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International audience; To ensure successful offspring ploidy, vertebrate oocytes must halt the cell cycle in meiosis II until sperm entry. Emi2 is essential to keep oocytes arrested until fertilization. However, how this arrest is implemented exclusively in meiosis II and not prematurely in meiosis I has until now remained enigmatic. Using mouse and frog oocytes, we show here that cyclin B3, an understudied B-type cyclin, is essential to keep Emi2 levels low in meiosis I. Direct phosphorylation of Emi2 at an evolutionarily highly conserved site by Cdk1/cyclin B3 targets Emi2 for degradation. In contrast, Cdk1/cyclin B1 is inefficient in Emi2 phosphorylation, and this provides a molecular explanation for the requirement of different B-type cyclins for oocyte maturation. Cyclin B3 degradation at exit from meiosis I enables Emi2 accumulation and thus timely arrest in meiosis II. Our findings illuminate the evolutionarily conserved mechanisms that control oocyte arrest for fertilization at the correct cell-cycle stage, which is essential for embryo viability.

Cohesin is a conserved, ring-shaped protein complex that topologically entraps DNA. This ability makes this member of the structural maintenance of chromosomes (SMC) complex family a central hub of chromosome dynamics regulation. Besides its essential role in sister chromatid cohesion, cohesin shapes the interphase chromatin domain architecture and plays important roles in transcriptional regulation and DNA repair. Cohesin is loaded onto chromosomes at centromeres, at the promoters of highly expressed genes, as well as at DNA replication forks and sites of DNA damage. However, the features that determine these binding sites are still incompletely understood. We recently described a role of the budding yeast RSC chromatin remodeler in cohesin loading onto chromosomes. RSC has a dual function, both as a physical chromatin receptor of the Scc2/Scc4 cohesin loader complex, as well as by providing a nucleosome-free template for cohesin loading. Here, we show that the role of RSC in sister chromatid cohesion is conserved in fission yeast. We discuss what is known about the broader conservation of the contribution of chromatin remodelers to cohesin loading onto chromatin.

Introduction: The Integrated Care Pathway (ICP) represents a multidisciplinary outline of anticipated care, placed in an appropriate timeframe, to support patients with specific conditions or symptoms. The aim of this paper is to define the ICP for patients with melanoma referring to the “Istituto Dermopatico dell’Immacolata-IRCCS di Roma e Villa Paola” (“Center”). Methods and results: A multidisciplinary group (oncologists, dermatologists, surgeons, pathologists etc.) was defined as well as a facilitator to act as a link between all experts. The first step of ICP development was a review of current practice for patients with melanoma referring to the Center. This first step had the scope to define the multidisciplinary process map (a “picture” of the care plan) for patients with melanoma. The process map defined: i) the activities performed during delivery of care to the patients, ii) the responsibilities for these activities and iii) potential problem areas or opportunities for improvements. The process map formed the basis of the final ICP document. Conclusion: The adoption of melanoma ICP will allow the multidisciplinary group to ensure that clinical guidelines and available evidence are incorporated into everyday practice. (Oncology, HTA & Market Access)

The coronavirus disease (COVID‐19), during its course, may involve several organs, including the skin with a petechial skin rash, urticaria and erythematous rash, or varicella‐like eruption, representing an additional effect of the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection, as commonly observed in other viral diseases. Considering that symptomatic patients with COVID‐19 generally undergo multidrug treatments, the occurrence of a possible adverse drug reaction presenting with cutaneous manifestations should be contemplated. Pleomorphic skin eruptions occurred in a 59‐year‐old Caucasian woman, affected by a stable form of chronic lymphocytic leukemia, and symptomatic SARS‐CoV‐2 infection, treated with a combination of hydroxychloroquine sulfate, darunavir, ritonavir, sarilumb, omeprazole, ceftriaxone, high‐flow oxygen therapy devices, filgrastim (Zarzio®) as a single injection, and enoxaparin. The patient stopped all treatment but oxygen and enoxaparin were continued and the patient received a high‐dose Desametasone with complete remission of dermatological impairment in 10 days. It is very important to differentially diagnose COVID‐19 disease‐related cutaneous manifestations, where is justified to continue the multidrug antiviral treatment, from those caused by an adverse drug reaction, where it would be necessary to identify the possible culprit drug and to start appropriate antiallergic treatment., The coronavirus disease (COVID‐19), during its course, may involve several organs, including the skin with a petechial skin rash, urticaria and erythematous rash, or varicella‐like eruption, representing an additional effect of the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection, as commonly observed in other viral diseases. Considering that symptomatic patients with COVID‐19 generally undergo multidrug treatments, the occurrence of a possible adverse drug reaction presenting with cutaneous manifestations should be contemplated. Pleomorphic skin eruptions occurred in a 59‐year‐old Caucasian woman, affected by a stable form of chronic lymphocytic leukemia, and symptomatic SARS‐CoV‐2 infection, treated with a combination of hydroxychloroquine sulfate, darunavir, ritonavir, sarilumb, omeprazole, ceftriaxone, high‐flow oxygen therapy devices, filgrastim (Zarzio®) as a single injection, and enoxaparin. The patient stopped all treatment but oxygen and enoxaparin and received a high‐dose Desametasone with complete remission of dermatological impairment in 10 days. It is very important to differentially diagnose COVID‐19 disease‐related cutaneous manifestations, where is justified to continue the multi‐drug antiviral treatment, from those caused by an adverse drug reaction, where it would be necessary to identify the possible culprit drug and to start appropriate antiallergic treatment.

The coronavirus disease (COVID-19), during its course, may involve several organs, including the skin with a petechial skin rash, urticaria and erythematous rash, or varicella-like eruption, representing an additional effect of the SARS-CoV-2 infection, as commonly observed in other viral diseases. Considering that symptomatic COVID-19 patients generally undergo multi-drug treatments, the occurrence of a possible adverse drug reaction (ADR) presenting with cutaneous manifestations should be contemplated. Pleomorphic skin eruptions occurred in a 59-year-old Caucasian woman, affected by a stable form of Chronic lymphocytic leukaemia, and symptomatic SARS-CoV.2 infection, treated with a combination of hydroxychloroquine sulphate, darunavir, ritonavir, sarilumb, omeprazole, ceftriaxone, high-flow oxygen therapy devices, filgrastim (Zarzio®) as a single injection, and enoxaparin. The patient stopped all treatment but oxygen and enoxaparin and received a high-dose Desametasone with complete remission of dermatological impairment in 10 days. It is very important to differentially diagnose COVID-19 disease-related cutaneous manifestations, where is justified to continue the multi-drug antiviral treatment, from those caused by an adverse drug reaction, where it would be necessary to identify the possible culprit drug and to start an appropriate treatment.

Variably reduced expression of the basement membrane component laminin-332 (α3aβ3γ2) causes junctional epidermolysis bullosa generalized intermediate (JEB-GI), a skin fragility disorder with an increased susceptibility to squamous cell carcinoma (SCC) development in adulthood. Laminin-332 is highly expressed in several types of epithelial tumors and is central to signaling pathways that promote SCC tumorigenesis. However, laminin-332 mutations and expression in individuals affected by JEB-GI and suffering from recurrent SCCs have been poorly characterized. We studied a JEB-GI patient who developed over a hundred primary cutaneous SCCs. Molecular analysis combined with gene expression studies in patient skin and primary keratinocytes revealed that the patient is a functional hemizygous for the p.Cys1171* mutant allele which is transcribed in a stable mRNA encoding for a β3 chain shortened of the last two C-terminal amino acids (Cys1171-Lys1172). The lack of the Cys1171 residue involved in the C-terminal disulphide bond to γ2 chain did not prevent assembly, secretion, and proteolytic processing of the heterotrimeric molecule. Immunohistochemistry of SCC specimens revealed accumulation of mutant laminin-332 at the epithelial-stromal interface of invasive front. We conclude that the C-terminal disulphide bond is a structural element crucial for laminin-332 adhesion function in-vivo. By saving laminin-332 amount, processing, and signaling role the p.Cys1171* mutation may allow intrinsic pro-tumorigenic properties of the protein to be conveyed, thus contributing to invasiveness and recurrence of SCCs in this patient.

Breast asymmetry can be congenital or developmental, however a tumorous growth may be the cause of this condition after puberty. A 19-year-old female presented with a slowly developing breast asymmetry pre-operatively diagnosed as Pseudoangiomatous Stromal Hyperplasia (PASH). The patient underwent tumour excision with breast gland remodelling. Postoperative course was uneventful.

Sezary syndrome (SS) is a rare and aggressive variant of Cutaneous T-Cell Lymphoma characterized by neoplastic distribution mainly involving blood, skin, and lymph-node. Although a role of the skin microenvironment in SS pathogenesis has long been hypothesized, its function in vivo is poorly characterized. To deepen this aspect, here we compared skin to blood-derived SS cells concurrently obtained from SS patients highlighting a greater proliferation-index and a PI3K/AKT/mTORC1 pathway activation level, particularly of mTOR protein, in skin-derived-SS cells. We proved that SDF-1 and CCL21 chemokines, both overexpressed in SS tissues, induce mTORC1 signaling activation, cell proliferation and Ki67 up-regulation in a SS-derived cell line and primary-SS cells. In a cohort of 43 SS cases, we observed recurrent copy number variations (CNV) of members belonging to this cascade, namely: loss of LKB1 (48%), PTEN (39%) and PDCD4 (35%) and gains of P70S6K (30%). These alterations represent druggable targets unraveling new therapeutic treatments as metformin here evaluated in vitro. Moreover, CNV of PTEN, PDCD4, and P70S6K, evaluated individually or in combination, are associated with reduced survival of SS patients. These data shed light on effects in vivo of skin-SS cells interaction underlying the prognostic and therapeutic relevance of mTORC1 pathway in SS.

Human keratinocytes were recently shown to respond to anti-EGFR (epidermal growth factor receptor) drugs with activation of an interferon-&kappa, driven autocrine loop, leading to enhanced expression of innate antiviral effectors and of the pro-inflammatory chemokines CXCL10 (C-X-C motif chemokine 10) and CCL2 (C-C motif ligand 2). Here we showed active type I interferon signaling in the skin lesions of cancer patients undergoing treatment with the anti-EGFR drug cetuximab. Strong nuclear positivity for Interferon Regulatory Factor 1 and phosphorylated Signal Transducer and Activator of Transcription 1, enhanced interferon-&kappa, expression and CXCL10 was associated to the epidermal compartment. Notably, 50 micromolar resveratrol and quercetin fully suppressed the low constitutive levels of type I interferon signaling and prevented its activation by the anti-EGFR cetuximab or gefitinib in cultured keratinocytes. In sensitized mice undergoing DNFB (2,4-dinitro-1-fluorobenzene)-induced contact hypersensitivity, local administration of gefitinib prior to elicitation further amplified hapten-induced type I interferon activation, tissue edema, and infiltration by T cells, whereas resveratrol or quercetin suppressed this inflammatory cascade. Overall, these data suggest that topical application of resveratrol or quercetin could be potentially effective in preventing pathological conditions due to overactivation of type I IFN (interferon)-driven circuits in the skin, including the inflammatory manifestations of anti-EGFR drug-induced skin-targeted toxicity.

Key Clinical Message We report on a case of Sweet's syndrome associated with multiple myeloma, as harbinger for disease relapse.

Interleukin-17 (IL-17) and IL-22 have been reported to play critical roles in autoimmunity and inflammation but information about their role in cancer is limited. In this study, we investigated the role of IL-17 and IL-22 in the progression of human skin basal-cell carcinoma (BCC) and squamous-cell carcinoma (SCC). We found that both tumor types are infiltrated with an high number of IL-17(+) and IL-22(+) T lymphocytes, as demonstrated by immunohistochemistry and by FACS analysis performed on peritumoral T-cell lines isolated from skin biopsies. In vitro studies demonstrated that proliferation and migration of the BCC- and SCC-cell lines M77015 and CAL27 were increased by IL-17 and IL-22. Moreover, IL-17, alone or in combination with TNF-α, was able to induce the production of two cytokines important for tumor progression, IL-6 and IL-8, in CAL27. We also showed that IL-17 upregulated NF-κB signaling, while IL-22 activated the STAT3 pathway and the antiapoptotic AKT protein in M77015 and CAL27. Finally, in vivo experiments demonstrated that IL-17 and IL-22 enhanced tumor growth in nude mice injected with CAL27. Altogether, our findings indicate that high levels of IL-22 and IL-17 in the BCC and SCC microenvironment promote tumor progression.

Caralluma fimbriata Wall. is currently used as a “natural slimming” food supplement, likely due to its content in pregnane glycosides. In the present study, a commercially available Caralluma fimbriata extract (Slimaluma®; CFE, 100 mg/kg) has been evaluated for its ability to affect the ingestive behaviour in female rats, also with reference to the modulation of the brain neuropeptides NPY and ORX.The interference of CFE with α-amylase and lipase enzymes has been investigated in vitro, as possible peripheral mechanism of action. Also, the chemical composition of CFE has been assessed by NMR and spectrophotometric analysis. Results from in vivo study showed that CFE induced effects neither on blood parameters, nor on liver and gut histomorphology. Interestingly, a reduction in body weight gain with an increase in water intake and hypothalamic levels of NPY and ORX peptides were found. Phytochemical analysis, showed CFE contained about 12% of pregnane glycosides and 1.3% of polyphenols. Present results suggest possible effects of C. fimbriata on ingestive behaviour, likely mediated by central and peripheral mechanisms.

Objectives: The outcome of patients with primary melanoma (PM) cannot be completely explained based on currently adopted clinical-histopathologic criteria. In this study, we evaluated the potential prognostic value of mismatch repair protein expression in PMs. Methods: We examined the immunohistochemical staining of mismatch repair proteins in 18 benign nevi and 101 stage I to III PMs and investigated their association with tumor clinicopathologic variables and melanoma mortality. Results: Expression of MSH2, MLH1, and PMS2 was high in benign nevi and reduced in a subset of PMs. Conversely, MSH6 expression was absent or extremely low in benign nevi and increased in a subset of PMs. In the multivariate analysis, including sex, age, Breslow thickness, and ulceration, high MSH6 expression in PMs (ie, immunostaining in >20% of tumor cells) was significantly associated with an increased risk of melanoma mortality (relative risk, 3.76; 95% confidence interval, 1.12–12.70). Conclusions: MSH6 protein expression can be a valuable marker to improve prognosis assessment in PMs.

An autoimmune hypothesis has been suggested for several disorders in childhood. The aim of the study was to clarify the role of the cerebellum in ADHD and to evaluate the possible association between anti-Yo antibodies and ADHD. The presence/absence of antibodies was tested by indirect immunofluorescence assay on 30 combined subtype ADHD children, on 19 children with other psychiatric disorders (Oppositional-defiant and Conduct Disorders, Dyslexia) and 27 healthy controls. Results showed a significant positive response to the anti-Yo antibody immunoreactivity in the Purkinje cells of the cerebellum of ADHD children, compared with the control group and the psychiatric non-ADHD children. This association points to an immune dysregulation and the involvement of the cerebellum in ADHD.

Fortes, Cristina Mastroeni, Simona Bonamigo, Renan Mannooranparampil, Thomas Marino, Claudia Michelozzi, Paola Passarelli, Francesca Boniol, Mathieu eng England 2015/02/04 06:00 Eur J Cancer Prev. 2016 Jan;25(1):34-40. doi: 10.1097/CEJ.0000000000000127.; International audience; Some studies have suggested that sun exposure plays a protective role in melanoma survival. This created a paradox as the known carcinogen can act as a cancer promoter and also as a survival enhancer. The aim of this study was to investigate the effect of sun exposure on melanoma mortality using both ambient sun exposure and individual data. A 10-year cohort study was carried out on primary cutaneous melanoma cases (n=972). Residential data were coupled with levels of ultraviolet radiation (UV) to provide a measure of individual exposure. Demographic, histological and clinical data were obtained for all participants. In a subsample, information on pigmentary characteristics, diet, medical history, phenotype and self-reported sun exposure was also collected. Survival analysis and Cox proportional hazards models were used to examine associations. No protective effect was found for UVB or individual sun exposure variables on melanoma mortality. However, an increased risk of mortality was found among patients with cutaneous melanoma located on the lower limbs and in the highest decile of UVB exposure (>/=3.298 J/cm) after controlling for sex, age and Breslow thickness (relative risk: 4.78; 95% confidence interval: 1.30-17.5). The increased risk of mortality for the highest decile of UVB was also confirmed in the subsample after controlling for sex, age, education, use of sun lamps, pigmentary characteristics and diet. The results of the study suggested no protective effect of sun exposure for melanoma mortality and showed that high sun exposure increases the risk of melanoma mortality among patients with melanomas located on the lower limbs.

Sezary Syndrome (SS) is an aggressive variant of cutaneous T cell lymphoma (CTCL) characterized by an accumulation of malignant T lymphocytes - the Sezary (SS) cells - in the skin, lymph nodes and blood. Until now, most studies on the characterization of this lymphoma have been performed on circulating SS cells because of the easy availability of tumor material from this compartment. For this reason, the role of skin microenvironment has not been characterized although it might play a fundamental role in the pathogenesis and in the disease progression of this neoplasia. According to this view, we recently demonstrated that PTEN downregulation, due to genetic loss and microRNA deregulation, commonly observed in SS patients, mainly activates AKT in skin-resident respect to blood-derived SS cells. This data underline how microenvironment signals amplify genetic abnormalities. Based on these observations, we investigated the effect of the skin microenvironment on SS cells in vivo. Comparing skin and blood-derived SS cells concurrently obtained from SS patients, we demonstrate that skin milieu promotes proliferation and PI3K/AKT/mTORC1 activation, a pathway that we prove to have multiple copy number (CN) alterations in our cohort of forty-three patients, thus indicating these genetic lesions as valid therapeutic targets to treat SS. At the end, we also demonstrate that the progressive increase of these CN alterations is associated to the shortening of overall survival of SS patients. Citation Format: Cristina Cristofoletti, Antonella Bresin, Mario Picozza, Maria Cristina Picchio, Francesca Passarelli, Francesca Monzo, Mauro Helmer Citterich, Alessandro Monopoli, Roberto Benucci, Maria Cantonetti, Alessandra Frezzolini, Enrico Scala, Elisabetta Caprini, Giandomenico Russo, Maria Grazia Narducci. The role of PI3 kinase pathway in the the skin of Sezary syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 761.

Summary We describe the landscape of genomic alterations in cutaneous melanomas through DNA, RNA, and protein-based analysis of 333 primary and/or metastatic melanomas from 331 patients. We establish a framework for genomic classification into one of four subtypes based on the pattern of the most prevalent significantly mutated genes: mutant BRAF , mutant RAS , mutant NF1 , and Triple-WT (wild-type). Integrative analysis reveals enrichment of KIT mutations and focal amplifications and complex structural rearrangements as a feature of the Triple-WT subtype. We found no significant outcome correlation with genomic classification, but samples assigned a transcriptomic subclass enriched for immune gene expression associated with lymphocyte infiltrate on pathology review and high LCK protein expression, a T cell marker, were associated with improved patient survival. This clinicopathological and multi-dimensional analysis suggests that the prognosis of melanoma patients with regional metastases is influenced by tumor stroma immunobiology, offering insights to further personalize therapeutic decision-making.

Aims: The role of the serotonin transporter gene (SLC6A4) in alcohol dependence (AD) is still unclear. In this paper,we have evaluated the association of theSLC6A4gene polymorphisms5-HTTLPR and rs25531 in AD and assessed the polymorphic patterns both in alcoholics and in healthy people of an Italian population. Methods: Genotyping of the 5-HTTLPR (L/S) and rs25531 (A/G) polymorphisms of the SLC6A4 gene was performed on 403 alcoholics outpatients and 427 blood donors. Results: Comparing AD and control populations and taking into account statistical correction for multiple testing, we found no statisticallysignificant differences for 5-HTTLPR (L/S) and rs25531 polymorphisms in terms of either genotypes or alleles frequencies. By univariate ANOVA, a statistically significant differencewas found in the onset of AD: the mean age of onset resulted to be of 25.4 years in males in respect to 28.1 in females. In particular in males, the early AD onset was different, in a statisticallysignificant manner, depending on the presence of at least one S or Lg allele (24.6 years) in respect to La homozygotes (27.5 years) (P= 0.03). Conclusions: These findings suggest that genetic factors contribute, together with gender and age, to the onset differences in alcohol-dependent phenotypes.

It has been proposed that S100B can be a marker for several pathological conditions including brain traumas, blood-brain barrier disruption, and ischemia. Because the hypothalamo-pituitary-adrenal axis is activated in these conditions, we investigated the role of glucocorticoids in the effects of stress on serum S100B. Restraint stress increased S100B levels in control and in adrenalectomized but not in corticosterone-injected rats. Adrenalectomy did not alter basal S100B. These results indicate a glucocorticoid-independent relationship between stress and S100B.

Rett syndrome (RTT) is a rare neurodevelopmental disorder, characterized by severe behavioral and physiological symptoms. Mutations in the methyl CpG-binding protein 2 gene (MECP2) cause >95% of classic cases, and currently there is no cure for this devastating disorder. The serotonin receptor 7 (5-HT7R) is linked to neuro-physiological regulation of circadian rhythm, mood, cognition, and synaptic plasticity. We presently report that 5-HT7R density is consistently reduced in cortical and hippocampal brain areas of symptomatic MeCP2–308 male mice, a RTT model. Systemic repeated treatment with LP-211 (0.25 mg/kg once/day for 7 days), a brain-penetrant selective 5-HT7R agonist, was able to rescue RTT-related defective performance: anxiety-related profiles in a Light/Dark test, motor abilities in a Dowel test, the exploratory behavior in the Marble Burying test, as well as memory in the Novelty Preference task. In the brain of RTT mice, LP-211 also reversed the abnormal activation of PAK and cofilin (key regulators of actin cytoskeleton dynamics) and of the ribosomal protein (rp) S6, whose reduced activation in MECP2 mutant neurons by mTOR is responsible for the altered protein translational control. Present findings indicate that pharmacological targeting of 5-HT7R improves specific behavioral and molecular manifestations of RTT, thus representing a first step toward the validation of an innovative systemic treatment. Beyond RTT, the latter might be extended to other disorders associated with intellectual disability.

The H1 haplotype of the MAPT gene influences the risk of PD and has been related to the development of PDD. We evaluated the influence of MAPT haplotypes on the expression of motor features in PD patients. We genotyped, for the MAPT haplotypes H1 and H2, a sample of 181 PD patients with distinct clinical subtypes: tremor dominant and non-tremor dominant (NTD). Our results indicate that the MAPT haplotypes contribute to the expression of motor features of PD. H1 homozygous PD patients are significantly more likely to present a NTD phenotype, a clinical subtype characterized by widespread pathological degeneration, than H2 carriers.

Introduction Sézary Syndrome (SS) is a rare and aggressive variant of Cutaneous-T-Cell Lymphoma (CTCL) characterized by the presence of malignant lymphocytes named Sezary (SS) cells in the skin, lymph nodes, and peripheral blood. With a poor prognosis, SS has not a specific therapy still available. As a role of skin in SS pathogenesis is not elucidated yet, here we study the contribution of this microenvironment by comparing matched skin and blood derived SS cells for tumor cell proliferation and activation levels of PI3k/AKT pathway. Using our previous SNP array data we also verified the genomic status of members belonging to this pathway in a large cohort of SS patients. Methods Expression of Ki67 proliferation marker was evaluated in perfect-paired blood and skin-paraffin biopsies obtained from eleven SS patients by flow cytometry analysis and immunohistochemistry respectively. KI67+ neoplastic cells were calculated as percentage within neoplastic CD4+ cells recognized by a co-staining with the specific TCRVb rearrangement. Phosphorylation levels of members of PI3k/AKT pathway were compared between matched circulating and skin resident SS cells proteins derived from 2 SS patients using an AKT kinase array (Cell Signaling). Affymetrix SNP6.0 arrays was used to investigate the copy number (CN) status of members of mTORC 1 pathway in a cohort in 37 SS samples derived from 23 SS patients and 3 cell lines. Results Skin derived SS cells showed a significant higher proliferation index (PI) respect to SS cells obtained from blood (12%±11 vs 1,24%+1,18; P = 0,00025). In order to identify the signals responsible for SS proliferation, we used a PI3K/AKT kinase array that revealed an enhanced phosphorylation levels of many components of this cascade, particularly of PRAS40 with a Fold change (Fc) of 6,15; GSK3a (Fc = 4.83), mTOR (Fc = 4.61) mP70S6K (Fc = 4.64) BAD (Fc = 7.09) and 4EBP1 (Fc = 5.40)in skin-SS cells respect to circulating ones. We next deepen our observations in mTORC1 signaling because of this and earlier observations made in CTCL cell lines. Using our SNP6 array data we have verified the genomic status of members of this pathway. Results obtained showed that P70S6K, the kinase downstream TORC1 that plays a crucial role in protein synthesis, showed a mono-allelic gain in 9 out 23 patients (39%) whereas PDCD4, a protein that inhibits protein translation displayed a mono-allelic loss in 10 of 23 individuals (43%). 4 of these patients showed a concomitant P70S6K gain and PDCD4 loss. Overall these genetic defects suggest that an abnormal protein synthesis can occur in these patients.. Conclusion: Our data demonstrate that skin microenvironment enhances SS cell proliferation index and activates mTORC1 signaling, an unbalanced pathway that reveals novel potential therapeutic targets for Sezary Syndrome. Citation Format: Cristina Cristofoletti, Mario Picozza, Antonella Bresin, Maria Cristina Picchio, Enrico Scala, Giuseppe A. Lombardo, Francesca Passarelli, Elisabetta Caprini, Giandomenico Russo, Maria Grazia Narducci. Skin microenvironment enhances proliferation index and activates mTORC 1 signaling in sezary syndrome. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 936.

Nociceptin/orphanin FQ (N/OFQ) peptide and its receptor are not only ubiquitously expressed in mammalian brain and spinal cord but are also abundant in limbic structures, particularly in the hippocampus. The widespread distribution of N/OFQ reflects the broad spectrum of its biological actions such as nociception, food intake, spontaneous locomotor activity, and learning and memory processes. Since the hippocampus is involved in the control of adrenocortical activity, its role in stress-related phenomena is well characterized. In male Wistar rats, we first examined the effects of acute restraint stress (120 min) on the brain immunohistochemical localization of N/OFQ. The analysis carried out on sections obtained at the onset of stress revealed enhanced expression of N/OFQ in CA1, CA3, and the dentate gyrus as well as increased plasma corticosterone concentrations. Next, we examined whether endogenous glucocorticoid hormone plays a role in the modulation of hippocampal N/OFQ expression in response to stress. To this end, rats were injected with corticosterone (1 mg/kg) or subjected to restraint stress 1 week after adrenalectomy. Two hours after corticosterone administration, plasma glucocorticoid concentrations were comparable to those observed after restraint stress, while N/OFQ expression had significantly increased in all the hippocampal subfields examined. By contrast, in adrenalectomized rats, stress did not modify protein expression. These results confirm that stress can affect N/OFQ expression and that glucocorticoids may constitute hormonal mediators of this complex interplay.

BACKGROUND: Cutaneous squamous cell carcinoma (SCC) is the second most frequent skin cancer among Caucasians. Advanced cases determine significant tissue destruction and they can metastasize. OBJECTIVES: To evaluate the role of GPs in timely diagnosis of SCC, examining the probability of delay before the diagnosis and treatment of SCC among patients having first seen a GP compared to patients having accessed directly a dermatologist. METHODS: We included a stratified sample of 308 SCC patients treated at a referral center in Italy. Medical records were reviewed and combined with patient interviews. RESULTS: Multivariable analysis has shown that patients who have first seen a GP have a significantly lower likelihood of long patient delay [odds ratio (OR) = 0.45; 95% confidence interval (95% CI) 0.21-0.94; P = 0.04) compared to patients having accessed directly a dermatologist. Treatment delay was not associated with the specialization of the first doctor seen for the lesion (OR = 0.52; 95% CI 0.15-1.84; P = 0.31). CONCLUSION: Our findings highlight the potential role of the GP in facilitating rapid access to appropriate health care.

Repeated administration of the dopamine D2/D3 agonist quinpirole (QNP) progressively increases non-regulatory water intake. This effect may model psychotic polydipsia, a potentially fatal but poorly understood condition. The growing evidence for a role of orexin in mediating arousal and cognition has linked this peptide to schizophrenia, hence we examined whether manipulations of dopaminergic and orexinergic systems, as well as of setting, would further characterize the model. Water intake was measured in rats sequentially tested in home and then operant conditioning setting, with chronic administration of D2 antagonist haloperidol (Hal) prior to QNP treatment. A group of rats similarly treated was also assessed for orexin A (OxA) expression in the cortex. Finally, the effect of the orexin-1 receptor antagonist SB-334867 on QNP-induced polydipsia was evaluated. In rats made polydipsic by QNP the amount of water drank during the first 4 h was strongly correlated with the degree of dissociation between appetitive and consummatory components of drinking behavior in the following hour of operant access to water. Hal 0.2 mg/kg prevented both polydipsia and the dissociation, while 0.1 mg/kg only blocked the dissociation. Chronic QNP treatment increased, in a Hal-reversible way, OxA expression in the somatosensory cortex (SI). Moreover, pretreatment with SB-334867 sped up and potentiated QNP-induced polydipsia. Results disclose compulsive components in QNP-induced polydipsia that are mediated by dopamine D2 receptors. QNP also regulates OxA expression in the SI, while the block of orexin-1 receptors enhances QNP-induced polydipsia. We suggest that dopamine and OxA play opposite roles in QNP-induced polydipsia.

Sporadic Parkinson's disease (PD) is a frequent neurodegenerative movement disorder. Both environmental and genetic factors have been studied in the etiology of PD. Among genetic factors, increasing evidences suggest that deletion/insertion (D/I) gene polymorphism of the angiotensin I-converting enzyme (ACE) may be involved in the pathogenesis of PD and in the occurrence of the adverse effects of chronic l -dopa therapy. We investigated this hypothesis by evaluating the frequency of the ACE gene D/I polymorphism in 120 Italian PD patients and 132 controls. Out of the 120 PD patients, 91 were under chronic l -dopa treatment. Our results revealed no difference in ACE I/D genotype (χ2 = 0.79, p = 0.66) and allele (χ2 = 0.34, p = 0.56) frequencies between PD and controls. We also failed to observe any significant association with the occurrence of l -dopa-induced adverse effects in long-term treated PD patients, thereby excluding the presence of an association between ACE I/D genotypes and the genetic susceptibility to PD and the development of adverse effect of chronic l -dopa therapy.

Bazex–Dupre–Christol syndrome is a rare X-linked genodermatosis characterized by early-onset nonmelanoma skin cancers, atrophoderma follicularis, hypotrichosis, hypohidrosis, and multiple milia. Its molecular basis remains unknown and nosologic classification is debated. We report a 5-year-old child presenting sparse hair, reduced sweating, ice-pick skin depressions of the dorsum of hands, facial and limb milia, perianal skin hyperpigmentation, and hyperpigmented papules of the axillae and neck. His mother showed similar features but lacked hair involvement. Histologic examination of a skin papule obtained from the index case revealed features consistent with trichoepithelioma. Our findings indicate that trichoepitheliomas are an early sign of Bazex–Dupre–Christol syndrome and may guide the diagnosis even before the development of basal cell carcinomas. The high frequency of hypotrichosis, hypohidrosis and dry skin in Bazex–Dupre–Christol syndrome indicates that it may be better classified as an ectodermal dysplasia. Comparison with other conditions combining features of ectodermal dysplasia and proneness to skin tumors suggests the involvement of a common pathogenic pathway implicated in both skin development and cancer.

Atorvastatin exerts beneficial vascular effects in diabetes, but the underlying mechanisms are yet to be elucidated. The aim of the present study was to determine whether Rac-1 is involved in the effect of atorvastatin on oxidative stress and vascular dysfunction. Using human aortic endothelial cells (HAECs) we evaluated the effect of high glucose levels on peroxide production by dihydrodichlorofluorescein and on Rac-1 activity using immunocytochemistry to detect Rac-1 translocation to the membrane. We evaluated vascular function, peroxide production by dihydroethidium and NADPH oxidase activity in vessels from atorvastatin-treated mice. Rac-1 activity was also assessed, both by immunoprecipitation of the Rac–p21-activated kinase complex and by analysis of Rac-1 translocation to the membrane. These experiments were also conducted in vessels infected with an adenoviral vector carrying a constitutively active mutant of Rac-1. In HAECs exposed to high glucose levels, atorvastatin prevented oxidative stress, and this protection was associated with impaired Rac-1 activation. This effect was also observed in a murine model of diabetes mellitus. More importantly, the addition of geranylgeranyl pyrophosphate (GGPP) blocked the effects of atorvastatin in both glucose-exposed HAECs and diabetic vessels. Atorvastatin failed to afford protection against vascular abnormalities in the presence of a constitutively active mutant of Rac-1. The results of this study demonstrate that the vascular antioxidant effect of atorvastatin in diabetes is mediated through inhibition of Rac-1 via a reduction in GGPP. Thus, selective Rac-1 inhibition should be considered in the design of novel pharmacological strategies to reduce the impact of diabetes mellitus on vascular function.

Endothelial cells from rat brain microvessels, human aortic artery and human umbilical vein were examined, together with ex vivo rat brain capillaries and rat aortic ring sections, for the expression of opioid receptor-like OP-4 mRNA and protein. High levels of mRNA expression and an immunopositive reaction for the receptor protein were detected in the endothelial cells from primary and from established in vitro cultures, as well as in the intima of ex vivo rat aortic rings, where the signal was limited to the endothelial layer. Interaction of the OP4 receptor with its physiological ligand nociceptin caused, in cultured endothelial cells, the activation of a mitogen-activated protein (MAP) kinase cascade. Taken together, these results show that the OP4 receptor is synthesised and functionally expressed in endothelial cells, presumably as a starting point for some vasoactive mechanism(s).

The involvement of metabotropic glutamate (mGlu) receptors in the induction of long-term potentiation (LTP) in vivo has been consistently documented. We have investigated whether LTP induction in the dentate gyrus of rats leads to changes in expression of mGlu2/3 or -5 receptor subtypes in the hippocampus. LTP was induced at the medial perforant path-dentate gyrus synapses, and mGlu receptor expression was examined by Western blot or in situ hybridization. An up-regulation of mGlu5 receptors was observed in the hippocampus both 24 and 48 h following LTP induction. This effect was restricted to the dentate gyrus and CA1 region, whereas no changes in mGlu5 receptor protein (but an increase in mRNA levels) were observed in the CA3 region. The increased expression of mGlu5 receptors was directly related to the induction of LTP, because it was not observed when tetanic stimulation was carried out in animals treated with the NMDA receptor antagonist, 2-amino-5-phosphonopentanoate (AP5). Western blot analysis also showed a reduced expression of mGlu2/3 receptors in the whole hippocampus 24 h after LTP induction, indicating that the increased expression of mGlu5 receptors was specific. These data suggest that an up-regulation of mGlu5 receptors is a component of the plastic changes that follow the induction of LTP at the perforant path-dentate gyrus synapse.

The aim of the present work was to verify whether an impairment of subtype 5 metabotropic glutamate receptor-mediated neurotransmission did occur in the aged striatum. To this end, the ability of the subtype 5 metabotropic glutamate receptor agonist, RS-2-chloro-5-hydroxyphenylglycine, to stimulate phosphoinositide hydrolysis and to potentiate N-methyl-d-aspartate-induced effects in striatal slices from young (3 months) and aged (24 months) rats was compared. The ability of RS-2-chloro-5-hydroxyphenylglycine to induce maximal phosphoinositide turnover and to potentiate N-methyl-d-aspartate effects was significantly reduced in slices from old vs. young rats. These changes were associated with a significant reduction in the expression of subtype 5 metabotropic glutamate receptor protein (-28.8%) and phospholipase C-beta1 (-55.8%) in old striata, while receptor messenger ribonucleic acid expression was unchanged. These results show that the signalling associated with subtype 5 metabotropic glutamate receptors undergoes significant age-related changes and that a reduced expression of subtype 5 metabotropic glutamate receptors and, more importantly, phospholipase C-beta1 may account for the functional decline of subtype 5 metabotropic glutamate receptors.

1. 1. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to investigate the effects of haloperidol treatment (1 mg/kg/day for 2, 7 or 21 consecutive days) on the expression of D1B and D3 dopamine receptor mRNAs in the rat lymphocytes. 2. 2. The expression of D1B receptor mRNA was significantly decreased after 2 days of treatment and progressively returned toward basal values at the end of treatment. Conversely, haloperidol failed to modify the expression of lymphocyte D3 receptor mRNA. 3. 3. These results indicate short-lasting dynamic changes of expression of lymphocyte D1B dopamine receptor mRNA by haloperidol and suggest that the effects of dopamine and dopaminergic drugs on the immune system might be mediated, at least in part, by direct interaction of these substances with dopamine receptors on lymphocyte membrane.

Changes in the local utilization of cerebral glucose resulting from administered drugs acting on the central nervous system can be evaluated quantitatively by the [14C]2-deoxyglucose method. We report the findings obtained by the [14C]2-deoxyglucose method that contribute to understanding the cerebral functional effects of drugs of abuse and discuss in particular the similarities between nicotine and other addictive drugs. A common consequence of the intravenous administration of psychomotor stimulants and opioids in the rat is the increase in glucose utilization in the shell of nucleus accumbens. This functional change is accompanied by increased local extracellular concentrations of dopamine. Altered functional activity and dopamine neurotransmission in the shell of the nucleus accumbens thus represent distinctive neurobiological markers of the addictive properties of several drugs, independently of the specific neurochemical mechanisms of action. It has recently been shown that the intravenous administration of a pulse of nicotine, at single-unit doses corresponding to those that maintain self-administration in the rat, produces neurochemical and metabolic changes in the shell of the nucleus accumbens that closely resemble those of psychomotor stimulants and opioids. The latter results demonstrate that nicotine shares with highly addictive drugs a distinct neurochemical and functional consequence. They therefore contribute to the neurochemical definition of the addictive nature of nicotine. These neurochemical and functional changes may contribute to the changes in expression of intracellular second messengers and neurotransmitter/receptor systems observed particularly in the shell following the administration of drugs of abuse.

The aim of the study was to measure the changes in cerebral energy metabolism and c-fos mRNA expression following challenge with heroin in drug-naive rats and in animals previously sensitized to the drug. Acute heroin administration to drug naive-rats produced a generalized metabolic depression. In contrast, challenge with heroin in drug-sensitized rats produced selective metabolic increases in structures belonging to the basal ganglia. These changes were accompanied by increased c-fos mRNA expression in the caudate-putamen nucleus. These results demonstrate that the process of sensitization to heroin is coupled to functional changes that are confined to the subcortical motor circuits of the basal ganglia.

The fractional release technique was applied to investigate the effects of single electroshock (ECS) on the opioid-mediated inhibition of dopamine release in rat striatal slices. Animals were submitted to ECS 24h before the experiments. The results demonstrate that pre-treatment with ECS suppresses the inhibition of dopamine release mediated by kappa opioid receptors. These data suggest that single ECS treatment modifies the sensitivity of the kappa opioid receptors located on the presynatic dopamine terminals in the rat striatum.

The [14C]2-deoxyglucose method was applied to measure the effects of the acute intravenous administration of morphine sulphate (0.2–0.4 mg/kg) on cerebral glucose utilization in rats. Morphine produced dose-dependent increases of glucose metabolism in the shell of the nucleus accumbens, without affecting functional activity in any other brain area. These results provide further evidence for the preferential effects of intravenously abused substances in the shell of the nucleus accumbens.

Heat shock treatment in PC12 cells induces the synthesis of two HSP70 transcripts of 2.55 and 3.05 kb in size, while only a 70-kDa protein is shown by SDS–PAGE. Using reverse transcription–polymerase chain reaction strategy, two mRNA species were amplified from heat-shocked PC12 cells and sequenced. Both cDNAs revealed the same open reading frame encoding a single predicted 641-amino-acid polypeptide of about 70 kDa. The two 3′ untranslated regions of the two mRNAs were completely different in both length and composition. Expression of the mRNA of the two corresponding genes and that of another rat HSP70 family member was investigated in PC12, Rat1 cells, and lung fibroblasts. Northern blot analysis revealed that the 2.55- and the 3.05-kb related gene transcripts are differentially expressed in the rat cell lines tested, while the third member of the subfamily is not induced. The single-copy nature of the three genes is also confirmed by Southern blot analysis.

Given the specific melanin-associated paramagnetic features, the Electron Spin Resonance (ESR, called also Electron Paramagnetic Resonance, EPR) analysis has been proposed as a potential tool for non-invasive melanoma diagnosis. However, studies comparing human melanoma tissues to the most appropriate physiological counterpart (nevi) have not been performed, and ESR direct correlation with melanoma clinical features has never been investigated. ESR spectrum was obtained from melanoma and non-melanoma cell-cultures as well as mouse melanoma and non-melanoma tissues and an endogenous ESR signal (g = 2.005) was found in human melanoma cells and in primary melanoma tissues explanted from mice, while it was always absent in non-melanoma samples. These characteristics of the measured ESR signal strongly suggested its connection with melanin. Quantitative analyses were then performed on paraffin-embedded human melanoma and nevus sections, and validated on an independent larger validation set, for a total of 112 sections (52 melanomas, 60 nevi). The ESR signal was significantly higher in melanomas (p=0.0002) and was significantly different between ‘‘Low Breslow’s and ‘‘High Breslow’s’’ depth melanomas (p,0.0001). A direct correlation between ESR signal and Breslow’s depth, expressed in millimetres, was found (R=0.57; p,0.0001). The eu/pheomelanin ratio was found to be significantly different in melanomas ‘‘Low Breslow’s’’ vs melanomas ‘‘High Breslow’s’’ depth and in nevi vs melanomas ‘‘High Breslow’s depth’’. Finally, ROC analysis using ESR data discriminated melanomas sections from nevi sections with up to 90% accuracy and p,0.0002. In the present study we report for the first time that ESR signal in human paraffin-embedded nevi is significantly lower than signal in human melanomas suggesting that spectrum variations may be related to qualitative melanin differences specifically occurring in melanoma cells. We therefore conclude that this ESR signal may represent a reliable marker for melanoma diagnosis in human histological sections.

The effect of electroshock on the brain levels of somatostatin mRNA were evaluated by in situ hybridization using a selective oligonucleotide probe. Rats were submitted to single or repeated (7 days, one session for each day) sessions of electroshock. There was a marked increase of the expression of somatostatin mRNA in the hippocampal formation, mostly in the multiform layer of the hilus of the dentate gyrus, following repeated but not single electroshock. Our findings show that repeated ECS is associated with increase in the synthesis of somatostatin. The results also support previous data indicating that the hippocampal formation is selectively affected by the treatment.

Local rates of cerebral glucose utilization (LCGU) were determined in adult rats submitted to electroconvulsive shock (ECS) treatment. One group of rats (n = 5) received a single ECS, a second group (n = 5) was treated once daily for 7 days. A third group (n = 5) of unshocked rats served as control. LCGU measurements were performed one day after either the single or the last ECS. Following a single ECS, most of the 45 brain regions examined exhibited lower rates of LCGU than controls, in contrast after repeated ECS treatment the metabolic activity results increased in segments of the hippocampus. The results indicate that repeated ECS treatment selectively increases metabolic rates within the hippocampus, a structure of the limbic system thought to be related to affective disorders and memory.

Background Several cellular functions relate to ion-channels activity. Physiologically relevant chains of events leading to angiogenesis, cell cycle and different forms of cell death, require transmembrane voltage control. We hypothesized that the unordered angiogenesis occurring in solid cancers and vascular malformations might associate, at least in part, to ion-transport alteration. Methods The expression level of several ion-channels was analyzed in human solid tumor biopsies. Expression of 90 genes coding for ion-channels related proteins was investigated within the Oncomine database, in 25 independent patients-datasets referring to five histologically-different solid tumors (namely, bladder cancer, glioblastoma, melanoma, breast invasive-ductal cancer, lung carcinoma), in a total of 3673 patients (674 control-samples and 2999 cancer-samples). Furthermore, the ion-channel activity was directly assessed by measuring in vivo the electrical sympathetic skin responses (SSR) on the skin of 14 patients affected by the flat port-wine stains vascular malformation, i.e., a non-tumor vascular malformation clinical model. Results Several ion-channels showed significantly increased expression in tumors (p