Improved burn wound healing using a bioactive peptide

[Excerpt] Antimicrobial peptides (AMPs) are good candidates to treat burn wounds, a major cause of morbidity, impaired life quality and resources consumption in developed countries. We took advantage of a commercially available hydrogel, Carbopol, a vehicle for topical administration that maintains a moist environment within the wound site. We hypothesized that the incorporation of LLKKK18 conjugated to dextrin would improve the healing process in rat burns. Whereas the hydrogel improves healing, LLKKK18 released from the dextrin conjugates further accelerates wound closure, and simultaneously improving the quality of healing. Indeed, the release of LLKKK18 reduces oxidative stress and inflammation (low neutrophil and macrophage infiltration and pro-inflammatory cytokines levels). Importantly, it induced a faster resolution of the inflammatory stage through early M2 macrophage recruitment. In addition, LLKKK18 stimulates angiogenesis (increased VEGF and microvessel development in vivo), potentially contributing to more effective transport of nutrients and cytokines. Moreover, collagen staining evaluated by Masson’s Trichrome was visually much more intense after treatment with LLKKK18, suggesting higher collagen deposition. (...)

Hemopressin: a novel bioactive peptide derived from the alpha1-chain of hemoglobin

Hemopressin (PVNFKFLSH), a novel bioactive peptide derived from the alpha1-chain of hemoglobin, was originally isolated from rat brain homogenates. Hemopressin causes hypotension in anesthetized rats and is metabolized in vivo and in vitro by endopeptidase 24.15 (EP24.15), neurolysin (EP24.16), and angiotensin-converting enzyme (ACE). Hemopressin also exerts an antinociceptive action in experimental inflammatory hyperalgesia induced by carrageenin or bradykinin via a mechanism that is independent of opioids. These findings suggest that this peptide may have important regulatory physiological actions in vivo.

Self-assembly of a dual functional bioactive peptide amphiphile incorporating both matrix metalloprotease substrate and cell adhesion motifs

We describe a bioactive lipopeptide that combines the capacity to promote the adhesion and subsequent self-detachment of live cells, using template-cell-environment feedback interactions. This self-assembling peptide amphiphile comprises a diene-containing hexadecyl lipid chain (C16e) linked to a matrix metalloprotease-cleavable sequence, Thr-Pro-Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln, and contiguous with a cell-attachment and signalling motif, Arg-Gly-Asp-Ser. Biophysical characterisation revealed that the PA self-assembles into 3 nm diameter spherical micelles above a critical aggregation concentration (cac). In addition, when used in solution at 5–150 nM (well below the cac), the PA is capable of forming film coatings that provide a stable surface for human corneal fibroblasts to attach and grow. Furthermore, these coatings were demonstrated to be sensitive to metalloproteases expressed endogenously by the attached cells, and consequently to elicit the controlled detachment of cells without compromising their viability. As such, this material constitutes a novel class of multi-functional coating for both fundamental and clinical applications in tissue engineering.

Purification and characterization of an antimicrobial peptide produced by Pseudomonas sp strain 4B

An antimicrobial peptide produced by a bacterium isolated from the effluent pond of a bovine abattoir was purified and characterized. The strain was characterized by biochemical profiling and 16S rDNA sequencing as Pseudomonas sp. The antimicrobial peptide was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. Direct activity on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was observed. A major band on SDS-PAGE suggested that the antimicrobial peptide has a molecular mass of about 30 kDa. The substance was inhibitory to a broad range of indicator strains, including pathogenic and food spoilage bacteria such as Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, among other. The partially purified antimicrobial substance remained active over a wide temperature range and was resistant to all proteases tested. This substance showed different properties than other antimicrobials from Pseudomonas species, suggesting a novel antimicrobial peptide was characterized.

Peptide separation of commercial fermented milk during refrigerated storage

Milk is an important source of bioactive compounds. Many of these compounds are released during fermentation and refrigerated storage. The aim of this study was to determine the release of peptides by lactic acid bacteria in commercial fermented milk during refrigerated storage. The size and profile of peptides were analyzed by polyacrylamide gel electrophoresis and sizeexclusion HPLC. During electrophoresis, it was observed that the peptides were released from caseins, whereas β-lactoglobulin was the whey protein with the highest degradation. HPLC analysis confirmed the pattern of peptide formation observed in electrophoresis. Two fractions lower than 2 kDa with aromatic amino acids in their structure were separated. These results were consistent with those reported for structures of peptides with antihypertensive activity. Therefore, the presence of aromatic amino acids in the peptide fractions obtained increases the likelihood of finding peptides with such activity in refrigerated commercial fermented milk. In conclusion, during cold storage, peptides with different molecular weights are released and accumulated. This could be due to the action of proteinases and peptidases of the proteolytic system in lactic acid bacteria.

Bioactive peptides from food proteins: new opportunities and challenges

Food proteins such as milk and soy are a rich source of bioactive peptides. In the last decade, research into this area has intensified and new bioactive peptide sequences have been discovered with a range of apparent biological functions; for example, antihypertensive, antioxidant, and antimicrobial effects and opiate-like qualities have been reported. These peptides could therefore lead to the development of important functional food products and ingredients for the prevention and even treatment of chronic diseases such as cardiovascular disease and cancer. Peptides can be produced by fermentation with dairy starters for instance, and by enzymatic hydrolysis with pancreatic and microbial enzymes. Further purification is typically carried out by membrane filtration and/or chromatographic methods. The production of novel bioactive peptides and their incorporation into functional food products poses several technological challenges as well as regulatory and marketing issues. Proof of efficacy is of paramount importance; this should be verified by conducting appropriate tests in vivo in animals and in humans. In addition, tests for cytotoxicity and allergenicity must be conducted. Despite all of these hurdles, scientific evidence is increasingly demonstrating the health benefits of diet-based disease prevention, and therefore new developments in this area are likely to continue both at the research and the commercialisation level.

Orpotrin: A novel vasoconstrictor peptide from the venom of the Brazilian Stingray Potamotrygon gr. orbignyi

Characterization of the peptide content of venoms has a number of potential benefits for basic research, clinical diagnosis, development of new therapeutic agents, and production of antiserum. In order to analyze in detail the peptides and small proteins of crude samples, techniques such as chromatography and mass spectrometry have been employed. The present study describes the isolation, biochemical characterization, and sequence determination of a novel peptide, named Orpotrin from the venom of Potamotrygon gr. orbignyi. The natural peptide was shown to be effective in microcirculatory environment causing a strong vasoconstriction. The peptide was fully sequenced by de novo amino acid sequencing with mass spectrometry and identified as the novel peptide. Its amino acid sequence, HGGYKPTDK, aligns only with creatine kinase residues 97-105, but has no similarity to any bioactive peptide. Therefore, possible production of this peptide from creatine kinase by limited proteolysis is discussed. Taken together, the results indicate the usefulness of this single-step approach for low molecular mass compounds in complex samples such as venoms. (c) 2006 Elsevier B.V. All rights reserved.

Microbially derived bioactive peptides to improve human health

This thesis describes a study of various methods to produce bioactive peptides. Initially, the generation of anti-Cronobacter spp. peptides by fermentation of milk protein is described. Lactobacillus johnsonii DPC6026 was used to generate two previously described antimicrobial peptides. Phenotypic analysis indicated unsatisfactory casein hydrolysis. The genome of the strain was sequenced and annotated. Results showed a number of unique features present, most notably a large symmetrical inversion of approximately 750kb in comparison with the human isolate L. johnsonii NCC 533. The data suggest significant genetic diversity and intra-species genomic rearrangements within the L. johnsonii spp.. Cronobacter spp. have emerged as pathogens of concern to the powdered infant formula industry. Chapters 3 and 4 of this thesis describe novel methods to generate two antimicrobial peptides, Caseicin A and B. In Chapter 3 a bank of Bacillus strains was generated and investigated for caseicin production. Following casein hydrolysis by specific B. cereus and B. thuringiensis strains the peptides of interest were generated. Chapter 4 describes a sterile enzymatic method to generate peptides from casein. Bioinformatic tools were used to predict enzymes capable of liberating caseicin peptides from casein. Hydrolysates were generated using suitable enzymes, examined and some were found to produce peptides with activity against Cronobacter spp.. This study establishes a potential industrial-grade method to generate antimicrobial peptides. Administration of GLP-1 leads to improved glycaemic control in diabetes patients. Generation of a recombinant lactic acid bacteria capable of producing a GLP-1 analogue is described in Chapter 5. In-vivo analysis confirmed insulinotropic activity. The results illustrate a method using bacteriocin producing cellular machinery to generate bioactive peptides. This thesis describes the generation of bioactive peptides by bacterial fermentation, tailored enzymatic hydrolysis and recombinant bacterial methods. The techniques described contribute to bioactive peptide research with regards novel methods of production and industrial scale-up.

Effect of biofunctionalized implant surface on osseointegration: a histomorphometric study in dogs

Among the different properties that influence bone apposition around implants, the chemical or biochemical composition of implant surface may interfere on its acceptance by the surrounding bone. The aim of this study was to investigate if a biofunctionalization of implant surface influences the bone apposition in a dog model and to compare it with other surfaces, such as a microstructured created by the grit-blasting/acid-etching process. Eight young adult male mongrel dogs had the bilateral mandibular premolars extracted and each one received 6 implants after 12 weeks, totaling 48 implants in the experiment. Four groups of implants were formed with the same microrough topography with or without some kind of biofunctionalization treatment. After histomorphometric analysis, it was observed that the modified microstructured surface with a "low concentration of the bioactive peptide" provided a higher adjacent bone density (54.6%) when compared to the other groups (microstructured + HA coating = 46.0%, microstructured only = 45.3% and microstructured + "high concentration of the bioactive peptide" = 40.7%), but this difference was not statistically significant. In conclusion, biofunctionalization of the implant surface might interfere in the bone apposition around implants, especially in terms of bone density. Different concentrations of bioactive peptide lead to different results.

Angiotensin II as an Inducer of Atherosclerosis: Evidence from Mouse Studies

Mechanisms responsible for atherosclerotic plaque development, destabilization, and rupture are still largely unknown. Angiotensin II, the main bioactive peptide of renin angiotensin system, has been shown to be critically involved in the pathogenesis of atherosclerosis and vulnerable plaque. Experimental studies in hypercholesterolemic mouse models with high circulating Angiotensin II levels, provide direct evidence that Angiotensin II induces plaque vulnerability partly by 1/ downregulating vascular expression of anti-atherosclerotic genes and/or upregulating expression of pro-atherosclerotic genes, and 2/ skewing the systemic lymphocyte Th1/Th2 balance towards a proinflammatory Th1 response in early disease phase. Further understanding the pro-atherosclerotic mechanisms of Angiotensin II and associated signaling pathways will help to design better therapeutic strategies for reducing the burden of atherosclerotic cardiovascular disease.

Ghrelin attenuates the growth of HO-8910 ovarian cancer cells through the ERK pathway

Ovarian cancer is one of the most common causes of death from gynecologic tumors and is an important public health issue. Ghrelin is a recently discovered bioactive peptide that acts as a natural endogenous ligand of the growth hormone secretagogue receptor (GHSR). Several studies have identified the protective effects of ghrelin on the mammalian reproductive system. However, little research has been done on the effects of ghrelin on ovarian cancer cells, and the underlying mechanisms of these effects. We sought to understand the potential involvement of mitogen-activated protein kinases (MAPKs) in ghrelin-mediated inhibition of growth of the ovarian line HO-8910. We applied different concentrations of ghrelin and an inhibitor of the ghrelin receptor (D-Lys3-GHRP-6) to HO-8910 cells and observed the growth rate of cells and changes in phosphorylation of the MAPKs ERK1/2, JNK and p38. We discovered that ghrelin-induced apoptosis of HO-8910 cells was though phosphorylated ERK1/2, and that this phosphorylation (as well as p90rsk phosphorylation) was mediated by the GHSR. The ERK1/2 pathway is known to play an essential part in the ghrelin-mediated apoptosis of HO-8910 cells. Hence, our study suggests that ghrelin inhibits the growth of HO-8910 cells primarily through the GHSR/ERK pathway.

Production optimization and characterization of immunomodulatory peptides obtained from fermented goat placenta

The goat placental immunomodulatory peptides were produced by fermentation with Aspergillus Niger. The objective of the present study was to investigate the effects of fermentation parameters (carbon source content, pH, and time) on spleen lymphocyte proliferation for the highest immune activity of the fermentation broth using response surface methodology (RSM). According to the data analysis by the Design-Expert® software, the stimulation index value (23.51%), which is the maximum immune activity, was obtained under the following conditions: content of carbon source 1.97 g·L-1, initial pH 5.0, and 74.43 h of fermentation time. Under the optimized fermentation conditions, at a certain concentration range, the fermentation broth produced a significant effect on the proliferation of mouse spleen lymphocytes. Ultrafiltration technique was performed to separate the fermentation broth with different MW (molecular weight). It was found that peptides in the range of <10 KDa were the main bioactivity fractions for the immunomodulatory and antioxidant activities.

The bioactivity of composite Fmoc-RGDS-collagen gels

The incorporation of small bioactive peptide motifs within robust hydrogels constitutes a facile procedure to chemically functionalise cell and tissue scaffolds. In this study, a novel approach to utilise Fmoc-linked peptide amphiphiles comprising the bio-functional cell-adhesion RGDS motif within biomimetic collagen gels was developed. The composite scaffolds thus created were shown to maintain the mechanical properties of the collagen gel while presenting additional bio-activity. In particular, these materials enhanced the adhesion and proliferation of viable human corneal stromal fibroblasts by 300% compared to nonfunctionalised gels. Furthermore, the incorporation of Fmoc-RGDS nanostructures within the collagen matrix significantly suppressed gel shrinkage resulting from the contractile action of encapsulated fibroblasts once activated by serum proteins. These mechanical and biological properties demonstrate that the incorporation of peptide amphiphiles provides a suitable and easy method to circumvent specific biomaterial limitations, such as cell-derived shrinkage, for improved performance in tissue engineering and regenerative medicine applications.

Potencial imunogênico dos curativos bioativos: aspectos imunhematológicos e leucoplaquetários

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)