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Pterygium, an ultraviolet radiation (UV)-related disease, is a relatively benign process, but since it displays tumor-like features, it has been proposed to be a neoplastic- like growth disorder. Vitamin D performs a number of functions in addition to calcium homeostasis, as inhibition of cell proliferation, activation of apoptotic pathways, and inhibition of angiogenesis. Since the antitumor actions of vitamin D are mediated primarily through the nuclear vitamin D receptor (VDR), the aim of the present study was to investigate vitamin D status in patients with pterygium and in control subjects, and VDR immunohistochemical expression in samples of pterygium and normal conjunctiva in order to evaluate a possible role of vitamin D pathway in the pathogenesis of the disease. Serum vitamin D concentration was measured among 41 patients with pterygium and 47 volunteers by an automated chemiluminescence immunoassay. Moreover, 23 formalin- fixed and paraffin-embedded pterygium biopsy samples and 24 conjunctiva specimens were treated for the immunohistochemical demonstration of VDR using the streptavidin-biotin alkaline phosphatase method. No differences were observed about vitamin D level between patient with pterygium and control group, but significant differences between VDR immunolocalization in pterygium and normal conjunctiva were observed (P=0.00001). In conjunctiva, the immunoreactivity, localized mainly in cytoplasm of epithelial cells, may probably demonstrate VDR regulation of cell growth, differentiation, and apoptosis, while in pterygium VDR co-localization in the nucleus and cytoplasm of epithelial cells may indicate alternative nuclear pathways by which vitamin D might exert its antiinflammatory and anti-proliferative effects by the regulation of gene expression.

PER3 gene polymorphisms have been associated with differences in human sleep-wake phenotypes, and sensitivity to light. The aims of this study were to assess: i) the frequency of allelic variants at two PER3 polymorphic sites (rs57875989 length polymorphism: PER34, PER35; rs228697 SNP: PER3C, PER3G) in relation to sleep-wake timing; ii) the effect of morning light on behavioural/circadian variables in PER34/PER34 and PER35/PER35 homozygotes. 786 Caucasian subjects living in Northern Italy donated buccal DNA and completed diurnal preference, sleep quality/timing and sleepiness/mood questionnaires. 19 PER3 4 /PER3 4 and 11 PER3 5 /PER3 5 homozygotes underwent morning light administration, whilst monitoring sleep-wake patterns and the urinary 6-sulphatoxymelatonin (aMT6s) rhythm. No significant relationship was observed between the length polymorphism and diurnal preference. By contrast, a significant association was observed between the PER3G variant and morningness (OR = 2.10), and between the PER3G-PER34 haplotype and morningness (OR = 2.19), for which a mechanistic hypothesis is suggested. No significant differences were observed in sleep timing/aMT6s rhythms between PER35/PER35 and PER34/PER34 subjects at baseline. After light administration, PER34/PER34 subjects advanced their aMT6s acrophase (p PER3 polymorphic variants/their combinations and both diurnal preference and the response to light.

The assessment of diurnal preference, or the preferred timing of sleep and activity, is generally based on comprehensive questionnaires such as the Horne-Ostberg (HO). The aim of the present study was to assess the reliability of a subject's self-classification as extremely morning (Self-MM), more morning than evening (Self-M), more evening than morning (Self-E) or extremely evening (Self-EE) type, based on the last question of the HO (Self-ME). A convenience sample of 461 subjects [23.8 ± 4.7 years; 322 females] completed a full sleep-wake assessment, including diurnal preference (HO), night sleep quality (Pittsburgh Sleep Quality Index, PSQI), daytime sleepiness (Karolinska Sleepiness Scale, KSS), and habitual sleep-wake timing (12 d sleep diaries; n = 296). Significant differences in HO total score were observed between Self-ME classes, with each class being significantly different from neighboring classes (p

ObjectiveThe purpose of the study was to examine whether the insertion (I) and/or deletion (D) polymorphism of ACE confers susceptibility to primary pterygium in Sardinian patients in a case–control study.Methods and resultsPolymorphism genotyping was performed by nested PCR using genomic DNA extracted from the whole peripheral blood of participants with (n=251) and without (n=260) pterygium. DD, ID and II genotype frequencies were: 48%, 39% and 13%, respectively, for patients with pterygium, and 15%, 40% and 44%, respectively, for the control group. A statistically significant difference was found between the pterygium and control groups for the ACE I/D polymorphism (pConclusionsStatistical analysis showed that the DD genotype is associated with an increased risk of developing pterygium, and with a good chance that the D allele may play an important role in the development of disease.

Hyperammonaemia is observed after prolonged, intense exercise, or in patients with hepatic failure. In the latter, it isassociatedwith a set of neurological and psychiatric abnormalities termedhepatic encephalopathy. The aimsof our study were: 1. to measure vigilance in a condition of induced hyperammonaemia; 2. to assess whether caffeine modulates the effects of hyperammonaemia on vigilance, if any. Ten healthy volunteers (28.5±5 years; 5 males) underwent three experimental sessions consisting of two-hourly measurements of capillary ammonia, subjective sleepiness (Karolinska Sleepiness Scale) and vigilance (Psychomotor Vigilance Task, PVT), in relation to the intake of breakfast (+/�coffee), an amino acid mixture which induces hyperammonaemia (amino acid challenge; AAC), and AAC+coffee (only for participants who had coffee with their standard breakfast). The AAC resulted in: 1. the expected increase in capillary ammonialevels,withhighest valuesatapproximately4hafter the administration; 2. a significant increase in subjective sleepiness ratings; 3. a sustained increase in PVT-based reaction times. When caffeine was administered after the AAC, both subjective sleepiness and the slowing in RTs were significantly milder than in the AAC-only condi- tion. In conclusion, acute hyperammonaemia induces an increase in subjective sleepiness and a sustained decrease in vigilance, which are attenuated by the administration of as ingle espresso coffee.

An early event in melanocytic tumor growth is the upregulation of Notch signaling. When an active form of Notch1 is overexpressed in primary human melanocytes, it increases cell growth, survival and invasive properties, promoting melanoma progression. Recent evidence suggested that tumor initiation and growth are driven by a subset of tumor-initiating cells termed cancer stem cells. Notch1 plays a predominant role in the maintenance of melanoblasts, including melanocyte stem cells, by preventing initiation of apoptosis. Moreover, the importance of Notch1 in the regulation of tumor angiogenesis is supported by growing evidence in various cancers. Nestin has been widely used as a marker for melanocyte stem cells as well as an angiogenic marker to evaluate neovascularity of endothelial cells in tumors. To gain an insight into the impact of Notch1 activation on the maintenance of melanocyte stem cells and angiogenesis in melanoma, the expression levels of activated Notch1 and nestin were analyzed by immunohistochemistry in 114 primary cutaneous melanomas and 35 lymph node metastases. Activated Notch1 and nestin expression was also evaluated in four dysplastic melanocytic nevi. This study provides evidence that activated Notch1 is overexpressed in cutaneous melanoma, in tumor cells as well as in microvessel endothelium, and that it can promote tumor angiogenesis. Indeed, the overexpression of activated Notch1 in both tumor and vascular endothelial cells was significantly associated with microvascular density in melanoma samples. Thus, activated Notch1 inhibitors may provide a therapeutic strategy in the treatment of melanoma by blocking tumor-associated vascularization.

Circadian rhythmicity and non-visual sensitivity to light can be assessed, in healthy subjects, by measuring the rhythm of the urinary melatonin metabolite 6-sulphatoxymelatonin (aMT6s) and by determining the response of plasma melatonin to nocturnal retinal light exposure, respectively. However, the validity of these techniques has not been assessed in disease states in which disruption of the circadian rhythm is known or suspected to occur. Thus, the aims of this study were as follows: (i) to assess the reliability of circadian aMT6s profile estimates derived from 36 h versus 56 h urine collections and (ii) to test different models for calculating melatonin suppression in response to light in healthy volunteers and patients with cirrhosis. Twenty patients with biopsy-proven cirrhosis and 10 matched healthy volunteers undertook: (i) separate 36 - and 56-h urine collections, under controlled conditions, for cosinor analysis of the urinary aMT6s profile; (ii) a melatonin suppression test, comprising of a baseline night, during which subjects were woken and asked to sit in front of a switched off light sphere, and an experimental night, identically executed, except that the light sphere was switched on and the subjects were exposed to white light (4.1 × 10(14) photons/cm(2)/s) for 30 min. Alternative approaches to the calculation of melatonin suppression were taken, with/without inclusion of the baseline night. Eighteen patients and eight healthy volunteers had matched analysable 36 - and 56-h urinary samples. Cosinor analysis showed a significant fit in 88% of the remaining 56 h collections, and 48% of the remaining 36-h collections. Thus, eight patients and five healthy volunteers had matched analysable samples for cosinor analysis. In the healthy volunteers, aMT6s profile indices obtained using the 36 - and the 56-h collections did not differ significantly. In contrast, considerably more variability was observed in patients [i.e. the difference in the aMT6s peak time was 0.5 ± 1.7 h (limits of agreement: -3.9; +2.9 h)]. No difficulties were encountered in obtaining suppression estimates by use of the experimental night only. In contrast, suppression estimates obtained by use of both nights were considered inaccurate in one (11%) healthy volunteer and in 5 (28%) patients, primarily because: (i) melatonin concentrations at the beginning of light administration were significantly different on baseline and experimental night; (ii) the rise in melatonin was inconsistent on baseline night; and (iii) the shape of the rising phase of melatonin was different on baseline and experimental night. In conclusion, shorter urine collections lead to a higher number of profiles with no significant cosinor fit, and differences in cosinor indices obtained from the 36 - and 56-h collections were considerable, especially in patients. Thus, 56-h collections are probably advisable. Use of both baseline and experimental nights to calculate melatonin suppression often resulted in increased variation and confounding, due to point oscillations in melatonin concentration and lack of repeatability of the melatonin profiles on the two nights. Thus, use of the experimental night only is probably advisable.

Pterygium is a common ocular surface disorder characterized by excessive cell proliferation, inflammation, fibrosis, angiogenesis and extracellular matrix remodeling. The Angiotensin converting enzyme (ACE or ACE I) is the major component of the Renin-angiotensin system (RAS) converting the inactive decapeptide Angiotensin I (Ang I) to the active octapeptide Angiotensin II (Ang II). Besides this 'classical role', it can act as transcriptional regulator in response to external stimuli that may lead to cell damage and tissue remodeling. Due to this role, it can be internalized into the nuclear compartment to act as transcriptional factor for proteins involved in the inflammatory response. The aim of the present study was to determine ACE expression and localization in pterygium and culture pterygium cells by immunohistochemistry. Our results are the first to demonstrate nuclear immunolocalization of ACE, more so in pterygium compared to conjunctiva epithelial cells in histological sections. ACE was not detected in the nuclei of subcultivated pterygium epithelial cells. The nuclear localization of ACE may be correlated with an anti-inflammatory path mediated by activation of its transcriptional role.

Nestin (a neuronal stem cell/progenitor cell marker of central nervous system development), vimentin (which is ubiquitously expressed in mesenchymal cells), and the glucocorticoid receptor (GR, which is involved in the immune response, cell proliferation, and apoptosis) have been shown to interact in embryonic and undifferentiated tissues in modulating cell proliferation. The aim of this study was to analyse nestin, vimentin and GR expression in tumour tissue (melanoma), and their association with clinicopathological variables, to evaluate any effect on tumour progression. Immunohistochemistry, double-label immunofluorescence and confocal laser scanning microscopy were performed on biopsy specimens of cutaneous melanoma from 81 patients. Fisher's and Pearson's tests showed a correlation between nestin, vimentin and subcellular GR location (P = 0.008). Their concomitant expression also correlated with Clark level and thickness (P = 0.02 and P = 0.029, respectively). Kaplan-Meier analysis revealed a poorer outcome for stage III and IV patients with associated expression of nestin, vimentin and cytoplasmic GR in tumour tissue (P = 0.02). These results suggest the presence in melanoma of growth mechanisms involving nestin, vimentin, and GR, similarly to that occurring in embryonic and undifferentiated cells, and may help in understanding tumour biology to provide a molecular basis for clinical therapies.