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Quaranta M.G.,Istituto Superiore di Sanita | Vincentini O.,Istituto Superiore di Sanita | Felli C.,Istituto Superiore di Sanita | Spadaro F.,Istituto Superiore di Sanita | And 4 more authors.
PLoS ONE | Year: 2011

Background: The mucosal tissues play a central role in the transmission of HIV-1 infection as well as in the pathogenesis of AIDS. Despite several clinical studies reported intestinal dysfunction during HIV infection, the mechanisms underlying HIV-induced impairments of mucosal epithelial barrier are still unclear. It has been postulated that HIV-1 alters enterocytic function and HIV-1 proteins have been detected in several cell types of the intestinal mucosa. In the present study, we analyzed the effect of the accessory HIV-1 Nef protein on human epithelial cell line. Methodology/Principal Findings: We used unstimulated or IFN-γ-stimulated Caco-2 cells, as a model for homeostatic and inflamed gastrointestinal tracts, respectively. We investigated the effect of exogenous recombinant Nef on monolayer integrity analyzing its uptake, transepithelial electrical resistance, permeability to FITC-dextran and the expression of tight junction proteins. Moreover, we measured the induction of proinflammatory mediators. Exogenous Nef was taken up by Caco-2 cells, increased intestinal epithelial permeability and upset the IFN-γ-induced reduction of transepitelial resistance, interfering with tight junction protein expression. Moreover, Nef inhibited IFN-γ-induced apoptosis and up-regulated TNF-α, IL-6 and MIP-3α production by Caco-2 cells while down-regulated IL-10 production. The simultaneous exposure of Caco-2 cells to Nef and IFN-γ did not affect cytokine secretion respect to untreated cells. Finally, we found that Nef counteracted the IFN-γ induced arachidonic acid cascade. Conclusion/Significance: Our findings suggest that exogenous Nef, perturbing the IFN-γ-induced impairment of intestinal epithelial cells, could prolong cell survival, thus allowing for accumulation of viral particles. Our results may improve the understanding of AIDS pathogenesis, supporting the discovery of new therapeutic interventions. © 2011 Quaranta et al. Source

Diatheva S.R.L. and Instituto Superiore Of Sanita | Date: 2012-12-21

An anti-CEA scFv having an uncleaved Pel B leader sequence is surprisingly stable and is highly specific for CEA and CEACAM1.

Fiori V.,Diatheva srl | Magnani M.,Urbino University
Annali dell'Istituto Superiore di Sanita | Year: 2012

Background: In this review, we focus our discussion on one class of carcinoembryonic antigen-related cell adhesion molecules, Carcinoembryonic antigen-related cell adhesion molecules 1 (CEACAM1). This has been observed in several malignant transformations to be subjected to complex mechanisms of modulation and dysregulation. Aims: Restoration of CEACAM1 expression in tumor cell lines often abolishes their oncogenicity in vivo, and therefore, this adhesion molecule has been regarded as a tumor suppressor. In contrast, de novo expression of CEACAM1 is found with the progression of malignancy and metastatic spread in a large array of cancer tissues which include melanoma, Non Small Cell Lung Carcinoma (NSCLC) as well as bladder, prostate, thyroid, breast, colon and gastric carcinomas. Discussion: We report and discuss the most significant findings confirming at immunohistochemical and clinical level the correlation between poor prognosis and expression of CEACAM1 on the cell surface of tumors. Source

Moricoli D.,Diatheva srl | Carbonella D.C.,Diatheva srl | Dominici S.,Diatheva srl | Fiori V.,Diatheva srl | And 12 more authors.
Applied Microbiology and Biotechnology | Year: 2016

Ewing’s sarcoma (EWS) is the second most common primary bone tumor in pediatric patients characterized by over expression of CD99. Current management consists in extensive chemotherapy in addition to surgical resection and/or radiation. Recent improvements in treatment are still overshadowed by severe side effects such as toxicity and risk of secondary malignancies; therefore, more effective strategies are urgently needed. The goal of this work was to develop a rapid, inexpensive, and “up-scalable” process of a novel human bivalent single-chain fragment variable diabody (C7 dAbd) directed against CD99, as a new therapeutic approach for EWS. We first investigated different Escherichia coli constructs of C7 dAbd in small-scale studies. Starting from 60 % soluble fraction, we obtained a yield of 25 mg C7 dAbd per liter of bacterial culture with the construct containing pelB signal sequence. In contrast, a low recovery of C7 dAbd was achieved starting from periplasmic inclusion bodies. In order to maximize the yield of C7 dAbd, large-scale fermentation was optimized. We obtained from 75 % soluble fraction 35 mg C7 dAbd per L of cell culture grown in a synthetic media containing 3 g/L of vegetable peptone and 1 g/L of yeast extract. Furthermore, we demonstrated the better efficacy of the cell lysis by homogenization versus periplasmic extraction, in reducing endotoxin level of the C7 dAbd. For gram-scale purification, a direct aligned two-step chromatography cascade based on binding selectivity was developed. Finally, we recovered C7 dAbd with low residual process-related impurities, excellent reactivity, and apoptotic ability against EWS cells. © 2015, Springer-Verlag Berlin Heidelberg. Source

Amagliani G.,Urbino University | Omiccioli E.,Diatheva srl | Brandi G.,Urbino University | Bruce I.J.,University of Kent | Magnani M.,Urbino University
Food Microbiology | Year: 2010

Seafood could become a source of bacterial pathogens by exposure to contaminated water or through processing practices, thus representing a public health hazard. Conventional culture-based analytical methods take several days to be completed, while the molecular rapid identification of bacterial pathogens is crucial for effective disease control. The developed application consist of a multiplex magnetic capture hybridisation (mMCH) assay for the simultaneous isolation of Salmonella spp. and Listeria monocytogenes DNA from seafood, using paramagnetic amino-modified nanoparticles with capture oligonucleotides, and a triplex Real-Time PCR with an Internal Amplification Control (IAC), in accordance with ISO 22174. The detection probability was 100% with 10 genome equivalents of each target species co-amplified in the same reaction. The complete molecular procedure was tested on raw and smoked salmon fillets artificially contaminated with known amounts of one or both target bacteria (1-103cfu/g), directly or after culture enrichment, and compared for equivalence with the standard methods. Results revealed a complete agreement between the two approaches, with a sensitivity of 1cfu/g, in enriched samples, and higher sensitivity (102-103cfu/g) of the molecular method in samples examined before culture enrichment. The proposed procedure was also able to identify a natural contamination by L. monocytogenes in smoked salmon with a considerable shortening of time. © 2010 Elsevier Ltd. Source

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