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

Virus-specific CD8(+) T cells are associated with declining viremia in acute human immunodeficiency virus (HIV)1 infection,but do not correlate with control of viremia in chronic infection,suggesting a progressive functional defect not measured by interferon gamma assays presently used. Here,we demonstrate that HIV-1-specific CD8(+) T cells proliferate rapidly upon encounter with cognate antigen in acute infection,but lose this capacity with ongoing viral replication. This functional defect can be induced in vitro by depletion of CD4(+) T cells or addition of interleukin 2-neutralizing antibodies,and can be corrected in chronic infection in vitro by addition of autologous CD4(+) T cells isolated during acute infection and in vivo by vaccine-mediated induction of HIV-1-specific CD4(+) T helper cell responses. These data demonstrate a loss of HIV-1-specific CD8(+) T cell function that not only correlates with progressive infection,but also can be restored in chronic infection by augmentation of HIV-1-specific T helper cell function. This identification of a reversible defect in cell-mediated immunity in chronic HIV-1 infection has important implications for immunotherapeutic interventions. View Publication

UNLABELLED: AC133 cells,a subpopulation of CD34+ hematopoietic stem cells,can transform into endothelial cells that may integrate into the neovasculature of tumors or ischemic tissue. Most current imaging modalities do not allow monitoring of early migration and incorporation of endothelial progenitor cells (EPCs) into tumor neovasculature. The goals of this study were to use magnetic resonance imaging (MRI) to track the migration and incorporation of intravenously injected,magnetically labeled EPCs into the blood vessels in a rapidly growing flank tumor model and to determine whether the pattern of EPC incorporation is related to the time of injection or tumor size. MATERIALS AND METHODS: EPCs labeled with ferumoxide-protamine sulfate (FePro) complexes were injected into mice bearing xenografted glioma,and MRI was obtained at different stages of tumor development and size. RESULTS: Migration and incorporation of labeled EPCs into tumor neovasculature were detected as low signal intensity on MRI at the tumor periphery as early as 3 days after EPC administration in preformed tumors. However,low signal intensities were not observed in tumors implanted at the time of EPC administration until tumor size reached 1 cm at 12 to 14 days. Prussian blue staining showed iron-positive cells at the sites corresponding to low signal intensity on MRI. Confocal microscopy showed incorporation into the neovasculature,and immunohistochemistry clearly demonstrated the transformation of the administered EPCs into endothelial cells. CONCLUSION: MRI demonstrated the incorporation of FePro-labeled human CD34+/AC133+ EPCs into the neovasculature of implanted flank tumors. View Publication

Mesenchymal stem cells (MSCs) modulate cardiac healing after myocardial injury through the release of paracrine factors,but the exact mechanisms are still unknown. One possible mechanism is through mobilization of endogenous cardiac stem cells (CSCs). This study aimed to test the pro-migratory effect of MSC conditioned medium (MSC-CM) on endogenous CSCs from human cardiac tissue. By using a three-dimensional collagen assay,we found that MSC-CM improved migration of cells from human cardiac tissue. Cell counts,perimeter and area measurements were utilized to quantify migration effects. To examine whether resident stem cells were among the migrating cells,specific stem cell properties were investigated. The migrating cells displayed strong similarities with resident Cardiac Atrial appendage Stem Cells (CASCs),including a clonogenic potential of ˜21.5% and expression of pluripotency associated genes like Oct-4,Nanog,c-Myc and Klf-4. Similar to CASCs,migrating cells demonstrated high aldehyde dehydrogenase activity and were able to differentiate towards cardiomyocytes. Receptor tyrosine kinase analysis and collagen assays performed with recombinant platelet derived growth factor (PDGF)-AA and Imatinib Mesylate,a PDGF receptor inhibitor,suggested a role for the PDGF-AA/PDGF receptor $$ axis in enhancing the migration process of CASCs. In conclusion,our findings demonstrate that factors present in MSC-CM improve migration of resident stem cells from human cardiac tissue. These data open doors towards future therapies in which MSC secreted factors,like PDGF-AA,can be utilized to enhance the recruitment of CASCs towards the site of myocardial injury. View Publication

OBJECTIVE: T lymphocytes play an important role in systemic sclerosis (SSc),a connective tissue disease characterized by inflammation,fibrosis,and vascular damage. While their precise role and antigen specificity are unclear,T cell-derived cytokines likely contribute to the induction of fibrosis. The aim of this study was to establish the role of cytokine dysregulation by T cells in the pathogenesis of SSc. METHODS: To identify relationships between a specific cytokine,T cell subset,and the disease course,we studied a large cohort of patients with diffuse cutaneous SSc (dcSSc) or limited cutaneous SSc (lcSSc). Using Luminex analysis and intracellular cytokine staining,we analyzed the intrinsic ability of CD4+ and CD8+ T cell subsets to produce cytokines following in vitro activation. RESULTS: High levels of the profibrotic type 2 cytokine interleukin-13 (IL-13) were produced following activation of peripheral blood effector CD8+ T cells from SSc patients as compared with normal controls or with patients with rheumatoid arthritis. In contrast,CD4+ T cells showed a lower and more variable level of IL-13 production. This abnormality correlated with the extent of fibrosis and was more pronounced in dcSSc patients than in lcSSc patients. CONCLUSION: Dysregulated IL-13 production by effector CD8+ T cells is important in the pathogenesis of SSc and is critical in the predisposition to more severe forms of cutaneous disease. Our study is the first to identify a specific T cell phenotype that correlates with disease severity in SSc and can be used as a marker of immune dysfunction in SSc and as a novel therapeutic target. View Publication

Laminin and fibronectin are glycoproteins that influence cell behavior and mediate cell/substratum adhesion. We have examined the interaction of these macromolecules with the serine protease plasminogen activator (PA) in two types of extracellular matrices; one produced by the murine Engelbreth-Holm-Swarm (EHS) tumor (Matrigel),and another by normal kidney epithelial cells in culture. Matrigel was found to contain significant quantities of tissue-type PA (tPA). Two of the major components of Matrigel,laminin and type IV collagen,were also examined. Tissue-type PA was associated with purified preparations of laminin; however,it was not found associated with type IV collagen. Normal kidney epithelial cells in culture secrete large amounts of urokinase (UK) and deposit a subepithelial matrix containing both laminin and fibronectin. These matrix macromolecules were isolated from the deposited matrix by immunoprecipitation,examined by zymography,and found to contain UK. The potential role of this interaction in the mechanisms of cell migration and matrix remodeling is discussed. View Publication

The glycogen synthase kinase 3 (GSK3) inhibitor,6-bromoindirubin-3'-oxime (BIO),is a key regulator of many signaling pathways to maintain pluripotency of human and mouse embryonic stem cells (ESCs). However,the effect of BIO on derivation of dairy goat male germline stem cells (mGSCs) remains unclear. The objectives of this study were to investigate whether BIO influences derivation of dairy goat mGSCs. Dairy goat mGSCs were cultured in mTeSR containing BIO medium and its effects on the proliferation ability of goat mGSCs (derived from goats ≤2 mo of age) were evaluated by 5-Bromo-2-deoxyuridine (BrdU) incorporation and alkaline phosphatase (AP) staining. Furthermore,its effects on maintenance of the undifferentiated state of mGSCs in late passages of cultures,as well as the capacity of mGSCs to differentiate into embryoid bodies (EBs) were examined. The presence of BIO increased the mitosis index and the number of AP positive colonies,as well as expression of pluripotent markers,Oct4,Nanog,Sox2,C-myc,Klf4,E-cadherin,and the proliferative markers,Pcna and C-myc. In contrast,there was no significant change in expression of apoptosis markers,P53,P21 and cyclin-related genes (Cyclin A,CDK2,Cyclin D1),as determined by RT-PCR analysis. When mGSCs were cultured in mTeSR medium containing BIO,EBs were formed,which were capable of further differentiating into various cell types found in the three embryonic germ layers,as determined by immunofluorescence and/or histologic staining. In conclusion,adding BIO to cultures BIO significantly promoted establishment of goat mGSC colonies and maintained their undifferentiated state. View Publication

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

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