U.S. patent application number 14/993814 was filed with the patent office on 2016-05-05 for methods for the treatment or prevention of systemic sclerosis.
The applicant listed for this patent is Stichting Katholieke Universiteit. Invention is credited to Timothy Ruben Dirk Jan Radstake.
Application Number | 20160121039 14/993814 |
Document ID | / |
Family ID | 41130516 |
Filed Date | 2016-05-05 |
United States Patent
Application |
20160121039 |
Kind Code |
A1 |
Radstake; Timothy Ruben Dirk
Jan |
May 5, 2016 |
METHODS FOR THE TREATMENT OR PREVENTION OF SYSTEMIC SCLEROSIS
Abstract
The disclosure is in the field of molecular immunology, more in
particular, in the field of the prevention or treatment of
autoimmune diseases, more in particular, systemic sclerosis or
scleroderma. The disclosure is based on the observation that SSC
patients have an elevated plasma level of CXCL4. This was found to
contribute to the pathogenesis of SSc, in particular, fibrosis.
When CXCL4 was neutralized in in vitro experiments, the fibrotic
effects could be neutralized. This led us to conclude that SSc may
be cured by reducing the plasma level of CXCL4. The disclosure,
therefore, relates to a method for treatment or prevention of
fibrosis in patients with scleroderma, wherein the plasma level of
CXCL4 is reduced.
Inventors: |
Radstake; Timothy Ruben Dirk
Jan; (Nijmegen, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Stichting Katholieke Universiteit |
Nijmegen |
|
NL |
|
|
Family ID: |
41130516 |
Appl. No.: |
14/993814 |
Filed: |
January 12, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14471968 |
Aug 28, 2014 |
9259466 |
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14993814 |
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13928557 |
Jun 27, 2013 |
8920802 |
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14471968 |
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13138608 |
Sep 8, 2011 |
8486407 |
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PCT/EP2010/052919 |
Mar 8, 2010 |
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13928557 |
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Current U.S.
Class: |
210/695 |
Current CPC
Class: |
A61M 1/3618 20140204;
A61K 38/17 20130101; A61K 39/3955 20130101; C07K 16/18 20130101;
A61K 2039/505 20130101; C07K 16/24 20130101; A61K 39/39541
20130101; C07K 2317/76 20130101; A61P 17/00 20180101; A61P 37/06
20180101; C07K 16/2851 20130101 |
International
Class: |
A61M 1/36 20060101
A61M001/36 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 8, 2009 |
EP |
09154582.2 |
Claims
1.-15. (canceled)
16. A method for treating a subject diagnosed as suffering from
diffuse cutaneous systemic sclerosis, the method comprising:
depleting plasmacytoid dendritic cells in the subject, thereby
treating diffuse cutaneous systemic sclerosis in the subject.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/471,968, filed Aug. 28, 2014, pending,
which is a continuation of U.S. patent application Ser. No.
13/928,557, filed Jun. 27, 2013, now U.S. Pat. No. 8,920,802,
issued Dec. 30, 2014, which is a continuation of U.S. patent
application Ser. No. 13/138,608, filed Sep. 8, 2011, now U.S. Pat.
No. 8,486,407, issued Jul. 16, 2013, which application is a
national phase entry under 35 U.S.C. .sctn. 371 of international
Patent Application PCT/EP2010/052919, filed Mar. 8, 2010, published
in English as International Patent Publication WO 2010/102983 A1 on
Sep. 16, 2010, which claims the benefit under Article 8 of the
Patent Cooperation Treaty to European Patent Application Serial No.
09154582.2, filed Mar. 8, 2009.
TECHNICAL FIELD
[0002] The disclosure is in the field of molecular immunology, more
in particular, in the field of the prevention or treatment of
autoimmune diseases, more in particular, systemic sclerosis or
scleroderma.
BACKGROUND
[0003] Scleroderma or Systemic Sclerosis (SSc) is an autoimmune
connective tissue disorder of unknown cause characterized by
microvascular injury, excessive fibrosis of the skin, and
distinctive visceral involvement including the heart, lung, kidneys
and gastrointestinal tract. Scleroderma is a progressive condition
in which fibrous tissue grows abnormally, causing the skin to
thicken and harden, often disfiguring and disabling patients. It
affects between 5,000-10,000 new persons annually (USA) and is
associated with a high morbidity and a poor prognosis. Overall,
scleroderma affects an estimated 300,000 Americans, most of them
women.
[0004] Forty percent of all scleroderma patients develop at least
moderate restrictive lung disease. A high proportion of scleroderma
patients (80 percent) develop lung involvement, either interstitial
lung disease and/or pulmonary hypertension, which are the leading
causes of death due to scleroderma. The mortality rate in
scleroderma patients with severe restrictive lung disease is about
30 percent within 10 years of onset.
[0005] The statistics underscore the need for effective treatment,
preferably at an early stage in the illness, to prevent progression
to severe interstitial lung disease. SSc is treated with oral
medications to halt the progression of disease. These drugs include
cyclophosphamide, high-dose prednisolone or even stem cell
transplantation, all having severe side effects. In SSc, drugs that
improve circulation, promote gastrointestinal function, preserve
kidney function, and control high blood pressure are also given.
Cyclophosphamide is an anti-cancer drug that suppresses the immune
system. This is the first drug that has been proven to alleviate
the most devastating effects of SSc. Patients taking
cyclophosphamide had a significant improvement in lung function and
a reduction in breathlessness.
[0006] Although many research groups work on the unraveling of SSc
pathogenesis, to date, the exact pathways underlying the
pathogenesis of SSc remain unknown. Currently, most of the research
is focused on the underlying pathways that cause fibroblast
activation. A deregulated growth and activation of fibroblasts has
often suggested to be implicated in SSc. However, a mode of action
that is responsible for this ongoing activation of fibroblasts has
not been identified.
DISCLOSURE
[0007] The disclosure is based on the observation that SSC patients
have an elevated plasma level of CXCL4. This was found to
contribute to the pathogenesis of SSc, in particular, fibrosis.
When CXCL4 was neutralized in in vitro experiments, the fibrotic
effects could be neutralized. This led to the conclusion that SSc
may be cured by reducing the plasma level of CXCL4. The disclosure,
therefore, relates to a method for treatment or prevention of
fibrosis in patients with scleroderma, wherein the plasma level of
CXCL4 is reduced.
[0008] It was also found that CXCL4 was produced by pDCs in the
blood circulation of patients with SSc. A method as described above
is, therefore, preferably performed by depleting pDCs from the
circulation of a patient with scleroderma.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1: Increased frequency of plasmacytoid DCs in SSc
patients. Using the DC enumeration kit (Miltenyi Biotec), SSc
patients showed a clearly increased frequency of plasmacytoid DCs
(pDCs, BDCA2+), whereas the frequency of myeloid DCs (mDCs, BDCA1+
or BDCA3+) was similarly distributed among groups. Patients with
diffuse cutaneous SSc especially had a markedly raised frequency of
pDCs that was most marked in those having a disease duration
shorter than 2 years.
[0010] FIG. 2: The increased frequency of pDCs is most pronounced
in diffuse SSc. Using magnetic bead isolation of pDCs (BDCA4+
cells), it is clear that patients with diffuse cutaneous SSc have a
significantly increased frequency of pDCs compared to those having
the limited cutaneous phenotype. In turn, patients having early
diffuse (<2 years of disease duration) SSc have significantly
higher frequencies of circulating pDCs than those with late diffuse
disease (>3 years of disease duration).
[0011] FIG. 3: CXCL4 in pDC supernatant and plasma from SSc
patients is markedly increased. CXCL4 levels were measured using
ELISA techniques in pDC supernatant (Panel A) derived from healthy
controls and patients with limited, late diffuse and early diffuse
SSc. CXCL4 levels are increased in all SSc patients but more
pronounced in patients having diffuse SSc, especially those having
early diffuse cutaneous SSc. Panel B displays the CXCL4 levels in
the plasma of healthy controls and SSc patients. CXCL4 levels in
both pDC supernatant as well as plasma closely reflect the
frequency of pDCs in the circulation.
[0012] FIG. 4: CXCL4 directly stimulates fibroblasts. Although
unstimulated skin fibroblasts do not express high levels of COMP,
co-incubation with SSc plasma resulted in a clear up-regulation of
COMP expression that was abrogated by heparinase, a natural CXCL4
antagonist. In contrast, plasma from healthy controls did not
induce the up-regulation of COMP.
[0013] FIG. 5: The addition of CXCL12 to SSc pDCs induces a
self-perpetuating activation loop. Although the IL-6 production by
pDCs from healthy controls could only be induced by the
co-incubation with CXCL4+CXCL12, SSc pDCs produced large amounts of
IL-6 upon the addition of CXCL12. This effect could be fully
abrogated by the addition of heparinase underscoring the CXCL4
dependency in this system. The addition of CXCL4 only did not have
a significant effect, suggesting the need for CXCL12 in the
CXCL4-dependent pDC activation loop.
[0014] FIG. 6: pDCs from SSc patients secrete higher levels of Type
I IFNs upon TLR-mediated stimulation. Upon stimulation of pDCs with
ligands for TLR2, TLR3, TLR4 and TLR9, pDCs from SSc patients
produce significantly more type I IFN compared with healthy
controls. More specifically, SSc patients with diffuse cutaneous
disease showed a markedly increased secretion of type I IFN.
[0015] FIG. 7: The augmented TLR response by pDC in SSc is
abrogated by neutralization of PF4. The administration of
heparinase, a natural CXCL4 antagonist, fully abrogates the
augmented TLR response as observed in SSc patients.
DETAILED DESCRIPTION
[0016] It was found that the frequency of plasmacytoid DCs (pDCs)
is markedly increased in SSc patients. This is illustrated in FIG.
1. In particular, the frequency of pDCs was increased in patients
with the diffuse cutaneous subtype of SSc. The frequency of pDCs
was highest in patients suffering from early diffuse SSc. The
number of pDCs in individual SSc patients, as compared to normal
healthy controls, is shown in FIG. 2.
[0017] It was also found that plasma from SSc patients contains
high levels of CXCL4. CXCL4 is a chemokine described to bind to
CXCR3, more specifically, the splice variant CXC3b, which is in
contrast to CXLC9, CXCL10 and CXCL11 that bind to CXCR3a (Lasagni
et al., J. Experimental Medicine 2003). CXCL4 is known to induce
apoptosis in endothelial cells leading to endothelial cell death.
In addition, CXCL4 is known to have potent anti-angiogenic
properties and known to induce fibroblast activation via activation
of monocyte-derived dendritic cells.
[0018] CXCL4 was measured in a standard ELISA assay and the results
are illustrated in FIG. 3, Panel A. It is shown that CXCL4 was
overproduced by pDCs of SSc patients since elevated levels of CXCL4
could also be detected in the supernatant of pDCs isolated from
early diffuse SSc patients. This is illustrated in FIG. 3, Panel B.
It was noted that the production of CXCL4 in pDC supernatant
closely mirrored the frequency of pDCs in plasma. Since CXCL4 was
originally identified as a sole platelet activation marker, the
possibility was excluded that platelets contaminated the pDCs
supernatants by measuring the .beta.-TG content, which was
negligible.
[0019] Hence, it was concluded that CXCL4 is over-produced by pDCs
in patients with SSc. This was confirmed in a whole proteome
analysis using SELDI-TOF. Analysis of pDC revealed that SSc pDCs
were solely committed to produce a few proteins that could be
identified as CXCL4 or CXCL4-precursor molecules.
[0020] It was also shown that CXCL4 plays a role in fibroblast
activation in SSc patients. To that end, the expression of known
fibroblast activation markers TGF.beta.R, COMP and .alpha.SMA in
healthy fibroblasts co-incubated with SSc plasma was measured. It
was found that SSc plasma induced the expression of these markers
for fibroblast activation significantly and in a dose-dependent
manner (FIG. 4). In addition, the plasma from healthy donors had no
effect and the effect of SSc plasma was abrogated using heparinase,
a natural antagonist of CXCL4. It was concluded that an elevated
level of pDCs in Ssc patients contributes to the fibrotic
process.
[0021] CXCL4 on its own was found to be unable to stimulate pDCs to
produce pro-inflammatory molecules; neither pDCs from normal
individuals nor pDCs from SSc patients produced pro-inflammatory
molecules. Only in the presence of CXCL12-specific stimulation of
pDCs derived from SSc patients could be observed. This is
illustrated in FIG. 6. Therein, the production of pro-inflammatory
molecules was measured in the presence of pDCs from healthy
individuals as compared to pDCs isolated from SSc patients.
[0022] pDCs from healthy controls were incubated with plasma from
SSc patients and healthy controls, after which the production of
pro-inflammatory molecules was measured. The addition of plasma
from SSc patients and healthy controls had no effect. pDCs from
healthy individuals were incubated, as well as from SSc patients
with SSc plasma and healthy control plasma, with and without the
presence of CXCL12. It was found that only the combination of SSc
plasma and CXCL12 led to a significantly increased level of IL-6
and IL-12. To assure that this effect was due to the
"auto-activation" of pDCs by endogenous CXCL4, CXCL4 was added to
the cultures of pDCs from healthy individuals with healthy control
serum and CXCL12. The addition of CXCL4 to these experiments
induced IL-6 and IL-12 secretion to the same levels as the
combination of SSc plasma and CXCL12 (FIG. 5). In addition, the
addition of heparinase fully abrogated the effect of SSc plasma and
CXCL12 on pDCs.
[0023] Without wanting to be bound by theory, it was hypothesized
that CXCL12 (also known as SDF-1) binds to CXCR4, thereby
initiating chemo-attractive properties aimed at the recruitment of
pDC toward the site of inflammation. CXCL12 is known to be highly
presented in the skin of SSc patients and likely to be produced by
local fibroblasts. In support of this hypothesis, it is mentioned
that monocyte-derived DCs were found to be insensitive to CXCL4
unless CXCL12 is present. It is currently unknown how CXCL12
sensitizes DCs to the effect of CXCL4 (Cipriani et al., Arthritis
Rheum. 2006 Sep., 54(9):3022-33).
[0024] It is known that pDCs are stimulated mostly via toll-like
receptor 9 (TLR9), via which they mainly make type I interferons
(IFNs). As such, pDCs are considered the main producers of this
cytokine.
[0025] Challenged by the question "what induces the increased CXCL4
production by pDCs," the role of toll-like receptors (TLR) was
investigated. These experiments showed that TLR3-, TLR7/8- and
TLR9-mediated stimulation induced CXCL4 secretion by pDCs.
Interestingly, DCs from SSc patients were found to produce much
more IFNs and IL6 than those from healthy controls (FIG. 6). A
clearly increased secretion of IFN.alpha. in lcSSc patients, ldSSc
patients and edSSc patients was observed.
[0026] This shows that the potentiated TLR response by pDCs is
caused by a CXCL4-dependent TLR augmentation. In support of this,
neutralization of CXCL4 led to a full abrogation of this TLR
augmentation, showing that CXL4 underlies this phenomenon (FIG.
7).
[0027] To further substantiate these findings, pDCs from healthy
controls were incubated with CXCL12, CXCL4 or its combination dose
dependently. These experiments demonstrated that CXCL4 in
combination with CXCL12 led to an augmentation of the TLR
response.
[0028] In conclusion, these observations show that CXCL4 (in
conjunction with CXCL12) plays a central role in the pathogenesis
of SSc. The mechanism for that is auto-activation of pDCs resulting
in a potentiated TLR response and further CXCL4 production.
[0029] Patients with SSc may, therefore, benefit from methods that
remove CXCL4 and/or pDCs, for instance, from the blood circulation.
That may be accomplished in a number of ways, which are known in
the art per se.
[0030] Patients with scleroderma may benefit from a treatment
wherein the effects of the CXCL4 produced in excess are neutralized
or counteracted, for instance, by removing CXCL4 from circulation.
In the alternative, CXCL4 may be prevented from binding to its
receptor. The disclosure, therefore, relates to a compound capable
of binding to CXCL4 for use in the treatment or prevention of
fibrosis in patients with scleroderma.
[0031] Compounds capable of binding to CXCL4 may be compounds
capable of binding to CXCL4 under physiological conditions.
[0032] Compounds capable of binding to CXCL4 per se are known in
the art. For instance, fragments of the receptor or fragments of
CXCL4 will be suitable compounds. In a preferred embodiment,
specific antibodies may be employed in order to remove CXCL4, for
instance, from the circulation. This may be accomplished by
plasmapheresis.
[0033] Antibodies against CXCL4 are available in the art. They have
been described, for instance, in Vandercapellen et al., J.
Leukocyte Biol. 82, 1519 (2007), and are commercially available
from R&D Systems, 614 McKinley Place NE, Minneapolis Minn.
55413.
[0034] Given the teachings of this disclosure, it will be within
the routine skills of a person skilled in the art to design and
develop materials and methods for removing or counteracting the
effects of CXCL4 and/or DCs such that patients with SSc may benefit
from these materials and methods.
[0035] For that purpose, CXCL4 antagonists may be advantageously
employed. Antagonists for CXCL4 are known to the skilled person. As
an example, heparinase is disclosed and exemplified herein.
[0036] In another embodiment, the disclosure also relates to a
compound capable of interfering with the in vivo production of
CXCL4 for treatment or prevention of fibrosis in patients with
scleroderma. Compounds capable of interfering with the in vivo
production of CXCL4 per se are known in the art. The disclosure,
therefore, also relates to a method for treatment or prevention of
fibrosis in patients with scleroderma by interfering with the in
vivo production of CXCL4. Such methods may encompass interference
at the level of transcription or translation of CXCL4. Such methods
are known in the art and may now advantageously be used in the
treatment of scleroderma.
[0037] A reduction in the level of CXCL4 in a patient's body may be
accomplished by a therapy in vivo. Alternatively, patients with SSc
may also benefit from in vitro methods that reduce the level of
CXCL4 in blood. Such may be done directly by applying a compound
capable of binding to CXCL4 and contacting that compound with blood
obtained from a patient with SSc. It may also be accomplished by
reducing the number of DC that produce the CXCL4 in patients with
scleroderma. This may also be accomplished in an in vitro method
wherein the previously isolated blood or plasma from a patient with
scleroderma is contacted with a compound or device capable of
reducing or depleting the number of pDCs in solution. The
disclosure, therefore, also relates to an in vitro method for
reducing the level of CXCL4 and/or the number of pDCs in blood
obtained from a patient with scleroderma. In more detail, the
disclosure relates to an in vitro method for reducing the level of
CXCL4 and/or the number of pDCs in a sample obtained from a patient
with scleroderma, comprising the steps of: [0038] a. providing the
sample obtained from a patient with scleroderma [0039] b.
contacting the sample with a compound capable of binding to CXCL4
[0040] c. allowing the compound to bind to CXCL4 in order to form a
complex [0041] d. removing the complex from the sample, thereby
reducing the level of CXCL4 and/or the number of pDCs.
[0042] Devices capable of reducing the level of CXCL4 and/or pDCs
in blood are known in the art. Preferably, CXCL4 and/or pDCs are
removed from circulation, for instance, using a commercially
available technique provided by Miltenyi (Jongbloed et al.,
Arthritis Res. Ther. 2006, 8(1):R15).
[0043] Miltenyi produces and markets such a device that may be
advantageously used in the new treatment according to the
disclosure. Hence, the disclosure relates to a device capable of
reducing the level of CXCL4 and/or pDCs in blood for the treatment
or prevention of fibrosis in patients with scleroderma.
[0044] Alternatively, a method according to the disclosure may also
comprise a step wherein the binding of CXCL4 to its receptor is
blocked or otherwise prevented or down-regulated or decreased. The
disclosure, therefore, also provides a method for treatment or
prevention of fibrosis in patients with scleroderma by interfering
with the binding of CXCL4 to its receptor.
EXAMPLES
Example 1: Isolation of PBMCs, Plasmacytoid DCs and Phenotypic
Characterization
[0045] PBMCs were isolated from heparinized venous blood (100 ml)
by using density-gradient centrifugation over FICOLL-PAQUE.RTM.
(Amersham Bioscience). Next, plasmacytoid DCs (pDCs) were isolated
using the BDCA4 magnetic isolation kit from Miltenyi Biotec
according to the manufacturer's protocol. All procedures were
carried out on ice and after the isolation of pDCs, these cells
were counted using counterchambers and directly transferred to RPMI
medium without any additions in a concentration of 1 million
cells/ml. After pelleting, the supernatant was collected and stored
at -80.degree. C. until further analysis. The cells were then
resuspended in RPMI supplemented with 2 nM L-glutamine, 100
U/.mu.L/ml penicillin/streptomycin (Life Technologies), and 10% FCS
(BioWhittaker), after which cells were brought into culture for
further analysis. Directly after isolation, a part of the BDCA4+
cells were resuspended in FACS buffer and were analyzed for the
expression of BDCA1, BDCA2, BDCA3, CD80, CD86, MHC and Fc.gamma.Rs
applying standard flow cytometry protocols. During the first
experiments, the pDCs diamond kit from Miltenyi Biotec was
exploited to replicate the data having so-called "untouched pDCs."
In all experiments, direct DC enumeration was performed using the
DC enumeration kit from Miltenyi Biotec according to the
manufacturer's protocol.
Example 2: Measurement of inflammatory mediators
[0046] Supernatant from unstimulated pDCs was stored after 1 and 24
hours of culture. In addition, pDCs were stimulated with ligands
specific for toll-like receptor 2 (TLR2, pam3Cys), TLR3 (Poly-IC),
TLR4 (LPS), TLR7/TLR8 (R848) and TLR9 (CPG) for 24 hours, after
which, supernatant was collected. In all these supernatants, the
levels of multiple inflammatory mediators (IL-8, TNF, IFN.gamma.,
IFN.alpha., MCP-1, MIP1.beta., IL-6, IL-1.beta., IL-12. IL-23,
IL-17F, IL-17A) were measured using Luminex technology as
previously described (Roelofs et al., Arthritis Rheum. 2005 Aug.,
52(8):2313-22; Roelofs et al., Ann. Rheum. Dis. 2009 Sep.,
68(9):1486-93, Epub 2008 Sep. 2; Wenink et al., J. Immunol, 2009
Oct. 1, 183(7):4509-20, Epub 2009 Sep. 4). To study the full
proteome in pDCs, the 1-hour spontaneous supernatants were used for
analysis in the SELDI-TOF. Exploiting this technique, 40
supernatants were tested originating from healthy controls (n=10)
and SSc patients having limited cutaneous (n=10), late diffuse
(n=10) and early diffuse (n=10) disease as previously described
(Radstake et al., PLos ONE 2009). The expression of CXCL4 and
.beta.-TG was measured by commercially available ELISAs used
following the manufacturer's protocols.
Example 3: Stimulation of Plasmacytoid DCs and Investigating the
Self-Perpetuating Loop
[0047] Since the literature suggests an important role for CXCL12
in the CXCL4-mediated activation of pDCs, the potential role of
this loop in SSc was investigated (van der Vliet et al., J.
Experimental Medicine 2003 Sep. 1, 198(5):823-3). To this aim, pDCs
from healthy controls and SSc patients were first co-cultured with
combinations of CXCL4 and/or CXCL12. During these experiments, pDCs
were cultured with RPMI only, RPMI/CXCL12, RPMI/CXCL12/CXCL4 and
RPMI/CXCL12/heparinase. In addition, the effect of CXCL4 on the TLR
pathways was investigated by pre-incubation of pDCs with CXCL4 or
the combination CXCL4/CXCL12, after which, pDCs were consecutively
stimulated with TLR ligands. After 24 hours of incubation, the
production of IFN.alpha., IL-12 and IL-6 was measured by
Luminex.
Example 4: CXCL4-Mediated Activation of Fibroblasts
[0048] Skin fibroblasts were isolated from healthy skin biopsies
and cultured according to standardized protocol as described
previously (Farina, Ann. Rheum. Dis. 2009 Mar., 68(3):435-41). When
confluent, fibroblasts were replated and cultured for an additional
24 hours to allow attachment to the culture plates. Subsequently,
fibroblasts were incubated with combinations of plasma from healthy
controls and SSc patients, plasma and heparinase and CXCL4/CXC12.
After 48 hours of culture, the expression of COMP was measured by
real-time PCR as previously described (Farina, Ann. Rheum. Dis.
2009 Mar., 68(3):435-41).
* * * * *