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Antimicrobial Peptides Secreted From Human Cryopreserved Viable Amniotic Membrane Contribute to its Antibacterial Activity.

ABSTRACT: Chronic wounds remain a large problem in the field of medicine and are often associated with risk of infection and amputation. Recently, a commercially available human cryopreserved viable amniotic membrane (hCVAM) has been shown to effectively promote wound closure and reduce wound-related infections. A sprevious study indicates that hCVAM can inhibit the growth of bacteria associated with chronic wounds. In the present study, we investigated the mechanism of hCVAM antimicrobial activity. Our data demonstrate that antimicrobial activities against common pathogens in chronic wounds such as P.aeruginosa, S.aureus and Methicillin-resistant S.aureus (MRSA) are mediated via the secretion of soluble factors by viable cells in hCVAM and that these factors are proteins in nature. Further, we show that genes for antimicrobial peptides (AMPs) including human beta-defensins (HBDs) are expressed by hCVAM and that expression levels positively correlate with antimicrobial activity of hCVAM. At the protein level, our data indicate that HBD2 and HBD3 are secreted by hCVAM and directly contribute to its activity against P. aeruginosa. These data provide evidence that soluble factors including AMPs are hCVAM antimicrobial agents and are consistent with a role for AMPs in mediating antimicrobial properties of the membrane.

SUBMITTER: Mao Y

PROVIDER: S-EPMC5651856 | biostudies-other | 2017 Oct

REPOSITORIES: biostudies-other

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Antimicrobial Peptides Secreted From Human Cryopreserved Viable Amniotic Membrane Contribute to its Antibacterial Activity.

Mao Yong Y Hoffman Tyler T Singh-Varma Anya A Duan-Arnold Yi Y Moorman Matthew M Danilkovitch Alla A Kohn Joachim J

Scientific reports 20171020 1

Chronic wounds remain a large problem in the field of medicine and are often associated with risk of infection and amputation. Recently, a commercially available human cryopreserved viable amniotic membrane (hCVAM) has been shown to effectively promote wound closure and reduce wound-related infections. A sprevious study indicates that hCVAM can inhibit the growth of bacteria associated with chronic wounds. In the present study, we investigated the mechanism of hCVAM antimicrobial activity. Our d ...[more]

PMID: 29057887

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Project description:IntroductionChronic wounds remain a major clinical challenge. Human cryopreserved viable amniotic membrane (hCVAM) is among the most successful therapies, but the mechanisms of action remain loosely defined. Because proper regulation of macrophage behavior is critical for wound healing with biomaterial therapies, we hypothesized that hCVAM would positively regulate macrophage behavior in vitro, and that soluble factors released from the hCVAM would be important for this effect.Materials and methodsPrimary human pro-inflammatory (M1) macrophages were seeded directly onto intact hCVAM or cultured in separation via transwell inserts (Soluble Factors) in the presence of pro-inflammatory stimuli (interferon-γ and lipopolysaccharide) to simulate the chronic wound environment. Macrophages were characterized after 1 and 6 days using multiplex gene expression analysis of 37 macrophage phenotype- and angiogenesis-related genes via NanoString™, and protein content from conditioned media collected at days 1, 3 and 6 was analyzed via enzyme linked immunosorbent assays.Results and discussionGene expression analysis showed that Soluble Factors promoted significant upregulation of pro-inflammatory marker IL1B on day 1 yet downregulation of TNF on day 6 compared to the M1 macrophage control. In contrast, intact hCVAM, which includes both extracellular matrix, viable cells, and soluble factors, promoted downregulation of pro-inflammatory markers TNF, CCL5 and CCR7 on day 1 and endothelial receptor TIE1 on day 6, and upregulation of the anti-inflammatory marker IL10 on day 6 compared to the M1 Control. Other genes related to inflammation and angiogenesis (MMP9, VEGF, SPP1, TGFB1, etc.) were differentially regulated between the Soluble Factors and intact hCVAM groups at both time points, though they were not expressed at significantly different levels compared to the M1 Control. Interestingly, Soluble Factors promoted increased secretion of the proinflammatory cytokine tumor necrosis factor-α (TNF-α), while direct contact with hCVAM inhibited secretion of TNF, relative to the M1 Control. Both Soluble Factors and intact hCVAM inhibited secretion of MMP9 and VEGF, pro-inflammatory proteins that are critical for angiogenesis and remodeling, compared to the M1 Control, with intact hCVAM having a stronger effect.ConclusionsIn a simulated pro-inflammatory environment, intact hCVAM has distinct anti-inflammatory effects on primary human macrophages, and direct macrophage contact with intact hCVAM is required for these effects. These findings are important for the design of next generation immunomodulatory biomaterials for wound repair and regenerative medicine that may include living cells, soluble factors, or a controlled drug delivery system.

Project description:BackgroundFor hallux rigidus, dorsal cheilectomy remains a treatment option even with advances in interposition techniques and devices. Cheilectomy aims to alleviate dorsal impingement and improve pain and function as well as range of motion. Cryopreserved umbilical cord allograft, with properties to mitigate inflammation and scar formation, has theoretical benefit for improving outcomes following cheilectomy. In this first prospective randomized and blinded cheilectomy study reported, we aimed to compare outcomes between cheilectomy alone and cheilectomy with umbilical cord allograft.MethodsPatients were randomized to cheilectomy alone (CA) or cheilectomy with cryopreserved umbilical cord (ie, amniotic membrane-umbilical cord [AM-UC]). Patients were evaluated with American Orthopaedic Foot & Ankle Society (AOFAS), Foot Function Index (FFI), and visual analog scale (VAS) pain outcomes collected preoperatively and at 6 months and 1 year postoperatively. In addition, radiographic range of motion data were collected using stress radiographs. Fifty-one patients (26 AM-UC, 25 CA) completed the study, with 5 bilateral surgeries in the AM-UC group and 2 in the CA group, totaling 31 and 27 feet, respectively.ResultsThe AM-UC group had statistically significant improved AOFAS and FFI scores at 1 year compared with the CA group, but there was no difference at 6 months. There was no significant difference between groups for VAS-pain scores at any time point, but overall VAS-pain improved in both groups from preoperative values. There was no significant difference in range of motion (total arc) between groups and changes in range of motion (total arc) in both groups from preoperative to 1 year postoperative were small.ConclusionWe present the results of the first randomized and blinded prospective study of cheilectomy surgery patients. When appropriately selected, cheilectomy remains a good option for patients with symptomatic hallux rigidus. Cryopreserved umbilical cord is a potential adjuvant to cheilectomy, with 1-year results showing improvements in functional outcome scores.Level of evidenceLevel II, prospective comparative study.

Project description:Antimicrobial activity of many AMPs can be improved by lysine-to-arginine substitution due to a more favourable interaction of arginine guanidinium moiety with bacterial membranes. In a previous work, the structural and functional characterization of an amphipathic antimicrobial peptide named RiLK1, including lysine and arginine as the positively charged amino acids in its sequence, was reported. Specifically, RiLK1 retained its β-sheet structure under a wide range of environmental conditions (temperature, pH, and ionic strength), and exhibited bactericidal activity against Gram-positive and Gram-negative bacteria and fungal pathogens with no evidence of toxicity on mammalian cells. To further elucidate the influence of a lysine-to-arginine replacement on RiLK1 conformational properties, antimicrobial activity and peptide-liposome interaction, a new RiLK1-derivative, named RiLK3, in which the lysine is replaced with an arginine residue, was projected and characterised in comparison with its parental compound. The results evidenced that lysine-to-arginine mutation not only did not assure an improvement in the antimicrobial potency of RiLK1 in terms of bactericidal, virucidal and fungicidal activities, but rather it was completely abolished against the hepatitis A virus. Therefore, RiLK1 exhibited a wide range of antimicrobial activity like other cationic peptides, although the exact mechanisms of action are not completely understood. Moreover, tryptophan fluorescence measurements confirmed that RiLK3 bound to negatively charged lipid vesicles with an affinity lower than that of RiLK1, although no substantial differences from the structural and self-assembled point of view were evidenced. Therefore, our findings imply that antimicrobial efficacy and selectivity are affected by several complex and interrelated factors related to substitution of lysine with arginine, such as their relative proportion and position. In this context, this study could provide a better rationalisation for the optimization of antimicrobial peptide sequences, paving the way for the development of novel AMPs with broad applications.

Project description:The self-assembly behavior and antimicrobial activity of two designed amphiphilic peptides, R3F3 and R4F4, containing short hydrophobic phenylalanine (F) and cationic arginine (R) sequences, are investigated. The conformation of the peptides was examined using circular dichroism and FTIR spectroscopy, which show that they have a disordered secondary structure. Concentration-dependent fluorescence assays show the presence of a critical aggregation concentration (cac) for each peptide. Above the cac, small-angle X-ray scattering (SAXS) and transmission electron microscopy (TEM) reveal a population of twisted tapes for R3F3 and nanosheets for R4F4. The interaction of the peptides with model bacterial membranes comprising mixtures of the lipids DPPG [1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol] and DPPE [1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine], was studied using SAXS and cryogenic-TEM. Analysis of the SAXS structure factor indicates that R3F3 interacts with lipid bilayers by inducing correlation between bilayers, whereas R4F4 interacts with the bilayers causing an increase in polydispersity of the vesicle wall thickness. Both peptides break vesicles with a 1:3 DPPG:DPPE composition, which is close to the ratio of PG and PE lipids observed in the lipid membrane of Pseudomonas aeruginosa, a pathogen responsible for serious infections and which has developed antimicrobial resistant strains. Both peptides show activity against this bacterium in planktonic form. Peptide R4F4 shows particularly strong bioactivity against this microbe, with a minimum inhibitory concentration (MIC) value in the range of concentrations where the peptide is cytocompatible. It was further shown to have activity against other Pseudomonas species including the common plant pathogen Pseudomonas syringae. Finally, we show that R4F4 inhibits the development of P. aeruginosa biofilms. This was examined in detail and a proposed mechanism involving binding of the signaling molecule c-di-GMP is suggested, based on circular dichroism spectroscopy studies and Congo red assays of extracellular polysaccharides produced by the stressed bacteria. Thus, R4F4 is a promising candidate antimicrobial peptide with activity against Pseudomonas species.

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Antimicrobial Peptides Secreted From Human Cryopreserved Viable Amniotic Membrane Contribute to its Antibacterial Activity.

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Antimicrobial Peptides Secreted From Human Cryopreserved Viable Amniotic Membrane Contribute to its Antibacterial Activity.

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