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Sample records for international kupffer cell

  1. Kupffer Cell Metabolism and Function

    PubMed Central

    Nguyen-Lefebvre, Anh Thu; Horuzsko, Anatolij

    2015-01-01

    Kupffer cells are resident liver macrophages and play a critical role in maintaining liver functions. Under physiological conditions, they are the first innate immune cells and protect the liver from bacterial infections. Under pathological conditions, they are activated by different components and can differentiate into M1-like (classical) or M2-like (alternative) macrophages. The metabolism of classical or alternative activated Kupffer cells will determine their functions in liver damage. Special functions and metabolism of Kupffer cells suggest that they are an attractive target for therapy of liver inflammation and related diseases, including cancer and infectious diseases. Here we review the different types of Kupffer cells and their metabolism and functions in physiological and pathological conditions. PMID:26937490

  2. Receptor-mediated endocytosis of carcinoembryonic antigen by rat liver Kupffer cells.

    PubMed

    Toth, C A; Thomas, P; Broitman, S A; Zamcheck, N

    1985-01-01

    In vivo, carcinoembryonic antigen (CEA) is removed from the circulation by the liver Kupffer cells. Immunologically identifiable CEA is transferred from these macrophages to the hepatocytes, where degradation is completed. Circulatory clearance of CEA is specific, rapid [t1/2 = 3.7 +/- 0.9 (S.D.) min], and saturable. In vitro, Kupffer cells take up CEA by a saturable process which is time/temperature dependent and colchicine sensitive. Isolated Kupffer cells endocytose CEA with an apparent Km of 6 X 10(-8) M. There are approximately 16,000 CEA binding sites per cell. Nonspecific cross-reacting antigen (NCA), a glycoprotein structurally similar to CEA, is recognized with lower affinity by the same receptor. Endocytosis is independent of the nonreducing terminal sugars on the molecule: CEA modified by Smith degradation inhibits Kupffer cell recognition of native CEA. Since performic acid oxidized CEA also inhibits endocytosis, receptor binding is similarly independent of intact protein conformation. Isolated Kupffer cells have mannose and/or N-acetyl glucosamine receptor activity but do not internalize CEA by that mechanism. Galactose-terminated glycoproteins impede CEA and NCA clearance in vivo but not Kupffer cell endocytosis in vitro. Radiolabeled CEA released from isolated Kupffer cells following endocytosis shows no apparent molecular weight change. However, the released CEA contains species with higher isoelectric points, suggesting that perhaps the removal of sialic acid and the resulting exposure of galactose residues mediate the subsequent transfer to the hepatocyte.

  3. High-affinity binding of fibronectin to cultured Kupffer cells

    SciTech Connect

    Cardarelli, P.M.; Blumenstock, F.A.; McKeown-Longo, P.J.; Saba, T.M.; Mazurkiewicz, J.E.; Dias, J.A. )

    1990-11-01

    Hepatic Kupffer cells are a major component of the reticuloendothelial or macrophage system. They were the first phagocytic cell type whose phagocytosis was shown to be influenced by plasma fibronectin, a dimeric opsonic glycoprotein. In the current study, the binding of soluble radioiodinated fibronectin purified from rat serum to isolated rat hepatic Kupffer cells was investigated using a cultured Kupffer cell monolayer technique. Binding was specific, since unlabeled purified fibronectin competed in a dose-dependent manner with the 125I-fibronectin for binding to the Kupffer cells. Addition of gelatin enhanced the binding of 125I-fibronectin to Kupffer cells. The phagocytosis of gelatinized-coated red cells by Kupffer cells was increased either by preopsonizing the target particles with purified fibronectin or by the addition of purified fibronectin to the culture medium. In contrast, exposure of the Kupffer cells to medium containing purified fibronectin followed by wash-removal of the fibronectin did not increase the uptake of gelatin-coated red blood cells, even though fibronectin was detected on the surface of the Kupffer cells by immunofluorescence. Trypsinized monolayers expressed decreased capacity to bind 125I-fibronectin as well as fibronectin-coated sheep erythrocytes. The binding of 125I-fibronectin-gelatin complexes was inhibited by excess unlabeled fibronectin. We calculated that specific high-affinity (Kd = 7.46 x 10(-9) M) binding sites for fibronectin exist on Kupffer cells. There are approximately 2,800-3,500 binding sites or putative fibronectin receptors per Kupffer cell. These sites appear to mediate the enhanced phagocytosis of gelatin-coated particles opsonized by fibronectin.

  4. Comparative studies of endotoxin uptake by isolated rat Kupffer and peritoneal cells.

    PubMed

    Fox, E S; Thomas, P; Broitman, S A

    1987-12-01

    The process of uptake of endotoxin by cells of the reticuloendothelial system is of current interest. Rabbit peritoneal macrophages have been used to study macrophage-endotoxin interactions and have suggested a receptor-mediated process. It is generally believed that the site of in vivo endotoxin clearance is the liver and that this clearance involves the Kupffer cell population. In the current report, the uptake characteristics of iodine-125-labeled Salmonella minnesota lipopolysaccharide (LPS) were compared in both isolated rat Kupffer cells and elicited rat peritoneal cells. Both types of cells were isolated from male Sprague-Dawley rats fed a semisynthetic AIN-76 5% saturated-fat diet either by peritoneal lavage for peritoneal cells or by collagenase perfusion followed by purification on a 17.5% metrizamide gradient for Kupffer cells. Hot phenol water-extracted S. minnesota LPS was labeled with iodine by the chloramine-T method following a reaction with methyl-p-hydroxybenzimidate. The in vitro uptake of [125I]LPS by Kupffer cells was unsaturable up to concentrations of 33.33 micrograms/ml, while peritoneal cells became saturated at between 16.67 and 25 micrograms of LPS per ml. Uptake by both types of cells could be inhibited by a 10-fold excess of unlabeled LPS. Kinetic experiments demonstrated that Kupffer cells were unsaturable after 60 min of incubation, while peritoneal cells were saturable after 40 min of incubation. Pretreatment with 75 mM colchicine inhibited uptake by peritoneal cells but not Kupffer cells, while pretreatment with 12 mM 2-deoxyglucose inhibited uptake by Kupffer cells but not peritoneal cells. These results are consistent with a process of receptor-mediated endocytosis for peritoneal cells, while Kupffer cells may internalize endotoxins by absorptive pinocytosis. These results suggest that studies of peritoneal cell-endotoxin interactions do not accurately describe the physiologic process within the liver, the major site for the

  5. Comparative studies of endotoxin uptake by isolated rat Kupffer and peritoneal cells.

    PubMed Central

    Fox, E S; Thomas, P; Broitman, S A

    1987-01-01

    The process of uptake of endotoxin by cells of the reticuloendothelial system is of current interest. Rabbit peritoneal macrophages have been used to study macrophage-endotoxin interactions and have suggested a receptor-mediated process. It is generally believed that the site of in vivo endotoxin clearance is the liver and that this clearance involves the Kupffer cell population. In the current report, the uptake characteristics of iodine-125-labeled Salmonella minnesota lipopolysaccharide (LPS) were compared in both isolated rat Kupffer cells and elicited rat peritoneal cells. Both types of cells were isolated from male Sprague-Dawley rats fed a semisynthetic AIN-76 5% saturated-fat diet either by peritoneal lavage for peritoneal cells or by collagenase perfusion followed by purification on a 17.5% metrizamide gradient for Kupffer cells. Hot phenol water-extracted S. minnesota LPS was labeled with iodine by the chloramine-T method following a reaction with methyl-p-hydroxybenzimidate. The in vitro uptake of [125I]LPS by Kupffer cells was unsaturable up to concentrations of 33.33 micrograms/ml, while peritoneal cells became saturated at between 16.67 and 25 micrograms of LPS per ml. Uptake by both types of cells could be inhibited by a 10-fold excess of unlabeled LPS. Kinetic experiments demonstrated that Kupffer cells were unsaturable after 60 min of incubation, while peritoneal cells were saturable after 40 min of incubation. Pretreatment with 75 mM colchicine inhibited uptake by peritoneal cells but not Kupffer cells, while pretreatment with 12 mM 2-deoxyglucose inhibited uptake by Kupffer cells but not peritoneal cells. These results are consistent with a process of receptor-mediated endocytosis for peritoneal cells, while Kupffer cells may internalize endotoxins by absorptive pinocytosis. These results suggest that studies of peritoneal cell-endotoxin interactions do not accurately describe the physiologic process within the liver, the major site for the

  6. Characterization of Kupffer cells in livers of developing mice

    PubMed Central

    2011-01-01

    Background Kupffer cells are well known macrophages of the liver, however, the developmental characteristics of Kupffer cells in mice are not well understood. To clarify this matter, the characteristics of Kupffer macrophages in normal developing mouse liver were studied using light microscopy and immunocytochemistry. Methods Sections of liver tissue from early postnatal mice were prepared using immunocytochemical techniques. The Kupffer cells were identified by their immunoreactivity to the F4/80 antibody, whereas endothelial cells were labelled with the CD-34 antibody. In addition, Kupffer cells and endothelial cells were labelled by systemically injected fluorescently labelled latex microspheres. Tissue slices were examined by fluorescence microscopy. Results Intravenous or intraperitonal injections of microspheres yielded similar patterns of liver cell labelling. The F4/80 positive Kupffer cells were labelled with both large (0.2 μm) and small (0.02 μm) diameter microspheres, while endothelial cells were labelled only with the smaller diameter microspheres. Microsphere labelling of Kupffer cells appeared stable for at least 6 weeks. Cells immunoreactive for F4/80 were identified as early as postnatal day 0, and these cells also displayed uptake of microspheres. Numbers of F4/80 Kupffer cells, relative to numbers of albumin positive hepatocytes, did not show a significant trend over the first 2 postnatal weeks. Conclusions Kupffer cells of the developing mouse liver appear quite similar to those of other mammalian species, confirming that the mouse presents a useful animal model for studies of liver macrophage developmental structure and function. PMID:21749715

  7. Perfluorochemical emulsions decrease Kupffer cell phagocytosis

    SciTech Connect

    Bottalico, L.A.; Betensky, H.T.; Min, Y.B.; Weinstock, S.B. )

    1991-07-01

    One drawback to using perfluorochemical emulsions as blood substitutes is that perfluorochemical particles are cleared from the blood by the reticuloendothelial system, primarily liver and spleen. The authors measured the impact of two perfluorochemical emulsions on clearance of colloidal carbon (less than 1 microns) and 51Cr-sheep red blood cells (about 8 microns) by the reticuloendothelial system in vivo and in the isolated perfused liver. Male rats were injected with 2 ml/100 gm body wt of Fluosol-DA or Oxypherol-ET for 4 consecutive days. Carbon (1 ml/100 gm body wt) or sheep red blood cells (0.05 ml of 5% vol/vol/100 gm body wt) were then injected intravenously (in vivo) or added to perfusate. Samples were taken at several time points for 1 hr. In the isolated perfused liver, carbon clearance was depressed by 25% 1 day after treatment. Rates returned to control levels by 12 days in Fluosol-DA-treated rats but remained depressed by 67% in Oxypherol-ET-treated rats. Sheep red blood cell (8 microns) clearance was two to five times slower than carbon clearance and depressed by 40% in livers from Fluosol-DA rats 1 day and 12 days after treatment. Added serum did not improve phagocytosis. In vivo carbon clearance remained normal in Fluosol-DA-treated rats but decreased by 74% in Oxypherol-ET-treated rats 1 day after treatment, returning to normal by 12 days. Clearance rates were similar in control rats in vivo and in the perfused liver. They conclude that the isolated perfused liver is a good model to measure liver clearance function. Although low doses of perfluorochemical emulsions may depress Kupffer cell phagocytosis, general reticuloendothelial system function is not significantly compromised.

  8. Endocytosis of heat-denatured albumin by cultured rat Kupffer cells

    SciTech Connect

    Brouwer, A.; Knook, D.L.

    1982-10-01

    Purified Kupffer cells were obtained by centrifugal elutriation of sinusoidal cells isolated by pronase treatment of the rat liver. The endocytosis of radioactively labeled heat-aggregated colloidal albumin (CA /sup 125/I) was investigated in maintenance cultures of the purified Kupffer cells. The endocytic capacity of the cells was studied during 4 days of culture. Maximum uptake was observed after 24 hr of culture, with a gradual decline during the following days. When the uptake was measured after incubation with increasing concentrations of CA /sup 125/I, a saturation effect was observed. This finding and the observed high rate of uptake are strong indications that receptor sites on the cell membrane are involved in the mechanism of endocytosis. The uptake of CA /sup 125/I by Kupffer cells was inhibited by the metabolic inhibitors fluoride and antimycin A, indicating that endocytosis of CA /sup 125/I is dependent on energy derived from both glycolysis and mitochondrial respiration. The mechanism of internalization may also require the action of microfilaments as well as intact microtubules, since both cytochalasin B and colchicine inhibited the uptake of CA /sup 125/I. The intracellular degradation of CA /sup 125/I by Kupffer cells was strongly inhibited by chloroquine but not by colchicine. The degradation of ingested CA /sup 125/I occurred within the Kupffer cell lysosomes.

  9. Uptake and modification of 125I-lipopolysaccharide by isolated rat Kupffer cells.

    PubMed

    Fox, E S; Thomas, P; Broitman, S A

    1988-01-01

    While it is generally believed that hepatic clearance of lipopolysaccharide involves Kupffer cells, the mechanism involved has not been fully elucidated. This study assesses this phenomenon in terms of in vitro uptake and post-uptake modification experiments with an 125I-labeled Salmonella minnesota lipopolysaccharide. 125I-Lipopolysaccharide was added to Kupffer cells in suspension cultures under a variety of conditions. In vitro uptake of 125I-Lipopolysaccharide was not saturable up to concentrations of 33.33 micrograms per ml. Kinetics experiments performed at 16.67 micrograms per ml demonstrated that Kupffer cells were unsaturable after 60 min of incubation. The kinetics of uptake could be inhibited, however, by incubation in the presence of a 10-fold excess of unlabeled lipopolysaccharide, indicating that a component of the uptake process may be limited. Energy dependence in this process was demonstrated by incubation in the presence of 1 mM 2-deoxyglucose which inhibited 125I-lipopolysaccharide uptake by approximately 30%. Pretreatment with 7.5 x 10(-5) M colchicine had no effect on kinetics, implying no role for the cell cytoskeleton in lipopolysaccharide uptake. These results are inconsistent with a receptor-mediated process as previously suggested. Modification of internalized label has been demonstrated by changes in buoyant density in CsCl isopyknic density gradients following overnight incubation with Kupffer cells. These results indicate that Kupffer cells clear bacterial endotoxin in vitro and post-uptake degradation occurs within 20 hr of incubation.

  10. Kupffer cell structure in the juvenile Nile crocodile, Crocodylus niloticus.

    PubMed

    van Wilpe, Erna; Groenewald, Hermanus Bernardus

    2014-01-01

    The morphology of Kupffer cells was examined in the liver of the juvenile Nile crocodile using light microscopy and transmission electron microscopy. Pleomorphic Kupffer cells were located in the sinusoids, in the space of Disse, in the hepatic parenchyma and often connected adjacent sinusoids. The cell surfaces were irregular due to the presence of filopodia and lamelliapodia with phagocytosis of white blood cells, red blood cells and thrombocytes being evident. The cells were in close contact with endothelial cells and pit cells in the sinusoidal lumen and with stellate cells in the space of Disse. The cytoplasm contained large phagosomes comprising a combination of ceroid pigment, melanosomes and siderosomes. The nuclei were often indented and eccentrically placed due to the presence of the phagosomes. Conspicuous clusters of membrane-bound tubular organelles with a filamentous or crystalline interior were observed in the cytoplasm. The clusters were sometimes separated into smaller groups around phagosomes. A clear zone existed between the limiting membrane and the interior of these tubular organelles with the electron-dense interior profiles being, respectively, circular, angular or divided. The tubular organelles have not previously been described in Kupffer cells and possibly represent lysosomes with specialized functions. Mitochondria, microtubules, Golgi profiles, granular and smooth endoplasmic reticulum, and a few cytoplasmic lipid droplets were also present. The presence of the tubular organelles and the occurrence of the Kupffer cells in different locations in the liver of the juvenile Nile crocodile are indicative of particularly active and mobile cells.

  11. Alcoholic hepatitis: The pivotal role of Kupffer cells

    PubMed Central

    Suraweera, Duminda B; Weeratunga, Ashley N; Hu, Robert W; Pandol, Stephen J; Hu, Richard

    2015-01-01

    Kupffer cells play a central role in the pathogenesis of alcoholic hepatitis (AH). It is believed that alcohol increases the gut permeability that results in raised levels of serum endotoxins containing lipopolysaccharides (LPS). LPS binds to LPS-binding proteins and presents it to a membrane glycoprotein called CD14, which then activates Kupffer cells via a receptor called toll-like receptor 4. This endotoxin mediated activation of Kupffer cells plays an important role in the inflammatory process resulting in alcoholic hepatitis. There is no effective treatment for AH, although notable progress has been made over the last decade in understanding the underlying mechanism of alcoholic hepatitis. We specifically review the current research on the role of Kupffer cells in the pathogenesis of AH and the treatment strategies. We suggest that the imbalance between the pro-inflammatory and the anti-inflammatory process as well as the increased production of reactive oxygen species eventually lead to hepatocyte injury, the final event of alcoholic hepatitis. PMID:26600966

  12. An intravascular immune response to Borrelia burgdorferi involves Kupffer cells and iNKT cells

    PubMed Central

    Lee, Woo-Yong; Moriarty, Tara J; Wong, Connie H Y; Zhou, Hong; Strieter, Robert M; van Rooijen, Nico; Chaconas, George; Kubes, Paul

    2016-01-01

    Here we investigate the dynamics of the hepatic intravascular immune response to a pathogen relevant to invariant natural killer T cells (iNKT cells). Immobilized Kupffer cells with highly ramified extended processes into multiple sinusoids could effectively capture blood-borne, disseminating Borrelia burgdorferi, creating a highly efficient surveillance and filtering system. After ingesting B. burgdorferi, Kupffer cells induced chemokine receptor CXCR3–dependent clustering of iNKT cells. Kupffer cells and iNKT cells formed stable contacts via the antigen-presenting molecule CD1d, which led to iNKT cell activation. An absence of iNKT cells caused B. burgdorferi to leave the blood and enter the joints more effectively. B. burgdorferi that escaped Kupffer cells entered the liver parenchyma and survived despite Ito cell responses. Kupffer cell–iNKT cell interactions induced a key intravascular immune response that diminished the dissemination of B. burgdorferi. PMID:20228796

  13. In vivo assessment of phagocytic properties of Kupffer cells

    SciTech Connect

    Reske, S.N.; Vyska, K.; Feinendegen, L.E.

    1981-05-01

    Three-compartment analysis was used to assess the kinetics of phagocytosis of Tc-99m-labeled human serum albumin microparticles (Tc-99m HSA-MM) in human Kupffer cells in vivo. The tracer turnover in these phagocytic cells could be described by a monoexponential accumulation with a two-stage elimination phase. Three-compartment analysis of the Tc-99m HSA-MM kinetics allowed us to quantify tracer attachment, phagocytosis, and degradation in Kupffer cells. The calculated time course of phagocytosis in ten control subjects proved to be identical to that of phagocytosis of various test substances in mouse macrophage monolayers (1). In addition, an impairment of particle turnover at the macrophage membrane, a significantly diminished (p less than 0.01) phagocytosis rate of the tracer, was observed in ten patients with various tumors.

  14. In vivo assessment of phagocytic properties of Kupffer cells

    SciTech Connect

    Reske, S.N.; Vyska, K.; Feinendegen, L.E.

    1981-05-01

    Three-compartment analysis was used to assess the kinetics of phagocytosis of Tc-99m-labeled human serum albumin microparticles (Tc-99m HSA-MM) in human Kupffer cells in vivo. The tracer turnover in these phagocytic cells could be described by a monoexponential accumulation with a two-stage elimination phase. Three-compartment analysis of the Tc-99m HSA-MM kinetics allowed us to quantify tracer attachment, phagocytosis, and degradation in Kupffer cells. The calculated time course of phagocytosis in ten control subjects proved to be identicl to that of phagocytosis of various test substances in mouse macrophage monolayers. In addition, an impairment of particle turnover at the macrophage membrane, a significantly diminished phagocytosis rate of the tracer, was observed in ten patients with various tumors.

  15. Kinetics of liver macrophages (Kupffer cells) in SIV-infected macaques

    SciTech Connect

    Ahsan, Muhammad H.; Gill, Amy F.; Alvarez, Xavier; Lackner, Andrew A.; Veazey, Ronald S.

    2013-11-15

    Since the liver drains antigens from the intestinal tract, and since the intestinal tract is a major site of viral replication, we examined the dynamics of liver macrophages (Kupffer cells) throughout SIV infection. Absolute numbers of Kupffer cells increased in the livers in acute infection, and in animals with AIDS. Significantly higher percentages of proliferating (BrdU+) Kupffer cells were detected in acute infection and in AIDS with similar trends in blood monocytes. Significantly higher percentages of apoptotic (AC3+) Kupffer cells were also found in acute and AIDS stages. However, productively infected cells were not detected in liver of 41/42 animals examined, despite abundant infected cells in gut and lymph nodes of all animals. Increased rates of Kupffer cell proliferation resulting in an increase in Kupffer cells without productive infection indicate SIV infection affects Kupffer cells, but the liver does not appear to be a major site of productive viral replication. - Highlights: • Kupffer cells increase in the liver of SIV-infected macaques. • Increased proliferation and apoptosis of Kupffer cells occurs in SIV infection. • Productively infected cells are rarely detected in the liver. • The liver is not a major site for SIV replication.

  16. Activation of human and mouse Kupffer cells by lipopolysaccharide is mediated by CD14.

    PubMed

    Su, Grace L; Goyert, Sanna M; Fan, Ming-Hui; Aminlari, Alireza; Gong, Ke Qin; Klein, Richard D; Myc, Andrzej; Alarcon, William H; Steinstraesser, Lars; Remick, Daniel G; Wang, Stewart C

    2002-09-01

    Upregulation of CD14 in Kupffer cells has been implicated in the pathogenesis of several forms of liver injury, including alcoholic liver disease. However, it remains unclear whether CD14 mediates lipopolysaccharide (LPS) signaling in this specialized liver macrophage population. In this series of experiments, we determined the role of CD14 in LPS activation of Kupffer cells by using several complementary approaches. First, we isolated Kupffer cells from human livers and studied the effects of anti-CD14 antibodies on LPS activation of these cells. Kupffer cells were incubated with increasing concentrations of LPS in the presence and absence of recombinant human LPS binding protein (LBP). With increasing concentrations of LPS, human Kupffer cell tumor necrosis factor-alpha (TNF-alpha) production (a marker for Kupffer cell activation) increased in a dose-dependent manner in the presence and absence of LBP. In the presence of anti-human CD14 antibodies, the production of TNF-alpha was significantly diminished. Second, we compared LPS activation of Kupffer cells isolated from wild-type and CD14 knockout mice. Kupffer cells from CD14 knockout mice produced significantly less TNF-alpha in response to the same amount of LPS. Together, these data strongly support a critical role for CD14 in Kupffer cell responses to LPS.

  17. Tobacco and e-cigarette products initiate Kupffer cell inflammatory responses.

    PubMed

    Rubenstein, David A; Hom, Sarah; Ghebrehiwet, Berhane; Yin, Wei

    2015-10-01

    Kupffer cells are liver resident macrophages that are responsible for screening and clearing blood of pathogens and foreign particles. It has recently been shown that Kupffer cells interact with platelets, through an adhesion based mechanism, to aid in pathogen clearance and then these platelets re-enter the general systemic circulation. Thus, a mechanism has been identified that relates liver inflammation to possible changes in the systemic circulation. However, the role that Kupffer cells play in cardiovascular disease initiation/progression has not been elucidated. Thus, our objective was to determine whether or not Kupffer cells are responsive to a classical cardiovascular risk factor and if these changes can be transmitted into the general systemic circulation. If Kupffer cells initiate inflammatory responses after exposure to classical cardiovascular risk factors, then this provides a potential alternative/synergistic pathway for cardiovascular disease initiation. We aimed to elucidate the prevalence of this potential pathway. We hypothesized that Kupffer cells would initiate a robust inflammatory response after exposure to tobacco cigarette or e-cigarette products and that the inflammatory response would have the potential to antagonize other salient cells for cardiovascular disease progression. To test this, Kupffer cells were incubated with tobacco smoke extracts, e-cigarette vapor extracts or pure nicotine. Complement deposition onto Kupffer cells, Kupffer cell complement receptor expression, oxidative stress production, cytokine release and viability and density were assessed after the exposure. We observed a robust inflammatory response, oxidative stress production and cytokine release after Kupffer cells were exposed to tobacco or e-cigarette extracts. We also observed a marginal decrease in cell viability coupled with a significant decrease in cell density. In general, this was not a function of the extract formulation (e.g. tobacco vs. e

  18. Biphasic control of polymorphonuclear cell migration by Kupffer cells. Effect of exposure to metabolic products of ethanol

    SciTech Connect

    Fainsilber, Z.; Feinman, L.; Shaw, S.; Lieber, C.S.

    1988-01-01

    In order to investigate the role of the Kupffer cells in the regulation of the inflammatory reaction seen in alcoholic hepatitis, rat liver Kupffer cells were cultured and exposed to products of ethanol metabolism. The resultant supernatants were tested to study their ability to stimulate or inhibit polymorphonuclear cell chemotaxis. Kupffer cells produced increased chemokinetic activity for human polymorphonuclear leukocytes; when incubated with soluble products of microsomal peroxidation, the Kupffer cells engendered more chemokinetic activity than that produced by untreated Kupffer cells. When Kupffer cells were incubated with acetaldehyde, the chemokinetic activity that appeared in the supernatant did not differ from control. Chemotaxis of polymorphonuclear cells was not observed when the Kupffer cell supernatants were tested by checkerboard analysis.

  19. Impact of myeloid-derived suppressor cell on Kupffer cells from mouse livers with hepatocellular carcinoma

    PubMed Central

    Lacotte, Stéphanie; Slits, Florence; Orci, Lorenzo A.; Meyer, Jeremy; Oldani, Graziano; Gonelle-Gispert, Carmen; Morel, Philippe; Toso, Christian

    2016-01-01

    ABSTRACT Kupffer cells represent the first line of defense against tumor cells in the liver. Myeloid-derived suppressor cells (MDSC) have recently been observed in the liver parenchyma of tumor-bearing animals. The present study investigates the function of the MDSC subsets, and their impact on Kupffer cell phenotype and function. RIL-175 mouse hepatocellular carcinoma (HCC) cells were injected into the median liver lobe of C57BL/6 mice. Three weeks later, the median lobe hosting the tumor nodule was removed, and Kupffer cells and MDSCs were sorted from the remaining liver. Mouse livers devoid of HCC served as control. Kupffer cells expressed less co-stimulatory CD86 and MHCII and more co-inhibitory CD274 molecules in HCC-bearing livers than in control livers. Corresponding to this phenotype, Kupffer cells from HCC-bearing mice were less efficient in their function as antigen-presenting cells. Three CD11b+ cell populations were identified and sorted from HCC-bearing mice. These cells had various phenotypes with different levels of MDSC-specific surface markers (Ly6Ghigh cells, Gr1high cells, and Ly6Clow cells), and may be considered as bonafide MDSCs given their suppression of antigen-specific T cell proliferation. Primary isolated Kupffer cells in co-culture with the three MDSC subsets showed a decrease in CCL2 and IL-18 secretion, and an increase in IL-10 and IL-1β secretion, and an increased expression of CD86, CD274, and MHCII. In conclusion, these data demonstrated the existence of three MDSC subsets in HCC-bearing animals. These cells altered Kupffer cell function and may decrease the migration and activation of anticancer effector cells in the liver. PMID:27999748

  20. Kupffer cells of cirrhotic rat livers sensitize colon cancer cells to Fas-mediated apoptosis.

    PubMed

    Song, E; Chen, J; Ouyang, N; Wang, M; Exton, M S; Heemann, U

    2001-05-04

    Metastasis of colorectal carcinomas rarely occurs in cirrhotic livers. Our study investigated the influence of activated Kupffer cells from cirrhotic rat livers on hepatic colonization and FasR-mediated apoptosis of colon cancer cells. A rat colon cancer cell line, RCN-9, was used to inoculate rat livers. Treatment with conditioned media of Kupffer cells isolated from CCl(4)-induced cirrhotic rat livers (cirrhotic KCM) significantly reduced the incidence of hepatic colonization of RCN-9 cells. In vitro cytotoxicity of Kupffer cells and tumour infiltrating lymphocytes (TILs) on RCN-9 cells was evaluated using [(3)H]-release assay. RCN-9 cells were resistant to cytotoxicity mediated by cirrhotic Kupffer cells, but were sensitized to TIL-mediated killing after treatment with cirrhotic KCM. The specific killing induced by TILs was FasR-mediated, as it was inhibited by ZB4, an antagonistic anti-FasR antibody. In agreement, cirrhotic KCM increased recombinant Fas ligand-induced apoptosis of RCN-9 cells, and up-regulated FasR expression on RCN-9 cells as evaluated by RT-PCR and flow cytometry. These findings suggest that Kupffer cells in cirrhotic livers sensitize metastatic colon cancer cells to FasR-mediated apoptosis by up-regulating the receptors, which thus prepare them to be eliminated by infiltrating lymphocytes.

  1. Hepatic Tumor Metastases Cause Enhanced PEGylated Liposome Uptake by Kupffer Cells.

    PubMed

    Ukawa, Masami; Fujiwara, Yukako; Ando, Hidenori; Shimizu, Taro; Ishida, Tatsuhiro

    2016-01-01

    Kupffer cells in livers bearing tumor metastases were found to have promoted tumor invasion and exacerbated the metastasis. This implies that the function of Kupffer cells might differ between animals bearing hepatic metastases and those that are healthy. Kupffer cells are considered responsible for the accumulation of liposomes in the liver. In this study, we hypothesized that the alteration in the function of Kupffer cells by hepatic metastasis would also affect the biodistribution of liposomes following intravenous administration. The hepatic accumulation and the blood concentration of PEGylated liposomes were compared between healthy mice and tumor-bearing mice. We noted that hepatic accumulation and elimination from the blood were significantly accelerated in tumor-bearing mice, indicating that our hypothesis was correct. In the tumor-bearing mice, the proportion of Kupffer cells taking up liposomes was significantly increased. Intravenous injection of oxaliplatin (l-OHP) containing PEGylated liposomes decreased the fraction of Kupffer cells, but this administration caused no injury to the hepatocytes. These results suggest that PEGylated liposomes containing l-OHP may have the potential to treat metastatic hepatic cancer-not only via the direct killing of the cancer cells but also via a reduction in tumor-supportive Kupffer cells.

  2. Alternative (M2) activation of Kupffer cells by PPARδ ameliorates obesity-induced insulin resistance

    PubMed Central

    Odegaard, Justin I.; Ricardo-Gonzalez, Roberto R.; Eagle, Alex Red; Vats, Divya; Morel, Christine R.; Goforth, Matthew H.; Subramanian, Vidya; Mukundan, Lata; Ferrante, Anthony W.; Chawla, Ajay

    2008-01-01

    SUMMARY Macrophage infiltration and activation in metabolic tissues underlie obesity-induced insulin resistance and type 2 diabetes. While inflammatory activation of resident hepatic macrophages potentiates insulin resistance, the functions of alternatively activated Kupffer cells in metabolic disease remain unknown. Here we show that, in response to the Th2 cytokine interleukin-4 (IL-4), peroxisome proliferator activated receptor δ (PPARδ) directs expression of the alternative phenotype in Kupffer cells and adipose tissue macrophages of lean mice. However, adoptive transfer of PPARδ null bone marrow into wild type mice only diminishes alternative activation of hepatic macrophages, causing hepatic dysfunction and systemic insulin resistance. Suppression of hepatic oxidative metabolism is recapitulated by treatment of primary hepatocytes with conditioned media from PPARδ null macrophages, indicating direct involvement of Kupffer cells in liver lipid metabolism. Taken together, these data suggest an unexpected beneficial role for alternatively activated Kupffer cells in metabolic syndrome and type 2 diabetes. PMID:18522831

  3. SELECTION WITH THE MAGNET AND CULTIVATION OF RETICULO-ENDOTHELIAL CELLS (KUPFFER CELLS)

    PubMed Central

    Rous, Peyton; Beard, J. W.

    1934-01-01

    Methods and apparatus are described where with living Kupffer cells can be procured from the liver of the rabbit and the dog for study and cultivation in vitro. Almost none of these cells can be dislodged from the normal liver by forcible perfusion; but after they have taken up finely particulate matter (India ink, iron oxide), they come away in great numbers. When they have phagocyted ferromagnetic iron oxide they can be selected with a magnet from amongst the blood elements present in suspension with them; and they are obtainable in quantity by this means. They do poorly when plated in a thin plasma clot, failing to multiply or to assume their characteristic shape; but they flourish when allowed to attach themselves to strands of lens paper bathed in serum that is frequently changed. Bacterial infection of serum cultures of Kupffer cells from normal rabbits and dogs occurs only as the result of secondary contamination of the materials, whereas it regularly develops in cultures from animals with fever induced by the injection of nucleic acid or of killed B. prodigiosus. Kupffer cells obtained under such conditions are abnormally active, and some can be washed out of the liver of sick animals in the absence of any preliminary phagocytosis of particulate matter. The facts have a bearing both on the conditions conducing to blood invasion and on the response of the Kupffer cells in the emergency. The characters of the isolated Kupffer cells and the results of tests of their presumptive functions will be described in later papers. PMID:19870267

  4. Inhibition of Kupffer Cell Autophagy Abrogates Nanoparticle-Induced Liver Injury.

    PubMed

    Zhu, Shasha; Zhang, Jiqian; Zhang, Li; Ma, Wentao; Man, Na; Liu, Yiming; Zhou, Wei; Lin, Jun; Wei, Pengfei; Jin, Peipei; Zhang, Yunjiao; Hu, Yi; Gu, Erwei; Lu, Xianfu; Yang, Zhilai; Liu, Xuesheng; Bai, Li; Wen, Longping

    2017-02-24

    The possible adverse effects of engineered nanomaterials on human health raise increasing concern as our research on nanosafety intensifies. Upon entry into a human body, whether intended for a theranostic purpose or through unintended exposure, nanomaterials tend to accumulate in the liver, leading to hepatic damage. A variety of nanoparticles, including rare earth upconversion nanoparticles (UCNs), have been reported to elicit hepatotoxicity, in most cases through inducing immune response or activating reactive oxygen species. Many of these nanoparticles also induce autophagy, and autophagy inhibition has been shown to decrease UCN-induced liver damage. Herein, using UCNs as a model engineered nanomaterial, this study uncovers a critical role for Kupffer cells in nanomaterial-induced liver toxicity, as depletion of Kupffer cells significantly exacerbates UCN-induced liver injury. Furthermore, UCN-induced prodeath autophagy in Kupffer cells, and inhibition of autophagy with 3-MA, a well-established chemical inhibitor of autophagy, enhances Kupffer cell survival and further abrogates UCN-induced liver toxicity. The results reveal the critical importance of Kupffer cell autophagy for nanoparticle-induced liver damage, and inhibition of autophagy may constitute a novel strategy for abrogating nanomaterial-elicited liver toxicity.

  5. Characterization of rat and human Kupffer cells after cryopreservation.

    PubMed

    Walbrun, Peter; Hellerbrand, Claus; Weiss, Thomas S; Netter, Susanne; Neumaier, Daniel; Gaebele, Erwin; Wiest, Reiner; Schoelmerich, Juergen; Froh, Matthias

    2007-04-01

    Kupffer cells (KC) are the resident macrophages of the liver and represent about 80% of the total fixed macrophage population. They are involved in disease states such as endotoxin shock, alcoholic liver diseases and other toxic-induced liver injury. They release physiologically active substances such as eicosanoids and inflammatory cytokines (IL-1, IL-6, TNFalpha), and produce free radical species. Thus, KC are attractive targets for anti-inflammatory therapies and potential candidates responsible for differences in inflammation in liver disease seen between different individuals. However, to perform parallel in vitro experiments with KC from different donors a suitable method for conservation of KC would be necessary. Therefore, the present study evaluated, whether rat and human KC can be frozen, stored and recovered without losing their functional integrity. Rat and human KC were isolated and either cultured under standard conditions (fresh KC) or cryopreserved in special freezing medium (cryopreserved KC). At least 24 h later, cryopreserved KC were thawed, brought into suspension and seeded in the same density as fresh cells for subsequent experiments. Viability of cultured KC was analyzed by trypan blue exclusion. LPS (or PBS as control) stimulation was performed at different time points and cytokine release was analyzed with IL-6 and TNFalpha ELISAs, respectively. Phagocytic capacity was investigated by using a specific phagocytosis assay and FACS analysis. The recovery rate after thawing was around 57% for rat and around 65% for human cryopreserved KC. The results indicate, that KC can successfully be cryopreserved with an adequate recovery rate of viable cells. The properties of fresh and frozen KC can also be compared after thawing. Freshly isolated and cryopreserved cultured KC showed near-normal morphology and did not differ in the cultivation profiles over a period of 72 h. One to three days after seeding, frozen rat or human KC also retained inducible

  6. Binding kinetics of monomeric and aggregated IgG to Kupffer cells and hepatocytes of mice.

    PubMed Central

    Sancho, J; González, E; Escanero, J F; Egido, J

    1984-01-01

    The binding kinetics of human monomeric IgG and stable heat-aggregated IgG (A-IgG) to Fc receptors of hepatocytes and Kupffer cells isolated from mice was studied. After injection of radiolabelled proteins the 60-70% of hepatic uptake was recovered in parenchymal cells (hepatocytes). In experiments in vitro the A-IgG bound in larger amounts to hepatocytes and Kupffer cells than monomeric IgG. The association rate constants of aggregates were somewhat higher for Kupffer cells than for hepatocytes whereas the percentage uptake of aggregates by Kupffer cells was only 5-15% of that of hepatocytes. The equilibrium constants of aggregates binding to both cells amounted to 0.4-1 X 10(8) M-1 for A-IgG compared with an equilibrium constant for monomeric IgG of 1-2 X 10(7)M-1. The maximum number of IgG and A-IgG molecules bound per cell was higher on hepatocytes (mean 14 X 10(6)) than on Kupffer cells (mean 2 X 10(5)) which is in agreement with the higher binding capacity of hepatocytes for these proteins observed in vivo and in vitro experiments. The ability to compete for receptor binding seemed to reside exclusively in the Fc portion of IgG since F(ab')2 fragments of IgG failed to inhibit labelled monomeric IgG or A-IgG. The receptor seems to be specific for IgG since unlabelled monomeric IgA demonstrated no binding inhibition of labelled IgG or A-IgG on hepatocytes and Kupffer cells. The overall results further suggest that hepatocytes might through Fc receptors play a collaborative role with the mononuclear phagocytic system in the clearance of circulating immune complexes. PMID:6237982

  7. Th2-Associated Alternative Kupffer Cell Activation Promotes Liver Fibrosis without Inducing Local Inflammation

    PubMed Central

    López-Navarrete, Giuliana; Ramos-Martínez, Espiridión; Suárez-Álvarez, Karina; Aguirre-García, Jesús; Ledezma-Soto, Yadira; León-Cabrera, Sonia; Gudiño-Zayas, Marco; Guzmán, Carolina; Gutiérrez-Reyes, Gabriela; Hernández-Ruíz, Joselín; Camacho-Arroyo, Ignacio; Robles-Díaz, Guillermo; Kershenobich, David; Terrazas, Luis I.; Escobedo, Galileo

    2011-01-01

    Cirrhosis is the final outcome of liver fibrosis. Kupffer cell-mediated hepatic inflammation is considered to aggravate liver injury and fibrosis. Alternatively-activated macrophages are able to control chronic inflammatory events and trigger wound healing processes. Nevertheless, the role of alternative Kupffer cell activation in liver harm is largely unclear. Thus, we evaluated the participation of alternatively-activated Kupffer cells during liver inflammation and fibrosis in the murine model of carbon tetrachloride-induced hepatic damage. To stimulate alternative activation in Kupffer cells, 20 Taenia crassiceps (Tc) larvae were inoculated into BALBc/AnN female mice. Six weeks post-inoculation, carbon tetrachloride or olive oil were orally administered to Tc-inoculated and non-inoculated mice twice per week during other six weeks. The initial exposure of animals to T. crassiceps resulted in high serum concentrations of IL-4 accompanied by a significant increase in the hepatic mRNA levels of Ym-1, with no alteration in iNOS expression. In response to carbon tetrachloride, recruitment of inflammatory cell populations into the hepatic parenchyma was 5-fold higher in non-inoculated animals than Tc-inoculated mice. In contrast, carbon tetrachloride-induced liver fibrosis was significantly less in non-inoculated animals than in the Tc-inoculated group. The latter showed elevated IL-4 serum levels and low IFN-γ concentrations during the whole experiment, associated with hepatic expression of IL-4, TGF-β, desmin and α-sma, as well as increased mRNA levels of Arg-1, Ym-1, FIZZ-1 and MMR in Kupffer cells. These results suggest that alternative Kupffer cell activation is favored in a Th2 microenvironment, whereby such liver resident macrophages could exhibit a dichotomic role during chronic hepatic damage, being involved in attenuation of the inflammatory response but at the same time exacerbation of liver fibrosis. PMID:22110380

  8. Kupffer cells-dependent inflammation in the injured liver increases recruitment of mesenchymal stem cells in aging mice

    PubMed Central

    Zong, Chen; Lai, Fobao; Zhu, Pengxi; Liu, Yu; Jiang, Jinghua; Yang, Yang; Gao, Lu; Ye, Fei; Zhao, Qiudong; Li, Rong; Han, Zhipeng; Wei, Lixin

    2016-01-01

    Mesenchymal stem cells (MSCs) repair tissue injury and may be used to treat immune associated diseases. In carbon tetrachloride (CCl4)-induced liver injury murine model, we administered MSCs. When MSCs were transmitted to young and old mice with liver injury, more MSCs were recruited in old mice. In old mice, inflammation, characterized by TNF-α and IL-6, was increased due to hyper-activation and hyper-function of Kupffer cells. Blocking Kupffer cells decreased MSCs migration in old mice. In vitro, Kupffer cells isolated from old mice secreted more inflammatory cytokines and chemokines. Thus, hyper-activation of Kupffer cells in old mice increased recruitment of MSCs after their therapeutic administration. PMID:26716516

  9. The Kupffer Cell Number Affects the Outcome of Living Donor Liver Transplantation from Elderly Donors

    PubMed Central

    Hidaka, Masaaki; Eguchi, Susumu; Takatsuki, Mitsuhisa; Soyama, Akihiko; Ono, Shinichiro; Adachi, Tomohiko; Natsuda, Koji; Kugiyama, Tota; Hara, Takanobu; Okada, Satomi; Imamura, Hajime; Miuma, Satoshi; Miyaaki, Hisamitsu

    2016-01-01

    Background There have been no previous reports how Kupffer cells affect the outcome of living donor liver transplantation (LDLT) with an elderly donor. The aim of this study was to elucidate the influence of Kupffer cells on LDLT. Methods A total of 161 adult recipients underwent LDLT. The graft survival, prognostic factors for survival, and graft failure after LDLT were examined between cases with a young donor (<50, n = 112) and an elderly donor (≥50, N = 49). The Kupffer cells, represented by CD68-positive cell in the graft, were examined in the young and elderly donors. Results In a multivariable analysis, a donor older than 50 years, sepsis, and diabetes mellitus were significant predictors of graft failure after LDLT. The CD68 in younger donors was significantly more expressed than that in elderly donors. The group with a less number of CD68-positive cells in the graft had a significantly poor survival in the elderly donor group and prognostic factor for graft failure. Conclusions The worse outcome of LDLT with elderly donors might be related to the lower number of Kupffer cells in the graft, which can lead to impaired recovery of the liver function and may predispose patients to infectious diseases after LDLT. PMID:27819035

  10. Effect of allyl alcohol on hepatic transporter expression: Zonal patterns of expression and role of Kupffer cell function

    SciTech Connect

    Campion, Sarah N.; Tatis-Rios, Cristina; Augustine, Lisa M.; Goedken, Michael J.; Rooijen, Nico van; Cherrington, Nathan J.; Manautou, Jose E.

    2009-04-01

    During APAP toxicity, activation of Kupffer cells is critical for protection from hepatotoxicity and up-regulation of multidrug resistance-associated protein 4 (Mrp4) in centrilobular hepatocytes. The present study was performed to determine the expression profile of uptake and efflux transporters in mouse liver following treatment with allyl alcohol (AlOH), a periportal hepatotoxicant. This study also investigated the role of Kupffer cells in AlOH hepatotoxicity, and whether changes in transport protein expression by AlOH are dependent on the presence of Kupffer cells. C57BL/6J mice received 0.1 ml clodronate liposomes to deplete Kupffer cells or empty liposomes 48 h prior to dosing with 60 mg/kg AlOH, i.p. Hepatotoxicity was assessed by plasma ALT and histopathology. Hepatic transporter mRNA and protein expression were determined by branched DNA signal amplification assay and Western blotting, respectively. Depletion of Kupffer cells by liposomal clodronate treatment resulted in heightened susceptibility to AlOH toxicity. Exposure to AlOH increased mRNA levels of several Mrp genes, while decreasing organic anion transporting polypeptides (Oatps) mRNA expression. Protein analysis mirrored many of these mRNA changes. The presence of Kupffer cells was not required for the observed changes in uptake and efflux transporters induced by AlOH. Immunofluorescent analysis revealed enhanced Mrp4 staining exclusively in centrilobular hepatocytes of AlOH treated mice. These findings demonstrate that Kupffer cells are protective from AlOH toxicity and that induction of Mrp4 occurs in liver regions away from areas of AlOH damage independent of Kupffer cell function. These results suggest that Kupffer cell mediators do not play a role in mediating centrilobular Mrp4 induction in response to periportal damage by AlOH.

  11. Effects of zinc oxide nanoparticles on Kupffer cell phagosomal motility, bacterial clearance, and liver function

    PubMed Central

    Watson, Christa Y; Molina, Ramon M; Louzada, Andressa; Murdaugh, Kimberly M; Donaghey, Thomas C; Brain, Joseph D

    2015-01-01

    Background Zinc oxide engineered nanoparticles (ZnO ENPs) have potential as nanomedicines due to their inherent properties. Studies have described their pulmonary impact, but less is known about the consequences of ZnO ENP interactions with the liver. This study was designed to describe the effects of ZnO ENPs on the liver and Kupffer cells after intravenous (IV) administration. Materials and methods First, pharmacokinetic studies were conducted to determine the tissue distribution of neutron-activated 65ZnO ENPs post-IV injection in Wistar Han rats. Then, a noninvasive in vivo method to assess Kupffer cell phagosomal motility was employed using ferromagnetic iron particles and magnetometry. We also examined whether prior IV injection of ZnO ENPs altered Kupffer cell bactericidal activity on circulating Pseudomonas aeruginosa. Serum and liver tissues were collected to assess liver-injury biomarkers and histological changes, respectively. Results We found that the liver was the major site of initial uptake of 65ZnO ENPs. There was a time-dependent decrease in tissue levels of 65Zn in all organs examined, refecting particle dissolution. In vivo magnetometry showed a time-dependent and transient reduction in Kupffer cell phagosomal motility. Animals challenged with P. aeruginosa 24 hours post-ZnO ENP injection showed an initial (30 minutes) delay in vascular bacterial clearance. However, by 4 hours, IV-injected bacteria were cleared from the blood, liver, spleen, lungs, and kidneys. Seven days post-ZnO ENP injection, creatine phosphokinase and aspartate aminotransferase levels in serum were significantly increased. Histological evidence of hepatocyte damage and marginated neutrophils were observed in the liver. Conclusion Administration of ZnO ENPs transiently inhibited Kupffer cell phagosomal motility and later induced hepatocyte injury, but did not alter bacterial clearance from the blood or killing in the liver, spleen, lungs, or kidneys. Our data show that

  12. Radioprotective effect of kupffer cell depletion on hepatic sinusoidal endothelial cells.

    PubMed

    Chen, Yi-Xing; Zeng, Zhao-Chong; Sun, Jing; Zhang, Zhen-Yu; Zeng, Hai-Ying; Hu, Wei-Xu

    2015-05-01

    Radiation-induced liver injury remains a clinical problem and data suggest that sinusoidal endothelial cells (SECs) are an important target. The purpose of this study was to determine whether the inhibition of Kupffer cells before exposure would protect SECs from radiation-induced injury. Sprague-Dawley rats were intravenously injected 24 h before irradiation with Kupffer cell inhibitor gadolinium chloride (GdCl3) (10 mg/kg body weight). Three groups of animals were treated: 1. control group (saline and sham irradiation); 2. GdCl3 + 30 Gy radiation group and 3. 30 Gy radiation only group. Specimens were collected at 2, 6, 12, 24 and 48 h after completion of each treatment. Liver tissue was assessed for inflammatory cytokine expression and radiation-induced SEC injury based on serum hyaluronic acid (HA) level, apoptosis and ultrastructural and histological analyses. The results showed that radiation exposure caused apoptosis of SECs, but not hepatocytes. Inflammatory cytokine expression, including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) expression, was significantly attenuated in the GdCl3 + 30 Gy radiation group, compared with the 30 Gy radiation-only group (P < 0.05). The GdCl3 + radiation-treated rats exhibited significantly lower levels of HA and SEC apoptosis than the radiation-treated only rats at early time points, and radiation-induced liver injury was also attenuated. In conclusion, we hypothesize that selective Kupffer cell inhibition by gadolinium chloride was shown to reduce apoptosis in SECs caused by irradiation of the live and protected the liver against radiation-induced injury.

  13. HGF Secreted by Activated Kupffer Cells Induces Apoptosis of Plasmodium-Infected Hepatocytes

    PubMed Central

    Gonçalves, Lígia Antunes; Rodo, Joana; Rodrigues-Duarte, Lurdes; de Moraes, Luciana Vieira; Penha-Gonçalves, Carlos

    2017-01-01

    Malaria liver stage infection is an obligatory parasite development step and represents a population bottleneck in Plasmodium infections, providing an advantageous target for blocking parasite cycle progression. Parasite development inside hepatocytes implies a gross cellular insult evoking innate host responses to counteract intra-hepatocytic infection. Using primary hepatocyte cultures, we investigated the role of Kupffer cell-derived hepatocyte growth factor (HGF) in malaria liver stage infection. We found that Kupffer cells from Plasmodium-infected livers produced high levels of HGF, which trigger apoptosis of infected hepatocytes through a mitochondrial-independent apoptosis pathway. HGF action in infected hepatocyte primary cultures results in a potent reduction of parasite yield by specifically sensitizing hepatocytes carrying established parasite exo-erythrocytic forms to undergo apoptosis. This apoptosis mechanism is distinct from cell death that is spontaneously induced in infected cultures and is governed by Fas signaling modulation through a mitochondrial-dependent apoptosis pathway. This work indicates that HGF and Fas signaling pathways are part of an orchestrated host apoptosis response that occurs during malaria liver stage infection, decreasing the success of infection of individual hepatocytes. Our results raise the hypothesis that paracrine signals derived from Kupffer cell activation are implicated in directing death of hepatocytes infected with the malaria parasite. PMID:28220125

  14. Inactivation of Kupffer Cells by Gadolinium Chloride Protects Murine Liver From Radiation-Induced Apoptosis

    SciTech Connect

    Du Shisuo; Qiang Min; Zeng Zhaochong; Ke Aiwu; Ji Yuan; Zhang Zhengyu; Zeng Haiying; Liu Zhongshan

    2010-03-15

    Purpose: To determine whether the inhibition of Kupffer cells before radiotherapy (RT) would protect hepatocytes from radiation-induced apoptosis. Materials and Methods: A single 30-Gy fraction was administered to the upper abdomen of Sprague-Dawley rats. The Kupffer cell inhibitor gadolinium chloride (GdCl3; 10 mg/kg body weight) was intravenously injected 24 h before RT. The rats were divided into four groups: group 1, sham RT plus saline (control group); group 2, sham RT plus GdCl3; group 3, RT plus saline; and group 4, RT plus GdCl3. Liver tissue was collected for measurement of apoptotic cytokine expression and evaluation of radiation-induced liver toxicity by analysis of liver enzyme activities, hepatocyte micronucleus formation, apoptosis, and histologic staining. Results: The expression of interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha was significantly attenuated in group 4 compared with group 3 at 2, 6, 24, and 48 h after injection (p <0.05). At early points after RT, the rats in group 4 exhibited significantly lower levels of liver enzyme activity, apoptotic response, and hepatocyte micronucleus formation compared with those in group 3. Conclusion: Selective inactivation of Kupffer cells with GdCl3 reduced radiation-induced cytokine production and protected the liver against acute radiation-induced damage.

  15. Hyaline droplets in Kupffer cells: a novel diagnostic clue for autoimmune hepatitis.

    PubMed

    Tucker, Suzanne M; Jonas, Maureen M; Perez-Atayde, Antonio R

    2015-06-01

    Pediatric autoimmune hepatitis (AIH) is relatively common and has a characteristic but relatively nonspecific histopathology with a usually prominent lymphoplasmacytic infiltrate. Herein, we describe for the first time the presence of characteristic hyaline droplets in the cytoplasm of Kupffer cells on routine hematoxylin and eosin (H&E) sections in AIH. The medical records and pathologic material over a 20-year period (1992 to 2012) were reviewed from children with AIH (n=30), hepatitis B virus (n=30), and hepatitis C virus (n=30) from the pathology files at Boston Children's Hospital. All children had percutaneous needle liver biopsies. We reviewed sections stained with H&E, PAS, and PAS with diastase for the presence of hyaline droplets in all 90 biopsies. We also performed immunohistochemical analysis for IgG, IgA, and IgD in 6 biopsies with AIH. Hyaline droplets were identified in Kupffer cells throughout the lobules in 15 of 30 biopsies (easily found in 13 and rare in 2); conversely, no droplets were identified in 15. Droplets were identified in 10 AIH type 1 biopsies, 1 in AIH type 2, 3 in overlap syndrome, and 1 in unclassified. Serum IgG levels, when available, were correlated with biopsy findings. Seventeen patients had serum IgG levels available for review. The average IgG level in patients without droplets in their biopsies was 1364 mg/dL, in contrast to 3424 mg/dL in patients with droplets (P=0.021). Immunohistochemical analysis performed in 6 biopsies revealed that droplets were nearly always positive for IgG, occasionally for IgA, and rarely for IgD. None of the biopsies in patients with hepatitis C contained hyaline droplets. One biopsy of a patient with hepatitis B revealed hyaline droplets; this biopsy had an unusually prominent plasmacytic infiltrate, and the patient was found to have an elevated IgG serum level and antibodies to smooth muscle actin. As far as we are aware, hyaline droplets in Kupffer cells on routine H&E sections have never been

  16. 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

  17. Kupffer cell proliferation and glucan-induced granuloma formation in mice depleted of blood monocytes by strontium-89

    SciTech Connect

    Yamada, M.; Naito, M.; Takahashi, K. )

    1990-03-01

    In mice with prolonged severe monocytopenia induced by selective irradiation of the bone marrow with the bone-seeking isotope 89Sr, the proliferative capacity of Kupffer cells was studied by immunohistochemistry with an anti-mouse macrophage monoclonal antibody, F4/80, ultrastructural peroxidase (PO) cytochemistry, and tritiated thymidine (3HTdR) autoradiography. The number and 3HTdR uptake of Kupffer cells were significantly increased in the splenectomized mice after severe monocytopenia had continued for more than 4 wk, and almost all the Kupffer cells showed a localization pattern of PO activity similar to that of resident macrophages in the liver of normal mice. In the glucan-induced granuloma formation in similar monocytopenic mice, Kupffer cells proliferated, conglomerated, and transformed into epithelioid cells, which fused together to become multinuclear giant cells. These results suggest that Kupffer cells are a self-renewing population by their own cell division and can participate actively in granulomatous inflammations in severely monocytopenic and intact mice.

  18. Kupffer cells induce Notch-mediated hepatocyte conversion in a common mouse model of intrahepatic cholangiocarcinoma

    PubMed Central

    Terada, Maiko; Horisawa, Kenichi; Miura, Shizuka; Takashima, Yasuo; Ohkawa, Yasuyuki; Sekiya, Sayaka; Matsuda-Ito, Kanae; Suzuki, Atsushi

    2016-01-01

    Intrahepatic cholangiocarcinoma (ICC) is a malignant epithelial neoplasm composed of cells resembling cholangiocytes that line the intrahepatic bile ducts in portal areas of the hepatic lobule. Although ICC has been defined as a tumor arising from cholangiocyte transformation, recent evidence from genetic lineage-tracing experiments has indicated that hepatocytes can be a cellular origin of ICC by directly changing their fate to that of biliary lineage cells. Notch signaling has been identified as an essential factor for hepatocyte conversion into biliary lineage cells at the onset of ICC. However, the mechanisms underlying Notch signal activation in hepatocytes remain unclear. Here, using a mouse model of ICC, we found that hepatic macrophages called Kupffer cells transiently congregate around the central veins in the liver and express the Notch ligand Jagged-1 coincident with Notch activation in pericentral hepatocytes. Depletion of Kupffer cells prevents the Notch-mediated cell-fate conversion of hepatocytes to biliary lineage cells, inducing hepatocyte apoptosis and increasing mortality in mice. These findings will be useful for uncovering the pathogenic mechanism of ICC and developing prevenient and therapeutic strategies for this refractory disease. PMID:27698452

  19. Accumulation and metabolism of iron-dextran by hepatocytes, Kupffer cells and endothelial cells in the neonatal pig liver.

    PubMed

    Caperna, T J; Failla, M L; Steele, N C; Richards, M P

    1987-02-01

    Treatment of newborn pigs with supplemental iron is a common procedure utilized to prevent neonatal anemia. The aim of this study was to investigate the hepatic distribution and intracellular metabolism of iron-dextran, a widely used colloidal-iron-carbohydrate preparation. Piglets were injected intramuscularly with iron-dextran (50 mg Fe/kg body wt) at 1 d of age. Hepatocytes and sinusoidal cells (Kupffer cells and endothelial cells) were isolated from iron-treated and control (uninjected) piglets at 2, 6 and 11 d of age. The concentrations of iron, copper and zinc in isolated cells were determined by atomic-absorption spectroscopy. In addition, the quantities of ferritin-protein and ferritin-iron were measured by immunoelectrophoresis and ion-exchange chromatography, respectively. At 2 d of age, the concentration (microgram/mg cell protein) of iron was 5-, 62- and 54-fold higher in hepatocytes, Kupffer cells and endothelial cells, respectively, isolated from iron-treated piglets than from control piglets. Hepatocytes, Kupffer cells and endothelial cells accumulated ferritin in response to iron-dextran treatment. Higher concentrations of ferritin-protein and ferritin-iron were present in Kupffer cells and endothelial cells than in hepatocytes at all times after treatment with iron-dextran. The percentage of cellular iron that was associated with ferritin, however, was greater in hepatocytes than in sinusoidal cells. Iron accumulated by all three liver cell types was mobilized to extrahepatic sites. Slight alterations in zinc and copper status of liver cells were evident at 11 d of age as a result of iron treatment.

  20. Susceptibility of chicken Kupffer cells to Chinese virulent infectious bursal disease virus.

    PubMed

    Ma, Haiyan; Zhao, Sufen; Ma, Yunfei; Guo, Xin; Han, Deping; Jia, Yuanyuan; Zhang, Weiwei; Teng, Kedao

    2013-06-28

    Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive avian disease caused by IBD virus (IBDV). Although the effects of IBDV on bursa of Fabricius in chickens have been well reported, the impacts of IBDV on liver after IBDV infection are still unclear. In the present study, specific pathogen free (SPF) chickens were experimentally inoculated with IBDV Chinese virulent strain BC6/85, and the cells in liver and bursa were examined by immunohistochemistry and transmission electron microscopy (TEM). The congestion of liver tissue and fatty degeneration of hepatocytes were characteristics of microscopical changes in chicken liver at 3 days post infection (d.p.i.), whereas there were follicular lymphoid necrosis, apoptosis, depletion, as well as edema and congestion in bursa. In addition, the number of IBDV-positive cells peaked at 4 d.p.i. in bursa and at 3 d.p.i. in liver, respectively. With respect to ultrastructural pathological changes of hepatocytes, mitochondria swelled and nucleus deformed into an irregular shape or its chromatin peripherally condensed which indicated that the hepatocyte was at the early stage of apoptosis, and the electron-lucent lipid droplets in a variety of sizes were observed within cytoplasm. Kupffer cells became "swollen-like" and the electron-density of their cytoplasm was lower than that of cells in uninfected group. Liver glycogen deposits significantly declined from 2 to 5 d.p.i. and recovered strongly at 6 d.p.i. More importantly, KLU01 (macrophage marker) positive (KUL01(+)) cells were infiltrated in bursa and liver in IBDV-exposed chickens by immunoperoxidase staining. To demonstrate the correlation between IBDV and macrophages in bursa and liver, we further investigated the colocalization of viral antigens and macrophages by double immunofluorescence labeling. At 4 d.p.i., the percentage of double positive cells (IBDV positive and KUL01(+) cells) accounted for 26.5 percent of the total IBDV positive

  1. Inhibition of allogeneic T-cell response by Kupffer cells expressing indoleamine 2,3-dioxygenase

    PubMed Central

    Yan, Mao-Lin; Wang, Yao-Dong; Tian, Yi-Feng; Lai, Zhi-De; Yan, Lv-Nan

    2010-01-01

    AIM: To explore the possibility and mechanism of inhibiting allogeneic T-cell responses by Kupffer cells (KC) pretreated with interferon-γ (IFN-γ) in vitro. METHODS: The expressions of indoleamine 2,3-dioxygenase (IDO) mRNA and FasL mRNA in KC pretreated with IFN-γ were studied with real-time polymerase chain reaction (PCR). The catabolism of tryptophan by IDO from KC was analyzed by high performance liquid chromatography. Allogeneic T-cell response was used to confirm the inhibition of KC in vitro. The proliferation of lymphocytes was detected using [3H] thymidine incorporation. Cell cycle and lymphocyte apoptosis were evaluated by flow cytometric assay. RESULTS: Real-time PCR revealed IDO mRNA and FasL mRNA expressions in KC pretreated with IFN-γ, and IDO catabolic effect was confirmed by a decrease in tryptophan and increase in kynurenine concentration. KC expressing IDO and FasL in BABL/c mice acquired the ability to suppress the proliferation of T-cells from C57BL/6, which could be blocked by addition of 1-methyl-tryptophan and anti-FasL antibody. KC expressing IDO could induce allogeneic T-cell apoptosis. CONCLUSION: In addition to Fas/FasL pathway, IDO may be another mechanism for KC to induce immune tolerance. PMID:20128035

  2. Steric stabilization of microspheres with grafted polyethylene oxide reduces phagocytosis by rat Kupffer cells in vitro.

    PubMed

    Harper, G R; Davies, M C; Davis, S S; Tadros, T F; Taylor, D C; Irving, M P; Waters, J A

    1991-09-01

    Sterically stabilized polyethylene oxide-polystyrene copolymer microspheres, (PS-PEO) and charge stabilized polystyrene (PS) microspheres of similar size (1 micron) were prepared in order to compare their uptake by cultured rat Kupffer cells isolated by centrifugal elutriation. The uptake of the sterically stabilized particles was found to be much less than that for the charge stabilized control. The uptake of microspheres stabilized with covalently grafted PEO was lower or equivalent to that of control microspheres stabilized by the adsorption of the non-ionic PEO-polypropylene oxide (PPO-PEO) surfactant Poloxamer 238 or Methoxy-PEO. Phagocytic uptake by Kupffer cells at low and body temperature (8 degrees C and 37 degrees C) demonstrated that PS-PEO particles showed both low adherence and low metabolic uptake. The adsorption of PEO, as Poloxamer 238, to particles with covalently attached or grafted PEO resulted in a synergistic reduction in uptake that was greater than the individual effects of grafting and adsorption alone (P less than or equal to 0.001). It is suggested that this combination produces a more effective steric barrier on the particle surface with the Poloxamer adsorbing to the surface between the grafted PEO chains. The relevance to drug targeting/carrier systems is discussed.

  3. Bone marrow-derived monocytes give rise to self-renewing and fully differentiated Kupffer cells

    PubMed Central

    Scott, Charlotte L.; Zheng, Fang; De Baetselier, Patrick; Martens, Liesbet; Saeys, Yvan; De Prijck, Sofie; Lippens, Saskia; Abels, Chloé; Schoonooghe, Steve; Raes, Geert; Devoogdt, Nick; Lambrecht, Bart N.; Beschin, Alain; Guilliams, Martin

    2016-01-01

    Self-renewing tissue-resident macrophages are thought to be exclusively derived from embryonic progenitors. However, whether circulating monocytes can also give rise to such macrophages has not been formally investigated. Here we use a new model of diphtheria toxin-mediated depletion of liver-resident Kupffer cells to generate niche availability and show that circulating monocytes engraft in the liver, gradually adopt the transcriptional profile of their depleted counterparts and become long-lived self-renewing cells. Underlining the physiological relevance of our findings, circulating monocytes also contribute to the expanding pool of macrophages in the liver shortly after birth, when macrophage niches become available during normal organ growth. Thus, like embryonic precursors, monocytes can and do give rise to self-renewing tissue-resident macrophages if the niche is available to them. PMID:26813785

  4. Critical Role of Kupffer Cell CD89 Expression in Experimental IgA Nephropathy

    PubMed Central

    Xu, Lijun; Li, Bingyu; Huang, Mengwen; Xie, Kun; Li, Dong; Li, You; Gu, Hua; Fang, Jianmin

    2016-01-01

    Although IgA nephropathy (IgAN) is the most common primary glomerulonephritis worldwide, its etiology remains only partly understood. It is clear that the pathogenesis of IgAN involves the formation of macromolecular IgA1 complexes and increased levels of serum IgA1 and IgA1-immune complexes(IC), due to defective IgA1 clearance. Previous studies suggest that the blood and tissue myeloid cell-expressed IgA Fc receptor (FcαR/CD89) mediates IgA-IC clearance and its dysfunction, via decreased activity or excessive levels of soluble FcαR/sCD89 induces IgAN. Such a mechanism requires robust stimulation of IgAN levels via forced expression of CD89. In the absence of unequivocal evidence supporting such a mechanism to date, we attempted to test the extent of CD89-evoked IgAN by generating a transgenic mouse strain expressing human CD89 under the control of murine CD14 promotor. No deposition of IgA-CD89 complexes or glomerulonephritis was detected, however. Further studies showed that elimination of murine IgA was mediated by Kupffer cells. In patients, however, CD89/IgA complexes were detected, and injection of patient IgA induced IgAN-like features in CD89 Tg mice. In transgenic mice, IgAN pathogenesis involves impaired clearance of abnormal IgA via CD89, primarily by the Kupffer cells. Conditional IgAN progression in CD89 transgenic mice thus reveals important aspects of IgAN pathogenesis. PMID:27437939

  5. Role of Kupffer cells in failure of fatty livers following liver transplantation and alcoholic liver injury.

    PubMed

    Thurman, R G; Gao, W; Connor, H D; Adachi, Y; Stachlewitz, R F; Zhong, Z; Knecht, K T; Bradford, B U; Mason, R P; Lemasters, J J

    1995-01-01

    Kupffer cells have been implicated in mechanisms of pathophysiology following liver transplantation. Recently, postoperative injury in ethanol-induced fatty liver has been evaluated because fatty livers often fail following transplantation. The low-flow, reflow liver perfusion model was used to study the role of Kupffer cells (KC) in reperfusion injury to fatty livers from rats fed a diet containing ethanol for 4-5 weeks. Treatment with GdCl3, which selectively destroys KC, decreased cell death significantly. Thus, destruction of KC minimized hepatic reperfusion injury, most likely by inhibiting free radical formation and improving microcirculation. Since it was demonstrated recently that destruction of KC prevented the hypermetabolic state observed with acute alcohol exposure, their involvement in events leading to alcohol-induced liver disease was investigated. In rats exposed to ethanol continuously via intragastric feeding for up to 4 weeks, GdCl3 treatment prevented elevation of aspartate aminotransferase (AST) and dramatically reduced the average hepatic pathological score. These results indicate that KC participate in the early phases of alcohol-induced liver injury. Endotoxaemia occurs in alcoholics and activates KC; therefore, we evaluated the effect of minimizing bacterial endotoxin by intestinal sterilization with the antibiotics polymyxin B and neomycin. Antibiotics diminished plasma endotoxin levels significantly and prevented ethanol-induced increases in AST values. These results indicate that endotoxin is involved in the mechanism of ethanol-induced liver injury. A six-line radical spectrum was detected with electron paramagnetic resonance spectroscopy in bile from alcohol-treated rats which was blocked by GdCl3. The free radical adducts had hyperfine coupling constants characteristic of lipid-derived free radical products. In conclusion, these studies demonstrate that KC are involved in reperfusion injury to ethanol-induced fatty livers and hepatic

  6. Biosynthesis of platelet-activating factor by cultured rat Kupffer cells stimulated with calcium ionophore A23187.

    PubMed Central

    Chao, W; Siafaka-Kapadai, A; Olson, M S; Hanahan, D J

    1989-01-01

    Cultured rat Kupffer cells synthesize and release platelet-activating factor (PAF) when stimulated with calcium ionophore A23187. The production of PAF is concentration- and time-dependent and, based upon [3H]serotonin release assays, approx. 1.0 pmol of PAF is formed per 8 x 10(6) cells during 10 min of ionophore stimulation. It is suggested that Kupffer cells are important cellular components which produce and release PAF in order to facilitate communication between hepatic sinusoidal and parenchymal cells. Further, it is suggested that such mediator production in response to reticulo-endothelial cell stimulation causes the hepatic glycogenolytic response previously in the isolated perfused rat liver. PMID:2494988

  7. Rat liver endothelial and Kupffer cell-mediated mutagenicity and polycyclic aromatic hydrocarbons and aflatoxin B sub 1

    SciTech Connect

    Steinberg, P.; Schlemper, B.; Molitor, E.; Platt, K.L.; Seidel, A.; Oesch, F. )

    1990-08-01

    The ability of isolated rat liver endothelial and Kupffer cells to activate benzo(a)pyrene (BP), trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene (DDBP), trans-1,2-dihydroxy-1,2-dihydrochrysene (DDCH), and aflatoxin B{sub 1} (AFB{sub 1}) to mutagenic metabolites was assessed by means of a cell-mediated bacterial mutagenicity assay and compared with the ability of parenchymal cells to activate these compounds. Endothelial and Kupffer cells from untreated rats were able to activate AFB{sub 1} and DDBP; DDBP was activated even in the absence of an NADPH-generating system. Pretreating the animals with Aroclor 1254 strongly enhanced the mutagenicity of the dihydrodiol, whereas the mutagenicity of AFB{sub 1} showed a slight increase. BP and DDCH were only activated by endothelial and Kupffer cells isolated from Aroclor 1254-pretreated rats. Parenchymal cells form untreated animals activated all four carcinogens tested; Aroclor 1254 enhanced the parenchymal cell-mediated mutagenicity of BP and DDCH but did not affect that of DDBP and clearly reduced that of AFB{sub 1}. The reduced mutagenicity of AFB{sub 1} correlates with the decrease in the amount of 2{alpha}-hydroxytestosterone formed when testosterone was incubated with parenchymal cell microsomes from Aroclor 1254-pretreated rats (compared with microsomes from untreated animals): the formation of 2{alpha}-hydroxytestosterone is specifically catalyzed by cytochrome P-450h, a hemoprotein thought to be involved in the activation of AFB{sub 1}. These results show that not only rat liver parenchymal cells, but also endothelial and Kupffer cells, activated several carcinogens to mutagenic metabolites.

  8. Graptopetalum Paraguayense Ameliorates Chemical-Induced Rat Hepatic Fibrosis In Vivo and Inactivates Stellate Cells and Kupffer Cells In Vitro

    PubMed Central

    Su, Li-Jen; Chang, Chia-Chuan; Yang, Chih-Hsueh; Hsieh, Shur-Jong; Wu, Yi-Chin; Lai, Jin-Mei; Tseng, Tzu-Ling; Huang, Chi-Ying F.; Hsu, Shih-Lan

    2013-01-01

    Background Graptopetalum paraguayense (GP) is a folk herbal medicine with hepatoprotective effects that is used in Taiwan. The aim of this study was to evaluate the hepatoprotective and antifibrotic effects of GP on experimental hepatic fibrosis in both dimethylnitrosamine (DMN)- and carbon tetrachloride (CCl4)-induced liver injury rats. Methods Hepatic fibrosis-induced rats were fed with the methanolic extract of GP (MGP) by oral administration every day. Immunohistochemistry, biochemical assays, and Western blot analysis were performed. The effects of MGP on the expression of fibrotic markers and cytokines in the primary cultured hepatic stellate cells (HSCs) and Kupffer cells, respectively, were evaluated. Results Oral administration of MGP significantly alleviated DMN- or CCl4-induced liver inflammation and fibrosis. High levels of alanine transaminase, aspartate transaminase, bilirubin, prothrombin activity and mortality rates also decreased in rats treated with MGP. There were significantly decreased hydroxyproline levels in therapeutic rats compared with those of the liver-damaged rats. Collagen I and alpha smooth muscle actin (α-SMA) expression were all reduced by incubation with MGP in primary cultured rat HSCs. Furthermore, MGP induced apoptotic cell death in activated HSCs. MGP also suppressed lipopolysaccharide-stimulated rat Kupffer cell activation by decreasing nitric oxide, tumor necrosis factor-α and interleukin-6 production, and increasing interleukin-10 expression. Conclusions The results show that the administration of MGP attenuated toxin-induced hepatic damage and fibrosis in vivo and inhibited HSC and Kupffer cell activation in vitro, suggesting that MGP might be a promising complementary or alternative therapeutic agent for liver inflammation and fibrosis. PMID:23335984

  9. Inflammatory conditions induce gap junctional communication between rat Kupffer cells both in vivo and in vitro

    PubMed Central

    Eugenín, Eliseo A.; González, Hernán E.; Sánchez, Helmuth A.; Brañes, María C.; Sáez, Juan C.

    2007-01-01

    Connexin43 (Cx43), a gap junction protein subunit, has been previously detected in Kupffer cells (KCs) during liver inflammation, however, KCs phagocytose cell debris that may include Cx43 protein, which could explain the detection of Cx43 in KCs. We determined that KCs express Cx43 and form gap junctions both in vivo and in vitro. In liver sections of animals treated with LPS, Cx43 was detected at ED2+ cells interfaces, indicating formation of GJ between KCs in vivo. In vitro, unstimulated KCs cultures did not form functional GJs, and expressed low levels of Cx43 that showed a diffuse intracellular distribution. In contrast, KCs treated with LPS plus IFN-γ, expressed a greater amount of Cx43 at both the, protein and mRNA levels, and showed Cx43 at cell-cell contacts associated with higher dye coupling. In conclusion, activation of KCs in vivo or in vitro resulted in enhanced Cx43 expression levels and formation of GJ that might play relevant roles during liver inflammation. PMID:17900549

  10. Kupffer cell blockade prevents induction of portal venous tolerance in rat cardiac allograft transplantation

    SciTech Connect

    Kamei, T.; Callery, M.P.; Flye, M.W. )

    1990-05-01

    Pretransplant portal venous (pv) administration of donor antigen induces allospecific partial tolerance. Although the involved mechanism has not been defined, antigen presentation by Kupffer cells (KC) in the liver is considered to be critical. We evaluated the effect of KC blockade on this pv tolerance induction in Buffalo (RT1b) rats receiving Lewis (RT1(1)) cardiac heterotopic allografts. Control rats received no treatment, while experimental animals received 25 X 10(6) ultraviolet B-irradiated (12,000 J/m2) donor spleen cells via either the iv (systemic intravenous) or the pv routes 7 days before transplantation. Gadolinium chloride (GdCl3), a rare earth metal known to inhibit KC phagocytosis, was given (7 mg/kg) 1 and 2 days before pv preimmunization. Cardiac graft prolongation was obtained by pv (MST = 13.3 +/- 1.9 days, n = 6, vs control = 7.3 +/- 0.5 days, n = 6; P less than 0.001) but not by iv preimmunization (7.7 +/- 0.7 days, n = 6, NS vs control). KC blockade abolished the pv tolerance, as indicated by abrogation of graft prolongation (PV + GdCl3 = 8.0 +/- 0.8 days, n = 6, NS vs control). These findings suggest that effective alloantigen uptake by KC in the liver is essential for the induction of pv tolerance in rat cardiac transplantation.

  11. Liver Monocytes and Kupffer Cells Remain Transcriptionally Distinct during Chronic Viral Infection

    PubMed Central

    van de Garde, Martijn D. B.; Movita, Dowty; van der Heide, Marieke; Herschke, Florence; De Jonghe, Sandra; Gama, Lucio; Boonstra, Andre

    2016-01-01

    Due to the scarcity of immunocompetent animal models for chronic viral hepatitis, little is known about the role of the innate intrahepatic immune system during viral replication in the liver. These insights are however fundamental for the understanding of the inappropriate adaptive immune responses during the chronic phase of the infection. We apply the Lymphocytic Choriomenigitis Virus (LCMV) clone 13 mouse model to examine chronic virus-host interactions of Kupffer cells (KC) and infiltrating monocytes (IM) in an infected liver. LCMV infection induced overt clinical hepatitis, with rise in ALT and serum cytokines, and increased intrahepatic F4/80 expression. Despite ongoing viral replication, whole liver transcriptome showed baseline expression levels of inflammatory cytokines, interferons, and interferon induced genes during the chronic infection phase. Transcriptome analyses of sorted KC and IMs using NanoString technology revealed two unique phenotypes with only minimal overlap. At the chronic viral infection phase, KC showed no increased transcription of activation markers Cd80 and Cd86, but an increased expression of genes related to antigen presentation, whereas monocytes were more activated and expressed higher levels of Tnf transcripts. Although both KCs and intrahepatic IM share the surface markers F4/80 and CD11b, their transcriptomes point towards distinctive roles during virus-induced chronic hepatitis. PMID:27812182

  12. A new Kupffer cell receptor mediating plasma clearance of carcinoembryonic antigen by the rat.

    PubMed Central

    Toth, C A; Thomas, P; Broitman, S A; Zamcheck, N

    1982-01-01

    Native human carcinoembryonic antigen is rapidly removed from the circulation by the rat liver Kupffer cell after intravenous injection. The molecule is subsequently transferred to the hepatocyte in an immunologically identifiable form. Carcinoembryonic antigen has a circulatory half-life of 3.7 (+/- 0.8) min, and cellular entry is by receptor-mediated endocytosis. Non-specific fluid pinocytosis and phagocytosis can be excluded as possible mechanisms by the kinetics of clearance and failure of colloidal carbon to inhibit uptake. Substances with known affinity for the hepatic receptors for mannose, N-acetylglucosamine, fucose and galactose all fail to inhibit carcinoembryonic antigen clearance. After two cycles of the Smith degradation, carcinoembryonic antigen is still able to inhibit clearance of the native molecule. Receptor specificity is apparently not dependent on those non-reducing terminal sugars of the native molecule. Performic acid-oxidized carcinoembryonic antigen also inhibits clearance of carcinoembryonic antigen in vivo. Receptor binding is not dependent on tertiary protein conformation. Non-specific cross-reacting antigen, a glycoprotein structurally similar to carcinoembryonic antigen, is cleared by the same mechanism. PMID:6896821

  13. A new Kupffer cell receptor mediating plasma clearance of carcinoembryonic antigen by the rat.

    PubMed

    Toth, C A; Thomas, P; Broitman, S A; Zamcheck, N

    1982-05-15

    Native human carcinoembryonic antigen is rapidly removed from the circulation by the rat liver Kupffer cell after intravenous injection. The molecule is subsequently transferred to the hepatocyte in an immunologically identifiable form. Carcinoembryonic antigen has a circulatory half-life of 3.7 (+/- 0.8) min, and cellular entry is by receptor-mediated endocytosis. Non-specific fluid pinocytosis and phagocytosis can be excluded as possible mechanisms by the kinetics of clearance and failure of colloidal carbon to inhibit uptake. Substances with known affinity for the hepatic receptors for mannose, N-acetylglucosamine, fucose and galactose all fail to inhibit carcinoembryonic antigen clearance. After two cycles of the Smith degradation, carcinoembryonic antigen is still able to inhibit clearance of the native molecule. Receptor specificity is apparently not dependent on those non-reducing terminal sugars of the native molecule. Performic acid-oxidized carcinoembryonic antigen also inhibits clearance of carcinoembryonic antigen in vivo. Receptor binding is not dependent on tertiary protein conformation. Non-specific cross-reacting antigen, a glycoprotein structurally similar to carcinoembryonic antigen, is cleared by the same mechanism.

  14. Targeting dexamethasone to Kupffer cells: effects on liver inflammation and fibrosis in rats.

    PubMed

    Melgert, B N; Olinga, P; Van Der Laan, J M; Weert, B; Cho, J; Schuppan, D; Groothuis, G M; Meijer, D K; Poelstra, K

    2001-10-01

    Kupffer cells (KC) play an important role in the pathogenesis of inflammatory liver diseases leading to fibrosis. Anti-inflammatory drugs are only effective when administered at high doses that may cause side effects. Therefore, dexamethasone coupled to mannosylated albumin (Dexa(5)-Man(10)-HSA) was designed by us to selectively deliver this anti-inflammatory drug to the KC. The effectiveness of Dexa(5)-Man(10)-HSA was studied both in organ cultures and fibrosis induced by bile duct ligation (BDL) in rats. Dexa(5)-Man(10)-HSA accumulated in livers of both healthy and fibrotic rats (67% +/- 5% and 70% +/- 9% of the dose, respectively) and uptake was found almost exclusively in KC. Active dexamethasone was liberated from its carrier, because Dexa(5)-Man(10)-HSA could effectively inhibit nitric oxide (NO) and tumor necrosis factor alpha (TNF-alpha) release in endotoxin-activated liver slices. In vivo, however, this was associated with increased collagen I and III depositions and enhanced tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA expression. This was accompanied by a decreased influx of reactive oxygen species (ROS) producing cells in the livers of BDL animals treated with Dexa(5)-Man(10)-HSA as compared with untreated BDL rats. Dexa(5)-Man(10)-HSA treatment also replenished the depleted glycogen stores in hepatocytes of BDL livers. In conclusion, our studies showed selective delivery of dexamethasone to KC with Dexa(5)-Man(10)-HSA. This conjugate reduced intrahepatic ROS in vivo and TNF-alpha production in vitro and prevented glycogen depletion in vivo, indicating effective pharmacologic targeting. Dexa(5)-Man(10)-HSA, however, also accelerated fibrogenesis, which was paralleled by TIMP-1 mRNA induction. Targeting of dexamethasone to KC provides evidence for a dual role of this cell type in fibrogenesis of BDL rats.

  15. Fc receptor mediated endocytosis of small soluble immunoglobulin G immune complexes in Kupffer and endothelial cells from rat liver.

    PubMed

    Løvdal, T; Andersen, E; Brech, A; Berg, T

    2000-09-01

    Soluble circulating immunoglobulin G immune complexes are mainly eliminated by the liver, predominantly by uptake in the Kupffer cells, but also the liver endothelial cells seem to be of importance. In the present study we have followed the intracellular turnover of immune complexes after Fc(gamma) receptor mediated endocytosis in cultured rat liver endothelial cells and Kupffer cells by means of isopycnic centrifugation, DAB cross-linking and morphological techniques. For the biochemical experiments the antigen, dinitrophenylated bovine serum albumin (BSA), was labeled with radioiodinated tyramine cellobiose that cannot cross biological membranes and therefore traps labeled degradation products at the site of formation. The endocytic pathway followed by immune complexes was compared with that followed by scavenger receptor ligands, such as formaldehyde treated BSA and dinitrophenylated BSA, and the mannose receptor ligand ovalbumin. Both Kupffer cells and liver endothelial cells took up and degraded the immune complexes, but there was a clear delay in the degradation of immune complexes as compared to degradation of ligands taken up via scavenger receptors. The kinetics of the endocytosis of scavenger receptor ligand was unaffected by simultaneous uptake of immune complexes. Experiments using both biochemical and morphological techniques indicated that the delayed degradation was due to a late arrival of the immune complexes at the lysosomes, which partly was explained by retroendocytosis of immune complexes. Electron microscopy studies revealed that the immune complexes were retained in the early endosomes that remained accessible to other endocytic markers such as ovalbumin. In addition, the immune complexes were seen in multivesicular compartments apparently devoid of other endocytic markers. Finally, the immune complexes were degraded in the same lysosomes as the ligands of scavenger receptors. Thus, immune complexes seem to follow an endocytic pathway that is

  16. Vanillin suppresses Kupffer cell-related colloidal carbon-induced respiratory burst activity in isolated perfused rat liver: anti-inflammatory implications.

    PubMed

    Galgani, José E; Núñez, Bárbara; Videla, Luis A

    2012-12-01

    The inhibition of NADPH oxidase has become a potential therapeutic target for oxidative stress-related diseases. We investigated whether vanillin modifies hepatic O(2) consumption associated with Kupffer cell functioning. The influence of vanillin on Kupffer cell functioning was studied in isolated perfused rat liver by colloidal carbon (CC) infusion (0.5 mg ml(-1)), concomitantly with sinusoidal efflux of lactate dehydrogenase (LDH) as an organ viability parameter. CC infusion increased the rate of O(2) consumption of the liver above basal values, an effect that represents the respiratory burst activity of Kupffer cells. However, CC-dependent respiratory burst activity was suppressed by previous infusion of 2 mM vanillin. Vanillin did not affect the liver CC uptake rate and liver sinusoidal efflux of LDH efflux. These findings, elicited by vanillin, were reproduced by the well-established NADPH oxidase inhibitor apocynin. In conclusion, vanillin suppresses the respiratory burst activity of Kupffer cells as assessed in intact liver, which may be associated with the inhibition of macrophage NADPH oxidase activity. Such a finding may have relevance in conditions underlying Kupffer cell-dependent up-regulation of the expression and release of pro-inflammatory mediators by redox-dependent mechanisms.

  17. Activation of Kupffer Cells Is Associated with a Specific Dysbiosis Induced by Fructose or High Fat Diet in Mice.

    PubMed

    Ferrere, Gladys; Leroux, Anne; Wrzosek, Laura; Puchois, Virginie; Gaudin, Françoise; Ciocan, Dragos; Renoud, Marie-Laure; Naveau, Sylvie; Perlemuter, Gabriel; Cassard, Anne-Marie

    2016-01-01

    The increase consumption of fructose in diet is associated with liver inflammation. As a specific fructan substrate, fructose may modify the gut microbiota which is involved in obesity-induced liver disease. Here, we aimed to assess whether fructose-induced liver damage was associated with a specific dysbiosis, especially in mice fed a high fat diet (HFD). To this end, four groups of mice were fed with normal and HFD added or not with fructose. Body weight and glucose sensitivity, liver inflammation, dysbiosis and the phenotype of Kupffer cells were determined after 16 weeks of diet. Food intake was increased in the two groups of mice fed with the HFD. Mice fed with HFD and fructose showed a higher infiltration of lymphocytes into the liver and a lower inflammatory profile of Kupffer cells than mice fed with the HFD without fructose. The dysbiosis associated with diets showed that fructose specifically prevented the decrease of Mouse intestinal bacteria in HFD fed mice and increased Erysipelotrichi in mice fed with fructose, independently of the amount of fat. In conclusion, fructose, used as a sweetener, induced a dysbiosis which is different in presence of fat in the diet. Consequently, the activation of Kupffer cells involved in mice model of HFD-induced liver inflammation was not observed in an HFD/fructose combined diet. These data highlight that the complexity of diet composition could highly impact the development of liver lesions during obesity. Specific dysbiosis associated with the diet could explain that the progressions of liver damage are different.

  18. Kupffer Cell Activation by Ambient Air Particulate Matter Exposure May Exacerbate Non-alcoholic Fatty Liver Disease

    PubMed Central

    Tan, Hui-Hui; Fiel, M. Isabel; Sun, Qinghua; Guo, Jinsheng; Gordon, Ronald E.; Chen, Lung-Chi; Friedman, Scott L.; Odin, Joseph A.; Allina, Jorge

    2009-01-01

    Due to increased obesity, non-alcoholic fatty liver disease (NAFLD) is now the most prevalent liver disease in the United States. NAFLD is considered a component of metabolic syndrome, a cluster of disorders that also includes diabetes mellitus, dyslipidemia, arteriosclerosis, and hypertension. Exposure to ambient air particulate matter with aerodynamic diameters < 2.5 µm (PM2.5) is a risk factor for arteriosclerosis as well as lung disease, but its effect on NAFLD is unknown. PM2.5 induces pulmonary dysfunction via toll-like receptor activation on alveolar macrophages. Toll-like receptor activation of Kupffer cells, resident hepatic macrophages, and subsequent pro-inflammatory cytokine production have been shown to play a key role in NAFLD progression. We hypothesized that PM2.5 exposure is a significant risk factor for progression of NAFLD. Thus, following exposure of male C57BL/6 mice fed high fat chow to concentrated air particulate matter (CAPs) or filtered air for 6 wk, progression of NAFLD was evaluated by standardized histological assessment of hepatic inflammation and fibrosis. In mice fed high fat chow, the hepatic inflammatory grade (3.00 ± 0.00 vs. 1.50 ± 0.71, p < 0.001) and fibrosis stage (1.00 ± 0.00 vs. 0.60 ± 0.52, p = 0.023) were both significantly higher in mice exposed to CAPs versus filtered air, respectively. Increased numbers of Kupffer cells contained PM in CAPs-exposed mice (2.00 ± 0.94 vs. 0.20 ± 0.42, respectively, p < 0.001). PM exposure increased IL-6 secretion up to seven fold in a dose-dependent manner by isolated wild-type but not TLR4−/− Kupffer cells (p < 0.050). Conclusion: Ambient PM2.5 exposure may be a significant risk factor for NAFLD progression. PMID:19908945

  19. Activation of Kupffer Cells Is Associated with a Specific Dysbiosis Induced by Fructose or High Fat Diet in Mice

    PubMed Central

    Ferrere, Gladys; Leroux, Anne; Wrzosek, Laura; Puchois, Virginie; Gaudin, Françoise; Ciocan, Dragos; Renoud, Marie-Laure; Naveau, Sylvie; Perlemuter, Gabriel; Cassard, Anne-Marie

    2016-01-01

    The increase consumption of fructose in diet is associated with liver inflammation. As a specific fructan substrate, fructose may modify the gut microbiota which is involved in obesity-induced liver disease. Here, we aimed to assess whether fructose-induced liver damage was associated with a specific dysbiosis, especially in mice fed a high fat diet (HFD). To this end, four groups of mice were fed with normal and HFD added or not with fructose. Body weight and glucose sensitivity, liver inflammation, dysbiosis and the phenotype of Kupffer cells were determined after 16 weeks of diet. Food intake was increased in the two groups of mice fed with the HFD. Mice fed with HFD and fructose showed a higher infiltration of lymphocytes into the liver and a lower inflammatory profile of Kupffer cells than mice fed with the HFD without fructose. The dysbiosis associated with diets showed that fructose specifically prevented the decrease of Mouse intestinal bacteria in HFD fed mice and increased Erysipelotrichi in mice fed with fructose, independently of the amount of fat. In conclusion, fructose, used as a sweetener, induced a dysbiosis which is different in presence of fat in the diet. Consequently, the activation of Kupffer cells involved in mice model of HFD-induced liver inflammation was not observed in an HFD/fructose combined diet. These data highlight that the complexity of diet composition could highly impact the development of liver lesions during obesity. Specific dysbiosis associated with the diet could explain that the progressions of liver damage are different. PMID:26731543

  20. Thyroid Hormone-Induced Cytosol-to-Nuclear Translocation of Rat Liver Nrf2 Is Dependent on Kupffer Cell Functioning

    PubMed Central

    Videla, Luis A.; Cornejo, Pamela; Romanque, Pamela; Santibáñez, Catherine; Castillo, Iván; Vargas, Romina

    2012-01-01

    L-3,3′,5-triiodothyronine (T3) administration upregulates nuclear factor-E2-related factor 2 (Nrf2) in rat liver, which is redox-sensitive transcription factor mediating cytoprotection. In this work, we studied the role of Kupffer cell respiratory burst activity, a process related to reactive oxygen species generation and liver homeostasis, in Nrf2 activation using the macrophage inactivator gadolinium chloride (GdCl3; 10 mg/kg i.v. 72 h before T3 [0.1 mg/kg i.p.]) or NADPH oxidase inhibitor apocynin (1.5 mmol/L added to the drinking water for 7 days before T3), and determinations were performed 2 h after T3. T3 increased nuclear/cytosolic Nrf2 content ratio and levels of heme oxygenase 1 (HO-1), catalytic subunit of glutamate cysteine ligase, and thioredoxin (Western blot) over control values, proteins whose gene transcription is induced by Nrf2. These changes were suppressed by GdCl3 treatment prior to T3, an agent-eliciting Kupffer-cell depletion, inhibition of colloidal carbon phagocytosis, and the associated respiratory burst activity, with enhancement in nuclear inhibitor of Nrf2 kelch-like ECH-associated protein 1 (Keap1)/Nrf2 content ratios suggesting Nrf2 degradation. Under these conditions, T3-induced tumor necrosis factor-α (TNF-α) response was eliminated by previous GdCl3 administration. Similar to GdCl3, apocynin given before T3 significantly reduced liver Nrf2 activation and HO-1 expression, a NADPH oxidase inhibitor eliciting abolishment of colloidal carbon-induced respiratory burst activity without altering carbon phagocytosis. It is concluded that Kupffer cell functioning is essential for upregulation of liver Nrf2-signaling pathway by T3. This contention is supported by suppression of the respiratory burst activity of Kupffer cells and the associated reactive oxygen species production by GdCl3 or apocynin given prior to T3, thus hindering Nrf2 activation. PMID:22649286

  1. 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.

  2. Effect of modulation of PPAR-γ activity on Kupffer cells M1/M2 polarization in the development of non-alcoholic fatty liver disease.

    PubMed

    Luo, Wenjing; Xu, Qinyu; Wang, Qi; Wu, Huimin; Hua, Jing

    2017-03-16

    Abnormal lipid-mediated hepatic inflammatory-immune dysfunction and chronic low grade inflammation play an important role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Macrophage polarization is an important mechanism for the regulation of inflammatory response. Since PPAR-γ has emerged as a master regulator of macrophage polarization, we aimed to investigate the lipid-induced macrophage/Kupffer cell polarization in vivo and in vitro, and explore the association between PPAR-γ activity and macrophages M1/M2 polarization shifting. Here we showed that long-term high-fat diet increased Kupffer cells content with M1-predominant phenotype and increasing production of pro-inflammatory cytokines. Saturated fatty acids polarized Kupffer cells/macrophages to an M1-predominant phenotype while n-3 PUFA polarized Kupffer cells/macrophages to an M2 phenotype, which was associated with activation of NF-κB signal pathway and PPAR-γ respectively. Furthermore, up-regulation of PPAR-γ shifted lipid-induced macrophages polarization from M1-predominant phenotype to M2 phenotype. Macrophages polarization switch was associated with the interaction between PPAR-γ and NF-κBp65 signal pathway. Rosiglitazone restored high-fat diet-induced imblance of Kupffer cells M1/M2 polarization and alleviated hepatic steatosis as well as local pro-inflammatory response. These findings suggest that manipulation of PPAR-γ activity has the potential to balance lipid-induced M1/M2 macrophage/Kupffer cell polarization, and leading to prevent the development of NAFLD.

  3. Effect of modulation of PPAR-γ activity on Kupffer cells M1/M2 polarization in the development of non-alcoholic fatty liver disease

    PubMed Central

    Luo, Wenjing; Xu, Qinyu; Wang, Qi; Wu, Huimin; Hua, Jing

    2017-01-01

    Abnormal lipid-mediated hepatic inflammatory-immune dysfunction and chronic low grade inflammation play an important role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Macrophage polarization is an important mechanism for the regulation of inflammatory response. Since PPAR-γ has emerged as a master regulator of macrophage polarization, we aimed to investigate the lipid-induced macrophage/Kupffer cell polarization in vivo and in vitro, and explore the association between PPAR-γ activity and macrophages M1/M2 polarization shifting. Here we showed that long-term high-fat diet increased Kupffer cells content with M1-predominant phenotype and increasing production of pro-inflammatory cytokines. Saturated fatty acids polarized Kupffer cells/macrophages to an M1-predominant phenotype while n-3 PUFA polarized Kupffer cells/macrophages to an M2 phenotype, which was associated with activation of NF-κB signal pathway and PPAR-γ respectively. Furthermore, up-regulation of PPAR-γ shifted lipid-induced macrophages polarization from M1-predominant phenotype to M2 phenotype. Macrophages polarization switch was associated with the interaction between PPAR-γ and NF-κBp65 signal pathway. Rosiglitazone restored high-fat diet-induced imblance of Kupffer cells M1/M2 polarization and alleviated hepatic steatosis as well as local pro-inflammatory response. These findings suggest that manipulation of PPAR-γ activity has the potential to balance lipid-induced M1/M2 macrophage/Kupffer cell polarization, and leading to prevent the development of NAFLD. PMID:28300213

  4. Kupffer Cells Undergo Fundamental Changes during the Development of Experimental NASH and Are Critical in Initiating Liver Damage and Inflammation

    PubMed Central

    Reid, D. T.; Reyes, J. L.; McDonald, B. A.; Vo, T.; Reimer, R. A.; Eksteen, B.

    2016-01-01

    Non-alcoholic fatty liver disease has become the leading liver disease in North America and is associated with the progressive inflammatory liver disease non-alcoholic steatohepatitis (NASH). Considerable effort has been made to understand the role of resident and recruited macrophage populations in NASH however numerous questions remain. Our goal was to characterize the dynamic changes in liver macrophages during the initiation of NASH in a murine model. Using the methionine-choline deficient diet we found that liver-resident macrophages, Kupffer cells were lost early in disease onset followed by a robust infiltration of Ly-6C+ monocyte-derived macrophages that retained a dynamic phenotype. Genetic profiling revealed distinct patterns of inflammatory gene expression between macrophage subsets. Only early depletion of liver macrophages using liposomal clodronate prevented the development of NASH in mice suggesting that Kupffer cells are critical for the orchestration of inflammation during experimental NASH. Increased understanding of these dynamics may allow us to target potentially harmful populations whilst promoting anti-inflammatory or restorative populations to ultimately guide the development of effective treatment strategies. PMID:27454866

  5. Central Insulin Action Activates Kupffer Cells by Suppressing Hepatic Vagal Activation via the Nicotinic Alpha 7 Acetylcholine Receptor.

    PubMed

    Kimura, Kumi; Tanida, Mamoru; Nagata, Naoto; Inaba, Yuka; Watanabe, Hitoshi; Nagashimada, Mayumi; Ota, Tsuguhito; Asahara, Shun-ichiro; Kido, Yoshiaki; Matsumoto, Michihiro; Toshinai, Koji; Nakazato, Masamitsu; Shibamoto, Toshishige; Kaneko, Shuichi; Kasuga, Masato; Inoue, Hiroshi

    2016-03-15

    Central insulin action activates hepatic IL-6/STAT3 signaling, which suppresses the gene expression of hepatic gluconeogenic enzymes. The vagus nerve plays an important role in this centrally mediated hepatic response; however, the precise mechanism underlying this brain-liver interaction is unclear. Here, we present our findings that the vagus nerve suppresses hepatic IL-6/STAT3 signaling via α7-nicotinic acetylcholine receptors (α7-nAchR) on Kupffer cells, and that central insulin action activates hepatic IL-6/STAT3 signaling by suppressing vagal activity. Indeed, central insulin-mediated hepatic IL-6/STAT3 activation and gluconeogenic gene suppression were impeded in mice with hepatic vagotomy, pharmacological cholinergic blockade, or α7-nAchR deficiency. In high-fat diet-induced obese and insulin-resistant mice, control of the vagus nerve by central insulin action was disturbed, inducing a persistent increase of inflammatory cytokines. These findings suggest that dysregulation of the α7-nAchR-mediated control of Kupffer cells by central insulin action may affect the pathogenesis of chronic hepatic inflammation in obesity.

  6. Activation and increase of radio-sensitive CD11b+ recruited Kupffer cells/macrophages in diet-induced steatohepatitis in FGF5 deficient mice

    PubMed Central

    Nakashima, Hiroyuki; Nakashima, Masahiro; Kinoshita, Manabu; Ikarashi, Masami; Miyazaki, Hiromi; Hanaka, Hiromi; Imaki, Junko; Seki, Shuhji

    2016-01-01

    We have recently reported that Kupffer cells consist of two subsets, radio-resistant resident CD68+ Kupffer cells and radio-sensitive recruited CD11b+ Kupffer cells/macrophages (Mφs). Non-alcoholic steatohepatitis (NASH) is characterized not only by hepatic steatosis but also chronic inflammation and fibrosis. In the present study, we investigated the immunological mechanism of diet-induced steatohepatitis in fibroblast growth factor 5 (FGF5) deficient mice. After consumption of a high fat diet (HFD) for 8 weeks, FGF5 null mice developed severe steatohepatitis and fibrosis resembling human NASH. F4/80+ Mφs which were both CD11b and CD68 positive accumulated in the liver. The production of TNF and FasL indicated that they are the pivotal effectors in this hepatitis. The weak phagocytic activity and lack of CRIg mRNA suggested that they were recruited Mφs. Intermittent exposure to 1 Gy irradiation markedly decreased these Mφs and dramatically inhibited liver inflammation without attenuating steatosis. However, depletion of the resident subset by clodronate liposome (c-lipo) treatment increased the Mφs and tended to exacerbate disease progression. Recruited CD11b+ CD68+ Kupffer cells/Mφs may play an essential role in steatohepatitis and fibrosis in FGF5 null mice fed with a HFD. Recruitment and activation of bone marrow derived Mφs is the key factor to develop steatohepatitis from simple steatosis. PMID:27708340

  7. Failure to demonstrate a major role for Kupffer cells and radiosensitive leukocytes in immunoglobulin-mediated elimination of Trypanosoma musculi

    SciTech Connect

    Kongshavn, P.A.; Shaw, K.; Ghadirian, E.; Ulczak, O. )

    1990-06-01

    Previous studies have indicated that elimination of parasitemia in Trypanosoma musculi infection is brought about by immunoglobulin G2a antibodies, C3, and an effector cell. Experiments were designed to identify the putative effector cell by using several approaches. Infected C5-deficient or C5-sufficient mice treated with silica particles or given 900 rads of radiation 3 days earlier effectively eliminated trypanosomes following administration of immune plasma (IP). Silica-treated, noninfected mice given T. musculi preincubated with IP also cleared the parasites. Radiolabeling studies revealed that uptake of the cleared trypanosomes by the liver in normal mice was relatively low and fell only slightly (19%) in silica-treated mice. In contrast, uptake of radiolabeled sheep erythrocytes by the liver was normally much higher and fell drastically (7%) in silica-treated mice. Mice were then immunocompromised by 900 rads of radiation, silica particles, and anti-platelet serum combined before IP-sensitized trypanosomes were given. Leukocyte and platelet counts were both reduced by 95% and sheep erythrocyte uptake by the liver fell from 77 to 5%; however, greater than 99% of the injected trypanosomes were cleared in these mice and uptake of radiolabeled trypanosomes by the liver was similar to that of normal mice. Lastly, in anesthetized mice in which Kupffer cells were excluded surgically from the circulation, greater than 99% of the IP-sensitized trypanosomes disappeared rapidly from the blood. Only 7% of the radiolabel was found in the liver versus 60% in sham-operated mice. The results are interpreted as showing that hepatic Kupffer cells play a minor role in the immune elimination of T. musculi. Likewise, radiosensitive leukocytes and platelets are unlikely to be sole candidates for the putative effector cell that mediates a cure of murine trypanosomiasis.

  8. Differential Effects of Kupffer Cell Inactivation on Inflammation and The Liver Sieve Following Caecal-Ligation and Puncture-Induced Sepsis in Mice.

    PubMed

    Gaddam, Ravinder Reddy; Fraser, Robin; Badiei, Alireza; Chambers, Stephen; Cogger, Victoria C; Le Couteur, David G; Bhatia, Madhav

    2017-04-01

    Sepsis remains a common clinical problem with significant mortality. Activation of the Kupffer cells during sepsis is associated with systemic inflammatory response and multiple organ failure. Kupffer cell activation also leads to structural changes in the liver sinusoidal endothelial cells (LSECs) during endotoxemia. However, these effects remain to be elucidated in caecal-ligation and puncture (CLP)-induced polymicrobial sepsis. To investigate the role of Kupffer cells on LSECs fenestrae and inflammation during CLP-induced sepsis, sepsis was induced by CLP and mice were treated with gadolinium chloride (GdCl3) before CLP-induced sepsis, to inactivate Kupffer cells. Mice were sacrificed after 8 h. Blood, liver, and lung tissues were collected and processed to measure LSECs fenestration, myeloperoxidase (MPO) activity, alanine transaminase (ALT) and aspartate aminotransferase (AST) activity, histological examination, and various cytokines/chemokines levels. LSECs fenestrae was studied using scanning electron micrographs of the LSECs. Strikingly, CLP mice treated with GdCl3 were protected against liver injury as evidenced by decreased LSECs defenestration and damage, MPO, ALT and AST activities, liver tissue damage, and inflammatory cytokines TNF-α, IL-6 and IL-1β, and chemokines MCP-1 and MIP-2α. However, CLP mice treated with GdCl3 had no protection against increased lung MPO activity, tissue damage, inflammatory cytokines, and chemokines. Treatment with GdCl3 also had no effect on the systemic inflammatory response as shown by no change in the circulatory inflammatory cytokines and chemokines following CLP-induced sepsis. Collectively, these data suggest that inactivation of Kupffer cells by GdCl3 protects the liver but had no effect on lung injury or inflammation and systemic inflammatory response following CLP-induced sepsis.

  9. Nucleation of platelets with blood-borne pathogens on Kupffer cells precedes other innate immunity and contributes to bacterial clearance.

    PubMed

    Wong, Connie H Y; Jenne, Craig N; Petri, Björn; Chrobok, Navina L; Kubes, Paul

    2013-08-01

    Through the use of intravital imaging of the liver, we demonstrate a collaborative role for platelets with Kupffer cells (KCs) in eradicating blood-borne bacterial infection. Under basal conditions, platelets, via the platelet-adhesion receptor GPIb, formed transient 'touch-and-go' interactions with von Willebrand factor (vWF) constitutively expressed on KCs. Bacteria such as Bacillus cereus and methicillin-resistant Staphylococcus aureus (MRSA) were rapidly caught by KCs and triggered platelets to switch from 'touch-and-go' adhesion to sustained GPIIb-mediated adhesion on the KC surface to encase the bacterium. Infected GPIbα-deficient mice had more endothelial and KC damage than did their wild-type counterparts, which led to more fluid leakage, substantial polycythemia and rapid mortality. Our study identifies a previously unknown surveillance mechanism by which platelets survey macrophages that rapidly converts to a critical host response to blood-borne bacteria.

  10. Nucleation of platelets with bloodborne pathogens on Kupffer cell precedes other innate immunity and contributes to bacterial clearance

    PubMed Central

    Wong, Connie H. Y.; Jenne, Craig N.; Petri, Björn; Chrobok, Navina L.; Kubes, Paul

    2016-01-01

    Using intravital imaging of the liver, we unveil a collaborative role for platelets with Kupffer cells (KCs) in eradicating bloodborne bacterial infections. Under basal conditions, platelets via glycoprotein Ib (GPIb) formed transient “touch-and-go” interactions with von Willebrand factor (vWF) constitutively expressed on KCs. Bacteria, such as Bacillus cereus and Methicillin-resistant Staphylococcus aureus (MRSA), were rapidly caught by KCs and triggered platelets to switch from “touch-and-go” to sustained GPIIb-mediated adhesion on the KC surface to encase the bacterium. Infected GpIbα−/− mice demonstrated increased endothelial and KC damage, leading to increased fluid leakage, significant polycythemia and rapid mortality. This study identifies a novel surveillance mechanism of intravascular macrophage by platelets that rapidly converts to a critical host response against bloodborne bacteria. PMID:23770641

  11. Puerarin ameliorates experimental alcoholic liver injury by inhibition of endotoxin gut leakage, Kupffer cell activation, and endotoxin receptors expression.

    PubMed

    Peng, Jing-Hua; Cui, Tuan; Huang, Fu; Chen, Liang; Zhao, Yu; Xu, Lin; Xu, Li-Li; Feng, Qin; Hu, Yi-Yang

    2013-03-01

    Puerarin, an isoflavone component extracted from Kudzu (Pueraria lobata), has been demonstrated to alleviate alcohol-related disorders. Our study examined whether puerarin ameliorates chronic alcoholic liver injury through inhibition of endotoxin gut leakage, the subsequent Kupffer cell activation, and endotoxin receptors expression. Rats were provided with the Liber-DeCarli liquid diet for 8 weeks. Puerarin (90 mg/kg or 180 mg/kg daily) was orally administered from the beginning of the third week until the end of the experiment. Chronic alcohol intake caused increased serum alanine aminotransferase, aspartate aminotransferase, hepatic gamma-glutamyl transpeptidase, and triglyceride levels as well as fatty liver and neutrophil infiltration in hepatic lobules as determined by biochemical and histologic assays. A significant increase of liver tumor necrosis factor α was detected by enzyme-linked immunosorbent assay. These pathologic effects correlated with increased endotoxin level in portal vein and upregulated protein expression of hepatic CD68, lipopolysaccharide-binding protein, CD14, Toll-like receptor 2, and Toll-like receptor 4. Meanwhile, the intestinal microvilli were observed to be sparse, shortened, and irregularity in distribution under the transmission electron microscope in conjunction with the downregulated intestinal zonula occludens-1 protein expression. These hepatic pathologic changes were significantly inhibited in puerarin-treated animals as were the endotoxin levels and hepatic CD68 and endotoxin receptors. Moreover, the pathologic changes in intestinal microvillus and the decreased intestinal zonula occludens-1 were also ameliorated with puerarin treatment. These results thus demonstrate that puerarin inhibition of endotoxin gut leakage, Kupffer cell activation, and endotoxin receptors expression is involved in the alleviation of chronic alcoholic liver injury in rats.

  12. Effect of CD16a, the surface receptor of Kupffer cells, on the growth of hepatocellular carcinoma cells

    PubMed Central

    LI, XIU-YUN; WU, LUN; LI, SHENG-WEI; ZHOU, WEN-BO; WANG, MENG-YUAN; ZUO, GUO-QING; LIU, CHANG-AN; DING, XIONG

    2016-01-01

    FcγRIIIa (CD16) is a low-affinity Fc receptor of IgG. As the idio-binding receptor of IgG Fc, it plays an important role in the antibody-dependent cellular cytotoxicity of natural killer cells. The aim of the present study was to investigate the distribution of Kupffer cells (KCs) and the expression of their surface receptor FcγRIIIa in hepatocellular carcinoma. Furthermore, we also aimed to observe the functional mechanism of FcγRIIIa. Immunohistochemical analysis was employed to study KCs and FcγRIIIa. In order to explore the role of FcγRIIIa in the growth of cancer cells, KCs and H22 tumor cells were co-cultured in different serum. The mRNA expression levels of tumor necrosis factor (TNF)-α and FcγRIIIa were analyzed by RT-qPCR; the TNF-α and FcγRIIIa protein expression levels were examined by enzyme-linked immunosorbent assay and western blot analysis, respectively. Our results showed that the number of Kuppfer cells in cancerous tissues (21.6±7.8) was lower than those in para-cancerous (68.8±9.1) tissues and adjacent normal hepatic tissues (62.0±1.9) (P<0.01); this decreased with the reduction in the differentiation degree of cancer (P<0.05). FcγRIIIa-positive cells were similar in morphology to KCs, and their distributive tendency was coincident (P<0.05). The increase in CD16a mRNA levels in the group treated with immune serum was 3.9-, 4.9- and 3.9-fold greater than that in the ordinary serum group at different time points, and CD16a protein expression also markedly increased (P<0.05). However, these effects were inhibited by the addition of anti-IgG Fc serum (P<0.05). The results of the present study suggested that FcγRIIIa resided in KCs, and it contributed to the inhibition of the growth of liver tumor cells. PMID:27082928

  13. No attenuation of ischemic and reperfusion injury in Kupffer cell-depleted, cold-preserved rat livers.

    PubMed

    Reinders, M E; van Wagensveld, B A; van Gulik, T M; Corssmit, N P; Frederiks, W M; Chamuleau, R A; van Rooijen, N; Obertop, H

    1997-02-15

    Activated Kupffer cells (KC) have been implicated in the damage sustained by preserved liver grafts during ischemia and reperfusion. The aim of this study was to compare ischemia/reperfusion injury in preserved, KC-depleted rat livers and preserved control livers, with special regard to sinusoidal endothelial cell (SEC) injury. Wistar rats were injected with liposome-encapsulated dichloromethylene diphosphonate, 48 hr before hepatectomy, to eliminate KC, or were withheld this pretreatment (controls). Livers were flushed with cold University of Wisconsin solution and after 0, 8, 16, or 24 hr of storage at 4 degrees C, were reperfused in a recirculation system with 200 ml of oxygenated Krebs-Henseleit solution at 37 degrees C for 90 min. Damage to SEC was measured by the uptake of hyaluronic acid (HA) from the perfusate and release of purine nucleoside phosphorylase (PNP). Perfusate samples were, furthermore, analyzed for aspartate aminotransferase (AST) and tumor necrosis factor-alpha. Carbon particles were infused in the perfusate to determine the phagocytotic capacity of KC. Biopsies were taken for histological examination and sections were stained with ED2 monoclonal antibodies to confirm the absence of KC. After 90 min of reperfusion, immediately after cold flush (t0), the uptake of HA was 72.2+/-2.3% and 69.3+/-1.3% in KC-depleted livers and in control livers, respectively (n.s.). After 8 hr of storage, HA uptake was 21.6+/-4.5% and 34.6+/-8.0%, respectively (n.s.). After 16 and 24 hr of storage and reperfusion, no uptake of HA was found in either KC-depleted or control livers, indicating abolished SEC function. PNP activities in the perfusate were higher in control livers (after 8 and 24 hr of storage), presumably due to release from damaged KC. No difference was found in AST and no tumor necrosis factor-alpha was measured in the perfusates of normal and KC-depleted livers. Electron microscopic studies showed that after 8 and 24 hr of storage and reperfusion

  14. Protective effects of branched-chain amino acids on hepatic ischemia-reperfusion-induced liver injury in rats: a direct attenuation of Kupffer cell activation.

    PubMed

    Kitagawa, Tomomi; Yokoyama, Yukihiro; Kokuryo, Toshio; Nagino, Masato

    2013-02-15

    We determined whether there is a protective effect of branched-chain amino acid (BCAA) on hepatic ischemia-reperfusion (I/R)-induced acute liver injury. Wister rats were divided into the following four groups: simple laparotomy with vehicle; simple laparotomy with BCAA (1 g/kg body wt orally); I/R (30 min clamp) with vehicle; and I/R with BCAA. Serum liver function tests and the gene expression of adhesion molecules (intercellular adhesion molecule and vascular cell adhesion molecule) and vasoconstrictor-related genes (endothelin-1) in the liver were examined. In the in vivo study, portal venous pressure, leukocyte adhesion, and hepatic microcirculation were evaluated. Furthermore, Kupffer cells were isolated and cultured with various concentrations of BCAA in the presence or absence of lipopolysaccharide (LPS). Increased levels of liver function tests following I/R were significantly attenuated by BCAA treatment. The increased expression of adhesion molecules and endothelin-1 was also significantly attenuated by BCAA treatment. Moreover, increased portal venous pressure, enhanced leukocyte adhesion, and deteriorated hepatic microcirculation following I/R were all improved by BCAA treatment. In the experiment using isolated Kupffer cells, the expression of interleukin-6, interleukin-1β, and endothelin-1 in response to LPS stimulation was attenuated by BCAA in a dose-dependent fashion. These results indicate that perioperative oral administration of BCAA has excellent therapeutic potential to reduce I/R-induced liver injury. These beneficial effects may result from the direct attenuation of Kupffer cell activation under stressful conditions.

  15. Co-culture of Hepatocytes and Kupffer Cells as an In Vitro Model of Inflammation and Drug-Induced Hepatotoxicity

    PubMed Central

    Rose, Kelly A.; Holman, Natalie S.; Green, Angela M.; Andersen, Melvin E.; LeCluyse, Edward L.

    2017-01-01

    Immune-mediated drug-induced hepatotoxicity is often unrecognized as a potential mode of action due to the lack of appropriate in vitro models. We have established an in vitro rat donor-matched hepatocyte and Kupffer cell co-culture (HKCC) model to study immune-related responses to drug exposure. Optimal cell culture conditions were identified for the maintenance of co-cultures based on cell longevity, monolayer integrity, and cytokine response after lipopolysaccharide (LPS) exposure. Hepatocyte monocultures and HKCCs were then used to test a subset of compounds associated with hepatotoxic effects with or without LPS. Cytokine levels and metabolic activity (cytochrome P450 3A [Cyp3A]) were measured after a 48-h exposure to monitor endotoxin-induced changes in acute phase and functional end points. LPS-activated HKCCs, but not hepatocyte monocultures, treated with trovafloxacin or acetaminophen, compounds associated with immune-mediated hepatotoxicity, showed LPS-dependent decreases in interleukin-6 production with concomitant increases in Cyp3A activity. Differential endotoxinand model-dependent alterations were observed in cytokine profiles and Cyp3A activity levels that corresponded to specific compounds. These results indicate the utility of the HKCC model system to discern compound-specific effects that may lead to enhanced or mitigate hepatocellular injury due to innate or adaptive immune responses. PMID:26869439

  16. Murine Kupffer Cells Are Protective in Total Hepatic Ischemia/Reperfusion Injury with Bowel Congestion through IL-10

    PubMed Central

    Ellett, Justin D.; Atkinson, Carl; Evans, Zachary P.; Amani, Zainab; Balish, Edward; Schmidt, Michael G.; van Rooijen, Nico; Schnellmann, Rick G.; Chavin, Kenneth D.

    2010-01-01

    Kupffer cells (KCs) are thought to mediate hepatocyte injury via their production of proinflammatory cytokines and reactive oxygen species in response to stress. In this study, we depleted KCs from the liver to examine their role in total warm hepatic ischemia/reperfusion (I/R) injury with bowel congestion. We injected 8-wk-old C57BL/10J mice with liposome-encapsulated clodronate 48 h before 35 min of hepatic ischemia with bowel congestion, followed by 6 or 24 h of reperfusion. KC-depleted animals had a higher mortality rate than diluent-treated animals and a 10-fold elevation in transaminase levels that correlated with increases in centrilobular necrosis. There was extensive LPS binding to the endothelial cells, which correlated with an upregulation of endothelial adhesion molecules in the KC-depleted animals versus diluent-treated animals. There was an increase in the levels of proinflammatory cytokines in KC-depleted animals, and a concomitant decrease in IL-10 levels. When KC-depleted mice were treated with recombinant IL-10, their liver damage profile in response to I/R was similar to diluent-treated animals, and endothelial cell adhesion molecules and proinflammatory cytokine levels decreased. KCs are protective in the liver subjected to total I/R with associated bowel congestion and are not deleterious as previously thought. This protection appears to be due to KC secretion of the potent anti-inflammatory cytokine IL-10. PMID:20400698

  17. Huangqi decoction inhibits apoptosis and fibrosis, but promotes Kupffer cell activation in dimethylnitrosamine-induced rat liver fibrosis

    PubMed Central

    2012-01-01

    Background Previously, Huangqi decoction (HQD) has been found to have a potential therapeutic effect on DMN-induced liver cirrhosis. Here, the mechanisms of HQD action against liver fibrosis were investigated in relation to hepatocyte apoptosis and hepatic inflammation regulation. Methods Liver fibrosis was induced by DMN administration for 2 or 4 weeks. Hepatocyte apoptosis and of Kupffer cells (KC) and hepatic stellate cells (HSC) interaction were investigated using confocal microscopy. The principle cytokines, fibrogenic proteins and apoptotic factors were investigated using western blot analysis. Results Compared with the DMN-water group, HQD showed decreased hepatocyte apoptosis and reduced expression of apoptotic effectors, cleaved-caspase-3, and fibrotic factors, such as smooth muscle α-actin (α-SMA), transforming growth factor beta-1 (TGF-β1). However, the KC marker CD68 increased significantly in DMN-HQD liver. Confocal microscopy demonstrated widespread adhesion of KCs to HSCs in DMN-water and DMN-HQD rats liver. Conclusions HQD exhibited positive protective effects against liver fibrosis; its mechanism of action was associated with protection from hepatocyte apoptosis and the promotion of CD68 expression in the devolopment of liver fibrosis to cirrhosis development. PMID:22531084

  18. Biofilm-Forming Methicillin-Resistant Staphylococcus aureus Survive in Kupffer Cells and Exhibit High Virulence in Mice

    PubMed Central

    Oyama, Takuto; Miyazaki, Motoyasu; Yoshimura, Michinobu; Takata, Tohru; Ohjimi, Hiroyuki; Jimi, Shiro

    2016-01-01

    Although Staphylococcus aureus is part of the normal body flora, heavy usage of antibiotics has resulted in the emergence of methicillin-resistant strains (MRSA). MRSA can form biofilms and cause indwelling foreign body infections, bacteremia, soft tissue infections, endocarditis, and osteomyelitis. Using an in vitro assay, we screened 173 clinical blood isolates of MRSA and selected 20 high-biofilm formers (H-BF) and low-biofilm formers (L-BF). These were intravenously administered to mice and the general condition of mice, the distribution of bacteria, and biofilm in the liver, lung, spleen, and kidney were investigated. MRSA count was the highest in the liver, especially within Kupffer cells, which were positive for acid polysaccharides that are associated with intracellular biofilm. After 24 h, the general condition of the mice worsened significantly in the H-BF group. In the liver, bacterial deposition and aggregation and the biofilm-forming spot number were all significantly greater for H-BF group than for L-BF. CFU analysis revealed that bacteria in the H-BF group survived for long periods in the liver. These results indicate that the biofilm-forming ability of MRSA is a crucial factor for intracellular persistence, which could lead to chronic infections. PMID:27376326

  19. Sympathetic nervous system promotes hepatocarcinogenesis by modulating inflammation through activation of alpha1-adrenergic receptors of Kupffer cells.

    PubMed

    Huan, Hong-Bo; Wen, Xu-Dong; Chen, Xue-Jiao; Wu, Lin; Wu, Li-Li; Zhang, Liang; Yang, Da-Peng; Zhang, Xia; Bie, Ping; Qian, Cheng; Xia, Feng

    2017-01-01

    The sympathetic nervous system (SNS) is known to play a significant role in tumor initiation and metastasis. Hepatocellular carcinoma (HCC) frequently occurs in cirrhotic livers after chronic inflammation, and the SNS is hyperactive in advanced liver cirrhosis. However, it remains unclear whether the SNS promotes hepatocarcinogenesis by modulating chronic liver inflammation. In this study, a retrospective pathological analysis and quantification of sympathetic nerve fiber densities (tyrosine hydroxylase, TH(+)) in HCC patients, and diethylnitrosamine (DEN)-induced hepatocarcinogenesis in rats were performed. Our data showed that high density of sympathetic nerve fibers and α1-adrenergic receptors (ARs) of Kupffer cells (KCs) were associated with a poor prognosis of HCC. Sympathetic denervation or blocking of α1-ARs decreased DEN-induced HCC incidence and tumor development. In addition, synergistic effects of interleukin-6 (IL-6) and transforming growth factor-beta (TGF-β) in hepatocarcinogenesis were observed. The suppression of the SNS reduced IL-6 and TGF-β expression, which suppressed hepatocarcinogenesis, and KCs play a key role in this process. After the ablation of KCs, IL-6 and TGF-β expression and the development of HCC were inhibited. This study demonstrates that sympathetic innervation is crucial for hepatocarcinogenesis and that the SNS promotes hepatocarcinogenesis by activating α1-ARs of KCs to boost the activation of KCs and to maintain the inflammatory microenvironment. These results indicate that sympathetic denervation or α1-ARs blockage may represent novel treatment approaches for HCC.

  20. Critical Roles of Kupffer Cells in the Pathogenesis of Alcoholic Liver Disease: From Basic Science to Clinical Trials

    PubMed Central

    Zeng, Tao; Zhang, Cui-Li; Xiao, Mo; Yang, Rui; Xie, Ke-Qin

    2016-01-01

    Alcoholic liver disease (ALD) encompasses a spectrum of liver injury ranging from steatosis to steatohepatitis, fibrosis, and finally cirrhosis. Accumulating evidences have demonstrated that Kupffer cells (KCs) play critical roles in the pathogenesis of both chronic and acute ALD. It has become clear that alcohol exposure can result in increased hepatic translocation of gut-sourced endotoxin/lipopolysaccharide, which is a strong M1 polarization inducer of KCs. The activated KCs then produce a large amount of reactive oxygen species (ROS), pro-inflammatory cytokines, and chemokines, which finally lead to liver injury. The critical roles of KCs and related inflammatory cascade in the pathogenesis of ALD make it a promising target in pharmaceutical drug developments for ALD treatment. Several drugs (such as rifaximin, pentoxifylline, and infliximab) have been evaluated or are under evaluation for ALD treatment in randomized clinical trials. Furthermore, screening pharmacological regulators for KCs toward M2 polarization may provide additional therapeutic agents. The combination of these potentially therapeutic drugs with hepatoprotective agents (such as zinc, melatonin, and silymarin) may bring encouraging results. PMID:27965666

  1. Amphiphilic core–shell nanoparticles containing dense polyethyleneimine shells for efficient delivery of microRNA to Kupffer cells

    PubMed Central

    Liu, Zuojin; Niu, Dechao; Zhang, Junyong; Zhang, Wenfeng; Yao, Yuan; Li, Pei; Gong, Jianping

    2016-01-01

    Efficient and targeted delivery approach to transfer exogenous genes into macrophages is still a great challenge. Current gene delivery methods often result in low cellular uptake efficiency in vivo in some types of cells, especially for the Kupffer cells (KCs). In this article, we demonstrate that amphiphilic core–shell nanoparticles (NPs) consisting of well-defined hydrophobic poly(methyl methacrylate) (PMMA) cores and branched polyethyleneimine (PEI) shells (denoted as PEI@PMMA NPs) are efficient nanocarriers to deliver microRNA (miRNA)-loaded plasmid to the KCs. Average hydrodynamic diameter of PEI@ PMMA NPs was 279 nm with a narrow size distribution. The NPs also possessed positive surface charges up to +30 mV in water, thus enabling effective condensation of negatively charged plasmid DNA. Gel electrophoresis assay showed that the resultant PEI@PMMA NPs were able to completely condense miRNA plasmid at a weight ratio of 25:1 (N/P ratio equal to 45:1). The Cell Counting Kit-8 assay and flow cytometry results showed that the PEI@PMMA/miRNA NPs displayed low cytotoxicity and cell apoptosis activity against the KCs. The maximum cell transfection efficiency reached 34.7% after 48 hours, which is much higher than that obtained by using the commercial Lipofectamine™ 2000 (1.7%). Bio-transmission electron microscope observation revealed that the PEI@PMMA NPs were mainly distributed in the cytoplasm of the KCs. Furthermore, when compared to the control groups, the protein expression of target nuclear factor κB P65 was considerably inhibited (P<0.05) both in vitro and in vivo. These results demonstrate that the PEI@PMMA NPs with a unique amphiphilic core–shell nanostructure are promising nanocarriers for delivering miRNA plasmid to KCs. PMID:27366061

  2. Long live the liver: immunohistochemical and stereological study of hepatocytes, liver sinusoidal endothelial cells, Kupffer cells and hepatic stellate cells of male and female rats throughout ageing.

    PubMed

    Marcos, Ricardo; Correia-Gomes, Carla

    2016-12-01

    Male/female differences in enzyme activity and gene expression in the liver are known to be attenuated with ageing. Nevertheless, the effect of ageing on liver structure and quantitative cell morphology remains unknown. Male and female Wistar rats aged 2, 6, 12 and 18 months were examined by means of stereological techniques and immunohistochemical tagging of hepatocytes (HEP), liver sinusoidal endothelial cells (LSEC), Kupffer cells (KC) and hepatic stellate cells (HSC) in order to assess the total number and number per gram of these cells throughout life. The mean cell volume of HEP and HSC, the lobular position and the collagen content of the liver were also evaluated with stereological techniques. The number per gram of HSC was similar for both genders and was maintained throughout ageing. The mean volume of HSC was also conserved but differences in the cell body and lobular location were observed. Statistically significant gender differences in HEP were noted in young rats (females had smaller and more binucleated HEP) but were attenuated with ageing. The same occurred for KC and LSEC, since the higher number per gram in young females disappeared in older animals. Liver collagen increased with ageing but only in males. Thus, the numbers of these four cell types are related throughout ageing, with well-defined cell ratios. The shape and lobular position of HSC change with ageing in both males and females. Gender dimorphism in HEP, KC and LSEC of young rat liver disappears with ageing.

  3. Depletion of Kupffer cells modulates ethanol-induced hepatocyte DNA synthesis in C57Bl/6 mice.

    PubMed

    Owumi, Solomon E; Corthals, Stacy M; Uwaifo, Anthony O; Kamendulis, Lisa M; Klaunig, James E

    2014-08-01

    Kupffer cells (KCs) are important in hepatic homeostasis and responses to xenobiotics. KCs are activated on interaction with endotoxin, releasing cytokines, and reactive oxygen species normally associated with increased gene expression, cellular growth, or hepatic injury. Ethanol-induced endotoxemia is one means of KC activation. We propose that KC depletion attenuates the effect of EtOH-induced endotoxemia to impact the hepatic growth response. Hepatic DNA synthesis was examined in KC competent (KC+) or KC-depleted (KC-) C57BL/6 mice fed EtOH-containing diet in the presence or absence of polyphenol-60 antioxidant. KC depletion was assessed by F4/80 antigen, and DNA synthesis was assessed by 5-bromo-2'-deoxyuridine incorporation. Tumor necrosis factor alpha (TNF-α) messenger RNA released was quantified by RT-PCR/electrophoresis. ERK1/2 phosphorylation was evaluated by Western blotting, and Nrf2 and CYP2E1protein were also assayed. Apoptosis and hepatic injury were examined by the Tunnel assay and hepatic transaminases in serum, respectively. Hepatic transaminases in serum (AST and ALT) were within normal range. Over 90% of KC was depleted by clodronate treatment. KC depletion decreased TNF-α mRNA release, ERK1/2 phosphorylation, and hepatocyte DNA synthesis. KC depletion is associated with increased numbers of apoptotic cells bodies in KC- mice. Antioxidant treatment decreased DNA synthesis, Nrf2, and CYP2E1 protein expression in EtOH-consuming mice. Our data indicate that upon ethanol exposure, KC participates in hepatic DNA synthesis and growth responses. Collectively, these observations suggest that KC depletion attenuates the downstream effect of ethanol-induced endotoxemia by reduced cytokine and reactive oxygen species production with its concomitant effect on MAPK-signaling pathway on hepatocyte DNA synthesis.

  4. Curative Effects of Thiacremonone against Acetaminophen-Induced Acute Hepatic Failure via Inhibition of Proinflammatory Cytokines Production and Infiltration of Cytotoxic Immune Cells and Kupffer Cells

    PubMed Central

    Kim, Yu Ri; Ban, Jung Ok; Yoo, Hwan Soo; Lee, Yong Moon; Yoon, Yeo Pyo; Eum, So Young; Jeong, Heon Sang; Yoon, Do-young; Han, Sang Bae; Hong, Jin Tae

    2013-01-01

    High doses of acetaminophen (APAP; N-acetyl-p-aminophenol) cause severe hepatotoxicity after metabolic activation by cytochrome P450 2E1. This study was undertaken to examine the preventive effects of thiacremonone, a compound extracted from garlic, on APAP-induced acute hepatic failure in male C57BL/6J. Mice received with 500 mg/kg APAP after a 7-day pretreatment with thiacremonone (10–50 mg/kg). Thiacremonone inhibited the APAP-induced serum ALT and AST levels in a dose-dependent manner, and markedly reduced the restricted area of necrosis and inflammation by administration of APAP. Thiacremonone also inhibited the APAP-induced depletion of intracellular GSH, induction of nitric oxide, and lipid peroxidation as well as expression of P450 2E1. After APAP injection, the numbers of Kupffer cells, natural killer cells, and cytotoxic T cells were elevated, but the elevated cell numbers in the liver were reduced in thiacremonone pretreated mice. The expression levels of I-309, M-CSF, MIG, MIP-1α, MIP-1β, IL-7, and IL-17 were increased by APAP treatment, which were inhibited in thiacremonone pretreated mice. These data indicate that thiacremonone could be a useful agent for the treatment of drug-induced hepatic failure and that the reduction of cytotoxic immune cells as well as proinflammatory cytokine production may be critical for the prevention of APAP-induced acute liver toxicity. PMID:23935693

  5. Transient Depletion of Kupffer Cells Leads to Enhanced Transgene Expression in Rat Liver Following Retrograde Intrabiliary Infusion of Plasmid DNA and DNA Nanoparticles

    PubMed Central

    Dai, Hui; Jiang, Xuan; Leong, Kam W.

    2011-01-01

    Abstract In this report, we have demonstrated that by temporarily removing Kupffer cells (KCs), the transgene expression levels mediated by retrograde intrabiliary infusion (RII) of plasmid DNA, polyethylenimine-DNA, and chitosan nanoparticles were enhanced by 1,927-, 131-, and 23,450-fold, respectively, in comparison with the respective groups without KC removal. KC removal also led to significantly prolonged transgene expression in the liver that received all three carriers. This increased transgene expression was correlated with significantly reduced serum tumor necrosis factor-α level as an indicator for KC activation. These results suggest that KC activation is a significant contributing factor to the lowered transgene expression by polycation-DNA nanoparticles delivered by RII. More importantly, the combination of RII and transient removal of KCs may be adopted as an effective approach to achieving high and persistent transgene expression in the liver mediated by nonviral nanoparticles. PMID:21091274

  6. Intravital imaging reveals improved Kupffer cell-mediated phagocytosis as a mode of action of glycoengineered anti-CD20 antibodies

    PubMed Central

    Grandjean, Capucine L.; Montalvao, Fabricio; Celli, Susanna; Michonneau, David; Breart, Beatrice; Garcia, Zacarias; Perro, Mario; Freytag, Olivier; Gerdes, Christian A.; Bousso, Philippe

    2016-01-01

    Anti-CD20 monoclonal antibodies (mAbs) represent an effective treatment for a number of B cell malignancies and autoimmune disorders. Glycoengineering of anti-CD20mAb may contribute to increased anti-tumor efficacy through enhanced antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADP) as reported by in vitro studies. However, where and how glycoengineered Ab may potentiate therapeutic responses in vivo is yet to be elucidated. Here, we have performed mouse liver transplants to demonstrate that the liver is sufficient to mediate systemic B cells depletion after anti-CD20 treatment. Relying on intravital two-photon imaging of human CD20-expressing mice, we provide evidence that ADP by Kupffer cells (KC) is a major mechanism for rituximab-mediated B cell depletion. Notably, a glycoengineered anti-mouse CD20 Ab but not its wild-type counterpart triggered potent KC-mediated B cell depletion at low doses. Finally, distinct thresholds for KC phagocytosis were also observed for GA101 (obinutuzumab), a humanized glycoengineered type II anti-CD20 Ab and rituximab. Thus, we propose that enhanced phagocytosis of circulating B cells by KC represents an important in vivo mechanism underlying the improved activity of glycoengineered anti-CD20 mAbs. PMID:27698437

  7. Intravital imaging reveals improved Kupffer cell-mediated phagocytosis as a mode of action of glycoengineered anti-CD20 antibodies.

    PubMed

    Grandjean, Capucine L; Montalvao, Fabricio; Celli, Susanna; Michonneau, David; Breart, Beatrice; Garcia, Zacarias; Perro, Mario; Freytag, Olivier; Gerdes, Christian A; Bousso, Philippe

    2016-10-04

    Anti-CD20 monoclonal antibodies (mAbs) represent an effective treatment for a number of B cell malignancies and autoimmune disorders. Glycoengineering of anti-CD20mAb may contribute to increased anti-tumor efficacy through enhanced antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADP) as reported by in vitro studies. However, where and how glycoengineered Ab may potentiate therapeutic responses in vivo is yet to be elucidated. Here, we have performed mouse liver transplants to demonstrate that the liver is sufficient to mediate systemic B cells depletion after anti-CD20 treatment. Relying on intravital two-photon imaging of human CD20-expressing mice, we provide evidence that ADP by Kupffer cells (KC) is a major mechanism for rituximab-mediated B cell depletion. Notably, a glycoengineered anti-mouse CD20 Ab but not its wild-type counterpart triggered potent KC-mediated B cell depletion at low doses. Finally, distinct thresholds for KC phagocytosis were also observed for GA101 (obinutuzumab), a humanized glycoengineered type II anti-CD20 Ab and rituximab. Thus, we propose that enhanced phagocytosis of circulating B cells by KC represents an important in vivo mechanism underlying the improved activity of glycoengineered anti-CD20 mAbs.

  8. Time course investigation of PPAR{alpha}- and Kupffer cell-dependent effects of WY-14,643 in mouse liver using microarray gene expression

    SciTech Connect

    Woods, Courtney G.; Kosyk, Oksana; Bradford, Blair U.; Ross, Pamela K.; Burns, Amanda M.; Cunningham, Michael L.; Qu Pingping; Ibrahim, Joseph G.; Rusyn, Ivan

    2007-12-15

    Administration of peroxisome proliferators to rodents causes proliferation of peroxisomes, induction of {beta}-oxidation enzymes, hepatocellular hypertrophy and hyperplasia, with chronic exposure ultimately leading to hepatocellular carcinomas. Many responses associated with peroxisome proliferators are nuclear receptor-mediated events involving peroxisome proliferators-activated receptor alpha (PPAR{alpha}). A role for nuclear receptor-independent events has also been shown, with evidence of Kupffer cell-mediated free radical production, presumably through NAPDH oxidase, induction of redox-sensitive transcription factors involved in cytokine production and cytokine-mediated cell replication following acute treatment with peroxisome proliferators in rodents. Recent studies have demonstrated, by using p47{sup phox}-null mice which are deficient in NADPH oxidase, that this enzyme is not related to the phenotypic events caused by prolonged administration of peroxisome proliferators. In an effort to determine the timing of the transition from Kupffer cell-to PPAR{alpha}-dependent modulation of peroxisome proliferator effects, gene expression was assessed in liver from Ppar{alpha}-null, p47{sup phox}-null and corresponding wild-type mice following treatment with 4-chloro-6-(2,3-xylidino)-pyrimidynylthioacetic acid (WY-14,643) for 8 h, 24 h, 72 h, 1 week or 4 weeks. WY-14,643-induced gene expression in p47{sup phox}-null mouse liver differed substantially from wild-type mice at acute doses and striking differences in baseline expression of immune related genes were evident. Pathway mapping of genes that respond to WY-14,643 in a time- and dose-dependent manner demonstrates suppression of immune response, cell death and signal transduction and promotion of lipid metabolism, cell cycle and DNA repair. Furthermore, these pathways were largely dependent on PPAR{alpha}, not NADPH oxidase demonstrating a temporal shift in response to peroxisome proliferators. Overall, this

  9. Subtoxic Concentrations of Hepatotoxic Drugs Lead to Kupffer Cell Activation in a Human In Vitro Liver Model: An Approach to Study DILI

    PubMed Central

    Kegel, Victoria; Pfeiffer, Elisa; Burkhardt, Britta; Liu, Jia L.; Zeilinger, Katrin; Nüssler, Andreas K.; Seehofer, Daniel; Damm, Georg

    2015-01-01

    Drug induced liver injury (DILI) is an idiosyncratic adverse drug reaction leading to severe liver damage. Kupffer cells (KC) sense hepatic tissue stress/damage and therefore could be a tool for the estimation of consequent effects associated with DILI. Aim of the present study was to establish a human in vitro liver model for the investigation of immune-mediated signaling in the pathogenesis of DILI. Hepatocytes and KC were isolated from human liver specimens. The isolated KC yield was 1.2 ± 0.9 × 106 cells/g liver tissue with a purity of >80%. KC activation was investigated by the measurement of reactive oxygen intermediates (ROI, DCF assay) and cell activity (XTT assay). The initial KC activation levels showed broad donor variability. Additional activation of KC using supernatants of hepatocytes treated with hepatotoxic drugs increased KC activity and led to donor-dependent changes in the formation of ROI compared to KC incubated with supernatants from untreated hepatocytes. Additionally, a compound- and donor-dependent increase in proinflammatory cytokines or in anti-inflammatory cytokines was detected. In conclusion, KC related immune signaling in hepatotoxicity was successfully determined in a newly established in vitro liver model. KC were able to detect hepatocyte stress/damage and to transmit a donor- and compound-dependent immune response via cytokine production. PMID:26491234

  10. Subtoxic Concentrations of Hepatotoxic Drugs Lead to Kupffer Cell Activation in a Human In Vitro Liver Model: An Approach to Study DILI.

    PubMed

    Kegel, Victoria; Pfeiffer, Elisa; Burkhardt, Britta; Liu, Jia L; Zeilinger, Katrin; Nüssler, Andreas K; Seehofer, Daniel; Damm, Georg

    2015-01-01

    Drug induced liver injury (DILI) is an idiosyncratic adverse drug reaction leading to severe liver damage. Kupffer cells (KC) sense hepatic tissue stress/damage and therefore could be a tool for the estimation of consequent effects associated with DILI. Aim of the present study was to establish a human in vitro liver model for the investigation of immune-mediated signaling in the pathogenesis of DILI. Hepatocytes and KC were isolated from human liver specimens. The isolated KC yield was 1.2 ± 0.9 × 10(6) cells/g liver tissue with a purity of >80%. KC activation was investigated by the measurement of reactive oxygen intermediates (ROI, DCF assay) and cell activity (XTT assay). The initial KC activation levels showed broad donor variability. Additional activation of KC using supernatants of hepatocytes treated with hepatotoxic drugs increased KC activity and led to donor-dependent changes in the formation of ROI compared to KC incubated with supernatants from untreated hepatocytes. Additionally, a compound- and donor-dependent increase in proinflammatory cytokines or in anti-inflammatory cytokines was detected. In conclusion, KC related immune signaling in hepatotoxicity was successfully determined in a newly established in vitro liver model. KC were able to detect hepatocyte stress/damage and to transmit a donor- and compound-dependent immune response via cytokine production.

  11. Kupffer cell inactivation by carbon monoxide bound to red blood cells preserves hepatic cytochrome P450 via anti-oxidant and anti-inflammatory effects exerted through the HMGB1/TLR-4 pathway during resuscitation from hemorrhagic shock.

    PubMed

    Ogaki, Shigeru; Taguchi, Kazuaki; Maeda, Hitoshi; Watanabe, Hiroshi; Ishima, Yu; Otagiri, Masaki; Maruyama, Toru

    2015-10-01

    Red blood cell (RBC) transfusions for controlling hemorrhaging induce systemic ischemia reperfusion, resulting in a decrease in hepatic cytochrome P450 (CYP) levels. Carbon monoxide (CO), when bound to red blood cells (CO-RBC) has the potential to protect the hepatic CYP protein to produce a resuscitative effect in a hemorrhagic shock rat model. The aim of this study was to investigate the mechanism by which CO-RBC resuscitation from a massive hemorrhage protects against a decrease in hepatic CYP. In the early phase (∼1h) after a hemorrhage and RBC resuscitation, hepatic CYP protein levels were significantly decreased with increasing hepatic free heme levels, but were maintained by a pre-treatment of gadolinium chloride (GdCl3), a Kupffer cell inhibitor, and Trolox, an anti-oxidant agent, as well as CO-RBC resuscitation. Under these conditions, the production of reactive oxygen species (ROS) derived from activated Kupffer cells was increased, but this increase was suppressed by CO-RBC resuscitation. At a late phase (6∼24h), CYP mRNA levels decreased after hemorrhage and RBC resuscitation, but not in the case of CO-RBC resuscitation. The increases in plasma IL-6 and TNF-α levels were decreased by CO-RBC resuscitation via the suppression of the toll-like receptor-4 (TLR-4) and the expression of the high mobility group box-1 (HMGB-1). Hepatic CYP protection after a hemorrhage and CO-RBC resuscitation can be attributed to the inactivation of Kupffer cells, resulting in the suppression of ROS production in the early phase and the suppression of inflammatory cytokine production via the TLR-4/HMGB-1signal pathway in the late phase.

  12. Metabolism of supplemental iron (Fe) by hepatocytes (HC), kupffer cells (KC) and endothelial cells (EC) in neonatal pig liver

    SciTech Connect

    Caperna, T.J.; Failla, M.L.

    1986-03-05

    Newborn pigs rapidly develop anemia unless treated with supplemental Fe. The authors have developed methods to isolate and culture the predominant cell types in porcine liver to investigate cellular distribution and metabolism of Fe supplements. One-day (d) old piglets were injected with Fe-dextran (50 mg Fe/kg) and liver cells were isolated from treated and age-matched control piglets 1, 5, and 10 d later. The concentration (..mu..g/mg cell protein) of Fe increased 62-, 54-, and 5-fold over controls in KC, EC, and HC, respectively, 1 d after Fe injection. Thereafter, accumulated Fe was mobilized from all 3 cell types. By 10 d HC mobilized > 85% of accumulated Fe, while Fe levels in KC and EC from treated pigs were at least 15-fold higher than control levels. In vitro studies confirmed the greater capacity of KC and EC to accumulate colloidal Fe compared to HC. The concentration of ferritin (Ft) to liver cells from control pigs was below 0.3 ..mu..g/mg cell protein. After treatment, Ft levels peaked in HC and KC on d 1 at 5.0 and 15.6 ..mu..g/mg cell protein, but in EC on d 5 at 13.3 ..mu..g/mg. Ferritin Fe represented 9% of total Fe in KC and EC at all times after treatment, but as much as 48% in HC at 1 d. Continued investigation of hepatic cellular metabolism of supplemental Fe provides a useful model for investigating the treatment of human neonatal anemia.

  13. Loss of lysosomal membrane protein NCU-G1 in mice results in spontaneous liver fibrosis with accumulation of lipofuscin and iron in Kupffer cells

    PubMed Central

    Kong, Xiang Y.; Nesset, Cecilie Kasi; Damme, Markus; Løberg, Else-Marit; Lübke, Torben; Mæhlen, Jan; Andersson, Kristin B.; Lorenzo, Petra I.; Roos, Norbert; Thoresen, G. Hege; Rustan, Arild C.; Kase, Eili T.; Eskild, Winnie

    2014-01-01

    Human kidney predominant protein, NCU-G1, is a highly conserved protein with an unknown biological function. Initially described as a nuclear protein, it was later shown to be a bona fide lysosomal integral membrane protein. To gain insight into the physiological function of NCU-G1, mice with no detectable expression of this gene were created using a gene-trap strategy, and Ncu-g1gt/gt mice were successfully characterized. Lysosomal disorders are mainly caused by lack of or malfunctioning of proteins in the endosomal-lysosomal pathway. The clinical symptoms vary, but often include liver dysfunction. Persistent liver damage activates fibrogenesis and, if unremedied, eventually leads to liver fibrosis/cirrhosis and death. We demonstrate that the disruption of Ncu-g1 results in spontaneous liver fibrosis in mice as the predominant phenotype. Evidence for an increased rate of hepatic cell death, oxidative stress and active fibrogenesis were detected in Ncu-g1gt/gt liver. In addition to collagen deposition, microscopic examination of liver sections revealed accumulation of autofluorescent lipofuscin and iron in Ncu-g1gt/gt Kupffer cells. Because only a few transgenic mouse models have been identified with chronic liver injury and spontaneous liver fibrosis development, we propose that the Ncu-g1gt/gt mouse could be a valuable new tool in the development of novel treatments for the attenuation of fibrosis due to chronic liver damage. PMID:24487409

  14. Subanesthetic Isoflurane Reduces Zymosan-Induced Inflammation in Murine Kupffer Cells by Inhibiting ROS-Activated p38 MAPK/NF-κB Signaling

    PubMed Central

    Wang, Hui; Wang, Lei; Li, Nan-lin; Li, Jun-tang; Yu, Feng; Zhao, Ya-li; Wang, Ling; Yi, Jun; Wang, Ling; Bian, Jie-fang; Chen, Jiang-hao; Yuan, Shi-fang; Wang, Ting; Lv, Yong-gang; Liu, Ning-ning; Zhu, Xiao-shan; Ling, Rui; Yun, Jun

    2014-01-01

    Volatile anesthetic isoflurane (ISO) has immunomodulatory effects. The fungal component zymosan (ZY) induces inflammation through toll-like receptor 2 or dectin-1 signaling. We investigated the molecular actions of subanesthetic (0.7%) ISO against ZY-induced inflammatory activation in murine Kupffer cells (KCs), which are known as the resident macrophages within the liver. We observed that ISO reduced ZY-induced cyclooxygenase 2 upregulation and prostaglandin E2 release, as determined by western blot and radioimmunoassay, respectively. ISO also reduced the production of tumor necrosis factor-α, interleukin-1β, IL-6, high-mobility group box-1, macrophage inflammatory protein-1α, macrophage inflammatory protein-2, and monocyte chemoattractant protein-1 as assessed by enzyme-linked immunosorbent assays. ISO blocked the ZY-induced nuclear translocation and DNA-binding activity of nuclear factor- (NF)-κB p65. Moreover, ISO attenuated ZY-induced p38 mitogen-activated protein kinase (MAPK) activation partly by scavenging reactive oxygen species (ROS); the interregulation that ROS activated p38 MAPK followed by NF-κB activation was crucial for the ZY-induced inflammatory responses in KCs. An in vivo study by peritoneal injection of ZY into BALB/C mice confirmed the anti-inflammatory properties of 0.7% ISO against ZY in KCs. These results suggest that ISO ameliorates ZY-induced inflammatory responses in murine KCs by inhibiting the interconnected ROS/p38 MAPK/NF-κB signaling pathways. PMID:25147596

  15. Mercury-Selenium Relationships in Liver of Guiana Dolphin: The Possible Role of Kupffer Cells in the Detoxification Process by Tiemannite Formation

    PubMed Central

    Lailson-Brito, José; Dorneles, Paulo Renato; Andrade, Leonardo; Azevedo, Alexandre de Freitas; Fragoso, Ana Bernadete; Vidal, Lara Gama; Costa, Marianna Badini; Bisi, Tatiana Lemos; Almeida, Ronaldo; Carvalho, Dario Pires; Bastos, Wanderley Rodrigues; Malm, Olaf

    2012-01-01

    Top marine predators present high mercury concentrations in their tissues as consequence of biomagnification of the most toxic form of this metal, methylmercury (MeHg). The present study concerns mercury accumulation by Guiana dolphins (Sotalia guianensis), highlighting the selenium-mediated methylmercury detoxification process. Liver samples from 19 dolphins incidentally captured within Guanabara Bay (Rio de Janeiro State, Brazil) from 1994 to 2006 were analyzed for total mercury (THg), methylmercury (MeHg), total organic mercury (TOrgHg) and selenium (Se). X-ray microanalyses were also performed. The specimens, including from fetuses to 30-year-old dolphins, comprising 8 females and 11 males, presented high THg (0.53–132 µg/g wet wt.) and Se concentrations (0.17–74.8 µg/g wet wt.). Correlations between THg, MeHg, TOrgHg and Se were verified with age (p<0.05), as well as a high and positive correlation was observed between molar concentrations of Hg and Se (p<0.05). Negative correlations were observed between THg and the percentage of MeHg contribution to THg (p<0.05), which represents a consequence of the selenium-mediated methylmercury detoxification process. Accumulation of Se-Hg amorphous crystals in Kupffer Cells was demonstrated through ultra-structural analysis, which shows that Guiana dolphin is capable of carrying out the demethylation process via mercury selenide formation. PMID:22860072

  16. Effects of the in vitro administered ethanol and lipopolysaccharide toxin on membrane properties, intracellular free calcium and phagocytic function of isolated rat kupffer cells

    SciTech Connect

    Victorov, A.; Smith, T.; Abril, E.; Hamlin, E.; Earnest, D. )

    1991-03-11

    Low concentrations of ethanol slightly stimulated phagocytosis of cultured Kupffer cells (KC), producing practically no effect on membrane microviscosity and cytosolic free (Ca{sup 2+}){sub i}. On the contrary, high concentrations of ethanol significantly suppressed phagocytic function, increased fluidity of membrane lipids and caused a sustained rise in (Ca{sup 2}){sub i}; above the resting level of 41-85 nM. Treatment of KC with colchicine and cytochalasin B dramatically destructurized the plasma membrane lipids. Short term preincubation of KC with high doses of alcohol stimulated the disordering effects of both drugs, suggesting direct interaction of ethanol with microtubule and microfilament structures. The authors hypothesize that ethanol impairs phagocytosis of KC by concerted actions on membrane lipid fluidity, cytosolic free Ca{sup 2+} and functioning of cytoskeleton. On the other hand, incubation of KC with low concentrations of lipopolysaccharide (LPS) produced no changes in (Ca{sup 2+}){sub i}; or plasma membrane fluidity but reduced by several fold the fluidizing effect of subsequently added ethanol. They suggested that low doses of LPS, by activating second messengers other than Ca{sup 2+}, alter the functioning of the cytoskeleton and cause reorganization of the plasma membrane thus making KC membranes more resistent to the fluidizing action of ethanol and partially restoring the phagocytic function.

  17. ETHANOL AND ARACHIDONIC ACID SYNERGIZE TO ACTIVATE KUPFFER CELLS AND MODULATE THE FIBROGENIC RESPONSE VIA TNFα, GSH, AND TGFβ-DEPENDENT MECHANISMS

    PubMed Central

    2015-01-01

    Aim because of the contribution of ethanol and polyunsaturated fatty acids (PUFAs) to alcoholic liver disease, we investigated whether chronic ethanol administration and arachidonic acid (AA) could synergistically mediate Kupffer cell (KC) activation and modulate the stellate cell (HSC) fibrogenic response. Results 1) Ethanol and AA effects on KC and HSC mono-cultures: cell proliferation, lipid peroxidation, H2O2, O2.−, NADPH oxidase activity, and TNFα were higher in KCethanol than in KCcontrol, and were enhanced by AA; HSCethanol proliferated faster, increased collagen, and showed higher GSH than HSCcontrol, with modest effects by AA. 2) AA effects on the control co-culture: we previously reported (1) the ability of KC to induce a pro-fibrogenic response in HSC via ROS-dependent mechanisms; we now show that AA further increases cell proliferation and collagen in the control co-culture. The latter was prevented by vitamin E (an antioxidant) and by diphenyleneiodonium (a NADPH oxidase inhibitor). 3) Ethanol effects on the co-cultures: co-culture with KCcontrol or KCethanol induced HSCcontrol and HSCethanol proliferation; however, the pro-fibrogenic response in HSCethanol was suppressed due to up-regulation of TNFα and GSH, which was prevented by a TNFα neutralizing Ab and by l-buthionine-sulfoximine, a GSH-depleting agent. 4) Ethanol plus AA effects on the co-cultures: AA lowered TNFα in the HSCcontrol co-cultures allowing for enhanced collagen deposition; furthermore, AA restored the pro-fibrogenic response in the HSCethanol co-cultures by counteracting the up-regulation of TNFα and GSH with a significant increase in GSSG and in pro-fibrogenic TGFβ. Conclusion these results unveil synergism between ethanol and AA to the mechanism whereby KC mediate ECM remodeling, and suggest that even if chronic ethanol consumption sensitizes HSC to up-regulate anti-fibrogenic signals, their effects are blunted by a second ‘hit’ such as AA. PMID:19003881

  18. Anti-TNF monoclonal antibodies prevent haemorrhage-induced suppression of Kupffer cell antigen presentation and MHC class II antigen expression.

    PubMed Central

    Ertel, W; Morrison, M H; Ayala, A; Perrin, M M; Chaudry, I H

    1991-01-01

    Kupffer cells (KC), by virtue of their ability to present antigen (AP) and express major histocompatibility complex (MHC) class II antigen (Ia), play a pivotal role in the host defence system against invading micro-organisms. Although haemorrhagic shock depresses the above KC functions, it is not known whether increased KC tumour necrosis factor (TNF) production and elevated TNF plasma levels following haemorrhage are responsible for it. To study this, C3H/HeN mice were pretreated intraperitoneally with either anti-murine TNF antibody (anti-TNF Ab) or saline. Twenty hours later mice were bled and maintained at a mean blood pressure of 35 mmHg for 60 min followed by adequate fluid resuscitation. Two and 24 hr later, plasma was collected and KC were isolated. AP was measured by co-culturing KC with the D10.G4.1 Th cell clone. Ia expression was determined by direct immunofluorescence. Interleukin (IL)-1, IL-6 and TNF levels in KC supernatants and plasma were measured with bioassays or ELISA. Haemorrhage increased circulating TNF levels by 215% at 2 hr and by 76% at 24 hr (P less than 0.05), which was prevented by pretreatment with anti-TNF Ab. Haemorrhage-induced increase of circulating IL-6 was abolished (P less than 0.05) at 2 hr but not at 24 hr in the anti-TNF Ab group. The suppression of KC AP (P less than 0.05) and Ia expression (P less than 0.05) due to haemorrhage was attenuated (P less than 0.05) in anti-TNF Ab-treated mice at 2 and 24 hr and KC IL-1 and TNF synthesis was further (P less than 0.01) increased. These results indicate that TNF plays a critical role in the initiation and regulation of KC AP, Ia expression, and cytokine production following haemorrhage. PMID:1748476

  19. Lipopolysaccharide (LPS) processing by Kupffer cells releases a modified LPS with increased hepatocyte binding and decreased tumor necrosis factor-alpha stimulatory capacity.

    PubMed

    Treon, S P; Thomas, P; Broitman, S A

    1993-02-01

    Normal physiological clearance of gut-derived endotoxin lipopolysaccharide [LPS] has been described previously; initially, there is uptake by Kupffer cells (KC), then release of modified LPS, followed by hepatocyte uptake. Previous work in our laboratories indicated that LPS is structurally modified with loss of carbohydrate prior to its release by KC. In this study, we functionally characterize KC modified LPS. KC-modified 125I-LPS was prepared from primary rat KC. Escherichia coli 0127:B8 native 125I-LPS or KC-modified 125I-LPS (40 ng) was incubated for 1 hr with 1 x 10E6 primary hepatocytes. The binding of KC-modified LPS was 4.33-fold higher than native LPS (P = 0.0024). Binding analysis studies were conducted to determine the region of KC-modified LPS responsible for enhanced hepatocyte binding. KC-modified Salmonella minnesota LPS was competed with 100-fold excess native or mutant (Ra, Rc, Rd, or Re) strains of LPS or Lipid A with no decrease to hepatocyte binding. S. minnesota-native 125I-LPS was compared with KC-modified 125I-LPS in a study to assess induction of tumor necrosis factor (TNF)-gamma by rat peritoneal macrophages. Native or KC-modified 125I-LPS (100 ng) was presented to 1 x 10E7 peritoneal macrophages for 6 hr. TNF-alpha was measured in supernatants using the WEHI-164 cytotoxicity assay. Native LPS induced 5.7-fold higher TNF-alpha levels than KC-modified LPS (P < 0.0001). The above data suggest that structural alterations in KC-modified LPS are accompanied by functional alterations resulting in enhanced hepatocyte binding and decreased TNF-alpha release. The latter result implies that an early step in LPS detoxification occurs in the KC in which LPS is modified to prevent elicitation of biologically active cytokines.

  20. Kupffer cells ameliorate hepatic insulin resistance induced by high-fat diet rich in monounsaturated fatty acids: the evidence for the involvement of alternatively activated macrophages

    PubMed Central

    2012-01-01

    Background Resident macrophages (Kupffer cells, KCs) in the liver can undergo both pro- or anti-inflammatory activation pathway and exert either beneficiary or detrimental effects on liver metabolism. Until now, their role in the metabolically dysfunctional state of steatosis remains enigmatic. Aim of our study was to characterize the role of KCs in relation to the onset of hepatic insulin resistance induced by a high-fat (HF) diet rich in monounsaturated fatty acids. Methods Male Wistar rats were fed either standard (SD) or high-fat (HF) diet for 4 weeks. Half of the animals were subjected to the acute GdCl3 treatment 24 and 72 hrs prior to the end of the experiment in order to induce the reduction of KCs population. We determined the effect of HF diet on activation status of liver macrophages and on the changes in hepatic insulin sensitivity and triacylglycerol metabolism imposed by acute KCs depletion by GdCl3. Results We found that a HF diet rich in MUFA itself triggers an alternative but not the classical activation program in KCs. In a steatotic, but not in normal liver, a reduction of the KCs population was associated with a decrease of alternative activation and with a shift towards the expression of pro-inflammatory activation markers, with the increased autophagy, elevated lysosomal lipolysis, increased formation of DAG, PKCε activation and marked exacerbation of HF diet-induced hepatic insulin resistance. Conclusions We propose that in the presence of a high MUFA content the population of alternatively activated resident liver macrophages may mediate beneficial effects on liver insulin sensitivity and alleviate the metabolic disturbances imposed by HF diet feeding and steatosis. Our data indicate that macrophage polarization towards an alternative state might be a useful strategy for treating type 2 diabetes. PMID:22439764

  1. Kupffer cell depletion protects against the steatosis, but not the liver damage, induced by marginal-copper, high-fructose diet in male rats

    PubMed Central

    Schuschke, Dale A.; Zhou, Zhanxiang; Zhong, Wei; Zhang, Jiayuan; Zhang, Xiang; Wang, Yuhua; McClain, Craig J.

    2015-01-01

    High-fructose feeding impairs copper status and leads to low copper availability, which is a novel mechanism in obesity-related fatty liver. Copper deficiency-associated hepatic iron overload likely plays an important role in fructose-induced liver injury. Excess iron in the liver is distributed throughout hepatocytes and Kupffer cells (KCs). The aim of this study was to examine the role of KCs in the pathogenesis of nonalcoholic fatty liver disease induced by a marginal-copper high-fructose diet (CuMF). Male weanling Sprague-Dawley rats were fed either a copper-adequate or a marginally copper-deficient diet for 4 wk. Deionized water or deionized water containing 30% fructose (wt/vol) was also given ad libitum. KCs were depleted by intravenous administration of gadolinium chloride (GdCl3) before and/or in the middle of the experimental period. Hepatic triglyceride accumulation was completely eliminated with KC depletion in CuMF consumption rats, which was associated with the normalization of elevated plasma monocyte chemoattractant protein-1 (MCP-1) and increased hepatic sterol regulatory element binding protein-1 expression. However, hepatic copper and iron content were not significantly affected by KC depletion. In addition, KC depletion reduced body weight and epididymal fat weight as well as adipocyte size. Plasma endotoxin and gut permeability were markedly increased in CuMF rats. Moreover, MCP-1 was robustly increased in the culture medium when isolated KCs from CuMF rats were treated with LPS. Our data suggest that KCs play a critical role in the development of hepatic steatosis induced by marginal-copper high-fructose diet. PMID:25813056

  2. A transgenic rat hepatocyte - Kupffer cell co-culture model for evaluation of direct and macrophage-related effect of poly(amidoamine) dendrimers.

    PubMed

    Jemnitz, Katalin; Bátai-Konczos, Attila; Szabó, Mónika; Ioja, Enikő; Kolacsek, Orsolya; Orbán, Tamás I; Török, György; Homolya, László; Kovács, Eszter; Jablonkai, István; Veres, Zsuzsa

    2017-02-01

    Increasing number of papers demonstrate that Kupffer cells (KCs) play a role in the development of drug induced liver injury (DILI). Furthermore, elevated intracellular Ca(2+) level of hepatocytes is considered as a common marker of DILI. Here we applied an in vitro model based on hepatocyte mono- and hepatocyte/KC co-cultures (H/KC) isolated from transgenic rats stably expressing the GCaMP2 fluorescent Ca(2+) sensor protein to investigate the effects of polycationic (G5), polyanionic (G4.5) and polyethylene-glycol coated neutral (G5 Peg) dendrimers known to accumulate in the liver, primarily in KCs. Following dendrimer exposure, hepatocyte homeostasis was measured by MTT cytotoxicity assay and by Ca(2+) imaging, while hepatocyte functions were studied by CYP2B1/2 inducibility, and bilirubin and taurocholate transport. G5 was significantly more cytotoxic than G4.5 for hepatocytes and induced Ca(2+) oscillation and sustained Ca(2+) signals at 1μM and10 μM, respectively both in hepatocytes and KCs. Dendrimer-induced Ca(2+) signals in hepatocytes were attenuated by macrophages. Activation of KCs by lipopolysaccharide and G5 decreased the inducibility of CYP2B1/2, which was restored by depleting the KCs with gadolinium-chloride and pentoxyphylline, suggesting a role of macrophages in the hindrance of CYP2B1/2 induction by G5 and lipopolysaccharide. In the H/KC, but not in the hepatocyte mono-culture, G5 reduced the canalicular efflux of bilirubin and stimulated the uptake and canalicular efflux of taurocholate. In conclusion, H/KC provides a good model for the prediction of hepatotoxic potential of drugs, especially of nanomaterials known to be trapped by macrophages, activation of which presumably contributes to DILI.

  3. The SMAC mimetic birinapant attenuates lipopolysaccharide-induced liver injury by inhibiting the tumor necrosis factor receptor-associated factor 3 degradation in Kupffer cells.

    PubMed

    Liu, Hongxiang; Liao, Rui; He, Kun; Zhu, Xiwen; Li, Peizhi; Gong, Jianping

    2017-03-09

    It was demonstrated that second mitochondria-derived activator of caspases (SMAC) mimetic inhibites tumor necrosis factor receptor-associated factor 3 (TRAF3) degradation and the mitogen-activated protein kinase (MAPK) signaling pathway activation induced by lipopolysaccharide (LPS) in vitro. However, the effect of Smac mimetic in vivo is not clear. The present study was to investigate the role of Smac mimetic in LPS-induced liver injury in mice and its possible mechanism. An animal model of LPS-induced liver injury was established by intraperitoneally injecting mice with 10mg/kg LPS pretreatment with or without Smac mimetic birinapant (30mg/kg body weight). Birinapant significantly improved the survival rate of endotoxemic mice (P<0.05) and attenuated LPS-induced liver pathologic damage and inflammatory response. IL-1 and TNF-α levels in the serum were markedly decreased in birinapant pretreatment mice compared with control mice (P<0.05).The cellular inhibitor of apoptosis protein 1 (cIAP1) expression in liver resident macrophage (Kupffer cells, KCs) was significantly decreased in the Birinapant group compared to the Vehicle group (P<0.05). At the same time, total TRAF3 protein abundance in KCs rapidly declined after LPS stimulation in the Vehicle group. However, it remained constant in the Birinapant group. Moreover, K48-linked polyubiquitination of TRAF3 in KCs was markedly impressed in the birinapant group compared with the control group. At last, the JNK and p38 MAPK activation in KCs was significantly inhibited by birinapant pretreatment (P<0.05). These results suggested that birinapant attenuated liver injury and improved survival rates in endotoxemic mice by inhibited the expression of cIAP1, degradation of TRAF3 and aviation of MAPK signaling pathway.

  4. [THE EXCESS OF PALMITIC FATTY ACID IN FOOD AS MAIN CAUSE OF LIPOIDOSIS OF INSULIN-DEPENDENT CELLS: SKELETAL MYOCYTES, CARDIO-MYOCYTES, PERIPORTAL HEPATOCYTES, KUPFFER MACROPHAGES AND B-CELLS OF PANCREAS].

    PubMed

    Titov, V N

    2016-02-01

    In phylogenesis, becoming of biologicalfunctions and biological reactions proceeds with the purpose ofpermanent increasing of "kinetic perfection ". The main role belongs to factors ofphysical, chemical and biological kinetics, their evaluation using systemic approach technique under permanent effect of natural selection. The late-in-phylogenesis insulin, proceeded with, in development of biological function of locomotion, specialization of insulin-dependent cells: skeletal myocytes, syncytium of cardiomyocytes, subcutaneous adipocytes, periportal hepatocytes, Kupffer's macrophages and β-cells of islets of pancreas. The insulin initiated formation of new, late in phylogenesis, large pool of fatty cells-subcutaneous adipocytes that increased kinetic parameters of biological function of locomotion. In realization of biological function of locomotion only adipocytes absorb exogenous mono unsaturated and saturated fatty acids in the form of triglycerides in composition of oleic and palmitic lipoproteins of very low density using apoE/B-100 endocytosis. The rest of insulin-dependent cells absorb fatty acids in the form of unesterified fatty acids from associates with albumin and under effect of CD36 of translocase offatty acids. The insulin in all insulin-depended cells inhibits biological reaction of lipolysis enhancing contributing into development of lipoidosis. The insulin expresses transfer offatty acids in the form of unsaturated fatty acids from adipocytes into matrix of mitochondria. The insulin supplies insulin-dependent cells with substrates for acquiring energy subject to that in pool of unsaturated fatty acids in adipocytes prevails hydrophobic palmitic unsaturated fatiy acid that slowly passes into matrix through external membrane ofmitochondria; oxidases of mitochondria so slowly implement its β-oxidation that content of exogenous palmitic unsaturatedfatty acid can't be higher than phylogenetic, physiological level - 15% of all amount offatty acids

  5. International Cell Exchange, 1994.

    PubMed

    Lau, M; Terasaki, P I; Park, M S

    1994-01-01

    1. We summarize typings of 40 cells for Class I antigens and 20 cultured cell lines for Class II antigens through the International Cell Exchange in 1994. Serologic Class II typings were compared with DNA typings for the same 20 cells. Two hundred eighty-one laboratories participated in the monthly Class I Serum Exchange. One hundred nineteen serology laboratories and 74 DNA laboratories reported Class II specificities on a monthly basis. 2. The average detection levels, as well as the high detection levels, were determined for 16 A-locus and 27 B-locus antigens. Mean detection rates of 95% or greater average detection were obtained for 12 A-locus and 10 B-locus antigens. Lower than 80% agreement was calculated for one A-locus antigen (A74) and 7 B-locus (B46, B48, B61, B67, B73, B75, B77) antigens. 3. We compared discrepancy rates of 10 A-locus and 7 B-locus antigens typed 3 times or more. The false-negative discrepancy rates, i.e. how often the antigen was missed, were greater for more of the B-locus specificities than for the A-locus antigens. B62, having the highest false-positive rate, tended to be overassigned. The discrepancy rates, especially the false-negative rate, for B70 were shown to decrease over a 7-year period. 4. In 1994, 8 laboratories attained records of total no misses for all analyzed antigens. Twelve laboratories had final records of only one discrepancy, and 5 laboratories had impressive perfect records (zero false negatives and false positives) for their yearly antigen reports. 5. Retyping of 12 Class I and 8 Class II reference cells showed improved detection of antigens. Results of a donor typed 4 times over 11 years demonstrated marked improvement, nearly doubling for A33, B38, and B75. Two cells first typed in 1991, then retyped in 1994, showed improved detection for Class II splits by serology and DNA typing. 6. We updated the list of sequenced Class I Exchange cells. Seven new cells were added as well as confirmatory sequence data for A

  6. ES Cell International & regenerative medicine.

    PubMed

    Colman, Alan; Alliston, Doug

    2006-03-01

    ES Cell International believes that human embryonic stem cells offer a renewable source material for the elaboration of a multitude of different tissues for use in human cell replacement therapy. There are many challenges to the successful implementation of this vision including technical, regulatory, financial and ethical hurdles. However with demographic changes towards an aging population in the richer countries, leading to growing demands of ways to combat degenerative disease, science would be remiss not to explore every potential medical solution.

  7. Sept6 is required for ciliogenesis in Kupffer's vesicle, the pronephros, and the neural tube during early embryonic development.

    PubMed

    Zhai, Gang; Gu, Qilin; He, Jiangyan; Lou, Qiyong; Chen, Xiaowen; Jin, Xia; Bi, Erfei; Yin, Zhan

    2014-04-01

    Septins are conserved filament-forming GTP-binding proteins that act as cellular scaffolds or diffusion barriers in a number of cellular processes. However, the role of septins in vertebrate development remains relatively obscure. Here, we show that zebrafish septin 6 (sept6) is first expressed in the notochord and then in nearly all of the ciliary organs, including Kupffer's vesicle (KV), the pronephros, eye, olfactory bulb, and neural tube. Knockdown of sept6 in zebrafish embryos results in reduced numbers and length of cilia in KV. Consequently, cilium-related functions, such as the left-right patterning of internal organs and nodal/spaw signaling, are compromised. Knockdown of sept6 also results in aberrant cilium formation in the pronephros and neural tube, leading to cilium-related defects in pronephros development and Sonic hedgehog (Shh) signaling. We further demonstrate that SEPT6 associates with acetylated α-tubulin in vivo and localizes along the axoneme in the cilia of zebrafish pronephric duct cells as well as cultured ZF4 cells. Our study reveals a novel role of sept6 in ciliogenesis during early embryonic development in zebrafish.

  8. Two T-box genes play independent and cooperative roles to regulate morphogenesis of ciliated Kupffer's vesicle in zebrafish.

    PubMed

    Amack, Jeffrey D; Wang, Xinghao; Yost, H Joseph

    2007-10-15

    The brain, heart and gastro-intestinal tract develop distinct left-right (LR) asymmetries. Asymmetric cilia-dependent fluid flow in the embryonic node in mouse, Kupffer's vesicle in zebrafish, notochordal plate in rabbit and gastrocoel roof plate in frog appears to be a conserved mechanism that directs LR asymmetric gene expression and establishes the orientation of organ asymmetry. However, the cellular processes and genetic pathways that control the formation of these essential ciliated structures are unknown. In zebrafish, migratory dorsal forerunner cells (DFCs) give rise to Kupffer's vesicle (KV), a ciliated epithelial sheet that forms a lumen and generates fluid flow. Using the epithelial marker atypical Protein Kinase C (aPKC) and other markers to analyze DFCs and KV cells, we describe a multi-step process by which DFCs form a functional KV. Using mutants and morpholinos, we show that two T-box transcription factors-No tail (Ntl)/Brachyury and Tbx16/Spadetail-cooperatively regulate an early step of DFC mesenchyme to epithelial transition (MET) and KV cell specification. Subsequently, each transcription factor independently controls a distinct step in KV formation: Tbx16 regulates apical clustering of KV cells and Ntl is necessary for KV lumen formation. By targeting morpholinos to DFCs, we show that these cell autonomous functions in KV morphogenesis are necessary for LR patterning throughout the embryo.

  9. Nanodiamond internalization in cells and the cell uptake mechanism

    NASA Astrophysics Data System (ADS)

    Perevedentseva, E.; Hong, S.-F.; Huang, K.-J.; Chiang, I.-T.; Lee, C.-Y.; Tseng, Y.-T.; Cheng, C.-L.

    2013-08-01

    Cell type-dependent penetration of nanodiamond in living cells is one of the important factors for using nanodiamond as cellular markers/labels, for drug delivery as well as for other biomedical applications. In this work, internalization of 100 nm nanodiamonds by A549 lung human adenocarcinoma cell, Beas-2b non-tumorigenic human bronchial epithelial cell, and HFL-1 fibroblast-like human fetal lung cell is studied and compared. The penetration of nanodiamond into the cells was observed using confocal fluorescence imaging and Raman imaging methods. Visualization of the nanodiamond in cells allows comparison of the internalization for diamond nanoparticles in cancer A549 cell, non-cancer HFL-1, and Beas-2b cells. The dose-dependent and time-dependent behavior of nanodiamond uptake is observed in both cancer as well as non-cancer cells. The mechanism of nanodiamond uptake by cancer and non-cancer cells is analyzed by blocking different pathways. The uptake of nanodiamond in both cancer and non-cancer cells was found predominantly via clathrin-dependent endocytosis. In spite of observed similarity in the uptake mechanism for cancer and non-cancer cells, the nanodiamond uptake for cancer cell quantitatively exceeds the uptake for non-cancer cells, for the studied cell lines. The observed difference in internalization of nanodiamond by cancer and non-cancer cells is discussed.

  10. Development of PEM fuel cell technology at international fuel cells

    SciTech Connect

    Wheeler, D.J.

    1996-04-01

    The PEM technology has not developed to the level of phosphoric acid fuel cells. Several factors have held the technology development back such as high membrane cost, sensitivity of PEM fuel cells to low level of carbon monoxide impurities, the requirement to maintain full humidification of the cell, and the need to pressurize the fuel cell in order to achieve the performance targets. International Fuel Cells has identified a hydrogen fueled PEM fuel cell concept that leverages recent research advances to overcome major economic and technical obstacles.

  11. [Sickle Cell Disease International Organization (SCDIO)].

    PubMed

    Ebakisse-Badassou, E

    2010-12-01

    A century after the first scientific description of sickle cell disease that had been known to African peoples under various names for more than three centuries, it is now time for this blood disorder and its associated sanitary and social disparities to come out of the shadows. Although sickle cell disease is the most widespread genetic disease in the world, public awareness remains low despite the considerable effort that has been made over the last decade. In order to improve understanding and management, the Sickle Cell Disease International Organization (SCDIO) has defined the following ambitious objectives: to implement effective action plans that must be based on the ability to sensitize minds about this disease; expand active support from public and private personalities and organizations; convince potential partners in all countries involved of the need for their active support of this important cause; raise funds that are indispensable to finance planned operations; provide effective organization to carry out priority initiatives aimed at lowering child mortality due to sickle cell disease in the world. To these ends, the SCDIO will continue in its advocacy role that will not stop until the resolutions are adopted and applied in all affected countries. The SCDIO will continue to prioritize the development of south/south partnerships. In view of the history of sickle cell disease, the major challenges for the next century will consist of prioritizing action to improve the quality of life of patients wherever they are and of developing research. To meet these challenges, we will need the involvement and support of the entire international community. We must all stand "United against sickle cell disease".

  12. Thanks to Cancer Cell International reviewers (2012)

    PubMed Central

    2013-01-01

    Contributing reviewers The editor and editorial board of Cancer Cell International would like to thank Angela Panther for her continued hard work throughout 2012 in keeping the journal going as our managing editor. We take this opportunity in early 2013 to thank all our reviewers who contributed to the journal in Volume 12 (2012). Last year saw submissions double, which will mean that in 2013 we will certainly have many more articles to publish, and hopefully our Impact Factor will rise above 2. PMID:23522244

  13. On-Demand Cell Internal Short Circuit Device

    NASA Technical Reports Server (NTRS)

    Darcy, Eric; Keyser, Matthew

    2014-01-01

    A device implantable in Li-ion cells that can generate a hard internal short circuit on-demand by exposing the cell to 60?C has been demonstrated to be valuable for expanding our understanding of cell responses. The device provides a negligible impact to cell performance and enables the instigation of the 4 general categories of cell internal shorts to determine relative severity and cell design susceptibility. Tests with a 18650 cell design indicates that the anode active material short to the aluminum cathode current collector tends to be more catastrophic than the 3 other types of internal shorts. Advanced safety features (such as shutdown separators) to prevent or mitigate the severity of cell internal shorts can be verified with this device. The hard short success rate achieved to date in 18650 cells is about 80%, which is sufficient for using these cells in battery assemblies for field-failure-relevant, cell-cell thermal runaway propagation verification tests

  14. Kupffer-phase findings of hepatic hemangiomas in contrast-enhanced ultrasound with sonazoid.

    PubMed

    Sugimoto, Katsutoshi; Moriyasu, Fuminori; Saito, Kazuhiro; Yoshiara, Hiroki; Imai, Yasuharu

    2014-06-01

    The aim of this study was to assess quantitatively the Kupffer-phase enhancement patterns of hepatic hemangiomas in contrast-enhanced ultrasound (CEUS) with Sonazoid. A total of 46 patients with 46 hepatic hemangiomas (17.1 ± 6.2 mm in diameter, 34 typical type and 12 high-flow type) underwent CEUS in the Kupffer phase. The lesion-to-liver contrast ratio in the Kupffer phase was quantitatively assessed for both types of hemangioma. Most of the hepatic hemangiomas, whether or not they were the high-flow type, were iso- to hypo-echoic relative to the surrounding liver parenchyma. The contrast ratio was -5.33 ± 6.70 dB for the high-flow hemangiomas and -4.54 ± 6.28 dB for the typical hemangiomas. There was no significant difference in contrast ratio between the two types of lesions (p = 0.73). All of the hemangiomas, whether of typical or high-flow type, are iso- to hypo-echoic relative to the surrounding liver parenchyma on Kupffer-phase imaging.

  15. Particle compositions with a pre-selected cell internalization mode

    NASA Technical Reports Server (NTRS)

    Decuzzi, Paolo (Inventor); Ferrari, Mauro (Inventor)

    2012-01-01

    A method of formulating a particle composition having a pre-selected cell internalization mode involves selecting a target cell having surface receptors and obtaining particles that have i) surface moieties, that have an affinity for or are capable of binding to the surface receptors of the cell and ii) a preselected shape, where a surface distribution of the surface moieties on the particles and the shape of the particles are effective for the pre-selected cell internalization mode.

  16. Development of large scale internal reforming molten carbonate fuel cell

    SciTech Connect

    Sasaki, A.; Shinoki, T.; Matsumura, M.

    1996-12-31

    Internal Reforming (IR) is a prominent scheme for Molten Carbonate Fuel Cell (MCFC) power generating systems in order to get high efficiency i.e. 55-60% as based on the Higher Heating Value (HHV) and compact configuration. The Advanced Internal Reforming (AIR) technology has been developed based on two types of the IR-MCFC technology i.e. Direct Internal Reforming (DIR) and Indirect Internal Reforming (DIR).

  17. Dynamics of Receptor-Mediated Nanoparticle Internalization into Endothelial Cells

    PubMed Central

    Gonzalez-Rodriguez, David; Barakat, Abdul I.

    2015-01-01

    Nanoparticles offer a promising medical tool for targeted drug delivery, for example to treat inflamed endothelial cells during the development of atherosclerosis. To inform the design of such therapeutic strategies, we develop a computational model of nanoparticle internalization into endothelial cells, where internalization is driven by receptor-ligand binding and limited by the deformation of the cell membrane and cytoplasm. We specifically consider the case of nanoparticles targeted against ICAM-1 receptors, of relevance for treating atherosclerosis. The model computes the kinetics of the internalization process, the dynamics of binding, and the distribution of stresses exerted between the nanoparticle and the cell membrane. The model predicts the existence of an optimal nanoparticle size for fastest internalization, consistent with experimental observations, as well as the role of bond characteristics, local cell mechanical properties, and external forces in the nanoparticle internalization process. PMID:25901833

  18. Fuel cell with internal flow control

    DOEpatents

    Haltiner, Jr., Karl J.; Venkiteswaran, Arun [Karnataka, IN

    2012-06-12

    A fuel cell stack is provided with a plurality of fuel cell cassettes where each fuel cell cassette has a fuel cell with an anode and cathode. The fuel cell stack includes an anode supply chimney for supplying fuel to the anode of each fuel cell cassette, an anode return chimney for removing anode exhaust from the anode of each fuel cell cassette, a cathode supply chimney for supplying oxidant to the cathode of each fuel cell cassette, and a cathode return chimney for removing cathode exhaust from the cathode of each fuel cell cassette. A first fuel cell cassette includes a flow control member disposed between the anode supply chimney and the anode return chimney or between the cathode supply chimney and the cathode return chimney such that the flow control member provides a flow restriction different from at least one other fuel cell cassettes.

  19. Sox17 and chordin are required for formation of Kupffer's vesicle and left-right asymmetry determination in zebrafish.

    PubMed

    Aamar, Emil; Dawid, Igor B

    2010-11-01

    Kupffer's vesicle (KV), a ciliated fluid-filled sphere in the zebrafish embryo with a critical role in laterality determination, is derived from a group of superficial cells in the organizer region of the gastrula named the dorsal forerunner cells (DFC). We have examined the role of the expression of sox17 and chordin (chd) in the DFC in KV formation and laterality determination. Whereas sox17 was known to be expressed in DFC, its function in these cells was not studied before. Further, expression of chd in these cells has not been reported previously. Targeted knockdown of Sox17 and Chd in DFC led to aberrant Left-Right (L-R) asymmetry establishment, as visualized by the expression of southpaw and lefty, and heart and pancreas placement in the embryo. These defects correlated with the formation of small KVs with apparently diminished cilia, consistent with the known requirement for ciliary function in the laterality organ for the establishment of L-R asymmetry.

  20. Internalization of ricin in Chinese hamster ovary cells.

    PubMed Central

    Ray, B; Wu, H C

    1981-01-01

    Internalization of ricin into Chinese hamster ovary cells has been investigated. Combined treatment with galactose and pronase at 0 degrees C resulted in a complete release of surface-bound [125I]ricin into the media. Galactose-pronase-resistant cell-bound [125I]ricin represents internalized ricin molecules inside the cells. The internalization process is time, temperature, and concentration dependent. The pH optimum of internalization of ricin is about pH 7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis has revealed that intact ricin molecules are internalized. Neither reduction nor proteolytic processing of ricin is required for the entry of ricin into Chinese hamster ovary cells. PMID:6965107

  1. Ovarian Tumor Cells Studied Aboard the International Space Station (ISS)

    NASA Technical Reports Server (NTRS)

    2001-01-01

    In August 2001, principal investigator Jeanne Becker sent human ovarian tumor cells to the International Space Station (ISS) aboard the STS-105 mission. The tumor cells were cultured in microgravity for a 14 day growth period and were analyzed for changes in the rate of cell growth and synthesis of associated proteins. In addition, they were evaluated for the expression of several proteins that are the products of oncogenes, which cause the transformation of normal cells into cancer cells. This photo, which was taken by astronaut Frank Culbertson who conducted the experiment for Dr. Becker, shows two cell culture bags containing LN1 ovarian carcinoma cell cultures.

  2. Internalization of ferromagnetic nanowires by different living cells

    PubMed Central

    Prina-Mello, Adriele; Diao, Zhu; Coey, John Michael David

    2006-01-01

    The ability of living cells, either adherent or suspended, to internalize nickel nanowires is demonstrated for MC3T3-E1, UMR106-tumour and Marrow-Stromal cells. Nanowires were produced by electrodeposition, 20 μm long and 200 nm in diameter. Cell separation and manipulation was achieved for the three cell types. Applied magnetic field successfully oriented the internalized nanowires but no clear anisotropy is induced on the adherent cells. Nanowires tend to bind to cytoplasm metalloproteins and trigger lysosome reorganization around the nucleus. This work demonstrates the applications of nanowires in adherent and suspended cells for cell separation and manipulation, and further explore into their role in nanobiotechnology. PMID:16953891

  3. Internal pigment cells respond to external UV radiation in frogs.

    PubMed

    Franco-Belussi, Lilian; Nilsson Sköld, Helen; de Oliveira, Classius

    2016-05-01

    Fish and amphibians have pigment cells that generate colorful skins important for signaling, camouflage, thermoregulation and protection against ultraviolet radiation (UVR). However, many animals also have pigment cells inside their bodies, on their internal organs and membranes. In contrast to external pigmentation, internal pigmentation is remarkably little studied and its function is not well known. Here, we tested genotoxic effects of UVR and its effects on internal pigmentation in a neotropical frog, Physalaemus nattereri We found increases in body darkness and internal melanin pigmentation in testes and heart surfaces and in the mesenterium and lumbar region after just a few hours of UVR exposure. The melanin dispersion in melanomacrophages in the liver and melanocytes in testes increased after UV exposure. In addition, the amount of melanin inside melanomacrophages cells also increased. Although mast cells were quickly activated by UVR, only longer UVR exposure resulted in genotoxic effects inside frogs, by increasing the frequency of micronuclei in red blood cells. This is the first study to describe systemic responses of external UVR on internal melanin pigmentation, melanomacrophages and melanocytes in frogs and thus provides a functional explanation to the presence of internal pigmentation.

  4. Characterization of Laminin Binding Integrin Internalization in Prostate Cancer Cells.

    PubMed

    Das, Lipsa; Anderson, Todd A; Gard, Jaime M C; Sroka, Isis C; Strautman, Stephanie R; Nagle, Raymond B; Morrissey, Colm; Knudsen, Beatrice S; Cress, Anne E

    2017-05-01

    Laminin binding integrins α6 (CD49f) and α3 (CD49c) are persistently but differentially expressed in prostate cancer (PCa). Integrin internalization is an important determinant of their cell surface expression and function. Using flow cytometry, and first order kinetic modeling, we quantitated the intrinsic internalization rates of integrin subunits in a single cycle of internalization. In PCa cell line DU145, α6 integrin internalized with a rate constant (kactual ) of 3.25 min(-1) , threefold faster than α3 integrin (1.0 min(-1) ), 1.5-fold faster than the vitronectin binding αv integrin (CD51) (2.2 min(-1) ), and significantly slower than the unrelated transferrin receptor (CD71) (15 min(-1) ). Silencing of α3 integrin protein expression in DU145, PC3, and PC3B1 cells resulted in up to a 1.71-fold increase in kactual for α6 integrin. The internalized α6 integrin was targeted to early endosomes but not to lamp1 vesicles. Depletion of α3 integrin expression resulted in redistribution of α6β4 integrin to an observed cell-cell staining pattern that is consistent with a suprabasal distribution observed in epidermis and early PIN lesions in PCa. Depletion of α3 integrin increased cell migration by 1.8-fold, which was dependent on α6β1 integrin. Silencing of α6 integrin expression however, had no significant effect on the kactual of α3 integrin or its distribution in early endosomes. These results indicate that α3 and α6 integrins have significantly different internalization kinetics and that coordination exists between them for internalization. J. Cell. Biochem. 118: 1038-1049, 2017. © 2016 Wiley Periodicals, Inc.

  5. Lysosomal and endosomal heterogeneity in the liver: A comparison of the intracellular pathways of endocytosis in rat liver cells

    SciTech Connect

    Kindberg, G.M.; Tolleshaug, H.; Gjoen, T.; Berg, T. )

    1991-02-01

    Air-filled albumin microspheres, asialoorosomucoid and formaldehyde-treated serum albumin are selectively taken up by endocytosis in rat liver Kupffer cells, parenchymal cells and endothelial cells, respectively. Intracellular transport and degradation of endocytosed material were studied by subcellular fractionation in sucrose and Nycodenz gradients after intravenous injection of the ligand. By using ligands labeled with 125I-tyramine-cellobiose, the subcellular distribution of labeled degradation products can be studied because they are trapped at the site of formation. The results show that the kinetics of intracellular transport are different in hepatic parenchymal, endothelial and Kupffer cells. In endothelial cells, the ligand is associated with two types of endosomes during the first minutes after internalization and then is transferred rapidly to the lysosomes. In parenchymal cells, 125I-tyramine-cellobiose-asialoorosomucoid was located in a relatively slowly sedimenting vesicle during the first minute after internalization and subsequently in denser endosomes. Degradation of 125I-tyramine-cellobiose-asialoorosomucoid in parenchymal cells started later than that of 125I-tyramine-cellobiose-formaldehyde-treated serum albumin in endothelial cells. Furthermore, the ligand seemed to be transferred relatively slowly from endosomes to lysosomes, and most of the undegraded ligand was in the endosomes. The rate-limiting step of proteolysis in parenchymal cells is probably the transport from endosomes to lysosomes. In Kupffer cells, most 125I-tyramine-cellobiose-microspheres are found as undegraded material in very dense endosomes up to 3 hr after injection. After 20 hr, most of the ligand is degraded in lysosomes distributed at a lower density than the endosomes in Nycodenz and sucrose gradients.

  6. Anion Recognition as a Supramolecular Switch of Cell Internalization

    PubMed Central

    Couceiro, José R.; Nitschke, Jonathan R.; Vázquez, M. Eugenio; Mascareñas, José L.

    2017-01-01

    The cell internalization of designed oligoarginine peptides equipped with six glutamic acid residues and an anionic pyranine at the N-terminus is triggered upon addition of a supramolecular host. This host binds specifically to the pyranine moiety, enabling the complex to traverse the cell membrane. Interestingly, none of the components, neither the host nor the guest, are able to cross the cell membrane on their own. PMID:27984855

  7. Internal reforming fuel cell assembly with simplified fuel feed

    DOEpatents

    Farooque, Mohammad; Novacco, Lawrence J.; Allen, Jeffrey P.

    2001-01-01

    A fuel cell assembly in which fuel cells adapted to internally reform fuel and fuel reformers for reforming fuel are arranged in a fuel cell stack. The fuel inlet ports of the fuel cells and the fuel inlet ports and reformed fuel outlet ports of the fuel reformers are arranged on one face of the fuel cell stack. A manifold sealing encloses this face of the stack and a reformer fuel delivery system is arranged entirely within the region between the manifold and the one face of the stack. The fuel reformer has a foil wrapping and a cover member forming with the foil wrapping an enclosed structure.

  8. Internal quantum efficiency analysis of solar cell by genetic algorithm

    SciTech Connect

    Xiong, Kanglin; Yang, Hui; Lu, Shulong; Zhou, Taofei; Wang, Rongxin; Qiu, Kai; Dong, Jianrong; Jiang, Desheng

    2010-11-15

    To investigate factors limiting the performance of a GaAs solar cell, genetic algorithm is employed to fit the experimentally measured internal quantum efficiency (IQE) in the full spectra range. The device parameters such as diffusion lengths and surface recombination velocities are extracted. Electron beam induced current (EBIC) is performed in the base region of the cell with obtained diffusion length agreeing with the fit result. The advantage of genetic algorithm is illustrated. (author)

  9. Cell Surface Protein Detection to Assess Receptor Internalization

    PubMed Central

    Czarnecka, Magdalena; Kitlinska, Joanna

    2017-01-01

    The migration of membrane receptors upon exposure to different stimulants/inhibitors is of great importance. Among others, the internalization of membrane receptors affects their accessibility to ligands and cell responsiveness to environmental cues. Experimentally, receptor internalization can be used as a measure of their activation. In our studies, we employed this approach to explore cross-talk between a seven transmembrane domain receptor for neuropeptide Y (NPY), Y5R, and a tyrosine kinase receptor for brain-derived neurotrophic factor (BDNF), TrkB. To this end, we measured the internalization of Y5R upon stimulation with the TrkB ligand, BDNF. Upon treatment with BDNF, the cells were exposed to a membrane impermeable, biotinylation reagent that selectively labels surface proteins. Subsequently, the biotinylated membrane proteins were affinity-purified on columns with avidin resins and analyzed by Western blot. Differences in the fraction of receptors present on the cell surface of control and ligand-treated cells served as a measure of their internalization and response to particular stimuli.

  10. Cell Penetrating Peptide-Modified Poly(Lactic-co-Glycolic Acid) Nanoparticles with Enhanced Cell Internalization

    PubMed Central

    Steinbach, Jill M.; Seo, Young-Eun; Saltzman, W. Mark

    2015-01-01

    The surface modification of nanoparticles (NPs) can enhance the intracellular delivery of drugs, proteins, and genetic agents. Here we studied the effect of different surface ligands, including cell penetrating peptides (CPPs), on the cell binding and internalization of poly(lactic-co-glycolic) (PLGA) NPs. Relative to unmodified NPs, we observed that surface-modified NPs greatly enhanced cell internalization. Using one CPP, MPG (unabbreviated notation), that achieved the highest degree of internalization at both low and high surface modification densities, we evaluated the effect of two different NP surface chemistries on cell internalization. After 2 hr, avidin-MPG NPs enhanced cellular internalization by 5 to 26-fold relative to DSPE-MPG NP formulations. Yet, despite a 5-fold increase in MPG density on DSPE- relative to Avidin-NPs, after 24 hr., both formulations resulted in similar internalization levels (48 and 64-fold, respectively). Regardless of surface modification, all NPs were internalized through an energy-dependent, clathrin-mediated process, and became dispersed throughout the cell. Overall both Avidin- and DSPE-CPP modified NPs significantly increased internalization and offer promising delivery options for applications in which internalization presents challenges to efficacious delivery. PMID:26602822

  11. Cell penetrating peptide-modified poly(lactic-co-glycolic acid) nanoparticles with enhanced cell internalization.

    PubMed

    Steinbach, Jill M; Seo, Young-Eun; Saltzman, W Mark

    2016-01-01

    The surface modification of nanoparticles (NPs) can enhance the intracellular delivery of drugs, proteins, and genetic agents. Here we studied the effect of different surface ligands, including cell penetrating peptides (CPPs), on the cell binding and internalization of poly(lactic-co-glycolic) (PLGA) NPs. Relative to unmodified NPs, we observed that surface-modified NPs greatly enhanced cell internalization. Using one CPP, MPG (unabbreviated notation), that achieved the highest degree of internalization at both low and high surface modification densities, we evaluated the effect of two different NP surface chemistries on cell internalization. After 2h, avidin-MPG NPs enhanced cellular internalization by 5 to 26-fold relative to DSPE-MPG NP formulations. Yet, despite a 5-fold increase in MPG density on DSPE compared to Avidin NPs, both formulations resulted in similar internalization levels (48 and 64-fold, respectively) after 24h. Regardless of surface modification, all NPs were internalized through an energy-dependent, clathrin-mediated process, and became dispersed throughout the cell. Overall both Avidin- and DSPE-CPP modified NPs significantly increased internalization and offer promising delivery options for applications in which internalization presents challenges to efficacious delivery.

  12. Nanostructured Monolinolein Miniemulsions as Delivery Systems: Role of the Internal Mesophase on Cytotoxicity and Cell Internalization.

    PubMed

    Guillot, Samuel; Méducin, Fabienne; Poljak, Kristina; Malard, Virginie; Foucault-Collet, Alexandra; Serieye, Sébastien; Pichon, Chantal

    2017-03-08

    Recent advances in nanoparticle systems for improved drug delivery display a great potential for the administration of active molecules. Here, lipid miniemulsions with various internal nanostructures were loaded with the chemotherapeutic agent Paclitaxel. The goal is to assess the impact of internal structures on their efficiency. Previously the structure, the stability and the physico-chemical properties of those carriers were characterized. Modalities of action were addressed by the evaluation of their effects on the tumor cells viability, their cellular uptake by flow cytometry and confocal microscopy detection of fluorescently labeled nanostructured miniemulsions. Nanostructured miniemulsions showed variations in the cell internalization process likely due to differences in the internal structure. All paclitaxel-loaded emulsions were active reservoirs from which Paclitaxel could be released, however bicontinuous cubosomes showed the best efficiency. Considering the fact that these delivery systems can offer a new life to bioactive compounds previously abandoned due to a low aqueous solubility, these data may represent an important step towards the development of new clinical therapeutic strategies against cancers.

  13. Lipopolysaccharide-induced multinuclear cells: Increased internalization of polystyrene beads and possible signals for cell fusion

    SciTech Connect

    Nakanishi-Matsui, Mayumi Yano, Shio; Futai, Masamitsu

    2013-11-01

    Highlights: •LPS induces multinuclear cells from murine macrophage-derived RAW264.7 cells. •Large beads are internalized by cells actively fusing to become multinuclear. •The multinuclear cell formation is inhibited by anti-inflammatory cytokine, IL10. •Signal transduction for cell fusion is different from that for inflammation. -- Abstract: A murine macrophage-derived line, RAW264.7, becomes multinuclear on stimulation with lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria. These multinuclear cells internalized more polystyrene beads than mononuclear cells or osteoclasts (Nakanishi-Matsui, M., Yano, S., Matsumoto, N., and Futai, M., 2012). In this study, we analyzed the time courses of cell fusion in the presence of large beads. They were internalized into cells actively fusing to become multinuclear. However, the multinuclear cells once formed showed only low phagocytosis activity. These results suggest that formation of the multinuclear cells and bead internalization took place simultaneously. The formation of multinuclear cells was blocked by inhibitors for phosphoinositide 3-kinase, phospholipase C, calcineurin, and c-Jun N-terminal kinase. In addition, interleukin 6 and 10 also exhibited inhibitory effects. These signaling molecules and cytokines may play a crucial role in the LPS-induced multinuclear cell formation.

  14. Ultrastable Nontoxic RNA Nanoparticles for Targeting Triple-Negative Breast Cancer Stem Cells

    DTIC Science & Technology

    2016-04-01

    nanoparticles to cancer cells; nanoscale size and shape which avoids rapid renal clearance and engulfment by lung macrophages and liver Kupffer cells; favorable...effectively block cancer progression and prevent metastases, while minimizing adverse side effects.13 Liver and other organ accumulations lead to low cancer...attractive for in vivo delivery applications, since it minimizes nonspecific cell entry, and entrapment by lung macrophages and liver Kupffer cells.11

  15. Internal Short Circuits in Lithium-Ion Cells for PHEVs

    SciTech Connect

    Sriramulu, Suresh; Stringfellow, Richard

    2013-05-25

    Development of Plug-in Hybrid Electric Vehicles (PHEVs) has recently become a high national priority because of their potential to enable significantly reduced petroleum consumption by the domestic transportation sector in the relatively near term. Lithium-ion (Li-ion) batteries are a critical enabling technology for PHEVs. Among battery technologies with suitable operating characteristics for use in vehicles, Li-ion batteries offer the best combination of energy, power, life and cost. Consequently, worldwide, leading corporations and government agencies are supporting the development of Li-ion batteries for PHEVs, as well as the full spectrum of vehicular applications ranging from mild hybrid to all-electric. In this project, using a combination of well-defined experiments, custom designed cells and simulations, we have improved the understanding of the process by which a Li-ion cell that develops an internal short progresses to thermal runaway. Using a validated model for thermal runaway, we have explored the influence of environmental factors and cell design on the propensity for thermal runaway in full-sized PHEV cells. We have also gained important perspectives about internal short development and progression; specifically that initial internal shorts may be augmented by secondary shorts related to separator melting. Even though the nature of these shorts is very stochastic, we have shown the critical and insufficiently appreciated role of heat transfer in influencing whether a developing internal short results in a thermal runaway. This work should lead to enhanced perspectives on separator design, the role of active materials and especially cathode materials with respect to safety and the design of automotive cooling systems to enhance battery safety in PHEVs.

  16. Multiple internalization pathways of polyelectrolyte multilayer capsules into mammalian cells.

    PubMed

    Kastl, Lena; Sasse, Daniel; Wulf, Verena; Hartmann, Raimo; Mircheski, Josif; Ranke, Christiane; Carregal-Romero, Susana; Martínez-López, José Antonio; Fernández-Chacón, Rafael; Parak, Wolfgang J; Elsasser, Hans-Peter; Rivera Gil, Pilar

    2013-08-27

    Polyelectrolyte multilayer (PEM) capsules are carrier vehicles with great potential for biomedical applications. With the future aim of designing biocompatible, effective therapeutic delivery systems (e.g., for cancer), the pathway of internalization (uptake and fate) of PEM capsules was investigated. In particular the following experiments were performed: (i) the study of capsule co-localization with established endocytic markers, (ii) switching-off endocytotic pathways with pharmaceutical/chemical inhibitors, and (iii) characterization and quantification of capsule uptake with confocal and electron microscopy. As result, capsules co-localized with lipid rafts and with phagolysosomes, but not with other endocytic vesicles. Chemical interference of endocytosis with chemical blockers indicated that PEM capsules enter the investigated cell lines through a mechanism slightly sensitive to electrostatic interactions, independent of clathrin and caveolae, and strongly dependent on cholesterol-rich domains and organelle acidification. Microscopic characterization of cells during capsule uptake showed the formation of phagocytic cups (vesicles) to engulf the capsules, an increased number of mitochondria, and a final localization in the perinuclear cytoplasma. Combining all these indicators we conclude that PEM capsule internalization in general occurs as a combination of different sequential mechanisms. Initially, an adsorptive mechanism due to strong electrostatic interactions governs the stabilization of the capsules at the cell surface. Membrane ruffling and filopodia extensions are responsible for capsule engulfing through the formation of a phagocytic cup. Co-localization with lipid raft domains activates the cell to initiate a lipid-raft-mediated macropinocytosis. Internalization vesicles are very acidic and co-localize only with phagolysosome markers, excluding caveolin-mediated pathways and indicating that upon phagocytosis the capsules are sorted to

  17. Isolation of Endothelial Cells and Vascular Smooth Muscle Cells from Internal Mammary Artery Tissue

    PubMed Central

    Moss, Stephanie C.; Bates, Michael; Parrino, Patrick E.; Woods, T. Cooper

    2007-01-01

    Analyses of vascular smooth muscle cell and endothelial cell function through tissue culture techniques are often employed to investigate the underlying mechanisms regulating cardiovascular disease. As diseases such as diabetes mellitus and chronic kidney disease increase a patient's risk of cardiovascular disease, the development of methods for examining the effects of these diseases on vascular smooth muscle cells and endothelial cells is needed. Commercial sources of endothelial cells and vascular smooth muscle cells generally provide minimal donor information and are in limited supply. This study was designed to determine if vascular smooth muscle cells and endothelial cells could be isolated from human internal mammary arteries obtained from donors undergoing coronary artery bypass graft surgery. As coronary artery bypass graft surgery is a commonly performed procedure, this method would provide a new source for these cells that when combined with the donor's medical history will greatly enhance our studies of the effects of complicating diseases on vascular biology. Internal mammary artery tissue was obtained from patients undergoing coronary artery bypass graft surgery. Through a simple method employing two separate tissue digestions, vascular smooth muscle cells and endothelial cells were isolated and characterized. The isolated vascular smooth muscle cells and endothelial cells exhibited the expected morphology and were able to be passaged for further analysis. The vascular smooth muscle cells exhibited positive staining for α-smooth muscle actin and the endothelial cells exhibited positive staining for CD31. The overall purity of the isolations was > 95%. This method allows for the isolation of endothelial cells and vascular smooth muscle cells from internal mammary arteries, providing a new tool for investigations into the interplay of vascular diseases and complicating diseases such as diabetes and kidney disease. PMID:21603530

  18. Adhesion and internalization differences of COM nanocrystals on Vero cells before and after cell damage.

    PubMed

    Gan, Qiong-Zhi; Sun, Xin-Yuan; Ouyang, Jian-Ming

    2016-02-01

    The adhesion and internalization between African green monkey kidney epithelial (Vero) cells (before and after oxidative damage by hydrogen peroxide) and calcium oxalate monohydrate (COM) nanocrystals (97±35nm) were investigated so as to discuss the molecular and cellular mechanism of kidney stone formation. Scanning electron microscope (SEM) was used to observe the Vero-COM nanocrystal adhesion; the nanocrystal-cell adhesion was evaluated by measuring the content of malonaldehyde (MDA), the activity of superoxide dismutase (SOD), the expression level of cell surface osteopontin (OPN) and the change of Zeta potential. Confocal microscopy and flow cytometry were used for the observation and quantitative analysis of crystal internalization. In the process of adhesion, the cell viability and the SOD activity declined, the MDA content, Zeta potential, and the OPN expression level increased. The adhesive capacity of injured Vero was obviously stronger than normal cells; in addition the injured cells promoted the aggregation of COM nanocrystals. The capacity of normal cells to internalize crystals was obviously stronger than that of injured cells. Cell injury increased adhesive sites on cell surface, thereby facilitating the aggregation of COM nanocrystals and their attachment, which results in enhanced risk of calcium oxalate stone formation.

  19. Polystyrene nanoparticles internalization in human gastric adenocarcinoma cells.

    PubMed

    Forte, Maurizio; Iachetta, Giuseppina; Tussellino, Margherita; Carotenuto, Rosa; Prisco, Marina; De Falco, Maria; Laforgia, Vincenza; Valiante, Salvatore

    2016-03-01

    The increase in the use of nanoparticles, as a promising tool for drug delivery or as a food additive, raises questions about their interaction with biological systems, especially in terms of evoked responses. In this work, we evaluated the kinetics of uptake of 44 nm (NP44) and 100 nm (NP100) unmodified polystyrene nanoparticles (PS-NPs) in gastric adenocarcinoma (AGS) cells, as well as the endocytic mechanism involved, and the effect on cell viability and gene expression of genes involved in cell cycle regulation and inflammation processes. We showed that NP44 accumulate rapidly and more efficiently in the cytoplasm of AGS compared to NP100; both PS-NPs showed an energy dependent mechanism of internalization and a clathrin-mediated endocytosis pathway. Dose response treatments revealed a non-linear curve. PS-NPs also affected cell viability, inflammatory gene expression and cell morphology. NP44 strongly induced an up-regulation of IL-6 and IL-8 genes, two of the most important cytokines involved in gastric pathologies. Our study suggests that parameters such as time, size and concentration of NPs must be taken carefully into consideration during the development of drug delivery systems based on NPs and for the management of nanoparticles associated risk factors.

  20. Inference of Internal Stress in a Cell Monolayer

    PubMed Central

    Nier, Vincent; Jain, Shreyansh; Lim, Chwee Teck; Ishihara, Shuji; Ladoux, Benoit; Marcq, Philippe

    2016-01-01

    We combine traction force data with Bayesian inversion to obtain an absolute estimate of the internal stress field of a cell monolayer. The method, Bayesian inversion stress microscopy, is validated using numerical simulations performed in a wide range of conditions. It is robust to changes in each ingredient of the underlying statistical model. Importantly, its accuracy does not depend on the rheology of the tissue. We apply Bayesian inversion stress microscopy to experimental traction force data measured in a narrow ring of cohesive epithelial cells, and check that the inferred stress field coincides with that obtained by direct spatial integration of the traction force data in this quasi one-dimensional geometry. PMID:27074687

  1. High performance internal reforming unit for high temperature fuel cells

    DOEpatents

    Ma, Zhiwen; Venkataraman, Ramakrishnan; Novacco, Lawrence J.

    2008-10-07

    A fuel reformer having an enclosure with first and second opposing surfaces, a sidewall connecting the first and second opposing surfaces and an inlet port and an outlet port in the sidewall. A plate assembly supporting a catalyst and baffles are also disposed in the enclosure. A main baffle extends into the enclosure from a point of the sidewall between the inlet and outlet ports. The main baffle cooperates with the enclosure and the plate assembly to establish a path for the flow of fuel gas through the reformer from the inlet port to the outlet port. At least a first directing baffle extends in the enclosure from one of the sidewall and the main baffle and cooperates with the plate assembly and the enclosure to alter the gas flow path. Desired graded catalyst loading pattern has been defined for optimized thermal management for the internal reforming high temperature fuel cells so as to achieve high cell performance.

  2. Stem cells in biology and disease - ESTOOLS International Symposium.

    PubMed

    Pistoi, Sergio

    2010-01-01

    ESTOOLS was the first EU-funded project entirely devoted to the study of human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). The final meeting of the consortium took place in Lisbon from 26 to 28 May 2010 and gathered 298 researchers from 27 countries. A dense programme of scientific sessions and outreach activities and an ethics workshop gave an all-round overview of the challenges, perspectives and ethical issues of this exciting field. The ESTOOLS consortium was launched in 2006 as a 4-year EU-funded 6th Framework (FP6) project, with a total funding of 12 million euro. The project has been the largest European consortium devoted to the study of human ESCs, teaming up 21 research groups in 10 countries. After the discovery of human induced pluripotent stem cells (iPSCs) by Shinya Yamanaka and James Thomson in 2007, the consortium opened a new workpackage dedicated to this groundbreaking field, and became the first to devote EU funding to study human iPSCs. The Lisbon meeting marked the end of the consortiums funding cycle, but it was more than a wrap-up of its overall work and achievements, providing a showcase of cutting-edge research in human pluripotent stem cells, with the participation of international leaders in the field.

  3. Right-elevated expression of charon is regulated by fluid flow in medaka Kupffer's vesicle.

    PubMed

    Hojo, Motoki; Takashima, Shigeo; Kobayashi, Daisuke; Sumeragi, Akira; Shimada, Atsuko; Tsukahara, Tatsuya; Yokoi, Hayato; Narita, Takanori; Jindo, Tomoko; Kage, Takahiro; Kitagawa, Tadao; Kimura, Tetsuaki; Sekimizu, Koshin; Miyake, Akimitsu; Setiamarga, Davin; Murakami, Ryohei; Tsuda, Sachiko; Ooki, Shinya; Kakihara, Ken; Naruse, Kiyoshi; Takeda, Hiroyuki

    2007-06-01

    Recent studies have revealed that a cilium-generated liquid flow in the node has a crucial role in the establishment of the left-right (LR) axis in the mouse. In fish, Kupffer's vesicle (KV), a teleost-specific spherical organ attached to the tail region, is known to have an equivalent role to the mouse node during LR axis formation. However, at present, there has been no report of an asymmetric gene expressed in KV under the control of fluid flow. Here we report the earliest asymmetric gene in teleost KV, medaka charon, and its regulation. Charon is a member of the Cerberus/DAN family of proteins, first identified in zebrafish. Although zebrafish charon was reported to be symmetrically expressed in KV, medaka charon displays asymmetric expression with more intense expression on the right side. This asymmetric expression was found to be regulated by KV flow because symmetric and up-regulated charon expression was observed in flow-defective embryos with immotile cilia or disrupted KV. Taken together, medaka charon is a reliable gene marker for LR asymmetry in KV and thus, will be useful for the analysis of the early steps downstream of the fluid flow.

  4. PEM fuel cell applications and their development at International Fuel Cells

    SciTech Connect

    Fuller, T.F.; Gorman, M.E.; Van Dine, L.L.

    1996-12-31

    International Fuel Cells (IFC) is involved with the full spectrum of fuel cell power plants including the development of Proton Exchange Membrane (PEM) fuel cell systems. The extensive background in systems, design, materials and manufacturing technologies has been brought to bear on the development of highly competitive PEM power plants. IFC is aggressively pursuing these opportunities and is developing low-cost designs for a wide variety of PEM fuel cell applications with special emphasis on portable power and transportation. Experimental PEM power plants for each of these applications have been successfully tested.

  5. Anisotropy of cell adhesive microenvironment governs cell internal organization and orientation of polarity

    PubMed Central

    Théry, Manuel; Racine, Victor; Piel, Matthieu; Pépin, Anne; Dimitrov, Ariane; Chen, Yong; Sibarita, Jean-Baptiste; Bornens, Michel

    2006-01-01

    Control of the establishment of cell polarity is an essential function in tissue morphogenesis and renewal that depends on spatial cues provided by the extracellular environment. The molecular role of cell–cell or cell–extracellular matrix (ECM) contacts on the establishment of cell polarity has been well characterized. It has been hypothesized that the geometry of the cell adhesive microenvironment was directing cell surface polarization and internal organization. To define how the extracellular environment affects cell polarity, we analyzed the organization of individual cells plated on defined micropatterned substrates imposing cells to spread on various combinations of adhesive and nonadhesive areas. The reproducible normalization effect on overall cell compartmentalization enabled quantification of the spatial organization of the actin network and associated proteins, the spatial distribution of microtubules, and the positioning of nucleus, centrosome, and Golgi apparatus. By using specific micropatterns and statistical analysis of cell compartment positions, we demonstrated that ECM geometry determines the orientation of cell polarity axes. The nucleus–centrosome orientations were reproducibly directed toward cell adhesive edges. The anisotropy of the cell cortex in response to the adhesive conditions did not affect the centrosome positioning at the cell centroid. Based on the quantification of microtubule plus end distribution we propose a working model that accounts for that observation. We conclude that, in addition to molecular composition and mechanical properties, ECM geometry plays a key role in developmental processes. PMID:17179050

  6. Applications of internal reforming molten carbonate fuel cells

    SciTech Connect

    Maru, H.C.; Baker, B.S.

    1986-03-01

    A modified version of the molten carbonate fuel cell (MCFC) is being developed which is capable of direct utilization of hydrocarbon fuels such as natural gas, methanol, alcohol, propane, coal-derived synthetic gas and others. This version is termed internal reforming MCFC or Direct Fuel Cell, DFC. The DFC provides an ideal match of heat and mass transfer requirements within the cell, and minimizes external processing equipment. Efficiencies as high as 55 to 60% can be expected, making DFC a unique and practical device. The overall system is expected to be simple and cost effective. Many attractive applications exist for the simple and highly efficient DFC generators. Natural gas fueled dispersed generators in the size range of 500kW to 10 Mw may provide early market entry. DFC applications to smaller-size on-site power plants, large coal-powered central stations and industrial cogeneration applications can follow once the technology is demonstrated and manufacturing base is established. 8 references, 3 figures, 2 table.

  7. International policy failures: cloning and stem-cell research.

    PubMed

    Tauer, Carol A

    In late 2003, two international bodies were unable to resolve disagreements that involved bioethical issues. First, the United Nations General Assembly failed to pass a treaty on reproductive cloning because of insistence by some countries that the treaty include a ban on cloning for research. In view of the importance of enacting prohibition of reproductive cloning, the two issues should be separated and each argued on its own merits. Relevant objections to separation of the two issues can be refuted. Second, the European Union (EU) failed to agree on conditions for funding stem-cell research because of the diversity of views and policies of the countries of the EU. Because a stalemate was reached, funding decisions in the next programme cycle will be made on an ad hoc basis. Scientists will not have information they need to plan research programmes, suggesting that clear guidelines, even if restrictive, are preferable to vague unpublicised criteria.

  8. Fuel cell crimp-resistant cooling device with internal coil

    DOEpatents

    Wittel, deceased, Charles F.

    1986-01-01

    A cooling assembly for fuel cells having a simplified construction whereby coolant is efficiently circulated through a conduit arranged in serpentine fashion in a channel within a member of such assembly. The channel is adapted to cradle a flexible, chemically inert, conformable conduit capable of manipulation into a variety of cooling patterns without crimping or otherwise restricting of coolant flow. The conduit, when assembled with the member, conforms into intimate contact with the member for good thermal conductivity. The conduit is non-corrodible and can be constructed as a single, manifold-free, continuous coolant passage means having only one inlet and one outlet. The conduit has an internal coil means which enables it to be bent in small radii without crimping.

  9. Cell surface glycoproteins of CHO cells. I. Internalization and rapid recycling

    SciTech Connect

    Raub, T.J.; Denny, J.B.; Roberts, R.M.

    1986-01-01

    The major cell surface proteins of Chinese hamster ovary (CHO) cells have been investigated after reacting cells at 4/sup 0/C with the membrane-impermeant reagent, trinitrobenzenesulfonate (TNBS). Immunoprecipitation and subsequent two-dimensional, sodiumdodecyl sulfate, polyacrylamide gel electrophoresis (SDS-PAGE) of proteins from derivatized cells that had been labelled previously with (/sup 3/H)D-glucosamine or (/sup 3/H)L-leucine showed that TNBS reacted with most of the high molecular weight (HMW) acidic glycoproteins that became labelled with iodine by the lactoperoxidase technique and that bind the lectin, wheat germ agglutinin (WGA). After warming the cells to allow endocytosis to proceed, molecule haptenized with trinitrophenol (TNP) groups were followed radio-chemically by means of (/sup 125/I)anti-DNP antibodies. Within 15 min at 37/sup 0/C, a steady-state between surface and cytoplasmic label was reached, with about 65% of the hapten located internally. Recycling of internalized TNP groups back to the cell surface also occurred rapidly (t/sub 1/2/ approx. 5 min). Our results are consistent with the view that the majority of plasma membrane glycoproteins are continuously being internalized and recycled at a high rate.

  10. The zebrafish Kupffer's vesicle as a model system for the molecular mechanisms by which the lack of Polycystin-2 leads to stimulation of CFTR.

    PubMed

    Roxo-Rosa, Mónica; Jacinto, Raquel; Sampaio, Pedro; Lopes, Susana Santos

    2015-10-02

    In autosomal dominant polycystic kidney disease (ADPKD), cyst inflation and continuous enlargement are associated with marked transepithelial ion and fluid secretion into the cyst lumen via cystic fibrosis transmembrane conductance regulator (CFTR). Indeed, the inhibition or degradation of CFTR prevents the fluid accumulation within cysts. The in vivo mechanisms by which the lack of Polycystin-2 leads to CFTR stimulation are an outstanding challenge in ADPKD research and may bring important biomarkers for the disease. However, hampering their study, the available ADPKD in vitro cellular models lack the three-dimensional architecture of renal cysts and the ADPKD mouse models offer limited access for live-imaging experiments in embryonic kidneys. Here, we tested the zebrafish Kupffer's vesicle (KV) as an alternative model-organ. KV is a fluid-filled vesicular organ, lined by epithelial cells that express both CFTR and Polycystin-2 endogenously, being each of them easily knocked-down. Our data on the intracellular distribution of Polycystin-2 support its involvement in the KV fluid-flow induced Ca(2+)-signalling. Mirroring kidney cysts, the KV lumen inflation is dependent on CFTR activity and, as we clearly show, the knockdown of Polycystin-2 results in larger KV lumens through overstimulation of CFTR. In conclusion, we propose the zebrafish KV as a model organ to study the renal cyst inflation. Favouring its use, KV volume can be easily determined by in vivo imaging offering a live readout for screening compounds and genes that may prevent cyst enlargement through CFTR inhibition.

  11. International Conference on the Cell and Molecular Biology of Chlamydomonas

    SciTech Connect

    Dr. Stephen Miller

    2010-06-10

    reported in the area of tool development, advances that conference attendees should be able to employ in their own labs to speed the analysis of gene function. In summary, support from DOE award SC0004085 helped to make the 2010 Conference on the Cell and Molecular Biology of Chlamydomonas an unqualified success. Thanks to that support it was possible to attract a new cohort of young investigators to this biennial conference. These young scientists benefited from the opportunity to present their results to, and to interact with, the international Chlamydomonas research community. The Chlamydomonas community benefited by learning about the advances reported by these scientists, and it will continue to benefit from the contributions these investigators will make as their training and careers progress.

  12. Experimental triggers for internal short circuits in lithium-ion cells

    NASA Astrophysics Data System (ADS)

    Orendorff, Christopher J.; Roth, E. Peter; Nagasubramanian, Ganesan

    2011-08-01

    Lithium-ion cell field failures due to internal short circuits are a significant concern to the entire lithium-ion cell market from consumer electronics to electric vehicles. While the probability of these failure events occurring is estimated to be very low (1 in 5-10 million), the consequences of a cell failure due to an internal short in a high energy battery system have the potential to be catastrophic. The statistical probability of one of these events is very low and they are difficult to predict and simulate in a laboratory using some external test; which makes cell failure due to an internal short circuit a unique challenge to overcome. Several of the experiments designed to simulate internal shorts have been adopted as testing protocols across the industry; in general, they do not accurately simulate an internal short. This work highlights our efforts to experimentally trigger an internal short circuit in a lithium-ion cell.

  13. B cell receptor-mediated internalization of salmonella: a novel pathway for autonomous B cell activation and antibody production.

    PubMed

    Souwer, Yuri; Griekspoor, Alexander; Jorritsma, Tineke; de Wit, Jelle; Janssen, Hans; Neefjes, Jacques; van Ham, S Marieke

    2009-06-15

    The present paradigm is that primary B cells are nonphagocytosing cells. In this study, we demonstrate that human primary B cells are able to internalize bacteria when the bacteria are recognized by the BCR. BCR-mediated internalization of Salmonella typhimurium results in B cell differentiation and secretion of anti-Salmonella Ab by the Salmonella-specific B cells. In addition, BCR-mediated internalization leads to efficient Ag delivery to the MHC class II Ag-loading compartments, even though Salmonella remains vital intracellularly in primary B cells. Consequently, BCR-mediated bacterial uptake induces efficient CD4(+) T cell help, which boosts Salmonella-specific Ab production. BCR-mediated internalization of Salmonella by B cells is superior over extracellular Ag extraction to induce rapid and specific humoral immune responses and efficiently combat infection.

  14. Internal reforming development for solid oxide fuel cells

    NASA Astrophysics Data System (ADS)

    Lee, A. L.

    1987-02-01

    Internal reforming of natural gas within a solid oxide fuel cell (SOFC) should simplify the overall system design and make the SOFC an attractive means for producing electrical power. This program was undertaken to investigate the catalytic properties of nickel cermets, which are prime candidates for SOFC anodes. The initial task in this program was an extensive literature search for information on steam reforming of light hydrocarbons. The second task was to modify and calibrate the reactor systems that were used in the experimental kinetic studies. Two systems were used in this investigation; a continuously stirred tank reactor system (CSTR) and a plug flow reactor system (PFR). In the third task, 16 nickel-zirconia cermets were prepared using four procedures, tape casting, Westinghouse slurry, incorporation of performers, and granulation. The catalytic behavior of three cermets was determined in the fourth task. The reaction was first order with respect to methane and -1.25 for steam. Ethane and propane in the feed did not affect the methane conversion rate. The cermet has a higher initial tolerance for sulfur than standard nickel reforming catalysts. The final task was a mechanistic study of the steam reforming reaction on nickel and nickel-zirconia catalysts.

  15. Bisphosphonates in Langerhans Cell Histiocytosis: An International Retrospective Case Series

    PubMed Central

    Chellapandian, Deepak; Makras, Polyzois; Kaltsas, Gregory; van den Bos, Cor; Naccache, Lamia; Rampal, Raajit; Carret, Anne-Sophie; Weitzman, Sheila; Egeler, R. Maarten; Abla, Oussama

    2016-01-01

    Background Bone is the most common organ of involvement in patients with Langerhans cell histiocytosis (LCH), which is often painful and associated with significant morbidity from pathological fractures. Current first-line treatments include chemotherapy and steroids that are effective but often associated with adverse effects, whereas the disease may reactivate despite an initial response to first-line agents. Bisphosphonates are osteoclast inhibitors that have shown to be helpful in treating bone lesions of LCH. To date, there are no large international studies to describe their role in treating bone lesions of LCH. Method We conducted a multicenter retrospective review of 13 patients with histologically proven LCH, who had received bisphosphonates either at diagnosis or at disease reactivation. Results Ten patients (77%) had a single system bone disease, and 3 (23%) had bone lesions as part of multisystem disease. Median follow-up time post-bisphosphonate therapy was 4.6 years (range, 0.8 to 8.2 years). Treatment with bisphosphonates was associated with significant pain relief in almost all patients. Twelve (92%) achieved resolution of active bone lesions, and 10 out of them had no active disease for a median of 3.5 years (range, 0.8 to 5 years). One patient did not respond. No major adverse effects were reported in this series. Conclusion Bisphosphonates are well-tolerated drugs that can significantly improve bone pain and induce remission in active bone LCH. Future prospective studies evaluating the role of bisphosphonates in LCH are warranted. PMID:27413525

  16. Cell internalization and traffic pathway of Clostridium botulinum type C neurotoxin in HT-29 cells.

    PubMed

    Uotsu, Nobuo; Nishikawa, Atsushi; Watanabe, Toshihiro; Ohyama, Tohru; Tonozuka, Takashi; Sakano, Yoshiyuki; Oguma, Keiji

    2006-01-01

    The bacterium Clostridium botulinum type C produces a progenitor toxin (C16S toxin) that binds to O-linked sugar chains terminating with sialic acid on the surface of HT-29 cells prior to internalization [A. Nishikawa, N. Uotsu, H. Arimitsu, J.C. Lee, Y. Miura, Y. Fujinaga, H. Nakada, T. Watanabe, T. Ohyama, Y. Sakano, K. Oguma, Biochem. Biophys. Res. Commun. 319 (2004) 327-333] [21]. Based on this, it was hypothesized that the C16S toxin is internalized via clathrin-coated pits. To examine this possibility, the internalized toxin was observed with a fluorescent antibody using confocal laser-scanning microscopy. The confocal images clearly indicated that the C16S toxin was internalized mainly via clathrin-coated pits and localized in early endosomes. The toxin was colocalized with caveolin-1 which is one of the components of caveolae, however, implying the toxin was also internalized via caveolae. The confocal images also showed that the neurotoxin transported to the endosome was transferred to the Golgi apparatus. However, the non-toxic components were not merged with the Golgi marker protein, TGN38, implying the neurotoxin was dissociated from progenitor toxin in endosomes. These results suggested that the C16S toxin was separated to the neurotoxin and other proteins in endosome and the neurotoxin was further transferred to the Golgi apparatus which is the center for protein sorting.

  17. Haemangioendothelioma (Kupffer cell angiosarcoma), myelofibrosis, splenic atrophy, and myeloma paraproteinaemia after parenteral thorotrast administration.

    PubMed Central

    Jennings, R C; Priestley, S E

    1978-01-01

    A case of thorotrastosis occurred 25 years after thorotrast angiography, with the previously unrecorded association of myeloma type paraproteinaemia. The relationship between haemangioendothelioma due to thorotrast and other vascular sarcomas of the liver is briefly reviewed. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 PMID:748384

  18. Global gyrokinetic particle-in-cell simulations of internal kink instabilities

    SciTech Connect

    Mishchenko, Alexey; Zocco, Alessandro

    2012-12-15

    Internal kink instabilities have been studied in straight tokamak geometry employing an electromagnetic gyrokinetic particle-in-cell (PIC) code. The ideal-MHD internal kink mode and the collisionless m=1 tearing mode have been successfully simulated with the PIC code. Diamagnetic effects on the internal kink modes have also been investigated.

  19. Protection of Candida parapsilosis from neutrophil killing through internalization by human endothelial cells

    PubMed Central

    Glass, Kyle A; Longley, Sarah J; Bliss, Joseph M; Shaw, Sunil K

    2015-01-01

    Candida parapsilosis is a fungal pathogen that is associated with hematogenously disseminated disease in premature neonates, acutely ill or immunocompromised patients. In cell culture, C. parapsilosis cells are actively and avidly endocytosed by endothelial cells via actin polymerization mediated by N-WASP. Here we present evidence that C. parapsilosis that were internalized by endothelial cells remained alive, and avoided being acidified or otherwise damaged via the host cell. Internalized fungal cells reproduced intracellularly and eventually burst out of the host endothelial cell. When neutrophils were added to endothelium and C. parapsilosis, they patrolled the endothelial surface and efficiently killed most adherent fungal cells prior to endocytosis. But after endocytosis by endothelial cells, internalized fungal cells evaded neutrophil killing. Silencing endothelial N-WASP blocked endocytosis of C. parapsilosis and left fungal cells stranded on the cell surface, where they were susceptible to neutrophil killing. These observations suggest that for C. parapsilosis to escape from the bloodstream, fungi may adhere to and be internalized by endothelial cells before being confronted and phagocytosed by a patrolling leukocyte. Once internalized by endothelial cells, C. parapsilosis may safely replicate to cause further rounds of infection. Immunosurveillance of the intravascular lumen by leukocytes crawling on the endothelial surface and rapid killing of adherent yeast may play a major role in controlling C. parapsilosis dissemination and infected endothelial cells may be a significant reservoir for fungal persistence. PMID:26039751

  20. Brucella abortus Choloylglycine Hydrolase Affects Cell Envelope Composition and Host Cell Internalization

    PubMed Central

    Marchesini, María Inés; Connolly, Joseph; Delpino, María Victoria; Baldi, Pablo C.; Mujer, Cesar V.; DelVecchio, Vito G.; Comerci, Diego J.

    2011-01-01

    Choloylglycine hydrolase (CGH, E.C. 3.5.1.24) is a conjugated bile salt hydrolase that catalyses the hydrolysis of the amide bond in conjugated bile acids. Bile salt hydrolases are expressed by gastrointestinal bacteria, and they presumably decrease the toxicity of host's conjugated bile salts. Brucella species are the causative agents of brucellosis, a disease affecting livestock and humans. CGH confers Brucella the ability to deconjugate and resist the antimicrobial action of bile salts, contributing to the establishment of a successful infection through the oral route in mice. Additionally, cgh-deletion mutant was also attenuated in intraperitoneally inoculated mice, which suggests that CGH may play a role during systemic infection other than hydrolyzing conjugated bile acids. To understand the role CGH plays in B. abortus virulence, we infected phagocytic and epithelial cells with a cgh-deletion mutant (Δcgh) and found that it is defective in the internalization process. This defect along with the increased resistance of Δcgh to the antimicrobial action of polymyxin B, prompted an analysis of the cell envelope of this mutant. Two-dimensional electrophoretic profiles of Δcgh cell envelope-associated proteins showed an altered expression of Omp2b and different members of the Omp25/31 family. These results were confirmed by Western blot analysis with monoclonal antibodies. Altogether, the results indicate that Brucella CGH not only participates in deconjugation of bile salts but also affects overall membrane composition and host cell internalization. PMID:22174816

  1. Brucella abortus choloylglycine hydrolase affects cell envelope composition and host cell internalization.

    PubMed

    Marchesini, María Inés; Connolly, Joseph; Delpino, María Victoria; Baldi, Pablo C; Mujer, Cesar V; DelVecchio, Vito G; Comerci, Diego J

    2011-01-01

    Choloylglycine hydrolase (CGH, E.C. 3.5.1.24) is a conjugated bile salt hydrolase that catalyses the hydrolysis of the amide bond in conjugated bile acids. Bile salt hydrolases are expressed by gastrointestinal bacteria, and they presumably decrease the toxicity of host's conjugated bile salts. Brucella species are the causative agents of brucellosis, a disease affecting livestock and humans. CGH confers Brucella the ability to deconjugate and resist the antimicrobial action of bile salts, contributing to the establishment of a successful infection through the oral route in mice. Additionally, cgh-deletion mutant was also attenuated in intraperitoneally inoculated mice, which suggests that CGH may play a role during systemic infection other than hydrolyzing conjugated bile acids. To understand the role CGH plays in B. abortus virulence, we infected phagocytic and epithelial cells with a cgh-deletion mutant (Δcgh) and found that it is defective in the internalization process. This defect along with the increased resistance of Δcgh to the antimicrobial action of polymyxin B, prompted an analysis of the cell envelope of this mutant. Two-dimensional electrophoretic profiles of Δcgh cell envelope-associated proteins showed an altered expression of Omp2b and different members of the Omp25/31 family. These results were confirmed by Western blot analysis with monoclonal antibodies. Altogether, the results indicate that Brucella CGH not only participates in deconjugation of bile salts but also affects overall membrane composition and host cell internalization.

  2. International review of cytology. Volume 109: A survey of cell biology

    SciTech Connect

    Bourne, G.; Jeon, K.W.; Friedlander, M.

    1987-01-01

    This book's contents are: Local Regulation of Testicular Function;Microtubules and DNA Replication;Differentiation of Spermatogenic Cells from Vertebrates in Vitro;The Developmental Program of Spermiogenesis in Drosophila: A Genetic Analysis;Cell Motility and Ionic Relations in Characean Cells as Revealed by Internal Perfusion and Other Cell Models;and The Culture of Oral Epithelium. Each chapter includes references.

  3. International Society for Stem Cell Research (ISSCR) Guidelines for the Conduct of Human Embryonic Stem Cell Research (December 2006).

    PubMed

    Dickens, Bernard M

    2008-03-01

    The International Society for Stem Cell Research (ISSCR) Guidelines for the Conduct of Human Embryonic Stem Cell Research, prepared by an international group of leading stem cell scientists, bioethicists and lawyers, build on the proposals previously drafted by agencies active in the conduct and funding of stem cell science. They aim not to duplicate existing codes of ethics in research with humans or animals, but to address special sensitivities about human embryo research and acquisition of ova, sperm, embryos and somatic tissues from often vulnerable or dependent donors. Guidelines concern the procurement of research materials, the derivation of stem cells, and banking , distribution and use of cells and cell lines derived from pre-implantation stages of human development. They categorize research that on ethical grounds requires special stem cell research oversight (SCRO), and indicate how that should be undertaken. They emphasize international collaboration in these activities, to maximize benefits to humanity.

  4. Analysis of an internally pressurized prismatic cell can. [In FORTRAN for IBM 360

    SciTech Connect

    Majumdar, S.

    1980-04-01

    This report contains an elastic stress and displacement analysis of a prismatic cell can subjected to internal pressure. A computer program was written to perform the analysis. The results show that, for the geometry chosen, the thicknesses of the cell-can walls and the magnitude of the internal pressure are the most important parameters that determine the stresses and deformations of the cell can. Recommendations for future studies are included. 21 figures, 1 table.

  5. Enhanced relative biological effectiveness of proton radiotherapy in tumor cells with internalized gold nanoparticles

    NASA Astrophysics Data System (ADS)

    Polf, Jerimy C.; Bronk, Lawrence F.; Driessen, Wouter H. P.; Arap, Wadih; Pasqualini, Renata; Gillin, Michael

    2011-05-01

    The development and use of sensitizing agents to improve the effectiveness of radiotherapy have long been sought to improve our ability to treat cancer. In this letter, we have studied the relative biological effectiveness of proton beam radiotherapy on prostate tumor cells with and without internalized gold nanoparticles. The effectiveness of proton radiotherapy for the killing of prostate tumor cells was increased by approximately 15%-20% for those cells containing internalized gold nanoparticles.

  6. Enhanced relative biological effectiveness of proton radiotherapy in tumor cells with internalized gold nanoparticles

    SciTech Connect

    Polf, Jerimy C.; Gillin, Michael; Bronk, Lawrence F.; Driessen, Wouter H. P.; Arap, Wadih; Pasqualini, Renata

    2011-05-09

    The development and use of sensitizing agents to improve the effectiveness of radiotherapy have long been sought to improve our ability to treat cancer. In this letter, we have studied the relative biological effectiveness of proton beam radiotherapy on prostate tumor cells with and without internalized gold nanoparticles. The effectiveness of proton radiotherapy for the killing of prostate tumor cells was increased by approximately 15%-20% for those cells containing internalized gold nanoparticles.

  7. Internal and ancestral controls of cell-generation times

    NASA Technical Reports Server (NTRS)

    Kubitschek, H. E.

    1969-01-01

    Lateral and longitudinal correlations between related cells reveal associations between the generation times of cells for an intermediate period /three generations in bacteral cultures/. Generation times of progeny are influenced by nongenetic factors transmitted from their ancestors.

  8. Experimental analysis of internal gas flow configurations for a polymer electrolyte membrane fuel cell stack

    NASA Astrophysics Data System (ADS)

    Friedl, A.; Fraser, S. D.; Baumgartner, W. R.; Hacker, V.

    The internal gas distribution system utilised for supplying fresh reactants and removing reaction products from the individual cells of a fuel cell stack can be designed in a parallel, a serial or a mixture of parallel and serial gas flow configuration. In order to investigate the interdependence between the internal stack gas distribution configuration and single cell as well as overall stack performance, a small laboratory-scale fuel cell stack consisting of identical unit cells was subject to operation with different gas distribution configurations and different operating parameters. The current/voltage characteristics measured with the different gas distribution configurations are analysed and compared on unit cell- as well as on stack-level. The results show the significant impact of the internal stack gas distribution system on operation and performance of the individual unit cells and the overall stack.

  9. Adjuvant therapy of Dukes' C colon cancer by intra-arterial P-32 colloid for internal radiation therapy of the liver

    SciTech Connect

    Grady, E.D.

    1984-09-01

    To prevent probable occult metastatic liver cancer from progressing to clinical disease, the author used internal radiation therapy as an effective adjuvant to surgical excision of primary Dukes' C colonic cancer. A calculated radiation dose of 5000 rads was delivered to the liver by injecting radioactive 32-P chromic phosphate colloid through the superior mesenteric and celiac arteries. When this was done, the colloid passed through the intestines and was mixed thoroughly with the blood and delivered to the liver by the portal vein. The Kupffer cells in the liver trapped the colloid, and a minimum amount passed through the liver and got into the general circulation. This kept the amount of colloid deposited in the bone marrow to a minimum. In a phase-I pilot study in which nine patients were treated, no serious side effects were noted. In eight patients, the liver has remained free of cancer for more than 1 year.

  10. Enhancing microparticle internalization by nonphagocytic cells through the use of noncovalently conjugated polyethyleneimine.

    PubMed

    Patiño, Tania; Nogués, Carme; Ibáñez, Elena; Barrios, Leonardo

    2012-01-01

    Development of micro- and nanotechnology for the study of living cells, especially in the field of drug delivery, has gained interest in recent years. Although several studies have reported successful results in the internalization of micro- and nanoparticles in phagocytic cells, when nonphagocytic cells are used, the low internalization efficiency represents a limitation that needs to be overcome. It has been reported that covalent surface modification of micro- and nanoparticles increases their internalization rate. However, this surface modification represents an obstacle for their use as drug-delivery carriers. For this reason, the aim of the present study was to increase the capability for microparticle internalization of HeLa cells through the use of noncovalently bound transfection reagents: polyethyleneimine (PEI) Lipofectamine™ 2000 and FuGENE 6(®). Both confocal microscopy and flow cytometry techniques allowed us to precisely quantify the efficiency of microparticle internalization by HeLa cells, yielding similar results. In addition, intracellular location of microparticles was analyzed through transmission electron microscopy and confocal microscopy procedures. Our results showed that free PEI at a concentration of 0.05 mM significantly increased microparticle uptake by cells, with a low cytotoxic effect. As determined by transmission electron and confocal microscopy analyses, microparticles were engulfed by plasma-membrane projections during internalization, and 24 hours later they were trapped in a lysosomal compartment. These results show the potential use of noncovalently conjugated PEI in microparticle internalization assays.

  11. From Banking to International Governance: Fostering Innovation in Stem Cell Research

    PubMed Central

    Isasi, Rosario; Knoppers, Bartha M.

    2011-01-01

    Stem cell banks are increasingly recognized as an essential resource of biological materials for both basic and translational stem cell research. By providing transnational access to quality controlled and ethically sourced stem cell lines, stem cell banks seek to foster international collaboration and innovation. However, given that national stem cell banks operate under different policy, regulatory and commercial frameworks, the transnational sharing of stem cell materials and data can be complicating. This paper will provide an overview of the most pressing challenges regarding the governance of stem cell banks, and the difficulties in designing regulatory and commercial frameworks that foster stem cell research. Moreover, the paper will shed light on the numerous international initiatives that have arisen to help harmonize and standardize stem cell banking and research processes to overcome such challenges. PMID:21904557

  12. Quantitation of alpha-factor internalization and response during the Saccharomyces cerevisiae cell cycle.

    PubMed Central

    Zanolari, B; Riezman, H

    1991-01-01

    The alpha-factor pheromone binds to specific cell surface receptors on Saccharomyces cerevisiae a cells. The pheromone is then internalized, and cell surface receptors are down-regulated. At the same time, a signal is transmitted that causes changes in gene expression and cell cycle arrest. We show that the ability of cells to internalize alpha-factor is constant throughout the cell cycle, a cells are also able to respond to pheromone throughout the cycle even though there is cell cycle modulation of the expression of two pheromone-inducible genes, FUS1 and STE2. Both of these genes are expressed less efficiently near or just after the START point of the cell cycle in response to alpha-factor. For STE2, the basal level of expression is modulated in the same manner. Images PMID:1656226

  13. Internalization of multiple cells during C. elegans gastrulation depends on common cytoskeletal mechanisms but different cell polarity and cell fate regulators

    PubMed Central

    Harrell, Jessica R.; Goldstein, Bob

    2010-01-01

    Understanding the links between developmental patterning mechanisms and force-producing cytoskeletal mechanisms is a central goal in studies of morphogenesis. Gastrulation is the first morphogenetic event in the development of many organisms. Gastrulation involves the internalization of surface cells, often driven by the contraction of actomyosin networks that are deployed with spatial precision -- both in specific cells and in a polarized manner within each cell. These cytoskeletal mechanisms rely on different cell fate and cell polarity regulators in different organisms. C. elegans gastrulation presents an opportunity to examine the extent to which diverse mechanisms may be used by dozens of cells that are internalized at distinct times within a single organism. We identified 66 cells that are internalized in C. elegans gastrulation, many of which were not known previously to gastrulate. To gain mechanistic insights into how these cells internalize, we genetically manipulated cell fate, cell polarity and cytoskeletal regulators and determined the effects on cell internalization. We found that cells of distinct lineages depend on common actomyosin-based mechanisms to gastrulate, but different cell fate regulators, and, surprisingly, different cell polarity regulators. We conclude that diverse cell fate and cell polarity regulators control common mechanisms of morphogenesis in C. elegans. The results highlight the variety of developmental patterning mechanisms that can be associated with common cytoskeletal mechanisms in the morphogenesis of an animal embryo. PMID:20875815

  14. The effect of internal stresses on solar cell efficiency

    NASA Technical Reports Server (NTRS)

    Weizer, Victor G.

    1987-01-01

    Diffusion induced stresses in silicon are shown to result in large localized changes in the minority carrier mobility which in turn have a significant effect on cell output. Evidence is given that both compressive and tensile stresses can be generated in either the emitter or the base region. Tensile stresses appear to be much more effective in altering cell performance. While most stress related effects appear to degrade cell efficiency, this is not always the case. Evidence is presented showing that arsenic induced stresses can result in emitter characteristics comparable to those found in the MINP cell without requiring a high degree of surface passivation.

  15. Development of internal reforming carbonate fuel cell stack technology

    SciTech Connect

    Farooque, M.

    1990-10-01

    Activities under this contract focused on the development of a coal-fueled carbonate fuel cell system design and the stack technology consistent with the system design. The overall contract effort was divided into three phases. The first phase, completed in January 1988, provided carbonate fuel cell component scale-up from the 1ft{sup 2} size to the commercial 4ft{sup 2} size. The second phase of the program provided the coal-fueled carbonate fuel cell system (CGCFC) conceptual design and carried out initial research and development needs of the CGCFC system. The final phase of the program emphasized stack height scale-up and improvement of stack life. The results of the second and third phases are included in this report. Program activities under Phase 2 and 3 were designed to address several key development areas to prepare the carbonate fuel cell system, particularly the coal-fueled CFC power plant, for commercialization in late 1990's. The issues addressed include: Coal-Gas Related Considerations; Cell and Stack Technology Improvement; Carbonate Fuel Cell Stack Design Development; Stack Tests for Design Verification; Full-Size Stack Design; Test Facility Development; Carbonate Fuel Cell Stack Cost Assessment; and Coal-Fueled Carbonate Fuel Cell System Design. All the major program objectives in each of the topical areas were successfully achieved. This report is organized along the above-mentioned topical areas. Each topical area has been processed separately for inclusion on the data base.

  16. Keratin cytoskeletons in epithelial cells of internal organs

    PubMed Central

    Sun, Tung-Tien; Shih, Chiaho; Green, Howard

    1979-01-01

    An antiserum against human epidermal keratins was used to detect keratins in frozen sections of various rabbit and human tissues by indirect immunofluorescence. Strong staining was observed in all stratified squamous epithelia (epidermis, cornea, conjunctiva, tongue, esophagus, vagina, and anus), in epidermal appendages (hair follicle, sebaceous gland, ductal and myoepithelial cells of sweat glands), as well as in Hassall's corpuscles of the thymus, indicating that all contain abundant keratins. No staining by the antiserum was observed in fibroblasts, muscle of any type, cartilage, blood vessel, nerve tissue, iris or lens epithelium, or the glomerular or tubular cells of the kidney. In contrast, the antiserum stained the cells of most epithelia of the intestinal tract, urinary tract (urethra, bladder, ureter, collecting ducts of kidney), female genital tract (cervix, cervical glands, uterus, and oviduct), and respiratory tract (trachea and bronchi). Epithelial cells of the fine ductal system in the pancreas and submaxillary gland also stained well. When primary cultures of epithelial cells derived from bladder, intestine, kidney, and trachea were grown on glass coverslips and stained with anti-keratin, fiber networks similar to those of cultured keratinocytes were observed. These results show that keratins constitute a cytoskeleton in epithelial cells of diverse morphology and embryological origin. The stability of keratin filaments probably confers the structural strength necessary for cells covering a free surface. Keratin staining can be used to obtain information about the origin of cell lines. Images PMID:111242

  17. Engineering of Targeted Nanoparticles for Cancer Therapy Using Internalizing Aptamers Isolated by Cell-Uptake Selection

    PubMed Central

    Xiao, Zeyu; Levy-Nissenbaum, Etgar; Alexis, Frank; Lupták, Andrej; Teply, Benjamin A.; Chan, Juliana M.; Shi, Jinjun; Digga, Elise; Cheng, Judy; Langer, Robert; Farokhzad, Omid C.

    2012-01-01

    One of the major challenges in the development of targeted nanoparticles (NPs) for cancer therapy is to discover targeting ligands that allow for differential binding and uptake by the target cancer cells. Using prostate cancer (PCa) as a model disease, we developed a cell-uptake selection strategy to isolate PCa-specific internalizing 2'-Omethyl RNA aptamers (Apts) for NP incorporation. Twelve cycles of selection and counter-selection were done to obtain a panel of internalizing Apts, which can distinguish PCa cells from non-prostate and normal prostate cells. After Apt characterization, size minimization, and conjugation of the Apts with fluorescently-labeled polymeric NPs, the NP-Apt bioconjugates exhibit PCa specificity and enhancement in cellular uptake when compared to non-targeted NPs lacking the internalizing Apts. Furthermore, when docetaxel, a chemotherapeutic agent used for the treatment of PCa, was encapsulated within the NP-Apt, a significant improvement in cytotoxicity was achieved in targeted PCa cells. Rather than isolating high-affinity Apts as reported in previous selection processes, our selection strategy was designed to enrich cancer-cell specific internalizing Apts. A similar cell-uptake selection strategy may be used to develop specific internalizing ligands for a myriad of other diseases and can potentially facilitate delivering various molecules, including drugs and siRNAs, into cells. PMID:22214176

  18. Nanotopology of cell adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM).

    PubMed

    Wagner, Michael; Weber, Petra; Baumann, Harald; Schneckenburger, Herbert

    2012-10-02

    Surface topology, e.g. of cells growing on a substrate, is determined with nanometer precision by Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM). Cells are cultivated on transparent slides and incubated with a fluorescent marker homogeneously distributed in their plasma membrane. Illumination occurs by a parallel laser beam under variable angles of total internal reflection (TIR) with different penetration depths of the evanescent electromagnetic field. Recording of fluorescence images upon irradiation at about 10 different angles permits to calculate cell-substrate distances with a precision of a few nanometers. Differences of adhesion between various cell lines, e.g. cancer cells and less malignant cells, are thus determined. In addition, possible changes of cell adhesion upon chemical or photodynamic treatment can be examined. In comparison with other methods of super-resolution microscopy light exposure is kept very small, and no damage of living cells is expected to occur.

  19. Internal configuration of prismatic lithium-ion cells at the onset of mechanically induced short circuit

    DOE PAGES

    Wang, Hsin; Simunovic, Srdjan; Maleki, Hosein; ...

    2016-01-01

    The response of Li-ion cells to mechanically induced internal electrical shorts is an important safety performance metric design. We assume that the battery internal configuration at the onset of electrical short influences the subsequent response and can be used to gauge the safety risk. We subjected a series of prismatic Li-ion cells to lateral pinching using 0.25", 0.5", 1", 2" and 3" diameter steel balls until the onset of internal short. The external aluminum enclosure froze the internal cell configuration at the onset of short and enabled us to cross-section the cells, and take the cross-section images. The images indicatemore » that an internal electric short is preceded by extensive strain partitioning in the cells, fracturing and tearing of the current collectors, and cracking and slipping of the electrode layers with multiple fault lines across multiple layers. These observations are at odds with a common notion of homogeneous deformation across the layers and strain hardening of electrodes that eventually punch through the separator and short the cell. The faults are akin to tectonic movements of multiple layers that are characteristic of granular materials and bonded aggregates. As a result, the short circuits occur after extensive internal faulting, which implies significant stretching and tearing of separators.« less

  20. Internal configuration of prismatic lithium-ion cells at the onset of mechanically induced short circuit

    SciTech Connect

    Wang, Hsin; Simunovic, Srdjan; Maleki, Hosein; Howard, Jason N.; Hallmark, Jerald A.

    2016-01-01

    The response of Li-ion cells to mechanically induced internal electrical shorts is an important safety performance metric design. We assume that the battery internal configuration at the onset of electrical short influences the subsequent response and can be used to gauge the safety risk. We subjected a series of prismatic Li-ion cells to lateral pinching using 0.25", 0.5", 1", 2" and 3" diameter steel balls until the onset of internal short. The external aluminum enclosure froze the internal cell configuration at the onset of short and enabled us to cross-section the cells, and take the cross-section images. The images indicate that an internal electric short is preceded by extensive strain partitioning in the cells, fracturing and tearing of the current collectors, and cracking and slipping of the electrode layers with multiple fault lines across multiple layers. These observations are at odds with a common notion of homogeneous deformation across the layers and strain hardening of electrodes that eventually punch through the separator and short the cell. The faults are akin to tectonic movements of multiple layers that are characteristic of granular materials and bonded aggregates. As a result, the short circuits occur after extensive internal faulting, which implies significant stretching and tearing of separators.

  1. Body Management: Mesenchymal Stem Cells Control the Internal Regenerator

    PubMed Central

    Hariri, Robert

    2015-01-01

    Summary It has been assumed that adult tissues cannot regenerate themselves. With the current understanding that every adult tissue has its own intrinsic progenitor or stem cell, it is now clear that almost all tissues have regenerative potential partially related to their innate turnover dynamics. Moreover, it appears that a separate class of local cells originating as perivascular cells appears to provide regulatory oversight for localized tissue regeneration. The management of this regeneration oversight has a profound influence on the use of specific cells for cell therapies as a health care delivery tool set. The multipotent mesenchymal stem cell (MSC), now renamed the medicinal signaling cell, predominantly arises from pericytes released from broken and inflamed blood vessels and appears to function as both an immunomodulatory and a regeneration mediator. MSCs are being tested for their management capabilities to produce therapeutic outcomes in more than 480 clinical trials for a wide range of clinical conditions. Local MSCs function by managing the body’s primary repair and regeneration activities. Supplemental MSCs can be provided from either endogenous or exogenous sources of either allogeneic or autologous origin. This MSC-based therapy has the potential to change how health care is delivered. These medicinal cells are capable of sensing their surroundings. Also, by using its complex signaling circuitry, these cells organize site-specific regenerative responses as if these therapeutic cells were well-programmed modern computers. Given these facts, it appears that we are entering a new age of cellular medicine. Significance This report is a perspective from an active scientist and an active entrepreneur and commercial leader. It is neither a comprehensive review nor a narrowly focused treatise. The broad themes and the analogy to the working component of a computer and that of a cell are meant to draw several important scientific principles and health

  2. Fuel cell stack with internal manifolds for reactant gases

    DOEpatents

    Schnacke, Arthur W.

    1985-01-01

    A fuel cell stack includes a plurality of plate-like fuel cells arranged along an axis generally parallel to cell thickness with electrically conductive separator plates between each pair of cells. A plurality of axial manifolds are provided at opposite sides of the stack in outer marginal portions beyond the edges of electrodes and electrolyte tiles. Sealing rings prevent cross-leakage of oxidant fuel gases through use of pairs of outwardly extending lips from opposite tile surfaces bonded to first and second electrode frames respectively. The frames provide transition between electrode edges and manifold perimeters. The pairs of extension lips are sealingly bonded together through an electrically insulative sealing ring with wedge shaped fastening members.

  3. Arf6 regulates EGF-induced internalization of E-cadherin in breast cancer cells.

    PubMed

    Xu, Rui; Zhang, Yujie; Gu, Luo; Zheng, Jianchao; Cui, Jie; Dong, Jing; Du, Jun

    2015-01-01

    E-cadherin internalization facilitates dissolution of adherens junctions and promotes tumor cell epithelial-mesenchymal transition (EMT) and migration. Our previous results have shown that Arf6 exerts pro-migratory action in breast cancer cells after EGF stimulation. Despite the fact that EGF signaling stimulates EMT of breast cancer cells, the effect of Arf6 on internalization of E-cadherin of breast cancer cells under EGF treatment remains to be determined. Here, we showed that EGF dose-dependently stimulated E-cadherin internalization by MCF-7 cells with the maximal effect at 50 ng/ml. Meanwhile, EGF treatment markedly increased Arf6 activation. Arf6 was involved in complexes of E-cadherin, and more E-cadherin was pulled down with Arf6 when the activity of the latter was increased. Immunoblotting and immunofluorescence assays showed that transfection breast cancer cells with Arf6-T27N or Arf6 siRNA suppressed EGF-induced E-cadherin internalization. Taken together, our study demonstrated that Arf6 activation plays a potential role in EGF-induced E-cadherin internalization, providing new mechanism underlying the effect of Arf6 on promoting breast cancer cell metastasis.

  4. Cell-surface metalloprotease ADAM12 is internalized by a clathrin- and Grb2-dependent mechanism.

    PubMed

    Stautz, Dorte; Leyme, Anthony; Grandal, Michael Vibo; Albrechtsen, Reidar; van Deurs, Bo; Wewer, Ulla; Kveiborg, Marie

    2012-11-01

    ADAM12 (A Disintegrin And Metalloprotease 12), a member of the ADAMs family of transmembrane proteins, is involved in ectodomain shedding, cell-adhesion and signaling, with important implications in cancer. Therefore, mechanisms that regulate the levels and activity of ADAM12 at the cell-surface are possibly crucial in these contexts. We here investigated internalization and subsequent recycling or degradation of ADAM12 as a potentially important regulatory mechanism. Our results show that ADAM12 is constitutively internalized primarily via the clathrin-dependent pathway and is subsequently detected in both early and recycling endosomes. The protease activity of ADAM12 does not influence this internalization mechanism. Analysis of essential elements for internalization established that proline-rich regions in the cytoplasmic domain of ADAM12, previously shown to interact with Src-homology 3 domains, were necessary for proper internalization. These sites in the ADAM12 cytoplasmic domain interacted with the adaptor protein growth factor receptor-bound protein 2 (Grb2) and knockdown of Grb2 markedly reduced ADAM12 internalization. These studies establish that internalization is indeed a mechanism that regulates ADAM cell surface levels and show that ADAM12 internalization involves the clathrin-dependent pathway and Grb2.

  5. Internal binding sites for MSH: Analyses in wild-type and variant Cloudman melanoma cells

    SciTech Connect

    Orlow, S.J.; Hotchkiss, S.; Pawelek, J.M. )

    1990-01-01

    Cloudman S91 mouse melanoma cells express both external (plasma membrane) and internal binding sites for MSH. Using 125I-beta melanotropin (beta-MSH) as a probe, we report here an extensive series of studies on the biological relevance of these internal sites. Cells were swollen in a hypotonic buffer and lysed, and a particulate fraction was prepared by high-speed centrifugation. This fraction was incubated with 125I-beta-MSH with or without excess nonradioactive beta-MSH in the cold for 2 hours. The material was then layered onto a step-wise sucrose gradient and centrifuged; fractions were collected and counted in a gamma counter or assayed for various enzymatic activities. The following points were established: (1) Specific binding sites for MSH were observed sedimenting at an average density of 50% sucrose in amelanotic cells and at higher densities in melanotic cells. (2) These sites were similar in density to those observed when intact cells were labeled externally with 125I-beta-MSH and then warmed to promote internalization of the hormone. (3) Most of the internal binding sites were not as dense as fully melanized melanosomes. (4) In control experiments, the MSH binding sites were not found in cultured hepatoma cells. (5) Variant melanoma cells, which differed from the wild-type in their responses to MSH, had reduced expression of internal binding sites even though their ability to bind MSH to the outer cell surface appeared normal. (MSH-induced responses included changes in tyrosinase, dopa oxidase, and dopachrome conversion factor activities, melanization, proliferation, and morphology.) (6) Isobutylmethylxanthine, which enhanced cellular responsiveness to MSH, also enhanced expression of internal binding sites. The results indicate that expression of internal binding sites for MSH is an important criterion for cellular responsiveness to the hormone.

  6. Harmonizing the international regulation of embryonic stem cell research: possibilities, promises and potential pitfalls.

    PubMed

    Campbell, Angela; Nycum, Gillian

    2005-01-01

    Despite near unanimous global opposition to human reproductive cloning, the United Nations has been unable to reach a consensus as to how cloning practices should be regulated at the international level. As a result, the U.N. objective of establishing binding international regulations governing cloning and stem cell research has yet to be achieved. Given the lack of consensus that exists within the global community on this topic, it seems that any attempt to harmonize the international regulation of cloning and stem cell science will face important obstacles. This paper seeks to illuminate the particular challenges to harmonizing international laws and policies related to stem cell research and human cloning, and to investigate potential methods for overcoming these challenges. By drawing on two other areas in which regulatory harmonization has been attempted, namely: environmental and human safety aspects of international trade, and pharmaceutical research and development, we study approaches to global regulatory harmonization. We conclude that while the challenges to harmonization are diverse and important, so too are the benefits of establishing uniformity in approaches to stem cell research worldwide. This paper proposes a model for harmonizing the regulation of stem cell research that focuses on broader norms and principles rather than specific rules. It further recommends that such harmonization should occur through a process initiated and developed by an independent international agency marked by diversity, both in terms of the cultural identities and perspectives represented, and the interdisciplinary expertise of its members.

  7. Quantification of surface tension and internal pressure generated by single mitotic cells

    NASA Astrophysics Data System (ADS)

    Fischer-Friedrich, Elisabeth; Hyman, Anthony A.; Jülicher, Frank; Müller, Daniel J.; Helenius, Jonne

    2014-08-01

    During mitosis, adherent cells round up, by increasing the tension of the contractile actomyosin cortex while increasing the internal hydrostatic pressure. In the simple scenario of a liquid cell interior, the surface tension is related to the local curvature and the hydrostatic pressure difference by Laplace's law. However, verification of this scenario for cells requires accurate measurements of cell shape. Here, we use wedged micro-cantilevers to uniaxially confine single cells and determine confinement forces while concurrently determining cell shape using confocal microscopy. We fit experimentally measured confined cell shapes to shapes obeying Laplace's law with uniform surface tension and find quantitative agreement. Geometrical parameters derived from fitting the cell shape, and the measured force were used to calculate hydrostatic pressure excess and surface tension of cells. We find that HeLa cells increase their internal hydrostatic pressure excess and surface tension from ~ 40 Pa and 0.2 mNm-1 during interphase to ~ 400 Pa and 1.6 mNm-1 during metaphase. The method introduced provides a means to determine internal pressure excess and surface tension of rounded cells accurately and with minimal cellular perturbation, and should be applicable to characterize the mechanical properties of various cellular systems.

  8. Quantification of surface tension and internal pressure generated by single mitotic cells.

    PubMed

    Fischer-Friedrich, Elisabeth; Hyman, Anthony A; Jülicher, Frank; Müller, Daniel J; Helenius, Jonne

    2014-08-29

    During mitosis, adherent cells round up, by increasing the tension of the contractile actomyosin cortex while increasing the internal hydrostatic pressure. In the simple scenario of a liquid cell interior, the surface tension is related to the local curvature and the hydrostatic pressure difference by Laplace's law. However, verification of this scenario for cells requires accurate measurements of cell shape. Here, we use wedged micro-cantilevers to uniaxially confine single cells and determine confinement forces while concurrently determining cell shape using confocal microscopy. We fit experimentally measured confined cell shapes to shapes obeying Laplace's law with uniform surface tension and find quantitative agreement. Geometrical parameters derived from fitting the cell shape, and the measured force were used to calculate hydrostatic pressure excess and surface tension of cells. We find that HeLa cells increase their internal hydrostatic pressure excess and surface tension from ≈ 40 Pa and 0.2 mNm(-1) during interphase to ≈ 400 Pa and 1.6 mNm(-1) during metaphase. The method introduced provides a means to determine internal pressure excess and surface tension of rounded cells accurately and with minimal cellular perturbation, and should be applicable to characterize the mechanical properties of various cellular systems.

  9. Relationship between stiffness, internal cell pressure and shape of outer hair cells isolated from the guinea-pig hearing organ.

    PubMed

    Chan, E; Ulfendahl, M

    1997-12-01

    The mechanical properties of outer hair cells are of importance for normal hearing, and it has been shown that damage of the cells can lead to a reduction in the hearing sensitivity. In this study, we measured the stiffness of isolated outer hair cells in hyper- and hypotonic conditions, and examined the change in stiffness in relation to the corresponding changes in internal cell pressure and cell shape. The results showed that the axial stiffness of isolated outer hair cells (30-90 microns in length, 8-12 microns in diameter), ranging from 0.13-5.39 mN m-1, was inversely related to cell length. Exposure to hyper- and hypotonic external media with a small percentage change in osmolality caused a similar magnitude of change in cell length and cell diameter, but an average 60% change in cell stiffness. Therefore, a moderate osmotic change in the external medium can lead to a significant alteration in cell stiffness. The findings thus indicate an important contribution of internal cell pressure to cell stiffness.

  10. Detailed Multi-dimensional Modeling of Direct Internal Reforming Solid Oxide Fuel Cells.

    PubMed

    Tseronis, K; Fragkopoulos, I S; Bonis, I; Theodoropoulos, C

    2016-06-01

    Fuel flexibility is a significant advantage of solid oxide fuel cells (SOFCs) and can be attributed to their high operating temperature. Here we consider a direct internal reforming solid oxide fuel cell setup in which a separate fuel reformer is not required. We construct a multidimensional, detailed model of a planar solid oxide fuel cell, where mass transport in the fuel channel is modeled using the Stefan-Maxwell model, whereas the mass transport within the porous electrodes is simulated using the Dusty-Gas model. The resulting highly nonlinear model is built into COMSOL Multiphysics, a commercial computational fluid dynamics software, and is validated against experimental data from the literature. A number of parametric studies is performed to obtain insights on the direct internal reforming solid oxide fuel cell system behavior and efficiency, to aid the design procedure. It is shown that internal reforming results in temperature drop close to the inlet and that the direct internal reforming solid oxide fuel cell performance can be enhanced by increasing the operating temperature. It is also observed that decreases in the inlet temperature result in smoother temperature profiles and in the formation of reduced thermal gradients. Furthermore, the direct internal reforming solid oxide fuel cell performance was found to be affected by the thickness of the electrochemically-active anode catalyst layer, although not always substantially, due to the counter-balancing behavior of the activation and ohmic overpotentials.

  11. Deciphering the internal complexity of living cells with quantitative phase microscopy: a multiscale approach

    NASA Astrophysics Data System (ADS)

    Martinez-Torres, Cristina; Laperrousaz, Bastien; Berguiga, Lotfi; Boyer-Provera, Elise; Elezgaray, Juan; Nicolini, Franck E.; Maguer-Satta, Veronique; Arneodo, Alain; Argoul, Françoise

    2015-09-01

    The distribution of refractive indices (RIs) of a living cell contributes in a nonintuitive manner to its optical phase image and quite rarely can be inverted to recover its internal structure. The interpretation of the quantitative phase images of living cells remains a difficult task because (1) we still have very little knowledge on the impact of its internal macromolecular complexes on the local RI and (2) phase changes produced by light propagation through the sample are mixed with diffraction effects by the internal cell bodies. We propose to implement a two-dimensional wavelet-based contour chain detection method to distinguish internal boundaries based on their greatest optical path difference gradients. These contour chains correspond to the highest image phase contrast and follow the local RI inhomogeneities linked to the intracellular structural intricacy. Their statistics and spatial distribution are the morphological indicators suited for comparing cells of different origins and/or to follow their transformation in pathologic situations. We use this method to compare nonadherent blood cells from primary and laboratory culture origins and to assess the internal transformation of hematopoietic stem cells by the transduction of the BCR-ABL oncogene responsible for the chronic myelogenous leukemia.

  12. Detailed Multi‐dimensional Modeling of Direct Internal Reforming Solid Oxide Fuel Cells

    PubMed Central

    Tseronis, K.; Fragkopoulos, I.S.; Bonis, I.

    2016-01-01

    Abstract Fuel flexibility is a significant advantage of solid oxide fuel cells (SOFCs) and can be attributed to their high operating temperature. Here we consider a direct internal reforming solid oxide fuel cell setup in which a separate fuel reformer is not required. We construct a multidimensional, detailed model of a planar solid oxide fuel cell, where mass transport in the fuel channel is modeled using the Stefan‐Maxwell model, whereas the mass transport within the porous electrodes is simulated using the Dusty‐Gas model. The resulting highly nonlinear model is built into COMSOL Multiphysics, a commercial computational fluid dynamics software, and is validated against experimental data from the literature. A number of parametric studies is performed to obtain insights on the direct internal reforming solid oxide fuel cell system behavior and efficiency, to aid the design procedure. It is shown that internal reforming results in temperature drop close to the inlet and that the direct internal reforming solid oxide fuel cell performance can be enhanced by increasing the operating temperature. It is also observed that decreases in the inlet temperature result in smoother temperature profiles and in the formation of reduced thermal gradients. Furthermore, the direct internal reforming solid oxide fuel cell performance was found to be affected by the thickness of the electrochemically‐active anode catalyst layer, although not always substantially, due to the counter‐balancing behavior of the activation and ohmic overpotentials. PMID:27570502

  13. Comparison of Several Methods for Determining the Internal Resistance of Lithium Ion Cells

    PubMed Central

    Schweiger, Hans-Georg; Obeidi, Ossama; Komesker, Oliver; Raschke, André; Schiemann, Michael; Zehner, Christian; Gehnen, Markus; Keller, Michael; Birke, Peter

    2010-01-01

    The internal resistance is the key parameter for determining power, energy efficiency and lost heat of a lithium ion cell. Precise knowledge of this value is vital for designing battery systems for automotive applications. Internal resistance of a cell was determined by current step methods, AC (alternating current) methods, electrochemical impedance spectroscopy and thermal loss methods. The outcomes of these measurements have been compared with each other. If charge or discharge of the cell is limited, current step methods provide the same results as energy loss methods. PMID:22219678

  14. Hypoxia Decreases Invasin-Mediated Yersinia enterocolitica Internalization into Caco-2 Cells

    PubMed Central

    Zeitouni, Nathalie E.; Dersch, Petra; Naim, Hassan Y.; von Köckritz-Blickwede, Maren

    2016-01-01

    Yersinia enterocolitica is a major cause of human yersiniosis, with enterocolitis being a typical manifestation. These bacteria can cross the intestinal mucosa, and invade eukaryotic cells by binding to host β1 integrins, a process mediated by the bacterial effector protein invasin. This study examines the role of hypoxia on the internalization of Y. enterocolitica into intestinal epithelial cells, since the gastrointestinal tract has been shown to be physiologically deficient in oxygen levels (hypoxic), especially in cases of infection and inflammation. We show that hypoxic pre-incubation of Caco-2 cells resulted in significantly decreased bacterial internalization compared to cells grown under normoxia. This phenotype was absent after functionally blocking host β1 integrins as well as upon infection with an invasin-deficient Y. enterocolitica strain. Furthermore, downstream phosphorylation of the focal adhesion kinase was also reduced under hypoxia after infection. In good correlation to these data, cells grown under hypoxia showed decreased protein levels of β1 integrins at the apical cell surface whereas the total protein level of the hypoxia inducible factor (HIF-1) alpha was elevated. Furthermore, treatment of cells with the HIF-1 α stabilizer dimethyloxalylglycine (DMOG) also reduced invasion and decreased β1 integrin protein levels compared to control cells, indicating a potential role for HIF-1α in this process. These results suggest that hypoxia decreases invasin-integrin-mediated internalization of Y. enterocolitica into intestinal epithelial cells by reducing cell surface localization of host β1 integrins. PMID:26731748

  15. Cystic fibrosis transmembrane conductance regulator and caveolin-1 regulate epithelial cell internalization of Pseudomonas aeruginosa

    PubMed Central

    Bajmoczi, Milan; Gadjeva, Mihaela; Alper, Seth L.; Pier, Gerald B.; Golan, David E.

    2009-01-01

    Patients with cystic fibrosis (CF) exhibit defective innate immunity and are susceptible to chronic lung infection with Pseudomonas aeruginosa. To investigate the molecular bases for the hypersusceptibility of CF patients to P. aeruginosa, we used the IB3-1 cell line with two defective CF transmembrane conductance regulator (CFTR) genes (ΔF508/W1282X) to generate isogenic stable, clonal lung epithelial cells expressing wild-type (WT)-CFTR with an NH2-terminal green fluorescent protein (GFP) tag. GFP-CFTR exhibited posttranslational modification, subcellular localization, and anion transport function typical of WT-CFTR. P. aeruginosa internalization, a component of effective innate immunity, required functional CFTR and caveolin-1, as shown by: 1) direct correlation between GFP-CFTR expression levels and P. aeruginosa internalization; 2) enhanced P. aeruginosa internalization by aminoglycoside-induced read through of the CFTR W1282X allele in IB3-1 cells; 3) decreased P. aeruginosa internalization following siRNA knockdown of GFP-CFTR or caveolin-1; and 4) spatial association of P. aeruginosa with GFP-CFTR and caveolin-1 at the cell surface. P. aeruginosa internalization also required free lateral diffusion of GFP-CFTR, allowing for bacterial coclustering with GFP-CFTR and caveolin-1 at the plasma membrane. Thus efficient initiation of innate immunity to P. aeruginosa requires formation of an epithelial “internalization platform” involving both caveolin-1 and functional, laterally mobile CFTR. PMID:19386787

  16. Cystic fibrosis transmembrane conductance regulator and caveolin-1 regulate epithelial cell internalization of Pseudomonas aeruginosa.

    PubMed

    Bajmoczi, Milan; Gadjeva, Mihaela; Alper, Seth L; Pier, Gerald B; Golan, David E

    2009-08-01

    Patients with cystic fibrosis (CF) exhibit defective innate immunity and are susceptible to chronic lung infection with Pseudomonas aeruginosa. To investigate the molecular bases for the hypersusceptibility of CF patients to P. aeruginosa, we used the IB3-1 cell line with two defective CF transmembrane conductance regulator (CFTR) genes (DeltaF508/W1282X) to generate isogenic stable, clonal lung epithelial cells expressing wild-type (WT)-CFTR with an NH(2)-terminal green fluorescent protein (GFP) tag. GFP-CFTR exhibited posttranslational modification, subcellular localization, and anion transport function typical of WT-CFTR. P. aeruginosa internalization, a component of effective innate immunity, required functional CFTR and caveolin-1, as shown by: 1) direct correlation between GFP-CFTR expression levels and P. aeruginosa internalization; 2) enhanced P. aeruginosa internalization by aminoglycoside-induced read through of the CFTR W1282X allele in IB3-1 cells; 3) decreased P. aeruginosa internalization following siRNA knockdown of GFP-CFTR or caveolin-1; and 4) spatial association of P. aeruginosa with GFP-CFTR and caveolin-1 at the cell surface. P. aeruginosa internalization also required free lateral diffusion of GFP-CFTR, allowing for bacterial coclustering with GFP-CFTR and caveolin-1 at the plasma membrane. Thus efficient initiation of innate immunity to P. aeruginosa requires formation of an epithelial "internalization platform" involving both caveolin-1 and functional, laterally mobile CFTR.

  17. AFBI assay – Aptamer Fluorescence Binding and Internalization assay for cultured adherent cells

    PubMed Central

    Thiel, William H.; Giangrande, Paloma H.

    2016-01-01

    The SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process allows for the enrichment of DNA or RNA aptamers from a complex nucleic acid library that are specific for a target molecule. The SELEX process has been adapted from identifying aptamers in vitro using recombinant target protein to cell-based methodologies (Cell-SELEX), where the targets are expressed on the surface of cells. One major advantage of Cell-SELEX is that the target molecules are maintained in a native confirmation. Additionally, Cell-SELEX may be used to discover novel therapeutic biomarkers by performing selections on diseased versus healthy cells. However, a caveat to Cell-SELEX is that testing of single aptamers identified in the selection is laborious, time-consuming, and expensive. The most frequently used methods to screen for aptamer binding and internalization on cells are flow cytometry and quantitative PCR (qPCR). While flow cytometry can directly assess binding of a fluorescently-labeled aptamer to a target, it requires significant starting material and is not easily scalable. qPCR-based approaches are highly sensitive but have non-negligible experiment-to-experiment variability due to the number of sample processing steps. Herein we describe a cell-based aptamer fluorescence binding and internalization (AFBI) assay. This assay requires minimal reagents and has few experimental steps/manipulations, thereby allowing for rapid screening of many aptamers and conditions simultaneously and direct quantitation of aptamer binding and internalization. PMID:26972784

  18. What Lies Beneath: Antibody Dependent Natural Killer Cell Activation by Antibodies to Internal Influenza Virus Proteins.

    PubMed

    Vanderven, Hillary A; Ana-Sosa-Batiz, Fernanda; Jegaskanda, Sinthujan; Rockman, Steven; Laurie, Karen; Barr, Ian; Chen, Weisan; Wines, Bruce; Hogarth, P Mark; Lambe, Teresa; Gilbert, Sarah C; Parsons, Matthew S; Kent, Stephen J

    2016-06-01

    The conserved internal influenza proteins nucleoprotein (NP) and matrix 1 (M1) are well characterised for T cell immunity, but whether they also elicit functional antibodies capable of activating natural killer (NK) cells has not been explored. We studied NP and M1-specific ADCC activity using biochemical, NK cell activation and killing assays with plasma from healthy and influenza-infected subjects. Healthy adults had antibodies to M1 and NP capable of binding dimeric FcγRIIIa and activating NK cells. Natural symptomatic and experimental influenza infections resulted in a rise in antibody dependent NK cell activation post-infection to the hemagglutinin of the infecting strain, but changes in NK cell activation to M1 and NP were variable. Although antibody dependent killing of target cells infected with vaccinia viruses expressing internal influenza proteins was not detected, opsonising antibodies to NP and M1 likely contribute to an antiviral microenvironment by stimulating innate immune cells to secrete cytokines early in infection. We conclude that effector cell activating antibodies to conserved internal influenza proteins are common in healthy and influenza-infected adults. Given the significance of such antibodies in animal models of heterologous influenza infection, the definition of their importance and mechanism of action in human immunity to influenza is essential.

  19. Internalization and intracellular trafficking of poly(propylene imine) glycodendrimers with maltose shell in melanoma cells.

    PubMed

    Filimon, A; Sima, L E; Appelhans, D; Voit, B; Negroiu, G

    2012-01-01

    The diagnosis and treatment of malignant melanoma by means of the formulation of active principles with dendrimeric nanoparticles is an area of great current interest. The identification and understanding of molecular mechanisms which ensure the integration of particular dendrimeric nanostructures in tumor cellular environment can provide valuable guidance in their coupling strategies with antitumor or diagnostic agents. Two structurally distinct maltose-shell modified 5th generation (G5) poly(propylene imine) (PPI) glycodendrimers fluorescently labeled, (a) with open maltose shell, cationic charged G5-PPI-OS and (b) with dense maltose shell and nearly neutral G5-PPI-DS, were tested in relation with several melanoma cell lines. We found that three melanoma cell lines internalize G5-PPI-DS structure more efficiently than non tumoral HEK297T cells. Furthermore, the internalization pathways of G5-PPI-OS and G5-PPI-DS are characteristic for each tumor cell phenotype and include more than one mechanism. As a general trend, large amounts of both G5-PPI-OS and G5-PPI-DS are internalized on cholesterol-dependent pathway in MJS primary melanoma cells and on non conventional pathways in SK28 metastatic melanoma cells. G5-PPI-OS, temporarily retained at plasma membrane in both cell lines, is internalized slower in metastatic than in primary phenotype. Unlike G5-PPI-OS, G5-PPI-DS is immediately endocytosed in both cell lines. The unconventional internalization pathway and trafficking, exclusively used by G5-PPI-DS in metastatic cells, is described at molecular level. The decay kinetics of fluorescent labeled G5-PPI-OS and G5-PPI-DS is distinct in the two cellular phenotypes. Both cationic and neutral maltose G5-PPI glycodendrimeric structures represent molecules based on which designing of new formulations for therapy or/and diagnosis of melanoma can be further developed.

  20. Internalization and degradation of human chorionic gonadotropin in ovine luteal cells: Kinetic studies

    SciTech Connect

    Ahmed, C.E.; Sawyer, H.R.; Niswender, G.D.

    1981-11-01

    Ovine luteal cells grown in suspensions and/or monolayer culture were used to study the rates of internalization and degradation of (/sup 125/I)hCG. At specified times after a 5- to 7-min exposure to (/sup 125/I)hCG, cells were treated with acidic buffer (pH 3.9) to elute membrane-bound hormone, which left the internalized radioactivity associated with the cell pellet. Radioactivity released into the medium during the incubation periods was subjected to 20% trichloroacetic acid and/or thin layer chromatography to monitor the extent of degradation of the radioactive hormone. Secretion of progesterone into the medium and exclusion of trypan blue were used to monitor the viability of the cells in each experiment. Radioactivity was lost from the plasma membrane with a tsub1/2 of 9.6 h, with approximately 85% of the radioactivity being lost within 24 h. Cell-associated radioactivity increased linearly with time to a plateau at 4 h, remained stable until 12 h, and then decreased between 12-24 h. The plateau between 4-12 h reflected an equilibrium between the (/sup 125/I)hCG which was internalized and degraded and the (/sup 125/I)hCG which was released into the medium. The degraded (/sup 125/I)hCG increased essentially linearly up to 24 h. These data suggest that the majority of (/sup 125/I)hCG bound to receptors in luteal cells is internalized and degraded. Less than 20% of the radioactivity bound initially to cells dissociated into the incubation medium and was trichloroacetic acid precipitable within 24 h. The internalization and degradation of (/sup 125/I)hCG was temperature dependent, with essentially no hCG internalized and/or degraded at 4C.

  1. The cell-type specific uptake of polymer-coated or micelle-embedded QDs and SPIOs does not provoke an acute pro-inflammatory response in the liver.

    PubMed

    Heine, Markus; Bartelt, Alexander; Bruns, Oliver T; Bargheer, Denise; Giemsa, Artur; Freund, Barbara; Scheja, Ludger; Waurisch, Christian; Eychmüller, Alexander; Reimer, Rudolph; Weller, Horst; Nielsen, Peter; Heeren, Joerg

    2014-01-01

    Semiconductor quantum dots (QD) and superparamagnetic iron oxide nanocrystals (SPIO) have exceptional physical properties that are well suited for biomedical applications in vitro and in vivo. For future applications, the direct injection of nanocrystals for imaging and therapy represents an important entry route into the human body. Therefore, it is crucial to investigate biological responses of the body to nanocrystals to avoid harmful side effects. In recent years, we established a system to embed nanocrystals with a hydrophobic oleic acid shell either by lipid micelles or by the amphiphilic polymer poly(maleic anhydride-alt-1-octadecene) (PMAOD). The goal of the current study is to investigate the uptake processes as well as pro-inflammatory responses in the liver after the injection of these encapsulated nanocrystals. By immunofluorescence and electron microscopy studies using wild type mice, we show that 30 min after injection polymer-coated nanocrystals are primarily taken up by liver sinusoidal endothelial cells. In contrast, by using wild type, Ldlr (-/-) as well as Apoe (-/-) mice we show that nanocrystals embedded within lipid micelles are internalized by Kupffer cells and, in a process that is dependent on the LDL receptor and apolipoprotein E, by hepatocytes. Gene expression analysis of pro-inflammatory markers such as tumor necrosis factor alpha (TNFα) or chemokine (C-X-C motif) ligand 10 (Cxcl10) indicated that 48 h after injection internalized nanocrystals did not provoke pro-inflammatory pathways. In conclusion, internalized nanocrystals at least in mouse liver cells, namely endothelial cells, Kupffer cells and hepatocytes are at least not acutely associated with potential adverse side effects, underlining their potential for biomedical applications.

  2. The cell-type specific uptake of polymer-coated or micelle-embedded QDs and SPIOs does not provoke an acute pro-inflammatory response in the liver

    PubMed Central

    Bargheer, Denise; Giemsa, Artur; Freund, Barbara; Scheja, Ludger; Waurisch, Christian; Eychmüller, Alexander; Reimer, Rudolph; Weller, Horst; Nielsen, Peter

    2014-01-01

    Summary Semiconductor quantum dots (QD) and superparamagnetic iron oxide nanocrystals (SPIO) have exceptional physical properties that are well suited for biomedical applications in vitro and in vivo. For future applications, the direct injection of nanocrystals for imaging and therapy represents an important entry route into the human body. Therefore, it is crucial to investigate biological responses of the body to nanocrystals to avoid harmful side effects. In recent years, we established a system to embed nanocrystals with a hydrophobic oleic acid shell either by lipid micelles or by the amphiphilic polymer poly(maleic anhydride-alt-1-octadecene) (PMAOD). The goal of the current study is to investigate the uptake processes as well as pro-inflammatory responses in the liver after the injection of these encapsulated nanocrystals. By immunofluorescence and electron microscopy studies using wild type mice, we show that 30 min after injection polymer-coated nanocrystals are primarily taken up by liver sinusoidal endothelial cells. In contrast, by using wild type, Ldlr -/- as well as Apoe -/- mice we show that nanocrystals embedded within lipid micelles are internalized by Kupffer cells and, in a process that is dependent on the LDL receptor and apolipoprotein E, by hepatocytes. Gene expression analysis of pro-inflammatory markers such as tumor necrosis factor alpha (TNFα) or chemokine (C-X-C motif) ligand 10 (Cxcl10) indicated that 48 h after injection internalized nanocrystals did not provoke pro-inflammatory pathways. In conclusion, internalized nanocrystals at least in mouse liver cells, namely endothelial cells, Kupffer cells and hepatocytes are at least not acutely associated with potential adverse side effects, underlining their potential for biomedical applications. PMID:25247125

  3. Embedded fiber-optic sensing for accurate internal monitoring of cell state in advanced battery management systems part 2: Internal cell signals and utility for state estimation

    NASA Astrophysics Data System (ADS)

    Ganguli, Anurag; Saha, Bhaskar; Raghavan, Ajay; Kiesel, Peter; Arakaki, Kyle; Schuh, Andreas; Schwartz, Julian; Hegyi, Alex; Sommer, Lars Wilko; Lochbaum, Alexander; Sahu, Saroj; Alamgir, Mohamed

    2017-02-01

    A key challenge hindering the mass adoption of Lithium-ion and other next-gen chemistries in advanced battery applications such as hybrid/electric vehicles (xEVs) has been management of their functional performance for more effective battery utilization and control over their life. Contemporary battery management systems (BMS) reliant on monitoring external parameters such as voltage and current to ensure safe battery operation with the required performance usually result in overdesign and inefficient use of capacity. More informative embedded sensors are desirable for internal cell state monitoring, which could provide accurate state-of-charge (SOC) and state-of-health (SOH) estimates and early failure indicators. Here we present a promising new embedded sensing option developed by our team for cell monitoring, fiber-optic (FO) sensors. High-performance large-format pouch cells with embedded FO sensors were fabricated. This second part of the paper focuses on the internal signals obtained from these FO sensors. The details of the method to isolate intercalation strain and temperature signals are discussed. Data collected under various xEV operational conditions are presented. An algorithm employing dynamic time warping and Kalman filtering was used to estimate state-of-charge with high accuracy from these internal FO signals. Their utility for high-accuracy, predictive state-of-health estimation is also explored.

  4. Cancer cell exosomes depend on cell-surface heparan sulfate proteoglycans for their internalization and functional activity.

    PubMed

    Christianson, Helena C; Svensson, Katrin J; van Kuppevelt, Toin H; Li, Jin-Ping; Belting, Mattias

    2013-10-22

    Extracellular vesicle (EV)-mediated intercellular transfer of signaling proteins and nucleic acids has recently been implicated in the development of cancer and other pathological conditions; however, the mechanism of EV uptake and how this may be targeted remain as important questions. Here, we provide evidence that heparan sulfate (HS) proteoglycans (PGs; HSPGs) function as internalizing receptors of cancer cell-derived EVs with exosome-like characteristics. Internalized exosomes colocalized with cell-surface HSPGs of the syndecan and glypican type, and exosome uptake was specifically inhibited by free HS chains, whereas closely related chondroitin sulfate had no effect. By using several cell mutants, we provide genetic evidence of a receptor function of HSPG in exosome uptake, which was dependent on intact HS, specifically on the 2-O and N-sulfation groups. Further, enzymatic depletion of cell-surface HSPG or pharmacological inhibition of endogenous PG biosynthesis by xyloside significantly attenuated exosome uptake. We provide biochemical evidence that HSPGs are sorted to and associate with exosomes; however, exosome-associated HSPGs appear to have no direct role in exosome internalization. On a functional level, exosome-induced ERK1/2 signaling activation was attenuated in PG-deficient mutant cells as well as in WT cells treated with xyloside. Importantly, exosome-mediated stimulation of cancer cell migration was significantly reduced in PG-deficient mutant cells, or by treatment of WT cells with heparin or xyloside. We conclude that cancer cell-derived exosomes use HSPGs for their internalization and functional activity, which significantly extends the emerging role of HSPGs as key receptors of macromolecular cargo.

  5. Diffuse thyroid metastases and bilateral internal jugular vein tumor thrombus from renal cell cancer.

    PubMed

    Jha, Priyanka; Shekhar, Mallika; Wan, Jennifer; Mari-Aparici, Carina

    2016-12-01

    Renal cell cancer rarely metastasizes to the thyroid gland, and it has been reported to present as a solitary mass. We present a case of diffuse thyroid cancer metastases from renal cell cancer. Bilateral internal jugular vein tumor thrombi were also present. To the best of our knowledge, this is the first description of diffuse thyroid metastases from renal cell cancer in the English literature. Renal cell cancer metastases should be considered in the differential of thyroid imaging abnormalities arising in the setting of known renal cell carcinoma, particularly late in the course of disease. This is frequently associated with internal jugular vein thrombi, which should be evaluated with an abnormal thyroid. Thyroglobulin levels are usually normal in such patients.

  6. NREL/NASA Internal Short-Circuit Instigator in Lithium Ion Cells

    SciTech Connect

    Keyser, Matthew; Long, Dirk; Pesaran, Ahmad; Darcy, Eric; Shoesmith, Mark; McCarthy, Ben

    2015-10-11

    Lithium-ion cells provide the highest specific energy (>280 Wh/kg) and energy density (>600 Wh/L) rechargeable battery building block to date with the longest life. Electrode/electrolyte thermal instability and flammability of the electrolyte of Li-ion cells make them prone to catastrophic thermal runaway under some rare internal short circuit conditions. Despite extensive QC/QA, standardized industry safety testing, and over 18 years of manufacturing experience, major recalls have taken place and incidents still occur. Many safety incidents that take place in the field originate due to an internal short that was not detectable or predictable at the point of manufacture. The Internal Short-Circuit Instigator can be used to study types of separators, non-flammable electrolytes, electrolyte additives, fusible tabs, propagation studies, and gas generation within a cell.

  7. A Summary of the International Workshops on Space Solar Cell Calibration and Measurement Techniques

    NASA Technical Reports Server (NTRS)

    Brinker, D. J.; Curtis, H. B.; Flood, D. J.; Jenkins, P.; Scheiman, D. A.

    1995-01-01

    The first two of a planned series of international workshops concerning space solar cell calibration and measurement techniques have been held within the past year. The need for these workshops arose from the increasing complexity of space solar cells coupled with the growing international nature of the market for space cells and arrays. The workshops, jointly sponsored by NASDA, ESA and NASA, have the objective of obtaining international agreement on standardized values for the AM0 spectrum and constant, recommendations for laboratory measurement practices and the establishment of a set of protocols for making interlaboratory comparison measurements. The results of the first two workshops, held in Waikiki, Hawaii, USA in 1994 and Madrid, Spain in 1995, are presented.

  8. Silica colloidal crystals as porous substrates for total internal reflection fluorescence microscopy of live cells.

    PubMed

    Velarde, Tomika R C; Wirth, Mary J

    2008-06-01

    Total internal reflection fluorescence (TIRF) microscopy is a powerful means of probing biological cells because it reduces autofluorescence, but the need for direct contact between the cell surface and the microscope slide hinders chemical access to the cell surface. In this work, a submicrometer crystalline layer of colloidal silica on the microscope coverslip is shown to allow TIRF microscopy while also allowing chemical access to the cell surface. A 750 nm layer of 165 nm silica colloidal crystals was sintered onto a fused silica coverslip, and Chinese hamster ovary cells were successfully grown on this surface. This cell line over-expresses the human delta-opioid receptor, which enabled probing of the binding of a labeled ligand to the receptors on the cell surface. Total internal reflection and chemical access to the cell surface are demonstrated. The range of angles for total internal reflection is reduced only by 1/3 due to the lower index of refraction of the colloidal multilayer relative to fused silica.

  9. Malignant H1299 tumour cells preferentially internalize iron-bound inositol hexakisphosphate.

    PubMed

    Helmis, Christina; Blechner, Christine; Lin, Hongying; Schweizer, Michaela; Mayr, Georg W; Nielsen, Peter; Windhorst, Sabine

    2013-10-22

    In colon enterocytes and in well-differentiated colon cancer CaCo-2 cells, InsP6 (inositol hexakisphosphate) inhibits iron uptake by forming extracellular insoluble iron/InsP6 complexes. In this study, we confirmed that CaCo-2 cells are not able to take up iron/InsP6 but, interestingly, found that the cells are able to internalize metal-free and Cr3+-bound InsP6. Thus, the inability of CaCo-2 cells to take up iron/InsP6 complexes seems to be due to the iron-bound state of InsP6. Since recently we demonstrated that the highly malignant bronchial carcinoma H1299 cells internalize and process InsP6, we examined whether these cells may be able to take up iron/InsP6 complexes. Indeed, we found that InsP6 dose-dependently increased uptake of iron and demonstrated that in the iron-bound state InsP6 is more effectively internalized than in the metal-free or Cr3+-bound state, indicating that H1299 cells preferentially take up iron/InsP6 complexes. Electron microscope and cell fraction assays indicate that after uptake H1299 cells mainly stored InsP6/iron in lysosomes as large aggregates, of which about 10% have been released to the cytosol. However, this InsP6-mediated iron transport had no significant effects on cell viability. This result together with our finding that the well-differentiated CaCo-2 cells did not, but the malignant H1299 cells preferentially took up iron/InsP6, may offer the possibility to selectively transport cytotoxic substances into tumour cells.

  10. Stem cell course in the Middle East: science diplomacy and international collaborations during the Arab spring.

    PubMed

    Sarkadi, Balazs; Schatten, Gerald

    2012-03-01

    In April 2011, an international advanced course and workshop entitled "Frontiers in Human Pluripotent Stem Cells" and an International Congress on Fertility and Genetics ( http://www.fertigen.com.jo/ConferenceDetails.aspx ) was held in Amman Jordan hosted by the Jordanian Society of Fertility and Genetics under the auspices of the International Cell Research Organization (ICRO), a UNESCO associated NGO. The Congress President Dr. Zaid Kilani, with Dr. Abdel Latif Abu Khadra, President of the Jordanian society for Fertility and Genetics, Dr. Rana Dajani of the Hashemite University of Jordan, and their Organizing Committee proved to be an excellent organizers and dedicated physician-scientists and, focusing on fertility, genetics and stem cells in a wide range of advanced therapeutic applications. Brilliant course participants included trainees, scientists and clinicians from the Greater Middle East. The lectures and practical sessions, presented by internationally acknowledged scientists, included overviews of recent achievements in pluripotent stem cell research, emphasizing the role of both the embryonic (ES) and induced pluripotent stem (iPS) cells. A major emphasis was placed on the clinical achievements in germ cell and umbilical cord stem cell transplantation issues, and on the potential of fast and successful prenatal and pre-implantation molecular genetics diagnostics. The organization of the stem cell course in the Holy Land especially emphasized that issues of "eternal life" and "rejuvenation" are already at hand--at least in the pluripotent stem cell research field. In the lively atmosphere of the course about 60 participants had heated discussions on the possibility and ethics of advanced prenatal diagnostics, and on regulatory issues reflecting the need of separation of clinically effective versus unapproved, unwarranted stem cell treatments. An open discussion of many ethical issues, reflecting profound differences in religion and medical tradition in

  11. Calcium oxalate monohydrate crystals internalized into renal tubular cells are degraded and dissolved by endolysosomes.

    PubMed

    Chaiyarit, Sakdithep; Singhto, Nilubon; Thongboonkerd, Visith

    2016-02-25

    Interaction between calcium oxalate crystals and renal tubular cells has been recognized as one of the key mechanisms for kidney stone formation. While crystal adhesion and internalization have been extensively investigated, subsequent phenomena (i.e. crystal degradation and dissolution) remained poorly understood. To explore these mechanisms, we used fluorescein isothiocyanate (FITC)-labelled calcium oxalate monohydrate (COM) crystals (1000 μg/ml of crystals/culture medium) to confirm crystal internalization into MDCK (Type II) renal tubular cells after exposure to the crystals for 1 h and to trace the internalized crystals. Crystal size, intracellular and extracellular fluorescence levels were measured using a spectrofluorometer for up to 48 h after crystal internalization. Moreover, markers for early endosome (Rab5), late endosome (Rab7) and lysosome (LAMP-2) were examined by laser-scanning confocal microscopy. Fluorescence imaging and flow cytometry confirmed that FITC-labelled COM crystals were internalized into MDCK cells (14.83 ± 0.85%). The data also revealed a reduction of crystal size in a time-dependent manner. In concordance, intracellular and extracellular fluorescence levels were decreased and increased, respectively, indicating crystal degradation/dissolution inside the cells and the degraded products were eliminated extracellularly. Moreover, Rab5 and Rab7 were both up-regulated and were also associated with the up-regulated LAMP-2 to form large endolysosomes in the COM-treated cells at 16-h after crystal internalization. We demonstrate herein, for the first time, that COM crystals could be degraded/dissolved by endolysosomes inside renal tubular cells. These findings will be helpful to better understand the crystal fate and protective mechanism against kidney stone formation.

  12. A novel configuration of microbial fuel cell stack bridged internally through an extra cation exchange membrane.

    PubMed

    Liu, Zhidan; Liu, Jing; Zhang, Songping; Su, Zhiguo

    2008-06-01

    This paper reports a novel configuration of stacked microbial fuel cells (MFCs) bridged internally through an extra cation exchange membrane (CEM). The MFC stack (MFC(stack)), assembled from two single MFCs (MFC(single)), resulted in double voltage output and half optimal external resistance. COD removal rate was increased from 32.4% to 54.5%. The performance improvement could be attributed to the smaller internal resistance and enhanced cations transfer. A result from a half cell study further confirmed the important role of the extra CEM. This study also demonstrated MFCs where the anode and cathode were sandwiched between two CEMs possessed significantly high power outputs.

  13. Trojan-Like Internalization of Anatase Titanium Dioxide Nanoparticles by Human Osteoblast Cells.

    PubMed

    Ribeiro, A R; Gemini-Piperni, S; Travassos, R; Lemgruber, L; Silva, R C; Rossi, A L; Farina, M; Anselme, K; Shokuhfar, T; Shahbazian-Yassar, R; Borojevic, R; Rocha, L A; Werckmann, J; Granjeiro, J M

    2016-03-29

    Dentistry and orthopedics are undergoing a revolution in order to provide more reliable, comfortable and long-lasting implants to patients. Titanium (Ti) and titanium alloys have been used in dental implants and total hip arthroplasty due to their excellent biocompatibility. However, Ti-based implants in human body suffer surface degradation (corrosion and wear) resulting in the release of metallic ions and solid wear debris (mainly titanium dioxide) leading to peri-implant inflammatory reactions. Unfortunately, our current understanding of the biological interactions with titanium dioxide nanoparticles is still very limited. Taking this into consideration, this study focuses on the internalization of titanium dioxide nanoparticles on primary bone cells, exploring the events occurring at the nano-bio interface. For the first time, we report the selective binding of calcium (Ca), phosphorous (P) and proteins from cell culture medium to anatase nanoparticles that are extremely important for nanoparticle internalization and bone cells survival. In the intricate biological environment, anatase nanoparticles form bio-complexes (mixture of proteins and ions) which act as a kind of 'Trojan-horse' internalization by cells. Furthermore, anatase nanoparticles-induced modifications on cell behavior (viability and internalization) could be understand in detail. The results presented in this report can inspire new strategies for the use of titanium dioxide nanoparticles in several regeneration therapies.

  14. Trojan-Like Internalization of Anatase Titanium Dioxide Nanoparticles by Human Osteoblast Cells

    PubMed Central

    Ribeiro, A. R.; Gemini-Piperni, S.; Travassos, R.; Lemgruber, L.; C. Silva, R.; Rossi, A. L.; Farina, M.; Anselme, K.; Shokuhfar, T.; Shahbazian-Yassar, R.; Borojevic, R.; Rocha, L. A.; Werckmann, J.; Granjeiro, J. M.

    2016-01-01

    Dentistry and orthopedics are undergoing a revolution in order to provide more reliable, comfortable and long-lasting implants to patients. Titanium (Ti) and titanium alloys have been used in dental implants and total hip arthroplasty due to their excellent biocompatibility. However, Ti-based implants in human body suffer surface degradation (corrosion and wear) resulting in the release of metallic ions and solid wear debris (mainly titanium dioxide) leading to peri-implant inflammatory reactions. Unfortunately, our current understanding of the biological interactions with titanium dioxide nanoparticles is still very limited. Taking this into consideration, this study focuses on the internalization of titanium dioxide nanoparticles on primary bone cells, exploring the events occurring at the nano-bio interface. For the first time, we report the selective binding of calcium (Ca), phosphorous (P) and proteins from cell culture medium to anatase nanoparticles that are extremely important for nanoparticle internalization and bone cells survival. In the intricate biological environment, anatase nanoparticles form bio-complexes (mixture of proteins and ions) which act as a kind of ‘Trojan-horse’ internalization by cells. Furthermore, anatase nanoparticles-induced modifications on cell behavior (viability and internalization) could be understand in detail. The results presented in this report can inspire new strategies for the use of titanium dioxide nanoparticles in several regeneration therapies. PMID:27021687

  15. Methods for Detecting Internalized, FM 1-43 Stained Particles in Epithelial Cells and Monolayers

    PubMed Central

    Bertrand, C. A.; Laboisse, C.; Hopfer, U.; Bridges, R. J.; Frizzell, R. A.

    2006-01-01

    The membrane dye FM 1-43 has frequently been used to quantify exocytosis in neurons. In epithelia, intense lateral intracellular space staining and fluctuations in baseline labeling produced inconsistent results. Membrane retrieved in the presence of FM 1-43 retains the dye, however, and cells that undergo compensatory endocytosis during and following evoked exocytosis contain punctate, fluorescent particles after washout of external stain. As an alternative measure of trafficking, we quantified the fluorescent puncta retained after dye washout and tested our method on both coverslip-grown cell clusters and filter-grown intact monolayers. Images for analysis were acquired using serial sectioning with either epifluorescence or confocal microscopy. Tests with an intestinal goblet cell line that exhibits basal and ATP-stimulated granule trafficking confirmed that 1), the algorithm identified the same number of internalized particles with either epifluorescence or confocal microscopy acquired images; 2), low density clusters exhibited significantly more internalized particles per cell than either filter-grown monolayers or high density clusters; 3), ATP stimulation significantly increased the number of internalized particles in all preparations; and 4), the number of particles internalized was comparable to capacitance measurements of exocytosis. This method provides a single technique for quantifying membrane trafficking in both monolayers and unpolarized cells. PMID:16935947

  16. Trojan-Like Internalization of Anatase Titanium Dioxide Nanoparticles by Human Osteoblast Cells

    NASA Astrophysics Data System (ADS)

    Ribeiro, A. R.; Gemini-Piperni, S.; Travassos, R.; Lemgruber, L.; C. Silva, R.; Rossi, A. L.; Farina, M.; Anselme, K.; Shokuhfar, T.; Shahbazian-Yassar, R.; Borojevic, R.; Rocha, L. A.; Werckmann, J.; Granjeiro, J. M.

    2016-03-01

    Dentistry and orthopedics are undergoing a revolution in order to provide more reliable, comfortable and long-lasting implants to patients. Titanium (Ti) and titanium alloys have been used in dental implants and total hip arthroplasty due to their excellent biocompatibility. However, Ti-based implants in human body suffer surface degradation (corrosion and wear) resulting in the release of metallic ions and solid wear debris (mainly titanium dioxide) leading to peri-implant inflammatory reactions. Unfortunately, our current understanding of the biological interactions with titanium dioxide nanoparticles is still very limited. Taking this into consideration, this study focuses on the internalization of titanium dioxide nanoparticles on primary bone cells, exploring the events occurring at the nano-bio interface. For the first time, we report the selective binding of calcium (Ca), phosphorous (P) and proteins from cell culture medium to anatase nanoparticles that are extremely important for nanoparticle internalization and bone cells survival. In the intricate biological environment, anatase nanoparticles form bio-complexes (mixture of proteins and ions) which act as a kind of ‘Trojan-horse’ internalization by cells. Furthermore, anatase nanoparticles-induced modifications on cell behavior (viability and internalization) could be understand in detail. The results presented in this report can inspire new strategies for the use of titanium dioxide nanoparticles in several regeneration therapies.

  17. Moesin Controls Clathrin-Mediated S1PR1 Internalization in T Cells

    PubMed Central

    Nomachi, Akira; Yoshinaga, Masanori; Liu, Jaron; Kanchanawong, Pakorn; Tohyama, Kiyoshi; Thumkeo, Dean; Watanabe, Takeshi; Narumiya, Shuh; Hirata, Takako

    2013-01-01

    The lipid mediator sphingosine 1-phosphate (S1P) regulates a wide range of cellular activities, including vascular maturation, angiogenesis, and immune-cell trafficking. Among the five known receptors for S1P (S1PR1-S1PR5), S1PR1 is a critical regulator of lymphocyte trafficking: its signaling is required for lymphocyte egress from lymphoid organs, while its down-modulation by agonist-induced internalization is a prerequisite for lymphocyte entry into lymphoid organs from the bloodstream. Despite the importance of S1PR1 down-regulation in determining lymphocyte behavior, the molecular mechanism of its internalization in lymphocytes has not been defined. Here we show that agonist-induced S1PR1 internalization in T cells occurs via clathrin-mediated endocytosis and is regulated by moesin, an ezrin-radixin-moesin (ERM) family member. In S1P-stimulated T cells, S1PR1 relocalized within clathrin-coated vesicles (CCVs) and early endosomes, and S1PR1 internalization was blocked when clathrin was pharmacologically inhibited. Stimulating moesin-deficient T cells with S1P failed to induce S1PR1 internalization and CCV formation. Furthermore, treating moesin-deficient mice with FTY720, an S1P receptor agonist known to internalize S1PR1, caused delayed lymphopenia, and lymphocytes isolated from FTY720-treated moesin-deficient mice still responded to S1P ex vivo in chemotaxis assays. These results reveal a novel role for moesin in regulating clathrin-dependent S1PR1 internalization through CCV formation. PMID:24358210

  18. Internalized Tau sensitizes cells to stress by promoting formation and stability of stress granules

    PubMed Central

    Brunello, Cecilia A.; Yan, Xu; Huttunen, Henri J.

    2016-01-01

    Stress granules are membrane-less RNA- and RNA-binding protein-containing complexes that are transiently assembled in stressful conditions to promote cell survival. Several stress granule-associated RNA-binding proteins have been associated with neurodegenerative diseases. In addition, a close link was recently identified between the stress granule core-nucleating protein TIA-1 and Tau. Tau is a central pathological protein in Alzheimer’s disease and other tauopathies, and misfolded, aggregated Tau is capable of propagating pathology via cell-to-cell transmission. Here we show that following internalization hyperphosphorylated extracellular Tau associates with stress granules in a TIA-1 dependent manner. Cytosolic Tau normally only weakly interacts with TIA-1 but mutations mimicking abnormal phosphorylation promote this interaction. We show that internalized Tau significantly delays normal clearance of stress granules in the recipient cells sensitizing them to secondary stress. These results suggest that secreted Tau species may have properties, likely related to its hyperphosphorylation and oligomerization, which promote pathological association of internalized Tau with stress granules altering their dynamics and reducing cell viability. We suggest that stress granules and TIA-1 play a central role in the cell-to-cell transmission of Tau pathology. PMID:27460788

  19. The cellular internalization of liposome encapsulated protoporphyrin IX by HeLa cells.

    PubMed

    Przybylo, Magdalena; Glogocka, Daria; Dobrucki, Jerzy W; Fraczkowska, Kaja; Podbielska, Halina; Kopaczynska, Marta; Borowik, Tomasz; Langner, Marek

    2016-03-31

    The proper lipid composition of liposomes designed to carry drugs determines their surface properties ensuring their accumulation within selected tissue. The electrostatic potential and surface topology of liposomes affect the internalization by single cells. The high-resolution imaging of cancer cells and the distribution of protoporphyrin-loaded liposomes within the cytoplasm and its dependence on the liposome surface properties are presented. In the paper, HeLa cells were used to investigate the uptake of porphyrin-loaded liposomes and liposomes alone by means of confocal and differential interference contrast microscopies. The effect of liposomes surface electrostatic potential and surface topology on their intracellular distribution was evaluated. The time evolution of the intracellular distribution of liposomes labelled with Rhodamine-PE was examined on HeLa cells. These studies allow for the identification of the liposome lipid composition so the efficient delivery of the active substance to cancer cells will be achieved. The obtained results showed that neutral PC-liposomes are the most efficiently internalized by HeLa cells. Moreover, results showed that properties of liposomes affect not only the internalization efficiency of the photosensitizer but also its distribution within the cells, as revealed by colocalization measurements.

  20. Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

    PubMed Central

    Munro, Kathryn M.; Kennedy, Matthew J.; Gunnersen, Jenny M.

    2014-01-01

    In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (“antibody feeding”) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available. PMID:24561550

  1. On-Orbit Measurement of Next Generation Space Solar Cell Technology on the International Space Station

    NASA Technical Reports Server (NTRS)

    Wolford, David S.; Myers, Matthew G.; Prokop, Norman F.; Krasowski, Michael J.; Parker, David S.; Cassidy, Justin C.; Davies, William E.; Vorreiter, Janelle O.; Piszczor, Michael F.; McNatt, Jeremiah S.

    2014-01-01

    On-orbit measurements of new photovoltaic (PV) technologies for space power are an essential step in the development and qualification of advanced solar cells. NASA Glenn Research Center will fly and measure several solar cells attached to NASA Goddards Robotic Refueling Mission (RRM), expected to be launched in 2014. Industry and government partners have provided advanced PV devices for evaluation of performance and environmental durability. The experiment is completely self-contained, providing its own power and internal data storage. Several new cell technologies including Inverted Metamorphic Multi-junction and four-junction cells will be tested.

  2. Poly(dimethylsiloxane) photonic microbioreactors and multiple internal reflection systems for real time cell screening

    NASA Astrophysics Data System (ADS)

    Ortiz, Pedro; Demming, Stefanie; Fernández-Rosas, Elisabet; Ibarlucea, Bergoi; Vila-Planas, Jordi; Büttgenbach, Stephanus; Llobera, Andreu

    2011-06-01

    In this paper two different photonic lab-on-a-chip devices for real-time cell screening are presented. We discuss, in parallel, screening of cell viability and cell count using a multiple total internal reflection (MIR) system, and a microbioreactor (MBR) for continuous cell culture screening. Each device takes advantage, in different ways, of the enhanced light-analyte interaction provided by the full-field approach. Their performance and how they represent an improvement to current methods, are also discussed herein. Both devices integrate photonic components and are fabricated by soft lithography (PDMS) replica molding of SU-8 masters, thus making them low-cost and easily mass produced.

  3. Membrane stress and internal pressure in a red blood cell freely suspended in a shear flow.

    PubMed Central

    Tran-Son-Tay, R; Sutera, S P; Zahalak, G I; Rao, P R

    1987-01-01

    Presented is an algorithm for the approximate calculation of the membrane stress distribution and the internal pressure of a steadily tank-treading red cell. The algorithm is based on an idealized ellipsoidal model of the tank-treading cell (Keller, S.R., and R. Skalak, 1982, J. Fluid Mech., 120:27-47) joined with experimental observations of projected length, width, and tank-treading frequency. The results are inexact because the membrane shape and velocity are assumed a priori, rather than being determined via appropriate material constitutive relations for the membrane; these results are, nevertheless, believed to be approximately correct, and show that internal pressure builds up slowly as cell elongation increases, rising more rapidly as the deformed cell approaches the limiting geometry of a prolate ellipsoid. The maximum shear stress resultant in the membrane was found to be below but approaching the yield point range at the highest shear rate applied. Images FIGURE 1 PMID:3607212

  4. NASA Glenn Research Center Solar Cell Experiment Onboard the International Space Station

    NASA Technical Reports Server (NTRS)

    Myers, Matthew G.; Wolford, David S.; Prokop, Norman F.; Krasowski, Michael J.; Parker, David S.; Cassidy, Justin C.; Davies , William E.; Vorreiter, Janelle O.; Piszczor, Michael F.; Mcnatt, Jeremiah S.; Spina, Danny C.

    2016-01-01

    Accurate air mass zero (AM0) measurement is essential for the evaluation of new photovoltaic (PV) technology for space solar cells. The NASA Glenn Research Center (GRC) has flown an experiment designed to measure the electrical performance of several solar cells onboard NASA Goddard Space Flight Center's (GSFC) Robotic Refueling Missions (RRM) Task Board 4 (TB4) on the exterior of the International Space Station (ISS). Four industry and government partners provided advanced PV devices for measurement and orbital environment testing. The experiment was positioned on the exterior of the station for approximately eight months, and was completely self-contained, providing its own power and internal data storage. Several new cell technologies including four-junction (4J) Inverted Metamorphic Multi-junction (IMM) cells were evaluated and the results will be compared to ground-based measurement methods.

  5. 15th International Symposium on Cells of the Hepatic Sinusoid, 2010.

    PubMed

    DeLeve, Laurie D; Jaeschke, Hartmut; Kalra, Vijay K; Asahina, Kinji; Brenner, David A; Tsukamoto, Hidekazu

    2011-07-01

    This is a meeting report of the presentations given at the 15th International Symposium on Cells of the Hepatic Sinusoid, held in 2010. The areas covered include the contributions of the various liver cell populations to liver disease, molecular and cellular targets involved in steatohepatitis, hepatic fibrosis and cancer and regenerative medicine. In addition to a review of the science presented at the meeting, this report provides references to recent literature on the topics covered at the meeting.

  6. [The micro-particles of blood plasma, micro-vesicles, exosomes, apoptotic bodies and Kupffer macrophage in liver: late in phylogenesis system of realization of biological function of endoecology].

    PubMed

    Titov, V N

    2014-07-01

    Probably, at early stages of phylogenesis and on the stage of first contacts of single cells in environment the mode of intercommunication was developed via formation of micro-vesicles. It is quite possible that this so complicated way was used during billions of years to develop the very early cenosises of functionally different cells. Later on, diffusion of humoral mediators within the framework of group of cells but without vesicles resulted in formation of very early regulated cenosises of various cells. In billions years, these paracrin regulated cenosises became structural and functional units of every organ. It is difficult to imagine that mode of early in phylogenesis humoral intercommunication of cells could preserve its significance in present conditions. At the same time, according to methodological approach of biological continuity in becoming of biological functions and biological reactions, micro-vesicles continue to function but with somehow different purposes. It is surmised that microparticles of blood plasma consist a heterogeneous population of micro-vesicles initially formed by cells, exosomes and apoptotic bodies. This population is a foundation of spontaneous, physical chemical formation of complexes in blood plasma on principles of absorption, hydrophobicity and ionic interaction of structural cells'components insoluble in water medium. It is assumed that presently formation of micro-particles in blood is functionally a kind of realization of phylogenetically late variant of biological function of endoecology, in inter-cellular medium, in local pool of intravascular blood plasma. This variant includes: a) microparticles, micro-vesicles, exosomes and apoptotic bodies as elements of biological reaction of support of "purity" of inter-cellular medium; b) in many respects highly specialized variant of phagocytosis of micro-particles by Kupffer macrophages in liver In the aggregate, this is a phylogenetically late medium of realization of

  7. Binding, internalization, and degradation of atrial natriuretic peptide in cultured vascular smooth muscle cells of rat

    SciTech Connect

    Hirata, Y.; Takata, S.; Tomita, M.; Takaichi, S.

    1985-11-15

    Binding, internalization, and degradation of /sup 125/I-labeled-rat atrial natriuretic peptide (rANP) were studied in cultured rat aortic vascular smooth muscle cells (VSMC). At 37 degrees C, /sup 125/I-labeled-rANP rapidly bound to VSMCs, but the cell-bound radioactivity rapidly decreased upon subsequent incubation, while the binding was slow at 4 degrees C, reaching to an apparent equilibrium after 6 hrs. The cell-bound /sup 125/I-labeled-rANP at 37 degrees C is rapidly dissociated from VSMC (t 1/2: approximately 40 min) with the appearance of degradaded product(s) of radioligand in the medium, whereas the degradation was minimal at 4 degrees C. This degradative process was blocked by inhibitors of metabolic energy production (azide, dinitrophenol), inhibitors of lysosomal cathepsins (leupeptin, pepstatin), and lysosomotropic agents (NH/sub 4/Cl, chloroquine, lidocaine, methylamine, dansylcadaverine), but not by inhibitors of serine or thiol proteases. /sup 125/I-labeled-rANP initially bound to the cell-surface was rapidly internalized, and delivered to lysosomal structures, which was confirmed by autoradiographic studies. These data indicate that rANP, after binding to the cell-surface receptors, is rapidly internalized into the cells through receptor-mediated endocytosis, and subsequently degradaded by lysosomal hydrolases.

  8. Internalization pathways into cancer cells of gadolinium-based radiosensitizing nanoparticles.

    PubMed

    Rima, Wael; Sancey, Lucie; Aloy, Marie-Thérèse; Armandy, Emma; Alcantara, Gustavo B; Epicier, Thierry; Malchère, Annie; Joly-Pottuz, Lucile; Mowat, Pierre; Lux, François; Tillement, Olivier; Burdin, Béatrice; Rivoire, Annie; Boulé, Christelle; Anselme-Bertrand, Isabelle; Pourchez, Jérémie; Cottier, Michèle; Roux, Stéphane; Rodriguez-Lafrasse, Claire; Perriat, Pascal

    2013-01-01

    Over the last few decades, nanoparticles have been studied in theranostic field with the objective of exhibiting a long circulation time through the body coupled to major accumulation in tumor tissues, rapid elimination, therapeutic potential and contrast properties. In this context, we developed sub-5 nm gadolinium-based nanoparticles that possess in vitro efficient radiosensitizing effects at moderate concentration when incubated with head and neck squamous cell carcinoma cells (SQ20B). Two main cellular internalization mechanisms were evidenced and quantified: passive diffusion and macropinocytosis. Whereas the amount of particles internalized by passive diffusion is not sufficient to induce in vitro a significant radiosensitizing effect, the cellular uptake by macropinocytosis leads to a successful radiotherapy in a limited range of particles incubation concentration. Macropinocytosis processes in two steps: formation of agglomerates at vicinity of the cell followed by their collect via the lamellipodia (i.e. the "arms") of the cell. The first step is strongly dependent on the physicochemical characteristics of the particles, especially their zeta potential that determines the size of the agglomerates and their distance from the cell. These results should permit to control the quantity of particles internalized in the cell cytoplasm, promising ambitious opportunities towards a particle-assisted radiotherapy using lower radiation doses.

  9. Hepatic non-parenchymal cells and extracellular matrix participate in oval cell-mediated liver regeneration

    PubMed Central

    Zhang, Wei; Chen, Xiao-Ping; Zhang, Wan-Guang; Zhang, Feng; Xiang, Shuai; Dong, Han-Hua; Zhang, Lei

    2009-01-01

    AIM: To elucidate the interaction between non-parenchymal cells, extracellular matrix and oval cells during the restituting process of liver injury induced by partial hepatectomy (PH). METHODS: We examined the localization of oval cells, non-parenchymal cells, and the extracellular matrix components using immunohistochemical and double immunofluorescent analysis during the proliferation and differentiation of oval cells in N-2-acetylaminofluorene (2-AAF)/PH rat model. RESULTS: By day 2 after PH, small oval cells began to proliferate around the portal area. Most of stellate cells and laminin were present along the hepatic sinusoids in the periportal area. Kupffer cells and fibronectin markedly increased in the whole hepatic lobule. From day 4 to 9, oval cells spread further into hepatic parenchyma, closely associated with stellate cells, fibronectin and laminin. Kupffer cells admixed with oval cells by day 6 and then decreased in the periportal zone. From day 12 to 15, most of hepatic stellate cells (HSCs), laminin and fibronectin located around the small hepatocyte nodus, and minority of them appeared in the nodus. Kupffer cells were mainly limited in the pericentral sinusoids. After day 18, the normal liver lobule structures began to recover. CONCLUSION: Local hepatic microenvironment may participate in the oval cell-mediated liver regeneration through the cell-cell and cell-matrix interactions. PMID:19195056

  10. Detection of internal structure by scattered light intensity: Application to kidney cell sorting

    NASA Technical Reports Server (NTRS)

    Goolsby, C. L.; Kunze, M. E.

    1985-01-01

    Scattered light measurements in flow cytometry were sucessfully used to distinguish cells on the basis of differing morphology and internal structure. Differences in scattered light patterns due to changes in internal structure would be expected to occur at large scattering angles. Practically, the results of these calculations suggest that in experimental situations an array of detectors would be useful. Although in general the detection of the scattered light intensity at several intervals within the 10 to 60 region would be sufficient, there are many examples where increased sensitivity could be acheived at other angles. The ability to measure at many different angular intervals would allow the experimenter to empirically select the optimum intervals for the varying conditions of cell size, N/C ratio, granule size and internal structure from sample to sample. The feasibility of making scattered light measurements at many different intervals in flow cytometry was demonstrated. The implementation of simplified versions of these techniques in conjunction with independant measurements of cell size could potentially improve the usefulness of flow cytometry in the study of the internal structure of cells.

  11. Development of AM 1.5 global measurement procedures and international cell measurement round robin

    NASA Technical Reports Server (NTRS)

    Mueller, R.

    1985-01-01

    The development of the capability for measurement under global irradiance spectral distribution is reported. The airmass 1.5 global measurement procedure is given. Also given is the procedure and justification for using the large area pulsed solar simulator (LAPSS). The status of the international round robin of reference cell measurements managed by the Commission of European Communities (CEC) is described.

  12. Binding and internalization of extracellular type-I phospholipase A2 in uterine stromal cells.

    PubMed Central

    Rossini, G P; Fayard, J M; Tessier, C; Laugier, C

    1996-01-01

    The cellular uptake of extracellular type-I phospholipase A2 (PLA2) was investigated in rat uterine stromal cells (UIII) in culture, which were found to express the high-affinity binding site for mammalian type-I PLA2, with a measured KD of 6.4 nM, a Bmax of 0.1-1 pmol/mg of DNA at 4 degrees C, and a molecular mass of about 200 kDa. When UIII cells were treated with type-I PLA2 at 37 degrees C, the ligand specifically associated with the cells increased, reaching a plateau after 90 min of incubation, whose level was about 5-fold higher than that measured if cells were maintained at 4 degrees C. We could determine that the PLA2 was bound to plasma membrane receptors which were responsible for internalization of the ligand, and that the binding sites were still suitable for binding at the level of plasma membrane during UIII cell incubation at 37 degrees C. Proteolysis of internalized PLA2 could be clearly detected only after 90 min of UIII cell incubation with the ligand at 37 degrees C, and most of the intracellular PLA2 consisted of the apparently intact 14 kDa enzyme. By cross-linking studies, we found that most of the internalized PLA2 was not associated with the receptor, supporting the conclusion that in our experimental system a single pool of membrane receptors for mammalian type-I PLA2 undergoes cycles of ligand binding, intracellular transfer and release of PLA2, followed by restoration of binding sites on the plasma membrane. We calculated that the rate of internalization of the ligand by one receptor molecule in UIII cells at 37 degrees C is about three molecules of type-I PLA2 per h. PMID:8645137

  13. Antitumor effects of calgranulin B internalized in human colon cancer cells

    PubMed Central

    Yoo, Byong Chul; Ku, Ja-Lok; Shin, Young-Kyoung; Cho, Jae Youl; Kim, Minjae; Kwon, Myung-Hee; Goh, Sung Ho; Chang, Hee Jin; Oh, Jae Hwan

    2016-01-01

    Calgranulin B is a small, calcium-binding protein expressed in neutrophils that is secreted into the tumor microenvironment in cancer cases. We previously showed that calgranulin B levels are increased in the stools of colorectal cancer patients. In patient tumor tissues, calgranulin B protein levels correlated with the presence of stromal inflammatory cells surrounding tumor cells, and calgranulin B promoter methylation was observed in both paired human tissues and colon cancer cell lines. Cell lines did not express calgranulin B, but in vitro studies showed that colon cancer cells internalized extracellular calgranulin B, while other types of cancer cells did not. Calgranulin B internalization led to reduced cell proliferation and increased apoptotic cell death. AKT and ERK signals were also increased after calgranulin B treatment, as were p53, β-catenin, E-cadherin and cleaved caspase-3 levels. Additionally, a human protein microarray identified aurora A kinase as a calgranulin B binding partner, and binding inhibited aurora A kinase activity in a dose-dependent manner. Our findings demonstrate the antitumor effects of calgranulin B in the inflammatory microenvironment and suggest that calgranulin B could be potentially efficacious in the treatment of colon cancer. PMID:26933915

  14. Ligand modified nanoparticles increases cell uptake, alters endocytosis and elevates glioma distribution and internalization.

    PubMed

    Gao, Huile; Yang, Zhi; Zhang, Shuang; Cao, Shijie; Shen, Shun; Pang, Zhiqing; Jiang, Xinguo

    2013-01-01

    Nanoparticles (NPs) were widely used in drugs/probes delivery for improved disease diagnosis and/or treatment. Targeted delivery to cancer cells is a highly attractive application of NPs. However, few studies have been performed on the targeting mechanisms of these ligand-modified delivery systems. Additional studies are needed to understand the transport of nanoparticles in the cancer site, the interactions between nanoparticles and cancer cells, the intracellular trafficking of nanoparticles within the cancer cells and the subcellular destiny and potential toxicity. Interleukin 13 (IL-13) peptide can specifically bind IL-13Rα2, a receptor that is highly expressed on glioma cells but is expressed at low levels on other normal cells. It was shown that the nanoparticels modification with the IL-13 peptide could improve glioma treatment by selectively increasing cellular uptake, facilitating cell internalization, altering the uptake pathway and increasing glioma localization.

  15. Effect of initial salt concentrations on cell performance and distribution of internal resistance in microbial desalination cells.

    PubMed

    Yang, Euntae; Choi, Mi-Jin; Kim, Kyoung-Yeol; Chae, Kyu-Jung; Kim, In S

    2015-01-01

    Microbial desalination cells (MDCs) are modified microbial fuel cells (MFCs) that concurrently produce electricity and desalinate seawater, but adding a desalination compartment and an ion-exchange membrane may increase the internal resistance (Ri), which can limit the cell performance. However, the effects of a desalination chamber and initial NaCl concentrations on the internal resistances and the cell performances (i.e. Coulombic efficiency (CE), current and power density) of MDCs have yet to be thoroughly explored; thus, the cell performance and Ri distributions of MDCs having different initial concentrations and an MFC having no desalination chamber were compared. In the MDCs, the current and power density generation increased from 2.82 mA and 158.2 mW/m2 to 3.17 mA and 204.5 mW/m2 when the initial NaCl concentrations were increased from 5 to 30 g/L, as a consequence of the internal resistances decreasing from 2432.0 to 2328.4 Ω. And even though the MFC has a lower Ri than the MDCs, lower cell performances (current: 2.59 mA; power density: 141.6 mW/m2 and CE: 62.1%) were observed; there was no effect of improved junction potential in the MFC. Thus, in the MDCs, the higher internal resistances due to the addition of a desalination compartment can be offset by reducing the electrolyte resistance and improving the junction potential at higher NaCl concentrations.

  16. Staphylococcus saprophyticus ATCC 15305 is internalized into human urinary bladder carcinoma cell line 5637.

    PubMed

    Szabados, Florian; Kleine, Britta; Anders, Agnes; Kaase, Martin; Sakinç, Türkân; Schmitz, Inge; Gatermann, Sören

    2008-08-01

    Invasion of bacteria into nonphagocytic host cells is an important pathogenicity factor for escaping the host defence system. Gram-positive organisms, for example Staphylococcus aureus and Listeria monocytogenes, are invasive in nonphagocytic cells, and this mechanism is discussed as an important part of the infection process. Uropathogenic Escherichia coli and Staphylococcus saprophyticus can cause acute and recurrent urinary tract infections as well as bloodstream infections. Staphylococcus saprophyticus shows strong adhesion to human urinary bladder carcinoma and Hep2 cells and expresses the 'Microbial Surface Components Recognizing Adhesive Matrix molecule' (MSCRAMM)-protein SdrI with collagen-binding activity. MSCRAMMs are responsible for adhesion and collagen binding in S. aureus and are discussed as an important pathogenicity factor for invasion. To investigate internalization in S. aureus, several fluorescence activated cell sorting (FACS) assays have been described recently. We used a previously described FACS assay, with slight modifications, in addition to an antibiotic protection assay and transmission electron microscopy to show that S. saprophyticus ATCC 15305 and the wild-type strain 7108 were internalized into the human urinary bladder carcinoma cell line 5637. The discovery of the internalization of S. saprophyticus may be an important step for understanding the pathogenicity of recurrent infections caused by this organism.

  17. Planar solid oxide fuel cell with staged indirect-internal air and fuel preheating and reformation

    DOEpatents

    Geisbrecht, Rodney A; Williams, Mark C

    2003-10-21

    A solid oxide fuel cell arrangement and method of use that provides internal preheating of both fuel and air in order to maintain the optimum operating temperature for the production of energy. The internal preheat passes are created by the addition of two plates, one on either side of the bipolar plate, such that these plates create additional passes through the fuel cell. This internal preheat fuel cell configuration and method reduce the requirements for external heat exchanger units and air compressors. Air or fuel may be added to the fuel cell as required to maintain the optimum operating temperature through a cathode control valve or an anode control valve, respectively. A control loop comprises a temperature sensing means within the preheat air and fuel passes, a means to compare the measured temperature to a set point temperature and a determination based on the comparison as to whether the control valves should allow additional air or fuel into the preheat or bypass manifolds of the fuel cell.

  18. Development of a Novel Test Method for On-Demand Internal Short Circuit in a Li-Ion Cell (Presentation)

    SciTech Connect

    Keyser, M.; Long, D.; Jung, Y. S.; Pesaran, A.; Darcy, E.; McCarthy, B.; Patrick, L.; Kruger, C.

    2011-01-01

    This presentation describes a cell-level test method that simulates an emergent internal short circuit, produces consistent and reproducible test results, can establish the locations and temperatures/power/SOC conditions where an internal short circuit will result in thermal runaway, and provides relevant data to validate internal short circuit models.

  19. Understanding internalization of rotavirus VP6 nanotubes by cells: towards a recombinant vaccine.

    PubMed

    Rodríguez, Mabel; Wood, Christopher; Sanchez-López, Rosana; Castro-Acosta, Ricardo M; Ramírez, Octavio T; Palomares, Laura A

    2014-05-01

    Rotavirus VP6 nanotubes are an attractive option for a recombinant vaccine against rotavirus disease. Protection against rotavirus infection and an adjuvant effect have been observed upon immunization with VP6 nanotubes. However, little information exists on how VP6 nanotubes interact with cells and trigger an immune response. In this work, the interaction between VP6 nanotubes and different cell lines was characterized. VP6 nanotubes were not cytotoxic to any of the animal or human cell lines tested. Uptake of nanotubes into cells was cell-line-dependent, as only THP1 and J774 macrophage cells internalized them. Moreover, the size and spatial arrangement of VP6 assembled into nanotubes allowed their uptake by macrophages, as double-layered rotavirus-like particles also displaying VP6 in their surface were not taken up. The internalization of VP6 nanotubes was inhibited by methyl-β-cyclodextrin, but not by genistein, indicating that nanotube entry is specific, depends on the presence of cholesterol in the plasma membrane, and does not require the activity of tyrosine kinases. The information generated here expands our understanding of the interaction of protein nanotubes with cells, which is useful for the application of VP6 nanotubes as a vaccine.

  20. A new approach to the internal thermal management of cylindrical battery cells for automotive applications

    NASA Astrophysics Data System (ADS)

    Worwood, Daniel; Kellner, Quirin; Wojtala, Malgorzata; Widanage, W. D.; McGlen, Ryan; Greenwood, David; Marco, James

    2017-04-01

    Conventional cooling approaches that target either a singular tab or outer surface of common format cylindrical lithium-ion battery cells suffer from a high cell thermal resistance. Under an aggressive duty cycle, this resistance can result in the formation of large in-cell temperature gradients and high hot spot temperatures, which are known to accelerate ageing and further reduce performance. In this paper, a novel approach to internal thermal management of cylindrical battery cells to lower the thermal resistance for heat transport through the inside of the cell is investigated. The effectiveness of the proposed method is analysed for two common cylindrical formats when subject to highly aggressive electrical loading conditions representative of a high performance electric vehicle (EV) and hybrid electric vehicle (HEV). A mathematical model that captures the dominant thermal properties of the cylindrical cell is created and validated using experimental data. Results from the extensive simulation study indicate that the internal cooling strategy can reduce the cell thermal resistance by up to 67.8 ± 1.4% relative to single tab cooling, and can emulate the performance of a more complex pack-level double tab cooling approach whilst targeting cooling at a single tab.

  1. Effects of Operating Conditions on Internal Resistances in Enzyme Fuel Cells Studied via Electrochemical Impedance Spectroscopy

    SciTech Connect

    Aaron, D; Borole, Abhijeet P; Yiacoumi, Sotira; Tsouris, Costas

    2012-01-01

    Enzyme fuel cells (EFCs) offer some advantages over traditional precious-metal-catalyzed fuel cells, such as polymer electrolyte membrane fuel cells (PEMFCs). However, EFCs exhibit far less power output than PEMFCs and have relatively short life spans before materials must be replaced. In this work, electrochemical impedance spectroscopy (EIS) is used to analyze the internal resistances throughout the EFC at a variety of operating conditions. EIS analysis is focused primarily on the resistances of the anode, solution/membrane, and cathode. Increased enzyme loading results in improved power output and reductions in internal resistance. Conditions are identified for which enzyme loading does not limit the EFC performance. EIS experiments are also reported for EFCs operated continuously for 2 days; power output declines sharply over time, while all internal resistances increase. Drying of the cathode and enzyme/mediator degradation are believed to have contributed to this behavior. Finally, experiments are performed at varying air-humidification temperatures. Little effect on internal resistances or power output is observed. However, it is anticipated that increased air humidification can improve longevity by delivering more water to the cathode. Improvements to the enzymatic cathode are needed for EFC development. These improvements need to focus on improving transport rather than increasing enzyme loading.

  2. Cell membrane integrity and internalization of ingested TiO(2) nanoparticles by digestive gland cells of a terrestrial isopod.

    PubMed

    Novak, Sara; Drobne, Damjana; Valant, Janez; Pipan-Tkalec, Živa; Pelicon, Primož; Vavpetič, Primož; Grlj, Nataša; Falnoga, Ingrid; Mazej, Darja; Remškar, Maja

    2012-05-01

    The present study was motivated by the paucity of reports on cellular internalization of ingested titanium dioxide (TiO(2)) nanoparticles (nano-TiO(2)). The model invertebrate (Porcellio scaber, Isopoda, Crustacea) was exposed to food dosed with nano-TiO(2) containing 100, 1,000, 3,000, or 5,000 µg nano-TiO(2) per gram of food. After 14 d of exposure, the amount of Ti in the entire body was analyzed by inductively coupled plasma-mass spectrometry, and elemental analyses of tissue cross sections were performed by particle induced X-ray emission. In addition, a series of toxicological markers including feeding parameters, weight change, and survival, as well as cytotoxic effects such as digestive gland cell membrane stability, were monitored. Internalization of ingested nano-TiO(2) by the isopod's digestive gland epithelial cells was shown to depend on cell membrane integrity. Cell membranes were found to be destabilized by TiO(2) particles, and at higher extracellular concentrations of nano-TiO(2), the nanoparticles were internalized.

  3. g-force induced giant efficiency of nanoparticles internalization into living cells

    NASA Astrophysics Data System (ADS)

    Ocampo, Sandra M.; Rodriguez, Vanessa; de La Cueva, Leonor; Salas, Gorka; Carrascosa, Jose. L.; Josefa Rodríguez, María; García-Romero, Noemí; Luis, Jose; Cuñado, F.; Camarero, Julio; Miranda, Rodolfo; Belda-Iniesta, Cristobal; Ayuso-Sacido, Angel

    2015-10-01

    Nanotechnology plays an increasingly important role in the biomedical arena. Iron oxide nanoparticles (IONPs)-labelled cells is one of the most promising approaches for a fast and reliable evaluation of grafted cells in both preclinical studies and clinical trials. Current procedures to label living cells with IONPs are based on direct incubation or physical approaches based on magnetic or electrical fields, which always display very low cellular uptake efficiencies. Here we show that centrifugation-mediated internalization (CMI) promotes a high uptake of IONPs in glioblastoma tumour cells, just in a few minutes, and via clathrin-independent endocytosis pathway. CMI results in controllable cellular uptake efficiencies at least three orders of magnitude larger than current procedures. Similar trends are found in human mesenchymal stem cells, thereby demonstrating the general feasibility of the methodology, which is easily transferable to any laboratory with great potential for the development of improved biomedical applications.

  4. g-force induced giant efficiency of nanoparticles internalization into living cells.

    PubMed

    Ocampo, Sandra M; Rodriguez, Vanessa; de la Cueva, Leonor; Salas, Gorka; Carrascosa, Jose L; Rodríguez, María Josefa; García-Romero, Noemí; Cuñado, Jose Luis F; Camarero, Julio; Miranda, Rodolfo; Belda-Iniesta, Cristobal; Ayuso-Sacido, Angel

    2015-10-19

    Nanotechnology plays an increasingly important role in the biomedical arena. Iron oxide nanoparticles (IONPs)-labelled cells is one of the most promising approaches for a fast and reliable evaluation of grafted cells in both preclinical studies and clinical trials. Current procedures to label living cells with IONPs are based on direct incubation or physical approaches based on magnetic or electrical fields, which always display very low cellular uptake efficiencies. Here we show that centrifugation-mediated internalization (CMI) promotes a high uptake of IONPs in glioblastoma tumour cells, just in a few minutes, and via clathrin-independent endocytosis pathway. CMI results in controllable cellular uptake efficiencies at least three orders of magnitude larger than current procedures. Similar trends are found in human mesenchymal stem cells, thereby demonstrating the general feasibility of the methodology, which is easily transferable to any laboratory with great potential for the development of improved biomedical applications.

  5. Cell tracking with caged xenon: using cryptophanes as MRI reporters upon cellular internalization.

    PubMed

    Klippel, Stefan; Döpfert, Jörg; Jayapaul, Jabadurai; Kunth, Martin; Rossella, Federica; Schnurr, Matthias; Witte, Christopher; Freund, Christian; Schröder, Leif

    2014-01-07

    Caged xenon has great potential in overcoming sensitivity limitations for solution-state NMR detection of dilute molecules. However, no application of such a system as a magnetic resonance imaging (MRI) contrast agent has yet been performed with live cells. We demonstrate MRI localization of cells labeled with caged xenon in a packed-bed bioreactor working under perfusion with hyperpolarized-xenon-saturated medium. Xenon hosts enable NMR/MRI experiments with switchable contrast and selectivity for cell-associated versus unbound cages. We present MR images with 10(3) -fold sensitivity enhancement for cell-internalized, dual-mode (fluorescence/MRI) xenon hosts at low micromolar concentrations. Our results illustrate the capability of functionalized xenon to act as a highly sensitive cell tracer for MRI detection even without signal averaging. The method will bridge the challenging gap for translation to in vivo studies for the optimization of targeted biosensors and their multiplexing applications.

  6. A high throughput method for quantification of cell surface bound and internalized chitosan nanoparticles.

    PubMed

    Tammam, Salma N; Azzazy, Hassan M E; Lamprecht, Alf

    2015-11-01

    Chitosan has become a popular polymer for drug delivery. It's hydro solubility and mild formulation conditions have made it an attractive polymer for macromolecular delivery. Accurate quantification of internalized chitosan nanoparticles (NPs) is imperative for fair assessment of the nano-formulation where it is important to determine the exact amount of drug actually being delivered into the cell, especially for macromolecular drugs where cellular entry is limited by molecule size and/or charge. The preferential affinity of wheat germ agglutinin tagged with fluorescein isothiocyanate (WGA-FITC) to chitosan is exploited in the development of a simple and rapid method for the differentiation between and quantification of cell surface bound and internalized chitosan NPs. The percentage of cell surface bound NPs could be easily determined and corrected NP uptake could be calculated accordingly. The developed method is applicable in several cell lines and has successfully been tested with NPs with different sizes (25 and 150nm) and with very low NP concentrations (20μg/mL). The method will allow for the correct evaluation of chitosan NP uptake and could be further used to evaluate chitosan based nanomedicine and provide guidelines on how to modify NPs for enhanced internalization, and improved drug delivery.

  7. Internalization and synaptogenic effect of GH in retinal ganglion cells (RGCs).

    PubMed

    Fleming, Thomas; Martínez-Moreno, Carlos G; Mora, Janeth; Aizouki, Miray; Luna, Maricela; Arámburo, Carlos; Harvey, Steve

    2016-08-01

    In the chicken embryo, GH gene expression occurs in the neural retina and retinal GH promotes cell survival and induces axonal growth of retinal ganglion cells. Neuroretinal GH is therefore of functional importance before the appearance of somatotrophs and the onset of pituitary GH secretion to the peripheral plasma (at ED15-17). Endocrine actions of pituitary GH in the development and function of the chicken embryo eye are, however, unknown. This possibility has therefore been investigated in ED15 embryos and using the quail neuroretinal derived cell line (QNR/D). During this research, we studied for the first time, the coexistence of exogenous (endocrine) and local GH (autocrine/paracrine) in retinal ganglion cells (RGCs). In ovo systemic injections of Cy3-labeled GH demonstrated that GH in the embryo bloodstream was translocated into the neural retina and internalized into RGC's. Pituitary GH may therefore be functionally involved in retinal development during late embryogenesis. Cy3-labelled GH was similarly internalized into QNR/D cells after its addition into incubation media. The uptake of exogenous GH was by a receptor-mediated mechanism and maximal after 30-60min. The exogenous (endocrine) GH induced STAT5 phosphorylation and increased growth associated protein 43 (GAP43) and SNAP-25 immunoreactivity. Ex ovo intravitreal injections of Cy3-GH in ED12 embryos resulted in GH internalization and STAT5 activation. Interestingly, the CY3-labeled GH accumulated in perinuclear regions of the QNR/D cells, but was not found in the cytoplasm of neurite outgrowths, in which endogenous retinal GH is located. This suggests that exogenous (endocrine) and local (autocrine/paracrine) GH are both involved in retinal function in late embryogenesis but they co-exist in separate intracellular compartments within retinal ganglion cells.

  8. Pancreatic cell immobilization in alginate beads produced by emulsion and internal gelation.

    PubMed

    Hoesli, Corinne A; Raghuram, Kamini; Kiang, Roger L J; Mocinecová, Dušana; Hu, Xiaoke; Johnson, James D; Lacík, Igor; Kieffer, Timothy J; Piret, James M

    2011-02-01

    Alginate has been used to protect transplanted pancreatic islets from immune rejection and as a matrix to increase the insulin content of islet progenitor cells. The throughput of alginate bead generation by the standard extrusion and external gelation method is limited by the rate of droplet formation from nozzles. Alginate bead generation by emulsion and internal gelation is a scaleable alternative that has been used with biological molecules and microbial cells, but not mammalian cells. We describe the novel adaptation of this process to mammalian cell immobilization. After optimization, the emulsion process yielded 90 ± 2% mouse insulinoma 6 (MIN6) cell survival, similar to the extrusion process. The MIN6 cells expanded at the same rate in both bead types to form pseudo-islets with increased glucose stimulation index compared to cells in suspension. The emulsion process was suitable for primary pancreatic exocrine cell immobilization, leading to 67 ± 32 fold increased insulin expression after 10 days of immobilized culture. Due to the scaleability and broad availability of stirred mixers, the emulsion process represents an attractive option for laboratories that are not equipped with extrusion-based cell encapsulators, as well as for the production of immobilized or encapsulated cellular therapeutics on a clinical scale.

  9. On the importance of modelling the internal spatial dynamics of biological cells.

    PubMed

    Sayyid, Faiz; Kalvala, Sara

    2016-07-01

    Spatial effects such as cell shape have very often been considered negligible in models of cellular pathways, and many existing simulation infrastructures do not take such effects into consideration. Recent experimental results are reversing this judgement by showing that very small spatial variations can make a big difference in the fate of a cell. This is particularly the case when considering eukaryotic cells, which have a complex physical structure and many subtle control mechanisms, but bacteria are also interesting for the huge variation in shape both between species and in different phases of their lifecycle. In this work we perform simulations that measure the effect of three common bacterial shapes on the behaviour of model cellular pathways. To perform these experiments we develop ReDi-Cell, a highly scalable GPGPU cell simulation infrastructure for the modelling of cellular pathways in spatially detailed environments. ReDi-Cell is validated against known-good simulations, prior to its use in new work. We then use ReDi-Cell to conduct novel experiments that demonstrate the effect that three common bacterial shapes (Cocci, Bacilli and Spirilli) have on the behaviour of model cellular pathways. Pathway wavefront shape, pathway concentration gradients, and chemical species distribution are measured in the three different shapes. We also quantify the impact of internal cellular clutter on the same pathways. Through this work we show that variations in the shape or configuration of these common cell shapes alter model cell behaviour.

  10. Proteomic characterization of the internalization of Opisthorchis viverrini excretory/secretory products in human cells.

    PubMed

    Chaiyadet, Sujittra; Smout, Michael; Laha, Thewarach; Sripa, Banchob; Loukas, Alex; Sotillo, Javier

    2016-02-09

    The association between liver fluke infection caused by Opisthorchis viverrini and cholangiocarcinoma (CCA - hepatic cancer of the bile duct epithelium) has been well established. Multiple mechanisms play a role in the development of CCA, but the excretory/secretory products released by O. viverrini (OvES) represent the major interface between the parasite and its host, and their uptake by biliary epithelial cells has been suggested to be responsible for proliferation of cholangiocytes, the cells that line the biliary epithelium. Despite recent progress in the study of the molecular basis of O. viverrini-host interactions, little is known about the effects that OvES induces upon internalization by host cells. In the present study we incubated non-cancerous human cholangiocytes (H69) and human colon cancer (CaCo-2) cells with OvES and performed a time-course quantitative proteomic analysis on the cells to determine the early changes induced by the parasite. Different KEGG pathways were altered in H69 cells compared to Caco-2 cells: glycolysis/gluconeogenesis and protein processing in the endoplasmic reticulum. In addition, the Reactome pathway analysis showed a predominance of proteins involved in cellular pathways related to apoptosis and apoptotic execution phase in H69 cells after incubation with OvES. The present study provides the first proteomic analysis to address the molecular mechanisms by which OvES products interact with host cells, and Sheds light on the cellular processes involved in O. viverrini-induced CCA.

  11. Differential cytotoxicity and internalization of graphene family nanomaterials in myocardial cells.

    PubMed

    Contreras-Torres, Flavio F; Rodríguez-Galván, Andrés; Guerrero-Beltrán, Carlos E; Martínez-Lorán, Erick; Vázquez-Garza, Eduardo; Ornelas-Soto, Nancy; García-Rivas, Gerardo

    2017-04-01

    Given the well-known physical properties of graphene oxide (GO), numerous applications for this novel nanomaterial have been recently envisioned to improve the performance of biomedical devices. However, the toxicological assessment of GO, which strongly depends on the used material and the studied cell line, is a fundamental task that needs to be performed prior to its use in biomedical applications. Therefore, the toxicological characterization of GO is still ongoing. This study contributes to this, aiming to synthesize and characterize GO particles and thus investigate their toxic effects in myocardial cells. Herein, GO particles were produced from graphite using the Tour method and subsequent mild reduction was carried out to obtain low-reduced GO (LRGO) particles. A qualitative analysis of the viability, cellular uptake, and internalization of particles was carried out using GO (~54% content of oxygen) and LRGO (~37% content of oxygen) and graphite. GO and LRGO reduce the viability of cardiac cells at IC50 of 652.1±1.2 and 129.4±1.2μg/mL, respectively. This shows that LRGO particles produce a five-fold increase in cytotoxicity when compared to GO. The cell uptake pattern of GO and LRGO particles demonstrated that cardiac cells retain a similar complexity to control cells. Morphological alterations examined with electron microscopy showed that internalization by GO and LRGO-treated cells (100μg/mL) occurred affecting the cell structure. These results suggest that the viability of H9c2 cells can be associated with the surface chemistry of GO and LRGO, as defined by the amount of oxygen functionalities, the number of graphitic domains, and the size of particles. High angle annular dark-field scanning transmission electron microscopy, dynamic light-scattering, Fourier-transform infrared, Raman, and X-ray photoelectron spectroscopies were used to characterize the as-prepared materials.

  12. Comparing national home-keeping and the regulation of translational stem cell applications: An international perspective.

    PubMed

    Sleeboom-Faulkner, Margaret; Chekar, Choon Key; Faulkner, Alex; Heitmeyer, Carolyn; Marouda, Marina; Rosemann, Achim; Chaisinthop, Nattaka; Chang, Hung-Chieh Jessica; Ely, Adrian; Kato, Masae; Patra, Prasanna K; Su, Yeyang; Sui, Suli; Suzuki, Wakana; Zhang, Xinqing

    2016-03-01

    A very large grey area exists between translational stem cell research and applications that comply with the ideals of randomised control trials and good laboratory and clinical practice and what is often referred to as snake-oil trade. We identify a discrepancy between international research and ethics regulation and the ways in which regulatory instruments in the stem cell field are developed in practice. We examine this discrepancy using the notion of 'national home-keeping', referring to the way governments articulate international standards and regulation with conflicting demands on local players at home. Identifying particular dimensions of regulatory tools - authority, permissions, space and acceleration - as crucial to national home-keeping in Asia, Europe and the USA, we show how local regulation works to enable development of the field, notwithstanding international (i.e. principally 'western') regulation. Triangulating regulation with empirical data and archival research between 2012 and 2015 has helped us to shed light on how countries and organisations adapt and resist internationally dominant regulation through the manipulation of regulatory tools (contingent upon country size, the state's ability to accumulate resources, healthcare demands, established traditions of scientific governance, and economic and scientific ambitions).

  13. Direct visualization of the phosphorylated epidermal growth factor receptor during its internalization in A-431 cells

    PubMed Central

    1987-01-01

    Epidermal growth factor (EGF) rapidly stimulates receptor autophosphorylation in A-431 cells. After 1 min the phosphorylated receptor can be identified at the plasma membrane using an anti- phosphotyrosine antibody. With further incubation at 37 degrees C, approximately 50% of the phosphorylated EGF receptor was internalized (t1/2 = 5 min) and associated with the tubulovesicular system and later with multivesicular bodies, but not the nucleus. During this period, there was no change in the extent or sites of phosphorylation. At all times the phosphotyrosine remained on the cytoplasmic side of the membrane, opposite to the EGF ligand identified by anti-EGF antibody. These data indicate that (a) the tyrosine-phosphorylated EGF receptor is internalized in its activated form providing a mechanism for translocation of the receptor kinase to substrates in the cell interior; (b) the internalized receptor remains intact for at least 60 min, does not associate with the nucleus, and does not generate any tyrosine-phosphorylated fragments; and (c) tyrosine phosphorylation alone is not the signal for receptor internalization. PMID:2447100

  14. Cell science and protein crystal growth research for the International Space Station.

    PubMed

    Sigler, P B; Stein, G S; Boskey, A L; Jones, N D; Kuriyan, J; Miller, W M; Shuler, M L; Wang, B C

    2000-09-14

    The recent National Research Council report, Future Biotechnology Research on the International Space Station, evaluates NASA's plans for research in cell science and protein crystal growth to be conducted on the International Space Station. This report concludes that the NASA biotechnology programs have the potential to significantly impact relevant scientific fields and to increase understanding and insight into fundamental biological issues. In order to realize the potential impacts, NASA must focus its research programs by selecting specific questions related to gravitational forces' role in cell behavior and by using the microgravity environment as a tool to determine the structure of macromolecules with important biological implications. Given the time and volume constraints associated with space-based experiments, instrumentation to be used on the space station must be designed to maximize the productivity of researchers, and NASA's recruitment of investigators and support for space station experiments should aim to encourage and facilitate cutting-edge research.

  15. Vitronectin enhances internalization of crocidolite asbestos by rabbit pleural mesothelial cells via the integrin alpha v beta 5.

    PubMed Central

    Boylan, A M; Sanan, D A; Sheppard, D; Broaddus, V C

    1995-01-01

    The mechanism by which pleural mesothelial cells, the likely progenitor cells of asbestos-induced mesothelioma, recognize and internalize crocidolite asbestos is unknown. Because incubation of asbestos fibers with serum increases their association with cells, we asked whether a protein coat on asbestos increased internalization of fibers via specific cellular receptors. Coating crocidolite with citronectin, but not with fibronectin or other proteins, increased fiber internalization by rabbit pleural mesothelial cells, as measured by a new technique using fluorescence confocal microscopy. Receptors for vitronectin, alpha v beta 3 and alpha v beta 5, were identified on mesothelial cells. Inhibiting vitronectin receptors by plating cells on a vitronectin substrate or incubating cells with excess soluble vitronectin reduced internalization of vitronectin-coated crocidolite. Inhibition of alpha v beta 5, but not alpha v beta 3, with blocking antibodies similarly reduced internalization. In addition, alpha v beta 5, but not alpha v beta 3, showed immunocytochemical colocalization with fibers. Of biologic relevance, coating crocidolite with serum also increased internalization via alpha v beta 5, an effect dependent on the vitronectin in serum. We conclude that pleural mesothelial cells recognize and internalize vitronectin- and serum-coated asbestos via the integrin alpha v beta 5. Since integrins initiate some of the same signaling pathways as does asbestos, our findings may provide insights into the mechanisms of asbestos-induced biologic effects. Images PMID:7560092

  16. Performance Evaluation of a Fused-Alkali Electrolyte, Internal-Reforming Fuel Cell

    DTIC Science & Technology

    1975-04-01

    obtained by permniaI con i nu nicat ions bKteteenilthe an hor andu ellployees of I’rototech, Inc-., and the Atlantic-Richifield Corporation . Thie author...Richfield Corporation ; but by the time Juda’s paper was presented, the two companies already had discontinued further development of the system. After this... communicatin . 5 INLET OUTLET FILLING HOLE FRAME DISTRIBUTION TUBE Figure 2. Internal stnicttare or ainod.Ie 6 c. Cell-Heating System. The oven was a

  17. Cell-free system responsible for internal radiolabeling of glycopeptidolipids of the Mycobacterium avium complex.

    PubMed Central

    Ramasesh, N; Wright, E L; Barrow, W W

    1992-01-01

    Internal radiolabeling of serotype-specific glycopeptidolipids with [14C]mannose was accomplished with a cell-free system derived from serotype 20 of the Mycobacterium avium complex. Similar radiolabeling was not apparent with a cell-free system derived from the rough colony variant, previously shown to be devoid of glycopeptidolipids. Although a comparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein analysis of the parent and rough variant strains revealed a close similarity, there were some proteins unique to the parent strain. Images PMID:1729193

  18. Internal voltage control of hydrogen-oxygen fuel cells: Feasibility study

    NASA Technical Reports Server (NTRS)

    Prokopius, P. R.

    1975-01-01

    An experimental study was conducted to assess the feasibility of internal voltage regulation of fuel cell systems. Two methods were tested. In one, reactant partial pressure was used as the voltage control parameter and in the other reactant total pressure was used for control. Both techniques were breadboarded and tested on a single alkaline-electrolyte fuel cell. Both methods were found to be possible forms of regulation, however, of the two the total pressure technique would be more efficient, simpler to apply and would provide better transient characteristics.

  19. Nanograting-based plasmon enhancement for total internal reflection fluorescence microscopy of live cells.

    PubMed

    Kim, Kyujung; Kim, Dong Jun; Cho, Eun-Jin; Suh, Jin-Suck; Huh, Yong-Min; Kim, Donghyun

    2009-01-07

    We investigated evanescent field enhancement based on subwavelength nanogratings for improved sensitivity in total internal reflection microscopy of live cells. The field enhancement is associated with subwavelength-grating-coupled plasmon excitation. An optimum sample employed a silver grating on a silver film and an SF10 glass substrate. Field intensity was enhanced by approximately 90% when measured by fluorescent excitation of microbeads relative to that on a bare prism as a control, which is in good agreement with numerical results. The subwavelength-grating-mediated field enhancement was also applied to live cell imaging of quantum dots, which confirmed the sensitivity enhancement qualitatively.

  20. Imaging with total internal reflection fluorescence microscopy for the cell biologist.

    PubMed

    Mattheyses, Alexa L; Simon, Sanford M; Rappoport, Joshua Z

    2010-11-01

    Total internal reflection fluorescence (TIRF) microscopy can be used in a wide range of cell biological applications, and is particularly well suited to analysis of the localization and dynamics of molecules and events near the plasma membrane. The TIRF excitation field decreases exponentially with distance from the cover slip on which cells are grown. This means that fluorophores close to the cover slip (e.g. within ~100 nm) are selectively illuminated, highlighting events that occur within this region. The advantages of using TIRF include the ability to obtain high-contrast images of fluorophores near the plasma membrane, very low background from the bulk of the cell, reduced cellular photodamage and rapid exposure times. In this Commentary, we discuss the applications of TIRF to the study of cell biology, the physical basis of TIRF, experimental setup and troubleshooting.

  1. On-Orbit Measurement of Next Generation Space Solar Cell Technology on the International Space Station

    NASA Technical Reports Server (NTRS)

    Wolford, David S.; Myers, Matthew G.; Prokop, Norman F.; Krasowski, Michael J.; Parker, David S.; Cassidy, Justin C.; Davies, William E.; Vorreiter, Janelle O.; Piszczor, Michael F.; McNatt, Jeremiah S.

    2015-01-01

    Measurement is essential for the evaluation of new photovoltaic (PV) technology for space solar cells. NASA Glenn Research Center (GRC) is in the process of measuring several solar cells in a supplemental experiment on NASA Goddard Space Flight Center's (GSFC) Robotic Refueling Mission's (RRM) Task Board 4 (TB4). Four industry and government partners have provided advanced PV devices for measurement and orbital environment testing. The experiment will be on-orbit for approximately 18 months. It is completely self-contained and will provide its own power and internal data storage. Several new cell technologies including four- junction (4J) Inverted Metamorphic Multijunction (IMM) cells will be evaluated and the results compared to ground-based measurements.

  2. Topography of Cells Revealed by Variable-Angle Total Internal Reflection Fluorescence Microscopy.

    PubMed

    Cardoso Dos Santos, Marcelina; Déturche, Régis; Vézy, Cyrille; Jaffiol, Rodolphe

    2016-09-20

    We propose an improved version of variable-angle total internal reflection fluorescence microscopy (vaTIRFM) adapted to modern TIRF setup. This technique involves the recording of a stack of TIRF images, by gradually increasing the incident angle of the light beam on the sample. A comprehensive theory was developed to extract the membrane/substrate separation distance from fluorescently labeled cell membranes. A straightforward image processing was then established to compute the topography of cells with a nanometric axial resolution, typically 10-20 nm. To highlight the new opportunities offered by vaTIRFM to quantify adhesion process of motile cells, adhesion of MDA-MB-231 cancer cells on glass substrate coated with fibronectin was examined.

  3. Meeting report of the first conference of the International Placenta Stem Cell Society (IPLASS)

    PubMed Central

    Parolini, O.; Alviano, F.; Betz, A.G.; Bianchi, D.W.; Götherström, C.; Manuelpillai, U.; Mellor, A.L.; Ofir, R.; Ponsaerts, P.; Scherjon, S.A.; Weiss, M.L.; Wolbank, S.; Wood, K.J.; Borlongan, C.V.

    2012-01-01

    The International Placenta Stem Cell Society (IPLASS) was founded in June 2010. Its goal is to serve as a network for advancing research and clinical applications of stem/progenitor cells isolated from human term placental tissues, including the amnio-chorionic fetal membranes and Wharton's jelly. The commitment of the Society to champion placenta as a stem cell source was realized with the inaugural meeting of IPLASS held in Brescia, Italy, in October 2010. Officially designated as an EMBO-endorsed scientific activity, international experts in the field gathered for a 3-day meeting, which commenced with “Meet with the experts” sessions, IPLASS member and board meetings, and welcome remarks by Dr. Ornella Parolini, President of IPLASS. The evening's highlight was a keynote plenary lecture by Dr. Diana Bianchi. The subsequent scientific program consisted of morning and afternoon oral and poster presentations, followed by social events. Both provided many opportunities for intellectual exchange among the 120 multi-national participants. This allowed a methodical and deliberate evaluation of the status of placental cells in research in regenerative and reparative medicine. The meeting concluded with Dr. Parolini summarizing the meeting's highlights. This further prepared the fertile ground on which to build the promising potential of placental cell research. The second IPLASS meeting will take place in September 2012 in Vienna, Austria. This meeting report summarizes the thought-provoking lectures delivered at the first meeting of IPLASS. PMID:21575989

  4. Selective internalization of self-assembled artificial oil bodies by HER2/neu-positive cells

    NASA Astrophysics Data System (ADS)

    Chiang, Chung-Jen; Lin, Li-Jen; Lin, Che-Chin; Chang, Chih-Hsiang; Chao, Yun-Peng

    2011-01-01

    A novel delivery carrier was developed using artificial oil bodies (AOBs). Plant seed oil bodies (OBs) consist of a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with the storage protein oleosin (Ole). Ole consists of a central hydrophobic domain with two amphiphatic arms that extrude from the surface of OBs. In this study, a bivalent anti-HER2/neu affibody domain (ZH2) was fused with Ole at the C terminus. After overproduction in Escherichia coli, the fusion protein (Ole-ZH2) was recovered to assemble AOBs. The size of self-assembled AOBs was tailored by varying the oil/Ole-ZH2 ratio and pH to reach a nanoscale. Upon co-incubation with tumor cells, the nanoscale AOBs encapsulated with a hydrophobic fluorescence dye were selectively internalized by HER2/neu-overexpressing cells and displayed biocompatibility with the cells. In addition, the ZH2-mediated endosomal entry of AOBs occurred in a time- and AOB dose-dependent manner. The internalization efficiency was as high as 90%. The internalized AOBs disintegrated at the non-permissive pH (e.g. in acidic endosomes) and the cargo dye was released. Results of in vitro study revealed a sustained and prolonged release profile. Taken together, our findings indicate the potential of AOBs as a delivery carrier.

  5. Cell Signaling, Internalization, and Nuclear Localization of the Angiotensin Converting Enzyme in Smooth Muscle and Endothelial Cells*

    PubMed Central

    Lucero, Héctor A.; Kintsurashvili, Ekaterina; Marketou, Maria E.; Gavras, Haralambos

    2010-01-01

    The angiotensin converting enzyme (ACE) catalyzes the extracellular formation of angiotensin II, and degradation of bradykinin, thus regulating blood pressure and renal handling of electrolytes. We have previously shown that exogenously added ACE elicited transcriptional regulation independent of its enzymatic activity. Because transcriptional regulation generates from protein-DNA interactions within the cell nucleus we have investigated the initial cellular response to exogenous ACE and the putative internalization of the enzyme in smooth muscle cells (SMC) and endothelial cells (EC). The following phenomena were observed when ACE was added to cells in culture: 1) it bound to SMC and EC with high affinity (Kd = 361.5 ± 60.5 pm) and with a low binding occupancy (Bmax = 335.0 ± 14.0 molecules/cell); 2) it triggered cellular signaling resulting in late activation of focal adhesion kinase and SHP2; 3) it modulated platelet-derived growth factor receptor-β signaling; 4) it was endocytosed by SMC and EC; and 5) it transited through the early endosome, partially occupied the late endosome and the lysosome, and was localized to the nuclei. The incorporation of ACE or a fragment of it into the nuclei reached saturation at 120 min, and was preceded by a lag time of 40 min. Internalized ACE was partially cleaved into small fragments. These results revealed that extracellular ACE modulated cell signaling properties, and that SMC and EC have a pathway for delivery of extracellular ACE to the nucleus, most likely involving cell surface receptor(s) and requiring transit through late endosome/lysosome compartments. PMID:20022959

  6. Glycolipid presentation to natural killer T cells differs in an organ-dependent fashion

    NASA Astrophysics Data System (ADS)

    Schmieg, John; Yang, Guangli; Franck, Richard W.; van Rooijen, Nico; Tsuji, Moriya

    2005-01-01

    It has been shown that dendritic cells (DCs) are able to present glycolipids to natural killer (NK) T cells in vivo. However, the essential role of DCs, as well as the role of other cells in glycolipid presentation, is unknown. Here, we show that DCs are the crucial antigen-presenting cells (APCs) for splenic NK T cells, whereas Kupffer cells are the key APCs for hepatic NK T cells. Both cell types stimulate cytokine production by NK T cells within 2 h of glycolipid administration, but only DCs are involved in the systemic, downstream responses to glycolipid administration. More specifically, CD8+ DCs produce IL-12 in response to glycolipid presentation, which stimulates secondary IFN- production by NK cells in different organs. Different APCs participate in glycolipid presentation to NK T cells in vivo but differ in their involvement in the overall glycolipid response. dendritic cell | Kupffer cell

  7. Data sharing in stem cell translational science: policy statement by the International Stem Cell Forum Ethics Working Party.

    PubMed

    Bredenoord, Annelien L; Mostert, Menno; Isasi, Rosario; Knoppers, Bartha M

    2015-01-01

    Data and sample sharing constitute a scientific and ethical imperative but need to be conducted in a responsible manner in order to protect individual interests as well as maintain public trust. In 2014, the Global Alliance for Genomics and Health (GA4GH) adopted a common Framework for Responsible Sharing of Genomic and Health-Related Data. The GA4GH Framework is applicable to data sharing in the stem cell field, however, interpretation is required so as to provide guidance for this specific context. In this paper, the International Stem Cell Forum Ethics Working Party discusses those principles that are specific to translational stem cell science, including engagement, data quality and safety, privacy, security and confidentiality, risk-benefit analysis and sustainability.

  8. Publishing SNP genotypes of human embryonic stem cell lines: policy statement of the International Stem Cell Forum Ethics Working Party.

    PubMed

    Knoppers, Bartha M; Isasi, Rosario; Benvenisty, Nissim; Kim, Ock-Joo; Lomax, Geoffrey; Morris, Clive; Murray, Thomas H; Lee, Eng Hin; Perry, Margery; Richardson, Genevra; Sipp, Douglas; Tanner, Klaus; Wahlström, Jan; de Wert, Guido; Zeng, Fanyi

    2011-09-01

    Novel methods and associated tools permitting individual identification in publicly accessible SNP databases have become a debatable issue. There is growing concern that current technical and ethical safeguards to protect the identities of donors could be insufficient. In the context of human embryonic stem cell research, there are no studies focusing on the probability that an hESC line donor could be identified by analyzing published SNP profiles and associated genotypic and phenotypic information. We present the International Stem Cell Forum (ISCF) Ethics Working Party's Policy Statement on "Publishing SNP Genotypes of Human Embryonic Stem Cell Lines (hESC)". The Statement prospectively addresses issues surrounding the publication of genotypic data and associated annotations of hESC lines in open access databases. It proposes a balanced approach between the goals of open science and data sharing with the respect for fundamental bioethical principles (autonomy, privacy, beneficence, justice and research merit and integrity).

  9. Internalization of targeted quantum dots by brain capillary endothelial cells in vivo.

    PubMed

    Paris-Robidas, Sarah; Brouard, Danny; Emond, Vincent; Parent, Martin; Calon, Frédéric

    2016-04-01

    Receptors located on brain capillary endothelial cells forming the blood-brain barrier are the target of most brain drug delivery approaches. Yet, direct subcellular evidence of vectorized transport of nanoformulations into the brain is lacking. To resolve this question, quantum dots were conjugated to monoclonal antibodies (Ri7) targeting the murine transferrin receptor. Specific transferrin receptor-mediated endocytosis of Ri7-quantum dots was first confirmed in N2A and bEnd5 cells. After intravenous injection in mice, Ri7-quantum dots exhibited a fourfold higher volume of distribution in brain tissues, compared to controls. Immunofluorescence analysis showed that Ri7-quantum dots were sequestered throughout the cerebral vasculature 30 min, 1 h, and 4 h post injection, with a decline of signal intensity after 24 h. Transmission electron microscopic studies confirmed that Ri7-quantum dots were massively internalized by brain capillary endothelial cells, averaging 37 ± 4 Ri7-quantum dots/cell 1 h after injection. Most quantum dots within brain capillary endothelial cells were observed in small vesicles (58%), with a smaller proportion detected in tubular structures or in multivesicular bodies. Parenchymal penetration of Ri7-quantum dots was extremely low and comparable to control IgG. Our results show that systemically administered Ri7-quantum dots complexes undergo extensive endocytosis by brain capillary endothelial cells and open the door for novel therapeutic approaches based on brain endothelial cell drug delivery.

  10. Glucose is a key driver for GLUT1-mediated nanoparticles internalization in breast cancer cells

    NASA Astrophysics Data System (ADS)

    Venturelli, Leonardo; Nappini, Silvia; Bulfoni, Michela; Gianfranceschi, Giuseppe; Dal Zilio, Simone; Coceano, Giovanna; Del Ben, Fabio; Turetta, Matteo; Scoles, Giacinto; Vaccari, Lisa; Cesselli, Daniela; Cojoc, Dan

    2016-02-01

    The mesenchymal state in cancer is usually associated with poor prognosis due to the metastatic predisposition and the hyper-activated metabolism. Exploiting cell glucose metabolism we propose a new method to detect mesenchymal-like cancer cells. We demonstrate that the uptake of glucose-coated magnetic nanoparticles (MNPs) by mesenchymal-like cells remains constant when the glucose in the medium is increased from low (5.5 mM) to high (25 mM) concentration, while the MNPs uptake by epithelial-like cells is significantly reduced. These findings reveal that the glucose-shell of MNPs plays a major role in recognition of cells with high-metabolic activity. By selectively blocking the glucose transporter 1 channels we showed its involvement in the internalization process of glucose-coated MNPs. Our results suggest that glucose-coated MNPs can be used for metabolic-based assays aimed at detecting cancer cells and that can be used to selectively target cancer cells taking advantage, for instance, of the magnetic-thermotherapy.

  11. Cell recycled culture of succinic acid-producing Anaerobiospirillum succiniciproducens using an internal membrane filtration system.

    PubMed

    Lee, Pyung-Cheon; Lee, Sang-Yup; Chang, Ho-Nam

    2008-07-01

    Cell recycled culture of succinic acid-producing Anaerobiospirillum succiniciproducens was anaerobically carried out using an internal membrane filter module in order to examine the physiological response of A. succiniciproducens to a high-cell-density environment. The optimal growth of A. succiniciproducens and its enhanced succinic acid productivity were observed under CO2-rich conditions, established by adding NaHCO3 and Na2CO3, in the cell recycled system. A. succiniciproducens grew up to 6.50 g-DCW/l, the highest cell concentration obtained so far, in cell recycled cultures. The cells did not change their morphology, which is known to be easily changed in unfavorable or stress environments. The maximum productivity of succinic acid was about 3.3 g/l/h, which is 3.3 times higher than those obtained in batch cultures. These results can serve as a guide for designing highly efficient cell recycled systems for succinic acid at a commercial level.

  12. Glucose is a key driver for GLUT1-mediated nanoparticles internalization in breast cancer cells

    PubMed Central

    Venturelli, Leonardo; Nappini, Silvia; Bulfoni, Michela; Gianfranceschi, Giuseppe; Dal Zilio, Simone; Coceano, Giovanna; Del Ben, Fabio; Turetta, Matteo; Scoles, Giacinto; Vaccari, Lisa; Cesselli, Daniela; Cojoc, Dan

    2016-01-01

    The mesenchymal state in cancer is usually associated with poor prognosis due to the metastatic predisposition and the hyper-activated metabolism. Exploiting cell glucose metabolism we propose a new method to detect mesenchymal-like cancer cells. We demonstrate that the uptake of glucose-coated magnetic nanoparticles (MNPs) by mesenchymal-like cells remains constant when the glucose in the medium is increased from low (5.5 mM) to high (25 mM) concentration, while the MNPs uptake by epithelial-like cells is significantly reduced. These findings reveal that the glucose-shell of MNPs plays a major role in recognition of cells with high-metabolic activity. By selectively blocking the glucose transporter 1 channels we showed its involvement in the internalization process of glucose-coated MNPs. Our results suggest that glucose-coated MNPs can be used for metabolic-based assays aimed at detecting cancer cells and that can be used to selectively target cancer cells taking advantage, for instance, of the magnetic-thermotherapy. PMID:26899926

  13. Effects of Sodium Octanoate on Innate Immune Response of Mammary Epithelial Cells during Staphylococcus aureus Internalization

    PubMed Central

    Alva-Murillo, Nayeli; Ochoa-Zarzosa, Alejandra; López-Meza, Joel E.

    2013-01-01

    Bovine mammary epithelial cells (bMECs) are capable of initiating an innate immune response to invading bacteria. Short chain fatty acids can reduce Staphylococcus aureus internalization into bMEC, but it has not been evaluated if octanoic acid (sodium octanoate, NaO), a medium chain fatty acid (MCFA), has similar effects. In this study we determined the effect of NaO on S. aureus internalization into bMEC and on the modulation of innate immune elements. NaO (0.25–2 mM) did not affect S. aureus growth and bMEC viability, but it differentially modulated bacterial internalization into bMEC, which was induced at 0.25–0.5 mM (~60%) but inhibited at 1-2 mM (~40%). Also, bMEC showed a basal expression of all the innate immune genes evaluated, which were induced by S. aureus. NaO induced BNBD4, LAP, and BNBD10 mRNA expression, but BNBD5 and TNF-α were inhibited. Additionally, the pretreatment of bMEC with NaO inhibited the mRNA expression induction generated by bacteria which coincides with the increase in internalization; only TAP and BNDB10 showed an increase in their expression; it coincides with the greatest effect on the reduction of bacterial internalization. In conclusion, NaO exerts a dual effect on S. aureus internalization in bMEC and modulates elements of innate immune response. PMID:23509807

  14. Gliotoxin promotes Aspergillus fumigatus internalization into type II human pneumocyte A549 cells by inducing host phospholipase D activation.

    PubMed

    Jia, Xiaodong; Chen, Fangyan; Pan, Weihua; Yu, Rentao; Tian, Shuguang; Han, Gaige; Fang, Haiqin; Wang, Shuo; Zhao, Jingya; Li, Xianping; Zheng, Dongyu; Tao, Sha; Liao, Wanqing; Han, Xuelin; Han, Li

    2014-06-01

    The internalization of Aspergillus fumigatus into lung epithelial cells is critical for the infection process in the host. Gliotoxin is the most potent toxin produced by A. fumigatus. However, its role in A. fumigatus internalization into the lung epithelial cells is still largely unknown. In the present study, the deletion of the gliP gene regulating the production of gliotoxin in A. fumigatus suppressed the internalization of conidia into the A549 lung epithelial cells, and this suppression could be rescued by the exogenous addition of gliotoxin. At lower concentrations, gliotoxin enhanced the internalization of the conidia of A. fumigatus into A549 cells; in contrast, it inhibited the phagocytosis of J774 macrophages in a dose-dependent manner. Under a concentration of 100 ng/ml, gliotoxin had no effect on A549 cell viability but attenuated ROS production in a dose-dependent manner. Gliotoxin significantly stimulated the phospholipase D activity in the A549 cells at a concentration of 50 ng/ml. This stimulation was blocked by the pretreatment of host cells with PLD1- but not PLD2-specific inhibitor. Morphological cell changes induced by gliotoxin were observed in the A549 cells accompanying with obvious actin cytoskeleton rearrangement and a moderate alteration of phospholipase D distribution. Our data indicated that gliotoxin might be responsible for modulating the A. fumigatus internalization into epithelial cells through phospholipase D1 activation and actin cytoskeleton rearrangement.

  15. The autophagy machinery restrains iNKT cell activation through CD1D1 internalization.

    PubMed

    Keller, Christian W; Loi, Monica; Ewert, Svenja; Quast, Isaak; Theiler, Romina; Gannagé, Monique; Münz, Christian; De Libero, Gennaro; Freigang, Stefan; Lünemann, Jan D

    2017-03-15

    Invariant natural killer T (iNKT) cells are innate T cells with powerful immune regulatory functions that recognize glycolipid antigens presented by the CD1D protein. While iNKT-cell-activating glycolipids are currently being explored for their efficacy to improve immunotherapy against infectious diseases and cancer, little is known about the mechanisms that control CD1D antigen presentation and iNKT cell activation in vivo. CD1D molecules survey endocytic pathways to bind lipid antigens in MHC class II containing compartments (MIICs) before recycling to the plasma membrane. Autophagosomes intersect with MIICs and autophagy-related proteins are known to support antigen loading for increased CD4(+) T cell immunity. Here, we report that mice with dendritic cell (DC)-specific deletion of the essential autophagy gene Atg5 showed better CD1D1-restricted glycolipid presentation in vivo. These effects led to enhanced iNKT cell cytokine production upon antigen recognition and lower bacterial loads during Sphingomonas paucimobilis infection. Enhanced iNKT cell activation was independent of receptor-mediated glycolipid uptake or costimulatory signals. Instead, loss of Atg5 in DCs impaired clathrin-dependent internalization of CD1D1 molecules via the adaptor protein complex 2 (AP2) and, thus, increased surface expression of stimulatory CD1D1-glycolipid complexes. These findings indicate that the autophagic machinery assists in the recruitment of AP2 to CD1D1 molecules resulting in attenuated iNKT cell activation, in contrast to the supporting role of macroautophagy in CD4(+) T cell stimulation.

  16. Non-genotoxic conditioning for hematopoietic stem cell transplantation using a hematopoietic-cell-specific internalizing immunotoxin

    PubMed Central

    Palchaudhuri, Rahul; Saez, Borja; Hoggatt, Jonathan; Schajnovitz, Amir; Sykes, David B; Tate, Tiffany A; Czechowicz, Agnieszka; Kfoury, Youmna; Ruchika, FNU; Rossi, Derrick J; Verdine, Gregory L; Mansour, Michael K; Scadden, David T

    2016-01-01

    Hematopoietic stem cell transplantation (HSCT) offers curative therapy for patients with hemoglobinopathies, congenital immunodeficiencies, and other conditions, possibly including AIDS. Autologous HSCT using genetically corrected cells would avoid the risk of graft-versus-host disease (GVHD), but the genotoxicity of conditioning remains a substantial barrier to the development of this approach. Here we report an internalizing immunotoxin targeting the hematopoietic-cell-restricted CD45 receptor that effectively conditions immunocompetent mice. A single dose of the immunotoxin, CD45–saporin (SAP), enabled efficient (>90%) engraftment of donor cells and full correction of a sickle-cell anemia model. In contrast to irradiation, CD45–SAP completely avoided neutropenia and anemia, spared bone marrow and thymic niches, enabling rapid recovery of T and B cells, preserved anti-fungal immunity, and had minimal overall toxicity. This non-genotoxic conditioning method may provide an attractive alternative to current conditioning regimens for HSCT in the treatment of non-malignant blood diseases. PMID:27272386

  17. Mycobacterium tuberculosis acquires iron by cell-surface sequestration and internalization of human holo-transferrin.

    PubMed

    Boradia, Vishant Mahendra; Malhotra, Himanshu; Thakkar, Janak Shrikant; Tillu, Vikas Ajit; Vuppala, Bhavana; Patil, Pravinkumar; Sheokand, Navdeep; Sharma, Prerna; Chauhan, Anoop Singh; Raje, Manoj; Raje, Chaaya Iyengar

    2014-08-28

    Mycobacterium tuberculosis (M.tb), which requires iron for survival, acquires this element by synthesizing iron-binding molecules known as siderophores and by recruiting a host iron-transport protein, transferrin, to the phagosome. The siderophores extract iron from transferrin and transport it into the bacterium. Here we describe an additional mechanism for iron acquisition, consisting of an M.tb protein that drives transport of human holo-transferrin into M.tb cells. The pathogenic strain M.tb H37Rv expresses several proteins that can bind human holo-transferrin. One of these proteins is the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Rv1436), which is present on the surface of M.tb and its relative Mycobacterium smegmatis. Overexpression of GAPDH results in increased transferrin binding to M.tb cells and iron uptake. Human transferrin is internalized across the mycobacterial cell wall in a GAPDH-dependent manner within infected macrophages.

  18. International round-robin inter-comparison of dye-sensitized and crystalline silicon solar cells

    NASA Astrophysics Data System (ADS)

    Chen, Chia-Yuan; Ahn, Seung Kyu; Aoki, Dasiuke; Kokubo, Junichi; Yoon, Kyung Hoon; Saito, Hidenori; Lee, Kyung Sik; Magaino, Shinichi; Takagi, Katsuhiko; Lin, Ling-Chuan; Lee, Kun-Mu; Wu, Chun-Guey; Zhou, Hong; Igari, Sanekazu

    2017-02-01

    An international round-robin inter-comparison of the spectral responsivity (SR) and current-voltage (I-V) characteristics for dye-sensitized solar cells (DSCs) and crystalline silicon solar cells is reported for the first time. The crystalline silicon cells with various spectral responsivities were also calibrated by AIST to validate this round-robin activity. On the basis of the remarkable consistency in Pmax (within ±1.4% among participants) and Isc (within ±1.2% compared to the primary calibration of AIST) of the silicon specimens, the discrepancy in the SR and photovoltaic parameters of five DSCs among three national laboratories can be verified and diagnosed. Recommendations about sample packages, SR and I-V measurement methods as well as the inter-comparison protocol for improving the performance characterization of the mesoscopic DSCs are presented according to the consolidated data and the experience of the participants.

  19. Characteristics of functionalized nano-hydroxyapatite and internalization by human epithelial cell

    NASA Astrophysics Data System (ADS)

    Yan-Zhong, Zhao; Yan-Yan, Huang; Jun, Zhu; Shai-Hong, Zhu; Zhi-You, Li; Ke-Chao, Zhou

    2011-11-01

    Hydroxyapatite is the main inorganic component of biological bone and tooth enamel, and synthetic hydroxyapatite has been widely used as biomaterials. In this study, a facile method has been developed for the fabrication of arginine-functionalized and europium-doped hydroxyapatite nanoparticles (Arg-Eu-HAP). The synthesized nanoparticles characterized by transmission electron microscopy, X-ray diffractometry, Fourier transform infrared, and Zeta potential analyzer. Its biological properties with DNA binding, cell toxicity, cell binding and intracellular distribution were tested by agarose gel electrophoresis assay, flow cytometry, and fluorescence microscope and laser scanning confocal microscope. The synthesized Arg-Eu-HAP could effectively bind DNA without any cytotoxicity and be internalized into the cytoplasm and perinuclear of human lung epithelial cells.

  20. Clathrin-dependent internalization of the angiotensin II AT₁A receptor links receptor internalization to COX-2 protein expression in rat aortic vascular smooth muscle cells.

    PubMed

    Morinelli, Thomas A; Walker, Linda P; Velez, Juan Carlos Q; Ullian, Michael E

    2015-02-05

    The major effects of Angiotensin II (AngII) in vascular tissue are mediated by AngII AT1A receptor activation. Certain effects initiated by AT1A receptor activation require receptor internalization. In rat aortic vascular smooth muscle cells (RASMC), AngII stimulates cyclooxygenase 2 protein expression. We have previously shown this is mediated by β-arrestin-dependent receptor internalization and NF-κB activation. In this study, a specific inhibitor of clathrin-mediated endocytosis (CME), pitstop-2, was used to test the hypothesis that clathrin-dependent internalization of activated AT1A receptor mediates NF-κB activation and subsequent cyclooxygenase 2 expression. Radioligand binding assays, real time qt-PCR and immunoblotting were used to document the effects of pitstop-2 on AngII binding and signaling in RASMC. Laser scanning confocal microscopy (LSCM) was used to image pitstop-2׳s effects on AT1 receptor/GFP internalization in HEK-293 cells and p65 NF-κB nuclear localization in RASMC. Pitstop-2 significantly inhibited internalization of AT1A receptor (44.7% ± 3.1% Control vs. 13.2% ± 8.3% Pitstop-2; n=3) as determined by radioligand binding studies in RASMC. Studies utilizing AT1A receptor/GFP expressed in HEK 293 cells and LSCM confirmed these findings. Pitstop-2 significantly inhibited AngII-induced p65 NF-κB phosphorylation and nuclear localization, COX-2 message and protein expression in RASMC without altering activation of p42/44 ERK or TNFα signaling. Pitstop-2, a specific inhibitor of clathrin-mediated endocytosis, confirms that internalization of activated AT1A receptor mediates AngII activation of cyclooxygenase 2 expression in RASMC. These data provide support for additional intracellular signaling pathways activated through β-arrestin mediated internalization of G protein-coupled receptors, such as AT1A receptors.

  1. Surface modification of PLGA nanoparticles by carbopol to enhance mucoadhesion and cell internalization.

    PubMed

    Surassmo, Suvimol; Saengkrit, Nattika; Ruktanonchai, Uracha Rungsardthong; Suktham, Kunat; Woramongkolchai, Noppawan; Wutikhun, Tuksadon; Puttipipatkhachorn, Satit

    2015-06-01

    Mucoadhesive poly (lactic-co-glycolic acid) (PLGA) nanoparticles having a modified shell-matrix derived from polyvinyl alcohol (PVA) and Carbopol (CP), a biodegradable polymer coating, to improve the adhesion and cell transfection properties were developed. The optimum formulations utilized a CP concentration in the range of 0.05-0.2%w/v, and were formed using modified emulsion-solvent evaporation technique. The resulting CP-PLGA nanoparticles were characterized in terms of their physical and chemical properties. The absorbed CP on the PLGA shell-matrix was found to affect the particle size and surface charge, with 0.05% CP giving rise to smooth spherical particles (0.05CP-PLGA) with the smallest size (285.90 nm), and strong negative surface charge (-25.70 mV). The introduction of CP results in an enhancement of the mucoadhesion between CP-PLGA nanoparticles and mucin particles. In vitro cell internalization studies highlighted the potential of 0.05CP-PLGA nanoparticles for transfection into SiHa cells, with uptake being time dependent. Additionally, cytotoxicity studies of CP-PLGA nanoparticles against SiHa cancer cells indicated that low concentrations of the nanoparticles were non-toxic to cells (cell viability >80%). From the various formulations studied, 0.05CP-PLGA nanoparticles proved to be the optimum model carrier having the required mucoadhesive profile and could be an alternative therapeutic efficacy carrier for targeted mucosal drug delivery systems with biodegradable polymer.

  2. From waste to energy: First experimental bacterial fuel cells onboard the international space station

    NASA Astrophysics Data System (ADS)

    de Vet, S. J.; Rutgers, R.

    2007-09-01

    Bacterial Fuel Cells are innovative energy systems that use bacteria to transform carbohydrates anaerobically into free electrons and waste products. The bacteria deposit the electrons on the anode and hence create a potential difference between the anode and the cathode, yielding a `bacterial battery'. This principle may be favourably influenced by enhanced bacterial productivity or bacterial growth in microgravity conditions, as is shown before in several other studies on bacteria in microgravity. Nonetheless, bacterial fuel cells have not been tested in space before. Currently foreseen applications are very promising for space flight and include waste disposal in manned space vehicles. This study describes a `space-first'test of bacterial fuel cells onboard the International Space Station using the Rhodoferax ferrireducens strain. We test if it is possible to use a bacterial fuel cell in 1g and under both simulated (RPM) and real microgravity conditions. Due to differences in magnitude of the output the data had to be normalized and cumulatively plotted. In all, it can be concluded that bacterial fuel cells show similar phases in the output under different gravitational conditions. Hence it can be concluded from a biological point of view that bacterial fuel cells do operate in space.

  3. Internal quantum efficiency improvement in polysilicon solar cells with porous silicon layer on the rear side

    NASA Astrophysics Data System (ADS)

    Trabelsi, Abdessalem; Zouari, Abdelaziz

    2016-01-01

    The present paper reports on a simulation study carried out to determine and optimize the effect of porous silicon (PS) layer at the rear side on the performance of thin polysilicon solar cells. It analytically solved the complete set of equations necessary to determine the contribution that this material has with regard to the internal quantum efficiency (IQE) of the cell when acting as a backside reflector. The contribution of the different regions of the cell, the increase in IQE, and the effects of high porosity and number of PS layers were derived and compared to conventional BSF solar cells. The findings revealed that the IQE of the solar cell with a PS layer at the backside was higher than that of conventional BSF, particularly in terms of medium and long wavelength range λ > 0.5 μm. This improvement was more significant with thin cells, large grain widths, and well-passivated grain boundaries. Furthermore, while the use of the PS layer had a significant effect on the contribution of the base, it exerted no effect on the contribution of the emitter and depletion regions. Overall, the maximum level of IQE improvement was recorded with three double-porosity structures in the PS layer, reaching a high porosity value of about 80 %.

  4. Clinicopathologic Characteristics of Angioimmunoblastic T-Cell Lymphoma: Analysis of the International Peripheral T-Cell Lymphoma Project

    PubMed Central

    Federico, Massimo; Rudiger, Thomas; Bellei, Monica; Nathwani, Bharat N.; Luminari, Stefano; Coiffier, Bertrand; Harris, Nancy L.; Jaffe, Elaine S.; Pileri, Stefano A.; Savage, Kerry J.; Weisenburger, Dennis D.; Armitage, James O.; Mounier, Nicholas; Vose, Julie M.

    2013-01-01

    Purpose The International Peripheral T-Cell Lymphoma Project was undertaken to better understand the subtypes of T-cell and natural killer (NK) –cell lymphomas. Patients and Methods Angioimmunoblastic T-cell lymphoma (AITL) was diagnosed according to the 2001 WHO criteria by a central review process consisting of panels of expert hematopathologists. Clinical, pathologic, immunophenotyping, treatment, and survival data were correlated. Results Of 1,314 patients, 243 (18.5%) were diagnosed with AITL. At presentation, generalized lymphadenopathy was noted in 76% of patients, and 89% had stages III to IV disease. Skin rash was observed in 21% of patients. Hemolytic anemia and hypergammoglobulinemia occurred in 13% and 30% of patients, respectively. Five-year overall and failure-free survivals were 33% and 18%, respectively. At presentation, prognostic models were evaluated, including the standard International Prognostic Index, which comprised the following factors: age ≥ 60 years, stages III to IV disease, lactic dehydrogenase (LDH) > normal, extranodal sites (ENSs) > one, and performance status (PS) ≥ 2; the Prognostic Index for Peripheral T-Cell Lymphoma, comprising: age ≥ 60 years, PS ≥ 2, LDH > normal, and bone marrow involvement; and the alternative Prognostic Index for AITL (PIAI), comprising: age > 60 years, PS ≥ 2, ENSs > one, B symptoms, and platelet count < 150 × 109/L. The simplified PIAI had a low-risk group (zero to one factors), with 5-year survival of 44%, and a high-risk group (two to five factors), with 5-year survival of 24% (P = .0065). Conclusion AITL is a rare clinicopathologic entity characterized by an aggressive course and dismal outcome with current therapies. PMID:22869878

  5. Loss of proliferation and antigen presentation activity following internalization of polydispersed carbon nanotubes by primary lung epithelial cells.

    PubMed

    Kumari, Mandavi; Sachar, Sumedha; Saxena, Rajiv K

    2012-01-01

    Interactions between poly-dispersed acid functionalized single walled carbon nanotubes (AF-SWCNTs) and primary lung epithelial (PLE) cells were studied. Peritoneal macrophages (PMs, known phagocytic cells) were used as positive controls in this study. Recovery of live cells from cultures of PLE cells and PMs was significantly reduced in the presence of AF-SWCNTs, in a time and dose dependent manner. Both PLE cells as well as PMs could take up fluorescence tagged AF-SWCNTs in a time dependent manner and this uptake was significantly blocked by cytochalasin D, an agent that blocks the activity of acto-myosin fibers and therefore the phagocytic activity of cells. Confocal microscopic studies confirmed that AF-SWCNTs were internalized by both PLE cells and PMs. Intra-trachially instilled AF-SWCNTs could also be taken up by lung epithelial cells as well as alveolar macrophages. Freshly isolated PLE cells had significant cell division activity and cell cycling studies indicated that treatment with AF-SWCNTs resulted in a marked reduction in S-phase of the cell cycle. In a previously standardized system to study BCG antigen presentation by PLE cells and PMs to sensitized T helper cells, AF-SWCNTs could significantly lower the antigen presentation ability of both cell types. These results show that mouse primary lung epithelial cells can efficiently internalize AF-SWCNTs and the uptake of nanotubes interfered with biological functions of PLE cells including their ability to present BCG antigens to sensitized T helper cells.

  6. 3D CFD ELECTROCHEMICAL AND HEAT TRANSFER MODEL OF AN INTERNALLY MANIFOLDED SOLID OXIDE ELECTROLYSIS CELL

    SciTech Connect

    Grant L. Hawkes; James E. O'Brien; Greg Tao

    2011-11-01

    A three-dimensional computational fluid dynamics (CFD) electrochemical model has been created to model high-temperature electrolysis cell performance and steam electrolysis in an internally manifolded planar solid oxide electrolysis cell (SOEC) stack. This design is being evaluated at the Idaho National Laboratory for hydrogen production from nuclear power and process heat. Mass, momentum, energy, and species conservation and transport are provided via the core features of the commercial CFD code FLUENT. A solid-oxide fuel cell (SOFC) model adds the electrochemical reactions and loss mechanisms and computation of the electric field throughout the cell. The FLUENT SOFC user-defined subroutine was modified for this work to allow for operation in the SOEC mode. Model results provide detailed profiles of temperature, operating potential, steam-electrode gas composition, oxygen-electrode gas composition, current density and hydrogen production over a range of stack operating conditions. Single-cell and five-cell results will be presented. Flow distribution through both models is discussed. Flow enters from the bottom, distributes through the inlet plenum, flows across the cells, gathers in the outlet plenum and flows downward making an upside-down ''U'' shaped flow pattern. Flow and concentration variations exist downstream of the inlet holes. Predicted mean outlet hydrogen and steam concentrations vary linearly with current density, as expected. Effects of variations in operating temperature, gas flow rate, oxygen-electrode and steam-electrode current density, and contact resistance from the base case are presented. Contour plots of local electrolyte temperature, current density, and Nernst potential indicate the effects of heat transfer, reaction cooling/heating, and change in local gas composition. Results are discussed for using this design in the electrolysis mode. Discussion of thermal neutral voltage, enthalpy of reaction, hydrogen production, cell thermal

  7. Lipid rafts are required for GLUT4 internalization in adipose cells

    PubMed Central

    Ros-Baró, Anna; López-Iglesias, Carmen; Peiró, Sandra; Bellido, David; Palacín, Manuel; Zorzano, Antonio; Camps, Marta

    2001-01-01

    It has been recently reported that insulin recruits a novel signaling machinery to lipid rafts required for insulin-stimulated GLUT4 translocation [Baumann, A., Ribon, V., Kanzaki, M., Thurmond, D. C., Mora, S., Shigematsu, S., Bickel, P. E., Pessin, J. E. & Saltiel, A. R. (2001) Nature 407, 202–207, 2000; Chiang, S. H., Baumann, C. A., Kanzaki, M., Thurmond, D. C., Watson, R. T., Neudauer, C. L., Macara, I. G., Pessin, J. E. & Saltiel, A. R. (2001) Nature 410, 944–948]. We have assessed the role of lipid rafts on GLUT4 traffic in adipose cells. High GLUT4 levels were detected in caveolae from adipocytes by two approaches, the mechanical isolation of purified caveolae from plasma membrane lawns and the immunogold analysis of plasma membrane lawns followed by freeze-drying. The role of lipid rafts in GLUT4 trafficking was studied by adding nystatin or filipin at concentrations that specifically disrupt caveolae morphology and inhibit caveolae function without altering clathrin-mediated endocytosis. These caveolae inhibitors did not affect the insulin-stimulated glucose transport. However, they blocked both the GLUT4 internalization and the down-regulation of glucose transport triggered by insulin removal in 3T3-L1 adipocytes. Our data indicate that lipid rafts are crucial for GLUT4 internalization after insulin removal. Given that high levels of GLUT4 were detected in caveolae from insulin-treated adipose cells, this transporter may be internalized from caveolae or caveolae may operate as an obligatory transition station before internalization. PMID:11593015

  8. Performance evaluation of a proof-of-concept 70 W internal reforming methanol fuel cell system

    NASA Astrophysics Data System (ADS)

    Avgouropoulos, G.; Schlicker, S.; Schelhaas, K.-P.; Papavasiliou, J.; Papadimitriou, K. D.; Theodorakopoulou, E.; Gourdoupi, N.; Machocki, A.; Ioannides, T.; Kallitsis, J. K.; Kolb, G.; Neophytides, S.

    2016-03-01

    A proof-of-concept 70 W Internal Reforming Methanol Fuel Cell (IRMFC) stack including Balance-of-Plant (BoP) was designed, assembled and tested. Advent TPS® high-temperature, polymer electrolyte membrane electrode assemblies were employed for fuel cell operation at 200 °C. In order to avoid phosphoric acid poisoning of the reformer, the anode electrocatalyst of each cell was indirectly adjoined, via a separation plate, to a highly active CuMnAlOx catalyst coated onto copper foam, which served as methanol reforming layer. The reformer was in-situ converting the methanol/steam feed to the required hydrogen (internal reforming concept) at 200 °C, which was readily oxidized at the anode electrodes. The operation of the IRMFC was supported through a number of BoP components consisting of a start-up subsystem (air blower, evaporator and monolithic burner), a combined afterburner/evaporator device, methanol/water supply and data acquisition units (reactants/products analysis, temperature control, flow control, system load/output control). Depending on the composition of the liquid MeOH/H2O feed streams, current densities up to 0.18 A cm-2 and power output up to 70 W could be obtained with remarkable repeatability. Specific targets for improvement of the efficiency were identified.

  9. The pluralization of the international: Resistance and alter-standardization in regenerative stem cell medicine

    PubMed Central

    Rosemann, Achim; Chaisinthop, Nattaka

    2016-01-01

    The article explores the formation of an international politics of resistance and ‘alter-standardization’ in regenerative stem cell medicine. The absence of internationally harmonized regulatory frameworks in the clinical stem cell field and the presence of lucrative business opportunities have resulted in the formation of transnational networks adopting alternative research standards and practices. These oppose, as a universal global standard, strict evidence-based medicine clinical research protocols as defined by scientists and regulatory agencies in highly developed countries. The emergence of transnational spaces of alter-standardization is closely linked to scientific advances in rapidly developing countries such as China and India, but calls for more flexible regulatory frameworks, and the legitimization of experimental for-profit applications outside of evidence-based medical care, are emerging increasingly also within more stringently regulated countries, such as the United States and countries in the European Union. We can observe, then, a trend toward the pluralization of the standards, practices, and concepts in the stem cell field. PMID:26983174

  10. The pluralization of the international: Resistance and alter-standardization in regenerative stem cell medicine.

    PubMed

    Rosemann, Achim; Chaisinthop, Nattaka

    2016-02-01

    The article explores the formation of an international politics of resistance and 'alterstandardization' in regenerative stem cell medicine. The absence of internationally harmonized regulatory frameworks in the clinical stem cell field and the presence of lucrative business opportunities have resulted in the formation of transnational networks adopting alternative research standards and practices. These oppose, as a universal global standard, strict evidence-based medicine clinical research protocols as defined by scientists and regulatory agencies in highly developed countries. The emergence of transnational spaces of alter-standardization is closely linked to scientific advances in rapidly developing countries such as China and India, but calls for more flexible regulatory frameworks, and the legitimization of experimental for-profit applications outside of evidence-based medical care, are emerging increasingly also within more stringently regulated countries, such as the United States and countries in the European Union. We can observe, then, a trend toward the pluralization of the standards, practices, and concepts in the stem cell field.

  11. Probiotic Microorganisms Inhibit Epithelial Cell Internalization of Botulinum Neurotoxin Serotype A

    PubMed Central

    Lam, Tina I.; Tam, Christina C.; Stanker, Larry H.; Cheng, Luisa W.

    2016-01-01

    Botulinum neurotoxins (BoNTs) are some of the most poisonous natural toxins known to man and are threats to public health and safety. Previous work from our laboratory showed that both BoNT serotype A complex and holotoxin can bind and transit through the intestinal epithelia to disseminate in the blood. The timing of BoNT/A toxin internalization was shown to be comparable in both the Caco-2 in vitro cell culture and in the oral mouse intoxication models. Probiotic microorganisms have been extensively studied for their beneficial effects in not only maintaining the normal gut mucosa but also protection from allergens, pathogens, and toxins. In this study, we evaluate whether probiotic microorganisms will block BoNT/A uptake in the in vitro cell culture system using Caco-2 cells. Several probiotics tested (Saccharomyces boulardii, Lactobacillus acidophilus, Lactobacillus rhamnosus LGG, and Lactobacillus reuteri) blocked BoNT/A uptake in a dose-dependent manner whereas a non-probiotic strain of Escherichia coli did not. We also showed that inhibition of BoNT/A uptake was not due to the degradation of BoNT/A nor by sequestration of toxin via binding to probiotics. These results show for the first time that probiotic treatment can inhibit BoNT/A binding and internalization in vitro and may lead to the development of new therapies. PMID:27999281

  12. Concepts in mature T-cell lymphomas - highlights from an international joint symposium on T-cell immunology and oncology().

    PubMed

    Herling, Marco; Rengstl, Benjamin; Scholtysik, René; Hartmann, Sylvia; Küppers, Ralf; Hansmann, Martin-Leo; Diebner, Hans H; Roeder, Ingo; Abken, Hinrich; Newrzela, Sebastian; Kirberg, Jörg

    2017-04-01

    Growing attention in mature T-cell lymphomas/leukemias (MTCL) is committed to more accurate and meaningful classifications, improved pathogenetic concepts and expanded therapeutic options. This requires considerations of the immunologic concepts of T-cell homeostasis and the specifics of T-cell receptor (TCR) affinities and signaling. Scientists from various disciplines established the CONTROL-T research unit and in an international conference on MTCL they brought together experts from T-cell immunity, oncology, immunotherapy and systems biology. We report here meeting highlights on the covered topics of diagnostic pitfalls, implications by the new WHO classification, insights from discovered genomic lesions as well as TCR-centric concepts of cellular dynamics in host defense, auto-immunity and tumorigenic clonal escape, including predictions to be derived from in vivo imaging and mathematical modeling. Presentations on novel treatment approaches were supplemented by strategies of optimizing T-cell immunotherapies. Work packages, that in joint efforts would advance the field of MTCL more efficiently, are identified.

  13. Composition and distribution of internal resistance in three types of microbial fuel cells.

    PubMed

    Liang, Peng; Huang, Xia; Fan, Ming-Zhi; Cao, Xiao-Xin; Wang, Cheng

    2007-12-01

    High internal resistance is a key problem limiting the power output of the microbial fuel cell (MFC). Therefore, more knowledge about the internal resistance is essential to enhance the performance of the MFC. However, different methods are used to determine the internal resistance, which makes the comparison difficult. In this study, three different types of MFCs were constructed to study the composition and distribution of internal resistance. The internal resistance (R(i)) is partitioned into anodic resistance (R(a)), cathodic resistance (R(c)), and ohmic resistance (R(Omega)) according to their origin and the design of the MFCs. These three resistances were then evaluated by the "current interrupt" method and the "steady discharging" method based on the proposed equivalent circuits for MFCs. In MFC-A, MFC-B, and MFC-C, the R(i) values were 3.17, 0.35, and 0.076 Omega m(2), the R(Omega) values were 2.65, 0.085, and 0.008 Omega m(2), the R(a) values were 0.055, 0.115, and 0.034 Omega m(2), and the R(c) values were 0.466, 0.15, and 0.033 Omega m(2), respectively. For MFC-B and MFC-C, the remarkable decrease in R(i) compared with the two-chamber MFC was mainly ascribed to the decline in R(Omega) and R(c). In MFC-C, the membrane electrodes' assembly lowered the ohmic resistance and facilitated the mass transport through the anode and cathode electrodes, resulting in the lowest R(i) among the three types.

  14. Results from an International Measurement Round Robin of III-V Triple Junction Solar Cells under Air Mass Zero

    NASA Technical Reports Server (NTRS)

    Jenkins, Phillip; Scheiman, Chris; Goodbody, Chris; Baur, Carsten; Sharps, Paul; Imaizumi, Mitsuru; Yoo, Henry; Sahlstrom, Ted; Walters, Robert; Lorentzen, Justin; Nocerino, John; Khan, Osman; Cravens, Robert; Valles, Juan; Toporow, Chantal; Gomez, Trinidad,; Bazan, Loreto Pazos; Bailey, Sheila

    2006-01-01

    This paper reports the results of an international measurement round robin of monolithic, triple-junction, GaInP/GaAs/Ge space solar cells. Eight laboratories representing national labs, solar cell vendors and space solar cell consumers, measured cells using in-house reference cells and compared those results to measurements made where each lab used the same set of reference cells. The results show that most of the discrepancy between laboratories is likely due to the quality of the standard cells rather than the measurement system or solar simulator used.

  15. Membrane with internal passages to permit fluid flow and an electrochemical cell containing the same

    NASA Technical Reports Server (NTRS)

    Cisar, Alan J. (Inventor); Gonzalez-Martin, Anuncia (Inventor); Hitchens, G. Duncan (Inventor); Murphy, Oliver J. (Inventor)

    1997-01-01

    The invention provides an improved proton exchange membrane for use in electrochemical cells having internal passages parallel to the membrane surface, an apparatus and process for making the membrane, membrane and electrode assemblies fabricated using the membrane, and the application of the membrane and electrode assemblies to a variety of devices, both electrochemical and otherwise. The passages in the membrane extend from one edge of the membrane to another and allow fluid flow through the membrane and give access directly to the membrane for purposes of hydration.

  16. Thin-film-based sensitivity enhancement for total internal reflection fluorescence live-cell imaging.

    PubMed

    Kim, Kyujung; Cho, Eun-Jin; Huh, Yong-Min; Kim, Donghyun

    2007-11-01

    We investigated experimentally the evanescent field enhancement based on dielectric thin films in total internal reflection microscopy. The sample employed two layers of Al2O3 and SiO2 deposited on an SF10 glass substrate. Field intensity enhancement measured by fluorescent excitation of microbeads relative to that of a control sample without dielectric films was polarization dependent, determined as 4.2 and 2.4 for TE and TM polarizations, respectively, and was in good agreement with numerical results. The thin-film-based field enhancement was also applied to live-cell imaging of quantum dots, which confirmed the sensitivity enhancement qualitatively.

  17. Internal Conductivity of Axons, Nerve Cell Bodies and Large Nonnervous Cells

    DTIC Science & Technology

    1975-03-01

    Thompson measured conductivity of packed frog muscle and determined that the in- ternal medium was as conductive as 0.13 to 0. 21 percent NaCl...solutions (resistivity 4. 3-6. 9 times that of frog Ringer). A more recent study on frog muscle using sim- ilar techniques reported...made to measure cytoplasmic conductivity of single i P, cells, beginning with Hartree and Hill who studied frog muscle fibers. They found

  18. International Society for the Advancement of Cytometry Cell Sorter Biosafety Standards

    PubMed Central

    Holmes, Kevin L.; Fontes, Benjamin; Hogarth, Philip; Konz, Richard; Monard, Simon; Pletcher, Charles H.; Wadley, Robert B.; Schmid, Ingrid; Perfetto, Stephen P.

    2014-01-01

    Flow cytometric cell sorting of biological specimens has become prevalent in basic and clinical research laboratories. These specimens may contain known or unknown infectious agents, necessitating precautions to protect instrument operators and the environment from biohazards arising from the use of sorters. To this end the International Society of Analytical Cytology (ISAC) was proactive in establishing biosafety guidelines in 1997 (Schmid et al., Cytometry 1997;28:99–117) and subsequently published revised biosafety standards for cell sorting of unfixed samples in 2007 (Schmid et al., Cytometry Part A J Int Soc Anal Cytol 2007;71A:414–437). Since their publication, these documents have become recognized worldwide as the standard of practice and safety precautions for laboratories performing cell sorting experiments. However, the field of cytometry has progressed since 2007, and the document requires an update. The new Standards provides guidance: (1) for laboratory design for cell sorter laboratories; (2) for the creation of laboratory or instrument specific Standard Operating Procedures (SOP); and (3) on procedures for the safe operation of cell sorters, including personal protective equipment (PPE) and validation of aerosol containment. PMID:24634405

  19. Characterization and modeling of radiation damages via internal radiative efficiency in multi-junction solar cells

    NASA Astrophysics Data System (ADS)

    Zhu, Lin; Yoshita, Masahiro; Nakamura, Tetsuya; Imaizumi, Mitsuru; Kim, Changsu; Mochizuki, Toshimitsu; Chen, Shaoqiang; Kanemitsu, Yoshihiko; Akiyama, Hidefumi

    2016-03-01

    In order to understand the radiation effects in space-used multi-junction solar cells, we characterized degradations of internal radiative efficiency (ηint i ) in respective subcells in InGaP/GaAs double-junction solar cells after 1-MeV electron irradiations with different electrons fluences (Φ) via absolute electroluminescence (EL) measurements, because ηint i purely represents material-quality change due to radiation damage, independently from cell structures. We analyzed the degradation of ηint i under different Φ and found that the data of ηint i versus Φ in moderate and high Φ regions are very similar and almost independent of subcell materials, while the difference in beginning-of-life qualities of InGaP and GaAs materials causes dominant difference in sub-cell sensitivity to the low radiation damages. Finally, a simple model was proposed to explain the mechanism in degradation of ηint i, and also well explained the degradation behavior in open-circuit voltage for these multi-junction solar cells.

  20. Proliferation of Listeria monocytogenes L-form cells by formation of internal and external vesicles

    PubMed Central

    Studer, Patrick; Staubli, Titu; Wieser, Noémi; Wolf, Patrick; Schuppler, Markus; Loessner, Martin J.

    2016-01-01

    L-forms are cell wall-deficient bacteria that divide through unusual mechanisms, involving dynamic perturbations of the cellular shape and generation of vesicles, independently of the cell-division protein FtsZ. Here we describe FtsZ-independent mechanisms, involving internal and external vesicles, by which Listeria monocytogenes L-forms proliferate. Using micromanipulation of single cells and vesicles, we show that small vesicles are formed by invagination within larger intracellular vesicles, receive cytoplasmic content, and represent viable progeny. In addition, the L-forms can reproduce by pearling, that is, generation of extracellular vesicles that remain transiently linked to their mother cell via elastic membranous tubes. Using photobleaching and fluorescence recovery, we demonstrate cytoplasmic continuity and transfer through these membranous tubes. Our findings indicate that L-forms' polyploidy and extended interconnectivity through membranous tubes contribute to the generation of viable progeny independently of dedicated division machinery, and further support L-forms as models for studies of potential multiplication mechanisms of hypothetical primitive cells. PMID:27876798

  1. N-glycosylated proteins are involved in efficient internalization of Klebsiella pneumoniae by cultured human epithelial cells.

    PubMed Central

    Fumagalli, O; Tall, B D; Schipper, C; Oelschlaeger, T A

    1997-01-01

    Klebsiella pneumoniae obtained from patients with urinary tract infections is able to invade cultured human epithelial cells. The internalization process is dependent upon both microfilaments and microtubules. To better understand the interaction of these invasive bacteria with the host cell receptor(s), bladder, lung, and ileocecal epithelial cells were infected with K. pneumoniae in the presence of various lectins possessing multiple glycan specificities. It was found that the N-acetylglucosamine (GlcNAc)-specific lectins concanavalin A, Datura stramonium agglutinin, and wheat germ agglutinin significantly inhibited the invasion of K. pneumoniae into these cells but did not interfere with the internalization of an invasive strain of Salmonella typhimurium. Conversely, internalization of K. pneumoniae but not S. typhimurium was also significantly inhibited when the bacteria were pretreated with GlcNAc or chitin hydrolysate, a GlcNAc polymer, prior to the gentamicin invasion assay. Other carbohydrates such as glucose, galactose, mannose, fucose, and N-acetylneuraminic acid had no inhibitory effects on K. pneumoniae uptake. Furthermore, internalization of K. pneumoniae but not S. typhimurium by HCT8 cells was also significantly inhibited when eukaryotic protein glycosylation was interrupted by tunicamycin or when host N-linked surface glycans were removed by pretreatment with N-glycosidase F. These studies suggest that a N-glycosylated protein receptor is involved in the internalization of K. pneumoniae by human epithelial cells in vitro. The results also indicate that internal GlcNAc residues might be a carbohydrate component of the receptor. PMID:9353018

  2. Propranolol restricts the mobility of single EGF-receptors on the cell surface before their internalization.

    PubMed

    Otero, Carolina; Linke, Max; Sanchez, Paula; González, Alfonso; Schaap, Iwan A T

    2013-01-01

    The epidermal growth factor receptor is involved in morphogenesis, proliferation and cell migration. Its up-regulation during tumorigenesis makes this receptor an interesting therapeutic target. In the absence of the ligand, the inhibition of phosphatidic acid phosphohydrolase activity by propranolol treatment leads to internalization of empty/inactive receptors. The molecular events involved in this endocytosis remain unknown. Here, we quantified the effects of propranolol on the mobility of single quantum-dot labelled receptors before the actual internalization took place. The single receptors showed a clear stop-and-go motion; their diffusive tracks were continuously interrupted by sub-second stalling events, presumably caused by transient clustering. In the presence of propranolol we found that: i) the diffusion rate reduced by 22 %, which indicates an increase in drag of the receptor. Atomic force microscopy measurements did not show an increase of the effective membrane tension, such that clustering of the receptor remains the likely mechanism for its reduced mobility. ii) The receptor got frequently stalled for longer periods of multiple seconds, which may signal the first step of the internalization process.

  3. Propranolol Restricts the Mobility of Single EGF-Receptors on the Cell Surface before Their Internalization

    PubMed Central

    Otero, Carolina; Linke, Max; Sanchez, Paula; González, Alfonso; Schaap, Iwan A. T.

    2013-01-01

    The epidermal growth factor receptor is involved in morphogenesis, proliferation and cell migration. Its up-regulation during tumorigenesis makes this receptor an interesting therapeutic target. In the absence of the ligand, the inhibition of phosphatidic acid phosphohydrolase activity by propranolol treatment leads to internalization of empty/inactive receptors. The molecular events involved in this endocytosis remain unknown. Here, we quantified the effects of propranolol on the mobility of single quantum-dot labelled receptors before the actual internalization took place. The single receptors showed a clear stop-and-go motion; their diffusive tracks were continuously interrupted by sub-second stalling events, presumably caused by transient clustering. In the presence of propranolol we found that: i) the diffusion rate reduced by 22 %, which indicates an increase in drag of the receptor. Atomic force microscopy measurements did not show an increase of the effective membrane tension, such that clustering of the receptor remains the likely mechanism for its reduced mobility. ii) The receptor got frequently stalled for longer periods of multiple seconds, which may signal the first step of the internalization process. PMID:24349439

  4. Actin dynamics at the living cell submembrane imaged by total internal reflection fluorescence photobleaching.

    PubMed Central

    Sund, S E; Axelrod, D

    2000-01-01

    Although reversible chemistry is crucial to dynamical processes in living cells, relatively little is known about relevant chemical kinetic rates in vivo. Total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP), an established technique previously demonstrated to measure reversible biomolecular kinetic rates at surfaces in vitro, is extended here to measure reversible biomolecular kinetic rates of actin at the cytofacial (subplasma membrane) surface of living cells. For the first time, spatial imaging (with a charge-coupled device camera) is used in conjunction with TIR/FRAP. TIR/FRAP imaging produces both spatial maps of kinetic parameters (off-rates and mobile fractions) and estimates of kinetic correlation distances, cell-wide kinetic gradients, and dependences of kinetic parameters on initial fluorescence intensity. For microinjected rhodamine actin in living cultured smooth muscle (BC3H1) cells, the unbinding rate at or near the cytofacial surface of the plasma membrane (averaged over the entire cell) is measured at 0.032 +/- 0.007 s(-1). The corresponding rate for actin marked by microinjected rhodamine phalloidin is very similar, 0.033 +/- 0.013 s(-1), suggesting that TIR/FRAP is reporting the dynamics of entire filaments or protofilaments. For submembrane fluorescence-marked actin, the intensity, off-rate, and mobile fraction show a positive correlation over a characteristic distance of 1-3 microm and a negative correlation over larger distances greater than approximately 7-14 microm. Furthermore, the kinetic parameters display a statistically significant cell-wide gradient, with the cell having a "fast" and "slow" end with respect to actin kinetics. PMID:10969025

  5. Identification of human somatostatin receptor 2 domains involved in internalization and signaling in QGP-1 pancreatic neuroendocrine tumor cell line.

    PubMed

    Cambiaghi, Valeria; Vitali, Eleonora; Morone, Diego; Peverelli, Erika; Spada, Anna; Mantovani, Giovanna; Lania, Andrea Gerardo

    2016-07-12

    Somatostatin exerts inhibitory effects on hormone secretion and cell proliferation via five receptor subtypes (SST1-SST5), whose internalization is regulated by β-arrestins. The receptor domains involved in these effects have been only partially elucidated. The aim of the study is to characterize the molecular mechanism and determinants responsible for somatostatin receptor 2 internalization and signaling in pancreatic neuroendocrine QGP-1 cell line, focusing on the third intracellular loop and carboxyl terminal domains. We demonstrated that in cells transfected with somatostatin receptor 2 third intracellular loop mutant, no differences in β-arrestins recruitment and receptor internalization were observed after somatostatin receptor 2 activation in comparison with cells bearing wild-type somatostatin receptor 2. Conversely, the truncated somatostatin receptor 2 failed to recruit β-arrestins and to internalize after somatostatin receptor 2 agonist (BIM23120) incubation. Moreover, the inhibitory effect of BIM23120 on cell proliferation, cyclin D1 expression, P-ERK1/2 levels, apoptosis and vascular endothelial growth factor secretion was completely lost in cells transfected with either third intracellular loop or carboxyl terminal mutants. In conclusion, we demonstrated that somatostatin receptor 2 internalization requires intact carboxyl terminal while the effects of SS on cell proliferation, angiogenesis and apoptosis mediated by somatostatin receptor 2 need the integrity of both third intracellular loop and carboxyl terminal.

  6. The importance of cellular internalization of antibody-targeted carbon nanotubes in the photothermal ablation of breast cancer cells

    NASA Astrophysics Data System (ADS)

    Marches, Radu; Mikoryak, Carole; Wang, Ru-Hung; Pantano, Paul; Draper, Rockford K.; Vitetta, Ellen S.

    2011-03-01

    Single-walled carbon nanotubes (CNTs) convert absorbed near infrared (NIR) light into heat. The use of CNTs in the NIR-mediated photothermal ablation of tumor cells is attractive because the penetration of NIR light through normal tissues is optimal and the side effects are minimal. Targeted thermal ablation with minimal collateral damage can be achieved by using CNTs attached to tumor-specific monoclonal antibodies (MAbs). However, the role that the cellular internalization of CNTs plays in the subsequent sensitivity of the target cells to NIR-mediated photothermal ablation remains undefined. To address this issue, we used CNTs covalently coupled to an anti-Her2 or a control MAb and tested their ability to bind, internalize, and photothermally ablate Her2 + but not Her2 - breast cancer cell lines. Using flow cytometry, immunofluorescence, and confocal Raman microscopy, we observed the gradual time-dependent receptor-mediated endocytosis of anti-Her2-CNTs whereas a control MAb-CNT conjugate did not bind to the cells. Most importantly, the Her2 + cells that internalized the MAb-CNTs were more sensitive to NIR-mediated photothermal damage than cells that could bind to, but not internalize the MAb-CNTs. These results suggest that both the targeting and internalization of MAb-CNTs might result in the most effective thermal ablation of tumor cells following their exposure to NIR light.

  7. Endothelial Cells Use a Formin-Dependent Phagocytosis-Like Process to Internalize the Bacterium Listeria monocytogenes

    PubMed Central

    Rengarajan, Michelle; Hayer, Arnold; Theriot, Julie A.

    2016-01-01

    Vascular endothelial cells act as gatekeepers that protect underlying tissue from blood-borne toxins and pathogens. Nevertheless, endothelial cells are able to internalize large fibrin clots and apoptotic debris from the bloodstream, although the precise mechanism of such phagocytosis-like uptake is unknown. We show that cultured primary human endothelial cells (HUVEC) internalize both pathogenic and non-pathogenic Listeria bacteria comparably, in a phagocytosis-like process. In contrast with previously studied host cell types, including intestinal epithelial cells and hepatocytes, we find that endothelial internalization of Listeria is independent of all known pathogenic bacterial surface proteins. Consequently, we exploited the internalization and intracellular replication of L. monocytogenes to identify distinct host cell factors that regulate phagocytosis-like uptake in HUVEC. Using siRNA screening and subsequent genetic and pharmacologic perturbations, we determined that endothelial infectivity was modulated by cytoskeletal proteins that normally modulate global architectural changes, including phosphoinositide-3-kinase, focal adhesions, and the small GTPase Rho. We found that Rho kinase (ROCK) is acutely necessary for adhesion of Listeria to endothelial cells, whereas the actin-nucleating formins FHOD1 and FMNL3 specifically regulate internalization of bacteria as well as inert beads, demonstrating that formins regulate endothelial phagocytosis-like uptake independent of the specific cargo. Finally, we found that neither ROCK nor formins were required for macrophage phagocytosis of L. monocytogenes, suggesting that endothelial cells have distinct requirements for bacterial internalization from those of classical professional phagocytes. Our results identify a novel pathway for L. monocytogenes uptake by human host cells, indicating that this wily pathogen can invade a variety of tissues by using a surprisingly diverse suite of distinct uptake mechanisms that

  8. Relationship between interstitial cells of Cajal, fibroblast-like cells and inhibitory motor nerves in the internal anal sphincter.

    PubMed

    Cobine, Caroline A; Hennig, Grant W; Kurahashi, Masaaki; Sanders, Kenton M; Ward, Sean M; Keef, Kathleen D

    2011-04-01

    Interstitial cells of Cajal (ICC) have been shown to participate in nitrergic neurotransmission in various regions of the gastrointestinal (GI) tract. Recently, fibroblast-like cells, which are positive for platelet-derived growth factor receptor α (PDGFRα(+)), have been suggested to participate additionally in inhibitory neurotransmission in the GI tract. The distribution of ICC and PDGFRα(+) cell populations and their relationship to inhibitory nerves within the mouse internal anal sphincter (IAS) are unknown. Immunohistochemical techniques and confocal microscopy were therefore used to examine the density and arrangement of ICC, PDGFRα(+) cells and neuronal nitric-oxide-synthase-positive (nNOS(+)) nerve fibers in the IAS of wild-type (WT) and W/W ( v ) mice. Of the total tissue volume within the IAS circular muscle layer, 18% consisted in highly branched PDGFRα(+) cells (PDGFRα(+)-IM). Other populations of PDGFRα(+) cells were observed within the submucosa and along the serosal and myenteric surfaces. Spindle-shaped intramuscular ICC (ICC-IM) were present in the WT mouse IAS but were largely absent from the W/W ( v ) IAS. The ICC-IM volume (5% of tissue volume) in the WT mouse IAS was significantly smaller than that of PDGFRα(+)-IM. Stellate-shaped submucosal ICC (ICC-SM) were observed in the WT and W/W ( v ) IAS. Minimum surface distance analysis revealed that nNOS(+) nerve fibers were closely aligned with both ICC-IM and PDGFRα(+)-IM. An even closer association was seen between ICC-IM and PDGFRα(+)-IM. Thus, a close morphological arrangement exists between inhibitory motor neurons, ICC-IM and PDGFRα(+)-IM suggesting that some functional interaction occurs between them contributing to inhibitory neurotransmission in the IAS.

  9. Internal carotid artery stenosis associated with giant cell arteritis: case report and discussion

    PubMed Central

    Zarar, Amna; Zafar, Taqi T; Khan, Asif A; Suri, M Fareed K; Qureshi, Adnan I

    2014-01-01

    Background Cerebrovascular ischemic events associated with giant cell arteritis (GCA) are uncommon and have been reported in 3%–4% of patients. We describe a case report of GCA associated with intracranial stenosis and review various angiographic findings. Case presentation A 66-year-old man presented with worsening headache and vision loss. A recent magnetic resonance angiogram of the head and neck showed multiple intracranial stenosis. Cerebrospinal fluid (CSF) analysis demonstrated increased protein of 135.6 mg/dL, with two white blood cells/µL. No bacteria were observed in the CSF on gram staining, and cultures were negative for bacterial growth. Erythrocyte sedimentation rate was noted to be 14 mm/h, and C-reactive protein was 1.514 mg/L at admission. Human immunodeficiency virus (HIV) and hepatitis panels were negative. On digital subtraction angiography, patient had predominantly narrowing and irregularities in petrous and cavernous segments of the internal carotid arteries bilaterally. The diagnosis of GCA was confirmed by temporal artery biopsy. He was treated with steroids, and a followup angiogram 6 weeks later showed minimal resolution of the angiographic findings. Patient reported complete resolution of headaches and visual loss. Conclusion Bilateral internal carotid arteries stenosis may be seen in patients presenting with typical symptoms of GCA and may persist after steroid treatment despite resolution of clinical symptoms. PMID:25566338

  10. Effects of internal and external environment on health and well-being: from cell to society.

    PubMed

    Tomljenović, Andrea

    2014-03-01

    Stem cell fate in cell culture depends on the composition of the culturing media. Every single cell in an organism is influenced by its microenvironment and surrounding cells. Biology, psychology, emotions, spirit, energy, lifestyle, culture, economic and political influences, social interactions in family, work, living area and the possibilities to expresses oneself and live full life with a sense of well-being have influence on people appearances. Disease is as much social as biological. It is a reaction of an organism to unbalancing changes in the internal environment caused by the changes in the external environment and/or by the structural and functional failures or unfortunate legacies. Health gradient in the society depends on the every day circumstances in which people live and work. The health of the population is an insight into the society. The problem facing medicine in the complex society of today cannot be resolved without the aid of social sciences, as cultural, social, ecological and mental processes affect physiological responses and health outcomes. Anthropology could be a bridge between biomedicine and social sciences and influence strategies in public health to prevent rather than cure and in education for fulfillment in life and improvement of society.

  11. Internal electrolyte supply system for reliable transport throughout fuel cell stacks

    DOEpatents

    Wright, Maynard K.; Downs, Robert E.; King, Robert B.

    1988-01-01

    An improved internal electrolyte supply system in a fuel cell stack employs a variety of arrangements of grooves and passages in bipolar plates of the multiplicity of repeating fuel cells to route gravity-assisted flowing electrolyte throughout the stack. The grooves route electrolyte flow along series of first paths which extend horizontally through the cells between the plates thereof. The passages route electrolyte flow along series of second paths which extend vertically through the stack so as to supply electrolyte to the first paths in order to expose the electrolyte to the matrices of the cells. Five different embodiments of the supply system are disclosed. Some embodiments employ wicks in the grooves for facilitating transfer of the electrolyte to the matrices as well as providing support for the matrices. Additionally, the passages of some embodiments by-pass certain of the grooves and supply electrolyte directly to other of the grooves. Some embodiments employ single grooves and others have dual grooves. Finally, in some embodiments the passages are connected to the grooves by a step which produces a cascading electrolyte flow.

  12. [Recent advancements in the treatment of renal cell carcinoma--focus on international guidelines].

    PubMed

    Biró, Krisztina; Küronya, Zsófia

    2010-12-01

    Recent advances in understanding the fundamental biology underlying clear-cell RCC have opened the door to a series of targeted agents, such as tyrosine kinase inhibitors (TKIs) or mTOR inhibitors. These new agents have become the standard of care in managing advanced clear-cell RCC. Choice of initial medical management in patients with metastatic clear-cell RCC should be guided by randomised studies. On the evidence available, the first-line therapy in patients with good- or intermediate-risk mRCC should be either sunitinib or pazopanib, or bevacizumab plus interferon. In selected patients sorafenib is an option, as is high-dose interleukin-2 if performance status is good. In patients with poor prognosis, temsirolimus is recommended. In cytokine refractory patients, sorafenib, when patients have progressed on a tyrosine kinase inhibitor everolimus is the agent of choice. Biró K, Küronya Z. Recent advancements in the treatment of renal cell carcinoma - focus on international guidelines.

  13. Osmotic properties of internally perfused barnacle muscle cells. I. Isosmotic conditions.

    PubMed

    Bitner, J B; Peña-Rasgado, C; Ruiz, J; Cardona, J; Rasgado-Flores, H

    2001-07-01

    Barnacle muscle cells regulate their volume when exposed to anisotonic conditions. Due to their large size, these cells can be internally perfused. Interestingly, perfused cells maintain their volume regulatory properties (17,21). Thus, the osmotic properties of barnacle muscle cells can be studied under conditions in which the intracellular and extracellular osmolalities, the membrane potential (V(M)), the cell volume and the intracellular pressure can all be measured simultaneously. In this manuscript we report the effect that various rates of isosmotic (1000 mOsm x kg H2O(-1)) intracellular perfusion have on cell volume, intracellular pressure, intracellular osmolality, V(M), and the apparent sarcolemmal hydraulic water permeability (L'p). Replacement of the cytosol with the perfusate at a perfusion rate of 0.83 microl x min(-1) took 120 min. During this transition period, the cell volume increased from 45.1+/-6.9 microl to 73.7+/-5.8 microl, the intracellular osmolality decreased from 1406+/-133 to 1188+/-64 mOsm x kg H2O(-1), and the intracellular pressure underwent a transient drop of 2.8 cm H2O. After 2.5 hr of continuous perfusion at 0.83 microl min(-1), the above mentioned parameters reached steady values: the L'p was 1.35 x 10(-5) cm x sec(-1) x Osm(-1) x kg H2O(-1); cell volume was 67.2+/-6 microl; the intracellular osmolality was 1052+/-10 mOsm x kg H2O(-1); the intracellular pressure was 5.6+/-0.4 cm H2O; V(M) depolarized slowly at a rate of 0.03 mV x min(-1). Stepwise increases in the rate of perfusion (from 0.83 to 3.18 microl min(-1)) produced reversible increases in the intracellular pressure, L'p and cell volume and decreases in intracellular osmolality. We conclude that intracellular perfusion: i/ produces a transient removal of intracellular osmotically active components; ii/ promotes sarcolemmal water filtration; iii/ induces a laminar flow of perfusate at the center of the cell, and iv/ enables calculations of sarcolemmal L'p values under

  14. Fluorescence intensity, lifetime, and anisotropy screening of living cells based on total internal reflection techniques

    NASA Astrophysics Data System (ADS)

    Bruns, Thomas; Angres, Brigitte; Steuer, Heiko; Strauss, Wolfgang S. L.; Schneckenburger, Herbert

    2009-02-01

    A setup for fluorescence measurements of surfaces of biological samples, in particular the plasma membrane of living cells, is described. The method is based on splitting of a laser beam and multiple total internal reflections (TIR) within the bottom of a microtiter plate, such that up to 96 individual samples are illuminated simultaneously by an evanescent electromagnetic field. Two different screening procedures for the detection of fluorescence arising from the plasma membrane of living cells by High Throughput Screening (HTS) and High Content Screening (HCS), are distinguished. In the first case a rapid measurement of large sample numbers based on fluorescence intensity, and in the second case a high content of information from a single sample based on the parameters fluorescence lifetime (Fluorescence Lifetime Screening, FLiS) and fluorescence anisotropy (Fluorescence Lifetime Polarization Screening, FLiPS) is achieved. Both screening systems were validated using cultivated cells incubated with different fluorescent markers (e. g. NBD-cholesterol) as well as stably transfected cells expressing a fluorescent membrane-associating protein. In addition, particularly with regard of potential pharmaceutical applications, the kinetics of the intracellular translocation of a fluorescent protein kinase c fusion protein upon stimulation of the cells was determined. Further, a caspase sensor based on Förster Resonance Energy Transfer (FRET) between fluorescent proteins was tested. Enhanced cyan fluorescent protein (ECFP) anchored to the inner leaflet of the plasma membrane of living cells transfers its excitation energy via a spacer (DEVD) to an enhanced yellow fluorescent protein (EYFP). Upon apoptosis DEVD is cleaved, and energy transfer is disrupted, as proven by changes in fluorescence intensity and decay times.

  15. Photovoltaic Engineering Testbed: A Facility for Space Calibration and Measurement of Solar Cells on the International Space Station

    NASA Technical Reports Server (NTRS)

    Landis, Geoffrey A.; Bailey, Sheila G.; Jenkins, Phillip; Sexton, J. Andrew; Scheiman, David; Christie, Robert; Charpie, James; Gerber, Scott S.; Johnson, D. Bruce

    2001-01-01

    The Photovoltaic Engineering Testbed ("PET") is a facility to be flown on the International Space Station to perform calibration, measurement, and qualification of solar cells in the space environment and then returning the cells to Earth for laboratory use. PET will allow rapid turnaround testing of new photovoltaic technology under AM0 conditions.

  16. Internalization of red blood cell-mimicking hydrogel capsules with pH-triggered shape responses.

    PubMed

    Kozlovskaya, Veronika; Alexander, Jenolyn F; Wang, Yun; Kuncewicz, Thomas; Liu, Xuewu; Godin, Biana; Kharlampieva, Eugenia

    2014-06-24

    We report on naturally inspired hydrogel capsules with pH-induced transitions from discoids to oblate ellipsoids and their interactions with cells. We integrate characteristics of erythrocytes such as discoidal shape, hollow structure, and elasticity with reversible pH-responsiveness of poly(methacrylic acid) (PMAA) to design a new type of drug delivery carrier to be potentially triggered by chemical stimuli in the tumor lesion. The capsules are fabricated from cross-linked PMAA multilayers using sacrificial discoid silicon templates. The degree of capsule shape transition is controlled by the pH-tuned volume change, which in turn is regulated by the capsule wall composition. The (PMAA)15 capsules undergo a dramatic 24-fold volume change, while a moderate 2.3-fold volume variation is observed for more rigid PMAA-(poly(N-vinylpyrrolidone) (PMAA-PVPON)5 capsules when solution pH is varied between 7.4 and 4. Despite that both types of capsules exhibit discoid-to-oblate ellipsoid transitions, a 3-fold greater swelling in radial dimensions is found for one-component systems due to a greater degree of the circular face bulging. We also show that (PMAA-PVPON)5 discoidal capsules interact differently with J774A.1 macrophages, HMVEC endothelial cells, and 4T1 breast cancer cells. The discoidal capsules show 60% lower internalization as compared to spherical capsules. Finally, hydrogel capsules demonstrate a 2-fold decrease in size upon internalization. These capsules represent a unique example of elastic hydrogel discoids capable of pH-induced drastic and reversible variations in aspect ratios. Considering the RBC-mimicking shape, their dimensions, and their capability to undergo pH-triggered intracellular responses, the hydrogel capsules demonstrate considerable potential as novel carriers in shape-regulated transport and cellular uptake.

  17. Short-term effects of ultrahigh concentration cationic silica nanoparticles on cell internalization, cytotoxicity, and cell integrity with human breast cancer cell line (MCF-7)

    NASA Astrophysics Data System (ADS)

    Seog, Ji Hyun; Kong, Bokyung; Kim, Dongheun; Graham, Lauren M.; Choi, Joon Sig; Lee, Sang Bok

    2015-01-01

    High concentrations of cationic colloidal silica nanoparticles (CCS-NPs) have been widely used for the enrichment of plasma membrane proteins. However, the interaction between the CCS-NPs and cells under the required concentration for the isolation of plasma membrane are rarely investigated. We evaluated the internalization and toxicity of the 15 nm CCS-NPs which were exposed at high concentrations with short time in human breast cancer cells (MCF-7) with transmission electron microscopy, energy dispersive X-ray spectroscopy, inductively coupled plasma atomic emission spectroscopy, and colorimetric assays. The NPs were observed throughout the cells, particularly in the cytoplasm and the nucleus, after short incubation periods. Additionally, the NPs significantly influenced the membrane integrity of the MCF-7 cells.

  18. A confocal microscopy image analysis method to measure adhesion and internalization of Pseudomonas aeruginosa multicellular structures into epithelial cells.

    PubMed

    Lepanto, Paola; Lecumberry, Federico; Rossello, Jéssica; Kierbel, Arlinet

    2013-10-24

    Formation of multicellular structures such as biofilms is an important feature in the physiopathology of many disease-causing bacteria. We recently reported that Pseudomonas aeruginosa adheres to epithelial cells rapidly forming early biofilm-like aggregates, which can then be internalized into cells. Conventional methods to measure adhesion/internalization, such as dilution plating for total cell-associated or antibiotic protected bacteria, do not distinguish between single and aggregated bacteria. We report a procedure that combining double bacteria labeling, confocal microscopy and image analysis allows identification and quantification of the number of adhered and internalized bacteria distinguishing between single and aggregated bacterial cells. A plugin for Fiji to automatically perform these procedures has been generated.

  19. A confocal microscopy image analysis method to measure adhesion and internalization of Pseudomonas aeruginosa multicellular structures into epithelial cells.

    PubMed

    Lepanto, Paola; Lecumberry, Federico; Rossello, Jéssica; Kierbel, Arlinet

    2014-02-01

    Formation of multicellular structures such as biofilms is an important feature in the physiopathology of many disease-causing bacteria. We recently reported that Pseudomonas aeruginosa adheres to epithelial cells rapidly forming early biofilm-like aggregates, which can then be internalized into cells. Conventional methods to measure adhesion/internalization, such as dilution plating for total cell-associated or antibiotic protected bacteria, do not distinguish between single and aggregated bacteria. We report a procedure that combining double bacteria labeling, confocal microscopy and image analysis allows identification and quantification of the number of adhered and internalized bacteria distinguishing between single and aggregated bacterial cells. A plugin for Fiji to automatically perform these procedures has been generated.

  20. Using Total Internal Reflection Fluorescence Microscopy To Visualize Rhodopsin-Containing Cells

    PubMed Central

    Keffer, J. L.; Sabanayagam, C. R.; Lee, M. E.; DeLong, E. F.; Hahn, M. W.

    2015-01-01

    Sunlight is captured and converted to chemical energy in illuminated environments. Although (bacterio)chlorophyll-based photosystems have been characterized in detail, retinal-based photosystems, rhodopsins, have only recently been identified as important mediators of light energy capture and conversion. Recent estimates suggest that up to 70% of cells in some environments harbor rhodopsins. However, because rhodopsin autofluorescence is low—comparable to that of carotenoids and significantly less than that of (bacterio)chlorophylls—these estimates are based on metagenomic sequence data, not direct observation. We report here the use of ultrasensitive total internal reflection fluorescence (TIRF) microscopy to distinguish between unpigmented, carotenoid-producing, and rhodopsin-expressing bacteria. Escherichia coli cells were engineered to produce lycopene, β-carotene, or retinal. A gene encoding an uncharacterized rhodopsin, actinorhodopsin, was cloned into retinal-producing E. coli. The production of correctly folded and membrane-incorporated actinorhodopsin was confirmed via development of pink color in E. coli and SDS-PAGE. Cells expressing carotenoids or actinorhodopsin were imaged by TIRF microscopy. The 561-nm excitation laser specifically illuminated rhodopsin-containing cells, allowing them to be differentiated from unpigmented and carotenoid-containing cells. Furthermore, water samples collected from the Delaware River were shown by PCR to have rhodopsin-containing organisms and were examined by TIRF microscopy. Individual microorganisms that fluoresced under illumination from the 561-nm laser were identified. These results verify the sensitivity of the TIRF microscopy method for visualizing and distinguishing between different molecules with low autofluorescence, making it useful for analyzing natural samples. PMID:25769822

  1. Staphylococcal enterotoxin type A internal deletion mutants: serological activity and induction of T-cell proliferation.

    PubMed Central

    Harris, T O; Hufnagle, W O; Betley, M J

    1993-01-01

    Previous findings indicate that the N-terminal region of staphylococcal enterotoxin type A (SEA) is required for its ability to induce T-cell proliferation. To better localize internal peptides of SEA that are important for induction of murine T-cell proliferation, SEA mutants that had internal deletions in their N-terminal third were constructed. A series of unique restriction enzyme sites were first engineered into sea; only one of these changes resulted in an amino acid substitution (the aspartic acid residue at position 60 of mature SEA was changed to a glycine [D60G]). Because the D60G substitution had no discernible effect on serological or biological activity, the sea allele encoding this mutant SEA was used to construct a panel of mutant SEAs lacking residues 3 to 17, 19 to 23, 24 to 28, 29 to 49, 50 to 55, 56 to 59, 61 to 73, 68 to 74, or 74 to 85. Recombinant plasmids with the desired mutations were constructed in Escherichia coli and transferred to Staphylococcus aureus. Staphylococcal culture supernatants containing the mutant SEAs were examined. Western immunoblot analysis with polyclonal anti-SEA antiserum revealed that each of the recombinant S. aureus strains produced a mutant SEA of the predicted size. All the mutant SEAs exhibited increased sensitivity to monkey stomach lavage fluid in vitro, which is consistent with these mutants having conformations unlike that of wild-type SEA or the SEA D60G mutant. In general, deletion of internal peptides had a deleterious effect on the ability to induce T-cell proliferation; only SEA mutants lacking either residues 3 to 17 or 56 to 59 consistently produced a statistically significant increase in the incorporation of [3H]thymidine. In the course of this work, two monoclonal antibodies that had different requirements for binding to SEA in Western blots were identified. The epitope for one monoclonal antibody was contained within residues 108 to 230 of mature SEA. Binding of the other monoclonal antibody to

  2. Direct internal reforming of hydrocarbon fuels in solid oxide fuel cells

    NASA Astrophysics Data System (ADS)

    Zhan, Zhongliang

    2005-07-01

    The direct operation of solid oxide fuel cells (SOFCs) on hydrocarbon fuels is desired since it could reduce power plant size, weight and complexity. The primary challenge is to find effective means through which anode-coking could be suppressed or avoided. Throughout the research, conventional Ni-anode supported SOFCs were employed because they provide high power densities and are being actively developed for commercial applications. Various strategies were used to reduce or avoid anode-coking during the SOFC operation. Firstly, air or CO2/H2O was added to hydrocarbon fuels, such that coking was less thermodynamically favorable, and the resulting internal partial oxidation or dry/steam reforming reactions provided H 2 and CO to the fuel cell. For example, for low hydrocarbons like propane, coke-free operation was achieved on 8% yttrium-stabilized zirconia (YSZ) electrolyte SOFCs via internal partial oxidation, yielding stable and high power densities, e.g. 0.7 W·cm-2 at 790°C. Secondly, a novel design for hydrocarbon fueled SOFCs was proposed, i.e. a separate supported catalyst (Ru-CeO2) layer was placed against the anode side. The catalyst layer provided good catalytic activity for the hydrocarbon reforming reactions, while the nickel-based anode was retained to provide excellent electrochemical activity for the oxidation of the hydrogen and carbon monoxide reforming products. For heavy hydrocarbons like iso-octane, the catalyst layer was crucial far allowing stable cell operation without coking. The lack of coking at the Ni-YSZ anode can be explained by reforming at the Ru-Ceria catalyst layer, which eliminated most of the hydrocarbon species before the fuel reached the anode. A key element of this strategy was the choice of a catalyst metal, Ru, that promotes hydrocarbon reforming but does not itself cause coking. Thirdly, reduced-temperature SOFCs with thin samarium-doped Ceria (SDC) electrolytes were developed; these devices have potentially improved

  3. High pressure sample cell for total internal reflection fluorescence spectroscopy at pressures up to 2500 bar

    NASA Astrophysics Data System (ADS)

    Koo, Juny; Czeslik, Claus

    2012-08-01

    Total internal reflection fluorescence (TIRF) spectroscopy is a surface sensitive technique that is widely used to characterize the structure and dynamics of molecules at planar liquid-solid interfaces. In particular, biomolecular systems, such as protein adsorbates and lipid membranes can easily be studied by TIRF spectroscopy. Applying pressure to molecular systems offers access to all kinds of volume changes occurring during assembly of molecules, phase transitions, and chemical reactions. So far, most of these volume changes have been characterized in bulk solution, only. Here, we describe the design and performance of a high pressure sample cell that allows for TIRF spectroscopy under high pressures up to 2500 bar (2.5 × 108 Pa), in order to expand the understanding of volume effects from the bulk phase to liquid-solid interfaces. The new sample cell is based on a cylindrical body made of Nimonic 90 alloy and incorporates a pressure transmitting sample cuvette. This cuvette is composed of a fused silica prism and a flexible rubber gasket. It contains the sample solution and ensures a complete separation of the sample from the liquid pressure medium. The sample solution is in contact with the inner wall of the prism forming the interface under study, where fluorescent molecules are immobilized. In this way, the new high pressure TIRF sample cell is very useful for studying any biomolecular layer that can be deposited at a planar water-silica interface. As examples, high pressure TIRF data of adsorbed lysozyme and two phospholipid membranes are presented.

  4. NREL/NASA Internal Short-Circuit Instigator in Lithium Ion Cells; NREL (National Renewable Energy Laboratory)

    SciTech Connect

    Long, Dirk; Ireland, John; Pesaran, Ahmad; Darcy, Eric; Shoesmith, Mark; McCarthy, Ben

    2013-11-14

    NREL has developed a device to test one of the most challenging failure mechanisms of lithium-ion (Li-ion) batteries -- a battery internal short circuit. Many members of the technical community believe that this type of failure is caused by a latent flaw that results in a short circuit between electrodes during use. As electric car manufacturers turn to Li-ion batteries for energy storage, solving the short circuit problem becomes more important. To date, no reliable and practical method exists to create on-demand internal shorts in Li-ion cells that produce a response that is relevant to the ones produced by field failures. NREL and NASA have worked to establish an improved ISC cell-level test method that simulates an emergent internal short circuit, is capable of triggering the four types of cell internal shorts, and produces consistent and reproducible results. Internal short circuit device design is small, low-profile and implantable into Li-ion cells, preferably during assembly. The key component is an electrolyte-compatible phase change material (PCM). The ISC is triggered by heating the cell above PCM melting temperature (presently 40 degrees C – 60 degrees C). In laboratory testing, the activated device can handle currents in excess of 300 A to simulate hard shorts (< 2 mohms). Phase change from non-conducting to conducting has been 100% successful during trigger tests.

  5. Outcome prediction of advanced mantle cell lymphoma by international prognostic index versus different mantle cell lymphoma indexes: one institution study.

    PubMed

    Todorovic, Milena; Balint, Bela; Andjelic, Bosko; Stanisavljevic, Dejana; Kurtovic, Nada Kraguljac; Radisavljevic, Ziv; Mihaljevic, Biljana

    2012-09-01

    The aim of this study was to evaluate the prognostic significance of international prognostic index (IPI), mantle cell lymphoma IPI (MIPI), simplified MIPI (sMIPI), and MIPI biological (MIPIb), as well as their correlation with immunophenotype, clinical characteristics, and overall survival (OS), in a selected group of 54 patients with advanced-stage mantle cell lymphoma (MCL), treated uniformly with CHOP. Seventeen patients had IV clinical stage (CS), while other 37 had leukemic phase at presentation. Diffuse type of marrow infiltration was verified in 68.5% and nodular in remainder patients. Extranodal localization (25.9%) included bowel (20.4%), pleural effusion, sinus, and palpebral infiltration. All of analyzed patients expressed typical MCL immunophenotypic profile: CD19(+)CD20(+)CD22(+)CD5(+)Cyclin-D1(+)FMC7(+)CD79b(+)smIg(+)CD38(+/-)CD23(-)CD10(-). Median OS of the whole group was 23 months, without significant differences between IV CS and leukemic phase patients. Thirty-two patients (59.3%) responded to initial treatment, 9 (16.7%) with complete and 23 (42.6%) with partial remission. Negative prognostic influence on OS had high IPI (P < 0.01), high sMIPI (P < 0.001), MIPI (P < 0.01), MIPIb (P < 0.01), extranodal localization (P < 0.01), and diffuse marrow infiltration (P < 0.01). Testing between randomly selected groups showed that patients with lower proportion of CD5(+) cells (<80%) correlated with cytological blastoid variant and had shorter survival comparing with the group with higher proportion of CD5(+) cells (>80%) (P < 0.01). Using univariate Cox regression, we proved that IPI, sMIPI, MIPI, and MIPIb had an independent predictive importance (P < 0.01) for OS in uniformly treated advanced MCL patients, although sMIPI prognostic significance was the highest (P < 0.001).

  6. Evaluation of hydrogen production and internal resistance in forward osmosis membrane integrated microbial electrolysis cells.

    PubMed

    Lee, Mi-Young; Kim, Kyoung-Yeol; Yang, Euntae; Kim, In S

    2015-01-01

    In order to enhance hydrogen production by facilitated proton transport through a forward osmosis (FO) membrane, the FO membrane was integrated into microbial electrolysis cells (MECs). An improved hydrogen production rate was obtained in the FO-MEC (12.5±1.84×10(-3)m(3)H2/m(3)/d) compared to that of the cation exchange membrane (CEM) - MEC (4.42±0.04×10(-3)m(3)H2/m(3)/d) during batch tests (72h). After an internal resistance analysis, it was confirmed that the enhanced hydrogen production in FO-MEC was attributed to the smaller charge transfer resistance than in the CEM-MEC (90.3Ω and 133.4Ω respectively). The calculation of partial internal resistance concluded that the transport resistance can be substantially reduced by replacing a CEM with a FO membrane; decrease of the resistance from 0.069Ωm(2) to 5.99×10(-4)Ωm(2).

  7. The Daniell cell, Ohm's law, and the emergence of the International System of Units

    NASA Astrophysics Data System (ADS)

    Jayson, Joel S.

    2014-01-01

    Telegraphy originated in the 1830s and 40 s and flourished in the following decades but with a patchwork of electrical standards. Electromotive force was for the most part measured in units of the predominant Daniell cell, but each telegraphy company had their own resistance standard. In 1862, the British Association for the Advancement of Science formed a committee to address this situation. By 1873, they had given definition to the electromagnetic system of units (emu) and defined the practical units of the ohm as 109 emu units of resistance and the volt as 108 emu units of electromotive force. These recommendations were ratified and expanded upon in a series of international congresses held between 1881 and 1904. A proposal by Giovanni Giorgi in 1901 took advantage of a coincidence between the conversion of the units of energy in the emu system (the erg) and in the practical system (the Joule). As it was, the same conversion factor existed between the cgs based emu system and a theretofore undefined MKS system. By introducing another unit X (where X could be any of the practical electrical units), Giorgi demonstrated that a self-consistent MKSX system was tenable without the need for multiplying factors. Ultimately, the ampere was selected as the fourth unit. It took nearly 60 years, but in 1960, Giorgi's proposal was incorporated as the core of the newly inaugurated International System of Units (SI). This article surveys the physics, physicists, and events that contributed to those developments.

  8. Exosome Adherence and Internalization by Hepatic Stellate Cells Triggers Sphingosine 1-Phosphate-dependent Migration.

    PubMed

    Wang, Ruisi; Ding, Qian; Yaqoob, Usman; de Assuncao, Thiago M; Verma, Vikas K; Hirsova, Petra; Cao, Sheng; Mukhopadhyay, Debabrata; Huebert, Robert C; Shah, Vijay H

    2015-12-25

    Exosomes are cell-derived extracellular vesicles thought to promote intercellular communication by delivering specific content to target cells. The aim of this study was to determine whether endothelial cell (EC)-derived exosomes could regulate the phenotype of hepatic stellate cells (HSCs). Initial microarray studies showed that fibroblast growth factor 2 induced a 2.4-fold increase in mRNA levels of sphingosine kinase 1 (SK1). Exosomes derived from an SK1-overexpressing EC line increased HSC migration 3.2-fold. Migration was not conferred by the dominant negative SK1 exosome. Incubation of HSCs with exosomes was also associated with an 8.3-fold increase in phosphorylation of AKT and 2.5-fold increase in migration. Exosomes were found to express the matrix protein and integrin ligand fibronectin (FN) by Western blot analysis and transmission electron microscopy. Blockade of the FN-integrin interaction with a CD29 neutralizing antibody or the RGD peptide attenuated exosome-induced HSC AKT phosphorylation and migration. Inhibition of endocytosis with transfection of dynamin siRNA, the dominant negative dynamin GTPase construct Dyn2K44A, or the pharmacological inhibitor Dynasore significantly attenuated exosome-induced AKT phosphorylation. SK1 levels were increased in serum exosomes derived from mice with experimental liver fibrosis, and SK1 mRNA levels were up-regulated 2.5-fold in human liver cirrhosis patient samples. Finally, S1PR2 inhibition protected mice from CCl4-induced liver fibrosis. Therefore, EC-derived SK1-containing exosomes regulate HSC signaling and migration through FN-integrin-dependent exosome adherence and dynamin-dependent exosome internalization. These findings advance our understanding of EC/HSC cross-talk and identify exosomes as a potential target to attenuate pathobiology signals.

  9. Exosome Adherence and Internalization by Hepatic Stellate Cells Triggers Sphingosine 1-Phosphate-dependent Migration*

    PubMed Central

    Wang, Ruisi; Ding, Qian; Yaqoob, Usman; de Assuncao, Thiago M.; Verma, Vikas K.; Hirsova, Petra; Cao, Sheng; Mukhopadhyay, Debabrata; Huebert, Robert C.; Shah, Vijay H.

    2015-01-01

    Exosomes are cell-derived extracellular vesicles thought to promote intercellular communication by delivering specific content to target cells. The aim of this study was to determine whether endothelial cell (EC)-derived exosomes could regulate the phenotype of hepatic stellate cells (HSCs). Initial microarray studies showed that fibroblast growth factor 2 induced a 2.4-fold increase in mRNA levels of sphingosine kinase 1 (SK1). Exosomes derived from an SK1-overexpressing EC line increased HSC migration 3.2-fold. Migration was not conferred by the dominant negative SK1 exosome. Incubation of HSCs with exosomes was also associated with an 8.3-fold increase in phosphorylation of AKT and 2.5-fold increase in migration. Exosomes were found to express the matrix protein and integrin ligand fibronectin (FN) by Western blot analysis and transmission electron microscopy. Blockade of the FN-integrin interaction with a CD29 neutralizing antibody or the RGD peptide attenuated exosome-induced HSC AKT phosphorylation and migration. Inhibition of endocytosis with transfection of dynamin siRNA, the dominant negative dynamin GTPase construct Dyn2K44A, or the pharmacological inhibitor Dynasore significantly attenuated exosome-induced AKT phosphorylation. SK1 levels were increased in serum exosomes derived from mice with experimental liver fibrosis, and SK1 mRNA levels were up-regulated 2.5-fold in human liver cirrhosis patient samples. Finally, S1PR2 inhibition protected mice from CCl4-induced liver fibrosis. Therefore, EC-derived SK1-containing exosomes regulate HSC signaling and migration through FN-integrin-dependent exosome adherence and dynamin-dependent exosome internalization. These findings advance our understanding of EC/HSC cross-talk and identify exosomes as a potential target to attenuate pathobiology signals. PMID:26534962

  10. Ligand- and cell-dependent determinants of internalization and cAMP modulation by delta opioid receptor (DOR) agonists.

    PubMed

    Charfi, Iness; Nagi, Karim; Mnie-Filali, Ouissame; Thibault, Dominic; Balboni, Gianfranco; Schiller, Peter W; Trudeau, Louis-Eric; Pineyro, Graciela

    2014-04-01

    Signaling bias refers to G protein-coupled receptor ligand ability to preferentially activate one type of signal over another. Bias to evoke signaling as opposed to sequestration has been proposed as a predictor of opioid ligand potential for generating tolerance. Here we measured whether delta opioid receptor agonists preferentially inhibited cyclase activity over internalization in HEK cells. Efficacy (τ) and affinity (KA) values were estimated from functional data and bias was calculated from efficiency coefficients (log τ/KA). This approach better represented the data as compared to alternative methods that estimate bias exclusively from τ values. Log (τ/KA) coefficients indicated that SNC-80 and UFP-512 promoted cyclase inhibition more efficiently than DOR internalization as compared to DPDPE (bias factor for SNC-80: 50 and for UFP-512: 132). Molecular determinants of internalization were different in HEK293 cells and neurons with βarrs contributing to internalization in both cell types, while PKC and GRK2 activities were only involved in neurons. Rank orders of ligand ability to engage different internalization mechanisms in neurons were compared to rank order of E max values for cyclase assays in HEK cells. Comparison revealed a significant reversal in rank order for cyclase E max values and βarr-dependent internalization in neurons, indicating that these responses were ligand-specific. Despite this evidence, and because kinases involved in internalization were not the same across cellular backgrounds, it is not possible to assert if the magnitude and nature of bias revealed by rank orders of maximal responses is the same as the one measured in HEK cells.

  11. Allogeneic Hematopoietic Cell Transplantation for Aggressive NK Cell Leukemia. A Center for International Blood and Marrow Transplant Research Analysis.

    PubMed

    Hamadani, Mehdi; Kanate, Abraham S; DiGilio, Alyssa; Ahn, Kwang Woo; Smith, Sonali M; Lee, Jong Wook; Ayala, Ernesto; Chao, Nelson; Hari, Parameswaran; Bolaños-Meade, Javier; Gress, Ronald; Smedegaard Anderson, Niels; Chen, Yi-Bin; Farooq, Umar; Schiller, Gary; Yared, Jean; Sureda, Anna; Fenske, Timothy S; Olteanu, Horatiu

    2017-02-01

    Aggressive NK cell leukemia (ANKL) is an exceedingly rare form of leukemia and carries a poor prognosis, with a median survival of only 2 months. Using the Center for International Blood and Marrow Transplant Research database, we evaluated outcomes of allogeneic hematopoietic cell transplantation (alloHCT) in patients with ANKL. Twenty-one patients with a centrally confirmed diagnosis of ANKL were included. Median patient age was 42 years and 15 patients (71%) were Caucasian. Fourteen patients (67%) were in complete remission (CR) at the time of alloHCT, and 5 patients had active disease. Median follow-up of survivors was 25 months (range, 12 to 116). The 2-year estimates of nonrelapse mortality, relapse/progression, progression-free (PFS), and overall survival (OS) were 21%, 59%, 20%, and 24%, respectively. The 2-year PFS of patients in CR at the time of alloHCT was significantly better than that of patients with active disease at transplantation (30% versus 0%; P = .001). The 2-year OS in similar order was 38% versus 0% (P < .001). In conclusion, this registry analysis that included majority non-Asian patient population shows that alloHCT can provide durable disease control in a subset of ANKL patients. Achieving CR before transplantation appears to be a prerequisite for successful transplantation outcomes.

  12. Cytotoxicity and variant cellular internalization behavior of water-soluble sulfonated nanographene sheets in liver cancer cells

    NASA Astrophysics Data System (ADS)

    Corr, Stuart J.; Raoof, Mustafa; Cisneros, Brandon T.; Kuznetsov, Oleksandr; Massey, Katheryn; Kaluarachchi, Warna D.; Cheney, Matthew A.; Billups, Edward W.; Wilson, Lon J.; Curley, Steven A.

    2013-05-01

    Highly exfoliated sulfonated graphene sheets (SGSs), an alternative to graphene oxide and graphene derivatives, were synthesized, characterized, and applied to liver cancer cells in vitro. Cytotoxicity profiles were obtained using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, WST-1[2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2 H-tetrazolium, and lactate dehydrogenase release colorimetric assays. These particles were found to be non-toxic across the concentration range of 0.1 to 10 μg/ml. Internalization of SGSs was also studied by means of optical and electron microscopy. Although not conclusive, high-resolution transmission and scanning electron microscopy revealed variant internalization behaviors where some of the SGS became folded and compartmentalized into tight bundles within cellular organelles. The ability for liver cancer cells to internalize, fold, and compartmentalize graphene structures is a phenomenon not previously documented for graphene cell biology and should be further investigated.

  13. S4(13)-PV cell-penetrating peptide induces physical and morphological changes in membrane-mimetic lipid systems and cell membranes: implications for cell internalization.

    PubMed

    Cardoso, Ana M S; Trabulo, Sara; Cardoso, Ana L; Lorents, Annely; Morais, Catarina M; Gomes, Paula; Nunes, Cláudia; Lúcio, Marlene; Reis, Salette; Padari, Kärt; Pooga, Margus; Pedroso de Lima, Maria C; Jurado, Amália S

    2012-03-01

    The present work aims to gain insights into the role of peptide-lipid interactions in the mechanisms of cellular internalization and endosomal escape of the S4(13)-PV cell-penetrating peptide, which has been successfully used in our laboratory as a nucleic acid delivery system. A S4(13)-PV analogue, S4(13)-PVscr, displaying a scrambled amino acid sequence, deficient cell internalization and drug delivery inability, was used in this study for comparative purposes. Differential scanning calorimetry, fluorescence polarization and X-ray diffraction at small and wide angles techniques showed that both peptides interacted with anionic membranes composed of phosphatidylglycerol or a mixture of this lipid with phosphatidylethanolamine, increasing the lipid order, shifting the phase transition to higher temperatures and raising the correlation length between the bilayers. However, S4(13)-PVscr, in contrast to the wild-type peptide, did not promote lipid domain segregation and induced the formation of an inverted hexagonal lipid phase instead of a cubic phase in the lipid systems assayed. Electron microscopy showed that, as opposed to S4(13)-PVscr, the wild-type peptide induced the formation of a non-lamellar organization in membranes of HeLa cells. We concluded that lateral phase separation and destabilization of membrane lamellar structure without compromising membrane integrity are on the basis of the lipid-driven and receptor-independent mechanism of cell entry of S4(13)-PV peptide. Overall, our results can contribute to a better understanding of the role of peptide-lipid interactions in the mechanisms of cell-penetrating peptide membrane translocation, helping in the future design of more efficient cell-penetrating peptide-based drug delivery systems.

  14. Mechanisms of nanoparticle internalization and transport across an intestinal epithelial cell model: effect of size and surface charge.

    PubMed

    Bannunah, Azzah M; Vllasaliu, Driton; Lord, Jennie; Stolnik, Snjezana

    2014-12-01

    This study investigated the effect of nanoparticle size (50 and 100 nm) and surface charge on their interaction with Caco-2 monolayers as a model of the intestinal epithelium, including cell internalization pathways and the level of transepithelial transport. Initially, toxicity assays showed that cell viability and cell membrane integrity were dependent on the surface charge and applied mass, number, and total surface area of nanoparticles, as tested in two epithelial cell lines, colon carcinoma Caco-2 and airway Calu-3. This also identified suitable nanoparticle concentrations for subsequent cell uptake experiments. Nanoparticle application at doses below half maximal effective concentration (EC₅₀) revealed that the transport efficiency (ratio of transport to cell uptake) across Caco-2 cell monolayers is significantly higher for negatively charged nanoparticles compared to their positively charged counterparts (of similar size), despite the higher level of internalization of positively charged systems. Cell internalization pathways were hence probed using a panel of pharmacological inhibitors aiming to establish whether the discrepancy in transport efficiency is due to different uptake and transport pathways. Vesicular trans-monolayer transport for both positively and negatively charged nanoparticles was confirmed via inhibition of dynamin (by dynasore) and microtubule network (via nocodazole), which significantly reduced the transport of both nanoparticle systems. For positively charged nanoparticles a significant decrease in internalization and transport (46% and 37%, respectively) occurred in the presence of a clathrin pathway inhibitor (chlorpromazine), macropinocytosis inhibition (42%; achieved by 5-(N-ethyl-N-isopropyi)-amiloride), and under cholesterol depletion (38%; via methyl-β-cyclodextrin), but remained unaffected by the inhibition of lipid raft associated uptake (caveolae) by genistein. On the contrary, the most prominent reduction in

  15. Sickle cell disease: an international survey of results of HLA-identical sibling hematopoietic stem cell transplantation.

    PubMed

    Gluckman, Eliane; Cappelli, Barbara; Bernaudin, Francoise; Labopin, Myriam; Volt, Fernanda; Carreras, Jeanette; Simões, Belinda Pinto; Ferster, Alina; Dupont, Sophie; de la Fuente, Josu; Dalle, Jean-Hugues; Zecca, Marco; Walters, Mark C; Krishnamurti, Lakshmanan; Bhatia, Monica; Leung, Kathryn; Yanik, Gregory; Kurtzberg, Joanne; Dhedin, Nathalie; Kuentz, Mathieu; Michel, Gerard; Apperley, Jane; Lutz, Patrick; Neven, Bénédicte; Bertrand, Yves; Vannier, Jean Pierre; Ayas, Mouhab; Cavazzana, Marina; Matthes-Martin, Susanne; Rocha, Vanderson; Elayoubi, Hanadi; Kenzey, Chantal; Bader, Peter; Locatelli, Franco; Ruggeri, Annalisa; Eapen, Mary

    2016-12-13

    Despite advances in supportive therapy to prevent complications of sickle cell disease (SCD), access to care is not universal. Hematopoietic cell transplantation is, to date, the only curative therapy for SCD, but its application is limited by availability of a suitable HLA-matched donor and lack of awareness of the benefits of transplant. Included in this study are 1000 recipients of HLA-identical sibling transplants performed between 1986 and 2013 and reported to the European Blood and Marrow Transplant, Eurocord and the Center for International Blood and Marrow Transplant Research. The primary endpoint was event-free survival, defined as being alive without graft failure; risk factors were studied using Cox regression model. The median age at transplantation was 9 years and the median follow-up, longer than 5 years. Most patients received a myeloablative conditioning regimen (n=873; 87%) and the remainder, reduced-intensity conditioning regimens (n=125; 13%). Bone marrow was the predominant stem cell source (n=839; 84%), while peripheral blood and cord blood progenitors were used in 73 (7%) and 88 patients (9%), respectively. The 5-year event-free and overall survival was 91.4% (95% CI 89.6%-93.3%) and 92.9% (95% CI 91.1%-94.6%), respectively. Event-free survival was lower with increasing age at transplantation (hazard ratio [HR] 1.09; p<0.001) and higher for transplantations performed after 2006 (HR 0.95, p=0.013). Twenty-three patients experienced graft failure; 70 patients (7%) died, the most common cause of death being infection. The excellent outcome of a cohort transplanted over 3 decades confirms the role of HLA-identical sibling transplantation for children and adults with SCD.

  16. Total internal reflection holographic microscopy (TIRHM) for quantitative phase characterization of cell-substrate adhesion

    NASA Astrophysics Data System (ADS)

    Ash, William Mason, III

    Total Internal Reflection Holographic Microscopy (TIRHM) combines near-field microscopy with digital holography to produce a new form of near-field phase microscopy. Using a prism in TIR as a near-field imager, the presence of microscopic organisms, cell-substrate interfaces, and adhesions, causes relative refractive index (RRI) and frustrated TIR (f-TIR) to modulate the object beam's evanescent wave phase front. Quantitative phase images of test specimens such as Amoeba proteus, Dictyostelium Discoideum and cells such as SKOV-3 ovarian cancer and 3T3 fibroblasts are produced without the need to introduce stains or fluorophores. The angular spectrum method of digital holography to compensate for tilt anamorphism due to the inclined TIR plane is also discussed. The results of this work conclusively demonstrate, for the first time, the integration of near-field microscopy with digital holography. The cellular images presented show a correlation between the physical extent of the Amoeba proteus plasma membrane and the adhesions that are quantitatively profiled by phase cross-sectioning of the holographic images obtained by digital holography. With its ability to quantitatively characterise cellular adhesion and motility, it is anticipated that TIRHM can be a tool for characterizing and combating cancer metastasis, as well as improving our understanding of morphogenesis and embryogenesis itself.

  17. CdSe quantum-dot-sensitized solar cell with ∼100% internal quantum efficiency.

    PubMed

    Fuke, Nobuhiro; Hoch, Laura B; Koposov, Alexey Y; Manner, Virginia W; Werder, Donald J; Fukui, Atsushi; Koide, Naoki; Katayama, Hiroyuki; Sykora, Milan

    2010-11-23

    We have constructed and studied photoelectrochemical solar cells (PECs) consisting of a photoanode prepared by direct deposition of independently synthesized CdSe nanocrystal quantum dots (NQDs) onto a nanocrystalline TiO(2) film (NQD/TiO(2)), aqueous Na(2)S or Li(2)S electrolyte, and a Pt counter electrode. We show that light harvesting efficiency (LHE) of the NQD/TiO(2) photoanode is significantly enhanced when the NQD surface passivation is changed from tri-n-octylphosphine oxide (TOPO) to 4-butylamine (BA). In the PEC the use of NQDs with a shorter passivating ligand, BA, leads to a significant enhancement in both the electron injection efficiency at the NQD/TiO(2) interface and charge collection efficiency at the NQD/electrolyte interface, with the latter attributed mostly to a more efficient diffusion of the electrolyte through the pores of the photoanode. We show that by utilizing BA-capped NQDs and aqueous Li(2)S as an electrolyte, it is possible to achieve ∼100% internal quantum efficiency of photon-to-electron conversion, matching the performance of dye-sensitized solar cells.

  18. Ethical and Regulatory Challenges with Autologous Adult Stem Cells: A Comparative Review of International Regulations.

    PubMed

    Lysaght, Tamra; Kerridge, Ian H; Sipp, Douglas; Porter, Gerard; Capps, Benjamin J

    2017-02-28

    Cell and tissue-based products, such as autologous adult stem cells, are being prescribed by physicians across the world for diseases and illnesses that they have neither been approved for or been demonstrated as safe and effective in formal clinical trials. These doctors often form part of informal transnational networks that exploit differences and similarities in the regulatory systems across geographical contexts. In this paper, we examine the regulatory infrastructure of five geographically diverse but socio-economically comparable countries with the aim of identifying similarities and differences in how these products are regulated and governed within clinical contexts. We find that while there are many subtle technical differences in how these regulations are implemented, they are sufficiently similar that it is difficult to explain why these practices appear more prevalent in some countries and not in others. We conclude with suggestions for how international governance frameworks might be improved to discourage the exploitation of vulnerable patient populations while enabling innovation in the clinical application of cellular therapies.

  19. Nickel-Hydrogen Battery Cell Life Test Program Update for the International Space Station

    NASA Technical Reports Server (NTRS)

    Miller, Thomas B.

    2000-01-01

    NASA and Boeing North America are responsible for constructing the electrical power system for the International Space Station (ISS), which circles the Earth every 90 minutes in a low Earth orbit (LEO). For approximately 55 minutes of this orbit, the ISS is in sunlight, and for the remaining 35 minutes, the ISS is in the Earth s shadow (eclipse). The electrical power system must not only provide power during the sunlight portion by means of the solar arrays, but also store energy for use during the eclipse. Nickel-hydrogen (Ni/H2) battery cells were selected as the energy storage systems for ISS. Each battery Orbital Replacement Unit (ORU) comprises 38 individual series-connected Ni/H2 battery cells, and there are 48 battery ORU s on the ISS. On the basis of a limited Ni/H2 LEO data base on life and performance characteristics, the NASA Glenn Research Center at Lewis Field commenced testing through two test programs: one in-house and one at the Naval Surface Warfare Center in Crane, Indiana.

  20. Are Soft Short Tests Good Indicators of Internal Li-ion Cell Defects?

    NASA Technical Reports Server (NTRS)

    Jeevarajan, J.; Chung, J.-S.; Jung, K.; Park, J.

    2013-01-01

    The self discharge test at full state of charge, may not be a good one to detect subtle defects since the li-ion chemistry has the highest self discharge at full state of charge. One should characterize self discharge versus storage time for each cell manufacturer/design to differentiate between normal self discharge and that due to a subtle manufacturing defect. The various soft short test methods indicate that if this test is carried out at full discharge (0% SOC) with all capacity removed (by lowering the current load in a stepwise manner to the same end of discharge voltage), then the cells need to be placed in storage for more than 72 hours to get a good analysis on the presence of subtle defects since it takes more than 72 hours to achieve voltage stabilization. If the cells are to be charged up even to a small percentage (ex. 1%), 72 hours are sufficient to determine issues. However, the pass/fail criteria should be based on a valid OCV decline. Less than 10 mV voltage decline is not a good method to detect subtle defects. As mentioned in the first bullet, self discharge is a competing reaction when a charge is introduced and hence a characterization of the self discharge versus storage time is required to fully correlate voltage decline to a failure due to a subtle defect. Soft short test method cannot be relied on for defect detection because cells with and without voltage decline seemed to have similar defects and characteristics. Screening methods such as internal resistance and capacity as well as a 3-sigma range for OCV, mass and dimensions should be used to screen out outliers. A very critical aspect in the understanding of subtle defects is to carry out destructive analysis of cells from every lot to confirm the quality of production and screen all cells and batteries in a stringent manner to have a high quality set of flight cells. Self Discharge Test: Fully charged cells shall be placed in Open circuit stand for 72 hours (OCV measurement twice a

  1. Polystyrene nanoparticles facilitate the internalization of impermeable biomolecules in non-tumour and tumour cells from colon epithelium

    NASA Astrophysics Data System (ADS)

    Cabeza, Laura; Cano-Cortés, Victoria; Rodríguez, María J.; Vélez, Celia; Melguizo, Consolación; Sánchez-Martín, Rosario M.; Prados, Jose

    2015-01-01

    Advanced colon cancer has a poor prognosis due to the limited effectiveness of current chemotherapies. Treatment failures may be avoided by the utilization of nanoparticles, which can enhance the effects of antitumor drugs, reduce their side effects and increase their directionality. Polystyrene nanoparticles have shown high biocompatibility and appropriate physicochemical properties and may represent a novel and more effective approach against colon cancer. In the present study, polystyrene nanoparticles were synthesized and fluorescently labelled, analyzing their cell internalization, intracellular localization and capacity to release transported molecules in tumour and non-tumour human colon cell lines (T84 and CCD-18). Flow cytometry and fluorescence microscopy studies demonstrated that polystyrene nanoparticles are an effective vehicle for the intracellular delivery of small molecules into colon epithelium cells. The percentage cell uptake was around 100 % in both T84 and CCD-18 cell lines after only 24 h of exposure and was cell confluence-independent. The polystyrene nanoparticles showed no cytotoxicity in either colon cell line. It was found that small molecules can be efficiently delivered into colon cells by using a disulphide bridge as release strategy. Analysis of the influence of the functionalization of the polystyrene nanoparticles surface on the internalization efficiency revealed some morphological changes in these cells. These results demonstrate that polystyrene nanoparticles may improve the transport of biomolecules into colon cells which could have a potential application in chemotherapeutic treatment against colon cancer.

  2. Rapid desensitization induces internalization of antigen-specific IgE on mouse mast cells

    PubMed Central

    Oka, Tatsuya; Rios, Eon J.; Tsai, Mindy; Kalesnikoff, Janet; Galli, Stephen J.

    2013-01-01

    Background Rapid desensitization transiently prevents severe allergic reactions, allowing administration of life-saving therapies in previously sensitized patients. However, the mechanisms underlying successful rapid desensitization are not fully understood. Objectives We sought to investigate whether the mast cell (MC) is an important target of rapid desensitization in mice sensitized to exhibit IgE-dependent passive systemic anaphylaxis in vivo and to investigate the antigen specificity and underlying mechanisms of rapid desensitization in our mouse model. Methods C57BL/6 mice (in vivo) or primary isolated C57BL/6 mouse peritoneal mast cells (PMCs; in vitro) were passively sensitized with antigen-specific anti–2,4-dinitrophenyl IgE, anti-ovalbumin IgE, or both. MCs were exposed over a short period of time to increasing amounts of antigen (2,4-dinitrophenyl–human serum albumin or ovalbumin) in the presence of extracellular calcium in vitro or by means of intravenous administration to sensitized mice in vivo before challenging the mice with or exposing the PMCs to optimal amounts of specific or irrelevant antigen. Results Rapidly exposing mice or PMCs to progressively increasing amounts of specific antigen inhibited the development of antigen-induced hypothermia in sensitized mice in vivo and inhibited antigen-induced PMC degranulation and prostaglandin D2 synthesis in vitro. Such MC hyporesponsiveness was induced antigen-specifically and was associated with a significant reduction in antigen-specific IgE levels on MC surfaces. Conclusions Rapidly exposing MCs to progressively increasing amounts of antigen can both enhance the internalization of antigen-specific IgE on the MC surface and also desensitize these cells in an antigen-specific manner in vivo and in vitro. PMID:23810240

  3. Cellular internalization of LiNbO3 nanocrystals for second harmonic imaging and the effects on stem cell differentiation

    NASA Astrophysics Data System (ADS)

    Li, Jianhua; Qiu, Jichuan; Guo, Weibo; Wang, Shu; Ma, Baojin; Mou, Xiaoning; Tanes, Michael; Jiang, Huaidong; Liu, Hong

    2016-03-01

    Second harmonic generation (SHG) nanocrystals have recently been reported to label cancer cells and other functional cell lines due to their unique double-frequency property. In this paper, we report for the first time the use of lithium niobate (LiNbO3, LN) nanocrystals as SHG labels for imaging stem cells. Rat mesenchymal stem cells (rMSCs) were labeled with LN nanocrystals in order to study the cellular internalization of the nanocrystals and the influence on stem cell differentiation. The results showed that LN nanocrystals were endocytosed by the rMSCs and the distribution of the internalized nanoparticles demonstrated a high consistency with the orientation of the actin filaments. Besides, LN-labeled rMSCs showed a concentration-dependent viability. Most importantly, rMSCs labeled with 50 μg per mL of LN nanocrystals retained their ability to differentiate into both osteogenic and adipogenic lineages. The results prove that LN nanocrystals can be used as a cytocompatible, near-infrared (NIR) light driven cell label for long-term imaging, without hindering stem cell differentiation. This work will promote the use of LN nanocrystals to broader applications like deep-tissue tracking, remote drug delivery and stem cell therapy.Second harmonic generation (SHG) nanocrystals have recently been reported to label cancer cells and other functional cell lines due to their unique double-frequency property. In this paper, we report for the first time the use of lithium niobate (LiNbO3, LN) nanocrystals as SHG labels for imaging stem cells. Rat mesenchymal stem cells (rMSCs) were labeled with LN nanocrystals in order to study the cellular internalization of the nanocrystals and the influence on stem cell differentiation. The results showed that LN nanocrystals were endocytosed by the rMSCs and the distribution of the internalized nanoparticles demonstrated a high consistency with the orientation of the actin filaments. Besides, LN-labeled rMSCs showed a concentration

  4. Further study of the intrinsic safety of internally shorted lithium and lithium-ion cells within methane-air

    PubMed Central

    Dubaniewicz, Thomas H.; DuCarme, Joseph P.

    2015-01-01

    National Institute for Occupational Safety and Health (NIOSH) researchers continue to study the potential for lithium and lithium-ion battery thermal runaway from an internal short circuit in equipment for use in underground coal mines. Researchers conducted cell crush tests using a plastic wedge within a 20-L explosion-containment chamber filled with 6.5% CH4-air to simulate the mining hazard. The present work extends earlier findings to include a study of LiFePO4 cells crushed while under charge, prismatic form factor LiCoO2 cells, primary spiral-wound constructed LiMnO2 cells, and crush speed influence on thermal runaway susceptibility. The plastic wedge crush was a more severe test than the flat plate crush with a prismatic format cell. Test results indicate that prismatic Saft MP 174565 LiCoO2 and primary spiral-wound Saft FRIWO M52EX LiMnO2 cells pose a CH4-air ignition hazard from internal short circuit. Under specified test conditions, A123 systems ANR26650M1A LiFePO4 cylindrical cells produced no chamber ignitions while under a charge of up to 5 A. Common spiral-wound cell separators are too thin to meet intrinsic safety standards provisions for distance through solid insulation, suggesting that a hard internal short circuit within these cells should be considered for intrinsic safety evaluation purposes, even as a non-countable fault. Observed flames from a LiMnO2 spiral-wound cell after a chamber ignition within an inert atmosphere indicate a sustained exothermic reaction within the cell. The influence of crush speed on ignitions under specified test conditions was not statistically significant. PMID:26139958

  5. Lattice cell and full core physics of internally cooled annular fuel in heavy water moderated reactors

    SciTech Connect

    Armstrong, J.; Hamilton, H.; Hyland, B.

    2013-07-01

    A program is underway at Atomic Energy of Canada Limited (AECL) to develop a new fuel bundle concept to enable greater burnups for PT-HWR (pressure tube heavy water reactor) cores. One option that AECL is investigating is an internally cooled annular fuel (ICAF) element concept. ICAF contains annular cylindrical pellets with cladding on the inner and outer diameters. Coolant flows along the outside of the element and through the centre. With such a concept, the maximum fuel temperature as a function of linear element rating is significantly reduced compared to conventional, solid-rod type fuel. The preliminary ICAF bundle concept considered in this study contains 24 half-metre long internally cooled annular fuel elements and one non-fuelled centre pin. The introduction of the non-fuelled centre pin reduces the coolant void reactivity (CVR), which is the increase in reactivity that occurs on voiding the coolant in accident scenarios. Lattice cell and full core physics calculations of the preliminary ICAF fuel bundle concept have been performed for medium burnups of approximately 18 GWd/tU using WIMS-AECL and reactor fuel simulation program (RFSP). The results will be used to assist in concept configuration optimization. The effects of radial and axial core power distributions, linear element power ratings, refuelling rates and operational power ramps have been analyzed. The results suggest that burnups of greater than 18 GWd/tU can be achieved in current reactor designs. At approximately 18 GWd/tU, expected maximum linear element ratings in a PT-HWR with online-refuelling are approximately 90 kW/m. These conditions would be prohibitive for solid-rod fuel, but may be possible in ICAF fuel given the reduced maximum fuel temperature as a function of linear element rating. (authors)

  6. The receptor and transporter for internalization of Clostridium botulinum type C progenitor toxin into HT-29 cells.

    PubMed

    Nishikawa, Atsushi; Uotsu, Nobuo; Arimitsu, Hideyuki; Lee, Jae-Chul; Miura, Yutaka; Fujinaga, Yukako; Nakada, Hiroshi; Watanabe, Toshihiro; Ohyama, Tohru; Sakano, Yoshiyuki; Oguma, Keiji

    2004-06-25

    Orally ingested botulinum toxin enters the circulatory system and eventually reaches the peripheral nerves, where it elicits a response of neurological dysfunction. In this study, we report the important findings concerning the mechanism of Clostridium botulinum type C progenitor toxin (C16S) endocytic mechanism. C16S toxin bound to high molecular weight proteins on the surface of human colon carcinoma HT-29 cells and was internalized, but not if the cells were pretreated with neuraminidase. Benzyl-GalNAc which inhibited O-glycosylation of glycoproteins also interfered in the toxin's ability to bind the cell surface. On the other hand, the toxin was internalized in spite of pretreatment of the cells with PPMP, an inhibitor of ganglioside synthesis. These results suggest that the glycoproteins, like mucin, fulfill the important roles of receptor and transporter of C16S toxin.

  7. Further Analyses of the NASA Glenn Research Center Solar Cell and Photovoltaic Materials Experiment Onboard the International Space Station

    NASA Technical Reports Server (NTRS)

    Myers, Matthew G.; Prokop, Norman F.; Krasowski, Michael J.; Piszczor, Michael F.; McNatt, Jeremiah S.

    2016-01-01

    Accurate air mass zero (AM0) measurement is essential for the evaluation of new photovoltaic (PV) technology for space solar cells. The NASA Glenn Research Center (GRC) has flown an experiment designed to measure the electrical performance of several solar cells onboard NASA Goddard Space Flight Center's (GSFC) Robotic Refueling Mission's (RRM) Task Board 4 (TB4) on the exterior of the International Space Station (ISS). Four industry and government partners provided advanced PV devices for measurement and orbital environment testing. The experiment was positioned on the exterior of the station for approximately eight months, and was completely self-contained, providing its own power and internal data storage. Several new cell technologies including four-junction (4J) Inverted Metamorphic Multi-Junction (IMM) cells were evaluated and the results will be compared to ground-based measurement methods.

  8. GapmeR cellular internalization by macropinocytosis induces sequence-specific gene silencing in human primary T-cells

    PubMed Central

    Fazil, Mobashar Hussain Urf Turabe; Ong, Seow Theng; Chalasani, Madhavi Latha Somaraju; Low, Jian Hui; Kizhakeyil, Atish; Mamidi, Akshay; Lim, Carey Fang Hui; Wright, Graham D.; Lakshminarayanan, Rajamani; Kelleher, Dermot; Verma, Navin Kumar

    2016-01-01

    Post-transcriptional gene silencing holds great promise in discovery research for addressing intricate biological questions and as therapeutics. While various gene silencing approaches, such as siRNA and CRISPR-Cas9 techniques, are available, these cannot be effectively applied to “hard-to-transfect” primary T-lymphocytes. The locked nucleic acid-conjugated chimeric antisense oligonucleotide, called “GapmeR”, is an emerging new class of gene silencing molecule. Here, we show that GapmeR internalizes into human primary T-cells through macropinocytosis. Internalized GapmeR molecules can associate with SNX5-positive macropinosomes in T-cells, as detected by super-resolution microscopy. Utilizing the intrinsic self-internalizing capability of GapmeR, we demonstrate significant and specific depletion (>70%) of the expression of 5 different endogenous proteins with varying molecular weights (18 kDa Stathmin, 80 kDa PKCε, 180 kDa CD11a, 220 kDa Talin1 and 450 kDa CG-NAP/AKAP450) in human primary and cultured T-cells. Further functional analysis confirms CG-NAP and Stathmin as regulators of T-cell motility. Thus, in addition to screening, identifying or verifying critical roles of various proteins in T-cell functioning, this study provides novel opportunities to silence individual or multiple genes in a subset of purified human primary T-cells that would be exploited as future therapeutics. PMID:27883055

  9. Direct observation of internalization and ROS generation of amyloid β-peptide in neuronal cells at subcellular resolution.

    PubMed

    Jiao, Yong; Zhang, Yi; Wei, Yibin; Liu, Zhiwei; An, Wenting; Guo, Maolin

    2012-11-05

    Seeing in many colors: Confocal images acquired using fluorescently labeled amyloid β-peptide revealed its efficient internalization by endocytosis into endosomes/lysosomes of human neuronal cells with a small portion reaching mitochondria, inducing marked cellular and mitochondrial reactive oxygen species production.

  10. International Institute for Collaborative Cell Biology and Biochemistry—History and Memoirs from an International Network for Biological Sciences

    PubMed Central

    Cameron, L. C.

    2013-01-01

    I was invited to write this essay on the occasion of my selection as the recipient of the 2012 Bruce Alberts Award for Excellence in Science Education from the American Society for Cell Biology (ASCB). Receiving this award is an enormous honor. When I read the email announcement for the first time, it was more than a surprise to me, it was unbelievable. I joined ASCB in 1996, when I presented a poster and received a travel award. Since then, I have attended almost every ASCB meeting. I will try to use this essay to share with readers one of the best experiences in my life. Because this is an essay, I take the liberty of mixing some of my thoughts with data in a way that it not usual in scientific writing. I hope that this sacrifice of the format will achieve the goal of conveying what I have learned over the past 20 yr, during which time a group of colleagues and friends created a nexus of knowledge and wisdom. We have worked together to build a network capable of sharing and inspiring science all over the world. PMID:24006381

  11. International institute for collaborative cell biology and biochemistry--history and memoirs from an international network for biological sciences.

    PubMed

    Cameron, L C

    2013-01-01

    I was invited to write this essay on the occasion of my selection as the recipient of the 2012 Bruce Alberts Award for Excellence in Science Education from the American Society for Cell Biology (ASCB). Receiving this award is an enormous honor. When I read the email announcement for the first time, it was more than a surprise to me, it was unbelievable. I joined ASCB in 1996, when I presented a poster and received a travel award. Since then, I have attended almost every ASCB meeting. I will try to use this essay to share with readers one of the best experiences in my life. Because this is an essay, I take the liberty of mixing some of my thoughts with data in a way that it not usual in scientific writing. I hope that this sacrifice of the format will achieve the goal of conveying what I have learned over the past 20 yr, during which time a group of colleagues and friends created a nexus of knowledge and wisdom. We have worked together to build a network capable of sharing and inspiring science all over the world.

  12. Intracellular distribution of TM4SF1 and internalization of TM4SF1-antibody complex in vascular endothelial cells

    SciTech Connect

    Sciuto, Tracey E.; Merley, Anne; Lin, Chi-Iou; Richardson, Douglas; Liu, Yu; Li, Dan; Dvorak, Ann M.; Dvorak, Harold F.; Jaminet, Shou-Ching S.

    2015-09-25

    Transmembrane-4 L-six family member-1 (TM4SF1) is a small plasma membrane-associated glycoprotein that is highly and selectively expressed on the plasma membranes of tumor cells, cultured endothelial cells, and, in vivo, on tumor-associated endothelium. Immunofluorescence microscopy also demonstrated TM4SF1 in cytoplasm and, tentatively, within nuclei. With monoclonal antibody 8G4, and the finer resolution afforded by immuno-nanogold transmission electron microscopy, we now demonstrate TM4SF1 in uncoated cytoplasmic vesicles, nuclear pores and nucleoplasm. Because of its prominent surface location on tumor cells and tumor-associated endothelium, TM4SF1 has potential as a dual therapeutic target using an antibody drug conjugate (ADC) approach. For ADC to be successful, antibodies reacting with cell surface antigens must be internalized for delivery of associated toxins to intracellular targets. We now report that 8G4 is efficiently taken up into cultured endothelial cells by uncoated vesicles in a dynamin-dependent, clathrin-independent manner. It is then transported along microtubules through the cytoplasm and passes through nuclear pores into the nucleus. These findings validate TM4SF1 as an attractive candidate for cancer therapy with antibody-bound toxins that have the capacity to react with either cytoplasmic or nuclear targets in tumor cells or tumor-associated vascular endothelium. - Highlights: • Anti-TM4SF1 antibody 8G4 was efficiently taken up by cultured endothelial cells. • TM4SF1–8G4 internalization is dynamin-dependent but clathrin-independent. • TM4SF1–8G4 complexes internalize along microtubules to reach the perinuclear region. • Internalized TM4SF1–8G4 complexes pass through nuclear pores into the nucleus. • TM4SF1 is an attractive candidate for ADC cancer therapy.

  13. Total saponins of Panax ginseng induces K562 cell differentiation by promoting internalization of the erythropoietin receptor.

    PubMed

    Zuo, Guowei; Guan, Tao; Chen, Dilong; Li, Chunli; Jiang, Rong; Luo, Chunyan; Hu, Xiaoshu; Wang, Yaping; Wang, Jianwei

    2009-01-01

    Ginseng is a commonly used herbal medicine with a wide range of therapeutic benefits. Total saponins of Panax ginseng (TSPG) is one of the main effective components of ginseng. Our previous studies have shown that TSPG could promote the production of normal blood cells and inhibition of the leukemia cell proliferation. However, whether ginseng can induce the differentiation of leukemia cells is still unclear. This study was to examine the effect of TSPG or the combination of erythropoietin (EPO) and TSPG on the erythroid differentiation of K562 cells, and their corresponding mechanisms regarding erythropoietin receptor (EPOR) expression. Under light and electron microscopes, the TSPG- or TSPG + EPO-treated K562 cells showed a tendency to undergo erythroid differentiation; early and intermediate erythroblast-like cells were observed. Hemoglobin and HIR2 expressions were significantly increased. As determined by Western blotting analysis, the EPOR protein level in the K562 cytoplasmic membrane was significantly decreased after TSPG treatment, while its cytoplasm level increased in a dose-dependent manner. However, the total cellular EPOR level was unchanged. These results indicate that TSPG-induced erythroid differentiation of K562 cells may be accompanied by the internalization of EPOR. Thus, our study suggests that treatment with a combination of TSPG and EPO may induce erythroid differentiation of K562 cells at least in part through induction of EPOR internalization.

  14. Ultrastructural changes in hepatic sinusoidal endothelial cells acutely exposed to colloidal iron.

    PubMed

    Bassett, Mark L; Dahlstrom, Jane E; Taylor, Matthew C; Koina, Mark E; Maxwell, Lesley; Francis, Douglas; Jain, Sanjiv; McLean, Allan J

    2003-07-01

    Hepatic sinusoidal endothelial cells form an important interface between the vascular system, represented by the sinusoids, and the space of Disse that surrounds the hepatocyte microvilli. This study aimed to assess the light microscopic and ultrastructural effects of acute exposure of hepatic sinusoidal endothelial cells to colloidal iron by injection of rats with iron polymaltose. Eight minutes after a single intravenous injection of iron polymaltose sinusoidal endothelial cells showed defenestration, and thickening and layering as assessed by transmission electron microscopy. Kupffer cells and stellate cells appeared activated. These changes were not observed in control animals, experiments using equivalent doses of maltose, or experiments using colloidal carbon except for Kupffer cell activation due to colloidal carbon. No significant light microscopic changes were seen in study or control animals. The findings indicate that acute exposure to colloidal iron causes changes in hepatic sinusoidal endothelial cells, stellate cells and Kupffer cells. This may be the result of a direct toxic effect of iron or increased production of reactive oxygen species. These observations suggest a possible mechanism for defenestration of sinusoidal endothelial cells in ageing and in disease states.

  15. High efficient radiation stable AlGaAs/GaAs solar cells with internal Bragg reflector

    NASA Technical Reports Server (NTRS)

    Andreev, V. M.; Kalinovsky, V. S.; Komin, V. V.; Kochnev, I. V.; Lantratov, V. M.; Shvarts, M. Z.

    1995-01-01

    An investigation of solar cells based on AlGaAs/GaAs heterostructures with an internal Bragg reflector as the back-surface reflector is presented. The Bragg reflector is grown by low pressure metalorganic chemical vapor deposition on n-GaAs substrates in a horizontal resistively heated reactor. The Bragg reflector with its maximum reflectance centered at a wavelength of 860 nm consists of 12 pairs of AlAs/GaAs layers. The resulting Bragg reflector has a thickness of 0.072 micrometers for AlAs and 0.059 micrometers for GaAs. The multi-layered quasi-dielectric stack selectively reflects weakly absorbed photons with energies near to the GaAs band gap for a second pass through the photoactive region, thus increasing the photocurrent. The use of the Bragg reflector allows the external quantum efficiency to be increased in the long wavelength of the spectrum. The use of the Bragg reflector and an antireflective coating and prismatic cover allowed an efficiency of 23.4 percent to be obtained.

  16. Cerebral X-linked adrenoleukodystrophy: the international hematopoietic cell transplantation experience from 1982 to 1999.

    PubMed

    Peters, Charles; Charnas, Lawrence R; Tan, Ye; Ziegler, Richard S; Shapiro, Elsa G; DeFor, Todd; Grewal, Satkiran S; Orchard, Paul J; Abel, Susan L; Goldman, Anne I; Ramsay, Norma K C; Dusenbery, Kathryn E; Loes, Daniel J; Lockman, Lawrence A; Kato, Shunichi; Aubourg, Patrick R; Moser, Hugo W; Krivit, William

    2004-08-01

    Cerebral X-linked adrenoleukodystrophy (X-ALD) is a disorder of very-long-chain fatty acid metabolism, adrenal insufficiency, and cerebral demyelination. Death occurs within 2 to 5 years of clinical onset without hematopoietic cell transplantation (HCT). One hundred twenty-six boys with X-ALD received HCT from 1982 to 1999. Survival, engraftment, and acute graft-versus-host disease were studied. Degree of disability associated with neurologic and neuropsychological function and cerebral demyelination were evaluated before and after HCT. Complete data were available and analyzed for 94 boys with cerebral X-ALD. The estimated 5- and 8-year survival was 56%. The leading cause of death was disease progression. Donor-derived engraftment occurred in 86% of patients. Demyelination involved parietal-occipital lobes in 90%, leading to visual and auditory processing deficits in many boys. Overall 5-year survival of 92% in patients with 0 or 1 neurologic deficits and magnetic resonance imaging (MRI) severity score less than 9 before HCT was superior to survival for all others (45%; P <.01). Baseline neurologic and neuropsychological function, degree of disability, and neuroradiologic status predicted outcomes following HCT. In this first comprehensive report of the international HCT experience for X-ALD, we conclude that boys with early-stage disease benefit from HCT, whereas boys with advanced disease may be candidates for experimental therapies.

  17. An Innovative Educational Program for International Hematopoietic Cell Transplant Pediatric Patients.

    PubMed

    MacWilliams, MariaEugenia Álvarez; Lunar, Estela Cabrera; Denzen, Ellen M

    2016-02-03

    Pediatric patients and their families confront a difficult journey with hematopoietic cell transplantation. Understanding complex information, navigation, and coping with emotional, physical, and social changes in the patient, parents and family nucleus life could be quite challenging for all. In Spanish-speaking countries where transplant is emerging as an available treatment option, healthcare professionals encounter the lack or limited quality resources to educate pediatric patients and their families on a complex treatment process. Also, the level of literacy of each family varies widely, making transplant education very difficult to address in a simple and engaging way that could benefit all levels of literacy and foster well-being in the family as a whole, so they are prepared to make better informed decisions and decrease stress in all members. Super Sam versus the Marrow Monsters is a DVD resource made by the National Marrow Donor Program® (NMDP)/Be The Match® intended as an educational tool, to be shared with our U.S. network transplant centers to use when educating their pediatric patients. Hospital Angeles Lomas in the State of Mexico-member of the NMDP international network-has adopted the DVD with their pediatric audience, with very successful results, and demonstrates the benefits of expanding the initial intended use.

  18. Design Study Conducted of a Stirred and Perfused Specimen Chamber for Culturing Suspended Cells on the International Space Station

    NASA Technical Reports Server (NTRS)

    Nelson, Emily S.; Kizito, John P.

    2003-01-01

    A tightly knit numerical/experimental collaboration among the NASA Ames Research Center, NASA Glenn Research Center, and Payload Systems, Inc., was formed to analyze cell culturing systems for the International Space Station. The Cell Culture Unit is a facility scheduled for deployment on the space station by the Cell Culture Unit team at Ames. The facility houses multiple cell specimen chambers (CSCs), all of which have inlets and outlets to allow for replenishment of nutrients and for waste removal. For improved uniformity of nutrient and waste concentrations, each chamber has a pair of counterrotating stir bars as well. Although the CSC can be used to grow a wide variety of organic cells, the current study uses yeast as a model cell. Previous work identified groundbased protocols for perfusion and stirring to achieve yeast growth within the CSC that is comparable to that for yeast cultures grown in a shaken Ehrlenmeyer flask.

  19. RGS2 modulates the activity and internalization of dopamine D2 receptors in neuroblastoma N2A cells.

    PubMed

    Luessen, Deborah J; Hinshaw, Tyler P; Sun, Haiguo; Howlett, Allyn C; Marrs, Glen; McCool, Brian A; Chen, Rong

    2016-11-01

    Dysregulated expression and function of dopamine D2 receptors (D2Rs) are implicated in drug addiction, Parkinson's disease and schizophrenia. In the current study, we examined whether D2Rs are modulated by regulator of G protein signaling 2 (RGS2), a member of the RGS family that regulates G protein signaling via acceleration of GTPase activity. Using neuroblastoma 2a (N2A) cells, we found that RGS2 was immunoprecipitated by aluminum fluoride-activated Gαi2 proteins. RGS2 siRNA knockdown enhanced membrane [(35)S] GTPγS binding to activated Gαi/o proteins, augmented inhibition of cAMP accumulation and increased ERK phosphorylation in the presence of a D2/D3R agonist quinpirole when compared to scrambled siRNA treatment. These data suggest that RGS2 is a negative modulator of D2R-mediated Gαi/o signaling. Moreover, RGS2 knockdown slightly increased constitutive D2R internalization and markedly abolished quinpirole-induced D2R internalization assessed by immunocytochemistry. RGS2 knockdown did not compromise agonist-induced β-arrestin membrane recruitment; however, it prevents β-arrestin dissociation from the membrane after prolonged quinpirole treatment during which time β-arrestin moved away from the membrane in control cells. Additionally, confocal microscopy analysis of β-arrestin post-endocytic fate revealed that quinpirole treatment caused β-arrestin to translocate to the early and the recycling endosome in a time-dependent manner in control cells whereas translocation of β-arrestin to these endosomes did not occur in RGS2 knockdown cells. The impaired β-arrestin translocation likely contributed to the abolishment of quinpirole-stimulated D2R internalization in RGS2 knockdown cells. Thus, RGS2 is integral for β-arrestin-mediated D2R internalization. The current study revealed a novel regulation of D2R signaling and internalization by RGS2 proteins.

  20. Decoupling Internalization, Acidification and Phagosmal-Endosomal/Iysosomal Phagocytosis of Internalin A coated Beads in epithelial cells

    SciTech Connect

    Blanchette, C D; Woo, Y; Thomas, C; Shen, N; Sulchek, T A; Hiddessen, A L

    2008-12-22

    Phagocytosis has been extensively examined in 'professional' phagocytic cells using pH sensitive dyes. However, in many of the previous studies, a separation between the end of internalization, beginning of acidification and completion of phagosomal-endosomal/lysosomal fusion was not clearly established, and in several cases, it was treated as a one-step process. In addition, very little work has been done to systematically examine phagosomal maturation in 'non-professional' phagocytic cells, such as epithelial cells. Therefore, in this study, we developed a simple and novel method to decouple and accurately measure particle internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in Madin-Darby Canine Kidney (MDCK) and Caco-2 epithelial cells. Our method was developed using a pathogen mimetic system consisting of polystyrene beads coated with Internalin A (InlA), a membrane surface protein from Listeria monocytogenes known to trigger receptor-mediated internalization. We achieved independent measurements of the rates of internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in epithelial cells by combining the InlA-coated beads (InlA-beads) with antibody quenching, pH sensitive dyes and endosomal/lysosomal dyes, as follows: the rate of InlA bead internalization was measured via antibody quenching of a pH independent dye (Alexa488) conjugated to InlA-beads, the rate at which phagosomes containing internalized InlA beads became acidified was measured using a pH dependent dye (FITC) conjugated to the beads and the rate of phagosomal-endosomal/lysosomal fusion was measured using a combination of unlabeled InlA-beads and an endosomal/lysosomal dye. By performing these independent measurements under identical experimental conditions, we were able to decouple the three processes and establish time scales for each. In a separate set of experiments, we also exploited the phagosomal acidification process to demonstrate

  1. Adhesion of CdTe quantum dots on model membranes and internalization into RBL-2H3 cells.

    PubMed

    Zhang, Mengmeng; Wei, Xiaoran; Ding, Lei; Hu, Jingtian; Jiang, Wei

    2017-03-08

    Quantum dots (QDs) have attracted broad attention due to their special optical properties and promising prospect in medical and biological applications. However, the process of QDs on cell membrane is worth further investigations because such process may lead to harmful effects on organisms and also important for QD application. In this study, adhesion of amino- and carboxyl-coated CdTe QDs (A-QDs and C-QDs) on cell membrane and the subsequent internalization are studied using a series of endocytosis-free model membranes, including giant and small unilamellar vesicles, supported lipid bilayers and giant plasma membrane vesicles (GPMVs). The adhered QD amounts on model membranes are quantified by a quartz crystal microbalance. The CdTe QD adhesion on model membranes is governed by electrostatic forces. Positively charged A-QDs adhere on GPMV surface and passively penetrate the plasma membrane via endocytosis-free mechanism, but negatively charged C-QDs cannot. Rat basophilic leukemia (RBL-2H3) cells are exposed to CdTe QDs to monitor the QD internalization process. Both A- and C-QDs are internalized by RBL-2H3 cells mainly via endocytosis. CdTe QDs do not accumulate on the plasma membrane of living cells due to the fast endocytosis and the weakened electrostatic attraction in biological medium, resulting in low chance of passive penetration. The suspended cells after trypsin digestion take more QDs than the adherent cells. A-QDs cause lower cell viability than C-QDs, probably because the approach of positively charged QDs to cells is favored and the smaller aggregates of A-QDs.

  2. Cooperative Strategies for Enhancing Performance of Photothermal Therapy (PTT) Agent: Optimizing Its Photothermal Conversion and Cell Internalization Ability.

    PubMed

    Du, Baoji; Ma, Chongbo; Ding, Guanyu; Han, Xu; Li, Dan; Wang, Erkang; Wang, Jin

    2017-01-23

    Photothermal conversion ability (PCA) and cell internalization ability (CIA) are two key factors for determining the performance of photothermal agents. The previous studies mostly focus on improving the PCA by exploring new photothermal nanomaterials. Herein, the authors take the hybrids of graphene and gold nanostar (GGN) as an example to investigate the gradually enhanced phototherapy effect by changing the PCA and CIA of photothermal therapy (PTT) agent simultaneously. Based on the GGN, the GGN and the reduced GGN protected by bovine serum albumin (BSA) or BSA-FA (folic acid) are prepared, which are named as GGNB, rGGNB, and rGGNB-FA, respectively. The rGGNB showed an enhanced PCA compared to GGNB, leading to strong cell ablation. On the other hand, the 1,2-dioleoyl-3-trimethylammoniumpropan (DOTAP) can activate the endocytosis and promote the CIA of rGGNB, further help rGGNB to be more internalized into the cells. Finally, rGGNB-FA with the target ability can make itself further internalized into the cells with the aid of DOTAP, which can significantly destroy the cancer cells even at the low laser density of 0.3 W cm(-2) . Therefore, a new angle of view is brought out for researching the PTT agents of high performance.

  3. Materials on the International Space Station - Forward Technology Solar Cell Experiment

    NASA Technical Reports Server (NTRS)

    Walters, R. J.; Garner, J. C.; Lam, S. N.; Vazquez, J. A.; Braun, W. R.; Ruth, R. E.; Lorentzen, J. R.; Bruninga, R.; Jenkins, P. P.; Flatico, J. M.

    2005-01-01

    This paper describes a space solar cell experiment currently being built by the Naval Research Laboratory (NRL) in collaboration with NASA Glenn Research Center (GRC), and the US Naval Academy (USNA). The experiment has been named the Forward Technology Solar Cell Experiment (FTSCE), and the purpose is to rapidly put current and future generation space solar cells on orbit and provide validation data for these technologies. The FTSCE is being fielded in response to recent on-orbit and ground test anomalies associated with space solar arrays that have raised concern over the survivability of new solar technologies in the space environment and the validity of present ground test protocols. The FTSCE is being built as part of the Fifth Materials on the International Space Station (MISSE) Experiment (MISSE-5), which is a NASA program to characterize the performance of new prospective spacecraft materials when subjected to the synergistic effects of the space environment. Telemetry, command, control, and communication (TNC) for the FTSCE will be achieved through the Amateur Satellite Service using the PCSat2 system, which is an Amateur Radio system designed and built by the USNA. In addition to providing an off-the-shelf solution for FTSCE TNC, PCSat2 will provide a communications node for the Amateur Radio satellite system. The FTSCE and PCSat2 will be housed within the passive experiment container (PEC), which is an approximately 2ft x2ft x 4in metal container built by NASA Langley Research Center (NASA LaRC) as part of the MISSE-5 program. NASA LaRC has also supplied a thin film materials experiment that will fly on the exterior of the thermal blanket covering the PCSat2. The PEC is planned to be transported to the ISS on a Shuttle flight. The PEC will be mounted on the exterior of the ISS by an astronaut during an extravehicular activity (EVA). After nominally one year, the PEC will be retrieved and returned to Earth. At the time of writing this paper, the

  4. Hump-shaped internal collection efficiency of degraded a-Si:H {ital p-i-n} solar cells

    SciTech Connect

    Smole, F.; Topic, M.; Furlan, J.; Kusian, W.

    1997-07-01

    Measured internal collection efficiency (ICE) characteristics of annealed and degraded a-Si:H p-i-n solar cells were used for an analysis of their internal behavior. Using the numerical simulator ASPIN, simulations were performed in order to fit and explain pronounced hump-shaped voltage-dependent ICE characteristics of degraded structures under weak short-wavelength illumination. Agreement with measured ICE characteristics for a degraded cell was obtained only if in addition to the introduction of light-induced dangling bond defect states, their capture cross sections were also increased, in particular the capture cross section for the charged defect states were increased. This caused a change in the occupancy of defect states at the p-i interface and front part of the i layer under forward biases. Consequently, the electric field in the front part of the cell was sustained under higher forward biases, resulting in recovery of the ICE. {copyright} {ital 1997 American Institute of Physics.}

  5. Cellular internalization, transcellular transport, and cellular effects of silver nanoparticles in polarized Caco-2 cells following apical or basolateral exposure.

    PubMed

    Imai, Shunji; Morishita, Yuki; Hata, Tomoyuki; Kondoh, Masuo; Yagi, Kiyohito; Gao, Jian-Qing; Nagano, Kazuya; Higashisaka, Kazuma; Yoshioka, Yasuo; Tsutsumi, Yasuo

    2017-03-11

    When considering the safety of ingested nanomaterials, it is important to quantitate their transfer across intestinal cells; however, little information exists about the effects of nanomaterial size or exposure side (apical versus basolateral epithelial surface) on nanomaterial transfer. Here, we examined cellular internalization and transcellular transport, and the effects of nanomaterials on Caco-2 monolayers after apical or basolateral exposure to Ag or Au nanoparticles with various sizes. After apical treatment, both internalization and transfer to the basolateral side of the monolayers were greater for smaller Ag nanoparticles than for larger Ag nanoparticles. In contrast, after basolateral treatment, larger Ag nanoparticles were more internalized than smaller Ag nanoparticles, but the transfer to the apical side was greater for smaller Ag nanoparticles. Au nanoparticles showed different rules of internalization and transcellular transport compared with Ag nanoparticles. Furthermore, the paracellular permeability of the Caco-2 monolayers was temporarily increased by Ag nanoparticles (5 μg/mL; diameters, ≤10 nm) following basolateral but not apical exposure. We conclude that the internalization, transfer, and effects of nanomaterials in epithelial cell monolayers depend on the size and composition of nanomaterials, and the exposure side.

  6. Structural/functional relationships between internal and external MSH receptors: modulation of expression in Cloudman melanoma cells by UVB radiation

    SciTech Connect

    Chakraborty, A.K.; Orlow, S.J.; Bolognia, J.L.; Pawelek, J.M. )

    1991-04-01

    Expression of internal receptors for MSH is an important criterion for responsiveness to MSH by Cloudman melanoma cells. Here, we show that internal and external receptors for MSH are of identical molecular weights (50-53 kDa) and share common antigenic determinants, indicating a structural relationship between the 2 populations of molecules. The internal receptors co-purified with a sub-cellular fraction highly enriched for small vesicles, many of which were coated. Ultraviolet B light (UVB) acted synergistically with MSH to increase tyrosinase activity and melanin content of cultured Cloudman melanoma cells, consistent with previous findings in the skin of mice and guinea pigs. Preceding the rise in tyrosinase activity in cultured cells, UVB elicited a decrease in internal MSH binding sites and a concomitant increase in external sites. The time frame for the UVB effects on MSH receptors and melanogenesis, 48 hours, was similar to that for a response to solar radiation in humans. Together, the results indicate a key role for MSH receptors in the induction of melanogenesis by UVB and suggest a potential mechanism of action for UVB: redistribution of MSH receptors with a resultant increase in cellular responsiveness to MSH.

  7. Challenges and Opportunities for International Cooperative Studies in Pediatric Hematopoeitic Cell Transplantation: Priorities of the Westhafen Intercontinental Group

    PubMed Central

    Schultz, Rudolph Kirk R.; Baker, Kevin Scott; Boelens, Jaap J.; Bollard, Catherine M.; Egeler, R. Maarten; Cowan, Mort; Ladenstein, Ruth; Lankester, Arjan; Locatelli, Franco; Lawitschka, Anita; Levine, John E.; Loh, Mignon; Nemecek, Eneida; Niemeyer, Charlotte; Prasad, Vinod K.; Rocha, Vanderson; Shenoy, Shalini; Strahm, Brigitte; Veys, Paul; Wall, Donna; Bader, Peter; Grupp, Stephan A.; Pulsipher, Michael A.; Peters, Christina

    2014-01-01

    More than 20% of allogeneic hematopoietic cell transplantations (HCTs) are performed in children and adolescents at a large number of relatively small centers. Unlike adults, at least one-third of HCTs in children are performed for rare, nonmalignant indications. Clinical trials to improve HCT outcomes in children have been limited by small numbers and these pediatric-specific features. The need for a larger number of pediatric HCT centers to participate in trials has led to the involvement of international collaborative groups. Representatives of the Pediatric Blood and Marrow Transplant Consortium, European Group for Blood and Marrow Transplantation’s Pediatric Working Group, International Berlin-Frankfurt-Munster (iBFm) Stem Cell Transplantation Committee, and Children’s Oncology Group’s Hematopoietic Stem Cell Transplantation Discipline Committee met on October 3, 2012, in Frankfurt, Germany to develop a consensus on the highest priorities in pediatric HCT. In addition, it explored the creation of an international consortium to develop studies focused on HCT in children and adolescents. This meeting led to the creation of an international HCT network, dubbed the Westhafen Intercontinental Group, to develop worldwide priorities and strategies to address pediatric HCT issues. This review outlines the priorities of need as identified by this consensus group. PMID:23883618

  8. Internal reforming characteristics of cermet supported solid oxide fuel cell using yttria stabilized zirconia fed with partially reformed methane

    NASA Astrophysics Data System (ADS)

    Momma, Akihiko; Takano, Kiyonami; Tanaka, Yohei; Negishi, Akira; Kato, Ken; Nozaki, Ken; Kato, Tohru; Ichigi, Takenori; Matsuda, Kazuyuki; Ryu, Takashi

    In order to investigate the internal reforming characteristics in a cermet supported solid oxide fuel cell (SOFC) using YSZ as the electrolyte, the concentration profiles of the gaseous species along the gas flow direction in the anode were measured. Partially reformed methane using a pre-reformer kept at a constant temperature is supplied to the center of the cell which is operated with a seal-less structure at the gas outlet. The anode gas is sucked in via silica capillaries to the initially evacuated gas tanks. The process is simultaneously carried out using five sampling ports. The sampled gas is analyzed by a gas chromatograph. Most of the measurements are made at the cell temperature (T cell) of 750 °C and at various temperatures of the pre-reformer (T ref) with various fuel utilizations (U f) of the cell. The composition of the fuel at the inlet of the anode was confirmed to be almost the same as that theoretically calculated assuming equilibrium at the temperature of the pre-reformer. The effect of internal reforming in the anode is clearly observed as a steady decrease in the methane concentration along the flow axis. The effect of the water-gas shift reaction is also observed as a decrease in the CO 2 concentration and an increase of CO concentration around the gas inlet region, as the water-gas shift reaction inversely proceeds when T cell is higher than T ref. The diffusion of nitrogen from the seal-less outermost edge is observed, and the diffusion is confirmed to be more significant as U f decreases. The observations are compared with the results obtained by the SOFC supported by lanthanum gallate electrolyte. With respect to the internal reforming performance, the cell investigated here is found to be more effective when compared to the previously reported electrolyte supported cell.

  9. Imaging-based analysis of liposome internalization to macrophage cells: Effects of liposome size and surface modification with PEG moiety.

    PubMed

    Lee, Jae Sun; Hwang, Sang Youn; Lee, E K

    2015-12-01

    Liposome is one of the frequently used carriers for active targeting systems in vivo. Such parameters as its size, surface charge, and surface modifiers are known to influence the liposome uptake by macrophage cells. In this study, we investigated the effects of liposome size and polyethylene glycol (PEG) surface modifier on the liposomal internalization to murine macrophage (RAW-264.7), by using an imaging analysis technique. Three different sized liposomes (100, 200, and 400 nm in nominal diameter) labeled with rhodamine fluorescence were used. Liposome internalization appeared to reach a pseudo-steady plateau in about 5h incubation, and most of the internalized liposomes were seen to accumulate in the cytosol including cellular extensions. The maximum fluorescent density from the internalized liposomes was similar between 100 nm and 200 nm liposomes. However, that of the larger 400 nm liposome was approximately 1.7 times higher than the others, confirming the previous report that the larger the liposomes are the higher the degree of internalization is. When the outside of the 200 nm liposomes was modified with biocompatible anchor molecule (BAM) consisting of PEG (ca. 2kD molecular weight) moiety, the endocytosis was indeed reduced by about 2.1-fold, despite the increase of the hydrodynamic size due to BAM conjugation. This fluorescence-based cellular imaging analysis can be used to quantitatively monitor and optimize cellular internalization systems.

  10. RD&D Cooperation for the Development of Fuel Cell, Hybrid and Electric Vehicles within the International Energy Agency: Preprint

    SciTech Connect

    Telias, G.; Day, K.; Dietrich, P.

    2011-01-01

    Annex XIII on 'Fuel Cell Vehicles' of the Implementing Agreement Hybrid and Electric Vehicles of the International Energy Agency has been operating since 2006, complementing the ongoing activities on battery and hybrid electric vehicles within this group. This paper provides an overview of the Annex XIII final report for 2010, compiling an up-to-date, neutral, and comprehensive assessment of current trends in fuel cell vehicle technology and related policy. The technological description includes trends in system configuration as well as a review of the most relevant components including the fuel cell stack, batteries, and hydrogen storage. Results from fuel cell vehicle demonstration projects around the world and an overview of the successful implementation of fuel cells in specific transport niche markets will also be discussed. The final section of this report provides a detailed description of national research, development, and demonstration (RD&D) efforts worldwide.

  11. An internal ribosome entry site (IRES) mutant library for tuning expression level of multiple genes in mammalian cells.

    PubMed

    Koh, Esther Y C; Ho, Steven C L; Mariati; Song, Zhiwei; Bi, Xuezhi; Bardor, Muriel; Yang, Yuansheng

    2013-01-01

    A set of mutated Encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) elements with varying strengths is generated by mutating the translation initiation codons of 10(th), 11(th), and 12(th) AUG to non-AUG triplets. They are able to control the relative expression of multiple genes over a wide range in mammalian cells in both transient and stable transfections. The relative strength of each IRES mutant remains similar in different mammalian cell lines and is not gene specific. The expressed proteins have correct molecular weights. Optimization of light chain over heavy chain expression by these IRES mutants enhances monoclonal antibody expression level and quality in stable transfections. Uses of this set of IRES mutants can be extended to other applications such as synthetic biology, investigating interactions between proteins and its complexes, cell engineering, multi-subunit protein production, gene therapy, and reprogramming of somatic cells into stem cells.

  12. Stomatal and pavement cell density linked to leaf internal CO2 concentration

    PubMed Central

    Šantrůček, Jiří; Vráblová, Martina; Šimková, Marie; Hronková, Marie; Drtinová, Martina; Květoň, Jiří; Vrábl, Daniel; Kubásek, Jiří; Macková, Jana; Wiesnerová, Dana; Neuwithová, Jitka; Schreiber, Lukas

    2014-01-01

    Background and Aims Stomatal density (SD) generally decreases with rising atmospheric CO2 concentration, Ca. However, SD is also affected by light, air humidity and drought, all under systemic signalling from older leaves. This makes our understanding of how Ca controls SD incomplete. This study tested the hypotheses that SD is affected by the internal CO2 concentration of the leaf, Ci, rather than Ca, and that cotyledons, as the first plant assimilation organs, lack the systemic signal. Methods Sunflower (Helianthus annuus), beech (Fagus sylvatica), arabidopsis (Arabidopsis thaliana) and garden cress (Lepidium sativum) were grown under contrasting environmental conditions that affected Ci while Ca was kept constant. The SD, pavement cell density (PCD) and stomatal index (SI) responses to Ci in cotyledons and the first leaves of garden cress were compared. 13C abundance (δ13C) in leaf dry matter was used to estimate the effective Ci during leaf development. The SD was estimated from leaf imprints. Key Results SD correlated negatively with Ci in leaves of all four species and under three different treatments (irradiance, abscisic acid and osmotic stress). PCD in arabidopsis and garden cress responded similarly, so that SI was largely unaffected. However, SD and PCD of cotyledons were insensitive to Ci, indicating an essential role for systemic signalling. Conclusions It is proposed that Ci or a Ci-linked factor plays an important role in modulating SD and PCD during epidermis development and leaf expansion. The absence of a Ci–SD relationship in the cotyledons of garden cress indicates the key role of lower-insertion CO2 assimilation organs in signal perception and its long-distance transport. PMID:24825295

  13. Role of NK, NKT cells and macrophages in liver transplantation

    PubMed Central

    Fahrner, René; Dondorf, Felix; Ardelt, Michael; Settmacher, Utz; Rauchfuss, Falk

    2016-01-01

    Liver transplantation has become the treatment of choice for acute or chronic liver disease. Because the liver acts as an innate immunity-dominant organ, there are immunological differences between the liver and other organs. The specific features of hepatic natural killer (NK), NKT and Kupffer cells and their role in the mechanism of liver transplant rejection, tolerance and hepatic ischemia-reperfusion injury are discussed in this review. PMID:27468206

  14. When the Cell Stress Society International became South American: meeting report of the IX International Workshop on the Molecular Biology of Stress Responses.

    PubMed

    Galigniana, Mario D

    2013-01-01

    The International Workshop on the Molecular Biology of the Stress Response organized by the Cell Stress Society International was held in Porto Alegre, Brazil, on May 27-30, 2012, as part of the development of the Latin American Chapter of the Society, a superb initiative headed by Drs. Antonio De Maio and Larry Hightower. The meeting took place in the wonderful facilities of the Pontifícia Universidade do Rio Grande do Sul (PUCRS) and was warmly chaired by Professor Cristina Bonorino. Thirty-four invited speakers presented their work to more than 200 scientists and, even more importantly, to 150 registered students, who were the main beneficiaries of the meeting. The first day of the workshop was dedicated to an educational program for students, young investigators, and participants who were unfamiliar with the field of molecular chaperones and the stress response. Speakers in this pre-workshop were Dr. Harm Kampinga, Dr. Lea Sistonen, Dr. Larry Hightower, Dr. Ivor Benjamin, Dr. Daniel Ciocca, and Dr. Linda Hendershot. Then, the scientific sessions discussed below followed.

  15. Evolutionary selection of new breast cancer cell-targeting peptides and phages with the cell-targeting peptides fully displayed on the major coat and their effects on actin dynamics during cell internalization

    PubMed Central

    Abbineni, Gopal; Modali, Sita; Safiejko-Mroczka, Barbara; Petrenko, Valery A.; Mao, Chuanbin

    2010-01-01

    Filamentous phage as a bacteria-specific virus can be conjugated with an anti-cancer drug and has been proposed to serve as a carrier to deliver drugs to cancer cells for targeted therapy. However, how cell-targeting filamentous phage alone affects cancer cell biology is unclear. Phage libraries provide an inexhaustible reservoir of new ligands against tumor cells and tissues that have potential therapeutic and diagnostic applications in cancer treatment. Some of these identified ligands might stimulate various cell responses. Here we identified new cell internalizing peptides (and the phages with such peptides fused to each of ~3900 copies of their major coat protein) using landscape phage libraries and for the first time investigated the actin dynamics when selected phages are internalized into the SKBR-3 breast cancer cells. Our results show that phages harboring VSSTQDFP and DGSIPWST peptides could selectively internalize into the SKBR-3 breast cancer cells with high affinity, and also show rapid involvement of membrane ruffling and re-arrangements of actin cytoskeleton during the phage entry. The actin dynamics was studied by using live cell and fluorescence imaging. The cell-targeting phages were found to enter breast cancer cells through energy dependent mechanism and phage entry interferes with actin dynamics, resulting in reorganization of actin filaments and increased membrane rufflings in SKBR-3 cells. These results suggest that, when phage enters epithelial cells, it triggers transient changes in the host cell actin cytoskeleton. This study also shows that using multivalent phage libraries considerably increases the repertoire of available cell-internalizing ligands with potential applications in targeted drug delivery, imaging, molecular monitoring and profiling of breast cancer cells. PMID:20735141

  16. Comparative analysis of the internalization of the macrophage receptor sialoadhesin in human and mouse primary macrophages and cell lines.

    PubMed

    De Schryver, Marjorie; Leemans, Annelies; Pintelon, Isabel; Cappoen, Davie; Maes, Louis; Caljon, Guy; Cos, Paul; Delputte, Peter L

    2016-11-21

    Sialoadhesin (Sn) is a surface receptor expressed on resident macrophages with the ability to bind with sialic acids. During inflammation, an upregulation of Sn is observed. Upon binding of monoclonal antibodies to Sn, the receptor becomes internalized and this has been observed in multiple species. The latter characteristic, combined with the strong upregulation of Sn on inflammatory macrophages and the fact that Sn-positive macrophages contribute to certain inflammatory diseases, makes Sn an interesting entry portal for phenotype-modulating or cytotoxic drugs. Such drugs or toxins can be linked to Sn-specific antibodies which should enable their targeted uptake by macrophages. However, the activity of such drugs depends not only on their internalization but also on the intracellular trafficking and final fate in the endolysosomal system. Although information is available for porcine Sn, the detailed mechanisms of human and mouse Sn internalization and subsequent intracellular trafficking are currently unknown. To allow development of Sn-targeted therapies, differences across species and cellular background need to be characterized in more detail. In the current report, we show that internalization of human and mouse Sn is dynamin-dependent and clathrin-mediated, both in primary macrophages and CHO cell lines expressing a recombinant Sn. In primary macrophages, internalized Sn-specific F(ab')2 fragments are located mostly in the early endosomes. With Fc containing Sn-specific antibodies, there is a slight shift towards lysosomal localization in mouse macrophages, possibly because of an interaction with Fc receptors. Surprisingly, in CHO cell lines expressing Sn, there is a predominant lysosomal localization. Our results show that the mechanism of Sn internalization and intracellular trafficking is concurrent in the tested species. The cellular background in which Sn is expressed and the type of antibody used can affect the intracellular fate, which in turn can

  17. Biochemical and microscopic evidence for the internalization and degradation of heparin-containing mast cell granules by bovine endothelial cells

    SciTech Connect

    Atkins, F.M.; Friedman, M.M.; Metcalfe, D.D.

    1985-03-01

    Incubation of (/sup 35/S)heparin-containing mast cell granules with cultured bovine endothelial cells was followed by the appearance of /sup 35/S-granule-associated radioactivity within the endothelial cells and a decrease in radioactivity in the extracellular fluid. These changes occurred during the first 24 hours of incubation and suggested ingestion of the mast cell granules by the endothelial cells. Periodic electron microscopic examination of the monolayers confirmed this hypothesis by demonstrating apposition of the granules to the plasmalemma of endothelial cells, which was followed by the engulfment of the granules by cytoplasmic projections. Under light microscopic examination, mast cell granules within endothelial cells then appeared to undergo degradation. The degradation of (/sup 35/S)heparin in mast cell granules was demonstrated by a decrease in the amount of intracellular (/sup 35/S)heparin proteoglycan after 24 hours and the appearance of free (/sup 35/S)sulfate in the extracellular compartment. Intact endothelial cells were more efficient at degrading (/sup 35/S)heparin than were cell lysates or cell supernatants. These data provide evidence of the ability of endothelial cells to ingest mast cell granules and degrade native heparin that is presented as a part of the mast cell granule.

  18. Internalization of mesoporous silica nanoparticles induces transient but not sufficient osteogenic signals in human mesenchymal stem cells

    SciTech Connect

    Huang, D.-M. Chung, T.-H.; Hung, Y.; Lu, F.; Wu, S.-H.; Mou, C.-Y.; Yao, M.; Chen, Y.-C.

    2008-09-01

    The biocompatibility of nanoparticles is the prerequisite for their applications in biomedicine but can be misleading due to the absence of criteria for evaluating the safety and toxicity of those nanomaterials. Recent studies indicate that mesoporous silica nanoparticles (MSNs) can easily internalize into human mesenchymal stem cells (hMSCs) without apparent deleterious effects on cellular growth or differentiation, and hence are emerging as an ideal stem cell labeling agent. The objective of this study was to thoroughly investigate the effect of MSNs on osteogenesis induction and to examine their biocompatibility in hMSCs. Uptake of MSNs into hMSCs did not affect the cell viability, proliferation and regular osteogenic differentiation of the cells. However, the internalization of MSNs indeed induced actin polymerization and activated the small GTP-bound protein RhoA. The MSN-induced cellular protein responses as believed to cause osteogenesis of hMSCs did not result in promotion of regular osteogenic differentiation as analyzed by cytochemical stain and protein activity assay of alkaline phosphatase (ALP). When the effect of MSNs on ALP gene expression was further examined by reverse transcriptase polymerase chain reaction, MSN-treated hMSCs were shown to have significantly higher mRNA expression than control cells after 1-hour osteogenic induction. The induction of ALP gene expression by MSNs, however, was absent in cells after 1-day incubation with osteogenic differentiation. Together our results show that the internalization of MSNs had a significant effect on the transient protein response and osteogenic signal in hMSCs, thereby suggesting that the effects of nanoparticles on diverse aspects of cellular activities should be carefully evaluated even though the nanoparticles are generally considered as biocompatible at present.

  19. Internalization and Down-Regulation of the ALK Receptor in Neuroblastoma Cell Lines upon Monoclonal Antibodies Treatment

    PubMed Central

    Mazot, Pierre; Cazes, Alex; Dingli, Florent; Degoutin, Joffrey; Irinopoulou, Théano; Boutterin, Marie-Claude; Lombard, Bérangère; Loew, Damarys; Hallberg, Bengt; Palmer, Ruth Helen; Delattre, Olivier

    2012-01-01

    Recently, activating mutations of the full length ALK receptor, with two hot spots at positions F1174 and R1275, have been characterized in sporadic cases of neuroblastoma. Here, we report similar basal patterns of ALK phosphorylation between the neuroblastoma IMR-32 cell line, which expresses only the wild-type receptor (ALKWT), and the SH-SY5Y cell line, which exhibits a heterozygous ALK F1174L mutation and expresses both ALKWT and ALKF1174L receptors. We demonstrate that this lack of detectable increased phosphorylation in SH-SY5Y cells is a result of intracellular retention and proteasomal degradation of the mutated receptor. As a consequence, in SH-SY5Y cells, plasma membrane appears strongly enriched for ALKWT whereas both ALKWT and ALKF1174L were present in intracellular compartments. We further explored ALK receptor trafficking by investigating the effect of agonist and antagonist mAb (monoclonal antibodies) on ALK internalization and down-regulation, either in SH-SY5Y cells or in cells expressing only ALKWT. We observe that treatment with agonist mAbs resulted in ALK internalization and lysosomal targeting for receptor degradation. In contrast, antagonist mAb induced ALK internalization and recycling to the plasma membrane. Importantly, we correlate this differential trafficking of ALK in response to mAb with the recruitment of the ubiquitin ligase Cbl and ALK ubiquitylation only after agonist stimulation. This study provides novel insights into the mechanisms regulating ALK trafficking and degradation, showing that various ALK receptor pools are regulated by proteasome or lysosome pathways according to their intracellular localization. PMID:22479414

  20. Differential receptor targeting of liver cells using 99mTc-neoglycosylated human serum albumins.

    PubMed

    Kim, Sungeun; Jeong, Jae Min; Hong, Mee Kyung; Jang, Ja-June; Lee, Jaetae; Lee, Dong Soo; Chung, June-Key; Lee, Myung Chul

    2008-01-01

    Neolactosyl human serum albumin (LSA) targets asialoglycoprotein receptor and shows high liver uptake due to accumulation in hepatocytes. Although neomannosyl human serum albumin (MSA) also shows high liver uptake, it has been reported to be taken up by Kupffer cells and endothelial cells. We compared the biological properties of LSA and MSA. 99mTc-LSA and 99mTc-MSA biodistribution in mice were investigated after intravenous injection. In vivo localization of rhodaminisothiocyanate (RITC)-LSA and fluoresceineisothiocyanate (FITC)-MSA were investigated in mouse liver. Excretion routes of 99mTc-LSA and 99mTc-MSA metabolites were examined. Both 99mTc-LSA and 99mTc-MSA showed high liver uptakes. RITC-LSA was taken up by hepatocytes whereas FITC-MSA was taken up by Kupffer cells and endothelial cells. 99mTc-MSA showed higher spleen and kidney uptakes than 99mTc-LSA. 99mTc-LSA metabolites excreted in urine and feces accounted for 44.4 and 50.0% of 99mTc-LSA injected, respectively, while 99mTc-MSA metabolites accounted for 51.5 and 10.3%, respectively. In conclusion, LSA is specifically taken up by hepatcytes while MSA by Kupffer cells and endothelial cells. After taken up by the liver, LSA is metabolized by the hepatocytes and then excreted through both the hepatobiliary tract and kidney, whereas MSA is metabolized by Kupffer cells and endoghelial cells and then excreted mainly through the kidney.

  1. Distinct CPT-induced deaths in lung cancer cells caused by clathrin-mediated internalization of CP micelles

    NASA Astrophysics Data System (ADS)

    Liu, Yu-Sheng; Cheng, Ru-You; Lo, Yu-Lun; Hsu, Chin; Chen, Su-Hwei; Chiu, Chien-Chih; Wang, Li-Fang

    2016-02-01

    We previously synthesized a chondroitin sulfate-graft-poly(ε-caprolactone) copolymer (H-CP) with a high content of poly(ε-caprolactone) (18.7 mol%), which self-assembled in water into a rod-like micelle to encapsulate hydrophobic camptothecin (CPT) in the core (micelle/CPT) for tumor-targeted drug delivery. As a result of the recognition of the micelle by CD44, the micelle/CPT entered CRL-5802 cells efficiently and released CPT efficaciously, resulting in higher tumor suppression than commercial CPT-11. In this study, H1299 cells were found to have a higher CD44 expression than CRL-5802 cells. However, the lower CD44-expressing CRL-5802 cells had a higher percentage of cell death and higher cellular uptake of the micelle/CPT than the higher CD44-expressing H1299 cells. Examination of the internalization pathway of the micelle/CPT in the presence of different endocytic chemical inhibitors showed that the CRL-5802 cells involved clathrin-mediated endocytosis, which was not found in the H1299 cells. Analysis of the cell cycle of the two cell lines exposed to the micelle/CPT revealed that the CRL-5802 cells arrested mainly in the S phase and the H1299 cells arrested mainly in the G2-M phase. A consistent result was also found in the evaluation of γ-H2AX expression, which was about three-fold higher in the CRL-5802 cells than in the H1299 cells. A near-infrared dye, IR780, was encapsulated into the micelle to observe the in vivo biodistribution of the micelle/IR780 in tumor-bearing mice. The CRL-5802 tumor showed a higher fluorescence intensity than the H1299 tumor at any tracing time after 1 h. Thus we tentatively concluded that CRL-5802 cells utilized the clathrin-mediated internalization pathway and arrested in the S phase on exposure to the micelle/CPT; all are possible reasons for the better therapeutic outcome in CRL-5802 cells than in H1299 cells.We previously synthesized a chondroitin sulfate-graft-poly(ε-caprolactone) copolymer (H-CP) with a high content of

  2. An analysis of myeloma plasma cell phenotype using antibodies defined at the IIIrd International Workshop on Human Leucocyte Differentiation Antigens.

    PubMed Central

    Jackson, N; Ling, N R; Ball, J; Bromidge, E; Nathan, P D; Franklin, I M

    1988-01-01

    Fresh bone marrow from 43 cases of myeloma and three cases of plasma cell leukaemia has been phenotyped both by indirect immune-rosetting and, on fixed cytospin preparations, by indirect immunofluorescence. Both clustered and unclustered B cell associated antibodies from the IIIrd International Workshop on Human Leucocyte Differentiation Antigens were used. The results confirm the lack of many pan-B antigens on the surface of myeloma plasma cells, i.e. CD19-23, 37, 39, w40. Strong surface reactivity is seen with CD38 antibodies and with one CD24 antibody (HB8). Weak reactions are sometimes obtained with CD9, 10 and 45R. On cytospin preparations CD37, 39 and w40 are sometimes weakly positive, and anti-rough endoplasmic reticulum antibodies are always strongly positive. Specific and surface-reacting antiplasma cell antibodies are still lacking. PMID:3048803

  3. Empirical analysis of contributing factors to heating in lithium-ion cells: Anode entropy versus internal resistance

    NASA Astrophysics Data System (ADS)

    Srinivasan, Rengaswamy; Carkhuff, Bliss G.

    2013-11-01

    Charging a battery beyond its maximum capacity can lead both to cell overheating and to the venting of gasses. A fundamental understanding of cell heating could lead to the development of real-time sensors that anticipate and avert catastrophic battery failure. Joule heating (also called ohmic or resistive heating) from cell internal resistance (Rint) dominates the overall thermal energy (ΔQ) generated during charging. Contrary to prior hypotheses, though, Joule heating does not appear to contribute to venting observed during overcharging. In this manuscript, we examine an alternate hypothesis, that heat released by the entropy change in the anode (ΔSanode) and the concomitant increase in the anode temperature (Tanode) triggers the venting. Using our recently developed non-invasive battery internal temperature (BIT) sensor to monitor Tanode, we separated the contributions of ΔSanode, Rint and the anode resistance (Ranode) to ΔQ. These quantities were tracked during constant current charging of a 18650 Lithium-ion cell, from zero state of charge (SoC) to overcharge. The resulting analysis suggests that anode entropy change is more important than resistive heating resulting from Ranode to the overall thermal energy. Anode entropy measurements, enabled by the BIT sensor, might serve as an alternative or adjunct method for anticipating and avoiding cell venting events.

  4. Slowing of velocity during isotonic shortening in single isolated smooth muscle cells. Evidence for an internal load

    PubMed Central

    1990-01-01

    In single smooth muscle cells, shortening velocity slows continuously during the course of an isotonic (fixed force) contraction (Warshaw, D.M. 1987. J. Gen. Physiol. 89:771-789). To distinguish among several possible explanations for this slowing, single smooth muscle cells were isolated from the gastric muscularis of the toad (Bufo marinus) and attached to an ultrasensitive force transducer and a length displacement device. Cells were stimulated electrically and produced maximum stress of 144 mN/mm2. Cell force was then reduced to and maintained at preset fractions of maximum, and cell shortening was allowed to occur. Cell stiffness, a measure of relative numbers of attached crossbridges, was measured during isotonic shortening by imposing 50-Hz sinusoidal force oscillations. Continuous slowing of shortening velocity was observed during isotonic shortening at all force levels. This slowing was not related to the time after the onset of stimulation or due to reduced isometric force generating capacity. Stiffness did not change significantly over the course of an isotonic shortening response, suggesting that the observed slowing was not the result of reduced numbers of cycling crossbridges. Furthermore, isotonic shortening velocity was better described as a function of the extent of shortening than as a function of the time after the onset of the release. Therefore, we propose that slowing during isotonic shortening in single isolated smooth muscle cells is the result of an internal load that opposes shortening and increases as cell length decreases. PMID:2121895

  5. Extracellularly occurring histone H1 mediates the binding of thyroglobulin to the cell surface of mouse macrophages.

    PubMed Central

    Brix, K; Summa, W; Lottspeich, F; Herzog, V

    1998-01-01

    Thyroglobulin is the major secretory protein of thyroid epithelial cells. Part of thyroglobulin reaches the circulation of vertebrates by transcytosis across the epithelial wall of thyroid follicles. Clearance of thyroglobulin from the circulation occurs within the liver via internalization of thyroglobulin by macrophages. Here we have analyzed the interaction of thyroglobulin with the cell surface of J774 macrophages with the aim to identify the possible thyroglobulin-binding sites on macrophages. Binding of thyroglobulin to J774 cells was saturated at approximately 100 nM thyroglobulin with a Kd of 50 nM, and it was competed by the ligand itself. Preincubation of J774 cells with thyroglobulin resulted in downregulation of thyroglobulin-binding sites, indicating internalization of thyroglobulin and its binding proteins. By affinity chromatography, two proteins from J774 cells were identified as thyroglobulin-binding proteins with an apparent molecular mass of approximately 33 kD. Unexpectedly, both proteins were identified as histone H1 by protein sequencing. The occurrence of histone H1 at the plasma membrane was further proven by biotinylation or immunolabeling of J774 cells. The in vitro interaction between histone H1 and thyroglobulin was analyzed by surface plasmon resonance that revealed a Kd at 46 nM. In situ, histone H1 was colocalized to FITC-Tg-containing endocytic compartments of Kupffer cells, i.e., liver macrophages. We conclude that histone H1 is detectable at the cell surface of macrophages where it serves as a thyroglobulin-binding protein and mediates thyroglobulin endocytosis. PMID:9664069

  6. Tyrosine phosphorylation of the insulin receptor is not required for receptor internalization: studies in 2,4-dinitrophenol-treated cells

    SciTech Connect

    Backer, J.M.; Kahn, C.R.; White, M.F.

    1989-05-01

    The relation between insulin-stimulated autophosphorylation of the insulin receptor and internalization of the receptor was studied in Fao rat hepatoma cells. Treatment of Fao cells with 2,4-dinitrophenol for 45 min depleted cellular ATP by 80% and equally inhibited insulin-stimulated receptor autophosphorylation, as determined by immunoprecipitation of surface-iodinated or (/sup 32/P)phosphate-labeled cells with anti-phosphotyrosine antibody. In contrast, internalization of the insulin receptor and internalization and degradation of /sup 125/I-labeled insulin by 2,4-dinitrophenol-treated cells were normal. These data show that autophosphorylation of the insulin receptor is not required for the receptor-mediated internalization of insulin in Fao cells and suggest that insulin receptor recycling is independent of autophosphorylation.

  7. Reliability Through Life of Internal Protection Devices in Small-Cell ABSL Batteries

    NASA Technical Reports Server (NTRS)

    Neubauer, Jeremy; Ng, Ka Lok; Bennetti, Andrea; Pearson, Chris; Rao, gopal

    2007-01-01

    This viewgraph presentation reviews a reliability analysis of small cell protection batteries. The contents include: 1) The s-p Topology; 2) Cell Level Protection Devices; 3) Battery Level Fault Protection; 4) Large Cell Comparison; and 5) Battery Level Testing and Results.

  8. Effects of Internalized Gold Nanoparticles with Respect to Cytotoxicity and Invasion Activity in Lung Cancer Cells

    PubMed Central

    Guo, Zhirui; Liu, Ying; Shen, Yujie; Zhou, Ping; Lu, Xiang

    2014-01-01

    The effect of gold nanoparticles on lung cancer cells is not yet clear. In this study, we investigated the cytotoxicity and cell invasion activity of lung cancer cells after treatment with gold nanoparticles and showed that small gold nanoparticles can be endocytosed by lung cancer cells and that they facilitate cell invasion. The growth of A549 cells was inhibited after treatment with 5-nm gold nanoparticles, but cell invasion increased. Endocytosed gold nanoparticles (size, 10 nm) notably promoted the invasion activity of 95D cells. All these effects of gold nanoparticles were not seen after treatment with larger particles (20 and 40 nm). The enhanced invasion activity may be associated with the increased expression of matrix metalloproteinase 9 and intercellular adhesion molecule-1. In this study, we obtained evidence for the effect of gold nanoparticles on lung cancer cell invasion activity in vitro. Moreover, matrix metalloproteinase 9 and intercellular adhesion molecule-1, key modulators of cell invasion, were found to be regulated by gold nanoparticles. These data also demonstrate that the responses of the A549 and 95D cells to gold nanoparticles have a remarkable relationship with their unique size-dependent physiochemical properties. Therefore, this study provides a new perspective for cell biology research in nanomedicine. PMID:24901215

  9. Reversal of diabetes by βTC3 cells encapsulated in alginate beads generated by emulsion and internal gelation.

    PubMed

    Hoesli, Corinne A; Kiang, Roger L J; Mocinecová, Dušana; Speck, Madeleine; Mošková, Daniela Jochec; Donald-Hague, Christine; Lacík, Igor; Kieffer, Timothy J; Piret, James M

    2012-05-01

    Encapsulation of insulin-producing cells in alginate beads could improve the treatment of type 1 diabetes by reducing or eliminating the need for immunosuppression. We have recently adapted an emulsion and internal gelation process to β-cell encapsulation. This process has the advantages of being well suited for m(3)/h production rates and allowing the use of increased alginate concentrations. Compared with 1.5% alginate beads generated by a standard extrusion process, 5% alginate emulsion-generated beads demonstrated greater in vitro stability and greater volumetric exclusion of antibody-sized pullulan. When βTC3 cells were transplanted into streptozotocin-induced allogeneic diabetic mice, a significant decrease in the blood glucose levels was seen within 2 days with the 5% emulsion-generated beads but not until >16 days with the 1.5% extrusion-generated beads. This was correlated with higher cell survival and lower graft-specific plasma immunoglobulin levels. These results suggest that higher-concentration alginate beads generated by emulsion and internal gelation have improved graft immunoprotection. The emulsion process is a promising and scalable technology for cellular therapies requiring immune isolation.

  10. Embedded fiber-optic sensing for accurate internal monitoring of cell state in advanced battery management systems part 1: Cell embedding method and performance

    NASA Astrophysics Data System (ADS)

    Raghavan, Ajay; Kiesel, Peter; Sommer, Lars Wilko; Schwartz, Julian; Lochbaum, Alexander; Hegyi, Alex; Schuh, Andreas; Arakaki, Kyle; Saha, Bhaskar; Ganguli, Anurag; Kim, Kyung Ho; Kim, ChaeAh; Hah, Hoe Jin; Kim, SeokKoo; Hwang, Gyu-Ok; Chung, Geun-Chang; Choi, Bokkyu; Alamgir, Mohamed

    2017-02-01

    A key challenge hindering the mass adoption of Lithium-ion and other next-gen chemistries in advanced battery applications such as hybrid/electric vehicles (xEVs) has been management of their functional performance for more effective battery utilization and control over their life. Contemporary battery management systems (BMS) reliant on monitoring external parameters such as voltage and current to ensure safe battery operation with the required performance usually result in overdesign and inefficient use of capacity. More informative embedded sensors are desirable for internal cell state monitoring, which could provide accurate state-of-charge (SOC) and state-of-health (SOH) estimates and early failure indicators. Here we present a promising new embedded sensing option developed by our team for cell monitoring, fiber-optic sensors. High-performance large-format pouch cells with embedded fiber-optic sensors were fabricated. The first of this two-part paper focuses on the embedding method details and performance of these cells. The seal integrity, capacity retention, cycle life, compatibility with existing module designs, and mass-volume cost estimates indicate their suitability for xEV and other advanced battery applications. The second part of the paper focuses on the internal strain and temperature signals obtained from these sensors under various conditions and their utility for high-accuracy cell state estimation algorithms.

  11. Cholesterol oxidation switches the internalization pathway of endothelin receptor type A from caveolae to clathrin-coated pits in Chinese hamster ovary cells.

    PubMed

    Okamoto, Y; Ninomiya, H; Miwa, S; Masaki, T

    2000-03-03

    We investigated the mechanism of endothelin receptor type A (ETA) internalization in Chinese hamster ovary cells using two assays; flow cytometric quantification of cell surface myc-ETA and in situ localization of Cy5-labeled ET-1. In both assays, agonist-dependent internalization of myc-ETA was inhibited by nystatin and filipin, both of which disrupt internalization via caveolae, whereas it was barely affected by chlorpromazine and hypertonic sucrose, both of which disrupt internalization via clathrin-coated pits. In addition to myc-ETA, ET-1 caused intracellular translocation of caveolin-1 and this translocation was also blocked by nystatin but not by chlorpromazine. These results strongly argue that ETA is internalized via caveolae but not clathrin-coated pits. Treatment of the cells with cholesterol oxidase reduced cellular cholesterol and caused intracellular translocation of caveolin-1 but did not affect cell surface localization of myc-ETA. In cholesterol oxidase-treated cells, however, both chlorpromazine and hypertonic sucrose effectively blocked ET-1-induced myc-ETA internalization and nystatin was less effective than in untreated cells. Accordingly, expression of a dominant negative form of beta-arrestin blocked myc-ETA internalization in cholesterol oxidase-treated cells but not in untreated cells. These results suggest that, in Chinese hamster ovary cells, 1) agonist-occupied ETA can be internalized either via caveolae or clathrin-coated pits; 2) of the two, the former is the default pathway; and 3) the oxidative state of cell surface cholesterol is one of the factors involved in the pathway selection.

  12. Pressure Regulator With Internal Ejector Circulation Pump, Flow and Pressure Measurement Porting, and Fuel Cell System Integration Options

    NASA Technical Reports Server (NTRS)

    Vasquez, Arturo

    2011-01-01

    An advanced reactant pressure regulator with an internal ejector reactant circulation pump has been developed to support NASA's future fuel cell power systems needs. These needs include reliable and safe operation in variable-gravity environments, and for exploration activities with both manned and un manned vehicles. This product was developed for use in Proton Exchange Membrane Fuel Cell (PEMFC) power plant reactant circulation systems, but the design could also be applied to other fuel cell system types, (e.g., solid-oxide or alkaline) or for other gas pressure regulation and circulation needs. The regulator design includes porting for measurement of flow and pressure at key points in the system, and also includes several fuel cell system integration options. NASA has recognized ejectors as a viable alternative to mechanical pumps for use in spacecraft fuel cell power systems. The ejector motive force is provided by a variable, high-pressure supply gas that travels through the ejector s jet nozzle, whereby the pressure energy of the fluid stream is converted to kinetic energy in the gas jet. The ejector can produce circulation-to-consumption-flow ratios that are relatively high (2-3 times), and this phenomenon can potentially (with proper consideration of the remainder of the fuel cell system s design) be used to provide completely for reactant pre-humidification and product water removal in a fuel cell system. Specifically, a custom pressure regulator has been developed that includes: (1) an ejector reactant circulation pump (with interchangeable jet nozzles and mixer sections, gas-tight sliding and static seals in required locations, and internal fluid porting for pressure-sensing at the regulator's control elements) and (2) internal fluid porting to allow for flow rate and system pressure measurements. The fluid porting also allows for inclusion of purge, relief, and vacuum-breaker check valves on the regulator assembly. In addition, this regulator could also

  13. Internal complementarities in the immune system: regulation of the expression of helper T-cell idiotypes.

    PubMed Central

    Martinez, C; Pereira, P; Bernabé, R; Bandeira, A; Larsson, E L; Cazenave, P A; Coutinho, A

    1984-01-01

    More than half of BALB/c helper T lymphocytes specific for 2,4,6-trinitrophenyl (TNP)-modified syngeneic spleen cells are inhibited in their proliferative responses to antigen-presenting cells and in their cooperation with B lymphocytes by monoclonal anti-idiotypic antibodies directed to a TNP-binding BALB/c myeloma protein (MOPC 460). This inhibition is specific for anti-TNP-self helper cells of BALB/c origin and is controlled by IgCh-linked genes, as it is not observed with CB.20 helper cells of the same specificity. In contrast, anti-TNP-self helper cells prepared from BALB/c mice that were chronically suppressed with anti-mu chain antibodies and possessed no B lymphocytes were not inhibited by anti-idiotypic antibodies. We conclude that the B-cell antibody repertoires contribute to the selection of the (idiotypic) T-helper-cell repertoires. PMID:6235521

  14. Detection of Staphylococcus aureus adhesion and biofilm-producing genes and their expression during internalization in bovine mammary epithelial cells.

    PubMed

    Pereyra, Elizabet A L; Picech, Florencia; Renna, María S; Baravalle, Celina; Andreotti, Carolina S; Russi, Romina; Calvinho, Luis F; Diez, Cristina; Dallard, Bibiana E

    2016-02-01

    Staphylococcus aureus is one of the most prevalent pathogens isolated from bovine mastitis, causing chronic intramammary infections (IMI) that limit profitable dairying. The course of infection is often associated with factors both related to the host and the bacterium. Aims of this study were to select S. aureus isolates from bovine IMI with different genotypic profiles harboring genes involved in adherence and biofilm production, to determine the behavior of these strains in contact with bovine mammary epithelial cells (MAC-T) and the expression of those genes during bacterial-cell early interactions. The genetic diversity of 20 S. aureus strains that were isolated from milk samples taken from cows with persistent-P and non-persistent-NP IMI was high, discriminated into 13 fingerprint groups. The occurrence of genes coding for S. aureus surface proteins (clfA, clfB, fnbA, fnbB, fib, cna) and biofilm formation (icaA, icaD, icaC, bap) and in vitro biofilm-forming ability was not related to strain clinical origin (NP or P). Internalization of S. aureus into MAC-T cells was strain-dependent and internalized bacteria overexpressed adherence and biofilm-forming genes compared with those that remained in the supernatant of co-cultures; particularly those genes encoding FnBPs and IcaD. Strains yielding highest invasion percentages were those able to overexpress fnBP, irrespectively of the presence of other evaluated genes. Strains from NP IMI showed a greater multiplication capacity in vitro compared with strains from P IMI. These results provide new insights about S. aureus differential gene expression of adhesion-internalization factors during early interaction with mammary epithelial cells.

  15. Mechanisms of modulation by internal protons of cyclic nucleotide-gated channels cloned from sensory receptor cells.

    PubMed Central

    Gavazzo, P; Picco, C; Menini, A

    1997-01-01

    We have examined the modulation by internal protons of cyclic nucleotide-gated (CNG) channels cloned from bovine olfactory receptor cells and retinal rods. CNG channels were studied in excised inside-out membrane patches from Xenopus laevis oocytes previously injected with the mRNA encoding for the subunit 1 of olfactory or rod channels. Channels were activated by cGMP or cAMP, and currents as a function of cyclic nucleotide concentrations were measured as pHi varied between 7.6 and 5.0. Increasing internal proton concentrations caused a partial blockage of the single-channel current, consistent with protonation of a single acidic site with a pK1 of 4.5-4.7, both in rod and in olfactory CNG channels. Channel gating properties were also affected by internal protons. The open probability at low cyclic nucleotide concentrations was greatly increased by lowering pHi, and the increase was larger when channels were activated by cAMP than by cGMP. Therefore, internal protons affected both channel permeation and gating properties, causing a reduction in single-channel current and an increase in open probability. These effects are likely to be caused by different titratable groups on the channel. PMID:9308192

  16. Efficient internalization of silica-coated iron oxide nanoparticles of different sizes by primary human macrophages and dendritic cells

    SciTech Connect

    Kunzmann, Andrea; Andersson, Britta; Vogt, Carmen; Feliu, Neus; Ye Fei; Gabrielsson, Susanne; Toprak, Muhammet S.; Buerki-Thurnherr, Tina; Laurent, Sophie; Vahter, Marie; Krug, Harald; Muhammed, Mamoun; Scheynius, Annika; Fadeel, Bengt

    2011-06-01

    Engineered nanoparticles are being considered for a wide range of biomedical applications, from magnetic resonance imaging to 'smart' drug delivery systems. The development of novel nanomaterials for biomedical applications must be accompanied by careful scrutiny of their biocompatibility. In this regard, particular attention should be paid to the possible interactions between nanoparticles and cells of the immune system, our primary defense system against foreign invasion. On the other hand, labeling of immune cells serves as an ideal tool for visualization, diagnosis or treatment of inflammatory processes, which requires the efficient internalization of the nanoparticles into the cells of interest. Here, we compare novel monodispersed silica-coated iron oxide nanoparticles with commercially available dextran-coated iron oxide nanoparticles. The silica-coated iron oxide nanoparticles displayed excellent magnetic properties. Furthermore, they were non-toxic to primary human monocyte-derived macrophages at all doses tested whereas dose-dependent toxicity of the smaller silica-coated nanoparticles (30 nm and 50 nm) was observed for primary monocyte-derived dendritic cells, but not for the similarly small dextran-coated iron oxide nanoparticles. No macrophage or dendritic cell secretion of pro-inflammatory cytokines was observed upon administration of nanoparticles. The silica-coated iron oxide nanoparticles were taken up to a significantly higher degree when compared to the dextran-coated nanoparticles, irrespective of size. Cellular internalization of the silica-coated nanoparticles was through an active, actin cytoskeleton-dependent process. We conclude that these novel silica-coated iron oxide nanoparticles are promising materials for medical imaging, cell tracking and other biomedical applications.

  17. Interactions of Aspergillus fumigatus with endothelial cells: internalization, injury, and stimulation of tissue factor activity.

    PubMed

    Lopes Bezerra, Leila M; Filler, Scott G

    2004-03-15

    Invasive aspergillosis causes significant mortality among patients with hematologic malignancies. This infection is characterized by vascular invasion and thrombosis. To study the pathogenesis of invasive aspergillosis, we investigated the interactions of Aspergillus fumigatus conidia and hyphae with endothelial cells in vitro. We found that both forms of the organism induced endothelial cell microfilament rearrangement and subsequent endocytosis. Conidia were endocytosed 2-fold more avidly than hyphae, and endocytosis was independent of fungal viability. Endocytosed conidia and hyphae caused progressive endothelial cell injury after 4 hours of infection. Live conidia induced more endothelial cell injury than did live hyphae. However, endothelial cell injury caused by conidia was dependent on fungal viability, whereas injury caused by hyphae was not, indicating that conidia and hyphae injure endothelial cells by different mechanisms. Neither live nor killed conidia increased tissue factor activity of endothelial cells. In contrast, both live and killed hyphae stimulated significant endothelial cell tissue factor activity, as well as the expression of tissue factor antigen on the endothelial cell surface. These results suggest that angioinvasion and thrombosis caused by A fumigatus hyphae in vivo may be due in part to endothelial cell invasion, induction of injury, and stimulation of tissue factor activity.

  18. Internalization: acute apoptosis of breast cancer cells using herceptin-immobilized gold nanoparticles

    PubMed Central

    Rathinaraj, Pierson; Al-Jumaily, Ahmed M; Huh, Do Sung

    2015-01-01

    Herceptin, the monoclonal antibody, was successfully immobilized on gold nanoparticles (GNPs) to improve their precise interactions with breast cancer cells (SK-BR3). The mean size of the GNPs (29 nm), as determined by dynamic light scattering, enlarged to 82 nm after herceptin immobilization. The in vitro cell culture experiment indicated that human skin cells (FB) proliferated well in the presence of herceptin-conjugated GNP (GNP–Her), while most of the breast cancer cells (SK-BR3) had died. To elucidate the mechanism of cell death, the interaction of breast cancer cells with GNP–Her was tracked by confocal laser scanning microscopy. Consequently, GNP–Her was found to be bound precisely to the membrane of the breast cancer cell, which became almost saturated after 6 hours incubation. This shows that the progression signal of SK-BR3 cells is retarded completely by the precise binding of antibody to the human epidermal growth factor receptor 2 receptor of the breast cancer cell membrane, causing cell death. PMID:25709498

  19. Intracellular Distribution of TM4SF1 and Internalization of TM4SF1-antibody Complex in Vascular Endothelial Cells

    PubMed Central

    Sciuto, Tracey E.; Merley, Anne; Lin, Chi-Iou; Richardson, Douglas; Liu, Yu; Li, Dan; Dvorak, Ann M.; Dvorak, Harold F.; Jaminet, Shou-Ching S.

    2015-01-01

    Transmembrane-4 L-six family member-1 (TM4SF1) is a small plasma membrane-associated glycoprotein that is highly and selectively expressed on the plasma membranes of tumor cells, cultured endothelial cells, and, in vivo, on tumor-associated endothelium. Immunofluorescence microscopy also demonstrated TM4SF1 in cytoplasm and, tentatively, within nuclei. With monoclonal antibody 8G4, and the finer resolution afforded by immuno-nanogold transmission electron microscopy, we now demonstrate TM4SF1 in uncoated cytoplasmic vesicles, nuclear pores and nucleoplasm. Because of its prominent surface location on tumor cells and tumor-associated endothelium, TM4SF1 has potential as a dual therapeutic target using an antibody drug conjugate (ADC) approach. For ADC to be successful, antibodies reacting with cell surface antigens must be internalized for delivery of associated toxins to intracellular targets. We now report that 8G4 is efficiently taken up into cultured endothelial cells by uncoated vesicles in a dynamin-dependent, clathrin-independent manner. It is then transported along microtubules through the cytoplasm and passes through nuclear pores into the nucleus. These findings validate TM4SF1 as an attractive candidate for cancer therapy with antibody-bound toxins that have the capacity to react with either cytoplasmic or nuclear targets in tumor cells or tumor-associated vascular endothelium. PMID:26241677

  20. Internalization of a novel, huge lectin from Ibacus novemdentatus (slipper lobster) induces apoptosis of mammalian cancer cells.

    PubMed

    Fujii, Y; Fujiwara, T; Koide, Y; Hasan, I; Sugawara, S; Rajia, S; Kawsar, S M A; Yamamoto, D; Araki, D; Kanaly, R A; Ogawa, Y; Fujita, H; Ozeki, Y

    2017-02-01

    An N-acetyl sugar-binding lectin (termed iNoL) displaying cytotoxic activity against human cancer cells was isolated from the slipper lobster Ibacus novemdentatus (family Scyllaridae). iNoL recognized monosaccharides containing N-acetyl group, and glycoproteins (e.g., BSM) containing oligosaccharides with N-acetyl sugar. iNoL was composed of five subunits (330, 260, 200, 140, and 30 kDa), which in turn consisted of 70-, 40-, and 30-kDa polypeptides held together by disulfide bonds. Electron microscopic observations and gel permeation chromatography indicated that iNoL was a huge (500-kDa) molecule and had a polygonal structure under physiological conditions. iNoL displayed cytotoxic (apoptotic) effects against human cancer cell lines MCF7 and T47D (breast), HeLa (ovarian), and Caco2 (colonic), through incorporation (internalization) into cells. The lectin was transported into lysosomes via endosomes. Its cytotoxic effect and incorporation into cells were inhibited by the co-presence of N-acetyl-D-mannosamine (ManNAc). Treatment of HeLa cells with iNoL resulted in DNA fragmentation and chromatin condensation, through activation of caspase-9 and -3. In summary, the novel crustacean lectin iNoL is incorporated into mammalian cancer cells through glycoconjugate interaction, and has cytotoxic (apoptotic) effects.

  1. Internalization of styryl dye FM1-43 in the hair cells of lateral line organs in Xenopus larvae.

    PubMed

    Nishikawa, S; Sasaki, F

    1996-07-01

    We used a fluorescent dye, FM1-43 to investigate mechanotransduction mechanisms in the hair cells of lateral line organs of Xenopus larvae. FM1-43 specifically labeled the hair cells. The photo-oxidation technique was performed with election microscopy to examine the labeling sites and their mechanisms. The results showed that the labeling sites were mitochondria and rough endoplasmic reticulum throughout the cytoplasm. Endocytic activity of the hair cells was limited to endosomes and small granules located at the apical part of the cells. Blockers of the mechanosensitive cation channel (neomycin, gentamicin, streptomycin, and amiloride) effectively inhibited FM1-43 labeling. One of the blockers, amiloride, was found to bind to hair cells when its fluorescence was examined. Divalent cations such as Mg2+ and Ca2+, but not monovalent cations such as Na+ and K+, inhibited FM1-43 labeling when they were added in excess amounts. These results suggest that FM1-43 internalizes hair cells via the putative mechanosensitive cation channel in the plasma membrane.

  2. Immune suppression of human lymphoid tissues and cells in rotating suspension culture and onboard the International Space Station.

    PubMed

    Fitzgerald, Wendy; Chen, Silvia; Walz, Carl; Zimmerberg, Joshua; Margolis, Leonid; Grivel, Jean-Charles

    2009-12-01

    The immune responses of human lymphoid tissue explants or cells isolated from this tissue were studied quantitatively under normal gravity and microgravity. Microgravity was either modeled by solid body suspension in a rotating, oxygenated culture vessel or was actually achieved on the International Space Station (ISS). Our experiments demonstrate that tissues or cells challenged by recall antigen or by polyclonal activator in modeled microgravity lose all their ability to produce antibodies and cytokines and to increase their metabolic activity. In contrast, if the cells were challenged before being exposed to modeled microgravity suspension culture, they maintained their responses. Similarly, in microgravity in the ISS, lymphoid cells did not respond to antigenic or polyclonal challenge, whereas cells challenged prior to the space flight maintained their antibody and cytokine responses in space. Thus, immune activation of cells of lymphoid tissue is severely blunted both in modeled and true microgravity. This suggests that suspension culture via solid body rotation is sufficient to induce the changes in cellular physiology seen in true microgravity. This phenomenon may reflect immune dysfunction observed in astronauts during space flights. If so, the ex vivo system described above can be used to understand cellular and molecular mechanisms of this dysfunction.

  3. Innate Response Activator (IRA) B Cells Reside in Human Tonsils and Internalize Bacteria In Vitro.

    PubMed

    Chiappini, Nico; Cantisani, Rocco; Pancotto, Laura; Ruggiero, Paolo; Rosa, Domenico; Manetti, Andrea; Romano, Antonio; Montagnani, Francesca; Bertholet, Sylvie; Castellino, Flora; Del Giudice, Giuseppe

    2015-01-01

    Innate response activator (IRA) B cells have been described in mice as a subset of B-1a B cells that produce granulocyte/macrophage colony-stimulating factor (GM-CSF) and have been found in the spleen upon activation. In humans, identification, tissue localization and functionality of these lymphocytes are poorly understood. We hypothesized that IRA B cells could reside in human palatine tonsils, which are a first line of defense from infection of the upper respiratory tract. In the present work, we used flow cytometry and confocal microscopy to identify and characterize human IRA (hIRA) B cells in tonsils. We show that CD19⁺CD20⁺GM-CSF⁺ B cells are present in the tonsils of all the subjects studied at a frequency ranging between ~0.2% and ~0.4% of the conventional CD19⁺CD20⁺GM-CSF⁻ B cells. These cells reside within the B cell follicles, are mostly IgM⁺IgD⁺, express CD5 and show phagocytic activity. Our results support a role for hIRA B cells in the effector immune response to infections in tonsils.

  4. Innate Response Activator (IRA) B Cells Reside in Human Tonsils and Internalize Bacteria In Vitro

    PubMed Central

    Pancotto, Laura; Ruggiero, Paolo; Rosa, Domenico; Manetti, Andrea; Romano, Antonio; Montagnani, Francesca; Bertholet, Sylvie; Castellino, Flora; Del Giudice, Giuseppe

    2015-01-01

    Innate response activator (IRA) B cells have been described in mice as a subset of B-1a B cells that produce granulocyte/macrophage colony-stimulating factor (GM-CSF) and have been found in the spleen upon activation. In humans, identification, tissue localization and functionality of these lymphocytes are poorly understood. We hypothesized that IRA B cells could reside in human palatine tonsils, which are a first line of defense from infection of the upper respiratory tract. In the present work, we used flow cytometry and confocal microscopy to identify and characterize human IRA (hIRA) B cells in tonsils. We show that CD19+CD20+GM-CSF+ B cells are present in the tonsils of all the subjects studied at a frequency ranging between ~0.2% and ~0.4% of the conventional CD19+CD20+GM-CSF- B cells. These cells reside within the B cell follicles, are mostly IgM+IgD+, express CD5 and show phagocytic activity. Our results support a role for hIRA B cells in the effector immune response to infections in tonsils. PMID:26066485

  5. Meeting report of the International Consortium of Stem Cell Networks' Workshop Towards Clinical Trials Using Stem Cells for Amyotrophic Lateral Sclerosis/Motor Neuron Disease.

    PubMed

    Chaddah, Maya R; Dickie, Brian G; Lyall, Drew; Marshall, Caroline J; Sykes, J Ben; Bruijn, Lucie I

    2011-09-01

    The International Consortium of Stem Cell Networks' (ICSCN) Workshop Towards Clinical Trials Using Stem Cells for Amyotrophic Lateral Sclerosis (ALS)/Motor Neuron Disease (MND) was held on 24-25 January 2011. Twenty scientific talks addressed aspects of cell derivation and characterization; preclinical research and phased clinical trials involving stem cells; latest developments in induced pluripotent (iPS) cell technology; industry involvement and investment. Three moderated panel discussions focused on unregulated ALS/MND treatments, and the state of the art and barriers to future progress in using stem cells for ALS/MND. This review highlights the major insights that emanated from the workshop around the lessons learned and barriers to progress for using stem cells for understanding disease mechanism, drug discovery, and as therapy for ALS/MND. The full meeting report is only available in the online version of the journal. Please find this material with the following direct link to the article: http://www.informahealthcare.com/als/doi/10.3109/17482968.2011.590992 .

  6. Interaction of Prevotella intermedia strain 17 leucine-rich repeat domain protein AdpF with eukaryotic cells promotes bacterial internalization.

    PubMed

    Sengupta, Dipanwita; Kang, Dae-Joong; Anaya-Bergman, Cecilia; Wyant, Tiana; Ghosh, Arnab K; Miyazaki, Hiroshi; Lewis, Janina P

    2014-06-01

    Prevotella intermedia is an oral bacterium implicated in a variety of oral diseases. Although internalization of this bacterium by nonphagocytic host cells is well established, the molecular players mediating the process are not well known. Here, the properties of a leucine-rich repeat (LRR) domain protein, designated AdpF, are described. This protein contains a leucine-rich region composed of 663 amino acid residues, and molecular modeling shows that it folds into a classical curved solenoid structure. The cell surface localization of recombinant AdpF (rAdpF) was confirmed by electron and confocal microscopy analyses. The recombinant form of this protein bound fibronectin in a dose-dependent manner. Furthermore, the protein was internalized by host cells, with the majority of the process accomplished within 30 min. The internalization of rAdpF was inhibited by nystatin, cytochalasin, latrunculin, nocodazole, and wortmannin, indicating that microtubules, microfilaments, and signal transduction are required for the invasion. It is noteworthy that preincubation of eukaryotic cells with AdpF increased P. intermedia 17 internalization by 5- and 10-fold for HeLa and NIH 3T3 fibroblast cell lines, respectively. The addition of the rAdpF protein was also very effective in inducing bacterial internalization into the oral epithelial cell line HN4, as well as into primary cells, including human oral keratinocytes (HOKs) and human umbilical vein endothelial cells (HUVECs). Finally, cells exposed to P. intermedia 17 internalized the bacteria more readily upon reinfection. Taken together, our data demonstrate that rAdpF plays a role in the internalization of P. intermedia 17 by a variety of host cells.

  7. Interaction of Prevotella intermedia Strain 17 Leucine-Rich Repeat Domain Protein AdpF with Eukaryotic Cells Promotes Bacterial Internalization

    PubMed Central

    Sengupta, Dipanwita; Kang, Dae-Joong; Anaya-Bergman, Cecilia; Wyant, Tiana; Ghosh, Arnab K.; Miyazaki, Hiroshi

    2014-01-01

    Prevotella intermedia is an oral bacterium implicated in a variety of oral diseases. Although internalization of this bacterium by nonphagocytic host cells is well established, the molecular players mediating the process are not well known. Here, the properties of a leucine-rich repeat (LRR) domain protein, designated AdpF, are described. This protein contains a leucine-rich region composed of 663 amino acid residues, and molecular modeling shows that it folds into a classical curved solenoid structure. The cell surface localization of recombinant AdpF (rAdpF) was confirmed by electron and confocal microscopy analyses. The recombinant form of this protein bound fibronectin in a dose-dependent manner. Furthermore, the protein was internalized by host cells, with the majority of the process accomplished within 30 min. The internalization of rAdpF was inhibited by nystatin, cytochalasin, latrunculin, nocodazole, and wortmannin, indicating that microtubules, microfilaments, and signal transduction are required for the invasion. It is noteworthy that preincubation of eukaryotic cells with AdpF increased P. intermedia 17 internalization by 5- and 10-fold for HeLa and NIH 3T3 fibroblast cell lines, respectively. The addition of the rAdpF protein was also very effective in inducing bacterial internalization into the oral epithelial cell line HN4, as well as into primary cells, including human oral keratinocytes (HOKs) and human umbilical vein endothelial cells (HUVECs). Finally, cells exposed to P. intermedia 17 internalized the bacteria more readily upon reinfection. Taken together, our data demonstrate that rAdpF plays a role in the internalization of P. intermedia 17 by a variety of host cells. PMID:24711565

  8. Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards.

    PubMed

    Porter, Joshua R; Telford, William G; Batchelor, Eric

    2017-02-25

    Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.

  9. In-situ monitoring of internal local temperature and voltage of proton exchange membrane fuel cells.

    PubMed

    Lee, Chi-Yuan; Fan, Wei-Yuan; Hsieh, Wei-Jung

    2010-01-01

    The distribution of temperature and voltage of a fuel cell are key factors that influence performance. Conventional sensors are normally large, and are also useful only for making external measurements of fuel cells. Centimeter-scale sensors for making invasive measurements are frequently unable to accurately measure the interior changes of a fuel cell. This work focuses mainly on fabricating flexible multi-functional microsensors (for temperature and voltage) to measure variations in the local temperature and voltage of proton exchange membrane fuel cells (PEMFC) that are based on micro-electro-mechanical systems (MEMS). The power density at 0.5 V without a sensor is 450 mW/cm(2), and that with a sensor is 426 mW/cm(2). Since the reaction area of a fuel cell with a sensor is approximately 12% smaller than that without a sensor, but the performance of the former is only 5% worse.

  10. Non-specific internalization of laser ablated pure gold nanoparticles in pancreatic tumor cell.

    PubMed

    Sobhan, M A; Sreenivasan, V K A; Withford, M J; Goldys, E M

    2012-04-01

    We investigate the intracellular uptake of 7.3 nm, 21.2 nm and 31.3 nm average size pure colloidal gold nanoparticles synthesized using femtosecond laser ablation technique in pure water. Dark-field imaging, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) was used to assess the uptake of these pure gold nanoparticles in the pancreatic tumor cell line. We show that these ligand-free gold nanoparticles are non-toxic to these cells. The nanoparticles and cell images indicated that unmodified gold nanoparticles interacted with the cells, despite negative surface charge on both the cells and the nanoparticles. We also demonstrate that the uptake of the gold nanoparticles is size-dependent.

  11. Internal nitrogen removal from sediments by the hybrid system of microbial fuel cells and submerged aquatic plants

    PubMed Central

    Xu, Peng; Xiao, En-Rong; Xu, Dan; Zhou, Yin; He, Feng; Liu, Bi-Yun; Zeng, Lei; Wu, Zhen-Bin

    2017-01-01

    Sediment internal nitrogen release is a significant pollution source in the overlying water of aquatic ecosystems. This study aims to remove internal nitrogen in sediment-water microcosms by coupling sediment microbial fuel cells (SMFCs) with submerged aquatic plants. Twelve tanks including four treatments in triplicates were designed: open-circuit (SMFC-o), closed-circuit (SMFC-c), aquatic plants with open-circuit (P-SMFC-o) and aquatic plants with closed-circuit (P-SMFC-c). The changes in the bio-electrochemical characteristics of the nitrogen levels in overlying water, pore water, sediments, and aquatic plants were documented to explain the migration and transformation pathways of internal nitrogen. The results showed that both electrogenesis and aquatic plants could facilitate the mineralization of organic nitrogen in sediments. In SMFC, electrogenesis promoted the release of ammonium from the pore water, followed by the accumulation of ammonium and nitrate in the overlying water. The increased redox potential of sediments due to electrogenesis also contributed to higher levels of nitrate in overlying water when nitrification in pore water was facilitated and denitrification at the sediment-water interface was inhibited. When the aquatic plants were introduced into the closed-circuit SMFC, the internal ammonium assimilation by aquatic plants was advanced by electrogenesis; nitrification in pore water and denitrification in sediments were also promoted. These processes might result in the maximum decrease of internal nitrogen with low nitrogen levels in the overlying water despite the lower power production. The P-SMFC-c reduced 8.1%, 16.2%, 24.7%, and 25.3% of internal total nitrogen compared to SMFC-o on the 55th, 82th, 136th, and 190th days, respectively. The smaller number of Nitrospira and the larger number of Bacillus and Pseudomonas on the anodes via high throughput sequencing may account for strong mineralization and denitrification in the sediments

  12. Internal nitrogen removal from sediments by the hybrid system of microbial fuel cells and submerged aquatic plants.

    PubMed

    Xu, Peng; Xiao, En-Rong; Xu, Dan; Zhou, Yin; He, Feng; Liu, Bi-Yun; Zeng, Lei; Wu, Zhen-Bin

    2017-01-01

    Sediment internal nitrogen release is a significant pollution source in the overlying water of aquatic ecosystems. This study aims to remove internal nitrogen in sediment-water microcosms by coupling sediment microbial fuel cells (SMFCs) with submerged aquatic plants. Twelve tanks including four treatments in triplicates were designed: open-circuit (SMFC-o), closed-circuit (SMFC-c), aquatic plants with open-circuit (P-SMFC-o) and aquatic plants with closed-circuit (P-SMFC-c). The changes in the bio-electrochemical characteristics of the nitrogen levels in overlying water, pore water, sediments, and aquatic plants were documented to explain the migration and transformation pathways of internal nitrogen. The results showed that both electrogenesis and aquatic plants could facilitate the mineralization of organic nitrogen in sediments. In SMFC, electrogenesis promoted the release of ammonium from the pore water, followed by the accumulation of ammonium and nitrate in the overlying water. The increased redox potential of sediments due to electrogenesis also contributed to higher levels of nitrate in overlying water when nitrification in pore water was facilitated and denitrification at the sediment-water interface was inhibited. When the aquatic plants were introduced into the closed-circuit SMFC, the internal ammonium assimilation by aquatic plants was advanced by electrogenesis; nitrification in pore water and denitrification in sediments were also promoted. These processes might result in the maximum decrease of internal nitrogen with low nitrogen levels in the overlying water despite the lower power production. The P-SMFC-c reduced 8.1%, 16.2%, 24.7%, and 25.3% of internal total nitrogen compared to SMFC-o on the 55th, 82th, 136th, and 190th days, respectively. The smaller number of Nitrospira and the larger number of Bacillus and Pseudomonas on the anodes via high throughput sequencing may account for strong mineralization and denitrification in the sediments

  13. Hematopoietic stem cell transplantation in thalassemia major and sickle cell disease: indications and management recommendations from an international expert panel.

    PubMed

    Angelucci, Emanuele; Matthes-Martin, Susanne; Baronciani, Donatella; Bernaudin, Françoise; Bonanomi, Sonia; Cappellini, Maria Domenica; Dalle, Jean-Hugues; Di Bartolomeo, Paolo; de Heredia, Cristina Díaz; Dickerhoff, Roswitha; Giardini, Claudio; Gluckman, Eliane; Hussein, Ayad Achmed; Kamani, Naynesh; Minkov, Milen; Locatelli, Franco; Rocha, Vanderson; Sedlacek, Petr; Smiers, Frans; Thuret, Isabelle; Yaniv, Isaac; Cavazzana, Marina; Peters, Christina

    2014-05-01

    Thalassemia major and sickle cell disease are the two most widely disseminated hereditary hemoglobinopathies in the world. The outlook for affected individuals has improved in recent years due to advances in medical management in the prevention and treatment of complications. However, hematopoietic stem cell transplantation is still the only available curative option. The use of hematopoietic stem cell transplantation has been increasing, and outcomes today have substantially improved compared with the past three decades. Current experience world-wide is that more than 90% of patients now survive hematopoietic stem cell transplantation and disease-free survival is around 80%. However, only a few controlled trials have been reported, and decisions on patient selection for hematopoietic stem cell transplantation and transplant management remain principally dependent on data from retrospective analyses and on the clinical experience of the transplant centers. This consensus document from the European Blood and Marrow Transplantation Inborn Error Working Party and the Paediatric Diseases Working Party aims to report new data and provide consensus-based recommendations on indications for hematopoietic stem cell transplantation and transplant management.

  14. Measurement of the internal pH of mast cell granules using microvolumetric fluorescence and isotopic techniques

    SciTech Connect

    De Young, M.B.; Nemeth, E.F.; Scarpa, A.

    1987-04-01

    The intragranular pH of isolated mast cell granules was measured. Because of the minute amounts of isolated granules available, two techniques were developed by modifying aminoacridine fluorescence and (/sup 14/C)methylamine accumulation techniques to permit measurements with microliter sample volumes. Granule purity was demonstrated by electron microscopy, ruthenium red exclusion, and biochemical (histamine, mast cell granule protease) analysis. The internal pH was determined to be 5.55 +/- 0.06, indicating that the pH environment within mast cell granules is not significantly different from that of previously studied granule types (i.e., chromaffin, platelet, pancreatic islet, and pituitary granules). Collapse of the pH gradient by NH+4 was demonstrated with both techniques. No evidence of Cl-/OH- or specific cation/H+ transport was found, and major chloride permeability could not be unequivocably demonstrated. Ca/sup 2 +/ and Cl- at concentrations normally present extracellularly destabilized granules in the presence of NH+4, but this phenomenon does not necessarily indicate a role for these ions in the exocytotic release of granule contents from intact cells. The pH measurement techniques developed for investigating the properties of granules in mast cells may be useful for studying other granules that can be obtained only in limited quantities.

  15. Stem Cell Gene Therapy for Fanconi Anemia: Report from the 1st International Fanconi Anemia Gene Therapy Working Group Meeting

    PubMed Central

    Tolar, Jakub; Adair, Jennifer E; Antoniou, Michael; Bartholomae, Cynthia C; Becker, Pamela S; Blazar, Bruce R; Bueren, Juan; Carroll, Thomas; Cavazzana-Calvo, Marina; Clapp, D Wade; Dalgleish, Robert; Galy, Anne; Gaspar, H Bobby; Hanenberg, Helmut; Von Kalle, Christof; Kiem, Hans-Peter; Lindeman, Dirk; Naldini, Luigi; Navarro, Susana; Renella, Raffaele; Rio, Paula; Sevilla, Julián; Schmidt, Manfred; Verhoeyen, Els; Wagner, John E; Williams, David A; Thrasher, Adrian J

    2011-01-01

    Survival rates after allogeneic hematopoietic cell transplantation (HCT) for Fanconi anemia (FA) have increased dramatically since 2000. However, the use of autologous stem cell gene therapy, whereby the patient's own blood stem cells are modified to express the wild-type gene product, could potentially avoid the early and late complications of allogeneic HCT. Over the last decades, gene therapy has experienced a high degree of optimism interrupted by periods of diminished expectation. Optimism stems from recent examples of successful gene correction in several congenital immunodeficiencies, whereas diminished expectations come from the realization that gene therapy will not be free of side effects. The goal of the 1st International Fanconi Anemia Gene Therapy Working Group Meeting was to determine the optimal strategy for moving stem cell gene therapy into clinical trials for individuals with FA. To this end, key investigators examined vector design, transduction method, criteria for large-scale clinical-grade vector manufacture, hematopoietic cell preparation, and eligibility criteria for FA patients most likely to benefit. The report summarizes the roadmap for the development of gene therapy for FA. PMID:21540837

  16. The current state of stem cell therapeutics: Canadian approaches in the international context.

    PubMed

    Noiseux, Nicolas; Marquis-Gravel, Guillaume; Mansour, Samer; Shahzad, Uswa; Stewart, Duncan J; Yau, Terrence M

    2014-11-01

    After ischemic injury, the endogenous repair mechanisms of the human heart are insufficient for meaningful tissue regeneration, so muscle lost is replaced by noncontractile scar tissue. Current treatments for ischemic cardiomyopathy improve quality of life and increase life expectancy, but cannot cure the underlying disease of cardiomyocyte loss. Cellular transplantation is emerging as a valuable therapeutic approach to heal the ischemic heart. Adult bone marrow stem cells are capable of differentiation, regeneration of infarcted myocardium, and induction of myogenesis and angiogenesis, ultimately leading to improved contractility. Positive results from animal studies have prompted several clinical trials to ascertain the safety and feasibility of cell therapy. However, despite all the excitement in stem cell research resulting from initial experimental data and preliminary clinical trials, the mixed results observed have raised many unanswered questions. A major obstacle to the identification of the optimal cell therapy is that the fate of the implanted cells and the nature of their beneficial effects are ill-defined. A better understanding is fundamental for the development of new therapeutic agents, and to optimize stem cell applications. Well-designed and powered double-blinded randomized studies are clearly needed to confirm promising findings from early studies. With several ongoing randomized trials directed toward evaluation of stem cell therapies in patients with acute or chronic ischemic cardiomyopathy, the Canadian initiative represents a milestone.

  17. Vectorization of biomacromolecules into cells using extracellular vesicles with enhanced internalization induced by macropinocytosis

    PubMed Central

    Nakase, Ikuhiko; Noguchi, Kosuke; Fujii, Ikuo; Futaki, Shiroh

    2016-01-01

    Extracellular vesicles (EVs, exosomes) are approximately 30- to 200-nm-long vesicles that have received increased attention due to their role in cell-to-cell communication. Although EVs are highly anticipated to be a next-generation intracellular delivery tool because of their pharmaceutical advantages, including non-immunogenicity, their cellular uptake efficacy is low because of the repulsion of EVs and negatively charged cell membranes and size limitations in endocytosis. Here, we demonstrate a methodology for achieving enhanced cellular EV uptake using arginine-rich cell-penetrating peptides (CPPs) to induce active macropinocytosis. The induction of macropinocytosis via a simple modification to the exosomal membrane using stearylated octaarginine, which is a representative CPP, significantly enhanced the cellular EV uptake efficacy. Consequently, effective EV-based intracellular delivery of an artificially encapsulated ribosome-inactivating protein, saporin, in EVs was attained. PMID:27748399

  18. Numerical study of thermoelectric characteristics of a planar solid oxide fuel cell with direct internal reforming of methane

    NASA Astrophysics Data System (ADS)

    Wang, Qiusheng; Li, Lijun; Wang, Cheng

    A three-dimensional mathematical thermo-fluid model coupling the electrochemical kinetics with fluid dynamics was developed to simulate the heat and mass transfer in planar anode-supported solid oxide fuel cell (SOFC). The internal reforming reactions and electrochemical reactions of carbon monoxide and hydrogen in the porous anode layer were analyzed. The temperature, species mole fraction, current density, overpotential loss and other performance parameters of the single cell unit were obtained by a commercial CFD code (Fluent) and external sub-routine. Results show that the current density produced by electrochemical reactions of carbon monoxide cannot be ignored, the cathode overpotential loss is the biggest one among the three overpotential losses, and that the proper decrease of the operating voltage leads to the increase of the current density, PEN structure temperature, fuel utilization factor, fuel efficiency and power output of the SOFC.

  19. Microglial cells (BV-2) internalize titanium dioxide (TiO2) nanoparticles: toxicity and cellular responses.

    PubMed

    Rihane, Naima; Nury, Thomas; M'rad, Imen; El Mir, Lassaad; Sakly, Mohsen; Amara, Salem; Lizard, Gérard

    2016-05-01

    Because of their whitening and photocatalytic effects, titanium dioxide nanoparticles (TiO2-NPs) are widely used in daily life. These NPs can be found in paints, plastics, papers, sunscreens, foods, medicines (pills), toothpastes, and cosmetics. However, the biological effect of TiO2-NPs on the human body, especially on the central nervous system, is still unclear. Many studies have demonstrated that the brain is one of the target organs in acute or chronic TiO2-NPs toxicity. The present study aimed to investigate the effect of TiO2-NPs at different concentrations (0.1 to 200 μg/mL) on murine microglial cells (BV-2) to assess their activity on cell growth and viability, as well as their neurotoxicity. Different parameters were measured: cell viability, cell proliferation and DNA content (SubG1 peak), mitochondrial depolarization, overproduction of reactive oxygen species (especially superoxide anions), and ultrastructural changes. Results showed that TiO2-NPs induced some cytotoxic effects with a slight inhibition of cell growth. Thus, at high concentrations, TiO2-NPs were not only able to inhibit cell adhesion but also enhanced cytoplasmic membrane permeability to propidium iodide associated with a loss of mitochondrial transmembrane potential and an overproduction of superoxide anions. No induction of apoptosis based on the presence of a SubG1 peak was detected. The microscopic observations also indicated that small groups of nanosized particles and micron-sized aggregates were engulfed by the BV-2 cells and sequestered as intracytoplasmic aggregates after 24-h exposure to TiO2-NPs. Altogether, our data show that the accumulation TiO2-NPs in microglial BV-2 cells favors mitochondrial dysfunctions and oxidative stress.

  20. Electrochemical fuel cell generator having an internal and leak tight hydrocarbon fuel reformer

    DOEpatents

    Dederer, Jeffrey T.; Hager, Charles A.

    1998-01-01

    An electrochemical fuel cell generator configuration is made having a generator section which contains a plurality of axially elongated fuel cells, each cell containing a fuel electrode, air electrode, and solid oxide electrolyte between the electrodes, in which axially elongated dividers separate portions of the fuel cells from each other, and where at least one divider also reforms a reformable fuel gas mixture prior to electricity generation reactions, the at least one reformer-divider is hollow having a closed end and an open end entrance for a reformable fuel mixture to pass to the closed end of the divider and then reverse flow and pass back along the hollowed walls to be reformed, and then finally to pass as reformed fuel out of the open end of the divider to contact the fuel cells, and further where the reformer-divider is a composite structure having a gas diffusion barrier of metallic foil surrounding the external walls of the reformer-divider except at the entrance to prevent diffusion of the reformable gas mixture through the divider, and further housed in an outer insulating jacket except at the entrance to prevent short-circuiting of the fuel cells by the gas diffusion barrier.

  1. Electrochemical fuel cell generator having an internal and leak tight hydrocarbon fuel reformer

    DOEpatents

    Dederer, J.T.; Hager, C.A.

    1998-03-31

    An electrochemical fuel cell generator configuration is made having a generator section which contains a plurality of axially elongated fuel cells, each cell containing a fuel electrode, air electrode, and solid oxide electrolyte between the electrodes, in which axially elongated dividers separate portions of the fuel cells from each other, and where at least one divider also reforms a reformable fuel gas mixture prior to electricity generation reactions, the at least one reformer-divider is hollow having a closed end and an open end entrance for a reformable fuel mixture to pass to the closed end of the divider and then reverse flow and pass back along the hollowed walls to be reformed, and then finally to pass as reformed fuel out of the open end of the divider to contact the fuel cells, and further where the reformer-divider is a composite structure having a gas diffusion barrier of metallic foil surrounding the external walls of the reformer-divider except at the entrance to prevent diffusion of the reformable gas mixture through the divider, and further housed in an outer insulating jacket except at the entrance to prevent short-circuiting of the fuel cells by the gas diffusion barrier. 10 figs.

  2. Anti-Inflammatory and Antimicrobial Effects of Estradiol in Bovine Mammary Epithelial Cells during Staphylococcus aureus Internalization.

    PubMed

    Medina-Estrada, Ivan; López-Meza, Joel E; Ochoa-Zarzosa, Alejandra

    2016-01-01

    17β-Estradiol (E2), the predominant sexual hormone in females, is associated with the modulation of the innate immune response (IIR), and changes in its levels at parturition are related to intramammary infections, such as mastitis. In bovine mammary epithelial cells (bMECs), E2 regulates differentiation and proliferation, but its immunomodulatory functions have not been explored. Staphylococcus aureus is the predominant pathogen causing mastitis, which can persist intracellularly in bMECs. The aim of this work was to analyze whether E2 modulates the IIR of bMECs during S. aureus internalization. bMECs treated with E2 (50 pg/mL, 24 h) reduced bacteria internalization (~50%). The host receptors α5β1 and TLR2 do not participate in this reduction. However, E2 activates ERα and modulates the IIR reducing the S. aureus induced-mRNA expression of TNF-α (~50%) and IL-1β (90%). E2 also decreased the secretion of these cytokines as well as IL-6 production; however, in infected bMECs, E2 induced the secretion of IL-1β. Furthermore, E2 upregulates the expression of the antimicrobial peptides DEFB1, BNBD5, and psoriasin S100A7 (~5-, 3-, and 6-fold, resp.). In addition, E2 induced the production of antimicrobial compounds in bMEC culture medium, which, together with the modulation of the IIR, could be related to the reduction of S. aureus internalization.

  3. Internally deleted WNV genomes isolated from exotic birds in New Mexico: function in cells, mosquitoes, and mice.

    PubMed

    Pesko, Kendra N; Fitzpatrick, Kelly A; Ryan, Elizabeth M; Shi, Pei-Yong; Zhang, Bo; Lennon, Niall J; Newman, Ruchi M; Henn, Matthew R; Ebel, Gregory D

    2012-05-25

    Most RNA viruses exist in their hosts as a heterogeneous population of related variants. Due to error prone replication, mutants are constantly generated which may differ in individual fitness from the population as a whole. Here we characterize three WNV isolates that contain, along with full-length genomes, mutants with large internal deletions to structural and nonstructural protein-coding regions. The isolates were all obtained from lorikeets that died from WNV at the Rio Grande Zoo in Albuquerque, NM between 2005 and 2007. The deletions are approximately 2kb, in frame, and result in the elimination of the complete envelope, and portions of the prM and NS-1 proteins. In Vero cell culture, these internally deleted WNV genomes function as defective interfering particles, reducing the production of full-length virus when introduced at high multiplicities of infection. In mosquitoes, the shortened WNV genomes reduced infection and dissemination rates, and virus titers overall, and were not detected in legs or salivary secretions at 14 or 21 days post-infection. In mice, inoculation with internally deleted genomes did not attenuate pathogenesis relative to full-length or infectious clone derived virus, and shortened genomes were not detected in mice at the time of death. These observations provide evidence that large deletions may occur within flavivirus populations more frequently than has generally been appreciated and suggest that they impact population phenotype minimally. Additionally, our findings suggest that highly similar mutants may frequently occur in particular vertebrate hosts.

  4. Report of the First International Consensus on Standardized Nomenclature of Antinuclear Antibody HEp-2 Cell Patterns 2014-2015.

    PubMed

    Chan, Edward K L; Damoiseaux, Jan; Carballo, Orlando Gabriel; Conrad, Karsten; de Melo Cruvinel, Wilson; Francescantonio, Paulo Luiz Carvalho; Fritzler, Marvin J; Garcia-De La Torre, Ignacio; Herold, Manfred; Mimori, Tsuneyo; Satoh, Minoru; von Mühlen, Carlos A; Andrade, Luis E C

    2015-01-01

    During the 12th International Workshop on Autoantibodies and Autoimmunity held in Sao Paulo, Brazil, on August 28, 2014, a full day session was devoted to establishing a consensus on the nomenclature of staining patterns observed in the antinuclear antibody (ANA) indirect immunofluorescence test on HEp-2 cells. The current report summarizes the collective agreements with input from the host Brazilian and international communities that represented research, clinical, and diagnostic service laboratories. Patterns are categorized in three major groups (nuclear, cytoplasmic, and mitotic patterns) and each pattern has been defined and described in detail. The consensus nomenclature and representative patterns are made available online at the international consensus on antinuclear antibody pattern (ICAP) website (www.ANApatterns.org). To facilitate continuous improvement and input, specific comments on ICAP are encouraged and these will be discussed in subsequent ICAP meetings. The ultimate goal with the establishment of the ICAP is to promote harmonization and understanding of autoantibody test nomenclature, as well as interpretation guidelines for ANA testing, thereby optimizing usage in patient care.

  5. Anti-Inflammatory and Antimicrobial Effects of Estradiol in Bovine Mammary Epithelial Cells during Staphylococcus aureus Internalization

    PubMed Central

    Medina-Estrada, Ivan; López-Meza, Joel E.

    2016-01-01

    17β-Estradiol (E2), the predominant sexual hormone in females, is associated with the modulation of the innate immune response (IIR), and changes in its levels at parturition are related to intramammary infections, such as mastitis. In bovine mammary epithelial cells (bMECs), E2 regulates differentiation and proliferation, but its immunomodulatory functions have not been explored. Staphylococcus aureus is the predominant pathogen causing mastitis, which can persist intracellularly in bMECs. The aim of this work was to analyze whether E2 modulates the IIR of bMECs during S. aureus internalization. bMECs treated with E2 (50 pg/mL, 24 h) reduced bacteria internalization (~50%). The host receptors α5β1 and TLR2 do not participate in this reduction. However, E2 activates ERα and modulates the IIR reducing the S. aureus induced-mRNA expression of TNF-α (~50%) and IL-1β (90%). E2 also decreased the secretion of these cytokines as well as IL-6 production; however, in infected bMECs, E2 induced the secretion of IL-1β. Furthermore, E2 upregulates the expression of the antimicrobial peptides DEFB1, BNBD5, and psoriasin S100A7 (~5-, 3-, and 6-fold, resp.). In addition, E2 induced the production of antimicrobial compounds in bMEC culture medium, which, together with the modulation of the IIR, could be related to the reduction of S. aureus internalization. PMID:27034592

  6. Estrogen Receptor Alpha Is Expressed in Mesenteric Mesothelial Cells and Is Internalized in Caveolae upon Freund's Adjuvant Treatment

    PubMed Central

    Balogh, Petra; Szabó, Arnold; Katz, Sándor; Likó, István; Patócs, Attila; L.Kiss, Anna

    2013-01-01

    Transformation of epithelial cells into connective tissue cells (epithelial-mesenchymal transition, EMT) is a complex mechanism involved in tumor metastasis, and in normal embryogenesis, while type II EMT is mainly associated with inflammatory events and tissue regenaration. In this study we examined type II EMT at the ultrastructural and molecular level during the inflammatory process induced by Freund's adjuvant treatment in rat mesenteric mesothelial cells. We found that upon the inflammatory stimulus mesothelial cells lost contact with the basal lamina and with each other, and were transformed into spindle-shaped cells. These morphological changes were accompanied by release of interleukins IL-1alpha, -1beta and IL-6 and by secretion of transforming growth factor beta (TGF-β) into the peritoneal cavity. Mesothelial cells also expressed estrogen receptor alpha (ER-α) as shown by immunolabeling at the light and electron microscopical levels, as well as by quantitative RT-PCR. The mRNA level of ER-α showed an inverse correlation with the secretion of TGF-β. At the cellular and subcellular levels ER-α was colocalized with the coat protein caveolin-1 and was found in the plasma membrane of mesothelial cells, in caveolae close to multivesicular bodies (MVBs) or in the membrane of these organelles, suggesting that ER-α is internalized via caveola-mediated endocytosis during inflammation. We found asymmetric, thickened, electron dense areas on the limiting membrane of MVBs (MVB plaques) indicating that these sites may serve as platforms for collecting and organizing regulatory proteins. Our morphological observations and biochemical data can contribute to form a potential model whereby ER-α and its caveola-mediated endocytosis might play role in TGF-β induced type II EMT in vivo. PMID:24244516

  7. Schistosoma mansoni soluble egg antigens are internalized by human dendritic cells through multiple C-type lectins and suppress TLR-induced dendritic cell activation.

    PubMed

    van Liempt, Ellis; van Vliet, Sandra J; Engering, Anneke; García Vallejo, Juan Jesus; Bank, Christine M C; Sanchez-Hernandez, Marta; van Kooyk, Yvette; van Die, Irma

    2007-04-01

    In schistosomiasis, a parasitic disease caused by helminths, the parasite eggs induce a T helper 2 cell (T(H)2) response in the host. Here, the specific role of human monocyte-derived dendritic cells (DCs) in initiation and polarization of the egg-specific T cell responses was examined. We demonstrate that immature DCs (iDCs) pulsed with schistosome soluble egg antigens (SEA) do not show an increase in expression of co-stimulatory molecules or cytokines, indicating that no conventional maturation was induced. The ability of SEA to affect the Toll-like receptor (TLR) induced maturation of iDCs was examined by copulsing the DCs with SEA and TLR-ligands. SEA suppressed both the maturation of iDCs induced by poly-I:C and LPS, as indicated by a decrease in co-stimulatory molecule expression and production of IL-12, IL-6 and TNF-alpha. In addition, SEA suppressed T(H)1 responses induced by the poly-I:C-pulsed DCs, and skewed the LPS-induced mixed response towards a T(H)2 response. Immature DCs rapidly internalized SEA through the C-type lectins DC-SIGN, MGL and the mannose receptor and the antigens were targeted to MHC class II-positive lysosomal compartments. The internalization of SEA by multiple C-type lectins may be important to regulate the response of the iDCs to TLR-induced signals.

  8. Super-resolution imaging-based single particle tracking reveals dynamics of nanoparticle internalization by live cells

    NASA Astrophysics Data System (ADS)

    Li, Yiming; Shang, Li; Nienhaus, G. Ulrich

    2016-03-01

    By combining super-resolution photoactivation localization microscopy with single particle tracking, we have visualized the endocytic process in the live-cell environment with nanoparticles (NPs) of different size and surface functionalization. This allowed us to analyze the dynamics of NPs interacting with cells with high spatial and temporal resolution. We identified two distinctly different types of pathways by which NPs are internalized via clathrin-coated pits (CCPs). Predominantly, NPs first bind to the membrane and, subsequently, CCPs form at this site. However, there are also instances where a NP diffuses on the membrane and utilizes a preformed CCP. Moreover, we have applied this new method to further explore the effects of size and surface functionalization on the NP dynamics on the plasma membrane and the ensuing endocytosis.By combining super-resolution photoactivation localization microscopy with single particle tracking, we have visualized the endocytic process in the live-cell environment with nanoparticles (NPs) of different size and surface functionalization. This allowed us to analyze the dynamics of NPs interacting with cells with high spatial and temporal resolution. We identified two distinctly different types of pathways by which NPs are internalized via clathrin-coated pits (CCPs). Predominantly, NPs first bind to the membrane and, subsequently, CCPs form at this site. However, there are also instances where a NP diffuses on the membrane and utilizes a preformed CCP. Moreover, we have applied this new method to further explore the effects of size and surface functionalization on the NP dynamics on the plasma membrane and the ensuing endocytosis. Electronic supplementary information (ESI) available: Experimental section, supporting figures and videos. See DOI: 10.1039/c6nr01495j

  9. Internal short circuit and accelerated rate calorimetry tests of lithium-ion cells: Considerations for methane-air intrinsic safety and explosion proof/flameproof protection methods

    PubMed Central

    Dubaniewicz, Thomas H.; DuCarme, Joseph P.

    2016-01-01

    Researchers with the National Institute for Occupational Safety and Health (NIOSH) studied the potential for lithium-ion cell thermal runaway from an internal short circuit in equipment for use in underground coal mines. In this third phase of the study, researchers compared plastic wedge crush-induced internal short circuit tests of selected lithium-ion cells within methane (CH4)-air mixtures with accelerated rate calorimetry tests of similar cells. Plastic wedge crush test results with metal oxide lithium-ion cells extracted from intrinsically safe evaluated equipment were mixed, with one cell model igniting the chamber atmosphere while another cell model did not. The two cells models exhibited different internal short circuit behaviors. A lithium iron phosphate (LiFePO4) cell model was tolerant to crush-induced internal short circuits within CH4-air, tested under manufacturer recommended charging conditions. Accelerating rate calorimetry tests with similar cells within a nitrogen purged 353-mL chamber produced ignitions that exceeded explosion proof and flameproof enclosure minimum internal pressure design criteria. Ignition pressures within a 20-L chamber with 6.5% CH4-air were relatively low, with much larger head space volume and less adiabatic test conditions. The literature indicates that sizeable lithium thionyl chloride (LiSOCl2) primary (non rechargeable) cell ignitions can be especially violent and toxic. Because ignition of an explosive atmosphere is expected within explosion proof or flameproof enclosures, there is a need to consider the potential for an internal explosive atmosphere ignition in combination with a lithium or lithium-ion battery thermal runaway process, and the resulting effects on the enclosure. PMID:27695201

  10. Internal short circuit and accelerated rate calorimetry tests of lithium-ion cells: Considerations for methane-air intrinsic safety and explosion proof/flameproof protection methods.

    PubMed

    Dubaniewicz, Thomas H; DuCarme, Joseph P

    2016-09-01

    Researchers with the National Institute for Occupational Safety and Health (NIOSH) studied the potential for lithium-ion cell thermal runaway from an internal short circuit in equipment for use in underground coal mines. In this third phase of the study, researchers compared plastic wedge crush-induced internal short circuit tests of selected lithium-ion cells within methane (CH4)-air mixtures with accelerated rate calorimetry tests of similar cells. Plastic wedge crush test results with metal oxide lithium-ion cells extracted from intrinsically safe evaluated equipment were mixed, with one cell model igniting the chamber atmosphere while another cell model did not. The two cells models exhibited different internal short circuit behaviors. A lithium iron phosphate (LiFePO4) cell model was tolerant to crush-induced internal short circuits within CH4-air, tested under manufacturer recommended charging conditions. Accelerating rate calorimetry tests with similar cells within a nitrogen purged 353-mL chamber produced ignitions that exceeded explosion proof and flameproof enclosure minimum internal pressure design criteria. Ignition pressures within a 20-L chamber with 6.5% CH4-air were relatively low, with much larger head space volume and less adiabatic test conditions. The literature indicates that sizeable lithium thionyl chloride (LiSOCl2) primary (non rechargeable) cell ignitions can be especially violent and toxic. Because ignition of an explosive atmosphere is expected within explosion proof or flameproof enclosures, there is a need to consider the potential for an internal explosive atmosphere ignition in combination with a lithium or lithium-ion battery thermal runaway process, and the resulting effects on the enclosure.

  11. Total internal reflection fluorescence microscopy study of spiral Ca2+ waves in single heart cell.

    PubMed

    Bai, Y; Tang, A; Wang, S; Zhu, X

    2008-03-01

    Spiral wave phenomena exist in many scales of nature and have attracted the attention of scientists from different fields. Although much work has been done on qualitative analysis of spiral waves, the mechanism of spiral waves' spontaneous formation and termination in living systems is still not clear. Here, by using total internal reflection fluorescence microscopy, we show the spiral waves of calcium signals in single rat cardiac myocytes and the simulation of the waves comprising calcium sparks. The mechanism of the formation and termination of spiral waves is attributable to the calcium release channels' refractory resulting from their stochastic release. We suggest that this mechanism can be adapted to other living systems.

  12. Accumulation of cell-penetrating peptides in large unilamellar vesicles: A straightforward screening assay for investigating the internalization mechanism.

    PubMed

    Swiecicki, Jean-Marie; Di Pisa, Margherita; Burlina, Fabienne; Lécorché, Pascaline; Mansuy, Christelle; Chassaing, Gérard; Lavielle, Solange

    2015-09-01

    The internalization of cell-penetrating peptides (CPPs) into liposomes (large unilamellar vesicles, LUVs) was studied with a rapid and robust procedure based on the quenching of a small fluorescent probe, 7-nitrobenz-2-oxa-1,3-diazole (NBD). Quenching can be achieved by reduction with dithionite or by pH jump. LUVs with different compositions of phospholipids (PLs) were used to screen the efficacy of different CPPs. In order to "validate" the composition of the membrane models, a control cationic peptide, which does not enter eukaryotic cells, was included in the study. It was found that pure DOPG or DOPG within ternary mixtures with cholesterol are the most appropriate models for studying CPP translocation. An anionic lipid, such as DOPG, is required for the adsorption of the basic peptides on the surface of LUVs. In addition, it acts as transfer agent through the lipid bilayer. A fluid phase and/or the presence of phase defects also appear mandatory for the internalization to occur. The neutralization of charges within an inverted micelle demonstrated in the case of DOPG and also proposed for a ternary mixture of PLs might not be the only mechanism for the CPP translocation. Finally, it is shown that oleic acid facilitates the entry inside LUVs in gel phase of a series of cationic peptides including CPPs and also the negative control peptide PKCi.

  13. The International Society of Urological Pathology (ISUP) grading system for renal cell carcinoma and other prognostic parameters.

    PubMed

    Delahunt, Brett; Cheville, John C; Martignoni, Guido; Humphrey, Peter A; Magi-Galluzzi, Cristina; McKenney, Jesse; Egevad, Lars; Algaba, Ferran; Moch, Holger; Grignon, David J; Montironi, Rodolfo; Srigley, John R

    2013-10-01

    The International Society of Urological Pathology 2012 Consensus Conference made recommendations regarding classification, prognostic factors, staging, and immunohistochemical and molecular assessment of adult renal tumors. Issues relating to prognostic factors were coordinated by a workgroup who identified tumor morphotype, sarcomatoid/rhabdoid differentiation, tumor necrosis, grading, and microvascular invasion as potential prognostic parameters. There was consensus that the main morphotypes of renal cell carcinoma (RCC) were of prognostic significance, that subtyping of papillary RCC (types 1 and 2) provided additional prognostic information, and that clear cell tubulopapillary RCC was associated with a more favorable outcome. For tumors showing sarcomatoid or rhabdoid differentiation, there was consensus that a minimum proportion of tumor was not required for diagnostic purposes. It was also agreed upon that the underlying subtype of carcinoma should be reported. For sarcomatoid carcinoma, it was further agreed upon that if the underlying carcinoma subtype was absent the tumor should be classified as a grade 4 unclassified carcinoma with a sarcomatoid component. Tumor necrosis was considered to have prognostic significance, with assessment based on macroscopic and microscopic examination of the tumor. It was recommended that for clear cell RCC the amount of necrosis should be quantified. There was consensus that nucleolar prominence defined grades 1 to 3 of clear cell and papillary RCCs, whereas extreme nuclear pleomorphism or sarcomatoid and/or rhabdoid differentiation defined grade 4 tumors. It was agreed upon that chromophobe RCC should not be graded. There was consensus that microvascular invasion should not be included as a staging criterion for RCC.

  14. Targeted tumor cell internalization and imaging of multifunctional quantum dot-conjugated immunoliposomes in vitro and in vivo.

    PubMed

    Weng, Kevin C; Noble, Charles O; Papahadjopoulos-Sternberg, Brigitte; Chen, Fanqing F; Drummond, Daryl C; Kirpotin, Dmitri B; Wang, Donghui; Hom, Yun K; Hann, Byron; Park, John W

    2008-09-01

    Targeted drug delivery systems that combine imaging and therapeutic modalities in a single macromolecular construct may offer advantages in the development and application of nanomedicines. To incorporate the unique optical properties of luminescent quantum dots (QDs) into immunoliposomes for cancer diagnosis and treatment, we describe the synthesis, biophysical characterization, tumor cell-selective internalization, and anticancer drug delivery of QD-conjugated immunoliposome-based nanoparticles (QD-ILs). Pharmacokinetic properties and in vivo imaging capability of QD-ILs were also investigated. Freeze-fracture electron microscopy was used to visualize naked QDs, liposome controls, nontargeted QD-conjugated liposomes (QD-Ls), and QD-ILs. QD-ILs prepared by insertion of anti-HER2 scFv exhibited efficient receptor-mediated endocytosis in HER2-overexpressing SK-BR-3 and MCF-7/HER2 cells but not in control MCF-7 cells as analyzed by flow cytometry and confocal microscopy. In contrast, nontargeted QD-Ls showed minimal binding and uptake in these cells. Doxorubicin-loaded QD-ILs showed efficient anticancer activity, while no cytotoxicity was observed for QD-ILs without chemotherapeutic payload. In athymic mice, QD-ILs significantly prolonged circulation of QDs, exhibiting a plasma terminal half-life ( t 1/2) of approximately 2.9 h as compared to free QDs with t 1/2 < 10 min. In MCF-7/HER2 xenograft models, localization of QD-ILs at tumor sites was confirmed by in vivo fluorescence imaging.

  15. Gradual internal methane reforming in intermediate-temperature solid-oxide fuel cells

    SciTech Connect

    Vernoux, P.; Guindet, J.; Kleitz, M.

    1998-10-01

    Gradual internal reforming is based on local coupling between steam reforming of the fuel which occurs on a catalyst and hydrogen electrochemical oxidation which occurs at the electrode triple-phase perimeter. In order to demonstrate the feasibility of this strategy, the catalytic and electrochemical properties of lanthanum chromite, pure and impregnated with ruthenium, were investigated. Ruthenium supported on lanthanum chromite exhibits very good catalytic activity for the steam reforming of methane. Full conversion of steam is obtained for ratios H{sub 2}O/CH{sub 4} even lower than 1 at 700 C. No carbon deposition could be detected after 100 h of operation. Electrochemical measurements, carried out by impedance spectroscopy on cone-shaped microelectrodes of lanthanum chromite, show that the overpotential resistance under H{sub 2}/H{sub 2}O is lower than under CO/CO{sub 2} and much lower than under CH{sub 4}/H{sub 2}O. In the presence of ruthenium, impedance diagrams under hydrogen and methane are fairly similar and gas analysis shows that some methane is reformed. This observation demonstrates that gradual internal reforming can be implemented. A detailed analysis of the electrode impedance diagrams shows that the so-called high-frequency semicircle is virtually independent of the nature of the atmosphere. This indicates that it is not directly related to any chemical or electrochemical step of the electrode reaction.

  16. Internalization and trafficking mechanisms of coxsackievirus B3 in HeLa cells

    SciTech Connect

    Chung, Sun-Ku; Kim, Joo-Young; Kim, In-Beom; Park, Sang-Ick; Paek, Kyung-Hee; Nam, Jae-Hwan . E-mail: jnam66@yahoo.com

    2005-03-01

    Coxsackievirus B3 (CVB3) is nonenveloped and has a single-stranded positive-sense RNA genome. CVB3 induces myocarditis and ultimately dilated cardiomyopathy. Although there are mounting evidences of an interaction between CVB3 particles and the cellular receptors, coxsackievirus and adenovirus receptor (CAR) and decay-accelerating factor (DAF), very little is known about the mechanisms of internalization and trafficking. In the present study, we used the CVB3 H3 strain, which is CAR-dependent but DAF-independent Woodruff variant and found that during entry, CVB3 particles were colocalized in clathrin, after interacting primarily with CAR, which was not recycled to the plasma membrane. We also found that CVB3 internalization was dependent on the function of dynamin, a large GTPase that has an essential role in endocytosis. Heat-shock cognate protein, Hsc70, which acts as a chaperone in the release of coat proteins from clathrin-coated vesicles (CCV), played a role in CVB3 trafficking processes. Moreover, endosomal acidification was crucial for CVB3 endocytosis. Finally, CVB3 was colocalized in early endosome autoantigen 1 (EEA1) molecules, which are involved in endosome-endosome tethering and fusion. In conclusion, these data together indicate that CVB3 uses clathrin-mediated endocytosis and is transcytosed to early endosomes.

  17. P