Genetic removal of tri-unsaturated fatty acids suppresses developmental and molecular phenotypes of an Arabidopsis tocopherol-deficient mutant. Whole-body mapping of malondialdehyde pools in a complex eukaryote.

Malondialdehyde (MDA) is a small, ubiquitous, and potentially toxic aldehyde that is produced in vivo by lipid oxidation and that is able to affect gene expression. Tocopherol deficiency in the vitamin E2 mutant vte2-1 of Arabidopsis thaliana leads to massive lipid oxidation and MDA accumulation shortly after germination. MDA accumulation correlates with a strong visual phenotype (growth reduction, cotyledon bleaching) and aberrant GST1 (glutathione S-transferase 1) expression. We suppressed MDA accumulation in the vte2-1 background by genetically removing tri-unsaturated fatty acids. The resulting quadruple mutant, fad3-2 fad7-2 fad8 vte2-1, did not display the visual phenotype or the aberrant GST1 expression observed in vte2-1. Moreover, cotyledon bleaching in vte2-1 was chemically phenocopied by treatment of wild-type plants with MDA. These data suggest that products of tri-unsaturated fatty acid oxidation underlie the vte2-1 seedling phenotype, including cellular toxicity and gene regulation properties. Generation of the quadruple mutant facilitated the development of an in situ fluorescence assay based on the formation of adducts of MDA with 2-thiobarbituric acid at 37 degrees C. Specificity was verified by measuring pentafluorophenylhydrazine derivatives of MDA and by liquid chromatography analysis of MDA-2-thiobarbituric acid adducts. Potentially applicable to other organisms, this method allowed the localization of MDA pools throughout the body of Arabidopsis and revealed an undiscovered pool of the compound unlikely to be derived from trienoic fatty acids in the vicinity of the root tip quiescent center.

Sentinel lymph node biopsy in nonmelanoma skin cancer patients.

The management of lymph nodes in nonmelanoma skin cancer patients is currently still debated. Merkel cell carcinoma (MCC), squamous cell carcinoma (SCC), pigmented epithelioid melanocytoma (PEM), and other rare skin neoplasms have a well-known risk to spread to regional lymph nodes. The use of sentinel lymph node biopsy (SLNB) could be a promising procedure to assess this risk in clinically N0 patients. Metastatic SNs have been observed in 4.5-28% SCC (according to risk factors), in 9-42% MCC, and in 14-57% PEM. We observed overall 30.8% positive SNs in 13 consecutive patients operated for high-risk nonmelanoma skin cancer between 2002 and 2011 in our institution. These high rates support recommendation to implement SLNB for nonmelanoma skin cancer especially for SCC patients. Completion lymph node dissection following positive SNs is also a matter of discussion especially in PEM. It must be remembered that a definitive survival benefit of SLNB in melanoma patients has not been proven yet. However, because of its low morbidity when compared to empiric elective lymph node dissection or radiation therapy of lymphatic basins, SLNB has allowed sparing a lot of morbidity and could therefore be used in nonmelanoma skin cancer patients, even though a significant impact on survival has not been demonstrated.

Expression of intercellular adhesion molecule-1 in UVA-irradiated human skin cells in vitro and in vivo.

Ultraviolet A (UVA) radiation represents an important oxidative stress to human skin and certain forms of oxidative stress have been shown to modulate intercellular adhesion molecule-1 (ICAM-1) expression. ICAM-1 has been shown to play an important part in many immune reactions and the perturbations of this molecule by ultraviolet radiation could have implications in many inflammatory responses. An enhancement immunohistochemical method with avidin/biotin was used for analysing the early effects of UVA radiation on human cell cultures and human skin (340-400 nm). Both in vitro and in vivo data show that ICAM-1 staining in epidermal keratinocytes, which was expressed constitutively, decreased in a UVA dose-dependent manner. The decrease was most noted at 3-6 h following UVA radiation with some ICAM-1 staining returning by 48 h post-UVA. ICAM-1 positive staining in the dermis was specific for vascular structures and was increased 24 h after UVA radiation. Cultured dermal fibroblasts exhibited ICAM-1 staining which increased slightly within 6-48 h post-UVA radiation. As epidermal ICAM-1 expression is depleted following UVA radiation and dermal expression increases due to an increase in the vascular structures, ICAM-1 provides a valuable marker following UVA radiation in human skin that can be readily measured in situ.

Loss of serum response factor in keratinocytes results in hyperproliferative skin disease in mice.

The transcription factor serum response factor (SRF) plays a crucial role in the development of several organs. However, its role in the skin has not been explored. Here, we show that keratinocytes in normal human and mouse skin expressed high levels of SRF but that SRF expression was strongly downregulated in the hyperproliferative epidermis of wounded and psoriatic skin. Keratinocyte-specific deletion within the mouse SRF locus during embryonic development caused edema and skin blistering, and all animals died in utero. Postnatal loss of mouse SRF in keratinocytes resulted in the development of psoriasis-like skin lesions. These lesions were characterized by inflammation, hyperproliferation, and abnormal differentiation of keratinocytes as well as by disruption of the actin cytoskeleton. Ultrastructural analysis revealed markedly reduced cell-cell and cell-matrix contacts and loss of cell compaction in all epidermal layers. siRNA-mediated knockdown of SRF in primary human keratinocytes revealed that the cytoskeletal abnormalities and adhesion defects were a direct consequence of the loss of SRF. In contrast, the hyperproliferation observed in vivo was an indirect effect that was most likely a consequence of the inflammation. These results reveal that loss of SRF disrupts epidermal homeostasis and strongly suggest its involvement in the pathogenesis of hyperproliferative skin diseases, including psoriasis.

Dose-dependent effect of atrial natriuretic peptide on blood pressure, heart rate, and skin blood flow of normal volunteers.

Synthetic atrial natriuretic peptide, containing 26 amino acids in the rat sequence, L-364, 343 (Ileu-ANP), was infused intravenously at increasing rates (1-40 micrograms/min) into four normal volunteers. Mean intraarterial blood pressure decreased and heart rate increased in cumulative-dose-dependent fashion. Skin blood flow as measured with a laser Doppler device rose already with a cumulative dose of 55 micrograms Ileu-ANP and further rises were directly related to dose. The only side effects observed were those accompanying symptomatic hypotension at higher doses. These findings provide strong evidence that Ileu-ANP acts as a vasodilator in normal volunteers.

Online teaching of inflammatory skin pathology by a French-speaking international university network

Introduction: Developments in technology, webbased teaching and whole slide imaging have broadened the teaching horizon in anatomic pathology. Creating online learning material including many types of media like radiologic images, videos, clinical and macroscopic photographs and whole slides imaging is now accessible to almost every university. Unfortunately, a major limiting factor to maintain and update the learning material is the amount of work, time and resources needed. In this perspective, a French national university network was initiated in 2011 to build mutualised online teaching pathology modules with clinical cases and tests. This network has been extended to an international level in 2012-2014 (Quebec, Switzerland and Ivory Coast). Method: One of the first steps of the international project was to build a learning module on inflammatory skin pathology intended for interns and residents of pathology and dermatology. A pathology resident from Quebec spent 6 weeks in France and Switzerland to develop the contents and build the module on an e-learning Moodle platform (http: //moodle.sorbonne-paris-cite.fr) under the supervision of two dermatopathologists (BV, MB). The learning module contains text, interactive clinical cases, tests with feedback, whole slides images (WSI), images and clinical photographs. For that module, the virtual slides are decentralized in 2 universities (Bordeaux and Paris 7). Each university is responsible of its own slide scanning, image storage and online display with virtual slide viewers. Results: The module on inflammatory skin pathology includes more than 50 web pages with French original content, tests and clinical cases, links to over 45 WSI and more than 50 micro and clinical photographs. The whole learning module is currently being revised by four dermatopathologists and two senior pathologists. It will be accessible to interns and residents in spring 2014. The experience and knowledge gained from that work will be transferred to the next international fellowship intern whose work will be aimed at creating lung and breast pathology learning modules. Conclusion: The challenges of sustaining a project of this scope are numerous. The technical aspect of whole-slide imaging and storage needs to be developed by each university or group. The content needs to be regularly updated, completed and its use and existence needs to be promoted by the different actors in pathology. Of the great benefits of that kind of project are the international partnerships and connections that have been established between numerous Frenchspeaking universities and pathologists with the common goals of promoting education in pathology and the use of technology including whole slide imaging. * The Moodle website is hosted by PRES Sorbonne Paris Cité, and financial supports for hardware have been obtained from UNF3S (http://www.unf3s.org/) and PRES Sorbonne Paris Cité. Financial support for international fellowships has been obtained from CFQCU (http://www.cfqcu.org/).

Biological activity of ectodysplasin A is conditioned by its collagen and heparan sulfate proteoglycan-binding domains.

Mutations in the TNF family ligand EDA1 cause X-linked hypohidrotic ectodermal dysplasia (XLHED), a condition characterized by defective development of skin appendages. The EDA1 protein displays a proteolytic processing site responsible for its conversion to a soluble form, a collagen domain, and a trimeric TNF homology domain (THD) that binds the receptor EDAR. In-frame deletions in the collagen domain reduced the thermal stability of EDA1. Removal of the collagen domain decreased its activity about 100-fold, as measured with natural and engineered EDA1-responsive cell lines. The collagen domain could be functionally replaced by multimerization domains or by cross-linking antibodies, suggesting that it functions as an oligomerization unit. Surprisingly, mature soluble EDA1 containing the collagen domain was poorly active when administered in newborn, EDA-deficient (Tabby) mice. This was due to a short stretch of basic amino acids located at the N terminus of the collagen domain that confers EDA1 with proteoglycan binding ability. In contrast to wild-type EDA1, EDA1 with mutations in this basic sequence was a potent inducer of tail hair development in vivo. Thus, the collagen domain activates EDA1 by multimerization, whereas the proteoglycan-binding domain may restrict the distribution of endogeneous EDA1 in vivo.

Immediate hip spica is as effective as, but more efficient than, flexible intramedullary nailing for femoral shaft fractures in pre-school children.

Flexible intramedullary nailing (FIN) is the gold standard treatment for femur fracture in school-aged children. It has been performed successfully in younger children, although Spica cast immobilisation (SCI) has been the most widely used strategy to date. METHOD: A retrospective analysis was performed between two comparable groups of children aged 1-4 years with a femoral shaft fracture. Two University hospitals, each using specific treatment guidelines, participated in the study: SCI in Group I (Basel, Switzerland) and FIN in Group II (Lausanne, Switzerland). RESULTS: Group I included 19 children with a median age of 26 months (range 12-46 months). Median hospital stay was 1 day (range 0-5 days) and casts were retained for a median duration of 21 days (range 12-29 days). General anaesthesia was used in six children and sedation in four. Skin breakdown secondary to cast irritation occurred in two children (10.5%). The median follow-up was 114 months (range 37-171 months). No significant malunion was noted. Group II included 27 children with a median age of 38.4 months (range 18.7-46.7 months). Median hospital stay was 4 days (range 1-13 days). All children required general anaesthesia for insertion and removal of the nails. Free mobilisation and full weight bearing were allowed at a median of 2 days (range 1-10 days) and 7 days (range 1-30 days), respectively, postoperatively. Nail exteriorisation was noted in three children (11%). The median follow-up was 16.5 months (range 8-172 months). No significant malunion was reported. CONCLUSIONS: Young children with a femoral shaft fracture treated by SCI or FIN had similarly favourable outcomes and complication rates. FIN allowed earlier mobilisation and full weight bearing. Compared to SCI, a greater number of children required general anaesthesia. In a pre-school child with a femoral shaft fracture, immediate SCI applied by a paediatric orthopaedic team following specific guidelines allowed early discharge from hospital with few complications.

Critical roles of the nuclear receptor PPARbeta (peroxisome-proliferator-activated receptor beta) in skin wound healing.

The PPARs (peroxisome-proliferator-activated receptors) alpha, beta/delta and gamma belong to the nuclear hormone receptor superfamily. While all three receptors are undetectable in adult mouse interfollicular epidermis, PPARbeta expression and activity is strongly re-activated by inflammatory stimuli during epidermal injury. The pro-inflammatory cytokine TNFalpha (tumour necrosis factor alpha) stimulates transcription of the PPARbeta gene via an activator protein-1 site in its promoter and it also triggers the production of PPARbeta ligands in keratinocytes. This increase of PPARbeta activity in these cells up-regulates the expression of integrin-linked kinase and 3-phosphoinositide-dependent kinase-1, which phosphorylates protein kinase B-alpha (Akt1). The resulting increase in Akt1 activity suppresses apoptosis and ensures the presence of a sufficient number of viable keratinocytes at the wound margin for re-epithelialization. Together, these observations reveal that PPARbeta takes on multiple roles and contributes favourably to the process of wound closure.

Regulation of epithelial-mesenchymal IL-1 signaling by PPARbeta/delta is essential for skin homeostasis and wound healing.

Skin morphogenesis, maintenance, and healing after wounding require complex epithelial-mesenchymal interactions. In this study, we show that for skin homeostasis, interleukin-1 (IL-1) produced by keratinocytes activates peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) expression in underlying fibroblasts, which in turn inhibits the mitotic activity of keratinocytes via inhibition of the IL-1 signaling pathway. In fact, PPARbeta/delta stimulates production of the secreted IL-1 receptor antagonist, which leads to an autocrine decrease in IL-1 signaling pathways and consequently decreases production of secreted mitogenic factors by the fibroblasts. This fibroblast PPARbeta/delta regulation of the IL-1 signaling is required for proper wound healing and can regulate tumor as well as normal human keratinocyte cell proliferation. Together, these findings provide evidence for a novel homeostatic control of keratinocyte proliferation and differentiation mediated via PPARbeta/delta regulation in dermal fibroblasts of IL-1 signaling. Given the ubiquitous expression of PPARbeta/delta, other epithelial-mesenchymal interactions may also be regulated in a similar manner.

Foot skin blood flow following infrainguinal revascularization for critical lower limb ischemia.

INTRODUCTION: The aim of this study was to assess the blood flow in the feet before and after lower limb revascularization using laser Doppler imaging (LDI). METHODS: Ten patients with critical lower limb ischemia were prospectively enrolled from June to October 2004. All patients underwent successful unilateral surgical interventions including above-knee bypass, distal bypass and endarterectomy. Skin blood flow (SBF) over the plantar surface of both forefeet and heels was measured by LDI 24h before and 10 days after revascularization, expressed in perfusion units (PU), and reported as mean+/-SD. RESULTS: Measurements in the forefoot and heel were similar. Before revascularization mean SBF was significantly lower in the ischemic foot (130+/-71 PU) compared to the contralateral foot (212+/-68 PU), p<0.05. After revascularization a significant increase of the SBF in the forefoot (from 135+/-67 to 202+/-86 PU, p=0.001) and hindfoot (from 148+/-58 to 203+/-83, p=0.001) was observed on the treatment side. However, a large decrease of the SBF was seen in forefoot and hindfoot on the untreated side (from 250+/-123 PU to 176+/-83 and from 208+/-116 to 133+/-40, p=0.001, respectively). CONCLUSION: This study confirms the benefits of revascularization in patients with nonhealing foot lesions due to critical limb ischemia. A significant increase of the SBF was observed on the treatment side. However, an unexpected decrease was observed on the untreated side.