Cristina La Torre

Production Conditions Affect the In Vitro Anti-Tumoral Effects of a High Concentration Multi-Strain Probiotic Preparation.

A careful selection of the probiotic agent, standardization of the dose and detailed characterization of the beneficial effects are essential when considering use of a probiotic for the dietary management of serious diseases. However, changes in the manufacturing processes, equipment or facilities can result in differences in the product itself due to the live nature of probiotics. The need to reconfirm safety and/or efficacy for any probiotic product made at a different factory is therefore mandatory. Recently, under the brand VSL#3®, a formulation produced by a manufacturer different from the previous one, has been commercialized in some European countries (the UK and Holland). VSL#3 is a high concentration multi-strain preparation which has been recognized by the main Gastroenterology Associations for the dietary management of pouchitis as well as ulcerative colitis. We have compared the “original” VSL#3 produced in USA with the “newfound” VSL#3 produced in Italy. According to our results, the “newfound” VSL#3 has 130–150% more “dead bacteria” compared to the “original” product, raising concerns for the well-known association between dead microbes with adverse effects. The abilities of bacterial lysates from the two formulations to influence in vitro viability and proliferation of different tumor cell lines also resulted different. The repair of previously scratched monolayers of various adherent tumor cell lines (i.e. HT1080, and Caco-2 cells) was inhibited more significantly by the “original” VSL#3 when compared to the “newfound” VSL#3. Tumor cell cycle profile, in particular cell cycle arrest and apoptotic death of the cancer cells, further confirms that the “original” VSL#3 has a better functional profile than the “newfound” VSL#3, at least in in vitro. Our data stress the importance of the production conditions for the “newfound” VSL#3 considering that this product is intended to be used for the dietary management of patients with very serious diseases, such as chronic inflammatory bowel diseases.

Immunophenotypic Characterization of Human Glioblastoma Stem Cells: Correlation With Clinical Outcome

Recently, glioma stem cells have been identified as the main cause of glioma propagation and recurrence and a number of several cell markers have been indicated as putative GSC markers. In the present work, a retrospective study to evaluate the prognostic potential of ability to generate GSCs in our series of 15 glioblastoma patients is described. β‐tubulin III, nestin, CD133, GFAP, and SOX‐2 marker expression, both in primary GBM cultures and in respective glioblastoma stem cells (GSCs), was evaluated by flow cytometric analysis. Our results demonstrated various expression levels of these markers in both cell cultures; of note, only those cells expressing SOX‐2 at greater than 30% levels were able to produce in vitro neurospheres. Moreover, statistical analysis revealed that the GSCs generation negatively affected overall survival (OS) (P = 0.000) and progression‐free survival (PFS) (P = 0.001). In addition, a very poor OS (P = 0.000) and PFS (P = 0.000) were observed among patients whose tumors expressed Ki67, evaluated by immunohistochemistry, and showed the ability to generate in vitro GSCs. Overall, the results suggest that in vitro GSCs generation associated to the expression of Ki67 and SOX‐2 may be useful to identify patients at risk of disease progression. J. Cell. Biochem. 116: 864–876, 2015.

In Vitro Evaluation of Different Methods of Handling Human Liposuction Aspirate and Their Effect on Adipocytes and Adipose Derived Stem Cells

Nowadays, fat tissue transplantation is widely used in regenerative and reconstructive surgery. However, a shared method of lipoaspirate handling for ensuring a good quality fat transplant has not yet been established. The study was to identify a method to recover from the lipoaspirate samples the highest number of human viable adipose tissue‐derived stem cells (hADSCs) included in stromal vascular fraction (SVF) cells and of adipocytes suitable for transplantation, avoiding an extreme handling. We compared the lipoaspirate spontaneous stratification (10‐20‐30 min) with the centrifugation technique at different speeds (90‐400‐1500 × g). After each procedure, lipoaspirate was separated into top oily lipid layer, liquid fraction, “middle layer”, and bottom layer. We assessed the number of both adipocytes in the middle layer and SVF cells in all layers. The histology of middle layer and the surface phenotype of SVF cells by stemness markers (CD105+, CD90+, CD45−) was analyzed as well. The results showed a normal architecture in all conditions except for samples centrifuged at 1500 × g. In both methods, the flow cytometry analysis showed that greater number of ADSCs was in middle layer; in the fluid portion and in bottom layer was not revealed significant expression levels of stemness markers. Our findings indicate that spontaneous stratification at 20 min and centrifugation at 400 × g are efficient approaches to obtain highly viable ADSCs cells and adipocytes, ensuring a good thickness of lipoaspirate for autologous fat transfer. Since an important aspect of surgery practice consists of gain time, the 400 × g centrifugation could be the recommended method when the necessary instrumentation is available. J. Cell. Physiol. 230: 1974–1981, 2015.

VSL#3 Probiotic Differently Influence IEC-6 Intestinal Epithelial Cell Status and Function

The data here reported introduce the wound‐healing assay as a tool for testing probiotics aimed at protecting gastrointestinal mucosal surfaces and to verify the consistency of their manufacturing. At the scope, we compared the in vitro effects of two multi‐strain high concentration formulations both commercialized under the same brand VSL#3 but sourced from different production sites (USA and Italy) on a non‐transformed small‐intestinal epithelial cell line, IEC‐6. The effects on cellular morphology, viability, migration, and H2O2‐induced damage, were assessed before and after the treatment with both VSL#3 formulations. While the USA‐sourced product (“USA‐made”) VSL#3 did not affect monolayer morphology and cellular density, the addition of bacteria from the Italy‐derived product (“Italy‐made”) VSL#3 caused clear morphological cell damage and strongly reduced cellularity. The treatment with “USA‐made” lysate led to a higher rate of wounded monolayer healing, while the addition of “Italy‐made” bacterial lysate did not influence the closure rate as compared to untreated cells. While lysates from “USA‐made” VSL#3 clearly enhanced the formation of elongated and aligned stress fibers, “Italy‐made” lysates had not similar effect. “USA‐made” lysate was able to cause a total inhibition of H2O2‐induced cytotoxic effect whereas “Italy‐made” VSL#3 lysate was unable to protect IEC‐6 cells from H2O2‐induced damage. ROS generation was also differently influenced, thus supporting the hypotesis of a protective action of “USA‐made” VSL#3 lysates, as well as the idea that “Italy‐made” formulation was unable to prevent significantly the H2O2‐induced oxidative stress.

Effects of transferrins and cytokines on nitric oxide production by an avian lymphoblastoid cell line infected with Marek's disease virus

Mareks disease virus (MDV), the causative agent of Mareks disease (MD), is a herpesvirus that infects poultry causing T lymphomas. Although vaccination may prevent lymphomas formation, it is not known whether it controls viral replication and spreading in the environment. Ovotransferrin (Otrf), a member of the transferrin family, is known to exert in vitro antiviral activity in primary cultures of chicken embryo fibroblasts (CEF). In addition Otrf is produced by CEF and by an avian lymphoblastoid cell line (MDCC-MSB1) following infection/reinfection with MDV. The present work was designed to investigate the effects of reinfection and of Otrf and lactoferrin (Lf) on the production by MDCC-MSB1 of nitric oxide (NO), a molecule naturally exerting an antiviral activity. These effects were also tested with two cytokines (IL-8 and IFN-gamma), alone and in association with transferrins. Synergy was found between Otrf and IFN-gamma, thus suggesting a possible role in a complementary or alternative strategy against MDV spreading.

Use of Probiotics for Dermal Applications

The concept of probiotic bacteria is considerably evolving. Clinical and experimental researches extensively document that beyond probiotic capacity to influence positively the intestinal functions, they can exert their benefits at the skin level thanks to their peculiar properties. Indeed, scientific and evidence-based reports strengthen the assumption that certain probiotics can contribute to modulate cutaneous microflora, lipid barrier, and skin immune system, leading to the preservation of the skin homeostasis. In this chapter, the most relevant evidences available from scientific literature as well as registered patents have been summarized in relation to actual or potential topical applications of probiotics in the field of dermatology. Altogether the evidences reported in this review afford the possibility of designing new strategies based on a topical approach for the prevention and treatment of cutaneous disorders.

NOS2 expression in glioma cell lines and glioma primary cell cultures: correlation with neurosphere generation and SOX-2 expression

Nitric oxide has been implicated in biology and progression of glioblastoma (GBM) being able to influence the cellular signal depending on the concentration and duration of cell exposure. NOS2 (inducible nitric oxide synthase) have been proposed as a component of molecular profile of several tumors, including glioma, one of the most aggressive primary brain tumor featuring local cancer stem cells responsible for enhanced resistance to therapies and for tumor recurrence. Here, we investigated the NOS2 mRNA expression by reverse transcription-PCR in human glioma primary cultures at several grade of malignancy and glioma stem cell (GSC) derived neurospheres. Glioma cell lines were used as positive controls both in terms of stemness marker expression that of capacity of generating neurospheres. NOS2 expression was detected at basal levels in cell lines and primary cultures and appeared significantly up-regulated in cultures kept in the specific medium for neurospheres. The immunofluorescence analysis of all cell cultures to evaluate the levels of SOX-2, a stemness marker aberrantly up-regulated in GBM, was also performed. The potential correlation between NOS2 expression and ability to generate neurospheres and between NOS2 and SOX-2 levels was also verified. The results show that the higher NOS2 expression is detected in all primary cultures able to arise neurosphere. A high and significant correlation between NOS2 expression and SOX-2 positive cells (%) in all cell cultures maintained in standard conditions has been observed. The results shed light on the potential relevance of NOS2 as a prognostic factor for glioma malignancy and recurrence.

Numerical methods for the semi-automatic analysis of multimodal wound healing images

Wound healing problem requires the analysis of tens of images from different microscopic systems. We describe a set of semi-automatic algorithms to analyze a variety of microscopy images used to study the wound healing process. The proposed suite, beside the phase contrast images, allows analyzing fluorescent microscopy images, inverted light microscopy images at different magnification and staining methods, or images obtained by scanning electron microscopy. The proposed software is designed in Matlab®. It is suggested to integrate it into the CellProfilerTM software, thus introducing new functionalities without losing the CellProfiler existing capabilities. The approach is efficient, easy-to-use, and enables biologists to comprehensively and quantitatively address many questions of the wound healing problem.

Nitric Oxide Chemical Donor Affects the Early Phases of In Vitro Wound Healing Process

An artificial wound in a confluent monolayer of human keratinocyte HaCaT cells or mouse embryo fibroblast Swiss NIH 3T3 cells was used to analyze the effects of the nitric oxide (NO) chemical donor, S‐nitroso‐N‐acetylpenicillamine (SNAP). SNAP exposure promoted an enhanced rate of wound closure and accelerated motility of both keratinocytes and fibroblasts compared to control cells. The wounded monolayer cultures of HaCaT and NIH 3T3 cells, treated with or without SNAP, were monitored under a phase contrast microscope. Structural and ultrastructural modifications were analyzed by scanning electron microscopy (SEM). The images were captured by a digital camera at different time points (0–28 h) and the wound area was analyzed through software included in Matlab®. As early as 15 min, SNAP induced significant cytoskeletal remodeling, as shown by immunostaining (phalloidin‐labelling), which in turn was associated with increased filopodium number and length rise. NO donor treatment also induced overexpression of Ki‐67 protein, a typical marker of cell proliferation, as shown by immunostaining. Both SNAP‐induced migration and proliferation were antagonized by the NO‐sensitive GC inhibitor 1H‐[1,2,4]oxadiazolo[‐4,3‐a]quinoxalin‐1‐one (ODQ), which suggests activation of the NO/cGMP signalling cascade in the observed SNAP‐induced effects in the early stages of the healing process. Moreover, we provide evidence that PPAR‐β antagonist (GSK0660) may interfere with NO‐mediated wound healing process. J. Cell. Physiol. 231: 2185–2195, 2016.

The Clinical Applications of Multifrequency Ultrasound Technology in Body Reshaping

The basic rules of UAL as described by Zocchi were followed without regard to other limitations such as ultrasonic energy site times, body areas, and level of tissue planes. US alters adipose tissue through micromechanical disruption and cavitation with minimal thermal effect. The cavitational effect is a dynamic phenomenon, triggered by the accomplishment of resonance frequency of cell membrane. Resonance frequency is dependent on the kind of tissue and surrounding environment characteristics that usually change continually during treatment. To use the better frequency in all conditions and to maintain the maximum effectiveness, the MAUL technology was introduced. This new technology uses last-generation microchips to determine the best resonance frequency every 10 s by measuring tissue humidity and impedance. By maintaining the better frequency during treatment, the new technology concentrates the entire ultrasound energy on the adipose cells minimizing the thermal effect on the cutaneous deeper layer and maximizing the lipoclastic effect.