ACNR • VOLUME 4 NUMBER 4 SEPTEMBER/OCTOBER 2004 1

MultipleThe Year in

SclerosisResearch

2010

The production of this supplement hasbeen sponsored by a grant from Merck Serono

ACNRADVANCES IN CLINICAL NEUROSCIENCE & REHABILITATION

SUPPLEMENT TO ACNR VOLUME 10 ISSUE 6 JANUARY/FEBRUARY 2011

ISSN 1473-9348

Foreword

Welcome to the secondACNR annual review of MS research

This is not a systematic collection.A few colleagues and friendshave kindly agreed to write up their favourite piece of multiplesclerosis research from 2010. (And some of my PhD students

were told they had to).There can be little doubt that the three most important papers this

year, at least for the short term,were the publication of the phase 3 trialsof cladribine and fingolimod in the NEJM.On the back of these,fingolimod has been licensed in the US and will shortly be accepted inthe UK, I imagine. I suspect cladribine will follow,but it is hard to saywhen as there have been some regulatory delays. So, these three papersget the coveted ACNR Prize for The Most Important MS ResearchPublished In 2010.

And the ACNR Prize for Best Bit Of Advocacy For Patients With MS In2010 goes to the UK MS Society who,alone amongst patient groups,refused to bow to mob pressure and spend its research budget onscanning the veins of people with multiple sclerosis.We are very grateful to Merck Serono for funding this supplement.

But the company had nothing to do with the choices of articles or theviews expressed.If my flippant tone annoys you,or if you have some comments on our

reviews,or feel we have missed a really important piece of MS researchin 2010,please do not hesitate to write in to ACNR.

Alasdair Coles, Cambridge, UK.

> Once your patients have reached EDSS 3.0 (which will be around the age of 40 yearsold), their subsequent disease course is pretty fixed > The genetic causes of multiplesclerosis will amount eventually to hundreds of genes with modest effects and possiblythousands with very small effects > Babies whose first trimester occurred during thewinter have a 1.3 times increased risk of multiple sclerosis > The MRI activity ofpatients with multiple sclerosis is increased during winter months > Vitamin D candirectly act on genes > A single T cell can have two sets of receptors, one recognising aforeign bug and one targeting the brain: activating the first can lead to brain inflammationvia the second > Interferon-beta only works in that subgroup of patients whosemultiple sclerosis is driven by “Th1” cytokines. For those inclined towards “Th17”, andpeople with neuromyelitis optica, interferon-beta exacerbates the disease >Neuromyelitis optica can present with vomiting (or hiccups) alone > Fingolimod, moreefficacious and more problematic than interferon-beta, will be the first oral treatment ofmultiple sclerosis ever licensed in the UK. There will, I predict, be big arguments overits price > Cladribine, similarish to fingolimod in efficacy and with a similarish level ofconcern over adverse effects, has been delayed by having to go through a second submissionto the regulators > Myelin peptide skin patches may be a treatment of the future >The first two trials of mesenchymal stem cell therapy (given iv in one and intrathecal in theother) were published this year. So far, it appears safe > Agonists at the retinoid Xreceptor promote remyelination in laboratory animals. Human trials are awaited >The published data on venous drainage of multiple sclerosis patients shows no differencefrom controls, except in Dr Zamboni’s hands.

2 SUPPLEMENT TO ACNR • VOLUME 10 NUMBER 6 JANUARY/FEBRUARY 2011

Simple Summary for the Stressed

Contents

Foreword ................................................................................................................................................................02

Epidemiology ................................................................................................................................................04

• A disease of two halves, Alasdair Coles ..................................................................................................................04

Genetical Things ......................................................................................................................................04

• Awesome study of identical twins, Maria Ban........................................................................................................04• GenomeWide Association Screening and Donald Rumsfeld, Steve Sawcer......................................................05

Vitamin D and Sunshine..............................................................................................................06

• Vitamin D meddling in our genes, Sreeram Ramagopalan and Gavin Giovannoni ..........................................06• It’s a seasonal thing, Neil Robertson........................................................................................................................07

Bugs and Viruses ......................................................................................................................................08• EBV and the brain: are we there yet? Ute Meier and Gavin Giovannoni ............................................................08• Viruses and multiple personality T cells, Orla Tuohy ............................................................................................08

Immune Bits and Bobs......................................................................................................................09• Yet another regulatory cell, Allison Curry ..............................................................................................................09• Why interferon-beta works for some but not others, Bruno Gran ........................................................................10• Dendritic cells: an unhealthy imbalance, Allison Curry........................................................................................11

Devic’s Disease ..........................................................................................................................................12

• A cause of vomiting and hiccups, Isabel Leite and Joanna Kitley ......................................................................12• Worrying epidemiology of neuromyelitis optica, Isabel Leite and Joanna Kitley ............................................12• Interferon-beta may exacerbate neuromyelitis optica, Isabel Leite and Joanna Kitley ....................................13

Astrocytes:Who They? ....................................................................................................................13• Do astrocytes regulate CNS inflammation? PatrickWaters....................................................................................13• Transporters: cassettes and media, Mike Zandi ......................................................................................................14• Th17 cells seek and destroy neurons, Denise Fitzgerald ......................................................................................14• Are histone mimics the future? Mike Zandi ..........................................................................................................15

Tablets and Trials......................................................................................................................................15• Disease-activity free status, Gavin Giovannoni ......................................................................................................15• Fingolimod has arrived! Martin Lee ........................................................................................................................16• Cladribine: we’ll have to wait a while, Martin Lee ................................................................................................17• Ocrelizumab and Ofatutumab; the new kids on the block, Ruth Dobson and Gavin Giovannoni..................17• Daclizumab: an improbable treatment, Orla Tuohy ..............................................................................................18• A skin patch to treat multiple sclerosis, Alasdair Coles ........................................................................................19• Update on natalizumab-associated PML, David Hunt and Gavin Giovannoni ..................................................19

Stem Cell Corner ....................................................................................................................................20• At last some mesenchymal stem cells into humans, Paolo Muraro. ....................................................................20

Repair..........................................................................................................................................................................21• Retinoids, rodents and remyelination, Suzanne Mosely ........................................................................................21• BDNF, in and outside the CNS,and repair? Suzanne Mosely ................................................................................22• Brain repair through neuroprotective autoimmunity in humans? Tom Button..................................................22

Last and Least ................................................................................................................................................23• Dem veins,dem veins,dem dry veins, Alasdair Coles ..........................................................................................23

SUPPLEMENT TO ACNR • VOLUME 10 NUMBER 6 JANUARY/FEBRUARY 2011 3

Epidemiology

A disease of two halves

It has become commonplace for people to

talk about multiple sclerosis being a disease

of two parts: first an inflammatory relapsing-

remitting phase and secondly a

neurodegenerative progressive phase.

Intuitively, the more relapses one has, the

more severe the subsequent progression

should be,but big studies in recent years

from London,Ontario, and Lyons have

suggested this is not the case.But when

does one phase end and the other begin?

This is important because the subtext is that

the neurodegenerative phase is untreatable.

Christian Confavreux has suggested in the

past that perhaps EDSS 4.0 (when patients

first have limitation of their walking,but are

still working full time) is the turning point:

once the disease causes this much disability

it becomes“amnestic”of its past history and

marches inexorably towards progressive

disability at a stereotyped rate.

Gilles Edan at Rennes is perhaps best

known for his promotion of mitoxantrone as

a treatment of multiple sclerosis.But he and

his team have been quietly registering cases

of multiple sclerosis since 1976,amassing

2054 patients, accounting for 26,273 patient-

years (1609 relapsing/445 progressive

onset).They hypothesise that the tipping

point is EDSS 3.0, significantly earlier than

the Lyons view.And it seems they might be

correct. For there is a wide variety of times

taken by people with multiple sclerosis to

get from disease onset to EDSS 3.0 (where

people are not disabled in any conventional

sense,but have some minor deficits).But

the duration of the phase from 3.0-6.0 is

nearly identical, taking from 6-9 years.They

also confirmed Christian Confavreux’s

finding that people tend to hit disability

milestones at the same age (EDSS 3.0 at 42

years old and EDSS 6.0 at 52 years old),

regardless of what their disease has been

like up until then.

At first glance, the 10th paper in the

iconic London (Ontario) series (which

started in 1989), involving 28,000 patient-

years,would appear to disagree.Ebers and

colleagues – going back on earlier

statements – suggest that the relapse

frequency in the first two years of the

disease can predict the extent of long-term

disability. For instance, those patients who

have more than three relapses in this time

will reach EDSS 6.0 13 years more quickly

than those who just had one.However,most

(but not all) of this variance is explained by

the differing time it takes patients to reach

EDSS 3.0; beyond that point,patients

disability follows a similar trajectory.

The big worry from these results, and

those of Christian Confavreux before, is the

possibility that immunotherapies may not

influence the long-term accumulation of

disability once patients reach EDSS 3.0.This

is by no means proven: observational

studies such as these can never do that.But

our treatment trials should be constructed

so that we can see if baseline EDSS above

or below 3.0 determines the long-term

disability outcome from treatment.

Leray E, Yaouanq J, Le Page E, Coustans M,

Laplaud D, Oger J, Edan G.

Evidence for a two-stage disability progression in

multiple sclerosis.

Brain. 2010 Jul;133(Pt 7):1900-13.

Scalfari A, Neuhaus A, Degenhardt A, Rice GP,

Muraro PA, Daumer M, Ebers GC.

The natural history of multiple sclerosis: a

geographically based study 10: relapses and

long-term disability.

Brain. 2010 Jul;133(Pt 7):1914-29.

Genetical ThingsEd: There is no doubt: all the money and

effort into genetics research on multiple

sclerosis is beginning to bear fruit. It has

taken decades, literally. But in 2009, we

saw an outpouring of new information on

genes that have something to do with the

risk of getting the disease. 2010 has been

a little quieter in terms of new genes. But

our reviewers have picked out two papers

which make important nuances to the

genetics story so far. And Steve Sawcer

hints at big news for 2011.

Awesome study ofidentical twins

Maria Ban, a Cambridge geneticist,

reviews the only paper on multiple

sclerosis to appear in that hallowed

journal, Nature, in 2010. Steve Hauser and

Stephen Kingsmore, at San Francisco and

Sante Fe, and a couple of football teams-

worth of authors studied three identical

twin pairs, discordant for multiple

sclerosis. The technological achievement

of the study is extraordinary; it shows just

how much can be done to investigate the

intricacies of gene translation and

transcription in the individual. Sadly,

though, nothing positively new was learnt

about multiple sclerosis.

Significant progress has been made in

identifying the genetic factors that lead to

susceptibility to MS,although much remains

to be understood of the complex aetiology

of this disease.The 30% concordance rate of

MS in monozygotic twins clearly indicates

that factors other than shared inherited

genetics contribute to the development of

MS. In a pioneering effort, the study by

Baranzini et al provides the first systematic

attempt in an autoimmune disease to

comprehensively screen the entire genome

of monozygotic twins to identify genetic

factors that may account for some of the

discordance in disease.

Given their role in the pathogenesis of

MS,CD4+ lymphocytes were isolated from

three monozygotic twin pairs discordant for

MS.Utilising next generation sequencing

technologies, they examined for differences

in genetic variants such as somatic

mutations, insertion/deletions and copy

number variations and also investigated

Journals

4 SUPPLEMENT TO ACNR • VOLUME 10 NUMBER 6 JANUARY/FEBRUARY 2011

2010 Prizes for MSResearch

– The John DystelPrize was awarded

by the AAN toDavid Hafler

– The KJ Zülch Prizefor basic neurological

research went toAlastair Compston and

Hans Lassmann– The Sobek Research

Prize was given toCatherine Lubetzki and

Rudolf Martini(The Charcot award was given

last year toJohn Prineas and is

awarded only every two years)

epigenetic changes, such as methylation,

which can alter a phenotype including gene

expression without an underlying change in

the DNA sequence.No outstanding

difference in genetic variants or methylation

patterns was found to account for the

discordance amongst the twin pairs.

The most interesting results came from

the study of the transcriptome [Ed: the

fancy word to describe the expression of

all the various RNA molecules].While no

differences in the expression of 19,000

genes was found in the CD4+ cells, as the

entire mRNA of each individual was re-

sequenced, this resolution allowed allele

specific expression to be measured.This

analysis provides a useful measure for the

‘real’ effects of an allele on gene expression

as it reduces the effects of other trans-acting

factors and environmental sources that can

impact on gene expression.Baranzini et al's

study identified that 43% of the tested

coding SNPs show a different direction or

degree of allelic imbalance in gene

expression between twin siblings.The

genetic or external factors that contribute to

this difference though remain to be

investigated.

Baranzini SE, Mudge J, van Velkinburgh JC,

Khankhanian P, Khrebtukova I, Miller NA, Zhang L,

Farmer AD, Bell CJ, Kim RW, May GD, Woodward

JE, Caillier SJ, McElroy JP, Gomez R, Pando MJ,

Clendenen LE, Ganusova EE, Schilkey FD,

Ramaraj T, Khan OA, Huntley JJ, Luo S, Kwok PY,

Wu TD, Schroth GP, Oksenberg JR, Hauser SL,

Kingsmore SF.

Genome, epigenome and RNA sequences of

monozygotic twins discordant for multiple

sclerosis.

Nature. 2010 Apr 29;464(7293):1351-6.

Genome WideAssociation Screeningand Donald Rumsfeld

Steve Sawcer, from Cambridge, explains

that we now know how much we do not

know about the genetics of multiple

sclerosis. And he should know. He

produced one of the first GWAS, back in

the day when you had do your PCRs by

hand. He ends optimistically, promising

that there will be news in 2011 of more

genes associated with multiple sclerosis.

So then what? We will all have to get our

biological thinking caps on...

Epidemiological studies have repeatedly

and consistently shown that genetic factors

have a marked influence on susceptibility;

however these studies lack the power to say

much about the genetic architecture

underlying the variation in individual risk.

Crude estimates of the relationship between

familial recurrence risk and the degree of

relatedness can be made and suggests that

risk is primarily determined by common

variants exerting modest effects, rather than

multiple rare variants of larger effect,but

such segregation analysis is unable to

provide answers to any of the more detailed

questions.How many genes are involved?

Do the relevant alleles act in a dominant or

recessive manner? Do these alleles interact

with each other?

Genome-Wide Association Studies (GWAS)

have begun to uncover the relevant genes but

to date the identified alleles only account for

a fraction of the observed heritability

(perhaps one quarter). In an effort to gain

some further insight into“the shape of things

to come”in the genetic analysis of multiple

sclerosis,we at the International Multiple

Sclerosis Genetics Consortium (IMSGC)

compared the data from two independent

GWAS and reported their finding in the

American Journal of Human Genetics.

Because genes influencing susceptibility

might lie in any part of the genome

screening for we them need to cover as

much of the genome as possible and

therefore necessarily has to involve many

thousands of Single Nucleotide

Polymorphisms (SNPs, variations in the DNA

sequence). In a typical GWAS it is usual to

test in the order of 250,000 to 1,000,000

SNPs.One problem with performing such a

large number of tests is that some will

inevitably show apparently significant

difference in allele frequency between

cases and controls just by chance,even if

there are no genes influencing

susceptibility.By definition the number of

such false positive results is of course just

related to the significance threshold

considered (the p-value). For example in a

study involving 500,000 SNPs we would

expect 5,000 false positives with a p-value of

< 10–2 (5,000 is 1% of 500,000), 500 with a p-

value of < 10–3 and 50 with a p-value of <

10–4 etc.One way of separating the wheat

from this chaff is to set a very high

significance threshold (typically 5x10–8).Any

SNP showing association with a p-value

exceeding this threshold is very likely to be

genuinely associated because it would be

highly unlikely to see such an extreme

difference by chance,even if you have

tested 500,000 SNPs.This logic is robust but

reality is even more intriguing. It turns out

that even when you remove all the SNPs

known to be associated with multiple

sclerosis there is a systematic tendency to

see more markers above any given

significance threshold than you would

expect by chance.This so called“genomic

inflation”occurs in almost all GWAS and has

been a focus of attention in the complex

genetics community.Unsurprisingly most of

this inflation results from subtle biases

which differentiate cases and controls but

are unrelated to the cause of the disease; in

particular differences in ancestry (so called

population stratification) and in genotyping

efficiency. In considering the implications of

genomic inflation one should remember

that such inflation is expected to occur in

all biomedical research employing the case

controls approach, from studies of

immunology through to clinical trials.The

only difference is that GWAS involve so

many tests that the phenomenon can be

observed and even measured. In the other

situations we have no way of knowing the

extent to which results have been

influenced by these inevitable and

confounding effects.While experimental

biases undoubtedly account for most of the

observed genomic inflation it is also

possible that undiscovered risk alleles could

Journals

SUPPLEMENT TO ACNR • VOLUME 10 NUMBER 6 JANUARY/FEBRUARY 2011 5

In September 2010,Roslyn Ekes died,aged 62. She both

had the disease andstudied it. She was

the patientcoordinator, and an

author, forDr Larry Jacobs’

pioneering studieson intrathecal

interferontreatment of

multiple sclerosis.

Journals

produce at least some of this inflation.

Following logic suggested by the

International Schizophrenia Consortium

(ISC) the IMSGC reasoned that if some of the

genomic inflation observed in their first

GWAS (reported in 2007) was due to as yet

undiscovered risk alleles that were exerting

individual effects on risk that were too weak

to be detected with extreme significance but

were acting together to produce some part

of the genomic inflation, then one would

expect the alleles over represented in the

cases from one GWAS to predict disease

status in a second independent GWAS.

Random ascertainment effects, such as

population stratification,and experimental

effects, such as differences in genotyping

efficiency,would not be expected to be

consistent and replicate between GWAS.

Testing this idea by comparing their original

GWAS data with that from an independent

study the IMSGC found highly significant

evidence (p= 10–18) for such an“en masse”

effect,which accounted for approximately

3% of the observed genomic inflation.

Moreover,we showed that this effect was

even apparent amongst those markers that

did not reach nominally significant

association individually. In other words when

taken together even those markers with p

values such as 0.2 in the original GWAS were

to a modest,but significant,extent able to

predict disease status in a second

independent GWAS. Inevitably the predictive

value was greatest amongst those markers

that had nominally significant association

(p<0.05) but fell short of genome wide

significance (5x10–8) but the fact that

prediction improved as less and less strongly

associated markers were included in the

analysis confirms that many risk alleles

underlie susceptibility.As we concluded

“These results statistically demonstrate a

polygenic component to MS susceptibility

and suggest that the risk alleles identified to

date represent just the tip of an iceberg of

risk variants likely to include hundreds of

modest effects and possibly thousands of

very small effects.”

Much remains to be discovered about the

genetic architecture underlying

susceptibility but these results suggest that

many common variants of modest effect

remain to be discovered as GWAS are

expanded and combined.We can expect a

new crop of susceptibility alleles in 2011.

International Multiple Sclerosis Genetics

Consortium (IMSGC), Bush WS, Sawcer SJ, de

Jager PL, Oksenberg JR, McCauley JL, Pericak-

Vance MA, Haines JL.

Evidence for polygenic susceptibility to multiple

sclerosis--the shape of things to come.

Am J Hum Genet. 2010 Apr 9;86(4):621-5

Vitamin D andSunshineEd: This year’s American Association of

Neurology meeting will be forever

remembered for the ash cloud. I was

trapped, with many other hundreds of

European neurologists, and have distinct

memories of enforced laziness conflicting

with scenes reminiscent (sort of) of that

last helicopter leaving the American

embassy in Saigon.... The meeting itself

was not the AAN at its best. I remain

appalled by the keynote Presidential

address, which the President devoted to

solemnly articulating that the main aim of

the organisation was to protect the

remuneration of US neurologists.

An uncharacteristic moment of self-

doubt came over me in one of the MS

teaching sessions, which was otherwise

great fun because I could play with an

interactive remote controller. We were

asked our views on prescribing vitamin D.

We were presented with options, such as

whether we measure vitamin D levels

before prescribing or not, and various

different dosing regimes. But the option I

needed, that I do not prescribe vitamin D,

was not available. It seems that there is

already a North American vitamin D

“feeding frenzy”, to quote Neil Robertson

below. I often wonder if there is some

special Harry Potter journal (“Neurology

and ¾”) to which I do not have access;

perhaps it is there that the phase 3 trials

demonstrating that vitamin D alters

multiple sclerosis disease activity have

been published? In fact, we are still at the

stage when a trial of 15 people with

vitamin D makes it into a journal like

PLoS One [Smolders 2010]. The patients

took, and mostly tolerated, 20 000 IU/d

vitamin D3 for 12 weeks. Very few

immunological effects were seen and none

on FOxP3 CD25hi Tregs. But, the

proportion of Tcells expressing IL-10

increased, and those producing IFN-

gamma decreased which is “good”. But

don’t we really want to know just whether

vitamin D has any impact on disability or

relapses? A stack of trials investigating

this come to an end in 2011, so perhaps

we will know soon.

It has become standard teaching that

multiple sclerosis incidence falls off as you

approach the Equator, amongst peoples of

similar genetic stock. This story took a hit

when Thiebault Moreau produced a new

analysis of the variation of multiple

sclerosis prevalence in France: no North-

South gradient was seen. It turns out

multiple sclerosis is more common in North

Eastern France and less common around

Paris, with South France having a medium

prevalence [Fromont, Brain 2010].

Vitamin D meddling inour genes

Sreeram Ramagopalan and George Ebers

have led the way in describing how

vitamin D might interact with the genetics

of multiple sclerosis. In their most recent

study, they identified 229 sites at which

vitamin D may bind and influence gene

expression. They found enrichment for

VDR binding amongst GWAS sites

6 SUPPLEMENT TO ACNR • VOLUME 10 NUMBER 6 JANUARY/FEBRUARY 2011

“The quicker and lighter you are the better”: on baking scones,by Richard Whittington, vitriolic cookery writer, who died this year,

having had multiple sclerosis for twenty years

Journals

associated with MS, type 1 diabetes,

Crohn’s disease, systemic lupus

erythematosus, rheumatoid arthritis,

chronic lymphocytic leukaemia, colorectal

cancer, hair colour, tanning, and height!

Sreeram Ramagopalan and Gavin

Giovannoni take up the story...

It has been estimated that over one billion

people worldwide lack vitamin D to some

degree due to dietary deficiency or

inadequate sun exposure. Initially thought

to play a restricted role in calcium

homeostasis, the pleiotropic actions of

vitamin D in biology and their clinical

significance are only now becoming

apparent.Vitamin D deficiency has been

associated with increased risk for several

diseases, including multiple sclerosis (MS).

Despite many research efforts, it is still

incompletely understood how vitamin D

acts at the molecular level to influence

disease susceptibility.

Vitamin D signalling occurs through

binding of activated vitamin D (calcitriol) to

the vitamin D receptor (VDR) which then

binds to specific genomic sequences

(vitamin D response elements,or VDREs) .

Binding of theVDR toVDREs influences

gene transcription.Using chromatin

immunoprecipitation followed by ‘next-

generation’DNA sequencing (ChIP-seq),we

generated a comprehensive genomic map

of VDR-DNA binding in lymphoblastoid cell

lines.Thousands of hitherto-unknown sites

of VDR binding across the genome were

identified,highlighting the widespread

actions of vitamin D. Intriguingly,VDR

binding sites were significantly enriched

near genes associated to MS identified from

genome-wide association (GWA) studies.

Notable genes influenced by vitamin D

included IRF8,CD226,CLEC16A,CD40 and

CTLA4.Several functional observations have

demonstrated that vitamin D influences the

innate and adaptive immune systems,and

the influence of vitamin D on the genes

identified in this study likely provides the

mechanism of these effects.

This study strongly supports the notion of

gene-vitamin D interactions in determining

MS susceptibility, in line with previous

epidemiological data.The support for a role

for vitamin D in MS is growing and raises

the idea of preventing MS.Based on the

data from the only longitudinal study of

serum vitamin D levels and MS,a large

proportion of MS cases in the U.S. and

Europe could be prevented by increasing

serum vitamin D levels to concentrations

commonly found in individuals in sunny

regions such as Africa. Further studies of

vitamin D in MS are therefore strongly

warranted.

Ramagopalan SV, Heger A, Berlanga AJ, Maugeri

NJ, Lincoln MR, Burrell A, Handunnetthi L,

Handel AE, Disanto G, Orton SM, Watson CT,

Morahan JM, Giovannoni G, Ponting CP, Ebers

GC, Knight JC.

A ChIP-seq defined genome-wide map of vitaminD

receptor binding: associations with disease and

evolution.

Genome Res. 2010 Oct;20(10):1352-60.

It’s a seasonal thing

Neil Robertson, a seasoned contributor to

the epidemiology of multiple sclerosis,

reviews two papers which show that there

is a smidgeon more bother from multiple

sclerosis in the winter. Firstly, if you are

an Australian baby, it is best if you can

plan your first three months in utero for

the summer months, to avoid a marginal

increase in the risk of eventually getting

multiple sclerosis. Secondly, if you already

have the disease, you can expect to get

more MRI activity over the winter. Of

course, the key issue for people with

multiple sclerosis is the extent to which

the factors responsible for these seasonal

changes are modifiable....

The past few years in MS research has

allowed an almost obsessional interest in

molecular genetics to waver, and turn a dim

spotlight on a re-evaluation of

environmental factors that may be of

importance on an individual or population

basis with a growing literature on the role of

vitamin D and ultraviolet radiation (UVR).

To some extent this is a re-invention of the

wheel and a re-interpretation of the well

described latitudinal gradient,but

technological advances and availability of

radiological techniques and robust

population statistics has allowed further

contemporary insights and in particular

how incidence and clinical

phenomenology in MS might be affected by

seasonal factors.

In the BMJ this year was a paper from an

Australian group exploring the relationship

of maternal UVR exposure,month of birth

and risk of multiple sclerosis. (The first

author, Judith Staples, is a medical student

who must be the envy of her peers).This

study is possible because of the huge

latitudinal span of Australia and the detailed

investment in disease epidemiology in the

early 1980s by Simon Hammond which has

since acted as a rich resource for MS

research in this (perhaps a lesson to other

regions, including the UK,who have

historically failed to invest in disease

registers or fully understand their value).

Using demographic data and

supplementary birth registration data the

authors examined the number of people

born with MS in each month relative to the

general population and examined

associations with ambient UV exposure over

the same period with variable lag of 1-9

months to represent potential impact at

various gestational ages.Relative to a

reference incidence rate for May-June, risk

of disease was higher in all other time

periods (1.23-1.34) and maximal in early

summer (November-December) [Ed: no

mistake, remember you are in Oz]. Further

examination of the prenatal exposure to

UVR demonstrated a strong inverse

association to disease incidence restricted

to the first trimester.

There is undoubtedly a danger of over-

interpreting these results and encouraging a

vitamin D feeding frenzy and it is important

to remember that this study demonstrated

association rather than aetiology. It may be

that vitamin D is a surrogate marker for a

number of other factors. In addition the

patterns observed are at odds with other

epidemiological data, including rising

incidence in western populations in

association with increasing UVR exposure.

But this study will serve to direct and

influence future research investment.

Prospective studies and northern

hemisphere corroboration would clearly be

of interest,but a sobering thought is that

even if we were able to modify risk of

disease on a population basis by ensuring

adequate vitamin D supplementation the

results might not be visible for 30 years.

Now if the complexities of interpretation

of historic population statistics leave you

less than convinced, then data from an

intensive MR programme may be more up

your street. In a heroic examination of 939

separate MRI brain examinations in a

heterogeneous cohort of 44 patients, a

group from Boston investigated the

prevalence of MS disease activity as

reflected by new T2 lesions and compared

SUPPLEMENT TO ACNR • VOLUME 10 NUMBER 6 JANUARY/FEBRUARY 2011 7

Journals

these to time of year and other climate data.

The results are beautifully illustrated and

the authors must be congratulated on the

extra investment in colour printing.

Although the patient numbers are small, the

biases which provide significant difficulty in

seasonal epidemiological studies are

effectively sidestepped by the objective MR

data which demonstrates the likelihood of

new T2 activity is 2-3 times higher in March-

August than during the rest of the year and

is most striking in patients with relapsing

disease.Although there have been one or

two earlier seasonal MR studies producing

variable results this is by far the most

convincing and the intensity of the

scanning remarkable and the patients

should be applauded for their commitment.

The importance of both these studies is

to underline that there are potentially

modifiable environmental factors that could

make significant impacts on not only

disease frequency but also clinical

expression. In addition the findings have

implications for trial design and service

delivery. So,MS really is a seasonal thing.

Staples J, Ponsonby AL, Lim L.

Low maternal exposure to ultraviolet radiation in

pregnancy, month of birth, and risk of multiple

sclerosis in offspring: longitudinal analysis.

British Medical Journal. 2010 Apr 29;340.

Meier DS, Balashov KE, Healy B, Weiner HL,

Guttmann CR.

Seasonal prevalence of MS disease activity.

Neurology. 2010 Aug 31;75(9):799-806.

Bugs andVirusesEd: The idea that multiple sclerosis may

be “triggered by a virus” has been around

for long time. As far as I know the earliest

proponent of an infective cause of the

disease was Pierre Marie, one of Charcot’s

residents, of CMT fame. My favourite MS-

virus story is how Cook and Dowling came

to suggest that canine distemper virus

might be responsible for multiple sclerosis

in 1977: they noted that three New York

sisters who lived together developed

multiple sclerosis after their dog had an

encephalopathy. The current candidate is

Epstein-Barr virus, for which there is lots

of uncontested evidence. But, in the last

few years, there has been some argy

bargey about whether or not EBV is in the

brains of people with multiple sclerosis.

The 2010 zeitgeist is that most people do

not find it. Ute Meier and Gavin

Giovannoni, from the Blizard Institute at

the Barts and the London, review one of

the key “no” papers, and bring to our

attention one of their own (unpublished)

studies, which was presented at the

International Society for

Neuroimmunology meeting.

EBV and the brain: arewe there yet?

No, the journey continues.Yet another US

publication by Jeffrey Bennett and

collaborators using real-time PCR failed to

detect active EBV infection in the brain of

patients with MS. In active MS plaques, a

small-non-coding EBV RNA (EBER-1) was

the only and rarely detected transcript.The

Denver group concluded that the MS brain

did not show any evidence of active EBV

infection.There are now two independent

PCR-based studies (Denver and Boston),

which conclude that active EBV infection is

not a characteristic feature of the MS brain.

However,how could latent infection play a

role?We and our collaborators in Oxford and

Abu Dhabi used in situ hybridisation to

detect EBER+ cells in active MS lesions.

However, this finding was not exclusive to the

MS brain as EBER+ cells were also found in

cases of stroke.We propose a more indirect

mechanism by which latent EBV infection

could contribute to neuroinflammation: that

these small RNAs bind to the toll-like

receptor 3 and thus stimulate IFN-alpha

production in active MS lesions.Could innate

activation, triggered by latent EBV infection,

be part of the game? Perhaps we have to

think differently - EBV might be more subtle

than we anticipated. It is a persistent virus

after all with the aim to co-exist with the host

rather than eradicate it.

The question remains – why do we get

discrepant results with different techniques?

Hans Lassmann invited all European and US

MS teams and several EBV researchers to a

July workshop inVienna.United under one

roof on neutral grounds they discussed the

different findings and techniques,exchanged

ideas,opinions and expertise.The Treaty of

Vienna has not been signed,but hopefully

productive EBV alliances were formed to

solve the conundrum of EBV and the brain.

Sargsyan SA, Shearer AJ, Ritchie AM, Burgoon

MP, Anderson S, Hemmer B, Stadelmann C,

Gattenlöhner S, Owens GP, Gilden D, Bennett JL.

Absence of Epstein-Barr virus in the brain and

CSF of patients with multiple sclerosis.

Neurology. 2010 Apr 6;74(14):1127-35.

Epub 2010 Mar 10.

Tzartos j, Khan G Vossenkamper A, Meager A,

Sefia E, Middledorp J, Giovannoni G, Meier UC.

Activation of innate immunity is a hallmark of the

active lesion in multiple sclerosis. J

Neuroimmunology. 228(1/2):163-4.

[Meeting Abstract].

Viruses and multiplepersonality T cells

Ed: Classical immunology has it that

viruses can trigger autoimmune disease by

mimicking a self-antigen, so that the

activated T cells get confused and attack

self, thinking it is the virus (“molecular

mimicry”) or that viruses cause such

widespread alarm in the immune system

that self-reactive T cells are non-

specifically wound up (“bystander

upregulation”). There is another way. Dan

Altmann had shown that a single T cell

can express two types of T cell receptor in

1995 at the Hammersmith; he argued that

8 SUPPLEMENT TO ACNR • VOLUME 10 NUMBER 6 JANUARY/FEBRUARY 2011

In January 2010, Fampridine (4-aminpyridine)was licensed by the FDA to improve walking

speed in people with multiple sclerosis;a UK decision is not expected until 2011.

It costs roughly $12,000 a year wholesale.

Journals

these aberrant cells had slipped through

the normal negative selection process.

Now, Joan Goverman and colleagues from

the University of Washington have done

some very clever experiments to show

that there can be a single CD8+ T cell

clone which responds to a virus with one

TCR and causes autoimmune

demyelination with the other. Orla Tuohy,

a clinical PhD student in Cambridge,

reports:

Ji et al in a paper published in Nature

Immunology describe a mechanism in

which viral infection can induce CNS

autoimmunity. The potential for T cells to

express more than one antigen-specific T

cell receptor (TCR) and trigger

autoimmunity has been previously

described. In this paper the authors use a

transgenic mouse model, that is MHC class 1

restricted and therefore amenable to the

study of CD8+ T cell-mediated interactions.

Although the CD4+ subsets have attracted

more interest and study in autoimmune

demyelination, the authors point out that

the increased presence of CD8+ myelin

antigen specific cells in MS warrants

consideration of this lymphocyte population

as a key player in disease pathogenesis. In

addition the high rate of serological

evidence of past EBV exposure in MS may

implicate viral involvement in disease

pathogenesis.

The transgenic mouse model they use

(“8.8”) is a MHC class I–restricted TCR-

transgenic model that generates CD8+ T

cells specific for myelin basic protein. The

authors found the 8.8 mouse was resistant

to traditional techniques to induce EAE

induction (which are generally MHC class 2

restricted,CD4+ cell-mediated).On the other

hand,EAE could be induced by vaccinating

with a recombinant MBP-expressing

vaccinia virus.

The key observations are that:

• The 8.8 mice also developed EAE when

they were infected with wild-type (non-

MBP expressing) vaccinia virus.Vaccinia

does not cross-react with MBP, so it

implies that the T cells in these animals

had newly generated TCRs to respond

to vaccinia,which then went on to

cause EAE.

• However, the 8.8 mice did not get EAE

with vaccinia, if they were made unable

to generate endogenous TCR chains by

knocking out Rag.

This led to the hypothesis that the disease

was caused by T cells expressing receptors

for vaccinia virus antigens and a second

TCR specific for myelin (and these TCRs

require Rag to be made),which could be

activated during viral infection and lead to

an autoimmune attack.

To support this hypothesis, evidence was

needed that the MBP-specific T cells

activated after wild-type vaccinia virus

infection,co-expressed endogenous TCRs

that recognised viral antigens. This could

account for the occurrence of disease in

Rag-competent mice versus Rag knockout

mice as Rag allows the generation of

endogenous TCR chains via incomplete

allelic exclusion in the transgenic MHC-

class 1 restricted mice models. The 8.8 mice

that still expressed Rag could potentially

express endogenous TCR chains that were

viral specific, in addition to their MBP-TCR.

Following wild-type vaccinia virus some

of the activated CD8+ T cells from 8.8 Rag

+/+ mice were seen to express an

endogenous TCR beta chain (beta 6). 8.8 T

cells expressing the TCR beta 6 chain

expressed more Interferon-gamma after

exposure to vaccinia-virus infection cells in-

vitro, than after MBP stimulation suggesting

the beta 6 chain was viral antigen reactive.

Along the way the authors excluded

molecular mimicry and bystander

activation as mechanisms for viral

inoculation to trigger disease in the 8.8

mouse model.

Through a series of carefully engineered

experiments, Ji et al have illustrated a

mechanism through which viral infection

could interact with genetic susceptibility to

trigger autoimmunity in a mouse model

with a genetically modified immune cell

repertoire. Whether this mechanism is

involved in autoimmune disease initiation

in the presence of a naturally occurring

immune system has yet to be proved.

Ji Q, Perchellet A, Goverman JM.

Viral infection triggers central nervous system

autoimmunity via activation of CD8+ T cells

expressing dual TCRs.

Nature Immunology. 2010 Jul;11(7):628-34.

Immune Bitsand Bobs

Yet another regulatorycell

Ed: Allison Curry, a post doc in my lab,

reviews a study from that prolific MS

immunologist, Jorge Correale, from the

Raúl Carrea Institute for Neurological

Research in Argentina. He makes the case

for the importance of the rather obscure

CD8 regulatory cell in multiple sclerosis.

You will remember that the CD4 regulatory

cell does not work properly in people with

multiple sclerosis. In fact, the failure of the

CD4 T reg to suppress aggressive T cells is

one of the very few consistent

immunological abnormalities in this

disease. My own take on this detailed

paper is that there is one important

element missing. I would like to know if the

CD8 regulatory cells in multiple sclerosis

are also intrinsically defective, compared to

controls, like their CD4 cousins.

The inflammatory process is a complex

network involving many cell types, their

related cytokines and signalling pathways.

We know that self reactive T cells exist in

the periphery,presumably having escaped

thymic clonal deletion,and that these must

be kept under tight surveillance in order to

prevent dys-regulation,potential

autoimmunity and disease.

SUPPLEMENT TO ACNR • VOLUME 10 NUMBER 6 JANUARY/FEBRUARY 2011 9

This year Jimmie Heuga died, founder ofThe Heuga Center for Multiple Sclerosis,

and 1964 bronze medallist in slalom at theWinter Olympics in Innsbruck, Austria.

Journals

Our understanding of T cell function and

regulation has expanded from initial CD4/8

phenotypic distinction through the

characteristic Th1/Th2 cytokine profiles to

the current focus on T regulatory cells (T

regs). Although CD8+ regulatory cells in MS

have previously received attention, recent

research has focused on existing and

emerging CD4 T reg populations.The role of

the CD8+ CD25+ Fox P3+ regulatory cell

(CD8 T reg) has been revisited by these

Argentinean MDs.

The authors focus on CD8 Treg in the

PBMC and CSF from 35 patients with RRMS

(15 patients in remission),15 controls and 10

patients suffering from other inflammatory

neurological diseases (OIND).Much of the

CD4 T reg data published in MS to date has

relied on precise isolation of CD4+ CD25 high

cells and their ability to suppress polyclonal

stimulation. Using limiting dilution

techniques,plus stimulation with Myelin Basic

Protein (MBP)/Myelin oligodendrocyte

(MOG) peptides,CD4 and CD8 T cells were

cloned from both the PBMC and CSF.CD8 T

regs were identified and shown to reduce the

responses of CD4 cells to their antigens.This

suppression was impaired in transwell

experiments which showed that CD8 cells

suppress by cell contact.The CD8 T regs seem

to do this via dendritic cells,using STAT3

signalling,rather than directly on CD4 T cells.

Finally FACS analysis of CSF and PBMC

showed reduced representation of CD8+ T

regs in patients during acute exacerbation

suggesting a reduction in CD8+ regulation at

potential sites of inflammation.

How CD8 T regs interact with CD4 T regs,

CD4 cells and the APC interact is a

fascinating puzzle.One can only

contemplate how future identification of

further target antigens may yet contribute to

solving the complicated,unfolding maze of

immune regulation.

Correale J, and Villa A.

The role of CD8+ CD25+ Foxp3+ regulatory T

cells in multiple Sclerosis.

Annals of Neurology 2010;67:625-38.

Why interferon-betaworks for some but notothers

Ed: This paper has certainly struck a

chord. Four of our reviewers wanted to

review it for the ACNR. In the end, I asked

Bruno Gran, a neuroimmunologist from

Nottingham, to comment. He gives it a

very fair write-up. It is certainly attractive,

because it uses the latest understanding of

multiple sclerosis immunology (around the

difference between Th1 and Th17 cells) to

explain why some people respond to the

standard licensed drug for multiple

sclerosis, interferon-beta. Personally, I am

more sanguine about this study, mainly

because each stage in the argument is

based on results from rather few animals

or patients, and it all looks a bit tidy. But

perhaps I am just jealous!

In the last 15 years,progress in MS

management has been remarkable.The

introduction of immunotherapies that

reduce the frequency of disease

exacerbations and,at least in the short-

medium term, the tendency to accrual of

disability,has been welcomed by patients

and,perhaps with less enthusiasm,by

clinical neurologists,whose ambitions are

often higher than patients’.The first of such

treatments was,of course, interferon-beta

(IFNb),a“type I interferon”whose main

physiological job may be to signal the

presence of viral infections. In addition,

IFNb is a useful immunomodulator,which

can reduce disease activity in perhaps two

thirds of the patients for whom it is

appropriately prescribed.

We are currently in a rather exciting phase

of MS treatment history as promising oral

treatment are being developed and

introduced into clinical practice.However,

reasonable concerns by regulatory agencies –

most recently raised for cladribine in Europe

– suggest that we may well be using injected

interferons,as well as Copaxone, for longer

than we have thought in the last couple of

years. It is then even more important to try

and understand why at least a third,but

possibly up to a half,of people with relapsing-

remitting MS do not respond to IFNb.If we

take a step back and consider how IFNb is

thought to work in MS,some humility is

called for.As in so many areas of medicine,

we have to admit that while some

mechanistic facts are reasonably clear,many

are not.Most will agree that the induction of

interleukin-10 (IL-10),a (usually) anti-

inflammatory immune molecule,and a

reduction of permeability of the inflamed

blood-brain barrier are involved.But what

about the effects on immune cells that are

thought to initiate autoimmune reactivity

against myelin? T helper 1 (Th1) and Th17

cells are considered most relevant in this

respect,due to their ability to induce CNS

inflammation in animal models of MS

(collectively called“experimental

autoimmune encephalomyelitis”,EAE),and to

their increased levels in the brain,spinal fluid,

and peripheral blood of people with MS.

In a study published in“Nature Medicine”

earlier this year, the Stanford team

addressed the role of the Th1 and Th17

“cytokine pathways” in how mice and

humans with autoimmune inflammatory

demyelination respond to IFNb. In a

nutshell,what they found is that when Th1

cells (producers of Interferon gamma, IFNg

and historically the first T helper subset to

be indicted with a role in MS pathogenesis)

are the key players in the induction of EAE,

IFNb helps. It reduces disease severity both

clinically and pathologically.However,when

Th17 cells (identified in 2005 as the most

likely“real culprit” in autoimmune

neuroinflammation) are the inducers of

EAE,not only does IFNb not help,but

disease is actually made worse.Technically,

the key experiments in the study involved

the use of“adoptive transfer EAE”, in which

mice are immunised with myelin peptides

and,at the time when a potent myelin-

reactive response develops, autoreactive T

10 SUPPLEMENT TO ACNR • VOLUME 10 NUMBER 6 JANUARY/FEBRUARY 2011

In 2010, the UK MS Society funded 12 newresearch projects costing more than £1.6

million. The US National MS Society funded34 new projects, costing $14.6 million.

Journals

cells are harvested from these mice and

cultured in vitro before transferring them to

recipient,“naive”mice.During in vitro

culture, these cells can be potently skewed

to a Th1 or a Th17 phenotype by adding

either IL-12 or IL-23 to the cultures.

Recipient mice will develop an MS-like

disease (with paralysis and CNS

inflammation) and can be treated with IFNb

to assess its effect on clinical,pathological

and immunological disease parameters. A

summary of the rather complex

immunology experiments reported is as

follows: in agreement with preliminary in

vitro studies in mouse T cells, in vivo studies

showed that suppression of experimental

disease by IFNb correlated nicely with the

ability of IFNb to induce IL-10 in the treated

mice. In fact, IL-10 production increased

after IFNb treatment of“Th1 EAE”and was

not affected by treatment of“Th17 EAE”. In

addition,when disease was induced with

Th1 cells, IFNb treatment reduced the

numbers of all types of inflammatory T

helper cells in the spinal cord, i.e., both Th1

and Th17.By contrast, IFNb given to mice

with Th17-induced disease was associated

with an increase in both cell types.Another

clear result was that the beneficial effects of

IFNb on Th1 EAE required the presence of

IFNg signalling, as mice deficient in IFNg

receptor were not protected.As most

cytokines, IFNg will have different or even

opposite effects in different biological

contexts, and these findings confirm that in

mouse EAE, IFNg is predominantly“good”.

Unfortunately, this is a well known example

of weak or absent“translational link” from

mice to men,as IFNg was found to

exacerbate MS in patients in the late 1980s

and has since been considered“bad”for MS.

But back to IL-17 and,perhaps as

importantly, to IL-10.Was there a link to

anything of relevance to clinical practice?

This is what the final part of the study starts

to look at,without going as far as in the

mouse studies,but pointing to an interesting

direction. In human immune cell cultures,

the authors showed that IFNb had little

effect on the differentiation of Th1 cells but,

by contrast, it did inhibit the development

of Th17 cells.More importantly, the

induction of IL-10 by IFNb in these cultures

was clear and potent only in the Th1 cells.

This suggests that if there is such a thing as

“Th1 MS”, it might be more susceptible to

IFNb treatment,because it is Th1 cells that

respond to IFNb by making IL-10.The same

concept would not apply to“Th17 MS”.

The study concludes by testing another

important hypothesis: could MS patients

who fail to respond to IFNb have a stronger

propensity to Th17 responses? To start

addressing this question, the authors

measured“pre-treatment cytokine profiles”

in the sera of individuals with MS before

starting treatment with IFNb and classified

them as either“responders”(12 subjects) or

“non responders”(14 subjects) based on

clinical parameters such as relapse

frequency,degree of disability, and the need

for steroid courses before and after at least

12 months of IFNb treatment.Cluster

analysis of the 28 cytokines studied showed

that a subgroup of non responders (6

subjects) had significantly elevated serum

concentrations of both IL-17F (one of the

two main isoforms of IL-17) and IFNb

(which is itself produced by the immune

system).Furthermore, there was a strong

correlation between serum levels of IL-17F

and IFNb not just in this subgroup,but also

in all non responders, responders, and

healthy controls.

In summary, the take-home message at

this stage is that if these findings are

confirmed in a considerably larger

population of patients, it might one day be

possible to use serum concentrations of IL-

17F and IFNb (or possibly other

“biomarkers”) to predict the therapeutic

success of IFNb therapy.With the caveat that

biomarker studies in MS have often been

promising,but eventually disappointing, this

line of investigation appears worth

pursuing.

Axtell RC, de Jong BA, Boniface K, van der Voort

LF, Bhat R, De Sarno P, Naves R, Han M, Zhong F,

Castellanos JG, Mair R, Christakos A, Kolkowitz I,

Katz L, Killestein J, Polman CH, de Waal Malefyt

R, Steinman L, Raman

T helper type 1 and 17 cells determine efficacy of

interferon-β in multiple sclerosis and experimental

encephalomyelitis.

Nature Medicine 2010;16(4):406-12.

Dendritic cells: anunhealthy imbalance

Ed: Heinz Wiendl from Wurzburg has had

another excellent year, with a stream of

important immunological papers on

multiple sclerosis. Allison Curry reviews

this very nice piece of work which shows

that the immunological fault in multiple

sclerosis is not just wayward T cells, but

their upstream bosses as well: the

dendritic cells. This is the family of cells

that sit in the blood (monocytes) or tissues

(macrophages, microglia and others) that

pick up debris and package them up

nicely for the T cells to see. Once the T

cells have recognised the antigen, the

dendritic cells tell the T cells what to do,

by secreting this or that cytokine.

Innate immunity represents a finite balance

between microbial recognition and the

peripheral T cell tolerance required to

minimise autoimmunity. Plasmoid dendritic

cells (pDC) are considered critical for this,

exerting both stimulatory and regulatory

SUPPLEMENT TO ACNR • VOLUME 10 NUMBER 6 JANUARY/FEBRUARY 2011 11

“A year in Lyon left him with fluent French, a taste for fine wine, and theability to ski with finesse ... Once asked what made him happy, he cited

teaching neurology residents, sharing a glass of wine with his wife beforedinner, making a correct diagnosis, and having a full tank of gas in his car.”

From the obituary of Hill Panitch, neurologist and early experimenter ofMS treatments, who died in 2010.

Journals

effects on T cells.Their contribution to

antigen presentation in vivo however,and

their contribution to autoimmune T cell

activation and localised inflammatory

reactions remains unclear. Not found in the

CNS under normal conditions,elevated pDC

have been reported under

neuroinflammatory conditions with impaired

maturation and altered regulatory functions

implicated in the pathogenesis of Multiple

Sclerosis (MS). Here,Schwab et al

demonstrate that pDC can be further

distinguished into two separate populations

(“pDC1 and pDC2”) based on their surface

markers,cytokine production,activation

criteria and ability to prime naïve allogenic T

cells.pDC 1(CD123 high CD86- MHC II low)

resemble immature DC whereas pDC2 exhibit

a more mature phenotype.The role of pDC1

and pDC2 in MS autoimmunity is contrasted

with autoimmune Myasthenia Gravis (MG).

The innate immune system relies on

conserved pattern recognition receptors to

distinguish pathogen associated molecular

patterns (PAMPs). One such PAMP is that of

the CpG oligonucleotides present in

microbial DNA often used to mimic DC

activation. In response to CpG activation,

pDC1 showed a 3.8 fold increase in

regulatory (TR1) cells whereas pDC2

caused a 4.8 fold elevated autoaggressive

(Th17) T cells.

To determine any contribution of these

two subsets to MS,pDC from the PBMC of 16

MS patients were investigated.The

pDC1:pDC2 ratio was low in MS patients,

compared to healthy controls although

overall numbers were comparable.The

impact of inverted pDC ratios in MS patients

on immune response priming was

investigated using HC and MS derived pDC

in allogenic T cell co-culture assays.MS pDC

not only stimulated higher levels of Th17

cells compared to HC but also the

production of IL-17+IFN+ double positive

cells absent in HC.These populations have

previously been identified as highly pro-

inflammatory and, in the proper antigenic

setting, an encephalitolytic subpopulation of

CD4+ cells. Interestingly,pDC1 numbers rise

in MS following 12 months of IFN beta

treatment.Changes were observed by two

months suggesting that the subset

imbalance observed during MS is not a

fixed abnormality and is amenable to

therapeutic modification.

Although IFN beta may act at a variety of

levels,of which the pDC may be just one,

this research highlights how immunotherapy

can act at a cellular level in modulating the

immunoregulatory dysfunctions of patients

with MS and gives an insight into

mechanisms both involved and required for

therapeutic intervention.

Schwab N, Zozulya A, Kieseier K, and Wiendl H.

An imbalance of two functionally and

phenotypically different subsets of plasmoid

Dendritic cells characterises the functional

immune regulation in multiple sclerosis.

Journal of Immunology 2010;184:5368-74.

Devic’s DiseaseEd: In 2010, 174 papers were published on

neuromyelitis optica; in 2000, the number

was only 15. This sudden interest has

largely been down to Vanda Lennon’s

discovery in 2004 of an antibody

associated with the illness, which once

and for all resolved the question of

whether neuromyelitis optica was a form

of multiple sclerosis or a distinct disease.

It has also allowed people to explore the

boundaries of the phenotype of

neuromyelitis optica, much in the same

way that anti-GQ1b antibodies have done

for Miller Fisher syndrome. Isabel Leite

and Joanna Kitley, from the

neuroimmunology group at Oxford, have

picked out three of this year’s crop of

papers on Devic’s disease, my favourite

being the largest series on the disease yet

published, from a survey of 25 French

centres including that of Christian

Confavreux, whose wife is the

granddaughter of Eugene Devic. This

confirms the clinical impression that

neuromyelitis optica is a nasty disabling

disease: for instance 30% of the patients

had unilateral visual loss (<6/60) and 13%

had bilateral severe visual loss. Patients

with a high early relapse rate do worse

and we are encouraged to treat them

early... but not with interferon-beta as both

the Japanese and Oxford experience is

that this may exacerbate the condition.

A cause of vomiting andhiccups

In 2008,Takahashi et al showed that

intractable hiccup and nausea could be a

clinical marker of aquaporin-4 (AQP4)

antibody mediated disease,which could

precede the neurological symptoms at first

presentation or exacerbation of

neuromyelitis optica.Those symptoms were

thought to be related to the autoimmune

attack on the AQP4 molecules of astrocytes

in the pericanal region in the medulla

oblongata,which includes the area postrema.

Recently,Apiwattanakul and colleagues

described 12 aquaporin-4 antibody positive

patients who had an initial presentation of

intractable vomiting that remained

unexplained despite extensive investigations,

until the appearance of typical clinical

manifestations of neuromyelitis optica.

Although some of the patients did have mild

neurological symptoms it was shown that

vomiting constitutes a well defined

presenting feature in neuromyelitis optica in

at least 12% of patients.There was a variable

interval between vomiting onset and optic

neuritis (optic neuritis) or longitudinally

extensive transverse myelitis (LETM).The

authors hypothesised that the structural and

molecular characteristics of the area

postrema,such as the presence of fenestrated

capillaries,may contribute to the entry of

circulating IgG/AQP4 antibodies into the

CNS.They also emphasised the unique

nature of lesions in the area postrema,by

illustrating the focal loss of AQP4 and the

presence of inflammatory cells,but unusually

no demyelination or necrosis,which may

explain the reversibility of clinical symptoms

and imaging abnormalities in this area.As

the initial site of antibody binding in some

patients with neuromyelitis optica, these

presenting symptoms may allow a ‘window-

of-opportunity’ for antibody testing and

treatment in patients that are found to be

aquaporin-4 antibody positive. As 75% of

these patients first presented to a

gastroenterologist it is important to make

these clinicians aware of the importance of

intractable nausea as an initial symptom of

neuromyelitis optica.

Apiwattanakul M, Popescu BF, Matiello M,

Weinshenker BG, Lucchinetti CF, Lennon VA,

McKeon A, Carpenter AF, Miller GM, Pittock SJ.

Intractable vomiting as the initial presentation of

neuromyelitis optica.

Annals of Neurology. 2010 Nov;68(5):757-61.

Worrying epidemiologyof neuromyelitis optica

This is the largest observational,

retrospective,multicentre study of

neuromyelitis optica (NMO) involving 125

patients.By applying the 2006 NMO

diagnostic criteria, excluding the criteria of

12 SUPPLEMENT TO ACNR • VOLUME 10 NUMBER 6 JANUARY/FEBRUARY 2011

Journals

NMO-IgG, the authors were able to make the

diagnosis of neuromyelitis optica in 90% of

cases,with the NMO-IgG indirect

immunofluorescence assay (IIF) mandatory

for the diagnosis of neuromyelitis optica in

only 10% of patients, confirming the power

of the clinical and imaging features in the

diagnosis and treatment of neuromyelitis

optica.The demographic and basic clinical

characteristics of the neuromyelitis optica

patients are in agreement with those of

previous studies,but it is important to

highlight that in more recent reports, the

range of onset age is even wider than found

in this paper; neuromyelitis optica patients

aged above 70 years at onset have been

diagnosed.Analyses of the course of

neuromyelitis optica and disability

underlined the high degree of disability

following a first attack of myelitis or optic

neuritis (optic neuritis).Nearly 50% of those

neuromyelitis optica patients were left with

a severe deficit after their first attack of

optic neuritis. Importantly, a shorter time to

residual visual acuity of ≤ 6/60 was found to

be associated with a high number of MRI

brain lesions,which was the only predictive

factor of poor evolution that this study

revealed. Interestingly, in our (Ed; that is the

Oxford] cohort of patients with aquaporin-4

(AQP4) antibody-positive disease,we have

also observed that brain disease is

associated with more severe optic neuritis

involvement,particularly in young and non-

Caucasian patients with neuromyelitis

optica (Leite,Palace unpublished data).

Finally, the sensitivity of their assay is lower

than other published methods.When

comparing clinical and imaging features of

antibody negative and antibody positive

patients the use of the most sensitive cell-

based assays may highlight further

differences and should aid the search for

new target antigens in neuromyelitis optica.

Collongues N, Marignier R, Zéphir H, Papeix C,

Blanc F, Ritleng C, Tchikviladzé M, Outteryck O,

Vukusic S, Fleury M, Fontaine B, Brassat D, Clanet

M, Milh M, Pelletier J, Audoin B, Ruet A, Lebrun-

Frenay C, Thouvenot E, Camu W, Debouverie M,

Créange A, Moreau T, Labauge P, Castelnovo G,

Edan G, Le Page E, Defer G, Barroso B, Heinzlef

O, Gout O, Rodriguez D, Wiertlewski S, Laplaud D,

Borgel F, Tourniaire P, Grimaud J, Brochet B,

Vermersch P, Confavreux C, de Seze J.

Neuromyelitis optica in France: a multicenter

study of 125 patients.

Neurology. 2010 Mar 2;74(9):736-42.

Interferon-beta mayexacerbateneuromyelitis optica

Whilst there is undoubtedly clinical overlap,

neuromyelitis optica (NMO) is now

generally considered to be an antibody

mediated autoimmune disease unlike

multiple sclerosis.A report by Shimuzu and

colleagues suggests that

immunomodulatory treatment may even

exacerbate neuromyelitis optica. In this

retrospective study of 56 patients diagnosed

with relapsing remitting MS (RRMS) and

treated with interferon beta (IFNβ), 7

patients experienced severe neurological

exacerbation shortly after initiating therapy.

Retrospectively, the diagnosis in these 56

patients was reclassified as neuromyelitis

optica in 14,high risk of neuromyelitis

optica in 6 and conventional RRMS in 36.

All 7 patients who deteriorated on IFNβ

were patients reclassified as neuromyelitis

optica, and all tested positive for the

aquaporin-4 antibody.The patients

experiencing exacerbations all developed

transverse myelitis with longitudinally

extensive signal change on spinal MRI.Two

patients also had optic neuritis, and two

developed new tumefactive brain lesions.

Exacerbations were severe,with EDSS

ranging from 7.5 to 9.5, and occurred within

90 days of starting IFNβ. We have recently

described a neuromyelitis optica patient

misdiagnosed with RRMS who experienced

an increase in relapses and also a dramatic

increase in the AQP4 antibody levels on

treatment with IFNβ,which reduced quickly

following conventional immunosuppressive

treatment (Palace Arch Neurol 2010).

Shimuzu and colleagues’ paper highlights

the need to test for aquaporin-4 antibodies

in all RRMS patients with atypical clinical

features such as poor spontaneous recovery

from relapses,dramatic response to steroid

therapy with exacerbation following steroid

withdrawal,unusually severe optic nerve or

spinal cord relapses and in those whose

brain imaging or CSF characteristics are not

classical for MS. Immunosuppressive

treatments that appear effective for

neuromyelitis optica [Ed: such as rituximab

and azathioprine] are also effective

treatments of multiple sclerosis.Thus, it is

sensible to treat those with an optico-spinal

MS phenotype,or borderline cases, as

though they had neuromyelitis optica to

avoid the potentially catastrophic

deterioration if treated with IFNβ.

Shimizu J, Hatanaka Y, Hasegawa M, Iwata A,

Sugimoto I, Date H, Goto J, Shimizu T, Takatsu M,

Sakurai Y, Nakase H, Uesaka Y, Hashida H,

Hashimoto K, Komiya T, Tsuji S.

IFNβ-1b may severely exacerbate Japanese optic-

spinal MS in neuromyelitis optica spectrum.

Neurology 2010;75:1423-7.

Astrocytes:Who They?

Do astrocytes regulateCNS inflammation?

Ed: I always thought that astrocytes were

pretty harmless. They don’t seem to do an

awful lot of anything really, just plodding

their foot processes around and looking

SUPPLEMENT TO ACNR • VOLUME 10 NUMBER 6 JANUARY/FEBRUARY 2011 13

Immunofluorescence binding of NMOpatient serum IgG to aquaporin-4(AQP4) expressed on the surface ofHEK cells. Live cells were identified bythe blue nuclear stain dapi and thepatient AQP4 specific antibodies weredetected by red anti-human IgGantibody.

This picture illustrates the diagnosticassay used to detect antibodies toAQP4, which is free of charge for allthe NHS patients from England andScotland. This is part of the Diagnosticand Advisory Service for NMO fundedby the National Commissioning Group,NHS, UK.

For further information regarding theservice, please, visit:http://www.oxfordradcliffe.nhs.uk/forclinicians/referrals/neurosciences/nmoclinic/nmoclinic.aspxor call 01865 234889.

Image courtesy of Patrick Waters,M Isabel Leite and Angela Vincent.

Journals

spidery. But it turns out that they can be

really quite aggressive, as this EAE study

from the Cleveland Clinic shows. Patrick

Waters, a neuroimmunologist in Oxford,

walks us through the data.

Neuromyelitis optica (NMO) is a severe

destructive antibody mediated

demyelinating disease of the CNS.A key

feature of NMO lesions is the early loss of

astrocytes, commonly demonstrated as loss

of GFAP stain by immunohistochemistry.

Other antibody mediated diseases of the

CNS whose antibody targets are on neurons

rather than astrocytes (e.g.NMDAR

encephalitis) are not destructive, suggesting

that the target cell may play a key role in

modifying CNS inflammation.

Kang et al use both a MOG 33-55 peptide

active immunisation and a Th17 T-cells

adoptive transfer EAE mouse model to

demonstrate that astrocytes, via the IL-17R,

play a key role in stimulating the effector

phase of Th17 induced CNS inflammation.

Previously they demonstrated that knocking

out the IL-17R signalling molecule Act1-/-

greatly reduced the severity and delayed the

onset of Th17 induced EAE.They have now

followed this using the Cre-lox transgenic

system to delete Act1 in different cell

lineages.They demonstrate that deletion of

Act1 in endothelial or myeloid cells does

not affect disease severity in their EAE

mouse model.However,using the NesCre

transgene they deleted Act1 from

neuroectodermal cells of the CNS

(astrocytes,oligodendrocytes and neurons)

and confirmed that these cells were the key

cell to the reduction in inflammation seen

in their previous knock-out animal.They

further demonstrated that the only cell type

in which the IL-17 induced chemokine

profile was impaired, in Act1-/-,was the

astrocytes, implicating them in recruitment

of leukocytes into the brain, inducing the

second wave of inflammation.

Although the destruction of astrocytes is

only one aspect of NMO, this paper

highlights their importance in Th17 induced

autoimmune disease in an animal model

which may shed some light on the

mechanism of pathology in this human

disease.

Kang Z, Altuntas CZ, Gulen MF, Liu C, Giltiay N,

Qin H, Liu L, Qian W, Ransohoff RM, Bergmann C,

Stohlman S, Tuohy VK, Li X.

Astrocyte-restricted ablation of interleukin-17-

induced Act1-mediated signaling ameliorates

autoimmune encephalomyelitis.

Immunity. 2010 Mar 26;32(3):414-25.

Transporters: cassettesand media

Ed: there is an ancient family of proteins

that act as sherpas across membranes,

both within and on the outside of cells.

They require energy in the form of ATP and

so have been named: the “ATP-binding

cassette transporter family” (great

creativity). Many are responsible for

bacterial multi-drug resistance. And a

defect in one of the members leads to X-

linked adrenoleukodystrophy. Elga de

Vries’ blood-brain barrier group in

Amsterdam now ask whether these

proteins do anything in multiple sclerosis.

Mike Zandi takes us through their Brain

paper. Once again, astrocytes are involved,

although it is hard to say whether they are

goodies or baddies in this set-up.

This study examined the expression and

function of members of the ATP-binding

cassette transporter family in multiple

sclerosis (MS) using post mortem tissue.

The study showed enhanced multidrug

resistance associated protein-1 (MRP-1) and

P-glycoprotein (P-gp) on reactive astrocytes,

in active and chronic MS lesions compared

to MS normal appearing white matter

(NAWM) and controls.The authors then

tried to address the issue of causality by

using two functional in vitro chemotaxis

assays.Conditioned media from astrocytes,

harvested from MS lesions, increased

monocyte migration across a human

endothelial cell model of the blood brain

barrier.Blocking MRP-1 and P-glycoprotein

(P-gp) activity reduced the migration in

both models,probably by reducing the

secretion of a chemokine,CCL2.

The usual problems of post mortem

studies stack the odds against the findings

being primary and relevant to early disease -

most of the 10 patients had longstanding

disease (range 7-39 years, and 2 with an

unknown disease duration) and died from

some major systemic insult (pneumonia) or

local disease (stroke).The lack of difference

between NAWM and healthy controls is

perhaps surprising.The specificity of

function of these diverse molecules remains

unclear – in PloS ONE last year (8;4:e8212),

the same group published a study

demonstrating reduced severity of MOG-

induced experimental autoimmune

encephalomyelitis through reduced

secretion of pro-inflammatory cytokines in a

P-gp knockout mouse.The authors conclude

reasonably that the generation of astrocyte-

specific P-gp knockout mice will be the

most fruitful step forward for further studies.

Kooij G, Mizee MR, van Horssen J, Reijerkerk A,

Witte ME, Drexhage JA, van der Pol SM, van Het

Hof B, Scheffer G, Scheper R, Dijkstra CD, van der

Valk P, de Vries HE.

Adenosine triphosphate-binding cassette

transporters mediate chemokine (C-C motif)

ligand 2 secretion from reactive astrocytes:

relevance to multiple sclerosis pathogenesis.

Brain epub 22 Dec 2010.

Th17 cells seek anddestroy neurons

Ed: Th17 cells are the new black. We

reviewed their history in last year’s

supplement. Basically we had all been

made to look very silly, because in the

past we had been measuring IL-12 with an

antibody which also detects IL-23, but we

did not realise it. So what we thought was

IL12 making “Th1” cells was in fact a

mixture of IL12 and IL23... and IL23 makes

a distinctive type of T cell called the

“Th17” cell. This year, everyone and his

dog has had a crack at working out what

exactly they do in multiple sclerosis. If

Frauke Zipp from Mainz is correct, the

news is bad. It seems Th17 cells have a

14 SUPPLEMENT TO ACNR • VOLUME 10 NUMBER 6 JANUARY/FEBRUARY 2011

Harry Potter author, JK Rowling, donated £10million to set up the Anne Rowling

Regenerative Neurology Clinic in Edinburgh.

Journals

particular predilection for neurons. Denise

Fitzgerald has chosen this paper; recently

recruited to Queen’s Belfast, she has been

deeply involved in working out what IL-27

(!) does in Th17 cells...

The role of Th17 cells in MS has been a hot

topic of research in recent years.Much of

the work has focused on how these cells

develop,what cytokines and transcription

factors they express and what effect Th17

cells have on other cells of the immune

system.However, this year, a research team

led by Frauke Zipp published a fascinating

study in mouse models,demonstrating

direct attack of neurons by Th17 cells.This is

particularly relevant to MS as axonal and

neuronal pathology is known to contribute

to permanent disability in MS.

Using a powerful imaging technique called

two-photon laser scanning microscopy and

reporter mouse models with green

fluorescent neurons and red fluorescent

immune cells, the authors were able to image

in vivo,neuroimmune interactions during

EAE.Typical immune cell infiltration was

observed in the CNS of mice with EAE.

Automated cell tracking during movies

allowed quantitative analysis of immune cell

locomotion and revealed a correlation

between slowing of T cell velocities and

clinical worsening/disease progression.

The authors went on to study the neuronal

interactions of Th1 and Th17 cells with and

without specificity for CNS antigen and

found that Th17 cells exhibited a greater

neuronal contact rate than Th1 cells and

interestingly, this was not restricted by CNS

antigen specificity. Indeed,Th17 cells

appeared to scan their microenvironment for

axon dysmorphology and varicosities and

establish lasting contact with axons while

Th1 cells appeared to predominantly form

random,short contacts with axons.What was

fascinating from the perspective of CD4+ T

cell biology was that T cell-axon interactions

did not appear to be mediated by antigen

presentation.Expression of MHC-II did not

co-localise with T cell-axon synapses and

contact was observed between myelin

specific T cells and the non-myelinated pre-

synaptic terminal of neuronal soma.

The team went on to show in vitro, that

Th17 cells formed neuronal synapses and

induced neuronal death, in contrast to Th1

and Th0 cells.Analysis of the synapse

formation revealed that MHC-II was not

required but that T cells re-orientated

cytoskeletal elements towards the synapse

and that the adhesion molecule LFA-1 was

involved.The mechanism of Th17-mediated

neuronal attack was shown to involve

glutamate-mediated excitotoxicity which

increased intracellular calcium in soma.

Taken together, these findings suggest a

completely novel pathogenic mechanism of

Th17 cells in CNS inflammation that likely

contributes to permanent disability.The next

step will be to translate these exciting

findings to human experimental systems

and potentially, to develop strategies to

disarm this destructive mechanism of Th17

cells and/or enhance neuroprotection to

withstand such attacks by T cells.

Siffrin V, Radbruch H, Glumm R, Niesner R,

Paterka M, Herz J, Leuenberger T, Lehmann SM,

Luenstedt S, Rinnenthal JL, Laube G, Luche H,

Lehnardt S, Fehling HJ, Griesbeck O, Zipp F.

In vivo imaging of partially reversible Th17 cell-

induced neuronal dysfunction in the course of

encephalomyelitis.

Immunity. 2010 Sep 24;33(3):424-36.

Are histone mimics thefuture?

Ed: Mike Zandi reads the runes of a

possible new therapeutic approach for

inflammatory disease in the future.

This paper is of interest as a demonstration of

a novel therapeutic approach.The

bromodomain and extra terminal domain

(BET) family of proteins ‘read’ the reversible

epigenetic marks left on proteins,such as a

phosphate here,or a methyl there,and relay

this information to histones which form

chromatin and influence gene expression.

The authors probed a synthetic library for

molecules which bound to the wide acetyl-

lysine pockets on BET,and named the

molecule with highest affinity I-BET.The

authors put the drug to the test in an in vivo

mouse model of lipopolysaccharide-induced

endotoxic shock and bacteria induced sepsis,

but the approach is hopefully generalisable to

other inflammatory diseases in neurology.

Taverna and Cole provide an editorial (pp

1050-51) and comparison with a similar

paper in the same issue applied to cancer.

Nicodeme E, Jeffrey KL, Schaefer U, Beinke S,

Dewell S, Chung CW, Chandwani R, Marazzi I,

Wilson P, Coste H, White J, Kirilovsky J, Rice CM,

Lora JM, Prinjha RK, Lee K, Tarakhovsky A.

Suppression of inflammation by a synthetic

histone mimic.

Nature. 2010 Dec 23;468(7327):1119-23.

Tablets andTrialsEd: What do we want multiple sclerosis

drugs to do? And how should we measure

that in clinical trials? It seems that almost

anything will have an effect on brain

scans, but very few agents can shift

disability over the long term by a real

amount. Gavin Giovannoni, a veteran of

multiple sclerosis trials, advocates for the

new no-prisoners outcome measure

(“DAF”) that was first proposed by Eva

Havrdova and the Tysabri people in 2009.

Gavin and colleagues have taken a

similar approach with the cladribine trial,

and presented it at the ECTRIMS meeting

this year.

Disease-activity freestatus

Disease activity-free (DAF) status is a

relatively new composite efficacy parameter

that incorporates clinical and radiological

measures of disease activity in multiple

sclerosis.DAF has been defined as no

clinical (no relapses and no sustained

disability progression) and radiological (no

gadolinium-enhancing lesions and no new

or enlarging T2-hyperintense lesions on

cranial MRI) disease activity over 2 years.

Achieving,and more importantly

maintaining, such a response over an

extended period of time is the expectation

of all disease-modifying therapies,

particularly with regard to emerging

therapies with novel mechanisms of action.

Post-hoc analysis of the phase 3

natalizumab vs.placebo (AFFIRM) study

revealed that 37% of natalizumab-treated

compared to 7% of placebo-treated subjects

were DAF over 2 years (p<0.0001)

(Havrdova Lancet Neurol 2009;8:254–260).

And a copy-cat post-hoc analysis of the

phase III oral cladribine (CLARITY) study

demonstrated that a greater proportion of

cladribine-treated patients remained DAF

over 96 weeks in both the 3.5 and 5.25

mg/kg treatment groups vs.placebo: 44.3%

and 46.0% versus 15.8%, respectively

(p<0.001).

Won’t it be interesting to see comparative

DAF analyses for fingolimod vs.placebo

(FREEDOMS) and alemtuzumab vs.

SUPPLEMENT TO ACNR • VOLUME 10 NUMBER 6 JANUARY/FEBRUARY 2011 15

Journals

interferon-beta-1a (CAMMS) studies? DAF is

an appealing concept and I predict that it

will become the primary outcome measure

of choice in the next generation of phase 3

studies in an era where placebo-controlled

trials will be unethical and impossible to

do.Will we eventually define DAF status of

>5, 10 or 15 years as a cure?

Giovannoni G, Comi G, Cook S, et al.

Analysis of sustained disease activity-free status in

patients with relapsing–remitting multiple sclerosis

treated with cladribine tablets, in the double-blind,

96-week CLARITY study.

Mult Scler. 2010;16(S7):S288(P825).

Fingolimod has arrived!

Ed: One of the most distinguished English

neurologists of the 19th century, Sir

Frederick Bateman, plied his trade at the

Norfolk & Norwich Hospital. His natural

successor, Martin Lee, summarises the

data from the three phase 3 studies that

emblazoned the NEJM in the early weeks

of 2010, and led to the licensing of one

drug (fingolimod)... but not of the other

(cladribine). 2011 will be remembered as

the year that people with multiple

sclerosis could first take a pill to control

their disease. As Martin emphasises, both

of these drugs show useful efficacy and

the key determinant of their future use

will be long-term safety, particularly with

respect to malignancy and viral

infections. These are low frequency

events, Martin makes the point that the

two deaths from disseminated herpes

infections with fingolimod occurred

around the time of corticosteroid use;

personally, I find it hard to think that is

really relevant (or if it is, it is of little

comfort, because plenty of multiple

sclerosis patients will need steroids after

fingolimod).

There has been surprisingly little fanfare

following the FDA’s approval of 0.5mg

Fingolimod (GilenyaTM) in September 2010

as the first oral therapy for patients with

relapsing MS.However with European

Licensing likely to follow soon and patient

demand expected to be high,Fingolimod is

unlikely to pass you by.

Fingolimod is a synthetic analogue of

myriocin (derived from Isaria sinclairii, an

entomopathogenic fungi) and has a

relatively novel mode of action as an

immunomodulator. It acts primarily by

preventing lymphocyte egress from lymph

nodes by the down-regulation of

lymphocyte sphingosine-1-phosphate (S1P)

receptors. In MS this is presumed to reduce

the circulation of auto-aggressive

lymphocytes.Pre-clinical studies have also

suggested there may be directly beneficial

effects within the CNS via S1P receptors

expressed at gap junctions and neural cells.

Recent licensing was based in the main

on two pivotal phase III trials reported side

by side in the New England Journal of

Medicine in February 2010.The first

‘FREEDOMS’ study involved 1272 relapsing

MS patients over 2 years comparing two

doses (0.5mgs and 1.25mgs) of Fingolimod

with placebo.Annualised relapse rates were

reduced by 54% and 60% respectively with a

significant reduction in the rate of disability

progression confirmed at 3 and 6 months.

All MRI surrogates were positively impacted,

including those linked with disability

including T1-black holes and brain atrophy.

The second ‘TRANSFORMS’ study was

similarly sized and compared the two doses

of Fingolimod with once weekly Avonex

over a year.Annualised relapse rates were

again significantly reduced (by 52% and

38%) compared with Avonex,with

significant but perhaps less marked MRI

parameter differences.Overall the results

suggest the efficacy of Fingolimod probably

lies somewhere between the standard DMT

therapies and Tysabri.This may be where

several of the new oral agents will sit in

terms of benefit and it is therefore their side

effect profile which may determine

licensing and treatment decisions.

Particular to Fingolimod is an association

with transient first dose bradycardia which

is usually asymptomatic and unless there is

a pre-existing cardiac history probably

unlikely to be troublesome in practice.

Macular oedema is also associated though

occurred in less than 1% and usually at the

higher dose which has not been licensed.

Concern regarding an increased

susceptibility to herpesvirus infections is

common to most of the drugs affecting

lymphocyte function/surveillance.A

reduction in peripheral lymphocyte counts

was universal with Fingolimod but

lymphocytopenia very rare.Overall

incidence of herpesvirus infection was not

greater in the 0.5mg group than controls but

two herpetic deaths did occur in the 1.25mg

study group in the TRANSFORMS study.The

first death was due to systemic varicella

zoster, in a patient with negative serology at

study entry who received high dose

intravenous methylprednisolone for a

relapse and was unlucky enough to be

exposed to a child with chicken pox.The

second death occurred from HSV

ACNR Prize for theBest MS Research in 2010

16 SUPPLEMENT TO ACNR • VOLUME 10 NUMBER 6 JANUARY/FEBRUARY 2011

The US Food and Drug Administration has approved Gilenya capsules(fingolimod) to reduce relapses and delay disability progression in patients withrelapsing forms of multiple sclerosis (MS). “Gilenya is the first oral drug that can

slow the progression of disability and reduce the frequency and severity ofsymptoms in MS, offering patients an alternative to currently available injectabletherapies,” said Russell Katz, MD, director of the Division of Neurology Products

in the FDA’s Center for Drug Evaluation and Research.FDA Press release, September 22nd 2010.

(The $48,000 per anum will cause our poor NHS to squeal a bit!)

Journals

encephalitis, also possibly complicated by

IV methylprednisolone use.

The other issue concerns the potential

risk of malignancy common amongst all

long-term immunomodulators.Overall

incidence of malignancy in these trials was

low and roughly equal across the three

treatment arms suggesting no increased risk

but long-term reporting will be required to

confirm this. It is difficult to know whether

three cases of melanoma picked up in the

0.5mg Fingolimod arm after dermatological

review was added to the study

retrospectively are statistically relevant.

In summary therefore it would probably

be fair to say that Fingolimod works, is likely

to be coming to a clinic near you soon and

will have high patient interest.An unknown

long-term safety profile, and funding issues

in the UK may initially temper the

excitement.

Kappos L et al. for the FREEDOMS study group.

A Placebo-controlled Trial of Oral Fingolimod in

Relapsing Multiple Sclerosis.

New Eng. J. Med. 2010 Feb 4;362(5):387-401.

Cohen JA et al. for the TRANSFORMS study

group.

Oral Fingolimod or Intramuscular Interferon for

Relapsing Multiple Sclerosis.

New Eng. J. Med. 2010 Feb 4;362(5):402-15.

Cladribine: we’ll have towait a while

You have to feel a little sorry for oral

Cladribine.Better known than Fingolimod,

with a favourable dosing regimen and

positive licensing decisions in Australia and

Russia, the recent thumbs down from the

European Medicines Agency was a big blow

and is unlikely to bode well for the pending

US licensing decision.European concerns

over long-term safety and a lack of

comparison with standard DMT therapy

appear to have thrown a spanner in the

works,at least while we wait for the results of

the resubmission to the regulatory agencies.

Cladribine is a deoxyadenosine analogue

that impairs DNA synthesis and repair

resulting in a relatively selective apoptosis of

CD4+ and CD8+ lymphocytes.Previous

studies of the intravenous preparation

showed predictably disappointing results in

fairly advanced MS but the results of the

phase III ‘CLARITY’ oral therapy trial

published in the same issue of the New

England Journal of Medicine as the

Fingolimod trials were anticipated to be

better. In this study, 1326 relapsing MS

patients with mild to moderate disability

were randomised to receive low or high

dose (3.5mg or 5.25mg/kg bodyweight)

cladribine given over 4 or 5 days once a

month for 4 months in year one with a

further 2 courses in year two or placebo.For

this study patients were excluded if they

had previously failed 2 or more DMT

therapies.

Results showed a reduction in annualised

relapse rate of 58% and 55% for low and high

dose groups respectively.Cladribine was also

associated with a 5% absolute reduction in

patients experiencing confirmed disability

progression at 3 months although the more

robust measure of 6 month confirmed

progression was not assessed.Surrogate MRI

parameters were reduced by around 75% in

the treatment arms.Overall the efficacy

results available look remarkably similar to

those of Fingolimod although disability

outcome measures were perhaps less

extensively assessed.

Lymphopenia occurred in 20-30% of

patients though counts below 0.2 x109/L

were unusual. Severe neutropenia occurred

in three patients which is of some concern.

Herpes zoster infections occurred in 20

cladribine patients and were all

dermatomal.No maligancies occurred in

the placebo group versus 10 in the

treatment group including 5 leiomyomas,a

melanoma and a pancreatic and ovarian

carcinoma.Chriocarcinoma also occurred

in one patient 9 months after study exit.

There were two possible deaths related to

cladribine in the study,one from metastatic

pancreatic carcinoma and one from

pancytopenia and reactivation of latent TB.

We can say therefore that cladribine

appears effective in MS but the balance

between benefit and risk remains

uncertain, at least in the eyes of the

European regulators. Lingering anxiety

regarding long-term cancer risk coupled

possibly with the potential risks of chronic

lymphopenia mean that for the near future

this is unlikely to be an option for patients

with MS in the UK.

Giovannoni G et al. for the CLARITY study group.

A Placebo-controlled trial of Oral Cladribine for

Relapsing Multiple Sclerosis.

New Eng. J. Med. 2010 feb 4; 362(5):416-26.

Ocrelizumab andOfatutumab; the newkids on the block

Ed: By now, most people interested in

multiple sclerosis will have heard that

rituximab is a pretty effective treatment of

the disease, at least as far as one phase 2

study shows. This might have come as a

surprise because this monoclonal antibody

depletes B cells, originally regarded as

“also-runs”, and leaves the evil T cells

alone. But, it turns out that (a) B cells can

be far more unpleasant than originally

thought and (b) T cells need B cells

around to be really nasty. So, it would

make sense to develop rituximab as a

treatment for multiple sclerosis.

Unfortunately its patent life is short, so the

pharmaceutical companies have invested

in newer, brighter versions of the

antibody.... iPhone 4 supersedes 3 sort of

thing. Ruth Dobson and Gavin Giovannoni

review the early (unpublished) news from

ECTRIMS on the results of these novel B

cell depleters.

Historically, research has focused on the

role of T-cells in multiple sclerosis (MS),

although recently attention has shifted to

the role of B-cells.Whilst previously, the

presence of B-cells within MS plaques was

thought to be secondary to T cell

dysregulation, it is now becoming clear that

these B-cells play an independent role in

disease, including antigen presentation,

cytokine production and the establishment

of follicle-like structures in the CNS

compartment.The role of B-cells in MS has

been further cemented by the success of

Rituximab,and more recently ocrelizumab

and ofatumumab,monoclonal antibodies

directed against the B cell surface marker

CD20 have undergone phase 2 clinical

trials.Both ocrelizumab and ofatumumab

are humanised monoclonal antibodies,

which in theory reduce side effects and

improve efficacy when compared to

rituximab,which is a chimeric antibody.

These exciting new advances in MS therapy

were discussed at the ECTRIMS meeting in

2010,and are briefly summarised.

The phase 2 trials of ocrelizumab

(Genentech and Biogen Idec) and

ofatutumab (Genmab) in RRMS evaluated

both efficacy,as demonstrated by MRI, and

safety.

ACNR Prize for theBest MS Research in 2010

SUPPLEMENT TO ACNR • VOLUME 10 NUMBER 6 JANUARY/FEBRUARY 2011 17

Journals

Ocrelizumab reduced the mean number

of gad-enhancing lesions at week 24 (the

primary endpoint) by 89% and 96% (600mg

and 2000mg total dose respectively)

compared to placebo (p<0.0001).The

percentage of patients free of new

gadolinium-enhancing lesions was lower in

the ocrelizumab groups compared to

placebo (76.5% and 69.2% vs 35.2%,

p<0.0001). There was no significant

difference in the number of new T2 lesions.

The clinical endpoint was also met: the

ocrelizumab group showed 80% and 73%

reduction in the annualised relapse rate

compared to placebo (600mg,p=0.0005,

2000mg,p=0.0014).There were no clear dose

differences.Ocrelizumab was generally well

tolerated,and there was no evidence of

opportunistic infections in the treatment

group.Worryingly one patient,who received

ocrelizumab,died of a systemic

inflammatory syndrome.

Three doses of ofatutumab were studied

– 100mg,300mg and 700mg.Ofatutumab

reduced the mean number of new

gadolinium-enhancing lesions (weeks 8-24)

to 0.04 compared to 9.69 in the placebo

group (relative risk reduction 99.8%,

p<0.001).There were no significant

differences between the treatment groups.

Similar reductions occurred for the

cumulative number of total Gd-enhancing

lesions and new and/or enlarging T2

lesions.No significant safety signals

emerged from this small trial.

It remains an exciting time for MS

therapeutics –“the new kids on the block”–

the new anti-B-cell therapies are a

therapeutic breakthrough and have put the

B cell centre stage.

Kappos L, Calabresi P, O’Connor P, Bar-Or A, Li D,

Barkhof F, Yin M, Glanzman R, Tinbergen J,

Hauser S.

Efficacy and safety of ocrelizumab in patients with

relapsing–remitting multiple sclerosis: results of a

phase II randomised placebo-controlled

multicentre trial.

Multiple Sclerosis 2010; 16: S7–S39, abstract 114.

Soelberg Sorensen P, Drulovic J, Havrdova E,

Lisby S, Graff O, Shackelford S. Magnetic

resonance imaging (MRI) efficacy of ofatumumab

in relapsing-remitting multiple sclerosis (RRMS) –

24-week results of a phase II study.

Multiple Sclerosis 2010; 16: S7–S39, abstract 136.

Daclizumab: animprobable treatment

Ed: the use of daclizumab as a treatment

of multiple sclerosis is a happy historical

accident. It binds to the CD25 molecule

which, as everyone knows, is expressed at

highest density on regulatory T cells.

Neutralising regulatory T cells is hardly

the way forward to treat multiple

sclerosis... But daclizumab has been

around for a lot longer than we have

known about Tregs. So it was first used,

mainly in transplantation, when people

thought that CD25 was just a marker of

activated T cells. Success in this setting led

to its use in multiple sclerosis, first by Bibi

Bielekova and colleagues at the NIH in the

early 2000s. She worked out that

daclizumab works not by suppressing

activated T cells or modifying Tregs, but –

bizarrely – by upregulating a curious

subgroup of natural killer cells defined by

high CD56 expression. Quite why this is

good news for multiple sclerosis is beyond

me. Orla Tuohy, a clinical PhD student

working with me, summarises the most

recent clinical trial.

The CHOICE study reported in Lancet

Neurology investigated the efficacy and

safety of daclizumab as add-on therapy in

patients with disease activity on beta-

interferon. Daclizumab,a monoclonal

antibody against CD25 (part of the IL2

receptor structure), is licensed in the US for

prevention of renal transplant rejection,and

has been used experimentally in

autoimmune uveitis and rheumatoid

arthritis. As CD25 is upregulated on

activated lymphocytes the initial assumption

was that blocking this component of the IL-2

receptor would block the function and/or

survival of the activated auto-reactive T cells

involved in MS pathogenesis. Further study

on the mechanism of action of daclizumab

however,has identified an upregulated

subpopulation of Natural Killer cells (CD56

bright cells), that may mediate the anti-

autoreactive T cell effect of daclizumab.

In multiple sclerosis daclizumab has

been used in open-label studies and in a

small phase 2 trial reported in Neurology in

2007. In the 2007 trial daclizumab reduced

radiological inflammatory lesions (the

primary endpoint),but the effect of

withdrawing concomitant beta-interferon

after six months of daclizumab led to

relapse in three patients.

The CHOICE study was designed as a

larger,multi-centre,phase 2 study in which

230 patients were randomised to one of

three groups; beta-interferon + placebo

(n=77),beta-interferon + high-dose

daclizumab (n=75),beta-interferon + low

dose daclizumab(n=78). The treatment

phase lasted 24 weeks,with an additional 48

weeks of follow-up during the wash-out

period.Patients included had either clinical

or radiological evidence of MS activity in

the year prior to enrolment despite

treatment with beta-interferon. Of the 230

patients included,92% had relapsing-

remitting MS,while the remaining 8% had

secondary progressive disease.Mean

interval since MS diagnosis was

approximately 6.5 years,with a mean EDSS

of 3.0 across the treatment groups. At

baseline the mean number of Gd-enhancing

lesions was higher in the low-dose

daclizumab group (2.75) than the Interferon

+ placebo (1.1) or Interferon + high dose

daclizumab group (0.8).The primary

endpoint was the total number of new or

enlarged contrast-enhancing lesions on MRI

brain scans done at four weekly intervals

between weeks 8 and 24.Clinical outcomes

(annual relapse rate,EDSS scores,MSFC

scores), and other radiological parameters

(change in T1-weighted lesions,T2-weighted

lesion changes), encompassing

inflammatory and neurodegenerative

markers, were included as secondary

18 SUPPLEMENT TO ACNR • VOLUME 10 NUMBER 6 JANUARY/FEBRUARY 2011

The top Google news hit for “multiple sclerosis”in 2010 was the assisted suicide at Dignitas of

Andrew Colgan, aged 42, a former marinebiologist who had the disease.

Journals

endpoints.On the immunology side, this

paper reproduced the finding of an

expansion of the CD56 bright population of

NK cells in daclizumab-treated patients (up

to 8-fold relative to interferon-treated

patients).

The only endpoint which reached

statistical significance was the reduction in

new and enlarged MRI contrast-enhancing

lesions between the high-dose daclizumab +

beta-interferon,and the beta-interferon +

placebo groups. There was no significant

difference in annual relapse rate between

groups,while mean EDSS scores remained

unchanged at the end of follow-up for all

groups.

From a safety perspective, serious adverse

events were more frequent in daclizumab

treated patients (13%) versus those on

interferon and placebo (5%) with infections

being the most common adverse events.

More concerning was the occurrence of

malignancy in two daclizumab-treated

patients,which were one case of breast

ductal carcinoma-in-situ, and a recurrence

of pseudomyxoma peritoneii.

Overall, high-dose daclizumab in

combination with interferon reduced MRI

activity markers relative to interferon alone,

an effect which seemed to wane after the

end of the 24 week daclizumab treatment

phase. The brief duration of the study may

have been insufficient to detect any

differences in clinical parameters. In the

current highly competitive field of MS

therapeutics,daclizumab needs to prove

that its effect extends beyond a reduction in

radiological markers of inflammation.

Wynn D, Kaufman M, Montalban X, Vollmer T,

Simon J, Elkins J, O'Neill G, Neyer L, Sheridan J,

Wang C, Fong A, Rose JW; CHOICE investigators.

Daclizumab in active relapsing multiple sclerosis

(CHOICE study): a phase 2, randomised,

double-blind, placebo-controlled, add-on trial with

interferon beta.

Lancet Neurology. 2010 April;9(4):381-90.

A skin patch to treatmultiple sclerosis

The editor writes:This is fantastic.Krys

Selmaj at Lodz in Poland has come up with

a really nifty idea.He picks up on the work

of DavidWraith and others,who have shown

that antigen-specific IL-10 producing

regulatory cells can be induced by injection

of peptides into the skin or by sniffing them

up the nose! He goes for the much more

elegant approach of applying myelin

peptides in skin patches.30 patients with

multiple sclerosis were treated with a

mixture of three peptides or control over

one year.And it worked! At least, it worked at

the immunological level: IL10 producing

regulatory T cells were induced. It is quite a

big step from here to actually controlling the

disease,but nonetheless,an exciting start.

Jurynczyk M, Walczak A, Jurewicz A,

Jesionek-Kupnicka D, Szczepanik M, Selmaj K.

Immune regulation of multiple sclerosis by

transdermally applied myelin peptides.

Ann Neurol. 2010 Nov;68(5):593-60.

Update on natalizumab-associated PML

Ed: When the history of multiple sclerosis

therapeutics is written, it will be divided

into two phases. First, before natalizumab-

PML, was an age of innocence, when any

disease-modifying agent was a miracle

cure offering unblemished promise. The

second, after PML was reported in

association with natalizumab, is now:

where we recognise the serious risks of

some of our treatments and – to put that in

context – we and our patients have to face

the seriousness of leaving multiple

sclerosis untreated. I think that is why

fingolimod is being received cautiously, so

differently from the fanfare which greeted

the arrival of the less efficacious

interferons. What we need is clarity about

the risks of this or that treatment... and to

be able to individualise risk as much as

possible. David Hunt, from Edinburgh,

commentates with Gavin Giovannoni on

the 2010 literature on PML risk after

natalizumab. In addition to their helpful

insights, it is worth saying that the risk of

PML after natalizumab does seem to relate

to duration of exposure: in the Lancet

Neurology report of 28 cases (of whom 8

died), the risk rises steadily towards

1:1000 at 36 months. Thereafter, it seems

superficially that the risk falls off, but the

confidence intervals are wide and there

were many fewer patients who have

received natalizumab this long. And, while

we are talking about the anti-adhesion

molecule antibody, Natalizumab, can I just

mention a very nice study by Heinz

Wiendl in PLoS One (Doerck 2010)

showing that blocking adhesion molecules

may not always be a good idea.

Regulatory T cells use LFA-1/ICAM-I (but

not VLA-4/VCAM-I, the Tysabri molecules)

to get into the CNS and downregulate

inflammation. If you stop them from

entering the brain, the disease lasts longer.

2010 saw the clinical experience with

natalizumab in multiple sclerosis pass

100,000 person-years and the tally of

associated PML cases rise to over 70.1 The

bottom line is that the risk of PML has not

changed much from the 1:1000 figure

originally suggested after the problem first

emerged in the phase 3 trials.However

analysis of these new cases has been

instructive on a number of fronts.2

Firstly, in contrast to PML associated with

other monoclonal antibodies such as

rituximab, survival is reasonably favourable

at approximately 80%,with some survivors

being able to return to work.2 It is likely that

this is due to a combination of early

detection and a reversal of the

pharmacodynamic effects of natalizumab

by plasma exchange,enabling a rapid

recovery of CNS immune surveillance.3

Much of the neurological damage to

survivors is mediated by a vigorous immune

reconstitution syndrome (PML-IRIS) akin to

that seen in HIV-associated PML after

HAART and optimisation of the

management of PML-IRIS will be important

to further improve outcome.

Secondly, a number of risk factors for the

development of PML are emerging.Prior

immunosuppressant use is associated with a

fourfold increase in risk.1 JC virus

seropositivity at baseline,as assessed with a

SUPPLEMENT TO ACNR • VOLUME 10 NUMBER 6 JANUARY/FEBRUARY 2011 19

The 5 year impactfactor of journals

recorded in 2009 (thelast information

available) was 9.98for Brain, 9.60 for

Annals of Neurology,7.20 for Neurology,4.7 for the JNNP.

Journals

novel 2-stage ELISA developed by Biogen,

has so far been positive in 17/17 patients

with natalizumab-associated PML who had

baseline serology available for assessment.4

One potential drawback is the 2.5% false-

negative rate of the ELISA plus evidence of

seroconversion on serial testing.However

these results suggest that JCV antibody

testing may be of potential clinical utility as

we learn more about how to risk-stratify

patients. In particular, a negative JCV

serology result may confer a much lower

risk of developing PML. In contrast, PCR-

based methods for virus detection in blood

and urine,when tested in a large

population,are a less promising prospect for

risk stratification.5

These results highlight the need for

ongoing vigilance for PML at both the

individual and population level to maximise

the benefits and minimise the risks

associated with this drug.

1. Bozic C, Cristiano LM, Hyde R. Utilization and

Safety of Natalizumab in Patients with Relapsing

Multiple Sclerosis. Presented at ECTRIMS;

October 13-16, 2010; Gothenburg, Sweden.P.893

2. Clifford DB, De Luca A, Simpson DM, Arendt G,

Giovannoni G, Nath A. Natalizumab-associated

progressive multifocal leukoencephalopathy in

patients with multiple sclerosis: lessons from 28

cases. Lancet Neurology, 2010;9:438-46.

3. Khatri BO, Man S, Giovannoni G, Koo AP, Lee

JC, Tucky B, Lynn F, Jurgensen S, Woodworth J,

Goelz S, Duda PW, Panzara MA, Ransohoff RM,

Fox RJ. Effect of plasma exchange in accelerating

natalizumab clearance and restoring leukocyte

function. Neurology, 2010;72:402-9.

4. Gorelik L, Lerner M, Bixler S, Crossman M,

Schlain B, Simon K, Pace A, Cheung A, Chen LL,

Berman M, Zein F, Wilson E, Yednock T, Sandrock

A, Goelz SE, Subramanyam M. Anti-JC virus

antibodies: Implications for PML Risk Stratification.

Annals of Neurology 2010;68:295-303.

5. Rudick RA, O'Connor PW, Polman CH,

Goodman AD, Ray SS, Griffith NM, Jurgensen SA,

Gorelik L, Forrestal F, Sandrock AW, E Goelz S.

Assessment of JC virus DNA in blood and urine

from natalizumab-treated patients. Annals of

Neurology 2010 Sep;68(3):304-10.

Stem CellCorner

At last somemesenchymal stemcells into humans

Ed: Perhaps the second most useful thing

that has happened in stem cell therapies

in 2010 is that Dr Troussel was struck off

the register. He is the charlatan who was

recruiting patients in this country to

travel to his “clinic” in Rotterdam to

receive supposed stem cell injections in

exchange for thousands of pounds. We

are now in the surreal situation that

“stem cell therapy” could mean anything

from dodgy deals in the lavatory of

ferries crossing to Ireland, or pointless

trips to unheard-of institutes in China or

Russia, to the most sophisticated of

modern medicine. This is compounded by

the fact that there are many different

types of stem cell treatments. Many

hundreds of patients with multiple

sclerosis have already had

haematopoietic stem cells to rescue them

from ablative chemotherapy. This is the

ultimate version of “rebooting” the

immune system. Paolo Muraro, at

Imperial, is a leading light in this high-

risk world and he reviews two papers for

us below. Another type of stem cell is the

mesenchymal cell derived from bone

marrow. There has been much talk in

recent years about whether these cells,

given intrathecally or perhaps

intravenously, can crawl into the brain

and repair damage. In 2010 it is looking

less likely, from the experimental

literature, that they do this; but they are

certainly immunosuppressive. Despite the

talk, there had been no published trials of

mesenchymal stem cell treatments in

multiple sclerosis...

The most useful thing to have happened

in 2010 is that two groups have done the

hard graft of giving mesenchymal stem

cells to people with multiple sclerosis in

controlled and regulated environments.

First out of the blocks was Neil Scolding’s

team in Bristol [Clin Pharmacol Ther.

2010 Jun;87(6):679-85] who showed that

intravenous injection of autologous bone

marrow cells was safe in 6 people with

progressive multiple sclerosis. Paolo

points us to another trial, in which

intrathecal therapy was tested. Finally,

there is much chatter about the potential

of neural precursor cells as a therapy of

multiple sclerosis; but, to my knowledge,

no clinical data yet.

Paolo Muraro writes: Stem cell therapies

attract great interest from the general public

and of course from patient communities.

Curiously, it often happens that advances in

basic aspects of stem cell biology are

reverberated by the mass media in a way that

misrepresent the results as immediately

applicable in the clinic.The consequences of

this perverted process are well known to

clinicians as well as to patient associations:

stem-cell related news are always followed by

a wave of patient enquiries about the

availability of clinical trials,only

20 SUPPLEMENT TO ACNR • VOLUME 10 NUMBER 6 JANUARY/FEBRUARY 2011

“Your conduct has unquestionably done lasting harm, if not physically, thenmentally and financially, to these patients and also to their families and supporters.

It is, therefore, undeniable that you have abused the position of trust afforded toyou. You continue to advocate untested and unproved treatments, using your statusas a registered doctor to reinforce your personal beliefs,” Professor Gomes da Costa

told Dr Toussel at his GMC hearing.BMJ 2010; 341:c5410 doi: 10.1136/bmj.c5410 (Published 30 September 2010)

Journals

outnumbered by questions on CCSVI

(though perhaps not for too much longer).

Quite a frustrating exercise for everyone

involved.So what about published clinical

trials of stem cells during 2010? I wish I could

tell you that the second decade of the

millennium started with milestone

publications;well, I’m afraid, I can’t.There

were no breakthroughs in 2010; however,

thankfully, it was a year of small, stepwise

progress.Why so? As things stand, trials of

stem cell therapies are being developed at

few academic departments, struggling to find

support and to navigate the complexities of

regulatory and ethical approval in an

evolving scenario of MS drugs and

biologicals.The products of this research are,

at best, small scale uncontrolled clinical

studies,which do not change current clinical

practice.Yet in my opinion these efforts

command respect and attention,albeit a

critical one.Thus, I have selected two articles

illustrating this principle.

Yamout B, Hourani R, Salti H, Barada W, El-Hajj T,

Al-Kutoubi A, Herlopian A, Baz EK, Mahfouz R,

Khalil-Hamdan R, Kreidieh NM, El-Sabban M,

Bazarbachi A.

Bone marrow mesenchymal stem cell

transplantation in patients with multiple sclerosis:

a pilot study.

J Neuroimmunol. 2010 Oct 8;227(1-2):185-9.

Several preclinical studies have described

highly desirable immunological and

neurotrophic effects of mesenchymal stem

cells (MSCs) on cultured cells in vitro and

in animal models of CNS autoimmunity

(experimental autoimmune

encephalomyelitis, EAE), yet evidence

describing their safety, tolerability and

efficacy as therapy for MS is lacking. In this

article,Yamout and colleagues report a

small open-label,uncontrolled clinical trial

of intrathecal injection of bone marrow-

derived mesenchymal stem cells in patients

with MS,which was primarily aimed at

establishing safety and tolerability.Ten

patients were treated in the study.One

patient developed a transient

encephalopathy with seizures,which

however resolved without sequelae.The

authors concluded that intrathecal

administration of autologous MSC was

feasible and relatively safe.Although the

lack of blinding and of a control arm

preclude drawing any conclusions about

efficacy, this is one of the first few original

reports of a clinical protocol utilising MSCs

for treatment of MS,and utilising

quantitative clinical and MRI outcome

measures.The authors must be applauded

for their courage and effort to develop a

novel therapy.Much stronger evidence (i.e.

larger controlled studies with longer follow-

up) remains needed to establish a treatment

effect of MSCs and to support an intrathecal

rather than peripheral (intravenous)

delivery of MSCs in MS.

Krasulová E, Trneny M, Kozák T, Vacková B,

Pohlreich D, Kemlink D, Kobylka P, Kovárová I,

Lhotáková P, Havrdová E.

High-dose immunoablation with autologous

haematopoietic stem cell transplantation in

aggressive multiple sclerosis: a single centre 10-

year experience.

Mult Scler. 2010 Jun;16(6):685-93.

This article describes long-term outcomes in

26 patients with severe, treatment refractory

MS who received autologous hematopoietic

transplantation in a clinical protocol jointly

carried out by the Neurology (Dr Eva

Havdrova’ and colleagues) and the

Haematology (Dr Thomas Kozak)

departments at the University Hospital in

Prague,Czech Republic.Although some

centres such as Thessaloniki have slightly

larger number of cases and much longer

follow-up (Professor Athanasios Fassas,

personal communication),as of the end of

2010 this article reports the longest post-

therapy follow-up evaluation (median of 5.5

years) carried out at a single-centre.

Reassuringly there was no mortality for a

treatment that has overall high risk (2-3%

according to the recent estimate by

Mancardi and Saccardi [Lancet Neurology

2008]).

The Prague case series by Krasulova et al.

makes a couple of additional interesting

points: first,a clear trend to a more

favourable outcome in relapsing-remitting

than in secondary progressive patients,

consistent with previous observations both in

alemtuzumab (Coles et al J Neurol 2006)

and in HSCT utilising a reduced intensity

conditioning (Burt et al Lancet Neurology

2009).Secondly,an association of better

responses was found with short disease

duration (<5years from diagnosis) and

younger age (<35), the latter being consistent

with a previous analysis of a European bone

marrow transplantation registry data.

Unfortunately the study falls short of

providing evidence-based information on

the treatment’s efficacy because of the lack

of a control group (undoubtedly a hard nut

to crack in trials of HSCT) and in view of

the much shorter duration of follow-up in

RR than in SPMS patients (again justifiable

from the historical perspective of the

evolving understanding of MS

pathophysiology and the development of

most immunotherapies).

Nonetheless, the Prague experience gives

us a first look into the long-term outcome in

patients who received autologous HSCT as

treatment aggressive, conventional

immunotherapy-refractory MS.As such it is a

valuable experience that can help design a

(much needed) controlled phase 3 trial of

HSCT in MS,and I congratulate the authors

for their tenacity.

Repair

Retinoids, rodents andremyelination

Ed: Robin Franklin, a colleague of mine in

Cambridge, gets this year’s ACNR Award

For The Most Impressive Performance By

An MS Scientist On The Today Radio 4

Programme. The occasion was the

SUPPLEMENT TO ACNR • VOLUME 10 NUMBER 6 JANUARY/FEBRUARY 2011 21

Tony Kennan, Chairman of the MS Society, was awarded a CBE in the Queen's New YearsHonours list for his services to healthcare. Rosemary Tocock, chairman of the MS Society’sReading and Wokingham branch and the Thames Valley Region, was made an MBE. She

said she was ‘gobsmacked’ when she received the letter.

Journals

publication of this Nature Neuroscience

article on a way to promote remyelination.

The science is formidable. And this is

important work: as we increasingly gain

control over the inflammatory phase of

multiple sclerosis, we will need to patch up

our patients with remyelinating therapies.

The media reaction was hopelessly ill-

informed. One of my patients showed me a

newspaper article which forgot to mention

that the “patients” in the study were furry

rodents! Suzanne Mosely, who is doing a

PhD with Robin, tells the story.

The recent development of more effective

immunomodulatory treatments in MS has

been welcomed with enthusiasm.However

none have,as of yet,been proven to enable

repair of demyelinated lesions.The Franklin

group has focused on the possibility of

therapeutically encouraging naturally-

occurring mechanisms of remyelination.By

studying changes in gene expression after

experimental demyelination in rodents, they

have identified a key signalling pathway

upregulated during remyelination: the

retinoid X receptor (RXR) pathway. In

particular,RXRγ was found to beupregulated in rodent oligodendrocyte

lineage cells in response to demyelination as

well as in active MS lesions in post-mortem

tissue.The functional role of this signalling

pathway was investigated by blocking RXR

expression using small interfering RNA

(siRNA),RXR antagonists and rxrg-/- mice.

Loss of this pathway caused impaired

oligodendrocyte precursor cell (OPC)

differentiation both in vitro and in vivo.

In order to see if this pathway could also

be used to enhance OPC differentiation and

hence remyelination, the RXR agonist 9-cis-

retinoic acid (9cRA) was added to OPC

cultures.This led to an increase in OPC

differentiation and, in cerebellar slices,was

found to boost remyelination. In vivo the

administration of 9cRA also yielded

promising results with an increase in

numbers of oligodendrocytes and

remyelinated axons.

Therapeutically enhancing the RXR

pathway could therefore offer the attractive

possibility of promoting remyelination in MS.

Huang JK, Jarjour AA, Oumesmar BN, Kerninon

C, Williams A, Krezel W, Kagechika H, Bauer J,

Zhao C, Evercooren AB, Chambon P, Ffrench-

Constant C, Franklin RJ.

Retinoid X receptor gamma signaling accelerates

CNS remyelination.

Nat Neurosci. 2011 Jan;14(1):45-53.

BDNF, in and outsidethe CNS, and repair?

Ed: Since the early 1990s, Michal Schwartzat the Weizman Institute has beenpromoting the idea that the peripheralimmune system can help protect thecentral nervous system from secondarydamage induced by trauma. She hasshown in animals that, in the recoveryfrom spinal cord injury for instance, theimmune system will generate anti-CNS Tcells which deliver goodies likeneurotrophins to the damaged area andpromote repair. Hence the name“neuroprotective autoimmunity”. Otherexperimental studies however suggest theimmune response to CNS trauma isharmful. In humans, there is very littleevidence either way. But the concept hasbeen picked up by those studying CNSinflammation. Could part of the CNS-specific immune response beneuroprotective? In 2010, two papers werepublished back to back on this topic inBrain. Suzanne Mosely reviews the first, amouth-wateringly elegant study from RalfGold’s team at Bochum, cleverlyemploying multiple knock-outs.

Could neurotrophins be beneficial in

multiple sclerosis? Brain-derived

neurotrophic factor (BDNF) in particular

is known to be produced by cells in the

CNS as well as infiltrating immune cells.

However its effect in vivo is still unclear,

with some studies pointing towards a

detrimental role (Javeri Clin Immunol

2010) and others supporting a more

beneficial effect (Makar J Neurosci 2008).

In order to clarify its role, Linker et al have

looked at an animal model of multiple

sclerosis in two conditional BDNF

knockout mice.They compare the effects

of a widespread loss of BDNF production

by astrocytes and some neurons in the

CNS versus a loss of BDNF limited to

infiltrating immune cells (T cells and

myeloid cells).

Firstly, widespread BDNF loss is shown

to lead to more severe disease and

increased axon damage.This would

suggest that endogenous CNS-derived

BDNF has a protective effect. Secondly,

although BDNF loss restricted to either T

cells or myeloid cells has no effect on

disease severity, an important protective

effect of immune cell-derived BDNF is

revealed by overcoming the redundancy

between both types of immune cell when

BDNF is knocked out of both T cells and

myeloid cells: these mice initially show

milder disease (due to weakened

inflammatory assault) but then the severity

of their disease continues to increase

whilst wild type mice show improved

symptoms.Thirdly, this study also supports

a role for BDNF in the mechanism of

action of the immunomodulatory

treatment glatiramer acetate.The

beneficial effects of this treatment are

suppressed in the immune cell double

knockout mouse despite CNS immune

infiltration being unchanged.To further

prove the neuroprotective ability of BDNF

independently of changes in the immune

response,mice were injected with BDNF-

secreting autoimmune cells after the initial

immune activation.This caused milder

disease and protection of naked axons.

These experiments strongly support the

concept of autoimmune infiltrating cells

exerting neuroprotective effects and show

their functional relevance in an animal

model of MS.

Linker RA, Lee DH, Demir S, Wiese S, Kruse N,

Siglienti I, Gerhardt E, Neumann H, Sendtner M,

Lühder F, Gold R

Functional role of brain-derived neurotrophic

factor in neuroprotective autoimmunity:

therapeutic implications in a model of multiple

sclerosis

Brain. 2010 Aug;133(Pt 8):2248-63.

Brain repair throughneuroprotectiveautoimmunity inhumans?

Ed: I am afraid we could not resist

plugging one our papers. Our group

studies Campath-1H, which we have just

about learnt to call “alemtuzumab” when

the owners, Genzyme, announced it was

to be called “Lemtrada” from now on.

They explain that this name is based on

the Spanish word for entrance, which

makes a lot of sense doesn’t it? Anyhow,

we have been investigating the possibility

that Michal Schwartz’s hypothesis

explains the improvement in disability we

see in our patients after Campath-1H

(oops sorry Lemtrada) treatment. Tom

Button, who is doing a PhD with me,

describes the paper.

22 SUPPLEMENT TO ACNR • VOLUME 10 NUMBER 6 JANUARY/FEBRUARY 2011

Journals

The monoclonal antibody alemtuzumab,

which binding to CD52 induces profound

and long lasting modulation of the

lymphocyte repertoire, is currently in phase

III trials for the treatment of multiple

sclerosis (http://care-ms.com). In 2008,

phase II data showed an impressive

reduction in relapse rate (74% relative to

interferon-β1a). This is in the context of a30% rate of thyroid autoimmunity and a 3%

risk of immune thrombocytopaenic

purpura. Importantly and uniquely,

disability in those treated with

alemtuzumab was reduced at 6 months, an

improvement sustained at 5 years (EDSS 1.9

reduced to 1.51) [presented at ECTRIMS].

These findings led to the hypothesis that

alemtuzumab might possess

neuroprotective properties.

This paper establishes a potential

mechanism by which these effects might be

exerted. The production of neurotrophic

factors by the immune cells of 15 patients

treated with alemtuzumab were studied in

detail. 12 months after treatment with

alemtuzumab,peripheral blood monocytes

in vitro secreted increased BDNF,CNTF,PDGF

and FGF (brain-derived neurotrophic factor,

ciliary neurotrophic factor,platelet derived

growth factor, fibroblast growth factor). This

response was maximal after stimulation with

MBP (myelin basic protein)(selected on the

basis of previous suggestion that

neuroprotective autoimmunity requires

reactivity to central nervous system

antigens),being less marked in unstimulated

or polyclonally stimulated cultures. [2]

To support the relevance of immune cell

derived factors, rat neurons were cultured in

media conditioned by MBP-stimulated

immune cells. Media from immune cells

derived from alemtuzumab treated patients

improved neuronal survival and axon length

at least 3-fold relative to healthy controls and

untreated patients. This effect was reversed

most strongly by blocking antibodies to BDNF.

The work is detailed and the proposition

that immunotherapies might have brain

repair activity is exciting, not least if it

proves to be a mechanism which might be

enhanced.

The limitations are acknowledged within

the paper. The sustained improvement in

disability from 0-6 months correlates with

altered neurotrophin production at 12

months shown to have efficacy in vitro on

murine neurons. It is not shown directly to

result from tissue repair. However, Jones et

al comment on the potential to investigate

this in a treatment model, a mouse

transgenic for human CD52.

Jones JL, Anderson JM, Phuah CL, Fox EJ,

Selmaj K, Margolin D, Lake SL, Palmer J,

Thompson SJ, Wilkins A, Webber DJ, Compston

DA, Coles AJ.

Improvement in disability after alemtuzumab

treatment of multiple sclerosis is associated with

neuroprotective autoimmunity.

Brain. 2010 Aug;133(Pt 8):2232-4.

Last and Least

Dem veins, dem veins,dem dry veins

The editor writes: It is hard for me to

summon up the energy to review this year’s

outpourings in response to Dr Zamboni’s

2009 claims that multiple sclerosis is due to

blockages of the cerebral veins and that it

may be cured by venous stenting.Partly, I am

fatigued by going over it all yet again (too

many clinics recently).Partly, I despair that so

much money is being spent refuting a daft

idea; so far the US and Canadian MS society

has spend $2.4 million funding independent

studies on“CCVSI”(chronic something

venous thingy...Zamboni’s term).Mainly, I am

cross that once again our vulnerable patients

are parting with serious money for unproven

dodgy treatments.For instance,people are

paying £7,990 pounds for the complete

package of scan and stenting at the“Essential

Health Clinic”in Glasgow

(http://www.essentialhealthclinic.com/webs

ite/index.php/clinic/ccsvi/treatment-

packages.html).

Anyhow,as far as I can see, there have

only been a few, small-scale, studies on

CCVSI published in 2010:

(Let me remind you that Zamboni

reported in the JNNP that 33/35 multiple

sclerosis cases, and no controls, had jugular

vein insufficiency).

• Frederik Barkhof in Amsterdam studied

20 patients and 20 controls with MR

venography and found anomalies of the

venous system in 10 patients and 8

controls

• Stephan Schreiber at the Charite in

Berlin found that 10/56 patients and

4/20 controls fulfilled at least one

“CCSVI criteria”.

• Jan Malm in Umea, Sweden, studied

cerebral blood and CSF flow,by MRI, in

21 multiple sclerosis patients and 20

controls.No difference was found.

• Zidavinov and Zamboni reported in

“International Angiology” that 16/16

patients fulfilled the diagnosis of CCSVI,

and none of 8 controls.This was

associated with higher iron

concentrations in the thalamus and

hippocampus, as measured by MRI in

the patients.

• In the same issue of the journal,

representatives of a private healthcare

facility in Poland report that ultrasound

abnormalities of cerebral veins were

found in 91% of 70 multiple sclerosis

patients (no controls).

I know what I think.....

Wattjes MP, van Oosten BW, de Graaf WL,

Seewann A, Bot JC, van den Berg R, Uitdehaag

BM, Polman CH, Barkhof F.

No association of abnormal cranial venous

drainage with multiple sclerosis: a magnetic

resonance venography and flow-quantification

study.

J Neurol Neurosurg Psychiatry. 2010 Oct 27.

Sundström P, Wåhlin A, Ambarki K, Birgander R,

Eklund A, Malm J.

Venous and cerebrospinal fluid flow in multiple

sclerosis: a case-control study.

Ann Neurol. 2010 Aug;68(2):255-9.

Doepp F, Paul F, Valdueza JM, Schmierer K,

Schreiber SJ.

No cerebrocervical venous congestion in patients

with multiple sclerosis.

Ann Neurol. 2010 Aug;68(2):173-83.

Zivadinov R, Schirda C, Dwyer MG, Haacke ME,

Weinstock-Guttman B, Menegatti E, Heininen-

Brown M, Magnano C, Malagoni AM, Wack DS,

Hojnacki D, Kennedy C, Carl E, Bergsland N,

Hussein S, Poloni G, Bartolomei I, Salvi F,

Zamboni P.

Chronic cerebrospinal venous insufficiency and

iron deposition on susceptibility-weighted imaging

in patients with multiple sclerosis: a pilot case-

control study.

Int Angiol. 2010 Apr;29(2):158-75.

Simka M, Kostecki J, Zaniewski M, Majewski E,

Hartel M.

Extracranial Doppler sonographic criteria of

chronic cerebrospinal venous insufficiency in the

patients with multiple sclerosis.

Int Angiol. 2010 Apr;29(2):109-14.

SUPPLEMENT TO ACNR • VOLUME 10 NUMBER 6 JANUARY/FEBRUARY 2011 23

“Zamboni forNobel Prize”

– from a MS blog

Alasdair Coles is co-editor of ACNR. He is a University Lecturer inNeuroimmunology at Cambridge University. He works on experimentalimmunological therapies in MS.

Mike Zandi is co-editor of ACNR. He is an Honorary Specialist Registrar inNeurology at Addenbrooke's Hospital, Cambridge and a Research Fellow atCambridge University. His research interests are in neuroimmunology,biomarkers and therapeutics in particular.

Cover image courtesy of David Menassa andAngela Vincent showing GFAP stain of culturedastrocytes, the cell targeted by anti-aquaporin-4antibodies in patients with neuromyelitis optica.

Paolo Muraro is a Clinical Reader in Neuroimmunology and HonoraryConsultant Neurologist at Imperial College London and Imperial CollegeHealthcare NHS Trust. He has an interest in the mechanisms of action ofimmune modifying treatments with a focus on haematopoietic stem celltransplantation (HSCT) in MS.

Ute C Meier studied at the Universities of Heidelberg and Oxford.Shemoved to London in 2007 to pursue her longstanding interest inneuroimmunology and leads the human immunology programme related totranslational research within the Neuroimmunology Group at the BlizardInstitute.

Sreeram Ramagopalan is a Junior Research Fellow at Somerville College,University of Oxford and a Post-Doctoral Research Fellow at the Barts and theLondon School of Medicine. For his research he studies the genetics andepidemiology of MS and is very interested in how gene-environmentinteractions influence the risk of developing MS.

Patrick Waters is a postdoctoral research scientist in the NeurosciencesGroup at the Nuffield Department of Neurosciences (Neurology), Universityof Oxford. His research interests include neuroimmunology, antigen discoveryand innate immunity.

Maria Ban is a Post-Doctoral Research Associate in the Department ofClinical Neurosciences, Cambridge University. Her research interest is inidentifying the genetic factors involved in susceptibility to MS.

David Hunt is based in Edinburgh and his research is focused onstem/precursor cells derived from non-neural tissues, such as skin and bonemarrow, and their relevance to neurological disease.

.

Joanna Kitley is a Specialist Registrar in Neurology and has recently beenappointed as Neuromyelitis Optica Clinical Fellow in the Department ofClinical Neurology, John Radcliffe Hospital, Oxford.

Martin Lee s a Consultant Neurologist and Hon Senior Lecturer at theNorfolk & Norwich University Hospital. His interests include imaging andclinical trials in MS.

Maria Isabel Leite is an Honorary Post-Doctoral Clinician Scientist inNeurology at John Radcliffe Hospital, Oxford. She trained in neurology inPorto, Portugal, where she worked as Consultant from 1992 to 2001, beforemoving to Oxford. Her clinical and research interests are in neuroimmunology,particularly in antibody mediated diseases. She works in the Oxford NMOteam as part of the activities of the National Commissioning GroupDiagnostic and Advisory Service for Neuromyelitis Optica.

Suzanne Mosely is a second year PhD student working with Dr AlasdairColes at the University of Cambridge. Her research project focuses on the roleof neurotrophins in beneficial effects of alemtuzumab treatment for MS.

Neil Robertson is Professor of Clinical Neurology at Cardiff University andthe University Hospital of Wales. He heads a clinical service for MS in southWales and has research interests in neuroepidemiology and biomarkers ofdisease.

Orla Tuohy is a neurology trainee, doing research towards a PhD in theDepartment of Clinical Neurosciences, Cambridge.

Ruth Dobson is a Clinical Research Fellow in the Neuroimmunology groupat the Blizard Institute of Cell and Molecular Science and an HonorarySpecialist Registrar at the Royal London Hospital. She is currently funded byan ABN/MS Society clinical fellowship. Her research focuses on factorsassociated with an increased risk of MS, and how they eventually lead toclinical disease.

Gavin Giovannoni was appointed to the Chair of Neurology, Blizard Institute ofCell and Molecular Science, Barts and The London School of Medicine and Dentistryand the Department of Neurology, Barts and The London NHS Trust in November2006. His clinical interests are MS and other inflammatory disorders of the centralnervous system. He is particularly interested in clinical issues related to optimisingMS disease modifying therapies. Other interests are immune-mediated movementdisorders.

Bruno Gran is Associate Professor of Neurology at Nottingham University.After postdoctoral training at the NIH and the University of Pennsylvania, hewas Assistant Professor of Neurology at Thomas Jefferson University inPhiladelphia. His current research focuses on the role of Toll-like receptors inMS and experimental autoimmune encephalomyelitis.

Tom Button is a neurology registrar who trained as an SHO in Yorkshire.He is currently undertaking research work in collaboration betweenAddenbrooke's Hospital, Cambridge, and the Institute of Neurology, London;studying patients treated with Alemtuzumab from the imaging of brain repairto its immunology.

Denise Fitzgerald is a Lecturer in Immunology at Queens University Belfastin Northern Ireland where she has recently started an MS-focused researchgroup. Her group works on CNS remyelination and the role of T cells in thepathogenesis of MS.

Allison Curry is a Senior Research Associate in the research team ofDr Alasdair Coles, the Department of Clinical Neurosciences at the Universityof Cambridge. Allison is a cellular immunologist and her research is focusedon the role of Alemtuzumab in MS.

Contributors