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Effects of UVR and UCA application were examined in relation to LC frequency within the epidermis. IVA1 (340-400nm) irradiation, or <i>trans</i>-UCA application, did not alter the LC numbers within the exposed site. UVB (280-315nm) irradiation and <i>cis</i>-UCA application depleted LC, and timecourses for LC depletion were established for both treatments. Injection of antibodies against either IL-1b or TNF-a before UVB irradiation or <i>cis</i>-UCA treatment completely abrogated their effects on LC numbers. Thus, the UVB-mediated reduction of LC is dependent on the cytokines IL-1b and TNF-a. Despite reports that UVA1 irradiation protects mice from subsequent immunosuppression by UVB exposure, UVA1 irradiation did not affect the decrease in LC numbers induced by UVB. Despite the differences in effects on LC frequency, both UVA1 and UVB induced an accumulation of DC within lymph nodes draining the site of irradiation. For both irradiation regimens, the accumulation of DC was dependent on IL-1b. This was identified by injecting neutralising IL-1b antibodies before irradiation, which inhibited the accumulation of DC within draining lymph nodes following irradiation. Similar experiments indicated that accumulation of DC following UVB irradiation, but not following UVA1 irradiation, was also dependent upon TNF-a. Induction of cytokines within irradiated skin was examined. UVB exposure increased the expression of IL-10 and TNF-a proteins at the irradiated site. Attempts to identify the source of these cytokines were inconclusive, as both keratinocyte (PAM-212) and melanocyte (B16) cell lines failed to secrete these cytokines following UVB irradiation. Intracellular stores of TNF-a decreased as the dose of radiation increased. The technique of reverse transcription-polymerase chain reaction (RT-PCR) was established to examine expression of cytokine mRNA in irradiated skin. Following UVB irradiation, TNF-a mRNA was upregulated and there was induction of IL-10 mRNA. UVA1 irradiation did not result in such changes. There were also differences in the timecourse of IL-1b mRNA upregulation.

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Coordinated cell migration is essential for tissue regeneration and· development. During migration cells sense various directional cues from the surrounding environment, and intracellular mechanisms process this extracellular information resulting in appropriate vectorial cell movement. During corneal epithelial maintenance, cells migrate centripetally from the limbal region towards the centre of the cornea. This centripetal movement is genetically controlled by Pax6 (Collinson et aI., 2004), one of the master genes in eye development (Kozmik, 2005). The guidance cues and cellular mechanisms controlling centripetal migration of corneal epithelial cells remain to be elucidated. Clarifying the mechanisms guiding cell migration in an experimentally accessible model of corneal epithelial cells would be relevant to mechanisms controlling vectorial cell migration in other cell types. In this project the roles of endogenous electrical and chemical cues in directing corneal epithelial cell migration were tested using in-vitro and ex-vivo culture systems in which migration was induce by wounding. It was ~hown that Pax6+1 - cells suffer from a 2 h wound healing delay, but at later time points, the migration is not significantly different from pax~/+ cells (Leiper et aI., 2006). Additionally Pax6+1 - cells showed defective glycoprotein trafficking which is als~ affecting tJ-le molecules not directly controlled by Pax6 at the transcriptional level and potentially increasing the number of molecular defects contributing to the Pax6+1 - phenotype.. An ex-vivo whole eye system was developed which subsequently demonstrated growth factor dependent woiInd healing defect of pax~/- cells, in contrast to Pax6+1+ epithelia which healed and migrated normally regardless externally supplied growth factors. Exogenous application ofEGF was found to restore wound healing in pax~/cells to wild-type levels. The extensive part of the work was focused on elucidating the role of electrical guidance in wound healing and migration. Recent publications have established roles for endogenous electric current in guiding epithelial ceIl migration (Zhao et aI., 2006)but until this project, there was no genetic model 'of defective 'wound-induced currents. paxrt'l- eyes were found to have compromised or reversed wound mediated currents and were therefore used as model to investigate the importance of electrical cues for normal wound healing. The study represented the most rigorous challenge yet to the hypothesised roles of endogenous electric fields, and surprisingly the analysis showed no significant correlation between electrical cues and directional migration, based on the observation that Pax6+1 - with defective signals healed at rates not significantly different from control wild type epithelia. Further investigation showed no significant correlation ofelectric current and wound healing even in Pax6+1+eyes. In searching for molecular mechanis1J1s controlling directional migration in applied EFs, Pax6+1 - cells were used as model because the cells previously showed compromised or reverse response to in-vitro applied EFs. Src was identified as a candidate signalling molecule playing an important role in mediating directional migration and additionally failure of pSrc to polarise in paxrt'l- cells in applied electric fields correlated with their impaired migration response. Finally wound healing acceleration by addition of Shh peptide was shown to be Pax6 dosage dependent. Investigation of Hh pathway components in Pax6+1+ and paxrt'l- corneal epithelial cells suggested important differences at the level of Gli transcription factors which may underlie the failure of paxrt'l- cell~ to show a migratory respOIise to exogenous Shh.

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No description available.