The ectonucleotidases CD39 and CD73 degrade immune stimulatory ATP to adenosine that inhibits T and NK cell responses via the A(2A) adenosine receptor (ADORA2A). This mechanism is used by regulatory T cells (T(reg)) that are associated with increased mortality in OvCA. Immunohistochemical staining of human OvCA tissue specimens revealed further aberrant expression of CD39 in 29/36 OvCA samples, whereas only 1/9 benign ovaries showed weak stromal CD39 expression. CD73 could be detected on 31/34 OvCA samples. While 8/9 benign ovaries also showed CD73 immunoreactivity, expression levels were lower than in tumour specimens. Infiltration by CD4(+) and CD8(+) T cells was enhanced in tumour specimens and significantly correlated with CD39 and CD73 levels on stromal, but not on tumour cells. In vitro, human OvCA cell lines SK-OV-3 and OaW42 as well as 11/15 ascites-derived primary OvCA cell cultures expressed both functional CD39 and CD73 leading to more efficient depletion of extracellular ATP and enhanced generation of adenosine as compared to activated T(reg). Functional assays using siRNAs against CD39 and CD73 or pharmacological inhibitors of CD39, CD73 and ADORA2A revealed that tumour-derived adenosine inhibits the proliferation of allogeneic human CD4(+) T cells in co-culture with OvCA cells as well as cytotoxic T cell priming and NK cell cytotoxicity against SK-OV3 or OAW42 cells. Thus, both the ectonucleotidases CD39 and CD73 and ADORA2A appear as possible targets for novel treatments in OvCA, which may not only affect the function of T(reg) but also relieve intrinsic immunosuppressive properties of tumour and stromal cells.

Background:Screening is an unsolved problem for ovarian cancer (OvCA). As late detection is equivalent to poor prognosis, we analysed whether OvCA patients show diagnostically meaningful microRNA (miRNA) patterns in blood cells.Methods:Blood-borne whole miRNome profiles from 24 patients with OvCA and 15 age- and sex-matched healthy controls were biostatistically evaluated.Results:Student's t-test revealed 147 significantly deregulated miRNAs before and 4 after Benjamini–Hochberg adjustment. Although these included miRNAs already linked to OvCA (e.g., miR-16, miR-155), others had never before been connected to specific diseases. A bioinformatically calculated miRNA profile allowed for discrimination between blood samples of OvCA patients and healthy controls with an accuracy of >76%. When only cancers of the serous subtype were considered and compared with an extended control group (n=39), accuracy, specificity and sensitivity all increased to >85%.Conclusion:Our proof-of-principle study strengthens the hypothesis that neoplastic diseases generate characteristic miRNA fingerprints in blood cells. Still, the obtained OvCA-associated miRNA pattern is not yet sensitive and specific enough to permit the monitoring of disease progression or even preventive screening. Microarray-based miRNA profiling from peripheral blood could thus be combined with other markers to improve the notoriously difficult but important screening for OvCA.

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The present study investigated the effect of tumor necrosis factor (TNF)-␣ on myocardial energy metabolism as estimated by myocardial oxygen consumption (MV O2). MV O2 of electrically stimulated isolated trabeculae of right ventricular Wistar rat myocardium was analyzed using a Clark-type oxygen probe. After the initial data collection in the absence of TNF-␣, measurements were repeated after TNF-␣ stimulation. In separate experiments, pretreatment with the nitric oxide (NO) synthase inhibitor N G -nitro-L-arginine methyl ester (L-NAME) or the ceramidase inhibitor n-oleoylethanolamine (NOE) was done to investigate NO/sphingosine-related effects. TNF-␣ impaired myocardial economy at increasing stimulation frequencies without altering baseline MV O2. Incubation with TNF-␣ in the presence of L-NAME further impaired myocardial economy. NOE preincubation abrogated the TNF-␣ effect on myocardial economy. Moreover, the negative inotropic effect of TNF-␣ was observed in NOE-pretreated but not L-NAME-pretreated muscle fibers. Exogenous sphingosine mimicked the TNF-␣ effect on mechanics and energetics. We conclude that TNF-␣ impairs the economy of chemomechanical energy transduction primarily through a sphingosine-mediated pathway. cytokines; nitric oxide; myocardial energy metabolism; tumor necrosis factor-␣

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