Interferon-gamma impairs maintenance and alters hematopoietic support of bone marrow mesenchymal stromal cells

Bone marrow (BM) mesenchymal stromal cells (MSCs) provide microenvironmental support to hematopoietic stem and progenitor cells (HSPCs). Culture-expanded MSCs are interesting candidates for cellular therapies due to their immunosuppressive and regenerative potential which can be further enhanced by pretreatment with interferon-gamma (IFN-γ). However, it remains unknown whether IFN-γ can also influence hematopoietic support by BM-MSCs. In this study, we elucidate the impact of IFN-γ on the hematopoietic support of BM-MSCs. We found that IFN-γ increases expression of interleukin (IL)-6 and stem cell factor by human BM-MSCs. IFN-γ-treated BM-MSCs drive HSPCs toward myeloid commitment in vitro, but impair subsequent differentiation of HSPC. Moreover, IFN-γ-ARE-Del mice with increased IFN-γ production specifically lose their BM-MSCs, which correlates with a loss of hematopoietic stem cells\u27 quiescence. Although IFN-γ treatment enhances the immunomodulatory function of MSCs in a clinical setting, we conclude that IFN-γ negatively affects maintenance of BM-MSCs and their hematopoietic support in vitro and in vivo

ATP Changes the Fluorescence Lifetime of Cyan Fluorescent Protein via an Interaction with His148

Recently, we described that ATP induces changes in YFP/CFP fluorescence intensities of Fluorescence Resonance Energy Transfer (FRET) sensors based on CFP-YFP. To get insight into this phenomenon, we employed fluorescence lifetime spectroscopy to analyze the influence of ATP on these fluorescent proteins in more detail. Using different donor and acceptor pairs we found that ATP only affected the CFP-YFP based versions. Subsequent analysis of purified monomers of the used proteins showed that ATP has a direct effect on the fluorescence lifetime properties of CFP. Since the fluorescence lifetime analysis of CFP is rather complicated by the existence of different lifetimes, we tested a variant of CFP, i.e. Cerulean, as a monomer and in our FRET constructs. Surprisingly, this CFP variant shows no ATP concentration dependent changes in the fluorescence lifetime. The most important difference between CFP and Cerulean is a histidine residue at position 148. Indeed, changing this histidine in CFP into an aspartic acid results in identical fluorescence properties as observed for the Cerulean fluorescent based FRET sensor. We therefore conclude that the changes in fluorescence lifetime of CFP are affected specifically by possible electrostatic interactions of the negative charge of ATP with the positively charged histidine at position 148. Clearly, further physicochemical characterization is needed to explain the sensitivity of CFP fluorescence properties to changes in environmental (i.e. ATP concentrations) conditions

Regeneration of myelin sheaths of normal length and thickness in the zebrafish CNS correlates with growth of axons in caliber

Demyelination is observed in numerous diseases of the central nervous system, including multiple sclerosis (MS). However, the endogenous regenerative process of remyelination can replace myelin lost in disease, and in various animal models. Unfortunately, the process of remyelination often fails, particularly with ageing. Even when remyelination occurs, it is characterised by the regeneration of myelin sheaths that are abnormally thin and short. This imperfect remyelination is likely to have implications for the restoration of normal circuit function and possibly the optimal metabolic support of axons. Here we describe a larval zebrafish model of demyelination and remyelination. We employ a drug-inducible cell ablation system with which we can consistently ablate 2/3rds of oligodendrocytes in the larval zebrafish spinal cord. This leads to a concomitant demyelination of 2/3rds of axons in the spinal cord, and an innate immune response over the same time period. We find restoration of the normal number of oligodendrocytes and robust remyelination approximately two weeks after induction of cell ablation, whereby myelinated axon number is restored to control levels. Remarkably, we find that myelin sheaths of normal length and thickness are regenerated during this time. Interestingly, we find that axons grow significantly in caliber during this period of remyelination. This suggests the possibility that the active growth of axons may stimulate the regeneration of myelin sheaths of normal dimensions

One piece of the puzzle: Population pharmacokinetics of FVIII during perioperative Haemate P®/Humate P® treatment in von Willebrand disease patients

Introduction: Many patients with von Willebrand disease (VWD) are treated on demand with von Willebrand factor and factor VIII (FVIII) containing concentrates present with VWF and/or FVIII plasma levels outside set target levels. This carries a risk for bleeding and potentially for thrombosis. Development of a population pharmacokinetic (PK) model based on FVIII levels is a first step to more accurate on-demand perioperative dosing of this concentrate

A FRET-based biosensor for measuring G alpha 13 activation in single cells

Contains fulltext : 187833.pdf (publisher's version ) (Open Access

Acridine orange staining reveals oligodendrocytes undergoing cell death following treatment with Mtz.

<p><b>A</b>. Representative images of spinal cords of DMSO- or Mtz-treated Tg(mbp:mCherry-NTR) larvae at 4dpt. Acridine orange labels cells undergoing cell death in green. In Mtz-treated animals several bright green puncta can be seen, and these colocalise with mCherry (yellow arrowheads). <b>B</b>. Quantification of cells that are positive for both mCherry and acridine orange, per four-somite stretch of spinal cord. At 0dpt, control mean: 0.56 ± 1.01 vs treated: 3.88 ± 1.25, p < 0.0001. The number of AO+ cells peaks at 4dpt, when the mean in controls was: 0.4 ± 0.89, vs treated: 6.5 ± 1.69, p < 0.0001. This then reduced so that at 7dpt, controls: 1.25 ± 1.16 vs treated: 0.86 ± 1.07, p = 0.510. All significances were obtained using multiple t tests per row (Holm-Sidak method). n = no less than 8. Scale bar: 20μm.</p

Remyelination occurs by 16 days post-treatment.

<p><b>A</b>. Electron micrographs of entire hemispinal cords of DMSO- or Mtz-treated Tg(mbp:mCherry-NTR) larvae). Scale bar: 3μm. <b>B.</b> Higher magnification electron micrographs of the ventral spinal cords. Scale bar: 1μm. <b>C.</b> Quantification of the numbers of myelinated axons per ventral hemi-spinal cord. Mean in controls: 80.88 ± 23.12 vs treated: 85.71 ± 22.01, p = 0.686. <b>D</b>. Quantification of the number of unmyelinated axons < 1μm in perimeter. Mean in controls: 10.25 ± 7.13 vs treated: 18.29 ± 12.47, p = 0.143. <b>E</b>. Quantification of the total number of axons with perimeter over 1μm. Mean in controls: 91.13 ± 25.39 vs treated: 104.0 ± 24.04 in Mtz-treated animals, p = 0.334. <b>F</b>. Quantification of the percentage of myelinated axons. Mean in controls: 88.75 ± 7.09% vs treated: 82.86 ± 9.72%, p = 0.199. n = 6 for controls, 5 for treated. Significances obtained from two-tailed Student’s t tests.<b>G</b>. G ratios plotted against axon diameters show that there is no difference in myelin thickness across axon sizes between control and Mtz-treated animals.</p

Oligodendrocyte numbers remain low in Mtz-treated Tg(mbp:mCherry-NTR) larvae for 7 days following treatment.

<p><b>A</b>. Representative images from the spinal cords of DMSO- and Mtz-treated Tg(mbp:mCherry-NTR) larvae at the time points indicated; GFP channel is shown. <b>B</b>. Quantification of oligodendrocyte numbers from a four-somite stretch of the spinal cord. At 0dpt, mean number of oligodendrocytes in controls: 69.94 ± 11.64 vs treated: 26.76 ± 7.20, p < 0.0001. At 1dpt, controls: 71.43 ± 12.09 vs treated: 37.75 ± 5.34, p < 0.0001. At 2pt, controls: 75.29 ± 6.16 vs treated: 37.22 ± 12.28, p < 0.0001. At 3dpt, controls: 79.29 ± 13.71 vs treated: 50.13 ± 15.78, p < 0.0001. At 4dpt, controls: 76.08 ± 8.92 vs treated: 42.95, p < 0.0001. At 5dpt, controls: 81.92 vs treated: 35.60 ± 10.88, p < 0.0001. At 6dpt, controls: 77.78 ± 10.54 vs treated: 35.64 ± in 11.3, p < 0.0001. At 7dpt, controls: 92.15 ± 9.76 vs treated: 44.25 ± 11.57, p < 0.0001. Statistical significances were determined by multiple t tests per row, without assuming equal standard deviations (Holm-Sidak method). n = no less than 7. Scale bar: 20μm.</p