L13: Autoimmune diseases 2 Flashcards
1
Q
Molecular mimicry - viral infection
A
- Presentation of viral peptides to a CD4 T cell via MHC 2, causing T cell activation
- The viral peptides happen to be similar to a host-derived peptide; the T cell would normally recognise these peptides, but would not react to them
- The process depends on having the correct MHC molecules to present this critical epitope that is common to both virus and host (inherited)
2
Q
Effect of T cell activation
A
- The activated T cell now reacts strongly to the self-peptide and initiates inflammation
3
Q
What are some examples of molecular mimicry
A
- Autoimmune haemolysis after mycoplasma pneumoniae
- Rheumatic fever
4
Q
What is autoimmune haemolysis
A
- Mycoplasma antigen has homology to ‘I’
- IgM antibody to mycoplasma may cause transient haemolysis
5
Q
Rheumatic fever
A
- Inflammatory disease occurring after streptococcal infection affecting heart, joints, skin and brain
- Anti-streptococcal antibodies believed to cross-react with connective tissue
- Even for these ‘best examples’ the target antigens are not well-defined; for other diseases, the paradigm remains rather theoretical
6
Q
Features of type 1 diabetes
A
- Lack of insulin impairs cellular update of glucose, leading to polyuria, polydypsia, polyphagia and weight loss
- Onset at any age, but typically childhood
- Disease prevalence around 0.8%; rising by around 5% per annum
- Treatment by injection of insulin and diet
7
Q
What is monogenic diabetes
A
- Can present with a similar phenotype but requires different management
8
Q
Type II diabetes mellitus
A
- Older onset, insulin secretion, ketoacidosis less likely and insulin not necessarily required
9
Q
Immunology of type 1 diabetes
A
- Islet cell antibodies detectable for months to years before the onset of clinical disease
- HLA associations
- Early pancreatic biopsy shows infiltration with CD4/8 T cells
- Although antibodies present, they do not appear to be directly relevant to destruction of the pancreas
- By the time patient has established diabetes, generally no active inflammation in pancreatic biopsy
10
Q
Relevance of genetics - type 1 dm
A
- Genetic background definitely important - concordance in monozygotic twins is close to 100% if they are observed for long enough
11
Q
Major defined genetic risk factors - T1DM
A
HLA class II alleles:
- DR3 or DR4 relative risk is 6
- DR3 and DR4 relative risk is 15
- Relative like coeliac disease, believed that these molecules are required to present relevant islet cell antigens to CD4 T cells
- Autoimmune response may occur if appropriate T cell receptors are present, together with other genetic and environmental co-factors
12
Q
Precipitating events
A
- Autoantibodies to islet cell antigens present for months-years before onset of clinical disease
- Gap between initiation of disease and its presentation makes identification of triggers difficult
- Much of the data is epidemiological
13
Q
Some evidence for coxsackie virus
A
- Stronger immune response to virus in cases compared to controls
- Viral infection can cause pancreatitis in mice and humans, and precipitate autoimmune diabetes in mouse models
- Protein 2C from coxsackie virus has homology with islet cell antigen glutamic acid decarboxylase (GAD)
14
Q
Development of AID
A
MHC background - critical for some diseases in determining which peptides can be presented
T cell receptor repertoire - critical in determining whether peptide-MHC complex can be recognised
Infection - May influence the activation of T cells and b cells that are potentially auto reactive
- Likely to be myriad of other genetic and environmental factors
15
Q
Autoimmune serology for diagnosis
A
Some autoantibodies have diagnostic value
- In some cases, the antibodies are pathogenic
- In others, they are simply a bystander effect
16
Q
Three methods of detection via autoantibodies
A
- Indirect immunofluorescence
- Solid-phase immunoassay
- Direct immunofluorescence
17
Q
Indirect immunofluorescence
A
1) Incubate - Patient serum containing (or not) relevant antibodies
2) Detect - Add detection antibody labelled with fluorescent marker
3) Read - Look for fluorescence under microscope
18
Q
What does the glass slide in indirect immunofluorescence contain
A
- Tissue of interest, harvested from animal source
19
Q
Link between ketoacidosis and type 1 dm
A
- Increases risk of ketoacidosis
20
Q
Treatment for type 1 DM
A
- Requires insulin
- Monogenic diabetes and type 2 diabetes require a different approach
21
Q
ELISA practical summary
A
- Antigen-coated well (wash)
- Specific antibody binds to antigen (wash)
- Enzyme-linked antibody binds to specific antibody (wash)
- Substrate is added and converted by enzyme into coloured product; the rate of colour formation is proportional to the amount of specific antibody
22
Q
Plate used in the ELISA practical
A
- The practical uses a plastic 96 well plate
23
Q
Antibody used in the ELISA practical
A
- The first step is to coat each well with tTG antibody
- To do this, tTG solution is simply added to the wells; because it’s a protein, it sticks to the plastic
- Excess tTG is then washed off
- The next step is to ‘block’ the well, using milk powder solution - this gets rid of the spare spaces on the plastic that biological molecules can bind to
- After blocking, excess milk solution is washed away (technique is important here)
24
Q
ELISA - principles of expt
A
- In the next step, your patient samples are added to the wells
- The samples are incubated, to allow time for tTG antibody in the samples to react with the tTG antibody
- Excess antibody is washed off
- If there was tTG antibody in the sample, it will now be captured by the immobilised tTG antigen - however, this reaction is of course invisible at this stage
25
What is a secondary antibody
- The secondary antibody is an anti-IgA antibody, which binds to IgA Fc regions
- It's covalently linked to an enzyme such as horse radish peroxidase
- Excess secondary antibody is washed away
26
What is added after the secondary antibody
- A substrate is then added
- The substrate reacts with the horse radish peroxidase that is bound to the secondary antibody, which in turn is bound to antibody tTG antibody that was present in the patient sample
- This produces a colour change, which you will be able to see with the naked in strong reactions
- The amount of colour change can be measured using a photocell
27
What should be set up along with the photocell in the ELISA practical
- A calibration curve
- This involves setting up wells containing known concentrations of tTG antibody
- The photocell readings from each well are used to create a standard curve, to which you can relate the values from your patient samples
- You will also run positive and negative controls, to show the assay has worked correctly
- Note that everything is run in triplicate, to improve precision
28
Production of solutions with tTG - ELISA
- A range of tTG concentrations are produced from solution A, which is neat rTG solution
- This is done by serial dilution, taking 500 microliters of A and adding to 500 microliters of buffer in B, then repeating through tubes C to H
29
Detecting antibodies bound to tissue: direct immunofluorescence
- Take a biopsy of affected tissue eg skin, kidney; if damage mediated by antibody, antibody will already be stuck to its antigen in the tissue
- Add detection antibody labelled with fluorescent marker
- Look for fluorescence under microscope using UV light
30
What is bullous skin disease: pemphigoid
- Thick-walled bullae, rarely on mucus membranes
- Fulfils criteria for antibody-mediated disease
- Target is antigen at dermo-epidermal junction
- Linear deposition of antibody, which activates complement producing skin dehiscence and tense blister
31
Coeliac disease diagnosis
- Antibodies binding to the endomysium of smooth muscle fibres found to have a strong disease association with coeliac disease
- Subsequently target antigen found to be tissue transglutaminase (tTG), which is now expressed in recombinant systems to provide antigen for modern immunoassays
- HLA typing also increasing utilised - absence of HLA DQ-2/8 makes coeliac disease very unlikely(ie high negative predictive value)
32
Where is vitamin B12 absorbed
- Terminal ileum
33
What does absorption of vitamin b12 require
- Absorption requires a co-factor called intrinsic factor which is secreted by the gastric parietal cells
34
Pernicious anaemia - pathology
- Pernicious anaemia describes autoimmune destruction of the gastric parietal cells
- Loss of intrinsic factor abrogates B12 absorption
- Liver stores around 2 years supply of B12
35
What happens once B12 stores are depleted
Once depleted, multiple possible manifestations:
- Anaemia
- Neurological
- Subfertility
36
Treatment of AID
Often preferable to treating the immunology:
- Immunosuppressive drugs are toxic
- By the time the disease is overt, the damage may already have been done and immunosuppression may be unhelpful
37
Examples of AID treatment
- Thyroxine for overactive thyroid
- Carbimazole, surgery or drugs for thyrotoxicosis
- Insulin for diabetes
- B12 for pernicious anaemia
However, in some AID, particularly non organ-specific, treatment of the immune system is the best option
38
Drugs used for immunomodulation
Used particularly for 'multi-system' autoimmune diseases
- Systemic corticosteroids
- Small molecule immunosuppressive drugs (eg methotrexate, azathioprine, ciclosporin)
- High-dose intravenous immunoglobulin (mechanism poorly understood)
39
What is plasmapharesis
- Removes antibodies from the bloodstream therefore may be useful in antibody-mediated diseases
