Cannabinoid receptor type 1 immunoreactivity and disease severity in human epithelial ovarian tumors.

OBJECTIVE: In light of recent findings indicating that endocannabinoid system has antitumor actions, our study aimed to localize it in the human epithelial ovarian tumors, highlighting the differences among benign, borderline, and invasive forms and correlating cannabinoid receptor type 1 (CB1R) expression with disease severity. STUDY DESIGN: We determined CB1R immunohistochemical expression in 66 epithelial ovarian tumors treated in the Department of Woman, Child, and General and Specialized Surgery, Second University of Naples, at S. Maria del Popolo degli Incurabili Hospital (Naples): 36 borderline ovarian tumors, the main target of interest being intermediate forms, 15 benign and 15 invasive ovarian tumors. RESULTS: The benign ovarian tumors showed a weak expression of CB1R in the 33% of the cases and moderate expression in the 67% of the cases. Borderline ovarian tumors had a similar trend. They showed weak CB1R expression in 28% of the cases, moderate expression in 53% of the cases, and strong expression in 19% of the cases. In contrast, invasive tumors showed a weak expression of CB1R in 7% of the cases, moderate expression in 20% of the cases, and strong expression in 73% of the cases. CONCLUSION: The recorded data show that the expression of CB1R increased from benign and borderline to malignant tumors. In the near future, endocannabinoid receptors might be used in clinical practice, alone or in combination with other markers, to identify or better characterize ovarian tumors, without considering the great opportunity that they might represent as therapeutic targets.

Candida spp. in oral cancer and oral precancerous lesions.

To assess the presence of Candida spp. in lesions of the oral cavity in a sample of patients with precancer or cancer of the mouth and evaluate the limitations and advantages of microbiological and histological methods, 103 subjects with precancerous or cancerous lesions and not treated were observed between 2007 and 2009. The presence of Candida in the lesions was analyzed by microbiological and histological methods. Cohen's k statistic was used to assess the agreement between culture method and staining techniques. Forty-eight (47%) patients had cancer and 55 (53%) patients had precancerous lesions. Candida spp. were isolated from 31 (30%) patients with cancerous lesions and 33 (32%) with precancerous lesions. C. albicans was the most frequent species isolated in the lesions. The k value showed a fair overall agreement for comparisons between culture method and PAS (0.2825) or GMS (0.3112). This study supports the frequent presence of Candida spp. in cancer and precancerous lesions of the oral cavity. Both microbiological investigations and histological techniques were reliable for detection of Candida spp. It would be desirable for the two techniques to be considered complementary in the detection of yeast infections in these types of lesions.

Primary extranodal non-Hodgkin's lymphoma of the vagina: a case report and a review of the literature.

Primary lymphoma of the female genital tract is very rare. We report the case of a 36-year-old woman who was referred to our hospital because of an indeterminate Pap smear test. The colposcopy showed a thickening of the posterior vaginal wall and various irregular ulcerated nodular lesions. Histological examination, immunohistochemistry and the staging procedures were conclusive of diffuse large B-cell lymphoma of the vagina, stage IEA. Complete remission was achieved after 6 cycles of immunopolychemotherapy (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone). No relapse has occurred during a follow-up of 71 months. Moreover, we reviewed the 62 previously reported cases of primary extranodal non-Hodgkin's lymphoma of the vagina, focusing on clinicopathological and therapeutic aspects, to better characterize this unusual disease.

Primary thyroid angiosarcoma: an unusual localization.

The finding of thyroid nodules is a very common occurrence in routine clinical practice. Approximately 5% to 7% of the entire population have thyroid nodules. Vascular lesions are one of the most controversial issues in thyroid pathology. These include benign lesions such as hemangiomas and, rarely, malignant tumors such as angiosarcomas or undifferentiated angiosarcomatoid carcinomas. In particular, angiosarcoma of the thyroid gland is a rare, highly aggressive malignant vascular tumor and in Italy the greatest geographical incidence of this lesion is witnessed near the Alps. Here, a case of thyroid angiosarcoma in a 71-year-old man with a history of goiter for about 20 years is described. The unusual localization of this lesion, the difficulties in reaching a definitive diagnosis for this particular histological type of primary tumor and a history of long-standing multinodular goiter in thyroid of an older man from outside the Alpine region prompted us to report this case of thyroid angiosarcoma mainly to discuss surgical, histopathological and immunohistochemical features.

Clinical considerations on the retroperitoneal liposarcomas.

AIM: It presents a clinical case of undifferentiated retroperitoneal liposarcoma with 5 years' recurrence from the first operation for the rarity of the occurrence, the problems related to surgery and complementary therapeutic approach. MATERIAL OF STUDY: Male patient aged 73 was operated for removal of retroperitoneal mass with involvement of the right kidney at the Second University of Naples in the 2003. In accordance with the interdisciplinary board, complementary therapy is not indicated and follow-up program. The successive controls were negative until at least 2007. Reoperation for recurrence in 2008 for the presence of massive bone formation occupying a large part in the right half of the abdomen at the sub-hepatic level. In both cases histological examination showed undifferentiated liposarcoma. DISCUSSION: It is of unknown etiology and only 25% occurs in well-differentiated cells are also more than 100 histological subtypes, 85% are malignant. The most affected is the male sex and from the beginning looks like malignancy. The trend of growth in general is slow, and in most cases tends to recur over time. The role of chemotherapy and radiation therapy is controversial. CONCLUSIONS: The peculiarity of our case is higher than the average survival despite advanced age and presence of recurrence. This confirms the importance of surgical treatment, thus offering the patient a chance of better long-term survival.

YY1 overexpression is associated with poor prognosis and metastasis-free survival in patients suffering osteosarcoma.

BACKGROUND: The polycomb transcription factor Yin Yang 1 (YY1) overexpression can be causally implicated in experimental tumor growth and metastasization. To date, there is no clinical evidence of YY1 involvement in outcome of patients with osteosarcoma. Prognosis of osteosarcoma is still severe and only few patients survive beyond five years. We performed a prospective immunohistochemistry analysis to correlate YY1 immunostaining with metastatic development and survival in a selected homogeneous group of patients with osteosarcoma. METHODS: We studied 41 patients suffering from osteosarcoma (stage II-IVa). Multivariate analysis was performed using Cox proportional hazard regression to evaluate the correlation between YY1 expression and both metastasis development and mortality. RESULTS: YY1 protein is not usually present in normal bone; in contrast, a high number of patients (61%) showed a high score of YY1 positive cells (51-100%) and 39% had a low score (10-50% positive cells). No statistical difference was found in histology, anatomic sites, or response to chemotherapy between the two degrees of YY1 expression. Cox regression analysis demonstrated that the highest score of YY1 expression was predictive of both low metastasis-free survival (HR = 4.690, 95%CI = 1.079-20.396; p = 0.039) and poor overall survival (HR = 8.353, 95%CI = 1.863-37.451 p = 0.006) regardless of the effects of covariates such as age, gender, histology and chemonecrosis. CONCLUSION: Overexpression of YY1 in primary site of osteosarcoma is associated with the occurrence of metastasis and poor clinical outcome.

Detrimental effects of Bartonella henselae are counteracted by L-arginine and nitric oxide in human endothelial progenitor cells.

The recruitment of circulating endothelial progenitor cells (EPCs) might have a beneficial effect on the clinical course of several diseases. Endothelial damage and detachment of endothelial cells are known to occur in infection, tissue ischemia, and sepsis. These detrimental effects in EPCs are unknown. Here we elucidated whether human EPCs internalize Bartonella henselae constituting a circulating niche of the pathogen. B. henselae invades EPCs as shown by gentamicin protection assays and transmission electron microscopy (TEM). Dil-Ac-LDL/lectin double immunostaining and fluorescence-activated cell sorting (FACS) analysis of EPCs revealed EPC bioactivity after infection with B. henselae. Nitric oxide (NO) and its precursor l-arginine (l-arg) exert a plethora of beneficial effects on vascular function and modulation of immune response. Therefore, we tested also the hypothesis that l-arg (1-30 mM) would affect the infection of B. henselae or tumor necrosis factor (TNF) in EPCs. Our data provide evidence that l-arg counteracts detrimental effects induced by TNF or Bartonella infections via NO (confirmed by DETA-NO and L-NMMA experiments) and by modulation of p38 kinase phosphorylation. Microarray analysis indicated several genes involved in immune response were differentially expressed in Bartonella-infected EPCs, whereas these genes returned in steady state when cells were exposed to sustained doses of l-arg. This mechanism may have broad therapeutic applications in tissue ischemia, angiogenesis, immune response, and sepsis.

Modification of the detrimental effect of TNF-α on human endothelial progenitor cells by fasudil and Y27632.

Exposure of human endothelial progenitor cells (EPCs) to tumor necrosis factor-α (TNF-α) reduced their number and biological activity. Yet, signal transduction events linked to TNF-α action are still poorly understood. To address this issue, we examined the possible effect of fasudil and Y27632, two inhibitors of Rho kinase pathway, which is involved in endothelial dysfunction, atherosclerosis, and in- flammation. Results demonstrated that incubation with fasudil starting from 50 µM but not Y27632 determined a dose-dependent improvement of EPC number during exposure to TNF-α (P < 0.05 vs. TNF-α alone). Analysis of the signal transduction pathway activated by TNF-α revealed that the increased expression of p-p38 was not significantly altered by fasudil. Instead, fasudil blocked the TNF-α induced phosphorylation of Erk1/2 (P < 0.05 vs. TNF-α) as well as the inhibitor of Erk1/2-specific phosphorylated form, i.e., PD98059 (P < 0.05 vs. TNF-α). These results were confirmed by analysis of these kinases by confocal microscopy. Finally, 2D-DIGE and MALDI-TOF/TOF analysis of EPCs treated with fasudil revealed increased expression levels of an actin-related protein and an adenylyl cyclase associated protein and decreased expression levels of proteins related to radical scavenger and nucleotide metabolism. These findings suggest that fasudil positively affects EPC number and that other major signals might take part to this complex pathway.

Oncocytic carcinoma of the accessory lobe of the parotid gland.

Oncocytic carcinoma is a rare tumor of the parotid gland. An additional case, characterized by an exceptional localization in the accessory lobe of the parotid gland, never reported to date in the literature, is described. The clinical and histologic difficulties in relationship to the diagnosis of a midcheek mass consisting of an unusual tumor are emphasized.

High glucose downregulates endothelial progenitor cell number via SIRT1.

Increasing evidence indicates that mammalian SIRT1 mediates calorie restriction and influences lifespan regulating a number of biological molecules such as FoxO1. SIRT1 controls the angiogenic activity of endothelial cells via deacetylation of FoxO1. Endothelial dysfunction and reduced new blood vessel growth in diabetes involve a decreased bioactivity of endothelial progenitor cells (EPCs) via repression of FoxO1 transcriptional activity. The relative contribution of SIRT1 with respect to the direct effects of high glucose on EPC number is poorly understood. We report that treatment of EPCs with high glucose for 3 days determined a consistent downregulation of EPC positive to DiLDL/lectin staining and, interestingly, this was associated with reduced SIRT1 expression levels and enzyme activity, and increased acetyl-FoxO1 expression levels. Moreover, EPCs responded to high glucose with major changes in the expression levels of cell metabolism-, cell cycle-, and oxidative stress-related genes or proteins. Proteomic analysis shows increased expression of nicotinamide phosphoribosyl transferase and mitochondrial superoxide dismutase whereas a glucose-related heat shock protein is reduced. These findings show that SIRT1 is a critical modulator of EPCs dysfunction during alteration of glucose metabolism.

Differential expression of cyclooxygenases in hypertrophic scar and keloid tissues.

Hypertrophic scar (HS) and keloid (KL) are two forms of an abnormal cutaneous scarring process, mainly characterized by excessive extracellular matrix deposition and fibroblast proliferation. Despite the increased understanding of the molecular and cellular events leading to HS and KL, the pathogenesis of these lesions remains poorly understood. A pivotal role in the formation of abnormal scars has been ascribed to transforming growth factor-beta, whose activity appears to be mediated through a link with pathways acting via cyclooxygenases (COX-1 and COX-2). To date, there is no report on the in vivo expression of COX-1 and COX-2 in human HS and KL tissues. Therefore, using immunohistochemistry and Western blot analysis, we investigated 36 cases of KL, 32 cases of HS, and 25 cases of normal skin in order to define the localization and distribution of COX-1 and COX-2 in the tissues of these scar lesions and the overlying epidermis. The results mainly show the following: (a) a significant overexpression of COX-1 in HS tissues and the overlying epidermis as compared with normal skin and KL tissues and (b) a significant overexpression of COX-2 in KL tissue and the overlying epidermis in contrast to normal skin and HS tissues. Our data support the hypothesis that both COXs are involved in the pathogenesis of scar lesions in different ways and, particularly, COX-1 in the formation of HS and COX-2 in the formation of KL. In addition, the overexpression of COX-1 and COX-2 in the epidermis overlying HS and KL tissues, respectively, underlines the importance of epithelial-mesenchymal interactions in the pathogenesis of scar lesions.

A scleroderma-like cutaneous syndrome associated with a marked Th2-type immune response occurring after a prosthetic joint implant.

A scleroderma-like cutaneous syndrome, occurring after implantation of a prosthetic knee joint in an elderly woman, is reported. This case did not seem to typically fit into any of the known scleroderma-like disorders of the skin described to date. The patient was shown to be sensitized to metals contained in the prosthesis and to mount a Th2-type immune response concomitantly with development of skin fibrosis. In particular, eosinophilia, markedly elevated serum IgE levels, in vitro spontaneous production of interleukin (IL)-4 by T lymphocytes, and elevated serum levels of Th2 cytokines (namely, IL-4, IL-5, and IL-13) were observed during the acute phase of illness. Since eosinophils and such Th2 cytokines as IL-13 also have recognized fibrogenic properties, it is speculated that the pathogenesis of skin fibrosis in this case could have been the direct and/or indirect consequence of the coexisting Th2-type immune response.

Silencing of YY1 downregulates RIZ1 promoter in human osteosarcoma.

RIZ1 isoform, but not RIZ2, is commonly silenced in many types of tumors. In osteosarcoma cells, RIZ1 protein is very abundant. The silencing of YY1 protein, a recent target gene in osteosarcoma cells, reduced the expression of RIZ1 protein. Here we show that RIZ1 overexpression is a transcriptional event documented by Western blot, RT-PCR, and promoter assays. YY1 protein binds and cooperates to positive regulation of the RIZ1 promoter and its presence reduced the dimethyl lysine 9 histone 3 by chromatin immunoprecipitation assays. These results indicate that overexpression of YY1 in osteosarcoma cells plays a key role in positive regulation of RIZ1. The coexpression of RIZ1/YY1 proteins suggests a tandem regulatory mechanism in human osteosarcoma cells and tissues.

Evidence of COX-1 and COX-2 expression in Kaposi's sarcoma tissues.

Cyclooxygenases (COXs) are enzymes catalysing prostaglandin synthesis and are implicated in the carcinogenesis of some cancer types. In addition, an important role of these enzymes in herpesvirus infections was demonstrated and it has recently been proposed that COX-2 may participate in herpesvirus-induced neoplasia such as Kaposi's sarcoma (KS). To date no immunohistochemical study has been performed to determine the identification of COX-1 and COX-2 in KS. We have investigated 35 cases of classic KS and 27 cases of epidemic KS form in order to study the distribution and localisation of COXs. We have examined by immunohistochemistry the expression of COX-1 and COX-2 in classic and epidemic forms of KS also in relationship to the characteristic morphological phases (patch, plaque and nodular stage) of KS and cell localisation by double immunostaining. Moreover, we have obtained COX-1 and COX-2 expression by Western blot analysis. Our results establish that (a) COX-1 and COX-2 are overexpressed significantly in classic and epidemic KS compared with control skin tissues (P<0.01 and P>0.03, respectively, for COX-1; P<0.01 and P>0.03, respectively, for COX-2); (b) the extent and intensity staining for both COXs were higher in classic than in epidemic form of KS. Our data support the hypothesis that both COXs may be involved in the pathogenesis of KS.

Comparison between total endothelial progenitor cell isolation versus enriched Cd133+ culture.

Endothelial progenitor cells (EPCs) play a role in endogenous neovascularization of ischaemic tissues. Isolation and characterization of EPCs from circulating mononuclear cells are important for developing targeted cellular therapies and reproducibility of data are the major scientific goals. Here we compared two currently employed isolation methods, i.e. from total peripheral blood mononuclear cells (PBMCs) and from enriched CD133(+) cells, by defining the cell morphology and functional activities. We show that EPCs from cultured PBMCs resulted in an adherent population of 23% +/- 4% merged cells positive for Dil-Ac-LDL and lectin, whereas the percentage of double positive cells in cultured CD133(+) enriched cells was 50% +/- 7% (P < 0.01). These data were obtained through a novel and a more complete method of analysis of cell calculations (specifically by dividing each microscope field into 120 subfields). When stimulated with tumour necrosis factor alpha (TNF)-alpha and glucose, cell number was reduced in EPCs from total PBMCs and, more consistently, in CD133(+) enriched cells. However, both cultured total PBMCs and CD133(+) enriched cells respond similarly to TNF-alpha or glucose-induced p38-phosphorylation. EPCs from both procedures show similar results in terms of phenotype and response to modulators of their functional activities. However, when the cell phenotype of CD133(+) enrichment-derived cells was compared with that of cells from the total PBMC, a significant increase in CD133(+) expression was observed (P < 0.01) This may have relevance during intervention studies using cultured EPCs.

Expression of transcription factor Yin Yang 1 in human osteosarcomas.

The transcription factor Yin Yang 1 (YY1) is known to be present in some human cancer cell lines and its expression correlates with immune-mediated apoptosis. By using Western blot analysis, we have shown that the YY1 protein is strongly expressed in human osteosarcoma cells and localised mainly in the nucleus. Moreover, by using immunohistochemistry and RT-PCR techniques, we have analysed the expression of YY1 protein in biopsies from human osteosarcomas. The YY1 protein was not detectable by immunohistochemistry in osteoid tissue. However, its expression was restricted to osteosarcoma tissues. These data were confirmed by densitometric analysis of RT-PCR for YY1 expression. Thus, YY1 gene activation appears to be an early event in the process of osteoblastic transformation and its detection may represent, together with the analysis of other established markers, a useful diagnostic tool in human osteosarcomas.

Persistent infarct-related artery occlusion is associated with an increased myocardial apoptosis at postmortem examination in humans late after an acute myocardial infarction.

BACKGROUND: Myocardial apoptosis persists beyond the acute phases of acute myocardial infarction (AMI) and is associated with left ventricular (LV) remodeling. Infarct-related artery (IRA) patency is considered a favorable prognostic factor after AMI and may be associated with more favorable LV remodeling because of reduced apoptosis at the site of AMI. The aim of this study was to assess the influence of IRA status on apoptotic rate (AR) in the hearts of subjects dying late after AMI. METHODS AND RESULTS: We used colocalization for in situ end-labeling of DNA fragmentation and immunohistochemistry for caspase-3 to calculate the AR at time of death (12 to 62 days after AMI) in 16 hearts with persistently occluded IRAs and in 8 hearts with patent IRAs. No significant differences were found when comparing the clinical characteristics of the 2 groups. Occluded IRA was associated with significantly higher AR at site of infarction (25.8% [interquartile range 20.9% to 28.5%] versus 2.3% [interquartile range 0.6% to 5.0%], P<0.001). This strong correlation between IRA occlusion and AR remained statistically significant even after correction for clinical characteristics such as sex, age, history of previous additional AMI or heart failure, transmural AMI, anterior AMI, fibrinolytic treatment, time from AMI to death, trauma as cause of death, and multivessel coronary disease (P=0.003). CONCLUSIONS: A significantly higher AR was associated with persistent IRA occlusion late post-AMI. These data may suggest that the post-AMI benefits observed with a patent IRA (the "open-artery hypothesis") may in part be due to reduced myocardial apoptosis.

Increased myocardial apoptosis in patients with unfavorable left ventricular remodeling and early symptomatic post-infarction heart failure.

OBJECTIVES: The purpose of this study was to evaluate a potential correlation between apoptotic rate (AR), post-infarction left ventricular (LV) remodeling, and clinical characteristics in subjects who died late (>or=10 days) after an acute myocardial infarction (AMI) with evidence of persistent occlusion of the infarct-related artery at autopsy. BACKGROUND: Apoptosis contributes to myocardiocyte loss in cardiac disease and may have a pathophysiologic role in post-infarction LV remodeling. METHODS: The AR was calculated at the site of infarction and in remote unaffected LV regions, using co-localization of in situ end labeling for deoxyribonucleic acid fragmentation and immunohistochemistry for caspase-3, in 14 subjects who died within two months after AMI. Correlation between AR and clinical characteristics such as age, site of AMI, transmural extension, multivessel coronary disease, and signs and/or symptoms of heart failure (HF), at the time of initial hospitalization for AMI or subsequently before death, was assessed using non-parametric statistical tests. Parameters of LV remodeling including diameters, free wall thickness, diameter-to-wall-thickness ratio, and mass were measured at gross examination at autopsy. Values are expressed as median (interquartile range). RESULTS: Among clinical variables, early symptomatic post-infarction HF (9 cases, 64%) was associated with nearly fourfold increased AR at the site of infarction (26.2% [24.5% to 28.8%] vs. 6.4% [1.9% to 13.3%], p = 0.001). Moreover, AR both at the site of infarction and in unaffected regions was significantly correlated with parameters of progressive LV remodeling (p < 0.05). CONCLUSIONS: Our data show that in patients dying >or=10 days after AMI, myocardial apoptosis is strongly associated with and may be a major determinant of unfavorable LV remodeling and early symptomatic post-infarction HF.

Nonrandom distribution of aberrant promoter methylation of cancer-related genes in sporadic breast tumors.

PURPOSE: In an effort to additionally determine the global patterns of CpG island hypermethylation in sporadic breast cancer, we searched for aberrant promoter methylation at 10 gene loci in 54 primary breast cancer and 10 breast benign lesions. EXPERIMENTAL DESIGN: Genomic DNA sodium bisulfate converted from benign and malignant tissues was used as template in methyl-specific PCR for BRCA1, p16, ESR1, GSTP1, TRbeta1, RARbeta2, HIC1, APC, CCND2, and CDH1 genes. RESULTS: The majority of the breast cancer (85%) showed aberrant methylation in at least 1 of the loci tested with half of them displaying 3 or more methylated genes. The highest frequency of aberrant promoter methylation was found for HIC1 (48%) followed by ESR1 (46%), and CDH1 (39%). Similar methylation frequencies were detected for breast benign lesions with the exception of the CDH1 gene (P = 0.02). The analysis of methylation distribution indicates a statistically significant association between methylation of the ESR1 promoter, and methylation at CDH1, TRbeta1, GSTP1, and CCND2 loci (P < 0.03). Methylated status of the BRCA1 promoter was inversely correlated with methylation at the RARbeta2 locus (P < 0.03). CONCLUSIONS: Our results suggest a nonrandom distribution for promoter hypermethylation in sporadic breast cancer, with tumor subsets characterized by aberrant methylation of specific cancer-related genes. These breast cancer subgroups may represent separate biological entities with potential differences in sensitivity to therapy, occurrence of metastasis, and overall prognosis.