Arch Dermatol Res DOI 10.1007/s00403-017-1792-6

ORIGINAL PAPER

Expression of Janus Kinase 1 in vitiligo & psoriasis before and after narrow band UVB: a case–control study Hanan Rabea Nada1 · Dina Ahmed El Sharkawy1 · Maha Fathy Elmasry1   · Laila Ahmed Rashed2 · Sally Mamdouh3 

Received: 26 February 2017 / Revised: 4 May 2017 / Accepted: 1 November 2017 © Springer-Verlag GmbH Germany, part of Springer Nature 2017

Abstract  Janus kinases (JAKs) are non-receptor protein tyrosine kinases that are expressed in many tissues. Once the JAKs are activated, a cascade of further signaling events is triggered involving phosphorylation of selected receptor chain tyrosines, binding of signal transducer and activator of transcription (STAT) proteins and phosphorylation of these STATs. Due to their ability to selectively modulate immune function, targeted JAK inhibitors are promising candidates for some skin diseases such as psoriasis and atopic dermatitis. The aim of this study was to assess the level of JAK1 in both vitiligo and psoriasis patients before and after treatment with NB-UVB which is considered a gold standard therapy for both diseases. This study was conducted on 10 patients with psoriasis, 10 patients with vitiligo and 10 controls. JAK1 levels before and after treatment with NB-UVB 311 nm (36 sessions) were measured using Western blot assay. The level of JAK1 was significantly higher in vitiligo and psoriasis patients than controls. There was a decline in the level of JAK1 after treatment, which was statistically significant. VASI and PASI scores of patients decreased after treatment with NB-UVB. In psoriatic patients, the JAK1 level positively correlated with the female participants, disease duration and PASI change. It was concluded that JAK1 plays a role in the pathogenesis of both vitiligo and psoriasis based on its upregulated level before treatment and downregulated level after treatment. This raises the possibility of

using the JAK1 inhibitors as targeted immunotherapy for vitiligo and psoriasis.

* Maha Fathy Elmasry [email protected]

Introduction

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Janus kinase (JAK) is a family of intracellular, non-receptor tyrosine kinases that transduce cytokine mediated signals via the JAK-signal transducer and activator of transcription (STAT) pathway. Approximately 2000 kinases are known, and more than 90 protein tyrosine kinases (PTKs) have been



Dermatology Department, Faculty of Medicine, Cairo University, Cairo, Egypt

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Medical Biochemistry and Molecular Biology Department, Faculty of Medicine, Cairo University, Cairo, Egypt

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Cairo, Egypt



Keywords  JAK · Psoriasis · Vitiligo · NB-UVB · JAK inhibitors Abbreviations CCL C-C motif chemokine ligand CNTF-R Ciliary neurotrophic factor-receptor CXCL Chemokine (C-X-C motif) ligand IFN-R Interferon receptor IL Interleukin JAK Janus kinase LIF-R Leukemia inhibitory factor-receptor NB-UVB Narrow band ultraviolet B NNT-1R/BSF3R Novel neurotrophin-1/B cell-stimulating factor-3 receptor OSM-R/ CT-1R Oncostatin M/Cardiotrophin-1-receptor PASI Psoriasis area and severity index PTK Protein tyrosine kinase STAT Signal transducer and activator of transcription Th17 T helper17 TNF-α Tumor necrosis factor-alpha TYK Tyrosine kinase VASI Vitiligo area and severity index

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found in the human genome [6]. Subsequent studies revealed that the JAK family differs markedly from other classes of PTKs by the presence of an additional kinase domain. To denote this unique structural feature, these kinases were renamed as ‘Janus kinases’ in reference to an ancient twofaced Roman God of gates and doorways. The members of this tyrosine kinase family include; Janus kinase 1 (JAK1), JAK2, JAK3, and tyrosine kinase 2 (TYK2) [27]. Numerous biochemical studies have indicated that JAK1 is involved in signaling by members of the interleukin (IL)-2 receptor family (IL-2R, IL-7R, IL-9R and IL-15R), the IL-4 receptor family (IL-4R, IL-13R), the gp130 receptor family (IL-6R, IL-11R, LIF-R, OSM-R/CT-1R, CNTF-R, NNT-1R/ BSF3R and Leptin-R) and class II cytokine receptors type I IFN-R, type II IFN-R, IL-10R [28].

signaling and downstream CXCL10 expression, thus giving rise to repigmentation in vitiligo [7]. Therefore, the aim of this study was to assess and compare the expression of JAK1 in the affected skin of vitiligo and psoriatic patients and normal healthy controls. It also aims to compare the level of JAK1 in vitiligo and psoriatic patients before and after treatment using NB-UVB 311 nm. The study also aims to assess the possible correlation between the level of JAK1 in vitiligo and psoriatic patients and the age and sex of the patients, the duration and severity of the disease, the VASI score in vitiligo patients, and the PASI score in psoriatic patients.

Role of JAK in psoriasis

This study is a prospective, case-control comparative study. It was approved by the Dermatology Research Ethics Committee and the Research Ethics Committee of the Faculty of Medicine, Cairo University.

Many of the inflammatory cytokines implicated in the pathogenesis of psoriasis utilize JAKs for signaling. TYK2 and JAK1 are critical for signal transduction for many of the cytokines upregulated in psoriatic lesions, such as IL-23, IL-12, IL-6, and IL-22 [16, 30]. Based on this receptor-signaling paradigm and the clinical efficacy of IL-23 inhibitors in psoriasis, JAKs and TYK2 enzymes have become attractive downstream targets in psoriasis research and drug discovery [9, 24]. Tofacitinib (JAK1 & JAK3 inhibitor) is being assessed by dermatologists for psoriasis because it blocks steps in the interleukin-17 (IL-17) signal transduction pathway [24]. The JAK1/STAT signal transduction pathway is used by many other immunologically active cytokines (e.g., IL-12, IL-23, IL-22, IL-6, IFN-γ, and type I interferons) [10]. Because IL-17 production by T cells is dependent on IL-23 stimulation, JAK-STAT pathway may be considered an upstream signaling mediator in the IL-23/Th17 axis [11, 18]. Therefore, tofacitinib may be useful in the treatment of psoriasis through different pathways [24]. Role of JAK in vitiligo Studies have shown that various cytokines including IFN-γ [5, 31], tumor necrosis factor α (TNF-α) [3, 29] and chemokine (C-C motif) ligand 22 (CCL22) [17] are differentially expressed in the lesional skin and serum of vitiligo patients than controls, indicating their roles in vitiligo. It has been found that IFN-γ bound receptor complex recruits JAK1 and JAK2 kinases, leading to the phosphorylation and nuclear translocation of STAT, which in turn transcriptionally activates downstream IFN-γ inducible genes [4]. It is suggested that the use of the JAK 1/3 inhibitor, tofacitinib effectively leads to blockade of interferon gamma

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Methods

Patients and controls The study was conducted on 10 patients with vitiligo, 10 patients with psoriasis and 10 healthy controls during the time interval between December 2015 and November 2016. Patients included in the study were all above 12 years of age. They suffered either from psoriasis vulgaris or non-segmental vitiligo and received no systemic or topical treatment at least 6 weeks prior to enrollment into the study. Psoriasis patients with erythrodermic psoriasis, pustular psoriasis or associated psoriatic arthritis, vitiligo patients with segmental or universal vitiligo were excluded from the study. Also excluded was any patient with a photosensitive disorder or any contraindication to phototherapy exposure, e.g., history or presence of malignant or premalignant skin lesions. Patients were divided into three equal study groups; Group A (vitiligo group), Group B (psoriasis group) and Group C (control group). Every patient was subjected to detailed history taking, skin examination to determine the distribution and clinical variant of the disease, skin type and percentage of body involvement using the rule of nines. Methodology A written informed consent was taken from every patient. PASI score [23] was done for all patients with psoriasis to assess the severity of psoriasis. VASI score [13] was done for all patients with vitiligo to assess the severity of vitiligo. Patients received 36 sessions of NB-UVB (311 nm) over a period of 3 months. Sessions were taken three times weekly on non-consecutive days. Clinical examination was done every session and the findings were recorded. A final VASI

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score was done in all vitiligo patients and a final PASI score in all psoriasis patients. NB-UVB was delivered by a UV cabin (Waldmann GmbH, Germany) equipped with an integrated UV photometer, having 16 TL-01/100 W Fluorescent lamps producing NB-UVB with a peak emission at 311 nm. Two (4 mm) punch skin biopsies were taken from the 20 patients; one prior to the initiation of phototherapy and the other after the last session (from the same site). One skin biopsy was taken from the skin of each of the 10 control healthy participants. Skin biopsy specimens were preserved in Radio-Immunoprecipitation Assay (RIBA) buffer. • Detection of JAK 1 protein expression using Western

blotting.

JAK1 was measured by a Western blot assay. Steps included: Protein extraction using RIBA buffer RIBA lysis buffer PL005 was provided by Bio BASIC INC. (Marhham Ontario L3R 8T4 Canada). The components of the RIBA buffer are demonstrated in Table 1. Procedure  The stored lysed samples were used to complete protein extraction. The lysate was kept on ice for 30  min on a shaker. Cell debris was removed by centrifugal ions at ~ 16,000×g for 30 min at 4 °C. Supernatant was transferred to a new tube for further protein concentration determination analysis. A Bradford Protein Assay Kit (SK3041) for quantitative protein analysis was provided by BIO BASIC INC. (Markham Ontario L3R 8T4 Canada). A Bradford assay was performed according to manufacture instructions. A 20 ug protein concentration of each sample was loaded with an equal volume of 2x Laemmli sample buffer. The components of 2x Laemmli sample loading buffer were 4% SDS, 10% 2-mercaptoethanol, 20% glycerol, Table 1  Components of RIBA lysis buffer: Components

Volume added to each 200 µl plasma (µl)

RIBA* Buffer 150 mM NaCl 1 0.0% NP-40 or 0. 1% Triton × 100 0.5% sodium deoxycholate 0.1% SDS (sodium dodecyl sulphate) 50 mMTris-HCl, pH was adjusted at 8.0 Protease Inhibitor Buffer Phosphatase Inhibitor Buffer

400

RIBA radio-immunoprecipitation assay

0.4 2.5

0.004% bromophenol blue and 0.125  M Tris HCl. The pH was checked and brought to 6.8. Each of the previous mixtures was boiled at 95 °C for 5 min to ensure the denaturation of protein before loading on polyacrylamide gel electrophoresis. Protein separation by electrophoresis Preparation of  PAGE gels  A sample was separated on a polyacrylamide gel; the procedure was abbreviated as SDS– PAGE (for Sodium Dodecyl Sulfate Polyacrylamide Gel Q Electrophoresis). The technique was a standard means for separating proteins according to their molecular weight. The polymerization was initiated by the addition of ammonium per sulfate along with Tetramethylethylenediamine (TEMED). In our study we used TGX Stain-Free™ FastCast™ Acrylamide Kit (SDS–PAGE) which was provided by BioRad Laboratories, TNC, USA Catalog. NO. 161–0181. The SDS–PAGE TGX Stain-Free FastCast was prepared according to manufacture instructions. Running buffer  A standard migration buffer (also called running buffer) for sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS- PAGE) was composed of 25 mM Tris base, I90 mM glycine and 0.1% SDS. The pH was checked to ensure it was around 8.3. Loading samples and  running the  gel  The prepared gel was submerged in an electrophoresis chamber. The prepared running buffer was poured in an electrophoresis chamber. Special gel loading tips were used to load the complete sample in a narrow well. 20 µg of total protein was loaded per mini-gel well. A molecular weight marker (BLUelf prestained protein ladder, Gene DireX, Taiwan, Cat No.PM0080500) was used to enable the determination of the protein size and also to monitor the progress of an electrophoresis run. The gel was run for 20  min at 50  V to allow sample migration into stacking layer. The voltage was increased to 100–150 V to allow protein migration and separation in the resolving layer to finish the run in about 1 h. Immediately, separation was visualized using stain-free technology and ChemiDoc TM imager. • Protein blotting (transfer of proteins from the gel to the

membrane).

The gel was assembled in a transfer sandwich as following from bottom to top (filter paper, PVDF membrane, gel and filter paper). No air bubbles were trapped in the sandwich. The membrane needed to be on the cathode and the gel on the anode. The sandwich was placed in the transfer tank with 1x transfer buffer, which is composed of 25mMTris,

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190 mM glycine and 20% methanol. The pH was checked and adjusted to pH 8.3 when necessary. The blot was run for 7 min at 25 V to allow protein bands to transfer from gel to membrane using Bio-Rad Trans-Blot Turbo. Immediately the blot separation was visualized and imaged using stain-free blot technology and ChemiDoc TM imager. • Blocking the membrane

Blocking the membrane prevents non-specific background binding of the primary and/or secondary antibodies to the membrane (which has a high capacity at binding proteins and therefore antibodies). The membrane was blocked in tris-buffered saline with Tween 20 (TEST) buffer and 3% bovine serum albumin (BSA) at room temperature for 1 h. • Incubation with the primary antibody

The primary antibodies for primary antibodies (JAK 1, 1:2000, Thermofisher, USA) were diluted in TEST. Incubation was done overnight in each primary antibody solution, against the blotted target protein, at 4 °C. The blot was rinsed 3–5 times for 5 min with TBST. Incubation was done in the HRP-conjugated secondary antibody (Goat antirabbit IgG- HRP-lmg Goat mab -Novus Biologicals) solution against the blotted target protein for 1 h at room temperature. The blot was rinsed 3–5 times for 5 min with TBST. • Imaging and data analysis quantitation

The chemiluminescent substrate (Clarity™ Western ECL substrate—BIO-RAD, USA cat#170-5060) was applied to the blot according to the manufacturer’s recommendation. Briefly, equal volumes were added from solution A (Clarity western luminal/enhancer solution) and solution B (peroxidase solution). The chemiluminescent signals were captured using a CCD camera-based imager. Image analysis software was used to read the band intensity of the target proteins

Table 2  Data of vitiligo patients, psoriasis patients and controls as regards age, sex, disease duration and extent:

Data Sex Age Duration (years) Extent SD standard deviation

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Males Females Range Mean ± SD Range Mean ± SD Range Mean ± SD

against a control sample after normalization by beta actin on the Chemi Doc MP imager. Statistical analysis The data were statistically described in terms of mean ± standard deviation (± SD), median and range, or frequencies (number of cases) and percentages when appropriate. A comparison of numerical variables between the study groups was done using Mann–Whitney U test for independent samples. Within the groups, comparison of numerical variables was done using Wilcoxon signed rank test for paired (matched) samples. To compare categorical data, a Chi-square (χ2) test was performed. An exact test was used instead when the expected frequency is less than 5. A correlation between various variables was done using the Spearman rank correlation equation. p values less than 0.05 were considered statistically significant. All statistical calculations were done using the computer program SPSS (Statistical Package for the Social Science; SPSS Inc., Chicago, IL, USA) release 15 for Microsoft Windows (2006).

Results: 1. Patients’ demographic data Table 2 summarizes the data of patients and controls regarding their sex, age, duration and extent of disease in patients. Comparison between vitiligo and psoriasis patients Upon comparing vitiligo patients with psoriasis patients, no significant difference was found between groups regarding the age of patients, disease duration and extent of the disease (p > 0.05).

Vitiligo

Psoriasis

Controls

3 (30%) 7 (70%) 18–60 40.20 ± 15.33 1–28 12.30 ± 9.19 5–60% 23.40 ± 17.61%

6 (60%) 4 (40%) 15–55 36.30 ± 11.71 0.25–17 7.28 ± 6.02 10–40% 20.70 ± 9.81%

4 (40%) 6 (60%) 18–55 36.30 ± 11.61

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Comparison between the level of JAK1 in vitiligo and psoriasis patients and the level of JAK1 in the healthy controls

Fig. 1  Comparison between mean values of JAK1 pre and JAK1 post in the vitiligo, psoriasis and control

Upon comparing the levels of JAK1 in vitiligo patients either before or after treatment and the level of JAK1 in controls, there was a statistically significant difference (p < 0.001). Additionally, upon comparing the levels of JAK1 in psoriasis patients either before or after treatment and the level of JAK1 in controls, there was a statistically significant difference (p < 0.001). Comparison between the JAK1 level in vitiligo and psoriasis groups

Fig. 2  Comparison between mean values of JAK1 change in vitiligo and psoriasis

There was a statistically minimal significant difference (p = 0.049) between the levels of JAK1 in the vitiligo group and psoriasis group before treatment. Otherwise, no statistically significant difference (p > 0.05) was found between the levels of JAK1 in the vitiligo group and psoriasis group after treatment. The JAK1 change in vitiligo patients with a mean of (− 5.6 ± 2.8) was apparently higher than the JAK1 change in psoriasis patients with a mean of (− 4.08 ± 2.72) (Fig. 2), but upon comparing them, there was no statistically significant difference (p > 0.05).

2. Level of JAK1 in vitiligo patients, psoriasis patients and controls

3. JAK correlations to other variables

• Vitiligo group The level of JAK1 before treatment

JAK1 level and sex and age of patients, duration and extent of the disease, VASI score and VASI change in patients either pre-treatment or post-treatment. • Psoriasis group A statistically significant difference was found between sex of patients and the JAK1 level pre-treatment. The JAK1 level was higher in females (p = 0.02) pre-treatment. There was no statistically significant difference found with JAK1 level post-treatment (p > 0.05). No correlations were found between the JAK1 level and age of patients, extent of the disease and PASI score either pre-treatment (p > 0.05) or post-treatment. The longer the duration of the disease, the higher was the JAK1 level pre-treatment (p = 0.002). But no correlations were found between the duration of the disease and the JAK1 level post-treatment. A statistically significant difference was found between the change in the JAK1 level and the change of the PASI score post-treatment (p = 0.008). • Control group No correlations were found between the JAK1 level and sex or age of controls.

ranged from 7.5 to 16.8 with a mean of 11.98 ± 2.62. The level of JAK1 after treatment ranged from 4.02 to 9.1 with a mean of 6.37 ± 1.73. Upon comparing between the levels of JAK1 in vitiligo patients before and after treatment, a statistically significant decrease was found (p = 0.005). There was a considerable decrease in the mean level of JAK1 of − 5.60 (Figs. 1, 2). • Psoriasis group The level of JAK1 before treatment ranged from 5.9 to 12.6 with a mean of 9.58 ± 2.04. The level of JAK1 after treatment ranged from 2.06 to 10.8 with a mean of 5.49 ± 3.11. Upon comparing between the levels of JAK1 in psoriasis patients before and after treatment, a statistically significant decrease was found (p = 0.005).There was a considerable decrease in the mean level of JAK1 of − 4.08 (Figs. 1, 2). • Control group The level of JAK1 in normal subjects ranged from 0.95 to 1.03 with a mean of 1.01 ± 0.06 (Fig. 1).

• Vitiligo group No correlations were found between the

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Discussion The JAK/STAT signal pathway controls a number of important biological responses, including immune functions, cellular growth, cellular differentiation and hematopoiesis [12]. Due to their ability to selectively modulate immune function, targeted JAK inhibitors are attractive candidates for some skin diseases such as psoriasis and atopic dermatitis [1, 15, 24]. In the current study, we attempted to assess the expression of JAK1 in the skin of vitiligo and psoriasis patients before and after treatment with NB-UVB 311 nm that is considered a gold standard therapeutic modality in both diseases. It was found that the level of JAK1 was significantly higher in vitiligo patients than controls. There was a statistically significant difference between the level of JAK1 before and after treatment with a lower JAK1 level after treatment. These findings suggest a possible role of JAK1 in the pathogenesis of vitiligo and the possible hope of using JAK1 inhibitors as a future treatment of vitiligo. No prior studies were found to prove or contradict our results. We found that tofacitinib citrate, an oral Janus kinase 1/3 inhibitor, was tried in a case of generalized vitiligo and has been reported to result in significant repigmentation that could support the study findings [7]. Additionally, oral ruxolitinib (JAK1/2 inhibitor), in one case report, was observed to cause rapid skin repigmentation in a patient with coexistent vitiligo and alopecia areata which also supports our findings [14]. In the current study, there was no significant correlation between the JAK1 level in relation to disease duration, disease extent, severity, VASI change or VASI score before and after treatment. This could point to the role played by JAK1 in the etiopathogenesis of vitiligo without being influenced by the extent or severity of the disease or any other parameters. No literature was found that matches or contradicts these results. In the present study, it was also found that the level of JAK1 was significantly higher in psoriatic patients than controls. There was a statistically significant difference between the level of JAK1 before and after treatment with a lower JAK1 level after treatment. These findings suggest a possible role of JAK1 in the pathogenesis of psoriasis and the possible hope of using JAK1 inhibitors as a future treatment of psoriasis. However, Andrés et al. reported that all JAK proteins except JAK3 were downregulated in lesional psoriatic skin compared to non-lesional psoriatic skin [2]. JAK3 is mainly expressed in lymphocytes, and therefore, its increase may be due to the increased infiltration of immunological cells in lesional psoriatic skin [20]. These results are contradictory to this study’s results, as it was found that the level of JAK1 was significantly higher in lesional skin of psoriatic patients

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than controls and that this level was significantly decreased after treatment. In support to this study’s findings are the results of the phase III clinical trials that proved the effectiveness of oral and topical formulations of tofacitinib in the treatment of plaque psoriasis and highlighted the possible role of JAK1/3 in the pathogenesis of psoriasis [8]. In a randomized, double-blind, placebo-controlled, dose-ranging phase 2b study, patients with moderate-to-severe psoriasis treated with baricitinib, an oral JAK1/2 inhibitor, for 12 weeks achieved significant improvements in PASI score [25]. Ruxolitinib, a small molecule inhibitor of JAK1 and JAK2, was safe, well tolerated, and exhibited clinical activity in the topical treatment of psoriasis [19, 26]. Additionally, Ludbrook et al. demonstrated that 12 weeks of treatment with the selective JAK1 inhibitor GSK2586184 resulted in clinical improvement in patients with moderateto-severe plaque-type psoriasis [22] which supports this study’s finding that JAK1 plays an important role in pathogenesis of psoriasis. JAK1 level is positively correlated with the sex of psoriatic patients as it showed significant higher values in female psoriatic patients in comparison to male ones. No prior studies that support or contradict this study’s findings were found. However, JAKs play an important role in adipose tissue development [21] and females usually have higher fat content if compared to males, which could be a probable explanation for this finding. Future studies with a larger scale number of patients are highly recommended to prove or contradict this observation. JAK1 level was also positively correlated with PASI change and disease duration (this was not evident in vitiligo). On the other hand, there was no significant correlation between the JAK1 level and the age, disease extent, severity, PASI score before and after treatment. Unfortunately, we could not explain these findings. Additionally, nothing in the literature matches or contradicts this study’s findings, which strongly advocates the importance of further research. In addition, the positive correlation of JAK1 level with PASI change and disease duration highlights the major role suggested for JAK1 in the pathogenesis of psoriasis and the possible use of the anti-JAK1 modalities in its treatment.

Conclusion In conclusion, JAK1 levels in vitiligo and psoriasis patients were higher than normal controls and these levels were downregulated after treatment. So we suggest that JAK1 plays a role in the pathogenesis of both vitiligo and psoriasis. This could also support the ideas of targeting JAK1 in both diseases and could open a new era for treatment of vitiligo and psoriasis by anti-JAK modalities.

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Limitations of the study 1. The small number of participated patients. 2. It was a short term study. Compliance with ethical standards  Conflict of interest  All the authors declare that they have no conflicts of interest. Funding  The authors did not receive any external funding for this work. Ethical approval  All procedures performed in the study were in accordance to the ethical standards of the Dermatology Research Ethical Committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent  A written informed consent was obtained from each patient for the participation in the study and photography.

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