The pathological anatomical collection of the Natural History Museum Vienna.

The pathological anatomical collection Vienna (Pathologisch-Anatomische Sammlung Wien; PASW) is a living and still growing research collection. It was established as early as 1796 as part of the Medical University of Vienna, acquired the status of an independent federal museum in 1971, and was assigned to the Natural History Museum Vienna in 2012. It houses a wide range of human wet and dry specimens and further objects, such as moulages, medical devices, microbiological and histological specimens, and a photo archive (approximately 50,000 objects), which, as a meaningful source, may contribute to disclosing not only aspects of the medical history and the development of corresponding museums in Vienna, but is also considered a collection of cultural and current scientific relevance, quite comparable to today's biobanks. Most of the tissue amassment represents wet organic specimens and human skeletons or skeletal elements representing, e.g., congenital and metabolic disorders, infectious diseases, injuries, neoplasms, or musculoskeletal diseases, basically collected as descriptive anatomical teaching aids. This article reviews the current medical issues on which research has been and is being conducted by including PASW specimens (hereby using the ICD-10 code), and the extent to and ethical conditions under which this important heritage could be used as a reference collection for clinical and bioanthropological (paleopathological and palaeoepidemiological) studies; finally, this article reflects on the value and future research prospects, taking into account different positions and the ongoing discussions in pathological anatomical human tissue collections.

Radiosensitizing effect of galunisertib, a TGF-ß receptor I inhibitor, on head and neck squamous cell carcinoma in vitro.

BACKGROUND: Resistance to radiation therapy poses a major clinical problem for patients suffering from head and neck squamous cell carcinoma (HNSCC). Transforming growth factor ß (TGF-ß) has emerged as a potential target. This study aimed to investigate the radiosensitizing effect of galunisertib, a small molecule TGF-ß receptor kinase I inhibitor, on HNSCC cells in vitro. METHODS: Three HNSCC cell lines were treated with galunisertib alone, or in combination with radiation. Of those three cell lines, one has a known inactivating mutation of the TGF-ß pathway (Cal27), one has a TGF-ß pathway deficiency (FaDu) and one has no known alteration (SCC-25). The effect on metabolic activity was evaluated by a resazurin-based reduction assay. Cell migration was evaluated by wound-healing assay, clonogenic survival by colony formation assay and cell cycle by FACS analysis. RESULTS: Galunisertib reduced metabolic activity in FaDu, increased in SCC-25 and had no effect on CAL27. Migration was significantly reduced by galunisertib in all three cell lines and showed additive effects in combination with radiation in CAL27 and SCC-25. Colony-forming capabilities were reduced in SCC-25 by galunisertib and also showed an additive effect with adjuvant radiation treatment. Cell cycle analysis showed a reduction of cells in G1 phase in response to galunisertib treatment. CONCLUSION: Our results indicate a potential antineoplastic effect of galunisertib in HNSCC with intact TGF-ß signaling in combination with radiation.

Multimodality imaging beyond CLEM: Showcases of combined in-vivo preclinical imaging and ex-vivo microscopy to detect murine mural vascular lesions.

In imaging, penetration depth comes at the expense of lateral resolution, which restricts the scope of 3D in-vivo imaging of small animals at micrometer resolution. Bioimaging will need to expand beyond correlative light and electron microscopy (CLEM) approaches to combine insights about in-vivo dynamics in a physiologically relevant 3D environment with ex-vivo information at micrometer resolution (or beyond) within the spatial, structural and biochemical contexts. Our report demonstrates the immense potential for biomedical discovery and diagnosis made available by bridging preclinical in-vivo imaging with ex-vivo biological microscopy to zoom in from the whole organism to individual structures and by adding localized spectroscopic information to structural and functional information. We showcase the use of two novel imaging pipelines to zoom into mural lesions (occlusions/hyperplasia and micro-calcifications) in murine vasculature in a truly correlative manner, that is using exactly the same animal for all integrated imaging modalities. This correlated multimodality imaging (CMI) approach includes well-established technologies such as Positron Emission Tomography (microPET), Autoradiography, Magnetic Resonance Imaging (microMRI) and Computed Tomography (microCT), and imaging approaches that are more novel in the biomedical setting, such as X-Ray Fluorescence Spectroscopy (microXRF) and High Resolution Episcopic Microscopy (HREM). Although the current pipelines are focused on mural lesions, they would also be beneficial in preclinical and clinical investigations of vascular diseases in general.

Bioelectrical signals improve cardiac function and modify gene expression of extracellular matrix components.

AIMS: Beyond the influence of stimulating devices on cardiac excitation, their use in treating patients with heart failure has positive effects on the myocardium at the molecular level. Electrical signals can induce a wide spectrum of effects in living tissue. Therefore, we sought to determine whether applying electrical microcurrent directly to failing hearts leads to functional improvement. METHODS AND RESULTS: Sixteen male spontaneously hypertensive rats (SHRs) with heart failure underwent application of a patch electrode to the left ventricular epicardium and placement of a subcutaneous counter electrode. The electrode delivered a 0.35 µA microcurrent to nine of the SHRs for 45 ± 3 days; the other seven SHRs were used as controls. At baseline and before the SHRs were humanely put to death, we measured the left ventricular ejection fraction (LVEF) and the thickness of the LV posterior wall during systole and diastole (LVPWs/d). We used quantitative PCR to determine extracellular matrix parameters [collagen I-III, matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinases 3 (TIMP3), TIMP4, connexins (Cxs) 40/43/45, transforming growth factor (TGF)-ß, and interleukin (IL)-6]. Among SHRs undergoing microcurrent application, LVEF normalized (mean decrease, 22.8%; P = 0.009), and LVPWs decreased (mean, 35.3%; P = 0.001). Compared with the control group, the SHRs receiving microcurrent exhibited a mean decrease in the gene expression of collagen I (10.6%, P = 0.003), TIMP3 (18.5%, P = 0.005), Cx43 (14.3%, P = 0.003), Cx45 (12.7%, P = 0.020), TGF-ß (13.0%, P = 0.005), and IL-6 (53.7%, P = 0.000). Microcurrent application induced no changes in the expression of collagen III, MMP-2, MMP-9, TIMP4, or Cx40. CONCLUSIONS: Applying microcurrent to the LV epicardium of SHRs leads to statistically significant functional improvement and alterations in the levels of inflammatory and extracellular matrix components.

Microcurrent stimulation promotes reverse remodelling in cardiomyocytes.

AIMS: It has been shown that electrical stimulation can improve tissue repair in patients. Imbalances in the extracellular matrix composition induce manifestation of heart failure. Here we investigated the application of microcurrent (MC) to modulate the expression of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) in cardiomyocytes in vitro and in vivo to reverse remodelling in the heart in spontaneous hypertensive rats (SHR). METHODS: Cardiomyocytes from young SHR (7 months) and old SHR (14 months) were stimulated in vitro and in vivo with MC. MMP and TIMP expression were analysed by qPCR and immunofluorescence to evaluate the modulation of MC treatment. RESULTS: Modulation of cardiomyocytes with MC enhances proliferation with no morphological changes in vitro. By electrical stimulation dual effects, increase and decrease, on MMP-2, MMP-9, TIMP-3, and TIMP-4 mRNA as well as protein expression were observed, depending on the age of the cardiomyocytes. In our in vivo study, MC down-regulated MMP-2, MMP-9, and TIMP-4 and increased TIMP-3 in young SHR. In old SHR MMP-2, MMP-9, and TIMP-4 were up-regulated, whereas TIMP-3 was unaffected. CONCLUSIONS: Our data indicate that treatment of MC can modulate the expression of MMPs and TIMPs in vitro and in vivo in SHR. Based on these results new treatments for heart failure could be developed.

Hypoxia-inducible factor-1ß (HIF-1ß) is upregulated in a HIF-1α-dependent manner in 518A2 human melanoma cells under hypoxic conditions.

Solid tumors include hypoxic areas due to excessive cell proliferation. Adaptation to low oxygen levels is mediated by the hypoxia-inducible factor (HIF) pathway promoting invasion, metastasis, metabolic alterations, chemo-resistance and angiogenesis. The transcription factor HIF-1, the major player within this pathway consists of HIF-1α and HIF-1ß. The alpha subunit is continuously degraded under normoxia and becomes stabilized under reduced oxygen supply. In contrast, HIF-1ß is generally regarded as constitutively expressed and being present in excess within the cell. However, there is evidence that the expression of this subunit is more complex. The aim of this study was to investigate the role of HIF-1ß in human melanoma cells. Among a panel of five different cell lines, in 518A2 cells exposed to the hypoxia-mimetic cobalt chloride HIF-1ß was rapidly elevated on protein level. Knockdown experiments performed under cobalt chloride-exposure and hypoxia revealed that this effect was mediated by HIF-1α. The non-canonical relationship between these subunits was further confirmed by pharmacologic inhibition of HIF-1α and by expression of a dominant-negative HIF mutant. Overexpression of HIF-1α showed a time delay in HIF-1ß induction, thus arguing for HIF-1ß de novo synthesis rather than protein stabilization by heterodimerization. A Hen's egg test-chorioallantoic membrane model of angiogenesis and invasion indicated a local expression of HIF-1ß and implies a biological relevance of these findings. In summary, this study demonstrates the HIF-1α-dependent regulation of HIF-1ß under hypoxic conditions for the first time. The results indicate a novel cell specific mechanism which might prevent HIF-1ß to become a limiting factor.

Combining light microscopy, dielectric spectroscopy, MALDI intact cell mass spectrometry, FTIR spectromicroscopy and multivariate data mining for morphological and physiological bioprocess characterization of filamentous organisms.

Along with productivity and physiology, morphological growth behavior is the key parameter in bioprocess design for filamentous fungi. Lacking tools for fast, reliable and efficient analysis however, fungal morphology is still commonly tackled by empirical trial-and-error techniques during strain selection and process development procedures. Bridging the gap, this work presents a comprehensive analytical approach for morphological analysis combining automated high-throughput microscopy, multi-frequency dielectric spectroscopy, MALDI intact cell mass spectrometry and FTIR spectromicroscopy. Industrial fed-batch production processes were investigated in fully instrumented, automated bioreactors using the model system Penicillium chrysogenum. Physiological process characterization was based on the determination of specific conversion rates as scale-independent parameters. Conventional light microscopic morphological analysis was based on holistic determination of time series for more than 30 morphological parameters and their frequency distributions over the respective parameter range by automated high-throughput light microscopy. Characteristic protein patterns enriched in specific morphological and physiological states were further obtained by MALDI intact cell mass spectrometry. Spatial resolution of molecular biomass composition was facilitated by FTIR spectromicroscopy. Real-time in situ monitoring of morphological process behavior was achieved by linking multi-frequency dielectric spectroscopy with above outlined off-line methods. Data integration of complementing orthogonal techniques for morphological and physiological analysis together with multivariate modeling of interdependencies between morphology, physiology and process parameters facilitated complete bioprocess characterization. The suggested approach will thus help understanding morphological and physiological behavior and, in turn, allow to control and optimize those complex processes.

Everolimus attenuates neointimal hyperplasia in cultured human saphenous vein grafts.

OBJECTIVE: Neointimal hyperplasia is the first step in a cascade leading to a reduced patency rate of saphenous vein grafts in comparison to arterial grafts in coronary artery bypass grafting. Using cultured human saphenous vein grafts as a model for coronary artery bypass grafting, we investigated if the mammalian target of rapamycin inhibitor everolimus attenuates neointimal hyperplasia. METHODS: Saphenous vein grafts from 10 patients undergoing coronary artery bypass grafting were processed as follows: from each patient, one segment served as baseline control at day 0. Two segments were cultured in a neointimal hyperplasia model separately. One received no treatment and the other everolimus (1 microM). All vein grafts underwent histomorphometric analysis, assessment of proliferation by Ki-67 immunostaining and quantification of phospho-S6 ribosomal protein using western blot analysis. RESULTS: Everolimus treatment resulted in reduced neointimal hyperplasia (thickness 3.7+/-1.2 microm) compared to untreated controls (10.1+/-2.5 microm, p=0.008). The intima/intima+media-ratio was reduced in the everolimus group (0.10+/-0.02) compared to untreated controls (0.24+/-0.07, p=0.008). The number of Ki-67 positive proliferating cells in everolimus treated vein grafts (15+/-7 cells/high power field) showed a tendency of reduction compared to untreated controls (36+/-20 cells/high power field, p=0.036). Finally, everolimus treatment resulted in downregulation of S6 ribosomal protein phosphorylation in comparison to untreated controls. CONCLUSION: Everolimus is able to reduce neointimal proliferation in cultured human saphenous vein grafts by inhibition of the mammalian target of rapamycin, even though different transfection methods are to be evaluated for a clinical application in coronary artery bypass grafting.

Colony-stimulating factor-1 transfection of myoblasts improves the repair of failing myocardium following autologous myoblast transplantation.

AIMS: Skeletal myoblasts are used in repair of ischaemic myocardium. However, a large fraction of grafted myoblasts degenerate upon engraftment. Colony-stimulating factor-1 (CSF-1) accelerates myoblast proliferation and angiogenesis. We hypothesized that CSF-1 overexpression improves myoblast survival and cardiac function in ischaemia-induced heart failure. METHODS AND RESULTS: Three weeks following myocardial infarction, rats developed heart failure and received intramyocardial injections of mouse CSF-1-transfected or untransfected primary autologous rat myoblasts, recombinant human CSF-1, mouse CSF-1 expressing plasmids, or culture medium. Tissue gene and protein expression was measured by quantitative RT-PCR (reverse transcription-polymerase chain reaction) and western blotting. Fluorescence imaging and immunocytochemistry were used to analyse myoblasts, endothelial cells, macrophages, and infarct wall thickening. Electrocardiograms were recorded online using a telemetry system. Left ventricular function was assessed by echocardiography over time, and improved significantly only in the CSF-1-overexpressing myoblast group. CSF-1-overexpression enhanced myoblast numbers and was associated with an increased infarct wall thickness, enhanced angiogenesis, increased macrophage recruitment and upregulated matrix metalloproteases (MMP)-2 and -12 in the zone bordering the infarction. Transplantation of CSF-1-overexpressing myoblasts did not result in major arrhythmias. CONCLUSION: Autologous intramyocardial transplantation of CSF-1 overexpressing myoblasts might be a novel strategy in the treatment of ischaemia-induced heart failure.

In vivo activity of a novel polymeric guanidine in experimental skin infection with methicillin-resistant Staphylococcus aureus.

The in vivo efficacy of the novel polymeric guanidine AKACID Plus was evaluated in a guinea pig model of experimental skin infection with methicillin-resistant Staphylococcus aureus (MRSA). Topical application of AKACID Plus at concentrations of > or =0.5% was as effective as mupirocin 2% cream in the treatment of superficial skin infection with MRSA.

Comparative activities of antibiotics against intracellular non-typeable Haemophilus influenzae.

INTRODUCTION: Non-typeable Haemophilus influenzae (NTHi) is a major bacterial pathogen of community-acquired respiratory tract infection and is usually found extracellularly, although studies have revealed that NTHi may possess the ability to invade human epithelial cells where it is then protected against attack by the local immune system and partly against the effect of antibiotics. The aim of the present study was to assess the ability of ampicillin, azithromycin, telithromycin, ciprofloxacin and moxifloxacin, five antibiotics in common clinical use, to kill NTHi within bronchial epithelial cells. METHODS: Confluent human bronchial epithelial cells were infected with NTHi 77, a particularly invasive clinical strain. Extracellular bacterial cells were killed with gentamicin and the intracellular bacteria were incubated with antibiotics at concentrations of 1 mg/l or 10 mg/l for 4 h or 8 h. Viable intracellular bacteria were counted after lysis of the epithelial cells. RESULTS: With the exception of ampicillin, all the antibiotics caused significant reduction of intracellular bacteria at concentrations of 10 mg/l and exposure for 4 h or at 1 mg/l for 8 h. At 1 mg/l, moxifloxacin eliminated 94% of intracellular NTHi after 4 h and 98% after 8 h; ciprofloxacin, azithromycin and telithromycin only achieved killing indices below 75 after 4 h but 86-90% killing after 8 h. At 10 mg/l, moxifloxacin, ciprofloxacin, telithromycin and azithromycin were able to achieve 99.7%, 96.3%, 86.7% and 74.7% eradication of intracellular bacteria, respectively, after exposure for 4 h. CONCLUSION: These results demonstrate the rapid antibacterial efficacy of moxifloxacin against intracellular NTHi in vitro. Moxifloxacin, which combines high extracellular and intracellular activities, could be an important tool in the treatment of recurrent respiratory tract infections.

Behavior of cardiomyocytes and skeletal muscle cells on different extracellular matrix components--relevance for cardiac tissue engineering.

Myocardial cell transplantation in patients with heart failure is emerging as a potential therapeutic option to augment the function of remaining myocytes. Nevertheless, further investigations on basic issues such as ideal cell type continue to be evaluated. Therefore, the aim of our studies was to compare the performance of skeletal muscle cells and cardiomyocytes with respect to their proliferation rate and viability on different extracellular matrix components (EMCs). Rat cardiomyocytes (RCM) and rat skeletal muscle cells (RSMC) were cultured on EMCs such as collagen type I, type IV, laminin, and fibronectin. The components were used as "single coating" as well as "double coating." Proliferation rates were determined by proliferation assays on days 1, 2, 4, and 8 after inoculation of the cells. The most essential result is that collagen type I enhances the proliferation rate of RSMC but decreases the proliferation of RCM significantly. This effect is independent of the second EMC used for the double-coating studies. Other EMCs also influence cellular behavior, whereas the sequence of the EMCs is essential. Results obtained in our studies reveal the significant different proliferation behavior of RCM and RSMC under identical conditions. As skeletal muscle cells are also used in heart tissue engineering models, these results are essential and should be investigated in further studies to prove the applicability of skeletal muscle cells for heart tissue engineering purposes.

MR imaging of the her2/neu and 9.2.27 tumor antigens using immunospecific contrast agents.

Molecular imaging of tumor antigens using immunospecific magnetic resonance (MR) contrast agents is a rapidly evolving field, which can potentially aid in early disease detection, monitoring of treatment efficacy, and drug development. In this study, we designed, synthetized, and tested in vitro two novel monocrystalline iron oxide nanoparticles (MION) conjugated to antibodies against the her2/neu tyrosine kinase receptor and the 9.2.27 proteoglycane sulfate. MION was synthetized by coprecipitation of iron II and iron III salts in 12-kD dextran solution; antibody coupling was performed by reductive amination. The relaxivity of the conjugates was 24.1-29.1 mM(-1) s(-1), with 1.8 to 2.1 antibody molecules per nanoparticle. A panel of cultured melanoma and mammary cell lines was used for testing. The cells were incubated with the particles at 16-32 microg Fe/ml in culture medium for 3 h at 37 degrees C, and investigated with immune fluorescence, transmission electron microscopy (TEM), MRI of cell suspensions in gelatine, and spectrophotometric iron determination. All receptor-positive cell lines, but not the controls, showed receptor-specific immune fluorescence, and strong changes in T(2) signal intensity at 1.5 T. The changes in 1/T(2) were between 1.5 and 4.6 s(-1) and correlated with the amount of cell-bound iron (R = 0.92). The relaxivity of cell-bound MION increased to 55.9 +/- 10.4 mM(-1) s(-1). TEM showed anti-9.2.27 conjugates binding to the plasma membrane, while the anti-her2/neu conjugates underwent receptor-mediated endocytosis. In conclusion, we obtained receptor-specific T(2) MR contrast with novel covalently bound, multivalent MION conjugates with anti-9.2.27 and anti-her2/neu to image tumor surface antigens. This concept can potentially be expanded to a large number of targets and to in vivo applications.

The mandatory CAM testing of cells and scaffolds for tissue engineering: benefits for the three Rs of cooperation with the vaccine industry.

In cooperation with BAXTER Vaccine AG, which supplies incubated special pathogen-free chicken eggs (including a full veterinary record), a permanent hen's egg chorio-allantoic membrane test (HET-CAM) unit has been established, where angiogenesis testing, cell culture, and digital and histological analyses are performed. At the Core Unit for Biomedical Research, the location of the animal testing facility of the Medical University Vienna, cell-scaffold constructs must be evaluated in vitro and in ovo prior to eventual in vivo tissue engineering experiments. The animal testing advisory committee requires that new test proposals are first evaluated by using cell culture and HET-CAM models. Approvals for in vivo experiments are postponed and not issued prior to in vitro/in ovo evaluation. Examples are presented of protocols planned for in vivo studies on cell seeded scaffolds, which were refined after in vitro/in ovo evaluations.

Glycoprotein 130 ligand oncostatin-M induces expression of vascular endothelial growth factor in human adult cardiac myocytes.

OBJECTIVE: In murine and rat cardiac myocytes the gp130 system transduces survival as well as hypertrophic signals and via induction of the expression of the potent angiogenic factor VEGF in these cells also indirectly contributes to cardiac repair processes through the development of new blood vessels. There are, however, species differences in receptor specificity and receptor crossreactivity in the gp130-gp130 ligand system. We asked whether gp130 signaling is also involved in the regulation of VEGF in human cardiac myocytes and if so which gp130 ligands are critical for such an effect. METHODS: Human adult cardiac myocytes (HACMs) were isolated from myocardial tissue and characterised by positive staining for myocardial actin, troponin-I and cardiotin. HACMs were treated with the gp130 ligands CT-1, IL-6, LIF or OSM and VEGF-1 was determined by a specific ELISA in the conditioned media of these cells. RT-PCR and Western blot analysis was used in order to detect gp130, IL-6-receptor, LIF-receptor or OSM-receptor specific protein and mRNA in human adult cardiac myocytes and for detection of VEGF-1 specific mRNA in cardiac myocytes after incubation with OSM. Pieces of myocardial tissue were incubated ex vivo in the presence and absence of OSM and VEGF was determined in supernatants of these cultures and immunohistochemistry was performed on the tissue using specific antibodies for VEGF-1. Immunohistochemistry was also employed to detect VEGF in sections from a healthy human heart and in a heart from a patient suffering from acute myocarditis. RESULTS: OSM, but not CT-1, IL-6 or LIF increased VEGF-1 production in human adult cardiac myocytes dose-dependently derived from five different donors. This selective stimulation of VEGF by gp130 ligands was also reflected by a specific receptor expression on these cells. We detected high levels of mRNA for gp130 and the OSM receptor in freshly isolated human cardiac myocytes but only low amounts of mRNA for the IL-6 receptor whereas mRNA for the LIF receptor was hardly detectable by RT-PCR. OSM receptor and IL-6 receptor were also detectable by Western blotting whereas LIF receptor was only present as a faint band. OSM also increased the expression of VEGF-1 mRNA in cardiac myocytes. When pieces of human myocardial tissue were incubated with the gp130 ligands in an ex vivo model only OSM resulted in an increase in VEGF-1 in the supernatants of these cultures. Furthermore, VEGF increased in tissue samples treated with OSM in cardiac myocytes as evidenced by immunohistochemistry. In addition, we found increased VEGF-1 expression in myocardial tissue from a patient suffering from acute myocarditis. CONCLUSION: The gp130-gp130 ligand system is also involved in VEGF regulation in human cardiac myocytes and OSM is the gp130 ligand responsible for this effect in the human system whereas LIF and CT-1 which had been shown to regulate VEGF expression in mouse and rat cardiac myocytes had no effect. Thus we have added OSM, which is produced by activated T lymphocytes and monocytes, to the list of regulatory molecules of VEGF production in the human heart. Our results lend further support to the notion that besides hypoxia, inflammation via induction of VEGF through autocrine or paracrine pathways plays a key role in (re)vascularisation of the myocardium.

Plasminogen activator inhibitor 1 expression is regulated by the inflammatory mediators interleukin-1alpha, tumor necrosis factor-alpha, transforming growth factor-beta and oncostatin M in human cardiac myocytes.

Accumulating evidence points towards a role for proteases and protease inhibitors in tissue remodelling and repair in a variety of organs. In particular-besides the matrix metalloprotease system-the plasminogen activator (PA)/plasmin system has been implicated in these processes in the heart. Urokinase type PA (u-PA) and PA inhibitor type 1 (PAI-1) seem to modulate cardiac rupture and infarct healing. In this study we aimed to investigate whether inflammatory mediators can regulate the expression of components of the PA/plasmin system in human adult cardiac myocytes (HACM). We could demonstrate that HACM, isolated from pieces of myocardial tissue by mechanical dispersion and characterized by positive immunostaining for the cardiac markers troponin I, tropomyosin, cardiotin and myocardial muscle-actin, in vitro express PAI-1 and tissue type PA (t-PA) whereas u-PA was not detectable in these cells. PAI-1 protein production was increased up to twofold by interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha) and up to fivefold by transforming growth factor-beta (TGF-beta) and oncostatin M (OSM). Similar changes were observed in PAI-1 transcript levels after cytokine treatment. t-PA production in HACM was not affected by these agonists. No effect of these cytokines on PAI-1 production in fibroblasts isolated from human myocardial tissue was seen. In an ex vivo model we could show that incubation of pieces of human myocardial tissue with these cytokines also resulted in an increase in PAI-1 in cardiac myocytes as evidenced by immuno-histochemistry. Furthermore we found increased PAI-1 expression in myocardial tissue from a patient suffering from acute myocarditis. Thus for the first time we provide evidence that inflammatory mediators modulate PAI-1 expression in human adult cardiac myocytes in vitro and ex vivo and could demonstrate that PAI-1 expression is increased in the in vivo setting under inflammatory conditions. If the effect on PAI-1 expression brought about by IL-1alpha, TNF-alpha, TGF-beta and OSM is not only operative under in vitro and ex vivo conditions but also in the in vivo setting one could speculate that these cytokines contribute to upregulation of PAI-1 in myocardial tissue and that PAI-1, when upregulated in myocardial tissue during inflammatory processes, could serve as a defence mechanism against excessive matrix degradation by proteases. Thus we propose a role for PAI-1 produced in the heart by cardiac myocytes in cardiac remodelling and repair processes.

[Good cell culture practice--implementation of a relational cell culture database]. / Good Cell Culture Practice - Entwicklung einer relationalen Datenbank für die Zellkultur.

The claim for cell culture to provide validable in vitro models for biomedical research postulates evasion of possible fatal record keeping errors. A prototype of a relational computer database for IBM-compatible personal computers using Microsoft(r) Windows 95/98/2000 and NT for administration of cell culture data has been developed using Microsoft(r) Access 98 (Microsoft Corporation, Redmond, USA), -Access Basic, -Visual Basic and Structured Query Language (SQL) (IBM Corporation, Armonk, USA), and was tested successfully. The modular software application manages the many aspects of cell culture laboratory record keeping like detailed information on tissue donor, primary cell isolation/cell line origin, immunohistochemical/molecular biological characterisation, cell countings at passaging/subcultivation/cell aliquotation and cryopreservation. One main feature is a collection of all methods performed at our cell culture laboratory, where linked tables and files store specific informations. Entries into the database are checked via validation rules for correctness to avoid mistakes. The developed prototype has been demonstrated to be an adaptable, reliable tool for improving quality of information storage according to Good Scientific Practice (GSP), Good Cell Culture Practice (GCCP) and general ISO certification trends.

Efficient carboplatin single therapy in a mouse model of human testicular nonseminomatous germ cell tumor.

PURPOSE: Cisplatin based combination therapy has shown excellent clinical results in patients with testicular nonseminomatous germ cell tumor but chemotherapy induced morbidity and reduced patient compliance are limiting factors in this regimen. To decrease cisplatin based combination therapy induced morbidity we examined carboplatin versus etoposide single therapy in an animal model. MATERIALS AND METHODS: A total of 180 SCID mice bearing testicular nonseminomatous germ cell tumor xenografts received 120 mg./kg. carboplatin as a single cycle, 60 or 30 mg./kg. carboplatin cycled twice, 80, 50 or 30 mg./kg. etoposide cycled twice, or Ringer solution as the control. An additional 20 sham treated and 20 untreated mice also served as controls. Histological and immunocytochemical testing, in vivo microscopy, vascular corrosion casting, serum tumor markers, complete blood count and real-time polymerase chain reaction were used to monitor therapy efficacy. RESULTS: Carboplatin at 60 mg./kg. cycled twice eradicated the tumor and significantly reduced vascular density and vascular endothelial growth factor-A messenger RNA (p <0.05). Elevated tumor markers returned to baseline after carboplatin administration. Therapy was well tolerated, resulting thrombocytopenia disappeared 6 weeks after therapy and the animals were tumor-free 6 months after treatment. Although 120 mg./kg. carboplatin eradicated the tumor, it resulted in extensive mortality and morbidity. Single treatment 30, 50 and 80 mg./kg. etoposide failed. CONCLUSIONS: Carboplatin single therapy was highly effective in our nonseminomatous germ cell tumor model and it may be examined in future clinical trials in patients with high risk stage I nonseminomatous germ cell cancer for reducing cisplatin based combination therapy induced morbidity. Vascular density and vascular endothelial growth factor messenger RNA were elevated in our animal model and deserve further study in nonseminomatous germ cell tumor cases as potential risk factors.