Function of host defence peptide LL-37 in human periodontal disease.

Objectives: Fibroblasts play a significant role as regulators of the host response in periodontal disease, responding to bacterial stimulation by producing an array of inflammatory cytokines and chemokines. LL-37, a host defence peptide, inhibits LPS-induced cytokine signalling in macrophages, suggesting an immunomodulatory role. The objective was to investigate the interaction between LL-37 and gingival fibroblasts – both its direct regulation of fibroblast activity and its effect on fibroblast response to LPS activation. Methods: Human gingival fibroblasts (HGFs) were incubated for 24 hours in the presence of either P. gingivalis LPS (10µg/ml) or E. coli LPS (10ng/ml) along with LL-37 (0-50 µg/ml). IL-6 and IL-8 production by HGFs in the conditioned medium was determined by ELISA. Western blot was performed to determine the effect of LL-37 on LPS -induced IκBα degradation in HGFs following LPS stimulation over 2 hours. DNA microarray analysis was performed on cell populations incubated for 6 hr in the presence or absence of the peptide. Confirmation of LL-37 effects on specific gene expression was obtained by QPCR. Results: At low concentrations (≤ 5 µg/ml) LL-37 significantly inhibited LPS-induced cytokine production by HGFs. At higher concentrations LL-37 induced IL-8 production independent of LPS. Addition of LL-37 blocked LPS-induced IκBα degradation in HGFs. Microarray analysis revealed that LL-37 (50µg/ml) upregulated a significant number of cytokines and chemokines by > 5 fold. Upregulation of five of these, CXCL1, CXCL2, CXCL3, IL-24 and IL-8 was confirmed by Q-PCR. Conclusion: The host defence peptide LL-37, the only known human cathelicidin, appears to have pleiotrophic effects in innate immunity. At least some of these are mediated through cytokine and chemokine signalling networks. The ability of LL-37 to reduce bacterial LPS-induced cytokine production in gingival fibroblasts, at low concentrations, suggests a potential therapeutic role in the management of periodontal disease.

Identification of Peptide Inhibitors of Enveloped Viruses Using Support Vector Machine

The peptides derived from envelope proteins have been shown to inhibit the protein-protein interactions in the virus membrane fusion process and thus have a great potential to be developed into effective antiviral therapies. There are three types of envelope proteins each exhibiting distinct structure folds. Although the exact fusion mechanism remains elusive, it was suggested that the three classes of viral fusion proteins share a similar mechanism of membrane fusion. The common mechanism of action makes it possible to correlate the properties of self-derived peptide inhibitors with their activities. Here we developed a support vector machine model using sequence-based statistical scores of self-derived peptide inhibitors as input features to correlate with their activities. The model displayed 92% prediction accuracy with the Matthew’s correlation coefficient of 0.84, obviously superior to those using physicochemical properties and amino acid decomposition as input. The predictive support vector machine model for self- derived peptides of envelope proteins would be useful in development of antiviral peptide inhibitors targeting the virus fusion process.

Development of polymeric–cationic peptide composite nanoparticles, a nanoparticle-in-nanoparticle system for controlled gene delivery

We report the formulation of novel composite nanoparticles that combine the high transfection efficiency of cationic peptide-DNA nanoparticles with the biocompatibility and prolonged delivery of polylactic acid–polyethylene glycol (PLA-PEG). The cationic cell-penetrating peptide RALA was used to condense DNA into nanoparticles that were encapsulated within a range of PLA-PEG copolymers. The composite nanoparticles produced exhibited excellent physicochemical properties including size <200 nm and encapsulation efficiency >80%. Images of the composite nanoparticles obtained with a new transmission electron microscopy staining method revealed the peptide-DNA nanoparticles within the PLA-PEG matrix. Varying the copolymers modulated the DNA release rate >6 weeks in vitro. The best formulation was selected and was able to transfect cells while maintaining viability. The effect of transferrin-appended composite nanoparticles was also studied. Thus, we have demonstrated the manufacture of composite nanoparticles for the controlled delivery of DNA.

Phylloseptin-PBa—A Novel Broad-Spectrum Antimicrobial Peptide from the Skin Secretion of the Peruvian Purple-Sided Leaf Frog (Phyllomedusa Baltea) Which Exhibits Cancer Cell Cytotoxicity

Antimicrobial peptides from amphibian skin secretion display remarkable broad-spectrum antimicrobial activity and are thus promising for the discovery of new antibiotics. In this study, we report a novel peptide belonging to the phylloseptin family of antimicrobial peptides, from the skin secretion of the purple-sided leaf frog, Phyllomedusa baltea, which was named Phylloseptin-PBa. Degenerate primers complementary to putative signal peptide sites of frog skin peptide precursor-encoding cDNAs were designed to interrogate a skin secretion-derived cDNA library from this frog. Subsequently, the peptide was isolated and identified using reverse phase HPLC and MS/MS fragmentation. The synthetic replicate was demonstrated to have activity against S. aureus, E. coli and C. albicans at concentrations of 8, 128 and 8 mg/L, respectively. In addition, it exhibited anti-proliferative activity against the human cancer cell lines, H460, PC3 and U251MG, but was less active against a normal human cell line (HMEC). Furthermore, a haemolysis assay was performed to assess mammalian cell cytotoxicity of Phylloseptin-PBa. This peptide contained a large proportion of α-helical domain, which may explain its antimicrobial and anticancer activities.

Intermedin and calcitonin gene-related peptide fail to shine in acute coronary syndrome

Aims: To measure levels of intermedin and calcitonin gene-related peptide (CGRP) in acute coronary syndrome (ACS) and to determine if they are elevated. 
Methods and results: 81 patients admitted with suspected ACS were enrolled into the study. 50 were confirmed ACS by ACC (2000) guidelines and 31 were in a control group as non-cardiac chest pain. Intermedin was nonsignificantly elevated 6.14 pg/ml vs 4.84 pg/ml b8 h in the ACS group; sensitivity 68%, specificity 63% on presenting sample. Intermedinwas significantly elevated in those patientswho had an initially negative troponin T (b0.03 ng/ml) on presentation, 6.67 pg/ml vs 4.84 pg/ml, p = 0.03. CGRP was significantly elevated in ACS patients, 8–b16 h after pain onset, 8.67 pg/ml vs 7.08 pg/ml, p= 0.036. However, it didn't aid diagnosis in initially negative troponin patients; sensitivity 61%, specificity 60% on presenting sample. Both intermedin and CGRP were elevated in STEMI patients on a first sample, but only intermedin was significantly elevated; 7.03 pg/ml vs 4.84 pg/ml, p =0.02 and 8.87 pg/ml vs 7.03 pg/ml p = 0.093, respectively. High sensitivity troponin T was significant elevated in the ACS group at b8 h (414.9 vs 17.22, p= 0.006) and at 8–b16 h (3325.27 vs 21.54, p = 0.02). 
Conclusions: Both intermedin and CGRP are detectable in human patients. Levels showa trend to elevation in ACS, with CGRP being significantly raised N8 h after pain onset. The degree of elevation will have limited clinical applicability.

Glucagon-like peptide-1 based therapies as a novel approach for chronic heart failure

Glucagon-like peptide-1 (GLP-1) is an endogenous peptide hormone whose metabolic effects have been exploited for glycaemic control in diabetes, but which also exerts important cardiovascular actions. We have recently reported that the GLP-1 mimetic, exendin-4, exerts clear benefits post-myocardial infarction via specific effects on extracellular matrix remodelling which is dysregulated in the diabetic heart (Robinson E et al, Basic Res Cardiol 2015; 110: 20), and have now shown similar cardioprotective actions in experimental diabetes, which are mediated via direct effects on infiltrating macrophages (Tate M et al, Basic Res Cardiol 2015; in press). Taken together with the apparent complexity of GLP-1 signalling and disappointing results of recent cardiovascular trials, our work strongly suggests that selective targeting of GLP-1 may be required in order to realise therapeutic benefit for both diabetic and non-diabetic heart failure patients. This is particularly important given the epidemic increase in the incidence of diabetes which is associated with a markedly enhanced susceptibility to cardiovascular stress.

Glucagon-like peptide-1 based therapies as a novel approach for chronic heart failure

Glucagon-like peptide-1 (GLP-1) is an endogenous peptide hormone whose metabolic effects have been exploited for glycaemic control in diabetes, but which also exerts important cardiovascular actions. We have recently reported that the GLP-1 mimetic, exendin-4, exerts clear benefits post-myocardial infarction via specific effects on extracellular matrix remodelling which is dysregulated in the diabetic heart (Robinson E et al, Basic Res Cardiol 2015; 110: 20), and have now shown similar cardioprotective actions in experimental diabetes, which are mediated via direct effects on infiltrating macrophages (Tate M et al, Basic Res Cardiol 2016; 111: 1). Taken together with the apparent complexity of GLP-1 signalling and disappointing results of recent cardiovascular trials, our work strongly suggests that selective targeting of GLP-1 may be required in order to realise therapeutic benefit for both diabetic and non-diabetic heart failure patients. This is particularly important given the epidemic increase in the incidence of diabetes which is associated with a markedly enhanced susceptibility to cardiovascular stress.

Plasma levels of intermedin (adrenomedullin-2) in healthy human volunteers and patients with heart failure

Intermedin/adrenomedullin-2 (IMD) is a member of the adrenomedullin/CGRP peptide family. Less is known about the distribution of IMD than for other family members within the mammalian cardiovascular system, particularly in humans. The aim was to evaluate plasma IMD levels in healthy subjects and patients with chronic heart failure. IMD and its precursor fragments, preproIMD25–56 and preproIMD57–92, were measured by radioimmunoassay in 75 healthy subjects and levels of IMD were also compared to those of adrenomedullin (AM) and mid-region proadrenomedullin45–92 (MRproAM45–92) in 19 patients with systolic heart failure (LVEF < 45%). In healthy subjects, plasma levels (mean + SE) of IMD (6.3 + 0.6 pg ml−1) were lower than, but correlated with those of AM (25.8 + 1.8 pg ml−1; r = 0.49, p < 0.001). Plasma preproIMD25–56 (39.6 + 3.1 pg ml−1), preproIMD57–92 (25.9 + 3.8 pg ml−1) and MRproAM45–92 (200.2 + 6.7 pg ml−1) were greater than their respective bioactive peptides. IMD levels correlated positively with BMI but not age, and were elevated in heart failure (9.8 + 1.3 pg ml−1, p < 0.05), similarly to MRproAM45–92 (329.5 + 41.9 pg ml−1, p < 0.001) and AM (56.8 + 10.9 pg ml−1, p < 0.01). IMD levels were greater in heart failure patients with concomitant renal impairment (11.3 + 1.8 pg ml−1) than those without (6.5 + 1.0 pg ml−1; p < 0.05). IMD and AM were greater in patients receiving submaximal compared with maximal heart failure drug therapy and were decreased after 6 months of cardiac resynchronization therapy. In conclusion, IMD is present in the plasma of healthy subjects less abundantly than AM, but is similarly correlated weakly with BMI. IMD levels are elevated in heart failure, especially with concomitant renal impairment, and tend to be reduced by high intensity drug or pacing therapy.

Quantitative structure-hydrophobicity relationships of molecular fragments and beyond

Quantitative structure-property relationship (QSPR) models were firstly established for the hydrophobic substituent constant (πX) using the theoretical descriptors derived solely from electrostatic potentials (EPSs) at the substituent atoms. The descriptors introduced are found to be related to hydrogen-bond basicity, hydrogen-bond acidity, cavity, or dipolarity/polarizability terms in linear solvation energy relationship, which endows the models good interpretability. The predictive capabilities of the models constructed were also verified by rigorous Monte Carlo cross-validation. Then, eight groups of meta- or para- disubstituted benzenes and one group of substituted pyridines were investigated. QSPR models for individual systems were achieved with the ESP-derived descriptors. Additionally, two QSPR models were also established for Rekker's fragment constants (foct), which is a secondary-treatment quantity and reflects average contribution of the fragment to logP. It has been demonstrated that the descriptors derived from ESPs at the fragments, can be well used to quantitatively express the relationship between fragment structures and their hydrophobic properties, regardless of the attached parent structure or the valence state. Finally, the relations of Hammett σ constant and ESP quantities were explored. It implies that σ and π, which are essential in classic QSAR and represent different type of contributions to biological activities, are also complementary in interaction site.

Naturally-occurring TGR5 agonists modulating glucagon-like peptide-1 biosynthesis and secretion

Selective GLP-1 secretagogues represent a novel potential therapy for type 2 diabetes mellitus. This study examined the GLP-1 secretory activity of the ethnomedicinal plant, Fagonia cretica, which is postulated to possess anti-diabetic activity. After extraction and fractionation extracts and purified compounds were tested for GLP-1 and GIP secretory activity in STC-1 pGIP/neo cells. Intracellular levels of incretin hormones and their gene expression were also determined. Crude F. cretica extracts stimulated both GLP-1 and GIP secretion, increased cellular hormone content, and upregulated gene expression of proglucagon, GIP and prohormone convertase. However, ethyl acetate partitioning significantly enriched GLP-1 secretory activity and this fraction underwent bioactivity-guided fractionation. Three isolated compounds were potent and selective GLP-1 secretagogues: quinovic acid (QA) and two QA derivatives, QA-3β-O-β-D-glycopyranoside and QA-3β-O-β-D-glucopyranosyl-(28→1)-β-D-glucopyranosyl ester. All QA compounds activated the TGR5 receptor and increased intracellular incretin levels and gene expression. QA derivatives were more potent GLP-1 secretagogues than QA. This is the first time that QA and its naturally-occurring derivatives have been shown to activate TGR5 and stimulate GLP-1 secretion. These data provide a plausible mechanism for the ethnomedicinal use of F. cretica and may assist in the ongoing development of selective GLP-1 agonists.

Host Defence Peptide LL-37; Effects of truncation on antimicrobial activity

Background: The oral cavity is an ideal environment for colonisation by micro-organisms. A first line of defence against microbial infection is the secretion of broad spectrum host defence peptides (HDPs). In the current climate of antibiotic resistance, exploiting naturally occurring HDPs or synthetic derivatives (mimetics) to combat infection is particularly appealing. The human cathelicidin, LL-37 is one such HDP expressed ubiquitously by epithelial cells and neutrophils. LL-37 exhibits the ability to bind lipopolysaccharide (LPS) and displays broad spectrum activity against a wide range of bacteria. The current study focuses on truncation of LL-37 and defining the antimicrobial and LPS binding activity of the resultant mimetics. Objectives: To assess the antimicrobial and LPS binding activity of LL-37 and three truncated mimetics (KE-18, EF-14 and KR-12). Methods: Peptides were synthesised in-house by Fmoc solid phase peptide synthesis or obtained commercially. Antimicrobial activity was determined using a radial diffusion assay and ability to bind LPS was determined by indirect ELISA. Results: LL-37 and mimetics displayed antimicrobial activity against Streptococcus mutans and Enterococcus Faecalis. KE-18 and KR-12 were shown to possess antimicrobial activity against both pathogens whereas EF-14 was the least antimicrobial. In terms of LPS binding, KE-18 and KR-12 were both effective whereas EF-14 showed the least activity of the three mimetics. Conclusion: Truncation of LL-37 can yield peptides which retain antimicrobial activities and have the ability to bind LPS. Interestingly in some cases the truncation of LL-37 produced mimetics with greater potency than the parent molecule in terms of antimicrobial activity and LPS binding. This work was funded by DEL and the Diabetes Wellness Foundation.

Effects of LL-37 Peptide Mimetics on Gingival Fibroblast Function

Host defence peptides, including the cathelicidin LL-37, play an important role in mucosal immunity, functioning as both antimicrobial agents and modulators of the inflammatory response. In the current climate of antibiotic resistance, the idea of using naturally occurring antimicrobial peptides, or their synthetic mimetics, to combat oral infection is particularly appealing. Objectives: The aim of this study was to investigate the effects of parent LL-37, and two peptide mimetics (KR-12 and KE-18), on cytokine expression and response to bacterial challenge by gingival fibroblasts. Methods: KR-12 and KE-18 are peptide mimetics of the biologically active, mid-region sequence of LL-37. The effects of commercially available LL-37, KR-12 and KE-18 on gingival fibroblast response to E coli and P gingivalis LPS challenge, analysed by IL-6 and IL-8 expression, were determined in cell culture by ELISA. The direct effects of each peptide on IL-6, IL-8, CXCL-1 and HGF expression were also determined by ELISA. The MTT assay was used to evaluate peptide effects on fibroblast viability. Results: LL-37 and KE-18, but not KR-12, inhibited LPS induction of inflammatory cytokine expression and directly stimulated CXCL-1 production by fibroblasts. All 3 peptides stimulated production of IL-8 and HGF. Neither LL-37 nor KE-12 affected cell viability, while KE-18, at higher concentrations, induced cell death. Conclusions: Shorter, peptide mimetics of LL-37, in particular KE-18, retain the immunomodulatory effects of the parent molecule and possess excellent potential as therapeutic agents in the treatment of oral infections including periodontal disease.

Identification and bioactivity evaluation of a novel bradykinin inhibitory peptide from the skin secretion of Chinese large odorous frog, Odorrana livida

A novel peptide was isolated from the skin secretion of Chinese large odorous frog, Odorrana livida, and was named as Rana-BI. The cDNA sequencing was obtained by 'shotgun' cloning. The amino acid sequence of the mature peptide was identified as Gly-Leu-Leu-Ser-Gly-Lys-Ser-Val-Lys-Gly-Ser-Ile-OH by automated Edman degradation, and the molecular weight of the peptide was confirmed to be 1144.68 Da by MALDI-TOF and liquid chromatography/MS. Subsequently, the bioactivity of synthetic peptide was evaluated by smooth muscle assay using isolated rat bladder preparation. It was demonstrated that Rana-BI inhibited the contraction of rat bladder induced by bradykinin. Comparing with other peptides by searching from database, the primary structure of Rana-BI showed high similarity with that of an antimicrobial peptide of Rana family (12/12 residues). These data revealed a novel biological function of this peptide