Regulatory networks contributing to psoriasis susceptibility.

The non-involved, healthy-looking skin of psoriatic patients displays inherent characteristics that make it prone to develop typical psoriatic symptoms. Our primary aim was to identify genes and proteins that are differentially regulated in the non-involved psoriatic and the normal epidermis, and to discover regulatory networks responsible for these differences. A cDNA microarray experiment was performed to compare the gene expression profiles of 4 healthy and 4 psoriatic non-involved epidermis samples in response to T-cell lymphokine induction in organotypic cultures. We identified 61 annotated genes and another 11 expressed transcripts that were differentially regulated in the psoriatic tissues. Bioinformatics analysis suggested that the regulation of cell morphology, development and cell death is abnormal, and that the metabolism of small molecules and lipids is differentially regulated in psoriatic epidermis. Our results indicate that one of the early steps of psoriasis pathogenesis may be the abnormal regulation of IL-23A and IL-1B genes in psoriatic keratinocytes.

Clinical relevance of autoantibodies in patients with autoimmune bullous dermatosis.

The authors present their experience related to the diagnosis, treatment, and followup of 431 patients with bullous pemphigoid, 14 patients with juvenile bullous pemphigoid, and 273 patients with pemphigus. The detection of autoantibodies plays an outstanding role in the diagnosis and differential diagnosis. Paraneoplastic pemphigoid is suggested to be a distinct entity from the group of bullous pemphigoid in view of the linear C3 deposits along the basement membrane of the perilesional skin and the "ladder" configuration of autoantibodies demonstrated by western blot analysis. It is proposed that IgA pemphigoid should be differentiated from the linear IgA dermatoses. Immunosuppressive therapy is recommended in which the maintenance dose of corticosteroid is administered every second day, thereby reducing the side effects of the corticosteroids. Following the detection of IgA antibodies (IgA pemphigoid, linear IgA bullous dermatosis, and IgA pemphigus), diamino diphenyl sulfone (dapsone) therapy is preferred alone or in combination. The clinical relevance of autoantibodies in patients with autoimmune bullous dermatosis is stressed.

Expression and functional studies on the noncoding RNA, PRINS.

PRINS, a noncoding RNA identified earlier by our research group, contributes to psoriasis susceptibility and cellular stress response. We have now studied the cellular and histological distribution of PRINS by using in situ hybridization and demonstrated variable expressions in different human tissues and a consistent staining pattern in epidermal keratinocytes and in vitro cultured keratinocytes. To identify the cellular function(s) of PRINS, we searched for a direct interacting partner(s) of this stress-induced molecule. In HaCaT and NHEK cell lysates, the protein proved to be nucleophosmin (NPM) protein as a potential physical interactor with PRINS. Immunohistochemical experiments revealed an elevated expression of NPM in the dividing cells of the basal layers of psoriatic involved skin samples as compared with healthy and psoriatic uninvolved samples. Others have previously shown that NPM is a ubiquitously expressed nucleolar phosphoprotein which shuttles to the nucleoplasm after UV-B irradiation in fibroblasts and cancer cells. We detected a similar translocation of NPM in UV-B-irradiated cultured keratinocytes. The gene-specific silencing of PRINS resulted in the retention of NPM in the nucleolus of UV-B-irradiated keratinocytes; suggesting that PRINS may play a role in the NPM-mediated cellular stress response in the skin.

The anti-apoptotic protein G1P3 is overexpressed in psoriasis and regulated by the non-coding RNA, PRINS.

Psoriasis Susceptibility-Related RNA Gene Induced by Stress (PRINS) is a non-coding RNA overexpressed in lesional and non-lesional psoriatic epidermis and induced by stress. Its function in healthy and psoriatic skin is still not known. Here, we report that PRINS regulates G1P3, a gene with anti-apoptotic effects in keratinocytes. siRNA-mediated inhibition of PRINS gene resulted in altered cell morphology and gene expression alterations, as demonstrated in a microarray experiment. One of the genes regulated by PRINS ncRNA was G1P3, an interferon-inducible gene with anti-apoptotic effects in cancer cells. Interestingly, we found that G1P3 was 400-fold upregulated in hyperproliferative lesional and ninefold upregulated in non-lesional psoriatic epidermis compared to healthy epidermis. In vitro, G1P3 protein levels were highest in proliferating keratinocytes and siRNA-mediated downregulation of G1P3 resulted in increased cell apoptosis. These data indicate that G1P3 inhibits spontaneous keratinocyte apoptosis and hence its high expression in psoriatic skin may contribute to the development of psoriatic lesions. We hypothesize that the deregulation of the PRINS ncRNA may contribute to psoriasis and results in decreased sensitivity to spontaneous keratinocyte apoptosis via the regulation of G1P3.

The altered expression of syndecan 4 in the uninvolved skin of venous leg ulcer patients may predispose to venous leg ulcer.

Syndecan 4 (SDC4), a heparan sulfate proteoglycan, and neuropilin 1 (NRP1), a transmembrane receptor, are both involved in normal wound healing, but little is known about their possible role in venous leg ulcer pathogenesis. We aimed to investigate whether there are any expression abnormalities and/or gene polymorphisms of SDC4 and NRP1 associated with venous leg ulcer. SDC4 showed significantly lower mRNA and protein expression in the uninvolved dermis of venous leg ulcer patients (n=15) compared with controls (n=15; p=0.0136), while NRP1 showed no expression abnormalities. None of the examined SDC4 and NRP1 polymorphisms showed a difference in their allelic distribution between leg ulcer patients (n=92) and controls (n=92). We hypothesize that SDC4 may play an essential role not only in the inflammation and tissue formation phases of normal wound healing, but its expression abnormalities observed in the uninvolved dermis of venous leg ulcer patients may contribute to venous leg ulcer development.

The Arg160Trp allele of melanocortin-1 receptor gene might protect against vitiligo.

Melanocortin-1 receptor (MC1R) and agouti signaling protein (ASIP) play pivotal roles in the regulation of human pigmentation. We aimed to study whether single nucleotide polymorphisms (SNPs) of the MC1R and ASIP genes contribute to the pathogenesis of the polygenic pigment skin disorder, vitiligo. The PCR-amplified, full-length MC1R gene was studied with sequence analysis, and the 3' untranslated region (3' UTR) SNP of ASIP was detected using restriction fragment length polymorphism. The allele frequency of the ASIP SNP did not show any difference between the skin type, hair color and eye color-matched 97 vitiligo patients and the 59 healthy control individuals. As one of the MC1R polymorphisms showed significantly higher incidence among fair-skinned individuals (Fitzpatrick I+II, n=140) than among dark-skinned individuals (Fitzpatrick III+IV, n=90), both vitiligo patients and controls were divided into two groups and the frequency of the MC1R alleles was studied separately in fair-skinned and dark-skinned subgroups of diseased and healthy groups. C478T, one of the MC1R SNPs studied in 108 fair-skinned vitiligo patients and in 70 fair-skinned healthy control individuals, showed a significant difference (P=0.0262, odds ratio [95% confidence interval]=3.6 [0.0046-0.1003]) in allele frequency between the two groups: the allele frequency was higher in the control group, suggesting protection against vitiligo. Computer prediction of antigenicity has revealed that the Arg160Trp amino acid change caused by this SNP results in a decrease in antigenicity of the affected peptide epitope.

[The prevalence of melanocytic naevi among teenagers]. / A festéksejtes anyajegyek elofordulása tinédzsereken.

UNLABELLED: Malignant melanoma is an increasing public health problem worldwide; accordingly, identification of the constitutional and environmental factors which contribute to the development of the disease, and hence identification of the individuals at high risk of melanoma, are indispensable steps in all primary prevention efforts. AIM: The objective of the present study was to assess the prevalence of different pigmented lesions among schoolchildren, and to investigate their relationship with phenotypic pigmentary characteristics, sun exposure and other factors. METHODS: A cross-sectional study was performed in two secondary schools in Szeged, Hungary. A total of 1320 schoolchildren, aged 14 to 18 years, underwent a whole-body skin examination. A standardized questionnaire was used to collect data on phenotypic, sun exposure and other variables. RESULTS: Between 1-10 common melanocytic naevi were found in 27% of the participants, and naevi numbers were in the range between 10-100 in 67%. 5.4% of them had more than 100 common melanocytic naevi. The prevalence of clinically atypical naevi was 24.3%. Congenital naevi were detected in 6.2% of the schoolchildren. A statistically significant association was found between the number of pigmented lesions and gender, hair colour, eye colour, skin phototype, the history of severe painful sunburns, and the family history of a large number of melanocytic naevi. CONCLUSIONS: Our study population displayed a markedly high prevalence of clinically atypical melanocytic naevi. Moreover, a considerable proportion of the investigated individuals had multiple common melanocytic naevi. Since the presence of large number of melanocytic naevi is a strong predictor for future melanoma development, health educational programmes on melanoma prevention should be aimed at young age groups.

First detection of the melanoma-predisposing proline-48-threonine mutation of p16 in Hungarians: was there a common founder either in Italy or in Hungary?

The P48T germ line mutation of p16 was detected in a Hungarian multiple primary melanoma patient (deceased at the age of 39) with no affected family members. Genetic analysis of the patient and his family revealed that the patient was homozygous for the mutation, whereas his parents (father currently aged 69 and mother 63), who are free from any malignancies and atypical moles, are both heterozygous for the mutation. Our data suggest that the P48T mutation of p16 is a strong melanoma-predisposing factor, but the fact that the heterozygous mutant parents have not yet exhibited melanoma or atypical moles indicates that the penetrance of this allele might depend on modifying factors. The rare P48T germ line mutation of p16 has been reported previously in only four independent studies, all in patients with Italian ancestry. Here, we first report the inheritance of the rare P48T mutation of CDKN2A in a Hungarian family with a homozygous multiple primary melanoma member and unaffected heterozygous family members. The question of whether the mutation detected in Hungary is the result of an independent event, or migration of the founder mutation occurred at some time in the past, necessitates further investigations.

Human adult epidermal melanocytes cultured without chemical mitogens express the EGF receptor and respond to EGF.

We describe a novel chemical mitogen-free in vitro culture technique for obtaining pure melanocyte cultures using normal human adult epidermis as a source. The culture medium consists equal parts of the commercially available Keratinocyte Basal and AIM-V media (both from Gibco), as basal medium, which is supplemented with fetal bovine serum, bovine pituitary extract and recombinant human epidermal growth factor (EGF). Melanocytes harvested from human adult skin proliferate extensively and can be passaged serially up to 10-15 times using this medium. We have verified the identity of the cultured cells by tyrosinase mRNA expression and TRP-1 protein staining. Moreover, we showed that autologous human serum alone, without additional supplements is able to provide sufficient growth support for the cultured cells in the basal medium, making this culture technique suitable for autologous melanocyte transplantation. In this culture system normal human adult melanocytes expressed both EGF receptor (EGFR) mRNA and protein and EGF showed a dose dependent mitogenic effect on the cells. EGF itself had no significant influence on EGFR mRNA expression.

Alzheimer's lymphocytes are resistant to ultraviolet B-induced apoptosis.

In the present pilot investigation, the susceptibility of T-lymphocytes from Alzheimer's disease (AD) subjects (n=22) and aged-matched, non-demented controls (CNT) (n=12) was examined with ultraviolet (UV) B light-induced apoptosis in vitro. The basal apoptotic ratios were similar in both groups. However, the AD lymphocytes displayed significantly (p<0.0001) lower apoptotic levels than those of the CNT lymphocytes at all of the applied UVB exposure doses (100, 200 and 300 mJ/cm(2)). These observations indicate that AD lymphocytes are more resistant than CNT lymphocytes to UVB irradiation.

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.

PUVA treatment of the nasal cavity improves the clinical symptoms of allergic rhinitis and inhibits the immediate-type hypersensitivity reaction in the skin.

We earlier reported that intranasal irradiation with the 308 nm xenon chloride (XeCl) ultraviolet-B laser and irradiation with a combination of ultraviolet-B (UVB), ultraviolet-A (UVA) and visible light (VIS) is highly effective in the treatment of allergic rhinitis and inhibit the immediate-type hypersensitivity reaction in the skin. Since photochemotherapy with 8-methoxypsoralen (8-MOP) plus UVA light (PUVA) is widely used in the treatment of different inflammatory skin disorders due to its immunosuppressive effect, in the present study we investigated the efficacy of intranasal PUVA treatment in allergic rhinitis and the effect of PUVA treatment on the skin prick test (SPT) reaction. An open study was performed in 17 patients with hay fever. Intranasal PUVA therapy was given four times weekly for 3 weeks. The treatment was started with a fluence of 0.5x of the individual minimal phototoxic dose (MPD) and the dosages were gradually increased. Evaluation was based on the symptom scores. The effect of PUVA treatment on the allergen-induced wheal formation was also studied in the SPT. PUVA treatment of the nasal cavity significantly decreased the nasal symptoms of the patients with allergic rhinitis. Treatment of the skin with PUVA also significantly suppressed the allergen-induced wheal formation in the SPT reaction. These data suggest that intranasal PUVA phototherapy is also an effective modality in the treatment of allergic rhinitis.

Microbial compounds induce the expression of pro-inflammatory cytokines, chemokines and human beta-defensin-2 in vaginal epithelial cells.

Vaginal epithelium has a powerful innate immune system that protects the female reproductive organs from bacterial and fungal infections. In the present study, we aimed to explore whether the Toll-like receptor (TLR) signaling pathway and the induction of pro-inflammatory cytokines and antimicrobial peptides could contribute to the protection against pathogenic microorganisms in vaginal epithelia, using an immortalized vaginal epithelial cell line PK E6/E7 as a model. We found that TLR2 and TLR4 receptors are expressed in vivo in the vaginal epithelia and in vitro in PK E6/E7 vaginal epithelial cell line. The Gram-negative cell wall compound lipopolysaccharide (LPS), the Gram-positive compound peptidoglycan (PGN), heat-killed Candida albicans and zymosan significantly (P<0.05) induced the expression of pro-inflammatory cytokines and chemokines such as TNF-alpha and IL-8/CXCL8 in vaginal epithelial cells. Furthermore, the expression and production of human beta-defensin-2 (hBD2), an antimicrobial peptide with chemotactic functions, was also up-regulated in PK E6/E7 cells after treatment with LPS, PGN or C. albicans. Treatment of vaginal epithelial cells with microbial compounds induced the activation and nuclear translocation of NF-kappaB transcription factor, a key element of innate and adaptive immune responses. In our work, we provide evidence that microbial compounds induce the production of pro-inflammatory cytokines, chemokines and antimicrobial peptides in vaginal epithelial cells. In vivo, vaginal epithelial cell-derived inflammatory mediators and antimicrobial peptides may play important roles in vaginal immune responses and in the elimination of pathogens from the female reproductive tract.

Autoantibodies to human alpha6 integrin in patients with bullous pemphigoid.

Bullous pemphigoid (BP) is characterized immunologically by tissue-bound and circulating autoantibodies targeting the hemidesmosomal proteins BP230 and BP180. Recent evidence suggests a pathophysiological role for autoantibodies against alpha6 integrin in the subepidermal blister formation of oral pemphigoid. The objective of our study was to investigate the presence of anti-alpha6 integrin antibodies in patients with classical BP. The autoantibody profiles of 30 patients with BP, 10 patients with pemphigus vulgaris, and 20 healthy persons were identified. With the use of PeptideStructure and PlotStructure software, four different antigenic epitopes for alpha6 integrin were predicted, and their fusion recombinant constructs were prepared in an E. coli expression system. Sera were tested for alpha6 integrin autoantibodies by an ELISA technique. Altogether, 52% of the patients with BP displayed circulating antibodies against at least one recombinant protein. Our findings provide the first evidence for the presence of anti-alpha6 integrin antibodies in patients with classical BP.

[Intranasal phototherapy for the treatment of allergic rhinitis]. / Intranasalis fototerápia az allergiãs rhinitis kezelésében.

INTRODUCTION: Allergic rhinitis is a frequent disease, accompanied by significant social-economic costs and a negative impact on the quality of life. Phototherapy has a profound immunosuppressive effect and is effectively used in the treatment of several immune mediated skin diseases such as atopic dermatitis. AIMS: The authors investigated the efficacy of intranasal phototherapy with a combination of low doses of ultraviolet-B, ultraviolet-A and visible light in allergic rhinitis. METHODS: A randomized, double-blind, placebo-controlled study was conducted in patients with a history of at least 2 years of moderate to severe ragweed-induced allergic rhinitis that was not controlled by anti-allergic drugs. Intranasal phototherapy was performed 3 times a week for 3 weeks. As placebo low intensity visible light was used. RESULTS: Phototherapy resulted in a significant improvement of clinical symptoms for nasal itching, rhinorrhea, sneezing and total nasal score. Scores for nasal obstruction slightly improved during phototherapy while a significant increased was found in the placebo group. In the overall efficacy assessment, both patients and investigators found phototherapy significantly more efficient than placebo. Phototherapy was well tolerated, the only side effect was the slight dryness of the nasal mucosa. CONCLUSIONS: These results suggest that intranasal phototherapy is effective for the treatment of allergic rhinitis, and opens up new opportunities for the treatment of immune-mediated mucosal diseases.

[Connection between uveal melanoma and dysplastic naevus syndrome]. / Az uvealis melanoma és a dysplasticus naevus-syndroma kapcsolata.

The dysplastic naevus syndrome increases the risk of cutaneous (RR: 4.36; CI: 1.84-10.36) as well as uveal melanoma (RR: 4.22; CI: 1.81-9.84). A significantly higher occurrence rate of conjunctival naevi (3.2% vs. 0%; p=0.029), choroidal naevi (5.2% vs. 0.7%; p=0.023) and iris freckles (17.1% vs. 5.6%; p=0.002) could be detected in the dysplastic naevus syndrome patients compared to subjects in the control group. The presence of cutaneous dysplastic naevi in uveal melanoma patients increases the risk of the prognostically worse--epitheloid or mixed--forms of uveal melanoma (RR: 5.97%; CI: 1.61-22.14), compared to patients without cutaneous atypical naevi.

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.

Efficacy of different UV-emitting light sources in the induction of T-cell apoptosis.

Ultraviolet B (UV-B) radiation is a modality widely used for the treatment of different skin diseases. One of the major mechanisms of UV-B immunosuppression in this treatment modality is thought to be an apoptosis-inducing effect on T cells infiltrating the skin. We examined the T-cell apoptosis-induction capacities of four different UV light sources, with and without UV filters. The xenon chloride (XeCl) laser proved to be the strongest apoptosis inducer. The use of a phtalic acid filter eliminated UV radiation almost completely below 300 nm, which resulted in a severe decrease in the apoptosis-inducing capacity of different UV-B sources. Using the results of the measurements with polychromatic UV light sources, the wavelength dependence of UV-B light for the induction of T-cell apoptosis was also determined. The regression line of the action spectrum demonstrated a continuous decrease from 290 to 311 nm. The apoptosis-inducing capacity of the XeCl laser was almost four times higher than the calculated value according to the action spectrum, which might be attributed to the high irradiance of the laser as compared with nonlaser light sources.

Ocular pigmented findings in patients with dysplastic naevus syndrome.

There is a growing body of evidence supporting the theory that cutaneous dysplastic naevus syndrome patients are at increased risk of developing not only skin but also uveal melanoma. The relationship between dysplastic naevus syndrome and ocular naevi needs to be clarified. In this study we investigated the ocular pigmented findings in patients with dysplastic naevus syndrome and compared the results with a control group (subjects without atypical moles) in order to investigate the frequency of ocular naevi among dysplastic naevi-bearing patients. A total of 152 dysplastic naevus syndrome patients were enrolled in our investigation. The control group consisted of 142 sex-, age- and skin type-matched healthy volunteers without cutaneous dysplastic naevi or skin melanoma. Conjunctival and uveal pigmented findings and iris colour were recorded during a detailed ophthalmic examination. A greater number of conjunctival naevi (3.2% versus 0%), iris naevi (5.2% versus 1.4%), iris freckles (17% versus 5.6%) and choroidal naevi (5.2% versus 0.7%) were detected in the dysplastic naevus syndrome group compared with the controls. The difference reached statistical significance in the case of conjunctival naevi, choroidal naevi and iris freckles. Our results confirm the hypothesis that dysplastic naevus syndrome patients might have overstimulation of their melanocytic system not only in the skin but also in the uvea, leading to increased benign (as well as rarely malignant) melanocytic proliferation. Dysplastic naevus syndrome patients should be screened by ophthalmologists because of the increased frequency of different ocular pigmented alterations.