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

OBJECTIVE True identity and specific set of markers to enrich endometrial stem cells still remains elusive. Present study was undertaken to further substantiate that very small embryonic-like stem cells (VSELs) are the true and elusive stem cells in adult mice endometrium. METHODS This was achieved by undertaking three sets of experiments. Firstly,SSEA-1+ and Oct-4??+??positive VSELs,sorted from GFP mice,were transplanted into the uterine horns of wild-type Swiss mice and GFP uptake was studied within the same estrus cycle. Secondly,uterine lumen was scratched surgically and OCT-4 expressing stem/progenitor cells were studied at the site of injury after 24-72 h. Thirdly,OCT-4????expression was studied in the endometrium and myometrium of adult mice after neonatal exposure to estradiol (20 µg/pup/day on days 5-7 after birth). RESULTS GFP??+??ve VSELs expressing SSEA-1 and Oct-4 engrafted and differentiated into the epithelial cells lining the lumen as well as the glands during the estrus stage when maximum remodeling occurs. Mechanical scratching activated tissue-resident,nuclear OCT-4 positive VSELs and slightly bigger 'progenitors' endometrial stem cells (EnSCs,cytoplasmic OCT-4) which underwent clonal expansion and further differentiated into luminal and glandular epithelial cells. Neonatal exposure to endocrine disruption resulted in increased numbers of OCT-4 positive VSELs/EnSCs in adult endometrium. DISCUSSION Results support the presence of functionally active VSELs in adult endometrium. VSELs self-renew and give rise to EnSCs that further differentiate into epithelial cells under normal physiological conditions. Also,VSELs are vulnerable to endocrine insults. To conclude VSELs are true and elusive uterine stem cells that maintain life-long uterine homeostasis and their dysregulation may result in various pathologies. View Publication

AbstractMyeloproliferative neoplasms (MPNs) are characterized by an increased production of blood cells due to the acquisition of mutations such as JAK2V617F. TGF‐β,whose secretion is increased in MPN patients,is known to negatively regulate haematopoietic stem cell (HSC) proliferation. Using an isogenic JAK2V617F or JAK2 wild‐type UT‐7 cell line we observed that JAK2V617F cells resist to TGF‐β antiproliferative activity. Although TGF‐β receptors and SMAD2/3 expressions are similar in both cell types,TGF‐β‐induced phosphorylation of SMAD2/3 is reduced in UT‐7 JAK2V617F cells compared with JAK2 WT cells. We confirmed that JAK2V617F mutated cells are resistant to the antiproliferative effect of TGF‐β in a competitive assay as we observed a positive selection of JAK2V617F cells when exposed to TGF‐β. Using cell lines,CD34‐positive cells from MPN patients and bone marrow cells from JAK2V617F knock‐in mice we identified a down regulation of the SHP‐1 phosphatase,which is required for the regulation of HSC quiescence by TGF‐β. The transduction of SHP‐1 cDNA (but not a phosphatase inactive cDNA) restores the antiproliferative effect of TGF‐β in JAK2V617F mutated cells. Finally,SC‐1,a known agonist of SHP‐1,antagonized the selection of JAK2V617F mutated cells in the presence of TGF‐β. In conclusion,we show a JAK2‐dependent down regulation of SHP‐1 in MPN patients' cells which is related to their resistance to the antiproliferative effect of TGF‐β. This may participate in the clonal selection of cancer cells in MPNs. View Publication

Airway inflammation is a critical feature of lower respiratory tract infections caused by viruses such as respiratory syncytial virus (RSV). A growing body of literature has demonstrated the importance of extracellular matrix (ECM) changes such as the accumulation of hyaluronan (HA) and versican in the subepithelial space in promoting airway inflammation; however,whether these factors contribute to airway inflammation during RSV infection remains unknown. To test the hypothesis that RSV infection promotes inflammation via altered HA and versican production,we studied an ex vivo human bronchial epithelial cell (BEC)/human lung fibroblast (HLF) co-culture model. RSV infection of BEC/HLF co-cultures led to decreased hyaluronidase expression by HLFs,increased accumulation of HA,and enhanced adhesion of U937 cells as would be expected with increased HA. HLF production of versican was not altered following RSV infection; however,BEC production of versican was significantly downregulated following RSV infection. In vivo studies with epithelial-specific versican-deficient mice [SPC-Cre(+) Vcan-/-] demonstrated that RSV infection led to increased HA accumulation compared to control mice which also coincided with decreased hyaluronidase expression in the lung. SPC-Cre(+) Vcan-/- mice demonstrated enhanced recruitment of monocytes and neutrophils in bronchoalveolar lavage fluid and increased neutrophils in the lung compared to SPC-Cre(-) RSV-infected littermates. Taken together,these data demonstrate that altered ECM accumulation of HA occurs following RSV infection and may contribute to airway inflammation. Additionally,loss of epithelial expression of versican promotes airway inflammation during RSV infection further demonstrating that versican's role in inflammatory regulation is complex and dependent on the microenvironment. View Publication

Loss or mutation of the TP53 tumor suppressor gene is not commonly observed in acute myeloid leukemia (AML),suggesting that there is an alternate route for cell transformation. We investigated the hypothesis that previously observed Bcl-2 family member overexpression suppresses wild-type p53 activity in AML. We demonstrate that wild-type p53 protein is expressed in primary leukemic blasts from patients with de novo AML using 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and phospho-specific flow cytometry. We found that p53 was heterogeneously expressed and phosphorylated in AML patient samples and could accumulate following DNA damage. Overexpression of antiapoptosis protein Bcl-2 in AML cells was directly correlated with p53 expression and phosphorylation on serine residues 15,46,and 392. Within those patients with the highest levels of Bcl-2 expression,we identified a mutation in FLT3 that duplicated phosphorylation site Y591. The presence of this mutation correlated with greater than normal Bcl-2 expression and with previously observed profiles of potentiated STAT and MAPK signaling. These results support the hypothesis that Flt3-mediated signaling in AML enables accumulation of Bcl-2 and maintains a downstream block to p53 pathway apoptosis. Bcl-2 inhibition might therefore improve the efficacy of existing AML therapies by inactivating this suppression of wild-type p53 activity. View Publication

We determined the embryonic origins of adult forebrain subventricular zone (SVZ) stem cells by Cre-lox fate mapping in transgenic mice. We found that all parts of the telencephalic neuroepithelium,including the medial ganglionic eminence and lateral ganglionic eminence (LGE) and the cerebral cortex,contribute multipotent,self-renewing stem cells to the adult SVZ. Descendants of the embryonic LGE and cortex settle in ventral and dorsal aspects of the dorsolateral SVZ,respectively. Both populations contribute new (5-bromo-2'-deoxyuridine-labeled) tyrosine hydroxylase- and calretinin-positive interneurons to the adult olfactory bulb. However,calbindin-positive interneurons in the olfactory glomeruli were generated exclusively by LGE-derived stem cells. Thus,different SVZ stem cells have different embryonic origins,colonize different parts of the SVZ,and generate different neuronal progeny,suggesting that some aspects of embryonic patterning are preserved in the adult SVZ. This could have important implications for the design of endogenous stem cell-based therapies in the future. View Publication

Mobilization of bone marrow-derived endothelial progenitor cells (EPC) might explain exercise-induced improvement of endothelial function. We assessed whether a maximal exercise bout could alter the number of circulating EPC in healthy subjects and whether this effect is related to their cardiovascular risk profile. Additionally,we investigated possible mediators of this effect,namely nitric oxide (NO) bioavailability and vascular endothelial growth factor (VEGF) release. Healthy subjects (group 1,n = 11; group 2,n = 14) performed a symptom-limited cardiopulmonary exercise test on a bicycle ergometer. Numbers of CD34+/kinase insert domain receptor (KDR)+ cells were determined by flow-cytometric analysis,either after magnetic separation of CD34+ cells (group 1) or starting from whole blood (group 2). Serum concentrations of VEGF and NO metabolites were measured by using ELISA. Following exercise,EPC increased by 76% (15.4 +/- 10.7 cells/ml vs. 27.2 +/- 13.7 cells/ml; P = 0.01) in group 1 and by 69% in group 2 (30.9 +/- 14.6 cells/ml vs. 52.5 +/- 42.6 cells/ml; P = 0.03). The increase in EPC correlated positively with LDL and total cholesterol/HDL ratio and negatively with peak oxygen consumption and oxygen consumption at anaerobic threshold. VEGF levels increased with exercise,with a strong trend toward significance (P = 0.055). NO levels remained unchanged. The present study demonstrates that a maximal bout of exercise induces a significant shift in CD34+ cells toward CD34+/KDR+ cells. This response was larger in subjects with a less favorable lipid profile. View Publication

Although natural Abs (NAbs) are present from birth,little is known about what drives their selection and whether they have housekeeping functions. The prototypic T15-NAb,first identified because of its protective role in infection,is representative of a special type of NAb response that specifically recognizes and forms complexes with apoptotic cells and which promotes cell-corpse engulfment by phagocytes. We now show that this T15-NAb IgM-mediated clearance process is dependent on the recruitment of C1q and mannose-binding lectin,which have known immune modulatory activities that also provide eat me" signals for enhancing phagocytosis. Further investigation revealed that the addition of T15-NAb significantly suppressed in vitro LPS-induced TNF-alpha and IL-6 secretion by the macrophage-like cell line� View Publication

In somatic cells,eroded telomeres can induce DNA double-strand break signaling,leading to a form of replicative senescence or apoptosis,both of which are barriers to tumorigenesis. However,cancer cells might display telomere dysfunctions which in conjunction with defects in DNA repair and apoptosis,enables them to circumvent these pathways. Chronic lymphocytic leukemia (CLL) cells exhibit telomere dysfunction,and a subset of these cells are resistant to DNA damage-induced apoptosis and display short telomeres. We show here that these cells exhibit significant resection of their protective telomeric 3' single-stranded overhangs and an increased number of telomere-induced foci containing gammaH2AX and 53BP1. Chromatin immunoprecipitation and immunofluorescence experiments demonstrated increased levels of telomeric Ku70 and phospho-S2056-DNA-PKcs,2 essential components of the mammalian nonhomologous end-joining DNA repair system. Notably,these CLL cells display deletions of telomeric signals on one or 2 chromatids in parallel with 11q22 deletions,or with 13q14 deletions associated with another chromosomal aberration or with a complex karyotype. Taken together,our results indicate that a subset of CLL cells from patients with an unfavorable clinical outcome harbor a novel type of chromosomal aberration resulting from telomere dysfunction. View Publication

Circulating tumor cells (CTCs) are important clinical indicators for prognosis and treatment efficacy. However,CTC investigation is hampered by their low number,making the establishment of permanent CTC lines very challenging. We derived and characterized nine CTC lines using blood samples from a patient with metastatic colorectal cancer collected before and after chemotherapy and targeted therapy,and during cancer progression. These cell lines displayed an intermediate epithelial/mesenchymal phenotype,stem-cell like characteristics,angiogenesis potential,an osteomimetic signature and the capacity to escape from the immune system. Moreover,they showed changes in mRNA and protein expression (e.g.,DEFA6,ABCB1 and GAL),whereas analysis of chromosomal copy number aberrations revealed no significant variation over time. These data indicate that although CTC lines derived from sequential blood samples during therapy have common traits,treatment-resistant CTC clones with distinct phenotypic characteristics are selected over time. View Publication

Current treatment options for advanced and hormone refractory prostate cancer are limited and responses to commonly used androgen pathway inhibitors are often unsatisfactory. Our recent results indicated that sodium ionophore monensin is one of the most potent and cancer-specific inhibitors in a systematic sensitivity testing of most known drugs and drug-like molecules in a panel of prostate cancer cell models. Because monensin has been extensively used in veterinary applications to build muscle mass in cattle,the link to prostate cancer and androgen signaling was particularly interesting. Here,we showed that monensin effects at nanomolar concentrations are linked to induction of apoptosis and potent reduction of androgen receptor mRNA and protein in prostate cancer cells. Monensin also elevated intracellular oxidative stress in prostate cancer cells as evidenced by increased generation of intracellular reactive oxygen species and by induction of a transcriptional profile characteristic of an oxidative stress response. Importantly,the antiproliferative effects of monensin were potentiated by combinatorial treatment with the antiandrogens and antagonized by antioxidant vitamin C. Taken together,our results suggest monensin as a potential well-tolerated,in vivo compatible drug with strong proapoptotic effects in prostate cancer cells,and synergistic effects with antiandrogens. Moreover,our data suggest a general strategy by which the effects of antiandrogens could be enhanced by combinatorial administration with agents that increase oxidative stress in prostate cancer cells. View Publication

The atypical cannabinoid Abn-CBD improves the inflammatory status in preclinical models of several pathologies,including autoimmune diseases. However,its potential for modulating inflammation in autoimmune type 1 diabetes (T1D) is unknown. Herein we investigate whether Abn-CBD can modulate the inflammatory response during T1D onset using a mouse model of T1D (non-obese diabetic- (NOD)-mice) and of beta cell damage (streptozotocin (STZ)-injected mice). Six-week-old female NOD mice were treated with Abn-CBD (0.1-1 mg/kg) or vehicle during 12 weeks and then euthanized. Eight-to-ten-week-old male C57Bl6/J mice were pre-treated with Abn-CBD (1 mg/kg of body weight) or vehicle for 1 week,following STZ challenge,and euthanized 1 week later. Blood,pancreas,pancreatic lymph nodes (PLNs) and T cells were collected and processed for analysis. Glycemia was also monitored. In NOD mice,treatment with Abn-CBD significantly reduced the severity of insulitis and reduced the pro-inflammatory profile of CD4+ T cells compared to vehicle. Concomitantly,Abn-CBD significantly reduced islet cell apoptosis and improved glucose tolerance. In STZ-injected mice,Abn-CBD decreased circulating proinflammatory cytokines and ameliorated islet inflammation reducing intra-islet phospho-NF-$\kappa$B and TXNIP. Abn-CBD significantly reduced 2 folds intra-islet CD8+ T cells and reduced Th1/non-Th1 ratio in PLNs of STZ-injected mice. Islet cell apoptosis and intra-islet fibrosis were also significantly reduced in Abn-CBD pre-treated mice compared to vehicle. Altogether,Abn-CBD reduces circulating and intra-islet inflammation,preserving islets,thus delaying the progression of insulitis. Hence,Abn-CBD and related compounds emerge as new candidates to develop pharmacological strategies to treat the early stages of T1D. View Publication

The limited ability of cytotoxic CD8+ T cells to infiltrate solid tumors and function within the tumor microenvironment presents a major roadblock to effective immunotherapy. Ion channels and Ca2+-dependent signaling events control the activity of T cells and are implicated in the failure of immune surveillance in cancer. Reduced KCa3.1 channel activity mediates the heightened inhibitory effect of adenosine on the chemotaxis of circulating T cells from head and neck squamous cell carcinoma (HNSCC) patients. Herein,we conducted experiments that elucidate the mechanisms of KCa3.1 dysfunction and impaired chemotaxis in HNSCC CD8+ T cells. The Ca2+ sensor calmodulin (CaM) controls multiple cellular functions including KCa3.1 activation. Our data showed that CaM expression is lower in HNSCC than healthy donor (HD) T cells. This reduction was due to an intrinsic decrease in the genes encoding CaM combined to the failure of HNSCC T cells to upregulate CaM upon activation. Furthermore,the reduction in CaM was confined to the plasma membrane and resulted in decreased CaM-KCa3.1 association and KCa3.1 activity (which was rescued by the delivery of CaM). IFN$\gamma$ production,also Ca2+- and CaM-dependent,was instead not reduced in HNSCC T cells,which maintained intact cytoplasmic CaM and Ca2+ fluxing ability. Knockdown of CaM in HD T cells decreased KCa3.1 activity,but not IFN$\gamma$ production,and reduced their chemotaxis in the presence of adenosine,thus recapitulating HNSCC T cell dysfunction. Activation of KCa3.1 with 1-EBIO restored the ability of CaM knockdown HD T cells to chemotax in the presence of adenosine. Additionally,1-EBIO enhanced INF$\gamma$ production. Our data showed a localized downregulation of membrane-proximal CaM that suppressed KCa3.1 activity in HNSCC circulating T cells and limited their ability to infiltrate adenosine-rich tumor-like microenvironments. Furthermore,they indicate that KCa3.1 activators could be used as positive CD8+ T cell modulators in cancers. View Publication