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ABSTRACT: The inflammatory response after prolonged ischemia and subsequent reperfusion leads to increased risk of primary organ dysfunction after cardiac transplantation. It has been demonstrated that the fibrin-derived peptide Bbeta(15-42) (also called FX06) reduces infarct size in coronary artery occlusion/reperfusion models by inhibition of leukocyte migration. Further, Bbeta(15-42) preserves endothelial barrier function. The purpose of this study was to investigate whether Bbeta(15-42) has a protective effect in cardiac allografts exposed to prolonged global ischemia and subsequent in vivo reperfusion.
Hearts of male Lewis rats were flushed and stored in cold Bretschneider preservation solution for 4 or 8 hr. Bbeta(15-42) was administered before being transplanted into syngeneic recipients. Serum samples were collected for troponin-T measurements. Hemodynamic performance was evaluated after a reperfusion period of 24 hr. Morphologic quantification of myocardial necrosis was performed in hearts exposed to 24 hr or 10 days of reperfusion.
Allografts from Bbeta(15-42) treated animals showed less myocardial necrosis (2.5% +/- 2.5% vs. 18.4% +/- 9.2%, P=0.0019) and decreased values of cardiac troponin-T (1.1 +/- 0.6 ng/mL vs. 2.7+/-2.3 ng/mL, P=0.0045), reduced number of infiltrating leukocytes (7.2 +/- 13.6 vs. 49.2 +/- 34.9 per high powerfield, P=0.0045), and superior cardiac output (78.1 +/- 1.8 mL/min vs. 21.7 +/- 4 mL/min, P = 0.0034). Hearts exposed to 0 and 4 hr of ischemia showed no severe signs of myocardial damage.
Bbeta(15-42) ameliorates the ischemia-reperfusion injury in transplanted hearts during extended cold ischemia by reduction of infiltrating leukocytes. This experimental protocol provides evidence that Bbeta(15-42) may play a useful role in organ preservation, but clinical evaluation is warranted.
Transplantation 04/2010; 89(7):824-9. · 4.00 Impact Factor
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ABSTRACT: Ischemia/reperfusion injury caused by cardioplegic arrest is still a major challenge in patients with reduced left ventricular function. We investigated the effect of chronic versus acute administration of the selective endothelin-A receptor antagonist (ERA) TBC-3214Na during ischemia/reperfusion in failing hearts.
Male Sprague-Dawley rats underwent coronary ligation. Three days after myocardial infarction (MI), 19 randomly assigned animals (ERA chronic) were administered TBC-3214Na continuously with their drinking water, 29 MI rats received placebo, and 3 rats died during the observation period. Six weeks after infarction, hearts were evaluated in a blood-perfused working heart model during 60 minutes of ischemia and 30 minutes of reperfusion. In 14 MI rats, TBC-3214Na (ERA acute) was added to the cardioplegic solution during ischemia. Thirteen MI rats served as control.
At a similar infarct size, postischemic recovery of cardiac output (ERA chronic: 91% +/- 10%, ERA acute: 86% +/- 11% vs control: 52% +/- 15%; P < .05) and external heart work (ERA chronic: 90% +/- 10%, ERA acute: 85% +/- 13% vs control: 51% +/- 17%; P < .05) was significantly enhanced in both TBC-3214Na-treated groups whereas recovery of coronary flow was only improved in ERA acute rats (ERA acute: 121% +/- 23% vs ERA chronic: 75% +/- 13%; control: 64% +/- 15%; P < .05). Blood gas measurements showed enhanced myocardial oxygen delivery and consumption with acute TBC-3214Na therapy. Additionally, high-energy phosphates (phosphocreatine) were significantly higher and transmission electron microscopy revealed less ultrastructural damage under acute TBC-3214Na administration.
Acute endothelin-A receptor blockade is superior to chronic blockade in attenuating ischemia/reperfusion injury in failing hearts. Therefore, acute endothelin-A receptor blockade might be an interesting option for patients with heart failure undergoing cardiac surgery.
The Journal of thoracic and cardiovascular surgery 05/2009; 137(4):1005-11, 1011e1. · 3.41 Impact Factor
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Nikolaus Wick,
Daniela Haluza,
Elisabeth Gurnhofer,
Ingrid Raab,
Marie-Theres Kasimir,
Michael Prinz,
Carl-Walter Steiner,
Christina Reinisch,
Anny Howorka,
Pietro Giovanoli,
Sabine Buchsbaum,
Sigurd Krieger,
Erwin Tschachler,
Peter Petzelbauer,
Dontscho Kerjaschki
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ABSTRACT: Expression of the lymphoendothelial marker membrane mucoprotein podoplanin (podo) distinguishes endothelial cells of both blood and lymphatic lineages. We have previously discovered two distinct subpopulations of lymphatic endothelial cells (LECs) in human skin that were defined by their cell surface densities of podoplanin and were designated LEC podo-low and LEC podo-high. LEC podo-low is restricted to lymphatic precollector vessels that originate from initial LEC podo-high-containing lymphatic capillaries and selectively express several pro-inflammatory factors. In addition to the chemokine receptor protein Duffy blood group antigen receptor for chemokines, these factors include the constitutively expressed chemokine CCL27, which is responsible for the accumulation of pathogenic CCR10+ T lymphocytes in human inflammatory skin diseases. In this study, we report that CCR10+ T cells accumulate preferentially both around and within CCL27+ LEC podo-low precollector vessels in skin biopsies of human inflammatory disease. In transmigration assays, isolated CCR10+ T lymphocytes are chemotactically attracted by LEC podo-low in a CCL27-dependent fashion, but not by LEC podo-high. These observations indicate that LEC podo-low-containing precollector vessels constitute a specialized segment of the initial lymphatic microvasculature, and we hypothesize that these LEC podo-low-containing vessels are involved in the trafficking of CCR10+ T cells during skin inflammation.
American Journal Of Pathology 10/2008; 173(4):1202-9. · 4.89 Impact Factor
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ABSTRACT: Platelet-derived growth factor (PDGF)-AA isoform and its receptor, PDGF-alpha receptor (PDGFRA) regulate tooth development and growth. We investigated the expression of both proteins in ameloblastomas, to contribute the understanding of the potential role of the PDGF/PDGFR system in this odontogenic neoplasm.
Twenty-nine specimens of ameloblastoma were analyzed for PDGF-AA and PDGFRA expression using immunohistochemistry. The proliferation activity was investigated with the MIB-1 antibody. Additionally, capillary sequencing of genomic DNA was performed to search for mutations in therapeutically relevant exons 12 and 18 of the PDGFRA gene.
PDGF-AA and PDGFRA expression were detectable in all cases with the exception of one tumor. However, protein expression levels did neither correlate with each other nor with MIB-1 expression. Unicystic ameloblastomas did not differ from solid tumors with regard to PDGF-AA, PDGFRA, and MIB-1 expression. One tumor revealed a somatic mutation of exon 12 of the PDGFRA gene.
PDGF-AA and PDGFRA proteins are regularly expressed in variable levels in ameloblastomas, and somatic mutations of exon 12 and exon 18 of the PDGFRA gene are rare findings.
Journal of Oral Pathology and Medicine 05/2008; 37(4):235-40. · 1.63 Impact Factor
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ABSTRACT: This study presents a potentially novel method of screening for pathogenetic factors in diabetic audiopathy by comparing the absolute plasma concentration of a microangiopathy biomarker, stromal cell-derived factor 1a (SDF-1a), with frequency-specific audiometric results.
Impaired hearing function in diabetic patients has, to date, remained a controversial and poorly understood theme with sparse clinical data. This is in contrast to more established components of the disease such as diabetic retinopathy, where diabetic microangiopathy is thought to be of pathogenetic relevance, and specific molecules such as SDF-1a have been assigned a relevant role.
Out patient clinic, Vienna General Hospital, Medical University of Vienna.
18 Type 2 diabetic patients and 18 nondiabetic controls.
Pure-tone audiometry and Freyburger number tests were used to evaluate hearing function. Blood plasma values of SDF-1a were analyzed using an enzyme-linked immunosorbent assay. Statistical comparison of functional audiometric data and the absolute SDF-1a values was performed for all frequencies.
A significantly higher plasma SDF-1a concentration (p < 0.005) in Type 2 diabetic patients, who also presented with higher pure-tone audiometry thresholds compared with nondiabetic subjects, was noted. Furthermore, an association between SDF-1a and audiometric performance, body mass index, and duration of diabetes was observed.
We hypothesize that diabetic microangiopathy and its biomarker SDF-1a should be considered as potential pathogenetic factors for altered diabetic hearing, warranting further investigation.
Otology & neurotology: official publication of the American Otological Society, American Neurotology Society [and] European Academy of Otology and Neurotology 04/2008; 29(6):739-44. · 1.44 Impact Factor
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ABSTRACT: To examine the prognostic relevance of c-kit expression in human osteosarcomas and to evaluate the mutation status in exon 9 and exon 11 of the c-kit gene.
c-kit expression was examined in 100 human osteosarcomas by immunohistochemistry using paraffin embedded tumour tissues, and capillary sequencing of genomic DNA was performed to search for mutations in exons 9 and 11 of the c-kit gene.
20 osteosarcomas showed c-kit expression ranging from 5% to 90% (mean 5.9%; SD 16.74%). Furthermore, DNA sequences of exon 9 and exon 11 of the c-kit gene were not altered in these tumours. Overall and disease free survival analysis did not reveal any differences between patients with osteosarcoma with c-kit expression and those with c-kit negative tumours.
C-kit expression is not a prognostic marker in patients with osteosarcoma. The protein expression is not linked to mutations in exon 9 or exon 11 of the c-kit gene. Therefore, these exons may not function as targets for treatment modalities based on the suppression of c-kit tyrosine kinase activity.
Journal of Clinical Pathology 08/2007; 60(7):804-7. · 2.31 Impact Factor
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Nikolaus Wick,
Pipsa Saharinen,
Juha Saharinen,
Elisabeth Gurnhofer,
Carl W Steiner,
Ingrid Raab,
Dejan Stokic,
Pietro Giovanoli,
Sabine Buchsbaum,
Aurea Burchard,
Stefan Thurner,
Kari Alitalo,
Dontscho Kerjaschki
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ABSTRACT: The in vivo functions of lymphatic endothelial cells depend on their microenvironment, which cannot be fully reproduced in vitro. Because of technical limitations, gene expression in uncultured, "ex vivo" lymphatic endothelial cells has not been characterized at the molecular level. We combined tissue micropreparation and direct cell isolation with DNA chip experiments to identify 159 genes differentiating human lymphatic endothelial cells from blood vascular endothelial cells ex vivo. The same analysis performed with cultured primary cells revealed that only 19 genes characteristic for lymphatic endothelium ex vivo retained this property upon culture, while 27 marker genes were newly induced. In addition, a set of panendothelial genes could be recognized. The propagation of lymphatic endothelial cells in culture stimulated transcription of genes associated with cell turnover, basic metabolism, and the cytoskeleton. On the other hand, there was downregulation of genes encoding extracellular matrix components, signaling via transmembrane tyrosine kinase pathways and the chemokine (C-C) ligand 21. Direct ex vivo analysis of the lymphatic endothelial cell transcriptome is helpful for the understanding of the physiology of the lymphatic vascular system and of the pathogenesis of its diseases.
Physiological Genomics 02/2007; 28(2):179-92. · 2.73 Impact Factor
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ABSTRACT: At present, the functional mechanism of acupuncture is not yet fully understood. Analysis of the subanatomic morphology of acupuncture points (APs) could help compensate for this shortcoming. In immunohistochemistry, the use of specific antibodies enables in situ characterization of the molecular profile of tissue microenvironments. Thus, as proof in principle for the utility of immunohistochemistry, we determined whether the nerve density in biopsies of autopsied skin of a selected standard AP differed from that of a control point (CP).
We analyzed pairs of skin samples from nine autopsy cases and studied the presence and density of soluble protein 100 (S-100), neuron-specific enolase (NSE), and neurofilament (NF) as markers of peripheral nerve structures. Cross-sections of nerves were counted by conventional microscopy and normalized to millimeters squared of subcutaneous fat, followed by statistical analyses for formal comparisons.
Immunohistochemistry could clearly identify myelinated peripheral nerves. The number of nerve structures expressing S-100 protein was significantly reduced in APs compared with CPs (0.020 1 0.005 vs. 0.061 +/- 0.014; P < 0.006). The same pattern was seen in staining of NSE (AP: 0.011 +/- 0.003 vs. CP: 0.045 +/- 0.011) and NF (AP: 0.011 +/- 0.004 vs. CP: 0.054 +/- 0.015; both P < 0.007).
In this study, we introduce immunohistochemistry as a suitable technology for acupuncture research. In addition, our findings demonstrate that a human AP is not necessarily associated with an increased but, rather, a significantly decreased number and density of subcutaneous nerve structures compared with skin biopsies from locations not recognized as effective for acupuncture. This pilot study, executed on a limited number of individuals and skin samples, justifies the application of immunohistochemistry on a larger collection of biopsy material.
American Journal of Physical Medicine & Rehabilitation 01/2007; 86(1):7-11. · 1.58 Impact Factor
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ABSTRACT: The expression of podoplanin, a small mucin-like protein, is upregulated in the invasive front of a number of human carcinomas. We have investigated podoplanin function in cultured human breast cancer cells, in a mouse model of pancreatic beta cell carcinogenesis, and in human cancer biopsies. Our results indicate that podoplanin promotes tumor cell invasion in vitro and in vivo. Notably, the expression and subcellular localization of epithelial markers are unaltered, and mesenchymal markers are not induced in invasive podoplanin-expressing tumor cells. Rather, podoplanin induces collective cell migration by filopodia formation via the downregulation of the activities of small Rho family GTPases. In conclusion, podoplanin induces an alternative pathway of tumor cell invasion in the absence of epithelial-mesenchymal transition (EMT).
Cancer Cell 05/2006; 9(4):261-72. · 26.57 Impact Factor
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Dontscho Kerjaschki,
Nicole Huttary,
Ingrid Raab,
Heinz Regele,
Katalin Bojarski-Nagy,
Gregor Bartel,
Stefan M Kröber,
Hildegard Greinix,
Agathe Rosenmaier,
Franz Karlhofer, Nikolaus Wick,
Peter R Mazal
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ABSTRACT: De novo lymphangiogenesis influences the course of different human diseases as diverse as chronic renal transplant rejection and tumor metastasis. The cellular mechanisms of lymphangiogenesis in human diseases are currently unknown, and could involve division of local preexisting endothelial cells or incorporation of circulating progenitors. We analyzed renal tissues of individuals with gender-mismatched transplants who had transplant rejection and high rates of overall lymphatic endothelial proliferation as well as massive chronic inflammation. Donor-derived cells were detected by in situ hybridization of the Y chromosome. We compared these tissues with biopsies of essentially normal skin and intestine, and two rare carcinomas with low rates of lymphatic endothelial proliferation that were derived from individuals with gender-mismatched bone marrow transplants. Here, we provide evidence for the participation of recipient-derived lymphatic progenitor cells in renal transplants. In contrast, lymphatic vessels of normal tissues and those around post-transplant carcinomas did not incorporate donor-derived progenitors. This indicates a stepwise mechanism of inflammation-associated de novo lymphangiogenesis, implying that potential lymphatic progenitor cells derive from the circulation, transmigrate through the connective tissue stroma, presumably in the form of macrophages, and finally incorporate into the growing lymphatic vessel.
Nature Medicine 03/2006; 12(2):230-4. · 22.46 Impact Factor
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ABSTRACT: It is a well known problem that standard techniques for analysing DNA chip data misspecify genes. In particular, genes that are confirmed to be active, often do not show up as potential candidates. This is possibly due to non-homogeneous distributions of expression levels over the whole expression range.
We introduce a method that allows the detection of genes based on a self-adaptive threshold. The threshold is determined for equally-populated expression bands by assuming a normal distribution of logarithms of expression level ratios. By specifying a significance level, the threshold is set according to 'local' expression statistics within a band. We call this method the relative variance method (RVM). We derive a test statistic for the RVM and compare it with other methods. On this statistical basis, we show that RVM is a complementary approach to the t-test, significance analysis of microarrays (SAM) or empirical Bayes analysis of microarrays (EBAM). The RVM should be particularly useful for experiments with small sample size.
Using a clinical dataset, we demonstrate that the RVM can correctly identify known marker genes, which are not found by the t-test, SAM or EBAM.
In situations with limited sample material and small number of replicates, as is often the case in clinical datasets, use of the proposed RVM provides a higher reliability of potential candidate genes.
Applied Bioinformatics 02/2006; 5(4):277-84.
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ABSTRACT: We analyze gene expression time-series data of yeast S. cerevisiae measured along two full cell-cycles. We quantify these data by using q-exponentials, gene expression ranking and a temporal mean-variance analysis. We construct gene interaction networks based on correlation coefficients and study the formation of the corresponding giant components and minimum spanning trees. By coloring genes according to their cell function we find functional clusters in the correlation networks and functional branches in the associated trees. Our results suggest that a percolation point of functional clusters can be identified on these gene expression correlation networks. Comment: 8 pages, 4 figures
Physics of Condensed Matter 09/2005; · 1.53 Impact Factor
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American Journal of Surgical Pathology 07/2005; 29(6):842. · 4.35 Impact Factor
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ABSTRACT: Low amounts of starting material are a significant limitation of gene-expression profiling of microprepared pathologic specimens. Linear RNA amplification has become the method of choice to overcome this problem. Thus, transcriptomal analyses by oligonucleotide-chips or cDNA microarrays are now feasible with labeled complementary RNA generated from total RNA samples in the lower nanogram range. However, in case of oligonucleotide-chips, it has been underestimated so far that individual complementary RNA molecules are shorter in length than and display a 3' bias in comparison to the sequence stretch represented by oligonucleotides on the chip. This can lead to incorrect interpretation of raw data. We have analyzed this problem testing ex vivo-microprepared endothelial cells with Affymetrix GeneChips U133A. Only a small subset of housekeeping genes showed adequate uniform hybridization. We developed a software tool for objective evaluation of oligonucleotide-chips based on automated analysis of as well as normalization to this subset of housekeeping genes. We analyzed the gene expression profile of microprepared lymphatic vascular endothelial cells. We show that optimized normalization prevented exclusion of angiopoietin-2, a lymphatic endothelial marker, from the lymphovascular transcriptome.
Diagnostic Molecular Pathology 10/2004; 13(3):151-9. · 2.26 Impact Factor
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ABSTRACT: Chicken embryo lethal orphan adenovirus (CELO) is used as a vector for expression of exogenous genes in mammalian cells. Here, we analyzed transcriptional alterations in mouse epithelial host cells following infection with CELO using cDNA microarray analysis. Sequence data characterization revealed that a major portion of CELO-induced genes contained short interspersed nuclear elements of the B2 subclass (B2 SINEs). In fact, we could identify SINEs and other repetitive sequences as contributing significantly to the cDNAs used for microarray construction. Moreover, we found that the CELO protein Gam1 was able to mediate transcriptional activation of these B2 SINE-containing RNAs. We hypothesize that upregulation of B2-SINE-containing RNAs could be a novel contribution of Gam1 to CELO host cell infection.
Journal of Molecular Biology 06/2003; 328(4):779-90. · 4.00 Impact Factor
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ABSTRACT: Epithelial cells of the mammary gland possess the inherent capacity to form epithelial monolayers in vitro. This requires coordination of cell migration, cell-cell contact formation, and cell proliferation. Using time-lapse phase contrast videomicroscopy we have observed mammary gland epithelial cells over different time scales. We show the generation of a complete polarized epithelial monolayer in real-time, starting from a few cells. We subsequently concentrated on the early stages of this process by tracking epithelial cells during phases of polarized migration. We performed migration analysis using fractal measures. With this technology the structure of seemingly random processes not accessible to the usual methods of linear analysis can be measured. As a control and proof of principle approach we applied infection of cells with an adenoviral vector, which is used as a gene targeting vector for many applications. Infection markedly influenced the patterns of migratory behavior. We, therefore, believe that time-lapse videomicroscopy in combination with fractal analysis can contribute to differential characterization of distinct cellular migration patterns. This will be useful in situations of long-term alterations in cell culture systems.
Histochemie 02/2003; 119(1):15-20. · 2.59 Impact Factor
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Ilja Vietor,
Santhosh K Vadivelu, Nikolaus Wick,
Robert Hoffman,
Matt Cotten,
Christian Seiser,
Irene Fialka,
Winfried Wunderlich,
Astrid Haase,
Gabriela Korinkova,
Gerald Brosch,
Lukas A Huber
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ABSTRACT: The mammalian SIN3 complex consists of histone deacetylases (HDAC1, HDAC2), several known proteins (SAP30, N-CoR) and as yet unidentified proteins. Here we show that the mouse tetradecanoyl phorbol acetate induced sequence 7 (TIS7) protein is a novel transcriptional co-repressor that can associate with the SIN3 complex. We have identified tis7 as a gene that is up-regulated upon loss of polarity in a mouse mammary gland epithelial cell line expressing an estrogen-inducible c-JunER fusion protein. In unpolarized cells, TIS7 protein levels increase and TIS7 translocates into the nucleus. Overexpression of tis7 causes loss of polarity and represses a set of genes, as revealed by cDNA microarray analysis. We have shown that TIS7 protein interacts with several proteins of the SIN3 complex (mSin3B, HDAC1, N-CoR and SAP30) by yeast two-hybrid screening and co-immunoprecipitations. TIS7 co-immunoprecipitated HDAC complex is enzymatically active and represses a GAL4-dependent reporter transcription. The transcriptional repression of endogenous genes by tis7 overexpression is HDAC dependent. Thus, we propose TIS7 as a transcriptional co-repressor affecting the expression of specific genes in a HDAC activity-dependent manner during cell fate decisions, e.g. scattering.
The EMBO Journal 10/2002; 21(17):4621-31. · 9.20 Impact Factor
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ABSTRACT: We propose a model for cell migration where epithelial cells are able to detect trajectories of other cells and try to follow them. As cells move along in 2D cell culture, they mark their paths by loosing tiny parts of cytoplasm. Any cell moving on a surface where other cells have moved before faces a network of cell trajectories, which it tries to restrict its motion onto. With the Tsallis modification of classical thermodynamics one can solve the relevant Fokker–Planck like equation and obtain experimentally testable distribution functions. We compare the model to experimental data of normal mammary epithelial cells and cells which have been genetically manipulated to change their cell–cell interaction.
Physica A: Statistical Mechanics and its Applications. 08/2002;
