Wk 14 - Immunology

Question 1

Antibody function

Answer 1

Depends on type of antibody (class) but they can:

• Formation of complexes by binding to an antigen and forming “clumps” which is then cleared away.
• Activation of complement (IgM and IgG only)
• Neutralisation
• Fc/Fc receptor-mediated functions (the long portion of the antibody which binds) - these re specific for IgA IgE and IgG. These receptors:
  Antibody-dependent killing (ADCC)
  Antibody-dependent phagocytosis
  Activation of mast cells
• Related to main sites at which they are found.

Question 2

IgA

Answer 2

Secreted in the gut
Prevents things binding to the surface of cells and causing infection in first instance.

Question 3

Antibody breakdown

Answer 3

Question 4

Antibody function by class and sub-class

Answer 4

Question 5

What do you know about IgE?

Answer 5

Minimal production unless atopic (sensitive to allergies and produce larger quantities). Important for antiparasitic activity and allergy.

Question 6

What do you know about IgA?

Answer 6

4.6g of IgA per day is produced!
Vast majority is in the gut SigA.
Present in mucous tears and saliva. Great for neutralisation.
Formed into a dimer which helps it to be secreted.
The receptor helps it to cross from the lymph into the gut.

Question 7

What do you know about IgG?

Answer 7

Four sub types 1, 2, 3 and 4.

Question 8

Somatic hyper mutation - changing binding sites and the b cell develops more antibody so has higher affinity for the antigen.

Class switching which generates the diff classes of antibody:
This happens during infection in the early stages - it will use IgM in early stage then IgG, IgA and IgE which have higher affinities for the antigen than the original IgM did.

Answer 8

Stages of infection can be determined by the presence of the particular antibody.

Question 9

How do we detect and measure the functions of antibodies with an ELISA?

Answer 9

Most common technique is ELISA.
The antibody is LINKED to an enzyme and uses a detection antibody to look for the reagent of interest (antibody to the pathogen or the pathogen itself).
Can insoluble antigens be used with ELISA? No.

Question 10

What is ELISA an acronym for?

Question 11

How do we detect and measure the functions of antibodies with immunohistochemistry?

Answer 11

Immunohistochemistry - are antibodies binding to the surface of tissues? The stage of disease can be detected by the antibodies produced. They are insoluble antigens so cannot be used with ELISA.

Question 12

How do we detect and measure the functions of antibodies with agglutination reactions?

Answer 12

Agglutination reactions:
Used in blood typing eg looking for an AB which can agglutinate blood.

Question 13

How do we detect and measure the functions of antibodies with immune complex formation?

Answer 13

Immune complex formation with agar and electrophoresis? CHECK THIS.

Question 14

How do we detect and measure the functions of antibodies with flow cytometry.

Answer 14

Flow cytometry

Question 15

Abnormal immune function

Answer 15

Abnormal immune function:

Varies in severity

Antibody deficiency – genetic. IgA def is the most common inherited deficiency. Is this why there is a genetic link to autoimmunity? The antibody response doesn’t produce IgA. IV immunoglobulin can be used to restore normal AB function.

Autoantibody – causes can be genetic and environmental. Deficiency in thymus. Environmental factors are common due to a pathogen response which is cross reactive with autoantigens. Molecular mimicry after initial infection with a pathogen. eg EBV.

Excess antibody creates immune complexes = chronic inflammation - chronic inflammation is the persistence of immune complexes being formed. Often lodged in the kidney and can result in renal disease. (CAN THIS BE LINKED TO GUT PERMEABILITY AND CASEIN AND GLIADIN LEAKS INTO BLOODSTREAM, CREATING MOLECULAR MIMICRY).

Neoplasms (bone marrow /leukaemia /lymph node) – may cause antibody excess (monotypic) or deficiency. Diverts and limits the devpt of other cells from bone marrow.

Question 16

Can there be insufficienceies in the complement?

Answer 16

Very rare.

Question 17

What are the functions of the complement system?

Answer 17

Function:
Cascade of factors C1-C9 triggered by antibody, bacteria or cell damage
Protease activity to generate a and b components
Sequence results in formation of membrane attack complex to lyse target cells.

Question 18

How may pathways are in the complement and can you name them?

Answer 18

Three:

Classical pathway ;

Lectin pathway:

Alternative pathway:

Question 19

What would the detection and measurement of the complement system achieve?

Answer 19

Could be supplemented.
A def would be v rare - infnats would be susceptible to recurrent infections (ENT and meningitis).
C2 def is most common.
Hyepractivity of complement - C1Q inhibitor def. this limits the inhibitor and the complement is hyperactive - sweeling odema, etc.

Question 20

Why measure complement?

Answer 20

Components of complement are int he liver. So lack of complement = problem of liver.
Chronic inflammtion = can run out of complement eg C4. Measuring would be important with this.

Question 21

Why use an activity assay?

Answer 21

ELISA gives levels of protein recognised by the antibodies used.
Not all protein is active so ELISA can be an overestimate of activity.
Antigens recognised by ELISA can be missing on an active protein so ELISA can be an underestimate of activity.

Question 22

Describe phagocyte types and their function:

Answer 22

Neutrophils
Recognise pathogens (directly and indirectly) with the antigen’s pathogen associated molecular pattern (PAMP).
Phagocytose and digest.

Monocytes and macrophages
Recognise pathogens (directly and indirectly) can be present the molecule after it has been digested.

Phagocytose and digest
Present antigens

Question 23

How can phagocytes be measured?

Answer 23

Activity of phagocytes can be assessed by
Uptake of labelled particles (microscopy or flow cytometry)
Generation of cytokines (ELISA)
Generation of cytotoxic molecules (e.g. generation of ROS such as superoxide or RNI such as nitrous oxide)
ROS – Fluorescence
RNI – Griess reagent
These can kill microorganism in the vacuoles in the macrophage.

Question 24

Name the phagocytes

Answer 24

Neutrophils, monocytes and macrophages

Question 25

GLWhat do phagocytes do?

Answer 25

Engulf
Kill
Digest

Question 26

How would NK cells be measured and what were they previously called?

Answer 26

LGL cells. Large granulor lipids?

Functions - to kill tumour cells and virus infected cells. reduces their experession of MHC class 1.
Add a radioactive label or enzyme to target (tumour) cells with NK cells and this will detect.

Question 27

A gentetic defect in NK cells can lead to an EBV or HPV overgrowth - it cant prevent the re-emergence. This can be reactivated and drive malignencies.

Answer 27

Genes
IRF8 (inteferon response factor)
RTEL1
FCGR3A

Question 28

What are the functions of T Cells?

Answer 28

CD8 T cells (cytotoxic T cells)
Killing of virus-infected cells.
Killing of cells with intracellular bacterial infection.
Killing of tumour cells.
Recognise peptides on the surface of infected cells.

CD4 T cells (helper T cells – Th)
Help for CD8 activity (Th1) produces alpha 1g
Help for antibody production (Th2). Produces IL4
These produce diff cytokines
[TH17?? Is this in the same subset as CD4Tcell?]

Question 29

What are the 3 methods for measuring T Cells

Answer 29

For CD8 T cells (cytotoxic T cells)
Cytotoxicity assays (as for NK cells)
Cytokine release (ELISA, ELISPOT)

For CD4 T cells (helper cells)
Proliferation assays (3H-thymidine, MTT, BrdU, CFSE /Violet)
CD4 cells will proliferate in response to an antigen (3H thymidine is in RDA not DNA so will stimuate cell proliferation).
Cytokine release (ELISA, ELISPOT).

Question 30

T cells and abnormal immune function

Answer 30

Genetic
Susceptibility to virus, fungus, bacterial and parasite infection.
Susceptibility to autoimmune disease.
Environmental.
Effects on T cells e.g. HIV.
Trigger for autoimmune disease.
Age.
Diminution of production with age - the thymus diminishes by age.
Re-emergence of latent infection e.g. TB, CMV, Shingles and other Herpesviruses.

Question 31

Name the effectors of the immune system

Answer 31

Antibody
Complement
Phagocytes
NK cells
T cells

Question 32

From slide 28

Question 33

Slide 31: Name some immune-related measurements in infectious disease

Answer 33

General tests such as CRP identify presence of infection
Specific tests in collaboration with microbiology.

Eg: antibody to Hepatitis B (first indication of infection in blood for ab takes months - it normally takes 2 weeks to make ab’s), antibody to SARS CoV2 (high IgG and high IgA response have poorer outcomes - the immune system is heightened as is causing the damage as a reaction to the pathogen therefore is important to measure the class and antigen).

Measure the class and the specificity to gauge the stage of infection and which antigens are recognised to guage the severity.

T cells
Measure by proliferation to stimulation
In HIV, CD4 positive t-lymphocytes decline and CD8 do what??? e.g. HIV
Tcell below 50/ml then the patient will develop AIDS.
So the marker function useful to measure here.

Functional assays
Complement - see if there is a deficiency in the system.
Phagocyte function

Question 34

IMMUNOCHEMISTRY:
Name some molecules which will give an immune status from the blood:

Answer 34

Paraprotein/Myeloma Screen (GAM; SPE; SFLC)
Albumin
B2Microglobulin - produced in higher quantities when inflamed. Produced from tumours.
CRP (C reactive protein)
Cystatin C
Serum protein electrophoresis
Immunofixation (presentation serum)
Immunofixation (presentation urine)
Immunoglobulins (IgG, IgA, IgM)
IgD
Plasma viscosity
Paraprotein quantitation by densitometry
Serum creatinine
Serum free kappa/lambda & ratio
Total protein

Question 35

ANTIBODY DEFICIENCY:
Name some molecules which will give an immune status from the blood:

Answer 35

Immunoglobulins (IgG, A and M)
IgG subclasses (IgG1,2,3 and 4)

Functional antibodies:
Tetanus
12 Pneumococcal serotypes,
Meningococcal C serotype,
Haemophilus Influenza b

Question 36

COMPLEMENT ASSAYS: Which assays are useful to run when suspecting a complement deficiency?

Answer 36

• C3/C4
• C1 esterase inhibitor (Immunochemical and functional)

Question 37

Why is it useful to measure analytes?

Answer 37

To define /refine autoimmune diagnosis:
- Many diseases share symptoms and autoantigens
e.g. Sjogrens syndrome and Rheumatoid Arthritis (RFAb)
- Antibody profile changes as disease progresses
e.g.Coeliac Disease May only have AB’s to gliadin early on in the disease rather than TGlutaminase.

To investigate immune deficiency:
- Complement deficiency (rare)
- Ig deficiency (1/500)
- CGD (neutrophil and macrophage function)

To investigate immune status:
- C-reactive protein (inflammation)
- β2-microglobulin (lymphocyte activation, renal function)
- IgE and Mast Cell Tryptase (allergy)

Question 38

Flow Cytometry

Answer 38

Cyto: cell meaning “hollow” or “container”.

Metry: suffix denoting the science of measuring.

Flow: pertains to liquid environment of cell sample and its passage through the cytometer.

Answer 39

It measures a SINGLE STREAM OF CELLS which passes through a laser beam, one cell at a time.

Question 40

M

Answer 40

Scatter - can be forward direction or a large deviation where the light has been refelcetd off of it.

Fleuroscence - natural or due to presence of a fleuro compound which we have introduced or created an attachmenet with binding a fleurochrome or antibody to a marker. The cell will emit fleuro light when eliminated.