Publications by authors named "Daniel Thieme"

9 Publications

  • Page 1 of 1

Lamin Cleavage: A Reliable Marker for Studying Staurosporine-Induced Apoptosis in Corneal Tissue.

Invest Ophthalmol Vis Sci 2017 11;58(13):5802-5809

Department of Ophthalmology, University Hospital, University of Erlangen-Nuremberg, Erlangen, Germany.

Purpose: The aims of this study were to identify a robust apoptosis marker suitable for both quantification and back-to-back analyses of programmed cell death and to define specific upstream targets for apoptosis in corneal cells.

Methods: Apoptotic cleavage of initiator caspases and their downstream targets such as lamins and poly-ADP ribose polymerase was investigated in human corneal endothelial cells (HCEC-12), keratocytes (HCK), epithelial cells (HCEp), and full-thickness corneas using Western blotting and confocal microscopy following apoptosis induction with staurosporine. We specifically focused on nuclear lamins, which have important structural and regulatory functions in the cell nucleus.

Results: The cleavage of lamin A in HCEC-12 was significantly increased following apoptotic induction compared with HCK. More importantly, lamin A cleavage was detected in a dose-dependent manner in full-thickness corneal tissue by both Western blot analysis and fluorescence microscopy. Our study also demonstrates that HCEp show approximately three-fold increase in caspase 6 cleavage compared with endothelial cells or keratocytes. The presence of cleaved caspase 9 was lower in endothelial cells compared with epithelial cells and keratocytes.

Conclusions: We successfully established lamin A cleavage as a quantifiable marker of apoptosis in both corneal cells and tissue. Quantification of lamin A cleavage by Western blotting followed by a back-to-back analysis with fluorescence microscopy was studied for the first time in the experimental (donor) corneal tissue. Screening of downstream apoptosis proteins and establishing cell type-specific protocols allowed us to identify possible targets (caspases, Apaf-1, etc.) for protective therapeutic approaches.
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http://dx.doi.org/10.1167/iovs.17-21830DOI Listing
November 2017

Optimized human platelet lysate as novel basis for a serum-, xeno-, and additive-free corneal endothelial cell and tissue culture.

J Tissue Eng Regen Med 2018 02 3;12(2):557-564. Epub 2017 Dec 3.

Department of Ophthalmology, Universitätsklinikum Erlangen/Nürnberg; Augenklinik, Erlangen, Germany.

The expansion of donor-derived corneal endothelial cells (ECs) is a promising approach for regenerative therapies in corneal diseases. To achieve the best Good Manufacturing Practice standard the entire cultivation process should be devoid of nonhuman components. However, so far, there is no suitable xeno-free protocol for clinical applications. We therefore introduce a processed variant of a platelet lysate for the use in corneal cell and tissue culture based on a Good Manufacturing Practice-grade thrombocyte concentrate. This processed human platelet lysate (phPL), free of any animal components and of anticoagulants such as heparin with a physiological ionic composition, was used to cultivate corneal ECs in vitro and ex vivo in comparison to standard cultivation with fetal calf serum (FCS). Human donor corneas were cut in quarters while 2 quarters of each cornea were incubated with the respective medium supplement. Three fields of view per quarter were taken into account for the analysis. Evaluation of phPL as a medium supplement in cell culture of immortalized EC showed a superior viability compared with FCS control with reduced cell proliferation. Furthermore, the viability during the expansion of primary cells is significantly (3-fold ±0.5) increased with phPL compared with FCS standard medium. Quartering donor corneas was traumatic for the endothelium and therefore resulted in increased EC loss. Interestingly, however, cultivation of the quartered pieces for 2 weeks in 0.1-mg/ml pHPL in Biochrome I showed a 21 (±10) % EC loss compared with 67 (±12) % EC loss when cultivated in 2% FCS in Biochrome I. The cell culture protocol with pHPL as FCS replacement seems to be superior to the standard FCS protocols with respect to EC survival. It offers a xeno-free and physiological environment for corneal endothelial cells. This alternative cultivation protocol could facilitate the use of EC for human corneal cell therapy.
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http://dx.doi.org/10.1002/term.2574DOI Listing
February 2018

Fetal Bovine Serum (FBS): Past - Present - Future.

ALTEX 2018 9;35(1):99-118. Epub 2017 Aug 9.

Division of Physiology, Medical University Innsbruck, Innsbruck, Austria.

The supplementation of culture medium with fetal bovine serum (FBS, also referred to as "fetal calf serum") is still common practice in cell culture applications. Due to a number of disadvantages in terms of quality and reproducibility of in vitro data, animal welfare concerns, and in light of recent cases of fraudulent marketing, the search for alternatives and the development of serum-free medium formulations has gained global attention. Here, we report on the 3rd Workshop on FBS, Serum Alternatives and Serum-free Media, where regulatory aspects, the serum dilemma, alternatives to FBS, case-studies of serum-free in vitro applications, and the establishment of serum-free databases were discussed. The whole process of obtaining blood from a living calf fetus to using the FBS produced from it for scientific purposes is de facto not yet legally regulated despite the existing EU-Directive 2010/63/EU on the use of animals for scientific purposes. Together with the above-mentioned challenges, several strategies have been developed to reduce or replace FBS in cell culture media in terms of the 3Rs (Refinement, Reduction, Replacement). Most recently, releasates of activated human donor thrombocytes (human platelet lysates) have been shown to be one of the most promising serum alternatives when chemically-defined media are not yet an option. Additionally, new developments in cell-based assay techniques, advanced organ-on-chip and microphysiological systems are covered in this report. Chemically-defined serum-free media are shown to be the ultimate goal for the majority of culture systems, and examples are discussed.
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http://dx.doi.org/10.14573/altex.1705101DOI Listing
August 2018

Adhesion and metabolic activity of human corneal cells on PCL based nanofiber matrices.

Mater Sci Eng C Mater Biol Appl 2017 Feb 26;71:764-770. Epub 2016 Oct 26.

Department of Ophthalmology, Universität Erlangen-Nürnberg, Schwabachanlage 6, 91054 Erlangen, Germany. Electronic address:

In this work, polycaprolactone (PCL) was used as a basic polymer for electrospinning of random and aligned nanofiber matrices. Our aim was to develop a biocompatible substrate for ophthalmological application to improve wound closure in defects of the cornea as replacement for human amniotic membrane. We investigated whether blending the hydrophobic PCL with poly (glycerol sebacate) (PGS) or chitosan (CHI) improves the biocompatibility of the matrices for cell expansion. Human corneal epithelial cells (HCEp) and human corneal keratocytes (HCK) were used for in vitro biocompatibility studies. After optimization of the electrospinning parameters for all blends, scanning electron microscopy (SEM), attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), and water contact angle were used to characterize the different matrices. Fluorescence staining of the F-actin cytoskeleton of the cells was performed to analyze the adherence of the cells to the different matrices. Metabolic activity of the cells was measured by cell counting kit-8 (CCK-8) for 20days to compare the biocompatibility of the materials. Our results show the feasibility of producing uniform nanofiber matrices with and without orientation for the used blends. All materials support adherence and proliferation of human corneal cell lines with oriented growth on aligned matrices. Although hydrophobicity of the materials was lowered by blending PCL, no increase in biocompatibility or proliferation, as was expected, could be measured. All tested matrices supported the expansion of human corneal cells, confirming their potential as substrates for biomedical applications.
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http://dx.doi.org/10.1016/j.msec.2016.10.058DOI Listing
February 2017

O2C Laser Doppler and Digital Photo Analysis for Treatment Evaluation of Beta-Glucan versus Provitamin Pantothenic Acid of Facial Burns.

Facial Plast Surg 2016 Apr 20;32(2):225-31. Epub 2016 Apr 20.

Department of Plastic, Reconstructive and Aesthetic Surgery, Hand Surgery, University Hospital Cologne-Merheim, Burns, Cologne, Germany.

Various creams are available for superficial second-degree burns (SSDB) of the face. We evaluated provitamin pantothenic acid versus β-glucan for SSDB of the face using the O2C laser Doppler system and digital photo analysis. Out of 20 patients (January to December 2012) with facial burns, 7 with SSDB of both cheeks were included to our study. Burned cheek wounds were treated using pantothenic acid or β-glucan. Digital photos of marked regions were taken daily from predefined distances. Microcirculation was measured at marked regions for 7 days at scheduled time points using the O2C laser Doppler. Data were evaluated using the SPSS program (SPSS Inc., Chicago, IL). Wounds treated with β-glucan showed faster reepithelialization. O2C laser Doppler measurements showed faster increase in SO2, microvascular perfusion, hemoglobin content, and blood flow. This correlated good with clinical Vancouver Scar Scale results. Although not statistically significant, β-glucan cream therapy of SSDB results in aesthetically superior outcome and faster reepithelialization.
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http://dx.doi.org/10.1055/s-0036-1579782DOI Listing
April 2016

The Dps protein of Escherichia coli is involved in copper homeostasis.

Microbiol Res 2010 Feb 20;165(2):108-15. Epub 2009 Feb 20.

Institute for Biology/Microbiology, Martin-Luther-University Halle, Germany.

The DNA-binding protein of starved cells (Dps) has two distinct cellular functions in Escherichia coli. The spherical Dps dodecamer can store iron and, predominantly in the stationary growth phase, high amounts of Dps protein protect the genome by binding non-specifically to DNA. In this study we investigated the role of Dps in copper homeostasis in growing cells of E. coli. Under reductive aerobic growth conditions that favor a redox shift from Cu(II) to Cu(I) or under anaerobiosis, cells deleted in dps were sensitive to low copper ion concentrations. Deletion of the DNA-binding N-terminus of Dps did not abrogate protection against copper toxicity indicating protection against copper stress is not directly related to DNA binding of Dps. The Dps protein is not a copper-storage protein in vitro or in vivo. In contrast, cells lacking Dps exhibited increased cellular copper concentrations compared to their wild-type parent. Furthermore, overproduction of Dps during growth phase resulted in decreased intracellular copper content under copper stress.
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http://dx.doi.org/10.1016/j.micres.2008.12.003DOI Listing
February 2010

Intraoperative consultation as an instrument of quality management.

World J Surg 2009 Jan;33(1):6-11; discussion 12-3

Department of Visceral Surgery, Asklepios Hospital Hamburg Altona, Paul-Ehrlich-Str. 1, 22763, Hamburg, Germany.

Background: This study was designed to investigate the effectiveness of intraoperative consultation (IOC) with regard to its feasibility for improving quality management. IOC comprises an assessment of major steps in the operative procedure by another surgeon requested by the operating surgeon to be present in the operating room in a consultative capacity during the operation.

Methods: Between January and June 2008, IOC was implemented in 1217 operations. Data on the frequency of the feasibility of IOC and on whether IOC led to decisions influencing the course of the procedure were measured.

Results: A total of 872 IOCs were performed: regular IOC in 708 cases (81%), and tactical IOC in 164 cases (19%). In 70 cases (8%), consultation resulted in minor changes, and in 59 cases (7%) major clinically relevant revision of the operative strategy was deemed necessary.

Conclusions: It was found that IOC can be performed in the majority of cases. In the case of tactical IOC in particular, a large number of treatment-relevant decisions are taken. This means that in the area of operative disciplines, IOC represents a potential preventive strategy within the framework of quality management.
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http://dx.doi.org/10.1007/s00268-008-9786-3DOI Listing
January 2009

Sandwich hybridization assay for sensitive detection of dynamic changes in mRNA transcript levels in crude Escherichia coli cell extracts in response to copper ions.

Appl Environ Microbiol 2008 Dec 24;74(24):7463-70. Epub 2008 Oct 24.

Institut für Biologie/Mikrobiologie, Martin-Luther-Universität, Halle-Wittenberg, Germany.

Transcript quantification techniques usually rely on purified mRNAs. We report here a solution-based sandwich hybridization assay for the quantification of mRNAs from Escherichia coli without the need of prior RNA isolation. This assay makes use of four DNA oligonucleotide probes adjacently hybridizing to target RNA in clarified cell extracts. Two helper probes facilitate the hybridization of a detection and a capture probe. The latter is biotin labeled, allowing binding to streptavidin-coated paramagnetic beads and the separation of the RNA-DNA hybrid from cellular constituents. Added antidigoxigenin Fab fragments conjugated to alkaline phosphatase bind to the digoxigenin-labeled detection probe, completing the sandwich of the paramagnetic bead, mRNA, probes, and alkaline phosphatase. The target transcript can be quantified by assessing phosphatase activity on a substrate that is converted into a fluorescent product. The amount of target mRNA is calculated from the fluorescence output and from a calibration curve for a known concentration of in vitro-synthesized target mRNA. This technique was used in time course experiments to investigate the expression of three genes responsible for the copper resistance of E. coli. The induction of gene expression by copper cations was rapid, but under aerobic conditions, the levels of expression returned to low, prestress levels within minutes. In anaerobiosis, high-level expression continued for at least 1 h. When cultures were shifted from anaerobiosis to aerobiosis, expression levels were diminished within minutes to prestress levels. The improved technique presented here is relatively simple, has very high degrees of sensitivity and robustness, is less laborious than other RNA quantification methods, and is not negatively affected by genomic DNA. These characteristics make it a powerful complementary application to genetic reporter fusions and to reverse transcription-PCR.
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http://dx.doi.org/10.1128/AEM.01370-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2607146PMC
December 2008

Linkage between catecholate siderophores and the multicopper oxidase CueO in Escherichia coli.

J Bacteriol 2004 Sep;186(17):5826-33

Department of Soil, Water, and Environmental Science, University of Arizona, Shantz Bldg. #38, Rm. 424, Tucson, AZ 85721, USA.

The multicopper oxidase CueO had previously been demonstrated to exhibit phenoloxidase activity and was implicated in intrinsic copper resistance in Escherichia coli. Catecholates can potentially reduce Cu(II) to the prooxidant Cu(I). In this report we provide evidence that CueO protects E. coli cells by oxidizing enterobactin, the catechol iron siderophore of E. coli, in the presence of copper. In vitro, a mixture of enterobactin and copper was toxic for E. coli cells, but the addition of purified CueO led to their survival. Deletion of fur resulted in copper hypersensitivity that was alleviated by additional deletion of entC, preventing synthesis of enterobactin. In addition, copper added together with 2,3-dihydroxybenzoic acid or enterobactin was able to induce a Phi(cueO-lacZ) operon fusion more efficiently than copper alone. The reaction product of the 2,3-dihydroxybenzoic acid oxidation by CueO that can complex Cu(II) ions was determined by gas chromatography-mass spectroscopy and identified as 2-carboxymuconate.
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http://dx.doi.org/10.1128/JB.186.17.5826-5833.2004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC516812PMC
September 2004