1. Part II project by Katherine Warre-Cornish
Inhibition of ganglioside biosynthesis by imino sugars reduces binding of Guillain-Barré Syndrome autoantibodies
Part II project by Katherine Warre-Cornish

2. Guillain Barré syndrome:
Auto-immune disease
Presence of anti-ganglioside antibodies
Causes immune system to attack neurons
Symptoms: limb-weakness and pain, impaired reflexes, coordination and respiration
85% of patients make a full recovery within 6 to 12 months
Mortality rate of approximately 5% and 7-15% of patients left with persistent minor symptoms
Incidence:1-3 cases in people per year
Current therapies:
plasma exchange (total exchange of about 5 plasma volumes)
high dose intravenous IgG

3. Causes of GBS
Antecedent bacterial infection (1-2 weeks before GBS onset)
Most commonly associated with Campylobacter jejuni
Haemophilus influenzae? Cytomegalovirus? Epstein-Barr?
Molecular mimicry
Lipo-oligosaccharide (LOS) is the principal cell surface structure that is recognised by the host.
Forms of LOS have been isolated with identical terminal sugar residues to various human gangliosides

4. Human gangliosides and C.jejuni lipo-oligo saccharides
Bacterial
Human
GM1-like LOS
Ceramide
GM1
Core-lipid A
GD1a-like LOS
Core-lipid A
Ceramide
GD1a
GD1c-like LOS
Core-lipid A
Ceramide
GQ1b
Glucose
Galactose
N-Acetylneuraminic acid
N-Acetylgalactosamine

5. Immune system attacks neurons with high levels of ganglioside
Due to cross reactivity with LOS, antibodies against self ganglioside are present in patients.
IgG antibodies to GM1 and GD1a strongly associated with GBS
IgG antibodies to GQ1b associated with Miller-Fischer syndrome (MFS) (caused similarly to GBS, different neurological symptoms)
Neurological features of the disease and sites of neurological damage are determined by the antibody specificities.

6. Is it possible to treat an auto-immune disease by metabolically decreasing levels of auto-epitope?

7. N-alkylated imino-sugars: inhibitors of ceramide specific glucosyltransferase
Glycolipid Synthesis
NB-DNJ and NB-DGJ are glucosyl transferase inhibitors
NB-DNJ: IC50 of 20.4uM
NB-DGJ: IC50 of 30uM
NB-DNJ/ NB-DGJ Inhibition
Butters et al, 2000, Chemical Reviews V100, 12, pp

8. Results

9. ELISA – determination of the degree of patient vs control sera antibody binding to gangliosides
GM1
GM2
GD1a
GQ1b
GD1b
12 patient sera (black dots) and 4 control sera (white dots) tested against 5 GSLs
Most significant differences between patient and control Ab binding observed with GM1 and GQ1b

10. ELISAs carried out using GM1 and GQ1b with increasing dilution factors of selected patient sera

11. Patient sera binding curves to GM1

12. Patient sera binding curve to GQ1b

13. Does inhibition of ganglioside biosynthesis reduce antibody binding?
Thin Layer Chromatography: demonstrate decrease in levels of antibody binding to GM1 in extract from drug treated cells
Glycolipids were extracted from RAW cells (high GM1 content) treated with a range of concentrations of drugs: NB-DNJ and NB-DGJ
Components were separated by TLC. Extracts were run parallel to GM1 standard.

14. Immuno-overlays: detection of patient antibody binding to TLC plate
Analogous to a western blot
TLC plates were incubated a 1:1000 dilution of patient sera
Plates were then incubated a 1:1000 dilution of secondary anti-human antibody, labelled with horse-radish peroxidase
Bands were detected by ECL
Horse-radish peroxidase
Goat anti-human antibody
Patient sera IgG
GM1

15. TLC - results
Orcinol staining (proportional to carbohydrate concentration) confirms high levels of GM1 in RAW cells
Conc(uM)
GM1 Standard 10 20 50
100
1000 10 50
100
1000
Drug:
NB-DNJ
NB-DGJ

16. TLC - results Patient sera Control sera Drug: NB-DGJ NB-DNJ Conc(uM)
GM1 Standard 10 20 50
100
1000
NB-DNJ
Conc(uM)
NB-DGJ

17. Metabolic inhibition reduces patient serum IgG binding.
Conclusions from TLC
Decrease in patient antibody binding as concentrations of NB-DNJ or NB-DGJ are increased.
Effect appears to be more pronounced with NB-DGJ than NB-DNJ
Physiologically, IgG binding is enhanced by glycolipid clustering. Relatively low concentrations of drug could disrupt this clustering (TLC- glycolipids are artificially clustered together)
Lower drug concentrations may be required than results suggest.

18. Current work: Can this approach be extended to other cell lines…?
Glycosphingolipids were extracted from PC12 cells treated with NB-DNJ and NB-DGJ
Extracted GSLs fluorescently labelled
Separated by HPLC
Spectra obtained
Levels of gangliosides quantified by measuring peak areas

19. HPLC results NB-DNJ
GQ1b
GD1b
GD1c
GM1a
Gb3

20. HPLC results NB-DGJ
GQ1b
GD1b
GD1c
GM1a
Gb3

21. General Conclusions
Use of a metabolic inhibitor of GSL synthesis could potentially be used as a treatment for Guillain-Barré syndrome
This depends on:
Levels of dosage required for decreased antibody binding – can these be tolerated physiologically
Turnover rate of gangliosides in the affected neurons?
NBDGJ appears better than NBDNJ
Further research:
additional, eg neuronal cell lines
Animal models – (GBS mice)

22. Acknowledgements
Dr Chris Scanlan
Dr Terry Butters