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101.
France Bruck Ludovic Belle Chantal Lechanteur Laurence de Leval Muriel Hannon Sophie Dubois Emilie Castermans Stephanie Humblet-Baron Souad Rahmouni Yves Beguin Alexandra Briquet Frédéric Baron 《Cytotherapy》2013,15(3):267-279
Background aimsGraft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation caused by donor T cells reacting against host tissues. Previous studies have suggested that mesenchymal stromal cells (MSCs) could exert potent immunosuppressive effects.MethodsThe ability of human bone marrow derived MSCs to prevent xenogeneic GVHD in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice and in NOD/SCID/interleukin-2Rγ(null) (NSG) mice transplanted with human peripheral blood mononuclear cells (PBMCs) was assessed.ResultsInjection of 200 × 106 human PBMCs intraperitoneally (IP) into sub-lethally (3.0 Gy) irradiated NOD/SCID mice also given anti-asialo GM1 antibodies IP 1 day prior and 8 days after transplantation induced lethal xenogeneic GVHD in all tested mice. Co-injection of 2 × 106 MSCs IP on day 0 did not prevent lethal xenogeneic GVHD induced by injection of human PBMCs. Similarly, injection of 30 × 106 human PBMCs IP into sub-lethally (2.5 Gy) irradiated NSG mice induced a lethal xenogeneic GVHD in all tested mice. Injection of 3 × 106 MSCs IP on days 0, 7, 14 and 21 did not prevent lethal xenogeneic GVHD induced by injection of human PBMCs.ConclusionsInjection of MSCs did not prevent xenogeneic GVHD in these two humanized mice models. 相似文献
102.
Karen Page David S. Guttery Nathalie Zahra Lindsay Primrose Shona R. Elshaw J. Howard Pringle Kevin Blighe Stephanie D. Marchese Allison Hills Laura Woodley Justin Stebbing R. Charles Coombes Jacqueline A. Shaw 《PloS one》2013,8(10)
Circulating nucleic acids (CNAs) are under investigation as a liquid biopsy in cancer. However there is wide variation in blood processing and methods for isolation of circulating free DNA (cfDNA) and microRNAs (miRNAs). Here we compare the extraction efficiency and reproducibility of 4 commercially available kits for cfDNA and 3 for miRNA using spike-in of reference templates. We also compare the effects of increasing time between venepuncture and centrifugation and differential centrifugation force on recovery of CNAs. cfDNA was quantified by TaqMan qPCR and targeted deep sequencing. miRNA profiles were assessed with TaqMan low-density arrays and assays. The QIAamp® DNA Blood Mini and Circulating nucleic acid kits gave the highest recovery of cfDNA and efficient recovery (>90%) of a 564bp spike-in. Moreover, targeted sequencing revealed overlapping cfDNA profiles and variant depth, including detection of HER2 gene amplification, using the Ion AmpliSeq™Cancer Hotspot Panel v2. Highest yields of miRNA and the synthetic Arabidopsis thaliana miR-159a spike-in were obtained using the miRNeasy Serum/Plasma kit, with saturation above 200 µl of plasma. miRNA profiles showed significant variation with increasing time before centrifugation (p<0.001) and increasing centrifugation force, with depletion of platelet associated miRNAs, whereas cfDNA was unaffected. However, sample replicates showed excellent reproducibility on TaqMan low density arrays (ρ = 0.96, p<0.0001). We also successfully generated miRNA profiles for plasma samples stored > 12 years, highlighting the potential for analysis of stored sample biobanks. In the era of the liquid biopsy, standardisation of methods is required to minimise variation, particularly for miRNA. 相似文献
103.
Genes encoding two proteins corresponding to elongation factor G (EF-G) were cloned from Pseudomonas aeruginosa. The proteins encoded by these genes are both members of the EFG I subfamily. The gene encoding one of the forms of EF-G is located in the str operon and the resulting protein is referred to as EF-G1A while the gene encoding the other form of EF-G is located in another part of the genome and the resulting protein is referred to as EF-G1B. These proteins were expressed and purified to 98% homogeneity. Sequence analysis indicated the two proteins are 90/84% similar/identical. In other organisms containing multiple forms of EF-G a lower degree of similarity is seen. When assayed in a poly(U)-directed poly-phenylalanine translation system, EF-G1B was 75-fold more active than EF-G1A. EF-G1A pre-incubate with ribosomes in the presence of the ribosome recycling factor (RRF) decreased polymerization of poly-phenylalanine upon addition of EF-G1B in poly(U)-directed translation suggesting a role for EF-G1A in uncoupling of the ribosome into its constituent subunits. Both forms of P. aeruginosa EF-G were active in ribosome dependent GTPase activity. The kinetic parameters (K
M) for the interaction of EF-G1A and EF-G1B with GTP were 85 and 70 μM, respectively. However, EF-G1B exhibited a 5-fold greater turnover number (observed k
cat) for the hydrolysis of GTP than EF-G1A; 0.2 s-1 vs. 0.04 s-1. These values resulted in specificity constants (k
cat
obs/K
M) for EF-G1A and EF-G1B of 0.5 x 103 s-1 M-1 and 3.0 x 103 s-1 M-1, respectively. The antibiotic fusidic acid (FA) completely inhibited poly(U)-dependent protein synthesis containing P. aeruginosa EF-G1B, but the same protein synthesis system containing EF-G1A was not affected. Likewise, the activity of EF-G1B in ribosome dependent GTPase assays was completely inhibited by FA, while the activity of EF-G1A was not affected. 相似文献
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107.
Intermittent hypoxia (IH) during sleep is a hallmark of sleep apnea, causing significant neuronal apoptosis, and cognitive and behavioral deficits in CNS regions underlying memory processing and executive functions. IH-induced neuroinflammation is thought to contribute to cognitive deficits after IH. In the present studies, we tested the hypothesis that IH would differentially induce inflammatory factor gene expression in microglia in a CNS region-dependent manner, and that the effects of IH would differ temporally. To test this hypothesis, adult rats were exposed to intermittent hypoxia (2 min intervals of 10.5% O2) for 8 hours/day during their respective sleep cycles for 1, 3 or 14 days. Cortex, medulla and spinal cord tissues were dissected, microglia were immunomagnetically isolated and mRNA levels of the inflammatory genes iNOS, COX-2, TNFα, IL-1β and IL-6 and the innate immune receptor TLR4 were compared to levels in normoxia. Inflammatory gene expression was also assessed in tissue homogenates (containing all CNS cells). We found that microglia from different CNS regions responded to IH differently. Cortical microglia had longer lasting inflammatory gene expression whereas spinal microglial gene expression was rapid and transient. We also observed that inflammatory gene expression in microglia frequently differed from that in tissue homogenates from the same region, indicating that cells other than microglia also contribute to IH-induced neuroinflammation. Lastly, microglial TLR4 mRNA levels were strongly upregulated by IH in a region- and time-dependent manner, and the increase in TLR4 expression appeared to coincide with timing of peak inflammatory gene expression, suggesting that TLR4 may play a role in IH-induced neuroinflammation. Together, these data indicate that microglial-specific neuroinflammation may play distinct roles in the effects of intermittent hypoxia in different CNS regions. 相似文献
108.
Stephanie S. Gervasi Jenny Urbina Jessica Hua Tara Chestnut Rick A. Relyea Andrew R. Blaustein 《EcoHealth》2013,10(2):166-171
The emerging fungal pathogen, Batrachochytrium dendrobatidis (Bd), has been associated with global amphibian population declines and extinctions. American bullfrogs (Lithobates catesbeianus) are widely reported to be a tolerant host and a carrier of Bd that spreads the pathogen to less tolerant hosts. Here, we examined whether bullfrogs raised from eggs to metamorphosis in outdoor mesocosms were susceptible to Bd. We experimentally exposed metamorphic juveniles to Bd in the laboratory and compared mortality rates of pathogen-exposed animals to controls (non-exposed) in two separate experiments; one using a Bd strain isolated from a Western toad and another using a strain isolated from an American bullfrog. We wanted to examine whether metamorphic bullfrogs were susceptible to either of these strains. We show that bullfrogs were susceptible to one strain of Bd and not the other. In both experiments, infection load detected in the skin decreased over time, suggesting that metamorphic bullfrogs from some populations may be inefficient long-term carriers of Bd. 相似文献
109.
Herbert Fluhr Julia Spratte Marike Bredow Stephanie Heidrich Marek Zygmunt 《Reproductive biology》2013,13(2):113-121
Apoptosis in the human endometrium plays an essential role for endometrial receptivity and early implantation. A dysbalance of pro- and anti-apoptotic events in the secretory endometrium seems to be involved in implantation disorders and consecutive pregnancy complications. However, little is known about the mechanisms regulating apoptosis-sensitivity in the human endometrium. Therefore this study was performed to identify molecular mechanisms underlying the resistance toward apoptosis in human endometrial stromal cells (ESCs). Human ESCs were isolated from hysterectomy specimens and used as undifferentiated cells or after decidualization in vitro. Cells were incubated with an activating anti-Fas antibody, tumor-necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), TNF-α and inhibitors of protein- and RNA-syntheses, a caspase-inhibitor and inhibitors of extracellular signal regulated kinase (Erk)1/2, nuclear factor (NF)-κB and Akt. Apoptosis was measured by flow cytometric detection of hypodiploid nuclei. Caspase-activity was detected by luminescencent assays. Several pro- and anti-apoptotic molecules and the activation of Erk1/2, NF-κB and Akt were analyzed by in-cell Western assays or flow cytometry. Inhibition of protein- and RNA-syntheses differentially sensitized human ESCs for death receptor-mediated apoptosis in a caspase-dependent manner, based on the up-regulation of the death receptors Fas and TRAIL-R2. The constitutive activity of Erk1/2 and NF-κB could be identified as a reason for the apoptosis-resistance of human ESCs. These results suggest the pro-survival signaling pathways Erk1/2 and NF-κB as key regulators of the sensitivity of human ESCs for death receptor-mediated apoptosis. The modulation of these pathways might play an important role in the physiology of implantation. 相似文献
110.
Ya-Yun Chu Mulugeta Nega Martina W?lfle Laure Plener Stephanie Grond Kirsten Jung Friedrich G?tz 《PLoS pathogens》2013,9(9)
The knowledge that many pathogens rely on cell-to-cell communication mechanisms known as quorum sensing, opens a new disease control strategy: quorum quenching. Here we report on one of the rare examples where Gram-positive bacteria, the ‘Staphylococcus intermedius group’ of zoonotic pathogens, excrete two compounds in millimolar concentrations that suppress the quorum sensing signaling and inhibit the growth of a broad spectrum of Gram-negative beta- and gamma-proteobacteria. These compounds were isolated from Staphylococcus delphini. They represent a new class of quorum quenchers with the chemical formula N-[2-(1H-indol-3-yl)ethyl]-urea and N-(2-phenethyl)-urea, which we named yayurea A and B, respectively. In vitro studies with the N-acyl homoserine lactone (AHL) responding receptor LuxN of V. harveyi indicated that both compounds caused opposite effects on phosphorylation to those caused by AHL. This explains the quorum quenching activity. Staphylococcal strains producing yayurea A and B clearly benefit from an increased competitiveness in a mixed community. 相似文献