Dr.

Burger-Kentischer, Anke

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Drug repurposing for the treatment of COVID-19: Targeting nafamostat to the lungs by a liposomal delivery system

2023 , Reus, Philipp , Guthmann, Hadar , Uhlig, Nadja , Agbaria, Majd , Issmail, Leila , Eberlein, Valentina , Nordling-David, Mirjam M. , Jbara-Agbaria, Doaa , Ciesek, Sandra , Bojkova, Denisa , Cinatl, Jindrich , Burger-Kentischer, Anke , Rupp, Steffen , Zaliani, Andrea , Grunwald, Thomas , Gribbon, Philip , Kannt, Aimo , Golomb, Gershon

Despite tremendous global efforts since the beginning of the COVID-19 pandemic, still only a limited number of prophylactic and therapeutic options are available. Although vaccination is the most effective measure in preventing morbidity and mortality, there is a need for safe and effective post-infection treatment medication. In this study, we explored a pipeline of 21 potential candidates, examined in the Calu-3 cell line for their antiviral efficacy, for drug repurposing. Ralimetinib and nafamostat, clinically used drugs, have emerged as attractive candidates. Due to the inherent limitations of the selected drugs, we formulated targeted liposomes suitable for both systemic and intranasal administration. Non-targeted and targeted nafamostat liposomes (LipNaf) decorated with an Apolipoprotein B peptide (ApoB-P) as a specific lung-targeting ligand were successfully developed. The developed liposomal formulations of nafamostat were found to possess favorable physicochemical properties including nano size (119-147 nm), long-term stability of the normally rapidly degrading compound in aqueous solution, negligible leakage from the liposomes upon storage, and a neutral surface charge with low polydispersity index (PDI). Both nafamostat and ralimetinib liposomes showed good cellular uptake and lack of cytotoxicity, and non-targeted LipNaf demonstrated enhanced accumulation in the lungs following intranasal (IN) administration in non-infected mice. LipNaf retained its anti-SARS-CoV 2 activity in Calu 3 cells with only a modest decrease, exhibiting complete inhibition at concentrations >100 nM. IN, but not intraperitoneal (IP) treatment with targeted LipNaf resulted in a trend to reduced viral load in the lungs of K18-hACE2 mice compared to targeted empty Lip. Nevertheless, upon removal of outlier data, a statistically significant 1.9-fold reduction in viral load was achieved. This observation further highlights the importance of a targeted delivery into the respiratory tract. In summary, we were able to demonstrate a proof-of-concept of drug repurposing by liposomal formulations with anti-SARS-CoV-2 activity. The biodistribution and bioactivity studies with LipNaf suggest an IN or inhalation route of administration for optimal therapeutic efficacy.

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The E. coli S30 lysate proteome: A prototype for cell-free protein production

2018 , Foshag, D. , Henrich, E. , Hiller, Ekkehard , Schäfer, Miriam , Kerger, Christian , Burger-Kentischer, Anke , Diaz-Moreno, I. , García-Mauriño, S.M. , Dötsch, V. , Rupp, Steffen , Bernhard, F.

Protein production using processed cell lysates is a core technology in synthetic biology and these systems are excellent to produce difficult toxins or membrane proteins. However, the composition of the central lysate of cell-free systems is still a "black box". Escherichia coli lysates are most productive for cell-free expression, yielding several mgs of protein per ml of reaction. Their preparation implies proteome fractionation, resulting in strongly biased and yet unknown lysate compositions. Many metabolic pathways are expected to be truncated or completely removed. The lack of knowledge of basic cell-free lysate proteomes is a major bottleneck for directed lysate engineering approaches as well as for assay design using non-purified reaction mixtures. This study is starting to close this gap by providing a blueprint of the S30 lysate proteome derived from the commonly used E. coli strain A19. S30 lysates are frequently used for cell-free protein production and represent the basis of most commercial E. coli cell-free expression systems. A fraction of 821 proteins was identified as the core proteome in S30 lysates, representing approximately a quarter of the known E. coli proteome. Its classification into functional groups relevant for transcription/translation, folding, stability and metabolic processes will build the framework for tailored cell-free reactions. As an example, we show that SOS response induction during cultivation results in tuned S30 lysate with better folding capacity, and improved solubility and activity of synthesized proteins. The presented data and protocols can serve as a platform for the generation of customized cell-free systems and product analysis.

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Interaction of Candida Species with the Skin

2017 , Kühbacher, Andreas , Burger-Kentischer, A. , Rupp, S.

The human skin is commonly colonized by diverse fungal species. Some Candida species, especially C. albicans, do not only reside on the skin surface as commensals, but also cause infections by growing into the colonized tissue. However, defense mechanisms at the skin barrier level are very efficient, involving residential non-immune and immune cells as well as immune cells specifically recruited to the site of infection. Therefore, the skin is an effective barrier against fungal infection. While most studies about commensal and pathogenic interaction of Candida species with host epithelia focus on the interaction with mucosal surfaces such as the vaginal and gastrointestinal epithelia, less is known about the mechanisms underlying Candida interaction with the skin. In this review, we focus on the ecology and molecular pathogenesis of Candida species on the skin and give an overview of defense mechanisms against C. albicans in this context. We also discuss new research avenues in dermal infection, including the involvement of neurons, fibroblasts, and commensal bacteria in both mouse and human model systems.

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Reduced cytotoxicity and enhanced bioactivity of cationic antimicrobial peptides liposomes in cell cultures and 3D epidermis model against HSV

2016 , Ron-Doitch, Sapir , Sawodny, Beate , Kühbacher, Andreas , Nordling, Mirjam M. , Samanta, Ayan , Phopase, Jaywant , Burger-Kentischer, Anke , Griffith, May , Golomb, Gershon , Rupp, Steffen

Cationic antimicrobial peptides (AMPs) are part of the innate immunity, and act against a wide variety of pathogenic microorganisms by perturbation of the microorganism's plasma membrane. Although attractive for clinical applications, these agents suffer from limited stability and activity in vivo, as well as non-specific interaction with host biological membranes, leading to cytotoxic adverse effects. We hypothesized that encapsulation of AMPs within liposomes could result in reduced cytotoxicity, and with enhanced stability as well as bioactivity against herpes simplex virus 1 (HSV-1). We formulated nano-sized liposomal formulations of LL-37 and indolicidin, and their physicochemical properties, cellular uptake, in vitro cytotoxicity and antiviral efficacy have been determined. Lower cytotoxicity of LL-37 liposomes was found in comparison to indolicidin liposomes attributed to the superior physicochemical properties, and to the different degree of interaction with the liposomal membrane. The disc-like shaped LL-37 liposomes (106.8 ± 10.1 nm, shelf-life stability of >1 year) were taken up more rapidly and to a significantly higher extent than the free peptide by human keratinocyte cell line (HaCaT), remained intact within the cells, followed by release of the active peptide within the cytoplasm and migration of the vesicles' lipids to the plasma membrane. LL-37 liposomes were found significantly less toxic than both the free agent and liposomal indolicidin. In the new 3D epidermis model (immortalized primary keratinocytes) liposomal LL-37 treatment (>20 mM), but not free LL-37, efficiently protected the epidermis, inhibiting HSV-1 infection. This positive antiviral effect was obtained with no cytotoxicity even at very high concentrations (400 mM). Thus, the antiviral activity of encapsulated LL-37 was significantly improved, expanding its therapeutic window. Liposomal LL-37 appears to be a promising delivery system for HSV therapy.

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Liposomal siRNA Formulations for the Treatment of Herpes Simplex Virus-1: In Vitro Characterization of Physicochemical Properties and Activity, and In Vivo Biodistribution and Toxicity Studies

2022 , Jbara-Agbaria, D. , Blondzik, Saskia , Burger-Kentischer, Anke , Agbaria, M. , Nordling-David, M.M. , Giterman, A. , Aizik, G. , Rupp, Steffen , Golomb, G.

Herpes simplex virus-1 (HSV-1) is highly contagious, and there is a need for a therapeutic means to eradicate it. We have identified an siRNA (siHSV) that knocks down gene expression of the infected cell protein 0 (ICP0), which is important in the regulation of HSV infection. The selected siHSV was encapsulated in liposomes to overcome its poor stability, increase cell permeability, and prolonging siRNA circulation time. Several siRNAs against ICP0 have been designed and identified. We examined the role of various parameters, including formulation technique, lipids composition, and ratio. An optimal liposomal siHSV formulation (LipDOPE-siHSV) was characterized with desirable physiochemical properties, in terms of nano-size, low polydispersity index (PDI), neutral surface charge, high siHSV loading, spherical shape, high stability in physiologic conditions in vitro, and long-term shelf-life stability (>1 year, 4â—¦C). The liposomes exhibited profound internalization by human keratinocytes, no cytotoxicity in cell cultures, no detrimental effect on mice liver enzymes, and a gradual endo-lysosomal escape. Mice biodistribution studies in intact mice revealed accumulation, mainly in visceral organs but also in the trigeminal ganglion. The therapeutic potential of siHSV liposomes was demonstrated by significant antiviral activity both in the plaque reduction assay and in the 3D epidermis model, and the mechanism of action was validated by the reduction of ICP0 expression levels.

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Computationally designed bispecific MD2/CD14 binding peptides show TLR4 agonist activity

2018 , Michaeli, Amit , Mezan, Shaul , Kühbacher, Andreas , Finkelmeier, Doris , Elias, Maayan , Zatsepin, Maria , Reed, Steven G. , Duthie, Malcolm S. , Rupp, Steffen , Lerner, Immanuel , Burger-Kentischer, Anke

Toll-like receptor 4 plays an important role in the regulation of the innate and adaptive immune response. The majority of TLR4 activators currently in clinical use are derivatives of its prototypic ligand LPS. The discovery of innovative TLR4 activators has the potential of providing new therapeutic immunomodulators and adjuvants. We used computational design methods to predict and optimize a total of 53 cyclic and linear peptides targeting myeloid differentiation 2 (MD2) and cluster of differentiation 14 (CD14), both coreceptors of human TLR4. Activity of the designed peptides was first assessed using NF-kB reporter cell lines expressing either TLR4/MD2 or TLR4/CD14 receptors, then binding to CD14 and MD2 confirmed and quantified using MicroScale Thermophoresis. Finally, we incubated select peptides in human whole blood and observed their ability to induce cytokine production, either alone or in synergy with LPS. Our data demonstrate the advantage of computational design for the discovery of new TLR4 peptide activators with little structural resemblance to known ligands and indicate an efficient strategy with which to identify TLR4 targeting peptides that could be used as easy-to-produce alternatives to LPS-derived molecules in a variety of settings.

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Central role for dermal fibroblasts in skin model protection against Candida albicans

2017 , Kühbacher, Andreas , Henkel, H. , Stevens, Philip , Grumaz, Christian , Finkelmeier, D. , Burger-Kentischer, A. , Sohn, K. , Rupp, S.

The fungal pathogen Candida albicans colonizes basically all human epithelial surfaces, including the skin. Under certain conditions, such as immunosuppression, invasion of the epithelia occurs. Not much is known about defense mechanisms against C. albicans in subepithelial layers such as the dermis. Using immune cell-supplemented 3D skin models we defined a new role for fibroblasts in the dermis and identified a minimal set of cell types for skin protection against C. albicans invasion. Dual RNA sequencing of individual host cell populations and C. albicans revealed that dermal invasion is directly impeded by dermal fibroblasts. They are able to integrate signals from the pathogen and CD4+ T cells and shift toward an antimicrobial phenotype with broad specificity that is dependent on Toll-like receptor 2 and interleukin 1β. These results highlight a central function of dermal fibroblasts for skin protection, opening new possibilities for treatment of infectious diseases.

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Phenomenological investigation of the cytotoxic activity of fucoidan isolated from Fucus vesiculosus

2019 , Zayed, Ahmed , Hahn, Thomas , Finkelmeier, Doris , Burger-Kentischer, Anke , Rupp, Steffen , Krämer, Roland , Ulber, Roland

The development of natural-based anti-tumor medicaments has acquired a great interest especially in the last few decades. Hence, cytotoxic activity of different fractions of fucoidan was evaluated. The fractions, produced from the total crude extract of the brown alga Fucus vesiculosus and purified by the recently-developed immobilized cationic dyes at different conditions, had different physicochemical properties and named fucoidan_1, fucoidan_6 and fucoidan_PDD. The activity of these fractions was studied in vitro against different kinds of cancerous mammalian cell lines including MCF-7 and Caco-2 and compared to their effects against skin primary fibroblasts. The results indicated a potent cytotoxic activity with regard to MCF-7 cells, while negligible (>1500 mg mL −1 ) towards primary fibroblasts. Moreover, higher general toxicity of crude fucoidan indicated that purification process succeeded to remove extraneous, co-extracted, cytotoxic compounds (e.g., polyphenols), which has a strong activity and possible interference in previously-published studies. Furthermore, a correlation was made between the cytotoxic activity and physico-chemical properties of fucoidan fractions, such as the sulfation degree and molecular weight. These findings reflected a real picture and expected low side effects regarding the cytotoxic activity of fucoidan purified by affinity chromatography.

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Immune cell-supplemented human skin model for studying fungal infections

2017 , Kühbacher, Andreas , Sohn, Kai , Burger-Kentischer, Anke , Rupp, Steffen

Human skin is a niche for various fungal species which either colonize the surface of this tissue as commensals or, primarily under conditions of immunosuppression, invade the skin and cause infection. Here we present a method for generation of a human in vitro skin model supplemented with immune cells of choice. This model represents a complex yet amenable tool to study molecular mechanisms of host-fungi interactions at human skin.

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Physicochemical and biological characterization of fucoidan from Fucus vesiculosus purified by dye affinity chromatography

2016 , Zayed, Ahmed , Muffler, Kai , Hahn, Thomas , Rupp, Steffen , Finkelmeier, Doris , Burger-Kentischer, Anke , Ulber, Roland

A comparative study concerning the physicochemical, monomeric composition and biological characters among different fucoidan fractions is presented. Common purification techniques for fucoidan usually involve many steps. During these steps, the important structural features might be affected and consequently alter its biological activities. Three purified fractions were derived from Fucus vesiculosus water extract which, afterwards, were purified by a recently-developed dye affinity chromatography protocol. This protocol is based on dye-sulfated polysaccharide interactions. The first two fractions were obtained from crude precipitated fucoidan at different pH values of the adsorption phase: pH 1 and 6. This procedure resulted in fucoidan_1 and 6 fractions. The other, third, fraction: fucoidan_M, however, was obtained from a buffered crude extract at pH 1, eliminating the ethanol precipitation step. All of the three fractions were then further evaluated. Results revealed that fucoidan_M showed the highest sulfur content (S%), 12.11%, with the lowest average molecular weight, 48 kDa. Fucose, galactose, and uronic acid/glucose dimers were detected in all fractions, although, xylose was only detected in fucoidan_1 and 6. In a concentration of 10 µg·mL−1, Fucoidan_6 showed the highest heparin-like anticoagulant activity and could prolong the APTT and TT significantly to 66.03 ± 2.93 and 75.36 ± 1.37 s, respectively. In addition, fucoidan_M demonstrated the highest potency against HSV-1 with an IC50 of 2.41 µg·mL−1. The technique proved to be a candidate for fucoidan purifaction from its crude extract removing the precipitation step from common purification protocols and produced different fucoidan qualities resulted from the different incubation conditions with the immobilized thiazine toluidine blue O dye.

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