Changes in total choline concentration in the breast of healthy fertile young women in relation to menstrual cycle or use of oral contraceptives: a 3-T 1H-MRS study

BACKGROUND: To evaluate changes in total choline (tCho) absolute concentration ([tCho]) in the breast of healthy fertile women in relation to menstrual cycle (MC) or use of oral contraceptives (OC). METHODS: After institutional review board approval, we prospectively evaluated 40 healthy fertile volunteers: 20 with physiological MC, aged 28 ± 3 years (mean ± standard deviation; nOC group); 20 using OC, aged 26 ± 3 years (OC group). Hormonal assays and water-suppressed single-voxel 3-T proton magnetic resonance spectroscopy (1H-MRS) were performed on MC days 7, 14, and 21 in the nOC group and only on MC day 14 in the OC group. [tCho] was measured versus an external phantom. Mann-Whitney U test and Spearman coefficient were used; data are given as median and interquartile interval. RESULTS: All spectra had good quality. In the nOC group, [tCho] (mM) did not change significantly during MC: 0.8 (0.3-2.4) on day 7, 0.9 (0.4-1.2) on day 14, and 0.4 (0.2-0.8) on day 21 (p = 0.963). In the OC group, [tCho] was 0.7 (0.2-1.7) mM. The between-groups difference was not significant on all days (p ≥ 0.411). All hormones except prolactin changed during MC (p ≤ 0.024). In the OC group, [tCho] showed a borderline correlation with estradiol (r = 0.458, p = 0.056), but no correlation with other hormones (p ≥ 0.128). In the nOC group, [tCho] negatively correlated with prolactin (r = -0.587, p = 0.006) on day 7; positive correlation was found with estradiol on day 14 (r = 0.679, p = 0.001). CONCLUSIONS: A tCho peak can be detected in the normal mammary gland using 3-T 1H-MRS. The [tCho] in healthy volunteers was 0.4-0.9 mM, constant over the MC and independent of OC use

Generation and characterization of a human single-chain fragment variable (scFv) antibody against cytosine deaminase from Yeast

<p>Abstract</p> <p>Background</p> <p>The ability of cytosine deaminase (CD) to convert the antifungal agent 5-fluorocytosine (5-FC) into one of the most potent and largely used anticancer compound such as 5-fluorouracil (5-FU) raised considerable interest in this enzyme to model gene or antibody – directed enzyme-prodrug therapy (GDEPT/ADEPT) aiming to improve the therapeutic ratio (benefit versus toxic side-effects) of cancer chemotherapy. The selection and characterization of a human monoclonal antibody in single chain fragment (scFv) format represents a powerful reagent to allow in <it>in vitro </it>and <it>in vivo </it>detection of CD expression in GDEPT/ADEPT studies.</p> <p>Results</p> <p>An enzymatic active recombinant CD from yeast (yCD) was expressed in E. coli system and used as antigen for biopanning approach of the large semi-synthetic ETH-2 antibody phage library. Several scFvs were isolated and specificity towards yCD was confirmed by Western blot and ELISA. Further, biochemical and functional investigations demonstrated that the binding of specific scFv with yCD did not interfere with the activity of the enzyme in converting 5-FC into 5-FU.</p> <p>Conclusion</p> <p>The construction of libraries of recombinant antibody fragments that are displayed on the surface of filamentous phage, and the selection of phage antibodies against target antigens, have become an important biotechnological tool in generating new monoclonal antibodies for research and clinical applications. The scFvH5 generated by this method is the first human antibody which is able to detect yCD in routinary laboratory techniques without interfering with its enzymatic function.</p

Cellular Localization and Functional Role of Phosphatidylcholine-Specific Phospholipase C in NK Cells

AbstractAlthough several classes of phospholipases have been implicated in NK cell-mediated cytotoxicity, no evidence has been reported to date on involvement of phosphatidylcholine-specific phospholipase C (PC-PLC) in NK activation by lymphokines and/or in lytic granule exocytosis. This study demonstrated the expression of two PC-PLC isoforms (Mr 40 and 66 kDa) and their IL-2-dependent distribution between cytoplasm and ectoplasmic membrane surface in human NK cells. Following cell activation by IL-2, cytoplasmic PC-PLC translocated from the microtubule-organizing center toward cell periphery, essentially by kinesin-supported transport along microtubules, while PC-PLC exposed on the outer cell surface increased 2-fold. Preincubation of NK cells with a PC-PLC inhibitor, tricyclodecan-9-yl-xanthogenate, strongly reduced NK-mediated cytotoxicity. In IL-2-activated cells, this loss of cytotoxicity was associated with a decrease of PC-PLC exposed on the cell surface, and accumulation of cytoplasmic PC-PLC in the Golgi region. Massive colocalization of PC-PLC-rich particles with perforin-containing granules was found in the cytoplasm of NK-activated (but not NK-resting) cells; both organelles clustered at the intercellular contact region of effector-target cell conjugates. These newly detected mechanisms of PC-PLC translocation and function support an essential role of this enzyme in regulated granule exocytosis and NK-mediated cytotoxicity

Exploring Cancer Metabolic Reprogramming Through Molecular Imaging

The inclusion of oncogene-driven reprogramming of energy metabolism within the list of cancer hallmarks (Hanahan and Weinberg, Cell 2000, 2011) has provided major impetus to further investigate the existence of a much wider metabolic rewiring in cancer cells, which not only includes deregulated cellular bioenergetics, but also encompasses multiple links with a more comprehensive network of altered biochemical pathways. This network is currently held responsible for redirecting carbon and phosphorus fluxes through the biosynthesis of nucleotides, amino acids, lipids and phospholipids and for the production of second messengers essential to cancer cells growth, survival and invasiveness in the hostile tumor environment. The capability to develop such a concerted rewiring of biochemical pathways is a versatile tool adopted by cancer cells to counteract the host defense and eventually resist the attack of anticancer treatments. Integrated efforts elucidating key mechanisms underlying this complex cancer metabolic reprogramming have led to the identification of new signatures of malignancy that are providing a strong foundation for improving cancer diagnosis and monitoring tumor response to therapy using appropriate molecular imaging approaches. In particular, the recent evolution of positron emission tomography (PET), magnetic resonance spectroscopy (MRS), spectroscopic imaging (MRSI), functional MR imaging (fMRI) and optical imaging technologies, combined with complementary cellular imaging approaches, have created new ways to explore and monitor the effects of metabolic reprogramming in cancer at clinical and preclinical levels. Thus, the progress of high-tech engineering and molecular imaging technologies, combined with new generation genomic, proteomic and phosphoproteomic methods, can significantly improve the clinical effectiveness of image-based interventions in cancer and provide novel insights to design and validate new targeted therapies. The Frontiers in Oncology Research Topic “Exploring Cancer Metabolic Reprogramming Through Molecular Imaging” focusses on current achievements, challenges and needs in the application of molecular imaging methods to explore cancer metabolic reprogramming, and evaluate its potential impact on clinical decisions and patient outcome. A series of reviews and perspective articles, along with original research contributions on humans and on preclinical models have been concertedly included in the Topic to build an open forum on perspectives, present needs and future challenges of this cutting-edge research area