Bioactivity and receptor selectivity of the TNF muteins.

<p>Mouse embryonic fibroblasts (MEF) from TNFR1^−/−/TNFR2^−/− mice stably transfected with the chimeric receptors TNFR1-Fas or TNFR2-Fas (<b>A</b>) or Kym-1 cells (<b>B</b>) were stimulated with wildtype human TNF (huTNF; •), scTNF_R2 (▪) or TNC-scTNF_R2 (▴). Where indicated, MEF TNFR2-Fas were pretreated with the ligand/receptor stabilizing monoclonal antibody 80M2 (open symbols; 1 µg/ml; 30 min) before TNF treatment. Cell viability was determined by crystal violet staining after 24 hours (n = 3, shown are the mean values ± SEM).</p

TNC-scTNF_R2 induces formation of TNFR2 signaling complexes.

<p>(<b>A,B</b>) R2 MEF were transfected with pTRAF2-eGFP. After 24 hours, cells were incubated with or without 80M2 (1 µg/ml) for 5 minutes on ice and subsequently incubated with scTNF_R2 or TNC-scTNF_R2 (10 ng/ml) for 10 minutes at 37°C. Then cells were fixed with 4% PFA, permeabilized with 0.1% Triton X-100 and localization of huTNFR2 (<b>A</b>) or huTNF (<b>B</b>) was detected with specific antibodies and Alexa-Fluor546-labeled secondary antibodies. Cell nuclei were visualized using DAPI. Pictures are optical sections obtained by confocal fluorescence microscopy (bar = 10 µm). White arrows indicate areas of colocalization.</p

TNC-scTNF_R2 induces neuroregeneration after H₂O₂-induced oxidative stress.

<p>(<b>A</b>) Differentiated LUHMES cells were incubated with different concentrations of hydrogen peroxide (H₂O₂) for one hour. Then cells were washed with medium und cultivated for additional 24 hours. Cell viability was measured using the MTT assay (n = 3; shown are the mean values of triplicate determinations ± SEM). (<b>B–E</b>) LUHMES cells were stimulated with H₂O₂ (100 µM). After one hour the cells were washed with medium und regenerated for the indicated time intervals in medium with or without TNC-scTNF_R2 (100 ng/ml). (<b>B</b>) LUHMES cells were regenerated for 24 hours and cell viability was measured using the MTT assay (n = 3, shown are the mean values ± SEM). (<b>C</b>) Cells were regenerated for one or three hours, fixed with 4%PFA, permeabilized with 0.1% Triton-X100 and β-III-tubulin was detected with specific antibodies. Cell nuclei were visualized using DAPI. Pictures are projections of eight optical sections (0.4 µm; bar = 50 µm). (<b>D</b>) Number of cells was determined by counting the nuclei (DAPI staining). (<b>E</b>) Cells were regenerated for one hour and apoptotic cells were identified by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-FITC nick end labeling (TUNEL). (D,E) At least 10 different image sections containing a minimum of 500 cells were used to determine the number of total and TUNEL-positive cells. *p values less than 0.05 (** p-value less than 0.001) were considered to be significant (n = 2, shown are the mean values ± SD).</p

TNC-scTNF_R2 protects neurons against catecholaminergic cell death.

<p>(<b>A</b>) Differentiated LUHMES cells were incubated with different concentrations of 6-hydroxydopamine (6-OHDA) for 20 hours. Cell viability was measured using the MTT assay (n = 3; shown are the mean values of triplicate determinations ± SEM). (<b>B</b>) LUHMES cells were stimulated with 6-OHDA (32 µM). Cells were stimulated with TNC-scTNF_R2 (100 ng/ml) one, two or four hours after 6-OHDA addition and incubated for a total time period of 20 hours. Cell viability was measured using the MTT assay (n = 3, shown are the mean values ± SEM). *p values less than 0.05 (** p-value less than 0.001) were considered to be significant.</p

TNC-scTNF_R2 induces TNFR2 signaling in R2 MEF.

<p>(<b>A–C</b>) R2 MEF were stimulated with TNC-scTNF_R2 (20 ng/ml) for the indicated times. (<b>A</b>) TNFR2 was immunoprecipitated using MR2-1 antibodies and protein G agarose. The precipitates were analyzed by immunoblot analysis using anti-huTNFR2 (HP9003) and anti-TRAF2 antibodies (NB = non-bound; B = bound). (<b>B</b>) Localization of NFκB p65 was visualized via immunofluorescence microscopy as shown in the upper panel and the number of nuclei showing NFκB translocation was quantified. At least 200 cells per experiment were analyzed (n = 3; shown are the mean values ± SEM of percent NFκB positive nuclei; bar = 20 µm). (<b>C</b>) Phospho-Akt (Ser473) levels in cell lysates were analyzed using immunoblot analysis. Akt was used as a loading control. Representative blot and bar graph show the quantification of the phospho-Akt (Ser473) band. *p values less than 0.05 versus untreated cells were considered to be significant (n = 3; shown are the mean values ± SEM).</p

Genetic engineering of the TNFR2-selective TNF muteins.

<p>(<b>A,B</b>) Schematic representation of the TNF variants used in this study. CT: cys-tag, HT: his-tag; TNF: huTNFR2-specific (D143N/A145R) TNF module (aa 80–233); L: GGGGS-linker; TNC: trimerization domain of human tenascin C (aa 110–139). (<b>C</b>) Coomassie staining and (<b>D</b>) immunoblot analysis of scTNF_R2 (1) and TNC-scTNF_R2 (2). Purified TNF variants were analyzed by 8% SDS-PAGE under reducing (r.) or non-reducing (n.r.) conditions and either stained with Coomassie or immunoblotted with anti-his-tag antibodies. (<b>E</b>) TNF muteins were analyzed by HPLC size exclusion chromatography using a BioSep-Sec-2000 column. Peak positions of relevant standard proteins are indicated (200 kDa; 67 kDa and 29 kDa).</p

Comparison of the NFκB translocation after stimulation with scTNF_R2 or TNC-scTNF_R2.

<p>Kym-1 cells were stimulated with scTNF_R2 or TNC-scTNF_R2 (10 ng/ml) for 10, 30 or 60 minutes. Cells were fixed with 4% PFA, permeabilized with 0.1% Triton-X100 and stained with anti-NFκB p65 antibodies and Alexa-Fluor546-labeled secondary antibodies. Cell nuclei were visualized using DAPI. Shown are representative images at point of time 30 minutes (upper panel) and the quantification of the number of NFκB p65-positive nuclei. At least 200 cells were analyzed per condition. *p values less than 0.01 versus untreated controls were considered to be significant (n = 2; shown are the mean values ± SD; bar = 20 µm).</p

TNC-scTNF_R2-mediated neuroregeneration is dependent on PI3K-PKB/Akt signaling.

<p>(<b>A</b>) LUHMES cells were stimulated with LY294002 (25 µM) for either 24 hours or for 1 hour. If stimulated for 1 hour, medium was exchanged to differentiation medium and cells were cultivated for further 23 hours. Cell viability was measured using the MTT assay (n = 3; shown are the mean values ± SEM). (<b>B,C</b>) LUHMES cells were stimulated with hydrogen peroxide (H₂O₂; 100 µM) with or without LY294002 (25 µM). After one hour the cells were washed with medium und regenerated in medium with or without TNC-scTNF_R2 (100 ng/ml). (<b>B</b>) Cells were incubated for 24 hours and cell viability was measured using the MTT assay (n = 3, shown are the mean values ± SEM). (<b>C</b>) Cells were regenerated for one hour, fixed with 4% PFA and apoptotic cells were identified by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-FITC nick end labeling (TUNEL). At least 5 different image sections containing a minimum of 250 cells were used to determine the number of total and TUNEL-positive cells (n = 3, shown are the mean values ± SEM). **p values less than 0.001 were considered to be significant.</p

Additional file 1: of Baden Prevention and Reduction of Incidence of Postoperative Delirium Trial (PRIDe): a phase IV multicenter, randomized, placebo-controlled, double-blind clinical trial of ketamine versus haloperidol for prevention of postoperative delirium

SPIRIT checklist. (DOC 115 kb

The Smac mimetic SM83 sensitizes oncogenic K-Ras expressing CRC cells to Db_αEGFR-scTRAIL.

<p>(a–d) Caco-2tet Ras^G12V cells were seeded into 3D cultures in the presence of doxycycline. (a) Three days later, cultures were left untreated or treated with 5 µM SM83 or 20 µM Z-VAD for 1 h prior to addition of 1 nM Db_αEGFR-scTRAIL. Viability was measured 24 h later by MTT assay and normalized to the untreated control (n = 3). (b) 24 h after treatment, cells were fixed and stained for DNA strand breaks. Tunel-positive cells were counted (n = 2). (c) Three days post seeding, cultures were left untreated (ut) or treated with 5 µM SM83. Cells were recovered from the cultures 24 h later and lysates were analyzed by immunoblotting. Shown is one representative blot of two independent experiments. Tubulin was detected as a loading control. (d) Quantification of Western blots from (c). Protein levels were normalized to the corresponding tubulin control; levels in the untreated cultures were set as 1 (n = 2). (e, f) HCT-116 and LoVo cells were grown in 3D cultures for six days, and then fixed and stained for F-actin and DNA (DAPI) (scale bar: 20 µm) (top panels). Three days post seeding, cultures were left untreated or pretreated with 5 µM SM83 prior to addition of 0.05 nM Db_αEGFR-scTRAIL. Viability was measured 24 h later by MTT assay and normalized to the untreated control (bottom panels) (n = 3).</p