Evaluating the cytotoxic doses of narrowband and broadband UVB in human keratinocytes, melanocytes, and fibroblasts

Background: No comparative and simultaneous in vitro studies have been performed to determine the cytotoxic dose of narrowband UVB (NBUVB) and broadband UVB (BBUVB) for keratinocytes, melanocytes, and fibroblasts. Culture medium was often replaced with phosphate-buffered saline (PBS) before UV irradiation; however, its amount differed across studies. We determined the cytotoxic doses of NBUVB and BBUVB and tested for changes in viability according to the amount of PBS.
Methods: We exposed cultured human keratinocytes, melanocytes, and fibroblasts to ultraviolet light in the range 12.5–1000 mJ/cm² for NBUVB and 1.25–100 mJ/cm² for BBUVB. The viability was assessed after 24 h. We also determined changes in viability at cytotoxic doses according to the amount of PBS (40, 80, and 120 μl/well in a 96-well plate).
Results: Cytotoxicity was observed at doses of 100, 200, and 400 mJ/cm² for NBUVB and 5, 10, and 25 mJ/cm² for BBUVB in keratinocytes, melanocytes, and fibroblasts, respectively. At cytotoxic doses, there was no change in viability according to the amount of PBS.
Conclusions: Fibroblasts are more resistant to UVB irradiation, irrespective of the amount of NBUVB and BBUVB, than keratinocytes and melanocytes. The amount of PBS during irradiation had no effect on viability.     

CaF2:Dy and CaF2 crystal-based UV dosimeters

Background/aims: Monitoring of ultraviolet (UV) exposure in humans is important, since UV has been implicated in the pathogenesis of skin cancer, skin ageing and immunosuppression. Biological and physical dosimeters are being developed to measure occupational and environmental UV radiation exposure. We studied the UV-dependent thermoluminescence in CaF₂:Dy and CaF₂ crystals and report on the development of a small personal UV dosimeter based on the thermoluminescent phenomenon.
Methods: CaF₂:Dy or CaF₂ was sensitized to UV by heating for 1-3 h to 750-950^oC on different supports (porcelain, steel, preheated steel, silicon, chromium, manganese, iron, cobalt, nickel, copper, Fe₂O₃, Fe₃O₄). Sensitized crystals were irradiated with UV of different energies and wavelengths. Thermoluminescence of irradiated crystals was measured at different temperatures.
Results: Maximal sensitivity of the crystals to UV was obtained after preheating to 900^oC on steel and manganese supports. Sensitivity could be improved further by prolonging heating time. CaF₂:Dy and CaF₂ were most sensitive to short-wave UVC and UVB radiation. Based on these findings we have constructed personal UVB and UVC dosimeters.
Conclusion: Development of personal UVC and UVB dosimeters based on UV-induced thermoluminescence in CaF₂:Dy and CaF₂ crystals is feasible. CaF₂:Dy and CaF₂ crystals are not sensitive enough to long-wave UV radiation to be used for construction of UVA dosimeters.     

Narrowband UVB (311 nm, TL01) phototherapy for pityriasis lichenoides

Background/purpose: Narrowband (NB) UVB (NB-UVB) phototherapy has recently demonstrated high levels of efficacy and tolerability in a variety of skin diseases. The purpose of the present study was to assess the efficacy of NB-UVB phototherapy in the management of pityriasis lichenoides (PL).
Methods: The therapeutic response in 31 PL patients (23 pityriasis lichenoides et varioliformis acuta; PLEVA, eight pityriasis lichenoides chronica; PLC) treated with NB-UVB phototherapy between 2000 and 2007 was assessed.
Results: NB-UVB treatment led to a complete response (CR) in 15 out of 23 PLEVA patients (65.2%) with a mean cumulative dose of 23 J/cm² after a mean number of 43.4 exposures and a partial response (PR) in eight patients (34.8%) with a cumulative dose of 15.6 J/cm² after a mean number of 32.3 exposures. NB-UVB treatment led to CR in seven out of eight PLC patients (87.5%) with a mean cumulative dose of 18.4 J/cm² after a mean number of 45.8 exposures and PR in one patient (12.5%) with a cumulative dose of 9.1 J/cm² after a mean number of 19 exposures. Relapses occurred in four PL patients within a mean time period of 6 months.
Conclusion: NB-UVB therapy is an effective, safe and practical alternative treatment modality for the management of PLEVA and PLC.     

Release of nickel from coins and deposition onto skin from coin handling--comparing euro coins and SEK

Background:  Nickel exposure is the most common cause of contact allergy. The role of contact with nickel-containing coins has been controversial.
Objectives:  To compare the release of nickel from 1 and 2 EUR coins (both composed of two alloys: Cu 75%, Zn 20%, Ni 5% and Cu 75%, Ni 25%) and Swedish 1 SEK coin (alloy: Cu 75%, Ni 25%) and to assess the deposition of nickel onto skin by coin handling.
Methods:  Nickel release was determined by immersion in artificial sweat (2 min, 1 hr, 24 hr, and 1 week). Deposition of nickel onto the skin was assessed in three subjects after 1-hr handling of 2 EUR and 1 SEK coins. Samples ( n  = 48) were taken from fingers and palms by acid wipe sampling and analysed by inductively coupled plasma mass spectrometry.
Results:  Amounts of nickel released by 1 week from 1 SEK, 1 EUR, and 2 EUR coins were 121, 86, and 99 μg/cm², respectively. Corresponding 2 min values were 0.11, 0.25, and 0.22 μg/cm². Nickel was deposited onto the skin by 1 hr coin handling (range 0.09–4.1 μg/cm²), the largest amounts were on fingers; similar amounts of nickel were deposited from 1 SEK and 2 EUR coins.
Conclusions:  Nickel is released from 1 and 2 EUR and 1 SEK coins at similar amounts. Nickel is deposited onto skin at substantial and similar amounts by coin handling. Acid wipe sampling is suitable for studies of skin exposure to nickel and in risk assessment.     

Modern tattoos cause high concentrations of hazardous pigments in skin

Background:  Modern tattoo colourants frequently consist of azo pigments that not only contain multiple impurities but also are originally produced for car paint and the dyeing of consumer goods.
Objective:  In order to be able to assess the health risk of tattoos, it is important to determine the pigment concentration in human skin.
Methods:  We tattooed excised pigskin and human skin with a common tattoo pigment (Pigment Red 22) under various conditions. After tattooing, we quantitatively extracted the pigment in order to determine the pigment concentration in skin.
Results:  The concentration of pigments ranged from about 0.60 to 9.42 mg/cm² of tattooed skin (mean value 2.53 mg/cm²) depending upon the size of the pigment crystals, the pigment concentration applied to the skin surface, and the respective procedure of tattooing.
Conclusion:  In conclusion, high concentrations of colourants are injected into the skin during tattooing and based upon this quantification, a risk assessment of tattooing ought to be carried out.     

Thioredoxin upregulation by 5-aminolaevulinic acid-based photodynamic therapy in human skin squamous cell carcinoma cell line

Background/purpose: 5-aminolaevulinic acid-based photodynamic therapy (ALA-PDT) is widely performed in the clinical setting for superficial skin cancers, giving favorable results, but residual tumor and recurrence occur occasionally. Thioredoxin is a common antioxidant that suppresses apoptosis and facilitates cell growth. We investigated the expression of thioredoxin following ALA-PDT in human skin squamous cell carcinoma cell line, HSC-5.
Methods: ALA-PDT was performed in HSC-5 cells using low-dose (5 J/cm², 100 mW/cm²) or high-dose (30 J/cm², 100 mW/cm²) irradiation, and the expression of thioredoxin was measured by Western blotting. An MTT assay was used to assess cell growth following a low dose of multiple irradiations. Cell death was examined by Western blotting for caspase-3 and PARP. Immunofluorescence double staining using annexin V and propidium iodine was also performed.
Results: Expression of thioredoxin was only observed following low-dose exposure ALA-PDT. Multiple low-dose exposure ALA-PDT significantly proliferated cell growth. With high-dose exposure ALA-PDT, caspase-3 and PARP expression were seen, and cell death due to apoptosis and/or necrosis was observed, but thioredoxin was barely detected.
Conclusion: Low-dose exposure ALA-PDT increased the expression of thioredoxin and facilitated the growth of HSC-5 cells.     

Transcutaneous pO2 imaging during tourniquet-induced forearm ischemia using planar optical oxygen sensors

Background: Oxygen-dependent quenching of luminescence using transparent planar sensor foils was shown to overcome the limitations of the polarographic electrode technique in an animal model. This method was then transferred to a clinical setting to measure the transcutaneous pO₂ (p_tcO₂).
Methods: In six healthy subjects, a cuff on the upper arm was occluded up to 20 mmHg above systolic pressure and released after 8 min. P_tcO₂ was measured at the lower arm every 30 s before, during, and up to 20 min after cuff occlusion (40 °C applied skin temperature) using luminescence lifetime imaging (LLI) of platinum(II)-octaethyl-porphyrin immobilized in a polystyrene matrix. For validation, the polarographic Clark electrode technique was applied in close proximity, and measurements were conducted simultaneously.
Results: P_tcO₂ measurements before (70.8±19.1 vs. 66.2±7.7 mmHg) and at the end of ischemic (2.7±1.2 vs. 3.6±1.7 mmHg) and reperfusion phases (72.2±3.6 vs. 68.4±8.9 mmHg) did not differ significantly using the Clark electrode vs. LLI. At both the initial ischemic and the reperfusion phases, the Clark electrode measured a faster decrease or increase, respectively, in p_tcO₂ because of the oxygen consumption occurring in this method.
Conclusion: The presented method provides accurate and reproducible p_tcO₂ values under changing microcirculatory conditions. The lack of oxygen consumption during measurement allows both a more realistic estimation of p_tcO₂ than compared with the gold standard and permanent use in regions with critical oxygen supply.     

Electronic UV dosimeters

Background/aims: The pathogenic role of ultraviolet (UV) in the development of skin cancer, skin ageing and immunosuppression makes it important to monitor human exposure to UV radiation. In a previous study we constructed UVB and UVC dosimeters based on a thermoluminescent phenomenon induced by UV in CaF₂:Dy and CaF₂ crystals. However, these dosimeters were insensitive to UVA radiation and readout was time-consuming. In the present study we aimed to develop an electronic dosimeter suitable for UVA, UVB and UVC. The principle of this dosimeter is a measure of accumulated electric current induced by UV on a photodetector.
Methods: Electric current induced by UV on a photodetector was accumulated in a Plessey's E-cell coulometer. A special reading device was constructed to quantify total charge of the coulometer. Sensitivity for UVA, UVB and UVC was achieved by the use of appropriate filters in front of the photodetector.
Results: The sensitivity of the electronic dosimeter increased with increasing wavelength of UV radiation; therefore, in UVB and UVC dosimeters the use of amplifiers was necessary. A linear response to UVA, UVB and UVC was achieved.
Conclusion: Dosimeters with a linear response to increasing doses of UVA, UVB and UVC have been constructed for personal monitoring of UV exposure.     

018 New source for evaluating PDT drugs in the laboratory

To test the hypothesis that UV could alter collagen susceptibility to interstitial collagenase, acid-extracted Skh-1 hairless mouse collagen samples were (un)irradiated with 0–140 J/cm² of radiation from bank of filtered FS lamp (UVB/UVA = 0.33, fluence rate = 0.81 mW/cm²). Subsequent to UV irradiation, collagen samples were coupled with fluorescein isothiocyanate (FITC) and assayed for its susceptibility to bacterial collagenase by monitoring the appearance of supernatant FITC fluorescence (a measure of lysed collagen) over time of incubation. As a reference. unirradiated commercial FITC – labeled collagen (Elastin Products) was similarly analyzed. Mouse collagen had a lower rate of cleavage than did the calf skin sample. Mouse collagen initial cleavage followed a quasi-linear time course over the first 5 h. Calf-skin collagen displayed a 'sigmoidal' time course, reminiscent of a cooperative mechanism. UV irradiation afforded no significant effect on the ability of collagenase to cleave mouse collagen, although a small effect could be discerned after 48 h (140 J/cm²). On the other hand, these samples exhibited significant chain degradation. cross-linking and loss of intrinsic collagen fluorescence on UV photolysis. It appears that the rate of cleavage depends on the superstructure of the collagen, and that the collagen fluorophores are not in proximity to the specific site of collagenase cleavage.
Supported in part by NIH/MBRS Grant #GM 08248 and RCMI Grant #RR 03034.     

Population reference intervals for minimal erythemal doses in monochromator phototesting

Background/purpose: Monochromator phototesting, to measure the minimal erythemal dose (MED), is useful in investigating patients with abnormal photosensitivity at different wavelengths. It relies on access to reliable, up-to-date data on the MED in normal individuals. The purpose of this study was to determine MED in normal subjects at different wavebands and compare these with historical controls.
Methods: The study group consisted of 415 normal individuals (349 males) of skin types I–III living in Scotland. Age range was 18–83 years (median 31 years). Phototesting was performed using a monochromator at prescribed wavelengths from 295 to 430 nm. All calibrations were traceable to the National Physical Laboratory. Quality systems were maintained to ISO 9001 and ISO 17025 international standards and ultraviolet (UV) measurements accredited by the United Kingdom Accreditation Service (UKAS).
Results: The 95% reference interval (99% confidence interval for this) ranged from 6.8 to 27 mJ/cm² at 295 nm to >82 000 mJ/cm² at 430 nm.
Conclusions: Results of the current investigation are broadly in agreement with values published 25 years ago by this centre. This validates the phototesting process based on the use of monochromators with attention to careful control of conditions during UV exposure and MED reading, supported by dosimetric calibration.     

Mitochondrial dysfunction and cellular stress progression after ultraviolet B irradiation in human keratinocytes

Background: Ultraviolet (UV) radiation is the major environmental harmful factor that affects human skin. UVB radiation is known to be a potent inducer of reactive oxygen species (ROS) production and has also been associated with the generation of nitric oxide (NO), all of which have been implicated in various skin disorders. It is well known that mitochondria can also be affected by UVB, leading to alterations in their membrane structure and permeabilization with cytochrome c release, which consequently affects the cell function. However, the loss of keratinocyte mitochondrial function generated by UVB, as well as its kinetics, has not been characterized completely.
Methods: We evaluated the effect of UVB irradiation on HaCat cells' mitochondrial function, assessed by membrane potential loss and superoxide anion (O₂^•−) production, correlating with apoptosis, p53 expression, ROS levels and NO production, 0, 6, 12, 24 and 48 h post-irradiation.
Results: HaCat cells progressed toward apoptotic cell death as the time post-irradiation increased, with the highest levels found 48 h after irradiation. Increased levels of ROS were observed 6 h after irradiation while high O₂^•− levels and mitochondrial membrane depolarization were detected 12 h post-UVB. Nevertheless, NO production was not significantly increased at any of the evaluated times.
Conclusions: The kinetics of mitochondrial dysfunction after UVB irradiation in human keratinocytes progressed in a time post-irradiation-dependent manner, and they are closely related to cell death. However, there are certain levels of apoptosis, although low, in the absence of mitochondrial alterations. In addition, our data suggest that ROS play a greater role in keratinocyte UVB damage than reactive nitrogen species.     

Hair removal with the long-pulse alexandrite and long-pulse Nd:YAG lasers is safe and well tolerated in children

Background.  Children with excessive hair may have severe psychological consequences. Laser hair removal in adults is known to be safe and well tolerated, but this is less well established in children.
Objective.  To describe our experience with laser hair removal in children, and to investigate the safety and tolerability of this procedure in children.
Methods.  The case records of 24 children aged < 16 years, who had received a minimum of three treatments for hair removal were analysed retrospectively. For patients with Fitzpatrick skin phototype II–IV, the lasers used were a long-pulse alexandrite (755 nm) with either continuous chilled-air cooling at fluences of 16–27 J/cm² or a long-pulse alexandrite with cryogen cooling at fluences of 16–32 J/cm². For patients with Fitzpatrick skin phototype IV–VI, lasers used were a long-pulse Nd:YAG (1064 nm) with a chilled contact sapphire tip at fluences of 20–35 J/cm² or a long-pulse Nd:YAG with cryogen cooling at fluences of 16–26 J/cm².
Results.  Mean age at first treatment was 12.3 years. Diagnoses were constitutional hirsutism (14 patients), polycystic ovarian syndrome (five), congenital melanocytic naevus (two), generalized hypertrichosis (two) and naevoid hypertrichosis (one). One patient required a general anaesthetic, eight required topical anaesthetic cream, and 15 did not require any form of anaesthesia. Intolerable discomfort requiring adjustment in fluence was the only recorded side-effect, affecting two cases. There were no incidences of blistering, infection, dyspigmentation or scarring.
Conclusion.  When administered appropriately, laser hair removal is safe and well tolerated in children aged < 16 years.     

Efficacy of three different laser wavelengths for in vitro wound healing

Background and objective: Despite contradictory reports on the effect of laser light on cell proliferation, studies have shown that appropriate doses and wavelengths of laser light are therapeutically beneficial in tissue repair and pain control. This study aimed to establish if the dose and/or wavelength influenced the biological responses of irradiated in vitro fibroblasts – 1 h after laser irradiation.
Materials and methods: This study aimed to establish cellular responses of normal and wounded human skin fibroblasts to helium-neon (632.8 nm), diode (830 nm) and Nd:YAG (1064 nm) laser irradiation using one exposure of 5 or 16 J/cm² on day 1 and again on day 4.
Results: Wounded cells exposed to 5 J/cm² using 632.8 nm showed an increase in cell migration and haptotaxis, a stable increase in the release of interleukin-6 (IL-6), a decrease in caspase 3/7 activity, an increase in ATP viability and an increase in cell proliferation – 1 h after the final exposure. The results confirm that changes in parameters such as ATP viability, cytokine expression (IL-6), cell proliferation (alkaline phosphatase enzyme activity) and DNA damage can be observed directly after the laser irradiation. The amount of DNA damage and cytotoxicity may be related to duration of the laser irradiation, which is dependent on the power density (mW/cm²) of each laser.
Conclusion: The results indicate that 5 J/cm² using 632.8 nm results in a stimulatory effect that is more effective than 830 and 1064 nm. The results suggest possible mechanisms by which the wavelength may potentially influence the cellular responses of wounded cells.     

Ultraviolet B radiation suppresses Langerhans cell migration in the dermis by down-regulation of alpha4 integrin

Background/Purpose: Ultraviolet B (UVB) radiation affects the migration and function of epidermal Langerhans cells (LC) and causes immunosuppression of contact hypersensitivity. It is known that LC leaves the epidermis after exposure to UVB. To know the behavior of LC in the dermis after UVB radiation, we studied the effect of UVB radiation on the expression of integrin families on freshly isolated or cultured murine LC. We also examined whether UVB radiation affects the migration of LC to secondary lymphoid tissue chemokine (SLC/6Ckine).
Methods: Integrin expressions of murine LC cultured in epidermal cell suspension were analyzed using flowcytometry. We used murine LC sorted flowcytometrically for binding assay to extracellular matrix and for migration assay to chemokine. Skin explant assay and immnohistochemical staining for 'cords formation' were performed as previously described.
Results: Twenty and 40 mJ/cm² of UVB radiation down-regulated the expression of α4 integrin on 24 h-cultured LC, but not that of α6, β1, or β4 integrin. The number of cultured LC adhered to fibronectin, a ligand for α4 integrin, was decreased after UVB irradiation, while that to laminin, a ligand for α6 integrin, was not influenced. UVB radiation reduced the number of migrating LC to SLC. Furthermore, skin sheet explant experiments showed that UVB radiation inhibited the 'cords' formation in dermal vessels of the 48 h-cultured skin.
Conclusions: These data suggest that UVB radiation may suppress the migration of LC from the dermis to lymphatic vessels. UVB radiation may downregulate the adherence of LC to dermal fibronectin and migration to SLC, and consequently suppress the migration of LC from the UVB-irradiated dermis to lymphatics.     

Deposition of nickel, chromium, and cobalt on the skin in some occupations - assessment by acid wipe sampling

Background:  Nickel, chromium, and cobalt are important skin sensitizers. Better knowledge about skin exposure is needed for more efficient prevention. We have previously developed acid wipe sampling for assessment of skin exposure to metals.
Objectives:  To apply the acid wipe sampling technique in some occupations where intense contact with metallic items occurs and to gather experience for the design of future workplace studies.
Methods:  18 volunteers (carpenters, locksmiths, cashiers, and secretaries as controls) participated. They performed their normal tasks during a job session for exposure. Samples were taken from fingers and palms by acid wipe sampling, and analysis of metals was performed by inductively coupled plasma mass spectrometer.
Results:  The metals were detected in all samples, and the amount of nickel was larger than that of chromium and cobalt. Fingers were more exposed than palms. 8-h exposure to nickel was calculated and was highest in locksmiths (mean 3.784 μg/cm², range 1.846–5.028 μg/cm²) followed by carpenters, cashiers, and secretaries.
Conclusions:  The acid wipe sampling technique is suitable for studies of skin exposure to nickel, chromium, and cobalt in the workplace. The sampling efficiency of acid wipe sampling is high. The amounts of nickel deposited on skin in carpenters, locksmiths, and cashiers are judged capable of eliciting allergic contact dermatitis.     

Polypodium leucotomos inhibits ultraviolet B radiation-induced immunosuppression

Background: An extract of the tropical fern Polypodium leucotomos (PL) administered orally to mice inhibits ultraviolet B (UVB) radiation-induced skin cancer formation. UVB-induced murine skin cancers occur, in part, because of UVB-induced immunosuppression. Thus, we examined whether PL inhibits UVB-suppression of the induction of contact hypersensitivity (CHS) locally or systemically.
Methods: C57BL/6 mice received standard drinking water or water-containing PL. In the local model, mice were shaved on the dorsum and exposed to 3500 J/m² of UVB radiation daily for 4 days. Control mice were not irradiated. After the last irradiation they were sensitized to oxazolone topically at the irradiated site. To examine the ability of PL to inhibit systemic UVB-induced immunosuppression, mice were given 10 000 J/m² of UVB radiation once and immunized at a non-exposed site 3 days later. Six days after immunization (in both models), mice were challenged on the ears with oxazolone and 24/48 h ear swelling assessed.
Results: PL in drinking water significantly reduced the inhibition of CHS observed with exposure to UVB radiation in both the local and systemic models.
Conclusions: The ability of PL to inhibit UVB radiation-induced immune suppression may explain, in part, its ability to inhibit UVR-induced skin cancer induction in mice.     

Responses of the 27-kDa heat shock protein to UVB irradiation in human epidermal melanocytes

Abstract:  Solar ultraviolet radiation (UVR) is a major environmental hazard for the skin, and UVB (280–320 nm) has been proposed to be a main factor for melanoma development. In response to sunlight exposure, the skin has adapted a number of innate resistance mechanisms. Among them is the small heat shock protein of 27 kDa (HSP27) known to play a role in the protection of cells from variety of environmental insults including UV irradiation. In this study, we demonstrated that UVB irradiation of cultured normal epidermal melanocytes initiates changes in HSP27 phosphorylation and localization. In unstressed melanocytes, HSP27 was present as the non-phosphorylated isoform. UVB irradiation with a physiological dose (7–25 mJ/cm²) resulted in the formation of a mono-phosphorylated isoform and sometimes a bi-phosphorylated isoform. The UVB-induced HSP27 phosphorylation was inhibited when melanocytes were treated with the antioxidant N -acetyl cysteine or inhibitor of p38 MAP kinase prior to UVB exposure, suggesting that UVB induced HSP27 phosphorylation through reactive oxygen species/p38 MAP kinase pathway. In response to UBV irradiation, HSP27 in melanocytes translocated from the cytoplasm to the nucleus. The HSP27 responses may provide some protective role against UVB-induced cell damage in the skin.     

The neurotrophin network in human skin

Acetylcholine (Ach) has been shown to be synthesized de novo and degraded in the human skin ( 1 ), while the presence of acetylcholinesterase (AchE), the degrading enzyme, was first mentioned in 1989 ( 2 ). It is now accepted that H₂O₂-related oxidative stress is a major player in the development/acceleration of vitiligo ( 3 ). This oxidative stress affects the recycling of the essential cofactor (6R)- l -erythro 5,6,7,8 tetrahydrobiopterin (6BH4) via H₂O₂-mediated oxidation of Trp and Met residues in the structure of pterin-4a carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR) ( 3 ). Only very recently, it was recognized that AchE is also affected and deactivated by 10⁻³  m H₂O₂ causing the accumulation of Ach in the epidermis of patients with vitiligo ( 4 ). One of the implications of oxidative stress in vitiligo includes pruritus, which was for a long time attributed to the presence of epidermal H₂O₂ in the 10⁻³  m range. In this context, a role for Ach has been suggested in association with pruritus, and therefore, we would like to suggest that Ach accumulation caused by H₂O₂-mediated deactivation of AchE may well initiate pruritus ( 5 ). The Ach accumulation can also explain the earlier documented decreased sweating in patients with vitiligo ( 6 ).