Dr.

Burger-Kentischer, Anke

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A helicase-primase drug candidate with sufficient target tissue exposure affects latent neural herpes simplex virus infections

2021 , Gege, Christian , Bravo, Fernando J. , Uhlig, Nadja , Hagmaier, Timo , Schmachtenberg,Rosanne , Elis, Julia , Burger-Kentischer, Anke , Finkelmeier, Doris , Hamprecht, Klaus , Grunwald, Thomas , Bernstein, David I. , Kleymann, Gerald

More than 50% of the world population is chronically infected with herpesviruses. Herpes simplex virus (HSV) infections are the cause of herpes labialis (cold sores), genital herpes, and sight-impairing keratitis. Less frequently, life-threatening disseminated disease (encephalitis and generalized viremia) can also occur, mainly in immunocompromised patients and newborns. After primary infection, HSV persists for life in a latent state in trigeminal or sacral ganglia and, triggered by diverse stimuli, disease recurs in more than 30% of patients up to several times a year. Current therapy with nucleoside analogs targeting the viral polymerase is somewhat effective but limited by poor exposure in the nervous system, and latent infections are not affected by therapy. Here, we report on an inhibitor of HSV helicase-primase with potent in vitro anti-herpes activity, a different mechanism of action, a low frequency of HSV resistance, and a favorable pharmacokinetic and safety profile. Improved target tissue exposure results in superior efficacy in preventing and treating HSV infection and disease in animal models as compared to standard of care. Therapy of primary HSV infections with drug candidate IM-250 {(S)-2-(2',5'-difluoro-[1,1'-biphenyl]-4-yl)-Nmethyl-N-(4-methyl-5-(S-methylsulfon-imidoyl)thiazol-2-yl)acetamide} not only reduces the duration of disease symptoms or time to healing but also prevents recurrent disease in guinea pigs. Treatment of recurrent infections reduces the frequency of recurrences and viral shedding, and, unlike nucleosidic drugs, IM-250 remains effective for a time after cessation of treatment. Hence, IM-250 has advantages over standard-of-care therapies and represents a promising therapeutic for chronic HSV infection, including nucleoside-resistant HSV.

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Teilvorhaben: Automatisierte Biosensoren, selbstlernende Monitoring-Tools und Konzepte für sichere Sensornetzwerke zur Erhöhung der Resilienz von Trinkwasserinfrastrukturen

2018 , Bernard, Thomas , Burger-Kentischer, Anke , Frick, Konstantin , Jacubasch, Andreas , Kerger, Christian , Kohl, Christina , Kühnert, Christian , Meier, David , Trick, Iris

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Discovery of new and diverse TLR9 receptor antagonists for regulating innate immune reactions

2015 , Goldblum, A. , Burger-Kentischer, A. , Mattes, Angela , Zatsepin, M.

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Identification and characterisation of novel antifungal compounds against fungal human pathogens

2012 , Keller, P.D. , Burger-Kentischer, A. , Finkelmeier, D. , Wiesmuller, K.H. , Lemuth, Karin , Hiller, Ekkehard , Engelhardt, I. , Muller, C. , Schroppel, K. , Bracher, F. , Rupp, S.

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Towards automation in biologics production via Raman micro-spectroscopy, laser-induced forward cell transfer and surface-enhanced Raman spectroscopy

2020 , Jaeckle, Elisabeth , Brauchle, Eva , Nottrodt, Nadine , Wehner, Martin , Lensing, Richard , Gillner, Arnold , Schenke-Layland, Katja , Bach, Monika , Burger-Kentischer, Anke

Mammalian cells have become the predominant expression system for the production of biopharmaceuticals due to their capabilities in posttranslational modifications. In recent years, the efficacy of these production processes has increased significantly through technical improvements. However, the state of the art in the development of producer cell lines includes many manual steps and is as such very time and cost consuming. In this study we developed a process combination of Raman micro-spectroscopy, laser-induced forward transfer (LIFT) and surface-enhanced Raman spectroscopy (SERS) as an automated machine system for the identification, separation and characterization of single cell-clones for biopharmaceutical production. Raman spectra showed clear differences between individual antibody-producing and non-producing chinese hamster ovary (CHO) cells after their stable transfection with a plasmid coding for an immunoglobulin G (IgG) antibody. Spectra of producing CHO c ells exhibited Raman signals characteristic for human IgG. Individual producing CHO cells were successfully separated and transferred into a multiwell plate via LIFT. Besides, changes in concentration of human IgG in solution were detected via SERS. SERS spectra showed the same peak patterns but differed in their peak intensity. Overall, our results show that identification of individual antibody-producing CHO cells via Raman micro-spectroscopy, cell separation via LIFT and determination of changes in concentrations of overexpressed protein via SERS are suitable and versatile tools for assembling a fully automated system for biopharmaceuticals manufacturing.

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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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B lymphocytes undergo TLR2-dependent apoptosis upon Shigella infection

2014 , Nothelfer, K. , Arena, E.T. , Pinaud, L. , Neunlist, M. , Mozeleski, B. , Belotserkovsky, I. , Parsot, C. , Dinadayala, P. , Burger-Kentischer, A. , Raqib, R. , Sansonetti, P.J. , Phalipon, A.

Antibody-mediated immunity to Shigella, the causative agent of bacillary dysentery, requires several episodes of infection to get primed and is short-lasting, suggesting that the B cell response is functionally impaired. We show that upon ex vivo infection of human colonic tissue, invasive S. flexneri interacts with and occasionally invades B lymphocytes. The induction of a type three secretion apparatus (T3SA)-dependent B cell death is observed in the human CL-01 B cell line in vitro, as well as in mouse B lymphocytes in vivo. In addition to cell death occurring in Shigella-invaded CL-01 B lymphocytes, we provide evidence that the T3SA needle tip protein IpaD can induce cell death in noninvaded cells. IpaD binds to and induces B cell apoptosis via TLR2, a signaling receptor thus far considered to result in activation of B lymphocytes. The presence of bacterial co-signals is required to sensitize B cells to apoptosis and to up-regulate tlr2, thus enhancing IpaD binding. Apoptotic B lymphocytes in contact with Shigella-IpaD are detected in rectal biopsies of infected individuals. This study therefore adds direct B lymphocyte targeting to the diversity of mechanisms used by Shigella to dampen the host immune response.

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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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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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An in vitro HSV-1 reactivation model containing quiescently infected PC12 cells

2013 , Hogk, I. , Kaufmann, M. , Finkelmeier, D. , Rupp, S. , Burger-Kentischer, A.

Advances in the understanding of the infection and reactivation process of herpes simplex type 1 (HSV-1) are generally gained by monolayer cultures or extensive and cost-intensive animal models. So far, no reliable in vitro skin model exists either to investigate the molecular mechanisms involved in controlling latency and virus reactivation or to test pharmaceuticals. Here we demonstrate the first in vitro HSV-1 reactivation model generated by using the human keratinocyte cell line HaCaT grown on a collagen substrate containing primary human fibroblasts. We integrated the unique feature of a quiescently infected neuronal cell line, the rat pheochromocytoma line PC12, within the dermal layer of the three-dimensional skin equivalent. Transmission electron microscopy, a cell-based TCID50 assay, and polymerase chain reaction analysis were used to verify cell latency. Thereby viral DNA could be detected, whereas extracellular as well as intracellular virus activity could not be found. Further, the infected PC12 cells show no spontaneous reactivation within the in vitro skin equivalent. In order to simulate a physiologically comparable HSV-1 infection, we achieved a specific and pointed reactivation of quiescently HSV-1 infected PC12 cells by UVB irradiation at 1000 mJ/cm2.

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