Note: Descriptions are shown in the official language in which they were submitted.
CA 02941460 2016-09-08
ERYTHROCYTE-DERIVED EXTRACELLULAR VESICLES AS A BIOMARKER FOR CLINICALLY
ASSESSING
PARKINSON'S DISEASE
The present description relates to Parkinson's disease. More particularly, the
present description relates to
the use of extracellular vesicles originating from erythrocytes as a biomarker
for clinically assessing Parkinson's
disease in a subject.
BACKGROUND
Parkinson's disease (PD) is one of the most common neurodegenerative disorders
affecting millions of
people worldwide. Definite diagnosis for PD can only be made postmortem, for
instance, by the characteristic
accumulation of the protein alpha-synuclein into Lewy body inclusions in
neurons. Currently, the diagnosis of PD is
based on fitting observed symptoms and their severity into clinical rating
scales such as the Unified Parkinson's
Disease Rating Scale (UPDRS) or the Hoehn & Yahr scale. Current clinical
assessments are subjective, however,
and would benefit from improved methods of clinically assessing PD,
particularly at early stages of the disease.
SUMMARY
The present description stems from the surprising discovery that erythrocytes
of Parkinson's disease (PD)
subjects produce more extracellular vesicles (EV) than erythrocytes from
corresponding control subjects, and that the
level of erythrocyte-derived extracellular vesicles (EEV) strongly correlates
with PD stage and treatment. In contrast,
no significant differences between PD and control blood samples were observed
in the number of EV originating from
other cell types (e.g., platelets, endothelial cells, monocytes, granulocytes,
and leukocytes), suggesting that the effect
is specific for EV originating from erythrocytes. Furthermore, the increase in
number of EEV and its strong correlation
with PD stage was not observed in erythrocytes from Huntington's disease
subjects, suggesting that these
observations are specific for PD. Thus, the present description relates to the
use of EEV as a biomarker for clinically
assessing Parkinson's disease.
Accordingly, the present description may relate to the following aspects:
1. An in vitro method for clinically assessing Parkinson's disease in a
human subject, said method comprising:
(a) quantifying the level of erythrocyte-derived extracellular vesicles (EEV)
in a blood sample from the
subject; and
(b) comparing the level of EEV to a suitable reference value indicative of the
presence, stage and/or
progression of Parkinson's disease, thereby clinically assessing Parkinson's
disease in the subject.
2. The method of aspect 1, wherein said EEV are CD235a+ extracellular
vesicles.
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3. The method of aspect 1 or 2, wherein said EEV are TSG101+, Rabs+,
CD9+, CD63+, CD81+, or any
combination thereof.
4. The method of any one of aspects 1 to 3, wherein said blood sample is
platelet-free-plasma.
5. The method of any one of aspects 1 to 4, wherein said EEV are
between about 20 nm and about 1000 nm in
diameter.
6. The method of any one of aspects 1 to 5, wherein said EEV are greater
than about 100 nm in diameter.
7. The method of any one of aspects 1 to 6, wherein said EEV are
quantified by flow cytometry, nanoparticle
tracking (NTA), or electron microscopy.
8. The method of any one of aspects 1 to 7, wherein clinically assessing
Parkinson's disease in the subject
comprises diagnosing Parkinson's disease in the subject.
9. The method of any one of aspects 1 to 8, wherein clinically assessing
Parkinson's disease in the subject
comprises staging Parkinson's disease in the subject.
10. The method of any one of aspects 1 to 9, wherein clinically assessing
Parkinson's disease in the subject
comprises monitoring the progression of Parkinson's disease in the subject.
11. The method of aspect 10, wherein steps (a) and (b) are repeated on a
further blood sample from the subject
.. obtained at a later point of time.
12. Erythrocyte-derived extracellular vesicles (EEV) from a human subject's
blood sample for use as a
biomarker for clinically assessing Parkinson's disease in said subject.
13. Use of erythrocyte-derived extracellular vesicles (EEV) from a human
subject's blood sample as a biomarker
for clinically assessing Parkinson's disease is said subject.
14. The EEV of aspect 12, or the use of aspect 13, wherein said EEV are
CD235a+ extracellular vesicles.
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15. The EEV of aspect 12 or 14, or the use of aspect 13 or 14, wherein said
EEV are TSG101+, Rabs+, CD9+,
CD63+, CD81+, or any combination thereof.
16. The EEV of any one of aspects 12, 14 and 15, or the use of any one of
aspects 13 to 15, wherein said EEV
are between about 20 nm and about 1000 nm in diameter.
17. The EEV of any one of aspects 12, 14, 15 and 16, or the use of any one
of aspects 13 to 16, wherein said
EEV are greater than about 100 nm in diameter.
18. The EEV of any one of aspects 12 and 14 to 17, or the use of any one of
aspects 13 to 17, wherein said
EEV are present in platelet-free plasma.
19. The EEV of any one of aspects 12 and 14 to 18, or the use of any one of
aspects 13 to 18, wherein said
clinical assessment comprises diagnosing Parkinson's disease in the subject.
20. The EEV of any one of aspects 12 and 14 to 19, or the use of any one of
aspects 13 to 19, wherein said
clinical assessment comprises staging Parkinson's disease in the subject.
21. The EEV of any one of aspects 12 and 14 to 20, or the use of any one of
aspects 13 to 20, wherein said
clinical assessment comprises monitoring the progression of Parkinson's
disease in the subject.
22.
A method for preparing a clinical human blood sample, said method
comprising: (a) identifying a human
subject as having or suspected of having Parkinson's disease; (b) obtaining a
blood sample comprising erythrocyte-
derived extracellular vesicles (EEVs) from the human subject having or
suspected of having Parkinson's disease; (c)
processing said blood sample by separating EEVs having a diameter of greater
than 100 nm from EEVs having a
diameter of less than 100 nm, thereby obtaining a processed blood sample
comprising EEVs of diameter greater
than 100 nm; and (d) quantifying the level of EEVs of diameter greater than
100 nm in the processed blood sample.
23. The method of aspect 22, wherein said EEV are CD235a+ extracellular
vesicles.
24. The method of aspect 22, wherein said EEV are TSG101+, Rabs+, CD9+,
CD63+, CD81+, or any
combination thereof.
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Date Recue/Date Received 2022-07-06
25. The method of any one of aspects 22 to 24, wherein said blood sample is
platelet-free-plasma.
26. The method of any one of aspects 22 to 25, wherein said EEV quantified
in (d) are between 100 nm and
1000 nm in diameter.
27. The method of any one of aspects 22 to 26, wherein said EEV are
quantified by flow cytometry, nanoparticle
tracking (NTA), or electron microscopy.
28. The method of any one of aspects 22 to 27, wherein steps (a) to (d) are
repeated on an additional blood
sample from the same subject obtained at a later point of time.
29. The method of any one of aspects 22 to 28, wherein the processed blood
sample enriched for extracellular
vesicles of diameter greater than 100 nm has a higher number of EEVs of
diameter greater than 100 nm than a
corresponding processed blood sample from a non-Parkinson's disease subject.
30. The method of any one of aspects 22 to 29, wherein the quantifying in
(d) further comprises separating the
EEVs from non-EEVs in the processed blood sample.
31. The method of any one of aspects 22 to 30, wherein the blood sample is
from a Parkinson's disease subject
having a Unified Parkinson's Disease Rating Scale (UPDRS) score lower than 35.
32. The method of any one of aspects 22 to 30, wherein the blood sample is
from a Parkinson's disease
subject having a UPDRS score between 35 and 75.
General Definitions
Headings, and other identifiers, e.g., (a), (b), (i), (ii), etc., are
presented merely for ease of reading the
specification and claims. The use of headings or other identifiers in the
specification or claims does not necessarily
require the steps or elements be performed in alphabetical or numerical order
or the order in which they are
presented.
The use of the word "a" or "an", when used in conjunction with the term
"comprising" in the claims and/or the
specification may mean "one" but it is also consistent with the meaning of
'one or more", "at least one", and "one or
more than one".
The term "about" is used to indicate that a value includes the standard
deviation of error for the device or
method being employed to determine the value. In general, the terminology
"about' is meant to designate a possible
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Date Recue/Date Received 2022-07-06
variation of up to 10%. Therefore, a variation of 1, 2, 3, 4, 5, 6, 7, 8, 9
and 10% of a value is included in the term
"about". Unless indicated otherwise, use of the term "about" before a range
applies to both ends of the range.
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As used in this specification and claim(s), the words "comprising" (and any
form of comprising, such as
"comprise" and "comprises"), "having" (and any form of having, such as "have"
and "has"), "including" (and any form
of including, such as "includes" and "include") or "containing" (and any form
of containing, such as "contains" and
"contain") are inclusive or open-ended and do not exclude additional, un-
recited elements or method steps.
Other objects, advantages and features of the present description will become
more apparent upon reading
of the following non-restrictive description of specific embodiments thereof,
given by way of example only with
reference to the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
In the appended drawings:
Figures 1A-1F show controls for flow cytometry in order to optimize the
detection of the extracellular
vesicles (EV). (A) To properly set the EV gate, fluorescent silica beads of
100 nm (Red), 500 nm (Blue) and 1000nm
(Yellow) were acquired on a flow cytometer Canto II modified with a FSC-PMT
small particles option. The EV gate
was used throughout the experiments. (B) Serial dilutions (1, 2, 4 and 10) of
erythrocyte-derived EV (EEV) to confirm
.. the linearity of the quantification. (C) FSC-PMT/SSC gates of platelet-free
plasma (PFP) stained with annexin V and
respective fluorochrome-conjugated antibodies directed against erythrocyte
(CD235a+), endothelial
(CD31+/CD41-)/platelets (CD41+) and leukocytes (CD14+CD45+, monocytes;
CD15+CD45+, granulocytes)-derived
EV. (D) Treatment with the ion chelator EDTA inhibited the binding of annexin
V to phosphatidylserine. (E) Minimal
background was observed using antibodies in absence of PFP. This background
was subtracted from all subsequent
EV quantifications. (F) EV sensitivity to 0.5% TritonTivI was assessed.
Abbreviations: AnnV, annexin V; FSC PMT-H,
forward scatter photomultiplier; PBS, phosphate buffered saline; PFP, platelet
free plasma; SSC-H, side scatter.
Figure 2A shows the correlations between the number of erythrocyte-derived
extracellular vesicles (EEV;
expressed as CD235a+ EV / total number of erythrocytes) and the Unified
Parkinson's Disease Rating Scale
(UPDRS) of subjects (n = 20). Subjects identified as "1" and "2" both
presented with the same UPDRS score of 37,
.. but subject "1" was not receiving any treatment for their PD symptoms,
while subject "2" was being treated with
levodopa. Subject "3" presented with a UPDRS score of 66, but was taking anti-
inflammatory medications to manage
arthritis.
Figure 2B shows the results of a similar analysis as in Figure 2A, but
performed on Huntington's disease
subjects (n = 42) using the Unified Huntington's Disease Rating Scale (UHDRS).
The numbers of EEV are expressed
as CD235a+ EV / total number of erythrocytes.
DETAILED DESCRIPTION
The present description stems from the surprising discovery that erythrocytes
of Parkinson's disease (PD)
subjects produce more extracellular vesicles (EV) than erythrocytes from
corresponding control subjects, and that the
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level of erythrocyte-derived extracellular vesicles (EEV) strongly correlates
with PD stage and PD treatment. This
effect was not observed for EV originating from other cell types (e.g.,
platelets, endothelial cells, monocytes,
granulocytes, and leukocytes). Furthermore, the increase in number of EEV and
its strong correlation with PD stage
was not observed in erythrocytes from Huntington's disease subjects,
suggesting that these observations are specific
for PD. Thus, the present description relates to the use of EEV as a biomarker
for clinically assessing Parkinson's
disease.
In some aspects the present description relates to a method for clinically
assessing Parkinson's disease in a
human subject using extracellular vesicles originating from erythrocytes as a
biomarker.
As used herein, the expression "clinically assessing" or "clinical assessment"
in the context of PD refers
to an evaluation of a subject's PD state, which may or may not occur in a
clinical setting, and which may or may not
be performed by a health care professional. For example, clinically assessing
may comprise screening and/or
diagnosing PD in a subject having or suspected of having PD, staging a
subject's PD, monitoring the progression of
PD in a subject, monitoring the effect of PD medication, or any combination
thereof.
As used herein, the term "biomarker" refers to a molecular indicator that is
associated with a particular
pathological or physiological state. For example, the expression "Parkinson's
disease biomarker" or "PD
biomarker" refers to a molecular indicator that is associated with the
presence, stage, and/or progression of PD in a
subject.
As used herein, the expression "extracellular vesicles" (EV) refers to
subcellular membrane vesicles found
in the extracellular environment (e.g., bodily fluids) that originate from
cells, and which range in size from about
20 nm to about 1000 nm. EV may comprise exosomes, microvesicles (MV),
multivesicular endosomes (MVE), or
vesicles produced by apoptotic bodies, or any combination thereof, as well as
other types of extracellular vesicles.
Whereas the majority of the circulating EV that are efficiently detected by
flow cytofluorometric assays are likely to be
MV, we do not completely exclude the potential contribution of larger exosomes
or vesicles produced by apoptotic
bodies. In some embodiments, the EV of the present description comprise
vesicles between about 30, 40, 50, 60, 70,
80, 90, or 100 nm to about 500, 600, 700, 800, 900, or 1000 nm in size. In
some embodiments, the EV of the present
description comprise vesicles between greater than 100 nm to 1000 nm in size.
In some embodiments, the EV of the
present description comprise vesicles between 150 nm to 1000 nm in size. All
EV are composed of membrane
proteins and lipids, as well as cytoplasmic components of the cell from which
they originate, such as mRNA and
miRNA, organelles or infectious particles (e.g., prions, virus). A variety of
methods may be used to determine the
origin of EV. For example, cell surface markers (e.g., with immunolabeling
and/or flow cytometry techniques) may be
used to identify, enrich/purify/isolate, and/or quantify EV according to their
cell of origin. Examples of such markers
include: CD235a+ (erythrocytes), CD31+/CD41- (endothelial cells), CD41+
(platelets), C045+ (leukocytes),
CD45+CD14+ (monocytes), and CD45+CD15+ (granulocytes). Of particular interest
for the present description are
markers that are present in (or specific for) EEV that may be used to
identify, enrich/purify/isolate, and/or quantify
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EEV from other types of EV. Examples of such EEV markers include endosome or
membrane-bonding proteins such
as TSG101 and Rabs (enriched in exosomes), tetraspanins such as CD9, CD63 and
CD81 (enriched in exosomes),
golgi and mitochondrial proteins (enriched in MVs and absent in exosomes)
(Lotvall et al., 2014).
As used herein, the expression "[marked+ EV" or "[marker]-positive" refers to
the presence or detectability
of that marker in an EV population of interest, regardless of whether that
marker is actually detected (e.g., using an
immunolabel). Conversely, the expression "[marked- EV" or "[marked-negative
EV" refers to the absence or lack of
detectability of that marker in an EV population of interest, regardless of
whether that marker is actually detected
(e.g., using an immunolabel). For example, the expression "CD235+ EV" or
"CD235a-positive EV" means EV that
comprise the marker CD235a (Glycophorin A).
In some embodiments, the method of the present description may comprise
identifying,
enriching/purifying/isolating, and/or quantifying EEV in a blood sample from
the subject. As used herein, the terms
"enriched", "purified", "isolated" and the like, refer to either removing
contaminants from a biological sample and/or
increasing the concentration of an analyte of interest in the sample, to an
extent that is not found in nature. In some
embodiments, identifying, enriching/purifying/isolating, and/or quantifying
EEV may be performed by flow cytometry,
or other methods such as nanoparticle tracking (NTA), biochemical approaches
and semi-quantitative electron
microscopy approaches. In some embodiments, the quantification of EEV may be
expressed as a relative value by
normalizing the number of EEV (e.g., in terms of the total number of
erythrocytes).
In some embodiments, the methods of the present description may further
comprise comparing the level of
EEV to a suitable reference value indicative of the presence, stage and/or
progression of Parkinson's disease,
thereby clinically assessing Parkinson's disease in the subject.
As used herein, the expression "reference value" means a control value or
range of values corresponding
to a known level or range of EEV associated with the presence, stage and/or
progression of Parkinson's disease. In
some embodiments, for example where the number of EEV has previously been
measured in a blood sample from a
subject, the reference value may be a value corresponding to the same
subject's previous reading (e.g., a baseline).
The term "suitable" in the expression "suitable reference value" reflects the
observations reported herein that the
number of EEV in blood samples from PD subjects may vary depending on, for
example, factors which may also
affect the EV and/or EEV levels. For example, it is reported herein that a
subject's EEV levels may be affected by
whether or not the subject is being treated for their PD symptoms, the number
of PD treatments being taken by the
subject, whether the subject has or previously had cancer, whether the subject
has or previously had diabetes, or
whether the subject is taking anti-inflammatory medication.
In some aspects, the blood samples may be processed to obtain platelet-free
plasma.
In some aspects, the present invention relates to erythrocyte-derived
extracellular vesicles (EEV) from a
human subject's blood sample for use as a biomarker for clinically assessing
Parkinson's disease in said subject. In
6
some aspects, the present invention relates to the use of erythrocyte-derived
extracellular vesicles (EEV) from a
human subject's blood sample as a biomarker for clinically assessing
Parkinson's disease is said subject.
Other objects, advantages and features of the present description will become
more apparent upon reading
the following non-restrictive description of specific embodiments thereof,
given by way of example only with reference
to the accompanying drawings.
EXAMPLES
Example 1 ¨ Methods
1.1 Participant recruitment and ethic statement
Human blood was obtained from two cohorts of participants. The first cohort
was composed of Parkinson's
disease (PD) patients and Controls, and the second cohort was composed of
Huntington's disease (HD) patients and
Controls. The demographics for both cohorts are shown in Table I. For the two
cohorts, the Controls were recruited
amongst the caregivers, spouses, family and friends of the patients.
Institutional review boards approved this study
(CHU de Quebec-Universite Laval, #A13-2-1096; CHUM, #14.228; Cambridge Central
Regional Ethics Committee,
REC #03/303; and Cambridge University Hospitals Foundation Trust Research and
Development department, R&D
#A085170) in accordance with the Declaration of Helsinki, and written informed
consent was obtained from all
participants. On the day of the blood collection, every participant filled in
a questionnaire on health issues and
medication.
Of note, participants excluded from the present EEV-related analyses included
those with diabetes and
those suffering or having suffered from cancer, because we observed a
significant PD-independent increase in EEV
concentration in the platelet-free plasma of these participants. Furthermore,
PFP samples with elevated free
hemoglobin (>45 000 ng/mL), potentially due to hemolysis at blood collection,
were also excluded from EEV-related
analyses.
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Table 1 - Participant demographics
Parkinson's disease (PD) cohort
PD Patients - Stages of disease
Ctrl
P value
Unknown Mild Moderate
Severe ,
n 37 7 12 33 8
.
Age 66.8 69.8 66.7 71.1
75.0* 0.04
Gender F (M) 18(19) 1(6) 6(6) 16(17) 0(8)
0.05 .
Disease severity
Hoehn & Yahr (n) 1 0.3 (12) 2 0.2 (33)
3 0.5 (8) <0.0001
UPDRS (n) 38 11(6) 52 . 19 (17)
73 20 (6) 0.02
ACE (n) 96 4 (6) 92 7 (17)
_ 84 14 (6) 0.13
MMSE (n) 29 2 (7) 29 1 (19)
26 3 (6) 0.01
BDI (n) 3 2 (6) 4 2(17)
13 7 (4) 0.03
Comorbidities
Asthma 3 1 1 1 5 0
0.71
Hypertension 10 1 2 10 3
_ 0.76 _
Diabetes 2 0 0 1 2
0.10
Cancer 5 0 3 4 , 1
0.64
Allergies 2 0 2 6 2
0.28
Depression 3 1 2 1 2
0.29
Hypercholesterolemia 5 0 1 6 1
0.73
Huntington's disease (HD) cohort
HD Patients - Stages of disease
Ctrl
P value
Pre-HD Stage 1 Stage 2 Stage 3 Stage
4 Stage 5 .
n 55 11 15 13 12 10 2
Age 55.0 37,5 53.1 54.2 58.3 58.1 55.5
0.02
, Gender F (M) 31(22) 6 (5) 5(10) 4(9) 8(4) 7 (3) 1(1)
0.26
Disease severity
UHDRS (n) 2.7(11) 15.7 (14) 34.5 (11)
42.9 (12) 55.9 (10) 67.5(2) <0.001
TFC (n) 13(16) 13(11) 12.5 (15) 7,8 (13) 4.3
(12) 1.6 (10) 0(2) <0.001
CAG (n) 28.3 (3) 41.1 (10) 42.3 (13) 42.6 (12)
43.7 (7) 44.3 (7) <0.001 -
BDS (n) 206 (10) 337 (13) _ 356 (12)
442 (7) 465 (7) <0.001 -
Comorbidities
Asthma 0 0 1 0 0 0 0
0.65
Hypertension 4 1 2 1 1 2 0
_ 0.92 ,
Diabetes 3 1 1 1 1 - 1 0
0.99
Cancer 0 0 0 0 0 0 0
Allergies 3 0 2 2 0 0
0 , 0.33
Depression 8 1 1 3 6 4 1
0.0497
Hypercholesterolemia 8 1 1 0 0 1 0
0.32
Abbreviations: ACE, Addenbrooke's cognitive examination; BDI, Beck depression
inventory; BDS, Burden of Disease
Score; CAG, Trinucleotide repeat; MMSE, Mini-Mental State Examination; UHDRS,
Unified Huntington's Disease
Rating Scale; TFC, Total Function Capacity.
1.2 Preparation of Platelet-free plasma (PFP) and extracellular vesicle
(EV) labeling
Citrated blood was centrifuged twice for 15 minutes at 2500g at room
temperature. Platelet-free plasma
(PFP) was harvested and stored at -80 C within 2 hours of collection following
guidelines suggested by Lacroix and
colleagues (Lacroix et al,, 2012).
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,
,
For all experiments, diluted annexin-V buffer (BD Pharmingen, Mississauga, ON,
Canada) and phosphate
buffered saline (PBS) were filtered on 0.22 pm pore size membranes. To
quantify the EV according to their cell of
origin, the following surface markers were used: CD235a+ (erythrocytes),
CD31+/CD41- (endothelial cells), CD41+
(platelets), CD45+ (leukocytes), CD45+CD14+ (monocytes), and CD45+CD15+
(granulocytes), with or without
annexin-V staining. PFP (5 pL) was incubated with Phenylalanyl-prolyl-arginyl
Chloromethyl Ketone (PPACK)
(Calbiochem, Etobicoke, ON, Canada) for 5 minutes, followed by a 30-minute
incubation with antibodies and
annexin-V, all at room temperature. The following antibodies were purchased at
BD Pharmingen and used
throughout the experiments: FITO-conjugated mouse anti-human CD235a (clone GA-
R2 (H1R2), 1/20), PE-
conjugated mouse anti-human CD31 (clone WM59, 1/100), V450-conjugated mouse
anti-human CD41a (clone HIP8,
1/20), APC mouse anti-human CD14 (clone M5E2, 1/10), PE-conjugated mouse anti-
human CD15 (clone HI98,
1/50), V450-conjugated mouse anti-human CD45 (clone HI30, 1/33), V450- and
PerCP-CyTM5.5-conjugated
annexin-V (1/33 and 1/10, respectively).
1.3 Flow cytometry quantification
For EV quantification, we used a FACS Canto II Special Order Research Product
equipped with a forward
scatter (FSC) coupled to a photomultiplier tube (FSC-PMT). Flow cytometer
performance tracking was carried out
daily using the BD cytometer setup and tracking beads (BD Biosciences, San
Jose, CA, USA). The size of the EV
was determined using fluorescent silicone beads of 100, 500 and 1000 nm.
Controls and optimization of the detection
method are presented in Figures 1A-1F. The settings for the EV detection were
determined as described previously
(Rousseau et al., 2015) using a threshold of 200 for SSC. Between PD and HD
analyses, the blue laser had to be
replaced for maintenance issues and therefore laser settings were reassessed.
For FSC-PMT, the assigned voltage
was 363 (PD) and 160 (HD) Volts. For SSC, the assigned voltage was 407 (PD)
and 300 (HD) Volts. All other
parameters were set between 450 and 500 Volts. The acquisition of EV was
performed at low speed with an
approximate rate of 10 pL/min. To determine background noise level, antibody
mixes were incubated in absence of
PFP sample and unlabeled PFP was used as a negative control.
1.4 Statistical analyses
All statistical analyses were performed using "The Statistics and Machine
Learning Toolbox" provided by
MathWorksTm under the MATLABTNA platform. The version used was MATLABOR2015a.
The analysis included the
scatter plot, the classical least-squares linear regression model, the R-
squared and p values, as well as Pearson's
goodness-of-fit model. Interval cut-off values were determined using a loop
program developed in MATLABTm.
Regression diagnostics, including residual behaviour and homoscedastivity,
were also obtained with the same
Toolbox.
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Example 2 ¨ Results
The cohorts studied here included Parkinson's disease (PD) (n=60) and
Huntington's disease (HD) patients
(n=52) of all stages (see Example 1.1), as well as their respective age- and
sex-matched healthy controls (n=37;
n=55, respectively). The demographics for both cohorts are shown in Table 1.
Full blood counts (erythrocytes,
lymphocytes, platelets, leukocytes, monocytes, neutrophils) and C-reactive
protein quantification were obtained for all
participants, but they did not reveal any differences between groups (data not
shown). Similarly, the hematocrit, the
mean corpuscular hemoglobin, as well as the mean corpuscular volume values
were similar between PD and control
groups (data not shown).
2.1 PD patients exhibit a disease-specific increase in erythrocyte-
derived EV
Platelet-free plasma (PFP) and extracellular vesicles (EV) were labeled and
quantified according to their cell
of origin for all participants, as described in Examples 1.2 and 1.3. Results
are summarized in Table 2A (PD
patients and controls) and Table 2B (HD patients and controls).
As shown in Table 2A, no significant differences between PD patient and
control samples were observed in
the number of EV originating from platelets, endothelial cells, monocytes,
granulocytes, and leukocytes. Similarly, as
shown in Table 2B, no significant differences between HD patient and control
samples were observed in the
concentrations EV originating from these same cell types.
Interestingly, a significant increase in erythrocyte-derived EV in patients
with PD was observed, as
compared to the control group (see values highlighted in black in Table 2A).
This increase in erythrocyte-derived EV
in patients with PD was disease-specific, as the same effect was not observed
in erythrocyte-derived EV in patients
with HD (Table 2B).
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Table 2A - Quantification of extracellular vesicles (EV) derived from
different cell types of PD patients and controls
CTRL PD
Cell type Markers Units P value
n Mean SEM n Mean SEM
CD41+PS- 37
7.88 1.68 59 10.3 1.33 0.27
CD41+PS+ 37 x 103/pL 15.2 3.20 59
17.9 2.53 0.51
Platelets CD41+CD31+ 37
1.51 0.69 59 1.99 0.54 0.59
CD41+ total 37 23.1 4.62 59 28.2
3.66 0.38
EV CD41+/platelet 35 0.106 0.021 57
0.125 0.016 0.49
CD31+CD41-PS- 37
15.8 8.04 59 11.7 6.37 0.75
Endothelial cells CD31+CD41-PS+ x 103/pL 37 0.91 0.13 59
0.92 0.10 0.96
CD31+CD41- total 37 16.7 8.03 59 12.6
6.36 0.75
CD45-CD14+ PS- 37 1.70 0.30 59 1.62
0.24 .. 0.85
CD45-CD14+ P5+ 37 1.20 4.00 59 5.84
3.17 0.50
CD45+CD14+ PS- x 103/4 37 0.16 0.04 59
0.14 0.03 0.74
Monocytes C045+CD14+PS+ 37
0.60 0.79 59 1.47 0.63 0.59
CD14+ total 37 3.66 4.88 59 9.06
3.87 0.60
EV CD14+ /monocyte 35 7.08 1.99 57 9.16
1.56 0.41
CD45-CD15+ PS- 37 12.3 7.96 59 16.7
6.30 0.92
CD45-CD15+ PS+ 37 2.21 0.77 59 1.39
0.61 0.47
C045+CD15+ PS- x 103/pL 37 0.55 0.36 59
1.15 0.29 0.20
Granulocytes
C045+CD15+PS+ 37
1.01 0.30 59 1.25 0.24 0.56
CD15+ total 37 16.0 8.83 59 20.6
6.99 0.91
EV CD15+ /granulocyte 35 3.70 0.64 57 3.16
0.50 .. 0.53
Leukocytes CD45+ total x 103/pL 37 10.4 2.21 59
13.8 1.75 0.26
CD235a+PS. 36
18.2 46.5 59 32.0 36.3 0.04
CD235a+PS+ x 103/pL 36 0.22 0.07 59
0.29 0.05 0.70
Erythrocytes
CD235a+ total 36 18.4 47.0 59 32.3
36.7 0.04
EV CD235a+ierythrocyte 34 0.0039
0.011 57 0.0069 0.008 0.04
Abbreviations: CD235a, glycophorin A; EV, extracellular vesicle; PD,
Parkinson's disease; PS, phosphatidylserine.
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CA 02941460 2016-09-08
Table 2B - Quantification of extracellular vesicles (EV) derived from
different cell types of HD patients and controls
7 CTRL HD pre-manifest HD
2i S. Markers Units
C") value
n Mean SEM n Mean SEM n Mean SEM
CD41+PS- 54 9.2 2.2 10 4.3 1.3 50
6.1 1.0 0.78
v.)
CD41+PS+ x 103/pL 54 19.3 4.8 10 7.1
2.0 50 12.4 2.4 074
CD41+ total 54 28.4 6.9 10 11.4 3.2 50
18.6 3.4 0.70
EV CD41+/platelet 53 0.12 0.03 10 0.05 0.02 48 0.08
0.01 0.34
7 CD31+CD41-PS- 54 1.4 0.3 10 0.6 0.2
50 1.2 0.2 0.31
0-
w
111 CD31+CD41-PS+ x 103/pL 54 0.68 0.16 10
0.25 0.06 50 0.46 0.09 0.59
-stii 4-)
CD31+CD41- total 54 2.1 0.4 10 0.8 0.2 50 1.7
0.3 0.26
CD45-CD14+ PS- 54 3.4 1.1 10 1.6 0.2 51 1.6 0.1
0.91
CD45-CD14+ PS+ 54 1.8 0.3 10 0.8 0.3 51 1.5 0.2
0.14
tft CD45+CD14+ PS- x 103/pL 54 0.18 0.07
10 0.069 0.016 51 0.056 0.008 0.34
CD45+CD 14+PS+ 54 0.62 0.12 10 0.24 0.06 51 0.55
0.14 0.12
0
CD14+ total 54 6.0 1.3 10 2.6 0.4 51
3.7 0.4 0.08
EV CD14+/monocyte 53 12.3 2.5 10 5.7 0.6 48 8.0 1.0
0.13
CD45-CD15+ PS- 54 1.2 0.1 10 1.2 0.3 51 1.5 0.2
0.33
CD45-CD15+ PS+ 54 0.12 0.04 10 0.18 0.08 51 0.22
0.11 0.33
g CD45+CD15+ PS- x 103/pL 54 0.20 0.05 10
0.07 0.02 51 0.15 0.04 0.64
-3 CD45+CD15+PS+ 54 0.25 0.05 10
0.13 0.06 51 0.20 0.04 0.39
CD15+ total 54 1.7 0.2 10 1.6 0.4 51
0,20 0.3 0.67
EV CD15+ /granulocyte 53 0.41 0.04 10 0.42 0.13
48 0.50 0.08 0.75
0 CD45+ total x 103/pL 54 33.4 2.7 10 31.6
5.3 51 31.7 2.4 0.88
CD235a+PS-
54 15.2 2.0 10 10.3 3.5 51 14.1 1.4 0.16
CD235a+PS+ x 103/pL 54 1.1 0.2 10 0.4 0.2
51 1.1 0.1 0.04
CD235a+ total 54 16.4 2.0 10 10.7 3.5 51
15.3 1.5 0.09
EV CD235a+/erythrocyte 54 0.0035 0.0005
10 0.0023 0.0008 50 0.0033 0.0003 0.11
Abbreviations: CD235a, glyc-ophorin A; EV, extracellular vesicle; HD,
Huntington's disease; PS, phosphatidylserine.
2.2
Increase in erythrocyte-derived EV in PD patient samples correlates with
PD progression and PD
treatment
To evaluate its suitability as a potential biomarker for monitoring PD
progression, we examined correlations
between the number of erythrocyte-derived EV (EEV) and two different PD
staging systems: the Hoehn & Yahr scale,
and Unified Parkinson's Disease Rating Scale (UPDRS). Strikingly, this
analysis revealed strong correlations
between the number of erythrocyte-derived EV and PD stage/progression, when
using either of these PD rating
scales. As shown in Figure 2A, strong correlations (correlations exceeding
0.8) were observed between the number
of erythrocyte-derived EV (expressed as CD135a+EV/total number of
erythrocytes) and patient UPDRS score, and
thus PD stages. Similar strong correlations were also observed using the Hoehn
& Yahr scale (data not shown),
12
CA 02941460 2016-09-08
'
although the UPDRS is being presented because of its greater sensitivity and
the recent publications validating this
approach (see Martinez-Martin et al., 2015). By stratifying the PD patients
according to their disease severity and/or
treatment, it was observed that "mild" PD patients with a UPDRS score lower
than 35 and taking 1 PD medication,
showed an increased number of EEV during disease evolution (correlation =
0.886). A very similar pattern
(correlation = 0.873) was observed for "moderate" PD patients with UPDRS
scores between 35 and 75 and taking
?.: 2 PD medications, with almost all data points falling within the
statistical confidence bounds. Of note, one patient
presenting with a UPDRS score of 66 exhibited a relatively low count of EEV,
but this patient was taking anti-
inflammatory medications to manage his arthritis, which may explain this
result (see Figure 2A, patient "3"). A lack of
available data precluded a similar analysis for patients presenting with
severe PD (UPDRS scores higher than 75).
Interestingly, two patients presenting with the same UPDRS score of 37 had
very different numbers of EEV.
The patient receiving treatment for their PD symptoms (see Figure 2A, patient
"2") exhibited much a lower number of
EEV than another patient having the same UPDRS score but who was not receiving
any treatment for their PD
symptoms (see Figure 2A, patient "1").
The above correlations observed with respect to the number of EEV in PD
patients was found to be disease-
specific, since a similar analysis performed in HD patients failed to reveal
the same strong correlations (see
Figure 2B).
We have thus identified at least two distinct groups of PD patients with
highly significant correlations to the
number of EV derived from erythrocytes, which relates to PD stage and/or PD
treatment (Figure 2A). Strikingly,
these correlations appear to be specific to PD, as similar correlations were
not observed in the cohort of HD patients
(of varying degrees of severity) in which we performed identical analyses
(Figure 2B).
REFERENCES
Lacroix et al, Journal of Thrombosis and Haemostasis (2012), 10:437-446.
Lotvall et al., J Extracell Vesicles (2014), 3:26913.
Martinez-Martinet al., Parkinsonism & related disorders (2015). 21(1):50-4.
Rousseau et al., PLoS One (2015), 10(1):e0116812.
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