Giovanna Piovani

Cytogenetic and molecular characterization of a de-novo t(2p;7p) translocation involving TNS3 and EXOC6B genes in a boy with a complex syndromic phenotype.

We describe a premature newborn child with left renal agenesis, right low functional kidney, altered chemical-clinical parameters, neutropenia, recurrent pulmonary infections, long bone diaphysis broadening, growth and developmental delay. Postnatal cytogenetic analysis revealed a 46,XY,t(2;7)(p13;p12) de-novo karyotype. The chromosome breakpoints were defined by FISH using BAC probes and initially restricted to about 123,000bp in 2p13 and delimited to 84,600bp in 7p12. Bioinformatic analysis of these genomic regions showed two genes that are involved in the rearrangement: exocyst C6B (EXOC6B) for chromosome 2 breakpoint and tensin3 (TNS3) for chromosome 7 breakpoint. A EXOC6B-TNS3 fusion transcript together with a reciprocal TNS3-EXOC6B chimeric RNA have been detected by RT-PCR performed on skin fibroblasts RNA of the proband. These data localize the chromosome 2 breakpoint within the first intron of EXOC6B, while the translocation event on chromosome 7 occurred in intron 15 of TNS3. We hypothesize that the phenotype observed in the patient results from one or several mechanisms including: haploinsufficiency of EXOC6B and TNS3 genes; a dominant negative effect exerted by the chimeric transcripts; a disregulation in the expression of other genes adjacent the breakpoints. Although no clear evidences exist supporting a role of any of the above mentioned mechanisms in the pathogenesis of the complex phenotype, immunofluorescence analysis of tensin1 in the patients fibroblasts suggests that the TNS3 gene haploinsufficiency results in a reduced expression of tensin1. These cells may be therefore a model for understanding the role and the organization of the tensin protein family.

Drug Loaded Gingival Mesenchymal Stromal Cells (GinPa-MSCs) Inhibit In Vitro Proliferation of Oral Squamous Cell Carcinoma

Human mesenchymal stromal cells (MSCs) have been widely investigated both for regenerative medicine and their antinflammatory/immunomodulatory capacity. However, their ability to home pathological tissues suggested the development of strategies for using MSCs as carrier to deliver drug into tumor microenvironment. MSCs obtained from different tissues can be loaded in vitro with anti-cancer drugs by a simple procedures. In this report, we studied MSCs isolated and expanded from gingival papilla (GinPa-MSCs), by testing their ability to uptake and release three important anti-neoplastic drugs: Paclitaxel (PTX), Doxorubicin (DXR) and Gemcitabine (GCB). The efficacy of drugs releasing GinPa-MSCs was studied on a pancreatic cancer cell line and confirmed in vitro against a line of tongue squamous cell carcinoma (SCC154). Our results demonstrated that GinPa-MSCs efficiently incorporate the drugs and then released them in active form and in sufficient amount to produce a dramatic inhibition of squamous cell carcinoma growth in vitro. If compared with other MSCs sources, the collection of GinPa-MSCs is poorly invasive and cells can be easily expanded and efficiently loaded with anti cancer drugs. In particular, gemcitabine loaded GinPa-MSCs provide a good “cell-mediated drug delivery system” for a future potential application in the context of the oral oncology.

Ropinirole and Pramipexole Promote Structural Plasticity in Human iPSC-Derived Dopaminergic Neurons via BDNF and mTOR Signaling

The antiparkinsonian ropinirole and pramipexole are D3 receptor- (D3R-) preferring dopaminergic (DA) agonists used as adjunctive therapeutics for the treatment resistant depression (TRD). While the exact antidepressant mechanism of action remains uncertain, a role for D3R in the restoration of impaired neuroplasticity occurring in TRD has been proposed. Since D3R agonists are highly expressed on DA neurons in humans, we studied the effect of ropinirole and pramipexole on structural plasticity using a translational model of human-inducible pluripotent stem cells (hiPSCs). Two hiPSC clones from healthy donors were differentiated into midbrain DA neurons. Ropinirole and pramipexole produced dose-dependent increases of dendritic arborization and soma size after 3 days of culture, effects antagonized by the selective D3R antagonists SB277011-A and S33084 and by the mTOR pathway kinase inhibitors LY294002 and rapamycin. All treatments were also effective in attenuating the D3R-dependent increase of p70S6-kinase phosphorylation. Immunoneutralisation of BDNF, inhibition of TrkB receptors, and blockade of MEK-ERK signaling likewise prevented ropinirole-induced structural plasticity, suggesting a critical interaction between BDNF and D3R signaling pathways. The highly similar profiles of data acquired with DA neurons derived from two hiPSC clones underpin their reliability for characterization of pharmacological agents acting via dopaminergic mechanisms.

Generation of induced pluripotent stem cells (iPSC) from an atrial fibrillation patient carrying a PITX2 p.M200V mutation

Atrial fibrillation (AF) is the most common sustained arrhythmia associated with several cardiac risk factors, but increasing evidences indicated a genetic component. Indeed, genetic variations of the specific PITX2 gene have been identified in patients with early-onset AF. To investigate the molecular mechanisms underlying AF, we reprogrammed to pluripotency polymorphonucleated leukocytes isolated from the blood of a patient carrying a PITX2 p.M200V mutation, using a commercially available non-integrating expression system. The generated iPSCs expressed pluripotency markers and differentiated toward cells belonging to the three embryonic germ layers. Moreover, the cells showed a normal karyotype and retained the PITX2 p.M200V mutation.

A standardized flow cytometry network study for the assessment of circulating endothelial cell physiological ranges

Circulating endothelial cells (CEC) represent a restricted peripheral blood (PB) cell subpopulation with high potential diagnostic value in many endothelium-involving diseases. However, whereas the interest in CEC studies has grown, the standardization level of their detection has not. Here, we undertook the task to align CEC phenotypes and counts, by standardizing a novel flow cytometry approach, within a network of six laboratories. CEC were identified as alive/nucleated/CD45negative/CD34bright/CD146positive events and enumerated in 269 healthy PB samples. Standardization was demonstrated by the achievement of low inter-laboratory Coefficients of Variation (CVL), calculated on the basis of Median Fluorescence Intensity values of the most stable antigens that allowed CEC identification and count (CVL of CD34bright on CEC ~ 30%; CVL of CD45 on Lymphocytes ~ 20%). By aggregating data acquired from all sites, CEC numbers in the healthy population were captured (medianfemale = 9.31 CEC/mL; medianmale = 11.55 CEC/mL). CEC count biological variability and method specificity were finally assessed. Results, obtained on a large population of donors, demonstrate that the established procedure might be adopted as standardized method for CEC analysis in clinical and in research settings, providing a CEC physiological baseline range, useful as starting point for their clinical monitoring in endothelial dysfunctions.