Tübingen, Germany
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Forensic proteome analysis: Protein signatures allow experts to match wounds to bullet holes. Credit: Journal of Proteome Research Tübingen researchers have developed a method which enables them to more accurately reconstruct crimes involving sharp blades or firearms. An interdisciplinary team from the Center for Bioinformatics Tübingen (ZBIT), Forensic Medicine, the Center for Quantitative Biology (QBiC) and bioanalysts from the Research Center for Ophthalmology (FIA) used traces of organic material found on bullets or other weapons used in crimes. Their findings are published in the latest edition of Journal of Proteome Research. Forensic medicine has long employed molecular biology to link, for instance, tiny amounts of organic material to a suspect. Yet such methods are less useful when it comes to finding out which shot or stab wound was the cause of death. They enable a weapon to be linked to the victim, but not the projectile to the wound. This is sometimes necessary for the full reconstruction of a crime and presentation of evidence - for example, in cases where several people were involved in a shoot-out. The study's lead authors Dr. Sascha Dammeier (FIA), Dr. Sven Nahnsen (QBiC) and Johannes Veit (ZBIT), used proteome analysis - which is based on mass spectrometry - to demonstrate that projectiles which have been fired through vital organs bear traces of organ-specific proteins. These protein "signatures" allow experts to identify which organ the projectile passed through - and even match a bullet to the wound it caused. The researchers initially tested the process on isolated cattle organs, which have typical protein patterns which can be statistically classified. Then the bullet metal was tested for relevant parameters. After experiments showed that a majority of the protein signatures were clearly in evidence, the method was put to the test in a real murder case. Unfortunately, the purely bioinformatical classification yielded no clear result due to contaminants. Yet the identification of organ-specific proteins on several projectiles enabled forensic scientists to match all wounds to projectiles using a process of elimination. And this led to a complete reconstruction of the crime. The method has been patented and licenced. The Tübingen researchers hope that it will be widely used to compile a forensic database which will successively make the protein signatures of all important organs - and their combinations - available to forensic experts, so that the signatures can be quickly and effectively identified. More information: Sascha Dammeier et al. Mass-Spectrometry-Based Proteomics Reveals Organ-Specific Expression Patterns To Be Used as Forensic Evidence, Journal of Proteome Research (2015). DOI: 10.1021/acs.jproteome.5b00704


Langenfeld A.,Center for Ophthalmology | Julien S.,Center for Ophthalmology | Schraermeyer U.,Center for Ophthalmology
Graefe's Archive for Clinical and Experimental Ophthalmology | Year: 2015

Purpose: Since adult rats are used in pre-clinical studies, and due to the necessity of investigating the side-effects of drugs on RPE cells in vitro, there is a great need for primary RPE cells from these animals. The aim of this study was to develop a reproducible and quantifiable method of isolation, culture, and maintenance of adult rat RPE cells. Moreover, potential differences between RPE cells from albino versus pigmented rats were also investigated. Methods: A total of 180 pigmented rats and 340 albino rats aged 6–14 weeks were used. RPE cells were isolated and cultured for several weeks by using three different methods: 1) growing directly on flat mounts, 2) after enzymatic isolation, and 3) after they spontaneously detached from the flat mounts and continued to grow on the plastic. Yield, cell survival, and morphological characteristics were investigated using light and electron microscopy as well as immunohistochemistry. Results: After 0 weeks, the yield of the first method was 30,000 cells/eye; after 2 weeks 18,000 cells/eye; and after 4 weeks 11,000 cells/eye. The yield of RPE cells was very low after enzymatic isolation in method 2 (0 weeks, 13.000 cells/eye; 2 weeks, 30,000 cells/eye; 4 weeks 38,000 cells/eye), whereas it was higher when the RPE cells spontaneously detached from the flat mounts and then continued to grow on the plastic in method three. (0 weeks, 30,000 cells/eye; 2 weeks, 314,000 cells/eye; 4 weeks, 659,000 cells/eye). The second method often showed contamination with fibroblasts, whereas the two other methods showed pure RPE cultures. The RPE cells were able to proliferate when using the second and the third method, but not when they were cultivated directly on the flat mounts (first method). Conclusion: The qualitative and quantitative best method for isolating adult rat RPE cells is the culture of RPE cells which spontaneously detach from flat mounts. No differences were observed between albino and pigmented RPE cells. © 2015, Springer-Verlag Berlin Heidelberg.


Julien S.,Center for Ophthalmology | Schraermeyer U.,Center for Ophthalmology
Neurobiology of Aging | Year: 2012

Lipofuscin is a cytologic hallmark of aging in metabolically active postmitotic cells including neurons, cardiac muscle cells, and the retinal pigment epithelium (RPE). High levels of lipofuscin are involved in the pathogenesis of age-related macular degeneration (AMD), the main cause of blindness in the elderly population in the western world. Degradation and exocytosis of lipofuscin by RPE cells have not been observed in vivo until now, and no drug is known to eliminate the intracellular amount of lipofuscin. Here, we show that in monkeys treated with a small molecule belonging to the tetrahydropyridoethers class (n = 36 of 48 monkeys), RPE cells significantly release lipofuscin. In 4 eyes, macrophages were detected which had taken up lipofuscin. They were located between the Bruch's membrane and the RPE, and in the choroid. The quantification of pigment granules was performed by transmission electron microscopy. Our findings open the way to develop therapeutic strategies to remove lipofuscin from RPE cells, which may have implications for the treatment of age-related macular degeneration in which lipofuscin accumulation in cells is a causative factor. © 2012 Elsevier Inc.


Julien S.,Center for Ophthalmology | Biesemeier A.,Center for Ophthalmology | Taubitz T.,Center for Ophthalmology | Schraermeyer U.,Center for Ophthalmology
British Journal of Ophthalmology | Year: 2014

Background: Since there is evidence that the Fc domain of antivascular endothelial growth factor drugs may cause unexpected consequences in retinal and choroidal vessels, the effects of intravitreal ranibizumab and aflibercept on monkey eyes were investigated. Methods: Four cynomolgus monkeys were intravitreally injected with 0.5 mg of ranibizumab and another four with 2 mg of aflibercept. Two untreated monkeys served as controls. Funduscopy, fluorescein angiography (FA), spectral-domain-optical coherence tomography (SD-OCT) and measurement of intraocular pressure (IOP) were performed. The eyes were inspected by light, fluorescence and electron microscopy. The diameter of the choriocapillaris (CC) was measured by morphometry, and the areas of the CC with free haemoglobin, CC fenestrations and endothelial thickness were quantified. Results: Analysis showed ranibizumab permeated the retina via intercellular clefts, whereas aflibercept was taken up by ganglion cells, cells of the inner and outer retinal layers and the retinal pigment epithelium (RPE). Stasis and haemolysis in the choriocapillaris and choroidal vessels were more frequent after aflibercept treatment, which caused hypertrophy and death of individual RPE cells. The area of the CC was significantly reduced after both drugs compared with controls, but the reduction of the CC endothelium thickness, number of fenestrations and the areas with haemolysis were more pronounced after aflibercept. Conclusions: Ranibizumab permeated the retina through intercellular spaces, whereas aflibercept was taken up by neuronal and RPE cells. Aflibercept induced protein complex formation and more haemolysis in the choriocapillaris, leading to individual RPE cell death. The clinical significance and relation of these findings to the Fc domain or to other characteristics of aflibercept remain to be investigated.


Julien S.,Center for Ophthalmology | Biesemeier A.,Center for Ophthalmology | Schraermeyer U.,Center for Ophthalmology
British Journal of Ophthalmology | Year: 2013

Purpose: By investigating the effects of intravitreal bevacizumab on retinal vessels of monkeys, we found that bevacizumab accumulated locally at high concentration within individual blood vessels. It formed electron-dense fibrous deposits between endothelial cells and erythrocytes or granulocytes inducing retinal vein thrombosis. To better characterise the observed deposits, we investigated in vitro whether these deposits result from a complex between bevacizumab, vascular endothelial growth factor (VEGF)-A165 and heparin. Methods: Cynomolgus monkeys were intravitreally injected with 1.25 mg bevacizumab. The eyes were enucleated between 1 and 14 days after injection and investigated by electron microscopy and immunohistochemistry. Human umbilical vein endothelial cells (HUVEC) were incubated with bevacizumab, VEGF-A 165 and heparin at different concentrations. Treatments with ranibizumab served as control. Bevacizumab and ranibizumab were detected immunohistochemically using Cy-3 or immunogold labelled antibodies. Results: Treated animals showed bevacizumab locally at high concentration within retinal blood vessels. Electrondense deposits inside retinal vessels and between erythrocytes were detected in three out of four treated monkeys. In vitro, many globular aggregates heavily stained with anti-human IgG were only observed with equimolar amounts (240 nM) of bevacizumab and VEGF-A165 and 0.2 U/ml heparin and not after ranibizumab treatment. The immunogold labelling specifically localised ultrastructurally the complexes formed between bevacizumab, VEGF-A165 and heparin at the surfaces of HUVEC cells. Conclusions: Heparin promotes bevacizumab immune complex deposition on to endothelial cells. Our in vitro results could explain the presence of deposits observed on endothelial veins in monkey eyes intravitreally injected with bevacizumab.


Arango-Gonzalez B.,Center for Ophthalmology
PloS one | Year: 2012

To explore the effect of ketamine-xylazine anesthesia on light-induced retinal degeneration in rats. Rats were anesthetized with ketamine and xylazine (100 and 5 mg, respectively) for 1 h, followed by a recovery phase of 2 h before exposure to 16,000 lux of environmental illumination for 2 h. Functional assessment by electroretinography (ERG) and morphological assessment by in vivo imaging (optical coherence tomography), histology (hematoxylin/eosin staining, TUNEL assay) and immunohistochemistry (GFAP and rhodopsin staining) were performed at baseline (ERG), 36 h, 7 d and 14 d post-treatment. Non-anesthetized animals treated with light damage served as controls. Ketamine-xylazine pre-treatment preserved retinal function and protected against light-induced retinal degeneration. In vivo retinal imaging demonstrated a significant increase of outer nuclear layer (ONL) thickness in the non-anesthetized group at 36 h (p<0.01) and significant reduction one week (p<0.01) after light damage. In contrast, ketamine-xylazine pre-treated animals showed no significant alteration of total retinal or ONL thickness at either time point (p>0.05), indicating a stabilizing and/or protective effect with regard to phototoxicity. Histology confirmed light-induced photoreceptor cell death and Müller cells gliosis in non-anesthetized rats, especially in the superior hemiretina, while ketamine-xylazine treated rats showed reduced photoreceptor cell death (TUNEL staining: p<0.001 after 7 d), thicker ONL and longer IS/OS. Fourteen days after light damage, a reduction of standard flash induced a-wave amplitudes and a-wave slopes (p = 0.01) and significant alterations in parameters of the scotopic sensitivity function (e.g. Vmax of the Naka Rushton fit p = 0.03) were observed in non-treated vs. ketamine-xylazine treated animals. Our results suggest that pre-treatment with ketamine-xylazine anesthesia protects retinas against light damage, reducing photoreceptor cell death. These data support the notion that anesthesia with ketamine-xylazine provides neuroprotective effects in light-induced cell damage.


Schraermeyer U.,Center for Ophthalmology | Julien S.,Center for Ophthalmology
Graefe's Archive for Clinical and Experimental Ophthalmology | Year: 2012

Background In this study, the effect of intravitreal injection of bevacizumab on choroidal blood vessels was examined in primate eyes. Methods Four Cynomolgus monkeys received an intravitreal injection of 1.25 mg bevacizumab. The eyes were enucleated on days 1, 4, 7 and 14. For each animal, one eye was embedded in paraffin whereas the other eye was embedded for electron microscopy. Seven untreated or PBS (phosphate buffered saline)-injected monkeys were used as controls. Results Thrombotic microangiopathy was found in the choriocapillaris and choroidal vessels of all eight injected eyes. Acute microangiopathy was characterized ultrastructurally as swelling of the endothelium, loss of fenestrations and complete collapse of the capillaries, and was commonly observed in bevacizumab-treated eyes. Quantitative analysis showed reduction of the lumina of the choriocapillaris in the eyes of three of the monkeys. Bevacizumab was frequently localized inside the blood vessels, often filling the entire breadth of the vessels, and formed clusters with blood cells. Death of photoreceptors occurred in two monkeys. Conclusions This study indicate that intravitreal injection of bevacizumab in monkeys induces activation of platelets, degranulation of thrombocytes and neutrophils, formation of immune complexes, thrombotic microangiopathy and alteration of the blood flow in choroidal vessels. © Springer-Verlag 2012.


Kustermann S.,Center for Ophthalmology | Hildebrandt H.,Hannover Medical School | Bolz S.,Center for Ophthalmology | Dengler K.,Center for Ophthalmology | And 2 more authors.
Journal of Comparative Neurology | Year: 2010

Polysialic acid (polySia) is a posttranslational modification of the neural cell adhesion molecule NCAM, which in the vertebrate brain is dynamically regulated during development and crucially involved in developmental and adult neurogenesis. In the fish retina, new neurons are persistently generated, but the possible contribution of polySia has not yet been addressed. Here we used immunohistochemistry with NCAM- and polySia-specific antibodies to study spatiotemporal expression patterns of NCAM and polySia in the developing and mature zebrafish retina. As early as 2.3 days postfertilization (dpf), NCAM but not poly-Sia was detected on cell somata and fibers of the developing retina. At 4.3 dpf polySia immunoreactivity first appeared in the ventral retina and was localized to the nascent outer nuclear layer (ONL). In mature zebrafish, polySia immunoreactivity in the ONL extended to the entire retina. Colocalization with rhodopsin-EGFP in transgenic zebrafish or the Müller glia-specific protein cellular retinaldehyde-binding protein (CRALBP) revealed that polySia immunoreactivity was confined to the compartment of radial Müller glia processes crossing the ONL and to a small band of processes positioned proximal to the horizontal cell layer of the mature retina. As shown by 5-bromo-2-deoxyuridine (BrdU) labeling, both newly generated rod precursors within the mature ONL and precursors of the marginal zone were polySia-negative. Thus, polySia-negative rod precursors of the mature zebrafish retina face a polySia-NCAM-positive microenvironment presented by radial Müller glia. In view of the prominent role of polySia in other neurogenic systems, this pattern indicates that polySia provides environmental cues that are relevant for the generation of new rods. © 2009 Wiley-Liss, Inc.


Durst W.,Center for Ophthalmology | Peters T.,Center for Ophthalmology | Wilhelm B.,Center for Ophthalmology
British Journal of Ophthalmology | Year: 2011

Aims: The aim of this work was to construct a lighting device that allows a uniform lighting of top illumination charts, as used for the examination of contrast vision and visual acuity. The relevant standards make strict demands for such charts with regard to the quality of the lighting, the absolute brightness and the uniformity of the luminous intensity distribution over the chart surface. If measurements are made under differing daylight conditions or if artificial light sources are installed in the examination room, as frequently practiced, then these requirements cannot be fulfilled. Methods: A lighting device was developed using daylight spectrum tubes installed on the right and left sides of the chart. The tubes were shielded by opaque diffusers to scatter the light. Results: The device produced an accurate and uniform distribution of luminance at 21 points over the chart that was not obtained with overhead lighting running parallel or horizontal to the chart. Conclusion: A uniform illumination of top illumination test charts was achieved allowing adherence to the standards for measuring acuity set out by the European Union and enabling comparison of such visual examinations in multi-centre clinical studies.


Schraermeyer U.,Center for Ophthalmology | Julien S.,Center for Ophthalmology
Expert Opinion on Biological Therapy | Year: 2013

Objective: Due to its low price, bevacizumab, which binds vascular endothelial growth factor, is currently used off-label for the treatment of over 50 different eye diseases and has been adopted worldwide despite the absence of serious preclinical data. This study examines the effects of intravitreal bevacizumab on monkey eyes with particular focus on choroidal and retinal vessels. Methods: Cynomolgus monkeys received an intravitreal injection of 1.25 mg bevacizumab with or without 125I labeling. The eyes were enucleated between 1 and 14 days after injection and were investigated by electron microscopy, immunocytochemistry, histochemistry or autoradiography. Untreated and phosphate buffered saline (PBS)-injected monkeys were used as controls. Results: Bevacizumab locally accumulated at high concentration within individual blood vessels. It formed electron-dense deposits inside retinal veins and between red and white blood cells, activated thrombocytes and induced retinal vein thrombosis. Retinal cells like Müller cells, astrocytes and microglia were also activated. High amounts of bevacizumab were found in retinal and choroidal vessels which may interfere with blood flow. Conclusions: The deposits on the retinal vein walls may provide a mechanistic basis for the observed retinal blood flow alterations after bevacizumab treatment in patients. © 2013 Informa UK, Ltd.

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