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Results: 11 to 20 of 96

Publication Record


Sustained Administration of β-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres.
Pasek RC, Kavanaugh TE, Duvall CL, Gannon MA
(2016) J Vis Exp :
MeSH Terms: Animals, Biocompatible Materials, Coculture Techniques, Drug Delivery Systems, Glycols, Humans, Insulin-Secreting Cells, Islets of Langerhans, Lactic Acid, Mice, Microspheres, Mitogens, Polyglycolic Acid
Show Abstract · Added March 14, 2018
The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the pancreatic β-cells. In addition, biomaterial particles, hydrogels, and scaffolds also provide a unique opportunity to administer sustained, controllable drug delivery to β-cells in culture and in transplanted tissue models. These technologies allow the study of candidate β-cell proliferation factors using intact islets and a translationally relevant system. Moreover, determining the effectiveness and feasibility of candidate factors for stimulating β-cell proliferation in a culture system is critical before moving forward to in vivo models. Herein, we describe a method to co-culture intact mouse islets with biodegradable compound of interest (COI)-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres for the purpose of assessing the effects of sustained in situ release of mitogenic factors on β-cell proliferation. This technique describes in detail how to generate PLGA microspheres containing a desired cargo using commercially available reagents. While the described technique uses recombinant human Connective tissue growth factor (rhCTGF) as an example, a wide variety of COI could readily be used. Additionally, this method utilizes 96-well plates to minimize the amount of reagents necessary to assess β-cell proliferation. This protocol can be readily adapted to use alternative biomaterials and other endocrine cell characteristics such as cell survival and differentiation status.
0 Communities
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13 MeSH Terms
Epithelial Coculture and l-Lactate Promote Growth of Helicobacter cinaedi under H2-Free Aerobic Conditions.
Schmitz JE, Taniguchi T, Misawa N, Cover TL
(2016) Appl Environ Microbiol 82: 6701-6714
MeSH Terms: Aerobiosis, Caco-2 Cells, Coculture Techniques, Epithelial Cells, HeLa Cells, Helicobacter, Helicobacter Infections, Humans, Hydrogen, Lactic Acid
Show Abstract · Added April 9, 2017
Helicobacter cinaedi is an emerging opportunistic pathogen associated with infections of diverse anatomic sites. Nevertheless, the species demonstrates fastidious axenic growth; it has been described as requiring a microaerobic atmosphere, along with a strong preference for supplemental H gas. In this context, we examined the hypothesis that in vitro growth of H. cinaedi could be enhanced by coculture with human epithelial cells. When inoculated (in Ham's F12 medium) over Caco-2 monolayers, the type strain (ATCC BAA-847) gained the ability to proliferate under H-free aerobic conditions. Identical results were observed during coculture with several other monolayer types (LS-174T, AGS, and HeLa). Under chemically defined conditions, 40 amino acids and carboxylates were screened for their effect on the organism's atmospheric requirements. Several molecules promoted H-free aerobic proliferation, although it occurred most prominently with millimolar concentrations of l-lactate. The growth response of H. cinaedi to Caco-2 cells and l-lactate was confirmed with a collection of 12 human-derived clinical strains. mRNA sequencing was next performed on the type strain under various growth conditions. In addition to providing a whole-transcriptome profile of H. cinaedi, this analysis demonstrated strong constitutive expression of the l-lactate utilization locus, as well as differential transcription of terminal respiratory proteins as a function of Caco-2 coculture and l-lactate supplementation. Overall, these findings challenge traditional views of H. cinaedi as an obligate microaerophile.
IMPORTANCE - H. cinaedi is an increasingly recognized pathogen in people with compromised immune systems. Atypical among other members of its bacterial class, H. cinaedi has been associated with infections of diverse anatomic sites. Growing H. cineadi in the laboratory is quite difficult, due in large part to the need for a specialized atmosphere. The suboptimal growth of H. cinaedi is an obstacle to clinical diagnosis, and it also limits investigation into the organism's biology. The current work shows that H. cinaedi has more flexible atmospheric requirements in the presence of host cells and a common host-derived molecule. This nutritional interplay raises new questions about how the organism behaves during human infections and provides insights for how to optimize its laboratory cultivation.
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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10 MeSH Terms
Androgen receptor differentially regulates the proliferation of prostatic epithelial cells in vitro and in vivo.
Yang S, Jiang M, Grabowska MM, Li J, Connelly ZM, Zhang J, Hayward SW, Cates JM, Han G, Yu X
(2016) Oncotarget 7: 70404-70419
MeSH Terms: Androgens, Animals, Cell Differentiation, Cell Line, Cell Proliferation, Coculture Techniques, Epithelial Cells, Humans, Male, Mice, Nude, Prostate, Proto-Oncogene Proteins c-myc, Rats, Receptors, Androgen, Stromal Cells
Show Abstract · Added November 1, 2018
Androgens regulate the proliferation and differentiation of prostatic epithelial cells, including prostate cancer (PCa) cells in a context-dependent manner. Androgens and androgen receptor (AR) do not invariably promote cell proliferation; in the normal adult, endogenous stromal and epithelial AR activation maintains differentiation and inhibits organ growth. In the current study, we report that activation of AR differentially regulates the proliferation of human prostate epithelial progenitor cells, NHPrE1, in vitro and in vivo. Inducing AR signaling in NHPrE1 cells suppressed cell proliferation in vitro, concomitant with a reduction in MYC expression. However, ectopic expression of AR in vivo stimulated cell proliferation and induced development of invasive PCa in tissue recombinants consisting of NHPrE1/AR cells and rat urogenital mesenchymal (UGM) cells, engrafted under renal capsule of adult male athymic mice. Expression of MYC increased in the NHPrE1/AR recombinant tissues, in contrast to the reduction seen in vitro. The inhibitory effect of AR signaling on cell proliferation in vitro were reduced by co-culturing NHPrE1/AR epithelial cells with prostatic stromal cells. In conclusion, these studies revealed that AR signaling differentially regulates proliferation of human prostatic epithelia cells in vitro and in vivo through mechanisms involving stromal/epithelial interactions.
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MeSH Terms
The Ste20 kinases SPAK and OSR1 travel between cells through exosomes.
Koumangoye R, Delpire E
(2016) Am J Physiol Cell Physiol 311: C43-53
MeSH Terms: Cell Communication, Cell Membrane, Coculture Techniques, Culture Media, Conditioned, Exosomes, HEK293 Cells, HeLa Cells, Humans, Luminescent Proteins, Microscopy, Fluorescence, Particle Size, Phosphorylation, Protein Transport, Protein-Serine-Threonine Kinases, Recombinant Proteins, Solute Carrier Family 12, Member 2, Tetraspanin 30, Time Factors, Transfection
Show Abstract · Added May 3, 2017
Proteomics studies have identified Ste20-related proline/alanine-rich kinase (SPAK) and oxidative stress response 1 (OSR1) in exosomes isolated from body fluids such as blood, saliva, and urine. Because proteomics studies likely overestimate the number of exosome proteins, we sought to confirm and extend this observation using traditional biochemical and cell biology methods. We utilized HEK293 cells in culture to verify the packaging of these Ste20 kinases in exosomes. Using a series of centrifugation and filtration steps of conditioned culture medium isolated from HEK293 cells, we isolated nanovesicles in the range of 40-100 nm. We show that these small vesicles express the tetraspanin protein CD63 and lack endoplasmic reticulum and Golgi markers, consistent with these being exosomes. We show by Western blot and immunogold analyses that these exosomes express SPAK, OSR1, and Na-K-Cl cotransporter 1 (NKCC1). We show that exosomes are not only secreted by cells, but also accumulated by adjacent cells. Indeed, exposing cultured cells to exosomes produced by other cells expressing a fluorescently labeled kinase resulted in the kinase finding its way into the cytoplasm of these cells, consistent with the idea of exosomes serving as cell-to-cell communication vessels. Similarly, coculturing cells expressing different fluorescently tagged proteins resulted in the exchange of proteins between cells. In addition, we show that both SPAK and OSR1 kinases entering cells through exosomes are preferentially expressed at the plasma membrane and that the kinases in exosomes are functional and maintain NKCC1 in a phosphorylated state.
Copyright © 2016 the American Physiological Society.
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19 MeSH Terms
Anti-proliferative effects of T cells expressing a ligand-based chimeric antigen receptor against CD116 on CD34(+) cells of juvenile myelomonocytic leukemia.
Nakazawa Y, Matsuda K, Kurata T, Sueki A, Tanaka M, Sakashita K, Imai C, Wilson MH, Koike K
(2016) J Hematol Oncol 9: 27
MeSH Terms: Antigens, CD34, Cell Line, Tumor, Cell Proliferation, Cells, Cultured, Coculture Techniques, Flow Cytometry, Humans, Immunotherapy, Adoptive, K562 Cells, Leukemia, Myelomonocytic, Juvenile, Ligands, Mutation, Receptors, Antigen, T-Cell, Receptors, Granulocyte-Macrophage Colony-Stimulating Factor, Recombinant Fusion Proteins, Stem Cell Factor, T-Lymphocytes, Thrombopoietin
Show Abstract · Added September 11, 2017
BACKGROUND - Juvenile myelomonocytic leukemia (JMML) is a fatal, myelodysplastic/myeloproliferative neoplasm of early childhood. Patients with JMML have mutually exclusive genetic abnormalities in granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR, CD116) signaling pathway. Allogeneic hematopoietic stem cell transplantation is currently the only curative treatment option for JMML; however, disease recurrence is a major cause of treatment failure. We investigated adoptive immunotherapy using GMR-targeted chimeric antigen receptor (CAR) for JMML.
METHODS - We constructed a novel CAR capable of binding to GMR via its ligand, GM-CSF, and generated piggyBac transposon-based GMR CAR-modified T cells from three healthy donors and two patients with JMML. We further evaluated the anti-proliferative potential of GMR CAR T cells on leukemic CD34(+) cells from six patients with JMML (two NRAS mutations, three PTPN11 mutations, and one monosomy 7), and normal CD34(+) cells.
RESULTS - GMR CAR T cells from healthy donors suppressed the cytokine-dependent growth of MO7e cells, but not the growth of K562 and Daudi cells. Co-culture of healthy GMR CAR T cells with CD34(+) cells of five patients with JMML at effector to target ratios of 1:1 and 1:4 for 2 days significantly decreased total colony growth, regardless of genetic abnormality. Furthermore, GMR CAR T cells from a non-transplanted patient and a transplanted patient inhibited the proliferation of respective JMML CD34(+) cells at onset to a degree comparable to healthy GMR CAR T cells. Seven-day co-culture of GMR CAR T cells resulted in a marked suppression of JMML CD34(+) cell proliferation, particularly CD34(+)CD38(-) cell proliferation stimulated with stem cell factor and thrombopoietin on AGM-S3 cells. Meanwhile, GMR CAR T cells exerted no effects on normal CD34(+) cell colony growth.
CONCLUSIONS - Ligand-based GMR CAR T cells may have anti-proliferative effects on stem and progenitor cells in JMML.
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18 MeSH Terms
Stromal matrix metalloproteinase 2 regulates collagen expression and promotes the outgrowth of experimental metastases.
Bates AL, Pickup MW, Hallett MA, Dozier EA, Thomas S, Fingleton B
(2015) J Pathol 235: 773-83
MeSH Terms: Actins, Animals, Cell Proliferation, Coculture Techniques, Collagen, Female, Fibroblasts, Gene Expression Profiling, Humans, Lung Neoplasms, Mammary Neoplasms, Experimental, Matrix Metalloproteinase 2, Mice, Inbred C57BL, Mice, Knockout, Spheroids, Cellular, Stromal Cells, Time Factors, Transfection, Transforming Growth Factor beta1, Vimentin
Show Abstract · Added January 20, 2015
Breast cancer survival rates decrease from 99% for patients with local disease to 25% for those with distant metastases. Matrix metalloproteinases (MMPs), including MMP2, are associated with metastatic progression. We found that loss of host MMP2 reduces the proliferation of experimental metastases in the lungs and identified fibroblasts in tumour-bearing lungs as the major source of MMP2. In vitro, spheroidal mammary tumour growth was increased by co-culture with control fibroblasts isolated from tumour-bearing lungs, but not when fibroblasts with stable Mmp2 knockdown were used. This result prompted us to assess whether MMP2 was responsible for a tumour-proliferative, activated fibroblast phenotype. To test this, we evaluated: (a) fibroblasts from wild-type tumour-bearing lungs, with or without shRNA-mediated MMP2 knockdown; and (b) normal, quiescent fibroblasts isolated from either WT or Mmp2(-/-) mice. Quantitative PCR revealed that Mmp2 knockdown attenuated expression of two markers of activation (α-smooth muscle actin and vimentin), but there was minimal expression in quiescent WT or Mmp2(-/-) fibroblasts, as expected. Placing quiescent fibroblasts under activating conditions led to increases in activation-associated transcripts in WT but not Mmp2(-/-) fibroblasts. Additionally, Mmp2 knockdown fibroblasts showed significantly decreased expression of the matrix transcripts collagen I, collagen IV and fibronectin. Addition of active TGFβ was sufficient to rescue the MMP2-dependent collagen I and IV expression, while MMP2-induced collagen expression was blocked by the addition of TGFβ1-neutralizing antibody. Gene expression data in stromal cells of human breast cancers reveal that MMP2 expression is also positively correlated with activation and matrix transcripts. Thus, we present a model whereby MMP2 production in tumour fibroblasts is important for TGFβ1 activity and subsequent activation of fibroblasts to a matrix-producing, proliferation-supportive phenotype. Overall, our results reveal a previously undefined role for MMP2 in metastatic outgrowth mediated by fibroblasts, and extend the mechanisms by which MMPs contribute to tumour progression.
Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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20 MeSH Terms
Dysfunctional high-density lipoproteins in children with chronic kidney disease.
Kaseda R, Jabs K, Hunley TE, Jones D, Bian A, Allen RM, Vickers KC, Yancey PG, Linton MF, Fazio S, Kon V
(2015) Metabolism 64: 263-73
MeSH Terms: Adolescent, Apoptosis, Biological Transport, Cardiovascular Diseases, Cell Adhesion, Cell Line, Cell Proliferation, Cells, Cultured, Chemotaxis, Child, Child, Preschool, Cholesterol, Coculture Techniques, Endothelium, Vascular, Human Umbilical Vein Endothelial Cells, Humans, Infant, Kidney Failure, Chronic, Lipoproteins, HDL, Macrophages, Renal Insufficiency, Chronic, Risk Factors, Severity of Illness Index, Tennessee
Show Abstract · Added February 12, 2015
OBJECTIVES - Our aim was to determine if chronic kidney disease (CKD) occurring in childhood impairs the normally vasoprotective functions of high-density lipoproteins (HDLs).
MATERIALS AND METHODS - HDLs were isolated from children with end-stage renal disease on dialysis (ESRD), children with moderate CKD and controls with normal kidney function. Macrophage response to HDLs was studied as expression of inflammatory markers (MCP-1, TNF-α, IL-1β) and chemotaxis. Human umbilical vein endothelial cells were used for expression of adhesion molecules (ICAM-1, VCAM-1, E-selectin) and adhesion. Cellular proliferation, apoptosis, and necrosis of endothelial cells were measured by MTS/PMS reagent-based assay, flow cytometry, and ELISA. Cholesterol efflux was assessed by gas chromatographic measurements of cholesterol in macrophages exposed to HDLs.
RESULTS - Compared with HDL(Control), HDL(CKD) and HDL(ESRD) heightened the cytokine response and disrupted macrophage chemotaxis. HDL(Control) reduced endothelial expression of ICAM-1, VCAM-1, E-selectin, whereas HDL(CKD) and HDL(ESRD) were less effective and showed reduced capacity to protect endothelial cells against monocyte adhesion. Compared with a dramatically enhanced endothelial proliferation following injurious stimulus by HDL(Control), neither HDL(CKD) nor HDL(ESRD) caused proliferative effects. HDLs of all three groups were equally protective against apoptosis assessed by flow cytometry and cleaved caspase-3 activity. Compared to HDL(Control), HDL(CKD) and HDL(ESRD) trended toward reduced capacity as cholesterol acceptors.
CONCLUSION - CKD in children impairs HDL function. Even in the absence of long-standing and concomitant risk factors, CKD alters specific HDL functions linked to control of inflammation and endothelial responses.
Copyright © 2015 Elsevier Inc. All rights reserved.
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24 MeSH Terms
Antibacterial gene transfer across the tree of life.
Metcalf JA, Funkhouser-Jones LJ, Brileya K, Reysenbach AL, Bordenstein SR
(2014) Elife 3:
MeSH Terms: Amino Acid Sequence, Anti-Bacterial Agents, Archaea, Bacteria, Coculture Techniques, Escherichia coli, Gene Transfer, Horizontal, Genes, Bacterial, Microbial Viability, Models, Molecular, Molecular Sequence Data, Muramidase, Phylogeny, RNA, Ribosomal
Show Abstract · Added October 8, 2015
Though horizontal gene transfer (HGT) is widespread, genes and taxa experience biased rates of transferability. Curiously, independent transmission of homologous DNA to archaea, bacteria, eukaryotes, and viruses is extremely rare and often defies ecological and functional explanations. Here, we demonstrate that a bacterial lysozyme family integrated independently in all domains of life across diverse environments, generating the only glycosyl hydrolase 25 muramidases in plants and archaea. During coculture of a hydrothermal vent archaeon with a bacterial competitor, muramidase transcription is upregulated. Moreover, recombinant lysozyme exhibits broad-spectrum antibacterial action in a dose-dependent manner. Similar to bacterial transfer of antibiotic resistance genes, transfer of a potent antibacterial gene across the universal tree seemingly bestows a niche-transcending adaptation that trumps the barriers against parallel HGT to all domains. The discoveries also comprise the first characterization of an antibacterial gene in archaea and support the pursuit of antibiotics in this underexplored group.
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14 MeSH Terms
Biocompatible mannosylated endosomal-escape nanoparticles enhance selective delivery of short nucleotide sequences to tumor associated macrophages.
Ortega RA, Barham WJ, Kumar B, Tikhomirov O, McFadden ID, Yull FE, Giorgio TD
(2015) Nanoscale 7: 500-10
MeSH Terms: Animals, Biocompatible Materials, Cell Line, Tumor, Cell Survival, Coculture Techniques, Drug Carriers, Endosomes, Female, Fluorescent Dyes, Lung, Lung Neoplasms, Macrophages, Mammary Neoplasms, Animal, Mannose, Mice, Mice, Inbred C57BL, Microscopy, Fluorescence, Nanoparticles, Ovarian Neoplasms, Polymers, RNA, Small Interfering, Transplantation, Homologous, Tumor Microenvironment
Show Abstract · Added December 17, 2014
Tumor associated macrophages (TAMs) can modify the tumor microenvironment to create a pro-tumor niche. Manipulation of the TAM phenotype is a novel, potential therapeutic approach to engage anti-cancer immunity. siRNA is a molecular tool for knockdown of specific mRNAs that is tunable in both strength and duration. The use of siRNA to reprogram TAMs to adopt an immunogenic, anti-tumor phenotype is an attractive alternative to ablation of this cell population. One current difficulty with this approach is that TAMs are difficult to specifically target and transfect. We report here successful utilization of novel mannosylated polymer nanoparticles (MnNP) that are capable of escaping the endosomal compartment to deliver siRNA to TAMs in vitro and in vivo. Transfection with MnNP-siRNA complexes did not significantly decrease TAM cell membrane integrity in culture, nor did it create adverse kidney or liver function in mice, even at repeated doses of 5 mg kg(-1). Furthermore, MnNP effectively delivers labeled nucleotides to TAMs in mice with primary mammary tumors. We also confirmed TAM targeting in the solid tumors disseminated throughout the peritoneum of ovarian tumor bearing mice following injection of fluorescently labeled MnNP-nucleotide complexes into the peritoneum. Finally, we show enhanced uptake of MnNP in lung metastasis associated macrophages compared to untargeted particles when using an intubation delivery method. In summary, we have shown that MnNP specifically and effectively deliver siRNA to TAMs in vivo.
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23 MeSH Terms
Structural basis for extracellular cis and trans RPTPσ signal competition in synaptogenesis.
Coles CH, Mitakidis N, Zhang P, Elegheert J, Lu W, Stoker AW, Nakagawa T, Craig AM, Jones EY, Aricescu AR
(2014) Nat Commun 5: 5209
MeSH Terms: Animals, Cell Differentiation, Chick Embryo, Coculture Techniques, Crystallization, Extracellular Matrix Proteins, Humans, Ligands, Mice, Neurogenesis, Neurons, Protein Binding, Protein Structure, Tertiary, Proteoglycans, Receptor, trkC, Receptor-Like Protein Tyrosine Phosphatases, Class 2, Signal Transduction, Synapses
Show Abstract · Added February 2, 2015
Receptor protein tyrosine phosphatase sigma (RPTPσ) regulates neuronal extension and acts as a presynaptic nexus for multiple protein and proteoglycan interactions during synaptogenesis. Unknown mechanisms govern the shift in RPTPσ function, from outgrowth promotion to synaptic organization. Here, we report crystallographic, electron microscopic and small-angle X-ray scattering analyses, which reveal sufficient inter-domain flexibility in the RPTPσ extracellular region for interaction with both cis (same cell) and trans (opposite cell) ligands. Crystal structures of RPTPσ bound to its postsynaptic ligand TrkC detail an interaction surface partially overlapping the glycosaminoglycan-binding site. Accordingly, heparan sulphate and heparin oligomers compete with TrkC for RPTPσ binding in vitro and disrupt TrkC-dependent synaptic differentiation in neuronal co-culture assays. We propose that transient RPTPσ ectodomain emergence from the presynaptic proteoglycan layer allows capture by TrkC to form a trans-synaptic complex, the consequent reduction in RPTPσ flexibility potentiating interactions with additional ligands to orchestrate excitatory synapse formation.
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18 MeSH Terms