A group of bacterial proteases, the Pro-Pro endopeptidases (PPEPs), pos-sess the unique ability to hydrolyze proline-proline bonds in proteins. Sincea protease’s function is largely determined by... Show moreA group of bacterial proteases, the Pro-Pro endopeptidases (PPEPs), pos-sess the unique ability to hydrolyze proline-proline bonds in proteins. Sincea protease’s function is largely determined by its substrate specificity,methods that can extensively characterize substrate specificity are valuabletools for protease research. Previously, we achieved an in-depth characteri-zation of PPEP prime-side specificity. However, PPEP specificity is alsodetermined by the non-prime-side residues in the substrate. To gain a morecomplete insight into the determinants of PPEP specificity, we character-ized the non-prime- and prime-side specificity of various PPEPs using acombination of synthetic combinatorial peptide libraries and mass spec-trometry. With this approach, we deepened our understanding of the P3-P30 specificities of PPEP-1 and PPEP-2, while identifying the endogenoussubstrate of PPEP-2 as the most optimal substrate in our library data. Fur-thermore, by employing the library approach, we investigated the alteredspecificity of mutants of PPEP-1 and PPEP-2. Additionally, we character-ized a novel PPEP from Anoxybacillus tepidamans, which we termed PPEP-4. Based on structural comparisons, we hypothesized that PPEP-4 displaysa PPEP-1-like prime-side specificity, which was substantiated by the experi-mental data. Intriguingly, another putative PPEP from Clostridioides diffi-cile, CD1597, did not display Pro-Pro endoproteolytic activity.Collectively, we characterized PPEP specificity in detail using our robustpeptide library method and, together with additional structural informa-tion, provide more insight into the intricate mechanisms that govern prote-ase specificity. Show less
The antibiotic management of catheter-related infections (CRIs) often fails owing to the emergence of antimicrobial-resistant strains and/or biofilm/persister apparitions. Thus, we investigated the... Show moreThe antibiotic management of catheter-related infections (CRIs) often fails owing to the emergence of antimicrobial-resistant strains and/or biofilm/persister apparitions. Thus, we investigated the efficacy of two novel antimicrobial agents, i.e., the synthetic peptide SAAP-148 and the novel antibiotic halicin, against Gram-negative bacteria (GNB) colonizing catheters. The antibacterial, anti-biofilm, and anti-persister activities of both agents were evaluated against Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae strains. The enrolled strains were isolated from catheters and selected based on their resistance to at least three antibiotic classes and biofilm formation potential. Furthermore, the hemolysis and endotoxin neutralization abilities of these agents were explored. The bactericidal activity of both agents was reduced in urine and plasma as compared to buffered saline. In a dose-dependent manner, SAAP-148 and halicin reduced bacterial counts in 24 h preformed biofilms on silicone elastomer discs and eliminated persisters originating from antibiotic-exposed mature 7-day biofilms, with halicin being less effective than SAAP-148. Importantly, SAAP-148 and halicin acted synergistically on E. coli and K. pneumoniae biofilms but not on A. baumannii biofilms. The peptide, but not halicin, decreased the production of IL-12p40 upon exposure to UV-killed bacteria. This preliminary study showed that SAAP-148 and halicin alone/in combination are promising candidates to fight GNB colonizing catheters. Show less
Proteases comprise the class of enzymes that catalyzesthe hydrolysisof peptide bonds, thereby playing a pivotal role in many aspects oflife. The amino acids surrounding the scissile bond determine... Show moreProteases comprise the class of enzymes that catalyzesthe hydrolysisof peptide bonds, thereby playing a pivotal role in many aspects oflife. The amino acids surrounding the scissile bond determine thesusceptibility toward protease-mediated hydrolysis. A detailed understandingof the cleavage specificity of a protease can lead to the identificationof its endogenous substrates, while it is also essential for the designof inhibitors. Although many methods for protease activity and specificityprofiling exist, none of these combine the advantages of combinatorialsynthetic libraries, i.e., high diversity, equimolar concentration,custom design regarding peptide length, and randomization, with thesensitivity and detection power of mass spectrometry. Here, we developedsuch a method and applied it to study a group of bacterial metalloproteasesthat have the unique specificity to cleave between two prolines, i.e.,Pro-Pro endopeptidases (PPEPs). We not only confirmed the prime-sidespecificity of PPEP-1 and PPEP-2, but also revealed some new unexpectedpeptide substrates. Moreover, we have characterized a new PPEP (PPEP-3)that has a prime-side specificity that is very different from thatof the other two PPEPs. Importantly, the approach that we presentin this study is generic and can be extended to investigate the specificityof other proteases. Show less
OP-145 and SAAP-148, two 24-mer antimicrobial peptides derived from human cathelicidin LL-37, exhibit killing efficacy against both Gram-positive and Gram-negative bacteria at comparable peptide... Show moreOP-145 and SAAP-148, two 24-mer antimicrobial peptides derived from human cathelicidin LL-37, exhibit killing efficacy against both Gram-positive and Gram-negative bacteria at comparable peptide concentrations. However, when it comes to the killing activity against Escherichia coli, the extent of membrane permeabilization does not align with the observed bactericidal activity. This is the case in living bacteria as well as in model membranes mimicking the E. coli cytoplasmic membrane (CM). In order to understand the killing activity of both peptides on a molecular basis, here we studied their mode of action, employing a combination of microbiological and biophysical techniques including differential scanning calorimetry (DSC), zeta potential measurements, and spectroscopic analyses. Various membrane dyes were utilized to monitor the impact of the peptides on bacterial and model membranes. Our findings unveiled distinct binding patterns of the peptides to the bacterial surface and differential permeabilization of the E. coli CM, depending on the smooth or rough/deep-rough lipopolysaccharide (LPS) phenotypes of E. coli strains. Interestingly, the antimicrobial activity and membrane depolarization were not significantly different in the different LPS phenotypes investigated, suggesting a general mechanism that is independent of LPS. Although the peptides exhibited limited permeabilization of E. coli membranes, DSC studies conducted on a mixture of synthetic phosphatidylglycerol/phosphatidylethanolamine/cardiolipin, which mimics the CM of Gram-negative bacteria, clearly demonstrated disruption of lipid chain packing. From these experiments, we conclude that depolarization of the CM and alterations in lipid packing plays a crucial role in the peptides' bactericidal activity. Show less
Bournez, C.; Riool, M.; Boer, L. de; Cordfunke, R.A.; Best, L. de; Leeuwen, R. van; ... ; Westen, G.J.P. van 2023
To combat infection by microorganisms host organisms possess a primary arsenal via the innate immune system. Among them are defense peptides with the ability to target a wide range of pathogenic... Show moreTo combat infection by microorganisms host organisms possess a primary arsenal via the innate immune system. Among them are defense peptides with the ability to target a wide range of pathogenic organisms, including bacteria, viruses, parasites, and fungi. Here, we present the development of a novel machine learning model capable of predicting the activity of antimicrobial peptides (AMPs), CalcAMP. AMPs, in particular short ones (<35 amino acids), can become an effective solution to face the multi-drug resistance issue arising worldwide. Whereas finding potent AMPs through classical wet-lab techniques is still a long and expensive process, a machine learning model can be useful to help researchers to rapidly identify whether peptides present potential or not. Our prediction model is based on a new data set constructed from the available public data on AMPs and experimental antimicrobial activities. CalcAMP can predict activity against both Gram-positive and Gram-negative bacteria. Different features either concerning general physicochemical properties or sequence composition have been assessed to retrieve higher prediction accuracy. CalcAMP can be used as an promising prediction asset to identify short AMPs among given peptide sequences. Show less
Bournez, C.; Riool, M.; Boer, L. de; Cordfunke, R.A.; Best, L. de; Leeuwen, R. van; ... ; Westen, G.J.P. van 2023
To combat infection by microorganisms host organisms possess a primary arsenal via the innate immune system. Among them are defense peptides with the ability to target a wide range of pathogenic... Show moreTo combat infection by microorganisms host organisms possess a primary arsenal via the innate immune system. Among them are defense peptides with the ability to target a wide range of pathogenic organisms, including bacteria, viruses, parasites, and fungi. Here, we present the development of a novel machine learning model capable of predicting the activity of antimicrobial peptides (AMPs), CalcAMP. AMPs, in particular short ones (<35 amino acids), can become an effective solution to face the multi-drug resistance issue arising worldwide. Whereas finding potent AMPs through classical wet-lab techniques is still a long and expensive process, a machine learning model can be useful to help researchers to rapidly identify whether peptides present potential or not. Our prediction model is based on a new data set constructed from the available public data on AMPs and experimental antimicrobial activities. CalcAMP can predict activity against both Gram-positive and Gram-negative bacteria. Different features either concerning general physicochemical properties or sequence composition have been assessed to retrieve higher prediction accuracy. CalcAMP can be used as an promising prediction asset to identify short AMPs among given peptide sequences. Show less
Synthetic antimicrobial and antibiofilm peptide (SAAP-148) commits significant antimicrobial activities against antimicrobial resistant (AMR) planktonic bacteria and biofilms. However, SAAP-148 is... Show moreSynthetic antimicrobial and antibiofilm peptide (SAAP-148) commits significant antimicrobial activities against antimicrobial resistant (AMR) planktonic bacteria and biofilms. However, SAAP-148 is limited by its low selectivity index, i.e., ratio between cytotoxicity and antimicrobial activity, as well as its bioavailability at infection sites. We hypothesized that formulation of SAAP-148 in PLGA nanoparticles (SAAP-148 NPs) improves the selectivity index due to the sustained local release of the peptide. The aim of this study was to investigate the physical and functional characteristics of SAAP-148 NPs and to compare the selectivity index of the formulated peptide with that of the peptide in solution. SAAP-148 NPs displayed favorable physiochemical properties [size = 94.1 +/- 23 nm, polydispersity index (PDI) = 0.08 +/- 0.1, surface charge = 1.65 +/- 0.1 mV, and encapsulation efficiency (EE) = 86.7 +/- 0.3%] and sustained release of peptide for up to 21 days in PBS at 37 degrees C. The antibacterial and cytotoxicity studies showed that the selectivity index for SAAP-148 NPs was drastically increased, by 10-fold, regarding AMR Staphylococcus aureus and 20-fold regarding AMR Acinetobacter baumannii after 4 h. Interestingly, the antibiofilm activity of SAAP-148 NPs against AMR S. aureus and A. baumannii gradually increased overtime, suggesting a dose-effect relationship based on the peptide's in vitro release profile. Using 3D human skin equivalents (HSEs), dual drug SAAP-148 NPs and the novel antibiotic halicin NPs provided a stronger antibacterial response against planktonic and cell-associated bacteria than SAAP-148 NPs but not halicin NPs after 24 h. Confocal laser scanning microscopy revealed the presence of SAAP-148 NPs on the top layers of the skin models in close proximity to AMR S. aureus at 24 h. Overall, SAAP-148 NPs present a promising yet challenging approach for further development as treatment against bacterial infections. Show less
The need for alternative treatment of multi-drug-resistant bacteria led to the increased design of antimicrobial peptides (AMPs). AMPs exhibit a broad antimicrobial spectrum without a distinct... Show moreThe need for alternative treatment of multi-drug-resistant bacteria led to the increased design of antimicrobial peptides (AMPs). AMPs exhibit a broad antimicrobial spectrum without a distinct preference for a specific species. Thus, their mechanism, disruption of fundamental barrier function by permeabilization of the bacterial cytoplasmic membrane is considered to be rather general and less likely related to antimicrobial resistance. Of all physico-chemical properties of AMPs, their positive charge seems to be crucial for their interaction with negatively charged bacterial membranes. Therefore, we elucidate the role of electrostatic interaction on bacterial surface neutralization and on membrane disruption potential of two potent antimicrobial peptides, namely, OP-145 and SAAP-148. Experiments were performed on Escherichia coli, a Gram-negative bacterium, and Enterococcus hirae, a Gram-positive bacterium, as well as on their model membranes. Zeta potential measurements demonstrated that both peptides neutralized the surface charge of E. coli immediately after their exposure, but not of E. hirae. Second, peptides neutralized all model membranes, but failed to efficiently disrupt model membranes mimicking Gram-negative bacteria. This was further confirmed by flow cytometry showing reduced membrane permeability for SAAP-148 and the lack of OP-145 to permeabilize the E. coli membrane. As neutralization of E. coli surface charges was achieved before the cells were killed, we conclude that electrostatic forces are more important for actions on the surface of Gram-negative bacteria than on their cytoplasmic membranes. Show less
Recently, using a deep learning approach, the novel antibiotic halicin was discovered. We compared the antibacterial activities of two novel bactericidal antimicrobial agents, i.e., the synthetic... Show moreRecently, using a deep learning approach, the novel antibiotic halicin was discovered. We compared the antibacterial activities of two novel bactericidal antimicrobial agents, i.e., the synthetic antibacterial and antibiofilm peptide (SAAP)-148 with this antibiotic halicin. Results revealed that SAAP-148 was more effective than halicin in killing planktonic bacteria of antimicrobial-resistant (AMR) Escherichia coli, Acinetobacter baumannii and Staphylococcus aureus, especially in biologically relevant media, such as plasma and urine, and in 3D human infection models. Surprisingly, SAAP-148 and halicin were equally effective against these bacteria residing in immature and mature biofilms. As their modes of action differ, potential favorable interactions between SAAP-148 and halicin were investigated. For some specific strains of AMR E. coli and S. aureus synergism between these agents was observed, whereas for other strains, additive interactions were noted. These favorable interactions were confirmed for AMR E. coli in a 3D human bladder infection model and AMR S. aureus in a 3D human epidermal infection model. Together, combinations of these two novel antimicrobial agents hold promise as an innovative treatment for infections not effectively treatable with current antibiotics. Show less
The development of antimicrobial agents against multidrug-resistant bacteria is an important medical challenge. Antimicrobial peptides (AMPs), human cathelicidin LL-37 and its derivative OP-145,... Show moreThe development of antimicrobial agents against multidrug-resistant bacteria is an important medical challenge. Antimicrobial peptides (AMPs), human cathelicidin LL-37 and its derivative OP-145, possess a potent antimicrobial activity and were under consideration for clinical trials. In order to overcome some of the challenges to their therapeutic potential, a very promising AMP, SAAP-148 was designed. Here, we studied the mode of action of highly cationic SAAP-148 in comparison with OP-145 on membranes of Enterococcus hirae at both cellular and molecular levels using model membranes composed of major constituents of enterococcal membranes, that is, anionic phosphatidylglycerol (PG) and cardiolipin (CL). In all assays used, SAAP-148 was consistently more efficient than OP-145, but both peptides displayed pronounced time and concentration dependences in killing bacteria and performing at the membrane. At cellular level, Nile Red-staining of enterococcal membranes showed abnormalities and cell shrinkage, which is also reflected in depolarization and permeabilization of E. hirae membranes. At the molecular level, both peptides abolished the thermotropic phase transition and induced disruption of PG/CL. Interestingly, the membrane was disrupted before the peptides neutralized the negative surface charge of PG/CL. Our results demonstrate that SAAP-148, which kills bacteria at a significantly lower concentration than OP-145, shows stronger effects on membranes at the cellular and molecular levels. Show less
Baart, V.M.; Manen, L. van; Bhairosingh, S.S.; Vuijk, F.A.; Iamele, L.; Jonge, H. de; ... ; Sier, C.F.M. 2021
Purpose Radical resection is paramount for curative oncological surgery. Fluorescence-guided surgery (FGS) aids in intraoperative identification of tumor-positive resection margins. This study aims... Show morePurpose Radical resection is paramount for curative oncological surgery. Fluorescence-guided surgery (FGS) aids in intraoperative identification of tumor-positive resection margins. This study aims to assess the feasibility of urokinase plasminogen activator receptor (uPAR) targeting antibody fragments for FGS in a direct comparison with their parent IgG in various relevant in vivo models. Procedures Humanized anti-uPAR monoclonal antibody MNPR-101 (uIgG) was proteolytically digested into F(ab')2 and Fab fragments named uFab2 and uFab. Surface plasmon resonance (SPR) and cell assays were used to determine in vitro binding before and after fluorescent labeling with IRDye800CW. Mice bearing subcutaneous HT-29 human colonic cancer cells were imaged serially for up to 120 h after fluorescent tracer administration. Imaging characteristics and ex vivo organ biodistribution were further compared in orthotopic pancreatic ductal adenocarcinoma (BxPc-3-luc2), head-and-neck squamous cell carcinoma (OSC-19-luc2-GFP), and peritoneal carcinomatosis (HT29-luc2) models using the clinical Artemis fluorescence imaging system. Results Unconjugated and conjugated uIgG, uFab2, and uFab specifically recognized uPAR in the nanomolar range as determined by SPR and cell assays. Subcutaneous tumors were clearly identifiable with tumor-to-background ratios (TBRs) > 2 after 72 h for uIgG-800F and 24 h for uFab2-800F and uFab-800F. For the latter two, mean fluorescence intensities (MFIs) dipped below predetermined threshold after 72 h and 36 h, respectively. Tumors were easily identified in the orthotopic models with uIgG-800F consistently having the highest MFIs and uFab2-800F and uFab-800F having similar values. In biodistribution studies, kidney and liver fluorescence approached tumor fluorescence after uIgG-800F administration and surpassed tumor fluorescence after uFab2-800F or uFab-800F administration, resulting in interference in the abdominal orthotopic mouse models. Conclusions In a side-by-side comparison, FGS with uPAR-targeting antibody fragments compared with the parent IgG resulted in earlier tumor visualization at the expense of peak fluorescence intensity. Show less
ObjectiveAntibodies targeting post-translationally modified proteins, such as anti-carbamylated protein antibodies (anti-CarP antibodies) are present in sera of rheumatoid arthritis (RA) patients.... Show moreObjectiveAntibodies targeting post-translationally modified proteins, such as anti-carbamylated protein antibodies (anti-CarP antibodies) are present in sera of rheumatoid arthritis (RA) patients. These autoantibodies associate with increased risk of RA development and with severity of joint destruction. It is not known which proteins in the RA joint are recognised by anti-CarP antibodies. Therefore, we investigated the presence and identity of carbamylated proteins in the human (inflamed) joint.MethodsWe obtained synovium, cartilage and synovial fluid from RA joints. Cartilage and synovium were obtained from controls. Samples were processed and used for immunohistochemistry or mass-spectrometric analysis to investigate the presence of carbamylated proteins. Anti-CarP antibody reactivity towards identified carbamylated proteins was tested by ELISA.ResultsImmunohistochemistry showed extensive staining of RA and control synovial tissue. Whole proteome analyses of the joint tissues revealed a large number of carbamylated peptidyllysine residues. We identified many carbamylated proteins in cartilage and were also able to detect carbamylation in synovial tissue and synovial fluid. Carbamylation was not exclusive to the RA joint and was also present in the joints of controls. Anti-CarP antibodies in the sera of RA patients were able to recognise the identified carbamylated proteins.ConclusionWe conclude that numerous carbamylated proteins are present in the RA joint. These carbamylated proteins can be recognised by anti-CarP antibodies, substantiating the notion that anti-CarP antibodies may play a role in the pathogenesis of RA. Show less
FiveC-glycosyl functionalized lysine building blocks, featuringC-glycosidic derivatives of alpha-rhamnose, alpha-mannose, alpha-galactose, beta-galactose, and beta-N-acetyl glucosamine have been... Show moreFiveC-glycosyl functionalized lysine building blocks, featuringC-glycosidic derivatives of alpha-rhamnose, alpha-mannose, alpha-galactose, beta-galactose, and beta-N-acetyl glucosamine have been designed and synthesized. These derivatives, equipped with acid-labile protecting groups, are eminently suitable for solid-phase synthesis of multivalent glycopeptides. The lysine building blocks were prepared fromC-allyl glycosides that underwent a Grubbs cross-metathesis with an acrylate, followed by a reduction of the C=C double bond in the resulting alpha,beta-unsaturated esters, and liberation of the carboxylate to allow condensation with a lysine side chain. The thus obtainedC-glycosides, five in total, were applied in the solid-phase peptide synthesis (SPPS) of three glycopeptides, showing the potential of the described building blocks in the assembly of well-defined mimics of homo- and heteromultivalent glycopeptides and glycoclusters. Show less
Purpose Aberrantly expressed glycans in cancer are of particular interest for tumor targeting. This proof-of-conceptin vivostudy aims to validate the use of aberrant Lewis glycans as target for... Show morePurpose Aberrantly expressed glycans in cancer are of particular interest for tumor targeting. This proof-of-conceptin vivostudy aims to validate the use of aberrant Lewis glycans as target for antibody-based, real-time imaging of gastrointestinal cancers. Procedures Immunohistochemical (IHC) staining with monoclonal antibody FG88.2, targeting Lewis(a/c/x), was performed on gastrointestinal tumors and their healthy counterparts. Then, FG88.2 and its chimeric human/mouse variant CH88.2 were conjugated with near-infrared fluorescent (NIRF) IRDye 800CW for real-time imaging. Specific binding was evaluatedin vitroon human gastrointestinal cancer cell lines with cell-based plate assays, flow cytometry, and immune-fluorescence microscopy. Subsequently, mice bearing human colon and pancreatic subcutaneous tumors were imagedin vivoafter intravenous administration of 1 nmol (150 mu g) CH88.2-800CW with the clinical Artemis NIRF imaging system using the Pearl Trilogy small animal imager as reference. One week post-injection of the tracer, tumors and organs were resected and tracer uptake was analyzedex vivo. Results IHC analysis showed strong FG88.2 staining on colonic, gastric, and pancreatic tumors, while staining on their normal tissue counterparts was limited. Next, human cancer cell lines HT-29 (colon) and BxPC-3 and PANC-1 (both pancreatic) were identified as respectively high, moderate, and low Lewis(a/c/x)-expressing. Using the clinical NIRF camera system for tumor-bearing mice, a mean tumor-to-background ratio (TBR) of 2.2 +/- 0.3 (Pearl: 3.1 +/- 0.8) was observed in the HT-29 tumors and a TBR of 1.8 +/- 0.3 (Pearl: 1.9 +/- 0.5) was achieved in the moderate expression BxPC-3 model. In both models, tumors could be adequately localized and delineated by NIRF for up to 1 week.Ex vivoanalysis confirmed full tumor penetration of the tracer and low fluorescence signals in other organs. Conclusions Using a novel chimeric Lewis(a/c/x)-targeting tracer in combination with a clinical NIRF imager, we demonstrate the potential of targeting Lewis glycans for fluorescence-guided surgery of gastrointestinal tumors. Show less
Riool, M.; Breij, A. de; Kwakman, P.H.S.; Schonkeren-Ravensbergen, E.; Boer, L. de; Cordfunke, R.A.; ... ; Zaat, S.A.J. 2020
Antimicrobial peptides are considered promising candidates for the development of novel antimicrobial agents to combat infections by multi-drug-resistant (MDR) bacteria. Here, we describe the... Show moreAntimicrobial peptides are considered promising candidates for the development of novel antimicrobial agents to combat infections by multi-drug-resistant (MDR) bacteria. Here, we describe the identification and characterization of the synthetic peptide TC19, derived from the human thrombocidin-l-derived peptide L3. Biophysical experiments into the interaction between TC19 and mimics of human and bacterial plasma membranes demonstrated that the peptide is highly selective for bacterial membranes. In agreement, TC19 combined low cytotoxicity towards human fibroblasts with efficient and rapid killing in human plasma of MDR strains of several bacterial species of the ESKAPE panel. In addition, TC19 induced minor resistance in vitro, neutralized pro-inflammatory activity of bacterial cell envelope components while displaying slight chemotactic activity for human neutrophils. Importantly, topical application of TC19-containing hypromellose gel significantly reduced numbers of viable methicillin-resistant Staphylococcus aureus (MRSA) and MDR Acinetobacter baumannii in a superficial wound infection in mice. Together, TC19 is an attractive candidate for further development as a novel agent against (MDR) bacterial skin wound infections. Show less
Background: We investigated the efficacy of a synthetic antimicrobial peptide SAAP-148, which was shown to be effective against Methicillin-resistant Staphylococcus aureus (MRSA) on tape-stripped... Show moreBackground: We investigated the efficacy of a synthetic antimicrobial peptide SAAP-148, which was shown to be effective against Methicillin-resistant Staphylococcus aureus (MRSA) on tape-stripped mice skin. Unexpectedly, SAAP-148 was not effective against MRSA in our pilot study using rats with excision wounds. Therefore, we investigated factors that might have contributed to the poor efficacy of SAAP-148. Subsequently, we optimised the protocol and assessed the efficacy of SAAP-148 in an adapted rat study.Methods: We incubated 100 mu L of SAAP-148 with 1 cm(2) of a wound dressing for 1 h and determined the unabsorbed volume of peptide solution. Furthermore, 10(5) colony forming units (CFU)/mL MRSA were exposed to increasing dosages of SAAP-148 in 50% (v/v) human plasma, eschar- or skin extract or PBS. After 30 min incubation, the number of viable bacteria was determined. Next, ex vivo skin models were inoculated with MRSA for 1 h and exposed to SAAP-148. Finally, excision wounds on the back of rats were inoculated with 10(7) CFU MRSA overnight and treated with SAAP-148 for 4 h or 24 h. Subsequently, the number of viable bacteria was determined.Results: Contrary to Cuticell, Parafilm and Tegaderm film, < 20% of peptide solution was recovered after incubation with gauze, Mepilex border and Opsite Post-op. Furthermore, in plasma, eschar- or skin extract > 20-fold higher dosages of SAAP-148 were required to achieve a 2-log reduction (LR) of MRSA versus SAAP-148 in PBS. Exposure of ex vivo models to SAAP-148 for 24 h resulted in a 4-fold lower LR than a 1 h or 4 h exposure period. Additionally, SAAP-148 caused a 1.3-fold lower mean LR at a load of 10(7) CFU compared to 10(5) CFU MRSA. Moreover, exposure of ex vivo excision wound models to SAAP-148 resulted in a 1.5-fold lower LR than for tape-stripped skin. Finally, SAAP-148 failed to reduce the bacterial counts in an adapted rat study.Conclusions: Several factors, such as absorption of SAAP-148 by wound dressings, components within wound exudates, re-colonisation during the exposure of SAAP-148, and a high bacterial load may contribute to the poor antimicrobial effect of SAAP-148 against MRSA in the rat model. Show less
Mishto, M.; Mansurkhodzhaev, A.; Ying, G.; Bitra, A.; Cordfunke, R.A.; Henze, S.; ... ; Liepe, J. 2019
Targeting CD8(+) T cells to recurrent tumor-specific mutations can profoundly contribute to cancer treatment. Some of these mutations are potential tumor antigens although they can be displayed by... Show moreTargeting CD8(+) T cells to recurrent tumor-specific mutations can profoundly contribute to cancer treatment. Some of these mutations are potential tumor antigens although they can be displayed by non-spliced epitopes only in a few patients, because of the low affinity of the mutated non-spliced peptides for the predominant HLA class I alleles. Here, we describe a pipeline that uses the large sequence variety of proteasome-generated spliced peptides and identifies spliced epitope candidates, which carry the mutations and bind the predominant HLA-I alleles with high affinity. They could be used in adoptive T cell therapy and other anti-cancer immunotherapies for large cohorts of cancer patients. As a proof of principle, the application of this pipeline led to the identification of a KRAS G12V mutation-carrying spliced epitope candidate, which is produced by proteasomes, transported by TAPs and efficiently presented by the most prevalent HLA class I molecules, HLA-A*02:01 complexes. Show less
Mishto, M.; Mansurkhodzhaev, A.; Ying, G.; Bitra, A.; Cordfunke, R.A.; Henze, S.; ... ; Liepe, J. 2019
Skin bacterial colonization/infection is a frequent cause of morbidity in patients with chronic wounds and allergic/inflammatory skin diseases. This study aimed to develop a novel approach to... Show moreSkin bacterial colonization/infection is a frequent cause of morbidity in patients with chronic wounds and allergic/inflammatory skin diseases. This study aimed to develop a novel approach to eradicate meticillin-resistant Staphylococcus aureus (MRSA) from human skin. To achieve this, the stability and antibacterial activity of the novel LL-37-derived peptide P10 in four ointments was compared. Results indicate that P10 is chemically stable and antibacterial in hypromellose gel and Softisan-containing cream, but not in Cetomacrogol cream (with or without Vaseline), at 4 degrees C for 16 months. Reduction in MRSA counts on Leiden human epidermal models (LEMs) by P10 in hypromellose gel was greater than that of the peptide in Cetomacrogol cream or phosphate buffered saline. P10 did not show adverse effects on LEMs irrespective of the ointment used, while Cetomacrogol with Vaseline and Softisan cream, but not hypromellose gel or Cetomacrogol cream, destroyed MRSA-colonized LEMs. Taking all this into account, P10 in hypromellose gel dose-dependently reduced MRSA colonizing the stratum corneum of the epidermis as well as biofilms of this bacterial strain on LEMs. Moreover, P10 dose-dependently reduced MRSA counts on ex-vivo human skin, with P10 in hypromellose gel being more effective than P10 in Cetomacrogol and Softisan creams. P10 in hypromellose gel is a strong candidate for eradication of MRSA from human skin. (C) 2019 Elsevier B.V. and International Society of Chemotherapy. All rights reserved. Show less
