MatTek-Related Presentations -
Society of Toxicology (SOT) Meeting - March 2002==========================
Abstract ID: 486
(1 of 4--Please scroll down to review all 4 Abstracts):
Expression of 15-lipoxygenase-2 in the EpiAirway™ in vitro human tracheal/bronchial epithelial model: regulation by TNF-α and INF-γ
P.J. Hayden1, G.R. Jackson1, T.J. Last1, M. Klausner1, J. Kubilus1, S.B. Shappell2
1MatTek Corporation, Ashland, MA, 2Department of Pathology, Vanderbilt University School of Medicine, Nashville, TN
(MatTek Technical Reference #256)
15-Hydroxyeicosatetraenoic acid (15[S]-HETE) is the predominate arachidonic acid (AA) metabolite produced by human tracheal/bronchial tissue. This product of the 15-lipoxygenase (15-LOX) enzyme is believed to play a significant role in human airway inflammation and respiratory diseases including cystic fibrosis and asthma. In the current work, RT-PCR and Western blotting techniques were utilized to investigate the expression of 15-LOX isozymes in EpiAirway, a commercially available in vitro model of human tracheal/bronchial epithelium. The effects of IL-1β, TNFα, the TH2 cytokines IL-4 and IL-13, the TH1 cytokine INFγ and various combinations of these cytokines were examined. The previously reported expression of 15-LOX-1, as well as induction of enhanced 15-LOX-1 expression by IL-4 and IL-13, was confirmed by RT-PCR. Additionally, 15-LOX-2 message and protein were detected in the EpiAirway model. TNFa was found to induce expression of 15-LOX-2 message and protein, while the TH1 cytokine INFg inhibited the TNFα-induced increase in both message and protein. Identical results were obtained in EpiAirway constructs derived from four separate airway cell donors, including one asthmatic donor. These results demonstrate, for the first time, 15-LOX-2 expression in human tracheal/bronchial epithelial cells. The induction of 15-LOX-2 by TNFα and inhibition by the TH1 cytokine INFγ suggest a possible important role of 15-LOX-2 in mediating human airways diseases such as asthma, which is characterized by increased TNFα and decreased TH1 (INFγ)/TH2 (IL-4, IL-13) ratios.
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Abstract ID: 1090
Identification of Potential In Vitro Endpoints for Skin Irritation Using Proteomic and Microarray Analysis
S.T. Fletcher1, J.H. Fentem1, D.A. Basketter1, D.P. Kelsell2, M. Philpott2, V.A. Baker1
1SEAC, Unilever, Bedfordshire, United Kingdom , 2Centre for Cutaneous Research, Queen Mary, University of London, United Kingdom
(MatTek Technical Reference #258)
Understanding the mechanistic basis of the human skin irritation response is key to the development of relevant in vitro test systems for the predictive identification of skin irritation hazards. Recent progress in the development of proteomic and microarray technologies means that tools for the identification and investigation of important biochemical events in the processes of skin irritation are now available. This study was designed to identify proteins (and genes encoding proteins) which may be involved in the skin irritation response, following exposure of a reconstructed human skin model (EpiDerm™ (MatTek)) to the skin irritant sodium lauryl sulphate (SLS). EpiDerm™ cultures were treated in triplicate with a non-cytotoxic dose of SLS (0.1mg/ml, as determined by the MTT assay and histological examination) for 15min, 1h, 2h, 4h and 24h. Proteomics was performed using 2D-gel electrophoresis (Multiphor II and DALT system, (APB)) in combination with mass spectroscopy to investigate the protein expression profile and identify proteins of interest. In addition microarray analysis was performed using DermArray (Research Genetics) cDNA arrays covering 5000 genes of relevance to skin biology. 67 proteins of potential interest were selected and identified from a range of 2D-gels (1st dimension : pH 4-7, 4-5, 5-6, 6-9 and 6-11). Of the proteins selected, 35 were upregulated, 19 downregulated and the expression of 4 remained unchanged following exposure to SLS. Data indicated that post-translational modification occurred at an early time point (15 min) for calmodulin-like skin protein and involucrin following exposure of EpiDerm™ to SLS. Epithelial cell marker protein demonstrated post-translational modification after 1h exposure to SLS. These results demonstrate the differential regulation of a number of proteins in response to a known irritant, which could represent potential new in vitro markers of skin irritation.
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Abstract ID: 1191
Assessment of Ocular Irritation Ranges of Market-Leading Cosmetic and Personal-Care Products Using an In Vitro Tissue Equivalent
N.E. McCain, R.R. Binetti, S.D. Gettings, B.C. Jones. Cell Biology & in vitro Toxicology, Avon Products, Inc., Suffern, NY
(MatTek Technical Reference #259)
The cosmetics and personal-care industry has focused considerable effort on the search for replacements for traditional animal-based safety tests. In vitro models have been found to be particularly useful for the assessment of eye irritation potential. We have used the MatTek EpiOcular™ OCL-200 tissue model (composed of human epidermal keratinocytes that differentiate and form a stratified squamous epithelium similar to corneal tissue) to establish ranges of in vitro ocular irritation scores for several categories of cosmetic and personal-care products. A significant advantage of the EpiOcular model is that it can be used to discriminate between the irritation potential of extremely mild products (most cosmetic and personal care products are formulated for inherent mildness). Materials were topically applied to the EpiOcular tissue equivalents (167L/cm2). Surfactant-based products were tested at a concentration of 10% to simulate "rinse-off" exposures; all other products were tested without dilution. Materials were tested using either the standard (4 hours) or extended (20 hours) exposure protocol based on their expected irritation potential (ie consideration of formula composition, product type, etc.). Cellular viability was used as a marker for irritation potential and measured at various time points by a MTT metabolism colorimetric assay. MTT was quantitated spectrophotometrically at 570nm and an ET50 (time to 50% loss of viability) calculated for each product. A range of in vitro ocular ET50 scores was then determined for each product category. The results obtained from this study provide a reference database of in vitro ocular irritation scores for a cross-section of currently marketed cosmetic and personal-care products. Comparison of the ET50 for new formulations with the range of ET50 scores established for currently marketed products of similar type is a useful benchmark of anticipated consumer acceptability under conditions of actual use.
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Abstract ID: 1848
Human Tissue Model For Vaginal Irritation Studies
M. Klausner1, J. Kubilus1, C.L. Cannon1, J.E. Sheasgreen1, E.C. Lonardo2
1R & D, MatTek Corporation, Ashland, MA , 2Medical Affairs, Johnson & Johnson, Personal Products Company, Skillman, NJ
(MatTek Technical Reference #255)
Recently, a tissue culture based model of the vaginal epithelium has been developed. Normal, human ectocervico-vaginal (ECV) epithelial cells were induced to form a three-dimensional tissue using specially formulated serum free medium. The in vitro tissue reproduces many of the histological, ultrastructural, and protein expression properties of native tissue, including inter-digitation of cells, glycogen production, and cytokeratin expression. Initial experiments investigated the use of this tissue model and the MTT tissue viability assay for predicting ECV irritation. Vaginal anti-fungal products, contraceptives and lubricants were exposed to the tissue model and the exposure time which causes a 50% reduction in tissue viability (ET-50) was determined. The ET-50s were compared to rabbit vaginal irritation scores for 7 products which are currently marketed or in clinical trials. Using an ET-50 cutoff of 9.0 hrs, the products could be successfully categorized into minimal or mild irritation classes. Based on these results and the problems associated with obtaining and the handling of human vaginal tissue, it is anticipated that the tissue model will be very useful in assisting product development scientists in developing safe, efficacious products.
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