Training Program at Medical School of Chieti, Italy

We describe the changes in medical training program offered at the G. D’Annunzio University Medical School in Chieti-Pescara, Italy, which took place over the last decade. The new curriculum differs from the previous one in several important aspects, including limited number of students admitted to school depending on the estimated needs for physicians, obligatory class attendance, student attendance in preclinical laboratories, formative credits as a measure of student activity, and elective subjects. Furthermore, all medical graduates are allowed to take the State exam to obtain the license to practice, which was not the case previously. As a result of these major changes, a higher number of students graduates in due time. The changes made in the medical education curriculum in Italy have enabled Italian medical graduates to work in European Community Hospitals, because their medical degree is recognized in other EU countries. The main motif that drives the Medical School in Chieti-Pescara is the achievement of high quality in medical education and biomedical research by creating as strong a relationship between education and research as possible

Circulating endothelial cells (CECs) in peripheral blood

CECs as well as bone-marrow-derived endothelial precursor cells (EPCs) are very rare events in the peripheral blood that have a high potential diagnostic value in different diseases which are characterized by cardiovascular problems and/or angiogenesis, e.g. cancer, ischemia and diabetes. Flow cytometry analysis of CECs is difficult because CECs are often discriminated using a combination of antigens with low, dull, or a continuum of cell surface expression. Since CECs can’t be characterized by a single marker, a combination of at least two markers is necessary. Therefore different combination of several endothelial markers (CD31, CD34, CD146, KDR and CD144) was used in order to get a more accurate discrimination of CECs. Such a test evidenced that KDR and CD144 were very weakly expressed on the CEC cell surface and could not be reliably analysed, while CD31, CD34 and CD146 were largely detected and therefore chosen for the panel. Dead cells, microparticles [1] and platelets were excluded from the analysis by using a DNA stain (Syto16) and a live/dead marker (NiRed). Leucocytes were excluded by gating CD45- cells. CD106 is expressed on endothelial cells after stimulation with cytokines and allows analysis of activated subsets of CECs

Lactobacillus paracasei Lp6 favors immune modulation induced by allergoid treatment in ragweed sensitized mice.

It has been hypothesized that lactic acid bacteria (LAB) could be used as adjuvant for specific immunotherapy (SIT), as various studies conducted on humans and animals converge to define LAB as anti-Th2 modulators and Treg inducers. In the present study we evaluated the effects of LAB, in particular Lactobacillus paracasei Lp6 (Lp6), in a mouse model of ragweed (RW) allergy. Groups of Balb/c mice, experimentally sensitized towards ragweed, were treated by viable Lp6 or by RW-allergoid with or without co-administration of Lp6. A control group was sham-sensitized with PBS and sham-treated with water and a group was sensitized with RW and treated with water. Serum IgE, RW-induced release of IFN-γ, IL-4 and IL-10 from splenocytes and the frequency of CD4CD25 regulatory T cells (Tregs) expressing Foxp3 or IL-10 were evaluated in various groups. RW-allergoid treatment induced a reduction of serum IgE, with a decrease in RW-induced release of IL-4, and an increase in IL-10 and IFN-γ, along with a significant change in the frequency of Tregs, both CD25+ and -. The joint RW-allergoid+Lp6 treatment induced the highest degree of suppression of allergen-driven IL-4, the greatest reduction of IL-4/IFN-γ and IL-4/IL-10 ratios and the most significant increase of Foxp3 and IL-10 expressing Tregs: The study shows that Lp6 strengthens the immune modulation induced by allergoid-SIT in RW-sensitized mice, essentially characterized by a differential induction of Tregs associated to a reduction of IL-4; data converge to define a role of SIT adjuvant for Lp6

A standardized multi-colour flow cytometry approach to characterize the many faces of peripheral circulating microparticles

Microparticles (MP) are small vescicles (<1 µm of diameter) released by several cell types and characterized by an integral plasma membrane expressing the phenotype of the cell from which they originate (Jayachandran et al., 2012). MP play a crucial role in a multitude of pathologies, Including inflammatory and autoimmune disease, diabetes, atherosclerosis, malignancies and cardiovascular disease. The role, as potential biomarker, attributed to circulating MP has been emphasized by the recent literature. In such context, multicolour flow cytometry has great potential In the MP studies (Lanuti et al., 2012). Unfortunately, consensus guidelines on MP identification and enumeration has not been reached yet, due to their small sizes. We purpose to identify, characterize and count MP from whole blood by a seven-colors flow cytometry protocol, with the aim to standardize such method and to allow the identification of both the whole compartment and different MP subpopulations (i.e. endothelial-, platelet- and leukocyte-derived MP). We optimized a novel flow cytometry protocol, using peripheral whole blood stained by a combinations of seven different antigens/probes. Different panel combinations, anticoagulants and storage conditions were evaluated to set the best protocol with the aim to obtain reliable and reproducible MP counts. MP enumeration was carried out by a single platform method by using TrueCount (BD Biosciences). Results demonstrated that the application of the newly optimized flow cytometry method here described, allows to obtain high reproducibility of MP enumeration, pointing out different MP subpopulations both in healthy donors and in different patients. This method may open new routes for the monitoring of MP numbers and phenotypes in different clinical setting

Subcellular localization and phosphorylation of phosphoinositide-phospholipase C γ1 correlate with breast cancer invasiveness

Activation of the enzyme phosphoinositide-phospholipase C γ1 (PLCγ1) is thought to play a critical role in both cytoskeletal changes and migration associated with the metastatic process. Activation of PLCγ1 by phosphorylation can occur downstream of many tyrosine kinase receptors including epidermal growth factor receptor, vascular endothelial growth factor receptor-2, c-MET, platelet-derived growth factor receptor, and also certain integrins. Activation induces hydrolysis of phosphatidylinositol 4,5-biphosphate to form the second messengers diacylglycerol and inositol-1,4,5-triphosphate, which in turn activate a number of signalling pathways. PLCγ1 is highly expressed in several tumours, including breast carcinomas in which the enzyme has been shown to be required for epidermal growth factor induced migration of breast cancer cells. In order to establish the significance of PLCγ1 subcellular localization and phosphorylation (PLCγ1-pY783 and PLCγ1-pY1253) in breast cancer, we compared, through the use of different methods, two different breast cancer models: the low-tumorigenic BT-474 cell line and MDA-MB-231 cell line which represents a more aggressiveness de-differentiated cell type, obtained from a pleural effusion from a patient

Proteome analysis of human Wharton's jelly cells during in vitro expansion

<p>Abstract</p> <p>Background</p> <p>The human umbilical cord contains mucoid connective tissue and fibroblast-like cells. These cells named Wharton's jelly cells, (WJCs) display properties similar to mesenchymal stem cells therefore representing a rich source of primitive cells to be potentially used in regenerative medicine.</p> <p>Results</p> <p>To better understand their self-renewal and potential <it>in vitro </it>expansion capacity, a reference 2D map was constructed as a proteomic data set. 158 unique proteins were identified. More than 30% of these proteins belong to cytoskeleton compartment. We also found that several proteins including Shootin1, Adenylate kinase 5 isoenzyme and Plasminogen activator-inhibitor 2 are no longer expressed after the 2^ndpassage of <it>in vitro </it>replication. This indicates that the proliferative potency of these cells is reduced after the initial stage of <it>in vitro </it>growing. At the end of cellular culturing, new synthesized proteins, including, ERO1-like protein alpha, Aspartyl-tRNA synthetase and Prolyl-4-hydroxylase were identified. It is suggested that these new synthesized proteins are involved in the impairment of cellular surviving during replication and differentiation time.</p> <p>Conclusions</p> <p>Our work represents an essential step towards gaining knowledge of the molecular properties of WJCs so as to better understand their possible use in the field of cell therapy and regenerative medicine.</p