Prostate-specific antigen: An unfamiliar protein in the human salivary glands.

OBJECTIVES: The presence of prostate-specific antigen (PSA) in saliva and salivary glands has been reported. Nevertheless, its release pathway in these glands remains to be elucidated. Here, we showed PSA subcellular distribution focusing on its plausible route in human salivary parenchyma. MATERIALS AND METHODS: Sections of parotid and submandibular glands were subjected to the immunohistochemical demonstration of PSA by the streptavidin-biotin method revealed by alkaline phosphatase. Moreover, ultrathin sections were collected on nickel grids and processed for immunocytochemical analysis, to visualize the intracellular distribution pattern of PSA through the observation by transmission electron microscopy. RESULTS: By immunohistochemistry, in both parotid and submandibular glands PSA expression was detected in serous secretory acini and striated ducts. By immunocytochemistry, immunoreactivity was retrieved in the cytoplasmic compartment of acinar and ductal cells, often associated with small cytoplasmic vesicles. PSA labeling appeared also on rough endoplasmic reticulum and in the acini's lumen. A negligible PSA labeling appeared in most of the secretory granules of both glands. CONCLUSIONS: Our findings clearly support that human parotid and submandibular glands are involved in PSA secretion. Moreover, based on the immunoreactivity pattern, its release in oral cavity would probably occur by minor regulated secretory or constitutive-like secretory pathways.

Synergic Action of Insulin-like Growth Factor-2 and miRNA-483 in Pterygium Pathogenesis.

Pterygium is a multifactorial disease in which UV-B is speculated to play a key role by inducing oxidative stress and phototoxic DNA damage. In search for candidate molecules that are useful for justifying the intense epithelial proliferation observed in pterygium, our attention has been focused on Insulin-like Growth Factor 2 (IGF-2), mainly detected in embryonic and fetal somatic tissues, which regulate metabolic and mitogenic functions. The binding between IGF-2 and its receptor Insulin-like Growth Factor 1 Receptor (IGF-1R) activates the PI3K-AKT pathway, which leads to the regulation of cell growth, differentiation, and the expression of specific genes. Since IGF2 is regulated by parental imprinting, in different human tumors, the IGF2 Loss of Imprinting (LOI) results in IGF-2- and IGF2-derived intronic miR-483 overexpression. Based on these activities, the purpose of this study was to investigate the overexpression of IGF-2, IGF-1R, and miR-483. Using an immunohistochemical approach, we demonstrated an intense colocalized epithelial overexpression of IGF-2 and IGF-1R in most pterygium samples (Fisher's exact test, p = 0.021). RT-qPCR gene expression analysis confirmed IGF2 upregulation and demonstrated miR-483 expression in pterygium compared to normal conjunctiva (253.2-fold and 12.47-fold, respectively). Therefore, IGF-2/IGF-1R co-expression could suggest their interplay through the two different paracrine/autocrine IGF-2 routes for signaling transfer, which would activate the PI3K/AKT signaling pathway. In this scenario, miR-483 gene family transcription might synergically reinforce IGF-2 oncogenic function through its boosting pro-proliferative and antiapoptotic activity.

MicroRNAs at the Crossroad of the Dichotomic Pathway Cell Death vs. Stemness in Neural Somatic and Cancer Stem Cells: Implications and Therapeutic Strategies.

Stemness and apoptosis may highlight the dichotomy between regeneration and demise in the complex pathway proceeding from ontogenesis to the end of life. In the last few years, the concept has emerged that the same microRNAs (miRNAs) can be concurrently implicated in both apoptosis-related mechanisms and cell differentiation. Whether the differentiation process gives rise to the architecture of brain areas, any long-lasting perturbation of miRNA expression can be related to the occurrence of neurodevelopmental/neuropathological conditions. Moreover, as a consequence of neural stem cell (NSC) transformation to cancer stem cells (CSCs), the fine modulation of distinct miRNAs becomes necessary. This event implies controlling the expression of pro/anti-apoptotic target genes, which is crucial for the management of neural/neural crest-derived CSCs in brain tumors, neuroblastoma, and melanoma. From a translational point of view, the current progress on the emerging miRNA-based neuropathology therapeutic applications and antitumor strategies will be disclosed and their advantages and shortcomings discussed.

Tyrosinase and nestin immunohistochemical expression in melanocytic nevi as a histopathologic pattern to trace melanocyte differentiation and nevogenesis.

While histological analysis represents a powerful tool for the classification of melanocytic lesions as benign or malignant, a clear-cut distinction between a nevus and a melanoma is sometimes a challenging step of the diagnostic process. The immunohistochemical detection of tyrosinase, cardinal melanogenic enzyme during melanocytic maturation, has often been helpful in formulating a differential diagnosis due to the peculiar staining pattern in nevocytes compared with melanoma cells. Tyrosinase distribution in nevi appears to overlap with the cytoarchitectural changes observable within these lesions, that result in epidermal or superficial dermal nevocytes being larger and strongly expressing melanocytic differentiation antigens, such as tyrosinase, compared with deeper dermal nevus cells. Our study aimed to evaluate the immunohistochemical expression pattern of tyrosinase in different histological types of acquired dysplastic melanocytic nevi, including junctional, compound, and intradermal nevi. Moreover, to estimate whether in nevocytes the expression of tyrosinase was associated with their differentiation state, we investigated the expression of two recognized markers of pluripotency, CD34 and nestin. In all examined nevi, our analysis revealed a remarkable immunoreactivity for tyrosinase in junctional and superficial dermal nevocytes and a decreasing gradient of staining in dermal nevocytes, up to become negative in deeper dermis. Meanwhile, junctional and dermal nevocytes were lacking in CD34 protein. Furthermore, nestin immunostaining showed an opposite distribution compared with tyrosinase, leading us to look into the tyrosinase/nestin expression pattern in melanocytic nevus as a tool to better understand the final stages of differentiation of melanocyte precursors toward their ultimate anatomical site into the epidermis.

Immunophenotypic characterization of telocyte-like cells in pterygium.

Purpose: Telocytes (TCs) are peculiar interstitial cells, characterized by their typical elongated and interconnected processes called telopodes. TCs are supposed to contribute to maintain tissue homeostasis but also to be involved in the pathophysiology of many disorders. The aim of the study was to identify TCs in pterygium, a chronic condition of bulbar conjunctiva, and to examine possible differences in TCs in terms of immunophenotype and/or localization between pterygium and normal conjunctiva, to evaluate the possible involvement of TCs in pathogenesis of pterygium. Methods: The analysis of the immunophenotype of TCs was performed on a group of 40 formalin-fixed and paraffin-embedded primary pterygium and ten bulbar conjunctiva samples. We examined with immunohistochemistry the expression of 11 commercially available antibodies (PDGFRα, CD34, c-kit, nestin, vimentin, α-SMA, laminin, S100, VEGF, CD133, and CD31) and with double immunofluorescence the concomitant expression of PDGFRα and CD34, and PDGFRα and nestin. In addition, we performed an ultrastructural study with transmission electron microscopy (TEM) on a group of five pterygium and three conjunctiva biopsy specimens. Results: TCs, ultrastructurally identified according to their "moniliform" prolongations, were localized underneath the epithelium along the basement membrane, around the vessels, and near the nerves and scattered in the stroma. In contrast, TCs, as fibroblasts, were almost absent in the fibrotic areas. In pterygium and normal conjunctiva, the TCs shared the same distribution pattern, except a marked TC hyperplasia detected in pterygium. Moreover, in pterygium, the immunohistochemical analysis of TCs showed a strong immunoreactivity to PDGFRα, CD34, and nestin. This result was confirmed with double immunofluorescence labeling, revealing that in pterygium stromal TCs always showed a PDGFRα+/nestin+ and PDGFRα+/CD34+ immunophenotype. Furthermore, moderate staining to vimentin and VEGF was detected, but only a small number of cells were weakly immunoreactive to laminin and S100. Only adventitial TCs of the perivascular sheaths exhibited strong immunoreactivity to α-SMA. Conversely, despite showing mild immunoreactivity to PDGFRα and CD34, the TCs in normal conjunctiva did not show any immunoreactivity to nestin and VEGF. Moreover, in pterygium and conjunctiva, the TCs were always negative for c-kit. Conclusions: Because of the distribution and immunophenotype, TCs in pterygium may represent a subpopulation of relatively immature cells with regenerative potential. In addition, the expression of nestin may suggest possible involvement of TCs as active players in the regeneration of ultraviolet-damaged stroma and vascular remodeling. The fibrotic transformation in the cicatricial area may stand for a breakdown of the regenerative process.

Role of epithelial-mesenchymal transition involved molecules in the progression of cutaneous melanoma.

Epithelial-mesenchymal transition (EMT) has been suggested to have a driving role in the acquisition of a metastatic potential by melanoma cells. Important hallmarks of EMT include both E-cadherin downregulation and increased expression of N-cadherin. This switch in distinct classes of adhesion molecules leads melanoma cells to lose contact with adjacent keratinocytes and interact instead with stromal fibroblasts and endothelial cells, thus promoting dermal and vascular melanoma invasion. Consequently, tumor cells migrate to distant host tissues and establish metastases. A key regulator in the induction of EMT in melanoma is the Notch1 signaling pathway that, when activated, is prompt to upregulate N-cadherin expression. By means of this strategy, melanoma cells gain enhanced survival, proliferation and invasion properties, driving the tumor toward a more aggressive phenotype. On the basis of these statements, the present study aimed to investigate the possible association between N-cadherin and Notch1 presence in primary cutaneous melanomas and lymph node metastases. Our results from immunohistochemical analysis confirmed a positive correlation between N-cadherin and Notch1 presence in the same tumor samples. Moreover, this study highlighted that a concomitant high expression of N-cadherin and Notch1, both in primary lesions and in lymph node metastases, predicts an adverse clinical outcome in melanoma patients. Therefore, N-cadherin and Notch1 co-presence can be monitored as a predictive factor in early- and advanced-stage melanomas and open additional therapeutic targets for the restraint of melanoma metastasis.

Nestin and vimentin colocalization affects the subcellular location of glucocorticoid receptor in cutaneous melanoma.

AIMS: 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. METHODS AND RESULTS: 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). CONCLUSIONS: 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.

Prevalence of human papillomavirus infection in women in Benin, West Africa.

BACKGROUND: Cervical cancer ranks as the first most frequent cancer among women in Benin. The major cause of cervical cancer now recognized is persistent infection of Human Papillomavirus (HPV). In Benin there is a lack of screening programs for prevention of cervical cancer and little information exists regarding HPV genotype distribution. METHODS: Cervical cells from 725 women were examined for the presence of viral DNA by means of a polymerase chain reaction (PCR) multiplex-based assay with the amplification of a fragment of L1 region and of E6/E7 region of the HPV genome, and of abnormal cytology by Papanicolaou method. The association between HPV status and Pap test reports was evaluated. Socio-demographic and reproductive characteristics were also related. RESULTS: A total of 18 different HPV types were identified, with a prevalence of 33.2% overall, and 52% and 26.7% among women with and without cervical lesions, respectively. Multiple HPV infections were observed in 40.2% of HPV-infected women. In the HPV-testing group, the odds ratio for the detection of abnormal cytology was 2.98 (95% CI, 1.83-4.84) for HPV positive in comparison to HPV negative women. High risk types were involved in 88% of infections, most notably HPV-59, HPV-35, HPV-16, HPV-18, HPV-58 and HPV-45. In multiple infections of women with cytological abnormalities HPV-45 predominated. CONCLUSIONS: This study provides the first estimates of the prevalence of HPV and type-specific distribution among women from Benin and demonstrates that the epidemiology of HPV infection in Benin is different from that of other world regions. Specific area vaccinations may be needed to prevent cervical cancer and the other HPV-related diseases.

SmartFlareTM is a reliable method for assessing mRNA expression in single neural stem cells.

BACKGROUND: One of the most challenging tasks of modern biology concerns the real-time tracking and quantification of mRNA expression in living cells. On this matter, a novel platform called SmartFlareTM has taken advantage of fluorophore-linked nanoconstructs for targeting RNA transcripts. Although fluorescence emission does not account for the spatial mRNA distribution, NanoFlare technology has grown a range of theranostic applications starting from detecting biomarkers related to diseases, such as cancer, neurodegenerative pathologies or embryonic developmental disorders. AIM: To investigate the potential of SmartFlareTM in determining time-dependent mRNA expression of prominin 1 (CD133) and octamer-binding transcription factor 4 (OCT4) in single living cells through differentiation. METHODS: Brain fragments from the striatum of aborted human fetuses aged 8 wk postconception were processed to obtain neurospheres. For the in vitro differentiation, neurospheres were gently dissociated with Accutase solution. Single cells were resuspended in a basic medium enriched with fetal bovine serum, plated on poly-L-lysine-coated glass coverslips, and grown in a lapse of time from 1 to 4 wk. Live cell mRNA detection was performed using SmartFlareTM probes (CD133, Oct4, Actin, and Scramble). All the samples were incubated at 37 °C for 24 h. For nuclear staining, Hoechst 33342 was added. SmartFlareTM CD133- and OCT4-specific fluorescence signal was assessed using a semiquantitative visual approach, taking into account the fluorescence intensity and the number of labeled cells. RESULTS: In agreement with previous PCR experiments, a unique expression trend was observed for CD133 and OCT4 genes until 7 d in vitro (DIV). Fluorescence resulted in a mixture of diffuse cytoplasmic and spotted-like pattern, also detectable in the contacting neural branches. From 15 to 30 DIV, only few cells showed a scattered fluorescent pattern, in line with the differentiation progression and coherent with mRNA downregulation of these stemness-related genes. CONCLUSION: SmartFlareTM appears to be a reliable, easy-to-handle tool for investigating CD133 and OCT4 expression in a neural stem cell model, preserving cell biological properties in anticipation of downstream experiments.

Erythropoietin is involved in angiogenesis in human primary melanoma.

In this study, the extent of angiogenesis, evaluated as microvascular volume density, immunoreactivity of tumour cells to erythropoietin (Epo) and of endothelial cells to Epo receptor (EpoR) have been correlated in human primary melanoma specimens. Results showed that Epo/EpoR expression correlate with angiogenesis and tumour thickness. These findings suggest that Epo is secreted by tumour cells and it affects vascular endothelial cells via its receptor and promotes angiogenesis in a paracrine manner, playing an important role in melanoma angiogenesis.

Absence of Polyphenol Oxidase in Cynomorium coccineum, a Widespread Holoparasitic Plant.

Polyphenol oxidase (PPO, E.C. 1.14.18.1) is a nearly ubiquitous enzyme that is widely distributed among organisms. Despite its widespread distribution, the role of PPO in plants has not been thoroughly elucidated. In this study, we report for the absence of PPO in Cynomorium coccineum, a holoparasitic plant adapted to withstand unfavorable climatic conditions, growing in Mediterranean countries and amply used in traditional medicine. The lack of PPO has been demonstrated by the absence of enzymatic activity with various substrates, by the lack of immunohistochemical detection of the enzyme, and by the absence of the PPO gene and, consequently, its expression. The results obtained in our work allow us to exclude the presence of the PPO activity (both latent and mature forms of the enzyme), as well as of one or more genes coding for PPO in C. coccineum. Finally, we discuss the possible significance of PPO deficiency in parasitic plants adapted to abiotic stress.

Correlation between NGF/TrkA and microvascular density in human pterygium.

Pterygium is a surface ocular lesion that is associated with chronic UV exposure. The primary effect is a solar actinic elastosis within the stroma. All the other changes are secondary. Pterygium is characterized by proliferation, inflammatory infiltrates, fibrosis, angiogenesis and extracellular matrix breakdown. The aim of this study was to correlate microvascular density and nerve growth factor (NGF)/NGF-receptor transmembrane tyrosine kinase (TrkA) expression in endothelial cells in human pterygium. Specimens of human pterygium obtained from 30 patients who had undergone surgical excision and of 10 normal bulbar conjunctiva were investigated immunohistochemically by using anti-CD31, anti-NGF and anti-TrkA antibodies. Results showed that endothelial cells in human pterygium are immunoreactive to both NGF and its receptor TrkA, and that this immunoreactivity is correlated to microvascular density. The results of this study suggest that an autocrine loop between NGF and its receptor TrkA is activated in pterygium and that it is involved in the angiogenic response taking place in this pathological condition. These data are in accord with recent evidences, which have clearly established that NGF plays a role as an angiogenic factor in several pathological conditions. Understanding the mechanism of angiogenesis in pterygium provides a basis for a rational approach to the development of anti-angiogenic therapy in patients affected by this disease.

Relationship between the expression of cyclooxygenase-2 and survivin in primary pterygium.

PURPOSE: To investigate the expression of cyclooxygenase-2 (COX-2) in a group of 93 Ecuadorian primary pterygia and to evaluate a possible association between COX-2 and survivin. METHODS: Primary pterygium samples were treated for the immunohistochemical evaluation of COX-2 and survivin. Mouse monoclonal antibody to COX-2 and rabbit polyclonal antibody to survivin were used. Statistical analysis was performed using the SPSS statistical software package, version 15.0. RESULTS: In our study, 63 (67.7%) primary pterygia samples were positive for COX-2 staining, and 70 (75.3%) specimens were positive for survivin expression. In the group of pterygia with survivin immunostaining, there were 55 (78.6%) samples with COX-2 expression. The staining of both COX-2 and survivin was localized in the lower and middle layers of the epithelium. When analyzed by Fisher's exact test, the expression of COX-2 showed a strong significant correlation with survivin (p=0.0002). CONCLUSIONS: These data, showing a significant correlation between COX-2 and survivin in primary pterygium, suggest that pterygium may originate through an anti-apoptotic mechanism.

Combinations of apoptosis and cell-cycle control biomarkers predict the outcome of human melanoma.

The deregulation of apoptosis is characteristic of human carcinogenesis. Survivin, an inhibitor of apoptosis, p53 and p16, two tumour suppressor proteins involved in cell cycle control, play a central role in apoptosis. The aim of this study was to investigate, in primary cutaneous melanoma from 68 patients, the expression of survivin with respect to p53 or p16; the association of these proteins, alone or in combination with clinicopathological features; and, most importantly, to elucidate the role of these markers in predicting survival. The level of survivin expression was significantly higher in the p53 positive group of melanomas compared with the p53 negative one, suggesting a cooperative effect in favouring the progression of melanoma, while no correlation was found between survivin and p16. Moreover, the altered expression of nuclear survivin, p53 and p16 were all associated with poor survival, as demonstrated by univariate analysis. However, these biomarkers have been shown to have superior predictive value when studied in combination (P<0.0001) rather than alone, while the risk of mortality grew progressively with increasing the number of altered biomarkers. These data suggest that the assessment of the combined marker status and number of altered markers in patients with melanoma provides important additional prognostic information that may help in patient selection for adjuvant therapies.

Vitamin D and vitamin D receptor in patients with ophthalmic pterygium.

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.

Oxidative stress in pterygium: relationship between p53 and 8-hydroxydeoxyguanosine.

PURPOSE: Ultraviolet (UV) radiation is known to cause oxidative DNA damage and is thought to be a major factor implicated in the pathogenesis of pterygium, a benign invasive lesion of the bulbar conjunctiva. Among all the photooxidative DNA products, 8-hydroxydeoxyguanosine (8-OHdG) is regarded as a sensitive and stable biomarker for evaluating the degree of DNA damage. The protein p53 is a major cell stress regulator that acts to integrate signals from a wide range of cellular stresses. UV radiation can cause mutations in the p53 tumor suppressor gene that, when inactivated through mutation and loss of heterozygosity, can lead to cell proliferation and genomic instability. In many types of UV-radiation damaged cells, p53 is overexpressed and immunohistochemically detectable. Recent data on tissues exposed to factors inducing oxidative stress have provided evidence of the concomitant presence of increased levels of 8-OHdG and protein p53. To verify a possible significant association between p53 and 8-OHdG, we examined a series of 31 Ecuadorian pterygia for the expression of the two markers. Moreover, we evaluated if clinical variables such as patient's age, gender, geographic location, and disease stage, might play a role affecting the 8-OHdG and p53 immunohistochemical staining results. METHODS: Primary pterygium samples were treated for immunohistochemical evaluations of 8-OHdG and p53 protein. Mouse monoclonal antibodies to 8-OHdG and p53 were used. Statistical analyses were performed using the SPSS 12 statistical software package. RESULTS: In our study, 21 (67.74%) pterygial samples were positive for 8-OHdG staining, 11 (35.48%) specimens were positive for p53 expression, and all negative control samples showed no staining. The staining for 8-OHdG was limited to the nuclei of the epithelial layer. No substantial staining was visible in the subepithelial fibrovascular layers. No differences in the pattern of staining between 8-OHdG and p53 were observed. All samples positive for p53 (11/31, 35.48%) were also positive for 8-OHdG immunostaining, and all specimens negative for 8-OHdG (10/31, 32.26%) were also negative for p53. When analyzed by Fisher's exact test, 8-OHdG expression was significantly associated with p53 positivity (p=0.0049). Student's t-test demonstrated statistically significant association between the expression of p53 and age (p=0.02). The correlation between the two markers and the other clinical variables revealed no statistically significant association. CONCLUSIONS: Although pterygium is a lesion with limited local invasion and an inability to metastasize, the concomitant presence of altered p53 in 8-OHdG-immunoreactive cells could provide evidence of apparent genetic instability, which is in contrast to its benign clinical course.

Activated Notch1 expression is associated with angiogenesis in cutaneous melanoma.

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.

Association between the ACE insertion/deletion polymorphism and pterygium in Sardinian patients: a population based case-control study.

OBJECTIVE: The 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 RESULTS: Polymorphism 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 (p<0.001). Moreover, a statistically significant difference was found between the DD and II groups (p<0.01; OR=10.49; 95% CI 6.18 to 17.79), DD+ID versus II group (p<0.01; OR=5.23; 95% CI 3.37 to 8.13) and DD versus ID groups (p<0.01; OR=3.21; 95% CI 2.04 to 5.04). CONCLUSIONS: Statistical 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.

Cytotoxic T-lymphocyte antigen 4 (CTLA4) +49AG and CT60 gene polymorphisms in Alopecia Areata: a case-control association study in the Italian population.

Alopecia Areata (AA) is an autoimmune disease characterized by well-circumscribed patches of hair loss especially from the scalp. Cytotoxic T-lymphocyte antigen 4 (CTLA4) gene, a negative regulator of T cells, has been associated with predisposition to most autoimmune disorders. We evaluated two CTLA4 functional single-nucleotide polymorphisms (SNPs) for potential association with Alopecia Areata in an Italian population using a case-control approach. We genotyped +49AG (rs231775) and CT60 (rs3087243) variants in 130 AA patients and 189 ethnically matched controls by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. CTLA4 +49AG analysis revealed no statistically significant difference in both the allele and genotype frequencies between patients and controls. As regarding CT60 SNP, we found that AA cases less frequently than healthy subjects carried A/A genotype with a higher prevalence of A/G and G/G genotypes (83.8 and 75.1 %; p = 0.041, OR = 0.58, 95 % CI 0.32-1.03), consistent with a dominant effect of G disease risk allele. In particular, it seemed to exert effects mainly in DQ7-negative patients with a less aggressive form of the disease. Haplotype analysis suggested that the G(+49AG), A(CT60) allelic combination was significantly related to a reduced disease risk (p = 0.014, OR = 0.28, 95 % 0.09-0.82). Altogether, our findings confirm that only CTLA4 CT60 polymorphism seems to be an important genetic determinant of Alopecia Areata development in Italian subjects.

Immunohistochemical analysis of angiotensin converting enzyme in Sardinian pterygium.

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.