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440395
2012
MSJ0010.1177/1352458512440395Poster Presentation AbstractsMultiple Sclerosis Journal
ABSTRACTS
Multiple Sclerosis Journal 2012; 18: 697–703
Poster Presentation Abstracts
P-1
Erk2 regulates oligodendrocyte myelination in vitro
Ferner AH, Xiao J, Wong AW, Kilpatrick TJ, Murray SS
Conclusions: These findings suggest that lysosomal storage has an
initial impact in reducing OL populations in preclinical disease before
vacuolation and the development of secondary inflammatory
neuropathology and neuronal loss exacerbate early myelin loss in late
disease.
Department of Anatomy and Neuroscience, University of Melbourne.
Objectives: Generation of the myelin sheath is crucial for central
nervous system (CNS) function. However, the nature of signals regulating
CNS myelination remain unclear. Brain-derived neurotrophic factor
(BDNF) knockout mice display a marked CNS hypomyelination,
implicating BDNF in myelination, although its precise role remains
elusive. Our objectives were to identify the downstream signalling
pathways activated by BDNF to promote myelination.
Methods: We used in vitro myelination assays consisting of co-cultures
of dorsal root ganglia neurons and oligodendrocyte precursor cells
(OPCs) to address this question.
Results: We identified that BDNF acts directly upon oligodendrocytes to
promote myelination in vitro. In addition, our data strongly suggest that
BDNF enhances CNS myelination via activating TrkB receptors, the
cognate receptor tyrosine kinase for BDNF, expressed on oligodendrocytes.
To investigate the intracellular signalling pathways regulated by BDNF to
promote myelination, we screened candidate downstream signalling
pathways. We found the activity of the MAPK/Erk signalling pathway
positively correlated with BDNF-induced myelination in vitro, and that
pharmacological inhibition of MAPK/Erk signalling blocked the
promyelinating effects of BNDF. OPCs infected with constitutively active
and dominant negative mutant constructs that regulate MAPK signalling
indicate Erk1/2 activation is critical for myelination in vitro. Furthermore,
over-expression of Erk1 or Erk2 in OPCs suggests that Erk2 is the MAPK
that plays a key role in oligodendrocytes to enhance myelination,
without affecting OPC survival, differentiation or proliferation.
Conclusion: Our data have identified a novel role for MAPK signalling,
through Erk2 within oligodendrocytes, that regulates CNS myelination.
P-2
Reduction of oligodendrocytes in the lysosomal storage
disease fucosidosis
Fletcher JL, Kondagari GS, Williamson P, Taylor RM
The Faculty of Veterinary Science, University of Sydney, Camperdown, NSW,
Australia
Objective: To determine how lysosomal storage impacts on
oligodendrocyte (OL)development and survival. It is not known if this is
due to lysosomal vacuolation with OLs or a secondary mechanism
interfering with the axon-glia interaction required for OLs to reach
maturity. In the lysosomal storage disorder fucosidosis, myelin loss
begins before the onset of clinical signs and is part of a pathogenic
cascade involving neuronal vacuolation, cell death and the activation of
neuroinflammatory pathways. Recent gene expression studies in canine
fucosidosis have shown that OL and myelin specific genes are significantly
downregulated in the cerebral cortex of 4 month old fucosidosis pups.
Method: To investigate this, brain tissue from fucosidosis affected and
age-matched control dogs were examined using immunohistochemistry
and electron microscopy (EM).
Results: In fucosidosis-affected dogs yet to show clinical signs of disease,
numerous vacuoles were identified in OLs by EM. Preliminary results
indicate that CNPase+ OLs are reduced in the cerebral cortex of affected
dogs in preclinical, early and late disease stages, explaining previous
findings of reduced expression of OL specific genes in preclinical disease.
Myelin loss becomes more severe in late disease and reductions in the
percentage of neurofilament staining in only late stage fucosidosis
affected dogs suggest that it is driven by axonal degeneration.
© 2012 SAGE Publications
P-3
A Strategy for the Generation and Characterization of
Monoclonal Isoform-Specific Antibodies against Glial
Fibrillary Acidic Protein
Nair B, Boyd S, Dang P, Woei NS, Orian JM
School of Mathematical Sciences, Monash University, Victoria; Department
of Biochemistry, La Trobe University, Bundoora, Victoria; Centre for
Neuroscience, The University of Melbourne, Victoria, Australia
Objectives: Glial fibrillary acidic protein (GFAP) is the most commonly
used astroglial marker. GFAP exists as a family of five isoforms (alpha,
beta, gamma, epsilon and kappa) which have been poorly characterized.
Our long-term aim is to gain an insight into mechanisms underlying the
range of interactions that astrocytes can engage in. Hence, a
bioinformatic analysis of GFAP isoforms in mouse, rat and human,
followed by biochemical analysis of GFAP isoforms in mouse was
performed.
Methods: The cerebral hemispheres (CH), cerebellum (CB), optic nerve
(ON), brain stem (BS) and spinal cord (SC) were harvested from adult
NOD/Lt and C57Bl/6 mice. Total proteome was extracted and subjected
to western blotting. Total RNA was extracted from corresponding regions
and PCR amplification of cDNA for each isoform performed. For the
bioinformatic analysis GFAP isoforms, protein sequences from human,
rat and mouse were retrieved from the National Centre for Biotechnology
Information (NCBI) database. Sequence conservation, hydropathies and
secondary structures of the GFAP isoforms within species and across
species were predicted.
Results: Western blotting and PCR analysis showed region-specific
differences in the profile of GFAP isoform expression in mouse. The
bioinformatic analysis revealed that GFAP alpha and delta isoforms have
relatively similar secondary structures and hydropathies but different
trends were observed for the kappa isoform.
Conclusions: The analysis allowed the development of a strategy to
generate isoform-specific monoclonal antibodies for GFAP that would
help investigate the specific role of each isoform in modulating astrocyte
structure-function relationship.
P-4
Quantification of GFAP Expression in Two EAE Variants
Reveals Contrasting Astrocytic Reactivity
D’Souza CS, Pham H, Doerrbecker J, Ramp AA, Gorasia DG, Purcell AW,
Ayers MM, Orian JM
Department of Biochemistry, La Trobe University, Victoria; Department
of Biochemistry and Molecular Biology, The Bio21 Molecular Science and
Biotechnology Institute, The University of Melbourne, Victoria; Department of
Pathology, The University of Melbourne, Victoria, Australia
Objectives: In view of the growing awareness of the early and immediate
response of astrocytes in neuroinflammation, we investigated astrocytic
reactivity from the earliest disease stage, within the EAE model.
Methods: EAE was induced in the NOD/Lt and C57Bl/6 mouse strains
(relapsing-remitting and chronic progressive respectively) using the
myelin oligodendrocyte glycoprotein (MOG) peptide as a neuroantigen.
Normal and mock-injected mice were used as controls. Spinal cord tissue
10.1177/1352458512440395
698
POSTER PRESENTATIONS
was isolated as it is an early site where active lesions were reproducibly
seen in preliminary immunohistochemical experiments.
Results: The mice were taken at clinical onset of disease with scores
ranging from 2.5 – 3. Initial survey of astrogliosis, by examining expression
of glial fibrillary acidic protein (GFAP) and aquaporin 4 (AQP-4), both
astrocytic markers, revealed opposite reactivity between EAE variants, with
increased expression of both markers in NOD/Lt mice and decreased in the
C57BL/6 mice, relative to controls. For validation of the astrocytic changes,
a complete proteome extraction of the spinal cord was carried out and
quantitative western blotting performed with polyclonal anti-GFAP
antibody. The resultant GFAP positive signals were normalized against
total protein loaded. The NOD/Lt samples showed a significant increase in
astrocytic reactivity of 12% between the mock injected and experimental
samples, whereas the C57Bl/6 indicated a relative decrease of 29% (1).
Conclusion: The data show that astrocytic responses vary within the
EAE paradigm. This implies that astrocytic reactivity needs to be included
as a major criterion in disease mapping and for the pre-clinical evaluation
of MS therapies.
(1) Pham H, Doerrbecker J, Ramp AA, D’Souza CS, Gorasia DG,
Purcell AW, Ayers MM, Orian JM (2011) Experimental autoimmune
encephalomyelitis (EAE) IN C57Bl/6 mice is not associated with astrogliosis. Journal of Neuroimmunology 232: 51-62
P-5
Predictors of time to first confirmed disability progression
after clinically isolated syndrome onset: an MSBase study
Jokubaitis VG, Spelman T, Meyniel C, Trojano M, Izquierdo G,
Grand’Maison F, Zwanikken C, Oreja-Guevara C, Tanner M, Rutherford
M, Butzkueven H on behalf of the MSBasis Investigators
Royal Melbourne Hospital, Melbourne, Australia
Background: The first 3-month confirmed expanded disability status
scale (EDSS) progression event after clinically isolated syndrome (CIS)
onset heralds the beginning of MS-related disability.
Objective: To assess the relationship of demographic, clinical, CSF, MRI
and treatment exposure criteria to time to first confirmed disability
progression after CIS in MS-specialist practice.
Methods: MSBasis is an observational prospective cohort study of
patients with CIS, enrolling patients from 59 MS centres worldwide. Data
were collected using iMed software and aggregated in MSBase. Baseline
data included patient profile, date of CIS, EDSS and MRI. Follow-up data
included relapses, treatment changes and EDSS. The effects of predictors
for time to 3-month confirmed progression were analysed using Cox
proportional hazards regression.
Results: Of 1950 CIS patients (mean follow-up of 2.4 years), 501 (26%)
had a first disability progression event. 402 patients had available
baseline brain MRI, 379 had a spinal MRI, 249 had CSF exam. Significant
baseline predictors of time to disability progression in multivariable
analysis were an EDSS >2 vs ≤2 (HR 1.26, p=0.031); age at CIS (HR 1.02/
year, p=0.017) and 2 or more abnormal Kurtzke Functional System
categories at baseline (HR 1.32, p=0.008). During follow-up, proportion
of total time on treatment significantly reduced the rate of progression
events (HR 0.58, p=0.002). No MRI or CSF findings were independent
predictors of time to progression.
Conclusion: Using survival analysis, the multicentre, multinational
MSBasis study has identified a number of independent predictors of first
3-month confirmed disability progression events, including a strong
protective effect of treatment.
P-6
Treatment persistence in the Australian relapsing remitting
MS population: Results from the MSBase Registry
Jokubaitis VG, Butzkueven H, Spelman T, Lechner-Scott J, Barnett M,
Shaw C, Vucic S, Slee M
Royal Melbourne Hospital, Melbourne, Australia
Background: Interferon-beta (IFNE) and glatiramer acetate (GA)
therapy in relapsing Multiple Sclerosis (RRMS) is potentially limited by
tolerability and treatment failure.
Multiple Sclerosis Journal 2012; 18: 697–703
Objective: To prospectively characterise treatment persistence in an
Australian MS population.
Methods: Australian tertiary referral centres participating in the MSBase
registry with >20 patients were included. Treatment utilisation,
persistence, and switch rates were prospectively assessed for RRMS
patients. Cox proportional hazards regression was used to compare
treatment persistence between drugs.
Results: 1618 patients were prospectively followed for a median of 3.4
years and included 1113 (68.6%) RRMS patients. The RRMS cohort
comprised 76.9% females and 23.1% males (median age 42.8 years and a
median age at diagnosis of 30.5 years). RRMS patients persisted on their
first disease-modifying therapy (DMT) for 1.8 years. Treatment
persistence on GA was shorter than on IFNE products (p<0.001). Patients
were followed on a subsequent DMT for 2.7 years, with no significant
differences in persistence between IFNE products or GA. However,
patients receiving natalizumab (NAT) persisted longer on treatment than
those on IFNE or GA (p<0.001). Sex was not predictive of treatment
persistence. The primary reason for treatment discontinuation for any
drug class was tolerability. Annualised switch rates were 9.5-12.5% for
IFNE, 11.6% for GA and 4.4% for NAT.
Conclusion: In this multicentre MS cohort, persistence on first DMT
was only 1.8 years, and higher on IFNE than GA. Treatment persistence
with a subsequent DMT was longer, suggesting that DMT switching can
improve persistence. Treatment intolerance was the principal factor
affecting persistence.
P-7
Matrilin-2 expression is up-regulated in murine Experimental
Autoimmune Encephalomyelitis (EAE) and human Multiple
Sclerosis lesions
Jonas AK, Gresle MM, Wagener R, Aszodi A, Kuhlmann T, Kilpatrick TJ,
Butzkueven H
Florey Neuroscience Institutes, University of Melbourne, Melbourne, Australia
Objective: In Multiple Sclerosis (MS) and its animal model, murine
experimental autoimmune encephalomyelitis (EAE), axonal injury is a
major determinant of irreversible neurological disability.
Using EAE, we aimed to characterize endogenous neuronal responses
to autoimmune injury, to identify beneficial responses that can be therapeutically augmented and harmful responses that can be inhibited.
Method: The majority of EAE inflammation is spinal, and affects axonal
projections from large motor neurons in the frontal motor cortex. We
hypothesized that these motoneurons respond to damage to their axons,
and conducted microarray studies on motor cortex enriched tissue from
EAE mice to investigate this.
Results: One of the most highly regulated genes was Matrilin-2 (Matn2).
Histological analyses showed that Matn2 was localized to the cytoplasm
of motoneurons in healthy and EAE brain and mRNA levels of Matn2
increased with EAE disease severity scores. An increased expression of
Matn2 by inflammation-damaged neurons was verified using neuron/
macrophage co-cultures. In EAE spinal cord, at peak disease, Matn2
expression was largely localized to the extracellular compartment,
particularly within areas of strong immune cell accumulation and axonal
injury. In chronic EAE lesional tissue, Matn2 further accumulated in the
extracellular compartment, however, additional intracellular staining was
detected in neurons, astrocytes and oligodendrocytes. Investigation of
Matn2 in lesions from MS brain biopsy and autopsy tissue demonstrated
similar expression patterns to those observed in EAE.
Conclusion: Matn2 has been identified as an endogenous neuronal
molecule that might potentially be involved in injury related responses
to neuroinflammatory disease.
P-8
Dendritic cell CCAAT/ enhancer binding protein delta
modulates Th17/Treg responses in an IL-10 dependent
manner
Mohammad MG, Tsai VWW, Tolhurst O, Breit SN, Sawchenko PE,
Brown DA
St Vincent’s Centre for Applied Medical Research, Sydney, Australia
msj.sagepub.com
POSTER PRESENTATIONS
Objective: CCAAT/enhancer binding protein delta (C/EBPd), a bZIPtranscription factor, is a recently identified regulator of innate immune
responses that is functionally active in central nervous system (CNS)
autoimmune disease. However, it has not been examined in this context.
This study aimed to define the expression characteristics and actions of
C/EBPd in experimental autoimmune encephalomylitis (EAE), an animal
model of multiple sclerosis.
Method and Results: The EAE affected CNS of C/EBPd +/+ mice showed
upregulation of C/EBPd associated with dendritic cells (DCs) and astocytes,
while EAE in C/EBPd-/- mice was significantly diminished. A chimeric BM
transplant model demonstrated that EAE severity is regulated by DC-C/
EBPd expression rather than its expression in resident CNS cells. The CNS
of C/EBPd -/- mice with EAE had decreased proinflammatory Th17 and
increased anti-inflammatory T-regulatory (Treg) T-cells numbers. These
same changes were found when C/EBPd -/- bone marrow derived DCs
(BMDCs) were directly injected into the CNS, where they recruited T-cells
and skewed their development to a more anti-inflammatory profile with
a reduced Th17:Treg ratio. To determine how C/EBPd acts in this context
we examined BMDC directed T-cell development. This showed that
decreased C/EBPd expression in DCs, even with its maintained expression
in all other cells, led to a reduced Th17:Treg ratio and increased IL-10
expression in DCs. Therefore, we inhibited IL-10 receptor signaling by
specific antibody therapy in vitro and in vivo. This treatment reversed the
effect of reduced DC-C/EBPd expression in both cases.
Conclusion: These results highlight DC expression of C/EBPd as an
important regulator of Th17:Treg balance mediated by IL-10.
P-9
Toll-like receptors signalling in Experimental Autoimmune
Encephalomyelitis
Miranda-Hernandez S, Fletcher JM, Gerlach N, Erik B, Chowdhury S,
Heinrich K, Baxter AG
Comparative Genomics Centre, James Cook University, Queensland, Australia
Objective: To analyze the importance of TLR signaling in EAE.
Methods: C57BL/6 and C57BL/6 deficient for TLR1, TLR2, TLR4, TLR6,
TLR9, TLR2/9, TLR4/9 and MyD88 female and male mice were immunized
with MOG35-55, Complete Freud Adjuvant (CFA), and Pertussis Toxin
(PTX) for active induction of EAE. Passive EAE was induced by adoptive
transfer of in vitro-activated leucocytes from spleen and lymph nodes of
mice with active MOG35-55/CFA+PTX–induced EAE to donors mice
(C57BL/6 to C57BL/6; C57BL/6 to C57BL6.Tlr9-/-; C57BL/6 to C57BL6.
Tlr2-/-; C57BL6.Tlr2-/- to C57BL6.Tlr2-/-; C57BL6.Tlr2-/- to C57BL/6 female
mice and C57BL/6 to C57BL6.Tlr2-/- male mice).
Results: C57BL/6.My1c88-/- male and female mice were completely
protected from the disease. C57BL/6.Tlr2-/- and C57BL/6.Tlr9-/- female
mice developed less severe active MOG35-55/CFA+PTX–induced EAE.
C57BL/6.Tlr2/9-/- mice of both sexes also decreased the clinical course of
active EAE but no more that the clinical course seen in single C57BL/6
TLR2 and TLR9 deficient mice. Less severe disease was associated with
milder CNS infiltration. The passive form of EAE confirmed protection
against the disease in the absence of TLR2 but not TLR9. The CNS of
C57BL/6.Tlr2-/- mice showed fewer infiltrating CD4+ T cells and reduced
IL17 secretion, increased splenic (CD62L+) CD4+CD25+Foxp3+ regulatory T cells
and IL6 was undetectable in plasma in the absence of TLR2.
Conclusions: Signalling via TLR2 contributes to the severity of CNS
inflammation in EAE.
699
using disease modifying drugs (DMD), principally interferon beta (IFN-E)
and glatiramer acetate (GA). However, up to half of patients do not
respond to these DMDs and determination of response status requires
clinical evaluation over a 1- 2 year follow-up period. To date, there is no
test that predicts the outcome of treatment. This necessitates discovery
of a reliable “biomarker” which will help identify patients as either
responders or non-responders to treatment before initiating DMD
therapy. Genetic traits associated with drug response can be quickly and
easily determined before initiation of treatment, and hold promise as
such a biomarker.
Objective: This project aims to identify polymorphisms associated with
response to IFN-E and GA treatment by performing a genome-wide
association study (GWAS). GWAS analysis for each drug will follow a two
stage protocol.
Methods: In Stage 1 genotype data will be analysed in conjunction with
clinical data from a subset of ANZgene patients (1,618 MS patients) to
screen the genome for response associated variations. In Stage 2 the 100
most significant associations from Stage 1 will be validated in
independent MS cohort who have received either treatment in question.
We will also attempt to build a prediction model for treatment outcome.
Lasso penalised regression in combination with cross-validation will be
used to specify a logistic regression like prediction model.
P-11
Do PLP1 mutations found in MS patients lead to the unfolded
protein response and apoptosis of PLP-expressing cells?
Moxey N, Greer J
University of Queensland Centre for Clinical Research, Herston,
Queensland, Australia
Objective: Myelin proteolipid protein (PLP) plays a major role in
myelin maintenance and structure. The gene for PLP, PLP1, is an
X-linked gene, with mutations manifesting in males as PelizaeusMerzbacher disease (PMD), where myelin production is highly disrupted.
Females are generally carriers and do not show any symptoms. Two
recent reports describe individuals diagnosed with MS who also carry
PLP1 mutations. These mutations differ from those that cause PMD.
How these mutations may relate to the development of MS in these
individuals is unclear. We know from research in PMD that mutant PLP
can accumulate within the body of oligodendrocytes, causing damage
and even death of these cells. This process is caused by the activation of
the unfolded protein response (UPR). This study investigates whether or
not the PLP1 mutations that have been found in MS patients can directly
damage oligodendrocytes.
Method: COS-7 and HOG oligodendroglioma cells will be transiently
transfected with native PLP1 or variants identified in patients with MS
(Leu30Val, Leu30Arg, and Arg136Trp).The pattern of PLP expression and
UPR activation will be investigated using immunofluorescent staining
and qRT-PCR arrays.
Results: Expression of the different constructs is currently underway in
COS-7 cells.
Conclusion: Results from this study will provide new insights into
oligodendrocytes and potentially into the role of PLP1 point mutations
in disease development for a subgroup of patients with MS.
P-12
P-10
Pharmacogenomics of interferon beta and glatiramer acetate
response in Australian MS Patients.
Mahurkar S, Moldovan M, Slee M, Butzkueven H, Taylor B, Broadley S,
Lechner-Scott J, O’Doherty C
University of South Australia; University of New South Wales; Flinders
University; University of Melbourne; Menzies Research Institute Tasmania;
Griffith University; University of Newcastle, Australia
Background: The current treatment approach in multiple sclerosis (MS)
is to maximise the patient’s quality of life by slowing disease progression
msj.sagepub.com
Analysis of the role of oligodendroglial TrkB in Central
Nervous System myelination
Wong AW, Xiao J, Kemper D, Kilpatrick TJ, Murray SS
Centre for Neuroscience and Department of Anatomy and Cell Biology and
Florey Neuroscience Institutes, University of Melbourne
Background: Myelin loss inhibits the saltatory conduction and is
detrimental to the normal functioning of the neurons, resulting in the
demyelinating disease such as Multiple Sclerosis. Re-myelination must be
sufficiently introduced into the lesions and such requires the
understanding of the molecular mechanisms that achieve myelination,
which are yet to be fully elucidated.
Multiple Sclerosis Journal 2012; 18: 697–703
700
POSTER PRESENTATIONS
Brain Derived Neurotrophic Factor (BDNF) knockout mice exhibited
CNS hypomyelination, indicating its importance in regulating CNS myelination. Using the in vitro myelination assay to replicate the myelination
process, BDNF promotes oligodendrocyte myelination in vitro. Further in
vitro analyses suggest that BDNF promotes oligodendrocyte myelination
via TrkB.
Methods: To verify these findings, we examined a mouse model of
conditional TrkB knockout in oligodendrocytes (TrkB fl/fl MBP cre).
Results: Analyses at P12 to P30 indicate that these mice exhibited
reduced expression of myelin protein in the CNS, compared to littermate
controls TrkB fl/fl but no difference in the density of mature
oligodendrocytes. Interestingly, a significant increase was concurrently
observed in the number of oligodendrocyte precursors, consistent with
the increased proliferative response observed in vitro.
Conclusion: The loss of myelin protein with a concurrent increase of
NG2 positive progenitor cells suggest that there is a potential
compensatory mechanism in this animal model.
P-13
A small peptide mimetic of BDNF promotes peripheral
myelination
Xiao J, Hughes R, Lim JY, Ivanusic JJ, Ferner A, Wong AW, Kilpatrick TJ,
Murray SS
Centre for Neuroscience, Department of Pharmacology, Florey Neuroscience
Institutes, Department of Anatomy and Cell Biology, The University of
Melbourne, Victoria, Australia
Objective: Current therapeutic interventions for peripheral demyelinating
neuropathies are sub-optimal, focusing on the inflammatory process rather
than directly influencing remyelination. As such, identifying factors that
facilitate axon-to-Schwann cell interactions which promote myelination
have therapeutic potential. We have previously demonstrated that brainderived neurotrophic factor (BDNF) exerts contrasting influences upon
myelination - acting through neuronally-expressed p75NTR to enhance
myelination, but inhibiting myelination via activation of neuronal TrkB.
Here we investigated whether the selective targeting of p75NTR signalling
is an approach that could exert a unified promyelinating response.
Methods: We have used the small cyclic pentapeptide cyclo-DPAKKR –
previously described by us that was designed to mimic the region of
BDNF that binds p75NTR. Myelination in vitro was assessed by using
dorsal root ganglion (DRG) neurons and Schwann cell myelinating cocultures. In vivo studies of peripheral myelination were performed on the
postnatal rat sciatic nerve following subcutaneous injection of exogenous
BDNF or cyclo-DPAKKR.
Results: Like BDNF, cyclo-DPAKKR promoted myelination of NGFdependent neurons in vitro, an effect dependent on the expression of
neuronal p75NTR. Importantly however, cyclo-DPAKKR enhanced
myelination of TrkB-expressing DRG neurons, whereas BDNF exerted an
inhibitory effect. In agreement with these in vitro findings, in vivo
injection of cyclo-DPAKKR significantly enhanced myelin protein
expression and increased the proportion of myelinated axons in sciatic
nerve during early postnatal development.
Conclusion: These results demonstrate that cyclo-DPAKKR effectively
promotes peripheral myelination both in vitro and in vivo, and by
extrapolation suggest that targeting p75NTR could be a useful approach
to promote peripheral myelin repair.
P-14
Western Australian Multiple Sclerosis Patients Exhibit a
Lower Prevalence of Helicobacter pylori Infection
Bennett KA, Qiu W, Tay A, Castley A, Wu J, Joseph JP, James I, Marshall
BJ, Carroll WM, Kermode AG
Centre for Neuromuscular and Neurological Disorders, University of Western
Australia, and Department of Neurology, Sir Charles Gairdner Hospital,
Queen Elizabeth II Medical Centre, Perth; Department of Neurology, the Third
Affiliated Hospital of Sun yat-sen University, Guangzhou, China; Centre for
Clinical Immunology & Biomedical Statistics, Murdoch University & Royal Perth
Hospital; Department of Clinical Immunology and Immunogenetics, Royal Perth
Hospital; Department of Neurology, Hospital Kuala Lumpur, Malaysia; The
Marshall Centre for Infectious Diseases, University of Western Australia
Multiple Sclerosis Journal 2012; 18: 697–703
Background: Infection with the bacterium Helicobacter pylori has a
reported role in many disease conditions. Relatively unclear is any
association it may have with immune-mediated, demyelination of
Multiple Sclerosis (MS). Two conflicting viewpoints currently dominate
the literature. A study in a Japanese cohort describes a lower H. pylori
seroprevalence in Conventional Multiple Sclerosis (CMS) patients than
in Optico-Spinal Multiple Sclerosis (OSMS) patients or controls (Li et al,
2007), suggesting a possible difference in prevalence according to disease
phenotype. Conversely, a Greek study reports a greater rate of infection
in MS overall (Kountouras et al, 2007).
Methods: In order to conclusively elucidate any correlation, enzymelinked immunoassay was performed on serum from 551 patients with
CMS from the Perth Demyelinating Diseases Database and 299 selected
community-based controls. Such numbers represent a much larger study
than the Japanese or Greek cohorts.
Results: H. pylori seropositivity was found to be considerably lower in
CMS patients than in controls, both overall (13.2% vs 21.4%) and in the
subgroup which were age and sex matched (15.1% vs 21.4%).
Conclusion: This may suggest a possible protective role for H. pylori
infection in Multiple Sclerosis. Alternatively, such findings may indicate
that H. pylori infection is a surrogate marker for the ‘hygiene hypothesis’,
a theory which postulates that infection in early life is essential to prime
the immune system and thus prevent allergic and autoimmune conditions
in later life (Cremonini & Gasbarrini, 2003). Further analyses will explore
H. pylori infection with clinical, imaging and laboratory phenotypes.
P-15
The temporal profile of optic nerve DTI measures and RNFL
thickness in the first year after acute optic neuritis
Van der Walt A, Kolbe SC, Wang E, Shuey N, Ahmadi G, Tsonis S,
Marriott M, Paine M, Mitchell P, Klistorner A, Egan GF, Butzkueven H,
Kilpatrick TJ
Centre for Neuroscience, University of Melbourne; The Royal Victorian Eye and
Ear Hospital, Melbourne; Royal Melbourne Hospital, Melbourne; The Sydney Eye
Hospital, Sydney, Australia; Department of Medicine, University of Melbourne
Objective: Optic nerve Diffusion Tensor Imaging (DTI) is pathologically
specific with axial diffusivity reflecting axonal loss and radial diffusivity
representing demyelination. We studied the longitudinal flux in DTI and
OCT parameters in the 12 months following optic neuritis (ON).
Methods: We tested 37 patients with acute ON at 1 week, 1,3,6 and 12
months. Ten matched controls (HC) were scanned twice. We used a 3T
MRI scanner with a 32-channel head-coil. Each optic nerve was scanned
individually using six direction DTI. OCT was performed using OCT-3
scanner (Stratus™) technology.
Results: HC Radial and Axial diffusivity had a respective intra-subject
coefficient of variance of 2.4% and 3%. Repeated measures ANOVA of
axial (p=0.33) and radial diffusivity (p=0.95) in the normal eye (UAE)
were not significant. Baseline axial diffusivity was significantly reduced
in the affected eye (AE) compared to UAE (p=0.009. The reduction in
axial diffusivity persisted at 1 month (p<0.001) and 3 months (p<0.05).
At 12 months, the AE axial diffusivity increased above that in the UAE
(p<0.0001). Radial diffusivity increased in the AE compared to UAE at 3
months (p=0.02), 6 months (p=0.003) and 12 months (p<0.0001). RNFL
oedema was seen at baseline (p=<0.0001) and 1 month (p=0.042). RNFL
thinning was seen at 6 months (p<0.0001) and 12 months (p<0.0001).
Axial and radial diffusivity did not correlate with RNFL thickness.
Conclusions: 1. Optic nerve DTI is reproducible and sensitive. 2.
Significant flux in optic nerve DTI and OCT parameters occur in the first
12 months after optic neuritis.
P-16
Baseline axial diffusivity predicts poor visual recovery at 6
months after acute optic neuritis.
Van der Walt A, Kolbe S, Wang E, Shuey N, Ahmadi G, Marriott M, Paine
M, Tsonis S, Mitchell P, Klistorner A, Egan G, Butzkueven H, Kilpatrick T
Centre for Neuroscience, University of Melbourne; The Royal Victorian Eye and
Ear Hospital, Melbourne; Royal Melbourne Hospital, Melbourne; The Sydney Eye
Hospital, Sydney, Australia; Department of Medicine, University of Melbourne
msj.sagepub.com
POSTER PRESENTATIONS
Objective: Diffusion tensor imaging (DTI) of the optic nerve may
provide new paraclinical markers of optic nerve integrity but needs to be
studied prospectively.
Methods: 35 patients with acute ON were tested at 1 week, 1,3 and 6
months post-acute optic neuritis (ON). We used a 3T MRI scanner with a
32-channel head-coil. Each optic nerve was scanned individually using
six direction DTI. OCT was performed using OCT-3 scanner (Stratus™)
technology. Clinical assessments included high contrast visual acuity
(VA) (Sloan 100%) and LCLA (Sloan 2.5% and 1.25%). Direct logistic
regression of 6 baseline factors (age, treatment with corticosteroids,
baseline asymmetry in high contrast letter acuity, axial and radial
diffusivity as well as average RNFL thickness asymmetry) on the likelihood
of predicting a poor visual outcome at 6 months was performed.
Results: Poor visual recovery at 6 months was defined as persistent
asymmetry in LCLA 2.5% between the affected and unaffected eye of
more than 31%. Three variables contributed independently to predicting
poor visual outcome: baseline asymmetry in high contrast VA (Odds
Ratio (OR) 1.06, 95% CI 1.004-1.112, p=0.03), RNFL thickness (0.96, 95%
CI 0.924-0.992, p=0.02) and asymmetry in baseline axial diffusivity
(0.840,95% CI 0.718-0.983, p=0.03).
Conclusion: Baseline Axial diffusivity asymmetry is a marker of
processes (such as axonal degeneration) that predict recovery but are
separate from the initial inflammatory dysfunction that is reflected in
RNFL oedema and decrease in vision after acute optic neuritis.
701
Methods: We tested 34 patients with acute ON at 1-week post onset and
again at 1, 3, 6 and 12 months. MR Imaging was performed on a 3T MRI
scanner. Ocular coherence tomography (OCT) was performed at each
time-point using OCT-3 scanner (Stratus™) technology to measure RNFL
thickness. Clinical assessments included high contrast visual acuity (VA)
(Sloan 100%) and Low contrast letter acuity (LCLA) (Sloan 2.5% and
1.25%). Spearman’s rho tests were performed on PASW statistic software
v.18. Asymmetry of MTR, Sloan VA (100%, 2.5%, 1.25%) and RNFL were
calculated using the expression: 100*(affected eye – unaffected eye ) /
unaffected eye (%).
Results: Significant correlations were observed between asymmetry of
MTR at 3-months and asymmetry of RNFL at 3 months (r=0.496, p=0.003),
6 months (r=0.395, p=0.028) and 12 months (r=0.445, p=0.009) after onset
of acute ON. In addition, significant correlations between asymmetry of
MTR at the 3-month time point and asymmetry of 2.5% Sloan at 3
(r=0.466, p=0.006) and 6 months (r=0.467, p=0.008) were observed. There
was a strong trend for a correlation between asymmetry of MTR at
3-months and 2.5% Sloan asymmetry at 12 months (r=0.342, p=0.052).
Conclusion: MTR is predominantly a marker of axonal loss. In addition,
MTR values at 3-months after acute ON predicts visual outcome using
Sloan 2.5% LCLA charts at 3 and 6 months.
P-19
P-17
Changes in the Gene Expression and Splicing in the CNS
during Pregnancy and Post-partum in the Rat
Inglis H, Narayanan R, Wallace R, Greer J, McCombe P
The Temporal Profile of Optic Nerve MTR Values in the First
Year After Acute Optic Neuritis
Wang Y, Van der Walt A, Kolbe S, Shuey N, Ahmadi G, Tsonis S, Marriott
M, Paine M, Mitchell P, Klistorner A, Egan GF, Butzkueven H, Kilpatrick TJ
The University of Queensland, Centre for Clinical Research; Queensland Brain
Institute, University of Queensland
Centre for Neuroscience, University of Melbourne; The Royal Victorian Eye and
Ear Hospital, Melbourne; Royal Melbourne Hospital, Melbourne; The Sydney Eye
Hospital, Sydney, Australia; Department of Medicine, University of Melbourne
Objective: To investigated the longitudinal change in Magnetization
Transfer Ratio (MTR) values after acute optic neuritis (ON) and establish
its use as a pathologically specific marker in MS.
Methods: We tested 34 patients with acute ON at 1-week post onset and
at 1, 3, 6 and 12 months. Eleven age and sex-matched controls (HC) were
scanned twice 4 weeks apart. MR imaging was performed on a 3T MR
scanner.
Results: Mean (± se) MTR of HC left and right optic nerves across two
time points demonstrated no inter-eye difference (Willks Lambda =
0.995, F(1,20) = 0.1, p = 0.755), and no time-related difference (Willks
Lambda = 0.883, F(1,20) = 2.658, p = 0.119). MTR asymmetry in HC was
averaged between the two scans for comparison with patients. Patient
MTR values were as follows: unaffected eye baseline: 43.51 ± 6.89; 1
month: 44.68 ± 7.53; 3 month: 45.09 ± 7.50; 6 month: 43.27 ± 6.32; 12
month: 42.51 ± 7.20. Affected eye baseline: 43.26 ± 7.18; 1 month: 42.70
± 6.97; 3 month: 40.88 ± 9.77; 6 month: 37.71 ± 7.77 and 12 month:
38.74 ± 7.24. Patient MTR asymmetry was significantly different from
HC at the 6-month time point only. Patient 6-month MTR asymmetry
was also significantly different from baseline MTR asymmetry.
Conclusion: Our study proved that optic nerve MTR is a reproducible
and sensitive technique. In addition, MTR measurement in the optic
nerve after optic neuritis is sensitive to changes in tissue integrity.
P-18
Optic nerve MTR at 3 months reflect RNFL thinning 12
months after onset of optic neuritis
Wang Y, Van der Walt A, Kolbe S, Shuey N, Ahmadi G, Tsonis S,
Marriott M, Paine M, Mitchell P, Klistorner A, Egan GF, Butzkueven H,
Kilpatrick TJ
Centre for Neuroscience, University of Melbourne; The Royal Victorian Eye and
Ear Hospital, Melbourne; Royal Melbourne Hospital, Melbourne; The Sydney
Eye Hospital, Sydney; Department of Medicine, University of Melbourne
Objective: To investigate whether the Magnetization Transfer Ratio
(MTR) early after onset of optic neuritis can predict axonal loss as
measured by retinal nerve fibre layer (RNFL) thinning.
msj.sagepub.com
A number of auto-immune diseases are attenuated during pregnancy.
Understanding how the differential expression of genes in a number of
significant pathways is able to produce a decrease in incidence and
severity of EAE in rats may provide insight into those with the potential
to be modified therapeutically to provide more specific treatment for demyelinating neuropathies such as MS.
Objective: To analyse gene expression in the CNS during pregnancy
and the immediate post-partum period.
Methods: Messenger RNA was extracted from the spinal cord tissue of
pregnant (e18), post-partum (e25) and normal healthy Lewis rats.
Samples were hybridized to Affimetrix GeneChip Rat Exon 1.0 ST Arrays.
These arrays facilitate Gene expression level analysis as well as exon
expression level analysis . Data was normalised and comparisons made
using Partek Genomics Suite software. Network and pathway analysis of
differentially regulated gene lists was then carried out using the Ingenuity
Pathway analysis system.
Results: We found changes in gene expression in the CNS during
pregnancy and post-partum including some in gene networks with the
potential to influence disease progression. Splice variations were also
found in a number of relevant genes.
Conclusion: There are a number of levels of transcriptional regulation
involved in cellular development and differentiation as well as response
to environmental stimuli. Epigenetic, heritable changes in gene activity
and expression occur without alteration in DNA sequence. We propose
that epigenetic modifications in genes as a result of pregnancy could
result in both the short and long term-effects of pregnancy on
autoimmune diseases such as MS.
P-20
Searching for rare variants conferring susceptibility to
multiple sclerosis
Lin R, Perreau V, Glazov E, McMorran B, Bahlo M, Brown M, Foote S,
Rubio J, Charlesworth J, Thomson R, Browning S, Martin N, Taylor B,
Stankovich J
Menzies Research Institute Tasmania, University of Tasmania; Centre for
Neuroscience, University of Melbourne, Melbourne; University of Queensland
Diamantina Institute, Brisbane; Department of Biostatistics, University
of Washington, Seattle, USA; Walter and Eliza Hall Institute of Medical
Research, Melbourne; GlaxoSmithKline, Uxbridge, UK; Queensland Institute
of Medical Research, Brisbane
Multiple Sclerosis Journal 2012; 18: 697–703
702
POSTER PRESENTATIONS
Objective: Genome-wide association (GWA) studies have revealed
about 60 loci associated with MS. Most associations are with common
variants near genes that play key roles in the immune system. However
rare variants may also influence susceptibility to MS (e.g. a rare variant in
the CYP27B1 gene was identified associated with MS recently).
Identification of rare variants may alter perspectives on the relative
importance of immunological and neurological factors in MS onset,
particularly as rare variants play an important role in other
neurodegenerative diseases such as ALS.
Methods: We used identity-by-descent (IBD) mapping to search for
long chromosomal segments (haplotypes) that cases have inherited
from distant common ancestors. By merging and cleaning four separate
GWA datasets (total 3252 cases, 5725 controls), we applied the newest
IBD mapping method, ‘Beagle fastIBD’, to identify regions of the
genome where more pairs of cases than controls have inherited shared
haplotypes.
Results: A strong linkage signal (LOD=4.65, p=1.9 x 10^-6) was observed
in a one megabase region on chromosome 19. Notable genes in the
region include ZNF274 involved in transcriptional regulation, and
ZSCAN4 involved in telomere maintenance. There is a cluster of 9 MS
cases from Tasmania, Victoria and New Zealand who have inherited the
same haplotype in this region; there are no such control clusters.
Conclusion: IBD mapping shows promise for the identification of
genomic segments containing rare disease susceptibility variants in
unrelated individuals. Even with the development of whole-genome
sequencing, it may help in prioritizing regions to look for rare variants.
Objectives: The interleukin-7 receptor alpha (IL7RD) gene controls
growth and differentiation of T cells as part of the receptor for potent
cytokines, interleukin-7 (IL-7) and thymic stromal lymphopoietin
(TSLP). Haplotype 2 (Hap2) is unequivocally associated with protection
from MS. Our ongoing work investigates how the common haplotypes
affect immune function.
Methods: Regulation of IL7RD haplotypes, including the response to
interferon-beta (IFNE) was determined in CD4 T cells and myeloid
subsets. Immunophenotype amongst carriers of the IL7RD haplotype was
examined.
Results: We and others have reported that Hap2 produces less of a
soluble isoform that can inhibit receptor signalling, an effect magnified
in dendritic cells. Hap2 is also associated with faster CD4 recovery from
lymphopenia following anti-retroviral therapy in HIV. We have
previously shown in T cells, and here report that also in dendritic cells,
IL7RD Hap4, homozygotes of which are associated with the highest MS
risk, are unresponsive to IFNE. A pilot study suggests increased IFNJ
response to stimulation in CD4 T cells of healthy control carriers of Hap4
(n = 18; p = 0.042).
Conclusions: Hap2 may protect from MS by increasing the strength of
IL7 or TSLP signals, which may reduce the T cell activation threshold or
direct Treg development, respectively. We predict that Hap2 will
influence CD4 recovery following T cell-depleting therapies for MS such
as alemtuzumab. Non-response of Hap4 to IFNE supports a model in
which Hap4 responds differently to viral infection, a putative
environmental risk factor for MS; and may have implications for IFNE
therapy.
P-21
P-23
Polymorphisms in the receptor tyrosine kinase MERTK gene
are associated with multiple sclerosis susceptibility
Ma GZM, Stankovich J, The Australia And New Zealand Multiple
Sclerosis Genetics Consortium (ANZgene), Kilpatrick TJ, Binder MD,
Field J
Multiple Sclerosis Division, Florey Neuroscience Institutes, University of
Melbourne, Victoria; Centre for Neuroscience, University of Melbourne,
Victoria; Menzies Research Institute, University of Tasmania, Hobart,
Tasmania
Objectives: Multiple sclerosis (MS) is a debilitating, chronic demyelinating disease of the central nervous system affecting over 2 million
people worldwide. The TAM family of receptor tyrosine kinases (Tyro3,
Axl and MerTK) have been implicated as important players during
demyelination in both animal models of MS and in the human disease.
We therefore conducted an association study to identify single nucleotide
polymorphisms (SNPs) within genes encoding the TAM receptors and
their ligands associated with MS.
Methods: Analysis of genotype data from a genome-wide association
study that consisted of 1618 MS cases and 3413 healthy controls
conducted by the Australia and New Zealand Multiple Sclerosis Genetics
Consortium (ANZgene) revealed several SNPs within the MERTK gene
(Entrez Gene ID: 10461) that showed suggestive association with MS. We
therefore interrogated 28 SNPs in MERTK in an independent replication
cohort of 1140 MS cases and 1140 healthy controls.
Results: We found 12 SNPs that replicated, with 7 SNPs showing
p-values of less than 10-5 when the discovery and replication cohorts
were combined.
Conclusion: All 12 replicated SNPs were in strong linkage disequilibrium
with each other. In combination, these data suggest the MERTK gene is a
novel risk gene for MS susceptibility.
P-22
Variation in the IL7R gene influences MS risk, response to
therapy and immune cell function
McKay F, Hoe E, Parnell G, Schibeci S, Rajasuriar R, Lewin S, Stewart G,
Booth D.
Institute for Immunology and Allergy Research, Westmead Millennium
Institute, University of Sydney; Infectious Diseases Unit, The Alfred Hospital,
Melbourne
Multiple Sclerosis Journal 2012; 18: 697–703
Genomic studies: a step further in unravelling Multiple
Sclerosis. What’s next?
Tajouri L
Faculty of Health Sciences and Medicine, Bond University, Gold Coast,
Queensland, Australia
Background: Multiple sclerosis (MS) is an autoimmune disease of the
central nervous system (CNS), characterized by zones of demyelination
and inflammatory plaques. Environmental factors might precipitate the
disease and include possibly vitamin D metabolism.
Objective: Genomic investigation of a MS population, using RFLP
analysis of three variations in the VDR gene were analysed for association
with MS susceptibility and pathology.
Method: Three VDR variants were investigated with genotypes detected
using the Taq I, Apa I and Fok I restriction enzymes.
Results: This study did show a positive association, specifically between
the functional variation in exon 9 of the VDR gene and MS (Taq I, F2=
7.22, P= 0.0072). Interestingly, the Apa I variant was also found to be
associated with MS (F2=4.2, P=0.04). The Taq I and Apa I variants were
also found to be in very strong and significant linkage disequilibrium
(D’=0.96, Pvalue < 0.0001) and their associations were more prominent
with the progressive forms of MS.
Conclusion: This study found a strong positive association of the Taq I
VDR marker in MS susceptibility. Both genotype and allele frequency
distributions for the Taq I variant were significantly different between
the MS and control populations. In addition, these findings demonstrate
that the rarer allele (t) is more predominant in MS cases than in controls.
P-24
The P2X7 receptor: Interaction with a HLA Class II allele
which modulates the autoantibody response in Multiple
Sclerosis
Cox MB, Scott RJ, Stankovich J, Kermode A, Cortes A, Brown M,
Lechner-Scott J, Wiley J and ANZgene
The Hunter Medical Research Institute, The University of Newcastle and
Hunter New England Health; The Menzies Research Institute, University of
Tasmania; University of Western Australia; The University of Queensland,
Diamantina Institute, 5 Florey Neuroscience Institute, University of
Melbourne
msj.sagepub.com
POSTER PRESENTATIONS
Objective: P2X7 is an ATP-gated receptor which is highly expressed in
monocyte/macrophages and microglia of the brain. Activation of this
receptor induces an inflammatory response, while prolonged activation
causes cell death. Major up-regulation of cell surface P2X7 receptors and
HLA Class II molecules occurs on differentiation of monocytes into
macrophages and dendritic cells. We explored a possible interaction
between these two proteins and MS oligoclonal band (OCB) status.
Methods: 283 MS patients (184 OCB+, 99 OCB-) and 116 controls from
two centres in Australia were genotyped for the rs2230912 polymorphisms
in P2RX7, whose minor allele tags a gain of function haplotype. The
P2XR7 variants were assessed for association with MS, OCB status, and
epistatic interactions with DRB1*1501. Statistical analysis was performed
in PLINK v1.07, using Benjamini and Hochberg step up FDR control.
Data from the ANZgene GWAS (3318 cases, 3643 controls) was used to
confirm the role of rs2230912 with MS risk.
Results: DRB1*1501 was over-represented in MS cases compared to
controls and in OCB+MS patients compared to OCB-MS patients. P2XR7
gain of function variant rs2230912G appears to be a risk factor for MS in
DRB1*1501 negative individuals. In OCB-MS patients, rs2230912G was
over-represented compared to OCB+MS cases.
Conclusion: The P2RX7 polymorphism appears to interact with
DRB1*1501 resulting in decreased risk of developing MS, although by
itself does not alter the risk of developing MS. The P2X7 receptor,
especially when the gain of function polymorphism is present, may
compete with DR-15 for membrane expression.
P-25
Brain imaging correlates of ocular motor dysfunction in MS
Fielding J, Kolbe S, Kilpatrick T, White O, Clough M, Egan G
msj.sagepub.com
703
School of Psychology and Psychiatry, Faculty of Medicine, Nursing and Health
Sciences, Monash University, Victoria
Objective: Ocular motor abnormalities are a common feature of MS,
reflecting changes to both lower-level motor and higher-order cognitive
control processes. We have previously demonstrated impaired inhibitory
control in MS, particularly for the antisaccade (AS) task which involves
the inhibition of a prepotent response in favour of a volitional
movement. To investigate the pathophysiological mechanisms
underlying this deficit, we investigated the micro/macro-structural brain
changes associated with AS errors using diffusion tensor (DTI) and
volumetric imaging.
Methods: 24 patients with RRMS performed the AS task, and underwent
DTI and high resolution T1-weighted imaging. For DTI, voxelwise
regression analysis was used to identify regions of significant covariance
between AS errors and fractional anisotropy (FA) or mean diffusivity
(MD). For volumetric imaging, T1 images were nonlinearly registered to
the MNI-152 template and regression analyses used to identify regions of
significant covariance between AS errors and regional atrophy.
Results: AS error rates correlated significantly with reduced FA and
increased MD in the cerebellum (FA throughout the cerebellum, MD
primarily along the midline). Grey matter atrophy was associated with
error in multiple cerebellar regions, particularly in the left hemisphere, as
well as the amygdalae bilaterally, and the paracingulate, opercular and
posterior parietal cortices.
Conclusions: In MS, inhibitory control, as reflected by AS errors, was
primarily associated with micro/macro-structural alterations to the
cerebellum. As inhibitory control is more typically attributed to (pre)
frontal brain regions, this research highlights the significance of
cerebellar integrity in maintaining a modulatory influence over higher
order cognitive control processes in MS.
Multiple Sclerosis Journal 2012; 18: 697–703
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