Nerve Growth Factor Expression and Its Receptors TrkA and p75NTR in Peri-Implantitis Lesions

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Article

Reference

Nerve Growth Factor Expression and Its Receptors TrkA and p75NTR in Peri-Implantitis Lesions

DOUILLARD, Thomas, MARTINELLI-KLÄY, C. P., LOMBARDI, Tommaso

Abstract

Fifteen biopsy specimens from patients with chronic peri-implantitis and 4 of healthy oral mucosa were immunostained with antibodies against NGF, TrkA, and p75NTR. The staining intensity and percentage of stained cells were semi-quantitatively evaluated and results were compared between the 2 groups

DOUILLARD, Thomas, MARTINELLI-KLÄY, C. P., LOMBARDI, Tommaso. Nerve Growth Factor Expression and Its Receptors TrkA and p75NTR in Peri-Implantitis Lesions. Implant Dentistry , 2016, vol. 25, no. 3, p. 373-379

DOI : 10.1097/ID.0000000000000418 PMID : 27064695

Available at:

http://archive-ouverte.unige.ch/unige:89710

Disclaimer: layout of this document may differ from the published version.

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Nerve Growth Factor Expression and Its Receptors TrkA and p75NTR in

Peri-Implantitis Lesions

T. Douillard, DDS,* C. P. Martinelli-Kläy, DDS, MSc, PhD,* and T. Lombardi, MD, DMD, PD*

P

eri-implantitis is defined as an inflammatory process with bone loss around the osseointegrated and functional implant.¹ It represents one of the major complications observed after the installation of a dental implant. When the inflammatory process is limited and involves only the mucosa, it is reversible and called mucositis.²

Differently from healthy bone re- modeling, where the action of osteoclasts is coupled with osteoblasts apposition, pathological bone resorption is associated with an increase of the number and the activity of osteoclasts. Classically, the phenomenon of bone resorption is regulated by the receptor activator of nuclear factor-kappaB ligand-osteoprotegerin (RANKL-OPG) system.³ How- ever, studies have shown other way of bone resorption independent from RANKL-OPG,⁴as the way of neurotrophins. The nerve growth factor (NGF) belongs to the family of neurotrophins.

The biological activity of NGF is medi- ated by interaction with two structurally independent transmembrane receptors.

It binds with high affinity to a tyrosine kinase receptor of 140 kDa (TrkA) and with low affinity to a glycoprotein receptor of 75 kDa (p75NTR).⁵ Although TrkA has a crucial role in neuronal survival and growth properties, p75NTR activates signaling cascades that can induce apoptosis and mediate neuronal growth inhibition. This receptor may also act as a Trk co-receptor increasing NGF-binding specificity and affinity to TrkA.⁶

NGF was initially studied for its role in the growth and survival of neurons.⁷Today, its role and scope are considered much more broadly. NGF

can act not only in nervous tissue but also in non-neuronal cells,⁷ including cutaneous keratinocytes.⁸ It has also been shown that NGF and its receptors are involved in inﬂammation⁹and bone resorption.^10,11In addition, NGF accel- erates wound healing in the skin of normal and diabetic mice¹²and plays a role in normal differentiation and survival of oral epithelium.¹³ Studies have also shown that NGF may promote osteogenesis^14,15during bone distraction.^16,17 Only few studies have examined NGF expression in periodontitis^11,18but none in peri-implantitis. Nevertheless, despite clinical and etiological

*Postgraduate in Oral Surgery, Laboratory of Oral & Maxillofacial Pathology, Oral Maxillofacial Pathology Unit, Division of Oral Maxillofacial Surgery, Department of Surgery, Geneva University Hospitals, Switzerland.

Reprint requests and correspondence to: Tommaso Lombardi, MD, DMD, PD, Laboratory of Oral &

Maxillofacial Pathology, Oral Maxillofacial Pathology Unit, Division of Oral Maxillofacial Surgery, Department of Surgery, Geneva University Hospitals, 19 rue Barthelemy-Menn, 1205 Geneva, Switzerland, Phone: +41 22 379 4034, Fax: +41 22 379 4082, E-mail:

Tommaso.Lombardi@unige.ch

ISSN 1056-6163/16/02503-373 Implant Dentistry

Volume 25Number 3

DOI: 10.1097/ID.0000000000000418

Background:Nerve growth factor (NGF) can, through its receptors TrkA and p75NTR, convey signals for cell survival or cell differentiation. These proteins are also involved in inﬂammation and in bone resorption. The aim of this study is to evaluate, for the ﬁrst time, the expression of NGF and its receptors TrkA and p75NTR in peri-implantitis lesions.

Materials and Methods:Fifteen biopsy specimens from patients with chronic peri-implantitis and 4 of healthy oral mucosa were immunostained with antibodies against NGF, TrkA, and p75NTR. The staining intensity and percentage of stained cells were semi-quantitatively evaluated and results were compared between the 2 groups.

Results: In the peri-implant pocket epithelium and gingival epithelium, NGF and TrkA expressions were similar to the healthy oral mucosa, however, a decreased expression of p75NTR was observed.

In all cases, more than 75% of the inﬂammatory cells stained positively for NGF and TrkA, and p75NTR was negatively expressed.

Conclusion:The intense expression of NGF and TrkA in the inﬂam- matory cell inﬁltrate associated with decreased expression of p75NTR in both gingival and pocket epithelium suggests that these proteins may have a role in peri-implantitis lesions.

(Implant Dent 2016;25:373–379) Key Words: nerve growth factor, TrkA, p75NTR, peri-implantitis, immunohistochemistry

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similarities between periodontitis and peri-implantitis, histopathologic differences remain critical.¹⁹

The objective of this study is to evaluate the expression of NGF and its receptors TrkA and p75NTR in peri- implantitis lesions.

MATERIALS AND METHODS

Specimens

This is a retrospective study carried out from parafﬁn blocks stored in the Laboratory of Oral & Maxillofacial Pathology, Oral Maxillofacial Pathol- ogy Unit, Division of Oral Maxillofa- cial Surgery, Department of Surgery, Geneva University HospitalsdHUG between 2000 and 2012. The agreement of the ethics committee of the Univer- sity Hospitals of Geneva was obtained (ref 12–063R/psy 12–003R).

All samples of gingival tissue with a diagnosis of chronic peri-implantitis were included in the study. The clinical diagnosis was confirmed by histological analysis. Samples showing no inflammatory infiltrate (and therefore incompatible with peri-implantitis) and samples showing no epithelial tissue

were excluded from the study. In total there were 15 cases in a group of 14 patients, 8 were women and 6 were men (mean age of 61 years, ranged from 29 to 85). Two out of the 15 cases were presented by the same person. Pocket epithelium of peri-implantary area was present in only 9 cases. Inﬂammation ranged from moderate to strong. A control group consisting of 4 biopsies of healthy mucosa was investigated.

Only samples of oral tissue with no histological signs of inﬂammation were included.

All tissue specimens wereﬁxed in 10% neutral buffered formalin and processed routinely to parafﬁn wax.

All parafﬁn blocks were cut and stained with hematoxylin and eosin for the diagnosis of peri-implantitis and to categorize the groups. NGF, TrkA, and p75 expression was evaluated by immunohistochemistry.

Immunohistochemistry

Three micrometres thick sections were deparafﬁnized in xylene, rehy- drated in graded alcohol and rinsed with distilled water. For antigen retrieval, slides were immersed in buffer Tris/

EDTA pH9 (S2367, Dako) and boiled three times for 5 minutes in the micro- wave (400 W). After 20 minutes of cooling, sections were rinsed with distilled water. Sections were immersed in a peroxidase-blocking solution (kit ab- 64261 for NGF and TrkA; kit ab-64259 for p75, AbCam, Cambridge Science Park, CB4 OFL, UK) for 20 minutes.

Then, tris-buffered saline solution (TBSd0.05 M Tris–HCl, 0.15 M NaCl, pH7.6, s3001 Dako Schweiz, Baar) was used for rinsing. After that, sections were immersed in protein- block solution (kit AbCam) for 1 hour for NGF and TrkA, for 30 minutes for p75NTR and then rinsed with TBS solution. The sections were incubated for 2 hours with primary antibodies at room temperature: polyclonal rabbit anti-NGF antibody (ab-6199, 1:200 dilution, AbCam); polyclonal rabbit anti-TrkA antibody (ab-37837, 1:100 dilution, AbCam); monoclonal mouse anti-p75NTR antibody (ab-3125, clone: NGFR5; 1:50 dilution, AbCam).

After rinsing with TBS, the sections were then incubated with secondary antibody for 15 minutes at room temperature: biotinylated goat anti-rabbit Table 1. NGF Expression in Gingival or Peri-Implantary Pocket Epithelium

Intensity Percentage

(−) (+) (++) (+++) ,75% 75%–89% $90%

Control (n¼4) 0 1 (25%) 2 (50%) 1 (25%) 0 0 4 (100%)

Gingiva (n¼15) 0 1 (6.7%) 11 (73.3%) 3 (20%) 0 0 15 (100%)

Pocket (n¼9) 0 2 (22.2%) 6 (66.7%) 1 (11.1%) 0 0 9 (100%)

Intensity is expressed as negative (−), weak (+), moderate (++), and strong (+++). The scores of percentage of positive immunostaining are classiﬁed as:,75%, 75%–89%, and$90%.

Fig. 1. Immunohistochemistry of NGF. Strong staining is observed in healthy oral mucosa (A), peri-implantitis gingival epithelium (B), and peri- implantitis pocket area epithelium (C). Immunoperoxidase,320.

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(ab-64261, AbCam) for NGF and TrkA; biotinylated goat anti-mouse (ab-64259, AbCam) for p75NTR. Neg- ative control was obtained by omitting the primary antibody from the assay and by replacing it with nonimmune rabbit serum (ab27472, AbCam) for NGF and TrkA, and with nonimmune mouse serum (Dako x0931, 1:25 dilution) for p75NTR. A standard avidin-biotin peroxidase technique was used to visualize the immune complexes. Polymer con- jugated to peroxidase (kit AbCam) was added and incubated for 15 minutes.

Between each step, several rinses with TBS were carried out. Visualization of the immunoreaction was completed using 3,3⁰-diaminobenzidine (DAB Chromogen, kit AbCam) for 5 minutes as the peroxidase chromogenic substrate forms and rinsed with distilled water.

The slides were then counterstained with Harris hematoxylin for 30 seconds, de- hydrated in graded alcohol, cleared in xylene and mounted. Brain sections were used as the positive control for NGF and TrkA, whereas nervousﬁbers were used as internal positive control for p75NTR (not shown).

Immunohistochemical Evaluation

Healthy mucosa, peri-implant pocket, and gingiva epithelium, as well as inﬂammatory cell inﬁltrate of the

pocket area were evaluated. A semi- quantitative analysis was performed using a light microscopy at a 403mag- niﬁcation to verify the stained cell proportion and the intensity of staining for NGF, TrkA, and p75NTR. The staining intensity was classiﬁed as negative, mild, moderate, and strong. The average percentage of labeled cells for NGF, TrkA, and p75NTR antibodies in the healthy mucosa epithelium (n¼4) were 95%, 95%, and 90%, respectively.

Therefore, the stained cell proportion was categorized as: (a) ,75% of stained cells; (a) from 75% to 89% of stained cells; (c)$90% of stained cells.

The inﬂammation was classiﬁed as weak, moderate, and strong.

Statistical Analysis

Chi-squared test was used for the analysis of intensity and percentage of stained cell between the groups.

A P value of ,0.05 was accepted as statistically signiﬁcant.

RESULTS

A total of 15 biopsies diagnosed as peri-implantitis were used to analyze the immunohistochemistry expression of the NGF, TrkA, and p75NTR proteins. Each specimen contained gingival and peri-implant pocket area, except

for 6 specimens that contained only gingival epithelium.

In healthy oral mucosa epithelium, NGF expression was positive for the whole epithelium extension in all cases with more than 90% of the labelled cells (Table1). The intensity of staining varied from weak to strong (1 sample weakd25%, 2 moderated50%, 1 strongd25%) (Table 1; Fig. 1A). The same staining was observed for TrkA in which the whole epithelium extension was labeled with more than 90% of stained cells proportion in all cases (Table 2). The intensity was mostly strong (75% of cases, Fig. 2A), except in 1 sample (25%), which presented moderate staining. For p75NTR, the expression was observed in the epithelium basal layer in both areas close to the base and top of rete ridges (Fig. 3A).

The stained cell proportion was over 75% (Table 3) in the 4 samples analyzed.

NGF and TrkA expressions were only observed in the cytoplasmic region (Figs. 1A and 2A), whereas p75NTR expression showed intense membrane staining and slight cytoplasmic staining (Fig. 3A). In peri-implantitis lesions, the percentage of stained cells for NGF in gingival epithelium was greater than 90% for all samples (Table 1). The average intensity of staining was Table 2. TrkA Expression in Gingival or Peri-Implantary Pocket Epithelium

Intensity Percentage

(−) (+) (++) (+++) ,75% 75%–89% $90%

Control (n¼4) 0 0 1 (25%) 3 (75%) 0 0 4 (100%)

Gingiva (n¼15) 0 2 (13.3%) 3 (20%) 10 (66.7%) 1 (6.7%) 1 (6.7%) 13 (86.6%)

Pocket (n¼9) 0 0 1 (11.1%) 8 (88.9%) 0 0 9 (100%)

Intensity is expressed as negative (−), weak (+), moderate (++), and strong (+++). The scores of percentage of positive immunostaining are classiﬁed as:,75%, 75%–89%, and$90%.

Fig. 2. Immunohistochemistry of TrkA. Strong staining is observed in healthy oral mucosa (A), in peri-implantitis gingival epithelium (B), and in peri-implantitis pocket epithelium (C). Immunoperoxidase,320.

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moderate in 11 cases (73.3%) but with a few variations: 20% of sections (3 cases) had strong intensity staining and 6.7% (1 case) presented mild intensity (Fig. 1B; Table 1). In the pocket epithelium, all cases showed a high percentage of staining (more than 90% of

stained cells, Table 1). The intensity was moderate in 6 cases (66.7%, Fig.

1C). One case (11.1%) showed strong intensity of staining and 2 cases (22.2%) showed slight intensity. For TrkA, we noted in 13 cases (86.6%) a high percentage of staining in the

gingival epithelium (more than 90% of stained cells). There were differences in 2 cases: one case (6.7%) showed less than 75% of stained cells and another (6.7%) between 75% and 89%

(Table 2). The intensity was strong in 10 cases (66.7%), moderate in 3 (20%) and slight in 2 (13.3%) (Table 2; Fig.

2B). In pocket epithelium, all cases had more than 90% of stained cells (Table 2). There was strong intensity of staining in 8 cases (88.9%) except for 1 case (11.1%) which showed moderate intensity (Table 2; Fig. 2C). There were no signiﬁcant differences between the peri-implantary pocket and gingival epithelium concerning the NGF and TrkA expressions, when compared with the healthy mucosa epithelium. For p75NTR, the expression was observed in the basal layer. In peri-implant gingival epithelium, we found a high percentage of stained cells: 8 cases (53.3%) had more than 90% of stained cells, 3 cases (20%) had between 75%

and 89% and 4 cases (26.7%) were lower than 75% (Table 3). Six samples (40%) presented the same type of staining as in the control group (intense membrane staining and slight cytoplasmic staining), whereas 9 cases (60%) had mainly slight cytoplasmic staining (Fig. 3B). There was a statistically sig- niﬁcant p75NTR expression relation between the control group and the peri-implantitis group (x² ¼ 15, P , 0.01). In the pocket epithelium (Table 3), 8 cases (88.9%) had more than 90% of stained cells and only one case (11.1%) showed a lower percentage, but still more than 75% of the positive cells. Four samples (44.4%) presented the same type of staining intensity as in the control group, whereas 5 cases (55.6%) had mainly slight cytoplasmic staining (Fig. 3C) suggesting a decrease of the p75NTR expression in the peri-implantitis. We also had a signiﬁcant relation between the peri-implantary pocket epithelium p75NTR expression and the control group (x²¼9,P,0.05).

The inﬂammatory cell inﬁltrate of the pocket area varied from moderate to intense and was mainly represented by lymphocytes, plasma cells, some macrophages, and occasionally neutrophils.

For NGF, the staining intensity of

Fig. 3. Immunohistochemistry of p75NTR. Strong membrane staining is only observed in the epithelium basal layer in both areas close to the base and top of rete ridges in healthy oral mucosa (AandA1). Slight cytoplasmic staining is observed in peri-implant gingival epithelium (BandB1) and pocket area epithelium (CandC1). p75NTR was not expressed in cells of inﬂammatory inﬁltrate (CandC1). Immunoperoxidase,320 (A–C) and340 (A1–C1).

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inflammatory cells ranged from slight (6.7%) or moderate (33.3%) to strong (60%). The percentage of stained cells was more than 90% in 14 cases (93.3%) except for 1 case (6.7%) where there was between 75% and 89% of labeled cells (Table 4). Several cell types in the connective tissue, including lymphocytes, plasmocytes, macrophages, and fibroblasts were positive for NGF (Fig. 1C). For TrkA, the staining was mainly strong in 12 cases (80%), whereas 3 cases (20%) had a moderate staining. The percentage was high with more than 90% of labeled cells in 14 cases (93.3%, Table 4). Lymphocytes, fibroblasts, plasmocytes, and macrophages were also positive for TrkA staining (Fig. 2C). P75NTR expression was negative in the inflammatory cell infiltrate (Fig. 3C).

D^ISCUSSION

According to the literature, NGF, TrkA, and p75NTR are expressed in healthy oral mucosa epithelium suggesting a role in normal differentiation and survival of oral epithelia, as well as in wound healing in the oral cavity.^13,20 In our study, we also found a positive expression for NGF, TrkA, and p75NTR in healthy oral mucosa. NGF and TrkA expressions were expressed by all epithelium layers, whereas p75NTR expression presented intense membrane staining and slight cytoplasmic staining only in the epithelium basal layer. Concerning TrkA intensity staining, some differences were observed with respect to the literature.

In fact, TrkA intense or weak to moderate staining were detected in basal/

parabasal cells and in granular/spinous cell layers, respectively,^13,21whereas in this study a strong staining was observed in the whole epithelium (75% of cases). In addition, our study showed NGF expression in whole epithelium, which differs from the literature, in which only NGF expression in the granular cell and upper spinous cell layer was observed. Nevertheless, pro- NGF expression was observed in the whole oral mucosa epithelium.^13,21 Differences in antibodies used and different methodologies used might con- tribute to these outcome discrepancies.

Table4.NGFandTrKAExpressionsbyInflammatoryCellsinthePeri-ImplantaryPocketArea NGFTrKA IntensityPercentageIntensityPercentage (−)(+)(++)(+++),75%75%–89%$90%(−)(+)(++)(+++),75%75%–89%$90% Inflammation Moderate/strong(n¼15)01(6.7%)5(33.3%)9(60%)01(6.7%)14(93.3%)003(20%)12(80%)01(6.7%)14(93.3%) InflammationisExpressedasModerate/Strong.Intensityisexpressedasnegative(−),weak(+),moderate(++),andstrong(+++).Thescoresofpercentageofpositiveimmunostainingareclassifiedas:,75%,75%–89%,and$90%.

Table3.p75NTRExpressioninGingivalorPeri-implantaryPocketEpithelium IntensityPercentage (−)(+)(++)(+++),75%75%–89%$90% Control(n¼4) M/C0004(100%)02(50%)2(50%) C0000000 Gingiva(n¼15) M/C0006(40%)4(26.7%)3(20%)8(53.3%) C09(60%)*00000 Pocket(n¼9) M/C0004(44.4%)01(11.1%)8(88.9%) C05(55.6%)*00000 Intensityisexpressedasnegative(−),weak(+),moderate(++),andstrong(+++).Thescoresofpercentageofpositiveimmunostainingareclassiﬁedas:,75%,75%–89%,and$90%. *SigniﬁcancewasEstablishedifP,0.05. C,Cytoplasm;M,Membrane.

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Our ﬁndings are in agreement with those showing that normal skin epithelium also expressed NGF in all epithelial cell layers.^13,22,23

In the peri-implant pocket epithelium and gingival epithelium, NGF and TrkA expressions were similar to the control group. However, the present study showed a decrease in p75NTR expression in both gingival and pocket epithelium. In a study on oral lichen planus and oral lichenoid reactions, none or quite weak cytoplasmic staining for p75NTR has been observed.²¹In addition, expression of the p75NTR was also significantly down-regulated in the skin of patients with urticaria, which could involve an increased mast cell reactivity and proliferation.²⁴In an experimental study, mice deficient of p75NTR (p75NTR knockout mice) developed severe or lethal disease and concomitant increased levels of inflammation in the central nervous system (CNS) suggesting a significant role for p75NTR in CNS endothelial cells during inflammation.²⁵

The receptor p75NTR belongs to tumor necrosis factor (TNF) receptors superfamily traditionally associated with inﬂammation and apoptosis.²⁶ Unlike TrKA chain which does not form homocomplex or heterocomplex, p75NTR exists as dimers and may be responsible for modulating responses of the TrK tyrosine kinase receptors.

Once bound to p75NTR, NGF is presented to aﬁrst TrKA chain and then it binds a second chain of TrKA forming an active homodimer and acti- vating subsequently the NGF signaling pathways.²⁷This protein has also been proposed as a potential tumor suppres- sor in carcinomas of prostate^28,29and gastric cancer³⁰ among others. In these cancers, p75NTR was down- regulated and its overexpression inhibited cell proliferation³¹and invasion,³⁰ and induced apoptosis.²⁹ It could be speculated that down regulation of this receptor could participate in the inﬂammation of peri- implantitis. Its role in peri-implant pocket epithelium proliferation and apoptosis is not known and require further investigations.

The inﬂammatory cell inﬁltrate of peri-implant pocket and gingival tissue

consisted mostly of lymphocytes, plasma cells, some macrophages, and occasionally neutrophils. The chronic inﬂammatory cells showed a positive staining for NGF and TrkA ranging from moderate to intense. A very high percentage (over 90%) of stained cells was also observed for both antigens.

Inflammatory cells did not express p75NTR protein. It has been shown that neutrophils, macrophages, and lymphocytes can secrete inflammatory mediators (eg, interleukin IL 1, 6, 11, and 17, tumor necrosis factor-alpha or TNF-a) leading to bone loss.³²Some studies have shown an increase of NGF in inflamed tissue.^33–35 Pro- inflammatory cytokines may upregulate NGF. It has been shown that the expression of NGF and TrkA mRNAs can be regulated by IL-1bin human periodontal ligament cells and fibroblast in vitro.²⁰Certain hormones (eg, oestrogen), inflammatory cytokines (eg, interferon-gamma), and NGF itself increase TrkA expression in different cell types.^36–39 All these data suggest that inflammatory signals upregulate or activate both NGF and TrkA expressions.¹¹ Moreover, some studies have demonstrated that NGF is capable of autocrine or paracrine activation of inflammatory cells.^40,41Expo- sure to NGF increases IL-6 gene expression in the primary human bone marrow stromal cell lines.⁴²NGF can also activate T-lymphocytes and induce migration of inflammatory cellular in- filtrates.⁴³Experimental investigations have also shown a relation between NGF expression and the periodontitis.¹¹ Polymerase chain reaction analysis showed a statistically significant up- regulation of NGF mRNA in rat induced periodontitis. Immunohisto- chemistry of trigeminal ganglion neurons innervating the gingival mucosa demonstrated increased expression of TrkA. In addition, anti-NGF treatment reduced IL-1b expression in gingiva and bilateral alveolar bone resorption.¹¹ Our results are in line with the only published study of NGF and TrkA in induced periodontitis. It seems therefore reasonable to assume that chronic periodontitis and chronic peri- implantitis share some physiopatholog- ical mechanisms. All these data suggest

that NGF and its receptors might act together in modulation of peri-implant inﬂammation and bone loss.

C^ONCLUSION

This is the ﬁrst study analyzing NGF and its receptors TrkA and p75NTR in chronic peri-implantitis.

The results obtained suggest that such proteins may play a role in pathogenesis of peri-implantitis. Other investigations are needed to further clarify their exact function.

DISCLOSURE

The authors claim to have no ﬁnancial interest, either directly or indirectly, in the products or informa- tion listed in the article.

A^PPROVAL

The agreement of the ethics committee of the University Hospitals of Geneva was obtained (ref 12–063R/psy 12–003R).

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