REVIEW ARTICLE

Immunotherapy of Multiple Sclerosis

The State of the Art

Dimitrios Karussis

� Springer International Publishing Switzerland 2013

Abstract It is widely accepted that the main common

pathogenetic pathway in multiple sclerosis (MS) involves

an immune-mediated cascade initiated in the peripheral

immune system and targeting CNS myelin. Logically,

therefore, the therapeutic approaches to the disease include

modalities aiming at downregulation of the various

immune elements that are involved in this immunologic

cascade. Since the introduction of interferons in 1993,

which were the first registered treatments for MS, huge

steps have been made in the field of MS immunotherapy.

More efficious and specific immunoactive drugs have been

introduced and it appears that the increased specificity for

MS of these new treatments is paralleled by greater effi-

cacy. Unfortunately, this seemingly increased efficacy has

been accompanied by more safety issues. The immuno-

therapeutic modalities can be divided into two main

groups: those affecting the acute stages (relapses) of the

disease and the long-term treatments that are aimed at

preventing the appearance of relapses and the progression

in disability. Immunomodulating treatments may also be

classified according to the level of the ‘immune axis’ where

they exert their main effect. Since, in MS, a neurodegen-

erative process runs in parallel and as a consequence of

inflammation, early immune intervention is warranted to

prevent progression of relapses of MS and the accumula-

tion of disability. The use of neuroimaging (MRI) tech-

niques that allow the detection of silent inflammatory

activity of MS and neurodegeneration has provided an

important tool for the substantiation of the clinical efficacy

of treatments and the early diagnosis of MS. This review

summarizes in detail the existing information on all the

available immunotherapies for MS, old and new, classifies

them according to their immunologic mechanisms of action

and proposes a structured algorithm/therapeutic scheme for

the management of the disease.

1 Introduction

Multiple sclerosis (MS) is a chronic disease of the central

nervous system (CNS) that is characterized by loss of motor

and sensory function, caused by immune-mediated inflam-

mation, demyelination, and subsequent neuronal/axonal dam-

age [1–3]. Clinically, most MS patients experience recurrent

episodes (relapses) of neurological impairment (relapsing-

remitting MS: RRMS). Early neurological dysfunction may

resolve spontaneously, partially or completely, but usually the

course of the disease becomes chronic and progressive (pri-

mary or secondary progressive MS: PPMS, SPMS), leading to

accumulating motor disability and cognitive deficits.

Histologically, perivenular inflammatory lesions involving

infiltrating mononuclear cells are evident in the earlier phases

of the disease, resulting in demyelinating plaques which are

the hallmark pathological feature of MS [2]. Inflammation

leads to damage or loss of oligodendrocytes and demyelina-

tion leads to disruption of the conduction of neuronal signals

in the affected regions. In the initial stages of MS, compen-

satory pathways (such as the upregulation of ion-channels in

the affected areas) may partially restore conduction and

reverse the neurological dysfunction. As the disease pro-

gresses, significant axonal loss and eventually neuronal

damage occurs [4] and the lost function becomes permanent

and non-reversible.

D. Karussis (&)

Department of Neurology, MS Center and the Agnes-Ginges

Center for Neurogenetics, Hadassah-Hebrew University

Hospital, Laboratory of Neuroimmunology, Hadassah,

Ein-Kerem, Jerusalem, IL 91120, Israel

e-mail: karus@cc.huji.ac.il

BioDrugs

DOI 10.1007/s40259-013-0011-z

It is widely accepted that the inflammatory process in

MS is caused or propagated by an autoimmune cascade,

involving mainly T cells that target myelin self-antigens [5,

6], possibly through mechanisms known as molecular

mimicry (cross-reactive antigens expressed by viruses or

other microorganisms and myelin components) [7]. An

alternative hypothesis is that myelin-specific T cells that

are present ‘naturally’ may expand to critical pathogenic

quantities [8] due to malfunctioning immunoregulatory

mechanisms (such as those involving the Th2, Th3 and

CD8? T cells and the regulatory T cells: Tr1 and Treg).

Although the autoimmune hypothesis is attractive and

supported by concrete data (including the efficacy of

immunomodulatory treatments in MS), the initial insult

which initiates the whole immune-mediated cascade is still

obscure. Environmental, genetic and infectious factors

seem to play an important role in MS pathogenesis, but it

seems that the role of any putative infectious agent is to

trigger/drive the autoimmune process [8], rather than to

serve as the primary target of infiltrating cells. In any case,

T cells of the Th1 and Th17 phenotype specific for myelin

antigenic epitopes seem to represent the common final

pathogenetic effector pathway, regardless of the initial

insult of the disease [9–11]. However, MS is not a

homogenous disease and it is now increasingly recognized

that it has several distinct immunopathological profiles,

including prominent humoral immune mechanisms in some

MS patients [12].

The initial stages of the autoimmune cascade in MS are

probably initiated in the peripheral immune system. Fol-

lowing activation by the macrophages and dendritic cells

(antigen-presenting cells: APC), Th1 and Th17 lympho-

cytes, which express the antigens specific for myelin,

T-cell receptors (TCR), proliferate and begin to express on

their membranes, adhesion molecules, and chemokine

receptors that enhance their ability to extravasate to the site

of inflammation in the CNS [13–18] (and to produce

interleukin [IL]-2, IL-17, tumor necrosis factor-alpha

[TNFa] and interferon-gamma [IFNc] [pro-inflammatory

cytokines]). This migration possibly follows ‘damage sig-

nals’ from the CNS tissue or is due to an opening/

destruction of the blood-brain barrier. Adhesion molecules

such as VLA4 are crucially important for this process and

their blockage may prevent the extravasation of the lym-

phocytes through the blood vessels’ endothelium [19, 20].

Interestingly, the best existing evidence that MS is indeed

an autoimmune disease and that the whole process is ini-

tiated in the periphery comes form the reported evidence of

high efficacy in MS (and the ‘rebound’ of MS activity

following their discontinuation) of medications that spe-

cifically block the migration of the lymphocytes into the

CNS (such as natalizumab which targets the CD40L mol-

ecule) [20].

B cells have traditionally been considered to play a

secondary T-cell-dependent role, producing antibodies that

may promote tissue destruction by recruiting macrophages

and through activation of the complement pathway [21].

Additionally, activated B cells can act as antigen-specific

APCs for T cells and produce costimulatory molecules that

influence the differentiation of T cells into Th1 or Th2 cells

[22]. Patients with MS have increased B-cell numbers in

the CNS, mainly memory cells and short-lived plasma-

blasts [23]. Plasmablasts persist in the cerebrospinal fluid

(CSF) throughout the course of MS, and the numbers of

these cells correlate with intrathecal immunoglobulin G

(IgG) synthesis (oligoclonal antibodies, one of the hall-

marks of MS diagnosis) and with active inflammatory

disease [23, 24]. Moreover, B cells, plasma cells, autoan-

tibodies, and complement have been detected in MS lesions

[25, 26], indicating their implication in demyelination.

Additional indications for antibody-mediated mechanisms

in MS come from the presence of ectopic lymphoid folli-

cles in the CNS of patients with MS [23, 27–29], especially

those with progressive disease. It appears, therefore, that as

the disease evolves into the progressive stage, the inflam-

mation becomes compartmentalized and predominantly

mediated by B cells. Further evidence for the role of B cells

in the immunopathology of MS is the efficacy of B-cell-

and antibody-directed therapies, such as plasmapheresis

and rituximab, in treating selected subgroups of MS

patients [30, 31]. The involvement of antibody-mediated

pathogenetic mechanisms is particularly pronounced in

variants of CNS demyelinating disease, such as neur-

omyelitic types of MS (neuromyelitis optica [NMO]-

spectrum of disorders) associated with anti-aquaporin

antibodies and long magnetic resonance imaging (MRI)

lesions in the cervical spine.

2 Multiple Sclerosis (MS) Immunotherapy

Logically, based on this widely accepted model for MS

immunopathogenesis described in Sect. 1 (Fig. 1), the

therapeutic approaches to the disease include modalities

aimed at downregulating the various immune elements that

are involved in the immunological cascade. Since the

introduction of interferons in 1993, which were the first

registered immunotherapies for MS, huge steps have been

made in the field of MS immunotherapy. The novel, at that

time, concept of ‘immunomodulation’ (referring to a spe-

cific dowregulation of the pathogenic immune cells

through induction of a shift of the immune response or

enhancement of the intrinsic regulatory immune networks),

as opposed to the generalized immunosuppression

approach, was initially introduced from the studies with

interferons and linomide, in the early 1990s [32–34].

D. Karussis

During the last two decades, since the advent of IFN, more

target-focused immunoactive drugs have been introduced

and it appears that these new treatments are more efficious

(Fig. 2). However, this seemingly increased efficacy of the

new immunomodulatory drugs has to be carefully inter-

preted as the clinical trials during the last decade included

MS patients with a lower relapse rate, and this fact com-

plicates the comparisons between old and new generation

drugs. In addition, more safety issues have arisen with

these new immunomodulators (Fig. 2).

Since, as mentioned in Sect. 1, a neurodegenerative

process runs in parallel and as a consequence of inflam-

mation, early immune intervention is warranted to prevent

the relapses of MS and the accumulation of disability. The

Th1

CD4+ CD25+ FOXP3

ThCD4

CD8B

cells

Th17

Th17

Th1B cells

CD8

IL4IL5IL10IL13

BAFFIL4, IL10

VCAMICAM

MMPs

IFNγTNFa

VLA4

LFA1

B7

CD40L

MHC+Myelin ags

CD40

CD80

TCR

IL23

IL6TGFB

(-)

IL17

BBB

NOTNFa

NOTNFa

Myelin damageMyelin

damage

Myelin damage

IL10

CD8 Cytotoxicity

HMyelin

Antibodiescomplement

Tregs

F

APC

Th2/Th3

VCAMICAMVCAMICAMVCAMICAMVCAMICAMVCAMICAMVCAMICAM

Microglial cells

Secondary activation

G

F

CB

DE

A

(-)

CN

SP

IS

Fig. 1 A model of multiple sclerosis (MS) immunopathogenesis; target

sites for immunotherapy. Levels of the immunopathogenetic mecha-

nisms of MS, which may represent targets for immune intervention:

Level A antigen presentation to lymphocytes and early lymphocyte

activation; Level B activation and proliferation/expansion of the

autoimmune, myelin reactive Th1 and Th17 lymphocytes; Level Cimmune regulation by regulatory cells (Tregs) or immunological (Th1 to

Th2) shift; Level D expansion of B cells and anti-myelin antibodies

production (humoral immunity); Level G production of proinflammatory

cytokines and mediators of tissue damage (such as TNFs and NO); LevelF migration or homing of the lymphocytes and entrance through the

BBB; Level H oligodendrocyte damage. APC antigen presenting cell,

BAFF B-cell activating factor, BBB blood brain barrier, CNS central

nervous system, FOXP3 forkhead box P3, ICAM intercellular adhesion

molecule, MMPs metaloproteinases, NO nitric oxide, Oligo oligoden-

drocytes, PIS peripheral immune system, TCR T-cell receptor, TNFatumor necrosis factor alpha, VLA4 very late antigen-4 (adhesion

molecule), VCAM vascular cell adhesion protein

β

Increasing intensity and target focusing

Eff

icac

y Safety

Fig. 2 Increasing intensity and target-focusing of new immunotherapies

is associated with more efficacy but also with more safety issues. GAglatiramer acetate, IFNb interferon beta, mAbs monoclonal antibodies

Immunotherapy of MS

use of neuroimaging (MRI) techniques that allow the

detection of silent inflammatory activity of MS and neu-

rodegeneration has provided an important tool for the

substantiation of the clinical efficacy of treatments and the

early diagnosis of MS. The introduction of new diagnostic

criteria [35] for clinically definite MS (CDMS) does not

necessitate—as in the past years—the occurrence of a

second clinical relapse, but only the dissemination in time

and space, reflected by MRI dynamic changes. This has

paved the path to the concept of early immunotherapy in

selected patients experiencing the first demyelinating epi-

sode, which is defined as clinically isolated syndrome

(CIS), or even (theoretically) in individuals with a sole

neuroradiological evidence of demyelination (radiologi-

cally isolated syndrome: RIS). However, the prognosis of

MS is highly variable and therefore additional clinical,

radiological and immunological surrogate biomarkers

should be used to define those cases with a bad prognosis in

which early immunotherapy at the stage of CIS or RIS may

be justified.

The immunotherapeutic modalities can be divided into

two main groups: those affecting the acute stages (relapses)

of the disease and the long-term treatments that aim at

preventing the appearance of relapses and the progression

in disability. Immunomodulating treatments may also be

classified according to the level of the ‘immune axis’ where

they exert their main effect (Fig. 1; Tables 1, 2).

Based on their molecular construction and their basic

mechanism of action, immunotherapeutic agents may be

also classified as: (i) cytotoxic drugs; (ii) synthetic

Table 1 Description of the mechanisms of action of immunotherapeutic agents in MS and their class evidence of efficacy

Immuno-

therapeutic agents

Basic mechanism of action Class evidence of

efficacy in MS

Corticosteroids • Induction of lipocortin-1 (annexin-1) synthesis, with resulting diminished eicosanoid

production

Class I–II (in attacks

of MS)

• Suppression of cyclooxygenase (COX-1 and COX-2) expression

• Inhibition of genes affecting production of cytokines IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8,

IFNc, TNFa, resulting in T-cell inhibition

Class II–III (as long-

term treatment)

• Downregulation of antibody production

• Downregulation of adhesion molecules expression

• Reduction of BBB permeability

IFNb • Reduction of T-cell activation and proliferation Class I in RRMS

• Downregulation of antigen presentation (reduction of MHC/HLA expression and blocking of

co-stimulatory signals from APCs

• Upregulation of CCR7, TGFb and IL-10 and enhancement of regulatory cell function Class I–II in SPMS with

relapses

• Induction of cytokine shift in favor of anti-inflammatory profile (Th1 to Th2 shift) Class II–III in other

SPMS

• Prevention of T-cell adhesion and extravasation across BBB (increased sVCAM and reduced

metalloproteinase-MMP9 production)

• Induction of Tregs None in PPMS

• Activation of B cells

• Neuroprotection

GA • Blockage of lymphocyte sensitization to MBP Class I in RRMS

• Downregulation of antigen presentation (competitive inhibition for binding with MHC/HLA)

• Induction of a Th2 shift None in SPMS or PPMS

• Induction of bystander suppression

• Induction of regulatory and CD8 suppressor GA-reactive cells

• Induction of a shift towards an M2 type of APCs

• Neuroprotective effects

Fingolimod • Action as a super agonist of the S1P1 receptor on lymphocytes, inducing its uncoupling/

internalization and intracellular lysosomal degradation, depriving CD4? and CD8? positive T

cells and B cells of the obligatory signal to egress from lymphoid organs and recirculate to

peripheral inflammatory tissues

Class I in RRMS

PLEX • Mechanical clearance of circulating antibodies Class II-III in relapses of

MS and NMO

D. Karussis

Table 1 continued

Immuno-

therapeutic agents

Basic mechanism of action Class evidence of

efficacy in MS

IVIG • Formation of immune complexes and blockage of circulating antibodies Class I in RRMS

• Interaction with Fc receptors on dendritic cells, which then mediate anti-inflammatory effects

• Activation of complement No evidence in

progressive MS

• Blockage of macrophages

• Direct suppression of T and B cells

Natalizumab • A humanized monoclonal antibody against cellular adhesion molecule a4-integrin, which is

crucial for lymphocyte migration to target organs

Class I in RRMS

• Prevents all activated lymphocytes from entering CNS through the BBB

Rituximab • Genetically engineered chimeric murine/human IgG1 monoclonal antibody that targets the

CD20 antigen, expressed only on pre-B and mature B cells

Class II in RRMS

• Rituximab lyses B cells via complement and antibody-dependent cellular cytotoxicity

Alemtuzumab • A recombinant humanized monoclonal antibody directed against cell surface glycoprotein

CD52

Class I in RRMS

• Depletion (antibody-dependent lysis) of all B and T cells

• Reduction of the number of monocytes, macrophages, NK cells, and part of granulocytes

Daclizumab • A humanized mAb that blocks IL-2 receptor alpha subunit (CD25) expressed on activated T

cells

Class II in RRMS

• Inhibition of T-cell expansion

• Expansion of CD56 NK cells which, in turn, inhibit T-cell survival

Cyclo-

phosphamide

• A nitrogen mustard alkylating agent from the oxazophorine group with broad cytotoxic and

immunosuppressive effects; its main metabolite phosphoramide mustard is only formed in

cells with low ALDH levels

Class II–III in

progressive cases

• Formation of DNA crosslinks at guanine positions, leading to cell death

• Suppression mostly of B cells

• Suppression of T cells and IFNc and IL-12 cytokine production

• Increase in IL-4 and IL-10 production and induction of a Th2 shift

Azathioprine • Inhibition of the first step in de novo purine biosynthesis, with resulting prevention of mitosis

and proliferation of fast dividing cells (especially lymphocytes), leading to decreased T-cell

number and reduced antibody synthesis

Class II in RRMS and

SPMS

Mitoxanthrone • A cytotoxic agent from the anthracenedione family; acts by intercalating with DNA and

inhibiting topoisomerase II

Class I in RRMS

• Reduction of the number of B cells

• Inhibition of the T helper cells and the Th1-related cytokine production

• Augmentation of T-cell suppressor activity

Methotrexate • A folate antagonist; diminishes proliferation of immune cells by inhibiting de novo purine and

pyrimidine synthesis

Class II–III in RRMS

• Decrease of neutrophil leukotriene synthesis the adenosine-mediated inflammation

• Inhibits AICAR transformylase, resulting in intracellular accumulation of AICAR, which

promotes release of the anti-inflammatory autocoid adenosine

• Suppression of antigen-dependent T-cell proliferation

Mycophenolate • Inhibition of purine synthesis and non-competitive inhibition of type II isomer of inosine

monophosphate dehydrogenase

Class III

• Depletion of guanosine and deoxyguanosine nucleotides in T and B lymphocytes, therefore

inhibiting their proliferation and immunoglobin production

• Suppression of dendritic cells maturation decreasing their capacity of antigen presentation to T

lymphocytes

Cyclosporine A • A calcineurin inhibitor that acts on IL-2 by inhibiting its production, leading to decreased

T-lymphocyte proliferation

Class III

Immunotherapy of MS

immunomodulators; (iii) monoclonal antibodies; (iv)

vaccines (T-cell vaccines, antigen vaccines); (v) oral tolerizing

agents; (vi) modalities that act as indirect immunosuppressants

(plasmapheresis, intravenous immunoglobulins: IVIG); and

(vii) cellular therapies (stem cells, progenitors).

2.1 Treatment of Acute Deterioration/Relapses of MS

2.1.1 Corticosteroids

The most widely used treatment, especially during an acute

MS relapse, is methylprednisolone or other corticosteroid

preparations.

In high, supraphysiologic doses, glucocorticoids

strongly downregulate inflammation. They suppress cellu-

lar immunity by inhibiting genes that affect the production

of the cytokines IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8,

and TNFc and by reducing the proliferative ability of T

cells [36–40]. Glucocorticoids also suppress humoral

immunity, causing B cells to express smaller amounts of

IL-2 and IL-2 receptors, thus diminishing their expansion

and antibody production.

Glucocorticoids downregulate all types of inflammatory

events by inducing the synthesis of lipocortin-1 (annexin-

1), which then binds to cell membranes preventing the

phospholipase A2 from coming into contact with its sub-

strate, arachidonic acid. This leads to diminished eicosa-

noid production. Cyclooxygenase (both COX-1 and COX-

2) expression is also suppressed, potentiating the effect.

Steroids reduce the permeability of the blood-brain barrier

[38, 41, 42] as reflected by the reduction in gadolinium

diethylenetriaminepentaacetic acid (Gd-DTPA) enhance-

ment in the MRI.

Results from the National Cooperative ACTH study

revealed that corticotropin (ACTH) may hasten recovery at

1 month after treatment [36]. Other studies have shown

comparable or superior results with methylprednisolone

[37, 43, 44]. Data from the Optic Neuritis Treatment Trial

showed that intravenous methylprednisolone is superior to

oral prednisone for the treatment of acute optic neuritis [39,

45]. However, the long term effect of both treatment reg-

imens on the visual acuity was marginal and there was no

significant ‘protection’ against later development of defi-

nite MS [39, 45]. A review of the trials with either intra-

venous methylprednisolone or ACTH showed the clear

benefit of steroids in MS exacerbations [46].

In patients with chronic progressive MS, repeated

intravenous infusions of methylprednisolone showed a

milder effect and failed to inhibit disease progression [47].

However, two more recent studies have shown a short-term

Table 1 continued

Immuno-

therapeutic agents

Basic mechanism of action Class evidence of

efficacy in MS

Cladribine • A synthetic purine nucleoside that acts as anti-metabolite, interfering with DNA synthesis and

repair and with immunosuppressive effects (mainly causing lymphocyte depletion)

Class I in RRMS

• Suppression mainly of the CD4? and CD8? T cells and B cells Class II in SPMS

• Prevention of lymphocyte migration through BBB

Dimethyl

fumarate

(BG12)

• Induction of anti-inflammatory effects Class I in RRMS

• Suppression of macrophages

• Activation of nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, the innate cellular

phase 2 detoxifying pathway-anti oxidant

• Th1 to Th2 shift

• Neuroprotection

Laquinimod • A synthetic immunomodulator Class I–II in RRMS

• Downregulation of antigen presentation

• Induction of a shift towards Th2

• Neuroprotective effects

Teriflunomide • An active metabolite of leflunomide Class I in RRMS

• Inhibition of dihydroorotate dehydrogenase, a key mitochondrial enzyme involved in new

pyrimidine synthesis for DNA replication

• Reduction of T- and B-cell activation and proliferation

AICAR 5-Aminoimidazole-4-carboxamide 1-b-D-ribofuranoside, ALDH aldehyde dehydrogenase, APCs antigen presenting cells, BBB blood–

brain barrier, GA glatiramer acetate, IFN interferon, IgG immunoglobulin gamma, IL interleukin, mAb monoclonal antibody, IVIG intravenous

immunoglobulins, MBP myelin basic protein, MMP matrix metalloproteinase, MS multiple sclerosis, NK natural killer, NMO neuromyelitis

optica, PLEX plasmapheresis, PPMS primary progressive MS, RRMS relapsing-remitting MS, SPMS secondary progressive MS, sVCAMvascular cell adhesion molecule, TGFb transforming growth factor beta, TNFa tumor necrosis factor alpha, Tregs regulatory T cells

D. Karussis

improvement following high dose intravenous methylpred-

nisolone [48] or a long-term beneficial effect by 4-monthly

intravenous methylprednisolone boosts which slowed the

development of T1 black holes and delayed whole-brain

atrophy and disability progression [49].

Despite these data and the documented transient beneficial

effect during the acute MS relapse, steroids do not seem to

significantly alter the natural course of the disease. Therefore,

their chronic use does not seem justified. In addition, chronic

steroid administration is associated with several adverse

effects, including osteoporosis, aseptic bone necrosis, hyper-

tension, hyperglycemia, cataracts, and psychotic events.

Despite wide agreement on the efficacy of steroidal

treatment in MS relapses, the optimal protocol for

Table 2 Mechanisms of action of immunotherapeutic agents in multiple sclerosis (MS)

Suppression of T

lymphocytes

Suppression

of antigen

presentation

Suppression of

B lymphocytes

and humoral

immunity

Suppression or

shift of

cytokines and

chemokines

Inhibition of

lymphocyte migration/

blockage of blood

brain barrier (BBB)

Induction

of immune

shift (Th1

to Th2)

Enhancement

of regulatory

cells

Levels of action

on the immune

cascade described

in Fig. 1

Corticosteroids ?? ?? ??? ?? ?? ? ? A, B, E, F, G

Interferon b (IFNb) ??? ?? - (probable

enhancement)

?? ? ??? ?? A, B, C, F, G

Glatiramer acetate

(GA)

?? ??? ?/- ? - ??? ? A, C, D, G

Fingolimod ??? (blockage

of their

eggerssion from

the lymph

nodes )

- ?? unknown ??? ? unknown B, D, E

Plasmapheresis - - ??? ?? - - - E

Intravenous

immunoglobulins

(IVIG)

? ? ??? ? - ? ?/- A, E

Natalizumab ?? (blockage of

their homing to

target organs)

- ?? (blockage

of their

homing to

target organs)

- ??? - - D, E, F

Rituximab - - ??? ? ? - - A, E

Alemtuzumab ??? - - ? ? ?/- - B, C, D, E, G

Daclizumab ??? - - ?? ? ? (from T

cells to

NK)

?? B, C, D

Cyclophosphamide ?? ? ??? ??? ? ?? ?? A, B, C, D, E

Azathioprine ?? ? ??? ?? ? ? ? A, B, C, D, E

Mitoxanthrone ??? ? ??? ?? - ? ?/- B, C, D, E

Methotrexate ?? ? ?? ? - - unknown A, B, C, D, E

Mycophenolate

(MMF)

??? ?? ??? ?/- - ?/- - B, C, D, E

Cyclosporine A

(CsA)

??? - ? ??? - - - (possible

suppression)

B, C, G

Cladribine ??? - ?? ? ?? - - B, C, D, E

Dimethyl fumarate

(BG12)

?? ?? ? ?? unknown ?? unknown A, B, G

Laquinimod ?? ??? ? (modulation) ?? ?? ?? ?? A, B, C, D, E

Teriflunomide ??? ??? ?? ?? unknown ?? ? B, C, D, E

Tarcolimus/

Sirolimus

??? - ? ??? ?/- unknown - B, D, E, G

?, mild effect; ??,moderate effect; ???, strong effect; ?/-, questionable effect

Level A Antigen presentation to lymphocytes and early lymphocyte activation: glatiramer acetate (GA), interferon b (IFNb), vaccines, laquinimod, steroids, rituximab. Levels Band D Activation and proliferation/expansion of the autoimmune, myelin reactive lymphocytes: chemotherapeutic/cytotoxic agents (azathiorpine, cyclophosphamide, meth-

otrexate, cyclosporine, mycophenolate, cladribine, mitoxanthrone, tarcolimus, teriflunomide); immunoablation and reseting of the immune system by hematopoietic stem cell

transplantation; lymphocyte depletion with monoclonal antibodies (alemtuzumab, rituximab, daclizumab); specific reduction of autoimmune myelin-responsive lymphocytes

with vaccination techniques (i.e., T-cell vaccination, myelin peptides vaccination). Levels C and D Regulatory cells or immunological shift: IFNb, GA, laquinimod, fingolimod,

alemtuzumab, teriflunomide, cyclophosphamide. Level E B cells and antibodies/humoral immunity: plasmapheresis, IVIG, anti-CD20 monoclonal antibodies, steroids,

cytotoxic drugs (azathiorpine, mycophenolate, cyclopshosphamide), alemtuzumab. Levels B and G Cytokine production: various immunosuppressants, corticosteroids, IFNb,

GA, fingolimod, teriflunomide, cyclosporine-A, laquinimod, cyclophosphamide, azathioprine, alemtuzumab. Level F Migration or homing of the lymphocytes and entrance

through the blood brain barrier: fingolimod, natalizumab, corticosteroids. Level H Oligodendrocyte damage and axonal/neuronal loss: neuroprotective modalities, stem cells,

dimethyl fumarate (BG12) and possibly (but largely unknown) many immunomodulating drugs (such as interferons, GA, laquinimod, fingolimod, natalizumab)

Immunotherapy of MS

administration of the drug is still not settled. Current pro-

tocols (mostly empirical) include 3, 5, or even 10 days of

1–2 g intravenous methylprednisolone with or without oral

tapering with prednisone. Oral administration of high dose

methylprednisolone appears to be equally efficient to

intravenous administration [50–52]. There are indications,

not based on published studies in MS but on experience in

other autoimmune diseases (such as systemic lupus ery-

thematosus: SLE), that abrupt discontinuation of intrave-

nous steroidal treatment in MS relapses may increase the

risk of a recurrence of the symptoms. Therefore, a tapering-

off protocol following high-dose intravenous methylpred-

nisolone appears preferable.

2.1.2 Plasmapheresis

Plasmapheresis, also known as therapeutic plasma

exchange (PLEX), is a procedure that separates the blood

components, exchanging the plasma (typically with donor

plasma or albumin), and returning the other components,

primarily red blood cells, to the patient. PLEX has been

proven to be effective in various autoimmune diseases,

including neurological ones, such as Gulliain–Barre syn-

drome, idiopathic thrombocytopenic purpura and myas-

thenia gravis [53–55].

Since the late 1980s, PLEX has been tried in several

studies on MS patients, but with inconsistent effects. This

may be related to (a) the small size of the studies, (b) the

lack of homogeneity in the treatment porotocols, (c) the use

of PLEX as an adjuvant therapy to other immunomodula-

tory modalities, and (d) the patient populations and type/

course of MS, which greatly varied among these studies

[56–59].

In a more recent and randomized trial, which repre-

sented the revival of PLEX as a treatment modality for MS,

PLEX (7 courses within 14 days) was for the first time

checked as a single modality in patients with various types

of acute CNS demyelinating diseases (including acute

disseminated encephalomyelitis and neuromyelitic types of

MS [NMO spectrum of disorders], either chronic or

relapsing) [31]. Clinical improvement was noted in 8 of the

19 (42.1 %) PLEX-treated patients compared with 1 of 17

(5.9 %) in the sham group. When the patients were swit-

ched from PLEX to sham and from sham to PLEX, the

clinical beneficial effects of PLEX noted in the first part of

the trial were strengthened in favor of PLEX.

A follow-up study by Keegan et al. [60] suggested that

patients with myelitis and Pattern II immunopathogenesis

(according to Lucchinetti et al. [12], a predominantly

antibody-mediated disease) had the best response to PLEX.

A recent retrospective analysis of 153 patients treated with

PLEX for a steroid-refractory CNS demyelinating episode,

showed that 59 % exhibited moderate to marked functional

neurological improvement within 6 months following

treatment. Plasma exchange was less effective for patients

with MS who subsequently developed a progressive dis-

ease course. Radiographic features associated with a ben-

eficial PLEX response were the presence of ring-enhancing

lesions and/or mass effect [61].

Logically, since NMO corresponds to the type II histo-

pathological phenotype of MS lesions, NMO patients are

expected to have an optimal response to PLEX. Indeed two

small open studies and a restrospective one in NMO

patients during an acute attack of the disease showed an

early and significant improvement following PLEX in most

of the treated patients [62–64].

In purely progressive MS, a study of combination

treatment with azathioprine and PLEX in eight patients

with SPMS led to the conclusion that PLEX did not

improve clinical outcomes [65], nor was there any signif-

icant difference in the number of MRI-enhancing lesions.

However, the total MS lesion load in MRI was significantly

lower and central motor conduction times decreased sig-

nificantly during PLEX treatment.

Recently, an American Academy of Neurology task

committee, upon evaluating the neurological indications of

PLEX, gave a level B recommendation for its use as sec-

ond-line treatment of steroid-resistant exacerbations in

relapsing forms of MS and of NMO [66].

2.2 Preventive Long-Term Immunotherapy of MS

2.2.1 The First Generation: The ‘Old Players’

2.2.1.1 Corticosteroids and Plasmapheresis Corticoste-

roids and PLEX are usually applied for the management of

acute exacerbations or deterioration phases of MS. How-

ever, as described above, these modalities may also be used

as chronic treatments in selected cases, usually in combi-

nation with other immunotherapeutic drugs.

2.2.1.2 Interferon Beta (IFN-b)

2.2.1.2.1 IFN-b in Relapsing MS. The type-I interferons

(IFNa and IFNb) were first used in the 1970s on the

grounds of their antiviral activity, which could possibly

help in the elimination of the putative viral agents involved

in MS pathogenesis. However, an initial trial with IFNc(type II) showed that the treatment actually promoted

relapses of MS [67]. IFNb, on the other hand, (initially

administered intrathecally) induced beneficial effects in

patients with MS [68]. Then IFNa was given subcutane-

ously in 12 MS patients and induced some positive clinical

effects [69].

In parallel, IFNb was shown to suppress experimental

autoimmune encephalomyelitis (EAE), the animal model

D. Karussis

of MS [70, 71], to inhibit T-cell activation [72], enhance

suppressor cell activity [73], reduce IFNc production and

MHC-II expression [74], increase IL-10 production [75],

downregulate antigen presentation, induce a Th1 to Th2 shift,

and reduce the permeability of the blood-brain barrier [76].

In the two large, pivotal, double-blind studies performed

in the US (which paved the way to a new era in MS

management and introduced a new generation of immu-

nomodulatory medications for MS) in patients with RRMS,

IFNb was found to significantly inhibit the activity of the

disease.

In the first trial, recombinant IFNb-1b (Betaferon�,

Schering/Bayer), given subcutaneously every other day,

was reported to reduce the relapse rate by 31 % (0.84/year

in the treated group vs 1.27/year in the placebo group),

especially at the high dose of 8 million IU, but without

significantly affecting the disability status [77]. IFNb-

treated patients had a significantly lower MRI T2 burden of

disease and a 75–80 % reduction in active scans [78]. This

effect was long-lasting even after 3–5 years of treatment

[79]. The treatment was generally well tolerated with the

exception of ‘flu-like’ symptoms (fever, chills, myalgias,

and fatigue) that occurred in the majority (76 %) of

patients, but tended to decrease after the first months of

treatment. Injection-site irritation was also very common.

About a third of the patients developed neutralizing anti-

bodies against IFNb, and this may be a limiting factor in

long-term treatment.

In a second study, recombinant glycosylated IFNb-1a

(Avonex�, Biogen), at a dose of 6 MIU intramuscularly

once per week, induced a reduction in relapse rate from

0.90/year (placebo) to 0.61/year. The treatment also

resulted in a decrease in the number and volume of Gd-

DTPA-enhancing lesions in the MRI and, most impor-

tantly, delayed the sustained progression of disability and

reduced the 1- and 2-year progression rates by about 40 %

[80].

A third type of recombinant IFNb (Rebif�, Merk-Ser-

ono) almost identical to Avonex�, was introduced later.

The first study with IFNb-1a given once weekly [81] pro-

vided evidence of IFNb-1a dose response in relapsing-

remitting MS, as reflected by the MRI outcomes. The

subsequent phase III trial, PRISMS [see Table 3 for full

trial names], with two doses of Rebif� (6 or 12 million IU

subcutaneously every other day), showed significant ben-

eficial effects in all the clinical and MRI parameters tested

in relapsing-remitting MS patients [82]. Clinical data on

533 patients revealed a significantly lower relapse rate in

the Rebif�-treated patients (risk reduction 27–33 % for

Rebif� 22 lg and 44 lg three times a week, respectively),

accompanied by a delay in the progression of disability.

The MRI analysis of the PRISMS study showed robust

effects in all MRI parameters [83]. Over the 2 years, the

placebo group had a median increase in disease burden of

10.9 %, whereas the 22 lg group and 44 lg group showed

median decreases of 1.2 % and 3.8 %, respectively. In the

subgroup undergoing initial monthly scanning, this reduc-

tion in activity became statistically significant 2 months

after the onset of treatment.

The original studies with IFNb were of 2 years duration.

The long-term effects of IFNb were evaluated in follow-up

open studies arising from the original pivotal ones. The

long-term data, although biased due to the fact that patients

continuing treatment and followed up are those who

responded to the treatment, show that in a great proportion,

the beneficial clinical effects were sustained for several

years [84]. In the PRISMS follow-up trial, patients origi-

nally assigned to Rebif� 44 lg subcutaneously three times

weekly showed lower Kurtzke Expanded Disability Status

Scale (EDSS) progression, relapse rate and T2 burden of

disease for up to 8 years compared with those in the late

treatment group [85]. In the longest follow-up study with

IFNb, early treatment with IFNb-1b (Betaferon�) proved,

in addition to the sustained clinical effect, to significantly

reduce the MS-related mortality rate [86].

2.2.1.2.2 Early Treatment with IFN-b in Clinically Iso-

lated Syndrome (CIS). It is only logical to assume that

IFNb may also delay the conversion to multiple sclerosis—

defined either by a second clinical episode or the accu-

mulation of new MRI lesions—in patients with CIS.

Clinical trials (CHAMPS, ETOMS, and BENEFIT) with

IFNb performed in such patients showed a reduction in the

conversion rate to CDMS over 2–3 years, from 45–50 % in

the placebo group to 28–35 % in the IFNb-treated patients

[87–89].

In an extension of the CHAMPS study, no gain in terms

of disability with Avonex� treatment could be detected at

5 years. However, this 5-year follow up showed that

patients who were assigned to the immediate/early IFN1a

treatment group had a significantly reduced risk of devel-

oping CDMS, that is, of developing a second demyelinat-

ing episode [90].

Similarly, the BENEFIT trial, the largest and perhaps

the best of the three IFN studies [89], showed a moderate

but significant effect of IFN on the accumulation of dis-

ability over 3 years favoring early initiation of treatment

[91].

The 5-year follow up of the same (BENEFIT) trial

showed a robust long-standing effect on the prevention of

CDMS. Early treatment with Betaferon� reduced the risk

of CDMS by 37 % compared with delayed treatment [92].

In a more recent study (REFLEX) [93], 517 patients

with CIS were randomly assigned to either subcutaneous

IFNb-1a (Rebif�) three times weekly, to subcutaneous

IFNb-1a (Rebif�) once weekly, or to placebo). The 2-year

Immunotherapy of MS

cumulative probability of McDonald MS was significantly

lower in both groups of patients treated with subcutaneous

IFNb-1a, with a clearly stronger beneficial effect of the

higher frequency regimen (62.5 %, vs 75.5 % and vs

85.8 %). 2-year rates of conversion to CDMS were lower

and similar for both IFNb-1a dosing regimens.

In all the previously mentioned trials, the most signifi-

cant effects of IFN were on the relapse rate (an approxi-

mately 30 % reduction) and on the MRI activity. The

effects on disability were less pronounced. Moreover, these

pivotal studies were fairly small (involving only a few

hundred patients) and of short duration (2–3 years). A

systematic review of the efficacy of IFNs in MS concluded

that there is evidence only for RRMS and this mainly in

terms of a reduction in relapse frequency during the first

(and less in the second) year of treatment, with no con-

vincing efficacy thereafter and no effect on the accumu-

lation of disability [94]. This surprising conclusion may be

related to the variability in the efficacy among the IFN-

treated patients, possibly caused by the pathological het-

erogeneity of MS (at least four distinct types have been

recognized) [12] and to the high numbers of drop-outs.

2.2.1.2.3 IFNb in Progressive MS. The European Study

Group showed that Betaferon� significantly increased (by

9–12 months) the time to confirmed disease progression

and to becoming wheelchair-bound compared with placebo

in patients with SPMS. Relapse rate and severity were also

lower in the Betaferon� group. This beneficial effect was

seen in patients with superimposed relapses and in patients

who had only progressive deterioration without relapses

[95].

As for MRI activity, the difference in total lesion vol-

ume between treatment groups was highly significant in

both trials. In the placebo group, there was an increase of

15 % from baseline to last scan, whereas in the IFN group,

Table 3 Trial names and acronyms

Acronym Trial name

ALLEGRO Safety and Efficacy of Orally Administered Laquinimod Versus Placebo for Treatment of Relapsing Remitting Multiple

Sclerosis (RRMS)

BECOME Betaseron vs Copaxone in MS with Triple-Dose Gadolinium and 3-T MRI Endpoints

BENEFIT Betaferon� in Newly Emerging Multiple Sclerosis for Initial Treatment

BEYOND Betaferon/Betaseron Efficacy Yielding Outcomes of a New Dose in Multiple Sclerosis

BRAVO Laquinimod Double-Blind Placebo-Controlled Study in RRMS Patients with a Rater Blinded Reference Arm of Interferon b-

1a (Avonex�)

CARE-MS Comparison of Alemtuzumab and Rebif� Efficacy in Multiple Sclerosis

CHAMPS Controlled High Risk Avonex� Multiple Sclerosis

CLARITY Cladribine Tablets Treating MS Orally

DEFINE Determination of the Efficacy and Safety of Oral Fumarate in Relapsing-Remitting MS

ETOMS Early Treatment of Multiple Sclerosis

EVIDENCE Evidence of Interferon Dose–Response-European North American Comparative Efficacy

FREEDOMS Efficacy and Safety of Fingolimod in Patients with Relapsing-Remitting Multiple Sclerosis

GLANCE Glatiramer Acetate and Natalizumab Combination Evaluation

HERMES Helping to Evaluate Rituxan in Relapsing–Remitting Multiple Sclerosis

IMPACT International Multiple Sclerosis Secondary Progressive Avonex� Clinical Trial

INCOMIN Independent Comparison of Interferon

MECOMBIN Methylprednisolone in Combination with Interferon beta-1a for Relapsing–Remitting Multiple Sclerosis

NORMIMS Nordic Trial of Oral Methylprednisolone as Add-On Therapy to Interferon beta-1a for Treatment of Relapsing–Remitting

Multiple Sclerosis

PRISMS Prevention of Relapses and Disability by Interferon beta-1a Subcutaneously in Multiple Sclerosis

REFLEX Rebif� Flexible Dosing in Early MS

REGARD Rebif� vs Glatiramer Acetate in Relapsing MS Disease

SENTINEL Safety and Efficacy of Natalizumab in Combination with Interferon beta-1a

SPECTRIMS Secondary Progressive Efficacy Clinical Trial of Recombinant Interferon-Beta-1a in MS

TEMSO Teriflunomide Multiple Sclerosis Oral

TENERE A Study Comparing the Effectiveness and Safety of Teriflunomide and Interferon Beta-1a in Patients With Relapsing Multiple

Sclerosis

TRANSFORMS Efficacy and Safety of Fingolimod in Patients With Relapsing-Remitting Multiple Sclerosis With Optional Extension Phase

D. Karussis

a reduction of 2 % was seen. In the frequent MRI sub-

group, treatment was associated with a significant 65 %

reduction in new lesion activity between months 1 and 6,

and a 78 % reduction from months 19 to 24 [96].

On the other hand, the North American trial on SPMS

failed to show any beneficial effect of Betaferon� on dis-

ease activity [97]. Although there was no significant dif-

ference in time to confirmed progression in EDSS compared

with the placebo group, there was improvement in sec-

ondary outcome measures including the number of super-

imposed clinical relapses and various MRI-activity criteria.

The differences found between the outcomes of these

two studies remain puzzling [98]. They might be related to

the different populations of MS patients included in these

two trials; the positive European study including more

patients with superimposed relapses than the American

trial, possibly indicating that IFNb is mainly effective in

the inflammatory active patients. This possibility is further

strengthened by the positive MRI results in both studies,

indicating that IFNb has an important beneficial impact

mainly on the inflammatory process in MS.

In another trial (SPECTRIMS) testing IFNb-1a in

SPMS, a total of 618 patients received subcutaneous Re-

bif�, 22 or 44 lg or placebo, three times weekly for

3 years. Treatment with IFNb-1a did not significantly

affect disability progression in this cohort, although sig-

nificant treatment benefit was observed on exacerbation-

related outcomes [99] and MRI activity [99]. The effect on

MRI parameters was more prominent in patients who had

at least one relapse in the 2 years prior to inclusion, again

indicating that the effects of IFN are mainly exerted on the

inflammatory components of the disease.

Finally, in a study with the third type of commercial

interferon (IMPACT) in 436 patients with SPMS [100],

although no differences in EDSS were detected, there were

significant beneficial effects on the Multiple Sclerosis

Functional Composite (MSFC) scale and on the relapse-

related parameters.

In PPMS there are only two randomized control trials

involving a total of 123 patients. IFNb treatment compared

with placebo did not show differences regarding the pro-

portion of patients with disease progression. However, the

total tested population was too small to allow definitive

conclusions to be drawn on the efficacy of the treatment in

PPMS patients [101].

In the first trial, 50 subjects with PPMS were random-

ized to weekly treatment with intramuscular Avonex� at

30 lg, 60 lg, or placebo for 2 years [102]. No effect was

seen on the primary endpoint (time to sustained progres-

sion in disability). Subjects on 30 lg Avonex� had a lower

rate of accumulation of T2 lesion load than the controls;

patients on 60 lg had a greater rate of ventricular

enlargement than the controls.

In the second double-blind study, involving IFNb-1b

(Betaferon�), 73 patients with primary progressive or

transitional MS were included [103]. Statistically signifi-

cant differences favoring treatment were observed for the

Multiple Sclerosis Functional Composite score and for

several secondary MRI parameters. Modest but beneficial

effects of IFNb-1b on clinical variables and brain atrophy

were observed 5 years after termination of this trial [104].

2.2.1.2.4 Comparative Efficacy of IFNs (Different Doses

of IFNb). Few studies have compared the efficacy of the

registered IFNs and investigated the optimal dosing of IFN.

The EVIDENCE trial [110], reported greater efficacy with

high-dose subcutaneous Rebif� (IFNb-1a) administration

three times weekly than with low-dose Avonex� (IFNb-1a)

once weekly, but the trial lasted only 24 weeks. 74.9 % of

patients receiving the high dose IFNb remained relapse

free compared with 63.3 % of those given the lower dose.

In addition, patients receiving Rebif� 44 lg three times

weekly had fewer active MRI lesions compared with those

receiving Avonex� once weekly. Injection-site reactions

were more frequent with Rebif� 44 lg three times weekly

(83 % vs 28 %), as were asymptomatic abnormalities of

liver enzymes. NAbs developed in 25 % of the Rebif�

44 lg three times weekly treated patients and in 2 % of

patients receiving Avonex� [110]. During the crossover

phase of the trial [111], a 50 % reduction in mean relapse

rates was observed in patients who switched from low-dose

once weekly IFNa to high-dose treatment, with significant

concomitant reductions in MRI activity.

Similarly, the 2-year INCOMIN trial [112] showed

greater efficacy with Betaferon� (IFNb-1b) given on

alternate days than with Avonex� given once weekly, in

terms of the percentage of relapse-free and progression-

free patients and the proportion of patients without new

MRI-documented lesions. The superiority of higher dose/

frequency of IFNb-1a (Rebif�) was also evidenced in the

REFLEX study [93], in patients with CIS.

On the other hand, the BECOME trial [113], showed no

significant differences between Betaferon� and subcuta-

neous Avonex� once weekly (Danish MS Group trial), in

all of the outcomes [114]. The practical relevance of these

findings is questionable, as there is no commercially

available IFN type given once a week subcutaneously.

[115, 116]

2.2.1.3 Glatiramer Acetate (GA)/Copaxone

2.2.1.3.1 GA in Relapsing-Remitting MS. GA (COP-1,

Copaxone�, Teva, Israel) is a synthetic co-polymer, com-

posed of alanine, glutamine, lysine and tyrosine, with some

immunologic similarities to the myelin basic protein

(MBP) molecule (as well as to other myelin antigens),

Immunotherapy of MS

without itself being encephalitogenic. COP-1 inhibited

EAE [117] and is presumed to block the MHC/TCR

complex and to downregulate the presentation of antigens

such as MBP, proteolipid protein (PLP) and myelin/oli-

godendrocyte glycoprotein (MOG), to T cells (competitive

inhibition) and/or induce regulatory cells. In a pilot con-

trolled study [118] in 50 patients with RRMS an impressive

reduction in the number of relapses in the GA-treated

group (annual relapse rate [ARR] 0.3 in the COP-1 group

vs 1.35 in the placebo group) was observed. A later and

larger pivotal, double-blind, 2-year trial involving 251

patients [119] showed that daily treatment with subcuta-

neous injections of GA induced a 29 % reduction in relapse

rate. The proportion of patients free of relapse or pro-

gression of disability at 2 years was no different between

the two groups, nor was the time to first relapse. MRI

monitoring was performed in one of the centers and

showed only marginal (and not statistically significant)

inhibition of the ‘MRI activity’ in the GA- treated patients.

Adverse effects were usually mild, including mainly

localized, injection-site reactions (at least once in 90 % of

the patients) and a systemic reaction occurring within

moments of GA administration (associated with chest pain,

palpitations or dyspnea lasting up to 30 min) in 15 % of the

patients.

The effect of GA on disease activity monitored with

MRI was evaluated in a separate randomized study that

included 239 patients. Treatment with GA led to a signif-

icant reduction in the total number of enhancing lesions

compared with placebo (-10.8 %) [120].

The efficacy of an oral preparation of GA (two doses)

was evaluated in a large trial including 1,651 RRMS

patients. The cumulative number of confirmed relapses did

not differ between the two active treatment groups and the

placebo group. No drug effect was detected for any of the

secondary and tertiary endpoints [121].

In a recent study [122], two doses of subcutaneously

administered GA (the regular dose of 20 mg and a higher

one of 40 mg per day) were compared in 569 patients. No

significant differences were observed in any of the clinical,

MRI, and safety parameters.

In long-term follow-up studies, in patients with RRMS

treated for up to 22 years with GA, 57 % maintained

improved or unchanged EDSS scores and the annualized

relapse rates remained low, indicating a long-term efficacy

of GA [123]. In the long-term treated patients, the long-

standing efficacy of GA was associated with an immuno-

logic shift which mainly included an anti-GA antibody

shift (decrease in IgG2 subtype) [124].

2.2.1.3.2 Neutralizing Antibodies Against Interferon. About

5–30 % of treated patients develop persistent neutralizing

antibodies, usually in the first year of treatment and more

commonly in those receiving IFNb-1b. Their presence is

associated with a reduction in treatment effect on relapse

activity.

An open-label study involving 78 patients with MS

treated with IFN [105] analyzed the impact of neutralizing

antibodies (NAbs) on the clinical efficacy of IFNb. The

incidence of persistent NAb? patients was 35 % for

Betaferon�, 20 % for Rebif�, and 3 % for Avonex�.

During IFNb treatment, both NAb? and NAb- patients

showed a reduction in relapse rate; the reduction (25 %)

was not significant in NAb ? patients but was significant

(67 %) in NAb- patients. This indicated a compromise in

IFN efficacy in those NAb? patients.

Data from the PRISMS study showed that persistent

NAbs, above 20 NU/mL, were present in 14 % of the

44 lg and 24 % of the 22 lg three-times-weekly group

over 4 years. Over the entire period of study, relapse and

disability measures were similar between the NAb? and

the NAb- patients. However, once NAbs developed, sig-

nificant differences were noted between the NAb? and

NAb- groups, particularly on MRI and relapse measures

[106].

In another study, NAbs were measured in 277 of 292

early-treated patients and detected at least once in 31.8 %

of the patients, with 60.2 % of them reverting to NAb

negativity by year 5. Time to CDMS, time to confirmed

disability progression, and annualized relapse rate did not

differ between NAb? and NAb- patients, but an increase

in newly active lesion number and T2 lesion volume and

conversion to McDonald MS were associated with NAb

positivity, which was more pronounced with higher titers

[107].

In a recent study, the frequency and impact of neutral-

izing antibodies (NAbs) to IFNb-1b (Betaferon�) on clin-

ical and radiographic outcomes was evaluated in the

patients from the BEYOND study. NAb ? titers were

detected in 37.0–40.7 % of the Betaferon�-treated patients.

Of these, 35 % reverted later to a NAb-seronegative.

NAb? status was not associated with a convincing impact

on any clinical outcome measure. By contrast, NAbs were

associated with a very consistent deleterious impact on

most MRI outcomes [108]. In the BECOME trial, a small,

but more sophisticated study, high levels of anti-IFNbantibodies, which resulted in diminished bioactivity (evi-

denced by the effect on gene expression), were correlated

with reduced therapeutic efficacy of IFNb, as reflected by

the MRI activity [109].

2.2.1.3.3 Early Treatment with GA (CIS). In a random-

ized, double-blind trial, 481 patients presenting with CIS,

and two or more T2-weighted brain lesions in the MRI,

were randomly assigned to treatment with subcutaneous

20 mg per day GA or placebo for up to 36 months, unless

D. Karussis

they converted to CDMS. GA reduced the risk of devel-

oping clinically definite MS by 45 % compared with pla-

cebo [125].

2.2.1.3.4 GA in Progressive MS. A double-blind, pla-

cebo-controlled, 3-year study involving 943 patients with

primary progressive MS tested the efficacy of GA in pro-

gressive disease. The trial was terminated after an interim

analysis by an independent data safety monitoring board

indicated no discernible treatment effect on primary out-

come. Intention-to-treat analyses of disability and MRI end

points showed a nonsignificant delay in time to sustained

accumulated disability in GA-treated patients, with sig-

nificant decreases in enhancing lesions in the first year. The

slow rate of disease activity in the control group may be

one of the explanations for these negative results [126,

127].

2.2.1.3.5 Comparative Efficacy of IFN vs GA. In a large

prospective randomized comparative study (BEYOND),

2,244 patients were enrolled. Patients were randomly

assigned 2:2:1 to receive one of two doses of Betaferon�

(250 lg or 500 lg) subcutaneously every other day or

20 mg GA subcutaneously every day. No differences in

relapse risk, EDSS progression, T1-hypointense lesion

volume, or normalized brain volume was observed between

the treatment groups. A 500 lg dose of IFNb-1b was not

more effective than the standard 250 lg dose, and both

doses had clinical effects similar to those of GA. The

overall tolerability to both drugs was similar [113, 115].

Similar results (no difference in efficacy between IFN and

GA) were obtained from the REGARD study in which

Rebif�, 44 lg three times weekly was used in the com-

parator group. In this study, the primary outcome was MRI

activity using a more sensitive way to detect active lesions

by triple dose gadolinium administration [116].

2.2.1.4 Intravenous Immunoglobulins (IVIG) Data from

an open pilot controlled study indicated that IVIG had

some efficacy in reducing the number of relapses in RRMS

[128]. However, two other trials failed to show any efficacy

of this treatment in patients with RRMS or progressive MS

[129, 130]. MRI monitoring in the latter trial did not reveal

any effect of IVIG treatment in preventing the appearance

of new lesions in the brain [131]. A pilot study examining

the issue of recovery of apparently fixed, irreversible

neurological deficits, by IVIG treatment, showed that this

treatment may enhance recovery in optic neuritis patients,

raising the possibility of remyelination induction [132]. A

later controlled study with monthly IVIG injections showed

a reduction of 59 % in the relapse rate in patients with

RRMS [133, 134]. There was also a statistically significant

difference between the placebo- and IVIG-treated groups

in the progression of disability (changes in EDSS score),

but the effects on MRI activity were not evaluated. In

general, there is some evidence pointing to a reduction in

relapse rate (weighted mean difference [WMD] -0.73) and

longer relapse-free periods during IVIG treatment.

In SPMS, two studies [135, 136] failed to show any

difference between the IVIG-treatment and the placebo

groups in terms of time to sustained disease progression. In

PPMS, a single study reported significantly fewer partici-

pants with EDSS progression in the IVIG-treated group

than in the placebo group, with a non-significant trend to a

delay in time to disease progression [136].

MRI data were reported in two RRMS studies [137, 138]

and in one on SPMS [135]. The RRMS results are con-

flicting. Lewanska et al. [137] reported a significant

reduction in both new lesions on T2 and in the number of

gadolinium-enhancing lesions on T1 in both IVIG-treat-

ment groups compared with placebo, accompanied by a

non-significant trend to reduced T2 lesion burden. In the

second study, no difference could be detected in any of the

MRI variables [138]. In SPMS, there was no evidence of

any beneficial effect of IVIG on the MRI parameters.

In an additional trial with clinical and MRI end-points,

significant differences were reported between the placebo

and the IVIG-treatment groups [139]. However, this study

suffers from considerable methodological difficulties, par-

ticularly with respect to blinding and allocation concealment.

Two additional trials [140, 141], of a rigorous method-

ological quality, were entirely negative across a wide range

of clinical and paraclinical outcome measures, including a

subset of participants undergoing serial MRI examinations.

The two trials included 122 participants with RRMS or

SPMS. Essentially, these trials tested without success the

hypothesis that IVIG would reverse established neurologic

impairments in MS.

Infusion-related side effects were reported in 4 % of the

participants [133, 135] and in 3.8 % (3.0 %) of the infu-

sions [142]. One study reported six participants with deep

venous thrombosis, four of whom also developed pul-

monary embolism in the treatment group. However, two of

these had a known coagulation defect. IVIG was extremely

poorly tolerated in one trial [138], with 11/21 (52 %)

experiencing severe cutaneous reactions. In the same study,

there was one case with hepatitis C viral infection and one

mortality from a pulmonary embolus [134–136, 142].

2.2.1.5 Immunosuppressants Several immunosuppres-

sive treatments have been tried in MS in the past decades,

with variable efficacy.

2.2.1.5.1 Azathioprine. Azathioprine, an antimetabolite

with broad spectrum immunosuppressive effects and one of

the main immunosuppressive cytotoxic substances

Immunotherapy of MS

available, is used extensively to control transplant rejec-

tion reactions. It is nonenzymatically cleaved to mercap-

topurine, which acts as a purine analog and an inhibitor of

DNA synthesis. By preventing the clonal expansion of

lymphocytes in the induction phase of the immune

response, it affects both cellular and humoral immunity

and is therefore efficient in the treatment of autoimmune

diseases. Azathioprine was initially tested in some pilot

clinical trials [142–144]. In the British and Dutch Multi-

ple Sclerosis Trial [143] it was shown to reduce the rate

of progression at 3 years. Worsening of the EDSS scale

was 0.62 in the azathioprine group and 0.80 in the pla-

cebo-treated patients.

In total, azathioprine was tested in five controlled trials

[143–146]. Both relapsing and remitting MS patients and

patients with progressive disease were included in these

five trials (RRMS = 388; SPMS = 188; PPMS = 62;

other progressive MS = 65). Almost half of the included

patients suffered from progressive disease and this is

unique in trials with such immunomodulating drugs (most

of which showed efficacy only in RRMS). In one of the

trials, only patients with progressive types of MS were

included [146]. In general, there were clear beneficial

effects of azathioprine treatment in the whole group

(including the progressive cases).

With regard to its efficacy on relapses, analysis of the

cumulative data from 698 patients (from these five trials)

showed that treatment with azathioprine was associated

with a significant reduction in the number of patients who

had relapses during the first year compared with placebo

(relative risk reduction [RRR] = 20 %), at 2 years

(RRR = 23 %) and at 3 years (RRR = 18 %) [147].

Cumulative data on disability progression rates, avail-

able from three trials [144, 146] involving 87 patients;

showed that the proportion of patients who progressed

during 2–3 years of treatment was 61 % (range of

46–83 %) in the placebo group and 34 % (range 30–43 %)

in the azathioprine group (a statistically significant RRR of

42 %).

Effects on MRI: In the only existing small study that

evaluated the effect of azathioprine on the MRI parameters

of MS [148], the MRI scans of 14 patients were followed

monthly for 6 months before and following the initiation of

azathioprine treatment. A reduction of more than 50 % in

both the gadolinium-enhancing lesions and the T2 lesions

was observed in almost all of the patients.

The azathioprine dose in the reported trials varied from

2 mg/kg to more than 4.4 mg/kg. The recommended dose

should be that which leads to a target white blood cell

count of 3,000–4,000 and an absolute lymphocyte count of

800–1,000. Mean cell volume, which should increase to

values of around 100, is also a reliable marker for drug

efficacy.

The side effects included gastrointestinal discomfort or

pain and a cutaneous rash (5 % for each) and a 9 % inci-

dence of liver enzyme elevation. Macrocytic anemia

occurred in 3 % of the azathioprine-treated patients. There

was a small but not significant absolute increase of 3.4 %

in the risk for malignancies from a 3-year course of aza-

thioprine and a slightly increased mortality rate in the

azathioprine groups [149]. In general, it is recommended

that the total cumulative dose of azathioprine should not

exceed 600 g per patient.

Azathioprine was also tested in a combination regimen.

In an open study, azathioprine given as an add-on treatment

(either with or without steroids) did not offer a significant

additional benefit to that of interferon treatment [150].

2.2.1.5.2 Cyclophosphamide. Cyclophosphamide is an

alkylating drug (a member of the nitrogen mustards), that

binds to DNA and interferes with mitosis and cell repli-

cation. Its immunologic actions include (i) suppression of

both cellular and humoral immunity through its effects on

B and T cells, with a somehow greater suppressive effect

on B cells; (ii) decreased secretion of IFNc and IL-12 by

monocytes [151, 152]; (iii) increased secretion of IL-4 and

IL-10; (iv) a shift from a Th1-type to a Th2-type cytokine

profile. Cyclophosphamide is commonly used as an anti-

neoplastic drug and for the treatment of severe systemic

autoimmune disorders, such as SLE [153, 154].

Initial pilot open studies back in the early 1970s, and later,

showed positive indications of the efficacy of cyclophos-

phamide in MS. In an uncontrolled, open-label trial, stabil-

ization of the disease for up to 5 years was observed in 69 %

of the 86 progressive MS patients treated with a short course

of cyclophosphamide (400 mg) and 1 g prednisone [155]. In

another open-label, prospective, randomized trial in 58

progressive MS patients, treatment with a short course of

high-dose intravenous cyclophosphamide and ACTH was

compared with ACTH alone and with low-dose cyclophos-

phamide plus ACTH and PLEX: 80 % of the patients in the

high-dose cyclophosphamide group stabilized or improved,

compared with 20–50 % in the two other groups.

Later, two large trials reported conflicting results. The

Northeast Cooperative Multiple Sclerosis Treatment

Group, randomizing a four-arm trial in 256 patients with

progressive MS [156], included two induction regimens of

intravenous cyclophosphamide and ACTH, with or without

subsequent intravenous boosters of cyclophosphamide

every other month. Although there were no differences in

disease stabilization between the two induction regimens,

the patients who received maintenance boosters of cyclo-

phosphamide had a significant delay in EDSS progression

compared with the patients who did not receive boosters.

The beneficial effect was more pronounced in patients of a

younger age. However the difference in stabilization rates

D. Karussis

(38 vs 24 % in 24 months) was small and disappeared by

36 months.

The Canadian Cooperative Multiple Sclerosis Group

[59] performed a single-blinded, placebo-controlled, mul-

ticenter trial including 168 patients with progressive MS

who received intravenous cyclophosphamide and oral

prednisone, or oral cyclophosphamide and oral prednisone

on alternate days, combined with weekly PLEX. The

control group received oral placebo and sham plasma

exchange. There was no significant difference in the time

to worsening of one or more points in the EDSS.

Since the early 1990s there have been several trials and

case reports with cyclophosphamide in patients with rapidly

worsening or treatment-refractory MS. An open-label

observational study in 95 patients with progressive MS

showed that 80 % of patients with SPMS who were treated

with monthly cyclophosphamide and intravenous pulses of

methylprednisolone had stable or improved EDSS scores at

12 months [157].

Gobbini et al. [303] reported a rapid reduction in gad-

olinium-enhancing lesions, in monthly brain MRI scans,

and clinical stability in five patients with rapidly deterio-

rating RRMS who were treated with monthly cyclophos-

phamide for 6 months followed by cyclophosphamide on

alternate months. In another open-label study of intrave-

nous cyclophosphamide given monthly to 14 patients with

RRMS who were rapidly deteriorating [158], the mean

EDSS scores were significantly lower than baseline at

follow-up (up to 18 months) and no relapses were reported.

In an open-label study of 24 patients with clinically active

and treatment-refractory MS, significant improvement in

EDSS scores, relapse rate, and MRI measures were docu-

mented in the patients treated with intravenous cyclo-

phosphamide plus intravenous methylprednisolone

(monthly in the first year and bimonthly in the second)

[159]. A retrospective, open-label review of 362 patients

with SPMS and 128 patients with PPMS who were given

12-monthly pulses of intravenous cyclophosphamide [160]

showed that compared with baseline, the EDSS score sta-

bilized or improved at month 12 in 78.6 % of the patients

with SPMS and in 73.5 % of the patients with PPMS. A

report of a single patient with RRMS who accidentally

received a dose of cyclophosphamide 3,800 mg (3.8 times

the normal dose) showed no evidence of clinical or MRI

disease activity for the next 7 years [161]. This observation

advocates the concept of autologous bone marrow trans-

plantation in MS. Finally, an open-label study in which 15

treatment-refractory MS patients were treated with cyclo-

phosphamide 200 mg/kg over 4 days showed a significant

stability in disease and improvement in quality of life after

15 months [162].

Several combination studies have been performed to test

the efficacy of cyclophosphamide combined with other

immunomodulators. In a randomized, multi-center trial of

59 patients with RRMS who did not respond to IFNb, the

combination of cyclophosphamide and IFNb-1a reduced

clinical disease activity and gadolinium-enhancing MRI

lesions in the brain [163]. In a smaller study, in ten patients

who had frequent and severe attacks despite IFNb therapy,

the addition of monthly intravenous cyclophosphamide led

to a significant reduction in the number of relapses, EDSS

score, and number of T2-weighted lesions. These benefits

were maintained for 36 months after cyclophosphamide

was discontinued [164].

Cyclophosphamide can cause several side effects,

including alopecia, nausea, vomiting, hemorrhagic cystitis,

leukopenia, myocarditis, infertility, and pulmonary inter-

stitial fibrosis.

2.2.1.5.3 Mitoxanthrone. Mitoxanthrone is an anthra-

cenedione antineoplastic drug that intercalates with DNA

and inhibits both DNA and RNA synthesis that was

developed in the 1970s. Mitoxanthrone inhibits B-cell

function, including antibody secretion, abates helper and

cytotoxic T-cell activity, and decreases the secretion of Th1

cytokines such as IFNc, TNFa, and IL-2 [165].

The first placebo-controlled trial of mitoxanthrone,

which was performed in 51 patients with RRMS randomly

assigned to monthly infusions of mitoxanthrone (8 mg/m2)

or saline for 1 year, showed a significant reduction in the

annual relapse rate and an increase in the proportion of

patients who were relapse-free at the end of 1 and 2 years,

but no differences in EDSS scores [166]. There was also a

trend to a reduction in the number of new brain MRI T2

lesions. In an unblinded, multi-center trial in 42 patients

with worsening RRMS and SPMS, Edan and co-workers

found that monthly infusion of intravenous mitoxanthrone

20 mg and intravenous methylprednisolone 1 g improved

the clinical and MRI parameters of MS activity over

6 months, as compared with those of the group on intra-

venous steroids alone [167]. In a double-blind trial of 49

patients with SPMS with superimposed relapses, who were

randomly assigned to receive 13 infusions of mitoxan-

throne 12 mg/m2) or intravenous methylprednisolone 1 g

over 32 months, neurological disability, relapse rate, and

the number of gadolinium-enhancing lesions in MRI were

significantly reduced in the mitoxanthrone group [168].

In a phase III trial, 194 patients with worsening RRMS

or SPMS were randomly assigned to receive either placebo

or intravenous mitoxanthrone 12 mg/m2 or 5 mg/m2 every

3 months for 2 years [169]. The higher dose of mitoxan-

throne was significantly more effective in the combined

clinical outcome measurement, consisting of five clinical

parameters (change in EDSS, change in ambulation index,

number of treated relapses, time to first treated relapse, and

change in neurological status). The results of this trial led

Immunotherapy of MS

to the regulatory approval of mitoxantrone as the first

immunosuppressive chemotherapeutic drug for patients

with MS.

Le Page and co-workers reported an observational study

on 100 patients with aggressive RRMS who were induced

with intravenous mitoxanthrone 20 mg and intravenous

prednisolone 1 g every month for 6 months [170]. This

was followed by mitoxanthrone maintenance therapy every

3 months, IFNb, GA, azathioprine, or methotrexate in 57

patients, with a mean follow-up of 3.8 years. In the year

after induction, the relapse rate, EDSS score, and MRI

activity were significantly decreased, the decrease sus-

tained for up to 5 years.

2.2.1.5.3.1 Combination Studies. Jeffery and co-workers

conducted an open-label, add-on, pilot study on ten active

disease MS patients despite at least 6 months of IFNbtreatment [171]. With the addition of mitoxanthrone

(12 mg/m2 for the first month, then 5 mg/m2 every month

for 2 months, and then 5 mg/m2 every third month), the

mean frequency and volume of gadolimium-enhancing

lesions decreased by 90 % and 96 %, respectively, after

7 months, and the relapse rate was reduced by 64 %. In

another open-label study, 27 consecutive patients with

clinically-active RRMS were treated with monthly infu-

sions of mitoxanthrone for 3–6 months followed by boosts

every 3 months, in combination with GA [172]. At a mean

follow-up of 36 months, EDSS scores and relapse rates

were significantly reduced compared with baseline.

As mentioned, mitoxanthrone is the only licensed

cytotoxic medication for MS (for patients with SPMS,

progressive relapsing or worsening RRMS but not for those

with PPMS) and is probably equally or more efficacious

than IFNs and GA, but its use is confined to cases with

sufficiently aggressive MS to justify its toxic effects. Mi-

toxanthrone causes cardiotoxicity (the cumulative allowed

dose should not exceed 120 mg/m2) and acute leukemia

(AML); the cumulative risk for both these adverse effects

is 0.2 %.

2.2.1.5.4 Methotrexate. Methotrexate, a folic acid ana-

log, is a potent immunosuppressant, whose mode of action

is predominantly through inhibition of dihydrofolate

reductase and thus thymidine biosynthesis, although there

are other anti-inflammatory effects which do not rely upon

this specific mechanism [173]. By inhibiting purine and

pyrimidine synthesis, methotrexate suppresses antigen-

dependent T-cell proliferation and promotion of adenosine-

mediated inflammation. It is used in the treatment of

autoimmune diseases (for example, rheumatoid arthritis or

Behcet’s disease) and in transplantations.

In a placebo-controlled study, methotrexate, at a low

dose, was reported to have a mild beneficial effect in

progressive MS [174, 175]. In that study, 60 patients were

treated with methotrexate (7.5 mg weekly) or with a pla-

cebo for 2 years. Methotrexate significantly reduced sus-

tained worsening, as assessed by a composite measure of

disability using the EDSS, the ambulation index, and two

arm function tests. However, the effect of this treatment

was less pronounced when changes in the ‘traditional’

measures, such as the EDSS score and the Ambulation

Index, were used as outcomes. The effect of methotrexate

on MRI activity was also marginal. Another trial with

methotrexate [176], included 44 participants, of whom 20

had RRMS and 24 suffered from progressive forms of MS.

The authors reported a trend to fewer relapses in the RRMS

group, but no difference in EDSS progression in the pro-

gressive group.

A recent study [177] evaluated the feasibility of intra-

thecal administration (up to eight treatments) of metho-

trexate in 121 progressive MS patients. No serious adverse

effects were noted during the study period. In 89 % of the

87 SPMS patients, EDSS scores were stable or improved. In

82 % of the four PPMS patients, EDSS scores were stable.

In general, methotrexate toxicity is usually low if

treatment is paralleled by folate substitution. However,

long-term methotrexate administration is associated with

serious side effects, including hepatic fibrosis.

2.2.1.5.5 Cyclosporin-A. Cyclosporin A, a cyclic fungal

peptide composed of 11 amino acids, is a calcineurin

inhibitor, similar in this aspect to tacrolimus. It has been in

use since 1983 and is one of the most widely administered

immunosuppressive drugs. It binds to the cytosolic protein

cyclophilin (an immunophilin) of immunocompetent lym-

phocytes, especially T-lymphocytes. This complex of

cyclosporin and cyclophilin inhibits calcineurin which,

under normal circumstances, induces the transcription of

IL-2, leading to the reduced function of effector T cells,

including a reduction in the frequency of regulatory T cells.

Cyclosporin A is used in the treatment of acute rejection

reactions, but has been increasingly substituted by newer

and less nephrotoxic immunosuppressants.

Cyclosporin A was studied in a 2-year, multicenter,

double-blind, clinical trial involving 547 progressive MS

patients [178] with an increase in EDSS of 1–3 grades in

the year prior to entry. Cyclosporine was given to 273

patients, placebo to 274. The mean increase in EDSS was

0.39 ± 1.07 for the cyclosporine-treated patients and

0.65 ± 1.08 in the placebo group. Cyclosporin A delayed

the time to becoming wheelchair-bound (relative risk:

0.765), but statistically significant effects were not

observed for ‘time to sustained progression’ or on a com-

posite score of ‘activities of daily living’.

The high rate of hypertension and nephrotoxicity

induced by this treatment limit its use. Calcineurin

D. Karussis

inhibitors and azathioprine have been linked also with post-

transplant malignancies and skin cancers.

2.2.1.5.6 Cladribine. The synthetic purine nucleoside

analog cladribine (2-chloro-20-deoxyadenosine) enters the

cell through purine nucleoside transporters and is phos-

phorylated by deoxycytidine kinase. Lymphocytes have

fairly high concentrations of this enzyme and low levels of

50 nucleotidase, leading to the preferential accumulation of

cladribine in lymphocytes [179]. Cladribine nucleotide

accumulation disturbs DNA synthesis and repair mecha-

nisms, resulting in lymphocyte depletion and long-lasting

lymphopenia. The drug targets mainly CD4? T cells,

CD8? T cells, and B cells [179]. Because cladribine can

penetrate the CNS, it interacts with cells in both the

peripheral circulation and in the CNS. The therapeutic

efficacy and safety of cladribine have been assessed in

several autoimmune disorders and the parenteral formula-

tion of cladribine is used as first-line treatment for hairy

cell leukemia.

Older studies in a total of 262 patients have shown the

efficacy of parenteral cladribine in RRMS and progressive

MS, both in clinical and MRI parameters [180–182]. In the

large and recent CLARITY trial, a placebo-controlled,

double-blind trial, a new preparation of oral cladribine (in

two doses) and a new treatment scheme, were tested in 1,326

patients with RRMS [183]. There was a significantly lower

annualized relapse rate in both cladribine groups compared

with the placebo group (0.14–0.15, vs 0.33), a higher relapse-

free rate (79.7–78.9 % vs 60.9 %), a lower risk of 3-month

sustained progression of disability, and significant reduc-

tions in brain lesion count upon MRI. Adverse events that

were more frequent in the cladribine groups included lym-

phocytopenia (21.6 % in the 3.5 mg group and 31.5 % in the

5.25 mg group) and herpes zoster infections. Neoplasms

arose in 6 (1.4 %) patients in the 3.5 mg/kg cladribine group

and 4 (\1 %) in the 5.25 mg/kg group, but in none of the

patients in the placebo group. Neoplasms included leio-

myomata (n = 5), cervical carcinoma in situ (n = 1), mel-

anoma (n = 1), ovarian carcinoma (n = 1), pancreatic

cancer (n = 1), and myelodysplasia (n = 1). These risks for

adverse events have probably been the main reason for

rejection of the drug as a registered treatment for MS by the

European and American authorities. A recently published

follow-up of the CALRITY study in 1,192 patients evaluated

the parameter of freedom from disease activity (no relapses,

no EDSS progression, no active or new lesions in the MRI) at

96 weeks. Of the patients in the two doses of cladribine

groups, 67–70 % were free of disease activity, versus 39 %

in the placebo group [184].

2.2.1.5.7 Mycophenolate Mofetil. Mycophenolate mofe-

til (MMF) belongs to the antimetabolite drug class and is a

pro-drug of its active metabolite, mycophenolic acid. By

depleting guanosine and deoxyguanosine nucleotides in T

and B lymphocytes, it inhibits their proliferation and,

hence, immunoglobin (Ig) production. MMF also sup-

presses dendritic cell maturation, decreasing its capacity of

antigen presentation to T lymphocytes. Small trials in

progressive MS suggest some efficacy, and its adminis-

tration appeared well tolerated [185, 186]. Interestingly, in

an open-label trial, the combination of MMF with IFNb-1a

exhibited superior efficacy, compared with IFNb-1a alone

[187]. In this study, 30 RRMS patients already treated with

IFNb-1a were given MMF at a progressive dose of 2 g per

day orally for 6 months. The annualized relapse rate was

0.57 ± 0.3 at the end of the study, as compared with

2.0 ± 0.7 at enrollment. No gadolinium-enhancing lesions

were detected upon MRI at the end of the study, as com-

pared with 11 active patients at baseline (a total of 35

lesions).

In a retrospective trial, MMF was shown to prevent

relapses in 24 NMO patients [188]. Adverse events inclu-

ded benign infectious diseases, insomnia and dizziness,

nausea and abdominal pains. Controlled and larger studies

are warranted.

2.2.1.5.8 Tacrolimus. Tacrolimus/FK506 (Prograf�, As-

tellas, US) is a product of the bacterium Streptomyces

tsukubaensis. It binds to the immunophilin FKBP1A, fol-

lowed by binding of the complex to calcineurin and the

inhibition of its phosphatase activity. In this way, it pre-

vents the cells from transitioning from the G0 to the G1

phase of the cell cycle. The drug, which is more potent than

cyclosporin and has less pronounced side effects, is used

primarily in transplantations.

A pilot clinical trial [189] with tacrolimus in chronic

progressive MS did not detect any significant change in the

proportion of circulating CD25?CD4? or CD45R?CD4?

cells over the study period. The side effects of tacrolimus

were mild and the overall degree of disability did not

deteriorate significantly in the 19 patients studied over the

12 months of tacrolimus administration.

2.2.1.6 Combination Immunotherapy The rationale for

combination immunotherapy (reviewed in [190]), comes

from the reported synergistic effects and increased efficacy

of such combinations of immunomodulatory agents in

other autoimmune diseases [191]. It is also justified by the

acknowledged complexity of MS immunopathogenesis,

which may necessitate the use of more than one drug to

interfere with the various distinct immune elements

involved at multiple levels of the immune axis, in order to

maximize treatment efficacy. However, owing to the

complex interactions and the delicate balance between the

various immune cells and cytokine/chemokine networks,

Immunotherapy of MS

unexpected or paradoxical effects may occur when differ-

ent drugs affecting the immune system are combined. Such

combinations must, therefore, be tested in clinical trials.

Small-size pilot studies have provided some positive

indications supporting the principle of combination treat-

ments. In one of them, Calabresi and colleagues [192]

added methotrexate 20 mg weekly to the existing therapy

with IFNb in 15 MS patients and reported a 40 % reduction

in gadolinium-enhancing lesion number (p = 0.02) after 4,

5, and 6 months, and a reduction in relapse rate from 0.73

to 0.27. A similar open-label trial of 12 participants com-

bined azathioprine 3 mg/kg with subcutaneous IFNb-1b

[193]. After the addition of azathioprine, there was a 65 %

reduction in gadolinium-enhancing MRI lesions after

6 months, compared with the baseline.

However, the results of the subsequently performed

larger controlled studies using IFN as basic therapy and

methotrexate, steroids, or azathioprine as add-ons, were

negative with the exception of the NORMIMS.

Specifically, in a 2-year study in which azathioprine or

azathioprine plus low dose oral prednisone were added to

IFNb, although a beneficial trend could be detected

favoring the triple therapy, these differences were not of

statistical significance [150].

In another trial involving 313 MS patients, methotrexate

20 mg weekly or methotrexate plus bi-monthly, 3-day,

intravenous, high-dose methylprednisolone were added to

weekly IFNb-1a. The combinations were generally safe and

well tolerated. Although the trends suggesting a modest

benefit were seen for some intravenous methylprednisolone

outcomes, the overall results did not point to a significant

benefit [194].

The MECOMBIN double-blind trial examined whether

the addition of methylprednisolone 500 mg/day orally for 3

consecutive days per month for 3–4 years to IFNb-1a could

provide any additional benefit in RRMS patients, in terms of

disability progression. A total of 341 patients were included

in this trial. Monthly pulses of methylprednisolone were not

shown to affect disability progression any more than IFNb-

1a treatment alone. Some positive effects, but only in sec-

ondary measurements, were detected [195]. In the NORM-

IMS (the only positive trial), a higher dose of IFNb-1a (44 lg

three times a week) was combined with 5-day monthly pulses

of oral methylprednisolone (200 mg each day). The combi-

nation led to a significant reduction in relapse rate [196].

Additional immunotherapeutic schemes tested included

combinations with natalizumab. In the SENTINEL trial,

natalizumab was added to weekly intramuscular IFNb. The

1-year annualized relapse rate was 0.82 in the monotherapy

group and 0.38 in the combination group, a 54 % reduction,

p \ 0.001). This effect persisted at 2 years. Additionally, the

two-year sustained disability was 29 % in the monotherapy

group and 23 % among participants who received

combination therapy, this representing a 24 % decrease

(p = 0.02). At 2 years, the percentage of relapse-free par-

ticipants was 32 % in the monotherapy group and 54 % in

the combination group (p \ 0.001).

The trial was terminated 1 month early because of two

cases of progressive multifocal leukoencephalopathy (PML)

in the natalizumab-Avonex� combination group [197].

GLANCE was a smaller study (n = 110) in which na-

talizumab was added to glatiramer acetate for 6 months.

For the imaging endpoints in this trial, there was a better

outcome in the combination group. In terms of relapses and

EDSS changes, the two groups did not differ significantly

(but, as mentioned above, this was a short study and its

primary outcome was MRI parameters). In both the above

trials, there was no natalizumab-alone group, which makes

it more difficult to draw definite conclusions on the com-

bination effect. These trials did not reveal any increased

danger for adverse events, and the two initially reported

cases of PML in SENTINEL seem to be related to natal-

izumab and not necessarily to the combination, since all the

(more than 200) later PML cases were in patients treated

with natalizumab alone. However, an increased risk for

PML from the combination treatment can not be ruled out.

Pilot studies combining cyclophosphamide with IFNbhave yielded encouraging results [164, 198, 199].

A randomized, double-blind, placebo-controlled trial of

cyclophosphamide as an add-on therapy to IFNb is in pro-

gress [164]. Treatment strategies entailing a rescue thera-

peutic scheme (see treatment algorithm) of strong

immunosuppression followed by maintenance treatment with

first-line immunomodulators have been suggested and vali-

dated. In one such approach, a short course of mitoxantrone

was used to induce immunosuppression, followed by

immunomodulation with IFNb or glatiramer acetate. Pilot

studies showed promising results with both combinations

[171, 172].

In the larger of the pilot open studies, 100 consecutive

patients with aggressive RRMS received mitoxanthrone

20 mg monthly combined with methylprednisolone 1 g for

6 months. During the 12 months following initiation of

mitoxantrone treatment, the annual relapse rate was

reduced by 91 % (with 78 % of the patients remaining

relapse-free), MRI activity was reduced by 89 %, the mean

EDSS decreased by 1.2 points and 64 % of the patients

improved by 1 point or more in the EDSS. In the longer

term, the ARR reduction was sustained and disability

remained improved after 5 years [200].

In a recent 3-year, randomized, controlled, clinical and

MRI study, 109 patients with aggressive MS (two or more

relapses in the previous 12 months and one or more gad-

olinium-enhancing MRI lesions) were randomized and

received mitoxanthrone monthly (12 mg/m2) combined

with methylprednisolone 1 g for 6 months followed by

D. Karussis

IFNb for the last 27 months, or IFNb for 3 years combined

with methylprednisolone 1 g monthly for the first

6 months. The time to worsening by at least one EDSS

point was delayed by 18 months in this group versus the

IFNb group (p \ 0.012). The 3-year risk of worsening

disability was reduced by 65 % in the mitoxanthrone res-

cue group compared with the IFN group. Mitoxanthrone-

treated patients had a reduced number of Gd-enhancing

lesions at month 9 and a slower accumulation of new T2

lesions at each time point [201].

Two small pilot studies did not detect any significant

beneficial effect of the addition of mycophenolate to IFNb-

1a treatment [202, 203]. In the second study, the MMF

group showed a trend to a lower accumulation of combined

active lesions in MRI.

Additional combination studies (examining the possibility

of enhancing the effect of GA or IFN by neuroprotective

agents) were also performed or are under way. A combination

of simvastatin, a cholesterol-lowering agent with putative

immunomodulatory and neuroprotective properties, with

IFNb-1a, did not offer any additional beneficial effect [204].

2.2.2 The New Generation of Immunotherapies

2.2.2.1 Antibodies Synthetically produced antibodies may

serve as a quick and potent immunosuppressive therapy.

2.2.2.1.1 Polyclonal Antibodies. Animal-derived poly-

clonal antibodies, such as antilymphocyte and antithymocyte

antigens, have been successfully used as adjuvant treatment

(to immunosuppressants in order to reduce their dose and

toxicity) for steroid-resistant acute rejection reaction and

aplastic anemia. Polyclonal antibodies inhibit T lympho-

cytes and cause their lysis (complement-mediated or cell-

mediated opsonization). In this way, polyclonal antibodies

inhibit cell-mediated immune reactions, including graft

rejection, delayed hypersensitivity, and graft-versus-host

disease.

Polyclonal antibodies affect all lymphocytes and cause

general immunosuppression, possibly leading to post-

transplant lymphoproliferative disorders or serious infec-

tions, especially by cytomegalovirus. Because of their high

immunogenicity, almost all patients have an acute reaction,

characterized by fever, rigor, and even anaphylaxis.

A small pilot study with antilymphocyte antigens in 12

MS patients showed some efficacy in progressive disease

[205], but in general the use of polyclonal antibodies is not

justified for MS due to the high toxicity, except in the

frame of autologous hematopoietic transplantation.

2.2.2.1.2 Monoclonal Antibodies. Monoclonal antibod-

ies are directed towards precisely-defined antigens.

Therefore, they cause fewer side effects.

2.2.2.1.2.1 Natalizumab. Pioneer animal studies in EAE

back in the early 1990s [19], demonstrated that an antibody

against the a4b1 integrin, a surface molecule of lympho-

cytes, could reduce lymphocytic infiltration and clinical

disease severity. These data led to clinical trials of a

humanized anti-a4 integrin antibody, natalizumab (Tysa-

bri, Biogen, US). Monthly infusions of this antibody

showed strong efficacy against placebo, reducing the

relapse rate at 1 year by 68 % and the chance of acquiring

fixed disability over 2 years by 42 % [20]. Moreover, na-

talizumab-treated patients were free of any disease activity

(relapses, progression of disability and new MRI activity)

by around 30 %. Although this efficacy appears higher than

that observed in the interferon and glatiramer acetate piv-

otal studies, such cross comparison between different

studies (of different design and MS patients populations) is

not scientifically sound. Only direct head-to-head pro-

spective trials (which do not exist at the present) can pro-

vide definite information on the relative efficacy of the

drugs. US and European authorities licensed natalizumab

for relapsing multiple sclerosis, but the drug was later

withdrawn from the market months when two cases of

PML were identified in a trial combining Avonex� (IFNb-

1a) with natalizumab [206]. As a result, natalizumab is now

licensed as monotherapy only for severe RRMS, or as

second-line immunotherapy for MS. Since licensing, many

(more than 200) additional cases of PML have been iden-

tified, almost exclusively among the patients treated for

more than 2 years with natalizumab. Three parameters

have been identified as being related to an increased risk of

PML: (i) duration of treatment for [2 years; (ii) previous

exposure to cytotoxic medications; and (iii) the presence of

anti-JC (John Cunningham) virus antibodies (which are

found in half of the MS and general population and indicate

previous exposure to the virus) [207]. All reported cases of

PML following natalizumab treatment were positive for the

JC virus antibodies. Introduction of the test for these

antibodies has helped in the selection of patients to be

treated with natalizumab and those in whom the treatment

should be discontinued due to a high risk of PML.

2.2.2.1.2.2 Alemtuzumab. Alemtuzumab is a humanized

monoclonal antibody against the CD52 antigen on T and B

lymphocytes [208] that produces rapid and sustained

lymphocyte depletion and is an approved therapy for B-cell

chronic lymphocytic leukemia.

The first trial of alemtuzumab in MS was performed by

Coles and co-workers, and was a rater-blinded, phase II

trial comparing two doses of the drug given once a year

with IFNb-1a (44 lg subcutaneously, three times a week)

in treatment-naive patients with RRMS [209]. The study

was suspended after immune thrombocytopenic purpura

developed in three patients, one of whom died.

Immunotherapy of MS

Alemtuzumab treatment reduced the relapse rate compared

with IFNb-1a by up to 74 %, and the chance of accumu-

lating disability by up to 71 %, over 3 years. MRI out-

comes were also significant, favoring alemtuzumab.

Adverse events in the alemtuzumab group included auto-

immunity (thyroid disorders and immune thrombocytope-

nic purpura [3 %]) and infections.

Two large phase III trials (CARE-MS I and II) have

been concluded and their preliminary results presented in

scientific meetings. CARE-MS I, a phase III trial in

patients with RRMS, showed a 55 % reduction in relapse

rate in the group treated with two annual cycles of ale-

mtuzumab as compared with Rebif over 2 years. Eight

percent of alemtuzumab-treated patients had a sustained

increase in their EDSS score (or worsening) compared with

11 % of those who received Rebif�. In CARE II, the

second randomized, phase III clinical trial, alemtuzumab

was compared with Rebif� in patients with RRMS who had

relapsed while on prior therapy. There was a 42 % reduc-

tion in the risk of sustained accumulation of disability in

patients treated with alemtuzumab, accompanied by a

49 % reduction in relapse rate, compared with Rebif� over

2 years.

In a recent open trial, alemtuzumab showed similarly

positive effects in 45 highly active RRMS patients who

experienced two or more relapses during 2 years prior to

admission to the study while receiving IFNb therapy. The

annualized relapse rate was reduced by 94 %, compared

with pre-treatment levels and 86 % of the patients showed

stable or improved scores on the EDSS [210]. The side

effects of alemtuzumab include a high incidence of auto-

immune diseases (22 %), especially autoimmune throm-

bocytopenia and thyroiditis (up to 33 %) [211].

2.2.2.1.2.3 Rituximab/Ocrelizumab. The accumulating

evidence on the involvement of B lymphocytes in the

pathophysiology of MS paved the way for B-cell-directed

therapies [212]. B-cell depletion affects antibody produc-

tion, cytokine networks, and B-cell-mediated antigen pre-

sentation and activation of T cells and macrophages [213].

Rituximab is a genetically engineered, chimeric murine/

human IgG1 monoclonal antibody that targets the CD20

antigen, a transmembrane phosphoprotein expressed only

by pre-B and mature B cells [214]. Rituximab lyses B cells

via complement and antibody-dependent cellular cytotox-

icity [214]. In MS patients, treatment with rituximab

resulted in depletion of CSF B cells after 24 weeks, asso-

ciated also with a reduction in CSF T cells [215].

Rituximab has shown significant efficacy in the sup-

pression of several autoimmune disorders such as SLE and

rheumatoid arthritis.

In a pilot open study, 26 MS patients received two

courses of rituximab 6 months apart, and were followed for

a total 72 weeks. No serious adverse events were noted, the

events limited to mild-to-moderate infusion-associated

reactions. Fewer new gadolinium-enhancing or T2 lesions

were seen starting from week 4 and through week 72.

Although this was not a trial designed to detect clinical

efficacy, there was an apparent reduction in relapses (which

was also observed over the 72 weeks compared with the

year before therapy (0.23 vs 1.27) [216]. An additional trial

included 30 MS patients with a relapse within the past

18 months, despite the use of an injectable disease-modi-

fying agent, and with at least one gadolinium-enhancing

lesion in any of three pretreatment MRIs. The patients were

treated with rituximab 375 mg/m2 weekly for four weeks.

A total 74 % of the post-treatment MRI scans were free of

gadolinium-enhancing lesions compared with 26 % at

baseline. The median number of gadolinium-enhancing

lesions was reduced from 1.0 to 0 and the mean number

from 2.81 per month to 0.33 after treatment (88 % reduc-

tion). The MSFC disability scores improved and the EDSS

remained stable [217].

A total of 104 patients with RRMS were included in the

only randomized, phase II, double-blind, placebo-con-

trolled trial (HERMES). Treatment with rituximab

1,000 mg, given once every 6 months, led to a significant

reduction of 91 % in gadolinium-enhancing lesions seen on

MRI, and in T2-weighted lesion volume, compared with

placebo. Patients in the rituximab group had a lower

annualized rate of relapses, which was significant at

24 weeks (0.37 vs 0.84) but not at 48 weeks (0.37 vs 0.72)

[218]. More patients in the rituximab group than in the

placebo group had adverse events within 24 h after the first

infusion, most of which were mild-to-moderate. After the

second infusion, the number of events was similar in the

two groups.

Rituximab treatment was also evaluated in 439 patients

with PPMS in a 96–122-week trial [219]. The patients

received two 1,000 mg intravenous rituximab or placebo

infusions every 24 weeks, through 96 weeks (4 courses).

The differences between the two groups in terms of time to

confirmed disease progression were not significant;

96-week rates: 38.5 %, placebo; 30.2 %, rituximab). From

baseline to week 96, rituximab patients had a significantly

lower increase in T2 lesion volume, although the brain

volume change was similar to that of the placebo group.

Subgroup analysis showed significant differences in time to

confirmed disease progression in patients aged \51 years

and those with gadolinium-enhancing lesions, compared

with placebo. Adverse events were comparable between

groups. However, serious infections occurred in 4.5 % of

the rituximab and\1.0 % of the placebo patients. Infusion-

related events, predominantly mild-to-moderate, were

more common with rituximab during the first course, and

decreased to rates comparable to placebo on successive

D. Karussis

courses. Additional phase III trials of rituximab are under

way.

Another, totally humanized, anti-CD20 monoclonal

antibody, ocrelizumab (at 2 doses) was also tested recently

[220] in 220 patients with RRMS in a multicenter, ran-

domized, double-blind, placebo-controlled study involving

79 centers in 20 countries. At week 24, the number of

gadolinium-enhancing lesions was 89 % lower in the

600 mg ocrelizumab group than in the placebo group, and

96 % lower in the 2,000 mg group, with similarly pro-

nounced effects in secondary relapse-related outcomes.

2.2.2.1.2.4 Daclizumab and Interleukin-2 Receptor-

Directed Antibodies. IL-2 is an important immune sys-

tem regulator necessary for clone expansion and survival of

activated T-lymphocytes. Its effects are mediated by the

trimer cell surface receptor IL-2a, consisting of the a, b,

and c chains. IL-2a (CD25) is expressed only by the

already-activated T-lymphocytes. Therefore, it is of special

significance for selective immunosuppressive treatment.

Two chimeric mouse/human anti-CD25 antibodies were

produced: basiliximab (Simulect�) and daclizumab (Zen-

apax�). These drugs act by binding the IL-2a receptor’s achain, preventing the IL-2-induced clonal expansion of

activated lymphocytes and shortening their survival. They

are used in the prophylaxis of acute organ rejection after

bilateral kidney transplantation, both being similarly

effective and with only a few side effects.

A pilot study with daclizumab provided some initial

indications of efficacy in reducing gadolinium-enhancing

lesions in nine MS patients [228]. In a randomized, double-

blind, placebo-controlled, phase II trial, subcutaneous

daclizumab was administered weekly or bi-weekly in

addition to IFNb therapy in 230 patients with RRMS. The

combination therapy was associated with a significant

reduction in gadolinium-enhancing lesions [221].

An additional recent study investigated whether dac-

lizumab monotherapy would reduce MRI active lesions in

16 untreated patients with highly active RRMS [222]. A

87.7 % reduction in brain gadolinium-enhancing lesions

and improvements in MSFC were observed in the treated

patients, accompanied by a significant expansion of CD56

(bright) natural killer (NK) cells in the peripheral blood and

CSF, with a resulting decrease in T cells/NK cells and B

cell/NK cell ratios and IL-12p40 in the CSF.

Phase III trials are needed to establish the efficacy and

safety of daclizumab as monotherapy and combination

therapy in patients with MS.

2.2.2.1.2.5 T-cell Receptor Directed Antibodies. Sup-

pression of the pathogenic, myelin-reactive T-lymphocytes

may be achieved by targeting their T-cell receptor mole-

cules. Muromonab-CD3 is a murine anti-CD3 monoclonal

antibody of the IgG2a type that prevents T-cell activation

and proliferation by binding the T-cell receptor complex

present on all differentiated T cells [223, 224]. As such, it

is one of the most potent immunosuppressive drugs and is

administered to control steroid- and/or polyclonal anti-

body-resistant acute organ rejection. As it acts more spe-

cifically than polyclonal antibodies, it is also used

prophylactically in transplantations.

The muromonab mechanism of action is only partially

understood. It is known that the molecule binds the TCR/

CD3 receptor complex. In the first few administrations this

causes a non-specific activation of T cells, leading to a

serious syndrome 30–60 min later. It is characterized by

fever, myalgia, headache, and arthralgia, which may

occasionally develop into a life-threatening reaction of the

cardiovascular system and the CNS. Following this initial

effect, CD3 blocks TCR-antigen binding and causes a

conformational change or the removal of the entire TCR3/

CD3 complex from the T-cell surface. The cross-binding of

CD3 molecules activates an intracellular signal leading to

T-cell anergy or apoptosis, unless the cells receive another

signal through a co-stimulatory molecule. CD3 antibodies

shift the balance from Th1 to Th2 cells [225].

The treated patients may develop neutralizing antibod-

ies, reducing the effectiveness of muromonab-CD3.

Muromonab-CD3 can cause excessive immunosuppres-

sion. Although CD3 antibodies act more specifically than

polyclonal antibodies, they compromise the cell-mediated

immunity significantly, predisposing the patient to oppor-

tunistic infections and malignancies.

An open trial with muromonab-CD3 was conducted in

13 patients with progressive MS and in three with severe

relapse [226]. Side effects were common and severe, and

included hypotension, nausea and vomiting, diarrhea,

fever, and myalgia. No new clinical or MRI activity of MS

was detected during the period of treatment, although many

patients deteriorated transiently in disability scores. At the

conclusion of follow up, only 14 patients had deteriorated

by 1.0 or more points in the EDSS (73 % stabilization

rate). Of those patients who deteriorated, two died of

complications of MS (EDSS 10). Only two patients showed

clinical improvement at the 1-year follow up. The attendant

toxicity of OKT3 makes it unlikely that it will play a major

role in the treatment of MS.

2.2.2.1.2.6 Cytokine-Directed Antibodies. TNFa is a

cytokine with pleiotropic actions, which can appear both as

a transmembrane protein and as a soluble cytokine (sTNF).

Both ligands interact with two different receptors, TNFR1

and TNFR2, which mediate their biological effects. TNFais involved in the pathogenesis of MS [227]. Treatment

with an antibody against TNFa suppresses EAE and was

beneficial in systemic inflammatory diseases in humans.

Immunotherapy of MS

Two rapidly progressive MS patients were treated with

intravenous infusions of a humanized mouse monoclonal

anti-TNFa antibody (cA2) in an open-label phase I safety

trial [228]. The number of gadolinium-enhancing lesions

increased transiently after each treatment in both patients,

and the CSF leukocyte counts and IgG index grew. These

findings suggest that the treatment caused immune activa-

tion and an increase in disease activity, halting the pro-

gramming of further clinical trials in MS.

However, it has been recently recognized that the two

distinct TNFa receptors mediate different effects: TNFR1

causes demyelination and TNFR2 may mediate remyeli-

nation. It may, therefore, be hypothesized that anti-TNFaagents which selectively inhibit sTNF or signals from

TNFR1 could be effective in treating MS [229].

2.2.2.1.2.7 Additional Monoclonal Antibodies. Addi-

tional monoclonal antibodies can be used to block other

specific cell-surface targets or delete particular lymphocyte

groups. Various pilot studies with such novel monoclonal

antibodies are under way.

2.2.2.2 Fingolimod Fingolimod (Gilenya�, Novartis,

Switzerland) modulates sphingosine-1-phosphate (S1P)

receptors and has strong immunoregulatory properties

[230]. In lymphocytes, S1P1 receptor internalization

appears to be a crucial step in their migration process. The

resulting neutralization of the S1P1 pathway does not

reduce lymphocyte activation but inhibits the egress of T

cells and B cells from the lymph nodes. Fingolimod

reduces the number of circulating memory T cells,

including those producing IL-17 cells (Th17 cells) by more

than 90 % [231].

Because fingolimod is lipophilic, it readily enters the

CNS, where it can bind to several S1P receptor subtypes on

different cell types, possibly leading to not well understood

neuroprotective effects. Fingolimod very efficiently sup-

pressed EAE [232]. Pilot clinical trials have indicated that

fingolimod is a highly effective treatment for RRMS. In a

6-month phase II, proof-of-concept randomized trial, 281

patients with active relapsing MS were given a single dose

of placebo or fingolimod 1.25 mg/day or 5.0 mg/day [233].

The numbers of gadolinium-enhancing MRI lesions were

reduced in the fingolimod groups compared with the pla-

cebo group. The relative reduction in ARR was 53 % in the

high-dose fingolimod group and 55 % in the low-dose

group.

The results of two phase III studies in patients with

RRMS (FREEDOMS, a placebo-controlled 24-month trial,

and TRANSFORMS, a 12-month trial with a comparator

IFNb-1a) showed similar to the phase II studies efficacy of

fingolimod over placebo, a clear superiority over IFNb-1a

and an acceptable safety profile. The US Food and Drug

Administration (FDA) approved fingolimod as first-line

treatment for RRMS, whereas the European Medicines

Agency (EMA) restricted its use as a second-line treatment

or a first-line treatment in patients with active disease.

A total of 1,272 patients with relapsing MS participated

in the FREEDOMS study [234] and 1,033 completed the

follow up. Compared with placebo, ARR (the primary

endpoint) was reduced by 60 % in the fingolimod 1.25 mg

group and by 54 % in the 0.5 mg group. Fingolimod also

significantly reduced the cumulative probability of the

3-month confirmed progression, according to the EDSS

(hazard ratio vs placebo 0.68 in the high-dose fingolimod

group and 0.70 in the low-dose fingolimod group). Addi-

tionally, the superiority of both fingolimod doses compared

with placebo was confirmed in all secondary MRI-related

endpoints.

There were no differences in the number of patients with

adverse events in the different study groups. Adverse

events related to fingolimod included bradycardia and

atrioventricular conduction block during the start of fin-

golimod administration, macular edema, elevated liver

enzyme levels, lymphocytopenia, and hypertension.

A total of 1,292 patients with active RRMS were

enrolled in the TRANSFORMS study [235], another phase

III trial. The duration of the core study was 12 months.

Participants were randomly assigned in a double-blind

manner to fingolimod 1.25 mg/day or 0.5 mg/day or to

IFNb-1a once a week. ARR was 0.33 in the IFNb-1a

group, 0.20 in the high-dose fingolimod group, and 0.16 in

the low-dose fingolimod group. Secondary MRI outcome

measures confirmed significant differences in favor of

fingolimod. No differences in the number of adverse events

between study groups were noted. Serious adverse events

and events leading to interruption of treatment, however,

arose most frequently in the high-dose fingolimod group.

Two patients died during treatment with high-dose fingo-

limod—one from a disseminated primary varicella zoster

infection and the other from herpes simplex encephalitis.

The 1-year extension of TRANSFORMS, in 882 par-

ticipants who completed 24 months of follow up, showed

persistent reductions in ARR in patients treated continu-

ously with fingolimod, whereas in those who were initially

given IFNb-1a, the ARR was significantly lower after

switching to fingolimod than in the initial year of the trial

[236].

Fingolimod treatment causes significant lymphopenia

but its effects on circulating lymphocytes are reversible,

with cell counts returning to normal within 4–6 weeks after

cessation of treatment. Safety issues including heart rate

changes, risk of herpes virus dissemination, macular

edema, elevated blood pressure, and the risk of cancer,

should be carefully considered. Long-term safety data are

warranted.

D. Karussis

Further trials, including one in patients with PPMS, are

under way.

2.2.2.3 Teriflunomide Teriflunomide is the active

metabolite of leflunomide [237], which is approved for use

in patients with rheumatoid arthritis. It reduces the activity

of the mitochondrial enzyme dihydroorotate dehydroge-

nase, which is crucial in pyrimidine synthesis. T-lympho-

cyte proliferation largely depends on pyrimidine synthesis.

However, because the drug induces only a small degree of

lymphocytopenia, these processes only partly account for

its effects. The results of a phase II trial of teriflunomide in

patients with relapsing MS showed a reduction in active

lesions on brain MRI scans [238]. The long-term follow-up

of the patients in this phase II study [239] further supported

the beneficial effect of the drug. A total of 147 patients

entered the open-label, 8-year extension phase: 42.2 % of

the patients discontinued the medication (9 % due to

treatment-emergent adverse events [TEAEs]). The most

common TEAEs were mild infections, fatigue, sensory

disturbances, and diarrhea. No serious opportunistic

infections occurred, with no discontinuations due to

infection. The annualized relapse rates remained low and

minimal disability progression was observed, with a dose-

dependent benefit with teriflunomide 14 mg for several

MRI parameters.

In a recent 2-year, randomized, controlled, phase III trial

(TEMSO) in 1,088 patients with active RRMS [240], both

teriflunomide doses (7 mg or 14 mg) significantly lowered

the ARR, with an RRR compared with placebo of 31.2 %

for the lower dose and 31.5 % for the higher dose. At

12 weeks, confirmed EDSS worsening was reduced by

29.8 % with teriflunomide 14 mg. The superiority of the

drug versus placebo was confirmed for a range of MRI

endpoints (39 % reduction in new lesion formation in the

7 mg, and 67 % in the 14 mg group). Both teriflunomide

doses were well tolerated, although diarrhea, nausea, and

liver enzyme elevation were recorded. Teriflunomide has

been suggested to have teratogenic, hepatotoxic, and bone

marrow suppressive effects.

Preliminary results from the TENERE head-to-head trial

revealed no statistical superiority between the Rebif� and

teriflunomide arms (7 mg and 14 mg) on risk of treatment

failure, the primary composite endpoint of the study. The

drug with the trade name Aubagio�, was recently licensed

by the FDA.

2.2.2.4 Laquinimod Laquinimod is a derivative of lino-

mide (Roquinimex�), which effectively suppressed EAE

and had a strong clinical effect in MS [32–34, 241, 242].

However, a phase III trial with linomide had to be termi-

nated because of unforeseen safety concerns [243]. Laqu-

inimod appears to be much better tolerated than linomide.

It seems to mediate its effects through downregulation of

antigen presentation, modulation of the pro-inflammatory

immune responses (induction of a cytokine shift towards

Th2 and Th3 cytokines) and interference with cell traf-

ficking, as well as possibly acting directly in the CNS to

limit demyelination and axonal injury [244]. The results of

two phase II studies showed that laquinimod reduced MRI-

monitored disease activity, based on assessment of the

number of gadolinium-enhancing T1 lesions and new T2

lesions, in patients with RRMS. The results of the first

clinical trial showed a 44 % reduction in the number of

active lesions at weeks 0–24 in patients treated with la-

quinimod 0.3 mg compared with placebo. In the second

study, the 0.6 mg dose resulted in a reduction of 40 % in

the number of gadolinium-enhancing T1 lesions in the last

4-monthly scans compared with placebo, whereas the

0.3 mg dose did not show any evidence of efficacy [245].

A total of 239 (93 %) patients completed an extension of

the phase IIb trial [246]. The number of gadolinium-

enhancing T1 lesions was reduced by 52 % in patients

switching from placebo to either dose of the active drug,

confirming the efficacy results of the core study.

The recently published phase III ALLEGRO study [247]

showed a 23 % reduction in ARR in the laquinimod group.

Additionally, the cumulative probability of 3-month con-

firmed EDSS worsening was reduced by 36 %. Laquini-

mod reduced the mean cumulative number of gadolinium-

enhancing lesions by 37 % and the mean cumulative

number of new T2 lesions by 30 %. Moreover, laquinimod

was associated with a 33 % reduction in the loss of brain

volume over 2 years and proved safe and well tolerated.

The most commonly reported adverse events were gastro-

intestinal side effects and back pain. The incidence of liver

enzyme elevation was higher in laquinimod-treated

patients. However, these elevations were transient,

asymptomatic, and reversible.

In a second phase III study (BRAVO), laquinimod at

0.6 mg was compared with placebo and IFNb-1a in about

1,200 patients with RRMS. The primary endpoint, reduc-

tion in ARR for laquinimod versus placebo, was not sig-

nificant, but secondary endpoints showed indications of

efficacy: a significant reduction in EDSS progression

(33.5 %) and in loss of brain volume (27.5 %).

These studies do support a mild beneficial effect of la-

quinimod in RRMS (probably somehow lower than that

observed with linomide) and may raise the question of

possible increased efficacy with a higher dose of the drug.

2.2.2.5 Dimethyl-fumarate (BG12) BG-12 is an oral

formulation of dimethyl fumarate. Both dimethyl-fumarate

and its primary metabolite, monomethyl-fumarate, induce

activation of the nuclear factor E2-related factor-2 path-

way, which protects against oxidative stress-related

Immunotherapy of MS

neuronal death and damage to myelin in the CNS. Several

neuroprotective and anti-inflammatory mechanisms have

been attributed to the drug, such as the expression of phase

II detoxification enzymes in astroglial and microglial cells

and a drug-induced shift towards a more anti-inflammatory

cytokine profile (induction of Th2-type cytokines and type

II dendritic cells) and adhesion molecule expression [248,

249]. In a pilot study in patients with RRMS, an oral for-

mulation of fumaric acid (Fumaderm�, Biogen Idec),

approved in Germany for the treatment of psoriasis,

reduced the number of gadolinium-enhancing lesions in

brain MRI scans [250].

Subsequently, three doses of BG-12 were tested against

placebo in a phase IIb study in 257 patients with RRMS

[251, 252]. Compared with placebo, BG-12 at 240 mg

three times daily reduced the number of gadolinium-

enhancing, new T2 and T1 lesions from week 12 to 24 by

69 %. A recently concluded phase III trial (DEFINE)

including 1,200 patients showed that BG-12 at 240 mg

twice or three times daily reduced by 49 % the proportion

of patients who relapsed, the ARR by 53 %, the number of

gadolinium-enhancing lesions by 90 %, and of new or

enlarging T2 lesions by 85 % compared with the placebo

group. The cumulative probability of 3-month confirmed

EDSS worsening was 38 % lower. No new significant

safety issues were reported.

2.2.2.6 Vaccines

2.2.2.6.1 T-cell Vaccination. The concept of T-cell vac-

cination (TCV) was first introduced in 1981 in the EAE rat

model, using activated and irradiated MBP-specific T-cell

lines and clones [253]. The rationale was that vaccination

with attenuated myelin-responsive T cells should induce an

immune response against those cells which are probably

the ones responsible for the inflammation and demyelina-

tion in MS. TCV was found to suppress EAE and induced

the production of regulatory T cells (CD4 or CD8) specific

for the anti-myelin T-cell receptors of the vaccine T cells—

anti-idiotypic regulation [254].

There are at least seven different, small, phase II

clinical trials with TCV at various stages of MS already

reported in the literature [255–260] and six additional

studies that are in progress or have been recently com-

pleted, but not yet reported (reviewed by Vandenbark and

Abulafia-Lapid [261]. These studies showed a consistent

reduction in MBP-specific T cells following TCV and

revealed some indications of clinical efficacy, as reflected

by the prolongation of the time to EDSS progression in

both the RRMS and the SPMS patients. In another open-

label TCV study, 20 RRMS patients were vaccinated with

MBP and MOG (panel of peptides) T-cell lines with

similar results [262].

Our group recently performed a double-blind, sham-

controlled clinical trial with TCV in 26 progressive MS

patients, using multiple autologous injections of a mixture

of attenuated T-cell lines reactive to three or more myelin

peptides. This study, which is the first controlled, double-

blind trial with TCV in progressive MS, demonstrated the

feasibility and safety of TCV in MS, and provided signif-

icant indications of clinical efficacy. The changes in EDSS

and relapse rates were significantly better in the TCV

group. Of the 17 patients in the TCV group, 16 (94.3 %)

were relapse-free during the year of study [304].

2.2.2.6.2 T-cell Receptor Vaccination. A similar

approach to TCV is to vaccinate with only the relevant

anti-myelin antigen- responsive TCR and not the whole T

cells. Based on a meta-analysis of 1,000 genes from

myelin-specific T cells from MS subjects, TCR genes/

proteins were identified as candidate epitopes for vacci-

nation. On the basis of successful therapeutic vaccination

in the EAE model, a trivalent TCR vaccine using three

CDR2 peptides emulsified in incomplete Freund’s adjuvant

were developed. Several blinded studies using both single

and multiple TCR peptides demonstrated that TCR vacci-

nation could induce high frequencies of TCR-specific T

cells, along with indications of clinical benefit. TCR-spe-

cific T cells that are expanded by vaccination in vivo may

include a mixture of regulatory cell types, including Th2,

Th3, and Tr1 cells [263].

In a small open trial, a TCR peptide vaccine from the V

beta 5.2 sequence (which is expressed in MS plaques and

on MBP-specific T cells) boosted peptide-reactive T cells

in patients with progressive MS. Vaccine responders had a

reduced MBP response and remained clinically stable

without side effects during 1 year of therapy, whereas

nonresponders had an increased MBP response and pro-

gressed clinically. Peptide-specific Th2 cells directly

inhibited MBP-specific Th1 cells in vitro through the

release of IL-10, implicating a bystander suppression

mechanism that holds promise for treatment of MS and

other autoimmune diseases [264].

2.2.2.6.3 Altered Peptide Vaccination. The concept of

partial activation of T cells by stimulation of TCR with

peptide analogs of antigens evolved in the early 1990s to

that of altered peptide ligands (APLs) [265]. Two phase II

trials using APL vaccination came out simultaneously in

2000. The larger, placebo-controlled, multicenter trial,

using three weekly subcutaneous injections of the altered

peptide NBI-5788 (5, 20, and 50 mg), planned to include

144 patients [266]. The trial was discontinued because of

the appearance of hypersensitivity reactions, mainly at

higher doses of APL. MRI analysis of the patients who

completed the trial showed a reduction of MRI activity at

D. Karussis

the lower (5 mg) dose of APL. Immunologically APL-

reactive T cells of the Th2 phenotype, which were cross-

reactive with the native MBP peptide, were detected. In a

smaller trial using only the higher dose (50 mg) of APL, in

which eventually only eight patients were enrolled, treat-

ment-related exacerbations led to its termination [267].

Considering the group as a whole, there were no clinical or

MRI differences before and after treatment with APL and

the treatment actually led to an increase in APL-reactive

T-cell lines, which were mainly skewed towards a Th1

phenotype.

2.2.2.7 Oral Tolerization with Myelin A unique approach

for specific downregulation of the myelin-reactive lym-

phocytes, which are considered the main responder cells

for the induction of demyelination, is oral tolerization

techniques. Based on animal studies [268, 269] and on the

recognition that oral administration of low doses of antigen

is an effective way of inducing tolerance, via the induction

of regulatory T cells capable of secreting Th2 and Th3

cytokines, trials of oral administration of bovine myelin

were initiated in the early 1990s. Administering whole

myelin had the advantage of circumventing the need to

identify the antigenic target for each particular patient. An

initial small-scale, 1-year, single-center, double-blind trial

with 30 early RRMS patients showed a trend to better

clinical outcomes, a reduction in MBP-reactive T cells in

myelin-treated patients, without any adverse effects [270].

A later study on patients in the continuation phase I/II trial

detected the presence in the peripheral blood of increased

frequencies of MBP and PLP-reactive T-cell lines with a

Th3 regulatory phenotype [271].

However, the phase III trial including 515 RRMS

patients did not confirm the efficacy of this therapy,

although only a single oral dose of myelin (300 mg, con-

taining 8 mg of MBP and 15 mg of PLP) was given, and a

large placebo effect was observed.

A different approach, the administration of synthetic

peptides derived from the sequence of myelin basic protein,

by intrathecal and intravenous routes, was used in an

attempt to tolerize patients. No adverse effects were

reported in two initial phase I trials with MBP peptides

75–95 [272, 273] in chronic progressive MS patients and

tolerance was inferred based on the reduction in CSF anti-

MBP antibodies, only after intravenous administration of

MBP85–96. A larger follow-up study using the MBP82–98

epitope included 56 patients, of which 41 received multiple

intravenous injections of MBP. Of the treated patients, 16

(39 %) failed to achieve long-term suppression of their

antibody levels, whereas 15 (36 %) achieved supression for

up to 1 year [274]. Unfortunately, no clinical or imaging

data were reported in these studies.

Finally, a multicenter study evaluated the safety, toler-

ability, and efficacy of a solubilized complex of DR2:

MBP84–102, which had been shown to reduce the prolif-

erative capacity of anti-MBP human T-cell lines, admin-

istered via three intravenous infusions in 33 SPMS patients

[275].

In the primary outcome measure—safety—there was an

exacerbation of MS in the low dose (2.0 mg/kg) group. All

clinical and MRI measures of efficacy did not show any

differences between the treatment and the placebo groups.

There was also no convincing evidence of reduced reac-

tivity to MBP or MOG peptides.

2.2.2.8 Stem Cells

2.2.2.8.1 Hematopoietic Stem Cell Transplanta-

tion. Hematopoietic stem cell transplantation (HSCT) for

MS (and autoimmune diseases in general) is based on the

principle of radical suppression of the immune system to

abrogate the inflammatory pathogenetic process in MS and

subsequently reset the immune system from scratch. The

autologous setting is usually used, where the immune

system is reconstituted by the patient’s own stem cells. The

rationale for this type of therapeutic approach in MS

derives from pivotal animal studies performed back in the

early 1990s [276, 277]. Open, uncontrolled clinical trials

have shown impressive effects on the suppression of

inflammation, induction of remission, and stabilization of

MS [278–281]. Worldwide, more than 500 patients with

MS have been treated with HSCT (reviewed by Karussis

and Vaknin-Dembinsky [282]). The different conditioning

protocols used in these studies complicate the uniform

interpretation of the data. In general, over the last 15 years,

more than 1,500 patients have received HSCT, mostly

autologous, for treatment of severe autoimmune diseases

(MS, systemic sclerosis, SLE, rheumatoid arthritis, juvenile

idiopathic arthritis and idiopathic cytopenic purpura). In

MS, an overall 85 % 5-year survival rate and 43 % pro-

gression-free survival have been recorded, with mean

100-day transplantation-related mortality ranging from 1 %

to 5 %. About 30 % of the patients had a complete

response, often durable despite full immune reconstitution.

2.2.2.8.2 Other Stem Cells. Pivotal studies with neuronal

stem cells have shown a significant beneficial clinical

effect in mice with EAE (the animal model of MS) [283–

285]. Subsequently, embryonic and other types of adult

stem cells, especially mesenchymal stem cells, were tested

in various models of EAE [286–288]. MSC injection, either

intravenous or into the CSF, strongly suppressed the clin-

ical and pathological signs of EAE and reversed disability.

Most importantly, remyelination was evident in these

Immunotherapy of MS

MSC-treated animals, accompanied by impressive neuro-

protection [288].

The additional scientific rationale for using MSC in EAE

and MS derives from the reported strong immunomodula-

tory effects of these cells [289–291]. Phase I/II safety

studies with MSC or bone marrow-derived cells have been

performed in MS [282, 292]. Overall, MSCs given intra-

venously or intrathecally were well tolerated, with some

preliminary evidence of efficacy [282]. In the latter study,

from our group, MSC were labeled with paramagnetic iron

particles and hypointense signals were detected in the cer-

vical spine MRI, providing some indication of the migration

of the injected cells into the CNS parenchyma. In addition,

in vivo immunomodulatory effects were shown to be

induced in these MSC-injected patients [282].

A more recent phase IIa study [293] in ten patients with

secondary progressive MS showed an improvement in

visual acuity and visual evoked response latency,

accompanied by an increase in optic nerve area, following

intravenous transplantation of autologous MSC. Although

no significant effects on other visual parameters, retinal

nerve fiber layer thickness, or optic nerve magnetization

transfer ratio were observed, this study provides a strong

indication of induction of tissue repair with MSC trans-

plantation in humans.

3 A Suggested Algorithm for MS Immunotherapy

MS is not an homogenous disease and distinct types of

immune pathogenesis seem to be involved in different

subgroups [12]. Its course and prognosis greatly varies

among patients. It would be therefore too pretentious to

suggest a common treatment algorithm that could fit all the

patients with MS. The use of neuroimaging markers (such

as the type, the number and activity of the lesions in MRI at

CIS

Highly active disease in MRI

McDonald criteria +Clinical-MRI

follow up every 3-6 m

McDonald criteria -

RIS

BIOMARKERS (OCB, anti-glycans etc)

Low activity in MRI

CDMS

Glatiramer acetate

High-dose IFNβ

Low-dose IFNβ

natalizumab, fingolimod, teriflunomide, light immunosuppressants (azathioprine, lowdose methotrexate,mycophenolate), chronic steroids,

plasmapheresis

Sub-optimal response or side

effects

Sub-optimal response or side effects

Sub-optimal response or side-effects or JCV abs

positivity

FIRST-LINE IMMUNOTHERAPIES

SECOND-LINE IMMUNOTHERAPIES

More aggressive immunosuppression: mitoxantrone, cyclophosphamide, other monoclonal antibodies (anti-

CD20, anti-CD52)

THIRD-LINE IMMUNOTHERAPIES

Experimental modalities/Clinical trials : TCV, laquinimod, HSCT, stem cells, etc

First line immunotherapies plus immunosuppressants

or steroids

COMBINATION IMMUNOTHERAPY

RESCUE IMMUNOTHERAPY

Sub-optimal response or side

effects

natalizumab, mitoxanthrone, steroid pulses, plasmapheresis, cyclophosphamide,

alemtuzumab, rituximab

MAINTENANCEIMMUNOTHERAPY

Active disease-fast prgression

Slowly progressive disease

azathioprine, steroid pulses, plasmapheresis, natalizumab

alemtuzumab, rituximab

Progressive MS

Highly active disease-with

relapses

Low activity

with relapses

Fig. 3 A suggested treatment algorithm for multiple sclerosis (MS)

immunotherapy. CDMS clinically definite MS, CIS clinically isolated

syndrome, HSCT hematopoietic stem cell transplantation, IFNbinterferon beta, JCV John Cunningham virus, MMF mycofenolate

mofenil, MTX methotrexate, NMO neuromyelitis optica, OCBoligoclonal antibodies, RIS radiologically isolated syndrome, TCVT-cell vaccination

D. Karussis

the early stages of MS) [294–296] and immunological bio-

markers (such as the presence of oligoclonal antibodies in the

CSF and anti-myelin antibodies and anti-glycan antibodies in

the serum, at the first stages of MS) [297–300] would help to

tailor/personalize immunotherapy for each individual case.

For instance, patients with type II MS immunopathogenesis,

i.e. more prominently antibody-mediated disease, or with the

neuromyelitic variants of demyelination (NMO spectrum of

demyelination, associated with anti-aquaporin antibodies),

may not only not respond well but even deteriorate under

some of the first-line treatments (such as IFNs). The unique

spinal cord extensive lesions in the NMO spectrum of

demyelinating diseases may also help to distinguish these

cases from classical MS. It would be therefore crucial to use

immunological and imaging biomarkers, not only to correctly

diagnose these MS variants, but also to identify the patients

with CIS or RIS who, according to their MRI findings, have

greater risks for disability progression (worse prognosis) or,

on the contrary, to predict a benign MS course. This will allow

the very early introduction of immunotherapy only in the

patients where such an early intervention is justified.

A summary of the existing knowledge from published and

ongoing trials and suggestions of some common treatment

recommendations is presented in Fig. 3. This ‘algorithm’

with all the above-mentioned reservations is also based on

previously published recommendations formulated by

expert forums (some of which the author of this review has

participated in) [301], and in which a suboptimal response to

therapies was defined and a treatment-escalation model for

the management of patients with such insufficient response

was suggested [301, 302]. This model is based on the prin-

ciple of starting with a ‘first-line’ treatment (IFNb or GA)

and then, if the response is not sufficient, escalate first to a

higher dose of IFN, then to natalizumab or fingolimod, and/

or to immunosuppressive medications. The suggested algo-

rithm incorporates an additional option for the severe MS

cases, that of short-term aggressive immunotherapy (cyclo-

phosphamide, mitoxanthrone, natalizumab, alemtuzumab,

rituximab, HSCT) followed by maintenance treatment with

the regular modalities as in the previous scheme. As already

mentioned, early treatment of CIS and RIS should be based

on identification of the patients with poor prognosis. Con-

cerning progressive MS, indications from the IFN (and

other) studies support the conception of dividing the pro-

gressive MS patients into those with active inflammatory

disease (manifested either by clinical relapses or by pro-

gression in disability or active lesions in MRI) and those with

no active inflammation (chronic, purely degenerative phase,

usually secondary to the inflammatory one; ‘burnt-out dis-

ease’). In the first group, it seems that immunotherapy

strategies may be more effective and in general should target

the putative compartmentalized inflammation, and espe-

cially B cells.

4 Conclusions

Until 1993, no specific immunotherapy was registered for

MS. Since the introduction of IFNb, huge steps have been

made and increasingly more specific and efficious (Fig. 2)

modalities have been introduced and registered. However,

along with the seemingly increasing efficacy of the newly

introduced and more targeted immunotherapies, more

safety issues have arisen. The cumulative experience

obtained from the numerous clinical trials in MS with

various types of immunotherapies allows one to draw the

conclusion that inflammation, most probably initiated in

the peripheral immune system, is crucial for the formation

of new demyelinating lesions and, with time, appears to be

the main cause for the resulting axonal damage and neu-

ronal tissue atrophy. Immunological therapies, ideally

applied as early as possible, might, therefore, prevent

progression of disability if given before the cascade of

events leading to irreversible tissue loss. The reported

lower efficacy of most of the known immunotherapies in

patients with progressive forms of MS might be explained

by the possibility that inflammation is less pronounced or

only compartmentalized in the CNS at these stages of the

disease. More effective modalities that exert strong local

immunomodulation in the CNS might be more beneficial

for progressive MS. The additional goals of future MS

therapy should include neuroprotective modalities and

techniques that may enhance neuroregeneration and re-

myelination. High expectations of this direction are

focused on stem cell-related treatments, but these have to

be substantiated in future controlled trials.

Disclosures Prof. Karussis has received honoraria from Biogen,

Teva, Serono, Novartis and Bayer for lectures. He has no conflicts of

interest that are directly relevant to the content of this article.

References

1. Compston A, Coles A. Multiple sclerosis. Lancet. 2008;

372(9648):1502–17.

2. Frohman EM, Racke MK, Raine CS. Multiple sclerosis: the

plaque and its pathogenesis. N Engl J Med. 2006;354(9):942–55.

3. Steinman L. Multiple sclerosis: a two-stage disease. Nat

Immunol. 2001;2(9):762–4.

4. Trapp BD, Peterson J, Ransohoff RM, Rudick R, Mork S, Bo L.

Axonal transection in the lesions of multiple sclerosis. N Engl J

Med. 1998;338(5):278–85.

5. Zhang J, Markovic-Plese S, Lacet B, Raus J, Weiner HL, Hafler

DA. Increased frequency of interleukin 2-responsive T cells

specific for myelin basic protein and proteolipid protein in

peripheral blood and cerebrospinal fluid of patients with multi-

ple sclerosis. J Exp Med. 1994;179(3):973–84.

6. Allegretta M, Nicklas JA, Sriram S, Albertini RJ. T cells

responsive to myelin basic protein in patients with multiple

sclerosis. Science. 1990;247(4943):718–21.

7. Wucherpfennig KW, Hafler DA. A review of T-cell receptors in

multiple sclerosis: clonal expansion and persistence of human

Immunotherapy of MS

T-cells specific for an immunodominant myelin basic protein

peptide. Ann N Y Acad Sci. 1995;756:241–58.

8. Venken K, Hellings N, Hensen K, Rummens JL, Stinissen P.

Memory CD4 ? CD127high T cells from patients with multiple

sclerosis produce IL-17 in response to myelin antigens. J Neu-

roimmunol. 2010;226(1–2):185–91.

9. Tesmer LA, Lundy SK, Sarkar S, Fox DA. Th17 cells in human

disease. Immunol Rev. 2008;223:87–113.

10. Korn T, Bettelli E, Oukka M, Kuchroo VK. IL-17 and Th17

Cells. Annu Rev Immunol. 2009;27:485–517.

11. Kebir H, Kreymborg K, Ifergan I, Dodelet-Devillers A, Cayrol

R, Bernard M, et al. Human TH17 lymphocytes promote blood-

brain barrier disruption and central nervous system inflamma-

tion. Nat Med. 2007;13(10):1173–5.

12. Lucchinetti C, Bruck W, Parisi J, Scheithauer B, Rodriguez M,

Lassmann H. Heterogeneity of multiple sclerosis lesions:

implications for the pathogenesis of demyelination. Ann Neurol.

2000;47(6):707–17.

13. Sharief MK, Noori MA, Ciardi M, Cirelli A, Thompson EJ.

Increased levels of circulating ICAM-1 in serum and cerebro-

spinal fluid of patients with active multiple sclerosis: correlation

with TNF-alpha and blood–brain barrier damage. J Neuroim-

munol. 1993;43(1–2):15–21.

14. Sharief MK, Thompson EJ. Correlation of interleukin-2 and

soluble interleukin-2 receptor with clinical activity of multiple

sclerosis. J Neurol Neurosurg Psychiatry. 1993;56(2):169–74.

15. Tsukada N, Matsuda M, Miyagi K, Yanagisawa N. Increased

levels of intercellular adhesion molecule-1 (ICAM-1) and tumor

necrosis factor receptor in the cerebrospinal fluid of patients

with multiple sclerosis. Neurology. 1993;43(12):2679–82.

16. Tsukada N, Miyagi K, Matsuda M, Yanagisawa N. Increased

levels of circulating intercellular adhesion molecule-1 in mul-

tiple sclerosis and human T-lymphotropic virus type I-associated

myelopathy. Ann Neurol. 1993;33(6):646–9.

17. Tsukada N, Matsuda M, Miyagi K, Yanagisawa N. Adhesion of

cerebral endothelial cells to lymphocytes from patients with

multiple sclerosis. Autoimmunity. 1993;14(4):329–33.

18. Washington R, Burton J, Todd RF 3rd, Newman W, Dragovic L,

Dore-Duffy P. Expression of immunologically relevant endo-

thelial cell activation antigens on isolated central nervous sys-

tem microvessels from patients with multiple sclerosis. Ann

Neurol. 1994;35(1):89–97.

19. Yednock TA, Cannon C, Fritz LC, Sanchez-Madrid F, Steinman

L, Karin N. Prevention of experimental autoimmune encepha-

lomyelitis by antibodies against alpha 4 beta 1 integrin. Nature.

1992;356(6364):63–6.

20. Polman CH, O’Connor PW, Havrdova E, Hutchinson M,

Kappos L, Miller DH, et al. A randomized, placebo-controlled

trial of natalizumab for relapsing multiple sclerosis. N Engl J

Med. 2006;354(9):899–910.

21. Hawker K. B-cell-targeted treatment for multiple sclerosis:

mechanism of action and clinical data. Curr Opin Neurol.

2008;21(Suppl. 1):S19–25.

22. Zouali M. B lymphocytes: chief players and therapeutic targets

in autoimmune diseases. Front Biosci. 2008;13:4852–61.

23. Cepok S, Rosche B, Grummel V, Vogel F, Zhou D, Sayn J, et al.

Short-lived plasma blasts are the main B cell effector subset during

the course of multiple sclerosis. Brain. 2005;128(Pt 7):1667–76.

24. Owens GP, Bennett JL, Gilden DH, Burgoon MP. The B cell

response in multiple sclerosis. Neurol Res. 2006;28(3):236–44.

25. Prineas JW, Graham JS. Multiple sclerosis: capping of surface

immunoglobulin G on macrophages engaged in myelin break-

down. Ann Neurol. 1981;10(2):149–58.

26. Archelos JJ, Storch MK, Hartung HP. The role of B cells and

autoantibodies in multiple sclerosis. Ann Neurol. 2000;47(6):

694–706.

27. Serafini B, Rosicarelli B, Magliozzi R, Stigliano E, Aloisi F.

Detection of ectopic B-cell follicles with germinal centers in the

meninges of patients with secondary progressive multiple scle-

rosis. Brain Pathol. 2004;14(2):164–74.

28. Krumbholz M, Theil D, Cepok S, Hemmer B, Kivisakk P,

Ransohoff RM, et al. Chemokines in multiple sclerosis:

CXCL12 and CXCL13 up-regulation is differentially linked to

CNS immune cell recruitment. Brain. 2006;129(Pt 1):200–11.

29. Magliozzi R, Howell O, Vora A, Serafini B, Nicholas R, Puo-

polo M, et al. Meningeal B-cell follicles in secondary progres-

sive multiple sclerosis associate with early onset of disease and

severe cortical pathology. Brain. 2007;130(Pt 4):1089–104.

30. Hemmer B, Hartung HP. Toward the development of rational

therapies in multiple sclerosis: what is on the horizon? Ann

Neurol. 2007;62(4):314–26.

31. Weinshenker BG, O’Brien PC, Petterson TM, Noseworthy JH,

Lucchinetti CF, Dodick DW, et al. A randomized trial of plasma

exchange in acute central nervous system inflammatory demy-

elinating disease. Ann Neurol. 1999;46(6):878–86.

32. Abramsky O, Lehmann D, Karussis D. Immunomodulation with

linomide: possible novel therapy for multiple sclerosis. Mult

Scler. 1996;2(4):206–10.

33. Karussis DM, Meiner Z, Lehmann D, Gomori JM, Schwarz A,

Linde A, et al. Treatment of secondary progressive multiple

sclerosis with the immunomodulator linomide: a double-blind,

placebo-controlled pilot study with monthly magnetic resonance

imaging evaluation. Neurology. 1996;47(2):341–6.

34. Karussis DM, Lehmann D, Slavin S, Vourka-Karussis U,

Mizrachi-Koll R, Ovadia H, et al. Inhibition of acute, experi-

mental autoimmune encephalomyelitis by the synthetic immu-

nomodulator linomide. Ann Neurol. 1993;34(5):654–60.

35. McDonald WI, Compston A, Edan G, Goodkin D, Hartung HP,

Lublin FD, et al. Recommended diagnostic criteria for multiple

sclerosis: guidelines from the International Panel on the diag-

nosis of multiple sclerosis. Ann Neurol. 2001;50(1):121–7.

36. Rose AS, Kuzma JW, Kurtzke JF, Namerow NS, Sibley WA,

Tourtellotte WW. Cooperative study in the evaluation of therapy

in multiple sclerosis. ACTH vs. placebo: final report. Neurology.

1970;20(5):1–59.

37. Abbruzzese JL, Rocco LE, Laskin OL, Skubitz KM, McGaug-

hey MD, Lipsky JJ. Prospective randomized double-blind

comparison of moxalactam and tobramycin in treatment of

urinary tract infections. Am J Med. 1983;74(4):694–9.

38. Barkhof F, Hommes OR, Scheltens P, Valk J. Quantitative MRI

changes in gadolinium-DTPA enhancement after high-dose

intravenous methylprednisolone in multiple sclerosis. Neurol-

ogy. 1991;41(8):1219–22.

39. Beck RW, Cleary PA, Anderson MM Jr. Keltner JL, Shults WT,

Kaufman DI, et al. A randomized, controlled trial of cortico-

steroids in the treatment of acute optic neuritis. The Optic

Neuritis Study Group. N Engl J Med. 1992;326(9):581–8.

40. Miller DH, Barkhof F, Berry I, Kappos L, Scotti G, Thompson

AJ. Magnetic resonance imaging in monitoring the treatment of

multiple sclerosis: concerted action guidelines. J Neurol Neu-

rosurg Psychiatry. 1991;54(8):683–8.

41. Miller DH, Thompson AJ, Morrissey SP, MacManus DG,

Moore SG, Kendall BE, et al. High dose steroids in acute

relapses of multiple sclerosis: MRI evidence for a possible

mechanism of therapeutic effect. J Neurol Neurosurg Psychiatry.

1992;55(6):450–3.

42. Kesselring J, Miller DH, MacManus DG, Johnson G, Milligan

NM, Scolding N, et al. Quantitative magnetic resonance imaging in

multiple sclerosis: the effect of high dose intravenous methyl-

prednisolone. J Neurol Neurosurg Psychiatry. 1989;52(1):14–7.

43. Barnes MP, Bateman DE, Cleland PG, Dick DJ, Walls TJ,

Newman PK, et al. Intravenous methylprednisolone for multiple

D. Karussis

sclerosis in relapse. J Neurol Neurosurg Psychiatry. 1985;48(2):

157–9.

44. Thompson AJ, Kennard C, Swash M, Summers B, Yuill GM,

Shepherd DI, et al. Relative efficacy of intravenous methyl-

prednisolone and ACTH in the treatment of acute relapse in MS.

Neurology. 1989;39(7):969–71.

45. Beck RW, Cleary PA, Trobe JD, Kaufman DI, Kupersmith MJ,

Paty DW, et al. The effect of corticosteroids for acute optic

neuritis on the subsequent development of multiple sclerosis.

The Optic Neuritis Study Group. N Engl J Med. 1993;329(24):

1764–9.

46. Filippini G, Brusaferri F, Sibley WA, Citterio A, Ciucci G,

Midgard R, et al. Corticosteroids or ACTH for acute exacer-

bations in multiple sclerosis. Cochrane Database Syst Rev.

2000;(4):CD001331.

47. Milligan NM, Newcombe R, Compston DA. A double-blind

controlled trial of high dose methylprednisolone in patients with

multiple sclerosis: 1. Clinical effects. J Neurol Neurosurg Psy-

chiatry. 1987;50(5):511–6.

48. Cazzato G, Mesiano T, Antonello R, Monti F, Carraro N, Torre

P, et al. Double-blind, placebo-controlled, randomized, cross-

over trial of high-dose methylprednisolone in patients with

chronic progressive form of multiple sclerosis. Eur Neurol.

1995;35(4):193–8.

49. Zivadinov R, Rudick RA, De Masi R, Nasuelli D, Ukmar M,

Pozzi-Mucelli RS, et al. Effects of IV methylprednisolone on brain

atrophy in relapsing-remitting MS. Neurology. 2001;57(7):1239–47.

50. Alam SM, Kyriakides T, Lawden M, Newman PK. Methyl-

prednisolone in multiple sclerosis: a comparison of oral with

intravenous therapy at equivalent high dose. J Neurol Neurosurg

Psychiatry. 1993;56(11):1219–20.

51. Sellebjerg F, Frederiksen JL, Nielsen PM, Olesen J. Double-

blind, randomized, placebo-controlled study of oral, high-dose

methylprednisolone in attacks of MS. Neurology. 1998;51(2):

529–34.

52. Martinelli V, Rocca MA, Annovazzi P, Pulizzi A, Rodegher M.

Martinelli Boneschi F, et al. A short-term randomized MRI

study of high-dose oral vs intravenous methylprednisolone in

MS. Neurology. 2009;73(22):1842–8.

53. Clark WF, Rock GA, Buskard N, Shumak KH, LeBlond P,

Anderson D, et al. Therapeutic plasma exchange: an update from

the Canadian Apheresis Group. Ann Intern Med. 1999;131(6):

453–62.

54. Lehmann HC, Hartung HP, Hetzel GR, Stuve O, Kieseier BC.

Plasma exchange in neuroimmunological disorders: Part 2.

Treatment of neuromuscular disorders. Arch Neurol. 2006;63(8):

1066–71.

55. Schroder A, Linker RA, Gold R. Plasmapheresis for neurolog-

ical disorders. Expert Rev Neurother. 2009;9(9):1331–9.

56. Gordon PA, Carroll DJ, Etches WS, Jeffrey V, Marsh L, Morrice

BL, et al. A double-blind controlled pilot study of plasma

exchange versus sham apheresis in chronic progressive multiple

sclerosis. Can J Neurol Sci. 1985;12(1):39–44.

57. Khatri BO, McQuillen MP, Harrington GJ, Schmoll D,

Hoffmann RG. Chronic progressive multiple sclerosis: double-

blind controlled study of plasmapheresis in patients taking

immunosuppressive drugs. Neurology. 1985;35(3):312–9.

58. Weiner HL, Dau PC, Khatri BO, Petajan JH, Birnbaum G,

McQuillen MP, et al. Double-blind study of true vs. sham

plasma exchange in patients treated with immunosuppression for

acute attacks of multiple sclerosis. Neurology. 1989;39(9):

1143–9.

59. The Canadian Cooperative Multiple Sclerosis Study Group. The

Canadian cooperative trial of cyclophosphamide and plasma

exchange in progressive multiple sclerosis. Lancet. 1991;337(8739):

441–6.

60. Keegan M, Konig F, McClelland R, Bruck W, Morales Y,

Bitsch A, et al. Relation between humoral pathological changes

in multiple sclerosis and response to therapeutic plasma

exchange. Lancet. 2005;366(9485):579–82.

61. Magana SM, Keegan BM, Weinshenker BG, Erickson BJ, Pit-

tock SJ, Lennon VA, et al. Beneficial plasma exchange response

in central nervous system inflammatory demyelination. Arch

Neurol. 2011;68(7):870–8.

62. Wang KC, Wang SJ, Lee CL, Chen SY, Tsai CP. The rescue

effect of plasma exchange for neuromyelitis optica. J Clin

Neurosci. 2011;18(1):43–6.

63. Watanabe S, Nakashima I, Misu T, Miyazawa I, Shiga Y,

Fujihara K, et al. Therapeutic efficacy of plasma exchange in

NMO-IgG-positive patients with neuromyelitis optica. Mult

Scler. 2007;13(1):128–32.

64. Bonnan M, Valentino R, Olindo S, Mehdaoui H, Smadja D,

Cabre P. Plasma exchange in severe spinal attacks associated

with neuromyelitis optica spectrum disorder. Mult Scler.

2009;15(4):487–92.

65. Sorensen PS, Wanscher B, Szpirt W, Jensen CV, Ravnborg M,

Christiansen P, et al. Plasma exchange combined with azathio-

prine in multiple sclerosis using serial gadolinium-enhanced

MRI to monitor disease activity: a randomized single-masked

cross-over pilot study. Neurology. 1996;46(6):1620–5.

66. Cortese I, Chaudhry V, So YT, Cantor F, Cornblath DR,

Rae-Grant A. Evidence-based guideline update: plasmapheresis

in neurologic disorders. Report of the therapeutics and tech-

nology assessment Subcommittee of the American Academy of

Neurology. Neurology. 2011;76(3):294–300.

67. Panitch HS, Hirsch RL, Haley AS, Johnson KP. Exacerbations

of multiple sclerosis in patients treated with gamma interferon.

Lancet. 1987;1(8538):893–5.

68. Jacobs L, O’Malley J, Freeman A, Ekes R. Intrathecal interferon

reduces exacerbations of multiple sclerosis. Science.

1981;214(4524):1026–8.

69. Panitch HS. Systemic alpha-interferon in multiple sclerosis.

Long-term patient follow-up. Arch Neurol. 1987;44(1):61–3.

70. Abreu SL. Suppression of experimental allergic encephalomy-

elitis by interferon. Immunol Commun. 1982;11(1):1–7.

71. Abreu SL. Interferon in experimental autoimmune encephalo-

myelitis (EAE): effects of exogeneous interferon on the antigen-

enhanced adoptive transfer of EAE. Int Arch Allergy Appl

Immunol. 1985;76(4):302–7.

72. Rudick RA, Carpenter CS, Cookfair DL, Tuohy VK, Ransohoff

RM. In vitro and in vivo inhibition of mitogen-driven T-cell

activation by recombinant interferon beta. Neurology.

1993;43(10):2080–7.

73. Noronha A, Toscas A, Jensen MA. Interferon beta augments

suppressor cell function in multiple sclerosis. Ann Neurol.

1990;27(2):207–10.

74. Ling PD, Warren MK, Vogel SN. Antagonistic effect of inter-

feron-beta on the interferon-gamma-induced expression of Ia

antigen in murine macrophages. J Immunol. 1985;135(3):

1857–63.

75. Porrini AM, Gambi D, Reder AT. Interferon effects on inter-

leukin-10 secretion. Mononuclear cell response to interleukin-10

is normal in multiple sclerosis patients. J Neuroimmunol.

1995;61(1):27–34.

76. Dhib-Jalbut S, Marks S. Interferon-beta mechanisms of action in

multiple sclerosis. Neurology. 2010;74(Suppl. 1):S17–24.77. Interferon beta-1b is effective in relapsing-remitting multiple

sclerosis. I. Clinical results of a multicenter, randomized, dou-

ble-blind, placebo-controlled trial. The IFNB Multiple Sclerosis

Study Group. Neurology. 1993;43(4):655–61.

78. Paty DW, Li DK. Interferon beta-1b is effective in relapsing-

remitting multiple sclerosis. II. MRI analysis results of a

Immunotherapy of MS

multicenter, randomized, double-blind, placebo-controlled trial.

UBC MS/MRI Study Group and the IFNB Multiple Sclerosis

Study Group. Neurology. 1993;43(4):662–7.

79. Interferon beta-1b in the treatment of multiple sclerosis: final

outcome of the randomized controlled trial. The IFNB Multiple

Sclerosis Study Group and The University of British Columbia

MS/MRI Analysis Group. Neurology. 1995;45(7):1277–85.

80. Jacobs LD, Cookfair DL, Rudick RA, Herndon RM, Richert JR,

Salazar AM, et al. Intramuscular interferon beta-1a for disease

progression in relapsing multiple sclerosis. The Multiple Scle-

rosis Collaborative Research Group (MSCRG). Ann Neurol.

1996;39(3):285–94.

81. Evidence of interferon beta-1a dose response in relapsing-

remitting MS: the OWIMS Study. The Once Weekly Interferon

for MS Study Group. Neurology. 1999;53(4):679–86.

82. Randomised double-blind placebo-controlled study of interferon

beta-1a in relapsing/remitting multiple sclerosis. PRISMS

(Prevention of Relapses and Disability by Interferon beta-1a

Subcutaneously in Multiple Sclerosis) Study Group. Lancet.

1998;352(9139):1498–504.

83. Li DK, Paty DW. Magnetic resonance imaging results of the

PRISMS trial: a randomized, double-blind, placebo-controlled

study of interferon-beta1a in relapsing-remitting multiple scle-

rosis. Prevention of relapses and disability by interferon-beta1a

subcutaneously in multiple sclerosis. Ann Neurol. 1999;46(2):

197–206.

84. Freedman MS. Long-term follow-up of clinical trials of multiple

sclerosis therapies. Neurology. 2011;76(1 Suppl. 1):S26–34.

85. PRISMS Study Group and the University of British Columbia

MS/MRI Analysis Group. PRISMS-4: long-term efficacy of

interferon-beta-1a in relapsing MS. Neurology. 2001;56(12):

1628–36.

86. Ebers GC, Traboulsee A, Li D, Langdon D, Reder AT, Goodin

DS, et al. Analysis of clinical outcomes according to original

treatment groups 16 years after the pivotal IFNB-1b trial.

J Neurol Neurosurg Psychiatry. 2010;81(8):907–12.

87. Jacobs LD, Beck RW, Simon JH, Kinkel RP, Brownscheidle

CM, Murray TJ, et al. Intramuscular interferon beta-1a therapy

initiated during a first demyelinating event in multiple sclerosis.

CHAMPS Study Group. N Engl J Med. 2000;343(13):898–904.

88. Filippi M, Rovaris M, Inglese M, Barkhof F, De Stefano N,

Smith S, et al. Interferon beta-1a for brain tissue loss in patients

at presentation with syndromes suggestive of multiple sclerosis:

a randomised, double-blind, placebo-controlled trial. Lancet.

2004;364(9444):1489–96.

89. Kappos L, Polman CH, Freedman MS, Edan G, Hartung HP,

Miller DH, et al. Treatment with interferon beta-1b delays

conversion to clinically definite and McDonald MS in patients

with clinically isolated syndromes. Neurology. 2006;67(7):

1242–9.

90. Kinkel RP, Kollman C, O’Connor P, Murray TJ, Simon J,

Arnold D, et al. IM interferon beta-1a delays definite multiple

sclerosis 5 years after a first demyelinating event. Neurology.

2006;66(5):678–84.

91. Kappos L, Freedman MS, Polman CH, Edan G, Hartung HP,

Miller DH, et al. Effect of early versus delayed interferon beta-

1b treatment on disability after a first clinical event suggestive of

multiple sclerosis: a 3-year follow-up analysis of the BENEFIT

study. Lancet. 2007;370(9585):389–97.

92. Kappos L, Freedman MS, Polman CH, Edan G, Hartung HP,

Miller DH, et al. Long-term effect of early treatment with

interferon beta-1b after a first clinical event suggestive of mul-

tiple sclerosis: 5-year active treatment extension of the phase 3

BENEFIT trial. Lancet Neurol. 2009;8(11):987–97.

93. Comi G, De Stefano N, Freedman MS, Barkhof F, Polman CH,

Uitdehaag BM, et al. Comparison of two dosing frequencies of

subcutaneous interferon beta-1a in patients with a first clinical

demyelinating event suggestive of multiple sclerosis (REFLEX):

a phase 3 randomised controlled trial. Lancet Neurol.

2012;11(1):33–41.

94. Rice GP, Incorvaia B, Munari L, Ebers G, Polman C, D’Amico

R, et al. Interferon in relapsing-remitting multiple sclerosis.

Cochrane Database Syst Rev. 2001;(4):CD002002.

95. Placebo-controlled multicentre randomised trial of interferon

beta-1b in treatment of secondary progressive multiple sclerosis.

European Study Group on interferon beta-1b in secondary pro-

gressive MS. Lancet. 1998;352(9139):1491–7.

96. Miller DH, Molyneux PD, Barker GJ, MacManus DG, Moseley

IF, Wagner K. Effect of interferon-beta1b on magnetic reso-

nance imaging outcomes in secondary progressive multiple

sclerosis: results of a European multicenter, randomized, dou-

ble-blind, placebo-controlled trial. European Study Group on

Interferon-beta1b in secondary progressive multiple sclerosis.

Ann Neurol. 1999;46(6):850–9.

97. Panitch H, Miller A, Paty D, Weinshenker B. Interferon beta-1b

in secondary progressive MS: results from a 3-year controlled

study. Neurology. 2004;63(10):1788–95.

98. Kappos L, Weinshenker B, Pozzilli C, Thompson AJ, Dahlke F,

Beckmann K, et al. Interferon beta-1b in secondary progressive

MS: a combined analysis of the two trials. Neurology. 2004;63(10):

1779–87.

99. Randomized controlled trial of interferon- beta-1a in secondary

progressive MS: clinical results. Neurology. 2001;56(11):

1496–504.

100. Cohen JA, Cutter GR, Fischer JS, Goodman AD, Heidenreich

FR, Kooijmans MF, et al. Benefit of interferon beta-1a on MSFC

progression in secondary progressive MS. Neurology. 2002;59(5):

679–87.

101. Rojas JI, Romano M, Ciapponi A, Patrucco L, Cristiano E.

Interferon beta for primary progressive multiple sclerosis.

Cochrane Database Syst Rev. 2010;(1):CD006643.

102. Leary SM, Thompson AJ. Interferon beta-1a in primary pro-

gressive multiple sclerosis. J Neurol Sci. 2003;206(2):215–6.

103. Montalban X, Sastre-Garriga J, Tintore M, Brieva L, Aymerich

FX, Rio J, et al. A single-center, randomized, double-blind,

placebo-controlled study of interferon beta-1b on primary pro-

gressive and transitional multiple sclerosis. Mult Scler.

2009;15(10):1195–205.

104. Tur C, Montalban X, Tintore M, Nos C, Rio J, Aymerich FX,

et al. Interferon beta-1b for the treatment of primary progressive

multiple sclerosis: five-year clinical trial follow-up. Arch Neu-

rol. 2011;68(11):1421–7.

105. Malucchi S, Sala A, Gilli F, Bottero R, Di Sapio A, Capobianco

M, et al. Neutralizing antibodies reduce the efficacy of betaIFN

during treatment of multiple sclerosis. Neurology. 2004;62(11):

2031–7.

106. Francis GS, Rice GP, Alsop JC. Interferon beta-1a in MS: results

following development of neutralizing antibodies in PRISMS.

Neurology. 2005;65(1):48–55.

107. Hartung HP, Freedman MS, Polman CH, Edan G, Kappos L,

Miller DH, et al. Interferon beta-1b-neutralizing antibodies

5 years after clinically isolated syndrome. Neurology.

2011;77(9):835–43.

108. Goodin DS, Hartung HP, O’Connor P, Filippi M, Arnason B,

Comi G, et al. Neutralizing antibodies to interferon beta-1b

multiple sclerosis: a clinico-radiographic paradox in the

BEYOND trial. Mult Scler. 2012;18(2):181–95.

109. Pachner AR, Warth JD, Pace A, Goelz S. Effect of neutralizing

antibodies on biomarker responses to interferon beta: the

INSIGHT study. Neurology. 2009;73(18):1493–500.

110. Panitch H, Goodin DS, Francis G, Chang P, Coyle PK,

O’Connor P, et al. Randomized, comparative study of interferon

D. Karussis

beta-1a treatment regimens in MS: The EVIDENCE Trial.

Neurology. 2002;59(10):1496–506.

111. Schwid SR, Panitch HS. Full results of the Evidence of Inter-

feron Dose-Response-European North American Comparative

Efficacy (EVIDENCE) study: a multicenter, randomized,

assessor-blinded comparison of low-dose weekly versus high-

dose, high-frequency interferon beta-1a for relapsing multiple

sclerosis. Clin Ther. 2007;29(9):2031–48.

112. Durelli L, Verdun E, Barbero P, Bergui M, Versino E, Ghezzi A,

et al. Every-other-day interferon beta-1b versus once-weekly

interferon beta-1a for multiple sclerosis: results of a 2-year

prospective randomised multicentre study (INCOMIN). Lancet.

2002;359(9316):1453–60.

113. Cadavid D, Cheriyan J, Skurnick J, Lincoln JA, Wolansky LJ,

Cook SD. New acute and chronic black holes in patients with

multiple sclerosis randomised to interferon beta-1b or glatiramer

acetate. J Neurol Neurosurg Psychiatry. 2009;80(12):1337–43.

114. Koch-Henriksen N, Sorensen PS, Christensen T, Frederiksen J,

Ravnborg M, Jensen K, et al. A randomized study of two

interferon-beta treatments in relapsing-remitting multiple scle-

rosis. Neurology. 2006;66(7):1056–60.

115. O’Connor P, Filippi M, Arnason B, Comi G, Cook S, Goodin D,

et al. 250 microg or 500 microg interferon beta-1b versus 20 mg

glatiramer acetate in relapsing-remitting multiple sclerosis: a

prospective, randomised, multicentre study. Lancet Neurol.

2009;8(10):889–97.

116. Mikol DD, Barkhof F, Chang P, Coyle PK, Jeffery DR, Schwid

SR, et al. Comparison of subcutaneous interferon beta-1a with

glatiramer acetate in patients with relapsing multiple sclerosis

(the REbif vs Glatiramer Acetate in Relapsing MS Disease

[REGARD] study): a multicentre, randomised, parallel, open-

label trial. Lancet Neurol. 2008;7(10):903–14.

117. Teitelbaum D, Webb C, Meshorer A, Arnon R, Sela M. Sup-

pression by several synthetic polypeptides of experimental

allergic encephalomyelitis induced in guinea pigs and rabbits

with bovine and human basic encephalitogen. Eur J Immunol.

1973;3(5):273–9.

118. Bornstein MB, Miller A, Slagle S, Weitzman M, Crystal H,

Drexler E, et al. A pilot trial of Cop 1 in exacerbating-remitting

multiple sclerosis. N Engl J Med. 1987;317(7):408–14.

119. Johnson KP, Brooks BR, Cohen JA, Ford CC, Goldstein J, Lisak

RP, et al. Copolymer 1 reduces relapse rate and improves dis-

ability in relapsing-remitting multiple sclerosis: results of a

phase III multicenter, double-blind placebo-controlled trial. The

Copolymer 1 Multiple Sclerosis Study Group. Neurology.

1995;45(7):1268–76.

120. Comi G, Filippi M, Wolinsky JS. European/Canadian multi-

center, double-blind, randomized, placebo-controlled study of

the effects of glatiramer acetate on magnetic resonance imaging-

measured disease activity and burden in patients with relapsing

multiple sclerosis. European/Canadian Glatiramer Acetate Study

Group. Ann Neurol. 2001;49(3):290–7.

121. Filippi M, Wolinsky JS, Comi G. Effects of oral glatiramer

acetate on clinical and MRI-monitored disease activity in

patients with relapsing multiple sclerosis: a multicentre, double-

blind, randomised, placebo-controlled study. Lancet Neurol.

2006;5(3):213–20.

122. Comi G, Cohen JA, Arnold DL, Wynn D, Filippi M. Phase III

dose-comparison study of glatiramer acetate for multiple scle-

rosis. Ann Neurol. 2011;69(1):75–82.

123. Miller A, Spada V, Beerkircher D, Kreitman RR. Long-term (up

to 22 years), open-label, compassionate-use study of glatiramer

acetate in relapsing-remitting multiple sclerosis. Mult Scler.

2008;14(4):494–9.

124. Karussis D, Teitelbaum D, Sicsic C, Brenner T. Long-term

treatment of multiple sclerosis with glatiramer acetate: natural

history of the subtypes of anti-glatiramer acetate antibodies and

their correlation with clinical efficacy. J Neuroimmunol.

2010;220(1–2):125–30.

125. Comi G, Martinelli V, Rodegher M, Moiola L, Bajenaru O,

Carra A, et al. Effect of glatiramer acetate on conversion to

clinically definite multiple sclerosis in patients with clinically

isolated syndrome (PreCISe study): a randomised, double-blind,

placebo-controlled trial. Lancet. 2009;374(9700):1503–11.

126. Wolinsky JS, Narayana PA, O’Connor P, Coyle PK, Ford C,

Johnson K, et al. Glatiramer acetate in primary progressive multiple

sclerosis: results of a multinational, multicenter, double-blind,

placebo-controlled trial. Ann Neurol. 2007;61(1):14–24.

127. Sajja BR, Narayana PA, Wolinsky JS, Ahn CW. Longitudinal

magnetic resonance spectroscopic imaging of primary progres-

sive multiple sclerosis patients treated with glatiramer acetate:

multicenter study. Mult Scler. 2008;14(1):73–80.

128. Achiron A, Pras E, Gilad R, Ziv I, Mandel M, Gordon CR, et al.

Open controlled therapeutic trial of intravenous immune glob-

ulin in relapsing-remitting multiple sclerosis. Arch Neurol.

1992;49(12):1233–6.

129. Cook AW, Nidzgorski F, Oygar A. Improvement of motor

function in multiple sclerosis by use of protopam chloride.

Acupunct Electrother Res. 1992;17(3):177–93.

130. Francis GS, Freedman MS, Antel JP. Failure of intravenous

immunoglobulin to arrest progression of multiple sclerosis: a

clinical and MRI based study. Mult Scler. 1997;3(6):370–6.

131. Arnold DL, Riess GT, Matthews PM, Francis GS, Collins DL,

Wolfson C, et al. Use of proton magnetic resonance spectros-

copy for monitoring disease progression in multiple sclerosis.

Ann Neurol. 1994;36(1):76–82.

132. van Engelen BG, Hommes OR, Pinckers A, Cruysberg JR,

Barkhof F, Rodriguez M. Improved vision after intravenous

immunoglobulin in stable demyelinating optic neuritis. Ann

Neurol. 1992;32(6):834–5.

133. Fazekas F, Deisenhammer F, Strasser-Fuchs S, Nahler G,

Mamoli B. Treatment effects of monthly intravenous immuno-

globulin on patients with relapsing-remitting multiple sclerosis:

further analyses of the Austrian Immunoglobulin in MS study.

Mult Scler. 1997;3(2):137–41.

134. Fazekas F, Deisenhammer F, Strasser-Fuchs S, Nahler G,

Mamoli B. Randomised placebo-controlled trial of monthly

intravenous immunoglobulin therapy in relapsing-remitting

multiple sclerosis. Austrian Immunoglobulin in Multiple Scle-

rosis Study Group. Lancet. 1997;349(9052):589–93.

135. Hommes OR, Sorensen PS, Fazekas F, Enriquez MM, Koelmel

HW, Fernandez O, et al. Intravenous immunoglobulin in sec-

ondary progressive multiple sclerosis: randomised placebo-

controlled trial. Lancet. 2004;364(9440):1149–56.

136. Pohlau D, Przuntek H, Sailer M, Bethke F, Koehler J, Konig N,

et al. Intravenous immunoglobulin in primary and secondary

chronic progressive multiple sclerosis: a randomized placebo

controlled multicentre study. Mult Scler. 2007;13(9):1107–17.

137. Lewanska M, Siger-Zajdel M, Selmaj K. No difference in effi-

cacy of two different doses of intravenous immunoglobulins in

MS: clinical and MRI assessment. Eur J Neurol. 2002;9(6):

565–72.

138. Fazekas F, Lublin FD, Li D, Freedman MS, Hartung HP, Rie-

ckmann P, et al. Intravenous immunoglobulin in relapsing-

remitting multiple sclerosis: a dose-finding trial. Neurology.

2008;71(4):265–71.

139. Sorensen PS, Wanscher B, Jensen CV, Schreiber K, Blinkenberg

M, Ravnborg M, et al. Intravenous immunoglobulin G reduces

MRI activity in relapsing multiple sclerosis. Neurology.

1998;50(5):1273–81.

140. Noseworthy JH, O’Brien PC, Weinshenker BG, Weis JA, Pett-

erson TM, Erickson BJ, et al. IV immunoglobulin does not

Immunotherapy of MS

reverse established weakness in MS. Neurology. 2000;55(8):

1135–43.

141. Noseworthy JH, O’Brien PC, Petterson TM, Weis J, Stevens L,

Peterson WK, et al. A randomized trial of intravenous immu-

noglobulin in inflammatory demyelinating optic neuritis. Neu-

rology. 2001;56(11):1514–22.

142. Achiron A, Gabbay U, Gilad R, Hassin-Baer S, Barak Y,

Gornish M, et al. Intravenous immunoglobulin treatment in

multiple sclerosis: effect on relapses. Neurology. 1998;50(2):

398–402.

143. Double-masked trial of azathioprine in multiple sclerosis. Brit-

ish and Dutch Multiple Sclerosis Azathioprine Trial Group.

Lancet. 1988;2(8604):179–83.

144. Goodkin DE, Bailly RC, Teetzen ML, Hertsgaard D, Beatty

WW. The efficacy of azathioprine in relapsing-remitting mul-

tiple sclerosis. Neurology. 1991;41(1):20–5.

145. Milanese C, La Mantia L, Salmaggi A, Eoli M. A double blind

study on azathioprine efficacy in multiple sclerosis: final report.

J Neurol. 1993;240(5):295–8.

146. Ellison GW, Myers LW, Mickey MR, Graves MC, Tourtellotte

WW, Syndulko K, et al. A placebo-controlled, randomized,

double-masked, variable dosage, clinical trial of azathioprine

with and without methylprednisolone in multiple sclerosis.

Neurology. 1989;39(8):1018–26.

147. Casetta I, Iuliano G, Filippini G. Azathioprine for multiple

sclerosis. J Neurol Neurosurg Psychiatry. 2009;80(2):131–2

(discussion 2).

148. Massacesi L, Parigi A, Barilaro A, Repice AM, Pellicano G,

Konze A, et al. Efficacy of azathioprine on multiple sclerosis

new brain lesions evaluated using magnetic resonance imaging.

Arch Neurol. 2005;62(12):1843–7.

149. Taylor L, Hughes RA, McPherson K. The risk of cancer from

azathioprine as a treatment for multiple sclerosis [letter]. Eur J

Neurol. 2004;11(2):141.

150. Havrdova E, Zivadinov R, Krasensky J, Dwyer MG, Novakova

I, Dolezal O, et al. Randomized study of interferon beta-1a, low-

dose azathioprine, and low-dose corticosteroids in multiple

sclerosis. Mult Scler. 2009;15(8):965–76.

151. Comabella M, Balashov K, Issazadeh S, Smith D, Weiner HL,

Khoury SJ. Elevated interleukin-12 in progressive multiple scle-

rosis correlates with disease activity and is normalized by pulse

cyclophosphamide therapy. J Clin Invest. 1998;102(4):671–8.

152. Smith DR, Balashov KE, Hafler DA, Khoury SJ, Weiner HL.

Immune deviation following pulse cyclophosphamide/methyl-

prednisolone treatment of multiple sclerosis: increased inter-

leukin-4 production and associated eosinophilia. Ann Neurol.

1997;42(3):313–8.

153. Brannagan TH 3rd, Pradhan A, Heiman-Patterson T, Winkelman

AC, Styler MJ, Topolsky DL, et al. High-dose cyclophospha-

mide without stem-cell rescue for refractory CIDP. Neurology.

2002;58(12):1856–8.

154. Boumpas DT, Fessler BJ, Austin HA 3rd. Balow JE, Klippel JH,

Lockshin MD. Systemic lupus erythematosus: emerging con-

cepts. Part 2: Dermatologic and joint disease, the antiphospho-

lipid antibody syndrome, pregnancy and hormonal therapy,

morbidity and mortality, and pathogenesis. Ann Intern Med.

1995;123(1):42–53.

155. Hommes OR, Prick JJ, Lamers KJ. Treatment of the chronic

progressive form of multiple sclerosis with a combination of

cyclophosphamide and prednisone. Clin Neurol Neurosurg.

1975;78(1):59–72.

156. Weiner HL, Mackin GA, Orav EJ, Hafler DA, Dawson DM,

LaPierre Y, et al. Intermittent cyclophosphamide pulse therapy

in progressive multiple sclerosis: final report of the Northeast

Cooperative Multiple Sclerosis Treatment Group. Neurology.

1993;43(5):910–8.

157. Hohol MJ, Olek MJ, Orav EJ, Stazzone L, Hafler DA, Khoury

SJ, et al. Treatment of progressive multiple sclerosis with pulse

cyclophosphamide/methylprednisolone: response to therapy is

linked to the duration of progressive disease. Mult Scler.

1999;5(6):403–9.

158. Khan OA, Zvartau-Hind M, Caon C, Din MU, Cochran M, Lisak

D, et al. Effect of monthly intravenous cyclophosphamide in

rapidly deteriorating multiple sclerosis patients resistant to

conventional therapy. Mult Scler. 2001;7(3):185–8.

159. Perini P, Gallo P. Cyclophosphamide is effective in stabilizing

rapidly deteriorating secondary progressive multiple sclerosis.

J Neurol. 2003;250(7):834–8.

160. Zephir H, de Seze J, Duhamel A, Debouverie M, Hautecoeur P,

Lebrun C, et al. Treatment of progressive forms of multiple

sclerosis by cyclophosphamide: a cohort study of 490 patients.

J Neurol Sci. 2004;218(1–2):73–7.

161. de Bittencourt PR, Gomes-da-Silva MM. Multiple sclerosis:

long-term remission after a high dose of cyclophosphamide.

Acta Neurol Scand. 2005;111(3):195–8.

162. Gladstone DE, Zamkoff KW, Krupp L, Peyster R, Sibony P,

Christodoulou C, et al. High-dose cyclophosphamide for mod-

erate to severe refractory multiple sclerosis. Arch Neurol.

2006;63(10):1388–93.

163. Smith DR, Weinstock-Guttman B, Cohen JA, Wei X, Gutmann

C, Bakshi R, et al. A randomized blinded trial of combination

therapy with cyclophosphamide in patients with active multiple

sclerosis on interferon beta. Mult Scler. 2005;11(5):573–82.

164. Patti F, Reggio E, Palermo F, Fiorilla T, Politi G, Nicoletti A,

et al. Stabilization of rapidly worsening multiple sclerosis for

36 months in patients treated with interferon beta plus cyclo-

phosphamide followed by interferon beta. J Neurol. 2004;

251(12):1502–6.

165. Fox EJ. Mechanism of action of mitoxantrone. Neurology.

2004;63(12 Suppl. 6):S15–8.

166. Millefiorini E, Gasperini C, Pozzilli C, D’Andrea F, Bastianello

S, Trojano M, et al. Randomized placebo-controlled trial of

mitoxantrone in relapsing-remitting multiple sclerosis:

24-month clinical and MRI outcome. J Neurol. 1997;244(3):

153–9.

167. Edan G, Miller D, Clanet M, Confavreux C, Lyon-Caen O,

Lubetzki C, et al. Therapeutic effect of mitoxantrone combined

with methylprednisolone in multiple sclerosis: a randomised

multicentre study of active disease using MRI and clinical cri-

teria. J Neurol Neurosurg Psychiatry. 1997;62(2):112–8.

168. van de Wyngaert FA, Beguin C, D’Hooghe MB, Dooms G,

Lissoir F, Carton H, et al. A double-blind clinical trial of

mitoxantrone versus methylprednisolone in relapsing, secondary

progressive multiple sclerosis. Acta Neurol Belg. 2001;101(4):

210–6.

169. Hartung HP, Gonsette R, Konig N, Kwiecinski H, Guseo A,

Morrissey SP, et al. Mitoxantrone in progressive multiple scle-

rosis: a placebo-controlled, double-blind, randomised, multi-

centre trial. Lancet. 2002;360(9350):2018–25.

170. Le Page E, Leray E, Taurin G, Coustans M, Chaperon J, Edan G.

Mitoxantrone as induction therapy in aggressive relapsing

remitting multiple sclerosis: a descriptive analysis of 100 con-

secutive patients. Rev Neurol (Paris). 2006;162(2):185–94.

171. Jeffery DR, Chepuri N, Durden D, Burdette J. A pilot trial of

combination therapy with mitoxantrone and interferon beta-1b

using monthly gadolinium-enhanced magnetic resonance imag-

ing. Mult Scler. 2005;11(3):296–301.

172. Ramtahal J, Jacob A, Das K, Boggild M. Sequential mainte-

nance treatment with glatiramer acetate after mitoxantrone is

safe and can limit exposure to immunosuppression in very

active, relapsing remitting multiple sclerosis. J Neurol.

2006;253(9):1160–4.

D. Karussis

173. Chan ES, Cronstein BN. Methotrexate: how does it really work?

Nat Rev Rheumatol. 2010;6(3):175–8.

174. Gray OM, McDonnell GV, Forbes RB. A systematic review of

oral methotrexate for multiple sclerosis. Mult Scler.

2006;12(4):507–10.

175. Goodkin DE, Rudick RA, VanderBrug Medendorp S, Daughtry

MM, Schwetz KM, Fischer J, et al. Low-dose (7.5 mg) oral

methotrexate reduces the rate of progression in chronic pro-

gressive multiple sclerosis. Ann Neurol. 1995;37(1):30–40.

176. Currier RD, Haerer AF, Meydrech EF. Low dose oral metho-

trexate treatment of multiple sclerosis: a pilot study. J Neurol

Neurosurg Psychiatry. 1993;56(11):1217–8.

177. Sadiq SA, Simon EV, Puccio LM. Intrathecal methotrexate

treatment in multiple sclerosis. J Neurol. 2010;257(11):1806–11.

178. Efficacy and toxicity of cyclosporine in chronic progressive

multiple sclerosis. A randomized, double-blinded, placebo-

controlled clinical trial. The Multiple Sclerosis Study Group.

Ann Neurol. 1990;27(6):591–605.

179. Leist TP, Weissert R. Cladribine: mode of action and implica-

tions for treatment of multiple sclerosis. Clin Neuropharmacol.

2011;34(1):28–35.

180. Sipe JC, Romine JS, Koziol JA, McMillan R, Zyroff J, Beutler

E. Cladribine in treatment of chronic progressive multiple

sclerosis. Lancet. 1994;344(8914):9–13.

181. Beutler E, Sipe JC, Romine JS, Koziol JA, McMillan R, Zyroff

J. The treatment of chronic progressive multiple sclerosis with

cladribine. Proc Natl Acad Sci USA. 1996;93(4):1716–20.

182. Romine JS, Sipe JC, Koziol JA, Zyroff J, Beutler E. A double-

blind, placebo-controlled, randomized trial of cladribine in

relapsing-remitting multiple sclerosis. Proc Assoc Am Phys.

1999;111(1):35–44.

183. Giovannoni G, Comi G, Cook S, Rammohan K, Rieckmann P,

Soelberg Sorensen P. A placebo-controlled trial of oral cladri-

bine for relapsing multiple sclerosis. N Engl J Med.

2010;362(5):416–26.

184. Giovannoni G, Cook S, Rammohan K, Rieckmann P, Sorensen

PS, Vermersch P, et al. Sustained disease-activity-free status in

patients with relapsing-remitting multiple sclerosis treated with

cladribine tablets in the CLARITY study: a post-hoc and sub-

group analysis. Lancet Neurol. 2011;10(4):329–37.

185. Ahrens N, Salama A, Haas J. Mycophenolate-mofetil in the

treatment of refractory multiple sclerosis. J Neurol.

2001;248(8):713–4.

186. Frohman EM, Brannon K, Racke MK, Hawker K. Mycophen-

olate mofetil in multiple sclerosis. Clin Neuropharmacol.

2004;27(2):80–3.

187. Vermersch P, Waucquier N, Michelin E, Bourteel H, Stojkovic

T, Ferriby D, et al. Combination of IFN beta-1a (Avonex) and

mycophenolate mofetil (Cellcept) in multiple sclerosis. Eur J

Neurol. 2007;14(1):85–9.

188. Jacob A, Matiello M, Weinshenker BG, Wingerchuk DM,

Lucchinetti C, Shuster E, et al. Treatment of neuromyelitis op-

tica with mycophenolate mofetil: retrospective analysis of 24

patients. Arch Neurol. 2009;66(9):1128–33.

189. Lemster B, Huang LL, Irish W, Woo J, Carroll PB, Abu-Elmagd

K, et al. Influence of FK 506 (tacrolimus) on circulating CD4?

T cells expressing CD25 and CD45RA antigens in 19 patients

with chronic progressive multiple sclerosis participating in an

open label drug safety trial. Autoimmunity. 1994;19(2):89–98.

190. Conway D, Cohen JA. Combination therapy in multiple scle-

rosis. Lancet Neurol. 2010;9(3):299–308.

191. Breedveld FC, Weisman MH, Kavanaugh AF, Cohen SB, Pav-

elka K, van Vollenhoven R, et al. The PREMIER study: a

multicenter, randomized, double-blind clinical trial of combi-

nation therapy with adalimumab plus methotrexate versus

methotrexate alone or adalimumab alone in patients with early,

aggressive rheumatoid arthritis who had not had previous

methotrexate treatment. Arthr Rheum. 2006;54(1):26–37.

192. Calabresi PA, Wilterdink JL, Rogg JM, Mills P, Webb A,

Whartenby KA. An open-label trial of combination therapy with

interferon beta-1a and oral methotrexate in MS. Neurology.

2002;58(2):314–7.

193. Pulicken M, Bash CN, Costello K, Said A, Cuffari C, Wilterdink

JL, et al. Optimization of the safety and efficacy of interferon

beta 1b and azathioprine combination therapy in multiple scle-

rosis. Mult Scler. 2005;11(2):169–74.

194. Cohen JA, Imrey PB, Calabresi PA, Edwards KR, Eickenhorst

T, Felton WL 3rd, et al. Results of the Avonex Combination

Trial (ACT) in relapsing-remitting MS. Neurology.

2009;72(6):535–41.

195. Ravnborg M, Sorensen PS, Andersson M, Celius EG, Jongen PJ,

Elovaara I, et al. Methylprednisolone in combination with

interferon beta-1a for relapsing-remitting multiple sclerosis (ME-

COMBIN study): a multicentre, double-blind, randomised, placebo-

controlled, parallel-group trial. Lancet Neurol. 2010;9(7):672–80.

196. Sorensen PS, Mellgren SI, Svenningsson A, Elovaara I,

Frederiksen JL, Beiske AG, et al. NORdic trial of oral Meth-

ylprednisolone as add-on therapy to Interferon beta-1a for

treatment of relapsing-remitting Multiple Sclerosis (NORMIMS

study): a randomised, placebo-controlled trial. Lancet Neurol.

2009;8(6):519–29.

197. GoodmanAD,Rossman H,Bar-Or A,MillerA,MillerDH,Schmierer

K, et al. GLANCE: results of a phase 2, randomized, double-blind,

placebo-controlled study. Neurology. 2009;72(9):806–12.

198. Patti F, Cataldi ML, Nicoletti F, Reggio E, Nicoletti A, Reggio

A. Combination of cyclophosphamide and interferon-beta halts

progression in patients with rapidly transitional multiple scle-

rosis. J Neurol Neurosurg Psychiatry. 2001;71(3):404–7.

199. Reggio E, Nicoletti A, Fiorilla T, Politi G, Reggio A, Patti F.

The combination of cyclophosphamide plus interferon beta as

rescue therapy could be used to treat relapsing-remitting mul-

tiple sclerosis patients: twenty-four months follow-up. J Neurol.

2005;252(10):1255–61.

200. Le Page E, Leray E, Taurin G, Coustans M, Chaperon J,

Morrissey SP, et al. Mitoxantrone as induction treatment in

aggressive relapsing remitting multiple sclerosis: treatment

response factors in a 5 year follow-up observational study of

100 consecutive patients. J Neurol Neurosurg Psychiatry.

2008;79(1):52–6.

201. Edan G, Comi G, Le Page E, Leray E, Rocca MA, Filippi M.

Mitoxantrone prior to interferon beta-1b in aggressive relapsing

multiple sclerosis: a 3-year randomised trial. J Neurol Neurosurg

Psychiatry. 2011;82(12):1344–50.

202. Etemadifar M, Kazemi M, Chitsaz A, Hekmatnia A, Tayari N,

Ghazavi A, et al. Mycophenolate mofetil in combination withinterferon beta-1a in the treatment of relapsing-remitting mul-

tiple sclerosis: a preliminary study. J Res Med Sci.

2011;16(1):1–5.

203. Frohman EM, Cutter G, Remington G, Gao H, Rossman H,

Weinstock-Guttman B, et al. A randomized, blinded, parallel-

group, pilot trial of mycophenolate mofetil (CellCept) compared

with interferon beta-1a (Avonex) in patients with relapsing-

remitting multiple sclerosis. Ther Adv Neurol Disord.

2010;3(1):15–28.

204. Sorensen PS, Lycke J, Eralinna JP, Edland A, Wu X, Freder-

iksen JL, et al. Simvastatin as add-on therapy to interferon beta-

1a for relapsing-remitting multiple sclerosis (SIMCOMBIN

study): a placebo-controlled randomised phase 4 trial. Lancet

Neurol. 2011;10(8):691–701.

205. Walker JE, Hoehn MM, Kashiwagi N. A trial of antilymphocyte

globulin in the treatment of chronic progressive multiple scle-

rosis. J Neurol Sci. 1976;29(2–4):303–9.

Immunotherapy of MS

206. Rudick RA, Stuart WH, Calabresi PA, Confavreux C, Galetta

SL, Radue EW, et al. Natalizumab plus interferon beta-1a for

relapsing multiple sclerosis. N Engl J Med. 2006;354(9):911–23.

207. Sorensen PS, Bertolotto A, Edan G, Giovannoni G, Gold R,

Havrdova E, et al. Risk stratification for progressive multifocal

leukoencephalopathy in patients treated with natalizumab. Mult

Scler. 2012;18(2):143–52.

208. Klotz L, Meuth SG, Wiendl H. Immune mechanisms of new

therapeutic strategies in multiple sclerosis—a focus on ale-

mtuzumab. Clin Immunol. 2012;142(1):25–30.

209. Coles AJ, Compston DA, Selmaj KW, Lake SL, Moran S,

Margolin DH, et al. Alemtuzumab vs. interferon beta-1a in early

multiple sclerosis. N Engl J Med. 2008;359(17):1786–801.

210. Fox EJ, Sullivan HC, Gazda SK, Mayer L, O’Donnell L, Melia

K, et al. A single-arm, open-label study of alemtuzumab in

treatment-refractory patients with multiple sclerosis. Eur J

Neurol. 2012;19(2):307–11.

211. Cossburn M, Pace AA, Jones J, Ali R, Ingram G, Baker K, et al.

Autoimmune disease after alemtuzumab treatment for multiple

sclerosis in a multicenter cohort. Neurology. 2011;77(6):573–9.

212. Lassmann H. New concepts on progressive multiple sclerosis.

Curr Neurol Neurosci Rep. 2007;7(3):239–44.

213. Duddy M, Bar-Or A. B-cells in multiple sclerosis. Int MS J.

2006;13(3):84–90.

214. Reff ME, Carner K, Chambers KS, Chinn PC, Leonard JE, Raab

R, et al. Depletion of B cells in vivo by a chimeric mouse human

monoclonal antibody to CD20. Blood. 1994;83(2):435–45.

215. Cross AH, Stark JL, Lauber J, Ramsbottom MJ, Lyons JA. Ritux-

imab reduces B cells and T cells in cerebrospinal fluid of multiple

sclerosis patients. J Neuroimmunol. 2006;180(1–2):63–70.

216. Bar-Or A, Calabresi PA, Arnold D, Markowitz C, Shafer S,

Kasper LH, et al. Rituximab in relapsing-remitting multiple

sclerosis: a 72-week, open-label, phase I trial. Ann Neurol.

2008;63(3):395–400.

217. Naismith RT, Piccio L, Lyons JA, Lauber J, Tutlam NT, Parks

BJ, et al. Rituximab add-on therapy for breakthrough relapsing

multiple sclerosis: a 52-week phase II trial. Neurology.

2010;74(23):1860–7.

218. Hauser SL, Waubant E, Arnold DL, Vollmer T, Antel J, Fox RJ,

et al. B-cell depletion with rituximab in relapsing-remitting

multiple sclerosis. N Engl J Med. 2008;358(7):676–88.

219. Hawker K, O’Connor P, Freedman MS, Calabresi PA, Antel J,

Simon J, et al. Rituximab in patients with primary progressive

multiple sclerosis: results of a randomized double-blind placebo-

controlled multicenter trial. Ann Neurol. 2009;66(4):460–71.

220. Kappos L, Li D, Calabresi PA, O’Connor P, Bar-Or A, Barkhof

F, et al. Ocrelizumab in relapsing-remitting multiple sclerosis: a

phase 2, randomised, placebo-controlled, multicentre trial.

Lancet. 2011;378(9805):1779–87.

221. Wynn D, Kaufman M, Montalban X, Vollmer T, Simon J,

Elkins J, et al. Daclizumab in active relapsing multiple sclerosis

(CHOICE study): a phase 2, randomised, double-blind, placebo-

controlled, add-on trial with interferon beta. Lancet Neurol.

2010;9(4):381–90.

222. Bielekova B, Richert N, Herman ML, Ohayon J, Waldmann TA,

McFarland H, et al. Intrathecal effects of daclizumab treatment

of multiple sclerosis. Neurology. 2011;77(21):1877–86.

223. Norman DJ. Mechanisms of action and overview of OKT3. Ther

Drug Monit. 1995;17(6):615–20.

224. Bonnefoy-Berard N, Revillard JP. Mechanisms of immunosup-

pression induced by antithymocyte globulins and OKT3. J Heart

Lung Transpl. 1996;15(5):435–42.

225. Reinke P, Schwinzer H, Hoflich C, Ode-Hakim C, Docke WD,

Frei U, et al. Selective in vivo deletion of alloactivated TH1

cells by OKT3 monoclonal antibody in acute rejection. Immunol

Lett. 1997;57(1–3):151–3.

226. Weinshenker BG, Bass B, Karlik S, Ebers GC, Rice GP. An

open trial of OKT3 in patients with multiple sclerosis. Neurol-

ogy. 1991;41(7):1047–52.

227. Ruddle NH. Tumor necrosis factor (TNF-alpha) and lympho-

toxin (TNF-beta). Curr Opin Immunol. 1992;4(3):327–32.

228. van Oosten BW, Barkhof F, Truyen L, Boringa JB, Bertelsmann

FW, von Blomberg BM, et al. Increased MRI activity and

immune activation in two multiple sclerosis patients treated with

the monoclonal anti-tumor necrosis factor antibody cA2. Neu-

rology. 1996;47(6):1531–4.

229. Caminero A, Comabella M, Montalban X. Tumor necrosis

factor alpha (TNF-alpha), anti-TNF-alpha and demyelination

revisited: an ongoing story. J Neuroimmunol. 2011;234(1–2):

1–6.

230. Chun J, Hartung HP. Mechanism of action of oral fingolimod

(FTY720) in multiple sclerosis. Clin Neuropharmacol. 2010;33(2):

91–101.

231. Mehling M, Lindberg R, Raulf F, Kuhle J, Hess C, Kappos L,

et al. Th17 central memory T cells are reduced by FTY720 in

patients with multiple sclerosis. Neurology. 2010;75(5):403–10.

232. Fujino M, Funeshima N, Kitazawa Y, Kimura H, Amemiya H,

Suzuki S, et al. Amelioration of experimental autoimmune

encephalomyelitis in Lewis rats by FTY720 treatment. J Phar-

macol Exp Ther. 2003;305(1):70–7.

233. Kappos L, Antel J, Comi G, Montalban X, O’Connor P, Polman

CH, et al. Oral fingolimod (FTY720) for relapsing multiple

sclerosis. N Engl J Med. 2006;355(11):1124–40.

234. Kappos L, Radue EW, O’Connor P, Polman C, Hohlfeld R,

Calabresi P, et al. A placebo-controlled trial of oral fingolimod

in relapsing multiple sclerosis. N Engl J Med. 2010;362(5):

387–401.

235. Cohen JA, Barkhof F, Comi G, Hartung HP, Khatri BO, Montalban

X, et al. Oral fingolimod or intramuscular interferon for relapsing

multiple sclerosis. N Engl J Med. 2010;362(5):402–15.

236. Khatri B, Barkhof F, Comi G, Hartung HP, Kappos L, Mon-

talban X, et al. Comparison of fingolimod with interferon beta-

1a in relapsing-remitting multiple sclerosis: a randomised

extension of the TRANSFORMS study. Lancet Neurol.

2011;10(6):520–9.

237. Palmer AM. Teriflunomide, an inhibitor of dihydroorotate

dehydrogenase for the potential oral treatment of multiple

sclerosis. Curr Opin Investig Drugs. 2010;11(11):1313–23.

238. O’Connor PW, Li D, Freedman MS, Bar-Or A, Rice GP, Con-

favreux C, et al. A phase II study of the safety and efficacy of

teriflunomide in multiple sclerosis with relapses. Neurology.

2006;66(6):894–900.

239. Confavreux C, Li DK, Freedman MS, Truffinet P, Benzerdjeb H,

Wang D, et al. Long-term follow-up of a phase 2 study of oral

teriflunomide in relapsing multiple sclerosis: safety and efficacy

results up to 8.5 years. Mult Scler. 2012;18(9):1278–89.

240. O’Connor P, Wolinsky JS, Confavreux C, Comi G, Kappos L,

Olsson TP, et al. Randomized trial of oral teriflunomide for

relapsing multiple sclerosis. N Engl J Med. 2011;365(14):

1293–303.

241. Karussis DM, Lehmann D, Slavin S, Kalland T, Vourka-

Karussis U, Mizrachi-Koll R, et al. Immunomodulation of

autoimmunity by linomide. Isr J Med Sci. 1995;31(1):38–41.

242. Lehmann D, Karussis D, Mizrachi-Koll R, Linde AS, Abramsky

O. Inhibition of the progression of multiple sclerosis by lino-

mide is associated with upregulation of CD4?/CD45RA? cells

and downregulation of CD4?/CD45RO? cells. Clin Immunol

Immunopathol. 1997;85(2):202–9.

243. Tan IL, Lycklama a Nijeholt GJ, Polman CH, Ader HJ, Barkhof

F. Linomide in the treatment of multiple sclerosis: MRI results

from prematurely terminated phase-III trials. Mult Scler.

2000;6(2):99–104.

D. Karussis

244. Giacomini PS, Bar-Or A. Laquinimod in multiple sclerosis. Clin

Immunol. 2012;142(1):38–43.

245. Comi G, Pulizzi A, Rovaris M, Abramsky O, Arbizu T, Boiko

A, et al. Effect of laquinimod on MRI-monitored disease activity

in patients with relapsing-remitting multiple sclerosis: a multi-

centre, randomised, double-blind, placebo-controlled phase IIb

study. Lancet. 2008;371(9630):2085–92.

246. Comi G, Abramsky O, Arbizu T, Boyko A, Gold R, Havrdova E,

et al. Oral laquinimod in patients with relapsing-remitting

multiple sclerosis: 36-week double-blind active extension of the

multi-centre, randomized, double-blind, parallel-group placebo-

controlled study. Mult Scler. 2010;16(11):1360–6.

247. Comi G, Jeffery D, Kappos L, Montalban X, Boyko A, Rocca

MA, et al. Placebo-controlled trial of oral laquinimod for mul-

tiple sclerosis. N Engl J Med. 2012;366(11):1000–9.

248. Linker RA, Lee DH, Ryan S, van Dam AM, Conrad R, Bista P,

et al. Fumaric acid esters exert neuroprotective effects in neur-

oinflammation via activation of the Nrf2 antioxidant pathway.

Brain. 2011;134(Pt 3):678–92.

249. Papadopoulou A, D’Souza M, Kappos L, Yaldizli O. Dimethyl

fumarate for multiple sclerosis. Expert Opin Investig Drugs.

2010;19(12):1603–12.

250. Schimrigk S, Brune N, Hellwig K, Lukas C, Bellenberg B, Rieks

M, et al. Oral fumaric acid esters for the treatment of active

multiple sclerosis: an open-label, baseline-controlled pilot study.

Eur J Neurol. 2006;13(6):604–10.

251. Kappos L, Gold R, Miller DH, Macmanus DG, Havrdova E,

Limmroth V, et al. Efficacy and safety of oral fumarate in

patients with relapsing-remitting multiple sclerosis: a multi-

centre, randomised, double-blind, placebo-controlled phase IIb

study. Lancet. 2008;372(9648):1463–72.

252. Kappos L, Gold R, Miller DH, Macmanus DG, Havrdova E,

Limmroth V, et al. Effect of BG-12 on contrast-enhanced

lesions in patients with relapsing-remitting multiple sclerosis:

subgroup analyses from the phase 2b study. Mult Scler.

2012;18(3):314–21.

253. Ben-Nun A, Wekerle H, Cohen IR. Vaccination against auto-

immune encephalomyelitis with T-lymphocyte line cells reac-

tive against myelin basic protein. Nature. 1981;292(5818):60–1.

254. Zhang J, Medaer R, Stinissen P, Hafler D, Raus J. MHC-

restricted depletion of human myelin basic protein-reactive T

cells by T cell vaccination. Science. 1993;261(5127):1451–4.

255. Correale J, Lund B, McMillan M, Ko DY, McCarthy K, Weiner

LP. T cell vaccination in secondary progressive multiple scle-

rosis. J Neuroimmunol. 2000;107(2):130–9.

256. Hermans G, Denzer U, Lohse A, Raus J, Stinissen P. Cellular

and humoral immune responses against autoreactive T cells in

multiple sclerosis patients after T cell vaccination. J Autoim-

mun. 1999;13(2):233–46.

257. Hermans G, Medaer R, Raus J, Stinissen P. Myelin reactive T

cells after T cell vaccination in multiple sclerosis: cytokine

profile and depletion by additional immunizations. J Neuroim-

munol. 2000;102(1):79–84.

258. Medaer R, Stinissen P, Truyen L, Raus J, Zhang J. Depletion of

myelin-basic-protein autoreactive T cells by T-cell vaccination:

pilot trial in multiple sclerosis. Lancet. 1995;346(8978):807–8.

259. Stinissen P, Zhang J, Medaer R, Vandevyver C, Raus J. Vac-

cination with autoreactive T cell clones in multiple sclerosis:

overview of immunological and clinical data. J Neurosci Res.

1996;45(4):500–11.

260. Zhang JZ, Rivera VM, Tejada-Simon MV, Yang D, Hong J, Li

S, et al. T cell vaccination in multiple sclerosis: results of a

preliminary study. J Neurol. 2002;249(2):212–8.

261. Vandenbark AA, Abulafia-Lapid R. Autologous T-cell vacci-

nation for multiple sclerosis: a perspective on progress. Bio-

Drugs. 2008;22(4):265–73.

262. Achiron A, Lavie G, Kishner I, Stern Y, Sarova-Pinhas I,

Ben-Aharon T, et al. T cell vaccination in multiple sclerosis

relapsing-remitting nonresponders patients. Clin Immunol.

2004;113(2):155–60.

263. Bourdette DN, Edmonds E, Smith C, Bowen JD, Guttmann CR,

Nagy ZP, et al. A highly immunogenic trivalent T cell receptor

peptide vaccine for multiple sclerosis. Mult Scler. 2005;11(5):

552–61.

264. Vandenbark AA, Chou YK, Whitham R, Mass M, Buenafe A,

Liefeld D, et al. Treatment of multiple sclerosis with T-cell

receptor peptides: results of a double-blind pilot trial. Nat Med.

1996;2(10):1109–15.

265. Fontoura P, Garren H, Steinman L. Antigen-specific therapies in

multiple sclerosis: going beyond proteins and peptides. Int Rev

Immunol. 2005;24(5–6):415–46.

266. Kappos L, Comi G, Panitch H, Oger J, Antel J, Conlon P, et al.

Induction of a non-encephalitogenic type 2 T helper-cell auto-

immune response in multiple sclerosis after administration of an

altered peptide ligand in a placebo-controlled, randomized phase

II trial. The Altered Peptide Ligand in Relapsing MS Study

Group. Nat Med. 2000;6(10):1176–82.

267. Bielekova B, Goodwin B, Richert N, Cortese I, Kondo T, Afshar

G, et al. Encephalitogenic potential of the myelin basic protein

peptide (amino acids 83–99) in multiple sclerosis: results of a

phase II clinical trial with an altered peptide ligand. Nat Med.

2000;6(10):1167–75.

268. Miller A, Lider O, Weiner HL. Antigen-driven bystander sup-

pression after oral administration of antigens. J Exp Med.

1991;174(4):791–8.

269. Weiner HL. Current issues in the treatment of human diseases

by mucosal tolerance. Ann N Y Acad Sci. 2004;1029:211–24.

270. Weiner HL, Mackin GA, Matsui M, Orav EJ, Khoury SJ,

Dawson DM, et al. Double-blind pilot trial of oral tolerization

with myelin antigens in multiple sclerosis. Science. 1993;259

(5099):1321–4.

271. Hohol MJ, Khoury SJ, Cook SL, Orav EJ, Hafler DA, Weiner

HL. Three-year open protocol continuation study of oral toler-

ization with myelin antigens in multiple sclerosis and design of a

phase III pivotal trial. Ann N Y Acad Sci. 1996;778:243–50.

272. Warren KG, Catz I. Administration of myelin basic protein

synthetic peptides to multiple sclerosis patients. J Neurol Sci.

1995;133(1–2):85–94.

273. Warren KG, Catz I, Wucherpfennig KW. Tolerance induction to

myelin basic protein by intravenous synthetic peptides con-

taining epitope P85 VVHFFKNIVTP96 in chronic progressive

multiple sclerosis. J Neurol Sci. 1997;152(1):31–8.

274. Warren KG, Catz I. Kinetic profiles of cerebrospinal fluid anti-

MBP in response to intravenous MBP synthetic peptide

DENP(85)VVHFFKNIVTP(96)RT in multiple sclerosis patients.

Mult Scler. 2000;6(5):300–11.

275. Goodkin DE, Shulman M, Winkelhake J, Waubant E, Andersson P,

Stewart T, et al. A phase I trial of solubilized DR2:MBP84-102

(AG284) in multiple sclerosis. Neurology. 2000;54(7):1414–20.

276. Karussis DM, Vourka-Karussis U, Lehmann D, Ovadia H,

Mizrachi-Koll R, Ben-Nun A, et al. Prevention and reversal of

adoptively transferred, chronic relapsing experimental autoim-

mune encephalomyelitis with a single high dose cytoreductive

treatment followed by syngeneic bone marrow transplantation.

J Clin Invest. 1993;92(2):765–72.

277. Karussis DM, Slavin S, Lehmann D, Mizrachi-Koll R, Abram-

sky O, Ben-Nun A. Prevention of experimental autoimmune

encephalomyelitis and induction of tolerance with acute

immunosuppression followed by syngeneic bone marrow

transplantation. J Immunol. 1992;148(6):1693–8.

278. Fassas A, Anagnostopoulos A, Kazis A, Kapinas K, Sakellari I,

Kimiskidis V, et al. Peripheral blood stem cell transplantation in

Immunotherapy of MS

the treatment of progressive multiple sclerosis: first results of a

pilot study. Bone Marrow Transpl. 1997;20(8):631–8.

279. Capello E, Saccardi R, Murialdo A, Gualandi F, Pagliai F,

Bacigalupo A, et al. Intense immunosuppression followed by

autologous stem cell transplantation in severe multiple sclerosis.

Neurol Sci. 2005;26(Suppl. 4):S200–3.

280. Mancardi GL, Murialdo A, Rossi P, Gualandi F, Martino G,

Marmont A, et al. Autologous stem cell transplantation as rescue

therapy in malignant forms of multiple sclerosis. Mult Scler.

2005;11(3):367–71.

281. Burt RK, Loh Y, Cohen B, Stefoski D, Balabanov R,

Katsamakis G, et al. Autologous non-myeloablative haemopoi-

etic stem cell transplantation in relapsing-remitting multiple

sclerosis: a phase I/II study. Lancet Neurol. 2009;8(3):244–53.

282. Karussis D, Vaknin-Dembinsky A. Hematopoietic stem cell

transplantation in multiple sclerosis: a review of the clinical

experience and a report of an international meeting. Expert Rev

Clin Immunol. 2010;6(3):347–52.

283. Einstein O, Karussis D, Grigoriadis N, Mizrachi-Kol R, Reinhartz

E, Abramsky O, et al. Intraventricular transplantation of neural

precursor cell spheres attenuates acute experimental allergic

encephalomyelitis. Mol Cell Neurosci. 2003;24(4):1074–82.

284. Ben-Hur T, Einstein O, Mizrachi-Kol R, Ben-Menachem O,

Reinhartz E, Karussis D, et al. Transplanted multipotential

neural precursor cells migrate into the inflamed white matter in

response to experimental autoimmune encephalomyelitis. Glia.

2003;41(1):73–80.

285. Pluchino S, Quattrini A, Brambilla E, Gritti A, Salani G, Dina G,

et al. Injection of adult neurospheres induces recovery in a chronic

model of multiple sclerosis. Nature. 2003;422(6933):688–94.

286. Zappia E, Casazza S, Pedemonte E, Benvenuto F, Bonanni I,

Gerdoni E, et al. Mesenchymal stem cells ameliorate experi-

mental autoimmune encephalomyelitis inducing T-cell anergy.

Blood. 2005;106(5):1755–61.

287. Aharonowiz M, Einstein O, Fainstein N, Lassmann H, Reubi-

noff B, Ben-Hur T. Neuroprotective effect of transplanted

human embryonic stem cell-derived neural precursors in an

animal model of multiple sclerosis. PLoS One. 2008;3(9):e3145.

288. Kassis I, Grigoriadis N, Gowda-Kurkalli B, Mizrachi-Kol R,

Ben-Hur T, Slavin S, et al. Neuroprotection and immunomod-

ulation with mesenchymal stem cells in chronic experimental

autoimmune encephalomyelitis. Arch Neurol. 2008;65(6):

753–61.

289. Nauta AJ, Fibbe WE. Immunomodulatory properties of mesen-

chymal stromal cells. Blood. 2007;110(10):3499–506.

290. Uccelli A, Pistoia V, Moretta L. Mesenchymal stem cells: a new

strategy for immunosuppression? Trends Immunol. 2007;28(5):

219–26.

291. Uccelli A, Moretta L, Pistoia V. Immunoregulatory function of

mesenchymal stem cells. Eur J Immunol. 2006;36(10):2566–73.

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

Al-Kutoubi A, et al. Bone marrow mesenchymal stem cell

transplantation in patients with multiple sclerosis: a pilot study.

J Neuroimmunol. 2010;227(1–2):185–9.

293. Connick P, Kolappan M, Crawley C, Webber DJ, Patani R,

Michell AW, et al. Autologous mesenchymal stem cells for the

treatment of secondary progressive multiple sclerosis: an open-

label phase 2a proof-of-concept study. Lancet Neurol. 2012;11(2):

150–6.

294. Miller D, Barkhof F, Montalban X, Thompson A, Filippi M.

Clinically isolated syndromes suggestive of multiple sclerosis,

part I: natural history, pathogenesis, diagnosis, and prognosis.

Lancet Neurol. 2005;4(5):281–8.

295. Barkhof F, Filippi M. MRI: the perfect surrogate marker for

multiple sclerosis? Nat Rev Neurol. 2009;5(4):182–3.

296. Barkhof F, Filippi M, Miller DH, Scheltens P, Campi A, Polman

CH, et al. Comparison of MRI criteria at first presentation to

predict conversion to clinically definite multiple sclerosis. Brain.

1997;120(Pt 11):2059–69.

297. Freedman M, Metzig C, Kappos L, Polman C, Edan G, Hartung

HP, et al. Predictive nature of IgM anti-alpha-glucose serum

biomarker for relapse activity and EDSS progression in CIS

patients: a BENEFIT study analysis. Mult Scler. 2012;18(7):

966–73.

298. Schwarz M, Spector L, Gortler M, Weisshaus O, Glass-Marmor

L, Karni A, et al. Serum anti-Glc(alpha1,4)Glc(alpha) antibodies

as a biomarker for relapsing-remitting multiple sclerosis.

J Neurol Sci. 2006;244(1–2):59–68.

299. Warren KG, Catz I, Johnson E, Mielke B. Anti-myelin basic

protein and anti-proteolipid protein specific forms of multiple

sclerosis. Ann Neurol. 1994;35(3):280–9.

300. Berger T, Rubner P, Schautzer F, Egg R, Ulmer H, Mayringer I,

et al. Antimyelin antibodies as a predictor of clinically definite

multiple sclerosis after a first demyelinating event. N Engl J

Med. 2003;349(2):139–45.

301. Karussis D, Biermann LD, Bohlega S, Boiko A, Chofflon M,

Fazekas F, et al. A recommended treatment algorithm in

relapsing multiple sclerosis: report of an international consensus

meeting. Eur J Neurol. 2006;13(1):61–71.

302. Treatment optimization in multiple sclerosis. Report of an

international consensus meeting. Eur J Neurol. 2004;11(1):43–7.

303. Gobbini MI, Smith ME, Richert ND, Frank JA, McFarland HF.

Effect of open label pulse cyclophosphamide therapy on MRI

measures of disease activity in five patients with refractory

relapsing-remitting multiple sclerosis. J Neuroimmunol. 1999;

99(1):142–9.

304. Karussis D, Shor H, Yachnin J, Lanxner N, Amiel M, Baruch K,

et al. T cell vaccination benefits relapsing progressive multiple

sclerosis patients: a randomized, double-blind clinical trial. PLoS

One. 2012;7(12):e50478. doi:10.1371/journal.pone.0050478

D. Karussis