Science.gov

Sample records for cell colony formation

  1. Study of budding yeast colony formation and its characterizations by using circular granular cell

    NASA Astrophysics Data System (ADS)

    Aprianti, D.; Haryanto, F.; Purqon, A.; Khotimah, S. N.; Viridi, S.

    2016-03-01

    Budding yeast can exhibit colony formation in solid substrate. The colony of pathogenic budding yeast can colonize various surfaces of the human body and medical devices. Furthermore, it can form biofilm that resists drug effective therapy. The formation of the colony is affected by the interaction between cells and with its growth media. The cell budding pattern holds an important role in colony expansion. To study this colony growth, the molecular dynamic method was chosen to simulate the interaction between budding yeast cells. Every cell was modelled by circular granular cells, which can grow and produce buds. Cohesion force, contact force, and Stokes force govern this model to mimic the interaction between cells and with the growth substrate. Characterization was determined by the maximum (L max) and minimum (L min) distances between two cells within the colony and whether two lines that connect the two cells in the maximum and minimum distances intersect each other. Therefore, it can be recognized the colony shape in circular, oval, and irregular shapes. Simulation resulted that colony formation are mostly in oval shape with little branch. It also shows that greater cohesion strength obtains more compact colony formation.

  2. Scatter factor influences the formation of prostate epithelial cell colonies on bone marrow stroma in vitro.

    PubMed

    Lang, S H; Clarke, N W; George, N J; Testa, N G

    1999-06-01

    Prostate cancer metastases form selectively in the bone marrow. Previously we demonstrated motility was important for the formation of primary prostatic epithelial cell colonies in bone marrow stroma (BMS) co-culture. In this study we looked at the influence of motility factors on the colony formation of epithelial cells derived from benign (bPEC) or malignant (mPEC) prostate tissue. After 7 days co-culture we found that anti-scatter factor consistently inhibited prostate epithelial cell colony formation on BMS (7/7 mPEC and 4/7 bPEC samples showed significant inhibition). Antibodies against bFGF and 5T4 did not significantly affect colony formation. Addition of fibroblast conditioned media (derived from benign prostates) to co-cultures stimulated the colony formation of bPEC (170%) and mPEC (252%). This stimulation was eliminated by depletion of SF from the conditioned media. Immunohistochemical staining found c-Met expression in 5/6 bPEC cultures and 7/9 mPEC cultures. When grown in BMS co-culture expression of c-Met was positive in 3/6 bPEC and 2/7 mPEC samples. In conclusion, scatter factor influences the in vitro formation of prostate epithelial cell colonies on BMS co-culture. PMID:10545020

  3. Colony formation and interleukin 2 production by leukaemic human T cells.

    PubMed Central

    Krajewski, A S; Dewar, A E; Seidelin, P H; Murray, R

    1983-01-01

    PHA-induced colony formation and interleukin 2 (IL-2) production were studied in four patients with T cell leukaemia (three cases OKT4+/T helper and one case OKT8+/T cytotoxic suppressor). Cases of T helper cell leukaemia showed colony formation that was comparable to normal purified blood T cells and was not dependent on the addition of conditioned medium, containing IL-2 activity, to cultures. In contrast the T suppressor cell leukaemia formed colonies only when cultures were supplemented with IL-2 containing medium. When IL-2 production by PHA stimulated cells was measured culture supernatants from the three T helper cell leukaemias all showed normal or high levels of activity, when compared to normal blood mononuclear cells, whereas the T suppressor cell leukaemia showed no activity. PMID:6604606

  4. Arsenic promotes centrosome abnormalities and cell colony formation in p53 compromised human lung cells

    SciTech Connect

    Liao Weiting; Lin Pinpin; Cheng, T.-S.; Yu, H.-S.; Chang, Louis W.

    2007-12-01

    Epidemiological evidence indicated that residents, especially cigarette smokers, in arseniasis areas had significantly higher lung cancer risk than those living in non-arseniasis areas. Thus, an interaction between arsenic and cigarette smoking in lung carcinogenesis was suspected. p53 dysfunction or mutation in lung epithelial cells was frequently observed in cigarette smokers. Our present study was to explore the differential effects by arsenic on H1355 cells (human lung adenocarcinoma cell line with mutation in p53), BEAS-2B (immortalized lung epithelial cell with functional p53) and pifithrin-{alpha}-treated BEAS-2B cells (p53-inhibited cells). These cells were treated with different doses of sodium arsenite (0, 0.1, 1, 5 and 10 {mu}M) for 48 h. A greater reduction in cell viability was observed in the BEAS-2B cells vs. p53 compromised cells (H1355 or p53-inhibited BEAS-2B). Similar observation was also made on 7-day cell survival (growth) study. TUNEL analysis confirmed that there was indeed a significantly reduced arsenite-induced apoptosis found in p53-compromised cells. Centrosomal abnormality has been attributed to eventual chromosomal missegregation, aneuploidy and tumorigenesis. In our present study, reduced p21 and Gadd45a expressions and increased centrosomal abnormality (atopic and multiple centrosomes) were observed in both arsenite-treated H1355 and p53-inhibited BEAS-2B cells as compared with similarly treated BEAS-2B cells. Increased anchorage-independent growth (colony formation) of BEAS-2B cells co-treated with pifithrin-{alpha} and 5 {mu}M sodium arsenite was also observed in soft agar. Our present investigation demonstrated that arsenic would act specifically on p53 compromised cells (either with p53 dysfunction or inhibited) to induce centrosomal abnormality and colony formation. These findings provided strong evidence on the carcinogenic promotional role of arsenic, especially under the condition of p53 dysfunction.

  5. Biophysical Properties of Scaffolds Modulate Human Blood Vessel Formation from Circulating Endothelial Colony-Forming Cells

    NASA Astrophysics Data System (ADS)

    Critser, Paul J.; Yoder, Mervin C.

    A functional vascular system forms early in development and is continually remodeled throughout the life of the organism. Impairment to the regeneration or repair of this system leads to tissue ischemia, dysfunction, and disease. The process of vascular formation and remodeling is complex, relying on local microenvironmental cues, cytokine signaling, and multiple cell types to function properly. Tissue engineering strategies have attempted to exploit these mechanisms to develop functional vascular networks for the generation of artificial tissues and therapeutic strategies to restore tissue homeostasis. The success of these strategies requires the isolation of appropriate progenitor cell sources which are straightforward to obtain, display high proliferative potential, and demonstrate an ability to form functional vessels. Several populations are of interest including endothelial colony-forming cells, a subpopulation of endothelial progenitor cells. Additionally, the development of scaffolds to deliver and support progenitor cell survival and function is crucial for the formation of functional vascular networks. The composition and biophysical properties of these scaffolds have been shown to modulate endothelial cell behavior and vessel formation. However, further investigation is needed to better understand how these mechanical properties and biophysical properties impact vessel formation. Additionally, several other cell populations are involved in neoangiogenesis and formation of tissue parenchyma and an understanding of the potential impact of these cell populations on the biophysical properties of scaffolds will also be needed to advance these strategies. This chapter examines how the biophysical properties of matrix scaffolds can influence vessel formation and remodeling and, in particular, the impact on in vivo human endothelial progenitor cell vessel formation.

  6. Formation and dissolution of bacterial colonies.

    PubMed

    Weber, Christoph A; Lin, Yen Ting; Biais, Nicolas; Zaburdaev, Vasily

    2015-09-01

    Many organisms form colonies for a transient period of time to withstand environmental pressure. Bacterial biofilms are a prototypical example of such behavior. Despite significant interest across disciplines, physical mechanisms governing the formation and dissolution of bacterial colonies are still poorly understood. Starting from a kinetic description of motile and interacting cells we derive a hydrodynamic equation for their density on a surface, where most of the kinetic coefficients are estimated from experimental data for N. gonorrhoeae bacteria. We use it to describe the formation of multiple colonies with sizes consistent with experimental observations. Finally, we show how the changes in the cell-to-cell interactions lead to the dissolution of the bacterial colonies. The successful application of kinetic theory to a complex far from equilibrium system such as formation and dissolution of living bacterial colonies potentially paves the way for the physical quantification of the initial stages of biofilm formation. PMID:26465495

  7. Formation and dissolution of bacterial colonies

    NASA Astrophysics Data System (ADS)

    Weber, Christoph A.; Lin, Yen Ting; Biais, Nicolas; Zaburdaev, Vasily

    2015-09-01

    Many organisms form colonies for a transient period of time to withstand environmental pressure. Bacterial biofilms are a prototypical example of such behavior. Despite significant interest across disciplines, physical mechanisms governing the formation and dissolution of bacterial colonies are still poorly understood. Starting from a kinetic description of motile and interacting cells we derive a hydrodynamic equation for their density on a surface, where most of the kinetic coefficients are estimated from experimental data for N. gonorrhoeae bacteria. We use it to describe the formation of multiple colonies with sizes consistent with experimental observations. Finally, we show how the changes in the cell-to-cell interactions lead to the dissolution of the bacterial colonies. The successful application of kinetic theory to a complex far from equilibrium system such as formation and dissolution of living bacterial colonies potentially paves the way for the physical quantification of the initial stages of biofilm formation.

  8. Cells capable of colony formation in the peripheral blood of man.

    PubMed

    McCredie, K B; Hersh, E M; Freireich, E J

    1971-01-22

    Colony-forming cells have been found in the peripheral blood of man and have been grown in vitro by use of a soft agar gel technique. It has been possible to collect these cells with a blood-cell separator in numbers similar to those found in the peripheral circulation. Repeat leukapheresis of the same donor does not reduce the number of circulating colony-forming cells. PMID:5538844

  9. Disrupting the PIKE-A/Akt interaction inhibits glioblastoma cell survival, migration, invasion and colony formation

    PubMed Central

    Qi, Q; He, K; Liu, X; Pham, C; Meyerkord, C; Fu, H; Ye, K

    2013-01-01

    The cyclin-dependent kinase 4 (CDK4) amplicon is frequently amplified in numerous human cancers including gliomas. PIKE-A, a proto-oncogene that is one of the important components of the CDK4 amplicon, binds to and enhances the kinase activity of Akt, thereby promoting cancer progression. To define the roles of the PIKE-A/Akt interaction in glioblastoma multiform (GBM) progression, we used biochemical protein/protein interaction (PPI) assays and live cell fluorescence-based protein complementation assays to search for small peptide antagonist from these proteins that were able to block their interaction. Here, we show that disruption of the interaction between PIKE-A and Akt by the small peptides significantly reduces glioblastoma cell proliferation, colony formation, migration and invasion. Disruption of PIKE-A/Akt association potently suppressed GBM cell proliferation and sensitized the cells to two clinical drugs that are currently used to treat GBM. Interestingly, GBM cells containing the CDK4 amplicon were more responsive to the inhibition of the PIKE-A/Akt interaction than GBM cells lacking this amplicon. Taken together, our findings provide proof-of-principle that blocking a PPI that is essential for cancer progression provides a valuable strategy for therapeutic discovery. PMID:22450747

  10. Enhancement of committed hematopoietic stem cell colony formation by nandrolone decanoate after sublethal whole body irradiation

    SciTech Connect

    Gallicchio, V.S.; Chen, M.G.; Watts, T.D.

    1984-11-01

    The ability of an anabolic steroid, nandrolone decanoate, to increase committed topoietic stem cell (CFU-gm, CFU-e, and BFU-e) colony formation after sublethal irradiation was evaluated. Immediately after receiving whole body irradiation and on the next two days, each mouse was injected intraperitoneally with nandrolone decanoate (1.25 mg) in propylene glycol. Irradiated control mice received only propylene glycol. Compared to controls, drug-treated mice showed marked peripheral blood leukocytosis and more stable packed red cell volume. Drug-treated mice also demonstrated increased erythropoiesis, as CFU-e/BFU-e concentrations from both marrow (9% to 581%) and spleen (15% to 797%) were elevated. Granulopoiesis was increased similarly, as CFU-gm concentrations from marrow (38% to 685%) and spleen (9% to 373%) were elevated. These results demonstrate that nandrolone decanoate enhances hematopoietic stem cell recovery after sublethal whole body irradiation. This suggests that following hematopoietic suppression, nandrolone decanoate may stimulate the recovery of hematopoiesis at the stem cell level and in peripheral blood.

  11. Abiotic factors in colony formation: effects of nutrition and light on extracellular polysaccharide production and cell aggregates of Microcystis aeruginosa

    NASA Astrophysics Data System (ADS)

    Yang, Zhen; Kong, Fanxiang

    2013-07-01

    Colony morphology is important for Microcystis to sustain a competitive advantage in eutrophic lakes. The mechanism of colony formation in Microcystis is currently unclear. Extracellular polysaccharide (EPS) has been reported to play an important role in cell aggregate formation of some phytoplankton. Microcystis aeruginosa was cultivated under varied abiotic conditions, including different nutrient, light, and temperature conditions, to investigate their effects on EPS production and morphological change. The results show that nutrient concentration and light intensity have great effects on EPS productionin M. aeruginosa. There was a considerable increase in EPS production after M. aeruginosa was cultivated in adjusted culture conditions similar to those present in the field (28.9 mg C/L, 1.98 mg N/L, 0.65 mg P/L, light intensity: 100 μmol/(m2 · s)). These results indicate that abiotic factors might be one of the triggers for colony formation in Microcystis.

  12. Convergence of bone morphogenetic protein and laminin-1 signaling pathways promotes proliferation and colony formation by fetal mouse pancreatic cells

    SciTech Connect

    Jiang Fangxu . E-mail: jiang@wehi.edu.au; Harrison, Leonard C.

    2005-08-01

    We previously reported that bone morphogenetic proteins (BMPs), members of the transforming growth factor superfamily, together with the basement membrane glycoprotein laminin-1 (Ln-1), promote proliferation of fetal pancreatic cells and formation of colonies containing peripheral insulin-positive cells. Here, we further investigate the cross-talk between BMP and Ln-1 signals. By RT-PCR, receptors for BMP (BMPR) (excepting BMPR-1B) and Ln-1 were expressed in the fetal pancreas between E13.5 and E17.5. Specific blocking antibodies to BMP-4 and -6 and selective BMP antagonists partially inhibited colony formation by fetal pancreas cells. Colony formation induced by BMP-6 and Ln-1 was completely abolished in a dose-dependent manner by blocking Ln-1 binding to its {alpha}{sub 6} integrin and {alpha}-dystroglycan receptors or by blocking the Ln-1 signaling molecules, phosphatidyl-inositol-3-kinase (P13K) and MAP kinase kinase-1. These results demonstrate a convergence of BMP and Ln-1 signaling through P13K and MAP kinase pathways to induce proliferation and colony formation in E15.5 fetal mouse pancreatic cells.

  13. Substrate Stiffness Affects Human Keratinocyte Colony Formation

    PubMed Central

    Zarkoob, Hoda; Bodduluri, Sandeep; Ponnaluri, Sailahari V.; Selby, John C.; Sander, Edward A.

    2015-01-01

    Restoration of epidermal organization and function in response to a variety of pathophysiological insults is critically dependent on coordinated keratinocyte migration, proliferation, and stratification during the process of wound healing. These processes are mediated by the reconfiguration of both cell-cell (desmosomes, adherens junctions) and cell-matrix (focal adhesions, hemidesmosomes) junctions and the cytoskeletal filament networks that they serve to interconnect. In this study, we investigated the role of substrate elasticity (stiffness) on keratinocyte colony formation in vitro during the process of nascent epithelial sheet formation as triggered by the calcium switch model of keratinocyte culture. Keratinocytes cultured on pepsin digested type I collagen coated soft (nominal E = 1.2 kPa) polyacrylamide gels embedded with fluorescent microspheres exhibited (i) smaller spread contact areas, (ii) increased migration velocities, and (iii) increased rates of colony formation with more cells per colony than did keratinocytes cultured on stiff (nominal E = 24 kPa) polyacrylamide gels. As assessed by tracking of embedded microsphere displacements, keratinocytes cultured on soft substrates generated large local substrate deformations that appeared to recruit adjacent keratinocytes into joining an evolving colony. Together with the observed differences in keratinocyte kinematics and substrate deformations, we developed two ad hoc analyses, termed distance rank (DR) and radius of cooperativity (RC), that help to objectively ascribe what we perceive as increasingly cooperative behavior of keratinocytes cultured on soft versus stiff gels during the process of colony formation. We hypothesize that the differences in keratinocyte colony formation observed in our experiments could be due to cell-cell mechanical signaling generated via local substrate deformations that appear to be correlated with the increased expression of β4 integrin within keratinocytes positioned

  14. Inhibition of colony formation in agarose of metastatic human breast carcinoma and melanoma cells by synthetic glycoamine analogs.

    PubMed

    Glinsky, G V; Mossine, V V; Price, J E; Bielenberg, D; Glinsky, V V; Ananthaswamy, H N; Feather, M S

    1996-05-01

    We studied the influence of 10 synthetic glycoamine analogs on colony formation in 0.3 and 0.9% agarose by metastatic human breast carcinoma (MDA-MB-435) and melanoma (TXM-13) cells. Nine synthetic analogs significantly inhibited the colony formation in 0.9% agarose of MDA-MB-435 human breast carcinoma cells; five compounds caused a 73-83% reduction of colony formation. Seven synthetic glycoamines caused a significant inhibition of colony formation in 0.9% agarose by TXM-13 melanoma cells with the inhibitory effect ranging from 71 to 87%. The 50% inhibition (I50) doses and relative activity rank of the compounds were similar for both breast carcinoma and melanoma cell lines. The murine B16 melanoma cell aggregation assay was employed to elucidate the potential mechanism(s) of the inhibitory activity of synthetic glycoamines. The relative activity ranks of the compounds based on the independently determined I50 doses for both cell aggregation and clonogenic growth assays were very similar for the four most active synthetic analogs and clearly indicated the importance of hydrophobic amino acid in mediating the bioactivity of synthetic glycoamines. In both experimental systems (clonogenic growth in agarose and cell aggregation assay) the leading compound was N-(1-deoxy-D-fructos-1-yl)-D-leucine (Fru-D-Leu) and the least active analog was N-(l-deoxy-D-fructos-1-yl)-glycine (Fru-Gly). These results show that synthetic glycoamines may act by competing for specific carbohydrate-lectin interactions, particularly those involving beta-galactoside-specific lectins expressed on metastatic cells. PMID:8674280

  15. Freeze-Drying of Mononuclear Cells Derived from Umbilical Cord Blood Followed by Colony Formation

    PubMed Central

    Natan, Dity; Nagler, Arnon; Arav, Amir

    2009-01-01

    Background We recently showed that freeze-dried cells stored for 3 years at room temperature can direct embryonic development following cloning. However, viability, as evaluated by membrane integrity of the cells after freeze-drying, was very low; and it was mainly the DNA integrity that was preserved. In the present study, we improved the cells' viability and functionality after freeze-drying. Methodology/Principal Findings We optimized the conditions of directional freezing, i.e. interface velocity and cell concentration, and we added the antioxidant EGCG to the freezing solution. The study was performed on mononuclear cells (MNCs) derived from human umbilical cord blood. After freeze-drying, we tested the viability, number of CD34+-presenting cells and ability of the rehydrated hematopoietic stem cells to differentiate into different blood cells in culture. The viability of the MNCs after freeze-drying and rehydration with pure water was 88%–91%. The total number of CD34+-presenting cells and the number of colonies did not change significantly when evaluated before freezing, after freeze-thawing, and after freeze-drying (5.4×104±4.7, 3.49×104±6 and 6.31×104±12.27 cells, respectively, and 31±25.15, 47±45.8 and 23.44±13.3 colonies, respectively). Conclusions This is the first report of nucleated cells which have been dried and then rehydrated with double-distilled water remaining viable, and of hematopoietic stem cells retaining their ability to differentiate into different blood cells. PMID:19381290

  16. Formation of Osteogenic Colonies on Well-Defined Adhesion Peptides by Freshly-Isolated Human Marrow Cells

    PubMed Central

    Au, Ada; Boehm, Cynthia A.; Mayes, Anne M.; Muschler, George F.; Griffith, Linda G.

    2007-01-01

    Bone graft performance can be enhanced by addition of connective tissue progenitors (CTPs) from fresh bone marrow in a manner that concentrates the CTP cell population within the graft. Here, we used small peptide adhesion ligands presented against an otherwise adhesion-resistant synthetic polymer background in order to illuminate the molecular basis for the attachment and colony formation by osteogenic CTPs from fresh human marrow, and contrast the behavior of fresh marrow to many commonly-used osteogenic cell sources. The linear GRGDSPY ligand was as effective as tissue culture polystyrene in fostering attachment of culture-expanded porcine CTPs. Although this GRGDSPY peptide was more effective than control peptides in fostering alkaline phosphatase-positive (AP) colony formation from primary human marrow in 5 of the 7 patients tested, GRGDSPY was as effective as the control glass substrate in only one patient of 7. Thus, the peptide appears capable of enabling osteoblastic development from only a subpopulation of CTPs in marrow. The bone sialoprotein-derived peptide FHRRIKA was ineffective in fostering attachment of primary culture-expanded pig CTPs, although it was as effective as GRGDSPY in fostering AP-positive colonies from fresh human marrow. This study provides insights into integrin-mediated behaviors of CTPs and highlights differences between freshly-isolated marrow and culture-expanded cells. PMID:17222453

  17. Loss of suppression of normal bone marrow colony formation by leukemic cell lines after differentiation is induced by chemical agents.

    PubMed

    Steinberg, H N; Tsiftsoglou, A S; Robinson, S H

    1985-01-01

    The human leukemic cell lines K562 and HL-60 were cocultured with normal bone marrow (BM) cells. Coculture with 10(4) K562 or HL-60 cells results in 50% inhibition of normal CFU-E and BFU-E colony formation. However, when the same number of K562 and HL-60 cells is first treated for two to five days with agents that induce their differentiation, a gradual loss in their capacity to inhibit CFU-E and BFU-E colony formation is observed. The inhibitory material in K562 cells is soluble and present in conditioned medium from cultures of these cells. The degree to which leukemic cell suppression of CFU-E and BFU-E growth is reversed is correlated with the time of exposure to the inducing agent. Suppression is no longer evident after five days of prior treatment with inducers. In fact, up to a 90% stimulation of CFU-E growth is observed in cocultures with K562 cells that have been pretreated with 30 to 70 mumol/L hemin for five days. K562 cells treated with concentrations of hemin as low as 30 mumol/L demonstrate increased hemoglobin synthesis and grow normally, but no longer have an inhibitory effect on CFU-E growth. Hence, reversal of normal BM growth inhibition must be caused by the more differentiated state of the K562 cells and not by a decrease in the number of these cells with treatment. Thus, induction of differentiation in cultured leukemic cells not only alters the malignant cell phenotype but also permits improved growth of accompanying normal marrow progenitor cells. Both are desired effects of chemotherapy. PMID:3838080

  18. Downregulation of ROS-FIG inhibits cell proliferation, colony-formation, cell cycle progression, migration and invasion, while inducing apoptosis in intrahepatic cholangiocarcinoma cells

    PubMed Central

    DENG, GANG; HU, CHENGHUAN; ZHU, LEI; HUANG, FEIZHOU; HUANG, WEI; XU, HONGBO; NIE, WANPIN

    2014-01-01

    Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer with poor responsiveness to existing drug therapies. Therefore, novel treatment strategies against ICC are required to improve survival. The aim of this study was to demonstrate the role of fused-in-glioblastoma-c-ros-oncogene1 (FIG-ROS) fusion gene in ICC. ROS was positively expressed in ICC tissues and HUCCT1 cells. Plasmids expressing ROS- and FIG-specific shRNAs were constructed and transfected into HUCCT1 cells. The results showed that single transfection of ROS- or FIG-specific shRNA inhibited HUCCT1 cell proliferation, colony formation, cell cycle progression, migration and invasion, while inducing apoptosis. Moreover, the co-inhibition of ROS- and FIG-specific shRNA exhibited stronger effects on HUCCT1 cell proliferation, apoptosis, colony formation, cell cycle progression, migration and invasion, when compared to single inhibition of ROS and FIG. Furthermore, findings of this study suggested that the AKT signaling pathway was involved in the ROS-FIG-mediated biological processes of HUCCT1 cells. In summary, the results suggest that FIG-ROS plays an oncogenic role in ICC. Additionally, ROS1-6290 and FIG-363 segments may become effective therapeutic targets for ICC harboring ROS-FIG fusion protein. PMID:24968753

  19. Ferulic acid decreases cell viability and colony formation while inhibiting migration of MIA PaCa-2 human pancreatic cancer cells in vitro.

    PubMed

    Fahrioğlu, Umut; Dodurga, Yavuz; Elmas, Levent; Seçme, Mücahit

    2016-01-15

    Novel and combinatorial treatment methods are becoming sought after entities in cancer treatment and these treatments are even more valuable for pancreatic cancer. The scientists are always on the lookout for new chemicals to help them in their fight against cancer. In this study, we examine the effects of ferulic acid (FA), a phenolic compound, on gene expression, viability, colony formation and migration/invasion in the cultured MIA PaCa-2 human pancreatic cancer cell. Cytotoxic effects of FA were determined by using trypan blue dye exclusion test and Cell TiterGlo (CTG) assay. IC50 dose in MIA PaCa-2 cells was detected as 500μM/ml at the 72nd hour. Expression profiles of certain cell cycle and apoptosis genes such as CCND1 (cyclin D1),CDK4, CDK6, RB, p21, p16, p53, caspase-3, caspase-9, caspase-8, caspase-10, Bcl-2, BCL-XL,BID, DR4,DR5,FADD,TRADD,PARP, APAF, Bax, Akt, PTEN, PUMA, NOXA, MMP2, MMP9, TIMP1 and TIMP2 were determined by real-time PCR. The effect of FA on cell viability was determined by CellTiter-Glo® Luminescent Cell Viability Assay. Additionally, effects of FA on colony formation and invasion were also investigated. It was observed that FA caused a significant decrease in the expression of CCND1, CDK 4/6, Bcl2 and caspase 8 and 10 in the MIA PaCa-2 cells while causing an increase in the expression of p53, Bax, PTEN caspase 3 and 9. FA was observed to decrease colony formation while inhibiting cell invasion and migration as observed by the BioCoat Matrigel Invasion Chamber guide and colony formation assays. In conclusion, FA is thought to behave as an anti-cancer agent by affecting cell cycle, apoptotic, invasion and colony formation behavior of MIA PaCa-2 cells. Therefore, FA is placed as a strong candidate for further studies aimed at finding a better, more effective treatment approach for pancreatic cancer. PMID:26516023

  20. Soft agar colony formation of bladder cells during carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)nitrosamine and application to detection of bladder cancer promoters.

    PubMed

    Hashimura, T; Kanamaru, H; Yoshida, O

    1987-05-01

    N-Butyl-N-(4-hydroxybutyl)nitrosamine (BBN) was given to male Fischer 344 rats at a dose of 0.05% in drinking water for 2, 4, 6 and 12 weeks, and double soft agar colony formation of the uroepithelial cells was determined periodically, during and after this administration. In the group administered BBN for 2 weeks, no significant colony growth was observed until week 8. In the group given BBN for 4 weeks, colony growth was observed at week 4 and the numbers of colonies remained constant until week 8. In the group given BBN for 6 weeks, significant colony growth was observed at weeks 6 and 8. In the group on BBN for 12 weeks, colonies grew from week 4 and significant numbers of colonies were observed from week 6, increasing up to week 10. Colony formation preceded papilloma development in the rat bladder, and was dependent on the duration of BBN administration. The effect of amino acids and sodium saccharin on colony formation was also evaluated. The rats were given 0.05% BBN for 3 weeks, followed immediately by the administration for 9 weeks of 2% L-tryptophan, 1% D-tryptophan, 2% L-leucine, 2% D-leucine, 2% DL-leucine, 2% L-isoleucine, 2% DL-isoleucine or 5% sodium saccharin in the diet. At week 12, the numbers of colonies were significantly higher in the groups given sodium saccharin, L-leucine, DL-leucine, L-isoleucine, DL-isoleucine and D-tryptophan. This method provides a potentially useful approach toward analyzing the early events in bladder carcinogenesis and may be applicable to detect new bladder carcinogens and promoters. PMID:3112059

  1. Matrix rigidity regulates spatiotemporal dynamics of Cdc42 activity and vacuole formation kinetics of endothelial colony forming cells

    PubMed Central

    Kim, Seung Joon; Wan, Qiaoqiao; Cho, Eunhye; Han, Bumsoo; Yoder, Mervin C.; Voytik-Harbin, Sherry L.; Na, Sungsoo

    2014-01-01

    Recent evidence has shown that endothelial colony forming cells (ECFCs) may serve as a cell therapy for improving blood vessel formation in subjects with vascular injury, largely due to their robust vasculogenic potential. The Rho family GTPase Cdc42 is known to play a primary role in this vasculogenesis process, but little is known about how extracellular matrix (ECM) rigidity affects Cdc42 activity during the process. In this study, we addressed two questions: Does matrix rigidity affect Cdc42 activity in ECFC undergoing early vacuole formation? How is the spatiotemporal activation of Cdc42 related to ECFC vacuole formation? A fluorescence resonance energy transfer (FRET)-based Cdc42 biosensor was used to examine the effects of the rigidity of three-dimensional (3D) collagen matrices on spatiotemporal activity of Cdc42 in ECFCs. Collagen matrix stiffness was modulated by varying the collagen concentration and therefore fibril density. The results showed that soft (150 Pa) matrices induced an increased level of Cdc42 activity compared to stiff (1 kPa) matrices. Time-course imaging and colocalization analysis of Cdc42 activity and vacuole formation revealed that Cdc42 activity was colocalized to the periphery of cytoplasmic vacuoles. Moreover, soft matrices generated faster and larger vacuoles than stiff matrices. The matrix-driven vacuole formation was enhanced by a constitutively active Cdc42 mutant, but significantly inhibited by a dominant-negative Cdc42 mutant. Collectively, the results suggest that matrix rigidity is a strong regulator of Cdc42 activity and vacuole formation kinetics, and that enhanced activity of Cdc42 is an important step in early vacuole formation in ECFCs. PMID:24393843

  2. Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells.

    PubMed

    Horibata, Sachi; Vo, Tommy V; Subramanian, Venkataraman; Thompson, Paul R; Coonrod, Scott A

    2015-01-01

    Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells. PMID:26067809

  3. Increased Potency of the PHSCN Dendrimer as an Inhibitor of Human Prostate Cancer Cell Invasion, Extravasation, and Lung Colony Formation

    PubMed Central

    Yao, Hongren; Zeng, Zhao-Zhu; Fay, Kevin S.; Staszewski, Evan D.; Veine, Donna M.; Livant, Donna L.

    2011-01-01

    Background Activated α5β1 integrin occurs specifically on tumor cells and on endothelial cells of tumor–associated vasculature, and plays a key role in invasion and metastasis. The PHSCN peptide (Ac-PHSCN-NH2) preferentially binds activated α5β1, to block invasion in vitro, and inhibit growth, metastasis and tumor recurrence in preclinical models of prostate cancer. In Phase I clinical trial, systemic Ac-PHSCN-NH2 monotherapy was well tolerated, and metastatic disease progression was prevented for 4–14 months in one third of treated patients. Results We have developed a significantly more potent derivative, the PHSCN-polylysine dendrimer (Ac-PHSCNGGK-MAP). Using in vitro invasion assays with naturally serum-free basement membranes, we observed that the PHSCN dendrimer was 130– to 1900–fold more potent than the PHSCN peptide at blocking α5β1–mediated invasion by DU 145 and PC-3 human prostate cancer cells, whether invasion was induced by serum, or by the Ac-PHSRN-NH2 peptide, under serum-free conditions. The PHSCN dendrimer was also approximately 800 times more effective than PHSCN peptide at preventing DU 145 and PC-3 extravasation in the lungs of athymic mice. Chou-Talalay analysis suggested that inhibition of both invasion in vitro and extravasation in vivo by the PHSCN dendrimer are highly synergistic. We found that many extravasated DU 145 and PC-3 cells go on to develop into metastatic colonies, and that a single pretreatment with the PHSCN dendrimer was 100–fold more affective than the PHSCN peptide at reducing lung colony formation. Conclusions Since many patients newly diagnosed with prostate cancer already have locally advanced or metastatic disease, the availability of a well-tolerated, nontoxic systemic therapy, like the PHSCN dendrimer, which prevents metastatic progression by inhibiting invasion, could be very beneficial. PMID:20339907

  4. The mycotoxin zearalenone enhances cell proliferation, colony formation and promotes cell migration in the human colon carcinoma cell line HCT116.

    PubMed

    Abassi, Haila; Ayed-Boussema, Imen; Shirley, Sarah; Abid, Salwa; Bacha, Hassen; Micheau, Olivier

    2016-07-01

    Zearalenone (ZEN) and Aflatoxin B1 (AFB1) are fungal secondary metabolites produced by Fusarium and Aspergillus genera, respectively. These mycotoxins are found world-wide as corn and wheat contaminants. AFB1 is probably the most toxic and carcinogenic mycotoxin. It has been demonstrated to be mutagenic, genotoxic, and hepatocarcinogenic. ZEN is a non-steroidal estrogenic mycotoxin that displays hepatotoxicity, immunotoxicity and genotoxicity. Its mutagenic and carcinogenic properties have so far remained controversial and questionable. Using the colon carcinoma cell line HCT116, we will show here that ZEN, at low concentrations, enhances cell proliferation, increases colony formation and fastens cell migration after wound healing. The highest effect of ZEN was observed at a concentration 10 times lower as compared to AFB1. Our findings suggest thus that this mycotoxin exhibits carcinogenesis-like properties in HCT116 cells. PMID:27084041

  5. Mesenchymal stromal cells, colony-forming unit fibroblasts, from bone marrow of untreated advanced breast and lung cancer patients suppress fibroblast colony formation from healthy marrow.

    PubMed

    Hofer, Erica Leonor; Labovsky, Vivian; La Russa, Vincent; Vallone, Valeria Fernández; Honegger, Alba Elizabeth; Belloc, Carlos Gabriel; Wen, Huei Chi; Bordenave, Raúl Horacio; Bullorsky, Eduardo Oscar; Feldman, Leonardo; Chasseing, Norma Alejandra

    2010-03-01

    We have shown that bone marrow (BM) from untreated advanced lung and breast cancer patients (LCP and BCP) have a reduced number of colony-forming unit fibroblasts (CFU-Fs) or mesenchymal stem cells (MSCs). Factors that regulate the proliferation and differentiation of CFU-F are produced by the patients' BM microenvironment. We have now examined whether conditioned media (CM) from patients' CFU-F-derived stromal cells also inhibits the colony-forming efficiency (CFE) of CFU-F in primary cultures from healthy volunteers (HV)-BM. Thus the number and proliferation potential of HV-CFU-F were also found to be decreased and similar to colony numbers and colony size of patients' CFU-F. Stromal cells from both of these types of colonies appeared relatively larger and lacked the characteristic spindle morphology typically seen in healthy stromal cells. We developed an arbitrary mesenchymal stromal cell maturational index by taking three measures consisting of stromal cell surface area, longitudinal and horizontal axis. All stromal indices derived from HV-CFU-F grown in patients' CM were similar to those from stromal elements derived from patients' CFU-F. These indices were markedly higher than stromal indices typical of HV-CFU-F cultured in healthy CM or standard medium [alpha-medium plus 20% heat-inactivated fetal bovine serum (FBS)]. Patients' CM had increased concentrations of the CFU-F inhibitor, GM-CSF, and low levels of bFGF and Dkk-1, strong promoters of self-renewal of MSCs, compared to the levels quantified in CM from HV-CFU-F. Moreover, the majority of patients' MSCs were unresponsive in standard medium and healthy CM to give CFU-F, indicating that the majority of mesenchymal stromal cells from patients' CFU-F are locked in maturational arrest. These results show that alterations of GM-CSF, bFGF, and Dkk-1 are associated with deficient cloning and maturation arrest of CFU-F. Defective autocrine and paracrine mechanisms may be involved in the BM microenvironments of

  6. Robust conversion of marrow cells to skeletal muscle with formation of marrow-derived muscle cell colonies: A multifactorial process

    SciTech Connect

    Abedi, Mehrdad; Greer, Deborah A.; Colvin, Gerald A.; Demers, Delia A.; Dooner, Mark S.; Harpel, Jasha A.; Weier, Heinz-Ulrich G.; Lambert, Jean-Francois; Quesenberry, Peter J.

    2004-01-10

    Murine marrow cells are capable of repopulating skeletal muscle fibers. A point of concern has been the robustness of such conversions. We have investigated the impact of type of cell delivery, muscle injury, nature of delivered cell, and stem cell mobilizations on marrow to muscle conversion. We transplanted GFP transgenic marrow into irradiated C57BL/6 mice and then injured anterior tibialis muscle by cardiotoxin. One month after injury, sections were analyzed by standard and deconvolutional microscopy for expression of muscle and hematopietic markers. Irradiation was essential to conversion although whether by injury or induction of chimerism is not clear. Cardiotoxin and to a lesser extent PBS injected muscles showed significant number of GFP+ muscle fibers while uninjected muscles showed only rare GFP+ cells. Marrow conversion to muscle was increased by two cycles of G-CSF mobilization and to a lesser extent with G-CSF and steel or GM-CSF. Transplantation of female GFP to male C57 BL/6 and GFP to Rosa26 mice showed fusion of donor cells to recipient muscle. High numbers of donor derived muscle colonies and up to12 percent GFP positive muscle cells were seen after mobilization or direct injection. These levels of donor muscle chimerism approach levels which could be clinically significant in developing strategies for the treatment of muscular dystrophies. In summary, the conversion of marrow to skeletal muscle cells is based on cell fusion and is critically dependent on injury. This conversion is also numerically significant and increases with mobilization.

  7. COLONY FORMATION ENHANCEMENT OF RAT TRACHEAL AND NASAL EPITHELIAL CELLS BY POLYACETATE, INDOLE ALKALOID, AND PHORBOL ESTER TUMOR PROMOTERS

    EPA Science Inventory

    The phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA), teleocidin, and two polyacetate tumor promoters (aplysiatoxin and debromoaplysiatoxin) have been tested for their effect on colony forming efficiency (CFE) of rat tracheal and nasal turbinate epithelial cells. In rat t...

  8. A novel small molecule STAT3 inhibitor, LY5, inhibits cell viability, colony formation, and migration of colon and liver cancer cells.

    PubMed

    Zhao, Chongqiang; Wang, Wenlong; Yu, Wenying; Jou, David; Wang, Yina; Ma, Haiyan; Xiao, Hui; Qin, Hua; Zhang, Cuntai; Lü, Jiagao; Li, Sheng; Li, Chenglong; Lin, Jiayuh; Lin, Li

    2016-03-15

    Signal Transducer and Activator of Transcription 3 (STAT3) is persistently activated in human liver and colon cancer cells and is required for cancer cell viability, survival and migration. Therefore, inhibition of STAT3 signaling may be a viable therapeutic approach for these two cancers. We recently designed a non-peptide small molecule STAT3 inhibitor, LY5, using in silico site-directed Fragment-based drug design (FBDD). The inhibitory effect on STAT3 phosphorylation, cell viability, migration and colony forming ability by LY5 were examined in human liver and colon cancer cells. We demonstrated that LY5 inhibited constitutive Interleukin-6 (IL-6)-induced STAT3 phosphorylation, STAT3 nuclear translocation, decreased STAT3 downstream targeted gene expression and induced apoptosis in liver and colon cancer cells. LY5 had little effect on STAT1 phosphorylation mediated by IFN-γ. Inhibition of persistent STAT3 phosphorylation by LY5 also inhibited colony formation, cell migration, and decreased the viability of liver cancer and colon cancer cells. Furthermore, LY5 inhibited STAT3 phosphorylation and suppressed colon tumor growth in a mouse model in vivo. Our results suggest that LY5 is a potent STAT3 inhibitor and may be a potential drug candidate for liver and colon cancer therapy. PMID:26883202

  9. A novel small molecule STAT3 inhibitor, LY5, inhibits cell viability, colony formation, and migration of colon and liver cancer cells

    PubMed Central

    Yu, Wenying; Jou, David; Wang, Yina; Ma, Haiyan; Xiao, Hui; Qin, Hua; Zhang, Cuntai; Lü, Jiagao; Li, Sheng; Li, Chenglong; Lin, Jiayuh; Lin, Li

    2016-01-01

    Signal Transducer and Activator of Transcription 3 (STAT3) is persistently activated in human liver and colon cancer cells and is required for cancer cell viability, survival and migration. Therefore, inhibition of STAT3 signaling may be a viable therapeutic approach for these two cancers. We recently designed a non-peptide small molecule STAT3 inhibitor, LY5, using in silico site-directed Fragment-based drug design (FBDD). The inhibitory effect on STAT3 phosphorylation, cell viability, migration and colony forming ability by LY5 were examined in human liver and colon cancer cells. We demonstrated that LY5 inhibited constitutive Interleukin-6 (IL-6)-induced STAT3 phosphorylation, STAT3 nuclear translocation, decreased STAT3 downstream targeted gene expression and induced apoptosis in liver and colon cancer cells. LY5 had little effect on STAT1 phosphorylation mediated by IFN-γ. Inhibition of persistent STAT3 phosphorylation by LY5 also inhibited colony formation, cell migration, and decreased the viability of liver cancer and colon cancer cells. Furthermore, LY5 inhibited STAT3 phosphorylation and suppressed colon tumor growth in a mouse model in vivo. Our results suggest that LY5 is a potent STAT3 inhibitor and may be a potential drug candidate for liver and colon cancer therapy. PMID:26883202

  10. NR4A1 promotes PDGF-BB-induced cell colony formation in soft agar.

    PubMed

    Eger, Glenda; Papadopoulos, Natalia; Lennartsson, Johan; Heldin, Carl-Henrik

    2014-01-01

    The fibroblast mitogen platelet-derived growth factor -BB (PDGF-BB) induces a transient expression of the orphan nuclear receptor NR4A1 (also named Nur77, TR3 or NGFIB). The aim of the present study was to investigate the pathways through which NR4A1 is induced by PDGF-BB and its functional role. We demonstrate that in PDGF-BB stimulated NIH3T3 cells, the MEK1/2 inhibitor CI-1040 strongly represses NR4A1 expression, whereas Erk5 downregulation delays the expression, but does not block it. Moreover, we report that treatment with the NF-κB inhibitor BAY11-7082 suppresses NR4A1 mRNA and protein expression. The majority of NR4A1 in NIH3T3 was found to be localized in the cytoplasm and only a fraction was translocated to the nucleus after continued PDGF-BB treatment. Silencing NR4A1 slightly increased the proliferation rate of NIH3T3 cells; however, it did not affect the chemotactic or survival abilities conferred by PDGF-BB. Moreover, overexpression of NR4A1 promoted anchorage-independent growth of NIH3T3 cells and the glioblastoma cell lines U-105MG and U-251MG. Thus, whereas NR4A1, induced by PDGF-BB, suppresses cell growth on a solid surface, it increases anchorage-independent growth. PMID:25269081

  11. NR4A1 Promotes PDGF-BB-Induced Cell Colony Formation in Soft Agar

    PubMed Central

    Eger, Glenda; Papadopoulos, Natalia; Lennartsson, Johan; Heldin, Carl-Henrik

    2014-01-01

    The fibroblast mitogen platelet-derived growth factor -BB (PDGF-BB) induces a transient expression of the orphan nuclear receptor NR4A1 (also named Nur77, TR3 or NGFIB). The aim of the present study was to investigate the pathways through which NR4A1 is induced by PDGF-BB and its functional role. We demonstrate that in PDGF-BB stimulated NIH3T3 cells, the MEK1/2 inhibitor CI-1040 strongly represses NR4A1 expression, whereas Erk5 downregulation delays the expression, but does not block it. Moreover, we report that treatment with the NF-κB inhibitor BAY11-7082 suppresses NR4A1 mRNA and protein expression. The majority of NR4A1 in NIH3T3 was found to be localized in the cytoplasm and only a fraction was translocated to the nucleus after continued PDGF-BB treatment. Silencing NR4A1 slightly increased the proliferation rate of NIH3T3 cells; however, it did not affect the chemotactic or survival abilities conferred by PDGF-BB. Moreover, overexpression of NR4A1 promoted anchorage-independent growth of NIH3T3 cells and the glioblastoma cell lines U-105MG and U-251MG. Thus, whereas NR4A1, induced by PDGF-BB, suppresses cell growth on a solid surface, it increases anchorage-independent growth. PMID:25269081

  12. The Stem Cell Marker CD133 Associates with Enhanced Colony Formation and Cell Motility in Colorectal Cancer

    PubMed Central

    Elsaba, Tarek M. A.; Martinez-Pomares, Luisa; Robins, Adrian R.; Crook, Simon; Seth, Rashmi; Jackson, Darryl; McCart, Amy; Silver, Andrew R.; Tomlinson, Ian P. M.; Ilyas, Mohammad

    2010-01-01

    CD133 is a membrane molecule that has been, controversially, reported as a CSC marker in colorectal cancer (CRC). In this study, we sought to clarify the expression and role of CD133 in CRC. Initially the size of the CD133−expressing (CD133+) population in eight well-described CRC cell lines was measured by flow cytometry and was found to range from 0% to >95%. The cell line HT29 has a CD133+ population of >95% and was chosen for functional evaluation of CD133 after gene knockdown by RNA interference. A time course assay showed that CD133 inhibition had no significant effect on cell proliferation or apoptosis. However, CD133 knockdown did result in greater susceptibility to staurosporine-induced apoptosis (p = 0.01) and reduction in cell motility (p<0.04). Since gene knockdown may cause “off-target” effects, the cell line SW480 (which has a CD133+ population of 40%) was sorted into pure CD133+ and CD133− populations to allow functional comparison of isogenic populations separated only by CD133 expression. In concordance with the knockdown experiments, a time course assay showed no significant proliferative differences between the CD133+/CD133− populations. Also greater resistance to staurosporine-induced apoptosis (p = 0.008), greater cell motility (p = 0.03) and greater colony forming efficiency was seen in the CD133+ population than the CD133− population in both 2D and 3D culture (p<0.0001 and p<0.003 respectively). Finally, the plasticity of CD133 expression in tumour cells was tested. Quantitative PCR analysis showed there was transcriptional repression in the CD133− population of SW480. Prolonged culture of a pure CD133− population resulted in re-emergence of CD133+ cells. We conclude that CD133 expression in CRCs is associated with some features attributable to stemness and that there is plasticity of CD133 expression. Further studies are necessary to delineate the mechanistic basis of these features. PMID:20502714

  13. Prolonged Proteasome Inhibition Cyclically Upregulates Oct3/4 and Nanog Gene Expression, but Reduces Induced Pluripotent Stem Cell Colony Formation

    PubMed Central

    Floyd, Elizabeth Z.; Staszkiewicz, Jaroslaw; Power, Rachel A.; Kilroy, Gail; Kirk-Ballard, Heather; Barnes, Christian W.; Strickler, Karen L.; Rim, Jong S.; Harkins, Lettie L.; Gao, Ru; Kim, Jeong

    2015-01-01

    Abstract There is ample evidence that the ubiquitin–proteasome system is an important regulator of transcription and its activity is necessary for maintaining pluripotency and promoting cellular reprogramming. Moreover, proteasome activity contributes to maintaining the open chromatin structure found in pluripotent stem cells, acting as a transcriptional inhibitor at specific gene loci generally associated with differentiation. The current study was designed to understand further the role of proteasome inhibition in reprogramming and its ability to modulate endogenous expression of pluripotency-related genes and induced pluripotent stem cells (iPSCs) colony formation. Herein, we demonstrate that acute combinatorial treatment with the proteasome inhibitors MG101 or MG132 and the histone deacetylase (HDAC) inhibitor valproic acid (VPA) increases gene expression of the pluripotency marker Oct3/4, and that MG101 alone is as effective as VPA in the induction of Oct3/4 mRNA expression in fibroblasts. Prolonged proteasome inhibition cyclically upregulates gene expression of Oct3/4 and Nanog, but reduces colony formation in the presence of the iPSC induction cocktail. In conclusion, our results demonstrate that the 26S proteasome is an essential modulator in the reprogramming process. Its inhibition enhances expression of pluripotency-related genes; however, efficient colony formation requires proteasome activity. Therefore, discovery of small molecules that increase proteasome activity might lead to more efficient cell reprogramming and generation of pluripotent cells. PMID:25826722

  14. Ultra-sensitive detection of tumorigenic cellular impurities in human cell-processed therapeutic products by digital analysis of soft agar colony formation.

    PubMed

    Kusakawa, Shinji; Yasuda, Satoshi; Kuroda, Takuya; Kawamata, Shin; Sato, Yoji

    2015-01-01

    Contamination with tumorigenic cellular impurities is one of the most pressing concerns for human cell-processed therapeutic products (hCTPs). The soft agar colony formation (SACF) assay, which is a well-known in vitro assay for the detection of malignant transformed cells, is applicable for the quality assessment of hCTPs. Here we established an image-based screening system for the SACF assay using a high-content cell analyzer termed the digital SACF assay. Dual fluorescence staining of formed colonies and the dissolution of soft agar led to accurate detection of transformed cells with the imaging cytometer. Partitioning a cell sample into multiple wells of culture plates enabled digital readout of the presence of colonies and elevated the sensitivity for their detection. In practice, the digital SACF assay detected impurity levels as low as 0.00001% of the hCTPs, i.e. only one HeLa cell contained in 10,000,000 human mesenchymal stem cells, within 30 days. The digital SACF assay saves time, is more sensitive than in vivo tumorigenicity tests, and would be useful for the quality control of hCTPs in the manufacturing process. PMID:26644244

  15. Inhibition of MEK and GSK3 supports ES cell-like domed colony formation from avian and reptile embryos.

    PubMed

    Nakanoh, Shota; Okazaki, Kenji; Agata, Kiyokazu

    2013-07-01

    As amniotes diversified, mammals may have modified mechanisms of cellular pluripotency along with the acquisition of a placenta. What then defined pluripotent states in the ancestral amniotes? To study the evolutionary background of pluripotency in amniotes, we tested the effects of extracellular effectors on primary culture cells from avian and reptile embryos in serum-free medium. When treated with a combination of a MEK inhibitor and a GSK3 inhibitor (2i condition), chicken early embryos formed domed colonies (DCs), which were morphologically indistinguishable from the colonies formed by mouse and rat naïve embryonic stem cells. However, no DCs formed when cells from further-developed embryos were cultured in the 2i condition, indicating that there is a clear boundary of DC-forming ability at around the stage of primitive streak elongation. Quail embryos at the blastoderm and cleavage stages also formed DCs in the 2i condition, which is consistent with the notion that the appearance of DCs corresponds with the presence of pluripotent cells in embryos. Gecko blastoderms also formed DCs in the 2i condition, but gastrulas did not. ERK activation by bFGF caused an effect opposite to that of the 2i condition, namely, it dispersed colonies of cells even from early embryos in all species examined. These results suggest that the regulation of pluripotency by FGF/ERK signaling may date back at least to the common ancestor of mammals, birds, and reptiles. However, gene expression analysis indicated the possibility that mammalian pluripotency transcription factors function differently in non-mammalian amniotes. PMID:23829214

  16. Evaluation of the effects of cryopreservation on viability, proliferation and colony formation of human spermatogonial stem cells in vitro culture.

    PubMed

    Mirzapour, T; Movahedin, M; Tengku Ibrahim, T A; Haron, A W; Nowroozi, M R

    2013-02-01

    Proliferation of spermatogonial stem cells (SSCs) in vitro system is very important. It can enhance SSCs numbers for success of transplantation and treatment of infertility in cancer patients. In this study, testicular cells that obtained from azoospermia patients (n=8) by enzymatic digestion were cryopreserved at the beginning and after 2 weeks of culture. Then, frozen-thawed SSCs were co-cultured on fresh Sertoli cells (experimental group 1), and frozen-thawed Sertoli cells (experimental group 2) for another 3 weeks. In control group, fresh SSCs were co-cultured on fresh Sertoli cells. Viability rate after enzymatic digestion was 93.4%±5.0. Frozen-thawed testicular cells after 2 weeks of culture had a significantly (P<0.05) higher percentage of living cells compared to frozen-thawed testicular cells at the beginning of culture (59.2±7.05 and 46.3±8.40 respectively). The number of colonies in the experimental group 1 was significantly higher than experimental group 2 (19.6±2.8 and 8.33±1.5, respectively, P<0.05). The diameter of the colonies in the experimental group 1 was significantly higher than control and experimental group 2 (P<0.05) after 3 weeks of culture (269.7±52.1, 204.34±24.1 and 112.52±23.5 μm, respectively). Cryopreservation technique will raise the possibility of banking SSCs for men who have a cancer-related illness and waiting for radiotherapy and/or chemotherapy. PMID:22621173

  17. Radial and Spiral Stream Formation in Proteus mirabilis Colonies

    PubMed Central

    Xue, Chuan; Budrene, Elena O.; Othmer, Hans G.

    2011-01-01

    The enteric bacterium Proteus mirabilis, which is a pathogen that forms biofilms in vivo, can swarm over hard surfaces and form a variety of spatial patterns in colonies. Colony formation involves two distinct cell types: swarmer cells that dominate near the surface and the leading edge, and swimmer cells that prefer a less viscous medium, but the mechanisms underlying pattern formation are not understood. New experimental investigations reported here show that swimmer cells in the center of the colony stream inward toward the inoculation site and in the process form many complex patterns, including radial and spiral streams, in addition to previously-reported concentric rings. These new observations suggest that swimmers are motile and that indirect interactions between them are essential in the pattern formation. To explain these observations we develop a hybrid model comprising cell-based and continuum components that incorporates a chemotactic response of swimmers to a chemical they produce. The model predicts that formation of radial streams can be explained as the modulation of the local attractant concentration by the cells, and that the chirality of the spiral streams results from a swimming bias of the cells near the surface of the substrate. The spatial patterns generated from the model are in qualitative agreement with the experimental observations. PMID:22219724

  18. Chiral pattern formation in compact microbial colonies

    NASA Astrophysics Data System (ADS)

    Korolev, Kirill; Bino George, Ashish

    Chirality is ubiquitous in biology from single molecules to entire populations. Yet, we are still lacking a detailed understanding of how chiral patterns emerge from cell competition and growth, even in simple microbial colonies. Although many microbes grow as dense colonies with no apparent chirality, recent experiments with Escherichia coli have demonstrated that internal dynamics in such populations can be in fact chiral. We show that there is a unique way to extend the commonly-used reaction-diffusion models of colony growth to account for the emergent chirality. This new model connects microscopic and macroscopic chirality and explains the origin of logarithmic spirals separating different sub-populations in a colony. We also show that chirality is substantially enhanced by the cooperation among the cells at the expansion frontier. In heterogeneous populations composed of strains with different chiralities and growth rates, our model predicts a very rich set of possible dynamics. For example, different chiralities can result in either sharp boundaries between the strains or promote their intermixing depending on the preferred twisting directions of the strains.

  19. MicroRNA-31 Is Overexpressed in Cutaneous Squamous Cell Carcinoma and Regulates Cell Motility and Colony Formation Ability of Tumor Cells

    PubMed Central

    Meisgen, Florian; Lohcharoenkal, Warangkana; Ståhle, Mona; Sonkoly, Enikö; Pivarcsi, Andor

    2014-01-01

    Cutaneous squamous cell carcinoma (cSCC) is a malignancy of epidermal keratinocytes that is responsible for approximately 20% of skin cancer-related death yearly. We have previously compared the microRNA (miRNA) expression profile of cSCC to healthy skin and found the dysregulation of miRNAs in human cSCC. In this study we show that miR-31 is overexpressed in cSCC (n = 68) compared to healthy skin (n = 34) and precancerous skin lesions (actinic keratosis, n = 12). LNA in situ hybridization revealed that miR-31 was specifically up-regulated in tumor cells. Mechanistic studies of inhibition of endogenous miR-31 in human metastatic cSCC cells revealed suppressed migration, invasion and colony forming ability, whereas overexpression of miR-31 induced these phenotypes. These results indicate that miR-31 regulates cancer-associated phenotypes of cSCC and identify miR-31 as a potential target for cSCC treatment. PMID:25068518

  20. Maintenance and induction of murine embryonic stem cell differentiation using E-cadherin-Fc substrata without colony formation

    NASA Astrophysics Data System (ADS)

    Meng, Qing-Yuan; Akaike, Toshihiro

    2013-03-01

    Induced embryonic stem (ES) cells are expected to be promising cell resources for the observation of the cell behaviors in developmental biology as well as the implantation in cell treatments in human diseases. A recombinant E-cadherin substratum was developed as a cell recognizable substratum to maintain the ES cells' self-renewal and pluripotency at single cell level. Furthermore, the generation of various cell lineages in different germ layers, including hepatic or neural cells, was achieved on the chimeric protein layer precisely and effectively. The induction and isolation of specific cell population was carried out with the enhancing effect of other artificial extracellular matrices (ECMs) in enzyme-free process. The murine ES cell-derived cells showed highly morphological similarities and functional expressions to matured hepatocytes or neural progenitor cells.

  1. Colony formation and morphology in Borrelia burgdorferi.

    PubMed

    Kurtti, T J; Munderloh, U G; Johnson, R C; Ahlstrand, G G

    1987-11-01

    Two strains of Borrelia burgdorferi, B31 and 297, formed colonies when plated onto Barbour-Stoenner-Kelly medium solidified with agarose (1.3%) and incubated in a candle jar at 34 degrees C. Colonies differing in morphology were observed in both strains after 2 to 3 weeks of incubation. Strain B31 colonies were either compact, round (mean diameter, 0.43 mm), and restricted to the surface of the agarose medium or diffuse (mean diameter, 1.80 mm) and penetrating into the solid medium. Strain 297 colonies (mean diameter, 1.43 mm) either showed a raised center surrounded by a diffuse ring of spirochetes or consisted of numerous small spirochetal aggregates. Both colony types expanded into the agarose medium. Scanning electron and light microscopy confirmed that the colonies were formed by spirochetes. Twisted tangles of intertwined spirochetes were visible on the surface, with numerous spherical bodies among them, especially in the central regions. At the periphery, the borreliae were more loosely packed, and individual coils were discernible. PMID:3693538

  2. Formation of complex bacterial colonies via self-generated vortices

    NASA Astrophysics Data System (ADS)

    Czirók, András; Ben-Jacob, Eshel; Cohen, Inon; Vicsek, Tamás

    1996-08-01

    Depending on the environmental conditions bacterial colonies growing on agar surfaces can exhibit complex colony formation and various types of collective motion. Experimental results are presented concerning the hydrodynamics (vortices, migration of bacteria in clusters) and colony formation of a morphotype of Bacillus subtilis. Some of these features are not specific to this morphotype but also have been observed in several other bacterial strains, suggesting the presence of universal effects. A simple model of self-propelled particles is proposed, which is capable of describing the hydrodynamics on the intermediate level, including the experimentally observed rotating disks of bacteria. The colony formation is captured by a complex generic model taking into account nutrient diffusion, reproduction, and sporulation of bacteria, extracellular slime deposition, chemoregulation, and inhomogeneous population. Our model also sheds light on some possible biological benefits of this ``multicellular behavior.''

  3. Diffusion-limited growth in bacterial colony formation

    NASA Astrophysics Data System (ADS)

    Matsushita, Mitsugu; Fujikawa, Hiroshi

    1990-09-01

    Colonies of bacterial species called Bacillus subtilis have been found to grow two-dimensionally and self-similarly on agar plates through diffusion-limited processes in a nutrient concentration field. We obtained a fractal dimension of the colony patterns of D=1.73±0.02, very close to that of the two-dimensional DLA model, and confirmed the existence of the screening effect of protruding main branches against inner ones in a colony, the repulsion between two neighboring colonies and the tendency to grow toward nutrient. These effects are all characteristic of the pattern formation in a Laplacian field. This finding implies the importance of physical properties of the environment for the morphology of bacterial colonies in general.

  4. 17β-Estradiol regulates cell proliferation, colony formation, migration, invasion and promotes apoptosis by upregulating miR-9 and thus degrades MALAT-1 in osteosarcoma cell MG-63 in an estrogen receptor-independent manner

    SciTech Connect

    Fang, Dengfeng; Yang, Hui; Lin, Jing; Teng, Yi; Jiang, Yingying; Chen, Jiao; Li, Yu

    2015-02-20

    In bone, different concentration of estrogen leads to various of physiological processes in osteoblast, such as the proliferation, migration, and apoptosis in an estrogen receptor-dependent manner. But little was known about the estrogen effects on osteosarcoma (OS). In this study, OS cell MG-63 was treated with low (1 nM) or high (100 nM) dose of 17β-Estradiol (E2) with the presence or absence of estrogen receptor α (ERα), for evaluating the E2 effects on proliferation, migration, invasion, colony formation and apoptosis. Consistent with a previous study, high dose of E2 treatment dramatically downregulated expressing level of long non-coding RNA metastasis associated lung adenocarcinoma transcript 1 (MALAT-1). The observation of upregulation of miR-9 after a high dose of E2 treatment indicated the cause of MALAT-1 reduction. Downregulation of MALAT-1 promoted the combination of SFPQ/PTBP2 complex. It was also observed that the proliferation, migration, invasion, colony formation and apoptosis of OS cells were remarkably affected by high dose of E2 treatment, but not by low dose, in an ERα independent manner. Furthermore, the abolishment of the effects on these physiological processes caused by ectopic expression of miR-9 ASOs suggested the necessity of miR-9 in MALAT-1 regulation. Here we found that the high dose of E2 treatment upregulated miR-9 thus posttranscriptionally regulated MALAT-1 RNA level in OS cells, and then the downregulation of MALAT-1 inhibited cell proliferation, migration, invasion and epithelial–mesenchymal transition (EMT) processes in the E2-dose dependent and ER-independent ways. - Highlights: • E2 affects osteosarcoma cell MG-63 in an Estrogen receptor-independent way. • High dose of E2 treatment upregulates miR-9 which target to MALAT-1 RNA. • Upregulated miR-9 degrades MALAT-1 and thus affects combination of SFPQ/PTBP2. • E2 treatment block cell proliferation, colony formation, mobility, and enhance apoptosis.

  5. Stage-dependent reduction in T colony formation in Hodgkin's disease. Coincidence with monocyte synthesis of prostaglandins.

    PubMed Central

    Bockman, R S

    1980-01-01

    Prostaglandin synthesis and T lymphocyte colony formation have been examined in previously untreated patients with Hodgkin's disease. Mononuclear cells have been isolated from peripheral blood and spleens of these patients. Significant augmentation in prostaglandin E levels were noted in the mononuclear cell cutures from Hodgkin's disease patients compared with controls (1.64 +/- 0.29 vs. 0.39 +/- 0.09 ng/10(6) cells, P < 0.005). Measured prostaglandin E levels increased with advancing stage of disease. Virtually all of the prostaglandins were synthesized by the adherent monocyte cell population. Prostaglandin E was the major product. Clonal expansion of a T lymphocyte precursor cell, which gives rise to colonies > 50 cells, was determined by a layered soft agar method. T colony formation was significantly reduced in patients with stage II, III, and IV disease. There were progressively reduced colony numbers seen with advancing stage of disease (609 +/- 209, 416 +/- 158, 207 +/- 58 compared with normals 2,274 +/- 360 colonies/10(6) cells plated; P < 0.005). The addition of inhibitors of endogenous prostaglandin synthesis resulted in significant augmentation of T colony number. However, a consistent relative decrease in T colony number was seen even when endogenous prostaglandin E synthesis was blocked. It would appear that both the prostaglandin-dependent and independent T colony precursor cells are lost with progressive stage of disease. A causative role of augmented prostaglandin synthesis in this stage-dependent reduction of T colony formation could not be established. PMID:6967491

  6. Lymphocyte culture: induction of colonies by conditioned medium from human lymphoid cell lines.

    PubMed

    Galbraith, R M; Goust, J M; Fudenberg, H H

    1977-12-01

    The presence of phytohemagglutinin or pokeweed mitogen in cultures of human peripheral blood mononuclear cells in agar is known to stimulate the formation of lymphoid colonies. We now report that similar colonies can be induced in the absence of plant lectins upon addition of filtered and ultracentrifuged conditioned medium (CM) obtained from certain human lymphoblastoid cell lines. Colony formation required at least 6 X 10(5) mononuclear cells per milliliter, and optimum results were obtained at concentrations of 1 X 10(6) cells/ml in the presence of 20% CM (50-500 colonies per 10(6) cells cultured). Individual cells within colonies displayed uniform morphological characteristics of lymphoid cells, and the majority formed rosettes with sheep erythrocytes, suggesting that they were of T-cell type. PMID:303689

  7. Studies of sector formation in expanding bacterial colonies

    NASA Astrophysics Data System (ADS)

    Golding, I.; Cohen, I.; Ben-Jacob, E.

    1999-12-01

    We study sector formation in expanding bacterial colonies grown on a substrate with low level of nutrient. Bursts of sectors are observed both during compact growth on soft agar and during branching growth on semi-solid agar. For theoretical studies of these bursts we employ two mathematical models we have used successfully in the past to study patterning of bacterial colonies: a discrete model and a continuous reaction-diffusion model. Using these models we investigate the amount of segregation achieved by a neutral mutation, as well as by mutations having some advantage over the wild type. We also study the effect of chemotaxis signaling on the sector formation.

  8. The PIM inhibitor AZD1208 synergizes with ruxolitinib to induce apoptosis of ruxolitinib sensitive and resistant JAK2-V617F-driven cells and inhibit colony formation of primary MPN cells

    PubMed Central

    Mazzacurati, Lucia; Lambert, Que T.; Pradhan, Anuradha; Griner, Lori N.; Huszar, Dennis; Reuther, Gary W.

    2015-01-01

    Classical myeloproliferative neoplasms (MPNs) are hematopoietic stem cell disorders that exhibit excess mature myeloid cells, bone marrow fibrosis, and risk of leukemic transformation. Aberrant JAK2 signaling plays an etiological role in MPN formation. Because neoplastic cells in patients are largely insensitive to current anti-JAK2 therapies, effective therapies remain needed. Members of the PIM family of serine/threonine kinases are induced by JAK/STAT signaling, regulate hematopoietic stem cell growth, protect hematopoietic cells from apoptosis, and exhibit hematopoietic cell transforming properties. We hypothesized that PIM kinases may offer a therapeutic target for MPNs. We treated JAK2-V617F-dependent MPN model cells as well as primary MPN patient cells with the PIM kinase inhibitors SGI-1776 and AZD1208 and the JAK2 inhibitor ruxolitinib. While MPN model cells were rather insensitive to PIM inhibitors, combination of PIM inhibitors with ruxolitinib led to a synergistic effect on MPN cell growth due to enhanced apoptosis. Importantly, PIM inhibitor mono-therapy inhibited, and AZD1208/ruxolitinib combination therapy synergistically suppressed, colony formation of primary MPN cells. Enhanced apoptosis by combination therapy was associated with activation of BAD, inhibition of downstream components of the mTOR pathway, including p70S6K and S6 protein, and activation of 4EBP1. Importantly, PIM inhibitors re-sensitized ruxolitinib-resistant MPN cells to ruxolitinib by inducing apoptosis. Finally, exogenous expression of PIM1 induced ruxolitinib resistance in MPN model cells. These data indicate that PIMs may play a role in MPNs and that combining PIM and JAK2 kinase inhibitors may offer a more efficacious therapeutic approach for MPNs over JAK2 inhibitor mono-therapy. PMID:26472029

  9. Colony-Stimulating Factor-1 Signaling Suppresses Renal Crystal Formation

    PubMed Central

    Taguchi, Kazumi; Kitamura, Hiroshi; Yasui, Takahiro; Naiki, Taku; Hamamoto, Shuzo; Ando, Ryosuke; Mizuno, Kentaro; Kawai, Noriyasu; Tozawa, Keiichi; Asano, Kenichi; Tanaka, Masato; Miyoshi, Ichiro; Kohri, Kenjiro

    2014-01-01

    We recently reported evidence suggesting that migrating macrophages (Mϕs) eliminate renal crystals in hyperoxaluric mice. Mϕs can be inflammatory (M1) or anti-inflammatory (M2), and colony-stimulating factor-1 (CSF-1) mediates polarization to the M2Mϕ phenotype. M2Mϕs promote renal tissue repair and regeneration, but it is not clear whether these cells are involved in suppressing renal crystal formation. We investigated the role of M2Mϕs in renal crystal formation during hyperoxaluria using CSF-1–deficient mice, which lack M2Mϕs. Compared with wild-type mice, CSF-1–deficient mice had significantly higher amounts of renal calcium oxalate crystal deposition. Treatment with recombinant human CSF-1 increased the expression of M2-related genes and markedly decreased the number of renal crystals in both CSF-1–deficient and wild-type mice. Flow cytometry of sorted renal Mϕs showed that CSF-1 deficiency resulted in a smaller population of CD11b+F4/80+CD163+CD206hi cells, which represent M2-like Mϕs. Additionally, transfusion of M2Mϕs into CSF-1–deficient mice suppressed renal crystal deposition. In vitro phagocytosis assays with calcium oxalate monohydrate crystals showed a higher rate of crystal phagocytosis by M2-polarized Mϕs than M1-polarized Mϕs or renal tubular cells. Gene array profiling showed that CSF-1 deficiency resulted in disordered M2- and stone-related gene expressions. Collectively, our results provide compelling evidence for a suppressive role of CSF-1 signaling in renal crystal formation. PMID:24578130

  10. Monoclonal origin of B lymphocyte colony-forming cells in spleen colonies formed by multipotential hemopoietic stem cells

    PubMed Central

    Lala, PK; Johnson, GR

    1978-01-01

    Spleen colonies produced by transplanting lethally irradiated mice with either 12 day fetal liver or adult bone marrow cells were found to contain B- lymphocyte colony-forming cells (BL-CFC) . The proportion of BL-CFC positive spleen colonies did not increase substantially between 8 and 14 days after transplantation, the range being 18-45 percent. However, the absolute number of BL-CFC per spleen colony varied considerably (between 1 and 10,318), although the majority of colonies contained less than 200 BL-CFC. Irrespective of the time after transplantation, smaller spleen colonies were found to have a higher frequency of BL-CFC than larger spleen colonies. To determine the possible clonal origin of BL-CFC from spleen colony- forming unit (CFU-S), CBA mice were injected with equal numbers of CBA and CBA T(6)/T(6) fetal liver or adult bone marrow cells. Analysis of 7-15-day spleen colonies demonstrated that 90 percent were either exclusively T(6) positive or T(6) negative and approximately equal numbers ofboth colony types were observed. B-lymphocyte colonies were grown and successfully karyotyped from 19 spleen colonies. When compared with the original spleen colony karyotype the B-lymphocyte colony cells karyotype was identical in all 19 cases. In 3 of the 19 colonies analyzed a mixture of T(6) positive and T(6) negative karyotypes was present and identical proportions of the karyotypes were present in the pooled B-lymphocyte colony cells and spleen colony cells. The data indicate that the B-lymphocyte colony-forming cells detected in spleen colonies are genuine members of the hemopoietic clone derived from the initiating hemopoietic stem cell (CFU-S). PMID:309918

  11. Periodic Colony Formation by Bacterial Species Bacillus subtilis

    NASA Astrophysics Data System (ADS)

    Wakita, Jun-ichi; Shimada, Hirotoshi; Itoh, Hiroto; Matsuyama, Tohey; Matsushita, Mitsugu

    2001-03-01

    We have investigated the periodic colony growth of bacterial species Bacillus subtilis. A colony grows cyclically with the interface repeating an advance (migration phase) and a rest (consolidation phase) alternately on a surface of semi-solid agar plate under appropriate environmental conditions, resulting in a concentric ring-like colony. It was found from macroscopic observations that the characteristic quantities for the periodic growth such as the migration time, the consolidation time and the terrace spacing do not depend so much on nutrient concentration Cn, but do on agar concentration Ca. The consolidation time was a weakly increasing function of Ca, while the migration time and the terrace spacing were, respectively, weakly and strongly decreasing function of Ca. Overall, the cycle (migration-plus-consolidation) time seems to be constant, and does not depend so much on both Cn and Ca. Microscopically, bacterial cells inside the growing front of a colony keep increasing their population during both migration and consolidation phases. It was also confirmed that their secreting surfactant called surfactin does not affect their periodic growth qualitatively, i.e., mutant cells which cannot secrete surfactin produce a concentric ring-like colony. All these results suggest that the diffusion of the nutrient and the surfactin are irrelevant to their periodic growth.

  12. The Rim101p/PacC pathway and alkaline pH regulate pattern formation in yeast colonies.

    PubMed

    Piccirillo, Sarah; White, Melissa G; Murphy, Jeffrey C; Law, Douglas J; Honigberg, Saul M

    2010-03-01

    Multicellular organisms utilize cell-to-cell signals to build patterns of cell types within embryos, but the ability of fungi to form organized communities has been largely unexplored. Here we report that colonies of the yeast Saccharomyces cerevisiae formed sharply divided layers of sporulating and nonsporulating cells. Sporulation initiated in the colony's interior, and this region expanded upward as the colony matured. Two key activators of sporulation, IME1 and IME2, were initially transcribed in overlapping regions of the colony, and this overlap corresponded to the initial sporulation region. The development of colony sporulation patterns depended on cell-to-cell signals, as demonstrated by chimeric colonies, which contain a mixture of two strains. One such signal is alkaline pH, mediated through the Rim101p/PacC pathway. Meiotic-arrest mutants that increased alkali production stimulated expression of an early meiotic gene in neighboring cells, whereas a mutant that decreased alkali production (cit1Delta) decreased this expression. Addition of alkali to colonies accelerated the expansion of the interior region of sporulation, whereas inactivation of the Rim101p pathway inhibited this expansion. Thus, the Rim101 pathway mediates colony patterning by responding to cell-to-cell pH signals. Cell-to-cell signals coupled with nutrient gradients may allow efficient spore formation and spore dispersal in natural environments. PMID:20038633

  13. Scaling of Traction Stresses with Size of Cohesive Cell Colonies

    NASA Astrophysics Data System (ADS)

    Mertz, Aaron F.; Banerjee, Shiladitya; Che, Yonglu; Marchetti, M. Christina; Horsley, Valerie; Dufresne, Eric R.

    2012-02-01

    We explore the mechanical properties of colonies of cohesive cells adherent on soft substrates. Specifically, we image the spatial distribution of traction stresses exerted by colonies of primary mouse keratinocytes on fibronectin-coated silicone gels. These cells have strong cell-cell adhesions mediated by E-cadherin. We observe that the work performed by a colony on its substrate is concentrated at the colony's periphery. The total work is strongly correlated to the geometrical size of the colony but not to number of cells. In other words, the mechanical output of a large single cell mimics that of a cohesive colony with the same overall size. We compare our findings to a recent theoretical model that treats the cohesive colony as an active gel.

  14. Active mechanics and geometry of adherent cells and cell colonies

    NASA Astrophysics Data System (ADS)

    Banerjee, Shiladitya

    2014-03-01

    Measurements of traction stresses exerted by adherent cells or cell colonies on elastic substrates have yielded new insight on how the mechanical and geometrical properties of the substrate regulate cellular force distribution, mechanical energy, spreading, morphology or stress ber architecture. We have developed a generic mechanical model of adherent cells as an active contractile gel mechanically coupled to an elastic substrate and to neighboring cells in a tissue. The contractile gel model accurately predicts the distribution of cellular and traction stresses as observed in single cell experiments, and captures the dependence of cell shape, traction stresses and stress ber polarization on the substrate's mechanical and geometrical properties. The model further predicts that the total strain energy of an adherent cell is solely regulated by its spread area, in agreement with recent experiments on micropatterned substrates with controlled geometry. When used to describe the behavior of colonies of adherent epithelial cells, the model demonstrates the crucial role of the mechanical cross-talk between intercellular and extracellular adhesion in regulating traction force distribution. Strong intercellular mechanical coupling organizes traction forces to the colony periphery, whereas weaker intercellular coupling leads to the build up of traction stresses at intercellular junctions. Furthermore, in agreement with experiments on large cohesive keratinocyte colonies, the model predicts a linear scaling of traction forces with the colony size. This scaling suggests the emergence of an effective surface tension as a scale-free material property of the adherent tissue, originating from actomyosin contractility.

  15. The induction of human peripheral blood lymphoid colonies by conditioned media from human tumour cell lines.

    PubMed Central

    Vesole, D H; Moore, G E

    1980-01-01

    Conditioned medium (CM) from 29 human tumour cell lines and 3 malignant pleural fluids were tested for their ability to stimulate lymphoid colony formation in semi-solid agar; 9 of 14 malignant melanomas, 3 of 6 colonic carcinomas, 2 of 5 ovarian carcinomas, 3 of 4 breast carcinomas and 1 of 3 pleural fluids from breast cancer patients contained colony-stimulating activity (CSA) for human peripheral blood lymphoid cells (PBL) in semi-solid agar. Conditioned media also stimulated PBL proliferation in liquid medium; these effects were dose dependent. With the exception of one pleural fluid, extensive dialysis of CM did not significantly increase colony formation; CM from two tumour cell lines demonstrated a significant decrease in the induction of colony formation after dialysis. PMID:6970165

  16. Two-Dimensionality of Yeast Colony Expansion Accompanied by Pattern Formation

    PubMed Central

    Chen, Lin; Noorbakhsh, Javad; Adams, Rhys M.; Samaniego-Evans, Joseph; Agollah, Germaine; Nevozhay, Dmitry; Kuzdzal-Fick, Jennie; Mehta, Pankaj; Balázsi, Gábor

    2014-01-01

    Yeasts can form multicellular patterns as they expand on agar plates, a phenotype that requires a functional copy of the FLO11 gene. Although the biochemical and molecular requirements for such patterns have been examined, the mechanisms underlying their formation are not entirely clear. Here we develop quantitative methods to accurately characterize the size, shape, and surface patterns of yeast colonies for various combinations of agar and sugar concentrations. We combine these measurements with mathematical and physical models and find that FLO11 gene constrains cells to grow near the agar surface, causing the formation of larger and more irregular colonies that undergo hierarchical wrinkling. Head-to-head competition assays on agar plates indicate that two-dimensional constraint on the expansion of FLO11 wild type (FLO11) cells confers a fitness advantage over FLO11 knockout (flo11Δ) cells on the agar surface. PMID:25504059

  17. Modeling cell-matrix traction forces in Keratinocyte colonies

    NASA Astrophysics Data System (ADS)

    Banerjee, Shiladitya

    2013-03-01

    Crosstalk between cell-cell and cell-matrix adhesions plays an essential role in the mechanical function of tissues. The traction forces exerted by cohesive keratinocyte colonies with strong cell-cell adhesions are mostly concentrated at the colony periphery. In contrast, for weak cadherin-based intercellular adhesions, individual cells in a colony interact with their matrix independently, with a disorganized distribution of traction forces extending throughout the colony. In this talk I will present a minimal physical model of the colony as contractile elastic media linked by springs and coupled to an elastic substrate. The model captures the spatial distribution of traction forces seen in experiments. For cell colonies with strong cell-cell adhesions, the total traction force of the colony measured in experiments is found to scale with the colony's geometrical size. This scaling suggests the emergence of an effective surface tension of magnitude comparable to that measured for non-adherent, three-dimensional cell aggregates. The physical model supports the scaling and indicates that the surface tension may be controlled by acto-myosin contractility. Supported by the NSF through grant DMR-1004789. This work was done in collaboration with Aaron F. Mertz, Eric R. Dufresne and Valerie Horsley (Yale University) and M. Cristina Marchetti (Syracuse University).

  18. Expression of the transcriptional repressor Gfi-1 is regulated by C/EBPα and is involved in its proliferation and colony formation inhibitory effects in p210BCR/ABL-expressing cells

    PubMed Central

    Lidonnici, Maria Rosa; Audia, Alessandra; Soliera, Angela Rachele; Prisco, Marco; Ferrari-Amorotti, Giovanna; Waldron, Todd; Donato, Nick; Zhang, Ying; Martinez, Robert V.; Holyoake, Tessa L.; Calabretta, Bruno

    2010-01-01

    Ectopic expression of C/EBPα in p210BCR/ABL-expressing cells induces granulocytic differentiation, inhibits proliferation and suppresses leukemogenesis. To dissect the molecular mechanisms underlying these biological effects, C/EBPα-regulated genes were identified by microarray analysis in 32D-p210BCR/ABL cells. One of the genes whose expression was activated by C/EBPα in a DNA binding-dependent manner in BCR/ABL-expressing cells is the transcriptional repressor Gfi-1. We show here that C/EBPα interacts with a functional C/EBP binding site in the Gfi-1 5′ flanking region and enhances the promoter activity of Gfi-1. Moreover, in K562 cells, RNAi-mediated downregulation of Gfi-1 expression partially rescued the proliferation inhibitory but not the differentiation inducing effect of C/EBPα. Ectopic expression of wild type Gfi-1 but not of a transcriptional repressor mutant (Gfi-1P2A) inhibited proliferation and markedly suppressed colony formation but did not induce granulocytic differentiation of BCR/ABL-expressing cells. By contrast, Gfi-1 shRNA-tranduced CD34+ CML cells were markedly more clonogenic than the scramble-transduced counterpart. Together, these studies indicate that Gfi-1 is a direct target of C/EBPα required for its proliferation and survival inhibitory effects in BCR/ABL-expressing cells. PMID:20924107

  19. Large area magnetic micropallet arrays for cell colony sorting.

    PubMed

    Cox-Muranami, Wesley A; Nelson, Edward L; Li, G P; Bachman, Mark

    2016-01-01

    A new micropallet array platform for adherent cell colony sorting has been developed. The platform consisted of thousands of square plastic pallets, 270 μm by 270 μm on each side, large enough to hold a single colony of cells. Each pallet included a magnetic core, allowing them to be collected with a magnet after being released using a microscope mounted laser system. The micropallets were patterned from 1002F epoxy resist and were fabricated on translucent, gold coated microscope slides. The gold layer was used as seed for electroplating the ferromagnetic cores within every individual pallet. The gold layer also facilitated the release of each micropallet during laser release. This array allows for individual observation, sorting and collection of isolated cell colonies for biological cell colony research. In addition to consistent release and recovery of individual colonies, we demonstrated stable biocompatibility and minimal loss in imaging quality compared to previously developed micropallet arrays. PMID:26606460

  20. Melanoma Cell Colony Expansion Parameters Revealed by Approximate Bayesian Computation

    PubMed Central

    Vo, Brenda N.; Drovandi, Christopher C.; Pettitt, Anthony N.; Pettet, Graeme J.

    2015-01-01

    In vitro studies and mathematical models are now being widely used to study the underlying mechanisms driving the expansion of cell colonies. This can improve our understanding of cancer formation and progression. Although much progress has been made in terms of developing and analysing mathematical models, far less progress has been made in terms of understanding how to estimate model parameters using experimental in vitro image-based data. To address this issue, a new approximate Bayesian computation (ABC) algorithm is proposed to estimate key parameters governing the expansion of melanoma cell (MM127) colonies, including cell diffusivity, D, cell proliferation rate, λ, and cell-to-cell adhesion, q, in two experimental scenarios, namely with and without a chemical treatment to suppress cell proliferation. Even when little prior biological knowledge about the parameters is assumed, all parameters are precisely inferred with a small posterior coefficient of variation, approximately 2–12%. The ABC analyses reveal that the posterior distributions of D and q depend on the experimental elapsed time, whereas the posterior distribution of λ does not. The posterior mean values of D and q are in the ranges 226–268 µm2h−1, 311–351 µm2h−1 and 0.23–0.39, 0.32–0.61 for the experimental periods of 0–24 h and 24–48 h, respectively. Furthermore, we found that the posterior distribution of q also depends on the initial cell density, whereas the posterior distributions of D and λ do not. The ABC approach also enables information from the two experiments to be combined, resulting in greater precision for all estimates of D and λ. PMID:26642072

  1. Melanoma Cell Colony Expansion Parameters Revealed by Approximate Bayesian Computation.

    PubMed

    Vo, Brenda N; Drovandi, Christopher C; Pettitt, Anthony N; Pettet, Graeme J

    2015-12-01

    In vitro studies and mathematical models are now being widely used to study the underlying mechanisms driving the expansion of cell colonies. This can improve our understanding of cancer formation and progression. Although much progress has been made in terms of developing and analysing mathematical models, far less progress has been made in terms of understanding how to estimate model parameters using experimental in vitro image-based data. To address this issue, a new approximate Bayesian computation (ABC) algorithm is proposed to estimate key parameters governing the expansion of melanoma cell (MM127) colonies, including cell diffusivity, D, cell proliferation rate, λ, and cell-to-cell adhesion, q, in two experimental scenarios, namely with and without a chemical treatment to suppress cell proliferation. Even when little prior biological knowledge about the parameters is assumed, all parameters are precisely inferred with a small posterior coefficient of variation, approximately 2-12%. The ABC analyses reveal that the posterior distributions of D and q depend on the experimental elapsed time, whereas the posterior distribution of λ does not. The posterior mean values of D and q are in the ranges 226-268 µm2h-1, 311-351 µm2h-1 and 0.23-0.39, 0.32-0.61 for the experimental periods of 0-24 h and 24-48 h, respectively. Furthermore, we found that the posterior distribution of q also depends on the initial cell density, whereas the posterior distributions of D and λ do not. The ABC approach also enables information from the two experiments to be combined, resulting in greater precision for all estimates of D and λ. PMID:26642072

  2. Phenotypic plasticity within yeast colonies: differential partitioning of cell fates.

    PubMed

    Piccirillo, Sarah; Kapros, Tamas; Honigberg, Saul M

    2016-05-01

    Across many phyla, a common aspect of multicellularity is the organization of different cell types into spatial patterns. In the budding yeast Saccharomyces cerevisiae, after diploid colonies have completed growth, they differentiate to form alternating layers of sporulating cells and feeder cells. In the current study, we found that as yeast colonies developed, the feeder cell layer was initially separated from the sporulating cell layer. Furthermore, the spatial pattern of sporulation in colonies depended on the colony's nutrient environment; in two environments in which overall colony sporulation efficiency was very similar, the pattern of feeder and sporulating cells within the colony was very different. As noted previously, under moderately suboptimal conditions for sporulation-low acetate concentration or high temperature-the number of feeder cells increases as does the dependence of sporulation on the feeder-cell transcription factor, Rlm1. Here we report that even under a condition that is completely blocked sporulation, the number of feeder cells still increased. These results suggest broader implications to our recently proposed "Differential Partitioning provides Environmental Buffering" or DPEB hypothesis. PMID:26743103

  3. High-Throughput Method for Automated Colony and Cell Counting by Digital Image Analysis Based on Edge Detection

    PubMed Central

    Choudhry, Priya

    2016-01-01

    Counting cells and colonies is an integral part of high-throughput screens and quantitative cellular assays. Due to its subjective and time-intensive nature, manual counting has hindered the adoption of cellular assays such as tumor spheroid formation in high-throughput screens. The objective of this study was to develop an automated method for quick and reliable counting of cells and colonies from digital images. For this purpose, I developed an ImageJ macro Cell Colony Edge and a CellProfiler Pipeline Cell Colony Counting, and compared them to other open-source digital methods and manual counts. The ImageJ macro Cell Colony Edge is valuable in counting cells and colonies, and measuring their area, volume, morphology, and intensity. In this study, I demonstrate that Cell Colony Edge is superior to other open-source methods, in speed, accuracy and applicability to diverse cellular assays. It can fulfill the need to automate colony/cell counting in high-throughput screens, colony forming assays, and cellular assays. PMID:26848849

  4. Human tumor clonogenic assay for carcinoma of the lung. II. Factors that influence colony formation in soft agar.

    PubMed

    Kanzawa, F; Matsushima, Y; Hamburger, A W; Ishihara, J; Sasaki, Y; Shimizu, E; Eguchi, K; Shinkai, T; Saijo, N; Miyazawa, N

    1987-01-01

    The human tumor clonogenic assay (HTCA) has potential value for studies of both the chemosensitivity and biology of human tumors. However, many technical problems including low plating efficiencies and the preparation of sufficient numbers of viable cells remain. In this study, an improved method for disaggregation of solid tumors increased the yield of single cells. Consequently, more than 10 anticancer drugs could be tested in 94 of 168 specimens (56%). Removal of peripheral blood lymphocytes from cell suspensions derived from effusions also improved colony formation. Adequate growth for sensitivity testing (greater than 30 colonies/plate) was obtained in 122 cases (73%), inadequate growth for drug evaluation (5-29 colonies/plate) in 29 cases (17%), and no colony formation (less than 5 colonies/plate) in 17 cases (10%) of the 168 viable samples. The cloning efficiencies of cells derived from primary tumors (median 0.015%) were higher than those of cells derived from metastatic tumors (0.012%), and they varied with the location of the metastatic site. Cloning efficiencies varied markedly from specimen to specimen, and were unaffected by tumor histology, grade of differentiation, patient age, stage of disease, or prior chemotherapy. The HTCA is promising as a potential tool for studying the biology of tumors. PMID:3601316

  5. Defect-Mediated Morphologies in Growing Cell Colonies

    NASA Astrophysics Data System (ADS)

    Doostmohammadi, Amin; Thampi, Sumesh P.; Yeomans, Julia M.

    2016-07-01

    Morphological trends in growing colonies of living cells are at the core of physiological and evolutionary processes. Using active gel equations, which include cell division, we show that shape changes during the growth can be regulated by the dynamics of topological defects in the orientation of cells. The friction between the dividing cells and underlying substrate drives anisotropic colony shapes toward more isotropic morphologies, by mediating the number density and velocity of topological defects. We show that the defects interact with the interface at a specific interaction range, set by the vorticity length scale of flows within the colony, and that the cells predominantly reorient parallel to the interface due to division-induced active stresses.

  6. Whole Cell Modeling: From Single Cells to Colonies

    PubMed Central

    Cole, John A.

    2015-01-01

    A great deal of research over the last several years has focused on how the inherent randomness in movements and reactivity of biomolecules can give rise to unexpected large-scale differences in the behavior of otherwise identical cells. Our own research has approached this problem from two vantage points – a microscopic kinetic view of the individual molecules (nucleic acids, proteins, etc.) diffusing and interacting in a crowded cellular environment; and a broader systems-level view of how enzyme variability can give rise to well-defined metabolic phenotypes. The former led to the development of the Lattice Microbes software – a GPU-accelerated stochastic simulator for reaction-diffusion processes in models of whole cells; the latter to the development of a method we call population flux balance analysis (FBA). The first part of this article reviews the Lattice Microbes methodology, and two recent technical advances that extend the capabilities of Lattice Microbes to enable simulations of larger organisms and colonies. The second part of this article focuses on our recent population FBA study of Escherichia coli, which predicted variability in the usage of different metabolic pathways resulting from heterogeneity in protein expression. Finally, we discuss exciting early work using a new hybrid methodology that integrates FBA with spatially resolved kinetic simulations to study how cells compete and cooperate within dense colonies and consortia. PMID:26989262

  7. Random mitotic activities across human embryonic stem cell colonies.

    SciTech Connect

    Jin, Q.; Duggan, R.; Dasa, S.; Li, F.; Chen, L.

    2010-08-01

    A systemic and quantitative study was performed to examine whether different levels of mitotic activities, assessed by the percentage of S-phase cells at any given time point, existed at different physical regions of human embryonic stem (hES) cell colonies at 2, 4, 6 days after cell passaging. Mitotically active cells were identified by the positive incorporation of 5-bromo-2-deoxyuridine (BrdU) within their newly synthesized DNA. Our data indicated that mitotically active cells were often distributed as clusters randomly across the colonies within the examined growth period, presumably resulting from local deposition of newly divided cells. This latter notion was further demonstrated by the confined growth of enhanced green florescence protein (EGFP) expressing cells amongst non-GFP expressing cells. Furthermore, the overall percentage of mitotically active cells remained constantly at about 50% throughout the 6-day culture period, indicating mitotic activities of hES cell cultures were time-independent under current growth conditions.

  8. Pattern Formation of Bacterial Colonies by Escherichia coli

    NASA Astrophysics Data System (ADS)

    Tokita, Rie; Katoh, Takaki; Maeda, Yusuke; Wakita, Jun-ichi; Sano, Masaki; Matsuyama, Tohey; Matsushita, Mitsugu

    2009-07-01

    We have studied the morphological diversity and change in bacterial colonies, using the bacterial species Escherichia coli, as a function of both agar concentration Ca and nutrient concentration Cn. We observed various colony patterns, classified them into four types by pattern characteristics and established a morphological diagram by dividing it into four regions. They are regions A [diffusion-limited aggregation (DLA)-like], B (Eden-like), C (concentric-ring), and D (fluid-spreading). In particular, we have observed a concentric-ring colony growth for E. coli. We focused on the periodic growth in region C and obtained the following results: (i) A colony grows cyclically with the growing front repeating an advance (migration phase) and a momentary rest (consolidation phase) alternately. (ii) The growth width L and the bulge width W in one cycle decrease asymptotically to certain values, when Ca is increased. (iii) L does not depend on Cn, while W is an increasing function of Cn. Plausible mechanisms are proposed to explain the experimental results, by comparing them with those obtained for other bacterial species such as Proteus mirabilis and Bacillus subtilis.

  9. Interrelationships between Colonies, Biofilms, and Planktonic Cells of Pseudomonas aeruginosa▿ †

    PubMed Central

    Mikkelsen, H.; Duck, Z.; Lilley, K. S.; Welch, M.

    2007-01-01

    Pseudomonas aeruginosa is a gram-negative bacterium and an opportunistic human pathogen that causes chronic infections in immunocompromised individuals. These infections are hard to treat, partly due to the high intrinsic resistance of the bacterium to clinically used antibiotics and partly due to the formation of antibiotic-tolerant biofilms. The three most common ways of growing bacteria in vitro are as planktonic cultures, colonies on agar plates, and biofilms in continuous-flow systems. Biofilms are known to express genes different from those of planktonic cells, and biofilm cells are generally believed to closely resemble planktonic cells in stationary phase. However, few, if any, studies have examined global gene expression in colonies. We used a proteomic approach to investigate the interrelationships between planktonic cells, colonies, and biofilms under comparable conditions. Our results show that protein profiles in colonies resemble those of planktonic cells. Furthermore, contrary to what has been reported previously, the protein profiles of biofilms were found to more closely resemble those of exponentially growing planktonic cells than those of planktonic cells in the stationary phase. These findings raise some intriguing questions about the true nature of biofilms. PMID:17220232

  10. Role of gas vesicles and intra-colony spaces during the process of algal bloom formation.

    PubMed

    Zhang, Yongsheng; Zheng, Binghui; Jiang, Xia; Zheng, Hao

    2013-06-01

    Aggregation morphology, vertical distribution, and algal density were analyzed during the algal cell floating process in three environments. The role of gas vesicles and intra-colony spaces was distinguished by algal blooms treated with ultrasonic waves and high pressure. Results demonstrated that the two buoyancy providers jointly provide buoyancy for floating algal cells. The results were also confirmed by force analysis. In the simulation experiment, the buoyancy acting on algal cells was greater than its gravity at sample ports 2 and 3 of a columnar-cultivated cell vessel, and intra-colony spaces were not detected. In Taihu Lake, gas vesicle buoyancy was notably less than total algal cell gravity. Buoyancy provided by intra-colony spaces exceeded total algal cell gravity at the water surface, but not at other water depths. In the Daning River, total buoyancies provided by the two buoyancy providers were less than total algal cell gravity at different water depths. PMID:23833817

  11. Colony Stimulating Factor-1 exerts direct effects on the proliferation and invasiveness of endometrial epithelial cells

    PubMed Central

    Aligeti, Sabitha; Kirma, Nameer B.; Binkely, Peter A; Schenken, Robert S.; Tekmal, Rajeshwar Rao

    2011-01-01

    Although macrophage colony stimulating factor (CSF-1) has been suggested to play a role in part in maintaining the chronic inflammatory response in endometriosis, our previous data suggest that CSF-1 may also play a role in early endometriosis lesion formation. In the current studies, we have examined this possibility and have shown that CSF-1, in an autocrine fashion, has a direct effect on endometrial epithelial cell proliferation and attachment to peritoneal mesothelial cells, early steps in endometriosis lesion formation on the peritoneum. PMID:21481374

  12. Burkholderia cenocepacia ShvR-regulated genes that influence colony morphology, biofilm formation, and virulence.

    PubMed

    Subramoni, Sujatha; Nguyen, David T; Sokol, Pamela A

    2011-08-01

    Burkholderia cenocepacia is an opportunistic pathogen that primarily infects cystic fibrosis (CF) patients. Previously, we reported that ShvR, a LysR regulator, influences colony morphology, virulence, and biofilm formation and regulates the expression of an adjacent 24-kb genomic region encoding 24 genes. In this study, we report the functional characterization of selected genes in this region. A Tn5 mutant with shiny colony morphology was identified with a polar mutation in BCAS0208, predicted to encode an acyl-coenzyme A dehydrogenase. Mutagenesis of BCAS0208 and complementation analyses revealed that BCAS0208 is required for rough colony morphology, biofilm formation, and virulence on alfalfa seedlings. It was not possible to complement with BCAS0208 containing a mutation in the catalytic site. BCAS0201, encoding a putative flavin adenine dinucleotide (FAD)-dependent oxidoreductase, and BCAS0207, encoding a putative citrate synthase, do not influence colony morphology but are required for optimum levels of biofilm formation and virulence. Both BCAS0208 and BCAS0201 contribute to pellicle formation, although individual mutations in each of these genes had no appreciable effect on pellicle formation. A mutant with a polar insertion in BCAS0208 was significantly less virulent in a rat model of chronic lung infection as well as in the alfalfa model. Genes in this region were shown to influence utilization of branched-chain fatty acids, tricarboxylic acid cycle substrates, l-arabinose, and branched-chain amino acids. Together, our data show that the ShvR-regulated genes BCAS0208 to BCAS0201 are required for the rough colony morphotype, biofilm and pellicle formation, and virulence in B. cenocepacia. PMID:21690240

  13. The isolation and culture of endothelial colony-forming cells from human and rat lungs.

    PubMed

    Alphonse, Rajesh S; Vadivel, Arul; Zhong, Shumei; Zong, Shumei; McConaghy, Suzanne; Ohls, Robin; Yoder, Mervin C; Thébaud, Bernard

    2015-11-01

    Blood vessels are crucial for the normal development, lifelong repair and homeostasis of tissues. Recently, vascular progenitor cell-driven 'postnatal vasculogenesis' has been suggested as an important mechanism that contributes to new blood vessel formation and organ repair. Among several described progenitor cell types that contribute to blood vessel formation, endothelial colony-forming cells (ECFCs) have received widespread attention as lineage-specific 'true' vascular progenitors. Here we describe a protocol for the isolation of pulmonary microvascular ECFCs from human and rat lung tissue. Our technique takes advantage of an earlier protocol for the isolation of circulating ECFCs from the mononuclear cellular fraction of peripheral blood. We adapted the earlier protocol to isolate resident ECFCs from the distal lung tissue. After enzymatic dispersion of rat or human lung samples into a cellular suspension, CD31-expressing cells are positively selected using magnetic-activated cell sorting and plated in endothelial-specific growth conditions. The colonies arising after 1-2 weeks in culture are carefully separated and expanded to yield pure ECFC cultures after a further 2-3 weeks. The resulting cells demonstrate the defining characteristics of ECFCs such as (i) 'cobblestone' morphology of cultured cell monolayers; (ii) acetylated low-density lipoprotein uptake and Ulex europaeus lectin binding; (iii) tube-like network formation in Matrigel; (iv) expression of endothelial cell-specific surface markers and the absence of hematopoietic or myeloid surface antigens; (v) self-renewal potential displayed by the most proliferative cells; and (vi) contribution to de novo vessel formation in an in vivo mouse implant model. Assuming typical initial cell adhesion and proliferation rates, the entire procedure can be completed within 4 weeks. Isolation and culture of lung vascular ECFCs will allow assessment of the functional state of these cells in experimental and human

  14. Agent Based Modelling Helps in Understanding the Rules by Which Fibroblasts Support Keratinocyte Colony Formation

    PubMed Central

    Sun, Tao; McMinn, Phil; Holcombe, Mike; Smallwood, Rod; MacNeil, Sheila

    2008-01-01

    Background Autologous keratincoytes are routinely expanded using irradiated mouse fibroblasts and bovine serum for clinical use. With growing concerns about the safety of these xenobiotic materials, it is desirable to culture keratinocytes in media without animal derived products. An improved understanding of epithelial/mesenchymal interactions could assist in this. Methodology/Principal Findings A keratincyte/fibroblast o-culture model was developed by extending an agent-based keratinocyte colony formation model to include the response of keratinocytes to both fibroblasts and serum. The model was validated by comparison of the in virtuo and in vitro multicellular behaviour of keratinocytes and fibroblasts in single and co-culture in Greens medium. To test the robustness of the model, several properties of the fibroblasts were changed to investigate their influence on the multicellular morphogenesis of keratinocyes and fibroblasts. The model was then used to generate hypotheses to explore the interactions of both proliferative and growth arrested fibroblasts with keratinocytes. The key predictions arising from the model which were confirmed by in vitro experiments were that 1) the ratio of fibroblasts to keratinocytes would critically influence keratinocyte colony expansion, 2) this ratio needed to be optimum at the beginning of the co-culture, 3) proliferative fibroblasts would be more effective than irradiated cells in expanding keratinocytes and 4) in the presence of an adequate number of fibroblasts, keratinocyte expansion would be independent of serum. Conclusions A closely associated computational and biological approach is a powerful tool for understanding complex biological systems such as the interactions between keratinocytes and fibroblasts. The key outcome of this study is the finding that the early addition of a critical ratio of proliferative fibroblasts can give rapid keratinocyte expansion without the use of irradiated mouse fibroblasts and bovine

  15. Microtable Arrays for Culture and Isolation of Cell Colonies

    PubMed Central

    Pai, Jeng-Hao; Xu, Wei; Sims, Christopher E.; Allbritton, Nancy L.

    2010-01-01

    Cell microarrays with culture sites composed of individually removable microstructures or micropallets have proven benefits for isolation of cells from a mixed population. The laser energy required to selectively remove these micropallets with attached cells from the array depends on the microstructure surface area in contact with the substrate. Laser energies sufficient to release micropallets greater than 100 μm resulted in loss of cell viability. A new 3-dimensional culture site similar in appearance to a table was designed and fabricated using a simple process that relied on a differential sensitivity of two photoresists to UV-mediated photopolymerization. With this design, the larger culture area rests on four small supports to minimize the surface area in contact with the substrate. Microtables up to 250 × 250 μm were consistently released with single 10 μJ pulses to each of the 4 support structures. In contrast, microstructures with a 150 × 150 μm surface area in contact with the substrate could not be reliably released at pulse energies up to 212 μJ. Cassie-Baxter wetting is required to provide a barrier of air to localize and sequester cells to the culture sites. A second asset of the design was an increased retention of this air barrier under conditions of decreased surface tension and after prolonged culture of cells. The improved air retention was due to the hydrophobic cavity created beneath the table and above the substrate which entrapped air when an aqueous solution was added to the array. The microtables proved an efficient method for isolating colonies from the array with 100% of selected colonies competent to expand following release from the array. PMID:20644916

  16. High nutrient concentration and temperature alleviated formation of large colonies of Microcystis: Evidence from field investigations and laboratory experiments.

    PubMed

    Zhu, Wei; Zhou, Xiaohua; Chen, Huaimin; Gao, Li; Xiao, Man; Li, Ming

    2016-09-15

    Correlations between Microcystis colony size and environmental factors were investigated in Meiliang Bay and Gonghu Bay of Lake Taihu (China) from 2011 to 2013. Compared with Gonghu Bay, both nutrient concentrations and Microcystis colony sizes were greater in Meiliang Bay. The median colony size (D50: 50% of the total mass of particles smaller than this size) increased from April to August and then decreased until November. In both bays, the average D50 of Microcystis colonies were <100 μm in spring, but colonies within moderate-size (100-500 μm) dominated in summer. The differences in colony size in Meiliang Bay and Gonghu Bay were probably due to horizontal drift driven by the prevailing south wind in summer. Redundancy analysis (RDA) of field data indicated that colony size was negatively related to nutrient concentrations but positively related to air temperature, suggesting that low nutrient concentrations and high air temperature promoted formation of large colonies. To validate the field survey, Microcystis colonies collected from Lake Taihu were cultured at different temperatures (15, 20, 25 and 30 °C) under high and low nutrient concentrations for 9 days. The size of Microcystis colonies significantly decreased when temperature was above 20 °C but had no significant change at 15 °C. The differences in temperature effects on colony formation shown from field and laboratory suggested that the larger colonies in summer were probably due to the longer growth period rather than the higher air temperature and light intensity. In addition, colony size decreased more significantly at high nutrient levels. Therefore, it could be concluded that high nutrient concentration and temperature may alleviate formation of large colonies of Microcystis. PMID:27262121

  17. Characteristics of rat megakaryocyte colonies and their progenitors in agar culture

    SciTech Connect

    Kellar, K.L.; Rolovic, Z.; Evatt, B.L.; Sewell, E.T.

    1985-11-01

    The characteristics of megakaryocyte colonies that develop from megakaryocyte progenitors of rat bone marrow stimulated by rat spleen-conditioned medium (SCM) in agar culture were investigated. Colony frequency was optimal on day 7 and increased relative to both the number of cells plated and the concentration of SCM used. Colonies were categorized as small cell and big cell. Small-cell colonies had a greater proliferative potential, with a mean of 25 cells/colony. Big-cell colonies averaged 15 cells/colony. The ratio of big-cell to small-cell colonies was 0.69 +/- 0.29. Granulocyte-macrophage colonies, which were also stimulated by SCM, accounted for 70% +/- 15% of the total colonies in the cultures. Cytocidal experiments with tritiated thymidine reduced megakaryocyte colony formation by 45% and granulocyte-macrophage colony formation by 21%. The properties of rat, mouse, and human megakaryocyte progenitors as assayed in vitro are compared.

  18. Heterogeneity in proliferative potential of ovine mesenchymal stem cell colonies.

    PubMed

    Rhodes, N P; Srivastava, J K; Smith, R F; Longinotti, C

    2004-04-01

    Bone marrow biopsies were taken from the iliac crest of 28 individual sheep from three different breeds, ranging in age from 4 months to 8 years and mesenchymal stem cells (MSCs) isolated using selection due to plastic adherence. Cells were cultured in medium that had been selected for its effect on observed MSC proliferation, until populations of greater than 50 million had been obtained from each biopsy. The identity of the isolated cell populations as progenitors of the mesenchymal lineage was verified by deriving both osteoblastic and chondrocytic phenotypes when cultured in osteogenic and chondrogenic medium supplements, respectively. The rate of cell proliferation for each marrow biopsy was measured at each passage and the number of initial stem cells in each sample estimated. There was no statistically significant correlation between the age of the sheep and MSC proliferative potential, or age and estimated initial MSC number. There was no apparent significant difference between proliferation rate and sheep breed and colonies established from frozen cells grew at similar rates to pre-frozen cells. Counter intuitively, there appeared to be a negatively correlated trend between proliferation rate and MSC concentration in the samples. It is concluded that no initial descriptive statistics of the marrow biopsies can assist in estimating the proliferative potential, and therefore the timing of future surgeries, of MSCs sampled for the purposes of tissue engineering. PMID:15332606

  19. The role of gravity in the nutrition and formation of Bacillus colonies

    NASA Astrophysics Data System (ADS)

    Puzyr, A.; Tirranen, L.; Krylova, T.

    The soil-like substrate is used to cultivate higher plants in man-made closed ecosystems. It allows increasing the closeness of the systems and decreasing the plant solid residues and human wastes. Unusual funnel-shaped bacterial colonies of Bacillus species have been observed during analysis of microflora of plant nutritional solution. The colonies have the following characteristics: a) the diameter of "funnel socket" (the biomass contacting with nutritional agar) is 10.0-15.0 mm; b) the thickness of "funnel socket" is 0.5-2.5 mm; c) the diameter of the middle part of the "funnel spout" (the biomass contacting with the gas phase) is 1,0-1,5 mm; d) the length of the "funnel spout" is 10.0-15.0 mm. In the socket and the middle part of the "funnel spout" there is a gas cavity which is most probably formed by bacterial gas metabolites. It has been shown that: i) the surface of these funnel-shaped colonies of Bacillus species is hydrophobic, as is the surface of other Bacillus species ( . brevis, B. cellulomonos, B. flavus, B.B formosus, B. subtilis); ii) the forms of colonies can be changed by varying the position of the growing biomass in relation to the gravitation forces. The experiment proved that the form of the "funnel sockets" and the length of the "funnel spouts" of the colonies are determined by hydrophobic air-contacting surface layer, which does not leak and stretches under the weight of accumulated water. A hypothesis has been suggested that the gravity force plays the role of a "pump" supplying and holding water within the colony. Thus, the water that comes under the gravity force contains dissolved nutrients and bacterial cells in the hydrophobic layer. These cells that are situated far away from the nutrient agar have no nutrient deficiency. The water accumulated by the colonies might be free water of agar media or it can be produced by metabolic disruption of medium fat. Hence, when growing a colony in agar media the water-soluble nutrient substances

  20. Accumulation of neutral mutations in growing cell colonies with competition

    PubMed Central

    Sorace, Ron; Komarova, Natalia L.

    2012-01-01

    Neutral mutations play an important role in many biological processes including cancer initiation and progression, the generation of drug resistance in bacterial and viral diseases as well as cancers, and the development of organs in multicellular organisms. In this paper we study how neutral mutants are accumulated in nonlinearly-growing colonies of cells subject to growth constraints such as crowding or lack of resources. We investigate different types of growth control which range from “division-controlled” to “death-controlled” growth (and various mixtures of both). In division-controlled growth, the burden of handling overcrowding lies with the process of cell-divisions, the divisions slow down as the carrying capacity is approached. In death-controlled growth, it is death rate that increases to slow down expansion. We show that division-controlled growth minimizes the number of accumulated mutations, and death-controlled growth corresponds to the maximum number of mutants. We check that these results hold in both deterministic and stochastic settings. We further develop a general (deterministic) theory of neutral mutations and achieve an analytical understanding of the mutant accumulation in colonies of a given size in the absence of back-mutations. The long-term dynamics of mutants in the presence of back-mutations is also addressed. In particular, with equal forward-and back-mutation rates, if division-controlled and a death-controlled types are competing for space and nutrients, cells obeying division-controlled growth will dominate the population. PMID:22940236

  1. Simple method for a cell count of the colonial Cyanobacterium, Microcystis sp.

    PubMed

    Joung, Seung-Hyun; Kim, Choong-Jae; Ahn, Chi-Yong; Jang, Kam-Yong; Boo, Sung Min; Oh, Hee-Mock

    2006-10-01

    The cell counting of colonial Microcystis spp. is a rather difficult and error-prone proposition, as this genus forms irregularly-shaped and irregularly-sized colonies, which are packed with cells. Thus, in order to facilitate a cell count, four methods of dividing the colonies into single cells were compared, including vortexing, sonication, TiO2 treatment, and boiling. As a result, the boiling method was determined to generate the greatest number of single cells from a colony, and all colonies were found to have divided completely after only 6 min of treatment. Furthermore, no significant cell destruction, which might alter the actual cell density, was detected in conjunction with the boiling method (P = 0.158). In order to compute the cell number more simply, the relationship between the colony size and the cell number was determined, via the boiling method. The colony volume, rather than the area or diameter was correlated more closely with the cell number (r2 = 0.727), thereby suggesting that the cell numbers of colonial Microcystis sp. can also be estimated effectively from their volumes. PMID:17082751

  2. A simple colony-formation assay in liquid medium, termed 'tadpoling', provides a sensitive measure of Saccharomyces cerevisiae culture viability.

    PubMed

    Welch, Aaron Z; Koshland, Douglas E

    2013-12-01

    Here we describe the first high-throughput amenable method of quantifying Saccharomyces cerevisiae culture viability. Current high-throughput methods of assessing yeast cell viability, such as flow cytometry and SGA analysis, do not measure the percentage viability of a culture but instead measure cell vitality or colony fitness, respectively. We developed a method, called tadpoling, to quantify the percentage viability of a yeast culture, with the ability to detect as few as one viable cell amongst ~10(8) dead cells. The most important feature of this assay is the exploitation of yeast colony formation in liquid medium. Utilizing a microtiter dish, we are able to observe a range of viability of 100% to 0.0001%. Comparison of tadpoling to the traditional plating method to measure yeast culture viability reveals that, for the majority of Saccharomyces species analyzed there is no significant difference between the two methods. In comparison to flow cytometry using propidium iodide, the high-throughput method of measuring yeast culture viability, tadpoling is much more accurate at culture viabilities < 1%. Thus, we show that tadpoling provides an easy, inexpensive, space-saving method, amenable to high-throughput screens, for accurately measuring yeast cell viability. PMID:24185677

  3. Pattern formation in a growing bacterial colony facilitated by extra-cellular polymeric substances

    NASA Astrophysics Data System (ADS)

    Ghosh, Pushpita; Mondal, Jagannath; Ben-Jacob, Eshel; Levine, Herbert

    2015-03-01

    Self-organization in bacterial colony is quite pervasive and diverse phenomena. Bacteria are known to self-organize into multicellular communities, commonly known as biofilms, in which microbial cells live in close association with a solid surface and are embedded in a self-produced extracellular polymeric substances(EPS). In such dense systems mechanical interactions among the structural components can be expected to significantly contribute to the morphological properties. By a simple particle-based simulation model of nonmotile rod-shaped bacterial cells and EPS secreted in a growing colony, we investigate how the combined mechanical effects can give rise naturally spatial heterogeneity observed in a biofilm. In our individual-based simulation model all the components interact mechanically via repulsive forces by pushing each other away as bacterial cells grow and divide consuming diffusing nutrient and produce EPS. We show that mechanical interactions control the collective behavior of the system, particularly, we show that the presence of non-adsorbing EPS leads spontaneous aggregation of bacterial cells by depletion attraction and generates phase separated patterns in a nonequilibrium growing colony.

  4. Involvement of allelopathy in the formation of monospecific colonies of ferns.

    PubMed

    Kato-Noguchi, Hisashi

    2015-05-01

    Some fern species often dominate plant communities by forming large monospecific colonies. However, the potential mechanism for this domination of the ferns remains obscure. Many plants secrete a wide range of compounds into the rhizosphere and change the chemical and physical properties of the rhizosphere soil. Through the secretion of compounds, such as allelopathic substances, plants inhibit the germination and growth of neighboring plants to compete more effectively for the resources. Ferns contain a variety of secondary metabolites and some of those compounds are released from the ferns into the rhizosphere soil, either as exudates from living ferns or by decomposition of fern residues in sufficient quantities to affect the germination and growth of neighboring plants as allelopathic substances. Therefore, allelopathic chemical interaction of the ferns with neighboring plants may play an important role in the formation of the monospecific colonies of the ferns. PMID:26058163

  5. Cooperation between human fibrocytes and endothelial colony forming cells increases angiogenesis via CXCR4 pathway

    PubMed Central

    Smadja, David M.; Dorfmüller, Peter; Bieche, Ivan; Guerin, Coralie; Badoual, Cécile; Boscolo, Elisa; Kambouchner, Marianne; Cazes, Aurélie; Mercier, Olaf; Humbert, Marc; Gaussem, Pascale; Bischoff, Joyce; Israël-Biet, Dominique

    2016-01-01

    Background Fibrotic diseases of the lung are associated with a vascular remodeling process. Fibrocytes (Fy) are a distinct population of blood-borne cells that coexpress hematopoietic cell antigens and fibroblast markers, which have been shown to contribute to organ fibrosis. The purpose of this study was to test the hypothesis that Fy might cooperate with endothelial colony forming cells to induce angiogenesis. Methods/Results We successfully isolated Fy from blood of idiopathic pulmonary fibrosis (IPF) patients, which were further characterized by flow cytometry, Reverse Transcriptase quantitative-PCR (RTQ-PCR), and confocal analysis. We investigated the interaction between Fy and cord blood derived endothelial colony forming cells (ECFC) angiogenic potential in vitro and in vivo in a Matrigel implant model. Compared to fibroblast culture media, secreted media from Fy increase ECFC proliferation and their differentiation ability via SDF-1/CXCR4 pathway. IPF-Fy co-implanted with human ECFC in a matrigel plug in immunodeficient mice formed functional microvascular beds, whereas fibroblasts did not. Evaluation of implants after 2 weeks revealed an extensive network of blood vessels containing erythrocytes. CXCR4 blockade significantly inhibited blood vessel formation in the implants. The clinical relevance of these data was confirmed by the high expression level of CXCR4 in vessels close to fibrotic areas in biopsy specimens from patients with IPF, in contrast to control lungs. Conclusions Circulating Fy might be contribute to the intense remodeling of the pulmonary vasculature in patients with IPF. PMID:25103869

  6. Sorting and Expansion of Murine Embryonic Stem Cell Colonies using Micropallet Arrays

    PubMed Central

    Shadpour, Hamed; Sims, Christopher E.; Thresher, Randy J.; Allbritton, Nancy L.

    2008-01-01

    Background Isolation of cell colonies is an essential task in most stem cell studies. Conventional techniques for colony selection and isolation require significant time, labor, and consumption of expensive reagents. New microengineered technologies hold the promise for improving colony manipulation by reducing the required manpower and reagent consumption. Methods Murine embryonic stem cells were cultured on arrays composed of releasable elements termed micropallets created from a biocompatible photoresist. Micropallets containing undifferentiated colonies were released using a laser-based technique followed by cell collection and expansion in culture. Results The micropallet arrays provided a biocompatible substrate for maintaining undifferentiated murine stem cells in culture. A surface coating of 0.025% gelatin was shown to be optimal for cell culture and collection. Arrays composed of surface roughened micropallets provided further improvements in culture and isolation. Colonies of viable stem cells were efficiently isolated and collected. Colonies sorted in this manner were shown to remain undifferentiated even after collection and further expansion in culture. Conclusions Qualitative and quantitative analyses of sorting, collection efficiency and cell viability after release and expansion of stem cell colonies demonstrated that the micropallet array technology is a promising alternative to conventional sorting methods for stem cell applications. PMID:19012319

  7. Manufacture of Clinical-Grade Human Clonal Mesenchymal Stem Cell Products from Single Colony Forming Unit-Derived Colonies Based on the Subfractionation Culturing Method.

    PubMed

    Yi, TacGhee; Kim, Si-na; Lee, Hyun-Joo; Kim, Junghee; Cho, Yun-Kyoung; Shin, Dong-Hee; Tak, Sun-Ji; Moon, Sun-Hwa; Kang, Ji-Eun; Ji, In-Mi; Lim, Huyn-Ja; Lee, Dong-Soon; Jeon, Myung-Shin; Song, Sun U

    2015-12-01

    Stem cell products derived from mesenchymal stem cells (MSCs) have been widely used in clinical trials, and a few products have been already commercialized. However, the therapeutic effects of clinical-grade MSCs are still controversial owing to mixed results from recent clinical trials. A potential solution to overcome this hurdle may be to use clonal stem cells as the starting cell material to increase the homogeneity of the final stem cell products. We have previously developed an alternative isolation and culture protocol for establishing a population of clonal MSCs (cMSCs) from single colony forming unit (CFU)-derived colonies. In this study, we established a good manufacturing practice (GMP)-compatible procedure for the clinical-grade production of human bone marrow-derived cMSCs based on the subfractionation culturing method. We optimized the culture procedures to expand and obtain a clonal population of final MSC products from single CFU-derived colonies in a GMP facility. The characterization results of the final cMSC products met our preset criteria. Animal toxicity tests were performed in a good laboratory practice facility, and showed no toxicity or tumor formation in vivo. These tests include single injection toxicity, multiple injection toxicity, biodistribution analysis, and tumorigenicity tests in vivo. No chromosomal abnormalities were detected by in situ karyotyping using oligo-fluorescence in situ hydridization (oligo-FISH), providing evidence of genetic stability of the clinical-grade cMSC products. The manufacture and quality control results indicated that our GMP methodology could produce sufficient clonal population of MSC products from a small amount of bone marrow aspirate to treat a number of patients. PMID:26421757

  8. Are self-ligating brackets related to less formation of Streptococcus mutans colonies? A systematic review

    PubMed Central

    do Nascimento, Leonard Euler Andrade Gomes; de Souza, Margareth Maria Gomes; Azevedo, Angela Rita Pontes; Maia, Lucianne Cople

    2014-01-01

    Objective To verify, by means of a systematic review, whether the design of brackets (conventional or self-ligating) influences adhesion and formation of Streptococcus mutans colonies. Methods Search strategy: four databases (Cochrane Central Register of Controlled Trials, Ovid ALL EMB Reviews, PubMed and BIREME) were selected to search relevant articles covering the period from January 1965 to December 2012. Selection Criteria: in first consensus by reading the title and abstract. The full text was obtained from publications that met the inclusion criteria. Data collection and analysis: Two reviewers independently extracted data using the keywords: conventional, self-ligating, biofilm, Streptococcus mutans, and systematic review; and independently evaluated the quality of the studies. In case of divergence, the technique of consensus was adopted. Results The search strategy resulted in 1,401 articles. The classification of scientific relevance revealed the high quality of the 6 eligible articles of which outcomes were not unanimous in reporting not only the influence of the design of the brackets (conventional or self-ligating) over adhesion and formation of colonies of Streptococcus mutans, but also that other factors such as the quality of the bracket type, the level of individual oral hygiene, bonding and age may have greater influence. Statistical analysis was not feasible because of the heterogeneous methodological design. Conclusions Within the limitations of this study, it was concluded that there is no evidence for a possible influence of the design of the brackets (conventional or self-ligating) over colony formation and adhesion of Streptococcus mutans. PMID:24713561

  9. Continuum Model of Collective Cell Migration in Wound Healing and Colony Expansion

    PubMed Central

    Arciero, Julia C.; Mi, Qi; Branca, Maria F.; Hackam, David J.; Swigon, David

    2011-01-01

    Collective cell migration plays an important role during wound healing and embryo development. Although the exact mechanisms that coordinate such migration are still unknown, experimental studies of moving cell layers have shown that the primary interactions governing the motion of the layer are the force of lamellipodia, the adhesion of cells to the substrate, and the adhesion of cells to each other. Here, we derive a two-dimensional continuum mechanical model of cell-layer migration that is based on a novel assumption of elastic deformation of the layer and incorporates basic mechanical interactions of cells as well as cell proliferation and apoptosis. The evolution equations are solved numerically using a level set method. The model successfully reproduces data from two types of experiments: 1), the contraction of an enterocyte cell layer during wound healing; and 2), the expansion of a radially symmetric colony of MDCK cells, both in the edge migration velocity and in cell-layer density. In accord with experimental observations, and in contrast to reaction-diffusion models, this model predicts a partial wound closure if lamellipod formation is inhibited at the wound edge and gives implications of the effect of spatially restricted proliferation. PMID:21281567

  10. Proliferation and colony-forming ability of peritoneal exudate cells in liquid culture.

    PubMed

    Stewart, C C; Lin, H S; Adles, C

    1975-05-01

    Peritoneal exudate cells, obtained from mice injected with thioglycollate medium and cultured in medium containing L-cell-conditioned medium, will proliferate in an exponential fashion for 18 days with a doubling time of 68 h. After a 2 h pulse of tritiated thymidine, labeled adherent cells increased to a maximum of 22-34% during the 1st and 2nd wk of culture. Increasing the cell concentration from 2 times 10-3 to 2 times 10-5 cells/culture reduced exponential growth to 10 days and the doubling time was increased to 81.6 h. Under these culture conditions, peritoneal exudate cells were shown to form colonies on the surface of culture dishes when plated at low density. The cells within the colony were shown to be macrophages using yeast and antibody-coated sheep erythrocytes as a test for phagocytic function. The plating efficiolonies arose from a single precursor cell. The adherent cell population contains the colony-forming precursors. These precursors can be stimulated to form colonies for at least 2 wk by the addition of conditioned medium to cultures at various times after plating. While very few colony-forming cells could be demonstrated in the unstimulated peritoneal lavage, their numbers begin to increase in the exudate 4 h after injection of thioglycollate medium and reach a maximum by day 3 and then decrease. Isolated colonies may be useful in studying the function of macrophages. PMID:1092793

  11. Impact of chlorine on the cell integrity and toxin release and degradation of colonial Microcystis.

    PubMed

    Fan, Jiajia; Rao, La; Chiu, Yi-Ting; Lin, Tsair-Fuh

    2016-10-01

    The occurrence of toxic cyanobacteria in drinking water sources is problematic for water authorities as they can impair drinking water quality. Chlorine as a commonly used oxidant in water treatment plants has shown the potential to lyse cyanobacterial cells, resulting in the release of secondary metabolites which are hard to be removed during conventional water treatment processes. The majority of cyanobacterial species such as Microcystis, often occur in colonial forms under natural conditions. However, previous studies have mainly focused on the influence of chlorination on individual cyanobacterial cells due to technique limitations. A syringe dispersion method combined with a fluorescence technique (SYTOX Green stain with flow cytometry), was successfully developed for the evaluation of cell integrity of colonial Microcystis. Chlorination of Microcystis-laden water was conducted at different chlorine dosages for different colonial sizes (<37, 37-270 and 270-550 μm). The results indicated that colonial Microcystis cells were more resistant to chlorine oxidation than individual cells, which may be attributed to protection from the cell-bound mucilage. There was a lag phase before cell rupture occurred and a Delayed Chick Watson Model describes the experimental data very well for the kinetics of cyanobacterial cell rupture. The growing colonial size caused increases in the lag phases but decreases in the cell lysis rates. Chlorination also induced the release of microcystins (MCs) from colonial Microcystis cells. In particular, increased levels of dissolved MCs were observed in Cheng Kung Lake (CKL) water. In summary, the reaction of chlorine with colonial cyanobacteria is more complicated than with individual cells. The efficiency of chlorine oxidation could be reduced by the cell-bound mucilage and natural water matrix. These observations may provide insights for water authorities to assess the risk to drinking water quality posed by chlorination under

  12. Early Developmental Program Shapes Colony Morphology in Bacteria

    PubMed Central

    Mamou, Gideon; Malli Mohan, Ganesh Babu; Rouvinski, Alex; Rosenberg, Alex; Ben-Yehuda, Sigal

    2016-01-01

    Summary When grown on a solid surface, bacteria form highly organized colonies, yet little is known about the earliest stages of colony establishment. Following Bacillus subtilis colony development from a single progenitor cell, a sequence of highly ordered spatiotemporal events was revealed. Colony was initiated by the formation of leading-cell chains, deriving from the colony center and extending in multiple directions, typically in a “Y-shaped” structure. By eradicating particular cells during these early stages, we could influence the shape of the resulting colony and demonstrate that Y-arm extension defines colony size. A mutant in ymdB encoding a phosphodiesterase displayed unordered developmental patterns, indicating a role in guiding these initial events. Finally, we provide evidence that intercellular nanotubes contribute to proper colony formation. In summary, we reveal a “construction plan” for building a colony and provide the initial molecular basis for this process. PMID:26904951

  13. Hepatic differentiation of human embryonic stem cells as microscaled multilayered colonies leading to enhanced homogeneity and maturation.

    PubMed

    Yao, Rui; Wang, Jingyu; Li, Xiaokang; Jung Jung, Da; Qi, Hao; Kee, Keh Kooi; Du, Yanan

    2014-11-12

    Directed differentiation of human embryonic stem cells (hESCs) towards hepatocyte-like cells on planar tissue culture plates has been extensively investigated with great promise to provide alternative cell sources for drug metabolism/toxicity testing. Recently, hepatic differentiation of hESCs in 3D configuration with better mimicry of embryonic liver development represents incremental efforts to improve the differentiation efficiency and cellular maturation. However, most of the present 3D differentiation configurations involved interruptive operations during the multi-staged differentiation process, which might impose unwanted influence on cellular differentiation. Most of the current researches resulted in generation of hepatocytes with high expression of AFP, which is minimally expressed in primary hepatocytes. Here, off-the-shelf micro-stencil arrays are developed to generate adherent multilayered colonies composed of hESCs-derived cells. Uninterrupted cellular differentiation and proliferation is achieved to recapitulate the continuous and multi-stage liver development. Compared with conventional 2D format, the micro-scaled multilayered colonies with uniform and defined sizes constrained within the microwells are composed of more homogenous and mature hepatocyte-like cells with significantly lowered AFP expression and elevated hepatic functions. The multilayered colonies as novel 3D configuration for hepatic differentiation of hESCs represent a significant step toward efficient generation of functional hepatocytes for regenerative medicine and drug discovery. PMID:25059765

  14. Automated Identification and Location Analysis of Marked Stem Cells Colonies in Optical Microscopy Images

    PubMed Central

    Paduano, Vincenzo; Tagliaferri, Daniela; Falco, Geppino; Ceccarelli, Michele

    2013-01-01

    Embryonic stem cells (ESCs) are characterized by two remarkable peculiarities: the capacity to propagate as undifferentiated cells (self-renewal) and the ability to differentiate in ectoderm, endoderm, and mesoderm derivatives (pluripotency). Although the majority of ESCs divide without losing the pluripotency, it has become evident that ESC cultures consists of multiple cell populations highlighted by the expression of early germ lineage markers during spontaneous differentiation. Hence, the identification and characterization of ESCs subpopulations represents an efficient approach to improve the comprehension of correlation between gene expression and cell specification status. To study markers of ESCs heterogeneity, we developed an analysis pipeline which can automatically process images of stem cell colonies in optical microscopy. The question we try to address is to find out the statistically significant preferred locations of the marked cells. We tested our algorithm on a set of images of stem cell colonies to analyze the expression pattern of the Zscan4 gene, which was an elite candidate gene to be studied because it is specifically expressed in subpopulation of ESCs. To validate the proposed method we analyzed the behavior of control genes whose pattern had been associated to biological status such as differentiation (EndoA), pluripotency (Pou5f1), and pluripotency fluctuation (Nanog). We found that Zscan4 is not uniformly expressed inside a stem cell colony, and that it tends to be expressed towards the center of the colony, moreover cells expressing Zscan4 cluster each other. This is of significant importance because it allows us to hypothesize a biological status where the cells expressing Zscan4 are preferably associated to the inner of colonies suggesting pluripotent cell status features, and the clustering between themselves suggests either a colony paracrine effect or an early phase of cell specification through proliferation. Also, the analysis on

  15. Automated identification and location analysis of marked stem cells colonies in optical microscopy images.

    PubMed

    Paduano, Vincenzo; Tagliaferri, Daniela; Falco, Geppino; Ceccarelli, Michele

    2013-01-01

    Embryonic stem cells (ESCs) are characterized by two remarkable peculiarities: the capacity to propagate as undifferentiated cells (self-renewal) and the ability to differentiate in ectoderm, endoderm, and mesoderm derivatives (pluripotency). Although the majority of ESCs divide without losing the pluripotency, it has become evident that ESC cultures consists of multiple cell populations highlighted by the expression of early germ lineage markers during spontaneous differentiation. Hence, the identification and characterization of ESCs subpopulations represents an efficient approach to improve the comprehension of correlation between gene expression and cell specification status. To study markers of ESCs heterogeneity, we developed an analysis pipeline which can automatically process images of stem cell colonies in optical microscopy. The question we try to address is to find out the statistically significant preferred locations of the marked cells. We tested our algorithm on a set of images of stem cell colonies to analyze the expression pattern of the Zscan4 gene, which was an elite candidate gene to be studied because it is specifically expressed in subpopulation of ESCs. To validate the proposed method we analyzed the behavior of control genes whose pattern had been associated to biological status such as differentiation (EndoA), pluripotency (Pou5f1), and pluripotency fluctuation (Nanog). We found that Zscan4 is not uniformly expressed inside a stem cell colony, and that it tends to be expressed towards the center of the colony, moreover cells expressing Zscan4 cluster each other. This is of significant importance because it allows us to hypothesize a biological status where the cells expressing Zscan4 are preferably associated to the inner of colonies suggesting pluripotent cell status features, and the clustering between themselves suggests either a colony paracrine effect or an early phase of cell specification through proliferation. Also, the analysis on

  16. Decreased level of cord blood circulating endothelial colony-forming cells in preeclampsia.

    PubMed

    Muñoz-Hernandez, Rocio; Miranda, Maria L; Stiefel, Pablo; Lin, Ruei-Zeng; Praena-Fernández, Juan M; Dominguez-Simeon, Maria J; Villar, Jose; Moreno-Luna, Rafael; Melero-Martin, Juan M

    2014-07-01

    Preeclampsia is a pregnancy-related disorder associated with increased cardiovascular risk for the offspring. Endothelial colony-forming cells (ECFCs) are a subset of circulating endothelial progenitor cells that participate in the formation of vasculature during development. However, the effect of preeclampsia on fetal levels of ECFCs is largely unknown. In this study, we sought to determine whether cord blood ECFC abundance and function are altered in preeclampsia. We conducted a prospective cohort study that included women with normal (n=35) and preeclamptic (n=15) pregnancies. We measured ECFC levels in the umbilical cord blood of neonates and characterized ECFC phenotype, cloning-forming ability, proliferation, and migration toward vascular endothelial growth factor-A and fibroblast growth factor-2, in vitro formation of capillary-like structures, and in vivo vasculogenic ability in immunodeficient mice. We found that the level of cord blood ECFCs was statistically lower in preeclampsia than in control pregnancies (P=0.04), a reduction that was independent of other obstetric factors. In addition, cord blood ECFCs from preeclamptic pregnancies required more time to emerge in culture than control ECFCs. However, once derived in culture, ECFC function was deemed normal and highly similar between preeclampsia and control, including the ability to form vascular networks in vivo. This study demonstrates that preeclampsia affects ECFC abundance in neonates. A reduced level of ECFCs during preeclamptic pregnancies may contribute to an increased risk of developing future cardiovascular events. PMID:24752434

  17. DECREASED LEVEL OF CORD BLOOD CIRCULATING ENDOTHELIAL COLONY-FORMING CELLS IN PREECLAMPSIA

    PubMed Central

    Muñoz-Hernandez, Rocio; Miranda, Maria L.; Stiefel, Pablo; Lin, Ruei-Zeng; Praena-Fernández, Juan M.; Dominguez-Simeon, Maria J.; Villar, Jose; Moreno-Luna, Rafael; Melero-Martin, Juan M.

    2014-01-01

    Preeclampsia is a pregnancy-related disorder associated with increased cardiovascular risk for the offspring. Endothelial colony-forming cells (ECFCs) are a subset of circulating endothelial progenitor cells that participate in the formation of vasculature during development. However, the effect of preeclampsia on fetal levels of ECFCs is largely unknown. In this study, we sought to determine whether cord blood ECFC abundance and function are altered in preeclampsia. We conducted a prospective cohort study that included women with normal (n=35) and preeclamptic (n=15) pregnancies. We measured ECFC levels in the umbilical cord blood of neonates and characterized ECFC phenotype, cloning-forming ability, proliferation and migration towards VEGF-A and FGF-2, in vitro formation of capillary-like structures, and in vivo vasculogenic ability in immunodeficient mice. We found that the level of cord blood ECFCs was statistically lower in preeclampsia than in control pregnancies (P = .04), a reduction that was independent of other obstetric factors. In addition, cord blood ECFCs from preeclamptic pregnancies required more time to emerge in culture than control ECFCs. However, once derived in culture, ECFC function was deemed normal and highly similar between preeclampsia and control, including the ability to form vascular networks in vivo. This study demonstrates that preeclampsia affects ECFC abundance in neonates. A reduced level of ECFCs during preeclamptic pregnancies may contribute to an increased risk of developing future cardiovascular events. PMID:24752434

  18. Role of gravity in the formation of bacterial colonies with a hydrophobic surface layer

    NASA Astrophysics Data System (ADS)

    Puzyr, A. P.; Tirranen, L. K.; Krylova, T. Y.; Borodina, E. V.

    A simple technique for determining hydrophobic-hydrophilic properties of bacterial colonies surface, which involves putting a drop of liquid with known properties (e.g. water, oil) on their surface, has been described. This technique allows quick estimate of wettability of bacterial colony surface, i.e. its hydrophobic-hydrophilic properties. The behaviour of water drops on colonies of bacteria Bacillus five strains (of different types) has been studied. It was revealed that 1) orientation in the Earth gravity field during bacterial growth can define the form of colonies with hydrophobic surface; 2) the form and size of the colony are dependent on the extention ability, most probably, of the hydrophobic layer; 3) the Earth gravity field (gravity) serves as a 'pump' providing and keeping water within the colony. We suppose that at growing colonies on agar media the inflow of water-soluble nutrient materials takes place both due to diffusion processes and directed water current produced by the gravity. The revealed effect probably should be taken into consideration while constructing the models of colonies growing on dense nutrient media. The easily determined hydrophobic properties of colonies surface can become a systematic feature after collecting more extensive data on the surface hydrophobic-hydrophilic properties of microorganism colonies of other types and species.

  19. Influence of Gelatin-Thrombin Matrix Tissue Sealant on Bacterial Colony Formation and Risk of Pelvic Infection.

    PubMed

    Jarrett, Michael J; Vázquez-Torres, Andres; Frank, Daniel N; McCollister, Bruce D; Henthorn, Patrick K; Ir, Diana; Sheeder, Jeanelle; Guy, Michael S; Anwar, Hiba Q; Behbakht, Kian

    2016-01-01

    Objective. Gelatin-thrombin matrix (GTM) tissue sealant use was previously identified as an independent predictor of pelvic infection following hysterectomies. We aim to elucidate contributing factors by assessing influence of GTM on bacterial colony formation and characterizing bacteria present at the vaginal cuff. Methods. Escherichia coli was incubated in phosphate-buffered saline (PBS) and pelvic washings with and without GTM to assess influence on colony formation. Pelvic washings of the vaginal cuff were collected from hysterectomies occurring from June through October 2015. In vitro techniques, 16S rRNA gene qPCR, and 16S amplicon sequencing were performed with washings to characterize bacteria at the vaginal cuff. Results. Mean bacterial colony formation in PBS was greater for E. coli incubated in the presence of GTM (1.48 × 10(7) CFU/mL) versus without (9.95 × 10(5) CFU/mL) following 20-hour incubation (p = 0.001). Out of 61 pelvic washings samples, 3 were culture positive (≥5000 CFU/mL) with Enterococcus faecalis. Conclusion. In vitro experiments support a facilitating role of GTM on colony formation of E. coli in PBS. However, given the negative results of surgical site washings following adequate disinfection, the role of GTM in promoting posthysterectomy pelvic infections may be limited. Analysis of pelvic washings revealed presence of E. faecalis, but results were inconclusive. Further studies are recommended. PMID:27199534

  20. Influence of Gelatin-Thrombin Matrix Tissue Sealant on Bacterial Colony Formation and Risk of Pelvic Infection

    PubMed Central

    Jarrett, Michael J.; Vázquez-Torres, Andres; Frank, Daniel N.; McCollister, Bruce D.; Henthorn, Patrick K.; Ir, Diana; Sheeder, Jeanelle; Guy, Michael S.; Anwar, Hiba Q.; Behbakht, Kian

    2016-01-01

    Objective. Gelatin-thrombin matrix (GTM) tissue sealant use was previously identified as an independent predictor of pelvic infection following hysterectomies. We aim to elucidate contributing factors by assessing influence of GTM on bacterial colony formation and characterizing bacteria present at the vaginal cuff. Methods. Escherichia coli was incubated in phosphate-buffered saline (PBS) and pelvic washings with and without GTM to assess influence on colony formation. Pelvic washings of the vaginal cuff were collected from hysterectomies occurring from June through October 2015. In vitro techniques, 16S rRNA gene qPCR, and 16S amplicon sequencing were performed with washings to characterize bacteria at the vaginal cuff. Results. Mean bacterial colony formation in PBS was greater for E. coli incubated in the presence of GTM (1.48 × 107 CFU/mL) versus without (9.95 × 105 CFU/mL) following 20-hour incubation (p = 0.001). Out of 61 pelvic washings samples, 3 were culture positive (≥5000 CFU/mL) with Enterococcus faecalis. Conclusion. In vitro experiments support a facilitating role of GTM on colony formation of E. coli in PBS. However, given the negative results of surgical site washings following adequate disinfection, the role of GTM in promoting posthysterectomy pelvic infections may be limited. Analysis of pelvic washings revealed presence of E. faecalis, but results were inconclusive. Further studies are recommended. PMID:27199534

  1. Establishment of a cell line of gallbladder carcinoma (GBK-1) producing human colony stimulating factor.

    PubMed

    Egami, H; Sakamoto, K; Yoshimura, R; Kikuchi, H; Akagi, M

    1986-02-01

    A cell line designated GBK-1 was established from a patient with anaplastic carcinoma of the gallbladder, marked neutrophilia and fever, and has been propagated for the past 18 months. The cells grew as a monolayer sheet with a doubling time of 43 hr. The GBK-1 cells were of a pleomorphic polygonal epitheloid shape and they were transplantable into nude mice. Mice bearing the tumor developed marked granulocytosis. In the culture supernatant of GBK-1 cells, high colony stimulating factor (CSF) activity was evident with both human bone marrow cells and C57BL mouse bone marrow cells, and granulocytic colonies, macrophage colonies or granulocyte-macrophage mixed colonies were produced. The CSF activity was distributed in the molecular weight range of 25,000 to 60,000 with two distinct peaks at molecular weights of approximately 50,000 and 30,000. CSF activity was inactivated by heat treatment at 70 degrees for 30 min. GBK-1 is a new human cell line that produces heat-labile human GM-CSF. PMID:3082828

  2. Identification and enrichment of colony-forming cells from the adult murine pituitary

    SciTech Connect

    Lepore, D.A.; Roeszler, K.; Wagner, J.; Ross, S.A.; Bauer, K.; Thomas, P.Q. , E-Mail: paul.thomas@mcri.edu.au

    2005-08-01

    Stem and progenitor cells have been identified in many adult tissues including bone marrow, the central nervous system, and skin. While there is direct evidence to indicate the activity of a progenitor cell population in the pituitary gland, this putative subpopulation has not yet been identified. Herein we describe the isolation and characterization of a novel clonogenic cell type in the adult murine pituitary, which we have termed Pituitary Colony-Forming Cells (PCFCs). PCFCs constitute 0.2% of pituitary cells, and generate heterogeneous colonies from single cells. PCFCs exhibit variable proliferative potential, and may exceed 11 population doublings in 14 days. Enrichment of PCFCs to 61.5-fold with 100% recovery can be obtained through the active uptake of the fluorescent dipeptide, {beta}-Ala-Lys-N{epsilon}-AMCA. PCFCs are mostly contained within the large, agranular subpopulation of AMCA{sup +} cells, and constitute 28% of this fraction, corresponding to 140.5-fold enrichment. Interestingly, the AMCA{sup +} population contains rare cells that are GH{sup +} or PRL{sup +}. GH{sup +} cells were also identified in PCFC single cell colonies, suggesting that PCFCs have the potential to differentiate into GH{sup +} cells. Together, these data show that the pituitary contains a rare clonogenic population which may correspond to the somatotrope/lactotrope progenitors suggested by previous experiments.

  3. Treprostinil indirectly regulates endothelial colony forming cell angiogenic properties by increasing VEGF-A produced by mesenchymal stem cells.

    PubMed

    Smadja, David M; Levy, Marilyne; Huang, Lan; Rossi, Elisa; Blandinières, Adeline; Israel-Biet, Dominique; Gaussem, Pascale; Bischoff, Joyce

    2015-10-01

    Pulmonary vasodilators and prostacyclin therapy in particular, have markedly improved the outcome of patients with pulmonary hypertension (PH). Endothelial dysfunction is a key feature of PH, and we previously reported that treprostinil therapy increases number and proliferative potential of endothelial colony forming cells (ECFC) isolated from PH patients' blood. In the present study, the objective was to determine how treprostinil contributes to the proangiogenic functions of ECFC. We examined the effect of treprostinil on ECFC obtained from cord blood in terms of colony numbers, proliferative and clonogenic properties in vitro, as well as in vivo vasculogenic properties. Surprisingly, treprostinil inhibited viability of cultured ECFC but did not modify their clonogenic properties or the endothelial differentiation potential from cord blood stem cells. Treprostinil treatment significantly increased the vessel-forming ability of ECFC combined with mesenchymal stem cells (MSC) in Matrigel implanted in nude mice. In vitro, ECFC proliferation was stimulated by conditioned media from treprostinil-pretreated MSC, and this effect was inhibited either by the use of VEGF-A blocking antibodies or siRNA VEGF-A in MSC. Silencing VEGF-A gene in MSC also blocked the pro-angiogenic effect of treprostinil in vivo. In conclusion, increased VEGF-A produced by MSC can account for the increased vessel formation observed during treprostinil treatment. The clinical relevance of these data was confirmed by the high level of VEGF-A detected in plasma from patients with paediatric PH who had been treated with treprostinil. Moreover, our results suggest that VEGF-A level in patients could be a surrogate biomarker of treprostinil efficacy. PMID:26062754

  4. Smooth muscle progenitor cells from peripheral blood promote the neovascularization of endothelial colony-forming cells

    SciTech Connect

    Joo, Hyung Joon; Seo, Ha-Rim; Jeong, Hyo Eun; Choi, Seung-Cheol; Park, Jae Hyung; Yu, Cheol Woong; Hong, Soon Jun; Chung, Seok; Lim, Do-Sun

    2014-07-11

    Highlights: • Two distinct vascular progenitor cells are induced from adult peripheral blood. • ECFCs induce vascular structures in vitro and in vivo. • SMPCs augment the in vitro and in vivo angiogenic potential of ECFCs. • Both cell types have synergistic therapeutic potential in ischemic hindlimb model. - Abstract: Proangiogenic cell therapy using autologous progenitors is a promising strategy for treating ischemic disease. Considering that neovascularization is a harmonized cellular process that involves both endothelial cells and vascular smooth muscle cells, peripheral blood-originating endothelial colony-forming cells (ECFCs) and smooth muscle progenitor cells (SMPCs), which are similar to mature endothelial cells and vascular smooth muscle cells, could be attractive cellular candidates to achieve therapeutic neovascularization. We successfully induced populations of two different vascular progenitor cells (ECFCs and SMPCs) from adult peripheral blood. Both progenitor cell types expressed endothelial-specific or smooth muscle-specific genes and markers, respectively. In a protein array focused on angiogenic cytokines, SMPCs demonstrated significantly higher expression of bFGF, EGF, TIMP2, ENA78, and TIMP1 compared to ECFCs. Conditioned medium from SMPCs and co-culture with SMPCs revealed that SMPCs promoted cell proliferation, migration, and the in vitro angiogenesis of ECFCs. Finally, co-transplantation of ECFCs and SMPCs induced robust in vivo neovascularization, as well as improved blood perfusion and tissue repair, in a mouse ischemic hindlimb model. Taken together, we have provided the first evidence of a cell therapy strategy for therapeutic neovascularization using two different types of autologous progenitors (ECFCs and SMPCs) derived from adult peripheral blood.

  5. Acceptance of mated Queens and Queen Cells in Colonies of Russian and Italian Honey Bees

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Requeening colonies is a standard beekeeping practice with both mated queens and queen cells. More beekeepers are requeening with Russian honey bees queens because of their significantly higher resistance to varroa and tracheal mites, their good honey production and their overwintering abilities. ...

  6. Suppression of the Formation of Polygenotypic Recombinant Colonies by a maf Mutation in Mating with Hfrh

    PubMed Central

    Ou, Jonathan T.; Kuo, Li-Mei

    1979-01-01

    W3011, a Cavalli-type Hfr (HfrC), was mated with F-KY9474, maf-1, which cannot maintain F or F-like plasmids, and with F-OU9474, Maf+, a spontaneous revertant of KY9474. The recombinant colonies obtained were 100% monogenotypic from KY9474 and 90% monogenotypic from OU9474. On the other hand, in matings with OU11, a Hayes-type Hfr (HfrH), and these two F- strains, recombinant colonies derived from KY9474 showed only 22% polygenotypic recombinant colonies; whereas, those derived from OU9474 showed a high production rate (57%) of polygenotypic recombinant colonies. Among the polygenotypic recombinant colonies derived from KY9474 maf-1, 50% contained three or more recombinant types. These were probably derived from a small fraction of Maf+ revertants in the KY9474 population, as suggested by the results of mating this strain with M80, an F' strain that contains an amber mutation in traH. These results support the hypothesis that the donor DNA fragments derived from an HfrH can undergo a limited replication in the recipient to produce polygenotypic recombinant colonies, whereas those derived from HfrC cannot. PMID:395025

  7. Colonial cell disaggregation and intracellular microcystin release following chlorination of naturally occurring Microcystis.

    PubMed

    He, Xuexiang; Wert, Eric C

    2016-09-15

    Colonial cell disaggregation and release of intracellular microcystin were evaluated following chlorine treatment of naturally occurring Microcystis. Microscopic observations of water samples collected from Lake Mead, Nevada, USA, confirmed the presence of colonial Microcystis with cells protected by an outer sheath up to 30 μm thick. During chlorination, two stages of cell decomposition were observed, stage 1: colonial cell disaggregation, and stage 2: unicellular degradation. Following a [Cl2]0:DOC0 ratio of 0.15 (t = 20 min, pH = 8.2-8.5) in unfiltered Lake Havasu samples, total particle count increased from (1.0 ± 0.11) × 10(5) to 4.2 × 10(5) particles/mL and fluorescent particle count increased from (1.2 ± 0.50) × 10(4) to 1.2 × 10(5) particles/mL, illustrating colonial cell disaggregation. Although total and fluorescent particles increased, the concentration of chlorophyll-a (Chl-a) decreased from 81 μg/L to 72 μg/L, and continued to decrease at higher [Cl2]0:DOC0 ratios. The preliminary second order rate constant for the reaction between Microcystis and chlorine in natural waters was estimated using either Chl-a (k = 15 M(-1) s(-1)) or fluorescence particle count (k = 38 M(-1) s(-1)) as an indicator of cell damage following colonial disaggregation (i.e., at [Cl2]0:DOC0 ratio ≥0.15). Complete release of intracellular microcystin-LR (MC-LR) was observed in both Lake Havasu and Lake Mead samples when applying a [Cl2]0:DOC0 ratio of 0.30 (t = 20 min), which was equivalent to a chlorine exposure of 8 min-mg/L for Lake Havasu samples. With chlorination, DOC increased by 3-18% indicating release of either colony-bound or cell-bound DOC. The results demonstrated the ability of chlorine to disaggregate/inactivate natural Microcystis colonies, and identified oxidation conditions resulting in complete release of intracellular MC-LR. PMID:27240297

  8. Maternal Body-Mass Index and Cord Blood Circulating Endothelial Colony-Forming Cells

    PubMed Central

    Lin, Ruei-Zeng; Miranda, Maria L.; Vallejo-Vaz, Antonio J.; Stiefel, Pablo; Praena-Fernández, Juan M.; Bernal-Bermejo, Jose; Jimenez-Jimenez, Luis M.; Villar, Jose; Melero-Martin, Juan M.

    2013-01-01

    Objective Endothelial colony-forming cells (ECFCs) are a subset of circulating endothelial progenitor cells that are particularly abundant in umbilical cord blood. We sought to determine whether ECFC abundance in cord blood is associated with maternal body-mass index (BMI) in non-pathological pregnancies. Study design We measured the level of ECFCs in the cord blood of neonates (n=27) born from non-obese healthy mothers with non-pathological pregnancies and examined whether ECFC abundance correlated with maternal BMI. We also examined the effect of maternal BMI on ECFC phenotype and function using angiogenic and vasculogenic assays. Results We observed variation in ECFC abundance among subjects and found a positive correlation between pre-pregnancy maternal BMI and ECFC content (r=0.51, P=0.007), which was independent of other obstetric factors. Despite this variation, ECFC phenotype and functionality were deemed normal and highly similar between subjects with maternal BMI <25 kg/m2 and BMI between 25–30 kg/m2, including the ability to form vascular networks in vivo. Conclusions This study underlines the need to consider maternal BMI as a potential confounding factor for cord blood levels of ECFCs in future comparative studies between healthy and pathological pregnancies. Endothelial colony-forming cells (ECFCs) are a subset of progenitor cells that circulate in peripheral blood and can give rise to endothelial cells (1,2), contributing to the formation of new vasculature and the maintenance of vascular integrity (3–5). The mechanisms that regulate the abundance of these cells in vivo remain poorly understood. ECFCs are rare in adult peripheral blood (1,2,10). In contrast, there is an elevated number of these cells in fetal blood during the third trimester of pregnancy (11–13). Emerging evidence indicates that deleterious conditions during fetal life can impair ECFC content and function. For instance, offspring of diabetic mothers have been shown to have

  9. [Effect of prodigiozan, levamisole and methyluracil on endogenous colony formation and absorptive function of the reticuloendothelial system].

    PubMed

    Riabchinskaia, L A

    1985-06-01

    The effect of prodigiosan, levamisole and methyluracil on the number of endogenic colonies in the spleen and absorptive function of the reticuloendothelial system (RES) was studied comparatively in intact mice and mice treated with prednisolone, cyclophosphamide and chloramphenicol. It was shown that prodigiosan stimulated the endogenic colony formation with all the administration schemes in both the intact animals and the animals treated with chloramphenicol. The stimulating effect of methyluracil was observed, when it was used prophylactically. The effect of levamisole was not consistent. A significant stimulating effect of prodigiosan on absorptive function of the RES in the intact animals and the animals treated with prednisolone was shown. Methyluracil increased absorption of staphylococci by the macrophages in the intact animals. The effect of levamisole on the intact animals was not observed, while in the animals treated with prednisolone and cyclophosphamide it stimulated the absorptive capacity of the RES. Levamisole did not change the effect of prodigiosan and methyluracil on endogenous colony formation when used therapeutically, prevented the prodigiosan effect and potentiated the effect of methyluracil on absorptive function of the RES. PMID:4062270

  10. Differences in estimates of cisplatin-induced cell kill in vitro between colorimetric and cell count/colony assays.

    PubMed

    Henriksson, Eva; Kjellén, Elisabeth; Wahlberg, Peter; Wennerberg, Johan; Kjellström, Johan H

    2006-01-01

    The aim of this study was to evaluate some bioassays that are different in principle: cell counting, colony forming assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), sulforhodamine B (SRB), crystal violet, and alamarBlue, with respect to their ability to measure cisplatin-induced cell death of in vitro-cultivated squamous cell carcinoma of the head and neck (SCCHN). Cisplatin was applied in concentrations of 1.0, 5.0, 10.0, 50.0, and 100 microM. The cells were incubated for 1 h, and the cell survival was measured 5 d after treatment. We found the colorimetric assays and cell counting to be comparable. The colony forming assay indicated a higher degree of cell kill compared with the other techniques. Measurement of cell survival after treatment with cisplatin can be done by use of any of the above tested assays. However, the majority of SCCHN cell lines available do not form colonies easily, or at all. Therefore, comparing the chemosensitivity between such cell lines is limited to alternative assays. In this respect, any of the tested colorimetric assays can be used. However, they seem to underestimate cell kill. Cell counting is also an alternative. This technique, however, is time consuming and operator dependent, as in the case of manual counting, or relatively expensive when counting is performed electronically, compared with the colorimetric assays. PMID:17316066

  11. Colony stimulating factor 1 is an extrinsic stimulator of mouse spermatogonial stem cell self-renewal

    PubMed Central

    Oatley, Jon M.; Oatley, Melissa J.; Avarbock, Mary R.; Tobias, John W.; Brinster, Ralph L.

    2009-01-01

    Summary Self-renewal and differentiation of spermatogonial stem cells (SSCs) provide the foundation for testis homeostasis, yet mechanisms that control their functions in mammals are poorly defined. We used microarray transcript profiling to identify specific genes whose expressions are augmented in the SSC-enriched Thy1+ germ cell fraction of mouse pup testes. Comparisons of gene expression in the Thy1+ germ cell fraction with the Thy1-depleted testis cell population identified 202 genes that are expressed 10-fold or higher in Thy1+ cells. This database provided a mining tool to investigate specific characteristics of SSCs and identify novel mechanisms that potentially influence their functions. These analyses revealed that colony stimulating factor 1 receptor (Csf1r) gene expression is enriched in Thy1+ germ cells. Addition of recombinant colony stimulating factor 1 (Csf1), the specific ligand for Csf1r, to culture media significantly enhanced the self-renewal of SSCs in heterogeneous Thy1+ spermatogonial cultures over a 63-day period without affecting total germ cell expansion. In vivo, expression of Csf1 in both pre-pubertal and adult testes was localized to clusters of Leydig cells and select peritubular myoid cells. Collectively, these results identify Csf1 as an extrinsic stimulator of SSC self-renewal and implicate Leydig and myoid cells as contributors of the testicular stem cell niche in mammals. PMID:19270176

  12. Curcumin Veto the Effects of Osteopontin (OPN) Specific Inhibitor on Leukemic Stem Cell Colony Forming Potential via Promotion of OPN Overexpression

    PubMed Central

    Mohammadi, Saeed; Ghaffari, Seyed H.; Shaiegan, Mojgan; Nikogoftar Zarif, Mahin; Nikbakht, Mohsen; Alimoghaddam, Kamran; Ghavamzadeh, Ardeshir

    2016-01-01

    Background: Acute myeloid leukemia (AML) is an immunophenotypically heterogeneous malignant disease, in which CD34 positivity is associated with poor prognosis. Osteopontin (OPN) plays different roles in physiologic and pathologic conditions like: survival, metastasis and cell protection from cytotoxic and apoptotic stimuli. Due to anti-apoptotic effect of OPN in normal and malignant cells, silencing of OPN leads to elevation of sensitivity towards chemotherapeutic agents and attenuates cancer cells migration and invasion. Therefore, the aim of this study was to evaluate OPN roles in modulating curcumin-mediated growth inhibitory on leukemic stem cells (LSCs) colony forming potential and survival in AML cell lines and primary CD34+/CD38- bone marrow-derived AML cells. Materials and Methods: Primary human CD34+/CD38- cells were isolated from bone marrow mononuclear cells of 10 AML patients at initial state of diagnosis, using a CD34 Multi sort kit. The growth inhibitory effects of curcumin (CUR) were evaluated by MTT and colony-formation assays. Apoptosis was analyzed by 7AAD assay in CD34+ KG-1, U937 cell lines and primary isolated cells. Short interfering RNA (siRNA) against OPN was used for OPN silencing in both cell lines and primary AML cells. Then, transfected cells were incubated with/without curcumin. The change in OPN gene expression was examined by Real-time PCR. Results: CUR inhibited proliferation and induced apoptosis in both KG-1 and U937 cells and also primary isolated AML cells. OPN silencing by siRNA increased the susceptibility of KG-1, U937 and primary CD34+/CD38- AML cells to apoptosis. Moreover, soft agar colony assays revealed that silencing of OPN with siRNA significantly decreased colony numbers in LSCs compared with the non-targeting group. Furthermore, CD34+/CD38- populations as a main LSCs compartment through OPN overexpression towards CUR treatment might be nullified the inhibitory effects of OPN siRNA on their survival and colony forming

  13. Colony Expansion of Socially Motile Myxococcus xanthus Cells Is Driven by Growth, Motility, and Exopolysaccharide Production

    PubMed Central

    Patra, Pintu; Kissoon, Kimberley; Cornejo, Isabel; Kaplan, Heidi B.; Igoshin, Oleg A.

    2016-01-01

    Myxococcus xanthus, a model organism for studies of multicellular behavior in bacteria, moves exclusively on solid surfaces using two distinct but coordinated motility mechanisms. One of these, social (S) motility is powered by the extension and retraction of type IV pili and requires the presence of exopolysaccharides (EPS) produced by neighboring cells. As a result, S motility requires close cell-to-cell proximity and isolated cells do not translocate. Previous studies measuring S motility by observing the colony expansion of cells deposited on agar have shown that the expansion rate increases with initial cell density, but the biophysical mechanisms involved remain largely unknown. To understand the dynamics of S motility-driven colony expansion, we developed a reaction-diffusion model describing the effects of cell density, EPS deposition and nutrient exposure on the expansion rate. Our results show that at steady state the population expands as a traveling wave with a speed determined by the interplay of cell motility and growth, a well-known characteristic of Fisher’s equation. The model explains the density-dependence of the colony expansion by demonstrating the presence of a lag phase–a transient period of very slow expansion with a duration dependent on the initial cell density. We propose that at a low initial density, more time is required for the cells to accumulate enough EPS to activate S-motility resulting in a longer lag period. Furthermore, our model makes the novel prediction that following the lag phase the population expands at a constant rate independent of the cell density. These predictions were confirmed by S motility experiments capturing long-term expansion dynamics. PMID:27362260

  14. Micronucleus formation induced by dielectric barrier discharge plasma exposure in brain cancer cells

    NASA Astrophysics Data System (ADS)

    Kaushik, Nagendra K.; Uhm, Hansup; Ha Choi, Eun

    2012-02-01

    Induction of micronucleus formation (cytogenetic damage) in brain cancer cells upon exposure of dielectric barrier discharge plasma has been investigated. We have investigated the influence of exposure and incubation times on T98G brain cancer cells by using growth kinetic, clonogenic, and micronucleus formation assay. We found that micronucleus formation rate directly depends on the plasma exposure time. It is also shown that colony formation capacity of cells has been inhibited by the treatment of plasma at all doses. Cell death and micronucleus formation are shown to be significantly elevated by 120 and 240 s exposure of dielectric barrier discharge plasma.

  15. Pre-B Cell Colony Enhancing Factor (PBEF), a Cytokine with Multiple Physiological Functions

    PubMed Central

    Sun, Zhongjie; Lei, Han; Zhang, Zhonge

    2013-01-01

    Pre-B cell colony enhancing factor (PBEF) is regarded as a proinflammatory cytokine. Named for its first discovered function as a pre-B cell colony enhancing factor, it has since been found to have many other functions relating to cell metabolism, inflammation, and immune modulation. It has also been found to have intracellular and extracellular forms, with the two overlapping in function. Most of the intracellular functions of PBEF are due to its role as a nicotinamide phosphoribosyltransferase (Nampt). It has been found in human endothelial cells, where it is able to induce angiogenesis through upregulation of VEGF and VEGFR and secretion of MCP-1. In human umbilical endothelial cells, PBEF increases levels of the protease MMP 2/9. PBEF has also been found in a variety of immune cells other than B cells and has been shown to inhibit apoptosis of macrophages. Extracellular PBEF has been shown to increase inflammatory cytokines, such as TNF-α, IL-1β, IL-16, and TGF-β1, and the chemokine receptor CCR3. PBEF also increases the production of IL-6, TNF-α, and IL-1β in CD14+ monocyctes, macrophages, and dendritic cells, enhances the effectiveness of T cells, and is vital to the development of both B and T lymphocytes. The purpose of this review is to summarize the recent advances in PBEF research. PMID:23787158

  16. Collection and expansion of single cells and colonies released from a micropallet array.

    PubMed

    Wang, Yuli; Young, Grace; Bachman, Mark; Sims, Christopher E; Li, G P; Allbritton, Nancy L

    2007-03-15

    The ability to selectively grow out individual cells possessing unique characteristics from within a mixed population is of widespread importance for biomedical investigations. Generation of genetically engineered cell lines, transformation studies, cell-based assays, and stem cell studies are examples where single-cell cloning is of immense value. The vast majority of mammalian cells grow adherent to a surface; therefore, positive selection followed by cloning of cells while the cells remain adherent to their growth surface is an important goal. We recently demonstrated a microfabricated cell array combined with laser-based release of individual array elements for positive selection of single cells. In the current work, a strategy to collect single cells for clonal expansion is described. The system enabled cloning of individual cells with 80-90% efficiency. Single cells were selected and cloned from small populations of fewer than 10,000 cells. Strategies used by cells to migrate from the pallets to form colonies on the surface of the collection device were examined. Implementation of encoded array elements made it possible to follow specific cells throughout the selection, collection, and cloning procedure. Thus, a particular cell can be identified by any number of imaging techniques, isolated, and clonally expanded to generate a homogeneous cell line or a pure sample for genetic or biochemical analysis. PMID:17288466

  17. Establishment and characterization of a human thyroid carcinoma cell line (HOTHC) producing colony stimulating factor.

    PubMed

    Ishiwata, Isamu; Ono, Isao; Kiguchi, Kazushige; Ishiwata, Chieko; Soma, Masayuki; Ishikawa, Hiroshi

    2005-09-01

    A cell line designated HOTHC was established from an anaplastic carcinoma (giant cell type) of the thyroid gland of 80-year-old woman. The HOTHC line grew rapidly in multilayer without contact inhibition, and more than 120 serial passages were made within 27 months. The cells were spindle or polygonal in shape and revealed neoplastic and pleomorphic features. These cells were characterized as containing coloid droplets and poorly developed rough-endoplasmic reticulum in the cytoplasm. Doubling time was about 24 hours and plating efficiency was about 70%. The karyotype exhibits hyperploidy and marker chromosomes, and the modal chromosome number ranged between 77-90. The HOTHC cells were transplanted into the subcutis of BALB/c nude mice and produced anaplatic carcinomas (giant cell type) resembling the original tumor. The HOTHC cells produced colony stimulating factor (CSF) and caused granulocytosis in the mice. PMID:17022149

  18. Machine Learning Approach to Automated Quality Identification of Human Induced Pluripotent Stem Cell Colony Images

    PubMed Central

    Haponen, Markus; Rasku, Jyrki

    2016-01-01

    The focus of this research is on automated identification of the quality of human induced pluripotent stem cell (iPSC) colony images. iPS cell technology is a contemporary method by which the patient's cells are reprogrammed back to stem cells and are differentiated to any cell type wanted. iPS cell technology will be used in future to patient specific drug screening, disease modeling, and tissue repairing, for instance. However, there are technical challenges before iPS cell technology can be used in practice and one of them is quality control of growing iPSC colonies which is currently done manually but is unfeasible solution in large-scale cultures. The monitoring problem returns to image analysis and classification problem. In this paper, we tackle this problem using machine learning methods such as multiclass Support Vector Machines and several baseline methods together with Scaled Invariant Feature Transformation based features. We perform over 80 test arrangements and do a thorough parameter value search. The best accuracy (62.4%) for classification was obtained by using a k-NN classifier showing improved accuracy compared to earlier studies. PMID:27493680

  19. A Novel Combinatorial Therapy With Pulp Stem Cells and Granulocyte Colony-Stimulating Factor for Total Pulp Regeneration

    PubMed Central

    Iohara, Koichiro; Murakami, Masashi; Takeuchi, Norio; Osako, Yohei; Ito, Masataka; Ishizaka, Ryo; Utunomiya, Shinji; Nakamura, Hiroshi; Matsushita, Kenji

    2013-01-01

    Treatment of deep caries with pulpitis is a major challenge in dentistry. Stem cell therapy represents a potential strategy to regenerate the dentin-pulp complex, enabling conservation and restoration of teeth. The objective of this study was to assess the efficacy and safety of pulp stem cell transplantation as a prelude for the impending clinical trials. Clinical-grade pulp stem cells were isolated and expanded according to good manufacturing practice conditions. The absence of contamination, abnormalities/aberrations in karyotype, and tumor formation after transplantation in an immunodeficient mouse ensured excellent quality control. After autologous transplantation of pulp stem cells with granulocyte-colony stimulating factor (G-CSF) in a dog pulpectomized tooth, regenerated pulp tissue including vasculature and innervation completely filled in the root canal, and regenerated dentin was formed in the coronal part and prevented microleakage up to day 180. Transplantation of pulp stem cells with G-CSF yielded a significantly larger amount of regenerated dentin-pulp complex compared with transplantation of G-CSF or stem cells alone. Also noteworthy was the reduction in the number of inflammatory cells and apoptotic cells and the significant increase in neurite outgrowth compared with results without G-CSF. The transplanted stem cells expressed angiogenic/neurotrophic factors. It is significant that G-CSF together with conditioned medium of pulp stem cells stimulated cell migration and neurite outgrowth, prevented cell death, and promoted immunosuppression in vitro. Furthermore, there was no evidence of toxicity or adverse events. In conclusion, the combinatorial trophic effects of pulp stem cells and G-CSF are of immediate utility for pulp/dentin regeneration, demonstrating the prerequisites of safety and efficacy critical for clinical applications. PMID:23761108

  20. Altered Proteome of Burkholderia pseudomallei Colony Variants Induced by Exposure to Human Lung Epithelial Cells

    PubMed Central

    Al-Maleki, Anis Rageh; Mariappan, Vanitha; Vellasamy, Kumutha Malar; Tay, Sun Tee; Vadivelu, Jamuna

    2015-01-01

    Burkholderia pseudomallei primary diagnostic cultures demonstrate colony morphology variation associated with expression of virulence and adaptation proteins. This study aims to examine the ability of B. pseudomallei colony variants (wild type [WT] and small colony variant [SCV]) to survive and replicate intracellularly in A549 cells and to identify the alterations in the protein expression of these variants, post-exposure to the A549 cells. Intracellular survival and cytotoxicity assays were performed followed by proteomics analysis using two-dimensional gel electrophoresis. B. pseudomallei SCV survive longer than the WT. During post-exposure, among 259 and 260 protein spots of SCV and WT, respectively, 19 were differentially expressed. Among SCV post-exposure up-regulated proteins, glyceraldehyde 3-phosphate dehydrogenase, fructose-bisphosphate aldolase (CbbA) and betaine aldehyde dehydrogenase were associated with adhesion and virulence. Among the down-regulated proteins, enolase (Eno) is implicated in adhesion and virulence. Additionally, post-exposure expression profiles of both variants were compared with pre-exposure. In WT pre- vs post-exposure, 36 proteins were differentially expressed. Of the up-regulated proteins, translocator protein, Eno, nucleoside diphosphate kinase (Ndk), ferritin Dps-family DNA binding protein and peptidyl-prolyl cis-trans isomerase B were implicated in invasion and virulence. In SCV pre- vs post-exposure, 27 proteins were differentially expressed. Among the up-regulated proteins, flagellin, Eno, CbbA, Ndk and phenylacetate-coenzyme A ligase have similarly been implicated in adhesion, invasion. Protein profiles differences post-exposure provide insights into association between morphotypic and phenotypic characteristics of colony variants, strengthening the role of B. pseudomallei morphotypes in pathogenesis of melioidosis. PMID:25996927

  1. Radiosensitivity of CD4 or CD8 positive human T-lymphocytes by an in vitro colony formation assay

    SciTech Connect

    Nakamura, N.; Kusunoki, Y.; Akiyama, M. )

    1990-08-01

    The recent development of an in vitro lymphocyte colony assay makes it possible to examine variations in the radiosensitivity of humans using peripheral blood lymphocytes (PBL) instead of the skin fibroblast assay. Our recent study showed that most of the colonies consisted of lymphocytes bearing CD4 or CD8 antigens. Since the fraction of CD4+ and CD8+ cells in PBL differs among individuals, we suspected that individual radiosensitivity might be biased by the different subset frequencies if the dose-survival curves of the CD4+ and CD8+ cells were different from each other. In the present study, CD4+ (helper/inducer T) and CD8+ (suppressor/cytotoxic T) lymphocytes were isolated from PBL and their dose-survival curves were determined. The results showed that the D10 (dose required to reduce the surviving fraction to 10%) was similar for these two types of cells (3.13 +/- 0.10 Gy (mean +/- SD) for CD4+, 3.34 +/- 0.50 Gy for CD8+ and 3.14 +/- 0.17 Gy for the unsorted cells), supporting the use of the whole PBL population for the screening of individuals with altered radiosensitivity.

  2. Subculture of Germ Cell-Derived Colonies with GATA4-Positive Feeder Cells from Neonatal Pig Testes

    PubMed Central

    Lee, Kyung Hoon; Lee, Won Young; Kim, Jin Hoi; Park, Chan Kyu; Do, Jeong Tae; Kim, Jae Hwan; Choi, Young Suk; Kim, Nam Hyung; Song, Hyuk

    2016-01-01

    Enrichment of spermatogonial stem cells is important for studying their self-renewal and differentiation. Although germ cell-derived colonies (GDCs) have been successfully cultured from neonatal pig testicular cells under 31°C conditions, the short period of in vitro maintenance (<2 months) limited their application to further investigations. To develop a culture method that allows for in vitro maintenance of GDCs for long periods, we subcultured the GDCs with freshly prepared somatic cells from neonatal pig testes as feeder cells. The subcultured GDCs were maintained up to passage 13 with the fresh feeder cells (FFCs) and then frozen. Eight months later, the frozen GDCs could again form the colonies on FFCs as shown in passages 1 to 13. Immunocytochemistry data revealed that the FFCs expressed GATA-binding protein 4 (GATA4), which is also detected in the cells of neonatal testes and total testicular cells, and that the expression of GATA4 was decreased in used old feeder cells. The subcultured GDCs in each passage had germ and stem cell characteristics, and flow cytometric analyses revealed that ~60% of these cells were GFRα-1 positive. In conclusion, neonatal pig testes-derived GDCs can be maintained for long periods with GATA4-expressing testicular somatic cells. PMID:26880974

  3. Computational image analysis of colony and nuclear morphology to evaluate human induced pluripotent stem cells.

    PubMed

    Tokunaga, Kazuaki; Saitoh, Noriko; Goldberg, Ilya G; Sakamoto, Chiyomi; Yasuda, Yoko; Yoshida, Yoshinori; Yamanaka, Shinya; Nakao, Mitsuyoshi

    2014-01-01

    Non-invasive evaluation of cell reprogramming by advanced image analysis is required to maintain the quality of cells intended for regenerative medicine. Here, we constructed living and unlabelled colony image libraries of various human induced pluripotent stem cell (iPSC) lines for supervised machine learning pattern recognition to accurately distinguish bona fide iPSCs from improperly reprogrammed cells. Furthermore, we found that image features for efficient discrimination reside in cellular components. In fact, extensive analysis of nuclear morphologies revealed dynamic and characteristic signatures, including the linear form of the promyelocytic leukaemia (PML)-defined structure in iPSCs, which was reversed to a regular sphere upon differentiation. Our data revealed that iPSCs have a markedly different overall nuclear architecture that may contribute to highly accurate discrimination based on the cell reprogramming status. PMID:25385348

  4. Identification of a murine high-proliferative-potential colony-forming cell (HPP-CFC) capable of producing a number of megakaryocytes and replating for secondary HPP-CFCs in culture.

    PubMed

    Han, Z C

    1994-04-01

    The high-proliferative-potential colony-forming cell (HPP-CFC) has been shown to be clearly heterogeneous. Hierarchical subpopulations can be identified by analyzing the kinetics of cell regeneration and the specific cellular requirement for cytokines. In this study, a new type of HPP-CFC, termed high-proliferative potential mixed colony-forming unit-megakaryocyte (HPP-mCFU-MK), was detected in murine bone marrow cell cultures. The HPP-mCFU-MK was able to form macroscopic colonies that fit the criteria of the HPP-CFC colony but contained a number of megakaryocytes. Its growth was stimulated by either aplastic anemia serum (AAS) or a combination of three or more recombinant hematopoietic growth factors but was not inhibited by transforming growth factor-beta 1 (TGF-beta 1) and platelet factor 4 (PF4) in vitro. The recloning of the 12-day-old HPP-mCFU-MK colonies picked up from AAS-stimulated primary cultures caused secondary formation of HPP-CFC colonies. These data suggest that HPP-mCFC-MK is a new subset of stem cell characterized by its ability to produce directly a number of megakaryocytes in response to multifactor stimulation, to generate a secondary set of HPP-CFC in replating culture, and to be unaffected by TGF-beta 1 and PF4 treatment. PMID:8145010

  5. Hypothalamic proline-rich polypeptide enhances bone marrow colony-forming cell proliferation and stromal progenitor cell differentiation.

    PubMed

    Galoyan, A A; Korochkin, L I; Rybalkina, E J; Pavlova, G V; Saburina, I N; Zaraiski, E I; Galoyan, N A; Davtyan, T K; Bezirganyan, K B; Revishchin, A V

    2008-01-01

    The AGAPEPAEPAQPGVY proline-rich peptide (PRP-1) was isolated from neurosecretory granules of the bovine neurohypophysis; it is produced by N. supraopticus and N. paraventricularis. It has been shown that PRP-1 has many potentially beneficial biological effects, including immunoregulatory, hematopoietic, antimicrobial, and antineurodegenerative properties. Here we showed that PRP increased colony-forming cell (CFC) proliferation in rat bone marrow (BM) cells in vivo. In PRP-treated rat BM, the CFU number at day 7 and day 14 was considerably increased in comparison with untreated rat BM and no difference was found at day 21 and day 28. The related peptide [arg8]vasopressin did not reveal CFC proliferation. PRP failed to farther increase CFC proliferation in vitro in BM obtained from PRP-treated or untreated rats. After 3-4 days of human BM stromal cell cultivation in the presence of 2-20 microg/ml PRP the appearance of cells expressing CD15, CD10, CD11a, CD11b, CD3, CD4, and CD16 surface antigens did not differ from the untreated cells. PRP increased the appearance of CD14-positive cells upon 3-4-day incubation with both adult and fetal BM stromal cells. Our results suggest a previously undescribed role for the hypothalamic peptide within neurosecretory hypothalamus-bone marrow humoral axis, because PRP enhances BM colony-forming cell proliferation and stromal cell differentiation. PMID:19177842

  6. Human Granulocyte Macrophage Colony-Stimulating Factor Enhances Antibiotic Susceptibility of Pseudomonas aeruginosa Persister Cells.

    PubMed

    Choudhary, Geetika S; Yao, Xiangyu; Wang, Jing; Peng, Bo; Bader, Rebecca A; Ren, Dacheng

    2015-01-01

    Bacterial persister cells are highly tolerant to antibiotics and cause chronic infections. However, little is known about the interaction between host immune systems with this subpopulation of metabolically inactive cells, and direct effects of host immune factors (in the absence of immune cells) on persister cells have not been studied. Here we report that human granulocyte macrophage-colony stimulating factor (GM-CSF) can sensitize the persister cells of Pseudomonas aeruginosa PAO1 and PDO300 to multiple antibiotics including ciprofloxacin, tobramycin, tetracycline, and gentamicin. GM-CSF also sensitized the biofilm cells of P. aeruginosa PAO1 and PDO300 to tobramycin in the presence of biofilm matrix degrading enzymes. The DNA microarray and qPCR results indicated that GM-CSF induced the genes for flagellar motility and pyocin production in the persister cells, but not the normal cells of P. aeruginosa PAO1. Consistently, the supernatants from GM-CSF treated P. aeruginosa PAO1 persister cell suspensions were found cidal to the pyocin sensitive strain P. aeruginosa PAK. Collectively, these findings suggest that host immune factors and bacterial persisters may directly interact, leading to enhanced susceptibility of persister cells to antibiotics. PMID:26616387

  7. Human Granulocyte Macrophage Colony-Stimulating Factor Enhances Antibiotic Susceptibility of Pseudomonas aeruginosa Persister Cells

    PubMed Central

    Choudhary, Geetika S.; Yao, Xiangyu; Wang, Jing; Peng, Bo; Bader, Rebecca A.; Ren, Dacheng

    2015-01-01

    Bacterial persister cells are highly tolerant to antibiotics and cause chronic infections. However, little is known about the interaction between host immune systems with this subpopulation of metabolically inactive cells, and direct effects of host immune factors (in the absence of immune cells) on persister cells have not been studied. Here we report that human granulocyte macrophage-colony stimulating factor (GM-CSF) can sensitize the persister cells of Pseudomonas aeruginosa PAO1 and PDO300 to multiple antibiotics including ciprofloxacin, tobramycin, tetracycline, and gentamicin. GM-CSF also sensitized the biofilm cells of P. aeruginosa PAO1 and PDO300 to tobramycin in the presence of biofilm matrix degrading enzymes. The DNA microarray and qPCR results indicated that GM-CSF induced the genes for flagellar motility and pyocin production in the persister cells, but not the normal cells of P. aeruginosa PAO1. Consistently, the supernatants from GM-CSF treated P. aeruginosa PAO1 persister cell suspensions were found cidal to the pyocin sensitive strain P. aeruginosa PAK. Collectively, these findings suggest that host immune factors and bacterial persisters may directly interact, leading to enhanced susceptibility of persister cells to antibiotics. PMID:26616387

  8. Increase in Bacterial Colony Formation from a Permafrost Ice Wedge Dosed with a Tomitella biformata Recombinant Resuscitation-Promoting Factor Protein.

    PubMed

    Puspita, Indun Dewi; Kitagawa, Wataru; Kamagata, Yoichi; Tanaka, Michiko; Nakatsu, Cindy H

    2015-01-01

    Resuscitation-promoting factor (Rpf) is a protein that has been found in a number of different Actinobacteria species and has been shown to promote the growth of active cells and resuscitate dormant (non-dividing) cells. We previously reported the biological activity of an Rpf protein in Tomitella biformata AHU 1821(T), an Actinobacteria isolated from a permafrost ice wedge. This protein is excreted outside the cell; however, few studies have investigated its contribution in environmental samples to the growth or resuscitation of bacteria other than the original host. Therefore, the aim of the present study was to determine whether Rpf from T. biformata impacted the cultivation of other bacteria from the permafrost ice wedge from which it was originally isolated. All experiments used recombinant Rpf proteins produced using a Rhodococcus erythropolis expression system. Dilutions of melted surface sterilized ice wedge samples mixed with different doses of the purified recombinant Rpf (rRpf) protein indicated that the highest concentration tested, 1250 pM, had a significantly (p <0.05) higher number of CFUs on agar plates after 8 d, approximately 14-fold higher than that on control plates without rRpf. 16S rRNA gene sequences revealed that all the colonies on plates were mainly related to Brevibacterium antiquum strain VKM Ac-2118 (AY243344), with 98-99% sequence identity. This species is also a member of the phylum Actinobacteria and was originally isolated from Siberian permafrost sediments. The results of the present study demonstrated that rRpf not only promoted the growth of T. biformata from which it was isolated, but also enhanced colony formation by another Actinobacteria in an environmental sample. PMID:25843055

  9. Acute leukemia after radiotherapy in a patient with Turcot's syndrome. Impaired colony formation in skin fibroblast cultures after irradiation

    SciTech Connect

    Li, F.P.; Little, J.B.; Bech-Hansen, N.T.; Paterson, M.C.; Arlett, C.; Garnick, M.B.; Mayer, R.J.

    1983-02-01

    Colonic polyposis and carcinoma developed in a woman with Turcot's syndrome at the age of 31 years; astrocytoma developed when she was 37. Her brother and sister had died of astrocytoma at the ages of 18 and 33 years, respectively. Progressive neutropenia developed in the patient three months after radiotherapy for her brain tumor and acute myelomonocytic leukemia 19 months after treatment. Three laboratories independently evaluated cultures of her skin fibroblasts for in vitro sensitivity to cell killing (loss of colony-forming ability) by x-rays. Survival assays consistently revealed slight but significant radiosensitivity in an early-passage (six to 10 doublings) fibroblast subculture. A later subculture (21 to 29 doublings) showed no abnormality, a possible effect of selective in vitro loss of radiosensitive cells.

  10. Bionomics and Formation of “Bonsai” Colonies With Long-Term Rearing of Coptotermes formosanus (Isoptera: Rhinotermitidae).

    PubMed

    Osbrink, W L A; Cornelius, M L; Showler, A T

    2016-04-01

    This laboratory study reports the ability of Formosan subterranean termite, Coptotermes formosanus Shiraki, colonies to survive for at least 9 yr while restricted to a sweater box. Colonies survived by limiting queen size and worker numbers, allowing these bonsai colonies to thrive. Queen physogastry appeared to plateau with 9-yr-old queens not larger than 6-yr-old queens, but nearly triple the size of 2-yr-old queens. Nine-year-old colony worker numbers were not greater than 6-yr-old colonies, but worker numbers were greater than in 2-yr-old colonies. Such colony survival under conditions of restricted resources provides a mechanism for re-infestation of areas following extensive area-wide control efforts. “Bonsai” colonies are relevant to the ability of marginalized colonies to avoid detection and then expand and invade into areas once the large, mature colonies are eliminated, and their potential to produce alates to start new C. formosanus colonies in areas which have been subjected to colony elimination programs impacts termite management strategies. PMID:26662736

  11. Exercise-induced norepinephrine decreases circulating hematopoietic stem and progenitor cell colony-forming capacity.

    PubMed

    Kröpfl, Julia M; Stelzer, Ingeborg; Mangge, Harald; Pekovits, Karin; Fuchs, Robert; Allard, Nathalie; Schinagl, Lukas; Hofmann, Peter; Dohr, Gottfried; Wallner-Liebmann, Sandra; Domej, Wolfgang; Müller, Wolfram

    2014-01-01

    A recent study showed that ergometry increased circulating hematopoietic stem and progenitor cell (CPC) numbers, but reduced hematopoietic colony forming capacity/functionality under normoxia and normobaric hypoxia. Herein we investigated whether an exercise-induced elevated plasma free/bound norepinephrine (NE) concentration could be responsible for directly influencing CPC functionality. Venous blood was taken from ten healthy male subjects (25.3+/-4.4 yrs) before and 4 times after ergometry under normoxia and normobaric hypoxia (FiO2<0.15). The circulating hematopoietic stem and progenitor cell numbers were correlated with free/bound NE, free/bound epinephrine (EPI), cortisol (Co) and interleukin-6 (IL-6). Additionally, the influence of exercise-induced NE and blood lactate (La) on CPC functionality was analyzed in a randomly selected group of subjects (n = 6) in vitro under normoxia by secondary colony-forming unit granulocyte macrophage assays. Concentrations of free NE, EPI, Co and IL-6 were significantly increased post-exercise under normoxia/hypoxia. Ergometry-induced free NE concentrations found in vivo showed a significant impairment of CPC functionality in vitro under normoxia. Thus, ergometry-induced free NE was thought to trigger CPC mobilization 10 minutes post-exercise, but as previously shown impairs CPC proliferative capacity/functionality at the same time. The obtained results suggest that an ergometry-induced free NE concentration has a direct negative effect on CPC functionality. Cortisol may further influence CPC dynamics and functionality. PMID:25180783

  12. Exercise-Induced Norepinephrine Decreases Circulating Hematopoietic Stem and Progenitor Cell Colony-Forming Capacity

    PubMed Central

    Mangge, Harald; Pekovits, Karin; Fuchs, Robert; Allard, Nathalie; Schinagl, Lukas; Hofmann, Peter; Dohr, Gottfried; Wallner-Liebmann, Sandra; Domej, Wolfgang; Müller, Wolfram

    2014-01-01

    A recent study showed that ergometry increased circulating hematopoietic stem and progenitor cell (CPC) numbers, but reduced hematopoietic colony forming capacity/functionality under normoxia and normobaric hypoxia. Herein we investigated whether an exercise-induced elevated plasma free/bound norepinephrine (NE) concentration could be responsible for directly influencing CPC functionality. Venous blood was taken from ten healthy male subjects (25.3+/−4.4 yrs) before and 4 times after ergometry under normoxia and normobaric hypoxia (FiO2<0.15). The circulating hematopoietic stem and progenitor cell numbers were correlated with free/bound NE, free/bound epinephrine (EPI), cortisol (Co) and interleukin-6 (IL-6). Additionally, the influence of exercise-induced NE and blood lactate (La) on CPC functionality was analyzed in a randomly selected group of subjects (n = 6) in vitro under normoxia by secondary colony-forming unit granulocyte macrophage assays. Concentrations of free NE, EPI, Co and IL-6 were significantly increased post-exercise under normoxia/hypoxia. Ergometry-induced free NE concentrations found in vivo showed a significant impairment of CPC functionality in vitro under normoxia. Thus, ergometry-induced free NE was thought to trigger CPC mobilization 10 minutes post-exercise, but as previously shown impairs CPC proliferative capacity/functionality at the same time. The obtained results suggest that an ergometry-induced free NE concentration has a direct negative effect on CPC functionality. Cortisol may further influence CPC dynamics and functionality. PMID:25180783

  13. Prolonged growth of a clinical Staphylococcus aureus strain selects for a stable small-colony-variant cell type.

    PubMed

    Bui, Long M G; Hoffmann, Peter; Turnidge, John D; Zilm, Peter S; Kidd, Stephen P

    2015-02-01

    An undetermined feature of Staphylococcus aureus pathogenesis is its persistence and then relapse of disease. This has been explained by its switch to alternative lifestyles, mainly as biofilm or small-colony variants (SCVs). Studying the native characteristics of SCVs has been problematic due to their reversion to the parental lifestyle. We have observed that for a number of S. aureus strains as they switch to an SCV lifestyle, there is the formation of an extracellular matrix. We focused our analysis on one strain, WCH-SK2. For bacterial survival in the host, the combination of low nutrients and the prolonged time frame forms a stress that selects for a specific cell type from the population. In this context, we used steady-state growth conditions with low nutrients and a controlled low growth rate for a prolonged time and with methylglyoxal. These conditions induced S. aureus WCH-SK2 into a stable SCV cell type; the cells did not revert after subculturing. Analysis revealed these cells possessed a metabolic and surface profile that was different from those of previously described SCVs or biofilm cells. The extracellular matrix was protein and extracellular DNA but not polysaccharide. The SCV cells induced expression of certain surface proteins (such as Ebh) and synthesis of lantibiotics while downregulating factors that stimulate the immune response (leucocidin, capsule, and carotenoid). Our data reveal cell heterogeneity within an S. aureus population and under conditions that resemble long-term survival in the host have identified a previously unnoticed S. aureus cell type with a distinctive metabolic and molecular profile. PMID:25385795

  14. Large-scale time-lapse microscopy of Oct4 expression in human embryonic stem cell colonies.

    PubMed

    Bhadriraju, Kiran; Halter, Michael; Amelot, Julien; Bajcsy, Peter; Chalfoun, Joe; Vandecreme, Antoine; Mallon, Barbara S; Park, Kye-Yoon; Sista, Subhash; Elliott, John T; Plant, Anne L

    2016-07-01

    Identification and quantification of the characteristics of stem cell preparations is critical for understanding stem cell biology and for the development and manufacturing of stem cell based therapies. We have developed image analysis and visualization software that allows effective use of time-lapse microscopy to provide spatial and dynamic information from large numbers of human embryonic stem cell colonies. To achieve statistically relevant sampling, we examined >680 colonies from 3 different preparations of cells over 5days each, generating a total experimental dataset of 0.9 terabyte (TB). The 0.5 Giga-pixel images at each time point were represented by multi-resolution pyramids and visualized using the Deep Zoom Javascript library extended to support viewing Giga-pixel images over time and extracting data on individual colonies. We present a methodology that enables quantification of variations in nominally-identical preparations and between colonies, correlation of colony characteristics with Oct4 expression, and identification of rare events. PMID:27286574

  15. Cooperation between human fibrocytes and endothelial colony-forming cells increases angiogenesis via the CXCR4 pathway.

    PubMed

    Smadja, David M; Dorfmüller, Peter; Guerin, Coralie L; Bieche, Ivan; Badoual, Cécile; Boscolo, Elisa; Kambouchner, Marianne; Cazes, Aurélie; Mercier, Olaf; Humbert, Marc; Gaussem, Pascale; Bischoff, Joyce; Israël-Biet, Dominique

    2014-11-01

    Fibrotic diseases of the lung are associated with a vascular remodelling process. Fibrocytes (Fy) are a distinct population of blood-borne cells that co-express haematopoietic cell antigens and fibroblast markers, and have been shown to contribute to organ fibrosis. The purpose of this study was to determine whether fibrocytes cooperate with endothelial colony-forming cells (ECFC) to induce angiogenesis. We isolated fibrocytes from blood of patient with idiopathic pulmonary fibrosis (IPF) and characterised them by flow cytometry, quantitative reverse transcriptase PCR (RTQ-PCR), and confocal microscopy. We then investigated the angiogenic interaction between fibrocytes and cord-blood-derived ECFC, both in vitro and in an in vivo Matrigel implant model. Compared to fibroblast culture medium, fibrocyte culture medium increased ECFC proliferation and differentiation via the SDF-1/CXCR4 pathway. IPF-Fy co-implanted with human ECFC in Matrigel plugs in immunodeficient mice formed functional microvascular beds, whereas fibroblasts did not. Evaluation of implants after two weeks revealed an extensive network of erythrocyte-containing blood vessels. CXCR4 blockade significantly inhibited this blood vessel formation. The clinical relevance of these data was confirmed by strong CXCR4 expression in vessels close to fibrotic areas in biopsy specimens from patients with IPF, by comparison with control lungs. In conclusion, circulating fibrocytes might contribute to the intense remodelling of the pulmonary vasculature in patients with idiopathic pulmonary fibrosis. PMID:25103869

  16. Human papillomavirus 16 E5 induces bi-nucleated cell formation by cell-cell fusion

    SciTech Connect

    Hu Lulin; Plafker, Kendra; Vorozhko, Valeriya; Zuna, Rosemary E.; Hanigan, Marie H.; Gorbsky, Gary J.; Plafker, Scott M.; Angeletti, Peter C.; Ceresa, Brian P.

    2009-02-05

    Human papillomaviruses (HPV) 16 is a DNA virus encoding three oncogenes - E5, E6, and E7. The E6 and E7 proteins have well-established roles as inhibitors of tumor suppression, but the contribution of E5 to malignant transformation is controversial. Using spontaneously immortalized human keratinocytes (HaCaT cells), we demonstrate that expression of HPV16 E5 is necessary and sufficient for the formation of bi-nucleated cells, a common characteristic of precancerous cervical lesions. Expression of E5 from non-carcinogenic HPV6b does not produce bi-nucleate cells. Video microscopy and biochemical analyses reveal that bi-nucleates arise through cell-cell fusion. Although most E5-induced bi-nucleates fail to propagate, co-expression of HPV16 E6/E7 enhances the proliferation of these cells. Expression of HPV16 E6/E7 also increases bi-nucleated cell colony formation. These findings identify a new role for HPV16 E5 and support a model in which complementary roles of the HPV16 oncogenes lead to the induction of carcinogenesis.

  17. Identification of vitamin B1 metabolism as a tumor-specific radiosensitizing pathway using a high-throughput colony formation screen

    PubMed Central

    Buffa, Francesca M.; Yu, Sheng; Ebner, Daniel V.; Howarth, Alison; Folkes, Lisa K.; Budwal, Balam; Chu, Kwun-Ye; Durrant, Lisa; Muschel, Ruth J.; McKenna, W. Gillies; Higgins, Geoff S.

    2015-01-01

    Colony formation is the gold standard assay for determining reproductive cell death after radiation treatment, since effects on proliferation often do not reflect survival. We have developed a high-throughput radiosensitivity screening method based on clonogenicity and screened a siRNA library against kinases. Thiamine pyrophosphokinase-1 (TPK1), a key component of Vitamin B1/thiamine metabolism, was identified as a target for radiosensitization. TPK1 knockdown caused significant radiosensitization in cancer but not normal tissue cell lines. Other means of blocking this pathway, knockdown of thiamine transporter-1 (THTR1) or treatment with the thiamine analogue pyrithiamine hydrobromide (PyrH) caused significant tumor specific radiosensitization. There was persistent DNA damage in cells irradiated after TPK1 and THTR1 knockdown or PyrH treatment. Thus this screen allowed the identification of thiamine metabolism as a novel radiosensitization target that affects DNA repair. Short-term modulation of thiamine metabolism could be a clinically exploitable strategy to achieve tumor specific radiosensitization. PMID:25788274

  18. Growth of a radiation-transformed clone of C3H 1OT1/2 cells into melanin-producing colonies

    SciTech Connect

    Szekely, J.G.; Raaphorst, G.P.; Lobreau, A.U.; Azzam, E.I.; Vadasz, J.A.

    1985-01-01

    When R25, a radiation-transformed clone of C3H 1OT1/2 cells, was plated in soft agarose, a fraction of the colonies became pigmented. The morphologies of the white and dark colonies and their cells were compared by optical, transmission and scanning electron microscopy. The transformed R25 cells had apparently differentiated into melanin-producing cells in the soft agarose, with the white colonies containing actively growing cells having only a few melanosomes, and the dark colonies being made up of stationary-phase cells filled with electronopaque melanosomes. Exposure of the R25 cells to 4.0 Gy of X-rays decreased the percentage of dark colonies, while exposure to 1% DMSO had no effect.

  19. Peptidoglycolipid nature of the superficial cell wall sheath of smooth-colony-forming mycobacteria.

    PubMed Central

    Barrow, W W; Ullom, B P; Brennan, P J

    1980-01-01

    The most superficial cell wall layer present in smooth-colony-forming mycobacteria was isolated from serovar 20 of the Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum (MAIS) serocomplex and examined chemically and by electron microscopy. Most (70 to 80%) of the fibrillar material consisted of an array of serologically active, acetylated C-myosidic peptidoglycoplipids with the basic structure (formula, see text) but in which the location of acetyl groups and the arrangement of monosaccharides have not been defined. Apparently, all serovars within the MAIS complex are characterized by structurally related superficies in which the monoglycosyl-lipopeptide portion is invariable but the oligosaccharide attachment is peculiar to each serovar. These unique inert structures may be an important factor in shielding the pathogen within phagolysosomes from lysosomal enzymes. Images PMID:7430072

  20. Growth of human hemopoietic colonies in response to recombinant gibbon interleukin 3: comparison with human recombinant granulocyte and granulocyte-macrophage colony-stimulating factor

    SciTech Connect

    Messner, H.A.; Yamasaki, K.; Jamal, N.; Minden, M.M.; Yang, Y.C.; Wong, G.G.; Clark, S.C.

    1987-10-01

    Supernatants of COS-1 cells transfected with gibbon cDNA encoding interleukin 3 (IL-3) with homology to sequences for human IL-3 were tested for ability to promote growth of various human hemopoietic progenitors. The effect of these supernatants as a source of recombinant IL-3 was compared to that of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) as well as to that of medium conditioned by phytohemagglutinin-stimulated leukocytes. The frequency of multilineage colonies, erythroid bursts, and megakaryocyte colonies in cultures containing the COS-1 cell supernatant was equivalent to the frequency observed in the controls and significantly higher than found in cultures plated with recombinant GM-CSF. G-CSF did not support the formation of multilineage colonies, erythroid bursts, and megakaryocyte colonies. In contrast, growth of granulocyte-macrophage colonies was best supported with GM-CSF, while recombinant IL-3 yielded colonies at lower or at best equivalent frequency. The simultaneous addition of higher concentrations of GM-CSF to cultures containing IL-3 in optimal amounts did not enhance the formation of multilineage colonies, erythroid bursts, and megakaryocyte colonies. However, the frequency of such colonies and bursts increased with GM-CSF when cultures were plated with suboptimal concentrations of IL-3. Growth of colonies within the granulocyte-macrophage lineage is optimally supported by GM-CSF and does not increase with further addition of IL-3.

  1. YfiBNR Mediates Cyclic di-GMP Dependent Small Colony Variant Formation and Persistence in Pseudomonas aeruginosa

    PubMed Central

    Malone, Jacob G.; Jaeger, Tina; Spangler, Christian; Ritz, Daniel; Spang, Anne; Arrieumerlou, Cécile; Kaever, Volkhard; Landmann, Regine; Jenal, Urs

    2010-01-01

    During long-term cystic fibrosis lung infections, Pseudomonas aeruginosa undergoes genetic adaptation resulting in progressively increased persistence and the generation of adaptive colony morphotypes. This includes small colony variants (SCVs), auto-aggregative, hyper-adherent cells whose appearance correlates with poor lung function and persistence of infection. The SCV morphotype is strongly linked to elevated levels of cyclic-di-GMP, a ubiquitous bacterial second messenger that regulates the transition between motile and sessile, cooperative lifestyles. A genetic screen in PA01 for SCV-related loci identified the yfiBNR operon, encoding a tripartite signaling module that regulates c-di-GMP levels in P. aeruginosa. Subsequent analysis determined that YfiN is a membrane-integral diguanylate cyclase whose activity is tightly controlled by YfiR, a small periplasmic protein, and the OmpA/Pal-like outer-membrane lipoprotein YfiB. Exopolysaccharide synthesis was identified as the principal downstream target for YfiBNR, with increased production of Pel and Psl exopolysaccharides responsible for many characteristic SCV behaviors. An yfi-dependent SCV was isolated from the sputum of a CF patient. Consequently, the effect of the SCV morphology on persistence of infection was analyzed in vitro and in vivo using the YfiN-mediated SCV as a representative strain. The SCV strain exhibited strong, exopolysaccharide-dependent resistance to nematode scavenging and macrophage phagocytosis. Furthermore, the SCV strain effectively persisted over many weeks in mouse infection models, despite exhibiting a marked fitness disadvantage in vitro. Exposure to sub-inhibitory concentrations of antibiotics significantly decreased both the number of suppressors arising, and the relative fitness disadvantage of the SCV mutant in vitro, suggesting that the SCV persistence phenotype may play a more important role during antimicrobial chemotherapy. This study establishes YfiBNR as an important

  2. Enhanced Viability of Endothelial Colony Forming Cells in Fibrin Microbeads for Sensor Vascularization

    PubMed Central

    Gandhi, Jarel K.; Zivkovic, Lada; Fisher, John P.; Yoder, Mervin C.; Brey, Eric M.

    2015-01-01

    Enhanced vascularization at sensor interfaces can improve long-term function. Fibrin, a natural polymer, has shown promise as a biomaterial for sensor coating due to its ability to sustain endothelial cell growth and promote local vascularization. However, the culture of cells, particularly endothelial cells (EC), within 3D scaffolds for more than a few days is challenging due to rapid loss of EC viability. In this manuscript, a robust method for developing fibrin microbead scaffolds for long-term culture of encapsulated ECs is described. Fibrin microbeads are formed using sodium alginate as a structural template. The size, swelling and structural properties of the microbeads were varied with needle gauge and composition and concentration of the pre-gel solution. Endothelial colony-forming cells (ECFCs) were suspended in the fibrin beads and cultured within a perfusion bioreactor system. The perfusion bioreactor enhanced ECFCs viability and genome stability in fibrin beads relative to static culture. Perfusion bioreactors enable 3D culture of ECs within fibrin beads for potential application as a sensor coating. PMID:26393602

  3. Colony aggregation and biofilm formation in xylem chemistry-based media for Xylella fastidiosa.

    PubMed

    Leite, Breno; Andersen, Peter Craig; Ishida, Maria Lucia

    2004-01-30

    Two chemically defined media based on xylem fluid chemistry were developed for Xylella fastidiosa. These media were tested and compared to chemically defined media XDM2, XDM4 and XF-26. New media were evaluated for the Pierce's disease (PD) strain UCLA-PD. Our media either was similar to the concentration of some amino acids found in the xylem fluid of the PD-susceptible Vitis vinifera cv. Chardonnay (medium CHARD2) or incorporated the tripeptide glutathione found in xylem fluid composition (medium 3G10-R). CHARD2 and 3G10-R are among the simplest chemically defined media available. Xylem fluid chemistry-based media supported X. fastidiosa growth and especially stimulated aggregation and biofilm formation. PMID:14757251

  4. CD34 expression modulates tube-forming capacity and barrier properties of peripheral blood-derived endothelial colony-forming cells (ECFCs).

    PubMed

    Tasev, Dimitar; Konijnenberg, Lara S F; Amado-Azevedo, Joana; van Wijhe, Michiel H; Koolwijk, Pieter; van Hinsbergh, Victor W M

    2016-07-01

    Endothelial colony-forming cells (ECFC) are grown from circulating CD34(+) progenitors present in adult peripheral blood, but during in vitro expansion part of the cells lose CD34. To evaluate whether the regulation of CD34 characterizes the angiogenic phenotypical features of PB-ECFCs, we investigated the properties of CD34(+) and CD34(-) ECFCs with respect to their ability to form capillary-like tubes in 3D fibrin matrices, tip-cell gene expression, and barrier integrity. Selection of CD34(+) and CD34(-) ECFCs from subcultured ECFCs was accomplished by magnetic sorting (FACS: CD34(+): 95 % pos; CD34(-): 99 % neg). Both fractions proliferated at same rate, while CD34(+) ECFCs exhibited higher tube-forming capacity and tip-cell gene expression than CD3(4-) cells. However, during cell culture CD34(-) cells re-expressed CD34. Cell-seeding density, cell-cell contact formation, and serum supplements modulated CD34 expression. CD34 expression in ECFCs was strongly suppressed by newborn calf serum. Stimulation with FGF-2, VEGF, or HGF prepared in medium supplemented with 3 % albumin did not change CD34 mRNA or surface expression. Silencing of CD34 with siRNA resulted in strengthening of cell-cell contacts and increased barrier function of ECFC monolayers as measured by ECIS. Furthermore, CD34 siRNA reduced tube formation by ECFC, but did not affect tip-cell gene expression. These findings demonstrate that CD34(+) and CD34(-) cells are different phenotypes of similar cells and that CD34 (1) can be regulated in ECFC; (2) is positively involved in capillary-like sprout formation; (3) is associated but not causally related to tip-cell gene expression; and (4) can affect endothelial barrier function. PMID:27043316

  5. T-lymphocyte colonies in the lymphoproliferative disorders.

    PubMed Central

    Dao, C; Marie, J P; Bernadou, A; Bilski-Pasquier, G

    1978-01-01

    Human lymphocytes from peripheral blood, bone marrow spleen and lymph nodes were cultured. Continuous phytoheamagglutinin (PHA) stimulation was used, first during a 24 h liquid preincubation, then during a 5 day culture in methylcellulose. In normal donors a rapid colony formation took place, with a mean of 124+/-82 colonies per 1 times 10(5) preincubated lymphocytes. Cells from such colonies were studied by cytology, scanning electron microscopy and rosette formation techniques; arguments favour the hypothesis that these could be T lymphocytes. Neither granulocytes nor macrophages could be grown, and no lymphoid colony formation occurred without PHA stimulation. The same technique was applied to patients with various lymphoproliferative disorders. Significant colony suppression was observed in nearly every case of chronic lymphatic leukaemia; the number of colonies was reduced in some patients with acute lymphatic leukaemia, lymphosarcoma, dysglobulinaemia and Hodgkin's disease. This lymphoid culture method should be applied to a larger number of patients to determine whether it has a classification value and/or prognostic significance. When colonies were grown in pathological states, rosette formation was identical to that of normal donors; colony formation could be due to persisting normal lymphocytes. Images Figure 2 Figure 3 PMID:309852

  6. Expression of Genes Related to Germ Cell Lineage and Pluripotency in Single Cells and Colonies of Human Adult Germ Stem Cells.

    PubMed

    Conrad, Sabine; Azizi, Hossein; Hatami, Maryam; Kubista, Mikael; Bonin, Michael; Hennenlotter, Jörg; Sievert, Karl-Dietrich; Skutella, Thomas

    2016-01-01

    The aim of this study was to elucidate the molecular status of single human adult germ stem cells (haGSCs) and haGSC colonies, which spontaneously developed from the CD49f MACS and matrix- (collagen-/laminin+ binding-) selected fraction of enriched spermatogonia. Single-cell transcriptional profiling by Fluidigm BioMark system of a long-term cultured haGSCs cluster in comparison to human embryonic stem cells (hESCs) and human fibroblasts (hFibs) revealed that haGSCs showed a characteristic germ- and pluripotency-associated gene expression profile with some similarities to hESCs and with a significant distinction from somatic hFibs. Genome-wide comparisons with microarray analysis confirmed that different haGSC colonies exhibited gene expression heterogeneity with more or less pluripotency. The results of this study confirm that haGSCs are adult stem cells with a specific molecular gene expression profile in vitro, related but not identical to true pluripotent stem cells. Under ES-cell conditions haGSC colonies could be selected and maintained in a partial pluripotent state at the molecular level, which may be related to their cell plasticity and potential to differentiate into cells of all germ layers. PMID:26649052

  7. Expression of Genes Related to Germ Cell Lineage and Pluripotency in Single Cells and Colonies of Human Adult Germ Stem Cells

    PubMed Central

    Conrad, Sabine; Azizi, Hossein; Hatami, Maryam; Kubista, Mikael; Bonin, Michael; Hennenlotter, Jörg; Sievert, Karl-Dietrich; Skutella, Thomas

    2016-01-01

    The aim of this study was to elucidate the molecular status of single human adult germ stem cells (haGSCs) and haGSC colonies, which spontaneously developed from the CD49f MACS and matrix- (collagen−/laminin+ binding-) selected fraction of enriched spermatogonia. Single-cell transcriptional profiling by Fluidigm BioMark system of a long-term cultured haGSCs cluster in comparison to human embryonic stem cells (hESCs) and human fibroblasts (hFibs) revealed that haGSCs showed a characteristic germ- and pluripotency-associated gene expression profile with some similarities to hESCs and with a significant distinction from somatic hFibs. Genome-wide comparisons with microarray analysis confirmed that different haGSC colonies exhibited gene expression heterogeneity with more or less pluripotency. The results of this study confirm that haGSCs are adult stem cells with a specific molecular gene expression profile in vitro, related but not identical to true pluripotent stem cells. Under ES-cell conditions haGSC colonies could be selected and maintained in a partial pluripotent state at the molecular level, which may be related to their cell plasticity and potential to differentiate into cells of all germ layers. PMID:26649052

  8. Granulocyte macrophage colony-stimulating factor and the intestinal innate immune cell homeostasis in Crohn's disease.

    PubMed

    Däbritz, Jan

    2014-03-01

    Current literature consolidates the view of Crohn's disease (CD) as a form of immunodeficiency highlighting dysregulation of intestinal innate immunity in the pathogenesis of CD. Intestinal macrophages derived from blood monocytes play a key role in sustaining the innate immune homeostasis in the intestine, suggesting that the monocyte/macrophage compartment might be an attractive therapeutic target for the management of CD. Granulocyte macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor that also promotes myeloid cell activation, proliferation, and differentiation. GM-CSF has a protective effect in human CD and mouse models of colitis. However, the role of GM-CSF in immune and inflammatory reactions in the intestine is not well defined. Beneficial effects exerted by GM-CSF during intestinal inflammation could relate to modulation of the mucosal barrier function in the intestine, including epithelial cell proliferation, survival, restitution, and immunomodulatory actions. The aim of this review is to summarize potential mechanistic roles of GM-CSF in intestinal innate immune cell homeostasis and to highlight its central role in maintenance of the intestinal immune barrier in the context of immunodeficiency in CD. PMID:24503766

  9. Conidial germination, infection structure formation, and early colony development of powdery mildew on poinsettia.

    PubMed

    Celio, G J; Hausbeck, M K

    1998-02-01

    ABSTRACT Powdery mildew disease on poinsettias (Euphorbia pulcherrima) growing in commercial greenhouses was first observed in the United States in 1990 and has become an economically significant problem for poinsettia growers in the Midwest and northern United States since 1992. The temporal development of infection structures produced by conidial germ tubes of the pathogen (Oidium sp.) and the effect of high temperature on their development were investigated using poinsettia leaf disks placed in humidity chambers. Observations were made using light microscopy and scanning electron microscopy. At 20 degrees C (85% relative humidity), conidia germinated and formed an appressorium within 6 h of inoculation. Germination over time followed a monomolecular curve (r(2) = 0.77, P cells. When the incubation temperature was 30 degrees C, conidial germination and development of secondary germ tubes and a haustorium were reduced. PMID:18944978

  10. Endoplasmic Reticulum Ca(2+) Handling and Apoptotic Resistance in Tumor-Derived Endothelial Colony Forming Cells.

    PubMed

    Poletto, Valentina; Dragoni, Silvia; Lim, Dmitry; Biggiogera, Marco; Aronica, Adele; Cinelli, Mariapia; De Luca, Antonio; Rosti, Vittorio; Porta, Camillo; Guerra, Germano; Moccia, Francesco

    2016-10-01

    Truly endothelial progenitor cells (EPCs) can be mobilized from bone marrow to support the vascular network of growing tumors, thereby sustaining the metastatic switch. Endothelial colony forming cells (ECFCs) are the only EPC subtype belonging to the endothelial phenotype and capable of incorporating within neovessels. The intracellular Ca(2+) machinery plays a key role in ECFC activation and is remodeled in renal cellular carcinoma-derived ECFCs (RCC-ECFCs). Particularly, RCC-ECFCs seems to undergo a drop in endoplasmic reticulum (ER) Ca(2+) concentration ([Ca(2+) ]ER ). This feature is remarkable when considering that inositol-1,4,5-trisphosphate (InsP3 )-dependent ER-to-mitochondria Ca(2+) transfer regulates the intrinsic apoptosis pathway. Herein, we sought to assess whether: (1) the [Ca(2+) ]ER and the InsP3 -induced ER-mitochondria Ca(2+) shuttle are reduced in RCC-ECFCs; and (2) the dysregulation of ER Ca(2+) handling leads to apoptosis resistance in tumor-derived cells. RCC-ECFCs displayed a reduction both in [Ca(2+) ]ER and in the InsP3 -dependent mitochondrial Ca(2+) uptake, while they expressed normal levels of Bcl-2 and Bak. The decrease in [Ca(2+) ]ER was associated to a remarkable ER expansion in RCC-ECFCs, which is a hallmark of ER stress, and did not depend on the remodeling of the Ca(2+) -transporting and the ER Ca(2+) -storing systems. As expected, RCC-ECFCs were less sensitive to rapamycin- and thapsigargin-induced apoptosis; however, buffering intracellular Ca(2+) levels with BAPTA dampened apoptosis in both cell types. Finally, store-operated Ca(2+) entry was seemingly uncoupled from the apoptotic machinery in RCC-ECFCs. Thus, the chronic underfilling of the ER Ca(2+) pool could confer a survival advantage to RCC-ECFCs and underpin RCC resistance to pharmacological treatment. J. Cell. Biochem. 117: 2260-2271, 2016. © 2016 Wiley Periodicals, Inc. PMID:26917354

  11. Inhibition of CD26 enzyme activity with pro-boropro stimulates rat granulocyte/macrophage colony formation and thymocyte proliferation in vitro.

    PubMed

    Bristol, L A; Bachovchin, W; Takács, L

    1995-06-15

    CD26 dipeptidyl peptidase (DPPIV) is involved in the regulation of proliferation of some hematopoietic and T-lineage cells. Here, we show that Pro-boropro a potent inhibitor of DPP activity has a costimulating effect in hematopoietic colony assays for macrophage and, to a lesser extent, for granulocyte colonies and has a stimulating effect in organ cultures of immature thymocytes. Based on these and other evidences, we propose that the mechanism by which CD26 regulates proliferation is associated with its DPP activity. PMID:7780144

  12. In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas.

    PubMed

    Tremblay, Jacob R; LeBon, Jeanne M; Luo, Angela; Quijano, Janine C; Wedeken, Lena; Jou, Kevin; Riggs, Arthur D; Tirrell, David A; Ku, H Teresa

    2016-01-01

    Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133(+)Sox9/EGFP(+) ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic q

  13. Cord blood-derived endothelial colony-forming cell function is disrupted in congenital diaphragmatic hernia.

    PubMed

    Fujinaga, Hideshi; Fujinaga, Hiroko; Watanabe, Nobuyuki; Kato, Tomoko; Tamano, Moe; Terao, Miho; Takada, Shuji; Ito, Yushi; Umezawa, Akihiro; Kuroda, Masahiko

    2016-06-01

    Vascular growth is necessary for normal lung development. Although endothelial progenitor cells (EPCs) play an important role in vascularization, little is known about EPC function in congenital diaphragmatic hernia (CDH), a severe neonatal condition that is associated with pulmonary hypoplasia. We hypothesized that the function of endothelial colony-forming cells (ECFCs), a type of EPC, is impaired in CDH. Cord blood (CB) was collected from full-term CDH patients and healthy controls. We assessed CB progenitor cell populations as well as plasma vascular endothelial growth factor (VEGF) and stromal cell-derived factor 1α (SDF1α) levels. CB ECFC clonogenicity; growth kinetics; migration; production of VEGF, SDF1α, and nitric oxide (NO); vasculogenic capacity; and mRNA expression of VEGF-A, fms-related tyrosine kinase 1 (FLT1), kinase insert domain receptor (KDR), nitric oxide synthase (NOS) 1-3, SDF1, and chemokine (C-X-C motif) receptor 4 (CXCR4) were also assessed. Compared with controls, CB ECFCs were decreased in CDH. CDH ECFCs had reduced potential for self-renewal, clonogenicity, proliferation, and migration. Their capacity for NO production was enhanced but their response to VEGF was blunted in CDH ECFCs. In vivo potential for de novo vasculogenesis was reduced in CDH ECFCs. There was no difference in CB plasma VEGF and SDF1α concentrations, VEGF and SDF1α production by ECFCs, and ECFC mRNA expression of VEGF-A, FLT1, KDR, NOS1-3, SDF1, and CXCR4 between CDH and control subjects. In conclusion, CB ECFC function is disrupted in CDH, but these changes may be caused by mechanisms other than alteration of VEGF-NO and SDF1-CXCR4 signaling. PMID:27130531

  14. Correlation between cell-cell contact formation and activation of protein kinase C in a human squamous cell carcinoma cell line.

    PubMed

    Nagao, S; Kitajima, Y; Nagata, K; Inoue, S; Yaoita, H; Nozawa, Y

    1989-02-01

    Formation of desmosomal cell-cell contact associated with reorganization of keratin intermediate filaments (KIFs) was observed when cultured cells of a cell line of human skin squamous cell carcinoma were transferred from low (0.07 mM) calcium to high (1.87 mM) calcium medium. At low calcium, cells were dispersed without desmosomal cell-cell contact and the KIFs were mostly concentrated around the nucleus. After 15 min of the transfer, cells contacted each other and formed small colonies and the KIFs initiated to show a radial arrangement. In addition to the cell-cell contact formation and rearrangement of KIFs, the transfer induced fourfold increase of particulate-associated protein kinase C (C-kinase) activity. When 12-O-tetradecanoyl phorbol-13-acetate (PMA), which specifically activates C-kinase, was added to the cells grown at low calcium medium, cell-cell contact formation and radial arrangement of KIF bundles almost identical to those induced by the transfer to high calcium medium were observed. These data suggest a correlation between an increase in C-kinase activity and formation of cell-cell contacts associated with rearrangements of KIFs. PMID:2465349

  15. Relationship between phase variation in colony morphology, intrastrain variation in cell wall physiology, and nasopharyngeal colonization by Streptococcus pneumoniae.

    PubMed Central

    Weiser, J N; Markiewicz, Z; Tuomanen, E I; Wani, J H

    1996-01-01

    Streptococcus pneumoniae undergoes phase variation in colony morphology, which has been implicated as a factor in the pathogenesis of pneumococcal disease. Phenotypic differences between opaque and transparent colony forms correlate with differences in rates of autolysis. This study examined whether differences in autolysis are caused by differences in expression of the major amidase, LytA, or the structure of its peptidoglycan substrate. No significant difference was detected by high-pressure liquid chromatography analysis of stem peptides released after treatment of purified peptidoglycan with amidase. Differences in the rate of digestion of purified cell walls, furthermore, did not correlate with susceptibility to autolysis. Lower levels of autolysis in opaque variants, however, was associated with decreased levels of immunodetectable LytA on colony immunoblots and Western blots (immunoblots). Diminished cell-surface-associated LytA in opaque variants was also demonstrated by whole-cell inhibition enzyme-linked immunosorbent assay. Since transparent variants have been shown both to colonize the nasopharynx more efficiently in an animal model and to express more surface-exposed LytA, it was determined whether LytA contributes to colonization in a neonatal rat model of pneumococcal carriage. Defined mutants in the lytA gene were used to show that there was no significant contribution by LytA to nasopharyngeal colonization in this model. Although the expression of LytA was shown to undergo phase variation in association with colony morphology, lytA mutants are still capable of phenotypic variation in colony morphology, which suggests that other factors are responsible for intrastrain differences which affect colonization. PMID:8675333

  16. Bioluminescence imaging of transplanted human endothelial colony-forming cells in an ischemic mouse model.

    PubMed

    Ding, Jie; Zhao, Zhen; Wang, Chao; Wang, Cong-Xiao; Li, Pei-Cheng; Qian, Cheng; Teng, Gao-Jun

    2016-07-01

    Ischemic strokes are devastating events responsible for high mortality and morbidity worldwide each year. Endothelial colony-forming cell (ECFC) therapy holds promise for stroke treatment; however, grafted ECFCs need to be monitored better understand their biological behavior in vivo, so as to evaluate their safety and successful delivery. The objectives of this study are to visualize the fate of infused human cord blood derived ECFCs via bioluminescence imaging (BLI) in an ischemic stroke mouse model and to determine the therapeutic effects of ECFC transplantation. ECFCs derived from human umbilical cord blood were infected with lentivirus carrying enhanced green fluorescent protein (eGFP) and firefly luciferase (Luc2) double fusion reporter gene. Labeled ECFCs were grafted into a photothrombotic ischemic stroke mouse model via intra-arterial injection though the left cardiac ventricle. The homing of infused cells and functional recovery of stroke mice were evaluated using BLI, neurological scoring, and immunohistochemistry. Significantly, BLI signals were highest in the brain on day 1 and decreased steadily until day 14. GFP-positive cells were also found surrounding infarct border zones in brain sections using immunohistochemical staining, suggesting that ECFCs properly homed to the ischemic brain tissue. Using a modified neurological severity score assay and histological analysis of brain slices with CD31 immunostaining in brain tissue, double cortin analysis, and the TdT-mediated dUTP nick end labeling (TUNEL) assay, we demonstrated functional restoration, improved angiogenesis, neurogenesis, and decreased apoptosis in ischemic mice after ECFC infusion. Collectively, our data support that ECFCs may be a promising therapeutic agent for stroke. PMID:27038754

  17. Role of Stem Cell Factor and Granulocyte-Colony Stimulating Factor in Remodeling during Liver Regeneration

    PubMed Central

    Meng, Fanyin; Francis, Heather; Glaser, Shannon; Han, Yuyan; DeMorrow, Sharon; Stokes, Allison; Staloch, Dustin; Venter, Julie; White, Melanie; Ueno, Yoshiyuki; Reid, Lola M.; Alpini, Gianfranco

    2011-01-01

    Functional pluripotent characteristics have been observed in specific subpopulations of hepatic cells that express some of the known cholangiocyte markers. Although evidence indicates that specific cytokines, granulocyte-macrophage colony stimulating factors (GM-CSF) and stem cell factor (SCF) may be candidate treatments for liver injury, the role of these cytokines in intrahepatic biliary epithelium remodeling is unknown. Thus, our aim was to characterize the specific cytokines that regulate the remodeling potentials of cholangiocytes after 70% partial hepatectomy (PH). The expression of the cytokines and their downstream signaling molecules was studied in rats after 70% PH by immunoblots, and in small and large murine cholangiocyte cultures (SMCCs and LMCCs) by immunocytochemistry and real-time PCR. There was a significant and stable increase in SCF and GM-CSF levels until 7 days after PH. Real-time PCR analysis revealed significant increases of key remodeling molecules, such as S100A4 and miR-181b after SCF plus GM-CSF administration in SMCCs. SMCCs produced significant amounts of soluble and bound SCF and GM-CSF in response to TGF-β. When SMCCs were incubated with TGF-β plus anti–SCF and GM-CSF antibodies, there was a significant decrease in S100A4 expression. Furthermore, treatment of SMCCs with SCF + GM-CSF significantly increased matrix metalloproteinases (MMP-2 and MMP-9) mRNA as well as miR-181b expression along with a reduction of metalloproteinase inhibitor 3 (TIMP-3). The levels of MMP-2, MMP-9 and miR-181b were also up-regulated in rat liver and isolated cholangiocytes after PH. CONCLUSION Our data suggest that altered expression of SCF and GM-CSF following PH can contribute to biliary remodeling (for example post-transplantation) by functional deregulation of activity of key signaling intermediates involved in cell expansion and multipotent differentiation. PMID:21932404

  18. Characterization of the Maize Stalk Rot Pathogens Fusarium subglutinans and F. temperatum and the Effect of Fungicides on Their Mycelial Growth and Colony Formation.

    PubMed

    Shin, Jong-Hwan; Han, Joon-Hee; Lee, Ju Kyong; Kim, Kyoung Su

    2014-12-01

    Maize is a socioeconomically important crop in many countries. Recently, a high incidence of stalk rot disease has been reported in several maize fields in Gangwon province. In this report, we show that maize stalk rot is associated with the fungal pathogens Fusarium subglutinans and F. temperatum. Since no fungicides are available to control these pathogens on maize plants, we selected six fungicides (tebuconazole, difenoconazole, fluquinconazole, azoxystrobin, prochloraz and kresoxim-methyl) and examined their effectiveness against the two pathogens. The in vitro antifungal effects of the six fungicides on mycelial growth and colony formation were investigated. Based on the inhibition of mycelial growth, the most toxic fungicide was tebuconazole with 50% effective concentrations (EC50) of <0.1 μg/ml and EC90 values of 0.9 μg/ml for both pathogens, while the least toxic fungicide was azoxystrobin with EC50 values of 0.7 and 0.5 μg/ml for F. subglutinans and F. temperatum, respectively, and EC90 values of >3,000 μg/ml for both pathogens. Based on the inhibition of colony formation by the two pathogens, kresoxim-methyl was the most toxic fungicide with complete inhibition of colony formation at concentrations of 0.1 and 0.01 μg/ml for F. subglutinans and F. temperatum, respectively, whereas azoxystrobin was the least toxic fungicide with complete inhibition of colony formation at concentrations >3,000 μg/ml for both pathogens. PMID:25506304

  19. Characterization of the Maize Stalk Rot Pathogens Fusarium subglutinans and F. temperatum and the Effect of Fungicides on Their Mycelial Growth and Colony Formation

    PubMed Central

    Shin, Jong-Hwan; Han, Joon-Hee; Lee, Ju Kyong; Kim, Kyoung Su

    2014-01-01

    Maize is a socioeconomically important crop in many countries. Recently, a high incidence of stalk rot disease has been reported in several maize fields in Gangwon province. In this report, we show that maize stalk rot is associated with the fungal pathogens Fusarium subglutinans and F. temperatum. Since no fungicides are available to control these pathogens on maize plants, we selected six fungicides (tebuconazole, difenoconazole, fluquinconazole, azoxystrobin, prochloraz and kresoxim-methyl) and examined their effectiveness against the two pathogens. The in vitro antifungal effects of the six fungicides on mycelial growth and colony formation were investigated. Based on the inhibition of mycelial growth, the most toxic fungicide was tebuconazole with 50% effective concentrations (EC50) of <0.1 μg/ml and EC90 values of 0.9 μg/ml for both pathogens, while the least toxic fungicide was azoxystrobin with EC50 values of 0.7 and 0.5 μg/ml for F. subglutinans and F. temperatum, respectively, and EC90 values of >3,000 μg/ml for both pathogens. Based on the inhibition of colony formation by the two pathogens, kresoxim-methyl was the most toxic fungicide with complete inhibition of colony formation at concentrations of 0.1 and 0.01 μg/ml for F. subglutinans and F. temperatum, respectively, whereas azoxystrobin was the least toxic fungicide with complete inhibition of colony formation at concentrations >3,000 μg/ml for both pathogens. PMID:25506304

  20. Expression of DDX27 contributes to colony-forming ability of gastric cancer cells and correlates with poor prognosis in gastric cancer

    PubMed Central

    Tsukamoto, Yoshiyuki; Fumoto, Shoichi; Noguchi, Tsuyoshi; Yanagihara, Kazuyoshi; Hirashita, Yuka; Nakada, Chisato; Hijiya, Naoki; Uchida, Tomohisa; Matsuura, Keiko; Hamanaka, Ryoji; Murakami, Kazunari; Seto, Masao; Inomata, Masafumi; Moriyama, Masatsugu

    2015-01-01

    Previously, we have reported that gain at chromosome 20q13 is the most common genomic copy number aberration in gastric cancer (GC) (29/30 cases), and that among the genes located in this region, we have identified DDX27, whose expression level shows the highest correlation with genomic copy number, as a candidate therapeutic target for GC. Here, we analyzed the clinicopathological significance of DDX27 using immunohistochemistry and studied its functions using knockdown assays. We found that DDX27 was frequently upregulated in GC tissues (98 of 140 cases, 70%), and significantly associated with venous invasion and liver metastasis. Furthermore, multivariate analysis of GC patients showed that high expression of DDX27 was independently associated with poorer prognosis. In functional assays, knockdown of DDX27 reduced the ability of GC cells to form colonies both on conventional plates and soft agar, but had little effect on their invasiveness. We also found that knockdown of DDX27 reduced the viability of GC cells through inhibition of cell cycle progression independently of apoptosis. Interestingly, DDX27 depletion induced accumulation of TP53 in a TP53 wild-type cell line, AGS, but not in a TP53-deleted cell line, 44As3, although DDX27 knockdown commonly reduced the viability of both, indicating the TP53-dependent and independent cell cycle control of DDX27. Thus, our results suggest that expression of DDX27 contributes to colony formation by GC cells through cell cycle control and may be a potential therapeutic target for GC patients with chromosome gain at 20q13. PMID:26693055

  1. Granulocyte/Macrophage Colony-stimulating Factor-dependent Dendritic Cells Restrain Lean Adipose Tissue Expansion.

    PubMed

    Pamir, Nathalie; Liu, Ning-Chun; Irwin, Angela; Becker, Lev; Peng, YuFeng; Ronsein, Graziella E; Bornfeldt, Karin E; Duffield, Jeremy S; Heinecke, Jay W

    2015-06-01

    The physiological roles of macrophages and dendritic cells (DCs) in lean white adipose tissue homeostasis have received little attention. Because DCs are generated from bone marrow progenitors in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF), we used GM-CSF-deficient (Csf2(-/-)) mice fed a low fat diet to test the hypothesis that adipose tissue DCs regulate the development of adipose tissue. At 4 weeks of age, Csf2(-/-) mice had 75% fewer CD45(+)Cd11b(+)Cd11c(+)MHCII(+) F4/80(-) DCs in white adipose tissue than did wild-type controls. Furthermore, the Csf2(-/-) mice showed a 30% increase in whole body adiposity, which persisted to adulthood. Adipocytes from Csf2(-/-) mice were 50% larger by volume and contained higher levels of adipogenesis gene transcripts, indicating enhanced adipocyte differentiation. In contrast, adipogenesis/adipocyte lipid accumulation was inhibited when preadipocytes were co-cultured with CD45(+)Cd11b(+)Cd11c(+)MHCII(+)F4/80(-) DCs. Medium conditioned by DCs, but not by macrophages, also inhibited adipocyte lipid accumulation. Proteomic analysis revealed that matrix metalloproteinase 12 and fibronectin 1 were greatly enriched in the medium conditioned by DCs compared with that conditioned by macrophages. Silencing fibronectin or genetic deletion of matrix metalloproteinase 12 in DCs partially reversed the inhibition of adipocyte lipid accumulation. Our observations indicate that DCs residing in adipose tissue play a critical role in suppressing normal adipose tissue expansion. PMID:25931125

  2. Ionizing Radiation Induces Macrophage Foam Cell Formation and Aggregation Through JNK-Dependent Activation of CD36 Scavenger Receptors

    SciTech Connect

    Katayama, Ikuo; Hotokezaka, Yuka; Matsuyama, Toshifumi; Sumi, Tadateru; Nakamura, Takashi

    2008-03-01

    Purpose: Irradiated arteries of cancer patients can be associated with atherosclerosis-like lesions containing cholesterol-laden macrophages (foam cells). Endothelial cell damage by irradiation does not completely explain the foam cell formation. We investigated the possible underlying mechanisms for ionizing radiation (IR)-induced foam cell formation. Methods and Materials: Human peripheral blood monocytes were activated by macrophage colony-stimulating factor and then treated with varying doses of IR in vitro in the absence of endothelial cells. Scavenger receptor expression and foam cell formation of IR-treated macrophages were investigated in the presence or absence of oxidized low-density lipoprotein. We also assessed the importance of mitogen-activated protein kinase activity in the macrophage colony-stimulating factor-activated human monocytes (macrophages) for the foam cell formation. Results: We found that IR treatment of macrophage colony-stimulating factor-activated human peripheral blood monocytes resulted in the enhanced expression of CD36 scavenger receptors and that cholesterol accumulated in the irradiated macrophages with resultant foam cell formation in the presence of oxidized low-density lipoprotein. Furthermore, when cultured on collagen gels, human macrophages formed large foam cell aggregates in response to IR. Antibodies against CD36 inhibited the IR-induced foam cell formation and aggregation, indicating that the IR-induced foam cell formation and the subsequent aggregation are dependent on functional CD36. In addition, we found that IR of human macrophages resulted in c-Jun N-terminal kinase activation and that c-Jun N-terminal kinase inhibition suppressed IR-induced CD36 expression and the subsequent foam cell formation and aggregation. Conclusion: Taken together, these results suggest that IR-induced foam cell formation is mediated by c-Jun N-terminal kinase-dependent CD36 activation.

  3. Granulocyte-Macrophage Colony-Stimulating Factor Priming plus Papillomavirus E6 DNA Vaccination: Effects on Papilloma Formation and Regression in the Cottontail Rabbit Papillomavirus-Rabbit Model

    PubMed Central

    Leachman, Sancy A.; Tigelaar, Robert E.; Shlyankevich, Mark; Slade, Martin D.; Irwin, Michele; Chang, Ed; Wu, T. C.; Xiao, Wei; Pazhani, Sundaram; Zelterman, Daniel; Brandsma, Janet L.

    2000-01-01

    A cottontail rabbit papillomavirus (CRPV) E6 DNA vaccine that induces significant protection against CRPV challenge was used in a superior vaccination regimen in which the cutaneous sites of vaccination were primed with an expression vector encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine that induces differentiation and local recruitment of professional antigen-presenting cells. This treatment induced a massive influx of major histocompatibility complex class II-positive cells. In a vaccination-challenge experiment, rabbit groups were treated by E6 DNA vaccination, GM-CSF DNA inoculation, or a combination of both treatments. After two immunizations, rabbits were challenged with CRPV at low, moderate, and high stringencies and monitored for papilloma formation. As expected, all clinical outcomes were monotonically related to the stringency of the viral challenge. The results demonstrate that GM-CSF priming greatly augmented the effects of CRPV E6 vaccination. First, challenge sites in control rabbits (at the moderate challenge stringency) had a 0% probability of remaining disease free, versus a 50% probability in E6-vaccinated rabbits, and whereas GM-CSF alone had no effect, the interaction between GM-CSF priming and E6 vaccination increased disease-free survival to 67%. Second, the incubation period before papilloma onset was lengthened by E6 DNA vaccination alone or to some extent by GM-CSF DNA inoculation alone, and the combination of treatments induced additive effects. Third, the rate of papilloma growth was reduced by E6 vaccination and, to a lesser extent, by GM-CSF treatment. In addition, the interaction between the E6 and GM-CSF treatments was synergistic and yielded more than a 99% reduction in papilloma volume. Finally, regression occurred among the papillomas that formed in rabbits treated with the E6 vaccine and/or with GM-CSF, with the highest regression frequency occurring in rabbits that received the combination

  4. Cationic star copolymers based on β-cyclodextrins for efficient gene delivery to mouse embryonic stem cell colonies.

    PubMed

    Loh, Xian Jun; Wu, Yun-Long

    2015-07-11

    A cationic star copolymer with a β-cyclodextrin core was developed for nonviral gene transfer to mouse embryonic stem cells (mESCs). The copolymer comprises poly(2-dimethyl aminoethyl methacrylate) as the cationic component and poly(2-hydroxyethyl methacrylate) as the non-toxic stealth component. These materials have very low toxicity and show highly efficient transfection to mESC colonies. PMID:26040469

  5. Order and instabilities in dense bacterial colonies

    NASA Astrophysics Data System (ADS)

    Tsimring, Lev

    2012-02-01

    The structure of cell colonies is governed by the interplay of many physical and biological factors, ranging from properties of surrounding media to cell-cell communication and gene expression in individual cells. The biomechanical interactions arising from the growth and division of individual cells in confined environments are ubiquitous, yet little work has focused on this fundamental aspect of colony formation. By combining experimental observations of growing monolayers of non-motile strain of bacteria Escherichia coli in a shallow microfluidic chemostat with discrete-element simulations and continuous theory, we demonstrate that expansion of a dense colony leads to rapid orientational alignment of rod-like cells. However, in larger colonies, anisotropic compression may lead to buckling instability which breaks perfect nematic order. Furthermore, we found that in shallow cavities feedback between cell growth and mobility in a confined environment leads to a novel cell streaming instability. Joint work with W. Mather, D. Volfson, O. Mondrag'on-Palomino, T. Danino, S. Cookson, and J. Hasty (UCSD) and D. Boyer, S. Orozco-Fuentes (UNAM, Mexico).

  6. Divergent branches of mitochondrial signaling regulate specific genes and the viability of specialized cell types of differentiated yeast colonies

    PubMed Central

    Rešetárová, Stanislava; Kučerová, Helena; Hlaváček, Otakar; Váchová, Libuše; Palková, Zdena

    2016-01-01

    Mitochondrial retrograde signaling mediates communication from altered mitochondria to the nucleus and is involved in many normal and pathophysiological changes, including cell metabolic reprogramming linked to cancer development and progression in mammals. The major mitochondrial retrograde pathway described in yeast includes three activators, Rtg1p, Rtg2p and Rtg3p, and repressors, Mks1p and Bmh1p/Bmh2p. Using differentiated yeast colonies, we show that Mks1p-Rtg pathway regulation is complex and includes three branches that divergently regulate the properties and fate of three specifically localized cell subpopulations via signals from differently altered mitochondria. The newly identified RTG pathway-regulated genes ATO1/ATO2 are expressed in colonial upper (U) cells, the cells with active TORC1 that metabolically resemble tumor cells, while CIT2 is a typical target induced in one subpopulation of starving lower (L) cells. The viability of the second L cell subpopulation is strictly dependent on RTG signaling. Additional co-activators of Rtg1p-Rtg3p specific to particular gene targets of each branch are required to regulate cell differentiation. PMID:26992228

  7. Radiation promotes invasiveness of non-small-cell lung cancer cells through granulocyte-colony-stimulating factor.

    PubMed

    Cui, Y-H; Suh, Y; Lee, H-J; Yoo, K-C; Uddin, N; Jeong, Y-J; Lee, J-S; Hwang, S-G; Nam, S-Y; Kim, M-J; Lee, S-J

    2015-10-16

    Despite ionizing radiation (IR) is being widely used as a standard treatment for lung cancer, many evidences suggest that IR paradoxically promotes cancer malignancy. However, its molecular mechanisms underlying radiation-induced cancer progression remain obscure. Here, we report that exposure to fractionated radiation (2 Gy per day for 3 days) induces the secretion of granulocyte-colony-stimulating factor (G-CSF) that has been commonly used in cancer therapies to ameliorate neutropenia. Intriguingly, radiation-induced G-CSF promoted the migratory and invasive properties by triggering the epithelial-mesenchymal cell transition (EMT) in non-small-cell lung cancer cells (NSCLCs). By irradiation, G-CSF was upregulated transcriptionally by β-catenin/TCF4 complex that binds to the promoter region of G-CSF as a transcription factor. Importantly, irradiation increased the stability of β-catenin through the activation of PI3K/AKT (phosphatidylinositol 3-kinase/AKT), thereby upregulating the expression of G-CSF. Radiation-induced G-CSF is recognized by G-CSFR and transduced its intracellular signaling JAK/STAT3 (Janus kinase/signal transducers and activators of transcription), thereby triggering EMT program in NSCLCs. Taken together, our findings suggest that the application of G-CSF in cancer therapies to ameliorate neutropenia should be reconsidered owing to its effect on cancer progression, and G-CSF could be a novel therapeutic target to mitigate the harmful effect of radiotherapy for the treatment of NSCLC. PMID:25639867

  8. Granulocyte/Macrophage Colony-stimulating Factor-dependent Dendritic Cells Restrain Lean Adipose Tissue Expansion*

    PubMed Central

    Pamir, Nathalie; Liu, Ning-Chun; Irwin, Angela; Becker, Lev; Peng, YuFeng; Ronsein, Graziella E.; Bornfeldt, Karin E.; Duffield, Jeremy S.; Heinecke, Jay W.

    2015-01-01

    The physiological roles of macrophages and dendritic cells (DCs) in lean white adipose tissue homeostasis have received little attention. Because DCs are generated from bone marrow progenitors in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF), we used GM-CSF-deficient (Csf2−/−) mice fed a low fat diet to test the hypothesis that adipose tissue DCs regulate the development of adipose tissue. At 4 weeks of age, Csf2−/− mice had 75% fewer CD45+Cd11b+Cd11c+MHCII+ F4/80− DCs in white adipose tissue than did wild-type controls. Furthermore, the Csf2−/− mice showed a 30% increase in whole body adiposity, which persisted to adulthood. Adipocytes from Csf2−/− mice were 50% larger by volume and contained higher levels of adipogenesis gene transcripts, indicating enhanced adipocyte differentiation. In contrast, adipogenesis/adipocyte lipid accumulation was inhibited when preadipocytes were co-cultured with CD45+Cd11b+Cd11c+MHCII+F4/80− DCs. Medium conditioned by DCs, but not by macrophages, also inhibited adipocyte lipid accumulation. Proteomic analysis revealed that matrix metalloproteinase 12 and fibronectin 1 were greatly enriched in the medium conditioned by DCs compared with that conditioned by macrophages. Silencing fibronectin or genetic deletion of matrix metalloproteinase 12 in DCs partially reversed the inhibition of adipocyte lipid accumulation. Our observations indicate that DCs residing in adipose tissue play a critical role in suppressing normal adipose tissue expansion. PMID:25931125

  9. Dynamics of Superoxide Production and Decay in Natural Trichodesmium Colonies from the Sargasso Sea: Implications for Cell Signaling

    NASA Astrophysics Data System (ADS)

    Hansel, C. M.; Buchwald, C.; Diaz, J. M.; Dyhrman, S.; Van Mooy, B. A. S.

    2014-12-01

    Reactive oxygen species (ROS) are key players in the biogeochemistry of the ocean, where they serve a critical role in the cycling of carbon and metals. Research in the past decade has introduced phytoplankton and, most recently, heterotrophic bacteria as significant sources of ROS, including superoxide, within both photic and aphotic regions of the ocean. ROS are both beneficial and detrimental to life. For instance, superoxide is a vital inter- and intra-cellular signaling molecule, yet at high concentrations it induces lipid peroxidation and initiates programmed cell death (PCD). In fact, superoxide has been implicated in PCD in the nitrogen-fixing diazotroph Trichodesmium, presumably leading to the demise of blooms within oligotrophic marine systems. Here, we explore the rates of superoxide production and decay by natural Trichodesmium populations obtained from various surface waters in the Sargasso Sea. We investigate also the role of light and colony density and morphology (puff v. raft) on superoxide fluxes. We find that Trichodesmium colonies produce extracellular superoxide at extremely high rates in the dark that are on par with those of the toxic raphidophyte Chattonella. The rates of superoxide production, however, rapidly decline with increasing cell density pointing to a role for superoxide in cell signaling in these organisms. We also find extremely rapid extracellular superoxide degradation by Trichodesmium. Together, this likely reflects a need for these organisms to maintain ROS at levels that will support signaling but below the threshold level that triggers PCD or oxidative damage. We also show differences in the effect of light on superoxide fluxes as a function of Trichodesmium colony morphology, suggesting differences in either colony physiology or associated bacterial symbionts. These findings point to complex physiological, ecological, and physical influences on ROS dynamics in phytoplankton that require further exploration.

  10. Interleukin 3 perfusion in W/Wv mice allows the development of macroscopic hematopoietic spleen colonies and restores cutaneous mast cell number

    SciTech Connect

    Ody, C.; Kindler, V.; Vassalli, P. )

    1990-07-01

    The genetically anemic W/Wv mice are characterized by the inability of their bone marrow cells to form macroscopic pluripotent hematopoietic colonies in the spleen of irradiated recipients upon transfer (colony-forming units). Furthermore, they almost totally lack mast cells, notably in the skin. In the present study, we have tested the effect of recombinant murine interleukin 3 (rmIL-3) on W/Wv mice hematopoiesis. Transfer of W/Wv bone marrow cells into lethally irradiated recipients perfused with rmIL-3 is followed by the appearance of macroscopic spleen colonies. Moreover, perfusion of rmIL-3 in W/Wv mice: (a) restores almost normal total numbers of hematopoietic precursors (colony-forming cells), but without modification of anemia; and (b) leads to the appearance of a normal number of mastocytes in the skin.

  11. Overexpression of granulocyte-macrophage colony-stimulating factor induces pulmonary granulation tissue formation and fibrosis by induction of transforming growth factor-beta 1 and myofibroblast accumulation.

    PubMed Central

    Xing, Z.; Tremblay, G. M.; Sime, P. J.; Gauldie, J.

    1997-01-01

    We have previously reported that transfer to rat lung of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene leads to high expression of GM-CSF between days 1 and 4 and granulation tissue formation followed by an irreversible fibrotic response starting from day 12 onward. In the current study, we investigated the underlying mechanisms. We found that GM-CSF overexpression did not enhance production of tumor necrosis factor-alpha in a significant manner at any time after GM-CSF gene transfer. However, the content of transforming growth factor-beta 1 in bronchoalveolar lavage fluid was markedly induced at day 4 and appeared to be maximal around day 7 and remained high at day 12. Macrophages purified from bronchoalveolar lavage fluid 7 days after GM-CSF gene transfer spontaneously released significant quantities of transforming growth factor-beta 1 protein in vitro. After peak transforming growth factor-beta 1 production was the emergence of alpha-smooth muscle actin-rich myofibroblasts. Accumulation of these cells was most prominent at day 12 within the granulation tissues and they were still present in fibrotic areas between days 12 and 24 and diminished markedly afterward. Thus, we provide the first in vivo evidence that tumor necrosis factor-alpha may be dissociated from participation in a fibrotic process in the lung and GM-CSF may play a more direct role in pulmonary fibrogenesis at least in part through its capability to induce transforming growth factor-beta 1 in macrophages and the subsequent emergence of myofibroblast phenotypes. This GM-CSF transgene lung model is useful for a stepwise dissection of both cellular and molecular events involved in pulmonary fibrosis. Images Figure 2 Figure 5 Figure 6 PMID:9006322

  12. Colonial America.

    ERIC Educational Resources Information Center

    Web Feet K-8, 2001

    2001-01-01

    Presents resources for grades K-8, on the subject of Colonial America. Describes Web sites; CD-ROMs and software; videos; books; audios; magazines; and professional resources. Includes two articles, "Native Americans in the Colonies," and "The Golden Age of Pirates," which also highlight resources. Presents a Web activity focusing on daily life in…

  13. Cooperatively Generated Stresslet Flows Supply Fresh Fluid to Multicellular Choanoflagellate Colonies

    NASA Astrophysics Data System (ADS)

    Roper, Marcus; Dayel, Mark J.; Pepper, Rachel E.; Koehl, M. A. R.

    2013-05-01

    The flagellated protozoan Salpingoeca rosetta is one of the closest relatives of multicellular animals. Unicellular S. rosetta can be induced to form multicellular colonies, but colonies swim more slowly than individual cells so the advantages conferred by colony formation are uncertain. Here we use theoretical models to show that hydrodynamic cooperation between cells can increase the fluid supply to the colony, an important predictor of feeding rate. Our results suggest that hydrodynamic benefits may have been an important selective factor in the evolution of early multicellular animals.

  14. Colony Size of Phaeocystis Antarctica (Prymnesiophyceae) as Influenced by Zooplankton Grazers

    EPA Science Inventory

    The haptophyte Phaeocystis antarctica is a dominant phytoplankton species in the Ross Sea, Antarctica, and exists as solitary cells and mucilaginous colonies that differ by several orders of magnitude in size. Recent studies with P. globosa suggested that colony formation and enl...

  15. Dynamic scaling analysis of two-dimensional cell colony fronts in a gel medium: A biological system approaching a quenched Kardar-Parisi-Zhang universality

    NASA Astrophysics Data System (ADS)

    Huergo, M. A. C.; Muzzio, N. E.; Pasquale, M. A.; González, P. H. Pedro; Bolzán, A. E.; Arvia, A. J.

    2014-08-01

    The interfacial two-dimensional spreading dynamics of quasilinear Vero cell colony fronts in methylcellulose (MC)-containing culture medium, under a constant average front displacement velocity regime, was investigated. Under comparable experimental conditions, the average colony front displacement velocity becomes lower than that reported for a standard culture medium. Initially, the presence of MC in the medium hinders both the colony spreading, due to a gradual change in the average size and shape of cells and their distribution in the colony, and the cell motility in the gelled medium. Furthermore, at longer culture times enlarged cells appear at random in the border region of the colony. These cells behave as obstacles (pinning sites) for the displacement of smaller cells towards the colony front. The dynamic scaling analysis of rough fronts yields the set of exponents α =0.63±0.04,β =0.75±0.05, and z =0.84±0.05, which is close to that expected for a quenched Kardar-Parisi-Zhang model.

  16. Dynamic scaling analysis of two-dimensional cell colony fronts in a gel medium: a biological system approaching a quenched Kardar-Parisi-Zhang universality.

    PubMed

    Huergo, M A C; Muzzio, N E; Pasquale, M A; Pedro González, P H; Bolzán, A E; Arvia, A J

    2014-08-01

    The interfacial two-dimensional spreading dynamics of quasilinear Vero cell colony fronts in methylcellulose (MC)-containing culture medium, under a constant average front displacement velocity regime, was investigated. Under comparable experimental conditions, the average colony front displacement velocity becomes lower than that reported for a standard culture medium. Initially, the presence of MC in the medium hinders both the colony spreading, due to a gradual change in the average size and shape of cells and their distribution in the colony, and the cell motility in the gelled medium. Furthermore, at longer culture times enlarged cells appear at random in the border region of the colony. These cells behave as obstacles (pinning sites) for the displacement of smaller cells towards the colony front. The dynamic scaling analysis of rough fronts yields the set of exponents α=0.63±0.04,β=0.75±0.05, and z=0.84±0.05, which is close to that expected for a quenched Kardar-Parisi-Zhang model. PMID:25215757

  17. Growing Yeast into Cylindrical Colonies

    PubMed Central

    Vulin, Clément; Di Meglio, Jean-Marc; Lindner, Ariel B.; Daerr, Adrian; Murray, Andrew; Hersen, Pascal

    2014-01-01

    Microorganisms often form complex multicellular assemblies such as biofilms and colonies. Understanding the interplay between assembly expansion, metabolic yield, and nutrient diffusion within a freely growing colony remains a challenge. Most available data on microorganisms are from planktonic cultures, due to the lack of experimental tools to control the growth of multicellular assemblies. Here, we propose a method to constrain the growth of yeast colonies into simple geometric shapes such as cylinders. To this end, we designed a simple, versatile culture system to control the location of nutrient delivery below a growing colony. Under such culture conditions, yeast colonies grow vertically and only at the locations where nutrients are delivered. Colonies increase in height at a steady growth rate that is inversely proportional to the cylinder radius. We show that the vertical growth rate of cylindrical colonies is not defined by the single-cell division rate, but rather by the colony metabolic yield. This contrasts with cells in liquid culture, in which the single-cell division rate is the only parameter that defines the population growth rate. This method also provides a direct, simple method to estimate the metabolic yield of a colony. Our study further demonstrates the importance of the shape of colonies on setting their expansion. We anticipate that our approach will be a starting point for elaborate studies of the population dynamics, evolution, and ecology of microbial colonies in complex landscapes. PMID:24853750

  18. Information use in colonial living.

    PubMed

    Evans, Julian C; Votier, Stephen C; Dall, Sasha R X

    2016-08-01

    Despite the fact that many animals live in groups, there is still no clear consensus about the ecological or evolutionary mechanisms underlying colonial living. Recently, research has suggested that colonies may be important as sources of social information. The ready availability of information from conspecifics allows animals to make better decisions about avoiding predators, reducing brood parasitism, migratory phenology, mate choice, habitat choice and foraging. These choices can play a large part in the development and maintenance of colonies. Here we review the types of information provided by colonial animals and examine the different ways in which decision-making in colonies can be enhanced by social information. We discuss what roles information might take in the evolution, formation and maintenance of colonies. In the process, we illustrate that information use permeates all aspects of colonial living. PMID:25882618

  19. Direct formate fuel cells: A review

    NASA Astrophysics Data System (ADS)

    An, L.; Chen, R.

    2016-07-01

    Direct formate fuel cells (DFFC), which convert the chemical energy stored in formate directly into electricity, are recently attracting more attention, primarily because of the use of the carbon-neutral fuel and the low-cost electrocatalytic and membrane materials. As an emerging energy technology, the DFFC has made a rapid progress in recent years (currently, the state-of-the-art power density is 591 mW cm-2 at 60 °C). This article provides a review of past research on the development of this type of fuel cell, including the working principle, mechanisms and materials of the electrocatalytic oxidation of formate, singe-cell designs and performance, as well as innovative system designs. In addition, future perspectives with regard to the development of this fuel cell system are also highlighted.

  20. Human endothelial colony-forming cells serve as trophic mediators for mesenchymal stem cell engraftment via paracrine signaling

    PubMed Central

    Lin, Ruei-Zeng; Moreno-Luna, Rafael; Li, Dan; Jaminet, Shou-Ching; Greene, Arin K.; Melero-Martin, Juan M.

    2014-01-01

    Endothelial colony-forming cells (ECFCs) are endothelial precursors that circulate in peripheral blood. Studies have demonstrated that human ECFCs have robust vasculogenic properties. However, whether ECFCs can exert trophic functions in support of specific stem cells in vivo remains largely unknown. Here, we sought to determine whether human ECFCs can function as paracrine mediators before the establishment of blood perfusion. We used two xenograft models of human mesenchymal stem cell (MSC) transplantation and studied how the presence of ECFCs modulates MSC engraftment and regenerative capacity in vivo. Human MSCs were isolated from white adipose tissue and bone marrow aspirates and were s.c. implanted into immunodeficient mice in the presence or absence of cord blood-derived ECFCs. MSC engraftment was regulated by ECFC-derived paracrine factors via platelet-derived growth factor BB (PDGF-BB)/platelet-derived growth factor receptor (PDGFR)-β signaling. Cotransplanting ECFCs significantly enhanced MSC engraftment by reducing early apoptosis and preserving stemness-related properties of PDGFR-β+ MSCs, including the ability to repopulate secondary grafts. MSC engraftment was negligible in the absence of ECFCs and completely impaired in the presence of Tyrphostin AG1296, an inhibitor of PDGFR kinase. Additionally, transplanted MSCs displayed fate-restricted potential in vivo, with adipose tissue-derived and bone marrow-derived MSCs contributing exclusive differentiation along adipogenic and osteogenic lineages, respectively. This work demonstrates that blood-derived ECFCs can serve as paracrine mediators and regulate the regenerative potential of MSCs via PDGF-BB/PDGFR-β signaling. Our data suggest the systematic use of ECFCs as a means to improve MSC transplantation. PMID:24982174

  1. Diatom Cell Size, Coloniality and Motility: Trade-Offs between Temperature, Salinity and Nutrient Supply with Climate Change

    PubMed Central

    Svensson, Filip; Norberg, Jon; Snoeijs, Pauline

    2014-01-01

    Reduction in body size has been proposed as a universal response of organisms, both to warming and to decreased salinity. However, it is still controversial if size reduction is caused by temperature or salinity on their own, or if other factors interfere as well. We used natural benthic diatom communities to explore how “body size” (cells and colonies) and motility change along temperature (2–26°C) and salinity (0.5–7.8) gradients in the brackish Baltic Sea. Fourth-corner analysis confirmed that small cell and colony sizes were associated with high temperature in summer. Average community cell volume decreased linearly with 2.2% per °C. However, cells were larger with artificial warming when nutrient concentrations were high in the cold season. Average community cell volume increased by 5.2% per °C of artificial warming from 0 to 8.5°C and simultaneously there was a selection for motility, which probably helped to optimize growth rates by trade-offs between nutrient supply and irradiation. Along the Baltic Sea salinity gradient cell size decreased with decreasing salinity, apparently mediated by nutrient stoichiometry. Altogether, our results suggest that climate change in this century may polarize seasonality by creating two new niches, with elevated temperature at high nutrient concentrations in the cold season (increasing cell size) and elevated temperature at low nutrient concentrations in the warm season (decreasing cell size). Higher temperature in summer and lower salinity by increased land-runoff are expected to decrease the average cell size of primary producers, which is likely to affect the transfer of energy to higher trophic levels. PMID:25279720

  2. Bone marrow cell mobilization by the systemic use of granulocyte colony-stimulating factor (GCSF) improves wound bed preparation.

    PubMed

    Iwamoto, Satori; Lin, Xiaofeng; Ramirez, Ron; Carson, Polly; Fiore, David; Goodrich, Jane; Yufit, Tatyana; Falanga, Vincent

    2013-12-01

    Innovative approaches are needed to accelerate the healing of human chronic wounds not responding to conventional therapies. An evolving and promising treatment is the use of stem cells. Our group has previously described the use of expanded (in vitro) autologous stem cells aspirated from human bone marrow and applied topically in a fibrin spray to human acute and chronic wounds. More recently, we have sought ways to mobilize stem cells directly from the bone marrow, without in vitro expansion. In this report, we show that systemic injections of granulocyte colony-stimulating factor (GCSF) can mobilize stem cells from bone marrow into the peripheral blood and then to the wound site. Our objectives were to optimize parameters for this method by using mouse models and proof of principle in a human chronic wound situation. Mice were injected for 5 days with 2 different formulations of GCSF and compared to control saline. To monitor stem cell mobilization, flow cytometric measurements of Sca-1 and c-Kit and colony-forming cell assays were performed. Full-thickness tail wounds in mice were created and monitored for healing, and polyvinyl alcohol sponges were implanted dorsally to assess collagen accumulation. To determine bone marrow stem cell homing to the wound site, chimeric mice transplanted with Green Fluorescent Protein bone marrow cells were scanned by live imaging. Additionally, as proof of principle, we tested the systemic GCSF approach in a patient with a nonhealing venous ulcer. Our findings lay the ground work and indicate that the systemic administration of GCSF is effective in mobilizing bone marrow stem cells into the peripheral blood and to the wound site. These findings are associated with an increased accumulation of collagen and promising results in terms of wound bed preparation and healing. PMID:24275756

  3. Mast cells mediate malignant pleural effusion formation

    PubMed Central

    Giannou, Anastasios D.; Marazioti, Antonia; Spella, Magda; Kanellakis, Nikolaos I.; Apostolopoulou, Hara; Psallidas, Ioannis; Prijovich, Zeljko M.; Vreka, Malamati; Zazara, Dimitra E.; Lilis, Ioannis; Papaleonidopoulos, Vassilios; Kairi, Chrysoula A.; Patmanidi, Alexandra L.; Giopanou, Ioanna; Spiropoulou, Nikolitsa; Harokopos, Vaggelis; Aidinis, Vassilis; Spyratos, Dionisios; Teliousi, Stamatia; Papadaki, Helen; Taraviras, Stavros; Snyder, Linda A.; Eickelberg, Oliver; Kardamakis, Dimitrios; Iwakura, Yoichiro; Feyerabend, Thorsten B.; Rodewald, Hans-Reimer; Kalomenidis, Ioannis; Blackwell, Timothy S.; Agalioti, Theodora; Stathopoulos, Georgios T.

    2015-01-01

    Mast cells (MCs) have been identified in various tumors; however, the role of these cells in tumorigenesis remains controversial. Here, we quantified MCs in human and murine malignant pleural effusions (MPEs) and evaluated the fate and function of these cells in MPE development. Evaluation of murine MPE-competent lung and colon adenocarcinomas revealed that these tumors actively attract and subsequently degranulate MCs in the pleural space by elaborating CCL2 and osteopontin. MCs were required for effusion development, as MPEs did not form in mice lacking MCs, and pleural infusion of MCs with MPE-incompetent cells promoted MPE formation. Once homed to the pleural space, MCs released tryptase AB1 and IL-1β, which in turn induced pleural vasculature leakiness and triggered NF-κB activation in pleural tumor cells, thereby fostering pleural fluid accumulation and tumor growth. Evaluation of human effusions revealed that MCs are elevated in MPEs compared with benign effusions. Moreover, MC abundance correlated with MPE formation in a human cancer cell–induced effusion model. Treatment of mice with the c-KIT inhibitor imatinib mesylate limited effusion precipitation by mouse and human adenocarcinoma cells. Together, the results of this study indicate that MCs are required for MPE formation and suggest that MC-dependent effusion formation is therapeutically addressable. PMID:25915587

  4. Influence of receptor activator of nuclear factor (NF)-kappaB ligand (RANKL), macrophage-colony stimulating factor (M-CSF) and fetal calf serum on human osteoclast formation and activity.

    PubMed

    Kreja, Ludwika; Liedert, Astrid; Schmidt, Carla; Claes, Lutz; Ignatius, Anita

    2007-10-01

    Human osteoclast (OC) formation and activity was studied in cultures of peripheral blood mononuclear cells (PBMNC) from six healthy donors after stimulation with fetal calf serum (FCS), under the influence of the receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) and the macrophage-colony stimulating factor (M-CSF). The results showed that selected FCS could stimulate OC formation without any medium supplementation with osteoclastogenic factors. The OC formation, investigated by quantification of multinucleated tartrate-resistant acid phosphatase-positive cells (TRAP+ cells), and the sensitivity of OC progenitors to RANKL and M-CSF, varied widely between individual donors. The OC resorption activity, measured in the "pit-assay" on dentine, was strictly dependent on the presence of RANKL and M-CSF in the medium and was also donor dependent. The considerable donor variability should be considered in culture studies investigating, e.g. the interactions of OC with biomaterials or the influence of cytokines, growth factors and drugs on osteoclastogenesis. PMID:18161075

  5. Hormonal regulation of secondary cell wall formation.

    PubMed

    Didi, Vojtěch; Jackson, Phil; Hejátko, Jan

    2015-08-01

    Secondary cell walls (SCWs) have critical functional importance but also constitute a high proportion of the plant biomass and have high application potential. This is true mainly for the lignocellulosic constituents of the SCWs in xylem vessels and fibres, which form a structured layer between the plasma membrane and the primary cell wall (PCW). Specific patterning of the SCW thickenings contributes to the mechanical properties of the different xylem cell types, providing the plant with mechanical support and facilitating the transport of solutes via vessels. In the last decade, our knowledge of the basic molecular mechanisms controlling SCW formation has increased substantially. Several members of the multi-layered regulatory cascade participating in the initiation and transcriptional regulation of SCW formation have been described, and the first cellular components determining the pattern of SCW at the subcellular resolution are being uncovered. The essential regulatory role of phytohormones in xylem development is well known and the molecular mechanisms that link hormonal signals to SCW formation are emerging. Here, we review recent knowledge about the role of individual plant hormones and hormonal crosstalk in the control over the regulatory cascades guiding SCW formation and patterning. Based on the analogy between many of the mechanisms operating during PCW and SCW formation, recently identified mechanisms underlying the hormonal control of PCW remodelling are discussed as potentially novel mechanisms mediating hormonal regulatory inputs in SCW formation. PMID:26002972

  6. OpenCFU, a New Free and Open-Source Software to Count Cell Colonies and Other Circular Objects

    PubMed Central

    Geissmann, Quentin

    2013-01-01

    Counting circular objects such as cell colonies is an important source of information for biologists. Although this task is often time-consuming and subjective, it is still predominantly performed manually. The aim of the present work is to provide a new tool to enumerate circular objects from digital pictures and video streams. Here, I demonstrate that the created program, OpenCFU, is very robust, accurate and fast. In addition, it provides control over the processing parameters and is implemented in an intuitive and modern interface. OpenCFU is a cross-platform and open-source software freely available at http://opencfu.sourceforge.net. PMID:23457446

  7. Bacterial lipopolysaccharide induces osteoclast formation in RAW 264.7 macrophage cells

    SciTech Connect

    Islam, Shamima; Hassan, Ferdaus; Tumurkhuu, Gantsetseg; Dagvadorj, Jargalsaikhan; Koide, Naoki; Naiki, Yoshikazu; Mori, Isamu; Yoshida, Tomoaki; Yokochi, Takashi . E-mail: yokochi@aichi-med-u.ac.jp

    2007-08-24

    Lipopolysaccharide (LPS) is a potent bone resorbing factor. The effect of LPS on osteoclast formation was examined by using murine RAW 264.7 macrophage cells. LPS-induced the formation of multinucleated giant cells (MGC) in RAW 264.7 cells 3 days after the exposure. MGCs were positive for tartrate-resistant acid phosphatase (TRAP) activity. Further, MGC formed resorption pits on calcium-phosphate thin film that is a substrate for osteoclasts. Therefore, LPS was suggested to induce osteoclast formation in RAW 264.7 cells. LPS-induced osteoclast formation was abolished by anti-tumor necrosis factor (TNF)-{alpha} antibody, but not antibodies to macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-{kappa}B ligand (RANKL). TNF-{alpha} might play a critical role in LPS-induced osteoclast formation in RAW 264.7 cells. Inhibitors of NF-{kappa}B and stress activated protein kinase (SAPK/JNK) prevented the LPS-induced osteoclast formation. The detailed mechanism of LPS-induced osteoclast formation is discussed.

  8. Granulocyte colony-stimulating factor reprograms bone marrow stromal cells to actively suppress B lymphopoiesis in mice

    PubMed Central

    Day, Ryan B.; Bhattacharya, Deepta; Nagasawa, Takashi

    2015-01-01

    The mechanisms that mediate the shift from lymphopoiesis to myelopoiesis in response to infectious stress are largely unknown. We show that treatment with granulocyte colony-stimulating factor (G-CSF), which is often induced during infection, results in marked suppression of B lymphopoiesis at multiple stages of B-cell development. Mesenchymal-lineage stromal cells in the bone marrow, including CXCL12-abundant reticular (CAR) cells and osteoblasts, constitutively support B lymphopoiesis through the production of multiple B trophic factors. G-CSF acting through a monocytic cell intermediate reprograms these stromal cells, altering their capacity to support B lymphopoiesis. G-CSF treatment is associated with an expansion of CAR cells and a shift toward osteogenic lineage commitment. It markedly suppresses the production of multiple B-cell trophic factors by CAR cells and osteoblasts, including CXCL12, kit ligand, interleukin-6, interleukin-7, and insulin-like growth factor-1. Targeting bone marrow stromal cells is one mechanism by which inflammatory cytokines such as G-CSF actively suppress lymphopoiesis. PMID:25814527

  9. Commensal Microbiota and CD8+ T Cells Shape the Formation of Invariant NKT Cells

    PubMed Central

    Wei, Bo; Wingender, Gerhard; Fujiwara, Daisuke; Chen, Diana YuHui; McPherson, Michael; Brewer, Sarah; Borneman, James; Kronenberg, Mitchell; Braun, Jonathan

    2012-01-01

    Commensal bacteria play an important role in formation of the immune system, but the mechanisms involved are incompletely understood. In this study, we analyze CD1d-restricted invariant NKT (iNKT) cells in germfree mice and in two colonies of C57BL/6 mice termed conventional flora and restricted flora (RF), stably bearing commensal microbial communities of diverse but distinct composition. In germfree mice, iNKT cells were moderately reduced, suggesting that commensal microbiota were partially required for the antigenic drive in maintaining systemic iNKT cells. Surprisingly, even greater depletion of iNKT cell population occurred in RF mice. This was in part attributable to reduced RF levels of intestinal microbial taxa (Sphingomonas spp.) known to express antigenic glycosphingolipid products. However, memory and activated CD8+ T cells were also expanded in RF mice, prompting us to test whether CD8+ T cell activity might be further depleting iNKT cells. Indeed, iNKT cell numbers were restored in RF mice bearing the CD8α−/− genotype or in adult wild-type RF mice acutely depleted with anti-CD8 Ab. Moreover, iNKT cells were restored in RF mice bearing the Prf1−/− phenotype, a key component of cytolytic function. These findings indicate that commensal microbiota, through positive (antigenic drive) and negative (cytolytic depletion by CD8+ T cells) mechanisms, profoundly shape the iNKT cell compartment. Because individuals greatly vary in the composition of their microbial communities, enteric microbiota may play an important epigenetic role in the striking differences in iNKT cell abundance in humans and therefore in their potential contribution to host immune status. PMID:20048124

  10. A hypothesis of target cell formation in sickle cell disease.

    PubMed

    Wong, P

    2016-08-01

    A fraction of erythrocytes appear as target cells in stained blood smears in sickle cell disease, due to a inheritance of the hemoglobin variant Hb S, polymerizing upon deoxygenation. These cells appear in a three dimension as thin cups. A process of their formation in this disease is proposed based on a band 3-based mechanism of the erythrocyte shape control, able to explain the erythrocyte echinocytosis by glucose depletion. It indicates that their formation is due to a stomatocytogenic slow outward transport of the dibasic form of endogenous Pi with an H(+) by band 3, promoted by the decrease of the Donnan ratio, which decreases cell pH and volume, attributed by a decrease of cell KCl concentration by the higher efflux of K(+)Cl(-) cotransport and Ca(2+) activation of the Gardos channel. Its implications are briefly discussed with respect to target cells per se, target cell formation in other hemoglobinopathies, acquired and inherited disorders of the lipid metabolism and dehydrated hereditary stomatocytosis as well as a stomatocyte presence in a double heterozygote of Hb S and Hb C and of an involvement of the process of target cell formation in acanthocytosis in acquired and inherited disorders. PMID:27372866

  11. Microfluidic Capture of Endothelial Colony-Forming Cells from Human Adult Peripheral Blood: Phenotypic and Functional Validation In Vivo

    PubMed Central

    Lin, Ruei-Zeng; Hatch, Adam; Antontsev, Victor G.; Murthy, Shashi K.

    2015-01-01

    Introduction: Endothelial colony-forming cells (ECFCs) are endothelial progenitors that circulate in peripheral blood and are currently the subject of intensive investigation due to their therapeutic potential. However, in adults, ECFCs comprise a very small subset among circulating cells, which makes their isolation a challenge. Materials and Methods: Currently, the standard method for ECFC isolation relies on the separation of mononuclear cells and erythrocyte lysis, steps that are time consuming and known to increase cell loss. Alternatively, we previously developed a novel disposable microfluidic platform containing antibody-functionalized degradable hydrogel coatings that is ideally suited for capturing low-abundance circulating cells from unprocessed blood. In this study, we reasoned that this microfluidic approach could effectively isolate rare ECFCs by virtue of their CD34 expression. Results: We conducted preclinical experiments with peripheral blood from four adult volunteers and demonstrated that the actual microfluidic capture of circulating CD34+ cells from unprocessed blood was compatible with the subsequent differentiation of these cells into ECFCs. Moreover, the ECFC yield obtained with the microfluidic system was comparable to that of the standard method. Importantly, we unequivocally validated the phenotypical and functional properties of the captured ECFCs, including the ability to form microvascular networks following transplantation into immunodeficient mice. Discussion: We showed that the simplicity and versatility of our microfluidic system could be very instrumental for ECFC isolation while preserving their therapeutic potential. We anticipate our results will facilitate additional development of clinically suitable microfluidic devices by the vascular therapeutic and diagnostic industry. PMID:25091645

  12. Granulocyte–Colony Stimulating Factor Promotes Liver Repair and Induces Oval Cell Migration and Proliferation in Rats

    PubMed Central

    PISCAGLIA, ANNA C.; SHUPE, THOMAS D.; OH, SEH–HOON; GASBARRINI, ANTONIO; PETERSEN, BRYON E.

    2011-01-01

    Background & Aims Hepatic regeneration is a heterogeneous phenomenon involving several cell populations. Oval cells are considered liver stem cells, a portion of which derive from bone marrow (BM). Recent studies have shown that granulocyte–colony stimulating factor (G-CSF) may be effective in facilitating liver repair. However, it remains unclear if G-CSF acts by mobilizing BM cells, or if it acts locally within the liver microenvironment to facilitate the endogenous restoration program. In the present study, we assessed the involvement of G-CSF during oval cell activation. Methods Dipeptidyl-peptidase-IV–deficient female rats received BM transplants from wild-type male donors. Four weeks later, rats were subjected to the 2-acetylaminofluorene/partial hepatectomy model of oval cell–mediated liver regeneration, followed by administration of either nonpegylated G-CSF or pegylated G-CSF. Control animals did not receive further treatments after surgery. The magnitude of oval cell reaction, the entity of BM contribution to liver repopulation, as well as the G-CSF/G-CSF–receptor expression levels were evaluated. In addition, in vitro proliferation and migration assays were performed on freshly isolated oval cells. Results Oval cells were found to express G-CSF receptor and G-CSF was produced within the regenerating liver. G-CSF administration significantly increased both the magnitude of the oval cell reaction, and the contribution of BM to liver repair. Finally, G-CSF acted as a chemoattractant and a mitogen for oval cells in vitro. Conclusions We have shown that G-CSF facilitates hepatic regeneration by increasing the migration of BM-derived progenitors to the liver, as well as enhancing the endogenous oval cell reaction. PMID:17681181

  13. Expression of human granulocyte colony stimulating factor (hG-CSF) in colon adenocarcinoma cell line (Caco-2).

    PubMed

    Jana, Snehasis; Patel, Hitesh

    2012-10-01

    Growth and progression of many cancer cells are mediated by alterations in the microenvironment often caused by an aberrant expression of growth factors and receptors. There is no report on expression of growth factor granulocyte colony-stimulating factor (G-CSF) in the experimental model, colon adenocarcinoma cell line (Caco2), that is commonly used in drug permeability assays. We hypothesize that in vitro, the Caco2 model is associated with a constitutive neo-expression of the hematopoietic G-CSF thereby causing an autocrine stimulation of Caco2 growth and proliferation in vitro. To test our hypothesis, we analyzed mRNA and protein expression of G-CSF in Caco2 cells using reverse transcriptase-PCR and SDS-PAGE. G-CSF mRNA and protein were detected in Caco2 cells. Expression of G-CSF protein was similar at different passages of this cell line. The expression of G-CSF has a significant role in the autocrine regulation of Caco2 cell growth and proliferation. PMID:22714276

  14. Solar cell contact formation using laser ablation

    DOEpatents

    Harley, Gabriel; Smith, David D.; Cousins, Peter John

    2014-07-22

    The formation of solar cell contacts using a laser is described. A method of fabricating a back-contact solar cell includes forming a poly-crystalline material layer above a single-crystalline substrate. The method also includes forming a dielectric material stack above the poly-crystalline material layer. The method also includes forming, by laser ablation, a plurality of contacts holes in the dielectric material stack, each of the contact holes exposing a portion of the poly-crystalline materiat layer; and forming conductive contacts in the plurality of contact holes.

  15. Solar cell contact formation using laser ablation

    DOEpatents

    Harley, Gabriel; Smith, David D.; Cousins, Peter John

    2015-07-21

    The formation of solar cell contacts using a laser is described. A method of fabricating a back-contact solar cell includes forming a poly-crystalline material layer above a single-crystalline substrate. The method also includes forming a dielectric material stack above the poly-crystalline material layer. The method also includes forming, by laser ablation, a plurality of contacts holes in the dielectric material stack, each of the contact holes exposing a portion of the poly-crystalline material layer; and forming conductive contacts in the plurality of contact holes.

  16. Solar cell contact formation using laser ablation

    DOEpatents

    Harley, Gabriel; Smith, David; Cousins, Peter

    2012-12-04

    The formation of solar cell contacts using a laser is described. A method of fabricating a back-contact solar cell includes forming a poly-crystalline material layer above a single-crystalline substrate. The method also includes forming a dielectric material stack above the poly-crystalline material layer. The method also includes forming, by laser ablation, a plurality of contacts holes in the dielectric material stack, each of the contact holes exposing a portion of the poly-crystalline material layer; and forming conductive contacts in the plurality of contact holes.

  17. Intravenous Administration of Endothelial Colony-Forming Cells Overexpressing Integrin β1 Augments Angiogenesis in Ischemic Legs.

    PubMed

    Goto, Kazuko; Takemura, Genzou; Takahashi, Tomoyuki; Okada, Hideshi; Kanamori, Hiromitsu; Kawamura, Itta; Watanabe, Takatomo; Morishita, Kentaro; Tsujimoto, Akiko; Miyazaki, Nagisa; Ushikoshi, Hiroaki; Kawasaki, Masanori; Mikami, Atsushi; Kosai, Ken-ichiro; Minatoguchi, Shinya

    2016-02-01

    When injected directly into ischemic tissue in patients with peripheral artery disease, the reparative capacity of endothelial progenitor cells (EPCs) appears to be limited by their poor survival. We, therefore, attempted to improve the survival of transplanted EPCs through intravenous injection and gene modification. We anticipated that overexpression of integrin β1 will enable injected EPCs to home to ischemic tissue, which abundantly express extracellular matrix proteins, the ligands for integrins. In addition, integrin β1 has an independent angiogenesis-stimulating function. Human endothelial colony-forming cells (ECFCs; late-outgrowth EPCs) were transduced using a lentiviral vector encoding integrin β1 (ITGB1) or enhanced green fluorescent protein (GFP). We then locally or systemically injected phosphate-buffered saline or the genetically modified ECFCs (GFP-ECFCs or ITGB1-ECFCs; 1 × 10(5) cells each) into NOD/Shi-scid, IL-2Rγnull mice whose right femoral arteries had been occluded 24 hours earlier. Upregulation of extracellular matrix proteins, including fibronectin, was apparent in the ischemic legs. Four weeks later, blood perfusion of the ischemic limb was significantly augmented only in the ITGB1-ECFC group. Scanning electron microscopy of vascular casts revealed increases in the perfused blood vessels in the ischemic legs of mice in the ITGB1-ECFC group and significant increases in the density of both capillaries and arterioles. Transplanted ECFC-derived vessels accounted for 28% ± 4.2% of the vessels in the ITGB1-ECFC group, with no cell fusion. Intravenous administration of ECFCs engineered to home to ischemic tissue appears to efficiently mediate therapeutic angiogenesis in a mouse model of peripheral artery disease. Significance: The intravenous administration of endothelial colony-forming cells (ECFCs) genetically modified to overexpress integrin β1 effectively stimulated angiogenesis in ischemic mouse hindlimbs. Transplanted ECFCs were

  18. Coupled Positive and Negative Feedbacks Produce Diverse Gene Expression Patterns in Colonies

    PubMed Central

    Mitarai, Namiko; Jensen, Mogens Høgh

    2015-01-01

    ABSTRACT Formation of patterns is a common feature in the development of multicellular organism as well as of microbial communities. To investigate the formation of gene expression patterns in colonies, we build a mathematical model of two-dimensional colony growth, where cells carry a coupled positive-and-negative-feedback circuit. We demonstrate that the model can produce sectored, target (concentric), uniform, and scattered expression patterns of regulators, depending on gene expression dynamics and nutrient diffusion. We reconstructed the same regulatory structure in Escherichia coli cells and found gene expression patterns on the surface of colonies similar to the ones produced by the computer simulations. By comparing computer simulations and experimental results, we observed that very simple rules of gene expression can yield a spectrum of well-defined patterns in a growing colony. Our results suggest that variations of the protein content among cells lead to a high level of heterogeneity in colonies. Importance Formation of patterns is a common feature in the development of microbial communities. In this work, we show that a simple genetic circuit composed of a positive-feedback loop and a negative-feedback loop can produce diverse expression patterns in colonies. We obtained similar sets of gene expression patterns in the simulations and in the experiments. Because the combination of positive feedback and negative feedback is common in intracellular molecular networks, our results suggest that the protein content of cells is highly diversified in colonies. PMID:25852158

  19. Colony Polymerase Chain Reaction with Schizosaccharomyces pombe.

    PubMed

    Murray, Johanne M; Watson, Adam T; Carr, Antony M

    2016-01-01

    When screening a large number of individual Schizosaccharomyces pombe strains by polymerase chain reaction (PCR), a rapid "colony PCR" approach may be used. Numerous colony PCR protocols are available, and fundamental to them all is that the colony must be fresh (grown overnight) and that as few cells as possible are used. In this protocol, we present three reliable methods for preparing S. pombe cells for colony PCR. PMID:27140919

  20. Collagen-IV supported embryoid bodies formation and differentiation from buffalo (Bubalus bubalis) embryonic stem cells

    SciTech Connect

    Taru Sharma, G.; Dubey, Pawan K.; Verma, Om Prakash; Pratheesh, M.D.; Nath, Amar; Sai Kumar, G.

    2012-08-03

    Graphical abstract: EBs formation, characterization and expression of germinal layers marker genes of in vivo developed teratoma using four different types of extracellular matrices. Highlights: Black-Right-Pointing-Pointer Collagen-IV matrix is found cytocompatible for EBs formation and differentiation. Black-Right-Pointing-Pointer Established 3D microenvironment for ES cells development and differentiation into three germ layers. Black-Right-Pointing-Pointer Collagen-IV may be useful as promising candidate for ES cells based therapeutic applications. -- Abstract: Embryoid bodies (EBs) are used as in vitro model to study early extraembryonic tissue formation and differentiation. In this study, a novel method using three dimensional extracellular matrices for in vitro generation of EBs from buffalo embryonic stem (ES) cells and its differentiation potential by teratoma formation was successfully established. In vitro derived inner cell masses (ICMs) of hatched buffalo blastocyst were cultured on buffalo fetal fibroblast feeder layer for primary cell colony formation. For generation of EBs, pluripotent ES cells were seeded onto four different types of extracellular matrices viz; collagen-IV, laminin, fibronectin and matrigel using undifferentiating ES cell culture medium. After 5 days of culture, ESCs gradually grew into aggregates and formed simple EBs having circular structures. Twenty-six days later, they formed cystic EBs over collagen matrix with higher EBs formation and greater proliferation rate as compared to other extracellular matrices. Studies involving histological observations, fluorescence microscopy and RT-PCR analysis of the in vivo developed teratoma revealed that presence of all the three germ layer derivatives viz. ectoderm (NCAM), mesoderm (Flk-1) and endoderm (AFP). In conclusion, the method described here demonstrates a simple and cost-effective way of generating EBs from buffalo ES cells. Collagen-IV matrix was found cytocompatible as it

  1. Granulocyte colony-stimulating factor (G-CSF) administration in individuals with sickle cell disease: time for a moratorium?

    PubMed

    Fitzhugh, Courtney D; Hsieh, Matthew M; Bolan, Charles D; Saenz, Carla; Tisdale, John F

    2009-01-01

    Granulocyte colony-stimulating factor (G-CSF) is used commonly in an attempt to reduce the duration of neutropenia and hospitalization in patients undergoing chemotherapy and to obtain hematopoietic stem cells (HSC) for transplantation applications. Despite the relative safety of administration of G-CSF in most individuals, including subjects with sickle cell trait, severe and life-threatening complications have been reported when used in individuals with sickle cell disease (SCD), including those who were asymptomatic and undiagnosed prior to administration. The administration of G-CSF has now been reported in a total of 11 individuals with SCD. Seven developed severe adverse events, including vaso-occlusive episodes, acute chest syndrome, multi-organ system failure and death. Precautions, including minimizing the peak white blood cell count, dividing or reducing the G-CSF dose and red blood cell transfusions to reduce sickle hemoglobin (HbS) levels, have been employed with no consistent benefit. These reported data indicate that administration of G-CSF in individuals with SCD should be undertaken only in the absence of alternatives and after full disclosure of the risks involved. Unless further data demonstrate safety, routine usage of G-CSF in individuals with SCD should be avoided. PMID:19513902

  2. Studies of chloroplast development in Euglena XV. Factors influencing the decay of photoreactivability of green colony formation.

    PubMed

    Hill, H Z; Schiff, J A; Epstein, H T

    1966-07-01

    When UV-treated cells of Euglena gracilis var. bacillaris are incubated in the dark in a nutrient medium which permits cell division, they lose the ability to be photoreactivated. The rate of this loss increases with the UV dose. For any given UV dose, the rate of decay increases with increasing growth rate. The same phenomena are observed in light-grown and in dark-grown cells, although the sensitivity to UV of the light-grown cells is smaller by a factor of 1.7. The kinetics of photoreactivation (PR) change during the decay of photoreactivability only if the cells are incubated in growth medium. A UV-inactivation curve for cells photoreactivated only after appreciable PR shows the same slope as that for untreated cells (number of UV-sensitive targets). These results are discussed from the point of view of possible models. PMID:19210965

  3. Macrophage colony-stimulating factor receptor marks and regulates a fetal myeloid-primed B-cell progenitor in mice

    PubMed Central

    Zriwil, Alya; Böiers, Charlotta; Wittmann, Lilian; Green, Joanna C. A.; Woll, Petter S.; Jacobsen, Sten Eirik W.

    2016-01-01

    Although it is well established that unique B-cell lineages develop through distinct regulatory mechanisms during embryonic development, much less is understood about the differences between embryonic and adult B-cell progenitor cells, likely to underpin the genetics and biology of infant and childhood PreB acute lymphoblastic leukemia (PreB-ALL), initiated by distinct leukemia-initiating translocations during embryonic development. Herein, we establish that a distinct subset of the earliest CD19+ B-cell progenitors emerging in the E13.5 mouse fetal liver express the colony-stimulating factor-1 receptor (CSF1R), previously thought to be expressed, and play a lineage-restricted role in development of myeloid lineages, and macrophages in particular. These early embryonic CSF1R+CD19+ ProB cells also express multiple other myeloid genes and, in line with this, possess residual myeloid as well as B-cell, but not T-cell lineage potential. Notably, these CSF1R+ myeloid-primed ProB cells are uniquely present in a narrow window of embryonic fetal liver hematopoiesis and do not persist in adult bone marrow. Moreover, analysis of CSF1R-deficient mice establishes a distinct role of CSF1R in fetal B-lymphopoiesis. CSF1R+ myeloid-primed embryonic ProB cells are relevant for infant and childhood PreB-ALLs, which frequently have a bi-phenotypic B-myeloid phenotype, and in which CSF1R-rearrangements have recently been reported. PMID:27207794

  4. Granulocyte colony-stimulating factor impairs CD8(+) T cell functionality by interfering with central activation elements.

    PubMed

    Bunse, C E; Tischer, S; Lahrberg, J; Oelke, M; Figueiredo, C; Blasczyk, R; Eiz-Vesper, B

    2016-07-01

    Besides mobilizing stem cells into the periphery, granulocyte colony-stimulating factor (G-CSF) has been shown to influence various types of innate and adaptive immune cells. For example, it impairs the effector function of cytotoxic T lymphocytes (CTLs). It is assumed that this effect is mediated indirectly by monocytes, regulatory T cells and immunomodulatory cytokines influenced by G-CSF. In this study, isolated G-CSF-treated CD8(+) T cells were stimulated antigen-dependently with peptide-major histocompatibility complex (pMHC)-coupled artificial antigen-presenting cells (aAPCs) or stimulated antigen-independently with anti-CD3/CD28 stimulator beads. By measuring the changes in interferon (IFN)-γ and granzyme B expression at the mRNA and protein level, we showed for the first time that G-CSF has a direct effect on CD8(+) CTLs, which was confirmed based on the reduced production of IFN-γ and granzyme B by the cytotoxic T cell line TALL-104 after G-CSF treatment. By investigating further elements affected by G-CSF in CTLs from stem cell donors and untreated controls, we found a decreased phosphorylation of extracellular-regulated kinase (ERK)1/2, lymphocyte-specific protein tyrosine kinase (Lck) and CD3ζ after G-CSF treatment. Additionally, miRNA-155 and activation marker expression levels were reduced. In summary, our results show that G-CSF directly influences the effector function of cytotoxic CD8(+) T cells and affects various elements of T cell activation. PMID:26990855

  5. Macrophage colony-stimulating factor receptor marks and regulates a fetal myeloid-primed B-cell progenitor in mice.

    PubMed

    Zriwil, Alya; Böiers, Charlotta; Wittmann, Lilian; Green, Joanna C A; Woll, Petter S; Jacobsen, Sten Eirik W; Sitnicka, Ewa

    2016-07-14

    Although it is well established that unique B-cell lineages develop through distinct regulatory mechanisms during embryonic development, much less is understood about the differences between embryonic and adult B-cell progenitor cells, likely to underpin the genetics and biology of infant and childhood PreB acute lymphoblastic leukemia (PreB-ALL), initiated by distinct leukemia-initiating translocations during embryonic development. Herein, we establish that a distinct subset of the earliest CD19(+) B-cell progenitors emerging in the E13.5 mouse fetal liver express the colony-stimulating factor-1 receptor (CSF1R), previously thought to be expressed, and play a lineage-restricted role in development of myeloid lineages, and macrophages in particular. These early embryonic CSF1R(+)CD19(+) ProB cells also express multiple other myeloid genes and, in line with this, possess residual myeloid as well as B-cell, but not T-cell lineage potential. Notably, these CSF1R(+) myeloid-primed ProB cells are uniquely present in a narrow window of embryonic fetal liver hematopoiesis and do not persist in adult bone marrow. Moreover, analysis of CSF1R-deficient mice establishes a distinct role of CSF1R in fetal B-lymphopoiesis. CSF1R(+) myeloid-primed embryonic ProB cells are relevant for infant and childhood PreB-ALLs, which frequently have a bi-phenotypic B-myeloid phenotype, and in which CSF1R-rearrangements have recently been reported. PMID:27207794

  6. Giant Cell Arteritis which Developed after the Administration of Granulocyte-colony Stimulating Factor for Cyclic Neutropenia.

    PubMed

    Umeda, Masataka; Ikenaga, Jin; Koga, Tomohiro; Michitsuji, Toru; Shimizu, Toshimasa; Fukui, Shoichi; Nishino, Ayako; Nakasima, Yoshikazu; Kawashiri, Sin-Ya; Iwamoto, Naoki; Ichinose, Kunihiro; Hirai, Yasuko; Tamai, Mami; Nakamura, Hideki; Origuchi, Tomoki; Kawakami, Atsushi

    2016-01-01

    A 78-year-old woman diagnosed with cyclic neutropenia 5 years previously had been treated with recombinant granulocyte-colony stimulating factor (G-CSF). She developed fever, tenderness and distension of temporal arteries after the treatment with G-CSF. Magnetic resonance imaging and ultrasonography revealed wall thickening of the temporal arteries. She was therefore diagnosed with giant cell arteritis (GCA). Small vessel vasculitis has been reported as a complication of G-CSF. However, the development of large vessel vasculitis after G-CSF treatment is quite rare. To our knowledge, the present case is the first report of GCA suspected to be associated with coexisting cyclic neutropenia and G-CSF treatment. PMID:27523011

  7. Organochlorine formation in magnesium electrowinning cells.

    PubMed

    Deutscher, R L; Cathro, K J

    2001-04-01

    The formation of organochlorines during the electrolytic production of magnesium was investigated using a laboratory-scale electrolytic cell having a graphite anode, a liquid aluminium alloy cathode, and a molten chloride electrolyte. The cell was operated at current densities ranging from 3000 to 10,000 A m(-2) and at temperatures ranging from 660 degrees C to 750 degrees C. Organochlorines were adsorbed from the cell off-gases onto silica gel, extracted with hexane, and determined by gas chromatography. All compounds identified were fully chlorinated aliphatic and aromatic compounds, the major components being hexachlorobutadiene, hexachlorobenzene, hexachloroethylene, and octachlorostyrene. The total amount of organochlorines per tonne of magnesium produced decreased with electrolysis time and with current density and increased with operating temperature; it was also dependent on the type of graphite employed. The output of organochlorines varied from 5 to 20 g t(-1) of magnesium. PMID:11297394

  8. Stromal cell-derived factor-1 enhances pro-angiogenic effect of granulocyte-colony stimulating factor

    PubMed Central

    Tan, Yaohong; Shao, Hongwei; Eton, Darwin; Yang, Zhe; Alonso-Diaz, Luis; Zhang, Hongkun; Schulick, Andrew; Livingstone, Alan S.; Yu, Hong

    2008-01-01

    Objective Granulocyte colony-stimulating factor (G-CSF) mobilizes bone marrow mononuclear cells into the peripheral circulation. Stromal cell-derived factor-1 (SDF-1) enhances the homing of progenitor cells mobilized from the bone marrow and augments neovascularization in ischemic tissue. We hypothesize that SDF-1 will boost the pro-angiogenic effect of G-CSF. Methods and results NIH 3T3 cells retrovirally transduced with SDF-1α gene (NIH 3T3/SDF-1) were used to deliver SDF-1 in vitro and in vivo. Endothelial progenitor cells (EPCs) co-cultured with NIH 3T3/SDF-1 cells using cell culture inserts migrated faster and were less apoptotic compared to those not exposed to SDF-1. NIH 3T3/SDF-1 (106 cells) were injected into the ischemic muscles immediately after resection of the left femoral artery and vein of C57BL/6J mice. G-CSF (25 μg/kg/day) was injected intraperitioneally daily for 3 days after surgery. Blood perfusion was examined using a laser Doppler perfusion imaging system. The perfusion ratio of ischemic/non-ischemic limb increased to 0.57±0.03 and 0.50±0.06 with the treatment of either SDF-1 or G-CSF only, respectively, 3 weeks after surgery, which was significantly higher than the saline-injected control group (0.41±0.01, P<0.05). Combined treatment with both SDF-1 and G-CSF resulted in an even better perfusion ratio of 0.69±0.08 (P<0.05 versus the single treatment groups). Mice were sacrificed 21 days after surgery. Immunostaining and Western blot assay of the tissue lysates showed that the injected NIH 3T3/SDF-1 survived and expressed SDF-1. CD34+ cells were detected with immunostaining, capillary density was assessed with alkaline phosphatase staining, and the apoptosis of muscle cells was viewed using an in situ cell death detection kit. More CD34+ cells, increased capillary density, and less apoptotic muscle cells were found in both G-CSF and SDF-1 treated group (P<0.05 versus other groups). Conclusion Combination of G-CSF-mediated progenitor cell

  9. An optimized colony forming assay for low-dose-radiation cell survival measurement

    SciTech Connect

    Zhu J.; Sutherland B.; Hu W.; Ding N.; Ye C.; Usikalu M.; Li S.; Hu B.; Zhou G.

    2011-11-01

    The aim of this study is to develop a simple and reliable method to quantify the cell survival of low-dose irradiations. Two crucial factors were considered, the same number of cells plated in each flask and an appropriate interval between cell plating and irradiation. For the former, we optimized cell harvest with trypsin, diluted cells in one container, and directly seeded cells on the bottom of flasks in a low density before irradiation. Reproducible plating efficiency was obtained. For the latter, we plated cells on the bottom of flasks and then monitored the processing of attachment, cell cycle variations, and the plating efficiency after exposure to 20 cGy of X-rays. The results showed that a period of 4.5 h to 7.5 h after plating was suitable for further treatment. In order to confirm the reliability and feasibility of our method, we also measured the survival curves of these M059K and M059J glioma cell lines by following the optimized protocol and obtained consistent results reported by others with cell sorting system. In conclusion, we successfully developed a reliable and simple way to measure the survival fractions of human cells exposed to low dose irradiation, which might be helpful for the studies on low-dose radiation biology.

  10. Accumulated lipids rather than the rigid cell walls impede the extraction of genetic materials for effective colony PCRs in Chlorella vulgaris

    PubMed Central

    2013-01-01

    Background Failure of colony PCRs in green microalga Chlorella vulgaris is typically attributed to the difficulty in disrupting its notoriously rigid cell walls for releasing the genetic materials and therefore the development of an effective colony PCR procedure in C. vulgaris presents a challenge. Results Here we identified that colony PCR results were significantly affected by the accumulated lipids rather than the rigid cell walls of C. vulgaris. The higher lipids accumulated in C. vulgaris negatively affects the effective amplification by DNA polymerase. Based on these findings, we established a simple and extremely effective colony PCR procedure in C. vulgaris. By simply pipetting/votexing the pellets of C. vulgaris in 10 ul of either TE (10 mM Tris/1 mM EDTA) or 0.2% SDS buffer at room temperature, followed by the addition of 10 ul of either hexane or Phenol:Chloroform:Isoamyl Alcohol in the same PCR tube for extraction. The resulting aqueous phase was readily PCR-amplified as genomic DNA templates as demonstrated by successful amplification of the nuclear 18S rRNA and the chloroplast rbcL gene. This colony PCR protocol is effective and robust in C. vulgaris and also demonstrates its effectiveness in other Chlorella species. Conclusions The accumulated lipids rather than the rigid cell walls of C. vulgaris significantly impede the extraction of genetic materials and subsequently the effective colony PCRs. The finding has the potential to aid the isolation of high-quality total RNAs and mRNAs for transcriptomic studies in addition to the genomic DNA isolation in Chlorella. PMID:24219401

  11. Spontaneous emergence of large-scale cell cycle synchronization in amoeba colonies

    NASA Astrophysics Data System (ADS)

    Segota, Igor; Boulet, Laurent; Franck, David; Franck, Carl

    2014-06-01

    Unicellular eukaryotic amoebae Dictyostelium discoideum are generally believed to grow in their vegetative state as single cells until starvation, when their collective aspect emerges and they differentiate to form a multicellular slime mold. While major efforts continue to be aimed at their starvation-induced social aspect, our understanding of population dynamics and cell cycle in the vegetative growth phase has remained incomplete. Here we show that cell populations grown on a substrate spontaneously synchronize their cell cycles within several hours. These collective population-wide cell cycle oscillations span millimeter length scales and can be completely suppressed by washing away putative cell-secreted signals, implying signaling by means of a diffusible growth factor or mitogen. These observations give strong evidence for collective proliferation behavior in the vegetative state.

  12. Spontaneous emergence of large-scale cell cycle synchronization in amoeba colonies.

    PubMed

    Segota, Igor; Boulet, Laurent; Franck, David; Franck, Carl

    2014-06-01

    Unicellular eukaryotic amoebae Dictyostelium discoideum are generally believed to grow in their vegetative state as single cells until starvation, when their collective aspect emerges and they differentiate to form a multicellular slime mold. While major efforts continue to be aimed at their starvation-induced social aspect, our understanding of population dynamics and cell cycle in the vegetative growth phase has remained incomplete. Here we show that cell populations grown on a substrate spontaneously synchronize their cell cycles within several hours. These collective population-wide cell cycle oscillations span millimeter length scales and can be completely suppressed by washing away putative cell-secreted signals, implying signaling by means of a diffusible growth factor or mitogen. These observations give strong evidence for collective proliferation behavior in the vegetative state. PMID:24732749

  13. Glucose induced fractal colony pattern of Bacillus thuringiensis.

    PubMed

    Roy, Manas K; Banerjee, Paromita; Sengupta, Tapas K; Dattagupta, Sushanta

    2010-08-01

    Growing colonies of bacteria on the surface of thin agar plates exhibit fractal patterns as a result of nonlinear response to environmental conditions, such as nutrients, solidity of the agar medium and temperature. Here, we examine the effect of glucose on pattern formation by growing colonies of Bacillus thuringiensis isolate KPWP1. We also present the theoretical modeling of the colony growth of KPWP1 and the associated spatio-temporal patterns. Our experimental results are in excellent agreement with simulations based on a reaction-diffusion model that describes diffusion-limited aggregation and branching, in which individual cells move actively in the periphery, but become immotile in the inner regions of the growing colony. We obtain the Hausdorff fractal dimension of the colony patterns: D(H.Expt)=1.1969 and D(H, R.D.=)1.1965, for experiment and reaction-diffusion model, respectively. Results of our experiments and modeling clearly show how glucose at higher concentration can prove to be inhibitory for motility of growing colonies of B. thuringiensis cells on semisolid support and be responsible for changes in the growth pattern. PMID:20553734

  14. Radioprotection of mice with interleukin-1: Relationship to the number of erythroid and granulocyte-macrophage colony-forming cells

    SciTech Connect

    Schwartz, G.N.; Patchen, M.L.; Neta, R.; MacVittie, T.J.

    1990-01-01

    This report presents the results of an investigation of changes in the number of erythroid and granulocyte-macrophage colony forming cells (GM-CFC) that had occurred in tissues of normal B6D2F1 mice 20 h after administration of a radioprotective dose (150 ng) of human recombinant interleukin-1 (rIL-1). Neutrophilia in the peripheral blood and changes in the tissue distribution of GM-CFC demonstrated that cells were mobilized from the bone marrow in response to rIL-1 injection. For example, 20 h after rIL-1 injection marrow GM-CFC numbers were 80% of the numbers in bone marrow from saline-injected mice. Associated with this decrease there was a twofold increase in the number of peripheral blood and splenic GM-CFC. Also, as determined by hydroxyurea injection, there was an increase in the number of GM-CFC in S phase of the cell cycle in the spleen, but not in the bone marrow. Data in this report suggest that when compared to the spleen, stimulation of granulopoiesis after rIL-1 injection is delayed in the bone marrow.

  15. Regulation of surface expression of the granulocyte/macrophage colony-stimulating factor receptor in normal human myeloid cells

    SciTech Connect

    Cannistra, S.A.; Groshek, P.; Griffin, J.D. ); Garlick, R.; Miller, J. )

    1990-01-01

    Recombinant human granulocyte/macrophage colony-stimulating factor (GM-CSF) exerts stimulatory effects on hematopoietic cells through binding to specific, high-affinity receptors. By using radiolabeled GM-CSF with high specific activity, the authors have investigated the factors and mechanisms that regulate GM-CSF receptor expression in normal human neutrophils, monocytes, and partially purified bone marrow myeloid progenitor cells. The neutrophil GM-CSF receptor was found to rapidly internalize in the presence of ligand through a mechanism that required endocytosis. Out of a large panel of naturally occurring humoral factors tested, only GM-CSF itself, tumor necrosis factor, and formyl-Met-Leu-Phe were found to down-regulate neutrophil GM-CSF receptor expression after a 2-hr exposure at biologically active concentrations. Since formyl-Met-Leu-Phe is known to stimulate neutrophil protein kinase C activity, they also tested the ability of protein kinase C agonists to modulate GM-CSF receptor expression. Phorbol 12-myristate 13-acetate, bryostatin-1, and 1,2-dioctanoylglycerol were found to induce rapid down-regulation of the GM-CSF receptor in neutrophils, monocytes, and partially purified myeloid progenitor cells, suggesting that this effect may be at least partially mediated by protein kinase C. These data suggest that certain activators of neutrophil function may negatively regulate their biological effects by inducing down-regulation of the GM-CSF receptor.

  16. Mobilization and collection of CD34+ cells for autologous transplantation of peripheral blood hematopoietic progenitor cells in children: analysis of two different granulocyte-colony stimulating factor doses

    PubMed Central

    Eid, Kátia Aparecida de Brito; Miranda, Eliana Cristina Martins; Aguiar, Simone dos Santos

    2015-01-01

    Introduction The use of peripheral hematopoietic progenitor cells (HPCs) is the cell choice in autologous transplantation. The classic dose of granulocyte-colony stimulating factor (G-CSF) for mobilization is a single daily dose of 10 μg/kg of patient body weight. There is a theory that higher doses of granulocyte-colony stimulating factor applied twice daily could increase the number of CD34+ cells collected in fewer leukapheresis procedures. Objective The aim of this study was to compare a fractionated dose of 15 μg G-CSF/kg of body weight and the conventional dose of granulocyte-colony stimulating factor in respect to the number of leukapheresis procedures required to achieve a minimum collection of 3 × 106 CD34+ cells/kg body weight. Methods Patients were divided into two groups: Group 10 – patients who received a single daily dose of 10 μg G-CSF/kg body weight and Group 15 – patients who received a fractioned dose of 15 μg G-CSF/kg body weight daily. The leukapheresis procedure was carried out in an automated cell separator. The autologous transplantation was carried out when a minimum number of 3 × 106 CD34+ cells/kg body weight was achieved. Results Group 10 comprised 39 patients and Group 15 comprised 26 patients. A total of 146 apheresis procedures were performed: 110 (75.3%) for Group 10 and 36 (24.7%) for Group 15. For Group 10, a median of three (range: 1–7) leukapheresis procedures and a mean of 8.89 × 106 CD34+ cells/kg body weight (±9.59) were collected whereas for Group 15 the corresponding values were one (range: 1–3) and 5.29 × 106 cells/kg body weight (±4.95). A statistically significant difference was found in relation to the number of apheresis procedures (p-value <0.0001). Conclusions To collect a minimum target of 3 × 106 CD34+ cells/kg body weight, the administration of a fractionated dose of 15 μg G-CSF/kg body weight significantly decreased the number of leukapheresis procedures performed. PMID:26041417

  17. A novel function of colony-stimulating factor 1 receptor in hTERT immortalization of human epithelial cells.

    PubMed

    Li, N F; Kocher, H M; Salako, M A; Obermueller, E; Sandle, J; Balkwill, F

    2009-02-01

    The receptor for macrophage colony-stimulating factor 1 receptor (CSF1R) is a product of the proto-oncogene c-fms and a member of the class III transmembrane tyrosine kinase receptor family. Earlier, we described increased mRNA expression of CSF1R in human telomerase reverse transcriptase (hTERT) immortalized human ovarian surface epithelial (IOSE) cell lines derived from a single donor. Here, we further describe that CSF1R is upregulated at both the mRNA and protein level in hTERT immortalized human normal OSE cells from two different donors and in hTERT immortalized human pancreatic ductal epithelial cells. CSF1R was not upregulated in hTERT immortalized epithelial clones that subsequently underwent senescence or in immortalized fibroblasts. Upon stimulation by the CSF1R ligand CSF1, the immortalized epithelial cell lines showed rapid internalization of CSF1R with concomitant down-modulation and colocalization of phosphorylated NFkappaBp65 with hTERT protein, hTERT translocation into the nucleus and the binding of c-Myc to the hTERT promoter region. Reducing the expression of CSF1R using short hairpin interfering RNA abolished these effects and also decreased cell survival and the number of population doublings under suboptimal culture conditions. The telomerase inhibitor GRN163L confirmed a role for telomerase in the cleavage of the intracellular domain of CSF1R. On the basis of these findings, we suggest that CSF1R may be a critical factor facilitating hTERT immortalization of epithelial cells. PMID:18997822

  18. Spatio-temporal morphology changes in and quenching effects on the 2D spreading dynamics of cell colonies in both plain and methylcellulose-containing culture media.

    PubMed

    Muzzio, N E; Pasquale, M A; Huergo, M A C; Bolzán, A E; González, P H; Arvia, A J

    2016-06-01

    To deal with complex systems, microscopic and global approaches become of particular interest. Our previous results from the dynamics of large cell colonies indicated that their 2D front roughness dynamics is compatible with the standard Kardar-Parisi-Zhang (KPZ) or the quenched KPZ equations either in plain or methylcellulose (MC)-containing gel culture media, respectively. In both cases, the influence of a non-uniform distribution of the colony constituents was significant. These results encouraged us to investigate the overall dynamics of those systems considering the morphology and size, the duplication rate, and the motility of single cells. For this purpose, colonies with different cell populations (N) exhibiting quasi-circular and quasi-linear growth fronts in plain and MC-containing culture media are investigated. For small N, the average radial front velocity and its change with time depend on MC concentration. MC in the medium interferes with cell mitosis, contributes to the local enlargement of cells, and increases the distribution of spatio-temporal cell density heterogeneities. Colony spreading in MC-containing media proceeds under two main quenching effects, I and II; the former mainly depending on the culture medium composition and structure and the latter caused by the distribution of enlarged local cell domains. For large N, colony spreading occurs at constant velocity. The characteristics of cell motility, assessed by measuring their trajectories and the corresponding velocity field, reflect the effect of enlarged, slow-moving cells and the structure of the medium. Local average cell size distribution and individual cell motility data from plain and MC-containing media are qualitatively consistent with the predictions of both the extended cellular Potts models and the observed transition of the front roughness dynamics from a standard KPZ to a quenched KPZ. In this case, quenching effects I and II cooperate and give rise to the quenched

  19. Branching instability in expanding bacterial colonies

    PubMed Central

    Giverso, Chiara; Verani, Marco; Ciarletta, Pasquale

    2015-01-01

    Self-organization in developing living organisms relies on the capability of cells to duplicate and perform a collective motion inside the surrounding environment. Chemical and mechanical interactions coordinate such a cooperative behaviour, driving the dynamical evolution of the macroscopic system. In this work, we perform an analytical and computational analysis to study pattern formation during the spreading of an initially circular bacterial colony on a Petri dish. The continuous mathematical model addresses the growth and the chemotactic migration of the living monolayer, together with the diffusion and consumption of nutrients in the agar. The governing equations contain four dimensionless parameters, accounting for the interplay among the chemotactic response, the bacteria–substrate interaction and the experimental geometry. The spreading colony is found to be always linearly unstable to perturbations of the interface, whereas branching instability arises in finite-element numerical simulations. The typical length scales of such fingers, which align in the radial direction and later undergo further branching, are controlled by the size parameters of the problem, whereas the emergence of branching is favoured if the diffusion is dominant on the chemotaxis. The model is able to predict the experimental morphologies, confirming that compact (resp. branched) patterns arise for fast (resp. slow) expanding colonies. Such results, while providing new insights into pattern selection in bacterial colonies, may finally have important applications for designing controlled patterns. PMID:25652464

  20. Organic Tandem Solar Cells: Design and Formation

    NASA Astrophysics Data System (ADS)

    Chen, Chun-Chao

    polyelectrolyte layer functioning as the surface dipole formation layer to provide better electrical contact with the photoactive layer. Due to the effectiveness of the conjugated polyelectrolyte layer, performance improvement was also observed. Furthermore, other issues regarding the semi-transparent tandem solar cells (e.g., photocurrent matching, exterior color tuning, and transparency tuning) are all explored to optimize best performance. In Chapter 5 and 6, the architectures of double- and triple-junction tandem solar cells are explored. Theoretically, triple-junction tandem solar cells with three photoactive absorbers with cascaded energy bandgaps have the potential to achieve higher performance, in comparison with double-junction tandem solar cells. Such expectations can be ascribed to the minimized carrier thermalization loss and further improved light absorption. However, the design of triple-junction solar cells often involves sophisticated multiple layer deposition as well as substantial optimization. Therefore, there is a lack of successful demonstrations of triple-junction solar cells outperforming the double-junction counterparts. To solve the incompatible issues related to the layer deposition in the fabrication, we proposed a novel architecture of inverted-structure tandem solar cells with newly designed interconnecting layers. Our design of interconnecting layers does not only focus on maintaining the orthogonal solution processing advantages, but also provides an excellent compatibility in the energy level alignment to allow different absorber materials to be used. Furthermore, we also explored the light management inside the double- and triple-junction tandem solar cells. The study of light management was carried out through optical simulation method based transfer matrix formalism. The intention is to obtain a balanced photocurrent output from each subcells inside the tandem solar cell, thus the minimal recombination loss at the contact of interconnecting

  1. A gene in the Pseudomonas syringae pv. tomato Hrp pathogenicity island conserved effector locus, hopPtoA1, contributes to efficient formation of bacterial colonies in planta and is duplicated elsewhere in the genome.

    PubMed

    Badel, J L; Charkowski, A O; Deng, W L; Collmer, A

    2002-10-01

    The ability of Pseudomonas syringae to grow in planta is thought to be dependent upon the Hrp (type III secretion) system and multiple effector proteins that this system injects into plant cells. ORF5 in the conserved effector locus of the P. syringae pv. tomato DC3000 Hrp pathogenicity island was shown to encode a Hrp-secreted protein and to have a similarly secreted homolog encoded in an effector-rich pathogenicity island located elsewhere in the genome. These putative effector genes were designated hopPtoA1 and hopPtoA2, respectively. DNA gel blot analysis revealed that sequences hybridizing with hopPtoA1 were widespread among P. syringae pathovars, and some strains, like DC3000, appear to have two copies of the gene. uidA transcriptional fusions revealed that expression of hopPtoA1 and hopPtoA2 can be activated by the HrpL alternative sigma factor. hopPtoA1 and hopPtoA1/hopPtoA2 double mutants were not obviously different from wild-type P. syringae pv. tomato DC3000 in their ability to produce symptoms or to increase their total population size in host tomato and Arabidopsis leaves. However, confocal laser-scanning microscopy of GFP (green fluorescent protein)-labeled bacteria in Arabidopsis leaves 2 days after inoculation revealed that the frequency of undeveloped individual colonies was higher in the hopPtoA1 mutant and even higher in the hopPtoA1/hopPtoA2 double mutant. These results suggest that hopPtoA1 and hopPtoA2 contribute redundantly to the formation of P. syringae pv. tomato DC3000 colonies in Arabidopsis leaves. PMID:12437299

  2. Dysregulation of VEGF-induced proangiogenic Ca2+ oscillations in primary myelofibrosis-derived endothelial colony-forming cells.

    PubMed

    Dragoni, Silvia; Reforgiato, Marta; Zuccolo, Estella; Poletto, Valentina; Lodola, Francesco; Ruffinatti, Federico Alessandro; Bonetti, Elisa; Guerra, Germano; Barosi, Giovanni; Rosti, Vittorio; Moccia, Francesco

    2015-12-01

    Endothelial progenitor cells could be implicated in the aberrant neoangiogenesis that occurs in bone marrow and spleen in patients with primary myelofibrosis (PMF). However, antivascular endothelial growth factor (VEGF) monotherapy had only a modest and transient effect in these individuals. Recently it was found that VEGF-induced proangiogenic intracellular Ca(2+) oscillations could be impaired in endothelial progenitor cells of subjects with malignancies. Therefore, we employed Ca(2+) imaging, wavelet analysis, and functional assays to assess whether and how VEGF-induced Ca(2+) oscillations are altered in PMF-derived endothelial progenitor cells. We focused on endothelial colony-forming cells (ECFCs), which are the only endothelial progenitor cell subtype capable of forming neovessels both in vivo and in vitro. VEGF triggers repetitive Ca(2+) spikes in both normal ECFCs (N-ECFCs) and ECFCs obtained from PMF patients (PMF-ECFCs). However, the spiking response to VEGF is significantly weaker in PMF-ECFCs. VEGF-elicited Ca(2+) oscillations are patterned by the interaction between inositol-1,4,5-trisphosphate-dependent Ca(2+) mobilization and store-operated Ca(2+) entry. However, in most PMF-ECFCs, Ca(2+) oscillations are triggered by a store-independent Ca(2+) entry pathway. We found that diacylglycerol gates transient receptor potential canonical 1 channel to trigger VEGF-dependent Ca(2+) spikes by recruiting the phospholipase C/inositol-1,4,5-trisphosphate signaling pathway, reflected as a decrease in endoplasmic reticulum Ca(2+) content. Finally, we found that, apart from being less robust and dysregulated as compared with N-ECFCs, VEGF-induced Ca(2+) oscillations modestly stimulate PMF-ECFC growth and in vitro angiogenesis. These results may explain the modest effect of anti-VEGF therapies in PMF. PMID:26432919

  3. CORONETTE KERATINOCYTE COLONY FORMATION IS SUPPORTED BY EPIDERMAL-DERMAL CELL INTERACTIONS IN THE BOVINE CLAW

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Delineating factors that orchestrate keratinocyte growth and differentiation in the claw is pivotal to understanding the quality of hoof horn production in health and disease. The specific objectives of this investigation were to establish an in vitro culture system for bovine coronette keratinocyt...

  4. Lipid nanocarriers containing sorafenib inhibit colonies formation in human hepatocarcinoma cells.

    PubMed

    Bondì, Maria Luisa; Botto, Chiara; Amore, Erika; Emma, Maria Rita; Augello, Giuseppa; Craparo, Emanuela Fabiola; Cervello, Melchiorre

    2015-09-30

    Here, the potential of two nanostructured lipid carriers (NLC) for controlled release of sorafenib was evaluated. The obtained systems showed characteristics suitable as drug delivery systems for the treatment of hepatocellular carcinoma (HCC) through parenteral administration. The use of a mixture between a solid lipid (tripalmitin) with a liquid lipid (Captex 355 EP/NF or Miglyol 812) to prepare NLC systems could give a higher drug loading capacity and a longer term stability during storage than that obtained by using only solid lipids. The obtained nanoparticles showed a nanometer size and high negative zeta potential values. Scansion electron microscopy (SEM) of the sorafenib loaded NLC revealed a spherical shape with a diameter <300 nm. In vitro biological studies demonstrated that sorafenib loaded into NLC had enhanced anti-tumor activity compared to that of free drug. This finding raises hope in terms of future drug delivery strategy of sorafenib loaded NLC, that can be useful for therapeutic application in HCC. PMID:26211902

  5. Pre-B-cell colony-enhancing factor protects against apoptotic neuronal death and mitochondrial damage in ischemia.

    PubMed

    Wang, Xiaowan; Li, Hailong; Ding, Shinghua

    2016-01-01

    We previously demonstrated that Pre-B-cell colony-enhancing factor (PBEF), also known as nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in mammalian NAD(+) biosynthesis pathway, plays a brain and neuronal protective role in ischemic stroke. In this study, we further investigated the mechanism of its neuroprotective effect after ischemia in the primary cultured mouse cortical neurons. Using apoptotic cell death assay, fluorescent imaging, molecular biology, mitochondrial biogenesis measurements and Western blotting analysis, our results show that the overexpression of PBEF in neurons can significantly promote neuronal survival, reduce the translocation of apoptosis inducing factor (AIF) from mitochondria to nuclei and inhibit the activation of capase-3 after glutamate-induced excitotoxicity. We further found that the overexpression of PBEF can suppress glutamate-induced mitochondrial fragmentation, the loss of mitochondrial DNA (mtDNA) content and the reduction of PGC-1 and NRF-1 expressions. Furthermore, these beneficial effects by PBEF are dependent on its enzymatic activity of NAD(+) synthesis. In summary, our study demonstrated that PBEF ameliorates ischemia-induced neuronal death through inhibiting caspase-dependent and independent apoptotic signaling pathways and suppressing mitochondrial damage and dysfunction. Our study provides novel insights into the mechanisms underlying the neuroprotective effect of PBEF, and helps to identify potential targets for ischemic stroke therapy. PMID:27576732

  6. Pre-B-cell colony-enhancing factor protects against apoptotic neuronal death and mitochondrial damage in ischemia

    PubMed Central

    Wang, Xiaowan; Li, Hailong; Ding, Shinghua

    2016-01-01

    We previously demonstrated that Pre-B-cell colony-enhancing factor (PBEF), also known as nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in mammalian NAD+ biosynthesis pathway, plays a brain and neuronal protective role in ischemic stroke. In this study, we further investigated the mechanism of its neuroprotective effect after ischemia in the primary cultured mouse cortical neurons. Using apoptotic cell death assay, fluorescent imaging, molecular biology, mitochondrial biogenesis measurements and Western blotting analysis, our results show that the overexpression of PBEF in neurons can significantly promote neuronal survival, reduce the translocation of apoptosis inducing factor (AIF) from mitochondria to nuclei and inhibit the activation of capase-3 after glutamate-induced excitotoxicity. We further found that the overexpression of PBEF can suppress glutamate-induced mitochondrial fragmentation, the loss of mitochondrial DNA (mtDNA) content and the reduction of PGC-1 and NRF-1 expressions. Furthermore, these beneficial effects by PBEF are dependent on its enzymatic activity of NAD+ synthesis. In summary, our study demonstrated that PBEF ameliorates ischemia-induced neuronal death through inhibiting caspase-dependent and independent apoptotic signaling pathways and suppressing mitochondrial damage and dysfunction. Our study provides novel insights into the mechanisms underlying the neuroprotective effect of PBEF, and helps to identify potential targets for ischemic stroke therapy. PMID:27576732

  7. Colony-stimulating factors and peripheral blood progenitor cell transplantation. Benefits and costs.

    PubMed

    Uyl-de Groot, C A; Huijgens, P C; Rutten, F F

    1996-07-01

    High dosage chemo- or radiotherapy followed by the administration of autologous bone marrow-derived stem cells [i.e. autologous bone marrow transplantation (ABMT)] is an established protocol for treating acute myeloid leukaemia and malignant lymphoma. The approach is also under investigation in the treatment of acute lymphocytic leukaemia, multiple myeloma and solid tumours. In all of these diseases, the optimisation of indications, conditioning schemes, stem cell harvest techniques and supportive care with growth factors is subject to continuous preclinical research and clinical phase II and III studies. Recently, the administration of peripheral blood stem cell preparations to cancer patients as rescue therapy after high dosage antitumour therapy has been received with much enthusiasm. At first glance, the technique looks rather easy to perform. The faster recovery of haemopoiesis, compared with ABMT, leads to shorter durations of hospitalisation. Moreover, in most studies, peripheral blood progenitor cell transplantation (PBPCT) resulted in fewer septic episodes, fewer intensive care admissions, fewer red blood cell and platelet transfusions, reduced use of anti-infectives and parenteral nutrition, and reduced hospital costs compared with ABMT. The overall conclusion is that the treatment costs of PBPCT are 15 to 30% lower than the treatment costs of ABMT. However, a formal comparison between PBPCT and ABMT, assessing the differences in toxicity, costs and quality of life, is still awaited. PMID:10160468

  8. Changes in different parameters, lymphocyte proliferation and hematopoietic progenitor colony formation in EAE mice treated with myelin oligodendrocyte glycoprotein.

    PubMed

    Doronin, Vasilii B; Parkhomenko, Taisiya A; Korablev, Alexey; Toporkova, Ludmila B; Lopatnikova, Julia A; Alshevskaja, Alina A; Sennikov, Sergei V; Buneva, Valentina N; Budde, Thomas; Meuth, Sven G; Orlovskaya, Irina A; Popova, Nelly A; Nevinsky, Georgy A

    2016-01-01

    Myelin oligodendrocyte glycoprotein (MOG) is an antigen of the myelin sheath, which may trigger immune cell responses and the production of auto-antibodies in multiple sclerosis (MS). In this study, we used MOG(35-55) -induced experimental autoimmune encephalomyelitis (EAE), a model of human MS, to assess the production of catalytically active immunoglobulin G (IgG) antibodies or abzymes which have been shown to be present in sera of patients with several autoimmune diseases. Here, we show that IgGs from the sera of control C57BL/6 mice are catalytically inactive. During development of EAE, a specific reorganization of the immune system of mice occurred leading to a condition which was associated with the generation of catalytically active IgGs hydrolysing DNA, myelin basic protein (MBP) and MOG which was associated with increased proteinuria, changes in differentiation of mice bone marrow hematopoietic stem cells (HSCs) and an increase in proliferation of lymphocytes in bone marrow, spleen and thymus as well as a significant suppression of cell apoptosis in these organs. The strongest alterations were found in the early disease phase (18-24 days after immunization) and were less pronounced in later EAE stages (40 days after EAE induction). We conclude that a significant increase in DNase and proteolytic activities of antibodies may be considered the earliest statistically significant marker of MOG-induced EAE in mice. The possible differences in immune system reorganizations during preclinical phases of the disease, acute and late EAE, leading to production of different auto-antibodies and abzymes as well other changes are discussed. PMID:26493273

  9. Stem cells are units of natural selection for tissue formation, for germline development, and in cancer development.

    PubMed

    Weissman, Irving L

    2015-07-21

    It is obvious that natural selection operates at the level of individuals and collections of individuals. Nearly two decades ago we showed that in multi-individual colonies of protochordate colonial tunicates sharing a blood circulation, there exists an exchange of somatic stem cells and germline stem cells, resulting in somatic chimeras and stem cell competitions for gonadal niches. Stem cells are unlike other cells in the body in that they alone self-renew, so that they form clones that are perpetuated for the life of the organism. Stem cell competitions have allowed the emergence of competitive somatic and germline stem cell clones. Highly successful germline stem cells usually outcompete less successful competitors both in the gonads of the genotype partner from which they arise and in the gonads of the natural parabiotic partners. Therefore, natural selection also operates at the level of germline stem cell clones. In the colonial tunicate Botryllus schlosseri the formation of natural parabionts is prevented by a single-locus highly polymorphic histocompatibility gene called Botryllus histocompatibility factor. This limits germline stem cell predation to kin, as the locus has hundreds of alleles. We show that in mice germline stem cells compete for gonad niches, and in mice and humans, blood-forming stem cells also compete for bone marrow niches. We show that the clonal progression from blood-forming stem cells to acute leukemias by successive genetic and epigenetic events in blood stem cells also involves competition and selection between clones and propose that this is a general theme in cancer. PMID:26195745

  10. Stem cells are units of natural selection for tissue formation, for germline development, and in cancer development

    PubMed Central

    Weissman, Irving L.

    2015-01-01

    It is obvious that natural selection operates at the level of individuals and collections of individuals. Nearly two decades ago we showed that in multi-individual colonies of protochordate colonial tunicates sharing a blood circulation, there exists an exchange of somatic stem cells and germline stem cells, resulting in somatic chimeras and stem cell competitions for gonadal niches. Stem cells are unlike other cells in the body in that they alone self-renew, so that they form clones that are perpetuated for the life of the organism. Stem cell competitions have allowed the emergence of competitive somatic and germline stem cell clones. Highly successful germline stem cells usually outcompete less successful competitors both in the gonads of the genotype partner from which they arise and in the gonads of the natural parabiotic partners. Therefore, natural selection also operates at the level of germline stem cell clones. In the colonial tunicate Botryllus schlosseri the formation of natural parabionts is prevented by a single-locus highly polymorphic histocompatibility gene called Botryllus histocompatibility factor. This limits germline stem cell predation to kin, as the locus has hundreds of alleles. We show that in mice germline stem cells compete for gonad niches, and in mice and humans, blood-forming stem cells also compete for bone marrow niches. We show that the clonal progression from blood-forming stem cells to acute leukemias by successive genetic and epigenetic events in blood stem cells also involves competition and selection between clones and propose that this is a general theme in cancer. PMID:26195745

  11. Chemotactic-based adaptive self-organization during colonial development

    NASA Astrophysics Data System (ADS)

    Cohen, Inon; Czirók, Andras; Ben-Jacob, Eshel

    1996-02-01

    Bacterial colonies have developed sophisticated modes of cooperative behavior which enable them to respond to adverse growth conditions. It has been shown that such behavior can be manifested in the development of complex colonial patterns. Certain bacterial species exhibit formation of branching patterns during colony development. Here we present a generic model to describe such patterning of swimming (tumbling) bacteria on agar surfaces. The model incorporates: (1) food diffusion, (2) reproduction and sporulation of the cells, (3) movement of the bacterial cells within a self-produced wetting fluid and (4) chemotactic signaling. As a plausible explanation for transitions between different branching morphologies, we propose an interplay between chemotaxis towards food, self-produced short range chemoattractant and long range chemorepellent.

  12. Structural and functional domains of the myb oncogene: requirements for nuclear transport, myeloid transformation, and colony formation.

    PubMed Central

    Ibanez, C E; Lipsick, J S

    1988-01-01

    The v-myb oncogene of avian myeloblastosis virus causes acute myelomonocytic leukemia in vivo and transforms only myeloid cells in vitro. Its product, p48v-myb, is a nuclear protein of unknown function. To determine structure-function relationships for this protein, we constructed a series of deletion mutants of v-myb, expressed them in retroviral vectors, and studied their biochemical and biological properties. We used these mutants to identify two separate domains of p48v-myb which had distinct roles in its accumulation in the cell nucleus. We showed that the viral sequences which normally encode both termini of p48v-myb were dispensible for transformation. In contrast, both copies of the highly conserved v-myb amino-terminal repeat were required for transformation. We also identified a carboxyl-terminal domain of p48v-myb which was required for the growth of v-myb-transformed myeloblasts in soft agar but not for morphological transformation. Images PMID:2835503

  13. Sox9 is a β-catenin-regulated transcription factor that enhances the colony-forming activity of squamous cell carcinoma cells.

    PubMed

    Li, Xue Mei; Piao, Yong Jun; Sohn, Kyung-Cheol; Ha, Jeong-Min; Im, Myung; Seo, Young-Joon; Whang, Kyu Uang; Lee, Jeung-Hoon; Lee, Young; Kim, Chang Deok

    2016-07-01

    Squamous cell carcinoma (SCC) is a common skin cancer, of which the incidence is relatively high, ranking second among the non‑melanoma skin cancers. It is known that numerous intracellular signal regulators are involved in the pathogenesis of SCC. The Wnt/β-catenin signaling pathway serves an important role in cancer development. However, the downstream effectors of β‑catenin remain to be clearly elucidated yet. The present study investigated the functional importance of Wnt/β‑catenin signaling in cutaneous SCC. β‑catenin expression was reduced using recombinant adenovirus expressing specific microRNA (miR). Knockdown of β‑catenin resulted in a marked reduction of the colony-forming activity of the SCC cells, SCC12. In an attempt to identify the β‑catenin downstream genes, it was found that Sox9 was regulated by β‑catenin in SCC12 cells. Overexpression of a constitutively active form of β‑catenin led to the induction of Sox9, while knockdown of β‑catenin resulted in downregulation of Sox9. When the expression of Sox9 was reduced using specific miR, colony-forming activity of the SCC12 cells was significantly reduced. When Sox9 was overexpressed in cells where β‑catenin was knocked down, it partially restored the colony‑forming potential. Taken together, the present results suggested that Sox9 is a β-catenin downstream transcription factor and is positively involved in SCC development. PMID:27151141

  14. Contact formation in gallium arsenide solar cells

    NASA Technical Reports Server (NTRS)

    Weizer, Victor G.; Fatemi, Navid S.

    1988-01-01

    Gold and gold-based alloys, commonly used as solar cell contact materials, are known to react readily with gallium arsenide. Experiments were performed to identify the mechanisms involved in these GaAs-metal interactions. It is shown that the reaction of GaAs with gold takes place via a dissociative diffusion process. It is shown further that the GaAs-metal reaction rate is controlled to a very great extent by the condition of the free surface of the contact metal, an interesting example of which is the previously unexplained increase in the reaction rate that has been observed for samples annealed in a vacuum environment as compared to those annealed in a gaseous ambient. A number of other hard-to-explain observations, such as the low-temperature formation of voids in the gold lattice and crystallite growth on the gold surface, are explained by invoking this mechanism.

  15. Lignin Formation in Wheat Coleoptile Cell Walls

    PubMed Central

    Whitmore, F. W.

    1971-01-01

    Four growth-influencing compounds—hydroxyproline, 2,2′-dipyridyl, 2-chloroethylphosphonic acid, and indoleacetic acid—were used to examine the relationship between lignin formation and growth of wheat coleoptile sections. Hydroxyproline and 2-chloroethylphosphonic acid, at low concentrations, inhibited growth and increased lignin content. Dipyridyl, which promoted coleoptile elongation, decreased lignin content. Indoleacetic acid caused a 300% increase in growth at 0.1 mm but resulted in lignin content no different from controls with no auxin. Chemical and anatomical evidence is given which indicates that lignin is present in the epidermal cell walls of the wheat coleoptile. It is thus possible that bonding between lignin and hemicellulose may have some influence on coleoptile growth. Images PMID:16657843

  16. Theoretical size controls of the giant Phaeocystis globosa colonies

    NASA Astrophysics Data System (ADS)

    Liu, Xiao; Smith, Walker O.; Tang, Kam W.; Doan, Nhu Hai; Nguyen, Ngoc Lam

    2015-06-01

    An unusual characteristic of the cosmopolitan haptophyte Phaeocystis globosa is its ability to form colonies of strikingly large size-up to 3 cm in diameter. The large size and the presence of a mucoid envelope are believed to contribute to the formation of dense blooms in Southeast Asia. We collected colonies of different sizes in shallow coastal waters of Viet Nam and conducted a series of measurements and experiments on individual colonies. Using these empirical data, we developed a simple carbon-based model to predict the growth and maximal size of P. globosa colonies. Our model suggests that growth of a colony from 0.2 cm to 1.4 cm (the maximal size in our samples) would take 16 days. This number, however, is strongly influenced by the maximal photosynthetic rate and other physiological parameters used in the model. The model also returns a specific growth rate of 0.30 d-1 for colonial cells, comparable to satellite estimates, but lower than have been measured for unicellular P. globosa in batch culture at similar temperatures. We attribute this low growth rate to not only the model uncertainties, but factors such as self-shading and diffusive limitation of nutrient uptake.

  17. Plerixafor and abbreviated-course granulocyte colony-stimulating factor for mobilizing hematopoietic progenitor cells in light chain amyloidosis.

    PubMed

    Dhakal, Binod; Strouse, Christopher; D'Souza, Anita; Arce-Lara, Carlos; Esselman, Jeanie; Eastwood, Daniel; Pasquini, Marcelo; Saber, Wael; Drobyski, William; Rizzo, J Douglas; Hari, Parameswaran N; Hamadani, Mehdi

    2014-12-01

    Cytokine-based mobilization in light chain (AL) amyloidosis is frequently complicated by fluid overload, weight gain, cardiac arrhythmias, and peri-mobilization mortality. We analyzed hematopoietic progenitor cells (HPC) mobilization outcomes in 49 consecutive AL amyloidosis patients at our institution between 2004 and 2013 with granulocyte colony-stimulating factor (G) (10 μg/kg/day) (n = 25) versus an institutional protocol to limit G exposure using plerixafor (P) (.24 mg/kg s.c. starting day 3 of G 10 μg/kg) (n = 24). G+P strategy yielded higher total CD34(+) cells/kg (12.8 × 10(6) versus 6.3 × 10(6); P < .001) and CD34(+) cells/kg collected on day 1 (10.8 × 10(6) versus 4.9 × 10(6), P = .004) compared with the G cohort. More G+P patients collected ≥5 × 10(6) CD34(+) HPCs/kg (22 versus 16, P = .02) and ≥ 10 × 10(6) CD34(+) HPCs/kg (13 versus 5, P = .01). Four patients (16%) had mobilization failure with G; none with G+P. Peri-mobilization weight gain was lower with G+P strategy (median weight gain 1 versus 7 pounds, P = .009). Numbers of apheresis sessions (median, 1 versus 1, P = .52), number of hospitalization days (median, 1.1 versus 1.6, P = .52), transfusions, use of intravenous antibiotics, and cardiac arrhythmias were similar. In conclusion, our study demonstrates that upfront use of G+P as a mobilization strategy results in superior HPC collection, no mobilization failures, and less weight gain than G alone. PMID:25111581

  18. Promoting the selection and maintenance of fetal liver stem/progenitor cell colonies by layer-by-layer polypeptide tethered supported lipid bilayer.

    PubMed

    Lee, I-Chi; Liu, Yung-Chiang; Tsai, Hsuan-Ang; Shen, Chia-Ning; Chang, Ying-Chih

    2014-12-10

    In this study, we designed and constructed a series of layer-by-layer polypeptide adsorbed supported lipid bilayer (SLB) films as a novel and label-free platform for the isolation and maintenance of rare populated stem cells. In particular, four alternative layers of anionic poly-l-glutamic acid and cationic poly-l-lysine were sequentially deposited on an anionic SLB. We found that the fetal liver stem/progenitor cells from the primary culture were selected and formed colonies on all layer-by-layer polypeptide adsorbed SLB surfaces, regardless of the number of alternative layers and the net charges on those layers. Interestingly, these isolated stem/progenitor cells formed colonies which were maintained for an 8 day observation period. Quartz crystal microbalance with dissipation measurements showed that all SLB-polypeptide films were protein resistant with serum levels significantly lower than those on the polypeptide multilayer films without an underlying SLB. We suggest the fluidic SLB promotes selective binding while minimizing the cell-surface interaction due to its nonfouling nature, thus limiting stem cell colonies from spreading. PMID:25243588

  19. Vitrified canine testicular cells allow the formation of spermatogonial stem cells and seminiferous tubules following their xenotransplantation into nude mice

    PubMed Central

    Lee, Kyung Hoon; Lee, Won Young; Kim, Dong Hoon; Lee, Seung Hoon; Do, Jung Tae; Park, Chankyu; Kim, Jae Hwan; Choi, Young Suk; Song, Hyuk

    2016-01-01

    Belgian Malinois (BM), one of the excellent military dog breeds in South Korea, is usually castrated before sexual maturation. Therefore, the transfer of their genetic features to the next generation is difficult. To overcome this, testicular cells from 4-month-old BMs were frozen. Testicular cells were thawed after 3 months and cultured in StemPro-34 medium. Spermatogonial stem cell (SSC) characteristics were determined by the transplantation of the cultured germ cell-derived colonies (GDCs) into empty testes, containing only several endogenous SSCs and Sertoli cells, of immunodeficient mice, 4 weeks after busulfan treatment. Following the implantation, the transplanted cells localized in the basement membrane of the seminiferous tubules, and ultimately colonized the recipient testes. Xenotransplantation of GDCs together with testicular somatic cells conjugated with extracellular matrix (ECM), led to the formation of de novo seminiferous tubules. These seminiferous tubules were mostly composed of Sertoli cells. Some germ cells were localized in the basement membrane of seminiferous tubules. This study revealed that BM-derived SSCs, obtained from the castrated testes, might be a valuable tool for the transfer of BM genetic features to the next generation. PMID:26907750

  20. Vitrified canine testicular cells allow the formation of spermatogonial stem cells and seminiferous tubules following their xenotransplantation into nude mice.

    PubMed

    Lee, Kyung Hoon; Lee, Won Young; Kim, Dong Hoon; Lee, Seung Hoon; Do, Jung Tae; Park, Chankyu; Kim, Jae Hwan; Choi, Young Suk; Song, Hyuk

    2016-01-01

    Belgian Malinois (BM), one of the excellent military dog breeds in South Korea, is usually castrated before sexual maturation. Therefore, the transfer of their genetic features to the next generation is difficult. To overcome this, testicular cells from 4-month-old BMs were frozen. Testicular cells were thawed after 3 months and cultured in StemPro-34 medium. Spermatogonial stem cell (SSC) characteristics were determined by the transplantation of the cultured germ cell-derived colonies (GDCs) into empty testes, containing only several endogenous SSCs and Sertoli cells, of immunodeficient mice, 4 weeks after busulfan treatment. Following the implantation, the transplanted cells localized in the basement membrane of the seminiferous tubules, and ultimately colonized the recipient testes. Xenotransplantation of GDCs together with testicular somatic cells conjugated with extracellular matrix (ECM), led to the formation of de novo seminiferous tubules. These seminiferous tubules were mostly composed of Sertoli cells. Some germ cells were localized in the basement membrane of seminiferous tubules. This study revealed that BM-derived SSCs, obtained from the castrated testes, might be a valuable tool for the transfer of BM genetic features to the next generation. PMID:26907750

  1. Deadly competition between sibling bacterial colonies

    NASA Astrophysics Data System (ADS)

    Be'Er, Avraham

    2011-03-01

    As a result of stress due to nutrient limitation or antibiotics, competing individual bacteria within a single colony may lyse sibling cells to release nutrients (cannibalism) or DNA (fratricide). However, we have recently shown that competition is not limited to individuals, but can occur at the colony level [A. Be'er et al., PNAS 106, 428 (2009); A. Be'er et al., PNAS 107, 6258 (2010).] In response to the presence of an encroaching sibling colony, Paenibacillus dendritiformis bacteria secrete a lethal protein, lysing cells at the interface between the colonies. Analysis of the proteins secreted by these competing sibling colonies, combined with a mathematical model, shows how colonies maintain their growth by self-regulating the secretion of two proteins: subtilisin (a well-known growth promoter), and Slf (a previously unknown protein, which is lethal). The results also explain why a single colony is not inhibited by its own secretions.

  2. Cell-cell interactions impacts on the rate of swarm expansion and the edge shape of a colony swarming Pseudomonas aeruginosa

    NASA Astrophysics Data System (ADS)

    Amiri, Aboutaleb; Tierra, Giordano; Xu, Zhiliang; Shrout, Joshua; Alber, Mark

    Collective motion has been observed by several bacterial species including the pathogenic bacterium P. aeruginosa. A flagellum at the pole is known to generate a self-propulsion motion. However, the role of type IV pili (TFP), distributed on the cell membrane, during swarming needs to be investigated in more details. In this work we introduce a model that combines the hydrodynamic and biophysical interactions in order to study the impact of the TFP interactions on swarming behavior of the colony. The model describes the motion and interactions of rod-shaped self propelled bacteria inside a thin liquid film. It also includes the equations describing the production and diffusion of surfactant rhamnolipids that is responsible for extraction of water from substrate, and Marangoni driven expansion of the thin liquid film by altering the surface tension. We show that TFP interactions are responsible for slower expansion rate of colonies of TFP deficient mutants compared to wild type. Experimental observations were used to calibrate the model and verify the model assumptions and predictions.

  3. The Alternative Role of Enterobactin as an Oxidative Stress Protector Allows Escherichia coli Colony Development

    PubMed Central

    Peralta, Daiana R.; Pomares, María Fernanda; de Cristóbal, Ricardo E.; Vincent, Paula A.

    2014-01-01

    Numerous bacteria have evolved different iron uptake systems with the ability to make use of their own and heterologous siderophores. However, there is growing evidence attributing alternative roles for siderophores that might explain the potential adaptive advantages of microorganisms having multiple siderophore systems. In this work, we show the requirement of the siderophore enterobactin for Escherichia coli colony development in minimal media. We observed that a strain impaired in enterobactin production (entE mutant) was unable to form colonies on M9 agar medium meanwhile its growth was normal on LB agar medium. Given that, neither iron nor citrate supplementation restored colony growth, the role of enterobactin as an iron uptake-facilitator would not explain its requirement for colony development. The absence of colony development was reverted either by addition of enterobactin, the reducing agent ascorbic acid or by incubating in anaerobic culture conditions with no additives. Then, we associated the enterobactin requirement for colony development with its ability to reduce oxidative stress, which we found to be higher in media where the colony development was impaired (M9) compared with media where the strain was able to form colonies (LB). Since oxyR and soxS mutants (two major stress response regulators) formed colonies in M9 agar medium, we hypothesize that enterobactin could be an important piece in the oxidative stress response repertoire, particularly required in the context of colony formation. In addition, we show that enterobactin has to be hydrolyzed after reaching the cell cytoplasm in order to enable colony development. By favoring iron release, hydrolysis of the enterobactin-iron complex, not only would assure covering iron needs, but would also provide the cell with a molecule with exposed hydroxyl groups (hydrolyzed enterobactin). This molecule would be able to scavenge radicals and therefore reduce oxidative stress. PMID:24392154

  4. Biological role of granulocyte macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) on cells of the myeloid lineage.

    PubMed

    Ushach, Irina; Zlotnik, Albert

    2016-09-01

    M-CSF and GM-CSF are 2 important cytokines that regulate macrophage numbers and function. Here, we review their known effects on cells of the macrophage-monocyte lineage. Important clues to their function come from their expression patterns. M-CSF exhibits a mostly homeostatic expression pattern, whereas GM-CSF is a product of cells activated during inflammatory or pathologic conditions. Accordingly, M-CSF regulates the numbers of various tissue macrophage and monocyte populations without altering their "activation" status. Conversely, GM-CSF induces activation of monocytes/macrophages and also mediates differentiation to other states that participate in immune responses [i.e., dendritic cells (DCs)]. Further insights into their function have come from analyses of mice deficient in either cytokine. M-CSF signals through its receptor (CSF-1R). Interestingly, mice deficient in CSF-1R expression exhibit a more significant phenotype than mice deficient in M-CSF. This observation was explained by the discovery of a novel cytokine (IL-34) that represents a second ligand of CSF-1R. Information about the function of these ligands/receptor system is still developing, but its complexity is intriguing and strongly suggests that more interesting biology remains to be elucidated. Based on our current knowledge, several therapeutic molecules targeting either the M-CSF or the GM-CSF pathways have been developed and are currently being tested in clinical trials targeting either autoimmune diseases or cancer. It is intriguing to consider how evolution has directed these pathways to develop; their complexity likely mirrors the multiple functions in which cells of the monocyte/macrophage system are involved. PMID:27354413

  5. Pre-B cell colony enhancing factor induces Nampt-dependent translocation of the insulin receptor out of lipid microdomains in A549 lung epithelial cells.

    PubMed

    Peng, Qianyi; Jia, Song Hui; Parodo, Jean; Ai, Yuhang; Marshall, John C

    2015-02-15

    Pre-B cell colony-enhancing factor (PBEF) is a highly conserved pleiotropic protein reported to be an alternate ligand for the insulin receptor (IR). We sought to clarify the relationship between PBEF and insulin signaling by evaluating the effects of PBEF on the localization of the IRβ chain to lipid rafts in A549 epithelial cells. We isolated lipid rafts from A549 cells and detected the IR by immunoprecipitation from raft fractions or whole cell lysates. Cells were treated with rPBEF, its enzymatic product nicotinamide adenine dinucleotide (NAD), or the Nampt inhibitor daporinad to study the effect of PBEF on IRβ movement. We used coimmunoprecipitation studies in cells transfected with PBEF and IRβ constructs to detect interactions between PBEF, the IRβ, and caveolin-1 (Cav-1). PBEF was present in both lipid raft and nonraft fractions, whereas the IR was found only in lipid raft fractions of resting A549 cells. The IR-, PBEF-, and Cav-1-coimmunoprecipitated rPBEF treatment resulted in the movement of IRβ- and tyrosine-phosphorylated Cav-1 from lipid rafts to nonrafts, an effect that could be blocked by daporinad, suggesting that this effect was facilitated by the Nampt activity of PBEF. The addition of PBEF to insulin-treated cells resulted in reduced Akt phosphorylation of both Ser⁴⁷³ and Thr³⁰⁸. We conclude that PBEF can inhibit insulin signaling through the IR by Nampt-dependent promotion of IR translocation into the nonraft domains of A549 epithelial cells. PBEF-induced alterations in the spatial geometry of the IR provide a mechanistic explanation for insulin resistance in inflammatory states associated with upregulation of PBEF. PMID:25516545

  6. Antibodies binding granulocyte-macrophage colony stimulating factor produced by cord blood-derived B cell lines immortalized by Epstein-Barr virus in vitro.

    PubMed

    Revoltella, R P; Laricchia Robbio, L; Liberati, A M; Reato, G; Foa, R; Funaro, A; Vinante, F; Pizzolo, G

    2000-09-15

    We detected natural antibodies (auto-Abs) binding human granulocyte-macrophage colony stimulating factor (GM-CSF) in umbilical cord blood (CB) (23 of 94 samples screened) and peripheral blood of women at the end of pregnancy (6 of 42 samples tested). To demonstrate that Abs detected in CB were produced by the fetus, CB mononuclear cells were infected with Epstein-Barr virus in vitro. Ten cell lines producing constitutively anti-recombinant human GM-CSF (rhGM-CSF) Abs were isolated and characterized. These cells displayed a male karyotype, an early activated B cell phenotype, coexpressed surface IgM and IgD, and secreted only IgM with prevailing lambda clonal restriction. Specific cell surface binding of biotinylated rhGM-CSF and high-level anti-rhGM-CSF IgM Ab production were typical features of early cell cultures. In late cell passages the frequency of more undifferentiated B cells increased. Serum Abs of either maternal or fetal origin or Abs produced in culture did not affect the granulocyte and macrophage colony stimulating activity of rhGM-CSF from bone marrow progenitors in soft agar, suggesting that the Abs produced were nonneutralizing. PMID:11069719

  7. Augmentation of erythroid burst formation by the addition of thymocytes and other myelo-lymphoid cells.

    PubMed

    Kanamaru, A; Durban, E; Gallagher, M T; Miller, S C; Trentin, J J

    1980-08-01

    Bone marrow from barrier-sustained specific pathogen-free (SPF) CBA and C57BL/6 mice gave relatively low numbers of BFU-E colonies in methylcellulose culture, as compared to conventional mice. Addition of thymocytes to the marrow cultures increased the yield of BFU-E colonies more than fourfold in SPF mice but only 1.5-fold in conventional mice. Colony size was also increased. Increased yield of BFU-E colonies was also obtained by co-culture of bone marrow with lymph node cells or with bone marrow or spleen cells from 900R whole-body irradiated mice. The effect appeared to be cellular rather than humoral. It was not reproduced by conditioned medium from thymus or pokeweed mitogen stimulated spleen cells. The helper effect of thymus cells was eliminated or reduced by freezing and thawing, or by 48 hours of incubation after irradiation. Treatment of bone marrow cells in vitro with anti-theta serum and complement did not decrease the number of BFU-E colonies. The putative helper cells appear not to be T cells, were non-adherent to the plastic culture dish, and were cortisone resistant and radioresistant. The low BFU-E colony yield from SPF mouse marrow is presumed to be largely the result of deficiency of these non-T helper cells in SPF bone marrow, rather than of BFU-E progenitor cells. PMID:6447706

  8. Focal Adhesion Kinase regulates cell-cell contact formation in epithelial cells via modulation of Rho

    SciTech Connect

    Playford, Martin P.; Vadali, Kavita; Cai Xinming; Burridge, Keith; Schaller, Michael D.

    2008-10-15

    Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase that plays a key role in cellular processes such as cell adhesion, migration, proliferation and survival. Recent studies have also implicated FAK in the regulation of cell-cell adhesion. Here, evidence is presented showing that siRNA-mediated suppression of FAK levels in NBT-II cells and expression of dominant negative mutants of FAK caused loss of epithelial cell morphology and inhibited the formation of cell-cell adhesions. Rac and Rho have been implicated in the regulation of cell-cell adhesions and can be regulated by FAK signaling. Expression of active Rac or Rho in NBT-II cells disrupted formation of cell-cell contacts, thus promoting a phenotype similar to FAK-depleted cells. The loss of intercellular contacts in FAK-depleted cells is prevented upon expression of a dominant negative Rho mutant, but not a dominant negative Rac mutant. Inhibition of FAK decreased tyrosine phosphorylation of p190RhoGAP and elevated the level of GTP-bound Rho. This suggests that FAK regulates cell-cell contact formation by regulation of Rho.

  9. Enhancement of the grafting efficiency of transplanted marrow cells by preincubation with interleukin-3 and granulocyte-macrophage colony-stimulating factor

    SciTech Connect

    Tavassoli, M.; Konno, M.; Shiota, Y.; Omoto, E.; Minguell, J.J.; Zanjani, E.D.

    1991-04-01

    To improve the grafting efficiency of transplanted murine hematopoietic progenitors, we briefly preincubated mouse bone marrow cells with interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF) ex vivo before their transplantation into irradiated recipients. This treatment was translated into an increase in the seeding efficiency of colony-forming unit-spleen (CFU-S) and CFU-GM after transplantation. Not only was the concentration of CFU-S in the tibia increased 2 and 24 hours after transplantation, but the total cell number and CFU-S and CFU-GM concentrations were persistently higher in IL-3- and GM-CSF-treated groups 1 to 3 weeks after transplantation. In addition, the survival of animals as a function of transplanted cell number was persistently higher in IL-3- and GM-CSF-treated groups compared with controls. The data indicate that the pretreatment of marrow cells with IL-3 and GM-CSF before transplantation increases the seeding efficiency of hematopoietic stem cells and probably other progenitor cells after transplantation. This increased efficiency may be mediated by upward modulation of homing receptors. Therefore, ex vivo preincubation of donor marrow cells with IL-3 and GM-CSF may be a useful tactic in bone marrow transplantation.

  10. Changes in numbers and types of mast cell colony-forming cells in the peritoneal cavity of mice after injection of distilled water: evidence that mast cells suppress differentiation of bone marrow-derived precursors

    SciTech Connect

    Kanakura, Y.; Kuriu, A.; Waki, N.; Nakano, T.; Asai, H.; Yonezawa, T.; Kitamura, Y.

    1988-03-01

    Two different types of cells in the peritoneal cavity of mice produce mast cell colonies in methylcellulose. Large mast cell colonies are produced by bone marrow-derived precursors resembling lymphoid cells by light microscopy (L-CFU-Mast), whereas medium and small mast cell colonies are produced by morphologically identifiable mast cells (M-CFU-Mast and S-CFU-Mast, respectively). In the present study we eradicated peritoneal mast cells by intraperitoneal (IP) injection of distilled water. The regeneration process was investigated to clarify the relationship between L-CFU-Mast, M-CFU-Mast, and S-CFU-Mast. After injection of distilled water, M-CFU-Mast and S-CFU-Mast disappeared, but L-CFU-Mast increased, and then M-CFU-Mast and S-CFU-Mast appeared, suggesting the presence of a hierarchic relationship. When purified peritoneal mast cells were injected two days after the water injection, the L-CFU-Mast did not increase. In the peritoneal cavity of WBB6F1-+/+ mice that had been lethally irradiated and rescued by bone marrow cells of C57BL/6-bgJ/bgJ (beige, Chediak-Higashi syndrome) mice, L-CFU-Mast were of bgJ/bgJ type, but M-CFU-Mast and S-CFU-Mast were of +/+ type. The injection of distilled water to the radiation chimeras resulted in the development of bgJ/bgJ-type M-CFU-Mast and then S-CFU-Mast. The presence of mast cells appeared to suppress the recruitment of L-CFU-Mast from the bloodstream and to inhibit the differentiation of L-CFU-Mast to M-CFU-Mast.

  11. Macrophage production during murine listeriosis: colony-stimulating factor 1 (CSF-1) and CSF-1-binding cells in genetically resistant and susceptible mice.

    PubMed Central

    Cheers, C; Stanley, E R

    1988-01-01

    The concentration of the macrophage-specific colony-stimulating factor (CSF-1) and the numbers of bone marrow and spleen cells with specific receptors for that factor have been investigated in a number of mouse strains under normal conditions and after infection with the facultative intracellular bacterium Listeria monocytogenes. The CSF-1 concentration in serum and tissue was markedly elevated in infected mice, the degree of stimulation reflecting the dose of L. monocytogenes. The CSF-1 titer did not correlate with genetic resistance or susceptibility of the mice to L. monocytogenes. In contrast to the effect of lipopolysaccharide, Listeria infection was able to increase the level of CSF-1 in the lipopolysaccharide nonresponder strain C3H/HeJ. In line with earlier findings on colony-forming cells, cells bearing receptors for CSF-1 in uninfected susceptible BALB/cJ mice were only half those in resistant C57BL/6J mice. After infection the majority of these cells disappeared from the bone marrow and spleen cells of both resistant and susceptible mice. The number of CSF-1 receptor-bearing cells in the normal bone marrow may determine the degree of resistance to L. monocytogenes. PMID:3262588

  12. Human recombinant granulocyte-macrophage colony-stimulating factor increases cell-to-cell adhesion and surface expression of adhesion-promoting surface glycoproteins on mature granulocytes.

    PubMed Central

    Arnaout, M A; Wang, E A; Clark, S C; Sieff, C A

    1986-01-01

    Human granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to inhibit migration of mature granulocytes and to enhance their antibody-dependent cellular cytotoxicity. We found that human recombinant GM-CSF also enhanced granulocyte-granulocyte adhesion and increased by two- to threefold the surface expression of Mo1 and LeuM5 (P150, 95), two members of a family of leukocyte adhesion molecules (Leu-CAM). Increased Mo1 surface expression occurred within 15 min at 37 degrees C and was maximal at the migration inhibitory concentration of 500 pM. One-half maximal rise in the expression of Mo1 on the cell surface occurred at 5 pM. The chemotactic peptide f-Met-Leu-Phe produced a comparable rise in surface Mo1 with one-half maximal expression occurring at 7 nM. Both GM-CSF and f-Met-Leu-Phe produced optimal granulocyte-granulocyte adhesion at 500 pM and 100 nM, respectively. This adhesion-promoting effect induced by either stimulus was inhibited by a mouse monoclonal antibody directed against Mo1 antigen. These data indicate that GM-CSF promotes cell-to-cell adhesion, presumably through enhanced expression of leukocyte adhesion molecules. This mechanism may explain, in part, the known effects of GM-CSF on the function of mature granulocytes. Images PMID:3090106

  13. The effects of vitamin D binding protein-macrophage activating factor and colony-stimulating factor-1 on hematopoietic cells in normal and osteopetrotic rats.

    PubMed

    Benis, K A; Schneider, G B

    1996-10-15

    Osteopetrosis is a heterogeneous group of bone disorders characterized by the failure of osteoclasts to resorb bone and by several immunological defects including macrophage dysfunction. Two compounds, colony-stimulating factor-1 (CSF-1) and vitamin D-binding protein-macrophage activating factor (DBP-MAF) were used in the present study to evaluate their effects on the peritoneal population of cells and on cells within the bone marrow microenvironment in normal and incisors absent (ia) osteopetrotic rats. Previous studies in this laboratory have demonstrated that administration of DBP-MAF to newborn ia animals results in a substantial increase in bone marrow cavity size due to upregulated osteoclast function. To study the effects of these compounds on the macrophage/osteoclast precursors, DBP-MAF, CSF-1, and the combination of these compounds were given to newborn ia and normal littermate animals. Both the normal and mutant phenotypes responded similarly when treated with these compounds. Rats exhibited a profound shift toward the macrophage lineage from the neutrophil lineage when compared with vehicle-treated control animals after treatment with these compounds. In the in vivo peritoneal lavage study, animals received injections of CSF-1, DBP-MAF or DBP-MAF/CSF-1 over a 4-week period. The various types of cells in the peritoneal cavity were then enumerated. The in vitro study consisted of cells isolated from the bone marrow microenvironment and cultured on feeder layers of CSF-1, DBP-MAF, or DBP-MAF/CSF-1 for colony enumeration. The increase in macrophage numbers at the expense of neutrophil numbers could be seen in both the in vivo and in vitro experiments. The macrophage/osteoclast and neutrophil lineages have a common precursor, the granulocyte/macrophage colony-forming cell (GM-CFC). With the addition of CSF-1, the GM-CFC precursor may be induced into the macrophage/osteoclast lineage rather than the granulocyte lineage. This increased pool of cells in the

  14. Lipocalin-2 inhibits osteoclast formation by suppressing the proliferation and differentiation of osteoclast lineage cells

    SciTech Connect

    Kim, Hyun-Ju; Yoon, Hye-Jin; Yoon, Kyung-Ae; Gwon, Mi-Ri; Jin Seong, Sook; Suk, Kyoungho; Kim, Shin-Yoon; Yoon, Young-Ran

    2015-06-10

    Lipocalin-2 (LCN2) is a member of the lipocalin superfamily and plays a critical role in the regulation of various physiological processes, such as inflammation and obesity. In this study, we report that LCN2 negatively modulates the proliferation and differentiation of osteoclast precursors, resulting in impaired osteoclast formation. The overexpression of LCN2 in bone marrow-derived macrophages or the addition of recombinant LCN2 protein inhibits the formation of multinuclear osteoclasts. LCN2 suppresses macrophage colony-stimulating factor (M-CSF)-induced proliferation of osteoclast precursor cells without affecting their apoptotic cell death. Interestingly, LCN2 decreases the expression of the M-CSF receptor, c-Fms, and subsequently blocks its downstream signaling cascades. In addition, LCN2 inhibits RANKL-induced osteoclast differentiation and attenuates the expression of c-Fos and nuclear factor of activated T cells c1 (NFATc1), which are important modulators in osteoclastogenesis. Mechanistically, LCN2 inhibits NF-κB signaling pathways, as demonstrated by the suppression of IκBα phosphorylation, nuclear translocation of p65, and NF-κB transcriptional activity. Thus, LCN2 is an anti-osteoclastogenic molecule that exerts its effects by retarding the proliferation and differentiation of osteoclast lineage cells. - Highlights: • LCN2 expression is regulated during osteoclast development. • LCN2 suppresses M-CSF-mediated osteoclast precursor proliferation. • LCN2 inhibits RANKL-induced osteoclast differentiation.

  15. Closure of supporting cell scar formations requires dynamic actin mechanisms.

    PubMed

    Hordichok, Andrew J; Steyger, Peter S

    2007-10-01

    In many vertebrate inner ear sensory epithelia, dying sensory hair cells are extruded, and the apices of surrounding supporting cells converge to re-seal the epithelial barrier between the electrochemically-distinct endolymph and perilymph. These cellular mechanisms remain poorly understood. Dynamic microtubular mechanisms have been proposed for hair cell extrusion; while contractile actomyosin-based mechanisms are required for cellular extrusion and closure in epithelial monolayers. The hypothesis that cytoskeletal mechanisms are required for hair cell extrusion and supporting cell scar formation was tested using bullfrog saccules incubated with gentamicin (6h), and allowed to recover (18h). Explants were then fixed, labeled for actin and cytokeratins, and viewed with confocal microscopy. To block dynamic cytoskeletal processes, disruption agents for microtubules (colchicine, paclitaxel) myosin (Y-27632, ML-9) or actin (cytochalasin D, latrunculin A) were added during treatment and recovery. Microtubule disruption agents had no effect on hair cell extrusion or supporting cell scar formation. Myosin disruption agents appeared to slow down scar formation but not hair cell extrusion. Actin disruption agents blocked scar formation, and largely prevented hair cell extrusion. These data suggest that actin-based cytoskeletal processes are required for hair cell extrusion and supporting cell scar formation in bullfrog saccules. PMID:17716843

  16. Predicting the Distribution of Spiral Waves from Cell Properties in a Developmental-Path Model of Dictyostelium Pattern Formation

    PubMed Central

    Geberth, Daniel; Hütt, Marc-Thorsten

    2009-01-01

    The slime mold Dictyostelium discoideum is one of the model systems of biological pattern formation. One of the most successful answers to the challenge of establishing a spiral wave pattern in a colony of homogeneously distributed D. discoideum cells has been the suggestion of a developmental path the cells follow (Lauzeral and coworkers). This is a well-defined change in properties each cell undergoes on a longer time scale than the typical dynamics of the cell. Here we show that this concept leads to an inhomogeneous and systematic spatial distribution of spiral waves, which can be predicted from the distribution of cells on the developmental path. We propose specific experiments for checking whether such systematics are also found in data and thus, indirectly, provide evidence of a developmental path. PMID:19593362

  17. Perivascular mast cells regulate vein graft neointimal formation and remodeling

    PubMed Central

    Grassia, Gianluca; Cambrook, Helen; Ialenti, Armando; MacRitchie, Neil; Carberry, Jaclyn; Lawrence, Catherine

    2015-01-01

    Objective. Emerging evidence suggests an important role for mast cells in vein graft failure. This study addressed the hypothesis that perivascular mast cells regulate in situ vascular inflammatory and proliferative responses and subsequent vein graft neointimal lesion formation, using an optimized local mast cell reconstitution method. Methods and Results. Neointimal hyperplasia was induced by insertion of a vein graft into the right carotid artery in wild type and mast cell deficient KitW−sh/W−sh mice. In some experiments, mast cells were reconstituted systemically (tail vein injection of bone marrow-derived mast cells) or locally (directly into the right neck area) prior to vein grafting. Vein graft neointimal lesion formation was significantly (P < 0.05) reduced in KitW−sh/W−sh mice. Mast cell deficiency reduced the number of proliferating cells, and inhibited L-selectin, CCL2, M-CSF and MIP-3α expression in the vein grafts. Local but not systemic mast cell reconstitution restored a perivascular mast cell population that subsequently promoted neointimal formation in mast cell deficient mice. Conclusion. Our data demonstrate that perivascular mast cells play a key role in promoting neointima formation by inducing local acute inflammatory and proliferative responses. These results suggest that ex vivo intraoperative targeting of mast cells may have therapeutic potential for the prevention of pathological vein graft remodeling. PMID:26312183

  18. Trichinella spiralis: nurse cell formation with emphasis on analogy to muscle cell repair

    PubMed Central

    Wu, Zhiliang; Sofronic-Milosavljevic, Lj; Nagano, Isao; Takahashi, Yuzo

    2008-01-01

    Trichinella infection results in formation of a capsule in infected muscles. The capsule is a residence of the parasite which is composed of the nurse cell and fibrous wall. The process of nurse cell formation is complex and includes infected muscle cell response (de-differentiation, cell cycle re-entry and arrest) and satellite cell responses (activation, proliferation and differentiation). Some events that occur during the nurse cell formation are analogous to those occurring during muscle cell regeneration/repair. This article reviews capsule formation with emphasis on this analogy. PMID:18710582

  19. Multipotent hematopoietic cell lines derived from C/EBPalpha(-/-) knockout mice display granulocyte macrophage-colony-stimulating factor, granulocyte- colony-stimulating factor, and retinoic acid-induced granulocytic differentiation.

    PubMed

    Collins, S J; Ulmer, J; Purton, L E; Darlington, G

    2001-10-15

    The transcription factor C/EBPalpha is an important mediator of granulocyte differentiation and regulates the expression of multiple granulocyte-specific genes including the granulocyte-colony-stimulating factor (G-CSF) receptor, neutrophil elastase, and myeloperoxidase. Indeed C/EBPalpha knockout mice display a profound block in granulocyte differentiation. To study this block in granulocytic differentiation in more detail, retroviral vector-mediated transduction of a dominant-negative retinoic acid receptor was used to establish hematopoietic growth factor-dependent, lympho-myeloid progenitor cell lines from the fetal livers of both the C/EBPalpha knockout animals (C/EBPalpha(-/-)) and their heterozygous littermates (C/EBPalpha(+/-)). Surprisingly, the C/EBPalpha(-/-) cell lines displayed significant spontaneous granulocytic differentiation, and this differentiation was markedly enhanced when the cells were stimulated with granulocyte macrophage (GM)-CSF. This GM-CSF-mediated differentiation was associated with the up-regulation of G-CSF receptor mRNA, and the combination of GM-CSF and G-CSF generated more than 95% mature neutrophils in the C/EBPalpha(-/-) cultures. The addition of all-trans retinoic acid also enhanced this granulocytic differentiation of the cultured C/EBPalpha(-/-) cells, indicating that the activated retinoic acid receptors can enhance granulocytic differentiation through a molecular pathway that is independent of C/EBPalpha. These studies clearly indicate that terminal granulocytic differentiation associated with the up-regulation of C/EBPalpha-responsive genes can occur in the absence of C/EBPalpha, and they indicate the existence of multiple independent molecular pathways potentially used by primitive hematopoietic precursors that can lead to the development of mature granulocytes. PMID:11588034

  20. Mathematical modeling of dormant cell formation in growing biofilm

    PubMed Central

    Chihara, Kotaro; Matsumoto, Shinya; Kagawa, Yuki; Tsuneda, Satoshi

    2015-01-01

    Understanding the dynamics of dormant cells in microbial biofilms, in which the bacteria are embedded in extracellular matrix, is important for developing successful antibiotic therapies against pathogenic bacteria. Although some of the molecular mechanisms leading to bacterial persistence have been speculated in planktonic bacterial cell, how dormant cells emerge in the biofilms of pathogenic bacteria such as Pseudomonas aeruginosa remains unclear. The present study proposes four hypotheses of dormant cell formation; stochastic process, nutrient-dependent, oxygen-dependent, and time-dependent processes. These hypotheses were implemented into a three-dimensional individual-based model of biofilm formation. Numerical simulations of the different mechanisms yielded qualitatively different spatiotemporal distributions of dormant cells in the growing biofilm. Based on these simulation results, we discuss what kinds of experimental studies are effective for discriminating dormant cell formation mechanisms in biofilms. PMID:26074911

  1. Suppression of T cell-induced osteoclast formation

    SciTech Connect

    Karieb, Sahar; Fox, Simon W.

    2013-07-12

    Highlights: •Genistein and coumestrol prevent activated T cell induced osteoclast formation. •Anti-TNF neutralising antibodies prevent the pro-osteoclastic effect of activated T cells. •Phytoestrogens inhibit T cell derived TNF alpha and inflammatory cytokine production. •Phytoestrogens have a broader range of anti-osteoclastic actions than other anti-resorptives. -- Abstract: Inhibition of T cell derived cytokine production could help suppress osteoclast differentiation in inflammatory skeletal disorders. Bisphosphonates are typically prescribed to prevent inflammatory bone loss but are not tolerated by all patients and are associated with an increased risk of osteonecrosis of the jaw. In light of this other anti-resorptives such as phytoestrogens are being considered. However the effect of phytoestrogens on T cell-induced osteoclast formation is unclear. The effect of genistein and coumestrol on activated T cell-induced osteoclastogenesis and cytokine production was therefore examined. Concentrations of genistein and coumestrol (10{sup −7} M) previously shown to directly inhibit osteoclast formation also suppressed the formation of TRAP positive osteoclast induced by con A activated T cells, which was dependent on inhibition of T cell derived TNF-α. While both reduced osteoclast formation their mechanism of action differed. The anti-osteoclastic effect of coumestrol was associated with a dual effect on con A induced T cell proliferation and activation; 10{sup −7} M coumestrol significantly reducing T cell number (0.36) and TNF-α (0.47), IL-1β (0.23) and IL-6 (0.35) expression, whereas genistein (10{sup −7} M) had no effect on T cell number but a more pronounced effect on T cell differentiation reducing expression of TNF-α (0.49), IL-1β (0.52), IL-6 (0.71) and RANKL (0.71). Phytoestrogens therefore prevent the pro-osteoclastic action of T cells suggesting they may have a role in the control of inflammatory bone loss.

  2. The Amana Colonies.

    ERIC Educational Resources Information Center

    Lilja, Marilyn

    Designed for use in Iowa elementary schools, this unit introduces students to Iowa's Amana Colonies. Four lessons cover the history and cultural heritage of the colonies, daily life in historical times, daily life in modern times, and the colonies as a corporate museum. Throughout the lessons, emphasis is placed on the values and organization of…

  3. Vaccination with Irradiated Tumor Cells Engineered to Secrete Murine Granulocyte-Macrophage Colony-Stimulating Factor Stimulates Potent, Specific, and Long-Lasting Anti-Tumor Immunity

    NASA Astrophysics Data System (ADS)

    Dranoff, Glenn; Jaffee, Elizabeth; Lazenby, Audrey; Golumbek, Paul; Levitsky, Hyam; Brose, Katja; Jackson, Valerie; Hamada, Hirofumi; Pardoll, Drew; Mulligan, Richard C.

    1993-04-01

    To compare the ability of different cytokines and other molecules to enhance the immunogenicity of tumor cells, we generated 10 retroviruses encoding potential immunomodulators and studied the vaccination properties of murine tumor cells transduced by the viruses. Using a B16 melanoma model, in which irradiated tumor cells alone do not stimulate significant anti-tumor immunity, we found that irradiated tumor cells expressing murine granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulated potent, long-lasting, and specific anti-tumor immunity, requiring both CD4^+ and CD8^+ cells. Irradiated cells expressing interleukins 4 and 6 also stimulated detectable, but weaker, activity. In contrast to the B16 system, we found that in a number of other tumor models, the levels of anti-tumor immunity reported previously in cytokine gene transfer studies involving live, transduced cells could be achieved through the use of irradiated cells alone. Nevertheless, manipulation of the vaccine or challenge doses made it possible to demonstrate the activity of murine GM-CSF in those systems as well. Overall, our results have important implications for the clinical use of genetically modified tumor cells as therapeutic cancer vaccines.

  4. Pretransplant Mobilization with Granulocyte Colony-Stimulating Factor Improves B-Cell Reconstitution by Lentiviral Vector Gene Therapy in SCID-X1 Mice

    PubMed Central

    Huston, Marshall W.; Riegman, Adriaan R.A.; Yadak, Rana; van Helsdingen, Yvette; de Boer, Helen; van Til, Niek P.

    2014-01-01

    Abstract Hematopoietic stem cell (HSC) gene therapy is a demonstrated effective treatment for X-linked severe combined immunodeficiency (SCID-X1), but B-cell reconstitution and function has been deficient in many of the gene therapy treated patients. Cytoreductive preconditioning is known to improve HSC engraftment, but in general it is not considered for SCID-X1 since the poor health of most of these patients at diagnosis and the risk of toxicity preclude the conditioning used in standard bone marrow stem cell transplantation. We hypothesized that mobilization of HSC by granulocyte colony-stimulating factor (G-CSF) should create temporary space in bone marrow niches to improve engraftment and thereby B-cell reconstitution. In the present pilot study supplementing our earlier preclinical evaluation (Huston et al., 2011), Il2rg−/− mice pretreated with G-CSF were transplanted with wild-type lineage negative (Lin−) cells or Il2rg−/− Lin− cells transduced with therapeutic IL2RG lentiviral vectors. Mice were monitored for reconstitution of lymphocyte populations, level of donor cell chimerism, and antibody responses as compared to 2 Gy total body irradiation (TBI), previously found effective in promoting B-cell reconstitution. The results demonstrate that G-CSF promotes B-cell reconstitution similar to low-dose TBI and provides proof of principle for an alternative approach to improve efficacy of gene therapy in SCID patients without adverse effects associated with cytoreductive conditioning. PMID:25222508

  5. Characterization of a cell-type-restricted negative regulatory activity of the human granulocyte-macrophage colony-stimulating factor gene.

    PubMed Central

    Fraser, J K; Guerra, J J; Nguyen, C Y; Indes, J E; Gasson, J C; Nimer, S D

    1994-01-01

    Human granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates the proliferation and maturation of normal myeloid progenitor cells and can also stimulate the growth of acute myelogenous leukemia (AML) blasts. GM-CSF is not normally produced by resting cells but is expressed by a variety of activated cells including T lymphocytes, macrophages, and certain cytokine-stimulated fibroblasts and endothelial cells. Production of GM-CSF by cultured AML cells has been demonstrated, and GM-CSF expression by normal myeloid progenitors has been postulated to play a role in myelopoiesis. We have investigated the regulation of expression of GM-CSF in AML cell lines, and our results demonstrate the presence of a strong constitutive promoter element contained within 53 bp upstream of the cap site. We have also identified a negative regulatory element located immediately upstream of the positive regulatory element (within 69 bp of the cap site) that is active in AML cell lines but not T cells or K562 CML cells. Competition transfection and mobility shift studies demonstrate that this activity correlates with binding of a 45-kDa protein. Images PMID:8114751

  6. Pretransplant mobilization with granulocyte colony-stimulating factor improves B-cell reconstitution by lentiviral vector gene therapy in SCID-X1 mice.

    PubMed

    Huston, Marshall W; Riegman, Adriaan R A; Yadak, Rana; van Helsdingen, Yvette; de Boer, Helen; van Til, Niek P; Wagemaker, Gerard

    2014-10-01

    Hematopoietic stem cell (HSC) gene therapy is a demonstrated effective treatment for X-linked severe combined immunodeficiency (SCID-X1), but B-cell reconstitution and function has been deficient in many of the gene therapy treated patients. Cytoreductive preconditioning is known to improve HSC engraftment, but in general it is not considered for SCID-X1 since the poor health of most of these patients at diagnosis and the risk of toxicity preclude the conditioning used in standard bone marrow stem cell transplantation. We hypothesized that mobilization of HSC by granulocyte colony-stimulating factor (G-CSF) should create temporary space in bone marrow niches to improve engraftment and thereby B-cell reconstitution. In the present pilot study supplementing our earlier preclinical evaluation (Huston et al., 2011), Il2rg(-/-) mice pretreated with G-CSF were transplanted with wild-type lineage negative (Lin(-)) cells or Il2rg(-/-) Lin(-) cells transduced with therapeutic IL2RG lentiviral vectors. Mice were monitored for reconstitution of lymphocyte populations, level of donor cell chimerism, and antibody responses as compared to 2 Gy total body irradiation (TBI), previously found effective in promoting B-cell reconstitution. The results demonstrate that G-CSF promotes B-cell reconstitution similar to low-dose TBI and provides proof of principle for an alternative approach to improve efficacy of gene therapy in SCID patients without adverse effects associated with cytoreductive conditioning. PMID:25222508

  7. Convective cell formation in a Z pinch

    NASA Astrophysics Data System (ADS)

    Kesner, J.

    2003-03-01

    Closed field line confinement systems can develop convective cells when the magnetohydrodynamic interchange stability criterion is violated. Using a previously derived set of reduced equations [V. P. Pastukhov and N. V. Chudin, Plasma Phys. Rep. 27, 907 (2001)] it is shown that a true steady state solution can exist. For an assumed large-scale vortex pattern, the plasma pressure profile that is implied by these convective flows as well as the nonlocal heat flux resulting from the convective flows is calculated.

  8. Single cell pattern formation and transient cytoskeletal arrays

    PubMed Central

    Bement, William M.; von Dassow, George

    2015-01-01

    A major goal of developmental biology is to explain the emergence of pattern in cell layers, tissues and organs. Developmental biologists now accept that reaction diffusion-based mechanisms are broadly employed in developing organisms to direct pattern formation. Here we briefly consider these mechanisms and then apply some of the concepts derived from them to several processes that occur in single cells: wound repair, yeast budding, and cytokinesis. Two conclusions emerge from this analysis: first, there is considerable overlap at the level of general mechanisms between developmental and single cell pattern formation; second, dynamic structures based on the actin cytoskeleton may be far more ordered than is generally recognized. PMID:24529246

  9. Nanopore formation in neuroblastoma cells following ultrashort electric pulse exposure

    NASA Astrophysics Data System (ADS)

    Roth, Caleb C.; Payne, Jason A.; Wilmink, Gerald J.; Ibey, Bennett L.

    2011-03-01

    Ultrashort or nanosecond electrical pulses (USEP) cause repairable damage to the plasma membranes of cells through formation of nanopores. These nanopores are able to pass small ions such as sodium, calcium, and potassium, but remain impermeable to larger molecules like trypan blue and propidium iodide. What remains uncertain is whether generation of nanopores by ultrashort electrical pulses can inhibit action potentials in excitable cells. In this paper, we explored the sensitivity of excitable cells to USEP using Calcium Green AM 1 ester fluorescence to measure calcium uptake indicative of nanopore formation in the plasma membrane. We determined the threshold for nanopore formation in neuroblastoma cells for three pulse parameters (amplitude, pulse width, and pulse number). Measurement of such thresholds will guide future studies to determine if USEP can inhibit action potentials without causing irreversible membrane damage.

  10. Formation of a Neurosensory Organ by Epithelial Cell Slithering.

    PubMed

    Kuo, Christin S; Krasnow, Mark A

    2015-10-01

    Epithelial cells are normally stably anchored, maintaining their relative positions and association with the basement membrane. Developmental rearrangements occur through cell intercalation, and cells can delaminate during epithelial-mesenchymal transitions and metastasis. We mapped the formation of lung neuroepithelial bodies (NEBs), innervated clusters of neuroendocrine/neurosensory cells within the bronchial epithelium, revealing a targeted mode of cell migration that we named "slithering," in which cells transiently lose epithelial character but remain associated with the membrane while traversing neighboring epithelial cells to reach cluster sites. Immunostaining, lineage tracing, clonal analysis, and live imaging showed that NEB progenitors, initially distributed randomly, downregulate adhesion and polarity proteins, crawling over and between neighboring cells to converge at diametrically opposed positions at bronchial branchpoints, where they reestablish epithelial structure and express neuroendocrine genes. There is little accompanying progenitor proliferation or apoptosis. Activation of the slithering program may explain why lung cancers arising from neuroendocrine cells are highly metastatic. PMID:26435104

  11. Podoplanin-positive periarteriolar stromal cells promote megakaryocyte growth and proplatelet formation in mice by CLEC-2.

    PubMed

    Tamura, Shogo; Suzuki-Inoue, Katsue; Tsukiji, Nagaharu; Shirai, Toshiaki; Sasaki, Tomoyuki; Osada, Makoto; Satoh, Kaneo; Ozaki, Yukio

    2016-03-31

    Megakaryopoiesis is the hierarchical differentiation of hematopoietic stem cells into megakaryocytes. Differentiating megakaryocytes undergo maturation characterized by endomitosis and produce numerous platelets through proplatelet formation. C-type lectin-like receptor 2 (CLEC-2) is a podoplanin (PDPN) receptor mainly expressed on platelets and megakaryocytes. Deletion of platelet/megakaryocyte CLEC-2 causes thrombocytopenia in mice; however, its contribution to megakaryopoiesis remains unknown. Here, we show that megakaryopoiesis is promoted through the CLEC-2/PDPN interaction in the vicinity of arterioles in the bone marrow (BM). We have also identified PDPN-expressing BM arteriolar stromal cells, tentatively termed as BM fibroblastic reticular cell (FRC)-like cells. Platelet/megakaryocyte-specific CLEC-2 conditional knockout (cKO) mice showed a decrease in the number of immature megakaryocytes. CLEC-2 wild-type megakaryocyte expansion was augmented in vitro by the addition of recombinant PDPN, but not cKO megakaryocytes. Moreover, megakaryocyte colonies were colocalized with periarteriolar BM FRC-like cells in the BM. Coculture of megakaryocytes with BM FRC-like cells augmented megakaryocyte expansion, which was dependent upon the CLEC-2/PDPN interaction. Furthermore, we found that the CLEC-2/PDPN interaction induces BM FRC-like cells to secrete chemokine (C-C motif) ligand 5 (CCL5) to facilitate proplatelet formation. These observations indicate that a reciprocal interaction between CLEC-2 on megakaryocytes and PDPN on BM FRC-like cells contributes to the periarteriolar megakaryopoietic microenvironment in mouse BM. PMID:26796360

  12. Laminin 5 regulates polycystic kidney cell proliferation and cyst formation.

    PubMed

    Joly, Dominique; Berissi, Sophie; Bertrand, Amélie; Strehl, Laetitia; Patey, Natacha; Knebelmann, Bertrand

    2006-09-29

    Renal cyst formation is the hallmark of autosomal dominant polycystic kidney disease (ADPKD). ADPKD cyst-lining cells have an increased proliferation rate and are surrounded by an abnormal extracellular matrix (ECM). We have previously shown that Laminin 5 (Ln-5, a alpha(3)beta(3)gamma(2) trimer) is aberrantly expressed in the pericystic ECM of ADPKD kidneys. We report that ADPKD cells in primary cultures produce and secrete Ln-5 that is incorporated to the pericystic ECM in an in vitro model of cystogenesis. In monolayers, purified Ln-5 induces ERK activation and proliferation of ADPKD cells, whereas upon epidermal growth factor stimulation blocking endogenously produced Ln-5 with anti-gamma(2) chain antibody reduces the sustained ERK activation and inhibits proliferation. In three-dimensional gel culture, addition of purified Ln-5 stimulates cell proliferation and cyst formation, whereas blocking endogenous Ln-5 strongly inhibits cyst formation. Ligation of alpha(6)beta(4) integrin, a major Ln-5 receptor aberrantly expressed by ADPKD cells, induces beta(4) integrin phosphorylation, ERK activation, cell proliferation, and cyst formation. These findings indicate that Ln-5 is an important regulator of ADPKD cell proliferation and cystogenesis and suggest that Ln-5 gamma(2) chain and Ln-5-alpha(6)beta(4) integrin interaction both contribute to these phenotypic changes. PMID:16870608

  13. Effects of transforming growth factor beta and epidermal growth factor on cell proliferation and the formation of bone nodules in isolated fetal rat calvaria cells.

    PubMed

    Antosz, M E; Bellows, C G; Aubin, J E

    1989-08-01

    When cells enzymatically isolated from fetal rat calvaria (RC cells) are cultured in vitro in the presence of ascorbic acid and Na beta-glycerophosphate, discrete three-dimensional nodules form with the histologic, immunohistochemical, and ultrastructural characteristics of bone (Bellows et al; Calcified Tissue International 38:143-154, 1986; Bhargava et al., Bone, 9:155-163, 1988). Quantitation of the number of bone nodules that forms provides a colony assay for osteoprogenitor cells present in the RC population (Bellows and Aubin, Develop. Biol., 133:8-13, 1989). Continuous culture with either epidermal growth factor (EGF) or transforming growth factor beta (TGF-beta) results in dose-dependent inhibition of bone nodule formation; however, the former causes increased proliferation and saturation density, while the latter reduces both parameters. Addition of EGF (48 h pulse, 2-200 ng/ml) to RC cells at day 1 after plating results in increased proliferation and population saturation density and an increased number of bone nodules formed. Similar pulses at confluence and in postconfluent multilayered cultures when nodules first begin forming (approx. day 11) inhibited bone nodule formation and resulted in a smaller stimulation of cell proliferation. Forty-eight hour pulses of TGF-beta (0.01-1 ng/ml) reduced bone nodule formation and proliferation at all times examined, with pulses on day 1 causing maximum inhibition. The effects of pulses with TGF-beta and EGF on inhibition of nodule formation are independent of the presence of serum in the culture medium during the pulse. The data suggest that whereas EGF can either stimulate or inhibit the formation of bone nodules depending upon the time and duration of exposure, TGF-B inhibits bone nodule formation under all conditions tested. Moreover, these effects on osteoprogenitor cell differentiation do not always correlate with the effects of the growth factors on RC cell proliferation. PMID:2787326

  14. Immunomodulation Induced by Stem Cell Mobilization and Harvesting in Healthy Donors: Increased Systemic Osteopontin Levels after Treatment with Granulocyte Colony-Stimulating Factor

    PubMed Central

    Melve, Guro Kristin; Ersvaer, Elisabeth; Akkök, Çiğdem Akalın; Ahmed, Aymen Bushra; Kristoffersen, Einar K.; Hervig, Tor; Bruserud, Øystein

    2016-01-01

    Peripheral blood stem cells from healthy donors mobilized by granulocyte colony-stimulating factor (G-CSF) and harvested by leukapheresis are commonly used for allogeneic stem cell transplantation. The frequency of severe graft versus host disease is similar for patients receiving peripheral blood and bone marrow allografts, even though the blood grafts contain more T cells, indicating mobilization-related immunoregulatory effects. The regulatory phosphoprotein osteopontin was quantified in plasma samples from healthy donors before G-CSF treatment, after four days of treatment immediately before and after leukapheresis, and 18–24 h after apheresis. Myeloma patients received chemotherapy, combined with G-CSF, for stem cell mobilization and plasma samples were prepared immediately before, immediately after, and 18–24 h after leukapheresis. G-CSF treatment of healthy stem cell donors increased plasma osteopontin levels, and a further increase was seen immediately after leukapheresis. The pre-apheresis levels were also increased in myeloma patients compared to healthy individuals. Finally, in vivo G-CSF exposure did not alter T cell expression of osteopontin ligand CD44, and in vitro osteopontin exposure induced only small increases in anti-CD3- and anti-CD28-stimulated T cell proliferation. G-CSF treatment, followed by leukapheresis, can increase systemic osteopontin levels, and this effect may contribute to the immunomodulatory effects of G-CSF treatment. PMID:27447610

  15. Immunomodulation Induced by Stem Cell Mobilization and Harvesting in Healthy Donors: Increased Systemic Osteopontin Levels after Treatment with Granulocyte Colony-Stimulating Factor.

    PubMed

    Melve, Guro Kristin; Ersvaer, Elisabeth; Akkök, Çiğdem Akalın; Ahmed, Aymen Bushra; Kristoffersen, Einar K; Hervig, Tor; Bruserud, Øystein

    2016-01-01

    Peripheral blood stem cells from healthy donors mobilized by granulocyte colony-stimulating factor (G-CSF) and harvested by leukapheresis are commonly used for allogeneic stem cell transplantation. The frequency of severe graft versus host disease is similar for patients receiving peripheral blood and bone marrow allografts, even though the blood grafts contain more T cells, indicating mobilization-related immunoregulatory effects. The regulatory phosphoprotein osteopontin was quantified in plasma samples from healthy donors before G-CSF treatment, after four days of treatment immediately before and after leukapheresis, and 18-24 h after apheresis. Myeloma patients received chemotherapy, combined with G-CSF, for stem cell mobilization and plasma samples were prepared immediately before, immediately after, and 18-24 h after leukapheresis. G-CSF treatment of healthy stem cell donors increased plasma osteopontin levels, and a further increase was seen immediately after leukapheresis. The pre-apheresis levels were also increased in myeloma patients compared to healthy individuals. Finally, in vivo G-CSF exposure did not alter T cell expression of osteopontin ligand CD44, and in vitro osteopontin exposure induced only small increases in anti-CD3- and anti-CD28-stimulated T cell proliferation. G-CSF treatment, followed by leukapheresis, can increase systemic osteopontin levels, and this effect may contribute to the immunomodulatory effects of G-CSF treatment. PMID:27447610

  16. Pulp stem cells: implication in reparative dentin formation.

    PubMed

    Dimitrova-Nakov, Sasha; Baudry, Anne; Harichane, Yassine; Kellermann, Odile; Goldberg, Michel

    2014-04-01

    Many dental pulp stem cells are neural crest derivatives essential for lifelong maintenance of tooth functions and homeostasis as well as tooth repair. These cells may be directly implicated in the healing process or indirectly involved in cell-to-cell diffusion of paracrine messages to resident (pulpoblasts) or nonresident cells (migrating mesenchymal cells). The identity of the pulp progenitors and the mechanisms sustaining their regenerative capacity remain largely unknown. Taking advantage of the A4 cell line, a multipotent stem cell derived from the molar pulp of mouse embryo, we investigated the capacity of these pulp-derived precursors to induce in vivo the formation of a reparative dentin-like structure upon implantation within the pulp of a rodent incisor or a first maxillary molar after surgical exposure. One month after the pulp injury alone, a nonmineralized fibrous matrix filled the mesial part of the coronal pulp chamber. Upon A4 cell implantation, a mineralized osteodentin was formed in the implantation site without affecting the structure and vitality of the residual pulp in the central and distal parts of the pulp chamber. These results show that dental pulp stem cells can induce the formation of reparative dentin and therefore constitute a useful tool for pulp therapies. Finally, reparative dentin was also built up when A4 progenitors were performed by alginate beads, suggesting that alginate is a suitable carrier for cell implantation in teeth. PMID:24698687

  17. Performance of direct formate-peroxide fuel cells

    NASA Astrophysics Data System (ADS)

    Li, Yinshi; Wu, Hao; He, Yaling; Liu, Yue; Jin, Lei

    2015-08-01

    We report the high-performance direct formate-peroxide fuel cells (DFPFCs) that consist of a cation-exchange membrane sandwiched between an alkaline formate anode and an acid peroxide cathode. Much attention has been paid to investigate the effects of different composite parameters and operating parameters, including catalyst loadings at both anode and cathode electrodes, operating temperatures, as well as the concentrations of both formate and electrolyte solutions. It is demonstrated that the optimization of both the electrode composition (anode 2.0 mgPd cm-2, cathode 2.0 mgPt cm-2) and the solution concentration (1.0 M HCOONa-3.0 M NaOH) enables the DFPFC to yield a peak power density as high as 591 mW cm-2 at 60 °C, which is about one times higher than that of state-of-the-art anion-exchange membrane direct formate fuel cells.

  18. Formation of dimethylthioarsenicals in red blood cells

    SciTech Connect

    Naranmandura, Hua; Suzuki, Kazuo T.

    2008-03-15

    The bladder and skin are the primary targets for arsenic-induced carcinogenicity in mammals. Thioarsenicals dimethylmonothioarsinic (DMMTA{sup V}) and dimethyldithioarsinic (DMDTA{sup V}) acids are common urinary metabolites, the former being much more toxic than non-thiolated dimethylarsinic acid (DMA{sup V}) and comparable to dimethylarsinous acid (DMA{sup III}) in epidermoid cells, suggesting that the metabolic production of thioarsenicals may be a risk factor for the development of cancer in these organs. To reveal their production sites (tissues/body fluids), we examined the uptake and transformation of the four dimethylated arsenicals by incubation with rat and human red blood cells (RBCs). Although DMA{sup V} and DMDTA{sup V} were not taken up by either type of RBCs, DMA{sup III} and DMMTA{sup V} were taken up by both (more efficiently by rat ones), though DMMTA{sup V} was taken up slowly, and then the arsenic transformed into DMDTA{sup V} was excreted from both types of animal RBCs. On the other hand, although DMA{sup III} taken up rapidly by rat RBCs was retained in the RBCs, that taken up by human RBCs was immediately transformed into DMMTA{sup V} and then excreted into the incubation medium without being retained in the RBCs. In a separate experiment, arsenic remaining in primary rat hepatocytes after incubation with 1.5 {mu}M DMA{sup III} was recovered from the incubation medium in the forms of DMA{sup V} and DMMTA{sup V} in the presence of human RBCs, but not in the presence of rat RBCs (in which the arsenic was bound to hemoglobin). Thus, DMMTA{sup V} was detected in the medium only in the presence of human RBCs and increased with incubation time. It was proposed that arsenic is excreted from hepatocytes into the bloodstream in the form of DMA{sup III} and then taken up by RBCs in humans, where it is transformed into DMMTA{sup V} and then excreted again into the bloodstream.

  19. Interleukin-33 is expressed in differentiated osteoblasts and blocks osteoclast formation from bone marrow precursor cells.

    PubMed

    Schulze, Jochen; Bickert, Thomas; Beil, F Timo; Zaiss, Mario M; Albers, Joachim; Wintges, Kristofer; Streichert, Thomas; Klaetschke, Kristin; Keller, Johannes; Hissnauer, Tim-Nicolas; Spiro, Alexander S; Gessner, Andre; Schett, Georg; Amling, Michael; McKenzie, Andrew N J; Horst, Andrea Kristina; Schinke, Thorsten

    2011-04-01

    Since the hematopoetic system is located within the bone marrow, it is not surprising that recent evidence has demonstrated the existence of molecular interactions between bone and immune cells. While interleukin 1 (IL-1) and IL-18, two cytokines of the IL-1 family, have been shown to regulate differentiation and activity of bone cells, the role of IL-33, another IL-1 family member, has not been addressed yet. Since we observed that the expression of IL-33 increases during osteoblast differentiation, we analyzed its possible influence on bone formation and observed that IL-33 did not affect matrix mineralization but enhanced the expression of Tnfsf11, the gene encoding RANKL. This finding led us to analyze the skeletal phenotype of Il1rl1-deficient mice, which lack the IL-33 receptor ST2. Unexpectedly, these mice displayed normal bone formation but increased bone resorption, thereby resulting in low trabecular bone mass. Since this finding suggested a negative influence of IL-33 on osteoclastogenesis, we next analyzed osteoclast differentiation from bone marrow precursor cells and observed that IL-33 completely abolished the generation of TRACP(+) multinucleated osteoclasts, even in the presence of RANKL and macrophage colony-stimulating factor (M-CSF). Although our molecular studies revealed that IL-33 treatment of bone marrow cells caused a shift toward other hematopoetic lineages, we further observed a direct negative influence of IL-33 on the osteoclastogenic differentiation of RAW264.7 macrophages, where IL-33 repressed the expression of Nfatc1, which encodes one of the key transciption factors of osteoclast differentiation. Taken together, these findings have uncovered a previously unknown function of IL-33 as an inhibitor of bone resorption. PMID:20939024

  20. Colony Fusion in a Parthenogenetic Ant, Pristomyrmex punctatus

    PubMed Central

    Satow, Show; Satoh, Toshiyuki; Hirota, Tadao

    2013-01-01

    In the ant Pristomyrmex punctatus Smith (Hymenoptera: Formicidae), all young workers lay a small number of eggs parthenogenetically. Some colonies consist of monoclonal individuals that provide high inclusive fitness, according to the kin selection theory. However, in some populations, a majority of the colonies contain multiple lineages. Intracolonial genetic variation of parthenogenetic ants cannot be explained by the multiple mating of single founderesses or by the foundation of a colony by multiple foundresses, which are the usual causes of genetically diverse colonies in social insects. Here, we hypothesized that the fusion of established colonies might facilitate the formation of multiclonal colonies. Colony fusion decreases indirect benefits because of the reduction in intracolonial relatedness. However, when suitable nesting places for overwintering are scarce, colony fusion provides a strategy for the survival of colonies. Here, ants derived from different colonies were allowed to encounter one another in a container with just one nesting place. Initially, high aggression was observed; however, after several days, no aggression was observed and the ants shared the nest. When the fused colonies were allowed to transfer to two alternative nests, ants from different colonies occupied the same nest. This study highlights the importance of limiting the number of nesting places in order to understand the genetic diversity of parthenogenetic ant colonies. PMID:23895053

  1. A Soluble Granulocyte Colony Stimulating Factor Decoy Receptor as a Novel Tool to Increase Hematopoietic Cell Homing and Reconstitution in Mice

    PubMed Central

    Fortin, Audrey; Benabdallah, Basma; Palacio, Lina; Carbonneau, Cynthia L.; Le, Oanh N.; Haddad, Élie

    2013-01-01

    The relative ineffectiveness of hematopoietic stem cells in reaching the bone marrow upon transplantation combined with the limited number of these cells available is a major reason for graft failure and delayed hematopoietic recovery. Hence, the development of strategies that could enhance homing is of high interest. Here, we provide evidence that homing is severely impaired postexposure to ionizing radiation (IR) in mice, an effect we found was time dependent and could be partially rescued using mesenchymal stromal cell (MSC) therapy. In an attempt to further increase homing, we took advantage of our observation that the granulocyte colony stimulating factor (G-CSF), a cytokine known to induce cell mobilization, is increased in the marrow of mice shortly after their exposure to IR. As such, we developed a truncated, yet functional, soluble G-CSF receptor (solG-CSFR), which we hypothesized could act as a decoy and foster homing. Using MSCs or conditioned media as delivery vehicles, we show that an engineered solG-CSFR has the potential to increase homing and hematopoietic reconstitution in mice. Altogether, our results provide novel findings at the interplay of IR and stromal cell therapy and present the regulation of endogenous G-CSF as an innovative proof-of-concept strategy to manipulate hematopoietic cell homing. PMID:23205715

  2. Formation of a cylindrical bridge in cell division

    NASA Astrophysics Data System (ADS)

    Citron, Daniel; Schmidt, Laura E.; Reichl, Elizabeth; Ren, Yixin; Robinson, Douglas; Zhang, Wendy W.

    2007-11-01

    In nature, the shape transition associated with the division of a mother cell into two daughter cells proceeds via a variety of routes. In the cylinder-thinning route, which has been observed in Dictyostelium and most animal cells, the mother cell first forms a broad bridge-like region, also known as a furrow, between two daughter cells. The furrow then rapidly evolves into a cylindrical bridge, which thins and eventually severs the mother cell into two. The fundamental mechanism underlying this division route is not understood. Recent experiments on Dictyostelium found that, while the cylinder-thinning route persists even when key actin cross-linking proteins are missing, it is disrupted by the removal of force-generating myosin-II proteins. Other measurements revealed that mutant cells lacking myosin-II have a much more uniform tension over the cell surface than wild-type cells. This suggests that tension variation may be important. Here we use a fluid model, previously shown to reproduce the thinning dynamics [Zhang & Robinson, PNAS 102, 7186 (2005)], to test this idea. Consistent with the experiments, the model shows that the cylinder formation process occurs regardless of the exact viscoelastic properties of the cell. In contrast to the experiments, a tension variation in the model hinders, rather then expedites, the cylinder formation.

  3. Purified murine granulocyte/macrophage progenitor cells express a high-affinity receptor for recombinant murine granulocyte/macrophage colony-stimulating factor

    SciTech Connect

    Williams, D.E.; Bicknell, D.C.; Park, L.S.; Straneva, J.E.; Cooper, S.; Broxmeyer, H.E.

    1988-01-01

    Purified recombinant murine granulocyte/macrophage colony-stimulating factor (GM-CSF) was labeled with /sup 125/I and used to examine the GM-CSF receptor on unfractionated normal murine bone marrow cells, casein-induced peritoneal exudate cells, and highly purified murine granulocyte/macrophage progenitor cells (CFU-GM). CFU-GM were isolated from cyclophosphamide-treated mice by Ficoll-Hypaque density centrifugation followed by counterflow centrifugal elutriation. The resulting population had a cloning efficiency of 62-99% in cultures containing conditioned medium from pokeweed mitogen-stimulated spleen cells and 55-86% in the presence of a plateau concentration of purified recombinant murine GM-CSF. Equilibrium binding studies with /sup 125/I-labeled GM-CSF showed that normal bone marrow cells, casein-induced peritoneal exudate cells, and purified CFU-GM had a single class of high-affinity receptor. Affinity crosslinking studies demonstrated that /sup 125/I-labeled GM-CSF bound specifically to two species of M/sub r/ 180,000 and 70,000 on CFU-GM, normal bone marrow cells, and peritoneal exudate cells. The M/sub r/ 70,000 species is thought to be a proteolytic fragment of the intact M/sub r/ 180,000 receptor. The present studies indicate that the GM-CSF receptor expressed on CFU-GM and mature myeloid cells are structurally similar. In addition, the number of GM-CSF receptors on CFU-GM is twice the average number of receptors on casein-induced mature myeloid cells, suggesting that receptor number may decrease as CFU-GM mature.

  4. Tracing haematopoietic stem cell formation at single-cell resolution.

    PubMed

    Zhou, Fan; Li, Xianlong; Wang, Weili; Zhu, Ping; Zhou, Jie; He, Wenyan; Ding, Meng; Xiong, Fuyin; Zheng, Xiaona; Li, Zhuan; Ni, Yanli; Mu, Xiaohuan; Wen, Lu; Cheng, Tao; Lan, Yu; Yuan, Weiping; Tang, Fuchou; Liu, Bing

    2016-05-26

    Haematopoietic stem cells (HSCs) are derived early from embryonic precursors, such as haemogenic endothelial cells and pre-haematopoietic stem cells (pre-HSCs), the molecular identity of which still remains elusive. Here we use potent surface markers to capture the nascent pre-HSCs at high purity, as rigorously validated by single-cell-initiated serial transplantation. Then we apply single-cell RNA sequencing to analyse endothelial cells, CD45(-) and CD45(+) pre-HSCs in the aorta-gonad-mesonephros region, and HSCs in fetal liver. Pre-HSCs show unique features in transcriptional machinery, arterial signature, metabolism state, signalling pathway, and transcription factor network. Functionally, activation of mechanistic targets of rapamycin (mTOR) is shown to be indispensable for the emergence of HSCs but not haematopoietic progenitors. Transcriptome data-based functional analysis reveals remarkable heterogeneity in cell-cycle status of pre-HSCs. Finally, the core molecular signature of pre-HSCs is identified. Collectively, our work paves the way for dissection of complex molecular mechanisms regulating stepwise generation of HSCs in vivo, informing future efforts to engineer HSCs for clinical applications. PMID:27225119

  5. Aggregation of red blood cells: From rouleaux to clot formation

    NASA Astrophysics Data System (ADS)

    Wagner, Christian; Steffen, Patrick; Svetina, Saša

    2013-06-01

    Red blood cells are known to form aggregates in the form of rouleaux. This aggregation process is believed to be reversible, but there is still no full understanding on the adhesion mechanism. There are at least two competing models, based either on bridging or on depletion. We review recent experimental results on the single cell level and theoretical analyses of the depletion model and of the influence of the cell shape on the adhesion strength. Another important aggregation mechanism is caused by activation of platelets. This leads to clot formation which is life-saving in the case of wound healing, but also a major cause of death in the case of a thrombus induced stroke. We review historical and recent results on the participation of red blood cells in clot formation.

  6. TRPV1 mediates cellular uptake of anandamide and thus promotes endothelial cell proliferation and network-formation

    PubMed Central

    Hofmann, Nicole A.; Barth, Sonja; Waldeck-Weiermair, Markus; Klec, Christiane; Strunk, Dirk; Malli, Roland; Graier, Wolfgang F.

    2014-01-01

    ABSTRACT Anandamide (N-arachidonyl ethanolamide, AEA) is an endogenous cannabinoid that is involved in various pathological conditions, including cardiovascular diseases and tumor-angiogenesis. Herein, we tested the involvement of classical cannabinoid receptors (CBRs) and the Ca2+-channel transient receptor potential vanilloid 1 (TRPV1) on cellular AEA uptake and its effect on endothelial cell proliferation and network-formation. Uptake of the fluorescence-labeled anandamide (SKM4-45-1) was monitored in human endothelial colony-forming cells (ECFCs) and a human endothelial-vein cell line (EA.hy926). Involvement of the receptors during AEA translocation was determined by selective pharmacological inhibition (AM251, SR144528, CID16020046, SB366791) and molecular interference by TRPV1-selective siRNA-mediated knock-down and TRPV1 overexpression. We show that exclusively TRPV1 contributes essentially to AEA transport into endothelial cells in a Ca2+-independent manner. This TRPV1 function is a prerequisite for AEA-induced endothelial cell proliferation and network-formation. Our findings point to a so far unknown moonlighting function of TRPV1 as Ca2+-independent contributor/regulator of AEA uptake. We propose TRPV1 as representing a promising target for development of pharmacological therapies against AEA-triggered endothelial cell functions, including their stimulatory effect on tumor-angiogenesis. PMID:25395667

  7. Long-Term Expansion in Platelet Lysate Increases Growth of Peripheral Blood-Derived Endothelial-Colony Forming Cells and Their Growth Factor-Induced Sprouting Capacity

    PubMed Central

    Tasev, Dimitar; van Wijhe, Michiel H.; Weijers, Ester M.; van Hinsbergh, Victor W. M.; Koolwijk, Pieter

    2015-01-01

    Introduction Efficient implementation of peripheral blood-derived endothelial-colony cells (PB-ECFCs) as a therapeutical tool requires isolation and generation of a sufficient number of cells in ex vivo conditions devoid of animal-derived products. At present, little is known how the isolation and expansion procedure in xenogeneic-free conditions affects the therapeutical capacity of PB-ECFCs. Results The findings presented in this study indicate that human platelet lysate (PL) as a serum substitute yields twice more colonies per mL blood compared to the conventional isolation with fetal bovine serum (FBS). Isolated ECFCs displayed a higher proliferative ability in PL supplemented medium than cells in FBS medium during 30 days expansion. The cells at 18 cumulative population doubling levels (CPDL) retained their proliferative capacity, showed higher sprouting ability in fibrin matrices upon stimulation with FGF-2 and VEGF-A than the cells at 6 CPDL, and displayed low β-galactosidase activity. The increased sprouting of PB-ECFCs at 18 CPDL was accompanied by an intrinsic activation of the uPA/uPAR fibrinolytic system. Induced deficiency of uPA (urokinase-type plasminogen activator) or uPAR (uPA receptor) by siRNA technology completely abolished the angiogenic ability of PB-ECFCs in fibrin matrices. During the serial expansion, the gene induction of the markers associated with inflammatory activation such as VCAM-1 and ICAM-1 did not occur or only to limited extent. While further propagation up to 31 CPDL proceeded at a comparable rate, a marked upregulation of inflammatory markers occurred in all donors accompanied by a further increase of uPA/uPAR gene induction. The observed induction of inflammatory genes at later stages of long-term propagation of PB-ECFCs underpins the necessity to determine the right time-point for harvesting of sufficient number of cells with preserved therapeutical potential. Conclusion The presented isolation method and subsequent cell

  8. Inhibitory Effects of Salinomycin on Cell Survival, Colony Growth, Migration, and Invasion of Human Non-Small Cell Lung Cancer A549 and LNM35: Involvement of NAG-1

    PubMed Central

    Arafat, Kholoud; Iratni, Rabah; Takahashi, Takashi; Parekh, Khatija; Al Dhaheri, Yusra; Adrian, Thomas E.; Attoub, Samir

    2013-01-01

    A major challenge for oncologists and pharmacologists is to develop more potent and less toxic drugs that will decrease the tumor growth and improve the survival of lung cancer patients. Salinomycin is a polyether antibiotic used to kill gram-positive bacteria including mycobacteria, protozoans such as plasmodium falciparum, and the parasites responsible for the poultry disease coccidiosis. This old agent is now a serious anti-cancer drug candidate that selectively inhibits the growth of cancer stem cells. We investigated the impact of salinomycin on survival, colony growth, migration and invasion of the differentiated human non-small cell lung cancer lines LNM35 and A549. Salinomycin caused concentration- and time-dependent reduction in viability of LNM35 and A549 cells through a caspase 3/7-associated cell death pathway. Similarly, salinomycin (2.5–5 µM for 7 days) significantly decreased the growth of LNM35 and A549 colonies in soft agar. Metastasis is the main cause of death related to lung cancer. In this context, salinomycin induced a time- and concentration-dependent inhibition of cell migration and invasion. We also demonstrated for the first time that salinomycin induced a marked increase in the expression of the pro-apoptotic protein NAG-1 leading to the inhibition of lung cancer cell invasion but not cell survival. These findings identify salinomycin as a promising novel therapeutic agent for lung cancer. PMID:23805285

  9. Secondary Sphere Formation Enhances the Functionality of Cardiac Progenitor Cells

    PubMed Central

    Cho, Hyun-Jai; Lee, Ho-Jae; Youn, Seock-Won; Koh, Seok-Jin; Won, Joo-Yun; Chung, Yeon-Ju; Cho, Hyun-Ju; Yoon, Chang-Hwan; Lee, Sae-Won; Lee, Eun Ju; Kwon, Yoo-Wook; Lee, Hae-Young; Lee, Sang Hun; Ho, Won-Kyung; Park, Young-Bae; Kim, Hyo-Soo

    2012-01-01

    Loss of cardiomyocytes impairs cardiac function after myocardial infarction (MI). Recent studies suggest that cardiac stem/progenitor cells could repair the damaged heart. However, cardiac progenitor cells are difficult to maintain in terms of purity and multipotency when propagated in two-dimensional culture systems. Here, we investigated a new strategy that enhances potency and enriches progenitor cells. We applied the repeated sphere formation strategy (cardiac explant → primary cardiosphere (CS) formation → sphere-derived cells (SDCs) in adherent culture condition → secondary CS formation by three-dimensional culture). Cells in secondary CS showed higher differentiation potentials than SDCs. When transplanted into the infarcted myocardium, secondary CSs engrafted robustly, improved left ventricular (LV) dysfunction, and reduced infarct sizes more than SDCs did. In addition to the cardiovascular differentiation of transplanted secondary CSs, robust vascular endothelial growth factor (VEGF) synthesis and secretion enhanced neovascularization in the infarcted myocardium. Microarray pathway analysis and blocking experiments using E-selectin knock-out hearts, specific chemicals, and small interfering RNAs (siRNAs) for each pathway revealed that E-selectin was indispensable to sphere initiation and ERK/Sp1/VEGF autoparacrine loop was responsible for sphere maturation. These results provide a simple strategy for enhancing cellular potency for cardiac repair. Furthermore, this strategy may be implemented to other types of stem/progenitor cell-based therapy. PMID:22713697

  10. The costs of peripheral blood progenitor cell reinfusion mobilised by granulocyte colony-stimulating factor following high dose melphalan as compared with conventional therapy in multiple myeloma.

    PubMed

    Uyl-de Groot, C A; Ossenkoppele, G J; van Riet, A A; Rutten, F F

    1994-01-01

    In a retrospective study, we calculated the treatment costs of 26 patients, who received either high dose melphalan combined with granulocyte colony-stimulating factor (G-CSF; filgrastim)(n = 7) or without G-CSF (n = 11) or alternatively, peripheral blood progenitor cell reinfusion (PBPC) mobilised by G-CSF following high dose melphalan. In comparison with the control group, a shortening of the pancytopenic period and platelet recovery was noticed in the PBPC group. This resulted in a reduction in hospital costs, diagnostics, laboratory services, total parenteral nutrition and transfusions. The average costs per treatment in the PBPC group amounted to about US$ 17,908 as compared to US$ 32,223 in the control group, implying a cost reduction of 44% when changing to PBPC reinfusion. PMID:7517149

  11. Endocytosis of cell surface material mediates cell plate formation during plant cytokinesis.

    PubMed

    Dhonukshe, Pankaj; Baluska, Frantisek; Schlicht, Markus; Hlavacka, Andrej; Samaj, Jozef; Friml, Jirí; Gadella, Theodorus W J

    2006-01-01

    Dividing plant cells perform a remarkable task of building a new cell wall within the cytoplasm in a few minutes. A long-standing paradigm claims that this primordial cell wall, known as the cell plate, is generated by delivery of newly synthesized material from Golgi apparatus-originated secretory vesicles. Here, we show that, in diverse plant species, cell surface material, including plasma membrane proteins, cell wall components, and exogenously applied endocytic tracers, is rapidly delivered to the forming cell plate. Importantly, this occurs even when de novo protein synthesis is blocked. In addition, cytokinesis-specific syntaxin KNOLLE as well as plasma membrane (PM) resident proteins localize to endosomes that fuse to initiate the cell plate. The rate of endocytosis is strongly enhanced during cell plate formation, and its genetic or pharmacological inhibition leads to cytokinesis defects. Our results reveal that endocytic delivery of cell surface material significantly contributes to cell plate formation during plant cytokinesis. PMID:16399085

  12. Formation and maintenance of nitrogen-fixing cell patterns in filamentous cyanobacteria.

    PubMed

    Muñoz-García, Javier; Ares, Saúl

    2016-05-31

    Cyanobacteria forming one-dimensional filaments are paradigmatic model organisms of the transition between unicellular and multicellular living forms. Under nitrogen-limiting conditions, in filaments of the genus Anabaena, some cells differentiate into heterocysts, which lose the possibility to divide but are able to fix environmental nitrogen for the colony. These heterocysts form a quasiregular pattern in the filament, representing a prototype of patterning and morphogenesis in prokaryotes. Recent years have seen advances in the identification of the molecular mechanism regulating this pattern. We use these data to build a theory on heterocyst pattern formation, for which both genetic regulation and the effects of cell division and filament growth are key components. The theory is based on the interplay of three generic mechanisms: local autoactivation, early long-range inhibition, and late long-range inhibition. These mechanisms can be identified with the dynamics of hetR, patS, and hetN expression. Our theory reproduces quantitatively the experimental dynamics of pattern formation and maintenance for wild type and mutants. We find that hetN alone is not enough to play the role as the late inhibitory mechanism: a second mechanism, hypothetically the products of nitrogen fixation supplied by heterocysts, must also play a role in late long-range inhibition. The preponderance of even intervals between heterocysts arises naturally as a result of the interplay between the timescales of genetic regulation and cell division. We also find that a purely stochastic initiation of the pattern, without a two-stage process, is enough to reproduce experimental observations. PMID:27162328

  13. Colletotrichum higginsianum Mitogen-Activated Protein Kinase ChMK1: Role in Growth, Cell Wall Integrity, Colony Melanization, and Pathogenicity.

    PubMed

    Wei, Wei; Xiong, Ying; Zhu, Wenjun; Wang, Nancong; Yang, Guogen; Peng, Fang

    2016-01-01

    Colletotrichum higginsianum is an economically important pathogen that causes anthracnose disease in a wide range of cruciferous crops. To facilitate the efficient control of anthracnose disease, it will be important to understand the mechanism by which the cruciferous crops and C. higginsianum interact. A key step in understanding this interaction is characterizing the mitogen-activated protein kinases (MAPK) signaling pathway of C. higginsianum. MAPK plays important roles in diverse physiological processes of multiple pathogens. In this study, a Fus3/Kss1-related MAPK gene, ChMK1, from C. higginsianum was analyzed. The results showed that the Fus3/Kss1-related MAPK ChMK1 plays a significant role in cell wall integrity. Targeted deletion of ChMK1 resulted in a hypersensitivity to cell wall inhibitors, reduced conidiation and albinistic colonies. Further, the deletion mutant was also unable to form melanized appressorium, a specialized infection structure that is necessary for successful infection. Therefore, the deletion mutant loses pathogenicity on A. thaliana leaves, demonstrating that ChMK1 plays an essential role in the early infection step. In addition, the ChMK1 deletion mutant showed an attenuated growth rate that is different from that of its homolog in Colletotrichum lagenarium, indicating the diverse roles that Fus3/Kss1-related MAPKs plays in phytopathogenic fungi. Furthermore, the expression level of three melanin synthesis associated genes were clearly decreased in the albinistic ChMK1 mutant compared to that of the wild type strain, suggesting that ChMK1 is also required for colony melanization in C. higginsianum. PMID:27536296

  14. Disruption of the pdhB Pyruvate Dehydrogenase Gene Affects Colony Morphology, In Vitro Growth and Cell Invasiveness of Mycoplasma agalactiae

    PubMed Central

    Hegde, Shivanand; Rosengarten, Renate; Chopra-Dewasthaly, Rohini

    2015-01-01

    The utilization of available substrates, the metabolic potential and the growth rates of bacteria can play significant roles in their pathogenicity. This study concentrates on Mycoplasma agalactiae, which causes significant economic losses through its contribution to contagious agalactia in small ruminants by as yet unknown mechanisms. This lack of knowledge is primarily due to its fastidious growth requirements and the scarcity of genetic tools available for its manipulation and analysis. Transposon mutagenesis of M. agalactiae type strain PG2 resulted in several disruptions throughout the genome. A mutant defective in growth in vitro was found to have a transposon insertion in the pdhB gene, which encodes a component of the pyruvate dehydrogenase complex. This growth difference was quite significant during the actively dividing logarithmic phase but a gradual recovery was observed as the cells approached stationary phase. The mutant also exhibited a different and smaller colony morphology compared to the wild type strain PG2. For complementation, pdhAB was cloned downstream of a strong vpma promoter and upstream of a lacZ reporter gene in a newly constructed complementation vector. When transformed with this vector the pdhB mutant recovered its normal growth and colony morphology. Interestingly, the pdhB mutant also had significantly reduced invasiveness in HeLa cells, as revealed by double immunofluorescence staining. This deficiency was recovered in the complemented strain, which had invasiveness comparable to that of PG2. Taken together, these data indicate that pyruvate dehydrogenase might be an important player in infection with and colonization by M. agalactiae. PMID:25799063

  15. Modulation of Decidual Macrophage Polarization by Macrophage Colony-Stimulating Factor Derived from First-Trimester Decidual Cells: Implication in Preeclampsia.

    PubMed

    Li, Min; Piao, Longzhu; Chen, Chie-Pein; Wu, Xianqing; Yeh, Chang-Ching; Masch, Rachel; Chang, Chi-Chang; Huang, S Joseph

    2016-05-01

    During human pregnancy, immune tolerance of the fetal semiallograft occurs in the presence of abundant maternal leukocytes. At the implantation site, macrophages comprise approximately 20% of the leukocyte population and act as primary mediators of tissue remodeling. Decidual macrophages display a balance between anti-inflammatory and proinflammatory phenotypes. However, a shift to an M1 subtype is reported in preeclampsia. Granulocyte-macrophage colony-stimulating-factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) are major differentiating factors that mediate M1 and M2 polarization, respectively. Previously, we observed the following: i) the preeclamptic decidua contains an excess of both macrophages and GM-CSF, ii) the preeclampsia-associated proinflammatory cytokines, IL-1β and tumor necrosis factor-α, markedly enhance GM-CSF and M-CSF expression in cultured leukocyte-free first-trimester decidual cells (FTDCs), iii) FTDC-secreted GM-CSF polarizes macrophages toward an M1 subtype. The microenvironment is a key determinant of macrophage phenotype. Thus, we examined proinflammatory stimulation of FTDC-secreted M-CSF and its role in macrophage development. Immunofluorescence staining demonstrated elevated M-CSF-positive decidual cell numbers in preeclamptic decidua. In FTDCs, IL-1β and tumor necrosis factor-α signal through the NF-κB pathway to induce M-CSF production, which does the following: i) enhances differentiation of and elevates CD163 expression in macrophages, ii) increases macrophage phagocytic capacity, and iii) inhibits signal-regulatory protein α expression by macrophages. These findings suggest that FTDC-secreted M-CSF modulates the decidual immune balance by inducing M2 macrophage polarization and phagocytic capacity in response to proinflammatory stimuli. PMID:26970370

  16. Colletotrichum higginsianum Mitogen-Activated Protein Kinase ChMK1: Role in Growth, Cell Wall Integrity, Colony Melanization, and Pathogenicity

    PubMed Central

    Wei, Wei; Xiong, Ying; Zhu, Wenjun; Wang, Nancong; Yang, Guogen; Peng, Fang

    2016-01-01

    Colletotrichum higginsianum is an economically important pathogen that causes anthracnose disease in a wide range of cruciferous crops. To facilitate the efficient control of anthracnose disease, it will be important to understand the mechanism by which the cruciferous crops and C. higginsianum interact. A key step in understanding this interaction is characterizing the mitogen-activated protein kinases (MAPK) signaling pathway of C. higginsianum. MAPK plays important roles in diverse physiological processes of multiple pathogens. In this study, a Fus3/Kss1-related MAPK gene, ChMK1, from C. higginsianum was analyzed. The results showed that the Fus3/Kss1-related MAPK ChMK1 plays a significant role in cell wall integrity. Targeted deletion of ChMK1 resulted in a hypersensitivity to cell wall inhibitors, reduced conidiation and albinistic colonies. Further, the deletion mutant was also unable to form melanized appressorium, a specialized infection structure that is necessary for successful infection. Therefore, the deletion mutant loses pathogenicity on A. thaliana leaves, demonstrating that ChMK1 plays an essential role in the early infection step. In addition, the ChMK1 deletion mutant showed an attenuated growth rate that is different from that of its homolog in Colletotrichum lagenarium, indicating the diverse roles that Fus3/Kss1-related MAPKs plays in phytopathogenic fungi. Furthermore, the expression level of three melanin synthesis associated genes were clearly decreased in the albinistic ChMK1 mutant compared to that of the wild type strain, suggesting that ChMK1 is also required for colony melanization in C. higginsianum. PMID:27536296

  17. Open-cell cloud formation over the Bahamas

    NASA Technical Reports Server (NTRS)

    2002-01-01

    What atmospheric scientists refer to as open cell cloud formation is a regular occurrence on the back side of a low-pressure system or cyclone in the mid-latitudes. In the Northern Hemisphere, a low-pressure system will draw in surrounding air and spin it counterclockwise. That means that on the back side of the low-pressure center, cold air will be drawn in from the north, and on the front side, warm air will be drawn up from latitudes closer to the equator. This movement of an air mass is called advection, and when cold air advection occurs over warmer waters, open cell cloud formations often result. This MODIS image shows open cell cloud formation over the Atlantic Ocean off the southeast coast of the United States on February 19, 2002. This particular formation is the result of a low-pressure system sitting out in the North Atlantic Ocean a few hundred miles east of Massachusetts. (The low can be seen as the comma-shaped figure in the GOES-8 Infrared image from February 19, 2002.) Cold air is being drawn down from the north on the western side of the low and the open cell cumulus clouds begin to form as the cold air passes over the warmer Caribbean waters. For another look at the scene, check out the MODIS Direct Broadcast Image from the University of Wisconsin. Image courtesy Jacques Descloitres, MODIS Land Rapid Response Team at NASA GSFC

  18. Proteoglycans support proper granule formation in pancreatic acinar cells.

    PubMed

    Aroso, Miguel; Agricola, Brigitte; Hacker, Christian; Schrader, Michael

    2015-10-01

    Zymogen granules (ZG) are specialized organelles in the exocrine pancreas which allow digestive enzyme storage and regulated secretion. The molecular mechanisms of their biogenesis and the sorting of zymogens are still incompletely understood. Here, we investigated the role of proteoglycans in granule formation and secretion of zymogens in pancreatic AR42J cells, an acinar model system. Cupromeronic Blue cytochemistry and biochemical studies revealed an association of proteoglycans primarily with the granule membrane. Removal of proteoglycans by carbonate treatment led to a loss of membrane curvature indicating a supportive role in the maintenance of membrane shape and stability. Chemical inhibition of proteoglycan synthesis impaired the formation of normal electron-dense granules in AR42J cells and resulted in the formation of unusually small granule structures. These structures still contained the zymogen carboxypeptidase, a cargo molecule of secretory granules, but migrated to lighter fractions after density gradient centrifugation. Furthermore, the basal secretion of amylase was increased in AR42J cells after inhibitor treatment. In addition, irregular-shaped granules appeared in pancreatic lobules. We conclude that the assembly of a proteoglycan scaffold at the ZG membrane is supporting efficient packaging of zymogens and the proper formation of stimulus-competent storage granules in acinar cells of the pancreas. PMID:26105026

  19. Micro-crack formation in direct methanol fuel cell electrodes

    NASA Astrophysics Data System (ADS)

    Li, Qing; Spernjak, Dusan; Zelenay, Piotr; Kim, Yu Seung

    2014-12-01

    This study focuses on the micro-crack formation of Nafion®-based membrane electrode assemblies (MEAs) after extended direct methanol fuel cell (DMFC) operation. All electrodes, both with metal-black and carbon-supported catalysts, contain some micro-cracks initially; the area covered by these cracks increases both in the anode and cathode after 100-hours of DMFC test. X-ray tomography shows an increase in the crack area in both anode and cathode that correlates with methanol feed concentration and methanol crossover. The MEAs with carbon-supported catalysts and thicker membrane are more resistant to the formation of micro-cracks compared to those with metal-black catalysts and thinner membrane, respectively. The impact of the micro-crack formation on cell performance and durability is limited over the 100-hour DMFC operation, with the long-term impact remaining unknown.

  20. In vitro myelin formation using embryonic stem cells

    PubMed Central

    Kerman, Bilal E.; Kim, Hyung Joon; Padmanabhan, Krishnan; Mei, Arianna; Georges, Shereen; Joens, Matthew S.; Fitzpatrick, James A. J.; Jappelli, Roberto; Chandross, Karen J.; August, Paul; Gage, Fred H.

    2015-01-01

    Myelination in the central nervous system is the process by which oligodendrocytes form myelin sheaths around the axons of neurons. Myelination enables neurons to transmit information more quickly and more efficiently and allows for more complex brain functions; yet, remarkably, the underlying mechanism by which myelination occurs is still not fully understood. A reliable in vitro assay is essential to dissect oligodendrocyte and myelin biology. Hence, we developed a protocol to generate myelinating oligodendrocytes from mouse embryonic stem cells and established a myelin formation assay with embryonic stem cell-derived neurons in microfluidic devices. Myelin formation was quantified using a custom semi-automated method that is suitable for larger scale analysis. Finally, early myelination was followed in real time over several days and the results have led us to propose a new model for myelin formation. PMID:26015546

  1. Spatial patterns in ant colonies.

    PubMed

    Theraulaz, Guy; Bonabeau, Eric; Nicolis, Stamatios C; Solé, Ricard V; Fourcassié, Vincent; Blanco, Stéphane; Fournier, Richard; Joly, Jean-Louis; Fernández, Pau; Grimal, Anne; Dalle, Patrice; Deneubourg, Jean-Louis

    2002-07-23

    The origins of large-scale spatial patterns in biology have been an important source of theoretical speculation since the pioneering work by Turing (1952) on the chemical basis of morphogenesis. Knowing how these patterns emerge and their functional role is important to our understanding of the evolution of biocomplexity and the role played by self organization. However, so far, conclusive evidence for local activation-long-range inhibition mechanisms in real biological systems has been elusive. Here a well-defined experimental and theoretical analysis of the pattern formation dynamics exhibited by clustering behavior in ant colonies is presented. These experiments and a simple mathematical model show that these colonies do indeed use this type of mechanism. All microscopic variables have been measured and provide the first evidence, to our knowledge, for this type of self-organized behavior in complex biological systems, supporting early conjectures about its role in the organization of insect societies. PMID:12114538

  2. The role of Kupffer cells in glucan-induced granuloma formation in the liver of mice depleted of blood monocytes by administration of strontium-89

    SciTech Connect

    Naito, M.; Takahashi, K. )

    1991-05-01

    In order to elucidate the role of Kupffer cells in granuloma formation in the liver of mice under a condition of severe monocytopenia induced by administration of strontium-89, granulomas were produced by particulate glucan injection and examined histopathologically, immunohistochemically, by ({sup 3}H)thymidine autoradiography, and in culture experiments. Hepatic granulomas were smaller, less numerous, and more irregularly shaped in the monocytopenic mice than in the control mice. The granulomas were composed of multinuclear giant cells, epithelioid cells, Kupffer cells, and T lymphocytes, but not monocytes or granulocytes. Kupffer cells were heavily labeled with ({sup 3}H)thymidine in the monocytopenic mice, particularly just before the stage of granuloma formation, and then clustered in the liver sinusoids. At 8 days, they formed granulomas, transformed into epithelioid cells, and transformed further into multinuclear giant cells. Although the culture of liver cell suspensions prepared from the livers of monocytopenic mice sustained diffuse proliferation of macrophages on a monolayer of mouse stromal cell line (ST2), no monocyte/macrophage colonies were formed. From these results, it is reasonable to conclude that Kupffer cells alone are activated in a condition without a supply of monocytes from peripheral blood; proliferate and cluster in the hepatic sinusoids; transform into peroxidase-negative macrophages, epithelioid cells, and multinuclear giant cells; and participate in granuloma formation in loco together with T lymphocytes.

  3. Local transplantation of granulocyte colony stimulating factor-mobilized CD34+ cells for patients with femoral and tibial nonunion: pilot clinical trial.

    PubMed

    Kuroda, Ryosuke; Matsumoto, Tomoyuki; Niikura, Takahiro; Kawakami, Yohei; Fukui, Tomoaki; Lee, Sang Yang; Mifune, Yutaka; Kawamata, Shin; Fukushima, Masanori; Asahara, Takayuki; Kawamoto, Atsuhiko; Kurosaka, Masahiro

    2014-01-01

    Most bone fractures typically heal, although a significant proportion (5%-10%) of fractures fail to heal, resulting in delayed union or persistent nonunion. Some preclinical evidence shows the therapeutic potential of peripheral blood CD34(+) cells, a hematopoietic/endothelial progenitor cell-enriched population, for bone fracture healing; however, clinical outcome following transplantation of CD34(+) cells in patients with fracture has never been reported. We report a phase I/IIa clinical trial regarding transplantation of autologous, granulocyte colony stimulating factor-mobilized CD34(+) cells with atelocollagen scaffold for patients with femoral or tibial fracture nonunion (n = 7). The primary endpoint of this study is radiological fracture healing (union) by evaluating anteroposterior and lateral views at week 12 following cell therapy. For the safety evaluation, incidence, severity, and outcome of all adverse events were recorded. Radiological fracture healing at week 12 was achieved in five of seven cases (71.4%), which was greater than the threshold (18.1%) predefined by the historical outcome of the standard of care. The interval between cell transplantation and union, the secondary endpoint, was 12.6 ± 5.4 weeks (range, 8-24 weeks) for clinical healing and 16.1 ± 10.2 weeks (range, 8-36 weeks) for radiological healing. Neither deaths nor life-threatening adverse events were observed during the 1-year follow-up after the cell therapy. These results suggest feasibility, safety, and potential effectiveness of CD34(+) cell therapy in patients with nonunion. PMID:24307697

  4. Autocrine protective mechanisms of human granulocyte colony-stimulating factor (G-CSF) on retinal ganglion cells after optic nerve crush.

    PubMed

    Huang, Shun-Ping; Fang, Kan-Tang; Chang, Chung-Hsing; Huang, Tzu-Lun; Wen, Yao-Tseng; Tsai, Rong-Kung

    2016-02-01

    This study investigated the role of autocrine mechanisms in the anti-apoptotic effects of human granulocyte colony-stimulating factor (G-CSF) on retinal ganglion cells (RGCs) after optic nerve (ON) crush. We observed that both G-CSF and G-CSF receptor (G-CSFR) are expressed in normal rat retina. Further dual immunofluorescence staining showed G-CSFR immunoreactive cells were colocalized with RGCs, Müller cells, horizontal and amacrine cells. These results confirm that G-CSF is an endogenous ligand in the retina. The semi-quantitative RT-PCR finding demonstrated the transcription levels of G-CSF and G-CSFR were up-regulated after ON crush injury. G-CSF treatment further increased and prolonged the expression level of G-CSFR in the retina. G-CSF has been shown to enhance transdifferentiation of the mobilized hematopoietic stem cells into tissue to repair central nervous system injury. We test the hypothesis that the hematopoietic stem cells recruited by G-CSF treatment can transdifferentiate into RGCs after ON crush by performing sublethal irradiation of the rats 5 days before ON crush. The flow cytometric analysis showed the number of CD34 positive cells in the peripheral blood is significantly lower in the irradiated, crushed and G-CSF-treated group than the sham control group or crush and G-CSF treated group. Nevertheless, the G-CSF treatment enhances the RGC survival after sublethal irradiation and ON crush injury. These data indicate that G-CSF seems unlikely to induce hematopoietic stem cell transdifferentiation into RGCs after ON crush injury. In conclusion, G-CSF may serve an endogenous protective signaling in the retina through direct activation of intrinsic G-CSF receptors and downstream signaling pathways to rescue RGCs after ON crush injury, exogenous G-CSF administration can enhance the anti-apoptotic effects on RGCs. PMID:26518178

  5. Outer Segment Formation of Transplanted Photoreceptor Precursor Cells

    PubMed Central

    Eberle, Dominic; Kurth, Thomas; Santos-Ferreira, Tiago; Wilson, John; Corbeil, Denis; Ader, Marius

    2012-01-01

    Transplantation of photoreceptor precursor cells (PPCs) into the retina represents a promising treatment for cell replacement in blinding diseases characterized by photoreceptor loss. In preclinical studies, we and others demonstrated that grafted PPCs integrate into the host outer nuclear layer (ONL) and develop into mature photoreceptors. However, a key feature of light detecting photoreceptors, the outer segment (OS) with natively aligned disc membrane staples, has not been studied in detail following transplantation. Therefore, we used as donor cells PPCs isolated from neonatal double transgenic reporter mice in which OSs are selectively labeled by green fluorescent protein while cell bodies are highlighted by red fluorescent protein. PPCs were enriched using CD73-based magnetic associated cell sorting and subsequently transplanted into either adult wild-type or a model of autosomal-dominant retinal degeneration mice. Three weeks post-transplantation, donor photoreceptors were identified based on fluorescent-reporter expression and OS formation was monitored at light and electron microscopy levels. Donor cells that properly integrated into the host wild-type retina developed OSs with the formation of a connecting cilium and well-aligned disc membrane staples similar to the surrounding native cells of the host. Surprisingly, the majority of not-integrated PPCs that remained in the sub-retinal space also generated native-like OSs in wild-type mice and those affected by retinal degeneration. Moreover, they showed an improved photoreceptor maturation and OS formation by comparison to donor cells located on the vitreous side suggesting that environmental cues influence the PPC differentiation and maturation. We conclude that transplanted PPCs, whether integrated or not into the host ONL, are able to generate the cellular structure for effective light detection, a phenomenon observed in wild-type as well as in degenerated retinas. Given that patients suffering from

  6. Dolomitized cells within chert of the Permian Assistência Formation, Paraná Basin, Brazil

    NASA Astrophysics Data System (ADS)

    Calça, Cléber P.; Fairchild, Thomas R.; Cavalazzi, Barbara; Hachiro, Jorge; Petri, Setembrino; Huila, Manuel Fernando Gonzalez; Toma, Henrique E.; Araki, Koiti

    2016-04-01

    Dolomitic microscopic structures in the form of microspheres, "horseshoe- shaped" objects, and thin botryoidal crusts found within microfossiliferous chert within stromatolites of the Evaporite Bed (EB) of the Permian Assistência Formation, Irati Subgroup, Paraná Basin, Brazil, have been investigated by means of optical microscopy, X-ray fluorescence, scanning electron microscopy, Raman spectrometry and energy-dispersive X-ray spectrometry. The microspheres were identified as dolomitized coccoidal cyanobacteria based on similarity in size, spheroidal and paired hemispheroidal morphologies and colonial habit to co-occurring silicified organic-walled cyanobacteria embedded within the same microfabric and rock samples. The co-occurrence of dolomite, pyrite framboids, and abundant dispersed carbonaceous material and silicified cells is consistent with a hypersaline depositional environment with abundant cyanobacterial mats and elevated Mg2 +/Ca2 + ratios and reducing conditions with active anoxic microbial processes near the water-(bio)sediment interface. The abundance of extracellular polymeric substances facilitated anoxic microbial processes (sulfate reduction), providing essential conditions for possible primary microbially induced dolomitization. In most of the dolomitized cells dolomite occurs only as an external layer; in fully dolomitized cells magnesium is richest in the outermost layer. Presumably, the dolomitization process was favored by the presence of anoxic microbial degraders and negatively charged functional groups at the surface of the cyanobacterial cells. Botryoidal dolomite rims of silica-filled fenestrae formed by a similar process and inherited the botryoidal morphology of the cell as originally lining the fenestrae. Silicification interrupted the dolomitization of the largely organic biosediment, mostly by permineralization, but locally by substitution, thereby preserving not only dolomitic microspheres, but also huge numbers of structurally

  7. In Vitro Ectopic Behavior of Porcine Spermatogonial Germ Cells and Testicular Somatic Cells.

    PubMed

    Lee, Kyung Hoon; Lee, Won Young; Do, Jung Tae; Park, Chan Kyu; Kim, Nam Hyung; Kim, Jin Hoi; Chung, Hak Jae; Kim, Dong Woon; Song, Hyuk

    2016-08-01

    Embryonic body-like colony formation is a unique pattern in male germ cell cultures, including spermatogonial stem cells. However, detailed information of the colony formation has not yet been sufficiently reported in male germ cell culture. To elucidate the formation of germ cell-derived colony (GDC), glial cell-derived neurotrophic factor receptor alpha-1 (GFRα-1)-positive pig germ cells were isolated using an immunomagnetic cell isolation method and labeled with red- or green-fluorescent dye. In GDC culture, red-fluorescent-labeled germ cells were evenly distributed in the wells from day 1 to 4, and they clustered together at the time of GDC formation on day 6. Interestingly, feeder cells migrated to the site of colony formation as spermatogonia carriers. Furthermore, when freshly prepared green-labeled GFRα-1-positive germ cells were added, mixed-fluorescent dye (red and green) colonies were observed. On bromodeoxyuridine (BrdU) treatment, 58% ± 3.13% of germ cells were positive to protein gene product 9.5 but negative to BrdU cells. Immunocytochemistry and reverse transcription-polymerase chain reaction results showed that cultured GDC cells were positive to stem cell- and pig germ cell-specific marker genes. In conclusion, in vitro formation of GDCs is mainly dependent on the aggregation of single germ cells as well as on the slow proliferation of germ cells. PMID:27328332

  8. Effect of Periodic Granulocyte Colony-Stimulating Factor Administration on Endothelial Progenitor Cells and Different Monocyte Subsets in Pediatric Patients with Muscular Dystrophies

    PubMed Central

    Sienkiewicz, Dorota; Grubczak, Kamil; Okurowska-Zawada, Bożena; Paszko-Patej, Grażyna; Miklasz, Paula; Singh, Paulina; Radzikowska, Urszula; Kulak, Wojciech

    2016-01-01

    Muscular dystrophies (MD) are heterogeneous group of diseases characterized by progressive muscle dysfunction. There is a large body of evidence indicating that angiogenesis is impaired in muscles of MD patients. Therefore, induction of dystrophic muscle revascularization should become a novel approach aimed at diminishing the extent of myocyte damage. Recently, we and others demonstrated that administration of granulocyte colony-stimulating factor (G-CSF) resulted in clinical improvement of patients with neuromuscular disorders. To date, however, the exact mechanisms underlying these beneficial effects of G-CSF have not been fully understood. Here we used flow cytometry to quantitate numbers of CD34+ cells, endothelial progenitor cells, and different monocyte subsets in peripheral blood of pediatric MD patients treated with repetitive courses of G-CSF administration. We showed that repetitive cycles of G-CSF administration induced efficient mobilization of above-mentioned cells including cells with proangiogenic potential. These findings contribute to better understanding the beneficial clinical effects of G-CSF in pediatric MD patients. PMID:26770204

  9. Granulocyte colony-stimulating factor does not enhance recruitment of bone marrow-derived cells in rats with acute myocardial infarction.

    PubMed

    Sato, Daisuke; Otani, Hajime; Fujita, Masanori; Shimazu, Takayuki; Yoshioka, Kei; Enoki, Chiharu; Minato, Naoki; Iwasaka, Toshiji

    2012-09-01

    Despite the potential benefit of granulocyte colony-stimulating factor (G-CSF) therapy in patients with acute myocardial infarction (MI), the efficacy of G-CSF in regenerating the heart after MI remains controversial. The authors hypothesize that the limited efficacy of G-CSF is related to its inhibitory effect on recruitment of bone marrow-derived cells (BMCs) to the infarcted tissue. MI was induced in rats with intrabone marrow-bone marrow transplantation from syngenic rats expressing green fluorescence protein to track BMCs. G-CSF was administered for five days after the onset of MI. G-CSF increased the number of CD45(+) cells in the peripheral circulation but did not increase their recruitment to the heart. G-CSF had no effect on myocardial stromal-derived factor-1 alpha and chemokine (C-X-C motif) receptor 4 (CXCR4) expression in mononuclear cells in the peripheral blood and CXCR4(+) cells in the heart. G-CSF had no effect on angiogenesis, myocardial fibrosis or left ventricular function four weeks after MI. These results suggest that G-CSF mobilizes BMCs to the peripheral circulation but does not increase recruitment to the infarcted myocardium despite preservation of the stromal-derived factor-1 alpha/CXCR4 axis. PMID:23620693

  10. Hypoxic tumor cell death and modulation of endothelial adhesion molecules in the regression of granulocyte colony-stimulating factor-transduced tumors.

    PubMed Central

    Colombo, M. P.; Lombardi, L.; Melani, C.; Parenza, M.; Baroni, C.; Ruco, L.; Stoppacciaro, A.

    1996-01-01

    C-26 colon adenocarcinoma cells transduced with the granulocyte colony-stimulating factor (G-CSF) gene form large tumors when injected into sublethally irradiated mice. These tumors regress when leukocyte function is reconstituted. Electron microscopy and immunocytochemical analysis of regressing C-26/G-CSF nodules indicates that tumor destruction is due mainly to hypoxia resulting from the functional loss of tumor vasculature and is only marginally due to direct cytolysis. Desegregation of basal lamina, cell swelling, and loss of junctions characterized the vessels within regressing tumors. Tumor cells were necrotic or filled with lipid vacuoles regardless of the distance from nearby vessels. Damage of tumor vasculature was dependent on the infiltrating leukocytes and the cytotoxic cytokines they produced. Locally produced interleukin-1 and tumor necrosis factor-alpha (TNF-alpha) induced vascular cellular adhesion molecule-1 (VCAM-1) and E-selectin on tumor vessels. Treatment with monoclonal antibodies to interferon-gamma (IFN-gamma) or TNF-alpha blocked tumor regression by inhibiting VCAM-1 and E-selectin expression on tumor-associated endothelial cells resulting in a reduced number of infiltrating leukocytes. Thus, C-26/G-CSF tumor regression presents features typical of hemorrhagic necrosis that occurs through the cytokines produced by infiltrating leukocytes in response to G-CSF. Images Figure 1 p477-a Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8579110

  11. Effect of Periodic Granulocyte Colony-Stimulating Factor Administration on Endothelial Progenitor Cells and Different Monocyte Subsets in Pediatric Patients with Muscular Dystrophies.

    PubMed

    Eljaszewicz, Andrzej; Sienkiewicz, Dorota; Grubczak, Kamil; Okurowska-Zawada, Bożena; Paszko-Patej, Grażyna; Miklasz, Paula; Singh, Paulina; Radzikowska, Urszula; Kulak, Wojciech; Moniuszko, Marcin

    2016-01-01

    Muscular dystrophies (MD) are heterogeneous group of diseases characterized by progressive muscle dysfunction. There is a large body of evidence indicating that angiogenesis is impaired in muscles of MD patients. Therefore, induction of dystrophic muscle revascularization should become a novel approach aimed at diminishing the extent of myocyte damage. Recently, we and others demonstrated that administration of granulocyte colony-stimulating factor (G-CSF) resulted in clinical improvement of patients with neuromuscular disorders. To date, however, the exact mechanisms underlying these beneficial effects of G-CSF have not been fully understood. Here we used flow cytometry to quantitate numbers of CD34+ cells, endothelial progenitor cells, and different monocyte subsets in peripheral blood of pediatric MD patients treated with repetitive courses of G-CSF administration. We showed that repetitive cycles of G-CSF administration induced efficient mobilization of above-mentioned cells including cells with proangiogenic potential. These findings contribute to better understanding the beneficial clinical effects of G-CSF in pediatric MD patients. PMID:26770204

  12. Accumulation of cells expressing macrophage colony-stimulating factor receptor gene in the ovary of a pregnant viviparous fish, Neoditrema ransonnetii (Perciformes, Embiotocidae).

    PubMed

    Ueda, Kazuki; Saito, Erina; Iwasaki, Kaoru; Tsutsui, Shigeyuki; Nozawa, Aoi; Kikuchi, Kiyoshi; Nakamura, Osamu

    2016-03-01

    Macrophage colony-stimulating factor receptor (M-CSFR), a member of the group of type III protein tyrosine kinase receptors, is expressed primarily by monocyte/macrophage lineage cells. In order to describe the distribution of macrophages at the maternal-fetal interface in Neoditrema ransonnetii, a viviparous fish species, M-CSFR cDNA was sequenced. Two sequences were obtained: NrM-CSFR1 (4381 bp, encoding 980 amino acids), and NrM-CSFR2 (3573 bp, encoding 1016 amino acids). Both the genes were expressed in the ovary of pregnant females. In situ hybridization revealed that a number of cells that were positive for NrM-CSFR1 and/or NrM-CSFR2 populated the ovigerous lamellae of the ovary during pregnancy. Following parturition, M-CSFR-positive cells disappeared from the subepithelial region of ovigerous lamellae, and were localized in perivascular tissues. These results suggest the role of M-CSFR-positive cells, which appear to be macrophages, in N. ransonnetii during pregnancy. PMID:26828262

  13. Induction of Specific Cellular and Humoral Responses against Renal Cell Carcinoma after Combination Therapy with Cryoablation and Granulocyte-Macrophage Colony Stimulating Factor: A Pilot Study

    PubMed Central

    Thakur, Archana; Littrup, Peter; Paul, Elyse N.; Adam, Barbara; Heilbrun, Lance K.; Lum, Lawrence G.

    2013-01-01

    Cryotherapy offers a minimally invasive treatment option for the management of both irresectable and localized prostate, liver, pulmonary and renal tumors. The anti-neoplastic effects of cryotherapy are mediated by direct tumor lysis and by indirect effects such as intracellular dehydration, pH changes, and microvascular damage resulting in ischemic necrosis. In this study, we investigated whether percutaneous cryoablation of lung metastasis from renal cell carcinoma (RCC) in combination with aerosolized granulocyte-macrophage colony stimulating factor (GM-CSF) can induce systemic cellular and humoral immune responses in 6 RCC patients. Peripheral blood mononuclear cells (PBMC) were sequentially studied up to 63 days post cryoimmunotherapy (CI). PBMC from pre and post CI were phenotyped for lymphocyte subsets and tested for cytotoxicity and IFNγ Elispots directed at RCC cells. Humoral responses were measured by in vitro antibody synthesis assay directed at RCC cells. The immune monitoring data showed that CI induced tumor specific CTL, specific in vitro anti-tumor antibody responses, and enhanced Th1 cytokine production in 4 out of 6 patients. More importantly, the magnitude of cellular and humoral anti-tumor response appears to be associated with clinical responses. These pilot data show that CI can induce robust and brisk cellular and humoral immune responses in metastatic RCC patients, but requires further evaluation in optimized protocols. PMID:21577139

  14. Comparative analysis of four lipoproteins from Mycoplasma mycoides subsp. mycoides Small Colony identifies LppA as a major T-cell antigen.

    PubMed

    Dedieu, Laurence; Totte, Philippe; Rodrigues, Valerie; Vilei, Edy M; Frey, Joachim

    2010-07-01

    Control of contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC), remains an important goal in Africa. Subunit vaccines triggering B and T-cell responses could represent a promising approach. To this aim, the T-cell immunogenicity of four MmmSC lipoproteins (LppA, LppB, LppC and LppQ), present in African strains and able to elicit humoral response, was evaluated. In vitro assays revealed that only LppA was recognized by lymph node lymphocytes taken from three cattle, 3 weeks after MmmSC exposure. Maintenance of the LppA-specific response, relying on CD4 T-cells and IFN gamma production, was then demonstrated 1 year after infection. LppA is thus an important target for the CD4 T-cells generated early after MmmSC infection and persisting in the lymph nodes of recovered cattle. Its role as a protective antigen and ability to in vivo trigger both arms of the host immune response remain to be evaluated. PMID:19187963

  15. Multiwell cell culture plate format with integrated microfluidic perfusion system

    NASA Astrophysics Data System (ADS)

    Domansky, Karel; Inman, Walker; Serdy, Jim; Griffith, Linda G.

    2006-01-01

    A new cell culture analog has been developed. It is based on the standard multiwell cell culture plate format but it provides perfused three-dimensional cell culture capability. The new capability is achieved by integrating microfluidic valves and pumps into the plate. The system provides a means to conduct high throughput assays for target validation and predictive toxicology in the drug discovery and development process. It can be also used for evaluation of long-term exposure to drugs or environmental agents or as a model to study viral hepatitis, cancer metastasis, and other diseases and pathological conditions.

  16. Registration of Colony Switchgrass

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Colony (Reg. No. __________, PI 658520) is a lowland cytotype of switchgrass (Panicum virgatum L.) developed and released by the USDA-ARS and North Carolina Agricultural Research Service. Colony was developed from three cycles of selection with random cross pollination occurring in cycle 0, index se...

  17. Polydatin Inhibits Formation of Macrophage-Derived Foam Cells

    PubMed Central

    Wu, Min; Liu, Meixia; Guo, Gang; Zhang, Wengao; Liu, Longtao

    2015-01-01

    Rhizoma Polygoni Cuspidati, a Chinese herbal medicine, has been widely used in traditional Chinese medicine for a long time. Polydatin, one of the major active ingredients in Rhizoma Polygoni Cuspidati, has been recently shown to possess extensive cardiovascular pharmacological activities. In present study, we examined the effects of Polydatin on the formation of peritoneal macrophage-derived foam cells in Apolipoprotein E gene knockout mice (ApoE−/−) and explored the potential underlying mechanisms. Peritoneal macrophages were collected from ApoE−/− mice and cultured in vitro. These cells sequentially were divided into four groups: Control group, Model group, Lovastatin group, and Polydatin group. Our results demonstrated that Polydatin significantly inhibits the formation of foam cells derived from peritoneal macrophages. Further studies indicated that Polydatin regulates the metabolism of intracellular lipid and possesses anti-inflammatory effects, which may be regulated through the PPAR-γ signaling pathways. PMID:26557864

  18. The High Effect of Chemomobilization with High-Dose Etopside + Granulocyte-Colony Stimulating Factor in Autologous Hematopoietic Peripheral Blood Stem Cell Transplantation: A Single Center Experience

    PubMed Central

    Yanmaz, Mustafa Teoman; Selvi, Ahmet; Usul, Cigdem

    2016-01-01

    Autologous hematopoietic stem cell transplantation (auto-HSCT) provides hematopoietic support after high-dose chemotherapy and is the standard of care for patients with multiple myeloma (MM), chemo sensitive relapsed high or intermediate grade non-Hodgkin’s lymphoma (NHL) and Hodgkin’s lymphoma (HL). However, yields of hematopoietic stem cells vary greatly between patients, and the optimal strategy to mobilize hematopoietic stem cells into peripheral blood for collection has not been defined yet. We investigated the efficacy and safety of chemo mobilization with an intermediate dose etoposide (VP-16; 200 mg/m2 on days 1-3) and granulocyte-colony stimulating factor (G-CSF)(5 µg/kg twice daily from day 4 through the final day of collection). We reviewed our institutional experience with 91 patients (71 MM, 12 HL, 8 NHL) mobilized with this regimen. VP-16 + G-CSF resulted in successful mobilization in 95.55% of the patients (on one patient stem cell collection with plerixafor was applied), including 76 patients (83.52%) whose stem cells were collected successfully in a single day. Collection was managed between min. D8 and max. D17. Patient age, gender, exposure to previous irradiation and chemotherapy, previous mobilization attempts, and disease characteristics were not considered during selection. Adverse effects of the regimen included supportive transfusions and fevers requiring hospitalization or intravenous antibiotics. VP-16 and G-CSF appears to be a safe and effective mobilization regimen for patients with multiple myeloma, non-Hodgkin’s lymphoma and Hodgkin’s lymphoma undergoing autologous stem cell transplantation, producing excellent stem cell yield with the majority of patients requiring 1 day of apheresis. PMID:27103979

  19. Characterization of Commercial Li-ion Cells in Pouch Format

    NASA Technical Reports Server (NTRS)

    Jeevarajan, Judith

    2014-01-01

    The li-ion pouch design cells exhibit similar behavior under off-nominal conditions as those in metal cans that do not have the internal safety devices. Safety should be well characterized before batteries are designed. Some of the li-ion pouch cell designs studied in this program reacted most violently to overcharge conditions at the medium rates but were tolerant to overcharge at very low rates. Some pouch cell designs have higher tolerance to vacuum exposures than some others. A comparison of the pouch material itself does not show a correlation between this tolerance and the number of layers or composition of the pouch indicating that this is a property of the electrode stack design inside the pouch. Reduced pressure (8 to 10 psi) test environments show that the extent of capacity degradation under reduced pressure environments is much less than that observed under vacuum conditions. Lithium-ion Pouch format cells are not necessarily true polymer cells.

  20. Granulocyte colony-stimulating factor inhibits CXCR4/SDF-1α signaling and overcomes stromal-mediated drug resistance in the HL-60 cell line

    PubMed Central

    SHENG, XIANFU; ZHONG, HUA; WAN, HAIXIA; ZHONG, JIHUA; CHEN, FANGYUAN

    2016-01-01

    Combining cytarabine, aclarubicin and granulocyte colony-stimulating factor (G-CSF) has demonstrated marked efficacy in the treatment of elderly and relapsed/refractory patients with acute myeloid leukemia (AML); however, the role of G-CSF remains poorly understood. The present study aimed to investigate the ability of G-CSF to overcome stromal-mediated drug resistance and the underlying molecular mechanism. Two types of co-culture models were established in the HS-5 human bone marrow/stromal and HL-60 human promyelocytic leukemia cell lines, in order to imitate the interactions between stromal and leukemia cells in vitro, which is mediated by the stromal cell-derived factor (SDF)-1α signaling axis. In the present study, HL-60 cells were attracted and adhered to HS-5 cells using migration assay and flow cytometry, respectively; however, these interactions were inhibited by treatment with G-CSF and/or the C-X-C chemokine receptor type 4 (CXCR4) antagonist, AMD3100. Co-culture with HS-5 cells, including direct and indirect contact, protected HL-60 cells against spontaneous apoptosis or drug-induced apoptosis; however, these protective effects were disrupted by treatment with G-CSF and/or AMD3100. Notably, G-CSF and/or AMD3100 did not alter cell viability or apoptosis when HL-60 cells were cultured with medium alone. In addition, G-CSF significantly reduced the expression levels of surface CXCR4 protein, total CXCR4 protein and CXCR4 mRNA, and significantly upregulated the expression of microRNA (miR)-146a. Conversely, AMD3100 significantly reduced surface CXCR4 expression levels, but not the total CXCR4, CXCR4 mRNA or miR-146a expression levels. The results of the present study suggested that interfering with the CXCR4/SDF-1α signaling axis via G-CSF inhibited the migration and adhesion of HL-60 cells to HS-5 cells and eliminated HS5 cell-mediated protective effects. Furthermore, G-CSF administration reduced CXCR4 expression levels by upregulating the expression of

  1. Botryllus schlosseri (Tunicata) whole colony irradiation: Do senescent zooid resorption and immunological resorption involve similar recognition events

    SciTech Connect

    Rinkevich, B.; Weissman, I.L. )

    1990-02-01

    The colonial tunicate Botryllus schlosseri undergoes cyclic blastogenesis where feeding zooids are senescened and resorbed and a new generation of zooids takes over the colony. When non-identical colonies come into direct contact, they either reject each other or fuse. Fusion is usually followed by the resorption of one of the partners in the chimera (immunological resorption). The striking morphological similarities between the two resorption phenomena suggest that both may involve tissue destruction following self-nonself recognition events. Here we attempt to modify these two events by whole colony gamma irradiation assays. Three sets of experiments were performed: (1) different doses of whole colony irradiation for determination of irradiation effects (110 colonies); (2) pairs of irradiated-nonirradiated isografts of clonal replicates for the potential of reconstruction of the irradiated partners (23 pairs); (3) chimeras of irradiated-nonirradiated partners for analysis of resorption hierarchy. Mortality increased with the irradiation dose. All colonies exposed to more than 5,000 rads died within 19 days, while no colony died below 2,000 rads. The average mortality periods, in days, for doses of 6,000-8,000, 5,000, and 2,500-4,000 rads were 14.4 +/- 3.1 (n = 24), 19.8 +/- 6.0 (n = 15), and 19.6 + 5.1 (n = 22), respectively. Younger colonies (3-6 months old) may survive radiation better than older ones (more than 13 months). Many morphological alterations were recorded in irradiated colonies: ampullar contraction and/or dilation, accumulation of pigment cells within ampullae, abnormal bleeding from blood vessels, sluggish blood circulation, necrotic zones, reduction in bud number, and irregularities in zooid and system structures. With doses of 3,000-4,000 rads and above, irradiation arrested the formation of new buds and interrupted normal takeover.

  2. Scanning electron microscope study of Pseudomonas putida colonies.

    PubMed Central

    Shapiro, J A

    1985-01-01

    Pseudomonas putida colonies were examined by scanning electron microscope. A variety of cell morphologies, multicellular arrangements, and extracellular materials were observed in the fixed material. Different regions of a single colony showed characteristic organizations of these architectural elements. In some cases, the detailed microstructure of the fixed colony surfaces observed by scanning electron microscopy could be correlated with macroscopic patterns visualized by histochemical staining and surface relief photography of live colonies. Extracellular materials were seen to extend onto the agar surface beyond the boundaries of the cell mass, and the final structures of these materials, after fixation and desiccation, were colony specific. The significance of these features of colony microstructure for formulating hypotheses about the control of colony morphogenesis is discussed. Images PMID:4066611

  3. Identification of a Colonial Chordate Histocompatibility Gene

    PubMed Central

    Voskoboynik, Ayelet; Newman, Aaron M.; Corey, Daniel M.; Sahoo, Debashis; Pushkarev, Dmitry; Neff, Norma F.; Passarelli, Benedetto; Koh, Winston; Ishizuka, Katherine J.; Palmeri, Karla J.; Dimov, Ivan K.; Keasar, Chen; Fan, H. Christina; Mantalas, Gary L.; Sinha, Rahul; Penland, Lolita; Quake, Stephen R.; Weissman, Irving L.

    2013-01-01

    Histocompatibility is the basis by which multicellular organisms of the same species distinguish self from non-self. Relatively little is known about the mechanisms underlying histocompatibility reactions in lower organisms. Botryllus schlosseri is a colonial urochordate, a sister group of vertebrates, that exhibits a genetically determined natural transplantation reaction, whereby self-recognition between colonies leads to formation of parabionts with a common vasculature, whereas rejection occurs between incompatible colonies. Using genetically defined lines, whole-transcriptome sequencing, and genomics, we identified a single gene that encodes self/non-self and determines “graft” outcomes in this organism. This gene is significantly upregulated in colonies poised to undergo fusion or rejection, is highly expressed in the vasculature, and is functionally linked to histocompatibility outcomes. These findings establish a platform for advancing the science of allorecognition. PMID:23888037

  4. Phenotypic analysis of bacterial colonies using laser light scatter and pattern-recognition techniques

    NASA Astrophysics Data System (ADS)

    Rajwa, Bartek; Bayraktar, Bulent; Banada, Padmapriya P.; Huff, Karleigh; Bae, Euiwon; Hirleman, E. Daniel; Bhunia, Arun K.; Robinson, J. Paul

    2008-02-01

    The formation of bacterial colonies and biofilms requires coordinated gene expression, regulated cell differentiation, autoaggregation, and intercellular communication. Therefore colonies of bacteria have been recognized as multicellular organisms or "superorganisms." It has consequently been postulated that the phenotype of colonies formed by microorganisms can be automatically recognized and classified using optical systems capable of collecting information related to cellular pattern formation and morphology of colonies. Recently we have reported a first practical implementation of such a system, capable of noninvasive, label-free classification and recognition of pathogenic Listeria species. The design employed computer-vision and pattern-recognition techniques to classify scatter patterns produced by bacterial colonies irradiated with laser light. Herein we report our efforts to extend this system to other genera of bacteria such as Salmonella, Vibrio, Staphylococcus, and E. coli. Application of orthogonal moments, as well as texture descriptors for image feature extraction, provides high robustness in the presence of noise. An improved pattern classification scheme based on an SVM algorithm provides better results than the previously employed neural network system. Low error rates determined by cross-validation, reproducibility of the measurements, and overall robustness of the recognition system prove that the proposed technology can be implemented in automated devices for bacterial detection.

  5. Ligand Mobility Modulates Immunological Synapse Formation and T Cell Activation

    PubMed Central

    Hsu, Chih-Jung; Hsieh, Wan-Ting; Waldman, Abraham; Clarke, Fiona; Huseby, Eric S.; Burkhardt, Janis K.; Baumgart, Tobias

    2012-01-01

    T cell receptor (TCR) engagement induces clustering and recruitment to the plasma membrane of many signaling molecules, including the protein tyrosine kinase zeta-chain associated protein of 70 kDa (ZAP70) and the adaptor SH2 domain-containing leukocyte protein of 76 kDa (SLP76). This molecular rearrangement results in formation of the immunological synapse (IS), a dynamic protein array that modulates T cell activation. The current study investigates the effects of apparent long-range ligand mobility on T cell signaling activity and IS formation. We formed stimulatory lipid bilayers on glass surfaces from binary lipid mixtures with varied composition, and characterized these surfaces with respect to diffusion coefficient and fluid connectivity. Stimulatory ligands coupled to these surfaces with similar density and orientation showed differences in their ability to activate T cells. On less mobile membranes, central supramolecular activation cluster (cSMAC) formation was delayed and the overall accumulation of CD3ζ at the IS was reduced. Analysis of signaling microcluster (MC) dynamics showed that ZAP70 MCs exhibited faster track velocity and longer trajectories as a function of increased ligand mobility, whereas movement of SLP76 MCs was relatively insensitive to this parameter. Actin retrograde flow was observed on all surfaces, but cell spreading and subsequent cytoskeletal contraction were more pronounced on mobile membranes. Finally, increased tyrosine phosphorylation and persistent elevation of intracellular Ca2+ were observed in cells stimulated on fluid membranes. These results point to ligand mobility as an important parameter in modulating T cell responses. PMID:22384241

  6. Neutrophil apoptosis: impact of granulocyte macrophage colony stimulating factor on cell survival and viability in chronic kidney disease and hemodialysis patients

    PubMed Central

    Zahran, Nariman; Sayed, Azza; William, Iman; Sabry, Omar; Rafaat, Manar

    2013-01-01

    Introduction Altered neutrophil apoptosis might be responsible for recurrent bacterial infections encountered in hemodialysis (HD) and chronic kidney disease (CKD) patients. This work was designed to assess the neutrophil apoptotic activity and the impact of implementation of granulocyte macrophage colony stimulating factor (GM-CSF), as a survival factor, on neutrophil apoptosis among these patients. Material and methods Twenty-five patients on regular HD along with 34 CKD patients on conservative treatment, as well as 15 healthy controls, were investigated for apoptotic rate via assessment of neutrophil expression of Annexin-V by flow cytometry, before and after 20 h culture in absence and presence of GM-CSF. Neutrophil viability was determined using light microscopy. The preservation of neutrophil activation in these patients was analyzed by flow cytometric CD18 neutrophil expression. Chronic inflammatory state was evaluated by estimating C-reactive protein (CRP) and soluble intercellular adhesion molecule-1 (sICAM-1). Obtained data were statistically analyzed. Results Compared to controls, both HD and CKD groups had a significant increase of Annexin-V and CD18 expression and significant decrease in neutrophil viability. Culture of their neutrophils with GM-CSF showed significant decrease of apoptosis accompanied by improvement of neutrophil viability compared to their cultured cells without GM-CSF. These patients also showed significant elevation of CRP and sICAM-1. Conclusions Granulocyte macrophage colony stimulating factor demonstrated an evident impact on improving in vitro neutrophil survival and viability in HD and CKD patients. Therefore, this may represent promising preventive and/or therapeutic strategies against infection frequently observed in these patients and causing morbidity and mortality. PMID:24482640

  7. Smooth Muscle Cell-targeted RNA Aptamer Inhibits Neointimal Formation.

    PubMed

    Thiel, William H; Esposito, Carla L; Dickey, David D; Dassie, Justin P; Long, Matthew E; Adam, Joshua; Streeter, Jennifer; Schickling, Brandon; Takapoo, Maysam; Flenker, Katie S; Klesney-Tait, Julia; Franciscis, Vittorio de; Miller, Francis J; Giangrande, Paloma H

    2016-04-01

    Inhibition of vascular smooth muscle cell (VSMC) proliferation by drug eluting stents has markedly reduced intimal hyperplasia and subsequent in-stent restenosis. However, the effects of antiproliferative drugs on endothelial cells (EC) contribute to delayed re-endothelialization and late stent thrombosis. Cell-targeted therapies to inhibit VSMC remodeling while maintaining EC health are necessary to allow vascular healing while preventing restenosis. We describe an RNA aptamer (Apt 14) that functions as a smart drug by preferentially targeting VSMCs as compared to ECs and other myocytes. Furthermore, Apt 14 inhibits phosphatidylinositol 3-kinase/protein kinase-B (PI3K/Akt) and VSMC migration in response to multiple agonists by a mechanism that involves inhibition of platelet-derived growth factor receptor (PDGFR)-β phosphorylation. In a murine model of carotid injury, treatment of vessels with Apt 14 reduces neointimal formation to levels similar to those observed with paclitaxel. Importantly, we confirm that Apt 14 cross-reacts with rodent and human VSMCs, exhibits a half-life of ~300 hours in human serum, and does not elicit immune activation of human peripheral blood mononuclear cells. We describe a VSMC-targeted RNA aptamer that blocks cell migration and inhibits intimal formation. These findings provide the foundation for the translation of cell-targeted RNA therapeutics to vascular disease. PMID:26732878

  8. Myeloid cell kinetics in mice treated with recombinant interleukin-3, granulocyte colony-stimulating factor (CSF), or granulocyte-macrophage CSF in vivo

    SciTech Connect

    Lord, B.I.; Molineux, G.; Pojda, Z.; Souza, L.M.; Mermod, J.J.; Dexter, T.M. )

    1991-05-15

    Myeloid cell kinetics in mice treated with pure hematopoietic growth factors have been investigated using tritiated thymidine labeling and autoradiography. Mice were injected subcutaneously with 125 micrograms/kg granulocyte colony-stimulating factor (G-CSF) (in some cases 5 micrograms/kg), or 10 micrograms/kg of granulocyte-macrophage CSF (GM-CSF), or interleukin-3 (IL-3) every 12 hours for 84 hours. {sup 3}HTdR labeling was performed in vivo after 3 days of treatment. G-CSF increased the peripheral neutrophil count 14-fold and increased the proportion and proliferation rate of neutrophilic cells in the marrow, suppressing erythropoiesis at the same time. Newly produced mature cells were released into the circulation within 24 hours of labeling, compared with a normal appearance time of about 96 hours. By contrast, GM-CSF and IL-3 had little effect on either marrow cell kinetics or on the rate of release of mature cells, although GM-CSF did stimulate a 50% increase in peripheral neutrophils. Monocyte production was also increased about eightfold by G-CSF and 1.5-fold by GM-CSF, but their peak release was only slightly accelerated. While the peripheral half-lives of the neutrophilic granulocytes were normal, those of the monocytes were dramatically reduced, perhaps due to sequestration in the tissues for functional purposes. The stimulated monocyte production in the case of G-CSF required an additional five cell cycles, a level that might have repercussions on the progenitor compartments.

  9. Geometry and mechanics of growing bacterial colonies

    NASA Astrophysics Data System (ADS)

    You, Zhihong; Pearce, Daniel; Sengupta, Anupam; Giomi, Luca

    Bacterial colonies are abundant on living and non-living surfaces, and are known to mediate a broad range of processes in ecology, medicine and industry. Although extensively researched - from single cells up to the population levels - a comprehensive biophysical picture, highlighting the cell-to-colony dynamics, is still lacking. Here, using numerical and analytical models, we study the mechanics of self-organization leading to the colony morphology of cells growing on a substrate with free boundary. We consider hard rods to mimic the growth of rod-shaped non-motile cells, and show that the colony, as a whole, does not form an ordered nematic phase, nor does it result in a purely disordered (isotropic) phase. Instead, different sizes of domains, in which cells are highly aligned at specific orientations, are found. The distribution of the domain sizes follows an exponential relation - indicating the existence of a characteristic length scale that determines the domain size relative to that of the colony. A continuum theory, based on the hydrodynamics of liquid crystals, is built to account for these phenomena, and is applied to describe the buckling transition from a planar to three-dimensional (3D) colony. The theory supports preliminary experiments conducted with different strains of rod shaped bacterial cells, and reveals that the buckling transition can be regulated by varying the cell stiffness and aspect ratio. This work proposes that, in addition to biochemical pathways, the spatio-temporal organization in microbial colonies is significantly tuned by the biomechanical and geometric properties of the microbes in consideration.

  10. Effect of supercooling and cell volume on intracellular ice formation.

    PubMed

    Prickett, Richelle C; Marquez-Curtis, Leah A; Elliott, Janet A W; McGann, Locksley E

    2015-04-01

    Intracellular ice formation (IIF) has been linked to death of cells cryopreserved in suspension. It has been assumed that cells can be supercooled by 2 to 10°C before IIF occurs, but measurements of the degree of supercooling that cells can tolerate are often confounded by changing extracellular temperature and solutions of different osmolality (which affect the cell volume). The purpose of this study was to examine how the incidence of IIF in the absence of cryoprotectants is affected by the degree of supercooling and cell volume. Human umbilical vein endothelial cells were suspended in isotonic (300 mOsm) and hypertonic (∼600 to 700 mOsm) solutions and exposed to supercooling ranging from 2 to 10°C before extracellular ice was nucleated. The number of cells undergoing IIF was examined in a cryostage (based on the darkening of cells upon intracellular freezing ("flashing")) as a function of the degree of supercooling, and cell survival post-thaw was assessed using a membrane integrity assay. We found that while the incidence of IIF increased with supercooling in both isotonic and hypertonic solutions, it was higher in the isotonic solution at any given degree of supercooling. Since cells in hypertonic solution were shrunken due to water efflux, we hypothesized that the difference in IIF behavior could be attributed to the decreased volume of cells in the hypertonic solution. Our results confirm that cells with a smaller diameter before extracellular ice nucleation have a decreased probability of IIF and suggest that cell volume could play a more significant role in the incidence of IIF than the extracellular ice nucleation temperature. PMID:25707695

  11. Valine-Resistance, a Potential Marker in Plant Cell Genetics. II. Optimization of Uv Mutagenesis and Selection of Valine-Resistant Colonies Derived from Tobacco Mesophyll Protoplasts.

    PubMed

    Grandbastien, M A; Bourgin, J P; Caboche, M

    1985-02-01

    The induction and selection of valine-resistant mutants from haploid tobacco (Nicotiana tabacum L.) mesophyll protoplast-derived cells have been studied. Using cells from an original mutant plant obtained previously, we performed reconstruction experiments in order to determine the best conditions for the recovery of resistant cells among a population of sensitive cells. Optimal selective conditions were shown to depend on various factors including cell density, time of addition of valine and seasonal variations affecting the mother plants.-Using cell densities of approximately 10( 4) cells/ml, we defined efficient selective conditions: more than 25% of the putative mutant clones selected from UV-mutagenized protoplasts were reproducibly confirmed to be valine resistant. Further characterization of some regenerated mutant plants indicated that valine-resistance was associated with an uptake deficiency, as in the case of the original mutant plant of the Val(r)-2 line used for reconstruction experiments. Spontaneous mutation rates for valine-resistance were below accurately detectable levels, i.e., less than 10(-6) per cell per generation. Induced mutation frequency varied nonlinearily with UV dose from 10(-5) to 5 x 10(-4) resistant clones per surviving colony. Two independent loci (vr2 and vr3) were previously shown to be involved in valine-resistance due to amino acid uptake deficiency. Haploid tobacco plants were produced through anther culture from an F(1) double-heterozygous plant obtained from a cross between the original mutant plant and a wild-type plant. Study of the level of resistance to valine of protoplast-derived cells allowed the classification of these haploid plants in four types: sensitive, resistant and two intermediary resistant types believed to result from the presence of a mutant allele at only one of the two loci involved. The frequencies of UV-induced mutations in cells derived from haploid plants of one of the intermediary types were

  12. Deformed wing virus implicated in overwintering honeybee colony losses.

    PubMed

    Highfield, Andrea C; El Nagar, Aliya; Mackinder, Luke C M; Noël, Laure M-L J; Hall, Matthew J; Martin, Stephen J; Schroeder, Declan C

    2009-11-01

    The worldwide decline in honeybee colonies during the past 50 years has often been linked to the spread of the parasitic mite Varroa destructor and its interaction with certain honeybee viruses. Recently in the United States, dramatic honeybee losses (colony collapse disorder) have been reported; however, there remains no clear explanation for these colony losses, with parasitic mites, viruses, bacteria, and fungal diseases all being proposed as possible candidates. Common characteristics that most failing colonies share is a lack of overt disease symptoms and the disappearance of workers from what appears to be normally functioning colonies. In this study, we used quantitative PCR to monitor the presence of three honeybee viruses, deformed wing virus (DWV), acute bee paralysis virus (ABPV), and black queen cell virus (BQCV), during a 1-year period in 15 asymptomatic, varroa mite-positive honeybee colonies in Southern England, and 3 asymptomatic colonies confirmed to be varroa mite free. All colonies with varroa mites underwent control treatments to ensure that mite populations remained low throughout the study. Despite this, multiple virus infections were detected, yet a significant correlation was observed only between DWV viral load and overwintering colony losses. The long-held view has been that DWV is relatively harmless to the overall health status of honeybee colonies unless it is in association with severe varroa mite infestations. Our findings suggest that DWV can potentially act independently of varroa mites to bring about colony losses. Therefore, DWV may be a major factor in overwintering colony losses. PMID:19783750

  13. Deformed Wing Virus Implicated in Overwintering Honeybee Colony Losses ▿

    PubMed Central

    Highfield, Andrea C.; El Nagar, Aliya; Mackinder, Luke C. M.; Noël, Laure M.-L. J.; Hall, Matthew J.; Martin, Stephen J.; Schroeder, Declan C.

    2009-01-01

    The worldwide decline in honeybee colonies during the past 50 years has often been linked to the spread of the parasitic mite Varroa destructor and its interaction with certain honeybee viruses. Recently in the United States, dramatic honeybee losses (colony collapse disorder) have been reported; however, there remains no clear explanation for these colony losses, with parasitic mites, viruses, bacteria, and fungal diseases all being proposed as possible candidates. Common characteristics that most failing colonies share is a lack of overt disease symptoms and the disappearance of workers from what appears to be normally functioning colonies. In this study, we used quantitative PCR to monitor the presence of three honeybee viruses, deformed wing virus (DWV), acute bee paralysis virus (ABPV), and black queen cell virus (BQCV), during a 1-year period in 15 asymptomatic, varroa mite-positive honeybee colonies in Southern England, and 3 asymptomatic colonies confirmed to be varroa mite free. All colonies with varroa mites underwent control treatments to ensure that mite populations remained low throughout the study. Despite this, multiple virus infections were detected, yet a significant correlation was observed only between DWV viral load and overwintering colony losses. The long-held view has been that DWV is relatively harmless to the overall health status of honeybee colonies unless it is in association with severe varroa mite infestations. Our findings suggest that DWV can potentially act independently of varroa mites to bring about colony losses. Therefore, DWV may be a major factor in overwintering colony losses. PMID:19783750

  14. Cytokine regulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) production by human retinal pigment epithelial cells

    PubMed Central

    Crane, I J; Kuppner, M C; Mckillop-Smith, S; Wallace, C A; Forrester, J V

    1999-01-01

    GM-CSF is an important regulator of macrophage, granulocyte and dendritic cell behaviour and function. These cell types have been implicated in the retinal damage characteristic of endogenous posterior uveitis. Dendritic cells in the choroid have access to retinal antigens processed by the retinal pigment epithelial (RPE) cells of the blood–retinal barrier and are thought to be candidates for the presentation of antigen in uveoretinitis. We therefore investigated the production of GM-CSF and its regulation in human RPE cells. IL-1β, tumour necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-β) all stimulated GM-CSF production by RPE cells and a combination of these cytokines increased GM-CSF production over five-fold compared with that with the individual cytokines alone. Interferon-gamma (IFN-γ) rapidly down-regulated these responses. IFN-γ did not appear to be acting directly on IL-1β or via the synthesis of another protein. GM-CSF mRNA expression showed the same pattern of response to these cytokines, indicating transcriptional or pre-transcriptional regulation, and there was no evidence that IFN-γ was acting by destabilizing GM-CSF mRNA. These results are generally important in understanding the ways in which cytokine regulation differs between different cell types and also more specifically for determining ways in which a cytokine with a significant role in the development of autoimmune uveoretinitis may be manipulated. PMID:9933455

  15. Aroma formation by immobilized yeast cells in fermentation processes.

    PubMed

    Nedović, V; Gibson, B; Mantzouridou, T F; Bugarski, B; Djordjević, V; Kalušević, A; Paraskevopoulou, A; Sandell, M; Šmogrovičová, D; Yilmaztekin, M

    2015-01-01

    Immobilized cell technology has shown a significant promotional effect on the fermentation of alcoholic beverages such as beer, wine and cider. However, genetic, morphological and physiological alterations occurring in immobilized yeast cells impact on aroma formation during fermentation processes. The focus of this review is exploitation of existing knowledge on the biochemistry and the biological role of flavour production in yeast for the biotechnological production of aroma compounds of industrial importance, by means of immobilized yeast. Various types of carrier materials and immobilization methods proposed for application in beer, wine, fruit wine, cider and mead production are presented. Engineering aspects with special emphasis on immobilized cell bioreactor design, operation and scale-up potential are also discussed. Ultimately, examples of products with improved quality properties within the alcoholic beverages are addressed, together with identification and description of the future perspectives and scope for cell immobilization in fermentation processes. PMID:25267117

  16. Unequal distribution of plastids during generative cell formation in Impatiens.

    PubMed

    van Went, J L

    1984-07-01

    This paper describes the unequal distribution of plastids in the developing microspores of Impatiens walleriana and Impatiens glandulifera which leads to the exclusion of plastids from the generative cell. During the development from young microspore to the onset of mitosis a change in the organization of the cytoplasm and distribution of organelles is gradually established. This includes the formation of vacuoles at the poles of the elongate-shaped microspores, the movement of the nucleus to a position near the microspore wall in the central part of the cell, and the accumulation of the plastids to a position near the wall at the opposite side of the cell. In Impatiens walleriana, the accumulated plastids are separated from each other by ER cisterns, and some mitochondria are also accumulated. In both Impatiens species, the portion of the microspore in which the generative cell will be formed is completely devoid of plastids at the time mitosis starts. PMID:24257638

  17. Induction of Monocyte Chemoattractant Proteins in Macrophages via the Production of Granulocyte/Macrophage Colony-Stimulating Factor by Breast Cancer Cells

    PubMed Central

    Yoshimura, Teizo; Imamichi, Tomozumi; Weiss, Jonathan M.; Sato, Miwa; Li, Liangzhu; Matsukawa, Akihiro; Wang, Ji Ming

    2016-01-01

    Monocyte chemoattractant protein-1 (MCP-1)/CCL2 plays an important role in the initiation and progression of cancer. We previously reported that in 4T1 murine breast cancer, non-tumor stromal cells, including macrophages, were the major source of MCP-1. In the present study, we analyzed the potential mechanisms by which MCP-1 is upregulated in macrophages infiltrating 4T1 tumors. We found that cell-free culture supernatants of 4T1 cells (4T1-sup) markedly upregulated MCP-1 production by peritoneal inflammatory macrophages. 4T1-sup also upregulated other MCPs, such as MCP-3/CCL7 and MCP-5/CCL12, but modestly upregulated neutrophil chemotactic chemokines, such as KC/CXCL1 or MIP-2/CXCL2. Physicochemical analysis indicated that an approximately 2–3 kDa 4T1 cell product was responsible for the capacity of 4T1-sup to upregulate MCP-1 expression by macrophages. A neutralizing antibody against granulocyte/macrophage colony-stimulating factor (GM-CSF), but not macrophage CSF, almost completely abrogated MCP-1-inducing activity of 4T1-sup, and recombinant GM-CSF potently upregulated MCP-1 production by macrophages. The expression levels of GM-CSF in 4T1 tumors in vivo were higher than other tumors, such as Lewis lung carcinoma. Treatment of mice with anti-GM-CSF antibody significantly reduced the growth of 4T1 tumors at the injection sites but did not reduce MCP-1 production or lung metastasis in tumor-bearing mice. These results indicate that 4T1 cells have the capacity to directly upregulate MCP-1 production by macrophages by releasing GM-CSF; however, other mechanisms are also involved in increased MCP-1 levels in the 4T1 tumor microenvironment. PMID:26834744

  18. Effects of Granulocyte-Macrophage Colony-Stimulating (GM-CSF) Factor on Corneal Epithelial Cells in Corneal Wound Healing Model.

    PubMed

    Rho, Chang Rae; Park, Mi-young; Kang, Seungbum

    2015-01-01

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that activates granulocyte and macrophage cell lineages. It is also known to have an important function in wound healing. This study investigated the effect of GM-CSF in wound healing of human corneal epithelial cells (HCECs). We used human GM-CSF derived from rice cells (rice cell-derived recombinant human GM-CSF; rhGM-CSF). An in vitro migration assay was performed to investigate the migration rate of HCECs treated with various concentrations of rhGM-CSF (0.1, 1.0, and 10.0 μg/ml). MTT assay and flow cytometric analysis were used to evaluate the proliferative effect of rhGM-CSF. The protein level of p38MAPK was analyzed by western blotting. For in vivo analysis, 100 golden Syrian hamsters were divided into four groups, and their corneas were de-epithelialized with alcohol and a blade. The experimental groups were treated with 10, 20, or 50 μg/ml rhGM-CSF four times daily, and the control group was treated with phosphate-buffered saline. The corneal wound-healing rate was evaluated by fluorescein staining at the initial wounding and 12, 24, 36, and 48 hours after epithelial debridement. rhGM-CSF accelerated corneal epithelial wound healing both in vitro and in vivo. MTT assay and flow cytometric analysis revealed that rhGM-CSF treatment had no effects on HCEC proliferation. Western blot analysis demonstrated that the expression level of phosphorylated p38MAPK increased with rhGM-CSF treatment. These findings indicate that rhGM-CSF enhances corneal wound healing by accelerating cell migration. PMID:26376304

  19. Effects of Granulocyte-Macrophage Colony-Stimulating (GM-CSF) Factor on Corneal Epithelial Cells in Corneal Wound Healing Model

    PubMed Central

    Rho, Chang Rae; Park, Mi-young; Kang, Seungbum

    2015-01-01

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that activates granulocyte and macrophage cell lineages. It is also known to have an important function in wound healing. This study investigated the effect of GM-CSF in wound healing of human corneal epithelial cells (HCECs). We used human GM-CSF derived from rice cells (rice cell-derived recombinant human GM-CSF; rhGM-CSF). An in vitro migration assay was performed to investigate the migration rate of HCECs treated with various concentrations of rhGM-CSF (0.1, 1.0, and 10.0 μg/ml). MTT assay and flow cytometric analysis were used to evaluate the proliferative effect of rhGM-CSF. The protein level of p38MAPK was analyzed by western blotting. For in vivo analysis, 100 golden Syrian hamsters were divided into four groups, and their corneas were de-epithelialized with alcohol and a blade. The experimental groups were treated with 10, 20, or 50 μg/ml rhGM-CSF four times daily, and the control group was treated with phosphate-buffered saline. The corneal wound-healing rate was evaluated by fluorescein staining at the initial wounding and 12, 24, 36, and 48 hours after epithelial debridement. rhGM-CSF accelerated corneal epithelial wound healing both in vitro and in vivo. MTT assay and flow cytometric analysis revealed that rhGM-CSF treatment had no effects on HCEC proliferation. Western blot analysis demonstrated that the expression level of phosphorylated p38MAPK increased with rhGM-CSF treatment. These findings indicate that rhGM-CSF enhances corneal wound healing by accelerating cell migration. PMID:26376304

  20. A full genomic characterization of the development of a stable Small Colony Variant cell-type by a clinical Staphylococcus aureus strain.

    PubMed

    Bui, Long M G; Kidd, Stephen P

    2015-12-01

    A key to persistent and recurrent Staphylococcus aureus infections is its ability to adapt to diverse and toxic conditions. This ability includes a switch into a biofilm or to the quasi-dormant Small Colony Variant (SCV). The development and molecular attributes of SCVs have been difficult to study due to their rapid reversion to their parental cell-type. We recently described the unique induction of a matrix-embedded and stable SCV cell-type in a clinical S. aureus strain (WCH-SK2) by growing the cells with limiting conditions for a prolonged timeframe. Here we further study their characteristics. They possessed an increased viability in the presence of antibiotics compared to their non-SCV form. Their stability implied that there had been genetic changes; we therefore determined both the genome sequence of WCH-SK2 and its stable SCV form at a single base resolution, employing Single Molecular Real-Time (SMRT) sequencing that enabled the methylome to also be determined. The genetic features of WCH-SK2 have been identified; the SCCmec type, the pathogenicity and genetic islands and virulence factors. The genetic changes that had occurred in the stable SCV form were identified; most notably being in MgrA, a global regulator, and RsbU, a phosphoserine phosphatase within the regulatory pathway of the sigma factor SigB. There was a shift in the methylomes of the non-SCV and stable SCV forms. We have also shown a similar induction of this cell-type in other S. aureus strains and performed a genetic comparison to these and other S. aureus genomes. We additionally map RNAseq data to the WCH-SK2 genome in a transcriptomic analysis of the parental, SCV and stable SCV cells. The results from this study represent the unique identification of a suite of epigenetic, genetic and transcriptional factors that are implicated in the switch in S. aureus to its persistent SCV form. PMID:26458527

  1. Blockade of Mast Cell Activation Reduces Cutaneous Scar Formation

    PubMed Central

    Ranzer, Matthew J.; Wilgus, Traci A.; DiPietro, Luisa A.

    2014-01-01

    Damage to the skin initiates a cascade of well-orchestrated events that ultimately leads to repair of the wound. The inflammatory response is key to wound healing both through preventing infection and stimulating proliferation and remodeling of the skin. Mast cells within the tissue are one of the first immune cells to respond to trauma, and upon activation they release pro-inflammatory molecules to initiate recruitment of leukocytes and promote a vascular response in the tissue. Additionally, mast cells stimulate collagen synthesis by dermal fibroblasts, suggesting they may also influence scar formation. To examine the contribution of mast cells in tissue repair, we determined the effects the mast cell inhibitor, disodium cromoglycate (DSCG), on several parameters of dermal repair including, inflammation, re-epithelialization, collagen fiber organization, collagen ultrastructure, scar width and wound breaking strength. Mice treated with DSCG had significantly reduced levels of the inflammatory cytokines IL-1α, IL-1β, and CXCL1. Although DSCG treatment reduced the production of inflammatory mediators, the rate of re-epithelialization was not affected. Compared to control, inhibition of mast cell activity caused a significant decrease in scar width along with accelerated collagen re-organization. Despite the reduced scar width, DSCG treatment did not affect the breaking strength of the healed tissue. Tryptase β1 exclusively produced by mast cells was found to increase significantly in the course of wound healing. However, DSCG treatment did not change its level in the wounds. These results indicate that blockade of mast cell activation reduces scar formation and inflammation without further weakening the healed wound. PMID:24465509

  2. Blockade of mast cell activation reduces cutaneous scar formation.

    PubMed

    Chen, Lin; Schrementi, Megan E; Ranzer, Matthew J; Wilgus, Traci A; DiPietro, Luisa A

    2014-01-01

    Damage to the skin initiates a cascade of well-orchestrated events that ultimately leads to repair of the wound. The inflammatory response is key to wound healing both through preventing infection and stimulating proliferation and remodeling of the skin. Mast cells within the tissue are one of the first immune cells to respond to trauma, and upon activation they release pro-inflammatory molecules to initiate recruitment of leukocytes and promote a vascular response in the tissue. Additionally, mast cells stimulate collagen synthesis by dermal fibroblasts, suggesting they may also influence scar formation. To examine the contribution of mast cells in tissue repair, we determined the effects the mast cell inhibitor, disodium cromoglycate (DSCG), on several parameters of dermal repair including, inflammation, re-epithelialization, collagen fiber organization, collagen ultrastructure, scar width and wound breaking strength. Mice treated with DSCG had significantly reduced levels of the inflammatory cytokines IL-1α, IL-1β, and CXCL1. Although DSCG treatment reduced the production of inflammatory mediators, the rate of re-epithelialization was not affected. Compared to control, inhibition of mast cell activity caused a significant decrease in scar width along with accelerated collagen re-organization. Despite the reduced scar width, DSCG treatment did not affect the breaking strength of the healed tissue. Tryptase β1 exclusively produced by mast cells was found to increase significantly in the course of wound healing. However, DSCG treatment did not change its level in the wounds. These results indicate that blockade of mast cell activation reduces scar formation and inflammation without further weakening the healed wound. PMID:24465509

  3. T Cell factor 1 represses CD8+ effector T cell formation and function.

    PubMed

    Tiemessen, Machteld M; Baert, Miranda R M; Kok, Lianne; van Eggermond, Marja C J A; van den Elsen, Peter J; Arens, Ramon; Staal, Frank J T

    2014-12-01

    The Wnt-responsive transcription factor T cell factor 1 (Tcf1) is well known for its role in thymic T cell development and the formation of memory CD8(+) T cells. However, its role in the initial phases of CD8(+) T effector cell formation has remained unexplored. We report that high levels of Wnt signaling and Tcf1 are operational in naive and memory CD8(+) T cells, whereas Wnt signaling and Tcf1 were low in effector CD8(+) T cells. CD8(+) T cells deficient in Tcf1 produce IFN-γ more rapidly, coinciding with increased demethylation of the IFN-γ enhancer and higher expression of the transcription factors Tbet and Blimp1. Moreover, virus-specific Tcf1(-/-) CD8(+) T cells show accelerated expansion in acute infection, which is associated with increased IFN-γ and TNF production and lower viral load. Genetic complementation experiments with various Tcf1 isoforms indicate that Tcf1 dosage and protein stability are critical in suppressing IFN-γ production. Isoforms lacking the β-catenin binding domain are equally effective in inhibiting CD8(+) effector T cell formation. Thus, Tcf1 functions as a repressor of CD8(+) effector T cell formation in a β-catenin/Wnt-independent manner. PMID:25355919

  4. Exocytosis of macrophage lysosomes leads to digestion of apoptotic adipocytes and foam cell formation[S

    PubMed Central

    Haka, Abigail S.; Barbosa-Lorenzi, Valéria C.; Lee, Hyuek Jong; Falcone, Domenick J.; Hudis, Clifford A.; Dannenberg, Andrew J.

    2016-01-01

    Many types of apoptotic cells are phagocytosed and digested by macrophages. Adipocytes can be hundreds of times larger than macrophages, so they are too large to be digested by conventional phagocytic processes. The nature of the interaction between macrophages and apoptotic adipocytes has not been studied in detail. We describe a cellular process, termed exophagy, that is important for macrophage clearance of dead adipocytes and adipose tissue homeostasis. Using mouse models of obesity, human tissue, and a cell culture model, we show that macrophages form hydrolytic extracellular compartments at points of contact with dead adipocytes using local actin polymerization. These compartments are acidic and contain lysosomal enzymes delivered by exocytosis. Uptake and complete degradation of adipocyte fragments, which are released by extracellular hydrolysis, leads to macrophage foam cell formation. Exophagy-mediated foam cell formation is a highly efficient means by which macrophages internalize large amounts of lipid, which may ultimately overwhelm the metabolic capacity of the macrophage. This process provides a mechanism for degradation of objects, such as dead adipocytes, that are too large to be phagocytosed by macrophages. PMID:27044658

  5. A Chimeric HIV-1 Envelope Glycoprotein Trimer with an Embedded Granulocyte-Macrophage Colony-stimulating Factor (GM-CSF) Domain Induces Enhanced Antibody and T Cell Responses*

    PubMed Central

    van Montfort, Thijs; Melchers, Mark; Isik, Gözde; Menis, Sergey; Huang, Po-Ssu; Matthews, Katie; Michael, Elizabeth; Berkhout, Ben; Schief, William R.; Moore, John P.; Sanders, Rogier W.

    2011-01-01

    An effective HIV-1 vaccine should ideally induce strong humoral and cellular immune responses that provide sterilizing immunity over a prolonged period. Current HIV-1 vaccines have failed in inducing such immunity. The viral envelope glycoprotein complex (Env) can be targeted by neutralizing antibodies to block infection, but several Env properties limit the ability to induce an antibody response of sufficient quantity and quality. We hypothesized that Env immunogenicity could be improved by embedding an immunostimulatory protein domain within its sequence. A stabilized Env trimer was therefore engineered with the granulocyte-macrophage colony-stimulating factor (GM-CSF) inserted into the V1V2 domain of gp120. Probing with neutralizing antibodies showed that both the Env and GM-CSF components of the chimeric protein were folded correctly. Furthermore, the embedded GM-CSF domain was functional as a cytokine in vitro. Mouse immunization studies demonstrated that chimeric EnvGM-CSF enhanced Env-specific antibody and T cell responses compared with wild-type Env. Collectively, these results show that targeting and activation of immune cells using engineered cytokine domains within the protein can improve the immunogenicity of Env subunit vaccines. PMID:21515681

  6. Epigenetic Regulation of Placenta-Specific 8 Contributes to Altered Function of Endothelial Colony-Forming Cells Exposed to Intrauterine Gestational Diabetes Mellitus.

    PubMed

    Blue, Emily K; Sheehan, BreAnn M; Nuss, Zia V; Boyle, Frances A; Hocutt, Caleb M; Gohn, Cassandra R; Varberg, Kaela M; McClintick, Jeanette N; Haneline, Laura S

    2015-07-01

    Intrauterine exposure to gestational diabetes mellitus (GDM) is linked to development of hypertension, obesity, and type 2 diabetes in children. Our previous studies determined that endothelial colony-forming cells (ECFCs) from neonates exposed to GDM exhibit impaired function. The current goals were to identify aberrantly expressed genes that contribute to impaired function of GDM-exposed ECFCs and to evaluate for evidence of altered epigenetic regulation of gene expression. Genome-wide mRNA expression analysis was conducted on ECFCs from control and GDM pregnancies. Candidate genes were validated by quantitative RT-PCR and Western blotting. Bisulfite sequencing evaluated DNA methylation of placenta-specific 8 (PLAC8). Proliferation and senescence assays of ECFCs transfected with siRNA to knockdown PLAC8 were performed to determine functional impact. Thirty-eight genes were differentially expressed between control and GDM-exposed ECFCs. PLAC8 was highly expressed in GDM-exposed ECFCs, and PLAC8 expression correlated with maternal hyperglycemia. Methylation status of 17 CpG sites in PLAC8 negatively correlated with mRNA expression. Knockdown of PLAC8 in GDM-exposed ECFCs improved proliferation and senescence defects. This study provides strong evidence in neonatal endothelial progenitor cells that GDM exposure in utero leads to altered gene expression and DNA methylation, suggesting the possibility of altered epigenetic regulation. PMID:25720387

  7. Second-line treatment of non small cell lung cancer by biweekly gemcitabine and docetaxel +/- granulocyte-macrophage colony stimulating factor and low dose aldesleukine.

    PubMed

    Correale, Pierpaolo; Tindara Miano, Salvatora; Remondo, Cinzia; Migali, Cristina; Rotundo, Maria Saveria; Macrì, Paolo; Tagliaferri, Pierosandro; Caraglia, Michele; Gotti, Giuseppe; Francini, Guido

    2009-03-15

    Background. The antitumor activity of a novel biweekly gemcitabine (G) + docetaxel (D) regimen +/- granulocyte-macrophage colony stimulating factor (GM-CSF) and aldesleukine (IL-2) has been evaluated in a phase II trial in advanced pretreated non-small-cell lung cancer (NSCLC). Results. The treatment was well tolerated. The 42.3% response rate exceeded the predefined target activity, while time to progression (TTP) and overall survival (OS) were 7 and 11.2 months, respectively. A greater objective response rate (58.3% vs 28.6%) and an increased number of eosinophils, basophils and activated mononuclear blood cells were observed in those patients who also received cytokine administration. Methods. Twenty-six NSCLC patients received second line G (1000 mg/m2) and D (75 mg/m2) every 15 days. 12/26 patients also received s.c. GM-CSF (100µg, days 2-6) and s.c. IL-2 (0.5MIU/ twice daily, days 7-14 and 16-29) by random selection. Conclusion. The biweekly GD regimen is a safe and active second-line treatment in NSCLC. Addition of immune-adjuvant cytokines' may enhance the activity of this therapeutic combination. PMID:19242101

  8. PU.1 (Spi-1) and C/EBP alpha regulate the granulocyte colony-stimulating factor receptor promoter in myeloid cells.

    PubMed

    Smith, L T; Hohaus, S; Gonzalez, D A; Dziennis, S E; Tenen, D G

    1996-08-15

    Cytokines, important for lineage commitment and differentiation during hematopoiesis, exert their influence by binding specific receptors. Receptor expression is tightly regulated and examining the factors that govern their expression will allow better understanding of the events that determine lineage commitment. The granulocyte colony-stimulating factor (G-CSF) receptor is expressed exclusively in myeloid cells and the placenta. We show here that the G-CSF receptor transcription start site is identical in each of these tissues. A 1,391-bp fragment of the G-CSF receptor promoter is both active in myeloid cell lines and tissue specific. We have also found two regions that are important for G-CSF receptor promoter activity. One region, located at bp -49, contains a GCAAT site that specifically binds the C/EBP alpha transcription factor in myeloid nuclear extracts. Mutation of this site prevents C/EBP alpha binding and reduces promoter activity by 60%. The other functionally important region of the G-CSF receptor promoter is in the 5' untranslated region, at bp +36 and +43, where there are two sites for the ets family member PU.1. Mutation of these sites prevents PU.1 binding and reduces promoter activity by 75%. These results reinforce the importance of both PU.1 and C/EBP alpha in the expression of myeloid-specific genes and neutrophil development. PMID:8695841

  9. Case Report: Combination Therapy with Mesenchymal Stem Cells and Granulocyte-Colony Stimulating Factor in a Case of Spinal Cord Injury

    PubMed Central

    Derakhshanrad, Nazi; Saberi, Hooshang; Tayebi Meybodi, Keyvan; Taghvaei, Mohammad; Arjmand, Babak; Aghayan, Hamid Reza; Kohan, Amir Hassan; Haghpanahi, Mohammad; Rahmani, Shahrokh

    2015-01-01

    Introduction: Various neuroregenerative procedures have been recently employed along with neurorehabilitation programs to promote neurological function after Spinal Cord Injury (SCI), and recently most of them have focused on the acute stage of spinal cord injury. In this report, we present a case of acute SCI treated with neuroprotective treatments in conjunction with conventional rehabilitation program. Methods: A case of acute penetrative SCI (gunshot wound), 40 years old, was treated with intrathecal bone marrow derived stem cells and parenteral Granulocyte-Colony Stimulating Factor (G-CSF) along with rehabilitation program. The neurological outcomes as well as safety issues have been reported. Results: Assessment with American Spinal Injury Association (ASIA), showed neurological improvement, meanwhile he reported neuropathic pain, which was amenable to oral medication. Discussion: In the acute setting, combination therapy of G-CSF and intrathecal Mesenchymal Stem Cells (MSCs) was safe in our case as an adjunct to conventional rehabilitation programs. Further controlled studies are needed to find possible side effects, and establish net efficacy. PMID:26649168

  10. Defective Self-Renewal and Differentiation of GBA-Deficient Neural Stem Cells Can Be Restored By Macrophage Colony-Stimulating Factor

    PubMed Central

    Lee, Hyun; Bae, Jae-sung; Jin, Hee Kyung

    2015-01-01

    Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the glucocerebrosidase gene (GBA), which encodes the lysosomal enzyme glucosylceramidase (GCase). Deficiency in GCase leads to characteristic visceral pathology and lethal neurological manifestations in some patients. Investigations into neurogenesis have suggested that neurodegenerative disorders, such as GD, could be overcome or at least ameliorated by the generation of new neurons. Bone marrow-derived mesenchymal stem cells (BM-MSCs) are potential candidates for use in the treatment of neurodegenerative disorders because of their ability to promote neurogenesis. Our objective was to examine the mechanism of neurogenesis by BM-MSCs in GD. We found that neural stem cells (NSCs) derived from a neuronopathic GD model exhibited decreased ability for self-renewal and neuronal differentiation. Co-culture of GBA-deficient NSCs with BM-MSCs resulted in an enhanced capacity for self-renewal, and an increased ability for differentiation into neurons or oligodendrocytes. Enhanced proliferation and neuronal differentiation of GBA-deficient NSCs was associated with elevated release of macrophage colony-stimulating factor (M-CSF) from BM-MSCs. Our findings suggest that soluble M-CSF derived from BM-MSCs can modulate GBA-deficient NSCs, resulting in their improved proliferation and neuronal differentiation. PMID:26282862

  11. The Colonial Adult Educator

    ERIC Educational Resources Information Center

    Long, Huey B.

    1975-01-01

    The adult evening schools of colonial America served a mixed clientele and provided instruction in a variety of subjects. Although most historians have described evening schoolmasters as incompetent frauds, research indicates that such descriptions are generally unjust and unfounded. (NHM)

  12. Robotic space colonies

    NASA Technical Reports Server (NTRS)

    Schenker, P.; Easter, R.; Rodriguez, G.

    2001-01-01

    This paper reviews recent advances in these technologies, with a particular focus on experimental state-of-the-art robot work crew system demonstrations at JPL, that are being conducted now to begin to realize the futuristic robotic colony vision.

  13. A motif within the N-terminal domain of TSP-1 specifically promotes the proangiogenic activity of endothelial colony-forming cells.

    PubMed

    Dias, Juliana Vieira; Benslimane-Ahmim, Zahia; Egot, Marion; Lokajczyk, Anna; Grelac, Françoise; Galy-Fauroux, Isabelle; Juliano, Luiz; Le-Bonniec, Bernard; Takiya, Cristina Maeda; Fischer, Anne-Marie; Blanc-Brude, Olivier; Morandi, Verônica; Boisson-Vidal, Catherine

    2012-10-15

    Thrombospondin-1 (TSP-1) gives rise to fragments that have both pro- and anti-angiogenic effects in vitro and in vivo. The TSP-HepI peptide (2.3 kDa), located in the N-terminal domain of TSP-1, has proangiogenic effects on endothelial cells. We have previously shown that TSP-1 itself exhibits a dual effect on endothelial colony-forming cells (ECFC) by enhancing their adhesion through its TSP-HepI fragment while reducing their proliferation and differentiation into vascular tubes (tubulogenesis) in vitro. This effect is likely mediated through CD47 binding to the TSP-1 C-terminal domain. Here we investigated the effect of TSP-HepI peptide on the angiogenic properties of ECFC in vitro and in vivo. TSP-HepI peptide potentiated FGF-2-induced neovascularisation by enhancing ECFC chemotaxis and tubulogenesis in a Matrigel plug assay. ECFC exposure to 20 μg/mL of TSP-HepI peptide for 18 h enhanced cell migration (p < 0.001 versus VEGF exposure), upregulated alpha 6-integrin expression, and enhanced their cell adhesion to activated endothelium under physiological shear stress conditions at levels comparable to those of SDF-1α. The adhesion enhancement appeared to be mediated by the heparan sulfate proteoglycan (HSPG) syndecan-4, as ECFC adhesion was significantly reduced by a syndecan-4-neutralising antibody. ECFC migration and tubulogenesis were stimulated neither by a TSP-HepI peptide with a modified heparin-binding site (S/TSP-HepI) nor when the glycosaminoglycans (GAGs) moieties were removed from the ECFC surface by enzymatic treatment. Ex vivo TSP-HepI priming could potentially serve to enhance the effectiveness of therapeutic neovascularisation with ECFC. PMID:22796565

  14. miR-155 Is Associated with the Leukemogenic Potential of the Class IV Granulocyte Colony-Stimulating Factor Receptor in CD34+ Progenitor Cells

    PubMed Central

    Zhang, HaiJiao; Goudeva, Lilia; Immenschuh, Stephan; Schambach, Axel; Skokowa, Julia; Eiz-Vesper, Britta; Blasczyk, Rainer; Figueiredo, Constança

    2014-01-01

    Granulocyte colony-stimulating factor (G-CSF) is a major regulator of granulopoiesis on engagement with the G-CSF receptor (G-CSFR). The truncated, alternatively spliced, class IV G-CSFR (G-CSFRIV) has been associated with defective differentiation and relapse risk in pediatric acute myeloid leukemia (AML) patients. However, the detailed biological properties of G-CSFRIV in human CD34+ hematopoietic stem and progenitor cells (HSPCs) and the potential leukemogenic mechanism of this receptor remain poorly understood. In the present study, we observed that G-CSFRIV–overexpressing (G-CSFRIV+) HSPCs demonstrated an enhanced proliferative and survival capacity on G-CSF stimulation. Cell cycle analyses showed a higher frequency of G-CSFRIV+ cells in the S and G2/M phase. Also, apoptosis rates were significantly lower in G-CSFRIV+ HSPCs. These findings were shown to be associated with a sustained Stat5 activation and elevated miR-155 expression. In addition, G-CSF showed to further induce G-CSFRIV and miR-155 expression of peripheral blood mononuclear cells isolated from AML patients. A Stat5 pharmacological inhibitor or ribonucleic acid (RNA) interference–mediated silencing of the expression of miR-155 abrogated the aberrant proliferative capacity of the G-CSFRIV+ HSPCs. Hence, the dysregulation of Stat5/miR-155 pathway in the G-CSFRIV+ HSPCs supports their leukemogenic potential. Specific miRNA silencing or the inhibition of Stat5-associated pathways might contribute to preventing the risk of leukemogenesis in G-CSFRIV+ HSPCs. This study may promote the development of a personalized effective antileukemia therapy, in particular for the patients exhibiting higher expression levels of G-CSFRIV, and further highlights the necessity of pre-screening the patients for G-CSFR isoforms expression patterns before G-CSF administration. PMID:25730818

  15. Constitutive Store-Operated Ca(2+) Entry Leads to Enhanced Nitric Oxide Production and Proliferation in Infantile Hemangioma-Derived Endothelial Colony-Forming Cells.

    PubMed

    Zuccolo, Estella; Bottino, Cinzia; Diofano, Federica; Poletto, Valentina; Codazzi, Alessia Claudia; Mannarino, Savina; Campanelli, Rita; Fois, Gabriella; Marseglia, Gian Luigi; Guerra, Germano; Montagna, Daniela; Laforenza, Umberto; Rosti, Vittorio; Massa, Margherita; Moccia, Francesco

    2016-02-15

    Clonal endothelial progenitor cells (EPCs) have been implicated in the aberrant vascular growth that features infantile hemangioma (IH), the most common benign vascular tumor in childhood that may cause ulceration, bleeding, and/or permanent disfigurement. Endothelial colony-forming cells (ECFCs), truly endothelial EPCs endowed with clonal ability and capable of forming patent vessels in vivo, remodel their Ca(2+) toolkit in tumor-derived patients to acquire an adaptive advantage. Particularly, they upregulate the proangiogenic store-operated Ca(2+) entry (SOCE) pathway due to the overexpression of its underlying components, that is, stromal interaction molecule 1 (Stim1), Orai1, and transient receptor potential canonical 1 (TRPC1). The present work was undertaken to assess whether and how the Ca(2+) signalosome is altered in IH-ECFCs by employing Ca(2+) and nitric oxide (NO) imaging, real-time polymerase chain reaction, western blotting, and functional assays. IH-ECFCs display a lower intracellular Ca(2+) release in response to either pharmacological (i.e., cyclopiazonic acid) or physiological (i.e., ATP and vascular endothelial growth factor) stimulation. Conversely, Stim1, Orai1, and TRPC1 transcripts and proteins are normally expressed in these cells and mediate a constitutive SOCE, which is sensitive to BTP-2, La(3+), and Pyr6 and recharges the intracellular Ca(2+) pool. The resting SOCE in IH-ECFCs is also associated to an increase in their proliferation rate and the basal production of NO compared to normal cells. Likewise, the pharmacological blockade of SOCE and NO synthesis block IH-ECFC growth. Collectively, these data indicate that the constitutive SOCE activation enhances IH-ECFC proliferation by augmenting basal NO production and sheds novel light on the molecular mechanisms of IH. PMID:26654173

  16. EFFECT OF PHORBOL ESTERS ON CLONAL CULTURES OF HUMAN, HAMSTER, AND RAT RESPIRATORY EPITHELIAL CELLS

    EPA Science Inventory

    The effect of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on the growth of epithelial cells from rat, hamster, and human respiratory tract has been measured by monitoring colony formation in culture. TPA and its active derivatives stimulated colony formation of ...

  17. Identification and characterization of a highly motile and antibiotic refractory subpopulation involved in the expansion of swarming colonies of Paenibacillus vortex

    PubMed Central

    Roth, Dalit; Finkelshtein, Alin; Ingham, Colin; Helman, Yael; Sirota-Madi, Alexandra; Brodsky, Leonid; Ben-Jacob, Eshel

    2013-01-01

    Summary Bacteria often use sophisticated cooperative behaviours, such as the development of complex colonies, elaborate biofilms and advanced dispersal strategies, to cope with the harsh and variable conditions of natural habitats, including the presence of antibiotics. Paenibacillus vortex uses swarming motility and cell-to-cell communication to form complex, structured colonies. The modular organization of P. vortex colony has been found to facilitate its dispersal on agar surfaces. The current study reveals that the complex structure of the colony is generated by the coexistence and transition between two morphotypes – ‘builders’ and ‘explorers’ – with distinct functions in colony formation. Here, we focused on the explorers, which are highly motile and spearhead colonial expansion. Explorers are characterized by high expression levels of flagellar genes, such as flagellin (hag), motA, fliI, flgK and sigD, hyperflagellation, decrease in ATP (adenosine-5′-triphosphate) levels, and increased resistance to antibiotics. Their tolerance to many antibiotics gives them the advantage of translocation through antibiotics-containing areas. This work gives new insights on the importance of cell differentiation and task distribution in colony morphogenesis and adaptation to antibiotics. PMID:23763278

  18. Multicellular group formation in response to predators in the alga Chlorella vulgaris.

    PubMed

    Fisher, R M; Bell, T; West, S A

    2016-03-01

    A key step in the evolution of multicellular organisms is the formation of cooperative multicellular groups. It has been suggested that predation pressure may promote multicellular group formation in some algae and bacteria, with cells forming groups to lower their chance of being eaten. We use the green alga Chlorella vulgaris and the protist Tetrahymena thermophila to test whether predation pressure can initiate the formation of colonies. We found that: (1) either predators or just predator exoproducts promote colony formation; (2) higher predator densities cause more colonies to form; and (3) colony formation in this system is facultative, with populations returning to being unicellular when the predation pressure is removed. These results provide empirical support for the hypothesis that predation pressure promotes multicellular group formation. The speed of the reversion of populations to unicellularity suggests that this response is due to phenotypic plasticity and not evolutionary change. PMID:26663204

  19. Regulation of lamellipodia formation and cell invasion by CLIP-170 in invasive human breast cancer cells.

    PubMed

    Suzuki, Katsuo; Takahashi, Kazuhide

    2008-04-01

    Lamellipodia formation necessary for cell invasion is regulated by Rac1. We report here that lamellipodia formation and three-dimensional invasion were significantly promoted by HGF and serum, respectively, in invasive human breast cancer cells. Rac1 formed a complex with CLIP-170, IQGAP1, and kinesin in serum-starved cells, and stimulation of the cells with HGF and serum caused the partial release of IQGAP1 and kinesin from Rac1-CLIP-170 complex. The HGF-induced release of the proteins and promotion of lamellipodia formation were inhibited by an inhibitor of PI3K. Moreover, downregulation of CLIP-170 by siRNA released IQGAP1 and kinesin from Rac1 and promoted lamellipodia formation and invasion, independent of HGF and serum. The results suggest that promotion of lamellipodia formation and invasion by HGF or serum requires PI3K-dependent release of IQGAP1 and kinesin from Rac1-CLIP-170 complex and that CLIP-170 prevents cells from the extracellular stimulus-independent lamellipodia formation and invasion by tethering IQGAP1 and kinesin to Rac1. PMID:18237546

  20. Cell collectivity regulation within migrating cell cluster during Kupffer's vesicle formation in zebrafish

    PubMed Central

    Matsui, Takaaki; Ishikawa, Hiroshi; Bessho, Yasumasa

    2015-01-01

    Although cell adhesion is thought to fasten cells tightly, cells that adhere to each other can migrate directionally. This group behavior, called “collective cell migration,” is observed during normal development, wound healing, and cancer invasion. Loss-of-function of cell adhesion molecules in several model systems of collective cell migration results in delay or inhibition of migration of cell groups but does not lead to dissociation of the cell groups, suggesting that mechanisms of cells staying assembled as a single cell cluster, termed as “cell collectivity,” remain largely unknown. During the formation of Kupffer's vesicle (KV, an organ of laterality in zebrafish), KV progenitors form a cluster and migrate together toward the vegetal pole. Importantly, in this model system of collective cell migration, knockdown of cell adhesion molecules or signal components leads to failure of cell collectivity. In this review, we summarize recent findings in cell collectivity regulation during collective migration of KV progenitor cells and describe our current understanding of how cell collectivity is regulated during collective cell migration. PMID:26000276

  1. Hydroxyl radical formation in phagocytic cells of the rat.

    PubMed

    Drath, D B; Karnovsky, M L; Huber, G L

    1979-01-01

    Polymorphonuclear leukocytes (PMN) and macrophages, harvested from the peritoneum and lung, release superoxide (O-.2) and hydrogen peroxide (H2O2) during phagocytosis. These two agents are thought to react with each other to produce a highly active oxidative substance known as hydroxyl radical (OH.). We present evidence suggesting that these radicals are generated by phagocytic cells of the rat. Our findings are based upon an assay where ethylene gas is generated from methional by the action of this radical. Ethylene generation was shown to be inhibited by superoxide dismutase, catalase, and scavengers of OH.. Of the cells examined, PMN generated the most ethylene from methional, exhibiting a fourfold increase during phagocytosis. Pulmonary and peritoneal macrophages caused smaller amounts of this gas to be formed. Regardless of cell type, an intact cell was required for ethylene generation. Zymosan appeared to be the most effective particle for all cells in ethylene formation from methional, although opsonization was critical only for PMN. Ethylene generation was dependent on cell concentration to an extent and increased with time. PMID:222719

  2. The cytoskeletal mechanisms of cell–cell junction formation in endothelial cells

    PubMed Central

    Hoelzle, Matthew K.; Svitkina, Tatyana

    2012-01-01

    The actin cytoskeleton and associated proteins play a vital role in cell–cell adhesion. However, the procedure by which cells establish adherens junctions remains unclear. We investigated the dynamics of cell–cell junction formation and the corresponding architecture of the underlying cytoskeleton in cultured human umbilical vein endothelial cells. We show that the initial interaction between cells is mediated by protruding lamellipodia. On their retraction, cells maintain contact through thin bridges formed by filopodia-like protrusions connected by VE-cadherin–rich junctions. Bridges share multiple features with conventional filopodia, such as an internal actin bundle associated with fascin along the length and vasodilator-stimulated phosphoprotein at the tip. It is striking that, unlike conventional filopodia, transformation of actin organization from the lamellipodial network to filopodial bundle during bridge formation occurs in a proximal-to-distal direction and is accompanied by recruitment of fascin in the same direction. Subsequently, bridge bundles recruit nonmuscle myosin II and mature into stress fibers. Myosin II activity is important for bridge formation and accumulation of VE-cadherin in nascent adherens junctions. Our data reveal a mechanism of cell–cell junction formation in endothelial cells using lamellipodia as the initial protrusive contact, subsequently transforming into filopodia-like bridges connected through adherens junctions. Moreover, a novel lamellipodia-to-filopodia transition is used in this context. PMID:22090347

  3. Vesicle Size Regulates Nanotube Formation in the Cell

    PubMed Central

    Su, Qian Peter; Du, Wanqing; Ji, Qinghua; Xue, Boxin; Jiang, Dong; Zhu, Yueyao; Lou, Jizhong; Yu, Li; Sun, Yujie

    2016-01-01

    Intracellular membrane nanotube formation and its dynamics play important roles for cargo transportation and organelle biogenesis. Regarding the regulation mechanisms, while much attention has been paid on the lipid composition and its associated protein molecules, effects of the vesicle size has not been studied in the cell. Giant unilamellar vesicles (GUVs) are often used for in vitro membrane deformation studies, but they are much larger than most intracellular vesicles and the in vitro studies also lack physiological relevance. Here, we use lysosomes and autolysosomes, whose sizes range between 100 nm and 1 μm, as model systems to study the size effects on nanotube formation both in vivo and in vitro. Single molecule observations indicate that driven by kinesin motors, small vesicles (100–200 nm) are mainly transported along the tracks while a remarkable portion of large vesicles (500–1000 nm) form nanotubes. This size effect is further confirmed by in vitro reconstitution assays on liposomes and purified lysosomes and autolysosomes. We also apply Atomic Force Microscopy (AFM) to measure the initiation force for nanotube formation. These results suggest that the size-dependence may be one of the mechanisms for cells to regulate cellular processes involving membrane-deformation, such as the timing of tubulation-mediated vesicle recycling. PMID:27052881

  4. Vesicle Size Regulates Nanotube Formation in the Cell.

    PubMed

    Su, Qian Peter; Du, Wanqing; Ji, Qinghua; Xue, Boxin; Jiang, Dong; Zhu, Yueyao; Lou, Jizhong; Yu, Li; Sun, Yujie

    2016-01-01

    Intracellular membrane nanotube formation and its dynamics play important roles for cargo transportation and organelle biogenesis. Regarding the regulation mechanisms, while much attention has been paid on the lipid composition and its associated protein molecules, effects of the vesicle size has not been studied in the cell. Giant unilamellar vesicles (GUVs) are often used for in vitro membrane deformation studies, but they are much larger than most intracellular vesicles and the in vitro studies also lack physiological relevance. Here, we use lysosomes and autolysosomes, whose sizes range between 100 nm and 1 μm, as model systems to study the size effects on nanotube formation both in vivo and in vitro. Single molecule observations indicate that driven by kinesin motors, small vesicles (100-200 nm) are mainly transported along the tracks while a remarkable portion of large vesicles (500-1000 nm) form nanotubes. This size effect is further confirmed by in vitro reconstitution assays on liposomes and purified lysosomes and autolysosomes. We also apply Atomic Force Microscopy (AFM) to measure the initiation force for nanotube formation. These results suggest that the size-dependence may be one of the mechanisms for cells to regulate cellular processes involving membrane-deformation, such as the timing of tubulation-mediated vesicle recycling. PMID:27052881

  5. Multimodal Approaches for Regenerative Stroke Therapies: Combination of Granulocyte Colony-Stimulating Factor with Bone Marrow Mesenchymal Stem Cells is Not Superior to G-CSF Alone

    PubMed Central

    Balseanu, Adrian Tudor; Buga, Ana-Maria; Catalin, Bogdan; Wagner, Daniel-Christoph; Boltze, Johannes; Zagrean, Ana-Maria; Reymann, Klaus; Schaebitz, Wolf; Popa-Wagner, Aurel

    2014-01-01

    Attractive therapeutic strategies to enhance post-stroke recovery of aged brains include methods of cellular therapy that can enhance the endogenous restorative mechanisms of the injured brain. Since stroke afflicts mostly the elderly, it is highly desirable to test the efficacy of cell therapy in the microenvironment of aged brains that is generally refractory to regeneration. In particular, stem cells from the bone marrow allow an autologous transplantation approach that can be translated in the near future to the clinical practice. Such a bone marrow-derived therapy includes the grafting of stem cells as well as the delayed induction of endogenous stem cell mobilization and homing by the stem cell mobilizer granulocyte colony-stimulating factor (G-CSF). We tested the hypothesis that grafting of bone marrow-derived pre-differentiated mesenchymal cells (BM-MSCs) in G-CSF-treated animals improves the long-term functional outcome in aged rodents. To this end, G-CSF alone (50 μg/kg) or in combination with a single dose (106 cells) of rat BM MSCs was administered intravenously to Sprague-Dawley rats at 6 h after transient occlusion (90 min) of the middle cerebral artery. Infarct volume was measured by magnetic resonance imaging at 3 and 48 days post-stroke and additionally by immunhistochemistry at day 56. Functional recovery was tested during the entire post-stroke survival period of 56 days. Daily treatment for post-stroke aged rats with G-CSF led to a robust and consistent improvement of neurological function after 28 days. The combination therapy also led to robust angiogenesis in the formerly infarct core and beyond in the “islet of regeneration.” However, G-CSF + BM MSCs may not impact at all on the spatial reference-memory task or infarct volume and therefore did not further improve the post-stroke recovery. We suggest that in a real clinical practice involving older post-stroke patients, successful regenerative therapies would have to be

  6. Pre-B cell colony enhancing factor (PBEF/NAMPT/Visfatin) and vascular endothelial growth factor (VEGF) cooperate to increase the permeability of the human placental amnion

    PubMed Central

    Astern, J.M.; Collier, A.C.; Kendal-Wright, C.E.

    2012-01-01

    Fluid efflux across the region of the amnion overlying the placenta is an essential component of the intramembranous absorption pathway that maintains amniotic fluid volume homeostasis. Dysregulation of this pathway may result in adverse pregnancy outcomes, however the factors controlling amnion permeability are unknown. Here, we report a novel mechanism that increases placental amnion permeability. Pre-B Cell Colony Enhancing Factor (PBEF) is a stress-responsive cytokine expressed by the human amnion, and is known to induce Vascular Endothelial Growth Factor (VEGF) production by other cell types. Interestingly, VEGF is up-regulated in the ovine amnion when intramembranous absorption is augmented. In this study, we show that PBEF induced VEGF secretion by primary human amniotic epithelial cells (AEC) derived from the placental amnion, as well as from the reflected amnion that lines the remainder of the gestational sac. Further, PBEF treatment led to the increased expression of VEGFR2 in placental AEC, but not reflected AEC. To test the hypothesis that PBEF and VEGF increase placental amnion permeability, we monitored the transfer of 2′,7′-dichlorofluorescein (DCF) from the fetal to the maternal side of human amnion explants. A treatment regimen including both PBEF and VEGF increased the rate of DCF transfer across the placental amnion, but not the reflected amnion. In summary, our results suggest that by augmenting VEGFR2 expression in the placental amnion, PBEF primes the tissue for a VEGF-mediated increase in permeability. This mechanism may have important implications in amniotic fluid volume control throughout gestation. PMID:23151382

  7. Changes in the proteomic profile during differentiation and maturation of human monocyte-derived dendritic cells stimulated with granulocyte macrophage colony stimulating factor/interleukin-4 and lipopolysaccharide.

    PubMed

    Pereira, Sandra Rodrigues; Faça, Vitor Marcel; Gomes, Glauce Gaspar; Chammas, Roger; Fontes, Aparecida Maria; Covas, Dimas Tadeu; Greene, Lewis Joel

    2005-04-01

    Dendritic cells (DCs) are highly specialized antigen-presenting cells that play an essential role in the immune response. We used the proteomic approach based on two-dimensional gel electrophoresis and mass spectrometry to identify the protein changes that occur during differentiation of DCs from monocytes (Mo) stimulated with granulocyte macrophage colony stimulating factor/interleukin-4 (GM-CSF/IL-4) and during the maturation of immature DCs stimulated with lipopolysaccharide. Sixty-three differentially expressed proteins (+/- two-fold) were unambiguously identified with sequence coverage greater than 20%. They corresponded to only 36 different proteins, because 11 were present as 38 electrophoretic forms. Some proteins such as tropomyosin 4 and heat shock protein 71 presented differentially expressed electrophoretic forms, suggesting that many of the changes in protein expression that accompany differentiation and maturation of DCs occur in post-translationally modified proteins. The largest differences in expression were observed for actin (21-fold in Mo), Rho GDP-dissociation inhibitor 2 (20-fold in Mo), vimentin (eight-fold in immature DCs), lymphocyte-specific protein 1 (12-fold in mature DCs) and thioredoxin (14-fold in mature DCs). Several proteins are directly related to functional and morphological characteristics of DCs, such as cytoskeletal proteins (cytoskeleton rearrangement) and chaperones (antigen processing and presentation), but other proteins have not been assigned specific functions in DCs. Only a few proteins identified here were the same as those reported in proteomic studies of DCs, which used different stimuli to produce the cells (GM-CSF/IL-4 and tumor necrosis factor-alpha). These data suggest that the DC protein profile depends on the stimuli used for differentiation and especially for maturation. PMID:15800872

  8. The Development of Murine Plasmacytoid Dendritic Cell Precursors Is Differentially Regulated by FLT3-ligand and Granulocyte/Macrophage Colony-Stimulating Factor

    PubMed Central

    Gilliet, Michel; Boonstra, Andre; Paturel, Carine; Antonenko, Svetlana; Xu, Xiu-Ling; Trinchieri, Giorgio; O'Garra, Anne; Liu, Yong-Jun

    2002-01-01

    Plasmacytoid predendritic cells or type 1 interferon (IFN)-producing cells (IPCs) have recently been identified in mice. Although culture systems giving rise to different murine dendritic cell subsets have been established, the developmental regulation of murine plasmacytoid IPCs and the culture conditions leading to their generation remain unknown. Here we show that large numbers of over 40% pure CD11c+CD11b−B220+Gr-1+ IPCs can be generated from mouse bone marrow cultures with FLT3-ligand. By contrast GM-CSF or TNF-α, which promote the generation of CD11c+CD11b+B220− myeloid DCs, block completely the development of IPCs. IPCs generated display similar features to human IPCs, such as the plasmacytoid morphology, the ability to produce large amounts of IFN-α in responses to herpes simplex virus, and the capacity to respond to ligands for Toll-like receptor 9 (TLR-9; CpG ODN 1668), but not to ligands for TLR-4 (lipopolysaccharide [LPS]). Unlike human IPCs which produce little IL-12p70, mouse IPCs produce IL-12p70 in response to CpG ODN 1668 and herpes simplex virus. This study demonstrates that the development of murine CD11c+CD11b−B220+Gr-1+ IPCs and CD11c+CD11b+B220− myeloid DCs is differentially regulated by FLT3-ligand and granulocyte/macrophage colony-stimulating factor. Human IPCs and mouse IPCs display different ability to produce IL-12p70. Large numbers of mouse IPCs can now be obtained from total bone marrow culture. PMID:11927638

  9. A microfluidic direct formate fuel cell on paper.

    PubMed

    Copenhaver, Thomas S; Purohit, Krutarth H; Domalaon, Kryls; Pham, Linda; Burgess, Brianna J; Manorothkul, Natalie; Galvan, Vicente; Sotez, Samantha; Gomez, Frank A; Haan, John L

    2015-08-01

    We describe the first direct formate fuel cell on a paper microfluidic platform. In traditional membrane-less microfluidic fuel cells (MFCs), external pumping consumes power produced by the fuel cell in order to maintain co-laminar flow of the anode stream and oxidant stream to prevent mixing. However, in paper microfluidics, capillary action drives flow while minimizing stream mixing. In this work, we demonstrate a paper MFC that uses formate and hydrogen peroxide as the anode fuel and cathode oxidant, respectively. Using these materials we achieve a maximum power density of nearly 2.5 mW/mg Pd. In a series configuration, our MFC achieves an open circuit voltage just over 1 V, and in a parallel configuration, short circuit of 20 mA absolute current. We also demonstrate that the MFC does not require continuous flow of fuel and oxidant to produce power. We found that we can pre-saturate the materials on the paper, stop the electrolyte flow, and still produce approximately 0.5 V for 15 min. This type of paper MFC has potential applications in point-of-care diagnostic devices and other electrochemical sensors. PMID:25546700

  10. Notch1-Dll4 signaling and mechanical force regulate leader cell formation during collective cell migration

    PubMed Central

    Riahi, Reza; Sun, Jian; Wang, Shue; Long, Min; Zhang, Donna D.; Wong, Pak Kin

    2015-01-01

    At the onset of collective cell migration, a subset of cells within an initially homogenous population acquires a distinct “leader” phenotype with characteristic morphology and motility. However, the factors driving leader cell formation as well as the mechanisms regulating leader cell density during the migration process remain to be determined. Here, we use single cell gene expression analysis and computational modeling to show that leader cell identity is dynamically regulated by Dll4 signaling through both Notch1 and cellular stress in a migrating epithelium. Time-lapse microscopy reveals that Dll4 is induced in leader cells after the creation of the cell-free region and leader cells are regulated via Notch1-Dll4 lateral inhibition. Furthermore, mechanical stress inhibits Dll4 expression and leader cell formation in the monolayer. Collectively, our findings suggest that a reduction of mechanical force near the boundary promotes Notch1-Dll4 signaling to dynamically regulate the density of leader cells during collective cell migration. PMID:25766473

  11. Granulocyte-colony stimulating factor for hematopoietic stem cell donation from healthy female donors during pregnancy and lactation: what do we know?

    PubMed

    Pessach, Ilias; Shimoni, Avichai; Nagler, Arnon

    2013-01-01

    BACKGROUND Hematopoietic growth factors (HGFs) are mostly used as supportive measures to reduce infectious complications associated with neutropenia. Over the past decade, the use of HGFs became a common method for mobilizing human CD34+ stem cells, either for autologous or allogeneic transplantation. However, since their introduction the long-term safety of the procedure has become a major focus of discussion and research. Most information refers to healthy normal donors and data concerning pregnant and lactating women are scarce. The clinical question, which is the core of this review, is whether stem cell donation, preceded by administration of granulocyte-colony stimulating factor (G-CSF) for mobilization, is a safe procedure for pregnant donors. METHODS Literature searches were performed in Pubmed for English language articles published before the end of May 2012, focusing on G-CSF administration during pregnancy, lactation and hematopoietic stem cell donation. Searches included animal and human studies. RESULTS Data from animals (n = 15 studies) and women (n = 46 studies) indicate that G-CSF crosses the placenta, stimulates fetal granulopoiesis, improves neonatal survival mostly for very immature infants, promotes trophoblast growth and placental metabolism and has an anti-abortive role. Granulocyte macrophage-CSF is a key cytokine in the maternal immune tolerance towards the implanted embryo and exerts protective long-term programming effects to preimplantation embryos. The available data suggest that probably CSFs should not be administered during the time of most active organogenesis (first trimester), except perhaps for the first week during which implantation takes place. Provided CSF is administered during the second and third trimesters, it appears to be safe, and pregnant women receiving the CSF treatment can become hematopoietic stem cell donors. There are also risks related to the anesthesia, which is required for the bone marrow aspiration. During

  12. Effects of acoustic and EHF impulses on multipotent stromal cells during formation of bone marrow containing heterotopic organs in tissue engineered constructions.

    PubMed

    Chaikhalyan, R K; Yusupov, V I; Gorskaya, Yu F; Kuralesova, A I; Gerasimov, Yu V; Sviridov, A P; Tambiev, A Kh; Vorob'eva, N N; Shishkova, A G Grosheva V V; Moskvina, I L; Bagratashvili, V N

    2015-03-01

    We studied the effects of physical factors (acoustic impulses of laser-induced hydrodynamics, AILIH, and EHF-radiation) on the formation of heterotopic bone marrow organs. Suspension of precipitated mouse bone marrow cells was exposed to AILIH and EHF or their combinations (AILIH+EHF, EHF+AILIH). The developed tissue engineering constructions (gelatin sponges containing 107 nucleated bone marrow cells exposed to physical factors) were transplanted under the renal capsule of syngeneic mice. Analysis of newly formed hemopoietic organs was performed after 3 and 5 months. The total amount of hemopoietic cells, number of multipotent stromal cells, efficiency of colony formation from these cells, and weight of bone capsule of the transplants were measured. Microscopic study showed that 5-month transplants were significantly larger than 3-month transplants and contained 3-fold more hemopoietic cells (20-fold in the AILIH+EHF group). The number of multipotent stromal cells was maximum in EHF+AILIH group (by 2.2 times higher than in the control) and minimum in AILIH+EHF group. Exposure to EHF+AILIH had most pronounced effect on the formation of the bone marrow transplants. The weight of bone capsules more rapidly increased in gelatin sponges of 3-month transplants of EHF+AILIH and AILIH groups. These data suggest that the studied physical factors can be used for acceleration of rehabilitation process. PMID:25778661

  13. Molecular Mechanisms for Vascular Development and Secondary Cell Wall Formation

    PubMed Central

    Yang, Jung Hyun; Wang, Huanzhong

    2016-01-01

    Vascular tissues are important for transporting water and nutrients throughout the plant and as physical support of upright growth. The primary constituents of vascular tissues, xylem, and phloem, are derived from the meristematic vascular procambium and cambium. Xylem cells develop secondary cell walls (SCWs) that form the largest part of plant lignocellulosic biomass that serve as a renewable feedstock for biofuel production. For the last decade, research on vascular development and SCW biosynthesis has seen rapid progress due to the importance of these processes to plant biology and to the biofuel industry. Plant hormones, transcriptional regulators and peptide signaling regulate procambium/cambium proliferation, vascular patterning, and xylem differentiation. Transcriptional regulatory pathways play a pivot role in SCW biosynthesis. Although most of these discoveries are derived from research in Arabidopsis, many genes have shown conserved functions in biofuel feedstock species. Here, we review the recent advances in our understanding of vascular development and SCW formation and discuss potential biotechnological uses. PMID:27047525

  14. mRNA retroposition in human cells: processed pseudogene formation.

    PubMed Central

    Maestre, J; Tchénio, T; Dhellin, O; Heidmann, T

    1995-01-01

    Using a sensitive assay for detection of reverse transcription events, we demonstrate that human HeLa cells can 'retropose', i.e. reverse transcribe and integrate, the mRNA of a naive reporter gene, at a low but detectable frequency. Furthermore, we show that the retroposed copies have all the hallmarks of the processed pseudogenes naturally found in the mammalian genome: they lack intron and 5' promoter sequence, they have acquired a 3' poly(A) tail, and they are flanked by short repeats (< 15 bp) of target DNA sequence. These results demonstrate that human cells possess an endogenous reverse transcription activity, which is not restricted to transcripts of transposable elements, and which is likely to be involved in the formation, still ongoing, of a large fraction of the eukaryotic genome. Images PMID:8557053

  15. Molecular Mechanisms for Vascular Development and Secondary Cell Wall Formation.

    PubMed

    Yang, Jung Hyun; Wang, Huanzhong

    2016-01-01

    Vascular tissues are important for transporting water and nutrients throughout the plant and as physical support of upright growth. The primary constituents of vascular tissues, xylem, and phloem, are derived from the meristematic vascular procambium and cambium. Xylem cells develop secondary cell walls (SCWs) that form the largest part of plant lignocellulosic biomass that serve as a renewable feedstock for biofuel production. For the last decade, research on vascular development and SCW biosynthesis has seen rapid progress due to the importance of these processes to plant biology and to the biofuel industry. Plant hormones, transcriptional regulators and peptide signaling regulate procambium/cambium proliferation, vascular patterning, and xylem differentiation. Transcriptional regulatory pathways play a pivot role in SCW biosynthesis. Although most of these discoveries are derived from research in Arabidopsis, many genes have shown conserved functions in biofuel feedstock species. Here, we review the recent advances in our understanding of vascular development and SCW formation and discuss potential biotechnological uses. PMID:27047525

  16. Entombed pollen: A new condition in honey bee colonies associated with increased risk of colony mortality

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Here we describe a new phenomenon, entombed pollen, which is highly associated with increased colony mortality. Entombed pollen appears as sunken, wax-covered cells amidst “normal”, uncapped cells of stored pollen, and the pollen contained within these cells is brick red in color. There appears to b...

  17. Cadmium stimulates osteoclast-like multinucleated cell formation in mouse bone marrow cell cultures

    SciTech Connect

    Miyahara, Tatsuro; Takata, Masakazu; Miyata, Masaki; Nagai, Miyuki; Sugure, Akemi; Kozuka, Hiroshi; Kuze, Shougo )

    1991-08-01

    Most of cadmium (Cd)-treated animals have been reported to show osteoporosis-like changes in bones. This suggests that Cd may promote bone loss by a direct action on bone. It was found that Cd stimulated prostaglandin E{sub 2}(PGE{sub 2}) production in the osteoblast-like cell, MC3T3-E1. Therefore, Cd stimulates bone resorption by increasing PGE{sub 2} production. Recently, several bone marrow cell culture systems have been developed for examining the formation of osteoclast-like multinucleated cells in vitro. As osteoblasts produce PGE{sub 2} by Cd-induced cyclooxygenase and may play an important role in osteoclast formation, the present study was undertaken to clarify the possibility that Cd might stimulate osteoclast formation in a mouse bone marrow culture system.

  18. Direct effects of caffeine on osteoblastic cells metabolism: the possible causal effect of caffeine on the formation of osteoporosis

    PubMed Central

    Tsuang, Yang-Hwei; Sun, Jui-Sheng; Chen, Li-Ting; Sun, Samuel Chung-Kai; Chen, San-Chi

    2006-01-01

    Background Caffeine consumption has been reported to decrease bone mineral density (BMD), increase the risk of hip fracture, and negatively influence calcium retention. In this study, we investigated the influence of caffeine on the osteoblasts behaviour. Method Osteoblasts derived from newborn Wistar-rat calvaria was used in this study. The effects of various concentrations of caffeine on bone cell activities were evaluated by using MTT assay. Alkaline phosphatase (ALP) staining, von Kossa staining and biochemical parameters including ALP, lactate dehydrogenase (LDH), prostaglandin E2 (PGE2) and total protein were performed at day 1, 3, and 7. DNA degradation analysis under the caffeine influence was also performed. Results and discussion The results showed that the viability of the osteoblasts, the formation of ALP positive staining colonies and mineralization nodules formation in the osteoblasts cultures decreased significantly in the presence of 10 mM caffeine. The intracellular LDH, ALP and PGE2 content decreased significantly, the LDH and PGE2 secreted into the medium increased significantly. The activation of an irreversible commitment to cell death by caffeine was clearly demonstrated by DNA ladder staining. Conclusion In summary, our results suggest that caffeine has potential deleterious effect on the osteoblasts viability, which may enhance the rate of osteoblasts apoptosis. PMID:17150127

  19. Colonial American Astronomy

    NASA Astrophysics Data System (ADS)

    Yeomans, Donald K.

    2007-12-01

    While a foundation of German scientific methods enabled the rapid growth of North American Astronomy in the nineteenth century, during the seventeenth and most of the eighteenth centuries, the colonial men of science looked only to the English mother country for scientific patronage and guidance. An essay on fundamental astronomy appeared in one of the annual colonial almanacs as early as 1656, telescopic observations were made about 1660 and the first original colonial astronomical work was published by Thomas Danforth on the comet of 1664. By 1671 the Copernican ideas were so espoused at Harvard College that a physics class refused to read a Ptolemaic textbook when it was assigned to them by a senior instructor. At least in the Cambridge-Boston area, contemporary colonialist had access to the most recent scientific publications from the mother country. Observations of the great comet of 1680 by the Almanac maker, John Foster, reached Isaac Newton and were used and gratefully acknowledged in his Principia. During the seventeenth century the colonial interest in astronomy was more intense than it was for other sciences but colonists still occupied a position in the scientific backwater when compared with contemporary European scientists. Nevertheless, the science of astronomy was successfully transplanted from England to North America in the seventeenth century.

  20. The Colonial Inheritance.

    ERIC Educational Resources Information Center

    Allen, Jack

    As the American nation developed through periods of rapid change and great cultural diversity, the American public school system served as a primary souce of national unity. The roots of comprehensive public education in America can be traced to the educational system developed by 17th century Puritan colonials. Although one of the central…

  1. Sailing to the Colonies.

    ERIC Educational Resources Information Center

    Carter, Dorothy S.

    1990-01-01

    Presents a class activity designed to foster an understanding of rules, develop analytical skills, and introduce students to early colonial history. Divides the class into groups who are sailing to the New World, and presents them with ethical and practical problems to be solved on board the ship. (RW)

  2. Stem Cell-Soluble Signals Enhance Multilumen Formation in SMG Cell Clusters.

    PubMed

    Maruyama, C L M; Leigh, N J; Nelson, J W; McCall, A D; Mellas, R E; Lei, P; Andreadis, S T; Baker, O J

    2015-11-01

    Saliva plays a major role in maintaining oral health. Patients with salivary hypofunction exhibit difficulty in chewing and swallowing foods, tooth decay, periodontal disease, and microbial infections. At this time, treatments for hyposalivation are limited to medications (e.g., muscarinic receptor agonists: pilocarpine and cevimeline) that induce saliva secretion from residual acinar cells as well as artificial salivary substitutes. Therefore, advancement of restorative treatments is necessary to improve the quality of life in these patients. Our previous studies indicated that salivary cells are able to form polarized 3-dimensional structures when grown on growth factor-reduced Matrigel. This basement membrane is rich in laminin-III (L1), which plays a critical role in salivary gland formation. Mitotically inactive feeder layers have been used previously to support the growth of many different cell types, as they provide factors necessary for cell growth and organization. The goal of this study was to improve salivary gland cell differentiation in primary cultures by using a combination of L1 and a feeder layer of human hair follicle-derived mesenchymal stem cells (hHF-MSCs). Our results indicated that the direct contact of mouse submandibular (mSMG) cell clusters and hHF-MSCs was not required for mSMG cells to form acinar and ductal structures. However, the hHF-MSC conditioned medium enhanced cell organization and multilumen formation, indicating that soluble signals secreted by hHF-MSCs play a role in promoting these features. PMID:26285810

  3. Protective layer formation on magnesium in cell culture medium.

    PubMed

    Wagener, V; Virtanen, S

    2016-06-01

    In the past, different studies showed that hydroxyapatite (HA) or similar calcium phosphates can be precipitated on Mg during immersion in simulated body fluids. However, at the same time, in most cases a dark grey or black layer is built under the white HA crystals. This layer seems to consist as well of calcium phosphates. Until now, neither the morphology nor its influence on Mg corrosion have been investigated in detail. In this work commercially pure magnesium (cp) was immersed in cell culture medium for one, three and five days at room temperature and in the incubator (37 °C, 5% CO2). In addition, the influence of proteins on the formation of a corrosion layer was investigated by adding 20% of fetal calf serum (FCS) to the cell culture medium in the incubator. In order to analyze the formed layers, SEM images of cross sections, X-ray Photoelectron Spectroscopy (XPS), X-ray diffraction (XRD), Energy Dispersive X-ray Spectroscopy (EDX) and Fourier Transformed Infrared Spectroscopy (FTIR) measurements were carried out. Characterization of the corrosion behavior was achieved by electrochemical impedance spectroscopy (EIS) and by potentio-dynamic polarization in Dulbecco's Modified Eagle's Medium (DMEM) at 37°C. Surface analysis showed that all formed layers consist mainly of amorphous calcium phosphate compounds. For the immersion at room temperature the Ca/P ratio indicates the formation of HA, while in the incubator probably pre-stages to HA are formed. The different immersion conditions lead to a variation in layer thicknesses. However, electrochemical characterization shows that the layer thickness does not influence the corrosion resistance of magnesium. The main influencing factor for the corrosion behavior is the layer morphology. Thus, immersion at room temperature leads to the highest corrosion protection due to the formation of a compact outer layer. Layers formed in the incubator show much worse performances due to completely porous structures. The

  4. Monoacylated Cellular Prion Protein Modifies Cell Membranes, Inhibits Cell Signaling, and Reduces Prion Formation*

    PubMed Central

    Bate, Clive; Williams, Alun

    2011-01-01

    Prion diseases occur following the conversion of the cellular prion protein (PrPC) into a disease related, protease-resistant isoform (PrPSc). In these studies, a cell painting technique was used to introduce PrPC to prion-infected neuronal cell lines (ScGT1, ScN2a, or SMB cells). The addition of PrPC resulted in increased PrPSc formation that was preceded by an increase in the cholesterol content of cell membranes and increased activation of cytoplasmic phospholipase A2 (cPLA2). In contrast, although PrPC lacking one of the two acyl chains from its glycosylphosphatidylinositol (GPI) anchor (PrPC-G-lyso-PI) bound readily to cells, it did not alter the amount of cholesterol in cell membranes, was not found within detergent-resistant membranes (lipid rafts), and did not activate cPLA2. It remained within cells for longer than PrPC with a conventional GPI anchor and was not converted to PrPSc. Moreover, the addition of high amounts of PrPC-G-lyso-PI displaced cPLA2 from PrPSc-containing lipid rafts, reduced the activation of cPLA2, and reduced PrPSc formation in all three cell lines. In addition, ScGT1 cells treated with PrPC-G-lyso-PI did not transmit infection following intracerebral injection to mice. We propose that that the chemical composition of the GPI anchor attached to PrPC modified the local membrane microenvironments that control cell signaling, the fate of PrPC, and hence PrPSc formation. In addition, our observations raise the possibility that pharmacological modification of GPI anchors might constitute a novel therapeutic approach to prion diseases. PMID:21212283

  5. Retinoic acid, local cell-cell interactions, and pattern formation in vertebrate limbs.

    PubMed

    Bryant, S V; Gardiner, D M

    1992-07-01

    Retinoic acid (RA), a derivative of vitamin A, has remarkable effects on developing and regenerating limbs. These effects include teratogenesis, arising from RA's ability to inhibit growth and pattern formation. They also include pattern duplication, arising as a result of the stimulation of additional growth and pattern formation. In this review we present evidence that the diverse effects of RA are consistent with a singular, underlying explanation. We propose that in all cases exogenously applied RA causes the positional information of pattern formation-competent cells to be reset to a value that is posterior-ventral-proximal with respect to the limb. The diversity of outcomes can be seen as a product of the mode of application of exogenous RA (global versus local) coupled with the unifying concept that growth and pattern formation in both limb development and limb regeneration are controlled by local cell-cell interactions, as formulated in the polar coordinate model. We explore the possibility that the major role of endogenous RA in limb development is in the establishment of the limb field rather than as a diffusible morphogen that specifies graded positional information across the limb as previously proposed. Finally, we interpret the results of the recent finding that RA can turn tail regenerates into limbs, as evidence that intercalary interactions may also be involved in the formation of the primary body axis. PMID:1628749

  6. Vitamin K2-induced cell growth inhibition via autophagy formation in cholangiocellular carcinoma cell lines.

    PubMed

    Enomoto, Masanobu; Tsuchida, Akihiko; Miyazawa, Keisuke; Yokoyama, Tomohisa; Kawakita, Hideaki; Tokita, Hiromi; Naito, Munekazu; Itoh, Masahiro; Ohyashiki, Kazuma; Aoki, Tatsuya

    2007-12-01

    Vitamin K2 (MK4) has antitumor effects on various types of cancer cell lines in vitro, and its efficacy has also been reported in clinical applications for patients with leukemia, myelodysplastic syndrome, and hepatocellular carcinoma (HCC). However, details of the mechanism of the antitumor effects of MK4 remain unclear. In the present study, we examined the antitumor effects of MK4 on cholangiocellular carcinoma (CCC) cell lines and its mechanism of action using the HL-60 leukemia cell line that exerts MK4-induced cell growth inhibition via apoptosis induction and cell cycle arrest as a control. MK4 exerted dose-dependent antitumor effects on all three types of CCC cell lines. However, apoptosis occurred in a smaller percentage of cells and there was less cell cycle arrest compared with other cancer cell lines studied previously, which suggested slight MK4-induced cell growth inhibition via apoptosis induction and cell cycle arrest. On the contrary, histopathological fidings showed a large number of cells containing vacuoles in their cytoplasm, and electron microscopic findings showed a large number of cytoplasmic autophagosomes and autolysosomes. These findings suggested evidence of autophagy-related cell death. Fluorescence microscopy following acridine orange staining revealed an increase in the number of cytoplasmic acidic vesicular organelles characteristic of autophagy. Moreover, there were few cells forming autophagic vesicles in the control group, while the percentage of cells containing vacuoles in the MK4-treated group increased with the duration of culture. These results suggested that, unlike in leukemia, gastric cancer, HCC, and other cancer cells, the antitumor effects of MK4 on CCC cells are induced via autophagy formation. PMID:17982686

  7. Myosin II-mediated cell shape changes and cell intercalation contribute to primitive streak formation

    PubMed Central

    Song, Feifei; Sang, Helen M.; Martin, René; Knölker, Hans-Joachim; MacDonald, Michael P; Weijer, Cornelis J

    2016-01-01

    Primitive streak formation in the chick embryo involves large scale highly coordinated flows of over 100.000 cells in the epiblast. These large scale tissue flows and deformations can be correlated with specific anisotropic cell behaviours in the forming mesendoderm through a combined light-sheet microscopy and computational analysis. Relevant behaviours include apical contraction, elongation along the apical-basal axis followed by ingression as well as asynchronous directional cell intercalation of small groups of mesendoderm cells. Cell intercalation is associated with sequential, directional contraction of apical junctions, the onset, localisation and direction of which correlate strongly with the appearance of active Myosin II cables in aligned apical junctions in neighbouring cells. Use of a class specific Myosin inhibitors and gene specific knockdowns show that apical contraction and intercalation are Myosin II dependent and also reveal critical roles for Myosin I and Myosin V family members in the assembly of junctional Myosin II cables. PMID:25812521

  8. Regulation of germinal center, B-cell memory, and plasma cell formation by histone modifiers.

    PubMed

    Good-Jacobson, Kim L

    2014-01-01

    Understanding the regulation of antibody production and B-cell memory formation and function is core to finding new treatments for B-cell-derived cancers, antibody-mediated autoimmune disorders, and immunodeficiencies. Progression from a small number of antigen-specific B-cells to the production of a large number of antibody-secreting cells is tightly regulated. Although much progress has been made in revealing the transcriptional regulation of B-cell differentiation that occurs during humoral immune responses, there are still many questions that remain unanswered. Recent work on the expression and roles of histone modifiers in lymphocytes has begun to shed light on this additional level of regulation. This review will discuss the recent advancements in understanding how humoral immune responses, in particular germinal centers and memory cells, are modulated by histone modifiers. PMID:25477884

  9. Automatic counting and classification of bacterial colonies using hyperspectral imaging

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Detection and counting of bacterial colonies on agar plates is a routine microbiology practice to get a rough estimate of the number of viable cells in a sample. There have been a variety of different automatic colony counting systems and software algorithms mainly based on color or gray-scale pictu...

  10. Hemp, an mbt domain-containing protein, plays essential roles in hematopoietic stem cell function and skeletal formation

    PubMed Central

    Honda, Hiroaki; Takubo, Keiyo; Oda, Hideaki; Kosaki, Kenjiro; Tazaki, Tatsuya; Yamasaki, Norimasa; Miyazaki, Kazuko; Moore, Kateri A.; Honda, Zen-ichiro; Suda, Toshio; Lemischka, Ihor R.

    2011-01-01

    To clarify the molecular pathways governing hematopoietic stem cell (HSC) development, we screened a fetal liver (FL) HSC cDNA library and identified a unique gene, hematopoietic expressed mammalian polycomb (hemp), encoding a protein with a zinc-finger domain and four malignant brain tumor (mbt) repeats. To investigate its biological role, we generated mice lacking Hemp (hemp−/−). Hemp−/− mice exhibited a variety of skeletal malformations and died soon after birth. In the FL, hemp was preferentially expressed in the HSC and early progenitor cell fractions, and analyses of fetal hematopoiesis revealed that the number of FL mononuclear cells, including HSCs, was reduced markedly in hemp−/− embryos, especially during early development. In addition, colony-forming and competitive repopulation assays demonstrated that the proliferative and reconstitution abilities of hemp−/− FL HSCs were significantly impaired. Microarray analysis revealed alterations in the expression levels of several genes implicated in hematopoietic development and differentiation in hemp−/− FL HSCs. These results demonstrate that Hemp, an mbt-containing protein, plays essential roles in HSC function and skeletal formation. It is also hypothesized that Hemp might be involved in certain congenital diseases, such as Klippel-Feil anomaly. PMID:21252303

  11. An improved alkaline direct formate paper microfluidic fuel cell.

    PubMed

    Galvan, Vicente; Domalaon, Kryls; Tang, Catherine; Sotez, Samantha; Mendez, Alex; Jalali-Heravi, Mehdi; Purohit, Krutarth; Pham, Linda; Haan, John; Gomez, Frank A

    2016-02-01

    Paper-based microfluidic fuel cells (MFCs) are a potential replacement for traditional FCs and batteries due to their low cost, portability, and simplicity to operate. In MFCs, separate solutions of fuel and oxidant migrate through paper due to capillary action and laminar flow and, upon contact with each other and catalyst, produce electricity. In the present work, we describe an improved microfluidic paper-based direct formate FC (DFFC) employing formate and hydrogen peroxide as the anode fuel and cathode oxidant, respectively. The dimensions of the lateral column, current collectors, and cathode were optimized. A maximum power density of 2.53 mW/cm(2) was achieved with a DFFC of surface area 3.0 cm(2) , steel mesh as current collector, 5% carbon to paint mass ratio for cathode electrode and, 30% hydrogen peroxide. The longevity of the MFC's detailed herein is greater than eight hours with continuous flow of streams. In a series configuration, the MFCs generate sufficient energy to power light-emitting diodes and a handheld calculator. PMID:26572774

  12. Light induced polaron formation in perovskite solar cell devices

    NASA Astrophysics Data System (ADS)

    Neukirch, Amanda; Nie, Wanyi; Blancon, Jean-Christophe; Appavoo, Kannatassen; Tsai, Hsinhan; Chhowalla, Manish; Alam, Muhammad; Sfeir, Matthew; Katan, Claudine; Even, Jacky; Crochet, Jared; Gupta, Gautum; Mohite, Aditya; Tretiak, Sergei

    The need for a low-cost, clean, and abundant source of energy has generated large amounts of research in solution processed solar cell materials. The lead halide perovskite has rapidly developed as a serious candidate for the active layer of photovoltaic devices. The efficiencies of devices made with this material have increased from 3.5% to over 20% in around 5 years. Despite the remarkable progress associated with perovskite materials, there are still fundamental questions regarding their lack of photo-stability over prolonged solar irradiation that need to be addressed. Recent experiments on photo-degradation under constant illumination have found fast self-healing by resting the device in the dark for less than 1 minute. Density functional theory and symmetry analysis show that localized charge states couple to local structural lattice distortions and methyl ammonium quasistatic configurations. Once translational symmetry is lost, additional bonding configurations become symmetry allowed, triggering localized charges in the vicinity over time under constant illumination, thus seeding the formation of macroscopic charged domains and preventing efficient charge extraction. Here we present an in-depth study of polaron formation and binding energy at the atomistic level.

  13. Fractionation of human bone marrow cell suspensions in nylon fiber columns: an efficient method for the removal of cells that produce colony stimulating factor (CSF).

    PubMed

    Sullivan, R; Gans, P J

    1980-09-01

    In this report, we describe an efficient technique for the extraction of CSF-producing cells from human marrow suspensions. Prior to plating in agar cultures, we incubated buoyant human marrow cells for 45 min in columns packed with nylon fiber or subjected the cells to two one-hour incubations in glass petri dishes. Recoveries of total cells, differential marrow elements, and committed granulocyte-monocyte progenitor cells (CFUc) were similar after each separative procedure. However, spontaneous CFUc proliferation was more effectively eliminated when cells were fractionated in nylon fiber columns. After the removal of cells which were adherent to glass, spontaneous CFUc proliferation in cultures containing no exogenous CSF accounted for 2.1% of total CFUc at a plating concentration of 10(5) cells/ml and 7.8% at a concentration of 3 X 10(5) cells/ml. After the fractionation of marrow cell suspensions in nylon fiber columns, spontaneous CFUc growth was completely obliterated at a plating concentration of 10(5) cells/ml, and at a concentration of 3 X 10(5) cells/ml accounted for only 0.09% of total CFUc. Further experiments were undertaken which demonstrated that buoyant marrow cells after incubation in nylon fiber columns may be employed to assay CSF in extremely dilute concentrations. Because of the simplicity and efficiency of this procedure, nylon fiber chromatography appears to be a highly useful technique for the rapid semi-purification of marrow suspensions for use in the assay of human CSF. PMID:6970676

  14. Myotube formation is affected by adipogenic lineage cells in a cell-to-cell contact-independent manner.

    PubMed

    Takegahara, Yuki; Yamanouchi, Keitaro; Nakamura, Katsuyuki; Nakano, Shin-ichi; Nishihara, Masugi

    2014-05-15

    Intramuscular adipose tissue (IMAT) formation is observed in some pathological conditions such as Duchenne muscular dystrophy (DMD) and sarcopenia. Several studies have suggested that IMAT formation is not only negatively correlated with skeletal muscle mass but also causes decreased muscle contraction in sarcopenia. In the present study, we examined w hether adipocytes affect myogenesis. For this purpose, skeletal muscle progenitor cells were transfected with siRNA of PPARγ (siPPARγ) in an attempt to inhibit adipogenesis. Myosin heavy chain (MHC)-positive myotube formation was promoted in cells transfected with siPPARγ compared to that of cells transfected with control siRNA. To determine whether direct cell-to-cell contact between adipocytes and myoblasts is a prerequisite for adipocytes to affect myogenesis, skeletal muscle progenitor cells were cocultured with pre- or mature adipocytes in a Transwell coculture system. MHC-positive myotube formation was inhibited when skeletal muscle progenitor cells were cocultured with mature adipocytes, but was promoted when they were cocultured with preadipocytes. Similar effects were observed when pre- or mature adipocyte-conditioned medium was used. These results indicate that preadipocytes play an important role in maintaining skeletal muscle mass by promoting myogenesis; once differentiated, the resulting mature adipocytes negatively affect myogenesis, leading to the muscle deterioration observed in skeletal muscle pathologies. PMID:24720912

  15. [Enhanced control of proliferation in telomerized cells].

    PubMed

    Egorov, E E; Moldaver, M V; Vishniakova, Kh S; Terekhov, S M; Dashinimaev, E B; Cheglakov, I B; Toropygin, I Iu; Iarygin, K N; Chumakov, P M; Korochkin, L I; Antonova, G A; Rybalkina, E Iu; Saburina, I N; Burnaevskiĭ, N S; Zelenin, A V

    2007-01-01

    Clones of telomerized fibroblasts of adult human skin have earlier been obtained. It was shown that despite their fast growth in mass cultures, these cells poorly form colonies. Conditioned medium, antioxidants, and reduced partial oxygen pressure enhanced their colony formation, but not to the level characteristic of the initial cells. The conditioned medium of telomerized cells enhanced colony formation to a much greater extent than that of the initial cells. A study of proteome of the telomerized fibroblasts has revealed changes in the activities of tens of genes. A general trend consists in weakening and increased lability of the cytoskeleton and in activation of the mechanisms controlling protein degradation. However, these changes are not very pronounced. During the formation of immortal telomerized cells, selection takes place, which appears to determine changes in the expression of some genes. It was proposed that a decrease in the capacity of telomerized cells for colony formation is due to increased requirements of these cells to cell-cell contacts. The rate of cell growth reached that characteristic of mass cultures only in the largest colonies. In this respect, the telomerized fibroblasts resembled stem cells: they are capable of self-maintenance, but "escape" to differentiation in the absence of the corresponding microenvironment (niche), which is represented by other fibroblasts. Non-dividing cells in the test of colony formation should be regarded as differentiated cells, since they have no features of degradation, preserve their viability, actively move, grow, phagocytized debris, etc. It was also shown that telomerization did not prevent differentiation of myoblasts and human neural stem cells. Thus, the results obtained suggest the existence of normal mechanisms underlying the regulation of proliferation in the telomerized cells, which opens possibilities of their use in cell therapy, especially in the case of autotransplantation to senior people

  16. 'Dark' cell formation under protein malnutrition: process of conversion and concept of 'semi-dark' type Purkinje cells.

    PubMed

    James, T J; Sharma, S P; Gupta, S K; Patro, I K

    1992-06-01

    This paper deals with some deleterious effects of protein malnourishment in rat cerebellum. Severe protein deprivation enhanced the formation of 'dark' cells in white rats. It is postulated that abnormal changes in the neuronal contents induced by nutritional stress play a vital role in the formation of the 'dark' cells through an intermediary stage, 'semi-dark' cells. Centrophenoxine a lipofuscinolytic agent, however, seems to interfere with the process of formation of 'dark' cells and/or helps reconversion of the 'dark' cells into the normal or 'light' type Purkinje cells. PMID:1506025

  17. Infection of Polarized Airway Epithelial Cells by Normal and Small-Colony Variant Strains of Staphylococcus aureus Is Increased in Cells with Abnormal Cystic Fibrosis Transmembrane Conductance Regulator Function and Is Influenced by NF-κB ▿

    PubMed Central

    Mitchell, Gabriel; Grondin, Gilles; Bilodeau, Ginette; Cantin, André M.; Malouin, François

    2011-01-01

    The infection of nonphagocytic host cells by Staphylococcus aureus and more particularly by small-colony variants (SCVs) may contribute to the persistence of this pathogen in the lungs of cystic fibrosis (CF) patients. The development of chronic infections is also thought to be facilitated by the proinflammatory status of CF airways induced by an activation of NF-κB. The aim of this study was to compare the infection of non-CF and CF-like airway epithelial cells by S. aureus strains (normal and SCVs) and to determine the impact of the interaction between cystic fibrosis transmembrane conductance regulator (CFTR) and NF-κB on the infection level of these cells by S. aureus. We developed an S. aureus infection model using polarized airway epithelial cells grown at the air-liquid interface and expressing short hairpin RNAs directed against CFTR to mimic the CF condition. A pair of genetically related CF coisolates with the normal and SCV phenotypes was characterized and used. Infection of both cell lines (non-CF and CF-like) was more productive with the SCV strain than with its normal counterpart. However, both normal and SCV strains infected more CF-like than non-CF cells. Accordingly, inhibition of CFTR function by CFTRinh-172 increased the S. aureus infection level. Experimental activation of NF-κB also increased the level of infection of polarized pulmonary epithelial cells by S. aureus, an event that could be associated with that observed when CFTR function is inhibited or impaired. This study supports the hypothesis that the proinflammatory status of CF tissues facilitates the infection of pulmonary epithelial cells by S. aureus. PMID:21708986

  18. Spheroid Formation of Hepatocarcinoma Cells in Microwells: Experiments and Monte Carlo Simulations.

    PubMed

    Wang, Yan; Kim, Myung Hee; Tabaei, Seyed R; Park, Jae Hyeok; Na, Kyuhwan; Chung, Seok; Zhdanov, Vladimir P; Cho, Nam-Joon

    2016-01-01

    The formation of spherical aggregates during the growth of cell population has long been observed under various conditions. We observed the formation of such aggregates during proliferation of Huh-7.5 cells, a human hepatocarcinoma cell line, in a microfabricated low-adhesion microwell system (SpheroFilm; formed of mass-producible silicone elastomer) on the length scales up to 500 μm. The cell proliferation was also tracked with immunofluorescence staining of F-actin and cell proliferation marker Ki-67. Meanwhile, our complementary 3D Monte Carlo simulations, taking cell diffusion and division, cell-cell and cell-scaffold adhesion, and gravity into account, illustrate the role of these factors in the formation of spheroids. Taken together, our experimental and simulation results provide an integrative view of the process of spheroid formation for Huh-7.5 cells. PMID:27571565

  19. Spheroid Formation of Hepatocarcinoma Cells in Microwells: Experiments and Monte Carlo Simulations

    PubMed Central

    Tabaei, Seyed R.; Park, Jae Hyeok; Na, Kyuhwan; Chung, Seok; Zhdanov, Vladimir P.

    2016-01-01

    The formation of spherical aggregates during the growth of cell population has long been observed under various conditions. We observed the formation of such aggregates during proliferation of Huh-7.5 cells, a human hepatocarcinoma cell line, in a microfabricated low-adhesion microwell system (SpheroFilm; formed of mass-producible silicone elastomer) on the length scales up to 500 μm. The cell proliferation was also tracked with immunofluorescence staining of F-actin and cell proliferation marker Ki-67. Meanwhile, our complementary 3D Monte Carlo simulations, taking cell diffusion and division, cell-cell and cell-scaffold adhesion, and gravity into account, illustrate the role of these factors in the formation of spheroids. Taken together, our experimental and simulation results provide an integrative view of the process of spheroid formation for Huh-7.5 cells. PMID:27571565

  20. Polymer Solar Cells: Solubility Controls Fiber Network Formation.

    PubMed

    van Franeker, Jacobus J; Heintges, Gaël H L; Schaefer, Charley; Portale, Giuseppe; Li, Weiwei; Wienk, Martijn M; van der Schoot, Paul; Janssen, René A J

    2015-09-16

    The photoactive layer of polymer solar cells is commonly processed from a four-component solution, containing a semiconducting polymer and a fullerene derivative dissolved in a solvent-cosolvent mixture. The nanoscale dimensions of the polymer-fullerene morphology that is formed upon drying determines the solar cell performance, but the fundamental processes that govern the size of the phase-separated polymer and fullerene domains are poorly understood. Here, we investigate morphology formation of an alternating copolymer of diketopyrrolopyrrole and a thiophene-phenyl-thiophene oligomer (PDPPTPT) with relatively long 2-decyltetradecyl (DT) side chains blended with [6,6]-phenyl-C71-butyric acid methyl ester. During solvent evaporation the polymer crystallizes into a fibrous network. The typical width of these fibers is analyzed by quantification of transmission electron microscopic images, and is mainly determined by the solubility of the polymer in the cosolvent and the molecular weight of the polymer. A higher molecular weight corresponds to a lower solubility and film processing results in a smaller fiber width. Surprisingly, the fiber width is not related to the drying rate or the amount of cosolvent. We have made solar cells with fiber widths ranging from 28 to 68 nm and found an inverse relation between fiber width and photocurrent. Finally, by mixing two cosolvents, we develop a ternary solvent system to tune the fiber width. We propose a model based on nucleation-and-growth which can explain these measurements. Our results show that the width of the semicrystalline polymer fibers is not the result of a frozen dynamical state, but determined by the nucleation induced by the polymer solubility. PMID:26306585

  1. Analysis of the cellulose synthase operon genes, bcsA, bcsB, and bcsC in Cronobacter species: Prevalence among species and their roles in biofilm formation and cell-cell aggregation.

    PubMed

    Hu, Lan; Grim, Christopher J; Franco, Augusto A; Jarvis, Karen G; Sathyamoorthy, Vengopal; Kothary, Mahendra H; McCardell, Barbara A; Tall, Ben D

    2015-12-01

    Cronobacter species are emerging food-borne pathogens that cause severe sepsis, meningitis, and necrotizing entercolitis in neonates and infants. Bacterial pathogens such as Escherichia coli and Salmonella species produce extracellular cellulose which has been shown to be involved in rugosity, biofilm formation, and host colonization. In this study the distribution and prevalence of cellulose synthase operon genes (bcsABZC) were determined by polymerase chain reaction (PCR) analysis in 231 Cronobacter strains isolated from clinical, food, environmental, and unknown sources. Furthermore, bcsA and bcsB isogenic mutants were constructed in Cronobacter sakazakii BAA894 to determine their roles. In calcofluor binding assays bcsA and bcsB mutants did not produce cellulose, and their colonial morphotypes were different to that of the parent strain. Biofilm formation and bacterial cell-cell aggregation were significantly reduced in bcsA and bcsB mutants compared to the parental strain. bcsA or bcsAB PCR-negative strains of C. sakazakii did not bind calcofluor, and produced less biofilm and cell-cell aggregation compared to strains possessing bcsAB genes. These data indicated that Cronobacter bcsABZC were present in all clinical isolates and most of food and environmental isolates. bcsA and bcsB genes of Cronobacter were necessary to produce cellulose, and were involved in biofilm formation and cell-cell aggregation. PMID:26338122

  2. Influence of cell-to-cell variability on spatial pattern formation.

    PubMed

    Greese, B; Wester, K; Bensch, R; Ronneberger, O; Timmer, J; Huulskamp, M; Fleck, C

    2012-08-01

    Many spatial patterns in biology arise through differentiation of selected cells within a tissue, which is regulated by a genetic network. This is specified by its structure, parameterisation and the noise on its components and reactions. The latter, in particular, is not well examined because it is rather difficult to trace. The authors use suitable local mathematical measures based on the Voronoi diagram of experimentally determined positions of epidermal plant hairs (trichomes) to examine the variability or noise in pattern formation. Although trichome initiation is a highly regulated process, the authors show that the experimentally observed trichome pattern is substantially disturbed by cell-to-cell variations. Using computer simulations, they find that the rates concerning the availability of the protein complex that triggers trichome formation plays a significant role in noise-induced variations of the pattern. The focus on the effects of cell noise yields further insights into pattern formation of trichomes. The authors expect that similar strategies can contribute to the understanding of other differentiation processes by elucidating the role of naturally occurring fluctuations in the concentration of cellular components or their properties. PMID:23039695

  3. Granulocyte colony-stimulating factor induces in vitro lymphangiogenesis

    SciTech Connect

    Lee, Ae Sin; Kim, Dal; Wagle, Susbin Raj; Lee, Jung Eun; Jung, Yu Jin; Kang, Kyung Pyo; Lee, Sik; Park, Sung Kwang; Kim, Won

    2013-07-12

    Highlights: •G-CSF induces tube formation, migration and proliferation of lymphatic cells. •G-CSF increases phosphorylation of MAPK and Akt in lymphatic endothelial cells. •MAPK and Akt pathways are linked to G-CSF-induced in vitro lymphangiogenesis. •G-CSF increases sprouting of a lymphatic ring. •G-CSF produces peritoneal lymphangiogenesis. -- Abstract: Granulocyte-colony stimulating factor (G-CSF) is reported to induce differentiation in cells of the monocyte lineage and angiogenesis in vascular endothelial cells, but its effects on lymphangiogenesis is uncertain. Here we examined the effects and the mechanisms of G-CSF-induced lymphangiogenesis using human lymphatic endothelial cells (hLECs). Our results showed that G-CSF induced capillary-like tube formation, migration and proliferation of hLECs in a dose- and time-dependent manner and enhanced sprouting of thoracic duct. G-CSF increased phosphorylation of Akt and ERK1/2 in hLECs. Supporting the observations, specific inhibitors of phosphatidylinositol 3′-kinase and MAPK suppressed the G-CSF-induced in vitro lymphangiogenesis and sprouting. Intraperitoneal administration of G-CSF to mice also stimulated peritoneal lymphangiogenesis. These findings suggest that G-CSF is a lymphangiogenic factor.

  4. Self-Renewal and High Proliferative Colony Forming Capacity of Late-Outgrowth Endothelial Progenitors Is Regulated by Cyclin-Dependent Kinase Inhibitors Driven by Notch Signaling.

    PubMed

    Patel, Jatin; Wong, Ho Yi; Wang, Weili; Alexis, Josue; Shafiee, Abbas; Stevenson, Alexander J; Gabrielli, Brian; Fisk, Nicholas M; Khosrotehrani, Kiarash

    2016-04-01

    Since the discovery of endothelial colony forming cells (ECFC), there has been significant interest in their therapeutic potential to treat vascular injuries. ECFC cultures display significant heterogeneity and a hierarchy among cells able to give rise to high proliferative versus low proliferative colonies. Here we aimed to define molecularly this in vitro hierarchy. Based on flow cytometry, CD34 expression levels distinguished two populations. Only CD34 + ECFC had the capacity to reproduce high proliferative potential (HPP) colonies on replating, whereas CD34- ECFCs formed only small clusters. CD34 + ECFCs were the only ones to self-renew in stringent single-cell cultures and gave rise to both CD34 + and CD34- cells. Upon replating, CD34 + ECFCs were always found at the centre of HPP colonies and were more likely in G0/1 phase of cell cycling. Functionally, CD34 + ECFC were superior at restoring perfusion and better engrafted when injected into ischemic hind limbs. Transcriptomic analysis identified cyclin-dependent kinase (CDK) cell cycle inhibiting genes (p16, p21, and p57), the Notch signaling pathway (dll1, dll4, hes1, and hey1), and the endothelial cytokine il33 as highly expressed in CD34 + ECFC. Blocking the Notch pathway using a γ-secretase inhibitor (DAPT) led to reduced expression of cell cycle inhibitors, increased cell proliferation followed by a loss of self-renewal, and HPP colony formation capacity reflecting progenitor exhaustion. Similarly shRNA knockdown of p57 strongly affected self-renewal of ECFC colonies. ECFC hierarchy is defined by Notch signalling driving cell cycle regulators, progenitor quiescence and self-renewal potential. Stem Cells 2016;34:902-912. PMID:26732848

  5. Colony image acquisition and genetic segmentation algorithm and colony analyses

    NASA Astrophysics Data System (ADS)

    Wang, W. X.

    2012-01-01

    Colony anaysis is used in a large number of engineerings such as food, dairy, beverages, hygiene, environmental monitoring, water, toxicology, sterility testing. In order to reduce laboring and increase analysis acuracy, many researchers and developers have made efforts for image analysis systems. The main problems in the systems are image acquisition, image segmentation and image analysis. In this paper, to acquire colony images with good quality, an illumination box was constructed. In the box, the distances between lights and dishe, camra lens and lights, and camera lens and dishe are adjusted optimally. In image segmentation, It is based on a genetic approach that allow one to consider the segmentation problem as a global optimization,. After image pre-processing and image segmentation, the colony analyses are perfomed. The colony image analysis consists of (1) basic colony parameter measurements; (2) colony size analysis; (3) colony shape analysis; and (4) colony surface measurements. All the above visual colony parameters can be selected and combined together, used to make a new engineeing parameters. The colony analysis can be applied into different applications.

  6. Recombinant human granulocyte colony-stimulating factor (rh-G-CSF) may accelerate hematopoietic recovery after HLA-identical sibling allogeneic peripheral blood stem cell transplantation.

    PubMed

    Ozcan, M; Ustün, C; Akçağlayan, E; Akan, H; Arslan, O; Ilhan, O; Beksaç, M; Gürman, G; Demirer, T; Arat, M; Celebi, H; Konuk, N; Uysal, A; Koç, H

    2001-03-01

    We studied the effects of recombinant human granulocyte colony-stimulating factor (G-CSF) on hematopoietic recovery and clinical outcome in patients undergoing allogeneic peripheral blood stem cell (PBSC) transplantation. Fifty-six patients with hematological malignancies who underwent allogeneic PBSC transplantation between 1995 and 1998 were entered into this study. Twenty-eight patients who received daily G-CSF from day +1 after allogeneic PBSC transplantation until the absolute neutrophil count (ANC) reached >0.5 x 10(9)/l for 3 consecutive days were compared with 28 patients (control group) who did not receive G-CSF in a non-randomized manner. The study group and the control group were comparable with respect to baseline patient and transplantation characteristics. Median times to ANC of >0.5 x 10(9)/l and 1 x 10(9)/l with or without G-CSF were 12 days (range 8-21), 13 days (10-32) (P = 0.04) and 13 days (9-21), 15 days (11-44) (P = 0.02), respectively. Median times to reach a platelet count of >20 x 10(9)/l with and without G-CSF were 11 days (0-20) and 13 days (9-26), respectively (P = 0.03). The incidence of febrile episodes was significantly lower with G-CSF, 75% vs 100% (P = 0.008). Patients receiving G-CSF had less grade III-IV mucositis than those who did not receive G-CSF (P = 0.01). There was also no increase in the incidence and severity of acute GVHD in patients using G-CSF (P = 0.22). Although the number of relapsing patients was greater in the G-CSF group (seven vs three patients), this was not statistically significant (P = 0.24). Disease-free and overall survival rates did not differ between the two groups (P = 0.58 and 0.53, respectively). The administration of G-CSF after allogeneic PBSC transplantation provided faster neutrophil and platelet engraftment associated with less severe mucositis and less febrile episodes. PMID:11313683

  7. Lateral inhibition-induced pattern formation controlled by the size and geometry of the cell.

    PubMed

    Seirin Lee, Sungrim

    2016-09-01

    Pattern formation in development biology is one of the fundamental processes by which cells change their functions. It is based on the communication of cells via intra- and intercellular dynamics of biochemicals. Thus, the cell is directly involved in biochemical interactions. However, many theoretical approaches describing biochemical pattern formation have usually neglected the cell's role or have simplified the subcellular process without considering cellular aspects despite the cell being the environment where biochemicals interact. On the other hand, recent experimental observations suggest that a change in the physical conditions of cell-to-cell contact can result in a change in cell fate and tissue patterning in a lateral inhibition system. Here we develop a mathematical model by which biochemical dynamics can be directly observed with explicitly expressed cell structure and geometry in higher dimensions, and reconsider pattern formation by lateral inhibition of the Notch-Delta signaling pathway. We explore how the physical characteristic of cell, such as cell geometry or size, influences the biochemical pattern formation in a multi-cellular system. Our results suggest that a property based on cell geometry can be a novel mechanism for symmetry breaking inducing cell asymmetry. We show that cell volume can critically influence cell fate determination and pattern formation at the tissue level, and the surface area of the cell-to-cell contact can directly affect the spatial range of patterning. PMID:27229622

  8. Nexrutine inhibits azoxymethane-induced colonic aberrant crypt formation in rat colon and induced apoptotic cell death in colon adenocarcinoma cells.

    PubMed

    Alam, Shamshad; Pal, Anu; Kumar, Rahul; Mir, Snober S; Ansari, Kausar M

    2016-08-01

    Colon cancer is the third most common cause of death in the United States. Therefore, new preventive strategies are warranted for preventing colon cancer. Nexrutine (NX), an herbal extract from Phellodendron amurense, has been shown to have anti-inflammatory, anti-microbial and anti-cancer activity for various tissue specific cancers, but its chemopreventive efficacy has not been evaluated against colon cancer. Here, we explored the mechanism of chemopreventive/chemotherapeutic efficacy of NX against colon cancer. We found that dietary exposure of NX significantly reduced the number of azoxymethane (AOM)-induced aberrant crypt foci (ACF) in rats. In addition, significant inhibition in AOM-induced cell proliferation and reduced expression of the inflammatory markers COX-2, iNOS as well as the proliferative markers PCNA and cyclin D1 were also seen. Moreover, NX exposure significantly enhanced apoptosis in the colon of AOM treated rats. Furthermore, in in vitro studies, NX (2.5, 5, 10 μg/ml, 48 h) decreased cell survival and colony formation while inducing G0/G1 cell cycle arrest and apoptosis in colon adenocarcinoma cells COLO205 and HCT-15. However, NX had minimal cytotoxic effect on IEC-6 normal rat intestinal cells, suggesting its high therapeutic index. NX treatment also modulates the level of Bax and Bcl-2 proteins along with cytochrome c release, cleavage and enhanced expression of poly (adenosine diphosphate-ribose) polymerase as well as the catalytic activity of caspase 3 and caspase 9 in both COLO205 and HCT-15 cells. Based on these in vivo and in vitro findings, we suggest that NX could be useful candidate agent for colon cancer chemoprevention and treatment. © 2015 Wiley Periodicals, Inc. PMID:26259065

  9. The role of biosimilar granulocyte colony stimulating factor (GCSF) Zarzio for progenitor cell mobilization and the treatment of therapy-induced neutropenia in adult hematopoietic stem cell transplantation.

    PubMed

    Severson, Cherie C

    2015-01-01

    Originator GCSF (Neupogen) has been used to mobilize progenitor stem cells and treat therapy-induced neutropenia in Canadian stem cell transplant settings for years. Although its benefit is not in question, viable alternatives are available. Biosimilar GCSF (Zarzio) is widely in use in Europe since 2009 and was recently approved in the U.S.for the same five indications as Neupogen. Zarzio is reported as safe, equally efficacious, more accessible and cost effective without negatively impacting patient outcomes. This paper summarizes the supporting evidence. PMID:26897866

  10. Formation of thin walled ceramic solid oxide fuel cells

    DOEpatents

    Claar, Terry D.; Busch, Donald E.; Picciolo, John J.

    1989-01-01

    To reduce thermal stress and improve bonding in a high temperature monolithic solid oxide fuel cell (SOFC), intermediate layers are provided between the SOFC's electrodes and electrolyte which are of different compositions. The intermediate layers are comprised of a blend of some of the materials used in the electrode and electrolyte compositions. Particle size is controlled to reduce problems involving differential shrinkage rates of the various layers when the entire structure is fired at a single temperature, while pore formers are provided in the electrolyte layers to be removed during firing for the formation of desired pores in the electrode layers. Each layer includes a binder in the form of a thermosetting acrylic which during initial processing is cured to provide a self-supporting structure with the ceramic components in the green state. A self-supporting corrugated structure is thus formed prior to firing, which the organic components of the binder and plasticizer removed during firing to provide a high strength, high temperature resistant ceramic structure of low weight and density.

  11. Fever in honeybee colonies

    NASA Astrophysics Data System (ADS)

    Starks, P. T.; Blackie, Caroline A.; Seeley, Thomas D.

    Honeybees, Apis spp., maintain elevated temperatures inside their nests to accelerate brood development and to facilitate defense against predators. We present an additional defensive function of elevating nest temperature: honeybees generate a brood-comb fever in response to colonial infection by the heat-sensitive pathogen Ascosphaera apis. This response occurs before larvae are killed, suggesting that either honeybee workers detect the infection before symptoms are visible, or that larvae communicate the ingestion of the pathogen. This response is a striking example of convergent evolution between this "superorganism" and other fever-producing animals.

  12. A microfluidic device for uniform-sized cell spheroids formation, culture, harvesting and flow cytometry analysis.

    PubMed

    Patra, Bishnubrata; Chen, Ying-Hua; Peng, Chien-Chung; Lin, Shiang-Chi; Lee, Chau-Hwang; Tung, Yi-Chung

    2013-01-01

    Culture of cells as three-dimensional (3D) aggregates, named spheroids, possesses great potential to improve in vitro cell models for basic biomedical research. However, such cell spheroid models are often complicated, cumbersome, and expensive compared to conventional Petri-dish cell cultures. In this work, we developed a simple microfluidic device for cell spheroid formation, culture, and harvesting. Using this device, cells could form uniformly sized spheroids due to strong cell-cell interactions and the spatial confinement of microfluidic culture chambers. We demonstrated cell spheroid formation and culture in the designed devices using embryonic stem cells, carcinoma cells, and fibroblasts. We further scaled up the device capable of simultaneously forming and culturing 5000 spheroids in a single chip. Finally, we demonstrated harvesting of the cultured spheroids from the device with a simple setup. The harvested spheroids possess great integrity, and the cells can be exploited for further flow cytometry assays due to the ample cell numbers. PMID:24396525

  13. Biofuel cells: Electro-enzymatic oxidation of formate using formate dehydrogenase, NAD{sup +}, diaphorase, benzyl viologen, and graphite

    SciTech Connect

    Palmmore, G.T.R.; Bertschy, H.; Bergens, S.H.; Whitesides, G.M.

    1993-12-31

    The authors have developed an effective electrochemical method to regenerate nicotinamide adenine dinucleotide (NAD{sup +}) from NADH. Diaphorase (D) was used to oxidize NADH to NAD{sup +} with the concomitant reduction of two equivalents of benzylviologen (BV{sup 2+}) to the mono cation, BV{sup +} to the mono cation, BV{sup +}. The BV{sup +} was then electrochemically oxidized to BV{sup 2+} at a carbon-felt anode: NADH + 2BV{sup 2+} --(D)--> NAD{sup +} + 2BV{sup +} 2BV{sup +}-->(anode)-->2BV{sup 2+}. This system was incorporated into an enzymatic fuel cell that used formate as fuel. Formate dehydrogenase (FDH) was used to oxidize formate to CO{sub 2} with the concomitant 2 e{sup {minus}}, 2H{sup +} reduction of NAD{sup +} to NADH. The NADH was then regenerated using the system described above. The anode of the fuel cell was used to oxidize BV{sup +} to BV{sup 2+}. Blackened Pt gauze as used as the cathode of the fuel cell. The working parameters of the formate/O{sub 2} biofuel cell will be presented including methods to develop this system into the more complex methanol/O{sub 2} biofuel cell.

  14. Myotube formation is affected by adipogenic lineage cells in a cell-to-cell contact-independent manner

    SciTech Connect

    Takegahara, Yuki; Yamanouchi, Keitaro Nakamura, Katsuyuki; Nakano, Shin-ichi; Nishihara, Masugi

    2014-05-15

    Intramuscular adipose tissue (IMAT) formation is observed in some pathological conditions such as Duchenne muscular dystrophy (DMD) and sarcopenia. Several studies have suggested that IMAT formation is not only negatively correlated with skeletal muscle mass but also causes decreased muscle contraction in sarcopenia. In the present study, we examined w hether adipocytes affect myogenesis. For this purpose, skeletal muscle progenitor cells were transfected with siRNA of PPARγ (siPPARγ) in an attempt to inhibit adipogenesis. Myosin heavy chain (MHC)-positive myotube formation was promoted in cells transfected with siPPARγ compared to that of cells transfected with control siRNA. To determine whether direct cell-to-cell contact between adipocytes and myoblasts is a prerequisite for adipocytes to affect myogenesis, skeletal muscle progenitor cells were cocultured with pre- or mature adipocytes in a Transwell coculture system. MHC-positive myotube formation was inhibited when skeletal muscle progenitor cells were cocultured with mature adipocytes, but was promoted when they were cocultured with preadipocytes. Similar effects were observed when pre- or mature adipocyte-conditioned medium was used. These results indicate that preadipocytes play an important role in maintaining skeletal muscle mass by promoting myogenesis; once differentiated, the resulting mature adipocytes negatively affect myogenesis, leading to the muscle deterioration observed in skeletal muscle pathologies. - Highlights: • We examined the effects of pre- and mature adipocytes on myogenesis in vitro. • Preadipocytes and mature adipocytes affect myoblast fusion. • Preadipocytes play an important role in maintaining skeletal muscle mass. • Mature adipocytes lead to muscle deterioration observed in skeletal muscle pathologies.

  15. The pros and cons of split-dose granulocyte colony-stimulating factor alone rather than a single high dose for hematopoietic progenitor cell mobilization in small children (< 15 kg) with solid tumors.

    PubMed

    Merlin, Etienne; Piguet, Christophe; Auvrignon, Anne; Rubie, Hervé; Deméocq, François; Kanold, Justyna

    2006-07-01

    Hematopoietic progenitor cells were mobilized in 34 children with solid tumors weighing < or = 15 kg using granulocyte colony-stimulating factor alone at the doses of 10, 20 or 2 x 12 microg/kg/day. The mobilization with 2 x 12 microg/kg/day was more efficient than that with 10 mg/kg/day. Although the superiority of the split-dose compared to the single, high daily dose (20 microg/kg/day) was not statistically significant, our results suggest that the 2 x 12 microg/kg/day regimen is interesting. PMID:16818292

  16. Cutting edge: Bcl6-interacting corepressor contributes to germinal center T follicular helper cell formation and B cell helper function.

    PubMed

    Yang, Jessica A; Tubo, Noah J; Gearhart, Micah D; Bardwell, Vivian J; Jenkins, Marc K

    2015-06-15

    CD4(+) germinal center (GC)-T follicular helper (Tfh) cells help B cells become long-lived plasma cells and memory cells. The transcriptional repressor Bcl6 plays a key role in GC-Tfh formation by inhibiting the expression of genes that promote differentiation into other lineages. We determined whether BCOR, a component of a Polycomb repressive complex that interacts with the Bcl6 BTB domain, influences GC-Tfh differentiation. T cell-targeted BCOR deficiency led to a substantial loss of peptide:MHC class II-specific GC-Tfh cells following Listeria monocytogenes infection and a 2-fold decrease following immunization with a peptide in CFA. The reduction in GC-Tfh cells was associated with diminished plasma cell and GC B cell formation. Thus, T cell-expressed BCOR is critical for optimal GC-Tfh cell differentiation and humoral immunity. PMID:25964495

  17. Living T9 glioma cells expressing membrane macrophage colony-stimulating factor produce immediate tumor destruction by polymorphonuclear leukocytes and macrophages via a "paraptosis"-induced pathway that promotes systemic immunity against intracranial T9 gliomas.

    PubMed

    Chen, Yijun; Douglass, Thomas; Jeffes, Edward W B; Xu, Qingcheng; Williams, Christopher C; Arpajirakul, Neary; Delgado, Christina; Kleinman, Michael; Sanchez, Ramon; Dan, Qinghong; Kim, Ronald C; Wepsic, H Terry; Jadus, Martin R

    2002-08-15

    Cloned T9-C2 glioma cells transfected with membrane macrophage colony-stimulating factor (mM-CSF) never formed subcutaneous tumors when implanted into Fischer rats, whereas control T9 cells did. The T9-C2 cells were completely killed within 1 day through a mechanism that resembled paraptosis. Vacuolization of the T9-C2 cell's mitochondria and endoplasmic reticulum started within 4 hours after implantation. By 24 hours, the dead tumor cells were swollen and terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL)-positive. Bcl2-transduced T9-C2 cells failed to form tumors in rats. Both T9 and T9-C2 cells produced cytokine-induced neutrophil chemoattractant that recruited the granulocytes into the tumor injection sites, where they interacted with the tumor cells. Freshly isolated macrophages killed the T9-C2 cells in vitro by a mechanism independent of phagocytosis. Nude athymic rats treated with antiasialo GM1 antibody formed T9-C2 tumors, whereas rats treated with a natural killer cell (NK)-specific antibody failed to form tumors. When treated with antipolymorphonuclear leukocyte (anti-PMN) and antimacrophage antibodies, 80% of nude rats formed tumors, whereas only 40% of the rats developed a tumor when a single antibody was used. This suggests that both PMNs and macrophages are involved in the killing of T9-C2 tumor cells. Immunocompetent rats that rejected the living T9-C2 cells were immune to the intracranial rechallenge with T9 cells. No vaccinating effect occurred if the T9-C2 cells were freeze-thawed, x-irradiated, or treated with mitomycin-C prior to injection. Optimal tumor immunization using mM-CSF-transduced T9 cells requires viable tumor cells. In this study optimal tumor immunization occurred when a strong inflammatory response at the injection of the tumor cells was induced. PMID:12149220

  18. Co-administration of plasmid-encoded granulocyte-macrophage colony-stimulating factor increases human immunodeficiency virus-1 DNA vaccine-induced polyfunctional CD4+ T-cell responses

    PubMed Central

    Santana, Vinicius Canato; Almeida, Rafael Ribeiro; Ribeiro, Susan Pereira; Ferreira, Luís Carlos de Souza; Kalil, Jorge; Rosa, Daniela Santoro; Cunha-Neto, Edecio

    2015-01-01

    T-cell based vaccines against human immunodeficiency virus (HIV) generate specific responses that may limit both transmission and disease progression by controlling viral load. Broad, polyfunctional, and cytotoxic CD4+T-cell responses have been associated with control of simian immunodeficiency virus/HIV-1 replication, supporting the inclusion of CD4+ T-cell epitopes in vaccine formulations. Plasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF) co-administration has been shown to induce potent CD4+ T-cell responses and to promote accelerated priming and increased migration of antigen-specific CD4+ T-cells. However, no study has shown whether co-immunisation with pGM-CSF enhances the number of vaccine-induced polyfunctional CD4+ T-cells. Our group has previously developed a DNA vaccine encoding conserved, multiple human leukocyte antigen (HLA)-DR binding HIV-1 subtype B peptides, which elicited broad, polyfunctional and long-lived CD4+ T-cell responses. Here, we show that pGM-CSF co-immunisation improved both magnitude and quality of vaccine-induced T-cell responses, particularly by increasing proliferating CD4+ T-cells that produce simultaneously interferon-γ, tumour necrosis factor-α and interleukin-2. Thus, we believe that the use of pGM-CSF may be helpful for vaccine strategies focused on the activation of anti-HIV CD4+ T-cell immunity. PMID:26602876

  19. Co-administration of plasmid-encoded granulocyte-macrophage colony-stimulating factor increases human immunodeficiency virus-1 DNA vaccine-induced polyfunctional CD4+ T-cell responses.

    PubMed

    Santana, Vinicius Canato; Almeida, Rafael Ribeiro; Ribeiro, Susan Pereira; Ferreira, Luís Carlos de Souza; Kalil, Jorge; Rosa, Daniela Santoro; Cunha-Neto, Edecio

    2015-12-01

    T-cell based vaccines against human immunodeficiency virus (HIV) generate specific responses that may limit both transmission and disease progression by controlling viral load. Broad, polyfunctional, and cytotoxic CD4+T-cell responses have been associated with control of simian immunodeficiency virus/HIV-1 replication, supporting the inclusion of CD4+ T-cell epitopes in vaccine formulations. Plasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF) co-administration has been shown to induce potent CD4+ T-cell responses and to promote accelerated priming and increased migration of antigen-specific CD4+ T-cells. However, no study has shown whether co-immunisation with pGM-CSF enhances the number of vaccine-induced polyfunctional CD4+ T-cells. Our group has previously developed a DNA vaccine encoding conserved, multiple human leukocyte antigen (HLA)-DR binding HIV-1 subtype B peptides, which elicited broad, polyfunctional and long-lived CD4+ T-cell responses. Here, we show that pGM-CSF co-immunisation improved both magnitude and quality of vaccine-induced T-cell responses, particularly by increasing proliferating CD4+ T-cells that produce simultaneously interferon-γ, tumour necrosis factor-α and interleukin-2. Thus, we believe that the use of pGM-CSF may be helpful for vaccine strategies focused on the activation of anti-HIV CD4+ T-cell immunity. PMID:26602876

  20. Colony image acquisition and segmentation

    NASA Astrophysics Data System (ADS)

    Wang, W. X.

    2007-12-01

    For counting of both colonies and plaques, there is a large number of applications including food, dairy, beverages, hygiene, environmental monitoring, water, toxicology, sterility testing, AMES testing, pharmaceuticals, paints, sterile fluids and fungal contamination. Recently, many researchers and developers have made efforts for this kind of systems. By investigation, some existing systems have some problems. The main problems are image acquisition and image segmentation. In order to acquire colony images with good quality, an illumination box was constructed as: the box includes front lightning and back lightning, which can be selected by users based on properties of colony dishes. With the illumination box, lightning can be uniform; colony dish can be put in the same place every time, which make image processing easy. The developed colony image segmentation algorithm consists of the sub-algorithms: (1) image classification; (2) image processing; and (3) colony delineation. The colony delineation algorithm main contain: the procedures based on grey level similarity, on boundary tracing, on shape information and colony excluding. In addition, a number of algorithms are developed for colony analysis. The system has been tested and satisfactory.

  1. Shear flow-induced formation of tubular cell protrusions in multiple myeloma cells

    PubMed Central

    Porat, Ziv; Yaron, Itamar; Katz, Ben-Zion; Kam, Zvi; Geiger, Benjamin

    2011-01-01

    Exposure of live cells to shear flow induces major changes in cell shape, adhesion to the extracellular matrix, and migration. In the present study, we show that exposure of cultured multiple myeloma (MM) cells to shear flow of 4–36 dynes/cm2 triggers the extension of long tubular protrusions (denoted FLow-Induced Protrusions, or FLIPs) in the direction of the flow. These FLIPs were found to be rich in actin, contain few or no microtubules and, apart from endoplasmic reticulum (ER)-like membranal structures, are devoid of organelles. Studying the dynamics of this process revealed that FLIPs elongate at their tips in a shear force-dependent manner, and retract at their bases. Examination of this force dependence revealed considerable heterogeneity in the mechanosensitivity of individual cells, most likely reflecting the diversity of the malignant B-cell population. The mechanisms underlying FLIP formation following mechanical perturbation, and their relevance to the cellular trafficking of MM cells, are discussed. PMID:21344380

  2. Generation of dendritic cells in vitro from peripheral blood mononuclear cells with granulocyte-macrophage-colony-stimulating factor, interleukin-4, and tumor necrosis factor-alpha for use in cancer immunotherapy.

    PubMed Central

    Morse, M A; Zhou, L J; Tedder, T F; Lyerly, H K; Smith, C

    1997-01-01

    OBJECTIVE: The purpose of the study was to characterize the requirements in terms of precursors, developmental pathways, and media for the generation of large numbers of mature dendritic cells (DC) under conditions acceptable for use in adjuvant, active immunotherapy strategies for surgically treated malignancies. SUMMARY BACKGROUND DATA: Although limited previously by the small numbers accessible, DC-based immunotherapies for malignancy have become more realistic with the development of methods for efficiently generating larger numbers of DC from peripheral blood mononuclear cells (PBMC) in vitro, but these methods rely on clinically unacceptable culture conditions (such as inclusion of fetal bovine serum), necessitating the development of methods for generating functionally equivalent DC in serum-free conditions. METHODS: Plastic-adherent PBMC (from healthy donors and patients with cancer) were incubated for 7 days with granulocyte-macrophage-colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) with and without tumor necrosis factor-alpha (TNF-alpha) in fetal bovine serum-containing and serum-free media and were analyzed by Wright's stain for morphology, flow cytometry for phenotype, and mixed lymphocyte reaction for allostimulatory function. RESULTS: Growth in either serum-containing or serum-free media supplemented with GM-CSF and IL-4 yielded a similarly heterogeneous population of cells, 6% to 10% of which had the morphology (large cells with thin projections), immunophenotype (including CD83+), and function of mature DC. Tumor necrosis factor-alpha significantly augmented the number of these mature DC, whereas preculture depletion of CD14+ PBMC virtually eliminated them. CONCLUSIONS: Generation of mature DC in the authors' serum-free clinically applicable conditions is similar to serum-containing conditions and requires CD14+ precursors, differentiation through a CD14-CD83- immature stage under the influence of GM-CSF and IL-4, and maturation into a

  3. Multinuclear giant cell formation is enhanced by down-regulation of Wnt signaling in gastric cancer cell line, AGS

    SciTech Connect

    Kim, Shi-Mun; Kim, Rockki; Ryu, Jae-Hyun; Jho, Eek-Hoon; Song, Ki-Joon; Jang, Shyh-Ing; Kee, Sun-Ho . E-mail: keesh@korea.ac.kr

    2005-08-01

    AGS cells, which were derived from malignant gastric adenocarcinoma tissue, lack E-cadherin-mediated cell adhesion but have a high level of nuclear {beta}-catenin, which suggests altered Wnt signal. In addition, approximately 5% of AGS cells form multinuclear giant cells in the routine culture conditions, while taxol treatment causes most AGS cells to become giant cells. The observation of reduced nuclear {beta}-catenin levels in giant cells induced by taxol treatment prompted us to investigate the relationship between Wnt signaling and giant cell formation. After overnight serum starvation, the shape of AGS cells became flattened, and this morphological change was accompanied by decrease in Myc expression and an increase in the giant cell population. Lithium chloride treatment, which inhibits GSK3{beta} activity, reversed these serum starvation effects, which suggests an inverse relationship between Wnt signaling and giant cell formation. Furthermore, the down-regulation of Wnt signaling caused by the over-expression of ICAT, E-cadherin, and Axin enhanced giant cell formation. Therefore, down-regulation of Wnt signaling may be related to giant cell formation, which is considered to be a survival mechanism against induced cell death.

  4. Collagen formation by transformed smooth muscle cells after arterial injury.

    PubMed

    Chidi, C C; DePalma, R G

    1981-01-01

    Twenty-five normocholesterolemic rabbits were sacrificed at intervals up to 60 days after the thoracic aortas were de-endothelialized. Ultrastructural studies of both the re-endothelialized and nonendothelialized intima were done. The smooth muscle cells in the re-endothelialized intima showed segmental structural changes typically associated with transformation to a secretory cell type; abundant accumulations of collagen were in juxtaposition with these cells. The nonendothelialized intima did not demonstrate similar smooth muscle cell changes and collagen accumulation. These observations suggest that regenerating endothelial cells and intimal smooth muscle cells interact to cause smooth muscle cell transformation and collagen accumulation during arterial repair. PMID:7455897

  5. Primary Bioassay of Human Myeloma Stem Cells

    PubMed Central

    Hamburger, Anne; Salmon, Sydney E.

    1977-01-01

    The ability to clone primary tumors in soft agar has proven useful in the study of the kinetics and biological properties of tumor stem cells. We report the development of an in vitro assay which permits formation of colonies of human monoclonal plasma cells in soft agar. Colony growth has been observed from bone marrow aspirates from 75% of the 70 patients with multiple myeloma or related monoclonal disorders studied. Growth was induced with either 0.02 ml of human type O erythrocytes or 0.25 ml of medium conditioned by the adherent spleen cells of mineral oil-primed BALB/c mice. 5-500 colonies appeared after 2-3 wk in culture yielding a plating efficiency of 0.001-0.1%. The number of myeloma colonies was proportional to the number of cells plated between concentrations of 105-106 and back-extrapolated through zero, suggesting that colonies were clones derived from single myeloma stem cells. Morphological, histochemical, and functional criteria showed the colonies to consist of immature plasmablasts and mature plasma cells. 60-80% of cells picked from colonies contained intracytoplasmic monoclonal immunoglobulin. Colony growth was most easily achieved from the bone marrow cells of untreated patients or those in relapse. Only 50% of bone marrow samples from patients in remission were successfully cultured. Tritiated thymidine suicide studies provided evidence that for most myeloma patients, a very high proportion of myeloma colony-forming cells was actively in transit through the cell cycle. Velocity sedimentation at 1 g showed myeloma stem cells sedimented in a broad band with a peak at 13 mm/h. Antibody to granulocyte colony-stimulating factor did not reduce the number or size of the colonies. Increased numbers of myeloma colonies were seen when the marrow was depleted of colony-stimulating factor elaborating adherent cells before plating. This bioassay should prove useful in studying the in vitro biological behavior of certain bone marrow-derived (B)-cell

  6. Mipu1 overexpression protects macrophages from oxLDL-induced foam cell formation and cell apoptosis.

    PubMed

    Qu, Shun-Lin; Fan, Wen-Jing; Zhang, Chi; Guo, Fang; Han, Dan; Pan, Wen-Jun; Li, Wei; Feng, Da-Ming; Jiang, Zhi-Sheng

    2014-12-01

    Mipu1 (myocardial ischemic preconditioning upregulated protein 1) is a novel N-terminal Kruppel-associated box (KRAB)/C2H2 zinc finger superfamily protein, that displays a powerful effect in protecting H9c2 cells from oxidative stress-induced cell apoptosis. The present study aims to investigate the effect of Mipu1 overexpression on oxidized low-density lipoprotein (oxLDL)-induced foam cell formation, cell apoptosis, and its possible mechanisms. New Zealand healthy rabbits were used to establish atherosclerosis model, and serum levels of triglycerides, total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol were detected by an automatic biochemical analyzer. Sudan IV staining was used to detect atherosclerotic lesions. The RAW264.7 macrophage cell line was selected as the experimental material. Oil red O staining, high-performance liquid chromatography, and Dil-labeled lipoprotein were used to detect cholesterol accumulation qualitatively and quantitatively, respectively. Flow cytometry was used to determine cell apoptosis. Real-time quantitative polymerase chain reaction (PCR) was used to detect the mRNA expression of the main proteins that are associated with the transport of cholesterol, such as ABCA1, ABCG1, SR-BI, and CD36. Western blot analysis was used to detect the protein expression of Mipu1. There were atherosclerotic lesions in the high-fat diet group with Sudan IV staining. High-fat diet decreased Mipu1 expression and increased CD36 expression significantly at the 10th week compared with standard-diet rabbits. Mipu1 overexpression decreased oxLDL-induced cholesterol accumulation, oxLDL uptake, cell apoptosis, and cleaved caspase-3. Mipu1 overexpression inhibited the oxLDL-induced CD36 mRNA and protein expression, but it did not significantly inhibit the mRNA expression of ABCA1, ABCG1, and SR-BI. Mipu1 overexpression inhibits oxLDL-induced foam cell formation and cell apoptosis. Mipu1 overexpression reduces the

  7. Synthesis of membrane-bound colony-stimulating factor 1 (CSF-1) and downmodulation of CSF-1 receptors in NIH 3T3 cells transformed by cotransfection of the human CSF-1 and c-fms (CSF-1 receptor) genes.

    PubMed Central

    Rettenmier, C W; Roussel, M F; Ashmun, R A; Ralph, P; Price, K; Sherr, C J

    1987-01-01

    NIH 3T3 cells cotransfected with the human c-fms proto-oncogene together with a 1.6-kilobase cDNA clone encoding a 256-amino-acid precursor of the human mononuclear phagocyte colony-stimulating factor CSF-1 (M-CSF) undergo transformation by an autocrine mechanism. The number of CSF-1 receptors on the surface of transformed cells was regulated by ligand-induced receptor degradation and was inversely proportional to the quantity of CSF-1 produced. A tyrosine-to-phenylalanine mutation at position 969 near the receptor carboxyl terminus potentiated its transforming efficiency in cells cotransfected by the CSF-1 gene but did not affect receptor downmodulation. CSF-1 was synthesized as an integral transmembrane glycoprotein that was rapidly dimerized through disulfide bonds. The homodimer was externalized at the cell surface, where it underwent proteolysis to yield the soluble growth factor. Trypsin treatment of viable cells cleaved the plasma membrane form of CSF-1 to molecules of a size indistinguishable from that of the extracellular growth factor, suggesting that trypsinlike proteases regulate the rate of CSF-1 release from transformed cells. The data raise the possibility that this form of membrane-bound CSF-1 might stimulate receptors on adjacent cells through direct cell-cell interactions. Images PMID:3039346

  8. The formation of electronically excited species in the human multiple myeloma cell suspension

    PubMed Central

    Rác, Marek; Sedlářová, Michaela; Pospíšil, Pavel

    2015-01-01

    In this study, evidence is provided on the formation of electronically excited species in human multiple myeloma cells U266 in the growth medium exposed to hydrogen peroxide (H2O2). Two-dimensional imaging of ultra-weak photon emission using highly sensitive charge coupled device camera revealed that the addition of H2O2 to cell suspension caused the formation of triplet excited carbonyls 3(R = O)*. The kinetics of 3(R = O)* formation in the real time, as measured by one-dimensional ultra-weak photon emission using low-noise photomultiplier, showed immediate enhancement followed by a slow decay. In parallel to the formation of 3(R = O)*, the formation of singlet oxygen (1O2) in U266 cells caused by the addition of H2O2 was visualized by the imaging of 1O2 using the green fluorescence of singlet oxygen sensor green detected by confocal laser scanning microscopy. Additionally, the formation of 1O2 after the addition of H2O2 to cell suspension was detected by electron paramagnetic resonance spin-trapping spectroscopy using 2,2,6,6-tetramethyl-4-piperidone. Presented results indicate that the addition of H2O2 to cell suspension results in the formation of 3(R = O)* and 1O2 in U266 cell suspension. The contribution of the cell-free medium to the formation of electronically excited species was discussed. PMID:25744165

  9. The formation of electronically excited species in the human multiple myeloma cell suspension.

    PubMed

    Rác, Marek; Sedlářová, Michaela; Pospíšil, Pavel

    2015-01-01

    In this study, evidence is provided on the formation of electronically excited species in human multiple myeloma cells U266 in the growth medium exposed to hydrogen peroxide (H2O2). Two-dimensional imaging of ultra-weak photon emission using highly sensitive charge coupled device camera revealed that the addition of H2O2 to cell suspension caused the formation of triplet excited carbonyls (3)(R = O)*. The kinetics of (3)(R = O)* formation in the real time, as measured by one-dimensional ultra-weak photon emission using low-noise photomultiplier, showed immediate enhancement followed by a slow decay. In parallel to the formation of (3)(R = O)*, the formation of singlet oxygen ((1)O2) in U266 cells caused by the addition of H2O2 was visualized by the imaging of (1)O2 using the green fluorescence of singlet oxygen sensor green detected by confocal laser scanning microscopy. Additionally, the formation of (1)O2 after the addition of H2O2 to cell suspension was detected by electron paramagnetic resonance spin-trapping spectroscopy using 2,2,6,6-tetramethyl-4-piperidone. Presented results indicate that the addition of H2O2 to cell suspension results in the formation of (3)(R = O)* and (1)O2 in U266 cell suspension. The contribution of the cell-free medium to the formation of electronically excited species was discussed. PMID:25744165

  10. Minimally oxidized LDL inhibits macrophage selective cholesteryl ester uptake and native LDL-induced foam cell formation[S

    PubMed Central

    Meyer, Jason M.; Ji, Ailing; Cai, Lei; van der Westhuyzen, Deneys R.

    2014-01-01

    Scavenger receptor-mediated uptake of oxidized LDL (oxLDL) is thought to be the major mechanism of foam cell generation in atherosclerotic lesions. Recent data has indicated that native LDL is also capable of contributing to foam cell formation via low-affinity receptor-independent LDL particle pinocytosis and selective cholesteryl ester (CE) uptake. In the current investigation, Cu2+-induced LDL oxidation was found to inhibit macrophage selective CE uptake. Impairment of selective CE uptake was significant with LDL oxidized for as little as 30 min and correlated with oxidative fragmentation of apoB. In contrast, LDL aggregation, LDL CE oxidation, and the enhancement of scavenger receptor-mediated LDL particle uptake required at least 3 h of oxidation. Selective CE uptake did not require expression of the LDL receptor (LDL-R) and was inhibited similarly by LDL oxidation in LDL-R−/− versus WT macrophages. Inhibition of selective uptake was also observed when cells were pretreated or cotreated with minimally oxidized LDL, indicating a direct inhibitory effect of this oxLDL on macrophages. Consistent with the effect on LDL CE uptake, minimal LDL oxidation almost completely prevented LDL-induced foam cell formation. These data demonstrate a novel inhibitory effect of mildly oxidized LDL that may reduce foam cell formation in atherosclerosis. PMID:24891335

  11. Effect of Emdogain enamel matrix derivative and BMP-2 on the gene expression and mineralized nodule formation of alveolar bone proper-derived stem/progenitor cells.

    PubMed

    Fawzy El-Sayed, Karim M; Dörfer, Christof; Ungefroren, Hendrick; Kassem, Neemat; Wiltfang, Jörg; Paris, Sebastian

    2014-07-01

    The objective of this study was to evaluate the effect of Emdogain (Enamel Matrix Derivative, EMD) and Bone Morphogenetic Protein-2 (BMP-2), either solely or in combination, on the gene expression and mineralized nodule formation of alveolar bone proper-derived stem/progenitor cells. Stem/progenitor cells were isolated from human alveolar bone proper, magnetically sorted using STRO-1 antibodies, characterized flowcytometrically for their surface markers' expression, and examined for colony formation and multilineage differentiation potential. Subsequently, cells were treated over three weeks with 100 μg/ml Emdogain (EMD-Group), or 100 ng/ml BMP-2 (BMP-Group), or a combination of 100 ng/ml BMP-2 and 100 μg/ml Emdogain (BMP/EMD-Group). Unstimulated stem/progenitor cells (MACS(+)-Group) and osteoblasts (OB-Group) served as controls. Osteogenic gene expression was analyzed using RTq-PCR after 1, 2 and 3 weeks (N = 3/group). Mineralized nodule formation was evaluated by Alizarin-Red staining. BMP and EMD up-regulated the osteogenic gene expression. The BMP Group showed significantly higher expression of Collagen-I, III, and V, Alkaline phosphatase and Osteonectin compared to MACS(+)- and OB-Group (p < 0.05; Two-way ANOVA/Bonferroni) with no mineralized nodule formation. Under in-vitro conditions, Emdogain and BMP-2 up-regulate the osteogenic gene expression of stem/progenitor cells. The combination of BMP-2 and Emdogain showed no additive effect and would not be recommended for a combined clinical stimulation. PMID:24080138

  12. Post-Colonial Theory and Action Research

    ERIC Educational Resources Information Center

    Parsons, Jim B.; Harding, Kelly J.

    2011-01-01

    This essay explores connections between post-colonial theory and action research. Post-colonial theory is committed to addressing the plague of colonialism. Action research, at its core, promises to problematize uncontested "colonial" hegemonies of any form. Both post-colonial theory and action research engage dialogic, critically reflective and…

  13. Student Discipline in Colonial America.

    ERIC Educational Resources Information Center

    Petry, John R.

    The basis for the severe discipline imposed on school children in colonial America, especially in the Puritan colonies, was the belief in original sin. The child was regarded as being born in sin and thus depraved and prone to sin. The purpose of education was to enable children to read the Bible and thus change the behavior which otherwise would…

  14. Colony-stimulating factor-1 (CSF-1) receptor-mediated macrophage differentiation in myeloid cells: a role for tyrosine 559-dependent protein phosphatase 2A (PP2A) activity.

    PubMed Central

    McMahon, K A; Wilson, N J; Marks, D C; Beecroft, T L; Whitty, G A; Hamilton, J A; Csar, X F

    2001-01-01

    M1 myeloid cells transfected with the wild-type (WT) colony-stimulating factor-1 (CSF-1) receptor (CSF-1R; M1/WT cells) undergo CSF-1-dependent macrophage differentiation. By mutation studies, we have provided prior evidence that tyrosine 559 in the CSF-1R cytoplasmic domain governs the Src-dependent differentiation pathway. Further components of this pathway were then sought. We report that the extent of CSF-1-mediated tyrosine phosphorylation of protein phosphatase 2A (PP2A), and the associated loss of its activity were reduced in M1 cells transfected with the CSF-1R with a tyrosine-to-phenylalanine mutation at position 559 (M1/559 cells), compared with the corresponding responses in CSF-1-treated M1/WT cells. This evidence for an involvement of a reduction in PP2A activity in the differentiation process was supported by the restoration of the defect in the CSF-1-mediated differentiation of M1/559 cells by the addition of the PP2A inhibitor, okadaic acid. It was also found that the degree of activation of extracellular-signal-regulated kinase (ERK) activities by CSF-1 was reduced in M1/559 cells, suggesting their involvement in the differentiation process. These data suggest that PP2A and ERK form part of the Src-dependent signal-transduction cascade governing CSF-1-mediated macrophage differentiation in M1 cells. PMID:11513742

  15. Spatial Arrangement of Legionella Colonies in Intact Biofilms from a Model Cooling Water System

    PubMed Central

    Taylor, Michael; Ross, Kirstin; Bentham, Richard

    2013-01-01

    There is disagreement among microbiologists about whether Legionella requires a protozoan host in order to replicate. This research sought to determine where in biofilm Legionellae are found and whether all biofilm associated Legionella would be located within protozoan hosts. While it is accepted that Legionella colonizes biofilm, its life cycle and nutritional fastidiousness suggest that Legionella employs multiple survival strategies to persist within microbial systems. Fluorescent in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM) demonstrated an undulating biofilm surface architecture and a roughly homogenous distribution of heterotrophic bacteria with clusters of protozoa. Legionella displayed 3 distinct spatial arrangements either contained within or directly associated with protozoa, or dispersed in loosely associated clusters or in tightly packed aggregations of cells forming dense colonial clusters. The formation of discreet clusters of tightly packed Legionella suggests that colony formation is influenced by specific environmental conditions allowing for limited extracellular replication. This work represents the first time that an environmentally representative, multispecies biofilm containing Legionella has been fluorescently tagged and Legionella colony morphology noted within a complex microbial system. PMID:24826074

  16. Spatial arrangement of legionella colonies in intact biofilms from a model cooling water system.

    PubMed

    Taylor, Michael; Ross, Kirstin; Bentham, Richard

    2013-01-01

    There is disagreement among microbiologists about whether Legionella requires a protozoan host in order to replicate. This research sought to determine where in biofilm Legionellae are found and whether all biofilm associated Legionella would be located within protozoan hosts. While it is accepted that Legionella colonizes biofilm, its life cycle and nutritional fastidiousness suggest that Legionella employs multiple survival strategies to persist within microbial systems. Fluorescent in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM) demonstrated an undulating biofilm surface architecture and a roughly homogenous distribution of heterotrophic bacteria with clusters of protozoa. Legionella displayed 3 distinct spatial arrangements either contained within or directly associated with protozoa, or dispersed in loosely associated clusters or in tightly packed aggregations of cells forming dense colonial clusters. The formation of discreet clusters of tightly packed Legionella suggests that colony formation is influenced by specific environmental conditions allowing for limited extracellular replication. This work represents the first time that an environmentally representative, multispecies biofilm containing Legionella has been fluorescently tagged and Legionella colony morphology noted within a complex microbial system. PMID:24826074

  17. Ammonia emissions from seabird colonies

    NASA Astrophysics Data System (ADS)

    Blackall, Trevor D.; Wilson, Linda J.; Theobald, Mark R.; Milford, Celia; Nemitz, Eiko; Bull, Jennifer; Bacon, Philip J.; Hamer, Keith C.; Wanless, Sarah; Sutton, Mark A.

    2007-05-01

    Ammonia emissions were measured from two entire seabird colonies with contrasting species assemblages, to ascertain the ammonia volatilisation potentials among seabird species in relation to their nesting behaviour. Emissions were calculated from downwind plume measurements of ammonia concentration using both inverse dispersion and tracer ratio methods. Measured colony emissions ranged 1-90 kg NH3 hour-1, and equated to 16 and 36% volatilization of excreted nitrogen for colonies dominated by ground/burrow nesting and bare rock nesting birds, respectively. The results were applied in a bioenergetics model with a global seabird database. Seabird colonies are found to represent the largest point sources of ammonia globally (up to ~6 Gg NH3 colony-1 year-1). Moreover the largest emissions occur mainly in remote environments with otherwise low NH3 emissions. These ammonia ``hot spots'' explain significant perturbations of the nitrogen cycle in these regions and add ~20% to oceanic ammonia emissions south of latitude 45°S.

  18. Sodium formate induces autophagy and apoptosis via the JNK signaling pathway of photoreceptor cells

    PubMed Central

    WANG, YING; XU, SHAO-LIN; XU, WEN-JING; YANG, HAI-YAN; HU, PING; LI, YU-XIN

    2016-01-01

    Incidents associated with methanol intoxication resulting from the consumption of fake wine occur not infrequently worldwide. Certain individuals are made blind due to methanol poisoning. The present study aimed to investigate the effects of sodium formate exposure on photoreceptor cells (661W cells). The 661W cells were exposed to sodium formate for 6–24 h and cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Subsequently, the 661W cells were exposed to 15 or 30 mM sodium formate for 24 h. The level of apoptosis was determined using Hoechst 33342/propidium iodide staining, visualizing the cells under a fluorescence microscope, and annexin V-fluorescein isothiocyanate staining, using flow cytometric analysis. Intracellular reactive oxygen species (ROS) were measured using 2′,7′-dichlorofluorescein diacetate (DCFH-DA) staining, followed by flow cytometric analysis. Autophagy of the 661W cells was measured by monodansylcadaverine staining. The activation of phosphorylated c-Jun N-terminal kinase (p-JNK), B-cell lymphoma (Bcl-2), Bcl-2-associated X protein, cleaved caspase-3, cleaved caspase-9 and microtubule-associated protein 1A/1B-light chain 3 (LC3) was assessed by western blotting. The effects of Z-VAD-fmk (a pan-caspase inhibitor) and SP600125 (a JNK inhibitor) on the viability of the sodium formate-induced 661W cells were determined using an MTT assay. Sodium formate treatment induced a decrease in the viability of the 661W cells in a time- and a dose-dependent manner. In addition, sodium formate at concentrations of 15 or 30 mM markedly increased the level of apoptosis and the ROS levels, as measured by DCFH-DA staining of the 661W cells. Additionally, 661W cells exposed to sodium formate for 24 h exhibited increased levels of p-JNK, Bax, cleaved caspase-3, cleaved caspase-9 and LC3II (the phosphatidylethanolamine-modified form of LC3), although the level of Bcl-2 was decreased

  19. In vivo formation of bone and hematopoietic territories by transplanted human bone marrow stromal cells generated in medium with and without osteogenic supplements

    PubMed Central

    Kuznetsov, Sergei A; Mankani, Mahesh H; Robey, Pamela Gehron

    2011-01-01

    Autologous transplantation of human bone marrow stromal cells (BMSCs) has been successfully used for bone reconstruction. However, in order to advance this approach into the mainstream of bone tissue engineering, the conditions for BMSC cultivation and transplantation must be optimized. In a recent report, cultivation with dexamethasone (Dex) significantly increased bone formation by human BMSCs in vivo. Based on this important conclusion, we analyzed the data accumulated by our laboratory where human BMSCs have been routinely generated using media both with and without a combination of two osteogenic supplements: Dex at 10-8M and ascorbic acid phosphate (AscP) at 10-4M. Our data demonstrate that for 22 out of 24 donors, BMSC strains propagated with and without Dex/AscP formed similar amounts of bone in vivo. Thus, human BMSCs do not appear to need to be induced to osteogenic differentiation ex vivo prior to transplantation. Similarly, for 12 of 14 donors, BMSC strains cultured with and without Dex/AscP formed hematopoietic territories to a comparable extent. While Dex/AscP did not increase bone formation, they significantly stimulated BMSC in vitro proliferation without affecting the number of BMSC colonies formed by the Colony Forming Units-Fibroblast. We conclude that for the substantial majority of donors, Dex/AscP have no effect on the ability of BMSCs to form bone and myelosupportive stroma in vivo. However, due to increased BMSC proliferation, the total osteogenic population obtained from a single marrow sample is larger after cultivation with Dex/AscP than without them. Secondary to increased BMSC proliferation, Dex/AscP may stimulate bone formation if BMSCs and/or the transplantation system are less than optimal. PMID:22052864

  20. Single cell motility and trail formation in populations of microglia

    NASA Astrophysics Data System (ADS)

    Lee, Kyoung Jin

    2009-03-01

    Microglia are a special type of glia cell in brain that has immune responses. They constitute about 20 % of the total glia population within the brain. Compared to other glia cells, microglia are very motile, constantly moving to destroy pathogens and to remove dead neurons. While doing so, they exhibit interesting body shapes, have cell-to-cell communications, and have chemotatic responses to each other. Interestingly, our recent in vitro studies show that their unusual motile behaviors can self-organize to form trails, similar to those in populations of ants. We have studied the changes in the physical properties of these trails by varying the cell population density and by changing the degree of spatial inhomogeneities (``pathogens''). Our experimental observations can be quite faithfully reproduced by a simple mathematical model involving many motile cells whose mechanical motion are driven by actin polymerization and depolymerization process within the individual cell body and by external chemical gradients.

  1. Effects of irradiated biodegradable polymer in endothelial cell monolayer formation

    NASA Astrophysics Data System (ADS)

    Arbeitman, Claudia R.; del Grosso, Mariela F.; Behar, Moni; García Bermúdez, Gerardo

    2013-11-01

    In this work we study cell adhesion, proliferation and cell morphology of endothelial cell cultured on poly-L-lactide acid (PLLA) modified by heavy ion irradiation. Thin films of PLLA samples were irradiated with sulfur (S) at energies of 75 MeV and gold (Au) at 18 MeV ion-beams. Ion beams were provided by the Tandar (Buenos Aires, Argentina) and Tandetron (Porto Alegre, Brazil) accelerators, respectively. The growth of a monolayer of bovine aortic endothelial cells (BAEC) onto unirradiated and irradiated surfaces has been studied by in vitro techniques in static culture. Cell viability and proliferation increased on modified substrates. But the results on unirradiated samples, indicate cell death (necrosis/apoptosis) with the consequent decrease in proliferation. We analyzed the correlation between irradiation parameters and cell metabolism and morphology.

  2. Enumeration of the colony-forming units–fibroblast from mouse and human bone marrow in normal and pathological conditions

    PubMed Central

    Kuznetsov, Sergei A.; Mankani, Mahesh H.; Bianco, Paolo; Robey, Pamela G.

    2009-01-01

    Bone marrow stromal cell populations, containing a subset of multipotential skeletal stem cells, are increasingly contemplated for use in tissue engineering and stem cell therapy, whereas their involvement in the pathogenetic mechanisms of skeletal disorders is far less recognized. We compared the concentrations of stromal clonogenic cells, colony forming units–fibroblast (CFU-Fs), in norm and pathology. Initially, culture conditions were optimized by demonstrating that fetal bovine serum heat inactivation could significantly repress colony formation. Using non-heat-inactivated fetal bovine serum, the concentration of CFU-Fs (colony-forming efficiency, CFE) ranged from 3.5 ± 1.0 to 11.5 ± 4.0 per 1 × 105 nucleated cells in five inbred mouse strains. In four transgenic lines with profound bone involvement, CFE was either significantly reduced or increased compared to wild-type littermates. In normal human donors, CFE decreased slightly with age and averaged 52.2 ± 4.1 for children and 32.3 ± 3.0 for adults. CFE was significantly altered in patients with several skeletal, metabolic, and hematological disorders: reduced in congenital generalized lipodystrophy, achondroplasia (SADDAN), pseudoachondroplasia, and Paget disease of bone and elevated in alcaptonuria and sickle cell anemia. Our findings indicate that under appropriate culture conditions, CFE values may provide useful insights into bone/bone marrow pathophysiology. PMID:19383412

  3. Foreign Body Giant Cell Formation Is Preceded by Lamellipodia Formation and Can Be Attenuated by Inhibition of Rac1 Activation

    PubMed Central

    Jay, Steven M.; Skokos, Eleni; Laiwalla, Farah; Krady, Marie-Marthe; Kyriakides, Themis R.

    2007-01-01

    Macrophages that are recruited to the site of implanted biomaterials undergo fusion to form surface-damaging foreign body giant cells. Exposure of peripheral blood monocytes to interleukin-4 can recapitulate the fusion process in vitro. In this study, we used interleukin-4 to induce multinucleation of murine bone marrow-derived macrophages and observed changes in cell shape, including elongation and lamellipodia formation, before fusion. Because cytoskeletal rearrangements are regulated by small GTPases, we examined the effects of inhibitors of Rho kinase (Y-32885) and Rac activation (NSC23766) on fusion. Y-32885 did not prevent cytoskeletal changes or fusion but limited the extent of multinucleation. NSC23766, on the other hand, inhibited lamellipodia formation and fusion in a dose-dependent manner. In addition, we found that in control cells, these changes were preceded by Rac1 activation. However, NSC23766 did not block the uptake of polystyrene microspheres. Likewise, short interfering RNA knockdown of Rac1 limited fusion without limiting phagocytosis. Thus, phagocytosis and fusion can be partially decoupled based on their susceptibility to NSC23766. Furthermore, poly(ethylene-co-vinyl acetate) scaffolds containing NSC23766 attenuated foreign body giant cell formation in vivo. These observations suggest that targeting Rac1 activation could protect biomaterials without compromising the ability of macrophages to perform beneficial phagocytic functions at implantation sites. PMID:17556592

  4. Formate: an Energy Storage and Transport Bridge between Carbon Dioxide and a Formate Fuel Cell in a Single Device.

    PubMed

    Vo, Tracy; Purohit, Krutarth; Nguyen, Christopher; Biggs, Brenna; Mayoral, Salvador; Haan, John L

    2015-11-01

    We demonstrate the first device to our knowledge that uses a solar panel to power the electrochemical reduction of dissolved carbon dioxide (carbonate) into formate that is then used in the same device to operate a direct formate fuel cell (DFFC). The electrochemical reduction of carbonate is carried out on a Sn electrode in a reservoir that maintains a constant carbon balance between carbonate and formate. The electron-rich formate species is converted by the DFFC into electrical energy through electron release. The product of DFFC operation is the electron-deficient carbonate species that diffuses back to the reservoir bulk. It is possible to continuously charge the device using alternative energy (e.g., solar) to convert carbonate to formate for on-demand use in the DFFC; the intermittent nature of alternative energy makes this an attractive design. In this work, we demonstrate a proof-of-concept device that performs reduction of carbonate, storage of formate, and operation of a DFFC. PMID:26510492

  5. Colony mapping: A new technique for monitoring crevice-nesting seabirds

    USGS Publications Warehouse

    Renner, H.M.; Renner, M.; Reynolds, J.H.; Harping, A.M.A.; Jones, I.L.; Irons, D.B.; Byrd, G.V.

    2006-01-01

    Monitoring populations of auklets and other crevice-nesting seabirds remains problematic, although numerous methods have been attempted since the mid-1960s. Anecdotal evidence suggests several large auklet colonies have recently decreased in both abundance and extent, concurrently with vegetation encroachment and succession. Quantifying changes in the geographical extent of auklet colonies may be a useful alternative to monitoring population size directly. We propose a standardized method for colony mapping using a randomized systematic grid survey with two components: a simple presence/absence survey and an auklet evidence density survey. A quantitative auklet evidence density index was derived from the frequency of droppings and feathers. This new method was used to map the colony on St. George Island in the southeastern Bering Sea and results were compared to previous colony mapping efforts. Auklet presence was detected in 62 of 201 grid cells (each grid cell = 2500 m2) by sampling a randomly placed 16 m2 plot in each cell; estimated colony area = 155 000 m2. The auklet evidence density index varied by two orders of magnitude across the colony and was strongly correlated with means of replicated counts of birds socializing on the colony surface. Quantitatively mapping all large auklet colonies is logistically feasible using this method and would provide an important baseline for monitoring colony status. Regularly monitoring select colonies using this method may be the best means of detecting changes in distribution and population size of crevice-nesting seabirds. ?? The Cooper Ornithological Society 2006.

  6. Nicotinate-Curcumin Impedes Foam