Publications by authors named "Irene Colavita"

12 Publications

  • Page 1 of 1

A novel smaller β-defensin-derived peptide is active against multidrug-resistant bacterial strains.

FASEB J 2021 12;35(12):e22026

Department of Molecular Medicine and Medical Biotechnologies, University of Naples Federico II, Naples, Italy.

Antibiotic resistance is becoming a severe obstacle in the fight against acute and chronic infectious diseases that accompany most degenerative illnesses from neoplasia to osteo-arthritis and obesity. Currently, the race is on to identify pharmaceutical molecules or combinations of molecules able to prevent or reduce the insurgence and/or progression of infectivity. Attempts to substitute antibiotics with antimicrobial peptides have, thus far, met with little success against multidrug-resistant (MDR) bacterial strains. During the last decade, we designed and studied the activity and features of human β-defensin analogs, which are salt-resistant, and hence active also under high salt concentrations as, for instance, in cystic fibrosis. Herein, we describe the design, synthesis, and major features of a new 21 aa long molecule, peptide γ2. The latter derives from the γ-core of the β-defensin natural molecules, a small fragment of these molecules still bearing high antibacterial activity. We found that peptide γ2, which contains only one disulphide bond, recapitulates most of the biological properties of natural human β-defensins and can also counteract both Gram-positive and Gram-negative MDR bacterial strains and biofilm formation. Moreover, it has great stability in human serum thereby enhancing its antibacterial presence and activity without cytotoxicity in human cells. In conclusion, peptide γ2 is a promising new weapon also in the battle against intractable infectious diseases.
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http://dx.doi.org/10.1096/fj.202002330RRDOI Listing
December 2021

Detection of colonic dysplasia in patients with ulcerative colitis using a targeted fluorescent peptide and confocal laser endomicroscopy: A pilot study.

PLoS One 2017 30;12(6):e0180509. Epub 2017 Jun 30.

CEINGE-Biotecnologie Avanzate, Naples, Italy.

Aim: Targeted molecular probes have been used to detect sporadic colonic dysplasia during confocal laser endomicroscopy (CLE) with promising results. This is a feasibility pilot study aiming to assess the potential role of CLE combined with a fluorescent-labeled peptide to stain and detect dysplasia associated with Ulcerative Colitis.

Method: A phage-derived heptapeptide with predicted high binding affinity for dysplastic tissue, was synthesized and labeled with fluorescein. Eleven lesions with suspected dysplasia at endoscopy were excised from nine patients with long-standing ulcerative colitis. Specimens were sprayed with the peptide and examined by CLE. The CLE images were then compared to the corresponding histological sections.

Results: At definitive histology, 4 lesions were diagnosed as inflammatory polyps, 6 as dysplastic lesions and one as invasive cancer. In inflammatory polyps, the fluorescence signal came from peri-cryptal spaces and crypt lumen due to passive accumulation of the peptide in these areas. Dysplasia was associated with active binding of the peptide to dysplastic colonocytes.

Conclusion: Ex vivo staining of ulcerative colitis-associated dysplasia using a fluorescent labeled molecular probe and CLE is feasible. In vivo studies on larger populations are required to evaluate the safety and the effective contribution of molecular probes in cancer surveillance of ulcerative colitis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0180509PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5493408PMC
October 2017

Host defense peptide-derived privileged scaffolds for anti-infective drug discovery.

J Pept Sci 2017 Apr 12;23(4):303-310. Epub 2017 Jan 12.

CEINGE-Biotecnologie Avanzate, Via Gaetano Salvatore 486, 80145, Naples, Italy.

'Privileged scaffolds' are molecular frameworks which have been successfully exploited for small molecule drug discovery. Peptide privileged scaffolds, featuring a strictly conserved multiple-disulfide framework and high variability in the rest of the sequence, display a broad range of biological effects, including antimicrobial and antiviral activity. Unlike small molecules, however, the cost of manufacturing these peptides is high, and their synthesis challenging. We previously described a simplified privileged scaffold corresponding to the γ-core of human β-defensin-3 (HBD3). The γ-core is a common structural signature found in virtually all host defense peptides (HDPs) stabilized by multiple disulfides, and we showed that for HBD3, it represents the evolutionary starting point of the full-length molecule and, thus, is itself a primordial HDP. Accordingly, we showed that the peptide folded rapidly and was stable in human serum, and displayed many of the biological activities of HBD3. We report here that in addition to the previously reported antibacterial activity on planktonic bacteria, the γ-core peptide is active against biofilm formation and maturation. We also show that it is readily cell penetrant, like HBD3, although with a different mechanism, which is independent from CD98. Overall, the potency of the single-disulfide, 23-amino acid γ-core is comparable with the full-length peptide across the whole spectrum of examined properties, and the peptide is not toxic to human cells. The HBD3 γ-core peptide may therefore represent the first example of an economically viable lead peptide derived from a HDP privileged scaffold. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/psc.2962DOI Listing
April 2017

An ancestral host defence peptide within human β-defensin 3 recapitulates the antibacterial and antiviral activity of the full-length molecule.

Sci Rep 2015 Dec 21;5:18450. Epub 2015 Dec 21.

CEINGE-Biotecnologie Avanzate, Via Gaetano Salvatore 486, 80145 Napoli, Italy.

Host defence peptides (HDPs) are critical components of innate immunity. Despite their diversity, they share common features including a structural signature, designated "γ-core motif". We reasoned that for each HDPs evolved from an ancestral γ-core, the latter should be the evolutionary starting point of the molecule, i.e. it should represent a structural scaffold for the modular construction of the full-length molecule, and possess biological properties. We explored the γ-core of human β-defensin 3 (HBD3) and found that it: (a) is the folding nucleus of HBD3; (b) folds rapidly and is stable in human serum; (c) displays antibacterial activity; (d) binds to CD98, which mediates HBD3 internalization in eukaryotic cells; (e) exerts antiviral activity against human immunodeficiency virus and herpes simplex virus; and (f) is not toxic to human cells. These results demonstrate that the γ-core within HBD3 is the ancestral core of the full-length molecule and is a viable HDP per se, since it is endowed with the most important biological features of HBD3. Notably, the small, stable scaffold of the HBD3 γ-core can be exploited to design disease-specific antimicrobial agents.
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http://dx.doi.org/10.1038/srep18450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4685272PMC
December 2015

Design and activity of a cyclic mini-β-defensin analog: a novel antimicrobial tool.

Int J Nanomedicine 2015 15;10:6523-39. Epub 2015 Oct 15.

Dipartimento di Farmacia, Università di Napoli Federico II, Naples, Italy ; Istituto di Biostrutture e Bioimmagini, CNR, Naples, Italy.

We have designed a cyclic 17-amino acid β-defensin analog featuring a single disulfide bond. This analog, designated "AMC" (ie, antimicrobial cyclic peptide), combines the internal hydrophobic domain of hBD1 and the C-terminal charged region of hBD3. The novel peptide was synthesized and characterized by nuclear magnetic resonance spectroscopy. The antimicrobial activities against gram-positive and gram-negative bacteria as well as against herpes simplex virus type 1 were analyzed. The cytotoxicity and serum stability were assessed. Nuclear magnetic resonance of AMC in aqueous solution suggests that the structure of the hBD1 region, although not identical, is preserved. Like the parent defensins, AMC is not cytotoxic for CaCo-2 cells. Interestingly, AMC retains the antibacterial activity of the parent hBD1 and hBD3 against Pseudomonas aeruginosa, Enterococcus faecalis, and Escherichia coli, and exerts dose-dependent activity against herpes simplex virus type 1. Moreover, while the antibacterial and antiviral activities of the oxidized and reduced forms of the parent defensins are similar, those of AMC are significantly different, and oxidized AMC is also considerably more stable in human serum. Taken together, our data also suggest that this novel peptide may be added to the arsenal of tools available to combat antibiotic-resistant infectious diseases, particularly because of its potential for encapsulation in a nanomedicine vector.
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http://dx.doi.org/10.2147/IJN.S89610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4610797PMC
August 2016

Membrane protein 4F2/CD98 is a cell surface receptor involved in the internalization and trafficking of human β-Defensin 3 in epithelial cells.

Chem Biol 2015 Feb 29;22(2):217-28. Epub 2015 Jan 29.

CEINGE-Biotecnologie Avanzate S.c.a r.l., Via Gaetano Salvatore 486, 80145 Naples, Italy; IRCCS-Fondazione SDN, Via Emanuele Gianturco 113, 80142 Naples, Italy. Electronic address:

Human β-defensins play a pivotal role in the innate immune response. Although expressed by and acting at epithelial surfaces, little is known about their specific interaction with epithelial structures. Here, we identify the transmembrane protein CD98 as a cell surface receptor involved in the internalization of human β-defensin 3 (hBD3) in human epithelial A549 cells. CD98 and hBD3 extensively colocalize on the basolateral domain of A549. While verifying their direct binding by fluorescence resonance energy transfer and surface plasmon resonance, we mapped the interaction to CD98 residues 304-414, i.e. to the region known to interact with the proteins of intestinal bacteria during colonic invasion. Treatment of A549 cells with hBD3 dramatically reduces CD98 expression and conversely, knockdown of CD98 expression impairs hBD3 cell surface binding and internalization. Competition for bacterial binding to CD98 and downregulation of CD98 expression may represent novel mechanisms for the antibacterial activity of hBD3.
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http://dx.doi.org/10.1016/j.chembiol.2014.11.020DOI Listing
February 2015

Selective strong synergism of Ruxolitinib and second generation tyrosine kinase inhibitors to overcome bone marrow stroma related drug resistance in chronic myelogenous leukemia.

Leuk Res 2014 Feb 15;38(2):236-42. Epub 2013 Nov 15.

Dipartimento di Medicina Clinica e Chirurgia, University of Naples Federico II, Italy; CEINGE Biotecnologie Avanzate, Napoli, Italy.

The IC50 of TKIs is significantly increased when BCR-ABL+ K562 cell line is cultured in stroma conditioned media produced by BM mesenchymal cells. In particular, while the Imatinib IC50 in the stromal co-cultures was well above the in vivo through levels of the drug, the IC50s of second generation TKIs were still below their through levels. Moreover, we provide a formal comparison of the synergy between first and second generation TKIs with the JAK inhibitor Ruxolitinib to overcome BM stroma related TKI resistance. Taken together, our data provide a rationale for the therapeutic combination of TKIs and Ruxolitinib with the aim to eradicate primary BCR-ABL+ cells homed in BM niches.
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http://dx.doi.org/10.1016/j.leukres.2013.11.006DOI Listing
February 2014

Identification of Annexin A1 interacting proteins in chronic myeloid leukemia KCL22 cells.

Proteomics 2013 Aug 16;13(16):2414-8. Epub 2013 Jul 16.

CEINGE-Biotecnologie Avanzate scarl, Napoli, Italy.

In the present study, we used a functional proteomic approach to identify Annexin A1 (Anxa1) interacting proteins in the Philadelphia-positive KCL22 cell line. We focused on Anxa1 because it is one of the major proteins upregulated in imatinib-sensitive KCL22S cells versus imatinib-resistant KCL22R. Our proteomic strategy revealed 21 interactors. Bioinformatic analysis showed that most of these proteins are involved in cell death processes. Among the proteins identified, we studied the interaction of Anxa1 with two phosphatases, Shp1 and Shp2, which were recently identified as biomarkers of imatinib sensitivity in patients affected by chronic myeloid leukemia. Our data open new perspectives in the search for annexin-mediated signaling pathways and may shed light on mechanisms of resistance to imatinib that are unrelated to Bcr-Abl activity. All mass spectrometry data have been deposited in the ProteomeXchange with identifier PXD000030.
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http://dx.doi.org/10.1002/pmic.201200444DOI Listing
August 2013

The secretome signature of colon cancer cell lines.

J Cell Biochem 2013 Nov;114(11):2577-87

Fondazione IRCCS SDN, Naples, Italy.

The definition of the secretome signature of a cancer cell line can be considered a potential tool to investigate tumor aggressiveness and a preclinical exploratory study required to optimize the search of cancer biomarkers. Dealing with a cell-specific secretome limits the contamination by the major components of the human serum and reduces the range of dynamic concentrations among the secreted proteins, thus favouring under-represented tissue-specific species. The aim of the present study is to characterize the secretome of two human colon carcinoma cell lines, CaCo-2 and HCT-GEO, in order to evaluate differences and similarities of two colorectal cancer model systems. In this study, we identified more than 170 protein species, 64 more expressed in the secretome of CaCo-2 cells and 54 more expressed in the secretome of HCT-GEO cells; 58 proteins were shared by the two systems. Among them, more than 50% were deemed to be secretory according to their Gene Ontology annotation and/or to their SignalP or SecretomeP scores. Such a characterization allowed corroborating the potential of a cell culture-based model in order to describe the cell-specific invasive properties and to provide a list of putative cancer biomarkers.
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http://dx.doi.org/10.1002/jcb.24600DOI Listing
November 2013

Chimeric beta-defensin analogs, including the novel 3NI analog, display salt-resistant antimicrobial activity and lack toxicity in human epithelial cell lines.

Antimicrob Agents Chemother 2013 Apr 28;57(4):1701-8. Epub 2013 Jan 28.

CEINGE-Biotecnologie Avanzate Scarl, Naples, Italy.

Human beta-defensins (hBDs) are crucial peptides for the innate immune response and are thus prime candidates as therapeutic agents directed against infective diseases. Based on the properties of wild-type hBD1 and hBD3 and of previously synthesized analogs (1C, 3I, and 3N), we have designed a new analog, 3NI, and investigated its potential as an antimicrobial drug. Specifically, we evaluated the antimicrobial activities of 3NI versus those of hBD1, hBD3, 1C, 3I, and 3N. Our results show that 3NI exerted greater antibacterial activity against Pseudomonas aeruginosa, Escherichia coli, and Enterococcus faecalis than did hBD1 and hBD3, even with elevated salt concentrations. Moreover, its antiviral activity against herpes simplex virus 1 was greater than that of hBD1 and similar to that of hBD3. Subsequently, we investigated the cytotoxic effects of all peptides in three human epithelial carcinoma cell lines: A549 from lung, CaCo-2 from colon, and Capan-1 from pancreas. None of the analogs significantly reduced cell viability versus wild-type hBD1 and hBD3. They did not induce genotoxicity or cause an increase in the number of apoptotic cells. Using confocal microscopy, we also investigated the localization of the peptides during their incubation with epithelial cells and found that they were distributed on the cell surface, from which they were internalized. Finally, we show that hBD1 and hBD3 are characterized by high resistance to serum degradation. In conclusion, the new analog 3NI seems to be a promising anti-infective agent, particularly given its high salt resistance--a feature that is relevant in diseases such as cystic fibrosis.
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http://dx.doi.org/10.1128/AAC.00934-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3623310PMC
April 2013

SHP-1 expression accounts for resistance to imatinib treatment in Philadelphia chromosome-positive cells derived from patients with chronic myeloid leukemia.

Blood 2011 Sep 5;118(13):3634-44. Epub 2011 Aug 5.

CEINGE Biotecnologie Avanzate, Naples, Italy.

We prove that the SH2-containing tyrosine phosphatase 1 (SHP-1) plays a prominent role as resistance determinant of imatinib (IMA) treatment response in chronic myelogenous leukemia cell lines (sensitive/KCL22-S and resistant/KCL22-R). Indeed, SHP-1 expression is significantly lower in resistant than in sensitive cell line, in which coimmunoprecipitation analysis shows the interaction between SHP-1 and a second tyrosine phosphatase SHP-2, a positive regulator of RAS/MAPK pathway. In KCL22-R SHP-1 ectopic expression restores both SHP-1/SHP-2 interaction and IMA responsiveness; it also decreases SHP-2 activity after IMA treatment. Consistently, SHP-2 knocking-down in KCL22-R reduces either STAT3 activation or cell viability after IMA exposure. Therefore, our data suggest that SHP-1 plays an important role in BCR-ABL-independent IMA resistance modulating the activation signals that SHP-2 receives from both BCR/ABL and membrane receptor tyrosine kinases. The role of SHP-1 as a determinant of IMA sensitivity has been further confirmed in 60 consecutive untreated patients with chronic myelogenous leukemia, whose SHP-1 mRNA levels were significantly lower in case of IMA treatment failure (P < .0001). In conclusion, we suggest that SHP-1 could be a new biologic indicator at baseline of IMA sensitivity in patients with chronic myelogenous leukemia.
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http://dx.doi.org/10.1182/blood-2011-03-341073DOI Listing
September 2011

Gaining insights into the Bcr-Abl activity-independent mechanisms of resistance to imatinib mesylate in KCL22 cells: a comparative proteomic approach.

Biochim Biophys Acta 2010 Oct 24;1804(10):1974-87. Epub 2010 Apr 24.

CEINGE Biotecnologie Avanzate Scarl, Napoli, Italy.

Imatinib mesylate is a potent inhibitor of Bcr-Abl tyrosine kinase, an oncoprotein that plays a key role in the development of chronic myeloid leukemia. Consequently, imatinib is used as front-line therapy for this disease. A major concern in imatinib treatment is the emergence of resistance to the drug. Here we used the imatinib-resistant KCL22R and imatinib-sensitive KCL22S cells in which none of the known resistance mechanisms has been detected and hence novel Bcr-Abl activity-independent mechanisms could be envisaged. We characterized proteins that were differentially expressed between the KCL22R and KCL22S cells. Using two-dimensional differential gel electrophoresis coupled with mass spectrometry and Western blot analysis we identified 51 differentially expressed proteins: 27 were over-expressed and 24 were under-expressed in KCL22R versus KCL22S cells. Several of these proteins are likely to be involved in such survival mechanisms as modulation of redox balance and activation of anti-apoptotic pathways mediated by NF-kappaB and Ras-MAPK signaling. The data reported may be useful for further studies on mechanisms of imatinib resistance and for the screening of biomarkers to develop new combinatorial therapeutic approaches.
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http://dx.doi.org/10.1016/j.bbapap.2010.04.009DOI Listing
October 2010
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