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

The rising incidence of autoimmune diseases such as multiple sclerosis (MS) in developed countries might be due to a more hygienic environment,particularly during early life. To investigate this concept,we developed a model of neonatal exposure to a common pathogen-associated molecular pattern,LPS,and determined its impact on experimental autoimmune encephalomyelitis (EAE). Mice exposed to LPS at 2 wk of age showed a delayed onset and diminished severity of myelin oligodendrocyte glycoprotein (MOG)-induced EAE,induced at 12 wk,compared with vehicle-exposed animals. Spinal cord transcript levels of CD3epsilon and F4/80 were lower in LPS- compared with PBS-exposed EAE animals with increased IL-10 levels in the LPS-exposed group. Splenic CD11c(+) cells from LPS-exposed animals exhibited reduced MHC class II and CD83 expression but increased levels of CD80 and CD86 both before and during EAE. MOG-treated APC from LPS-exposed animals stimulated less T lymphocyte proliferation but increased expansion of CD4(+)FoxP3(+) T cells compared with APC from PBS-exposed animals. Neuropathological studies disclosed reduced myelin and axonal loss in spinal cords from LPS-exposed compared with PBS-exposed animals with EAE,and this neuroprotective effect was associated with an increased number of CD3(+)FoxP3(+) immunoreactive cells. Analyses of human brain tissue revealed that FoxP3 expression was detected in lymphocytes,albeit reduced in MS compared with non-MS patients' brains. These findings support the concept of early-life microbial exposure influencing the generation of neuroprotective regulatory T cells and may provide insights into new immunotherapeutic strategies for MS. View Publication

Purpose: We determined whether elimination of CD105+ cells in the tumor microenvironment (TME) with anti-CD105 antibodies enhanced anti-disialoganglioside (GD2) antibody dinutuximab therapy of neuroblastoma when combined with activated natural killer (aNK) cells.Experimental Design: The effect of MSCs and monocytes on antibody-dependent cellular cytotoxicity (ADCC) mediated by dinutuximab with aNK cells against neuroblastoma cells was determined in vitro. ADCC with anti-CD105 mAb TRC105 and aNK cells against MSCs,monocytes,and endothelial cells,which express CD105,was evaluated. Anti-neuroblastoma activity in immunodeficient NSG mice of dinutuximab with aNK cells without or with anti-CD105 mAbs was determined using neuroblastoma cell lines and a patient-derived xenograft.Results: ADCC mediated by dinutuximab with aNK cells against neuroblastoma cells in vitro was suppressed by addition of MSCs and monocytes,and dinutuximab with aNK cells was less effective against neuroblastomas formed with coinjected MSCs and monocytes in NSG mice than against those formed by tumor cells alone. Anti-CD105 antibody TRC105 with aNK cells mediated ADCC against MSCs,monocytes,and endothelial cells. Neuroblastomas formed in NSG mice by two neuroblastoma cell lines or a patient-derived xenograft coinjected with MSCs and monocytes were most effectively treated with dinutuximab and aNK cells when anti-human (TRC105) and anti-mouse (M1043) CD105 antibodies were added,which depleted human MSCs and murine endothelial cells and macrophages from the TME.Conclusions: Immunotherapy of neuroblastoma with anti-GD2 antibody dinutuximab and aNK cells is suppressed by CD105+ cells in the TME,but suppression is overcome by adding anti-CD105 antibodies to eliminate CD105+ cells. View Publication

Neurogenesis in the subventricular zone (SVZ) is regulated by diffusible factors and cell-cell contacts. In vivo,SVZ stem cells are associated with the abluminal surface of blood vessels and such interactions are thought to regulate their neurogenic capacity. SVZ neural stem cells (NSCs) have been described to contact endothelial-derived laminin via α6β1 integrin. To elucidate whether heterocellular contacts with brain endothelial cells (BEC) regulate SVZ cells neurogenic capacities,cocultures of SVZ neurospheres and primary BEC,both obtained from C57BL/6 mice,were performed. The involvement of laminin-integrin interactions in SVZ homeostasis was tested in three ways. Firstly,SVZ cells were analyzed following incubation of BEC with the protein synthesis inhibitor cycloheximide (CHX) prior to coculture,a treatment expected to decrease membrane proteins. Secondly,SVZ cells were cocultured with BEC in the presence of an anti-α6 integrin neutralizing antibody. Thirdly,BEC were cultured with β1-/- SVZ cells. We showed that contact with BEC supports,at least in part,proliferation and stemness of SVZ cells,as evaluated by the number of BrdU positive (+) and Sox2+ cells in contact with BEC. These effects are dependent on BEC-derived laminin binding to α6β1 integrin and are decreased in cocultures incubated with anti-α6 integrin neutralizing antibody and in cocultures with SVZ β1-/- cells. Moreover,BEC-derived laminin sustains stemness in SVZ cell cultures via activation of the Notch and mTOR signaling pathways. Our results show that BEC/SVZ interactions involving α6β1 integrin binding to laminin,contribute to SVZ cell proliferation and stemness. View Publication

Sphingolipids,particularly gangliosides,are enriched in neuronal membranes where they have been implicated as mediators of various regulatory events. We recently provided evidence that sphingolipid synthesis is necessary to maintain neuronal growth by demonstrating that in hippocampal neurons,inhibition of ceramide synthesis by Fumonisin B1 (FB1) disrupted axonal outgrowth (Harel,R. and Futerman,A. H. (1993) J. Biol. Chem. 268,14476-14481). We now analyze further the relationship between neuronal growth and sphingolipid metabolism by examining the effect of an inhibitor of glucosylceramide synthesis,D-threo-1-phenyl-2-decanoylamino-3-morpholino-1- propanol (PDMP) and by examining the effects of both FB1 and PDMP at various stages of neuronal development. No effects of FB1 or PDMP were observed during the first 2 days in culture,but by day 3 axonal morphology was significantly altered,irrespective of the time of addition of the inhibitors to the cultures. Cells incubated with FB1 or PDMP had a shorter axon plexus and less axonal branches. FB1 appeared to cause a retraction of axonal branches between days 2 and 3,although long term incubation had no apparent effect on neuronal morphology or on the segregation of axonal or dendritic proteins. In contrast,incubation of neurons with conduritol B-epoxide,an inhibitor of glucosylceramide degradation,caused an increase in the number of axonal branches and a corresponding increase in the length of the axon plexus. A direct correlation was observed between the number of axonal branch points per cell and the extent of inhibition of either sphingolipid synthesis or degradation. These results suggest that sphingolipids play an important role in the formation or stabilization of axonal branches. View Publication

An in vivo transplantation system has been used to evaluate the developmental capacities of specific mouse mammary epithelial cell populations. Specifically,mouse mammary epithelial cells with distinctly limited developmental potentials have been identified using this procedure. Two distinct epithelial cell progenitors have been identified by experiments designed to determine whether basal lobular and ductal phenotypes could develop independently under conditions imposed by a limiting dilution. The prediction that these separate epithelial progenitors must exist was based upon the results from transplantation experiments carried out in epithelium-divested mammary fat pads of syngeneic mice with mammary epithelium from two different transgenic mouse models. The results presented here demonstrate the following points: 1) lobular,i.e. secretory,progenitor cells are present as distinct entities among the mammary epithelial cells found in immature virgin female mice; 2) similarly,ductal epithelial progenitors are present within the same population; 3) lobular progenitors are present in greater numbers,although both cell populations are extremely small; 4) as expected,some inocula produce outgrowths with simultaneous development of both lobular and ductal phenotypes--it is not known whether this indicates cooperative interaction between the two epithelial progenitors or signals the presence of a third progenitor type capable of producing both ductular and lobular committed daughters; 5) these findings have important consequences in the design of experiments aimed at testing the effects of known and putative mammary oncogenes and tumor suppressor genes,using techniques which include cellular transformation in vitro followed by in vivo cultivation and evaluation. View Publication

BACKGROUND The epidermal growth factor receptor (EGFR) and Notch signaling pathways have been implicated in self-renewal of normal breast stem cells. We investigated the involvement of these signaling pathways in ductal carcinoma in situ (DCIS) of the breast. METHODS Samples of normal breast tissue (n = 15),pure DCIS tissue of varying grades (n = 35),and DCIS tissue surrounding an invasive cancer (n = 7) were used for nonadherent (i.e.,mammosphere) culture. Mammosphere cultures were treated at day 0 with gefitinib (an EGFR inhibitor),DAPT (N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester) (a gamma-secretase inhibitor),or Notch 4-neutralizing antibody. Mammosphere-forming efficiency (MFE) was calculated by dividing the number of mammospheres of 60 microm or more formed by the number of single cells seeded and is expressed as a percentage. The Notch 1 intracellular domain (NICD) was detected immunohistochemically in paraffin-embedded DCIS tissue from 50 patients with at least 60 months of follow-up. All statistical tests were two-sided. RESULTS DCIS had a greater MFE than normal breast tissue (1.5% versus 0.5%,difference = 1%,95% confidence interval [CI] = 0.62% to 1.25%,Ptextless.001). High-grade DCIS had a greater MFE than low-grade DCIS (1.6% versus 1.09%,difference = 0.51%,95% CI = 0.07% to 0.94%,P = .01). The MFE of high-grade DCIS treated with gefitinib in the absence of exogenous EGF was lower than that of high-grade DCIS treated with mammosphere medium lacking gefitinib and exogenous EGF (0.56% versus 1.36%,difference 0.8%,95% CI = 0.33% to 1.4%,P = .004). Increased Notch signaling as detected by NICD staining was associated with recurrence at 5 years (P = .012). DCIS MFE was reduced when Notch signaling was inhibited using either DAPT (0.89% versus 0.51%,difference = 0.38%,95% CI = 0.2% to 0.6%,Ptextless.001) or a Notch 4-neutralizing antibody (0.97% versus 0.2%,difference = 0.77%,95% CI = 0.52% to 1.0%,Ptextless.001). CONCLUSION We describe a novel primary culture technique for DCIS. Inhibition of the EGFR or Notch signaling pathways reduced DCIS MFE. View Publication

The bioorthogonal keto group has attracted interest for the site-specific chemical conjugation of recombinant proteins under mild conditions,e.g. with aminooxy-functionalised fluorescent probes,radiometal chelates,toxins or polymers. However,the cotranslational incorporation of the corresponding non-canonical amino acid p-acetyl-L-phenylalanine (Apa) into proteins expressed in Escherichia coli by means of amber suppression using a previously described system with a mutated tRNA and an engineered tyrosyl-tRNA synthetase from Methanococcus jannaschii shows limited efficiency and considerable promiscuity towards endogenous amino acids. Employing a one-plasmid system that encodes all three components required for selection,i.e. the modified aminoacyl-tRNA synthetase (aaRS),the cognate amber suppressor tRNA and the enhanced green fluorescent protein equipped with an amber stop codon and serving as reporter,we have generated an Apa-specific aaRS&tRNA pair with considerably improved efficiency (17-fold increased expression) and also fidelity (6-fold). To this end,both the aaRS and the tRNA were subjected to doped random mutagenesis and selection in altogether four evolutionary cycles using fluorescence-activated bacterial cell sorting as well as automated screening of microcultures. The resulting aaRS&tRNA pair was applied to the functionalisation of an Anticalin with specificity towards oncofetal fibronectin by introducing a keto group at a permissible site for subsequent conjugation with a fluorescent dye,thus allowing visualisation of this tumour target under the microscope. View Publication

Recent developments in understanding how the functional phenotype of the innate immune system is programmed has led to paradigm-shifting views on immunomodulation. These advances have overturned two long-held dogmas: (1) only adaptive immunity confers immunological memory; and,(2) innate immunity lacks specificity. This work describes the observation that innate immune effector cells appear to be differentially recruited to specific pathological sites when mobilized by distinct inactivated bacterial-based stimuli administered subcutaneously. The studies presented suggest that the immune system,upon detecting the first signs of a potential infection by a specific pathogen,tends to direct its resources to the compartment from which that pathogen is most likely originating. The findings from this work puts forth the novel hypothesis that the immunotherapeutic efficacy of a microbial-based stimulus for innate immune mobilization depends on the correct selection of the microbial species used as the stimulant and its relationship to the organ in which the pathology is present. View Publication

One important function of endothelial cells in glioblastoma multiforme (GBM) is to create a niche that helps promote self-renewal of cancer stem-like cells (CSLC). However,the underlying molecular mechanism for this endothelial function is not known. Since activation of NOTCH signaling has been found to be required for propagation of GBM CSLCs,we hypothesized that the GBM endothelium may provide the source of NOTCH ligands. Here,we report a corroboration of this concept with a demonstration that NOTCH ligands are expressed in endothelial cells adjacent to NESTIN and NOTCH receptor-positive cancer cells in primary GBMs. Coculturing human brain microvascular endothelial cells (hBMEC) or NOTCH ligand with GBM neurospheres promoted GBM cell growth and increased CSLC self-renewal. Notably,RNAi-mediated knockdown of NOTCH ligands in hBMECs abrogated their ability to induce CSLC self-renewal and GBM tumor growth,both in vitro and in vivo. Thus,our findings establish that NOTCH activation in GBM CSLCs is driven by juxtacrine signaling between tumor cells and their surrounding endothelial cells in the tumor microenvironment,suggesting that targeting both CSLCs and their niche may provide a novel strategy to deplete CSLCs and improve GBM treatment. View Publication

Availability of tumor and non-tumor patient-derived models would promote the development of more effective therapeutics for non-small cell lung cancer (NSCLC). Recently,conditionally reprogrammed cells (CRC) methodology demonstrated exceptional potential for the expansion of epithelial cells from patient tissues. However,the possibility to expand patient-derived lung cancer cells using CRC protocols is controversial. Here,we used CRC approach to expand cells from non-tumoral and tumor biopsies of patients with primary or metastatic NSCLC as well as pulmonary metastases of colorectal or breast cancers. CRC cultures were obtained from both tumor and non-malignant tissues with extraordinary high efficiency. Tumor cells were tracked in vitro through tumorigenicity assay,monitoring of tumor-specific genetic alterations and marker expression. Cultures were composed of EpCAM+ lung epithelial cells lacking tumorigenic potential. NSCLC biopsies-derived cultures rapidly lost patient-specific genetic mutations or tumor antigens. Similarly,pulmonary metastases of colon or breast cancer generated CRC cultures of lung epithelial cells. All CRC cultures examined displayed epithelial lung stem cell phenotype and function. In contrast,brain metastatic lung cancer biopsies failed to generate CRC cultures. In conclusion,patient-derived primary and metastatic lung cancer cells were negatively selected under CRC conditions,limiting the expansion to non-malignant lung epithelial stem cells from either tumor or non-tumor tissue sources. Thus,CRC approach cannot be applied for direct therapeutic testing of patient lung tumor cells,as the tumor-derived CRC cultures are composed of (non-tumoral) airway basal cells. View Publication

The identification and targeted therapy of cells involved in hepatocellular carcinoma (HCC) recurrence remain challenging. Here,we generated a monoclonal antibody against recurrent HCC,1B50-1,that bound the isoform 5 of the α2δ1 subunit of voltage-gated calcium channels and identified a subset of tumor-initiating cells (TICs) with stem cell-like properties. A surgical margin with cells detected by 1B50-1 predicted rapid recurrence. Furthermore,1B50-1 had a therapeutic effect on HCC engraftments by eliminating TICs. Finally,α2δ1 knockdown reduced self-renewal and tumor formation capacities and induced apoptosis of TICs,whereas its overexpression led to enhanced sphere formation,which is regulated by calcium influx. Thus,α2δ1 is a functional liver TIC marker,and its inhibitors may serve as potential anti-HCC drugs. View Publication

Graphene quantum dots (GQDs) have gained significant attention in various biomedical applications. The physicochemical properties of these nanoparticles,including toxic effects,are largely determined by their surface modifications. Previous studies have demonstrated high in vitro cytotoxicity of the hydroxylated GQDs (OH-GQDs). The focus of this study was on the intestinal toxicity of OH-GQDs. Briefly,C57BL/6J mice were given daily oral gavage of 0.05,0.5 or 5 mg/kg OH-GQD for 7 days,and the indices of intestinal damage were evaluated. Higher doses of the OH-GQDs caused significant intestinal injuries,such as enhanced intestinal permeability,shortened villi and crypt loss. The number of Lgr5+ intestinal stem cells also decreased dramatically upon OH-GQDs exposure,which also inhibited the Ki67+ proliferative progenitor cells. In addition,an increased number of crypt cells harboring the oxidized DNA base 8-OHdG and $\gamma$H2AX foci were also detected in the intestines of OH-GQD-treated mice. Mechanistically,the OH-GQDs up-regulated both total and phosphorylated p53. Consistent with this,the average number of TUNEL+ and cleaved caspase-3+ apoptotic intestinal epithelial cells were significantly increased after OH-GQDs treatment. Finally,a 3-dimensional organoid culture was established using isolated crypts,and OH-GQDs treatment significantly reduced the size of the surviving intestinal organoids. Taken together,the intestinal toxicity of the OH-GQDs should be taken into account during biomedical applications. View Publication