搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

Marburg virus (MARV) and Ebola virus (EBOV) belong to the family Filoviridae. MARV causes severe disease in humans with high fatality. We previously isolated a large panel of monoclonal antibodies (mAbs) from B cells of a human survivor with previous naturally acquired MARV infection. Here,we characterized functional properties of these mAbs and identified non-neutralizing mAbs targeting the glycoprotein (GP) 2 portion of the mucin-like domain (MLD) of MARV GP,termed the wing region. One mAb targeting the GP2 wing,MR228,showed therapeutic protection in mice and guinea pigs infected with MARV. The protection was mediated by the Fc fragment functions of MR228. Binding of another GP2 wing-specific non-neutralizing mAb,MR235,to MARV GP increased accessibility of epitopes in the receptor-binding site (RBS) for neutralizing mAbs,resulting in enhanced virus neutralization by these mAbs. These findings highlight an important role for non-neutralizing mAbs during natural human MARV infection. View Publication

Type III interferon-lambda (IFN-$\lambda$) plays a critical role against infection,particularly in mucosal infection in the respiratory and gastrointestinal tract. Our study and other previous studies have shown that porcine IFN-$\lambda$ more efficiently curtails the infection of porcine epidemic diarrhea virus (PEDV) in the intestine epithelia than type I IFN,whereas IFN-$\lambda$3 exerts a more potent effect than IFN-$\lambda$1. However,the underlying mechanism remains elusive,and in particular,the transcriptional profile induced by IFN-$\lambda$3 has not been reported. Here,to resolve the mechanism responsible for the disparity between IFN-$\lambda$3 and type I IFN in anti-mucosal virus infection,we compared the transcription profiles induced by the two IFNs in porcine intestinal epithelial (IPEC-J2) cells by RNA-Seq. Our results showed that the pretreatment of IPEC-J2 cells with IFN-$\lambda$3 resulted in the differential expression of 983 genes. In contrast,IFN-$\alpha$ only modified the expression of 134 genes,and 110 of these genes were also observed in the response to IFN-$\lambda$3. A transcriptional enrichment analysis indicated that IFN-$\lambda$3 or IFN-$\alpha$ regulates multiple cellular processes and that IFN-$\lambda$3 activates more robust signaling pathways,particularly the antiviral Jak-STAT signaling pathway,than IFN-$\alpha$. Furthermore,we verified the RNA-Seq results through an RT-qPCR analysis of IPEC-J2 cells and porcine enteroids. Moreover,transient expression of the porcine rsad2 and mx2 genes among the top 10 genes induced by IFN-$\lambda$3 significantly inhibited PEDV infection. Collectively,the data showed that IFN-$\lambda$3 induces a unique transcriptional profile that does not completely overlap with that induced by IFN-$\alpha$ and strongly elicits a set of genes responsible for the antiviral activity of IFN-$\lambda$3. These findings provide important knowledge regarding the elicited ISGs of type I and III IFNs in restricting porcine intestinal viral infection. View Publication

The mechanism of Fas antigen-mediated apoptosis is at present unclear. We show here that the 100,000 x g supernatant from cell lysates prepared from anti-Fas-stimulated JUR-KAT T cells,induces chromatin fragmentation in isolated nuclei with concomitant morphological changes typically seen in apoptosis. The formation of this apoptotic nuclei promoting activity (ANPA) in JURKAT T cells after Fas antigen ligation was blocked by the serine protease inhibitors,TPCK and DCI,and by the interleukin 1-beta-converting enzyme inhibitor,VAD-FMK. In addition,chromatin degradation and morphological changes mediated by the ANPA in isolated nuclei were inhibited by TPCK,but not by DCI or VAD-FMK. These results suggest that Fas-mediated apoptosis in T cells involves the activation of a cascade of proteases. View Publication

Although arsenic trioxide (As(2)O(3)) is an effective therapy in acute promyelocytic leukemia (APL),its use in other malignancies is limited by the toxicity of concentrations required to induce apoptosis in non-APL tumor cells. We looked for agents that would synergize with As(2)O(3) to induce apoptosis in malignant cells,but not in normal cells. We found that trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid),a widely known antioxidant,enhances As(2)O(3)-mediated apoptosis in APL,myeloma,and breast cancer cells. Treatment with As(2)O(3) and trolox increased intracellular oxidative stress,as evidenced by heme oxygenase-1 (HO-1) protein levels,c-Jun terminal kinase (JNK) activation,and protein and lipid oxidation. The synergistic effects of trolox may be specific to As(2)O(3),as trolox does not add to toxicity induced by other chemotherapeutic drugs. We explored the mechanism of this synergy using electron paramagnetic resonance and observed the formation of trolox radicals when trolox was combined with As(2)O(3),but not with doxorubicin. Importantly,trolox protected nonmalignant cells from As(2)O(3)-mediated cytotoxicity. Our data provide the first evidence that trolox may extend the therapeutic spectrum of As(2)O(3). Furthermore,the combination of As(2)O(3) and trolox shows potential specificity for tumor cells,suggesting it may not increase the toxicity associated with As(2)O(3) monotherapy in vivo. View Publication

OBJECTIVES An imbalance between neutrophil extracellular trap (NET) formation and degradation has been described in systemic lupus erythematosus (SLE),potentially contributing to autoantigen externalisation,type I interferon synthesis and endothelial damage. We have demonstrated that peptidylarginine deiminase (PAD) inhibition reduces NET formation and protects against lupus-related vascular damage in the New Zealand Mixed model of lupus. However,another strategy for inhibiting NETs--knockout of NOX2--accelerates lupus in a different murine model,MRL/lpr. Here,we test the effects of PAD inhibition on MRL/lpr mice in order to clarify whether some NET inhibitory pathways may be consistently therapeutic across models of SLE. METHODS NET formation and autoantibodies to NETs were characterised in lupus-prone MRL/lpr mice. MRL/lpr mice were also treated with two different PAD inhibitors,Cl-amidine and the newly described BB-Cl-amidine. NET formation,endothelial function,interferon signature,nephritis and skin disease were examined in treated mice. RESULTS Neutrophils from MRL/lpr mice demonstrate accelerated NET formation compared with controls. MRL/lpr mice also form autoantibodies to NETs and have evidence of endothelial dysfunction. PAD inhibition markedly improves endothelial function,while downregulating the expression of type I interferon-regulated genes. PAD inhibition also reduces proteinuria and immune complex deposition in the kidneys,while protecting against skin disease. CONCLUSIONS PAD inhibition reduces NET formation,while protecting against lupus-related damage to the vasculature,kidneys and skin in various lupus models. The strategy by which NETs are inhibited will have to be carefully considered if human studies are to be undertaken. View Publication

Human embryonic stem cell-derived beta cells offer a promising cell-based therapy for diabetes. However,efficient stem cell to beta cell differentiation has proven difficult,possibly due to the lack of cross-talk with the appropriate mesenchymal niche. To define organ-specific niche signals,we isolated pancreatic and gastrointestinal stromal cells,and analyzed their gene expression during development. Our genetic studies reveal the importance of tightly regulated Hedgehog signaling in the pancreatic mesenchyme: inactivation of mesenchymal signaling leads to annular pancreas,whereas stroma-specific activation of signaling via loss of Hedgehog regulators,Sufu and Spop,impairs pancreatic growth and beta cell genesis. Genetic rescue and transcriptome analyses show that these Sufu and Spop knockout defects occur through Gli2-mediated activation of gastrointestinal stromal signals such as Wnt ligands. Importantly,inhibition of Wnt signaling in organoid and human stem cell cultures significantly promotes insulin-producing cell generation,altogether revealing the requirement for organ-specific regulation of stromal niche signals. View Publication

Peroxynitrite toxicity is a major cause of neuronal injury in stroke and neurodegenerative disorders. The mechanisms underlying the neurotoxicity induced by peroxynitrite are still unclear. In this study,we observed that TPEN [N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine],a zinc chelator,protected against neurotoxicity induced by exogenous as well as endogenous (coadministration of NMDA and a nitric oxide donor,diethylenetriamine NONOate) peroxynitrite. Two different approaches to detecting intracellular zinc release demonstrated the liberation of zinc from intracellular stores by peroxynitrite. In addition,we found that peroxynitrite toxicity was blocked by inhibitors of 12-lipoxygenase (12-LOX),p38 mitogen-activated protein kinase (MAPK),and caspase-3 and was associated with mitochondrial membrane depolarization. Inhibition of 12-LOX blocked the activation of p38 MAPK and caspase-3. Zinc itself induced the activation of 12-LOX,generation of reactive oxygen species (ROS),and activation of p38 MAPK and caspase-3. These data suggest a cell death pathway triggered by peroxynitrite in which intracellular zinc release leads to activation of 12-LOX,ROS accumulation,p38 activation,and caspase-3 activation. Therefore,therapies aimed at maintaining intracellular zinc homeostasis or blocking activation of 12-LOX may provide a novel avenue for the treatment of inflammation,stroke,and neurodegenerative diseases in which the formation of peroxynitrite is thought to be one of the important causes of cell death. View Publication

Pluripotent cancer stem cells play a pivotal role in inducing phenotypic plasticity across various cancer types,including bladder cancer. This plasticity,crucial for cancer progression,is largely regulated by epigenetic modifications including N6-methyladenosine (m6A) in RNAs. However,the role of the m6A reader protein YTHDC2 in this process remains poorly understood. In this study,we uncovered that the depletion of YTHDC2 significantly increased the pool of bladder cancer stem cells (BCSCs),resulting in a phenotypic shift towards a more invasive subtype of bladder cancer. This shift was characterized by enhanced proliferation,migration,invasion,and self-renewal capabilities of cancer cells,highlighting YTHDC2’s function as a tumor suppressor. Mechanistically,YTHDC2 recognized and bound to m6A-modified SOX2 mRNA,resulting in translational inhibition of SOX2. In conclusion,our study identifies YTHDC2 as a tumor suppressor in bladder cancer through inhibiting SOX2-mediated cell pluripotency and underscores the therapeutic potential of targeting the YTHDC2-SOX2 axis in bladder cancer. View Publication

Transforming growth factor beta1 (TGF-beta1) is a potent fibrotic factor responsible for the synthesis of extracellular matrix. TGF-beta1 acts through the TGF-beta type I and type II receptors to activate intracellular mediators,such as Smad proteins,the p38 mitogen-activated protein kinase (MAPK),and the extracellular signal-regulated kinase pathway. We expressed the kinase domain of the TGF-beta type I receptor [activin receptor-like kinase (ALK)5] and the substrate,Smad3,and determined that SB-431542 is a selective inhibitor of Smad3 phosphorylation with an IC50 of 94 nM. It inhibited TGF-beta1-induced nuclear Smad3 localization. The p38 mitogen-activated protein kinase inhibitors SB-203580 and SB-202190 also inhibit phosphorylation of Smad3 by ALK5 with IC50 values of 6 and 3 microM,respectively. This suggests that these p38 MAPK inhibitors must be used at concentrations of less than 10 microM to selectively address p38 MAPK mechanisms. However,the p38 MAPK inhibitor SB-242235 did not inhibit ALK5. To evaluate the relative contribution of Smad signaling and p38 MAPK signaling in TGF-beta1-induced matrix production,the effect of SB-431542 was compared with that of SB-242235 in renal epithelial carcinoma A498 cells. All compounds inhibited TGF-beta1-induced fibronectin (FN) mRNA,indicating that FN synthesis is mediated in part via the p38 MAPK pathway. In contrast,SB-431542,but not the selective p38 MAPK inhibitor SB-242235,inhibited TGF-beta1-induced collagen Ialpha1 (col Ialpha1). These data indicate that some matrix markers that are stimulated by TGF-beta1 are mediated via the p38 MAPK pathway (i.e.,FN),whereas others seem to be activated via ALK5 signaling independent of the p38 MAPK pathway (i.e.,col Ialpha1). View Publication