Claudia Röhl

Virostatic potential of micro–nano filopodia-like ZnO structures against herpes simplex virus-1

Herpes simplex virus type-1 (HSV-1) entry into target cell is initiated by the ionic interactions between positively charged viral envelop glycoproteins and a negatively charged cell surface heparan sulfate (HS). This first step involves the induction of HS-rich filopodia-like structures on the cell surface that facilitate viral transport during cell entry. Targeting this initial first step in HSV-1 pathogenesis, we generated different zinc oxide (ZnO) micro-nano structures (MNSs) that were capped with multiple nanoscopic spikes mimicking cell induced filopodia. These MNSs were predicted to target the virus to compete for its binding to cellular HS through their partially negatively charged oxygen vacancies on their nanoscopic spikes, to affect viral entry and subsequent spread. Our results demonstrate that the partially negatively charged ZnO-MNSs efficiently trap the virions via a novel virostatic mechanism rendering them unable to enter into human corneal fibroblasts - a natural target cell for HSV-1 infection. The anti-HSV-1 activity of ZnO MNSs was drastically enhanced after creating additional oxygen vacancies under UV-light illumination. Our results provide a novel insight into the significance of ZnO MNSs as the potent HSV-1 inhibitor and rationalize their development as a novel topical agent for the prevention of HSV-1 infection.

The effect of activated microglia on astrogliosis parameters in astrocyte cultures

In the diseased central nervous system, astrogliosis is accompanied by microglial activation. Depending on the context of their activation, reactive astrocytes are involved in neuronal survival and regeneration in an either protective or impedimental way. Major reactive changes of astrocytes in vivo are the upregulation of the intermediate filaments GFAP (glial fibrillary acidic protein) and vimentin with accompanying cellular hypertrophy and/or hyperplasia. To examine the involvement of activated microglia in the onset and maintenance of astrogliosis, we used an in vitro model of purified cultures of astrocytes and assessed as parameters for astrogliosis GFAP, vimentin, astroglial hypertrophy and cell growth after treatment with medium conditioned by LPS (lipopolysaccarides)-stimulated microglia. Furthermore, IL-6 as a typically upregulated cytokine in proinflammatory processes in the brain was determined in treated astrocytes. GFAP, the classical marker for astrogliosis, was downregulated on its protein and in parallel with vimentin on its mRNA level. The expression of actin, another cytoskeleton protein used as control, remained unchanged. Ultrastructural studies of astroglial intermediate filaments supported these findings. No hypertrophy was found. Nevertheless, LPS-activated microglia stimulated astrocytes as demonstrated by an increased cell number and an enhanced mRNA expression of IL-6. Resting microglia did not change any of the determined parameters. Our results suggest that the role of activated microglia in astrogliotic processes following injury of the brain has to be reevaluated, as microglia in their activated state might support the onset of astrogliosis on the one hand, but might delay or reduce subsequent glial scar formation on the other hand.

Toxicity of Functional Nano-Micro Zinc Oxide Tetrapods: Impact of Cell Culture Conditions, Cellular Age and Material Properties

With increasing production and applications of nanostructured zinc oxide, e.g., for biomedical and consumer products, the question of safety is getting more and more important. Different morphologies of zinc oxide structures have been synthesized and accordingly investigated. In this study, we have particularly focused on nano-micro ZnO tetrapods (ZnO-T), because their large scale fabrication has been made possible by a newly introduced flame transport synthesis approach which will probably lead to several new applications. Moreover, ZnO-T provide a completely different morphology then classical spherical ZnO nanoparticles. To get a better understanding of parameters that affect the interactions between ZnO-T and mammalian cells, and thus their biocompatibility, we have examined the impact of cell culture conditions as well as of material properties on cytotoxicity. Our results demonstrate that the cell density of fibroblasts in culture along with their age, i.e., the number of preceding cell divisions, strongly affect the cytotoxic potency of ZnO-T. Concerning the material properties, the toxic potency of ZnO-T is found to be significantly lower than that of spherical ZnO nanoparticles. Furthermore, the morphology of the ZnO-T influenced cellular toxicity in contrast to surface charges modified by UV illumination or O2 treatment and to the material age. Finally, we have observed that direct contact between tetrapods and cells increases their toxicity compared to transwell culture models which allow only an indirect effect via released zinc ions. The results reveal several parameters that can be of importance for the assessment of ZnO-T toxicity in cell cultures and for particle development.

Activated Microglia Modulate Astroglial Enzymes Involved in Oxidative and Inflammatory Stress and Increase the Resistance of Astrocytes to Oxidative Stress In Vitro

Neuropathological processes in the central nervous system are commonly accompanied by an activation of microglia and astrocytes. The involvement of both cell populations in the onset and progress of neurological disorders has been widely documented, implicating both beneficial and detrimental influences on the neural tissue. Nevertheless, little is known about the interplay of these glial cell populations, especially under diseased conditions. To examine the effects of activated microglia on astrocytes purified rat astroglial cell cultures were treated with medium conditioned by purified quiescent (MCM[−]) or lipopolysaccharide (LPS)‐activated rat microglia (MCM[+]) and subjected to a comparative proteome analysis based on two‐dimensional gel electrophoresis. No significant down regulation of proteins was observed. The majority of the 19 proteins identified by means of nano HPLC/ESI‐MS/MS in the 12 most prominent protein spots significantly overexpressed (≥2‐fold) in MCM[+] treated astrocytes are involved in inflammatory processes and oxidative stress response: superoxide dismutases (Sod), peroxiredoxins, glutathione S‐transferases (Gst), nucleoside diphosphate kinase B, argininosuccinate synthase (Ass), and cellular retinol‐binding protein I (Rbp1). Sod2, Rbp1, Gstp1, and Ass were also significantly increased on the mRNA level determined by quantitative RT‐PCR. The upregulation of antioxidative enzymes in astrocytes was accompanied by a higher resistance to oxidative stress induced by H2O2. These results show that activated microglia change the expression of antioxidative proteins in astrocytes and protect them against oxidative stress, which might be an effective way to increase the neuroprotective potential of astrocytes under pathological conditions associated with oxidative stress and inflammation.

Inhibition of bacterial adhesion to live human cells: Activity and cytotoxicity of synthetic mannosides

Bacterial adhesion to glycosylated surfaces is a key issue in human health and disease. Inhibition of bacterial adhesion by suitable carbohydrates could lead to an anti‐adhesion therapy as a novel approach against bacterial infections. A selection of five α‐mannosides has been evaluated as inhibitors of bacterial adhesion to the polysaccharide mannan, as well as to the surface of live human HT‐29 cells. Cell toxicity studies were performed to identify the therapeutic window for a potential in vivo‐application of the tested carbohydrates. A previously published mannosidic squaric acid diamide was shown to be exceptionally effective as inhibitor of the bacterial lectin FimH.

Phenolic Antioxidant Compounds Produced by in Vitro. Cultures of Rosemary (Rosmarinus officinalis.) and Their Anti-inflammatory Effect on Lipopolysaccharide-Activated Microglia

Abstract Different culture types (shoot culture, callus culture, and cell suspension culture) of Rosmarinus officinalis. L. were established and their ability to biosynthesize the phytochemicals carnosic acid, carnosol, and rosmarinic acid were assessed and compared with subcultures of the same stem plant over time. Furthermore, we examined the antioxidative effect of extracts from different culture types by measuring their scavenging activity on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals as well as their anti-inflammatory potential on neonatal microglial rat cells activated by means of the inflammogen lipopolysaccharide (LPS) taking nitric oxide (NO) as parameter. Our results show that during an investigation period of 17 months, the shoot culture accumulated varying amounts of carnosic acid and carnosol, which were also present in callus culture but about 20- to 80-fold lower than in the shoot culture. In suspension culture, only carnosic acid and no carnosol could be detected. The level of carnosic acid in suspension culture was threefold less than detected for the callus culture on average. The amount of rosmarinic acid produced in shoot culture and callus culture were comparable, whereas in suspension culture higher concentrations of rosmarinic acid could be measured than in shoot and callus culture. Thus, the content of carnosic acid, carnosol, and rosmarinic acid in the extracts depended on the differentiation grade of the cell culture type. The DPPH radical-scavenging activity of the extracts depended on the amount of all three phytochemicals, in particular of rosmarinic acid. The anti-inflammatory character of the extracts was mainly based on their carnosic acid content.

The organotin compounds trimethyltin (TMT) and triethyltin (TET) but not tributyltin (TBT) induce activation of microglia co-cultivated with astrocytes

The organotin compounds trimethyltin (TMT), triethyltin (TET) and tributyltin (TBT) show different organotoxicities in vivo. While TMT and TET induce a strong neurotoxicity accompanied by microglial and astroglial activation, TBT rather effects the immune system. Previously, we have shown in an in vitro co-culture model that microglial cells can be activated by TMT in the presence of astrocytes. In this study, we wanted to investigate (a) if the neurotoxic organotin compound TET can also activate microglial cells in vitro similar to TMT and (b) if differences between the neurotoxicants TMT and TET on the one side and TBT on the other exist concerning microglial activation. Therefore, purified microglial and astroglial cell cultures from neonatal rat brains were treated either alone or in co-cultures for 24h with different concentrations of TMT, TET or TBT and the basal cytotoxicity and nitric oxide formation was determined. Furthermore, morphological changes of astrocytes were examined. Our results show that microglial activation can be increased in subcytolethal concentrations, but only in the presence of astrocytes and not in microglial cell cultures alone. This increase was induced by the neurotoxicants TMT and TET but not by TBT. Taken together, the differing microglia activating effect of the organotin compounds may contribute to the differing neurotoxic potential of this group of chemicals in vivo. In addition, our results emphasize the need for co-culture systems when studying interactions between different cell types for toxicity assessment.

Huge increase of therapeutic window at a bioactive silver/titania nanocomposite coating surface compared to solution.

A silver containing coating used in the human body, e.g., on an implant should be both effectively antimicrobial and non-cytotoxic to human cells. It is generally believed that the biologic effect originates from silver ions released from the coating. Nanocomposites with well controlled Ag filling factor were prepared by co-sputtering, and the silver surface concentration and the silver release were determined by XPS and ICP-MS, respectively. Here we show that only a small therapeutic window exists for dissolved silver but the therapeutic window is largely increased at the surface. While the toxicity observed for mammalian cells in contact with the bioactive Ag/TiO2 nanocomposite surface and for silver ions in solution is rather similar the antimicrobial activity is drastically enhanced at the surface. A model is proposed to explain the strong increase of the antimicrobial activity at the surface. The present results not only question well-established tests for antimicrobial activity but they are also important for the design of antimicrobial coatings, e.g., for biomedical devices.