1. Development and Validation of a High- Performance Liquid Chromatography– Tandem Mass Spectrometry Method for the Simultaneous Determination of Irinotecan and Its Main Metabolites in Human Plasma and Its Application in a Clinical Pharmacokinetic StudyGiuseppe Toffoli, Elena Marangon, Elisa Mazzega, Bianca Posocco, 2015, original scientific article Abstract: Irinotecan is currently used in several cancer regimens mainly in colorectal cancer (CRC). This drug has a narrow therapeutic range and treatment can lead to side effects, mainly neutropenia and diarrhea, frequently requiring discontinuing or lowering the drug dose. A wide inter-individual variability in irinotecan pharmacokinetic parameters and pharmacodynamics has been reported and associated to patients’ genetic background. In particular, a polymorphism in the UGT1A1 gene (UGT1A1*28) has been linked to an impaired detoxification of SN-38 (irinotecan active metabolite) to SN-38 glucuronide (SN-38G) leading to increased toxicities. Therefore, therapeutic drug monitoring of irinotecan, SN-38 and SN-38G is recommended to personalize therapy. In order to quantify simultaneously irinotecan and its main metabolites in patients’ plasma, we developed and validated a new, sensitive and specific HPLC–MS/MS method applicable to all irinotecan dosages used in clinic. This method required a small plasma volume, addition of camptothecin as internal standard and simple protein precipitation. Chromatographic separation was done on a Gemini C18 column (3 μM, 100 mm x 2.0 mm) using 0.1% acetic acid/bidistilled water and 0.1% acetic acid/acetonitrile as mobile phases. The mass spectrometer worked with electrospray ionization in positive ion mode and selected reaction monitoring. The standard curves were linear (R20.9962) over the concentration ranges (10–10000 ng/mL for irinotecan, 1–500 ng/mL for SN-38 and SN-38G and 1–5000 ng/mL for APC) and had good back-calculated accuracy and precision. The intra- and inter-day precision and accuracy, determined on three quality control levels for all the analytes, were always <12.3% and between 89.4% and 113.0%, respectively. Moreover, we evaluated this bioanalytical method by re-analysis of incurred samples as an additional measure of assay reproducibility. This method wassuccessfully applied to a pharmacokinetic study in metastatic CRC patients enrolled in a genotype-guided phase Ib study of irinotecan administered in combination with 5-fluorouracil/ leucovorin and bevacizumab.
Found in: osebi
Keywords: Pharmacokinetic, pharmacodynamics, phase I clinical study, colorectal cancer, Mass spectrometry, pharmacogenetics
Published: 21.03.2017; Views: 3782; Downloads: 0
 Fulltext (1018,06 KB) |
2. Ex Vivo Molecular Rejuvenation Improves the Therapeutic Activity of Senescent Human Cardiac Stem Cells in a Mouse Model of Myocardial InfarctionElisa Avolio, Giuseppe Gianfranceschi, Angela Caragnano, Emmanouil Athanasakis, Rajesh Katare, Daniela Cesselli, Marco Meloni, Anita Palma, Arianna Barchiesi, Carlo Vascotto, Barbara Toffoletto, Elisa Mazzega, Nicoletta Finato, Giuseppe Aresu, Ugolino Livi, Costanza Emanueli, Giacinto Scoles, Carlo Alberto Beltrami, Paolo Madeddu, Antonio Paolo Beltrami, 2014, original scientific article Abstract: Cardiac stem cells (CSC) from explanted decompensated hearts (E-CSC) are, with respect to those obtained from healthy donors (D-CSC), senescent and functionally impaired. We aimed to identify alterations in signaling pathways that are associated with CSC senescence. Additionally, we inves- tigated if pharmacological modulation of altered pathways can reduce CSC senescence in vitro and enhance their reparative ability in vivo. Measurement of secreted factors showed that E-CSC release larger amounts of proinflammatory cytokine IL1b compared with D-CSC. Using blocking antibodies, we verified that IL1b hampers the paracrine protective action of E-CSC on cardiomyo- cyte viability. IL1b acts intracranially inducing IKKb signaling, a mechanism that via nuclear factor-jB upregulates the expression of IL1b itself. Moreover, E-CSC show reduced levels of AMP protein kinase (AMPK) activating phosphorylation. This latter event, together with enhanced IKKb signaling, increases TORC1 activity, thereby impairing the autophagic flux and inhibiting the phos- phorylation of Akt and cAMP response element-binding protein. The combined use of rapamycin and resveratrol enhanced AMPK, thereby restoring downstream signaling and reducing IL1b secretion. These molecular corrections reduced E-CSC senescence, re-establishing their protective activity on cardiomyocytes. Moreover ex vivo treatment with rapamycin and resveratrol improved E-CSC capacity to induce cardiac repair upon injection in the mouse infarcted heart, leading to reduced cardiomyocyte senescence and apoptosis and increased abundance of endog- enous c-Kit1 CSC in the peri-infarct area. Molecular rejuvenation of patient-derived CSC by short pharmacologic conditioning boosts their in vivo reparative abilities. This approach might prove useful for refinement of CSC-based therapies.
Found in: osebi
Keywords: Stem cells, Myocardial infarction, Cellular senescence, Heart failure
Published: 21.03.2017; Views: 3366; Downloads: 0
Fulltext (1,48 MB) |
3. Analysis of mechanosensing in human cardiac stem cellsElisa Mazzega, Eliana Pomarè, Angela Caragnano, Sebastian Martewicz, Nicola Elvassore, Antonio Paolo Beltrami, Ugo Livi, 2015, published scientific conference contribution abstract Abstract: Objectives: We have shown that age and pathology impair the biological properties of stem cells isolated from human hearts (CSC) and functional assays showed differences between CSC isolated from normal (DCSC) and end-stage failing (ECSC) hearts. As alterations of mechanical properties of the myocardium, such as stiffening and increased wall stress, are crucial features of cardiac remodeling, this work addresses the biological effects exerted on CSC by mechanical stimuli. Materials and methods: DCSC and ECSC were cultured under defined conditions to mimic specific features of the pathologic condition: increased mechanical loading (up to 15%, cyclic at 1 Hz), differential substrate stiffness (ranging from 1 to 231 kPa), differential cell densities. After 24, 48 and 72 h, cells were fixed and stained for analysis of proliferation and subcellular localization of YAP or lysed for RT-PCR analysis.Results: Cyclic stretch was significantly associated with both increased proliferation of DCSC (n = 6, p<0.0001) and ECSC (n = 4, p = 0.003), and with a significant reduction of nuclear localized YAP (nYAP) as a function of time (p<0.05). However, while significant correlation between cell density and decreased nYAP (p = 0.003, r2 = 0.37) characterized ECSC, this was not evident for unstretched DCSC, suggesting a less stringent regulation of contact inhibition in DCSC. These data were further confirmed by seeding cells at differential density. As opposed to what previously shown for epithelial cell lines, DCSC did not reduce nYAP positivity as a function of cell density, when grown in serum containing medium, suggesting that soluble factors present in the serum could maintain the nuclear localization of YAP, independently from the cell density. In line, serum significantly increased the nYAP expressing cells in DCSC, while a significant positive correlation between cell density and nYAP positivity can be demonstrated in DCSC cultured in serum free medium. RT-PCR for YAP-regulated targets confirmed immuno- fluorescence data. Furthermore, independently from the pathologic status, cyclic stretch was significantly associated with a persistent YAP signaling at high cell density. Besides, tension and assembly of cytoskeletal network, induced by increasing substrate stiffness, correlates with nYAP (p<0.05) and YAP transcriptional activation (p<0.05). Conclusions: D- and ECSC differ in their mechanosensing properties. However, in the first cell type, nYAP localization is dictated by the combined action of paracrine factors and cytoskeletal tension, thus reducing the contact inhibition effect. This finding is in line with a more primitive phenotype of SC isolated from normal hearts.
Found in: osebi
Keywords: Stem cells, human cardiac stem cells, mechanosensing, mechanotransduction, heart failure, YAP
Published: 21.03.2017; Views: 3630; Downloads: 0
Fulltext (65,67 KB) |
4. Cardiac stem cell aging and heart failureDaniela Cesselli, Aneta Aleksova, Elisa Mazzega, Angela Caragnano, Antonio Paolo Beltrami, 2017, review article Abstract: A side effect of the medical improvements of the last centuries is the progressive aging of the world population, which is estimated to reach the impressive number of 2 billion people with more than 65 years by 2050. As a consequence, age-related diseases, such as heart failure, will affect more and more patients in the next years. To understand the biological bases of these diseases will be a crucial task in order to find better treatments, and possibly slow age-related morbidity and mortality.
Cardiac stem cells have been at the center of a heated debate and their potential involvement in cardiac homeostasis has been questioned. In this review, we summarize evidence obtained by independent groups, on different animal models and humans, that strongly support the important role played by immature, cardiac resident cells in the cardioprotection against heart failure.
Found in: osebi
Keywords: Aging, Heart failure, Cardiomyocyte turnover, Stem cells, Cell senescence, cKit, Sca1, PDGFRα, Cardiospheres
Published: 28.03.2017; Views: 3661; Downloads: 0
Fulltext (1,43 MB) |
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9. Toxins in microalgaeTanja Batkovič, Mojca Zotler, Meta Križaj, Jan David, Dani Bratuž, Anže Kuraj, 2017, final research report Abstract: The aim of the project is to develop a detection system for the toxic algae Alexandrium minutum which can be than used as part of biosensor. First, we will isolate a single-domain antibody from a pre-immune library, then subclone its sequence in different vectors and produce it. Finally, we will design alternative ELISA methods and choose the most suitable to quantify the microalgae in water samples.
Found in: osebi
Summary of found: ...produce it. Finally, we will design alternative ELISA methods and choose the most suitable to...
Keywords: Alexandrium minutum, mikroalge, biosenzorji ELISA
Published: 02.11.2018; Views: 2938; Downloads: 0
Fulltext (5,00 MB) |
10. Recombinant nanobodies as cheap and customizable reagents for unicellular algae detectionElisa Mazzega, Ario De Marco, Marina Cabrini, Alfred Beran, published scientific conference contribution abstract Abstract: At the present, the identification of planktonic species in coastal water mainly relies on light microscopy
observations. This kind of analyses is performed by highly trained personnel, requires lab equipment and long
processing time. High-throughput and easy-to-perform methods are instead highly needed for routine costal and
ballast water monitoring.
Immuno-reagents are widely employed in the medical field for routine diagnostics, where they provide the necessary
sensitivity and specificity, as for example for cancer subtype characterization. Reagents of similar grade are so far not
widely available for both diagnostics and basic research of microalgae. We describe the first successful isolation of a
single-domain antibody (nanobody or VHH) from a pre-immune library, its engineering into application-ready
reagents, and its inexpensive production as recombinant fusion protein.
Alexandrium minutum was chosen as a model organism to test the feasibility of the procedure. The procedure foresees
the panning of a pre-immune phage library of VHHs that was used for in vitro selection against directly the target cells.
Monoclonal nanobodies specific for A. minutum cells were identified and optimized for recombinant production as
fusion with fluorescent proteins in bacterial hosts. Such fluorescently-tagged VHHs were validated by
immunofluorescence and cytofluorimetry for their selectivity by testing unicellular algal species that can be found in
the same environment of A. minutum. Two nanobodies were found to be highly specific for the target cells, were able
to bind also cysts of A. minutum and they gave no cross-reaction, even for a not-toxic strain of the closely related A.
tamutum.
Different tags can be then fused to the selected nanobodies and used instead of the fluorescent proteins to obtain a
reagent immediately applicable to further techniques, such as cell Enzyme Linked Immuno Sorbent Assay (ELISA) or
biosensor surface functionalization. The newly produced reagents can be applied for direct whole-cell detection in
seawater, bypassing the need of cell processing required for DNA or RNA diagnostics, and can be used for both alive
and fixed cells, guaranteeing the possibility to check old samples and to perform confirmatory morphological studies.
Found in: osebi
Summary of found: ...as cell Enzyme Linked Immuno Sorbent Assay ( ELISA) or
biosensor surface functionalization. The newly produced...
Keywords: Nanobodies, toxic algae, detection, recombinant reagent, naive library, phage display
Published: 21.12.2018; Views: 2968; Downloads: 0
Fulltext (4,97 MB) |
