Effect of poly (I:C) and HCV RNA containing cryoprecipitates on the expression of TNFα and TNF receptors; effect of TNFα on the expression of TNF receptors and viral receptors.

<p>HMEC were stimulated without (basal) and with poly (I:C) (10 µg/ml) for different time intervals (3, 6, 9, 12, 24, 48 hours) and expression of TNFα (A), TNF receptor 1 (TNFR1) (B) and TNF receptor 2 (TNFR2) (C) was analyzed by RT-PCR. (D, E) HMEC were transfected with siRNA for TLR3, RIG-I and MDA5 as well as unspecific RNA as negative control as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113351#s2" target="_blank">Materials and Methods</a> and stimulated with poly (I:C) (10 µg/ml) for 12 hours. Expression of TNFα (D) and TNFR2 (E) was analyzed by RT-PCR. Protein expression of TNFR2 after different time intervals of poly (I:C) stimulation (3, 6, 9, 12, 48 hours) was confirmed by western blot (F). HMEC were stimulated with different concentrations (100×10⁶ geq/ml, 200×10⁶ geq/ml) of HCV RNA containing cryoprecipitates for 12 hours and mRNA expression of TNFR2 (G) was analyzed by RT-PCR. HMEC were stimulated with TNFα for different time intervals (3, 6, 9, 12, 24 hours) and expression of TNFα (H), TNFR1 (I), TNFR2 (J), TLR3 (K), RIG-I (L) and MDA5 (M) was analyzed by RT-PCR. Results are given as means ± SD of three experiments done in parallel for each condition and rRNA served as the reference gene. Comparable results were obtained in two series of independent experiments. Statistically significant differences to the control are depicted with *  = p≤0.05.</p

Effect of HCV RNA containing cryoprecipitates on the expression of cytokines, chemokines and adhesion molecules.

<p>HMEC were stimulated with different concentrations (100×10⁶ geq/ml, 200×10⁶ geq/ml) of HCV RNA containing cryoprecipitates (HCV) for 12 hours and mRNA expression of selected cytokines and chemokines IL-6 (A), IL-8 (C), IP-10 (E) and adhesion molecule ICAM-1 (G) was analyzed by RT-PCR. Protein synthesis of IL-6 (B), IL-8 (D) and IP-10 (F) was also confirmed by ELISA. Results are given as means ± SD of three experiments done in parallel for each condition and rRNA served as the reference gene. Comparable results were obtained in two series of independent experiments. Statistically significant differences to the control are depicted with *  = p≤0.05.</p

Effect of poly (I:C) on vascular inflammation in vivo.

<p>C57Bl/6 mice were systemically treated with poly (I:C) (200 µg i.p.) and white blood cell counts were perfomed after 48 hours (A, B). At the same time leukocyte rolling (C) and adhesion (D) in vivo as well as extravasation of leukocytes (E) were investigated in postcapillary venules of the mouse cremaster muscle by intravital microscopy or in cremaster muscle whole mounts respectively. After repeated ploly (I:C) treatment (100 µg every other day for 2 weeks) vascular inflammation was investigated in lung tissue as described (F) and spleen weight was measured (G). Results are given as means ± SEM (n = 4–6 animals). Statistically significant differences to the control are depicted with *  = p≤0.05.</p

Amplification of poly (I:C)-mediated induction of cytokines, chemokines and adhesion molecules by TNFα.

<p>HMEC were grown in culture medium for 24 hours and additionally incubated in medium (basal) for different time intervals (6, 12, 24 hours) or grown in culture medium for 24 hours and additionally incubated with TNFα (TNFα) for different time intervals (6, 12, 24 hours) or incubated with poly (I:C) for 24 hours and then grown in medium (poly (I:C)) or stimulated with TNFα (poly (I:C) + TNFα) for different time intervals (6, 12, 24 hours). mRNA expression of selected cytokines and chemokines IL-6 (A), IL-8 (C), IP-10 (E), RANTES (G), MCP-1 (H), IFN-β (I), MCSF (J), ICAM-1 (K) and VCAM-1 (L) was analyzed by RT-PCR. Protein synthesis of selected targets IL-6 (B), IL-8 (D) and IP-10 (F) was also confirmed by ELISA. Results are given as means ± SD of three experiments done in parallel for each condition and rRNA served as the reference gene. Comparable results were obtained in two series of independent experiments. Statistically significant differences to the control are depicted with * = p≤0.05, ** = p≤0.01.</p

Role of TNFR2 in poly (I:C) induced vascular inflammation.

<p>WT and TNFR2^-/- mice were sham treated or treated with poly (I:C) 200 µg i.p. as described and expression of TLR3 and TNFR2 was analyzed by RT-PCR. Expression of TLR3 (A) and TNFR2 (B) was measured in lung, kidney, aorta and cremaster muscle tissue after 48 hours. At the same time point white blood cell counts were performed and effects of poly (I:C) on trauma-induced leukocyte rolling and adherence in vivo were investigated in cremaster muscle venules by intravital microscopy (C–E). Statistically significant differences to the control are depicted with *  = p≤0.05.</p

Age-dependent differences in DNA damage after in vitro CT exposure

<p><b>Purpose:</b> Age dependent radiation sensitivity for DNA damage after in vitro blood exposure by computer tomography (CT) was investigated.</p> <p><b>Materials and methods:</b> Radiation biomarkers (dicentrics and gammaH2AX) in blood samples of newborns, children under five years and adults after sham exposure (0 mGy), low-dose (41 mGy) and high-dose (978 mGy) in vitro CT exposure were analyzed.</p> <p><b>Results:</b> Significantly higher levels of dicentric induction were found for the single and combined newborns/children group compared to adults, by a factor of 1.48 (95% CI 1.30–1.68), after exposure to 978 mGy. Although a significant dose response for damage induction and dose-dependent repair was found, the gammaH2AX assay did not show an age-dependent increase in DNA damage in newborns/children compared to adults. This was the case for the gammaH2AX levels after repair time intervals of 30 minutes and 24 hours, after correcting for the underlying background damage. For the low dose of 41 mGy, the power of the dicentric assay was also not sufficient to detect an age-dependent effect in the sample size investigated.</p> <p><b>Conclusion:</b> A 1.5-fold increased level of dicentric aberrations is detected in newborns and children under five years after 1 Gy radiation exposure.</p