Superhydrophobic lab-on-chip measures secretome protonation state and provides a personalized risk assessment of sporadic tumour.

Secretome of primary cultures is an accessible source of biological markers compared to more complex and less decipherable mixtures such as serum or plasma. The protonation state (PS) of secretome reflects the metabolism of cells and can be used for cancer early detection. Here, we demonstrate a superhydrophobic organic electrochemical device that measures PS in a drop of secretome derived from liquid biopsies. Using data from the sensor and principal component analysis (PCA), we developed algorithms able to efficiently discriminate tumour patients from non-tumour patients. We then validated the results using mass spectrometry and biochemical analysis of samples. For the 36 patients across three independent cohorts, the method identified tumour patients with high sensitivity and identification as high as 100% (no false positives) with declared subjects at-risk, for sporadic cancer onset, by intermediate values of PS. This assay could impact on cancer risk management, individual's diagnosis and/or help clarify risk in healthy populations.

Innate immunity in cutaneous melanoma.

The skin immune system is composed of a vast network of immune cells, including lymphocytes, macrophages, neutrophils, dendritic cells and Langerhans cells, which not only are involved in inflammatory responses but also contribute to homeostatic function and may participate in the various steps of carcinogenesis. Many studies support the notion that innate immunity has a key role in the development, growth and prognosis of cutaneous malignant melanoma (MM), through the release of pro- and/or anti-inflammatory cytokines and tumour growth factors. The tumour environment in a major subset of cutaneous MM shows evidence of a T cell-infiltrated phenotype, but there is less known about the presence and the phenotype of other immune system cells. Response to immunotherapy is largely correlated with the presence of T cells in the tumour microenvironment, while the regulation exerted by stromal components such as macrophages and mast cells has been less investigated. In the current report, we review the recent literature, focusing our attention on the role of macrophages, dendritic cells, mast cells and natural killer cells in orchestrating MM progression, to better understand tumour immunobiology. The identification of new therapeutic targets and the application of approaches aimed at modulating crosstalk between immune and tumour cells, could have a crucial impact on immunotherapy and result in better clinical outcome. We hope this review will be helpful in cutaneous MM research.

Elastofibroma dorsi: a histochemical and immunohistochemical study.

Elastofibroma dorsi (ED) is considered a member of a heterogeneous group of benign fibrous (fibroblastic or myofibroblastic) soft-tissue tumors, frequently localized in the periscapular region in middle aged or older individuals. However, the pathogenesis of ED is still unclear and many authors believe that ED results from a reactive hyperproliferation of fibroblastic tissue, while others suggest that it may be a consequence of a mechanical friction. In our study, we examined 11 cases of ED using histochemical and immunohistochemical methods, in order to extend the knowledge about extracellular matrix composition and histopathogenesis of ED. From the results it appeared that stroma and interspersed spindle cells of ED were positive for both periostin and tenascin-C. Mast cells tryptase-positive were also abundant throughout the lesion. The perivascular distribution of periostin and tenascin-C, associated with the CD34 positivity, suggest that endothelial-mesenchymal transition events can account for neovascularization and production of fibroelastic tissue characteristic of elastofibroma. Our data obtained in endothelial cells cultures demonstrated that elastin production is higher when the status of confluence of the cells is low. So, we can assume that such a phenomenon is a characteristic of mesenchymal/endothelial cells CD34 positive, in which elastin production results to be inversely proportional to the vascular differentiation of cellular elements. In the light of these considerations, we think that a cancerous nature of ED is unlikely. Overall, our study report, for the first time, a detailed description of extracellular matrix composition in ED, suggesting that a mechanical strain-dependent reactivation of periostin and tenascin-C expression, as well as of elastin deposition, could be responsible for development of ED.

Glucose tolerance, insulin sensitivity and insulin release in European non-diabetic carriers of a polymorphism upstream of CDKN2A and CDKN2B.

AIMS/HYPOTHESIS: The aim of this study was to investigate the association of the rs10811661 polymorphism near the CDKN2B/CDKN2A genes with glucose tolerance, insulin sensitivity and insulin release in three samples of white people with European ancestry. METHODS: Sample 1 comprised 845 non-diabetic offspring of type 2 diabetes patients recruited in five European centres participating in the EUGENE2 study. Samples 2 and 3 comprised, respectively, 864 and 524 Italian non-diabetic participants. All individuals underwent an OGTT. Screening for the rs10811661 polymorphism was performed using a TaqMan allelic discrimination assay. RESULTS: The rs10811661 polymorphism did not show a significant association with age, BMI and insulin sensitivity. Participants carrying the TT genotype showed a significant reduction in insulin release, measured by an OGTT-derived index, compared with carriers of the C allele, in the three samples. When these results were pooled with those of three published studies, and meta-analysed with a random-effects model, the T allele was significantly associated with reduced insulin secretion (-35.09 [95% CI 14.68-55.52], p = 0.0008 for CC+CT vs TT; and -29.45 [95% CI 9.51-49.38], p = 0.0038, for the additive model). In addition, in our three samples, participants carrying the TT genotype exhibited an increased risk for impaired glucose tolerance (IGT) compared with carriers of the C allele (OR 1.55 [95% CI 1.20-1.95] for the meta-analysis of the three samples). CONCLUSIONS/INTERPRETATION: Our data, together with the meta-analysis of previously published studies, show that the rs10811661 polymorphism is associated with impaired insulin release and IGT, suggesting that this variant may contribute to type 2 diabetes by affecting beta cell function.

C-reactive protein induces phosphorylation of insulin receptor substrate-1 on Ser307 and Ser 612 in L6 myocytes, thereby impairing the insulin signalling pathway that promotes glucose transport.

AIMS/HYPOTHESIS: C-reactive protein (CRP) is associated with insulin resistance and predicts development of type 2 diabetes. However, it is unknown whether CRP directly affects insulin signalling action. To this aim, we determined the effects of human recombinant CRP (hrCRP) on insulin signalling involved in glucose transport in L6 myotubes. MATERIALS AND METHODS: L6 myotubes were exposed to endotoxin-free hrCRP and insulin-stimulated activation of signal molecules, glucose uptake and glycogen synthesis were assessed. RESULTS: We found that hrCRP stimulates both c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK)1/2 activity. These effects were paralleled by a concomitant increase in IRS-1 phosphorylation at Ser(307) and Ser(612), respectively. The stimulatory effects of hrCRP on IRS-1 phosphorylation at Ser(307) and Ser(612) were partially reversed by treatment with specific JNK and ERK1/2 inhibitors, respectively. Exposure of L6 myotubes to hrCRP reduced insulin-stimulated phosphorylation of IRS-1 at Tyr(632), a site essential for engaging p85 subunit of phosphatidylinositol-3 kinase (PI-3K), protein kinase B (Akt) activation and glycogen synthase kinase-3 (GSK-3) phosphorylation. These events were accompanied by a decrease in insulin-stimulated glucose transporter (GLUT) 4 translocation to the plasma membrane, glucose uptake and glucose incorporation into glycogen. The inhibitory effects of hrCRP on insulin signalling and insulin-stimulated GLUT4 translocation were reversed by treatment with JNK inhibitor I and the mitogen-activated protein kinase inhibitor, PD98059. CONCLUSIONS/INTERPRETATION: Our data suggest that hrCRP may cause insulin resistance by increasing IRS-1 phosphorylation at Ser(307) and Ser(612) via JNK and ERK1/2, respectively, leading to impaired insulin-stimulated glucose uptake, GLUT4 translocation, and glycogen synthesis mediated by the IRS-1/PI-3K/Akt/GSK-3 pathway.

Proteomic analysis of human thyroid cell lines reveals reduced nuclear localization of Mn-SOD in poorly differentiated thyroid cancer cells.

Differential protein arrays between nuclear extracts of human thyroid cell lines obtained from tumors with different degree of differentiation were exploited to define molecular alterations occurring during thyroid tumor progression. Nuclear extracts from the well differentiated TPC-1 (from papillary carcinoma) and the poorly differentiated ARO (from anaplastic carcinoma) cells showed an overall similar pattern of protein expression as revealed by two-dimensional gel electrophoresis analysis. However, manganese-superoxide dismutase (Mn-SOD) was clearly identified by mass spectrometry procedures as significantly less expressed in ARO compared to TPC-1 cells. A reduced expression of Mn-SOD in the nuclear compartment was confirmed by Western blot and immunofluorescence analysis. A similar expression pattern of nuclear Mn-SOD was detected by immunohistochemistry in human thyroid tumors, with the lowest or absent detection in anaplastic carcinomas. Moreover, the levels of nuclear Mn-SOD in tumor cells were lower than in the normal thyrocytes. These data indicate that an altered nuclear expression of Mn-SOD parallels, together with changes in other elements of the antioxidant protective system, the loss of differentiation occurring during the progression of thyroid tumors.

Novel somatic MEN1 gene alterations in sporadic primary hyperparathyroidism and correlation with clinical characteristics.

Primary hyperparathyroidism (pHPT) is a common endocrine disease that in more than 95% of cases is sporadic and only in some cases is caused by inherited disorders, isolated or as part of multiple endocrine neoplasia (MEN1 and 2). Somatic mutations of MEN1 gene have also been described in sporadic parathyroid tumors. In our study, we examined the presence of alterations in MEN1 gene in a series of 39 patients who had undergone surgery for sporadic pHPT (35 with parathyroid adenoma or hyperplasia, 4 with a carcinoma). A genotype-phenotype correlation was also analysed. After DNA extraction from paraffin-embedded tissues, we amplified by PCR and sequenced the exons 2-10 of the MEN1 gene. Somatic MEN1 mutations were detected in 6 of the 35 patients with a benign parathyroid lesion examined (17.1%), whereas no alterations were found in the carcinomas. Four novel MEN1 gene mutations were identified as follows: one frameshift mutation (222insT, exon 2), one frameshift deletion (912delTA, exon 5), one in-frame deletion (835del18, exon 4) and one missense mutation (P291A, exon 6). In addition, one missense mutation (L89R, exon 2) and one nonsense mutation (Q536X, exon 10) were previously reported. Moreover, two polymorphisms were also found: one allele carried a R171Q polymorphism (1/39 tumors), while a D418D polymorphism (GAC/GAT) was found in 15 and 8 tumors in hetero (CT) and homozygosity (TT), respectively. In no case (mutations and/or polymorphisms) did we find a genotype-phenotype correlation. In conclusion, our data demonstrate the presence of somatic alterations of the MEN1 tumor suppressor gene in about one fifth of benign sporadic parathyroid tumors. The absence of a genotype-phenotype correlation, however, suggests the involvement of other genetic/epigenetic factors for the full expression of the disease.

Expression of adenylyl cyclase types III and VI in human hyperfunctioning thyroid nodules.

Hyperfunctioning thyroid nodules are characterized by the presence of spontaneous somatic mutations responsible for constitutive activation of the cAMP pathway. However, alterations affecting other elements of the cAMP signaling system may counteract the effects of the mutations. In this study, the expression of the adenylyl cyclase (AC) types III and VI was investigated by Western blot in 18 hyperfunctioning thyroid nodules; in 12 samples, we also assessed the presence of TSH receptor (TSHR) or gsp mutations and levels of AC VI and III mRNA. We found that the expression of nodular AC VI (but not AC III) was significantly lower (85.1% of normal, P=0.014) than the expression of both adenylyl cycles types of perinodular tissue from the same patients. Slightly, but not significant differences were detected in nodules with or without mutations and AC protein levels generally showed correlation with the levels of the transcripts detected by RT-PCR. In addition, AC III and AC VI expression levels within a given nodule were characterized by a significant positive correlation. These findings indicate that a diminished expression of AC type VI may be part of the mechanisms occurring in the hyperfunctioning nodules, independently of the presence of TSHR or gsp mutations, which influence the resulting phenotype.