Week 6 Flashcards
What is an autoimmune response?
A response directed against self-antigens that causes tissue damage or altered physiological function resulting from the autoimmune response.
What is attacked in terms of an autoimmune disease?
Usually the host proteins but can be nucleic acids.
What is self tolerance?
The bodies protective mechanism that prevents the development of autoimmune diseases.
In self tolerance, describe gene rearrangement.
Random
What does the random rearrangement of genes in self tolerance lead to?
Generation of lymphocytes that are specific for self-antigens.
What happens to self-reactive T and B cells during self tolerance?
They are destroyed or down-regulated.
Where does central tolerance occur?
Thymus (T cells) or Bone Marrow (B cells)
What is the mechanism of central self tolerance?
Clonal deletion
What is the site of peripheral self tolerance?
Everywhere in the body.
What is the mechanism for peripheral self tolerance?
Anergy, cell death or immune deviation.
Where do T cells originate and develop?
Originate in the bone marrow but develop in the Thymus.
In central self tolerance, what is the first step?
Antigen presenting cells present self-peptides to T cells and the cells can then undergo selection or apoptosis.
In central self tolerance, which cells survive positive selection?
Only the cells whose receptors interactions with self-MHC and self-peptide low affinity survive.
In central self tolerance, what happens in negative selection?
Cells that bind with high affinity to the self-MHC and peptide are deleted.
In central self tolerance, what cells undergo apoptosis?
Cells that fail to interact with self MHC and peptide.
What may happen to some self-reactive T cells that encounter self antigens during central self tolerance?
Some self-reactive T cells that encounter self-antigens in the Thymus develop into T regulatory cells followed by B cell initiated changes in receptors in the bone marrow.
Why is T cell tolerance often regarded as more important than B cell tolerance?
CD4 T cells have a central role in controlling nearly all immune responses. Whereas most B cells will not be able to produce autoantibodies unless the receive appropriate T cell help.
What happens to B cells in the bone marrow, whose receptors are cross-linked by self-antigens?
They are deleted or become self-tolerant.
What happens to T cell’s that do not have any T cell receptors expressed during self tolerance?
Cellular death
What happens to T cells which have T cell receptors with no recognition of self MHC during self tolerance?
Death by default
What happens to T cells which have T cell receptors with strong recognition of self MHC in self tolerance?
Negative selection (signalled death).
What happens to T cells which have T cell receptors with weak recognition of self MHC in self tolerance.
Positive selection
What happens to T cells if they are positively selected for during self tolerance?
Survival and maturation
What are the processes that can occur to peripheral auto reactive cells in peripheral self tolerance?
Clonal detection
Clonal anergy
Regulation and supression
What may cause clonal deletion of self-reactive cells in peripheral self-tolerance?
Physical elimination or apoptosis of cells.
What may cause clonal anergy of self-reactive cells in peripheral self tolerance ?
Lack of co-stimulation or low affinity antigen binding leads to cells not being activated. This is achieved through signalling blocks and/ or inhibitory receptors.
What is immune privilege in terms of peripheral tolerance?
Limited lymphatic drainage, low levels of MHC class I expressed and expression of fasL to kill infiltrating T cells.
Why may expression of fasL cause immune privelledge?
This kills infiltrating T cells and protects the eyes and such like.
How do B cells become anergic during peripheral self tolerance?
When exposed to large amounts of soluble antigen, they down regulate their surface IgM and become anergic.
What is meant by the term anergic?
Lack of immune reaction to antigens.
How do B cells carry out apoptosis during peripheral self-tolerance?
B cells up-regulate the Fasomolecules on their surface and then interact with Fas-ligans-bearing cells resulting in apoptosis.
What happens to B cells which bind to large amounts of soluble antigen with low affinity binding?
They become activated to re-expresses RAG-1 and RAG-2 genes. These genes cause them to undergo DNA recombination and change their antigen specificity.
What are the steps of autoimmunity?
Susceptibility phase
Initiation phase
Propagation Phase
Regulation / Resolution phase
When does the susceptibility phase occur in autoimmunity and what happens during this phase?
Before disease
One or several preconditions for later initiation are satisfied. These tend to be genetic factors or altered immune signalling thresholds that cause disease.
What influences the susceptibility phase of autoimmunity?
Genetic factors that impair regulation and human leukocyte antigen associations.
When does the initiation phase of autoimmunity begin and what indicates its onset?
Begins before the onset of clinical disease and is marked by the presence of an autoimmune response.
What happens (in general) in the initiation phase of autoimmunity?
Hidden epitopes are revealed and new epitopes are created.
What are the possible mechanisms of the initiation phase of autoimmunity?
Post-translational modification.
Proteolytic cleavage of intracellular antigens during cell death and mutation.
Truncations.
Splicings.
What molecular mimicry is used in the initiation phase of autoimmunity?
Pro-inflammatory signals with infection can activate low-affinity, self-reactive T and B cells.
What marks the onset of the propagation phase of autoimmunity?
The propagation of specific immune responses.
What often initiates the propagation phase of autoimmunity?
Tissue damage provides a further drive in the autoimmune response.
What happens during the resolution/regulation phase of autoimmunity?
Immunoregulatory pathways are activated activated, potentially resulting in a natural inhibition or resolution of the clinical disease.
What are the 2 different categories of autoimmune disease?
Organ specific
Non-organ specific
What methods are often used to detect autoantibodies?
Immunofluorescence Radioimmunoassay ELISA Immunoblot Multiplex Immunoprecipitation reactions
What are the types of immunoprecipitation reaction?
Agglutination
Double immunodiffusion (DID)
Counterimmunoelectrophoresis (CIE)
What can be used as antigens in lab tests for autoantibodies ?
Cells
Tissues
Purified native or recombinant antigens.
How are cells prepared for antigen testing in terms of testing for autoantibodies?
Cells are extracted via chemical or mechanical extraction.
Chemical: Extracted using a lysis buffer that can preserve or destroy the protein structure.
Mechanical: Extraction by homogenisation or freeze thaw cycles.
How are tissues prepared for antigen testing in terms of testing for autoantibodies?
And how are they analysed for antibodies?
They are frozen or embedded in paraffin and analysed with monoclonal antibodies using patient serum to determine if they have antibodies.
How are native or recombinant antigens prepared for antigen testing in terms of testing for autoantibodies?
They are coated to plates, beds, magnetic particles, micro spheres or micro spots and then purified.
Why must native or recombinant antigens be purified to be used in lab tests?
This is crucial to maintaining conformational epitopes, tertiary and quaternary structure and post translational modifications.
Also increases the sensitivity and specificity of antibody tests.
What is a qualitative result?
Has a positive or negative result.
What is a semiquantitative result?
Dilutions or titres (e.g. 1 in 40)
What is a quantitative result?
Numerical value
What is hybridoma in terms of antisera?
A single clone of B cells are fused with myeloma cells which immortalises them.
What is polyclonal antisera purified by?
Anitbodies
What is monoclonal antisera purified by?
Hybridoma procedure
What are the advantages and disadvantages of polyclonal antisera being used in immunoassays?
+ Cheap, quick production
- Low reproducibility
What are the advantages and disadvantages of monoclonal antisera being used in immunoassays?
+ Reproducible, specific properties
- Expensive, slow production
Name some qualitative assays for antigen/antibody reaction
Immunoprecipitation
Immunodiffusion
Counter-
Immunoelectrophoresis
Immunofluorescence Microscopy
Outline what happens during immunoprecipitation
A specific antigen will react with a specific antibody to form a precipitate.
The immune complex formed is usually insoluble and so can be seen.
What antigen types are used for immunoprecipitation?
Purified native or recombinant
What is RF in terms of immunoprecipitation?
An antibody which reacts with the Fc region of IgG antibodies.
Outline what happens during immunodiffusion.
Electrophoretic diffusion of antigen through a gel towards an anode (+)and the counter flow of antibody towards a cathode (-) when an electric current is applied across the Gel. The antibody and antigen cause a precipitation line if they bind.
What are the positives of immunodiffusion?
+ Use cheap, purified antigen, high specificity, can test several antigens at once, faster than double diffusion, easy to perform.
What are the negatives of immunodiffusion?
- only a modest sensitivity, requires a large volume of control sera, time consuming, small work loads only, requires source of purified antigen.
What gel is usually used for immunodiffusion?
Agar or polyacrylamide gel
What is the overall purpose of direct immunofluorescent microscopy?
To investigate Ig and complement deposition in tissues.
Also provides histological information.
What is the overall purpose of indirect immunofluorescent microscopy?
Tests for antibodies in patients serum to help diagnose and monitor autoimmune diseases.
What samples must be used for direct immunofluorescent microscopy?
Tissue biopsy samples
What sample types must be used for indirect immunofluorescent microscopy?
Tissue selections or cells
Outline how immunofluorescent microscopy works.
Samples are stained with a fluorochrome. A filter or mercury bulb emits blue light which illuminates the slide from above.
Wha can be used as a source of blue light in terms of immunofluorescent microscopy?
The filter or high pressure mercury bulbs.
What type of immunofluorescent microscopy is used for antinuclear antibodies?
Indirect
Name some types of quantitative assaay.
ELISA
Immunoblotting
Multiplex technologies
What does ELISA stand for?
Enzyme Linked Immunosorbent Assay
What are ELISA tests used for?
To detect auto-antibodies.
Outline how an indirect ELISA test is carried out
Antigen coated well.
Specific antibody added from patient sera.
May bind.
Enzyme-conjugated secondary antibody added.
Substrate is added for fluorescent conjugation.
Stock solution is added.
Blue colour indicates positive result.
Optical density measured.
What indicates a positive result in an ELISA test?
Blue colour
What are the advantages of the ELISA test?
Easily automated, more objective and less technical than immunofluorescence.
What are the disadvantages of the ELISA test?
Sometimes generate less information than IF.
Boarder-line values may leave room for interpretation.
Less sensitive than IF overall.
What is another name for Immuoblotting?
Western Blotting
In immunoblotting, what factors are incubated?
PVDF or nitrocellulose membrane with patient serum rather than with purified antibodies.
Outline the process of immunoblotting
1) Load and separate protein samples.
2) Electrophoretically transfer fractionated proteins onto PVDF membrane.
3) Block the membrane with neutral protein.
4) Incubate the membrane with primary antibody specific to the target protein.
5) Incubate the membrane with HRP-labeled secondary antibody specific to primary antibody.
6) Incubate the blot with chemiluminescent HRP substrate and expose to film.
What are multiplex technologies used for?
Detection of multiple specific antibodies as separate entities at the same time.
Used in clinical laboratory’s for a variety of assays.
What are the different types of multiples technologies?
Planar arrays (Microspots on slides, microplanes and nitrocellulose membranes)
Suspension Assays - Luminex
What are the issues with multiplex technologies only using a small amount of antigen?
This increases the susceptibility to interference.
How does Bio-plex work?
- Beads are infused with varying ratios of fluorescent dyes, creating 100 unique bead sets.
- Beads in each set are coated with a ligand.
- Beads sets are mixed into a single well to allow the simultaneous detection of multiple analytes in a single sample.
- The bound antibodies are detected using secondary-fluorescent labelled antibodies.
- The bead-antibody mixture passes though the detector where lasers are used to identify the bead analyse.
During bio-plex what is used to determine the results?
The concentration of analyte
What are antinuclear antibodies?
Antibodies directed against the nuclear components of the cell
What are antinuclear antibodies used for?
Screening tool for differentiating many connective tissue diseases.
What cell line is used for indirect immunofluorescence for antinuclear antibodies?
Hep-2 cell immortalised cell line.
What are the advantages of Hep-2 over rodent tissue in terms of indirect immunofluorescence?
Much more sensitive substrate so allow identification of many patterns of standing.
Nuclei are much larger and therefore more visible.
What is required in terms of indirect immunofluorescence?
Cell monolayer to allow clearer visualisation of all nuclei.
What are the patterns visualised by homogenous nuclear results during immunofluorescence of antinuclear antibodies?
Diffuse uniform fluorescence of interphase nuclei and mitotic chromatin.
What are the patterns visualised by fine speckled results during immunofluorescence of antinuclear antibodies?
Fine granular striating and no staining of nucleoli or condensed chromatin in metaphase cells.
What are the patterns visualised by HEp2000-Ro60 results during immunofluorescence of antinuclear antibodies?
Prominent staining of the nuceoli in 10-20% of the interphase nuclei.
What are the patterns visualised by coarse speckles results during immunofluorescence of antinuclear antibodies?
Dense large speckles
No staining of nucleoli and condensed chromatin in metaphase cells.
What causes the large, dense speckles in coarse speckled immunofluorescence results?
Heterogeous nuclear riboproteins which co-localise with nuclear RNPs and precursor mRNA.
What type of disease is antibody phospholipid syndrome?
Autoimmune disease
What are the clinical presentations of anti-phospholipid syndrome?
Vascular thrombosis
Pregnancy morbidity
What are the serological indicators of anti-phospholipid syndrome?
Presence of anti-phospholipid antibodies.
Pro-thrombotic phenotype with prolonger clotting in vitro.
What is the target of anti-phospholipid antibodies?
Phospholipid binding proteins or phospholipid protein complexes.
What are the risk factors for development of Rheumatoid Arthritis?
- Obesity
- Smoking
- High red meat consumption
- A previous blood transfusion
- An adverse pregnancy outcome
- Family history of RA
What is used to detect anti-neutrophil cytoplasmic antibodies?
Immunofluorescence screening followed by ELISA
What is MPO?
A critical enzyme in the conversion of hydrogen peroxide to hypochlorous acid.
What is vasculitis?
Inflammation of the blood vessel wall.
What do Anti-neutrophil cytoplasms antibodies cause ?
Damage to the eyes, ears and nose.
In what diseases are anti-neutrophil cytoplasmic antibodies found?
Small vessel vasculitis.
Glomerulonephritis.
Pulmonary haemorrhage.
How are perinuclear anti-neutrophil cytoplasmic antibodies identified?
During ethanol fixation, antigens which are more cationic migrate and localise around the nucleus as they are attracted by the partial negative charges on DNA content. Antibody staining therefore results in fluorescence of the region around the nucleus.
What type of staining would the presence of cytoplasmic anti-neutrophil antibodies cause and on what detection method?
Granular cytoplasmic staining when an ANCA detection method is carried out.
What are endocrine glands?
Glands which secrete hormones directly into the blood.
What does the release of Thyrotopin cause?
It simulates the synthesis and release of thyroid stimulating hormones from the anterior pituitary.
Where is Thyrotropin synthesises?
Hypothalamus
Where is Thyroid stimulating hormone released from?
The anterior pituitary
What effects does the release of Thyroid Stimulating Hormone have?
Stimulates the thyroid gland growth and thyroid hormone synthesis by enhancing synthesis of the iodide transporter, thyroid peroxidase and thyroglobulin.
What type of feedback system is thyroid production?
Negative
What are the symptoms of hypothyroidism?
Lack of energy Weight gain Constipation Sensitivity to cold Dry skin and hair Poor concentration Decrease circulation and respiration
What are the symptoms of hyperthyroidism?
Anxiety Increased perspiration Heat intolerance Tremor Hyperactivity Palpitations Weight loss Frequent bone movements
What cases Hashimoto’s thyroiditis?
Weak human leukocyte antigen associations.
What are the clinical features of Hasimoto’s hyrofitis?
Hypothyroid
T-cell destruction
What is the most common antibody associated with Hashimoto’s disease?
Anti-thyroid peroxidase antibodies
What are the clinical symptoms of Grave’s disease?
Hyperthyroid
Exophthalmos
T cell destruction
What is exophthalmos ?
An abnormal protrusion of the eyeball in the orbit when observed from the side.
What receptor antibodies are most common in Grave’s disease?
Anti-TSH receptor antibodies
What is used to detect hypo/hyper throidism?
Anti-TPO antibody detection.
Outline how Anti-TPO detection works?
Particle agglutination - microsomal extract attached to coloured beads added to serial dilutions of serum. Agglutination if antibody is present.
Outline how an enzyme-linked immunosorbent assay works.
TPO coated wells, serum, anti-human Ig conjugated with horse-radish peroxidase, TMB substrate, reaction stopped with HCl, optical density measure. Colour formed is proportional to the initial concentration of antibody present in a patient sample.
Outline how an Immunocap works
TPO bound to solid phase, serum added, IgG-beta-galactoside-labelled secondary antibody, substrate added.
Outline how an Immunlite works.
Antigen coats beads and monoclonal murine anti-IgG antibodies are conjugated with alkaline phosphatase.
What is autoimmune diabetes mellitus also called?
Insulin dependent diabetes mellitus type 1
What causes Autoimmune diabetes mellitus type 1?
Autoimmune mediated destruction of the B cells of the pancreas.
What is the age of onset of type 1 diabetes?
Below 30 years
What is the age of onset of type 2 diabetes?
30-60 years
What happens to the body weight in people with type 1 and 2 diabetes?
Type 1 - thin
Type 2 - obese
How common is ketosis in type 1 and 2 in diabetes?
1 - common
2 - uncommon
What is ketosis?
Ketosis is a state the body goes into if it needs to break down body fat for energy. The state is marked by raised levels of ketones in the blood which can be used by the body as fuel.
What is the treatment for type 1 diabetes?
Insulin
What is the treatment for type 2 diabetes?
Diet
Oral hypoglycaemic agents
Insulin (occasionally).
What is used to detect autoimmune diabetes detection?
Anti-GAD immunofluorescence on primate pancreas.
ELISA
What is Primary Biliary Cirrhosis and what causes it?
An autoimmune liver disease caused by the destruction of small to medium bile duct leading to progressive cholestasis.
What is Cholestasis?
Blockage of bile flow from the liver to the duodenum.
What clinical characteristics indicate primary biliary cirrhosis?
Raised alkaline phosphatase, bilirubin and hypercholesterolaemia.
Anti-mitochondria antibodies.
Polyclonal increase in IgM.
What does raised alkaline phosphatase indicate?
Primary bilirubin chirrhosis
Hepatocellular disease
What characterises Xanthomas and Xanthelasmas ?
Accumulations of lipid-laden macrophages (collections of cholesterol under the skin).
What issues can anti-neuronal antibodies give?
Demyelinated diseases
Channelopathies
Paraneoplastic syndromes
When do the symptoms of Guilian Barre syndrome peak?
After 4 weeks
What are the symptoms of Guilian Barre Syndrome?
Progressive motor weakness
Muscle weakness and maybe even paralysis.
What is PR3?
Protinase that is a component of the neutrophil granules and secretory vesicles.
