Phenotypic and functional characterization of primary murine endothelial cells after in vivo irradiation

The inner cellular lining of all blood vessels consists of a monolayer of endothelial cells (ECs) that have a low proliferation rate in healthy tissue. Under pathological conditions such as wound healing, inflammation and in growing tumors, the proliferation rate of ECs is elevated. Radiotherapy is commonly used for the local control of solid tumors. During radiotherapy of patients with thoracic tumors, the surrounding healthy tissue including ECs of heart and lung may become damaged which in turn can change the proliferation rate of these resting ECs. This damage may increase the risk to develop cardiac diseases in patients after thoracic irradiation at later time-points. It is hypothesized that the protein profile of resting and growing primary ECs is different. To address this question, viable primary ECs are required. Established methods allow the isolation of ECs only from very young mice at low yields and purities. The analysis of late radiation-induced effects on primary ECs requires the availability of primary ECs from old mice. I succeeded to establish a novel method for the isolation of viable primary ECs at high purity from non-proliferating (heart from young and old mice), proliferating benign (repair blastema) and malignant (tumor) tissues at high yields. These ECs were characterized phenotypically, functionally and with respect to their gene expression profiling under static and physiological flow conditions. The expression density of proliferation markers such as endoglin and VE-cadherin is higher on isolated ECs of proliferating tissues from repair blastema and tumor compared to non-proliferating normal tissues from heart and lung. The expression density of the progenitor marker mucosialin is elevated on tumor-derived ECs, but not on those of repair blastemas. The inflammatory markers PECAM-1, ICAM-1 and ICAM-2 were found to be elevated on ECs of repair blastema and tumor compared to ECs from heart and lung. Further, I could show that tumor ECs are larger, have a significantly higher migration capacity and distribute in a more chaotic pattern in cell culture compared to ECs derived from normal tissues. Tube formation assays showed that tumor ECs have a smaller number of branching points and loops compared to that of normal ECs. In contrast to normal tissue ECs, tumor-derived ECs show no tendency to align under flow conditions. The results suggest that increased expression of surface molecules on ECs in proliferating tissues contributes to a loss of EC function that might be responsible for a chaotic tumor vasculature. In contrast to the short-term effects in tumors and repair blastemas induced by proliferation, irradiation can cause long-term effects in heart and lung ECs. Local thorax irradiation of mice resulted in a temporary and differential up-regulation of proliferation markers such as HCAM, integrin β3, endoglin, VE-cadherin and VEGFR-2 on ECs after 8 Gy at later time-points. The progenitor marker mucosialin is increased on lung ECs 15 to 20 weeks after irradiation. Inflammatory markers such as PECAM-1, ICAM-1, ICAM-2 and VCAM-1 started to increase 10 weeks after thorax irradiation with 8 Gy. Interestingly, ICAM-1 and VCAM-1 remained up-regulated even 20 weeks after thorax irradiation on heart and lung ECs. The persistent increase of both markers ICAM-1 and VCAM-1 after irradiation may suggest a predisposition for the development of atherosclerotic plaques in heart and lung ECs at later time points. Shear stress on ECs induced by blood flow is disturbed in heart capillaries with atherosclerotic plaques as well as in tumor vasculature. mRNA expression analysis of genes from heart ECs under normal flow conditions and pathological static conditions in vitro show significant changes related to extracellular organisation, cell membrane function, signaling, hemostasis, metabolism and smooth muscle contraction. Moreover, it was observed that mRNA expression of the inflammation markers Pecam1, Icam1 and Icam2 was higher on heart ECs under static conditions. The protein expression of these inflammatory markers was also elevated on ECs from pathologic conditions. These results provide the basis for subsequent investigations on the role of irradiation on gene expression profiles of ECs derived from normal and tumor tissues

Phenotypic and functional characterization of primary murine endothelial cells after in vivo irradiation

The inner cellular lining of all blood vessels consists of a monolayer of endothelial cells (ECs) that have a low proliferation rate in healthy tissue. Under pathological conditions such as wound healing, inflammation and in growing tumors, the proliferation rate of ECs is elevated. Radiotherapy is commonly used for the local control of solid tumors. During radiotherapy of patients with thoracic tumors, the surrounding healthy tissue including ECs of heart and lung may become damaged which in turn can change the proliferation rate of these resting ECs. This damage may increase the risk to develop cardiac diseases in patients after thoracic irradiation at later time-points. It is hypothesized that the protein profile of resting and growing primary ECs is different. To address this question, viable primary ECs are required. Established methods allow the isolation of ECs only from very young mice at low yields and purities. The analysis of late radiation-induced effects on primary ECs requires the availability of primary ECs from old mice. I succeeded to establish a novel method for the isolation of viable primary ECs at high purity from non-proliferating (heart from young and old mice), proliferating benign (repair blastema) and malignant (tumor) tissues at high yields. These ECs were characterized phenotypically, functionally and with respect to their gene expression profiling under static and physiological flow conditions. The expression density of proliferation markers such as endoglin and VE-cadherin is higher on isolated ECs of proliferating tissues from repair blastema and tumor compared to non-proliferating normal tissues from heart and lung. The expression density of the progenitor marker mucosialin is elevated on tumor-derived ECs, but not on those of repair blastemas. The inflammatory markers PECAM-1, ICAM-1 and ICAM-2 were found to be elevated on ECs of repair blastema and tumor compared to ECs from heart and lung. Further, I could show that tumor ECs are larger, have a significantly higher migration capacity and distribute in a more chaotic pattern in cell culture compared to ECs derived from normal tissues. Tube formation assays showed that tumor ECs have a smaller number of branching points and loops compared to that of normal ECs. In contrast to normal tissue ECs, tumor-derived ECs show no tendency to align under flow conditions. The results suggest that increased expression of surface molecules on ECs in proliferating tissues contributes to a loss of EC function that might be responsible for a chaotic tumor vasculature. In contrast to the short-term effects in tumors and repair blastemas induced by proliferation, irradiation can cause long-term effects in heart and lung ECs. Local thorax irradiation of mice resulted in a temporary and differential up-regulation of proliferation markers such as HCAM, integrin β3, endoglin, VE-cadherin and VEGFR-2 on ECs after 8 Gy at later time-points. The progenitor marker mucosialin is increased on lung ECs 15 to 20 weeks after irradiation. Inflammatory markers such as PECAM-1, ICAM-1, ICAM-2 and VCAM-1 started to increase 10 weeks after thorax irradiation with 8 Gy. Interestingly, ICAM-1 and VCAM-1 remained up-regulated even 20 weeks after thorax irradiation on heart and lung ECs. The persistent increase of both markers ICAM-1 and VCAM-1 after irradiation may suggest a predisposition for the development of atherosclerotic plaques in heart and lung ECs at later time points. Shear stress on ECs induced by blood flow is disturbed in heart capillaries with atherosclerotic plaques as well as in tumor vasculature. mRNA expression analysis of genes from heart ECs under normal flow conditions and pathological static conditions in vitro show significant changes related to extracellular organisation, cell membrane function, signaling, hemostasis, metabolism and smooth muscle contraction. Moreover, it was observed that mRNA expression of the inflammation markers Pecam1, Icam1 and Icam2 was higher on heart ECs under static conditions. The protein expression of these inflammatory markers was also elevated on ECs from pathologic conditions. These results provide the basis for subsequent investigations on the role of irradiation on gene expression profiles of ECs derived from normal and tumor tissues

Immunotherapeutic targeting of membrane Hsp70-expressing tumors using recombinant human granzyme B

Background: We have previously reported that human recombinant granzyme B (grB) mediates apoptosis in membrane heat shock protein 70 (Hsp70)-positive tumor cells in a perforin-independent manner

Irradiation-Induced Up-Regulation of HLA-E on Macrovascular Endothelial Cells Confers Protection against Killing by Activated Natural Killer Cells

BACKGROUND: Apart from the platelet/endothelial cell adhesion molecule 1 (PECAM-1, CD31), endoglin (CD105) and a positive factor VIII-related antigen staining, human primary and immortalized macro- and microvascular endothelial cells (ECs) differ in their cell surface expression of activating and inhibitory ligands for natural killer (NK) cells. Here we comparatively study the effects of irradiation on the phenotype of ECs and their interaction with resting and activated NK cells. METHODOLOGY/PRINCIPAL FINDINGS: Primary macrovascular human umbilical vein endothelial cells (HUVECs) only express UL16 binding protein 2 (ULBP2) and the major histocompatibility complex (MHC) class I chain-related protein MIC-A (MIC-A) as activating signals for NK cells, whereas the corresponding immortalized EA.hy926 EC cell line additionally present ULBP3, membrane heat shock protein 70 (Hsp70), intercellular adhesion molecule ICAM-1 (CD54) and HLA-E. Apart from MIC-B, the immortalized human microvascular endothelial cell line HMEC, resembles the phenotype of EA.hy926. Surprisingly, primary HUVECs are more sensitive to Hsp70 peptide (TKD) plus IL-2 (TKD/IL-2)-activated NK cells than their immortalized EC counterpatrs. This finding is most likely due to the absence of the inhibitory ligand HLA-E, since the activating ligands are shared among the ECs. The co-culture of HUVECs with activated NK cells induces ICAM-1 (CD54) and HLA-E expression on the former which drops to the initial low levels (below 5%) when NK cells are removed. Sublethal irradiation of HUVECs induces similar but less pronounced effects on HUVECs. Along with these findings, irradiation also induces HLA-E expression on macrovascular ECs and this correlates with an increased resistance to killing by activated NK cells. Irradiation had no effect on HLA-E expression on microvascular ECs and the sensitivity of these cells to NK cells remained unaffected. CONCLUSION/SIGNIFICANCE: These data emphasize that an irradiation-induced, transient up-regulation of HLA-E on macrovascular ECs might confer protection against NK cell-mediated vascular injury

Global prevalence and genotype distribution of hepatitis C virus infection in 2015 : A modelling study

Publisher Copyright: © 2017 Elsevier LtdBackground The 69th World Health Assembly approved the Global Health Sector Strategy to eliminate hepatitis C virus (HCV) infection by 2030, which can become a reality with the recent launch of direct acting antiviral therapies. Reliable disease burden estimates are required for national strategies. This analysis estimates the global prevalence of viraemic HCV at the end of 2015, an update of—and expansion on—the 2014 analysis, which reported 80 million (95% CI 64–103) viraemic infections in 2013. Methods We developed country-level disease burden models following a systematic review of HCV prevalence (number of studies, n=6754) and genotype (n=11 342) studies published after 2013. A Delphi process was used to gain country expert consensus and validate inputs. Published estimates alone were used for countries where expert panel meetings could not be scheduled. Global prevalence was estimated using regional averages for countries without data. Findings Models were built for 100 countries, 59 of which were approved by country experts, with the remaining 41 estimated using published data alone. The remaining countries had insufficient data to create a model. The global prevalence of viraemic HCV is estimated to be 1·0% (95% uncertainty interval 0·8–1·1) in 2015, corresponding to 71·1 million (62·5–79·4) viraemic infections. Genotypes 1 and 3 were the most common cause of infections (44% and 25%, respectively). Interpretation The global estimate of viraemic infections is lower than previous estimates, largely due to more recent (lower) prevalence estimates in Africa. Additionally, increased mortality due to liver-related causes and an ageing population may have contributed to a reduction in infections. Funding John C Martin Foundation.publishersversionPeer reviewe

European New Car Assessment Programme (Euro NCAP)

Um die Automobilhersteller zu animieren, mehr als die gesetzlich geforderte Sicherheit anzubieten, haben strengere Versuche im Rahmen des Verbraucherschutzes in den letzten Jahren nicht nur in den USA, sondern auch in Europa deutlich an Bedeutung zugenommen. Besonders Initiativen aus England ist es zu verdanken, dass sich heute die Testverfahren nach dem sogenannten Euro NCAP, dem European New Car Assessment Programme, durchgesetzt haben. Diese Entwicklung wurde auch von der Europäischen Kommission unterstützt. Ziel des Euro NCAP ist es, die unabhängige und objektive Bewertung des Sicherheitsniveaus von Fahrzeugen zu einer transparenten und leicht verständlichen Verbraucherberatung zu fördern. Weiterhin sollen objektive Bewertungsverfahren entwickelt werden, um die Fahrzeughersteller zu ermutigen, die Fahrzeuge sicherer zu machen. Im Beitrag wird auf die Struktur des Euro NCAP sowie auf seine Arbeitsweise eingegangen. Vorgestellt werden ferner bisherige Testphasen mit Fahrzeugen deutscher Hersteller oder Tochtergesellschaften sowie deren Bewertung

Tests on the crabbed barrier

Bei der Untersuchung des Insassenschutzes von Pkw's bei Seitenkollisionen wurden als seitlich auftreffende Fahrzeuge neben Pkw auch unterschiedliche Barrierenformen verwendet. Um die Zahl der Prüfungen im Typgenehmigungsverfahren für neue Pkw-Modelle gering zu halten, wurde von der europäischen Automobilindustrie ein Vorschlag zur Harmonisierung europäischer und amerikanischer Lösungen entwickelt. Es wurden zwei Versuchsserien zur Beurteilung des Quergeschwindigkeitseinflusses mit Fahrzeugen europäischer Größenverhältnisse durchgeführt sowie eine Serie mit einer Versuchskonfiguration, die zur Harmonisierung vorgeschlagen wurde. Die Untersuchungsergebnisse zeigen, dass die Kombination einer mobilen deformierbaren Barriere mit Bewegungsparametern zur Simulation eines Querbewegungseinflusses durch kinematische Umkehrung am gestoßenen Fahrzeug nur zu einem geringen Teil die gleiche Belastung bewirkt, wie bei der Kollision mit zwei bewegten Fahrzeugen.For some years now, the Federal Highway Research Institute (BASt) has been carrying out work on the protection of private car passengers in lateral collisions. In addition to private cars, various barrier forms have been used as side-striking vehicles in a number of research projects with various objectives. The BASt has also played e considerable role in the development and validation of the EEVC barrier face. Efforts in Europe and the U.S.A. to improve the protection of private car passengers in lateral collisions have led to the separate develop-ment of lateral collision test procedures. The European solutions (CCMC and EEVC) are very similar with respect to the test tool, test parameters and evaluation and represent practicable test procedures. The American proposal not only differs from the European solutions with respect to the design of the test tool (mass and width of the barrier front), but it also considers the proper motion of the side-struck vehicle during an angular collision. In an effort to keep down the number of tests in the type approval test procedure for net/ private cars, a proposal for harmonisation which contains elements of both the European and American solutions has been elaborated and put up for discussion by the European car industry. In addition to considering data from tests to evaluate the European test proposals, this investigation also conducted two series of tests with vehicles of the size driven in Europe (VW Golf, DB W 123) to determine the influence of transversal velocity, and a series with a test configuration suggested for the harmonisation process. The tests were evaluated with regard to the load on the vehicles (measurement of acceleration and deformation) and on the dummies (measurement of the acceleration in the head, chest and pelvis) and the results then compared with one another. Compared with the tests with a stationary vehicle, the tests with moving vehicles produced a lower deformation and, as a function of the differences in mass, a higher load on the driver dummy in the side-struck vehicle. A direct comparison of the tests with the EEVC barrier with impact angles of 90-° and 63-° revealed considerable differences in the kinematic behaviour of the vehicles, similar deformations to the struck vehicle and a considerably lower load on the front dummy. If one considers the results and the differences which came to light, it is possible to establish that combining a mobile deformable barrier with the movement parameters for simulating the influence of transversal movement by kinematic inversion (Umkehrung) on the struck vehicle produces the same load as a collision between two moving vehicles in only a few cases. Simulation of the transversal movement is not essential for taking into account the peculiarities of a real lateral collision, i.e. for adapting the test procedure; this can also be achieved by altering other parameters. A useful - and the easiest - measure involves increasing the ground clearance. The test can also be made "harder" by increasing the test velocity or mass or by partially increasing the rigidity of the deformation elements, i.e. the test can be adjusted to the higher dummy loads experienced during a lateral collision between two moving vehicles. The necessity to make the test "harder" has to be proofed in comparable tests with the EUROSID which is now in development because in the here described tests Hybrid II-dummies were used