**2. Methods**

inpostpubertalindividuals);twoormoreNFsofanytype,oroneplexiformNF;axillaryfreckling; optic glioma; two or more Lisch nodules within the iris; a distinctive osseous lesion; or firstdegree relative diagnosed with NF1 according to the preceding criteria [2]. While significant advances in understanding both the pathoetiology and genetics of NF1 have been made in the lastdecade, no therapeutic modalities are currently available for NF1patients; although several studies are currently examining various agents specifically directed at plexiform NF, such as

We have previously investigated the effects of vitamin D3 (VD3) and its analogs on skin lesions of patients with NF1 and observed inhibited growth of fibroblasts primarily isolated from NFs (previously called fibroblastic cells) [4]. We next performed in vivo experiments in which we found topical application of a VD3 analog onto a CALM grafted to nude mouse skin inhibited uptake of bromodeoxyuridine into cells on the basal layer of grafted epidermis [5]. In addition, cell density of NF tissues subcutaneously grafted onto the skin of nude mice decreased significantly with direct local injection of a VD3 analog [6]. Consequently, as an initial human study, we evaluated the effect of 1 month of VD3 analog application twice a day to an NF1 patient's large pigmented plaque. We observed moderate improvement of the applied macule, including a remarkable decrease in intensity of Fontana Masson staining in the epidermal basal layer [6]. We extended our study to examine whether VD3 or its analogs inhibit growth of Schwann cells and mast cells isolated from primary NFs. We also investigated the capacity of these agents to inhibit growth of human epidermal melanocytes. Our results indicated that with the exception of Schwann cells, all cell types examined were inhibited by VD3 and its

From a molecular aspect, the NF1 gene encodes a Ras GTPase-activating protein called neurofibromin; mutations in this gene affect Ras-mitogen-activated protein kinase (MAPK) signaling [7]. Inhibition of this signaling, also known as the Ras/Raf/MEK/ERK or MEK pathway, has also been shown to be efficacious in treating human NF [8]. Therefore, we examined inhibitory effects of a mechanistic target of rapamycin (mTOR) inhibitor (rapamycin) and a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor (lovastatin) [9] on growth of Schwann cells isolated from primary NFs. Our results demonstrated that these

We examined the potential of other therapeutic modalities to be combined with VD3 for even greater improvement of skin lesions in patients with NF1. Previous reports indicate narrowband ultraviolet B (NB-UVB) irradiation directly induces conversion of vitamin D to VD3 in various cell types including human keratinocytes [10, 11]; thus, we analyzed whether longterm whole body NB-UVB irradiation increased serum levels of VD3 in NF1 patients. We also observed the effects of irradiation on patient skin lesions by photographing treatment areas before and after irradiation procedures. Our results showed that 6 months of irradiation or more increased patient VD3 serum levels significantly. This increase was accompanied by changes in the patients' skin color including lightening of generalized hyperpigmentation. It has been established that laser treatments have little effect on pigmented skin lesions, such as CALMs or small freckling, of patients with NF1. Instead, we investigated the effects of laser or intense pulsed-radio frequency (IPL-RF) irradiation in combination with topical application

clinical trials for sirolimus and imatinib mesylate [3].

162 A Critical Evaluation of Vitamin D - Clinical Overview

agents significantly inhibited Schwann cell growth in vitro.

analogs in vitro.

#### **2.1. Cell culture**

Primary fibroblasts isolated from NFs of patients with NF1 were cultured as previously described [4]. Human epidermal melanocytes (Melanocel 1; Kurabo, Japan) were cultured according to the manufacturer's instructions. Isolation of Schwann cells and mast cells was performed as previously described [12]. Briefly, NF pieces were dissociated in Dulbecco's Modified Eagle's Medium (DMEM; Thermo Fisher Scientific K.K., Yokohama, Japan) containing collagenase (Life Technologies, Carlsbad, CA) and dispase (Roche Diagnostics, Basel, Switzerland), and then cells were resuspended in DMEM containing fetal calf serum, antibiotics, 3-iso-butyl-L-methlxanthine (Sigma, St Louis, MO), β-heregulin (Wako, Japan), forskolin (Sigma) and insulin (Sigma). Isolated Schwann cells were seeded onto culture flasks coated with poly-L-Lysine (Sigma) and laminin (Life Technologies). To isolate mast cells, NF pieces were incubated in a digestion buffer containing Hank's Balanced Salt Solution (Life Technologies), collagenase (Worthington Biochemical, Lakewood, NJ), hyaluronidase (Worthington Biochemical), and DNase (Sigma). Cells were layered over 75% Percoll® (Sigma) and centrifuged, and then nucleated cells were collected from the buffer-Percoll interface. Percollenriched gradient cells were suspended in AIM-V® Medium (Life Technologies) containing human stem cell factor (PeproTech, Rocky Hill, NJ). The purity of Schwann or mast cell populations was assessed with S-100 or toluidine blue staining, respectively, and a purity grade of more than 90% was confirmed for both cell-type isolations. Cells were used after 2–3 passages.

#### **2.2. Treatment of cells with VD3 and its analogs with or without excimer light**

Fibroblasts from NFs and human epidermal melanocytes were seeded at a density of 2 × 104 cells/ml onto 35-mm cell culture dishes for 2 days. Next, cells were treated for 3 days with 10−9–10−7 M calcitriol (VD3, 1α,25-dihydroxyvitamin D3; Enzo Life Sciences, Farmingdale, NY), tacalcitol [1,24-dyhydroxyvitamin D3; Teijin, Japan], or 22-oxacalcitriol (OCT; 1α,25-dihydroxy-22-oxavitamin D3; Chugai Pharmaceuticals, Japan). Cells were trypsinized and then dissociated cells were collected by centrifugation and counted by Coulter counter. Melanocytes treated with agents were also labeled with 1 μCi of 3 [H]-thymidine (3 [H]-TdR); incorporation into melanocytes was counted by liquid scintillation counter, as previously described for counting fibroblasts isolated from NFs [4]. Both cell types were exposed to excimer light (308 nm) at doses of 150–300 mJ/cm2 in the presence of calcitriol and tacalcitol and were then cultured for 3 days. Excimer light (308-nm UVB) was used to irradiate culture dishes instead of 308 nm of UVB because the tip of the excimer light was small and handy and it allowed for precise irradiation of an expected dose. In the following experiments, isolated primary fibroblasts (three patients), mast cells (five patients), and Schwann cells (six patients) were cultured as described above and then treated with 0.1 μM calcitriol or tacalcitol for 3 days. Cells irradiated with excimer light in the presence or absence of agents were cultured for 3 days after irradiation. Floating mast cells were centrifuged before counting. Mean counts of triplicate measurements were determined.

#### **2.3. Treatment of cells with rapamycin and/or lovastatin**

Isolated primary Schwann cells and fibroblasts from NFs were seeded at a density of 1.0– 2.0 × 104 cells/ml onto 35-mm culture dishes. After 2 days, cells were treated with rapamycin (0.1–100 nM), lovastatin (0.1–10 μM), or a combination of both for 3 days. Number of cells per dish was evaluated by Coulter counter and mean counts of triplicate measurements were determined for each dish, as previously described [13].

#### **2.4. NB-UVB irradiation**

In the dermatology outpatient clinic at Fukuoka University Hospital, NF1 patients who had complained of itching or painful sensation were administered whole body NB-UVB irradiation (312 ± 2 nm) at doses of 0.2–0.5 J/cm2 once weekly or biweekly (every 2 weeks). Approximately, half of these patients presented with complicating atopic dermatitis. During irradiation, serum VD3 levels of patients who had received NB-UVB irradiation for more than 18 months were measured and compared with those of patients without any treatment. Next, nine patients who had not received any previous treatment were irradiated with NB-UVB for 6 months and serum VD3 levels were measured before and after irradiation for a kinetic study. Patients not showing an increase in serum VD3 levels, even after 6 months of irradiation, were further irradiated for more than a year to study whether additional irradiation increased serum VD3 levels. Special Laboratory References (Tokyo, Japan) measured serum VD3 concentration of patient serum samples using a VD3 Radioimmunoassay Kit (Immunodiagnostic Systems, UK) in accordance with the manufacturer's instructions. As a preliminary clinical investigation, we also examined whether increases in serum (and possibly skin) VD3 levels brought about by long-term NB-UVB irradiation bestowed any benefit to NF1 patient skin lesions [10, 11]. We photographed the examined areas before and after irradiation, especially focusing on generalized pigmentation of the skin.

#### **2.5. IPL-RF or laser irradiation with or without topical VD3 analogs**

We investigated combinations of topical VD3 analogs with various forms of irradiation including NB-UVB, IPL-RF (Aurora; Syneron Medical, Israel), Q-switched Ruby (JMEC, Japan), and neodymium-doped yttrium-aluminum-garnet (Nd:YAG) laser (MedLight C6; Cynosure, Westford, MA) treatments. IPL-RF irradiation was performed according to previously described methods [14, 15]. Laser toning procedures using a Q-switched Nd:YAG laser were carried out during 5–10 sessions over 1- to 2-week interval with 2.5 J/cm2 at 5 Hz. Clinical improvement of skin lesions was determined by photographs or using a colorimeter (Crystaleye; Olympus, Japan) before and after treatments.

All clinical studies were performed with prior approval from an ethics committee at Fukuoka University Hospital, and all enrolled patients agreed to the studies and provided informed consent.
