11 research outputs found
Reduced indoleamine 2,3-dioxygenase enzyme activity and expression under hypoxic conditions.
<p>(A–C) Determination of the kynurenine production in different cells after IDO induction by IFN-γ <i>in vitro</i>. Glioblastoma cells 86HG39 (A), HeLa cells (B) or human foreskin fibroblasts (HFF) (C) were stimulated with IFN-γ (500 U/mL) or left unstimulated in IMDM cell culture medium containing additional L-tryptophan (100 µg/mL). The 72 h incubation period was carried out at normoxia (20% O<sub>2</sub>), (10% O<sub>2</sub>) or hypoxia (1% O<sub>2</sub>). The kynurenine content in the cell culture supernatants was determined by optical density at 492 nm +/− SEM, using Ehrlichs reagent. A significant inhibition of kynurenine production as compared to the stimulated normoxia positive control is marked with asterisks, n = 3. (D-F) Western Blot analysis of 86HG39 (D), HeLa (E) or HFF lysates (F) stimulated with IFN-γ (0–500 U/mL) under normoxia or hypoxia for 24 h. Protein expression of IDO, phosphorylated STAT1 (pSTAT1) and β-actin was detected. (G-L) Densitometric analysis of the Western Blots shown in D–F. Relative protein expression of IDO and pSTAT1 with reference to the β-actin protein expression.</p
Tanycytes control the hormonal output of the hypothalamic-pituitary-thyroid axis
The hypothalamic-pituitary-thyroid (HPT) axis regulates a wide range of physiological processes. Here the authors show that hypothalamic tanycytes play a role in the homeostatic regulation of the HPT axis; activation of TRH signaling in tanycytes elevates their intracellular Ca2+ via Gαq/11 pathway, ultimately resulting in reduced TRH release into the pituitary vessels
IDO-mediated antimicrobial effect is lost under low oxygen concentrations.
<p>(A) Growth of <i>Staphylococcus aureus</i> in supernatants of unstimulated or IFN-γ stimulated (500 U/mL) human fibroblasts, incubated for 72 h under different oxygen concentrations (1%–20% O<sub>2</sub>). Bacterial growth was determined 24 h after infection by optical density at 620 nm +/− SEM. (B) Proliferation of <i>Toxoplasma gondii</i> in HFF cells that have been pre-stimulated with 500 U/mL IFN-γ or not for 72 h under different oxygen concentrations (1%–20% O<sub>2</sub>). Then the cells were infected with the parasites and the Toxoplasma proliferation was determined after 48 h by the incorporation of <sup>3</sup>H-uracil. (C) Replication of herpes simplex virus type 1 (HSV1) in pre-stimulated (500 U/mL IFN-γ or not) HFF cells that have been incubated for 72 h under normoxia (20% O<sub>2</sub>) or hypoxia (1% O<sub>2</sub>). After pre-stimulation cells were infected with HSV1 and viral replication was detected after additional 72 h via real-time PCR. A significant inhibition of bacterial, parasitic and viral growth, respectively, as compared to the stimulated normoxia positive control is marked with an asterisk (*), n = 3.</p
Inhibition of indoleamine 2,3-dioxygenase enzyme activity by different inhibitors.
<p>(A) Determination of the kynurenine production in HFF cells after IDO induction by IFN-γ (100 U/mL). The cells were incubated for 72 h under normoxia (20% O<sub>2</sub>) or hypoxia (1% O<sub>2</sub>) and treated with different amounts of the JAK2 inhibitor BSK805 (0–2 µM). (B) Kynurenine production of HFF cells after IDO induction by IFN-γ (100 U/mL). The cells have been incubated for 72 h under normoxia (20% O<sub>2</sub>) or hypoxia (1% O<sub>2</sub>) with different amounts of the proteasome inhibitor MG-132 (0–1 µM) or the sumoylation inhibitors Anancardic Acid (0–10 µM) or Ginkgolic Acid (0–10 µM). The kynurenine content in the cell culture supernatants was determined by optical density at 492 nm +/− SEM, using Ehrlichs reagent. A significant inhibition of kynurenine production as compared to the stimulated normoxia positive control is marked with asterisks. In B the addition of inhibitors did not result in a significant increase of the kynurenine production in cells incubated under hypoxia, this is marked by n.s. (not significant), n = 3.</p
IDO-mediated immunoregulatory effect is lost under hypoxia.
<p>(A, B) Co-stimulation assay of 1.5×10<sup>5</sup> PBMC and 0–10000 HFF cells/well. T-cells were activated by the anti-CD3 antibody OKT3. The T-cell proliferation in the absence (A) or presence (B) of supplemented tryptophan was determined by incorporation of [<sup>3</sup>H] thymidine. Data are given as T cell proliferation in % of the positive control (stimulated T cells under normoxia) +/− SEM. A significant increase of T-cell proliferation as compared to the positive normoxia control is marked with an asterisks, n = 3.</p
IFN-γ signalling and cell survival under different oxygen conditions.
<p>(A) FACS analysis of unstimulated or IFN-γ stimulated (100 U/mL) HeLa cells that have been incubated for 72 h under normoxia (20% O<sub>2</sub>) or hypoxia (1% O<sub>2</sub>) in cell culture medium with supplemental L-tryptophan (100 µg/mL). Cells were stained for HLA-DR. (B-D) Cell viability assay. Indicated cell numbers of 86HG39 glioblastoma cells (B), HeLa cells (C) or human foreskin fibroblasts (HFF; D) were incubated under normoxia or hypoxia (24–72 h). Then alamarBlue was added to the cells and the reducing power of living cells in the samples was measured quantitatively via absorbance at OD<sub>570</sub>/OD<sub>600</sub>+/− SD. Data of one representative experiment, performed in triplicates. (E) FACS analysis of unstimulated or IFN-γ stimulated (100 U/mL) HeLa cells, incubated for 72 h under normoxia, hypoxia or anoxia in cell culture medium with supplemental L-tryptophan (100 µg/mL). Cells were stained for DAPI as indicator for cell survival. PFA-fixed cells served as positive control for cell death. (F) Kynurenine amount in supernatants of unstimulated or IFN-γ stimulated (500 U/mL) HeLa cells in the presence of different amounts of L-tryptophan (0, 6 or 50 µg/mL). Cells were incubated for 72 h in normoxia or hypoxia and subsequently reoxygenated for 48 h in normoxia. Then the kynurenine content in the cell culture supernatants was determined by optical density at 492 nm +/− SEM, using Ehrlichs reagent. A significant inhibition of kynurenine production as compared to the stimulated normoxia positive control is marked with asterisks, n = 3.</p
Human Engineered Heart Tissue: Analysis of Contractile Force
Analyzing contractile force, the most important and best understood function of cardiomyocytes in vivo is not established in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). This study describes the generation of 3D, strip-format, force-generating engineered heart tissues (EHT) from hiPSC-CM and their physiological and pharmacological properties. CM were differentiated from hiPSC by a growth factor-based three-stage protocol. EHTs were generated and analyzed histologically and functionally. HiPSC-CM in EHTs showed well-developed sarcomeric organization and alignment, and frequent mitochondria. Systematic contractility analysis (26 concentration-response curves) reveals that EHTs replicated canonical response to physiological and pharmacological regulators of inotropy, membrane-and calcium-clock mediators of pacemaking, modulators of ion-channel currents, and proarrhythmic compounds with unprecedented precision. The analysis demonstrates a high degree of similarity between hiPSC-CM in EHT format and native human heart tissue, indicating that human EHTs are useful for preclinical drug testing and disease modeling
Differentiation of cardiomyocytes and generation of human engineered heart tissue
Since the advent of the generation of human induced pluripotent stem cells (hiPSCs), numerous protocols have been developed to differentiate hiPSCs into cardiomyocytes and then subsequently assess their ability to recapitulate the properties of adult human cardiomyocytes. However, hiPSC-derived cardiomyocytes (hiPSC-CMs) are often assessed in single-cell assays. A shortcoming of these assays is the limited ability to characterize the physiological parameters of cardiomyocytes, such as contractile force, due to random orientations. This protocol describes the differentiation of cardiomyocytes from hiPSCs, which occurs within 14 d. After casting, cardiomyocytes undergo 3D assembly. This produces fibrin-based engineered heart tissues (EHTs) - in a strip format - that generate force under auxotonic stretch conditions. 10-15 d after casting, the EHTs can be used for contractility measurements. This protocol describes parallel expansion of hiPSCs; standardized generation of defined embryoid bodies, growth factor and small-molecule-based cardiac differentiation; and standardized generation of EHTs. To carry out the protocol, experience in advanced cell culture techniques is required
Author Correction: Differentiation of cardiomyocytes and generation of human engineered heart tissue (Nature Protocols, (2017), 12, 6, (1177-1197), 10.1038/nprot.2017.033)
Please note that since the original publication of this paper, the author David Letuffe-Brenière has changed his name to David Brenière-Letuff