Cerium oxide nanoparticles reduce the accumulation of autofluorescent deposits in light-induced retinal degeneration: Insights for age-related macular degeneration.

Accumulation of lipofuscin deposits in the retinal pigment epithelium (RPE) is one of the main events involved in age-related macular degeneration and its increase together with RPE dysfunction, blood retinal barrier disruption and photoreceptors death progressively leads to blindness. Lipofuscin is the main autofluorescent (AF) component of the retina and therapies to counteract its deposition are a main goal to be achieved, since effective treatments have not yet been identified. Here, we first investigated the spatio-temporal pattern of AF deposits accumulation in the light-damage model of age-related macular degeneration. Afterward, we tested the ability of cerium oxide nanoparticles, a well known anti-oxidant agent, to counteract AF granules accumulation. The treatment was performed both before and after the induction of the degeneration. AF granules were quantified by confocal microscopy on whole mounted retinas. We demonstrated that the acute light-damage increases the accumulation of AF deposits in the hot spot retina in terms of number of granules and percentage of occupied area, with a peak 7 days after the exposure. Remarkably, cerium oxide nanoparticles showed a strong efficacy in preventing the formation of AF deposits when they were injected 3 days before light exposure. Moreover, when the treatment was performed 7 days after light exposure, nanoceria activity was found to be effective also in reducing the amount of the AF granules still deposited up to 60 days. These important results represent the very first evidence about the ability of cerium oxide nanoparticles to counteract AF deposits accumulation in retinal degeneration, laying the foundations for the development of a new therapy possibly targeting lipofuscin in AMD.

Retinal long term neuroprotection by Cerium Oxide nanoparticles after an acute damage induced by high intensity light exposure.

Cerium Oxide nanoparticles are antioxidant agents with autoregenerative radical scavenging activities, effective in preventing degeneration of photoreceptors of an albino rat when intravitreally injected prior to exposure to high intensity light. In this study, we performed a post injury administration of nanoceria and a long term analysis of their neuroprotective properties in order to better simulate the therapeutic treatment as it is carried out on patients with age related macular degeneration, and while photoreceptor degeneration is ongoing. We also injected nanoceria labelled with fluorescein isothiocianate in order to analyze their persistence after a single administration in a damaged retina and to investigate how long they both maintain their neuroprotective properties and where they localize in the retina. We demonstrated that after a single intravitreal injection, nanoceria remained in the retina for a long time and retained their neuroprotective properties. All these data form excellent bases for future clinical applications.

Electronic structure investigation of biphenylene films.

Photoelectron Spectroscopy (PS) and Near-Edge X-ray Absorption Fine Structure (NEXAFS) spectroscopy have been used to investigate the occupied and empty density of states of biphenylene films of different thicknesses, deposited onto a Cu(111) crystal. The obtained results have been compared to previous gas phase spectra and single molecule Density Functional Theory (DFT) calculations to get insights into the possible modification of the molecular electronic structure in the film induced by the adsorption on a surface. Furthermore, NEXAFS measurements allowed characterizing the variation of the molecular arrangement with the film thickness and helped to clarify the substrate-molecule interaction.

Development of molecularly imprinted polymeric nanofibers by electrospinning and applications to pesticide adsorption.

Novel polystyrene-based molecularly imprinted polymer nanofibers were synthesized through the electrospinning technique. The molecularly imprinted polymers were prepared using a non-covalent approach and atrazine as template. For comparison, nonimprinted polymer nanofibers were also synthesized. The morphology of the synthesized nanofibers was characterized using scanning electron microscopy. The adsorption of pesticides, atrazine, atrazine desisopropyl, atraton, carboxin, linuron, and chlorpyrifos was studied under equilibrium (batch) conditions. To describe the adsorption capability of the synthesized polymers, Langmuir and Freundlich models were used. The Freundlich model provided a better mathematical approximation of the sorption characteristic for polymers nanofibers. To evaluate the adsorption capacity in the presence of interferents experiments on river water samples spiked with a mixture of six pesticides were also performed. The results obtained for the highest concentration levels investigated, show a greater amount of pesticide adsorbed on molecularly imprinted polymers and non-imprinted polymers compared to those obtained using commercial stationary phases used as reference.

Why do acute myocardial infarction patients not call an ambulance? An interview with patients presenting to hospital with acute myocardial infarction symptoms.

BACKGROUND: Studies from overseas indicate that patients with acute myocardial infarction (AMI) symptoms often fail to use the emergency services as recommended, thereby depriving themselves from life-saving treatment in case of cardiac arrest and delaying the time to myocardial reperfusion in the presence of a coronary occlusion. AIMS: To compare patients brought in by ambulance to those not brought in by ambulance and to question why some patients do not use the emergency services when presenting to hospital with AMI symptoms. METHODS: Prospective interview and follow up of consecutive patients presenting with AMI symptoms to the emergency department of a tertiary hospital in a metropolitan area within a 1-month period. RESULTS: Of the 215 patients presenting to the emergency department, 113 (53%) arrived by private transportation. Sixty (53%) of these felt their symptoms did not warrant calling the ambulance, 17 (15%) had first consulted their general practitioner. The private transport group accounted for 28% of documented AMI. CONCLUSIONS: A large proportion of patients with AMI symptoms refrain from calling the emergency services because they do not consider themselves critically ill. Education programmes appear to be warranted because more appropriate use of emergency services will save lives.

Ozone adsorption on carbon nanotubes: the role of Stone-Wales defects.

First-principles calculations within the density functional theory have been performed in order to investigate ozone adsorption on carbon nanotubes. Particular emphasis is placed on the effects of Stone-Wales-like defects on the structural and electronic properties of (i) ideal tubes and (ii) tubes in the presence of ozone. Our results show that structural deformations induced on the pure carbon nanotubes by Stone-Wales defects are similar, as expected, to those induced on graphite; for the (10,0) tube, the semiconducting character is kept, though with a small reduction of the band gap. As for the ozone adsorption, the process on ideal nanotubes is most likely physisorption, though slightly stronger if compared to other previously studied molecules and consistent with the strong oxydizing nature of O(3). However, when ozone adsorbs on Stone-Wales defects, a strong chemisorption occurs, leading to relevant structural relaxations and to the formation of a CO covalent bond; this is consistent with experimental observations of CO functional groups, as well as of the liberation of CO gas phase and of the formation of C vacancies, thus explaining the consumption of the nanotube film upon ozone exposure.

Electronic structure of crystalline copper phthalocyanine.

The electronic structure of copper-phthalocyanine (CuPc) has been studied both experimentally and theoretically. Experiments have been performed on alpha and beta crystalline phases, using photoemission spectroscopy to probe core levels and valence band spectra. Different photon energies have been used, in order to probe different sample depths. Only minor differences have been observed in the experimental data on the two different phases, except for a small charge effect on the beta phase crystal. First-principles calculations have been performed using the density functional for molecular and three-dimensional periodic solids (Dmol(3)) code on both the single CuPc molecule and the beta phase, allowing the identification of the different atomic and angular contributions to the experimental density of states. In particular, the highest occupied molecular level is mainly due to Cu and N states. The comparison between theoretical data obtained for the CuPc in the beta phase and in the single molecule shows that the interchain interaction between the molecules is negligible, whereas slightly stronger intrachain interactions occur.

A parathyroid-hormone-related-protein (PTH-rP)-specific cytotoxic T cell response induced by in vitro stimulation of tumour-infiltrating lymphocytes derived from prostate cancer metastases, with epitope peptide-loaded autologous dendritic cells and low-dose IL-2.

Bone metastases are one of the most common events in patients with prostate carcinoma. PTH-rP, a protein produced by prostate carcinoma and other epithelial cancers, is a key agent for the development of bone metastases. A PTH-rP-derived peptide, designated PTR-4 was identified, which is capable to bind HLA-A2.1 molecules and to generate PTH-rP-specific cytotoxic T cell (CTL) lines from healthy HLA-A2.1(+) individual peripheral-blood-mononuclear-cells (PBMC). In this model, we investigated the in vitro possibility of generating an efficient PTH-rP specific CTL response by cyclical stimulations with IL-2 and PTR-4 peptide-pulsed autologous dendritic cells (DC), of HLA-A2.1(+) tumour infiltrating lymphocytes (TIL) derived from a patient with metastatic prostate carcinoma. A T cell line generated in this way (called TM-PTR-4) had a CD3(+), CD5(+), CD4(-), CD8(+), CD45(Ro+), CD56(-) immunophenotype and a HLA-A2.1 restricted cytotoxic activity to PTR-4-peptide pulsed CIR-A2 (HLA-A2.1(+)) target cells, PTH-rP(+)/HLA-A2.1(+) CIR-A2 transfected with PTH-rP gene, prostate carcinoma LNCaP cells, and autologous metastatic prostate cancer cells (M-CaP). These lymphocytes were not cytotoxic to HLA-A2.1(+) targets not producing PTH-rP, such as peptide-unpulsed CIR-A2 and colon carcinoma SW-1463, cell lines. Our results provide evidence that PTR-4 peptide-pulsed autologous DC may break the tolerance of human TIL against the autologous tumour by inducing a PTH-rP-specific CTL immune reaction. In conclusion PTR-4 peptide-pulsed autologous DC may be a promising approach for vaccine-therapy and antigen-specific CTL adoptive immunotherapy of hormone-resistant prostrate cancer.

A novel mimetic antigen eliciting protective antibody to Neisseria meningitidis.

Molecular mimetic Ags are of considerable interest as vaccine candidates. Yet there are few examples of mimetic Ags that elicit protective Ab against a pathogen, and the functional activity of anti-mimetic Abs has not been studied in detail. As part of the Neisseria meningitidis serogroup B genome sequencing project, a large number of novel proteins were identified. Herein, we provide evidence that genome-derived Ag 33 (GNA33), a lipoprotein with homology to Escherichia coli murein transglycosylase, elicits protective Ab to meningococci as a result of mimicking an epitope on loop 4 of porin A (PorA) in strains with serosubtype P1.2. Epitope mapping of a bactericidal anti-GNA33 mAb using overlapping peptides shows that the mAb recognizes peptides from GNA33 and PorA that share a QTP sequence that is necessary but not sufficient for binding. By flow cytometry, mouse antisera prepared against rGNA33 and the anti-GNA33 mAb bind as well as an anti-PorA P1.2 mAb to the surface of eight of nine N. meningitidis serogroup B strains tested with the P1.2 serosubtype. Anti-GNA33 Abs also are bactericidal for most P1.2 strains and, for susceptible strains, the activity of an anti-GNA33 mAb is similar to that of an anticapsular mAb but less active than an anti-P1.2 mAb. Anti-GNA Abs also confer passive protection against bacteremia in infant rats challenged with P1.2 strains. Thus, GNA33 represents one of the most effective immunogenic mimetics yet described. These results demonstrate that molecular mimetics have potential as meningococcal vaccine candidates.

Mimicking the nicotinic receptor binding site by a single chain Fv selected by competitive panning from a synthetic phage library.

We have developed a novel competitive method to select from a phage display library a single chain Fv which is able to mimic the alpha-bungarotoxin binding site of the muscle nicotinic receptor. The single chain Fv was selected from a large synthetic library using alpha-bungarotoxin-coated magnetic beads. Toxin-bound phages were then eluted by competition with affinity purified nicotinic receptor. Recognition of the toxin by the anti-alpha-bungarotoxin single chain Fv was very similar to that of the receptor, such as indicated by the epitope mapping of alpha-bungarotoxin through overlapping synthetic peptides. Moreover, several positively charged residues located in the toxin second loop and in the C-terminal region were found to be critical, to a similar extent, for toxin recognition by the single chain Fv and the receptor. However, although the anti-alpha-bungarotoxin single chain Fv seems to mimic the toxin binding site of the nicotinic receptor, it does not bind other nicotinic agonists or antagonists. Our results suggest that competitive selection of anti-ligand antibody phages can allow the production of receptor-mimicking molecules directly and exclusively targeted at one specific ligand. Since physiologically and pharmacologically different ligands can produce opposite effects on receptor functions, such selective ligand decoys can have important therapeutic applications.

Mimotopes of the nicotinic receptor binding site selected by a combinatorial peptide library.

Peptide libraries allow selecting new molecules, defined as mimotopes, which are able to mimic the structural and functional features of a native protein. This technology can be applied for the development of new reagents, which can interfere with the action of specific ligands on their target receptors. In the present study we used a combinatorial library approach to produce synthetic peptides mimicking the snake neurotoxin binding site of nicotinic receptors. On the basis of amino acid sequence comparison of different alpha-bungarotoxin binding receptors, we designed a 14 amino acid combinatorial synthetic peptide library with five invariant, four partially variant, and five totally variant positions. Peptides were synthesized using SPOT synthesis on cellulose membranes, and binding sequences were selected using biotinylated alpha-bungarotoxin. Each variant position was systematically identified, and all possible combinations of the best reacting amino acids in each variant position were tested. The best reactive sequences were identified, produced in soluble form, and tested in BIACORE to compare their kinetic constants. We identified several different peptides that can inhibit the binding of alpha-bungarotoxin to both muscle and neuronal nicotinic receptors. Peptide mimotopes have a toxin-binding affinity that is considerably higher than peptides reproducing native receptor sequences.

Epitope focus, clonal composition and Th1 phenotype of the human CD4 response to the secretory mycobacterial antigen Ag85.

Lymphoproliferation of healthy donors was tested against mycobacterial antigens (PPD, Ag85, Ag85 peptides). All PPD responders recognized the secretory antigen Ag85 and the peptide specificity for Ag85B was defined. Peptide 91-108 was recognized by 85% of donors. In addition, all CD4 T cell lines generated from 12 donors against PPD or Ag85 responded to 91-108. When this peptide was used to generate T cell lines, the cells responded also to tuberculins from atypical mycobacterial species. Thus the cross-reactive peptide behaved as quasi-universal. The analysis of TCR-BV gene usage by cell lines showed that most Ag85-specific T cells correspond to 91-108-specific clonotypes. Intracytoplasmic staining of cell lines after phorbol myristate acetate stimulation resulted in dominance of interferon-gamma (IFN-gamma)-IL-4 double-positive cells, whereas antigen stimulation resulted in production of IFN-gamma only. The data show that peptide 91-108 is the major focus of the CD4 response to mycobacterial antigens in peripheral blood mononuclear cells and in T cell lines from PPD responders.

Selection of catalytically active biotin ligase and trypsin mutants by phage display.

Phage display has been shown to facilitate greatly the selection of polypeptides with desired properties by establishing a direct link between the polypeptide and the gene that encodes it. However, selection for catalytic activities displayed on phage remains a challenge, since reaction products diffuse away from the enzyme and make it difficult to recover catalytically active phage-enzymes. We have recently described a selection methodology in which the reaction substrate (and eventually the reaction product) is anchored on calmodulin-tagged phage-enzymes by means of a calmodulin binding peptide. Phage displaying a catalytic activity are physically isolated by means of affinity reagents specific for the product of reaction. In this study, we investigated the efficiency of selection for catalysis by phage display, using a ligase (the Escherichia coli biotin ligase BirA) and an endopeptidase (the rat trypsin His57--> Ala mutant) as model enzymes. These enzymes could be displayed on phage as fusion proteins with calmodulin and the minor coat protein pIII. Both the display of functional enzyme and the efficiency of selection for catalysis were significantly improved by using phage vectors, rather than phagemid vectors. In model selection experiments, phage displaying BirA were consistently enriched (between 4-fold and 800-fold) per round of panning, relative to negative controls. Phage displaying the trypsin His57-->Ala mutant, a relatively inefficient endopeptidase which cleaves a specific dipeptide sequence, were enriched (between 15-fold and 2000-fold), relative to negative controls. In order to improve the catalytic properties of the trypsin His57-->Ala mutant, we constructed a combinatorial phage display library of trypsin mutants. Selection of catalytically active phage-enzymes was evidentiated by increasing phage titres at the different rounds of panning relative to negative control selections, but mutants with catalytic properties superior to those of trypsin His57-->Ala mutant could not be isolated. The results obtained provide evidence that catalytic activities can be recovered using phage display technology, but stress the importance of both library design and stringent biopanning conditions for the recovery of novel enzymes.

Highly fluorescent protein labeling using dendritic peptide derivatives.

Non-specific fluorescent dyes and photosensitizers are routinely used in clinical practice for the photodetection and photoablation of superficial lesions. Future applications in photomedicine are likely to rely on the selective delivery of photoactive compounds to diseased areas, using specific targeting agents such as antibodies. This fact underlines the need for methods that allow the chemically defined conjugation of several photoactive molecules to a single protein 'vehicle', with full retention of binding affinity. Here, we present methods for the site-specific fluorescent labeling of proteins using dendritic peptides, which had been chemically modified with multiple molecules of fluorescein. Branched peptide derivatives can be stably conjugated to proteins either by reaction with suitable free reactive groups or by using the high-affinity non-covalent interaction between calmodulin and a specific binding peptide. Chemical modification of proteins with one, two or four molecules of fluorescein resulted in a proportional increase in protein fluorescence.

Natural analogue peptides of an HIV-1 GP120 T-helper epitope antagonize response of GP120-specific human CD4 T-cell clones.

Neutralizing antibodies and specific cytotoxic T lymphocytes (CTL) may contribute to controlling viral spread, and ideally, to virus clearance in HIV infection. Both effector mechanisms depend on specific CD4 T-helper (Th) cells. Nevertheless, HIV hypervariability facilitates appearance of escape mutants for antibodies and for CTL responses. Here we also show that natural mutations (i.e., from sequences of different HIV strains) in an immunodominant Th epitope recognized by human CD4 clones specific for the envelope glycoprotein gp120 escape CD4 T-cell recognition. Furthermore, several natural analogue peptides exert an antagonistic function by inhibiting proliferative response of T cells specific to gp120 with a wild-type sequence. If similar events occur in vivo, they may represent an additional escape mechanism for HIV. In fact, antagonism for CD4 Th response may occur during superinfection with a different strain, or with the appearance of a variant carrying a mutated antagonistic sequence. In both cases, impaired Th cell function could lead to reduced immune control of HIV infection by interfering with CTL and antibody response.

Antagonistic activity of HIV-1 T helper peptides flanked by an unrelated carrier protein.

Antagonism is the ability of a modified antigenic peptide (altered peptide ligand, APL) to prevent CD4 T cell activation by the original peptide. Here we show that antagonistic activity can be conferred to peptides of HIV envelope glycoprotein gp120 and reverse transcriptase p66 by adding flanking polypeptide sequences at the C or at the N terminus by genetic engineering, rather than by introducing substitutions by synthesis. The glutathione S-transferase (GST)-peptide system has been used to produce molecules that display the peptide at the appropriate end of the GST carrier. When the gp120 peptide 191-205 (pep24) was expressed at the C terminus of GST (GST-24), antigenicity of specific human CD4 T cells was maintained. In contrast, when the peptide was expressed at the N terminus of GST (24-GST), antigenicity was abolished and antagonistic activity was introduced. Similar results were obtained with a p66-derived peptide at the C terminus of the GST carrier. Antagonism was (1) specific; proliferation of a CD4 T cell line from the same donor responding to the envelope glycoprotein of another retrovirus, HTLV-1, was not affected; (2) reversible; proliferative response was rescued in T cells exposed to antigen-presenting cells (APC) pulsed with the antagonist; (3) dominant; T cells cultured with APC pulsed with the agonist and with APC pulsed with the antagonist did not proliferate. The carrier could be cleaved by proteolysis while the antagonistic activity was preserved. Thus a minimal sequence that confers antagonistic activity can be engineered or synthesized with peptides to antagonize undesired CD4 responses as an alternative to the use of APL.

On the structural stability of a small bioactive peptide of potential use in biotechnology.

A tridecapeptide with the sequence CCEICCNPACFGC has been synthesized to reproduce the active moiety of a heat stable enterotoxin from Vibrio cholerae. The proton NMR analysis indicates, for the active synthetic fragment, a rigid secondary structure stabilised by three disulfide bridges. Such a rigid peptide, suitably detoxified and activated, could be a good candidate to be used as a carrier for linear bioactive peptides or other functional groups.

Antigenicity and immunogenicity of the V3 domain of HIV type 1 glycoprotein 120 expressed on the surface of Streptococcus gordonii.

Five different V3 domains of HIV-1 gp120 were expressed on the surface of the gram-positive bacterium Streptococcus gordonii, a model live vector for vaccine delivery. Sera of HIV-1-infected individuals and human monoclonal antibodies specifically recognized the gp120 sequences on the bacterial surface. Recombinant V3 from the reference HIV-1 strain MN was also shown to retain a conformation that allowed reaction with a conformation-specific monoclonal antibody. A V3-specific serum antibody response was detected in mice immunized both by subcutaneous injection and by vaginal colonization. V3-specific IgG2a antibodies, suggestive of a Th1 response, were found in the sera of mice colonized by recombinant bacteria.

A strategy for the isolation of catalytic activities from repertoires of enzymes displayed on phage.

We have aimed at developing a general methodology for the isolation of enzymatic activities from large repertoires of protein displayed on the surface of a filamentous phage. When selecting for protein binders by phage display, phage particles with suitable specificities are physically isolated by affinity capture and amplified by bacterial infection. Selection for catalysis mediated by enzymes displayed on filamentous phage is more difficult, as reaction products (which represent the biochemical memory of the reaction catalysed by the phage particle) diffuse away after the reaction is complete. We reasoned that if we were able to anchor the reaction products on the phage surface, the catalytically active phages could then be physically isolated using specific anti-product affinity reagents. We achieve the conditional anchoring of reaction substrates and products on phage by displaying enzyme-calmodulin chimeric proteins on filamentous phage as gene III fusions. Such phage particles can be targeted in a stable fashion (koff<10(-4) s(-1)) by chemical derivatives of a calmodulin-binding peptide. The peptide-phage complexes are stable in purification procedures such as capture with magnetic beads and polyethylene glycol precipitation, and can be conditionally dissociated by addition of calcium chelators. Glutathione-S-transferase and an endopeptidase were used in model selection experiments to demonstrate that it is possible to isolate catalytic activities from calmodulin-tagged enzymes displayed on filamentous phage, with enrichment factors >50 per round of selection.

CD and NMR structural characterization of ceratotoxins, natural peptides with antimicrobial activity.

Antibacterial properties of the secretion from the female reproductive accessory glands of medfly Ceratitis capitata are mostly ascribed to the presence of two peptides, ceratotoxin A and B, which exhibit a strong activity against gram-positive and gram-negative bacterial strains, and show sequence and function homology with cecropins, melittin, and magainins. CD experiments performed in different solvents indicate the presence of a significant content of helical structures in organic solvent. Two-dimensional nmr results for ceratotoxin A in methanol show a helical behavior for the 8-25 region of the peptide. A ramachandran classification of each residue for the structures obtained from distance geometry calculations lead to the definition of four structural families in which the central segment 10-19 is always helical and differences refer to residues 8-9 and 19-23. A sequence analysis of the two ceratotoxins and a systematic search on the protein data bank revealed the occurrence of a KX-hydrophobic-hydrophobic-P motif that seems to be important for helix stabilization.