In vitro models for investigating keratinocyte responses to ultraviolet B radiation

This thesis describes the use of 2- and 3-dimensional cell-based models for studying how skin cells respond to ultraviolet radiation. These methods were used to investigate skin damage and repair after exposure to radiation in the context of skin cancer development. Interactions between different skin cell types were demonstrated as being significant in protecting against ultraviolet radiation-induced skin damage. This has important implications in understanding how skin cancers occur, as well as in the development of new strategies to prevent and treat them.

Characterisation of a human skin equivalent model to study the effects of ultraviolet B radiation on keratinocytes

The incidences of skin cancers resulting from chronic ultraviolet radiation (UVR) exposure are on the incline both in Australia and globally. Hence, the cellular and molecular pathways associated with UVR-induced photocarcinogenesis urgently need to be elucidated, in order to develop more robust preventative and treatment strategies against skin cancers. In vitro investigations into the effects of UVR (in particular the highly-mutagenic UVB wavelength) have, to date, mainly involved the use of cell culture and animal models. However, these models possess biological disparities to native skin, which to some extent have limited their relevance to the in vivo situation. To address this, we characterised a 3-dimensional, tissue-engineered human skin equivalent (HSE) model (consisting of primary human keratinocytes cultured on a dermal-derived scaffold) as a representation of a more physiologically-relevant platform to study keratinocyte responses to UVB. Significantly, we demonstrate that this model retains several important epidermal properties of native skin. Moreover, UVB-irradiation of the HSE constructs was shown to induce key markers of photodamage in the HSE keratinocytes, including the formation of cyclobutane pyrimidine dimers, the activation of apoptotic pathways, the accumulation of p53 and the secretion of inflammatory cytokines. Importantly, we also demonstrate that the UVB-exposed HSE constructs retain the capacity for epidermal repair and regeneration following photodamage. Together, our results demonstrate the potential of this skin equivalent model as a tool to study various aspects of the acute responses of human keratinocytes to UVB radiation damage.

In vitro investigations on the effect of dermal fibroblasts on keratinocyte responses to ultraviolet B radiation

Exposure to ultraviolet radiation is closely linked to the development of skin cancers in humans. The ultraviolet B (UVB) radiation wavelength (280-320 nm), in particular, causes DNA damage in epidermal keratinocytes, which are linked to the generation of signature premalignant mutations. Interactions between dermal fibroblasts and keratinocytes play a role in epidermal repair and regeneration after UVB-induced damage. To investigate these processes, established two and three-dimensional culture models were utilized to study the impact of fibroblast-keratinocyte crosstalk during the acute UVB response. Using a coculture system it was observed that fibroblasts enhanced keratinocyte survival and the repair of cyclobutane pyrimidine dimers (CPDs) after UVB radiation exposure. These findings were also mirrored in irradiated human skin coculture models employed in this study. Fibroblast coculture was shown to play a role in the expression and activation of members of the apoptotic cascade, including caspase-3 and Bad. Interestingly, the expression and phosphorylation of p53, a key player in the regulation of keratinocyte cell fate postirradiation, was also shown to be influenced by fibroblast-produced factors. This study highlights the importance of synergistic interactions between fibroblasts and keratinocytes in maintaining a functional epidermis while promoting repair and regeneration following UVB radiation-induced damage.

Luminescence studies of Ce : YAG using vacuum ultraviolet synchrotron radiation

Photoluminescence spectrum of Ce:YAG single crystal was studied employing vacuum ultraviolet (VUV) synchrotron radiation. Intrinsic absorption edge at about 52,000 cm(-1) was observed in the absorption spectrum. From the VUV excitation spectrum, the energy of the highest d-component of 53,191 cm(-1) (188 nm) for the Ce3+ ions in YAG was obtained at 300 K. The disappearance of the third 5d level at 37,735 cm(-1) (265 nm) in absorption and excitation spectra in our samples may be due to the impurity Fe3+ ions absorption. (C) 2006 Elsevier Ltd. All rights reserved.

Generation of bright, extreme-ultraviolet harmonic radiation from a krypton fluoride laser

We present results of experiments studying the efficiency of high harmonic generation from a gas target using the TITANIA krypton fluoride laser at the Rutherford Appleton Laboratory. The variation of harmonic yield for the 7th to 13th harmonics (355-191 Angstrom) is studied as a function of the backing pressure of a solenoid valve gas jet and of the axial position of the laser focus relative to the centre of the gas jet nozzle. Harmonic energies up to 1 mu J were produced in helium and neon targets from laser energies of approximately 200 mJ. This corresponds to absolute conversion efficiencies of up to 5 x 10(-6).

The effect of pantethine and ultraviolet-B radiation on the development of lenticular opacity in the emory mouse.

PURPOSE: Few studies have examined the impact of long-term treatments or exposures on the development of cataract in maturity-onset animal models. We studied the effect of treatment with D-pantethine and exposure to ultraviolet-B (UVB) radiation on the development of lenticular opacity in the Emory mouse. METHODS: A total of 164 Emory mice were randomized by litter at weaning to exposure to UVB light at 12 mJ/cm(2) for 6 hr/day (UV) or usual room light (A), and within litter, were further randomized to bi-weekly intra-peritoneal injections of 0.8 g/kg pantethine (T) or no treatment (C). Retro illumination lens photos were taken at 2, 4, 6, 8, and 10 months after weaning, and graded in masked fashion. The animals were sacrificed at 10 months and the lenses analyzed for total pantethine and total cysteamine. RESULTS: Lens pantethine and cysteamine levels were significantly (P < 0.001) higher for the T as compared to C litters. Mean cataract grade increased monotonically over time for all four groups. Unadjusted mean grade for the AT group at 8 (1.32) and 10 (1.86) months appeared lower than for the other groups (AC: 2.17, 2.39; UVC: 1.77, 2.40; UVT: 1.88, 2.37). However, the mean grade for the pantethine-treated litters did not differ significantly from the untreated litters except at 2 months (when untreated litters had significantly lower grades), when adjusting for UV treatment, gender and litter effect. No significant difference in cataract score existed between UV-exposed and ambient litters. Mortality was higher among pantethine-treated (hazard ratio = 1.8, p = 0.05) and UV-exposed animals (hazard ratio = 1.8, p = 0. 03) than among the untreated and unexposed litters. CONCLUSION: Significantly increased lens levels of pantethine are achieved with long-term intra-peritoneal dosing. The impact of pantethine on the progression of lenticular opacity in the Emory mouse is less than has been reported in other models. This level of chronic UVB exposure appeared to have no effect on the development of cataract in this model.

Solar ultraviolet-B radiation and vitamin D: a cross-sectional population-based study using data from the 2007 to 2009 Canadian Health Measures Survey

Background: Exposure to solar ultraviolet-B (UV-B) radiation is a major source of vitamin D3. Chemistry climate models project decreases in ground-level solar erythemal UV over the current century. It is unclear what impact this will have on vitamin D status at the population level. The purpose of this study was to measure the association between ground-level solar UV-B and serum concentrations of 25-hydroxyvitamin D (25(OH)D) using a secondary analysis of the 2007 to 2009 Canadian Health Measures Survey (CHMS). Methods: Blood samples collected from individuals aged 12 to 79 years sampled across Canada were analyzed for 25(OH)D (n=4,398). Solar UV-B irradiance was calculated for the 15 CHMS collection sites using the Tropospheric Ultraviolet and Visible Radiation Model. Multivariable linear regression was used to evaluate the association between 25(OH)D and solar UV-B adjusted for other predictors and to explore effect modification. Results: Cumulative solar UV-B irradiance averaged over 91 days (91-day UV-B) prior to blood draw correlated significantly with 25(OH)D. Independent of other predictors, a 1 kJ/m 2 increase in 91-day UV-B was associated with a significant 0.5 nmol/L (95% CI 0.3-0.8) increase in mean 25(OH)D (P =0.0001). The relationship was stronger among younger individuals and those spending more time outdoors. Based on current projections of decreases in ground-level solar UV-B, we predict less than a 1 nmol/L decrease in mean 25(OH)D for the population. Conclusions: In Canada, cumulative exposure to ambient solar UV-B has a small but significant association with 25(OH)D concentrations. Public health messages to improve vitamin D status should target safe sun exposure with sunscreen use, and also enhanced dietary and supplemental intake and maintenance of a healthy body weight.

Growth and physiological responses of sunflower plants exposed to ultraviolet-B radiation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Sensitivity of yellow passion fruit plants to ultraviolet-B radiation

The objective of this work was to evaluate the effects of UV-B radiation on the vegetative growth and on the gas exchange characteristics of passion fruit plants (Passiflora edulis) grown in greenhouse. The average unweighted UV-B radiation near the apex of the plants was 8 W m-2 for the UV-B treatment (high UV-B), and 0.8 W m-2 for the control plants (low UV-B). Plants were irradiated with UV-B for 7 hours per day, centered on solar noon, during 16 days. High UV-B radiation resulted in lower shoot dry matter accumulation per plant. The content of UV-B absorbing compounds and anthocyanins was increased in the plants exposed to high UV-B radiation, when compared with the control. UV-B radiation did not affect stomatal conductance or transpiration rate, but reduced photosynthesis and instantaneous water‑use efficiency, and increased intercellular CO2 concentration. The accumulation of UV-B-absorbing compounds and anthocyanins did not effectively shield plants from supplementary UV-B radiation, since the growth and photosynthetic processes were significantly reduced.

The effects of ultraviolet B radiation on immune responses to {\it Borrelia burgdorferi}, the causative agent of Lyme disease

Ultraviolet B (UVB) radiation, in addition to being carcinogenic, is also immunosuppressive. Immunologically, UVB induces suppression locally, at the site of irradiation, or systemically, by inducing the production of a variety of immunosuppressive cytokines. Systemic effects include suppression of delayed-type hypersensitivity (DTH) responses to a variety of antigens (e.g. haptens, proteins, bacterial antigens, or alloantigens). One of the principal mediators of UV-induced immune suppression is the T helper-2 (Th2) cytokine interleukin-10 (IL-10); this suggests that UV irradiation induces suppression by shifting the immune response from a Th1 (cellular) to a Th2 (humoral) response. These "opposing" T helper responses are usually mutually exclusive, and polarized Th1 or Th2 responses may lead to either protection from infection or increased susceptibility to disease, depending on the infectious agent and the route of infection.^ This study examines the effects of UVB irradiation on cellular and humoral responses to Borrelia burgdorferi (Bb), the causative agent of Lyme disease (LD) in both immunization and infectious disease models; in addition, it examines the role of T cells in protection from and pathology of Bb infection. Particular emphasis is placed on the Bb-specific antibody responses following irradiation since UVB effects on humoral immunity are not fully understood. Mice were irradiated with a single dose of UV and then immunized (in complete Freund's adjuvant) or infected with Bb (intradermally at the base of the tail) in order to examine both DTH and antibody responses in both systems. UVB suppressed the Th1-associated antibodies IgG2a and IgG2b in both systems, as well as the DTH response to Bb in a dose dependent manner. Injection of anti-IL-10 antibody into UV-irradiated mice within 24 h after UV exposure restored the DTH response, as well as the Th1 antibody (IgG2a and IgG2b) response. In addition, injecting recombinant IL-10 mimicked some of the effects of UV radiation.^ Bb-specific Th1 T cell lines (BAT2.1-2.3) were generated to examine the role of T cells in Lyme borreliosis. All lines were CD4$\sp+,$ $\alpha\beta\sp+$ and proliferated specifically in response to Bb. The BAT2 cell lines not only conferred a DTH response to naive C3H recipients, but reduced the number of organisms recovered from the blood and tissues of mice infected with Bb. Furthermore, BAT2 cell lines protected mice from Bb-induced periarthritis. ^

Investigation of mechanisms involved in ultraviolet B radiation-induced, T cell -mediated immune suppression

Cutaneous exposure to ultraviolet-B radiation (UVR) results in the suppression of cell-mediated immune responses such as contact hypersensitivity (CHS) and delayed-type hypersensitivity (DTH). This modulation of immune responses is mediated by local or systemic mechanisms, both of which are associated with the generation of antigen-specific suppressor T lymphocytes (Ts). UV-induced Ts have been shown to be CD3+CD4+CD8 − T cells that control multiple immunological pathways. However, the precise mechanisms involved in the generation and function of these immunoregulatory cells remain unclear. We investigated the cellular basis for the generation of UV-induced Ts lymphocytes in both local and systemic models of immune suppression, and further examined the pleiotrophic function of these immunoregulatory cells. ^ We used Thy1.1 and Thy1.2 congenic mice in a draining lymph node (DLN) cell transfer model to analyze the role played by epidermal Langerhans cells in the generation of Ts cells. We demonstrate that T cells tightly adhered to antigen-presenting cells (APC) from UV-irradiated skin are the direct progenitors of UV-induced Ts lymphocytes. Our studies also reveal that UV-induced DNA-damage in the form of cyclobutyl pyrimidine dimers (CPD) in the epidermal APC is crucial for the altered maturation of these adherent T cells into Ts. ^ We used TCR transgenic mice in an adoptive transfer model and physically tracked the antigen-specific clones during immune responses in unirradiated versus UV-irradiated mice. We demonstrate that UV-induced Ts and effector TDTH cells share the same epitope specificity, indicating that both cell populations arise from the same clonal progenitors. UVR also causes profound changes in the localization and proliferation of antigen-specific T cells during an immune response. Antigen-specific T cells are not detectable in the DLNs of UV-irradiated mice after 3 days post-immunization, but are found in abundance in the spleen. In contrast, these clones continue to be found in the DLNs and spleens of normal animals several days post-immunization. Our studies also reveal that a Th2 cytokine environment is essential for the generation of Ts in UV-irradiated mice. ^ The third part of our study examined the pleiotrophic nature of UV-induced Ts. We used a model for the induction of both cellular and humoral responses to human gamma-globulin (HGG) to demonstrate that UV-induced Ts lymphocytes can suppress DTH as well as antibody responses. (Abstract shortened by UMI.) ^

Stability of nonrelativistic quantum mechanical matter coupled to the (ultraviolet cutoff) radiation field

We announce a proof of H-stability for the quantized radiation field, with ultraviolet cutoff, coupled to arbitrarily many non-relativistic quantized electrons and static nuclei. Our result holds for arbitrary atomic numbers and fine structure constant. We also announce bounds for the energy of many electrons and nuclei in a classical vector potential and for the eigenvalue sum of a one-electron Pauli Hamiltonian with magnetic field.

Ultraviolet-B Radiation Effects on Water Relations, Leaf Development, and Photosynthesis in Droughted Pea Plants1

The effects of ultraviolet-B (UV-B) radiation on water relations, leaf development, and gas-exchange characteristics in pea (Pisum sativum L. cv Meteor) plants subjected to drought were investigated. Plants grown throughout their development under a high irradiance of UV-B radiation (0.63 W m−2) were compared with those grown without UV-B radiation, and after 12 d one-half of the plants were subjected to 24 d of drought that resulted in mild water stress. UV-B radiation resulted in a decrease of adaxial stomatal conductance by approximately 65%, increasing stomatal limitation of CO2 uptake by 10 to 15%. However, there was no loss of mesophyll light-saturated photosynthetic activity. Growth in UV-B radiation resulted in large reductions of leaf area and plant biomass, which were associated with a decline in leaf cell numbers and cell division. UV-B radiation also inhibited epidermal cell expansion of the exposed surface of leaves. There was an interaction between UV-B radiation and drought treatments: UV-B radiation both delayed and reduced the severity of drought stress through reductions in plant water-loss rates, stomatal conductance, and leaf area.

Folate degradation due to ultraviolet radiation : possible implications for human health and nutrition

Folate is essential for human health in the prevention of megaloblastic anaemia and neural tube birth defects as well as roles in cardiovascular disease and cancer. Therefore research into environmental factors that may impact folate status, such as solar ultraviolet radiation, is of great health significance. In vitro studies have shown that ultraviolet (UV) radiation can degrade folate and folic acid in human blood and this has been confirmed in several human studies. Despite these findings, there is a dearth of epidemiological research into investigating the relationship between folate status and the links to solar UV exposure.