Autoantibodies in the CNS

Question 1

general facts

Answer 1

• 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
• Can be against intracellular protein  T-cell will kill cell (example: cerebellar encephalitis)

Question 2

NMDAr encephalitis

Answer 2

• Teratomas express NMDA receptors at the cell membrane
• BUT only ~50% of NMDAR encephalitis cases are related to tumors
• Antibodies against NMDA receptor 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
• Favours internalisation and degradation

Question 3

Ig crossing BBB

Answer 3

• 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

Question 4

diapedesis

Answer 4

1. floating
2. tethering via selectins
3. adhesion and crawling via integrins (ICAM)
4. diapedesis

Question 5

witebsky’s postulates

Answer 5

1. clinical manifestation detectable in blood/tissue
2. target a protein
3. antibody transfer must replicate the disease
4. elimination of autoimmunity must improve symptoms

Question 6

ELISA

Answer 6

• 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

Question 7

immunohistochemistry

Answer 7

1. Apply serum with suspected antibody to rat brain
2. Apply secondary antibody with biotin
3. Add enzyme to create reaction
4. Add substrate
5. Analyse
   - Gives insight into location of antigen

Question 8

cell based assay

Answer 8

1. Lumbar puncture  collect CSF
2. Produce plasmids that contain the gene for mGluR1 and a strong promotor
3. Grow Hek2-cells to desired cell density
4. Transfect Hek2-cells with the plasmid DNA
5. Wait for the Hek2 cells to highly express mGluR1
6. Add the CSF to the Hek2 cells
7. Then wash away the CSF
8. Add anti-human-antibody with fluorescent marker
9. Wash
10. Add commercial anti-mGLUR1 with fluorescent marker (other colour)
11. 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

Question 9

neuromyotonia

Answer 9

1. Antibodies against VGKC
2. No repolarization after AP
3. 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

Question 10

radioimmunoassay - neuromyotonia

Answer 10

1. Harvest dendrotoxin
2. Harvest and homogenize animal brain (contains VGKC)
3. Radiolabel (iodine) the dendrotoxin
4. Dendrotoxin is added to brain to label the VGKC
5. Patient serum added
6. Precipitate all antibodies (using secondary antibody) by crosslinking
7. 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)

Question 11

Morvan’s syndrome

Answer 11

• Same VGKC antibodies but now found in the CNS
• High levels of antibodies (lower  only neuromyotonia)
• Psychiatric and neurological issues
• VGKC complex is not just 1 protein but is a few together
• LGI1 (reversible limbic encephalitis), CASPR2 (most common) and Contactin2 can be targeted

Question 12

PERM

Answer 12

• 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

Question 13

hypersensitivity reactions

Answer 13

1. Allergies  IgE (asthma)
2. Cytotoxic and antibody-dependent  IgM but mostly IgG1/3 (MG, rheumatoid arthritis)
3. Immune complex disease  IgG (Lupus)
4. Delayed-type hypersensitivity cell-mediated immune memory response, antibody-independent  T-cells (MS)

Question 14

IgG isotypes

Answer 14

IgG1 and 3 activate complement
IgG4 does fab arm exchange