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ABSTRACT Objective to prospectively evaluate the ability of post-graduate students enrolled in a laparoscopy program of the Institute for Teaching and Research to complete single port total nephrectomies. Materials and Methods 15 post-graduate students were enrolled in the study, which was performed using the SILStm port system for single-port procedures. All participants were already proficient in total nephrectomies in animal models and performed a left followed by a right nephrectomy. Analyzed data comprised incision size, complications, and the time taken to complete each part of the procedure. Statistical significance was set at p

Background: Convolutional Neural Networks can be effectively used only when data are endowed with an intrinsic concept of neighbourhood in the input space, as is the case of pixels in images. We introduce here Ph-CNN, a novel deep learning architecture for the classification of metagenomics data based on the Convolutional Neural Networks, with the patristic distance defined on the phylogenetic tree being used as the proximity measure. The patristic distance between variables is used together with a sparsified version of MultiDimensional Scaling to embed the phylogenetic tree in a Euclidean space., Results: Ph-CNN is tested with a domain adaptation approach on synthetic data and on a metagenomics collection of gut microbiota of 38 healthy subjects and 222 Inflammatory Bowel Disease patients, divided in 6 subclasses. Classification performance is promising when compared to classical algorithms like Support Vector Machines and Random Forest and a baseline fully connected neural network, e.g. the Multi-Layer Perceptron., Conclusion: Ph-CNN represents a novel deep learning approach for the classification of metagenomics data. Operatively, the algorithm has been implemented as a custom Keras layer taking care of passing to the following convolutional layer not only the data but also the ranked list of neighbourhood of each sample, thus mimicking the case of image data, transparently to the user.

Background: Mesenchymal stromal cells (MSCs) have been largely investigated, in the past decade, as potential therapeutic strategies for various acute and chronic pathological conditions. MSCs isolated from different sources, such as bone marrow (BM), umbilical cord tissue (UCT) and adipose tissue (AT), share many biological features, although they may show some differences on cumulative yield, proliferative ability and differentiation potential. The standardization of MSCs growth and their functional amplification is a mandatory objective of cell therapies. The aim of this study was to evaluate the cumulative yield and the ex vivo amplification potential of MSCs obtained from various sources and different subjects, using defined culture conditions with a standardized platelet lysate (PL) as growth stimulus., Methods: MSCs isolated from BM, UCT and AT and expanded in human PL were compared in terms of cumulative yield and growth potential per gram of starting tissue. MSCs morphology, phenotype, differentiation potential, and immunomodulatory properties were also investigated to evaluate their biological characteristics., Results: The use of standardized PL-based culture conditions resulted in a very low variability of MSC growth. Our data showed that AT has the greater capacity to generate MSC per gram of initial tissue, compared to BM and UCT. However, UCT-MSCs replicated faster than AT-MSCs and BM-MSCs, revealing a greater proliferation capacity of this source irrespective of its lower MSC yield. All MSCs exhibited the typical MSC phenotype and the ability to differentiate into all mesodermal lineages, while BM-MSCs showed the most prominent immunosuppressive effect in vitro., Conclusions: The adoption of standardized culture conditions may help researchers and clinicians to reveal particular characteristics and inter-individual variability of MSCs sourced from different tissues. These data will be beneficial to set the standards for tissue collection and MSCs clinical-scale expansion both for cell banking and for cell-based therapy settings.

Alternatives to the use of fetal bovine serum (FBS) have been investigated to ensure xeno-free growth condition. In this study we evaluated the efficacy of human platelet lysate (PL) as a substitute of FBS for the in vitro culture of some human cell lines. PL was obtained by pools of pathogen inactivated human donor platelet (PLT) concentrates. Human leukemia cell lines (KG-1, K562, JURKAT, HL-60) and epithelial tumor cell lines (HeLa and MCF-7) were cultured with either FBS or PL. Changes in cell proliferation, viability, morphology, surface markers and cell cycle were evaluated for each cell line. Functional characteristics were analysed by drug sensitivity test and cytotoxicity assay. Our results demonstrated that PL can support growth and expansion of all cell lines, although the cells cultured in presence of PL experienced a less massive proliferation compared to those grown with FBS. We found a comparable percentage of viable specific marker-expressing cells in both conditions, confirming lineage fidelity in all cultures. Functionality assays showed that cells in both FBS- and PL-supported cultures maintained their normal responsiveness to adriamycin and NK cell-mediated lysis. Our findings indicate that PL is a feasible serum substitute for supporting growth and propagation of haematopoietic and epithelial cell lines with many advantages from a perspective of process standardization, ethicality and product safety.

Background: Supplements to support clinical-grade cultures of mesenchymal stem cells (MSC) are required to promote growth and expansion of these cells. Platelet lysate (PL) is a human blood component which may replace animal serum in MSC cultures being rich in various growth factors. Here, we describe a plasma poor pathogen-free platelet lysate obtained by pooling 12 platelet (PLT) units, to produce a standardized and safe supplement for clinical-grade expansion of MSC., Methods: PL lots were obtained by combining 2 6-unit PLT pools in additive solution (AS) following a transfusional-based procedure including pathogen inactivation (PI) by Intercept technology and 3 cycles of freezing/thawing, followed by membrane removal. Three PI-PL and 3 control PL lots were produced to compare their ability to sustain bone marrow derived MSC selection and expansion. Moreover, two further PL, subjected to PI or not, were also produced starting from the same initial PLT pools to evaluate the impact of PI on growth factor concentration and capacity to sustain cell growth. Additional PI-PL lots were used for comparison with fetal bovine serum (FBS) on MSC expansion. Immunoregulatory properties of PI-PL-generated MSC were documented in vitro by mixed lymphocyte culture (MLC) and peripheral blood mononuclear cells (PBMC) mitogen induced proliferation., Results: PI-PL and PL control lots had similar concentrations of 4 well-described growth factors endowed with MSC stimulating ability. Initial growth and MSC expansion by PI-PL and PL controls were comparable either using different MSC populations or in head to head experiments. Moreover, PI-PL and PL control sustained similar MSC growth of frozen/thawed MSC. Multilineage differentiation of PI-derived and PI-PL-derived MSC were maintained in any MSC cultures as well as their immunoregulatory properties. Finally, no direct impact of PI on growth factor concentration and MSC growth support was observed, whereas the capacity of FBS to sustain MSC expansion in basic medium was irrelevant as compared to PL and PI-PL., Conclusion: The replacement of animal additives with human supplements is a basic issue in MSC ex vivo production. PI-PL represents a standardized, plasma-poor, human preparation which appears as a safe and good candidate to stimulate MSC growth in clinical-scale cultures.

Background Aims: We have recently shown that thymoglobulin (TG) efficiently expands cytokine-induced killer (CIK) cells in combination with interferon (IFN)-γ and interleukin (IL)-2 (ITG2 protocol). It is presently unknown whether the infusion of autologous immune effector cells generated by TG, IFN-γ and IL-2 is feasible and safe., Methods: Five patients with advanced and/or refractory solid tumors were enrolled in the present phase I/II study. Peripheral blood mononuclear cells (PBMC) collected by leukapheresis were stimulated under good manufacturing practice (GMP)-conditions with IFN-γ, followed by TG and IL-2. After 2-3 weeks in culture, a median of 4.65 × 10(6) immune effector cells per kilogram of recipient's body weight was obtained and infused intravenously. The median time from enrollment into the study to infusion of the expanded CIK cells was 30 days., Results: ITG2 efficiently expanded immune effector cells that comprised both conventional natural killer (NK) cells and CD3(+) CD16(+) CD56(+) CIK cells. One patient with advanced melanoma died because of disease progression before the infusion of CIK cells. The target dose of at least 2.5 × 10(6) CIK cells/kg of recipient's body weight was reached in four out of five evaluable patients. CIK cells were administered intravenously without any measurable toxicity. In vitro, CIK cells exerted lytic activity against cervical cancer cells. The median survival was 4.5 months (range 1-13) from the first infusion of CIK cells., Conclusions: This study has highlighted the feasibility and safety of the administration of CIK cells generated with the ITG2 protocol. Whether CIK cells can help control disease burden in patients with advanced malignancies will be determined in future clinical trials.

Background: The aim of this study was to evaluate the quality of red blood cell concentrates obtained from donated whole blood, selected for transfusion therapy of thalassaemic patients, by measuring the following parameters: haemoglobin, haematocrit, percentage haemolysis, residual leucocyte count and residual protein content., Materials and Methods: Overall 345 red cell concentrates were evaluated, of which 205 had been filtered in-line pre-storage and washed and 140 were buffy coat-depleted and used within 2 days of collection. Of the buffy coat-depleted concentrates, 62 were leucodepleted and 78 washed and leucodepleted post-storage all within 2 days of collection. The off-line filters used for the leucodepletion were gamma-irradiated polyester with a pore size of 200 μm. The washing procedure was automated (Haemonetics ACP 215, Braintree, MA, USA). The haematological parameters were evaluated by a blood cell counter (Coulter, Ramsey, IL, USA) and the white blood cell count by cytofluorimetry (FACScan)., Results: Ninety-five percent (194/205) of the red cell concentrates that had been filtered pre-storage and washed, 92% (57/62) of the red cell concentrates that had been leucodepleted post-storage and 94% (73/78) of the those subjected to both treatments had normal values of haemoglobin (>40 g/unit), haematocrit (between 50-70%), percentage haemolysis (<0.8/unit), white cell count (<1×10(6)) and residual protein content (<0.5 g/L). Five percent (11/205) of the red cell concentrates that had been filtered pre-storage and washed, 8% (5/62) of those leucodepleted post-storage after 2 days and 6% (5/78) of those that underwent both procedures had a haemoglobin content <40 g/unit and a haematocrit <50%., Conclusions: The preparation procedures had been carried out satisfactorily; nevertheless, transfusion therapy with some "low dose" normal units could be less effective and might, therefore, result in greater transfusion requirements in patients receiving such units.

Background: Cytokine-induced killer (CIK) cells are typically differentiated in vitro with interferon (IFN)-γ and αCD3 monoclonal antibodies (mAb), followed by the repeated provision of interleukin (IL)-2. It is presently unknown whether thymoglobulin (TG), a preparation of polyclonal rabbit γ immunoglobulins directed against human thymocytes, can improve the generation efficiency of CIK cells compared with αCD3 mAb in a clinical-grade culture protocol., Methods: Peripheral blood mononuclear cells (PBMC) from 10 healthy donors and 4 patients with solid cancer were primed with IFN-γ on day 0 and low (50 ng/ml), intermediate (250 ng/ml) and high (500 ng/ml) concentrations of either αCD3 mAb or TG on day 1, and were fed with IL-2 every 3 days for 21 days. Aliquots of cells were harvested weekly to monitor the expression of representative members of the killer-like immunoglobulin receptor (KIR), NK inhibitory receptor, NK activating receptor and NK triggering receptor families. We also quantified the frequency of bona fide regulatory T cells (Treg), a T-cell subset implicated in the down-regulation of anti-tumor immunity, and tested the in vitro cytotoxic activity of CIK cells against NK-sensitive, chronic myeloid leukaemia K562 cells., Results: CIK cells expanded more vigorously in cultures supplemented with intermediate and high concentrations of TG compared with 50 ng/ml αCD3 mAb. TG-driven CIK cells expressed a constellation of NK activating/inhibitory receptors, such as CD158a and CD158b, NKp46, NKG2D and NKG2A/CD94, released high quantities of IL-12p40 and efficiently lysed K562 target cells. Of interest, the frequency of Treg cells was lower at any time-point compared with PBMC cultures nurtured with αCD3 mAb. Cancer patient-derived CIK cells were also expanded after priming with TG, but they expressed lower levels of the NKp46 triggering receptor and NKG2D activating receptor, thus manifesting a reduced ability to lyse K562 cells., Conclusions: TG fosters the generation of functional CIK cells with no concomitant expansion of tumor-suppressive Treg cells. The culture conditions described herein should be applicable to cancer-bearing individuals, although the differentiation of fully functional CIK cells may be hindered in patients with advanced malignancies.

Defects in chromosome segregation play a critical role in producing genomic instability and aneuploidy, which are associated with congenital diseases and carcinogenesis. We recently provided evidence from immunofluorescence and electron microscopy studies that merotelic kinetochore orientation is a major mechanism for lagging chromosomes during mitosis in PtK1 cells. Here we investigate whether human primary fibroblasts exhibit similar errors in chromosome segregation and if at least part of lagging chromosomes may arise in cells entering anaphase in the presence of mono-oriented chromosomes. By using in situ hybridization with alphoid probes to chromosome 7 and 11 we showed that loss of a single sister is much more frequent than loss of both sisters from the same chromosome in anatelophases from human primary fibroblasts released from a nocodazole-induced mitotic arrest, as predicted from merotelic orientation of single kinetochores. Furthermore, the lagging of pairs of separated sisters was higher than expected from random chance indicating that merotelic orientation of one sister may promote merotelic orientation of the other. Kinetochores of lagging chromosomes in anaphase human cells were found to be devoid of the mitotic checkpoint phosphoepitopes recognized by the 3F3/2 antibody, suggesting that they attached kinetochore microtubules prior to anaphase onset. Live cell imaging of H2B histone-GFP-transfected cells showed that cells with mono-oriented chromosomes never enter anaphase and that lagging chromosomes appear during anaphase after chromosome alignment occurs during metaphase. Thus, our results demonstrate that the mitotic checkpoint efficiently prevents the possible aneuploid burden due to mono-oriented chromosomes and that merotelic kinetochore orientation is a major limitation for accurate chromosome segregation and a potentially important mechanism of aneuploidy in human cells.

In this work we have used the inhibitor of F-actin polymerisation cytochalasin B (Cyt B) to test the hypothesis that the contractile ring and the central spindle are mutually interdependent structures in mammalian mitotic cells. Double fluorescence staining of alpha-tubulin and F-actin was employed to analyse anaphase and telophase figures from asynchronously growing cultures and prometaphase-synchronised cells. Testing for the presence of the central spindle and contractile ring in human primary fibroblasts, human hepatoma cells and Chinese hamster cells after Cyt B treatment showed that both structures were simultaneously absent in over 60% of treated anaphases and 80% of telophases. Experiments on resumption of cytokinesis in cleavage-arrested cells further showed that Cyt B-treated human fibroblasts proceeded to cleavage within minutes after removal of the drug from the medium, concomitant with the re-formation of both cellular structures in cleaving cells. These data suggest that the presence of a correctly assembled contractile ring is essential for the formation and persistence of the central spindle during ana-telophase and provide further support for the idea of a strong co-operative interaction between these two structures during cytokinesis.