A mouse organotypic tissue culture model for autosomal recessive congenital ichthyosis.

BACKGROUND: Autosomal recessive congenital ichthyoses (ARCIs) are keratinization disorders caused by impaired skin barrier function. Mutations in the genes encoding the lipoxygenases 12R-LOX and eLOX-3 are the second most common cause of ARCIs. In recent years, human skin equivalents recapitulating the ARCI phenotype have been established. OBJECTIVES: To develop a murine organotypic tissue culture model for ARCI. METHODS: Epidermal keratinocytes were isolated from newborn 12R-LOX-deficient mice and cocultivated with mouse dermal fibroblasts embedded in a scaffold of native collagen type I. RESULTS: With this experimental set-up the keratinocytes formed a well-organized multilayered stratified epithelium resembling skin architecture in vivo. All epidermal layers were present and the keratinocytes within showed the characteristic morphological features. Markers for differentiation and maturation indicated regular epidermal morphogenesis. The major components of epidermal structures were expressed, and were obviously processed and assembled properly. In contrast to their wild-type counterparts, 12R-LOX-deficient skin equivalents showed abnormal vesicular structures in the upper epidermal layers correlating with altered lipid composition and increased transepidermal water loss, comparable with 12R-LOX-deficient mice. CONCLUSIONS: The mouse skin equivalents faithfully recapitulate the 12R-LOX-deficient phenotype observed in vivo, classifying them as appropriate in vitro models to study molecular mechanisms involved in the development of ARCI and to evaluate novel therapeutic agents. In contrast to existing human three-dimensional skin models, the generation of these murine models is not constrained by a limited supply of material and does not depend on in vitro expansion and/or genetic manipulations that could result in inadvertent genotypic and phenotypic alterations.

A new approach for calibration of laser ablation inductively coupled plasma mass spectrometry using thin layers of spiked agarose gels as references.

Calibration of analytical methods using laser ablation for sample introduction is often problematic. The availability of matrix-adapted standard materials is a crucial factor in the analysis of biological samples in particular. In this work a method for preparation of thin-film references for LA-ICP-MS is presented which is inexpensive, relatively simple and generally practicable. Aqueous solutions of agarose spiked with defined amounts of the analytes were cast on a carrier and then dried. When the thin-film references were characterized the average thickness of the films was 0.03 mm in the centre of the film and the relative standard deviation was 8%. Nebulization ICP-MS analysis after acid digestion of the agarose film was used to investigate the effectiveness of the spiking procedure. Recovery of the spiked elements was frequently in the range 90-110% (for rare earth elements 97-102%). Laser ablation ICP-MS analysis was used to investigate the distribution of the spiked elements in the film. When the laser was scanned across the gel the measured intensities were not constant, but had a peak-shaped profile with a flat top. Use of this flat-top region for analytical purposes, after its characterization by laser ablation ICP-MS, is proposed. Analysis of cell cultures was carried out by direct laser ablation-ICP-MS with the calibration method described. The results were in accordance with values previously achieved by nebulization ICP-MS.

Gain of 11q/cyclin D1 overexpression is an essential early step in skin cancer development and causes abnormal tissue organization and differentiation.

Non-melanoma skin cancers, in particular keratoacanthomas (KAs) and squamous cell carcinomas (SCCs), have become highly frequent tumor types especially in immune-suppressed transplant patients. Nevertheless, little is known about essential genetic changes. As a paradigm of 'early' changes, that is, changes still compatible with tumor regression, we studied KAs by comparative genomic hybridization and show that gain of chromosome 11q is not only one of the most frequent aberration (8/18), but in four tumors also the only aberration. Furthermore, 11q gain correlated with amplification of the cyclin D1 locus (10/14), as determined by fluorescence in situ hybridization, and overexpression of cyclin D1 protein (25/31), as detected by immunohistochemistry. For unraveling the functional consequence, we overexpressed cyclin D1 in HaCaT skin keratinocytes. These cells only gained little growth advantage in conventional and in organotypic co-cultures. However, although the control vector-transfected cells formed a well-stratified and orderly differentiated epidermis-like epithelium, they showed deregulation of tissue architecture with an altered localization of proliferation and impaired differentiation. The most severe phenotype was seen in a clone that additionally upregulated cdk4 and p21. These cells lacked terminal differentiation, exhibited a more autonomous growth in vitro and in vivo and even formed tumors in two injection sites with a growth pattern resembling that of human KAs. Thus, our results identify 11q13 gain/cyclin D1 overexpression as an important step in KA formation and point to a function that exceeds its known role in proliferation by disrupting tissue organization and thereby allowing abnormal growth.

Id proteins: novel targets of activin action, which regulate epidermal homeostasis.

Activin is a member of the transforming growth factor beta (TGF-beta) family, which plays a crucial role in skin morphogenesis and wound healing. To gain insight into the underlying mechanisms of action, we searched for activin-regulated genes in cultured keratinocytes. One of the identified target genes encodes Id1, a negative regulator of helix-loop-helix transcription factors. We show that Id1, Id2, and Id3 are strongly downregulated by activin in keratinocytes in vitro and in vivo. To determine the role of Id1 in keratinocyte biology, we generated stable HaCaT keratinocyte cell lines overexpressing this protein. Our results revealed that enhanced levels of Id1 do not affect proliferation of keratinocytes in monoculture under exponential culture conditions or in response to activin or TGF-beta1. However, in three-dimensional organotypic cultures, Id1-overexpressing HaCaT cells formed a hyperthickened and disorganized epithelium that was characterized by enhanced keratinocyte proliferation, abnormal differentiation, and an increased rate of apoptosis. These results identify an important function of Id1 in the regulation of epidermal homeostasis.

The effects of Aspergillus fumigatus challenge on exhaled and nasal NO levels.

Several studies have previously shown that exposure to indoor air microbes from moisture-damaged buildings can cause adverse health effects. Aspergillus fumigatus is one of the best-documented moulds causing health problems to those exposed. In this study, inhalation of a commercial A. fumigatus solution was assessed, to establish if it would have effects on fractional exhaled (FeNO) and nasal (FnNO) nitric oxide levels and on lung function. The results were compared with placebo challenge. A total of 28 subjects were divided into three study groups: group 1 had been exposed to occupational mould; group 2 consisted of atopic subjects; and group 3 was a control group. Some 3 h after A. fumigatus challenge, there was a considerable increase in FeNO, and a significant difference was observed between the A. fumigatus and placebo inhalations. The difference was seen in all study groups. No such differences were found in the levels of FnNO or nitrite in nasal lavage fluid. Subjects reported significantly more frequent respiratory tract symptoms after the A. fumigatus inhalation compared with placebo challenge. In conclusion, it was shown here that inhalation challenge of Aspergillus fumigatus elevated fractional exhaled nitric oxide levels. An increase in fractional exhaled nitric oxide may serve as an indicator of respiratory inflammation of acute mould exposure.

Continuous-flow fractionation of trace metals in environmental solids using rotating coiled columns. Some kinetic aspects and applicability of three-step BCR leaching schemes.

The applicability of the three-step BCR leaching scheme to the continuous-flow fractionation of trace metals (TM) using rotating coiled columns (RCC) has been investigated taking soil and sediment reference samples (SRM-2710, CRM-601, BCR-701) as examples. A particulate sample was retained in the rotating column as the stationary phase under the action of centrifugal forces while different eluents, used according to the original and optimised BCR protocols, were continuously pumped through. The whole procedure required 3-4 h instead of at least 50 h needed for the traditional sequential extraction. It has been shown that in comparison with batch sequential extraction procedures (SEP), the recoveries of Cd, Zn, Cu, and Pb at the first stage (most mobile and potentially dangerous acid soluble forms) are somewhat higher, if a dynamic extraction in RCC is used. Nevertheless, the distribution patterns for TM in the first two leachable fractions (acid soluble and reducible forms) are similar in most cases. Since no heating is used in RCC, the recoveries of TM at the third stage (when hydrogen peroxide is applied to oxidize the organic matter) may be incomplete and matrix-dependent. The effect of eluent volume and flow rate on the recovery of TM in different forms has been investigated. It has been shown that the kinetics of heavy metal leaching vary significantly with samples. Hence, investigating the elution profiles can provide additional important information for risk assessment of TM mobilization.

Species-specific isotope-ratio measurements of volatile tin and antimony compounds using capillary GC-ICP-time-of-flight MS.

The analytical performance of an axial inductively-coupled-plasma time-of-flight mass spectrometer (ICP-TOFMS) as a detector for fast transient chromatographic signals resulting from the coupling to capillary gas chromatography (CGC) was investigated. A cryotrapping GC-ICP-TOFMS method for the determination of volatile metal(loid) compounds (VOMs) in gases was used and the suitability of the TOF mass analyzer for multielemental speciation analysis and multi-isotope ratio determinations was studied in terms of accuracy and precision. Isotope ratios 118Sn/120Sn and 121Sb/123Sb have been determined in in-house gas standard atmospheres in Tedlar bags at two different levels (100 pg and 1 ng) for different elemental species (SnH4, MeSnH3, Me2SnH2, Me3SnH, BuSnH3, SbH3, and MeSbH2). A limitation arising from counting statistics in both detection modes could be shown. A solution containing rhodium (10 ng mL(-1)) and cadmium (40 ng mL(-1)) was introduced simultaneously to the GC outlet. Rhodium acts as a continuous internal standard and Cd is used for mass-bias correction (by measuring the 111Cd/113Cd ratio). The detection system in both pulse counting and analog mode was examined. The best attainable precision was established for Me2SnH2 (analog mode, 12 replicates, 1 ng, RSD 0.34%, accuracy 0.31%) whereas most other species ranged between 0.4 and 0.5% RSD if higher concentrations were used. The limitations of the pulse counting system are clearly seen, with peak heights of more than 2000 counts reaching saturation (for an integration time of 100 ms), which reduces the accuracy of isotope ratio determinations. A dozen VOM could be detected in an aged landfill gas sample; several unidentified Sn compounds were present. Although their isotope ratios are within the confidence value of the standards, it is not yet clear if the acquired precision is good enough to identify isotopic fractionation of metal(loid)s through biovolatilization processes. With the precision achieved, the combination of cryotrapping GC and ICP-TOFMS is a powerful tool for monitoring volatile multi-element species in multi-tracer experiments and isotope dilution methodology.

Organotypic cocultures with genetically modified mouse fibroblasts as a tool to dissect molecular mechanisms regulating keratinocyte growth and differentiation.

Organotypic cocultures of keratinocytes and fibroblasts generate a normal epidermis irrespective of the species and tissue origin of fibroblasts. The use of mouse fibroblasts and human keratinocytes facilitates the identification of the origin of compounds involved in epidermal tissue reconstitution and growth regulation. Moreover, the functional significance for the keratinocyte phenotype of genetically modified fibroblasts from transgenic or knockout mice, even those exhibiting an embryonic lethal phenotype, can be studied in such heterologous in vitro tissue equivalents. Here we communicate results of such studies revealing the antagonistic function of mouse fibroblasts defective in the AP-1 constituents c-Jun and JunB, respectively, on human keratinocyte growth and differentiation. Furthermore, the hematopoietic growth factor granulocyte macrophage-colony stimulating factor has been identified as a novel regulator of keratinocyte growth and differentiation. As will be reported in detail elsewhere both granulocyte macrophage-colony stimulating factor and keratinocyte growth factor have been identified as major mediators of fibroblast-keratinocyte interactions and their expression is induced via AP-1 by interleukin-1 released by the epithelial cells. Thus, these heterologous cocultures provide a novel promising tool for elucidating molecular mechanisms of epithelial-mesenchymal interactions and their consequences on epithelial cell proliferation and differentiation.

Keratinocyte growth regulation in defined organotypic cultures through IL-1-induced keratinocyte growth factor expression in resting fibroblasts.

Balanced keratinocyte proliferation and differentiation resulting in regular tissue organization strictly depend on dermal support. Organotypic cultures represent biologically relevant in vitro models to study the molecular mechanism of the underlying dermal-epidermal interactions. To mimic the state of resting fibroblasts in the dermis, postmitotic (irradiated) fibroblasts were incorporated in the collagen matrix, where they typically support epidermal proliferation and tissue organization. In coculture with keratinocytes, fibroblasts exhibit an enhanced expression of keratinocyte growth factor and the interleukin-1 receptor (type I), which further increase with culture time. In cocultured keratinocytes, keratinocyte growth factor receptor as well as RNA expression and protein release of interleukin-1alpha and interleukin-1beta are upregulated. We hypothesized that the modulated cytokine expression represents a basic mechanism for keratinocyte growth regulation. The functional significance of this double paracrine pathway, i.e., induction of keratinocyte growth factor expression in fibroblasts by keratinocytes via release of interleukin-1, was confirmed by interfering with both signaling elements: (i) interleukin-1-neutralizing antibodies and interleukin-1 receptor antagonist significantly inhibited keratinocyte growth factor release, keratinocyte proliferation, and tissue formation comparable to the effect produced by keratinocyte-growth-factor-blocking antibodies; (ii) addition of keratinocyte growth factor to cocultures with inactivated interleukin-1 pathway completely reverted growth inhibition; (iii) in organotypic cocultures with subthreshold fibroblast numbers both interleukin-1 and keratinocyte growth factor restored the impaired epidermal morphogenesis. Thus, epidermal tissue regeneration in organotypic cocultures is mainly regulated by keratinocyte-derived interleukin-1 signaling, which induces keratinocyte growth factor expression in cocultured fibroblasts. This demonstrates a novel role for interleukin-1 in skin homeostasis substantiating data from wound healing studies in vivo.

Biomonitoring of airborne inorganic and organic pollutants by means of pine tree barks. I. Temporal and spatial variations.

Scots pine (Pinus sylvestris L.) bark samples were collected at two field sites (Neuglobsow, Rösa) and in different years between 1987 and 1996 in the east of Germany. The barks were analyzed with respect to the following inorganic and organic substances: Al, As, B, Ca, Cd, Ce, Cr, Cu, Fe, Hg, Mo, NH4+, Ni, NO3-, PO4(3)-, Pb, Sr, SO4(2)-, Ti, V, W, Zr, Zn, benzo[a]pyrene, fluoranthene, pyrene, alpha-hexachlorocyclohexane (alpha-HCH) and dichlorodiphenyltrichloroethane (DDT). In addition to bark samples from the site Rösa, 53 test sites were investigated in the Nature Park Dübener Heide. Here, the analysis of the barks aimed at discovering spatial patterns of the above-mentioned substances. Since 1991, most of the determined substances (e.g. sulfate, nitrate, calcium, lead, benzo[a]pyrene, alpha-HCH) show decreased concentration values in bark samples from both sites. Temporal variations reflect substantial infra-structural changes in eastern Germany, especially at Rösa and in the industrial region around the cities Leipzig, Halle, and Bitterfeld. Moreover, nitrate concentrations in barks are increasing since 1995. The trend can be explained with increased nitrogen emissions from motor traffic and livestock farms. Spatial patterns of sulphate and ammonia reflect inputs from power plants and agriculture in pine stands of the Nature Park Dübener Heide. The results show that barks of pine trees can be used as biomonitoring tools to indicate and characterize depositions of airborne organic and inorganic pollutants.

Organotypic keratinocyte cocultures in defined medium with regular epidermal morphogenesis and differentiation.

Skin equivalents formed by keratinocytes cocultured with fibroblasts embedded in collagen lattices represent promising tools for mechanistic studies of skin physiology, for pharmacotoxicologic testing, and for the use as skin substitutes in wound treatment. Such cultures would be superior in defined media to avoid interference with components of serum or tissue extracts. Here we demonstrate that a defined medium (supplemented keratinocyte defined medium) supports epidermal morphogenesis in organotypic cocultures equally well as serum-containing medium (mixture of Ham's F12 and Dulbecco's modified Eagle's medium), as documented by hallmarks of the epidermal phenotype studied by immunofluorescence and electron microscopy. In both cases regularly structured, orthokeratinized epithelia evolved with similar kinetics. Morphology in mixture of Ham's F12 and Dulbecco's modified Eagle's medium was slightly hyperplastic, and keratins 1 and 10 synthesis less co-ordinated than in supplemented keratinocyte defined medium, but a consistently inverted sequence of expression of keratins 1 and 10 was found in either medium. The late differentiation markers filaggrin, involucrin, keratin 2e, and transglutaminase 1 corresponded in their typical distribution in upper suprabasal layers. Keratin 16 persisted under both conditions indicating the activated epidermal state. Keratinocyte proliferation was comparable in both media, whereas fibroblast multiplication and proliferation was delayed and reduced in supplemented keratinocyte defined medium. In both media, ultrastructural features of epidermal differentiation as well as reconstitution of a basement membrane occurred similarly. Immature lamellar bodies and cytoplasmatic vacuoles, however, indicated an impaired lipid metabolism in supplemented keratinocyte defined medium. Nevertheless, these defined organotypic cocultures provide a suitable basis for in vitro skin models to study molecular mechanisms of tissue homeostasis and for use in pharmacotoxicologic testing.

Epidermal differentiation and basement membrane formation by HaCaT cells in surface transplants.

The immortal human keratinocyte line HaCaT has been employed in many studies as paradigm for epidermal keratinocytes. In order to demonstrate its potential to form stable epidermal structures in response to connective tissue, this was challenged in surface transplants on nude mice, where normal keratinocytes rebuild a typical epidermis within two weeks. During the initial regeneration phase (day 1-4) multilayered but poorly organized epithelia formed with proliferating cells in all layers in analogy to normal keratinocytes. Similarly, with tissue consolidation (around day 7) proliferation was reduced and restricted to cells in basal position marked by keratin K14 and beta1-integrin immunostaining. The strong suprabasal reaction for K1 and K10, the appearance of the late markers K2e, filaggrin and loricrin as well as the polarized distribution of alpha2beta1 and alpha3beta1 indicated advancing tissue normalization (day 14). Keratinization further improved at around three weeks switching from the initial parakeratotic to the regular orthokeratotic type which was prominent at six weeks. Accordingly, most ultrastructural features typical for epidermis or normal keratinocyte grafts were detectable including a complete basement membrane (BM) with regular attachment structures. Matrix- and BM-components appeared sequentially with marked linear deposition of laminin-5 (day 4) followed by accumulation of collagen-IV and 'classical' BM-laminin between one and two weeks. With the general codistribution of integrin alpha6beta4 and BM-molecules (day 14) collagen-VII lining of BM became prominent, while epithelium and host connective tissue were still separated by the collagen matrix. In accordance with the delayed orthokeratinization, wound-matrix molecules (fibronectin, tenascin) persisted longer than in normal keratinocyte transplants. Finally, grafts of long-term passaged (no. 310) cells demonstrated a remarkable stability in the expression of epidermal markers. Thus, the immortalized HaCaT cells reveal a generally high competence to realize an epidermal phenotype in a natural environment and appear therefore qualified for in vitro studies on structural and regulatory aspects of keratinocyte physiology and pathology.

Dynamics of basement membrane formation by keratinocyte-fibroblast interactions in organotypic skin culture.

The cutaneous basement membrane zone, composed of numerous macromolecules, plays a multifunctional role in tissue regeneration and maintenance. To elucidate the cellular origin and dynamics of basement membrane formation, de novo synthesis, deposition, and ultrastructural assembly of its components were analyzed in organotypic cultures of adult skin keratinocytes on collagen gels with or without collagen-embedded dermal cells. Collagen IV and laminin-1 deposition occurred only in the presence of mesenchymal cells: patchy at day 4 and continuous after 1 week. Chain-specific mRNA expression started at day 2 in both keratinocytes and fibroblasts. It steadily increased up to day 10, however, with a reciprocal induction pattern, mRNA abundance shifting from keratinocytes to fibroblasts. On the other hand, laminin-5 staining was first observed at day 4, but in keratinocyte both mono- and cocultures. This was followed by nidogen, which was detected in cocultures but also in dermal monocultures. Laminin-5 protein persisted throughout day 21, whereas nidogen steadily increased in intensity. Expression kinetics revealed high levels of laminin-5 transcripts early and in keratinocytes only, whereas nidogen was expressed later and predominantly in fibroblasts. Although basement membrane protein deposition was continuous at day 14, the ultrastructural organization was still fragmentary, eventually normalizing at 3 weeks. These data demonstrate a dynamic interaction and cooperation of epithelial and mesenchymal skin cells in basement membrane formation. This interaction is supposedly mediated via diffusible factors. Our findings further extend the scope of epithelial-mesenchymal interactions stressing that both cell compartments are essential to constitute a tissue-specific extracellular matrix structure.

Plasminogen activator inhibitor type 2 is expressed in keratinocytes during re-epithelialization of epidermal defects.

Plasminogen activation is observed in the human epidermis during re-epithelialization of epidermal defects. The activation reaction depends on plasminogen activators (PAs) associated with re-epithelializing keratinocytes. PA inhibitor type 2 (PAI-2) is thought to be a major epidermal PA inhibitor in keratinocytes. However, no data are available on the expression of PAI-2 in keratinocytes during epidermal regeneration. We have therefore analysed PAI-2 at the mRNA and protein level in keratinocyte cultures as well as in epidermal lesions in which re-epithelializing keratinocytes were apparent. We found that PAI-2 expression at the mRNA and protein level was negatively correlated with the cell density in regular keratinocyte cultures. In organotypic cocultures, in which the transition from a re-epithelializing to a sedentary phenotype can be studied, PAI-2 was most strongly expressed in early cultures prior to formation of a differentiated epidermis-like structure. We found a strong expression of PAI-2 in keratinocytes that re-epithelialized dermal burn wounds or lesions caused by the autoimmune blistering disease pemphigus vulgaris. Our results suggest that not only PAs, but also a major PA inhibitor, PAI-2, are expressed in keratinocytes that are actively involved in re-epithelialization.

5-Lipoxygenase expression in Langerhans cells of normal human epidermis.

We studied expression of the 5-lipoxygenase (5-LO) pathway in normal human skin. In situ hybridization revealed a 5-LO mRNA-containing epidermal cell (EC) population that was predominantly located in the midportion of the spinous layer, in outer hair root sheaths, and in the epithelial compartment of sebaceous glands. Examination of skin specimens by immunohistochemistry and of primary ECs by flow cytometry mapped the 5-LO protein exclusively to Langerhans cells (LCs). The LC 5-LO protein was largely found in the nuclear matrix, in nuclear envelopes, and perinuclear regions as indicated by in situ confocal laser scan microscopy. Reverse transcription-PCR and immunoblot analyses of purified primary EC populations further indicated that LCs are major 5-LO expressing cells. Enriched primary LCs were also found to contain 5-LO activating protein (FLAP), leukotriene (LT) C4 synthase, and LTA4 hydrolase. By contrast, 5-LO, FLAP, and LTC4 synthase were undetectable or largely reduced, but LTA4 hydrolase transcripts and protein were identified in ECs depleted of LCs. These data show that naive LCs are major, and possibly the sole, 5-LO pathway expressing cells in the normal human epidermis.

Integrin and basement membrane normalization in mouse grafts of human keratinocytes--implications for epidermal homeostasis.

Integrin patterns and formation of basement membrane (BM) were investigated in correlation to epidermal growth and differentiation during skin regeneration in human keratinocyte transplants on nude mice. Immuno-fluorescence and transmission electron microscopy (TEM) showed that different stages of tissue reconstruction were characterized by a sequence of coordinated events. Features of the initial tissue activation, with rapid keratinocyte proliferation around day 4, including cells in a suprabasal position, were: (1) a marked increase in and extended distribution of the integrin chains alpha 2, alpha 3, beta 1 and alpha 6, while beta 4 already showed a preferential basal location; (2) de novo expression of alpha 5 and alpha v; and (3) marked deposition of laminin-5 and nidogen but low levels of other BM components. Tissue normalization during the 2nd week, initiated by a drastic decrease in the number of proliferating cells after day 4, now strictly in basal position, was signified: by (1) orthotopic staining for basal-type keratins (K5, K14) together with a regular pericellular alpha 2 beta 1 and alpha 3 beta 1 distribution, (2) linear, balanced deposition of BM components (e.g. laminin-1, type IV collagen) and (3) colocalization of integrin alpha 6 beta 4 and bullous pemphigoid antigen. Simultaneously at 7 days hemidesmosomes and a defined BM had developed (TEM), becoming continuous at 14 days. This coincided with the regular distribution of suprabasal keratins (K1, K10) as well as intermediate (involucrin) and late differentiation markers (filaggrin, loricrin). Type-VII collagen deposition, still irregular at 14 days, became continuous at 22 days together with developing BM-anchoring fibrils indicating final tissue consolidation. This model mimics principal stages of epidermal wound healing in human skin and implies a linkage between BM assembly, integrin distribution and the compartment of proliferation competent cells, which in turn determines the onset of differentiation. Thus, apart from the balance of diffusible growth regulators, this positional control of keratinocytes, largely accomplished by integrin-matrix interactions, seems to be prerequisite to establishment and maintenance of tissue homeostasis.

Determination of traces of heavy metals (Mn, Cu, Zn, Cd and Pb) in microsamples of teeth material by ETV-ICP-MS.

Heavy metal levels in tooth compartments would appear to be a suitable indicator of long-term exposure. A method has been developed to assess the heavy metal levels (Mn, Cu, Zn, Cd and Pb) in tooth compartments like dentin and enamel. For the simultaneous determination of these elements in small dentin sample amounts (in some cases less than 1 mg) inductively coupled plasma mass spectrometry with electrothermal vaporization was the method of choice. Sample preparation, method development and first results of the application of the method for the analysis of tooth compartments have been described. Variation in the elemental concentrations depending on the kind of tooth and the sampling position have been observed. The method is useful for epidemiological studies of the heavy metal exposure of children.

Differential expression of urokinase-type plasminogen activator (uPA), its receptor (uPA-R), and inhibitor type-2 (PAI-2) during differentiation of keratinocytes in an organotypic coculture system.

Cultured keratinocytes resemble migrating keratinocytes under conditions of reepithelialization during wound healing. Such keratinocytes express urokinase-type plasminogen activator (uPA) and its specific receptor (uPA receptor). Receptor-bound uPA activates plasminogen, thus providing plasmin for pericellular proteolysis. uPA is regulated by the plasminogen activator inhibitors PAI-1 and PAI-2. As indicated by immunohistology, neither uPA nor uPA receptor is expressed in normal epidermis. Thus, the down-regulation of uPA and uPA-receptor expression in keratinocytes appears to be an important event in epidermal healing and restoration of a normal epidermal tissue architecture. We have addressed this matter by using a culture and differentiation system for keratinocytes in vitro. Keratinocytes were grown in organotypic cocultures for 4, 7, and 14 days. Frozen sections were analyzed with indirect immunofluorescence staining and overlay zymography, the latter detecting activity of plasminogen activators. While tPA and PAI-1 stainings were consistently negative over the entire observation period, uPA and uPA receptor were expressed by basal keratinocytes at Days 4 and 7, but not at Day 14. Accordingly, overlay zymography revealed uPA activity at Days 4 and 7. PAI-2 was found throughout the entire observation period, but with varying distribution: at Days 4 and 7 all suprabasal keratinocytes stained positive for PAI-2. At Day 14, PAI-2-specific stainings were confined to the uppermost cells of the stratum spinosum. Our data demonstrate that uPA and uPA receptor, which are up-regulated in cultured keratinocytes, are down-regulated upon restoration of an epidermis-like structure. The distribution of PAI-2 varied over the observation period and at Day 14 resembled the distribution of PAI-2 in normal epidermis. Taken together, keratinocytes in organotypic coculture behave like keratinocytes in healing wounds in vivo with respect to the expression of the plasminogen activator system.

Expression of 5-lipoxygenase in differentiating human skin keratinocytes.

We studied the expression of arachidonate 5-lipoxygenase (5-LO) in a cell line of human keratinocytes (HaCaT) and in normal human skin keratinocytes in tissue culture. In undifferentiated keratinocytes 5-LO gene expression was low or undetectable as determined by 5-LO mRNA, protein, cell-free enzyme activity, and leukotriene production in intact cells. However, after shift to culture conditions that promote conversion of prokeratinocytes into a more differentiated phenotype, 5-LO gene expression was markedly induced in HaCaT cells and, to a lesser extent, in normal keratinocytes. These results show that 5-LO gene expression is an intrinsic property of human skin keratinocytes.