Inhibition of Ganglioside Biosynthesis by Imino Sugars Reduces Binding of Guillain-Barré Syndrome Autoantibodies. Rhea McGarry Glycobiology Department Oxford University Trinity College

Introduction Autoimmune Disease The immune system must be able to discriminate between self- and non-self antigens, in order to prevent self destruction. This is termed self- tolerance, the loss of which results in autoimmune disease. Antibody mediated autoimmune disease, such as GBS, are driven by a loss of tolerance in both the B and T lymphocytes, as a consequence of molecular mimicry between self and non-self lipo-oligosaccharides (LOS).

Molecular mimicry and autoimmunity The adaptive immune response which continuously monitors for infection may generate a response against an epitope that is very similar, both to the microbe and the host. This results in an attack on the host tissue by the same mechanism which has been activated to eliminate the pathogen. In this case, molecular mimicry is occurring as the epitope is an intrinsic part of the body and so constant synthesis of antigen prevents clearance and consequently sustained B cell activation and antibody production.

GBS and C. jejuni Some form of infection was found to precede nearly 90% of GBS cases (Koga et al., 2001). The gram-negative bacteria Campylobacter jejuni is a leading cause of acute gastroenteritis in developed countriesSome form of infection was found to precede nearly 90% of GBS cases (Koga et al., 2001). The gram-negative bacteria Campylobacter jejuni is a leading cause of acute gastroenteritis in developed countries The mimicry is occurring between C. jejuni lipo- oligosaccharides and peripheral nerve gangliosides. The most frequent anti-Ganglioside antibodies were of the class IgG, against GM1, GM1b, GD1a and GalNAc-GD1a (Willison et al., 2002). Antibodies reactive to GM3 and GD1b have also been identified (Usuki et al., 2006). Core-lipid ACeramide Bacterial GM1-like LOS Human GM1

GBS pathology and treatment Guillian-Barré syndrome is characterised by symmetrical limb weakness and loss of tendon flexes and has 3 subtypes. GBS is a self-limiting disease which occurs 1-3 weeks after infection. Muscle weakness can reach nadir within 4 weeks, followed by partial or complete recovery over weeks to months. Therapies include plasma exchange and high-dose intravenous IgG. The therapeutic effect of plasma exchange is related to the removal or dilution of circulating factors. Therapy is often not effective. Treatments do have side effects, including skin rashes and transient liver disturbances and the efficiency of both methods are limited.

Imino sugars as inhibitors An alternative method, which was developed in my study, involves metabolically reducing the level of auto-epitopes. This is done by inhibiting the glycosphingolipid (GSL) biosynthetic pathway using imino sugars which inhibit the enzyme ceramide-specific glucosyltransferase (CSGT).

Butters et al, 2000, Chemical Reviews, V100, 12, p

Study aim The aim of my study was to show the therapeutic potential of NB-DNJ and NB- DGJ and NP-IDJ as drugs for Guillain- Barré syndrome through reduction of the levels of ganglioside auto-epitopes present in the cell.

GBS sera binding to RAW cells healthyGBS+ve

GBS sera binding to RAW cells can be reduced by NB-DNJ GBS+ve + NB-DNJ (1mM)

GBS sera binding to RAW cells can be reduced by NB-DNJ

Can the binding be explained by GM1 depletion Method: Extract GSLs from cells (+/- NB-DNJ) Remove carbohydrate with ceramide glycanase Label reducing terminus with 2-AA Run labelled sugars on RP-HPLC and measure retention time.

Inhibition of GM1 antigen by NB-DNJ 200 μ M 100 μ M 50 μ M 25 μ M 5μM5μM 0μM0μM NB-DNJ GD1a GM1a GD1a GM1a 50% reduction in GM1 at 5  M NB-DNJ

Application of method to a range of clinical samples 10 samples with GBS-like symptoms.. From John Radcliffe Hospital ELISA plates coated with GM1 Probed with serial dilution of sera from patients Detected with anti-human-HRP

Some, but not all “GBS” sera show reactivity to GM1 by ELISA

But… GBS+ve Pat6 GBS-ve Healthy control

Summary/explanation for inconsistent results Why might some patient sera with similar antigenic profiles (by ELISA) react differently to cell surface? GM1 not presented at the cell surface, as is on ELISA plate. Complex and clustering may be inhibiting binding of antibodies. Some patient sera more sensitive to effects of this than others. Future experiments to resolve this..

Would need fairly high doses of NB-DNJ (>10uM) to achieve >50% reduction in antigen So, may need to try other drugs for actual therapy.. Hence compared to NB-DGJ and NP-IDJ N-pentyl-idonojirimycin

NB-DGJ GM2 GD2 GM2 GD2

NP-IDJ GM2 GD2

NB-DNJ GD2 GM2

Reduction in gangliosides with: NB-DNJ, NB-DGJ, NP-IDJ 0µM200µM 0µM

Summary I was able to show using GSL analysis, that the concentration of gangliosides and neutral glycolipids were reduced on introduction of imino sugar drugs. I was unable to replicate initial observations with patient 8, with the new patient sera, showing the reduction in binding at the cell surface due to reduced ganglioside synthesis, caused by the introduction of the drug. Further experiments are needed to distinguish between GM1-reactive sera types.