Journal of Neuroimmunology 264 (2013) 8–13

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Journal of Neuroimmunology

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Cladribine interferes with IL-1β synaptic effects in experimentalmultiple sclerosis

Alessandra Musella a,b,1, Georgia Mandolesi a,b,1, Antonietta Gentile a,b, Silvia Rossi a,b, Valeria Studer a,b,Caterina Motta a,b, Helena Sepman a, Diego Fresegna a,b, Nabila Haji a, Andrea Paolillo c,Giuseppe Matarese d, Diego Centonze a,b,⁎a Fondazione Santa Lucia, Centro Europeo per la Ricerca sul Cervello (CERC), 00143 Rome, Italyb UOC Neurologia, Dipartimento di Medicina dei Sistemi, Università Tor Vergata, 00133 Rome, Italyc Neurology Therapeutic Area, Merck Serono, Rome, Italyd Istituto di Endocrinologia e Oncologia Sperimentale, Consiglio Nazionale delle Ricerche (IEOS-CNR), 80131 Napoli, Italy

⁎ Corresponding author at: UOC Neurologia, DipartimUniversità Tor Vergata, Via Montpellier 1, 00133 Rome,fax: +39 06 7259 6006.

E-mail address: centonze@uniroma2.it (D. Centonze).1 Equally contributing first authors.

0165-5728/$ – see front matter © 2013 Elsevier B.V. All rhttp://dx.doi.org/10.1016/j.jneuroim.2013.08.009

a b s t r a c t

a r t i c l e i n f o

Article history:Received 30 June 2013Received in revised form 16 August 2013Accepted 20 August 2013

Keywords:EAEEPSCIL-1βMicrogliaStriatumCladribine

Alterations of glutamate-mediated synaptic transmission occur in bothmultiple sclerosis (MS) and experimentalautoimmune encephalomyelitis (EAE), the animal model of MS. Here we investigated whether intracerebroven-tricular (Icv) administration of cladribine has effects on EAE.Icv infusion of cladribine reduced the clinical deficits of EAEmice and reversed EAE-induced enhancement of ex-citatory postsynaptic current (sEPSC) frequency, a neurophysiological measure of glutamatergic synaptopathyassociatedwith central inflammation. Cladribine failed to interferewith EAE-inducedmicroglial and astroglial ac-tivation, but blocked EAE synaptic alterations by interfering with interleukin-1β effects.Cladribine possesses neuroprotective properties in experimental MS that are independent of its peripheral im-munosuppressant action.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Multiple sclerosis (MS) is a chronic inflammatory disorder thataffects the central nervous system, leading to progressive and irrevers-ible neuronal degeneration. The current pharmacological treatment ofMS largely relies upon medications that interfere with the abnormalimmune response at the basis of the disease, but neuroprotection ishighly desirable in MS as in classical neurodegenerative disorders. Themechanisms of the neurodegenerative damage in MS are only incom-pletely understood, but evidence exists that deficits of synaptic trans-mission might be involved (Pitt et al., 2000; Sarchielli et al., 2003;Clements et al., 2008; Centonze et al., 2009; Rossi et al., 2011).

Cladribine (2-chloro-2′-deoxyadenosine, CdA) is a purine ana-log, responsible for the toxic accumulation of triphosphorylateddeoxyadenosine in lymphocytes, resulting in reduction of thesecells in the periphery. The therapeutic efficacy of cladribine hasbeen assessed in several autoimmune disorders, and its parenteralformulation is currently used as first-line treatment for hairy-cell

ento di Medicina dei Sistemi,Italy. Tel.: +39 06 7259 6010;

ights reserved.

leukemia. The efficacy of cladribine in MS has been evaluated in theCLARITY clinical trial, the first completed phase 3 trial of an oral treat-ment for relapsing–remitting MS (Gasperini et al., 2010; Giovannoniet al., 2010; Cook et al., 2011;Warnke et al., 2012), leading to the regula-tory approval in Russia and Australia. Because of safety concerns, thedrug did not obtain licensing in the USA and Europe.

In the CLARITY study, cladribine treatment resulted in significantreductions in both number of active brain lesions and rate of clinicalrelapses, and also in significantly lower probability of disability progres-sion (Giovannoni et al., 2010, 2011). This latter information might sug-gest neuroprotective effects of cladribine, but the possible effects of thiscompound in the central nervous system have never been addressed sofar.

Thus, in the present investigation we explored the effects of intra-cerebroventricular (Icv) infusion of cladribine on the clinical and synapticabnormalities associated with experimental autoimmune encephalomy-elitis (EAE), whichmodels MS inmice. Our results provide evidence thatcladribine possesses neuroprotective properties in experimental MS byinterfering with the effects of interleukin-1β (IL-1β) at central synapses.Icv cladribine, in fact, reduced the clinical deficits of EAE mice, andreversed EAE- and IL-1β-induced enhancement of excitatory postsyn-aptic current (sEPSC) frequency, a neurophysiological measure ofglutamatergic synaptopathy associated with central inflammation(Rossi et al., 2010, 2012a; Grasselli et al., 2013).

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2. Materials and methods

All effortsweremade tominimize animal suffering and to reduce thenumber of mice used, in accordance with the European CommunityCouncil Directive of November 24, 1986 (86/609/EEC).

2.1. EAE induction and clinical score

As described (Centonze et al., 2009; Mandolesi et al., 2012), chronicrelapsing EAE was induced in C57BL/6 female mice (n = 28, 8 weeksold) by subcutaneous immunization with 300 μl of 200 μg MOG (35–55) (Multiple Peptide System) in incomplete Freund's adjuvant con-taining 8 mg ml-1 Mycobacterium tuberculosis (strain H37Ra; Difco).Pertussis toxin (Sigma) (500 ng) was injected on the day of the immu-nization and again two days later. Body weight and clinical score (0 =healthy; 1 = limp tail; 2 = ataxia and/or paresis of hindlimbs; 3 = pa-ralysis of hindlimbs and/or paresis of forelimbs; 4 = tetraparalysis;5 = moribund or death) were recorded daily.

Mice treated with complete Freund's adjuvant without MOG andpertussis toxin were used as the healthy control group (n = 20). Allanimals were housed, four per cage with food and water ad libitum, ona 12 h light/dark cycle with lights on at 06:00 h and controlled (22–23 °C) temperature. In some experiments, cladribine (supplied byMerck Serono) was given once daily by gavage at a dose of 0.4 mg/mouse started the same day of the immunization.

2.2. Minipump implantation and continuous intracranial infusions

One week before immunization, some EAE mice were implantedwith a minipump under ketamine (100 mg/kg) anesthesia, in order toallow continuous Icv infusion of either vehicle (n = 8) or cladribine(0.24 mg/mouse) (n = 8) for two weeks. ALZET osmotic minipumps(model 1004; DURECT Corporation, Cupertino, CA) connected via acatheter tube to the intracranial cannula (ALZET Brain Infusion Kits 3)delivered the vehicle or cladribine into the right lateral ventricle at acontinuous rate of 0.11 μl/h. The coordinates used for Icvminipump im-plantation were: anteroposterior: −0.4 mm; lateral: −1 mm; depth:2.5 mm from the skull.

2.3. Electrophysiology

Micewere killed by cervical dislocation under halothane anesthesia,and corticostriatal coronal slices (200 μm) were prepared from freshtissue blocks of the brain with the use of a vibratome (Centonze et al.,2009; Rossi et al., 2012a). Single slices were then transferred to arecording chamber and submerged in a continuously flowing artificialCSF (ACSF) (34 °C, 2–3 ml/min) gassed with 95% O2–5% CO2. The com-position of the control ACSF was (in mM): 126 NaCl, 2.5 KCl, 1.2 MgCl2,1.2 NaH2PO4, 2.4 CaCl2, 11 Glucose, and 25 NaHCO3.

Recording pipettes were advanced towards individual striatal cellsin the slice under positive pressure and visual control (WinVision2000, Delta Sistemi, Italy) and, on contact, tight GΩ seals were madeby applying negative pressure. The membrane patch was then rup-tured by suction and membrane current and potentially monitoredusing an Axopatch 1D patch clamp amplifier (Axon Instruments,Foster City, CA, USA). Whole-cell access resistances measured in volt-age clamp were in the range of 5–20 MΩ. Whole-cell patch clamprecordings were made with borosilicate glass pipettes (1.8 mm o.d.;4–8 MΩ), in voltage-clamp mode, at the holding potential (HP) of−80 mV.

To study spontaneous glutamate-mediated excitatory postsynapticcurrents (sEPSCs), the recordingpipetteswerefilledwith an internal so-lution of the following composition (mM): K+-gluconate (125), NaCl(10), CaCl2 (1.0), MgCl2 (2.0), 1,2-bis (2-aminophenoxy) ethane-N,N,N,N-tetraacetic acid (BAPTA; 0.5), N-(2-hydroxyethyl)-piperazine-N-s-ethanesulfonic acid (HEPES; 19), guanosine triphosphate (GTP; 0.3),

and Mg-adenosine triphosphate (Mg-ATP; 2.0), adjusted to pH 7.3with KOH. Bicuculline (10 μM) was added to the perfusing solution toblock GABAA-mediated transmission.

Synaptic events were stored by using P-CLAMP 9.2 (Axon Instru-ments) and analyzed off line on a personal computer withMini Analysis5.1 (Synaptosoft, Leonia, NJ, USA) software. The detection threshold ofsEPSCs was set at twice the baseline noise. The fact that no false eventswould be identified was confirmed by visual inspection for each exper-iment. Offline analysis was performed on spontaneous synaptic eventsrecorded during fixed time epochs (1–2 min, 3–5 samplings), sampledevery 5 or 10 min.

Only cells that exhibited stable frequencies in control (less than 20%changes during the control samplings) were taken into account. Drugswere applied by dissolving them to the desired final concentration inthe bathing ACSF. Drugs were (in μM): CNQX (10), MK-801 (30)(from Tocris Cookson, Bristol, UK), Bicuculline (10) (from Sigma-RBI,St. Louis, USA) and IL-1β (30 ng/ml) (from PeproTech, USA).

2.4. Immunohistochemistry and confocal microscopy

EAE-vehicle, EAE-cladribine and CFA-vehicle mice were deeplyanesthetized at 21 dpi with avertine and perfused through the aortawith ice-cold 4% paraformaldehyde. Brains were post-fixed for at least4 h at 4 °C and equilibrated with 30% sucrose overnight. Thirtymicrometer-thick coronal corticostriatal sections were permeabilizedin PBS with Triton-X 0.25% (TPBS). All following incubations wereperformed in TPBS. Sections from mice were pre-incubated with 10%normal donkey serum solution for 1 h at room temperature and incu-bated eitherwith the primary antibody anti-glialfibrillary acidic protein(GFAP) (1:500) or with the primary antibody anti-ionized calciumbinding adaptormolecule 1 (Iba1) overnight at 4 °C. After beingwashedfor three times, 10 min each, sections were incubated either with thesecondary antibody Alexa-488-conjugated donkey anti-rabbit (1:200,Invitrogen, USA) or with the secondary antibody cy3-conjugated don-key anti-rabbit (1:200, Jackson, USA) for 2 h at RT, then rinsed andDAPI counterstained.

Sections were mounted with Vecta-Shield (Vector Labs, USA) onpoly-L-lysine-coated slides, air-dried and coverslipped. Images wereacquired using a LSM5 Zeiss confocal laser-scanner microscope (Zeiss,Göttingen, Germany). For the analysis of astroglial density, immuno-staining stacks of the striatum were acquired by using a 20× objective(zoom 0.5×, pixel resolution 1024 × 1024, 5 μm Z-step, pinhole of 1airy unit) The stacks were z-projected and exported in TIFF file formatby the NIH ImageJ software (http://rsb.info.nih.gov/ij/). The settingsfor the acquisition of Iba1 signal were the following: 20× objective,zoom 1×, pixel resolution 1024 × 1024, 5.51 μm Z-stack and pinholeof 1 airy unit. GFAP – either Iba1 – positive cells were counted on thez-projections. Their number was divided by the surface of striatal sec-tions to obtain respectively the astroglial and microglial densities. Mea-surements were repeated for 7–9 images from 5 sections per animal(n = 3 mice per group).

2.5. Statistical analysis

For each type of experiment and time point, at least four mice eachgroupwere employed, unless otherwise specified. For electrophysiolog-ical data throughout the text “n” refers to the number of cells, unlessotherwise specified. One to six cells per animal were recorded. Datawere presented as the mean ± S.E.M. The significance level wasestablished at p b 0.05.

Statistical analysis was performed using a paired or unpairedStudent's t-test. Multiple comparisons were analyzed by one-wayANOVA followed by Tukey HSD and contingency analyses by Chi-square test.

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3. Results

3.1. Effect of Icv infusion of cladribine on the clinical score of EAE mice

To shed some light on possible direct effects of cladribine in thebrain, thedrugwas delivered by Icv injections in EAEmice. Icv cladribineinduced a less severe disease comparedwith EAEmice receiving contin-uous Icv infusion of vehicle. In particular, the development of neurolog-ical signs was significantly inhibited in the EAE group treated withcladribine, as only 25% of EAE-cladribine mice showed clinical deficitsin comparison with the vehicle-treated mouse group, where 75% ofthe animals developed an EAE symptom (n = 8 animals for eachgroup; p b 0,05, Chi square test) (Fig. 1).

These data strongly indicate that cladribine has direct neuroprotectiveeffects, which are independent of its immunosuppressive action onperipheral lymphocytes.

Fig. 2. Effects of Icv injection and oral treatment with cladribine on striatal glutamatergic

3.2. Icv infusion of cladribine prevents EAE-induced synaptic alterations

Icv cladribine was also able to prevent EAE-induced enhancement ofspontaneous excitatory postsynaptic currents (sEPSCs). As previouslydemonstrated (Centonze et al., 2009; Rossi et al., 2010), in fact, thefrequency of glutamate-mediated sEPSCs in medium spiny neuronsincreased in the acute phase of EAE mice treated with vehicle (con-trol mice: 2.4 ± 0.11 Hz; EAE-mice 20 dpi: 4.2 ± 0.25 Hz; n = 8;p b 0.01). Treatment with Icv cladribine completely prevented thealteration of sEPSC frequency recorded at the same time point (EAE-cladribine mice, 20 dpi: 2.8 ± 0.18 Hz n = 11 p N 0.05 respect to con-trol mice). Amplitude of sEPSCs was conversely unchanged in eachexperimental group (control mice: 10.2 ± 0.51 pA; EAE mice: 11.1 ±0.25 pA; EAE-cladribine mice: 10.8 ± 0.64 pA) (Fig. 2).

These data indicate an action of cladribine at glutamatergic synapses,potentially able to reduce the excitotoxic damage associated with EAE(Smith et al., 2000; Zhu et al., 2003; Rossi et al., 2010; Ziehn et al., 2010).

transmission. (A) Icv infusion of cladribine rescued the alterations of sEPSC frequency,recorded from EAE-striatal MSNs (20–25 dpi). (B) Amplitudewas unchanged in all exper-imental groups. (C) Electrophysiological traces of voltage-clamp recordings show thereduction of sEPSC frequency in a neuron from EAE-cladribine mice (20 dpi), comparedto EAE-vehicle (20 dpi) and control mice. (One-way ANOVA **p b 0.01). (B) Oral treat-ment with cladribine was able to reduce the sEPSC frequency in EAE mice, compared toEAE mice receiving vehicle (unpaired t-test, **p b 0.01), without altering the amplitude.Data are expressed as means ± S.E.M.

3.3. Effect of oral cladribine on glutamate synapses

Cladribine is administered orally in MS patients, and to concludetherefore that the treatment can be neuroprotective, it is important toshow that oral cladribine can pass the blood–brain barrier to affectglutamatergic transmission. Indeed, oral cladribine was able to preventEAE-induced enhancement of sEPSC frequency (EAE-vehicle 20–27 dpi:4.8 ± 0.32 Hz; EAE-cladribine mice: 2.7 ± 0.21 Hz; n: at least 10 neu-rons from each experimental group p b 0.01) without affecting sEPSCamplitude (EAE mice: 11.1 ± 0.25 pA; EAE-cladribine mice: 10.8 ±0.64 pA), thereby mimicking the effect of the Icv treatment (Fig. 2).

Fig. 1. Effects of in vivo cladribine on EAE score. (A, B) Icv injection of cladribine induced a less sterms of development of neurological signs (Chi-square test, *p b 0.05).

3.4. Effects of Icv cladribine on astroglial and microglial proliferation

Inflammatory cytokines released from activated microglial andastroglial cells are responsible of altered synaptic transmission in EAE(Centonze et al., 2009; Rossi et al., 2011, 2012a). Moreover, both

evere disease, in EAEmice compared to EAEmice receiving vehicle, statistically different in

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microgliosis and astrogliosis have been described among the striatal pa-renchyma of EAE mice (Centonze et al., 2009; Haji et al., 2012; Grasselliet al., 2013). Thus, to uncover a possible effect of cladribine onmicrogliaand astroglia we investigated the proliferation rate of these cells byimmunofluorescence for Iba-1 (marker of microglia/macrophage cells)and GFAP (marker of astroglia) in EAE mice treated or not with Icvcladribine.

Immunofluorescence analysis of both microglial and astroglialdensities in the striatum performed during the acute phase of thedisease failed to reveal significant effects of in vivo treatment ofcladribine on both cell types. While the proliferation of both microglia(Fig. 3A, red staining) and astroglia (Fig. 3C, green staining) in theEAE-vehicle striata compared to CFA-vehicle was evident, the stainingfor Iba-1 and GFAP in EAE-cladribine striatum was very similar to thatobserved in EAE-vehicle striata (Fig. 3A-C) . In fact, neither Iba-1 norGFAP immunostainings were significantly different in terms of numberof cells in the striatum of EAE mice (25 dpi) treated or not withcladribine, as shown by the cell density quantification reported inFig. 3B–D (Iba1 density: CFA-vehicle 13.43 ± 1.84, EAE-vehicle32.11 ± 2.02, EAE-cladribine 30.92 ± 2.01; GFAP density: CFA-vehicle55.38 ± 3.90, EAE-vehicle 97.56 ± 10.15, EAE-cladribine 114.2 ±8.82; p N 0.001 post-hoc comparison CFA-vehicle vs EAE-vehicle andCFA-vehicle vs EAE-cladribine for both of the analysis).

These data suggest that cladribine exerts its protection against gluta-mate excitotoxicity without interfering with microglial and astroglialproliferation, which characterize the striatum of mice undergoingneuroinflammation.

3.5. Effect of cladribine on synaptic transmission

Potential candidates capable to influence synaptic transmissionduring neuroinflammatory diseases are soluble mediators, released bybrain infiltrating T lymphocytes and activated microglia and astroglia.IL-1β is one of the major pro-inflammatory cytokine involved in EAE-

Fig. 3. In vivo cladribine treatment does not affect proliferation of bothmicroglial and astroglial ipositive cell densities (red, counterstainedwith DAPI-blue) among striata of control (CFA-vehicland EAE-cladribine striata. Scale bar: 50 μm. (B) Histogram showing the quantification of Iba1-pcomparison: ***p N 0.001). (C) Astroglial cell density, identified by GFAP immunostaining (greevehicle, but is unchanged compared to EAE-cladribine. Scale bar: 50 μm. (D) The quantificationEAE striatum (Tukey post hoc comparison: ***p N 0.001).

induced brain damage (Zhao et al., 1998; Mandolesi et al., 2012), andrecently found to mediate the excessive synaptic excitation duringEAE, as it enhances the frequency of sEPSCs (Musumeci et al., 2011;Rossi et al., 2012a).

Thus, to clarify the mechanism implicated in the neuroprotectiveand synaptic effects of cladribine, we studied the interference of thiscompound with IL-1β effects on glutamate synaptic transmission. Bathapplication of cladribine (10 μM) was unable to alter per se sEPSCfrequency (n = 7; paired t-test p N 0.1), but fully prevented the synap-tic effects of IL-1β on glutamate transmission. Application of IL-1β incontrol slices was in fact able to increase sEPSC frequency (120.8 ±7.2% of pre-drug values, paired t-test p = 0.03; n = 5), as previouslydemonstrated in striatal slices (Musumeci et al., 2011; Rossi et al.,2012a). Pre-incubation with cladribine (5–8 min) fully abolished theeffect of this cytokine (sEPSC frequency: 101.3 ± 8.2% of pre IL-1β,paired t-test p N 0.05; n = 5) (Fig. 4).

4. Discussion

The present study demonstrated that the clinical and synapticdefects of EAE mice can be significantly attenuated by cladribine,suggesting that treatment with this pharmacological agent could affordneuroprotective effects in patients withMS. A direct action of cladribineon central neurons is involved in this neuroprotective effect, as bothclinical score and synaptic transmission alterations seen in EAE micewere rescued by Icv injections of this drug.

Based on a series of experimental findings, excessive signaling atglutamate synapses has been proposed as a crucial intermediate steplinking inflammation and neurodegeneration in EAE and in MS(Centonze et al., 2010). Glutamate levels are in fact higher in the cere-brospinal fluid (Stover et al., 1997; Sarchielli et al., 2003) and in thebrains of MS patients (Srinivasan et al., 2005; Cianfoni et al., 2007),and glutamate receptor antagonists exert beneficial effects in EAE(Wallström et al., 1996; Bolton and Paul, 1997; Pitt et al., 2000; Smith

n the striatumof EAEmice. (A) Immunostaining of striatal sections showing different Iba1-e) and EAE-vehicle animals. No visible differences can be appreciated between EAE-vehicleositive cells counted on striatal sections of the three experimental groups (Tukey post hocn, counterstained with DAPI-blue), is increased in EAE-vehicle striatum compared to CFA-of the GFAP positive cells confirms the absence of an effect of cladribine on astrogliosis in

Fig. 4.Effect of IL-1β potentiation on the glutamatergic frequency. (A) The increase of sEPSC frequencymediated by IL-1βwas abolished in slices pre-incubatedwith cladribine. (B) Exampleof electrophysiological traces of sEPSC, before and after incubation of IL-1β, that show the increase of frequency only in control condition.

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et al., 2000; Centonze et al., 2009; Grasselli et al., 2013) and inMS (Plaut,1987). Themechanism bywhich effector T lymphocytes activated in theperiphery against myelin antigens lead to synaptic alterations andneurodegeneration in the central nervous system of EAE mice hasbeen partially elucidated in previous studies. Inflammatory infiltratescomposed of CD3+ T cells and activated microglial cells are in factresponsible for the release of a number of proinflammatory cytokines,which cause significant adaptations of synaptic transmission. In ourEAE model, we have recently demonstrated that activated CD3+ Tcells have the ability of producing and releasing high concentrations ofIL-1β (Mandolesi et al., 2013), and that IL-1β both enhances glutamatetransmission (Rossi et al., 2012a) and reduces GABA signaling(Mandolesi et al., 2012; Rossi et al., 2012b), thereby favoring excitotoxicneuronal damage.

The described effects of cladribine on the synaptic hyperexcitabilityaccompanying EAE do not seem to involve an action on microglial orastroglial activation, and cladribine is instead mediated by the interfer-ence with the effect of IL-1β at glutamatergic synapses. In this respect, itis worth mentioning that pharmacological compounds able to interferewith IL-1β synaptic effects in EAEmice are likely to exert neuroprotectiveeffects also in MS patients. We have in fact recently demonstrated thatboth the IL-1β enhancement of glutamate transmission and the IL-1βdependent reduction of GABA signaling typical of EAE can be replicatedin control brain tissues incubated with the cerebrospinal fluid (CSF) ofMS patients (Rossi et al., 2012a, 2012b). Thus, these results indicatethat in the course of MS proinflammatory cytokines such as IL-1β arereleased from infiltrating T cells and from activated microglial cells at aconcentration high enough to diffuse within the CSF circulation in MSpatients, and to cause a widespread alteration of synaptic transmissionand finally neuronal death (Rossi et al., 2012a, 2012b).

Further investigations are needed to clarify the mechanisms at thebasis of cladribine effects on synaptic and neuronal functioning duringEAE. This information might be helpful to better define the relevanceof this agent as a neuroprotective compound in MS, as well as in otherdisorders in which excitotoxicity processes play a role.

Conflicts of interest

Dr. Centonze acted as an Advisory Board member of Merck-Serono,Teva, Bayer Schering, Biogen Idec, Novartis, Almirall, and GW Pharma-ceuticals, and received funding for traveling and honoraria for speakingor consultation fees fromMerck Serono, Teva, Novartis, Bayer Schering,Sanofi-aventis, and Biogen Idec. He is the principal investigator in clini-cal trials for Novartis, Merck Serono, Teva, Bayer Schering, Sanofi-aventis, Biogen Idec, and Roche.

Dr. Silvia Rossi received honoraria for writing from Bayer Scheringand funding for traveling from Novartis, Teva, and Merck Serono. She

acted as an Advisory Board member of Biogen Idec and is involved asa sub-investigator in clinical trials for Novartis, Merck Serono, Teva,Bayer Schering, Sanofi-aventis, Biogen Idec, and Roche.

Dr Andrea Paolillo is an employee of Merck Serono, Rome, Italy.The other authors have no conflict of interest.

Acknowledgments

The present investigation was supported by a grant from MerckSerono to DC.

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