Immunogenicity of liposomes containing lipid core peptides and the adjuvant Quil A

Purpose. The purpose of this study was to investigate the immunogenicity of liposomes containing mannosylated lipid core peptide (manLCP) constructs, both in vitro and in vivo, with or without the addition of the immune stimulating adjuvant Quil A. Methods. Mouse bone marrow dendritic cells (BMDC) were cultured with liposome formulations for 48 h, and the resulting level of BMDC activation was determined by flow cytometry. BMDC pulsed with liposome formulations were incubated with 5,6-carboxyfluoroscein diacetate succinimidyl ester-labeled T cells for 72 h and the resulting T cell proliferation was determined by flow cytometry. To investigate the immunogenicity of formulations in vivo, groups of C57Bl/6J mice were immunized by subcutaneous injection, and the resulting antigen-specific cytotoxic and protective immune responses toward tumor challenge evaluated. Results. Despite being unable to demonstrate the activation of BMDC, BMDC pulsed with liposomes containing manLCP constructs were able to stimulate the proliferation of naive T cells in vitro. However, in vivo only liposomes containing both manLCP and Quil A were able to stimulate a strong antigen-specific cytotoxic immune response. Liposomes containing manLCP and Quil A within the same particle were able to protect against the growth of tumor cells to a similar level as if the antigen was administered in alum with CD4 help. Conclusion. ManLCPs administered in liposomes are able to stimulate strong cytotoxic and protective immune responses if Quil A is also incorporated as an adjuvant.

Prediction of HLA-DQ3.2 beta ligands: evidence of multiple registers in class II binding peptides

Motivation: While processing of MHC class II antigens for presentation to helper T-cells is essential for normal immune response, it is also implicated in the pathogenesis of autoimmune disorders and hypersensitivity reactions. Sequence-based computational techniques for predicting HLA-DQ binding peptides have encountered limited success, with few prediction techniques developed using three-dimensional models. Methods: We describe a structure-based prediction model for modeling peptide-DQ3.2 beta complexes. We have developed a rapid and accurate protocol for docking candidate peptides into the DQ3.2 beta receptor and a scoring function to discriminate binders from the background. The scoring function was rigorously trained, tested and validated using experimentally verified DQ3.2 beta binding and non-binding peptides obtained from biochemical and functional studies. Results: Our model predicts DQ3.2 beta binding peptides with high accuracy [area under the receiver operating characteristic (ROC) curve A(ROC) > 0.90], compared with experimental data. We investigated the binding patterns of DQ3.2 beta peptides and illustrate that several registers exist within a candidate binding peptide. Further analysis reveals that peptides with multiple registers occur predominantly for high-affinity binders.

Protease-activated receptor-2 peptides activate neurokinin-1 receptors in the mouse isolated trachea

Protective roles for protease-activated receptor-2 (PAR2) in the airways including activation of epithelial chloride (Cl-) secretion are based on the use of presumably PAR(2)-selective peptide agonists. To determine whether PAR(2) peptide-activated Cl- secretion from mouse tracheal epithelium is dependent on PAR(2), changes in ion conductance across the epithelium [short-circuit current (I-SC)] to PAR(2) peptides were measured in Ussing chambers under voltage clamp. In addition, epithelium and endothelium-dependent relaxations to these peptides were measured in two established PAR(2) bioassays, isolated ring segments of mouse trachea and rat thoracic aorta, respectively. Apical application of the PAR(2) peptide SLIGRL caused increases in I-SC, which were inhibited by three structurally different neurokinin receptor-1 (NK1R) antagonists and inhibitors of Cl- channels but not by capsaicin, the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP(8-37), or the nonselective cyclooxygenase inhibitor indomethacin. Only high concentrations of trypsin caused an increase in I-SC but did not affect the responses to SLIGRL. Relaxations to SLIGRL in the trachea and aorta were unaffected by the NK1R antagonist nolpitantium (SR 140333) but were abolished by trypsin desensitization. The rank order of potency for a range of peptides in the trachea I-SC assay was 2-furoyl-LIGRL > SLCGRL > SLIGRL > SLIGRT > LSIGRL compared with 2-furoyl-LIGRL > SLIGRL > SLIGRT > SLCGRL (LSIGRL inactive) in the aorta relaxation assay. In the mouse trachea, PAR(2) peptides activate both epithelial NK1R coupled to Cl- secretion and PAR(2) coupled to prostaglandin E-2-mediated smooth muscle relaxation. Such a potential lack of specificity of these commonly used peptides needs to be considered when roles for PAR(2) in airway function in health and disease are determined.

Synthesis of 'difficult' peptides free of aspartimide and related products, using peptoid methodology

We developed an efficient, cost effective strategy for Fmoc-based solid phase synthesis of 'difficult' peptides and/or peptides containing Asp/Asn-Gly sequences, free of aspartimide and related products, using a peptoid methodology for the preparation of N-substituted glycines.

The cyclotides and related macrocyclic peptides as scaffolds in drug design

The applicability of linear peptides as drugs is potentially limited by their susceptibility to proteolytic cleavage and poor bioavailability. Cyclotides are macrocyclic cystine-knotted mini-proteins that have a broad range of bioactivities and are exceptionally stable, being resistant to chemical, thermal and enzymatic degradation. The general limitations of peptides as drugs can potentially be overcome by using the cyclotide framework as a scaffold onto which new activities may be engineered. The potential use of cyclotides and related peptide scaffolds for drug design is evaluated herein, with reference to increasing knowledge of the structures and sequence diversity of natural cyclotides and the emergence of new approaches in protein engineering.

The effects of freshwater to seawater transfer on circulating levels of angiotensin II, C-type natriuretic peptide and arginine vasotocin in the euryhaline elasmobranch, Carcharhinus leucas

This study examined the effect of transfer to increased environmental salinity on the circulating levels of angiotensin II (ANG II), C-type natriuretic peptide (CNP), and arginine vasotocin (AVT) in the euryhaline elasmobranch, Carcharhinus letteas. Plasma levels of ANG 11 and CNP were significantly increased in C. leucas chronically acclimated to seawater (SW) in comparison to freshwater (FW) acclimated fish. There was no difference in plasma AVT levels. Acute transfer of FW fish to 75% SW induced an increase in plasma ANG II levels within 12 h, and subsequent transfer from 75 to 100% SW further increased plasma ANG 11 levels at both 24 and 72 h. No change in plasma CNP was observed during acute transfer to increased salinity. However, a significant increase in plasma AVT levels was observed following 96 h in 75% SW and 24 h in 100% SW. In chronically SW acclimated C leucas plasma osmolality, sodium, chloride, and Urea were all significantly higher than FW acclimated fish but there was no difference in haematocrit. Acute transfer of C letteas to 75% SW induced a significant increase in plasma osmolality, sodium and urea concentrations within 96 h of transfer. Subsequent transfer from 75 to 100% SW induced a further increase in these variables within 24 h in addition to a significant increase in plasma chloride above control levels. Haematocrit did not differ between the experimental and control groups throughout the acute study. Circulating levels of ANG 11 were significantly correlated to plasma, sodium, chloride, and urea concentrations during acclimation to SW. Conversely, circulating levels of CNP and AVT did not correlate to plasma osmolytes, however, CNP was significantly correlated to haematocrit during acclimation to seawater. (c) 2005 Elsevier Inc. All rights reserved.

Population-based detection of systolic and diastolic dysfunction with amino-terminal pro-B-type natriuretic peptide

Background There is limited information regarding the clinical utility of amino-terminal pro-B-type natriuretic pepticle (NT-proBNP) for the detection of left ventricular (LV) dysfunction in the community. We evaluated predictors of circulating NT-proBNP levels and determined the utility of NT-proBNP to detect systolic and diastolic LV dysfunction in older adults. Methods. A population-based sample of 1229 older adults (mean age 69.4 years, 50.1% women) underwent echocardiographic assessment of cardiac structure and function and measurement of circulating NT-proBNP levels. Results Predictors of NT-proBNP included age, female sex, body mass index, and cardiorenal parameters (diastolic dysfunction [DID] severity; LV mass and left atrial volume; right ventricular overload; decreasing ejection fraction [EF] and creatinine clearance). The performance of NT-proBNP to detect any degree of LV dysfunction, including mild DID, was poor (area under the curve 0.56-0.66). In contrast, the performance of NT-proBNP for the detection of EF 0.90 regardless of age and sex; history of hypertension, diabetes, coronary artery disease; or body mass category. The ability of NT-proBNP to detect EF

Detecting and sorting targeting peptides wtih neural networks and support vector machines

This paper presents a composite multi-layer classifier system for predicting the subcellular localization of proteins based on their amino acid sequence. The work is an extension of our previous predictor PProwler v1.1 which is itself built upon the series of predictors SignalP and TargetP. In this study we outline experiments conducted to improve the classifier design. The major improvement came from using Support Vector machines as a "smart gate" sorting the outputs of several different targeting peptide detection networks. Our final model (PProwler v1.2) gives MCC values of 0.873 for non-plant and 0.849 for plant proteins. The model improves upon the accuracy of our previous subcellular localization predictor (PProwler v1.1) by 2% for plant data (which represents 7.5% improvement upon TargetP).