Now showing 1 - 10 of 18
  • Publication
    A helicase-primase drug candidate with sufficient target tissue exposure affects latent neural herpes simplex virus infections
    ( 2021)
    Gege, Christian
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    Bravo, Fernando J.
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    Hagmaier, Timo
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    Schmachtenberg,Rosanne
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    Elis, Julia
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    Hamprecht, Klaus
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    Bernstein, David I.
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    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.
  • Publication
    Towards automation in biologics production via Raman micro-spectroscopy, laser-induced forward cell transfer and surface-enhanced Raman spectroscopy
    ( 2020)
    Jaeckle, Elisabeth
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    Brauchle, Eva
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    Schenke-Layland, Katja
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    Bach, Monika
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    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.
  • Publication
    Low-energy electron irradiation efficiently inactivates the gram-negative pathogen rodentibacter pneumotropicus
    ( 2020) ;
    Bayer, Lea
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    Kähl, Sophie
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    Haji, Rukiya M.
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    Baums, Christoph Georg
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    Casado, Javier Portillo
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    Schönfelder, Jessi
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    Bacterial pathogens cause severe infections worldwide in livestock and in humans, and antibiotic resistance further increases the importance of prophylactic vaccines. Inactivated bacterial vaccines (bacterins) are usually produced via incubation of the pathogen with chemicals such as formaldehyde, which is time consuming and may cause loss of immunogenicity due to the modification of structural components. We evaluated low-energy electron irradiation (LEEI) as an alternative method to generate a bacterin. Rodentibacter pneumotropicus, an invasive Gram-negative murine pathogen, was inactivated with LEEI and formaldehyde. LEEI resulted in high antigen conservation, and LPS activity was significantly better maintained when compared with formaldehyde treatment. Immunization of mice with LEEI-inactivated R. pneumotropicus elicited a strong immune response with no detectable bacterial burden upon sublethal challenge. The results of this study suggest the inactivation of bacteria with LEEI as an alternative, fast and efficient method to generate bacterial vaccines with increased efficacy.
  • Publication
    Phenomenological investigation of the cytotoxic activity of fucoidan isolated from Fucus vesiculosus
    ( 2019)
    Zayed, Ahmed
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    Krämer, Roland
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    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.
  • Patent
    Verfahren und Vorrichtung zur Bereitstellung einer eine gewünschte Zielprotein-Expression aufweisenden Zelllinie
    ( 2018) ; ; ;
    Riester, Dominik
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    Bach, Monika
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    Brauchle, Eva
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    Schenke-Layland, Katja
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    Nguyen, Phuong-Ha
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    Hofer, Katharina
    Die Erfindung betrifft ein Verfahren zur Bereitstellung einer eine gewünschte Zielprotein-Expression aufweisenden Zelllinie sowie eine Vorrichtung zur Selektion von eine gewünschte Zielprotein-Expression aufweisenden Zelllinien.
  • Publication
    Computationally designed bispecific MD2/CD14 binding peptides show TLR4 agonist activity
    ( 2018)
    Michaeli, Amit
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    Mezan, Shaul
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    Kühbacher, Andreas
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    Elias, Maayan
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    Zatsepin, Maria
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    Reed, Steven G.
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    Duthie, Malcolm S.
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    Lerner, Immanuel
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    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.
  • Publication
    Immune cell-supplemented human skin model for studying fungal infections
    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.
  • Publication
    Reduced cytotoxicity and enhanced bioactivity of cationic antimicrobial peptides liposomes in cell cultures and 3D epidermis model against HSV
    ( 2016)
    Ron-Doitch, Sapir
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    Sawodny, Beate
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    Kühbacher, Andreas
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    Nordling, Mirjam M.
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    Samanta, Ayan
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    Phopase, Jaywant
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    Griffith, May
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    Golomb, Gershon
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    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.
  • Publication
    Physicochemical and biological characterization of fucoidan from Fucus vesiculosus purified by dye affinity chromatography
    ( 2016)
    Zayed, Ahmed
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    Muffler, Kai
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    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.