Phenotypic plasticity of melanocytes derived from human adult skin.

We previously described a novel in vitro culture technique for dedifferentiated human adult skin melanocytes. Melanocytes cultured in a defined, cholera toxin and PMA free medium became bipolar, unpigmented, and highly proliferative. Furthermore, TRP-1 and c-Kit expression disappeared and EGFR receptor and nestin expression were induced in the cells. Here, we further characterized the phenotype of these dedifferentiated cells and by comparing them to mature pigmented melanocytes we detected crucial steps in their phenotype change. Our data suggest that normal adult melanocytes easily dedifferentiate into pluripotent stem cells given the right environment. This dedifferentiation process described here for normal melanocyte is very similar to what has been described for melanoma cells, indicating that phenotype switching driven by environmental factors is a general characteristic of melanocytes that can occur independent of malignant transformation.

Melanoma-Derived BRAF(V600E) Mutation in Peritumoral Stromal Cells: Implications for in Vivo Cell Fusion.

Melanoma often recurs in patients after the removal of the primary tumor, suggesting the presence of recurrent tumor-initiating cells that are undetectable using standard diagnostic methods. As cell fusion has been implicated to facilitate the alteration of a cell's phenotype, we hypothesized that cells in the peritumoral stroma having a stromal phenotype that initiate recurrent tumors might originate from the fusion of tumor and stromal cells. Here, we show that in patients with BRAF(V600E) melanoma, melanoma antigen recognized by T-cells (MART1)-negative peritumoral stromal cells express BRAF(V600E) protein. To confirm the presence of the oncogene at the genetic level, peritumoral stromal cells were microdissected and screened for the presence of BRAF(V600E) with a mutation-specific polymerase chain reaction. Interestingly, cells carrying the BRAF(V600E) mutation were not only found among cells surrounding the primary tumor but were also present in the stroma of melanoma metastases as well as in a histologically tumor-free re-excision sample from a patient who subsequently developed a local recurrence. We did not detect any BRAF(V600E) mutation or protein in the peritumoral stroma of BRAF(WT) melanoma. Therefore, our results suggest that peritumoral stromal cells contain melanoma-derived oncogenic information, potentially as a result of cell fusion. These hybrid cells display the phenotype of stromal cells and are therefore undetectable using routine histological assessments. Our results highlight the importance of genetic analyses and the application of mutation-specific antibodies in the identification of potentially recurrent-tumor-initiating cells, which may help better predict patient survival and disease outcome.

Different activations of toll-like receptors and antimicrobial peptides in chronic rhinosinusitis with or without nasal polyposis.

Both up- and down-regulation of the Toll-like receptors (TLRs) and antimicrobial peptides (AMPs) of the sinonasal mucosa have already been associated with the pathogenesis of chronic rhinosinusitis with (CRSwNP) or without (CRSsNP) nasal polyps. The objective of this study was to determine the expression of all known TLR and several AMP genes and some selected proteins in association with allergy, asthma and aspirin intolerance (ASA) in CRS subgroups. RT-PCR was applied to measure the mRNA expressions of 10 TLRs, four defensins, lysozyme, cathelicidin and lactoferrin (LTF) in sinonasal samples from patients with CRSsNP (n = 19), CRSwNP [ASA(-): 17; ASA(+): 7] and in control subjects (n = 12). Protein expressions were detected with immunohistochemistry (n = 10). Statistical analysis was done with the Kruskal-Wallis ANOVA, Mann-Whitney U, and Student t test. TLR2, TLR5, TLR6, TLR7, TLR8, TLR9, ß-defensins 1 and 4, cathelicidin and LTF mRNA expressions were significantly (p < 0.05) increased in CRSwNP, whereas only TLR2 and LTF were up-regulated in CRSsNP compared to controls. There was no statistical difference in respect of allergy, aspirin intolerance and smoking between CRSsNP, ASA(-) and ASA(+) CRSwNP patients. TLR2, TLR3, TLR4, LTF, ß defensin 2 and lysozyme protein expressions were found to be elevated in macrophages of CRSwNP samples (p < 0.05). Gene expression analysis showed markedly different expressions in CRSwNP (6 out of 10 TLR and 4 out of 7 AMP genes were up-regulated) compared to CRSsNP (1/10, 1/7). The distinct activation of the innate immunity may support the concept that CRSsNP and CRSwNP are different subtypes of CRS. These findings were found to be independent from allergy, asthma, smoking, aspirin intolerance and systemic steroid application.

A conserved linkage group on chromosome 6, the 8.1 ancestral haplotype, is a predisposing factor of chronic rhinosinusitis associated with nasal polyposis in aspirin-sensitive Hungarians.

Inflammation plays a central role in the pathogenesis of chronic rhinosinusitis (CRS), and TNFα is a key pro-inflammatory cytokine in the pathogenesis of this disease. In our previous studies, we showed that the TNFA -308A allele is a genetic predisposition factor in a subgroup of aspirin-sensitive (ASA+) CRS patients suffering from nasal polyps (NP) in the Hungarian population. To determine whether the TNF -308A allele or the presence of a complex, extended ancestral haplotype (8.1AH) located on chromosome 6 is responsible for the previously observed genetic effect, we performed a case-control study for examining the frequency of 8.1AH carriers in controls and in subgroups of CRS patients. Our novel observations demonstrate that the presence of the 8.1AH may be responsible for the development of severe forms of CRS (CRSwNP, ASA+) and strengthen the clinical observation that CRS patients can be classified into clinically and genetically different subgroups.

The -308 G>A SNP of TNFA is a factor predisposing to chronic rhinosinusitis associated with nasal polyposis in aspirin-sensitive Hungarian individuals: conclusions of a genetic study with multiple stratifications.

Single nucleotide polymorphisms (SNPs) of the tumour necrosis factor alpha (TNFα) gene (TNFA) have been extensively studied and shown to be associated with an increased risk of the development of various chronic inflammatory diseases. Inflammation has been demonstrated to play a central role in the pathogenesis of chronic rhinosinusitis (CRS), and TNFα is a key pro-inflammatory cytokine with important functions in these processes. In order to determine whether the well-known TNFA -308 G>A SNP has a role in a genetic predisposition to CRS in the Hungarian population, we analyzed our genomic collection containing control and CRS patient samples in a case-control study, and compared the genotype and allele frequencies. There was no significant difference in the observed genotype or allele frequencies between the controls and the total CRS group. However, after careful stratification of the patient group on the basis of the observed clinical symptoms, we found a significantly higher carriage rate of the rare A allele-containing genotypes among the CRS patients with nasal polyposis (NP) who also exhibited sensitivity to aspirin (acetylsalicylic acid, ASA(+)). It is concluded that genetic variants of the TNFA gene may affect the risk of CRS in a clinically well-defined group of CRSNP(+)ASA(+) patients in the Hungarian population. Our results also emphasize that the group of CRS patients is not homogenous in that patients exhibiting different clinical symptoms exist. Their carried genetic predisposing factors, and as a result, the exact molecular events leading to the development of various forms of CRS, may also differ.

The expressions of ABCC4 and ABCG2 xenobiotic transporters in human keratinocytes are proliferation-related.

Xenobiotic transporters of the ATP-binding cassette (ABC) protein superfamily play important roles in maintaining the biochemical barrier of various tissues, but their precise functions in the skin are not yet known. Screening of the expressions of the known xenobiotic transporter genes in two in vitro keratinocyte differentiation models revealed that the ABCC4 and ABCG2 transporters are highly expressed in proliferating keratinocytes, their expressions decreasing along with differentiation. Abrogation of the ABCC4 and ABCG2 protein functions by siRNA-mediated silencing and chemical inhibition did not affect the proliferation of HaCaT cells. In contrast, disruption of the ABCG2 function had no effect on normal human epidermal keratinocyte proliferation, while the inhibition of ABCC-type transporters by probenecid resulted in a striking decrease in the proliferation of the cells. These results indicate that, besides their possible therapy-modulating effects, xenobiotic transporters may contribute significantly to other keratinocyte functions, such as cell proliferation.

Neonatal blue light phototherapy and melanocytic nevi: a twin study.

BACKGROUND: Neonatal blue light phototherapy (NBLP) has been widely and successfully used for the treatment of neonatal jaundice to reduce the plasma concentration of bilirubin and, hence, to prevent kernicterus. Only a few and controversial data are available in the literature as to how NBLP influences melanocytic nevus development. OBJECTIVE: Our goal was to conduct a twin study with the aim of better understanding the role of NBLP in melanocytic nevus development. We also investigated the roles of other environmental and constitutional factors in nevus formation. METHODS: Fifty-nine monozygotic and dizygotic twins were included in this cross-sectional study. One of the twin members received NBLP, and the other did not. A whole-body skin examination was performed to determine the density of melanocytic skin lesions. The prevalence of benign pigmented uveal lesions was evaluated during a detailed ophthalmologic examination. A standardized questionnaire was used to assess data relating to constitutional, sun-exposure, and other variables. To search for possible gene-environmental interactions involved in the appearance of pigmented lesions, the melanocortin 1 receptor variants and the I439V polymorphism of histidine ammonia-lyase genes were also determined in the enrolled twins. RESULTS: NBLP was associated with a significantly higher prevalence of both cutaneous and uveal melanocytic lesions. No association was found between the examined gene polymorphisms and the number of pigmented alterations in the examined study group. CONCLUSIONS: Our data suggest that NBLP could well be a risk factor for melanocytic nevus development. Phototherapy with blue-light lamps is a standard and essential therapeutic modality in neonatal care; therefore, additional in vivo and in vitro studies are necessary to establish its potential long-term adverse effects.

The rs3761548 polymorphism of FOXP3 is a protective genetic factor against allergic rhinitis in the Hungarian female population.

We aimed to study whether forkhead box P3 (FOXP3) polymorphisms contribute to allergic rhinitis (AR) in a Central-European population, the Hungarians, similarly as it was found in Han Chinese. A case-control study was performed and the genotype distribution of the rs3761548 FOXP3 polymorphism was analyzed separately in females and in males. The results demonstrated that females homozygous for the rare FOXP3 rs3761548 allele (A/A) are protected against AR; otherwise, females who are either wild types (C/C) or heterozygote carriers (C/A) of the rare allele are more susceptible to AR (OR [95%CI] = 2.089 [1,095; 3.988]). We were able to confirm the findings of Zhang et al. in a geographically and ethnically distinct population, the Hungarians, and revealed that the rs3761548 SNP is a marker of a haplotype in these two populations, but not in Sub-Saharan Africans, suggesting that this haplotype was fixed after early modern humans left Africa.

Corrosive effects of fluoride on titanium: investigation by X-ray photoelectron spectroscopy, atomic force microscopy, and human epithelial cell culturing.

High fluoride (F(-)) concentrations and acidic pH impair the corrosion resistance of titanium (Ti). Effects of F(-)-containing caries-preventive prophylactic rinses, and gels on Ti were investigated by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Human epithelial cell attachment and proliferation were investigated by dimethylthiazol-diphenyl tetrazolium bromide (MTT) and protein content assays. Aqueous 1% NaF solution (3800 ppm F(-), pH 4.5) or high (12,500 ppm) F(-) content gel (pH 4.8) strongly corroded the surface and modified its composition. XPS revealed formation of a strongly bound F(-)-containing complex (Na(2)TiF(6)). AFM indicated an increase in roughness (R(a)) of the surfaces: 10-fold for the NaF solution and smaller for the gel or a mouthwash (250 ppm F(-), pH 4.4). MTT revealed that cell attachment was significantly increased by the gel, but was not disturbed by either the mouthwash or the NaF. Cell proliferation determined by MTT decreased significantly only for the NaF-treated samples; protein content assay experiments showed no such effect. This study indicates that epithelial cell culturing results can depend on the method used, and the adverse effects of a high F(-) concentration and low pH should be considered when prophylactic gels are applied by patients with Ti implants or other dental devices.

Effects of the neuropeptides substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide and galanin on the production of nerve growth factor and inflammatory cytokines in cultured human keratinocytes.

Neuropeptides released from the cutaneous sensory nerve endings have neurotransmitter and immunoregulatory roles; they exert mitogenic actions and can influence the functions of different cell types in the skin. The aims of this study were a systematic investigation of the effects of the neuropeptides substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and galanin (GAL) on the inflammatory cytokine production (IL-1alpha, IL-8 and TNF-alpha) of the keratinocytes, and a study of their role in the production and secretion of nerve growth factor (NGF) and its precursor molecule (proNGF). Cultures of normal human keratinocytes were treated with 10(-8)M SP, CGRP, VIP or GAL for 30 min. After different time intervals, cells were harvested for total RNA isolation; in addition, cell lysates and supernatants were collected. The effects of the neuropeptides on the mRNA expressions of the different cytokines and NGF were investigated by Q-RT-PCR and the protein levels were studied by means of ELISA assays and Western blotting. Each of the four neuropeptides induced increases in the expressions of IL-1alpha, IL-8 and TNF-alpha mRNA. Increases appeared in the amount of the IL-1alpha protein in the supernatants of neuropeptide-treated cells, and the IL-8 secretion was mildly elevated, while secretion of TNF-alpha remained undetectable. The four neuropeptides increased the NGF mRNA expression to different extents. In the cell lysates of the keratinocytes, only proNGF could be detected, its concentration in the neuropeptide-treated cells being approximately twice that in the time-matched controls. Both control cultures and neuropeptide-treated cultures were found to secrete proNGF and mature NGF, but neuropeptide-treated cell cultures produced markedly higher (3-7-fold) amounts of NGF-like immunoreactive materials. The results demonstrated that neuropeptides released from cutaneous nerves after an injurious stimulus are able to induce an upregulation of IL-1alpha and IL-8 production; they are additionally able to influence the expressions of proNGF/NGF and their secretion from the keratinocytes. These findings may contribute toward an understanding of the neural influence on skin health and disease.

Galanin receptor expression in cultured human keratinocytes and in normal human skin.

Galanin (GAL) is a biologically active neuropeptide that is widely distributed in the nervous system. GAL exerts diverse action via the GAL receptors (GALR1, GALR2, and GALR3), which belong in the superfamily of G-protein-coupled transmembrane receptors. In human skin, GAL-like immunoreactivity has been reported in free nerve endings and fibers of the dermis. The extraneuronal expression of GAL has also been demonstrated. Although the GALRs are essential for biological functions, the expressions of different GALR subtypes in cultured human keratinocytes have not yet been investigated. The aim of our study was to investigate the mRNA and protein expressions of the different GALRs in the HaCaT immortalized keratinocyte cell line and in cultured human keratinocytes. When reverse transcription (RT)-polymerase chain reaction (PCR) was used with different GALR-specific primers, only GALR2 mRNA was identified in cultured HaCaT cells and keratinocytes. Sequencing of the PCR products proved the presence of GALR2 mRNA in the keratinocytes. The presence of GALR2 protein was next investigated, using a polyclonal antibody against human GALR2. Both the HaCaT cells and the cultured keratinocytes displayed specific immunohistochemical staining, with higher intensity on the surface of the keratinocytes. Immunohistochemical investigations of normal human skin specimens revealed that GALR2 was expressed with high intensity in the basal layer of the epidermis and also around the hair follicles in the dermis. GAL treatment of the keratinocytes resulted in an increase in cytosolic Ca2+ concentration, suggesting that GALR2 is a functional receptor. Further studies are necessary to clarify the biological effects of GAL in the skin.

Identification and characterization of a novel, psoriasis susceptibility-related noncoding RNA gene, PRINS.

To identify genetic factors contributing to psoriasis susceptibility, gene expression profiles of uninvolved epidermis from psoriatic patients and epidermis from healthy individuals were compared. Besides already characterized genes, we identified a cDNA with yet unknown functions, which we further characterized and named PRINS (Psoriasis susceptibility-related RNA Gene Induced by Stress). In silico structural and homology studies suggested that PRINS may function as a noncoding RNA. PRINS harbors two Alu elements, it is transcribed by RNA polymerase II, and it is expressed at different levels in various human tissues. Real time reverse transcription-PCR analysis showed that PRINS was expressed higher in the uninvolved epidermis of psoriatic patients compared with both psoriatic lesional and healthy epidermis, suggesting a role for PRINS in psoriasis susceptibility. PRINS is regulated by the proliferation and differentiation state of keratinocytes. Treatment with T-lymphokines, known to precipitate psoriatic symptoms, decreased PRINS expression in the uninvolved psoriatic but not in healthy epidermis. Real time reverse transcription-PCR analysis showed that stress signals such as ultraviolet-B irradiation, viral infection (herpes simplex virus), and translational inhibition increased the RNA level of PRINS. Gene-specific silencing of PRINS by RNA interference revealed that down-regulation of PRINS impairs cell viability after serum starvation but not under normal serum conditions. Our findings suggest that PRINS functions as a noncoding regulatory RNA, playing a protective role in cells exposed to stress. Furthermore, elevated PRINS expression in the epidermis may contribute to psoriasis susceptibility.

Proliferating keratinocytes are putative sources of the psoriasis susceptibility-related EDA+ (extra domain A of fibronectin) oncofetal fibronectin.

The extra domain A of fibronectin (EDA+ oncofetal isoform of fibronectin was recently reported to be overexpressed in psoriatic uninvolved epidermis. It has been proposed that the abnormal presence of EDA+ oncofetal protein at the dermal-epidermal junction in the uninvolved skin may provide the "psoriatic" environment in which keratinocytes are in a preactivated state with regard to mitogenic signals (e.g., T cell lymphokines). To determine the possible sources of cellular fibronectin in the non-lesional psoriatic skin, we aimed to investigate whether keratinocytes could produce the EDA+ oncofetal form of fibronectin. RT-PCR studies revealed that both cultured normal keratinocytes and HaCaT cells express the EDA+ splice variant of fibronectin mRNA, and in HaCaT cells the EDA+/EDA- transcript ratio was elevated compared with normal keratinocytes. Cultured keratinocytes and HaCaT cells showed intracytoplasmic staining with an EDA+ fibronectin-specific antibody and among the positively stained cells many showed mitosis. Using RT-PCR, western blot analysis, and flow cytometry, we showed that in synchronized HaCaT cells the amount of both total fibronectin and its EDA+ isoform change with the proliferation/differentiation state of HaCaT cells and peak in highly proliferating cells. We show that in short-term ex vivo cultures, a small population of EDA+ fibronectin containing cell population appear among psoriatic uninvolved, but not normal epidermal cells. We also demonstrate that cell attachment has a strong influence on the expression of both total and EDA+ fibronectin. Our results suggest that proliferating keratinocytes could be the sources of the psoriasis susceptibility-related EDA+ oncofetal fibronectin in the epidermis.

Atrazine resistance entails a limited xanthophyll cycle activity, a lower PSII efficiency and an altered pattern of excess excitation dissipation.

Atrazine-resistant (AR) weeds have a modified D1 protein structure, with a Ser264-->Gly mutation on the D1 protein, near the plastoquinone binding niche. The photosynthetic performance, the light response of the xanthophyll cycle and chlorophyll fluorescence quenching-related parameters were compared in attached leaves of susceptible (S) and AR biotypes of the C3 dicot Chenopodium album L., Epilobium adenocaulon Hausskn., Erigeron canadensis L., Senecio vulgaris L. and Solanum nigrum L. and the C4 dicot Amaranthus retroflexus L. grown under natural high-light conditions. No significant difference in CO2 assimilation rate per leaf area unit was found between the S and AR biotypes of the investigated C3 plants, whereas the AR biotype of A. retroflexus exhibited a relatively poor photosynthetic performance. The D1 protein mutant plants expressed a reduced activity of light-stimulated zeaxanthin formation. Neither the lower violaxanthin de-epoxidase activity nor the depletion of ascorbate seems to be the cause of the lower in vivo zeaxanthin formation in the AR plants. All the D1 mutant weeds had limited light-induced non-photochemical (NPQ) and photochemical (qP) quenching capacities, and displayed a higher photosensitivity, as characterized by the ratio (1-qP)/NPQ and a higher susceptibility to photoinhibition. Analysis of the chlorophyll fluorescence parameters showed that a lower proportion of excitation energy was allocated to PSII photochemistry, while a higher excess of excitation remained in the AR weeds relative to the S plants.