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BACKGROUND: The Langerhans cell (LC) hypothesis suggests that cutaneous T-cell lymphomas (CTCL) are diseases of chronic T-cell stimulation by LC-mediated antigen presentation. OBJECTIVE: To investigate a broad panel of CTCL and cutaneous B-cell lymphomas (CBCL) for the spatial association of langerin(+) dendritic cells (DC) with T and B cells in the skin, respectively. METHODS: Fifty-five specimens of CTCL and 10 of CBCL were double-stained with monoclonal antibodies against langerin and CD3 or CD20, respectively, and evaluated by confocal laser scan microscopy. RESULTS: Dermal infiltrates in mycosis fungoides (n = 38), primary cutaneous CD4+ small/medium-sized pleomorphic T-cell lymphoma (n = 3) and primary cutaneous peripheral T-cell lymphoma, unspecified (n = 3) were characterized by a high frequency of dermal langerin(+) DCs. These cells were exclusively present in the malignant infiltrates. No direct co-localization of CD3 and langerin could be resolved. Dermal langerin(+) cells were detected only in one of six primary cutaneous anaplastic large cell lymphomas (C-ALCL), characterized by epidermotropism. In other C-ALCL cases (five of six), in lymphomatoid papulosis (n = 3), subcutaneous panniculitis-like T-cell lymphoma (n = 2), and all variants of CBCL no dermal langerin(+) DCs could be found. CONCLUSIONS: Langerin(+) DCs are abundant in the dermal infiltrates of T-cell lymphomas with specific involvement of the epidermis. This might indicate that immature LC and neoplastic T cells interact and gives rise to further studies to characterize the phenotype of the langerin(+) cell population described here and its role in the pathology of CTCL.

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Heat shock proteins (hsp) are expressed in all cells and organisms. Their expression is induced by heat shock (temperatures above 42 degrees C) and other forms of pathophysiological stress. Elevated levels of hsp protect cells from further stress exposure. Hsp are expressed intracellularly. They are highly conserved throughout evolution indicating hsp being necessary for survival under potentially harmful environmental conditions. Hsp are divided into families according to their molecular weight. The majority of hsp function as molecular chaperones. Chaperone function is characterized by binding to other proteins and mediating their folding, transport and interaction with other molecules. In human epidermis hsp are abundantly expressed and have been linked with functions in cell differentiation and photobiology. Recent research has mainly focused on the 27 and 72 kD hsp that are constitutively expressed in human keratinocytes. ultraviolet radiation (UV)-induced cell death and sunburn cell formation can be inhibited by previous heat shock exposure and UV itself can induce hsp expression. The expression of the 27 kD hsp (hsp27) in epidermal keratinocytes in situ and in culture correlates with differentiation. Expression of hsp27 increases simultaneously with keratinocyte differentiation. For that reason, hsp27 is described as a marker of epidermal differentiation. Changes in the expression and inducibility of hsp have been linked with ageing. In the skin, recent data indicate that hsp72 expression remains remarkably stable with intrinsic ageing. In contrast, levels of hsp27 have been found to be elevated in sun-protected aged skin indicating a link between hsp27 expression and age-dependent epidermal alterations. Regulation of hsp can be modified by pharmacological intervention and the development of safe topical and systemic treatments for the prevention of skin damage and disorders of keratinocyte differentiation can be expected for the future.

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BACKGROUND: hsp27 is a member of the small heat shock protein family. Its expression in epidermal keratinocytes in situ and in tissue culture correlates with differentiation. Experimental evidence points to the fact that hsp27 is a molecular chaperone and is involved in the regulation of cell growth and differentiation. OBJECTIVES: To investigate whether epidermal hsp27 through its chaperone function plays a role in the assembly of keratin filaments and the cornified cell envelope. METHODS: We performed double staining immunofluorescence and immunogold microscopy on normal human skin (n = 15). We analysed the colocalization of hsp27 with actin, keratins and proteins of the cornified cell envelope (loricrin, filaggrin, transglutaminase 1). RESULTS: Actin staining did not reveal detectable colocalization with hsp27. For keratins, transglutaminase, loricrin and filaggrin colocalization was found in more than 60% of the samples. Colocalization was confined to a narrow subcorneal layer with varying patterns of expression. Electron microscopy revealed that loricrin and filaggrin colocalize with hsp27 indirectly through binding to intermediate filaments. CONCLUSIONS: These results provide morphological evidence that in normal human skin hsp27 might act as a chaperone of cornification. Investigations of the molecular hsp27 interactions with the proteins of the cornified cell envelope are necessary to gain further insight into terminal keratinocyte differentiation and disorders of keratinization.

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Treatment with 8-methoxypsoralen plus ultraviolet A radiation and extracorporeal photochemotherapy (photopheresis) are widely used for the treatment of psoriasis and other inflammatory skin diseases, graft-versus-host disease, and mycosis fungoides. As the ratio of Th1 and Th2 cells appears to be critical for pathogenesis and progression of these disorders the effect of psoralen plus ultraviolet A on Th1 and Th2 cytokine production by CD4+ lymphocytes was investigated. Human peripheral blood lymphocytes were incubated in the presence of anti-CD3, rh-IL2, and rh-IL4 for 48 h. After subsequent stimulation with rh-IL2 and rh-IL4 for 72 h cells were treated with 8-methoxypsoralen (100, 500, 1000 ng per ml) plus ultraviolet A (2 J per cm2) and incubated for a further period of 5 h in the presence of ionomycine, phorbol-12-myristate acetate and monensin. Fluorescence-activated cell sorter analysis revealed a significant reduction of interleukin-2- and interferon-gamma-producing CD4+ cells upon psoralen plus ultraviolet A treatment depending on the concentration of 8-methoxypsoralen. In contrast, interleukin-4-producing CD4+ cells were increased, indicating a shift from Th1 to a Th2 cell cytokine profile upon psoralen plus ultraviolet A treatment. These results indicate that 8-methoxypsoralen photochemotherapy of lymphocytes is able to modulate their Th1/Th2 distribution. Inhibition of Th1 cytokine expression by psoralen plus ultraviolet A might help to explain its beneficial effects in the treatment of Th1 dominated skin diseases.

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Merkel cell carcinoma was first described in 1972 by Toker and is an aggressive neuroendocrine skin tumor with a high metastatic potential. Merkel cell carcinoma is thought to derive from the neuroendocrine (Merkel) cells of the skin, although in contrast to fetal and especially adult Merkel cells, Merkel cell carcinomas express high levels of the Bcl-2 oncoprotein. Bcl-2 is capable of blocking programmed cell death and has been shown to play an important role in normal cell turnover, tumor biology, and chemoresistance. High Bcl-2 expression leading to prolonged survival of cells may therefore be of importance in the biological and clinical characteristics of Merkel cell carcinoma. In a SCID mouse xenotransplantation model for human Merkel cell carcinoma, we investigated the influence of the bcl-2 antisense oligonucleotide G3139 (Genta) on tumor growth in comparison with control oligonucleotides or cisplatin. Bcl-2 antisense treatment, targeting the first six codons of the bcl-2 mRNA, resulted in either a dramatic reduction of tumor growth or complete remission, whereas reverse sequence and two-base mismatch control oligonucleotides or cisplatin had no significant antitumor effects compared with saline-treated controls. Apoptosis was enhanced 2.4-fold in the bcl-2 antisense treated tumors compared with the saline-treated group, and no other treatment showed a comparable increase in apoptosis. Our findings suggest that bcl-2 antisense treatment may be a novel approach to improve treatment outcome of human Merkel cell carcinoma.

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The 72-kD heat shock protein (hsp72) belongs to a family of stress inducible proteins (heat shock proteins, hsp) and its expression is associated with increased survival of cells in culture following exposure to ultraviolet radiation (UV). Hsp72 can be induced by a number of stresses, including heat, cold, and toxic chemicals. The purpose of this study was to evaluate whether UV is able to activate transcription of hsp72. The human fibrosarcoma cell line HT1080 was used for these experiments because hsp72 is not detectable in these cells under normal culture conditions. Cells were exposed to UVA and UVB using a solar simulating source and hsp72 was determined in whole cell extracts by immunoblotting. For inhibition of mRNA and protein synthesis cordycepin (20 microg/ml) and cycloheximide (10 microg/ml) were added to the cultures, respectively. UVA-induced lipid peroxidation was inhibited by alpha-tocopherol and butylated hydroxytoluene (BHT). UVA but not UVB induced hsp72 with maximal expression at 40 J/cm2, 8-12 h after exposure. Induction was blocked by cordycepin as well as by cycloheximide indicating that both, mRNA and protein synthesis, are required for UVA-induction of hsp72. Inhibition of cell lipid peroxidation with alpha-tocopherol and BHT had no effect on hsp72 expression. These results suggest that induction of hsp72 is part of an adaptive response mechanism in human cells to UV-related stress.

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Exposure to ultraviolet (UV) radiation and photochemotherapy induces apoptotic cell death in epidermal cells. In this study annexin V binding and propidium iodide (PI) uptake have been measured by flow cytometry to evaluate UV-induced cell death in the human squamous cell carcinoma-derived cell line A 431. Physiological and therapeutical relevant doses of UVA, UVA1, UVB, narrow-band UVB (311 nm) and photochemotherapy using 100 ng/ml of 8-methoxypsoralen (8-MOP) with UVA or UVA1 (PUVA or PUVA1) have been applied. Doses ranged from 8 to 96 J/cm2 for UVA1 and UVA, from 8 to 128 mJ/cm2 for UVB, from 256 to 4096 mJ/cm2 for narrow-band UVB (311 nm) and from 1 to 16J/cm2 for photochemotherapy. Results show that the amount of annexin V binding, a measure of early apoptosis, as well as PI uptake, a parameter of ultimate cell death, are strictly correlated with the applied UV dose. Peak values of annexin V-positive cells are noted 12 h after UV exposure in all protocols and are followed by an increase of PI-uptaking cells with peak values at 24 h after UVA and UVA1, and 48 h after PUVA, PUVA1, UVB and narrow-band UVB. To compare the effect of different wavelengths and light sources, dose equivalents are calculated based on the induction of 50% cell death (as determined by PI uptake). The equivalents are 96 J/cm2 for UVA and UVA1, 16 J/cm2 for PUVA and PUVA1, 256 mJ/cm2 for UVB and 2048 mJ/cm2 for narrow-band UVB. Our results establish annexin V/PI double staining as an appropriate method for the quantification of UV-induced cell death. Moreover, they provide a basis for further investigations concerning mechanisms and modifications of UV-induced apoptosis.

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Since pentoxifylline (PTX) was recently recognized as a substance with antiinflammatory capacities, we studied the in vivo and in vitro effect of PTX on the expression of the intercellular adhesion molecule-1 (ICAM-1) on human monocytes. For this purpose four healthy volunteers were treated with PTX (5 x 400 mg/day) for 2 days. Monocytes were isolated before and after PTX treatment and ICAM-1 expression was investigated. As shown by fluorescence-activated cell sorter (FACS) analysis, cultured monocytes isolated after oral application of PTX expressed significantly decreased amounts of ICAM-1 when compared with monocytes collected prior to oral PTX application. Northern blot analysis revealed reduced amounts of ICAM-1 mRNA in monocytes derived from volunteers after oral PTX treatment in comparison with monocytes isolated before oral PTX administration. Similarly, in monocytes treated with PTX (200 micrograms/ml) in vitro ICAM-1 was found decreased both at the protein and mRNA level in comparison with untreated cells. The inhibitory effect of PTX on ICAM-1 expression in monocytes could be reversed by the addition of exogenous tumour necrosis factor-alpha (TNF-alpha; 200 U/ml) suggesting that ICAM-1 down-regulation is mediated secondary to TNF-alpha suppression by PTX. The specific role of TNF-alpha in mediating ICAM-1 expression in cultured monocytes could be confirmed by the finding that a neutralizing anti-TNF-alpha antibody partially down-regulated ICAM-1 expression. The observed suppressive in vivo and in vitro effects of PTX on ICAM-1 expression in monocytes may contribute to the recently described antiinflammatory effects of PTX, e.g. in sepsis or allergic contact dermatitis.

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The soluble tumour necrosis factor receptor I (sTNFRI, p55) is produced at similar levels by both immortalized (A431, HaCaT, KB) and primary normal human keratinocytes (HNK), whereas the soluble TNFR II (sTNFR II, p75) appears to be specifically released only by immortalized human keratinocytes. The purpose of this study was to investigate whether the increase in p75 secretion by immortalized human keratinocytes is due to an increased shedding of the receptor from the cell membrane, or is related to increased steady-state levels of p75 mRNA. FACS analysis showed that levels of membranous p75 decreased in a time-dependent manner in immortalized cells cultured for 1, 3, 6, 12 and 24 h, while remaining unchanged in HNK throughout. Northern blot analysis showed that after 12 h of culture, when p75 expression was decreased on the cell membrane of all immortalized cells, there was no significant difference in steady state levels of p75 mRNA between immortalized keratinocytes and HNK. Supernatants of immortalized cells, cultured for 24 h contained distinct levels of p75, while levels of p75 in supernatants of HNK were under the detection limit, confirming that the p75 decrease on the cell membrane results from increased p75 shedding from the cell membrane of immortalized cells. In contrast to p75, p55 was continuously expressed on the cell membrane of normal and immortalized keratinocytes without significant variation throughout the entire 24-h culture period and was similarly shed by both cell types. These results suggest that immortalized keratinocytes are specifically activated for shedding of p75 from the cell membrane. Since p75 has a high affinity for TNF, the release of this receptor may imply a direct role in the escape of malignant/transformed keratinocytes from the TNF-mediated immune response.

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Most tumor cells produce both tumor necrosis factor (TNF) receptors, the M(r) 55,000 TNFRI and the M(r) 75,000 TNFRII, but they are mostly resistant to TNF-alpha-induced cytotoxicity. To gain further insight into the escape mechanisms of tumor cells from the harmful effect of TNF-alpha, we investigated the production of TNF-binding proteins (TNF-BPI, TNF-BPII, both M(r) 30,000) by malignant and normal epidermal cells and studied their functional role in TNF-alpha-induced cytotoxicity. Malignant human keratinocytes (A431, KB, HaCaT) and malignant human melanoma cells (KRFM) produced significant levels of both TNF-BPI and TNF-BPII on stimulation with phorbol myristate acetate. In contrast, normal human keratinocytes (HNK) and normal human melanocytes (HNM) released TNF-BPI but not TNF-BPII. The specific production of TNF-BPII in concert with TNF-BPI by the malignant cell lines revealed an inhibitory effect of supernatants on recombinant human TNF-alpha-mediated cytotoxicity of the TNF-dependent murine cell line L929, while supernatants of normal epidermal cells had no effect. Preincubation of supernatants with anti-TNF-BPI monoclonal antibody htr-9 or anti-TNF-BPII monoclonal antibody utr-1 reversed this inhibitory effect additively, indicating that the production of both TNF-BPs is necessary to protect cells from TNF-alpha-mediated cytotoxicity. A TNF-alpha scavanging effect of TNF-BPs resulting in subsequent inhibition of TNF-alpha binding to L929 cells could be demonstrated by ligand blotting and fluorescence-activated cell sorting analysis. Thus the production of TNF-BPII by epidermal tumor cells in concert with TNF-BPI appears to demonstrate a specific mechanism by which malignant epithelial cells escape from TNF-alpha-mediated cytotoxicity.

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Pentoxifylline (PTX) is a methylxanthine compound known to inhibit the production of tumour necrosis factor-alpha (TNF-alpha), which is an important inflammatory mediator. There is also recent evidence that PTX may influence other inflammatory cytokines, such as interleukin-1 (IL-1) and IL-6. Due to the therapeutic implications, the present study addressed the in vivo effects of PTX on the release of TNF-alpha, IL-1 beta, IL-6 and IL-8 by human peripheral blood mononuclear cells (PBMC). When PBMC were obtained from healthy volunteers ingesting 5 x 400 mg PTX orally for 2 days, the ability of PBMC cultured for 24 hr to release TNF-alpha was significantly reduced, while secretion of IL-1 beta, IL-6 and IL-8 was not affected. However, when PBMC were obtained from the same individuals 5 days after PTX had been stopped, the release of all four cytokines was significantly suppressed. This effect appeared to be exerted at the transcriptional level, since Northern blot analysis revealed reduced cytokine transcripts. In order to gain more insight into the effect of PTX on cytokine release, PBMC were obtained from normal volunteers, either stimulated with lipopolysaccharide (LPS) or left unstimulated, and subsequently incubated in vitro with PTX for 48 hr. Under these conditions, only TNF-alpha was found to be reduced by PTX, while IL-1 beta and IL-8 were not affected, IL-6 was even enhanced. However, when PBMC were incubated with PTX for 24 hr, PTX removed thereafter by medium change and cells further cultured, the production not only of TNF-alpha but also of IL-1 beta, IL-6 and IL-8 was reduced, demonstrating that PTX exerts diverse (inhibitory) effects on cytokine release by PBMC.

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