FITC-labeled Peptide Research Articles

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Classification of allele overlapping peptide-binding specificities has become an important issue in vaccine design with direct implications concerning population coverage. In this study, we used a combinatoric approach not only to discover epitope/HLA combinations suitable to be utilized in peptide binding assays, but also to evaluate the degree of overlapping peptide binding capacities among the alleles tested. A set of 20 different FITC-labeled reference peptides was applied in combination with over 50 sHLA alleles to determine their binding capabilities. During the process, each peptide candidate was incubated with activated sHLA, and the peptide/HLA interaction was monitored over time using Fluorescence Polarization (FP). This assay combination allows the direct measurement of the ratio between free and bound labeled ligand in solution without any separation steps. The technique of FP is based on the principal that if a fluorescent-labeled peptide binds to the sHLA molecule of higher molecular weight, polarization values will increase due to the slower molecular rotation of the bound probe. Data analysis showed that the majority of peptide candidates were capable of binding with various degrees of overlap, reflecting the ability of HLA class I alleles to share the binding of identical peptides. The approach resulted in the identification of over 40 new peptide/HLA combinations applicable for epitope discovery and characterization. However, comparison to published binding motifs and supertype classifications showed several inconsistencies indicating the need of more refinement in their definition. Our approach suggests that broadly cross-reactive peptide epitopes can be identified and greatly enhance the effectiveness of future vaccine designs by providing a more extensive population coverage. They will also have a profound benefit in the understanding of antigenic peptide selection, degeneration and discrimination during T-cell mediated immune responses.

Entry of a cationic lytic-type peptide into the cytoplasm via endocytosis-dependent and -independent pathways in human glioma U251 cells

Cationic lytic-type peptides have been studied for clinical application in various infections and cancers. This study aimed to determine the functions of our specially designed lytic peptide. To investigate the functional mechanism at the cell membrane level, we used giant unilayer vesicles (GUVs) mimicking cell membranes. In GUVs treated with FITC-labeled lytic peptide (lytic-FITC), fluorescence increased in a time-dependent manner. However, no inner fluorescence was detected in GUVs treated with lytic peptide and calcein. Next, distribution of lytic-FITC peptide on the cell membrane and in the cytoplasm was examined in a living human glioma U251 cell line. In the immunocytochemical study, some lytic peptide stains colocalized with early endosome antigen protein 1 (EEA-1). In cells treated with lytic peptide, the immunofluorescence intensity of lytic peptide increased in a concentration and treatment time-dependent manner. Cytotoxic activity of lytic peptide decreased after pretreatment with the endocytosis inhibitors cytochalasin D, chlorpromazine and amiloride. These findings suggest that lytic peptide exerts cytotoxic activity after cellular uptake via an endocytosis pathway. In conclusion, the influx mechanism of lytic peptide was shown to include not only disintegration and pore formation at the cell membrane, but also cell entry via endocytosis dependent and independent pathways.

The relative roles of charge and a recognition peptide in luminal targeting of colorectal cancer by fluorescent polyacrylamide

Real time detection of biomarkers at the mucosal level is imperative for the prevention and efficient treatment of colorectal cancer.Cationized polyacrylamide (CPAA) with increasing charge densities was prepared by radical polymerization of acrylamide and different mol% ratios of N-acryloyl, N′-(tert-butyl-carbonyl) diaminoethane. The NIR fluorophore derivative of IR-783, IR-783-S-Ph-COOH, was attached to the CPAA to give CPAA-783. After selecting the optimal IR-783-S-Ph-COOH ratio that avoids quenching, the preferential binding of the polymer was tested in SW-620, SW-480, HT-29, and LS-174T cancer cells. The optimal polymeric product was tested in situ in gut sac preparations of the dimethylhydrazine induced rat model.To increase the detection capabilities of CPAA-783, the FITC-labeled peptide EPPT1, that targets the cell transmembrane underglycosylated MUC-1 (uMUC-1), was conjugated to the polymer to obtain CPAA-783-EPPT1. The dually labeled modified polymer was tested in HT-29 and LS-174T cells (over expressing uMUC-1), followed by an examination in an orthotopic mouse model.CPAA-783 preferentially bound to the cancer cells, depending on CRC staging. The best binding occurred when the fraction of the cationic monomer was 100mol%, labeled with 0.75mol% of IR-783-S-Ph-COOH. An increase in the recognition of the dually labeled polymeric product, CPAA-783-EPPT1, towards HT-29 and LS-174T cells occurred in the lowest EPPT1molar ratio (0.63mol%) only, probably due to quenching phenomena and steric hindrance. Similar observation was obtained in the orthotopic mice.It is concluded that fluorescently tagged CPAA can be used for the detection of malignant tissues in colorectal cancer after luminal instillation. Dually targeted CPAA with EPPT1is feasible, but requires further optimization.

Crystal Structures of the Scaffolding Protein LGN Reveal the General Mechanism by Which GoLoco Binding Motifs Inhibit the Release of GDP from Gαi

GoLoco (GL) motif-containing proteins regulate G protein signaling by binding to Gα subunit and acting as guanine nucleotide dissociation inhibitors. GLs of LGN are also known to bind the GDP form of Gα(i/o) during asymmetric cell division. Here, we show that the C-terminal GL domain of LGN binds four molecules of Gα(i)·GDP. The crystal structures of Gα(i)·GDP in complex with LGN GL3 and GL4, respectively, reveal distinct GL/Gα(i) interaction features when compared with the only high resolution structure known with GL/Gα(i) interaction between RGS14 and Gα(i1.) Only a few residues C-terminal to the conserved GL sequence are required for LGN GLs to bind to Gα(i)·GDP. A highly conserved "double Arg finger" sequence (RΨ(D/E)(D/E)QR) is responsible for LGN GL to bind to GDP bound to Gα(i). Together with the sequence alignment, we suggest that the LGN GL/Gα(i) interaction represents a general binding mode between GL motifs and Gα(i). We also show that LGN GLs are potent guanine nucleotide dissociation inhibitors.

A distinct effect of transient and sustained upregulation of cellular factor XIII in the goldfish retina and optic nerve on optic nerve regeneration

Unlike in mammals, fish retinal ganglion cells (RGCs) have a capacity to repair their axons even after optic nerve transection. In our previous study, we isolated a tissue type transglutaminase (TG) from axotomized goldfish retina. The levels of retinal TG (TGR) mRNA increased in RGCs 1–6weeks after nerve injury to promote optic nerve regeneration both in vitro and in vivo. In the present study, we screened other types of TG using specific FITC-labeled substrate peptides to elucidate the implications for optic nerve regeneration. This screening showed that the activity of only cellular coagulation factor XIII (cFXIII) was increased in goldfish optic nerves just after nerve injury. We therefore cloned a full-length cDNA clone of FXIII A subunit (FXIII-A) and studied temporal changes of FXIII-A expression in goldfish optic nerve and retina during regeneration. FXIII-A mRNA was initially detected at the crush site of the optic nerve 1h after injury; it was further observed in the optic nerve and achieved sustained long-term expression (1–40days after nerve injury). The cells producing FXIII-A were astrocytes/microglial cells in the optic nerve. By contrast, the expression of FXIII-A mRNA and protein was upregulated in RGCs for a shorter time (3–10days after nerve injury). Overexpression of FXIII-A in RGCs achieved by lipofection induced significant neurite outgrowth from unprimed retina, but not from primed retina with pretreatment of nerve injury. Addition of extracts of optic nerves with injury induced significant neurite outgrowth from primed retina, but not from unprimed retina without pretreatment of nerve injury. The transient increase of cFXIII in RGCs promotes neurite sprouting from injured RGCs, whereas the sustained increase of cFXIII in optic nerves facilitates neurite elongation from regrowing axons.

Striatal-enriched Protein-tyrosine Phosphatase (STEP) Regulates Pyk2 Kinase Activity

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase family and is highly expressed in brain and hematopoietic cells. Pyk2 plays diverse functions in cells, including the regulation of cell adhesion, migration, and cytoskeletal reorganization. In the brain, it is involved in the induction of long term potentiation through regulation of N-methyl-d-aspartate receptor trafficking. This occurs through the phosphorylation and activation of Src family tyrosine kinase members, such as Fyn, that phosphorylate GluN2B at Tyr(1472). Phosphorylation at this site leads to exocytosis of GluN1-GluN2B receptors to synaptic membranes. Pyk2 activity is modulated by phosphorylation at several critical tyrosine sites, including Tyr(402). In this study, we report that Pyk2 is a substrate of striatal-enriched protein-tyrosine phosphatase (STEP). STEP binds to and dephosphorylates Pyk2 at Tyr(402). STEP KO mice showed enhanced phosphorylation of Pyk2 at Tyr(402) and of the Pyk2 substrates paxillin and ASAP1. Functional studies indicated that STEP opposes Pyk2 activation after KCl depolarization of cortical slices and blocks Pyk2 translocation to postsynaptic densities, a key step required for Pyk2 activation and function. This is the first study to identify Pyk2 as a substrate for STEP.

In Vivo Applied Positively Charged FITC-Labeled Peptide Conjugates Show Artificial Relocation in Frozen Sections

Our original aim was to evaluate in vivo nuclear uptake of a conjugate containing the SV40 T Antigen nuclear localization sequence (NLS), 6 aminohexanoic acids alternating with 7 arginines, the fluorescence dye fluorescein isothiocyanate (FITC) and the gadolinium chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). During the confocal laser scanning microscopy examination of frozen sections of the kidneys of mice injected intravenously with the conjugate we found unexpected conjugate relocation. In the dry frozen sections no nuclear staining was found. After covering the sections with physiological saline and a coverslip, the conjugate was relocated to the cell nuclei. Causation of this effect by FITC and DOTA could be excluded with appropriate controls. In the assumption that other positively charged conjugates behave in the same manner we tested two further conjugates in vivo. An octaarginine coupled to a lysine carrying FITC on the -amino-group was found in the cell nuclei of the frozen kidney sections before and after covering the slices with saline. By contrast, a conjugate containing myristoylated HIV-Tat was found not to stain the cell nuclei but only the cytoplasm before and after covering with saline. This demonstrates that not all positively charged conjugates are relocated to the cell nucleus after covering the frozen sections with saline. This was confirmed by direct incubation of frozen sections from untreated kidneys with the same conjugates and additional positively charged FITC conjugates. By coincubating the FITC conjugates with a cytoplasm directed rhodamine conjugate on untreated kidney sections we found that such conjugates could be used as nuclear FITC counterstain. In summary, one should bear in mind that positively charged conjugates applied in vivo can relocate artificially after covering with saline. Keywords: Morphological artifacts, Frozen Sections, FITC-Labeled Peptide Conjugates, Artificial Relocation, SV40 T Antigen nuclear localization sequence, nuclear uptake, FITC conjugates, DOTA, glioma cells, N-terminally myristoylated HIV 1 Tat peptide sequence

Vena cava and aortic smooth muscle cells express transglutaminases 1 and 4 in addition to transglutaminase 2

Transglutaminase (TG) function facilitates several vascular processes and diseases. Although many of these TG-dependent vascular processes have been ascribed to the function of TG2, TG2 knockout mice have a mild vascular phenotype. We hypothesized that TGs besides TG2 exist and function in the vasculature. Biotin-pentylamide incorporation, a measure of general TG activity, was similar in wild-type and TG2 knockout mouse aortae, and the general TG inhibitor cystamine reduced biotin-pentylamine incorporation to a greater extent than the TG2-specific inhibitor Z-DON, indicating the presence of other functional TGs. Additionally, 5-hydroxytryptamine-induced aortic contraction, a TG-activity-dependent process, was decreased to a greater extent by general TG inhibitors vs. Z-DON (maximum contraction: cystamine = abolished, monodansylcadaverine = 28.6 ± 14.9%, and Z-DON = 60.2 ± 15.2% vehicle), providing evidence for the importance of TG2-independent activity in the vasculature. TG1, TG2, TG4, and Factor XIII (FXIII) mRNA in rat aortae and vena cavae was detected by RT-PCR. Western analysis detected TG1 and TG4, but not FXIII, in rat aortae and vena cavae and in TG2 knockout and wild-type mouse aortae. Immunostaining confirmed the presence of TG1, TG2, and TG4 in rat aortae and vena cavae, notably in smooth muscle cells; FXIII was absent. K5 and T26, FITC-labeled peptide substrates specific for active TG1 and TG2, respectively, were incorporated into rat aortae and vena cavae and wild-type, but not TG2 knockout, mouse aortae. These studies demonstrate that TG2-independent TG activity exists in the vasculature and that TG1 and TG4 are expressed in vascular tissues.

A graphene oxide-peptide fluorescence sensor for proteolytically active prostate-specific antigen

This paper presents a novel sensor to detect proteolytically active prostate-specific antigen (PSA) by assembling a purpose-designed FITC-labeled peptide with graphene oxide (GO). The fluorescence of the dye-labeled peptide was quenched in the presence of GO. Reaction of the sensor with PSA cleaves the peptide, leading to the release of the dye moiety and a great increase in fluorescence intensity in a dose- and time-dependent manner, and PSA can be quantified accordingly. This approach is simple compared to existing methods since the GO-peptide-based sensor is easily assembled and detection can be achieved without the involvement of complicated procedures. Moreover, the applicability of the method has been demonstrated by detecting PSA in spiked urine samples.

Screening of cell‐penetrating peptides using mRNA display

Cell-penetrating peptides (CPPs) are attractive vectors for in vivo and in vitro cellular uptake. Their use is, however, limited by insufficient understanding of their preference for a target cell. Here, a new CPP screening method is presented that uses mRNA display. After incubating the target cell lines, such as human embryonic kidney 293 (HEK 293) and HeLa cells, with an mRNA display library for 3 h at 37°C, the CPP-mRNA nucleotide conjugates were harvested. These were amplified with PCR and subsequently sequenced. The screened CPPs for each cell line were identified after four rounds of selection. Among them, two peptides, MAMPGEPRRANVMAHKLEPASLQLR NSCA (CPPK) and MAPQRDTVGGRTTPPSWGPAKAQLRNSCA (CPPL) were selected, and the FITC-labeled peptides were evaluated for their ability to penetrate cells. The screened CPPs were superior to polyarginine (R(11) ), which is widely used as a standard peptide and shows good cell penetration efficiency. Our method can be applied to other target cells for which CPPs have not yet been elucidated.

Microtubule Affinity Regulating Kinase Activity in Living Neurons Was Examined by a Genetically Encoded Fluorescence Resonance Energy Transfer/Fluorescence Lifetime Imaging-based Biosensor

Protein kinases of the microtubule affinity regulating kinase (MARK)/Par-1 family play important roles in the establishment of cellular polarity, cell cycle control, and intracellular signal transduction. Disturbance of their function is linked to cancer and brain diseases, e.g. lissencephaly and Alzheimer disease. To understand the biological role of MARK family kinases, we searched for specific inhibitors and a biosensor for MARK activity. A screen of the ChemBioNet library containing ~18,000 substances yielded several compounds with inhibitory activity in the low micromolar range and capable of inhibiting MARK activity in cultured cells and primary neurons, as judged by MARK-dependent phosphorylation of microtubule-associated proteins and its consequences for microtubule integrity. Four of the compounds share a 9-oxo-9H-acridin-10-yl structure as a basis that will serve as a lead for optimization of inhibition efficiency. To test these inhibitors, we developed a cellular biosensor for MARK activity based on a MARK target sequence attached to the 14-3-3 scaffold protein and linked to enhanced cyan or teal and yellow fluorescent protein as FRET donor and acceptor pairs. Transfection of the teal/yellow fluorescent protein sensor into neurons and imaging by fluorescence lifetime imaging revealed that MARK was particularly active in the axons and growth cones of differentiating neurons.

Stability Screening of Arrays of Major Histocompatibility Complexes on Combinatorially Encoded Flow Cytometry Beads

The binding and stabilization capacity of potential T cell epitopes to class I MHC molecules form the basis for their immunogenicity and provide fundamental insight into factors that dictate cellular immune responses. We have developed a versatile high throughput cell-free method to measure MHC stability by capturing a variety of MHC products on the surface of streptavidin-coated particles followed by flow cytometry analysis. Arrays of peptide-MHC combinations, generated by exchanging conditional ligand-loaded MHC, could be probed in a single experiment, thus combining the molecular precision of biochemically purified MHCs with high content multiparametric flow cytometry-based assays. Semiquantitative determination of the peptide affinity for the restriction element could also be accomplished through competition experiments using this bead-based assay. Furthermore, the generated peptide-MHC reagents could directly be applied to antigen-specific CD8(+) T lymphocyte analysis. The combinatorial labeling of beads allowed straightforward identification by their unique fluorescent signatures and provided a convenient means for extended assay multiplexing.

Antifungal activity of novel synthetic peptides by accumulation of reactive oxygen species (ROS) and disruption of cell wall against Candida albicans

In the present work, we investigated the antifungal activity of two de novo designed, antimicrobial peptides VS2 and VS3, incorporating unnatural amino acid α,β-dehydrophenylalanine (ΔPhe). We observed that the low-hemolytic peptides could irreversibly inhibit the growth of various Candida species and multidrug resistance strains at MIC80 values ranging from 15.62μM to 250μM. Synergy experiments showed that MIC80 of the peptides was drastically reduced in combination with an antifungal drug fluconazole. The dye PI uptake assay was used to demonstrate peptide induced cell membrane permeabilization. Intracellular localization of the FITC-labeled peptides in Candida albicans was studied by confocal microscopy and FACS. Killing kinetics, PI uptake assay, and the intracellular presence of FITC-peptides suggested that growth inhibition is not solely a consequence of increased membrane permeabilization. We showed that entry of the peptide in Candida cells resulted in accumulation of reactive oxygen species (ROS) leading to cell necrosis. Morphological alteration in Candida cells caused by the peptides was visualized by electron microscopy. We propose that de novo designed VS2 and VS3 peptides have multiple detrimental effects on target fungi, which ultimately result in cell wall disruption and killing. Therefore, these peptides represent a good template for further design and development as antifungal agents.

Multiple Triphenylphosphonium Cations as a Platform for the Delivery of a Pro-Apoptotic Peptide

Triphenyl phosphonium cations (TPPs) are delocalized lipophilic cations that accumulate in the mitochondria of cells. We have explore the effect of increasing the number of TPPs on delivery of a cell-impermeable pro-apoptotic peptide to intact cells. The pro-apoptotic peptide D-(KLAKLAK)(2) (KLA) was extended with 0-3 L-Lysines modified at their ε-amine with TPP. Peptides were studied in HeLa cells to determine their cytotoxic activity and cellular uptake. In HeLa cells, the increased cytotoxicity correlates with the number of TPPs; the peptide with 3 TPP molecules (3-KLA) exerts the highest cytotoxic activity. This FITC-labeled peptide is found to accumulate in intact HeLa cells, whereas peptides with 0-2 TPPs are not detected at the same peptide concentration. Mitochondria-dependent apoptosis of HeLa cells in the presence of 3-KLA was followed by propidium iodide, Annexin-V and DiOC fluorescence by FACS. A facile synthetic methodology has been presented for the delivery of a biologically active peptide into mitochondria of intact cells by attaching multiple TPP moieties to the peptide. This approach was shown to dramatically increase biological activity of the peptide as a pro-apoptotic agent.

Targeted imaging of colorectal dysplasia in living mice with fluorescence microendoscopy

We validate specific binding activity of a fluorescence-labeled peptide to colorectal dysplasia in living mice using a miniature, flexible, fiber microendoscope that passes through the instrument channel of an endoscope. The microendoscope delivers excitation light at 473 nm through a fiber-optic bundle with outer diameter of 680 µm to collect en face images at 10 Hz with 4 µm lateral resolution. We applied the FITC-labeled peptide QPIHPNNM topically to colonic mucosa in genetically engineered mice that spontaneously develop adenomas. More than two-fold greater fluorescence intensity was measured from adenomas compared to adjacent normal-appearing mucosa. Images of adenomas showed irregular morphology characteristic of dysplasia.

Mammalian cell toxicity and candidacidal mechanism of Arg- or Lys-containing Trp-rich model antimicrobial peptides and their d-enantiomeric peptides

In this study, we investigated the mammalian cell toxicity and candidacidal mechanism of Arg- or Lys-containing Trp-rich model antimicrobial peptides (K 6L 2W 3 and R 6L 2W 3) and their d-enantiomeric peptides (K 6L 2W 3- d and R 6L 2W 3- d). Arg-containing peptides were more toxic to human erythrocytes and mammalian cells as compared to Lys-containing peptides. Arg-containing peptides is slightly more hydrophobic than Lys-containing counterparts, as judged from their reverse phase-high performance liquid chromatography (RP-HPLC) retention time. These results suggested that a little difference in hydrophobicity of these peptides affect their hemolytic activity and mammalian cell toxicity. Interestingly, K 6L 2W 3 and K 6L 2W 3- d almost similar mammalian cell cytotoxicity, whereas R 6L 2W 3- d showed much higher cytotoxicity as compared to R 6L 2W 3. A low ability to facilitate fluorescent marker escape from Candida albicans membrane-mimicking vesicles suggested that the major target site of Lys-containing peptides may be not the cell membrane but the cytoplasm of C. albicans. Confocal laser-scanning microscopy revealed that FITC-labeled Lys-containing peptides penetrated the cell wall and cell membrane and accumulated inside the cells, whereas FITC-labeled Arg-containing peptides did not penetrate but associated with the membranes. Collectively, our results suggested that the ultimate target site of action of Arg-containing peptides and Lys-containing peptides may be the membrane and the cytoplasm of C. albicans, respectively.

Uptake mechanisms of EGFR-targeted TiO2 nanoparticles.

e13583 Background: We are developing TiO2 nanoconjugates (NCs) that can be used as therapeutic and diagnostic agents. Nanoscale TiO2 can be surface conjugated with various molecules and has the unique ability to induce reactive oxygen species after radiation activation. The two major questions that we wish to answer are (1) how are NCs internalized by cells and (2) is targeting of NCs using a small peptide achievable. To address these questions, we have created NCs targeted to epidermal growth factor receptor (EGFR) which is enriched in many epithelial cancers. Methods: 3 nm TiO2 nanoparticles (NPs) were synthesized by low temperature alkaline hydrolysis. EGFR targeted NCs were created by conjugating an eleven amino acid EGF peptide to NPs through a dopamine linker. Cell growth and immunocytochemistry were done as usual. X-ray Fluorescence Microscopy (XFM) was performed at the 2-ID-E beamline of the Advanced Photon Source at Argonne National Laboratories. Results: Previous reports have shown that a part of epidermal growth factor (EGF) known as the B-loop is required for binding EGFR. We selected eleven amino acids from this region and synthesized FITC-labeled, dopamine-conjugated peptides. These peptides were then used to treat two isogenic colon cancer cell lines, one with high EGFR expression (SW480) and one with low EGFR expression (SW620). Immunofluorescent imaging of treated cells showed that FITC-labeled peptides colocalized with EGFR labeled with fluorescent antibodies on the cell surface and within the cytoplasm. The synthetic EGF peptides were then conjugated to NPs via dopamine to create EGFR targeted NCs. These NCs also colocalized with EGFR in treated cells by immunofluorescence imaging. This was confirmed using XFM which allows for direct detection of titanium as well as gold. These images showed that EGFR labeled with gold conjugated antibodies colocalized with titanium at the cell surface. Conclusions: TiO2 NCs are a promising vehicle for the delivery of therapeutic and diagnostic agents to certain cell types. Conjugating biomolecules that promote specific uptake and retention in tumor cells will allow for controlled drug delivery. In fact, we have shown that EGFR targeted NCs can successfully colocalize with EGFR on the surface of colon cancer cells. No significant financial relationships to disclose.

Bactericidal effect of bovine lactoferrin, LFcin, LFampin and LFchimera on antibiotic-resistant Staphylococcus aureus and Escherichia coli

Increased prevalence of antibiotic-resistant bacteria has become a major threat to the health sector worldwide due to their virulence, limited therapeutic options and distribution in both hospital and community settings. Discovery and development of new agents to combat antibiotic-resistant bacteria is thus needed. This study therefore aimed to evaluate the ability of bovine lactoferrin (LF), peptides from two antimicrobial domains lactoferricin B (LFcin17-30) and lactoferrampin (LFampin265-284) and a chimeric construct (LFchimera) containing both peptides, as potential bactericidal agents against clinical isolates of antibiotic-resistant Staphylococcus aureus and Escherichia coli. Results in kinetics of growth show that LF chimera and peptides inhibited the growth of both bacterial species. By confocal microscopy and flow cytometry it was observed that LF and FITC-labeled peptides are able to interact with these bacteria and cause membrane permeabilization, as monitored by propidium iodide staining, these effects were decreased by preincubation with lipopolysaccharide in E. coli. By electron microscopy, a clear cellular damage was observed in bacteria after treatments with LFchimera and peptides, suggesting that interaction and membrane disruption are probably involved as a mechanism of action. In conclusion, results show that LFchimera, LF and peptides have potential as bactericidal agents in the antibiotic-resistant strains of S. aureus and E. coli and also the work strongly suggest that LFcin17-30 and LFampin265-284 acts synergistically with antibiotics against multidrug resistant EPEC and MRSA in vitro.

780 Quantitative In Vivo Assessment of Vascular Permeability in Human Colonic Mucosa Using Confocal Endomicroscopy: Clinical Implications for Colonic Neoplasia

Background: Peptides are promising molecular probes for detecting over expressed cell surface targets in the digestive tract. We have previously demonstrated their In Vivo use with colonic dysplasia on confocal microendoscopy, and have developed the sequence ASYNYDA to target Barrett's dysplasia. Aims: We aim to quantify the fluorescence intensity and determine the diagnostic accuracy of this target peptide for Barrett's esophagus. Methods: Biopsy specimens of esophageal mucosa were obtained from human subjects in regions of endoscopically apparent squamous epithelium, intestinal metaplasia, and adenocarcinoma. The tissues were incubated with the FITC-labeled target peptide at a concentration of 100 μmol for 10 minutes. Unbound peptide was removed by rinsing in PBS. The specimens were embedded in OCT freezing compound, cut in 10 μm sections, and counterstained with DAPI. An adjacent serial section was cut and stained with H&E for routine histology. Confocal fluorescence images were collected from each section, and were analyzed for area, mean gray value, standard deviation, min/max gray values, and integrated density using NIH Image J. Results: We evaluated a total of 52 confocal fluorescence images from 26 biopsy specimens collected from 18 subjects, including 20 squamous, 12 intestinal metaplasia, 8 high-grade dysplasia, and 12 adenocarcinoma. The mean intensity for squamous, intestinal metaplasia, high-grade dysplasia and adenocarcinoma were 0.1±0.1, 1.9±3.2, 4.41±2.2, and 9.8±13.1, respectively. Fig A shows binding of the FITC-labeled peptide ASYNYDA to esophageal adenocarcinoma. Fig B shows locations of cell nuclei in blue, identified by DAPI stain, and overlay in Fig C demonstrates peptide binding to cell surface targets. An intensity threshold of 0.73 produced a detection sensitivity and specificity of 85% and 88%, respectively for either high-grade dysplasia or adenocarcinoma. Conclusions: We demonstrate preferential binding of the peptide ASYNYDA to intra-epithelial neoplasia in Barrett's esophagus on confocal fluorescence images, demonstrating a promising novel molecular probe to perform targeted detection on surveillance with confocal microendoscopy.

779 Targeting of Intra-Epithelial Neoplasia in Barrett's Esophagus with Fluorescence-Labeled Peptide

Background: Peptides are promising molecular probes for detecting over expressed cell surface targets in the digestive tract. We have previously demonstrated their In Vivo use with colonic dysplasia on confocal microendoscopy, and have developed the sequence ASYNYDA to target Barrett's dysplasia. Aims: We aim to quantify the fluorescence intensity and determine the diagnostic accuracy of this target peptide for Barrett's esophagus. Methods: Biopsy specimens of esophageal mucosa were obtained from human subjects in regions of endoscopically apparent squamous epithelium, intestinal metaplasia, and adenocarcinoma. The tissues were incubated with the FITC-labeled target peptide at a concentration of 100 μmol for 10 minutes. Unbound peptide was removed by rinsing in PBS. The specimens were embedded in OCT freezing compound, cut in 10 μm sections, and counterstained with DAPI. An adjacent serial section was cut and stained with H&E for routine histology. Confocal fluorescence images were collected from each section, and were analyzed for area, mean gray value, standard deviation, min/max gray values, and integrated density using NIH Image J. Results: We evaluated a total of 52 confocal fluorescence images from 26 biopsy specimens collected from 18 subjects, including 20 squamous, 12 intestinal metaplasia, 8 high-grade dysplasia, and 12 adenocarcinoma. The mean intensity for squamous, intestinal metaplasia, high-grade dysplasia and adenocarcinoma were 0.1±0.1, 1.9±3.2, 4.41±2.2, and 9.8±13.1, respectively. Fig A shows binding of the FITC-labeled peptide ASYNYDA to esophageal adenocarcinoma. Fig B shows locations of cell nuclei in blue, identified by DAPI stain, and overlay in Fig C demonstrates peptide binding to cell surface targets. An intensity threshold of 0.73 produced a detection sensitivity and specificity of 85% and 88%, respectively for either high-grade dysplasia or adenocarcinoma. Conclusions: We demonstrate preferential binding of the peptide ASYNYDA to intra-epithelial neoplasia in Barrett's esophagus on confocal fluorescence images, demonstrating a promising novel molecular probe to perform targeted detection on surveillance with confocal microendoscopy.