Antibacterial Efficacy of a Chitosan-Based Hydrogel Modified With Epsilon-Poly-l-Lysine Against Pseudomonas aeruginosa in a Murine-Infected Burn Wound Model.

INTRODUCTION: Because antibiotic resistance is increasing worldwide and the leading cause of death in burn patients is an infection, an urgent need exists for nonantibiotic approaches to eliminate multidrug-resistant bacteria from burns to prevent their systemic dissemination and sepsis. We previously demonstrated the significant antibiofilm activity of a chitosan (CS) hydrogel containing the antimicrobial peptide epsilon-poly-l-lysine (EPL) against multidrug-resistant Pseudomonas aeruginosa using ex vivo porcine skin. In this study, we evaluated the in vivo antibacterial efficacy of a CS/EPL hydrogel against P. aeruginosa in a murine burn wound infection model. MATERIALS AND METHODS: Full-thickness burns were created on the dorsum using a heated brass rod and were inoculated with bioluminescent, biofilm-forming P. aeruginosa (Xen41). Mice were treated with CS/EPL, CS, or no hydrogel applied topically 2 or 24 hours after inoculation to assess the ability to prevent or eradicate existing biofilms, respectively. Dressing changes occurred daily for 3 days, and in vivo bioluminescence imaging was performed to detect and quantitate bacterial growth. Blood samples were cultured to determine systemic infection. In vitro antibacterial activity and cytotoxicity against human primary dermal fibroblasts, keratinocytes, and mesenchymal stem cells were also assessed. RESULTS: CS/EPL treatment initiated at early or delayed time points showed a significant reduction in bioluminescence imaging signal compared to CS on days 2 and 3 of treatment. Mice administered CS/EPL had fewer bloodstream infections, lower weight loss, and greater activity than the untreated and CS groups. CS/EPL reduced bacterial burden by two orders of magnitude in vitro and exhibited low cytotoxicity against human cells. CONCLUSION: A topical hydrogel delivering the antimicrobial peptide EPL demonstrates in vivo efficacy to reduce but not eradicate established P. aeruginosa biofilms in infected burn wounds. This biocompatible hydrogel shows promise as an antimicrobial barrier dressing for the sustained protection of burn wounds from external bacterial contamination.

Priming With Toll-Like Receptor 3 Agonist Poly(I:C) Enhances Content of Innate Immune Defense Proteins but Not MicroRNAs in Human Mesenchymal Stem Cell-Derived Extracellular Vesicles.

Mesenchymal stem cells (MSCs) help fight infection by promoting direct bacterial killing or indirectly by modulating the acute phase response, thereby decreasing tissue injury. Recent evidence suggests that extracellular vesicles (EVs) released from MSCs retain antimicrobial characteristics that may be enhanced by pretreatment of parent MSCs with the toll-like receptor 3 (TLR3) agonist poly(I:C). Our aim was to determine whether poly(I:C) priming can modify EV content of miRNAs and/or proteins to gain insight into the molecular mechanisms of their enhanced antimicrobial function. Human bone marrow-derived MSCs were cultured with or without 1 µg/ml poly(I:C) for 1 h and then conditioned media was collected after 64 h of culture in EV-depleted media. Mass spectrometry and small RNA next-generation sequencing were performed to compare proteomic and miRNA profiles. Poly(I:C) priming resulted in 49 upregulated EV proteins, with 21 known to be important in host defense and innate immunity. In contrast, EV miRNA content was not significantly altered. Functional annotation clustering analysis revealed enrichment in biological processes and pathways including negative regulation of endopeptidase activity, acute phase, complement and coagulation cascades, innate immunity, immune response, and Staphylococcus aureus infection. Several antimicrobial peptides identified in EVs remained unaltered by poly(I:C) priming, including dermcidin, lactoferrin, lipocalin 1, lysozyme C, neutrophil defensin 1, S100A7 (psoriasin), S100A8/A9 (calprotectin), and histone H4. Although TLR3 activation of MSCs improves the proteomic profile of EVs, further investigation is needed to determine the relative importance of particular functional EV proteins and their activated signaling pathways following EV interaction with immune cells.

Antibiofilm activity of chitosan/epsilon-poly-L-lysine hydrogels in a porcine ex vivo skin wound polymicrobial biofilm model.

As antibiotic resistance continues to increase globally, there is an urgency for novel, non-antibiotic approaches to control chronic drug-resistant infections, particularly those associated with polymicrobial biofilm formation in chronic wounds. Also needed are clinically relevant polymicrobial biofilm models that can be utilized to assess the efficacy of innovative therapeutics against mature biofilms. We successfully developed a highly reproducible porcine ex vivo skin wound polymicrobial biofilm model using clinical isolates of multidrug-resistant Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and Candida albicans. This ex vivo biofilm model was then used to assess the antimicrobial and antibiofilm properties of an easily fabricated chitosan hydrogel incorporating the natural antimicrobial peptide epsilon-poly-L-lysine. Antimicrobial activity was evaluated against planktonic cultures in vitro and against mature biofilms ex vivo. The antibiofilm efficiency of the hydrogels was especially pronounced against Pseudomonas aeruginosa, whose counts were reduced by 99.98% after 2 hours in vitro and by 99.94% after treatment for 24 hours when applied to 24 hour ex vivo polymicrobial wound biofilms. The activity of the hydrogels was lower against Staphylococcus aureus and ineffective against Candida albicans. Gram, Hucker-Twort staining of paraffin sections revealed balanced polymicrobial communities in mature 48 hour untreated biofilms. Treatment of 48 or 72 hour biofilms for 2 or 3 days with hydrogels that were applied within 5 hours after inoculation resulted in an impressive 96% and 97% reduction in biofilm thickness compared to untreated biofilms, respectively (P < .001). Likewise, topical gel treatment for 24 hours reduced biofilm thickness by 84% and 70%, respectively, when applied to mature biofilms at 24 and 48 hours after inoculation (P < .001). Thus, this ex vivo wound biofilm model provides a useful means to assess the efficacy of novel treatments to prevent and eradicate polymicrobial biofilms consisting of multidrug-resistant Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and Candida albicans.

Oxidative Stress Alters Angiogenic and Antimicrobial Content of Extracellular Vesicles and Improves Flap Survival.

Extracellular vesicles (EVs) secreted from adipose-derived mesenchymal stem cells (ADSCs) (ADSC-EVs) improve flap survival after ischemia-reperfusion injury. Exposure of parent ADSCs to oxidative stress has been shown to enhance this effect, but mechanisms are unclear. We aimed to determine whether angiogenesis-promoting protein and microRNA (miRNA) content is altered in EVs after preconditioning with hydrogen peroxide (H2O2 ADSC-EVs) and whether H2O2 ADSC-EVs can increase viability of random pattern skin flaps. METHODS: EVs secreted by human ADSCs were isolated after culture in EV-depleted medium ± H2O2. Nanoparticle tracking analysis determined size and concentration of purified EVs. Mass spectrometry and small RNA next-generation sequencing were performed to compare proteomic and miRNA profiles. ADSC-EVs, H2O2 ADSC-EVs, or vehicle were injected into random pattern skin flaps of BALB/c mice (4-5 mice per group). Viable and necrotic areas were measured on day 7, and tissues underwent histologic analysis. RESULTS: Angiogenic and antimicrobial protein content of EVs was altered with H2O2 preconditioning. Functional enrichment analysis identified constitutive photomorphogenesis 9 signalosome (known to direct vascular endothelial growth factor production) as the major enriched Gene Ontology term unique to H2O2 ADSC-EVs. Two miRNAs were increased, and 12 (including 10 antiangiogenic miRNAs) were reduced in H2O2 ADSC-EVs. Enhanced viability (P < 0.05) of flaps treated with H2O2 ADSC-EVs compared with vehicle corresponded to increased capillary density in the H2O2 group (P < 0.001). CONCLUSION: Altered protein and miRNA content in ADSC-EVs after H2O2 pretreatment likely contributes to enhanced therapeutic effects on flap survival observed in preclinical models.

Inhibition of microRNA-124-3p as a novel therapeutic strategy for the treatment of Gulf War Illness: Evaluation in a rat model.

Gulf War Illness (GWI) is a chronic, multisymptom illness that continues to affect up to 30% of veterans deployed to the Persian Gulf during the 1990-1991 Gulf War. After nearly 30 years, useful treatments for GWI are lacking and underlying cellular and molecular mechanisms involved in its pathobiology remain poorly understood, although exposures to pyridostigmine bromide (PB) and pesticides are consistently identified to be among the strongest risk factors. Alleviation of the broad range of symptoms manifested in GWI, which involve the central nervous system, the neuroendocrine system, and the immune system likely requires therapies that are able to activate and inactivate a large set of orchestrated genes. Previous work in our laboratory using an established rat model of GWI identified persistent elevation of microRNA-124-3p (miR-124) levels in the hippocampus whose numerous gene targets are involved in cognition-associated pathways and neuroendocrine function. This study aimed to investigate the broad effects of miR-124 inhibition in the brain 9 months after completion of a 28-day exposure regimen of PB, DEET (N,N-diethyl-3-methylbenzamide), permethrin, and mild stress by profiling the hippocampal expression of genes known to play a critical role in synaptic plasticity, glucocorticoid signaling, and neurogenesis. We determined that intracerebroventricular infusion of a miR-124 antisense oligonucleotide (miR-124 inhibitor; 0.05-0.5 nmol/day/28 days), but not a negative control oligonucleotide, into the lateral ventricle of the brain caused increased protein expression of multiple validated miR-124 targets and increased expression of downstream target genes important for cognition and neuroendocrine signaling in the hippocampus. Off-target cardiotoxic effects were revealed in GWI rats receiving 0.1 nmol/day as indicated by the detection in plasma of 5 highly elevated protein cardiac injury markers and 6 upregulated cardiac-enriched miRNAs in plasma exosomes determined by next-generation sequencing. Results from this study suggest that in vivo inhibition of miR-124 function in the hippocampus is a promising, novel therapeutic approach to improve cognition and neuroendocrine dysfunction in GWI. Additional preclinical studies in animal models to assess feasibility and safety by developing a practical, noninvasive drug delivery system to the brain and exploring potential adverse toxicologic effects of miR-124 inhibition are warranted.

Evaluation of a Novel Hybrid Viable Bioprosthetic Mesh in a Model of Mesh Infection.

BACKGROUND: The reported incidence of mesh infection in contaminated operative fields is as high as 30% regardless of material used. Our laboratory previously showed that augmenting acellular bioprosthetic mesh with allogeneic mesenchymal stem cells (MSC) enhances resistance to bacterial colonization in vivo and preserves mesh integrity. This study's aim was to determine whether augmentation of non-crosslinked porcine dermis (Strattice) with commercially available, cryopreserved, viable MSC-containing human placental tissue (Stravix) similarly improves infection resistance after inoculation with Escherichia coli (E. coli) using an established mesh infection model. METHODS: Stravix was thawed per manufacturer's instructions and 2 samples were tested for cell viability using a Live/Dead Cell assay at the time of surgery. Rats (N = 20) were implanted subcutaneously with 1 piece of Strattice and 1 piece of hybrid mesh (Strattice + Stravix sutured at the corners). Rats were inoculated with either sterile saline or 106 colony-forming units of E. coli before wound closure (n = 10 per group). At 4 weeks, explants underwent microbiologic and histologic analyses. RESULTS: In E. coli-inoculated animals, severe or complete mesh degradation concurrent with abscess formation was observed in 100% (10/10) hybrid meshes and 90% (9/10) Strattice meshes. Histologic evaluation determined that meshes inoculated with E. coli exhibited severe acute inflammation, which correlated with bacterial recovery (P < 0.001). Viability assays performed at the time of surgery failed to verify the presence of numerous live cells in Stravix. CONCLUSIONS: Stravix cryopreserved MSC-containing human umbilical tissue does not improve infection resistance of a bioprosthetic mesh in vivo in rats after inoculation with E. coli.

Bone Marrow-Derived Mesenchymal Stem Cells Enhance Bacterial Clearance and Preserve Bioprosthetic Integrity in a Model of Mesh Infection.

BACKGROUND: The reported incidence of mesh infection in contaminated operative fields is as high as 30% regardless of the material used. Recently, mesenchymal stem cells (MSCs) have been shown to possess favorable immunomodulatory properties and improve tissue incorporation when seeded onto bioprosthetics. The aim of this study was to evaluate whether seeding noncrosslinked bovine pericardium (Veritas Collagen Matrix) with allogeneic bone marrow-derived MSCs improves infection resistance in vivo after inoculation with Escherichia coli (E. coli). METHODS: Rat bone marrow-derived MSCs at passage 3 were seeded onto bovine pericardium and cultured for 7 days before implantation. Additional rats (n = 24) were implanted subcutaneously with MSC-seeded or unseeded mesh and inoculated with 7 × 10(5) colony-forming units of E. coli or saline before wound closure (group 1, unseeded mesh/saline; group 2, unseeded mesh/E. coli; group 3, MSC-seeded mesh/E. coli; 8 rats per group). Meshes were explanted at 4 weeks and underwent microbiologic and histologic analyses. RESULTS: MSC-seeded meshes inoculated with E. coli demonstrated superior bacterial clearance and preservation of mesh integrity compared with E. coli-inoculated unseeded meshes (87.5% versus 0% clearance; p = 0.001). Complete mesh degradation concurrent with abscess formation was observed in 100% of rats in the unseeded/E. coli group, which is in contrast to 12.5% of rats in the MSC-seeded/E. coli group. Histologic evaluation determined that remodeling characteristics of E. coli-inoculated MSC-seeded meshes were similar to those of uninfected meshes 4 weeks after implantation. CONCLUSIONS: Augmenting a bioprosthetic material with stem cells seems to markedly enhance resistance to bacterial infection in vivo and preserve mesh integrity.

Long-term epigenetic alterations in a rat model of Gulf War Illness.

Gulf War Illness (GWI) is a chronic, multisymptom illness that affects 25% of the 700,000 US veterans deployed to the Persian Gulf during the 1990-1991 Gulf War. Central nervous system impairments are among the most common symptoms reported, including memory dysfunction and depression. After 25 years, the diagnosis remains elusive, useful treatments are lacking, and the cause is poorly understood, although exposures to pyridostigmine bromide (PB) and pesticides are consistently identified to be among the strongest risk factors. Epigenetic changes including altered microRNA (miRNA) expression and DNA methylation play an important role in learning, memory, and emotion regulation and have been implicated in various neurological disorders. In this study, we used an established rat model of GWI to determine whether 1) chronic alterations in miRNA expression and global DNA methylation and DNA hydroxymethylation are mechanisms involved in the pathobiology of GWI, and 2) plasma exosome small RNAs may serve as potential noninvasive biomarkers of this debilitating disease. One year after a 28-day exposure regimen of PB, DEET (N,N-diethyl-3-methylbenzamide), permethrin, and mild stress, expression of 84 mature miRNAs and global 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) content were analyzed in the brains of GWI rats and vehicle controls by PCR array and enzyme-linked immunosorbent assay, respectively. Plasma exosome RNA next-generation sequencing analysis was performed in pooled samples to discover potential noninvasive biomarkers. We found that combined exposure to low doses of GW-related chemicals and mild stress caused epigenetic modifications in the brain that persisted one year after exposure, including increased expression of miR-124-3p and miR-29b-3p in the hippocampus and regional alterations in global 5mC and 5hmC content. GW-relevant exposures also induced the differential expression of two piwi-interacting RNAs (piRNAs) in circulation (piR-007899 and piR-019162). Results from this study implicate a role for epigenetic alterations in GWI. Evaluation of the diagnostic potential of plasma exosome RNAs in veterans with GWI is warranted.

Investigation of a novel molecular descriptor for the lead optimization of 4-aminoquinazolines as vascular endothelial growth factor receptor-2 inhibitors: application for quantitative structure-activity relationship analysis in lead optimization.

We investigated the use of infrared vibrational frequency of ligands as a potential novel molecular descriptor in three different molecular target and chemical series. The vibrational energy of a ligand was approximated from the sum of infrared (IR) absorptions of each functional group within a molecule and normalized by its molecular weight (MDIR). Calculations were performed on a set of 4-aminoquinazolines with similar docking scores for the VEGFR2/KDR receptor. 4-Aminoquinazolines with MDIR values ranging 192-196 provided compounds with KDR inhibitory activity. The correlation of KDR inhibitory activity was similarly observed in a separate chemical series, the pyrazolo[1,5-a]pyrimidines. Initial exploration of this molecular descriptor supports a tool for rapid lead optimization in the 4-aminoquinazoline chemical series and a potential method for scaffold hopping in pursuit of new inhibitors.

A novel connexin43-interacting protein, CIP75, which belongs to the UbL-UBA protein family, regulates the turnover of connexin43.

The degradation of connexin43 (Cx43) has been reported to involve both lysosomal and proteasomal degradation pathways; however, very little is known about the mechanisms regulating these Cx43 degradation pathways. Using yeast two-hybrid, glutathione S-transferase pull-down, and co-immunoprecipitation approaches, we have identified a novel Cx43-interacting protein of approximately 75 kDa, CIP75. Laser confocal microscopy showed that CIP75 is located primarily at the endoplasmic reticulum, as indicated by the calnexin marker, with Cx43 co-localization in this perinuclear region. CIP75 belongs to the UbL (ubiquitin-like)-UBA (ubiquitin-associated) domain-containing protein family with a N-terminal UbL domain and a C-terminal UBA domain. The UBA domain of CIP75 is the main element mediating the interaction with Cx43, whereas the CIP75-interacting region in Cx43 resides in the PY motif and multiphosphorylation sites located between Lys 264 and Asn 302. Interestingly, the UbL domain interacts with the S2/RPN1 and S5a/RPN10 protein subunits of the regulatory 19 S proteasome cap subunit of the 26 S proteasome complex. Overexpression experiments suggested that CIP75 is involved in the turnover of Cx43 as measured by a significant stimulation of Cx43 degradation and reduction in its half-life with the opposite effects on Cx43 degradation observed in small interference RNA knockdown experiments.

Novel rab GAP-like protein, CIP85, interacts with connexin43 and induces its degradation.

Gap junctions play critical roles in tissue function and homeostasis. Connexin43 (Cx43) is a major gap junction protein expressed in the mammalian heart and other tissues and may be regulated by its interaction with other cellular proteins. Using the yeast two-hybrid screen, we identified a novel Cx43-interacting protein of 85-kDa, CIP85, which contains a single TBC, SH3, and RUN domain, in addition to a short coiled coil region. Homologues containing this unique combination of domains were found in human, D. melanogaster, and C. elegans. CIP85 mRNA is expressed ubiquitously in mouse and human tissues. In vitro interaction assays and in vivo co-immunoprecipitation experiments confirmed the interaction of endogenous CIP85 with Cx43. In vitro interaction experiments using CIP85 mutants with in-frame deletions of the TBC, SH3, and RUN domains indicated that the SH3 domain of CIP85 is involved in its interaction with Cx43. Conversely, analysis of Cx43 mutants with proline to alanine substitutions in the two proline-rich regions of Cx43 revealed that the P(253)LSP(256) motif is an important determinant of the ability of Cx43 to interact with CIP85. Laser-scanning confocal microscopy showed that CIP85 colocalized with Cx43 at the cell periphery, particularly in areas reminiscent of gap junction plaques. The functional importance of the interaction between CIP85 and Cx43 was suggested by the observation that CIP85 appears to induce the turnover of Cx43 through the lysosomal pathway.

Connexin43 PDZ2 binding domain mutants create functional gap junctions and exhibit altered phosphorylation.

Connexin43 (Cx43) is the most abundantly expressed gap junction protein. The C-terminal tail of Cx43 is important for regulation of gap junctions via phosphorylation of specific tyrosine and serine residues and through interactions with cellular proteins. The C-terminus of Cx43 has been shown to interact with the PDZ2 domain of the tight and adherens junction associated zona occludens 1 (ZO-1) protein. Analysis of the PDZ2 binding domain of Cx43 indicated that positions -3 and -2, and the final hydrophobic amino acid at the C-terminus, are critical for ZO-1 binding. In addition, the C-termini of connexins 40 and 45, but not Cx32, interacted with ZO-1. To evaluate the functional significance of the Cx43-ZO-1 interaction, Cx43 wild type (Cx43wt) and mutants lacking either the C-terminal hydrophobic isoleucine (Cx43deltaI382) or the last five amino acids (Cx43delta378-382), required for ZO-1 binding in vitro, were introduced into a Cx43-deficient MDCK cell line. In vitro binding studies and coimmunoprecipitation assays indicated that these Cx43 mutants failed to interact with ZO-1. Confocal and deconvolution microscopy revealed that a fraction of Cx43wt colocalized with ZO-1 at the plasma membrane. A similar colocalization pattern was observed for the Cx43deltaI382 and Cx43 delta378-382 mutants, which were translocated to the plasma membrane and formed functional gap junction channels. The wt and mutant Cx43 appeared to have similar turnover rates. However, the P2 and P3 phosphoisoforms of the Cx43 mutants were significantly reduced compared to Cx43wt. These studies indicated that the interaction of Cx43 with ZO-1 may contribute to the regulation of Cx43 phosphorylation.