Publications by authors named "Artur Schmidtchen"

139 Publications

Development of an Experimental Ex Vivo Wound Model to Evaluate Antimicrobial Efficacy of Topical Formulations.

Int J Mol Sci 2021 May 10;22(9). Epub 2021 May 10.

Division of Dermatology and Venereology, Department of Clinical Sciences, Lund University, SE-22184 Lund, Sweden.

Wound infections are considered a major cause for wound-associated morbidity. There is a high demand for alternative, robust, and affordable methods that can provide relatable and reproducible results when testing topical treatments, both in research and in the pharmaceutical industry. Here we present an ex vivo wound infection model using porcine skin and a burn wounding method, allowing for the efficacy evaluation of topical antimicrobial formulations. Utilizing this model, we demonstrate the potential of topical treatments after infecting the wounds with clinically significant bacteria, and . We show that the method is compatible with several analytical tools used to analyze infection and antimicrobial effects. Both bacterial strains successfully infected the wound surface, as well as deeper regions of the tissue. Quantification of viable bacteria on the wound surface and in the tissue, longitudinal measurements of bioluminescence, fluorescence microscopy, and scanning electron microscopy were used to confirm the effects of antibacterial treatments. Furthermore, we show that biofilms are formed on the wound surface, indicating that the demonstrated method mirrors typical in vivo infections.
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http://dx.doi.org/10.3390/ijms22095045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8126222PMC
May 2021

The role of full-length apolipoprotein E in clearance of Gram-negative bacteria and their endotoxins.

J Lipid Res 2021 May 18:100086. Epub 2021 May 18.

Division of Dermatology and Venereology, Institution of Clinical Sciences, Lund University, SE-22184 Lund, Sweden. Electronic address:

Apolipoprotein E (apoE) is a well-known lipid-binding protein that plays a main role in the metabolism and transport of lipids. More recently, apoE-derived peptides have been shown to exert antimicrobial effects. Here, we investigated the anti-bacterial activity of apoE using in vitro assays, advanced imaging techniques, and in vivo mouse models . The formation of macromolecular complexes of apoE and endotoxins from Gram-negative bacteria was explored using gel shift assays, transmission electron microscopy , and circular dichroism spectroscopy followed by calculation of the α-helical content. The binding affinity of apoE to endotoxins was also confirmed by fluorescent spectroscopy detecting the quenching and shifting of tryptophan intrinsic fluorescence. We showed that apoE exhibits antibacterial activity particularly against Gram-negative bacteria such as Pseudomonas aeruginosa and Escherichia coli. ApoE protein folding was affected by binding of bacterial endotoxins components such as lipopolysaccharide (LPS) and Lipid A , yielding similar increases in apoE αhelical content. Moreover, high molecular-weight complexes of apoE were formed in the presence of LPS, but not to the same extent as with Lipid A. Together, our results demonstrate that interaction of apoE with Gram-negative bacteria and their endotoxins leads to the structural changes in apoE and the formation of aggregate-like complexes. The protein interaction provides a molecular explanation for the observed antimicrobial effects of apoE, which binds to LPS on the bacterial surface and leads to cell membrane damage and bacterial death.
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http://dx.doi.org/10.1016/j.jlr.2021.100086DOI Listing
May 2021

Peptide-coated polyurethane material reduces wound infection and inflammation.

Acta Biomater 2021 May 2. Epub 2021 May 2.

Division of Dermatology and Venereology, Department of Clinical Sciences, Lund University, Lund, SE 22184, Sweden. Electronic address:

There is an urgent need for treatments that not only reduce bacterial infection that occurs during wounding but that also target the accompanying excessive inflammatory response. TCP-25, a thrombin-derived antibacterial peptide, scavenges toll-like receptor agonists such as endotoxins and lipoteichoic acid and prevents toll-like receptor-4 dimerization to reduce infection-related inflammation in vivo. Using a combination of biophysical, cellular, and microbiological assays followed by experimental studies in mouse and pig models, we show that TCP-25, when delivered from a polyurethane (PU) material, exerts anti-infective and anti-inflammatory effects in vitro and in vivo. Specifically, TCP-25 killed the common wound pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, in both in vitro and in vivo assays. Furthermore, after its release from the PU material, the peptide retained its capacity to induce its helical conformation upon endotoxin interaction, yielding reduced activation of NF-κB in THP-1 reporter cells, and diminished accumulation of inflammatory cells and subsequent release of IL-6 and TNF-α in subcutaneous implant models in vivo. Moreover, in a porcine partial thickness wound infection model, TCP-25 treated infection with S. aureus, and reduced the concomitant inflammatory response. Taken together, these findings demonstrate a combined antibacterial and anti-inflammatory effect of TCP-25 delivered from PU in vitro, and in mouse and porcine in vivo models of localized infection-inflammation. STATEMENT OF SIGNIFICANCE: Local wound infections may result in systemic complications and can be difficult to treat due to increasing antimicrobial resistance. Surgical site infections and biomaterial-related infections present a major challenge for hospitals. In recent years, various antimicrobial coatings have been developed for infection prevention and current concepts focus on various matrices with added anti-infective components, including various antibiotics and antiseptics. We have developed a dual action wound dressing concept where the host defense peptide TCP-25, when delivered from a PU material, targets both bacterial infection and the accompanying inflammation. TCP-25 PU showed efficacy in in vitro and experimental wound models in mouse and minipigs.
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http://dx.doi.org/10.1016/j.actbio.2021.04.045DOI Listing
May 2021

Probing Skin Barrier Recovery on Molecular Level Following Acute Wounds: An In Vivo/Ex Vivo Study on Pigs.

Biomedicines 2021 Mar 31;9(4). Epub 2021 Mar 31.

Department of Biomedical Science, Faculty of Health and Society, Malmö University, SE-205 06 Malmö, Sweden.

Proper skin barrier function is paramount for our survival, and, suffering injury, there is an acute need to restore the lost barrier and prevent development of a chronic wound. We hypothesize that rapid wound closure is more important than immediate perfection of the barrier, whereas specific treatment may facilitate perfection. The aim of the current project was therefore to evaluate the quality of restored tissue down to the molecular level. We used Göttingen minipigs with a multi-technique approach correlating wound healing progression in vivo over three weeks, monitored by classical methods (e.g., histology, trans-epidermal water loss (TEWL), pH) and subsequent physicochemical characterization of barrier recovery (i.e., small and wide-angle X-ray diffraction (SWAXD), polarization transfer solid-state NMR (PTssNMR), dynamic vapor sorption (DVS), Fourier transform infrared (FTIR)), providing a unique insight into molecular aspects of healing. We conclude that although acute wounds sealed within two weeks as expected, molecular investigation of stratum corneum (SC) revealed a poorly developed keratin organization and deviations in lipid lamellae formation. A higher lipid fluidity was also observed in regenerated tissue. This may have been due to incomplete lipid conversion during barrier recovery as glycosphingolipids, normally not present in SC, were indicated by infrared FTIR spectroscopy. Evidently, a molecular approach to skin barrier recovery could be a valuable tool in future development of products targeting wound healing.
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http://dx.doi.org/10.3390/biomedicines9040360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8065685PMC
March 2021

Cell-Free DNA Promotes Thrombin Autolysis and Generation of Thrombin-Derived C-Terminal Fragments.

Front Immunol 2021 24;12:593020. Epub 2021 Feb 24.

Division of Dermatology and Venereology, Department of Clinical Sciences, Lund University, Lund, Sweden.

Cell-free DNA (cfDNA) is the major structural component of neutrophil extracellular traps (NETs), an innate immune response to infection. Antimicrobial proteins and peptides bound to cfDNA play a critical role in the bactericidal property of NETs. Recent studies have shown that NETs have procoagulant activity, wherein cfDNA triggers thrombin generation through activation of the intrinsic pathway of coagulation. We have recently shown that thrombin binds to NETs and consequently can alter the proteome of NETs. However, the effect of NETs on thrombin is still unknown. In this study, we report that DNA binding leads to thrombin autolysis and generation of multiple thrombin-derived C-terminal peptides (TCPs) Employing a 25-residue prototypic TCP, GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE), we show that TCPs bind NETs, thus conferring mutual protection against nuclease and protease degradation. Together, our results demonstrate the complex interplay between coagulation, NET formation, and thrombin cleavage and identify a previously undisclosed mechanism for formation of TCPs.
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http://dx.doi.org/10.3389/fimmu.2021.593020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7943729PMC
February 2021

Real-time Imaging of LPS-induced Local Inflammation and Drug Deposition in NF-κB Reporter Mice.

Bio Protoc 2020 Aug 20;10(16):e3724. Epub 2020 Aug 20.

Division of Dermatology and Venereology, Department of Clinical Sciences, Lund University, SE-22184 Lund, Sweden.

Wound, biomaterial, and surgical infections are all characterized by a localized and excessive inflammation, motivating the development of methods focused on the analysis of local immune events. However, current inflammation models, such as the commonly used models of endotoxin-induced inflammation are based on systemic, usually intraperitoneal, administration of lipopolysaccharide (LPS), causing endotoxin shock. Here, we describe a model of LPS-induced local inflammation in NF-κB-RE-Luc reporter mice. LPS, alone or with added therapeutic substances, is delivered locally via a hydrogel which is deposited subcutaneously, providing a spatially defined environment, enabling bioimaging analyses of local NF-κB activation. Evaluation of drug efficacy can be analyzed longitudinally in the same mouse, and using fluorescently labeled drugs, local drug deposition can be simultaneously analyzed, and correlated to the site of inflammation. Finally, the protocol can also be used to study retention and systemic release of the drug from locally deposited gels and other biomaterials.
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http://dx.doi.org/10.21769/BioProtoc.3724DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7842668PMC
August 2020

Bioinformatic Analysis of the Wound Peptidome Reveals Potential Biomarkers and Antimicrobial Peptides.

Front Immunol 2020 3;11:620707. Epub 2021 Feb 3.

Division of Dermatology and Venereology, Department of Clinical Sciences, Lund University, Lund, Sweden.

Wound infection is a common and serious medical condition with an unmet need for improved diagnostic tools. A peptidomic approach, aided by mass spectrometry and bioinformatics, could provide novel means of identifying new peptide biomarkers for wound healing and infection assessment. Wound fluid is suitable for peptidomic analysis since it is both intimately tied to the wound environment and is readily available. In this study we investigate the peptidomes of wound fluids derived from surgical drainages following mastectomy and from wound dressings following facial skin grafting. By applying sorting algorithms and open source third party software to peptidomic label free tandem mass spectrometry data we provide an unbiased general methodology for analyzing and differentiating between peptidomes. We show that the wound fluid peptidomes of patients are highly individualized. However, differences emerge when grouping the patients depending on wound type. Furthermore, the abundance of peptides originating from documented antimicrobial regions of hemoglobin in infected wounds may contribute to an antimicrobial wound environment, as determined by analysis. We validate our findings by compiling literature on peptide biomarkers and peptides of physiological significance and cross checking the results against our dataset, demonstrating that well-documented peptides of immunological significance are abundant in infected wounds, and originate from certain distinct regions in proteins such as hemoglobin and fibrinogen. Ultimately, we have demonstrated the power using sorting algorithms and open source software to help yield insights and visualize peptidomic data.
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http://dx.doi.org/10.3389/fimmu.2020.620707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7888259PMC
February 2021

Concentration- and pH-Dependent Oligomerization of the Thrombin-Derived C-Terminal Peptide TCP-25.

Biomolecules 2020 11 19;10(11). Epub 2020 Nov 19.

Division of Dermatology and Venereology, Department of Clinical Sciences, Lund University, 22241 Lund, Sweden.

Peptide oligomerization dynamics affects peptide structure, activity, and pharmacodynamic properties. The thrombin C-terminal peptide, TCP-25 (GKYGFYTHVFRLKKWIQKVIDQFGE), is currently in preclinical development for improved wound healing and infection prevention. It exhibits turbidity when formulated at pH 7.4, particularly at concentrations of 0.3 mM or more. We used biochemical and biophysical approaches to explore whether the peptide self-associates and forms oligomers. The peptide showed a dose-dependent increase in turbidity as well as α-helical structure at pH 7.4, a phenomenon not observed at pH 5.0. By analyzing the intrinsic tryptophan fluorescence, we demonstrate that TCP-25 is more stable at high concentrations (0.3 mM) when exposed to high temperatures or a high concentration of denaturant agents, which is compatible with oligomer formation. The denaturation process was reversible above 100 µM of peptide. Dynamic light scattering demonstrated that TCP-25 oligomerization is sensitive to changes in pH, time, and temperature. Computational modeling with an active 18-mer region of TCP-25 showed that the peptide can form pH-dependent higher-order end-to-end oligomers and micelle-like structures, which is in agreement with the experimental data. Thus, TCP-25 exhibits pH- and temperature-dependent dynamic changes involving helical induction and reversible oligomerization, which explains the observed turbidity of the pharmacologically developed formulation.
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http://dx.doi.org/10.3390/biom10111572DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7699335PMC
November 2020

Thrombin-derived C-terminal fragments aggregate and scavenge bacteria and their proinflammatory products.

J Biol Chem 2020 03 7;295(11):3417-3430. Epub 2020 Feb 7.

Department of Clinical Sciences, Division of Dermatology and Venereology, Lund University, Lund SE-22184, Sweden; Department of Biomedical Sciences, Copenhagen Wound Healing Center, Bispebjerg Hospital, University of Copenhagen, Copenhagen DK-2400, Denmark.

Thrombin-derived C-terminal peptides (TCPs), including a major 11-kDa fragment (TCP96), are produced through cleavage by human neutrophil elastase and aggregate lipopolysaccharide (LPS) and the Gram-negative bacterium However, the physiological roles of TCP96 in controlling bacterial infections and reducing LPS-induced inflammation are unclear. Here, using various biophysical methods, molecular modeling, microbiological and cellular assays, and animal models, we examined the structural features and functional roles of recombinant TCP96 (rTCP96) in the aggregation of multiple bacteria and the Toll-like receptor (TLR) agonists they produce. We found that rTCP96 aggregates both Gram-negative and Gram-positive bacteria, including and , and their cell-wall components LPS, lipid A, and lipoteichoic acid (LTA). The Gram-negative bacteria and were particularly sensitive to aggregation-induced bacterial permeabilization and killing. As a proof of concept, we show that rTCP96 reduces LPS-induced NF-κB activation in human monocytes, as well as in mouse models of LPS-induced subcutaneous inflammation. Moreover, in a mouse model of subcutaneous inoculation with , rTCP96 reduced bacterial levels. Together, these results link TCP-mediated aggregation of endotoxins and bacteria to attenuation of inflammation and bacterial levels .
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http://dx.doi.org/10.1074/jbc.RA120.012741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7076200PMC
March 2020

A dual-action peptide-containing hydrogel targets wound infection and inflammation.

Sci Transl Med 2020 01;12(524)

Division of Dermatology and Venereology, Department of Clinical Sciences, Lund University, SE-22184 Lund, Sweden.

There is a clinical need for improved wound treatments that prevent both infection and excessive inflammation. TCP-25, a thrombin-derived peptide, is antibacterial and scavenges pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide, thereby preventing CD14 interaction and Toll-like receptor dimerization, leading to reduced downstream immune activation. Here, we describe the development of a hydrogel formulation that was functionalized with TCP-25 to target bacteria and associated PAMP-induced inflammation. In vitro studies determined the polymer prerequisites for such TCP-25-mediated dual action, favoring the use of noncharged hydrophilic hydrogels, which enabled peptide conformational changes and LPS binding. The TCP-25-functionalized hydrogels killed Gram-positive and Gram-negative bacteria in vitro, as well as in experimental mouse models of subcutaneous infection. The TCP-25 hydrogel also mediated reduction of LPS-induced local inflammatory responses, as demonstrated by analysis of local cytokine production and in vivo bioimaging using nuclear factor κB (NF-κB) reporter mice. In porcine partial thickness wound models, TCP-25 prevented infection with and reduced concentrations of proinflammatory cytokines. Proteolytic fragmentation of TCP-25 in vitro yielded a series of bioactive TCP fragments that were identical or similar to those present in wounds in vivo. Together, the results demonstrate the therapeutic potential of TCP-25 hydrogel, a wound treatment based on the body's peptide defense, for prevention of both bacterial infection and the accompanying inflammation.
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http://dx.doi.org/10.1126/scitranslmed.aax6601DOI Listing
January 2020

A C-terminal peptide of TFPI-1 facilitates cytosolic delivery of nucleic acid cargo into mammalian cells.

Biochim Biophys Acta Biomembr 2020 02 28;1862(2):183093. Epub 2019 Oct 28.

Lee Kong Chian School of Medicine, Nanyang Technological University Singapore, 59 Nanyang Drive, Singapore 636921, Singapore. Electronic address:

Efficient intracellular nucleic acid delivery into mammalian cells remains a long-standing challenge owing to poor cell permeability and uptake of naked nucleic acids across the cell membrane and limited cargo stability. Conventional delivery methods have several drawbacks, such as cytotoxicity, limited cell-type applicability, low efficiency, hindrances that limit the potential of oligonucleotide delivery in functional genomics, therapeutics and diverse research applications. Thus, new approaches that are robust, safe, effective and valid across multiple cell types are much needed. Here, we demonstrate that GGL27, a TFPI-1-derived novel cationic host defence peptide, facilitates the delivery of nucleic acid cargo into the cytosol of a range of mammalian cells. The GGL27 peptide is non-cytotoxic and is internalized in a broad range of mammalian cell-types, including transformed cell lines and primary cells. GGL27 spontaneously forms complexes with nucleic acids of variable sizes, protects them from nuclease degradation, and delivers cargo effectively. Together, our observations demonstrate the versatile cell-penetrating property of GGL27, providing an excellent template for developing a simple, non-toxic peptide-based cytosolic delivery tool for wide use in biomedical research.
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http://dx.doi.org/10.1016/j.bbamem.2019.183093DOI Listing
February 2020

Inflammation Biomarkers and Correlation to Wound Status After Full-Thickness Skin Grafting.

Front Med (Lausanne) 2019 12;6:159. Epub 2019 Jul 12.

Division of Dermatology, Department of Clinical Sciences, Skane University Hospital, Lund University, Lund, Sweden.

A surgical site infection (SSI) is believed to be the result of an exaggerated inflammatory response. Examine the relationship between clinical status and inflammation biomarkers in full-thickness skin grafting wounds. Twenty patients planned for facial full-thickness skin grafting were enrolled. A week after surgery, all graft wounds were clinically assessed using a 3-step scale for inflammation (low, moderate, high). All wounds were swabbed for routine microbiological analysis and assessment of numbers of aerobic bacteria. Tie-over dressings from all patients were collected and used for wound fluid extraction and subsequent analysis of MMPs, cytokines, and NF-κB inducing activity. Wounds with a high degree of inflammation contained increased total MMP activity ( ≤ 0.05) in their corresponding fluids. Likewise, the level of the cytokines IL-1ß, IL-8, IL-6, TNF-α was analyzed, and particularly IL-1ß was discriminatory for highly inflamed wounds ( ≤ 0.01). Moreover, bacterial loads were increased in highly inflamed wounds compared to wounds with a low degree of inflammation ( ≤ 0.01). NF-κB activation in the monocytic cell line THP-1 was significantly higher when these cells were stimulated by wound fluids with a high degree of inflammation ( ≤ 0.01). Growth of in wounds did not vary between wounds with different degrees of inflammation (chi-square 3.8, = 0.144). Biomarkers analyzed from tie-over dressings correlated to clinical wound healing in full-thickness skin grafting.
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http://dx.doi.org/10.3389/fmed.2019.00159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6640317PMC
July 2019

Multiscale modeling of innate immune receptors: Endotoxin recognition and regulation by host defense peptides.

Pharmacol Res 2019 09 24;147:104372. Epub 2019 Jul 24.

Bioinformatics Institute, A⁎STAR (Agency for Science, Technology and Research), Singapore; Department of Biological Sciences, National University of Singapore, Singapore. Electronic address:

The innate immune system provides a first line of defense against foreign microorganisms, and is typified by the Toll-like receptor (TLR) family. TLR4 is of particular interest, since over-stimulation of its pathway by excess lipopolysaccharide (LPS) molecules from the outer membranes of Gram-negative bacteria can result in sepsis, which causes millions of deaths each year. In this review, we outline our use of molecular simulation approaches to gain a better understanding of the determinants of LPS recognition, towards the search for novel immunotherapeutics. We first describe how atomic-resolution simulations have enabled us to elucidate the regulatory conformational changes in TLR4 associated with different LPS analogues, and hence a means to rationalize experimental structure-activity data. Furthermore, multiscale modelling strategies have provided a detailed description of the thermodynamics and intermediate structures associated with the entire TLR4 relay - which consists of a number of transient receptor/coreceptor complexes - allowing us trace the pathway of LPS transfer from bacterial membranes to the terminal receptor complex at the plasma membrane surface. Finally, we describe our efforts to leverage these computational models, in order to elucidate previously undisclosed anti-inflammatory mechanisms of endogenous host-defense peptides found in wounds. Collectively, this work represents a promising avenue for the development of novel anti-septic treatments, inspired by nature's innate defense strategies.
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http://dx.doi.org/10.1016/j.phrs.2019.104372DOI Listing
September 2019

Degradable dendritic nanogels as carriers for antimicrobial peptides.

J Colloid Interface Sci 2019 Oct 13;554:592-602. Epub 2019 Jul 13.

Department of Pharmacy, Uppsala University, SE-75123 Uppsala, Sweden; Department of Pharmacy, University of Copenhagen, DK-2100 Copenhagen, Denmark.

In the present study, we investigate degradable anionic dendritic nanogels (DNG) as carriers for antimicrobial peptides (AMPs). In such systems, the dendritic part contains carboxylic acid-based anionic binding sites for cationic AMPs, whereas linear poly(ethylene glycol) (PEG) chains form a shell for promotion of biological stealth. In order to clarify factors influencing membrane interactions of such systems, we here address effects of nanogel charge, cross-linking, and degradation on peptide loading/release, as well as consequences of these factors for lipid membrane interactions and antimicrobial effects. The DNGs were found to bind the AMPs LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) and DPK-060 (GKHKNKGKKNGKHNGWKWWW). For the smaller DPK-060 peptide, loading was found to increase with increasing nanogel charge density. For the larger LL-37, on the other hand, peptide loading was largely insensitive to nanogel charge density. In line with this, results on the secondary structure, as well as on the absence of stabilization from proteolytic degradation by the nanogels, show that the larger LL-37 is unable to enter into the interior of the nanogels. While 40-60% nanogel degradation occurred over 10 days, promoted at high ionic strength and lower cross-linking density/higher anionic charge content, peptide release at physiological ionic strength was substantially faster, and membrane destabilization not relying on nanogel degradation. Ellipsometry and liposome leakage experiments showed both free peptide and peptide/DNG complexes to cause membrane destabilization, indicated also by antimicrobial activities being comparable for nanogel-bound and free peptide. Finally, the DNGs were demonstrated to display low toxicity towards erythrocytes even at peptide concentrations of 100 µM.
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http://dx.doi.org/10.1016/j.jcis.2019.07.028DOI Listing
October 2019

[Superficial surgical site infections in dermatologic surgery].

Lakartidningen 2019 Mar 26;116. Epub 2019 Mar 26.

Skanes universitetssjukhus Lund - Dermatology Lund, Sweden Skanes universitetssjukhus Lund - Dermatology Lund, Sweden.

Surgical site infections (SSIs) in dermatologic surgery are rare, but when they do occur they can cause unnecessary suffering in patients, delayed healing, and result in poor scar cosmesis. The etiology and pathogenesis of SSIs in dermatologic surgery are not completely understood and most current preventative measures lack strong scientific evidence. Focusing on dermatologic surgery, this article provides an updated overview of the subject with data summarizing relevant studies.
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March 2019

Carbonic anhydrases in human keratinocytes and their regulation by all-trans retinoic acid and 1α,25-dihydroxyvitamin D.

Exp Dermatol 2019 08 3;28(8):976-980. Epub 2019 Jul 3.

Lee Kong Chian School of Medicine, Nanyang Technological University Singapore, Singapore, Singapore.

Carbonic anhydrases (CAs) are ubiquitously expressed enzymes and catalyse an important physiological reaction of interconverting the hydration of carbon dioxide to bicarbonates, which is crucial for maintaining acid/base equilibrium in certain tissues. While 15 different isoforms of CAs are present in various cell types in human tissues, their expression pattern in the epidermis remains to be investigated. Here, we report the expression of 5 CA isoforms (CAII, CAIX, CAXI, CAXII and CAXIII) in human primary keratinocytes. Further, we demonstrate that the expression of CAII and CAIX in these cells is significantly up-regulated by the biologically active metabolites of vitamin A (all-trans retinoic acid) and vitamin D (1α,25-dihydroxyvitamin D ), respectively. Taken together, apart from providing new information on the expression of CAs in the skin, our results highlight a previously undisclosed connection between vitamin A and CAII expression and vitamin D and CAIX expression.
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http://dx.doi.org/10.1111/exd.13976DOI Listing
August 2019

Interaction of Laponite with Membrane Components-Consequences for Bacterial Aggregation and Infection Confinement.

ACS Appl Mater Interfaces 2019 May 17;11(17):15389-15400. Epub 2019 Apr 17.

Department of Pharmacy , Uppsala University , SE-75123 Uppsala , Sweden.

The antimicrobial effects of Laponite nanoparticles with or without loading of the antimicrobial peptide LL-37 was investigated along with their membrane interactions. The study combines data from ellipsometry, circular dichroism, fluorescence spectroscopy, particle size/ζ potential measurements, and confocal microscopy. As a result of the net negative charge of Laponite, loading of net positively charged LL-37 increases with increasing pH. The peptide was found to bind primarily to the outer surface of the Laponite nanoparticles in a predominantly helical conformation, leading to charge reversal. Despite their net positive charge, peptide-loaded Laponite nanoparticles did not kill Gram-negative Escherichia coli bacteria or disrupt anionic model liposomes. They did however cause bacteria flocculation, originating from the interaction of Laponite and bacterial lipopolysaccharide (LPS). Free LL-37, in contrast, is potently antimicrobial through membrane disruption but does not induce bacterial aggregation in the concentration range investigated. Through LL-37 loading of Laponite nanoparticles, the combined effects of bacterial flocculation and membrane lysis are observed. However, bacteria aggregation seems to be limited to Gram-negative bacteria as Laponite did not cause flocculation of Gram-positive Bacillus subtilis bacteria nor did it bind to lipoteichoic acid from bacterial envelopes. Taken together, the present investigation reports several novel phenomena by demonstrating that nanoparticle charge does not invariably control membrane destabilization and by identifying the ability of anionic Laponite nanoparticles to effectively flocculate Gram-negative bacteria through LPS binding. As demonstrated in cell experiments, such aggregation results in diminished LPS-induced cell activation, thus outlining a promising approach for confinement of infection and inflammation caused by such pathogens.
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http://dx.doi.org/10.1021/acsami.9b03527DOI Listing
May 2019

A novel derivative of the fungal antimicrobial peptide plectasin is active against Mycobacterium tuberculosis.

Tuberculosis (Edinb) 2018 12 30;113:231-238. Epub 2018 Oct 30.

Department of Microbiology, Immunology and Glycobiology, Institution of Laboratory Medicine, Lund University, Lund, Sweden. Electronic address:

Tuberculosis has been reaffirmed as the infectious disease causing most deaths in the world. Co-infection with HIV and the increase in multi-drug resistant Mycobacterium tuberculosis strains complicate treatment and increases mortality rates, making the development of new drugs an urgent priority. In this study we have identified a promising candidate by screening antimicrobial peptides for their capacity to inhibit mycobacterial growth. This non-toxic peptide, NZX, is capable of inhibiting both clinical strains of M. tuberculosis and an MDR strain at therapeutic concentrations. The therapeutic potential of NZX is further supported in vivo where NZX significantly lowered the bacterial load with only five days of treatment, comparable to rifampicin treatment over the same period. NZX possesses intracellular inhibitory capacity and co-localizes with intracellular bacteria in infected murine lungs. In conclusion, the data presented strongly supports the therapeutic potential of NZX in future anti-TB treatment.
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http://dx.doi.org/10.1016/j.tube.2018.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6289163PMC
December 2018

Prevalence of chronic wounds in the general population: systematic review and meta-analysis of observational studies.

Ann Epidemiol 2019 01 12;29:8-15. Epub 2018 Nov 12.

Centre for Population Health Sciences (CePHaS), Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore.

Purpose: Chronic wounds are a major public health challenge, but little is known about the true burden with studies reporting different estimates because of disparities in study designs and measurement methods. This hampers efficient resource allocation, planning, and improvement of wound care.

Methods: Our study aimed to pool prevalence estimates from a global perspective by systematically carrying out searches in MEDLINE, EMBASE, Cochrane, CINAHL, Global Health, and PsycINFO databases for articles reporting the prevalence of chronic wounds in adults, from January 2000 to June 2018. The included publications had to define wound chronicity by duration (≥3 weeks), and/or labeling the wounds as chronic, complex, or hard-to-heal.

Results: Seventeen studies met the inclusion criteria, and 11 studies analyzing chronic wounds in the general population were included in random effects meta-analyses to calculate pooled prevalence. Chronic wounds of mixed etiologies (n = 3) showed a pooled prevalence of 2.21 per 1000 population, and for chronic leg ulcers (n = 9), the prevalence was estimated at 1.51 per 1000 population.

Conclusions: Our findings, aligned to previous studies reporting point prevalence of chronic wounds identified within the healthcare system, showed that the vast majority of chronic wounds in epidemiological studies are made up by chronic leg ulcers.
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http://dx.doi.org/10.1016/j.annepidem.2018.10.005DOI Listing
January 2019

The humanistic and economic burden of chronic wounds: A systematic review.

Wound Repair Regen 2019 01 2;27(1):114-125. Epub 2018 Dec 2.

Centre for Population Health Sciences (CePHaS), Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore.

Chronic wounds are a health problem that have devastating consequences for patients and contribute major costs to healthcare systems and societies. To understand the magnitude of this health issue, a systematic review was undertaken. Searches were conducted in MEDLINE, EMBASE, EBM Reviews and Cochrane library, CINAHL, EBSCO, PsycINFO, and Global Health databases for articles published between 2000 and 2015. Included publications had to target adults (≥18 years of age), state wound chronicity (≥3 weeks) and/or label the wounds as chronic, complex, hard-to-heal, or having led to an amputation. The review excluded studies that did not present data on generic health-related quality of life and/or cost data, case studies, randomized controlled trials, economic modeling studies, abstracts, and editorials. Extracted data were summarized into a narrative synthesis, and for a few articles using the same health-related quality of life instrument, average estimates with 95% confidence intervals were calculated. Thirty articles met the inclusion criteria. Findings revealed that health-related quality of life was lowest for physical pathologies, and based on average estimates were scores most inferior in the domain physical role for both patients with chronic wounds and for those with wound-related amputations. The cost burden was mainly attributed to amputations for patients also comorbid with diabetes, where the cost for hospitalization ranged from US$12,851 to US$16,267 (median) for this patient group. Patients with chronic wounds have poor health-related quality of life in general and wound-related costs are substantial. Development and implementation of wound management strategies that focus on increasing health-related quality of life and effectively reduce costs for this patient group are urgently needed.
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http://dx.doi.org/10.1111/wrr.12683DOI Listing
January 2019

TFPI-2 Protects Against Gram-Negative Bacterial Infection.

Front Immunol 2018 11;9:2072. Epub 2018 Sep 11.

Division of Dermatology and Venereology, Department of Clinical Sciences Lund, Lund University, Lund, Sweden.

Tissue factor pathway inhibitor-2 (TFPI-2) has previously been characterized as an endogenous anticoagulant. TFPI-2 is expressed in the vast majority of cells, mainly secreted into the extracellular matrix. Recently we reported that EDC34, a C-terminal peptide derived from TFPI-2, exerts a broad antimicrobial activity. In the present study, we describe a previously unknown antimicrobial mode of action for the human TFPI-2 C-terminal peptide EDC34, mediated via binding to immunoglobulins of the classes IgG, IgA, IgE, and IgM. In particular the interaction of EDC34 with the Fc part of IgG is of importance since this boosts interaction between the immunoglobulin and complement factor C1q. Moreover, we find that the binding increases the C1q engagement of the antigen-antibody interaction, leading to enhanced activation of the classical complement pathway during bacterial infection. In experimental murine models of infection and endotoxin challenge, we show that TFPI-2 is up-regulated in several organs, including the lung. Correspondingly, mice are more susceptible to pulmonary bacterial infection. No anti-coagulant role of TFPI-2 was observed in these models . Furthermore, , the mouse TFPI-2-derived C-terminal peptide VKG24, a homolog to human EDC34 is protective against systemic bacterial infection. Moreover, in sputum from cystic fibrosis patients TFPI-2 C-terminal fragments are generated and found associated with immunoglobulins. Together our data describe a previously unknown host defense mechanism and therapeutic importance of TFPI-2 against invading Gram-negative bacterial pathogens.
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http://dx.doi.org/10.3389/fimmu.2018.02072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6141739PMC
September 2019

Bacteria Display Differential Growth and Adhesion Characteristics on Human Hair Shafts.

Front Microbiol 2018 7;9:2145. Epub 2018 Sep 7.

Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore.

Apart from the skin surface, hair represents a significant tissue component with a capacity of bacterial interactions. New information can be obtained about hair function through the characterization of bacterial adherence, colonization, and responses to hair shafts In this proof-of-principle study, we examine the growth kinetics of Gram-positive and and Gram-negative and in the presence of human hair shafts. We explore the ability of these bacteria to adhere to and colonize hair shaft surfaces, as well as the resulting impact on the hair's surface morphology. We show that hair shafts inhibit the growth of Gram-positive and , while the growth kinetics of and remain unaffected. Scanning electron microscope analysis and steeping studies show that and to adhere to and colonize on human hair shafts without significantly affecting the hair shaft's surface morphology. produced a substantial amount of biofilm on the hair shaft surfaces, while specifically inhabited the edges of the cuticle scales. Taken together, our results demonstrate differences in bacterial responses to human hair shafts, which may provide novel insights into hair and scalp health.
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http://dx.doi.org/10.3389/fmicb.2018.02145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6137140PMC
September 2018

Thrombin and Plasmin Alter the Proteome of Neutrophil Extracellular Traps.

Front Immunol 2018 9;9:1554. Epub 2018 Jul 9.

Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore.

Neutrophil extracellular traps (NETs) consist of a decondensed DNA scaffold decorated with neutrophil-derived proteins. The proteome of NETs, or "NETome," has been largely elucidated . However, components such as plasma and extracellular matrix proteins may affect the NETome under physiological conditions. Here, using a reductionistic approach, we explored the effects of two proteases active during injury and wounding, human thrombin and plasmin, on the NETome. Using high-resolution mass spectrometry, we identified a total of 164 proteins, including those previously not described in NETs. The serine proteases, particularly thrombin, were also found to interact with DNA and bound to NETs . Among the most abundant proteins were those identified previously, including histones, neutrophil elastase, and antimicrobial proteins. We observed reduced histone (H2B, H3, and H4) and neutrophil elastase levels upon the addition of the two proteases. Analyses of NET-derived tryptic peptides identified subtle changes upon protease treatments. Our results provide evidence that exogenous proteases, present during wounding and inflammation, influence the NETome. Taken together, regulation of NETs and their proteins under different physiological conditions may affect their roles in infection, inflammation, and the host response.
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http://dx.doi.org/10.3389/fimmu.2018.01554DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6046383PMC
July 2018

Structural basis for endotoxin neutralisation and anti-inflammatory activity of thrombin-derived C-terminal peptides.

Nat Commun 2018 07 17;9(1):2762. Epub 2018 Jul 17.

Lee Kong Chian School of Medicine, Nanyang Technological University, 59 Nanyang Drive, Singapore, 636921, Singapore.

Thrombin-derived C-terminal peptides (TCPs) of about 2 kDa are present in wounds, where they exert anti-endotoxic functions. Employing a combination of nuclear magnetic resonance spectroscopy (NMR), biophysical, mass spectrometry and cellular studies combined with in silico multiscale modelling, we here determine the bound conformation of HVF18 (HVFRLKKWIQKVIDQFGE), a TCP generated by neutrophil elastase, in complex with bacterial lipopolysaccharide (LPS) and define a previously undisclosed interaction between TCPs and human CD14. Further, we show that TCPs bind to the LPS-binding hydrophobic pocket of CD14 and identify the peptide region crucial for TCP interaction with LPS and CD14. Taken together, our results demonstrate the role of structural transitions in LPS complex formation and CD14 interaction, providing a molecular explanation for the previously observed therapeutic effects of TCPs in experimental models of bacterial sepsis and endotoxin shock.
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http://dx.doi.org/10.1038/s41467-018-05242-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6050251PMC
July 2018

Peptide-Loaded Microgels as Antimicrobial and Anti-Inflammatory Surface Coatings.

Biomacromolecules 2018 08 18;19(8):3456-3466. Epub 2018 Jul 18.

Division of Dermatology and Venereology, Department of Clinical Sciences , Lund University , SE-22184 Lund , Sweden.

Here we report on covalently immobilized poly(ethyl acrylate- co-methacrylic acid) microgels loaded with the host defense peptide KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYTLR), which is derived from human heparin cofactor II, as well as its poly(ethylene glycol)-conjugated (PEGylated) version, KYE28PEG. Peptide loading and release, as well as the consequences of these processes on the microgel and peptide properties, were studied by in situ ellipsometry, confocal microscopy, zeta potential measurements, and circular dichroism spectroscopy. The results show that the microgel-peptide interactions are electrostatically dominated, thus promoted at higher microgel charge density, while PEGylation suppresses peptide binding. PEGylation also enhances the α-helix induction observed for KYE28 upon microgel incorporation. Additionally, peptide release is facilitated at physiological salt concentration, particularly so for KYE28PEG, which illustrates the importance of electrostatic interactions. In vitro studies on Escherichia coli show that the microgel-modified surfaces display potent antifouling properties in both the absence and presence of the incorporated peptide. While contact killing dominates at low ionic strength for the peptide-loaded microgels, released peptides also provide antimicrobial activity in bulk at a high ionic strength. Additionally, KYE28- and KYE28PEG-loaded microgels display anti-inflammatory effects on human monocytes. Taken together, these results not only show that surface-bound microgels offer an interesting approach for local drug delivery of host defense peptides but also illustrate the need to achieve high surface loads of peptides for efficient biological effects.
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http://dx.doi.org/10.1021/acs.biomac.8b00776DOI Listing
August 2018

Collagen VI Contains Multiple Host Defense Peptides with Potent In Vivo Activity.

J Immunol 2018 08 20;201(3):1007-1020. Epub 2018 Jun 20.

Division of Infection Medicine, Department of Clinical Sciences, Lund University, 221 84 Lund, Sweden.

Collagen VI is a ubiquitous extracellular matrix component that forms extensive microfibrillar networks in most connective tissues. In this study, we describe for the first time, to our knowledge, that the collagen VI von Willebrand factor type A-like domains exhibit a broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria in human skin infections in vivo. In silico sequence and structural analysis of VWA domains revealed that they contain cationic and amphipathic peptide sequence motifs, which might explain the antimicrobial nature of collagen VI. In vitro and in vivo studies show that these peptides exhibited significant antibacterial activity against , , and through membrane disruption. Our findings shed new light on the role of collagen VI-derived peptides in innate host defense and provide templates for development of peptide-based antibacterial therapies.
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http://dx.doi.org/10.4049/jimmunol.1700602DOI Listing
August 2018

Influence of pH on the activity of thrombin-derived antimicrobial peptides.

Biochim Biophys Acta Biomembr 2018 11 6;1860(11):2374-2384. Epub 2018 Jun 6.

Lee Kong Chian School of Medicine, Nanyang Technological University Singapore, 59 Nanyang Drive, 636921, Singapore. Electronic address:

The wound environment is characterized by physiological pH changes. Proteolysis of thrombin by wound-derived proteases, such as neutrophil elastase, generates antimicrobial thrombin-derived C-terminal peptides (TCPs), such as HVF18 (HVFRLKKWIQKVIDQFGE). Presence of such TCPs in human wound fluids in vivo, as well as the occurrence of an evolutionarily conserved His residue in the primary amino acid sequence of TCPs, prompted us to investigate the pH-dependent antibacterial action of HVF18, as well as of the prototypic GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE). We show that protonation of this His residue at pH 5.5 increases the antibacterial activity of both TCPs against Gram-negative Escherichia coli by membrane disruption. Physiological salt level (150 mM NaCl) augments antibacterial activity of GKY25 but diminishes for the shorter HVF18. Replacing His with Leu or Ser in GKY25 abolishes the His protonation-dependent increase in antibacterial activity at pH 5.5, whereas substitution with Lys maintains activity at neutral (pH 7.4) and acidic pH. Interestingly, both TCPs display decreased binding affinities to human CD14 with decreasing pH, suggesting a likely switch in mode-of-action, from anti-inflammatory at neutral pH to antibacterial at acidic pH. Together, the results demonstrate that apart from structural prerequisites such as peptide length, charge, and hydrophobicity, the evolutionarily conserved His residue of TCPs influences their antibacterial effects and reveals a previously unknown aspect of TCPs biological action.
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http://dx.doi.org/10.1016/j.bbamem.2018.06.002DOI Listing
November 2018

A Thermodynamic Funnel Drives Bacterial Lipopolysaccharide Transfer in the TLR4 Pathway.

Structure 2018 08 17;26(8):1151-1161.e4. Epub 2018 May 17.

Bioinformatics Institute (BII), Agency for Science, Technology and Research (A(∗)STAR), Matrix 07-01, 30 Biopolis Street, Singapore, 138671 Singapore; Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore. Electronic address:

The Gram-negative bacterial outer membrane contains lipopolysaccharide, which potently stimulates the mammalian innate immune response. This involves a relay of specialized complexes culminating in transfer of lipopolysaccharide from CD14 to Toll-like receptor 4 (TLR4) and its co-receptor MD-2 on the cell surface, leading to activation of downstream inflammatory responses. In this study we develop computational models to trace the TLR4 cascade in near-atomic detail. We demonstrate through rigorous thermodynamic calculations that lipopolysaccharide molecules traversing the receptor cascade fall into a thermodynamic funnel. An affinity gradient for lipopolysaccharide is revealed upon extraction from aggregates or realistic bacterial outer membrane models and transfer through CD14 to the terminal TLR4/MD-2 receptor-co-receptor complex. We subsequently assemble viable CD14/TLR4/MD-2 oligomers at the plasma membrane surface, and observe lipopolysaccharide exchange between CD14 and TLR4/MD-2. Collectively, this work helps to unravel the key structural determinants governing endotoxin recognition in the TLR4 innate immune pathway.
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http://dx.doi.org/10.1016/j.str.2018.04.007DOI Listing
August 2018

Toll-like Receptor 3 Agonist, Polyinosinic-polycytidylic Acid, Upregulates Carbonic Anhydrase II in Human Keratinocytes.

Acta Derm Venereol 2018 Aug;98(8):762-765

Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore 636921, Singapore.

Carbonic anhydrases are ubiquitously expressed enzymes that reversibly hydrate carbon dioxide to bicarbonate and protons. While the main function of carbonic anhydrases is to regulate pH and osmotic balance, their involvement in other physiological processes remains to be explored. This study analysed changes in mRNA and protein levels of carbonic anhydrase II in human primary keratinocytes treated with various toll-like receptor agonists and cytokines. A significant upregulation of carbonic anhydrase II at the mRNA and protein levels was observed upon treatment with polyinosinic-polycytidylic acid, a toll-like receptor 3 agonist. Furthermore, in agreement with the increased expression of carbonic anhydrase II in atopic dermatitis skin, carbonic anhydrase II was upregulated by the Th2 cytokines interleukins -4 and -13. In conclusion, these results suggest a potential role of carbonic anhydrase II in Th2-dependent and toll-like receptor 3-induced pathways in inflammatory skin conditions.
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http://dx.doi.org/10.2340/00015555-2963DOI Listing
August 2018

Identification of Antibacterial Components in Human Hair Shafts.

Acta Derm Venereol 2018 Jul;98(7):708-710

Lee Kong Chian School of Medicine, Nanyang Technological University, 59 Nanyang Drive, Singapore 636921, Singapore.

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http://dx.doi.org/10.2340/00015555-2952DOI Listing
July 2018