Survival response in the skin

Apoptosis is defined as a programmed cell death process operating in multicellular organisms in order to maintain proper homeostasis of tissues. Caspases are among the best characterized proteases to execute apoptosis although lately many studies have associated them with non-apoptotic functions. In the laboratory an antiapoptotic pathway relying on caspase-3 activation and RasGAP has been described in vitro. RasGAP bears two conserved caspase-3 cleavage sites. Under low stress conditions, RasGAP is first cleaved by low caspase-3 activity generating an N terminal fragment (fragment N) that induces a potent anti-apoptotic response mediated by the Ras/PI3K/Akt pathway. High levels of active caspase-3, associated with increased stress conditions, induce further cleavage of fragment N abrogating this anti-apoptotic response. In the present work I studied the functionality of fragment N-mediated protection in physiological conditions as well as the mechanism by which fragment N induces an anti-apoptotic response, with a focus on survivin, an inhibitor of apoptosis. During my work in the laboratory I found that mice lacking caspase-3 or unable to cleave RasGAP (KI mice) are deficient in Akt activation and more sensitive to apoptosis than wild-type mice in response to stress. This higher sensitivity to stress led to augmented tissue damage, highlighting the importance of this pathway in protection against low stress. In parallel I focused on the study of survivin expression in the skin in response to UV-B light and I found that survivin is induced in the cytoplasm of keratinocytes in response to stress where it may fulfill a cyto-protective role. However fragment N had no effect on survivin expression. In addition, cytoplasmic survivin was increased in keratinocytes exposed to UV-B light, whether RasGAP is cleaved (WT mice) or not (KI mice), indicating that survivin is not involved in fragment N mediated protection. Altogether these data indicate that fragment N is pivotal for cell protection against pathophysiologic damage and can encourage the development of therapies aimed to strengthen the resistance of cells against aggressive treatments. Importantly, this finding contributes to the characterization of how caspase-3 can be activated without inducing cell death, although further studies need to be conducted in order to completely characterize this pro-survival molecular mechanism.

Autologous keratinocyte suspension in platelet concentrate accelerates and enhances wound healing - a prospective randomized clinical trial on skin graft donor sites: platelet concentrate and keratinocytes on donor sites.

BACKGROUND: Wound healing involves complex mechanisms, which, if properly chaperoned, can enhance patient recovery. The abilities of platelets and keratinocytes may be harnessed in order to stimulate wound healing through the formation of platelet clots, the release of several growth factors and cytokines, and cell proliferation. The aim of the study was to test whether autologous keratinocyte suspensions in platelet concentrate would improve wound healing. The study was conducted at the Lausanne University Hospital, Switzerland in 45 patients, randomized to three different topical treatment groups: standard treatment serving as control, autologous platelet concentrate (PC) and keratinocytes suspended in autologous platelet concentrate (PC + K). Split thickness skin graft donor sites were chosen on the anterolateral thighs of patients undergoing plastic surgery for a variety of defects. Wound healing was assessed by the duration and quality of the healing process. Pain intensity was evaluated at day five. RESULTS: Healing time was reduced from 13.9 ± 0.5 days (mean ± SEM) in the control group to 7.2 ± 0.2 days in the PC group (P < 0.01). An addition of keratinocytes in suspension further reduced the healing time to 5.7 ± 0.2 days. Pain was reduced in both the PC and PC + K groups. Data showed a statistically detectable advantage of using PC + K over PC alone (P < 0.01). CONCLUSION: The results demonstrate the positive contribution of autologous platelets combined with keratinocytes in stimulating wound healing and reducing pain. This strikingly simple approach could have a significant impact on patient care, especially critically burned victims for whom time is of the essence. CLINICAL TRIAL REGISTRY INFORMATION: Protocol Record Identification Number: 132/03Registry URL: http://www.clinicaltrials.gov.

Altered expression of the transcription factor Mef2c during retinal degeneration in Rpe65-/- mice.

Purpose. To investigate the role of the myocyte enhancer factor 2 (Mef2) transcription factor family in retinal diseases, Mef2c expression was assessed during retinal degeneration in the Rpe65(-/-) mouse model of Leber's congenital amaurosis (LCA). Mef2c-dependent expression of photoreceptor-specific genes was further addressed. Methods. Expression of Mef2 members was analyzed by oligonucleotide microarray, quantitative PCR (qPCR) and in situ hybridization. Mef2c-dependent transcriptional activity was assayed by luciferase assay in HEK293T cells. Results. Mef2c was the only Mef2 member markedly downregulated during retinal degeneration in Rpe65(-/-) mice. Mef2c mRNA level was decreased by more than 2 fold at 2 and 4 months and by 3.5 fold at 6 months in retinas of Rpe65(-/-) mice. Downregulation of Mef2c at the protein level was confirmed in Rpe65(-/-) retinas. The decrease in Mef2c mRNA levels in the developing Rpe65(-/-) retinas, from post-natal day (P)13 onward, was concomitant with the decreased expression of the rod-specific transcription factors Nrl and Nr2e3. Nrl was further shown to drive Mef2c transcriptional activity, supporting a physiological role for Mef2c in the retina. In addition, Mef2c appeared to act as a transcriptional repressor of its own expression, as well as those of the retina-specific retinal G-protein coupled receptor (Rgr), rhodopsin and M-opsin genes. Conclusions. These findings highlight the early altered regulation of the rod-specific transcriptional network in Rpe65-related disease. They further indicate that Mef2c may act as a novel transcription factor involved in the development and the maintenance of photoreceptor cells.

Epidémiologie des cancers épithéliaux de la peau [Epidemiology of epithelial skin cancers]

With no less than 15,000 estimated new cases diagnosed per year, non melanomatous carcinomas are the commonest cutaneous cancers in the Swiss population. About 1 in 3 new cancer case is a basal (BCC) or a squamous cell carcinoma (SCC). Incidence rates are steadily increasing, faster for BCC than SCC. Rates are higher for men than women and increase exponentially with age. Systematic population-based registration of non melanomatous skin cancers faces many challenges that few cancer registries can meet. Rates of these cancers in Switzerland are among the highest in Europe. Primary and secondary nationwide prevention campaigns have been carried out for nearly 20 years with a focus on the deadliest cutaneous cancer: melanoma. However, detection of non melanomatous skin cancers benefits from these campaigns since prevention messages and means of early detection are similar for melanomas and other skin cancers.

Human skin in vitro permeation of bentazon and isoproturon formulations with or without protective clothing suit.

Skin exposures to chemicals may lead, through percutaneous permeation, to a significant increase in systemic circulation. Skin is the primary route of entry during some occupational activities, especially in agriculture. To reduce skin exposures, the use of personal protective equipment (PPE) is recommended. PPE efficiency is characterized as the time until products permeate through material (lag time, Tlag). Both skin and PPE permeations are assessed using similar in vitro methods; the diffusion cell system. Flow-through diffusion cells were used in this study to assess the permeation of two herbicides, bentazon and isoproturon, as well as four related commercial formulations (Basagran(®), Basamais(®), Arelon(®) and Matara(®)). Permeation was measured through fresh excised human skin, protective clothing suits (suits) (Microchem(®) 3000, AgriSafe Pro(®), Proshield(®) and Microgard(®) 2000 Plus Green), and a combination of skin and suits. Both herbicides, tested by itself or as an active ingredient in formulations, permeated readily through human skin and tested suits (Tlag < 2 h). High permeation coefficients were obtained regardless of formulations or tested membranes, except for Microchem(®) 3000. Short Tlag, were observed even when skin was covered with suits, except for Microchem(®) 3000. Kp values tended to decrease when suits covered the skin (except when Arelon(®) was applied to skin covered with AgriSafe Pro and Microgard(®) 2000), suggesting that Tlag alone is insufficient in characterizing suits. To better estimate human skin permeations, in vitro experiments should not only use human skin but also consider the intended use of the suit, i.e., the active ingredient concentrations and type of formulations, which significantly affect skin permeation.

Skin cancer in survivors of childhood and adolescent cancer.

The incidence of basal cell carcinoma (BCC) has been related to ionizing radiation, particularly for exposure occurring at young age. In this study, we considered the incidence of second skin neoplasms in long-term survivors from childhood cancer. We considered second primary cancers occurring among 776 subjects (436 males, 340 females) with first primary cancer diagnosed before age 20 years, between 1974 and 2001, in the Swiss Cantons of Vaud and Neuchâtel (786,000 inhabitants). Five BCC were observed versus 0.43 expected (standardized incidence ratio: 11.6, 95% confidence interval: 3.7-27.1). No case of cutaneous squamous cell carcinoma, nor of malignant melanoma was observed. The estimated radiation doses at 1mm through the skin ranged between 7 and 27 Sv. These data confirm that BCC are strongly related to ionizing radiation exposure in childhood. All the BCC were located within the radiation field, thus indicating that ionizing radiation is the key aetiological factor, even in the absence of any meaningful interaction with UV.

Reducing the clinical burden of ranibizumab treatment for neovascular age-related macular degeneration using an individually planned regimen.

AIMS: The purpose of this study was to clinically validate an individually planned treatment regimen for neovascular age-related macular degeneration (nAMD), termed, observe and plan. This regimen was based on the predictability of an individual's need for retreatment and aimed to reduce the clinical burden, while obtaining good functional results. METHODS: This was a prospective case series that included 104 patients (115 eyes) with treatment-naive nAMD. Following three loading doses of ranibizumab, monthly observation visits allowed the disease recurrence interval to be determined. The recurrence interval was reduced by 2 weeks to give the retreatment interval for the next three injections. Periodical control visits (at least every 6 months) allowed the effectiveness of the treatment to be assessed and individual intervals adjusted. RESULTS: Mean visual acuity (VA) improved by 8.7 and 9.8 letters in months 3 and 12, respectively. The mean number of injections during the 12-month study was 7.8, while the mean number of ophthalmic examinations between months 3 and 12 was 3.97. The mean treatment interval after the loading doses was 1.97 months. CONCLUSIONS: The observe-and-plan regimen significantly improved VA. This was obtained with fewer clinic visits compared with other regimens, which could ease the burden of nAMD treatment. TRIAL REGISTRATION NUMBER: Commission cantonale (VD) d'éthique de la recherché Clinique, Université de Lausanne, Protocole 351/11.

Allergy to betalactam antibiotics in children: a prospective follow-up study in retreated children after negative responses in skin and challenge tests.

BACKGROUND: Up to 10% of the patients in whom suspected betalactam hypersensitivity (HS) has been excluded by skin and challenge tests report suspected allergic reactions during subsequent treatments with the same or very similar betalactams. It has been suggested that the reactions may result from a resensitization induced by the challenge performed at the time of the allergological work-up. However, most patients did not undergo a second allergological work-up, to determine if the reactions resulted from betalactam HS or not. OBJECTIVES: We aimed to determine if children diagnosed nonallergic to betalactams have tolerated subsequent treatments with the initially suspected and/or other betalactams, and, in case of a reaction, if the reaction resulted from betalactam HS. Methods: We sent a questionnaire concerning the clinical history of their children to the parents of 256 children previously diagnosed nonallergic to betalactams. A second allergological work-up was performed in the children reporting suspected allergic reactions during subsequent treatments with the same and/or other betalactams. Skin tests were performed with the soluble form of the suspected (or very similar) betalactams and other betalactams from the same and other classes. Skin test responses were assessed at 15-20 min (immediate), 6-8 h (semi-late) and 48-72 h (late). Oral challenge (OC) was performed in children with negative skin tests, either at the hospital (immediate and accelerated reactions), or at home (delayed reactions). RESULTS: A response was obtained from 141 children (55.3%). Forty-eight (34%) of those children had not been treated with the betalactams for whom a diagnosis of allergy had been ruled out previously. Seven (7.5%) of the 93 children who had been treated again reported suspected allergic reactions. Skin tests and OC were performed in six of those children, and gave negative results in five children. In one child previously diagnosed nonallergic to amoxicillin associated with clavulanic acid, we diagnosed a delayed HS to clavulanic acid and a serum sickness-like disease to cefaclor. Thus, the frequency of reactions resulting from betalactam HS in children with negative skin and challenge tests is very low, and does not exceed 2.1% (2/93) if we consider that the child which refused a second allergological work-up is really allergic to betalactams. CONCLUSION: Our results in a very large number of children show that reactions presumed to result from betalactam HS are rare in children in whom the diagnosis of betalactam allergy has been ruled out previously. Moreover, they suggest that, as shown for the initial reactions, most of the reactions during subsequent treatments are rather a consequence of the infectious diseases for whom betalactams have been prescribed than a result of betalactam HS. Finally, they suggest that the risk of resensitization by OC is very low, and do not support the notion that skin testing should be repeated in children diagnosed nonallergic to betalactams.

Role of PPARβ in keratinocyte adhesion and migration during skin wound healing

RESUME L'homéostasie du tissu cutané est assurée par des interactions étroites entre les cellules le composant et par l'équilibre entre la différenciation et la prolifération des kératinocytes devant permettre un renouvellement constant du tissu. Après une blessure, les kératinocytes environnant la zone blessée sont activés par des cytokines. Ils acquièrent alors un phénotype migratoire qui s'accompagne d'une modulation de l'activité protéolytique de la matrice extra cellulaire, d'une modulation de la dynamique du cytosquelette d'active, de la polarisation de la cellule, de l'affaiblissement des contacts entre cellules et de changements dans leurs contacts avec la matrice extra cellulaire. PPARβ est un facteur de transcription activé par les acides gras et leurs dérivés. Il appartient à la famille des récepteurs nucléaires aux hormones et son expression est avérée dans les kératinocytes des follicules pileux et dans les kératinocytes inter-folliculaires activés par la blessure cutanée. Le rôle de PPARβ dans la peau est principalement lié à son effet protecteur contre l'apoptose ainsi qu'à son implication dans l'équilibre dynamique entre la prolifération et la différentiation des kératinocytes. L'objet de ce travail fut de déterminer le rôle de PPARβ dans les processus d'adhésion et de migration des kératinocytes activés durant la régénération de l'épithélium blessé. Nous avons montré que les souris dépourvues du gène codant pour PPARβ ont de sévères imperfections affectant la morphologie de l'épithélium. Ce phénotype est corrélé à la modulation imparfaite du réseau d'active chez les souris dépourvues de PPARβ, à un défaut de localisation de l'intégrine α3 impliquée dans les complexes induisant la migration cellulaire, ainsi qu'à la modulation de l'expression d'acteurs majeurs affectant l'activité protéolytique de la matrice extra cellulaire. En conclusion, nos résultats montrent que PPARβ est impliqué dans le contrôle de la dynamique du cytosquelette d'active et la polarisation des kératinocytes activés. PPARβ étant impliqué dans l'acquisition d'un phénotype migratoire, il est légitime de se demander s'il intervient de même dans d'autres types cellulaires, par exemple dans la transition épithéliale-mésenchymateuse durant le développement, ou encore la progression de cellules tumorales. SUMMARY Highly coordinated intercellular interactions and single cell metabolism ensure cell and tissue maintenance of the skin. Healing of a skin wound involves keratinocyte activation by cytokines and growth factors. Activated keratinocytes acquire a motile phenotype that requires extracellular matrix remodeling and subsequent ligand activation through proteolytic activity, as well as cytoskeletal reorganisation induced by the release of cell-cell junctions and by the signalling relayed via integrin receptors and their cytoplasmic adaptors. PPARβ is a transcription factor activated by polyunsaturated fatty acids and fatty acid derivatives which belong to the nuclear hormone receptor superfamily. It is expressed in activated keratinocytes where it plays an essential role in protecting them from apoptosis. In addition, it plays an important function in hair follicle morphogenesis at the time of elongation, via the regulation of the balance between keratinocyte differentiation and proliferation. The aim of the present work was to determine if PPARβ is also involved in the regulation of migration and adhesion properties of keratinocytes during skin wound healing. We have shown that wounded PPARβ null mice display severe abnormalities of the keratinocyte migratory layer as shown at the histological level and using three-dimensional reconstruction. This altered migratory phenotype is correlated to altered dynamic of the actin cytoskeleton network, impaired α3 integrin localisation in migrating keratinocytes and changes in the expression of a key actor involved in extracellular matrix proteolytic activity. These results show that PPARβ is implicated in the fine tuning of the actin network organisation and the polarisation of activated keratinocytes following an epithelial wound. Whether these mechanisms are also controlled by PPARβ in other cell types during epithelial mesenchymal transition or tumour cell progression is an interesting question to rise.

Loss of ATF2 function leads to cranial motoneuron degeneration during embryonic mouse development.

The AP-1 family transcription factor ATF2 is essential for development and tissue maintenance in mammals. In particular, ATF2 is highly expressed and activated in the brain and previous studies using mouse knockouts have confirmed its requirement in the cerebellum as well as in vestibular sense organs. Here we present the analysis of the requirement for ATF2 in CNS development in mouse embryos, specifically in the brainstem. We discovered that neuron-specific inactivation of ATF2 leads to significant loss of motoneurons of the hypoglossal, abducens and facial nuclei. While the generation of ATF2 mutant motoneurons appears normal during early development, they undergo caspase-dependent and independent cell death during later embryonic and foetal stages. The loss of these motoneurons correlates with increased levels of stress activated MAP kinases, JNK and p38, as well as aberrant accumulation of phosphorylated neurofilament proteins, NF-H and NF-M, known substrates for these kinases. This, together with other neuropathological phenotypes, including aberrant vacuolisation and lipid accumulation, indicates that deficiency in ATF2 leads to neurodegeneration of subsets of somatic and visceral motoneurons of the brainstem. It also confirms that ATF2 has a critical role in limiting the activities of stress kinases JNK and p38 which are potent inducers of cell death in the CNS.