Publications by authors named "Anna Rita Taddei"

44 Publications

Molecular, Cellular and Functional Analysis of TRγ Chain along the European Sea Bass Development.

Int J Mol Sci 2021 Mar 25;22(7). Epub 2021 Mar 25.

Department for Innovation in Biological, Agro-Food and Forest Systems, University of Tuscia, Largo dell'Università, 01100 Viterbo, Italy.

In jawed vertebrates, adaptive immune responses are enabled by T cells. Two lineages were characterized based on their T cell receptor (TcR) heterodimers, namely αβ or γδ peptide chains, which display an Ig domain-type sequence that is somatically rearranged. γδ T cells have been less extensively characterized than αβ and teleost fish, in particular, suffer from a severe scarcity of data. In this paper, we worked on the well-known model, the European sea bass to broaden the understanding of teleost γδ-T cells. The T cell receptor chain (TR) γ transcript was expressed at a later developmental stage than TRβ, suggesting a layered appearance of fish immune cells, and the thymus displayed statistically-significant higher mRNA levels than any other organ or lymphoid tissue investigated. The polyclonal antibody developed against the TRγ allowed the localization of TRγ-expressing cells in lymphoid organs along the ontogeny. Cell positivity was investigated through flow cytometry and the highest percentage was found in peripheral blood leukocytes, followed by thymus, gut, gills, spleen and head kidney. Numerous TRγ-expressing cells were localized in the gut mucosa, and the immunogold labelling revealed ultrastructural features that are typical of T cells. At last, microalgae-based diet formulations significantly modulated the abundance of TRγ cells in the posterior intestine, hinting at a putative involvement in nutritional immunity. From a comparative immunological perspective, our results contribute to the comprehension of the diversity and functionalities of γδ T cells during the development of a commercially relevant marine teleost model.
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http://dx.doi.org/10.3390/ijms22073376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8036326PMC
March 2021

Retinal damage in a new model of hyperglycemia induced by high-sucrose diets.

Pharmacol Res 2021 Apr 11;166:105488. Epub 2021 Feb 11.

Department for Innovation in Biological, Agro-food and Forest Systems (DIBAF), Università degli Studi della Tuscia, largo dell'Università snc, 01100 Viterbo, Italy. Electronic address:

Loss of retinal neurons may precede clinical signs of diabetic retinopathy (DR). We studied for the first time the effects of hyperglycemia on the visual system of the fruit fly Drosophila melanogaster to characterize a model for glucose-induced retinal neurodegeneration, thus complementing more traditional vertebrate systems. Adult flies were fed with increased high-sucrose regimens which did not modify the locomotion ability, muscle phenotype and mobility after 10 days. The increased availability of dietary sucrose induced hyperglycemia and phosphorylation of Akt in fat tissue, without significant effects on adult growth and viability, consistent with the early phase of insulin signaling and a low impact on the overall metabolic profile of flies at short term. Noteworthy, high-sucrose diets significantly decreased Drosophila responsiveness to the light as a consequence of vision defects. Hyperglycemia did not alter the gross anatomical architecture of the external eye phenotype although a progressive damage of photosensitive units was observed. Appreciable levels of cleaved caspase 3 and nitrotyrosine were detected in the internal retina network as well as punctate staining of Light-Chain 3 and p62, and accumulated autophagosomes, indicating apoptotic features, peroxynitrite formation and autophagy turnover defects. In summary, our results in Drosophila support the view that hyperglycemia induced by high-sucrose diets lead to eye defects, apoptosis/autophagy dysregulation, oxidative stress, and visual dysfunctions which are evolutionarily conserved, thus offering a meaningful opportunity of using a simple in vivo model to study the pathophysiology of neuroretinal alterations that develop in patients at the early stages of DR.
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http://dx.doi.org/10.1016/j.phrs.2021.105488DOI Listing
April 2021

Chemical Investigation and Screening of Anti-Proliferative Activity on Human Cell Lines of Pure and Nano-Formulated Lavandin Essential Oil.

Pharmaceuticals (Basel) 2020 Oct 29;13(11). Epub 2020 Oct 29.

Department of Drug Chemistry and Technology, Sapienza University, 00185 Roma RM, Italy.

Lavandin essential oil (LEO), a natural sterile hybrid obtained by crossbreeding is mainly composed by active components belonging to the family of terpenes endowed with relevant anti-proliferative activity, which can be enhanced by proper application of nanotechnology. In particular, this study reports the chemical characterization and the screening of the anti-proliferative activity on different human cell lines of pure and nano-formulated lavandin essential oil (EO). LEO and its formulation (NanoLEO) were analyzed by HS/GC-MS (Headspace/Gas Chromatography-Mass Spectrometry) to describe and compare their chemical volatile composition. The most abundant compounds were linalool and 1,8-cineole (LEO: 28.6%; 27.4%) (NanoLEO: 60.4%; 12.6%) followed by α-pinene (LEO: 9.6%; NanoLEO: 4.5%), camphor (LEO: 6.5%; NanoLEO: 7.0%) and linalyl acetate (LEO: 6.5%; NanoLEO: 3.6%). The cytotoxic effects of LEO and NanoLEO were investigated on human neuroblastoma cells (SHSY5Y), human breast adenocarcinoma cells (MCF-7), human lymphoblastic leukemia cells (CCRF CEM), human colorectal adenocarcinoma cells (Caco-2) and one normal breast epithelial cell (MCF10A) by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide)-assay. Caco-2, MCF7 and MCF10A normal cells resulted more resistant to the treatment with LEO, while CCRF-CEM and SHSY5Y cells were more sensitive. The antiproliferative effect of LEO resulted amplified when the essential oil was supplied as nanoformulation, mainly in Caco-2 cells. Scanning and transmission electron microscopy investigations were carried out on Caco-2 cells to outline at ultrastructural level possible affections induced by LEO and NanoLEO treatments.
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http://dx.doi.org/10.3390/ph13110352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7692866PMC
October 2020

Defects of full-length dystrophin trigger retinal neuron damage and synapse alterations by disrupting functional autophagy.

Cell Mol Life Sci 2021 Feb 4;78(4):1615-1636. Epub 2020 Aug 4.

Department for Innovation in Biological, Agro-Food and Forest Systems (DIBAF), Università degli Studi della Tuscia, largo dell'Università snc, 01100, Viterbo, Italy.

Dystrophin (dys) mutations predispose Duchenne muscular disease (DMD) patients to brain and retinal complications. Although different dys variants, including long dys products, are expressed in the retina, their function is largely unknown. We investigated the putative role of full-length dystrophin in the homeostasis of neuro-retina and its impact on synapsis stabilization and cell fate. Retinas of mdx mice, the most used DMD model which does not express the 427-KDa dys protein (Dp427), showed overlapped cell death and impaired autophagy. Apoptotic neurons in the outer plexiform/inner nuclear layer and the ganglion cell layer had an impaired autophagy with accumulated autophagosomes. The autophagy dysfunction localized at photoreceptor axonal terminals and bipolar, amacrine, and ganglion cells. The absence of Dp427 does not cause a severe phenotype but alters the neuronal architecture, compromising mainly the pre-synaptic photoreceptor terminals and their post-synaptic sites. The analysis of two dystrophic mutants of the fruit fly Drosophila melanogaster, the homozygous Dys and Dys, lacking functional large-isoforms of dystrophin-like protein, revealed rhabdomere degeneration. Structural damages were evident in the internal network of retina/lamina where photoreceptors make the first synapse. Both accumulated autophagosomes and apoptotic features were detected and the visual system was functionally impaired. The reactivation of the autophagosome turnover by rapamycin prevented neuronal cell death and structural changes of mutant flies and, of interest, sustained autophagy ameliorated their response to light. Overall, these findings indicate that functional full-length dystrophin is required for synapsis stabilization and neuronal survival of the retina, allowing also proper autophagy as a prerequisite for physiological cell fate and visual properties.
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http://dx.doi.org/10.1007/s00018-020-03598-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7904721PMC
February 2021

A case of acute renal failure with multiple origins of the renal injury.

CEN Case Rep 2020 11 4;9(4):437-441. Epub 2020 Jul 4.

Nephrology and Dialysis Unit, Belcolle Hospital, Via Sammartinese, Snc, 01100, Viterbo, Italy.

Acute kidney injury (AKI) is an abrupt and usually reversible decline in the glomerular filtration rate (GFR). Patients with AKI must be evaluated promptly to determine cause. Different disorders can BE associated with AKI, and biopsy is the most accurate instrument for diagnosis of different types of diseases. We report a case of 69-year-old woman. In history, type II diabetes mellitus and arterial hypertension admitted to our hospital for the evaluation of leg pain, asthenia, diarrhea, and malaise. She was in the treatment with metformin and ARB. Laboratory data revealed renal failure: serum creatinine (Scr 16.5 mg/dl, BUN 280 mg/dl) hyperkalemia and severe anemia (Hb 7.8 g/dl). Renal ultrasound displayed preserved kidneys size. An X-ray of backbone showed fracture. She underwent hemodialysis in urgency regimen. After some days, urine output began to improve up to 1200 cc/24 h. we find proteinuria in nephrotic range. Renal function remained compromised (sCr 8.5 mg/dl, BUN 150 mg/dl). For the evaluation of renal disease, the patient underwent a kidney biopsy. Histological examination findings showed overlapping changes composed of three concurrent pathologic findings: cast nephropathy, diabetes, and light chain deposition disease. After the renal biopsy, therapy with bortezomib, thalidomide, and steroid were administered. At the same time, plasma exchange was carried out. Clinical response occurred with partial recovery of renal function (Scr 3.5 mg/dl eGFR), and dialysis treatment was stopped.
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http://dx.doi.org/10.1007/s13730-020-00505-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7502086PMC
November 2020

Apoptotic Effects on HL60 Human Leukaemia Cells Induced by Lavandin Essential Oil Treatment.

Molecules 2020 Jan 26;25(3). Epub 2020 Jan 26.

Department of Drug Chemistry and Technology, Sapienza University, 00185 Rome, Italy.

Recent scientific investigations have reported a number of essential oils to interfere with intracellular signalling pathways and to induce apoptosis in different cancer cell types. In this paper, Lavandin Essential Oil (LEO), a natural sterile hybrid obtained by cross-breeding  ×  was tested on human leukaemia cells (HL60). Based on the MTT results, the reduced cell viability of HL60 cells was further investigated to determine whether cell death was related to the apoptotic process. HL60 cells treated for 24 h with LEO were processed by flow cytometry, and the presence of Annexin V was measured. The activation of caspases-3 was evaluated by western blot and immunofluorescence techniques. Treated cells were also examined by scanning and transmission electron microscopy to establish the possible occurrence of morphological alterations during the apoptotic process. LEO main compounds, such as linalool, linalyl acetate, 1,8-cineole, and terpinen-4-ol, were also investigated by MTT and flow cytometry analysis. The set of obtained results showed that LEO treatments induced apoptosis in a dose-dependent, but not time-dependent, manner on HL60 cells, while among LEO main compounds, both terpinen-4-ol and linalyl acetate were able to induce apoptosis.
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http://dx.doi.org/10.3390/molecules25030538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7036901PMC
January 2020

The meningococcal vaccine antigen GNA2091 is an analogue of YraP and plays key roles in outer membrane stability and virulence.

FASEB J 2019 11 23;33(11):12324-12335. Epub 2019 Aug 23.

GlaxoSmithKline (GSK) Vaccines, Siena, Italy.

GNA2091 is one of the components of the 4-component meningococcal serogroup B vaccine (4CMenB) vaccine and is highly conserved in all meningococcal strains. However, its functional role has not been fully characterized. Here we show that is part of an operon and is cotranscribed with the , and adjacent genes, and a similar but reduced operon arrangement is conserved in many other gram-negative bacteria. Deletion of the gene causes an aggregative phenotype with a mild defect in cell separation; differences in the outer membrane composition and phospholipid profile, in particular in the phosphoethanolamine levels; an increased level of outer membrane vesicles; and deregulation of the zinc-responsive genes such as . Finally, the ∆2091 strain is attenuated with respect to the wild-type strain in competitive index experiments in the infant rat model of meningococcal infection. Altogether these data suggest that GNA2091 plays important roles in outer membrane architecture, biogenesis, homeostasis, and in meningococcal survival , and a model for its role is discussed. These findings highlight the importance of GNA2091 as a vaccine component.-Seib, K. L., Haag, A. F., Oriente, F., Fantappiè, L., Borghi, S., Semchenko, E. A., Schulz, B. L., Ferlicca, F., Taddei, A. R., Giuliani, M. M., Pizza, M., Delany, I. The meningococcal vaccine antigen GNA2091 is an analogue of YraP and plays key roles in outer membrane stability and virulence.
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http://dx.doi.org/10.1096/fj.201900669RDOI Listing
November 2019

Fish-derived antimicrobial peptides: Activity of a chionodracine mutant against bacterial models and human bacterial pathogens.

Dev Comp Immunol 2019 07 18;96:9-17. Epub 2019 Feb 18.

Department for Innovation in Biological, Agrofood and Forest Systems, University of Tuscia, Viterbo, Italy. Electronic address:

The increasing resistance to conventional antibiotics is an urgent problem that can be addressed by the discovery of new antimicrobial drugs such as antimicrobial peptides (AMPs). AMPs are components of innate immune system of eukaryotes and are not prone to the conventional mechanisms that are responsible of drug resistance. Fish are an important source of AMPs and, recently, we have isolated and characterized a new 22 amino acid residues peptide, the chionodracine (Cnd), from the Antarctic icefish Chionodraco hamatus. In this paper we focused on a new Cnd-derived mutant peptide, namely Cnd-m3a, designed to improve the selectivity against prokaryotic cells and the antimicrobial activity against human pathogens of the initial Cnd template. Cnd-m3a was used for immunization of rabbits, which gave rise to a polyclonal antibody able to detect the peptide. The interaction kinetic of Cnd-m3a with the Antarctic bacterium Psychrobacter sp. (TAD1) was imaged using a transmission electron microscopy (TEM) immunogold method. Initially the peptide was associated with the plasma membrane, but after 180 min of incubation, it was found in the cytoplasm interacting with a DNA target inside the bacterial cells. Using fluorescent probes we showed that the newly designed mutant can create pores in the outer membrane of the bacteria E. coli and Psychrobacter sp. (TAD1), confirming the results of TEM analysis. Moreover, in vitro assays demonstrated that Cnd-m3a is able to bind lipid vesicles of different compositions with a preference toward negatively charged ones, which mimics the prokaryotic cell. The Cnd-m3a peptide showed quite low hemolytic activity and weak cytotoxic effect against human primary and tumor cell lines, but high antimicrobial activity against selected Gram - human pathogens. These results highlighted the high potential of the Cnd-m3a peptide as a starting point for developing a new human therapeutic agent.
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http://dx.doi.org/10.1016/j.dci.2019.02.012DOI Listing
July 2019

Characterization of Collagen/Lipid Nanoparticle-Curcumin Cryostructurates for Wound Healing Applications.

Macromol Biosci 2019 05 15;19(5):e1800446. Epub 2019 Feb 15.

Univ. Grenoble Alpes, CEA-LETI, 17 rue des martyrs, 38054, Grenoble cedex 9, France.

Curcumin-loaded collagen cryostructurates have been devised for wound healing applications. Curcumin displays strong antioxidant, antiseptic, and anti-inflammatory properties, while collagen is acknowledged for promoting cell adhesion, migration and differentiation. However, when curcumin is loaded directly into collagen hydrogels, it forms large molecular aggregates and clogs the matrix pores. A double-encapsulation strategy is therefore developed by loading curcumin into lipid nanoparticles (LNP), and embedding these particles inside collagen scaffolds. The resulting collagen/LNP cryostructurates have an optimal fibrous structure with ≈100 µm average pore size for sustaining cell migration. Results show that collagen is structurally unaltered and that nanoparticles are homogeneously distributed amidst collagen fibers. Hydrogels soaked in saline buffer release about 20 to 30% of their nanoparticles content within 24 h, while achieved 100% release after 25 days. When exposed to NIH 3T3 fibroblasts, these hydrogels provide a satisfactory scaffold for cell interaction as early as 4 h after seeding, with no cytotoxic counter effect. These positive features make the collagen/lipid cryostructurates a promising material for further use in wound healing.
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http://dx.doi.org/10.1002/mabi.201800446DOI Listing
May 2019

Dysfunctional autophagy induced by the pro-apoptotic natural compound climacostol in tumour cells.

Cell Death Dis 2018 12 19;10(1):10. Epub 2018 Dec 19.

Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), Università degli Studi della Tuscia, Viterbo, Italy.

Autophagy occurs at a basal level in all eukaryotic cells and may support cell survival or activate death pathways. Due to its pathophysiologic significance, the autophagic machinery is a promising target for the development of multiple approaches for anti-neoplastic agents. We have recently described the cytotoxic and pro-apoptotic mechanisms, targeting the tumour suppressor p53, of climacostol, a natural product of the ciliated protozoan Climacostomum virens. We report here on how climacostol regulates autophagy and the involvement of p53-dependent mechanisms. Using both in vitro and in vivo techniques, we show that climacostol potently and selectively impairs autophagy in multiple tumour cells that are committed to die by apoptosis. In particular, in B16-F10 mouse melanomas climacostol exerts a marked and sustained accumulation of autophagosomes as the result of dysfunctional autophagic degradation. We also provide mechanistic insights showing that climacostol affects autophagosome turnover via p53-AMPK axis, although the mTOR pathway unrelated to p53 levels plays a role. In particular, climacostol activated p53 inducing the upregulation of p53 protein levels in the nuclei through effects on p53 stability at translational level, as for instance the phosphorylation at Ser15 site. Noteworthy, AMPKα activation was the major responsible of climacostol-induced autophagy disruption in the absence of a key role regulating cell death, thus indicating that climacostol effects on autophagy and apoptosis are two separate events, which may act independently on life/death decisions of the cell. Since the activation of p53 system is at the molecular crossroad regulating both the anti-autophagic action of climacostol and its role in the apoptosis induction, it might be important to explore the dual targeting of autophagy and apoptosis with agents acting on p53 for the selective killing of tumours. These findings also suggest the efficacy of ciliate bioactive molecules to identify novel lead compounds in drug discovery and development.
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http://dx.doi.org/10.1038/s41419-018-1254-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6315039PMC
December 2018

A New Species of the γ-Proteobacterium Francisella, F. adeliensis Sp. Nov., Endocytobiont in an Antarctic Marine Ciliate and Potential Evolutionary Forerunner of Pathogenic Species.

Microb Ecol 2019 Apr 5;77(3):587-596. Epub 2018 Sep 5.

Departamento de Microbiología, Universidad de Sevilla, Av Reina Mercedes 6, 41012, Seville, Spain.

The study of the draft genome of an Antarctic marine ciliate, Euplotes petzi, revealed foreign sequences of bacterial origin belonging to the γ-proteobacterium Francisella that includes pathogenic and environmental species. TEM and FISH analyses confirmed the presence of a Francisella endocytobiont in E. petzi. This endocytobiont was isolated and found to be a new species, named F. adeliensis sp. nov.. F. adeliensis grows well at wide ranges of temperature, salinity, and carbon dioxide concentrations implying that it may colonize new organisms living in deeply diversified habitats. The F. adeliensis genome includes the igl and pdp gene sets (pdpC and pdpE excepted) of the Francisella pathogenicity island needed for intracellular growth. Consistently with an F. adeliensis ancient symbiotic lifestyle, it also contains a single insertion-sequence element. Instead, it lacks genes for the biosynthesis of essential amino acids such as cysteine, lysine, methionine, and tyrosine. In a genome-based phylogenetic tree, F. adeliensis forms a new early branching clade, basal to the evolution of pathogenic species. The correlations of this clade with the other clades raise doubts about a genuine free-living nature of the environmental Francisella species isolated from natural and man-made environments, and suggest to look at F. adeliensis as a pioneer in the Francisella colonization of eukaryotic organisms.
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http://dx.doi.org/10.1007/s00248-018-1256-3DOI Listing
April 2019

Iodoxybenzoic Acid Supported on Multi Walled Carbon Nanotubes as Biomimetic Environmental Friendly Oxidative Systems for the Oxidation of Alcohols to Aldehydes.

Nanomaterials (Basel) 2018 Jul 10;8(7). Epub 2018 Jul 10.

Department of Ecology and Biology, University of Tuscia, Largo dell'Università, 01100 Viterbo (VT), Italy.

Iodoxybenzoic acid (IBX) supported multi walled carbon nanotube (MWCNT) derivatives have been prepared as easily recyclable solid reagents. These compounds have been shown to be able to mimic the alcohol dehydrogenases and monooxygenases promoted oxidation of aromatic alcohols to corresponding aldehydes. Their reactivity was found to be dependent on the degree of functionalization of MWCNTs as well as from the chemical properties of the spacers used to bind IBX on the surface of the support. Au-decorated MWCNTs and the presence of longer spacers resulted in the optimal experimental conditions. A high conversion of the substrates and yield of desired products were obtained.
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http://dx.doi.org/10.3390/nano8070516DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071043PMC
July 2018

Candidate Enzymes for Saffron Crocin Biosynthesis Are Localized in Multiple Cellular Compartments.

Plant Physiol 2018 07 29;177(3):990-1006. Epub 2018 May 29.

Italian National Agency for New Technologies, Energy and Sustainable Economic Development, 00123 Rome, Italy

Saffron is the dried stigmas of and is the most expensive spice in the world. Its red color is due to crocins, which are apocarotenoid glycosides that accumulate in the vacuole to a level up to 10% of the stigma dry weight. Previously, we characterized the first dedicated enzyme in the crocin biosynthetic pathway, carotenoid cleavage dioxygenase2 (CsCCD2), which cleaves zeaxanthin to yield crocetin dialdehyde. In this work, we identified six putative () genes expressed in stigmas. Heterologous expression in showed that only one of corresponding proteins (CsALDH3I1) was able to convert crocetin dialdehyde into the crocin precursor crocetin. CsALDH3I1 carries a carboxyl-terminal hydrophobic domain, similar to that of the membrane-associated apocarotenoid dehydrogenase YLO-1. We also characterized the UDP-glycosyltransferase CsUGT74AD1, which converts crocetin to crocins 1 and 2'. In vitro assays revealed high specificity of CsALDH3I1 for crocetin dialdehyde and long-chain apocarotenals and of CsUGT74AD1 for crocetin. Following extract fractionation, CsCCD2, CsALDH3I1, and CsUGT74AD1 were found in the insoluble fraction, suggesting their association with membranes or large insoluble complexes. Analysis of protein localization in both stigmas and following transgene expression in leaves revealed that CsCCD2, CsALDH3I, and CsUGT74AD1 were localized to the plastids, the endoplasmic reticulum, and the cytoplasm, respectively, in association with cytoskeleton-like structures. Based on these findings and current literature, we propose that the endoplasmic reticulum and cytoplasm function as transit centers for metabolites whose biosynthesis starts in the plastid and are accumulated in the vacuole.
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http://dx.doi.org/10.1104/pp.17.01815DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6053014PMC
July 2018

Microvesicles shed from fibroblasts act as metalloproteinase carriers in a 3-D collagen matrix.

J Circ Biomark 2016 Jan-Dec;5:1849454416663660. Epub 2016 Nov 7.

Department of Innovation in Biological, Agrifood and Forestry Systems, Tuscia University, Viterbo, Italy.

This study shows that fibroblasts migrating into a collagen matrix release numerous microvesicles into the surrounding medium. By spreading in regions of the matrix far distant from cells of origin, microvesicles carry metalloproteinase 9 (MMP-9) to act upon the collagen fibrils. As a result, the collagen matrix is gradually transformed from a laminar to a fibrillar type of architecture. As shown by western blots and gelatin zymography, MMP-9 is secreted as a 92 kDa precursor and activated upon release of 82 kDa product into the culture medium. Activation is more efficient under three-dimensional than in two-dimensional culturing conditions. While MMP-9 labeling is associated with intraluminal vesicles clustered inside the microvesicles, the microvesicle's integrin β1 marker is bound to the outer membrane. The intraluminal vesicles are recruited from the cortical cytoplasm and eventually released following uploading inside the microvesicle. Here, we propose that fusion of the intraluminal vesicles with the outer microvesicle's membrane could work as a mechanism controlling the extent to which MMP-9 is first activated and then released extracellularly.
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http://dx.doi.org/10.1177/1849454416663660DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5548308PMC
November 2016

Ultrastructural investigation on fibroblast interaction with collagen scaffold.

J Biomed Mater Res A 2016 Jan 1;104(1):272-82. Epub 2015 Oct 1.

Department for Innovation in Biological, Agrifood and Forestry Systems, Tuscia University, Viterbo, 01100, Italy.

Collagen-based scaffolds are used as temporary or permanent coverings to help wound healing. Under natural conditions, wound healing is affected by such factors as cell types, growth factors and several components of the extracellular matrix. Due to the complexity of the cell-to-matrix interaction, many cell based mechanisms regulating wound healing in vivo are not yet properly understood. However, the whole process can be partially simulated in vitro to determine how cells interact with the collagen scaffold in relation to such features as physico-chemical properties, matrix architecture and fiber stability. Under these conditions, cell migration into the collagen matrix can be easily assessed and causally correlated with these features. In this study, we aimed at providing a structural analysis of how NIH3T3 fibroblasts migrate and proliferate in vitro when seeded on a native type-I collagen scaffold. To this end, samples were collected at regular time intervals and analyzed by light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Through this experimental approach we demonstrate that collagen is gradually frayed into progressively thinner fibrils as fibroblasts migrate into the matrix, embrace the collagen fibers with long filopodia and form large intracellular vacuoles. A key role in this process is also played by microvesicles shed from the fibroblast plasma membrane and spread over long distances inside the collagen matrix. These observations indicate that a native type-I equine collagen provides favorable conditions for simulating collagen processing in vitro and eventually for unraveling the mechanisms controlling cell uptake and intracellular degradation.
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http://dx.doi.org/10.1002/jbm.a.35563DOI Listing
January 2016

An Innovative Pseudotypes-Based Enzyme-Linked Lectin Assay for the Measurement of Functional Anti-Neuraminidase Antibodies.

PLoS One 2015 12;10(8):e0135383. Epub 2015 Aug 12.

GSK, Research Center, Via Fiorentina 1, 53100, Siena, Italy; GSK, Peter Merian Strasse, 4056, Basel, Switzerland.

Antibodies (Ab) to neuraminidase (NA) play a role in limiting influenza infection and might help reduce the disease impact. The most widely used serological assay to measure functional anti-NA immune responses is the Enzyme-Linked Lectin Assay (ELLA) which relies on hemagglutinin (HA) mismatched virus reassortants, or detergent treated viruses as the NA source to overcome interference associated with steric hindrance of anti-HA Ab present in sera. The difficulty in producing and handling these reagents, which are not easily adapted for screening large numbers of samples, limits the routine analysis of functional anti-NA Ab in clinical trials. In this study, we produced influenza lentiviral pseudoparticles (PPs) containing only the NA antigen (NA-PPs) with a simple two-plasmid co-transfection system. NA-PPs were characterized and tested as an innovative source of NA in the NA inhibition (NI) assay. Both swine A/California/07/2009 (H1N1) and avian A/turkey/Turkey/01/2005 (H5N1) N1s within NA-PPs retained their sialidase activity and were specifically inhibited by homologous and N1 subtype-specific, heterologous sheep sera. Moreover, A/California/07/2009 N1-PPs were a better source of NA compared to whole live and detergent treated H1N1 viruses in ELLA, likely due to lack of interference by anti-HA Ab, and absence of possible structural modifications caused by treatment with detergent. This innovative assay is safer and applicable to all NAs. Taken together, these results highlight the potential of NA-PPs-based NI assays to be developed as sensitive, flexible, easy to handle and scalable serological tests for routine NA immune response analysis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0135383PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4534301PMC
May 2016

Growth Factors Cross-Linked to Collagen Microcarriers Promote Expansion and Chondrogenic Differentiation of Human Mesenchymal Stem Cells.

Tissue Eng Part A 2015 Oct 17;21(19-20):2618-28. Epub 2015 Sep 17.

1 Biomedical Laboratories, Swiss Paraplegic Research , Nottwil, Switzerland .

Tissue engineering is a field in progressive expansion and requires constant updates in methods and devices. One of the central fields is the development of biocompatible, biodegradable, and injectable scaffolds, such as collagen microcarriers. To enhance cell attachment and produce a cost-effective cell culture solution with local stimulation of cells, basic fibroblast growth factor (bFGF) or transforming growth factor-β1 (TGF-β1) was covalently immobilized on microcarriers either by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) or riboflavin/UV (RB/UV) light-mediated cross-linking. Collagen microcarriers cross-linked with bFGF or TGF-β1 were used for expansion and chondrogenic differentiation of human mesenchymal stem cells (MSCs). Evaluation methods included cell viability test, chondrogenic marker expression (aggrecan and collagen type I and type II), histological detection of proteoglycans, and immunohistochemical analysis. Cross-linking strengthened the collagen structure of the microcarriers and reduced collagenase-mediated degradation. MSCs effectively proliferated on microcarriers cross-linked with bFGF, especially by EDC/NHS cross-linking. Chondrogenic differentiation of MSCs was induced by TGF-β1 cross-linked on microcarriers, promoting gene expression and protein accumulation of aggrecan and collagen type I and type II, as well as proteoglycans. Cross-linking by RB/UV enhanced chondrogenesis more than any other group. In addition, cross-linking reduced scaffold shrinkage exerted by MSCs during chondrogenesis, a desirable feature for microcarriers if used as tissue defect filler. In conclusion, cross-linking of bFGF or TGF-β1 to collagen microcarriers supported in vitro proliferation and chondrogenesis, respectively. If translated in vivo and in clinical practice, such approach might lead a step closer to development of a cost-effective and locally acting device for cell-based therapy.
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http://dx.doi.org/10.1089/ten.TEA.2015.0029DOI Listing
October 2015

The Human Pathogen Streptococcus pyogenes Releases Lipoproteins as Lipoprotein-rich Membrane Vesicles.

Mol Cell Proteomics 2015 Aug 27;14(8):2138-49. Epub 2015 May 27.

From the ‡Novartis Vaccines and Diagnostics (a GSK company), Via Fiorentiina 1, 53100 Siena, Itlay;

Bacterial lipoproteins are attractive vaccine candidates because they represent a major class of cell surface-exposed proteins in many bacteria and are considered as potential pathogen-associated molecular patterns sensed by Toll-like receptors with built-in adjuvanticity. Although Gram-negative lipoproteins have been extensively characterized, little is known about Gram-positive lipoproteins. We isolated from Streptococcus pyogenes a large amount of lipoproteins organized in vesicles. These vesicles were obtained by weakening the bacterial cell wall with a sublethal concentration of penicillin. Lipid and proteomic analysis of the vesicles revealed that they were enriched in phosphatidylglycerol and almost exclusively composed of lipoproteins. In association with lipoproteins, a few hypothetical proteins, penicillin-binding proteins, and several members of the ExPortal, a membrane microdomain responsible for the maturation of secreted proteins, were identified. The typical lipidic moiety was apparently not necessary for lipoprotein insertion in the vesicle bilayer because they were also recovered from the isogenic diacylglyceryl transferase deletion mutant. The vesicles were not able to activate specific Toll-like receptor 2, indicating that lipoproteins organized in these vesicular structures do not act as pathogen-associated molecular patterns. In light of these findings, we propose to name these new structures Lipoprotein-rich Membrane Vesicles.
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http://dx.doi.org/10.1074/mcp.M114.045880DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4528243PMC
August 2015

Reproductive biology in Anophelinae mosquitoes (Diptera, Culicidae): Fine structure of the female accessory gland.

Arthropod Struct Dev 2015 Jul 18;44(4):378-87. Epub 2015 Apr 18.

Dipartimento per l'Innovazione nei sistemi Biologici, Agroalimentari e Forestali, Università della Tuscia, 01100 Viterbo, Italy. Electronic address:

The morphology and ultrastructure of female accessory reproductive glands of Anopheles maculipennis s.s., Anopheles labranchiae and Anopheles stephensi were investigated by light and electron microscopy. The reproductive system in these species is characterized by two ovaries, two lateral oviducts, a single spermatheca and a single accessory gland. The gland is globular and has a thin duct which empties into the vagina, near the opening of the spermathecal duct. Significant growth of the accessory reproductive gland is observed immediately after blood meal, but not at subsequent digestion steps. At ultrastructural level, the gland consists of functional glandular units belonging to type 3 ectodermal glands. The secretory cells are elongated and goblet shaped, with most of their cytoplasm and large nucleus in the basal part, close to the basement lamella. Finely fibrous electron-transparent material occupies the secretory cavity that is in contact with the end of a short efferent duct (ductule) emerging from the gland duct. The present study is the first detailed description of female accessory gland ultrastructure in Anophelinae and provides insights into the gland's functional role in the reproductive biology of these insects.
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http://dx.doi.org/10.1016/j.asd.2015.04.003DOI Listing
July 2015

Lactobacillus rhamnosus lowers zebrafish lipid content by changing gut microbiota and host transcription of genes involved in lipid metabolism.

Sci Rep 2015 Mar 30;5:9336. Epub 2015 Mar 30.

Dipartimento di Scienze della Vita e dell'Ambiente, Università Politecnica delle Marche, Ancona, Italy.

The microbiome plays an important role in lipid metabolism but how the introduction of probiotic communities affects host lipid metabolism is poorly understood. Using a multidisciplinary approach we addressed this knowledge gap using the zebrafish model by coupling high-throughput sequencing with biochemical, molecular and morphological analysis to evaluate the changes in the intestine. Analysis of bacterial 16S libraries revealed that Lactobacillus rhamnosus was able to modulate the gut microbiome of zebrafish larvae, elevating the abundance of Firmicutes sequences and reducing the abundance of Actinobacteria. The gut microbiome changes modulated host lipid processing by inducing transcriptional down-regulation of genes involved in cholesterol and triglycerides metabolism (fit2, agpat4, dgat2, mgll, hnf4α, scap, and cck) concomitantly decreasing total body cholesterol and triglyceride content and increasing fatty acid levels. L. rhamnosus treatment also increased microvilli and enterocyte lengths and decreased lipid droplet size in the intestinal epithelium. These changes resulted in elevated zebrafish larval growth. This integrated system investigation demonstrates probiotic modulation of the gut microbiome, highlights a novel gene network involved in lipid metabolism, provides an insight into how the microbiome regulates molecules involved in lipid metabolism, and reveals a new potential role for L. rhamnosus in the treatment of lipid disorders.
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http://dx.doi.org/10.1038/srep09336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4378510PMC
March 2015

Structure and membrane interactions of chionodracine, a piscidin-like antimicrobial peptide from the icefish Chionodraco hamatus.

Biochim Biophys Acta 2015 Jun 6;1848(6):1285-93. Epub 2015 Mar 6.

Department for Innovation in Biological, Agro-food, and Forest Systems, University of Tuscia, Viterbo 01100, Italy. Electronic address:

Chionodracine (Cnd) is a 22-residue peptide of the piscidin family expressed in the gills of the Chionodraco hamatus as protection from bacterial infections. Here, we report the effects of synthetic Cnd on both Psychrobacter sp. TAD1 and Escherichia coli bacteria, as well as membrane models. We found that Cnd perforates the inner and outer membranes of Psychrobacter sp. TAD1, making discrete pores that cause the cellular content to leak out. Membrane disruption studies using intrinsic and extrinsic fluorescence spectroscopy revealed that Cnd behaves similarly to other piscidins, with comparable membrane partition coefficients. Membrane accessibility assays and structural studies using NMR in detergent micelles show that Cnd adopts a canonical topology of antimicrobial helical peptides, with the hydrophobic face toward the lipid environment and the hydrophilic face toward the bulk solvent. The analysis of Cnd free energy of binding to vesicles with different lipid contents indicates a preference for charged phospholipids and a more marked binding to native E. coli extracts. Taken with previous studies on piscidin-like peptides, we conclude that Cnd first adsorbs to the membrane, and then forms pores together with membrane fragmentation. Since Cnd has only marginal hemolytic activity, it constitutes a good template for developing new antimicrobial agents.
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http://dx.doi.org/10.1016/j.bbamem.2015.02.030DOI Listing
June 2015

Antiproliferative activity of yatein isolated from Austrocedrus chilensis against murine myeloma cells: cytological studies and chemical investigations.

Pharm Biol 2015 Mar 25;53(3):378-85. Epub 2014 Nov 25.

Departamento de Acuicultura, Facultad de Ciencias del Mar, Universidad Católica del Norte , Coquimbo , Chile .

Context: Fitzroya cupressoides (Molina) I. M. Johnst. and Austrocedrus chilensis (D. Don) Pic.Serm. & Bizzarri are two Chilean Cupressaceae that are naturally resistant to biodegradation. Secondary metabolites from these species display a variety of biological activities.

Objective: To evaluate the antiproliferative activity of two lignans, a diterpene and a flavonol isolated from A. chilensis and F. cupressoides, to elucidate their cytological effects on P3X murine myeloma cells.

Materials And Methods: The antiproliferative activity of yatein, isotaxiresinol, ferruginol, and isorhamnetin was evaluated in vitro using the MTT assay. The effect of yatein at the cellular level, due to its high antiproliferative activity was evaluated. P3X cells treated for 24 h with 12.5 and 25 µg/mL of yatein were also examined at the cytological level using immunofluorescence and scanning and transmission electron microscopy.

Results: Yatein, a lignan isolated from A. chilensis, potentially inhibited P3X murine myeloma cell proliferation, resulting in approximately 75% cell death in response to a 25 µg/mL treatment with the lignan. P3X cells lost membrane integrity at the nuclear and cytoplasmic levels, including organelles, in response to yatein treatment (12.5 µg/mL), and we observed changes in the cytoplasmic organization and distribution of microtubules. The other compounds tested had low activity.

Discussion And Conclusions: Yatein is a lignan precursor of podophyllotoxin, a key agent in anticancer drugs. Due to its structural similarities to podophyllotoxin, yatein could have similar cytoplasmic target(s), such as the microtubular apparatus. These findings suggest that yatein may be of potential pharmacological interest and warrants further investigation in human cell lines.
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http://dx.doi.org/10.3109/13880209.2014.922588DOI Listing
March 2015

SpyAD, a moonlighting protein of group A Streptococcus contributing to bacterial division and host cell adhesion.

Infect Immun 2014 Jul 28;82(7):2890-901. Epub 2014 Apr 28.

Novartis Vaccines and Diagnostics Srl, Siena, Italy.

Group A streptococcus (GAS) is a human pathogen causing a wide repertoire of mild and severe diseases for which no vaccine is yet available. We recently reported the identification of three protein antigens that in combination conferred wide protection against GAS infection in mice. Here we focused our attention on the characterization of one of these three antigens, Spy0269, a highly conserved, surface-exposed, and immunogenic protein of unknown function. Deletion of the spy0269 gene in a GAS M1 isolate resulted in very long bacterial chains, which is indicative of an impaired capacity of the knockout mutant to properly divide. Confocal microscopy and immunoprecipitation experiments demonstrated that the protein was mainly localized at the cell septum and could interact in vitro with the cell division protein FtsZ, leading us to hypothesize that Spy0269 is a member of the GAS divisome machinery. Predicted structural domains and sequence homologies with known streptococcal adhesins suggested that this antigen could also play a role in mediating GAS interaction with host cells. This hypothesis was confirmed by showing that recombinant Spy0269 could bind to mammalian epithelial cells in vitro and that Lactococcus lactis expressing Spy0269 on its cell surface could adhere to mammalian cells in vitro and to mice nasal mucosa in vivo. On the basis of these data, we believe that Spy0269 is involved both in bacterial cell division and in adhesion to host cells and we propose to rename this multifunctional moonlighting protein as SpyAD (Streptococcus pyogenes Adhesion and Division protein).
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http://dx.doi.org/10.1128/IAI.00064-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4097626PMC
July 2014

Degradation of silicone rubber causes Provox 2 voice prosthesis malfunctioning.

J Voice 2014 Mar 4;28(2):250-4. Epub 2013 Dec 4.

Department of Sensory Organs, University "Sapienza" of Rome, Rome, Italy.

Objective/hypothesis: Provox 2 voice prosthesis requires periodic replacement due to biofilm proliferation which causes malfunctioning of the valve. The aim of this study was to show that Provox 2 voice prosthesis malfunctioning is due not only to valve obstruction caused by biofilm but also to the silicone variations.

Design Methods: Prospective study on the malfunction of Provox2 voice prostheses.

Methods: Through photographic and electron microscopic assessment, the authors studied nine Provox 2 voice prostheses, which were removed due to malfunctioning.

Results: Findings revealed that the silicone undergoes a degenerative process, thus causing the surface to become rough, deformed, swollen, and translucent. Furthermore, electron microscopy confirmed the presence of immune system cells and biofilm on the prosthesis surface and their role in creating a structural nonhomogenous structure in the silicone, which is deformed due to the presence of "crests" caused by material degeneration.

Conclusion: The degenerative process of the silicone seems to be related to the oxygen present in the trachea and esophagus and to the production of oxygen-free radicals on the biofilm's part and the immune system.
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http://dx.doi.org/10.1016/j.jvoice.2013.08.012DOI Listing
March 2014

Methyl carnosate, an antibacterial diterpene isolated from Salvia officinalis leaves.

Nat Prod Commun 2013 Apr;8(4):429-30

Department for the Innovation in Biological, Agro-food and Forest systems, Laboratory of Plant Cytology and Biotechnology, Tuscia University, Largo dell'Università blocco D, 01100 Viterbo, Italy.

Ethanolic extracts of Salvia officinalis leaves demonstrated antibacterial activity against Bacillus cereus. Fractionation of the extracts led to the isolation of the most active antibacterial compound, which, from spectroscopic and LC-MS evidence, was proved to be the diterpene, methyl carnosate.
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April 2013

In vitro toxicity of silver nanoparticles to kiwifruit pollen exhibits peculiar traits beyond the cause of silver ion release.

Environ Pollut 2013 Aug 20;179:258-67. Epub 2013 May 20.

Dipartimento BiGeA, Università di Bologna, Via Irnerio 42, 40126 Bologna, Italy.

The vast use of silver nanoparticles (AgNPs) mandates thorough investigation of their impact on biosystems at various levels. The cytotoxicity of PVP coated-AgNPs to pollen, the aploid male gametophyte of higher plants, has been assessed here for the first time. The negative effects of AgNPs include substantial decreases in pollen viability and performance, specific ultrastructural alterations, early changes in calcium content, and unbalance of redox status. Ag⁺ released from AgNPs damaged pollen membranes and inhibited germination to a greater extent than the AgNPs themselves. By contrast, the AgNPs were more potent at disrupting the tube elongation process. ROS deficiency and overproduction were registered in the Ag⁺- and AgNP-treatment, respectively. The peculiar features of AgNP toxicity reflected their specific modes of interaction with pollen surface and membranes, and the dynamic exchange between coating (PVP) and culture medium. In contrast, the effects of Ag⁺ were most likely induced through chemical/physicochemical interactions.
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http://dx.doi.org/10.1016/j.envpol.2013.04.021DOI Listing
August 2013

Is Montgomery tracheal Safe-T-Tube clinical failure induced by biofilm?

Otolaryngol Head Neck Surg 2013 Aug 6;149(2):269-76. Epub 2013 May 6.

Department of Sensory Organs, Sapienza University of Rome, Italy.

Objectives: Montgomery Safe-T-Tube deterioration and early biofilm colonization may explain the discomfort claimed by many patients and clinical failures. The aim of the study was to analyze the deterioration of Montgomery Safe-T-Tube morphological and mechanical properties in vivo in 16 patients by using microbiological methods, optical and electron microscopy, and engineering tests.

Study Design: Prospective controlled study at a single medical center.

Setting: University hospital.

Subjects And Methods: The study, conducted from April 2007 to February 2012 at the "Sapienza" University of Rome, was designed to collect 2 Montgomery Safe-T-Tubes from each patient. The first was removed 3 to 15 days after insertion (group A) and the second at least 90 days after (group B). Specimens underwent microbiologic assays, electron microscopic analysis, immunocytologic analysis, and mechanical tests.

Results: Microorganisms were not isolated in 2 group A cases (12%), whereas they were in all group B cases. Biofilm was identified in 11 of 16 (69%) group A samples and in 16 of 16 (100%) group B samples (P = .0149) using scanning electron microscopy. Immunohistochemistry showed monocyte-granulocyte line cells producing interleukin-1β on the external surfaces of Montgomery Safe-T-Tubes. The tensile test showed that the wear related to the longer period of use makes Montgomery Safe-T-Tubes more rigid than newer ones.

Conclusion: Early biofilm colonization takes place in Montgomery Safe-T-Tubes in most cases. The mechanical decay could be justified in part by the destructive biofilm activity and by the release of inflammatory effectors and enzymes.
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http://dx.doi.org/10.1177/0194599813488752DOI Listing
August 2013

The stimulation of dendritic cells by amyloid beta 1-42 reduces BDNF production in Alzheimer's disease patients.

Brain Behav Immun 2013 Aug 8;32:29-32. Epub 2013 Apr 8.

Department of Clinical and Behavioral Neurology, IRCCS Santa Lucia Foundation, Via Ardeatina 306, 00179 Rome, Italy.

Dendritic cells (DCs), the main actors of immune responses and inflammation, may play a role in Alzheimer's disease (AD). Recent studies demonstrate that monocyte-derived DCs (MDDCs), generated in vitro in the presence of amyloid β1-42 peptide (Aβ1-42), show a functional alteration and an increased production of inflammatory molecules. Accordingly, MDDCs from AD patients show a more pronounced pro-inflammatory profile than DCs obtained from control subjects. In this study, we aimed at further investigating DC role in AD. Thus, we analyzed the in vitro effect of Aβ1-42 treatment on already differentiated DCs from AD patients, as compared to control subjects. We found that Aβ1-42 significantly decreases the expression of brain-derived neurotrophic factor (BDNF) in DCs derived from AD patients but not from control subjects. Thus, possibly due to their Aβ-induced reduction of neurotrophic support to neurons, DCs from AD patients might contribute to brain damage by playing a part in Aβ-dependent neuronal toxicity.
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http://dx.doi.org/10.1016/j.bbi.2013.04.001DOI Listing
August 2013

Is biofilm the cause of chronic otitis externa?

Laryngoscope 2011 Dec;121(12):2626-33

Department of Otorhinolaryngology, Audiology and Phoniatrics G. Ferreri ,University Sapienza, Rome, Italy.

Objectives/hypothesis: This study was undertaken in two phases. In the first phase, we considered patients affected by chronic external otitis treated either by chemical ear peeling (CEP) or by antibiotic/steroid treatment to compare the clinical and microbiological outcomes. In the second phase, we compared the microscopic findings observed in the CEP samples of patients affected by chronic otitis externa's acute exacerbation or by acute otitis externa to demonstrate the role of biofilm in the pathogenesis of chronic otitis externa.

Study Design: Prospective, double-blind, controlled study.

Methods: In phase 1 we compared clinical and microbiological data collected from two groups of 25 patients with chronic otitis externa treated by CEP or by conventional antibiotic/steroid treatment. In phase 2 we compared the results of the optical and electron microscopic analysis of specimens obtained by performing CEP in two groups of patients (25 with chronic otitis externa exacerbation and 15 with acute otitis externa).

Results: In phase 1 the disease control rate yielded markedly better results when treated with CEP. In phase 2 biofilms were identified in 23 of the 25 patients with chronic otitis externa exacerbation (92%) and in only three acute external otitis cases (20%).

Conclusions: CEP is a simple and effective method for the treatment of chronic external otitis. The removal of the bacterial biofilm has a high correlation with a long-term clinical remission.
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http://dx.doi.org/10.1002/lary.22348DOI Listing
December 2011

A naturally occurring single-residue mutation in the translocator domain of Neisseria meningitidis NhhA affects trimerization, surface localization, and adhesive capabilities.

Infect Immun 2011 Nov 15;79(11):4308-21. Epub 2011 Aug 15.

Novartis Vaccines and Diagnostics, Via Fiorentina, 1, 53100 Siena, Italy.

Neisseria meningitidis NhhA (Neisseria hia/hsf homologue A) is an oligomeric outer membrane protein belonging to the family of trimeric autotransporter adhesins. NhhA mediates the interaction of N. meningitidis with human epithelial cells and components of the extracellular matrix. The recombinant protein is able to induce bactericidal antibodies and hence has also been considered a potential vaccine candidate. In this study, we analyzed the production of NhhA in a large panel of N. meningitidis strains belonging to different serogroups and clonal complexes. We found that trimeric NhhA was produced at different levels by the various strains tested. In some strains belonging to the clonal complex ST41/44, the protein is detectable only as a monomer. Sequencing of the nhhA gene and generation of complementing strains in different genetic backgrounds have proved that a single mutation (Gly to Asp) in the translocator domain affected both trimerization and surface localization of NhhA. In vitro infection assays showed that this mutation impairs meningococcal NhhA-mediated adhesion, suggesting that strains carrying the mutation may rely on different strategies or molecules to mediate interaction with host cells. Finally, we demonstrated that N. meningitidis ST41/44 strains producing the mutated form did not induce killing mediated by NhhA-specific bactericidal antibodies. Our data help to elucidate the secretion mechanisms of trimeric autotransporters and to understand the contribution of NhhA in the evolutionary process of host-Neisseria interactions. Also, they might have important implications for the evaluation of NhhA as a vaccine candidate.
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http://dx.doi.org/10.1128/IAI.00198-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3257927PMC
November 2011