LC 2 - auto-Ab in the CNS Flashcards
1
Q
general facts
A
- Auto-antibody mediated encephalitis
- Involved in psychosis
- 4% of patients only have psychiatric symptoms
- Can be directed against neuronal surface protein –> leads to protein destruction while leaving the cell alive by Ab mechanisms
- Can be against intracellular protein –> T-cell will kill cell (example: cerebellar encephalitis) and the Abs are non-pathogenic
2
Q
NMDAr encephalitis
A
- Teratomas express NMDA receptors at the cell membrane
- BUT only ~50% of NMDAR encephalitis cases are related to tumors
- Antibodies against NMDA receptor (IgG1/3) and/or antibody producing B-cells pass the BBB and induce an increased internalization of NMDR without killing the target cells
- Anti-NMDAr Ig causes:
- Blocking of receptors
- Reduced surface diffusion –> declustering
- Favours internalisation and degradation –> antigenic modulation
- complement is not very likely to be involved in CNS (only 1% of what is present in plasma)
3
Q
Ig crossing BBB
A
- Yes, but to a limited extend.
- Enter with rate of 0.018 mg/min
- turnover of 0.0036/min (comparable to the CSF turnover; four times per day)
- Immunoglobulin G (IgG) levels in the brain of around 1% of the plasma levels and IgG composes 9.8% of the protein in the CSF
- Mechanism unknown. FcRn transport (transcytosis)?
- ApoE plays a role in tight junction functionality
- Moreover: intrathecal synthesis after diapedesis of B cells –> MS
4
Q
diapedesis
A
- floating –> attracted to chemokines (gradient)
- tethering via selectins
- adhesion and crawling via integrins (ICAM)
- diapedesis
5
Q
witebsky’s postulates
A
- clinical manifestation detectable in blood/tissue
- target a protein
- antibody transfer must replicate the disease
- elimination of autoimmunity must improve symptoms
6
Q
ELISA
A
- Enzyme-linked Immunosorbent Assay (ELISA)
1. Antigen coated on the plate
2. Sample applied
3. Antigen specific antibody binds antigen
4. Secondary antibody recognizes and binds primary antibody
5. Marker: enzyme, that transforms a chemical substrate into a coloured dye = colorimetric detection - Advantages: quantitative, robust, low-tech
- Disadvantages: requires soluble antigen, epitopes might be denatured during processing
7
Q
immunohistochemistry - tissue based assay
A
- Apply serum with suspected antibody to rat brain
- Apply secondary antibody with biotin
- Add enzyme to create reaction
- Add substrate
- Analyse
- Gives insight into location of antigen but not used to identify antigen –> cell based assay
8
Q
cell based assay
A
- Lumbar puncture to collect CSF
- Produce plasmids that contain the gene for mGluR1 and a strong promotor
- Grow Hek2-cells to desired cell density
- Transfect Hek2-cells with the plasmid DNA
- Wait for the Hek2 cells to highly express mGluR1
- Add the CSF to the Hek2 cells
- Then wash away the CSF
- Add anti-human-antibody with fluorescent marker
- Wash
- Add commercial anti-mGLUR1 with fluorescent marker (other colour)
- If there is a signal that overlaps with the colour from step 10 that means that there was indeed anti-mGluR1 in the CSF of this patient
- if you test for an intracellular Ab you must permeabalise the cells (triton-X)
9
Q
neuromyotonia
A
- Antibodies against VGKC
- No repolarization after AP
- Continuous hyper-excitability
- Similar to dendrotoxin
- Shaker gene in mutant drosophilae results in similar K-channel blocking and hyper-excitability
- Ion channels/receptors can be affected by toxins or by autoantibodies or by mutations
- PNS, if CNS –> morvan’s
10
Q
radioimmunoassay - neuromyotonia
A
- Harvest dendrotoxin
- Harvest and homogenize animal brain (contains VGKC)
- Radiolabel (iodine) the dendrotoxin
- Dendrotoxin is added to brain to label the VGKC
- Patient serum added
- Precipitate all antibodies (using secondary antibody) by crosslinking
- If there is a signal after washing there were anti-VGKC in the serum
- Antibodies are polyclonal so they do no have to compete al lot with the dendrotoxin (might miss a few)
11
Q
Morvan’s syndrome
A
- Same VGKC antibodies but now found in the CNS
- High levels of antibodies (lowe: only neuromyotonia)
- Psychiatric and neurological issues
- VGKC complex is not just 1 protein but is a few together –> the channel itself is not targetted
- LGI1 (reversible limbic encephalitis), CASPR2 (most common) and Contactin2 can be targeted
12
Q
PERM
A
- IgG1/3 Antibodies against GLyR (inhibitory receptor)
- Channel function:
- ligand-gated ion channels
- present throughout the brain, but are most abundant in the spinal cord and brainstem.
- also the target of the alkaloid strychnine, –> generalized muscle spasms and cramps
- effector mechanisms:
- GlyR antibodies degrade their target by antigenic modulation
- Large proportion of the GlyR antibodies are of the IgG1 and IgG3 isotypes –> activate complement on GlyR-expressing cells (depending on distribution density)
13
Q
hypersensitivity reactions
A
- Allergies –> IgE (asthma)
- Cytotoxic and antibody-dependent –> IgM but mostly IgG1/3 (MG, rheumatoid arthritis)
- Immune complex disease –> IgG (Lupus)
- Delayed-type hypersensitivity cell-mediated immune memory response, antibody-independent –> T-cells (MS)
14
Q
IgG isotypes
A
IgG1 and 3 activate complement
IgG4 does fab arm exchange
15
Q
anti-GAD in autoimmunity
A
- Anti-glutamic acid decarboxylase (GAD) antibodies
o Glu –> GABA conversion is inhibited leading to reduction in GABA production and inhibited inhibitory signaling
This possibly contributes to psychosis, in other cases it just serves as biomarker - Present in a range of autoimmune diseases (stiff person syndrome, cerebellar ataxia, limbic encephalitis, epilepsy and oculomotor dysfunction)
- Non-pathological intracellular protein
o Serves as biomarker - In neurological diseases: high Ab titers
- In diabetes: Low Ab titers
