Soc. of Invest. Dermatology (SID)
2002 Meeting Presentations
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Presentation #1 of 6:
(SID Final ID# 373)
Proteomic and Microarray Investigation to Identify Potential In Vitro Endpoints for Skin Irritation. Fletcher, Samantha1; Fentem, Julia1; Basketter, David1; Kelsell, David2; Philpott, Mike2; Baker, Val1. 1SEAC, Unilever, Sharnbrook, UK; 2Centre for Cutaneous Research, Queen Mary, London, UK.
To enable development of relevant in vitro test systems for identification of skin irritation, an understanding of the mechanistic basis of the response is required. Recent progress in development of proteomic and microarray technologies provides tools required for identification and investigation of biochemical events that occur within the skin irritation response. This study was aimed at the identification of proteins (and genes encoding proteins) which may be involved in this response, following exposure of a human skin model (EpiDerm(MatTek)) to the irritant sodium lauryl sulphate (SLS). EpiDerm cultures were treated in triplicate with non-cytotoxic dose of SLS (0.1mg/ml, determined by histology and MTT) for 15min, 1h, 2h, 4h and 24h. 2D-gel electrophoresis (Multiphor II and DALT system, APB) and mass spectrometry (Bruker) was performed to investigate the protein expression profile and identify proteins of interest. Western blotting and ELISA were used to confirm modifications for selected proteins of interest. In addition microarray analysis was performed on EpiDerm samples (treated as described), using DermArray (Research Genetics) covering 4000 human genes of relevance to skin biology. From the proteomic experiments 67 proteins of potential interest were selected and identified from a range of 2D-gels (1st dimension: pH4-7, 4-5, 5-6, 6-9 and 6-11). Of the proteins selected, 35 proteins were up-regulated, 19 down-regulated and the expression of 4 remained unchanged (controls). The data indicated that post-translational modification occurred at an early time point (15min) for calmodulin-like skin protein and involucrin following exposure of EpiDerm to SLS. The results demonstrate the differential regulation of a number of proteins in response to the skin irritant SLS which could provide potential new in vitro markers of skin irritation.
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Presentation #2 of 6:
(SID Final ID# 316)
Full Thickness EpiDermTM, a Dermal-Epidermal Skin Model To Study Epithelial-Mesenchymal Interactions. J. Kubilus, P.J. Hayden, T.J. Last, S. Ayehunie, K.L. Bellavance, S.D. Lamore, M. Klausner. MatTek Corporation, Ashland, MA.
It has long been recognized that significant paracrine signaling occurs between dermal fibroblasts and epidermal keratinocytes. Further, it is understood that such interactions likely modulate the response of skin to materials which may lead to contact irritant or allergic responses. Also, dermis related phenomena such as photoaging and photodamage, wound healing and cancer progression are ideally studied in a dermis-containing tissue. To address these issues, we have recently developed a second-generation skin model consisting of a fibroblast-containing dermal/ keratinocyte-containing epidermal, full thickness tissue.
In contradistinction to previous dermal/epidermal models, the new tissue is cultured in an easily manipulated cell culture insert and the tissue extends from wall-to-wall. In terms of ease of use, these characteristics greatly facilitate testing of potential allergens or irritants in that direct topical application is possible. Topical exposure to the common surfactant, 1% Triton X-100, results in MTT tissue viability dose response curves which fall within the normal range of the keratinocyte-only tissue, EpiDerm . Currently, in order to produce a standardized, reproducible organotypic tissue, all lots of EpiDerm are compared to a reference database of Effective Time-50 (ET-50) values, i.e. the time of exposure after which viability is reduced to 50% following exposure to 100 µL of Triton X-100. The database average (184 tissue lots) is 6.74 ± 0.99 hours (± 1 standard deviation); initial lots of the full thickness tissue, tested in an identical manner, averaged 7.14 ± 1.33 hours (n=3). Histological cross-sections of the full thickness tissue show an epidermal layer which is very similar to EpiDerm and native epidermis atop a fibroblast-containing collagen matrix dermis-like layer. Based on these initial results, investigation of the tissue response to stimuli specifically affecting the dermis or epidermal/dermal "crosstalk" may prove rather informative.
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Presentation #3 of 6:
(SID Final ID# 102)
Expression of Cyclooxygenase in the EpiDermTM Human Skin Model: Regulation by Ultraviolet Radiation and Cytokines. P.J. Hayden, T.J. Last, B. Burnham and M. Klausner. MatTek Corporation, Ashland MA.
Cyclooxygenase-2 (COX-2) appears to play an important role in skin carcinogenesis caused by solar ultraviolet radiation (UVR): UVR induces COX-2 expression in human skin, COX-2 activity is upregulated in skin cancers and COX inhibitors reduce UVR-induced tumor formation. In this report, we use RT-PCR, Western blotting and ELISA to study the effect of UVR on COX message, protein and metabolites, respectively, in the EpiDerm in vitro human skin equivalent. There are two isoforms of COX, COX-1 (constitutive) and COX-2 (inducible). COX enzymes metabolize arachadonic acid (AA) to eicosanoid products including prostaglandin E2 (PGE2) thromboxane A2 and PGI2, which are thought to contribute to skin carcinogenesis. COX may also metabolize AA to 8-isoprostane (8IP), a novel mitogenic prostaglandin previously thought to be formed only via free radical pathways. EpiDerm tissues expressed COX-1 message and protein constitutively, and secreted PGE2 and 8IP into the culture medium. COX-2 message and protein were also detectable. UVR enhanced COX-2 expression without effecting COX-1, and increased production of both PGE2 and 8IP. COX inhibitors reduced production of PGE2 and 8IP to below baseline levels, indicating a metabolic rather than free radical mechanism of formation. Additional experiments were conducted to study regulation of COX expression by cytokines in the absence of UVR. IL-1α, IL-1β or TNFα induced expression of COX-2 without effect on COX-1, and increased production of PGE2 and 8IP. Combinations of IL-1α or IL-1β with TNFα produced additive PGE2 and 8IP production. Thus, EpiDerm tissues behave similarly to in vivo human skin with respect to regulation of COX-2 by solar UVR and cytokines. COX-dependent production of 8IP by human skin equivalents is demonstrated for the first time, suggesting a potential role for this mitogenic metabolite in skin carcinogenesis. Finally, the results indicate that in vitro human skin equivalents are useful models for study of COX regulation by UVR, and related aspects of human skin carcinogenesis.
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Presentation #4 of 6:
(SID Final ID# 707)
Use of EpiDermTM as an inflammatory model for pre-clinical screening. T.J. Last, P. Hayden, B. Burnham, M. Klausner and J. Kubilus. MatTek Corporation, Ashland, MA 01721.
Organotypic skin models are becoming widely used by the pharmaceutical industry as an alternative to animal testing for screening potential therapeutics for safety and efficacy. The purpose of this study is to test if MatTek's EpiDerm skin model can be induced at the molecular level to initiate an inflammatory response. EpiDerm is a highly differentiated, three-dimensional epidermal tissues composed solely of keratinocytes. Skin keratinocytes are known to be involved with the progression of numerous inflammatory skin diseases such as psoriasis and contact dermatitis. Molecular markers of these skin diseases include intercellular adhesion molecule-1 (ICAM-1), inducible nitric oxide synthase (iNOS) and interleukin 20 (IL-20).
In the present work, EpiDerm tissues were treated with a panel of cytokines including IFN-γ, IL-1α, IL-1β, TNFα, and various combinations thereof. IL-20, iNOS and ICAM-1 message levels of the tissues were examined by RT-PCR. IL-20 message levels are significantly up-regulated by treatment with some of these cytokines. Inducible nitric oxide synthase was also up-regulated. However, there were temporal differences in response between the two genes. ICAM-1 levels were also enhanced following cytokine treatment and detectable soluble ICAM levels were present in the supernatant, as analyzed by ELISA.
In conclusion, this study shows that genes important in inflammatory skin response can be regulated in EpiDerm tissues by the various cytokines studied. Furthermore, these results suggest that EpiDerm can be used as in vitro skin model for testing therapeutics that target suppression or inhibition of inflammatory responses in epidermis.
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Presentation #5 of 6:
(SID Final ID# 786)
Results Using MelanoDermTM, an Epidermal Model Containing Functional Melanocytes, Demonstrate Efficacy of Novel Skin Lightening Formulations. M. Klausner, J. Kubilus, P.J. Hayden, T.J. Last, G. DePaoliAmbrosi*. MatTek Corporation, Ashland, MA, *General Topics, s.r.l., San Felice del Benaco, Italy.
To minimize the cost of human clinical studies and shorten the time to market, we have used the MelanoDerm skin model (MatTek Corporation) to screen novel skin lightening formulations that we have recently developed. The formulations were applied topically to MelanoDerm tissues 5 times over an 8-day period. Tissue samples were fixed for histological analysis on Days 4, 7, 11, and 14; others were frozen on Days 11 and 14 to measure melanin content. Visual observation of the tissues showed that the tissues progressively darken over the 14-day culture period and that the 4 formulations tested resulted in significantly decreased pigmentation versus the negative control (untreated) tissues. This was borne out by melanin assay results that showed decreases of 64-77% in melanin content versus the controls. Histological analysis showed that the tissues maintained normal epidermal morphology over the experimental period and that the reduction in pigmentation was not due to cytotoxicity. Using en face microscopic observation, the melanocytes were difficult to visualize in the treated tissues up through Day 11. However, 6 days after the final lightener application (Day 14), the melanocytes in tissues treated with 3 of the 4 formulations were highly pigmented and dendritic, similar to the melanocytes contained within the control tissues. Based on these results, we conclude that 3 of the 4 formulations dramatically inhibit melanogenesis without cytotoxicity. These and initial, positive clinical results lead us to believe that the formulations will be commercially useful in skin lightening products.
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Presentation #6 of 6:
(SID Final ID# 314)
Gingival Tissue Model For Prediction of Irritancy of Oral Care Products. J. Kubilus, M. Klausner, B. Breyfogle. MatTek Corporation, Ashland, MA.
The development of human reconstructed tissues of the oral cavity that express in vivo-like differentiated characteristics would be important to better understand and develop therapies for gum disease, oral cancer, lesions due to smokeless tobacco, and gingivitis, amongst other diseases of the oral mucosa. In vitro tissue models would be used to assess the biocompatibility of new dental materials, dentifrices, and oral care consumer products and would be useful in determining pharmaceutical delivery through the oral mucosa. To meet these needs, we have recently developed a tissue culture model of the gingival epithelium.
Gingival cells were harvested from tissue explants, expanded in monolayer culture, and induced to form a three-dimensional tissue using serum free medium. Histologically, the tissue is 8-12 cells layers thick with cells becoming increasingly squamous as the apical surface is approached. The tissue is cultured in easily manipulated cell culture inserts and the tissue extends from wall-to-wall. In terms of ease of use, these characteristics greatly facilitate testing of oral care products in that direct topical application is possible. To assess the reproducibility of the tissue, tissues were exposed to the common surfactant, 1% Triton X-100 (TRI), and viability dose response curves were constructed using the MTT assay. An Effective Time-50 (ET50), i.e. the time of exposure after which viability is reduced to 50%, was determined. The ET50 for 3 lots of tissue averaged 112 ± 12.7 minutes. In terms of resistance to surfactant injury by TRI, the gingival tissue is more susceptible to damage compared with the EpiDermTM skin model (ET50 = 6.74 ± 0.99 hrs) but more robust than the EpiOcularTM tissue model (ET50= 24.9 ± 6.3 mins). Exposure to sensitive and normal strength dentifrices showed that viability of the tissue was compromised to a greater degree by the normal strength toothpastes. Based on these initial results, further investigation of the tissue model's ability to predict potential gingival irritation from oral care products is warranted.
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