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Background: Amifostine is a pharmaceutical agent that is used clinically to counteract the side-effects of chemotherapy and radiotherapy. It acts as a free radical scavenger that protects against harmful DNA cross-linking. The purpose of this study was to determine the effect of amifostine on the development of skin cancer in xeroderma pigmentosum (XP) mice exposed to ultraviolet B radiation (UVB). Methods: Twenty-five XP mice were equally divided into five groups. Group 1 (control) received no amifostine and no UVB exposure. Group 2 also received no amifostine, but was exposed to UVB at a dose of 200 mJ/cm2 every other day. The remaining groups were subjected to the same irradiation, but were given amifostine at a dose of 50 mg/kg (group 3), 100 mg/kg (group 4), or 200 mg/kg (group 5) immediately prior to each exposure. Results: No tumours were seen in the control group. The animals in group 2 (no amifostine) developed squamous cell carcinoma (SCC) at 3.5-4.5 months (mean 3.9 months). Groups 3 and 4 (low- and medium-dose amifostine) developed SCC at 4.0-7.0 months (mean 5.3 months), representing a statistically significant delay in tumour presentation (p = 0.04). An even greater delay was seen in group 5 (highdose amifostine), which developed SCC at 7.0-9.0 months (mean 8.5 months, p < 0.001 versus groups 3 and 4). Ocular keratitis developed in all animals except the unexposed controls and the high-dose treatment group. Conclusion: Treatment with amifostine significantly delays the onset of skin cancer and prevents ocular keratitis in UVB-exposed XP mice. [ABSTRACT FROM AUTHOR]

Ubiquitin-specific protease 15 (USP15) is a widely expressed deubiquitylase that has been implicated in diverse cellular processes in cancer. Here we identify topoisomerase II (TOP2A) as a novel protein that is regulated by USP15. TOP2A accumulates during G2 and functions to decatenate intertwined sister chromatids at prophase, ensuring the replicated genome can be accurately divided into daughter cells at anaphase. We show that USP15 is required for TOP2A accumulation, and that USP15 depletion leads to the formation of anaphase chromosome bridges. These bridges fail to decatenate, and at mitotic exit form micronuclei that are indicative of genome instability. We also describe the cell cycle-dependent behaviour for two major isoforms of USP15, which differ by a short serine-rich insertion that is retained in isoform-1 but not in isoform-2. Although USP15 is predominantly cytoplasmic in interphase, we show that both isoforms move into the nucleus at prophase, but that isoform-1 is phosphorylated on its unique S229 residue at mitotic entry. The micronuclei phenotype we observe on USP15 depletion can be rescued by either USP15 isoform and requires USP15 catalytic activity. Importantly, however, an S229D phospho-mimetic mutant of USP15 isoform-1 cannot rescue either the micronuclei phenotype, or accumulation of TOP2A. Thus, S229 phosphorylation selectively abrogates this role of USP15 in maintaining genome integrity in an isoform-specific manner. Finally, we show that USP15 isoform-1 is preferentially upregulated in a panel of non-small cell lung cancer cell lines, and propose that isoform imbalance may contribute to genome instability in cancer. Our data provide the first example of isoform-specific deubiquitylase phospho-regulation and reveal a novel role for USP15 in guarding genome integrity.

PurposeHypertrophic cardiomyopathy (HCM) is considered a hereditary autosomal dominant condition, but genetic testing is positive in only half of patients. In patients with negative genetic tests, the inheritance pattern and utility of family screening are unclear.MethodsSubjects with HCM were prospectively enrolled in a registry. A survey at a median follow-up of 4 years determined the yield of family screening.ResultsThe outcome of cardiac screening on 267 family members was reported by 120 survey respondents. Subjects with positive genetic test or family history (n=74, 62%) reported an HCM diagnosis in 34 of 203 first-degree relatives who were screened (17%). Affected family members were diagnosed at a mean age of 30-39 years, and 22 of 34 experienced HCM-related adverse events (65%). Gene test-negative subjects with no prior family history of HCM (n=46, 38%) reported an HCM diagnosis in only 2 of 64 first-degree relatives who were screened (3%, p<0.001). These two individuals were diagnosed at age >40 years without HCM-related adverse events.ConclusionHypertrophic cardiomyopathy is a heterogeneous disorder, only half of which tracks with a Mendelian inheritance pattern. Negative genetic testing and family history indicates a more complex genetic basis corresponding to low risk for family members.

Introduction: Chronic wounds remain a major clinical challenge. Human cryopreserved viable amniotic membrane (hCVAM) is among the most successful therapies, but the mechanisms of action remain loosely defined. Because proper regulation of macrophage behavior is critical for wound healing with biomaterial therapies, we hypothesized that hCVAM would positively regulate macrophage behavior in vitro , and that soluble factors released from the hCVAM would be important for this effect., Materials and Methods: Primary human pro-inflammatory (M1) macrophages were seeded directly onto intact hCVAM or cultured in separation via transwell inserts (Soluble Factors) in the presence of pro-inflammatory stimuli (interferon- γ and lipopolysaccharide) to simulate the chronic wound environment. Macrophages were characterized after 1 and 6 days using multiplex gene expression analysis of 37 macrophage phenotype- and angiogenesis-related genes via NanoString™, and protein content from conditioned media collected at days 1, 3 and 6 was analyzed via enzyme linked immunosorbent assays., Results and Discussion: Gene expression analysis showed that Soluble Factors promoted significant upregulation of pro-inflammatory marker IL1B on day 1 yet downregulation of TNF on day 6 compared to the M1 macrophage control. In contrast, intact hCVAM, which includes both extracellular matrix, viable cells, and soluble factors, promoted downregulation of pro-inflammatory markers TNF , CCL5 and CCR7 on day 1 and endothelial receptor TIE1 on day 6, and upregulation of the anti-inflammatory marker IL10 on day 6 compared to the M1 Control. Other genes related to inflammation and angiogenesis ( MMP9 , VEGF , SPP1 , TGFB1 , etc .) were differentially regulated between the Soluble Factors and intact hCVAM groups at both time points, though they were not expressed at significantly different levels compared to the M1 Control. Interestingly, Soluble Factors promoted increased secretion of the proinflammatory cytokine tumor necrosis factor- α (TNF- α ), while direct contact with hCVAM inhibited secretion of TNF, relative to the M1 Control. Both Soluble Factors and intact hCVAM inhibited secretion of MMP9 and VEGF, pro-inflammatory proteins that are critical for angiogenesis and remodeling, compared to the M1 Control, with intact hCVAM having a stronger effect., Conclusions: In a simulated pro-inflammatory environment, intact hCVAM has distinct anti-inflammatory effects on primary human macrophages, and direct macrophage contact with intact hCVAM is required for these effects. These findings are important for the design of next generation immunomodulatory biomaterials for wound repair and regenerative medicine that may include living cells, soluble factors, or a controlled drug delivery system., Competing Interests: CONFLICT OF INTEREST KLS discloses a potential conflict of interest: this study was funded in large part by Osiris Therapeutics, Inc. The study was designed by KLS and CEW, with some input from Osiris with respect to the potential impact of different experiments. Employees from Osiris had no part in interpretation of the study’s results. CEW, TY, MC, and WD declare that they have no conflicts of interest.

Background: Trials using inadequate levels of blinding may report larger effect sizes than blinded studies. It has been suggested that blinded outcome assessment in open trials may in some cases be undertaken by assessments of photographs. The aim of this paper is to explore the effect of using different methods to assess the primary outcome in the EVerT (Effective Verruca Treatments) trial. It also aims to give an overview of the experiences of using digital photographs within the trial., Methods: We undertook a secondary analysis to explore the effect of using three different methods to assess the primary outcome in the EVerT trial: assessment of digital photographs by blinded healthcare professionals; blinded healthcare professional assessment at the recruiting site and patient self-report. The verruca clearance rates were calculated using the three different methods of assessment. A Cohen's kappa measure of inter-rater agreement was used to assess the agreement between the methods. We also investigated the experiences of healthcare professionals using digital photographs within the trial., Results: Digital photographs for 189 out of 240 (79 %) patients in the trial were received for outcome assessment. Of the 189 photographs, 30 (16 %) were uninterpretable. The overall verruca clearance rates were 21 % (43/202,) using the unblinded patient self-reported outcome, 6 % (9/159,) using blinded assessment of digital photographs and 14 % (30/210,) using blinded outcome assessment at the site., Conclusions: Despite differences in the clearance rates found using different methods of outcome assessment, this did not change the original conclusion of the trial, that there is no evidence of a difference in effectiveness between cryotherapy and salicylic acid. Future trials using digital photographs should consider individual training needs at sites and have a backup method of assessment agreed a priori., Trial Registration: ISRCTN Registry ISRCTN18994246.

Background: Corns are a common foot problem affecting a large proportion of the population. This study describes the characteristics of corns experienced by 201 participants taking part in a randomised controlled trial to investigate associations between demographic and corn parameters on pain, foot related disability and quality of life (QoL)., Methods: Pain from the main (index) corn was measured using a visual analogue scale (VAS); foot related disability was assessed with the Foot Disability Questionnaire (now known as the Manchester Foot Pain and Disability Index) and quality of life was recorded with the EQ-5D questionnaire. The effect of demographic and corn parameters on the pain and quality of life outcomes was assessed with analysis of variance (ANOVA) methods. The effect of the same factors on a linear combination of the foot-related disability outcome measures was assessed using multivariate ANOVA methods. Pain was also tested for its mediating properties on the causal pathway between the independent variables and quality of life., Results: The mean pain score was 5.29 points on a 10 cm VAS, with females reporting substantively higher pain levels than males. Age affected foot-related disability, with lower levels on all domains of the MFPDI reported in older participants; each year of advancing age was associated with falls of: 0.009 points on the Concern about Appearance (CA) domain; 0.047 points on the Functional Limitation (FL) domain and 0.048 points on the Pain Intensity (PI) domain. Sex and corn type also affected disability, with higher scores reported by females and participants with plantar corns., Conclusions: The effect of pain was shown to mediate the relationship between sex and foot-related disability. The presence of plantar corns has a more detrimental effect on QoL than dorsal/inter-digital corns., Trial Registration: ISRCTN 13166839.

Background: Corns are a common foot problem and surveys have indicated that between 14-48% of people suffer from them. Many of these will seek podiatry treatment, however there is little evidence to indicate which current treatments provide long term resolution. This study compared 'usual' treatment (enucleation with a scalpel) with the application of 40% salicylic acid plasters to corns to investigate which is the most effective in terms of clinical, economic and patient-centred outcomes., Methods: A parallel-group randomised controlled trial was carried out in two centres where adults who presented with one or more corns and who met the inclusion criteria were allocated to either 'usual' scalpel debridement or corn plaster treatment. All participants had measurements of corn size, pain using a 100 mm visual analogue scale (VAS) and health-related quality of life (EQ-5D) measures by an independent podiatrist, blind to treatment allocation at baseline, 3, 6, 9 and 12 months., Results: 202 participants were randomised to receive scalpel debridement or corn plaster treatment (101 in each group). At 3 months 34% (32/95) of corns had completely resolved in the corn plaster group compared with 21% (20/94) in the scalpel group (p = 0.044), and 83% (79/95) had reduced in size in the corn plaster group compared with 56% (53/94) in the scalpel group (p < 0.001). At 12 months, time to corn recurrence was longer in the corn plaster group (p < 0.001). Pain from the corns was significantly lower in the corn plaster group at 3 months (p < 0.001) and EQ-5D scores changed (improved), from baseline, by 0.09 (SD ±0.31) and 0.01 (SD ±0.25) points in the corn plaster and scalpel groups respectively (p = 0.056). By month 12, EQ-5D scores had changed by 0.12 and -0.05 in the corn plaster and scalpel groups respectively (p = 0.005). The EQ-5D, VAS scores and the four domains of the Foot Disability Scale were similar in both groups at 3 and 12 months. The economic analysis indicated that corn plasters were a cost effective intervention., Conclusions: The use of corn plasters was associated with a higher proportion of resolved corns, a prolonged time to corn recurrence, less pain and reduced corn size over the first 6 months in comparison with 'usual' scalpel treatment and this intervention was cost effective. Used under supervision of a podiatrist on appropriate patients, corn plasters offer an effective alternative to scalpel debridement.

Background: Randomised controlled trials are widely accepted as the gold standard method to evaluate medical interventions, but they are still open to bias. One such bias is the effect of patient's preference on outcome measures. The aims of this study were to examine whether patients' treatment preference affected clearance of plantar warts and explore whether there were any associations between patients' treatment preference and baseline variables in the EverT trial., Methods: Two hundred and forty patients were recruited from University podiatry schools, NHS podiatry clinics and primary care. Patients were aged 12 years and over and had at least one plantar wart which was suitable for treatment with salicylic acid and cryotherapy. Patients were asked their treatment preference prior to randomisation. The Kruskal-Wallis test was performed to test the association between preference group and continuous baseline variables. The Fisher's exact test was performed to test the association between preference group and categorical baseline variables. A logistic regression analysis was undertaken with verruca clearance (yes or no) as the dependent variable and treatment, age, type of verruca, previous treatment, treatment preference as independent variables. Two analyses were undertaken, one using the health professional reported outcome and one using the patient's self reported outcomes. Data on whether the patient found it necessary to stop the treatment to which they had been allocated and whether they started another treatment were summarised by treatment group., Results: Pre-randomisation preferences were: 10% for salicylic acid; 42% for cryotherapy and 48% no treatment preference. There was no evidence of an association between treatment preference group and either patient (p=0.95) or healthcare professional (p=0.46) reported verruca clearance rates. There was no evidence of an association between preference group and any of the baseline variables except gender, with more females expressing a preference for salicylic acid (p=0.004). There was no evidence that the number of times salicylic acid was applied was different between the preference groups at one week (p=0.89) or at three weeks (p=0.24). Similarly, for the number of clinic visits for cryotherapy (p=0.71) CONCLUSIONS: This secondary analysis showed no evidence to suggest that patients' baseline preferences affected verruca clearance rates or adherence with the treatment., Trial Registration: Current Controlled Trials ISRCTN18994246 and National Research Register N0484189151.

Endorepellin, the C-terminal module of perlecan, negatively regulates angiogenesis counter to its proangiogenic parental molecule. Endorepellin (the C-terminal domain V of perlecan) binds the α2β1 integrin on endothelial cells and triggers a signaling cascade that leads to disruption of the actin cytoskeleton. Here, we show that both perlecan and endorepellin bind directly and with high affinity to both VEGF receptors 1 and 2, in a region that differs from VEGFA-binding site. In both human and porcine endothelial cells, this interaction evokes a physical down-regulation of both the α2β1 integrin and VEGFR2, with concurrent activation of the tyrosine phosphatase SHP-1 and downstream attenuation of VEGFA transcription. We demonstrate that endorepellin requires both the α2β1 integrin and VEGFR2 for its angiostatic activity. Endothelial cells that express α2β1 integrin but lack VEGFR2, do not respond to endorepellin treatment. Thus, we provide a new paradigm for the activity of an antiangiogenic protein and mechanistically explain the specificity of endorepellin for endothelial cells, the only cells that simultaneously express both receptors. We hypothesize that a mechanism such as dual receptor antagonism could operate for other angiostatic fragments.

Background and Purpose: Sleep apnea is very common after stroke and is associated with poor outcome. Supine sleep is known to exacerbate apneas in the general sleep apnea population. We therefore investigated the pattern of sleep positions in the acute stroke period., Methods: Inpatients with acute ischemic stroke underwent full polysomnography that included continuous monitoring of sleep positions. Sleep apnea severity was measured using the apnea-hypopnea index (AHI). Stroke severity was measured by the NIH Stroke Scale (NIHSS) at the time of study enrollment by certified study personnel. Percent total sleep time spent in the supine position was calculated and compared by stroke severity based on a median split of NIHSS using a Wilcoxon rank-sum test., Results: Of the 30 patients, the median age was 67. The median AHI was 23 (IQR: 6, 47). Twenty-two patients (73%) had sleep apnea with an AHI >/=5. The vast majority of sleep time among the stroke cases was spent supine, with a median percent sleep time spent supine of 100 (IQR: 62, 100). The majority (63%) of subjects spent no time asleep in any of the nonsupine positions (prone, left, right). Median percent sleep time supine was 100 (IQR: 100, 100) in those with a higher NIHSS and 63 (IQR: 51, 100) in those with a lower NIHSS (P<0.01)., Conclusions: Given the high prevalence of supine sleep identified, research into positional therapy for stroke patients with sleep apnea seems warranted.

Unlabelled: Osteoporosis public health measures are hindered by the inability to easily identify subclinical disease. We have now estimated state-specific osteoporosis prevalences using a simple formula (OST Index) to analyze age and weight of 62,882 older women; the prevalences determined are similar to those based on BMD. This new method has potential use for guiding implementation of osteoporosis prevention/treatment programs., Introduction: Although osteoporosis-related fractures are a major U.S. public health issue, population-based prevention programs have not yet been developed. One contributing factor has been lack of a suitable screening test to detect asymptomatic high-risk individuals., Materials and Methods: We estimated state-specific prevalences of postmenopausal osteoporosis using the Osteoporosis Self-Assessment Tool Index (OST Index; [self-reported weight in kg - age] x 0.2) to analyze data from 62,882 women >or=50 yr of age who participated in the 2002 Behavioral Risk Factor Surveillance System (BRFSS). The OST Index, designed to assess an individual's risk of disease, has previously been shown to have modest positive and high negative predictive value for osteoporosis defined by BMD criteria. Based on this index, women from each state were distributed among high-, moderate-, and low-risk OST categories. Calculated percentages for each category were weighted to U.S. Census Bureau population projections for 2002. By adjusting results to reflect previously validated percentages of women with osteoporosis in each risk category, we estimated the prevalence of postmenopausal osteoporosis in each state., Results: Our calculated weighted prevalence estimates agreed closely with those of the National Osteoporosis Foundation derived from actual femoral neck BMD measurements obtained in the third National Health and Nutrition Examination Survey (1988-1994) and projected to U.S. census state population predictions for 2002. Comparison of unweighted BRFSS-OST results and NHANES BMD data revealed similar percentages of osteoporosis among all women >or=50 yr of age (BRFSS, 18.5%; NHANES, 18.0%; p = 0.47) and also among white women (BRFSS, 19.0%; NHANES, 20.0%; p = 0.28). However, the percentages of osteoporosis among blacks and Hispanics did not correspond, at least partly because of the lack of race-specific reference standards for BMD measurements and OST index ranges., Conclusions: Analysis of readily available BRFSS data with the OST index formula is a simple, no-cost technique that provides state prevalence estimates of postmenopausal osteoporosis that could be used to guide allocation of resources to statewide osteoporosis prevention programs.

The use of digital wound images would allow remote consultation between patients, physicians, or other caregivers over the Internet. To evaluate the efficacy and validity of digital images for assessment of wounds, we compared the ability and reliability of surgeons to diagnose and make treatment suggestions using either digital or slide images of several types of wounds. Twenty-four wound images on 35-mm slides were selected for use in this study. Each slide image was digitized at 24-bit color with a resolution of 640 pixels horizontal by 425 pixels vertical, and stored as a JPEG file. Six physicians, of varying experience, examined the images, first in digital format and then later in the original slide form. Each observer assessed each wound for condition and possible treatment options by filling out a questionnaire using a series of yes/no questions. For all observers, there was an 87% agreement of answers between digital and slide images (p = 0.004). The agreement between the digital and slide images was also measured for each individual observer using a kappa coefficient. The agreement kappa correlated with the experience of the observer, with the kappa values ranging from greater than 0.8 (almost perfect agreement) for an experienced woundcare professional to just greater than 0.5 (moderate agreement) for a surgical intern. With this study, we have demonstrated the feasibility of distance wound consultation using digital images of a quality consistent with consumer-grade digital photography.