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

Angiotensin I-converting enzyme (ACE) inhibitors can affect hematopoiesis by several mechanisms including inhibition of angiotensin II formation and increasing plasma concentrations of AcSDKP (acetyl-N-Ser-Asp-Lys-Pro),an ACE substrate and a negative regulator of hematopoiesis. We tested whether ACE inhibition could decrease the hematopoietic toxicity of lethal or sublethal irradiation protocols. In all cases,short treatment with the ACE inhibitor perindopril protected against irradiation-induced death. ACE inhibition accelerated hematopoietic recovery and led to a significant increase in platelet and red cell counts. Pretreatment with perindopril increased bone marrow cellularity and the number of hematopoietic progenitors (granulocyte macrophage colony-forming unit [CFU-GM],erythroid burst-forming unit [BFU-E],and megakaryocyte colony-forming unit [CFU-MK]) from day 7 to 28 after irradiation. Perindopril also increased the number of hematopoietic stem cells with at least a short-term reconstitutive activity in animals that recovered from irradiation. To determine the mechanism of action involved,we evaluated the effects of increasing AcSDKP plasma concentrations and of an angiotensin II type 1 (AT1) receptor antagonist (telmisartan) on radioprotection. We found that the AT1-receptor antagonism mediated similar radioprotection as the ACE inhibitor. These results suggest that ACE inhibitors and AT1-receptor antagonists could be used to decrease the hematopoietic toxicity of irradiation. View Publication

BACKGROUND The ability to site-specifically conjugate a protein to a payload of interest (e.g.,a fluorophore,small molecule pharmacophore,oligonucleotide,or other protein) has found widespread application in basic research and drug development. For example,antibody-drug conjugates represent a class of biotherapeutics that couple the targeting specificity of an antibody with the chemotherapeutic potency of a small molecule drug. While first generation antibody-drug conjugates (ADCs) used random conjugation approaches,next-generation ADCs are employing site-specific conjugation. A facile way to generate site-specific protein conjugates is via the aldehyde tag technology,where a five amino acid consensus sequence (CXPXR) is genetically encoded into the protein of interest at the desired location. During protein expression,the Cys residue within this consensus sequence can be recognized by ectopically-expressed formylglycine generating enzyme (FGE),which converts the Cys to a formylglycine (fGly) residue. The latter bears an aldehyde functional group that serves as a chemical handle for subsequent conjugation. RESULTS The yield of Cys conversion to fGly during protein production can be variable and is highly dependent on culture conditions. We set out to achieve consistently high yields by modulating culture conditions to maximize FGE activity within the cell. We recently showed that FGE is a copper-dependent oxidase that binds copper in a stoichiometric fashion and uses it to activate oxygen,driving enzymatic turnover. Building upon that work,here we show that by supplementing cell culture media with copper we can routinely reach high yields of highly converted protein. We demonstrate that cells incorporate copper from the media into FGE,which results in increased specific activity of the enzyme. The amount of copper required is compatible with large scale cell culture,as demonstrated in fed-batch cell cultures with antibody titers of 5 g textperiodcentered L(-1),specific cellular production rates of 75 pg textperiodcentered cell(-1) textperiodcentered d(-1),and fGly conversion yields of 95-98 %. CONCLUSIONS We describe a process with a high yield of site-specific formylglycine (fGly) generation during monoclonal antibody production in CHO cells. The conversion of Cys to fGly depends upon the activity of FGE,which can be ensured by supplementing the culture media with 50 uM copper(II) sulfate. View Publication

OBJECTIVE: We used genetically engineered mouse hematopoietic progenitor cells (HPCs) to investigate the therapeutic effects of human apoAI on atherosclerosis in apoE(-/-) mice. METHODS AND RESULTS: Lentiviral constructs expressing either human apoAI (LV-apoAI) or green fluorescent protein (LV-GFP) cDNA under a macrophage specific promoter (CD68) were generated and used for ex vivo transduction of mouse HPCs and macrophages. The transduction efficiency was textgreater25% for HPCs and textgreater70% for macrophages. ApoAI was found in the macrophage culture media,mostly associated with the HDL fraction. Interestingly,a significant increase in mRNA and protein levels for ATP binding cassette A1 (ABCA1) and ABCG1 were found in apoAI-expressing macrophages after acLDL loading. Expression of apoAI significantly increased cholesterol efflux in wild-type and apoE(-/-) macrophages. HPCs transduced with LV-apoAI ex vivo and then transplanted into apoE(-/-) mice caused a 50% reduction in atherosclerotic lesion area compared to GFP controls,without influencing plasma HDL-C levels. CONCLUSIONS: Lentiviral transduction of apoAI into HPCs reduces atherosclerosis in apoE(-/-) mice. Expression of apoAI in macrophages improves cholesterol trafficking in wild-type apoE-producing macrophages and causes upregulation of ABCA1 and ABCG1. These novel observations set the stage for a cell therapy approach to atherosclerosis regression,exploiting the cooperation between apoE and apoAI to maximize cholesterol exit from the plaque. View Publication

Selective and regulatable expansion of transduced cells could augment gene therapy for many disorders. The activation of modified growth factor receptors via synthetic chemical inducers of dimerization allows for the coordinated growth of transduced cells. This system can also provide information on specific receptor-mediated signaling without interference from other family members. Although several receptor subunits have been investigated in this context,little is known about the precise molecular events associated with dimerizer-initiated signaling. We have constructed and expressed an AP20187-regulated KDR chimeric receptor in human TF1 cells and analyzed activation of this gene switch using functional,biochemical,and microarray analyses. When deprived of natural ligands,GM-CSF,interleukin-3,or erythropoietin,AP20187 prevented apoptosis of transduced TF1 cells,induced dose-dependent proliferation,and supported long-term growth. In addition,AP20187 stimulation activated the signaling molecules associated with mitogen-activated protein kinase and phosphatidyl-inositol 3-kinase/Akt pathways. Microarray analysis determined that a number of transcripts involved in a variety of cellular processes were differentially expressed. Notably,mRNAs affiliated with heat stress,including Hsp70 and Hsp105,were up-regulated. Functional assays showed that Hsp70 and Hsp105 protected transduced TF1 cells from apoptosis and premature senescence,in part through regulation of Akt. These observations delineate specific roles for kinase insert domain-containing receptor,or KDR,signaling and suggest strategies to endow genetically modified cells with a survival advantage enabling the generation of adequate cell numbers for therapeutic outcomes. View Publication

For antibody-based therapy of cancer,monoclonal antibodies (mAbs) of human origin are superior to mouse,mouse/human chimeric or humanized mAbs,because of their minimum immunogenicity to humans and their efficient collaboration with human effector cells. In the present study,human mAbs were prepared against a pancarcinoma antigen,MK-1 (Ep-CAM),using a genetically-engineered mouse (KM mouse) that contains the human immunoglobulin genes. Spleen cells from KM mice,immunized with recombinant MK-1,were fused with P3-U1 mouse myeloma cells. Of 44 anti-MK-1 clones analyzed,two were of IgG4 and the others of IgM clones. Although the two IgG4 clones were suggested to recognize the same antigenic determinant or two closely located determinants,their VK regions were encoded by different light-chain genes while their VH sequences were identical. The two IgG4 and one of the IgM clones tested revealed antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity,respectively,against MK-1-expressing cells in vitro,suggesting that these fully human mAbs produced against MK-1 and their V-region genes,which are applicable for the preparation of engineered antibody fragments that may be useful for antibody-based therapy of cancer. View Publication

The aryl hydrocarbon receptor (AhR) mediates the carcinogenicity of a family of environmental contaminants,the most potent being 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Increased incidence of lymphoma and leukemia in humans is associated with TCDD exposure. Although AhR activation by TCDD has profound effects on the immune system,precise cellular and molecular mechanisms have yet to be determined. These studies tested the hypothesis that alteration of marrow populations following treatment of mice with TCDD is due to an effect on hematopoietic stem cells (HSCs). Treatment with TCDD resulted in an increased number and proliferation of bone marrow (BM) populations enriched for HSCs. There was a time-dependent decrease in B-lineage cells with a concomitant increase in myeloid populations. The decrease in the B-cell lineage colony-forming unit-preB progenitors along with a transient increase in myeloid progenitors were consistent with a skewing of lineage development from lymphoid to myeloid populations. However,HSCs from TCDD-treated mice exhibited diminished capacity to reconstitute and home to marrow of irradiated recipients. AhR messenger RNA was expressed in progenitor subsets but is downregulated during HSC proliferation. This result was consistent with the lack of response following the exposure of 5-fluorouracil-treated mice to TCDD. The direct exposure of cultured BM cells to TCDD inhibited the growth of immature hematopoietic progenitor cells,but not more mature lineage-restricted progenitors. Overall,these data are consistent with the hypothesis that TCDD,through AhR activation,alters the ability of HSCs to respond appropriately to signals within the marrow microenvironment. View Publication

Long-chain bases are present in the oral cavity. Previously we determined that sphingosine,dihydrosphingosine,and phytosphingosine have potent antimicrobial activity against oral pathogens. Here,we determined the cytotoxicities of long-chain bases for oral cells,an important step in considering their potential as antimicrobial agents for oral infections. This information would clearly help in establishing prophylactic or therapeutic doses. To assess this,human oral gingival epithelial (GE) keratinocytes,oral gingival fibroblasts (GF),and dendritic cells (DC) were exposed to 10.0-640.0 μM long-chain bases and glycerol monolaurate (GML). The effects of long-chain bases on cell metabolism (conversion of resazurin to resorufin),membrane permeability (uptake of propidium iodide or SYTOX-Green),release of cellular contents (LDH),and cell morphology (confocal microscopy) were all determined. GE keratinocytes were more resistant to long-chain bases as compared to GF and DC,which were more susceptible. For DC,0.2-10.0 μM long-chain bases and GML were not cytotoxic; 40.0-80.0 μM long-chain bases,but not GML,were cytotoxic; and 80.0 μM long-chain bases induced cellular damage and death in less than 20 min. The LD50 of long-chain bases for GE keratinocytes,GF,and DC were considerably higher than their minimal inhibitory concentrations for oral pathogens,a finding important to pursuing their future potential in treating periodontal and oral infections. View Publication

Proof of drug-target engagement in physiologically-relevant contexts is a key pillar of successful therapeutic target validation. We developed two orthogonal technologies,the cellular thermal shift assay (CETSA) and a covalent chemical probe reporter approach (harnessing sulfonyl fluoride tyrosine labeling and subsequent click chemistry) to measure the occupancy of the mRNA-decapping scavenger enzyme DcpS by a small molecule inhibitor in live cells. Enzyme affinity determined using isothermal dose response fingerprinting (ITDRFCETSA) and the concentration required to occupy 50% of the enzyme (OC50) using the chemical probe reporter assay were very similar. In this case,the chemical probe method worked well due to the long offset kinetics of the reversible inhibitor (determined using a fluorescent dye-tagged probe). This work suggests that CETSA could become the first choice assay to determine in-cell target engagement due to its simplicity. View Publication