bluuebunny/crossref_metadata_2025 huggingface dataset:
Prevention of anergy induction in cloned T cells by interleukin 12
Authors: Becker Jürgen C. , Bröcker Eva‐Bettina
Published: April 2006

Abstract A variety of tumors are potentially immunogenic but do not stimulate an effective antitumor immune response in vivo. Tumors may be capable of delivering antigen‐specific signals to T cells, but may not deliver the costimulatory signals necessary for full activation of T cells. In this regard, we recently reported that a human melanoma cell line (sMC) expressing MHC class II, was able to induce clonal anergy in a specific, MHC‐restricted CD‐4+ T cell clone (sTC3). We used this system to investigate the influence of interleukin (IL)‐12 on induction of this T cell unresponsiveness. The presence of 10 to 100 U IL‐12 during the induction phase of anergy leads to a primary proliferative response of sTC3, which was significantly higher than that induced by IL‐12 alone; however, in the absence of IL‐12 no proliferation was seen during the induction of anergy. Subsequent optimal stimulation of IL‐12 treated cells, but not of those cultured without IL‐12, led to substantial IL‐2 production and cell proliferation. This indicates that induction of the unresponsive state could be inhibited by IL‐12. In addition, we have recently demonstrated that anergic T cell clones can produce high amounts of 1L‐10 and that this event was correlated with their impaired ability to produce IL‐2. This marked induction of IL‐10 can be suppressed if IL‐12 is present during initiation of unresponsiveness. However, IL‐12 was not able to prime the T cell clone, sTC3, to become resistant against the anergizing stimulus, as this cytokine was only effective when present at the time of anergy induction. These findings indicate that IL‐12 is very effective in preventing anergy when present at the same time as the anergizing stimulus, but is unable to prime T cells to resist anergy induction in this system.
IL‐10 secretion of allergen‐specific skin‐derived T cells correlates positively with that of the Th2 cytokines IL‐4 and IL‐5*
Authors: Gutgesell Carsten , Yssel Hans , Scheel Dagmar , Gerdes Johannes , Neumann Christine
Published: April 2006

Abstract In atopic individuals, allergen‐specific CD4+ T lymphocytes often belong to the T‐helper 2 (Th2) subset as they secrete the marker cylokincs interleukin‐4 (IL‐4) and IL‐5 but not intcrfcron‐y (INF‐y). IL‐10 is a cytokine the production of which, in the mouse system has been described to be restricted to the Th2 subset, but in the human was found to be produced by both ThI and Th2 T cell clones (TCC). We have recently shown that house dust mite antigen (Dermatophagoides pteron‐yssinus)‐specific TCC isolated from skin of patients with atopic dermatitis have a more polarized Th2 cytokine production profile than TCC obtained from the peripheral blood of these patients. In this study, we report that skin‐derived TCC secrete more IL‐10, IL‐4 and IL‐5, than TCC isolated from the blood of the same individual (p < 0.05). The difference was more significant with specific TCC than with non‐specific TCC. Furthermore, there was a positive correlation between the production of IL‐10 and that of IL‐4 and IL‐5, respectively. In addition, the amount of IL‐4 and IL‐5 secreted by specific TCC from the skin correlated positively. These results were confirmed by the detection of mRNA by PCR. Finally, our data confirm that in human blood‐derived TCC IL‐10 secretion is not related to a particular cytokine production profile. We suggest that the skin of AD provides an unique environment for the development of aTh2‐likc secretion pattern not only with respect to IL‐4 and IL‐5 but also regarding IL‐10.
Heat‐stable antigen is expressed by murine keratinocytes and delivers costimulatory signals in T‐cell activation
Authors: Erdmann Gabicla , Saloga Joachim , Mohamadzadeh Mansour , Becker Detlef , Knop Jürgen , Enk Alexander H.
Published: April 2006

Abstract Heat‐stable antigen (HSA), expressed by various antigen‐presenting cells (APC), has been described as a costimulatory molecule for CD4+ T cells. Recently, we observed that HSA also serves as an important costimulatory molecule on epidermal Langerhans cells (LC). During these studies, low levels of HSA staining were also detected on normal murine keratinocytes (KC). To investigate whether HSA also is involved in T‐cell activation by KC, normal murine KC or the spontaneously transformed KC cell‐line PAM 212 were treated with PDB or PMA to induce HSA‐expression. FACS analyses showed induction of HSA expression on normal murine KC, as well as PAM 212 cells. In functional assays PDB or PMA‐treated normal or transformed KC were far more potent inducers of primary allogeneic T‐cell responses than untreated KC. Addition of anti‐HSA‐specific mAb 20C9 specifically inhibited the costimulatory activity of KC, an effect that was even more pronounced when CTLA‐4Ig was added to the cultures. Cleavage of HSA on KC surfaces by a phosphoinositol‐specific phospholipase C (PI‐PLC) also significantly inhibited the costimulatory capacity of KC for naive CD4+ T cells. In aggregate, our data indicate that expression of HSA on activated KC contributes to the capacity of these cells to induce proliferation of allogeneic T cells.
Keratinocyte degeneration in human facial skin: Documentation of new ultrastructural markers for photodamage and their improvement during topical tretinoin therapy
Authors: Yamamoto Osamu , Bhawan Jag , Hara Masahiro , Gilchrest Barbara A.
Published: April 2006

Abstract We examined epidermal impairment in photodamaged Caucasian skin by light and electron microscopy and observed two types of degeneration of the basal and suprabasal keratinocytes. The first was an electron‐lucent degeneration predominantly seen in the periphery of the cells. The marked lucent degeneration occurred in 14% of the basal and suprabasal keratinocytes, predominantly in cells immediately adjacent to melanocytes. In skin specimens with a large number of such damaged keratinocytes, bleb‐like keratinocytic protrusions or electron‐lucent intercellular structures were also seen. Many vacuolar structures were observed just under the dermoepidermal junction, occupying 9% of the junction length. These structures were produced by hernialion of the degenerative portion of the basal and suprabasal keratinocytes, and appeared to be phagocytized by dermal macrophages. The vacuolar alterations in the basal layer and dermoepidermal junction previously reported at the light microscopic level probably represent these intercellular lucent structures, bleb‐like protrusions and vacuole‐like structures at the electron microscopic level. The second type, dark‐staining keratinocytes, probably representing an extensive degenerative process, constituted 4% of the basal and suprabasal keratinocytes. After 12 months of topical trelinoin treatment, dramatic improvement of both degenerative processes of the keratinocytes was noted.
Quantitative screening of human stratum corneum lipids by sequential one‐dimensional thin layer chromatography
Authors: Schürer Nanna Y. , Schliep Viola , Barlag Kerstin
Published: April 2006

Abstract There is a need in many studies on the epidermal permeability barrier for practical and reliable separation of all major fractions of human stratum corneum lipids. Various methods have been described including thin layer chromatography, high performance thin layer chromatography and iatroscan. However, none of these methods seems to be applicable for an inexpensive, rapid and reliable analysis of a large number of samples. Here, such a method for the separation and quantification of all major stratum corneum lipid fractions is presented. This method employs the one‐dimensional separation of stratum corneum lipids using thin layer chromatography. The systems used herein are based upon various modifications of the solvents, solvent ratio and developing distance of each system. For quantification of chromatographed and charred lipids a Desaga densitometer is used. The system described here had been successfully applied to 550 samples of human stratum corneum lipids.
Effect of beta‐carotene supplementation on the human sunburn reaction
Authors: Garmyn Marjan , Ribaya‐Mercado Judy D. , Russel Robert M. , Bhawan Jag , Gilchrest Barbara A.
Published: April 2006

Abstract Beta‐carotene, a quencher of excited species such as singlet oxygen and free radicals, has been reported to protect against cutaneous photodamage, including sunburn acutely and photocarcinogenesis chronically. The present double blind placebo‐controlled study examines the ef‐tect of beta‐carotene supplementation on the human sunburn response and specifically on the induction of sunburn cells at the time of peak reaction intensity (24 h) after a single solar simulated light exposure 3 times the individually determined minimal erythema dose (MED). Administered orally either as a single 120 mg dose to dietarily restricted subjects or for 23 d as a daily 90 mg supplement to subjects on standard diets, beta‐carotene increased plasma and skin levels of beta‐carotene compared to both pretreatment levels and placebo‐treated controls, but provided no clinically or histologically detectable protection against a 3 MED sunburn reaction. Thus, these data suggest that oral beta‐carotene supplementation is unlikely to modify the severity of cutaneous photodamage in normal individuals to a clinically meaningful degree.
Detection of autoantibodies against bullous pemphigoid and pemphigus antigens by an enzyme‐linked immunosorbent assay using the bacterial recombinant proteins
Authors: Ide Akiko , Hashimoto Takashi , Amagai Masayuki , Tanaka Masaru , Nishikawa Takeji
Published: April 2006

Abstract To develop a new method to evaluate autoantibodies in various autoimmune bullous skin diseases, we examined reactivity of bullous pemphigoid (BP) and pemphigus vulgaris (PV) patients' sera with partial bacterial fusion proteins of the 230 kD BP antigen (BPAG1) and PV antigen, respectively, by an enzyme‐linked immunosorbent assay (ELISA), and compared the results with those of immunoblotting. We used two fusion proteins derived from the mouse BPAG1 and a fusion protein derived from the amino‐terminus (EC 1‐2) of PV antigen. For both BP and PV sera, the ELISA scores were well correlated with the reactivities on immunoblot assays. The present study indicates that ELISA using recombinant antigen proteins in various autoimmune bullous skin diseases will be a new useful technique to detect the autoantibodies. However, development of recombinant proteins with the entire molecule and correct conformation will be necessary to establish a perfect ELISA system in the future.
Interleukin‐8 and melanoma growth‐stimulating activity (GRO) are induced by ultraviolet B radiation in human keratinocyte cell lines
Authors: Venner Thomas J. , Sauder Daniel N. , Feliciani Claudio , Mckenzie Roderick C.
Published: April 2006

Abstract Ultraviolet radiation can induce the transcription and release of cytokines From keratinocytes (KC's). These cytokines have the potential to modulate local and systemic immunologic responses. In this paper we report that northern blotting showed that human KC and KC lines expressed a 1.2‐1.4 kb transcript for the chemokine and melanoma growth‐stimulatory protein, GRO‐α and that ultraviolet B radiation (UVB) could upregulate the expression of GRO‐α mRNA and protein in the KC line A431. The GRO‐α gene response to UVB was maximal at 48h post‐irradiation with 70 J/m2. Reverse transcription‐polymerase chain reaction (RT‐PCR) revealed a 4.5‐fold increase in GRO‐α mRNA over basal levels (p<0.001). GRO‐α protein was measured in the culture media by enzyme‐linked immunosorbent assay (ELISA). Media from unirradiated cultures contained 1166±83 pg/ml GRO‐α protein. After UVB, a time‐dependent increase in GRO‐α protein was seen in the culture media from 6‐48h. At 48h post‐irradiation the GRO‐α protein content was 27583±678 pg/ml, or 23 times the basal level. This protein release could be inhibited by 70% when the cells were pre‐incubated with 10 μg/ml interleukin‐1 receptor antagonist (IL‐1RA). We also show that another potent leukocyte chemoattractant, Interleukin‐8 (IL‐8), was induced in A431 cells by UVB. The induction of IL‐8 mRNA began as early as 3h post‐irradiation, when it reached twice basal levels (p<0.05) and reached 4.5‐fold basal levels at 48h post‐irradiation (p<0.005). Analysis of the conditioned media from the irradiated cells by ELISA revealed that IL‐8 protein accumulation was increased from 162± 16 pg/ml (unirradiated cultures) to 993± 120 pg/ml 24h post‐irradiation. The accumulation increased to 1383± 178 pg/ml, or 9‐fold the basal level by 48h post‐irradiation. Thus keratinocytes are capable of generating chemoattractant factors which enhance melanoma growth after UVB irradiation, which may contribute to the inflammatory infiltrate found in the dermis of sunburned skin.
Ultrastructural effects of topical tretinoin on dermo‐epidermal junction and papillary dermis in photodamaged skin. A controlled study
Authors: Yamamoto O. , Bhawan J. , Solares G. , Tsay A. W , Gilchrest B. A.
Published: April 2006

Abstract We examined the effects of daily topical application of 0.05% tretinoin cream on photodamaged Caucasian facial skin by electron microscopy. Specimens obtained pretreatment, after 6 and 12 months of tretinoin therapy (20 patients), and after 6 months of vehicle treatment (5 patients) were compared in a blinded fashion, with special attention to the dermoepidermal junction and papillary dermis. Baseline specimens disclosed various degrees of damage including reduplication of basal lamina, smudging and sparsity of collagen fibers, and nodular arrangement of degenerated microfibrils in the papillary dermis. No significant changes were observed at 6 months in the papillary dermis of either tretinoin‐treated or vehicle‐treated patients. After 12 months of tretinoin treatment, however, disorganized collagen fibers, which were conspicuous in 11 patients at baseline, were replaced by new well‐organized collagen fibers in a wavy pattern in 6 patients. In addition, the amount of nodularly degenerated microfibrillar material decreased in 15 of 18 patients with this finding at baseline. In contrast, no significant change was noted in the number of anchoring fibrils per unit length of the lamina densa. These observations provide further evidence that topical treatment with 0.05% tretinoin produces papillary dermal reconstruction, for which more than 6 months of application were required.
Rapid purification of human Langerhans cells using paramagnetic microbeads
Authors: Simon Jan C. , Dittmar Henning C. , Schopf Erwin , Wilting Jörg , Christ Bodo , De Roche Roland
Published: April 2006

Abstract Detailed studies on the biology of Langerhans cells (LC), which account for only 1‐3% of all epidermal cells, require isolation from their cutaneous symbionts. Several techniques of LC isolation have been reported, including positive enrichment with mAb coupled to immuno‐magnetic beads. The disadvantage of this technique is the size of the beads (= 2‐5 μm), which can interfere with subsequent phenotypic and functional analyses. This limitation prompted us to test wheter paramagnetic microbeads (15 nm) employed by the MACS™ system could be used to purify LC from human skin. To isolate fresh LC (fLC), epidermal cell suspensions (EC) were stained with anti‐CD la mAb and with appropriate secondary reagents conjugated to microbeads and to FITC. They were then passed over a separation column and exposed to a strong magnetic field. Thereafter both CD la‐depleted and CD la‐enriched cells were collected. Cultured LC (cLC) were isolated by staining 72‐h cultured EC with anti‐HLA‐DR mAb followed by the same isolation procedure. Using this technique, we could routinely isolate viable EC that were 45‐88% CD la or HLA‐DR+ as determined by FACS. Two‐color FACS analysis demonstrated the majority of MACS‐purified cells to be CDla+/HLA‐DR+, indicating that they were indeed LC. By transmission electron microscopy (TEM), the MACS‐purified CDla+/HLA‐DR+ cells showed typical ultrastructural characteristics of LC. Furthermore, MACS‐purified fLC or cLC were functionally intact, because they stimulated the proliferation of alloreactive T cells in a primary, one‐way, mixed epidermal cell leukocyte reaction (MECLR). We conclude that MACS‐separation is an efficient and rapid method to isolate human fLC and cLC of high purity and unimpaired function.
Growth and pigmentation in genetically related Cloudman S91 melanoma cell lines treated with 3‐isobutyl‐1‐methyl‐xanthine and β‐melanocyte‐stimulating hormone
Authors: Cieszka Krystyna A. , Hill Helene Z. , Hill George J. , Plonka Przemyslaw M.
Published: April 2006

Abstract 4 clonal sublines of Cloudman S91 melanoma cells, S91/mel, S91/13, S91/6 and S91/amel, were evaluated for changes in growth, pigment content and plating efficiency during and after treatment with a cyclic‐AMP phosphodiesterase inhibitor‐melanin‐stimulating agent, 3‐isobutyl‐l‐methyl‐xanthine (IBMX) plus β‐melanocyte stimulating hormone (β‐MSH) or IBMX alone. After combined treatment, increases in melanin content on day 3 were 48, 27, 11, and 2 pg/cell in the four cell lines respectively. In each case IBMX alone was less effective than IBMX plus β‐MSH. Doubling time increased and plating efficiency decreased with increased melanization. The increases in doubling time and decreases in plating efficiency were cell line dependent. The greatest rate of increase in doubling time and decrease in plating efficiency as a function of melanin content were seen in S91/amel, which produced the least pigment. The lowest rates of increase/decrease were seen in S91/mel, which produced the most pigment. Melanin pigment induced in the cells was classified as etimelanin by EPR determination. The differential response to induction of pigmentation makes these cell lines suitable models for comparative studies on the role of melanin in pigment cell biology.
Diminished expression of the extracellular domain of bullous pemphigoid antigen 2 (BPAG2) in the epidermal basement membrane of patients with generalized atrophic benign epidermolysis bullosa
Authors: Pohla‐Gubo G. , Lazarova Z. , Giudice G. J. , Liebert M. , Grassegger A. , Hintner H. , Yancey Kim. B.
Published: April 2006

Abstract Generalized atrophic benign epidermolysis bullosa (GABEB) is a nonlethal form of junctional epidermolysis bullosa characterized by generalized skin and mucosal blisters that heal with atrophy; other features include alopecia, nail dystrophy, large melanocytic nevi, and autosomal recessive inheritance. The specific aim of this study was to identify an abnormality in epidermal basement membrane adhesion molecules in well characterized GABEB patients that would explain why these subjects' epidermis separates from their epidermal basement membrane. Cryostat sections of nonlesional skin from 8 GABEB patients in 5 different families as well as skin from normal volunteers (controls) were studied by indirect iminunofluorescence microscopy using rabbit antiserum directed against a BPAG1 fusion protein or monoclonal antibodies directed against the extracellular domain of BPAG2 (HD18 and 233), epiligrin (P1E1), laminin 5 (GB3), types IV and VII collagen, or integrin subunits α2, α3, β, α6, or β4. In these studies, monoclonal antibodies HD18 and 233 showed no reactivity and diminished reactivity, respectively, to the epidermal BM of all GABEB patients. Interestingly, in one patient, the absent or diminished reactivities of monoclonal anti‐BPAG2 antibodies were limited to well demarcated portions of an otherwise intact epidermal basement membrane. Moreover, BPAG1, epiligrin, laminin 5, types IV and VII collagen, and all integrin subunits under study were expressed in the same manner in both GABEB and normal human skin. These findings identify an abnormality in the extracellular domain of BPAG2 in the skin of GABEB patients. BPAG2 (type XVII collagen) is a transmembrane, hemidesmo‐some‐associated molecule whose extracellular domain resides at the exact level where blisters develop in the skin of patients with GABEB. Impairment of this adhesion molecule may play a key role in the pathogenesis of this inherited subepidermal bullous disease.
Mast cells in the cytokine network: the what, where from and what for
Authors: Czarnetzki Beate M. , Grabbe Jürgcn , Kolde Gerhard , Krüger‐Krasagakes Sabine , Welker Pia , Zuberbier Torsten
Published: April 2006

Abstract The basic understanding of mast cell ontogeny and function has been fundamentally changed in recent years with observations that the cells produce and respond to a broad range of cytokines. These rapidly accruing data and their potential significance were discussed at the recent symposium “Mast Cells in the Cytokine Network”, and the overview lectures of most speakers are summarized in this special journal issue. In the present introductory manuscript, the organizers of the meeting discuss data fundamental to an understanding of the topic and highlight aspects of special interest. They consider mast cells to be defined most reliably by their unique ultrastructure since the cells are highly heterogeneous in dependence of the species studied, their tissue location, their stage of development and probably also in relation to cytokines. Most other characteristics of mast cells are shared with diverse other cell types. Murine mast cell development is induced by several cytokines. These factors are mostly ineffective in human cells except for stem cell factor which causes mast cell development from CD34+/c‐kit+ progenitors. There is however recent evidence thai fibroblasts and keratinocytes produce additional growth factors for human mast cells. Regarding cytokine secretion, most molecules known so far are produced by both murine and human mast cells. The cells furthermore bear receptors for several cytokines, enabling them to respond in an autocrine and paracrine fashion. Mast cells may thus function within a complex cytokine network, affecting physiological as well as immunological and inflammatory processes.
Cytokine secretion by human mast cells
Authors: Krüger‐Krasagakes Sabine , Czarnetzki Beate M.
Published: April 2006

Abstract Mast cells have been traditionally viewed as effector cells of immediate‐type hypersensitivity reactions. Besides this, mast cell activation and degranulation have been associated with various biologically and clinically important functions. Results of the past few years suggest that mast cells are involved in the development of late‐phase reactions and influence other chronic inflammatory responses through the generation and secretion of various multipotcnlial cylokines.
Expression of adhesion molecules and their ligands in contact allergy
Authors: Wahbi Abdel‐hadi , Marcusson Jan A. , Sundqvist Karl‐Gösta
Published: April 2006

Abstract Sequential biopsies from skin lesions induced by nickel sulphate and sodium lauryl sulphate, respectively, were investigated with respect to expression of extracellular matrix proteins and adhesion molecules on lymphocytes, endothelial cells, and keratinocytes. The majority of the infiltrating lymphocytes expressed VLA‐4, LFA‐1, CD44 and ICAM‐1, a variable fraction expressed Leu‐8 and VLA‐5, and few or no cells were positive for VLA‐1, VLA‐2 and VLA‐6. Noteworthy, was that the infiltrating cells showed a substantial amount of fibronectin but relatively small or negliglible presence of laminin, collagen type IV, IgG, IgA. IgM, and albumin. The fibronectin was associated with cell bodies as well as the area surrounding infiltrating cells. The number of infiltrating cells was larger in biopsies from nickel‐sulphate induced lesions and the infiltrates contained more fibroneclin than biopsies from lesions induced by sodium lauryl sulphate. However, at the single‐cell level, the expression of VLA antigens, LFA‐1, CD44 and ICAM‐1 was similar in both groups. The endothelial cells of skin biopsies from nickel‐sulphate‐induced lesions showed a stronger expression of VCAM‐1, ELAM‐1 and ICAM‐I compared to biopsies from sodium lauryl sulphate‐induced lesions. In the biopsies from nickel sulphate‐induced lesions, the keratinocytes showed a tendency to less VLA‐6 expression. These results suggest that fibroneclin plays a role in lymphocyte extravasation or extravascular lymphocyte migration.
Improved method for rhesus monkey or rat skin preparation and cryosectioning for topical or transdermal skin distribution studies
Authors: Fong Eva K. , Frank John D. , Cartwright Mark E. , Loper Alice E.
Published: April 2006

Abstract A method for preparing skin biopsies for cryosectioning was developed to accurately obtain samples from specific areas of the dermis, while minimizing contamination with epidermal tissue. Routine preparation of 6 mm punch biopsies from freshly excised, full‐thickness skin produced contraction and folding of the edges of the biopsy prior to mounting for snap‐freezing and cryosectioning. Sample orientation was ruined, and cryosections were heterogeneous with respect to dermal structures and/or to dermal and epidermal layers. Biopsy artifacts were prevented by prefreezing skin over dry ice prior to taking biopsies. The biopsies were held frozen on dry ice until they were mounted on cryostat pegs with flattened, frozen OCT surfaces; then they were snap‐frozen in chilled OCT in an isopentane bath cooled with liquid nitrogen. The method for determining skin level homogeneity of cryosections consisted of taking 10 μm cryosections for histology between sections sampled for drug level analysis. The histological sections were fixed in 5% acetic acid in methanol and stained with hematoxylin and eosin to define the skin layers and structures associated with each sample for analysis. Histological sections from prefrozen skin had fewer processing artifacts, and dermal cryosections free of epidermal contamination were dramatically increased compared to the routine procedure.
Superantigens
Authors: Saloga Joachim , Gelfand Erwin W. , Knop Jürgen
Published: April 2006

Abstract Superantigens are potent modulators of the immune system. Some of their biological and immunological properties are reviewed here with special attention to their potential significance for cutaneous inflammation, specific skin immune responses and skin diseases.
Modulation of keratinocyte growth factor (KGF) mRNA expression in human dermal fibroblasts grown in monolayer or within a collagen matrix
Authors: Le Panse Rozen , Bouchard Brigitte , Lebreton Corinne , Coulomb Bernard
Published: April 2006

Abstract In this study, we analysed the modulation of keratinocyte growth factor (KGF) mRNA expression in human dermal fibroblasts cultured either in monolayer or within a collagen matrix (dermal equivalent). In monolayer cultures, KGF expression by quiescent fibroblasts was stimulated by different growth substances such as serum, epidermal growth factor and basic fibroblast growth factor. Moreover, we demonstrated that the induction of this gene was mediated by at least 2 different signalling pathways involving protein kinase C (PKC) and cAMP. In dermal equivalents, we observed that the collagen matrix negatively modulated KGF mRNA expression. Indeed, among the growth substances used, only the serum slightly stimulated KGF expression. Nevertheless, as in monolayers, this induction involved at least PKC and cAMP signalling pathways. As the collagen matrix can modulate fibroblast growth, we also studied KGF expression in growing fibroblasts from either monolayer cultures or dermal equivalents. We then showed that this collagen matrix negatively influenced KGF expression independently of the proliferative state of fibroblasts. All these results underline the fact that KGF mRNA expression by human dermal fibroblasts is induced by different substances; however this expression can be modulated by fibroblast‐matrix interactions.
Tattooing increases the number of Langerhans cells in skin: an immunocytochemical study
Authors: Karanth Suryanarayana S.
Published: April 2006

Abstract Tattooing is an act of permanent marking of the skin with indelible patterns by pricking and inserting pigments. Langerhans cells (LCS) are dendritic cells normally present in suprabasal layers of the epidermis of the skin. To assess whether there were any effects caused by the tattooing on Langerhans cell population and cutaneous nerves, skin from affected areas (n=15) was compared with controls (n=10). Frozen sections were immunostained with antisera to S‐100. No discernible change either in distribution or in number of Langerhans cells and nerves was seen upon comparison with control skin taken from different areas, but all of the specimens taken from affected areas had a significant increase in the number of Langerhans cells (p < 0.001) even after several years of tattooing with no change in the cutaneous nerves. Thus, the study shows persistent stimulation of Langerhans cell population in tattooed skin.
In vivo cytokine and receptor gene expression during the rat hair growth cycle
Authors: Little Julic C. , Redwood Katherine L. , Granger Stewart P. , Jenkins Gail
Published: April 2006

Abstract A number of cytokines have previously been localised within the developing and adult hair follicle, however, the role they play in producing a mature hair follicle remains unknown. In an attempt to identify dermal papilla specific cytokines and thus those that may have an important controlling role, cytokine gene expression profiles, obtained by reverse transcriplase‐polymerase chain reaction (RT‐PCR), were compared between whole anagen rat hair follicles, passage 2 dermal papillae (a cell type with hair inductive capacity), and footpad fibroblasts (a non‐hair inducing cell type). Based on this qualitative data, we were unable to identify a dermal papilla specific gene. The analysis of the pattern and timing of cytokine gene expression during the hair cycle is likely to be more informative. A semi‐quantitative RT‐PCR technique was therefore developed for studying trends in the level of in vivo expression of the following cytokines and their receptors from early anagen to early catagen in the rat hair growth cycle: insulin‐like growth factor I, transforming growth factor β1, tumour necrosis factor, and basic fibroblast growth factor. These genes were found to be differentially expressed and this was correlated with their possible functions in controlling the hair growth cycle, providing valuable insights into the role of cytokines in regulating the hair growth process.
Tyrosinase may protect human melanocytes from the cytotoxic effects of the superoxide an ion
Authors: Valverde P. , Manning P. , Todd C. , McNeil C.J. , Thody A. J.
Published: April 2006

Abstract Reactive oxygen species (ROS) are potentially cytotoxic and several mechanisms have evolved to protect against their damaging effects. In melanocytes, tyrosinase may have such a role by utilising the superoxide anion (O2−)in the production of melanin. In the present study, we have examined the cytotoxic effects of O2− and hydrogen peroxide (H2O2) in human melanocytes both before and following the activation of tyrosinase. Xanthine oxidase (XO, 5–150 mU · ml−1) and glucose oxidase (GO, 0.1‐20 mU · ml−1) were used to generate the O2− and H2O2 respectively, and the cytotoxic effects assessed by measuring cell survival using the 3‐[4,5‐dimethylthiazol‐2‐yl]‐2.5 diphenyltetrazolium (MTT) assay. 3 h later, dose related decreases in melanocyte survival were seen. Similar experiments with keratinocytes and libroblasts showed that these cells were more resistant to the cytotoxic effects of O2− than were the melanocytes. The effect of increasing tyrosinase activity was examined by growing the melanocytes in the presence of an analogue of melano‐cyte‐stimulating hormone (MSH) Nle4DPhe7α‐MSH (10−8 M), for 48 h. This increased tyrosinase activity, melanin content, the ability to trap O2− and the resistance of the melanocytes to the cytoloxic effects of this ROS. but failed to alter their susceptibility to the damaging effects of H2O. Nle4DPhe7α‐MSH had no effect on the resistance of keratinocytes and libroblasts to either O2− or H202. After 3 h, XO. as opposed to GO. also increased the melanin content of human melanocytes; this effect was not accompanied by an increase in tyrosinase activity. The present results suggest that tyrosinase may utilise O2− to produce melanin and that this process may protect melanocytes from the potentially damaging effects of this ROS.
Nickel chloride/chlorides of divalent metal interactions in nickel‐sensitive subjects
Authors: Santucci B. , Cannistraci C. , Cristaudo A. , Picardo M.
Published: April 2006

Abstract To verify if the counter‐ion Cl− permits the same interactions between nickel and divalent metals with physicochemical similarities as the counter‐ion SO4− does, 50 sensitive subjects to nickel sulfate 5% pet. who previously gave positive patch test reactions either to 8 μl of aq. nickel sulfate 0.1 M or to 8 μl of aq. nickel chloride 0.1 M, or to both, were patch retested simultaneously to 8 μl of respectively, aq. nickel sulfate 0.1 M and aq. nickel chloride 0.1 M, and to 8 (il of aq. mixed solutions containing, respectively, nickel chloride 0.1 M +magnesium chloride 0.3 M, nickel chloride 0.1 M+ zinc chloride 0.3 M, nickel chloride 0.1 M+zinc chloride 0.5 M, nickel chloride 0.1 M + manganese chloride 0.3 M, and nickel chloride 0.1 M + manganese chloride O.5 M. Whilst 4 subjects gave a positive patch test response to only nickel sulphate. 8 gave a positive response to nickel chloride alone and the remaining 38 gave a concomitant positive response to both. In all subjects who gave positive responses to nickel chloride, the chlorides of divalent metals were not able to inhibit or reduce the positive reaction. 25 healthy subjects patch tested to both single salts and mixed solutions, and all gave negative responses. 9 of the 50 subjects, 4 who previously gave positive reactions to only nickel chloride 0.1 M, and 5 with concomitant reactions of equal intensity to both nickel chloride and nickel sulfate 0.1 M, were patch retested simultaneously to 8 μ1 of, respectively, aq. nickel sulfate 0.1 M, aq. nickel chloride 0.1 M and aq. mixed solutions containing nickel sulfate (0.1 M) mixed with sulfates (0.3 M) and nickel chloride (0.1 M) mixed with chlorides of Mg, Zn, Mn (0.3 M). Whilst the mixed sulfate solutions were able to reduce nickel sulfale, 0.1 M patch test positive reactions, those containing chlorides, at all concentrations tested, did not inhibit the nickel chloride reactions in any of the subjects. The results of the tests to chlorides, compared to those reached on testing to sulfates of the same metals, lead us to hypothesize that the anion probably affects the uptake and local tissue distribution of the metal, modulating in this way, together with the individual cutaneous ligands, its effects.
Epidermal innervation correlates with severity of photodamage
Authors: Toyoda M. , Hara M. , Bhawan J.
Published: April 2006

Abstract Intraepidermal nerve fibers were studied by electron microscopy in chronically photodamaged preauricular skin and in paired sun‐protected postauricular sites of 20 Caucasian women aged 56–70 years. As previously reported, basal keratinocytes in the sun‐exposed skin showed various degrees of degenerative changes including intracellular vacuolar structures and widened intercellular spaces. Neurites were frequently closely apposed to basal keratinocytes in preauricular sun‐exposed skin, but were observed less than 10% as often in sun‐protected postauricular skin. When degree of epidermal photodamage was quantified by means of the number of degenerated keratinocytes per 100 keratinocytes in the basal layer, the number of intraepidermal nerve libers was significantly correlated by linear regression analysis to the severity of epidermal photodamage (r=0.913) independent of anatomical sites. These results demonstrate for the first time a correlation between degree of epidermal innervation and chronic photodamage and suggest the possibility of neural involvement in the pathophysiology and/or repair of photodamaged skin.
Human keratin diseases:
Authors: Cordon Laura D. , McLean W H. Irwin
Published: April 2006

Abstract Keratins are heteropolymeric proteins which form the intermediate filament cytoskeleton in epithelial cells. Since 1991. mutations in several keratin genes have been found to cause a variety of human diseases affecting the epidermis and other epithelial structures. Epidermolysis bullosa simplex (EBS) was the First mechanobullous disease for which the underlying genetic lesion was found, with mutations in both the K5 and K14 genes rendering basal epidermal keratinocytes less resilient to trauma, resulting in skin fragility. The site of mutation in the keratin protein correlates with phenotypic severity in this disorder. Since mutations were identified in the basal cell keratins, the total number of keratin genes associated with diseases has risen to eleven. The rod domains of suprabasal keratins K1 and K 10 are mutated in bullous congenital ichthyosiform erythroderma (BC1E; also called epidermolytic hyperkeratosis, EH) and mosaicism for K 1/K 10 mutations results in a nevoid distribution of EH. An unusual mutation in the VI domain of K1 has also been found to cause diffuse non‐epidermolytic palmoplantar keratoderma (DNEPPK.). Mutations in palmoplantar specific keratin K9 cause epidermolytic palmoplantar keratoderma (EPPK) and mutations in the late differentiation suprabasal keratin K2e cause iehthyosis bullosa of Siemens (IBS). In the last year or so, mutations were discovered in differentiation specific keratins K6a and K16 causing pachyonychia congenita type 1 and K1 7 mutations occur in pachyonychia congenita type 2. K16 and K17 mutations have also been reported to produce phenotypes with little or no nail changes: K16 mutations can present as focal non‐epidermolytic palmoplantar keratoderma (NEPPK) and K17 mutations can result in a phenotype resembling steatocystoma multiplex. Recently, mutation of mucosal keratin pair K4 and K13 has been shown to underlie white sponge nevus (WSN). This year, the first mutations in a keratin‐associated protein, plectin, were shown to cause a variant of epidermolysis bullosa associated with late‐onset muscular dystrophy (MD‐EBS). An unusual mutation has been identified in K.5 which is responsible for EBS with mottled pigmentation and genetic linkage analysis suggests that the hair disorder monilethrix is likely to be due to a mutation in a hair keratin. The study of keratin diseases has led to a better understanding of the importance of the intermediate filament cytoskeleton and associated connector molecules in maintaining the structural integrity of the epidermis and other high stress epithelial tissues, as well as allowing diagnosis at the molecular level thus facilitating prenatal testing for this heterogeneous group of genodermatoses.
Attachment, spreading and migration of melanoma cells on vitronectin
Authors: van Leeuwen Robert L. , Yoshinaga Iara G. , Akasaka Toshihide , Dekker Sybren K. , Vermeer Bert Jan , Byers H. Randolph
Published: April 2006

Abstract Recent in situ studies suggest the αvβ3 integrin is a tumour progression marker in melanoma. We analyzed 5 human melanoma cell lines for their expression of the vitronectin binding αvβ3 and αvβ5, integrins using flow cytometry. The role of these receptors in cell attachment, spreading and migration was investigated using attachment assays, video time lapse spreading and migration assays and with function blocking monoclonal antibodies. Cell lines derived from later stages of tumor progression exhibited high levels of αvβ3 expression, whereas no similar correlation with αvβ5 expression was identified. Cell attachment, spreading and migration response on vitronectin correlated well with the expression level of the αvβ3 but not the αvβ5 vitronectin receptor. Blocking of the αvβ3 integrin resulted in a significant decrease in cell attachment, spreading and motility whereas the function blocking antibody against the αvβ5 integrin only inhibited cell attachment in cell lines with the highest level of expression of this integrin. Taken together, our study indicates that the level of expression of the αvβ3 and αvβ5 integrins is heterogeneous in melanoma cell lines and that the αvβ5 integrin, if present, may function only during the initial cell attachment whereas the αvβ3 plays an important rôle in cell spreading and cell migration as well.
The expression of cytokines, growth factors and ICAM‐1 in the healing of human cutaneous xenografts on nude mice
Authors: Matsumoto Katsuo , Robb Edward , Warden Glenn , Nordlund James
Published: April 2006

Abstract We postulate that wound healing is an orderly process mediated by a programmed expression of cytokines and growth factors. We suggest that these factors are produced in a consistent sequence, in regulated quantities and eliminated when their function is complete. We report here the results of studies on several cytokines, growth factors and the intercellular adhesion molecule expressed during the healing of human skin grafted onto athymic nude mice. Signs of healing of grafts were visible clinically around 3–5 days post‐graft and were completed by 4 weeks post‐graft. During the Ist 2 weeks, we observed the following, (i) K‐14 keratin was prominent throughout the entire epidermis. Thereafter it was limited to basal cell layers, (ii) Langerhans cells were not detectable wilh anti‐human CDIa antibodies during the first week of healing but were clearly detectable 2 weeks post‐graft, (iii) DOPA (dihydroxy phenyl‐alanine) positive melanocytes gradually increased with time. The epidermis 21 to 28 days post‐graft clinically and histologically seemed to be morphologically intact. Interleukin‐l (IL‐I) was clearly detected in some basal cells of the epidermis, especially in melanoeytes and some keratinocytes during the early stage of healing. Transforming growth factor‐α: (TGF‐(α) was detected in epidermis first in melanocytes and some kera‐tinocytes shortly after grafting and again in the late stage of healing. It was also found in some dermal cells. Its expression coincided wilh kera‐tinocyte proliferation and melanocyte migration. TGF‐β was strongly ex‐pressed in the epidermis and dermis after the first week post graft, (iv) ICAM‐1 was transiently expressed only at the onset of healing. We previously reported that pro‐opiomelanocortin and its derivatives MSH/ACTH are expressed strongly during the healing of human xenografts. The 4 additional molecules which are the subject of this report all are ex‐pressed in healing human skin in a predictable sequence and quantity (intensity of stain). Together these data support our hypothesis that healing is a highly regulated process mediated by numerous cytokines.
Differences in responses of interleukin‐1 and tumor necrosis factor α production and secretion to cyclosporin‐A and ultraviolet B‐irradiation by normal and transformed keratinocyte cultures
Authors: Marionnet Anne Valère , Chardonnet Yvette , Viac Jacqueline , Schmitt Daniel
Published: April 2006

Abstract Among epidermal cytokines, IL‐1 and TNFα are involved in inflammatory skin reactions and suspected of modulation by immuno‐suppressive treatment (e.g., cyclosporin A, CsA) or UVB‐irradiation, 2 mediators probably being involved in epithelial carcinogenesis. We evaluated the effects of 8 μg/ml CsA and 100 J/m2 UVB‐irradiation on the production and secretion of IL‐1 and TNFα on normal human epidermal keratinocytes (NHK) and epidermal keratinocyte cell lines either spontaneously transformed (HaCaT) or transformed by human papillomavirus (HPV) type 16 or 18 (EK I6 and EK 18), by using FLISA test. Normal and immortalized keratinocytes constitutively produced and released IL‐lα IL‐lβ and IL‐1 receptor antagonist (IL‐IRA) but IL‐I synthesis by NHK was significantly higher than by cell lines. All the cells spontaneously excreted low amounts of TNFα. Different responses to treatments were evidenced between NHK and cell lines. CsA modified significantly the production and secretion of ILI in most cells whereas slight changes were observed with TNFα secretion. UVB irradiation had no effect on the intracellular ILI pool of any cells but increased the release of IL1 and TNFα. The association CsA‐UVB did not result in additive effects on synthesis and secretion of IL1; the release of TNFα by the cells remained poor except for EK18 cells. Taken together, these results show that, in immortalized keratinocytes, the IL‐1 and TNFα expression was differently affected by treatments wilh CsA and/or UVB‐irradiation as compared to NHK. In addition, spontaneously transformed keratinocytcs. HaCaT, reacted differently from HPV‐transformed keratinocyles, EK I6 and EK I8.
Parallel intraindividual evaluation of the vasoconstrictory action and the anti‐allergic activity of topical corticosteroids
Authors: Seidenari S. , Di Nardo A. , Mantovani L. , Giannetti A.
Published: April 2006

Abstract The human skin blanching assay is a well established method for ranking the efficacy of corticosteroids after epicutaneous application. Vasoconstriction is a pharmacological activity, which correlates well with the clinical efficacy, the intensity of skin blanching after a single application under occlusion corresponding, generally, to the clinical efficacy after repealed application without occlusion. However, in studies dealing with the comparison between the vasoconstriction assay and the evaluation of the clinical effects on inflammatory skin diseases, some exceptions to this correlation have been reported. Therefore, in a pre‐clinical phase, it would be useful to combine the blanching assay with at least one anti‐inflammatory assay. In the present study the blanching assay and the allergic contact dermatitis inhibition test were performed in parallel, in order to compare the two testing procedures in the same group of subjects, utilizing standardized study designs supported by objective means of evaluation. Three commercial preparations of corticosteroids containing clobetasol propionate (CP), clobetasone butyrate (CB) and hydrocortisone acetate (HA), respectively, were employed both to treat nickel‐induced positive patch test responses on the volar forearms, and to perform a vasoconstrictor assay on normal forearm skin in 16 nickel‐sensitised healthy volunteers. For evaluating skin blanching, we employed colorimetric measurements, whereas for the quantitative determination of the inhibition of the intensity of allergic patch test reactions, 20 MHz B‐scanning supported by image analysis was used. Both colorimetric and echogenicity values enabled us to distinguish between the three corticosteroids (at the 17 h evaluation and the 64 h assessment, respectively). A fair correlation was noted between colorimetric and echogenicity values. Both testing procedures ranked the three corticosteroids in the expected order. Corticosteroid preparations should be compared using methods which allow different effects to be simultaneously monitored, without involving a high number of patients. We are proposing this double procedure for the parallel intraindividual evaluation of the vasoconstrictory action and the anti‐allergic activity of topical steroids.