U.S. patent application number 16/277565 was filed with the patent office on 2019-06-13 for marker for the classification, diagnosis and treatment of scoliosis.
This patent application is currently assigned to Chu Sainte-Justine. The applicant listed for this patent is Chu Sainte-Justine. Invention is credited to Marie - Yvonne Akoume Ndong, Alain Moreau.
Application Number | 20190178896 16/277565 |
Document ID | / |
Family ID | 52627658 |
Filed Date | 2019-06-13 |
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United States Patent
Application |
20190178896 |
Kind Code |
A1 |
Moreau; Alain ; et
al. |
June 13, 2019 |
MARKER FOR THE CLASSIFICATION, DIAGNOSIS AND TREATMENT OF
SCOLIOSIS
Abstract
A method of classifying a subject having idiopathic scoliosis
(IS) or at risk of developing IS comprising: determining the
cellular response to Gi stimulation in a cell sample from the
subject in the presence of OPN; determining the cellular response
to Gi stimulation in a cell sample from the subject in the absence
of OPN; and comparing the cellular response obtained in the
presence of OPN with the cellular response obtained in the absence
of OPN, whereby the comparing step enables the classification of
the subject into one IS functional group. Also provided is the use
of the foregoing method to classify borderline subjects and kits
for applying the methods.
Inventors: |
Moreau; Alain; (Montreal,
CA) ; Akoume Ndong; Marie - Yvonne; (Montreal,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Chu Sainte-Justine |
Montreal |
|
CA |
|
|
Assignee: |
Chu Sainte-Justine
Montreal
CA
|
Family ID: |
52627658 |
Appl. No.: |
16/277565 |
Filed: |
February 15, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14917757 |
Mar 9, 2016 |
10247737 |
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PCT/CA2014/050853 |
Sep 9, 2014 |
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16277565 |
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61879314 |
Sep 18, 2013 |
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61875162 |
Sep 9, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2310/14 20130101;
A61K 38/19 20130101; C12N 2320/30 20130101; A61F 5/02 20130101;
A61F 5/026 20130101; A61K 31/713 20130101; G01N 33/6893 20130101;
G01N 2333/47 20130101; G01N 33/48728 20130101; G01N 2800/10
20130101; G01N 2800/52 20130101; C12N 15/1138 20130101; A61K
31/4045 20130101; A61K 45/06 20130101; A61K 33/04 20130101; G01N
2333/726 20130101; G01N 2800/50 20130101; A61K 31/4045 20130101;
A61K 2300/00 20130101; A61K 31/713 20130101; A61K 2300/00 20130101;
A61K 33/04 20130101; A61K 2300/00 20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68; A61K 38/19 20060101 A61K038/19; A61K 45/06 20060101
A61K045/06; A61K 31/713 20060101 A61K031/713; A61F 5/02 20060101
A61F005/02; G01N 33/487 20060101 G01N033/487; A61K 33/04 20060101
A61K033/04; C12N 15/113 20060101 C12N015/113; A61K 31/4045 20060101
A61K031/4045 |
Claims
1. A method of classifying a subject having idiopathic scoliosis
(IS) or at risk of developing IS or a method of determining the
risk of developing IS in a subject comprising: (A) a) contacting a
cell sample from the subject with i) an agonist of cluster I or
cluster II GiPCR; and ii) Pertussis toxin (PXT); b) determining the
cellular response to Gi stimulation; and c) classifying said
subject into a IS functional group according to the subject's
cellular response profile; or (B) a) contacting a cell sample from
the subject with an agonist of a cluster II GiPCR; b) determining
the cellular response to Gi protein stimulation by cellular
impedance; and c) classifying the subject into a IS functional
group according to the subject's cellular impedance profile.
2. The method of claim 1: i. 1(A), wherein step (c) comprises (i)
classifying said subject into a first functional group when the
cellular response is above the cellular response of a control
sample; or (ii) classifying said subject into a second functional
group when the cellular response is substantially comparable to the
cellular response of a control sample; or ii. 1(B), wherein step
(c) comprises: (i) classifying the subject into a first IS
functional group when said cellular impedance profile comprises a
phase of negative impedance followed by a phase of positive
impedance; or (ii) classifying the subject into a second IS
functional group when the cellular impedance profile does not
comprise a phase of negative impedance.
3. The method of claim 1, wherein said cluster I GiPCR is
lysophosphatidic acid receptor (LPAR), melatonin receptor (MT1 or
MT2), mu-opioid receptor (OR) or adenosine receptor (AR) or wherein
said cluster II GiPCR is cannabinoid receptor (CB2R) or a
.alpha.2-Adrenoreceptor (.alpha.2AR).
4. The method of claim 3, wherein said agonist of cluster I GiPCR
is melatonin, iodomelatonin, phenylmelatonin, LPA, DAMGO or NECA or
wherein said agonist of cluster II GiPCR is CB65 or UK14304.
5. The method of claim 1(A), wherein the cellular response to Gi
stimulation is determined by measuring cellular impedance,
preferably by cellular dielectric spectroscopy.
6. The method of claim 1, further comprising classifying borderline
subjects into one IS functional group by using an alternative
classification method, and wherein said alternative classification
method is preferably performed prior to step (a).
7. The method of claim 6, wherein the alternative classification
method comprises: (i) determining the difference between cellular
responses to Gi and Gs protein stimulations in the cell sample from
the subject; (ii) determining the magnitude of a cellular response
to Gi stimulation in the cell sample relative to a control sample;
(iii) measuring cAMP concentration; (iv) determining the
phosphorylation state of Gi.alpha. proteins in the cell sample; or
(v) determining cellular proliferation of the cell sample relative
to a control sample.
8. The method of claim 7, wherein (i) determining the difference
between cellular responses to Gi and Gs protein stimulations
comprises (i a) measuring cAMP cellular concentration produced by
each of Gi and Gs protein stimulations; (i b) measuring cellular
impedance, wherein said cellular impedance is preferably measured
by cellular dielectric spectroscopy (CDS); (i c) determining the
.DELTA.G in the sample from the subject; or (i d) by determining a
ratio between cellular responses to Gi and Gs protein stimulations
(Gi/Gs).
9. The method of claim 8, wherein: in (i c) the .DELTA.G is
determined by measuring cellular impedance and the cellular
impedance is measured by CDS, and wherein i) a .DELTA.G below -10
is indicative that the subject belongs to IS functional group FG1;
ii) a .DELTA.G above -10 and below +10 is indicative that the
subject belongs to IS functional group FG2; and iii) a .DELTA.G
above +10 is indicative that the subject belongs to IS functional
group FG3; or in (i d) i) a ratio below about 0.5 is indicative
that the subject belongs to IS functional group FG1; ii) a ratio
between about 0.5 and 1.5, is indicative that the subject belongs
to IS functional group FG2; and iii) a ratio above 1.5 is
indicative that the subject belongs to IS functional group FG3.
10. The method of claim 7, wherein: in (ii):(ii a) a reduction of
between about 60-90% of the cellular response to Gi stimulation
relative to control is indicative that the subject belongs to IS
functional group FG1; (ii b) a reduction of between about 40-60% of
the cellular response to Gi stimulation relative to control is
indicative that the subject belongs to IS functional group FG2; and
(ii c) a reduction between about 10-40% of the cellular response to
Gi stimulation relative to control is indicative that the subject
belongs to IS functional group FG3; in (iii):(iii a) an increase in
cAMP concentration relative to baseline is indicative that the
subject belongs to IS functional group FG1, (iii b) no significant
or weak reduction in cAMP concentration relative to baseline is
indicative that the subject belongs to IS functional group FG2; and
(iii c) a reduction in cAMP concentration relative to baseline is
indicative that the subject belongs to IS functional group FG3; in
(iv): (iv a) an increase in the level of serine phosphorylated
Gi.alpha.1, Gi.alpha.2 and Gi.alpha.3 proteins as compared to a
control sample is indicative that the subject belongs to IS
functional group FG1; (iv b) an absence of serine phosphorylation
in Gi.alpha.3 proteins or a level of serine phosphorylation on
Gi.alpha.3 proteins comparable or lower to the level of serine
phosphorylation in Gi.alpha.3 proteins in a control sample is
indicative that the subject belongs to IS functional group FG2; and
(iv c) an absence of serine phosphorylation in Gi.alpha.1 proteins
or a level of serine phosphorylation on Gi.alpha.1 proteins
comparable to the level of serine phosphorylation in Gi.alpha.1
proteins in a control sample is indicative that the subject belongs
to IS functional group FG3; or in (v): (v a) a reduction in
cellular proliferation of about 50% or more relative to control is
indicative that the subject belongs to IS functional group FG1; (v
b) a reduction in cellular proliferation between about 25% and 45%
relative to control is indicative that the subject belongs to IS
functional group FG2; and (v c) a reduction in cellular
proliferation of about 25% or less relative to control is
indicative that the subject belongs to IS functional group FG3.
11. The method of claim 10, wherein in (ii), the magnitude of a
cellular response to Gi stimulation is determined by measuring
cellular impedance, preferably by cellular dielectric spectroscopy
(CDS).
12. A method of selecting a preventive measure, treatment or
follow-up schedule for a subject suffering from IS or at risk of
developing IS comprising classifying the subject using the method
as defined in claim 1; wherein a) when the subject is classified as
belonging to the FG1 functional group: i) the level of OPN in said
subject is increased; ii) the level of HA in said subject is
increased; iii) the level of CD44 in said subject is decreased;
and/or iv) the frequency of assessment of scoliosis progression is
decreased; b) when the subject is classified as belonging to the
FG2 functional group: i) the level of OPN is said subject is
decreased; ii) the level of HA in said subject is decreased; and/or
iii) the level of CD44 in said subject is increased; iv) the
frequency of assessment of scoliosis progression is increased;
and/or v) the subject is prescribed corrective surgery prior to
reaching a scoliosis with a cobb angle of 45 degree; c) when the
subject is classified as belonging to the FG3 functional group: i)
the level of OPN is said subject is decreased; ii) the level of HA
in said subject is decreased; iii) the level of CD44 in said
subject is increased; and/or v) the frequency of assessment of
scoliosis progression is decreased.
13. The method of claim 12, wherein: (aa) increasing the level of
OPN comprises administering OPN; applying pulsative compressive
pressure to a body part of the subject or applying low intensity
pulsed ultrasound to a body part of the subject; (bb) increasing
the level of HA comprises administering to the subject HA
supplements or complying to a HA-rich diet and wherein decreasing
the level of HA comprises complying to a HA-poor diet; (cc)
decreasing OPN level comprises brace treatment, accupoint heat
sensitive moxibustion, heat therapy with pad, thermal bath,
electroacupuncture, administering melatonin, administering selenium
or administering PROTANDIM; or (dd) decreasing the frequency of
assessment of scoliosis progression comprises performing less than
22 radiological examinations within 3 years following
classification.
14. The method of claim 1, wherein (i) the subject is a subject
pre-diagnosed with IS; (ii) the subject is asymptomatic; and/or
(iii) the subject has at least one family member suffering from
IS.
15. The method of claim 1, wherein the cell sample comprises
osteoblasts, chondrocytes, myoblasts and/or peripheral blood
mononuclear cells (PBMCs), preferably fresh PBMCs comprising
lymphocytes.
16. The method of claim 1, wherein said Gi stimulation further
comprises contacting the cell with GP Ant-2.
17. The method of claim 7, wherein the Gs stimulation comprises
contacting the cells with isoproterenol.
18. A kit for classifying a subject having idiopathic scoliosis
(IS) or at risk of developing IS or for predicting the risk of
developing IS comprising: (A) a) Pertussis toxin (PTX); and b) a
cluster I GiPCR and/or a cluster II receptor agonist; and
optionally, (B) c) a ligand for Gs stimulation; d) one or more
antibodies for detecting Gi.alpha. phosphorylation; and/or e)
instructions for classifying the subject or for predicting the risk
of developing IS.
19. The kit of claim 18, wherein the cluster I GiPCR is
lysophosphatidic acid receptor (LPAR), mu-opioid receptor (OR) or
adenosine receptor (AR) or melatonin receptor and the cluster II
GiPCR is cannabinoid receptor (CB2R) or a .alpha.2-Adrenoreceptor
(.alpha.2AR), and/or wherein the agonist is melatonin,
iodomelatonin, phenylmelatonin, LPA, DAMGO, NECA, CB65 or
UK14304.
20. A composition for classifying a subject having idiopathic
scoliosis (IS) or at risk of developing IS or for determining the
risk of developing IS in a subject comprising: (A) a) cell sample
from the subject; b) Pertussis toxin (PTX); and c) a cluster I
GiPCR and/or a cluster II GiPCR agonist; and optionally, (B) d) a
ligand for Gs stimulation; and/or e) one or more antibodies for
detecting Gi.alpha. phosphorylation.
21. The composition of claim 20, wherein the cluster I GiPCR is
LPAR, OR, A1R or melatonin receptor and the cluster II GiPCR is
CB2R or .alpha.2AR, and/or wherein the agonist is melantonin,
iodomelatonin, phenylmelatonin, LPA, DAMGO, NECA, CB65 or UK14304.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 14/917,757 filed on Sep. 9, 2014, now allowed,
which is a National Phase Entry of PCT application Ser. No.
PCT/CA2014/050853 filed on Sep. 9, 2014 and published in English
under PCT Article 21(2), which itself claims benefit of U.S.
provisional application Ser. No. 61/875,162, filed on Sep. 9, 2013
and of U.S. provisional application Ser. No. 61/879,314, filed on
Sep. 18, 2013. All documents above are incorporated herein in their
entirety by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to markers for idiopathic
scoliosis diagnosis and prognosis. More specifically, the present
invention is concerned with methods of identifying subjects at risk
of developing scoliosis and methods of classifying subjects having
or at risk of developing scoliosis according to their GiPCR
mediated cellular responses in the presence of osteopontin
(OPN).
BACKGROUND OF THE INVENTION
[0003] Idiopathic Scoliosis (IS) (e.g., Infantile Idiopathic
Scoliosis, Juvenile Idiopathic Scoliosis or Adolescent Idiopathic
Scoliosis (AIS)) is a spine deformity of unknown cause generally
defined as a lateral curvature greater than 10 degrees accompanied
by a vertebral rotation.sup.1. The condition affects 4% of the
paediatric population and is most commonly diagnosed between the
ages of 9 to 13 years.sup.2,3,4. The diagnosis is primarily of
exclusion and is made only after ruling out other causes of spinal
deformity such as vertebral malformation, neuromuscular or
syndromic disorders. Traditionally, the trunkal asymmetry is
revealed by Adams forward bending test and measured with
scoliometer during physical examination.sup.5. The diagnosis can
then be confirmed by radiographic observation of the curve and the
angle measurement using the Cobb method.sup.6.
[0004] Once diagnosed, the primary concern for physicians in
managing scoliotic children is whether the curve will progress.
Indeed, the curve progression is often unpredictable and is more
frequently observed among girls than in boys.sup.7. If untreated,
the curve can progress dramatically, creating significant physical
deformity and even cardiopulmonary problems. These manifestations
become life threatening when the curve exceeds 70 degrees.sup.8,9.
The current treatment options to prevent or stop curve progression
include bracing and surgery. In general, bracing is recommended for
curves between 25 and 40 degrees, while surgery is reserved for
curve greater than 45 degrees or curves that are unresponsive to
bracing.
[0005] Today in the United States there are approximately one
million children between ages 10 and 16 with some degree of IS and
about 100 000 children in Canada are diagnosed with IS. The total
cost of diagnosis and monitoring of the scoliotic children by X-ray
exposure is over $2.5 billion dollars annually in North America.
Approximately, 10% of children diagnosed with idiopathic scoliosis
have curve progression requiring corrective surgery.sup.10. About
29,000 scoliosis surgeries are done every year in North America,
resulting in significant psychological and physical morbidity.
[0006] Currently, there is no approved method or test available to
identify subjects at risk of developing IS or to predict which
affected individuals will show a curve progression that will
require surgery. Therefore, the application of current treatments,
such as bracing or surgical correction, is delayed until a
significant deformity is detected or until a significant
progression is clearly demonstrated, resulting in a delayed, less
than optimal treatment and often important psychological sequels
(Scoliosis Research Society) Morbidity & Mortality Committee
annual Report 1997). All diagnosed children are subjected to
multiple radiographs over several years, usually until they reach
skeletal maturity. It is estimated that the typical patients with
scoliosis will have approximately 22 radiological examinations over
a 3-year period.sup.11. Because of the potential risk of multiple
radiographic examinations, the alternative approaches that could
allow performing the prognosis of idiopathic scoliosis without
exposing children to ionizing radiation are strongly desirable.
[0007] The major limitation in developing prognostic tests that
could facilitate treatment choices for patients is the
heterogeneous nature of IS. At the clinical level, the
heterogeneity of IS is clearly illustrated by the variability of
curve patterns, localisations and curve magnitude even in families
with multiple affected members. In absence of reliable IS
phenotypes, there is a need to understand better the molecular
changes associated with disease onset and spinal deformity
progression. Molecular definition of disease is rapidly replacing
traditional pathology-based disease descriptions in part because of
its utility in identifying the optimal treatment regimen for
patients.
[0008] In this regard, the present inventors have previously
discovered that scoliotic patients and subjects at risk of
developing scoliosis are less responsive to Gi protein (inhibitory
guanine nucleotide binding protein in G protein coupled receptors
(GPCRs) also known as G.sub.i alpha subunit) stimulation when
compared with healthy control subjects. The presence of a general
differential Gi-signaling dysfunction allowed to stratify/classify
patients into three functional groups (FG1, FG2 and FG3)
representing distinct biological endophenotypes. This impairment
was detected in all cell types tested including bone-forming cells;
muscle-forming cells and blood cells (e.g., PBMCs). Furthermore,
because the response impairment is generalized and not specific to
a particular receptor, any Gi-PCR agonist can be used to classify
subjects.
[0009] A first classification method is based on the percentage of
degree of reduction (inhibitory response) relative to control
group. The classification ranges were fixed between about 10 and
40% (or below 40%) of reduction of response relative to control
group for FG3, about 40 and 60% for FG2 and above about 60% (e.g.,
between about 60% and 90%) for FG1. The same the classification
ranges can be expressed as the percentage of maximal response
relative to the control (as opposed to the % of reduction of
response relative to the control). In such a case, the ranges are
fixed between about 10-40% for FG1, about 40 and 60% for FG2 and
about 60-90% for FG3. Both classification ranges can be used
interchangeably (See Moreau et al., 2004; Akoume et al., 2010;
Azeddine et al., 2007; Letellier et al., 2008; WO2003/073102,
WO2010/040234 to Moreau, which are incorporated herein by reference
in their entirety).
[0010] More recently, the present inventors have modified this
approach by demonstrating that the three functional groups can
clearly be distinguished according to the profile of imbalance
between response to Gi and Gs stimulation (i.e. Gi response minus
Gs response or Gi/Gs-See PCT/CA2014/050562, which is incorporated
herein by reference). It was found that the response to Gi
stimulation predominated in FG3, while no substantial imbalance (or
a very small imbalance) was observed in FG2. In contrast, FG1
exhibited predominance for response to Gs stimulation. In addition,
evidence was provided to the effect that patients belonging to the
FG2 endophenotype are more at risk of progressing to the point of
needing surgery.sup.17.
[0011] The differences in Gi-mediated cellular response observed
among the three endophenotypes is (at least partly) a consequence
of differences observed at the level of Gi protein phosphorylation.
When Gi.alpha. proteins are phosphorylated they become inactive.
The inventors have shown that the degree of serine phosphorylation
of Gi.alpha. proteins can alternatively be used to classify
subjects into a specific functional group. In FG1 subjects, all
Gi.alpha. proteins (Gi.alpha.1-3) are phosphorylated and their
level of serine phosphorylation is substantially higher than in
control subjects. In FG2 Gi.alpha.1 and Gi.alpha.2 are
phosphorylated, the level of Gi.alpha.1 and Gi.alpha.2
phosphorylation is higher than in control subjects and most
Gi.alpha.3 proteins are not phosphorylated and thus, remain
functional. Finally, in FG3 subjects Gi.alpha.2 and Gi.alpha.3 are
phosphorylated, their level of phosphorylation is higher than in
control subjects and most Gi.alpha.1 are not phosphorylated and
thus remain functional.sup.18.
[0012] The assessment of an imbalance between Gi and Gs coupled
receptor signaling (as opposed to the assessment of a Gi-coupled
receptor signaling impairment), greatly simplifies the risk
assessment and endophenotype (functional group) assessment by
eliminating the need of a reference signal from a control subject.
The establishment of a reference signal is often difficult and may
sometimes constitute an obstacle because the control subject(s)
from whom the reference signal is derived should preferably match
with age, gender and medication, if any.
[0013] Although the methods of diagnosing subjects suffering from
scoliosis or at risk of developing scoliosis described above
provide significant advantages, certain subjects are more difficult
to classify into a functional group (FG1, FG2 or FG3) because their
response to Gi stimulation or degree of Gi/Gs imbalance approaches
the cut-off values defined for the classification. Furthermore,
determining the phosphorylation level/pattern of Gi.alpha. proteins
may be more difficult to implement in a clinical setting.
Therefore, there is a need for novel alternative or complementary
methods for classifying subjects having diseases involving spinal
deformities (e.g., scoliosis, such as IS) and for diagnosing a
predisposition to scoliosis.
[0014] The present description refers to a number of documents, the
content of which is herein incorporated by reference in their
entirety.
SUMMARY OF THE INVENTION
[0015] Accordingly, there are provided novel alternative methods of
identifying subjects at risk of developing IS and of classifying IS
subjects into a specific IS functional group. The methods can be
used alone or in combination with one or more previous methods to
increase the specificity of the functional group identification
which in turn can increase the specificity and sensitivity of risk
prediction and/or facilitate/improve the application of preventive
or treatment measures. Once a subject is identified as belonging to
a given functional group, treatment and preventive measures can be
adapted to his/her specific endophenotype.
[0016] Applicants demonstrate herein for the first time a
differential effect of osteopontin (OPN) on the response to Gi
stimulation among functional groups in IS. Subjects suffering from
IS can be classified into a particular functional group based on
their Gi mediated response to OPN. Furthermore, subjects at risk of
developing IS may also be identified and classified. Indeed, it was
found that in the presence of OPN, the response to Gi stimulation
increases in FG1, while it decreases in FG2 and FG3. Furthermore,
the response to Gi stimulation decreases to a higher extent in the
FG2 than in the FG3 functional group.
[0017] In a second aspect, Applicants show herein that IS subjects
or subjects at risk of developing IS can be classified according to
their impedance signature (i.e., the shape of their impedance
curve) in response to GiPCR stimulation with four Gi-coupled
receptor clusters (GiPCR clusters). By using cluster II GiPCR
agonists (e.g., agonists to the CB2R .alpha.2AR, .alpha.2BR and
.alpha.2C receptors), it is possible to rapidly distinguish
subjects belonging to the FG1 functional group over those belonging
to the FG2 and FG3 functional groups. Indeed, subjects belonging to
the FG1 group show a characteristic negative impedance phase in
response to Gi-PCR cluster II stimulation, which is not present for
subjects belonging to the FG2 and FG3 groups. Hence, this method
can also be useful to identify or classify subjects which are
difficult to identify or classify using other methods such as
subjects which have a Gi cellular response considered to be
borderline between two groups.
[0018] In a third aspect, the present inventors have found that IS
subjects or subjects at risk of developing IS can also be
classified according to their Gi-mediated response in the presence
of inhibitors/antagonists of Gi proteins such as Pertussis Toxin
(PTX) and GP Ant-2, which inactivates Gi/G.sub.o proteins. The IC50
value for GP Ant-2 is smaller for FG1 subjects than for FG3
subjects. FG2 subjects have an IC50 value for GP-Ant 2 which is
between that of FG1 and FG3 subjects. Similar results were obtained
with the G-protein antagonist PTX, although the differences were
less important. Unexpectedly, at high concentrations of PTX
subjects belonging to the FG1 functional group can also be rapidly
distinguished over FG2 and FG3 subjects based on their response to
cluster I and/or cluster II GiPCR agonists. Indeed, at high PTX
concentrations, stimulation by cluster I or cluster II agonists
results in a cellular response which is substantially higher than
the response observe in FG2 and FG3 subjects (as opposed to a
lower/decreased response normally observed at lower concentrations
of PTX). This difference/shift in the FG1 response was not observed
with the GP Ant-2 antagonist.
[0019] Accordingly, in an aspect there is provided a method of
classifying a subject having idiopathic scoliosis (IS) or at risk
of developing IS comprising: (a) determining the cellular response
to Gi stimulation in a cell sample from the subject in the presence
of OPN; (b) determining the cellular response to Gi stimulation in
a cell sample from the subject in the absence of OPN; and (c)
comparing the cellular response obtained in the presence of OPN
with the cellular response obtained in the absence of OPN, whereby
the comparing step enables the classification of the subject into
one IS functional group.
[0020] In a specific embodiment of the method, an increase in
cellular response following Gi-stimulation in the presence of OPN
in the sample is indicative that the subject belongs to functional
group FG1; and wherein a decrease in cellular response following
Gi-stimulation in the presence of OPN is indicative that the
subject belongs to functional group FG2 or FG3.
[0021] In another specific embodiment, step (c) comprises
determining the Fold effect (Fe) of OPN on the cellular response to
Gi stimulation. In another specific embodiment, a Fe value above
about 100% is indicative that the subject belongs to IS functional
group FG1; a Fe value below about 50% is indicative that the
subject belongs to IS functional group FG2 and a Fe value below
about 95% and above about 50% is indicative that the subject
belongs to IS functional group FG3. In another specific embodiment,
the method is used to classify subjects found to be borderline
according to a previously used alternative classification method,
into one IS functional group.
[0022] The present invention also concerns a method of classifying
a subject having idiopathic scoliosis (IS) or at risk of developing
IS comprising: (a) contacting a cell sample from the subject with
i) an agonist of cluster I or cluster II GiPCR; and ii) Pertussis
toxin (PXT); (b) Determining the cellular response to Gi
stimulation; and (c) Classifying the subject into a IS functional
group according to the subject's cellular response profile. In an
embodiment, step (c) comprises (i) classifying the subject into a
first functional group when the cellular response is above the
cellular response of a control sample; or (ii) classifying the
subject into a second functional group when the cellular response
is substantially comparable to the cellular response of a control
sample. In an embodiment, the cluster I GiPCR is lysophosphatidic
acid receptor (LPAR), melatonin receptor (MT1 or MT2), mu-opioid
receptor (OR) or adenosine receptor (AR). In an embodiment, the
agonist is melatonin, iodomelatonin, phenylmelatonin, LPA, DAMGO or
NECA. In a particular embodiment, the cluster II GiPCR is
cannabinoid receptor (CB2R) or a .alpha.2-Adrenoreceptor
(.alpha.2AR). In an embodiment, the agonist is CB65 or UK14304.
[0023] In another specific embodiment of the above methods, the
cellular response to Gi stimulation is determined by measuring
cellular impedance. In another specific embodiment, the cellular
impedance is measured by cellular dielectric spectroscopy.
[0024] The present invention further provides a method of
classifying a subject having idiopathic scoliosis IS or at risk of
developing IS comprising: (a) contacting a cell sample from the
subject with an agonist of a cluster II GiPCR; (b) determining the
cellular response to Gi protein stimulation by cellular impedance;
and (c) classifying the subject into a IS functional group
according to the subject's cellular impedance profile. In an
embodiment, step (c) comprises: (i) classifying the subject into a
first IS functional group when the cellular impedance profile
comprises a phase of negative impedance followed by a phase of
positive impedance; and (ii) classifying the subject into a second
IS functional group when the cellular impedance profile does not
comprise a phase of negative impedance. In an embodiment the
cluster II GiPCR is cannabinoid receptor (CB2R) or a
.alpha.2-Adrenoreceptor (.alpha.2AR). In an embodiment, the agonist
is CB65 or UK14304.
[0025] In another specific embodiment, the methods of the present
invention further comprise classifying borderline subjects into one
IS functional group by using an alternative classification method.
In another specific embodiment, the alternative classification
method is performed prior to step (a) of the method.
[0026] In another specific embodiment, the alternative
classification method comprises determining the difference between
cellular responses to Gi and Gs protein stimulations in the cell
sample from the subject. In another specific embodiment,
determining the difference between cellular responses to Gi and Gs
protein stimulations comprises measuring cAMP cellular
concentration produced by each of Gi and Gs protein
stimulations.
[0027] In another specific embodiment, determining the difference
between cellular responses to Gi and Gs protein stimulations
comprises measuring cellular impedance. In another specific
embodiment, the cellular impedance is measured by cellular
dielectric spectroscopy (CDS). In another specific embodiment,
determining the difference between cellular responses to Gi and Gs
protein stimulations is performed by determining the .DELTA.G in
the sample from the subject. In another specific embodiment, the
.DELTA.G is determined by measuring cellular impedance and the
cellular impedance is measured by CDS, and wherein i) a .DELTA.G
below -10 is indicative that the subject belongs to IS functional
group FG1; ii) a .DELTA.G above -10 and below +10 is indicative
that the subject belongs to IS functional group FG2; and iii) a
.DELTA.G above +10 is indicative that the subject belongs to IS
functional group FG3.
[0028] In another specific embodiment, determining the difference
between cellular responses to Gi and Gs protein stimulations is
performed by determining a ratio between cellular responses to Gi
and Gs protein stimulations (Gi/Gs). In another specific
embodiment, i) a ratio below about 0.5 is indicative that the
subject belongs to IS functional group FG1; ii) a ratio between
about 0.5 and 1.5, is indicative that the subject belongs to IS
functional group FG2; and iii) a ratio above 1.5 is indicative that
the subject belongs to IS functional group FG3
[0029] In another specific embodiment, the alternative method
comprises determining the magnitude of a cellular response to Gi
stimulation in the cell sample relative to a control sample. In
another specific embodiment, i) a reduction of between about 60-90%
of the cellular response to Gi stimulation relative to control is
indicative that the subject belongs to IS functional group FG1; ii)
a reduction of between about 40-60% of the cellular response to Gi
stimulation relative to control is indicative that the subject
belongs to IS functional group FG2; and iii) a reduction between
about 10-40% of the cellular response to Gi stimulation relative to
control is indicative that the subject belongs to IS functional
group FG3. In another specific embodiment, i) a reduction above 60%
of the cellular response to Gi stimulation relative to control is
indicative that the subject belongs to IS functional group FG1; ii)
a reduction of between about 40-60% of the cellular response to Gi
stimulation relative to control is indicative that the subject
belongs to IS functional group FG2; and iii) a reduction below
about 40% of the cellular response to Gi stimulation relative to
control is indicative that the subject belongs to IS functional
group FG3. In another specific embodiment, the magnitude of a
cellular response to Gi stimulation is determined by measuring
cellular impedance. In another specific embodiment, cellular
impedance is measured by cellular dielectric spectroscopy (CDS). In
another specific embodiment, the alternative method comprises
measuring cAMP concentration. In another specific embodiment, i) an
increase in cAMP concentration relative to baseline is indicative
that the subject belongs to IS functional group FG1, ii) no
significant or weak reduction in cAMP concentration relative to
baseline is indicative that the subject belongs to IS functional
group FG2; and iii) a reduction in cAMP concentration relative to
baseline is indicative that the subject belongs to IS functional
group FG3.
[0030] In another specific embodiment, the alternative method
comprises determining the phosphorylation state of Gi.alpha.
proteins in the cell sample. In another specific embodiment, i) the
presence of serine phosphorylated Gi.alpha.1, Gi.alpha.2 and
Gi.alpha.3 proteins (or of an increase in or substantially higher
level of serine phosphorylated Gi.alpha.1, Gi.alpha.2 and
Gi.alpha.3 proteins as compared to a control sample from a subject
not having IS or not at risk of developing IS) is indicative that
the subject belongs to IS functional group FG1; ii) an absence of
serine phosphorylation in Gi.alpha.3 proteins (or a level of serine
phosphorylation in Gi.alpha.3 proteins comparable or lower to the
level of serine phosphorylation in Gi.alpha.3 proteins in a control
sample from a subject not having IS or not at risk of developing
IS) is indicative that the subject belongs to IS functional group
FG2; and iii) an absence of serine phosphorylation in Gi.alpha.1
proteins (or a level of serine phosphorylation in Gi.alpha.1
protein comparable or lower to the level of serine phosphorylation
in Gi.alpha.1 proteins in a control sample from a subject not
having IS or not at risk of developing IS) is indicative that the
subject belongs to IS functional group FG3.
[0031] In another specific embodiment, the alternative method
comprises determining cellular proliferation of the cell sample
relative to a control sample. In another specific embodiment, i) a
reduction in cellular proliferation of about 50% or more relative
to control is indicative that the subject belongs to IS functional
group FG1; ii) a reduction in cellular proliferation between about
25% and 45% relative to control is indicative that the subject
belongs to IS functional group FG2; and iii) a reduction in
cellular proliferation of about 25% or less relative to control is
indicative that the subject belongs to IS functional group FG3.
[0032] In accordance with another aspect of the present invention,
there is provided a method of predicting the risk of developing IS
comprising: a) determining the cellular response to Gi stimulation
in a cell sample from the subject in the presence of OPN; b)
determining the cellular response to Gi stimulation in a cell
sample from the subject in the absence of OPN; and c) comparing the
cellular response obtained in the presence of OPN with the cellular
response obtained in the absence of OPN, whereby the comparing step
enables the prediction of the risk of developing IS. In a specific
embodiment, an increase in cellular response following
Gi-stimulation in the presence of OPN in the sample is indicative
that the subject belongs to functional group FG1; and wherein a
decrease in cellular response following Gi-stimulation in the
presence of OPN is indicative that the subject belongs to
functional group FG2 or FG3 and wherein identification of the
subject as belonging to the FG2 functional group indicates that the
subject is at risk of developing severe IS. In another specific
embodiment, step (c) comprises determining the Fold effect (Fe) of
OPN on the cellular response to Gi stimulation. In another specific
embodiment, a Fe value above about 100% is indicative that the
subject belongs to IS functional group FG1; a Fe value below about
50% is indicative that the subject belongs to IS functional group
FG2 and a Fe value below about 95% and above about 50% is
indicative that the subject belongs to IS functional group FG3.
[0033] The present invention further relates to method of
determining the risk of developing IS in a subject comprising: (a)
contacting a cell sample from the subject with i) an agonist of
cluster I or cluster II GiPCR; and ii) Pertussis toxin (PXT); (b)
determining the cellular response to Gi stimulation; and (c)
classifying the subject into a IS functional group according to the
subject's cellular response profile. In an embodiment, step (c)
comprises (i) classifying the subject into a first functional group
when the cellular response is above the cellular response of a
control sample; or (ii) classifying the subject into a second
functional group when the cellular response is substantially
comparable to the cellular response of a control sample. In a
particular embodiment, the cluster I GiPCR is lysophosphatidic acid
receptor (LPAR), melatonin receptor (MT1 or MT2), mu-opioid
receptor (OR) or adenosine receptor (AR). In an embodiment, the
agonist is melatonin, iodomelatonin, phenylmelatonin, LPA, DAMGO or
NECA. In an embodiment, the cluster II GiPCR is cannabinoid
receptor (CB2R) or a .alpha.2-Adrenoreceptor (.alpha.2AR). In a
particular embodiment, the agonist is CB65 or UK14304. In an
embodiment, the identification of the subject as belonging to the
FG2 functional group indicates that the subject is at risk of
developing severe IS.
[0034] In another specific embodiment, the cellular response to Gi
stimulation is determined by measuring cellular impedance. In
another specific embodiment, the cellular impedance is measured by
cellular dielectric spectroscopy.
[0035] The present invention also relates to a method of
determining the risk of developing IS in a subject comprising: (a)
contacting a cell sample from the subject with an agonist of a
cluster II GiPCR; (b) determining the cellular response to Gi
protein stimulation by cellular impedance; and (c) classifying the
subject into a IS functional group according to the subject's
cellular impedance profile. In an embodiment, step (c) comprises:
(i) classifying the subject into a first IS functional group when
the cellular impedance profile comprises a phase of negative
impedance followed by a phase of positive impedance; and (ii)
classifying the subject into a second IS functional group when the
cellular impedance profile does not comprise a phase of negative
impedance. In an embodiment the cluster II GiPCR is cannabinoid
receptor (CB2R) or a .alpha.2-Adrenoreceptor (.alpha.2AR). In an
embodiment, the identification of the subject as belonging to the
FG2 functional group indicates that the subject is at risk of
developing severe IS.
[0036] In another specific embodiment, the method further comprises
classifying borderline subjects into one IS functional group by
using an alternative classification method. In another specific
embodiment, the alternative classification method is performed
prior to step (a) of the method of the present invention. In
another specific embodiment, the alternative classification method
comprises determining the difference between cellular responses to
Gi and Gs protein stimulations in the cell sample from the subject.
In another specific embodiment, determining the difference between
cellular responses to Gi and Gs protein stimulations comprises
measuring cAMP cellular concentration produced by each of Gi and Gs
protein stimulations. In another specific embodiment, determining
the difference between cellular responses to Gi and Gs protein
stimulations comprises measuring cellular impedance. In another
specific embodiment, the cellular impedance is measured by cellular
dielectric spectroscopy (CDS). In another specific embodiment,
determining the difference between cellular responses to Gi and Gs
protein stimulations is performed by determining the .DELTA.G in
the sample from the subject. In another specific embodiment, the
.DELTA.G is determined by measuring cellular impedance and the
cellular impedance is measured by CDS, and wherein i) a .DELTA.G
below -10 is indicative that the subject belongs to IS functional
group FG1; ii) a .DELTA.G above -10 and below +10 is indicative
that the subject belongs to IS functional group FG2; and iii) a
.DELTA.G above +10 is indicative that the subject belongs to IS
functional group FG3.
[0037] In another specific embodiment, determining the difference
between cellular responses to Gi and Gs protein stimulations is
performed by determining a ratio between cellular responses to Gi
and Gs protein stimulations (Gi/Gs). In another specific
embodiment, i) a ratio below about 0.5 is indicative that the
subject belongs to IS functional group FG1; ii) a ratio between
about 0.5 and 1.5, is indicative that the subject belongs to IS
functional group FG2; and iii) a ratio above 1.5 is indicative that
the subject belongs to IS functional group FG3.
[0038] In another specific embodiment, the alternative method
comprises determining the magnitude of a cellular response to Gi
stimulation in the cell sample relative to a control sample. In
another specific embodiment, i) a reduction of between about 60-90%
of the cellular response to Gi stimulation relative to control is
indicative that the subject belongs to IS functional group FG1; ii)
a reduction of between about 40-60% of the cellular response to Gi
stimulation relative to control is indicative that the subject
belongs to IS functional group FG2; and iii) a reduction between
about 10-40% of the cellular response to Gi stimulation relative to
control is indicative that the subject belongs to IS functional
group FG3. In another specific embodiment, i) a reduction above 60%
of the cellular response to Gi stimulation relative to control is
indicative that the subject belongs to IS functional group FG1; ii)
a reduction of between about 40-60% of the cellular response to Gi
stimulation relative to control is indicative that the subject
belongs to IS functional group FG2; and iii) a reduction below
about 40% of the cellular response to Gi stimulation relative to
control is indicative that the subject belongs to IS functional
group FG3.
[0039] In another specific embodiment, the magnitude of a cellular
response to Gi stimulation is determined by measuring cellular
impedance. In another specific embodiment, cellular impedance is
measured by cellular dielectric spectroscopy (CDS). In another
specific embodiment, the alternative method comprises measuring
cAMP concentration. In another specific embodiment, i) an increase
in cAMP concentration relative to baseline is indicative that the
subject belongs to IS functional group FG1, ii) no significant or
weak reduction in cAMP concentration relative to baseline is
indicative that the subject belongs to IS functional group FG2; and
iii) a reduction in cAMP concentration relative to baseline is
indicative that the subject belongs to IS functional group FG3.
[0040] In another specific embodiment, the alternative method
comprises determining the phosphorylation state of Gi.alpha.
proteins in the cell sample. In another specific embodiment, i) the
presence of serine phosphorylated Gi.alpha.1, Gi.alpha.2 and
Gi.alpha.3 proteins (or of a substantially higher level of serine
phosphorylated Gi.alpha.1, Gi.alpha.2 and Gi.alpha.3 proteins as
compared to a control sample not having IS or not at risk of
developing IS) is indicative that the subject belongs to IS
functional group FG1; ii) an absence of serine phosphorylation in
Gi.alpha.3 proteins (or a level of serine phosphorylation in
Gi.alpha.3 protein comparable or lower to the level of serine
phosphorylation in Gi.alpha.3 proteins in a control sample from a
subject not having IS or not at risk of developing IS) is
indicative that the subject belongs to IS functional group FG2; and
iii) an absence of serine phosphorylation in Gi.alpha.1 proteins
(or a level of serine phosphorylation in Gi.alpha.1 proteins
comparable or lower to the level of serine phosphorylation in
Gi.alpha.1 proteins in a control sample from a subject not having
IS or not at risk of developing IS) is indicative that the subject
belongs to IS functional group FG3. In another specific embodiment,
the alternative method comprises determining cellular proliferation
of the cell sample relative to a control sample. In another
specific embodiment, i) a reduction in cellular proliferation of
about 50% or more relative to control is indicative that the
subject belongs to IS functional group FG1; ii) a reduction in
cellular proliferation between about 25% and 45% relative to
control is indicative that the subject belongs to IS functional
group FG2; and iii) a reduction in cellular proliferation of about
25% or less relative to control is indicative that the subject
belongs to IS functional group FG3.
[0041] In accordance with another aspect of the present invention,
there is provided a method of selecting a preventive measure,
treatment or follow-up schedule for a subject suffering from IS or
at risk of developing IS comprising classifying the subject using
one or more of the above-noted methods: a) When the subject is
classified as belonging to the FG1 functional group: i) the level
of OPN in said subject is increased; ii) the level of HA in said
subject is increased; iii) the level of CD44 in said subject is
decreased; and/or iv) the frequency of assessment of scoliosis
progression is decreased; b) when the subject is classified as
belonging to the FG2 functional group: i) the level of OPN is said
subject is decreased; ii) the level of HA in said subject is
decreased; and/or iii) the level of CD44 in said subject is
increased; iv) the frequency of assessment of scoliosis progression
is increased; and/or v) the subject is prescribed corrective
surgery prior to reaching a scoliosis with a cobb angle of 45
degree; or c) when the subject is classified as belonging to the
FG3 functional group: i) the level of OPN is said subject is
decreased; ii) the level of HA in said subject is decreased; iii)
the level of CD44 in said subject is increased; and/or iv) the
frequency of assessment of scoliosis progression is decreased.
[0042] In an embodiment, increasing the level of OPN comprises
performing massages such as by applying pulsative compressive
pressure to a body part of the subject. In an embodiment,
increasing the level of OPN comprises administering OPN or a
fragment thereof or derivative thereof to the subject. In an
embodiment, increasing the level of OPN comprises applying low
intensity pulsed ultrasound (LIPUS) to the subject. In a specific
embodiment, increasing the level of HA comprises administering to
the subject HA supplements or complying to a HA-rich diet. In a
specific embodiment, decreasing the level of HA comprises complying
to a HA-poor diet. In an embodiment decreasing OPN level comprises
brace treatment. In an embodiment decreasing OPN level comprises
administering to the subject selenium supplements, melatonin or
PROTANDIM.TM.. In an embodiment decreasing OPN level comprises
accupoint heat sensitive moxibustion, heat therapy with pad,
thermal bath and electroacupuncture. In a specific embodiment
decreasing the frequency of assessment of scoliosis progression
comprises performing less than 22 radiological examinations within
the 3 years following classification.
[0043] In a specific embodiment of the methods, the subject is a
subject pre-diagnosed with IS. In another specific embodiment, the
subject is asymptomatic. In another specific embodiment, the
subject is a subject having at least one family member suffering
from IS. In another specific embodiment, the cell sample comprises
osteoblasts, chondrocytes, myoblasts and/or peripheral blood
mononuclear cells (PBMCs). In another specific embodiment, the cell
sample comprises PBMCs. In another specific embodiment, the PBMCs
comprise lymphocytes. In another specific embodiment, the PBMCs are
frozen PBMCs. In another specific embodiment, the PBMCs are fresh
PBMCs. In another specific embodiment, the Gi stimulation comprises
contacting the cells with somatostatin. In another specific
embodiment, the Gi-stimulation comprises contacting the cells with
a cluster I, cluster II, cluster III and/or cluster IV receptor
agonist. In an embodiment the cluster I receptor is the melatonin
receptor (MT1 or MT2), lysophosphatidic acid receptor (LPAR),
mu-opioid receptor (OR) or the adenosine receptor (AR). In an
embodiment, the cluster I agonist is LPA, DAMGO or NECA. In an
embodiment, the cluster II receptor is .alpha.2 adrenergic receptor
or the type 2 cannabinoid receptor (CB2R). In an embodiment, the
cluster II agonist is CB65 or UK14304. In an embodiment, the
cluster III receptor is the somatostatin receptor (SSTR) or the
formyl peptide receptor 2 (FPR2). In an embodiment, the cluster III
agonist is somatostatin or MMK1. In an embodiment, the cluster iv
receptor is the Apelin receptor (APJR), the 5-HT1 receptor (5-HT1R)
or the domapine D2 receptor (D2R). In an embodiment, the cluster iv
agonist is Apelin-17, BP554 or quinpirole. In another specific
embodiment, the Gi stimulation comprises contacting the cells with
a cluster I or cluster II agonist. In another specific embodiment,
the Gs stimulation comprises contacting the cells with
isoproterenol. In another specific embodiment, the IS is Adolescent
idiopathic scoliosis (AIS).
[0044] In accordance with another aspect of the present invention,
there is provided a kit for classifying a subject having idiopathic
scoliosis (IS) or at risk of developing IS or for predicting the
risk of developing IS comprising: OPN; and one or more ligands for
Gi stimulation.
[0045] In another aspect of the present invention, there is
provided a kit for classifying a subject having idiopathic
scoliosis (IS) or at risk of developing IS or for predicting the
risk of developing IS comprising: a cluster II GiPCR (e.g., CB2R or
.alpha.2AR) agonist (e.g., CB65 or UK14304). In an embodiment, the
kit further comprises at least one of i) a cluster iv GiPCR (e.g.,
APJR, 5-HT1R or D2R) agonist (e.g., Apelin-17 BP554 or quinpirole);
ii) OPN; iii) PTX and iv) instructions for classifying the subject
or for predicting the risk of developing IS.
[0046] In another aspect of the present invention, there is
provided a kit for classifying a subject having idiopathic
scoliosis (IS) or at risk of developing IS or for predicting the
risk of developing IS comprising: i) a cluster I GiPCR (e.g., LPAR,
OR, A1R or melatonin receptor) and/or cluster II GiPCR (e.g., CB2R
or .alpha.2AR) agonist (e.g., melatonin, LPA, DAMGO, NECA, CB65 or
UK14304); and ii) PTX. In an embodiment, the kit further comprises
at least one of i) a cluster III GiPCR agonist (e.g., somatostatin,
MMK1); ii) a cluster IV GiPCR agonist (Apelin-17, BP554 or
quinpirole); iii) OPN; and/or v) instructions for classifying the
subject or for predicting the risk of developing IS.
[0047] In an embodiment, the above-noted kits are for predicting
the risk of developing IS.
[0048] In another specific embodiment, the above-noted kits further
comprise: a ligand for Gs stimulation; one or more antibodies for
detecting Gi.alpha. phosphorylation (e.g., an antibody specific for
Gi.alpha.1, Gi.alpha.2 and/or Gi.alpha.3); hyaluronic acid (HA)
and/or instructions for classifying the subject or for predicting
the risk of developing IS.
[0049] In an embodiment, the above-mentioned method is performed on
more than one receptor coupled to a G, protein. In another
embodiment, the above-mentioned method is performed using more than
one ligand specific for a receptor coupled to a G.sub.i protein. In
another specific embodiment, each ligand is specific to a different
receptor coupled to a G.sub.i protein (e.g., 2, 3, 4, 5 or 6
ligands).
[0050] Other objects, advantages and features of the present
invention 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
[0051] In the appended drawings:
[0052] FIG. 1 shows an embodiment of a design for cell seeding in
accordance with the present invention;
[0053] FIG. 2 shows an embodiment for a design for dispensing
compounds in accordance with the present invention;
[0054] FIG. 3 shows the percent (%) viability and cell
concentration obtained in an illustrative experiment described in
Examples 1 and 2 as determined using an automated cell counter and
viability analyzer;
[0055] FIG. 4 illustrates the .DELTA.G functional classification.
The graph illustrates the difference between responses to Gi and Gs
(i.e. .DELTA.G) stimulation obtained in PBMCs from patients with
IS. Values were measured by the CDS-based system in response to 10
.mu.M of Somatostatin and isoproterenol. Each point represents the
.DELTA.G of both responses in duplicate;
[0056] FIG. 5 shows the effect of rOPN on the cellular response to
Gi stimulation in PBMCs. Cells were serum-starved for 18 h in the
presence or absence of 0.5 .mu.g/mL rOPN and then stimulated with
10 .mu.M of somatostatin to initiate Gi-mediated cellular response.
Data in the graph were generated from maximum-minimum impedance and
correspond to the average of response in duplicate;
[0057] FIGS. 6A-6D show the functional status (i.e., the magnitude
of the cellular response to GiPCR-stimulation) of Gi proteins in
PBMCs from control and scoliotic subjects. PBMCs from control and
scoliotic subjects were exposed to increasing concentrations of
somatostatin to stimulate Gi proteins via endogenous somatostatin
receptor. The cellular response was measured by CDS-based system as
described in Example 1. Curves were generated from maximum-minimum
impedance. Each curve represents the non-linear regression
performed with GraphPad.TM. Prism 5.0 software. Data were
normalized to maximal response in cells from control subjects and
each point corresponds to the average of response in duplicate. As
shown, the percentage of maximal response relative to the control
following Gi stimulation are below 40% (e.g., between about 10-40%
for FG1), about 40 and 60% for FG2 and above 60% (e.g., about
60-90% for FG3).
[0058] FIG. 7 shows the distribution of functional groups among
different phases of scoliosis. A large cohort of scoliotic patients
comprising 794 moderate cases (curvatures between 10 and 44
degrees) and 162 severe cases (curvature greater than 45 degrees)
regularly followed at Sainte-Justine Hospital, were classified
according to their degree of imbalance between response to Gi and
Gs stimulation (.DELTA.G). Responses were measured by the CDS-based
system in response to 10 .mu.M of somatostatin and
isoproterenol;
[0059] FIGS. 8A-8G show a list of known Gi-protein coupled
receptors;
[0060] FIGS. 9A-9DDD show a list of known ligands to Gi-protein
coupled receptors;
[0061] FIGS. 10A-10J show that IS subjects or subjects at risk of
developing IS can be classified according to their impedance
signature in response to GiPCR stimulation in four receptor
clusters. Impedance signatures of various GiPCR agonists in AIS
osteoblasts reveal 4 distinct clusters. Cells were stimulated with
10 .mu.M of (FIG. 10A) LPA, (FIG. 10B) DAMGO, (FIG. 10C) NECA,
(FIG. 10D) CB65, (FIG. 10E) UK14304, (FIG. 10F) Somatostatin, (FIG.
10G) MMK1, (FIG. 10H) Apelin-17, (FIG. 10I) BP554 or (FIG. 10J)
Quinpirole. The targeted endogenous receptors are shown in
parentheses. The impedance represented in y-axis as dziec was
measured by the Cellkey.TM. system every 2 sec. Data are
representative of impedance signature in osteoblasts from 12
individuals tested three times in duplicate for each group;
[0062] FIGS. 11A-11B show dual melatonin signaling in AIS. (FIG.
11A) Effect of 16 h pre-treatment with 1 .mu.g/mL PTX on kinetic
signature of melatonin. (FIG. 11B) Effect of 16 h pre-treatment
with varying concentration of PTX on impedance response to 10 .mu.M
melatonin normalized to the response in the presence of vehicle.
Data are expressed as mean .+-.SEM of experiments performed three
times in duplicate for n=12 patients per group. *P<0.05,
**P<0.01, ***P<0.001, versus control group based on one-way
ANOVA followed by in post-doc test of Dunnett;
[0063] FIGS. 12A-12J show the variations in the effect of PTX on
response to various selective agonists of Gi-coupled receptors in
FG1 subjects, depending on the receptor cluster. Osteoblasts from
control subjects or AIS patients of different groups were
pre-incubated with varying concentrations of PTX for 16 h prior
stimulation with 10 .mu.M of specific synthetic agonist. The tested
agonists and targeted receptors are indicated in each panel. Data
were normalized to response in the presence of vehicle and are
expressed as mean.+-.SEM of experiments performed three times in
duplicate for n=12 patients per group. *P<0.05, **P<0.01,
***P<0.001, versus control group based on one-way ANOVA followed
by in post-doc test of Dunnett;
[0064] FIGS. 13A-13B show the validation of the Gs and Gq siRNA
effectiveness in AIS osteoblasts. (FIG. 13A) Total RNA extracted
from control and AIS osteoblasts transfected with scramble, Gs or
Gq siRNA and nontranfected (NT) cells, were subjected to qPCR
analysis, using .beta.-actin as internal control. Data are
expressed as mean.+-.SEM of n=12 patients for each group. *
P<0.05, **P<0.01, ***P<0.001, versus NT cells based on
one-way ANOVA followed by in post-doc test of Dunnett. (FIG. 13B)
Total cell lysates from cells transfected with scramble, Gs or Gq
siRNA, were subjected to western blot analysis, using antibody to
.alpha.-tubulin as control. Bands shown are representative of
results obtained with osteoblasts from 12 different patients for
each AIS group.
[0065] FIGS. 14A-14P show the differential effects of Gs and Gq
knockdown by siRNA method on the biphasic impedance signature of
GiPCR agonists among AIS groups. Osteoblasts from control subjects
and AIS patients of each functional group were transfected with
scramble siRNA, Gs siRNA, or Gq siRNA. Efficiency of siRNA in
control and AIS groups was verified with qPCR and Western blot
analyses 48 hours after transfection, and response to stimulation
with GiPCR agonist of cluster I, cluster II, cluster III and
cluster IV was evaluated by challenging cells with 10 .mu.M of
(FIG. 14A-14D) LPA, (FIG. 14EA-14H) CB65, (FIG. 14I-14L)
Somatostatin and (FIG. 14M-14P) Apelin-17, respectively. Results
from control subjects are shown in FIGS. 14A, 14E, 14I, 14M; in FG1
subjects are shown in FIGS. 14B, 14F, 14J, 14N; in FG2 subjects are
shown in FIGS. 14C, 14G, 14K, 14O; and in FG3 subjects are shown in
FIGS. 14D, 14H, 14L, 14P. The impedance represented in y-axis as
dziec was measured every 2 sec. Data are representative of
impedance signatures generated by CellKey.TM. system in osteoblasts
from 12 individuals tested three times in duplicate for each
group.;
[0066] FIGS. 15A-15B summarize alternative methods to classify IS
subjects or subjects at risk of developing IS; and
[0067] FIGS. 16A-16H provide examples of Examples of clusters I to
IV GiPCR ligands (e.g., full or partial agonists).
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0068] The Applicants have tested on a large number of
pre-classified subjects (by one or more of previous stratification
methods (e.g., response to Gi-agonist stimulation (cAMP production
inhibition, impedance modifications (by cellular dielectric
spectroscopy), etc.)) the effect of OPN on Gi response.
[0069] They have found that depending on the functional group
(e.g., FG1, FG2 or FG3), subjects at risk of suffering from
scoliosis (e.g., already diagnosed IS subjects or suspected IS
subjects) have a different Gi-dependent cellular response in the
presence of OPN. In the FG1 group the presence of OPN increases the
cellular response following Gi stimulation, while in groups FG2 and
FG3, the Gi-dependent cellular response is decreased in the
presence of OPN. Groups FG2 and FG3 can be further distinguished
based on the extent of the OPN effect.
[0070] Applicants have also found that subjects can further be
distinguished based on their impedance profiles following Gi
protein stimulation in four GiPCR clusters (I, II, III and IV). The
impedance profile for each cluster has a different shape. In
addition, in the case of GiPCR cluster II, only FG1 subject show an
impedance profile comprising a characteristic negative impedance
phase followed by a positive phase, thereby enabling to easily
distinguish FG1 subjects from FG2 and FG3 subjects.
[0071] Furthermore, results presented herein show that FG1 subjects
can further be distinguished over FG2 and FG3 subjects based on
their cellular response to GiPCR cluster I and/or II stimulation in
the presence of high concentration of PTX.
[0072] The methods of the present invention can be used to classify
subjects already diagnosed with IS or to identify subject at risk
of developing IS. For example asymptomatic subjects predisposed to
IS (e.g., a subject having at least one family member suffering
from IS) can be tested and their risk of developing scoliosis
determined based e.g., on their impedance profile following Gi
protein stimulation with Gi PCR cluster I, II, III and/or IV
agonist (or ligand) or based on their Gi response in the presence
of i) OPN; or ii) PTX;. Their risk of developing severe scoliosis
and of requiring surgery can also be determined based on their
classification into a specific functional group. The present
methods can be used alone or preferably in combination with one or
more alternative methods of identifying the risk of developing IS
and/or classifying subjects into one particular functional group
(endophenotype). Combining the assessment of OPN effect, PTX effect
(e.g., for cluster I and II GiPCR cellular response) or cluster II
GiPCR stimulation on Gi cellular response with another method of
identifying the risk of developing IS and/or classifying IS
subjects allows to greatly improve the specificity of the method by
allowing the classification of otherwise borderline subjects into
one specific functional group (i.e., when one classification method
alone did not enable to distinguish between two groups (e.g.,
between FG1 and FG2 or FG2 and FG3)). Thus, the precision of prior
classification tests based on the response to Gi stimulation (or
Gi/Gs imbalance) can thus further be improved by demonstrating a
differential effect of osteopontin (OPN); or PTX on the response to
Gi stimulation among functional groups or by looking at the
impedance profiles in response to Gi protein stimulation in the
presence of cluster I, II, III or iv GiPCR agonists.
[0073] The following predictive algorithm Table 6 is used in
accordance with the present invention for the selection of agonists
and GPCRs (Clusters I, II, III, IV) corresponding to functional
classification of idiopathic scoliosis patients.
TABLE-US-00001 TABLE 6 Cluster I Cluster II Cluster III Cluster IV
FG1 LPA/Gs+ CB65/Gs+ Somatostatin/Gs+ Apelin-17/Gs- and Gq+ and Gq-
FG2 LPA/Gq+ CB65/Gs- Somatostatin/Gq+ Apelin-17/Gs- and Gq- and Gq-
FG3 LPA/Gq+ CB65/Gs- Somatostatin/Gs+ Apelin-17/Gs- and Gq- and Gq+
and Gq- Cluster Gs permissive Gs permissive Mainly Gq permissive
Not permissive characteristics (coupled) only (coupled) only
(coupled) for all groups (not coupled) for FG1 for FG1 with weak Gs
interaction for Gq and Gs Gq permissive (coupling) for FG1 and
(coupled) only FG3 groups for FG2 and FG3
[0074] Cluster I GiPCR (and their ligands (e.g., agonists)) are
coupled to Gs only for FG1, while being coupled to Gq for FG2 and
FG3. Cluster II GiPCR (and their ligands (e.g., agonists)) are
coupled to Gs, for FG1 only. Cluster III GiPCR (and their ligands
(e.g., agonists)) are mainly coupled to Gq but also show a weak
interaction with Gs for FG1 and FG3 groups. Cluster IV GiPCR (and
ligands (e.g., agonists)) are not coupled to Gq and Gs.
[0075] Non-limiting examples of GICPR and their agonist for cluster
I, II, III and IV are given in Table 7 below.
[0076] Accordingly, the present invention provides a method of
classifying a subject having IS or at risk of developing IS (and/or
of predicting the risk of developing IS) comprising: (a)
determining the cellular response to Gi stimulation in a cell
sample from the subject in the presence of OPN; (b) determining the
cellular response to Gi stimulation in a cell sample from the
subject in the absence of OPN; (c) comparing the cellular response
obtained in the presence of OPN with the cellular response obtained
in the absence of OPN; whereby the comparing step enables the
classification of the subject into one IS functional group (and/or
enables predicting the risk of developing IS).
[0077] In the above method based on the effect of OPN,
classification into a specific functional group can be performed as
follows. For example, when detecting an increase in cellular
response following Gi-stimulation in the presence of OPN (when
compared to in the absence of OPN), the subject is classified into
the FG1 functional group; whereas when detecting a decrease in
cellular response following Gi-stimulation in the presence of OPN
(when compared to in the absence of OPN), the subject is classified
into the FG2 or FG3 functional group depending on the extent of the
OPN effect (the FG2 functional group being more sensitive to
OPN).
[0078] Optionally and advantageously, the above-described methods
may further comprise determining the fold effect (Fe) of OPN on
Gi-mediated response which allows to more effectively distinguish
between groups. The fold effect of OPN is determined by dividing
the average of response magnitude to Gi stimulation in presence of
OPN (RmGiOPN) with the average of response magnitude to Gi
stimulation in the absence of OPN (RmGi) using the following
formula:
Fe=100.times.(RmGiOPN/RmGi)
[0079] According to the above formulae, a Fe value above about 100%
is indicative that the subject belongs to IS functional group FG1;
a Fe value below about 50% is indicative that the subject belongs
to IS functional group FG2 and a Fe value below about 95% and above
about 50% is indicative that the subject belongs to IS functional
group FG3.
[0080] The present invention also provides a method of classifying
a subject having idiopathic scoliosis (IS) or at risk of developing
IS (and/or of predicting the risk of developing IS) comprising: a)
Contacting a cell sample from the subject with i) an agonist of
cluster I or cluster II GiPCR; and ii) Pertussis toxin (PXT); b)
Determining the cellular response to Gi protein stimulation; and c)
Classifying the subject into a IS functional group according to the
subject's cellular response profile.
[0081] Subjects can be classified into a specific functional group
based on their response curve in the presence of increasing amounts
of PTX. For all functional groups (i.e., FG1, FG2 and FG3), the
response curve showed a left shift with respect to the control
sample (i.e., the IC50 for PTX was smaller than in control
samples). This characteristic can be used to identify subjects at
risk of developing scoliosis.
[0082] Following GiPCR cluster I or cluster II agonist stimulation,
the response curve in the presence of PTX is characterized by a
first phase in which the response decreases with increasing amounts
of PTX followed by a second phase where the response increases with
increasing amounts of PTX (V shape curve). In the second phase of
the response, the % of response relative to administration of a
control vehicle is above that of the corresponding % of response
for control (subjects not having IS or not at risk of developing
IS), FG2 and FG3 subjects. Hence, a magnitude of cellular response
in the presence of high concentration of PTX that is above that of
a control (subjects not having IS or not at risk of developing IS
as well as FG2 and FG3 subjects) indicates that the subjects
belongs to the FG1 functional group.
[0083] By "high PTX concentration" is meant a concentration of PXT
at which the GiPCR-mediated cellular response is almost completely
inhibited by PTX in cells from subjects not having IS or not at
risk of developing IS. For example, a high PTX concentration is a
concentration at which the % of the GiPCR-mediated response is from
about 0% (i.e., background noise) to 20%, from about 0% to 15% or
from about 0% to 10%, preferably below 5% of the corresponding
cellular response in the presence of a vehicle (instead of PTX).
The high PTX concentration may also be expressed relative to the
IC50 value for PTX in control samples. For example, a high PTX
concentration in accordance with the present invention is a
concentration that is at least 3.times., 5.times., 6.times.,
8.times., 9.times., 10.times., 75.times., 100.times., 150.times.,
200.times., 250.times., 500.times., 750.times. or 1000.times. the
concentration of PTX at which 50% of the cellular response is
normally inhibited (IC50) in control cells (e.g., cells from
subjects not having IS or not at risk of developing IS). In an
embodiment, the high PTX concentration is 6.times. the IC50
concentration. In a particular embodiment, the high PTX
concentration is 0.5 ug/ml). As one skilled in the art can
appreciate from the results presented herein, the higher the
concentration of PTX, the greater the difference in the cellular
response between FG1 subjects and control/FG2/FG3 subjects.
[0084] In another aspect, the present invention concerns a method
of classifying a subject having idiopathic scoliosis IS or at risk
of developing IS comprising: a) Contacting a cell sample from the
subject with an agonist of a cluster II GiPCR; b) Determining the
cellular response to Gi protein stimulation by cellular impedance;
and c) Classifying the subject into a IS functional group according
to the subject's cellular impedance profile.
[0085] Classification into a specific functional group can also be
made by looking at the shape of the impedance profile following
stimulation with one or more of clusters I, II, III and IV GiPCR
agonists relative to control samples. Each GiPCR cluster gives a
shape of impedance curve (i.e, an impedance signature) which is
different from the other clusters. In addition, among a given GiPCR
cluster, controls, FG1, FG2 and FG3 subjects have an impedance
profile which is different from the other functional groups and
from control, healthy subjects. Unexpectedly and advantageously,
following stimulation with cluster II GiPCR agonists, subjects
belonging to the FG1 or FG2/FG3 functional groups can be easily
classified/identified by the sole shape of their impedance curve in
the absence of any control sample. Indeed, GiPCR cluster II agonist
stimulation of samples from subjects belonging to the FG1
functional group results in an impedance curve which begins with a
negative impedance phase followed by a phase of positive impedance.
This negative impedance phase is absent in FG2 and FG3 subjects,
thereby allowing to unambiguously identify FG1 subjects.
[0086] All classification methods described herein can be used for
i) predicting the risk of developing a scoliosis in a subject, ii)
for selecting an appropriate preventive measure or treatment iii)
for identifying compounds useful in the prevention or treatment of
scoliosis or for simply classifying a subject into a specific
functional group for clinical trials or other studies in which the
effect of a given molecule or treatment may differ between
functional groups.
[0087] The above described methods may be used alone or in
combination and may also be used in addition to at least one
other/alternative method of classification and/or method of
predicting the risk of developing IS (e.g., method using the
.DELTA.G to classify the subject and/or predict the risk of
developing IS). The at least one other method may be used prior to
or after using one or more of the above-described methods. In a
specific embodiment, the at least one other/alternative method is
used prior to using the one or more above-described methods. When
the at least one other/alternative method is used prior to using
the one or more above-described methods, the above-described
methods may advantageously be used (alone or in combination) to
classify subjects that had borderline values (i.e. could not be
clearly classified into one functional group) by using the
other/alternative method (e.g., subjects having a .DELTA.G of about
10 or about -10 using CDS) into a IS functional group. When the
other/alternative method is used after the above-cited one or more
methods, it may advantageously be used to classify subjects that
had borderline values (i.e. could not be clearly classified into
one functional group) by using the above-described method (e.g.,
subjects having Fe value of about 50% or between about 95 and about
100%) into a IS functional group.
[0088] As used herein the terms "borderline subjects" refer to
subjects that could not be classified with sufficient confidence
(i.e., specificity) into one specific IS functional group using a
specific method of classifying IS subjects or where there is a need
to simply confirm to which specific functional group a subject
belongs.
[0089] OPN also has an effect on the cellular response to Gi
stimulation in normal cells. In normal cells, OPN will decrease the
cellular response to Gi-stimulation through its interaction with
integrins (e.g., .alpha..sub.5.beta..sub.1). The use of a control
sample (e.g., sample from a normal healthy subject) is therefore
preferred for identification of at least FG3 subjects based on the
above method since these subjects have a Gi cellular response that
is closest to normal subjects and a response to Gi-stimulation that
is least modified in the presence of OPN. The same observation is
made when classifying subjects in accordance with their impedance
profile or in the presence of a GiPCR antagonists. When used for
predicting the risk of developing a scoliosis in asymptomatic
patients, FG3 subjects may thus be difficult to unambiguously
identify if no comparison is made with normal subjects. Preferably,
a control a sample is used for identification of FG2 and FG3
subjects when predicting the risk of developing a scoliosis.
[0090] Hence, in certain embodiments of the above-mentioned
methods, an additional step of further comparing the results with a
control sample (e.g., one or more samples from healthy subject(s)
or an equivalent "control value" derived from samples from control
subjects) may be useful where, for example, asymptomatic subjects
are tested for classifying subjects or predicting the risk of
developing scoliosis. This step may however be omitted if an
optimal concentration of OPN (i.e., concentration showing no or
weak effect on normal cells) has been selected or if only certain
functional groups need to be identified (e.g., FG1 and FG2). A
control sample (e.g., sample from normal subject) may also not be
necessary when the above methods are used as a classification
method for borderline subjects in conjunction with (e.g., prior to)
an alternative classification method as explained below. In those
cases, the alternative method has already determined with a control
that the subject has an IS.
[0091] As indicated above, Applicants have discovered that
combining one or more methods of the present invention (e.g., the
detection of the OPN effect on Gi cellular response) with another
classification method allows to more precisely classify subjects
having values that are borderline between two functional groups or
fall outside the above OPN Fe ranges (e.g., a Fe value of about 50%
or between about 95 and about 100%). Any alternative method of
classifying a subject into a functional subgroup may be used in
accordance with the present invention. For example, confirmation
can be performed by classifying the subject into one IS functional
group by i) determining the difference (.DELTA.G) or ratio (e.g.,
Gi/Gs) between cellular responses to Gi and Gs protein stimulations
in the cell sample from the subject; ii) determining the magnitude
of a cellular response to GiPCR-stimulation in the cell sample
relative to a control sample (using GiPCR agonist stimulation and
optionally antagonists of GiPCR cellular response) ; iii)
determining the phosphorylation states of Gi proteins; or iv)
determining the cell (e.g., osteoblasts, chondrocytes, myoblasts)
proliferation (described in Moreau et al., 2004; Akoume et al.,
2010; Azeddine et al., 2007; Letellier et al., 2008; and
WO2003/073102; WO2010/040234 and PCT/CA2014/050562 and U.S.
61/879,314 to Moreau, all incorporated herein by reference).
[0092] Preferably, the confirmation is performed by determining the
difference between cellular responses to Gi and Gs protein
stimulations (.DELTA.G) in the cell sample from the subject. The
.DELTA.G is determined by subtracting the average of response
magnitude to Gi stimulation (RmGi) from the average of response
magnitude to Gs stimulation (RmGs) using the following formula:
.DELTA.G=RmGi-RmGs.
[0093] According to the .DELTA.G classification, response to Gi
stimulation predominates in FG3, while no apparent (i.e., no
substantial) imbalance is observed in FG2 subjects. In contrast,
FG1 subjects exhibit predominance for response to Gs stimulation. A
value of .DELTA.G below -10 is indicative that the subject belongs
to IS subgroup FG1; a value of .DELTA.G above -10 and below +10 is
indicative that the subject belongs to IS subgroup FG2; and a value
of .DELTA.G above +10 is indicative that the subject belongs to IS
subgroup FG3.
[0094] Alternatively, the difference between Gi and Gs responses
may be expressed as a ratio of response to Gi vs. Gs stimulation
(Gi/Gs). The FG3 group shows a predominance of response to Gi
stimulation (i.e. a Gi/Gs ratio of more than about 1.5), there is
no substantial imbalance observed in the FG2 group (i.e. a Gi/Gs
ratio of between about 0.5 and 1.5) and the FG1 group exhibits a
predominance of response to Gs stimulation (i.e. a Gi/Gs ratio of
less than about 0.5).
[0095] In the case where classification is resolved by determining
the magnitude of a cellular response to Gi stimulation in the cell
sample relative to a control sample: i) a cellular response to Gi
stimulation lower than the control sample by about 60% (e.g., 60%
to 90%) is indicative that the subject belongs to IS subgroup FG1;
ii) a cellular response to Gi stimulation lower than the control
sample by about 40 to 60% is indicative that the subject belongs to
IS subgroup FG2; and iii) a cellular response to Gi stimulation
lower than the control sample by less than about (i.e., at most)
40% (or by about 10 to 40%) is indicative that the subject belongs
to IS subgroup FG3. The above classification is based on the
percentage of degree of reduction relative to control group.
Alternatively, the classification can be expressed as the
percentage of maximal response relative to the control. In such an
embodiment, i) a maximal response below 40% of the control (e.g.,
between about 10 and 40%) is indicative that the subject belongs to
IS subgroup FG1; ii) a maximal response between about 40 and 60% of
the control is indicative that the subject belongs to IS subgroup
FG2; and iii) a maximal response above about 60% of the control (or
between about 60 and 90%) is indicative that the subject belongs to
IS subgroup FG3. Because the Gi-mediated defective signaling is due
to reduced Gi-protein activity, the magnitude of a cellular
response to Gi-stimulation for each endophenotype (i.e., FG1, FG2
and FG3 subgroups) can also be assessed by determining (e.g.,
measuring) the effect of a GPCR antagonist (e.g., GP Ant-2 or PTX)
or GiPCR activator (e.g., mastoparan-7). For example, inhibition
curves of GPCR antagonist GP Ant-2 on response to various selective
agonists of Gi-coupled receptors (from clusters i to iv) give
relative inhibition profiles (e.g., IC50 FG1>IC50 FG2>IC50
FG3) which reflect the magnitude of Gi-mediated response relative
to control observed for agonist stimulation. GiPCR activator
Mastoparan-7 produces Gi protein-response profiles in control and
IS (FG1, FG2 and FG3) groups similar to that observed for any other
Gi-protein agonists with similar degree (%) of Gi-mediated response
relative to the control group. The magnitude of a cellular response
to Gi stimulation (as measured following Gi-stimulation and/or
inhibition) can be determined by various methods including but not
limited to determination of impedance (e.g., CDS), Time-Resolved
fluorescence (TRF), Time-Resolved-Florescence Resonance Energy
Transfer (TR-FRET), Enzyme Fragment complementation (EFC),
melanophore phenotype and optical biosensor.
[0096] In certain subjects, scoliosis develops rapidly over a short
period of time to the point of requiring a corrective surgery
(often when the deformity reaches a Cobb's angle
.gtoreq.45.degree.). Current courses of action available from the
moment a scoliosis such as IS is diagnosed (when scoliosis is
apparent) include observation (including periodic x-rays, when
Cobb's angle is around 10-25.degree.), orthopedic devices (such as
bracing, when Cobb's angle is around) 25-30.degree.), and surgery
(Cobb's angle over 45.degree.). Thus, a more reliable determination
of the risk of progression (through better classification) could
enable to 1) select an appropriate diet to remove certain food
products identified as contributors to scoliosis in certain
subjects (e.g., Hyaluronic acid (HA) for FG1 and FG2 subjects); 2)
select the best therapeutic agent o treatment or preventive measure
(an inhibitor of OPN expression or activity (e.g., neutralizing
antibody specific to OPN, sCD44 or RGD peptide or derivative
thereof, long term brace treatment which reduce OPN level,
melatonin, selenium, PROTANDIM) in the case of FG2 and FG3 subjects
or a stimulator of OPN expression or activity in the case of FG1
subjects (e.g., HA supplements or HA-rich diet, antibody against
CD44 etc.); 3) select the least invasive available treatment such
as postural exercises (e.g., massages (e.g., 30-90 minutes
pulsative compressive pressure applied locally) or low intensity
pulsed ultrasound (LIPUS) which increase OPN level for FG1
subjects, orthopedic device (brace) or other treatment or
preventive measure (e.g., acupoint heat sensitive moxibustion, heat
therapy with pad, thermal bath, electroacupuncture) which decrease
OPN level, for FG2 and FG3 subjects, or less invasive surgeries or
surgeries without fusions (a surgery that does not fuse vertebra
and preserves column mobility) and/or 4) the best follow-up
schedule (e.g., increasing or decreasing the number of follow-up
visit to the doctor during for example a 3, 6 or 12 month period or
increasing or decreasing the number of x-rays during for example a
3, 6 or 12 month period).
[0097] Applicants have determined that subjects classified in the
FG2 functional group are more at risk of developing severe
scoliosis (i.e., a scoliosis which will require corrective surgery,
or which will progress more rapidly). Furthermore, as disclosed
herein and in co-pending WO2015032004 to Moreau et al., OPN, which
is an important factor in scoliosis progression and development,
has opposite effects in FG1 subjects as compared to FG2 and FG3
subjects. Applicant's have found that OPN has a protective effect
in FG1 subjects by increasing the Gi-mediated cellular response in
these subjects (i.e. OPN is able to help compensate the Gi
signalling defect present in scoliosis subjects or subject at risk
of developing scoliosis). Conversely, FG2 and FG3 subjects are
sensitive to OPN i.e., that OPN further reduces the Gi-mediated
response in these subjects and thereby further contributes
(enhance) scoliosis development and progression. OPN' s effect is
more pronounced in FG2 subjects, which are qualified as being
"hypersensitive to OPN.
[0098] The present inventors have also previously established that
OPN' s inhibitory action on Gi-mediated cell signalling is due (at
least partly) to its interaction with integrins (e.g.,
.alpha..sub.5.beta..sub.1). This interaction can be modulated by
the presence of CD44, another OPN receptor. A mutation in CD44 (CT
mutation) has been shown to further increase the sensitivity of
scoliotic subjects to the damaging effects of OPN (see
PCT/CA2014/050569, to Moreau). CD44 (e.g., soluble CD44) can
compete with integrins for OPN binding and therefore indirectly
regulate OPN' s effect on Gi-mediated cell signaling. Furthermore,
PIPK1.gamma. activity increases the affinity of integrins for OPN
through FAK and Src. In turn, PIP1K.gamma. activity is regulated by
PTP.mu.. PIPK1.gamma. has been shown to be upregulated while
PTP.mu. has been shown to be downregulated in cells from IS
subjects (see WO2014201560 to Moreau et al.).
[0099] Accordingly, the present invention provides a method of
predicting the risk of developing severe IS comprising determining
the effect of OPN on the cellular response to GiPCR stimulation by
comparing the cellular response obtained in the presence of OPN
with the cellular response obtained in the absence of OPN and
classifying the subject into one IS functional, wherein
classification into the FG2 functional group is indicative that the
subject is at risk of developing severe IS. Corresponding methods
of predicting the risk of developing scoliosis are provided based
on i) the impedance signature in response to Gi protein stimulation
(e.g., cluster II receptor agonists) and ii) the cellular response
to Gi protein stimulation in the presence of PTX. The present
invention also encompasses selecting the most efficient and least
invasive known preventive action, treatment or follow-up schedule
in view of the determined classification and risk of developing
scoliosis.
[0100] Accordingly, the present invention provides a method of
selecting a preventive action, treatment or follow-up schedule for
a subject suffering from IS comprising classifying the subject
using at least one of the above described classification
methods.
[0101] As discussed above, in the FG1 subgroup, OPN has a
protective effect on scoliosis development and progression.
Accordingly, in these subjects it is desirable to increase OPN
level or bioavailability rather than to decrease it. Preventive and
treatment measures in FG1 subjects should thus aim at increasing
OPN level or activity. Conversely, FG2 and FG3 subjects are
particularly sensitive to OPN. In these subjects high OPN levels
are undesirable and can increase the risk of scoliosis development.
Preventive and treatment measures in FG2 and FG3 subjects should
thus aim at decreasing OPN level or activity.
[0102] Hyaluronic Acid (HA) is known to compete with OPN for
binding to CD44 (another OPN receptor) and thus to increase the
level of OPN available to bind to integrins. Accordingly, one way
of increasing OPN's activity (e.g., binding to integrins) is by
increasing the amount of HA in the subject which in turn increases
OPN bioavailability. This can be done for example by taking HA
supplements or by increasing HA intake or HA synthesis by favoring
certain food. Non-limiting examples of food with high HA content or
which stimulates/support HA production include, meat and meat
organs (e.g., veal, lamb, beef and gizzards, livers, hearts and
kidneys), fish, poultry (including meat fish and poultry broths),
soy (including soy milk), root vegetables containing starch
including potatoes and sweet potatoes, satoimo (Japanese sweet
potato), imoji (Japanese sweet potato), Konyaku concoction (root
vegetable concoction. Fruits and vegetables rich in vitamin C,
magnesium or zinc are also useful as they support the synthesis of
HA by the body. Non-limiting examples of food rich in vitamin C
include lemons, oranges, limes, grapefruit, guava, mango, cherries,
kiwi, blueberries, raspberries, all varieties of grapes, parsley
and thyme. Fruits and vegetables rich in magnesium include apples,
bananas, tomatoes, avocados, pineapples, melons, peaches, pears,
spinach, cauliflower, broccoli, asparagus, green lettuce, Brussels
sprouts and green beans. Non-limiting examples of food rich in zinc
include pumpkins, yeast, peanuts, whole grains, beans, and brown
rice.
[0103] Conversely, FG2 and FG3 subjects should decrease or maintain
lower levels of HA and therefore should comply with a HA-poor diet
(by avoiding one or more of the above foods).
[0104] Other non-limiting examples of treatment or preventive
measures which increase OPN expression (e.g., circulating OPN
levels) or activity and which may be beneficial to FG1 subjects
include low intensity pulsed ultrasound (LIPUS) and the application
of massages/pulsative compressive pressure as described in U.S.
Ser. No. 13/822,982.
[0105] Other non-limiting examples of treatment or preventive
measures which decrease OPN expression (e.g., circulating OPN
levels) or activity and which may be beneficial to FG2 and/or FG3
subjects include acupoint heat sensitive moxibustion, heat
therapies with pad, thermal bath, electroacupuncture, melatonin,
selenium (as supplements or by complying to a selenium-rich diet),
PROTANDIM (nutraceutical cocktail known to reduce plasma or serum
OPN levels and used as a natural anti-oxidant mix), etc, which have
been shown to decrease OPN levels.
[0106] Other ways to increase or decrease the level or activity OPN
include the examples provided in Table 1, below.
TABLE-US-00002 TABLE 1 Non-limiting examples of treatment and
preventive measures according to the functional endophenotype.
Treatment or Functional group Preventive measure FG1 FG2 FG3
Comments Increase in OPN yes no no OPN has a protective level or
activity effect in FG1 subjects but constitute a risk factor in FG2
and FG3 subjects. Increase in sCD44 no yes yes Decreases OPN's
level bioavailability to integrins Incease HA level yes avoid avoid
HA increases OPN's bioavailability Decrease HA level avoid yes yes
HA increases OPN's bioavailability Brace treatment Avoid or close
yes yes Long term brace treatment monitoring of decreases OPN level
(see OPN level required. US 61/879,314) Massages (e.g., pulsative
yes avoid avoid Increase OPN compressive pressure) level (see US
13/822,982) Src inhibitors -- yes yes Involved in the activation of
PIPK1.gamma. FAK inhibitors -- yes yes Involved in the activation
of PIPK1.gamma. RGD peptides -- yes yes Inhibit the binding of OPN
to integrins Decreasing PIPK1.gamma. -- yes yes Increases binding
of level or activity integrins to OPN (e.g., inhibitors) Increasing
the level -- yes yes Involves in the or activity of PTP.mu.
dephosphorylation (inhibition) of PIPK1.gamma. Early corrective no
yes no FG2 subjects have an surgery (before 45 increased risk of
severe degrees Cobb angle) scoliosis (assessment of sCD44 levels,
OPN levels and presence of mutation in CD44 which decreases
affinity toward OPN are also important in making decision)
[0107] In an embodiment, when the subject is classified as
belonging to the FG2 functional group the subject is prescribed
corrective surgery prior to reaching a 45 degree scoliosis (e.g.,
prior to reaching 30, 32, 34, 36, 38, 40, 41, 42, 43, 44 degree
scoliosis); and wherein when the subject belongs to the FG1 or FG2
functional group, the subject has a decreased risk of curve
progression over a 45.degree. angle and the subject is prescribed
fewer than 22 radiological examinations within the next 3 years
following classification.
[0108] The present invention also encompasses kits for classifying
subjects having IS or at risk of developing IS; kits for predicting
the risk of developing IS and kits for predicting the risk of
developing severe IS. Such kits may comprise 1. (a) recombinant OPN
(rOPN); (b) one or more ligands (e.g., agonists) for stimulating
GiPCRs (e.g., Cluster I, II, III or iv ligands (e.g., agonists),
Mastoparan, etc.); or 2. a) PTX; b) one or more ligands for
stimulating GiPCRs (e.g., Cluster I, II, III or IV ligands (e.g.,
agonists). Optionally the kits may comprise (c) i) one or more
ligands for stimulating GsPCRs; ii) one or more ligands for
inhibiting GiPCR (e.g., antagonists, GP Ant-2) and/or (ii) one or
more antibody for detecting Gi.alpha. phosphorylation, together
with instructions for using the kit.
Definitions
[0109] For clarity, definitions of the following terms in the
context of the present invention are provided.
[0110] As used herein the terms "at risk of developing a scoliosis"
or "at risk of developing IS" refer to a genetic or metabolic
predisposition of a subject to develop a scoliosis (i.e. spinal
deformity) and/or a more severe scoliosis at a future time (i.e.,
curve progression of the spine). For instance, an increase of the
Cobb's angle of a subject (e.g., from 40.degree. to 50.degree. or
from 18.degree. to 25.degree. is a "development" of a scoliosis.
The terminology "a subject at risk of developing a scoliosis"
includes asymptomatic subjects which are more likely than the
general population to suffer in a future time of a scoliosis such
as subjects (e.g., children) having at least one parent, sibling or
family member suffering from a scoliosis. Among others, age
(adolescence), gender and other family antecedent are factors that
are known to contribute to the risk of developing a scoliosis and
are used to evaluate the risk of developing a scoliosis. Also
included in the terminology "a subject at risk of developing a
scoliosis" are subjects already diagnosed with IS but which are at
risk to develop a more severe scoliosis (i.e. curve
progression).
[0111] As used herein, the terms "severe scoliosis", "severe IS" or
"severe progression" is an increase of the Cobb's angle to
45.degree. or more, potentially at a younger age.
[0112] As used herein the term "subject" is meant to refer to any
mammal including human, mouse, rat, dog, chicken, cat, pig, monkey,
horse, etc. In a particular embodiment, it refers to a human.
[0113] As used herein the term "treating" or "treatment" in
reference to scoliosis is meant to refer to at least one of a
reduction of Cobb's angle in a preexisting spinal deformity,
improvement of column mobility, preservation/maintenance of column
mobility, improvement of equilibrium and balance in a specific
plan; maintenance/preservation of equilibrium and balance in a
specific plan; improvement of functionality in a specific plan,
preservation/maintenance of functionality in a specific plan,
cosmetic improvement, and combination of any of the above.
[0114] As used herein the term "preventing" or "prevention" in
reference to scoliosis is meant to refer to a at least one of a
reduction in the progression of a Cobb's angle in a patient having
a scoliosis or in an asymptomatic patient, a complete prevention of
apparition of a spinal deformity, including changes affecting the
rib cage and pelvis in 3D, or a combination of any of the
above.
[0115] As used herein the terms "follow-up schedule" is meant to
refer to future medical visits a subject diagnosed with a scoliosis
or at risk of developing a scoliosis is prescribed once the
diagnosis or risk evaluation is made. For example, when a subject
is identified as being at risk of developing a severe scoliosis or
at risk of rapid curve progression (e.g., a subject classified as
belonging to the FG2 subgroup in accordance with the present
invention), the number of medical visits (e.g., to the orthopedist)
is increased and/or the number of x-rays in a given period (e.g.,
3, 6 or 12 months) is increased. On the other hand, when a subject
is identified as having a lower risk of curve progression or rapid
curve progression (e.g., subject being classified as belonging to
the F1 or FG3 subgroup) the number of medical visits or x-rays may
be decreased to less than the average (e.g., less than 22 x-rays
over a 3 year period or less than 1 visit every 3 months, 6 months
or 12 months).
[0116] As used herein, the terminology "blood sample" is meant to
refer to blood, plasma or serum.
[0117] As used herein, the terminology "cell sample" is meant to
refer to a sample containing cells expressing the desired GPCR(s)
in sufficient amount to detect a cellular response in accordance
with the present invention. The cells in the cell sample may be any
type of cells as long as they express the desired GPCR to be
tested. The cells used herein naturally express one or more
receptors coupled to G, proteins and were selected in part for
their accessibility for collection from subjects. Hence, cells such
as osteoblasts, osteoclasts, peripheral blood mononuclear cell
(PBMC) (inherently including principally lymphocytes but also
monocytes) and myoblasts are advantageously accessible and may
conveniently be used in the methods of the present invention. Blood
cells (e.g., PBMCs, platelets (thrombocytes), etc.) in particular
are particularly accessible and provide for a more rapid testing.
Any blood cell can be used for the methods of the present invention
so long as it possesses at least one GPCR receptor coupled to a Gi
protein. The cells can be fresh or frozen and may or may not have
been cultured (expanded) prior to testing. The "sample" may be of
any origin including blood, saliva, tears, sputum, urine, feces,
biopsy (e.g., muscle biopsy), as long as it contains cells
expressing the desired GPCR(s).
[0118] The methods of the invention may be performed using cell
expressing one or more receptor(s) coupled to a G, and/or Gs
proteins. "Receptor" as used herein refers to wild-type receptors
as well as to fragments and/or variants thereof that retains the
activity (i.e. GPCR-mediated activity) of the wild-type receptors.
FIGS. 8A-8G present a non-exhaustive list of GiPCRs suitable for
use in the method of the present invention.
[0119] As used herein the terminology "control sample" is meant to
refer to a sample from which it is possible to make a comparison
and to classify/stratify subjects into a specific functional group.
In an embodiment, a "control sample" is a sample that does not
originate from a subject known to have scoliosis or known to be a
likely candidate for developing a scoliosis (e.g., idiopathic
scoliosis (e.g., Infantile Idiopathic Scoliosis, Juvenile
Idiopathic Scoliosis or Adolescent Idiopathic Scoliosis (AIS))). In
another embodiment, the control sample is from one or more subjects
of the group FG1, FG2 or FG3. In the context of the present
invention, "a control sample" also includes a "control vaule" or
"reference signal" derived from one or more control samples from
one or more subjects. In methods for predicting the risk of
developing scoliosis in a subject that is pre-diagnosed with
scoliosis, the sample may also come from the subject under scrutiny
at an earlier stage of the disease or disorder. In an embodiment,
the control sample is a cell of the same type (e.g., both the test
sample and the reference sample(s) are e.g., lymphocytes,
osteoblasts, myoblasts or chondrocytes) as that from the subject.
Of course, multiple control samples derived from different
categories of subjects (e.g., FG1, FG2, FG3 and healthy subjects)
can be used in the methods of the present invention. As used
herein, the term "reference signal" or "control value" is meant to
refer to a signal (e.g., fluorescence, impedance, cAMP
concentration or any other measurable signal) that serves as a
reference in predicting the risk of developing scoliosis or in
establishing classification of a subject into a particular
functional group. A reference signal can be obtained by using one
or more samples from suitable control subjects (subjects not having
a scoliosis or not at risk of developing a scoliosis, FG1, FG2 or
FG3 subjects depending on the method). The reference signal may
originate from a single control subject ((i.e., a normal healthy
subject or a subject already classified in a given endophenotype
group) or may be derived from a group of control subjects (i.e.,
equivalent to the average response in control subjects).
[0120] "GPCRs" or "G protein-coupled receptors" constitute a large
protein family of receptors that sense molecules outside the cell
and activate inside signal transduction pathways leading to various
cellular responses.
[0121] The G protein-coupled receptor is activated by an external
signal in the form of a ligand (e.g., agonists). This creates a
conformational change in the receptor, causing activation of a G
protein. Further effect depends on the type of G protein. Four
types of G proteins exist : G.alpha.s (Gs), G.alpha.i/o (Gi),
G.alpha.q/11, and G.alpha.12/13. G.alpha.s (Gs) is a heterotrimeric
G protein subunit that activates the cAMP-dependent pathway by
activating adelylate cyclase. G.alpha.i/o (Gi) is a heterotrimeric
G protein subunit that inhibits the cAMP-dependent pathway by
inhibiting adelylate cyclase. As supported herein by the
identification of four difference GiPCR receptor cluster, G
protein-coupled receptors are not necessarily exclusively coupled
to a particular type of G protein (e.g., Gi or Gs) but may interact
with other G proteins (e.g., Gi or Gs) depending on the
conditions.
[0122] The ligands (e.g., agonists and antagonists) that bind and
activate (inhibit) these receptors include light-sensitive
compounds, odors, pheromones, hormones, and neurotransmitters, and
vary in size from small molecules to peptides to large proteins. As
used herein, the term "ligand" includes any molecule (synthetic or
natural) capable of binding to GPCR to modulate (increase or
decrease) its cellular response. Accordingly, the term "ligand" in
the expression "GiPCR ligand" or "GsPCR ligand" includes agonists
of GiPCR, agonists of GsPCR, antagonists of GiPCR and antagonists
of GsPCR. Any molecule can be used in accordance to the present
invention as long as it allows measuring the magnitude of the
cellular responses involving Gi or GsPCR.
[0123] There are two principal signal transduction pathways
involving the G protein-coupled receptors: the cAMP signal pathway
and the phosphatidylinositol signal pathway. When a ligand binds to
the GPCR it causes a conformational change in the GPCR, which
allows it to act as a guanine nucleotide exchange factor (GEF). The
GPCR can then activate an associated G-protein by exchanging its
bound GDP for a GTP. The G-protein's a subunit, together with the
bound GTP, can then dissociate from the .beta. and .gamma. subunits
to further affect intracellular signaling proteins or target
functional proteins directly depending on the a subunit type.
[0124] The effector of both the Gs and Gi pathways is the
cyclic-adenosine monophosphate (cAMP) generating enzyme adenylate
cyclase, or AC. AC catalyzes the conversion of cytosolic adenosine
triphosphate (ATP) to cAMP, and all are directly stimulated by
G-proteins of the Gs class. Conversely, interaction with G.alpha.
subunits of the Gi type inhibits AC from generating cAMP. Thus, a
GPCR coupled to G.alpha.s counteracts the actions of a GPCR coupled
to G.alpha.i/o, and vice versa. The level of cytosolic cAMP may
then determine the activity of various ion channels as well as
members of the ser/thr specific protein kinase A (PKA) family Thus,
cAMP is considered a second messenger and PKA a secondary
effector.
[0125] Accordingly, as used herein the term "GiPCR" refers to a
GPCR preferably (i.e., mainly) coupled to the G.alpha.i proteins
(e.g., Gi.alpha.1, Gi.alpha.2 and Gi.alpha.3) which when stimulated
by a ligand (i.e., an agonist to the GiPCR receptor) inhibits
(reduces) the production of cAMP in a cell. The methods of the
present invention may be performed by stimulating any GiPCR and
more than one GiPCR at the same time (1, 2, 3, 4 GiPCRs). GPCRs
coupled to the G.sub.i protein include, for example, CD47,
serotonin receptors (5-HT), adenosine receptors, adrenergic
receptors, cannabinoid receptors, histamine receptors,
prostaglandin receptors and dopamine receptors. Non-limiting
examples of GiPCRs suitable for use in the method of the present
invention are presented in FIGS. 8A-8G. Furthermore, any ligand of
a given GiPCR may be used in accordance with the present invention
as long as it leads to the activation of the Gi protein and
reduction of cellular cAMP. Multiple ligands (agonists and
antagonists) may also be used in accordance with the present
invention. Ligands (e.g., synthetic or natural) of GiPCRs are well
known in the art and several of these ligands are commercially
available. FIGS. 9A-9DDD present a non-exhaustive list of GiPCR
ligands suitable for use in the method of the present invention. In
an embodiment, the above-mentioned receptor coupled to a G.sub.i
protein is a melatonin receptor, a serotonin receptor, a
somatostatin receptor, an Apelin receptor, a dopaminergic receptor,
a lysophosphatidic acid receptor, a formyl peptide receptor, an
.alpha.-adrenergic receptor, an adenosine receptor, a cannabinoid
receptor or any combination thereof. In a further embodiment, the
above-mentioned receptor is LPAR, A1R, MT2R, 5-HT1AR, .alpha.2-ADR,
A3R, OR, D2R, SSTR, APJ, FPR2 or CB2.
[0126] In a specific embodiment, the above-mentioned ligand is a
known agonist of the receptor. In an embodiment, the
above-mentioned ligand is (a) melatonin for the MT2 receptor, (b)
1-[3-(3,4-Methylenedioxyphenoxy)propyl]-4-phenyl-piperazine maleate
(known as BP554 maleate) for the 5-HT1A receptor, (c)
5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (known
as UK14304) for the .alpha.2-AD receptor; (d)
1-Deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-.beta.-
-D-ribofuranuronamide (known as IB-MECA) for the A3 receptor;
Lysophosphatidic acid (known as LPA) for the LPA receptor;
(2S)-2-[[2-[[(2R)-2-[[(2S)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]pro-
panoyl]amino]acetyl]-methylamino]-N-(2-hydroxyethyl)-3-
phenylpropanamide (known as DMAGO) for the mu-opioid receptor,
1-(6-Amino-9H-purin-9-yl)-1-deoxy-N-ethyl-.beta.-D-ribofuranuronamide
(known as NECA) for the adenosine receptors (e.g., A1, A2a and A3);
somatostatin for the SSTR receptor; peptide MMK-1 (LESIFRSLLFRVM)
for the FPR2 receptor; Apelin-17 for the APJR receptor; (4aR,
8aR)-5-propyl-4,4a,5,6,7,8,8a,9-octahydro-1H-pyrazolo[3
,4-g]quinolone (known as quinpirole) for the D2 and D3 receptors
and/or (e)
N-Cyclohexyl-7-chloro-1-[2-(4-morpholinyl)ethyl]quinolin-4(1H)-one-3-carb-
oxamide (known as CB65) for the CB2 receptor.
[0127] Similarly, as used herein the term "GsPCR" refers to a GPCR
preferably (i.e., mainly) coupled to the G.alpha.s protein which
when stimulated by a ligand (i.e., an agonist to the GsPCR
receptor) leads to the activation of the cAMP-dependent pathway
thereby increasing the level of cAMP in a cell. When assessing an
imbalance between Gi and Gs cellular responses in accordance with
the methods of the present invention the activity (cellular
response) of any GsPCR may assessed. Also the activity (cellular
response) of more than one GsPCR may be assessed at the same time
(1, 2, 3, 4 GsPCRs). Non-limiting examples of GsPCRs that can be
used in accordance with the present invention include receptors
types 5-HT4 and 5-HT7, ACTH receptor, Adenosine receptor types A2a
and A2b, Arginine vasopressin receptor 2, .beta.-adrenergic
receptors types .beta.1, .beta.2 and .beta.3, Calcitonin receptor,
Calcitonin gene-related peptide receptor, corticotropin-releasing
hormone receptor, Dopamine receptors D1-like family (D1 and D5),
FSH-receptor, Gastric inhibitory polypeptide receptor, Glucagon
receptor, Histamine H2 receptor, Luteinizing
hormone/choriogonadotropin receptor, melanocortin receptor: MC1R,
MC2R (aka ACTH receptor), MC3R, MC4R, MCSR, Parathyroid hormone
receptor 1, Prostaglandin receptor types D2 and 12, secretin
receptor and thyrotropin receptor. Furthermore, any ligand of a
given GsPCR may be used in accordance with the present invention as
long as it leads to the activation of the Gs protein and production
of cAMP. Of course, the effect of antagonists to GsPCR may also
alternatively be used to determine the magnitude of the cellular
response to GsPCR stimulation and the use of antagonists is within
the scope of the present invention. For example, the IC50 could be
calculated to assess magnitude of GsPCR response within a cell.
Antagonists to GsPCR are well-known and the skilled practitioner
knows how to assess the magnitude of response to GsPCR response in
the presence of antagonists. Multiple ligands (agonists and
antagonists) for GsPCR may also be used in accordance with the
present invention. Ligands (e.g., synthetic or natural) of GsPCRs
are well known in the art and several of these ligands are
commercially available.
[0128] As used herein, the expression "cellular response" in
"determining the cellular response to Gi/Gs stimulation" for
example is meant to refer to any detectable cellular
response/reaction due to Gi or GsPCR stimulation and/or inhibition
(agonist/antagonist ligand binding). For example, any cellular
response linked to the activation/inhibition of the cAMP-dependent
pathway may be used to assess Gi/GsPCR cellular response in
accordance with the present invention. Cellular response to
Gi/GsPCR stimulation includes but is not limited to cAMP
inhibition/activation; Gi and/or Gs proteins phosphorylation
pattern and/or levels, kinases phosphorylation pattern and/or
levels (PKA, PKC, CAMK1, CK, CAMK2), cellular proliferation levels,
changes in protein-protein interactions and cellular impedance
modifications (detected by cellular dielectric spectroscopy (CDS)).
All of these cellular responses can be assessed following Gi or
Gs-PCR stimulation with any agonists in the presence or absence of
antagonists as long as it allows to determine the relative level of
GiPCR and/or GsPCR cellular response in order to distinguish
between the three endophenotypes (FG1, FG2 and FG3). For example,
as disclosed herein, the magnitude of GiPCR response can be
assessed and the endophenotypes distinguished based on their
relative cellular response in the presence of GP Ant-2 or PTX,
which both inhibit GPCRs. FIGS. 15A-15B provide examples of
cellular responses expected when stimulating GiPCRs in the presence
and absence of OPN.
[0129] "GiPCR/GsPCR stimulation" or "Gi/Gs stimulation" refers to
GiPCR or GsPCR activation (ligand binding) leading to a
cAMP-dependent cellular response. As noted above Gi/Gs stimulation
is achieved by contacting a cell expressing a given GiPCR/GsPCR
with its cognate ligand(s).
[0130] The changes in the magnitude of the signal induced by the
ligand (i.e., changes in cellular response(s) induced by e.g. an
agonist or antagonist) may be detected using any suitable methods.
Methods for measuring the magnitude or intensity of the signal
(e.g., intracellular response) mediated through GiPCRs are well
known in the art. The magnitude of the signal may be determined,
for example, by measuring the level of a molecule, such as a second
messenger (e.g., cAMP, Ca.sup.2+) or a gene product (e.g., mRNA or
protein) whose level is modulated following triggering of the
receptor by a ligand. The magnitude of the signal may also be
determined, for example, by measuring changes in protein-protein
interactions (e.g., by fluorescence resonance energy transfer
(FRET); Time Resolved (TR)-FRET or bioluminescence resonance energy
transfer (TR-BRET)) following triggering of the receptor by a
ligand. Other methods to measure the magnitude or intensity of the
signal mediated through GiPCRs include, for example, measurement of
cAMP levels (Medhurst et al., 2003. In: J Neurochem., 84),
measurement of thallium flux using GIRK-thallium Flux Assay
(Niswender et al., 2008; In: Mol Pharmacol. 73(4)), Patch-clamp
(Saugstad et al., 1996. In: J. Neurosci. 16), measurement of
GTP.gamma.S binding using [.sup.35S] GTP.gamma.S labelling assay
(Riobo et al., 2006. In: Proc Natl Acad Sci USA, 103), and
measurement of the changes in impedance (Peters et al., 2007. In: J
Biomol. Screen. 12: 312-9). Other non-limiting examples of suitable
methods include Enzyme Fragment Complementation (EFC), Time
Resolved Florescence (TRF), melanophore phenotype and optical
biosensor. In an embodiment, the change in magnitude of the signal
is determined using the changes in impedance that occurs in the
cell following receptor triggering (e.g., cellular dielectric
spectroscopy (CDS)). Such measurement may be made, for example,
using the real-time cell electronic sensing (RT-CES.TM.) technology
(ACEA Biosciences Inc., San Diego, Calif., USA) (Huang et al.,
Analyst, 2008, 133(5): 643-648; Solly et al., Assay Drug Dev.
Technol., 2004, 2(4): 363-372) or using the CellKey.TM. technology
(MDS Sciex, Concord, Ontario, Canada) according to the method
described below. In a preferred embodiment, the magnitude of a
cellular response to Gi or Gs stimulation is determined by
(TR)-FRET, EFC, TRF, melanophore phenotype, optical biosensor or
CDS.
[0131] In an embodiment, a lower or higher signal refers to a
difference of at least about 5%, or 10%, in further embodiments at
least about 15%, 20%, 25%, 30%, 35%, 40%, 45% ,50%, 55%, 60%, 65%,
70% 75%, 80%, 85%, 90%, 95% 100%, 120%, 130%, 140%, 150%, 160%,
170%, 180%, 190% or 200% between the signal obtained with the test
sample (sample obtained from the subject being tested) relative to
the reference (control) signal. In an embodiment, a substantially
identical signal refers to a signal that differs by less than 10%,
in further embodiments by less than 9%, 8%, 7%, 6% or 5%, as
compared to the reference signal.
[0132] In an embodiment, the methods are performed in a format
suitable for high throughput assays, e.g., 96- or 384-well format,
and suitable robots, (e.g., pipetting robots), and instrumentation
may be used. In an embodiment, the assay will be assayed in plates
(e.g., 96-wells, 384-wells, etc.) containing the test sample and
one or more samples.
[0133] As used herein, the terminology "fold effect" or "Fe", when
used in the context of the present invention refers to the effect
of OPN on the magnitude of the Gi-mediated cellular response. The
fold effect (Fe) of OPN on Gi-mediated response is calculated by
dividing the average of response magnitude to Gi stimulation in
presence of OPN (RmGiOPN) with the average of response magnitude to
Gi stimulation in the absence of OPN (RmGi) using the following
formula:
Fe=100.times.(RmGiOPN/RmGi).
[0134] The articles "a," "an" and "the" are used herein to refer to
one or to more than one (i.e., to at least one) of the grammatical
object of the article.
[0135] The term "including" and "comprising" are used herein to
mean, and are used interchangeably with, the phrases "including but
not limited to" and "comprising but not limited to".
[0136] The terms "such as" are used herein to mean, and is used
interchangeably with, the phrase "such as but not limited to".
[0137] The present invention is illustrated in further details by
the following non-limiting examples.
[0138] The classification protocols described below details
embodiments of the experimental and analytical procedure for a
cell-based assay developed in Applicants' laboratory as a
functional test to predict the risk of developing idiopathic
scoliosis in asymptomatic and IS subjects (e.g., AIS). In a first
aspect of the present invention, the assay comprises the evaluation
of the functional status (relative cellular response) of Gi and Gs
proteins in cells (e.g., peripheral blood mononuclear cells
(PBMCs)) by e.g., changes in impedance (e.g., cellular dielectric
spectroscopy (CDS) assessed using e.g., CellKey.TM. apparatus) and
in the classification of children into functional groups (FG1, FG2,
FG3) with respect to the difference between the degree of response
to Gi and Gs proteins stimulation (.DELTA.G). Of course, any other
suitable methods of assessing Gi and Gs cellular response (e.g.,
TR-FRET; EFC, TRF, melanophore phenotype and optical biosensor) can
be used in accordance with the present methods. The classification
is further confirmed by the determination of the effect of
osteopontin (OPN) on the subject's cellular response to Gi
stimulation.
[0139] In accordance with the technique followed in Examples 1 and
2 below, approximately a volume of 10 mL of blood is required to
extract PBMCs by Ficoll-gradient and cells are then stored in
liquid nitrogen. The optimal number of PBMCs (about
1.5.times.10.sup.5 cells or more) to perform the assay is obtained
after about two days of cell culture. Essentially, cells are first
incubated with phytohemmaglutinin (PHA). After 24 h incubation, the
medium is replaced by a PHA-free culture medium for an additional
24 h prior to cell seeding and OPN treatment. Cells are then
spectroscopically screened for their response to somatostatin and
isoproterenol which activate Gi and Gs proteins, respectively,
through their cognate receptors. Both somatostatin and
isoproterenol are simultaneously injected with an integrated
fluidics system and the cells' responses are monitored for 15
min.
[0140] The assay can be performed with fresh or frozen PBMCs
(conserved frozen for up to one year) and the procedure is
completed within 4 days. Since using freshly isolated PBMCs may be
cumbersome when testing large number of individuals, frozen PBMCs
may optimally be used and offer a more practical alternative in
clinical setting. In addition, the use of frozen PBMCs allows
simultaneous analysis, within a single assay run, of PBMCs samples
from multiple time points during a longitudinal study. To maximize
assay reproducibility, Applicants recommend avoiding freeze-thaw
cycle and using the frozen sample only once. The procedure is very
simple, allowing for accurate detection of defective Gi protein
function in a short time. Using this procedure, asymptomatic and
scoliotic children can be easily classified to better predict their
clinical outcome without any danger for their health. However, when
performing classification according to the degree of maximum
response to Gi stimulation relative to the healthy control
subjects.sup.12, the fact that these control subjects should, not
only match with age and gender of scoliotic or asymptomatic
children, but also not be on any medication, may constitute an
important obstacle for the recruitment of control subjects.
Therefore, performing classification by examining the degree of
imbalance between response to Gi and Gs protein stimulation (i.e.
average of response magnitude to Gi stimulation (RmGi) minus the
average of response magnitude to Gs stimulation (RmGs)) in the same
individual is ideal to eliminate the necessity of using control
subjects.
[0141] The use of the CDS-based system to perform this prognostic
test advantageously simultaneously provides Gi- and Gs-mediated
cellular response in the same assay.
[0142] Certain patients will exhibit borderline values in the
.DELTA.G CDS-based assay (or other classification methods based on
e.g., Gi cellular response detection) making them difficult to
classify, as illustrated by results presented in FIG. 4. Applicants
have discovered that OPN induces a Gi-mediated cellular response
that varies between the three functional groups. Applicants have
found that in the presence of OPN, the response to Gi stimulation
increases in functional FG1, while it decreases in FG2, and, to a
lesser extent, in FG3.
[0143] The present invention is illustrated in further, details by
the following non-limiting examples.
EXAMPLE 1
Materials and Methods
[0144] Reagents and equipment. Table 2 presents the reagents,
equipment and their sources used in an embodiment of the method of
the present invention.
TABLE-US-00003 TABLE 2 Name Company Catalog Number Comments RPMI
Wisent Inc 350-005-CL FBS Therno Scientific SH3007103 Hyclone DMSO
Sigma Aldrich D2650 Ficoll-Plaque GE Healthcare 17144003
Antibiotic-Antimycotic Invitrogen 15240-062 Phytohemagglutinin
(PHA) Invitrogen (Gibco) 10576-015 Recombinant Human Osteopontin R
& D Systems, Inc 1433-OP/CF Somatostatin Tocris 1157
Isoproterenol Tocris 1743 PBS Wisent Inc 311-010-CL Sterile pipette
tips Axygen Scientific 301-06-451 Sterile Eppendorf tubes Ultident
24-MCT-150-C 50 mL conical tubes VWR International 89039-658
Cellkey .TM. Small sample 96W Molecular Devices 1026496 microplate
Cellkey .TM. tips Cybio OL3800-25-559N Pre-cut pierceable seals
Excel Scientific, Inc. XP-100 Equipment Automated cell counter
Beckman Coulter 731050 (Vicell XR) Cell culture hood Forma
Scientific 1284 Class II Liquid Nitrogen storage Thermo Scientific
CY5093570 Water bath VWR International 89032-204 Standard light
microscope Leica Microsystems DMIL LED Cell culture incubator
Thermo Scientific 51019557 5% CO.sub.2 at 37.degree. C. Low speed
centrifuge Thermo Scientific 75004364 Cellkey .TM. system Molecular
Devices 1019185
[0145] The solutions are prepared according to Table 3. The
balanced salt solution (BSS) is kept at room temperature and all
other solutions at 4.degree. C. until the time of use. Cold media
is warmed to 37.degree. C. in a water bath for a few minutes before
using.
TABLE-US-00004 TABLE 3 Solutions Solution A Anhydrous D-glucose
0.1% CaCL.sub.22H2O 0.05 mM MgCL.sub.2 0.98 mM KCL 5.4 mM Tris 145
mM Solution B NaCL 140 mM Balanced Salt Solution A 1 volume
Solution (BSS) Solution B 9 volume Complete media RPMI-1640 500 mL
Antibiotic-antimycotic 1% FBS 10% Supplementary RPMI-1640 50 mL
media Antibiotic-antimycotic 1% FBS 40% Freezing media RPMI-1640 50
mL Antibiotic-antimycotic 1% FBS 40% DMSO 20% PHA media RPMI-1640
500 mL Antibiotic-antimycotic 1% FBS 10% Phytohemaglutinin 1%
[0146] 1. Preparation of solutions: 1. Prepare solutions according
to Table 3 above. 2. Keep balanced salt solution (BSS) at room
temperature and all other solutions at 4.degree. C. until the time
of use. 3. Heat media to 37.degree. C. in the water bath for a few
minutes before using.
[0147] 2. Preparation and storage of PBMCs. 1. Collect 10 mL of
whole blood in EDTA-treated collection tubes to prepare two
aliquots of PBMCs using 5 mL for each aliquot. 2. Transfer 5 mL of
whole blood from the EDTA-treated collection tube to a 50 mL tube.
3. Add an equal volume of BSS and mix sample by gentle pipetting up
and down. 4. Place 3 mL of Ficoll in two 15 mL Falcon tubes. 5.
Carefully layer 4.5 mL of diluted blood mixture over the Ficoll in
each tube. 6. Let the tubes rest for up to 5 minutes to favor a
clear separation of the blood and Ficoll. 7. Centrifuge the tubes
at 400.times.g for 30 minutes at room temperature with no brake. 8.
Carefully remove the tubes from the centrifuge so as to not disturb
the layering. The PBMCs are visible at the BSS/Ficoll interface. 9.
Harvest the cloudy layer of PBMCs at the interface of both tubes
with a pipette and transfer to a new 50 mL tube. 10. Add 20 mL of
complete media. 11. Centrifuge the tube at 288.times.g for 7
minutes at room temperature. 12. Remove the supernatant by
aspiration. 13. Re-suspend the cell pellet in 500 .mu.L of
supplementary media. 14. Add an equal volume of freezing media. 15.
Transfer the cell suspension to a cryovial. 16. Place the cryovial
into a cryofreezing container with isopropanol. 17. Store the
freezing container at -80 .degree. C. overnight. 18. Transfer the
frozen PBMCs aliquot to liquid nitrogen for long-term storage.
[0148] 3. Functional Assay: Day 1: 1. Place PBMCs aliquot from
liquid nitrogen in water bath at 37.degree. C. for a minute or
until defrosted. 2. Transfer the cell suspension to a 50 mL tube
with a sterile pipette. 3. Add 15 mL of Complete media and spin the
cells down at 200.times.g for 5 minutes at room temperature. 4.
Remove the supernatant by aspiration. 5. Gently suspend cell pellet
in 1 mL of PHA media. 6. Complete the volume to 20 mL with the same
media. 7. Cap the tube loosely to allow air to enter. 8. Leave the
tube overnight at 37 .degree. C. in a CO.sub.2 incubator to allow
quiescent lymphocytes to transform into rapidly-proliferating
lymphoblasts.
[0149] Day 2: 1. Take the tube out of the incubator, screw the caps
completely and spin the cells down at 200.times.g for 5 min at room
temperature. 2. Remove the supernatant by aspiration. 3. Gently
suspend cell pellet in 1 mL of complete media. 4. Complete the
volume to 20 mL with the same media. 5. Cap the tube loosely to
allow air to enter. 6. Leave the tube overnight at 37.degree. C. in
a CO.sub.2 incubator to expand cell numbers.
[0150] Day 3: 1. Take the tube out of the incubator, screw the caps
completely and spin the cells down at 200.times.g for 5 mM at room
temperature. 2. Remove the supernatant by aspiration. 3. Wash cells
twice with 10 mL of RPMI-1640 (media developed for growing human
normal and neoplastic leukocytes (peripheral blood lymphocytes)) by
centrifugation at 200.times.g for 5 mM at room temperature. 4.
Gently re-suspend the cell pellet in 600 .mu.L of RPMI-1640. 5.
Measure the cell concentration and viability, using an automated
cell counter and viability analyzer. 6. Add appropriate volume of
RPMI-1640 to adjust to a cell concentration of 1.5.times.10.sup.5
cell/20 .mu.L. 7. Treat cells with recombinant OPN (rOPN) or
vehicle (PBS). 7.1 Transfer 100 .mu.L of cell suspension to two
sterile 1.5 ml eppendorf tubes. 7.2 Add rOPN in one tube to a final
concentration of 0.5 .mu.g/mL. 7.3 Add an equal volume of PBS in
the second tube. 7.4 Gently mix each condition by pipetting up and
down twice using a sterile pipette set at 100 .mu.L. 8. Prepare the
small sample 96-well microplate. 8.1 Add 5 .mu.L of RPMI-1640 to
each well. 8.2 Centrifuge the plate at 200.times.g for 3 min to
remove any air bubbles. 9. Seed the untreated cells as well as
cells treated with rOPN or PBS. 9.1 Before transferring cells from
tube to microplate, gently pipette up and down once to ensure a
uniform suspension of cells. 9.2 Add 40 .mu.L of cell suspension
per well in quadruplicate for untreated cells, in duplicate for
rOPN or PBS treated cells. Refer to FIG. 1 for the design. This
design allows 12 patients to be tested on the same microplate. 9.3
Leave the cell plate under the sterile hood for 5 minutes to allow
cells to rest and settle evenly to the bottom of the well before
placing in the incubator. 9.4 Incubate the plate for 18 h at
37.degree. C. in a CO.sub.2 incubator to optimise the effect of
OPN.
[0151] Day 4: 1. Run the plate with compounds. 1.1 Take the plate
out of the incubator and leave it at room temperature for around 30
mM 1.2 Prepare 1 mL of 100 .mu.M of Somatostatin (which activates
Gi-mediated cellular response) and isoproterenol (which activates
Gs-mediated cellular response) in RPMI-1640 by adding 10 .mu.L of
stock solution (10 mM) in 990 .mu.L of RPMI-1640. 1.3 Fill the
compound plate by dispensing 20 .mu.L in appropriate wells as
indicated in FIG. 2. 1.4 Cover the compound plate with a pre-cut
pierceable seal to avoid change in compound concentration due to
evaporation before or during incubation in the CDS-based system.
1.5 Load cell plate, pipette tips and compound plate into the
CDS-based system. 1.6 Name the plate in the CDS-based instrument
software. 1.7 Select the appropriate protocol. 1.8 The integrated
fluidics system simultaneously adds the compounds to all wells by
injecting 5 .mu.L per well to achieve a final concentration of 10
.mu.M in a total volume of 50 .mu.L. 1.9 The CDS-based system
automatically collects the data for 15 min after compound
addition.
[0152] Data Analysis: 1. Select low and high ranges of frequencies
to use when calculating extracted values for the non-adherent
cells. 2. Select drift correction to correct the linear change in
baseline impedance measurements over time. 3. Select data filtering
to reduce variations in the kinetic response measurement due to
electronic noise and compound addition. 4. Select the Max-Min
method for the full analysis time. 5. Export data to Excel under
the plate format option. 6. Calculate delta G (.DELTA.G) by
subtracting the average of response magnitude to Gi stimulation
(RmGi) from the average of response magnitude to Gs stimulation
(RmGs) using the following formula:
.DELTA.G=RmGi-RmGs
[0153] 7. Calculate the percentage of the fold effect (Fe) of OPN
on Gi-mediated response by dividing the average of response
magnitude to Gi stimulation in presence of OPN (RmGiOPN) with the
average of response magnitude to Gi stimulation in presence of PBS
(RmGiPBS) using the following formula:
Fe=100.times.(RmGiOPN/RmGiPBS)
[0154] Refer to Table 4 to classify patients.
TABLE-US-00005 TABLE 4 Dynamic ranges Functional Dynamic ranges
with .DELTA.G Groups with Fe .DELTA.G < -10 FG1 Fe > 100% -10
< .DELTA.G < +10 FG2 Fe < 50% .DELTA.G > +10 FG3 50%
< Fe < 95%
EXAMPLE 2
IS Subjects or Sujects at Risk of Developing IS can be Classified
According to their Response to OPN Stimulation
[0155] Cell viability was comparable between all samples with
values consistent in the range of 86 and 96%. In contrast, high
variations were noted in cell numbers among samples (FIG. 3). Of
the 32 pre-classified samples used, two had insufficient number of
cells and have not been further classified. The functional
classification of all patient samples used had been previously
determined using one or more alternative classification method
(e.g., cAMP-detection, impedance modification, etc.). An
illustration of the functional classification according to the
.DELTA.G determined with CDS is showed in FIG. 4. The vertical axis
of this figure is divided into three sections delineating the
functional groups with dynamic ranges established as >+10 for
FG3, between +10 and -10 for FG2 and finally <-10 for FG1. Among
30 patients tested 14, 6 and 5 patients were clearly classified
into groups FG3, FG2 and FG1, respectively, while five patients
notably 345, 353, 370, 371 and 382 had borderlines values (see FIG.
4).
[0156] The evaluation of the OPN effect on the response to Gi
stimulation revealed that OPN increased the response in patients
353 and 371. In contrast, the response was reduced by more than 50%
in patients 345 and 382 and by less than 50% in patient 370
following rOPN treatment (FIG. 5). According to the classification
criteria (Table 4) it was possible to categorize patients 353 and
371 in FG1, patients 345 and 382 in FG2, and patient 370 in
FG3.
[0157] In parallel, all patients were screened for their response
to Gi protein stimulation and compared to control subjects. As
expected, the latter classified the borderline subjects in the same
functional group as the method determining the Gi mediated response
in the presence or absence OPN ("Gi/OPN") did (FIGS. 6A-6D). The
classification of a large cohort of scoliotic patients regularly
followed in our special clinic at Sainte-Justine Hospital has
revealed that the three functional groups were similarly
distributed among moderate cases, while the FG2 was predominant
among severe cases (FIG. 7), identifying patients categorized into
this functional group as more at risk for severe progression of the
disease and indicating that this classification test can be useful
in the prognosis of IS.
EXAMPLE 3
IS Subjects or Sujects at Risk of Developing IS can be Classified
According to their Impedance Signature in Response to GiPCR
Stimulation in Four Distinct Receptor Clusters
[0158] In order to demonstrate that to determine if the reduced
ability of Gi proteins to promote signal transduction in AIS is a
generalized impairment and is not restricted to melatonin
receptors, we performed a comparative study with various synthetic
compounds activating selectively other receptors coupled to Gi
proteins. A total of ten compounds were tested and the
representative refraction index curves generated by each of these
compounds in control and AIS osteoblasts are illustrated in FIGS.
10A-10J). Analysis of impedance signature revealed that the tested
compounds fell into four distinct clusters. In the cluster I (FIGS.
10A-10C), compounds elicit shapes of impedance profiles similar to
those obtained with melatonin, consisting of a biphasic shape of
impedance in all three AIS groups with a negative phase of larger
extent for FG1. In cluster II (FIGS. 10D and 10E), the compounds
elicit negative response only in FG1. In cluster III (FIGS. 10F and
10G), the compounds elicit a relatively short transient negative
phase of a similar extent in all three AIS groups, while in cluster
IV (FIGS. 10H-10J), the compounds totally lack this feature and
elicit complete positive impedance in all AIS groups. Despite these
differences in the shape of impedance profiles, the regression
analysis of concentration-response curve of each tested compound
revealed no significant difference in EC50 values between control
and AIS groups (Table 5), while all groups were clearly
distinguished by the amplitude of their maximum response (data not
shown). In each case, the three AIS functional groups were less
responsive than the control group. The reduction degree for each
functional group relative to the control group was similar to that
obtained with melatonin. This suggests that AIS patients can be
classified using agonists of any GiPCR, with respect to the range
of values as established with a melatonin receptor agonist.
[0159] Furthermore, based on the impedance profiles provided in
FIGS. 10D-10E for cluster II agonists, it is possible to rapidly
distinguish subjects belonging to the FG1 functional group over
those belonging to the FG2 and FG3 functional groups. Indeed,
subjects belonging to the FG1 functional group show a
characteristic reduction in impedance, which is not present for the
FG2 and FG3 groups.
TABLE-US-00006 TABLE 5 Potency of various GiPCR agonists for
impedance response in osteoblasts from control and AIS patients
Control FG1 FG2 FG3 EC.sub.50 (nM) EC.sub.50 (nM) EC.sub.50 (nM)
EC.sub.50 (nM) Melatonin 33.4 .+-. 8.4 42.3 .+-. 9.4 48.68 .+-. 6.4
45.79 .+-. 5.3 LPA 8.52 .+-. 1.5 8.78 .+-. 0.6 8.54 .+-. 1.4 8.59
.+-. 1.2 DAMGO 18.78 .+-. 2.2 18.67 .+-. 2.1 19.11 .+-. 2.3 19.88
.+-. 2.4 NECA 20.85 .+-. 2.2 20.88 .+-. 1.7 20.83 .+-. 1.2 22.25
.+-. 2.2 CB65 13.91 .+-. 1.2 13.97 .+-. 1.2 13.7 .+-. 1.2 13.95
.+-. 1.3 UK14304 16.48 .+-. 2.3 18.52 .+-. 4.2 16.65 .+-. 1.6 17.8
.+-. 1.3 Somatostatin 31.12 .+-. 3.3 31.23 .+-. 3.1 31.3 .+-. 2.6
30.98 .+-. 2.3 MMK1 48.95 .+-. 4.3 50.51 .+-. 2.3 50.45 .+-. 5.2
51.58 .+-. 2.3 Apelin-17 32.50 .+-. 2.3 32.45 .+-. 2.6 32.67 .+-.
1.9 39.1 .+-. 2.4 BP554 22.30 .+-. 1.7 22.33 .+-. 2.3 22.50 .+-.
1.6 22.37 .+-. 1.5 Quinpirole 43.57 .+-. 4.1 45.39 .+-. 4.2 47.1
.+-. 3.2 48.7 .+-. 5.1
EXAMPLE 4
IS Subjects or Sujects at Risk of Developing IS can be Classified
According to their Response to GiPCR Stimulation in the Presence of
PTX
[0160] The effect of PTX on response to various selective agonists
of Gi-coupled receptors was tested in AIS endophenotype groups FG1,
FG2 and FG3 in four gene clusters.
[0161] The amount of functional Gi proteins was selectively
decreased by incubating osteoblasts with pertussis toxin (PTX) and
Melatonin. Results showed that treatment with PTX did not alter the
initial drop of the impedance response to melatonin in AIS groups
but dramatically reduced the positive component as well in control
as in AIS groups (FIG. 11A).
[0162] The concentration-response curve describing the maximum
impedance response showed that at low concentrations, PTX inhibited
the response to melatonin in control and AIS groups, while at high
concentrations, this treatment selectively increased response in
FG1 (FIG. 11B). These results support a relationship between the
divergent defective melatonin signaling and reduced Gi protein
activity among AIS groups and raise the possibility of a
compensatory Gi-independent signaling pathway in AIS patients
classified in FG1 group.
[0163] To determine if the reduced ability of Gi proteins to
promote signal transduction in AIS is restricted to melatonin
receptors, a comparative study using various agonists belonging to
the four previously identified receptor clusters (see Example 3) in
the presence of PTX was performed.
[0164] As shown in FIGS. 12A-12J, following treatment with PTX at
higher concentrations, only receptor agonists of clusters I and II
(FIGS. 12A-12E) elicited increased response in FG1 as observed
following melatonin receptor stimulation, while response to
receptor agonists of clusters III and IV in contrast, were
abolished in all groups (FIGS. 12F-12J). This supports the notion
of a compensatory Gi-independent signaling in FG1 but independent
of the receptor.
[0165] Based on these results, FG1 functional group could be easily
distinguished by their response to cluster I and cluster II
agonists in the presence of PTX. This test could advantageously be
used to stratify borderline subjects (which may not be identified
as belonging to the FG1 or FG2 functional group with sufficient
confidence (high specificity) using other known methods or other
GiPCR ligands.
[0166] Whether this signaling defect is confined in osteoblasts was
examined by extending the analysis on myoblasts and PBMCs. It was
found that both cell types exhibited a response pattern similar to
that obtained with osteoblasts following stimulation with each of
the tested compounds (data not shown). These findings are strongly
indicative that the defective Gi-mediated signaling is a
generalized impairment expressed in AIS.
EXAMPLE 5
Differential Effects of Gs and Gq Knockdown by SiRNA Method on the
Biphasic Impedance Signature of giPCR Agonists Among AIS Groups
[0167] All Gi.sub.1, Gi.sub.2, Gi.sub.3, Gs and scrambled siRNA
were obtained from Ambion (Ambion USA). The sequences used for gene
silencing are shown in supplemental Table 3. Osteoblasts from
control subjects and AIS patients were transiently transfected in
serum-free medium, using Lipofectamine RNAiMAX reagent (Invitrogen)
according to the manufacturer's instructions and functional
experiments were performed 48 h post transfection. The gene
knockdown was evaluated by quantitative real-time PCR (qPCR).
[0168] The possibility that the disparity in the shape of impedance
among AIS groups in response to GiPCR activation implicates a
component of Gs or Gq proteins-dependent response was then examined
For this purpose, the small interference RNA (siRNA) approach to
knockdown Gs or Gq examined For this purpose, the small
interference RNA (siRNA) approach was used to knockdown Gs or Gq
proteins prior to stimulate cells. Efficiency of siRNA was
confirmed by qPCR (FIG. 13A) and western blot analyses (FIG. 13B).
Results illustrated in FIGS. 14A-14P show that Gs or Gq protein
deletion has no effect on the impedance signature of GiPCR agonists
in control group (FIGS. 14A, 14E, 14I, 14M) for any agonist
cluster. In contrast, in FG1 (FIGS. 14B, 14F, 14J, 14N), the
negative phase was completely abrogated by the deletion of Gs
protein for clusters I and II and by the deletion of Gq protein for
cluster III, while the positive phase remained unaffected by these
deletions in all clusters. In FG2 subgroup (FIGS. 14C, 14G, 14K,
14O), the deletion of Gs protein was without effect on the negative
phase in any clusters, while deletion of Gq protein led to the loss
of negative phase without affecting the positive phase. Similar
observations were noticed in FG3 (FIGS. 14D, 14H, 14L, 14P). These
results suggest that Gs and Gq protein-dependent responses are
integrated in the biphasic impedance signature of GiPCR in AIS. It
appears that reduced Gi proteins associated with this disease
favours the functional duality of these receptors and that the dual
coupling to Gi and Gs protein is exclusively favoured in FG1.
[0169] The scope of the claims should not be limited by the
preferred embodiments set forth in the examples but should be given
the broadest interpretation consistent with the description as a
whole.
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