International Journal of

Molecular Sciences

Article
Myelin Basic Protein in Oligodendrocyte-Derived Extracellular
Vesicles as a Diagnostic and Prognostic Biomarker in Multiple
Sclerosis: A Pilot Study
Cristina Agliardi 1 , Franca Rosa Guerini 1, * , Milena Zanzottera 1 , Elisabetta Bolognesi 1 , Silvia Picciolini 1 ,
Domenico Caputo 1 , Marco Rovaris 1 , Maria Barbara Pasanisi 1 and Mario Clerici 1,2

1 IRCCS Fondazione Don Carlo Gnocchi, 20147 Milan, Italy

2 Pathophysiology and Transplantation Department, University of Milan, 20122 Milan, Italy
* Correspondence: fguerini@[Link]; Tel.: +39-0240308376

Abstract: Approximately 15% of multiple sclerosis (MS) patients develop a progressive form of
disease from onset; this condition (primary progressive-PP) MS is difficult to diagnose and treat,
and is associated with a poor prognosis. Extracellular vesicles (EVs) of brain origin isolated from
blood and their protein cargoes could function as a biomarker of pathological conditions. We verified
whether MBP and MOG content in oligodendrocytes-derived EVs (ODEVs) could be biomarkers of
MS and could help in the differential diagnosis of clinical MS phenotypes. A total of 136 individuals
(7 clinically isolated syndrome (CIS), 18 PPMS, 49 relapsing remitting (RRMS)) and 70 matched healthy
controls (HC) were enrolled. ODEVs were enriched from serum by immune-capture with anti-MOG
antibody; MBP and MOG protein cargoes were measured by ELISA. MBP concentration in ODEVs
was significantly increased in CIS (p < 0.001), RRMS (p < 0.001) and PPMS (p < 0.001) compared to HC
and was correlated with disease severity measured by EDSS and MSSS. Notably, MBP concentration in
ODEVs was also significantly augmented in PPMS compared to RRMS (p = 0.004) and CIS (p = 0.03).
Logistic regression and ROC analyses confirmed these results. A minimally invasive blood test
Citation: Agliardi, C.; Guerini, F.R.;
measuring the concentration of MBP in ODEVs is a promising tool that could facilitate MS diagnosis.
Zanzottera, M.; Bolognesi, E.;
Picciolini, S.; Caputo, D.; Rovaris, M.;
Pasanisi, M.B.; Clerici, M. Myelin
Keywords: multiple sclerosis; primary progressive multiple sclerosis; extracellular vesicles; exosomes;
Basic Protein in oligodendrocytes; MOG; MBP; biomarkers
Oligodendrocyte-Derived
Extracellular Vesicles as a Diagnostic
and Prognostic Biomarker in Multiple
Sclerosis: A Pilot Study. Int. J. Mol. 1. Introduction
Sci. 2023, 24, 894. [Link]
Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central
10.3390/ijms24010894
nervous system (CNS) [1] that predominantly affects young adults. The disease has a preva-
Academic Editor: Francesco Bruno lence of 50–300/100.000 people, and about 2–3 million people are estimated to live with
MS globally [2]. Proteins within the CNS are the targets of autoimmune responses in MS.
Received: 28 November 2022
Thus, myelin is a multilamellar sheath necessary to insulate neurons’ axons and increase
Revised: 28 December 2022
the rate at which action potentials are passed along; it is formed by the elaboration of oligo-
Accepted: 30 December 2022
dendrocyte processes around axons [3]. The main proteinaceous components of myelin,
Published: 3 January 2023
myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), and proteolipid
protein (PLP), are effective autoantigens and targets of autoimmunity in MS [4–6].
MS is characterized by a highly variable clinical course that spans from minimally
Copyright: © 2023 by the authors. disabling to severe forms resulting in progressive, irreversible clinical and cognitive deficits
Licensee MDPI, Basel, Switzerland. and limited response to therapy [7]. Relapsing remitting (RR) MS patients are patients
This article is an open access article who experience episodes of neurological dysfunction with or without residual disability.
distributed under the terms and About 15–30% of RRMS patients will develop progressive disability and become secondary
conditions of the Creative Commons progressive (SP) MS patients. Finally, about 15% of patients develop primary progressive
Attribution (CC BY) license (https:// (PP) MS, a worsening neurologic function from the onset of symptoms, without relapses
[Link]/licenses/by/ or remissions [8]. The identification of easily reproducible and minimally invasive blood-
4.0/).

Int. J. Mol. Sci. 2023, 24, 894. [Link] [Link]

Int. J. Mol. Sci. 2023, 24, 894 2 of 10

based biomarkers would facilitate the early classification of MS patients into these different
clinical subtypes, allowing the optimization of therapeutic and rehabilitative strategies.
Extracellular vesicles of brain origin can cross the blood brain barrier (BBB) and can
be isolated from peripheral blood [9]; their cargoes protein could function as biomarkers
of pathological conditions. Myelin oligodendrocyte glycoprotein (MOG) is expressed at
the RNA level predominantly in oligodendrocytes ([Link]
0000204655-MOG/single+cell+type (accessed on 16 September 2022)) and can be used
to identify oligodendrocyte-derived extracellular vesicles (ODEVs) [10]. Using a newly
developed protocol, we enriched ODEVs from the serum of subjects with clinically isolated
syndrome (CIS), i.e., a single episode of CNS dysfunction suggestive of MS, RRMS, PPMS,
and healthy controls (HC) to verify whether ODEVs MBP and MOG concentrations could
be used as a diagnostic and prognostic biomarker for MS.

2. Results
2.1. Cohort Characteristics
The clinical and demographic characterization of all the individuals enrolled in the
study is presented Table 1. A total of 67 MS patients (49 RRMS and 18 PPMS) had a
confirmed diagnosis of MS according to the 2017 McDonald diagnostic criteria [11]. Seven
subjects were defined as CIS. There were some statistical differences in gender distribu-
tion among groups (see Table 1). When MS patients were considered as a unique group
(RRMS + PPMS), the differences lapsed. Age at onset was significantly higher in PPMS
compared to RRMS (p < 0.01). As expected, both expanded disability status scale (EDSS)
and Multiple Sclerosis Severity Score (MSSS) scores were higher in PPMS compared to
RRMS and CIS patients (see Table 1). Finally, the mean age of all the groups of individuals
was comparable.

Table 1. Study cohort demographic and clinical characteristics.

Study Cohort
CIS RRMS PPMS HC
Number (n) 7 49 18 62
Females (n, %) 2 (28.6) & 32 (65.3) &,¥ 5 (38.5) ¥,# 32 (51.6) #
Age (years ± SD) 44.14 ± 7.10 45.80 ± 9.46 54.00 ± 12.53 51.14 ± 12.29
Disease duration (years ± SD) 10.14 ± 6.69 14.71 ± 9.02 12.44 ± 11.98 n.a.
Age at onset (years ± SD) 34.00 ± 3.37 31.08 ± 9.37 * 41.56 ± 12.26 * n.a.
EDSS (mean ± SD) 1.57 ± 1.51 $ 2.62 ± 1.94 £ 5.08 ± 1.40 $,£ n.a.
MSSS (mean ± SD) 1.38 ± 1.25 ◦ 2.72 ± 2.32 § 6.97 ± 2.06 ◦ ,§ n.a.
& p = 0.01; ¥ p < 0.01; # p = 0.04; * p < 0.01; $ p < 0.001; £ p < 0.001; ◦ p < 0.001; § p < 0.001.

2.2. HLA-DRB1*15 Genotyping

The presence of the HLA-DRB1*15 allele either in heterozygosity or homozigosity
was evaluated in all individuals. HLA-DRB1*15 was significantly more often seen in MS
patients (CIS + RRMS + PPMS) compared to HC (p = 2.8 × 10−5 , Odds ratio (OR) = 10.58,
2.82–67.99 95% confidence interval (CI)), confirming the association between the disease
and this allele.

2.3. ODEVs Characterization

Enriched ODEVs were characterized according to the international society of Extracel-
lular vesicles (ISEV) directions regarding minimal information for studies of extracellular
vesicles (MISEV) [12]. A representative developed membrane of Exo-Check Exosome
Antibody Array is shown in Figure 1A. Exosomal-associated markers (CD63, CD81, ALIX,
FLOT1, ICAM1, EpCam, ANXA5, and TSG101) were all present in enriched ODEVs lysate
(ODEVs), as were the two positive controls. The absence of the GM130 cis-Golgi marker
evidences absence of cellular contamination. The dimensions and morphology of isolated
extravesicles was analyzed by immuno-gold transmission electron microscopy (TEM).
 extracellular vesicles (MISEV) [12]. A representative developed membrane of Exo-Check
Exosome Antibody Array is shown in Figure 1A. Exosomal-associated markers (CD63,
CD81, ALIX, FLOT1, ICAM1, EpCam, ANXA5, and TSG101) were all present in enriched
ODEVs lysate (ODEVs), as were the two positive controls. The absence of the GM130 cis-
Int. J. Mol. Sci. 2023, 24, 894 Golgi marker evidences absence of cellular contamination. The dimensions 3 and of 10
morphology of isolated extravesicles was analyzed by immuno-gold transmission
electron microscopy (TEM). A heterogeneous population of extravesicles, in terms of size
and shape, was detected.
A heterogeneous Immunogold
population labelinginwith
of extravesicles, termsantiofOMG mAb
size and confirmed
shape, ODEVs
was detected.
enrichment: black punctate regions indicate a positive staining
Immunogold labeling with anti OMG mAb confirmed ODEVs enrichment: black punctateon ODEVs membrane
surface
regions (Figure
indicate1B). Quantitative
a positive stainingmeasurements of enriched
on ODEVs membrane ODEVs
surface were 1B).
(Figure conducted in
Quantita-
five representative samples from the three groups: HC, PPMS, and
tive measurements of enriched ODEVs were conducted in five representative samples RRMS. The mean
concentration
from the threeofgroups:
NDEVsHC, afterPPMS,
an ANOVA test resulted
and RRMS. The mean in comparable
concentrationamong groups after
of NDEVs (HC
=an6.3ANOVA
× 1010 particles/mL,
test resultedstandard deviation
in comparable (SD): 2.6
among × 1010(HC
groups particles/mL;
= 6.3 × 10PPMS = 6.0 × 1010
10 particles/mL,
particles/mL, SD: 2.2(SD):
standard deviation × 102.6
9 particles/mL; RRMS = 1.1
× 1010 particles/mL; PPMS × 10=116.0
particles/mL, SD: 1.2 × 10
× 1010 particles/mL, 10
SD:
particles/mL) (p = 0.10) RRMS
2.2 × 109 particles/mL; (Figure=1C). 1.1 ×NDEVs’ mean dimension
1011 particles/mL, was×comparable
SD: 1.2 among
1010 particles/mL)
groups as well (HC = 74.2 ± 12.3 nm; PPMS = 88.9 ± 3.2 nm; RRMS
(p = 0.10) (Figure 1C). NDEVs’ mean dimension was comparable among groups as well = 82.5 ± 8.5 nm) (p =
0.10)
(HC =(Figure
74.2 ± 1C).
12.3 nm; PPMS = 88.9 ± 3.2 nm; RRMS = 82.5 ± 8.5 nm) (p = 0.10) (Figure 1C).

Figure [Link]
Figure ODEVs characterization. (A): Exo-Check™
characterization. ExosomeExosome
(A): Exo-Check™ Antibody Array on an Array
Antibody exemplificative
on an
exemplificative
ODEVs lysate. In ODEVs lysate.
the image are In the image
visible are associated
exosomal visible exosomal
markers:associated markers: ICAM1
FLOT1 (flotillin-1), FLOT1
(flotillin-1),
(intercellular ICAM1 (intercellular
adhesion molecule 1), adhesion molecule 1), ALIX
ALIX (programmed (programmed
cell death cell protein),
6 interacting death 6 interacting
CD81 and
protein),
CD63 (tetraspanins), EpCAM (epithelial cell adhesion molecule), ANXA5 (annexin A5), ANXA5
CD81 and CD63 (tetraspanins), EpCAM (epithelial cell adhesion molecule), TSG101
(annexin A5), TSG101 (tumor susceptibility gene 101) and controls (2 positive assay control, negative
(tumor susceptibility gene 101) and controls (2 positive assay control, negative control: blank and
control: blank and GM130: cis-golgi matrix protein: control for cellular contamination). (B):
GM130: cis-golgi matrix protein: control for cellular contamination). (B): Immuno-gold (OMGp
Immuno-gold (OMGp antigen detected) TEM micrograph of an exemplificative ODEVs
antigen detected)
preparation. TEM100
Scale bar: micrograph of an exemplificative
nm. (C): Representative ODEVs preparation.
size distribution Scale bar:tracking
graph of nanoparticle 100 nm.
(C): Representative
analysis (NTA) that size distribution
shows graph of nanoparticle
size and concentration tracking
of enriched ODEVs analysis (NTA) from
in a sample that shows
an RRMSsize
and concentration
patient; a frame ofoftheenriched
video is ODEVs in a sample
also shown. Meanfrom an RRMS
ODEVs patient; a (particles/mL)
concentration frame of the video
± SDis and
also
shown.
mean Mean diameter
ODEVs ODEVs concentration
(nm) ± SD obtained by NTA ±
(particles/mL) SD andfrom
analysis meanfiveODEVs
ODEVsdiameter
samples (nm)
from± the
SD
three conditions
obtained by NTA (HC, PPMS,from
analysis and five
RRMS).
ODEVsANOVA testsfrom
samples p values are reported.
the three conditions (HC, PPMS, and
RRMS). ANOVA tests p values are reported.

2.4. MBP Concentration in Enriched ODEVs Is Increased in MS Patients

The MBP and MOG concentration in enriched ODEVs from HC, CIS, RRMS, and
PPMS was analyzed by a sandwich enzyme-linked immunosorbent assay (ELISA) and was
not normally distributed as per the results of Kolmogorov–Smirnov analyses. The Kruskal-
Wallis test was then applied; results showed the presence of a statistically significant
difference in MBP concentration among groups (p < 0.0001) (Figure 2A). In particular, the
Dwass–Steel–Critchlow–Fligner test for pairwise comparisons indicated that the MBP con-
centration was significantly lower in HC (median: 17.85 pg/mL, interquartile range (IQR):
 significant difference in MBP concentration among groups (p < 0.0001) (Figure 2A). In
particular, the Dwass–Steel–Critchlow–Fligner test for pairwise comparisons indicated
that the MBP concentration was significantly lower in HC (median: 17.85 pg/mL,
interquartile range (IQR): 14.00–24.39 pg/mL) compared to CIS (median: 78.73 pg/mL,
IQR: 65.78–91.20 pg/mL; p < 0.001 vs. HC), RRMS (median: 83.76 pg/mL, IQR: 71.05–95.03
Int. J. Mol. Sci. 2023, 24, 894 4 of 10
pg/mL; p < 0.001 vs. HC) and PPMS (median: 136.44 pg/mL, IQR: 86.32–225.67 pg/mL; p <
0.001 vs. HC) (Figure 2A). Moreover, the MBP concentration in ODEVs was significantly
increased in PPMS compared to RRMS (p = 0.004) and CIS (p = 0.03) (Figure 2A), whereas
14.00–24.39 pg/mL) compared to CIS (median: 78.73 pg/mL, IQR: 65.78–91.20 pg/mL;
no psignificant
< 0.001 vs. differences emerged83.76
HC), RRMS (median: when CIS was
pg/mL, IQR:compared
71.05–95.03topg/mL;
[Link],
< 0.001 vs. HC)if CIS
subjects
and PPMS (median: 136.44 pg/mL, IQR: 86.32–225.67 pg/mL; p < 0.001 vs. HC) in
and RRMS patients were grouped (RRMS + CIS), the difference ODEVs
(Figure 2A).MPB
concentration
Moreover, thebetween PPMS (median:
MBP concentration in ODEVs136.44 pg/mL, IQR:
was significantly 86.32–225.67
increased pg/mL) and
in PPMS compared
to RRMS (p = 0.004) and CIS (p = 0.03) (Figure 2A), whereas no
RRMS + CIS (median: 82.16; IQR: 68.09–93.51 pg/mL) patients was even more evident (p <significant differences
0.01)emerged
(Figurewhen
2B). CIS was compared to RRMS. Interestingly, if CIS subjects and RRMS pa-
tients were grouped
A logistic regression(RRMS + CIS),was
analysis theperformed
difference innext,
ODEVs MPB concentration
considering the disease between
phenotype
PPMS (median: 136.44 pg/mL, IQR: 86.32–225.67 pg/mL) and RRMS + CIS (median: 82.16;
(CIS, RRMS, and PPMS) or the condition of HC as the dependent variable, and MBP
IQR: 68.09–93.51 pg/mL) patients was even more evident (p < 0.01) (Figure 2B).
concentrations in ODEVs, as well as gender, age, and presence of at least one HLA-
A logistic regression analysis was performed next, considering the disease phenotype
DRB1*15 allele
(CIS, RRMS, and asPPMS)
covariates. The model
or the condition of HCwas applied
as the on the
dependent overall
variable, andgroup of the 136
MBP concen-
subjects
trationsenrolled
in ODEVs, in as
thewell
study. Results
as gender, age,confirmed
and presencetheofsignificant contribution of
at least one HLA-DRB1*15 ODEVs
allele
MBP concentration
as covariates. The in discriminating
model was appliedbetween MS and
on the overall healthy
group of the condition
136 subjects (overall
enrolled model
fit: in
χ the
2 = 113.61;
study. pResults
< 0.0001); the other
confirmed thecovariates
significantwere not statistically
contribution of ODEVs significant. The model
MBP concentra-
2 = 113.61;
hadtion theinpower
discriminating between
to correctly MS and
classify healthy
90.83% ofcondition
cases. The(overall
samemodel fit: χregression
logistic was
p < 0.0001); the other covariates were not statistically significant. The
applied considering the clinical diagnosis of either RRMS + CIS or PPMS as a dependent model had the power
to correctly classify 90.83% of cases. The same logistic regression was applied considering
variable and the same above-mentioned variables as covariates. The contribution of MBP
the clinical diagnosis of either RRMS + CIS or PPMS as a dependent variable and the same
concentration in ODEVs was statistically significant (p = 0.003), as were gender (p = 0.05)
above-mentioned variables as covariates. The contribution of MBP concentration in ODEVs
andwas agestatistically
(p = 0.004), in discriminating
significant (p = 0.003),between the two(pdiagnoses
as were gender = 0.05) and(overall
age (p =model0.004), fit:
in χ =
2

40.91; p < 0.0001).between the two diagnoses (overall model fit: χ2 = 40.91; p < 0.0001).
discriminating
In In
contrast
contrastwith theseresults,
with these results,thethe
MOG MOG protein
protein was detected
was detected in enriched
in enriched ODEVs from ODEVs
from bothboth
MS MS patients
patients andbut
and HC, HC, nobut no statistically
statistically significant
significant differencesdifferences
among groups among groups
could
couldbe observed (data(data
be observed not shown).
not shown).

Figure
Figure2. 2.
MBP MBP ininenriched
enrichedODEVs
ODEVs inin HC,
HC,CIS,
CIS,RR-MS,
RR-MS, andand PP-MS.
PP-MS. (A): (A): Multiple
Multiple comparison
comparison
graphs
graphs ofofMBP concentrationin in
MBP concentration enriched
enriched ODEVs,ODEVs, respectively
respectively in RRMS,
in HC, CIS, HC, CIS, RRMS,subjects;
and PPMS and PPMS
subjects;
all dataallare
data are plotted,
plotted, and and
and median median and interquartile
interquartile range (IQR)range (IQR) areThe
are reported. reported.
reportedThe reported
global
global p values of the differences between the groups of subjects was calculated by Kruskal–Wallis
p values of the differences between the groups of subjects was calculated by Kruskal–Wallis test
for non-parametric distributions. p values of post hoc Dwass–Steel–Critchlow–Fligner for pairwise
comparisons are also reported. (B): Multiple comparison graphs of MBP concentration in enriched
ODEVs, respectively in HC, (CIS + RRMS) and PP-MS subjects; all data are plotted; median and
interquartile range (IQR) are reported. The reported global p values of the differences between the
groups of subjects was calculated by Kruskal–Wallis test for non-parametric distributions. p values of
post hoc Dwass–Steel–Critchlow–Fligner for pairwise comparisons are also reported.
 test for non-parametric distributions. p values of post hoc Dwass–Steel–Critchlow–Fligner for
pairwise comparisons are also reported. (B): Multiple comparison graphs of MBP concentration in
enriched ODEVs, respectively in HC, (CIS + RRMS) and PP-MS subjects; all data are plotted; median
and interquartile range (IQR) are reported. The reported global p values of the differences between
the groups of subjects was calculated by Kruskal–Wallis test for non-parametric distributions. p
Int. J. Mol. Sci. 2023, 24, 894 5 of 10
values of post hoc Dwass–Steel–Critchlow–Fligner for pairwise comparisons are also reported.

2.5. ROC Curve Analysis

[Link]
ROC Curve Analysis characteristic (ROC) curve analysis was applied next to evaluate
operating
Receiver
the possible operating value
diagnostic characteristic
of ODEVs (ROC)MBPcurve analysis was applied
concentration. When next
MBPtoinevaluate
ODEVs was
the possible diagnostic value of ODEVs MBP concentration. When MBP in ODEVs was
analyzed in HC vs. MS patients (considering together CIS, RRMS, and PPMS) an ROC
analyzed in HC vs. MS patients (considering together CIS, RRMS, and PPMS) an ROC area
area under the curve (AUC) = 0.963 (0.916–0.988 95% confidence interval (CI)) with 97.84%
under the curve (AUC) = 0.963 (0.916–0.988 95% confidence interval (CI)) with 97.84% sen-
sensitivity and
sitivity and 100.00%
100.00% specificity
specificity (p <(p < 0.0001)
0.0001) waswas obtained
obtained (Figure
(Figure 3A). When
3A). When ROC curve
ROC curve
analysis
analysiswas
wasapplied consideringthe
applied considering theclinical
clinical diagnosis
diagnosis (RRMS
(RRMS + CIS+ vs.
CISPPMS),
vs. PPMS),
an ROC an ROC
AUC
AUC== 0.795 (0.685–0.880
0.795 (0.685–0.880 95%
95% CI) with
CI) with 66.67%66.67% sensitivity
sensitivity and
and 87.50% 87.50%(pspecificity
specificity < 0.0001) (p <
0.0001) was obtained
was obtained (Figure (Figure
3B). 3B).

Figure
Figure 3. 3.
ROCROCcurvecurve (A):(A):
analysis.
analysis. ROCROC curves
curves of MBP
of MBP in enriched
in enriched ODEVs:
ODEVs: HC [Link].(CIS
MS+ RRMS
(CIS + RRMS + PPMS). AUC and p value are reported. (B): ROC curves of MBP in enriched
+ PPMS). AUC and p value are reported. (B): ROC curves of MBP in enriched ODEVs: PPMS vs. ODEVs:
PPMS
(CIS vs. (CISAUC
+ RRMS). + RRMS).
and pAUC andare
value p value are reported.
reported.
2.6. Correlation with Clinical Scores
2.6. Correlation with Clinical Scores
Pearson’s correlations showed that MBP concentration in ODEVs was correlated both
Int. J. Mol. Sci. 2023, 24, 894 withPearson’s
EDSS (p <correlations showed
0.01, coefficient that MBP
of correlation concentration
= 0.32, in ODEVs
0.09–0.51 95% was correlated
CI) and MSSS 12 both
(p 6<of0.01,
with EDSS (p
coefficient of <correlation
0.01, coefficient of correlation
= 0.32, 0.09–0.51 = 0.32,
95% CI) scores0.09–0.51 95% CI) and MSSS (p < 0.01,
(Figure 4A,B).
coefficient of correlation = 0.32, 0.09–0.51 95% CI) scores (Figure 4A,B).

Figure 4. (A):
Figure 4. (A):Correlation
Correlationbetween
between MBP
MBP in ODEVs
in ODEVs and clinical
and clinical [Link]. Bivariate
Bivariate Pearson’s
Pearson’s correla-
correlation between MBP concentration in enriched ODEVs and EDSS. (B): Bivariate Pearson’s
tion between MBP concentration in enriched ODEVs and EDSS. (B): Bivariate Pearson’s correlation cor-
relation between MBP concentration in enriched ODEVs and MSSS.
between MBP concentration in enriched ODEVs and MSSS.

3. Discussion
In the present case-control pilot study, we show that quantification of MBP as a cargo
protein inside enriched oligodendrocyte extracellular vesicles (ODEVs) isolated from se-
rum allows identification of MS, as it discriminates between MS patients and healthy con-
Int. J. Mol. Sci. 2023, 24, 894 6 of 10

3. Discussion
In the present case-control pilot study, we show that quantification of MBP as a cargo
protein inside enriched oligodendrocyte extracellular vesicles (ODEVs) isolated from serum
allows identification of MS, as it discriminates between MS patients and healthy controls in
a highly sensible and specific way. Moreover, and more interestingly, the amount of MBP
in ODEVs can differentiate between MS patients with a clinical diagnosis of either PPMS
or RRMS. Finally, we show that MBP concentration in ODEVs is also positively correlated
with MS severity, as evaluated by the EDSS and MSSS scales.
The spectrum of MS disease spans from non-disabling forms to forms characterized
by progressive, irreversible, clinical, and cognitive deficits and limited response to stan-
dard treatment [7]. PPMS has unfortunately a poor prognosis, and therapies approved
for RRMS have little or no benefits in PPMS patients, even if treatment efficacy for two
molecules—rituximab [13] and ocrelizumab [14]—has been reported. A prompt diagnosis
of the different clinical MS phenotypes in the initial stages of disease could be of utmost
importance for therapeutic decisions. Knowing more in advance the progressive prognosis
of patients could aid in treatment and rehabilitation decision-making and could help to
minimize costs for the sanitary system [15]. The diagnosis of MS is currently based on
the integration of clinical, imaging, and laboratory findings [11]; age, sex, spinal cord
lesions, and the extent of brain abnormalities on MRI are predictors of outcome across
the different MS clinical phenotypes. Notably, though, easily accessible and non-invasive
biomarkers to diagnose the disease or to characterize it are not currently available; ODEVs
MBP quantification could be a useful tool.
MBP was firstly isolated in the early 1960s and constitutes 30% of total CNS myelin
proteins, [16] it is the most widely studied myelin protein in MS as it is the main target of MS-
associated autoimmune responses. The added value of testing MBP in CSF for MS diagnosis
was suggested to be low [17], but recently it was reported as a potential biomarker of
disability progression in SPMS [18]. Oligodendrocytes are known to secrete large quantities
of exosomes expressing myelin proteins in vitro [19], and exosomes of CNS origin are
known to cross the BBB [9]. Galazka et al. recently reported that MOG and other myelin
proteins can be detected in serum exosomes of both MS patients, especially during clinical
exacerbation, and non-MS subjects [20]. They analyzed total EVs preparations obtained
from serum instead of enriched ODEVs and compared the concentrations of myelin proteins,
including MBP and MOG, in RRMS, secondary progressive (SP)MS and HC. Results
showed that MOG was increased in EVs from secondary progressive (SP)MS and RRMS
during clinical exacerbations compared to healthy controls; notably, no differences in MBP
concentration were detected among groups. The apparent discrepancy between Galazka’s
and our results could be due to the fact that in our case, the source of myelin proteins was
enriched ODEVs instead of total serum EVs. Moreover, the enrolled groups of MS patients
were different in the two studies: We did not study SPMS and RRMS patients during
clinical relapse; Galazka, on the other hand, did not analyze PPMS patients. Galazka et al.
also found that serum exosomes were able to induce the proliferation of MOG-recognizing
transgenic TCR T lymphocytes. This observation led to the hypothesis that the augmented
amounts of CNS myelin proteins and peptides seen in EVs of MS patients could result in
the maintaining and the amplification of autoimmune reactions and/or the modulation
of immune effector mechanisms in response to the spread of such autoantigens [20]. Data
herein confirm that CNS proteins can be detected in ODEVs isolated from serum and
indicate that the measurement of one of such proteins, MBP, could have a diagnostic and
prognostic value.
Interestingly, and although needing to be confirmed in larger cohorts, results showed
that the MBP concentration in ODEVs is comparable in CIS and RRMS patients. Because
CIS can, but does not always evolve into RRMS over time, it will be interesting to verify
in longitudinal studies whether the MBP concentration in ODEVs will be able to predict
which CIS patients will develop RRMS.
Int. J. Mol. Sci. 2023, 24, 894 7 of 10

The study has some limits: first of all, the small sample size. Moreover, we do not
know if different pharmacological treatments could affect MBP concentrations in ODEVs.
It will be necessary to confirm these results in larger cohorts of patients; it will be
interesting to design follow-up studies on patients in the initial phases of the disease in order
to verify whether MBP concentration in ODEVs will allow the precocious identification of
different clinical MS phenotypes.

4. Material and Methods

4.1. Study Cohort
A total of 136 individuals were included in this case-control study. Sixty-seven MS
patients and 7 individuals with a CIS diagnosis were enrolled by the Multiple Sclerosis
Unit of the IRCCS Fondazione Don Carlo Gnocchi, Milan. MS patients were classified by
a neurologist according to the revised McDonald’s diagnostic criteria [11] in two clinical
subgroups: 18 of them were diagnosed as being affected by PPMS; the remaining 49 patients
had a diagnosis of RRMS and they were all in remission phase at the time of blood collection.
At the time of enrollment, demographic and clinical features (age, age at onset, disease
duration, EDSS [21], pharmacological treatments) were registered in a pseudo anonymized
database. MSSS was calculated as described by Roxburgh [22]. A group of 70 healthy
subjects were enrolled as healthy controls (HC) among hospital staff. Exclusion criteria for
HC were any autoimmune and/or neurological conditions and age <35 years in order to
reduce the risk to introduce false-negative subjects. Informed consent was obtained from
all the individuals prior to inclusion in the study. The study was conducted according
to the guidelines of the Declaration of Helsinki and was approved by the institutional
review board of the Don Carlo Gnocchi ONLUS Foundation, Milan (Protocol number:
#11_27/06/2019). Demographic and clinical data of study population are reported in
Table 1.

4.2. Serum Collection

Blood samples collection and preparation were conducted in a standardized way:
25 mL of peripheral whole blood was collected using a serum separator tube (SST II Ad-
vance, BD Vacutainer® ). Samples were allowed to clot for 10–20 min at room temperature.
Tubes were then centrifuged at 1500× g for 10 min, and serum was collected, aliquoted,
and stored at −80 ◦ C.

4.3. ODEVs Enrichment

ODEVs were enriched from 250 µL of serum by adjusting a previously published
double step method [23]. Briefly, after addition of 150 µL of calcium- and magnesium-free
Dulbecco balanced salt solution (PBS) and 15 µL of Halt™ Protease and Phosphatase In-
hibitor Cocktail (Thermo Fisher scientific, Waltham, MA, USA) samples were centrifuged
at 3000× g for 30 min at 4 ◦ C; 126 µL of ExoQuick® exosome precipitation solution (System
Bioscience, Palo Alto, CA, USA) was added to supernatants and the mix was centrifuged at
1500× g for 30 min at 4 ◦ C. Pellets were suspended in 350 µL of PBS, 50 µL of 3% bovine
serum albumin (BSA), and 2 µg of anti-oligodendrocyte myelin glycoprotein (MOG) bi-
otinylated antibody (Bioss antibodies, Boston, MA, USA). After an incubation of 1 h at room
temperature, 10 µL of Pierce™ Streptavidin UltraLink™ Resin and 40 µL of 3% BSA were
added; samples were then incubated for 30 min at room temperature. After centrifugation
at 800× g for 10 min at 4 ◦ C and removal of supernatants, pellets were suspended in 100 µL
of 0.05 M glycine-HCl (pH 3.0) and vortexed 10 s. After centrifugation at 4000× g for10 min
at 4 ◦ C, 25 µL of 10% BSA, 10 µL of Tris-HCl and 15 µL of Halt™ Protease and Phosphatase
Inhibitor Cocktail (Thermo Fisher scientific, Waltham, MA, USA) was added. Twenty µL
of intact enriched ODEVs preparations was frozen and stored at 80 ◦ C for downstream
applications, the remaining extravesicles were lysed by adding 365 µL of mammalian
protein extraction reagent (M-PER™, Thermo Fisher scientific, Waltham, MA, USA), and
Int. J. Mol. Sci. 2023, 24, 894 8 of 10

15 µL of protease/phosphatase inhibitors. Lysates of enriched ODEVs were subjected to

2 freeze-thaw cycles and stored at −80 ◦ C.

4.4. ODEVs Characterization

Lysates of enriched ODEVs were tested for the expression of canonical exosome
markers by a commercial kit based on Western blotting (Exo-check Exosome Antibody
Array, System Bioscience, Palo Alto, CA, USA) following manufacturer’s instructions. It
consists of 12 pre-printed spots, including 8 antibodies for known exosome markers (CD63,
CD81, ALIX, FLOT1, ICAM1, EpCam, ANXA5 and TSG101), and 4 controls (two positive
control signals, a background control, and the GM130 cis-Golgi marker that monitors for
cellular contamination). Membranes were developed using the Clarity Max Western ECL
Substrate (Bio-Rad, Hercules, CA, USA) and imaged by ChemiDoc™ Gel Imaging System
(Bio-Rad, Hercules, CA, USA).
Immuno-gold TEM imaging was performed as follows: 5 µL of exosomes suspensions
was adsorbed on 200 mesh thin-film formvar-carbon coated TEM grids for 10 min and
excess was blotted on filter paper. Two washing steps on 50-µL drops of wash buffer
(0.1% BSA in PBS) were performed. TEM grids were then incubated in a wet chamber
at room temperature for 3 h on 50 µL drops containing Primary Antibody (anti OMG
for ODEVs) diluted 1:100 in wash buffer. Additional washing steps on 50-µL drops of
wash buffer were performed. TEM grids were then incubated for 1 h on 30 µL drops of
1:50 dilutions of anti-rabbit or anti-mouse IgG-gold conjugate (10-nm particle size) in wash
buffer. Washing steps on five drops of wash buffer followed by five washes on water drops
were performed. Negative staining was done on 1% filtered aqueous solution of Uranyl
Acetate drops for 10 s before drying. TEM micrographs were acquired by means of the
JEOL JEM 2100Plus Transmission Electron Microscope (JEOL, Tokyo, Japan) operating
with an acceleration voltage of 200 kV and equipped with an 8 megapixel Gatan (Gatan,
Pleasanton, CA, USA) Rio Complementary Metal-Oxide-Superconductor (CMOS) camera.
To evaluate size and concentration of ODEVs nanoparticles, tracking analysis (NTA)
was performed for five representative samples from each group (HC, PPMS, and RRMS)
on a NanoSight NS300 device equipped with a blue 488 nm laser, a flow-cell top-plate,
and a syringe pump to enable analysis in constant flow (Malvern Panalytical, Malvern,
UK). Samples were diluted 1:250 in 1X filtered PBS. Three videos of 60 s were acquired per
sample. Mean sizes were calculated by integrating the data from three records. Data were
analyzed using the NTA software v. 3.4 with the detection threshold 5 to track as many
particles as possible with minimal background.

4.5. Immunoenzymatic Dosages

MBP and MOG protein concentration in undiluted lysates from enriched ODEVs was
measured by sandwich ELISA using commercial kits (cat n◦ : MBS2502574 and MBS928110
respectively, MyBiosource, San Diego, CA, USA), according to manufacturers’ instructions.
Standard curves and samples were run in duplicate.

4.6. HLA-DRB1*15 Genotyping

The presence of HLA-DRB1*15 alleles, either in heterozygous or homozygous form,
was inferred by the genotyping of the tag SNP rs3135388 [24] by allelic discrimination real-
time PCR with the TaqMan™ C__27464665_30 probe (Thermo Fisher scientific, Waltham,
MA, USA). PCR consisted of a hot start at 95 ◦ C for 10 min followed by 40 cycles of 94 ◦ C for
15 s and 60 ◦ C for 1 min. Fluorescence detection took place at 60 ◦ C. Assays were performed
in 10 µL reactions using 1 µL of DNA at 50 ng/µL, using TaqMan™ Genotyping Master Mix
(Thermo Fisher scientific, Waltham, MA, USA) on 96-well plates using a CFX96 instrument
(Bio-Rad, Hercules, CA, USA). Control samples representing all possible genotypes and a
negative control were included in each reaction.
Int. J. Mol. Sci. 2023, 24, 894 9 of 10

4.7. Statistical Analysis

Differences in demographic and clinical variables between HC, CIS, RRMS, and PPMS
patients were evaluated by Chi-square and Student’s t test. Kolmogorov–Smirnov test
showed that both MBP and MOG protein concentrations were not normally distributed
in the present cohort. Non-parametric Mann–Whitney U test or Kruskal–Wallys test were
then performed to compare MBP and MOG proteins’ concentration between different
groups. Post hoc Dwass–Steel–Critchlow–Fligner test was applied for pairwise compar-
isons. NTA results (ODEVs concentration and dimension) were analyzed by ANOVA
tests in order to highlight possible differences amongst groups of patients. The discrim-
inatory ability of enriched ODEVs biomarkers to distinguish between different groups
was analyzed by using receiver ROC analyses with calculation of the AUC, sensitivity
and specificity as well as 95% CI. In general, an AUC between 0.7 and 0.8 is considered
“acceptable”, an AUC between 0.8 and 0.9 is considered “excellent”, and more than 0.9
is considered “outstanding” [25]. Logistic regressions were applied considering the dif-
ferent conditions of illness as dependent variable, and ODEVs MBP concentrations, age,
gender, and HLADRB1*15 positivity as covariates. Bivariate Pearson’s correlations were
applied in order to investigate possible correlations between biological biomarkers and
clinical features of patients. In all cases, differences were considered statistically significant
when p ≤ 0.05. MedCalc® software (MedCalc® , 14.10.2, Belgium), SPSS software (v.27,
IBM, USA), jamovi (version 2.2, [Link] (accessed on 6 October 2022))
and OpenEpi ([Link] (accessed on 6 October 2022)) were used for
statistical analyses.

Author Contributions: Conceptualization, C.A. and M.C.; methodology, C.A.; formal analysis, C.A.
and F.R.G.; investigation, C.A., M.Z., E.B. and S.P.; resources, D.C., M.R. and M.B.P.; writing—original
draft preparation, C.A.; writing—review and editing, C.A., F.R.G., M.C., M.R., M.B.P. and D.C.;
supervision, M.C.; project administration, C.A. and M.C.; funding acquisition, M.C. All authors have
read and agreed to the published version of the manuscript.
Funding: This research was funded by Italian Ministry of Health (Ricerca Corrente 2022) and was also
partially supported by grants from the Fondazione Alessandro e Vincenzo Negroni Prati Morosini
and Fondazione Romeo ed Enrica Invernizzi.
Institutional Review Board Statement: The study was conducted according to the guidelines of the
Declaration of Helsinki, and approved by the Institutional Ethics Committee of IRCCS Fondazione
Don Carlo Gnocchi (Protocol number: #11_27/06/2019).
Informed Consent Statement: Written informed consent was obtained from all subjects involved in
the study.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: We thank Melissa Saibene for TEM imaging.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Thompson, A.J.; Baranzini, S.; Geurts, J.; Hemmer, B.; Ciccarelli, O. Multiple sclerosis. Lancet 2018, 391, 1622–1636. [CrossRef]
[PubMed]
2. Browne, P.; Chandraratna, D.; Angood, C.; Tremlett, H.; Baker, C.; Taylor, B.V.; Thompson, A.J. Atlas of Multiple Sclerosis 2013: A
growing global problem with widespread inequity. Neurology 2014, 83, 1022–1024. [CrossRef] [PubMed]
3. Dubois-Dalcq, M.; Armstrong, R. The cellular and molecular events of central nervous system remyelination. Bioessays 1990, 12,
569–576. [CrossRef] [PubMed]
4. Krogsgaard, M.; Wucherpfennig, K.W.; Cannella, B.; Hansen, B.E.; Svejgaard, A.; Pyrdol, J.; Ditzel, H.; Raine, C.; Engberg, J.;
Fugger, L. Visualization of myelin basic protein (MBP) T cell epitopes in multiple sclerosis lesions using a monoclonal antibody
specific for the human histocompatibility leukocyte antigen (HLA)-DR2-MBP 85-99 complex. J. Exp. Med. 2000, 191, 1395–1412.
[CrossRef] [PubMed]
Int. J. Mol. Sci. 2023, 24, 894 10 of 10

5. Kerlero de Rosbo, N.; Milo, R.; Lees, M.B.; Burger, D.; Bernard, C.C.; Ben-Nun, A. Reactivity to myelin antigens in multiple
sclerosis. Peripheral blood lymphocytes respond predominantly to myelin oligodendrocyte glycoprotein. J. Clin. Investig. 1993,
92, 2602–2608. [CrossRef]
6. Correale, J.; McMillan, M.; McCarthy, K.; Le, T.; Weiner, L.P. Isolation and characterization of autoreactive proteolipid protein-
peptide specific T-cell clones from multiple sclerosis patients. Neurology 1995, 45, 1370–1378. [CrossRef]
7. Filippi, M.; Bar-Or, A.; Piehl, F.; Preziosa, P.; Solari, A.; Vukusic, S.; Rocca, M.A. Multiple sclerosis. Nat. Rev. Dis. Prim. 2018, 4, 43.
[CrossRef]
8. Miller, D.H.; Leary, S.M. Primary-progressive multiple sclerosis. Lancet Neurol. 2007, 6, 903–912. [CrossRef]
9. Shi, M.; Liu, C.; Cook, T.J.; Bullock, K.M.; Zhao, Y.; Ginghina, C.; Li, Y.; Aro, P.; Dator, R.; He, C.; et al. Plasma exosomal
α-synuclein is likely CNS-derived and increased in Parkinson’s disease. Acta Neuropathol. 2014, 128, 639–650. [CrossRef]
10. Ohmichi, T.; Mitsuhashi, M.; Tatebe, H.; Kasai, T.; Ali El-Agnaf, O.M.; Tokuda, T. Quantification of brain-derived extracellular
vesicles in plasma as a biomarker to diagnose Parkinson’s and related diseases. Park. Relat. Disord. 2019, 61, 82–87. [CrossRef]
11. Thompson, A.J.; Banwell, B.L.; Barkhof, F.; Carroll, W.M.; Coetzee, T.; Comi, G.; Correale, J.; Fazekas, F.; Filippi, M.; Freedman,
M.S.; et al. Diagnosis of multiple sclerosis: 2017 revisions of the McDonald criteria. Lancet Neurol. 2018, 17, 162–173. [CrossRef]
12. Théry, C.; Witwer, K.W.; Aikawa, E.; Alcaraz, M.J.; Anderson, J.D.; Andriantsitohaina, R.; Antoniou, A.; Arab, T.; Archer, F.;
Atkin-Smith, G.K.; et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): A position statement of the
International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J. Extracell. Vesicles. 2018, 7, 1535750.
[CrossRef] [PubMed]
13. Hawker, K.; O’Connor, P.; Freedman, M.S.; Calabresi, P.A.; Antel, J.; Simon, J.; Hauser, S.; Waubant, E.; Vollmer, T.; Panitch, H.;
et al. Rituximab in patients with primary progressive multiple sclerosis: Results of a randomized double-blind placebo-controlled
multicenter trial. Ann. Neurol. 2009, 66, 460–471. [CrossRef] [PubMed]
14. Montalban, X.; Hauser, S.L.; Kappos, L.; Arnold, D.L.; Bar-Or, A.; Comi, G.; de Seze, J.; Giovannoni, G.; Hartung, H.P.; Hemmer,
B.; et al. Ocrelizumab versus Placebo in Primary Progressive Multiple Sclerosis. N. Engl. J. Med. 2017, 376, 209–220. [CrossRef]
[PubMed]
15. Rocca, M.A.; Sormani, M.P.; Rovaris, M.; Caputo, D.; Ghezzi, A.; Montanari, E.; Bertolotto, A.; Laroni, A.; Bergamaschi, R.;
Martinelli, V.; et al. Long-term disability progression in primary progressive multiple sclerosis: A 15-year study. Brain 2017, 140,
2814–2819. [CrossRef] [PubMed]
16. Einstein, E.R.; Robertson, D.M.; Dicaprio, J.M.; Moore, W. The isolation from bovine spinal cord of a homogeneous protein with
encephalitogenic activity. J. Neurochem. 1962, 9, 353–361. [CrossRef]
17. Greene, D.N.; Schmidt, R.L.; Wilson, A.R.; Freedman, M.S.; Grenache, D.G. Cerebrospinal fluid myelin basic protein is frequently
ordered but has little value: A test utilization study. Am. J. Clin. Pathol. 2012, 138, 262–272. [CrossRef] [PubMed]
18. Herman, S.; Khoonsari, P.E.; Tolf, A.; Steinmetz, J.; Zetterberg, H.; Åkerfeldt, T.; Jakobsson, P.J.; Larsson, A.; Spjuth, O.; Burman, J.;
et al. Integration of magnetic resonance imaging and protein and metabolite CSF measurements to enable early diagnosis of
secondary progressive multiple sclerosis. Theranostics 2018, 8, 4477–4490. [CrossRef]
19. Fabriek, B.O.; Zwemmer, J.N.; Teunissen, C.E.; Dijkstra, C.D.; Polman, C.H.; Laman, J.D.; Castelijns, J.A. In vivo detection of
myelin proteins in cervical lymph nodes of MS patients using ultrasound-guided fine-needle aspiration cytology. J. Neuroimmunol.
2005, 161, 190–194. [CrossRef]
20. Galazka, G.; Mycko, M.P.; Selmaj, I.; Raine, C.S.; Selmaj, K.W. Multiple sclerosis: Serum-derived exosomes express myelin
proteins. Mult. Scler. 2018, 24, 449–458. [CrossRef]
21. Kurtzke, J.F. Rating neurologic impairment in multiple sclerosis: An expanded disability status scale (EDSS). Neurology 1983, 33,
1444–1452. [CrossRef] [PubMed]
22. Roxburgh, R.H.; Seaman, S.R.; Masterman, T.; Hensiek, A.E.; Sawcer, S.J.; Vukusic, S.; Achiti, I.; Confavreux, C.; Coustans, M.; le
Page, E.; et al. Multiple Sclerosis Severity Score: Using disability and disease duration to rate disease severity. Neurology 2005, 64,
1144–1151. [CrossRef] [PubMed]
23. Goetzl, E.J.; Schwartz, J.B.; Abner, E.L.; Jicha, G.A.; Kapogiannis, D. High complement levels in astrocyte-derived exosomes of
Alzheimer disease. Ann. Neurol. 2018, 83, 544–552. [CrossRef] [PubMed]
24. de Bakker, P.I.; McVean, G.; Sabeti, P.C.; Miretti, M.M.; Green, T.; Marchini, J.; Ke, X.; Monsuur, A.J.; Whittaker, P.; Delgado, M.;
et al. A high-resolution HLA and SNP haplotype map for disease association studies in the extended human MHC. Nat. Genet.
2006, 38, 1166–1172. [CrossRef]
25. Mandrekar, J.N. Receiver operating characteristic curve in diagnostic test assessment. J. Thorac. Oncol. 2010, 5, 1315–1316.
[CrossRef]

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