Reagents used were CD3ε PE (145-C11), CD11b PE (M1/70), CD45R PE (RA3-6B2), CD117 eFluor780 (2B8), Sca-1 PerCP-Cy5.5 (D7; eBiosciences), CD4 PE (H129.19), CD8a PE (53–6.7), CD19 PE (1D3), Gr-1 PE (RB6-8C5), NK1.1 PE (PK136), and Ter119 PE (Ter119; BD Pharmingen). Create New Account. S2 E). There were no significant changes in the frequencies of YFP+ liver ECs while performing an indexed analysis comparing resected and regenerated liver lobes of six individual mice (Fig. Augustin), and SFB-TR209, “Hepatocellular carcinoma” (TP C3 to H.G. 3 G). The regenerated livers of Rag2−/−γc−/−KitW/Wv mice showed a similar vascular microstructure as WT controls 10 d after PHx, suggesting that liver regeneration was not impaired in Rag2−/−γc−/−KitW/Wv mice (Fig. I.3) Communication. The majority of previous studies investigated the contribution of BMDMCs in irradiation-conditioned bone marrow chimeric mice (Asahara et al., 1997; Mathews et al., 2004; Zhang et al., 2014). Neurosci., 20 December 2019 ... (Regierungspräsidium Karlsruhe, Germany) in accordance with the local Animal Welfare Act and the European Communities Council Directives (2010/63/EU and 2012/707/EU; Ref. Embryonic tissue was used for genotyping. 1 mo later, bone marrow chimeric mice (Fig. Likewise, resident ECs were previously reported to mediate adult neovascularization following cardiac injury (He et al., 2017) and during tumor progression (Peters et al., 2005; Purhonen et al., 2008). Quantitative PCR analysis revealed higher expression of Bax, an apoptotic activator, in livers of irradiated but not partially hepatectomized mice compared with nonirradiated and partially hepatectomized mice (Fig. In case of chemotherapy 150 l (0.5 mg/ml) cisplatin were injected intraperitoneally (i.p.) VI.1) Information about recurrence. In brief, female CB‐17 SCID mouse (7 weeks) were purchased from Charles River Laboratories (Arbresle, France) and inoculated subcutaneously with NCl … Brief Research Report ARTICLE Front. In concordance with the other two nonmyeloablative models, the data demonstrate that liver vasculature depended on differentiated resident ECs during PHx-induced liver regeneration. In contrast to most studies that use multiple cocaine-cue conditioning sessions, … Regierungspräsidium Karlsruhe Délivrance le oct. 2014. Quantitative PCR was performed with SYBR Green Master Mix (Invitrogen). Afterward, mice were bled at regular intervals, and plasma ALT/AST levels were monitored. Future studies involving stringent and mechanistic preclinical experimental approaches will need to address these questions to possibly establish a scientific rationale for bone marrow cell–involved stem cell therapies to interfere with liver dysfunction. A summary of the experimental procedure is depicted in Figure Figure1 1. Interestingly, a subset of liver ECs coexpressing CD157 and CD200 was recently identified as tissue-resident vascular endothelial stem cells that are capable of local clonal expansion and thereby can support neovascularization during liver repair (Wakabayashi et al., 2018). Primers (Bax: 5′-AGACAGGGGCCTTTTTGCTAC-3′, forward, and 5′-AATTCGCCGGAGACACTCG-3′, reverse; Icam1: 5′-CTGGATCTCAGGCCGCAAG-3′, forward, and 5′-TGTCGAGCTTTGGGATGGTAG-3′, reverse) were ordered from Sangon Biotech. BRIEF OVERVIEW OF FOREST MANAGEMENT IN BADEN-WÜRTTEMBERG. 3 D). Augustin); and the National Science Foundation of China (31571203 to J. Hu). }, B 462, Rettungstreppenhauserweiterung Tunnel Gernsbach. S2 A). WE ARE WHERE THE FORESTS ARE Landratsamt Alb-Donau-Kreis Schillerstrasse 30 . Scale bars, 100 µm. To induce liver regeneration, both mice of the operated pairs were subjected to two-thirds PHx. All animal studies were approved by the local ethics committee (Regierungspraesidium Karlsruhe, Germany), approval number 35-9185.81/G-203/10, date of approval 7 April 2011. In brief, after the incubation period with the test compounds, MTT-solution (10 mg/ml in PBS) was added (10 μl/well). Electrochemotherapy … Mice receiving 9 Gy whole-body irradiation in the presence of a liver shield had a strong reduction of GFP+ BMDMCs in their livers as compared with mice irradiated without a liver shield (Fig. All stainings were performed in 5% FCS on ice for 30 min with optimal dilutions of commercially prepared antibodies. We hypothesized that the observed DNA damage might cause EC apoptosis. S1 D). local Animal Care and Use Committee (Regierungspräsidium Karlsruhe, Germany). Dass nachweislich keine schweren Verfehlungen begangen wurden, die die Zuverlässigkeit als Bewerber in Frage stellen. To expand and mobilize endogenous BMDMCs, Neulasta (PEGylated G-CSF), a clinically approved agent for mobilizing hematopoietic progenitor cells (Hopman and DiPersio, 2014), was injected in WT mice on day 2 following PHx (Fig. On day 2 after PHx or sham operation, the NSG mice were injected intravenously with suspension of bone marrow cells (consisting of 20,000 LSK cells). See more of Europe Direct Karlsruhe im Regierungspräsidium on Facebook. Yet, when comparing the livers of PHx and sham-operated animals, there was no significant incorporation of YFP+ cells among total liver ECs (Fig. For transfer experiments, lymphocyte-deficient Values of P < 0.05 were considered statistically significant. Animal preparation and handling. Thus, the data from both chronic liver damage models coherently demonstrate that intact endothelium is self-sufficient for tissue repair. S2 F). For Video 1, a semiautomatic surface rendering module in Imaris (Bitplane) was used to create three-dimensional volumetric surface objects corresponding either to individual cells or to the liver vascular system. BRIEF OVERVIEW OF FOREST MANAGEMENT These two certificates have been awarded to the state operated ForstBW for their sustainable forestry management practices. Write a Review. However, in a recent randomized, controlled phase 2 trial involving 81 patients with compensated liver cirrhosis, administration of G-CSF alone or in combination with hematopoietic stem cell (HSC) infusion failed to improve liver function or to ameliorate fibrosis (Newsome et al., 2018). Likewise, the phase 2 REALISTIC trial with 81 randomly assigned liver cirrhosis patients concluded that the addition of G-CSF and stem cell infusion did not improve liver dysfunction as compared with standard care alone (Newsome et al., 2018). Sections were then incubated with primary antibodies overnight at 4°C, followed by fluorophore-conjugated secondary antibodies for 2 h at room temperature. All procedures were performed according to the Guide of the Care and Use of Laboratory Animals published by the National Academy of Sciences and were approved by the local authorities (Regierungspräsidium Karlsruhe AZ: 35-9185.81/G—19/12, 35-9185.81/G—229/12). Blood samples were taken from the Vena facialis after 4 hours of fasting during the day cycle. / B. Eng. Mice were kept at constant temperature (22 °C) on a 12-hour light/dark cycle and were fed with standard laboratory diet (2.98 kcal/g; Altromin, Hannover, Germany) and water ad libitum. Claim your business to immediately update business information, track page views, and more! The wreck rests several dozen … In brief, magnetic beads (Dynabeads; Dynal) were coated with anti-CD31 antibodies (Mec13.3; BD Biosciences). These definite experiments revealed that BMDMCs do not incorporate into the liver vasculature under nonvascular-damaging conditions. After hearing B, the Regierungspräsidium Karlsruhe (Regional Council, Karlsruhe, Germany) determined, by decision of 25 November 2014, adopted on the basis of Paragraph 6(5) of the FreizügG/EU in conjunction with Article 28(3)(a) of Directive 2004/38, that B had lost his right of entry to and residence in Germany. Finally, the percentage of GFP+ or YFP+ cells in the liver EC population was measured using either Beckman Coulter Cytoflex or BD Canto-II. Abiturprüfungen 2020: Im Regierungsbezirk Karlsruhe gehen 11.872 Schülerinnen und Schüler an den Start; Abiturientinnen und Abiturienten konnten aufgrund aktueller Corona … (D) Experimental outline for transplantation of LSK cells into Rag2−/−γc−/−KitW/Wv mice. doi: https://doi.org/10.1084/jem.20180008. By continuing to use our website, you are agreeing to, https://creativecommons.org/licenses/by-nc-sa/4.0/, Pumping blood with self-reliance and cooperation, https://doi.org/10.1016/j.stem.2017.07.004, https://doi.org/10.1016/j.exphem.2010.04.007, https://doi.org/10.1634/stemcells.22-6-994, https://doi.org/10.1126/science.275.5302.964, https://doi.org/10.1146/annurev-cellbio-092910-154002, https://doi.org/10.1016/j.jhep.2015.02.040, https://doi.org/10.1046/j.1365-2249.1998.00726.x, https://doi.org/10.1053/j.gastro.2009.05.009, https://doi.org/10.1146/annurev-immunol-032414-112019, https://doi.org/10.1016/j.blre.2014.01.001, https://doi.org/10.1016/j.mayocp.2013.10.023, https://doi.org/10.1016/j.stem.2018.07.011, https://doi.org/10.1016/S2468-1253(17)30326-6, https://doi.org/10.1371/journal.pone.0053719, https://doi.org/10.1016/j.stem.2018.01.010, https://doi.org/10.1097/MOH.0000000000000041, A novel antiangiogenic and vascular normalization therapy targeted against human CD160 receptor, The oxysterol–CXCR2 axis plays a key role in the recruitment of tumor-promoting neutrophils, Organ-specific lymphatic vasculature: From development to pathophysiology. How to Reach Us. All traced CD133+ cells were negative for CD45 and EC markers. Schuljahresbeginn 2020/2021 - Regierungspräsidium Karlsruhe informiert über Veränderungen bei Schülerzahlen, Unterrichtsversorgung und schulartbezogene Entwicklungen im Regierungsbezirk Karlsruhe. "button": "button-icon" Circulating liver EC progenitors were reported to express CD133 (Harb et al., 2009), and the infusion of CD133+ BMDMCs could accelerate liver regeneration (Wang et al., 2012). Zoomed-in images are shown on the right. Further, staining for the proliferation marker Ki67 demonstrated that the proliferating ECs in Para-WT mice were GFP– resident ECs (Fig. Two-tailed Student’s t test. Microarray data were normalized and analyzed with the Chipster software. Embryonic tissue was used for genotyping. Cell. Representative images of liver sections of bone marrow chimeric mice costained with GFP, CD45, and CD31 (EC-specific surface marker). Whole bone marrow cells were isolated from PanRosaYFP mice as described above. Surgical anaesthesia was induced by a short carbon dioxide narcosis followed by 40 mg/kg intramuscular ketamine (Ketanest ® S 25 mg/ml, Parke-Davis, Berlin, … In summary, using a wide array of lineage-tracing tools, the present study was aimed at unveiling the source of newly formed vessels during liver regeneration. Scale bars, 50 µm. Animal Handling . Regierungspräsidium Karlsruhe, Referat 47.2 – Baureferat Mitte Schlossplatz 4-6 76131 Karlsruhe Baden-Württemberg, Auskünfte über Vergabeunterlagen bis 23.1.2018 möglich. mRNA levels were normalized to β-actin expression. All animal experiments were approved by the local animal ethics committee (Regierungspräsidium Karlsruhe; Ministerium für Landwirtschaft, Umwelt und ländliche Räume, Kiel, Germany). — zur Ausführung von Leistungen in den letzten drei abgeschlossenen Geschäftsjahren, die mit der zu vergebenden Leistung vergleichbar sind. Thereby, it uniquely enabled us to trace ECs in newly formed blood vessels of the regenerating liver. In certain experiments, recombinant murine TNFα (R&D systems, Minneapolis, USA) was injected intrascrotally at a dose of 500 ng per mouse 3 hours before intravital microscopy. Search for other works by this author on: Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China, University of Chinese Academy of Sciences, Beijing, China, State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China, German Cancer Consortium, Heidelberg, Germany, Deutsche Forschungsgemeinschaft Claim This Business. Fig. In brief, inclusion criteria were study enrollment within 24 h after symptom onset. Our data clearly suggest that stem cell infusion/mobilization therapies do not physically contribute to the regeneration of liver vasculature in mice with healthy remaining vasculature. Augustin, and J. Hu wrote the manuscript. RM, GK, KK, and RP: data acquisition. Languages in which tenders or requests to participate may be submitted: Minimum time frame during which the tenderer must maintain the tender, Body responsible for mediation procedures, Service from which information about the review procedure may be obtained, https://www.vergabe24.de/vergabeunterlagen/54321-Tender-1602be37cb4-15c4279d45d073d4, Information on certificates required in public procurement in the EU. Approval for experiments involving animals was granted by the local Animal Welfare Committee (Regierungspraesidium Karlsruhe, Germany, reference numbers … Add photos. (A) Experimental outline of the parabiotic model. Not Now. We are most grateful for the excellent technical support of the Flow Cytometry, Genomics, Light Microscopy, and Laboratory Animal Facilities of the German Cancer Research Center and Interdisciplinary Research Center of Biology and Chemistry. — 4 x Wasserhaltung inkl. The procurement documents are available for unrestricted and full direct access, free of charge, at: Additional information can be obtained from the abovementioned address. BRIEF OVERVIEW OF FOREST MANAGEMENT These two certificates have been awarded to the state operated ForstBW for their sustainable forestry management practices. All animal experiments are approved by the legal authority on animal welfare (Regierungspräsidium Karlsruhe, G-256/15) and were in accordance with the guidelines issued by the Federation of European Laboratory Animal Science Associations (FELASA) 29.Prior to any micro-CT examinations, ten C57BL/6J mice were placed in an acrylic glass box and anesthesia was delivered via … After sedation with azaperon (5 mg/kg IM; Elanco, … Bisected kidney basins were used as enrichment. The number of GFP+ ECs in PHx mice was ∼1.8× higher as compared with the sham-operated mice (Fig. (A) Experimental outline of PHx-induced liver regeneration in C57BL/6N mice. Both expression systems allow for spatial and temporal control of gene activities, and, in the case of … "to" : ["more", "e-mail", "sms", "facebook","twitter","googleplus","linkedin"], All animal experiments were approved by the institutional and governmental Animal Care and Use Committees (IRCBC-2016-02 to J. Hu and G220/11, G213/17, and G219/17 to H.G. FORSTBW MANAGEMENT Headquarter Ministry of Rural Affairs and Consumer Protection Kernerplatz 10 . In brief, 2.5 × 10 6 END cells were incubated with 200 μM CoCl 2 for 4 h. Cells were cross-linked in 1% formaldehyde for 20 min at RT. Division of Vascular Oncology and Metastasis Research, German Cancer Research Center Heidelberg (DKFZ-ZMBH Alliance), Heidelberg, Germany, European Center for Angioscience, Medical Faculty Mannheim, Heidelberg University, Heidelberg, Germany, Faculty of Biosciences, Heidelberg University, Heidelberg, Germany. Schematic overview of in vivo experiments. H.G. However, neither LSK cells in the bone marrow nor mononuclear cells in the peripheral blood were YFP labeled (Fig. Livers were fixed in 4% PFA overnight. All stainings were performed in 5% FCS (in PBS) on ice for 30 min with optimal dilutions of commercially prepared antibodies. (C) The ratio of YFP+ ECs in the livers of sham-operated and PHx mice was analyzed by FACS (mean ± SD, n = 4–5 mice). Electrophysiological studies were performed in 17 anesthetized pigs of both sexes (<6 months of age; body weight 30–45 kg). However, following irradiation-induced EC damage, bone marrow–derived mononuclear cells were recruited and incorporated into the vasculature. Animals were … However, a subsequent randomized controlled trial involving 58 patients with decompensated alcoholic liver disease resulted in no additional benefit from autologous BMDMC infusion combined with standard medical therapy as compared with standard medical therapy alone (Spahr et al., 2013). K. Jiang and H. He contributed to the parabiosis experiments, cell isolation, and FACS analysis. In line with a previous report (Verbiest et al., 2016), the NSG mice successfully accepted the allograft, as there were ∼15% YFP+ cells among total circulating CD45+ cells in the peripheral blood on day 10 following PHx (Fig. The microarray data with the description are deposited under GEO accession no. Mice were bled, and plasma ALT/AST levels were monitored during the course of the experiment. We develop and publish International Standards. Nevertheless, they play a central role in maintaining tissue homeostasis (Hu et al., 2014; Rafii et al., 2016; Augustin and Koh, 2017). Total RNA was extracted from the livers of irradiated or hepatectomized mice using a Trizol isolation system (Thermo Scientific) according to the manufacturer’s instructions. This causes an early cytopathic effect on hepatocytes and a secondary adaptive immune response against infected hepatocytes during later stages of the experiment, as illustrated by enhanced plasma ALT/AST values (Fig. S2 contains validation data for different nonmyeloablative models employed to lineage trace liver ECs after PHx. "service" : "share", To fuse the blood circulation of two independent mice, female WT and CAG-GFP mice were subjected to parabiotic surgery as described previously (Kamran et al., 2013). 3 H). Dass sich das Unternehmen bei der Berufsgenossenschaft angemeldet hat. Next, VECad-CreERT2xRosa26-YFPfl/fl mice with labeled ECs were injected with 1011 viral particles of empty replication-deficient adenovirus. Further, flow cytometry analysis revealed no significant changes in the frequencies of YFP+ liver ECs between mice treated with either oil or CCl4 (Fig. Get Directions +49 721 9267404. B was therefore ordered to leave that Member State within one month of the entry into force … In brief, mice were anaesthetized with a mixture of ketamine (100 mg/kg body weight) and xylazine (10 mg/kg body weight). Regierungspräsidium Karlsruhe Délivrance le oct. 2014. Sections were blocked and permeabilized with PBS containing 10% normal donkey serum and 0.3% Triton for 2 h at room temperature. Log In. Thus, these data demonstrate that bone marrow–derived cells did not physically incorporate into the regenerating liver vasculature after PHx under nonvascular-damaging conditions. Dead cells were excluded by staining with Sytox Blue (Invitrogen). Two-tailed Student’s t test. Surprisingly, though, GFP+ ECs were also detectable in livers of sham-operated mice (Fig. Das … Der Antrag auf Entschädigung kann über die Seite www.ifsg-online.de direkt online gestellt werden. 1 F). Yet, the source of new endothelial cells (ECs) remains elusive. / FH / … Interestingly, Icam1 was highly upregulated in livers of irradiated mice as compared with the PHx group (Fig. Photos and Videos. 4 check-ins. This work was supported by grants from the Deutsche Forschungsgemeinschaft for projects SFB-873, “Maintenance and differentiation of stem cells in development and disease” (TP B6 to H.G. The institutional ethics committee (“Tierschutzausschuss”) of Heidelberg Pharma GmbH reviewed and enforced the general compliance with animal welfare of the facility. For FACS analysis, DAPI+ dead cells were first excluded, and CD45−CD31+CD146+ populations were defined as liver ECs. Whole thoracic irradiation was administrated to 8–10 weeks old female C57BL/6 mice as described previously. 72074 Tübingen Tel +49 7071 602-0 Mail … 4 C). All animal studies were performed in accordance with regulations for animal welfare and approved by the responsible agency (Regierungspraesidium Karlsruhe, Land Baden-Wuerttemberg, Karlsruhe, Germany) under the following numbers: G-264/12, G-75/13, G-29/15, G-179/16 (The last two digits represent the year of … All experiments were performed in accordance with the institutional guidance for the care and use of laboratory animals. The study was conducted in accordance with national guidelines for the use of experimental animals and the protocols were approved by the governmental committees (Regierungspraesidium Karlsruhe, Germany). The care and experimental use of animals were in accordance with institutional guidelines and in compliance with the Association for Research in Vision and Ophthalmology statement. 1 A; and Video 1), suggesting that bone marrow–recruited cells had incorporated into the liver vasculature independent of the PHx-induced regenerative burst, possibly as a result of irradiation-induced vascular damage. Fish Keeping, Embryo Handling and Drug Exposure. After hearing B, the Regierungspräsidium Karlsruhe (Regional Council, Karlsruhe, Germany) determined, by decision of 25 November 2014, adopted on the basis of Paragraph 6(5) of the FreizügG/EU in conjunction with Article 28(3)(a) of Directive 2004/38, that B had lost his right of entry to and residence in Germany. Briefly, liver ECs were isolated from irradiation-conditioned bone marrow chimeric mice for surface marker expression (DAPI− CD45− CD31+ CD146+). By analyzing the regeneration of the liver vasculature in irradiation-based myeloablative and nonmyeloablative bone marrow transplantation mouse models, we discovered that neoangiogenesis in livers with intact endothelium was solely mediated by proliferation of resident ECs. Thereafter, RNA was isolated with the Arcturus PicoPure RNA Isolation Kit (Life Technologies), and RNA quality and quantity were analyzed on an Agilent Bioanalyzer.