Decellularised tissues obtained by a CO2-philic detergent and supercritical CO2.

Tissue decellularisation has gained much attention in regenerative medicine as an alternative to synthetic materials. In decellularised tissues, biological cues can be maintained and provide cellular environments still unmet by synthetic materials. Supercritical CO2 (scCO2 ) has recently emerged as a promising alternative decellularisation technique to aggressive detergents; in addition, scCO2 provides innate sterilisation. However, to date, decellularisation with scCO2 is limited to only a few tissue types with low cellular density. In the current study, a scCO2 technique to decellularise high density tissues, including articular cartilage, tendon and skin, was developed. Results showed that most of the cellular material was removed, while the sample structure and biocompatibility was preserved. The DNA content was reduced in cartilage, tendon and skin as compared to the native tissue. The treatment did not affect the initial tendon elastic modulus [reduced from 126.35 ± 9.79 MPa to 113.48 ± 8.48 MPa (p 〉 0.05)], while it reduced the cartilage one [from 12.06 ± 2.14 MPa to 1.17 ± 0.34 MPa (p 〈 0.0001)]. Interestingly, cell adhesion molecules such as fibronectin and laminin were still present in the tissues after decellularisation. Bovine chondrocytes were metabolically active and adhered to the surface of all decellularised tissues after 1 week of cell culture. The developed method has the potential to become a cost-effective, one-step procedure for the decellularisation of dense tissues.

Zone-dependent mechanical properties of human articular cartilage obtained by indentation measurements.

Emerging 3D printing technology permits innovative approaches to manufacture cartilage scaffolds associated with layer-by-layer mechanical property adaptation. However, information about gradients of mechanical properties in human articular cartilage is limited. In this study, we quantified a zone-dependent change of local elastic modulus of human femoral condyle cartilage by using an instrumented indentation technique. From the cartilage superficial zone towards the calcified layer, a gradient of elastic modulus values between 0.020 ± 0.003 MPa and 6.44 ± 1.02 MPa was measured. To validate the tissue quality, the histological tissue composition was visualized by glycosaminoglycan and collagen staining. This work aims to introduce a new protocol to investigate the zone-dependent mechanical properties of graded structures, such as human articular cartilage. From this knowledge, better cartilage repair strategies could be tailored in the future.

Bench-top validation testing of selected immunological and molecular Renibacterium salmoninarum diagnostic assays by comparison with quantitative bacteriological culture.

No gold standard assay exhibiting error-free classification of results has been identified for detection of Renibacterium salmoninarum, the causative agent of salmonid bacterial kidney disease. Validation of diagnostic assays for R. salmoninarum has been hindered by its unique characteristics and biology, and difficulties in locating suitable populations of reference test animals. Infection status of fish in test populations is often unknown, and it is commonly assumed that the assay yielding the most positive results has the highest diagnostic accuracy, without consideration of misclassification of results. In this research, quantification of R. salmoninarum in samples by bacteriological culture provided a standardized measure of viable bacteria to evaluate analytical performance characteristics (sensitivity, specificity and repeatability) of non-culture assays in three matrices (phosphate-buffered saline, ovarian fluid and kidney tissue). Non-culture assays included polyclonal enzyme-linked immunosorbent assay (ELISA), direct smear fluorescent antibody technique (FAT), membrane-filtration FAT, nested polymerase chain reaction (nested PCR) and three real-time quantitative PCR assays. Injection challenge of specific pathogen-free Chinook salmon, Oncorhynchus tshawytscha (Walbaum), with R. salmoninarum was used to estimate diagnostic sensitivity and specificity. Results did not identify a single assay demonstrating the highest analytical and diagnostic performance characteristics, but revealed strengths and weaknesses of each test.

Bone regeneration and stem cells.

This invited review covers research areas of central importance for orthopaedic and maxillofacial bone tissue repair, including normal fracture healing and healing problems, biomaterial scaffolds for tissue engineering, mesenchymal and foetal stem cells, effects of sex steroids on mesenchymal stem cells, use of platelet-rich plasma for tissue repair, osteogenesis and its molecular markers. A variety of cells in addition to stem cells, as well as advances in materials science to meet specific requirements for bone and soft tissue regeneration by addition of bioactive molecules, are discussed.

Effective Management Of Topical Nosocomial Aspergillus Spp. Infections In Three Severely Burned Patients.

Nosocomial opportunistic fungal infections by Aspergillus spp. represent increasing morbidity and mortality factors for severely burned patients, who are fragile and immunocompromised. Voriconazole (VRC), a modern antifungal drug, is used as a first-line therapy against systemic mold and yeast infections. Little has been published about the place, relative importance and efficacy of voriconazole in the treatment protocols involving Aspergillus spp. in Burn Centers. The objective of the present work was to assess the place and importance of voriconazole for the treatment of burn patients presenting superficial Aspergillus spp. infections. We performed a retrospective evaluation of VRC treatment in three severely burned patients with superficial nosocomial Aspergillus spp. infections in our Burn Center. Results showed that VRC allowed for control and cure of topical nosocomial Aspergillus spp. infections. In two cases, treatment with VRC had to be discontinued because of hepatotoxicity. In two cases, following or during systemic treatment with VRC, a 1% terbinafine cream was applied to resolve the infection in order to continue standard wound management. Overall, VRC has been shown to be an effective antifungal agent and is an alternative to amphotericin B to fight Aspergillus spp. infections developing in the wounds of severely burned patients.

La survenue d'une aspergillose chez les patients gravement brûlés, dès lors immunodéprimés, est une cause de morbidité et de mortalité. Le voriconazole (VRC) est un antifongique utilisé en première intention dans le traitement des infections à moisissures. La littérature est pauvre au sujet de son utilisation dans l'aspergillose chez le brûlé. Cette étude a pour but de l 'évaluer dans le traitement de l'aspergillose cutanée chez le brûlé et a consisté en l'évaluation rétrospective de la prise en charge de trois patients de notre CTB, gravement brûlés et victimes d'une aspergillose cutanée. VRC en a permis la guérison, mais a dû être suspendu 2 fois en raison d'une toxicité hépatique. Dans 2 cas, il a été associé à de la crème de terbinafine à 1%. Le traitement habituel a pu être repris après guérison de l'aspergillose. Globalement, VRC semble efficace et représente une alternative à l'amphotéricine B dans le traitement de l'aspergillose cutanée chez les brûlés.

Identification of the molecular target for the suppression of contact hypersensitivity by ultraviolet radiation.

This study was conducted to explore the involvement of DNA damage in the suppression of contact hypersensitivity (CHS) by UV irradiation. The opossum, Monodelphis domestica, was used because cells of these marsupials have an enzyme that is activated by visible light (photoreactivating enzyme) and repairs ultraviolet radiation (UVR)-induced pyrimidine dimers in DNA. A single dose of 1,500 J/m2 of UVB (280-320 nm) radiation, representing 2 minimal erythema doses, was administered to the dorsal skin of opossums. This treatment prevented the opossums from developing a CHS response to dinitrofluorobenze (DNFB) applied either at the site of irradiation or an unirradiated site. In addition, this dose of UVR decreased the number of ATPase+ epidermal Langerhans cells in the dorsal epidermis to approximately 3% of that in unirradiated skin at the time of DNFB application. Treatment of the animals with wavelengths that activate the repair enzyme (320-500 nm, photoreactivating light, PRL) for 120 min immediately after UV irradiation inhibited the UVR-induced suppression of CHS almost completely. Exposure to PRL before UVR did not prevent UVR-induced suppression of CHS. PRL treatment after UV irradiation also prevented the decrease in the number of ATPase+ Langerhans cells. Measurements of lesions in DNA indicated that PRL treatment removed around 85% of the UVR-induced pyrimidine dimers. These data provide direct evidence that DNA, and most likely, the pyrimidine dimer, is the primary molecular target for the UVB-induced suppression of contact hypersensitivity to haptens applied to irradiated or unexposed skin.

Whole-cell bioprocessing of human fetal cells for tissue engineering of skin.

Current restrictions for human cell-based therapies have been related to technological limitations with regards to cellular proliferation capacity (simple culture conditions), maintenance of differentiated phenotype for primary human cell culture and transmission of communicable diseases. Cultured primary fetal cells from one organ donation could possibly meet the exigent and stringent technical aspects for development of therapeutic products. Master and working cell banks from one fetal organ donation (skin) can be developed in short periods of time and safety tests can be performed at all stages of cell banking. For therapeutic use, fetal cells can be used up to two thirds of their life-span in an out-scaling process and consistency for several biological properties includes protein concentration, gene expression and biological activity. As it is the intention that banked primary fetal cells can profit from the prospected treatment of hundreds of thousands of patients with only one organ donation, it is imperative to show consistency, tracability and safety of the process including donor tissue selection, cell banking, cell testing and growth of cells in out-scaling for the preparation of whole-cell tissue-engineering products.

Acceptation of Folk Medicine and its "secrets" in a Swiss Burn Centre.

In Switzerland 'Secret' is a folk medicine called upon for burns. It has belonged to UNESCO's intangible cultural heritage since 2012. It is supposed to ease pain and accelerate the healing process of burns. As the practice is widely used in the population, this observational study investigated the opinion of caregivers and patients from the National Burn Center of Lausanne. Qualitative observational study based on a survey including ten questions aimed at identifying the professionals' perception of the phenomenon. Questions were developed from repeated encounters in the burn center. Data collection took five months. Thirty-six healthcare professionals (HP) and 12 selected patients (or parents for minors) discharged after burns were interviewed on a voluntary basis: all of the HPs knew about 'Secret' from the workplace, and 26 from home: 33 were convinced that it might be useful and reduce pain. The perceived efficiency of the practice (36 respondents) differs depending on professional category and personal experience. Only one HP considered the practice to be dangerous. The nurses and auxiliary nurses expressed that it should be used more widely. The 12 patients considered it as a complementary step, not a replacement for medical care. Health professionals globally considered this practice safe and helpful. The patients were interested in using parallel approaches and were careful about their expectations. This openness is probably an indication that HPs believe that acceptance of the culture and beliefs of patients and their families might positively affect response to treatment, whatever the burn size.

Il existe en Suisse une médecine traditionnelle dénommée « secret ¼ dédiée aux brûlures (supposée avoir des effets analgésiques et cicatrisants) inscrite au patrimoine immatériel de l'UNESCO depuis 2012. Dans la mesure où elle est très largement utilisée, nous avons conduit une étude observationnelle sur l'opinion qu'en ont les soignants et les patients du CTB national de Lausanne. Nous avons utilisé un questionnaire à dix items, développé après des entretiens plus informels. Trente six professionnels et 12 patients (ou parents quand le patient était mineur), interrogés après leur sortie, ont volontairement participé à l'étude. Tous les professionnels avaient entendu parler de « secret ¼ soit au travail soit chez eux (26). Trente trois étaient persuadés de son utilité analgésique, 1 seul le considérant comme dangereux. Cette opinion varie selon la catégorie professionnelle et l'expérience personnelle, les infirmières et aide- soignantes estimant qu'il devrait être plus largement utilisé. Les patients estimaient que « secret ¼ était un adjuvant ne devant pas remplacer la prise en charge médicalisée. Les professionnels considéraient que « secret ¼ est simple et utile. Les patients étaient intéressée par cette approche parallèle, tout en gardant une certaine retenue quant à ce qu'ils pouvaient en attendre. Cette ouverture d'esprit suggère que les professionnels pensent que la prise en compte de la culture et des croyances des patients et de leur famille peut promouvoir l'efficacité du traitement conventionnel, quelle que soit la surface brûlée.

Effect of temporal onsets of mechanical loading on bone formation inside a tissue engineering scaffold combined with cell therapy.

Several approaches to combine bone substitutes with biomolecules, cells or mechanical loading have been explored as an alternative to the limitation and risk-related bone auto- and allo-grafts. In particular, human bone progenitor cells seeded in porous poly(L-lactic acid)/tricalcium phosphate scaffolds have shown promising results. Furthermore, the application of mechanical loading has long been known to be a key player in the regulation of bone architecture and mechanical properties. Several in vivo studies have pointed out the importance of its temporal offset. When an early mechanical loading was applied a few days after scaffold implantation, it was ineffective on bone formation, whereas a delayed mechanical loading of several weeks was beneficial for bone tissue regeneration. No information is reported to date on the effectiveness of applying a mechanical loading in vivo on cell-seeded scaffold with respect to bone formation in a bone site. In our study, we were interested in human bone progenitor cells due to their low immunogenicity, sensitivity to mechanical loading and capacity to differentiate into osteogenic human bone progenitor cells. The latest capacity allowed us to test two different bone cell fates originating from the same cell type. Therefore, the general aim of this study was to assess the outcome on bone formation when human bone progenitor cells or pre-differentiated osteogenic human bone progenitor cells are combined with early and delayed mechanical loading inside bone tissue engineering scaffolds. Scaffolds without cells, named cell-free scaffold, were used as control. Surprisingly, we found that (1) the optimal solution for bone formation is the combination of cell-free scaffolds and delayed mechanical loading and that (2) the timing of the mechanical application is crucial and dependent on the cell type inside the implanted scaffolds.

Oxidant stress leads to transcriptional activation of the human heme oxygenase gene in cultured skin fibroblasts.

Treatment of cultured human skin fibroblasts with near-UV radiation, hydrogen peroxide, and sodium arsenite induces accumulation of heme oxygenase mRNA and protein. In this study, these treatments led to a dramatic increase in the rate of RNA transcription from the heme oxygenase gene but had no effect on mRNA stability. Transcriptional activation, therefore, appears to be the major mechanism of stimulation of expression of this gene by either oxidative stress or sulfydryl reagents.

Induction of heme oxygenase: a general response to oxidant stress in cultured mammalian cells.

Accumulation of heme oxygenase mRNA is strongly stimulated by treatment of cultured human skin fibroblasts with ultraviolet radiation, hydrogen peroxide, or the sulfhydryl reagent sodium arsenite (S. M. Keyse and R. M. Tyrrell. Proc. Natl. Acad. Sci. USA, 86: 99-103, 1989). Since this will result in a transient reduction in the prooxidant state of cells, the phenomenon may represent an important inducible antioxidant defense mechanism. To examine the generality of the response, we have measured the accumulation of the specific mRNA in a variety of human and mammalian cell types after inducing treatments. Induction by sodium arsenite is observed in all additional human cell types tested. This includes primary epidermal keratinocytes and lung and colon fibroblasts as well as established cell lines such as HeLa, TK6 lymphoblastoid, and transformed fetal keratinocytes. Strong induction of heme oxygenase mRNA is also observed following sodium arsenite treatment of cell lines of rat, hamster, mouse, monkey, and marsupial origin. The agents which lead to induction in cultured human skin fibroblasts fall into two categories: (a) those which are oxidants or can generate active intermediates (ultraviolet A radiation, hydrogen peroxide, menadione, and the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate); (b) agents which are known to interact with or modify cellular glutathione levels (buthionine sulfoximine, sodium arsenite, iodoacetamide, diamide, and cadmium chloride). These observations strongly support the hypothesis that induction of the enzyme is a general response to oxidant stress in mammalian cells and are consistent with the possibility that the cellular redox state plays a key role.

Photoreactivation of ultraviolet radiation-induced skin and eye tumors of Monodelphis domestica.

Chronic exposure of the opossum Monodelphis domestica to UV radiation (UVR) leads to the formation of cutaneous and corneal tumors. Groups of shaved opossums were exposed 3 times/week to: (a) UVR alone; (b) UVR followed immediately by 1 h of photoreactivating light (PRL) (320-700 nm); (c) 1 h of PRL followed by UVR; and (d) 1 h of PRL alone. Exposures were terminated after 70 weeks of treatment. Analysis of data plotted as probability of tumor formation versus weeks from first exposure shows that post-UVR exposure to PRL significantly (P less than 0.005) delayed the time to appearance of cutaneous tumors from a 50% probability of tumor formation at 73 weeks for those animals exposed to UVR alone to 128 weeks for those animals exposed to PRL after UVR. Pre-UVR exposure to PRL delayed the appearance of tumors by 6 weeks when compared to the UVR alone group, but the difference between the two groups was not statistically significant. The yield (number of tumors/surviving animal) of cutaneous tumors at 70 and 110 weeks following initiation of treatments also was significantly less in those animals exposed to PRL after, but not before, UVR. Based on the specificity of the PR repair pathway to act only on pyrimidine dimers, these results suggest that dimers are involved in the induction of cutaneous tumors. The results obtained with the induction of corneal tumors are more difficult to interpret. While exposure to PRL significantly delayed the appearance of corneal tumors, the magnitude of the effect was the same regardless of whether the PRL was given before or after each UVR exposure.

Hypersensitivity of skin fibroblasts from basal cell nevus syndrome patients to killing by ultraviolet B but not by ultraviolet C radiation.

Basal cell nevus syndrome (BCNS) is an autosomal dominant genetic disorder in which the afflicted individuals are extremely susceptible to sunlight-induced skin cancers, particularly basal cell carcinomas. However, the cellular and molecular basis for BCNS is unknown. To ascertain whether there is any relationship between genetic predisposition to skin cancer and increased sensitivity of somatic cells from BCNS patients to killing by UV radiation, we exposed skin fibroblasts established from unexposed skin biopsies of several BCNS and age- and sex-matched normal individuals to either UV-B (280-320 nm) or UV-C (254 nm) radiation and determined their survival. The results indicated that skin fibroblasts from BCNS patients were hypersensitive to killing by UV-B but not UV-C radiation as compared to skin fibroblasts from normal individuals. DNA repair studies indicated that the increased sensitivity of BCNS skin fibroblasts to killing by UV-B radiation was not due to a defect in the excision repair of pyrimidine dimers. These results indicate that there is an association between hypersensitivity of somatic cells to killing by UV-B radiation and the genetic predisposition to skin cancer in BCNS patients. In addition, these results suggest that DNA lesions (and repair processes) other than the pyrimidine dimer are also involved in the pathogenesis of sunlight-induced skin cancers in BCNS patients. More important, the UV-B sensitivity assay described here may be used as a diagnostic tool to identify presymptomatic individuals with BCNS.

Human Fetal Progenitor Tenocytes for Regenerative Medicine.

Tendon injuries are very frequent and affect a wide and heterogeneous population. Unfortunately, the healing process is long with outcomes that are not often satisfactory due to fibrotic tissue appearance, which leads to scar and adhesion development. Tissue engineering and cell therapies emerge as interesting alternatives to classical treatments. In this study, we evaluated human fetal progenitor tenocytes (hFPTs) as a potential cell source for treatment of tendon afflictions, as fetal cells are known to promote healing in a scarless regenerative process. hFPTs presented a rapid and stable growth up to passage 9, allowing to create a large cell bank for off-the-shelf availability. hFPTs showed a strong tenogenic phenotype with an excellent stability, even when placed in conditions normally inducing cells to differentiate. The karyotype also indicated a good stability up to passage 12, which is far beyond that necessary for clinical application (passage 6). When placed in coculture, hFPTs had the capacity to stimulate human adult tenocytes (hATs), which are responsible for the deposition of a new extracellular matrix during tendon healing. Finally, it was possible to distribute cells in porous or gel scaffolds with an excellent survival, thus permitting a large variety of applications (from simple injections to grafts acting as filling material). All of these results are encouraging in the development of an off-the-shelf cell source capable of stimulating tendon regeneration for the treatment of tendon injuries.

Burn patient care lost in good manufacturing practices?

Application of cell therapies in burn care started in the early 80s in specialized hospital centers world-wide. Since 2007, cell therapies have been considered as "Advanced Therapy Medicinal Products" (ATMP), so classified by European Directives along with associated Regulations by the European Parliament. Consequently, regulatory changes have transformed the standard linear clinical care pathway into a more complex one. It is important to ensure the safety of cellular therapies used for burn patients and to standardize as much as possible the cell sources and products developed using cell culture procedures. However, we can definitely affirm that concentrating the bulk of energy and resources on the implementation of Good Manufacturing Practice (GMP) alone will have a major negative impact on the care of severely burned patients world-wide. Developing fully accredited infrastructures and training personnel (required by the new directives), along with obtaining approval for clinical trials to go ahead, can be a lengthy process.We discuss whether or not these patients could benefit from cell therapies provided by standard in-hospital laboratories, thus avoiding having to meet rigid regulations concerning the use of industrial pharmaceutical products. "Hospital Exemption" could be a preferred means to offer burn patients a customized and safe product, as many adaptations may be required throughout their treatment pathway. Patients who are in need of rapid treatment will be the ones to suffer the most from regulations intended to help them.

L'utilisation de la « thérapie cellulaire ¼ au profit des patients brûlés s'est mise en place au début des années 1980 dans de nombreux centres, répartis de par le monde. Depuis 2007, les produits utilisés ont fait l'objet de directives européennes. De ce fait, la prise en charge directe du patient est devenue un parcours semé d'embûches. S'il est important d'assurer au patient l'utilisation de produits dérivés de culture cellulaire de qualité, fabriqués selon des procédés reproductibles, il est évident que la mise en place dans les unités des « Bonnes Pratiques de Fabrication ¼ entraînera des dépenses de temps et d'énergie qui auront inévitablement un impact négatif sur la prise en charge du patient très gravement brûlé. En outre, la mise à niveau de l'infrastructure et la formation du personnel (exigées par les directives actuelles) ainsi que l'obtention des essais cliniques nécessaires à l'autorisation d'utilisation de ces produits peuvent s'avérer très longues. Nous argumentons la possibilité de fabriquer ces produits de culture cellulaire dans des laboratoires hospitaliers classiques en évitant la très lourde procédure destinée principalement à l'industrie pharmaceutique. Une « exemption hospitalière ¼ pourrait être un moyen d'offrir aux brûlés une thérapeutique adaptée et sécurisée, dans la mesure où des adaptations personnalisées peuvent être nécessaires au long de leur traitement. Les patients ayant un besoin vital d'un traitement urgent seront ceux qui pâtiront le plus d'une loi sensée les protéger.

Peptide-decorated chitosan derivatives enhance fibroblast adhesion and proliferation in wound healing.

RGD peptide sequences are known to regulate cellular activities by interacting with α5ß1, αvß5 and αvß3 integrin, which contributes to the wound healing process. In this study, RGDC peptide was immobilized onto chitosan derivative 1,6-diaminohexane-O-carboxymethyl-N,N,N-trimethyl chitosan (DAH-CMTMC) to display RGDC-promoting adhesion for enhanced wound healing. The efficiency of N-methylation, O-carboxymethylation and spacer grafting was quantitatively and qualitatively analyzed by (1)H NMR and FTIR, yielding 0.38 degree of substitution for N-methylation and >0.85 for O-carboxymethylation. The glass transition temperatures for chitosan derivatives were also studied. Peptide immobilization was achieved through sulfhydryl groups using sulfosuccinimidyl (4-iodoacetyl)amino-benzoate (sulfo-SIAB method). RGDC immobilized peptide onto DAH-CMTMC was found to be about 15.3 µg/mg of chitosan derivative by amino acid analysis (AAA). The significant increase of human dermal fibroblast (HDF) viability in vitro over 7 days suggests that RGDC-functionalized chitosan may lead to enhanced wound healing (viability >140%). Moreover, bio-adhesion and proliferation assays confirmed that coatings of RGDC-functionalized chitosan derivatives exhibit in vitro wound healing properties by enhancing fibroblast proliferation and adhesion. These results showed that RGDC peptide-functionalized chitosan provides an optimal environment for fibroblast adhesion and proliferation.

Stability Enhancement Using Hyaluronic Acid Gels for Delivery of Human Fetal Progenitor Tenocytes.

Tendon afflictions are very common, and their negative impact is high both at the workplace and in leisure activities. Tendinopathies are increasing in prevalence and can lead to tendon ruptures, where healing is a long process with outcomes that are often disappointing. Human fetal progenitor tenocytes (hFPTs) have been recently tested in vitro as a potential cell source to stimulate tendon regeneration. The aim of the present study was to compare different commercial hyaluronic acid (HA) gels, which could be used to resuspend hFPTs in a formulation that would allow for good delivery of the cells. No medium or growth supplement was used in the formulation in order to make it therapeutically dispensable. These conditions are stringent for cells, but surprisingly, we found that different formulations could allow a good survival for up to 3 days when stored at 4°C (refrigerator stable). The gels must allow a good survival of the cells in parallel with a good stability of the preparation over time and sufficient viscosity to remain in place if deposited on a wounded location. Moreover, the cells must conserve their ability to attach and to proliferate. hFPTs were able to survive and to recover from all of the tested gels, but some products showed some advantages over others in terms of survival and viscosity. Finally, the Ostenil Tendon HA gel fulfilled all of the requirements and presented the best compromise between a good survival and sufficient rheological characteristics to create an interesting cell delivery system.

Photoreversal of the ultraviolet radiation-induced disappearance of ATPase-positive Langerhans cells in the epidermis of Monodelphis domestica.

The present study was undertaken to explore the possible causes of ultraviolet radiation (UVR)-induced disappearance of ATPase-positive, epidermal Langerhans cells (LC). Monodelphis domestica was used because it has the capacity for photoreactivation of UVR-induced pyrimidine dimers in epidermal DNA. Single, 330 J/m2 (ears) or 500 J/m2 (back) UVR exposures (FS-40 sunlamps) reduced the numbers of ATPase-positive epidermal LC in M. domestica ears to approximately 15% of those in unirradiated ears and approximately 37% of those in unirradiated dorsal skin. Immediate 90-minute exposures to photoreactivating light (PRL, 320-400 nm) post-UVR reversed the effects of UVR, resulting in ATPase-positive LC numbers not being significantly different from controls. Exposure to PRL immediately preceeding UVR did not prevent ATPase-positive LC disappearance. The photoreactivation of UVR-induced ATPase-positive LC disappearance indicates that DNA damage (pyrimidine dimers) is involved in the loss of ATPase-positive LC.

Ultraviolet radiation-induced histopathologic changes in the skin of the marsupial Monodelphis domestica. II. Quantitative studies of the photoreactivation of induced hyperplasia and sunburn cell formation.

Induction of sunburn cells (SBCs) and hyperplasia of the epidermis of the marsupial Monodelphis domestica by ultraviolet radiation (UVR) has been studied. A dose of 500 J/m2 (approximately 1 minimal erythemal dose) from an FS-40 sunlamp induced measurable numbers of SBCs with a peak number at 32-48 h post-UVR exposure of skin to photoreactivating light suppressed the induction of SBCs by approximately 75%. Pre-UVR exposure to photoreactivating light had no effect on the induction of SBCs. Induction of hyperplasia also was suppressed to a similar extent by post-UVR photoreactivation treatment. These studies identify pyrimidine dimers in DNA as the major photoproduct involved in the induction of SBCs and hyperplasia in M. domestica by UVR.

Evidence that ferritin is UV inducible in human skin: part of a putative defense mechanism.

As ferritin has been identified as an important factor in antioxidant defense in cultured human skin cells we evaluated the presence of ferritin in human skin in vivo and the modifications following irradiation with UVA I, UVA I + II, and solar simulating light by immunohistochemical analysis. We report that the putative protective protein ferritin is regularly present in the basal layer of unirradiated epidermis in vivo and that the induction of ferritin was dependent on wavelength and cell type. Following UVA I radiation, ferritin increased both in epidermal and in dermal tissue. The same response occurred, although to a lesser extent, with UVA I + II but did not occur following solar simulating radiation. Quantitative analysis for ferritin in cultured keratinocytes and fibroblasts from seven individuals following each UV spectra were also assessed by enzyme-linked immunosorbent assay. The induction of ferritin by UV was highly dependent on the waveband and cell type. UVA I and UVA I + II radiations induced ferritin expression in dermal fibroblasts up to 260% and 200% over basal levels, respectively. Solar simulating radiation produced only a small induction of approximately 130% over basal ferritin levels in dermal fibroblasts. Ferritin increased in cultured fibroblasts as early as 3 h post-UVA with a peak at 6 h that remained until 48 h; there was no observable qualitative or quantitative increase seen in the undifferentiated cultured epidermal keratinocytes. Our findings indicate that the putative defense system of ferritin exists in human skin in vivo and its induction is dependent on UV spectra and cell type. The increased concentrations of this antioxidant in human skin following acute UV radiation could afford increased protection against subsequent oxidative stress.