Normal Human Skin Cells Research Articles

Molecular diagnosis of feline sporotrichosis

SPOROTRICHOSIS is a chronic infection caused by Sporothrix schenckii, and usually presents as a fixed cutaneous or lymphocutaneous infection in animals (Kwon-Chung and Benett 1992, Rosser and Dunstan 1998). Since a number of diseases can resemble sporotrichosis quite closely (KwonChung and Bennett 1992), it should be diagnosed definitively on the basis of histopathological and mycological findings. Direct microscopic examination is rapid, but can be quite insensitive and is reliant on the operator’s skills. Kano and others (2003) developed a PCR method to detect S schenckii directly in biopsy samples from human patients with sporotrichosis using specific oligonucleotide primers based on the chitin synthase 1 (CHS1) gene of S schenckii (Kano and others 2001). The primer pair (S2 and R2) did not amplify DNAs from other pathogenic fungi (Aspergillus fumigatus, Blastomyces dermatitidis, Candida species, Cryptococcus neoformans, dermatophytes, Malassezia species and Histoplasma capsulatum), bacteria (Staphylococcus aureus), normal human skin cells or normal feline skin cells (Kano and others 2001). The sensitivity of the PCR was equivalent to the detection of 10 pg of S schenckii genomic DNA (Kano and others 2001). This short communication describes a study to identify S schenckii DNA directly from biopsy specimens obtained from a case of feline sporotrichosis. A 23-month-old male crossbred cat, weighing 5·4 kg, was referred to the Gifu University Animal Medical Center with abscesses and a draining puncture wound on the left foreleg and right hindleg which had been present for seven months. The lesions had been drained surgically and treated with chitin and chitosan. A subcutaneous nodule, 18 x 6 x 15 mm in size, was found on the right hindleg. Histopathological analysis of a biopsy of the nodule revealed granulomatous inflammation and many globular and ovoid budding yeast cells (Fig 1), with no asteroid bodies. Therefore, molecular diagnosis of sporotrichosis was performed. DNA was extracted from the biopsy. The tissue sample (approximately 100 mg) was digested at 37°C for 16 hours with 100 μg of proteinase K in 400 μl of a lysis buffer containing 0·1mM Tris-HCl (pH 8·0) and 0·3 per cent 2-mercaptoethanol. The DNA was obtained by phenol and chloroform extraction and then dissolved in Tris-EDTA buffer (10mM Tris-HCl [pH 8·0] and 1mM EDTA) and used directly for PCR amplification. A sample (200 ng) of DNA was amplified by PCR in a 30 μl reaction mixture containing 10mM Tris-HCl (pH 8·3), 50mM potassium chloride, 1·5mM magnesium chloride, 0·001 per cent gelatin, 200μM of each deoxynucleoside triphosphate, 1·0 U Taq polymerase (Takara) and 0·5 μg of the primer pair. To detect S schenckii rapidly, species-specific primers for PCR based on its CHS1 gene sequence were used (Kano and others 2001, 2003). The sequence of the forward primer (S2) was 5′-TGGGCGTCTACCAAGAGGGTATTGC-3′ (nucleotides 173 to 197 of the S schenckii CHS1 gene) (GenBank accession number L24908) and the sequence of the reverse primer (R2) was 5′-GCACATGGGCTCAAGATCAAAGGCC-3′ (nucleotides 468 to 492 of the S schenckii CHS1 gene). With these primers, a 318 base pair (bp) fragment of the CHS1 gene was expected to be amplified. The PCR amplification was carried out for 40 cycles consisting of template denaturation (one minute at 94°C), primer annealing (two minutes at 68°C) and polymerisation (two minutes at 72°C). Then, 10 μl of PCR products were separated in a 2 per cent (w/v) agarose gel, stained with ethidium bromide and visualised with ultraviolet light. The S2-R2 primer pair amplified approximately 320 bp fragments of the DNA from the clinical specimen and a control sample of S schenckii DNA (Fig 2). The primer pair did not amplify 320 bp fragments from normal feline skin DNA (Fig 2). Therefore, the case was diagnosed molecularly as sporotrichosis. The biopsy material from the nodule on the right hindleg was cultured on sunflower seed agar at 27°C for one week and then examined morphologically. The clinical isolate developed a black colony with a grey aerial surface after incubation for two weeks on sunflower seed agar. Microscopic examination of the isolate revealed one-cell, dark brown, thick-walled, subglobular, elliptical to cylindrical, smooth and hyaline conidia, which were 2 to 3 x 3 to 6 μm in size, produced on hyphae and in a bouquet at the tip of conidiophores. The conidiophores were slender and tapered from 1 to 2 μm diameter at the base to 0·5 to 1 μm at the tip. Since these findings were consistent with the description of S schenckii by Kwon-Chung and Bennett (1992), the isolate was identified as S schenckii. The cat was treated with itraconazole (Itrizole; Janssen Pharmaceutical) administered orally at a dose of 15 mg/kg once a day. After two months of treatment, the nodule had healed and the itraconazole was discontinued. The cat was in Veterinary Record (2005) 156, 484-485

Characterization of ATF3 Induction after Ionizing Radiation in Human Skin Cells

The epidermis is a physiological barrier that protects an organism against pathogens and chemical or physical damage. The degree of skin injuries caused by radiation influences the subsequent survival from and prognosis of such injuries. Recently, we have identified, using microarray technology, genes regulated by high-dose radiation exposure in normal human epidermal keratinocytes. Our results suggest the possible role of ATF3 in the apoptosis pathway in radiation injuries caused by high-dose radiation exposure in human skin. In the present study, we characterized ATF3 induction after X-ray irradiation in normal human skin cells. Our results showed that the induction of ATF3 mRNA is rapid, transient, and at least in part mediated through a transcriptional mechanism after X-irradiation of normal human keratinocytes. In addition, ATF3 was induced and accumulated in the nuclei of keratinocytes upon treatment with an apoptosis inducer, anisomycin. Our results also suggested that the induction mechanism of ATF3 mRNA by X-irradiation may be different not only between epidermal keratinocytes and dermal fibroblasts, but also between dermal fibroblasts and lung fibroblasts.

Larger yield of cyclobutane dimers than 8-oxo-7,8-dihydroguanine in the DNA of UVA-irradiated human skin cells

Exposure to solar ultraviolet light is the major cause of most skin cancers. While the genotoxic properties of UVB radiation are now well understood, the DNA damaging processes triggered by less energetic but more abundant UVA photons remain to be elucidated. Evidence has been provided for the induction of oxidative lesions to cellular DNA including strand breaks and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo). Formation of cyclobutane pyrimidine dimers (CPDs) has also been reported, mostly in rodent cells. In order to gain insights into the relevance of the latter photoproducts in UVA-mutagenesis of human skin, we quantified the level of 8-oxodGuo and CPDs within primary cultures of normal fibroblasts and keratinocytes using specific chromatographic assays. The yield of formation of CPDs was found to be higher than that of 8-oxodGuo in both cell types. In addition, CPDs were mostly TT derivatives, and neither (6-4) photoproducts nor Dewar valence isomers were detected. These observations are reminiscent of results obtained in rodent cells and suggest that a photosensitized triplet energy transfer occurs and that this reaction is more efficient than photooxidation of DNA components. The predominant formation of CPDs with respect to oxidative damage within normal human skin cells exposed to UVA radiation should be taken into account in photoprotection strategies.

Expression of C-type lectin receptors by subsets of dendritic cells in human skin.

C-type lectins are cell surface receptors that recognize carbohydrate structures which are often part of microbial pathogens. Several of these molecules are expressed on dendritic cells and are involved in antigen uptake. Expression of C-type lectins on dendritic cells of the human skin, i.e. Langerhans cells of the epidermis and dermal dendritic cells, has been incompletely studied to date. We therefore investigated C-type lectins in situ and on dendritic cells obtained by migration from skin explants by immunofluorescence and flow cytometry. Emphasis was laid on expression patterns of DEC-205/CD205 and BDCA-2, a marker for plasmacytoid dendritic cells. Langerhans cells in situ expressed low levels of DEC-205. Expression was upregulated upon maturation in skin explant organ culture. Most dermal dendritic cells were found to be positive for DEC-205 and DC-SIGN/CD209. Few BDCA-2-expressing cells were found in most skin samples. They were located in small groups in the dermis close beneath the basement membrane. The vast majority of all types of dendritic cells in normal human skin was of immature phenotype, i.e. did not express DC-LAMP/CD208. It is concluded that normal appearing human skin harbors different subsets of dendritic cells including few scattered BDCA-2-expressing cells, presumably plasmacytoid dendritic cells, expressing variable sets of C-type lectin receptors. This may critically contribute to the capacity of the skin immune system to flexibly respond to the world of microbial pathogens.

Matrix Metalloproteinase‐1 Production Observed After Solar‐Simulated Radiation Exposure is Assumed by Dermal Fibroblasts but Involves a Paracrine Activation Through Epidermal Keratinocytes¶

ABSTRACTChronic exposure of human skin to solar UV radiation leads to serious dermal damages, a hallmark of photoaging. In vivo, acute UV radiation has been shown previously to induce various matrix‐degrading proteases. Among them, matrix metalloproteinase‐1 (MMP‐1) has been suggested to be involved in skin photodamage. The purpose of this study was to investigate the effects of solar‐simulated radiation (SSR) on MMP‐1 production in normal human skin cells. SSR exposure of human skin reconstructed in vitro comprising both a differentiated epidermis and a fibroblast‐populated dermal equivalent led to an increase in MMP‐1 production, which was abolished when epidermis was removed immediately after SSR exposure. In addition, SSR exposure of differentiated keratinocytes grown on an acellular collagen gel did not induce MMP‐1 production. Experiments on cell cultures grown on plastic confirmed that keratinocytes failed, in contrast with fibroblasts, to produce MMP‐1 in response to SSR exposure. However, when conditioned medium from SSR‐exposed keratinocytes was added to human fibroblasts in culture, MMP‐1 production was induced. Altogether, these data show that MMP‐1 production observed after SSR exposure involved the release of soluble epidermal factors, which could modulate its production by dermal fibroblasts.

Matrix metalloproteinase-1 production observed after solar-simulated radiation exposure is assumed by dermal fibroblasts but involves a paracrine activation through epidermal keratinocytes.

Chronic exposure of human skin to solar UV radiation leads to serious dermal damages, a hallmark of photoaging. In vivo, acute UV radiation has been shown previously to induce various matrix-degrading proteases. Among them, matrix metalloproteinase-1 (MMP-1) has been suggested to be involved in skin photodamage. The purpose of this study was to investigate the effects of solar-simulated radiation (SSR) on MMP-1 production in normal human skin cells. SSR exposure of human skin reconstructed in vitro comprising both a differentiated epidermis and a fibroblast-populated dermal equivalent led to an increase in MMP-1 production, which was abolished when epidermis was removed immediately after SSR exposure. In addition, SSR exposure of differentiated keratinocytes grown on an acellular collagen gel did not induce MMP-1 production. Experiments on cell cultures grown on plastic confirmed that keratinocytes failed, in contrast with fibroblasts, to produce MMP-1 in response to SSR exposure. However, when conditioned medium from SSR-exposed keratinocytes was added to human fibroblasts in culture, MMP-1 production was induced. Altogether, these data show that MMP-1 production observed after SSR exposure involved the release of soluble epidermal factors, which could modulate its production by dermal fibroblasts.

Molecular responses to photogenotoxic stress induced by the antibiotic lomefloxacin in human skin cells: from DNA damage to apoptosis.

Photo-unstable chemicals sometimes behave as phototoxins in skin, inducing untoward clinical side-effects when exposed to sunlight. Some drugs, such as psoralens or fluoroquinolones, can damage genomic DNA, thus increasing the risk of photocarcinogenesis. Here, lomefloxacin, an antibiotic from the fluoroquinolone family known to be involved in skin tumor development in photoexposed mice, was studied using normal human skin cells in culture: fibroblasts, keratinocytes, and Caucasian melanocytes. When treated cells were exposed to simulated solar ultraviolet A (320-400 nm), lomefloxacin induced damage such as strand breaks and pyrimidine dimers in genomic DNA. Lomefloxacin also triggered various stress responses: heme-oxygenase-1 expression in fibroblasts, changes in p53 status as shown by the accumulation of p53 and p21 proteins or the induction of MDM2 and GADD45 genes, and stimulation of melanogenesis by increasing the tyrosinase activity in melanocytes. Lomefloxacin could also lead to apoptosis in keratinocytes exposed to ultraviolet A: caspase-3 was activated and FAS-L gene was induced. Moreover, keratinocytes were shown to be the most sensitive cell type to lomefloxacin phototoxic effects, in spite of the well-established effectiveness of their antioxidant equipment. These data show that the phototoxicity of a given drug can be driven by different mechanisms and that its biologic impact varies according to cell type.

Bio-artificial skin composed of gelatin and (1→3), (1→6)- β-glucan

Porous scaffolds composed of gelatin and β-glucan were prepared using the freeze-drying method. The scaffold had an inter-connected pore structure with average pore size of 90–150 μm. Results for the contact angle and cell attachment revealed that a high gelatin content was suitable for cellular attachment and distribution in two- or three-dimensional fibroblast cultures, because the gelatin had acidic residues, and arginine–glycine–aspartic acid groups. To prepare a stratified wound dressing to mimic the normal human skin, fibroblasts and keratinocyte cells were isolated from a child's foreskin, and were co-cultured in gelatin/ β-glucan scaffolds were cross-linked using 1-ethyl-(3-3-dimethylaminopropyl) carbodiimide hydrochloride. An in vivo study showed that after 1 week, the artificial dermis containing the fibroblasts enhanced the re-epithelialization of a full-thickness skin defect rather than the acellular scaffold.

Human macrophage lectin specific for galactose/N-acetylgalactosamine is a marker for cells at an intermediate stage in their differentiation from monocytes into macrophages.

We studied the expression of a human macrophage lectin specific for galactose/N-acetylgalactosamine (hMGL) during macrophage differentiation. The expression of hMGL during the in vitro differentiation induced by human serum was examined by immunostaining and Western blotting with a specific mAb, MLD-1, as well as with RT-PCR analysis. hMGL was detected on cells at an intermediate stage of differentiation. These cells were round, slightly larger in size (12.7 +/- 0.2 microm) than monocytes (9.8 +/- 0.1 microm) and expressed the macrophage marker CD14, but lacked the dendritic cell marker CD1a. The highest levels of expression occurred after 2-4 days of culture. At this time point, MLD-1 prominently stained 20-40% of the cells. Monocytes cultured for 16 h or fully differentiated monocyte-derived macrophages were negative or weak for hMGL expression. Similar transient expression was also observed during granulocyte macrophage colony stimulating factor- or macrophage colony stimulating factor-dependent macrophage differentiation. The lectin was characterized as a functional endocytic receptor for glycosylated macromolecules, since the uptake of carbohydrate polymers was partially inhibited by the addition of MLD-1. The distribution of hMGL(+) cells in normal human skin was found by immunostaining to be mainly in the upper dermis distant from vascular structures. More than 90% of the hMGL(+) cells were double stained with anti-CD68 mAb and constituted approximately 20% of the CD68(+) cells. We suggest that the dermal hMGL(+) cells are a subset of differentiated cells derived from monocytes and that hMGL is a unique marker for cells at an intermediate stage of macrophage differentiation.

CD40 ligation‐induced cytokine production in human skin explants is partly mediated via IL‐1

CD40 ligation by CD40 ligand(+) CD4(+) T cells has been claimed to be involved in inflammatory responses in human skin. However, these data are derived from in vitro cell culture systems and immunohistochemistry, and the mechanisms involved have not been fully elucidated. We previously observed that cells in intact normal human skin secrete high levels of IL-6 and IL-8 upon stimulation with IL-1 beta. In vitro studies have shown that CD40 ligation on human keratinocytes results in the production of IL-6 and IL-8 as well. We used a novel tissue culture system with intact normal human skin, and show that antibody ligation of CD40 results in the induction of several pro- and anti-inflammatory cytokines. IL-6, IL-8, tumor necrosis factor (TNF)-alpha, IL-12 and IL-1 beta were induced upon CD40 ligation and IFN-gamma stimulation, while IL-10 could be induced by CD40 ligation alone and was reduced again by the addition of IFN-gamma. Since CD40 ligation on monocytes and dendritic cells in vitro results in the secretion of IL-1, which is pre-stored in high concentrations in normal human keratinocytes, we subsequently investigated whether CD40 induced IL-6 and IL-8 production in skin is mediated via IL-1. Indeed IL-1 receptor antagonist inhibited the CD40 ligation-induced IL-6 and IL-8 production, while TNF-alpha and IL-10 production were not affected. These data show that CD40 ligation-induced secretion of IL-6 and IL-8, but not TNF-alpha and IL-10, is partially mediated via IL-1 and that IL-1 plays a prominent role in the inflammatory response initiated by CD40 ligation in intact human skin.

Biomarkers of Human Skin Cells Identified Using DermArray DNA Arrays and New Bioinformatics Methods

Biomarker genes of human skin-derived cells were identified by new simple bioinformatic methods and DNA microarray analysis utilizing in vitro cultures of normal neonatal human epidermal keratinocytes, melanocytes, and dermal fibroblasts. A survey of 4405 human cDNAs was performed using DermArray DNA microarrays. Biomarkers were rank ordered by “likelihood ratio” algorithms and stringent selection criteria that have general applicability for analyzing a minimum of three RNA samples. Signature biomarker genes (up-regulated in one cell type) and anti-signature biomarker genes (down-regulated in one cell type) were determined for the three major skin cell types. Many of the signature genes are known biomarkers for these cell types. In addition, 17 signature genes were identified as ESTs, and 22 anti-signature biomarkers were discovered. Quantitative RT–PCR was used to verify nine signature biomarker genes. A total of 158 biomarkers of normal human skin cells were identified, many of which may be valuable in diagnostic applications and as molecular targets for drug discovery and therapeutic intervention.

Effect of xenogenic repair enzymes on photoimmunology and photocarcinogenesis

Exposure to ultraviolet B (UVB) radiation leads to an increased generation of UVB-induced skin damage in humans. The most important UVB-induced side effects are UVB-induced immunosuppression and photocarcinogenesis and there is a large body of evidence that cyclobutane pyrimidine dimers (CPD) induced by UVB radiation play a pivotal role in both processes. The topical application of DNA repair enzymes is a new innovative strategy to reduce the amount of CPDs in human skin. Two different methods have recently been established. The use of T4 endonuclease V was of clinical efficacy in protecting patients with a nucleotide excision repair defect from premalignant and malignant skin lesions. Application of photolyase, a xenogenic enzyme which has been found in different organisms is also capable of removing UVB-induced CPD from normal human skin cells in vivo and appears to be more effective than T4 endonuclease V in damage removal. Photolyase encapsulated in liposomes may have in the near future a broad use as an active ingredient in modern skin care products.

In Vivo Detection of Small Subsurface Melanomas in Athymic Mice Using Noninvasive Fiber Optic Confocal Imaging

Fiber optic confocal imaging, following intravenous administration of fluorescently labeled antibodies and Texas Red-dextran, enabled in vivo detection of melanoma and surrounding blood vessels in athymic mice. Human melanoma cells (three cell lines) and cultured normal human skin cells were implanted intradermally into the haunch skin of anesthetized athymic BALB/C mice and allowed to grow to a maximum size of 2 mm diameter. Using three different fluorescein-isothiocyanate-labeled antimelanoma antibodies, single channel confocal images of melanoma cells were obtained in vivo. Using noninvasive techniques, the overall in vivo melanoma detection rate for tumors within 0.2 mm of the skin surface was 84% (27 of 32 tumors). Normal cultured human skin cells were found to have little or no fluorescence after administration of the fluorescein-isothiocyanate-labeled antibodies and tumors were not labeled by an isotype control antibody. Dual channel imaging of the implanted melanoma tumor and surrounding dermal vasculature in vivo showed increased blood vessel density at the melanoma site. Conventional immunoperoxidase histology confirmed that fiber optic confocal imaging was able to detect melanoma tumors up to 0.2 mm below the skin surface, in vivo.

Comparative in vitro cytotoxicity of ethyl acrylate and tripropylene glycol diacrylate to normal human skin and lung cells.

The potential for occupational exposure to the esters of acrylic acid (acrylates) is considerable, and, thus, requires a greater understanding of the their toxicity. Confluent (70-90%) cultures of normal human epidermal keratinocytes (NHEK), dermal fibroblasts (NHDF). or bronchial epithelium (NHBE) were exposed to the monofunctional ethyl acrylate (EA), the multifunctional tripropylene glycol diacrylate (TPGDA), or TPGDA monomer in a radiation curable lacquer (Lacquer A) at equimolar dosages in order to determine human in vitro cytotoxicity. Viability of the cells after 2-24-h exposure to the representative monofunctional or multifunctional acrylate or solvent control was used to calculate an index of acute cytotoxicity (50% inhibitory dose; ID50) and to determine the shape of the dose-response curves. TPGDA, Lacquer A, and EA were equally cytotoxic (ID50 is approximately equal to 0.1 micromol/cm2) to NHEK at equimolar doses. TPGDA or Lacquer A were more cytotoxic (is approximately equal to 100X) to NHDF or NHBE than EA. Sequential exposure of UV(A) and TPGDA to NHEK indicate the potential for a synergistic cytotoxic response. These findings are consistent with observed decreases in free sulfhydryl groups (e.g., glutathione or cysteine) that parallel the dose-response-related decreases in viability. logether, these data suggest possible differences in toxicity between the monofunctional EA and multifunctional TPGDA to NHEK, NHDF. or NHBE, possibly due to the difference in the number of functional acrylate groups and/or physicochemical differences (e.g., vapor pressure) between the acrylates investigated.

Analysis of the TCRBV Repertoire of T Cells in Normal, Human Skin: Evidence for a Restricted Diversity

Alpha beta T cells constitute an important component in the first line of immunologic defense in human skin. In order to determine the local selection forces driving T cell diversity, we studied the T cell receptor repertoire in normal human skin and compared it with that of matched blood samples. Using semiquantitative reverse transcription-polymerase chain reaction the expression of T cell receptor beta-chain V genes was determined. The majority of skin, but not blood T cells, revealed a bias towards usage of T cell receptor beta-chain V2 and V6. Whereas sequencing of T cell receptor beta-chain V2 and V6 polymerase chain reaction products showed a heterogeneous clonal distribution within these beta-chain V gene families, the analysis of other selected either over- or underrepresented beta-chain V gene families (BV3, BV12, BV13S1, BV17) revealed numerous identical T cell receptor beta-chain V transcript sequences that were not detected in blood. Restricted T cell receptor diversity in terms of beta-chain V gene preferences or clonal expansion was observed in skin samples of donors from all ages (0.5-87 y). Hence, the repertoire of T cells in normal human skin is apparently subjected to skin-specific selection throughout life. According to our data, this process could involve superantigens, which favor polyclonal accumulation of T cells using certain beta-chain V genes, as well as antigens, which induce clonal T cell expansion. Our results furthermore indicate, that T cell receptor beta-chain V repertoire restrictions do not necessarily result from disease-associated activation of the skin immune system, but could reflect regular mechanisms of immunologic homeostasis within the epithelial surface of the body.

Expression of the pro-apoptotic caspase 3/CPP32 in cutaneous basal and squamous cell carcinomas.

Apoptosis, a process of programmed cell death, plays an important role in normal and pathologic tissue homeostasis. The apoptotic cascade is triggered by caspases, a family of cystein proteases, among which caspase 3 (CPP32) seems to be the most directly related to apoptosis. Until now, few, if any, data exist on the role of CPP32 in skin tumors. We studied by immunohistochemistry, Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) the expression of CPP32 in specimens of normal human skin, basal and squamous cell carcinomas. CPP32 (both at the protein and mRNA level) was detected within epidermal and adnexal keratinocytes. CPP32 was also expressed in tumor masses of squamous cell carcinomas and more weakly in basal cell carcinomas; no correlation was found between CPP32 expression and depth of tumor invasion. CPP32 was occasionally expressed in tumor-infiltrating lymphocytes and peritumor endothelial cells. The expression of CPP32 in normal skin and in tumors arising from it suggests that this enzyme is involved in their homeostasis; its precise functional significance awaits further investigation.

Production of interferon-beta in a culture of fibroblast cells on some polymeric films.

Normal human skin (NB1-RGB) cells were cultured in the presenceof polyinosinic and polycytidylic acids, diethylaminoethyldextran, cycloheximide and actinomycin D, which induced humaninterferon-beta. The simplest induction method, that requiredonly polyinosinic and polycytidylic acids and diethylaminoethyldextran was found to give the highest production ofinterferon-beta by the cells. The cell growth and productionof interferon-beta were investigated for NB1-RGB cellscultured on silk fibroin, poly(gamma-methyl-L-glutamate),poly(gamma-benzyl-L-glutamate) and collagen films prepared bythe Langmuir-Blodgett (LB) and casting methods. The cell densityof NB1-RGB cells cultured on the LB films was found to be higherthan that on the cast films made of the same polymer. Thisindicates that not only the chemical structure of the polymersused for the preparation of the films but the preparationmethods of the films, i.e., casting and LB methods, are also astrong factor affecting the cell growth. The production ofinterferon-beta per unit number of cells was found to behigher on the cast films than that on the LB films made of thesame polymer. This is explained by the fact that the optimalsuppressed growth of NB1-RGB cells on the cast films leads tothe enhanced production of interferon-beta on the cast filmscompared to those on the LB films prepared by the same polymer.

Immunohistochemical analysis of DNA mismatch repair enzyme hMSH-2 in normal human skin and basal cell carcinomas.

We have analysed the expression and distribution of the DNA mismatch repair enzyme hMSH-2 in normal skin and basal cell carcinomas. hMSH-2 protein was investigated immunohistochemically (normal human skin: n = 10; basal cell carcinomas: n = 16) on frozen sections using a highly sensitive streptavidin-peroxidase technique and a specific mouse monoclonal antibody (clone FE11). In normal human skin, we found nuclear immunoreactivity for hMSH-2 in epidermal keratinocytes of the basal and first 1-3 suprabasal cell layers. All basal cell carcinomas analysed revealed strong nuclear imunoreactivity that was pronounced in peripheral tumour cells and cells of the palisade. Expression of hMSH-2 protein was consistently and strongly upregulated in tumour cells of the carcinomas as compared to adjacent unaffected epidermis or epidermis of normal human skin. Twelve of the sixteen carcinomas analysed revealed no visual correlation in comparing the labelling patterns for hMSH-2 with the labelling pattern for the proliferation marker Ki-67. Our findings indicate that (a) hMSH-2 is expressed in human epidermal keratinocytes, predominantly in lower cell layers of the viable epidermis; (b) expression of hMSH-2 protein is strongly upregulated in basal cell carcinomas as compared to unaffected epidermis; (c) the level of hMSH-2 proteins in the carcinomas is not exclusively regulated by the proliferative activity of these tumour cells; (d) inactivating mutations of the hMSH-2 gene may in the carcinomas not be involved in the carcinogenesis or microsatellite instability secondary to replication errors; (e) expression of hMSH-2 may be of importance for the genetic stability of basal cell carcinomas in vivo.

Gli1 Protein is Expressed in Basal Cell Carcinomas, Outer Root Sheath Keratinocytes and a Subpopulation of Mesenchymal Cells in Normal Human Skin

Genetic studies of patients with the nevoid basal cell carcinoma syndrome have led to the recognition of the importance of the hedgehog signaling pathway in the development of basal cell carcinomas of the skin. Although hedgehog signaling is known to be important in hair follicle development, the function of this pathway in adult skin and the mechanism by which activation of this pathway leads to basal cell carcinoma development remain to be established. The Gli1 family of transcription factors mediates hedgehog signaling in mammalian cells and we have shown in previous studies that Gli1 mRNA is differentially expressed in basal cell carcinomas. Using antibodies to epitopes on the N and C terminal regions of Gli1 we show now that Gli1 protein is present in basal cell carcinomas and that the protein is mainly localized to the cytoplasmic compartment. Focal nuclear staining was seen in a small number of basal cell carcinomas with the C terminal antibody which suggest that nuclear localization is not dependent on loss of the C terminus of Gli1 due to proteolysis. Strong Gli1 immunostaining was seen in the outer root sheath keratinocytes of some hair follicles, a subpopulation of mesenchymal cells in the vicinity of the bulge region of adult hair follicles and the dermal sheath cells of developing hair follicles. Quantitation of Gli1 mRNA in basal cell carcinomas using northern blot analysis indicates that Gli1 is highly expressed in basal cell carcinomas. This suggests that the lower intensity of Gli1 immunostaining in basal cell carcinoma islands relative to outer root sheath keratinocytes is not simply a reflection of differences in gene expression. The continued expression of Gli1 in adult hair follicles and in the mesenchyme of adult human skin suggest that Hh signaling may play a part in hair cycling and in epidermal mesenchymal interactions important in normal skin maintenance.

Ethanol-Modulated Cytokine Production and Expression in Skin Cells Exposed to Methotrexate

Evidence suggests a link between alcohol consumption, psoriasis and the response of psoriatic patients to methotrexate (MTX) therapy. Ethanol (EtOH) may play a role in the pathogenesis of psoriasis by upregulating the expression and inducing the local secretion of proinflammatory cytokines, e.g. interleukins IL-1α, IL-6, chemokine IL-8 and tumor necrosis factor alpha (TNF-α). We investigated whether EtOH or MTX or their combination influence the secretion of these cytokines using normal human primary skin cells (NHPSC) and epidermoid cell line A431. The objectives of this study were: (1) to quantify the differences in cellular changes induced by MTX, (2) to measure the effect of EtOH on MTX toxicity and (3) to determine the relationship between MTX and EtOH exposure and production of proinflammatory cytokines. NHPSC and A431 were incubated with 0–10 mM MTX or α-MEM (control) in the presence or absence of 40 mM EtOH. A formazan 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was used as a marker for cell viability (control was 100%). Significance was calculated by ANOVA. Cytokine release into media was quantitated by ELISA. After 24 h of MTX exposure, the release of IL-1α was unchanged. IL-6 increased 1.7 times in both cultures, and IL-8 increased 1.7 times in NHPSC and 2.1 times in A431. TNF-α release increased twice in A431 but not in NHPSC. Human recombinant IL-1α and IL-6 for 24 h had no effect, while TNF-α reduced cytoviability by 30% in NHPSC and 22% in A431. Anti-TNF-α reversed the effect produced by TNF-α in NHPSC and reduced it in A431 (11.8%, p < 0.05). We concluded that in vitro in normal human primary keratinocytes, toxicity and inflammatory responses are enhanced by EtOH.