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Porotto M.,Cornell University | Rockx B.,National Institute of Allergy and Infectious Diseases | Yokoyama C.C.,Cornell University | Talekar A.,Cornell University | And 8 more authors.
PLoS Pathogens | Year: 2010

In the paramyxovirus cell entry process, receptor binding triggers conformational changes in the fusion protein (F) leading to viral and cellular membrane fusion. Peptides derived from C-terminal heptad repeat (HRC) regions in F have been shown to inhibit fusion by preventing formation of the fusogenic six-helix bundle. We recently showed that the addition of a cholesterol group to HRC peptides active against Nipah virus targets these peptides to the membrane where fusion occurs, dramatically increasing their antiviral effect. In this work, we report that unlike the untagged HRC peptides, which bind to the postulated extended intermediate state bridging the viral and cell membranes, the cholesterol tagged HRC-derived peptides interact with F before the fusion peptide inserts into the target cell membrane, thus capturing an earlier stage in the F-activation process. Furthermore, we show that cholesterol tagging renders these peptides active in vivo: the cholesterol-tagged peptides cross the blood brain barrier, and effectively prevent and treat in an established animal model what would otherwise be fatal Nipah virus encephalitis. The in vivo efficacy of cholesterol-tagged peptides, and in particular their ability to penetrate the CNS, suggests that they are promising candidates for the prevention or therapy of infection by Nipah and other lethal paramyxoviruses.


Cavaliere P.,University of Naples Federico II | Cavaliere P.,Institute Pasteur Paris | Pagano B.,University of Naples Federico II | Granata V.,CEINGE | And 4 more authors.
Nucleic Acids Research | Year: 2013

Prion protein (PrP) is involved in lethal neurodegenerative diseases, and many issues remain unclear about its physio-pathological role. Quadruplex-forming nucleic acids (NAs) have been found to specifically bind to both PrP cellular and pathological isoforms. To clarify the relevance of these interactions, thermodynamic, kinetic and structural studies have been performed, using isothermal titration calorimetry, surface plasmon resonance and circular dichroism methodologies. Three quadruplex-forming sequences, d(TGGGGT), r(GGAGGAGGAGGA), d(GGAGGAGGAGGA), and various forms of PrP were selected for this study. Our results showed that these quadruplexes exhibit a high affinity and specificity toward PrP, with KD values within the range 62÷630 nM, and a weaker affinity toward a PrP-β oligomer, which mimics the pathological isoform. We demonstrated that the NA quadruplex architecture is the structural determinant for the recognition by both PrP isoforms. Furthermore, we spotted both PrP N-terminal and C-terminal domains as the binding regions involved in the interaction with DNA/RNAs, using several PrP truncated forms. Interestingly, a reciprocally induced structure loss was observed upon PrP-NA interaction. Our results allowed to surmise a quadruplex unwinding-activity of PrP, that may have a feedback in vivo. © 2012 The Author(s). Published by Oxford University Press.


Iolascon A.,CEINGE | Iolascon A.,University of Naples Federico II | Russo R.,CEINGE | Russo R.,University of Naples Federico II | Delaunay J.,University Paris - Sud
Current Opinion in Hematology | Year: 2011

Purpose of Review: Congenital dyserythropoietic anemias (CDAs) are rare hereditary disorders characterized by ineffective erythropoiesis and by distinct morphological abnormalities of erythroblasts in the bone marrow. Characteristic morphological aberrations were the cornerstone of diagnosis, but following the identification of several causative genes, the molecular approach could represent a rapid tool for the identification of these conditions. This review presents advances in diagnosis and classification of CDAs. Recent Findings: The classification of CDAs has long been based on morphological features. Now, the discovery of some of the responsible genes allows reconsideration of part of the classification. The first CDA partly accounted for genetically has been CDA 1, through the discovery in 2002 of the gene responsible, CDAN1, encoding codanin-1. Recently, the dramatic identification of the genes responsible for CDA II, SEC23B, and for a hitherto unnamed CDA, KLF1, took place. SEC23B encodes SEC23B which is a component of the coated vesicles transiting from the endoplasmic reticulum to the cis compartment of the Golgi apparatus. A unique mutation in KLF1, which encodes the erythroid transcription factor KLF1, causes major ultrastructural abnormalities, the persistence of embryonic and fetal hemoglobins, and the absence of some red cell membrane proteins. Summary: Studies of genotype-phenotype relationship, as has already been done for CDA II, will allow a more accurate prognosis. Identification of the responsible genes has opened new vistas for research on CDAs. © 2011 Wolters Kluwer Health | Lippincott Williams and Wilkins.


Vercauteren K.,Microbiology and Immunology | Van Den Eede N.,Microbiology and Immunology | Mesalam A.A.,Microbiology and Immunology | Belouzard S.,University of Lille Nord de France | And 12 more authors.
Hepatology | Year: 2014

Hepatitis C virus (HCV)-induced endstage liver disease is currently a major indication for liver transplantation. After transplantation the donor liver inevitably becomes infected with the circulating virus. Monoclonal antibodies (mAbs) against the HCV coreceptor scavenger receptor class B type I (SR-BI) inhibit HCV infection of different genotypes, both in cell culture and in humanized mice. Anti-SR-BI mAb therapy is successful even when initiated several days after HCV exposure, supporting its potential applicability to prevent HCV reinfection of liver allografts. However, HCV variants with reduced SR-BI dependency have been described in the literature, which could potentially limit the use of SR-BI targeting therapy. In this study we show, both in a preventative and postexposure setting, that humanized mice infected with HCV variants exhibiting increased in vitro resistance to SR-BI-targeting molecules remain responsive to anti-SR-BI mAb therapy in vivo. A 2-week antibody therapy readily cleared HCV RNA from the circulation of infected humanized mice. We found no evidence supporting increased SR-BI-receptor dependency of viral particles isolated from humanized mice compared to cell culture-produced virus. However, we observed that, unlike wild-type virus, the in vitro infectivity of the resistant variants was inhibited by both human high density lipoprotein (HDL) and very low density lipoprotein (VLDL). The combination of mAb1671 with these lipoproteins further increased the antiviral effect. Conclusion: HCV variants that are less dependent on SR-BI in vitro can still be efficiently blocked by an anti-SR-BI mAb in humanized mice. Since these variants are also more susceptible to neutralization by anti-HCV envelope antibodies, their chance of emerging during anti-SR-BI therapy is severely reduced. Our data indicate that anti-SR-BI receptor therapy could be an effective way to prevent HCV infection in a liver transplant setting. © 2014 by the American Association for the Study of Liver Diseases.


Lacek K.,CEINGE | Vercauteren K.,Ghent University | Grzyb K.,University of Gdansk | Naddeo M.,CEINGE | And 11 more authors.
Journal of Hepatology | Year: 2012

Background & Aims: Hepatitis C virus (HCV)-induced end-stage liver disease is currently the major indication for liver transplantation in the Western world. After transplantation, the donor liver almost inevitably becomes infected by the circulating virus and disease progression is accelerated in immune suppressed transplant patients. The current standard therapy, based on pegylated interferon and ribavirin, induces severe side effects and is often ineffective in this population. Therefore, new strategies to prevent graft re-infection are urgently needed. We have previously shown that monoclonal antibodies (mAbs) against the HCV co-receptor scavenger receptor class B type I (SR-BI/Cla1) inhibit infection by different HCV genotypes in cell culture. Methods: Using phage display libraries, we have generated a large set of novel human mAbs against SR-BI and evaluated their effectiveness in preventing HCV infection and direct cell-to-cell spread in vitro and in vivo using uPA-SCID mice with a humanized liver. Results: Eleven human monoclonal antibodies were generated that specifically recognize SR-BI. Two antibodies, mAb8 and mAb151, displayed the highest binding and inhibitory properties and also interfered with direct cell-to-cell spread in vitro. Studies in humanized mice showed that both antibodies were capable of preventing HCV infection and could block intrahepatic spread and virus amplification when administered 3 days after infection. Interestingly, anti-SR-BI therapy was effective against an HCV variant that escaped the control of the adaptive immune response in a liver transplant patient. Conclusions: The anti-SR-BI mAbs generated in this study may represent novel therapeutic tools to prevent HCV re-infection of liver allografts. © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.


Bucci C.,University of Salerno | Zingone F.,University of Salerno | Russo I.,University of Salerno | Morra I.,University of Salerno | And 5 more authors.
Clinical Gastroenterology and Hepatology | Year: 2013

Background & Aims: Nonceliac gluten-sensitive (NCGS) patients report intestinal and extra-intestinal symptoms shortly after ingesting gluten; these symptoms disappear on gluten-free diets, although these patients have no serologic markers of celiac disease or intestinal damage. In fact, there is no evidence for mucosal or serologic modifications in those individuals. We investigated immunologic responses of duodenal mucosa samples and peripheral blood basophils, isolated from NCGS patients, after exposure to gliadin. Methods: Participants underwent a complete clinical evaluation to exclude celiac disease while on a gluten-containing diet, a skin prick test to exclude wheat allergy, and upper endoscopy (n= 119) at 2 tertiary medical centers in Italy. Patients were considered to have NCGS based on their symptoms and the current definition of the disorder. Subjects were assigned to the following groups: patients with celiac disease on gluten-free diets (n= 34), untreated patients with celiac disease (n= 35), patients with NCGS (n= 16), or controls (n= 34). Duodenal biopsy samples collected during endoscopy were incubated with gliadin peptides, and levels of inflammatory markers were assessed. Peripheral blood basophils were extracted and incubated with gliadin peptides or a mix of wheat proteins; activation was assessed based on levels of CD203c, CD63, and CD45. Results: Duodenal mucosa samples collected from 69 patients with celiac disease showed markers of inflammation after incubation with gliadin. Some, but not all, markers of inflammation were detected weakly in biopsy samples from 3 controls and 3 NCGS patients ( P= .00 for all markers). There were no significant increases in the levels of CD63 and CD203c in NCGS patients. Conclusions: Unlike the duodenal mucosa from patients with celiac disease, upon incubation with gliadin, mucosa from patients with NCGS does not express markers of inflammation, and their basophils are not activated by gliadin. The in vitro gliadin challenge therefore should not be used to diagnose NCGS. © 2013 AGA Institute.


Valentino T.,University of Naples Federico II | Palmieri D.,University of Naples Federico II | Vitiello M.,University of Naples Federico II | Simeone A.,CEINGE | And 6 more authors.
Journal of Cellular Physiology | Year: 2013

PATZ1 is an emerging cancer-related gene coding for a POZ/AT-hook/kruppel Zinc finger transcription factor, which is lost or misexpressed in human neoplasias. Here, we investigated its role in development exploring wild-type and Patz1-knockout mice during embryogenesis. We report that the Patz1 gene is ubiquitously expressed at early stages of development and becomes more restricted at later stages, with high levels of expression in actively proliferating neuroblasts belonging to the ventricular zones of the central nervous system (CNS). The analysis of embryos in which Patz1 was disrupted revealed the presence of severe defects in the CNS and in the cardiac outflow tract, which eventually lead to a pre-mature in utero death during late gestation or soon after birth. Moreover, the Patz1-null mice showed a general growth retardation, which was consistent with the slower growth rate and the increased susceptibility to senescence of Patz1-/- mouse embryonic fibroblasts (MEFs) compared to wild-type controls. Therefore, these results indicate a critical role of PATZ1 in the control of cell growth and embryonic development. © 2012 Wiley Periodicals, Inc.


Puca F.,University of Naples Federico II | Colamaio M.,University of Naples Federico II | Federico A.,University of Naples Federico II | Gemei M.,CEINGE | And 6 more authors.
Oncotarget | Year: 2014

High-mobility group A1 (HMGA1) proteins are architectural chromatinic proteins, abundantly expressed during embryogenesis and in most cancer tissues, but expressed at low levels or absent in normal adult tissues. Several studies have demonstrated that HMGA1 proteins play a causal role in neoplastic cell transformation. The aim of this study was to investigate the role of these proteins in the control of cancer stem cells (CSCs), which have emerged as a preferred target in cancer therapy, because of their role in cancer recurrence. We observed that HMGA1 is overexpressed in colon tumour stem cell (CTSC) lines compared to normal and colon cancer tissues. We demonstrated that HMGA1 silencing in CTSCs increases stem cell quiescence and reduces self-renewal and sphere-forming efficiency (SFE). The latter, together with the upregulation and asymmetric distribution of NUMB, is indicative of the recovery of an asymmetric division pattern, typical of normal stem cells. We further found that HMGA1 transcriptionally regulates p53, which is known to control the balance between symmetric and asymmetric divisions in CSCs. Therefore, our data indicate a critical role for HMGA1 in regulating both self-renewal and the symmetric/asymmetric division ratio in CSCs, suggesting that blocking HMGA1 function may be an effective anti-cancer therapy. © 2008-2014 Impact Journals, LLC.


Iolascon A.,CEINGE | Iolascon A.,University of Naples Federico II | Heimpel H.,University of Ulm | Wahlin A.,Umeå University | And 2 more authors.
Blood | Year: 2013

The congenital dyserythropoietic anemias (CDAs) are hereditary disorders characterized by distinct morphologic abnormalities of marrow erythroblasts. The unveiling of the genes mutated in the major CDA subgroups (I-CDAN1 and II-SEC23B) has now been completed with the recent identification of the CDA III gene ( KIF23). KIF23 encodes mitotic kinesin-like protein 1, which plays a critical role in cytokinesis, whereas the cellular role of the proteins encoded by CDAN1 and SEC23B is still unknown. CDA variants with mutations in erythroid transcription factor genes (KLF1 and GATA-1 ) have been recently identified. Molecular diagnosis of CDA is now possible in most patients. © 2013 by The American Society of Hematology.


Pagliara V.,University of Catanzaro | Adornetto A.,University of Calabria | Mammi M.,University of Catanzaro | Masullo M.,Parthenope University of Naples | And 4 more authors.
Biochimica et Biophysica Acta - Molecular Cell Research | Year: 2014

Protease Nexin-1 (PN-1) or Serpine2 is a physiological regulator of extracellular proteases as thrombin and urokinase (uPA) in the brain. Besides, PN-1 is also implicated in some human cancers and further identified as a substrate for Matrix Metalloproteinase (MMP)-9, a key enzyme in tumor invasiveness. Our aim was to study the role of PN-1 in the migration and invasive potential of glioma cells, using the rat C6 glioma cell line as stable clones transfected with pAVU6. +. 27 vector expressing PN-1 short-hairpin RNA. We find that PN-1 knockdown enhanced the in vitro migration and invasiveness of C6 cells which also showed a strong gelatinolytic activity by in situ zymography. PN-1 silencing did not alter prothrombin whereas increased uPA, MMP-9 and MMP-2 expression levels and gelatinolytic activity in a conditioned medium from stable C6 cells. Selective inhibitors for MMP-9 (Inhibitor I), MMP-2 (Inhibitor III) or exogenous recombinant PN-1 added to the culture medium of C6 silenced cells restored either the migration and invasive ability or gelatinolytic activity thus validating the specificity of PN-1 silencing strategy. Phosphorylation levels of extracellular signal-related kinases (Erk1/2 and p38 MAPK) involved in MMP-9 and MMP-2 signaling were increased in PN-1 silenced cells. This study shows that PN-1 affects glioma cell migration and invasiveness through the regulation of uPA and MMP-9/2 expression levels which contribute to the degradation of extracellular matrix during tumor invasion. © 2014 Elsevier B.V.

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