bluuebunny/crossref_metadata_2025 huggingface dataset:
Predictive testing of metalworking fluids: a comparison of 2 cumulative human irritation models and correlation with epidemiological data
Authors: Wigger‐Alberti W. , Hinnen U. , Elsner P.
Published: April 2006

Metalworking fluids (MWF) have been reported as being an important cause of irritant contact dermatitis in metal workers. Our purpose was to determine whether the irritancy of different MWF assessed by 2 different types of predictive human in vivo tests could be compared with epidemiological data, 3 water‐based MWF were tested in the same panel of subjects. Reactions were assessed by a visual score (VS), evaporimetry to evaluate the transepidermal water loss (TEWL) and chromametry to quantify erythema. Test 1: MWF were applied with Finn Chambers on the volunteers' mid‐back, removed after 1 day of exposure, and reapplied for a further 2 days Test 2: Cumulative irritant contact dermatitis was induced using a repetitive irritation test for 2 weeks (omitting weekends) for 6 h per day. We observed an increase in VS, TEWL, and erythema for all MWF, with the same irritancy ranking in both test models. Differentiation of the substances was better in the D1/D3 test. The experimental results partially correlated with the epidemiological data, Considering the shorter application time and the better discrimination of irritancy, we prefer the D1/D3 model as a predictive test of MWF irritancy. Our results might aid development of a Standardized test to reduce cumulative skin irritation in metal workers.
National rates and regional differences in sensitization to allergens of the standard series
Authors: Schnuch A. , Geier J. , Uter W. , Frosch P.J. , Lehmacher W. , Aberer W. , Agathos M. , Arnold R. , Fuchs Th. , Laubstein B. , Lischka G. , Pietrzyk P.M. , Rakoski J. , Richter G. , Rueff F.
Published: April 2006

Sensitization rates to contact allergens vary between centers and are influenced by sex and age. Eliminating the latter 2 factors by standardization of data by age and sex, the present analysis address possible differences between centers remaining after elimination of these confounders, and analyzes other factors which might influence rates, e.g. the MOAHL index. Overall standardized rates were well within the range reported in previous studies and may be regarded as representing the rates of the “patch test population” in Central Europe (e.g. nickel sulfate 12.9%, fragrance mix 10.5″. balsam of Peru 7.3%. thimerosal 5.6%). For this analysis, data of those departments which contributed more than 2000 patients or of those with extreme proportion concerning sex, age and occupational cases were selected. Patients from these 10 departments differed considerably will regard to the items of the MOAHL index and with regard to standardized rates. The items of the MOAHL index proved to be suitable for describing different patch test populations and for explaining some differences between centers. Only ‘atopic dermatitis’ seems to have little influence on (standardized) rates. Face dermatitis is not yet represented in the MOAHL index, but should be included, together with age > 40 years, in an extended index (acronym: MOAHLFA). Regional allergen exposure (with striking differences between East Germany, West Germany and to a lesser extent Austria) seems to have a great influence on the sensitization pattern observed in a department. In addition, sociological factors may influence sensitization rates, which is exemplified by high rates of nickel allergy in a socially delined subgroup. Future studies should focus on these factors, as well as on factors concerning patch test practices and quality control.
Allergy to phosphorus sesquisulfide: 0.5% pet. is a more appropriate concentration for patch testing
Authors: Abdullah A. , Bourke J. F. , Foulds I. S.
Published: April 2006

Patch testing was performed with phosphorus sesquisulfide P4S3 in 2 groups containing equal numbers of patients using different concentrations (0.5% P4S3 in pet. and 1% P4S3 in pet., the usual suggested test concentration as recommended by the International Contact Dermatitis Research Group). We found that there was a statistically significant increase in the number of clinically irrelevant irritant reactions in the group tested with the concentration (χ2= 16, P < 0.0004). We recommend that patch testing with phosphorous sesquisulfide should he at a reduced concentration of 0.5% pet.
Irritancy of antiseptics tested by repeated open exposures on the human skin, evaluated by non‐invasive methods
Authors: Tupker R. A. , Schuur J. , Coenraads P. J.
Published: April 2006

The aim of the study was to test the irritancy of 6 antiseptics in an open exposure model. The following agents were tested in their normal use concentrations using open exposures. 2 × daily for 4 days in 20 subjects: chlorhexidine 4%(CH), chlorhexidine 0.5% in ethanol 70%(CE), ethanol 70%(ET), iodine 1% in ethanol 70% (IE), povidone‐iodine 10% (PI) and sodium hypochlorite 0.25% (SH). Responses were evaluated by visual scoring, subjective irritancy scoring, stratum corneum hydration (Corneometer), transepidermal water loss and laser Doppler flowmetry. Exposure to SH had to be discontinued after 4 applications because of severe subjective irritation. The same held true for IE (7 applications), whereas the other agents were exposed 8 ×. All evaluation methods showed SH to be significantly more irritating than IE, which was in turn more irritating than CH. CE, ET and PI. Thus, it can be concluded that CH, CE, ET and PI were nonirritating in this open exposure model.
Visual assessment of human skin irritation: a sensitive and reproducible tool
Authors: Basketter David , Reynolds Fiona , Rowson Matt , Talbot Claire , Whittle Ed
Published: April 2006

Human volunteer studies of skin irritation have been curried out for decades, both for research and for safety evaluation purposes. For the majority of this time, and consequently for the majority of the studies, assessment of the skin reactions has been made visually. Typical endpoints include erythema, oedema, dryness and scaling, sonic or all of which would he rated on a simple scale, eg 0, ±, +, ++, +++. Such approaches can he criticized as subjective, of poor reproducibility, lacking in sensitivity and highly variable between observers and/or institutions. In consequence, instrumental methods of assessment have been strongly promoted and do indeed offer several advantages, not least their objectivity, However, ii is possible to use the human eye, which is a very sensitive tool, to make detailed, accurate and reproducible descriptions of skin irritation reactions To achieve this, it is necessary to give prolonged and thorough training to each observer. In this paper, 3 examples of human volunteer studies, in which different pairs of trained observers independently carried out double blinded scoring of the irritation reactions, are reported. The grading patterns produced were almost identical: statistical analysis showed that properly trained observers are in fact able to reliably measure a grade of erythema to within ±1 on a 10 point scale: 97.6% of scores were within 2 grade points on this scale. These results provide evidence that visual scoring can be sensitive reliable and reproducible within a testing institution.
Occupational dermatitis in Danish gardeners and greenhouse workers
Authors: Paulsen E. , Søgaard J. , Andersen K. E.
Published: April 2006

Floristry is considered a hazardous occupation from a dermatological point of view, but there are relatively few epidemiological studies to support this notion. The present investigation set out to assess the prevalence of and association between occupational dermatitis and possible risk factors in Danish gardeners and greenhouse workers. A cross‐sectional study, based on a postal questionnaire and subsequent examination and patch testing of those who had occupational eczema from their present work or occupational problems with Compositae, was carried out in 1958 gardeners and greenhouse workers. The response rate was 84.6% and among 301 persons invited for further examination, 253 (84.1%) attended. The lifetime prevalence of occupational eczema in floristry, defined as dermatitis lasting more than 24 h, was 19.6% (95% confidence intervals 17.8–21.3%) among all potential respondents. Occupational mucosal symptoms, working with Compositae plants and a history of previous occupational eczema in floristry were significantly associated with an increased risk of occupational eczema. Sex, age and atopy did not seem to be important risk factors. The results confirm that floristry is a dermatologically hazardous occupation and emphasize the importance of eliciting agents in the working environment rather than personal inherent factors.
B‐scanning evaluation with image analysis of psoriatic skin
Authors: Di Nardo A. , Seidenari S. , Giannetti A.
Published: April 2006

Abstract Ten psoriatic plaques from different subjects were investigated by 20 MHz ultrasound B scanning. Lesions were evaluated before therapy, and after 7 and 15 days of topical treatment with anthralin. Recordings from lesional skin were compared with those from clinically non‐involved contralateral skin. Images were assessed by means of a new image analysis program, allowing the conversion of the B‐scan image color scale into a numerical amplitude scale, the selection and highlighting of amplitude bands, and the quantification of objects reflecting in a definite amplitude range. B‐scanning evaluation of the psoriatic plaque revealed a thickening of the epidermis and dermis, the presence of parallel acoustic shadows in the dermis, and a characteristic hypo‐echogenic band corresponding to the papillary dermis. Image processing allowed an exact quantification of the progressive reduction in thickness of the epidermis and dermis and of the hypo‐echogenic band observed at skin sites which had been treated with topical substances. Thus, the echographic method with image analysis allows the evaluation of three different parameters of response to topical treatment of psoriatic skin.
Spleen dendritic cells exhibit altered morphology and increased allostimulatory capacity after short‐term culture*
Authors: Girolomoni Giampiero , Zambruno Giovanna , Manca Vania , Bergstresser Paul R. , Giannetti Alberto , Cruz Ponciano D.
Published: April 2006

Abstract la‐bearing dendritic cells (DC) are a class of bone marrow‐derived antigen‐presenting cells that appear to possess an increased capacity to stimulate resting T lymphocytes. DC from different tissues share several morphologic, phenotypic and functional attributes. For example, freshly isolated DC from spleen resemble phenotypically and functionally freshly isolated Langerhans cells (LC) from epidermis; in addition, during short‐term culture both DC and LC undergo several parallel changes including modifications affecting phenotype, capacity to present protein antigens, and ability to route surface la molecules into intracellular acidic compartments (J Immunol 1990: 145: 2820–2826). In the present study we show, using immunoelectron microscopy with anti‐la and anti‐33Dl monoclonal antibodies, freshly isolated DC in suspension to have a smooth cell surface with few and short cytoplasmic projections. By contrast, cultured DC display conspicuous bulbous cytoplasmic protrusions. In addition, spleen DC following culture for 24–48 hours exhibit an increased ability to stimulated allogeneic T lymphocytes in the primary mixed leukocyte reaction. These changes, similar to those described for freshly isolated and cultured LC respectively, further substantiate the close relationship between DC and LC.
Differential localization of ICAM‐1 and HLA‐DR expression on epidermal basal surface in mycosis fungoides and lichenoid reaction
Authors: Imayama Shuhei , Furumura Minao , Hori Yoshiaki
Published: April 2006

Abstract Scanning electron microscopy with immunogold labeling revealed that epidermal keratinocytes expressed ICAM‐1 (intercellular adhesion molecule‐1) and HLA‐DR molecules on their surfaces in patterns that differed in mycosis fungoides (MF) and lichenoid reaction (LR). ICAM‐1 molecules, visualized as deposits of gold particle, were visualized as clusters adjacent to the junctions interconnecting the keratinocytes of MF lesions. LFA‐1 (leukocyte function‐associated antigen‐1) molecules were seen as granules on the surfaces of all infiltrates, most of which also expressed ICAM‐1. HLA‐DR molecules were seen continuously along the borders of the individual keratinocytes, thus producing a cobblestone appearance on the epidermal undersurface. In contrast, ICAM‐1 and HLA‐DR were found only sparsely on the undersurface of the epidermis from LR. These findings may help to explain the differing histological features of MF and LR: ICAM‐1 molecules present on the intercellular junctions of MF epidermis lead the LFA‐1‐bearing cells to migrate into the interspaces, thus producing epidermotropism. These cells aggregate by means of co‐expressed ICAM‐1 to thus produce Pautrier's microabscess. In LR, the minimal expression of ICAm‐1 on the epidermal undersurface leaves most infiltrates within the dermis, thus producing a band‐like infiltrate.
T‐cell receptor γδ‐positive peripheral T‐cell lymphomas presenting in the skin:
Authors: Ralfkiaer Elisabeth , Wollf‐Sneedorff Annette , Thomsen Kristian , Geisler Christian , Vejlsgaard Gunhild Lange
Published: April 2006

Abstract Examination of biopsy samples from 62 patients with – or with suspected – cutaneous T‐cell lymphoma (CTCL) revealed 2 cases in which the neoplastic cells were positive for the T‐cell receptor (TCR) γδ complex. One patient had mycosis fungoides and 1 patient had a pleomor‐phic lymphoma of medium and large‐cell type. Both cases showed aggressive courses with dissemination to internal organs and short survival times. The phenotypic examination showed that the neoplastic cells were positive with TCRδ1, CD3, CD25, CD29, CD45RO and CD54. No staining was seen with antibodies against framework determinants or variable regions on the TCR αβ heterodimer. Negative reactions were also seen with CD4, CD8, CD5, CD7, CD16, CD30 and CD57. It is concluded that rare CTCL express TCR γδ chains. These malignancies may originate from the TCR γδ‐positive T cells seen in normal skin, and it is possible that their recognition may be important for clinical reasons.
Heterogeneity of cytokine production by human malignant melanoma cells
Authors: Armstrong Cheryl A. , Tara David C. , Hart Charles E. , Köck Andreas , Luger Thomas A. , Ansel John C.
Published: April 2006

Abstract Recent investigations indicate that malignant melanoma cells can produce distinct cytokines. While differences in the production of single cytokines have been observed among different melanoma cell lines, the extent of variability in the production of single and multiple cytokines between individual melanoma cell lines has not been as thoroughly investigated. A heterogeneity in melanoma cell cytokine production could have important implications for the biology of this aggressive neoplasm since certain cytokines may act as autocrine growth factors or be potent modulators of host immune response to the developing tumor. The purpose of this study is to assess the cytokine production profile of two widely available human melanoma cell lines, A375 and G361. The A375 cell line constirutively expressed the mRNA for IL‐1α, IL‐1β and PDGF‐A, with increased expression of these cytokines after induction with PMA. GM‐CSF mRNA was expressed by the A375 melanoma line only after induction with PMA. No IL‐6 mRNA was detected in the A375 melanoma cell line. The cell culture supernatants from the A375 cells likewise contained a parallel increase in IL‐1 activity as determined in the D10 bioassay and secreted GM‐CSF and PDGF‐AA as measured by ELISA. In contrast, the G361 cell line did not express IL‐1, GM‐CSF or PDGF‐A mRNA (constitutively or after PMA induction) but expressed only IL‐6 mRNA and secreted IL‐6 activity after PMA induction. These results demonstrate a significant heterogeneity in the production of IL‐1α, IL‐1β, IL‐6, GM‐CSF, and PDGF in two distinct melanoma cell lines. This study demonstrates that individual melanoma cell lines express and secrete multiple cytokines both constitutively and after stimulation with PMA. The immunomodulating and mitogenic properties of these melanoma‐derived cytokines may have implications in determining the biologic behavior of different malignant melanomas.
Isolation of plasma membranes from keratinocytes: A newly developed method allows the sensitive detection of the membrane antigen pattern in normal and psoriatic skin
Authors: Licht A. , Bauer Ch. , Stadlcr R.
Published: April 2006

Abstract Distinct differences of proteins in the plasma membranes have been described in psoriatic keratinocytes as compared with normal epidermis. These changes are presumably involved in the pathogenesis of psoriasis. To identify and distinguish glycosylated proteins of the plasma membranes of keratinocytes, a method for mild preparation was developed that avoids the use of degrading or digestive enzymes. After cell lysis, three steps of centrifugation were performed, including the use of a sucrose step gradient. A fine‐vesicular membrane fraction was obtained. Using marker enzymes for cell compartments, no contamination of cell nuclei or mitochondria and only 0.4% of endoplasmic reticulum was detectable in the final membrane fraction. Based on this preparation, disc‐polyacrylamide‐gel electrophoresis, followed by Western blotting, were performed. Staining with five different lectins to visualize glycosylated proteins allowed 75 different membrane glycoproteins to be distinguished. The patterns of normal, transformed (HaCaT), foreskin and psoriatic keratinocytes after cell culture with one passage were compared. Up to six proteins per lectin staining were expressed differently in psoriatic as compared to normal keratinocytes. Psoriatic cells shared similarities with highly proliferative foreskin cells, but not with transformed HaCaT cells. Main alterations of glycosylation were detected in the fucose content. In conclusion, the method developed for isolation of plasma membranes allows selective and sensitive examination of plasma membranes of normal and pathological keratinocytes. The glycosylation patterns observed suggest that distinct membrane proteins may be involved in the pathogenesis of psoriasis.
Induction of inflammatory cytokines in murine keratinocytes upon in vivo stimulation with contact sensitizers and tolerizing analogues
Authors: Haas Jürgen , Lipkow Thilo , Mohamadzadeh Mansour , Kolde Gerhard , Knop Jürgen
Published: April 2006

Abstract In order to elucidate the role of keratinocytes (KCs) in the induction of contact sensitivity, we applied various contact sensitizers [2,4‐dinitrofluorobenzene (DNFB), urushiol, 3‐n‐pentadecyleatechol (PDC), 4‐ethoxymethylene‐2‐phenyloxazol‐5‐one (oxazolone)] and tolerizing compounds [2,4‐dinitrothiocyanobenzene (DNTB), 5‐methyl‐3‐n‐pentadecyl‐catechol (5‐Me‐PDC)] onto the earskin of non‐sensitized Balb/c mice. In addition, we applied croton oil as a non‐sensitizing, but stimulatory agent. Cytokine production was demonstrated by Northern blot hybridization of the total cellular RNA extracted from epidermal cells depleted by Langerhans cells and Thy 1+ dendritic cells using radiolabeled DNA probes encoding for the murine cytokines IL‐lα, ‐2, ‐3, ‐4, TNFα, IFNτ, GM‐CSF and G‐CSF. From all cytokines tested, TNFα and IL‐lα were markedly increased upon in vivo stimulation with contact sensitizers and also after application of croton oil. Both light and electron microscopic immunostaining with a polyclonal and monoclonal antibody demonstrated the presence of TNFα in the epidermis. This staining was most pronounced in KCs of the suprabasal epidermis upon application of contact sensitizers or croton oil, but not with tolerizing analogues. Using a functional assay significantly more TNFα was found in the supenatants of KCs treated in vitro with DNFB or LPS than with DNTB. GM‐CSF was found in untreated epidermis as well as in stimulated cells. The results suggest that the sensitizing properties of contact sensitizers may partly be dependent on their ability to induce proinflammatory mediators. The induction and release of TNF7α and IL‐lα in KCs by contact sensitizers may play an important role in the early response to immunogenic or inflammatory signals in vivo, whereby tolerance induction seems to be less dependent on these cytokines.
Early development of human Merkel cells
Authors: Moll I. , Moll R.
Published: April 2006

Abstract Human fetal Merkel cells are now generally considered to be epidermal derivatives. Previous studies using antibodies against the simple epithelial cytokeratins (CKs), 8 and 18, have demonstrated the presence of these cells in the epidermis at as early as fetal week 10 to 12. Using antibodies against CK 20 whose expression within the skin is restricted to Merkel cells, we applied immunofluorescence and immunoperoxidase microscopy to analyze earlier embryonic and fetal human skin (wk 7 to 9). We were able to demonstrate the first Merkel cells at as early as fetal wk 8, i.e., at the same time as the epidermis starts to develop an intermediate, third layer, characterized by the expression of CKs 1,10, and 11. Most of these early Merkel cells were localized above the basal layer. Their shape was round to oval, dendrites being infrequent and short. At fetal wk 9, Merkel cells were considerably more numerous. These results persuasively argue for a much earlier fetal development of Merkel cells within the epidermis than previously thought. A hypothesis concerning the differentiation of Merkel cells from embryonic basal keratinocytes is discussed.
Altered regulation of collagen metabolism in scleroderma fibroblasts grown within three‐dimensional collagen gels
Authors: Mauch Cornelia , Kozlowska Ewa , Eckes Beate , Krieg Thomas
Published: April 2006

Abstract In systemic scleroderma (SSc) excessive deposition of collagen leads to fibrosis of various tissues including the skin. Previous studies have demonstrated that scleroderma fibroblasts in explant monolayer cultures are heterogeneous with respect to their levels of collagen synthesis. The critical role played by the extracellular matrix (ECM) in the modulation of fibroblast metabolism prompted us to study the regulation of collagen synthesis in scleroderma fibroblasts grown within three‐dimensional collagen gels, a culture system representing more physiological conditions than monolayer cultures. Normal fibroblasts grown in this system dramatically reduce their collagen synthesis as compared to monolayer cultures. Quantification of total protein and collagen synthesis showed that scleroderma fibroblasts did not demonstrate the down regulation of collagen synthesis as observed in control fibroblasts, resulting in a much higher collagen synthesis in scleroderma fibroblasts compared to controls. However, also in this system scleroderma fibroblasts were heterogeneous in their response to the collagenous lattice. Ten strains were investigated, of which 3 were indistinguishable from controls, while 7 maintained higher levels of collagen production. In addition, our data showed that the changes in collagen synthesis on the protein level were accompanied by respective up‐ or downregulalion on the mRNA level. These results indicate that an altered response to the surrounding ECM is an important factor in the disturbed regulation of connective tissue synthesis in scleroderma fibroblasts observed in vivo.
T‐cell receptor gene rearrangement studies as a diagnostic tool in lymphoproliferative skin diseases
Authors: Lessin Stuart R. , Rook Alain H.
Published: April 2006

Abstract The growth of our knowledge in T‐cell biology, in particular the molecular biology of the T‐cell receptor (TCR). has provided a means to molecularly characterize lymphoproliferative diseases of the skin based on the presence or absence of a clonal population of T lymphocytes. TCR gene rearrangement studies, by Southern blot analysis, have aided the investigative dermatologist in gaining insights into the pathogenesis and clonal evolution of lymphoproliferative skin diseases. In addition, the application of TCR gene rearrangement studies as a diagnostic aid in the evaluation of lymphoproliferative skin diseases has been introduced into clinical dermatology. Despite its enormous research value. TCR gene rearrangement studies presently have limited applications as an independent diagnostic tool. However, as our knowledge and experience grows and as the application of new techniques provides us with greater detection sensitivity and specificity, the diagnostic utility of TCR gene rearrangement studies will be enhanced.
Molecular analysis of the γδ T‐cell receptor repertoire in normal human skin and in Oriental Cutaneous Leishmaniasis
Authors: Alaibac M. , Daga A. , Harms G. , Morris J. , Yu R. C. H. , Zwingerberger K. , Chu A. C.
Published: April 2006

Abstract The extent of diversity of the γδ T‐cell receptor (TCR) in normal human skin and Oriental Cutaneous Leishmaniasis (OCL) was examined by molecular analysis of the variable (V) δ gene segment, junctional (J) δ gene segment and junctional regions. To examine the expression of TCR δ genes, segments of γδ T lymphocytes, DNA isolated from normal human skin and from OCL were subjected to enzymatic gene amplification by the polymerase chain reaction (PCR) method using TCR Vδ‐and Jδ‐specific oligonucleotides as primers. PCR amplification using these primers indicated that the Vδ2 gene segment was predominantly used by γδ T lymphocytes in both normal human skin and OCL. To determine the extent of junctional diversity in the δ gene of γδ T cells in normal human skin and OCL, we sequenced the nucleic acid sequences corresponding to the V δ2/Jδ1 junctional regions. Sequence analysis of junctional regions demonstrated broad junctional diversity in normal skin but only limited diversity in OCL. Our findings support the hypothesis that skin γδ T lymphocytes may derive from a fetal subset of γδ T lymphocytes that leaves the thymus early and colonizes (he periphery. The limited junctional diversity demonstratd in OCL lesions indicates that γδ T cells can undergo oligoclonal expansion following recognition of a specific ligand and supports the idea that junctional regions are important in the recognition of antigenic determinant.
1,25‐dihydroxyvitamin D3 and the vitamin D analogue KH1060 induce hyperproliferation in normal mouse epidermis
Authors: Lützow‐Holm C. , De Angelis P. , Grosvik H. , Clausen O. P. F.
Published: April 2006

Abstract 1,25‐dihydroxyvitamin D3 (calcitriol) affects differentiation and proliferation of epidermal keratinocytes in vitro and in vivo. We have studied the topical effects of calcitriol (0.08 2.0 μg/ml) and of a new vitamin D analogue, the epi‐20‐analogue KH1060 (0.4 –2.0 μg/ml) on epidermal proliferation in normal hairless mice. Epidermis was examined at intervals from 4 h to 8 days after a single‐dose application. The mitotic rate was assessed by the stathmokinetic method and hyperplasia was scored in histological sections. Cell cycle parameters were measured by bivariale bromodeoxyuridine (BrdUrd)/DNA flow cytometry on isolated epidermal basal cells after pulse‐labelling with BrdUrd. Both calcitriol and KM 1060 induced a dose‐ and time‐dependent increase in the mitotic rate and in hyperplasia, the latter drug being the most effective. Calcitriol and KH1060 induced changes in the cell cycle traverse compatible with the regenerative reaction seen after other hyperplasiogens, but with an additionally increased accumulation of cells in the G2 phase. This is similar to that seen after topical application of retinoic acid to mouse skin. Our results are thus in contrast to the anti‐proliferative effects of calcitriol observed in vitro and following treatment of the hyperproliferative disease psoriasis with calcitriol as well as other vitamin D analogues.
ICAM‐1 and LFA‐1 on mouse epidermal Langerhans cells and spleen dendritic cells identify disparate requirements for activation of KLH‐specific CD4+ Th1 and Th2 clones*
Authors: Simon Jan C. , Girolomoni Giampiero , Edelbaum Dale , Bergstresser Paul R. , Cruz Ponciano D.
Published: April 2006

Abstract Expression of the adhesion molecules ICAM‐1 and LFA‐1 (CD11a/CD18) on mouse epidermal Lungerhans cells (LC) and on spleen dendritic cells (DC) from BALB/c mice was examined by staining with specific mAb and was evaluated by flow cytometry. LC were shown to express both ICAM‐1 and LFA‐1, whereas spleen DC expressed only LFA‐1. The contribution of these adhesion molecules to LC‐ or DC‐induced activation of keyhole limpet hemocyanin (KLH)‐specific, lad‐restricted, Th1 or Th2 clones was investigated in mAb blocking studies. At optimal doses, anti‐CD1la or anti‐CD18 mAb completely inhibited Th1 proliferation induced by either LC or DC. Anti‐ICAM‐1 also abrogated Th1 proliferation induced by LC, but only moderately reduced Th1 proliferation induced by DC. Inhibition in these experiments was specific, since isotype‐matched control Ab against other Ag constitutively expressed on LC (NLDC 145) or DC (33D1) had no effect on Th1 proliferation. In marked contradistinction, the capacity of LC to present KLH to our Th2 clones was resistant to treatment with the same mAb against ICAM‐1, CD11a or CD18. We conclude that interactions between ICAM‐1 and LFA‐1 on epidermal LC and LFA‐1 on spleen DC with their respective ligands on our Th1 clones are required for optimal presentation of protein Ag to Th1. Our results also indicate that neither ICAM‐1 nor LFA‐1 is required for the analogous activation of our Th2 clones by LC.
Demonstration of the high‐affinity IgE receptor (FcɛRI) on Langerhans cells of oral mucosa
Authors: Haas N. , Hamann K. , Grabbe J. , Czarnetzki B. M.
Published: April 2006

Abstract Langerhans cells in the skin have recently been shown to bind IgE molecules via a high‐affinity IgE receptor. Using two specific antibodies, 29C6 and 6F7, against the α‐chain of the high‐affinity IgE receptor we here demonstrate that Langerhans cells express this receptor in oral mucosa. A specific antibody. Tül, against the low‐affinity IgE receptor showed only low expression of this receptor. High‐affinity binding for IgE may be important for induction and support of Langerhans cell‐dependent transepithelial IgE‐mediated allergic reactions and inflammation.
Lipid composition and synthesis of HaCaT cells, an immortalized human keratinocyte line, in comparison with normal human adult keratinocytes
Authors: Schürer Nanna , Köhne Angela , Schliep Viola , Barlag Kerstin , Goerz Günter
Published: April 2006

Abstract Cultured keratinocytes are frequently employed for studies of epidermal lipid metabolism. Interpretation of experimental data may be complicated by donor to donor variability, the relatively short culture lifetime and variations between passages, problems thai are not encountered to the same extent with immortalized cell lines. The present study was undertaken to compare the lipid composition and synthesis of normal human adult keratinocytes (NHAK) with HaCaT cells, a long‐lived. spontaneously immortalized human keratinocyte line, in relation to proliferation and differentiation. No differences between the two cell types were observed: a) in total lipid content; b) in the distribution of major lipid classes during growth at 50%, 75% and 100% confluence: c) in cultures grown at 0.6 mM calcium, at which differentiation is retarded, or at 1.6 mM calcium, at which some differentiation takes place; d) in the incorporation of [14C] acetate into cellular lipids at confluence, or e) in the fatty acid composition of major cellular lipid classes. At 100% confluence NHAK and HaCaT cells differ in their cholesterol metabolism. At all stages of growth, cholesterol synthesis in HaCaT cells is more LDL‐dependent than in NHAK. Furthermore, NHAK become less LDL‐dependent at confluence whereas HaCaT cells do not. HaCaT cells also revealed a significantly larger fraction of phosphatidyl‐ethanolamine, ‐serine and ‐inositol at 0.6 mM calcium concentration than NHAK. These findings suggest that HaCaT cells do not differentiate as well as NHAK in vitro and may therefore serve as a model for the study of lipid metabolism in cells defective in terminal differentiation.
Cellular retinoic acid‐binding proteins (CRABP)
Authors: Piletta P. , Saurat J.‐H.
Published: April 2006

Abstract Mammalian cell cytoplasm contains at least two proteins which bind retinoic acid (RA): CRABP I and CRABP II. Produced by separate cenes, they differ in their affinity for RA, their transcriplional regulation by R A, their tissue distribution, and possibly their function. They intervene, probably at different stages, in the “intracine” metabolic process which controls the amount of biologically active ligaml that is available for binding to the nuclear receptors of RA.
Expression of epidermal integrins in human organotypic keratinocyte cultures
Authors: Limat Alain , Klein C. Ebcrhard
Published: April 2006

Abstract In organotypic coculture of human epidermal keralinocyles (HEK) or follicular outer root sheath (ORS) cells with human dermal fibroblasts, a stratified epithelium develops which in many regards re‐sembles inleifollicular epidermis. The epithelium growing on type I collagen gels in the absence of a preformed basement membrane itself produces only low or moderate amounts of laminin and collagen type IV, so that a well‐structured basement membrane cannot be formed. This results in loose and insufficient anchoring of basal cells in the collagen gel, frequently leading to cleft formation at the junction. Because integrins are important receptors for cell‐cell and cell‐matrix adhesion of keratino‐cytes which under certain circumstances may also influence epidermal differentiation, we studied their expression under this culture condition which provides adhesional stress but leaves epidermal differentiation largely unaltered. The localization of integrins differed markedly from that in normal epidermis or normal outer root sheath since all integrin chains were polarized to the epithelium‐collagen I interface. Thus, not only the 7.6 and β4‐chains showed preferential expression at the basal attachment site of keratinocytes as in normal epidermis, but also the α2‐, α3‐, βM‐chains which in normal epidermis under “steady stale” conditions appeal‐primarily involved in cell‐cell interaction of keralinocytes and are preferen‐tially expressed at the lateral sides of their plasma membranes. Interest‐ingly, the altered expression of integrins in organotypic cultures is not accompanied by significant disturbances in terminal differentiation.
γ‐melanocyte stimulating hormone (γ‐MSH)‐like immunoreactivity is present in certain normal human keratinocytes
Authors: Johansson Ollc , Yue Liu Peng
Published: April 2006

Abstract Using ihe indirect immunohistochemical approach, the occurrence of γ‐melanocyte stimulating hormone (γ‐MSH)‐like immunoreactivily in human normal keratinocytes is described. The positive cells were observed in each layer of the epidermis (except stratum corneum) and often, at the level of the stratum spinosum, also around the orifices of cutaneous accessory organs, such as sweat glands and sebaceous glands/ hair follicles. Combining these data with our previous investigations, the results support the possibility that locally produced γ‐MSH could be involved in cutaneous immune response, pigmentation and epithelial pro‐liferation, as well as neuromodulation.
Calcium‐independent increases in pericellular plasminogen activator activity in pemphigus vulgaris*
Authors: Crameri Flavío M. , Suter Maja M.
Published: April 2006

AbstractIntracellular free calcium ([Ca2+]i), an important second messenger, plays a crucial role in a variety of biochemical reactions leading to cell activation and protein secretion. This study examines the potential role of (Ca2+)iin mediating increases in pericellular plasminogen activator activity of canine keratinocytes observed upon binding of human pemphigus vulgaris IgG (hPV IgG). Using the calcium‐sensitive fluorescent probe fura‐2 and digital video fluorescence imaging microscopy, [Ca2+]ilevels were determined in individual keratinocytes for up to 29 minutes after addition of 0.1–5 mg/ml hPV IgG to monolayers of subconfluent and confluent cultures. Extracellular ATP (a known [Ca2+]i‐agonist in canine keratinocytes) and normal human IgG (nh IgG) served as positive and negative controls, respectively. HPV IgG and nh IgG failed to induce significant increases in [Ca2+]iwhereas 500 μM ATP induced a rapid, 3‐ to 12‐fold transient increase above resting levels. Binding of hPV IgG to these keratinocyte cultures was demonstrated by immunoflurescence at the end of selected experiments. ATP stimulation of cultures previously treated with hPV IgG showed normal responsiveness and more than 90% of the cells were still viable at the end of [Ca2plus;]iimaging, thus demonstrating that failure to respond to hPV IgG was not due to an experimental artifact. Plasminogen activator activity in supernatants of confluent cultures incubated with 0.1– 1 mg/ml hPV IgG or nh IgG and harvested at various time intervals was dependent on the IgG dose used and increased steadily over time. Increases in activity were 47–92% higher in cultures treated with hPV IgG than those incubated with the same dose of nh IgG. Taken together, these results suggest that hPV IgG‐mediated increases in pericellular uPA activity occur via pathways independent of Ca2+mobilization.
Protein kinase C agonist and antagonist effects in normal human epidermal keratinocytes
Authors: Matsui Mary Steidl , Illarda Isabella , Wang Nianci , DeLeo Vincent A.
Published: April 2006

Abstract Several lines of evidence implicate protein kinase C (PKC) in the development of basal cell and squamous cell carcinomas, tumors which originate from epidermal keratinocytes. To examine PKC in a model relevant to human skin, we exposed normal human epidermal keratinocytes (NHEK) in serum‐free media to a variety of PKC agonists and antagonists. NHEK PKC activity increased up to 10‐fold within the 1st hour of exposure to tetradecanoyl phorbol acetate (TPA), and gradually returned to control values within 72 h. TPA‐induced PKC activity was enhanced by pretreatment of cultures with protein and RNA synthesis inhibitors. TPA‐induced growth arrest and differentiation was antagonized by staurosporine. Down‐regulation by bryostatin pretreatment blocked TPA‐stimulated differentiation. Our overall conclusion is that activation of PKC in cultured human keratinocytes is required for differentiation. These results are crucial to the analysis of compounds suspected of promoting or inhibiting epidermal tumors.
UVB radiation induces phosphorylation of the epidermal growth factor receptor, decreases EGF binding and blocks EGF induction of ornithine decarboxylase gene expression in SV40‐transformed human keratinocytes
Authors: Zheng Zai S. , Chen Rong Z. , Prystowsky Janet H.
Published: April 2006

Abstract We previously demonstrated that epidermal growth factor (EGF) induces a several‐fold increase in ornithine decarboxylase (ODC) activity and the steady‐state level of ODC mRNA in cultured SV40‐transformed human keratinocytes (1). Pretreatment of cell cultures with ultraviolet B (UVB) radiation resulted in a reduction of EGF‐induced ODC activity. To determine whether UVB inhibits the accumulation of ODC mRNA by EGF, cells were pretreated with 20 mJ/cm2 UVB or sham‐irradiated and then incubated with 100 ng/ml EGF. Northern blot analysis revealed that UVB irradiation entirely blocked the EGF induction of ODC mRNA. Since the binding of EGF to its plasma membrane receptor is the first step in initiating a biological response, the effect of UVB on EGF binding was evaluated. UVB treatment of cultured keratinocytes resulted in an immediate and dose‐dependent reduction of EGF binding. Scatchard analysis revealed thai the reduction of EGF binding was due to a 52% decrease in the number of available receptors, from 6.2 × 104/cell to 3.0 × 104/cell. However, UVB decreased the EGF‐binding affinity very little (Kd = 0.60 nM in control and Kd=0.75 nM in UVB‐treated Z114 cells). In addition, UVB did not alter the rate of EGF internalization. These data suggest that UVB blocks the signal transduction pathway of EGF that is involved in regulation of ODC gene expression. Immunoblot analysis of extracts from irradiated cells showed that UVB induced tyro‐sine phosphorylation of EGFR and that the quantity of EGFR protein was unaffected by UVB treatment. Phosphorylation of EGFR may be responsible for decreased binding of EGF to its receptor.
The wounded dermal equivalent offers a simplified model for studying wound repair in vitro
Authors: Genever Paul G. , Wood Edward J. , Cunliffe William J.
Published: April 2006

Abstract Dermal equivalents (DEs), fabricated by seeding fibroblasts into a collagen lattice, may be used as in vitro models for studying wounding and the remodelling processes observed in vivo. We investigated fibroblast responses to a wound stimulus in vitro by making small wounds in DEs. We found that, following wounding, fibroblasts appeared to migrate towards and into the wound defect and appeared to initiate the closure of the wound by bringing together the cut collagen surfaces over a period of 5–11 days. Fibroblast movement into the wound defects was significantly stimulated in the presence of EGF and PDGF (2.8‐ and 3.5‐fold respectively) but not TGF‐β. Cell proliferation in wounded DEs was up to 21% greater than in non‐wounded DEs and cell numbers were stimulated further by the addition of TGF‐β, EGF and PDGF (1.5‐. 1.7‐ and 1.8‐fold respectively). Wounded DEs also displayed a 2.1‐fold increase in latent collagenase production followed by a 1.3‐fold increase in active collagenase levels compared to non‐wounded DEs. Staining actin fibers within fibroblasts using rhodamine‐phalloidin showed that fibroblasts in DEs were under tension, but that this tension was lost upon wounding. Subsequently the stress fibers reappeared concomitantly with the observed “healing” process. Additionally a continuous cell‐cell actin cable purse‐string developed around the entire wound edge which may be involved in wound closure. The findings suggest that the wounded dermal equivalent offers a valuable model for studying wound healing in vitro.
Detection of Propionibacterium acnes polypeptides which have stimulated an immune response in acne patients but not in normal individuals
Authors: Holland K. T. , Holland D. B. , Cunliffe W. J. , Cutcliffe A. G.
Published: April 2006

Abstract Patient and normal volunteer sera were used as probes in two‐dimensional PAGE of P. acnes culture supernatant fluid and cell extracts to determine whether specific P. acnes polypeptides were associated with the immune reaction in acne. Eight polypeptides, Mr 20 to 131 × 103, pi 4.7 to 6.5 in the cell extract, and 7 polypeptides Mr 10 to 24 kD, pI 4.8 to 7.5 in the culture supernatant fluid were specifically highlighted by patient sera and not volunteer sera. These polypeptides were not related to described extracellular enzymes of P. acnes. It is possible that these polypeptides are involved in the induction of acne.
Inhibition of melanoma cell directional migration in vitro via different cellular targets
Authors: Fink‐Puches Regina , Helige Christine , Kerl Helmut , Smolle Josef , Tritthart Helmut A.
Published: April 2006

Abstract In malignant melanoma active movement of cancer cells is considered to be essential for tissue invasion. Various mechanisms, such as the Ca2+‐calmodulin‐proteinkinase C cascade or G‐protein‐dependent processes are considered to play a role in tumor cell functions. The assay of directional migration, combined with computer‐assisted image analysis, was used to evaluate the antimigratory efficacy of drugs interfering with different steps of signal (ransduction pathways. Treatment with different compounds showed a more or less concentration‐dependent reduction of migration rates: The Ca2+‐channel blockers verapamil and devapamil showed a slight reduction of molility. The effect was more pronounced when the calmodulin antagonist flunarizine was used or the proteinkinase C inhibitors dequalinium, tamoxifen and H‐7 were applied. A marked inhibition of molility was found with the G‐protein antagonist L 651582. Thus, our results indicate that different signal transduclion pathways are involved in the regulation of directional migration of K1735‐M2 melanoma cells.
Chemiluminescent restriction fragment length polymorphism analysis (DNA fingerprinting) to identify keratinocytes from two donors in co‐culture
Authors: Young David M. , Summers William C. , Cuono Charles B.
Published: April 2006

Abstract The transplantation of allogeneic cells such as pancreatic islet cells, bone marrow, and keratinocytes for skin replacement is in growing use clinically. Present techniques which are used to distinguish transplanted allogeneic cells from phenolypically identical host cells in chimeric tissue have serious limitations. This study assesses the feasibility of using Chemiluminescent restriction fragment length polymorphism (RFLP) analysis to distinguish between keratinocyte cell lines from 2 different donors grown in co‐culture. We evaluated the sensitivity of this technique in tissue cultured cells in order to define its usefulness in clinical situations. RFLP analysis readily detects heterologous DNA comprising only 10% of a 5 μg sample. This was a detection threshold of 500 ng of high molecular wefght DNA. With standard extraction methods, this represents the DNA obtained from 4000 cells. This new chemiluminescent technique is as sensitive as older techniques which employ radioactive probes, but avoids the hazards of handling radioactive material. Using this RFLP analysis, the presence of transplanted allogeneic cells can be readily delected in small biopsy specimens. This technique has wide potential application in allogeneic cellular transplantation investigations.
Distribution of monohydroxy fatty acids in specific human epidermal phospholipids
Authors: Grøn Brian , Iversen Lars , Zihoh Vincent , Kragballe Knud
Published: April 2006

Abstract Monohydroxy derivatives of polyunsaturated fatty acids such a s arachidonic acid (AA) and linoleic acid (LA) can inodulate inflamation and epidermal proliferation. The purpose o f this study was to determine the in vito distribution orthc AA derivatives; 12‐ and 15‐hydroxy‐ eicosatetraenoic acid (1 2‐HETE and 15‐HETE) and the LA derivatives; 9‐ and 13‐liydroxyotadecadienoic acid (9‐IHODE and 13‐HODE) in specific phospholipids of normal humoii skin. Lipids were extracted from 6 normal keratoine skin biopsies and phospholipids were separated into the major classes by two‐dimensional thin layer chroiiiatograpliy. Monohydroxy fatty acids (MHFAs) released from specific phospholipids after treatment with phospholipase A2 were identified by reversed phase and straight phase high‐performance liquid chromatography and UV‐absorption spec‐ tra. Unesterified MIHFAs were deteriiiined in a similar way. 9‐MODE, 13‐HODE and 15‐IHETE were detectable in pliospliatidylcholamine (PC), pliospliatidylinositol (PI) and pliosphatidylethanolamine (PE). Interestingly. 12‐NETE was not detectable in these phospholipids, although the Linesteriried 12‐HETE was detectable in amounts similar to uncstcrilied 15‐HETE. Eskrilied I 5‐HETE was equally distributed between PI arid PC, in which IS‐HETE was predominant, accounting for 60% and 69% of the total MHFAs, respectively (p < 0.05). These results demonstrate that the LA dcrivntives 9‐MODE and 13‐HODE. as well as the AA derivative 15‐1HETE, are esterified to PC, PI and PE of normal human epidermis in vito. The possibility remains that 9‐HODE. 13‐HODE anrl 15‐HETE, may incdiatc their biological cfrccts by being incorporated into specific I pli osplioli picis.