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

Recent advances in immunometabolism link metabolic changes in stimulated macrophages to production of IL-1β,a crucial cytokine in the innate immune response to Mycobacterium tuberculosis. To investigate this pathway in the host response to M. tuberculosis,we performed metabolic and functional studies on human alveolar macrophages,human monocyte-derived macrophages,and murine bone marrow-derived macrophages following infection with the bacillus in vitro. M. tuberculosis infection induced a shift from oxidative phosphorylation to aerobic glycolysis in macrophages. Inhibition of this shift resulted in decreased levels of proinflammatory IL-1β and decreased transcription of PTGS2,increased levels of anti-inflammatory IL-10,and increased intracellular bacillary survival. Blockade or absence of IL-1R negated the impact of aerobic glycolysis on intracellular bacillary survival,demonstrating that infection-induced glycolysis limits M. tuberculosis survival in macrophages through induction of IL-1β. Drugs that manipulate host metabolism may be exploited as adjuvants for future therapeutic and vaccination strategies. View Publication

BACKGROUND Transplantation of endothelial progenitor cells (EPCs) improves neovascularization after ischemia,but patients with coronary artery disease (CAD) or diabetes mellitus show a reduced number of EPCs and impaired functional activity. Therefore,we investigated the effects of risk factors,such as glucose and TNF-alpha,on the number of EPCs in vitro to elucidate the underlying mechanisms. METHODS AND RESULTS EPCs of patients or healthy subjects were isolated from peripheral blood. Incubation with glucose or TNF-alpha dose-dependently reduced the number of EPCs (79.9+/-1.3% and 74.3+/-8.1% of control; Ptextless0.05,respectively). This reduction was not caused by apoptosis. TNF-alpha and glucose induced a dose- and time-dependent activation of the p38 MAP kinase,the downstream kinase mitogen- and stress-activated kinase 1,and the transcription factor cAMP-responsive element-binding protein (CREB),in EPCs. Moreover,EPCs from CAD patients had significantly higher basal p38-phosphorylation levels (1.83+/-0.2-fold increase; Ptextless0.05) compared with healthy subjects. The inhibition of the p38-kinase by SB203580 or infection with a dominant negative p38 kinase adenovirus significantly increased basal number of EPCs (136.7+/-6.3% and 142.9+/-18% versus control,respectively). Likewise,ex vivo cultivation of EPCs from patients with CAD with SB203580 significantly increased the number of EPCs and partially reversed the impaired capacity for neovascularization of EPCs in vivo (relative blood flow: 0.40+/-0.03 versus 0.64+/-0.08,Ptextless0.05). The increased numbers of EPCs by SB203580 were associated with an augmentation of EPC proliferation and a reduction of cells expressing the monocytic marker proteins CD14 and CD64,suggesting that p38 regulates proliferation and differentiation events. CONCLUSIONS These results demonstrate that p38 MAP kinase plays a pivotal role in the signal transduction pathways regulating the number of EPCs ex vivo. SB203580 can prevent the negative effects of TNF-alpha and glucose on the number of EPCs and may be useful to improve the number of EPCs for potential cell therapy. View Publication

First developed for hematologic disorders,the concept of cancer stem cells (CSCs) was expanded to solid tumors,including colorectal cancer (CRC). The traditional model of colon carcinogenesis includes several steps that occur via mutational activation of oncogenes and inactivation of tumor suppressor genes. Intestinal epithelial cells exist for a shorter amount of time than that required to accumulate tumor-inducing genetic changes,so researchers have investigated the concept that CRC arises from the long-lived stem cells,rather than from the differentiated epithelial cells. Colon CSCs were originally identified through the expression of the CD133 glycoprotein using an antibody directed to its epitope AC133. It is not clear if CD133 is a marker of colon CSCs-other cell surface markers,such as epithelial-specific antigen,CD44,CD166,Musashi-1,CD29,CD24,leucine-rich repeat-containing G-protein-coupled receptor 5,and aldehyde dehydrogenase 1,have been proposed. In addition to initiating and sustaining tumor growth,CSCs are believed to mediate cancer relapse after chemotherapy. How can we identify and analyze colon CSCs and what agents are being designed to kill this chemotherapy-refractory population? View Publication

Development of resistance to therapy continues to be a serious clinical problem in breast cancer management. Cancer stem/progenitor cells have been shown to play roles in resistance to chemo�? and radiotherapy. Here,we examined their role in the development of resistance to the oestrogen receptor antagonist tamoxifen. Tamoxifen�?resistant cells were enriched for stem/progenitors and expressed high levels of the stem cell marker Sox2. Silencing of the SOX2 gene reduced the size of the stem/progenitor cell population and restored sensitivity to tamoxifen. Conversely,ectopic expression of Sox2 reduced tamoxifen sensitivity in vitro and in vivo. Gene expression profiling revealed activation of the Wnt signalling pathway in Sox2�?expressing cells,and inhibition of Wnt signalling sensitized resistant cells to tamoxifen. Examination of patient tumours indicated that Sox2 levels are higher in patients after endocrine therapy failure,and also in the primary tumours of these patients,compared to those of responders. Together,these results suggest that development of tamoxifen resistance is driven by Sox2�?dependent activation of Wnt signalling in cancer stem/progenitor cells. View Publication

Current laboratory methods used to passage adherent human pluripotent stem cells (hPSCs) are labor intensive,result in reduced cell viability and are incompatible with larger scale production necessary for many clinical applications. To meet the current demand for hPSCs,we have developed a new non-enzymatic passaging method using sodium citrate. Sodium citrate,formulated as a hypertonic solution,gently and efficiently detaches adherent cultures of hPSCs as small multicellular aggregates with minimal manual intervention. These multicellular aggregates are easily and reproducibly recovered in calcium-containing medium,retain a high post-detachment cell viability of 97%±1% and readily attach to fresh substrates. Together,this significantly reduces the time required to expand hPSCs as high quality adherent cultures. Cells subcultured for 25 passages using this novel sodium citrate passaging solution exhibit characteristic hPSC morphology,high levels (textgreater80%) of pluripotency markers OCT4,SSEA-4,TRA-1-60 andTRA-1-81,a normal G-banded karyotype and the ability to differentiate into cells representing all three germ layers,both in vivo and in vitro. View Publication

The decreased proportion of antigen-inexperienced,na{\{i}}ve T cells is a hallmark of aging in both humans and mice and contributes to reduced immune responses particularly against novel and re-emerging pathogens. Na{\"{i}}ve T cells depend on survival signals received during their circulation among the lymph nodes by direct contacts with stroma in particular fibroblastic reticular cells. Macroscopic changes to the architecture of the lymph nodes have been described but it is unclear how lymph node stroma are altered with age and whether these changes contribute to reduced na{\"{i}}ve T cell maintenance. Here using 2-photon microscopy we determined that the aged lymph node displayed increased fibrosis and correspondingly that na{\"{i}}ve T-cell motility was impaired in the aged lymph node especially in proximity to fibrotic deposition. Functionally adoptively transferred young na{\"{i}}ve T-cells exhibited reduced homeostatic turnover in aged hosts supporting the role of T cell-extrinsic mechanisms that regulate their survival. Further we determined that early development of resident fibroblastic reticular cells was impaired which may correlate to the declining levels of na{\"{i}}ve T-cell homeostatic factors observed in aged lymph nodes. Thus our study addresses the controversy as to whether aging impacts the composition lymph node stroma and supports a model in which impaired differentiation of lymph node fibroblasts and increased fibrosis inhibits the interactions necessary for na{\"{i}}ve T cell homeostasis." View Publication