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The presence of T regulatory (Treg) cells in the tumor microenvironment is associated with poor prognosis and resistance to therapies aimed at reactivating anti-tumor immune responses. Therefore,depletion of tumor-infiltrating Tregs is a potential approach to overcome resistance to immunotherapy. However,identifying Treg-specific targets to drive such selective depletion is challenging. CCR8 has recently emerged as one of these potential targets. Here,we describe GS-1811,a novel therapeutic monoclonal antibody that specifically binds to human CCR8 and is designed to selectively deplete tumor-infiltrating Tregs. We validate previous findings showing restricted expression of CCR8 on tumor Tregs,and precisely quantify CCR8 receptor densities on tumor and normal tissue T cell subsets,demonstrating a window for selective depletion of Tregs in the tumor. Importantly,we show that GS-1811 depleting activity is limited to cells expressing CCR8 at levels comparable to tumor-infiltrating Tregs. Targeting CCR8 in mouse tumor models results in robust anti-tumor efficacy,which is dependent on Treg depleting activity,and synergizes with PD-1 inhibition to promote anti-tumor responses in PD-1 resistant models. Our data support clinical development of GS-1811 to target CCR8 in cancer and drive tumor Treg depletion in order to promote anti-tumor immunity. View Publication

The phosphatidylinositol-3 kinase (PI3K) pathway plays a central role in regulating numerous biologic processes,including survival,adhesion,migration,metabolic activity,proliferation,differentiation,and end cell activation through the generation of the potent second messenger PI-3,4,5-trisphosphate (PI-3,4,5-P(3)). To ensure that activation of this pathway is appropriately suppressed/terminated,the ubiquitously expressed 54-kDa tumor suppressor PTEN hydrolyzes PI-3,4,5-P(3) to PI-4,5-P(2),whereas the 145-kDa hematopoietic-restricted SH2-containing inositol 5'-phosphatase SHIP (also known as SHIP1),the 104-kDa stem cell-restricted SHIP sSHIP,and the more widely expressed 150-kDa SHIP2 break it down to PI-3,4-P(2). In this review,we focus on the properties of these phospholipid phosphatases and summarize recent data showing that the activities of these negative regulators often are modulated by simply altering their protein levels. We also highlight the critical role that SHIP plays in lipopolysaccharide-induced macrophage activation and in endotoxin tolerance. View Publication

Differences in clinical outcome of simian immunodeficiency virus (SIV) infection in disease-resistant African sooty mangabeys (SM) and disease-susceptible Asian rhesus macaques (RM) prompted us to examine the role of regulatory T cells (Tregs) in these two animal models. Results from a cross-sectional study revealed maintenance of the frequency and absolute number of peripheral Tregs in chronically SIV-infected SM while a significant loss occurred in chronically SIV-infected RM compared to uninfected animals. A longitudinal study of experimentally SIV-infected animals revealed a transient increase in the frequency of Tregs from baseline values following acute infection in RM,but no change in the frequency of Tregs occurred in SM during this period. Further examination revealed a strong correlation between plasma viral load (VL) and the level of Tregs in SIV-infected RM but not SM. A correlation was also noted in SIV-infected RM that control VL spontaneously or in response to antiretroviral chemotherapy. In addition,immunofluorescent cell count assays showed that while Treg-depleted peripheral blood mononuclear cells from RM led to a significant enhancement of CD4+ and CD8+ T-cell responses to select pools of SIV peptides,there was no detectable T-cell response to the same pool of SIV peptides in Treg-depleted cells from SIV-infected SM. Our data collectively suggest that while Tregs do appear to play a role in the control of viremia and the magnitude of the SIV-specific immune response in RM,their role in disease resistance in SM remains unclear. View Publication

While it is now acknowledged that CD4+ T cells expressing CD25 and Foxp3 (Treg cells) regulate immune responses and,consequently,influence the pathogenesis of infectious diseases,the regulatory response mediated by Treg cells upon infection by Trypanosoma cruzi was still poorly characterized. In order to understand the role of Treg cells during infection by this protozoan parasite,we determined in time and space the magnitude of the regulatory response and the phenotypic,functional and transcriptional features of the Treg cell population in infected mice. Contrary to the accumulation of Treg cells reported in most chronic infections in mice and humans,experimental T. cruzi infection was characterized by sustained numbers but decreased relative frequency of Treg cells. The reduction in Treg cell frequency resulted from a massive accumulation of effector immune cells,and inversely correlated with the magnitude of the effector immune response as well as with emergence of acute immunopathology. In order to understand the causes underlying the marked reduction in Treg cell frequency,we evaluated the dynamics of the Treg cell population and found a low proliferation rate and limited accrual of peripheral Treg cells during infection. We also observed that Treg cells became activated and acquired a phenotypic and transcriptional profile consistent with suppression of type 1 inflammatory responses. To assess the biological relevance of the relative reduction in Treg cells frequency observed during T. cruzi infection,we transferred in vitro differentiated Treg cells at early moments,when the deregulation of the ratio between regulatory and conventional T cells becomes significant. Intravenous injection of Treg cells dampened parasite-specific CD8+ T cell immunity and affected parasite control in blood and tissues. Altogether,our results show that limited Treg cell response during the acute phase of T. cruzi infection enables the emergence of protective anti-parasite CD8+ T cell immunity and critically influences host resistance. View Publication

BackgroundStem-cell-derived therapy is a promising option for tissue regeneration. Human iPSC-derived progenitors of smooth muscle cells (pSMCs) exhibit limited proliferation and differentiation,which minimizes the risk of tumor formation while restoring smooth muscle cells (SMCs). Up to 29% of women suffer from recurrence of vaginal prolapse after prolapse surgery. Therefore,there is a need for therapies that can restore vaginal function. SMCs contribute to vaginal tone and contractility. We sought to examine whether human pSMCs can restore vaginal function in a rat model.MethodsFemale immunocompromised RNU rats were divided into 5 groups: intact controls (n?=?12),VSHAM (surgery?+?saline injection,n?=?35),and three cell-injection groups (surgery?+?cell injection using pSMCs from three patients,n?=?14/cell line). The surgery to induce vaginal injury was analogous to prolapse surgery. Menopause was induced by surgical ovariectomy. The vagina,urethra,bladder were harvested 10 weeks after surgery (5 weeks after cell injection). Organ bath myography was performed to evaluate the contractile function of the vagina,and smooth muscle thickness was examined by tissue immunohistochemistry. Collagen I,collagen III,and elastin mRNA and protein expressions in tissues were assessed.ResultsVaginal smooth muscle contractions induced by carbachol and KCl in the cell-injection groups were significantly greater than those in the VSHAM group. Collagen I protein expression in the vagina of the cell-injections groups was significantly higher than in the VSHAM group. Vaginal elastin protein expression was similar between the cell-injection and VSHAM groups. In the urethra,gene expression levels of collagen I,III,and elastin were all significantly greater in the cell-injection groups than in the VSHAM group. Collagen I,III,and elastin protein expression of the urethra did not show a consistent trend between cell-injection groups and the VSHAM group.ConclusionsHuman iPSC-derived pSMCs transplantation appears to be associated with improved contractile function of the surgically injured vagina in a rat model. This is accompanied by changes in extracellular protein expression the vagina and urethra. These observations support further efforts in the translation of pSMCs into a treatment for regenerating the surgically injured vagina in women who suffer recurrent prolapse after surgery.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03900-3. View Publication

Tumor angiogenesis is essential for malignant growth and metastasis. Bone marrow (BM)-derived endothelial progenitor cells (EPC) contribute to angiogenesis-mediated tumor growth. EPC ablation can reduce tumor growth; however,the lack of a marker that can track EPCs from the BM to tumor neovasculature has impeded progress in understanding the molecular mechanisms underlying EPC biology. Here,we report the use of transgenic mouse and lentiviral models to monitor the BM-derived compartment of the tumor stroma; this approach exploits the selectivity of the transcription factor inhibitor of DNA binding 1 (Id1) for EPCs to track EPCs in the BM,blood,and tumor stroma,as well as mature EPCs. Acute ablation of BM-derived EPCs using Id1-directed delivery of a suicide gene reduced circulating EPCs and yielded significant defects in angiogenesis-mediated tumor growth. Additionally,use of the Id1 proximal promoter to express microRNA-30-based short hairpin RNA inhibited the expression of critical EPC-intrinsic factors,confirming that signaling through vascular endothelial growth factor receptor 2 is required for EPC-mediated tumor biology. By exploiting the selectivity of Id1 gene expression in EPCs,our results establish a strategy to track and target EPCs in vivo,clarifying the significant role that EPCs play in BM-mediated tumor angiogenesis. View Publication

CXCL12-induced chemotaxis and adhesion to VCAM-1 decrease as B cells differentiate in the bone marrow. However,the mechanisms that regulate CXCL12/CXCR4-mediated signaling are poorly understood. We report that after CXCL12 stimulation of progenitor B cells,focal adhesion kinase (FAK) and PI3K are inducibly recruited to raft-associated membrane domains. After CXCL12 stimulation,phosphorylated FAK is also localized in membrane domains. The CXCL12/CXCR4-FAK pathway is membrane cholesterol dependent and impaired by metabolic inhibitors of G(i),Src family,and the GTPase-activating protein,regulator of G protein signaling 1 (RGS1). In the bone marrow,RGS1 mRNA expression is low in progenitor B cells and high in mature B cells,implying developmental regulation of CXCL12/CXCR4 signaling by RGS1. CXCL12-induced chemotaxis and adhesion are impaired when FAK recruitment and phosphorylation are inhibited by either membrane cholesterol depletion or overexpression of RGS1 in progenitor B cells. We conclude that the recruitment of signaling molecules to specific membrane domains plays an important role in CXCL12/CXCR4-induced cellular responses. View Publication

As cyclin-dependent kinases (CDKs) regulate cell cycle progression and RNA transcription,CDKs are attractive targets for creating cancer cell treatments. In this study we investigated the effects of the small molecular agent NU6140 (inhibits CDK2 and cyclin A interaction) on human embryonic stem (hES) cells and embryonal carcinoma-derived (hEC) cells via the expression of transcription factors responsible for pluripotency. A multiparameter flow cytometric method was used to follow changes in the expression of NANOG,OCT4,and SOX2 together in single cells. Both hES and hEC cells responded to NU6140 treatment by induced apoptosis and a decreased expression of NANOG,OCT4,and SOX2 in surviving cells. A higher sensitivity to NU6140 application in hES than hEC cells was detected. NU6140 treatment arrested hES and hEC cells in the G2 phase and inhibited entry into the M phase as evidenced by no significant increase in histone 3 phosphorylation. When embryoid bodies (EBs) formed from NU6104 treated hES cells were compared to EBs from untreated hES cells differences in ectodermal,endodermal,and mesodermal lineages were found. The results of this study highlight the importance of CDK2 activity in maintaining pluripotency of hES and hEC cells and in differentiation of hES cells. View Publication

Memory T-cells are essential to maintain long-term immunological memory. It is widely thought that the bone marrow (BM) plays an important role in the long-term maintenance of memory T-cells. There is controversy however on the longevity and recirculating kinetics of BM memory T-cells. While some have proposed that the BM is a reservoir for long-lived,non-circulating memory T-cells,it has also been suggested to be the preferential site for memory T-cell self-renewal. In this study,we used in vivo deuterium labeling in goats to simultaneously quantify the average turnover rates-and thereby expected lifespans-of memory T-cells from BM,blood and lymph nodes (LN). While the fraction of Ki-67 positive cells,a snapshot marker for recent cell division,was higher in memory T-cells from blood compared to BM and LN,in vivo deuterium labeling revealed no substantial differences in the expected lifespans of memory T-cells between these compartments. Our results support the view that the majority of memory T-cells in the BM are self-renewing as fast as those in the periphery,and are continuously recirculating between the blood,BM,and LN. View Publication

Stem cells exhibit a promiscuous gene expression pattern. We show herein that the early embryo and adult MSCs express B-cell receptor component mRNAs. To examine possible bearings of these genes on the expressing cells,we studied immunoglobulin mu chain-deficient mice. Pregnant mu chain-deficient females were found to produce a higher percentage of defective morulae compared with control females. Structure analysis indicated that the mu mRNA species found in embryos and in mesenchyme consist of the constant region of the mu heavy chain that encodes a recombinant 50-kDa protein. In situ hybridization localized the constant mu gene expression to loose mesenchymal tissues within the day-12.5 embryo proper and the yolk sac. In early embryo and in adult mesenchyme from mu-deficient mice,delta replaced mu chain,implying a possible requirement of these alternative molecules for embryo development and mesenchymal functions. Indeed,overexpression of the mesenchymal-truncated mu heavy chain in 293T cells resulted in specific subcellular localization and in G(1) growth arrest. The lack of such occurrence following overexpression of a complete,rearranged form of mu chain suggests that the mesenchymal version of this mRNA may possess unique functions. View Publication

Retinoic acid (RA) is used to treat leukemia and other cancers through its ability to promote cancer cell differentiation. Strategies to enhance the anticancer effects of RA could deepen and broaden its beneficial therapeutic applications. In this study,we describe a receptor cross-talk system that addresses this issue. RA effects are mediated by RAR/RXR receptors that we show are modified by interactions with the aryl hydrocarbon receptor (AhR),a protein functioning both as a transcription factor and a ligand-dependent adaptor in an ubiquitin ligase complex. RAR/RXR and AhR pathways cross-talk at the levels of ligand-receptor and also receptor-promoter interactions. Here,we assessed the role of AhR during RA-induced differentiation and a hypothesized convergence at Oct4,a transcription factor believed to maintain stem cell characteristics. RA upregulated AhR and downregulated Oct4 during differentiation of HL-60 promyelocytic leukemia cells. AhR overexpression in stable transfectants downregulated Oct4 and also decreased ALDH1 activity,another stem cell-associated factor,enhancing RA-induced differentiation as indicated by cell differentiation markers associated with early (CD38 and CD11b) and late (neutrophilic respiratory burst) responses. AhR overexpression also increased levels of activated Raf1,which is known to help propel RA-induced differentiation. RNA interference-mediated knockdown of Oct4 enhanced RA-induced differentiation and G(0) cell-cycle arrest relative to parental cells. Consistent with the hypothesized importance of Oct4 downregulation for differentiation,parental cells rendered resistant to RA by biweekly high RA exposure displayed elevated Oct4 levels that failed to be downregulated. Together,our results suggested that therapeutic effects of RA-induced leukemia differentiation depend on AhR and its ability to downregulate the stem cell factor Oct4. View Publication

Elevated numbers of primitive Philadelphia chromosome-positive (Ph+) progenitors,including long-term culture-initiating cells (LTC-IC) as well as colony-forming cells (CFC),have been previously described in the blood of patients with chronic myeloid leukemia (CML) in chronic phase with high white blood cell counts. In the present study,which focused primarily on an analysis of circulating progenitors present in such patients at diagnosis,we discovered the frequent and occasionally exclusive presence of circulating normal (Ph-) LTC-IC,often at levels above those seen for LTC-IC in the blood of normal individuals. The presence of detectable numbers of circulating Ph- LTC-IC was independent of the fact that the same peripheral blood samples also contained elevated numbers of predominantly or exclusively Ph+ CFC. Interestingly,both the Ph+ and Ph- LTC-IC in these samples were CD34+CD71- and variably CD38- and Thy-1+,as previously documented for LTC-IC in normal marrow. Thus,neither CD38 nor Thy-1 expression was useful for discriminating between Ph+ and Ph- LTC-IC in mixed populations. Nevertheless,an association of these phenotypes with LTC-IC function did allow highly enriched (textgreater 5% pure) suspensions of either Ph+ or Ph- LTC-IC to be obtained from selected samples of CML blood in which the initial LTC-IC population was either predominantly Ph+ or Ph-,respectively. These findings suggest that the mechanisms causing mobilization of leukemic stem cells in untreated CML patients may affect their normal counterparts. They also indicate a possible new source of autologous cells for the support of intensive therapy of CML patients. Finally,they provide a method for obtaining the most highly purified populations of Ph+ LTC-IC described to date. This method should be useful for further analyses of the molecular activities of these very primitive neoplastic cells. View Publication