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Objective: To evaluate the effects of an intervention using the robot device PARO on visuospatial hemineglect and activities of daily living, and its acceptance during stroke rehabilitation. PARO is an interactive robotic toy with the appearance of a baby seal, which can move, produce sounds, and react to speech and touch., Design: A randomized controlled trial., Setting: Hospital for neurorehabilitation., Subjects: Patients above 60 years old who have suffered their first stroke within the previous three months with left hemineglect ( n  = 39)., Interventions: The PARO group ( n  = 21) was exposed to PARO over a period of two weeks, three times per week. The participants of the control group ( n  = 18) were read to aloud., Outcome Measure: Visuospatial hemineglect was measured by a cancellation test and a Line Bisection Test, and independence in the activities of daily living was assessed by Scores of Independence Index for Neurological and Geriatric Rehabilitation (SINGER) test. The acceptance of PARO was also evaluated. Data were collected blinded at three times: baseline (T0), after two weeks of interventions (T1), and after additional two weeks as follow-up (T2)., Results: Improvement of hemineglect at T1 and T2 was significantly higher in the PARO group (T1: mean (SD) = 6.23 (3.81); T2: mean (SD) = 7.85 (3.68)) compared to the control group (T1: mean (SD) = 2.66 (4.19); T2: mean (SD) = 3.33 (4.16)) (T1: P  < 0.05; T2: P  < 0.05)., Conclusion: The study showed that the use of the PARO is well accepted and can help to improve neglect symptoms in patients with subacute stroke.

Fish oil rich in docosahexaenoic acid (DHA) has beneficial effects on human health. Omega-3 polyunsaturated fatty acids are precursors of eicosanoids and docosanoids, signaling molecules that control inflammation and immunity, and their dietary uptake improves a range of disorders including cardiovascular diseases, ulcerative colitis, rheumatoid arthritis, and psoriasis. The unsaturated nature of these fatty acids, however, makes them prone to oxidation, especially when they are incorporated into (membrane) phospholipids. The skin is an organ strongly exposed to oxidative stress, mainly due to solar ultraviolet radiation. Thus, increased levels of PUFA in combination with oxidative stress could cause increased local generation of oxidized lipids, whose action spectrum reaches from signaling molecules to reactive carbonyl compounds that can crosslink biomolecules. Here, we investigated whether PUFA supplements to fibroblasts are incorporated into membrane phospholipids and whether an increase of PUFA within phospholipids affects the responses of the cells to UV exposure. The redox-sensitive transcription factor Nrf2 is the major regulator of the fibroblast stress response to ultraviolet radiation or exposure to oxidized lipids. Here we addressed how Nrf2 signaling would be affected in PUFA-supplemented human dermal fibroblasts and mouse dermal fibroblasts from Nrf2-deficient and wild type mice. We found, using HPLC-tandem MS, that DHA supplements to culture media of human and murine fibroblasts were readily incorporated into phospholipids and that subsequent irradiation of the supplemented cells with UVA resulted in an increase in 1-palmitoyl-2-(epoxyisoprostane-E2)-sn-glycero-3-phosphorylcholine and Oxo-DHA esterified to phospholipid, both of which are Nrf2 agonists. Also, induction of Nrf2 target genes was enhanced in the DHA-supplemented fibroblasts after UVA irradiation. In Nrf2-deficient murine fibroblasts, the expression of the target genes was, as expected, decreased, but surprisingly, expression of TNFα and MMP13 was strongly induced in DHA-supplemented, UVA-irradiated cells. Also, Nrf2-deficient cells had increased levels of oxidized phospholipids relative to the unoxidized precursors after UVA irradiation. Our data suggest that under ultraviolet stress a functioning Nrf2 system is required to prevent DHA-induced inflammation and matrix degradation in dermal fibroblasts., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Survival after childhood cancer has improved substantially over recent decades. Although cancer in childhood is rare increasingly effective treatments have led to a growing number of long-term survivors. It is estimated that there are between 300,000 and 500,000 childhood cancer survivors in Europe. Such good survival prospects raise important questions relating to late effects of treatment for cancer. Research has shown that the majority will suffer adverse health outcomes and premature mortality compared with the general population. While chronic health conditions are common among childhood cancer survivors, each specific type of late effect is very rare. Long-term effects must be considered particularly when addressing complex multimodality treatments, and taking into account the interaction between aspects of treatment and genotype. The PanCare Network was set up across Europe in order to effectively answer many of these questions and thereby improve the care and quality of life of survivors. The need for a structured long-term follow-up system after childhood cancer has been recognised for some time and strategies for implementation have been developed, first nationally and then trans-nationally, across Europe. Since its first meeting in Lund in 2008, the goal of the PanCare Network has been to coordinate and implement these strategies to ensure that every European survivor of childhood and adolescent cancer receives optimal long-term care. This paper will outline the structure and work of the PanCare Network, including the results of several European surveys, the start of two EU-funded projects and interactions with relevant stakeholders and related projects., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Autophagy is the central cellular mechanism for delivering organelles and cytoplasm to lysosomes for degradation and recycling of their molecular components. To determine the contribution of autophagy to melanocyte (MC) biology, we inactivated the essential autophagy gene Atg7 specifically in MCs using the Cre-loxP system. This gene deletion efficiently suppressed a key step in autophagy, lipidation of microtubule-associated protein 1 light chain 3 beta (LC3), in MCs and induced slight hypopigmentation of the epidermis in mice. The melanin content of hair was decreased by 10-15% in mice with autophagy-deficient MC as compared with control animals. When cultured in vitro, MCs from mutant and control mice produced equal amounts of melanin per cell. However, Atg7-deficient MCs entered into premature growth arrest and accumulated reactive oxygen species (ROS) damage, ubiquitinated proteins, and the multi-functional adapter protein SQSTM1/p62. Moreover, nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent expression of NAD(P)H dehydrogenase, quinone 1, and glutathione S-transferase Mu 1 was increased, indicating a contribution of autophagy to redox homeostasis in MCs. In summary, the results of our study suggest that Atg7-dependent autophagy is dispensable for melanogenesis but necessary for achieving the full proliferative capacity of MCs.

Antimicrobial peptides (AMPs), an important part of the innate immune system, are crucial for defense against invading microorganisms. Whereas AMPs have been extensively studied in adult skin, little is known about the impact of AMPs in the developing human skin. We therefore compared the expression and regulation of AMPs in fetal, neonatal, and adult keratinocytes (KCs) in vitro. The constitutive expression of human β-defensin-2 (HBD-2), HBD-3, S100 protein family members, and cathelicidin was significantly higher in KCs from fetal skin than in KCs from postnatal skin. The capacity to further increase AMP production was comparable between prenatal and postnatal KCs. Analysis of skin equivalents (SEs) revealed a strong constitutive expression of S100 proteins in fetal but not in neonatal and adult SEs. The elevated AMP levels correlated with reduced H3K27me3 (tri-methyl-lysine 27 on histone H3) levels and increased expression of the histone demethylase JMJD3. Knockdown of JMJD3 in fetal KCs elevated H3K27me3 levels and significantly downregulated the expression of HBD-3, S100A7, S100A8, S100A9, and cathelicidin. Our data indicate a crucial contribution of histone modifications in the regulation of AMP expression in the skin during ontogeny. The elevated AMP expression in prenatal skin might represent an essential defense strategy of the unborn.

The skin is exposed to environmental insults such as UV light that cause oxidative damage to macromolecules. A centerpiece in the defense against oxidative stress is the Nrf2 (nuclear factor (erythroid-derived-2)-like 2)-mediated transcriptional upregulation of antioxidant and detoxifying enzymes and the removal of oxidatively damaged material. Autophagy has an important role in the intracellular degradation of damaged proteins and entire organelles, but its role in the epidermis has remained elusive. Here, we show that both UVA and UVA-oxidized phospholipids induced autophagy in epidermal keratinocytes. Oxidative stressors induced massive accumulation of high-molecular-weight protein aggregates containing the autophagy adaptor protein p62/SQSTM1 in autophagy-deficient (autophagy-related 7 (ATG7) negative) keratinocytes. Strikingly, even in the absence of exogenous stress, the expression of Nrf2-dependent genes was elevated in autophagy-deficient keratinocytes. Furthermore, we show that autophagy-deficient cells contained significantly elevated levels of reactive oxidized phospholipids. Thus, our data demonstrate that autophagy is crucial for both the degradation of proteins and lipids modified by environmental UV stress and for limiting Nrf2 activity in keratinocytes. Lipids that promote inflammation and tissue damage because of their reactivity and signaling functions are commonly observed in aged and diseased skin, and thus targeting autophagy may be a promising strategy to counteract the damage promoted by excessive lipid oxidation.

We report on the first documented outbreak of leptospirosis in Austria. In July 2010, four cases of serologically confirmed leptospirosis occurred in athletes after a triathlon held in Langau. Heavy rains preceded the triathlon (rainfall: 22 mm). The index case (Patient A) was a 41-year-old previously healthy male, who was admitted to hospital A on July 8 with a four-day history of fever up to 40°C that began 14 days after attending the triathlon event. On July 7, patient B, a 42-year-old male, was admitted to the same hospital, with signs and symptoms of kidney failure. Hemodialysis was performed every other day for 3 weeks. While the serum drawn on the day of admission was negative for antibodies against Leptospira, a specimen from July 28 tested positive with Leptospira interrogans. On July 11, patient C, a 40-year-old male, was admitted to hospital B for nephritis. On July 14, patient D, a 44-year-old male, was admitted to hospital C with a ten days history of intermittent fever, mild dry cough and headache. Our report underlines that in Austria recreational users of bodies of freshwater must be aware of an existing risk of contracting leptospirosis, particularly after heavy rains. The suppressive influence of a triathlon on the immune system is well documented and therefore an outbreak in this population group can be seen as a sensitive indicator concerning possible risk for the general population.

Background and Purpose: 5-aminosalicylate (5-ASA) raises levels of 6-thioguanine nucleotides (6-TGN), the active metabolites of thiopurines such as azathioprine (AZA). Changes in levels of each individual TGN - 6-thioguanosine mono-, di- and triphosphate (6-TGMP, 6-TGDP, 6-TGTP) - and of 6-methylmercaptopurine ribonucleotides (6-MMPR) after 5-ASA are not known., Experimental Approach: Effects of increasing 5-ASA doses on AZA metabolites were investigated prospectively in 22 patients with inflammatory bowel disease in 4-week study periods. Patients started with 2 g 5-ASA daily, and then were increased to 4 g daily and followed by a washout period. Thiopurine doses remained unchanged throughout the entire study. Levels of 6-TGMP, 6-TGDP, 6-TGTP and 6-MMPR as well as of 5-ASA and N-acetyl-5-aminosalicylic acid (N-Ac-5-ASA) were determined each study period., Key Results: Median baseline levels in 17 patients of 6-TGDP, 6-TGTP and 6-MMPR were 52, 319 and 1676 pmol per 8 x 10(8) red blood cells respectively. After co-administration of 2 g 5-ASA daily, median 6-TGDP and 6-TGTP levels increased but median 6-MMPR levels were unchanged. Increasing 5-ASA to 4 g daily did not affect median 6-TGDP and 6-TGTP levels, but median 6-MMPR levels decreased. After discontinuation of 5-ASA, both 6-TGDP and 6-TGTP levels decreased and median 6-MMPR levels increased. The 6-TGTP/(6-TGDP+6-TGTP)-ratio did not change during the study, but 6-MMPR/6-TGN ratios decreased., Conclusions and Implications: Individual 6-TGN metabolites increased after addition of 5-ASA, but 6-MMPR-levels and the 6-MMPR/6-TGN ratios decreased. Further studies are needed to decide whether this pharmacokinetic interaction would result in improvement of efficacy and/or increased risk of toxicity of AZA.

6-Thioguanine nucleotides are the sum of 6-thioguanosine 5'-monophosphate (TGMP), -diphosphate (TGDP), and -triphosphate (TGTP) representing essential metabolites involved in drug action of thiopurines. Elevated levels of TGDP have been associated with poor response to azathioprine therapy in patients with inflammatory bowel disease. The conversion of TGDP to TGTP is supposed to be catalyzed by nucleoside diphosphate kinase (NDPK). The aim of this work was to investigate simultaneously individual 6-thioguanosine phosphate levels and NDPK activity in red blood cells (RBCs) of patients on azathioprine therapy. Ion-pair high-performance liquid chromatography methods with fluorescence and ultraviolet detection were applied to quantify individual levels of 6-thioguanosine 5'-phosphates and NDPK activity, respectively, in RBCs. Recombinantly expressed NDPK isoforms A and B were unequivocally identified to catalyze the formation of TGTP (30.6 +/- 3.88 nmol x min x mg for NDPK A versus 41.2 +/- 1.05 nmol x min x mg for NDPK B). Comprehensive analyses on the stability of TGMP, TGDP, and TGTP and the reproducibility of NDPK activity in RBCs were performed to provide a reliable sampling protocol for clinical practice. Of note, isolation of RBCs within 6 hours followed by immediate storage at -80 degrees C is crucial for prevention of degradation of 5'-phosphates. In a clinical study of 37 patients on azathioprine, TGTP was the predominant 6-thioguanosine phosphate in RBCs. In contrast, three patients showed TGTP/(TGDP + TGTP) ratios of 57.2%, 64.3%, and 66% corresponding to elevated TGDP levels. NDPK activity ranged from 4.1 to 11.3 nmol x min x mg hemoglobin. No correlation between NDPK activity and the 6-thioguanosine phosphate levels was found. The question whether interindividual variability of NDPK activity may explain differences in 6-thioguanosine 5'-phosphates levels has to be investigated in a prospective large-scale study.

Background: Spectrophotometric intracutaneous analysis (SIAscopy) is a multispectral imaging technique that is used to identify 'suspicious' (i.e. potentially malignant) pigmented skin lesions for further investigation. The MoleMate system is a hand-held scanner that captures SIAscopy images that are then classified by the clinician using a computerized diagnostic algorithm designed for the primary health care setting. The objectives of this study were to test the effectiveness of a computer program designed to train health care workers to identify the diagnostic features of SIAscopy images and compare the results of a group of Australian and a group of English general practitioners (GPs)., Methods: Thirty GPs recruited from the Perth (Western Australia) metropolitan area completed the training program at a workshop held in March 2008. The accuracy and speed of their pre- and post-test scores were then compared with those of a group of 18 GPs (including 10 GP registrars) who completed a similar program at two workshops held in Cambridge (U.K.) in March and April, 2007., Results: The median test score of the Australian GPs improved from 79.5% to 86.5% (median increase 5.5%; p < 0.001) while the median test score of the English GPs improved from 74.5% to 86.5% (median increase 9.5%; p < 0.001). The Australian GPs had significantly higher pre-test scores but there were no significant differences in post-test scores between the Australian and English GPs or between the GPs and GP registrars. There was no significant difference in scores between GPs with previous dermoscopy experience or dermatology training., Conclusion: Most of the SIAscopy features can be learnt to a reasonable degree of accuracy with this brief computer training program. Although the Australian GPs scored higher in the pre-test, both groups had similar levels of accuracy and speed in interpreting the SIAscopy features after completing the program. Scores were not affected by previous dermoscopy experience or dermatology training, which suggests that the MoleMate system is relatively easy to learn.

Background: An accurate diabetes register enables a general practice to effectively monitor and manage the services for their patients with diabetes. This pilot project builds on the National Primary Care Collaboratives Program (a quality improvement programme for general practice) as the first change principle for managing chronic diseases., Objectives: The main aim of the project was to improve the systems management of electronic registers of people with diabetes in the general practice setting. The pilot project assessed the uptake, awareness and confidence levels amongst practice staff in improving the diabetes register., Method: This was completed by conducting a survey of general practitioners and practice nurses within one general practice in Perth, Western Australia. In addition, focus groups per and post intervention were facilitated to obtain practice staff's views upon the issues around maintaining an efficient and updated patient register within a busy practice setting., Results: By the end of the project the general practice had an established diabetes register with defined and agreed practice systems.

Caspase-14, a protease involved in skin barrier formation, is specifically expressed in epidermal keratinocytes (KCs). Here, we mapped three start sites of transcription of the human caspase-14 gene and analyzed the upstream chromosomal region for promoter activity. Reporter gene assays identified a core promoter region proximal to the first exon and a distal regulatory region which differentially suppressed promoter activity in KC and other cells. Sequence elements in the proximal promoter were bound by the transcription factors AP-1 (JunB, c-Jun, JunD, Fra-1 and Fra-2) and NFkappaB (p50 and RelB). Our data reveal the basic organization of the human caspase-14 promoter and suggest an important role of AP-1 and NFkappaB in the transcriptional control of caspase-14.

The family of the organic anion transporting polypeptides forms an increasing group of uptake transport proteins with a wide substrate spectrum. Although the expression of some members of this group, such as organic anion transporting polypeptide (OATP)-A or C, is limited to special tissues (such as liver or brain), the organic anion transporting polypeptide 2B1 (OATPB/SLCO2B1) is expressed in many organs, including liver, placenta, mammary gland, brain, and intestine. However, little is known about its function in those tissues because only a limited number of compounds, such as dehydroepiandrosterone-sulfate (DHEAS) and estrone-3-sulfate (E3S), have been characterized as OATP2B1 substrates. To further elucidate the role of OATP2B1 on steroid transport, we examined the influence of steroid hormones on OATP2B1-mediated E3S and DHEAS uptake using OATP2B1-overexpressing Madin-Darby canine kidney II cells. We identified unconjugated androgens (e.g., testosterone) as potent inhibitors for OATP2B1. In contrast, gestagenes such as progesterone enhanced E3S uptake in a concentration-dependent manner to up to 300% of the control, accompanied by a significant decrease in the OATP2B1 K(m) value for E3S (control, K(m) = 14 microM; in the presence of 31.6 muM progesterone, K(m) = 3.6 microM). Moreover, we demonstrated that testosterone and progesterone are not substrates of OATP2B1, indicating an allosteric mechanism for the observed effects. Furthermore, we showed that progesterone enhances the OATP2B1-dependent pregnenolone sulfate transport. Taken together, the results indicate functional modification of OATP2B1-mediated E3S and DHEAS as well as pregnenolone sulfate transport through steroid hormones such as progesterone. These effects can have physiological consequences for the organ-specific uptake of steroids.