
1 4. From here activated B cell has two fates:
- SOME become a plasmoblast, move to medulla (extra follicular
- Moves to follicle, form germinal centres (follicular response)
3

T-zone
Extra follicular response
• B cells migrate to medullary cords of lymph nodes (or red pulp if in spleen)
• Become plasmoblasts and proliferate many times before differentiating
• After about 3 days (fast!), differentiate into non-dividing plasma cells
• Short-lived
• Secrete antibodies at high rate, mainly IgM, some IgG and IgA – low affinity
• Antibodies produced locally (i.e. in medulla/redpulp) which are full of macrophages. Low affinity antibodies
need to be close to pathogen so can be phagocytosed by macrophages

Follicular response
• B cells activated in the T zone migrate to B follicles
• B blasts differentiate into centroblasts
• A process of somatic hypermutation occurs in the immunoglobulin variable genes – this is the process immune system
adapts specifically to the antigen. Use of AID enzyme (Activation Induced Deaminase)
(Genetic rearrangement will either increase or decrease antibodies affinities )
• Centroblasts differentiate into centrocytes (non-dividing) and re-express mutant immunoglobulin
• Follicular dendritic cells present intact antigens to centerocytes, which compete to capture the antigen
• Centrocytes present parts of captured antigen to follicular helper T cells (Tfh)
 IF centrocyte has mutant Ig receptor of lower affinity, no rescue signal from Tfh and die by apoptosis, happens to most!
 IF centrocytes have a mutant Ig receptor of higher affinity, receive rescue signals undergo more somatic hypermutation
• Iterative cycles accumulate beneficial mutation  produce very high affinity antibodies.
• Tfh (and Th2) also help induce class switching, change in constant region of heavy chain (e.g. IgM/IgD  IgA/IgG/IgE)
• Selected cells become plasma cells and memory cells. Plasma cells produce antibodies systematically and are long-lived
Natural Antibodies
(Probably first secreted antibodies to evolve)
• Produced spontaneously, in the absence of the pathogen, antibodies will still be produced
• Innate antibodies – high avidity and low affinity e.g. IgM
• Important in providing resistance to common pathogens in the first 24-48 hours

Produced by B1 cells
• Found in the peritoneal and pleural cavities
• Do not require secondary lymphoid organs for maintenance
• B1 cells are produced in the foetal liver
• And then bone marrow in early life
• Production appears to cease after infancy therefore self-sustaining
• Number of different antigen receptors is less diverse than recirculating B cells as only a portion of V segments
used. Junctional diversity is not increased by the action of Tdt either.
Functions of Antibodies
1. Opsonisation – Fc of Ig binds to Fc receptor on phagocyte (e.g. neutrophil and
extracell. Bacteria)
2. Neutralisation – Toxins neutralised, antibodies block entrance of bacteria and viruses
into cells – need high affinity to be effective
3. Immune complexes – lattice of Ig and Antigen, limits diffusion of
antigen and ultimately removed and destroy
4. Complement activation – via classical pathway
5. Direct cellular activation – e.g. Fc of IgE binds to Fcε1R on mast cells  degranulation.
6. Antibody dependent cell mediated cytotoxicity – labelling cells to be killed (not for
phagocytosis). Ig bind to antigens on the surface of infected cells. Binds to FcγRIII of
Natural Killer cells. Also IgE and Eosinophils and FceRI
Releases perforin and granzyme  apoptosis. Also cytokines (interferon), activates
phagocytes.
Infectious Organisms and Immune
Reactions
• Prions
• Fungi
• Bacteria: extracellular and intracellular
• Viruses
• Parasites
 Prions
• Mutant protein (no DNA/RNA) induces misfolding of normal host proteins in an exponential chain reaction
• Resistant to disinfectants and heat!
• E.g. Creutzfeldt-Jakob disease (CJD)
• Proteins fold to form amyloid fibres which aggregated  degeneration of brain function

Fungi
• Single or multicellular, eukaryotic organisms
• Most grow as hyphae, cylindrical interconnected tubes
• Only a few studies species
• Include yeasts, moulds and mushrooms
• Can cause serious disease in immunocompromised e.g. Aspergillus

Bacteria
• Most bacteria are not harmful. Only very few harmful all the time, some opportunistic
and some are commensal and help us
• No nucleus as prokaryotes, instead have a nucleoid. No membrane mound organelles e.g.
mitochondria etc . Have appendages
• May have a capsule – reduces opsonisation and protects from phagocytosis
• Shape of bacteria used as measure of classification e.g. ‘coccus’ is round: staphylococci,
streptococci, or classified on Gram stain….
• Classified as gram positive or gram negative:
Gram staining uses crystal violet stain
Lipopolysaccharide and iodine. After washing with
alcohol,
(Gram +ve have • Gram +ve bacteria remain purple
Lipoteichoic acid, as they retain the crystal violet
can cause septic complex
shock) • Gram –ve bacteria will go
colourless
Can counter stain with safranain Gram
positive purpole and negative pink
Cause disease:
1. Use of appendages called pili or fimbrae to attach to host cells.
2. Type 3 secretion systems inject effector molecule to host cell in invasion. Effector molecules alter cytoskeleton and promote
bacterial update
3. Acquire nutrients – e.g. the use of siderophores which have a higher affinity to iron than host carriers such as lactoferrin and
transferrin
4. Evade immune system – e.g. inhibit phagocytosis or complement
5. Damage host directly: produce toxin that damages membrane, e.g.C. tetani and c. botulinum
6. Damage host indirectly: over activate immune system (E.g. LPS) or molecular mimicry where microbial components are similar
to the hosts so antibodies produced towards pathogen, cross react with bodily tissues e.g. group A streptococcus and rheumatic
fever, damaging lungs, heart and kidney and acute glomerulonephritis
Also conjugation – transfer of resistance genes on plasmids between bacteria of the same or different species  antibiotic
resistance. Inappropriate use of antibiotics (e.g. over prescribing, duration of course) is leading to resistance!
Intracellular bacteria
Advantages of living in host cell:
• Help avoid host defences – e.g. complement, antibodies, drugs
• Continuous supply of nutrients for pathogen
Need to overcome innate anti-microbial killing mechanisms of the phagocyte, for example acidification, oxygen derived
products (e.g. O2- H2O2), toxic nitrogen oxides (e.g. NO), antimicrobial peptides, enzymes (e.g. lysozymes) etc

Bacteria can block/evade anti-microbial mechanisms:

How to bacteria get in?

Utilise the early steps of phagocytosis, and evolved mechanisms for disrupting or avoiding
later steps. For instance:
• Rupture phagosome in the early stages and escape into cytosol. Not exposed to
lysosome contents e.g. Listeria monocytogenes
• OR can block the fusion of the phagosome and cell lysosome. Will exist in a vacuole
eg. Mycobacterium tuberculosis
Fighting Intracellular bacteria:
If pathogen exists in a vacuole:
• Block fusion of phagosome and lysosome –e.g. Mtb
• primary response is by Th1 CD4 cells
• TCR of primed Th1 binds to MHC II molecule on macrophage
• Th1 releases IFNγ and TNFα. Also signalling through CD40/CD40L
• Increase macrophages antigen presenting ability: increase MHC I and II, CD40, CD80/86
• Increases antimicrobial activity: increase reactive oxygen intermediates, NO, lysosome numbers and
enzymes
• In TB, IFNγ overcomes pathogen induced block of phagosome lysosome fusion
• Clinical application – anti-TNFα therapy, eg from rheumatoid arthritis block this, latent disease TB
activates

If pathogen exists in the cytosol:

• Pathogen escapes from early phagosome into cytosol e.g. Listeria monocytogenes
• Primary response is by cytotoxic CD8 T cells
• TCR of Primed T CD8 cell interacts thought MHC I on macrophage
• T cell releases perforin, granzyme and granulysin – directly toxic to intracellular bacteria
• Also signalling through Fas/FasL

However not mutually exclusive!

Viruses
• Obligate intracellular parasites
• VERY SIMPLE – A nucleic acid genome, in a protein shell called a capsid. May have a membranous envelope.
• Not cells, cannot make energy or utilise nutrients – dependent on cells to function

Classified based on genetic material called the Baltimore scheme - 7 groups:

1. Double stranded DNA e.g Herpes simplex virus
2. Single stranded DNA e.g. parovirus
3. Double standard RNA e.g. rotavirus
4. Single stranded sense RNA e.g. polio, HCV
5. Single stranded anti sense RNA e.g. influenza
6. Single stranded sense RNA with reverse transcriptase e.g. HIV  retroviruses
7. Double stranded DNA with reverse transcriptase e.g. HBV  DNA retroviruses

Basic life:
Attach (determined by tropism) Replication  Assembly  Release of new viruses by budding or lysis
Except retroviruses which made a DNA reverse transcript for their RNA first.
Immune response to Viruses
• Prodromal symptoms: non-specific “flu-like”, headache, fatigue, fever due to the innate immune system, e.g. IL-1, IL-6
• Viral infections leads to cascade of interferon induced responses (use of PPRs)
• Stimulates release of type 1 interferon (IFNα and IFNβ) which stimulates interferon stimulated genes (ISG)
• Expression of these proteins refers cells resistant to viral infection, limiting the spread of infection

Other mechanisms:
1. Viral infected cells present peptides on MHC I to CD8 cytotoxic cells  apoptosis
2. Viruses may have down regulated MHC expression  activates natural killer cells  kill infected cells
3. Antibody production directly block virions entrance to cell, endocytosis, release into cytoplasm and aggregate viruses
4. Anti-body directed cell cytotoxicity – Natural killer cells have FcγRIII, binds to Fc portion of IgG antibodies which are
coating infected cells.

Infected cell
Anti-Helminth Immunity
Helminth = parasite
Too large for phagocytosis
Immunity mediated through Eosinophils releasing preformed granules

Process:
• First line of defence: mast cell degranulation  increase blood flow
to area, increase recruitment of immune cells
• IgE mediated degranulation of mast cells produces IL-5 – recruits and
mobilises eosinophils
• Eosinophils bind to Fc portion of IgE via FceRI,
• Eosinophils release preformed mediators:
- Major Basic Protein (MPB) – very toxic, alkaline
- Proteases, ribonucleases, peroxidases – penetrate surface of
helminth
- Reactive oxygen and nitrogen radicals
• Eosinophils release IL-13 which leads to goblet cell hyperplasia
(increase numbers)  increase mucus and contraction of smooth
muscle. Together lead to expulsion and eradication of worm
Clinical Application of Immunology
• Tolerance and Autoimmunity
• Immunodeficiency
• Vaccination
• Allergy
Tolerance
Rearrangement of variable regions (V,D,J regions) in BCR and TCR gene (by somatic recombination) is random
Therefore some receptors generated will recognise self –antigens and some will recognise nothing.

B cell Tolerance:
• B cells develop in the bone marrow
• No recognition of self-antigen: survival
• If B cell recognises antigen in the bone marrow, cell is eliminated
• Also, a lack of T cell help in T dependent response

T cell Tolerance: Central tolerance

• T cells are produced in the bone marrow and move to the thymus where they mature
• First T cells in the cortex where positive selection occurs: does T cell recognise MHC?
• If not, death by neglect
• If T cells can recognise MHC, they are selected and differentiate. Gene rearrangement occurs as they pass into medulla
• At cortico-meduallary junction: macrophages prevent damaged cells entering the medulla, (DC offer some –ve selection)
• The T cells undergo negative selection in the medulla, how strongly do TCRs bind to MHC complexes
• Too strong  negatively selected and die by apoptosis
• If does not bind too strongly, will develop into a mature CD4 or CD8 T cell

Tolerance to tissue specific proteins achieved through AIRE gene: AutoImmuneRegulator – ‘promiscuous gene expression’
Peripheral T Cell Tolerance:
T regulatory Cells
• Key Molecules: CD4 and CD25, express transcription factor FoxP3
• Suppress autoreactive lymphocytes that have escaped negative selection by:
- secreting inhibitory cytokines: IL-10 and TGF-β
- CD-25 competes for IL-2, IL-2 is a growth factor for self-reactive T cells
- Possibly contact dependents
Inappropriate antigen presentation
• When dendritic cell primes T cell, there are additional danger signals. Danger signals are not given when self-
antigen
MHC II restriction
• MHC II is only found on B cells, macrophages, monocytes and dendritic cells, means most cells can not directly
present their own proteins to CD4 T cells
Threshold response
• T cells have capacity to regulate themselves
- CD28 provides positive T cell activation signals
- CTLA-4 provides negative activation signals – if removed, severe autoimmune disease
Ignorance
• Certain sites of the body are immune privilege – suppression of immune responses by cytokines e.g. TGF-β and
active deletion of reactive cells
Autoimmunity: Loss of Tolerance
1. Loss of ignorance:
Penetrating injury to eye means immune cells have direct access to previously hidden antigens. Immune response is bilateral,
contralateral eye is damaged as involved in severe immune response – sympathetic ophthalmia
2. Regulatory T cells:
Fail to supress self-reactive T-helper cells. E.g. lack of CTLA-4, lack of negative activation signals
3. Inappropriate antigen presentation
New antigenic determinant is associated with self-antigen, new antigenic adjuvant may trigger danger signals e.g. collagen
and dead bacteria. (like an adjuvant in a vaccine)

How to autoantibodies causes disease?

1. Complement dependent lysis of target cell
- Paroxysmal cold haemaglobinuria (type of haemolytic anaemia). Mediated by complement fixing IgG
2. Opsonisation
- Most forms of haemolytic anaemia
3. Immune complexes
- Glomerlonephritis in SLE – immune complexes have a net positive charge, deposit in -ve charged basement membrane
4. Receptor Blockage
- Myasthenia Gravis – against acetylcholine receptors  skeletal muscle fatigue and weakness
- Pernicious anaemia – anti-intrinsic factor
5. Receptor Stimulation
- Graves disease  hyperthyroidism as mimic actions of TSH
 Immunodeficiency
Main points:
Deficiency in different components of immune system lead to different types of diseases in different places
Deficiencies can be primary or secondary
Can be quantitative (reduced production of a cell type) or qualitative (normal number but function defective)

Neutrophil Deficiency
• Extracellular bacteria and fungal infections, skin/mucosal surfaces (e.g. staphylococcus/aspergillus), omphalitis, abscesses,
cellulitis - from first months of life
• Neutropenia due to: congenital neutropenia or 2ndry: cytotoxic drugs (chemoTx), haematological malignancies, autoimmune
• Leukocyte adhesion deficiency – defect with integrins, cannot migrate into tissue. Inflammation but no Pus and high NP count

• Chronic granulomatous disease – inability of neutrophil to produce oxidative burst – no superoxide radial produced
• Secondary qualitative causes: steroids (prednisolone), other immunosuppressants metabolic e.g. renal failure, toxic and
metabolites not cleared
 Complement Deficiency
A low C4
• C4 is at the start of the classical pathway
• C4 made up of 4 alleles, relatively common for 1/2 to be null partial deficiency
• May have a low C4 due to overactivation of classical pathway due to:
1. C1 Esterase Inhibitor deficiency
• C1 esterase  C1
• Inhibitor reduces how much C1 produced  reducing activation C1 Esterase
• Therefore deficiency of this  overactivtion of classical pathway, C4 used up.
• C1 esterase inhibitor is also part of bradykinin pathway, bradykinin  oedema
• Classical feature: potentially life-threatening non-itchy swelling
• Autosomal dominant
2. Immune complex disease (acquired disease)
• E.g. lupus, rheumatoid vasculitis, ANCA positive vasculitis
• Defect in classical pathway  infections of encapsulated bacteria and immune complex disease (as also functions as
preventing formation of immune complexes!)
• Defect in early alternative pathway: associated with Neisseria infections
• Defect in mannose-Binding pathway: accessory mechanism of complement, only show if there is a problem elsewhere too
A low C3
• Most severe, recurrent meningitis, severe recurrent bacterial infections
C9 deficiency
• Neisseria infections – Neisseria meningitidis and Neisseria gonorrhoeae, and other pyogenic infecitons
Antibody/B cell deficiency
• Encapsulated bacteria, leading to recurrent injections of respiratory tract – sinitis, bronchitis, pneumonia, bronchiectasis
• Don’t show until 6 months as this is when maternal IgG drop (although if premature, this is much shorter period)
Primary causes:
- Agammaglobinaemia – B cell maturation arrest as absence of Brutons tyrosine Kinase (Btk). Autosomal recessive or X-linked
- Hyper IgM syndrome – failure to class switch as reduced effective communication between B and T cells due to defect in
CD40/CD40L – no somatic hypermutation
Secondary causes:
- Reduced production: marrow suppression, myeloma, CLL
- Reduced function: immunosuppression: steroids and cytotoxics
- Increased loss – protein loss e.g. nephrotic syndrome, Rituximab
Management with immunoglobulin replacement therapy
T cell deficiency
• Intracellular bacteria, viruses, fungi affecting respiratory and GI tract
• Show in first few months  failure to thrive
• Many pathogens are of low pathogenicity:
- Fungi: Pneumocystis Carinii – pneumonia, candida
- Bacteria: Atypical mycobactieria, salmonella (Extracellular)
- Viruses: Varicella, HSV, CMV
- Parasite: cryptosporidium (diarrhoea)
Primary causes:
• SCID (variety of gene defects) and DiGeorge syndrome
Secondary causes: HIV, leukaemia/lymphoma, immunosuppression for preventing graft rejection
Management: no live vaccines, prophylactic antibiotics, Ig replacement, stem cell transplant is needed
Vaccination
Artificial ways of introducing memory to a pathogen. Memory allows for faster, larger adaptive immune response

Passive Vaccination:
- Uses preformed elements of the immune system, therefore immediate protection (not 2 week lag for GC follicles)
- E.g. Maternal IgG is transferred to foetal blood, provides protection for first 4-6 months
- Immunoprophylaxis: rabies, VZV, rhesus D – use serum from a survivor
- However, short lasting half-life 14-21 days.

Active vaccination:
- Inject an organism (whole or subunit) to artificially induce immune memory:
1. Whole can be live-attenuated – i.e. disabled/altered so don’t cause disease. Most immunogenic, cannot be given to
immunosuppressed patients. Risk of reactivation.
eg. BCG, yellow fever, VZV, MMR
2. Whole dead – no risk reactivation
e.g. rabies, whole-cell pertussis
3. Sub-unit eg. Inactivated toxin (e.g. diphtheria, tetanus), polysaccharide (streptococcus, meningococcus)
Conjugate vaccines
• Problem: polysaccharide capsules of bacteria are poorly immunogenic, relies on TI T2 response  low affinity, poor memory
• Most at risk are those under 2 years – as this response doesn’t fully develop until 5 years
• Solution: conjugate polysaccharide to protein in the vaccine.

T cells recognise peptide part presented to them by APC

B cell receptor (Ig) recognise polysaccharide
B cell internalises and cleaves. Present peptide on MHC II
T cell can provide help

e.g. Hib – Hib influenza

Prevenar 13-valent - Pneumococcus
Men C ACWY – Meningococcus C

Adjuvants
• Problem: some proteins are poorly immunogenic alone
• Give adjuvant that enhance immunogenicity of substance
given with it
Mechanisms:
- Convert soluble antigens into particulates e.g. Alum
- Include bacteria/products – upregulate co-receptors and
cytokines, danger signals
- TLR agonists – being developed as tumour vaccine
 Allergy
Hypersensitivity: Reactions of the immune system that are detrimental to the host
Types of hypersensitivity reactions:
Type 1: Allergy, IgE mediated, mast cell degranulation, Eosinophil activation, immediate  anaphylaxis. E.g. food, hayfever
Type II: Self IgG auto-antibodies to own cells and surfaces to cause damage e.g. Autoimmune haemolytic anaemia
Type III: Involves immune complexes  activation of complement causes damage e.g. vasculitis
Type IV: T cell mediated immune response (delayed hypersensitivity) e.g. contact dermatitis, mantoux test
Other reactions: chemical/irritant response, food intolerance, side effects of drugs
Type 1: Allergy
Sensitisation:
• Process of body recognising allergen
• On initial exposure, body decides substance is harmful e.g. presence of other
inflammatory or unusual way of presentations (food substances through inflamed skin)
• During TD response, Th2 cells produce IL-4 and IL-13 to initiate class switching to IgE in
germinal centres (also basophils)
• NOTE IgE switching is inhibited by IFN gamma produced by Th1 cells
• High affinity IgE will cause mast cell degranulation when exposed to allergen
Early mediators: Histamine, Kallikrien ( bradykinin), serotonin, proteases, tryptase  low BP, bronchoconstriction and others
Late mediators: Prostaglandins, leukotrienes, cytokines produced  late phase response – 1 in 5

Treatment: Intra-muscular adrenaline for early phase, steroids and antihistamines to prevent late phase
Cause? Hygiene hypothesis, immune system shifts from Th1 (IFNγ) to Th2 (IL-4 and IL-9  mast cells, IL-4 and IL-13  IgE)
Haematology
• Haematopoiesis
• Red Cells and anaemia
• Clotting
Haematopoiesis
• Forming new blood cells
• Around 1010 erythrocytes an hour, 108 – 109 leukocytes an hour can can be greatly amplified on stress and demand (e.g.
infection, after blood loss)
1. Originate from haematopoietic stem cells (HSC) which are multipotent as can differentiate into all cells of all blood lineages
2. Or divide into myeloid or lymphoid lineage – lineage commitment
3. Then terminal differentiation at the progenitor and precursor level
• Process requires cellular and soluble growth factor support and organisation as different niches in the bone marrow
environment
• HSC have CD34 as a marker
When and Where does haematopoiesis occur?
• Starts 17 days after fertilisation
• Haematopoiesis occurs in transient primitive waves in the extra-embryonic yolk sac
• Definitive HSCs start to appear in the aorta-gonad-mesonphros (AGM) region of the developing liver
• HSC migrate to the foetal liver (placenta and liver) between 2-7months.
• Then to the bone marrow between 5-9 months
• After birth, takes place in medullary cavities. Infants: virtually all bone. Adults: axial skeleton (skull, sternum and vertebral
column)
• There is red and yellow marrow. Red marrow is where active haematopoiesis occurs, has a rich blood supply. Yellow fat cells
Extramedullary haematopoiesis can occur in the liver and spleen when bone marrow is compromised or exceeded e.g.
myelopfibrosis, untreated thalassaemia
Regulation
• Platelets regulated by thrombopoietin (TPO), 90% produced by liver. Therefore in liver failure platelets numbers may
fall (and also post chemotherapy).
Clinical use:
- Treat immune thrombocytopenic patients (platelets destroyed by immune system) when resistant to other
treatment
- Potential use in thrombocytopenia in myelodysplasia and post-chemotherapy

• Granulocytes regulated by G-SCF. Can be given as subcutaneous injections

Clinical use:
-neutropenic patients (e.g.chemotherapy, congenital neutropenia).
-Also mobilises HSC into the peripheral blood for HSC transplant

• Monocytes regulated by GM-CSF

• Erythrocytes regulated by Erythropoietin (Epo) 90% produced by the kidney, 10% in liver. Main stimulus is hypoxia.
Clinical use:
- Patients with chronic renal failure who are anaemic
- Pre-autologous blood transfusions
- Jehovah’s witnesses instead of transfusions
- some cases of myelodysplasia
- ALSO DRUG OF ABUSE in professional athletes to increase oxygen carrying capacity
Assessments of Haematopoiesis
• Full blood count
• Blood film – Reticulocytes (precursor of erythrocytes) are nucleated cells and not normally found in peripheral
circulation (make up 1-2% of red cells). Presence of nucleated red cells in peripheral circulation suggests
erythropoiesis is being stimulated as more red cells required – e.g. haemolytic anaemia

Bone marrow testing

• Bone marrow trephine- a core of bone marrow is used to examine cellularity and architecture of bone marrow,
including extracellular matrix
• Bone marrow aspirate – used to assess morphology and numbers of haematopoietic cells at different stages of
differentiation, help identify abnormal malignant infiltrates

Haematological Malignancies
• Leukaemia: Cancers of haematopoietic cells. Start in the bone marrow  spread to involve blood, LN, spleen
• Lymphomas: Cancers of mature lymphoid cells in the LN/spleen  spread to involve the blood and bone marrow
• Myeloma: cancers of plasma cells in the bone marrow
• Myelodysplasia – “abnormal growth of bone”
AML
Leukaemia classified as:
- Acute or chronic: Acute – maturation arrest, cells do not mature ‘blasts’
- By lineage: Myeloid or lymphoid
- Therefore: AML, ALL, CML, CLL
Haemoglobin and Red Cells
• Carries oxygen, from lungs to tissues, and return of carbon dioxide
• Achieved through Haemoglobin, in adult HbA
• HbF is fetal haemoglobin, (2 alpha and 2 gamma chains)
Fe2+
(Ferrous)

Red cell features:

• Life span: 120 days
• No nucleus
• No mitochondria – anaerobic glycolysis via
Increase Decrease embden-Meyerhof Pathway for energy
affinity affinity generation
• Concave in shape
• Flexible – to pass through capillaries#
• NADH reduces ferric to ferrous
• 2,3-DPG used to regulate Hb oxygen affinity
Anaemia
• Reduction in haemoglobin, red cell count or haematocrit (% of blood cells that are RBCs). Defined as:
- Men <13g/dL
-Woman <12g/dL
Relates to plasma volume – e.g. in pregnancy this increases, therefore artificial decrease Hb concentration would
look like anaemia. Therefore anaemia defined lower as <11g/L.
If blood loss, artificial elevation of Hb concentration would mask anaemia. Takes 24 hours for anaemia to be evident
after acute major blood loss.

Symptoms: Signs:
• Fatigue • Pallor
• Palpitations • Tachycardia
• Headaches • Bounding pulse
• Drowsiness • Flow murmur
• Shortness of breath, esp on exertion • Signs of heart failure
• Angina • Rare: Koilonychia – rounded finger nails
Note: signs and symptoms are variable depending on • Rare: Angular stomatistis – corners of lips
- Hb level and cause inflamed, red and sore
- Rate of onset
- Co-existence of other medical conditions e.g. cardiac and pulmonary disease may exacerbate symptoms
- Concurrent changes – e.g. in 2,3 DPG
Ways to classify anaemia include: Aetiology or Mean cell volume (Microcytic (small RBC <80fl), Marcocytic >100fl, or
normocytic (80 – 100fl)
Causes of Microcytic Anaemia:
• Iron deficiency
• Haemoglobinopathies: Thalassaemia
• Anaemia of chronic disease (less common)
1. Iron deficient anaemia
Causes include: 2. Thalassaemia:
- Dietary deficiency - Imbalance of α- globulin and β-globulin protein
- Chronic blood loss - GI blood loss, need to rule out - Autosomal recessive, major – homozygous, minor – carrier
malignancy, Aspirin? Also menstrual blood loss and
pregnancy Β-Thalassaemia: reduced B chain production
- Excess α chains precipitate in normoblasts leading to
Diagnosis involves: ineffective erythoropoises
- Ferritin levels, low in IDA (ferritin is inert storage of iron in - Haemolysis and extramedullary haemoatopoeisis
cells) - Shows at 6 months as this is when HbF reduces
- Soluble transferrin receptor assay, reduced transferrin - Repeated blood transfusions needed iron overload
saturation in IDA (transferrin is iron storage in blood) complications
- Investigation of GI tract – e.g. endoscopoy/colonoscopy
α-Thalassamia: reduced α chain production
Treatment: Ferrous sulphate. - More complicated as 4 genes (i.e. two inherited from each
Dietary sources include green veg, eggs, red meat and bread parent). 2 copies OK.
Causes of Normocytic Anaemia:
• Anaemia of chronic disease
e.g. in malignancy, chronic inflammatory disease ( rheumatoid arthritis) or chronic infection (e.g. TB)
inflammatory cytokines increase hepcidin which blocks ferroportin transporter Iron is locked in reticulo-endothelium
system and iron can’t be used
Diagnosis: ferritin is normal
Treatment: Successfully treat underlying disease
• Acute blood loss (after 24 hours)
• Mixed deficiency anaemia
• Chronic renal failure – low Epo
• Sickle cell anaemia (a type of haemolytic anaemia) (next slide)
• Bone marrow failure/ineffective erythropoiesis (Also macro)
- e.g. aplastic anaemia, myelodysplasia, post-chemotherapy

Cause of Macrocytic Anaemia:

• Haemolytic anaemia ( more on next slide)
• B12 deficiency
- Absorbed in ileum. Require intrinsic factor produce by gastric parietal cells.
- Causes: pernicious anaemia (autoimmune condition with Ab against parietal cells or intrinsic factor, gastrectomy,
malabsorption, congenital absence of intrinsic factor, vegans – B12 is confined to animal produce
• Folate deficient
- Absorbed in duodenum or jejenum. Causes – malabsorption and poor diet. Found in green veg and liver
• Alcoholism
• Myelofibrosis
• Drugs
Haemolytic Anaemia: Macrocytic anaemia
• Destruction of red cells may take place within the circulation (intra-vascular) or outside (extra-vascular) – mainly spleen
Causes:
- Stimulus maybe outside the membrane – e.g. auto-antibody against red, nothing wrong with red cells themselves
- Problem with the membrane causes red cells to be haemolysed
- Problem with the red cell itself causes cell to be haemolysed

1. Auto immune haemolytic anaemia

• Antibody against red cell is present in the plasma, increases destruction of red cells
• Coombs test is positive
• Causes: idiopathic, lymphoma, drugs
2. Hereditary spherocytosis
• Problem with red cell membrane means cells are sphere-
shaped and fragile
• Destroyed by the spleen
• Autosomal dominant
3. Sickle cell disease: Normocytic Anaemia
- Variant of haemoglobin HbS, in B globulin gene. Swap valine for glutamic acid at position
6
- HbS polymerises deforms red blood cells into a sickle shape during deoxygenation
leading to chronic heamolysis and vaso-occlusive crisis (obstructs capillaries – painful
- Autosomal recessive
- Treatment - exchange transfusion and hydroxycarbamide
Haemostasis
Haemostasis: Process that stops bleeding following injury to a blood vessel, involves platelets and clotting factors
Platelets
• Lifespan 8- 12 days von willebrand factor (vWF)
• Has glycoprotein receptors: GPIb and GPIIb/IIIa - Largest protein in blood
• Dense granules: mediators of platelet activation e.g. - Endothelail cells of blood vessles produce vWF
serotonin, ADP, catecholamines
• Alpha granules: clotting factors (e.g. V, VIII, fibrinogen) and
Haemostatic response by platelets
others e.g. PDGF, platelet factor 4
• Glycogen granules: energy source 1. Local vasoconstriction at the site of injury
2. vWF binds to sub-endothelial collagen
3. vWF multimers stretch out into flowing flood and bind to
platelets via GPIb receptor (weak bond)
4. Platelets roll along vWF to the site of injury
5. Interaction upregulates GPIIb/IIIa receptor on platelet
6. vWF can bridge platelets via GPIIb/IIa
7. Interaction of vWF with GPIIb/IIIa receptor is a strong bond
8. Causes release of ADP from endothelial cells which
activates platelet metabolic pathways, increasing Ca2+ levels
9. Platelets release granule contents
10. Overall induces platelet aggregation and coagulation
Coagulation Cascade
• Involves protease enzymes and their cofactors
• Serine protease enzymes: FVII, FIX, FX, FXI and prothrombin (FII)
• Cofactors: FV and FVIII
• Also Fibrinogen
Produced in the liver, released as inactive precursor form

Disorders leading to bleeding tendencies

Hereditary
• Clotting factor deficiencies
- Haemophilia A (deficiency of FVIII)
- Haemophilia B (deficiency of FIX)
• Von Willebrand disease – deficiency of vWF
• Platelet disorders:
- Bernard-Soulier syndrome (deficiency of GPIb)
- Glanzmann Thrombasthenia (deficiency of GPIIb/IIIa)

Acquired
• Thrombocytopenia (low platelets)
- Autoimmune, Bone marrow failure, chemotherapy
• Liver disease
• Warfarin therapy inhibits vitamin K in synthesis of factors VII, IX, X and prothrombin
• Disseminated Intravascular Coagulation (DIC)
Prevention of Clot formation
i.e. inhibit coagulation: through platelets and clotting factors
• Antithrombin circulates in the blood
• Protein C/S system
• Tissue Factor Pathway inhibitor (TFPI)
• Synthesis of release of prostacyclin and NO which inhibits
platelets
Protein C/S system
• Thrombin (on clot) binds to thrombomodulin on intact endothelium cell
• Complex enables binding of Protein C  activated through cleaving by TFPI
thrombin
• Protein C with Co-factors protein S and inactive FV Antithrombin
Protein C
• Protein C cleaves FV and FVIII neutralises
Conditions predisposing to thrombosis:
Hereditary:
• Factor V Leiden Mutation – FV resistant to neutralisation by Protein C
• Prothrombin gene mutation  increased amounts  more fibrin
• Deficiencies in antithrombin, protein C and Protein S
Acquired:
• Lupus anticoagulant, antibody in circulation (so called as in lab associated with anticoagulation)
• Myeloproliferative disorders – e.g. essential thrombocythaemia
• Malignancy, surgery, long-haul surgery, prolonged periods of immobility, pregnancy
Fibrinolysis
• Process by which fibrin clot is degraded: involves plasmin
1. Thrombin (on clot) acts on endothelial cell to release tissue plasminogen activator (tPA)
2. tPA incorportated into clot, allows binding of plasminogen
3. tPA converts plasminogen to plasmin
4. Plasmin initiates the proteolytic dissolution of the clot resulting in fibrin degradation products
Laboratory Tests to investigate Haemostatic system:

Prothrombin time (PT)

- Citrated plasma incubated with TF
 extrinsic pathway
Activated Partial Thromboplastin time (APTT)
- Citrated plasma incubated with contact activator
 intrinsic pathway

Interpretation:
• Prolonged PT alone – FVIII deficiency (hereditary or ‘mild’ liver disease/vitamin K def)
• Prolonged APTT alone – FVIII, FIX, FXI, FXII
remix with normal plasma to determine if deficiency or antibody present (e.g. lupus anticoagulant)
• Both prolonged – FX, FV, prothrombin, fibrinogen deficiency – part of common pathway

Other test are platelet count, fibrinogen level, von Willebrand actor level, Platelet Aggregation studies
Anatomy
• Lymphatics and Lymph nodes
• Spleen
• Thymus
• MALT and Tonsils
The lymphatics system
Fluid leaves the circulation at the arteriolar end of capillaries
 Excess fluid into the lymph system.
 Afferent lymphatics, LN, efferent lymphatics  circulation
(Immune cells likely to leave at post capillary venules)

R lymph.
Duct
Drains right
half of Thoracic duct in posterior
thorax mediastinum drains onto L Thoracic duct drains
subclavian vein, at its union all of body except
with left internal jugular vein the right thorax

Cyterna chyli
expansion of distal end
of thoracic duct.
Contains fat (as GI
system drained, white)
Key Facts of Lymph nodes and drainage
Superficial lymph system drains in parallel with vein – e.g. skin, mucous membranes, serous linings of cavities
Deep lymph system drain sin parallel with arteries – e.g. thoracic duct enters thorax at T12 with aorta

Lymph nodes:
• Superficial inguinal LN drain: skin of LL, scrotum and lower abdomen.  Drain into external iliac nodes
• Deep inguinal LN drain: deep lower limb and testes  para-aortic nodes.
• Mammary glands drain into axillary LN (75%) and parasternal LN (20-25%)
• Cervical lymph nodes – drain scalp, face, nasal cavity and pharynx

Virchov’s Node – LN situated at the site where thoracic duct drains into the left subclavian vein. Enlarged is a signed
of intra-abdominal malignancy – especially gastric malignancy

Palpation of lymph nodes:

• Size: about 2cm in femoral region, 1cm in neck
• Soft: normal
• Rubbery: lymphoma
• Hard and not painful: malignant
• Tender: infection
Spleen
Glorified LN, with a few extra bits
• Acts as a LN as had white pulp: interactions with B and T cells
• Extras: recycles red blood cells in red pulp – lined with macrophages
• Important in fighting certain bacterial infections eg pneumococcal
 post splenectomy, greater risk to these infections

Anatomical location
• Left upper quadrant, between the 9th and 11th ribs
• Covered by diaphragm and ribs
• Anterior is stomach
• Posterior is diaphragm, ribs 9 – 11
• Inferior is left colic flexure
• Medially is left kidney and tail of pancreas Blood supply is splenic Artery, arising from Coeliac trunk.
Drained by portal vein into hepatic portal vein

Thymus
• Function: T cell maturation
• Anterior to thoracic cavity, posterior to the manubrium of the sternum
• Approximately at level T4-T6
• Posterior is the aorta, trachea and oesophagus
• Most active in early life and reflected in its weight – decreases in weight
past puberty
MALT: Mucosa-associated Lymphoid
Tissue
• These are patches of lymphoid tissue in the mucosa of the GI tract, breast, thyroid
• Present where body is exposed to bacteria – e.g. gut and mouth
• Contain T and B cells, IgA antibodies and some macrophages to maintain mucosal integrity and immunity
• Peyer’s Patches – form of MALT in the ileum. Part of barrier to prevent pathogens entering the blood

Tonsils: Waldeyer’s Ring

An arrangement of lymphoid MALT tissue in the pharynx – form a ring.
• 1 Pharyngeal tonsil – superior to the uvula in the nasopharynx
• 2 Tubal tonsils – located at the entrance of Eustachian tube in the oropharynx
• 2 palatine tonsils – located in the oropharynx
• 1 Sublingual tonsils – posterior surface of tongue as it descends in the
oropharynx
Problems:
• Adenoiditis – inflammation of pharyngeal tonsil – obstruct passage of air from
the nasal cavities  breath through mouth
• Enlargement of Tubal tonsils  swelling and closure of Eustachian tube.
Impairment of hearing and may lead to middle ear infection
• Enlargement of palatine tonsils – dysphagia, obstructive sleep apnoea
• Enlargement of lingual tonsils – sore throat, dysphagia, globus sensation,
obstructive sleep apnoea,
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