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b r a i n r e s e a r c h 1 4 8 8 ( 2 0 1 2 ) 9 2 – 1 0 3

0006-8993/$ - see frohttp://dx.doi.org/10

nCorrespondenceE-mail address:

1 Current addres

Research Report

Immunization with a peptide of Semliki Forest viruspromotes remyelination in experimentalautoimmune encephalomyelitis

Foroozan Mokhtariana,c,d,n, Farinaz Safavia,1, Ehsan Sarafraz-Yazdib

aDepartment of Cell Biology, SUNY Downstate, USAbDepartment of Pathology, SUNY Downstate, USAcDepartment of Neurology, SUNY Stony Brook, USAdSUNY Stony Brook Incubator, USA

a r t i c l e i n f o

Article history:

Accepted 23 September 2012

Remyelination is one of the elusive topics in treatment of multiple sclerosis (MS). Our previous

studies have shown that Semliki Forest virus (SFV)-infected d-knock-out (KO) mice did not

Available online 29 September 2012

Keywords:

Experimental Autoimmune

Encephalitis (EAE)

Semliki Forest virus (SFV)

E2 peptide2

IFA (incomplete Freund’s Adjuvant)

Demyelination

Remyelination

Treatment

Antibody

Oligodendrocyte

OPC (Oligodendrocyte Precursor

Cell)

Astrocyte

nt matter & 2012 Elsevie.1016/j.brainres.2012.09.0

to: SUNY Stonybrook IncfmokhtarianSUNY@aol.cs: Department of Neurol

a b s t r a c t

exhibit the extensive remyelination, seen in wild type (WT) B6 mice, after viral clearance and

demyelination. The Remyelination in SFV-infected WT mice started on day 15 and was

completed by day 35 post-infection (pi), whereas the KO mice remained partially demyelinated

through day 42 pi. Treatment with E2 peptide2 in incomplete Freund’s adjuvant (IFA), resulted in

higher antibody production and earlier remyelination in SFV-infected KO (day 28 pi), than WT

mice. This finding suggested that anti-E2 peptide2 antibody could play a part in remyelination.

In the current study, the effect of E2 peptide2 treatment was evaluated in the experimental

autoimmune encephalomyelitis (EAE) model. Mice with established EAE were treated with E2

peptide2 in IFA to develop antibody. Treated EAE mice made significantly higher anti-E2

peptide2 antibody than untreated EAE group. Average clinical disease scores were significantly

lower in peptide treated compared to untreated EAE mice. Furthermore, histopathological and

immunohistochemical studies demonstrated increased remyelinating areas and higher number

of activated oligodendrocytes and astrocytes, in treated compared to untreated EAE groups.

Moreover, the anti-E2 peptide2 antibody showed higher binding to the myelinated areas of

treated than untreated EAE mice. We conclude that treatment with, or antibody to, SFV E2

peptide2 triggers some mechanism that promotes remyelination.

& 2012 Elsevier B.V. All rights reserved.

r B.V. All rights reserved.38

ubator, 4603 Middle Country Road, Calverton, NY 11933, USA. Fax: þ1 718 745 5335.om (F. Mokhtarian).ogy, Thomas Jefferson University, USA.

1. Introduction

In multiple sclerosis (MS), autoimmune-mediated damage to

myelin within the CNS leads to progressive disability primarily

due to limited endogenous repair of demyelination (Storch and

Lassmann, 1997; Lucchinetti et al., 1998). While treatments are

available to limit demyelination (Vennegoor et al., 2011;

Vandenbroeck et al., 2010; Miller and Jezewski, 2006; Stuve

et al., 2006; Wolinsky et al., 2007; Hassen et al., 2006) and axonal

damage (Hassen et al., 2008), therapies that induce remyelination

b r a i n r e s e a r c h 1 4 8 8 ( 2 0 1 2 ) 9 2 – 1 0 3 93

constitute a new paradigm (Miller et al., 1995; Freedman et al.,

2011; Rudick et al., 2008; Mi et al., 2009). Our E2 peptide2

treatment protocol in the chronic-progressive EAE model has

produced very encouraging remyelinating results and if these

results can be reproduced in MS patients, this new clinical

paradigm would present a potential curative treatment for MS.

EAE is an autoimmune animal model of MS, driven

by activation of auto-aggressive myelin specific T cells

(Mokhtarian et al., 1984; Sobel et al., 1994; Mendel et al., 1995;

Beraud et al., 1986; Zamvil et al., 1986; Amor et al., 1994). In

contrast, there is some evidence that certain natural human

antibodies may mediate remyelination and repair in EAE (Miller

et al., 1997; Asakura and Rodriguez, 1998; Warrington et al.,

2000). It has also been shown that this antibody may play a role

in remyelination in other demyelinating animal models such as

the Theiler’s murine encephalomyelitis (Miller et al., 1994) and

Lysolecithin-induced demyelinating models (Pavelko et al.,

1998). It has also been reported that antibodies reactive with

myelin basic protein (MBP) promoted CNS remyelination

(Rodriguez et al., 1996). Glatiramer Acetate (GA), one of the

established treatments for MS, may act in part through an

antibody mediated repair mechanism (Ure and Rodriguez,

2002). Although treatment with GA downregulates certain

immune functions, passive transfer of GA reactive T cells and

anti GA antibody can facilitate the repair of demyelinated

lesions in mice with EAE (Ure and Rodriguez, 2002). These

findings indicate that antibodies may have a potential role in

repair and remyelination.

We have previously shown that antibody production against

one of the SFV E2 epitopes (E2 peptide2), induces disease

recovery and promotes remyelination in SFV-infected d-KO mice

(Safavi et al., 2011). Treatment of these mice with E2 peptide2

induced higher antibody response and accelerated recovery and

remyelination in these mice, indicating the possible role of anti-

E2 peptide2 antibody in these processes. In order to further

investigate the role of E2 peptide2 antibody in remyelination,

EAE model has been used in the current study. The results have

suggested that treatment with E2 peptide2, and the antibody to

this peptide, improved remyelination and recovery from chronic

progressive EAE animal model. The immunoregulatory mechan-

isms exerted by anti-E2 peptide2 antibody, and the role of

activated oligodendrocytes and astrocytes, leading to improved

remyelination, have been discussed.

2. Results

2.1. Induction of EAE and treatment with E2 peptide2

C57BL/6J (B6) mice were inoculated with MOG 35-55 in CFA and

treated with E2 peptide2, as outlined in Experimental procedure.

To investigate the role of E2 peptide2 treatment in remyelina-

tion, scores of clinical disease, antibody production and de-and

remyelination were evaluated in treated and untreated groups.

2.2. Clinical scores of EAE in untreated mice is higherthan in peptide treated EAE mice

Untreated MOG 35-55-inoculated mice developed neurologi-

cal signs of EAE, such as floppy tail and hind limb weakness,

starting at 10 days (D10) post-EAE inoculation of (pEi). The

effect of E2 peptide2 treatment before the onset of clinical

scores in EAE, was evaluated. Treatment with E2 peptide2/IFA

of EAE mice was performed on D5 pEi. Two separate experi-

ments were conducted with total number of 12 untreated EAE

and 12 D5 E2 peptide2-treated EAE mice. Daily clinical score

evaluation showed that early peptide treatment inhibited the

appearance of clinical scores of EAE in treated mice, and

these mice did not show any disease symptoms, even at later

days following treatment (data not shown).

Next, one group of EAE mice were treated with E2 peptide2/

IFA on day 12 pEi, at which time at least 50% of mice showed

visible signs of disease. Neurological signs of EAE increased in

severity in all mice and reached a peak by day 19 pEi (Fig. 1).

Treatment with 0.5 mg of E2 peptide2/IFA led to the improve-

ment of clinical symptoms of EAE mice and prevented the

progression of this disease. There was a significant difference in

the clinical scores between the untreated and treated EAE

groups on day 19 pEi (Po0.001) (Fig. 1). The difference between

the two groups became smaller, but remained significant on

day 26 (Po0.05), and remained so up to day 42 pEi (po0.05).

There was no significant difference between average clinical

score of untreated EAE and saline/IFA-treated EAE mice (data

not shown). Control mice injected with only CFA or only E2

peptide2/IFA did not show any disease symptoms.

2.3. Anti-E2 peptide2 antibody-treated EAE mice displayless severe clinical disease than untreated EAE mice

EAE mice were also treated with purified IgG of anti-E2

peptide2 antibody (0.5 mg/injection) on D12, D17 and D22

pEi, in two different experiments. All anti-E2-peptide2

antibody-treated EAE mice developed lower clinical scores

than untreated EAE mice, during the time of treatment

(po0.05, on day 18 pEi) (Fig. 2). However, following secession

of antibody injection on D22 pEi, clinical scores increased by

D28 pEi, in all antibody-treated EAE mice and reached the

same level as untreated EAE mice (Fig. 2).

2.4. Remyelination is more extensive in E2 peptide2-treated EAE than untreated EAE mice

Initially, using H&E staining of spinal cord sections of all groups

at 2, 4 and 6 weeks pEi, perivascular cuffs and parenchymal

infiltration of mononuclear cells were seen in the spinal cords of

both untreated and treated EAE mice. The extent of the inflam-

matory response was more severe in untreated than in E2

peptide2-treated EAE mice on all weeks tested (data not shown).

In order to be able to analyze de- and remyelination

accurately in untreated and E2 peptide2-treated EAE mice,

toluidine blue stained one-micron sections of lumbar spinal

cords of all mice were compared after 6 weeks pEi (Fig. 3).

First, a section of lumbar spinal cord of a normal control

mouse is shown for comparison. Note the darkly stained

myelin sheaths around the cross sections of nerve fibers

(Fig. 3A). Similar section from untreated EAE mice at 6 weeks

pEi, showed the presence of myelin debris, indicating mas-

sive destruction of white matter throughout the field, and

leading to demyelinated fibers (Fig. 3B, see arrows). Lumbar

spinal cord sections from E2 peptide2-treated EAE mice

Comparison of average clinical scores in untreated and Ab treated EAE mice

00.20.40.60.8

11.21.41.61.8

22.22.42.62.8

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42Day post EAE induction

Clin

ical

sco

re

Ab treated EAE Untreated EAE

Fig. 2 – Comparison of average clinical scores in untreated and anti-E2 peptide2 antibody- treated EAE mice. EAE was induced

as described in Experimental procedure. Two separate experiments were performed and in each experiment mice were

divided into untreated EAE, anti-E2 peptide2 antibody-treated EAE, saline treated EAE and nave control groups. There were

six mice in the first two groups, and two mice in the last two groups. Treated group received the antipeptide antibody on

days 12, 17 and 22 pEi. Animals were observed daily for clinical manifestations of disease and were scored on a scale of 0 to

6, as described in Experimental procedure. Average clinical scores of untreated4antibody-treated EAE mice on day 18

(po0.05).

Comparison of average clinical scores in untreated and treated EAE mice

0

0.5

1

1.5

2

2.5

0Day post EAE induction

Clin

ical s

core

Untreated EAE mice Treated EAE mice CFA control mice

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

Fig. 1 – Comparison of average clinical scores in untreated and E2 peptide2/IFA-treated EAE mice. To induce EAE all mice were

injected subcutaneously with MOG 35-55/CFA, as described in Experimental procedure. Three separate experiments were

performed and in each experiment, mice were divided into untreated EAE, E2 peptide2/IFA-treated EAE, saline/IFA-treated

EAE and CFA only, E2 peptide2/IFA only and nave control groups. There were 6–8 mice in each group in all experiments.

Treated received the peptide on days 12, 17 and 22 pEi. Animals were observed daily for clinical manifestations of disease

and were scored on a scale of 0 to 6, as described in Experimental procedure. Average clinical scores of untreated4peptide-

treated EAE mice, on 19 (po0.001), day 26 (po0.05) and day 42 (po0.01) pEi.

b r a i n r e s e a r c h 1 4 8 8 ( 2 0 1 2 ) 9 2 – 1 0 394

showed fewer demyelinated fibers compared to untreated

EAE mice (Fig. 3C), and also revealed thinly remyelinated

fibers (Fig. 3C, see arrows), whereas no evidence of remyeli-

nated fibers was seen in the sections of untreated EAE mice

(Fig. 3B). As seen in the magnified region of each one of the

panels in Fig. 3, the intense dark ring of myelin is clearly

visible in the normal sample (A). In contrast, the darkly

stained ring of myelin is virtually undetectable in EAE sample

(B). The re-appearance of a thin ring of myelin indicates

remyelination (C). Thus, remyelination, as evidenced by the

presence of fibers surrounded by a thin layer of myelin was

only present in E2 peptide2-treated EAE mice and virtually

absent in untreated group throughout the experiment.

2.5. Antibody responses of E2 peptide2-treated EAE to E2peptide2 is higher than untreated EAE mice

Sera obtained from untreated and E2 peptide2-treated EAE

mice, reacted with MOG 35-55 and the two E2 peptides;

peptide1 and peptide2, in ELISA assays, when tested at 6

Fig. 3 – Demyelination and remyelination in untreated and E2 peptide2/IFA-treated EAE mice. All figures come from one micron

epoxy sections taken from lumbar spinal cord tissues, fixed in 2.5% glutaraldehyde/1% osmium tetroxide, embedded in epoxy

resin, stained with toluidine blue and photographed by light microscopy. (A) Cross section of normally myelinated fibers in

ventral column of lumbar spinal cord tissue is shown for comparison. (B) Lumbar spinal cord, untreated EAE mice, 6 weeks post-

EAE induction (pEi). Lesions of demyelination (arrows and outline magnified) and residual inflammatory activity are seen in

ventral column of lumbar spinal cord. (C) Lumbar spinal cord, peptide-treated EAE mice, 6 weeks pEi. In contrast to (B), The fibers

display remyelination characterized by thin myelin sheets in cross-section fibers (arrows and outline magnified).

Fig. 4 – Comparison of anti-E2 peptide1, anti-E2 peptide2 and anti-MOG 35-55 antibody responses (7SEM) in untreated and

E2 peptide2/IFA—treated EAE mice at 6 weeks pEi. Sera were used at dilution of 1:80. Amount of antibody is shown as mean

optical density minus background (OD) of sera from three experiments7standard error of the mean (SEM).�Anti-E2 peptide2

antibody of treated4than untreated EAE mice (po0.05).

b r a i n r e s e a r c h 1 4 8 8 ( 2 0 1 2 ) 9 2 – 1 0 3 95

weeks pEi. Sera reacted strongly with MOG 35-55 in both

groups. Each time 3–4-serum samples were used and the

average of three different experiments is shown in Fig. 4. The

anti-E2 peptide2 antibody was higher but not significantly

different after 4 weeks, but became significantly higher in

treated than in untreated EAE mice after 6 weeks pEi (po0.05)

b r a i n r e s e a r c h 1 4 8 8 ( 2 0 1 2 ) 9 2 – 1 0 396

(Fig. 4). There was no difference between the reactivity of sera

from untreated and treated EAE, to E2 peptide1, used as

control. This result indicated that E2 peptide2-treatment

effectively increased the amount of antibody to this peptide

in the EAE mice.

2.6. Activated astrocytes are more frequent in spinalcords of E2 peptide2-treated EAE than untreated EAE mice

In order to evaluate activated astrocytes, sections of lumbar

spinal cords from both untreated and E2 peptide2-treated EAE

groups were stained with antibody to the marker for astro-

cytes, glial fibrillary acidic protein or GFAP. Average fluores-

cent density was measured and compared by Image J

software (NIH) in ventral column of spinal cord. The average

of three experiments is shown in bar graph (Fig. 5). E2

peptide2-treated EAE mice showed significantly higher aver-

age fluorescent density in the ventral column of spinal cords

than untreated EAE mice (po0.01). This indicated that treat-

ment with E2 peptide2 might play a role in the increased

Untreated EAE

0200000400000600000800000

10000001200000140000016000001800000

Integrity De

Fig. 5 – Activated astrocytes in untreated and E2 peptide2/IFA

untreated and treated EAE mice, 6 weeks pEi, were stained wit

mouse immunoglobulin. Average density of immunofluorescen

was measured with Image J software, and photographed by flu

mice, 6 weeks pEi. Activated astrocytes are seen as high densit

(B) Lumbar spinal cord, treated EAE mice, 6 weeks pEi. Highe

lumbar spinal cord 40� . The average of three experiments is s

mice4untreated EAE mice (po0.01). (For interpretation of the ref

the web version of this article.)

activation of astrocytes, which may in turn activate oligoden-

drocytes and indirectly promote remyelination in treated

EAE group.

2.7. Significant increase in the number ofoligodendrocytes is observed in peptide-treated compared tountreated EAE mice

To measure the number of oligodendrocytes, these cells were

stained with the antibody to an oligodendrocyte marker

(CNPase), using DAB immunohistochemistry method. The num-

ber of oligodendrocytes was counted in ventral column of

lumbar spinal cord sections from untreated and E2 peptide2-

treated EAE mice, as described in Experimental procedure. The

results are shown in Fig. 6, and in a magnified representative

region in the corner, as well. The average of three experiments is

depicted in a bar graph (Fig. 6). There was a significantly higher

number of oligodendrocytes in treated than in untreated group

(po0.01). This result demonstrated that E2 peptide2-treatment

led to higher proliferation and/or increased migration of

Peptide -Treated EAE

nsity

EAE

EAE teated

-treated EAE mice. Sections of lumbar spinal cords from

h GFAP antibody and immunofluorescence-conjugated-anti-

se in activated astrocytes, in untreated and treated EAE mice

orescent microscopy. (A) Lumbar spinal cord, untreated EAE

y green color in ventral column of lumbar spinal cord 40� .

r activation is observed in astrocytes in ventral column of

hown in bar graph. Average integrity density in treated EAE

erences to color in this figure legend, the reader is referred to

Untreated EAE Peptide-Treated EAE

Comparison of average oligodendrocytenumber in EAE and treated EAE mice

0

2

4

6

8

10

12

aver

age

num

ber o

f olig

oden

droc

ytes

EAE miceTreated EAE mice

Fig. 6 – Oligodendrocytes in untreated and E2 peptide2/IFA treated EAE mice. Sections of lumbar spinal cords were stained

with CNPase antibody, biotinylated anti-mouse immunoglobulin, and avidin–HRP, and photographed by light microscopy.

(A) Lumbar spinal cord, untreated EAE mice, 6 weeks pEi. 40� and magnified at the corner. (B) Lumbar spinal cord, E2

peptide2-treated EAE mice, 6 weeks pEi. 40� and magnified at the corner. The average of three experiments is shown in bar

graph. Average number of oligodendrocytes in treated4untreated EAE mice (po0.01).

b r a i n r e s e a r c h 1 4 8 8 ( 2 0 1 2 ) 9 2 – 1 0 3 97

oligodendrocytes into demyelinated areas and consequently to

increased remyelination in treated EAE mice.

2.8. Higher number of oligodendrocyte progenitor cells(OPC) are shown in spinal cords of E2 peptide2-treated thanuntreated EAE mice

Sections from lumbar spinal cords of both E2 peptide2-

treated and untreated EAE mice were stained with antibody

to O4 (the marker for OPC), by DAB immunohistochemistry

method, in order to evaluate the number of OPC in these

mice. O4-positive cells were more prominent in the ventral

column of lumbar spinal cords of treated EAE than untreated

EAE mice after four weeks pEi (Fig. 7), but not after 6 weeks

pEi. Thus, peptide treatment may also increase proliferation

of OPC, which may lead to increased number of oligoden-

drocytes and consequently to increased remyelination in E2

peptide2-treated EAE group.

2.9. Binding of anti-E2 peptide2 antibody to the whitematter of spinal cords

Sections of the ventral column of spinal cords from E2

peptide2-treated EAE mice, stained with antipeptide

antibody, showed higher fluorescent staining intensity

than those of untreated EAE mice (Fig. 8, see arrows).

There were clear intensity differences in FITC staining, in

the white matter, between the sections from untreated-

(upper panel) and the peptide-treated EAE mice (lower

panel). Also, the stain in untreated sample was relatively,

uniformly green and not significantly above the back-

ground (left: upper panel), whereas in the peptide-treated

sample (left: lower panel) the green stain was differentially

and much more intensely in the white matter, as

compared to both the gray matter of and the white

matter of the untreated sample. This indicated a specific

localization and accumulation of the peptide in the

white matter of the treated mice during remyelina-

tion. Higher magnification of FITC-stained sections of

some representative areas of untreated (right: upper

panel) and peptide-treated (right: lower panel) samples,

as shown in the right panels of Fig. 8, also indicated a

higher intensity staining in the treated mice. Thus,

higher binding of anti-E2 peptide2 antibody to the

white matter in the CNS of treated mice may have led to

higher activation of astrocytes and oligodendrocytes in the

white matter, leading to remyelination in the treated

EAE group.

EA

EP

epti

de T

reat

edE

AE

Unt

reat

ed

20X Magnification4X Magnification

Fig. 8 – Sections of lumbar spinal cords from both untreated and E2 peptide2/IFA-treated EAE groups were stained with anti-

E2 peptide2 as primary antibody. The sections were then reacted with goat anti-rabbit FITC secondary antibody, as labeled

on the confocal micrograph. Lumbar spinal cords of untreated (upper panels) and peptide-treated EAE mice (lower panels) are

shown at 6 weeks pEi. Higher FITC intensity of anti-peptide antibody stained section of ventral column of lumbar spinal cord

is shown in at lower (left panel), and at higher magnification (right panel), 4� and 20� , respectively, as marked.

Untreated EAE Peptide-Treated EAE

Fig. 7 – Oligodendrocyte progenitor cells in untreated and E2 peptide2/IFA-treated EAE mice. Sections of lumbar spinal cords

were stained with O4 antibody, biotinylated anti-mouse immunoglobulin, and avidin–HRP, and photographed by light

microscopy. (A) Lumbar spinal cord, untreated EAE mice, 4 weeks pEi. (B) Lumbar spinal cord, E2 peptide2-treated EAE mice,

4 weeks pEi. 40� and magnified at the corner.

b r a i n r e s e a r c h 1 4 8 8 ( 2 0 1 2 ) 9 2 – 1 0 398

3. Discussion

EAE is a conventional autoimmune animal model of MS. In

EAE, autoreactive T cells specific for myelin proteins/peptides,

enter the CNS, cause inflammation and recruit macrophages,

resulting in the destruction of myelin (Mokhtarian et al., 1984;

Sobel et al., 1994; Mendel et al., 1995; Beraud et al., 1986;

Zamvil et al., 1986; Amor et al., 1994). While it is generally

accepted that B cells are incapable of initiating EAE, some

b r a i n r e s e a r c h 1 4 8 8 ( 2 0 1 2 ) 9 2 – 1 0 3 99

antibodies are able to contribute to the demyelination process

(Stefferl et al., 2000). In contrast, there is some evidence that B

cells and antibodies may have regulatory or remyelinating

roles in animal models of MS (Antel and Bar-Or, 2006; Miller

et al., 1994, 1997; Wolf et al., 1996; Racke, 2008), albeit using

different mechanisms.

We have recently shown that SFV-infected dKO mice dis-

played a higher percentage of clinical sickness than WT, and

unlike WT mice, displayed inefficient remyelination (Safavi

et al., 2011). This difference appears to be due to low antibody

response to SFV E2 peptide2 in gd KO, compared to WT mice.

Immunization (treatment) with the latter peptide induced

antibody to this peptide, which promoted recovery and

regulated remyelinating mechanisms (Safavi et al., 2011). In

current study, using the EAE model, we have found that

treatment with E2 peptide2 suppressed the progression of

EAE. Unlike in the viral models of CNS such as SFV and

Theiler’s virus, there is no clear remyelination in chronic

progressive mouse model of EAE and other mouse EAE

models (Fazakerley et al., 1997; Hassen et al., 2006, 2008).

Our experiments also confirmed that in chronic progressive

EAE model of mice, there was no completion of demyelina-

tion, only a progressive pathological evidence of myelin

destruction and no evidence of remyelination in the CNS

during the 6 weeks period of observation. The areas sugges-

tive of remyelination were only evident in the E2 peptide2-

treated mice

Using the Theiler’s virus (Miller et al., 1994; Mitsunaga

et al., 2002; Warrington et al., 2000) and EAE (Miller et al.,

1997; Asakura and Rodriguez, 1998) models of demyelination,

it has been shown that human IgM autoantibodies

(Warrington et al., 2000; Paz Soldan et al., 2003; Rodriguez

et al., 2009), and a mouse IgM monoclonal antibody raised

against myelin basic protein (Rodriguez et al., 1996), when

injected into animals with chronic demyelination, induced

myelin repair. These antibodies also play some role in

remyelination in other demyelinating animal models such

as the Lysolecithin-induced demyelinating model (Pavelko

et al., 1998).

Similar to E2 peptide2, Glatiramer Acetate (GA), one of the

established treatments for MS (Miller and Jezewski, 2006;

Munari et al., 2004; Wolinsky et al., 2007), may also act in part

through a largely antibody mediated repair mechanism

(Ure and Rodriguez, 2002). GA is an immunogenic mixture

of synthetic MBP peptides that reduces exacerbations in MS

(Teitelbaum et al., 1997). It has been reported that GA

(Copolymer 1) may affect the immune system via different

mechanisms including induction of suppressor T cells spe-

cific to shared components of MBP and copolymer1

(Teitlebaum et al., 1991), and competition with various

myelin-associated antigens for the activation of effector

T cells (Arnon et al., 1996). Similar to GA, the existence of

homology between a portion of E2 peptide of SFV and a

peptide of MBP (Mokhtarian et al., 1999) may be the under-

lying mechanism for the remyelinating effect of the E2

peptide2 treatment on EAE. The significance of this cross

reactivity to the field of MS and other autoimmune inflam-

matory diseases of the CNS rely on the concept of molecular

mimicry, as we have previously described (Mokhtarian et al.,

1999), and has been proven to be credible in several

autoimmune diseases (Fujinami and Oldstone, 1985; Ercolini

and Miller, 2005, 2006; Mamula et al., 1994; Miyazaki et al.,

1995). Some binding of the anti-E2 peptide2 antibody to the

white matter of EAE mice is seen due to shared MBP and E2

peptide2 sequences. The anti-E2 peptide2 antibody, however,

appears to bind these shared sequences, in the CNS of

peptide-treated EAE mice during remyelination, at a higher

intensity than to those of untreated EAE mice without

remyelination, indicating the appearance of more MBP and

myelin during remyelination. Staining for MBP has been

routinely used as an indicator for remyelination in many

studies. Treatment with a synthetic MBP peptide, MBP8298,

delayed disease progression in an HLA Class II-defined cohort

of patients with progressive multiple sclerosis (Freedman

et al., 2011).

In separate experiments, we have shown that treatment

with anti-E2 peptide2 antibody could improve clinical dis-

ease in EAE mice, although not as effectively as the peptide

treatment did. It would be of our interest to use a higher

affinity anti-E2 peptide2 antibody to investigate its immu-

noregulatory or remyelinating mechanisms on recovery of

EAE disease. Similarly, treatment with GA downregulates

certain immune functions and passive transfer of GA

reactive T cells and anti GA antibody can facilitate the

repair of demyelinated lesions in mice with EAE (Ure and

Rodriguez, 2002).

It has been reported that intravenous immunoglobulin

(IVIG), an established therapy for guillan bare syndrome, can

also be beneficial in treatment of MS. It may regulate the

immune system at humoral and cellular level (Soelberg

Sorensen, 2008). In addition, IVIG might have remyelinating

effect through an influence on the function of oligodendrocyte

precursor cells possibly by protecting these cells from injury

(Stangel et al., 2000). The remyelinating autoantibodies appear

to work directly at the level of glial cells. They may bind to

oligodendrocytes and directly stimulate these cells to increase

remyelination (Ciric et al., 2004). In the current study, elevated

amount of serum anti-E2 peptide2 antibody in peptide-treated

EAE mice correlated with remyelination in treated compared

with untreated EAE mice. Furthermore, higher number of

oligodendrocytes and oligodendrocyte progenitor cells (OPC)

in treated EAE mice suggested that the production of specific

antibodies after peptide treatment could have led to more

migration of oligodendrocytes into demyelinated areas and

proliferation leading to remyelination in treated EAE mice. It

has been suggested that for a successful remyelination, OPCs

must proliferate, migrate to sites of demyelination and

mature into myelinating oligodendrocytes (Belmadani et al.,

2006; Mi et al., 2009). More data is needed, however, to show

the exact mechanism of remyelination, and whether remye-

lination is a direct consequence of activation of oligodendro-

cytes and OPCs. We are now using an in vitro culture of

oligodendrocytes to investigate the effect of antipeptide anti-

body on the activation of these cells leading to remyelination.

Antibody to LINGO1, a negative regulator of oligodendrocyte

differentiation and myelination, induced differentiation of

oligodendrocyte precursor cells and promoted central nervous

system remyelination (Rudick et al., 2008). This effect helped

the recovery of mice with EAE. A monoclonal antibody against

LINGO1 is in phase 1 clinical trial (Rudick et al., 2008).

b r a i n r e s e a r c h 1 4 8 8 ( 2 0 1 2 ) 9 2 – 1 0 3100

Our studies also indicated that treatment with E2 peptide2

might be able to further activate the astrocytes, which could

indirectly promote remyelinating effect of oligodendrocytes

in treated EAE mice. Accordingly, astrocytes derived factors

and chemokines promote OPC migration (Belmadani et al.,

2006), proliferation (Valerio et al., 2002) and maturation

(Zhang et al., 2006). Furthermore, astrocytes derived factors

promote the survival of oligodendrocyte precursor cells (Nair

et al., 2008).

In summary, our studies have shown that treatment with

SFV E2 peptide2, or antipeptide antibody, led to immunomo-

dulation, improvement of clinical disease, and detectable

remyelination, in mice with EAE. The remyelination appeared

to be as a result of increased oligodendrocyte number (and

OPC), and oligodendrocyte and astrocyte activation that led to

repair and remyelination in the CNS of EAE mice. Generally,

there are different proposed mechanisms for antibody-

mediated remyelination. One of the proposed mechanisms

would be binding of anti-E2 peptide2 antibody to glia and

direct enhancement of oligodendrocyte proliferation and

migration. Another mechanism could be an immunoregula-

tory role of this antibody, which may have led to production

of some cytokines and growth factors that promote immu-

noregulation and consequently remyelination.

4. Experimental procedure

4.1. Induction of EAE

C57BL/6J female mice (7- to 8-wk-old) were purchased from the

Jackson Laboratories (Bar Harbor, MD) and housed according to

Institutional Animal Care and Use Committee (IACUC) regula-

tions in an Association for Assessment Accreditation of Labora-

tory Animal Care (AAALAC) certified facility.

EAE was induced by injecting each mouse subcutaneously

(sc) in each flank with a 100 ml emulsion containing 300 mg

MOG 35-55 in complete Freund’s adjuvant (CFA) (Sigma,

St. Louis, MO). Pertussis toxin (100 ng) (Sigma) was adminis-

tered intraperitoneally (i.p.) at the time of immunization on

days 0 and 2, as previously described (Mokhtarian et al., 1999;

Hassen et al., 2006).

4.2. Treatment with E2 peptide2

Animals were separated into two groups, experimental and

control. Three experimental groups consisted of (1) EAE mice

each treated sc with 500 mg of E2 peptide2/IFA (Sigma), as

previously described (Safavi et al., 2011) (treated EAE; n¼7), (2)

mice immunized with MOG/CFA only (untreated EAE; n¼7)

and (3) EAE mice treated with saline/IFA (n¼5). EAE mice were

randomly assigned to different subgroups after induction of

EAE. Treatment was initially began on day 5, when no mice

exhibited any signs of EAE, and later changed to day 12, after

the onset of the clinical signs were established. Treatment

with E2 peptide2/IFA was repeated on days 17 and 22 post-

EAE induction (pEi).

Three control groups consisted of (1) normal mice injected

with E2 peptide2 in IFA (n¼5), (2) normal mice injected with

CFA only (n¼5) and (3) normal control mice (n¼4).

4.3. Antibody transfer

In order to treat EAE mice with anti-E2 peptide2 antibody,

polyclonal rabbit antibody to this peptide was synthesized

(Strategic Diagnostics, Newark, DE). The final concentration

of this antibody was 9.1 mg/ml of IgG. After EAE induction,

animals were separated as above into experimental and

control groups. Each experimental mice were injected i.p.

with 0.5 mg/0.2 ml of antibody on days 12, 17 and 22 pEi.

4.4. Clinical evaluation of EAE disease

All mice were evaluated for the extent of neurological deficits

on a daily basis up to the termination of the experiment at 6

weeks pEi, by two independent investigators. Clinical severity

was assessed on a scale of 0–6 as follows: 0¼no abnormality;

1¼mild hind limb weakness (some difficulty righting them-

selves when turned on their back); 2¼moderate hind limb

weakness, sometimes associated with floppy tail; 3¼paresis

of hind limbs accompanied by some forelimb weakness,

sometimes more marked on one limb or one side, but not

complete paralysis; 4¼hind limb paralysis accompanied by

moderate forelimb weakness; 5¼paralysis of hind limbs,

associated with forelimb paralysis still able to move; and

6¼quadriplegia, moribund (Mokhtarian et al., 1984;

Mokhtarian and Swoveland, 1987).

4.5. Synthesis of peptides

The SFV epitopes, E2 peptide1 and E2 peptide2, and MOG

35-55 were synthesized (Department of Biophysics, JHU,

School of Hygiene and Public health, Balto, MD), as previously

described (Mokhtarian et al., 1999) and used to determine

antibody responses in different EAE groups (Smith-Norowitz

et al., 2000). In ELISA experiments MOG 35-55 and E2 peptide1

were used as positive and negative controls respectively.

4.6. Histopathology

In order to be able to detect de- and remyelination accurately,

CNS tissues were obtained for histopathology from untreated

and treated EAE mice at 6 weeks pEi. For this, mice were

anesthetized with ether and perfused through the heart with

cold Trump’s fixative. Optic nerve, brain and spinal cord and

spinal nerve roots were dissected out. Samples were taken from

lumbar spinal cord (L1–L4), post-fixed in cold 1% osmic acid,

embedded in Epon, and 1-mm epoxy sections were cut from all

levels. Slides were stained with 1% toluidine blue and read by

observers blinded to the code, as previously described

(Mokhtarian et al., 2003).

4.7. Enzyme-linked immunosorbent assay (ELISA)

In each experiment, sera from 3–4 untreated EAE and 3–4

treated EAE mice were collected at 0, 2, 4 and 6 weeks pEi.

The reactivity of the sera to MOG 35-55 and the two E2

peptides (1 and 2), were measured with indirect ELISA. Briefly,

Immunolon 2HB 96 well microtiter plates (VWR, Bridge Port, NJ)

were coated with 1 mg/well of each antigen in 100 ml/well

carbonate buffer overnight at room temperature. After washing,

b r a i n r e s e a r c h 1 4 8 8 ( 2 0 1 2 ) 9 2 – 1 0 3 101

plates were blocked and serum samples added. After washing,

biotinylated goat anti-mouse Igs (BD Pharmingen, Torreyana,

CA), Streptavidin conjugated to Horse Radish Peroxidase (Vector

Laboratories, Burlingame, CA), and OPD substrate (Sigma, St.

Louis, MO), were used as previously described (Mokhtarian

et al., 1999). The absorbance at 450 nm was measured using a

Vmax kinetic microplate reader (Molecular Devices, Sunnyvale,

CA), and results expressed as optical density units [OD]. Values

were considered to be positive when they were 40.3 OD units

and/or exceeded the mean value plus three standard deviations

of antibody response on day 0 pEi (Smith-Norowitz et al., 2000).

4.8. Immunohistochemistry

Tissues specimens were fixed in 4% paraformaldehyde and

embedded in paraffin. Lumbar spinal cord tissues were cut in

8-mm-thick sections and immunohistochemical staining was

performed with an avidin–biotin technique, as previously

described with some modifications (Goldschmidt et al.,

2009). Briefly, after deparaffinization, intrinsic peroxidase

activity was blocked by incubation with 5% H2O2 in

phosphate-buffered saline (PBS) for 10 min. Nonspecific anti-

body binding was blocked with 5% goat serum in PBS for 1 h.

Microwave pretreatment for better antigen retrieval was

performed for CNP, GFAP and O4. The primary antibodies

were mouse anti-GFAP (1:100 dilution; Invitrogen Carlsbad,

CA, USA), mouse anti-O4 (1:100; Sigma-Aldrich, St. Louis, MO,

USA), and mouse anti-CNPase (1:100; Sigma, St. Louis, MO,

USA). Secondary antibodies for CNP and O4 were biotinylated

anti-mouse immunoglobulin, and avidin-HRP (1:200; Sigma-

Aldrich, St. Louis, MO, USA).

For GFAP, immunofluorescence-conjugated-anti-mouse

immunoglobulin was used. The density of immunofluores-

cense in activated astrocytes, in untreated and treated EAE

mice was measured with Image J software, after staining with

anti-GFAP antibody. In order to count oligodendrocytes, three

random 200�200 mm2 fields in ventral column of spinal cord

were selected and the number of cells that had been stained

with anti-CNPase antibody was counted. Average number of

oligodendrocytes was calculated in five different sections

from two animals, in each untreated EAE and treated EAE

groups of mice. The same method was used to count

oligodendrocyte progenitor cells (OPC) after staining with

O4 antibody.

4.9. Immunofluorescense study using anti-E2 peptide2antibody

Binding of the anti-E2 peptide2 antibody to the white matter was

determined by immunofluorescent staining: sections of

the lumbar spinal cords of normal, E2 peptide2-treated

and untreated EAE mice, were stained with polyclonal rabbit

anti-E2 peptide2 antibody followed by FITC-conjugated goat

anti-rabbit antibody. The slides were viewed by fluorescent

microscope. To eliminate the background, the level of laser beam

was adjusted for non-specific binding of FITC-conjugate, using

sample stained without the specific antibody. The same excita-

tion was then used for all stained samples. Three to four

untreated and 3–4 E2 peptide2 treated mice were used.

4.10. Statistics

Clinical results, peptides antibody responses and histological

studies were all performed four or five times and were

analyzed for statistical significance by Student’s t-test, using

InStat v.2.01.

Acknowledgment

We are very grateful to Dr. Cedric S. Raine for his valuable

guidance, advice and overall help in the immunopathological

studies, without whom this work would have been incom-

plete. We also thank Dr. Joseph Michl for the use of his

laboratory for antibody staining experiments. This work was

supported by funds from the New York Immunology Labora-

tory, LLC.

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