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

During drug development,measurement of suitable pharmacodynamic biomarkers is key to establishing in vivo drug activity. Binding of monoclonal antibody (mAb) therapeutics to soluble target proteins often results in elevated serum levels of their target antigen,and measuring total (free and bound) concentration of the target antigen can be an important means of demonstrating that the mAb has reached its specific target. However,accurately measuring soluble circulating antigen in preclinical or clinical samples in the presence of a therapeutic mAb presents a bioanalytical challenge. Particularly in the case of low molecular weight and/or multimeric targets,epitopes for capture and detection of the target by reagent antibodies can be obscured by bound therapeutic mAb. Lymphotoxin-alpha (LTα) is a cytokine in the TNF superfamily that has been implicated in the pathophysiology of autoimmune disease,and is a therapeutic target for neutralizing mAb. During preclinical safety studies in cynomolgus macaques,we encountered difficulties in measuring total LTα in serum of dosed animals. When serum LTα trimer was saturated with the anti-LTα mAb,binding of two reagent antibodies,as required for a classic sandwich ELISA,was not feasible,and dissociation methods were also found to be unsuitable. We therefore developed an approach in which excess anti-LTα mAb was added to the in vitro assay system to fully saturate all binding sites,and an anti-idiotypic antibody was used to detect bound therapeutic antibody. Using this method,total LTα could be accurately measured in cynomolgus macaque serum,and was observed to increase with increasing anti-LTα therapeutic mAb dose. Additional in vitro studies demonstrated that the method worked equally well in human serum. This assay strategy will be useful for quantifying total concentrations of other small and/or multimeric target proteins in the presence of a therapeutic antibody. View Publication

The growth of human megakaryocyte progenitors from human bone marrow (BM) cells was compared using a methylcellulose semisolid assay supplemented either by normal human plasma or by a serum-free medium. Far better growth of megakaryocyte colonies from CD34+ BM cells stimulated by interleukin 3 (IL-3) and interleukin 6 (IL-6) was observed in serum-free medium compared with human plasma supplemented cultures. These results were confirmed in liquid cultures using the same serum-free medium composition. The megakaryocytes were identified by using an immunocytochemical procedure after labeling with an anti-GPIIb-IIIa monoclonal antibody. High percentages (15 to 20%) of megakaryocytes were present in serum-free cultures stimulated by IL-3 alone or combined with IL-6. The absolute number of megakaryocytes in serum-free medium exceeds by 3.3 (IL-3 plus IL-6) to 4.4 (IL-3 alone) times the corresponding number of megakaryocytes observed in human plasma supplemented cultures. The optimal concentration of IL-3 alone was 5 ng/ml,and an optimal synergistic effect of IL-6 (5 ng/ml) was obtained when combined with a suboptimal dose of IL-3 (1 ng/ml). The poor growth of megakaryocyte colonies from CD34+ BM cells in human plasma suggested the presence of an inhibitory factor. When a neutralizing monoclonal antibody against transforming growth factor beta (TGF beta) is present in human plasma supplemented cultures of CD34+ BM cells,the number of megakaryocyte colonies is increased to the level observed in corresponding serum-free cultures. The high efficiency of this serum-free medium to promote the growth of human megakaryocytes will be useful to study the effects of regulators and platelet agonists acting on human megakaryocytes,without interference from factors in the serum. View Publication

There is a medical need for an agent with the positive effects of estrogen on bone and the cardiovascular system,but without the negative effects on reproductive tissue. Raloxifene (LY139481 HCI) is a benzothiophene derivative that binds to the estrogen receptor and inhibits the effects of estrogen on the uterus. In an ovariectomized (OVX) rat model we investigated the effects of raloxifene on bone loss (induced by estrogen deficiency),serum lipids,and uterine tissue. After oral administration of raloxifene for 5 wk (0.1-10 mg/kg per d) to OVX rats,bone mineral density in the distal femur and proximal tibia was significantly greater than that observed in OVX controls (ED50 of 0.03-0.3 mg/kg). Serum cholesterol was lower in the raloxifene-treated animals,which had a minimal effective dose of 0.1 mg/kg and an approximate oral ED50 of 0.2 mg/kg. The effects of raloxifene on bone and serum cholesterol were comparable to those of a 0.1-mg/kg per d oral dose of ethynyl estradiol. Raloxifene diverged dramatically from estrogen in its lack of significant estrogenic effects on uterine tissue. Ethynyl estradiol produced a marked elevation in a number of uterine histologic parameters (e.g.,epithelial cell height,stromal eosinophilia). These data suggest that raloxifene has promise as an agent with beneficial bone and cardiovascular effects in the absence of significant uterine effects. View Publication

The human induced pluripotent stem cell (hiPSC) provides a breakthrough approach that helps overcoming ethical and allergenic challenges posed in application of neural stem cells (NSCs) in targeted cancer gene therapy. However,the tumor-tropic capacity of hiPSC-derived NSCs (hiPS-NSCs) still has much room to improve. Here we attempted to promote the tumor tropism of hiPS-NSCs by manipulating the activity of endogenous miR-199a/214 cluster that is involved in regulation of hypoxia-stimulated cell migration. We first developed a baculovirus-delivered CRISPR interference (CRISPRi) system that sterically blocked the E-box element in the promoter of the miR-199a/214 cluster with an RNA-guided catalytically dead Cas9 (dCas9). We then applied this CRISPRi system to hiPS-NSCs and successfully suppressed the expression of miR-199a-5p,miR-199a-3p,and miR-214 in the microRNA gene cluster. Meanwhile,the expression levels of their targets related to regulation of hypoxia-stimulated cell migration,such as HIF1A,MET,and MAPK1,were upregulated. Further migration assays demonstrated that the targeted inhibition of the miR-199a/214 cluster significantly enhanced the tumor tropism of hiPS-NSCs both in vitro and in vivo. These findings suggest a novel application of CRISPRi in NSC-based tumor-targeted gene therapy. View Publication

BACKGROUND & AIMS Wnt signaling regulates multiple aspects of intestinal physiology,including stem cell maintenance. Paneth cells support stem cells by secreting Wnt,but little is known about the exact sources and primary functions of individual Wnt family members. METHODS We analyzed intestinal tissues and cultured epithelial cells from adult mice with conditional deletion of Wnt3 (Vil-CreERT2;Wnt3fl/fl mice). We also analyzed intestinal tissues and cells from Atoh1 mutant mice,which lack secretory cells. RESULTS Unexpectedly,Wnt3 was dispensable for maintenance of intestinal stem cells in mice,indicating a redundancy of Wnt signals. By contrast,cultured crypt organoids required Paneth cell-derived Wnt3. Addition of exogenous Wnt,or coculture with mesenchymal cells,restored growth of Vil-CreERT2;Wnt3fl/fl crypt organoids. Intestinal organoids from Atoh1 mutant mice did not grow or form Paneth cells; addition of Wnt3 allowed growth in the absence of Paneth cells. Wnt signaling had a synergistic effect with the Lgr4/5 ligand R-spondin to induce formation of Paneth cells. Mosaic expression of Wnt3 in organoids using a retroviral vector promoted differentiation of Paneth cells in a cell-autonomous manner. CONCLUSIONS Wnt is part of a signaling loop that affects homeostasis of intestinal stem and Paneth cells in mice. Wnt3 signaling is required for growth and development of organoid cultures,whereas nonepithelial Wnt signals could provide a secondary physiological source of Wnt. View Publication

Human rhinovirus (HRV)-induced respiratory infections are associated with elevated levels of IFN-gamma-inducible protein 10 (IP-10),which is an enhancer of T lymphocyte chemotaxis and correlates with symptom severity and T lymphocyte number. Increased IP-10 expression is exhibited by airway epithelial cells following ex vivo HRV challenge and requires intracellular viral replication; however,there are conflicting reports regarding the necessity of type I IFN receptor ligation for IP-10 expression. Furthermore,the involvement of resident airway immune cells,predominantly bronchoalveolar macrophages,in contributing to HRV-stimulated IP-10 elaboration remains unclear. In this regard,our findings demonstrate that ex vivo exposure of human peripheral blood monocytes and bronchoalveolar macrophages (monocytic cells) to native or replication-defective HRV serotype 16 (HRV16) resulted in similarly robust levels of IP-10 release,which occurred in a time- and dose-dependent manner. Furthermore,HRV16 induced a significant increase in type I IFN (IFN-alpha) release and STAT1 phosphorylation in monocytes. Neutralization of the type I IFN receptor and inhibition of JAK or p38 kinase activity strongly attenuated HRV16-stimulated STAT1 phosphorylation and IP-10 release. Thus,this work supports a model,wherein HRV16-induced IP-10 release by monocytic cells is modulated via autocrine/paracrine action of type I IFNs and subsequent JAK/STAT pathway activity. Our findings demonstrating robust activation of monocytic cells in response to native and/or replication-defective HRV16 challenge represent the first evidence indicating a mechanistic disparity in the activation of macrophages when compared with epithelial cells and suggest that macrophages likely contribute to cytokine elaboration following HRV challenge in vivo. View Publication

Umbilical cord blood (CB) is a valuable source of stem cells for transplantation,but CB transplantations are frequently complicated by delayed platelet engraftment. The reasons underlying this are unclear. We hypothesized that CB- and peripheral-blood (PB)-derived megakaryocytes (MKs) respond differently to the adult hematopoietic microenvironment and to thrombopoietin (Tpo). To test this,we cultured CB- and PB-CD34(+) cells in adult bone marrow stromal conditioned media (CM) or unconditioned media (UCM) with increasing concentrations of recombinant Tpo and compared the effects of these conditions on CB-versus PB-MKs. PB-MKs reached highest ploidy in response to UCM + 100 ng/mL rTpo,and the addition of CM inhibited their maturation. In contrast,CB-MKs reached highest ploidy in CM without rTpo,and high rTpo concentrations (textgreater 0.1 ng/mL) inhibited their maturation. This is the first evidence that human neonatal and adult MKs have substantially different biologic responses to Tpo and potentially to other cytokines. View Publication