Introduction The aim of this study was to compare the effects of tumour necrosis factor-alpha (TNF-α) and interleukin-1-beta (IL-1β) on protease and catabolic cytokine and receptor gene expression in normal and degenerate human nucleus pulposus cells in alginate culture.

Excessive ultraviolet radiation (UVR) exposure of the skin is associated with adverse clinical outcomes. Although both exogenous sunscreens and endogenous tissue components (including melanins and tryptophan-derived compounds) reduce UVR penetration, the role of endogenous proteins in absorbing environmental UV wavelengths is poorly defined. Having previously demonstrated that proteins which are rich in UVR-absorbing amino acid residues are readily degraded by broadband UVB-radiation (containing UVA, UVB and UVC wavelengths) here we hypothesised that UV chromophore (Cys, Trp and Tyr) content can predict the susceptibility of structural proteins in skin and the eye to damage by physiologically relevant doses (up to 15.4 J/cm2) of solar UVR (95% UVA, 5% UVB). We show that: i) purified suspensions of UV-chromophore-rich fibronectin dimers, fibrillin microfibrils and β- and γ-lens crystallins undergo solar simulated radiation (SSR)-induced aggregation and/or decomposition and ii) exposure to identical doses of SSR has minimal effect on the size or ultrastructure of UV chromophore-poor tropoelastin, collagen I, collagen VI microfibrils and α-crystallin. If UV chromophore content is a factor in determining protein stability in vivo, we would expect that the tissue distribution of Cys, Trp and Tyr-rich proteins would correlate with regional UVR exposure. From bioinformatic analysis of 244 key structural proteins we identified several biochemically distinct, yet UV chromophore-rich, protein families. The majority of these putative UV-absorbing proteins (including the late cornified envelope proteins, keratin associated proteins, elastic fibre-associated components and β- and γ-crystallins) are localised and/or particularly abundant in tissues that are exposed to the highest doses of environmental UVR, specifically the stratum corneum, hair, papillary dermis and lens. We therefore propose that UV chromophore-rich proteins are localised in regions of high UVR exposure as a consequence of an evolutionary pressure to express sacrificial protein sunscreens which reduce UVR penetration and hence mitigate tissue damage.

The dermal elastic fibre network is the primary effector of skin elasticity, enabling it to extend and recoil many times over the lifetime of the individual. Fibrillin‐rich microfibrils (FRMs) constitute integral components of the elastic fibre network, with their distribution showing differential deposition in the papillary dermis across individuals of diverse skin ethnicity. Despite these differential findings in histological presentation, it is not known if skin ethnicity influences FRM ultrastructure. FRMs are evolutionarily highly conserved from jellyfish to man and, regardless of tissue type or species, isolated FRMs have a characteristic ‘beads‐on‐a‐string’ ultrastructural appearance, with an average inter‐bead distance (or periodicity) of 56 nm. Here, skin biopsies were obtained from the photoprotected buttock of healthy volunteers (18‐27 years; African: n = 5; European: n = 5), and FRMs were isolated from the superficial papillary dermis and deeper reticular dermis and imaged by atomic force microscopy. In the reticular dermis, there was no significant difference in FRM ultrastructure between European and African participants. In contrast, in the more superficial papillary dermis, inter‐bead periodicity was significantly larger for FRMs extracted from European participants than from African participants by 2.20 nm (p < .001). We next assessed whether these differences in FRM ultrastructure were present during early postnatal development by characterizing FRMs from full‐thickness neonatal foreskin. Analysis of FRM periodicity identified no significant difference between neonatal cohorts (p = .865). These data suggest that at birth, FRMs are developmentally invariant. However, in adults of diverse skin ethnicity, there is a deviation in ultrastructure for the papillary dermal FRMs that may be acquired during the passage of time from child to adulthood. Understanding the mechanism by which this difference in papillary dermal FRMs arises warrants further study.

28Langerhans cells (LC) are sentinels of skin's immune system, their loss from epidermis 29 contributing to UVR-suppression of cell mediated immunity (CMI). Omega-3 polyunsaturated 30 fatty acids can show potential to abrogate UVR-suppression of CMI in mice and humans, 31 potentially through modulation of LC migration. Our objectives were to examine if 32 eicosapentaenoic acid (EPA) ingestion influences UV-mediated effects on epidermal LC 33 numbers and levels of immunomodulatory mediators including prostaglandin (PG)D 2 , which 34 is expressed by LC. 35In a double-blind randomised controlled study, healthy individuals took 5g EPA-rich 36 (n=40) or control (n=33) lipid for 12-weeks; UVR exposed and unexposed skin samples were 37 taken pre-and post-supplementation. Epidermal LC numbers were assessed by 38 immunofluorescence for CD1a, and skin blister fluid PG and cytokines quantified by LC-39 MS/MS and Luminex assay, respectively. Pre-supplementation, UVR reduced mean (SEM) 40LC number/mm 2 from 913 (28) to 322 (40) (p<0.001), and mean PGD 2 level by 37% from 8.1 41 (11.6) to 5.1 (5.6) pg/µl; p<0.001), while IL-8 level increased (p<0.001). Despite confirmation 42 of EPA bioavailability in red blood cells and skin in the active group, no between-group effect 43 of EPA was found on UVR-modulation of LC numbers, PGD 2 or cytokine levels post-44 supplementation. 45

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The efficacy of topical sunscreens is currently assessed by crude, costly and time consuming in vivo assays. We have previously demonstrated that components of the dermal extracellular matrix (ECM), rich in UV-absorbing amino acids, are susceptible to damage by solar simulated radiation (SSR) in vitro. Here we developed an in vitro method to test the ability of sunscreens to protect fibrillin-rich microfibrils (FRM) and fibronectin, key components of the dermal ECM from UV-induced damage. Solutions of FRM or fibronectin were irradiated without protection, in the presence of a vehicle or a commercially-available flat-spectrum sunscreen. The effect of SSR on molecular structure was determined by atomic force microscopy (FRM) and SDS-PAGE (fibronectin). Following irradiation, FRM periodicity became bi-modally distributed (peaks: 40nm & 59nm) compared to the unimodal distribution in unexposed controls (peak: 50nm). Irradiation in the presence of flat-spectrum sunscreen protected against this change, maintaining the unimodal distribution. SSR induced significant aggregation of fibronectin (p=0.005), which was abrogated by sunscreen. These results demonstrate that this in vitro assay system is sufficiently sensitive to act as an initial/additional screen of sunscreen efficacy. We conclude that sunscreen can reduce UV-mediated damage of key dermal ECM in vitro and thereby prevent remodelling associated with photoageing.

Fibrillin-rich microfibrils (FRMs) constitute integral components of the dermal elastic fibre network with a distinctive ultrastructural 'beads-on-a-string' appearance that can be visualised using atomic force microscopy and characterised by measurement...

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