Inhibition of Ganglioside Biosynthesis by Imino Sugars Reduces Binding of Guillain-Barré Syndrome...
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Transcript of Inhibition of Ganglioside Biosynthesis by Imino Sugars Reduces Binding of Guillain-Barré Syndrome...
Inhibition of Ganglioside Biosynthesis by Imino Sugars Reduces Binding of
Guillain-Barré Syndrome Autoantibodies.
Rhea McGarryGlycobiology Department
Oxford UniversityTrinity College
Introduction
• Autoimmune DiseaseThe 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 Some form of infection was found to precede nearly 90% of GBS cases (Koga et al., 2001). The gram-negative bacteria cases (Koga et al., 2001). The gram-negative bacteria Campylobacter jejuni Campylobacter jejuni is a leading cause of acute gastroenteritis in is a leading cause of acute gastroenteritis in developed countries 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 A Ceramide
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, p4683-4696
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
healthy GBS+ve
GBS sera binding to RAW cells can be reduced by NB-DNJ
GBS+veGBS+ve
+ NB-DNJ (1mM)
GBS sera binding to RAW cells can be reduced by NB-DNJ
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[NB-DNJ]
Mea
n F
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ence
(F
ITC
)
GBS
Geo Mean H
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
200μM
100μM
50μM
25μM
5μM
0μM
NB-DNJ
GD1aGM1a
GD1aGM1a
50% reduction in GM1 at 5M 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
0
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0.0001 0.001 0.01 0.1
Serum Dilution
p1
p2
p3
p4
p5
p6
p7
p8
p9
p10
hc
bl
Some, but not all “GBS” sera show reactivity to GM1 by ELISA
But…
GBS+ve Pat6GBS-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
HO OH
CH2OH
HO
(CH2)4CH3
N-pentyl-idonojirimycin
NB-DGJ
GM2GD2
GM2 GD2
NP-IDJ
GM2
GM2
GD2
GD2
32.00
SATIN - Fluorescencem
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SATIN - Fluorescence
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SATIN - Fluorescence
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Minutes
10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.0032.00
SATIN - Fluorescencem
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SATIN - Fluorescence
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Minutes
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NB-DNJ
GD2
GD2
GM2
GM2
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NB-DNJ NB-DGJ NP-IDJ
Reduction in gangliosides with: NB-DNJ, NB-DGJ, NP-IDJ
0µM 200µM 200µM 200µM0µM 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.