Inhibition of c-kit tyrosine kinase by imatinib mesylate
induces apoptosis in mast cells in rheumatoid synovia: a
potential approach to the treatment of arthritis
A Juurikivi*, C Sandler*, K A Lindstedt, P T Kovanen, T Juutilainen, M J Leskinen, T Ma ¨ki, K K Eklund
See end of article for
Dr K K Eklund, Division of
Hospital, Kasarmikatu 11–
13, 00130 Helsinki,
28 December 2004
Ann Rheum Dis 2005;64:1126–1131. doi: 10.1136/ard.2004.029835
Background: Mast cells have been implicated in the pathogenesis of arthritis, but elucidation of their
precise role has been hampered by a lack of efficient and selective inhibitors of their function.
Objective: To elucidate the role of mast cells in the pathogenesis of rheumatoid arthritis (RA) and to assess
whether apoptosis of cultured and synovial tissue mast cells can be induced by inhibiting mast cell growth
factor receptor, c-kit tyrosine kinase.
Methods and results: Double staining with tumour necrosis factor (TNF) a and tryptase antibodies showed
the presence of TNFa positive mast cells in human rheumatoid synovial tissue. Selective activation of mast
cells by anti-IgE resulted in production of TNFa in synovial tissue cultures. Inhibition of the c-kit tyrosine
kinase with imatinib mesylate (1.0–10 mmol/l) induced profound apoptosis in cultured mast cells as
judged by typical apoptotic morphology, increased number of apoptotic nucleosomes, and activation of
caspases 8 and 9. Importantly, imatinib also induced apoptosis of mast cells in explant cultures of synovial
tissue obtained from patients with RA as judged by a TUNEL assay. Inhibition of c-kit tyrosine kinase was
accompanied by significant reduction of TNFa production in synovial tissue cultures.
Conclusion: Mast cells may have a role in the pathogenesis of RA, and inhibition of c-kit may be a new
means of inhibiting mast cell activity and of abrogating the contribution of mast cells to synovial
inflammation in RA.
human rheumatoid synovium, and their number correlates
with the activity of the disease.2In addition, strong evidence
has indicated that mast cells have an important role in the
pathogenesis of experimental arthritis. In an autoantibody
induced mouse model of arthritis, infusion of serum from an
autoimmune mouse into normal mice containing mast cells
induced erosive arthritis, whereas almost no signs of inflam-
mation were seen in mast cell deficient mice (W/Wv).3
Susceptibility to autoantibody induced arthritis could be
restored in the W/Wvmice by mast cell engraftment.3The
mechanisms by which mast cells may play a part in the
pathogenesis of arthritis involve secretion of potent proinflam-
matory cytokines, such as tumour necrosis factor (TNF) a,
which has been implicated in the pathogenesis of arthritis, as
well as secretion of other mediators of inflammation and tissue
destruction, such as matrix metalloproteinases.4
For the growth and survival of human mast cells, stem cell
factor (SCF; also named c-kit ligand, steel factor, or mast cell
growth factor) is essential.5SCF is the ligand for the c-kit
receptor (c-kit, CD117), which is a transmembrane glyco-
protein and a member of the receptor tyrosine kinase
subclass III family.6–8c-Kit is expressed by haematopoietic
progenitor cells, germ cells, and some human tumour cells,
but of the mature immune system cells, only mast cells
express c-kit in significant numbers.9 10
Because c-kit tyrosine kinase is essential for the growth
and survival of human mast cells, the inhibition of c-kit
signalling might prevent mast cell activity in the synovium.
Imatinib mesylate (also known as STI-571 or Gleevec) is a
relatively specific inhibitor of tyrosine kinases, including
ncreasing evidence suggests that mast cells have a role in
the pathogenesis of rheumatoid arthritis (RA).1Thus,
increased numbers of mast cells have been found in the
ABL, BCR-ABL, platelet derived growth factor (PDGF)
receptor tyrosine kinases, and c-kit. Originally, imatinib
mesylate was used in the treatment of chronic myeloid
leukaemia11 12and later also in the treatment of certain c-kit
positive tumours.13The results of a recent preliminary clinical
study,14and also a recent case report,15suggest that imatinib
mesylate may possess considerable antirheumatic efficacy.
The mechanisms underlying the antirheumatic effects of
imatinib mesylate are not known, but may involve the
inhibition of mast cell activity. Therefore, we have studied the
role of mast cells in RA and the effects of imatinib mesylate
on cultured mast cells and on mast cells residing in synovial
Human mast cell leukaemia cell line (HMC-1) cells express
constitutively activated mutant c-kit polypeptide.16 17HMC-1
cells were cultured in Iscove’s modified Dulbecco’s medium
(Gibco, BRL) supplemented with 10% fetal calf serum (FCS;
Gibco, BRL), 1% non-essential MEM amino acids (Gibco,
BLR), 0.5% penicillin-streptomycin (Gibco, BRL), and 0.004%
Mouse bone marrow derived mast cells (mBMMC) were
obtained by culturing bone marrow cells from the femurs and
tibias of BALB/c mice for 3 weeks in enriched culture
medium (RPMI 1640 containing 10% FCS (Gibco, BRL), 1%
non-essential MEM amino acids (Gibco, BRL), 0.5% peni-
cillin-streptomycin (Gibco, BRL), and 0.004% monothio-
glycerol (Sigma, St Louis USA) supplemented with 50%
Abbreviations: ELISA, enzyme linked immunosorbent assay; FCS, fetal
calf serum; HuMC, human mast cells; IL, interleukin; mBMMC, mouse
bone marrow derived mast cells; PDGF, platelet derived growth factor;
RA, rheumatoid arthritis; SCF, stem cell factor; TNF, tumour necrosis
factor; WCM, WEHI-conditioned media
*The first two authors contributed equally to the study.
WEHI-3B-conditioned medium.18The human mast cells
(HuMC) were derived from cord blood mononuclear cells as
described previously.19HuMC were cultured in Iscove’s
modified Dulbecco’s medium supplemented with 10% FCS.
During the first 1–3 weeks the cells were cultured in the
presence of 80 ng/ml human recombinant SCF, 50 ng/ml
interleukin (IL) 6, and 10 ng/ml IL10 (R&D Systems), in the
following 4–8 weeks in the presence of 80 ng/ml SCF, and
after 8 weeks in the presence of 80 ng/ml SCF and 10 ng/ml
Cell viability and proliferation
Cell viability was assessed by directly counting the cells after
trypan blue staining. HMC-1 cells were cultured at a density
of450 000 cells/ml andmBMMC
250 000 cells/ml. The cells were incubated at 37˚C and trypan
blue-excluding cells were counted 5 days after addition of
Apoptosis assays and immunohistochemistry
The apoptosis of HMC-1 cells and HuMC was determined by
measuring the amount of apoptotic nucleosomes after 20–
24 hours’ incubation at 37˚C in the presence of different
concentrations of imatinib mesylate with an enzyme linked
immunosorbent assay (ELISA) assay kit according to the
manufacturer’s instructions (Roche Diagnostics). The enzy-
matic activities of caspases 3 and 9 were measured in HMC-1
cells after incubation of HMC-1 cells with imatinib mesylate
for 6 hours using a fluorometric assay kit according to the
results were adjusted according to the protein content of
the samples. Each experiment was carried out in triplicate.
Apoptosis in cryosections of synovial tissue was examined
with the ApopTag apoptosis detection kit. Mast cells were
identified by staining for tryptase using mouse antihuman
mast cell tryptase (clone AA1, Dako) and mouse elite kit
(Vector Laboratories) or Alexa-fluor (594 or 488) goat
antimouse antibodies (Molecular Probes). TNFa was detected
with polyclonal goat antibodies against TNFa (Santa Cruz)
and Alexa-fluor (594) rabbit antigoat antibodies (Molecular
Probes). Specificity was controlled by omitting the primary
Synovial tissue culture
Synovial tissue was obtained after informed consent during
knee synovectomy or arthroplasty of seven patients fulfilling
the revised criteriaof
Rheumatology.20The study was approved by the ethical
committee of surgery of the Helsinki University Central
Hospital. Fat, bone, cartilage, and fibrous tissues were
removed and synovial tissue was cut into small pieces of
approximately 2 mm3each. Thereafter pieces were randomly
picked, weighed, placed into wells of a 12 well plate (18–20
explants, approximately 300 mg tissue per well), and the
exact total weight of synovial tissue in each well recorded.
After washing with Hanks’s buffered salt solution, the
explants were transferred into 1.5 ml of Dulbecco’s modified
Eagle’s medium. After incubation for 1 hour with imatinib
mesylate, rabbit IgG antibody for human IgE (150 mg/ml/
well) or control non-specific rabbit IgG (150 mg/ml/well;
Dako) was added to activate the mast cells. Cultures were
incubated at 37˚C in 5% CO2for 20–24 hours. To verify the
activation, a sample was taken for histamine assay after
incubation for 1 hour and stored at 220˚C. After incubation
for 20–24 hours, the explants were embedded in the OCT
compound containing mould, which was then frozen in
the AmericanCollege of
Imatinib mesylate concentration (µmol/l)
Number of viable cells
(2.5 × 105/ml)
101 0.10.01 Control
Imatinib mesylate concentration (µmol/l)
Number of viable cells
00.01 0.11 10
Imatinib mesylate concentration (µmol/l)
Apoptotic nucleosomes (A)
CAM101 0.1 0.01Control
Imatinib mesylate concentration (µmol/l)
1 0.1 0.010
imatinib mesylate for 5 days. Imatinib was added on day 0 and trypan blue-excluding viable cells were counted on day 5. Mean (SEM) of four
experiments. (B) Imatinib induces apoptosis of mBMMC only when cultured in the presence of SCF. mBMMC were cultured in the presence of SCF or IL3
(WEHI-conditioned media (WCM)) and imatinib (0.01–10 mmol/l) for 5 days, and the number of trypan blue excluding cells was assessed (n=4).
(C) HMC-1 cells were incubated with the indicated concentrations of imatinib and the amount of apoptotic nucleosomes was measured 24 hours later
with an ELISA. Mean (SEM) of four absorbance readings shown. Positive control, camphotericin (CAM) is a strong inducer of apoptosis. (D) Caspase
activation by imatinib. HMC-1 cells were incubated with indicated concentrations of imatinib for 6 hours and the activity of caspase 3 and 9 was
measured by a fluorometric assay. Mean (SEM) of three experiments. *p,0.05 compared with controls, results analysed with analysis of variance for
Imatinib induces apoptosis of cultured mast cells by inhibiting c-kit. (A) Human mast cell line (HMC-1) cells cultured with increasing doses of
Mast cell apoptosis in rheumatoid synovia 1127
liquid nitrogen and stored at 270˚C. Culture media were
aliquoted and stored at –20˚C for later analysis. The amount
of TNFa released was measured in the supernatants by an
ELISA (R&D Systems) according to the manufacturer’s
The overall significance of differences between experimental
groups was analysed with Friedmann’s non-parametric
repeated measurements analysis of variance (InStat 3 for
Macintosh, Graph Pad software Inc). The differences were
considered to be significant for p,0.05, in which case the
differences between individual experiment groups were
further tested with Dunn’s multiple comparisons test. The
data are shown as means (SEM).
Inhibition of c-kit tyrosine kinase induces apoptosis of
cultured mast cells
In the presence of increasing concentrations of the c-kit
inhibitor, imatinib mesylate, the number of cultured trans-
formed human mast cell line cells (HMC-1) was significantly
decreased (fig 1A). A considerable decrease in cell numbers
was already seen after 5 days of culture in the presence of
imatinib mesylate (0.1 mmol/l), and at concentrations of 1
and 10 mmol/l imatinib mesylate only a few living cells were
detected. Similarly, in the presence of an increasing
concentration of imatinib mesylate, DNA synthesis, mea-
sured as the incorporation of [3H]thymidine into HMC-1
cells, was decreased (data not shown).
To show that the effects of imatinib mesylate were
mediated through c-kit, we next studied mBMMC. The
viability of mBMMC can be sustained either with SCF (c-kit
dependent pathway) or with IL3 (IL3 dependent pathway).21
mBMMC generated in IL3 containing WEHI-conditioned
media (WCM) were taken and, after washing, half of the
cells were further cultured in the presence of SCF and the
other half in the presence of IL3 containing WCM; increasing
concentrations of imatinib mesylate were added to both
cultures. When the mBMMC were cultured in the presence of
SCF and imatinib mesylate, there was a strong reduction in
the cell number at 1 mmol/l and higher imatinib concentra-
tions (fig 1B). In contrast, when mBMMC were cultured in
the presence of IL3, imatinib mesylate did not significantly
reduce the cell number, suggesting that imatinib mesylate
specifically affected the c-kit dependent survival pathway.
The experiment also shows that imatinib mesylate, in
addition to inhibiting the c-kit in HMC-1 cells, inhibits the
c-kit tyrosine kinase of normal mouse mast cells.
To verify that the observed reduction in cell number was
caused by apoptosis, the amount of apoptotic nucleosomes in
HMC-1 cells treated with imatinib mesylate was measured. A
concentration of imatinib mesylate as low as 0.1 mmol/l
induced a significant increase in the amount of apoptotic
nucleosomes (fig 1C). The number of apoptotic nucleosomes
at an imatinib concentration of 1 mmol/l was higher than in
the presence of camphotericin (5 mg/ml), which was used as
a positive control and which is a strong inducer of apoptosis
(fig 1C). The induction of apoptosis in HMC-1 cells by
imatinib mesylate was further verified using additional
methods, including annexin binding and TUNEL staining,
which confirmed the finding (data not shown). Furthermore,
the inhibition of c-kit tyrosine kinase by imatinib mesylate
(0.1 and 1 mmol/l) in HMC-1 cells resulted in significant
activation of both caspase 3 and caspase 9 (fig 1D). Thus,
inhibition of SCF/c-kit signalling in HMC-1 cells activates a
metabolic cascade, which induces cell death by apoptosis.
Because c-kit is constitutively activated in HMC-1 cells
owing to a dominant mutation,16we wanted to verify that
imatinib mesylate can induce apoptosis in normal human
mast cells, in which the c-kit mediated cell survival pathway
is dependent on the presence of SCF. Furthermore, it is not
known whether the presence of other cytokines, besides IL3
in mouse mast cells, can rescue human mast cells from c-kit
inhibitor induced apoptosis. When HuMC were cultured in
the presence of SCF and an increasing concentration of
imatinib mesylate, the number of apoptotic nucleosomes was
significantly increased (fig 2A). The induction of human
mast cell apoptosis by imatinib mesylate was further verified
by studying nuclear fragmentation (insert in fig 2A), TUNEL
staining (fig 2B), and annexin binding (data not shown).
Interestingly, in the presence of another cytokine (IL6), in
addition to SCF, HuMC are more resistant to apoptosis
Mast cell activation increases levels of TNFa in
The above results show that imatinib mesylate mediated
inhibition of c-kit tyrosine kinase can induce apoptosis in
cultured mBMMC, HMC-1 cells, and huMC. However, in
inflamed synovial tissues there are also other potential
cytokine and growth factor pathways in addition to the
(A) HuMC were cultured for 20–24 hours in the presence of either SCF
or SCF and IL6. Thereafter, the amount of apoptotic nucleosomes was
assessed with an ELISA. Data represent mean (SEM) amounts of
apoptotic nucleosomes as absorbance readings of three experiments.
*p,0.05 compared with controls. The insert shows the typical apoptotic
morphology of imatinib treated HuMC. (B) HuMC were cultured in SCF
and with imatinib (10 mmol/l) for 24 hours. Cytospin slides were made
and apoptosis was studied using the TUNEL assay. The green colour in
the control panel and imatinib treated panel show the TUNEL positive
cells. The blue colour (DAPI (49, 69-diamino-2-phenylindole-
dihydrochloride) stain) demonstrates the presence of equal numbers of
cells. Results of two experiments are shown.
Imatinib induces apoptosis of non-malignant HuMC.
1128Juurikivi, Sandler, Lindstedt, et al
shows mast cells in synovial tissue as brown staining cells. (B) Synovial tissue stained with anti-tryptase antibody and visualised with fluorescent label
(green colour). (C) Synovial tissue stained with anti-TNFa antibodies (red). (D) When anti-tryptase and anti-TNFa stainings are merged, the resulting
yellow colour shows the cells that are positive for both anti-tryptase (mast cells) and TNFa. Representative stainings of three experiments are shown.
TNFa positive mast cells can be seen in the synovial tissue of control explant cultures. (A) Synovial tissue stained with anti-tryptase antibody
Histamine release (ng/mg wet tissue)
TNFα production (% of anti-IgE stimulated)
concentrations of imatinib were added to synovial tissue cultures. Mast cells were activated by adding anti-IgE. Addition of non-specific IgG was used
as a control for the specificity of anti-IgE. (A) TNFa produced by the synovial tissue culture was measured 24 hours after activation of mast cells with an
ELISA. Release of TNFa is shown as the percentage change from the TNFa produced by the IgE activated sample. (B) Histamine released from mast cells
measured 1 hour after addition of anti-IgE. Mean (SEM) of results obtained from four patients is shown. *p,0.05 compared with the activated control.
The specific activation of mast cells in synovial tissue results in the production of TNFa, which is partly inhibited by imatinib. The indicated
Mast cell apoptosis in rheumatoid synovia1129
SCF/c-kit pathway, which can support the viability of tissue
mast cells. Furthermore, during continuing inflammatory
reactions, mast cells are likely to be activated, which renders
them less susceptible to apoptosis. Therefore, we next studied
synovial tissue obtained from the knee joints of patients with
RA. The inflamed synovial tissue contained a large number of
tryptase positive mast cells (brown-reddish stained (fig 3A)
or green when stained with fluorescent secondary antibodies,
fig 3B). The synovial tissue also showed TNFa positive
cellular staining (fig 3C), which was colocalised to tryptase
positive cells—that is, to mast cells (fig 3D). It is worth
noting that the strong TNFa positive staining in mast cells, as
compared with other TNFa producing cells, notably macro-
phages, is due to the ability of mast cells to store TNFa in
their intracellular granules.
To study the effect of mast cell activation in synovial tissue,
anti-IgE, a specific mast cell activator, was added to the
synovial tissue in culture. Significant release of TNFa (0.65,
1.41, 14.3, and 26.0 pg/mg wet tissue) was seen in all the
samples studied 24 hours after mast cell activation with anti-
IgE, as compared with control synovial tissue, which did not
release detectable amounts of TNFa (fig 4A). Importantly, in
the presence of increasing amounts of imatinib mesylate,
decreasing amounts of TNFa were released (fig 4A). One
hour after the addition of anti-IgE, an increased amount of
histamine was also detected in the incubation media,
indicating mast cell activation. The amount of TNFa and
histamine released upon mast cell activation correlated also
with each other (Spearman’s correlation, rs=0.73). However,
in contrast with TNFa, imatinib did not inhibit IgE induced
acute histamine release (fig 4B). Therefore we studied the
histamine release also in cultured mast cells, and the results
showed that imatinib had no clear dose dependent effect on
IgE induced histamine release in huMC, or compound 48/80
induced histamine release in rat peritoneal mast cells (data
not shown). The above results show that selective activation
of synovial mast cells increases TNFa levels in synovial tissue,
and this can be partly inhibited by imatinib mesylate,
probably through induction of mast cell apoptosis.
Inhibition of c-kit tyrosine kinase results in apoptosis
of synovial tissue mast cells
Finally, we studied whether imatinib mesylate could induce
apoptosis of mast cells in synovial tissue. In untreated
synovial tissue obtained from a patient with RA, only
occasional apoptotic mast cells were observed (fig 5D-F).
However, after incubation of the synovial tissue for 24 hours
in the presence of imatinib mesylate (1 mmol/l), several
apoptotic cells could be seen, most of which were mast cells
(fig 5A and B). Most mast cells in the rheumatoid synovial
tissue were rendered apoptotic during the incubation with
imatinib mesylate (fig 5C). These experiments show that
inhibition of c-kit tyrosine kinase induces apoptosis not only
in cultured mast cells but also in those mast cells which
reside in RA synovial tissue.
TNFa has a central role in the pathogenesis of RA, which is
demonstrated by the good clinical efficacy of anti-TNFa
treatments.22Here we show that mast cells, in addition to
monocytes/macrophages, are also a significant source of
TNFa in the synovial tissue. Indeed, selective IgE mediated
activation of synovial mast cells resulted in the production
and release of significant amounts of TNFa in synovial tissue.
IgE mediated activation of mast cells was used here as it
offers an opportunity to activate mast cells specifically, and
the purpose of these experiments was to show that mast cells
can bring about significant inflammatory response. In
rheumatoid synovia it is more likely that mast cells are
activated by mechanisms other than IgE mediated activation
such as by immune complexes and complement components,
C5a in particular.23The results of the present study and other
immunohistochemical studies on synovial tissue24indicate
that mast cells in RA synovium are in an activated state.
Thus, irrespective of the way in which this activation occurs,
these findings strongly support the view that mast cells do
contribute significantly to the production and secretion of
TNFa in RA, and, thus may have a significant role in the
pathogenesis of this disease.
Inhibition of c-kit tyrosine kinase with imatinib mesylate
induced apoptosis in HMC-1 cells, which suggests that the
cells harbour the mutation at the codon 560 of the c-kit
receptor, shown to be sensitive to imatinib.25Inhibition of
c-kit by imatinib induced apoptosis also in normal cultured
mouse and HuMC. The apoptosis was c-kit dependent, as no
apoptosis occurred in mBMMC in the presence of IL3 only.
Moreover, apoptosis occurred at low imatinib mesylate
concentrations, corresponding to those typically found in
patients treated with this drug.26The sensitivity of cultured
mast cells to imatinib mesylate has been observed pre-
viously.27–29However, here we show for the first time that
apoptosis of mature mast cells cultured with cytokines other
than SCF and, importantly, apoptosis of mast cells residing in
synovial tissue, can be induced by inhibiting c-kit. This
the presence of imatinib mesylate (1 mmol/l). The appearance of apoptotic cells was studied 24 hours later, using a TUNEL assay. The green colour
indicates apoptotic cells and red the tryptase positive mast cells. When the anti-tryptase and TUNEL pictures are merged, the apoptotic mast cells
appear as red cells with a yellow spot. The arrows indicate individual apoptotic mast cells. Synovial tissue from four separate patients with RA was
analysed with a TUNEL assay, and representative samples are shown.
Inhibition of c-kit by imatinib induces apoptosis of mast cells in synovial tissue. Synovial tissue obtained from patients with RA was cultured in
1130Juurikivi, Sandler, Lindstedt, et al
finding is important as in synovial tissue, and in inflamed Download full-text
synovial tissue in particular, the mast cells are exposed to a
myriad of cytokines and growth factors capable of supporting
mast cell growth.
Recently, the results of a pilot clinical study suggested that
imatinib mesylate may have significant antirheumatic
activity.14One of the patients who participated in the initial
clinical study has now continued treatment with imatinib for
24 months and the disease activity of his RA has remained
very low (Eklund et al, unpublished results). In addition, a
recent case report described a patient with chronic myeloid
leukaemia and concomitant RA whose RA symptoms clearly
improved during imatinib treatment.15The reason for this
potentially antirheumatic activity of imatinib is not clear, but
it might be related to the inhibition of the synoviocyte PDGF
receptor tyrosine kinase or the inhibition of c-kit in synovial
tissue or it might be mediated by inhibition of some as yet
unidentified tyrosine kinase.
We are currently studying the effect of inhibiting synovio-
cyte PDGF receptor on the growth and proliferation of
synoviocytes. The only c-kit positive cells in synovial tissue
have been shown to be mast cells.30Here we show that the
function of synovial mast cells can be inhibited and their
apoptosis induced by inhibiting c-kit. As mast cells are
involved in many aspects of the inflammatory reaction, it is
conceivable that inhibition of c-kit, resulting in inhibition of
mast cell activity, might result in attenuation of the
inflammatory reaction in arthritic joints.
If the imatinib mesylate induced apoptosis and depletion of
tissue mast cells can also be accomplished in vivo, it might
represent a new means of examining the significance of mast
cells in RA. In addition, induction of mast cell apoptosis
through inhibition of c-kit might be the basis for new
targeted treatments of RA and of other diseases associated
with inappropriate mast cell activity.
The expert technical help of Jaana Tuomikangas, Suvi Ma ¨kinen, and
Mari Jokinen is greatly appreciated.
This study was supported by the grants from Finnish Medical
Foundation (KKE) and EVO research funds. Imanitib mesylate was
kindly provided by Novartis Pharma.
A Juurikivi, C Sandler, T Ma ¨ki, K K Eklund, Department of Medicine,
Division of Rheumatology, Helsinki University Central Hospital, Helsinki,
K A Lindstedt, P T Kovanen, M J Leskinen, Wihuri Research Institute,
T Juutilainen, Department of Orthopaedic Surgery, Helsinki University
Central Hospital, Helsinki, Finland
One of the authors (KKE) has served as a consultant to Novartis Pharma.
The other authors have nothing to disclose.
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