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Rudin Pistulli,
Sebastian König,
Stefanie Drobnik,
Daniel Kretzschmar,
Ilonka Rohm,
Michael Lichtenauer,
Michael Fritzenwanger,
Gerhard Mall,
Gita Mall, Hans-Reiner Figulla,
Atilla Yilmaz
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ABSTRACT: AIMS: Dendritic cells (DCs) are sentinels of the immune system-their role in myocardial disease is unknown as yet. We investigated their myocardial presence in human dilated cardiomyopathy (DCM). METHODS AND RESULTS: Endomyocardial biopsies from 72 patients with DCM (EF ∼30%), as well as myocardial specimens from 18 suicide or accident victims were immunohistochemically analysed for myeloid and plasmacytoid DCs, antigen-presenting cells (APCs), and other leucocytes; also tissue fibrosis and apoptosis were histologically quantified. The myocardial viral genome was identified through polymerase chain reaction, and patients underwent clinical follow-up in 3-6 months. We found myocardial DCs of all examined subtypes and maturation stages (fascin, CD11c, CD209, CD83, and CD304), as well as markers for APCs (HLA-DR and CD40) and T-cell activation (CD69) to be significantly decreased in DCM compared with controls. In contrast, regulatory T cells (the GITR epitope), apoptosis (by TUNEL reaction and immunostaining with BCL-2), and a DC chemokine receptor (CCR7) were overexpressed, while no significant differences were observed for macrophages (CD68). Immature myeloid and plasmacytoid DCs strongly correlated with endothelial progenitor cells (CD34), which were similarly reduced in DCM, and inversely correlated with fibrosis. Myeloid DCs were especially reduced in virus-positive biopsies, and their numbers correlated with positive change in EF (ΔEF) at follow-up. CONCLUSION: Myocardial DCs are reduced in heart biopsies of symptomatic DCM patients. Such a reduction correlates with an unfavourable short-term outcome in terms of EF, and could result from myocardial tissue damage, cellular death, and insufficient vascularization in chronic heart failure.
European Journal of Heart Failure 04/2013; · 4.90 Impact Factor
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ABSTRACT: Embryonic stem (ES) cells are a powerful model for the development of cells responsible for cellular immune response. Therefore we analyzed the defense- and phagocytic capacity of Embryoid Bodies (EBs) derived from ES cells using in vitro inflammatory conditions caused by Escherichia coli (E. coli). Further we used this phagocytic activity to purify activated immune cells. Our data show that spontaneously differentiated 18-day-old EBs of the cell line CGR8 contained immune cells which were positive for CD45, CD68, CD11b, F4/80 and CD19. Exposure of these EBs to E. coli with defined infection doses of bacterial colony forming units (CFUs) led to a significant time-dependent reduction of CFUs, indicating immune responses exerted by EBs. This was paralleled by an up-regulation of inflammatory cytokines, i.e. IL-1β and TNF-α. Western Blot analysis of infected EBs indicated an up-regulation of CD14 and cytochrome b-245 heavy chain (NOX2). Silencing of NOX2 significantly reduced the antibacterial capacity of EBs which was partially explained by reduction of F4/80 positive cells. To identify, isolate and further cultivate phagocytic active cells from differentiated EBs, a co-cultivation assay of differentiated ES cells with green fluorescent protein (GFP)-labeled E. coli was established. Co-localization of GFP-labeled E. coli with cells positive for CD45, CD68 and F4/80 revealed time-dependent phagocytotic uptake which was underlined by a co-localization with LysoTracker-Red® dye as well as pre-incubation with Cytochalasin D. In conclusion, a primitive immune response with efficient phagocytosis was responsible for the anti¬bacterial capacity of differentiated EBs.
Stem cells and development 02/2013; · 4.15 Impact Factor
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Biomedizinische Technik/Biomedical Engineering 09/2012; · 0.53 Impact Factor
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Biomedizinische Technik/Biomedical Engineering 08/2012; · 0.53 Impact Factor
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ABSTRACT: Embolization of atherosclerotic debris from the rupture of a vulnerable atherosclerotic plaque occurs iatrogenically during percutaneous coronary interventions (PCI) and can induce myocardial necrosis. These microembolizations are detected as high intensity transient signals (HITS) using intracoronary Doppler technology.
In the presented study we will test if abciximab (ReoPro®) infusion reduces high intensity transient signals in patients with stable angina pectoris undergoing PCI in comparison to standard therapy alone.
The High Intensity Transient Signals ReoPro® (HITS-RP) study will enroll 60 patients. It is a prospective, single center, randomized, double-blinded, controlled trial. The study is designed to compare the efficacy of intravenous abciximab administration for reduction of microembolization during elective PCI. Patients will be randomized in a 1:1 fashion to abciximab or placebo infusion. The primary end point of the HITS-RP-Study is the number of HITS during PCI measured by intracoronary Doppler wire. Secondary endpoints are bleeding complications, elevation of cardiac biomarkers or ECG changes after percutaneous coronary interventions, changes in coronary flow velocity reserve, hs-CRP elevation, any major adverse cardio-vascular event during one month follow-up.
The HITS-RP-Study addresses important questions regarding the efficacy of intravenous abciximab administration in reducing microembolization and periprocedural complications in stable angina pectoris patients undergoing PCI.
The trial is registered under http://www.drks-neu.uniklinik-freiburg.de/drks_web/:DRKS00000603.
Cardiovascular Ultrasound 05/2012; 10:21. · 1.26 Impact Factor
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ABSTRACT: Hintergrund:
Die Mikrozirkulation bei kritisch Kranken ist zunehmend in das Interesse intensivmedizinischer Forschung gerückt, da hierdurch
frühzeitig Veränderungen der Gewebeperfusion sichtbar werden. Bei Kurzzeitanästhesie konnte kürzlich ein negativer Einfluss
bei der Infusion von Propofol auf die Mikrozirkulation nachgewiesen werden. Der Einfluss von Propofol auf den Mikrofluss bei
internistischen Intensivpatienten unter Langzeitanästhesie ist bislang unklar.
Patienten und Methodik:
Bei 28 Patienten (elf mit Propofol) der eigenen internistischen Intensivstation wurde mittels Sidestream-Darkfield-Mikroskopie
der sublinguale Mikrofluss visualisiert, aufgezeichnet und quantifiziert (0: „no flow“; 1: „intermittent flow“; 2: „sluggish
flow“; 3: „continuous flow“).
Ergebnisse:
Bei stabilen internistischen Intensivpatienten konnten in sublingualen Gefäßen (10–100 μm) gute Mikroflüsse nachgewiesen werden,
bei vielen Patienten nahezu kontinuierliche Flüsse. Dabei unterschieden sich Patienten mit und ohne Propofol nicht.
Schlussfolgerung:
Bei stabilen Patienten zeigte sich in dieser kleinen Kohorte kein Einfluss von Propofol im Langzeiteinsatz auf den Mikrofluss.
Die bekannte Interaktion zwischen Propofol und Mikrozirkulation sollte der Intensivmediziner dennoch kennen und den Einsatz
dieser Präparate bei Patienten mit Schock zu vermeiden versuchen. Der Einsatz von Propofol bei hämodynamisch kompromittierten
Patienten sollte in zukünftigen Studien untersucht werden.
Background:
Microcirculation has become a major focus of research in critical care medicine due to its growing clinical relevance detecting
changes in organ perfusion at an early stage. A negative impact of propofol infusion on microcirculation during short-term
anesthesia was described recently. The influence of long-term sedation with propofol on microflow of critical care patients
is still unclear.
Patients and Methods:
Microflow was analyzed using sidestream darkfield microscopy of sublingual mucosa in 28 patients of whom eleven received continuous
infusion of propofol. According to current guidelines, microflow was recorded digitally. Quantitative analysis was performed
offline in a semiquantitative way (0: no flow; 1: intermittent flow; 2: sluggish flow; 3: continuous flow).
Results:
Good microflow rates were detected in sublingual vessels (10–100 μm) in hemodynamically stable, medical intensive care patients.
In the majority of cases, continuous flow profiles were recorded. There was no difference in flow rates between patients with
and without propofol therapy.
Conclusion:
In hemodynamically stable intensive care patients, long-term therapy with propofol did not affect sublingual microflow in
this small cohort. However, intensive care physicians should keep such possible interactions in mind avoiding administration
of these substances in patients with manifested shock. The effects of propofol in hemodynamically impaired patients should
be evaluated in further studies.
04/2012; 104(5):336-342.
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Daniel Kretzschmar,
Stefan Betge,
Alexander Windisch,
Rudin Pistulli,
Ilonka Rohm,
Michael Fritzenwanger,
Christian Jung,
Katja Schubert,
Bernhard Theis,
Iver Petersen,
Stefanie Drobnik,
Gita Mall, Hans-Reiner Figulla,
Atilla Yilmaz
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ABSTRACT: DC (dendritic cells) play an important role in the immune system. They invade peripheral tissues to detect harmful antigens, inducing a local immune response. Studies suggest that DCPs (dendritic cell precursors) might be reduced in AMI (acute myocardial infarction); however, the reason for their reduction is unknown yet. In the present study, circulating mDCPs (myeloid DCPs), pDCPs (plasmacytoid DCPs), tDCPs (total DCPs) and serum levels of TNFα (tumour necrosis factor α), IL (interleukin)-2, -4, -5, -6, -10 and -12 were analysed by flow cytometry in blood of patients with NSTEMI [non-STEMI (ST-segment elevation myocardial infarction)] (n=44) and STEMI (n=34) compared with controls with excluded CAD (coronary artery disease) (n=45). Post-mortem myocardial specimens of patients with AMI (n=12) and healthy myocardium of accident victims (n=10) were immunostained for mDCs (myeloid dendritic cells) T-cells and macrophages. Compared with controls, in patients with AMI a significant decrease in circulating mDCPs, pDCPs and tDCPs was observed (each P<0.0001). The extent of the decrease was higher in STEMI than NSTEMI patients. Serum levels were significantly higher in patients with AMI compared with controls for IL-6, -10, -12 and TNFα (each P<0.03). Immunostaining revealed significantly higher number of DCs, T-cells and macrophages (each P<0.002) in infarcted than control myocardium. We show that circulating DCPs are significantly reduced in AMI, with a pronounced reduction in STEMI patients. This was accompanied by a significant increase of inflammatory serum cytokines in patients with AMI. Immunohistochemical analysis unravelled that the reduction of circulating DCPs might be due to recruitment into the infarcted myocardium.
Clinical Science 04/2012; 123(6):387-98. · 4.61 Impact Factor
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ABSTRACT: AIMS: To investigate the effects of static magnetic fields (MFs) on cardiomyogenesis of mouse embryonic stem (ES) cell-derived embryoid bodies and Flk-1(+) cardiac progenitor cells and to assess the impact of cytosolic calcium [Ca(2+)](c) and reactive oxygen species (ROS). METHODS AND RESULTS: Embryoid bodies and ES cell-derived Flk-1(+) cardiovascular progenitor cells were exposed to static MFs. The expression of cardiac genes was evaluated by RT-PCR; sarcomeric structures were assessed by immunohistochemistry; intracellular ROS and [Ca(2+)](c) of ES cells were examined by H(2)DCF-DA- and fluo-4-based microfluorometry. Treatment of embryoid bodies with MFs dose-dependent increased the number of contracting foci and cardiac areas as well as mRNA expression of the cardiac genes MLC2a, MLC2v, α-MHC and β-MHC. In Flk-1(+) cells MFs (1mT) elevated both [Ca(2+)](c) and ROS, increased expression of the cardiogenic transcription factors Nkx-2.5 and GATA-4 as well as cardiac genes. This effect was due to Ca(2+) influx, since extracellular Ca(2+) chelation abrogated ROS production and MF-induced cardiomyogenesis. Furthermore absence of extracellular calcium impaired sarcomere structures. Neither the phospholipase C inhibitor U73122 nor thapsigargin inhibited MF-induced increase in [Ca(2+)](c) excluding involvement of intracellular calcium stores. ROS were generated through NAD(P)H oxidase, since NOX-4 but not NOX-1 and NOX-2 mRNA was upregulated upon MF exposure. Ablation of NOX-4 by sh-RNA and treatment with the NAD(P)H oxidase inhibitor diphenylen iodonium (DPI) totally abolished MF-induced cardiomyogenesis. CONCLUSION: The ability of static MFs to enhance cardiomyocyte differentiation of ES cells allows high throughput generation of cardiomyocytes without pharmacological or genetic modification.
International journal of cardiology 03/2012; · 7.08 Impact Factor
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ABSTRACT: Heart rate turbulence, deceleration capacity (DC), and symbolic dynamics (SD) are promising novel domains of autonomic indices representing the multidimensional qualities of autonomic heart rate dynamics.
The aim of this study was to test the impact of these novel indices in predicting early AF recurrence within the first month after electrical cardioversion (CV).
In 45 patients with AF, standard Holter recordings were commenced immediately after CV. Holter-based indices were retrospectively analyzed using computerized algorithms. The best indices were applied in a multivariate model to select the optimal combination set that correctly classified patients who developed early AF recurrence.
Early AF recurrence occurred in 25 vs 20 patients with stable sinus rhythm. The set with the highest predictive power consisted of DC, turbulence onset, VLF/P, and PTH19 as a parameter of SD. The receiver operating curve analysis applied to this optimum set produced an area under the curve of 0.86, thus correctly classifying patients with 95.0% specificity and 76.0% sensitivity.
The analysis of novel multidimensional Holter-based autonomic indices after CV appears of clinical value because the procedure identifies patients with high risk of early AF recurrence. Furthermore, it indicates a substantial alteration of autonomic regulation.
Journal of electrocardiology 03/2012; 45(2):116-22. · 1.08 Impact Factor
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International journal of cardiology 09/2011; 152(3):417-8. · 7.08 Impact Factor
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Caroline Bartsch,
Mohamed M Bekhite,
Anne Wolheim,
Madeleine Richter,
Carola Ruhe,
Bianka Wissuwa,
Anja Marciniak,
Jörg Müller,
Regine Heller, Hans-Reiner Figulla,
Heinrich Sauer,
Maria Wartenberg
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ABSTRACT: Ascorbic acid (AA) increases cardiomyogenesis of embryonic stem (ES) cells. Herein we show that treatment of mouse ES cells with AA enhanced cardiac differentiation accompanied by an upregulation of the NADPH oxidase isoforms NOX2 and NOX4, phosphorylation of endothelial nitric oxide synthase (eNOS), and cyclic GMP (cGMP) formation, indicating that reactive oxygen species (ROS) as well as nitric oxide (NO) may be involved in cardiomyogenesis. In whole mount embryoid bodies as well as isolated Flk-1-positive (Flk-1(+)) cardiovascular progenitor cells ROS elevation by AA was observed in early stages of differentiation (Days 4-7), and absent at Day 10. In contrast NO generation following incubation with AA was absent at Day 4 and increased at Days 7 and 10. AA-mediated cardiomyogenesis was blunted by the NADPH oxidase inhibitors diphenylen iodonium (DPI) and apocynin, the free radical scavengers N-(2-mercaptopropionyl)-glycine (NMPG) and ebselen, and the NOS inhibitor L-NAME. Downregulation of NOX4 by short hairpin RNA (shRNA) resulted in significant inhibition of cardiomyogenesis and abolished the stimulation of MHC-ß and MLC2v gene expression observed on AA treatment. Our data demonstrate that AA stimulates cardiomyocyte differentiation from ES cells by signaling pathways that involve ROS generated at early stages and NO at late stages of cardiomyogenesis.
Free radical biology & medicine 07/2011; 51(2):432-43. · 5.42 Impact Factor
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ABSTRACT: VEGF-, phosphoinositide 3-kinase (PI3K)- and protein kinase C (PKC)-regulated signaling in cardiac and vascular differentiation was investigated in mouse ES cells and in ES cell-derived Flk-1⁺ cardiovascular progenitor cells. Inhibition of PI3K by wortmannin and LY294002, disruption of PI3K catalytic subunits p110α and p110δ using short hairpin RNA (shRNA), or inhibition of p110α with compound 15e and of p110δ with IC-87114 impaired cardiac and vascular differentiation. By contrast, TGX-221, an inhibitor of p110β, and shRNA knockdown of p110β were without significant effects. Antagonists of the PKC family, i.e. bisindolylmaleimide-1 (BIM-1), GÖ 6976 (targeting PKCα/βII) and rottlerin (targeting PKCδ) abolished vasculogenesis, but not cardiomyogenesis. Inhibition of Akt blunted cardiac as well as vascular differentiation. VEGF induced phosphorylation of PKCα/βII and PKCδ but not PKCζ. This was abolished by PI3K inhibitors and the VEGFR-2 antagonist SU5614. Furthermore, phosphorylation of Akt and phosphoinositide-dependent kinase-1 (PDK1) was blunted upon inhibition of PI3K, but not upon inhibition of PKC by BIM-1, suggesting that activation of Akt and PDK1 by VEGF required PI3K but not PKC. In summary, we demonstrate that PI3K catalytic subunits p110α and p110δ are central to cardiovasculogenesis of ES cells. Akt downstream of PI3K is involved in both cardiomyogenesis and vasculogenesis, whereas PKC is involved only in vasculogenesis.
Journal of Cell Science 06/2011; 124(Pt 11):1819-30. · 6.11 Impact Factor
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Anja Baldinger,
Bernhard R Brehm,
Petra Richter,
Torsten Bossert,
Katja Gruen,
Khosro Hekmat,
Hartwig Kosmehl,
Dario Neri, Hans-Reiner Figulla,
Alexander Berndt,
Marcus Franz
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ABSTRACT: Aortic valve stenosis (AVS) and coronary artery disease (CAD) are accompanied by changes in the cardiac extra cellular matrix (cECM) including the re-expression of oncofetal fibronectin (Fn) and tenascin-C (Tn-C) variants. Human antibodies against these variants are usable for targeted therapy. Aim of the study was the comparative analysis of cECM remodelling in tissue samples from right atrial auricle (RAA) and left ventricular septum (LVS). RAA and LVS specimens from 30 patients (17 × AVS; 13 × AVS+CAD) were analysed with respect to histological changes and ECM remodelling using PCR based ECM gene expression profiling. Re-expression of ED-A(+) Fn and A1(+) Tn-C was investigated on the mRNA and on the protein level. For immunofluorescence, human recombinant small immunoprotein (SIP) format antibodies were used. There was a positive correlation of the grade of histological changes in RAA and corresponding LVS samples (r = 0.695). ECM gene expression levels were higher in LVS compared to RAA. For 24 genes, a corresponding relevant (>2.5-fold) up- or down-regulation in RAA and LVS occurred. Using SIP antibodies, a positive correlation of protein deposition levels in RAA and corresponding LVS (r = 0.818) could be shown for ED-A(+) Fn. Cardiac tissue remodelling is likely a process involving the entire heart reflected by intra-individually comparable histology and cECM changes in RAA and LVS samples. ED-A(+) Fn might be an excellent target for an antibody-mediated delivery of diagnostic or therapeutic agents. The RAA is a valuable and representative tool to evaluate cardiac remodelling and to plan individualized therapy.
Histochemie 05/2011; 135(5):427-41. · 2.59 Impact Factor
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ABSTRACT: Heparin-induced thrombocytopenia (HIT) related to fondaparinux has been rarely reported, although the ability of fondaparinux to cross-react with heparin antibodies has been often a subject of debate. A patient previously exposed to unfractionated heparin and low-molecular-weight heparin (LMWH) was diagnosed with HIT. During treatment with fondaparinux for 5 consecutive days, his thrombocytopenia significantly deteriorated. A functional platelet activation test in vitro showed clear platelet activation after serum exposure with fondaparinux. After discontinuation of fondaparinux, the platelet count was rapidly reestablished. Fondaparinux cross-reacted with heparin antibodies in this case of HIT, resulting in a deterioration of thrombocytopenia. The implication of this drug in HIT was observed clinically and demonstrated in vitro using a platelet activation test.
Blood coagulation & fibrinolysis: an international journal in haemostasis and thrombosis 11/2010; 22(1):76-8. · 1.25 Impact Factor
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Clinical Research in Cardiology 07/2010; 99(7):463-6. · 2.95 Impact Factor
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ABSTRACT: We investigated the safety, feasibility and efficacy of the Occlutech devices for patent foramen ovale (PFO) and atrial septal defect (ASD) closure in a prospective trial.
The retention discs of the PFO device (23 x 25 mm, 27 x 30 mm) are connected by a 3 mm waist in the center with only one right atrial side central pin. The left atrial disc is produced either with a single or double flat layer, which allows a significant reduction of meshwork material. The ASD occluder (6-40 mm, 3 mm increments) has only one central pin on the right atrial side. Indications for closure included cryptogenic stroke with evidence of a PFO on transesophageal echocardiography (TEE) or an ASD II. The devices were implanted in 29 patients with PFO and in 12 patients with ASD II (fluoroscopy and TEE). An echocardiographic follow-up examination was performed after 1, 2 and 6 months.
The devices were successfully implanted in all 41 patients. There were no periprocedural complications. One patient with ASD II died of recurrent myocardial infarction without evidence of cardioembolic origin. TEE studies showed a residual shunt in 11.2% after 60 days in patients with PFO and a left-to-right shunt in 9.1% of the remaining patients with ASD II. After 180 days only 1 patient with PFO had a right-to-left shunt (3.7%). A residual shunt in the patients with ASD was not observed.
The novel Occlutech devices appear to be safe, feasible and effective for PFO and ASD closure, with a significant reduction of the meshwork and absence of left atrial central pin.
The Journal of invasive cardiology 04/2010; 22(4):182-7. · 1.84 Impact Factor
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ABSTRACT: Embryonic stem (ES) cells spontaneously differentiate capillary-like structures as well as leucocytes such as monocytes/macrophages, neutrophils, natural killer (NK) cells and cytototoxic T lymphocytes. The interplay between vasculogenesis and leucocyte differentiation as well as the population of tumour tissues with ES cell-derived leucocytes and endothelial cells is, however, not sufficiently specified. In the present study, gene expression of the cell surface markers CD68 and CD14 (expressed on monocytes and macrophages), Mac-1 (CD11b) (expressed on granulocytes, monocytes and NK cells) and CD16 (expressed on neutrophils) was investigated in murine CGR8 ES cells in relation to the endothelial cell markers CD31 and vascular endothelial (VE)-cadherin. Expression of leucocyte markers increased from day 7-8 of cell culture on. Furthermore, addition of macrophage colony-stimulating factor to the cell culture medium resulted in a threefold increase in the number of CD68(+) monocytes/macrophages. Treatment of embryoid bodies with lipopolysaccharide (LPS) up-regulated CD14 thus suggesting functionality of the CD14 LPS receptor. Differentiation of vascular structures positive for CD31 and VE-cadherin preceded leucocyte differentiation by 2 days (i.e. from day 5-6 on) suggesting that vasculogenesis may be a determinant of leucocyte differentiation. Consequently the Flk-1 antagonist SU5416 which inhibits vasculogenesis of ES cells significantly blunted leucocyte differentiation. Confrontation culture of embryoid bodies with multicellular breast tumour spheroids initiated significant increase of leucocyte cell numbers and invasion of leucocytes into the tumour tissue. In summary our data demonstrate that during ES cell differentiation vasculogenesis precedes leucocyte differentiation, and point towards the direction that leucocyte cell invasion into tumour tissue may initiate the pro-inflammatory microenvironment necessary for tumour vascularization.
Journal of Cellular and Molecular Medicine 01/2010; 14(1-2):303-12. · 4.13 Impact Factor
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ABSTRACT: Obesity and related diseases are dramatically increasing problems, particularly in children and adolescents. We determined circulating levels of different interleukin (IL)-1 family members in normal weight and overweight adolescents.
Seventy male, Caucasian adolescents (13-17 years) were recruited. Thirty-five had a body-mass index (BMI) above the 90th age-specific percentile. IL-1alpha, IL-1beta, IL-1 receptor antagonist (IL-1ra), and IL-18 were determined using multiplex-technology.
IL-18 concentrations were higher in the overweight group compared to normal weight (161.6 +/- 40.7 pg/ml versus 134.7 +/- 43.4 pg/ml, P = .009). Concentrations of circulating IL-1beta levels were below the detection threshold. IL-18 (R2:0.355, P < .01) and IL-1ra (R2:0.287, P < .05) correlated with BMI, whereas IL-1alpha did not.
Accumulating data indicate the importance of the endocrine function of adipose tissue for the pathophysiological consequences of obesity-related co-morbidities. Since IL-18 is involved in the pathogenesis of different cardiovascular diseases, we conclude that IL-18 may represent a link between obesity and related co-morbidities in children and adolescents.
Mediators of Inflammation 01/2010; 2010:958403. · 3.26 Impact Factor
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ABSTRACT: It is well established that leukotrienes (LTs), products of the 5-lipoxygenase (5-LO) pathway, participate in inflammatory tissue reactions and immune responses. In the present study, the impact of the 5-LO pathway on vasculogenesis of mouse embryonic stem (ES) cells was investigated.
Immunohistochemistry studies demonstrated that 5-LO(+) cells first appeared at day 6 of embryoid body (EB) formation from ES cells. 5-LO(+)/CD68(+) as well as 5-LO(+)/CD45(+) cells were prominent at day 10 of EB differentiation. Real-time PCR and western blot analysis revealed all constituents of the 5-LO pathway. High performance liquid chromatography analyses indicated the synthesis of LTB(4) and LTD(4) in conformity with induction of the 5-LO pathway. Furthermore, Flk-1(+)/CD105(+) cells displayed calcium- (Ca(2+)) transients in response to LTB(4), whereas CD11b(+) cells responded to LTD(4). Treatment of EBs with LTB(4) and LTD(4) resulted in phosphorylation of the mitogen-activated protein kinase ERK1/2. Pharmacological inhibition of the 5-LO pathway and stable shRNA targeting of 5-LO-activating protein decreased capillary cell areas positive for PECAM-1.
Our data demonstrate that the 5-LO pathway emerges early during ES cell differentiation into cells of the myeloid lineage and that LTs play an until now unrecognized role in vascular development of ES cells.
Cardiovascular research 12/2009; 86(1):37-44. · 5.80 Impact Factor
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ABSTRACT: ABC transporters like P-glycoprotein (P-gp/ABCB1) are membrane proteins responsible for the transport of toxic compounds out of non-malignant cells and tumor tissue. Aim: To investigate the effect of glycolysis and the tissue redox state on P-gp expression in multicellular tumor spheroids derived from prostate adenocarcinoma cells (DU-145), glioma cells (Gli36), and the human cervix carcinoma cell line KB-3-1 transfected with a P-gp-EGFP fusion gene that allows monitoring of P-gp expression in living cells. During cell culture of DU-145, Gli36, and KB-3-1 tumor spheroids P-gp expression was observed as well as increased lactate and decreased pyruvate levels and expression of glycolytic enzymes. Inhibition of glycolysis for 24 h by either iodoacetate (IA) or 2-deoxy-D-glucose (2-DDG) downregulated P-gp expression which was reversed upon coincubation with the radical scavenger ebselen as shown by semi-quantitative immunohistochemisty in DU-145 and Gli36 tumor spheroids, and by EGFP fluorescence in KB-3-1 tumor spheroids. Consequently endogenous ROS generation in DU-145 tumor spheroids was increased in the presence of either IA or 2-DDG, which was abolished upon coincubation with ebselen. Exogenous addition of pyruvate significantly reduced ROS generation, increased P-gp expression as well as efflux of the P-gp substrate doxorubicin. Doxorubicin transport was significantly blunted by 2-DDG and IA, indicating that inhibition of glycolysis reversed the multidrug resistance phenotype. In summary our data demonstrate that P-gp expression in tumor spheroids is closely related to the glycolytic metabolism of tumor cells and can be downregulated by glycolysis inhibitors via mechanisms that involve changes in the cellular redox state.
Journal of Cellular Biochemistry 11/2009; 109(2):434-46. · 2.87 Impact Factor
