**3. Results**

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

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 measure-

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

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 gener-

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

once weekly or biweekly (every 2 weeks). Approximately,

at 5 Hz. Clinical

triplicate measurements were determined.

164 A Critical Evaluation of Vitamin D - Clinical Overview

**2.4. NB-UVB irradiation**

(312 ± 2 nm) at doses of 0.2–0.5 J/cm2

alized pigmentation of the skin.

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

ments were determined for each dish, as previously described [13].

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

were carried out during 5–10 sessions over 1- to 2-week interval with 2.5 J/cm2

We previously reported that VD3 and its analogs inhibited growth of primary fibroblasts isolated from NFs, whereby around 30–50% of fibroblast growth inhibition was observed with 0.1 μM calcitriol, tacalcitol, or OCT [4]. This inhibition rate almost corresponded to that of isolated primary normal human fibroblasts. A decreased number of fibroblasts was accompanied by 50–70% inhibition of 3 [H]-TdR uptake, observed as early as 2–4 days after addition of agents to culture medium [4]. We also tested whether growth of human epidermal melanocytes was inhibited by VD3 and its analogs and found similar levels of cell growth inhibition and 3 [H]-TdR uptake (**Figure 1**).

**Figure 1.** Growth inhibition of human melanocytes with addition of calcitriol, tacalcitol, or OCT. After treatment, number of cells was counted with a Coulter counter and 3 [H]-TdR uptake was measured by liquid scintillation. Percent inhibition of cell growth (A) and 3 [H]-TdR uptake (B) are shown.

We observed growth inhibition in fibroblasts from NFs and human melanocytes was increased by 10−9 to 10−7 M calcitriol or tacalcitol in combination with excimer light irradiation. Indeed, calcitriol and tacalcitol inhibited growth of both cell types in a dose-dependent manner, and this inhibition was further augmented by around 20–30% with 150–300 mJ/cm2 of excimer light irradiation (**Figure 2**). Recent progress in cell culture techniques allowed us to isolate and culture Schwann and mast cells from NFs with a purity of more than 90%, as described in the Methods section. Primary fibroblasts, mast cells, and Schwann cells were isolated from NFs and examined for how they responded to 0.1 μM calcitriol or tacalcitol and/or excimer light irradiation. Growth of mast cells was inhibited by 16–20% with 0.1 μM calcitriol or tacalcitol; whereas with excimer light irradiation at a dose of 300 mJ/cm2 , this value rose with statistical significance to 26% (almost the same as that of fibroblasts). Also, a small additive inhibition was observed with 0.1 μM calcitriol or tacalcitol and excimer light irradiation at a dose of 300 mJ/cm2 in mast cells. With regard to Schwann cells, neither VD3 nor tacalcitol had an effect on their growth, an unexpected result. Excimer light irradiation inhibited growth of Schwann cells by approximately 38% in a statistically significant manner (**Figure 3**).

**Figure 2.** Inhibition of cell growth by calcitriol or tacalcitol in combination with excimer light irradiation. Fibroblasts isolated from NFs and human melanocytes were irradiated with excimer light in the presence or absence of calcitriol or tacalcitol, and number of cells per dish was counted. Data for fibroblasts (A) and melanocytes (B) are shown.

We observed growth inhibition in fibroblasts from NFs and human melanocytes was increased by 10−9 to 10−7 M calcitriol or tacalcitol in combination with excimer light irradiation. Indeed, calcitriol and tacalcitol inhibited growth of both cell types in a dose-dependent manner, and this inhibition was further augmented by around 20–30% with 150–300 mJ/cm2 of excimer light irradiation (**Figure 2**). Recent progress in cell culture techniques allowed us to isolate and culture Schwann and mast cells from NFs with a purity of more than 90%, as described in the Methods section. Primary fibroblasts, mast cells, and Schwann cells were isolated from NFs and examined for how they responded to 0.1 μM calcitriol or tacalcitol and/or excimer light irradiation. Growth of mast cells was inhibited by 16–20% with 0.1 μM calcitriol or tacalcitol;

significance to 26% (almost the same as that of fibroblasts). Also, a small additive inhibition was observed with 0.1 μM calcitriol or tacalcitol and excimer light irradiation at a dose of

on their growth, an unexpected result. Excimer light irradiation inhibited growth of Schwann

**Figure 2.** Inhibition of cell growth by calcitriol or tacalcitol in combination with excimer light irradiation. Fibroblasts isolated from NFs and human melanocytes were irradiated with excimer light in the presence or absence of calcitriol or

tacalcitol, and number of cells per dish was counted. Data for fibroblasts (A) and melanocytes (B) are shown.

in mast cells. With regard to Schwann cells, neither VD3 nor tacalcitol had an effect

, this value rose with statistical

whereas with excimer light irradiation at a dose of 300 mJ/cm2

166 A Critical Evaluation of Vitamin D - Clinical Overview

cells by approximately 38% in a statistically significant manner (**Figure 3**).

300 mJ/cm2

**Figure 3.** Inhibitory effects of excimer light irradiation with or without calcitriol or tacalcitol treatment on the growth of fibroblasts (A), mast cells (B), and Schwann cells (C). \**P* < 0.05 and \*\**P* < 0.01 vs. control cells. Values represent mean ± standard deviation.

As we did not see any effect of VD3 or its analogs on Schwann cells, we investigated whether rapamycin or lovastatin inhibited growth of these cells using isolated primary fibroblasts as a control. Growth of primary Schwann cells isolated from NFs was inhibited by 0.1–10 nM rapamycin or 0.1–10 μM lovastatin in a dose-dependent manner. Furthermore, combination of 0.3 nM rapamycin with 1 μM lovastatin resulted in a considerable additive inhibitory effect on the growth of Schwann cells. Growth inhibition rates for fibroblasts with either rapamycin or lovastatin were consistently less than those of Schwann cells (**Figure 4**). We also examined whether a combination of rapamycin and/or lovastatin with VD3 augmented growth inhibitory effects on Schwann cells and fibroblasts. Our experimental results showed that addition of 0.1 μM calcitriol slightly diminished the growth inhibitory effect of 0.3 nM of rapamycin and 1 μM of lovastatin. In contrast, the combination of these agents showed additive effects on fibroblast growth inhibition (data not shown).

**Figure 4.** Significant growth inhibition of Schwann cells(A) and fibroblasts(B) isolated from NFs by 0.3 nM rapamycin and/or 1 μM lovastatin.

We investigated whether long-term whole body NB-UVB irradiation increased serum VD3 levels in patients with NF1 and found that patients irradiated once weekly or biweekly (0.2– 0.5 J/cm2 dose per irradiation) for at least 18 months had significantly increased serum VD3 levels compared with patients receiving no treatment (**Figure 5**). A kinetic analysis of this increase subsequently carried out in nine patients revealed that 6 months of irradiation was enough to significantly increase serum VD3 levels (**Figure 6**). Finally, we also observed that significant increases in serum VD3 levels occurred when irradiation with NB-UVB was continued for more than 1 year in patients previously demonstrating low serum VD3 levels after an initial 6 months of irradiation (**Figure 7**).

**Figure 5.** Increase in serum VD3 levels in NF1 patients irradiated with NB-UVB at doses of 0.2–0.5 J/cm2 once weekly or biweekly for more than 18 months; levels compared with untreated patients (\**P* < 0.05).

**Figure 4.** Significant growth inhibition of Schwann cells(A) and fibroblasts(B) isolated from NFs by 0.3 nM rapamycin

We investigated whether long-term whole body NB-UVB irradiation increased serum VD3 levels in patients with NF1 and found that patients irradiated once weekly or biweekly (0.2–

levels compared with patients receiving no treatment (**Figure 5**). A kinetic analysis of this increase subsequently carried out in nine patients revealed that 6 months of irradiation was enough to significantly increase serum VD3 levels (**Figure 6**). Finally, we also observed that significant increases in serum VD3 levels occurred when irradiation with NB-UVB was continued for more than 1 year in patients previously demonstrating low serum VD3 levels

**Figure 5.** Increase in serum VD3 levels in NF1 patients irradiated with NB-UVB at doses of 0.2–0.5 J/cm2

or biweekly for more than 18 months; levels compared with untreated patients (\**P* < 0.05).

once weekly

dose per irradiation) for at least 18 months had significantly increased serum VD3

and/or 1 μM lovastatin.

168 A Critical Evaluation of Vitamin D - Clinical Overview

after an initial 6 months of irradiation (**Figure 7**).

0.5 J/cm2

**Figure 6.** Time course of serum VD3 levels in NF1 patients irradiated with NB-UVB. Serum VD3 levels of patients before and after 6 months of NB-UVB irradiation were measured (\**P* < 0.05).

**Figure 7.** Increase in serum VD3 levels in NF1 patients who had not shown an increase in VD3 levels, even after an initial 6 months of NB-UVB irradiation, after continuing irradiation treatment for more than 1 year thereafter. Statistical analysis was performed using a paired *t*-test (\*\*\**P* < 0.001).

We examined whether CALMs, small pigmented freckles, or generalized hyperpigmentation improved in 10 patients after more than 6 months of NB-UVB irradiation (once weekly or biweekly). Using photography, we observed general hyperpigmentation in most patients receiving NB-UVB irradiation became brighter; although effects on CALMs and small pigmented spots were not apparent within this period (**Figure 8**). A questionnaire completed by the patients indicated that 80% (8/10) believed they had either excellent or fair improvement of pigmented skin lesions, and all of the patients were satisfied with the results of NB-UVB irradiation.

**Figure 8.** Photographs of three typical cases, showing lightened skin color of generalized hyperpigmentation after NB-UVB irradiation at doses of 0.3–0.5 J/cm2 weekly or biweekly for more than 6 months.

Next, the effects of combining IPL-RF irradiation with topical OCT application on CALMs and multiple small pigmented spots were investigated. We IPL-RF irradiated multiple pigmented spots on the body of a 27-year-old female patient. Multiple pigmented spots received either OCT only or IPL-RF irradiation only for comparison to the area treated with both IPL-RF and OCT. Remarkable improvement of multiple pigmented spots was observed after six IPL-RF irradiation procedures and continuous topical OCT application over a 4-month period (**Figure 9**). In addition, the area treated only with IPL-RF irradiation showed similar improvements to the combination treatment area; however, areas treated with OCT only or OCT plus IPL-RF exhibited increased lightness of skin appearance, as measured by colorimeter [14]. We then treated an additional eight NF1 patients with this combination therapy and observed a moderate to good response with regard to lightening of pigmented spots in six patients [15].

biweekly). Using photography, we observed general hyperpigmentation in most patients receiving NB-UVB irradiation became brighter; although effects on CALMs and small pigmented spots were not apparent within this period (**Figure 8**). A questionnaire completed by the patients indicated that 80% (8/10) believed they had either excellent or fair improvement of pigmented skin lesions, and all of the patients were satisfied with the results of NB-UVB

**Figure 8.** Photographs of three typical cases, showing lightened skin color of generalized hyperpigmentation after NB-

Next, the effects of combining IPL-RF irradiation with topical OCT application on CALMs and multiple small pigmented spots were investigated. We IPL-RF irradiated multiple pigmented

weekly or biweekly for more than 6 months.

UVB irradiation at doses of 0.3–0.5 J/cm2

irradiation.

170 A Critical Evaluation of Vitamin D - Clinical Overview

**Figure 9.** Effects of combining IPL-RF irradiation with topical OCT treatment on multiple pigmented spots on the torso of a 27-year-old female NF1 patient. Six courses of irradiation with a light fluence of 15–25 J/cm2 and RF energy 15– 25 J/cm2 were carried out over a 4-month period. Irradiation of 70–100 shots/100 cm2 was performed once every 2 or 4 weeks. Photographs shown were taken before (A) and 6 months after (B) treatment.

With regard to the effects of topical VD3 analogs in combination with laser irradiation on CALMs, we experienced one case of a young male patient who had a conspicuous CALM on his nose. The patient had previously been treated with Q-switched Ruby laser irradiation in a cosmetic clinic for a long time without success before he visited our dermatology clinic at Fukuoka University Hospital. The pigmented macule was first treated only with topical OCT application twice a day for 10 months. After that, Q-switched Ruby laser irradiation (8 J/cm2 ) was performed twice every 3 months along with continuing topical OCT application. The CALM showed fair improvement after the initial 10 months of topical OCT application and then virtually disappeared over the course of the next 6 months with combination treatment (**Figure 10**).

Recently, an irradiation procedure known as laser toning has been applied to alleviate facial melasma of women for cosmetic purposes [16, 17]. Given that our previous experience using laser toning with an Nd:YAG laser for melasma brought about fairly good results, we applied this procedure to CALMs and small pigmented freckles on seven NF1 patients in combination with topical tacalcitol application. Side-by-side therapy with either laser toning alone or in combination with tacalcitol treatment was adopted. A typical case, a 17-year-old female patient with NF1, is shown in **Figure 11**.

**Figure 10.** CALM on the nose of a 20-year-old male NF1 patient treated with OCT in combination with Q-switched Ruby laser irradiation. A) Before treatment, (B) after 10 months of topical OCT application twice a day, and (C) after 6 months of Q-switched Ruby laser irradiation twice every 3 months in combination with topical OCT treatment.

**Figure 11.** Side-by-side treatment of hyperpigmentation and small pigmented spots using Nd:YAG laser toning with or without topical tacalcitol ointment. Pigmented lesions of a 17-year-old female patient were treated seven times with laser toning only (right side) or seven times with laser toning in combination with topical tacalcitol ointment (left side). Pigmentation of her left side was more improved compared with the right side.

Her facial pigmented spots were treated with either laser toning alone (right side) or laser toning plus tacalcitol treatment (left side, **Figure 11**). Hyperpigmentation around her mouth and small pigmented spots around her neck became lighter on the left side compared with those on the right. Since laser toning with an Nd:YAG laser exerts effects mainly on epidermal melanocytes, this treatment was thought to have little beneficial effect on CALMs. However, side-by-side therapy revealed that a combination of laser toning with topical tacalcitol application caused more lightening in the examined area compared with laser toning treatments alone (**Figure 12**).

**Figure 12.** Side-by-side treatment of a CALM using Nd:YAG laser toning with or without topical tacalcitol ointment. A CALM of a 28-year-old male patient was treated five times with laser toning only (upper half) or five times with laser toning and topical tacalcitol ointment (lower half).
