Low wintertime pre-diagnostic vitamin D status is associated with an increased risk of internal malignancies in kidney transplant recipients.

Low serum 25-hydroxyvitamin D (25OHD) concentrations have been associated with increased cancer risk, but the relative importance of seasonality, i.e. high summer concentrations versus low winter concentrations, is unclear. We investigated this issue in a high risk group: kidney transplant recipients with known increased risk of cancer and low vitamin D statuses. We examined the relationship between registered concentrations of 25OHD binned by quarter and subsequent risk of internal malignancy or cutaneous squamous cell carcinoma in 1112 kidney transplant recipients. Hazard ratios for internal malignancies were significantly increased with lower pre-diagnostic 25OHD concentrations in the first quarter of the year (January-March); a 1.4 fold increase (95%CI 1.1;1.7) per 10 nmol L-1 decrease in 25OHD. Except for women in April-June (1.3 (1.01;1.7) per 10 nmol L-1 decrease) pre-diagnostic 25OHD concentrations in the other quarters were not statistically significantly associated with internal malignancies. Higher 25OHD concentrations tended to be associated with the development of cutaneous squamous cell carcinomas, independent of the time of the year. Our study indicates that low wintertime 25OHD concentrations are associated with an increased risk of internal malignancies and that transplant recipients may benefit from wintertime vitamin D supplementation. Our findings need further corroboration, but suggest that the lowest concentrations of vitamin D, which occur in winter, are important for the risk of internal malignancies.

Psoriasis and daily low-emission phototherapy: effects on disease and vitamin D level.

BACKGROUND/PURPOSE: Hospital-based phototherapy is a widely accepted treatment modality in psoriasis patients. It, however, requires several hospital visits weekly, interfering with (school)work. Home ultraviolet (UV) treatment has been proven effective before but is only available in certain countries, and safety aspects play a part in reluctancy to prescribe this treatment. Patients, however, are usually keen on the use of phototherapy as it is effective and gives them the possibility of reducing the amount of topical treatment needed. In this study, we assess the effectivity of a low-emission UV device used daily. METHODS: Sixty-two patients were treated for 6 months either with daily low-emission UV treatment and mometasone ointment 0.1% or with mometasone ointment 0.1% alone. Psoriasis severity scores, quality of life, vitamin D level, and blood pressure were monitored every 2 months during the study. RESULTS: Patients treated with daily low-emission UV treatment showed a significant improvement in psoriasis severity, quality of life, amount of steroid ointment used, and vitamin D levels. CONCLUSION: Daily low-emission UV therapy is an effective treatment for psoriasis patients, diminishing the amount of steroid ointment needed and improving disease activity, quality of life, and vitamin D scores. Further investigation, however, is necessary.

In situ detection of HY-specific T cells in acute graft-versus-host disease-affected male skin after sex-mismatched stem cell transplantation.

HY-specific T cells are presumed to play a role in acute graft-versus-host disease (aGVHD) after female-to-male stem cell transplantation (SCT). However, infiltrates of these T cells in aGVHD-affected tissues have not yet been reported. We evaluated the application of HLA-A2/HY dextramers for the in situ detection of HY-specific T cells in cryopreserved skin biopsy specimens. We applied the HLA-A2/HY dextramers on cryopreserved skin biopsy specimens from seven male HLA-A2(+) pediatric patients who underwent stem cell transplantation with confirmed aGVHD involving the skin. The dextramers demonstrated the presence of HY-specific T cells. In skin biopsy specimens of three male recipients of female grafts, 68% to 78% of all skin-infiltrating CD8(+) T cells were HY-specific, whereas these cells were absent in biopsy specimens collected from sex-matched patient-donor pairs. Although this study involved a small and heterogeneous patient group, our results strongly support the hypothesis that HY-specific T cells are actively involved in the pathophysiology of aGVHD after sex-mismatched stem cell transplantation.

The effects of a mid-winter 8-week course of sub-sunburn sunbed exposures on tanning, vitamin D status and colds.

Like UV irradiation, which generates vitamin D(3) in the skin, the hormonally active metabolite, 1,25-dihydroxyvitamin D(3), boosts innate immunity against viruses and bacteria. Epidemiologic studies have found high vitamin D levels to be associated with lower risk of infections of the upper respiratory tract (colds). We have therefore performed an intervention study in 105 young adults (ages 18-30 years; 91% female) over a mid-winter 8-week period (January-March 2010). The participants were randomised to 3 groups: (A) subjected to 3 times a week sub-sunburn sunbed exposure (n = 35), (B) daily vitamin D supplementation, @ 1000 IU (n = 37), and (C) a control group without any intervention (n = 33). The mean serum level of 25-hydroxyvitamin D (25(OH)D) dropped from 62 to 55 nmol l(-1) in group C, while these levels rose from 62 to 109 and from 58 to 93 nmol l(-1) in groups A and B, respectively (p < 0.001). The skin on the chest darkened significantly in group A (mean difference in lightness, L*, equalled -5.7, p < 0.001), correlating significantly, but weakly, with increases in 25(OH)D (3.3 nmol l(-1) per unit drop in L*, R(2) = 0.17, p = 0.014). The percentage of self-reported colds with proper signs and symptoms was only slightly and not significantly reduced in groups A and B in comparison to group C: 57 and 51 versus 67%, respectively. Hence, the sub-sunburn sunbed treatment was effective in tanning and increasing the 25(OH)D serum level, more so than 1000 IU per day, but had no appreciable effect on colds.

Exogenous Addition of Minor H Antigen HA-1+ Dendritic Cells to Skin Tissues Ex Vivo Causes Infiltration and Activation of HA-1-Specific Cytotoxic T Cells.

T cells specific for hematopoietic system restricted minor Histocompatibility (H) antigens target normal and malignant hematopoietic cells. Thus, cellular immune responses against the latter miHAS eradicate the recipient's hematopoiesis including residual leukemic cells after HLA-matched minor H antigen-mismatched stem-cell transplantation (SCT). However, there are controversial reports on the role of HA-1 in the development of graft-versus-host-disease (GVHD) as well. Here, we address the behavior of HA-1-specific cytotoxic T cells (CTLs) in an ex vivo in situ skin explant model wherein HA-1-expressing dendritic cells (DCs) were added as antigen-presenting cells (APCs). Infiltration and activation of HA-1 CTLs occurred only in those cases where both HLA-A2 and HA-1 were expressed, either by the skin or by the DCs, or by the combination of HLA-A2(+) skin and HA-1(+) DCs. These results point toward the role of recipient's HA-1(+) DCs in the chimeric patient suffering from GVHD after HA-1-mismatched SCT. Although in our model the infiltrated and activated CTLs did not cause skin tissue destruction, our results provide a first step in understanding the reported association of HA-1 mismatching with clinical GVHD.

Hydrophilic and lipophilic moisturizers have similar penetration profiles but different effects on SC water distribution in vivo.

Dry skin is often treated with hydrophilic and/or lipophilic moisturizers. Hydrophilic moisturizers must penetrate the stratum corneum (SC) deeply to function properly, whereas lipophilic moisturizers should remain in the upper SC layers. In this study, both types of moisturizers were applied on volunteers for 3 h, after which the relative amount of moisturizer and the water distribution in the SC were determined using attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy in combination with tape-stripping. The results show that while hydrophilic moisturizers penetrate much more readily than lipophilic moisturizers, the latter are abundantly present in the upper regions of the SC. It was also observed that a 3-h treatment with lipophilic moisturizer did not result in increased water levels in the SC, whereas hydrophilic moisturizers retained water where they are located. The results suggest that upon prolonged application, adequate amounts of moisturizer can be obtained in those regions where they may cause moisturization in the central part of the SC. However, a single application of 3 h is probably too short to exert increased hydration as measured with ATR-FTIR.

Preclinical studies with 5,10,15-Tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride for the photodynamic treatment of superficial mycoses caused by Trichophyton rubrum.

Dermatophytes are fungi that cause infections of keratinized tissues. We have recently demonstrated the susceptibility of the dermatophyte Trichophyton rubrum to photodynamic treatment (PDT) with 5,10,15-Tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) in 5 mm citric acid/sodium citrate buffer (pH 5.2, formulation I). In this work, we examined the penetration of Sylsens B in healthy and with T. rubrum infected skin and we investigated the susceptibility of T. rubrum to PDT using formulation I and UVA-1 radiation (340-550 nm). Skin penetration studies were performed with formulations I and II (Sylsens B in PBS, pH 7.4) applied on dermatomed skin, human stratum corneum (SC), disrupted SC by T. rubrum growth and SC pretreated with a detergent. No penetration was observed in healthy skin. Disruption of SC by preceding fungal growth caused Sylsens B penetration at pH 7.4, but not at pH 5.2. However, chemically damaged SC allowed Sylsens B to penetrate also at pH 5.2. UVA-1 PDT was applied ex vivo during two fungal growth stages of two T. rubrum strains (CBS 304.60 and a clinical isolate). Both strains could be killed by UVA-1 alone (40 J/cm(2)). Combined with formulation I (1 and 10 microm Sylsens B for, respectively, CBS 304.60 and the clinical isolate), only 18 J/cm(2) UVA-1 was required for fungal kill. Therefore, PDT with 10 microm Sylsens B (formulation I) and 18 J/cm(2) UVA-1 could be considered as effective and safe. This offers the possibility to perform clinical studies in future.

The hunt for natural skin whitening agents.

Skin whitening products are commercially available for cosmetic purposes in order to obtain a lighter skin appearance. They are also utilized for clinical treatment of pigmentary disorders such as melasma or postinflammatory hyperpigmentation. Whitening agents act at various levels of melanin production in the skin. Many of them are known as competitive inhibitors of tyrosinase, the key enzyme in melanogenesis. Others inhibit the maturation of this enzyme or the transport of pigment granules (melanosomes) from melanocytes to surrounding keratinocytes. In this review we present an overview of (natural) whitening products that may decrease skin pigmentation by their interference with the pigmentary processes.

Increased melanogenesis is a risk factor for oxidative DNA damage--study on cultured melanocytes and atypical nevus cells.

Melanin synthesis is an oxygen-dependent process that acts as a potential source of reactive oxygen species (ROS) inside pigment-forming cells. The synthesis of the lighter variant of melanin, pheomelanin, consumes cysteine and this may limit the capacity of the cellular antioxidative defense. We show that tyrosine-induced melanogenesis in cultured normal human melanocytes (NHM) is accompanied by increased production of ROS and decreased concentration of intracellular glutathione. Clinical atypical (dysplastic) nevi (DN) regularly contain more melanin than do normal melanocytes (MC). We also show that in these cultured DN cells three out of four exhibit elevated synthesis of pheomelanin and this is accompanied by their early senescence. By using various redox-sensitive molecular probes, we demonstrate that cultured DN cells produce significantly more ROS than do normal MC from the same donor. Our experiments employing single-cell gel electrophoresis (comet assay) usually reveal higher fragmentation of DNA in DN cells than in normal MC. Even if in some cases the normal alkaline comet assay shows no differences in DNA fragmentation between DN cells and normal MC, the use of the comet assay with formamidopyrimidine DNA glycosylase can disclose that the DNA of the cultured DN cells harbor more oxidative damage than the DNA of normal MC from the same person.

In vivo assessment of safety of microneedle arrays in human skin.

Microneedle arrays are promising devices for the delivery of drugs and vaccines into or the skin. However, little is known about the safety of the microneedles. In this study we obtained insight in the ability of microneedles to disrupt the skin barrier, which was evaluated by transepidermal water loss (TEWL). We also determined the safety in terms of skin irritation (skin redness and blood flow) and pain sensation. We applied microneedle arrays varying in length and shape on the ventral forearms of 18 human volunteers. An effect of needle length was observed, as TEWL and redness values after treatment with solid microneedle arrays of 400 microm were significantly increased compared to 200 microm. The blood flow showed a similar trend. Needle design also had an effect. Assembled microneedle arrays induced higher TEWL values than the solid microneedle arrays, while resulting in less skin irritation. However, for all microneedles the irritation was minimal and lasted less than 2h. In conclusion, the microneedle arrays used in this study are able to overcome the barrier function of the skin in human volunteers, are painless and cause only minimal irritation. This opens the opportunity for dermal and transdermal delivery of drugs and vaccines.

Susceptibility to UV-A and UV-B provocation does not correlate with disease severity of polymorphic light eruption.

OBJECTIVE: To examine whether the ease of disease provocation by UV-A and/or UV-B radiation correlates with clinical features of polymorphic light eruption (PLE), including those indicative of disease severity. DESIGN: Intervention study. PATIENTS: One hundred forty-three patients with PLE. INTERVENTIONS: Provocation testing with broadband UV-A and UV-B lamps. Additionally, a range of clinical characteristics of the disorder, including a 5-item PLE severity score, was assessed by questionnaire. MAIN OUTCOME MEASURES: Percentage of PLE rash induction by UV-A and UV-B provocation, differences between the skin types, and correlation between the results of provocation and a range of clinical characteristics of the disorder, including a 5-item PLE severity score. RESULTS: Rash provocation was seen in 78.3% of patients after UV-A and in 46.7% after UV-B exposure. Neither UV-A nor UV-B provocation showed a significant association with the total 5-item severity score. The UV-B reactivity was associated with a high score on the severity item "number of months affected per year" (P = .04), whereas UV-A responsiveness showed a tendency for association with facial involvement (P = .06). CONCLUSION: The objective assessment of UV-A or UV-B susceptibility in this large group of patients showed no significant relationship with clinical disease severity.

Acitretin induces capillary leak syndrome in a patient with pustular psoriasis.

Capillary leak syndrome is a rare and potentially life-threatening condition caused by a shift of intravascular fluid and proteins to the interstitial space. We describe a patient with pustular psoriasis in whom capillary leak syndrome developed after the start of acitretin. Immediate withdrawal of retinoic acid is necessary and corticosteroid therapy should be considered.

Tyrosine-induced melanogenesis shows differences in morphologic and melanogenic preferences of melanosomes from light and dark skin types.

The quality, quantity and distribution of melanosomes in epidermis play a crucial role in the determination of skin color and its sensitivity to UV radiation. Melanocyte cultures originating from individuals with light and dark skin types were grown in media with varying concentration of L-tyrosine. Melanosomal melanin content and the size of the organelles were measured after subcellular fractionation. In light-skin type cells, increased melanin production resulted in a more elliptical shape of melanosomes. In melanosomes that constitutively produce more melanin, the tyrosine-induced melanogenesis caused enlargement in all dimensions. X-ray microanalysis provided evidence that the increase in sulfur content induced by high tyrosine concentration was more prominent in the melanosomes from light skin types. A ratio between pheomelanin and eumelanin found in light-skin type melanosomes by HPLC was increased more markedly than that in melanosomes from dark skin melanocytes. These findings suggest that the melanocytes of light-skinned individuals exhibit a preference for pheomelanogenesis. Pheomelanin production is a thiol-consuming process and that might increase the risk of oxidation stress in these cells. This fact, together with the limited ability of pheomelanin to absorb UV radiation may lead to an elevated skin cancer risk among light-skinned individuals.

Disturbed melanin synthesis and chronic oxidative stress in dysplastic naevi.

Dysplastic naevi (DN) are a known risk factor for malignant melanoma. Their occurrence is closely connected with the degree of skin pigmentation. People with a light complexion are more likely to develop DN than dark-skinned individuals. We examined the proposition that DN exhibit altered melanin formation, which may be involved in their malignant transformation. X-ray microanalysis was used to study the composition of melanosomes from DN and to compare the results with those obtained from melanomas, banal (dermal) naevi and normal cutaneous melanocytes. We analysed sulphur (an indicator of phaeomelanin) and two metals, iron and calcium, involved in oxidative stress. FACS analysis of dihydrorhodamine-123-labelled cells was employed to quantify differences in the production of radical oxygen species in DN cells and normal skin melanocytes. A significantly higher sulphur content was found in melanosomes from DN cells and melanoma cells when compared with normal melanocytes and naevus cells from banal naevi. In addition, melanosomes of DN cells and melanoma cells contained higher amounts of iron and calcium. In the case of calcium, this was associated with a significantly elevated cytoplasmic concentration. FACS analysis showed that DN cells exhibited higher concentrations of radical oxygen species than normal skin melanocytes from the same individuals. We propose that increased phaeomelanogenesis in DN cells is connected with oxidative imbalance, which is reflected by increased intracellular concentrations of reactive oxygen species and raised calcium and iron concentrations. We show that the metabolic alterations in DN cells resemble those found in melanoma cells. Our findings provide support for the idea that DN cells are true precursor lesions of melanoma.

Homozygous germline mutation of CDKN2A/p16 and glucose-6-phosphate dehydrogenase deficiency in a multiple melanoma case.

CDKN2A is regarded as a major melanoma susceptibility gene. A 19 bp deletion has been detected within Dutch families with familial atypical multiple mole-melanoma syndrome. Genetic analysis revealed two individuals with germline deletions in both copies of CDKN2A. One of them did not develop atypical naevi or melanoma, but died of adenocarcinoma at the age of 54 years. This report describes the results of the investigation of the second p16-null individual, who was also found to have glucose-6-phosphate dehydrogenase (G-6-PD) deficiency and who has developed many atypical naevi and seven melanomas. Using electron microscopic techniques, striking alterations in melanosomal structures and deviations in their sulphur, iron and calcium composition indicating a strong preference for phaeomelanogenesis and increased oxidative stress were found in the naevus cells of the patient. Using an in vitro model, we demonstrated that leaking melanin precursors may strongly enhance oxidative DNA damage through iron release from ferritin. We conclude that the homozygous p16 deletion is not sufficient for the development of a dysplastic naevus phenotype and melanoma. However, when an additional modifying factor, such as G-6-PD deficiency, increases the level of oxidative DNA damage in melanin-producing cells, the risk of developing atypical naevi and their malignant transformation may increase significantly.

Photodynamic and Nail Penetration Enhancing Effects of Novel Multifunctional Photosensitizers Designed for The Treatment of Onychomycosis.

Novel multifunctional photosensitizers (MFPSs), 5,10,15-tris(4-N-methylpyridinium)-20-(4-phenylthio)-[21H,23H]-porphine trichloride (PORTH) and 5,10,15-tris(4-N-methylpyridinium)-20-(4-(butyramido-methylcysteinyl)-hydroxyphenyl)-[21H,23H]-porphine trichloride (PORTHE), derived from 5,10,15-Tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) and designed for treatment of onychomycosis were characterized and their functionality evaluated. MFPSs should function as nail penetration enhancer and as photosensitizer for photodynamic treatment (PDT) of onychomycosis. Spectrophotometry was used to characterize MFPSs with and without 532 nm continuous-wave 5 mW cm(-2) laser light (± argon/mannitol/NaN3 ). Nail penetration enhancement was screened (pH 5, pH 8) using water uptake in nails and fluorescence microscopy. PDT efficacy was tested (pH 5, ± argon/mannitol/NaN3 ) in vitro with Trichophyton mentagrophytus microconida (532 nm, 5 mW cm(-2) ). A light-dependent absorbance decrease and fluorescence increase were found, PORTH being less photostable. Argon and mannitol increased PORTH and PORTHE photostability; NaN3 had no effect. PDT (0.6 J cm(-2) , 2 µm) showed 4.6 log kill for PORTH, 4.4 for Sylsens B and 3.2 for PORTHE (4.1 for 10 µm). Argon increased PORTHE, but decreased PORTH PDT efficacy; NaN3 increased PDT effect of both MFPSs whereas mannitol increased PDT effect of PORTHE only. Similar penetration enhancement effects were observed for PORTH (pH 5 and 8) and PORTHE (pH 8). PORTHE is more photostable, effective under low oxygen conditions and thus realistic candidate for onychomycosis PDT.

The susceptibility of dermatophytes to photodynamic treatment with special focus on Trichophyton rubrum.

Owing to the accessibility of skin to light, many applications of photodynamic treatment (PDT) have been developed within dermatology. The recent increase of dermatological antimicrobial PDT investigations is related to the growing problem of bacterial and fungal resistance to antibiotics. This review focuses on the susceptibility of dermatophytic fungi, in particular Trichophyton rubrum, to PDT and shows its potential usefulness in treatment of clinical dermatophytoses. There are no data indicating significant differences in PDT susceptibility between various dermatophytes and it is unlikely that treatment problems of especially T. rubrum with current antimycotics would occur in case of PDT. Red light 5-aminolevulinic acid-mediated PDT is after repeated sessions successful in in vivo treatment of onychomycosis (fungal nail infection) caused by various dermatophytes. Regarding skin dermatophytoses, UVA-1 PDT with cationic porphyrins appears to be safe and efficient. Most effective toward T. rubrum ex vivo is 5,10,15-tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) when combined with UVA-1 radiation or red light; this creates the possibility of efficiently treating nail infections and remaining spores in hair follicles. If the promising in vitro and ex vivo results could be transferred to clinical practice, then PDT has a good prospect to become a worthy alternative to established antifungal drugs.

Morphological changes of the dermatophyte Trichophyton rubrum after photodynamic treatment: a scanning electron microscopy study.

Treatment strategies for superficial mycosis caused by the dermatophyte Trichophyton rubrum consist of the use of topical or oral antifungal preparations. We have recently discovered that T. rubrum is susceptible to photodynamic treatment (PDT), with 5,10,15-tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) as a photosensitizer. The susceptibility appeared to depend on the fungal growth stage, with PDT efficacy higher with microconidia when compared to mycelia. The aim of this study was to investigate, with the use of scanning electron microscopy, the morphological changes caused by a lethal PDT dose to T. rubrum when grown on isolated human stratum corneum. Corresponding dark treatment and light treatment without photosensitizer were used as controls. A sub-lethal PDT dose was also included in this investigation The morphologic changes were followed at various time points after the treatment of different fungal growth stages. Normal fungal growth was characterized by a fiber-like appearance of the surface of the hyphae and microconidia with the exception of the hyphal tips in full mycelia and the microconidia shortly after attachment to the stratum corneum. Here, densely packed globular structures were observed. The light dose (108 J/cm2) in the absence of Sylsens B, or the application of the photosensitizer in the absence of light, caused reversible fungal wall deformations and bulge formation. However, after a lethal PDT, a sequence of severe disruptions and deformations of both microconidia and the mycelium were observed leading to extrusion of cell material and emptied fungal elements. In case of a non-lethal PDT, fungal re-growth started on the remnants of the treated mycelium.

Investigation of conditions involved in the susceptibility of the dermatophyte Trichophyton rubrum to photodynamic treatment.

BACKGROUND: Photodynamic treatment (PDT) refers to a treatment with light-activated agents (photosensitizers) in combination with visible light and molecular oxygen. Recently, we have demonstrated that the porphyrins, 5,10,15-tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) and deuteroporphyrin monomethylester (DP mme) are excellent photosensitizers to be used against Trichophyton rubrum both in vitro and ex vivo. OBJECTIVES AND METHODS: The objective of this study was to investigate the key factors involved in PDT efficacy of both photosensitizers in an ex vivo situation during different fungal growth stages using a recently developed ex vivo model. The study focused on the influence of pH and ion strength of incubation media, photochemical properties of the photosensitizers (spectra and singlet oxygen production), and the effect of several scavengers of reactive oxygen species (sodium azide, histidine, mannitol) and phenylmethylsulphonylfluoride (keratinase inhibitor) on the PDT efficacy. RESULTS AND CONCLUSIONS: The results show that an optimal pH and low concentrations of calcium are crucial for a selective binding of Sylsens B to the fungus, leading to an increased PDT efficacy. This selective binding to T. rubrum cannot be accomplished for DP mme. It can be concluded that the prerequisite for successful treatment is a use of a low molarity solution of pH 5, supplemented with a chelating agent and a keratinase activity-repressing agent. Under these conditions, PDT with Sylsens B inactivates, initially via singlet oxygen, effectively the fungus in different fungal growth stages.