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Salvatore M.,Instituto Superiore Of Sanita | Floridia G.,Instituto Superiore Of Sanita | Amato A.,Instituto Superiore Of Sanita | Censi F.,Instituto Superiore Of Sanita | And 13 more authors.
Clinical Biochemistry | Year: 2016

Objectives: Sweat chloride test is the gold standard test for cystic fibrosis (CF) diagnosis. In 2014 the Istituto Superiore di Sanità established the Italian pilot external quality assessment program for CF sweat test (IEQA-ST). Design and methods: Ten laboratories, included among the 33 Italian CF Referral Centers, were selected and enrolled on the basis of their attitude to perform sweat test (ST) analysis by using methods recommended by the Italian Guidelines. They received three different sweat-like samples (normal, borderline and pathologic chloride concentration), with mock clinical indications, for analysis according to routine procedures. Assessment, performed by a panel of experts, covered analytical performance, interpretation and reporting of results; categories of "poor" and "satisfactory" performance were not defined. All data were managed through a web utility. Results: The program identified important areas of interest and, in some case, of concern. It is important to underline that results are referred to a small proportion, i.e. about 30%, of Italian laboratories performing CF ST in the context of the Referral Centers. Conclusions: Data collected highlight the importance of participation in EQA programs as it may improve laboratory/clinical performance; our study represents a model for the setting up of a large-scale EQA scheme for ST. © 2016 The Canadian Society of Clinical Chemists.


Porcile C.,University of Molise | Di Zazzo E.,University of Molise | Monaco M.L.,CEINGE Advanced Biotechnology Scarl | D'Angelo G.,University of Molise | And 13 more authors.
Journal of Cellular Physiology | Year: 2014

Adiponectin (Acrp30) is an adipocyte-secreted hormone with pleiotropic metabolic effects, whose reduced levels were related to development and progression of several malignancies. We looked at the presence of Acrp30 receptors in human glioblastomas (GBM), hypothesizing a role for Acrp30 also in this untreatable cancer. Here we demonstrate that human GBM express Acrp30 receptors (AdipoR1 and AdipoR2), which are often co-expressed in GBM samples (70% of the analyzed tumors). To investigate the effects of Acrp30 on GBM growth, we used human GBM cell lines U87-MG and U251, expressing both AdipoR1 and AdipoR2 receptors. In these cells, Acrp30 treatment inhibits DNA synthesis and cell proliferation rate, inducing arrest in G1 phase of the cell cycle. These effects were correlated to a sustained activation of ERK1/2 and Akt kinases, upon Acrp30 treatment. Our results suggest that Acrp30 may represent a novel endogenous negative regulator of GBM cell proliferation, to be evaluated for the possible development of novel pharmacological approaches. © 2014 Wiley Periodicals, Inc.


Pavone L.M.,University of Naples Federico II | Del Vecchio P.,University of Naples Federico II | Mallardo P.,University of Naples Federico II | Altieri F.,University of Naples Federico II | And 13 more authors.
Molecular BioSystems | Year: 2013

Gastrokine-1 (GKN1), a protein expressed in normal gastric tissue, but absent in gastric cancer tissues and derived cell lines, has recently emerged as a potential biomarker for gastric cancer. To better establish the molecular properties of GKN1, the first protocol for the production of mature human GKN1 in the expression system of Pichia pastoris was settled. The recombinant protein showed anti-proliferative properties specifically on gastric cancer cell lines thus indicating that it was properly folded. Characterization of structural and biochemical properties of recombinant GKN1 was achieved by limited proteolysis analysis, circular dichroism and fluorescence spectroscopy. The analysis of GKN1 primary structure coupled to proteolytic experiments highlighted that GKN1 was essentially resistant to proteolytic enzymes and showed the presence of at least a disulphide bond between Cys61 and one of the other three Cys (Cys122, Cys145 and Cys159) of the molecule. The secondary structure analysis revealed a prevailing β-structure. Spectroscopic and calorimetric investigations on GKN1 thermal denaturation pointed out its high thermal stability and suggested a more complex than a two-state unfolding process. The resulting protein was endowed with a globular structure characterized by domains showing different stabilities toward chemical and physical denaturants. These results are in agreement with the prediction of GKN1 secondary structure and a three-dimensional structure model. Our findings provide the basis for the development of new pharmaceutical compounds of potential use for gastric cancer therapy. © 2013 The Royal Society of Chemistry.


Smaldone G.,National Research Council Italy | Smaldone G.,University of Naples Federico II | Falanga A.,Molecular Diagnostics and Pharmaceuticals Scarl | Capasso D.,University of Naples Federico II | And 11 more authors.
International Journal of Nanomedicine | Year: 2013

A genetically modified recombinant gH625-c-prune was prepared through conjugation of c-prune with gH625, a peptide encompassing 625-644 residues of the glycoprotein H of herpes simplex virus 1, which has been proved to possess the ability to carry cargo molecules across cell membranes. C-prune is the C-terminal domain of h-prune, overexpressed in breast, colorectal, and gastric cancers, interacting with multiple partners, and representing an ideal target for inhibition of cancer development. Its C-terminal domain results in an intrinsically disordered domain (IDD), and the peculiar properties of gH625 render it an optimal candidate to act as a carrier for this net negatively charged molecule by comparison with the positively charged TAT. A characterization of the recombinant gH625-c-prune fusion protein was conducted by biochemical, cellular biology and confocal microscopy means in comparison with TAT-c-prune. The results showed that the gH625-c-prune exhibited the ability to cross biomembranes, opening a new scenario on the use of gH625 as a novel multifunctional carrier. © 2013 Smaldone et al, publisher and licensee Dove Medical Press Ltd.


PubMed | The Second University of Naples, University of Naples Federico II and CEINGE Advanced Biotechnology s.c.ar.l
Type: | Journal: Pharmacological research | Year: 2016

Epidemiological data suggest that obesity represent an important risk factor for asthma, but the link between excess fat and airway hyperresponsiveness (AHR) and inflammation is not fully understood. Recently, a key role in physiopathologic conditions of lungs has been given to adiponectin (Acrp30). Acrp30 is one of the most expressed adipokines produced and secreted by adipose tissue, showing an intriguing relationship with metabolism of sphingolipids. Sphingosine-1-phosphate (S1P) has been proposed as an important inflammatory mediator implicated in the pathogenesis of airway inflammation and asthma. In the present study we analyze the effects of recombinant Acrp30 administration in an experimental model of S1P-induced AHR and inflammation. The results show that S1P is able to reduce endogenous Acrp30 serum levels and that recombinant Acrp30 treatment significantly reduce S1P-induced AHR and inflammation. Moreover, we observed a reduction of Adiponectin receptors (AdipoR1, AdipoR2 and T-cadherin) expression in S1P treated mice. Treatment with recombinant Acrp30 was able to restore Acrp30 serum levels and adiponectin receptors expression. These results could indicate the ability of S1P to modulate the Acrp30 action, by modulating not only the serum levels of the protein, but also its receptors. Taken together, these data suggest that adiponectin could represent a possible biomarker in obesity-associated asthma.


PubMed | Ceinge Advanced Biotechnology S.C.ar.l., Ohio State University and University of Naples Federico II
Type: Journal Article | Journal: Oncotarget | Year: 2016

Breast cancer is the most common cancer in women worldwide. A new promising anti-cancer therapy involves the use of monoclonal antibodies specific for target tumor-associated antigens (TAAs). A TAA of interest for immunotherapy of Triple Negative Breast Cancer (TNBC) is nucleolin (NCL), a multifunctional protein, selectively expressed on the surface of cancer cells, which regulates the biogenesis of specific microRNAs (miRNAs) involved in tumor development and drug-resistance. We previously isolated a novel human anti-NCL scFv, called 4LB5, that is endowed with selective anti-tumor effects. Here we report the construction and characterization of a novel immunoRNase constituted by 4LB5 and a human pancreatic RNase (HP-RNase) called 4LB5-HP-RNase. This immunoRNase retains both the enzymatic activity of human pancreatic RNase and the specific binding of the parental scFv to a panel of surface NCL-positive breast cancer cells. Notably, 4LB5-HP-RNase dramatically and selectively reduced the viability and proliferation of NCL-positive tumor cells in vitro and in vivo. Specifically, it induced apoptosis and reduced the levels of the tumorigenic miRNAs miR-21, -221 and -222. Thus, this novel immunoagent could be a valuable tool for the treatment of TNBC patients ineligible for currently available targeted treatments.

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