Otras apuestas (Concursos, Bolsa y otros)

We therefore conducted studies using human epidermal equivalents which have been shown to closely follow differentiation patterns of human skin in vivo (Auxenfans et al., 2009; Eckhart et al., 2000; Stark et al., 1999) and exhibit inducible metabolic activity by AhR agonists (Harris et al., 2002a; Harris et al., 2002b). We tested the hypothesis that the changes in epidermal equivalent differentiation and stratum corneum compaction observed in response to TCDD are associated with the potency and/or residency of the AhR ligand. We also aimed to test whether these changes could be blocked by AhR knock down and whether we could develop a system to test how these changes relate to AhR degradation.

Previously human epidermal equivalents have been used in studies of AhR activation by 1 or 10nM TCDD (Geusau et al., 2005; Loertscher et al., 2001b). Loertscher et al show early induction of terminal differentiation in epidermal equivalents formed with a spontaneously immortalized human keratinocyte cell line NIKS, which were shown to have the same differentiation and apoptosis pathways as normal primary keratinocytes (Allen-Hoffmann et al., 2000). They show histological changes resulting from treatment with 10nM TCDD for 7 days by showing early development of the cornified layer and by IHC, early and aberrant expression of filaggrin, involucrin and TGM-1. They also

observed an increase in thickness in the ratio of keratinized layers to non-keratinized layers, which they attribute to increased and early onset of terminal differentiation

resulting from TCDD treatment. To rule out the effects of cell death on culture thickness, the authors used IHC staining for active caspase-3 and TUNEL assays, which showed no evidence of TCDD-dependent increase in apoptosis. They concluded that early onset of terminal differentiation was the main mechanism for phenotypic effects in their epidermal equivalent model (Loertscher et al., 2001b). However, this paper is based on an epidermal equivalent model consisting of immortalised keratinocytes as opposed to primary keratinocytes. The author claims to have carried out the phenotypic

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experiments on epidermal equivalents from primary keratinocytes showing the same results, but these data are not shown. Conclusions on terminal differentiation and cell death cannot be reliably extrapolated from a model consisting of immortalized

keratinocytes, as they may not undergo normal cell death, they have been

characterised for differentiation and apoptosis, but not necrosis or autophagy. This must be kept in mind while considering the conclusions from this paper. Geusau et al. show similar effects as Loertscher et al. but in epidermal equivalents produced using primary human keratinocytes. Again, they show that TCDD treatment resulted in increased thickness of the stratum corneum and parakeratosis, decreased VCL thickness (basal, spinous and granular layer as indicated in Figure 4.1) and loss of granular layer. However, differentiation markers did not consistently indicate increased/early differentiation; Western blots were used to show increased involucrin, but also an accumulation of profilaggrin, resulting from a block in processing to filaggrin, and decreased caspase 14 (and processing to its mature form), a protease involved in

terminal differentiation. This paper concludes that TCDD induced aberrant differentiation and blocked final stages of terminal differentiation. The model described in this paper was more relevant to chloracne because it consisted of primary cells, but some of the results are not consistent with other literature. These inconsistencies may be because of cell and species variability in extrapolating results in the literature to this primary human keratinocyte model. Loertscher et al. suggest that TCDD induced non-apoptotic cell death may contribute to the phenotype, but did not define the mechanism of cell death. We go on to confirm that apoptosis was not induced by TCDD in epidermal equivalents and show novel data that autophagy, a mechanism of both cell survival and cell death, is induced by TCDD.

The metabolic capacity of human epidermal equivalents has been studied, but has not been correlated to phenotype, differentiation or homeostasis in the cells (Harris et al., 2002ab; Harris et al., 2002b). Harris et al showed that treatment of epidermal

equivalents, primary human keratinocytes in monolayer culture and ex vivo follicles and epidermis with β-NF or 3-MC induced CYP1A1 and glutathione-s-transferase (GST) activity. GST activity was higher in epidermal equivalents compared to ex vivo samples, and CYP1A1 activity in epidermal equivalents was batch dependent but CYP1A1 could be induced in ex vivo samples and keratinocyte cultures (but was not present in non- induced samples). CYP1A1 and GST activity increased with keratinocyte monolayer

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confluence, in agreement with the literature (Harris et al., 2002a). These papers confirm metabolic function in epidermal equivalents and that it is inducible by AhR activation.

One of the primary benefits for using the epidermal equivalent model in this project is to allow treatment of ex vivo human keratinocytes in a model relevant to human skin, with AhR ligands. The characteristics of this model allow cell differentiation and homeostasis to be observed by development and phenotype (H&E)(Ponec et al., 2000). Formation of the epidermal equivalent is dependent upon normal homeostasis in cell culture, with a normal balance of proliferation, differentiation and cell death. Any changes in this homeostasis can be seen in changes to the epidermal equivalent phenotype by H&E (thinner VCL, parakeratosis, holes in cell layers) and these can be further defined by IHC to provide specific measurement and localisation of proteins.