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Sample records for international kupffer cell

  1. Amphibia Kupffer cells.

    PubMed

    Sichel, Giovanni; Scalia, Marina; Corsaro, Concetta

    2002-06-15

    Amphibia Kupffer cells (i.e., liver resident macrophages) show many common characteristics when compared with Mammalia Kupffer cells: filopodia, microvillous-like structures, lamellipodia, fuzzy coat, coated vesicles, bristled vacuoles, nonspecific esterase activity, and pinocytotic and phagocytic activity are present both in Amphibia and Mammalia Kupffer cells. On the other hand, some differences are present between Kupffer cells of both zoological classes: phagocytosed red cells and their derivatives, iron-protein complexes, and lipofuscin bodies are normally present in Amphibia Kupffer cells, but absent in the same cells of healthy mammals. Worm-like structures are not seen in Amphibia and endogenous peroxidase activity is very weak in these animals compared with Mammalia. The most important difference lies in the ability of Amphibia Kupffer cells to produce melanins: in fact the tyrosinase gene is expressed, "melanosome centers" are present, and dopa oxidase activity is demonstrable.

  2. 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

  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. 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.

  5. 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.

  6. Malaria circumsporozoite protein inhibits the respiratory burst in Kupffer cells.

    PubMed

    Usynin, Ivan; Klotz, Christian; Frevert, Ute

    2007-11-01

    After transmission by infected mosquitoes, malaria sporozoites rapidly travel to the liver. To infect hepatocytes, sporozoites traverse Kupffer cells, but surprisingly, the parasites are not killed by these resident macrophages of the liver. Here we show that Plasmodium sporozoites and recombinant circumsporozoite protein (CSP) suppress the respiratory burst in Kupffer cells. Sporozoites and CSP increased the intracellular concentration of cyclic adenosyl mono-phosphate (cAMP) and inositol 1,4,5-triphosphate in Kupffer cells, but not in hepatocytes or liver endothelia. Preincubation with cAMP analogues or inhibition of phosphodiesterase also inhibited the respiratory burst. By contrast, adenylyl cyclase inhibition abrogated the suppressive effect of sporozoites. Selective protein kinase A (PKA) inhibitors failed to reverse the CSP-mediated blockage and stimulation of the exchange protein directly activated by cAMP (EPAC), but not PKA inhibited the respiratory burst. Both blockage of the low-density lipoprotein receptor-related protein (LRP-1) with receptor-associated protein and elimination of cell surface proteoglycans inhibited the cAMP increase in Kupffer cells. We propose that by binding of CSP to LRP-1 and cell surface proteoglycans, malaria sporozoites induce a cAMP/EPAC-dependent, but PKA-independent signal transduction pathway that suppresses defence mechanisms in Kupffer cells. This allows the sporozoites to safely pass through these professional phagocytes and to develop inside neighbouring hepatocytes.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. THE FATE OF VACCINIA VIRUS ON CULTIVATION IN VITRO WITH KUPFFER CELLS (RETICULO-ENDOTHELIAL CELLS)

    PubMed Central

    Beard, Joseph W.; Rous, Peyton

    1938-01-01

    The pathogenic activity of vaccinia virus is in large part suppressed when it is mixed with living Kupffer cells or clasmatocytes in the test-tube and injected intradermally. Vaccinia increases in quantity when introduced into cultures of Kupffer cells in vitro, and survives in immediate association with these elements. No antiviral principle is elaborated by them under such conditions. PMID:19870763

  12. 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.

  13. 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.

  14. PEGylated IL-10 Activates Kupffer Cells to Control Hypercholesterolemia

    PubMed Central

    Chan, Ivan H.; Van Hoof, Dennis; Abramova, Marina; Bilardello, Melissa; Mar, Elliot; Jorgensen, Brett; McCauley, Scott; Bal, Harminder; Oft, Martin; Van Vlasselaer, Peter

    2016-01-01

    Interleukin-10 (IL-10) is a multifunctional cytokine that exerts potent context specific immunostimulatory and immunosuppressive effects. We have investigated the mechanism by which PEGylated rIL-10 regulates plasma cholesterol in mice and humans. In agreement with previous work on rIL-10, we report that PEGylated rIL-10 harnesses the myeloid immune system to control total plasma cholesterol levels. We have discovered that PEG-rMuIL-10’s dramatic lowering of plasma cholesterol is dependent on phagocytotic cells. In particular, PEG-rHuIL-10 enhances cholesterol uptake by Kupffer cells. In addition, removal of phagocytotic cells dramatically increases plasma cholesterol levels, suggesting for the first time that immunological cells are implicitly involved in regulating total cholesterol levels. These data suggest that treatment with PEG-rIL-10 potentiates endogenous cholesterol regulating cell populations not currently targeted by standard of care therapeutics. Furthermore, we show that IL-10’s increase of Kupffer cell cholesterol phagocytosis is concomitant with decreases in liver cholesterol and triglycerides. This leads to the reversal of early periportal liver fibrosis and facilitates the restoration of liver health. These data recommend PEG-rIL-10 for evaluation in the treatment of fatty liver disease and preventing its progression to non-alcoholic steatohepatitis. In direct confirmation of our in vivo findings in the treatment of hypercholesterolemic mice with PEG-rMuIL-10, we report that treatment of hypercholesterolemic cancer patients with PEG-rHuIL-10 lowers total plasma cholesterol by up to 50%. Taken together these data suggest that PEG-rIL-10’s cholesterol regulating biology is consistent between mice and humans. PMID:27299860

  15. 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. PMID:26830481

  16. Importance of Kupffer Cells in the Development of Acute Liver Injuries in Mice

    PubMed Central

    Tsutsui, Hiroko; Nishiguchi, Shuhei

    2014-01-01

    Kupffer cells reside within the liver sinusoid and serve as gatekeepers. They produce pro- and anti-inflammatory cytokines and other biologically important molecules upon the engagement of pattern recognition receptors such as Toll-like receptors. Kupffer cell-ablated mice established by in vivo treatment with clodronate liposomes have revealed many important features of Kupffer cells. In this paper, we review the importance of Kupffer cells in murine acute liver injuries and focus on the following two models: lipopolysaccharide (LPS)-induced liver injury, which is induced by priming with Propionibacterium acnes and subsequent challenge with LPS, and hypercoagulability-mediated acute liver failure such as that in concanavalin A (Con A)-induced hepatitis. Kupffer cells are required for LPS sensitization induced by P. acnes and are a major cellular source of interleukin-18, which induces acute liver injury following LPS challenge. Kupffer cells contribute to Con A-induced acute liver failure by initiating pathogenic, intrasinusoidal thrombosis in collaboration with sinusoidal endothelial cells. The mechanisms underlying these models may shed light on human liver injuries induced by various etiologies such as viral infection and/or abnormal metabolism. PMID:24802875

  17. Triglycerides potentiate the inflammatory response in rat Kupffer cells.

    PubMed

    Budick-Harmelin, Noga; Dudas, Jozsef; Demuth, Julia; Madar, Zecharia; Ramadori, Giuliano; Tirosh, Oren

    2008-12-01

    Accumulation of fat in the liver, also known as steatosis, may lead to inflammation and tissue damage. Kupffer cells (KCs) are the resident macrophages of the liver and have an important role in inflammatory reactions. The inflammatory response of isolated rat KCs to endotoxin in the presence of lipids was investigated in this study. KCs were treated with lipopolysaccharide (LPS) and triglycerides (TGs) alone or in combination. TGs had no effect on the expression of pro-inflammatory mediators, but adding TGs to LPS enhanced the induction of inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF), compared with LPS treatment alone. Increased DNA binding of NF-kappaB transcription factor was seen on simultaneous exposure of the cells to TGs and LPS, which was accompanied by decreased intracellular ROS production and increased GSH levels. The inflammation-potentiating effect of TGs on iNOS expression was abolished on NF-kappaB inhibition. This enhanced inflammatory response might indicate a contribution of lipids to the inflammatory conditions in the fatty liver by increased activation of KCs. PMID:18710323

  18. Triglycerides potentiate the inflammatory response in rat Kupffer cells.

    PubMed

    Budick-Harmelin, Noga; Dudas, Jozsef; Demuth, Julia; Madar, Zecharia; Ramadori, Giuliano; Tirosh, Oren

    2008-12-01

    Accumulation of fat in the liver, also known as steatosis, may lead to inflammation and tissue damage. Kupffer cells (KCs) are the resident macrophages of the liver and have an important role in inflammatory reactions. The inflammatory response of isolated rat KCs to endotoxin in the presence of lipids was investigated in this study. KCs were treated with lipopolysaccharide (LPS) and triglycerides (TGs) alone or in combination. TGs had no effect on the expression of pro-inflammatory mediators, but adding TGs to LPS enhanced the induction of inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF), compared with LPS treatment alone. Increased DNA binding of NF-kappaB transcription factor was seen on simultaneous exposure of the cells to TGs and LPS, which was accompanied by decreased intracellular ROS production and increased GSH levels. The inflammation-potentiating effect of TGs on iNOS expression was abolished on NF-kappaB inhibition. This enhanced inflammatory response might indicate a contribution of lipids to the inflammatory conditions in the fatty liver by increased activation of KCs.

  19. Liver injury in hypervitaminosis A: Evidence for activation of Kupffer cell function

    SciTech Connect

    Sim, W.L.W.

    1988-01-01

    The most important and novel finding of this work was enhanced liver Kupffer cell phagocytic and metabolic function by hypervitaminosis A. An animal model of hypervitaminosis A was developed in male Sprague-Dawley rats gavaged with 250,000 I.U. retinol/kg body weight/day for 3 weeks. Presence of hypervitaminosis A was indicated by characteristic changes in the fur coat, presence of brittle bones and spontaneous fractures and a significant increase in plasma and liver concentrations of retinyl palmitate while retinol levels remained the same as in controls. Hypervitaminosis A did not cause severe liver abnormalities as reflected by normal plasma glutamate pyruvate transaminase activity and bilirubin. The main change was a marked increase in size of the fat or Vitamin A storing cells. Measurement of clearance from blood of indocyanine green and {sup 99m}Tc-disofenin indicated this hepatocyte function was normal. Kupffer cell phagocytic function was enhanced in hypervitaminosis A as determined by clearance from blood of {sup 99m}Tc-sulfur colloid. In vitro, there was also evidence that treatment with high doses of Vitamin A activated or enhanced Kupffer cell function. Kupffer cells from control and Vitamin A treated rats were isolated by enzymatic dispersion, purified by centrifugal elutriation, and placed in culture. Activation was indicated by (1) increased phagocytosis of {sup 51}Cr-labeled opsonized sheep red blood cells (2) enhanced release of superoxide anion and (3) enhanced production of tumor cytolytic factor by Kupffer cells from Vitamin A treated rats.

  20. Interactions between macrophage/Kupffer cells and hepatocytes in surgical sepsis

    SciTech Connect

    West, M.A.

    1988-01-01

    Experiments were performed to investigate the role of Kupffer cell/macrophage interactions with hepatocytes in modulating liver function during infections using direct in vitro cocultivation of rat macrophages or Kupffer cells with rat hepatocytes. Protein synthesis was assayed as a sensitive indicator of integrated hepatocellular function by measuring {sup 3}H-leucine incorporation into hepatocyte protein. Septic stimuli such as lipoploysaccharide and killed bacteria were added to cocultures of hepatocytes and macrophages or Kupffer cells and the responses compared to hepatocytes alone. Information about the types of proteins synthesized by hepatocytes under various culture conditions was determined using polyacrylamide gel electrophoresis and autoradiography. These experiments showed that septic stimuli alter the amount and type of protein synthesized by hepatocytes and had no direct effect on hepatocytes in the absence of macrophages or Kupffer cells. The mediator(s) appears to be a heat labile, soluble monokine(s) which is distinct from interleukin-1 or tumor necrosis factor. The important role of Kupffer cells/macrophages in mediating alterations in hepatocellular function in sepsis may ultimately improve patient care.

  1. 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

  2. [Isolation and purification of primary Kupffer cells from mouse liver].

    PubMed

    Sun, Chao; Luo, Qingbo; Lu, Xiuxian; Zheng, Daofeng; He, Diao; Wu, Zhongjun

    2016-08-01

    Objective To isolate and purify Kupffer cells (KCs) from BALB/c mice by an efficient method of low-speed centrifugation and rapid adherence. Methods The mouse liver tissue was perfused in situ and digested with 0.5 g/L collagenase type IV in vitro by water bath. Then, through the low-speed centrifugation, KCs were separated from the mixed hepatocytes, and purified by rapid adherent characteristics. Finally, the production and activity of KCs obtained by this modified method were compared with those isolated by Percoll density gradient centrifugation. We used F4/80 antibody immunofluorescence technique to observe morphological features of KCs, flow cytometry (FCM) to detect the expression of F4/80 antibody and the ink uptake test to observe the phagocytic activity. Moreover, using FCM, we evaluated the expressions of molecules associated with antigen presentation, including major histocompatibility complex class II (MHC II), CD40, CD86 and CD68 on the surface of KCs subjected to hypoxia/reoxygenation (H/R) modeling. And, ELISA was conducted to measure tumor necrosis factor-α (TNF-α) production of the cultured KCs following H/R. Results The yield of KCs was (5.83±0.54)×10(6) per mouse liver and the survival rate of KCs was up to 92% by low-speed centrifugation and rapid adherent method. Compared with Percoll density gradient centrifugation [the yield of KCs was (2.19±0.43)×10(6) per liver], this new method significantly improved the yield of KCs. F4/80 immunofluorescence showed typical morphologic features of KCs such as spindle or polygon shapes and FCM identified nearly 90% F4/80 positive cells. The phagocytic assay showed that lots of ink particles were phagocytosed into the isolated cells. KC H/R models expressed more MHC II, CD40 and CD86 and produced more TNF-α participating in inflammation. Conclusion The efficient method to isolate and purify KCs from BALB /c mice has been successfully established. PMID:27412929

  3. [Isolation and purification of primary Kupffer cells from mouse liver].

    PubMed

    Sun, Chao; Luo, Qingbo; Lu, Xiuxian; Zheng, Daofeng; He, Diao; Wu, Zhongjun

    2016-08-01

    Objective To isolate and purify Kupffer cells (KCs) from BALB/c mice by an efficient method of low-speed centrifugation and rapid adherence. Methods The mouse liver tissue was perfused in situ and digested with 0.5 g/L collagenase type IV in vitro by water bath. Then, through the low-speed centrifugation, KCs were separated from the mixed hepatocytes, and purified by rapid adherent characteristics. Finally, the production and activity of KCs obtained by this modified method were compared with those isolated by Percoll density gradient centrifugation. We used F4/80 antibody immunofluorescence technique to observe morphological features of KCs, flow cytometry (FCM) to detect the expression of F4/80 antibody and the ink uptake test to observe the phagocytic activity. Moreover, using FCM, we evaluated the expressions of molecules associated with antigen presentation, including major histocompatibility complex class II (MHC II), CD40, CD86 and CD68 on the surface of KCs subjected to hypoxia/reoxygenation (H/R) modeling. And, ELISA was conducted to measure tumor necrosis factor-α (TNF-α) production of the cultured KCs following H/R. Results The yield of KCs was (5.83±0.54)×10(6) per mouse liver and the survival rate of KCs was up to 92% by low-speed centrifugation and rapid adherent method. Compared with Percoll density gradient centrifugation [the yield of KCs was (2.19±0.43)×10(6) per liver], this new method significantly improved the yield of KCs. F4/80 immunofluorescence showed typical morphologic features of KCs such as spindle or polygon shapes and FCM identified nearly 90% F4/80 positive cells. The phagocytic assay showed that lots of ink particles were phagocytosed into the isolated cells. KC H/R models expressed more MHC II, CD40 and CD86 and produced more TNF-α participating in inflammation. Conclusion The efficient method to isolate and purify KCs from BALB /c mice has been successfully established.

  4. Local proliferation and extrahepatic recruitment of liver macrophages (Kupffer cells) in partial-body irradiated rats

    SciTech Connect

    Bouwens, L.; Knook, D.L.; Wisse, E.

    1986-06-01

    The relative significance of local proliferation and extrahepatic recruitment of Kupffer cells was investigated by partial-body irradiation before the induction of macrophage hyperplasia by zymosan. There was no difference in growth of the Kupffer cells population between nonirradiated rats and rats irradiated with the liver shielded, whereas irradiation of the liver with the rest of the body (bone marrow) shielded resulted in strong inhibition of growth (-61%). Splenectomy combined with bone marrow irradiation inhibited growth to a lesser extent as compared to liver irradiation (-38%). Monocyte and other leukocyte numbers were strongly reduced in peripheral blood and their accumulation in the liver was completely prevented by bone marrow irradiation. Our results demonstrate that local proliferation of resident Kupffer cells represents the predominant source for their increased number during hyperplasia.

  5. Prolactin inhibits the increased cytokine gene expression in Kupffer cells following haemorrhage.

    PubMed

    Zhu, X H; Zellweger, R; Ayala, A; Chaudry, I H

    1996-02-01

    Kupffer cells are an important source of proinflammatory cytokines and contribute to the systemic inflammatory response observed following haemorrhagic shock. The systemic release of cytokines, such as TNF-alpha, IL-1 beta, IL-6, etc., has been associated with the decreased host immune and organ dysfunction following hypotension. Studies indicate that anterior pituitary hormone prolactin (PRL) plays an important role in the regulation of lymphocyte proliferation and macrophage function in vivo, as well as in vitro. However, it is not known what effects PRL administration has on Kupffer cells proinflammatory mediator release following haemorrhage. Therefore, it was the aim of this study to determine the effect of in vivo PRL administration on cytokine gene expression in Kupffer cells after haemorrhage. To study this, C3H/HeN male mice were bled to and maintained at a mean arterial pressure of 35 mmHg for 60 minutes, then resuscitated with shed blood, and segregated into two groups: one group was treated with PRL (100 micrograms/25 g body weight subcutaneously) while the other group received saline-vehicles. This was followed with lactated Ringer's solution (2 x the volume of shed blood). Two hours thereafter, the animals were sacrificed, Kupffer cells were isolated and stimulated with or without 10 micrograms/ml LPS for 1 hour. Total RNA was extracted and cytokine mRNA was detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The results demonstrated that haemorrhage markedly increased the level of mRNA for IL-1 beta, IL-6, TGF-beta and TNF-beta in Kupffer cells. However, in vivo PRL treatment significantly decreased the cytokine gene expression in Kupffer cells following haemorrhage. This indicates that PRL may be useful in blunting the systemic inflammatory response associated with cell and organ depression following shock.

  6. 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

  7. 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.

  8. Kupffer cells suppress hepatocarcinogenesis and metastasis in tumor orthotopic implanted Kunming mice.

    PubMed

    Li, X Y; Wang, M Y; Zhang, J Y; Li, J Z; Gong, J P; Zhang, Wei

    2013-01-01

    In this research, we used GdCl3 (gadolinium chloride) to restrain the function of Kupffer cells and assessed effects on hepatocarcinogenesis and metastasis in the Kunming mouse. A 0.25% GdCl3 solution (10 mg/kg b.w.) was infused via the vena caudalis of each mouse 1 week before inoculation of H22 cells and was continued once per three days. Then we observed the follow indexes 3 weeks after injection of H22 cells: tumor weight, histologic characteristics of tumor tissue by light microscopy, ultramicrostructure of Kupffer cells under the electron microscope, distribution and number of Kupffer cells by histochemical staining, and TNF-α and IFN-γ levels in blood-serum and liver tissue by ELISA and RT-PCR. MMP-2 protein expression was tested by immunohistochemistry. The GdCl3 pretreatment had no effect on the quantity of Kupffer cells, but clearly restrained their functions, with decrease of TNF-α and IFN-γ levels and elevation of MMP2. Tumor immunity functions were markedly suppressed and tumor growth was accelerated with appearance of metastasis. Furthermore, survival time of trial mice was shortened.

  9. 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.

  10. Effects of glycine on phagocytosis and secretion by Kupffer cells in vitro

    PubMed Central

    Wu, Hui-Wen; Yun, Ke-Ming; Han, De-Wu; Xu, Rui-Ling; Zhao, Yuan-Chang

    2012-01-01

    AIM: To investigate the effects and mechanisms of action of glycine on phagocytosis and tumor necrosis factor (TNF)-α secretion by Kupffer cells in vitro. METHODS: Kupffer cells were isolated from normal rats by collagenase digestion and Percoll density gradient differential centrifugation. After culture for 24 h, Kupffer cells were incubated in fresh Dulbecco's Modification of Eagle’s Medium containing glycine (G1: 1 mmol/L, G2: 10 mmol/L, G3: 100 mmol/L and G4: 300 mmol/L) for 3 h, then used to measure phagocytosis by a bead test, TNF-α secretion after lipopolysaccharide stimulation by radioactive immunoassay, and microfilament and microtubule expression by staining with phalloidin-fluorescein isothiocyanate (FITC) or a monoclonal anti-α tubulin-FITC antibody, respectively, and evaluated under a ultraviolet fluorescence microscope. RESULTS: Glycine decreased the phagocytosis of Kupffer cells at both 30 min and 60 min (P < 0.01, P < 0.05). The numbers of beads phagocytosed by Kupffer cells in 30 min were 16.9 ± 4.0 (control), 9.6 ± 4.1 (G1), 12.1 ± 5.7 (G2), 8.1 ± 3.2 (G3) and 7.5 ± 2.0 (G4), and were 22.5 ± 7.9 (control), 20.1 ± 5.8 (G1), 19.3 ± 4.8 (G2), 13.5 ± 4.7 (G3) and 9.2 ± 3.1 (G4) after 60 min. TNF-α secretion by Kupffer cells in G1 (0.19 ± 0.03), G2 (0.16 ± 0.04), G3 (0.14 ± 0.03) and G4 (0.13 ± 0.05) was significantly less than that in controls (0.26 ± 0.03, P < 0.01), and the decrease in secretion was dose-dependent (P < 0.05). Microfilaments of Kupffer cells in G2, G3 and G4 groups were arranged in a disorderly manner. The fluorescence densities of microtubules in G1 (53.4 ± 10.5), G2 (54.1 ± 14.6), G3 (64.9 ± 12.1) and G4 (52.1 ± 14.2) were all lower than those in the controls (102.2 ± 23.7, P < 0.01), but the decrease in microtubule fluorescence density was not dose-dependant. CONCLUSION: Glycine can decrease the phagocytosis and secretion by Kupffer cells in vitro, which may be related to the changes in the expression of

  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. Influence of Kupffer cell inactivation on cycloheximide-induced hepatic injury.

    PubMed

    Kumagai, Kazuyoshi; Kiyosawa, Naoki; Ito, Kazumi; Yamoto, Takashi; Teranishi, Munehiro; Nakayama, Hiroyuki; Manabe, Sunao

    2007-11-30

    In our previous study, we found that cycloheximide (CHX) induces hepatocellular necrosis as well as hepatocellular apoptosis. This article evaluates the role of Kupffer cells on cycloheximide-induced hepatic injury using gadolinium chloride (GdCl(3)) for the inhibition of Kupffer cells. One group of rats was treated with CHX (CHX group), and another was treated with GdCl(3) before being treated with the same dose of CHX (GdCl(3)/CHX group). The necrotic change in the GdCl(3)/CHX group was exacerbated under the induction of hepatocellular apoptosis by the CHX treatment. A substantial diminution of the number of ED1- or ED2-positive cells was demonstrated in the GdCl(3)/CHX group compared to the CHX group. In addition, the degree of decrease in ED2-positive cells was more apparent than that in ED1-positive cells. Increases in the mRNA levels of IL-10 and Stat3 were observed in the CHX group, but not in the GdCl(3)/CHX group. On the other hand, the hepatic mRNA levels of chemokines and adhesion molecules such as Ccl20, LOX-1, and E-selectin were significantly increased only in the GdCl(3)/CHX group. Thus, Kupffer cell inactivation by the GdCl(3) treatment leads to a loss of the capacity to produce IL-10, supposedly resulting in the enhancement of pro-inflammatory cytokine activities such as tumor necrosis factor (TNF) signaling. These events are suggested to be a factor of the inflammatory exacerbation in the livers of the GdCl(3)/CHX group. In conclusion, Kupffer cells may play a role in protecting hepatic necroinflammatory changes by releasing anti-inflammatory cytokines following the hepatocellular apoptosis resulting from CHX treatment. PMID:17900782

  13. CD18/ICAM-1-dependent oxidative NF-kappaB activation leading to nitric oxide production in rat Kupffer cells cocultured with syngeneic hepatoma cells.

    PubMed Central

    Kurose, I; Saito, H; Miura, S; Ebinuma, H; Higuchi, H; Watanabe, N; Zeki, S; Nakamura, T; Takaishi, M; Ishii, H

    1997-01-01

    Previous studies have indicated that nitric oxide (NO) released from Kupffer cells modulates biological viability of cocultured hepatoma cells. This study was designed to evaluate the mechanisms by which Kupffer cells synthesize and release NO in reponse to cocultured hepatoma cells. Kupffer cells isolated from male Wistar rats were cocultured with rat hepatoma cell line, AH70 cells. The sum of nitrite and nitrate levels increased in the culture medium of Kupffer cells with AH70 cells as compared with those of Kupffer cells or AH70 cells alone. Increased expressions of iNOS and iNOS mRNA in Kupffer cells cocultured with AH70 cells were detected by an immunofluorescence staining and a fluorescence in situ hybridization study, respectively. A fluorescence in situ DNA-protein binding assay revealed that NF-kappaB activation occurs in Kupffer cells and activated NF-kappaB moved into the nuclei preceding to an increased production of NO. Oxidative stress indicated by dichlorofluorescein fluorescence was observed in Kupffer cells cocultured with AH70 cells. An increased calcium mobilization indicated as increased fluo-3-associated fluorescence was also induced in Kupffer cells after coculture with AH70 cells. Monoclonal antibodies directed against rat CD18 and ICAM-1, as well as TMB-8, a calcium inhibitor, prevented the calcium mobilization, active oxygen production, and NF-kappaB activation in addition to the increased production of NO. Pyrrolidine dithiocarbamate, an inhibitor of oxidative NF-kappaB activation, diphenylene iodonium, an NADPH oxidase inhibitor, and quinacrine, a phospholipase A2 inhibitor, significantly attenuated the increase in dichlorofluorescein fluorescence, NF-kappaB activation, and NO production. Therefore, this study suggests that CD18/ICAM-1-dependent cell-to-cell interaction with hepatoma cells causes calcium mobilization and oxidative activation of NF-kappaB, which may lead to the increased production of NO in Kupffer cells. PMID:9062344

  14. Diminished organelle motion in murine Kupffer cells during the erythrocytic stage of malaria

    PubMed Central

    Bellows, Charles F.; Molina, Ramon M.; Brain, Joseph D.

    2011-01-01

    Parasitized erythrocytes are ingested by murine hepatic macrophages during malaria infection. We non-invasively monitored how this altered the motion of intracellular phagosomes in Kupffer cells using magnetometry. Submicrometric γFe2O3 particles were injected prior to malaria infection. They were cleared from the blood, primarily by Kupffer cells, and retained within their phagosomes. The mice were periodically magnetized. After removing this external magnet, the aligned iron particles created a remnant magnetic field (RMF) which then decayed (relaxation), reflecting the motion of particle-containing phagosomes. After baseline measurements of relaxation, the mice were injected intravenously with Plasmodium chabaudi-parasitized or normal murine red blood cells (RBCs). During the next 15 days, relaxation measurements, parasitaemia and haematocrit values were monitored. At 6 days post injection with 3 × 107 parasitized RBCs, relaxation rates had decreased. At this time, all mice had parasitaemias greater than 58 per cent and haematocrits less than 20 per cent. At day 7, while the parasitaemias were declining, the rate of relaxation continued to decrease. Throughout the experiment, relaxation remained constant in animals injected with normal RBCs. Electron microscopy revealed Kupffer cells filled with damaged and parasitized erythrocytes, and haemoglobin degradation pigment. We conclude that ingestion and metabolism of parasitized erythrocytes by liver macrophages during malaria infection decreases their organelle motion with likely consequences of compromised host defences. PMID:21068031

  15. 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.

  16. Purinergic signaling via P2Y receptors up-mediates IL-6 production by liver macrophages/Kupffer cells.

    PubMed

    Ishimaru, Makiko; Yusuke, Negishi; Tsukimoto, Mitsutoshi; Harada, Hitoshi; Takenouchi, Takato; Kitani, Hiroshi; Kojima, Shuji

    2014-06-01

    Resident macrophages in the liver (Kupffer cells) produce various cytokines and chemokines, and have important roles in hepatitis and liver fibrosis. The cells are activated by various factors, for example lipopolysaccharide (LPS), which is an endotoxin and is high in the blood of patients with liver cirrhosis. Involvement of P2 receptors in the release of pro-inflammatory cytokines from Kupffer cells is little. In this study, we investigated purinergic signaling in the release of pro-inflammatory cytokines, such as IL-6 and TNF-α, from liver Kupffer cells of C57BL/6 mice (KUP5 cells). KUP5cells were isolated from C57BL/6 mice and cultivated with Dulbecco's modified Eagle's medium. The cells were stimulated with LPS. LPS-induced IL-6 production by KUP5 cells was suppressed significantly by pretreatments with non-selective P2 antagonist suramin, P2Y13antagonist MRS2211, and ecto-nucleotidase, whereas P2Y receptor agonists, significantly increased the IL-6 production. P2Y13knockdown reduced LPS-induced IL-6 production, but by less than 50%. These results would suggest that P2Y receptors including P2Y13and others, may involves in LPS-induced IL-6 production in Kupffer cells, leading to the liver inflammation. Therefore, we first showed the importance of purinergic signaling via P2Y receptors in the activation of Kupffer cells and liver injury, which is worthwhile in drug development for liver diseases. PMID:24849676

  17. 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

  18. 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.

  19. 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

  20. Interactions between isolated hepatocytes and Kupffer cells in iron metabolism: a possible role for ferritin as an iron carrier protein.

    PubMed

    Sibille, J C; Kondo, H; Aisen, P

    1988-01-01

    Like the rat peritoneal macrophage, the isolated Kupffer cell is capable of processing and releasing iron acquired by phagocytosis of immunosensitized homologous red blood cells. When erythrophagocytosis is restrained to levels which do not affect cell viability, about one red cell per macrophage, close to 50% of iron acquired from red cells is released within 24 hr in the form of ferritin. Immunoradiometric assay of the extracellular medium indicates that 160 ng ferritin are released by 10(6) Kupffer cells after 24-hr incubation at 37 degrees C. Iron release is temperature-dependent, the rate at 37 degrees C being nearly 5-fold greater than at 4 degrees C. As estimated by sucrose-gradient ultracentrifugation, ferritin released by the erythrophagocytosing Kupffer cell averages 2,400 iron atoms per molecule. When reincubated with isolated hepatocytes, this released ferritin is rapidly taken up by the cells. Via this process, hepatocytes may accumulate more than 160,000 iron atoms per cell per min. Such accumulation is not impeded by the presence of iron-loaded transferrin in the culture medium, but is markedly depressed by rat liver ferritin. In contrast to the conservation of transferrin during its interaction with hepatocytes, the protein shell of the ferritin molecule is rapidly degraded into trichloroacetic acid-soluble fragments. Ferritin-mediated transfer of iron from Kupffer cells to hepatocytes may help explain the resistance of the liver to iron deficiency as well as the liver's susceptibility to iron overload. PMID:3356411

  1. Establishment of c-myc-immortalized Kupffer cell line from a C57BL/6 mouse strain.

    PubMed

    Kitani, Hiroshi; Sakuma, Chisato; Takenouchi, Takato; Sato, Mitsuru; Yoshioka, Miyako; Yamanaka, Noriko

    2014-01-01

    We recently demonstrated in several mammalian species, a novel procedure to obtain liver-macrophages (Kupffer cells) in sufficient numbers and purity using a mixed primary culture of hepatocytes. In this study, we applied this method to the C57BL/6 mouse liver and established an immortalized Kupffer cell line from this mouse strain. The hepatocytes from the C57BL/6 adult mouse liver were isolated by a two-step collagenase perfusion method and cultured in T25 culture flasks. Similar to our previous studies, the mouse hepatocytes progressively changed their morphology into a fibroblastic appearance after a few days of culture. After 7-10 days of culture, Kupffer-like cells, which were contaminants in the hepatocyte fraction at the start of the culture, actively proliferated on the mixed fibroblastic cell sheet. At this stage, a retroviral vector containing the human c-myc oncogene and neomycin resistance gene was introduced into the mixed culture. Gentle shaking of the culture flask, followed by the transfer and brief incubation of the culture supernatant, resulted in a quick and selective adhesion of Kupffer cells to a plastic dish surface. After selection with G418 and cloning by limiting dilutions, a clonal cell line (KUP5) was established. KUP5 cells displayed typical macrophage morphology and were stably passaged at 4-5 days intervals for more than 5 months, with a population doubling time of 19 h. KUP5 cells are immunocytochemically positive for mouse macrophage markers, such as Mac-1, F4/80. KUP5 cells exhibited substantial phagocytosis of polystyrene microbeads and the release of inflammatory cytokines upon lipopolysaccharide stimulation. Taken together, KUP5 cells provide a useful means to study the function of Kupffer cells in vitro. PMID:25379377

  2. Accessory function of Kupffer cells in the antigen-specific blastogenic response of an L3T4+ T-lymphocyte clone to Listeria monocytogenes.

    PubMed Central

    Gregory, S H; Wing, E J

    1990-01-01

    The function of Kupffer cells in the development of protective immunity to infection by Listeria monocytogenes is controversial. To determine their role in antilisterial host defenses, Kupffer cells were separated from other nonparenchymal cells of the liver by centrifugation on a metrizamide gradient followed by adherence to glass or plastic. The resultant highly enriched Kupffer cell population supported the antigen-specific blastogenic response [( 3H]thymidine incorporation) of cloned L3T4+ T lymphocytes to L. monocytogenes in vitro. Blastogenesis was dependent upon the duration of the incubation period, the concentration of the antigen, and the number of Kupffer cells in culture. Maximum reactivity was greater than that observed when the same T-cell population was incubated with adherent peritoneal exudate cells and antigen under optimal conditions. The addition of antibodies specific for murine interleukin-1 beta to cocultures of Kupffer cells and T lymphocytes eliminated the antigen-stimulated incorporation of [3H]thymidine, indicating a requirement for interleukin-1. Analysis of the culture supernatants demonstrated that, in addition to interleukin-1, granulocyte-macrophage colony-stimulating factor, interleukin-6, and gamma interferon were elaborated in cocultures containing cloned T lymphocytes, Kupffer cells, and antigen. These results suggest that Kupffer cells may serve a critical role in the development of immunity to infection by L. monocytogenes in vivo. Images PMID:2114361

  3. Cytochrome P4502E1 inhibitor, chlormethiazole, decreases lipopolysaccharide-induced inflammation in rat Kupffer cells with ethanol treatment

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To investigate the role of Cytochrome P4502E1 in sensitizing Kupffer cells to lipopolysaccharide (LPS)-mediated inflammation after ethanol induction. Sprague-Dawley rats were fed a liquid ethanol diet, control diet or ethanol diet supplemented with CYP2E1 inhibitor, chlormethiazole (CMZ), for 4'week...

  4. Hexokinase II binding to mitochondria is necessary for Kupffer cell activation and is potentiated by ethanol exposure.

    PubMed

    Shulga, Nataly; Pastorino, John G

    2014-09-19

    Ethanol exposure promotes the development of steatohepatitis, which can progress to end stage liver disease. Kupffer cells have been documented to play a key role in the genesis and progression of alcoholic liver disease with ethanol exposure enhancing Kupffer cell activation. In the present study, we identified the binding of hexokinase II to the mitochondria as a requirement for LPS-induced activation of Kupffer cells and its potentiation by ethanol. LPS and ethanol exposure induced a reduction in sirtuin-3 activity. In turn, the decline of sirtuin-3 activity led to the activation of cyclophilin-D, which mediated an increased binding of hexokinase II to the mitochondria. Suppression of cyclophilin-D expression or enforced detachment of hexokinase II from the mitochondria abrogated the LPS- and ethanol-induced stimulation of Kupffer cells, preventing NADPH oxidase and inflammasome activation. Moreover, activation of AMP-activated protein kinase restored sirtuin-3 activity, thereby preventing LPS and ethanol from stimulating the binding of hexokinase II to the mitochondria and precluding NADPH oxidase and inflammasome activation.

  5. Effect of praseodymium nitrate on hepatocytes and Kupffer cells in the rat.

    PubMed

    Tuchweber, B; Trost, R; Salas, M; Sieck, W

    1976-12-01

    Intravenous administration of the rare earth metal salt, praseodymium nitrate, induced hepatic damage in the rat, as assessed by morphologic examination (light and electron microscopy) and biochemical parameters (serum glutamic-pyruvic transaminase (EC 2.6.1.2) and glutamic-oxalacetic transaminase (EC 2.6.1.1) activity as well as hepatic triglyceride content). Praseodymium hepatotoxicity was only attained with lower doses (10, 20, or 40 mg/kg), whereas a larger dose (80 mg/kg) was inactive in this respect. As detected by electron microscopy, lower doses of the metal salt caused hepatocytic alterations consisting of degranulation and dilatation of rough endoplasmic reticulum, accumulation of smooth endoplasmic reticulum as well as numerous lipid droplets. No abnormalities were detected in the cell organelles following administration of a large dose of the metal salt; however, vacuoles containing markedly electron-dense material were seen in the cytoplasm of the hepatocytes and the sinusoidal Kupffer cells.

  6. 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

  7. Iron-dependent activation of NF-kappaB in Kupffer cells: a priming mechanism for alcoholic liver disease.

    PubMed

    Xiong, Shigang; She, Hongyun; Sung, Chin K; Tsukamoto, Hidekazu

    2003-06-01

    Alcoholic liver disease is associated with hepatic iron accumulation, and iron supplementation exacerbates alcoholic liver disease, suggesting the pathogenic role of iron in alcoholic liver disease. We have tested a hypothesis that iron plays a signaling role in activation of redox-sensitive nuclear factor-kappa B (NF-kappaB) and that increased iron content results in heightened expression of proinflammatory cytokines in Kupffer cells because of this signaling. In cultured Kupffer cells isolated from normal rats, treatment with a lipophilic iron chelator, 1,2-dimethyl-3-hydroxypyrid-4-one (L1), markedly reduced lipopolysaccharide (LPS)-induced NF-kappaB activation and expression of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6. Kupffer cells, isolated from rats with experimentally induced alcoholic liver disease, had significant increases in nonheme iron content, NF-kappaB binding, and mRNA expression for TNF-alpha and macrophage inflammatory protein-1. Ex vivo L1 treatment normalized all these parameters. Addition of ferrous iron to cultured normal rat Kupffer cells increased I-kappa B kinase (IKK) activity at 15 min and NF-kappaB binding at 30 min. L1 pretreatment completely abrogated both effects. Moreover, the iron treatment increased TNF-alpha release and TNF-alpha promoter activity in a NF-kappaB-dependent manner. Ferrous iron also transiently decreased cytoplasmic I-kappa B-alpha (IkappaB-alpha), with concomitant increases in nuclear p65 protein and DNA binding of p65/p50. Taken together, these results support the existence of iron-dependent signaling for activation of IKK/NF-kappaB in Kupffer cells, and this iron signaling serves as a target for a potential priming effect for the pathogenesis of experimental alcoholic liver disease.

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

    PubMed

    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.

  9. 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

  10. Targeting Kupffer cells in non-alcoholic fatty liver disease/non-alcoholic steatohepatitis: Why and how?

    PubMed Central

    Lanthier, Nicolas

    2015-01-01

    Mechanisms for non-alcoholic steatohepatitis (NASH) development are under investigation in an era of increased prevalence of obesity and metabolic syndrome. Previous findings have pointed to the role of adipose tissue, adipose tissue macrophages and their secretory products in the development of a chronic inflammatory status inducing insulin resistance and a higher risk of liver steatosis called non-alcoholic fatty liver disease. The activation of resident macrophages [Kupffer cells (KC)] and the recruitment of blood derived monocytes/macrophages into the diseased liver have now been identified as key elements for disease initiation and progression. Those cells could be activated through gut flora modifications and an altered gut barrier function but also through the internalization of toxic lipid compounds in adjacent hepatocytes or in KC themselves. Due to the role of activated KC in insulin resistance, fibrosis development and inflammation amplification, they became a target in clinical trials. A shift towards an anti-inflammatory KC phenotype through peroxisome proliferator activator-receptorδ agonists, an inhibition of macrophage recruitment through anti-C-C chemokine receptor 2 action and a specific blocking of internalization of toxic lipoxidation or glycation compounds into KC by galectin-3 receptor inhibitors are now under investigation in human NASH. PMID:26380042

  11. 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.

  12. 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

  13. LXRα represses LPS-induced inflammatory responses by competing with IRF3 for GRIP1 in Kupffer cells.

    PubMed

    Miao, Chun-Mu; He, Kun; Li, Pei-Zhi; Liu, Zuo-Jin; Zhu, Xi-Wen; Ou, Zhi-Bing; Ruan, Xiong-Zhong; Gong, Jian-Ping; Liu, Chang-An

    2016-06-01

    Liver X receptors (LXRs) in the nucleus play important roles in lipid metabolism and inflammation. The mechanism of LXR regulation of the LPS-induced Toll-like receptor 4 (TLR4) inflammatory signaling pathway remains to be elucidated. C57/BL6 mice were randomly divided into four groups: control, T0901317 (a LXRs agonist), LPS and T0901317+LPS. Additionally, Kupffer cells isolated from male C57/BL6 mice were divided into the same four groups. A decreased amount of inflammatory cells infiltrated the portal areas and the hepatic sinusoids in the livers of mice in the T0901317+LPS group than in those of mice in the LPS group. In the T0901317+LPS group, the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and tumor necrosis factor alpha (TNF-α) were lower, while the serum level of interleukin-10 (IL-10) was higher. In vitro, Kupffer cells pretreated with T0901317 for 24h presented reduced TNF-α, interferon-beta (IFN-β) and interleukin-1 beta (IL-1β) levels, while the IL-10 level increased; however, the mRNA and protein expression levels of interferon regulatory factor 3 (IRF3) and glucocorticoid receptor-interacting protein 1 (GRIP1) were not significantly reduced. The co-IP data illustrated that LXRα bound to GRIP1 specifically in the T0901317+LPS group, while less IRF3 was bound to GRIP1 in the T0901317+LPS group than in the LPS group. Furthermore, the DNA-binding activity of NF-κB was decreased by pretreating Kupffer cells with T0901317 for 24h. These results suggest that activated LXRα competes with IRF3 for GRIP1 binding, thus repressing IRF3 and NF-κB transcriptional activity and inhibiting the inflammatory response initiated by LPS in Kupffer cells. PMID:27085678

  14. Advanced glycation end products are eliminated by scavenger-receptor-mediated endocytosis in hepatic sinusoidal Kupffer and endothelial cells.

    PubMed Central

    Smedsrød, B; Melkko, J; Araki, N; Sano, H; Horiuchi, S

    1997-01-01

    Long-term incubation of proteins with glucose leads to the formation of advanced glycation end products (AGE). Physiological aspects of the catabolism of non-enzymically glycated proteins were studied in vivo and in vitro. AGE-modified BSA (AGE-BSA) was a mixture of high-Mr (cross-linked), monomeric and low-Mr (fragmented) AGE-BSA. After intravenous administration in rat, all three fractions of AGE-BSA accumulated extremely rapidly and almost exclusively in liver. Uptake in liver endothelial, Kupffer and parenchymal cells accounted for approx. 60%, 25% and 10-15% respectively of hepatic elimination. Both cross-linked and monomeric AGE-BSA were efficiently taken up and degraded in cultures of purified liver endothelial and Kupffer cells. Endocytosis of AGE-BSA by these cells was inhibited by several ligands for the scavenger receptor. Although 125I-Hb was not endocytosed in vitro, 125I-AGE-Hb was effectively endocytosed by a mechanism that was subject to inhibition by AGE-BSA. Endocytosis of N-terminal propeptide of type I procollagen, a physiological ligand for the scavenger receptor, was effectively inhibited by AGE-Hb and AGE-BSA. We conclude that AGE-modification renders macromolecules susceptible for elimination via the scavenger receptor of both liver endothelial and Kupffer cells. PMID:9065778

  15. Fatty acid binding protein 7 regulates phagocytosis and cytokine production in Kupffer cells during liver injury.

    PubMed

    Miyazaki, Hirofumi; Sawada, Tomoo; Kiyohira, Miwa; Yu, Zhiqian; Nakamura, Keiji; Yasumoto, Yuki; Kagawa, Yoshiteru; Ebrahimi, Majid; Islam, Ariful; Sharifi, Kazem; Kawamura, Saki; Kodama, Takanori; Yamamoto, Yui; Adachi, Yasuhiro; Tokuda, Nobuko; Terai, Shuji; Sakaida, Isao; Ishikawa, Toshizo; Owada, Yuji

    2014-09-01

    Kupffer cells (KCs) are involved in the progression of liver diseases such as hepatitis and liver cancer. Several members of the fatty acid binding proteins (FABPs) are expressed by tissue macrophages, and FABP7 is localized only in KCs. To clarify the role of FABP7 in the regulation of KC function, we evaluated pathological changes of Fabp7 knockout mice during carbon tetrachloride-induced liver injury. During liver injury in Fabp7 knockout mice, serum liver enzymes were increased, cytokine expression (tumor necrosis factor-α, monocyte chemoattractant protein-1, and transforming growth factor-β) was decreased in the liver, and the number of KCs in the liver necrotic area was significantly decreased. Interestingly, in the FABP7-deficient KCs, phagocytosis of apoptotic cells was impaired, and expression of the scavenger receptor CD36 was markedly decreased. In chronic liver injury, Fabp7 knockout mice showed less fibrogenic response to carbon tetrachloride compared with wild-type mice. Taken together, FABP7 is involved in the liver injury process through its regulation of KC phagocytic activity and cytokine production. Such modulation of KC function by FABP7 may provide a novel therapeutic approach to the treatment of liver diseases.

  16. The activity of mouse Kupffer cells following intravenous injection of T4 bacteriophage

    PubMed Central

    Inchley, C. J.

    1969-01-01

    The response of macrophages from the livers and spleens of mice given a single immunizing dose of T4 bacteriophage has been studied. Following their rapid removal from the circulation, phage particles were found to be concentrated in the liver to a level twelve times that for the spleen. Investigation of the fate of ingested phage showed that it was disposed of more rapidly in the liver than in the spleen, as measured by the disappearance of viable T4 particles and by the loss of radioactive label following injection of [131I]T4. It was also found that antigen-containing Kupffer cells could elicit little or no antibody synthesis on transfer into normal syngeneic recipients, or on incubation with lymphoid cells in vitro. It is suggested that these macrophages differ from other components of the reticulo-endothelial system in their treatment of T4 antigen, and may be concerned mainly with its breakdown and disposal rather than with providing a stimulus for the initiation of antibody synthesis. PMID:5370053

  17. 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.

  18. Kupffer cells modulate hepatic fatty acid oxidation during infection with PR8 influenza.

    PubMed

    Tarasenko, Tatyana N; Singh, Larry N; Chatterji-Len, Milani; Zerfas, Patricia M; Cusmano-Ozog, Kristina; McGuire, Peter J

    2015-11-01

    In response to infection, patients with inborn errors of metabolism may develop a functional deterioration termed metabolic decompensation. The biochemical hallmarks of this disruption of metabolic homeostasis are disease specific and may include acidosis, hyperammonemia or hypoglycemia. In a model system previously published by our group, we noted that during influenza infection, mice displayed a depression in hepatic mitochondrial enzymes involved in nitrogen metabolism. Based on these findings, we hypothesized that this normal adaptation may extend to other metabolic pathways, and as such, may impact various inborn errors of metabolism. Since the liver is a critical organ in inborn errors of metabolism, we carried out untargeted metabolomic profiling of livers using mass spectrometry in C57Bl/6 mice infected with influenza to characterize metabolic adaptation. Pathway analysis of metabolomic data revealed reductions in CoA synthesis, and long chain fatty acyl CoA and carnitine species. These metabolic adaptations coincided with a depression in hepatic long chain β-oxidation mRNA and protein. To our surprise, the metabolic changes observed occurred in conjunction with a hepatic innate immune response, as demonstrated by transcriptional profiling and flow cytometry. By employing an immunomodulation strategy to deplete Kupffer cells, we were able to improve the expression of multiple genes involved in β-oxidation. Based on these findings, we are the first to suggest that the role of the liver as an immunologic organ is central in the pathophysiology of hepatic metabolic decompensation in inborn errors of metabolism due to respiratory viral infection.

  19. Soluble CLEC2 Extracellular Domain Improves Glucose and Lipid Homeostasis by Regulating Liver Kupffer Cell Polarization

    PubMed Central

    Wu, Xinle; Zhang, Jun; Ge, Hongfei; Gupte, Jamila; Baribault, Helene; Lee, Ki Jeong; Lemon, Bryan; Coberly, Suzanne; Gong, Yan; Pan, Zheng; Rulifson, Ingrid C.; Gardner, Jonitha; Richards, William G.; Li, Yang

    2015-01-01

    The polarization of tissue resident macrophages toward the alternatively activated, anti-inflammatory M2 phenotype is believed to positively impact obesity and insulin resistance. Here we show that the soluble form of the extracellular domain (ECD) of C-type lectin-like receptor 2, CLEC2, regulates Kupffer cell polarization in the liver and improves glucose and lipid parameters in diabetic animal models. Over-expression of Fc-CLEC2(ECD) in mice via in vivo gene delivery, or injection of recombinant Fc-CLEC2(ECD) protein, results in a reduction of blood glucose and liver triglyceride levels and improves glucose tolerance. Furthermore, Fc-CLEC2(ECD) treatment improves cytokine profiles and increases both the M2 macrophage population and the genes involved in the oxidation of lipid metabolism in the liver. These data reveal a previously unidentified role for CLEC2 as a regulator of macrophage polarity, and establish CLEC2 as a promising therapeutic target for treatment of diabetes and liver disease. PMID:26151067

  20. Cell-surface arylsulfatase A and B on sinusoidal endothelial cells, hepatocytes, and Kupffer cells in mammalian livers.

    PubMed

    Mitsunaga-Nakatsubo, Keiko; Kusunoki, Shinichiro; Kawakami, Hayato; Akasaka, Koji; Akimoto, Yoshihiro

    2009-06-01

    Arylsulfatase A (ARSA) and B (ARSB) have been regarded as lysosomal enzymes because of their hydrolytic activity on synthetic aromatic substrates and the lysosomal localization of their enzymatic activity. Using sea urchin embryos, we previously demonstrated that the bulk of ARS is located on the cell surface of the epithelium, colocalizing with sulfated polysaccharides, and that it does not exhibit enzymatic activity. To examine whether ARSA and ARSB exist on the cell surface in mammalian tissues, we raised antibodies against ARSA and ARSB and examined immunohistochemically their localization in the liver using light and electron microscopy. Here we show that mammalian ARSA and ARSB exist on the cell surface of sinusoidal endothelial cells, hepatocytes, and sinusoidal macrophages (Kupffer cells), as well as in the lysosome. They are also colocalized with heparan sulfate proteoglycan. These results suggest that ARSA and ARSB also may function in the cell surface of mammals. This is the first report to show cell-surface localization of ARS in mammalian somatic cells. The extracellular localization of ARS will provide new insight for human ARS deficiency disorders, such as metachromatic leukodystrophy and mucopolysaccharidosis VI.

  1. Origin and kinetics of Kupffer cells during an acute inflammatory response

    PubMed Central

    Dulk, M. M. C. Diesselhoff-Den; Crofton, R. W.; Van Furth, R.

    1979-01-01

    The course of the increased number of liver macrophages and the origin of these cells were studied after intravenous stimulation by zymosan, stilboestrol, or corynebacterium. The macrophages were isolated by digestion of the liver with pronase and DNAase. Zymosan doubled the number of liver macrophages per gram of liver and led to a four- to five-fold increase in the number of blood monocytes, whereas stilboestrol induced a four-fold increase of liver macrophages and a two-fold increase of blood monocytes. Corynebacterium administration gave a two-fold rise in the number of liver macrophages and a six-fold increase of blood monocytes. The in vitro labelling index of the liver macrophages showed a transient but marked increase after the administration of zymosan or stilboestrol, but returned to approximately normal values 4 days after the stimulus. Hydrocortisone given 48 h before zymosan prevented this increase in the in vitro labelling index of liver macrophages, thus demonstrating that the mononuclear phagocytes labelled in vitro had recently been recruited from the bone marrow to the liver. Stimulation by stilboestrol or zymosan of mice labelled with [3H]-thymidine caused an increase in the number of labelled liver macrophages and blood monocytes as compared with the numerical course of the labelled Kupffer cells and monocytes in untreated mice. It may be concluded that this increase is attributable to the increased influx into the circulation of labelled monocytes from the bone marrow, which in turn migrate to the liver in larger numbers than are seen in the normal steady state. Local proliferation could not have been responsible for the increased number of labelled liver macrophages, because free [3H]-thymidine was no longer available when the stimulus was applied. Evidence supporting the bone marrow origin of the increased number of monocytes and liver macrophages after intravenous stimulation was provided by the course of the number of monocytes and liver

  2. Anti-inflammatory effects of propofol on lipopolysaccharides-treated rat hepatic Kupffer cells.

    PubMed

    Li, Sen; Wang, Chun-xia; Liu, Nai-Zheng; Liu, Ping

    2015-03-01

    This study is set to explore the role of commonly used intravenous anesthetic propofol on the inflammatory response of rat liver Kupffer cells (KCs) induced by lipopolysaccharides (LPS). The isolated KCs were cultured at the density of 1 × 10(5)/ml, divided into five groups randomly after 48 h culture: group C, control group; group L, KCs were treated with 1 μg/ml LPS for 24 h; groups P1, P2, P3, KCs were pretreated with propofol at low (25 μM), medium (50 μM), high (100 μM) concentration for 2 h, respectively, and then were stimulated with 1 μg/ml LPS for 24 h. The expressions of tumor necrosis factor-α (TNF-α) mRNA and interleukin-1β (IL-1β) mRNA of every group were measured by RT-PCR. Nuclear NF-ΚB p65 was determined by Western blot. The concentrations of IL-1β and TNF-α in supernatant were measured by ELISA. Compared with the group C, TNF-α mRNA and IL-1β mRNA in group L were significantly up-regulated and NF-ΚB p65 was significantly up-regulated after LPS treatment (P < 0.05). Meanwhile, TNF-α and IL-1β were also significantly increased (P < 0.05). With propofol the mRNA expressions of aforementioned inflammatory mediators were significantly down-regulated and NF-ΚB p65 was significantly inhibited in group P2 and P3 (P < 0.05), compared with group L. However, low propofol concentration did not exhibit any effect (group P1, P > 0.05). Propofol at medium and high concentration can counteract the LPS-induced inflammatory response in KCs by regulating NF-ΚB p65 protein expression. PMID:25296958

  3. Kupffer cell blockade improves the endotoxin-induced microcirculatory inflammatory response in obstructive jaundice.

    PubMed

    Abrahám, Szabolcs; Szabó, Andrea; Kaszaki, József; Varga, Renáta; Eder, Katalin; Duda, Erno; Lázár, György; Tiszlavicz, László; Boros, Mihály; Lázár, György

    2008-07-01

    Cholestasis predisposes to hypersensitivity to LPS, leading to potential septic complications. We set out to characterize the involvement of Kupffer cell (KC) activation in the hepatic microcirculatory and structural consequences of obstructive jaundice in the presence and absence of acute endotoxemia. The hepatic microcirculatory consequences of 3-day extrahepatic bile duct ligation (BDL) were assessed in rats. The contributions of changes in hepatic perfusion, leukocyte influx, and proinflammatory cytokine release to the development of hepatic structural damage were also determined. Furthermore, the corresponding consequences of BDL in combination with acute (2-h) endotoxemia (1 mg kg(-1) LPS, i.v.) were compared with those observed after LPS alone. In a second series, the same protocols were applied in identical groups of rats where the KC function was inhibited with 24-h gadolinium chloride pretreatment (10 mg kg(-1), i.v.). Bile duct ligation induced minor inflammatory reactions but caused a marked reduction in hepatic sinusoidal perfusion and severe histological damage. LPS treatment, however, elicited an approximately 5-fold increase in leukocyte adherence in the central venules and pronounced IL-6 and TNF-alpha release, but without significant structural damage. The combination of BDL with LPS enhanced the perfusion failure, leukocyte sticking/deposition, and proinflammatory cytokine release; most of these changes can be effectively ameliorated by gadolinium chloride. In conclusion, when obstructive jaundice is followed by a second hit of LPS, perfusion failure, liver inflammation, and structural damage are enhanced, the KCs playing a decisive role in this scenario. Therapeutic strategies aimed at KC blockade can potentially reduce the risk of inflammatory complications in cholestasis. PMID:18562926

  4. Kupffer cells are associated with apoptosis, inflammation and fibrotic effects in hepatic fibrosis in rats.

    PubMed

    Liu, Cheng; Tao, Qing; Sun, Mingyu; Wu, Jim Z; Yang, Wengang; Jian, Ping; Peng, Jinghua; Hu, Yiyang; Liu, Chenghai; Liu, Ping

    2010-12-01

    Hepatocellular apoptosis, hepatic inflammation, and fibrosis are prominent features in chronic liver diseases. However, the linkage among these processes remains mechanistically unclear. In this study, we examined the apoptosis and activation of Kupffer cells (KCs) as well as their pathophysiological involvement in liver fibrosis process. Hepatic fibrosis was induced in rats by dimethylnitrosamine (DMN) or carbon tetrachloride (CCl4) treatment. KCs were isolated from normal rats and incubated with lipopolysaccharide (LPS) or from fibrotic rats. The KCs were stained immunohistochemically with anti-CD68 antibody, a biomarker for KC. The level of expression of CD68 was analyzed by western blot and real-time PCR methods. The apoptosis and pathophysiological involvement of KCs in the formation of liver fibrosis were studied using confocal microscopy. The mRNA and protein expression of CD68 were significantly increased in DMN- and CCL4-treated rats. Confocal microscopy analysis showed that CD68-positive KCs, but not α-smooth muscle actin (SMA)-positive cells, underwent apoptosis in the liver of DMN- and CCL4-treated rats. It was also revealed that the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and CD68-double-positive apoptotic KCs located in the portal or fibrotic septa area were situated next to hepatic stellate cells (HSCs). Tumor necrosis factor-α (TNF-α) and KC co-localized in the liver in the neighbor of HSCs. The double α-SMA- and collagen type I-positive cells predominantly existed in fibrotic septa, and those cells were co-localized clearly with CD68-positive cells. Interestingly, some CD68 and Col (1) double positive, but completely negative for α-SMA, were found in the portal areas and hepatic sinusoids; this phenomenon was also validated in primary isolated KCs after 6 h LPS exposure or fibrotic rats in vitro. These results show that KCs are associated with hepatocellular apoptosis, inflammation, and fibrosis process in a liver

  5. 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.

  6. Development of a cytokine-producing immortalized murine Kupffer cell line.

    PubMed

    Wang, Zheng-Yu; Burlak, Christopher; Klaunig, James E; Kamendulis, Lisa M

    2014-12-01

    Kupffer cells (KC) play a critical role in both liver physiology and the pathogenesis of various liver diseases. Isolated primary KC have a limited lifespan in culture, and due to the relatively low number obtained, limit their study in vitro. Here, a cytokine-producing immortalized KC (ImKC) line was established from transgenic mice that express the thermolabile mutant tsA58 of the Simian virus 40 large T antigen under the control of the H-2k(b) promoter. Primary KC were obtained using a three step procedure: liver perfusion, centrifugal elutriation, and sorting for F4/80⁺ cells. ImKC were identified within the small-intermediate population of KC that maintained stable expression of F4/80, and the surface antigens CD11b, CD14 and TLR4. ImKC grow at IFNγ-independent manner at 37°C and exhibited a doubling time of ∼24 h when cultured in RPMI 1640 with 5% FBS. Our observations indicate that both activation of telomerase and expression of P53 are markedly increased, suggesting that enhanced telomerase activity and P53 expression may contribute to the immortalization of this cell population. ImKC cells maintained a high capacity to phagocytose FITC-latex beads, and bind/phagocytose erythrocytes. In addition, similar to primary KC, ImKC responded to stimulation with lipopolysaccharide (LPS: 0.1-1μg/ml) by upregulating mRNA levels of TNFα (23-fold), IL-6 (28-fold), and IL-1β (1459-fold), as measured by qRT-PCR. Protein levels of TNFα and IL-6 were also increased, 10-fold and 12-fold, respectively. Reactive oxygen species (ROS) and nitric oxide (NO) production were significantly enhanced in ImKC following an LPS challenge. Furthermore, LPS elicited a marked increase in mitogen activated protein kinase (MAPK) phospho-(ERK1/2, JNK) and NF-κB p50 with decreased IκBα in ImKC, as assessed by Western blot. Collectively, these results demonstrate that the ImKC line retains critical characteristics of primary KC, and thus provides a useful tool to assess the role of

  7. Silibinin protects OTA-mediated TNF-alpha release from perfused rat livers and isolated rat Kupffer cells.

    PubMed

    Al-Anati, Lauy; Essid, Ebtisam; Reinehr, Roland; Petzinger, Ernst

    2009-04-01

    We studied the inhibitory effect of silibinin on ochratoxin A (OTA) and LPS-mediated tumor necrosis factor alpha (TNF-alpha) release and the leakage of cytotoxic markers glutamate dehydrogenase (GLDH) and lactate dehydrogenase (LDH), from isolated blood-free perfused rat livers, and from isolated pure rat Kupffer cells. In the recirculation perfusion model at the end point 90 min, 2.5 micromol/L OTA released 2600 pg/mL TNF-alpha without effects on liver vitality. LPS at 0.1 microg/mL induced 3000 pg TNF-alpha/mL with slight leakage of GLDH and LDH. Under similar experimental conditions, the addition of silibinin 10 min prior to OTA and LPS showed dose-dependent protection against OTA or LPS-induced hepatic TNF-alpha release. High-dose of silibinin (12.5 microg/mL) also completely restored GLDH and LDH levels in the perfusate. Pretreatment of isolated Kupffer cells with 0.02, 0.1, 0.5, 2.5, and 12.5 microg silibinin/mL 30 min prior to OTA reduced OTA-induced TNF-alpha levels to 90, 70, 25, 25, and 25% at 4 h, respectively, and abrogated any TNF-alpha release at 24 h. Similarly, in the presence of silibinin LPS-induced TNF-alpha levels decreased at 4 h to 71, 57, 18, 22, and 18%, respectively. However, after 24 h of LPS exposition the protection by silibinin vanished and TNF-alpha partially recurred into the incubation medium under LPS. In summary, silibinin had hepatoprotective effects against OTA- or LPS-mediated TNF-alpha release and also reduced the cytotoxicity of both toxins. Isolated Kupffer cells were even more sensitive to the protective effect than perfused livers and responded to very low concentrations of silibinin with a strong inhibition of toxins-mediated TNF-alpha release. PMID:19156713

  8. 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

  9. 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.

  10. Kupffer cell-independent acute hepatocellular oxidative stress and decreased bile formation in post-cold-ischemic rat liver.

    PubMed

    Kumamoto, Y; Suematsu, M; Shimazu, M; Kato, Y; Sano, T; Makino, N; Hirano, K I; Naito, M; Wakabayashi, G; Ishimura, Y; Kitajima, M

    1999-12-01

    The purpose of this study was to examine distribution and time history of oxidative stress during the hyperacute period of reperfusion in the liver grafts undergoing cold ischemia and to investigate roles of Kupffer cells as a potential oxidant source. Rat livers were harvested at 4 degrees C in University of Wisconsin solution and followed by reperfusion with Krebs-Henseleit buffer under monitoring bile excretion. To investigate oxidative changes, laser-confocal microfluorography was performed in reperfused livers preloaded with dichlorodihydrofluorescein diacetate succinimidyl ester, a fluorescence precursor sensing intracellular hydroperoxide generation. Livers undergoing the 16-hour cold storage displayed an impaired recovery of bile acid-dependent bile output concurrent with a marked increase in hydroperoxide generation in hepatocytes, which occurred as early as 5 minutes after the onset of reperfusion, whereas the status of lobular perfusion was well maintained. Pretreatment with liposome-encapsulated dichloromethylene diphosphonate, a Kupffer cell-depleting reagent, did neither alter the reperfusion-induced periportal oxidative changes nor improve the recovery of bile output in the graft. On the other hand, EPCK, a hepatotropic antioxidant composed of vitamin E phosphate ester bound to vitamin C, not only diminished the oxidative changes but also improved the reduction of bile acid-dependent bile output. Furthermore, the reagent was capable of inhibiting H(2)O(2)-induced oxidative stress in cultured hepatocytes. These results suggest that hepatocytes constitute a major site of the oxidative insult triggered through Kupffer cell-independent mechanisms and serve as an important cellular component to be protected by antioxidant therapeutics.

  11. myo-Inositol is an osmolyte in rat liver macrophages (Kupffer cells) but not in RAW 264.7 mouse macrophages.

    PubMed Central

    Warskulat, U; Weik, C; Häussinger, D

    1997-01-01

    The role of myo-inositol as an osmolyte was studied in cultured rat liver macrophages (Kupffer cells). Hyperosmotic exposure of Kupffer cells stimulated myo-inositol uptake and led to an increase in the mRNA levels for the sodium/myo-inositol cotransporter (SMIT). Conversely, hypo-osmotic (205 m-osM) exposure diminished myo-inositol uptake when compared with normo-osmotic (305 m-osM) control incubations. The hyperosmolarity-induced SMIT mRNA increase was counteracted by added myo-inositol or betaine. In contrast with Kupffer cells, there was only a slight hyperosmotic stimulation of myo-inositol uptake in RAW 264.7 mouse macrophages, and the myo-inositol transporter (SMIT) mRNA was not detectable. Further, a slight stimulation of taurine uptake and an increase in taurine transporter (TAUT) mRNA level by hyperosmolarity was observed in RAW 264.7 cells, whereas hypo-osmolarity led to a decrease in taurine uptake and TAUT mRNA level. When Kupffer cells were preloaded with myo-inositol, hypo-osmotic exposure led to a rapid efflux of myo-inositol from the cells. Myo-inositol efflux was also stimulated by phagocytosis of latex particles; however, latex was without effect on the hyperosmolarity-induced increase of SMIT mRNA levels. The results suggest a role of myo-inositol as an osmolyte in rat Kupffer cells but not in RAW 264.7 mouse macrophages. The functional relevance of this osmolyte strategy might lie in the maintenance of cell volume homeostasis during phagocytosis in Kupffer cells; however, the interplay with the other osmolytes betaine and taurine remains to be established. PMID:9337881

  12. A standardized aqueous extract of Anoectochilus formosanus ameliorated thioacetamide-induced liver fibrosis in mice: the role of Kupffer cells.

    PubMed

    Wu, Jin-Bin; Chuang, Hin-Ru; Yang, Li-Chan; Lin, Wen-Chuan

    2010-01-01

    Anoectochilus formosanus is used in traditional folk medicine as an hepatoprotective agent. The purpose of this study was to investigate the effects of a standardized aqueous extract of A. formosanus (SAEAF) on thioacetamide (TAA)-induced liver fibrosis. An in vitro study showed that the inhibitive effect of kinsenoside, a major component of SAEAF, on tumor necrosis factor alpha (TNF-alpha) secretion from Kupffer cells might be derived at least partly from downregulation of LPS-receptor Toll-like receptor 4 (TLR4) signaling. Hepatic fibrosis was produced by TAA (200 mg/kg, i.p.) 3 times per week for 12 weeks. Mice in the three TAA groups were treated daily with distilled water and SAEAF (1.0, 0.2 g/kg) via gastrogavage throughout the experimental period. The mice that received the SAEAF treatment had significantly reduced plasma alanine aminotransferase activity, relative liver weights, and hepatic hydroxyproline contents. A histological examination also confirmed that SAEAF reduced the degree of fibrosis caused by TAA treatment. RT-PCR analysis showed that SAEAF treatment reduced mRNA expression of collagen (alpha1)(I), lipopolysaccharide-binding protein, CD14, TLR4, and TNF receptor 1. An immunohistochemical examination also indicated that SAEAF reduced the number of CD68-positive cells (macrophages). In conclusion, oral administration of SAEAF significantly reduced TAA-induced hepatic fibrosis in mice, probably through inhibition of hepatic Kupffer cell activation. PMID:20378990

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

    PubMed

    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

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

    PubMed

    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.

  15. 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

  16. Activated Kupffer cells play an important role in intra-hepatic Th1-associated necro-inflammation in Concanavalin A-induced hepatic injury in mice.

    PubMed

    Morita, Atsuhiro; Itoh, Yoshito; Toyama, Tetsuya; Fujii, Hideki; Nishioji, Kenichi; Kirishima, Toshihiko; Makiyama, Akiko; Yamauchi, Norihito; Okanoue, Takeshi

    2003-10-01

    BACKGROUND/AIMS: To examine whether or not activated Kupffer cells play an important role in intra-hepatic Th1-associated necro-inflammation in Concanavalin A (Con A)-induced hepatic injury in mice. METHODS: Con A was administered to Balb/c mice pretreated with or without gadolinium chloride (GdCl(3)). Kupffer cell activation was evaluated by their ability to produce superoxide anions in situ under liver perfusion with nitro blue tetrazolium (NBT). Hepatic concentration of cytokines was measured by ELISA and the mRNA expression of CXC chemokine receptor 3 (CXCR3) was evaluated by RT-PCR. Immunohistochemical detection of CD4 positive lymphocytes in the liver was also performed. RESULTS: GdCl(3)-pretreatment significantly (P<0.01) reduced the serum levels of alanine aminotransferase (ALT) in Con A-treated mice. Formazan deposition in Kupffer cells, the hepatic concentration of tumor necrosis factor-alpha and interferon-gamma, the mRNA expression of CXCR3 and the CD4 positive lymphocytes in the liver were decreased in GdCl(3)-pretreated mice as compared with those without GdCl(3)-pretreatment (P<0.05, respectively). CONCLUSIONS: Activated Kupffer cells, which produce superoxide anions, are involved in Con A-induced hepatic necro-inflammation in mice possibly through the activation of Th1-associated immune response mediated by CD4 and/or CXCR3 positive cells recruited into the liver.

  17. 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.

  18. 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

  19. Suppression of Kupffer cell function prevents cadmium induced hepatocellular necrosis in the male Sprague-Dawley rat.

    PubMed

    Sauer, J M; Waalkes, M P; Hooser, S B; Kuester, R K; McQueen, C A; Sipes, I G

    1997-08-15

    Exposure of humans to toxic metals and metalloids is a major environmental problem. Many metals, such as cadmium, can be hepatotoxic. However, the mechanisms by which metals cause acute hepatic injury are in many cases unknown. Previous reports suggest a major role for inflammation in acute cadmium induced hepatotoxicity. In initial experiments we found that a non-hepatotoxic dose of cadmium chloride (CdCl2; 2.0 mg/kg, i.v.) markedly increased the clearance rate of colloidal carbon from the blood, which is indicative of enhanced phagocytic activity by Kupffer cells (resident hepatic macrophages). Thus. the objective these studies was to determine the involvement of Kupffer cells in cadmium induced liver injury by inhibiting their function with gadolinium chloride (GdCl3). Male Sprague-Dawley rats were administered GdCl3 (10 mg/kg, i.v.) followed 24 h later by a single dose of CdCl2 (3.0 and 4.0 mg/kg, i.v.). Twenty four hours after CdCl2 administration animals were killed and the degree of liver toxicity was assessed using plasma alanine aminotransferase (ALT), as well as light microscopy. Cadmium chloride administration produced multifocal hepatocellular necrosis and increased plasma ALT activity. Pretreatment with GdCl3 significantly reduced both the morphological changes and hepatic ALT release caused by CdCl2. However, the protection was specific to the liver, and did not alter CdCl2 induced testicular injury, as determined by histopathological damage. In many cases, the inducible cadmium-binding protein, metallothionein (MT) is often an essential aspect of the acquisition of cadmium tolerance in the liver. Although cadmium caused a dramatic induction of hepatic MT (32-fold), GdCl3 caused only a minor increase (2-fold). Combined CdCl2 and GdCl3 treatment did not induce levels to an extent greater than CdCl2 alone. As expected, GdCl3 also caused a slight increase in the amount of cadmium associated with the liver. In cultured hepatocytes isolated from GdCl3

  20. 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.

  1. 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.

  2. 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

  3. Erythrophagocytosis by Liver Macrophages (Kupffer Cells) Promotes Oxidative Stress, Inflammation, and Fibrosis in a Rabbit Model of Steatohepatitis

    PubMed Central

    Otogawa, Kohji; Kinoshita, Kohji; Fujii, Hideki; Sakabe, Masahide; Shiga, Ryoko; Nakatani, Kazuki; Ikeda, Kazuo; Nakajima, Yuji; Ikura, Yoshihiro; Ueda, Makiko; Arakawa, Tetsuo; Hato, Fumihiko; Kawada, Norifumi

    2007-01-01

    Nonalcoholic steatohepatitis (NASH) is a progressive fibrotic disease, the pathogenesis of which has not been fully elucidated. Here, we report a molecular aspect of this disease elucidated using rabbits fed a cholesterol-rich high-fat diet and exhibiting insulin resistance. The liver in this model showed steatohepatitis with fibrosis and high mRNA expression for some cytokines, heme oxygenase-1, transforming growth factor-β1, and collagen α1(I). Erythrocytes isolated from the model showed marked fragility and the externalization of phosphatidylserine (PS) on the outer leaflet of the membrane and were frequently engulfed by Kupffer cells/macrophages in the hepatic sinusoids. Expression of milk fat globule-epidermal growth factor (EGF)-factor 8, a PS-binding protein, was augmented in the liver. In culture, RAW 264.7 cells engulfed erythrocytes oxidized by tert-butyl hydroperoxide, a process that was inhibited by anti-milk fat globule-EGF-factor 8 antibody. In addition, PS-positive erythrocytes appeared entrapped in the model liver in ex vivo perfusion experiments. Finally, in specimens from NASH patients, the aggregation of erythrocytes in inflammatory hepatic sinusoids was notable. These results indicate that the engulfment of PS-externalized, apoptotic signal-positive, erythrocytes by hepatic macrophages may lead to the deposition of iron derived from hemoglobin in the liver and be involved in the pathogenesis of steatohepatitis. PMID:17322381

  4. Characterization of a receptor for oxidized low-density lipoproteins on rat Kupffer cells: similarity to macrosialin.

    PubMed Central

    Van Velzen, A G; Da Silva, R P; Gordon, S; Van Berkel, T J

    1997-01-01

    Rat liver Kupffer cell membranes contain a protein that recognizes specifically oxidized low-density lipoproteins (oxLDL). Visualization after blotting under reducing conditions indicates that the receptor is a monomeric protein, with an estimated molecular mass of 115-120 kDa. N-Glycosidase F and endoglycosidase F treatment resulted in a fall in estimated molecular mass of 24 and 11 kDa respectively, whereas O-glycosidase was ineffective. No effect on the extent of interaction with oxLDL was noticed, suggesting that glycans are not essential for ligand recognition. Using a polyclonal antibody to mouse macrosialin, we visualized macrosialin on blot, and compared this glycoprotein with the oxLDL-binding protein. It appears that the two glycoproteins have a similar molecular mass and are comparably affected by treatment with the different glycosidases. Incubation with trypsin resulted in a reduction in the estimated molecular mass of about 25 kDa for both the oxLDL-binding protein and macrosialin. These results indicate that the oxLDL-binding protein and macrosialin are identical, suggesting a role for macrosialin in modified LDL catabolism. PMID:9065757

  5. 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

  6. 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

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

    PubMed

    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.

  8. 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.

  9. Large-pore mesoporous silica nanospheres as vehicles for delivering TRAF3-shRNA plasmids to Kupffer cells.

    PubMed

    Zhang, Junyong; Guo, Shipeng; Zhang, Wenfeng; Niu, Dechao; Gong, Jianping

    2016-01-01

    The currently available techniques for transferring exogenous genes into macrophages, especially the targeted import of exogenous genes into Kupffer cells (KCs) in vivo, are inefficient and achieve only low targeting. Novel Large-Pore Mesoporous Silica Nanospheres (LPMSNs) may be a promising gene transfection agent for KCs because of their superior biodegradation and hypotoxic characteristics, as well as their ability to retain the biological function of KCs and the high loading-rate of exogenous plasmid. LPMSNs were able to completely adsorb shRNA-TRAF3 (tumor necrosis factor receptor-associated factor-3) plasmid at a mass ratio as low as 30:1, and exhibited a low cytotoxicity for KCs. LPMSNs were detected in KC cytoplasm in vitro, and transmission electron microscopy (TEM) revealed that they were present only in KCs in liver tissue in vivo. The max KC transfection efficiency with LPMSNs was 34.8± 0.07%, as evaluated using flow cytometry, and the protein and mRNA levels of TRAF3 were significantly inhibited (P < 0.05) by shRNA-TRAF3 plasmid transfection after 24 h in vitro and 48 h in vivo. In conclusion, KC targeted transfection was achieved successfully by LPMSNs carrying shRNA-TRAF3 plasmids in vitro and vivo. The protein and mRNA levels of TRAF3 were suppressed significantly. These results suggest that LPMSNs are a promising vehicle for delivering exogenous genes into KCs in vitro and vivo. PMID:26631959

  10. Role of p38 mitogen-activated protein kinase in Kupffer cell secretion of the proinflammatory cytokines after burn trauma.

    PubMed

    Chen, Xu-Lin; Xia, Zhao-Fan; Wei, Duo; Han, Sheng; Ben, Dao-Feng; Wang, Guang-Qing

    2003-09-01

    This study was designed to investigate the role of p38 mitogen-activated protein (MAP) kinase on Kupffer cells (KCs) secretion of proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta and hepatic injury following burn trauma. Sprague-Dawley rats were randomized into four groups: (1) sham burn rats given vehicle, (2) sham burn rats given the p38 MAP kinase inhibitor SB203580 (10mg/kg i.v., 15min and 12h after sham burn), (3) rats given a 30% total body surface area (TBSA) full-thickness burn and fluid resuscitation plus vehicle, and (4) burn rats given injury and fluid resuscitation plus SB203580. Rats from each group were killed at 24h post-burn to examine plasma aspartate transaminase (AST) and alanine transaminase (ALT) and KCs were isolated. The KCs secretion of TNF-alpha and IL-1beta and p38 MAP kinase activity (by Western blot analysis) were also examined. These studies showed by more significant activation of p38 MAP kinase in KCs harvested from burn rats than from shams. Burn trauma resulted in hepatic dysfunction and promoted KCs secretion of TNF-alpha and IL-1beta. SB203580 inhibited p38 MAP kinase activity, reduced KCs secretion of proinflammatory cytokines, and alleviated burn-mediated hepatic dysfunction. These data suggest p38 MAP kinase activation is one important aspect of the signaling event that may mediate the KCs secretion of proinflammatory cytokines TNF-alpha and IL-1beta following burn trauma.

  11. Selective role for tumor necrosis factor-α, but not interleukin-1 or Kupffer cells, in down-regulation of CYP3A11 and CYP3A25 in livers of mice infected with a noninvasive intestinal pathogen.

    PubMed

    Kinloch, Ryan D; Lee, Choon-Myung; van Rooijen, Nico; Morgan, Edward T

    2011-08-01

    Hepatic cytochrome P450 (P450) gene and protein expression are modulated during inflammation and infection. Oral infection of C57BL/6 mice with Citrobacter rodentium produces mild clinical symptoms while selectively regulating hepatic P450 expression and elevating levels of proinflammatory cytokines. Here, we explored the role of cytokines in the regulation of hepatic P450 expression by orally infecting tumor necrosis factor-α (TNFα) receptor 1 null mice (TNFR1-/-), interleukin-1 (IL1) receptor null mice (IL1R1-/-), and Kupffer cell depleted mice with C. rodentium. CYP4A mRNA and protein levels and flavin monooxygenase (FMO)3 mRNA expression levels were down-regulated, while CYP2D9 and CYP4F18 mRNAs remained elevated during infection in wild-type, receptor knockout, and Kupffer cell depleted mice. CYPs 3A11 and 3A25 mRNA levels were down-regulated during infection in wild-type mice but not in TNFR1-/- mice. Consistent with this observation, CYPs 3A11 and 3A25 were potently down-regulated in mouse hepatocytes treated with TNFα. Oral infection of IL1R1-/- mice and studies with mouse hepatocytes indicated that IL1 does not directly regulate CYP3A11 or CYP3A25 expression. Uninfected mice injected with clodronate liposomes had a significantly reduced number of Kupffer cells in their livers. Infection increased the Kupffer cell count, which was attenuated by clodronate treatment. The P450 mRNA and cytokine levels in infected Kupffer cell depleted mice were comparable to those in infected mice receiving no clodronate. The results indicate that TNFα is involved in the regulation of CYPs 3A11 and 3A25, but IL1β and Kupffer cells may not be relevant to hepatic P450 regulation in oral C. rodentium infection.

  12. Selective Role for Tumor Necrosis Factor-α, but Not Interleukin-1 or Kupffer Cells, in Down-Regulation of CYP3A11 and CYP3A25 in Livers of Mice Infected with a Noninvasive Intestinal Pathogen

    PubMed Central

    Kinloch, Ryan D.; Lee, Choon-Myung; van Rooijen, Nico; Morgan, Edward T.

    2011-01-01

    Hepatic cytochrome P450 (P450) gene and protein expression are modulated during inflammation and infection. Oral infection of C57BL/6 mice with Citrobacter rodentium produces mild clinical symptoms while selectively regulating hepatic P450 expression and elevating levels of proinflammatory cytokines. Here, we explored the role of cytokines in the regulation of hepatic P450 expression by orally infecting tumor necrosis factor-α (TNFα) receptor 1 null mice (TNFR1−/−), interleukin-1 (IL1) receptor null mice (IL1R−/−), and Kupffer cell depleted mice with C. rodentium. CYP4A mRNA and protein levels and flavin monooxygenase (FMO)3 mRNA expression levels were down-regulated, while CYP2D9 and CYP4F18 mRNAs remained elevated during infection in wild-type, receptor knockout, and Kupffer cell depleted mice. CYPs 3A11 and 3A25 mRNA levels were down-regulated during infection in wild-type mice but not in TNFR1−/− mice. Consistent with this observation, CYPs 3A11 and 3A25 were potently down-regulated in mouse hepatocytes treated with TNFα. Oral infection of IL1R−/− mice and studies with mouse hepatocytes indicated that IL1 does not directly regulate CYP3A11 or CYP3A25 expression. Uninfected mice injected with clodronate liposomes had a significantly reduced number of Kupffer cells in their livers. Infection increased the Kupffer cell count, which was attenuated by clodronate treatment. The P450 mRNA and cytokine levels in infected Kupffer cell depleted mice were comparable to those in infected mice receiving no clodronate. The results indicate that TNFα is involved in the regulation of CYPs 3A11 and 3A25, but IL1β and Kupffer cells may not be relevant to hepatic P450 regulation in oral C. rodentium infection. PMID:21570957

  13. Altered Endothelin-1 Signaling in Production of Thromboxane A2 in Kupffer Cells from Bile Duct Ligated Rats

    PubMed Central

    Miller, Andrew M; Zhang, Jian X

    2009-01-01

    Kupffer cells (KCs), the liver resident macrophages accounting for 80–90% of the total population of fixed tissue macrophages in the body, not only play a key role in host defense via removing particulate materials from the portal circulation, but may also contribute to the pathogenesis of various liver diseases. We have previously demonstrated that KCs play an important role in controlling portal hypertension and hepatocellular injury via releasing thromboxane A2 (TXA2) in early fibrosis induced by one-week bile duct ligation (BDL). Production of TXA2 is controlled by cytosolic phospholipase A2 (cPLA2) that is activated by the interaction of entothelin-1 (ET-1) with its G-protein coupled ET receptor B (ETBR). However, the signaling pathways that contribute to the ET-1-induced activation of cPLA2 and production of TXA2 in KCs in the normal healthy or injured livers are not yet clear, which are investigated in the present study using isolated KCs from one-week BDL or sham rats. The pharmacological inhibition of cPLA2 or chelation of intracellular calcium abrogated the ET-1 induction of TXA2 from KCs. Compared to those from sham rats, KCs from BDL animals displayed a significantly enhanced responsiveness of p38 MAPK to ET-1, increased ETBR and Gαi subunit but decreased Gαq and Gα11 expression. Inhibition of ERK1/2 or Gq signaling abrogated significantly the ET-1 induction of TXA2 in sham KCs but only slightly in BDL KCs. In contrast, inhibition of p38 MAPK and Gi signaling markedly attenuated the ET-1 induction of TXA2 in BDL KCs but had no effect in sham KCs. Lastly, inhibition of PLC or PKC abrogated ET-1 induction of TXA2 in KCs from both sham and BDL groups. The hepatic stress (such as BDL) induces significant modifications in the receptor and intermediates of ET-1 signaling in KC and subsequently alters ET-1 signaling mechanisms, particularly a shift from Gq induced signaling to Gi induced signaling, in the activation of cPLA2 and production of TXA2 in

  14. Polaris and Polycystin-2 in dorsal forerunner cells and Kupffer's vesicle are required for specification of the zebrafish left-right axis.

    PubMed

    Bisgrove, Brent W; Snarr, Brian S; Emrazian, Anoush; Yost, H Joseph

    2005-11-15

    Recently, it has become clear that motile cilia play a central role in initiating a left-sided signaling cascade important in establishing the LR axis during mouse and zebrafish embryogenesis. Two genes proposed to be important in this cilia-mediated signaling cascade are polaris and polycystin-2 (pkd2). Polaris is involved in ciliary assembly, while Pkd2 is proposed to function as a Ca(2+)-permeable cation channel. We have cloned zebrafish homologues of polaris and pkd2. Both genes are expressed in dorsal forerunner cells (DFCs) from gastrulation to early somite stages when these cells form a ciliated Kupffer's vesicle (KV). Morpholino-mediated knockdown of Polaris or Pkd2 in zebrafish results in misexpression of left-side-specific genes, including southpaw, lefty1 and lefty2, and randomization of heart and gut looping. By targeting morpholinos to DFCs/KV, we show that polaris and pkd2 are required in DFCs/KV for normal LR development. Polaris morphants have defects in KV cilia, suggesting that the laterality phenotype is due to problems in cilia function per se. We further show that expression of polaris and pkd2 is dependent on the T-box transcription factors no tail and spadetail, respectively, suggesting that these genes have a previously unrecognized role in regulating ciliary structure and function. Our data suggest that the functions of polaris and pkd2 in LR patterning are conserved between zebrafish and mice and that Kupffer's vesicle functions as a ciliated organ of asymmetry. PMID:16216239

  15. Salmonella choleraesuis and Salmonella typhimurium associated with liver cells after intravenous inoculation of rats are localized mainly in Kupffer cells and multiply intracellularly.

    PubMed

    Nnalue, N A; Shnyra, A; Hultenby, K; Lindberg, A A

    1992-07-01

    Male Sprague-Dawley rats were inoculated intravenously with Salmonella choleraesuis or Salmonella typhimurium and used over 3 consecutive days to produce highly enriched (greater than 95% homogenous) preparations of Kupffer and mononuclear cells (KC), liver endothelial cells (LEC), and hepatocytes. The methods involved collagenase perfusion of the liver in situ, differential centrifugation of liver cells over a Percoll gradient, and selective attachment of the cells to plastic or to culture dishes coated with collagen. The different cell preparations were then assayed for the number and location, intracellular or extracellular, of associated viable bacteria. Most of the viable bacteria recovered were associated with KC and were mainly intracellular. The intracellular bacteria in KC from rats infected with either bacterial strain increased about 20- to 50-fold over 2 days. Some of the bacteria associated with LEC and in some experiments with hepatocytes also survived treatment with gentamicin and increased in number with time. Intracellular bacteria were readily visualized in KC by light microscopy and transmission electron microscopy. On rare occasions, bacteria were seen within LEC from rats infected with S. choleraesuis but not from those infected with S. typhimurium. Microcolonies of S. typhimurium but not of S. choleraesuis were occasionally found on the surface of some LEC. Bacteria were not seen within or on the surface of hepatocytes by transmission or scanning electron microscopy. The integration of microscopic and viability data suggested that most intracellular S. choleraesuis organisms in KC had been killed whereas most intracellular S. typhimurium organisms were viable.

  16. Kupffer cell stimulation with Corynebacterium parvum reduces some cytochrome P450-dependent activities and diminishes acetaminophen and carbon tetrachloride-induced liver injury in the rat.

    PubMed

    Raiford, D S; Thigpen, M C

    1994-11-01

    Chemical activation of Kupffer cells in vivo by vitamin A or latex beads is associated with a worsening of hepatic injury induced by the P450-dependent hepatotoxins acetaminophen (ACET) and carbon tetrachloride (CCl4) and by the P450-independent toxin galactosamine (GLN). Immunostimulants such as Corynebacterium parvum (CP) also activate Kupffer cells, but do so while prompting release of soluble mediators which depress microsomal oxidative activities in cultured hepatocytes. Therefore, we sought to characterize the effects of CP on hepatic injury in vivo due to ACET and CCl4 while employing GLN as a control. Hepatic microsomal oxidative activity and glutathione (GSH) disposition were examined since each influences susceptibility to injury from ACET or CCl4. Rats were given CP 28 mg/kg i.v. 5 days before challenge with hepatotoxicant. Hepatic injury was assessed 24 hr after hepatotoxicant administration by measurement of serum alanine aminotransferase (ALT) activity and review of histological sections. Livers from parallel groups of rats were used to prepare microsomal and cytosolic fractions, to measure tissue GSH, or for perfusion to assess GSH efflux. Significant reductions in injury due to ACET or CCl4 were observed while injury due to GLN was potentiated. Serum ALT levels after ACET were 3000 +/- 620 in controls vs 170 +/- 45 IU/liter in the CP-treated group and ALT levels after CCl4 were 3100 +/- 500 in controls vs 1700 + 450 IU/liter in the CP-treated group. In contrast, serum ALT levels after GLN were 920 +/- 230 in controls vs 1700 +/- 370 in the CP-treated group. Patterns of hepatic injury observed on histological sections were those characteristic for each toxin and the severity of injury correlated well with alterations in serum ALT levels for each agent. Hepatic microsomal fractions from rats pretreated with CP showed significantly diminished total cytochrome P450 content as well as reduced activity for two P450IIE1 substrates, p-nitrophenol and 7

  17. Intrahepatic endothelial and Kupffer cells involved in immunosuppressive cytokines and natural killer (NK)/NK T cell disorders in viral acute hepatitis

    PubMed Central

    Jacques, A; Bleau, C; Martin, J-P; Lamontagne, L

    2008-01-01

    During acute viral hepatitis, the intrahepatic tolerance sustained by immunosuppressive cytokines such as interleukin (IL)-4, IL-10, transforming growth factor (TGF)-β and prostaglandin E2 (PGE2), produced by Kupffer cells (KC), liver sinusoidal endothelial cells (LSEC), natural killer (NK) T cells and natural regulatory T cells may be disturbed. NK cells are recruited normally in the liver and produce interferon (IFN)-γ to control viral replication. The use of mouse hepatitis virus type 3 (MHV3) attenuated variants showing selected tropisms for KC or LSEC have allowed determining their roles in the disturbances of immune tolerance during viral hepatitis. Groups of C57BL/6 mice were infected with the pathogenic L2-MHV3 (KC+, LSEC+), low attenuated 51·6-MHV3 (KC+, LSEC−) or high attenuated CL12-MHV3 (KC−, LSEC−) variants for the first 3 days. Results showed that IL-10, TGF-β and PGE2 production in the liver decreased in L2-MHV3-infected mice and increased in 51·6-MHV3- and CL12-MHV3-infected mice. The ratio of IFN-γ/IL-4 in liver decreased in L2-MHV3-infected mice, while it was not (or low) altered in mice infected with the attenuated MHV3 variant mice. Phenotypic analysis of intrahepatic mononuclear cells revealed that apoptotic NK and NK T cells increased in mice infected with the L2-MHV3, but were minor in 51·6-MHV3- and CL12-MHV3-infected mice. The numbers of CD4+ forkhead box P3+ cells increased in the livers from low pathogenic CL12-MHV3 and YAC-MHV3-infected mice. These results indicate that viral permissivity of KC and LSEC is involved in the decrease of IL-10 and PGE2, while KC may play an additional role in the apoptosis of NK and NK T cells during acute viral hepatitis. PMID:18336588

  18. 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

  19. Kupffer cells potentiate liver sinusoidal endothelial cell injury in sepsis by ligating programmed cell death ligand-1

    PubMed Central

    Hutchins, Noelle A.; Wang, Fei; Wang, Yvonne; Chung, Chun-Shiang; Ayala, Alfred

    2013-01-01

    PD-1 and PD-L1 have been reported to provide peripheral tolerance by inhibiting TCR-mediated activation. We have reported that PD-L1−/− animals are protected from sepsis-induced mortality and immune suppression. Whereas studies indicate that LSECs normally express PD-L1, which is also thought to maintain local immune liver tolerance by ligating the receptor PD-1 on T lymphocytes, the role of PD-L1 in the septic liver remains unknown. Thus, we hypothesized initially that PD-L1 expression on LSECs protects them from sepsis-induced injury. We noted that the increased vascular permeability and pSTAT3 protein expression in whole liver from septic animals were attenuated in the absence of PD-L1. Isolated LSECs taken from septic animals, which exhibited increased cell death, declining cell numbers, reduced cellular proliferation, and VEGFR2 expression (an angiogenesis marker), also showed improved cell numbers, proliferation, and percent VEGFR2+ levels in the absence of PD-L1. We also observed that sepsis induced an increase of liver F4/80+PD-1+-expressing KCs and increased PD-L1 expression on LSECs. Interestingly, PD-L1 expression levels on LSECs decreased when PD-1+-expressing KCs were depleted with clodronate liposomes. Contrary to our original hypothesis, we document here that increased interactions between PD-1+ KCs and PD-L1+ LSECs appear to lead to the decline of normal endothelial function—essential to sustain vascular integrity and prevent ALF. Importantly, we uncover an underappreciated pathological aspect of PD-1:PD-L1 ligation during inflammation that is independent of its normal, immune-suppressive activity. PMID:23766529

  20. Mesenchymal stromal cell-dependent reprogramming of Kupffer cells is mediated by TNF-α and PGE2 and is crucial for liver transplant tolerance.

    PubMed

    You, Yu; Zhang, Jiqin; Gong, Jianping; Chen, Yupei; Li, Yue; Yang, Kang; Liu, Zuojin

    2015-07-01

    The role of mesenchymal stromal cells (MSCs) in the modulation of liver transplant tolerance has attracted significant interest. However, the interaction between MSCs and Kupffer cells (KCs) has received little attention, and the effect of this interaction on liver transplant tolerance remains unclear. KCs were cultured in the presence and absence of MSCs. After 24 h, cells were treated with lipopolysaccharide (LPS), after which the production of cytokines and the expression of surface antigens were measured for cell function identification. Moreover, the effects of the KCs and the prostaglandin E2 (PGE2) levels produced by the MSCs were determined using an experimental rat liver transplantation model. Blood and liver samples were collected at three time points after transplantation for further analysis. After LPS treatment, when compared with the KC single cultures, the expression of pro-inflammatory cytokines (IL-1β, IL-6, MHC-II, CD40, CD80, and CD86) in the coculture system was down-regulated, whereas the expression of anti-inflammatory cytokines (TGF-β, IL-4, PGE2, and IL-10) was markedly increased. These data indicate that MSCs can reprogram the phenotype of KCs. However, KCs treated with miR/TNF-α (tumor necrosis factor) plasmid prior to coculture to inhibit the production of TNF-α resulted in an inhibition of the reprogramming effect of MSCs. Moreover, overexpression of PGE2 in MSCs increased the effect of MSCs on KC reprogramming. After rat liver transplantation, allograft recipients that received MSCs showed better allograft tolerance when compared with rats in which KC function was inhibited. Furthermore, rats treated with MSCs overexpressing PGE2 demonstrated the best liver tolerance of all of the groups tested. MSCs reprogram the phenotype of KCs through TNF-α and PGE2, and this process is crucial for the immunomodulatory function of MSCs in liver transplantation. PMID:25982496

  1. Mesenchymal stromal cell-dependent reprogramming of Kupffer cells is mediated by TNF-α and PGE2 and is crucial for liver transplant tolerance.

    PubMed

    You, Yu; Zhang, Jiqin; Gong, Jianping; Chen, Yupei; Li, Yue; Yang, Kang; Liu, Zuojin

    2015-07-01

    The role of mesenchymal stromal cells (MSCs) in the modulation of liver transplant tolerance has attracted significant interest. However, the interaction between MSCs and Kupffer cells (KCs) has received little attention, and the effect of this interaction on liver transplant tolerance remains unclear. KCs were cultured in the presence and absence of MSCs. After 24 h, cells were treated with lipopolysaccharide (LPS), after which the production of cytokines and the expression of surface antigens were measured for cell function identification. Moreover, the effects of the KCs and the prostaglandin E2 (PGE2) levels produced by the MSCs were determined using an experimental rat liver transplantation model. Blood and liver samples were collected at three time points after transplantation for further analysis. After LPS treatment, when compared with the KC single cultures, the expression of pro-inflammatory cytokines (IL-1β, IL-6, MHC-II, CD40, CD80, and CD86) in the coculture system was down-regulated, whereas the expression of anti-inflammatory cytokines (TGF-β, IL-4, PGE2, and IL-10) was markedly increased. These data indicate that MSCs can reprogram the phenotype of KCs. However, KCs treated with miR/TNF-α (tumor necrosis factor) plasmid prior to coculture to inhibit the production of TNF-α resulted in an inhibition of the reprogramming effect of MSCs. Moreover, overexpression of PGE2 in MSCs increased the effect of MSCs on KC reprogramming. After rat liver transplantation, allograft recipients that received MSCs showed better allograft tolerance when compared with rats in which KC function was inhibited. Furthermore, rats treated with MSCs overexpressing PGE2 demonstrated the best liver tolerance of all of the groups tested. MSCs reprogram the phenotype of KCs through TNF-α and PGE2, and this process is crucial for the immunomodulatory function of MSCs in liver transplantation.

  2. 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

  3. 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

  4. The cellular and proteomic response of primary and immortalized murine Kupffer cells following immune stimulation diverges from that of monocyte-derived macrophages.

    PubMed

    Tweedell, Rebecca; Tao, Dingyin; Dinglasan, Rhoel R

    2015-01-01

    Kupffer cells (KCs) are the first line of defense in the liver against pathogens, yet several microbes successfully target the liver, bypass immune surveillance, and effectively develop in this tissue. Our current, albeit poor, understanding of KC-pathogen interactions has been largely achieved through the study of primary cells, requiring isolation from large numbers of animals. To facilitate the study of KC biology, an immortalized rat KC line 1, RKC1, was developed. We performed a comparative global proteomic analysis of RKC1 and primary rat KCs (PRKC) to characterize their respective responses to lipopolysaccharide-mediated immune stimulation. We identified patent differences in the proteomic response profile of RKC1 and PRKC to lipopolysaccharide. We observed that PRKC upregulated more immune function pathways and exhibited marked changes in cellular morphology following stimulation. We consequently analyzed the cytoskeletal signaling pathways of these cells in light of the fact that macrophages are known to induce cytoskeletal changes in response to pathogens. Our findings suggest that KCs respond differently to inflammatory stimulus than do monocyte-derived macrophages, and such data may provide insight into how pathogens, such as the malaria parasite, may have evolved mechanisms of liver entry through KCs without detection.

  5. 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.

  6. 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.

  7. Chronic ethanol feeding modulates inflammatory mediators, activation of nuclear factor-κB, and responsiveness to endotoxin in murine Kupffer cells and circulating leukocytes.

    PubMed

    Maraslioglu, Miriam; Oppermann, Elsie; Blattner, Carolin; Weber, Roxane; Henrich, Dirk; Jobin, Christian; Schleucher, Elke; Marzi, Ingo; Lehnert, Mark

    2014-01-01

    Chronic ethanol abuse is known to increase susceptibility to infections after injury, in part, by modification of macrophage function. Several intracellular signalling mechanisms are involved in the initiation of inflammatory responses, including the nuclear factor-κB (NF-κB) pathway. In this study, we investigated the systemic and hepatic effect of chronic ethanol feeding on in vivo activation of NF-κB in NF-κB(EGFP) reporter gene mice. Specifically, the study focused on Kupffer cell proinflammatory cytokines IL-6 and TNF-α and activation of NF-κB after chronic ethanol feeding followed by in vitro stimulation with lipopolysaccharide (LPS). We found that chronic ethanol upregulated NF-κB activation and increased hepatic and systemic proinflammatory cytokine levels. Similarly, LPS-stimulated IL-1 β release from whole blood was significantly enhanced in ethanol-fed mice. However, LPS significantly increased IL-6 and TNF-α levels. These results demonstrate that chronic ethanol feeding can improve the responsiveness of macrophage LPS-stimulated IL-6 and TNF-α production and indicate that this effect may result from ethanol-induced alterations in intracellular signalling through NF-κB. Furthermore, LPS and TNF-α stimulated the gene expression of different inflammatory mediators, in part, in a NF-κB-dependent manner. PMID:24623963

  8. Chronic ethanol feeding modulates inflammatory mediators, activation of nuclear factor-κB, and responsiveness to endotoxin in murine Kupffer cells and circulating leukocytes.

    PubMed

    Maraslioglu, Miriam; Oppermann, Elsie; Blattner, Carolin; Weber, Roxane; Henrich, Dirk; Jobin, Christian; Schleucher, Elke; Marzi, Ingo; Lehnert, Mark

    2014-01-01

    Chronic ethanol abuse is known to increase susceptibility to infections after injury, in part, by modification of macrophage function. Several intracellular signalling mechanisms are involved in the initiation of inflammatory responses, including the nuclear factor-κB (NF-κB) pathway. In this study, we investigated the systemic and hepatic effect of chronic ethanol feeding on in vivo activation of NF-κB in NF-κB(EGFP) reporter gene mice. Specifically, the study focused on Kupffer cell proinflammatory cytokines IL-6 and TNF-α and activation of NF-κB after chronic ethanol feeding followed by in vitro stimulation with lipopolysaccharide (LPS). We found that chronic ethanol upregulated NF-κB activation and increased hepatic and systemic proinflammatory cytokine levels. Similarly, LPS-stimulated IL-1 β release from whole blood was significantly enhanced in ethanol-fed mice. However, LPS significantly increased IL-6 and TNF-α levels. These results demonstrate that chronic ethanol feeding can improve the responsiveness of macrophage LPS-stimulated IL-6 and TNF-α production and indicate that this effect may result from ethanol-induced alterations in intracellular signalling through NF-κB. Furthermore, LPS and TNF-α stimulated the gene expression of different inflammatory mediators, in part, in a NF-κB-dependent manner.

  9. 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

  10. Down-Regulated Receptor Interacting Protein 140 Is Involved in Lipopolysaccharide-Preconditioning-Induced Inactivation of Kupffer Cells and Attenuation of Hepatic Ischemia Reperfusion Injury

    PubMed Central

    Ji, Li; Jie, Xu; Yue, Li; Kang, Yang; Jianping, Gong; Zuojin, Liu

    2016-01-01

    Background Lipopolysaccharide (LPS) preconditioning is known to attenuate hepatic ischemia/reperfusion injury (I/RI); however, the precise mechanism remains unclear. This study investigated the role of receptor-interacting protein 140 (RIP140) on the protective effect of LPS preconditioning in hepatic I/RI involving Kupffer cells (KCs). Methods Sprague—Dawley rats underwent 70% hepatic ischemia for 90 minutes. LPS (100 μg/kg) was injected intraperitoneally 24 hours before ischemia. Hepatic injury was observed using serum and liver samples. The LPS/NF-κB (nuclear factor-κB) pathway and hepatic RIP140 expression in isolated KCs were investigated. Results LPS preconditioning significantly inhibited hepatic RIP140 expression, NF-κB activation, and serum proinflammatory cytokine expression after I/RI, with an observation of remarkably reduced serum enzyme levels and histopathologic scores. Our experiments showed that protection effects could be effectively induced in KCs by LPS preconditioning, but couldn’t when RIP140 was overexpressed in KCs. Conversely, even without LPS preconditioning, protective effects were found in KCs if RIP140 expression was suppressed with siRNA. Conclusions Down-regulated RIP140 is involved in LPS-induced inactivation of KCs and hepatic I/RI attenuation. PMID:27723769

  11. Selective depletion or blockade of Kupffer cells leads to enhanced and prolonged hepatic transgene expression using high-capacity adenoviral vectors.

    PubMed

    Schiedner, Gudrun; Hertel, Sabine; Johnston, Marion; Dries, Volker; van Rooijen, Nico; Kochanek, Stefan

    2003-01-01

    Tissue macrophages, in particular hepatic Kupffer cells (KCs), contribute to early inflammatory responses following adenoviral vector administration. This study evaluates the effect of selective and transient (3 days) depletion of KCs by a single injection of clodronate liposomes on the in vivo performance of high-capacity adenoviral (HC-Ad) vectors. In KC-depleted C57BL/6 and C3H mice increased and stabilized hAAT levels were observed following intravenous injection of HC-Ad vectors expressing human alpha-1 anti-trypsin (hAAT) either from the hAAT promoter or from the human cytomegalovirus promoter. Comparable increases in hAAT levels were obtained in mice preinjected with a transcriptionally silent HC-Ad vector. Interestingly, in the majority of animals of both strains depletion of KCs was sufficient to prevent the generation of anti-hAAT antibodies, resulting in prolonged transgene expression. Thus, short-term and selective depletion of hepatic macrophages at the same time significantly increased hepatic transgene expression and reduced the humoral immune response to the transgenic protein.

  12. Gut Bacteria Drive Kupffer Cell Expansion via MAMP-Mediated ICAM-1 Induction on Sinusoidal Endothelium and Influence Preservation-Reperfusion Injury after Orthotopic Liver Transplantation

    PubMed Central

    Corbitt, Natasha; Kimura, Shoko; Isse, Kumiko; Specht, Susan; Chedwick, Lisa; Rosborough, Brian R.; Lunz, John G.; Murase, Noriko; Yokota, Shinichiro; Demetris, Anthony J.

    2014-01-01

    Bacteria in the gut microbiome shed microbial-associated molecule patterns (MAMPs) into the portal venous circulation, where they augment various aspects of systemic immunity via low-level stimulation. Because the liver is immediately downstream of the intestines, we proposed that gut-derived MAMPs shape liver immunity and affect Kupffer cell (KC) phenotype. Germ-free (GF), antibiotic-treated (AVMN), and conventional (CL) mice were used to study KC development, function, and response to the significant stress of cold storage, reperfusion, and orthotopic transplantation. We found that a cocktail of physiologically active MAMPs translocate into the portal circulation, with flagellin (Toll-like receptor 5 ligand) being the most plentiful and capable of promoting hepatic monocyte influx in GF mice. In MAMP-deficient GF or AVMN livers, KCs are lower in numbers, have higher phagocytic activity, and have lower major histocompatibility complex II expression. MAMP-containing CL livers harbor significantly increased KC numbers via induction of intercellular adhesion molecule 1 on liver sinusoidal endothelium. These CL KCs have a primed yet expected phenotype, with increased major histocompatibility complex class II and lower phagocytic activity that increases susceptibility to liver preservation/reperfusion injury after orthotopic transplantation. The KC number, functional activity, and maturational status are directly related to the concentration of gut-derived MAMPs and can be significantly reduced by broad-spectrum antibiotics, thereby affecting susceptibility to injury. PMID:23159949

  13. Chronic Ethanol Feeding Modulates Inflammatory Mediators, Activation of Nuclear Factor-κB, and Responsiveness to Endotoxin in Murine Kupffer Cells and Circulating Leukocytes

    PubMed Central

    Oppermann, Elsie; Jobin, Christian; Schleucher, Elke; Marzi, Ingo

    2014-01-01

    Chronic ethanol abuse is known to increase susceptibility to infections after injury, in part, by modification of macrophage function. Several intracellular signalling mechanisms are involved in the initiation of inflammatory responses, including the nuclear factor-κB (NF-κB) pathway. In this study, we investigated the systemic and hepatic effect of chronic ethanol feeding on in vivo activation of NF-κB in NF-κBEGFP reporter gene mice. Specifically, the study focused on Kupffer cell proinflammatory cytokines IL-6 and TNF-α and activation of NF-κB after chronic ethanol feeding followed by in vitro stimulation with lipopolysaccharide (LPS). We found that chronic ethanol upregulated NF-κB activation and increased hepatic and systemic proinflammatory cytokine levels. Similarly, LPS-stimulated IL-1β release from whole blood was significantly enhanced in ethanol-fed mice. However, LPS significantly increased IL-6 and TNF-α levels. These results demonstrate that chronic ethanol feeding can improve the responsiveness of macrophage LPS-stimulated IL-6 and TNF-α production and indicate that this effect may result from ethanol-induced alterations in intracellular signalling through NF-κB. Furthermore, LPS and TNF-α stimulated the gene expression of different inflammatory mediators, in part, in a NF-κB-dependent manner. PMID:24623963

  14. 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.

  15. Alteration of Kupffer cell function and morphology by low melt point paraffin wax in female Fischer-344 but not Sprague-Dawley rats.

    PubMed

    Hoglen, N C; Regan, S P; Hensel, J L; Younis, H S; Sauer, J M; Steup, D R; Miller, M J; Waterman, S J; Twerdok, L E; Sipes, I G

    1998-11-01

    This study was conducted to compare the effects of 60-day dietary exposure (2%) to low melt point paraffin wax (LMPW) on both general liver morphology and Kupffer cell (KC) function and morphology in female F-344 and Sprague-Dawley (SD) rats. Livers from only F-344 rats fed LMPW had granuloma formation/lymphoid cell aggregates with small areas of necrosis. Significant increases in serum alanine and aspartate aminotransferase as well as gamma-glutamyltransferase activities were detected only in treated F-344 rats. Additionally, detectable amounts of LMPW were present only in livers of treated F-344 rats. Because KC can be involved in granuloma formation, their morphology and function were examined. Electron microscopy revealed the presence of large, irregularly shaped, membrane-associated vacuoles in cells isolated from F-344 rats exposed to LMPW. These vacuoles were not seen in KC from control rats and rarely detected in KC isolated from LMPW-exposed SD rats. Moreover, indices of KC function including phagocytic activity and nitric oxide and superoxide anion production were significantly increased by KC isolated from F-344 rats exposed to LMPW (1.6-, 36-, and 2.2-fold increases, respectively) over untreated controls. In contrast, LPS-stimulated production of TNF and LTB4 was significantly decreased only in KC of LMPW-fed F-344 rats. No significant changes in these functions were observed in KC isolated from SD rats exposed to LMPW or from KC isolated from control F-344 or SD rats. These data provide evidence that dietary LMPW alters the morphology and functional capacity of KC of F-344 but not SD rats and these changes may ultimately lead to granuloma formation.

  16. Chinese medicinal formula, Qinggan Huoxue Recipe protects rats from alcoholic liver disease via the lipopolysaccharide-Kupffer cell signal conduction pathway.

    PubMed

    Wu, Tao; Liu, Tao; Zhang, Li; Xing, Lian-Jun; Zheng, Pei-Yong; Ji, Guang

    2014-08-01

    The Chinese medicinal formula, Qinggan (QG) Huoxue (HX) Recipe (R) exerts a range of pharmacological effects, including reversible steatosis, decreased levels of inflammatory cytokines and lipid peroxidation resistance. The aim of the present study was to determine the specific mechanisms of QGHXR hepatoprotection through the lipopolysaccharide-Kupffer cell (LPS-KC) signal conduction pathway in rats with alcoholic liver disease (ALD). ALD rats were exposed to the compound factors, QGR and HXR. Hematoxylin and eosin staining was conducted to evaluate the pathological changes in the liver following QGHXR treatment and an enzyme-linked immunosorbent assay was performed to measure the content of tumor necrosis factor (TNF)-α in the plasma. Immunohistochemical staining was conducted to examine the expression of cell differentiation antigen (CD) 68 and 14. In addition, western blot analysis and reverse transcription-polymerase chain reaction were used to measure the expression of Toll-like receptor 4 (TLR4), phosphorylated-extracellular regulated protein kinases (p-ERK), nuclear factor (NF)-κB, CD14 and TNF-α. Following stimulation with the compound factors, the rats exhibited increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, as well as marked pathological changes. Furthermore, the related molecules in the LPS-KC pathway were upregulated and QGHXR was identified to be effective in the LPS-KC signal conduction pathway in the ALD rats. QGHXR was superior to QGR and HXR in reducing the serum ALT and AST levels, regulating CD14, TLR4, NF-κB, ERK and TNF-α as well as improving the pathological changes. The results indicated that QGHXR therapy may provide a novel strategy for treating ALD via regulation of the related molecules in the LPS-KC signaling pathway. PMID:25009584

  17. The role of intracellular high-mobility group box 1 in the early activation of Kupffer cells and the development of Con A-induced acute liver failure.

    PubMed

    Yang, Qiao; Liu, Yanning; Shi, Yu; Zheng, Min; He, Jiliang; Chen, Zhi

    2013-10-01

    Acute liver failure (ALF) is a highly complex syndrome characterized by devastating activation of early activation of Kupffer cells (KCs) has been implicated in the pathogenesis of ALF. However, the factors regulating KC early activation are virtually unexplored. The aim of present study was to determine the role of the intracellular high-mobility group box 1 (HMGB1) in modulating the early activation of KCs during ALF. The intravenous injection of Concanavalin A (Con A) was used to establish a mouse model of ALF. The dynamic pro-inflammatory properties and MHC II expression of KCs were measured by qRT-PCR and flow cytometry. HMGB1 expression in KCs was measured by qRT-PCR and Western blotting. The immunofluorescence was implemented to determine the relocation of HMGB1 in KCs, and the siRNA against HMGB1 was utilized to assess the impact of HMGB1 on KC pro-inflammatory properties. The peak of pro-inflammatory cytokines production and MHC II expression in KCs appeared at the early stage of ALF. The up-regulation of HMGB1 expression and the translocation of HMGB1 in KCs were in parallel with the early activation of KCs. The blockade of intracellular HMGB1 expression caused by siRNA significantly inhibited the production of KC-derived pro-inflammatory cytokines, and led to a down-regulation of MAP kinase activation in KCs. The self-derived HMGB1 is an "early alarmin" of KC activation during Con A-induced ALF. HMGB1 might be a potential target for cell-specific strategy in ALF.

  18. Chinese medicinal formula, Qinggan Huoxue Recipe protects rats from alcoholic liver disease via the lipopolysaccharide-Kupffer cell signal conduction pathway

    PubMed Central

    WU, TAO; LIU, TAO; ZHANG, LI; XING, LIAN-JUN; ZHENG, PEI-YONG; JI, GUANG

    2014-01-01

    The Chinese medicinal formula, Qinggan (QG) Huoxue (HX) Recipe (R) exerts a range of pharmacological effects, including reversible steatosis, decreased levels of inflammatory cytokines and lipid peroxidation resistance. The aim of the present study was to determine the specific mechanisms of QGHXR hepatoprotection through the lipopolysaccharide-Kupffer cell (LPS-KC) signal conduction pathway in rats with alcoholic liver disease (ALD). ALD rats were exposed to the compound factors, QGR and HXR. Hematoxylin and eosin staining was conducted to evaluate the pathological changes in the liver following QGHXR treatment and an enzyme-linked immunosorbent assay was performed to measure the content of tumor necrosis factor (TNF)-α in the plasma. Immunohistochemical staining was conducted to examine the expression of cell differentiation antigen (CD) 68 and 14. In addition, western blot analysis and reverse transcription-polymerase chain reaction were used to measure the expression of Toll-like receptor 4 (TLR4), phosphorylated-extracellular regulated protein kinases (p-ERK), nuclear factor (NF)-κB, CD14 and TNF-α. Following stimulation with the compound factors, the rats exhibited increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, as well as marked pathological changes. Furthermore, the related molecules in the LPS-KC pathway were upregulated and QGHXR was identified to be effective in the LPS-KC signal conduction pathway in the ALD rats. QGHXR was superior to QGR and HXR in reducing the serum ALT and AST levels, regulating CD14, TLR4, NF-κB, ERK and TNF-α as well as improving the pathological changes. The results indicated that QGHXR therapy may provide a novel strategy for treating ALD via regulation of the related molecules in the LPS-KC signaling pathway. PMID:25009584

  19. The UII/UT system mediates upregulation of proinflammatory cytokines through p38 MAPK and NF-κB pathways in LPS-stimulated Kupffer cells.

    PubMed

    Liu, Liang Ming; Liang, Dong Yu; Ye, Chang Gen; Tu, Wen Juan; Zhu, Tong

    2015-01-01

    The urotensin II (UII)/UII receptor (UT) system is closely related to immune inflammation. In acute liver failure (ALF), the UII/UT system can promote the production and release of proinflammatory cytokines, inducing an inflammatory injury response in liver tissue. However, the mechanism by which the hepatic UII/UT system promotes proinflammatory cytokine production and release is not clear. To solve this problem, we used primary Kupffer cells (KCs) as the model system in the current study. The results showed that after lipopolysaccharide (LPS) stimulation, KCs showed significantly increased expression and release of UII/UT and proinflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β). Pretreatment with urantide, which is a UT receptor antagonist, significantly inhibited the LPS-stimulated expression and release of UII/UT, TNF-α, and IL-1β by KCs. In addition, LPS stimulation induced nuclear p38 mitogen-activated protein kinase (MAPK) protein phosphorylation and expression of the nuclear nuclear factor κB (NF-κB) p65 subunit in KCs and enhanced the binding activity of NF-κB to DNA molecules, whereas urantide pretreatment significantly inhibited the LPS-stimulated nuclear expression and activity of these molecules in KCs. Therefore, our conclusion is that the UII/UT system mediates LPS-stimulated production and release of proinflammatory cytokine by KCs, and this mediating effect at least partially relies on the inflammatory signaling pathway molecules p38 MAPK and NF-κB. PMID:25803040

  20. Kupffer-cell-expressed transmembrane TNF-α is a major contributor to lipopolysaccharide and D-galactosamine-induced liver injury.

    PubMed

    Yang, Peng; Zhou, Wenjing; Li, Chenxi; Zhang, Meng; Jiang, Yaping; Jiang, Rui; Ba, Hongping; Li, Cheng; Wang, Jing; Yin, Bingjiao; Gong, Feili; Li, Zhuoya

    2016-02-01

    Tumor necrosis factor (TNF)-α exists in two bioactive forms, a 26-kDa transmembrane form (tmTNF-α) and a 17-kDa soluble form (sTNF-α). sTNF-α has been recognized as a key regulator of hepatitis; however, serum sTNF-α disappears in mice during the development of severe liver injury, and high levels of serum sTNF-α do not necessarily result in liver damage. Interestingly, in a mouse model of acute hepatitis, we have found that tmTNF-α expression on Kupffer cells (KCs) significantly increases when mice develop severe liver injury caused by lipopolysaccharide (LPS)/D-galactosamine (D-gal), and the level of tmTNF-α expression is positively related to the activity of serum transaminases. Therefore, we hypothesized that KC-expressed tmTNF-α constitutes a pathomechanism in hepatitis and have explored the role of tmTNF-α in this disease model. Here, we have compared the impact of KCs(tmTNFlow) and KCs(tmTNFhigh) on acute hepatitis in vivo and ex vivo and have further demonstrated that KCs(tmTNFhigh), rather than KCs(tmTNFlow), not only exhibit an imbalance in secretion of pro- and anti-inflammatory cytokines, favoring inflammatory response and exacerbating liver injury, but also induce hepatocellular apoptosis via tmTNF-α and the expression of another pro-apoptotic factor, Fas ligand. Our data suggest that KC(tmTNFhigh) is a major contributor to liver injury in LPS/D-gal-induced hepatitis. PMID:26267221

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

    PubMed

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

    2015-06-01

    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.

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

    PubMed

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

    2015-06-01

    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

  3. Association between liver X receptor-α and neuron-derived orphan nuclear receptor-1 in Kupffer cells of C57BL/6 mice during inflammation.

    PubMed

    He, Kun; Dai, Zhuo-Ya; Li, Pei-Zhi; Zhu, Xi-Wen; Gong, Jian-Ping

    2015-10-01

    The liver X receptor (LXR) isoform LXR‑α has a significant role in lipid metabolism and innate immunity. Overexpression of neuron‑derived orphan nuclear receptor‑1 (NOR‑1) in macrophages reduces the synthesis of inflammatory cytokines and chemokines. However, to date, the mechanisms via which NOR‑1 inhibits lipopolysaccharide (LPS)‑induced inflammation in Kupffer cells (KCs) via LXR‑α have not been elucidated. T0901317 is the most potent LXR‑α ligand, leading to its activation. In the present study, KCs were isolated from C57BL/6 mice and randomly divided into five groups: Control, T0901317, LPS, LPS + T0901317 and LPS + T0901317 + NOR‑1 small hairpin (sh)RNA groups. In order to investigate the role of NOR‑1 in inflammation, shRNA targeting NOR‑1 was used to specifically knock down NOR‑1 mRNA in KCs. The expression levels of LXR‑α and NOR‑1 in KCs were determined by reverse transcription quantitative polymerase chain reaction and western blot analyses. The protein levels of tumor necrosis factor (TNF)‑α and interleukin (IL)‑10 in the supernatant of KCs were evaluated by ELISA. The results revealed that LXR‑α expression in the T0901317 group was higher than that in the control group; furthermore, LXR‑α expression was higher in KCs treated with LPS + T0901317 compared with that in KCs treated with LPS only. The expression levels of NOR‑1 in each group showed a similar trend. shRNA targeting of NOR‑1 suppressed the mRNA expression of NOR‑1, but had no influence on LXR‑α mRNA expression. NOR‑1 protein expression was augmented in the LPS + T0901317 group compared with that in the LPS + T09 + shRNA group. In the supernatant of KCs, the TNF‑α levels in the LPS + T0901317 group were lower than those in the LPS group, whereas the IL‑10 levels were higher in the LPS + T0901317 group compared with those in the LPS group. The results of the present study suggested that ligand T0901317 promotes LXR‑α expression

  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. Three-dimensional flow in Kupffer's Vesicle.

    PubMed

    Montenegro-Johnson, T D; Baker, D I; Smith, D J; Lopes, S S

    2016-09-01

    Whilst many vertebrates appear externally left-right symmetric, the arrangement of internal organs is asymmetric. In zebrafish, the breaking of left-right symmetry is organised by Kupffer's Vesicle (KV): an approximately spherical, fluid-filled structure that begins to form in the embryo 10 hours post fertilisation. A crucial component of zebrafish symmetry breaking is the establishment of a cilia-driven fluid flow within KV. However, it is still unclear (a) how dorsal, ventral and equatorial cilia contribute to the global vortical flow, and (b) if this flow breaks left-right symmetry through mechanical transduction or morphogen transport. Fully answering these questions requires knowledge of the three-dimensional flow patterns within KV, which have not been quantified in previous work. In this study, we calculate and analyse the three-dimensional flow in KV. We consider flow from both individual and groups of cilia, and (a) find anticlockwise flow can arise purely from excess of cilia on the dorsal roof over the ventral floor, showing how this vortical flow is stabilised by dorsal tilt of equatorial cilia, and (b) show that anterior clustering of dorsal cilia leads to around 40 % faster flow in the anterior over the posterior corner. We argue that these flow features are supportive of symmetry breaking through mechano-sensory cilia, and suggest a novel experiment to test this hypothesis. From our new understanding of the flow, we propose a further experiment to reverse the flow within KV to potentially induce situs inversus.

  6. 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

  7. International Cell Exchange: 1992.

    PubMed

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

    1992-01-01

    1. This is a review of 1992 typing of 40 cells for Class I antigens and 18 cultured cell lines for Class II antigens through the International Cell Exchange. Serological typings were compared with DNA typing reports for Class II specificities. Presently, 290 laboratories participate in the monthly Class I exchange. Class II results were received monthly from 166 serology laboratories and from 36 DNA laboratories. 2. In 1992, 11 of the 16 A-locus antigens attained 95% or greater average detection. Nine of the 27 B-locus antigens showed 95% or better mean agreement levels. Antigens such as B46 and B70 continued to show improvement in detection in a 5-year period. 3. We compared discrepancy rates of 7 A-locus and 8 B-locus antigens typed 3 times or more. The rates for the B-locus specificities, especially for percentages of false negatives (ie, how often the antigen assignment was missed), continued to be greater than those for the A-locus antigens. Nevertheless, the discrepancy rates of B35 and B70 decreased dramatically during the last 5 years. 4. We showed the number of laboratories with the total of false negatives and false positives. Nine laboratories achieved perfect records (0 false negatives and false positives) for all analyzed antigens in 1992. 5. Results of retyping of 3 donors over several years were shown to indicate improved antigen detection. 6. Recently recognized HLA-specificities, such as A2403 and B5102, were shown as cell variants studied in previous cell exchanges. Variants of B15, B16, and B40 families were presented, as well as several new A-locus antigens. 7. The low and high rates, in addition to the average detection levels, were indicated for a total of 27 (18 DR and 9 DQ) Class II specificities by serology and by DNA typings. Eight of the 15 DR/DRB1 specificities attained 90% or better average agreement by both serology and DNA. Three of the 9 DQ antigens achieved 90% or better average detection by both methods. 8. Confirmation by DNA

  8. Exploring Kupffer's Vescicle Through Self Propelled Particle Simulations

    NASA Astrophysics Data System (ADS)

    Lundy, Kassidy; Dasgupta, Agnik; Amack, Jeff; Manning, M. Lisa

    Early development is an important stage in the formation of functional, relatively healthy organisms. In zebrafish embryos, a transient organ in the tailbud called Kupffer's Vescicle (KV) is responsible for the initial left-right (L-R) asymmetry that results in asymmetric organ and tissue placement in the adult zebrafish. Originating as a collection of symmetrically organized monociliated cells, the KV experiences a shift in cell shapes over time that leaves more cells on the anterior or top side of the KV. This arrangement helps to generate a stronger counter-clockwise fluid flow across the anterior side of the organ, which is required for L-R asymmetry. In seeking to understand the source of the shape changes occurring within the KV, we simulate a Self Propelled Particle (SPP) model that includes parameters for cell polarization and speed. We model the KV as a large particle moving in a straight line with constant velocity to mimic the physical forces of the notochord acting on this organ, and we model the surrounding tailbud cells as smaller, slower active particles with an orientation that changes over time due to rotational noise. Our goal is to calculate the forces exerted on the KV by the surrounding tissue, to see if they are sufficient to explain the shape changes we observe in the KV that lead to L-R asymmetry.

  9. 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.

  10. Internal Cell Manipulation Using Infrared Laser Traps

    NASA Astrophysics Data System (ADS)

    Ashkin, A.; Dziedzic, J. M.

    1989-10-01

    The ability of infrared laser traps to apply controlled forces inside of living cells is utilized in a study of the mechanical properties of the cytoplasm of plant cells. It was discovered that infrared traps are capable of plucking out long filaments of cytoplasm inside cells. These filaments exhibit the viscoelastic properties of plastic flow, necking, stress relaxation, and set, thus providing a unique way to probe the local rheological properties of essentially unperturbed living cells. A form of internal cell surgery was devised that is capable of making gross changes in location of such relatively large organelles as chloroplasts and nuclei. The utility of this technique for the study of cytoplasmic streaming, internal cell membranes, and organelle attachment was demonstrated.

  11. Internal cell manipulation using infrared laser traps

    SciTech Connect

    Ashkin, A.; Dziedzic, J.M. )

    1989-10-01

    The ability of infrared laser traps to apply controlled forces inside of living cells is utilized in a study of the mechanical properties of the cytoplasm of plant cells. It was discovered that infrared traps are capable of plucking out long filaments of cytoplasm inside cells. These filaments exhibit the viscoelastic properties of plastic flow, necking, stress relaxation, and set, thus providing a unique way to probe the local rheological properties of essentially unperturbed living cells. A form of internal cell surgery was devised that is capable of making gross changes in location of such relatively large organelles as chloroplasts and nuclei. The utility of this technique for the study of cytoplasmic streaming, internal cell membranes, and organelle attachment was demonstrated.

  12. Internal cell manipulation using infrared laser traps.

    PubMed Central

    Ashkin, A; Dziedzic, J M

    1989-01-01

    The ability of infrared laser traps to apply controlled forces inside of living cells is utilized in a study of the mechanical properties of the cytoplasm of plant cells. It was discovered that infrared traps are capable of plucking out long filaments of cytoplasm inside cells. These filaments exhibit the viscoelastic properties of plastic flow, necking, stress relaxation, and set, thus providing a unique way to probe the local rheological properties of essentially unperturbed living cells. A form of internal cell surgery was devised that is capable of making gross changes in location of such relatively large organelles as chloroplasts and nuclei. The utility of this technique for the study of cytoplasmic streaming, internal cell membranes, and organelle attachment was demonstrated. Images PMID:2813368

  13. 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.

  14. [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".

  15. Organized chaos in Kupffer's vesicle: How a heterogeneous structure achieves consistent left-right patterning

    PubMed Central

    Smith, DJ; Montenegro-Johnson, TD; Lopes, SS

    2014-01-01

    Successful establishment of left-right asymmetry is crucial to healthy vertebrate development. In many species this process is initiated in a ciliated, enclosed cavity, for example Kupffer's vesicle (KV) in zebrafish. The microarchitecture of KV is more complex than that present in the left-right organizer of many other species. While swirling flow in KV is recognized as essential for left-right patterning, its generation, nature and conversion to asymmetric gene expression are only beginning to be fully understood. We recently [Sampaio, P et al. Dev Cell 29:716–728] combined imaging, genetics and fluid dynamics simulation to characterize normal and perturbed ciliary activity, and their correlation to asymmetric charon expression and embryonic organ fate. Randomness in cilia number and length have major implications for robust flow generation; even a modest change in mean cilia length has a major effect on flow speed to due to nonlinear scaling arising from fluid mechanics. Wildtype, and mutant embryos with normal liver laterality, exhibit stronger flow on the left prior to asymmetric inhibition of charon. Our discovery of immotile cilia, taken with data on morphant embryos with very few cilia, further support the role of mechanosensing in initiating and/or enhancing flow conversion into gene expression. PMID:25454897

  16. Organized chaos in Kupffer's vesicle: how a heterogeneous structure achieves consistent left-right patterning.

    PubMed

    Smith, D J; Montenegro-Johnson, T D; Lopes, S S

    2014-01-01

    Successful establishment of left-right asymmetry is crucial to healthy vertebrate development. In many species this process is initiated in a ciliated, enclosed cavity, for example Kupffer's vesicle (KV) in zebrafish. The microarchitecture of KV is more complex than that present in the left-right organizer of many other species. While swirling flow in KV is recognized as essential for left-right patterning, its generation, nature and conversion to asymmetric gene expression are only beginning to be fully understood. We recently [Sampaio, P et al. Dev Cell 29:716-728] combined imaging, genetics and fluid dynamics simulation to characterize normal and perturbed ciliary activity, and their correlation to asymmetric charon expression and embryonic organ fate. Randomness in cilia number and length have major implications for robust flow generation; even a modest change in mean cilia length has a major effect on flow speed to due to nonlinear scaling arising from fluid mechanics. Wildtype, and mutant embryos with normal liver laterality, exhibit stronger flow on the left prior to asymmetric inhibition of charon. Our discovery of immotile cilia, taken with data on morphant embryos with very few cilia, further support the role of mechanosensing in initiating and/or enhancing flow conversion into gene expression.

  17. Organized chaos in Kupffer's vesicle: how a heterogeneous structure achieves consistent left-right patterning.

    PubMed

    Smith, D J; Montenegro-Johnson, T D; Lopes, S S

    2014-01-01

    Successful establishment of left-right asymmetry is crucial to healthy vertebrate development. In many species this process is initiated in a ciliated, enclosed cavity, for example Kupffer's vesicle (KV) in zebrafish. The microarchitecture of KV is more complex than that present in the left-right organizer of many other species. While swirling flow in KV is recognized as essential for left-right patterning, its generation, nature and conversion to asymmetric gene expression are only beginning to be fully understood. We recently [Sampaio, P et al. Dev Cell 29:716-728] combined imaging, genetics and fluid dynamics simulation to characterize normal and perturbed ciliary activity, and their correlation to asymmetric charon expression and embryonic organ fate. Randomness in cilia number and length have major implications for robust flow generation; even a modest change in mean cilia length has a major effect on flow speed to due to nonlinear scaling arising from fluid mechanics. Wildtype, and mutant embryos with normal liver laterality, exhibit stronger flow on the left prior to asymmetric inhibition of charon. Our discovery of immotile cilia, taken with data on morphant embryos with very few cilia, further support the role of mechanosensing in initiating and/or enhancing flow conversion into gene expression. PMID:25454897

  18. 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

  19. 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.

  20. 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.

  1. Dynamics of receptor-mediated nanoparticle internalization into endothelial cells.

    PubMed

    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.

  2. 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).

  3. Fuel cell with internal flow control

    SciTech Connect

    Haltiner, Jr., Karl J.; Venkiteswaran, Arun

    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.

  4. The 1993 cell typings of the International Cell Exchange.

    PubMed

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

    1993-01-01

    1. This is a summary of the typings for 40 cells for Class I antigens and 20 cultured cell lines for Class II antigens through the International Cell Exchange. Serological typings were compared with DNA typing reports for Class II specificities. Presently, 283 laboratories participate in the monthly Class I exchange. Class II results were received from 124 serology labs and 81 DNA labs on a monthly basis. 2. In 1993, 12 A-locus antigens were typed and 8 specificities reached levels of 95% or greater average detection. Thirteen of the 33 B-locus antigens showed 95% or better mean agreement levels. There was an improvement in detection of B76 and B7801. 3. Discrepancy rates of 7 A-locus and 9 B-locus antigens typed 3 or more times were compared with the overall rates for each respective locus. The discrepancy rate of false negatives, ie, how often the antigen was missed for the recognized B-locus specificities, continued to be greater than those for the A-locus antigens. The discrepancy rates, especially the percent false-positive, decreased for A33 during the recent 6-year period. 4. We showed the number of labs with their total of false-negatives and false-positives. Twelve labs attained a final total of no misses for all antigens. In 1993, 11 labs achieved impressive perfect records (zero false negative and false positive) for all analyzed antigens. 5. Retyping results of 2 donors showed improved antigen detection, particularly of A2403, B70, and B76. 6. Eleven cells typed in previous cell exchanges as having new or rare variants were sequenced recently. The B*5102 and B*5901 cells were retyped as reference cells. A new A-locus variant detected in previous exchanges was recently confirmed by sequence work as A*8001. New variants of B5 and B22 were discussed. 7. In addition to the mean detection rates, the low and high levels were determined for 15 broad (11 DR & 4 DQ) specificities by serology and compared with those attained for the respective generic (low

  5. 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.

  6. Hepatic lipase is localized at the parenchymal cell microvilli in rat liver.

    PubMed Central

    Breedveld, B; Schoonderwoerd, K; Verhoeven, A J; Willemsen, R; Jansen, H

    1997-01-01

    Hepatic lipase (HL) is thought to be located at the vascular endothelium in the liver. However, it has also been implicated in the binding and internalization of chylomicron remnants in the parenchymal cells. In view of this apparent discrepancy between localization and function, we re-investigated the localization of HL in rat liver using biochemical and immunohistochemical techniques. The binding of HL to endothelial cells was studied in primary cultures of rat liver endothelial cells. Endothelial cells bound HL in a saturable manner with high affinity. However, the binding capacity accounted for at most 1% of the total HL activity present in the whole liver. These results contrasted with earlier studies, in which non-parenchymal cell (NPC) preparations had been found to bind HL with a high capacity. To study HL binding to the different components of the NPC preparations, we separated endothelial cells, Kupffer cells and blebs by counterflow elutriation. Kupffer cells and endothelial cells showed a relatively low HL-binding capacity. In contrast, the blebs, representing parenchymal-cell-derived material, had a high HL-binding capacity (33 m-units/mg of protein) and accounted for more than 80% of the total HL binding in the NPC preparation. In contrast with endothelial and Kupffer cells, the HL-binding capacity of parenchymal cells could account for almost all the HL activity found in the whole liver. These data strongly suggest that HL binding occurs at parenchymal liver cells. To confirm this conclusion in situ, we studied HL localization by immunocytochemical techniques. Using immunofluorescence, we confirmed the sinusoidal localization of HL. Immunoelectron microscopy demonstrated that virtually all HL was located at the microvilli of parenchymal liver cells, with a minor amount at the endothelium. We conclude that, in rat liver, HL is localized at the microvilli of parenchymal cells. PMID:9020876

  7. Experimental study of the microbial fuel cell internal resistance

    NASA Astrophysics Data System (ADS)

    Zhang, Pei-Yuan; Liu, Zhong-Liang

    The internal resistance, including activation loss internal resistance (AIR), ohmic loss internal resistance (OIR) and concentration loss internal resistance (CIR), is an important parameter that determines the performance of microbial fuel cells (MFCs). The experimental investigations were completed to estimate the contributions of these three components to the internal resistance. The internal resistance is found to vary with electric current, although it is almost a constant for the current is within a certain region. The largest component of the internal resistance is CIR except for small currents. The AIR decreases quickly for small current and reduces its decreasing rate as the current increases and approaches to a constant. The OIR is constant over the whole current range. The experiments also disclose that increasing the limiting current and reducing the concentration loss are both important for improving the MFC performance.

  8. 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. PMID:26944494

  9. 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.

  10. Internalization of Aspergillus fumigatus conidia by epithelial and endothelial cells.

    PubMed Central

    Paris, S; Boisvieux-Ulrich, E; Crestani, B; Houcine, O; Taramelli, D; Lombardi, L; Latgé, J P

    1997-01-01

    The internalization of conidia of the opportunistic fungus Aspergillus fumigatus by primary cell cultures of nonprofessional phagocytes was investigated. This study is the first to show that A. fumigatus conidia were able to be engulfed by tracheal epithelial, alveolar type II, and endothelial cells. PMID:9119494

  11. Cell-Internalization SELEX: Method for Identifying Cell-Internalizing RNA Aptamers for Delivering siRNAs to Target Cells

    PubMed Central

    Thiel, William H.; Thiel, Kristina W.; Flenker, Katie S.; Bair, Tom; Dupuy, Adam J.; McNamara, James O.; Miller, Francis J.; Giangrande, Paloma H.

    2015-01-01

    After a decade of work to address cellular uptake, the principal obstacle to RNAi-based therapeutics, there is now well-deserved, renewed optimism about RNAi-based drugs. Phase I and II studies have shown safe, strong, and durable-gene knockdown (80–90 %, lasting for a month after a single injection) and/or clinical benefit in treating several liver pathologies. Although promising, these studies have also highlighted the need for robust delivery techniques to develop RNAi therapeutics for treating other organ systems and diseases. Conjugation of siRNAs to cell-specific, synthetic RNA ligands (aptamers) is being proposed as a viable solution to this problem. While encouraging, the extended use of RNA aptamers as a delivery tool for siRNAs awaits the identification of RNA aptamer sequences capable of targeting and entering the cytoplasm of many different cell types. We describe a cell-based selection process for the rapid identification and characterization of RNA aptamers suited for delivering siRNA drugs into the cytoplasm of target cells. This process, termed “cell-internalization SELEX (Systematic Evolution of Ligands by Exponential Enrichment),” entails the combination of multiple sophisticated technologies, including cell culture-based SELEX procedures, next-generation sequencing (NGS), and novel bioinformatics tools. PMID:25319652

  12. 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.

  13. Translocation and Endocytosis for Cell-penetrating Peptide Internalization

    PubMed Central

    Jiao, Chen-Yu; Delaroche, Diane; Burlina, Fabienne; Alves, Isabel D.; Chassaing, Gérard; Sagan, Sandrine

    2009-01-01

    Cell-penetrating peptides (CPPs) share the property of cellular internalization. The question of how these peptides reach the cytoplasm of cells is still widely debated. Herein, we have used a mass spectrometry-based method that enables quantification of internalized and membrane-bound peptides. Internalization of the most used CPP was studied at 37 °C (endocytosis and translocation) and 4 °C (translocation) in wild type and proteoglycan-deficient Chinese hamster ovary cells. Both translocation and endocytosis are internalization pathways used by CPP. The choice of one pathway versus the other depends on the peptide sequence (not the number of positive changes), the extracellular peptide concentration, and the membrane components. There is no relationship between the high affinity of these peptides for the cell membrane and their internalization efficacy. Translocation occurs at low extracellular peptide concentration, whereas endocytosis, a saturable and cooperative phenomenon, is activated at higher concentrations. Translocation operates in a narrow time window, which implies a specific lipid/peptide co-import in cells. PMID:19833724

  14. Internalization of cystatin C in human cell lines.

    PubMed

    Ekström, Ulf; Wallin, Hanna; Lorenzo, Julia; Holmqvist, Bo; Abrahamson, Magnus; Avilés, Francesc X

    2008-09-01

    Altered protease activity is considered important for tumour invasion and metastasis, processes in which the cysteine proteases cathepsin B and L are involved. Their natural inhibitor cystatin C is a secreted protein, suggesting that it functions to control extracellular protease activity. Because cystatins added to cell cultures can inhibit polio, herpes simplex and coronavirus replication, which are intracellular processes, the internalization and intracellular regulation of cysteine proteases by cystatin C should be considered. The extension, mechanism and biological importance of this hypothetical process are unknown. We investigated whether internalization of cystatin C occurs in a set of human cell lines. Demonstrated by flow cytometry and confocal microscopy, A-431, MCF-7, MDA-MB-453, MDA-MB-468 and Capan-1 cells internalized fluorophore-conjugated cystatin C when exposed to physiological concentrations (1 microm). During cystatin C incubation, intracellular cystatin C increased after 5 min and accumulated for at least 6 h, reaching four to six times the baseline level. Western blotting showed that the internalized inhibitor was not degraded. It was functionally intact and extracts of cells exposed to cystatin C showed a higher capacity to inhibit papain and cathepsin B than control cells (decrease in enzyme activity of 34% and 37%, respectively). The uptake of labelled cystatin C was inhibited by unlabelled inhibitor, suggesting a specific pathway for the internalization. We conclude that the cysteine protease inhibitor cystatin C is internalized in significant quantities in various cancer cell lines. This is a potentially important physiological phenomenon not previously described for this group of inhibitors.

  15. 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)

  16. Monoclonal antibody to human endothelial cell surface internalization and liposome delivery in cell culture.

    PubMed

    Trubetskaya, O V; Trubetskoy, V S; Domogatsky, S P; Rudin, A V; Popov, N V; Danilov, S M; Nikolayeva, M N; Klibanov, A L; Torchilin, V P

    1988-02-01

    A monoclonal antibody (mAb), E25, is described that binds to the surface of cultured human endothelial cells. Upon binding E25 is rapidly internalized and digested intracellularly. Selective liposome targeting to the surface of the cells is performed using a biotinylated E25 antibody and an avidin-biotin system. Up to 30% of the cell-adherent liposomal lipid is internalized.

  17. 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.

  18. 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.

  19. External and internal triggers of cell death in yeast.

    PubMed

    Falcone, Claudio; Mazzoni, Cristina

    2016-06-01

    In recent years, yeast was confirmed as a useful eukaryotic model system to decipher the complex mechanisms and networks occurring in higher eukaryotes, particularly in mammalian cells, in physiological as well in pathological conditions. This article focuses attention on the contribution of yeast in the study of a very complex scenario, because of the number and interconnection of pathways, represented by cell death. Yeast, although it is a unicellular organism, possesses the basal machinery of different kinds of cell death occurring in higher eukaryotes, i.e., apoptosis, regulated necrosis and autophagy. Here we report the current knowledge concerning the yeast orthologs of main mammalian cell death regulators and executors, the role of organelles and compartments, and the cellular phenotypes observed in the different forms of cell death in response to external and internal triggers. Thanks to the ease of genetic manipulation of this microorganism, yeast strains expressing human genes that promote or counteract cell death, onset of tumors and neurodegenerative diseases have been constructed. The effects on yeast cells of some of these genes are also presented.

  20. Prognostic Value of Homotypic Cell Internalization by Nonprofessional Phagocytic Cancer Cells

    PubMed Central

    Schwegler, Manuela; Wirsing, Anna M.; Schenker, Hannah M.; Ott, Laura; Ries, Johannes M.; Büttner-Herold, Maike; Fietkau, Rainer; Putz, Florian; Distel, Luitpold V.

    2015-01-01

    Background. In this study, we investigated the prognostic role of homotypic tumor cell cannibalism in different cancer types. Methods. The phenomenon of one cell being internalized into another, which we refer to as “cell-in-cell event,” was assessed in 416 cases from five head and neck cancer cohorts, as well as one anal and one rectal cancer cohort. The samples were processed into tissue microarrays and immunohistochemically stained for E-cadherin and cleaved caspase-3 to visualize cell membranes and apoptotic cell death. Results. Cell-in-cell events were found in all of the cohorts. The frequency ranged from 0.7 to 17.3 cell-in-cell events per mm2. Hardly any apoptotic cells were found within the cell-in-cell structures, although apoptotic cell rates were about 1.6 to two times as high as cell-in-cell rates of the same tissue sample. High numbers of cell-in-cell events showed adverse effects on patients' survival in the head and neck and in the rectal cancer cohorts. In multivariate analysis, high frequency was an adverse prognostic factor for overall survival in patients with head and neck cancer (p = 0.008). Conclusion. Cell-in-cell events were found to predict patient outcomes in various types of cancer better than apoptosis and proliferation and might therefore be used to guide treatment strategies. PMID:26504802

  1. Proceedings: international regulatory considerations on development pathways for cell therapies.

    PubMed

    Feigal, Ellen G; Tsokas, Katherine; Viswanathan, Sowmya; Zhang, Jiwen; Priest, Catherine; Pearce, Jonathan; Mount, Natalie

    2014-08-01

    Regenerative medicine is a rapidly evolving field that faces novel scientific and regulatory challenges. In September 2013, the International Workshop on Regulatory Pathways for Cell Therapies was convened to discuss the nature of these challenges and potential solutions and to highlight opportunities for potential convergence between different regulatory bodies that might assist the field's development. The workshop discussions generated potentially actionable steps in five main areas that could mitigate cell therapy development pathway risk and accelerate moving promising therapies to patients. These included the need for convergence of regulatory guidelines on donor eligibility and suitability of lines for use in clinical trials and subsequent commercialization for cell therapies to move forward on a global basis; the need to challenge and encourage investigators in the regenerative medicine field to share information and provide examples of comparability studies related to master cell banks; the need for convergence of guidelines across regulatory jurisdictions on requirements for tumorigenicity studies, based on particular cell types and on biodistribution studies; the need to increase transparency in sharing clinical trial information more broadly and disseminating results more rapidly; and the need to establish a forum for sharing the experiences of various approaches being developed to expedite regulatory approvals and access for patients to innovative cell and regenerative therapies in the different regulatory jurisdictions and to assess their potential strengths and weaknesses.

  2. Proceedings: International Regulatory Considerations on Development Pathways for Cell Therapies

    PubMed Central

    Tsokas, Katherine; Viswanathan, Sowmya; Zhang, Jiwen; Priest, Catherine; Pearce, Jonathan; Mount, Natalie

    2014-01-01

    Regenerative medicine is a rapidly evolving field that faces novel scientific and regulatory challenges. In September 2013, the International Workshop on Regulatory Pathways for Cell Therapies was convened to discuss the nature of these challenges and potential solutions and to highlight opportunities for potential convergence between different regulatory bodies that might assist the field’s development. The workshop discussions generated potentially actionable steps in five main areas that could mitigate cell therapy development pathway risk and accelerate moving promising therapies to patients. These included the need for convergence of regulatory guidelines on donor eligibility and suitability of lines for use in clinical trials and subsequent commercialization for cell therapies to move forward on a global basis; the need to challenge and encourage investigators in the regenerative medicine field to share information and provide examples of comparability studies related to master cell banks; the need for convergence of guidelines across regulatory jurisdictions on requirements for tumorigenicity studies, based on particular cell types and on biodistribution studies; the need to increase transparency in sharing clinical trial information more broadly and disseminating results more rapidly; and the need to establish a forum for sharing the experiences of various approaches being developed to expedite regulatory approvals and access for patients to innovative cell and regenerative therapies in the different regulatory jurisdictions and to assess their potential strengths and weaknesses. PMID:25038248

  3. Cell Damage in Light Chain Amyloidosis: FIBRIL INTERNALIZATION, TOXICITY AND CELL-MEDIATED SEEDING.

    PubMed

    Marin-Argany, Marta; Lin, Yi; Misra, Pinaki; Williams, Angela; Wall, Jonathan S; Howell, Kyle G; Elsbernd, Laura R; McClure, Megan; Ramirez-Alvarado, Marina

    2016-09-16

    Light chain (AL) amyloidosis is an incurable human disease characterized by the misfolding, aggregation, and systemic deposition of amyloid composed of immunoglobulin light chains (LC). This work describes our studies on potential mechanisms of AL cytotoxicity. We have studied the internalization of AL soluble proteins and amyloid fibrils into human AC16 cardiomyocytes by using real time live cell image analysis. Our results show how external amyloid aggregates rapidly surround the cells and act as a recruitment point for soluble protein, triggering the amyloid fibril elongation. Soluble protein and external aggregates are internalized into AC16 cells via macropinocytosis. AL amyloid fibrils are shown to be highly cytotoxic at low concentrations. Additionally, caspase assays revealed soluble protein induces apoptosis, demonstrating different cytotoxic mechanisms between soluble protein and amyloid aggregates. This study emphasizes the complex immunoglobulin light chain-cell interactions that result in fibril internalization, protein recruitment, and cytotoxicity that may occur in AL amyloidosis. PMID:27462073

  4. 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. PMID:26585375

  5. Inference of Internal Stress in a Cell Monolayer.

    PubMed

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

    2016-04-12

    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

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. International renal-cell cancer study. II. Analgesics.

    PubMed

    McCredie, M; Pommer, W; McLaughlin, J K; Stewart, J H; Lindblad, P; Mandel, J S; Mellemgaard, A; Schlehofer, B; Niwa, S

    1995-01-27

    There has been concern about the role of analgesics in the development of renal-cell cancer, although a few studies have reported moderately elevated risks with regular or long-term use. In a large international case-control study of renal-cell cancer we examined, among other hypotheses, the effect of phenacetin-containing and of other types of analgesics: paracetamol (acetaminophen), salicylates (mainly aspirin) and pyrazolones (e.g., antipyrine or phenazone). Relative risks, adjusted for the effects of age, sex, body-mass index, tobacco smoking and study centre, were not significantly increased with intake of phenacetin, either when lifetime consumption was categorized at the level of > or = 0.1 kg or when subjects were subdivided further by amount. Nor were paracetamol, salicylates or pyrazolones linked with renal-cell cancer. No consistently increasing risks with consumption level was found. The lack of association was not altered by restricting analgesic use to that which occurred 5 or 10 years before the defined "cut-off" date or when analysis was restricted to exclusive users of a particular type of analgesic. Neither was the risk influenced by the rate of consumption or whether the consumption had occurred at a young age. Our study provides clear evidence that aspirin is unrelated to renal-cell cancer risk, and our findings do not support the hypothesis that analgesics containing phenacetin or paracetamol increase the risk, although the number of "regular" users and the amount of these types of analgesic consumed were too small to confidently rule out a minor carcinogenic effect of phenacetin and paracetamol.

  12. Human endothelial progenitor cells internalize high-density lipoprotein.

    PubMed

    Srisen, Kaemisa; Röhrl, Clemens; Meisslitzer-Ruppitsch, Claudia; Ranftler, Carmen; Ellinger, Adolf; Pavelka, Margit; Neumüller, Josef

    2013-01-01

    Endothelial progenitor cells (EPCs) originate either directly from hematopoietic stem cells or from a subpopulation of monocytes. Controversial views about intracellular lipid traffic prompted us to analyze the uptake of human high density lipoprotein (HDL), and HDL-cholesterol in human monocytic EPCs. Fluorescence and electron microscopy were used to investigate distribution and intracellular trafficking of HDL and its associated cholesterol using fluorescent surrogates (bodipy-cholesterol and bodipy-cholesteryl oleate), cytochemical labels and fluorochromes including horseradish peroxidase and Alexa Fluor® 568. Uptake and intracellular transport of HDL were demonstrated after internalization periods from 0.5 to 4 hours. In case of HDL-Alexa Fluor® 568, bodipy-cholesterol and bodipy-cholesteryl oleate, a photooxidation method was carried out. HDL-specific reaction products were present in invaginations of the plasma membrane at each time of treatment within endocytic vesicles, in multivesicular bodies and at longer periods of uptake, also in lysosomes. Some HDL-positive endosomes were arranged in form of "strings of pearl"- like structures. HDL-positive multivesicular bodies exhibited intensive staining of limiting and vesicular membranes. Multivesicular bodies of HDL-Alexa Fluor® 568-treated EPCs showed multilamellar intra-vacuolar membranes. At all periods of treatment, labeled endocytic vesicles and organelles were apparent close to the cell surface and in perinuclear areas around the Golgi apparatus. No HDL-related particles could be demonstrated close to its cisterns. Electron tomographic reconstructions showed an accumulation of HDL-containing endosomes close to the trans-Golgi-network. HDL-derived bodipy-cholesterol was localized in endosomal vesicles, multivesicular bodies, lysosomes and in many of the stacked Golgi cisternae and the trans-Golgi-network Internalized HDL-derived bodipy-cholesteryl oleate was channeled into the lysosomal intraellular

  13. 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 Central

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

    2015-01-01

    ABSTRACT 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 Ca2+-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. PMID:26432887

  14. 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.

  15. Gastrulation in the spider Zygiella x-notata involves three distinct phases of cell internalization.

    PubMed

    Chaw, R Crystal; Vance, Emily; Black, Steven D

    2007-12-01

    The cell movements of gastrulation were analyzed in embryos of the spider Zygiella x-notata, using time-lapse video, cell tracing, and improved histology. Cells are internalized near the center of the germ disc in three distinct phases. First, cumulus mesenchyme cells ingress and migrate as a group beneath the superficial layer. Second, mass internalization through a blastopore yields a diffusely organized deep layer. Third, superficial cells accumulate at the center of the germ disc to form the caudal bud. The floor is internalized, and the caudal bud moves over the nascent dorsal field to form the caudal lobe. This pattern of gastrulation differs from the canonical pattern described in the historical literature: (1) the cumulus of Z. x-notata is completely formed before any other cells internalize; and (2) the caudal lobe is formed by means of the caudal bud, which is a locus of cell internalization.

  16. 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.

  17. Patentability of Parthenogenic Stem Cells: International Stem Cell Corporation v. Comptroller General of Patents.

    PubMed

    Mansnérus, Juli

    2015-06-01

    The European Court of Justice (ECJ) has recently issued a ruling in Case C-364/13 International Stem Cell Corporation v. Comptroller General of Patents Designs and Tademarks (Case) that aims at harmonising the patenting practices regarding interpretation of Article 6.2.c of Directive 98/44/EC (Biotech Patent Directive) in respect of patentability of human parthenogenic stem cells (hpSCs). The Case alters the patenting regime for human embryonic stem cell (hESC) applications, by stating that moral restrictions against hESC-patents are only applicable to such cells derived from embryos that had the potential to develop into a human being. Consequently, hpSC-based inventions may be patentable in Europe. This Case represents a leap forward to striking a balance between protecting human dignity and integrity whilst granting patent incentives for biomedical research.

  18. International renal-cell cancer study. IV. Occupation.

    PubMed

    Mandel, J S; McLaughlin, J K; Schlehofer, B; Mellemgaard, A; Helmert, U; Lindblad, P; McCredie, M; Adami, H O

    1995-05-29

    The relationship between renal-cell cancer (RCC) and occupation was investigated in an international multicenter population-based case-control study. Study centers in Australia, Denmark, Germany, Sweden and the United States interviewed 1732 incident RCC cases and 2309 controls. Significant associations were found with employment in the blast-furnace or the coke-oven industry [relative risk (RR), 1.7; 95% confidence interval (CI), 1.1-2.7], the iron and steel industry (RR, 1.6; 95% CI, 1.2-2.2) and exposure to asbestos (RR, 1.4; 95% CI, 1.1-1.8), cadmium (RR, 2.0; 95% CI, 1.0-3.9), dry-cleaning solvents (RR, 1.4; 95% CI, 1.1-1.7), gasoline (RR, 1.6; 95% CI, 1.2-2.0) and other petroleum products (RR, 1.6; 95% CI, 1.3-2.1). Asbestos, petroleum products and dry-cleaning solvents appear to merit further investigation, in view of the relationship between risk and duration of employment or exposure and after adjustment for confounding. There was a negative association between RCC and education, but it was not consistent across all centers. Overall, the results of our multicenter case-control study suggest that occupation may be more important in the etiology of RCC than indicated by earlier studies. PMID:7768630

  19. 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

  20. 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.

  1. International renal-cell cancer study. I. Tobacco use.

    PubMed

    McLaughlin, J K; Lindblad, P; Mellemgaard, A; McCredie, M; Mandel, J S; Schlehofer, B; Pommer, W; Adami, H O

    1995-01-17

    The relationship between renal-cell cancer (RCC) and tobacco use was investigated in an international, multicenter, population-based case-control study. Coordinated studies were conducted in Australia, Denmark, Germany, Sweden and the United States using a shared protocol and questionnaire. A total of 1,732 cases (1,050 men, 682 women) and 2,309 controls (1,429 men, 880 women) were interviewed for the study. No association was observed between risk and use of cigars, pipes or smokeless tobacco. A statistically significant association was observed for cigarette smoking, with current smokers having a 40% increase in risk [relative risk (RR) = 1.4, 95% confidence interval (CI) 1.2-1.7]. Risk increased with intensity (number of cigarettes) and duration (years smoked). Among current smokers the RR for pack-years rose from 1.1 (95% CI 0.8-1.5) for < 15.9 pack years to 2.0 (95% CI 1.6-2.7) for > 42 pack years (p for trend < 0.001). Long-term quitters (> 15 years) experienced a reduction in risk of about 15-25% relative to current smokers. Those who started smoking late (> 24 years of age) had about two-thirds the risk of those who started young (< or = 12 years of age). Overall, the findings of this pooled analysis confirm that cigarette smoking is a causal factor in the etiology of RCC.

  2. Phytosterols Promote Liver Injury and Kupffer Cell Activation in Parenteral Nutrition–Associated Liver Disease

    PubMed Central

    El Kasmi, Karim C.; Anderson, Aimee L.; Devereaux, Michael W.; Vue, Padade M.; Zhang, Wujuan; Setchell, Kenneth D. R.; Karpen, Saul J.; Sokol, Ronald J.

    2014-01-01

    Parenteral nutrition–associated liver disease (PNALD) is a serious complication of PN in infants who do not tolerate enteral feedings, especially those with acquired or congenital intestinal diseases. Yet, the mechanisms underlying PNALD are poorly understood. It has been suggested that a component of soy oil (SO) lipid emulsions in PN solutions, such as plant sterols (phytosterols), may be responsible for PNALD, and that use of fish oil (FO)–based lipid emulsions may be protective. We used a mouse model of PNALD combining PN infusion with intestinal injury to demonstrate that SO-based PN solution causes liver damage and hepatic macrophage activation and that PN solutions that are FO-based or devoid of all lipids prevent these processes. We have furthermore demonstrated that a factor in the SO lipid emulsions, stigmasterol, promotes cholestasis, liver injury, and liver macrophage activation in this model and that this effect may be mediated through suppression of canalicular bile transporter expression (Abcb11/BSEP, Abcc2/MRP2) via antagonism of the nuclear receptors Fxr and Lxr, and failure of up-regulation of the hepatic sterol exporters (Abcg5/g8/ABCG5/8). This study provides experimental evidence that plant sterols in lipid emulsions are a major factor responsible for PNALD and that the absence or reduction of plant sterols is one of the mechanisms for hepatic protection in infants receiving FO-based PN or lipid minimization PN treatment. Modification of lipid constituents in PN solutions is thus a promising strategy to reduce incidence and severity of PNALD. PMID:24107776

  3. Methods for Evaluating Cell-Specific, Cell-Internalizing RNA Aptamers

    PubMed Central

    Hernandez, Luiza I.; Flenker, Katie S.; Hernandez, Frank J.; Klingelhutz, Aloysius J.; II, James O. McNamara; Giangrande, Paloma H.

    2013-01-01

    Recent clinical trials of small interfering RNAs (siRNAs) highlight the need for robust delivery technologies that will facilitate the successful application of these therapeutics to humans. Arguably, cell targeting by conjugation to cell-specific ligands provides a viable solution to this problem. Synthetic RNA ligands (aptamers) represent an emerging class of pharmaceuticals with great potential for targeted therapeutic applications. For targeted delivery of siRNAs with aptamers, the aptamer-siRNA conjugate must be taken up by cells and reach the cytoplasm. To this end, we have developed cell-based selection approaches to isolate aptamers that internalize upon binding to their cognate receptor on the cell surface. Here we describe methods to monitor for cellular uptake of aptamers. These include: (1) antibody amplification microscopy, (2) microplate-based fluorescence assay, (3) a quantitative and ultrasensitive internalization method (“QUSIM”) and (4) a way to monitor for cytoplasmic delivery using the ribosome inactivating protein-based (RNA-RIP) assay. Collectively, these methods provide a toolset that can expedite the development of aptamer ligands to target and deliver therapeutic siRNAs in vivo. PMID:23894227

  4. 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

  5. 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

  6. 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.

  7. 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.

  8. Cell Internalization Studies of Gadofullerene-(ZME-018) Immunoconjugates into A375m Melanoma Cells12

    PubMed Central

    Berger, Christopher Scott; Marks, John W; Bolskar, Robert D; Rosenblum, Michael G; Wilson, Lon J

    2011-01-01

    Fullerene (C60)-monoclonal antibody (mAb) immunoconjugates have been determined to internalize into target cells using water-soluble Gd3+ ion-filled metallofullerenes (Gd@C60[OH]x). Two separate conjugations of Gd@C60(OH)x with the antibody ZME-018 and a murine antibody mixture (MuIgG) were performed in a 1:5 mAb/Gd@C60 ratio. Characterization of the immunoconjugates was established using inductively coupled plasma mass spectrometry (ICP-MS) for Gd3+ and UV-Vis spectrometry (for Gd@C60 + C60). Once conjugated, enzyme-linked immunosorbent assays showed little change in the specific binding of ZME-018. Each immunoconjugate was exposed to two cancer cell lines, A375m (antigen positive), and T24, bladder carcinoma (antigen negative). Internalization levels of the immunoconjugate were determined at various time points during 24 hours by harvesting and digesting the cells with 70% HNO3 for Gd3+ ion analysis by ICP-MS. These results are the first to demonstrate the practicality of a targeted cancer therapy based on fullerene immunotherapy. PMID:22190999

  9. 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.

  10. 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.

  11. 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.

  12. Cell surface heparan sulfate proteoglycans mediate the internalization of PDX-1 protein.

    PubMed

    Ueda, Michiko; Matsumoto, Shinichi; Hayashi, Shuji; Kobayashi, Naoya; Noguchi, Hirofumi

    2008-01-01

    Although islet transplantation is a promising therapeutic option for the treatment of type 1 diabetes, the shortage of suitable donor tissues remains a major obstacle. Pancreatic stem/progenitor cells residing within the ductal epithelium have been used to generate human islet-like clusters, but there is no efficient strategy for facilitating differentiation of progenitor cells into insulin-producing cells. A previous study reported that exogenous PDX-1 protein can be transduced into pancreatic stem/progenitor cells and induce differentiation of the cells into insulin-producing cells without requiring gene transfer technology. This study provides genetic and biochemical evidence that cell membrane heparan sulfate proteoglycans are required for extracellular PDX-1 internalization. Heparin, one of the soluble glycosaminoglycans (GAGs), inhibited PDX-1 internalization, while chondroitin sulfate A, B, and C caused only very limited inhibition. Cell treatment with heparinase-III demonstrated impaired PDX-1 internalization, while treatment with chondroitinase ABC, or with chondroitinase AC, was completely ineffective in inhibiting PDX-1 internalization. Different mutant cell lines originating from CHO K1 cells and defective in GAG biosynthesis were also examined. PDX-1 internalization was significantly reduced in both pgs A-745 mutant cells, which are defective in a enzyme that initiates GAG synthesis, and pgs B-618 cells, which produce about 15% of the amount of GAGs synthesized by wild-type cells. These data indicate that cell-surface heparan sulfate proteoglycans are required for PDX-1 internalization and that PDX-1 protein transduction could be a valuable strategy for inducing insulin expression in pancreatic stem/progenitor cells without requiring gene transfer technology.

  13. Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry.

    PubMed

    Pohl, Marie O; Stertz, Silke

    2015-01-01

    Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed protocol for measuring relative changes in the amount of viral particles that attach to A549 cells, a human lung epithelial cell line, as well as in the amount of particles that are internalized into the cell. We use biotinylated virus which can be easily detected following staining with Cy3-labeled streptavidin (STV-Cy3). We describe the growth, purification and biotinylation of A/WSN/33, a widely used IAV laboratory strain. Cold-bound biotinylated IAV particles on A549 cells are stained with STV-Cy3 and measured using flow cytometry. To investigate uptake of viral particles, cold-bound virus is allowed to internalize at 37 °C. In order to differentiate between external and internalized viral particles, a blocking step is applied: Free binding spots on the biotin of attached virus on the cell surface are bound by unlabeled streptavidin (STV). Subsequent cell permeabilization and staining with STV-Cy3 then enables detection of internalized viral particles. We present a calculation to determine the relative amount of internalized virus. This assay is suitable to measure effects of drug-treatments or other manipulations on attachment or internalization of IAV. PMID:26575457

  14. Effect of β-cyclodextrin on the internalization of nanoparticles into intestine epithelial cells.

    PubMed

    García-González, Lorena; Yépez-Mulía, Lilián; Ganem, Adriana

    2016-01-01

    The influence of β-cyclodextrin on the interaction and internalization of PLGA nanoparticles into intestine epithelial cells was assessed. For this purpose β-cyclodextrin was adsorbed on PLGA nanoparticles. Interaction of nanoparticles with Caco-2 cells, determined by fluorescence, was expressed as the number of particles per cell. Confocal microscopy confirmed the localization of the particles in the cell monolayer. The results showed that adsorption of β-cyclodextrin on the surface of PLGA nanoparticles reduces interaction with mucin, enhancing in this way the internalization into the Caco-2 cells.

  15. Cell Internal Treatable Microplasma Jets in Cancer Therapies

    NASA Astrophysics Data System (ADS)

    Kim, Jae Young; Wei, Yanzhang; Li, Jinhua; Kim, Sung-O.

    2011-10-01

    We developed a 15- μm-sized, single-cellular-level, and cell-manipulatable microplasma jet device with a microcapillary glass tip and described its potential in physical cancer therapies. The microcapillary tip is a funnel shaped glass tube and its nozzle has an inner diameter of 15 μm and an outer diameter of 20 μm with 20 capillary angle. The electrical and optical properties of this plasma jet and apoptosis results of cultured murine B16F0 melanoma tumor cells and CL.7 fibroblast cells treated with the plasma jets were described. In spite of the small inner diameter and the low gas flow rate of the microplasma jet device, the generated plasma jets are stable enough to treat tumor cells. The microplasma jet was observed inducing apoptosis in cultured murine melanoma tumor cells in a dose-dependent manner. Furthermore, the percentage of apoptotic cells of murine melanoma tumor cells induced by this plasma device was approximately 2.5 times bigger than that of murine fibroblast cells as indicated by an Annex V-FITC method. This highly precise plasma medicine, which enables new directed cancer therapies, can be combined with current cell manipulation and cell culturing technologies without much difficulty.

  16. 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

  17. 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.

  18. 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.

  19. The spleen pigment cells in some amphibia.

    PubMed

    Scalia, Marina; Di Pietro, Cinzia; Poma, Mariangela; Ragusa, Marco; Sichel, Giovanni; Corsaro, Concetta

    2004-04-01

    It was demonstrated that the spleen pigment cells of Amphibia are macrophages: they show an ultrastructurally distinctive morphology, are able to phagocytose and react positively for non-specific esterases. These pigmented macrophages express mRNA for tyrosinase and also they show dopa oxidase activity; therefore they are able to synthesize melanins, as Kupffer cells do.

  20. Fc gamma receptor IIb on target B cells promotes rituximab internalization and reduces clinical efficacy.

    PubMed

    Lim, Sean H; Vaughan, Andrew T; Ashton-Key, Margaret; Williams, Emily L; Dixon, Sandra V; Chan, H T Claude; Beers, Stephen A; French, Ruth R; Cox, Kerry L; Davies, Andrew J; Potter, Kathleen N; Mockridge, C Ian; Oscier, David G; Johnson, Peter W M; Cragg, Mark S; Glennie, Martin J

    2011-09-01

    The anti-CD20 mAb rituximab is central to the treatment of B-cell malignancies, but resistance remains a significant problem. We recently reported that resistance could be explained, in part, by internalization of rituximab (type I anti-CD20) from the surface of certain B-cell malignancies, thus limiting engagement of natural effectors and increasing mAb consumption. Internalization of rituximab was most evident in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), but the extent of internalization was heterogeneous within each disease. Here, we show that the inhibitory FcγRIIb on target B cells promotes this process and is largely responsible for the observed heterogeneity across a range of B-cell malignancies. Internalization correlated strongly with FcγRIIb expression on normal and malignant B cells, and resulted in reduced macrophage phagocytosis of mAb-coated targets. Furthermore, transfection of FcγRIIb into FcγRIIb negative Ramos cells increased internalization of rituximab in a dose-dependent manner. Target-cell FcγRIIb promoted rituximab internalization in a cis fashion and was independent of FcγRIIb on neighboring cells. It became phosphorylated and internalized along with CD20:anti-CD20 complexes before lysosomal degradation. In MCL patients, high FcγRIIb expression predicted less durable responses after rituximab-containing regimens. Therefore, target-cell FcγRIIb provides a potential biomarker of response to type I anti-CD20 mAb.

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. Actomyosin-based Self-organization of cell internalization during C. elegans gastrulation

    PubMed Central

    2012-01-01

    Background Gastrulation is a key transition in embryogenesis; it requires self-organized cellular coordination, which has to be both robust to allow efficient development and plastic to provide adaptability. Despite the conservation of gastrulation as a key event in Metazoan embryogenesis, the morphogenetic mechanisms of self-organization (how global order or coordination can arise from local interactions) are poorly understood. Results We report a modular structure of cell internalization in Caenorhabditis elegans gastrulation that reveals mechanisms of self-organization. Cells that internalize during gastrulation show apical contractile flows, which are correlated with centripetal extensions from surrounding cells. These extensions converge to seal over the internalizing cells in the form of rosettes. This process represents a distinct mode of monolayer remodeling, with gradual extrusion of the internalizing cells and simultaneous tissue closure without an actin purse-string. We further report that this self-organizing module can adapt to severe topological alterations, providing evidence of scalability and plasticity of actomyosin-based patterning. Finally, we show that globally, the surface cell layer undergoes coplanar division to thin out and spread over the internalizing mass, which resembles epiboly. Conclusions The combination of coplanar division-based spreading and recurrent local modules for piecemeal internalization constitutes a system-level solution of gradual volume rearrangement under spatial constraint. Our results suggest that the mode of C. elegans gastrulation can be unified with the general notions of monolayer remodeling and with distinct cellular mechanisms of actomyosin-based morphogenesis. PMID:23198792

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

    DOE PAGESBeta

    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 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

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

    NASA Astrophysics Data System (ADS)

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

    2016-02-01

    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. The short circuits occur after extensive internal faulting, which implies significant stretching and tearing of separators.

  8. 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.

  9. Fuel cell stack with internal manifolds for reactant gases

    DOEpatents

    Schnacke, A.W.

    1983-10-12

    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.

  10. [International approaches to the regulation of cell therapy products].

    PubMed

    Piatigorskaia, N V; Tulina, M A; Aladysheva, Zh I; Beregovykh, V V

    2013-01-01

    This article is a review of the main methods and approaches used in regulation of cell therapy products in the United States of America, Canada, European Union, Australia, Japan and South Korea. Intensive developments ofscientific and technological aspects in stem cell and tissue engineering have led to the wide use of human cells and tissues for the treatment of various diseases and injuries of organs and tissues. Drug regulatory agencies of different countries are working on implementation of a risk-based legal framework with some common features. In many countries there is a multilevel control system that assures quality and safety of used cell products. Competent authorities establish strict requirements both to safety of the products and to the implemented standards of good laboratory, manufacturing, clinical and tissue practices. PMID:24340637

  11. 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.

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

    PubMed Central

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

    2014-01-01

    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. PMID:25169063

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. Comparison of several methods for determining the internal resistance of lithium ion cells.

    PubMed

    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

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

    PubMed

    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.

  19. 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

  20. International analysis of the frequency and outcomes of NK/T-cell lymphomas.

    PubMed

    William, Basem M; Armitage, James O

    2013-03-01

    Peripheral T-cell and NK-cell lymphomas are uncommon disorders accounting for 10-15% of all non-Hodgkin lymphomas (NHL). The NHL classification project represents the first attempt to systematically study the distribution of NHL subtypes based on a collaborative international effort and it confirmed the wide geographic variation in the frequency of different subtypes of PTCL. Subsequently, the International T-cell Lymphoma Project (ITLP), the largest collaborative international effort to date, reported prevalence and outcomes of 1314 cases of PTCL from 22 institutions worldwide with central pathology review. The ITLP consortium launched a prospective study, the T-cell project, in September 2006 aimed at collecting an exhaustive clinical and biologic data set on 1000 patients with PTCL for better definition of prognostic factors that would influence outcomes of these patients. This review aims to describe the difference in frequency and outcomes for various subtypes of PTCL based on these studies.

  1. Short tandem repeat profiling provides an international reference standard for human cell lines

    PubMed Central

    Masters, John R.; Thomson, Jim A.; Daly-Burns, Bernadette; Reid, Yvonne A.; Dirks, Wilhelm G.; Packer, Phil; Toji, Lorraine H.; Ohno, Tadao; Tanabe, Hideyuki; Arlett, Colin F.; Kelland, Lloyd R.; Harrison, Maureen; Virmani, Arvind; Ward, Timothy H.; Ayres, Karen L.; Debenham, Paul G.

    2001-01-01

    Cross-contamination between cell lines is a longstanding and frequent cause of scientific misrepresentation. Estimates from national testing services indicate that up to 36% of cell lines are of a different origin or species to that claimed. To test a standard method of cell line authentication, 253 human cell lines from banks and research institutes worldwide were analyzed by short tandem repeat profiling. The short tandem repeat profile is a simple numerical code that is reproducible between laboratories, is inexpensive, and can provide an international reference standard for every cell line. If DNA profiling of cell lines is accepted and demanded internationally, scientific misrepresentation because of cross-contamination can be largely eliminated. PMID:11416159

  2. 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

  3. Gadolinium-chelate nanoparticle entrapped human mesenchymal stem cell via photochemical internalization for cancer diagnosis.

    PubMed

    Kim, Kyoung Sub; Park, Wooram; Na, Kun

    2015-01-01

    To improve the gadolinium (Gd) internalization efficiency in stem cells, gadolinium-chelate nanoparticles were prepared from a pullulan derivative (pullulan-deoxycholic acid (DOCA)-diethylene triamine pentaacetic (DTPA)-Gd conjugate; PDDG) and then the PDDG was entrapped into human mesenchymal stem cells (hMSCs) by the photochemical-internalization (PCI) method for cancer diagnosis via the cancer homing property of hMSCs. The internalization efficiency of Gd in hMSCs was significantly increased to 98 ± 4 pg Gd/cell from 32 ± 2 pg Gd/cell via the PCI method. Moreover, the Gd-entrapped hMSCs revealed a low exocytosis ratio of gadolinium-chelate nanoparticles during cell division in vitro and a high cellular labeling efficiency for at least 21 days in vivo. The cancer-targeting and diagnosis effect of the Gd-entrapped hMSCs were confirmed in a small CT26 tumor-bearing mice model. The stem cells detected an early tumor (∼3 mm(3)) within 2 h using 4.7-T MR and optical imaging. The results demonstrated that the PCI-mediated internalization of Gd-incorporated nanoparticles into hMSCs is a promising protocol for efficient cell labeling and tracking.

  4. Undifferentiated HL-60 cells internalize an antitumor alkyl ether phospholipid more rapidly than resistant K562 cells.

    PubMed

    Tsutsumi, T; Tokumura, A; Kitazawa, S

    1998-02-01

    In this study, we confirmed a previous finding that 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (methyl-PAF) expresses higher antineoplastic activity against the promyelocytic leukemia cell line HL-60, than against the erythroleukemic cell line K562, and intended to clarify the reason for this. Using an albumin back-exchange method, we measured the rates of binding and internalization of [3H]methyl-PAF by HL-60 and K562 cells. We found that methyl-PAF associated very rapidly and to similar extents with the two types of cells at low concentrations of extracellular bovine serum albumin, but that when bound to the cell surface, it was internalized into HL-60 cells faster than into K562 cells. The internalization of methyl-PAF by HL-60 cells was concentration-independent, intracellular ATP-independent and susceptible to thiol group-modifying reagents and cytochalasin B. Thus the inward transbilayer movement of methyl-PAF seems to occur by cytochalasin B-sensitive protein-mediated mechanism based on passive diffusion not requiring energy, in which SH-groups of protein play a critical role. We also found that the internalization of 1-hexadecanoyl-2-(4,4-difluoro-5,7- dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphocholine (Bodipy-C5-PC), whose structure resembles that of methyl-PAF, into HL-60 cells was faster than that into K562 cells. Using a combination of an albumin back-exchange method and observation by confocal laser scanning microscopy, we next examined the intracellular distribution of this fluorescent phospholipid probe after its internalization. Intracellular membranes, especially those peripheral to nuclei, were fluorescence-labeled in both HL-60 and K562 cells, but fluorescence of the nuclear membranes was weak, suggesting that this probe seems mainly to accumulate in intracellular granules, and may interact directly with several key enzymes for phospholipid metabolism, leading to cell injury. Because the difference between

  5. Internalization of Vectored Liposomes in a Culture of Poorly Differentiated Tumor Cells.

    PubMed

    Mel'nikov, P A; Baklaushev, V P; Gabashvili, A N; Nukolova, N V; Levinsky, A B; Chehonin, V P

    2016-08-01

    Internalization of liposomal nanocontainers conjugated with monoclonal antibodies to VEGF, VEGFR2 (KDR), and proteins overproduced in the tumor tissue was studied in vitro on cultures of poorly differentiated tumor cells. Comparative analysis of accumulation of vectored liposomes in the tumor cells was performed by evaluating co-localization of labeled containers and cell organelles by laser scanning confocal microscopy. We observed nearly 2 times more active penetration and accumulation of liposomes vectored with antibodies in the tumor cells in comparison with non-vectored liposomes. Selective clathrin-dependent penetration of vectored liposomes into tumor cells was demonstrated by using pharmacological agents inhibiting endocytosis. PMID:27590766

  6. 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

  7. 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. PMID:27428437

  8. Identification of internalizing human single chain antibodies targeting brain tumor sphere cells

    PubMed Central

    Zhu, Xiaodong; Bidlingmaier, Scott; Hashizume, Rintaro; James, C. David; Berger, Mitchel S.; Liu, Bin

    2010-01-01

    Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain tumor and there is no curative treatment to date. Resistance to conventional therapies and tumor recurrence pose major challenges to treatment and management of this disease, and therefore new therapeutic strategies need to be developed. Previous studies by other investigators have shown that a subpopulation of GBM cells can grow as neurosphere-like cells when cultured in restrictive media, and exhibit enhanced tumor initiating ability and resistance to therapy. We report here the identification of internalizing human single chain antibodies (scFvs) targeting GBM tumor sphere cells. We selected a large naive phage antibody display library on the glycosylation-dependent CD133 epitope-positive subpopulation of GBM cells grown as tumor spheres and identified internalizing scFvs that target tumor sphere cells broadly, as well as scFvs that target the CD133 positive subpopulation. These scFvs were found to be efficiently internalized by GBM tumor sphere cells. One scFv GC4 inhibited self-renewal of GBM tumor sphere cells in vitro. We have further developed a full-length human IgG1 based on this scFv and found that it potently inhibits proliferation of GBM tumor sphere cells and GBM cells grown in regular non-selective media. Taken together, these results show that internalizing human scFvs targeting brain tumor sphere cells can be readily identified from a phage antibody display library, which could be useful for further development of novel therapies that target subpopulations of GBM cells to combat recurrence and resistance to treatment. PMID:20587664

  9. Intracellular killing of mastitis pathogens by penethamate hydriodide following internalization into mammary epithelial cells.

    PubMed

    Almeida, R A; Patel, D; Friton, G M; Oliver, S P

    2007-04-01

    Penethamate hydriodide was highly effective in killing Streptococcus uberis, Streptococcus dysgalactiae subsp. dysgalactiae and Staphylococcus aureus that internalized mammary epithelial cells. At higher concentrations (32 microg/mL to 32 mg/mL), killing rates ranged from 85% to 100%. At lower concentrations (0.032 microg/mL to 3.2 microg/mL), killing rates ranged from 0 to 80%. Results of this proof-of-concept study demonstrated that: (1) penethamate hydriodide is capable of entering mammary epithelial cells and killing intracellular mastitis pathogens without affecting mammary epithelial cell viability, (2) the in vitro model used is capable of quantifying the fate of mastitis pathogens internalized into mammary epithelial cells, and (3) this in vitro model can be used to determine the effectiveness of antibiotics at killing bacteria within the cytoplasm of mammary epithelial cells.

  10. An E-cadherin-mediated hitchhiking mechanism for C. elegans germ cell internalization during gastrulation

    PubMed Central

    Chihara, Daisuke; Nance, Jeremy

    2012-01-01

    Gastrulation movements place endodermal precursors, mesodermal precursors and primordial germ cells (PGCs) into the interior of the embryo. Somatic cell gastrulation movements are regulated by transcription factors that also control cell fate, coupling cell identity and position. By contrast, PGCs in many species are transcriptionally quiescent, suggesting that they might use alternative gastrulation strategies. Here, we show that C. elegans PGCs internalize by attaching to internal endodermal cells, which undergo morphogenetic movements that pull the PGCs into the embryo. We show that PGCs enrich HMR-1/E-cadherin at their surfaces to stick to endoderm. HMR-1 expression in PGCs is necessary and sufficient to ensure internalization, suggesting that HMR-1 can promote PGC-endoderm adhesion through a mechanism other than homotypic trans interactions between the two cell groups. Finally, we demonstrate that the hmr-1 3′ untranslated region promotes increased HMR-1 translation in PGCs. Our findings reveal that quiescent PGCs employ a post-transcriptionally regulated hitchhiking mechanism to internalize during gastrulation, and demonstrate a morphogenetic role for the conserved association of PGCs with the endoderm. PMID:22675206

  11. B Cell Antigen Receptor Signaling and Internalization Are Mutually Exclusive Events

    PubMed Central

    Hou, Ping; Araujo, Elizabeth; Zhao, Tong; Zhang, Miao; Massenburg, Don; Veselits, Margaret; Doyle, Colleen; Dinner, Aaron R; Clark, Marcus R

    2006-01-01

    Engagement of the B cell antigen receptor initiates two concurrent processes, signaling and receptor internalization. While both are required for normal humoral immune responses, the relationship between these two processes is unknown. Herein, we demonstrate that following receptor ligation, a small subpopulation of B cell antigen receptors are inductively phosphorylated and selectively retained at the cell surface where they can serve as scaffolds for the assembly of signaling molecules. In contrast, the larger population of non-phosphorylated receptors is rapidly endocytosed. Each receptor can undergo only one of two mutually exclusive fates because the tyrosine-based motifs that mediate signaling when phosphorylated mediate internalization when not phosphorylated. Mathematical modeling indicates that the observed competition between receptor phosphorylation and internalization enhances signaling responses to low avidity ligands. PMID:16719564

  12. 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

  13. The effect of internal air bleed on CO poisoning in a proton exchange membrane fuel cell

    NASA Astrophysics Data System (ADS)

    Wang, Wentao

    It is found that carbon monoxide (CO) poisoning could be mitigated by increasing only cathode backpressure for a proton exchange membrane fuel cell (PEMFC) with ultra-thin membranes (≤25 μm). This mitigation can be explained by a heterogeneous oxidation of CO on a Pt-Ru/C anode by the permeated O 2 which is known as "internal air bleed" in his paper. A steady-state model which accounts for this internal air bleed has been developed to model the Pt-Ru/C anode polarization data when 50 ppm CO in H 2 is used as anode feed gas. The modeling results show that the mitigation of CO poisoning by the internal air bleed even exists at ambient conditions for a PEMFC with an ultra-thin membrane. Therefore, the effect of internal air bleed must be considered for modeling fuel cell performance or anode polarization data if an ultra-thin membrane and a low level of CO concentration are used for a Pt-Ru/C anode. An empirical relationship between the amount of internal air bleed used for the mitigation of CO poisoning and the fraction of free Pt sites is provided to facilitate the inclusion of an internal air bleed term in the modeling of anode polarization and the fuel cell performance.

  14. 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.

  15. Binding, internalization, and degradation of basic fibroblast growth factor in human microvascular endothelial cells

    SciTech Connect

    Bikfalvi, A.; Dupuy, E.; Inyang, A.L.; Tobelem, G. ); Fayein, N.; Courtois, Y. ); Leseche, G. )

    1989-03-01

    The binding, internalization, and degradation of basic fibroblast growth factor (bFGF) in human omental microvascular endothelial cells (HOME cells) were investigated. Binding studies of bFGF in human endothelial cells have not yet been reported. Basic FGF bound to HOME cells. The number of low-affinity binding sites was found to be variable. Washing the cells with 2 M phosphate-buffered saline removed completely {sup 125}I-bFGF bound to low-affinity binding sites but decreased also the high-affinity binding. The majority of the surface-bound {sup 125}I-bFGF was removed by washing the cells with acetic acid buffer at pH 3. At this temperature, degradation of the internalized ligand was followed after 1 hour by the appearance of three major bands of 15,000 10,000, and 8,000 Da and was inhibited by chloroquine. These results demonstrated two classes of binding sites for bFGF in HOME cells; the number of high-affinity binding sites being larger than the number reported for bovine capillary endothelial cells. The intracellular processing of bFGF in HOME cells seems to be different from that of heparin binding growth factor-1 in murine lung capillary endothelial cells and of eye-derived growth factor-1 in Chinese hamster fibroblasts.

  16. Internalization and re-expression of antigens of human melanoma cells following exposure to monoclonal antibody

    SciTech Connect

    Wang, B.S.; Lumanglas, A.L.; Silva, J.; Ruszala-Mallon, V.; Durr, F.E.

    1987-04-15

    Modulation of the surface membrane of human Sk-Mel-28 melanoma cells by monoclonal antibody (MoAb) 96.5 recognizing p97 determinants was examined using direct radioimmunoassay and indirect fluorescent antibody-staining techniques. It was determined that the majority of /sup 111/In-labeled antibody that remained associated with cells after a 24-hr incubation at 37 degrees C had been internalized because MoAb 96.5 was no longer visible on the cell surface. A second treatment of these cells with the same antibody 24 hr later not only increased the cell-associated radioactivity, reflecting an increase of total antibody bound, but also rendered these cells membrane immunofluorescent again, indicating the re-expression of surface antigens. Autoradiographs of the electrophoretically analyzed membrane components of Sk-Mel-28 cells further demonstrated the appearance of newly synthesized 97-kDa proteins that were immunoprecipitable with MoAb 96.5. Taken together, the present findings suggest that p97 antigens undergo endocytosis in Sk-Mel-28 cells following exposure to MoAb 96.5. However, the same antigens were regenerated and expressed on the cell surface within a period of 24 hr. The re-expression of tumor cell surface antigen following initial internalization of the MoAb-antigen complex may have implications for diagnosis and therapy.

  17. 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.

  18. A light and electron microscope study on the origin of Foà-Kurloff cells.

    PubMed

    Kittas, C; Parsons, M A; Henry, L

    1979-06-01

    Numerous Foà-Kurloff (FK) cells have been found in the circulation, spleen, bone marrow, thymus and placenta of guinea pigs under endogenous or exogenous oestrogenic stimulation. The origin of these cells is obscure. In the present experiment, the distribution of FK cells in organs other than those stated above was studied following gonadectomy and hexoestrol administration in guinea pigs of both sexes for either 1 or 10 weeks. More FK cells than those expected from the vascularity of the organ were found in the liver and lungs of male and female animals. The number of FK cells in these organs was larger after the long-term treatment with hexoestrol and it was accompanied by a significant decrease in the number of Kupffer cells of the liver. The latter observation is in contrast to previous reports of increased numbers of Kupffer cells in the liver of oestrogen-receiving mice, a species not producing FK cells. These observations suggest a relation between the two types of cells. No transformation of mature Kupffer cells into FK cells was seen with the electron microscope. However, other findings suggest that both Kupffer cells and FK cells may well be derived from a common precursor of MPS in the bone marrow.

  19. 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

  20. 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

    2016-01-01

    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

  1. 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

  2. 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.

  3. 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.

  4. Structural Features Facilitating Tumor Cell Targeting and Internalization by Bleomycin and Its Disaccharide

    PubMed Central

    2016-01-01

    We have shown previously that the bleomycin (BLM) carbohydrate moiety can recapitulate the tumor cell targeting effects of the entire BLM molecule, that BLM itself is modular in nature consisting of a DNA-cleaving aglycone which is delivered selectively to the interior of tumor cells by its carbohydrate moiety, and that there are disaccharides structurally related to the BLM disaccharide which are more efficient than the natural disaccharide at tumor cell targeting/uptake. Because BLM sugars can deliver molecular cargoes selectively to tumor cells, and thus potentially form the basis for a novel antitumor strategy, it seemed important to consider additional structural features capable of affecting the efficiency of tumor cell recognition and delivery. These included the effects of sugar polyvalency and net charge (at physiological pH) on tumor cell recognition, internalization, and trafficking. Since these parameters have been shown to affect cell surface recognition, internalization, and distribution in other contexts, this study has sought to define the effects of these structural features on tumor cell recognition by bleomycin and its disaccharide. We demonstrate that both can have a significant effect on tumor cell binding/internalization, and present data which suggests that the metal ions normally bound by bleomycin following clinical administration may significantly contribute to the efficiency of tumor cell uptake, in addition to their characterized function in DNA cleavage. A BLM disaccharide-Cy5** conjugate incorporating the positively charged dipeptide d-Lys-d-Lys was found to associate with both the mitochondria and the nuclear envelope of DU145 cells, suggesting possible cellular targets for BLM disaccharide–cytotoxin conjugates. PMID:25905565

  5. Internalization of Escherichia Coli O157:H7 by Bovine Rectal Epithelial Cells

    PubMed Central

    Sheng, Haiqing; Wang, Jing; Lim, Ji Youn; Davitt, Christine; Minnich, Scott A.; Hovde, Carolyn J.

    2011-01-01

    Escherichia coli O157:H7 (O157) causes human diarrheal disease and healthy cattle are its primary reservoir. O157 colonize the bovine epithelial mucosa at the recto-anal junction (RAJ). Previous studies show that O157 at this site are not eliminated by aggressive interventions including applications of O157-specific lytic bacteriophages and other bactericidal agents. We hypothesize that some O157 at the RAJ mucosa are protected from these killing agents by host cell internalization. To test this hypothesis, rectal biopsies from O157 culture positive and negative cattle were analyzed by fluorescent microscopy and subjected to gentamicin protection assays. GFP-labeled bacteria were found located deep within the tissue crypts and a small number of O157 were recovered from rectal biopsies after gentamicin treatment. Primary bovine rectal epithelial (PBRE) cell cultures were incubated with O157 and subjected to gentamicin protection assays. Strains ATCC 43895, 43894, Sakai, and WSU180 entered the PBRE cells with different levels of efficiency ranging from 0.18 to 19.38% of the inocula. Intracellular bacteria were confirmed to be within membrane-bounded vacuoles by electron microscopy. Cytochalasin D curtailed internalization of O157 indicating internalization was dependent on eukaryotic microfilament assembly. Strain ATCC 43895 exhibited the highest efficiency of internalization and survived for at least 24 h within PBRE cells. Deletion mutation of intimin or its receptor in ATCC 43895 did not reduce bacterial internalization. This strain produced more biofilm than the others tested. Retrospective analysis of cattle challenged with two O157 strains, showed ATCC 43895, the most efficient at host cell internalization, was most persistent. PMID:21687423

  6. Internal dynamics of a living cell nucleus investigated by dynamic light scattering

    NASA Astrophysics Data System (ADS)

    Suissa, M.; Place, C.; Goillot, E.; Freyssingeas, E.

    2008-08-01

    Recent progresses in cellular biology have shown that the nucleus of a living cell is a structured integration of many functional domains with a complex spatial organization. This organization, as well as molecular and biochemical processes, is time regulated. In the past years many investigations have been performed using fluorescent microscopy techniques to study the internal dynamics of the nucleus of a living cell. These investigations, however, have never focussed on the global internal dynamics of the nucleus, which is still unknown. In this article we present an original light scattering experimental device that we built to investigate this dynamics during biological processes. By means of this experimental set-up, we investigated the global dynamics of the nucleus of a living cell treated with a DNA replication inhibitor. This dynamics presents different and independent kinds of relaxation well separated in time that vary as a function of the cell cycle phases.

  7. Impact of sub-cell internal luminescence yields on energy conversion efficiencies of tandem solar cells: A design principle

    SciTech Connect

    Zhu, Lin Kim, Changsu; Yoshita, Masahiro; Chen, Shaoqiang; Sato, Shintaroh; Mochizuki, Toshimitsu; Akiyama, Hidefumi; Kanemitsu, Yoshihiko

    2014-01-20

    To develop a realistic design principle, we calculated the maximum conversion efficiency η{sub sc} and optimized sub-cell band-gap energies E{sub g} in double-junction tandem solar cells via a detailed-balance theory, paying particular attention to their dependence on internal luminescence quantum yields y{sub int} of the top and bottom sub-cell materials. A strong drop in the maximum η{sub sc} occurs when y{sub int} slightly drops from 1 to 0.9, where the drop in y{sub int} of the bottom cell causes a stronger effect than that of the top cell. For low values of y{sub int}, the maximum η{sub sc} has a simple logarithmic dependence on the geometric mean of the two sub-cells'y{sub int}.

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

    PubMed

    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

    2016-07-01

    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

  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. [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

  11. 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.

  12. A short story of Victor Hensen and a cell of the internal ear.

    PubMed

    Raica, M

    2012-01-01

    Cells of Hensen of the internal ear are known from more than 100 years and investigations on their function(s) are still waiting for an answer. They were first described by the German scientist from Kiel, Christian Andreas Victor Hensen. This short historical review gives some details about the life, scientific activity and perspectives opened by the work of Victor Hensen. PMID:23188454

  13. Internal noise induced pattern formation and spatial coherence resonance for calcium signals of diffusively coupled cells

    NASA Astrophysics Data System (ADS)

    Wang, Maosheng; Sun, Runzhi; Huang, Wanxia; Tu, Yubing

    2014-01-01

    The effects of internal noise in a square-lattice Höfer calcium oscillation system have been studied numerically in the context of chemical Langevin equations. It was found that spatial pattern can be induced by internal noise and, interestingly, an optimal internal noise strength (or optimal cell size) exists which maximizes the spatial coherence of pattern, indicating the occurrence of spatial coherence resonance. The effects of control parameter and coupling strength on system’s spatial coherence have also been investigated. We found that larger internal noise strength is needed to induce spatial pattern for a small control parameter or a stronger coupling strength, and spatial coherence can be enhanced by coupling.

  14. 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

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

    PubMed Central

    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. PMID:23982586

  16. 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.

  17. General overview of the Seventh International Symposium on Stem Cell Therapy and Cardiovascular Innovations.

    PubMed

    Gutiérrez, Enrique; Sanz-Ruiz, Ricardo; Alvarez, Eugenia Vázquez; Villa, Adolfo; Fernández, Lucia; Vázquez, Sandra; Lorenzo, José; Fernández-Santos, Eugenia; Sánchez, Pedro L; Fernández-Avilés, Francisco

    2011-04-01

    The Seventh International Symposium on Stem Cell Therapy and Cardiovascular Innovations was held in Madrid on the 6th and 7th of May 2010. Gathering for the seventh consecutive year the most relevant researchers and opinion leaders on cardiovascular cell therapy, it has become the most important worldwide event on this field. A comprehensive review of the last developments on cell therapy, surgery for heart failure and tissue engineering was made, and the results of three clinical trials were reported. The Symposium was dedicated to the memory of Professor Helmut Drexler.

  18. 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.

  19. 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.

  20. Mechanistic aspects of fluorescent gold nanocluster internalization by live HeLa cells.

    PubMed

    Yang, Linxiao; Shang, Li; Nienhaus, G Ulrich

    2013-02-21

    We have studied cellular uptake of ultrasmall fluorescent gold nanoclusters (AuNCs) by HeLa cells by confocal fluorescence microscopy in combination with quantitative image analysis. Water solubilized, lipoic acid-protected AuNCs, which had an overall hydrodynamic diameter of 3.3 nm and emitted fluorescence in the near-infrared region at ∼700 nm, were observed to accumulate on the cell membrane prior to internalization. The internalization mechanisms were analyzed using inhibitors known to interfere with specific pathways. Cellular uptake of AuNCs is energy-dependent and involves multiple mechanisms: clathrin-mediated endocytosis and macropinocytosis appear to play a significant role, whereas the caveolin-mediated pathway contributes only to a lesser extent. Co-labeling of different cell organelles showed that intracellular trafficking of AuNCs mainly follows through endosomal pathways. The AuNCs were ultimately transferred to lysosomes; they were completely excluded from the nucleus even after 24 h.

  1. 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.

  2. 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.

  3. 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.

  4. Carbon nanotubes enhance the internalization of drugs by cancer cells and decrease their chemoresistance to cytostatics.

    PubMed

    Mahmood, M; Xu, Y; Dantuluri, V; Mustafa, T; Zhang, Y; Karmakar, A; Casciano, D; Ali, S; Biris, A

    2013-02-01

    Etoposide is a semisynthetic, chemotherapeutic drug widely recommended to treat an extensive range of human cancers. Our studies indicate that, while etoposide is capable of killing human cancer cells, exposure to single-walled carbon nanotubes (SWCNTs) and etoposide results in enhanced cell death that appears to be synergistic and not merely additive. In this study, we used high pressure liquid chromatography and mass spectrometry to quantify the internal effective dose of etoposide when the human pancreatic cancer cell (PANC-1) was exposed to the combination of these agents. Our results unequivocally indicate that SWCNTs improve etoposide uptake and increase its capacity to kill cancer cells. We suggest that a combination of SWCNTs and etoposide may prove to be a more efficient chemotherapeutic protocol, especially because of the potential to lower toxic drug doses to levels that may be useful in decreasing adverse side effects, as well as in lowering the probability of inducing chemoresistance in exposed cancer cells.

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

    PubMed Central

    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

    2015-01-01

    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. PMID:26477718

  6. 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.

  7. 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.

  8. Carbon nanotubes enhance the internalization of drugs by cancer cells and decrease their chemoresistance to cytostatics

    NASA Astrophysics Data System (ADS)

    Mahmood, M.; Xu, Y.; Dantuluri, V.; Mustafa, T.; Zhang, Y.; Karmakar, A.; Casciano, D.; Ali, S.; Biris, A.

    2013-02-01

    Etoposide is a semisynthetic, chemotherapeutic drug widely recommended to treat an extensive range of human cancers. Our studies indicate that, while etoposide is capable of killing human cancer cells, exposure to single-walled carbon nanotubes (SWCNTs) and etoposide results in enhanced cell death that appears to be synergistic and not merely additive. In this study, we used high pressure liquid chromatography and mass spectrometry to quantify the internal effective dose of etoposide when the human pancreatic cancer cell (PANC-1) was exposed to the combination of these agents. Our results unequivocally indicate that SWCNTs improve etoposide uptake and increase its capacity to kill cancer cells. We suggest that a combination of SWCNTs and etoposide may prove to be a more efficient chemotherapeutic protocol, especially because of the potential to lower toxic drug doses to levels that may be useful in decreasing adverse side effects, as well as in lowering the probability of inducing chemoresistance in exposed cancer cells.

  9. 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

    2015-01-01

    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. PMID:26477718

  10. Uniaxial deformation of open-cell aluminum foam: the role of internal damage

    SciTech Connect

    San Marchi, C.; Despois, J.-F.; Mortensen, A

    2004-06-07

    Internal damage accumulation is measured and shown to play a role in the mechanical response of replicated pure Al and Al-12Si open-cell foams. This internal damage is quantified by measuring the reduction in the foam's stiffness with strain. The brittle Si second phase fractures during deformation of Al-12Si foam, resulting in damage accumulation rates an order of magnitude greater than for pure Al foam. Elementary damage mechanics is used to relate the measured rate of damage accumulation to the foam's tensile failure strain. The analysis and experimental results highlight in particular the strong embrittling influence of brittle second phases within the foam, such as Si.

  11. Dvr1 transfers left-right asymmetric signals from Kupffer's vesicle to lateral plate mesoderm in zebrafish.

    PubMed

    Peterson, Annita G; Wang, Xinghao; Yost, H Joseph

    2013-10-01

    An early step in establishing left-right (LR) symmetry in zebrafish is the generation of asymmetric fluid flow by Kupffer's vesicle (KV). As a result of fluid flow, a signal is generated and propagated from the KV to the left lateral plate mesoderm, activating a transcriptional response of Nodal expression in the left lateral plate mesoderm (LPM). The mechanisms and molecules that aid in this transfer of information from the KV to the left LPM are still not clear. Here we provide several lines of evidence demonstrating a role for a member of the TGFβ family member, Dvr1, a zebrafish Vg1 ortholog. Dvr1 is expressed bilaterally between the KV and the LPM. Knockdown of Dvr1 by morpholino causes dramatically reduced or absent expression of southpaw (spaw, a Nodal homolog), in LPM, and corresponding loss of downstream Lefty (lft1 and lft) expression, and aberrant brain and heart LR patterning. Dvr1 morphant embryos have normal KV morphology and function, normal expression of southpaw (spaw) and charon (cha) in the peri-KV region and normal expression of a variety of LPM markers in LPM. Additionally, Dvr1 knockdown does not alter the capability of LPM to respond to signals that initiate and propagate spaw expression. Co-injection experiments in Xenopus and zebrafish indicate that Dvr1 and Spaw can enhance each other's ability to activate the Nodal response pathway and co-immunoprecipitation experiments reveal differential relationships among activators and inhibitors in this pathway. These results indicate that Dvr1 is responsible for enabling the transfer of a left-right signal from KV to the LPM. PMID:23791819

  12. Dvr1 transfers left-right asymmetric signals from Kupffer's vesicle to lateral plate mesoderm in zebrafish.

    PubMed

    Peterson, Annita G; Wang, Xinghao; Yost, H Joseph

    2013-10-01

    An early step in establishing left-right (LR) symmetry in zebrafish is the generation of asymmetric fluid flow by Kupffer's vesicle (KV). As a result of fluid flow, a signal is generated and propagated from the KV to the left lateral plate mesoderm, activating a transcriptional response of Nodal expression in the left lateral plate mesoderm (LPM). The mechanisms and molecules that aid in this transfer of information from the KV to the left LPM are still not clear. Here we provide several lines of evidence demonstrating a role for a member of the TGFβ family member, Dvr1, a zebrafish Vg1 ortholog. Dvr1 is expressed bilaterally between the KV and the LPM. Knockdown of Dvr1 by morpholino causes dramatically reduced or absent expression of southpaw (spaw, a Nodal homolog), in LPM, and corresponding loss of downstream Lefty (lft1 and lft) expression, and aberrant brain and heart LR patterning. Dvr1 morphant embryos have normal KV morphology and function, normal expression of southpaw (spaw) and charon (cha) in the peri-KV region and normal expression of a variety of LPM markers in LPM. Additionally, Dvr1 knockdown does not alter the capability of LPM to respond to signals that initiate and propagate spaw expression. Co-injection experiments in Xenopus and zebrafish indicate that Dvr1 and Spaw can enhance each other's ability to activate the Nodal response pathway and co-immunoprecipitation experiments reveal differential relationships among activators and inhibitors in this pathway. These results indicate that Dvr1 is responsible for enabling the transfer of a left-right signal from KV to the LPM.

  13. The activation of the sodium pump in pig red blood cells by internal and external cations.

    PubMed

    Brand, S C; Whittam, R

    1985-05-30

    A study has been made with pig red blood cells of the activation of the sodium pump by internal and external cations. Cell Na and K concentrations were altered using a PCMBS cation loading procedure. The procedure was characterised for resultant ionic conditions, maintenance of ATP levels and fragility. The activation of the sodium pump by external K was measured in cells suspended in choline (Na-free) solutions. External Cs was used as a substitute for K and elicited lower rates of pump activity. Both the Vmax and apparent Km for 42K influx and 134Cs influx increased as internal Na concentration was raised (within the non-saturating range). Vmax/apparent Km ratios for cation influx were constant. Raising external Cs concentration exerted a similar influence on pump activation by internal Na: both the maximum pump velocity and the apparent Na-site dissociation constant (K'Na) increased. The results provide evidence for a transmembrane connection between cation binding sites on opposite faces of the membrane and are consistent with a consecutive model for the sodium pump in pig red blood cells. PMID:2581622

  14. RNAi Screen Reveals an Abl Kinase-Dependent Host Cell Pathway Involved in Pseudomonas aeruginosa Internalization

    PubMed Central

    Pielage, Julia F.; Powell, Kimberly R.; Kalman, Daniel; Engel, Joanne N.

    2008-01-01

    Internalization of the pathogenic bacterium Pseudomonas aeruginosa by non-phagocytic cells is promoted by rearrangements of the actin cytoskeleton, but the host pathways usurped by this bacterium are not clearly understood. We used RNAi-mediated gene inactivation of ∼80 genes known to regulate the actin cytoskeleton in Drosophila S2 cells to identify host molecules essential for entry of P. aeruginosa. This work revealed Abl tyrosine kinase, the adaptor protein Crk, the small GTPases Rac1 and Cdc42, and p21-activated kinase as components of a host signaling pathway that leads to internalization of P. aeruginosa. Using a variety of complementary approaches, we validated the role of this pathway in mammalian cells. Remarkably, ExoS and ExoT, type III secreted toxins of P. aeruginosa, target this pathway by interfering with GTPase function and, in the case of ExoT, by abrogating P. aeruginosa–induced Abl-dependent Crk phosphorylation. Altogether, this work reveals that P. aeruginosa utilizes the Abl pathway for entering host cells and reveals unexpected complexity by which the P. aeruginosa type III secretion system modulates this internalization pathway. Our results furthermore demonstrate the applicability of using RNAi screens to identify host signaling cascades usurped by microbial pathogens that may be potential targets for novel therapies directed against treatment of antibiotic-resistant infections. PMID:18369477

  15. 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.

  16. Role of the Tet38 Efflux Pump in Staphylococcus aureus Internalization and Survival in Epithelial Cells

    PubMed Central

    Truong-Bolduc, Q. C.; Bolduc, G. R.; Medeiros, H.; Vyas, J. M.; Wang, Y.

    2015-01-01

    We previously identified the protein Tet38 as a chromosomally encoded efflux pump of Staphylococcus aureus that confers resistance to tetracycline and certain unsaturated fatty acids. Tet38 also contributes to mouse skin colonization. In this study, we discovered a novel regulator of tet38, named tetracycline regulator 21 (TetR21), that bound specifically to the tet38 promoter and repressed pump expression. A ΔtetR21 mutant showed a 5-fold increase in tet38 transcripts and an 8-fold increase in resistance to tetracycline and fatty acids. The global regulator MgrA bound to the tetR21 promoter and indirectly repressed the expression of tet38. To further assess the full role of Tet38 in S. aureus adaptability, we tested its effect on host cell invasion using A549 (lung) and HMEC-1 (heart) cell lines. We used S. aureus RN6390, its Δtet38, ΔtetR21, and ΔmgrA mutants, and a Δtet38 ΔtetR21 double mutant. After 2 h of contact, the Δtet38 mutant was internalized in 6-fold-lower numbers than RN6390 in A549 and HMEC-1 cells, and the ΔtetR21 mutant was internalized in 2-fold-higher numbers than RN6390. A slight increase of 1.5-fold in internalization was found for the ΔmgrA mutant. The growth patterns of RN6390 and the ΔmgrA and ΔtetR21 mutants within A549 cells were similar, while no growth was observed for the Δtet38 mutant. These data indicate that the Tet38 efflux pump is regulated by TetR21 and contributes to the ability of S. aureus to internalize and replicate within epithelial cells. PMID:26324534

  17. Somites in zebrafish doubly mutant for knypek and trilobite form without internal mesenchymal cells or compaction.

    PubMed

    Henry, C A; Hall, L A; Burr Hille, M; Solnica-Krezel, L; Cooper, M S

    2000-09-01

    In vertebrates, paraxial mesoderm is partitioned into repeating units called somites. It is thought that the mechanical forces arising from compaction of the presumptive internal cells of prospective somites cause them to detach from the unsegmented presomitic mesoderm [1-3]. To determine how prospective somites physically segregate from each other, we used time-lapse microscopy to analyze the mechanics underlying early somitogenesis in wild-type zebrafish and in the mutants trilobite(m209) (tri), knypek(m119) (kny), and kny;tri, which are defective in convergent extension during gastrulation. Formation of somite boundaries in all of these embryos involved segregation, local alignment, and cell-shape changes of presumptive epitheloid border cells along nascent intersomitic boundaries. Although kny;tri somites formed without convergence of the presomitic mesoderm and were composed of only two cells in their anteroposterior (AP) dimension, they still exhibited AP intrasegmental polarity. Furthermore, morphogenesis of somite boundaries in these embryos proceeded in a manner similar to that in wild-type embryos. Thus, intersomitic boundary formation in zebrafish involves short-range movements of presumptive border cells that do not require mechanical forces generated by internal cells or compaction of the presomitic mesoderm. PMID:10996075

  18. Binding and internalization of nerve growth factor by PC12 cells

    SciTech Connect

    Kasaian, M.T.

    1987-01-01

    The interaction of nerve growth factor (NGF) with its cell surface receptors has been studied using both fluorescent- and radio-labelled NGF. The fluorescence studies were done by flow cytometry, and gave information about the concentration dependence and time course of NGF binding to rat pheochromocytoma cells (PC12) and human melanoma cells (A875). /sup 125/I-NGF was used to study the fate of NGF in PC12 cells following its association with cell surface receptors. Variations of the PC12 binding assay were used to distinguish ligand bound to fast and slowly dissociating receptors at the cell surface, internalized ligand, and cytoskeletally-associated NGF. Ligand uptake into each of these pools was followed in untreated cells, as well as in cells exposed to colchicine and/or cytochalasin B to disrupt the cytoskeleton. NGF degradation was also followed in these cells, and chloroquine was used to inhibit this process. In a separate project, NGF activity was assayed in samples of human amniotic fluid and cerebrospinal fluid (CSF). A range of activities was found in these samples, with the CSF samples containing somewhat more activity than the amniotic fluid samples.

  19. A homogeneous fluorescence-based method to measure antibody internalization in tumor cells.

    PubMed

    Gong, Haibiao; Urlacher, Teresa

    2015-01-15

    We have developed a simple fluorescence-based method to monitor antibody internalization. Panitumumab was dual-labeled with the fluorophore IRDye 800CW and quencher IRDye QC-1 to yield the biomolecular probe Pan800QC. The fluorescence of IRDye 800CW is quenched by IRDye QC-1 on the same intact antibody. After incubation with epidermal growth factor receptor (EGFR)-expressing cells, internalization of Pan800QC was detected by an increase in fluorescence signal due to enzymatic digestion of the antibody and separation of IRDye 800CW and IRDye QC-1. By optimizing reaction conditions, a signal-to-background ratio of 8.5 was obtained. This homogeneous assay can be applied in the characterization and screening of internalizing antibodies. PMID:25245185

  20. 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). PMID:26921839

  1. 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).

  2. Binding of Host Factors Influences Internalization and Intracellular Trafficking of Streptococcus uberis in Bovine Mammary Epithelial Cells

    PubMed Central

    Almeida, Raul A.; Dunlap, John R.; Oliver, Stephen P.

    2010-01-01

    We showed that internalization of Streptococcus uberis into bovine mammary epithelial cells occurred through receptor- (RME) and caveolae-mediated endocytosis (CME). We reported also that treatment of S. uberis with host proteins including lactoferrin (LF) enhanced its internalization into host cells. Since the underlying mechanism(s) involved in such enhancement was unknown we investigated if preincubation of S. uberis with host proteins drives internalization of this pathogen into host cells through CME. Thus, experiments involving coculture of collagen-, fibronectin-, and LF-pretreated S. uberis with bovine mammary epithelial cells treated with RME and CME inhibitors were conducted. Results showed that internalization of host proteins-pretreated S. uberis into mammary epithelial cells treated with RME inhibitors was higher than that of untreated controls. These results suggest that pretreatment with selected host proteins commits S. uberis to CME, thus avoiding intracellular bactericidal mechanisms and allowing its persistence into bovine mammary epithelial cells. PMID:20614000

  3. 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. PMID:20971701

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

    PubMed Central

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

    2010-01-01

    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. PMID:20971701

  5. 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. PMID:27653490

  6. General overview of the Sixth International Symposium on Stem Cell Therapy and Cardiovascular Innovations.

    PubMed

    Vázquez-Alvarez, Ma Eugenia; Sanz-Ruiz, Ricardo; Gutiérrez, Enrique; Villa, Adolfo; Fernández, Ma Eugenia; Vázquez, Sandra; José Lorenzo, Ma; Fernández, Lucía; Pascual, Isaac; Sánchez, Pedro L; Fernández-Avilés, Francisco

    2010-02-01

    Being one of the main stem cell therapy meetings of the year, the Sixth International Symposium on Stem Cell Therapy and Cardiovascular Innovations was held on April 23rd-24th, 2009, at the Auditorium of the High Council of Scientific Research of Spain (CSIC) in Madrid. Gathering the most prestigious basic researchers and clinical experts in the field of cardiovascular regenerative medicine, the aim of the meeting was to discuss the available evidence and the recent contributions from preclinical investigators, cardiologists, and cardiac surgeons in a participative translational fashion. The role of young "clinician scientists" was reinforced with a special poster session and three awards. The main conclusions of the symposium were (1) that standardization, larger clinical trials, and true translational research are needed, and (2) that new-allogeneic-stem cell products, biotechnological devices, and cell-based bioartificial organs are potentially exciting options for the future. PMID:20560031

  7. 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.

  8. 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.

  9. International Society for Cell and Gene Therapy of Cancer 2009 Annual Meeting held in Cork, Ireland.

    PubMed

    Guinn, Barbara; Casey, Garrett; Möller, Mecker G; Kasahara, Noriyuki; O'Sullivan, Gerald C; Peng, Kah-Whye; Tangney, Mark

    2010-01-01

    The International Society for Cell and Gene Therapy (ISCGT) of Cancer annual meeting was held from September 2 through September 4, 2009, in Cork, Ireland ( www.iscgt2009.com ). The conference was held in conjunction with the Irish Society for Gene and Cell Therapy third annual meeting, and brought together scientists and clinicians from around the world in a country developing its knowledge economy. Next year's ISCGT meeting will be held in Doha, the capital of Qatar ( www.iscgt.net ), from September 27 through September 29, 2010.

  10. 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.

  11. Mechanistic aspects of fluorescent gold nanocluster internalization by live HeLa cells

    NASA Astrophysics Data System (ADS)

    Yang, Linxiao; Shang, Li; Nienhaus, G. Ulrich

    2013-01-01

    We have studied cellular uptake of ultrasmall fluorescent gold nanoclusters (AuNCs) by HeLa cells by confocal fluorescence microscopy in combination with quantitative image analysis. Water solubilized, lipoic acid-protected AuNCs, which had an overall hydrodynamic diameter of 3.3 nm and emitted fluorescence in the near-infrared region at ~700 nm, were observed to accumulate on the cell membrane prior to internalization. The internalization mechanisms were analyzed using inhibitors known to interfere with specific pathways. Cellular uptake of AuNCs is energy-dependent and involves multiple mechanisms: clathrin-mediated endocytosis and macropinocytosis appear to play a significant role, whereas the caveolin-mediated pathway contributes only to a lesser extent. Co-labeling of different cell organelles showed that intracellular trafficking of AuNCs mainly follows through endosomal pathways. The AuNCs were ultimately transferred to lysosomes; they were completely excluded from the nucleus even after 24 h.We have studied cellular uptake of ultrasmall fluorescent gold nanoclusters (AuNCs) by HeLa cells by confocal fluorescence microscopy in combination with quantitative image analysis. Water solubilized, lipoic acid-protected AuNCs, which had an overall hydrodynamic diameter of 3.3 nm and emitted fluorescence in the near-infrared region at ~700 nm, were observed to accumulate on the cell membrane prior to internalization. The internalization mechanisms were analyzed using inhibitors known to interfere with specific pathways. Cellular uptake of AuNCs is energy-dependent and involves multiple mechanisms: clathrin-mediated endocytosis and macropinocytosis appear to play a significant role, whereas the caveolin-mediated pathway contributes only to a lesser extent. Co-labeling of different cell organelles showed that intracellular trafficking of AuNCs mainly follows through endosomal pathways. The AuNCs were ultimately transferred to lysosomes; they were completely excluded

  12. Detection of single photoluminescent diamond nanoparticles in cells and study of the internalization pathway.

    PubMed

    Faklaris, Orestis; Garrot, Damien; Joshi, Vandana; Druon, Frédéric; Boudou, Jean-Paul; Sauvage, Thierry; Georges, Patrick; Curmi, Patrick A; Treussart, François

    2008-12-01

    Diamond nanoparticles are promising photoluminescent probes for tracking intracellular processes, due to embedded, perfectly photostable color centers. In this work, the spontaneous internalization of such nanoparticles (diameter 25 nm) in HeLa cancer cells is investigated by confocal microscopy and time-resolved techniques. Nanoparticles are observed inside the cell cytoplasm at the single-particle and single-color-center level, assessed by time-correlation intensity measurements. Improvement of the nanoparticle signal-to-noise ratio inside the cell is achieved using a pulsed-excitation laser and time-resolved detection taking advantage of the long radiative lifetime of the color-center excited state as compared to cell autofluorescence. The internalization pathways are also investigated, with endosomal marking and colocalization analyses. The low colocalization ratio observed proves that nanodiamonds are not trapped in endosomes, a promising result in prospect of drug delivery by these nanoparticles. Low cytotoxicity of these nanoparticles in this cell line is also shown. PMID:18989862

  13. Mannosylation of Virus-Like Particles Enhances Internalization by Antigen Presenting Cells

    PubMed Central

    Al-Barwani, Farah; Young, Sarah L.; Baird, Margaret A.; Larsen, David S.; Ward, Vernon K.

    2014-01-01

    Internalization of peptides by antigen presenting cells is crucial for the initiation of the adaptive immune response. Mannosylation has been demonstrated to enhance antigen uptake through mannose receptors, leading to improved immune responses. In this study we test the effect of surface mannosylation of protein-based virus-like particles (VLP) derived from Rabbit hemorrhagic disease virus (RHDV) on uptake by murine and human antigen presenting cells. A monomannoside and a novel dimannoside were synthesized and successfully conjugated to RHDV VLP capsid protein, providing approximately 270 mannose groups on the surface of each virus particle. VLP conjugated to the mannoside or dimannoside exhibited significantly enhanced binding and internalization by murine dendritic cells, macrophages and B cells as well as human dendritic cells and macrophages. This uptake was inhibited by the inclusion of mannan as a specific inhibitor of mannose specific uptake, demonstrating that mannosylation of VLP targets mannose receptor-based uptake. Consistent with mannose receptor-based uptake, partial retargeting of the intracellular processing of RHDV VLP was observed, confirming that mannosylation of VLP provides both enhanced uptake and modified processing of associated antigens. PMID:25122183

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

    PubMed

    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. PMID:21135463

  15. Effects of the properties of short peptides conjugated with cell-penetrating peptides on their internalization into cells.

    PubMed

    Matsumoto, Ryo; Okochi, Mina; Shimizu, Kazunori; Kanie, Kei; Kato, Ryuji; Honda, Hiroyuki

    2015-01-01

    Peptides, especially intracellular functional peptides that can play a particular role inside a cell, have attracted attention as promising materials to control cell fate. However, hydrophilic materials like peptides are difficult for cells to internalize. Therefore, the screening and design of intracellular functional peptides are more difficult than that of extracellular ones. An effective high-throughput screening system for intracellular functional peptides has not been reported. Here, we demonstrate a novel peptide array system for screening intracellular functional peptides, in which both cell-penetrating peptide (CPP) domain and photo-cleavable linkers are used. By using this screening system, we determined how the cellular uptake properties of CPP-conjugated peptides varied depending on the properties of the conjugated peptides. We found that the internalization ability of CPP-conjugated peptides varied greatly depending on the property of the conjugated peptides, and anionic peptides drastically decreased the uptake ability. We summarized our data in a scatter diagram that plots hydrophobicity versus isoelectric point (pI) of conjugated peptides. These results define a peptide library suitable for screening of intracellular functional peptides. Thus, our system, including the diagram, is a promising tool for searching biological active molecules such as peptide-based drugs. PMID:26256261

  16. International Stem Cell Collaboration: How Disparate Policies between the United States and the United Kingdom Impact Research

    PubMed Central

    Solnick, Rachel E.; Ecklund, Elaine Howard; Matthews, Kirstin R. W.

    2011-01-01

    As the scientific community globalizes, it is increasingly important to understand the effects of international collaboration on the quality and quantity of research produced. While it is generally assumed that international collaboration enhances the quality of research, this phenomenon is not well examined. Stem cell research is unique in that it is both politically charged and a research area that often generates international collaborations, making it an ideal case through which to examine international collaborations. Furthermore, with promising medical applications, the research area is dynamic and responsive to a globalizing science environment. Thus, studying international collaborations in stem cell research elucidates the role of existing international networks in promoting quality research, as well as the effects that disparate national policies might have on research. This study examined the impact of collaboration on publication significance in the United States and the United Kingdom, world leaders in stem cell research with disparate policies. We reviewed publications by US and UK authors from 2008, along with their citation rates and the political factors that may have contributed to the number of international collaborations. The data demonstrated that international collaborations significantly increased an article's impact for UK and US investigators. While this applied to UK authors whether they were corresponding or secondary, this effect was most significant for US authors who were corresponding authors. While the UK exhibited a higher proportion of international publications than the US, this difference was consistent with overall trends in international scientific collaboration. The findings suggested that national stem cell policy differences and regulatory mechanisms driving international stem cell research in the US and UK did not affect the frequency of international collaborations, or even the countries with which the US and UK most

  17. 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.

  18. 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).

  19. Cellular Internalization Mechanism and Intracellular Trafficking of Filamentous M13 Phages Displaying a Cell-Penetrating Transbody and TAT Peptide

    PubMed Central

    Shin, Seung-Min; Pham, Chuong D.; Choi, Dong-Ki; Kwon, Myung-Hee; Kim, Yong-Sung

    2012-01-01

    Cellular internalization of bacteriophage by surface-displayed cell penetrating peptides has been reported, though the underlying mechanism remains elusive. Here we describe in detail the internalization mechanism and intracellular trafficking and stability of filamentous M13 phages, the cellular entry of which is mediated by surface-displayed cell-penetrating light chain variable domain 3D8 VL transbody (3D8 VL-M13) or TAT peptide (TAT-M13). Recombinant 3D8 VL-M13 and TAT-M13 phages were efficiently internalized into living mammalian cells via physiologically relevant, energy-dependent endocytosis and were recovered from the cells in their infective form with the yield of 3D8 VL-M13 being higher (0.005∼0.01%) than that of TAT-M13 (0.001∼0.005%). Biochemical and genetic studies revealed that 3D8 VL-M13 was internalized principally by caveolae-mediated endocytosis via interaction with heparan sulfate proteoglycans as cell surface receptors, whereas TAT-M13 was internalized by clathrin- and caveolae-mediated endocytosis utilizing chondroitin sulfate proteoglycans as cell surface receptors, suggesting that phage internalization occurs by physiological endocytotic mechanism through specific cell surface receptors rather than non-specific transcytotic pathways. Internalized 3D8 VL-M13 phages routed to the cytosol and remained stable for more than 18 h without further trafficking to other subcellular compartments, whereas TAT-M13 phages routed to several subcellular compartments before being degraded in lysosomes even after 2 h of internalization. Our results suggest that the internalizing mechanism and intracellular trafficking of filamentous M13 bacteriophages largely follow the attributes of the displayed cell-penetrating moiety. Efficient internalization and cytosolic localization of 3D8 VL transbody-displayed phages will provide a useful tool for intracellular delivery of polar macromolecules such as proteins, peptides, and siRNAs. PMID:23251631

  20. Co-migration and internalization of transferrin and its receptor on K562 cells

    PubMed Central

    1983-01-01

    The incorporation of iron into human cells involves the binding of diferric transferrin to a specific cell surface receptor. We studied the process of endocytosis in K562, a human erythroid cell line, by using tetramethylrhodamine isothiocyanate-labeled transferrin (TRITC- transferrin) and fluorescein isothiocyanate-labeled Fab fragments of goat antireceptor IgG preparation (FITC-Fab-antitransferrin receptor antibody). Because the antireceptor antibody and transferrin bind to different sites on the transferrin receptor molecule it was possible to simultaneously and independently follow ligand and receptor. At 4 degrees C, the binding of TRITC-transferrin or FITC-Fab antitransferrin receptor antibody exhibited diffuse membrane fluorescence. At 20 degrees C, the binding of TRITC-transferrin was followed by the rapid formation of aggregates. However, the FITC-Fab antitransferrin receptor did not show similar aggregation at 20 degrees C unless transferrin was present. In the presence of transferrin, the FITC-Fab antitransferrin receptor antibody formed aggregates at the same sites and within the same time period as TRITC transferrin, indicating co-migration. Although the diffuse surface staining of either label was removed by proteolysis, the larger aggregates were not susceptible to enzyme degradation, indicating that they were intracellular. The internal location of the aggregates was also demonstrated using permeabilized cells that had been preincubated with transferrin and fixed with 4% paraformaldehyde. These cells showed aggregated receptor in the interior of the cell when reacted with fluorescein-labeled antibody to the receptor. This indicated that the transferrin and the transferrin receptor co-internalize and migrate to the same structures within the cell. PMID:6309864

  1. Total internal reflection fluorescence (TIRF) microscopy. I. Modelling cell contact region fluorescence.

    PubMed

    Reichert, W M; Truskey, G A

    1990-06-01

    Total Internal Reflection Fluorescence (TIRF) is a powerful technique for visualizing focal and close contacts between the cell and the surface. Practical application of TIRF has been hampered by the lack of straightforward methods to calculate separation distances. The characteristic matrix theory of thin dielectric films was used to develop simple exponential approximations for the fluorescence excited in the cell-substratum contact region during a TIRF experiment. Two types of fluorescence were examined: fluorescently labeled cell membranes, and a fluorescent water-soluble dye. By neglecting the refractive index of the cell membrane, the fluorescence excited in the cell membrane was modelled by a single exponential function while the fluorescence in the membrane/substratum water gap followed a weighted sum of two exponentials. The error associated with neglecting the cell membrane for an incident angle of 70 degrees never exceeded 2.5%, regardless of the cell-substratum separation distance. Comparisons of approximated fluorescence intensities to more exact solutions of the fluorescence integrals for the three-phase model indicated that the approximations are accurate to about 1% for membrane/substratum gap thicknesses of less than 50 nm if the cytoplasmic and water gap refractive indices are known. The intrinsic error of this model in the determination of membrane/substratum separations was 10% as long as the uncertainties in the water gap and cytoplasmic refractive indices were less than 1%.

  2. Heparan Sulfate Proteoglycans Promote Telomerase Internalization and MHC Class II Presentation on Dendritic Cells.

    PubMed

    Galaine, Jeanne; Kellermann, Guillaume; Guillaume, Yves; Boidot, Romain; Picard, Emilie; Loyon, Romain; Queiroz, Lise; Boullerot, Laura; Beziaud, Laurent; Jary, Marine; Mansi, Laura; André, Claire; Lethier, Lydie; Ségal-Bendirdjian, Evelyne; Borg, Christophe; Godet, Yann; Adotévi, Olivier

    2016-09-01

    Telomerase is a prototype-shared tumor Ag and represents an attractive target for anticancer immunotherapy. We have previously described promiscuous and immunogenic HLA-DR-restricted peptides derived from human telomerase reverse transcriptase (hTERT) and referred as universal cancer peptide (UCP). In nonsmall cell lung cancer, the presence of spontaneous UCP-specific CD4 T cell responses increases the survival of chemotherapy-responding patients. However, the precise mechanisms of hTERT's uptake, processing, and presentation on MHC-II molecules to stimulate CD4 T cells are poorly understood. In this work, by using well-characterized UCP-specific CD4 T cell clones, we showed that hTERT processing and presentation on MHC-II involve both classical endolysosomal and nonclassical cytosolic pathways. Furthermore, to our knowledge, we demonstrated for the first time that hTERT's internalization by dendritic cells requires its interaction with surface heparan sulfate proteoglycans. Altogether, our findings provide a novel mechanism of tumor-specific CD4 T cell activation and will be useful for the development of novel cancer immunotherapies that harness CD4 T cells. PMID:27481844

  3. 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

  4. 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.

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

    PubMed

    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

  6. Internalization of Sambucus nigra agglutinins I and II in insect midgut CF-203 cells.

    PubMed

    Shahidi-Noghabi, Shahnaz; Van Damme, Els J M; De Vos, Winnok H; Smagghe, Guy

    2011-04-01

    In this project, the uptake mechanisms and localization of two lectins from Sambucus nigra, further referred to as S. nigra agglutinin (SNA)-I and SNA-II, into insect midgut CF-203 cells were studied. SNA-I is a chimeric lectin belonging to the class of ribosome-inactivating proteins, whereas SNA-II is a hololectin devoid of enzymatic activity. Internalization of the fluorescein isothiocyanate-labeled lectin was investigated using confocal microscopy. Both lectins were internalized into the cytoplasm of CF-203 cells at similar rates. Preexposure of the insect midgut cells to specific inhibitors of clathrin- and caveolae-mediated endocytosis resulted in an inhibition of lectin uptake in CF-203 cells and caspase-induced cytotoxicity caused by SNA-I and SNA-II, confirming the involvement of both endocytosis pathways. Further studies demonstrated that the uptake mechanism(s) for both lectins required phosphoinositide 3-kinases, but did not depend on the actin cytoskeleton. Since the hololectin SNA-II apparently uses a similar endocytosis pathway as the chimerolectin SNA-I, it can be concluded that the endocytosis process mainly relies on the carbohydrate-binding activity of the lectins under investigation. © 2011 Wiley Periodicals, Inc. PMID:21254203

  7. Internalization and cellular pools of never growth factor in pheochromocytoma (PC12) cells

    SciTech Connect

    Neet, K.E.; Kasaian, M.

    1987-05-01

    Nerve Growth Factor (NGF) binds to a cell surface receptor on responsive neuronal cells to initiate cell maintenance and/or differentiation regimes. The purpose of these studies was to define quantitatively the fate of NGF in PC12 cells with respect to various cellular compartments in a single series of biochemical experiments. Different binding methodologies were evaluated in suspension and on plates. 50 pM SVI-NGF was bound to rat PC12 cells in suspension for 30 min at 37, followed by various methods and combinations of methods to remove subsets of bound ligand. Distinction could be made between NGF bound to fast vs. slow cell surface receptors, NGF bound to slow receptors at the cell surface vs. cell interior, and detergent-soluble vs. cytoskeletally-attached NGF. These treatments defined the relative size of five pools, including the fast receptor (65%), two intracellular compartments (12% and 3%) susceptible to nonionic detergent, and a detergent-stable intracellular pool of ligand (16%). At 37 the cold chase stable and the acid stable pools were about the same size because of rapid internalization, but the slow receptor was measurable at 4. Inhibitors were used to define the route of NGF through the cell from the plasma membrane to degradation. Chloroquine caused accumulation of NGF only in pools that were not associated with the cytoskeleton, implicating this compartment in supplying ligand to the lysosome. Results with cytochalasin B and colchicine and suggested both microfilament and microtubule pathways in NGF degradation. A model for the movement of NGF through the cell was developed based on these observations.

  8. Robust patterning of gene expression based on internal coordinate system of cells.

    PubMed

    Ogawa, Ken-ichiro; Miyake, Yoshihiro

    2015-06-01

    Cell-to-cell communication in multicellular organisms is established through the transmission of various kinds of chemical substances such as proteins. It is well known that gene expression triggered by a chemical substance in individuals has stable spatial patterns despite the individual differences in concentration patterns of the chemical substance. This fact reveals an important property of multicellular organisms called "robustness", which allows the organisms to generate their forms while maintaining proportion. Robustness has been conventionally accounted for by the stability of solutions of dynamical equations that represent a specific interaction network of chemical substances. However, any biological system is composed of autonomous elements. In general, an autonomous element does not merely accept information on the chemical substance from the environment; instead, it accepts the information based on its own criteria for reaction. Therefore, this phenomenon needs to be considered from the viewpoint of cells. Such a viewpoint is expected to allow the consideration of the autonomy of cells in multicellular organisms. This study aims to explain theoretically the robust patterning of gene expression from the viewpoint of cells. For this purpose, we introduced a new operator for transforming a state variable of a chemical substance from an external coordinate system to an internal coordinate system of each cell, which describes the observation of the chemical substance by cells. We then applied this operator to the simplest reaction-diffusion model of the chemical substance to investigate observation effects by cells. Our mathematical analysis of this extended model indicates that the robust patterning of gene expression against individual differences in concentration pattern of the chemical substance can be explained from the viewpoint of cells if there is a regulation field that compensates for the difference between cells seen in the observation results

  9. Effect of surface charge of magnetite nanoparticles on their internalization into breast cancer and umbilical vein endothelial cells.

    PubMed

    Osaka, Tetsuya; Nakanishi, Takuya; Shanmugam, Sangaraju; Takahama, Shintaro; Zhang, Hong

    2009-07-01

    Internalization of magnetite nanoparticles with diameter of approximately 40 nm into normal and cancer cells was examined by microscopic observation and flow cytometry. Magnetite nanoparticles were synthesized by hydrolysis in an aqueous solution containing ferrous chloride with organic amines as a base. It was demonstrated that the difference in surface charge of magnetite nanoparticles brought about the difference in uptake efficiency. The nanoparticles with positive charge showed higher internalization into human breast cancer cells than the nanoparticles with negative charge, while the degree of internalization of the positively- and negatively-charged nanoparticles into human umbilical vein endothelial cells (HUVEC) was almost the same.

  10. Internalization and Trafficking of Cell Surface Proteoglycans and Proteoglycan-Binding Ligands

    PubMed Central

    Payne, Christine K.; Jones, Sara A.; Chen, Chen; Zhuang, Xiaowei

    2009-01-01

    Using multi-color live cell imaging in combination with biochemical assays we have investigated an endocytic pathway mediated by cell surface proteoglycans, primary receptors for many cationic ligands. We have characterized this pathway for a variety of proteoglycan-binding ligands including cationic polymers, lipids, and polypeptides. Following clathrin- and caveolin-independent, but flotillin- and dynamin-dependent internalization, proteoglycan-bound ligands associate with flotillin-1-positive vesicles and are efficiently trafficked to late endosomes. The route to late endosomes differs considerably from that following clathrin-mediated endocytosis. The proteoglycan-dependent pathway to late endosomes does not require microtubule-dependent transport or PI(3)K-dependent sorting from early endosomes. The pathway taken by these ligands is identical to that taken by an antibody against heparan sulfate proteoglycans, suggesting this mechanism may be used generally by cell surface proteoglycans and proteoglycan-binding ligands without secondary receptors. PMID:17394486

  11. 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.

  12. 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 %.

  13. 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.

  14. 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

  15. A planar anode-supported Solid Oxide Fuel Cell model with internal reforming of natural gas

    NASA Astrophysics Data System (ADS)

    Chinda, P.; Chanchaona, S.; Brault, P.; Wechsatol, W.

    2011-05-01

    Solid Oxide Fuel Cells (SOFCs) are of great interest due to their high energy efficiency, low emission level, and multiple fuel utilization. SOFC can operate with various kinds of fuels such as natural gas, carbon monoxide, methanol, ethanol, and hydrocarbon compounds, and they are becoming one of the main competitors among environmentally friendly energy sources for the future. In this study, a mathematical model of a co-flow planar anode-supported solid oxide fuel cell with internal reforming of natural gas has been developed. The model simultaneously solves mass, energy transport equations, and chemical as well as electrochemical reactions. The model can effectively predict the compound species distributions as well as the cell performance under specific operating conditions. The main result is a rather small temperature gradient obtained at 800 °C with S/C = 1 in classical operating conditions. The cell performance is reported for several operating temperatures and pressures. The cell performance is specified in terms of cell voltage and power density at any specific current density. The influence of electrode microstructure on cell performance was investigated. The simulation results show that the steady state performance is almost insensitive to microstructure of cells such as porosity and tortuosity unlike the operating pressure and temperature. However, for SOFC power output enhancement, the power output could be maximized by adjusting the pore size to an optimal value, similarly to porosity and tortuosity. At standard operating pressure (1 atm) and 800 °C with 48% fuel utilization, when an output cell voltage was 0.73 V, a current density of 0.38 A cm-2 with a power density of 0.28 W cm-2 was predicted. The accuracy of the model was validated by comparing with existing experimental results from the available literature.

  16. Evidence of involvement of the mannose receptor in the internalization of Streptococcus pneumoniae by Schwann cells

    PubMed Central

    2014-01-01

    Background The ability of S. pneumoniae to generate infections depends on the restrictions imposed by the host’s immunity, in order to prevent the bacterium from spreading from the nasopharynx to other tissues, such as the brain. Some authors claim that strains of S. pneumoniae, which fail to survive in the bloodstream, can enter the brain directly from the nasal cavity by axonal transport through the olfactory and/or trigeminal nerves. However, from the immunological point of view, glial cells are far more responsive to bacterial infections than are neurons. This hypothesis is consistent with several recent reports showing that bacteria can infect glial cells from the olfactory bulb and trigeminal ganglia. Since our group previously demonstrated that Schwann cells (SCs) express a functional and appropriately regulated mannose receptor (MR), we decided to test whether SCs are involved in the internalization of S. pneumoniae via MR. Results Immediately after the interaction step, as well as 3 h later, the percentage of association was approximately 56.5%, decreasing to 47.2% and 40.8% after 12 and 24 h, respectively. Competition assays by adding a 100-fold excess of mannan prior to the S. pneumoniae infection reduced the number of infected cells at 3 and 24 h. A cytochemistry assay with Man/BSA-FITC binding was performed in order to verify a possible overlap between mannosylated ligands and internalized bacteria. Incubation of the SCs with Man/BSA-FITC resulted in a large number of intracellular S. pneumoniae, with nearly complete loss of the capsule. Moreover, the anti-pneumococcal antiserum staining colocalized with the internalized man/BSA-FITC, suggesting that both markers are present within the same endocytic compartment of the SC. Conclusions Our data offer novel evidence that SCs could be essential for pneumococcal cells to escape phagocytosis and killing by innate immune cells. On the other hand, the results also support the idea that SCs are

  17. Internalization and cytotoxicity effects of carbon-encapsulated iron nanoparticles in murine endothelial cells: Studies on internal dosages due to loaded mass agglomerates.

    PubMed

    Cywinska, Monika A; Bystrzejewski, Michal; Poplawska, Magdalena; Kosmider, Anita; Zdanowski, Robert; Lewicki, Slawomir; Fijalek, Zbigniew; Ostrowska, Agnieszka; Bamburowicz, Magdalena; Cieszanowski, Andrzej; Grudzinski, Ireneusz P

    2016-08-01

    Carbon-encapsulated iron nanoparticles (CEINs) qualified as metal-inorganic hybrid nanomaterials offer a potential scope for an increasing number of biomedical applications. In this study, we have focused on the investigation of cellular fate and resulting cytotoxic effects of CEINs synthesized using a carbon arc route and studied in murine endothelial (HECa-10) cells. The CEIN samples were characterized as pristine (the mean diameter between 47 and 56nm) and hydrodynamic (the mean diameter between 270 and 460nm) forms and tested using a battery of methods to determine the cell internalization extent and cytotoxicity effects upon to the exposures (0.0001-100μg/ml) in HECa-10 cells. Our studies evidenced that the incubation with CEINs for 24h is accompanied with substantial changes of Zeta potential in cells which can be considered as a key factor for affecting the membrane transport, cellular distribution and cytotoxicity of these nanoparticles. The results demonstrate that CEINs have entered the endothelial cell through the endocytic pathway rather than by passive diffusion and they were mainly loaded as agglomerates on the cell membrane and throughout the cytoplasm, mitochondria and nucleus. The studies show that CEINs induce the mitochondrial and cell membrane cytotoxicities in a dose-dependent manner resulting from the internal dosages due to CEIN agglomerates. Our results highlight the importance of the physicochemical characterization of CEINs in studying the magnetic nanoparticle-endothelial cell interactions because the CEIN mass agglomerates can sediment more or less rapidly in culture models. PMID:27107485

  18. 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.

  19. Cells Behave Distinctly Within Sponges and Hydrogels Due to Differences of Internal Structure

    PubMed Central

    Zhang, Jingjing; Yang, Zheng; Li, Chao; Dou, Yana; Li, Yijiang; Thote, Tanushree; Wang, Dong-an

    2013-01-01

    Different forms of biomaterials, including microspheres, sponges, hydrogels, and nanofibers, have been broadly used in cartilage regeneration; however, effects of internal structures of the biomaterials on cells and chondrogenesis remain largely unexplored. We hypothesized that different internal structures of sponges and hydrogels led to phenotypic disparity of the cells and may lead to disparate chondrogenesis. In the current study, the chondrocytes in sponges and hydrogels of chitosan were compared with regard to cell distribution, morphology, gene expression, and production of extracellular matrix. The chondrocytes clustered or attached to the materials with spindle morphologies in the sponges, while they distributed evenly with spherical morphologies in the hydrogels. The chondrocytes proliferated faster with elevated gene expression of collagen type I and down-regulated gene expression of aggracan in sponges, when compared with those in the hydrogels. However, there was no significant difference of the expression of collagen type II between these two scaffolds. Excretion of both glycosaminoglycan (GAG) and collagen type II increased with time in vitro, but there was no significant difference between the sponges and the hydrogels. There was no significant difference in secretion of GAG and collagen type II in the two scaffolds, while the levels of collagen type I and collagen type X were much higher in sponges compared with those in hydrogels during an in vivo study. Though the chondrocytes displayed different phenotypes in the sponges and hydrogels, they led to comparable chondrogenesis. An optimized design of the biomaterials could further improve chondrogenesis through enhancing functionalities of the chondrocytes. PMID:23614637

  20. Cells behave distinctly within sponges and hydrogels due to differences of internal structure.

    PubMed

    Zhang, Jingjing; Yang, Zheng; Li, Chao; Dou, Yana; Li, Yijiang; Thote, Tanushree; Wang, Dong-an; Ge, Zigang

    2013-10-01

    Different forms of biomaterials, including microspheres, sponges, hydrogels, and nanofibers, have been broadly used in cartilage regeneration; however, effects of internal structures of the biomaterials on cells and chondrogenesis remain largely unexplored. We hypothesized that different internal structures of sponges and hydrogels led to phenotypic disparity of the cells and may lead to disparate chondrogenesis. In the current study, the chondrocytes in sponges and hydrogels of chitosan were compared with regard to cell distribution, morphology, gene expression, and production of extracellular matrix. The chondrocytes clustered or attached to the materials with spindle morphologies in the sponges, while they distributed evenly with spherical morphologies in the hydrogels. The chondrocytes proliferated faster with elevated gene expression of collagen type I and down-regulated gene expression of aggracan in sponges, when compared with those in the hydrogels. However, there was no significant difference of the expression of collagen type II between these two scaffolds. Excretion of both glycosaminoglycan (GAG) and collagen type II increased with time in vitro, but there was no significant difference between the sponges and the hydrogels. There was no significant difference in secretion of GAG and collagen type II in the two scaffolds, while the levels of collagen type I and collagen type X were much higher in sponges compared with those in hydrogels during an in vivo study. Though the chondrocytes displayed different phenotypes in the sponges and hydrogels, they led to comparable chondrogenesis. An optimized design of the biomaterials could further improve chondrogenesis through enhancing functionalities of the chondrocytes.

  1. 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

  2. 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.

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

    PubMed

    Ros-Baro, A; Lopez-Iglesias, C; Peiro, S; Bellido, D; Palacin, M; Zorzano, A; Camps, M

    2001-10-01

    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.

  4. 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.

  5. 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

  6. A methodology for thermodynamic simulation of high temperature, internal reforming fuel cell systems

    NASA Astrophysics Data System (ADS)

    Matelli, José Alexandre; Bazzo, Edson

    This work presents a methodology for simulation of fuel cells to be used in power production in small on-site power/cogeneration plants that use natural gas as fuel. The methodology contemplates thermodynamics and electrochemical aspects related to molten carbonate and solid oxide fuel cells (MCFC and SOFC, respectively). Internal steam reforming of the natural gas hydrocarbons is considered for hydrogen production. From inputs as cell potential, cell power, number of cell in the stack, ancillary systems power consumption, reformed natural gas composition and hydrogen utilization factor, the simulation gives the natural gas consumption, anode and cathode stream gases temperature and composition, and thermodynamic, electrochemical and practical efficiencies. Both energetic and exergetic methods are considered for performance analysis. The results obtained from natural gas reforming thermodynamics simulation show that the hydrogen production is maximum around 700 °C, for a steam/carbon ratio equal to 3. As shown in the literature, the found results indicate that the SOFC is more efficient than MCFC.

  7. The evolution of policy issues in stem cell research: an international survey.

    PubMed

    Caulfield, Timothy; Rachul, Christen; Zarzeczny, Amy

    2012-12-01

    Stem cell research remains a tremendously promising yet controversial field of study. It continues to attract considerable public interest and generate discussion and debate. However, while the high profile of this field has endured, the tone and nature of the discourse that drives this profile appears to be changing. In order to get a better sense of how these potential shifts are perceived by individuals directly embedded in the field, we conducted an international internet survey of members of the stem cell research community. Our participants included individuals publishing on both scientific and ethical, legal and social issues topics. We explored the degree to which participants perceived that key policy issues were becoming more or less contentious over time. We queried views regarding the effect of regulatory frameworks on emerging stem cell research technologies and the extent to which participants experience pressure related to clinical translation. We also explored participants' relationships with industry, experience with patents and perceptions regarding the emphasis placed on the potential economic benefits of stem cell research. Our results suggest that while traditional debates such as those surrounding the moral status of the embryo remain, other issues more closely associated with clinical translation and commercialization are perceived as becoming increasingly contentious. This survey provides useful insight into the perspectives of a sample of active researchers working in countries around the world as well as an opportunity to reflect on the likely direction of future stem cell policy debates.

  8. Direct ethanol solid oxide fuel cell operating in gradual internal reforming

    NASA Astrophysics Data System (ADS)

    Nobrega, S. D.; Galesco, M. V.; Girona, K.; de Florio, D. Z.; Steil, M. C.; Georges, S.; Fonseca, F. C.

    2012-09-01

    An electrolyte supported solid oxide fuel cell (SOFC) using standard electrodes, doped-lanthanum manganite cathode and Ni-cermet anode, was operated with direct (anhydrous) ethanol for more than 100 h, delivering essentially the same power output as running on hydrogen. A ceria-based layer provides the catalytic activity for the gradual internal reforming, which uses the steam formed by the electrochemical oxidation of hydrogen for the decomposition of ethanol. Such a concept opens up the way for multi-fuel SOFCs using standard components and a catalytic layer.

  9. 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.

  10. Red blood cell deformation in shear flow. Effects of internal and external phase viscosity and of in vivo aging.

    PubMed Central

    Pfafferott, C; Nash, G B; Meiselman, H J

    1985-01-01

    Shear deformation of young and old human red blood cells was examined over a range of shear stresses and suspending phase viscosities (eta o) using a cone-plate Rheoscope. The internal viscosities (eta i) of these cell types differ, and further changes in internal viscosity were induced by alteration of suspension osmolality and hence cell volume. For low suspending viscosities (0.0555 or 0.111 P) old cells tended to tumble in shear flow, whereas young cells achieved stable orientation and deformed. Changes in osmolality, at these external viscosities, altered the percentage of cells deforming, and for each cell type threshold osmolalities (Osm-50) were determined where 50% of cells deformed. The threshold osmolalities were higher for younger cells than for older cells, but the internal viscosities of the two cell types were similar at their respective Osm-50. Threshold osmolalities were also higher for the higher external viscosity, but the ratio of internal to external viscosities (i.e., eta i/eta o) was nearly constant for both external viscosities. Deformation of stably oriented cells increased with increasing shear stress and approached a value limited by cell surface area and volume. For isotonic media, over a wide range of external viscosities and shear stresses, deformation was greater for younger cells than for older cells. However, deformation vs. shear stress data for the two cell types became nearly coincident if young cells were osmotically shrunk to have their internal viscosity close to that for old cells. Increases in external viscosity, at constant shear stress, caused greater deformation for all cells. This effect of external viscosity was not equal for young and old cells; the ratio of old/young cell deformation increased with increasing eta o. However, if deformation was plotted as a function of the ratio lambda = eta i/eta o, at constant shear stress, young and old cell data followed similar paths. Thus the ratio lambda is a major determinant

  11. 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

  12. Impact of National and International Stem Cell Banking Initiatives on progress in the field of cell therapy: IABS-JST Joint Workshop: Summary for Session 5.

    PubMed

    Aoi, Takashi; Stacey, Glyn

    2015-09-01

    In order to assure the quality and safety of future advanced cell therapies it is vital to ensure that source materials including the donor cells have been assessed and demonstrated as suitable for use in the development and manufacture of such new medicines. Here we provide a brief overview of the key issues in the delivery of quality controlled and safety tested seed stocks of human pluripotent stem cell lines to support stem cell research and the development of advanced cell therapies. We also reflect on the importance of national and internationally coordinated cell banking systems in this process in order to promote more efficient development of cell therapies.

  13. Development of internal manifold heat exchanger (IMHEX reg sign ) molten carbonate fuel cell stacks

    SciTech Connect

    Marianowski, L.G.; Ong, E.T.; Petri, R.J.; Remick, R.J.

    1991-01-01

    The Institute of Gas Technology (IGT) has been in the forefront of molten carbonate fuel cell (MCFC) development for over 25 years. Numerous cell designs have been tested and extensive tests have been performed on a variety of gas manifolding alternatives for cells and stacks. Based upon the results of these performance tests, IGT's development efforts started focusing on an internal gas manifolding concept. This work, initiated in 1988, is known today as the IMHEX{reg sign} concept. MCP has developed a comprehensive commercialization program loading to the sale of commercial units in 1996. MCP's role is in the manufacture of stack components, stack assembly, MCFC subsystem testing, and the design, marketing and construction of MCFC power plants. Numerous subscale (1 ft{sup 2}) stacks have been operated containing between 3 and 70 cells. These tests verified and demonstrated the viability of internal manifolding from technical (no carbonate pumping), engineering (relaxed part dimensional tolerance requirements), and operational (good gas sealing) aspects. Simplified fabrication, ease of assembly, the elimination of external manifolds and all associated clamping requirements has significantly lowered anticipated stack costs. Ongoing 1 ft{sup 2} stack testing is generating performance and endurance characteristics as a function of system specified operating conditions. Commercial-sized, full-area stacks (10 ft{sup 2}) are in the process of being assembled and will be tested in November. This paper will review the recent developments the MCFC scale-up and manufacture work of MCP, and the research and development efforts of IGT which support those efforts. 17 figs.

  14. Lupus risk variants in the PXK locus alter B-cell receptor internalization

    PubMed Central

    Vaughn, Samuel E.; Foley, Corinne; Lu, Xiaoming; Patel, Zubin H.; Zoller, Erin E.; Magnusen, Albert F.; Williams, Adrienne H.; Ziegler, Julie T.; Comeau, Mary E.; Marion, Miranda C.; Glenn, Stuart B.; Adler, Adam; Shen, Nan; Nath, Swapan; Stevens, Anne M.; Freedman, Barry I.; Tsao, Betty P.; Jacob, Chaim O.; Kamen, Diane L.; Brown, Elizabeth E.; Gilkeson, Gary S.; Alarcón, Graciela S.; Reveille, John D.; Anaya, Juan-Manuel; James, Judith A.; Moser, Kathy L.; Criswell, Lindsey A.; Vilá, Luis M.; Alarcón-Riquelme, Marta E.; Petri, Michelle; Scofield, R. Hal; Kimberly, Robert P.; Ramsey-Goldman, Rosalind; Binjoo, Young; Choi, Jeongim; Bae, Sang-Cheol; Boackle, Susan A.; Vyse, Timothy J.; Guthridge, Joel M.; Namjou, Bahram; Gaffney, Patrick M.; Langefeld, Carl D.; Kaufman, Kenneth M.; Kelly, Jennifer A.; Harley, Isaac T. W.; Harley, John B.; Kottyan, Leah C.

    2015-01-01

    Genome wide association studies have identified variants in PXK that confer risk for humoral autoimmune diseases, including systemic lupus erythematosus (SLE or lupus), rheumatoid arthritis and more recently systemic sclerosis. While PXK is involved in trafficking of epidermal growth factor Receptor (EGFR) in COS-7 cells, mechanisms linking PXK to lupus pathophysiology have remained undefined. In an effort to uncover the mechanism at this locus that increases lupus-risk, we undertook a fine-mapping analysis in a large multi-ancestral study of lupus patients and controls. We define a large (257kb) common haplotype marking a single causal variant that confers lupus risk detected only in European ancestral populations and spans the promoter through the 3′ UTR of PXK. The strongest association was found at rs6445972 with P < 4.62 × 10−10, OR 0.81 (0.75–0.86). Using stepwise logistic regression analysis, we demonstrate that one signal drives the genetic association in the region. Bayesian analysis confirms our results, identifying a 95% credible set consisting of 172 variants spanning 202 kb. Functionally, we found that PXK operates on the B-cell antigen receptor (BCR); we confirmed that PXK influenced the rate of BCR internalization. Furthermore, we demonstrate that individuals carrying the risk haplotype exhibited a decreased rate of BCR internalization, a process known to impact B cell survival and cell fate. Taken together, these data define a new candidate mechanism for the genetic association of variants around PXK with lupus risk and highlight the regulation of intracellular trafficking as a genetically regulated pathway mediating human autoimmunity. PMID:25620976

  15. 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

  16. 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.

  17. Endothelial Cells Use a Formin-Dependent Phagocytosis-Like Process to Internalize the Bacterium Listeria monocytogenes.

    PubMed

    Rengarajan, Michelle; Hayer, Arnold; Theriot, Julie A

    2016-05-01

    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

  18. 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

  19. 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.

  20. International renal-cell-cancer study. VI. the role of medical and family history.

    PubMed

    Schlehofer, B; Pommer, W; Mellemgaard, A; Stewart, J H; McCredie, M; Niwa, S; Lindblad, P; Mandel, J S; McLaughlin, J K; Wahrendorf, J

    1996-06-11

    A number of medical conditions have been linked with renal-cell cancer, although the evidence is not consistent in every case. In a large international case-control study of renal-cell cancer, we examined, among other hypotheses, associations with a personal history of certain medical conditions and a family history of cancer of the kidney or thyroid. Relative risks (RR), adjusted for the effects of age, gender, body-mass index, tobacco smoking and study centre, were significantly increased by a history of kidney stones or thyroid or kidney disease. The RR were not altered by additional adjustment for hypertension, or when diagnoses were restricted to those made at least 5 or 10 years before 1987 (the usual "cut-off" date). The link with kidney injury is particularly likely to be affected by recall bias. Increased RR of borderline significance were found for kidney infection (RR, 1.2) and diabetes (RR, 1.4). Having one first-degree relative with kidney cancer was associated with a significantly increased risk of renal-cell cancer (RR, 1.6; 95% Cl, 1.1-2.4). Seven cases reported 2 first-degree relatives with kidney cancer. No controls had first-degree relatives with kidney cancer. None of our participants reported having von Hippel-Lindau disease. The data suggests that a few conditions of the kidney are strongly associated with renal-cell cancer and that heredity plays a role in a small proportion of cases.

  1. Regulation of Mct1 by cAMP-dependent internalization in rat brain endothelial cells.

    PubMed

    Smith, Jeffrey P; Uhernik, Amy L; Li, Lun; Liu, Zejian; Drewes, Lester R

    2012-10-22

    In the cerebrovascular endothelium, monocarboxylic acid transporter 1 (Mct1) controls blood-brain transport of short chain monocarboxylic and keto acids, including pyruvate and lactate, to support brain energy metabolism. Mct1 function is acutely decreased in rat brain cerebrovascular endothelial cells by β-adrenergic signaling through cyclic adenosine monophosphate (cAMP); however, the mechanism for this acute reduction in transport capacity is unknown. In this report, we demonstrate that cAMP induces the dephosphorylation and internalization of Mct1 from the plasma membrane into caveolae and early endosomes in the RBE4 rat brain cerebrovascular endothelial cell line. Additionally, we provide evidence that Mct1 constitutively cycles through clathrin vesicles and recycling endosomes in a pathway that is not dependent upon cAMP signaling in these cells. Our results are important because they show for the first time the regulated and unregulated vesicular trafficking of Mct1 in cerebrovascular endothelial cells; processes which have significance for better understanding normal brain energy metabolism, and the etiology and potential therapeutic approaches to treating brain diseases, such as stroke, in which lactic acidosis is a key component.

  2. 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. PMID:25937384

  3. 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.

  4. Internalization of Red Blood Cell-Mimicking Hydrogel Capsules with pH-Triggered Shape Responses

    PubMed Central

    2015-01-01

    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. PMID:24848786

  5. Using total internal reflection fluorescence microscopy to visualize rhodopsin-containing cells.

    PubMed

    Keffer, J L; Sabanayagam, C R; Lee, M E; DeLong, E F; Hahn, M W; Maresca, J A

    2015-05-15

    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

  6. Using total internal reflection fluorescence microscopy to visualize rhodopsin-containing cells.

    PubMed

    Keffer, J L; Sabanayagam, C R; Lee, M E; DeLong, E F; Hahn, M W; Maresca, J A

    2015-05-15

    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.

  7. 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

  8. 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.

  9. Hemin-dependent induction and internalization of CD38 in K562 cells.

    PubMed

    Yalcintepe, Leman; Ercelen, Sebnem; Adin-Cinar, Suzan; Badur, Selim; Tiryaki, Demir; Bermek, Engin

    2003-10-01

    The cell surface antigen, CD38, is a bifunctional ecto-enzyme, which is predominantly expressed on hematopoietic cells during differentiation. In the present study, it is shown that hemin treatment of K562 cells gives rise to induction of enzymatic activities inherent to CD38. GDP-ribosyl cyclase activity, an indicator of CD38, increased initially in response to hemin in a time-dependent manner, reached a maximum level on the 5th day and, thereafter, declined sharply to the initial level. The increase in NAD(+) glycohydrolase and ADP-ribose uptake activities followed a similar time course. However, the decline in the latter activities after the 5th day of induction appeared to be rather slow in contrast to GDP-ribosyl cyclase activity. The time course of these changes was well correlated with the FACScan findings obtained by use of anti-CD38 monoclonal antibody. SDS-PAGE and Western blot analyses by use of the monoclonal antibody OKT10 revealed a transient hemin-dependent appearance of a 43 kDa membrane protein with maximum signal intensity on the first 4 days of incubation. There was subsequently a gradual decrease on the 5th day, concomitant with a reciprocal increase in activity of the internalized protein fraction. The results together indicated that hemin-induced expression of CD38 was followed by its down-regulation.

  10. 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.

  11. The effect of simultaneous internal tamponade on fluid compartmentalization and its relationship to cell proliferation.

    PubMed

    De Molfetta, V; Bottoni, F; Arpa, P; Vinciguerra, P; Zenoni, S

    1992-01-01

    To determine whether the residual free spaces within the vitreous chamber that result after vitreoretinal surgery and internal tamponade may be avoided, and to verify whether such compartmentalization is of real importance in the recurrence of postoperative proliferative vitreoretinopathy (PVR), the use of simultaneous double filling with polydimethylsiloxane (PDMS) and fluorosilicone (FSiO) in the repair of complicated retinal detachment is evaluated in 12 selected cases. Initial retinal reattachment was achieved in all cases. PVR recurred in 10 patients (83%), 6 of whom showed partial retinal detachment. Inferior and superior postoperative residual free spaces were abolished by this procedure, but a new residual fluid space was created, lying horizontally between the bubbles and expanding in a triangular shape nasal to the optic disc and temporal to the macula. Overall, 9 of 10 eyes with PVR after surgery had proliferation involving these areas. These findings support the concept that compartmentalization is of major importance in determining postoperative cell proliferation.

  12. High internal quantum efficiency in fullerene solar cells based on crosslinked polymer donor networks

    NASA Astrophysics Data System (ADS)

    Liu, Bo; Png, Rui-Qi; Zhao, Li-Hong; Chua, Lay-Lay; Friend, Richard H.; Ho, Peter K. H.

    2012-12-01

    The power conversion efficiency of organic photovoltaic cells depends crucially on the morphology of their donor-acceptor heterostructure. Although tremendous progress has been made to develop new materials that better cover the solar spectrum, this heterostructure is still formed by a primitive spontaneous demixing that is rather sensitive to processing and hence difficult to realize consistently over large areas. Here we report that the desired interpenetrating heterostructure with built-in phase contiguity can be fabricated by acceptor doping into a lightly crosslinked polymer donor network. The resultant nanotemplated network is highly reproducible and resilient to phase coarsening. For the regioregular poly(3-hexylthiophene):phenyl-C61-butyrate methyl ester donor-acceptor model system, we obtained 20% improvement in power conversion efficiency over conventional demixed biblend devices. We reached very high internal quantum efficiencies of up to 0.9 electron per photon at zero bias, over an unprecedentedly wide composition space. Detailed analysis of the power conversion, power absorbed and internal quantum efficiency landscapes reveals the separate contributions of optical interference and donor-acceptor morphology effects.

  13. High internal quantum efficiency in fullerene solar cells based on crosslinked polymer donor networks.

    PubMed

    Liu, Bo; Png, Rui-Qi; Zhao, Li-Hong; Chua, Lay-Lay; Friend, Richard H; Ho, Peter K H

    2012-01-01

    The power conversion efficiency of organic photovoltaic cells depends crucially on the morphology of their donor-acceptor heterostructure. Although tremendous progress has been made to develop new materials that better cover the solar spectrum, this heterostructure is still formed by a primitive spontaneous demixing that is rather sensitive to processing and hence difficult to realize consistently over large areas. Here we report that the desired interpenetrating heterostructure with built-in phase contiguity can be fabricated by acceptor doping into a lightly crosslinked polymer donor network. The resultant nanotemplated network is highly reproducible and resilient to phase coarsening. For the regioregular poly(3-hexylthiophene):phenyl-C(61)-butyrate methyl ester donor-acceptor model system, we obtained 20% improvement in power conversion efficiency over conventional demixed biblend devices. We reached very high internal quantum efficiencies of up to 0.9 electron per photon at zero bias, over an unprecedentedly wide composition space. Detailed analysis of the power conversion, power absorbed and internal quantum efficiency landscapes reveals the separate contributions of optical interference and donor-acceptor morphology effects.

  14. 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). PMID:25841189

  15. 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).

  16. 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.

  17. Dynamics of internalization and recycling of the prometastatic membrane type 4 matrix metalloproteinase (MT4-MMP) in breast cancer cells.

    PubMed

    Truong, Alice; Yip, Cassandre; Paye, Alexandra; Blacher, Silvia; Munaut, Carine; Deroanne, Christophe; Noel, Agnès; Sounni, Nor Eddine

    2016-02-01

    Membrane type 4 matrix metalloproteinase (MT4-MMP) [matrix metalloproteinase (MMP) 17] is a GPI-anchored membrane-type MMP expressed on the cell surface of human breast cancer cells. In triple-negative breast cancer cells, MT4-MMP promotes primary tumour growth and lung metastases. Although the trafficking and internalization of the transmembrane membrane type 1 MMP have been extensively investigated, little is known about the regulatory mechanisms of the GPI-anchored MT4-MMP. Here, we investigated the fate and cellular trafficking of MT4-MMP by analysing its homophilic complex interactions, internalization and recycling dynamics as compared with an inert form, MT4-MMP-E249A. Oligomeric and dimeric complexes were analysed by cotransfection of cells with FLAG-tagged or Myc-tagged MT4-MMP in reducing and nonreducing immunoblotting and coimmunoprecipitation experiments. The trafficking of MT4-MMP was studied with an antibody feeding assay and confocal microscopy analysis or cell surface protein biotinylation and western blot analysis. We demonstrate that MT4-MMP forms homophilic complexes at the cell surface, and internalizes in early endosomes, and that some of the enzyme is either autodegraded or recycled to the cell surface. Our data indicate that MT4-MMP is internalized by the clathrin-independent carriers/GPI-enriched early endosomal compartments pathway, a mechanism that differs from that responsible for the internalization of other membrane-type MMP members. Although MT4-MMP localizes with caveolin-1, MT4-MMP internalization was not affected by inhibitors of caveolin-1 or clathrin endocytosis pathways, but was reduced by CDC42 or RhoA silencing with small interfering RNA. We provide a new mechanistic insight into the regulatory mechanisms of MT4-MMP, which may have implications for the design of novel therapeutic strategies for metastatic breast cancer.

  18. 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.

  19. Insights into the Internalization and Retrograde Trafficking of Dengue 2 Virus in BHK-21 Cells

    PubMed Central

    Kaur, Jasmine; Shah, Paresh S.; Cecilia, D.

    2011-01-01

    Background Dengue virus (DENV) enters cells via endocytosis, traffics to perinuclear (PN) region, the site of morphogenesis and exits by exocytosis. This study aims to understand the role of dynamin II, endosomes, microtubules (MT) and dynein in the early events of DENV replication. Findings Using double immunoflourescence labelling of DENV-2 infected BHK-21 cells it was observed that the surface envelope (E) protein of the virion associated with dynamin II from 0–30 min post infection (p.i.). The sphincter like array of dynamin II supported its pinchase-like activity. The association with endosomes was observed from 0 min at cell periphery to 30 min in the perinuclear (PN) region, suggesting that internalization continued for 30 min. Association of E protein with alpha-tubulin was observed from 8 h indicating that it was the newly translated protein that trafficked on the MT. Dynein was found to associate with the E protein from 4 h in the cytoplasm to 48 h in the PN region and dissociate at 72 h. Association of E protein with dynein was confirmed by immunoprecipitation. Overexpression of dynamitin, which disrupts the dynein complex, resulted in loss of trafficking of viral E and core proteins. The findings corroborated with the growth kinetics assessed by quantitation of viral RNA in infected BHK-21 cells. The detection of E protein at 4 h–8 h correlated with detectable increase in viral RNA from 8 h. The detection of high concentrations of E protein in the PN region at 24–48 h coincided with release of virus into the supernatant starting from 36 h p.i. The dissociation of dynein from E protein by 72 h was coincident with maximum release of virus, hinting at a possible negative feedback for viral protein translation. Conclusion The study shows for the first time the association of dynamin II with DENV-2 during entry and dynein dependent retrograde trafficking of DENV proteins on microtubules. PMID:21991304

  20. 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

  1. Cytotoxicity and variant cellular internalization behavior of water-soluble sulfonated nanographene sheets in liver cancer cells

    PubMed Central

    2013-01-01

    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)-2H-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. PMID:23639042

  2. 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

  3. Integrin activation and internalization on soft ECM as a mechanism of induction of stem cell differentiation by ECM elasticity

    PubMed Central

    Du, Jing; Chen, Xiaofei; Liang, Xudong; Zhang, Guangyao; Xu, Jia; He, Linrong; Zhan, Qingyuan; Feng, Xi-Qiao; Chien, Shu; Yang, Chun

    2011-01-01

    The mechanism by which ECM elasticity induces lineage specification of stem cells has not been clearly understood. Integrins are well-documented mechanosensors that are positioned at the beginning of the sensing pathway. By using an antibody specifically recognizing the active conformation of β1 integrin, we observed that β1 integrin activation in bone marrow mesenchymal stem cells (BMMSCs) was induced by soft substrate to a significantly greater degree than by stiff substrate. In contrast, however, the level of cell surface integrin on soft substrate was significantly lower than that on stiff substrate. Soft substrate markedly enhanced the internalization of integrin, and this internalization was mediated mainly through caveolae/raft-dependent endocytosis. The inhibition of integrin internalization blocked the neural lineage specification of BMMSCs on soft substrate. Furthermore, soft substrate also repressed the bone morphogenetic protein (BMP)/Smad pathway at least partially through integrin-regulated BMP receptor endocytosis. A theoretical analysis based on atomic force microscopy (AFM) data indicated that integrin–ligand complexes are more easily ruptured on soft substrate; this outcome may contribute to the enhancement of integrin internalization on soft substrate. Taken together, our results suggest that ECM elasticity affects integrin activity and trafficking to modulate integrin BMP receptor internalization, thus contributing to stem cell lineage specification. PMID:21593411

  4. Stem cell research in the Greater Middle East: the importance of establishing policy and ethics interoperability to foster international collaborations.

    PubMed

    Flynn, Jesse M; Matthews, Kirstin R W

    2010-06-01

    While fossil fuel reserves have strengthened the economies of numerous countries in the Greater Middle East (GME) for decades, multiple nations within this region are now increasingly investing in internal science and engineering programs as a mechanism to develop more extensive knowledge-based economies. One of these newly pursued disciplines is stem cell research. Nations such as Saudi Arabia and Qatar have founded nascent programs while Iran, Turkey, and Israel are more established in the field. The extent to which these investments have been productive, as measured by publication quantity and impact, remains unknown. Here we assess the state of stem cell research in the GME, report on the policy and ethical considerations facing the region, and determine the impact of international research collaborations in this area. In the majority of the region, there is no legal framework regulating stem cell research. Instead, scientists often rely on religious decrees outlining acceptable practices. These guidelines do not provide the necessary structure to foster international collaborations with nations that have enacted formal laws recognized worldwide. Our results illustrate that international collaborations in the GME produce publications of greater impact despite the fact that political tensions and issues unrelated to science have the potential to dramatically hinder cross-border relationships in the region. Overall, we conclude that the national governments of countries within the GME have the unique opportunity to establish stem cell research policies which confer interoperability between nations to foster crucial international collaborations throughout the region. PMID:20198516

  5. Stem cell research in the Greater Middle East: the importance of establishing policy and ethics interoperability to foster international collaborations.

    PubMed

    Flynn, Jesse M; Matthews, Kirstin R W

    2010-06-01

    While fossil fuel reserves have strengthened the economies of numerous countries in the Greater Middle East (GME) for decades, multiple nations within this region are now increasingly investing in internal science and engineering programs as a mechanism to develop more extensive knowledge-based economies. One of these newly pursued disciplines is stem cell research. Nations such as Saudi Arabia and Qatar have founded nascent programs while Iran, Turkey, and Israel are more established in the field. The extent to which these investments have been productive, as measured by publication quantity and impact, remains unknown. Here we assess the state of stem cell research in the GME, report on the policy and ethical considerations facing the region, and determine the impact of international research collaborations in this area. In the majority of the region, there is no legal framework regulating stem cell research. Instead, scientists often rely on religious decrees outlining acceptable practices. These guidelines do not provide the necessary structure to foster international collaborations with nations that have enacted formal laws recognized worldwide. Our results illustrate that international collaborations in the GME produce publications of greater impact despite the fact that political tensions and issues unrelated to science have the potential to dramatically hinder cross-border relationships in the region. Overall, we conclude that the national governments of countries within the GME have the unique opportunity to establish stem cell research policies which confer interoperability between nations to foster crucial international collaborations throughout the region.

  6. Technique for internal channelling of hydroentangled nonwoven scaffolds to enhance cell penetration.

    PubMed

    Durham, Elaine R; Ingham, Eileen; Russell, Stephen J

    2013-08-01

    An important requirement in thick, high-porosity scaffolds is to maximise cellular penetration into the interior and avoid necrosis during culture in vitro. Hitherto, reproducible control of the pore structure in nonwoven scaffolds has proved challenging. A new, channelled scaffold manufacturing process is reported based on water jet entanglement of fibres (hydroentangling) around filamentous template to form a coherent scaffold that is subsequently removed. Longitudinally-oriented channels were introduced within the scaffold in controlled proximity using 220 µm diameter cylindrical templates. In this case study, channelled scaffolds composed of poly(l-lactic acid) were manufactured and evaluated in vitro. Environmental scanning electron microscope and µCT (X-ray microtomography) confirmed channel openings in the scaffold cross-section before and after cell culture with human dermal fibroblasts up to 14 weeks. Histology at week 11 indicated that the channels promoted cell penetration and distribution within the scaffold interior. At week 14, cellular matrix deposition was evident in the internal channel walls and the entrances remained unoccluded by cellular matrix suggesting that diffusion conduits for mass transfer of nutrient to the scaffold interior could be maintained.

  7. 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.

  8. 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.

  9. Technique for internal channelling of hydroentangled nonwoven scaffolds to enhance cell penetration

    PubMed Central

    Durham, Elaine R; Ingham, Eileen; Russell, Stephen J

    2013-01-01

    An important requirement in thick, high-porosity scaffolds is to maximise cellular penetration into the interior and avoid necrosis during culture in vitro. Hitherto, reproducible control of the pore structure in nonwoven scaffolds has proved challenging. A new, channelled scaffold manufacturing process is reported based on water jet entanglement of fibres (hydroentangling) around filamentous template to form a coherent scaffold that is subsequently removed. Longitudinally-oriented channels were introduced within the scaffold in controlled proximity using 220 µm diameter cylindrical templates. In this case study, channelled scaffolds composed of poly(l-lactic acid) were manufactured and evaluated in vitro. Environmental scanning electron microscope and µCT (X-ray microtomography) confirmed channel openings in the scaffold cross-section before and after cell culture with human dermal fibroblasts up to 14 weeks. Histology at week 11 indicated that the channels promoted cell penetration and distribution within the scaffold interior. At week 14, cellular matrix deposition was evident in the internal channel walls and the entrances remained unoccluded by cellular matrix suggesting that diffusion conduits for mass transfer of nutrient to the scaffold interior could be maintained. PMID:22532409

  10. Psychrotrophic Streptomyces spp. cells immobilisation in alginate microspheres produced by emulsification-internal gelation.

    PubMed

    Cotârleţ, Mihaela; Dima, Stefan; Bahrim, Gabriela

    2014-01-01

    The objective of the investigations was the optimisation of the parameters for cold-adapted Streptomyces MIUG 4 Alga strain cells immobilisation using emulsification-internal gelation technique in calcium alginate microspheres and testing their ability to produce cold-active β-amylase. By Box-Behnken design and response surface methodology, the effects of independent variables were established, which included sodium alginate concentration (A), sodium alginate:living cell ratio (B) and the Span 80 concentration (C) upon microspheres formation and their functionality. Mean diameter of formed microspheres with immobilised biomass and cold-active β-amylase production were chosen as dependent variables in order to increase the yield of starch hydrolysis. Diameters of microspheres <25.5 μm provided large yield of cold-active β-amylase comparing with microspheres with bigger diameter. A 1.5-fold increase in the substrate hydrolysis yield was achieved using the immobilised biocatalyst compared with the crude enzyme extract, after 96 h of substrate bioconversion.

  11. CdSe Quantum-Dot-Sensitized Solar Cell with ~100% Internal Quantum Efficiency

    SciTech Connect

    Fuke, Nobuhiro; Hoch, Laura B.; Koposov, Alexey Y.; Manner, Virginia W.; Werder, Donald J.; Fukui, Atsushi; Koide, Naoki; Katayama, Hiroyuki; Sykora, Milan

    2010-10-20

    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 TiO2 film (NQD/TiO2), aqueous Na2S or Li2S electrolyte, and a Pt counter electrode. We show that light harvesting efficiency (LHE) of the NQD/TiO2 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/TiO2 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 Li2S 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.

  12. "Spot and hop": internal referencing for surface plasmon resonance imaging using a three-dimensional microfluidic flow cell array.

    PubMed

    Eddings, Mark A; Eckman, Josh W; Arana, Carlos A; Papalia, Giuseppe A; Connolly, John E; Gale, Bruce K; Myszka, David G

    2009-02-15

    We have developed a novel referencing technique for surface plasmon resonance imaging systems referred to as "spot and hop." The technique enables internal referencing for individual flow cells in a parallel processing microfluidic network. Internal referencing provides the ability to correct for nonspecific binding and instrument drift, significantly improving data quality at each region of interest. The performance of a 48-flow-cell device was demonstrated through a series of studies, including "rise and fall" time, ligand preconcentration, ligand immobilization, analyte binding, and regeneration tests. Interfacing parallel processing fluidics with imaging systems will significantly expand the throughput and applications of array-based optical biosensors while retaining high data quality.

  13. 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

  14. A Cell Internalizing Antibody Targeting Capsid Protein (p24) Inhibits the Replication of HIV-1 in T Cells Lines and PBMCs: A Proof of Concept Study

    PubMed Central

    Ali, Syed A.; Teow, Sin-Yeang; Omar, Tasyriq Che; Khoo, Alan Soo-Beng; Choon, Tan Soo; Yusoff, Narazah Mohd

    2016-01-01

    There remains a need for newer therapeutic approaches to combat HIV/AIDS. Viral capsid protein p24 plays important roles in HIV pathogenesis. Peptides and small molecule inhibitors targeting p24 have shown to inhibit virus replication in treated cell. High specificity and biological stability of monoclonal antibodies (mAbs) make them an attractive contender for in vivo treatments. However, mAbs do not enter into cells, thus are restricted to target surface molecules. This also makes targeting intracellular HIV-1 p24 a challenge. A mAb specific to p24 that can internalize into the HIV-infected cells is hypothesized to inhibit the virus replication. We selected a mAb that has previously shown to inhibit p24 polymerization in an in vitro assay and chemically conjugated it with cell penetrating peptides (CPP) to generate cell internalizing anti-p24 mAbs. Out of 8 CPPs tested, κFGF-MTS -conjugated mAbs internalized T cells most efficiently. At nontoxic concentration, the κFGF-MTS-anti-p24-mAbs reduced the HIV-1 replication up to 73 and 49% in T-lymphocyte and PBMCs respectively. Marked inhibition of HIV-1 replication in relevant cells by κFGF-MTS-anti-p24-mAbs represents a viable strategy to target HIV proteins present inside the cells. PMID:26741963

  15. 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

  16. CFD analysis of a solid oxide fuel cell with internal reforming: Coupled interactions of transport, heterogeneous catalysis and electrochemical processes

    NASA Astrophysics Data System (ADS)

    Janardhanan, Vinod M.; Deutschmann, Olaf

    Direct internal reforming in solid oxide fuel cell (SOFC) results in increased overall efficiency of the system. Present study focus on the chemical and electrochemical process in an internally reforming anode supported SOFC button cell running on humidified CH 4 (3% H 2 O). The computational approach employs a detailed multi-step model for heterogeneous chemistry in the anode, modified Butler-Volmer formalism for the electrochemistry and Dusty Gas Model (DGM) for the porous media transport. Two-dimensional elliptic model equations are solved for a button cell configuration. The electrochemical model assumes hydrogen as the only electrochemically active species. The predicted cell performances are compared with experimental reports. The results show that model predictions are in good agreement with experimental observation except the open circuit potentials. Furthermore, the steam content in the anode feed stream is found to have remarkable effect on the resulting overpotential losses and surface coverages of various species at the three-phase boundary.

  17. 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)

  18. Langerhans cell histiocytosis in adults. Report from the International Registry of the Histiocyte Society.

    PubMed

    Aricò, M; Girschikofsky, M; Généreau, T; Klersy, C; McClain, K; Grois, N; Emile, J-F; Lukina, E; De Juli, E; Danesino, C

    2003-11-01

    Langerhans cell histiocytosis (LCH), characterised by the infiltration of one or more organs by large mononuclear cells, can develop in persons of any age. Although the features of this disease are well described in children, they remain poorly defined in adults. From January 2000 to June 2001, 274 adults from 13 countries, with biopsy-proven adult LCH, were registered with the International Histiocyte Society Registry. Information was collected about clinical presentation, family history, associated conditions, cigarette smoking and treatment, to assist in future management decisions in patients aged 18 years and older. There were slightly more males than females (143:126), and the mean ages at the onset and diagnosis of disease were 33 years (standard deviation (S.D.) 15 years) and 35 years (S.D. 14 years), respectively. 2 patients had consanguineous parents, and 1 had a family history of LCH; 129 reported smoking (47.1%); 17 (6.2%) had been diagnosed with different types of cancer. Single-system LCH, found in 86 patients (31.4%), included isolated pulmonary involvement in 44 cases; 188 patients (68.6%) had multisystem disease; 81 (29.6%) had diabetes insipidus. Initial treatment consisted of vinblastine administered with or without steroids, to 82 patients (29.9%), including 9 who had received it with etoposide, which was the sole agent given to 19 patients. 236 patients were considered evaluable for survival. At a median follow-up of 28 months from diagnosis, 15 patients (6.4%) had died (death rate, 1.5/100 person years, 95% Confidence Interval (95% CI) 0.9-2.4). The probability of survival at 5 years postdiagnosis was 92.3% (95% CI 85.6-95.9) overall, 100% for patients with single-system disease (n=37), 87.8% (95% CI 54.9-97.2) for isolated pulmonary disease (n=34), and 91.7% (95% CI 83.6-95.9) for multisystem disease (n=163). Survival did not differ significantly among patients with multisystem disease, with or without liver or lung involvement) 5-year

  19. Study of internal short in a Li-ion cell I. Test method development using infra-red imaging technique

    NASA Astrophysics Data System (ADS)

    Ramadass, Premanand; Fang, Weifeng; Zhang, Zhengming (John)

    2014-02-01

    A new controlled test method has been developed to simulate the occurrence of internal short in Li-ion cells. Two different internal short kinds namely aluminum shorting to anode and cathode shorting to anode has been studied with this test method at several states of charge. Infra-red imaging technique has been adopted to analyze the thermal propagation for both the short kinds. As a comparison, the most commonly adopted nail penetration test was also conducted and analyzed using IR-imaging. The instantaneous rise in temperature referred as temperature spike upon incurring internal short was able to be captured using the IR imaging for the anode-aluminum short kind and the magnitude of such temperature spike was found to be proportional to SOC of the cell.

  20. Kinetics of methanol-steam reformation in an internal reforming fuel cell

    NASA Astrophysics Data System (ADS)

    Samms, S. R.; Savinell, R. F.

    A study of the kinetics of the methanol-steam reformation reaction within an idealized tube reactor and within a non-ideal internal reforming fuel cell (IRFC) was performed. Kinetic expressions were calculated from the reaction rate data obtained from the tube reactor by least squares fitting to general power law model, as well as to a mechanism-based model put forth by Peppley et al. [Appl. Catal. A 179 (1999) 21; Appl. Catal. A 179 (1999) 31] assuming isothermal plug flow behavior. Reaction rate data obtained from an IRFC with and without an H 3PO 4 containing membrane electrode assembly (MEA) was compared to the reaction rates predicted by the kinetic model. It was found that methanol conversion rates in the IRFC were significantly less than would be for an ideal plug flow reactor (PFR) with an equal amount of catalyst due to the non-ideal flow through the reactor bed. However, despite the non-ideal flow caused by the design compromises inherent in an IRFC and the resulting drop in effective catalyst activity, it was projected that for fuel cell systems with a current density greater than 400 mA cm -2, the IRFC would require less catalyst mass than a traditional system with external reformer. This is the result of an experimentally verified accelerated methanol conversion rate in the IRFC caused by the extraction of H 2 from the reforming reactor bed. Long-term stability of the IRFC due to acid leaching still needs to be addressed. Additionally, the extraction of H 2 from the reformer bed, which occurs in the IRFC, introduces concerns of failure due to coking.

  1. 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.

  2. 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.

  3. The Phosphoinositide-3-Kinase–Akt Signaling Pathway Is Important for Staphylococcus aureus Internalization by Endothelial Cells

    PubMed Central

    Oviedo-Boyso, Javier; Cortés-Vieyra, Ricarda; Huante-Mendoza, Alejandro; Yu, Hong B.; Valdez-Alarcón, Juan J.; Bravo-Patiño, Alejandro; Cajero-Juárez, Marcos; Finlay, B. Brett; Baizabal-Aguirre, Víctor M.

    2011-01-01

    Internalization of Staphylococcus aureus in bovine endothelial cells (BEC) is increased by tumor necrosis factor alpha stimulation and NF-κB activation. Because the phosphoinositide-3-kinase (PI3K)–Akt signaling pathway also modulates NF-κB activity, we considered whether the internalization of S. aureus by BEC is associated with the activity of PI3K and Akt. We found a time- and multiplicity of infection-dependent phosphorylation of Akt on Ser473 in BEC infected with S. aureus. This phosphorylation was inhibited by LY294002 (LY), indicating the participation of PI3K. Inhibition of either PI3K with LY or wortmannin, or Akt with SH-5, strongly reduced the internalization of S. aureus. Transfection of BEC with a dominant-negative form of the Akt gene significantly decreased S. aureus internalization, whereas transfection with the constitutively active mutant increased the number of internalized bacterium. Inhibition of PDK1 activity with OSU-03012 did not affect the level of S. aureus internalization, demonstrating that phosphorylation of Akt on Thr308 is not important for this process. Compared to the untreated control, the adherence of S. aureus to the surface of BEC was unaltered when cells were transfected or incubated with the pharmacological inhibitors. Furthermore, Akt activation by internalized S. aureus triggered a time-dependent phosphorylation of glycogen synthase kinase-3α (GSK-3α) on Ser21 and GSK-3β on Ser9 that was partially inhibited with SH-5. Finally, treatment of BEC with LY prior to S. aureus infection inhibited the NF-κB p65 subunit phosphorylation on Ser536, indicating the involvement of PI3K. These results suggest that PI3K-Akt activity is important for the internalization of S. aureus and phosphorylation of GSK-3α, GSK-3β, and NF-κB. PMID:21844240

  4. 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.

  5. 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

  6. Cladribine and cytarabine in refractory multisystem Langerhans cell histiocytosis: results of an international phase 2 study

    PubMed Central

    Bernard, Frederic; van Noesel, Max; Barkaoui, Mohamed; Bardet, Odile; Mura, Rosella; Arico, Maurizio; Piguet, Christophe; Gandemer, Virginie; Armari Alla, Corinne; Clausen, Niels; Jeziorski, Eric; Lambilliote, Anne; Weitzman, Sheila; Henter, Jan Inge; Van Den Bos, Cor

    2015-01-01

    An international phase 2 study combining cladribine and cytarabine (Ara-C) was initiated for patients with refractory, risk-organ–positive Langerhans cell histiocytosis (LCH) in 2005. The protocol, comprising at least two 5-day courses of Ara-C (1 g/m2 per day) plus cladribine (9 mg/m2 per day) followed by maintenance therapy, was administered to 27 patients (median age at diagnosis, 0.7 years; median follow-up, 5.3 years). At inclusion, all patients were refractory after at least 1 course of vinblastine (VBL) plus corticosteroid, all had liver and spleen involvement, and 25 patients had hematologic cytopenia. After 2 courses, disease status was nonactive (n = 2), better (n = 23), or stable (n = 2), with an overall response rate of 92%. Median disease activity scores decreased from 12 at the start of therapy to 3 after 2 courses (P < .0001). During maintenance therapy, 4 patients experienced reactivation in risk organs. There were 4 deaths; 2 were related to therapy toxicity and 2 were related to reactivation. All patients experienced severe toxicity, with World Health Organization grade 4 hematologic toxicity and 6 documented severe infections. The overall 5-year survival rate was 85% (95% confidence interval, 65.2%-94.2%). Thus, the combination of cladribine/Ara-C is effective therapy for refractory multisystem LCH but is associated with high toxicity. PMID:26194764

  7. Recurrent internal tandem duplications of BCOR in clear cell sarcoma of the kidney.

    PubMed

    Roy, Angshumoy; Kumar, Vijetha; Zorman, Barry; Fang, Erica; Haines, Katherine M; Doddapaneni, HarshaVardhan; Hampton, Oliver A; White, Simon; Bavle, Abhishek A; Patel, Nimesh R; Eldin, Karen W; John Hicks, M; Rakheja, Dinesh; Leavey, Patrick J; Skapek, Stephen X; Amatruda, James F; Nuchtern, Jed G; Chintagumpala, Murali M; Wheeler, David A; Plon, Sharon E; Sumazin, Pavel; Parsons, D Williams

    2015-01-01

    The X-linked BCL-6 co-repressor (BCOR) gene encodes a key constituent of a variant polycomb repressive complex (PRC) that is mutated or translocated in human cancers. Here we report on the identification of somatic internal tandem duplications (ITDs) clustering in the C terminus of BCOR in 23 of 27 (85%) pediatric clear cell sarcomas of the kidney (CCSK) from two independent cohorts. We profile CCSK tumours using a combination of whole-exome, transcriptome and targeted sequencing. Identical ITD mutations are found in primary and relapsed tumour pairs but not in adjacent normal kidney or blood. Mutant BCOR transcripts and proteins are markedly upregulated in ITD-positive tumours. Transcriptome analysis of ITD-positive CCSKs reveals enrichment for PRC2-regulated genes and similarity to undifferentiated sarcomas harbouring BCOR-CCNB3 fusions. The discovery of recurrent BCOR ITDs defines a major oncogenic event in this childhood sarcoma with significant implications for diagnostic and therapeutic approaches to this tumour. PMID:26573325

  8. Recurrent internal tandem duplications of BCOR in clear cell sarcoma of the kidney

    PubMed Central

    Roy, Angshumoy; Kumar, Vijetha; Zorman, Barry; Fang, Erica; Haines, Katherine M.; Doddapaneni, HarshaVardhan; Hampton, Oliver A.; White, Simon; Bavle, Abhishek A.; Patel, Nimesh R.; Eldin, Karen W.; John Hicks, M.; Rakheja, Dinesh; Leavey, Patrick J.; Skapek, Stephen X.; Amatruda, James F.; Nuchtern, Jed G.; Chintagumpala, Murali M.; Wheeler, David A.; Plon, Sharon E.; Sumazin, Pavel; Parsons, D. Williams

    2015-01-01

    The X-linked BCL-6 co-repressor (BCOR) gene encodes a key constituent of a variant polycomb repressive complex (PRC) that is mutated or translocated in human cancers. Here we report on the identification of somatic internal tandem duplications (ITDs) clustering in the C terminus of BCOR in 23 of 27 (85%) pediatric clear cell sarcomas of the kidney (CCSK) from two independent cohorts. We profile CCSK tumours using a combination of whole-exome, transcriptome and targeted sequencing. Identical ITD mutations are found in primary and relapsed tumour pairs but not in adjacent normal kidney or blood. Mutant BCOR transcripts and proteins are markedly upregulated in ITD-positive tumours. Transcriptome analysis of ITD-positive CCSKs reveals enrichment for PRC2-regulated genes and similarity to undifferentiated sarcomas harbouring BCOR–CCNB3 fusions. The discovery of recurrent BCOR ITDs defines a major oncogenic event in this childhood sarcoma with significant implications for diagnostic and therapeutic approaches to this tumour. PMID:26573325

  9. 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.

  10. 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

  11. 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

  12. Internalization and presentation of myelin antigens by the brain endothelium guides antigen-specific T cell migration

    PubMed Central

    Lopes Pinheiro, Melissa A; Kamermans, Alwin; Garcia-Vallejo, Juan J; van het Hof, Bert; Wierts, Laura; O'Toole, Tom; Boeve, Daniël; Verstege, Marleen; van der Pol, Susanne MA; van Kooyk, Yvette; de Vries, Helga E; Unger, Wendy WJ

    2016-01-01

    Trafficking of myelin-reactive CD4+ T-cells across the brain endothelium, an essential step in the pathogenesis of multiple sclerosis (MS), is suggested to be an antigen-specific process, yet which cells provide this signal is unknown. Here we provide direct evidence that under inflammatory conditions, brain endothelial cells (BECs) stimulate the migration of myelin-reactive CD4+ T-cells by acting as non-professional antigen presenting cells through the processing and presentation of myelin-derived antigens in MHC-II. Inflamed BECs internalized myelin, which was routed to endo-lysosomal compartment for processing in a time-dependent manner. Moreover, myelin/MHC-II complexes on inflamed BECs stimulated the trans-endothelial migration of myelin-reactive Th1 and Th17 2D2 cells, while control antigen loaded BECs did not stimulate T-cell migration. Furthermore, blocking the interaction between myelin/MHC-II complexes and myelin-reactive T-cells prevented T-cell transmigration. These results demonstrate that endothelial cells derived from the brain are capable of enhancing antigen-specific T cell recruitment. DOI: http://dx.doi.org/10.7554/eLife.13149.001 PMID:27336724

  13. 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.}

  14. BODIPY-labeled DC-SIGN-targeting glycodendrons efficiently internalize and route to lysosomes in human dendritic cells.

    PubMed

    Ribeiro-Viana, Renato; García-Vallejo, Juan J; Collado, Daniel; Pérez-Inestrosa, Ezequiel; Bloem, Karien; van Kooyk, Yvette; Rojo, Javier

    2012-10-01

    Glycodendrons bearing nine copies of mannoses or fucoses have been prepared by an efficient convergent strategy based on Cu(I) catalyzed azide-alkyne cycloaddition (CuAAC). These glycodendrons present a well-defined structure and have an adequate size and shape to interact efficiently with the C-type lectin DC-SIGN. We have selected a BODIPY derivative to label these glycodendrons due to its interesting physical and chemical properties as chromophore. These BODIPY-labeled glycodendrons were internalized into dendritic cells by mean of DC-SIGN. The internalized mannosylated and fucosylated dendrons are colocalized with LAMP1, which suggests routing to lysosomes. The interaction of these glycodendrons with DC-SIGN at the surface of dendritic cells did not induce maturation of the cells. Signaling analysis by checking different cytokines indicated also the lack of induction the expression of inflammatory and noninflammatory cytokines by these second generation glycodendrons. PMID:22920925

  15. Concise review: isolation and characterization of cells from human term placenta: outcome of the first international Workshop on Placenta Derived Stem Cells.

    PubMed

    Parolini, Ornella; Alviano, Francesco; Bagnara, Gian Paolo; Bilic, Grozdana; Bühring, Hans-Jörg; Evangelista, Marco; Hennerbichler, Simone; Liu, Bing; Magatti, Marta; Mao, Ning; Miki, Toshio; Marongiu, Fabio; Nakajima, Hideaki; Nikaido, Toshio; Portmann-Lanz, C Bettina; Sankar, Venkatachalam; Soncini, Maddalena; Stadler, Guido; Surbek, Daniel; Takahashi, Tsuneo A; Redl, Heinz; Sakuragawa, Norio; Wolbank, Susanne; Zeisberger, Steffen; Zisch, Andreas; Strom, Stephen C

    2008-02-01

    Placental tissue draws great interest as a source of cells for regenerative medicine because of the phenotypic plasticity of many of the cell types isolated from this tissue. Furthermore, placenta, which is involved in maintaining fetal tolerance, contains cells that display immunomodulatory properties. These two features could prove useful for future cell therapy-based clinical applications. Placental tissue is readily available and easily procured without invasive procedures, and its use does not elicit ethical debate. Numerous reports describing stem cells from different parts of the placenta, using nearly as numerous isolation and characterization procedures, have been published. Considering the complexity of the placenta, an urgent need exists to define, as clearly as possible, the region of origin and methods of isolation of cells derived from this tissue. On March 23-24, 2007, the first international Workshop on Placenta Derived Stem Cells was held in Brescia, Italy. Most of the research published in this area focuses on mesenchymal stromal cells isolated from various parts of the placenta or epithelial cells isolated from amniotic membrane. The aim of this review is to summarize and provide the state of the art of research in this field, addressing aspects such as cell isolation protocols and characteristics of these cells, as well as providing preliminary indications of the possibilities for use of these cells in future clinical applications. PMID:17975221

  16. Single-Cell Metabolite Profiling of Stalk and Glandular Cells of Intact Trichomes with Internal Electrode Capillary Pressure Probe Electrospray Ionization Mass Spectrometry.

    PubMed

    Nakashima, Taiken; Wada, Hiroshi; Morita, Satoshi; Erra-Balsells, Rosa; Hiraoka, Kenzo; Nonami, Hiroshi

    2016-03-15

    In this report, we developed the pressure probe electrospray ionization-mass spectrometry with internal electrode capillary (IEC-PPESI-MS) which enables high spatial-resolution cell sampling, precise postsampling manipulation, and high detection sensitivity. Using this technique, a comparative in situ single-cell metabolite profiling of stalk and glandular cells, the two adjacent cell types comprising a trichome unit in tomato plants (Solanum lycopersicum L.), were performed to clarify the extent of metabolic differentiation between two cell types as well as among different types of trichomes. Owing to high sensitivity of the system, less than a picoliter cell sap from a single stalk cell sufficiently yielded a number of peaks of amino acids, organic acids, carbohydrates, and flavonoids. The minimal cell sap removal from a stalk cell without severe disturbance of trichome structure enabled sequential analysis of adjacent glandular cell on the same trichome, which showed the presence of striking differences in metabolite compositions between two adjacent cell types. Comparison among different types of trichome also revealed significant variations in metabolite profiles, particularly in flavonoids and acyl sugars compositions. Some metabolites were found only in specific cell types or particular trichome types. Although extensive metabolomics analysis of glandular cells of tomato trichomes has been previously documented, this is the first report describing cell-to-cell variations in metabolite compositions of stalk and glandular cells as well as in different trichome types. Further application of this technique may provide new insights into distinct metabolism in plant cells displaying variations in shape, size, function and physicochemical properties. PMID:26845634

  17. Single-Cell Metabolite Profiling of Stalk and Glandular Cells of Intact Trichomes with Internal Electrode Capillary Pressure Probe Electrospray Ionization Mass Spectrometry.

    PubMed

    Nakashima, Taiken; Wada, Hiroshi; Morita, Satoshi; Erra-Balsells, Rosa; Hiraoka, Kenzo; Nonami, Hiroshi

    2016-03-15

    In this report, we developed the pressure probe electrospray ionization-mass spectrometry with internal electrode capillary (IEC-PPESI-MS) which enables high spatial-resolution cell sampling, precise postsampling manipulation, and high detection sensitivity. Using this technique, a comparative in situ single-cell metabolite profiling of stalk and glandular cells, the two adjacent cell types comprising a trichome unit in tomato plants (Solanum lycopersicum L.), were performed to clarify the extent of metabolic differentiation between two cell types as well as among different types of trichomes. Owing to high sensitivity of the system, less than a picoliter cell sap from a single stalk cell sufficiently yielded a number of peaks of amino acids, organic acids, carbohydrates, and flavonoids. The minimal cell sap removal from a stalk cell without severe disturbance of trichome structure enabled sequential analysis of adjacent glandular cell on the same trichome, which showed the presence of striking differences in metabolite compositions between two adjacent cell types. Comparison among different types of trichome also revealed significant variations in metabolite profiles, particularly in flavonoids and acyl sugars compositions. Some metabolites were found only in specific cell types or particular trichome types. Although extensive metabolomics analysis of glandular cells of tomato trichomes has been previously documented, this is the first report describing cell-to-cell variations in metabolite compositions of stalk and glandular cells as well as in different trichome types. Further application of this technique may provide new insights into distinct metabolism in plant cells displaying variations in shape, size, function and physicochemical properties.

  18. Hematopoietic cell transplantation for mucopolysaccharidosis patients is safe and effective: results after implementation of international guidelines.

    PubMed

    Aldenhoven, Mieke; Jones, Simon A; Bonney, Denise; Borrill, Roisin E; Coussons, Mary; Mercer, Jean; Bierings, Marc B; Versluys, Birgitta; van Hasselt, Peter M; Wijburg, Frits A; van der Ploeg, Ans T; Wynn, Robert F; Boelens, Jaap Jan

    2015-06-01

    Allogeneic hematopoietic cell transplantation (HCT) is the only treatment able to prevent progressive neurodegenerative disease in a selected group of mucopolysaccharidosis (MPS) disorders. However, its use was historically limited by the high risk of graft failure and transplantation-related morbidity and mortality. Therefore, since 2005 new international HCT guidelines for MPS disorders were proposed. The survival and graft outcomes of MPS patients receiving HCT according to these guidelines in 2 European centers of expertise were evaluated. Two consecutive conditioning regimens were used, busulfan/cyclophosphamide or fludarabine/busulfan-based, both with exposure-targeted i.v. busulfan. A noncarrier matched sibling donor (MSD), matched unrelated cord blood (UCB), or matched unrelated donor (MUD) were considered to be preferred donors. If not available, a mismatched UCB donor was used. Participants were 62 MPS patients (56 MPS type I-Hurler, 2 MPS type II, 2 MPS type III, and 2 MPS type VI) receiving HCT at median age 13.5 months (range, 3 to 44). Forty-one patients received a UCB donor, 17 MSD, and 4 MUD. High overall survival (95.2%) and event-free survival (90.3%) were achieved with only low toxicity: 13.3% acute graft-versus-host disease aGVHD) grades II to IV and 14.8% chronic GVHD (1.9% extensive). A mismatched donor predicted for lower event-free survival (P = .04). A higher age at HCT was a predictor for both aGVHD (P = .001) and chronic GVHD (P = .01). The use of a mismatched donor was a predictor for aGVHD (P = .01). Higher rates of full-donor chimerism were achieved in successfully transplanted UCB recipients compared with MSD/MUD (P = .002). If complying with the international HCT guidelines, HCT in MPS patients results in high safety and efficacy. This allows extension of HCT to more attenuated MPS types. Because a younger age at HCT is associated with reduction of HCT-related toxicity, newborn screening may further increase safety. PMID

  19. 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.

  20. Effects of niacin on Staphylococcus aureus internalization into bovine mammary epithelial cells by modulating NF-κB activation.

    PubMed

    Wei, Zhengkai; Fu, Yunhe; Zhou, Ershun; Tian, Yuan; Yao, Minjun; Li, Yimeng; Yang, Zhengtao; Cao, Yongguo

    2014-01-01

    Niacin is a precursor of coenzymes NAD and NADP and plays a critical role in electron transfer during the metabolic process. In addition to its nutrimental function, niacin has long been used for the treatment of lipid disorders and cardiovascular disease. However, the effect of niacin on Staphylococcus aureus (S. aureus) internalization into bovine mammary epithelial cells (bMEC) remains unclear. Here we sought to examine the effect of niacin on S. aureus internalization into bovine mammary epithelial cells (bMEC) and to investigate the potential mechanism. In this study, the growth of S. aureus supplemented with niacin (0.5-2 mM) was monitored turbidimetrically at 600 nm for 24 h and cell viability was measured by MTT assay. Gentamicin protection assay was carried out to determine the effect of niacin on S. aureus internalization into bMEC. To determine the potential mechanism, tracheal antimicrobial peptide (TAP) and β-defensin (BNBD5) expressions were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The activation of nuclear factor-kappa B (NF-κB) was determined by Western blotting. The results showed that niacin (0.5-2 mM) did not affect S. aureus growth and bMEC viability, whereas it inhibits S. aureus internalization ranging from 13% to 42% and down-regulated the mRNA expression of TAP and BNBD5 compared to the control group. No exactly relationship was discovered between S. aureus internalization into bMEC and antimicrobial peptide expression, while niacin inhibited S. aureus-induced NF-κB activation in a dose manner. These dates suggest that inhibiting NF-κB activation may be the potential mechanism of niacin on modulating S. aureus internalization into bMEC.

  1. 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.

  2. 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. PMID:24349195

  3. Renal cell carcinoma and synchronous thyroid metastasis with neoplastic thrombosis of the internal jugular vein: report of a case.

    PubMed

    Matei, Deliu-Victor; Brescia, Antonio; Nordio, Andrea; Spinelli, Matteo Giulio; Melegari, Sara; Cozzi, Gabriele; Andrioli, Massimiliano; Salvatori, Pietro

    2011-12-01

    A case of thyroid metastasis of a renal clear cell carcinoma is presented. The fine-needle aspiration cytology pointed out the primary tumor origin. The patient underwent robot-assisted radical nephrectomy and contextual thyroidectomy. During the operative procedure, a neoplastic thrombus extending from the thyroid metastasis and protruding into the internal jugular vein was found. As a result, thrombectomy and ligation of the internal jugular vein were required. In cases of single synchronous thyroid metastases form RCC, radical surgery should be advisable. Robotic approach allows to associate major surgery procedures, as nephrectomy, with radical metastasectomy.

  4. Adhesion of living cells revealed by variable-angle total internal reflection fluorescence microscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

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

    2016-02-01

    Total Internal Reflection Fluorescence Microscopy (TIRFM) is a widespread technique to study cellular process occurring near the contact region with the glass substrate. In this field, determination of the accurate distance from the surface to the plasma membrane constitutes a crucial issue to investigate the physical basis of cellular adhesion process. However, quantitative interpretation of TIRF pictures regarding the distance z between a labeled membrane and the substrate is not trivial. Indeed, the contrast of TIRF images depends on several parameters more and less well known (local concentration of dyes, absorption cross section, angular emission pattern…). The strategy to get around this problem is to exploit a series of TIRF pictures recorded at different incident angles in evanescent regime. This technique called variable-angle TIRF microscopy (vaTIRFM), allowing to map the membrane-substrate separation distance with a nanometric resolution (10-20 nm). vaTIRFM was developed by Burmeister, Truskey and Reichert in the early 1990s with a prism-based TIRF setup [Journal of Microscopy 173, 39-51 (1994)]. We propose a more convenient prismless setup, which uses only a rotatable mirror to adjust precisely the laser beam on the back focal plane of the oil immersion objective (no azimuthal scanning is needed). The series of TIRF images permit us to calculate accurately membrane-surface distances in each pixel. We demonstrate that vaTIRFM are useful to quantify the adhesion of living cells for specific and unspecific membrane-surface interactions, achieved on various functionalized substrates with polymers (BSA, poly-L-lysin) or extracellular matrix proteins (collagen and fibronectin).

  5. 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

  6. (−)-Epigallocatechin gallate causes internalization of the epidermal growth factor receptor in human colon cancer cells

    PubMed Central

    Adachi, Seiji; Nagao, Tomokazu; To, Satoshi; Joe, Andrew K.; Shimizu, Masahito; Matsushima-Nishiwaki, Rie; Kozawa, Osamu; Moriwaki, Hisataka; Maxfield, Frederick R.; Weinstein, I.Bernard

    2008-01-01

    We recently found that the inhibitory effect of (−)-epigallocatechin gallate (EGCG) on epidermal growth factor (EGF) binding to the epidermal growth factor receptor (EGFR) is associated with alterations in lipid organization in the plasma membrane of colon cancer cells. Since changes in lipid organizations are thought to play a role in the trafficking of several membrane proteins, in this study we examined the effects of EGCG on cellular localization of the EGFR in SW480 cells. Treatment of the cells for 30 min with as little as 1 μg/ml of EGCG caused a decrease in cell surface-associated EGFRs and this was associated with internalization of EGFRs into endosomal vesicles. Similar effects were seen with a green fluorescent protein (GFP)–EGFR fusion protein. As expected, the EGFR protein was phosphorylated at tyrosine residues, ubiquitinated and partially degraded when the cells were treated with EGF, but treatment with EGCG caused none of these effects. The loss of EGFRs from the cell surface induced by treating the cells with EGF for 30 min persisted for at least 2 h. However, the loss of EGFRs from the cell surface induced by temporary exposure to EGCG was partially restored within 1–2 h. These studies provide the first evidence that EGCG can induce internalization of EGFRs into endosomes, which can recycle back to the cell surface. This sequestrating of inactivated EGFRs into endosomes may explain, at least in part, the ability of EGCG to inhibit activation of the EGFR and thereby exert anticancer effects. PMID:18586691

  7. International stem cell tourism and the need for effective regulation. Part I: Stem cell tourism in Russia and India: clinical research, innovative treatment, or unproven hype?

    PubMed

    Cohen, Cynthia B; Cohen, Peter J

    2010-03-01

    Persons with serious and disabling medical conditions have traveled abroad in search of stem cell treatments in recent years. However, weak or nonexistent oversight systems in some countries provide insufficient patient protections against unproven stem cell treatments, raising concerns about exposure to harm and exploitation. The present article, the first of two, describes and analyzes stem cell tourism in Russia and India and addresses several scientific/medical, ethical, and policy issues raised by the provision of unproven stem cell-based treatments within them. The distinction between treatment based on proven clinical research and "innovative treatment" is addressed and the authors conclude that the innovations at issue constitute neither. Regulatory measures need to be developed or strengthened in accord with internationally accepted standards in such countries to protect those seeking stem cell treatments. PMID:20506693

  8. 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.

  9. 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

  10. 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.

  11. Internalization of the Extracellular Full-Length Tau Inside Neuro2A and Cortical Cells Is Enhanced by Phosphorylation.

    PubMed

    Wauters, Mathilde; Wattiez, Ruddy; Ris, Laurence

    2016-08-19

    Tau protein is mainly intracellular. However, several studies have demonstrated that full-length Tau can be released into the interstitial fluid of the brain. The physiological or pathological function of this extracellular Tau remains unknown. Moreover, as evidence suggests, extracellular Tau aggregates can be internalized by neurons, seeding Tau aggregation. However, much less is known about small species of Tau. In this study, we hypothesized that the status of phosphorylation could alter the internalization of recombinant Tau in Neuro2A and cortical cells. Our preliminary results revealed that the highly phosphorylated form of Tau entered the cells ten times more easily than a low phosphorylated one. This suggests that hyperphosphorylated Tau protein could spread between neurons in pathological conditions such as Alzheimer's disease.

  12. Internalization of the Extracellular Full-Length Tau Inside Neuro2A and Cortical Cells Is Enhanced by Phosphorylation

    PubMed Central

    Wauters, Mathilde; Wattiez, Ruddy; Ris, Laurence

    2016-01-01

    Tau protein is mainly intracellular. However, several studies have demonstrated that full-length Tau can be released into the interstitial fluid of the brain. The physiological or pathological function of this extracellular Tau remains unknown. Moreover, as evidence suggests, extracellular Tau aggregates can be internalized by neurons, seeding Tau aggregation. However, much less is known about small species of Tau. In this study, we hypothesized that the status of phosphorylation could alter the internalization of recombinant Tau in Neuro2A and cortical cells. Our preliminary results revealed that the highly phosphorylated form of Tau entered the cells ten times more easily than a low phosphorylated one. This suggests that hyperphosphorylated Tau protein could spread between neurons in pathological conditions such as Alzheimer’s disease. PMID:27548242

  13. Internalization of the Extracellular Full-Length Tau Inside Neuro2A and Cortical Cells Is Enhanced by Phosphorylation.

    PubMed

    Wauters, Mathilde; Wattiez, Ruddy; Ris, Laurence

    2016-01-01

    Tau protein is mainly intracellular. However, several studies have demonstrated that full-length Tau can be released into the interstitial fluid of the brain. The physiological or pathological function of this extracellular Tau remains unknown. Moreover, as evidence suggests, extracellular Tau aggregates can be internalized by neurons, seeding Tau aggregation. However, much less is known about small species of Tau. In this study, we hypothesized that the status of phosphorylation could alter the internalization of recombinant Tau in Neuro2A and cortical cells. Our preliminary results revealed that the highly phosphorylated form of Tau entered the cells ten times more easily than a low phosphorylated one. This suggests that hyperphosphorylated Tau protein could spread between neurons in pathological conditions such as Alzheimer's disease. PMID:27548242

  14. Surface Phosphatidylserine Is Responsible for the Internalization on Microvesicles Derived from Hypoxia-Induced Human Bone Marrow Mesenchymal Stem Cells into Human Endothelial Cells

    PubMed Central

    Liu, Chaozhong; Wang, Lisheng; Xiao, Fengjun; Zhang, Hongchao

    2016-01-01

    Background Previous data have proven that microvesicles derived from hypoxia-induced mesenchymal stem cells (MSC-MVs) can be internalized into endothelial cells, enhancing their proliferation and vessel structure formation and promoting in vivo angiogenesis. However, there is a paucity of information about how the MSC-MVs are up-taken by endothelial cells. Methods MVs were prepared from the supernatants of human bone marrow MSCs that had been exposed to a hypoxic and/or serum-deprivation condition. The incorporation of hypoxia-induced MSC-MVs into human umbilical cord endothelial cells (HUVECs) was observed by flow cytometry and confocal microscopy in the presence or absence of recombinant human Annexin-V (Anx-V) and antibodies against human CD29 and CD44. Further, small interfering RNA (siRNA) targeted at Anx-V and PSR was delivered into HUVECs, or HUVECs were treated with a monoclonal antibody against phosphatidylserine receptor (PSR) and the cellular internalization of MVs was re-assessed. Results The addition of exogenous Anx-V could inhibit the uptake of MVs isolated from hypoxia-induced stem cells by HUVECs in a dose- and time-dependent manner, while the anti-CD29 and CD44 antibodies had no effect on the internalization process. The suppression was neither observed in Anx-V siRNA-transfected HUVECs, however, addition of anti-PSR antibody and PSR siRNA-transfected HUVECs greatly blocked the incorporation of MVs isolated from hypoxia-induced stem cells into HUVECs. Conclusion PS on the MVs isolated from hypoxia-induced stem cells is the critical molecule in the uptake by HUVECs. PMID:26808539

  15. Internalization of cycloheptaamylose-dansyl chloride complex during labelling of surface membrane in living Paramecium aurelia cells.

    PubMed

    Giordano, P A; Wyroba, E; Bottiroli, G

    1985-01-01

    Internalization of cycloheptaamylose-dansyl chloride complex during surface labelling of living long-term starved Paramecium aurelia cells has been observed. This process may be inhibited by pretreatment of the ciliates with dichloroisoproterenol. Uptake of cycloheptaamylose-dansyl chloride may be visualized only after UV preirradiation: the appearance of orange-fluorescing vacuoles of diameter 2.3-4.5 micron may then be observed. Microspectrographic analysis performed on the cells and dansyl derivatives indicates that this fluorescence is produced by a photochemical reaction of dansyl chloride - released from CDC complex inside the digestive vacuoles-under the influence of UV irradiation.

  16. Effect of internal structure of collagen/hydroxyapatite scaffold on the osteogenic differentiation of mesenchymal stem cells.

    PubMed

    Chen, Guobao; Lv, Yonggang; Dong, Chanjuan; Yang, Li

    2015-01-01

    Consisting of seed cells and scaffold, regenerative medicine provides a new way for the repair and regeneration of tissue and organ. Collagen/hydroxyapatite (HA) biocomposite scaffold is highlighted due to its advantageous features of two major components of bone matrix: collagen and HA. The aim of this study is to investigate the effect of internal structure of collagen/HA scaffold on the fate of rat mesenchymal stem cells (MSCs). The internal structure of collagen/HA scaffold was characterized by micro-CT. It is found that the porosity decreased while average compressive modulus increased with the increase of collagen proportion. Within the collagen proportion of 0.35%, 0.5% and 0.7%, the porosities were 89.08%, 78.37% and 75.36%, the pore sizes were 140.6±75.5 μm, 133.9±48.4 μm and 160.7±119.6 μm, and the average compressive moduli were 6.74±1.16 kPa, 8.82±2.12 kPa and 23.61±8.06 kPa, respectively. Among these three kinds of scaffolds, MSCs on the Col 0.35/HA 22 scaffold have the highest viability and the best cell proliferation. On the contrary, the Col 0.7/HA 22 scaffold has the best ability to stimulate MSCs to differentiate into osteoblasts in a relatively short period of time. In vivo research also demonstrated that the internal structure of collagen/HA scaffold has significant effect on the cell infiltration. Therefore, precise control of the internal structure of collagen/HA scaffold can provide a more efficient carrier to the repair of bone defects.

  17. 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.

  18. 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

  19. 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.

  20. Internalization and processing of Bacillus anthracis lethal toxin by toxin-sensitive and -resistant cells.

    PubMed

    Singh, Y; Leppla, S H; Bhatnagar, R; Friedlander, A M

    1989-07-01

    Anthrax lethal toxin consists of two separate proteins, protective antigen and lethal factor (LF). Certain macrophages and a mouse macrophage-like cell line, J774A.1, are lysed by low concentrations of lethal toxin. In contrast, another macrophage cell line, IC-21, and all other cell types tested were resistant to this toxin. To discover the basis for this difference, each step in the intoxication process was examined. No differences between sensitive and resistant cells were found in receptor binding or proteolytic activation of protective antigen, steps that are required prior to LF binding. To determine whether resistance results from a defect in translocation to the cytosol, we introduced LF into J774A.1 and IC-21 cells and a nonmacrophage cell line (L6 myoblast) by osmotic lysis of pinocytic vesicles. Only J774A.1 cells were lysed; no effect was observed in IC-21 and L6 cells. These results suggest that resistant cells either lack the intracellular target of LF or fail to process LF to an active form. The relatively low potency of LF introduced into J774A.1 cells by osmotic lysis suggests that protective antigen may also be required at a stage subsequent to endocytosis. PMID:2500434

  1. 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

  2. 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.

  3. Monitoring penetratin interactions with lipid membranes and cell internalization using a new hydration-sensitive fluorescent probe.

    PubMed

    Zamotaiev, Oleksandr M; Postupalenko, Viktoriia Y; Shvadchak, Volodymyr V; Pivovarenko, Vasyl G; Klymchenko, Andrey S; Mély, Yves

    2014-09-28

    A new fluorescent label N-[4′-(dimethylamino)-3-hydroxyflavone-7-yl]-N-methyl-β-alanine (7AF) was synthesized. Due to two electron donor groups at the opposite ends of the chromophore, an excited state intramolecular proton transfer (ESIPT) resulting in a dual emission was observed even in highly polar media and its fluorescence quantum yield was found to be remarkably high in a broad range of solvents including water. As a consequence, this label exhibits a remarkable sensitivity to the hydration of its environment, which is observed as a color switch between the emission of the ESIPT product (T* form) and that of the normal N* form. The 7AF label was coupled to the N-terminus of penetratin, a cell penetrating peptide, in order to study its interactions with lipid membranes and internalization inside the cells. As expected, the binding of penetratin to lipid membranes resulted in a dramatic switch in the relative intensity of its two emission bands as compared to its emission in buffer. Our studies with different lipid compositions confirmed the preference of penetratin to lipid membranes of the liquid disordered phase. After incubation of low concentrations of labeled penetratin with living cells, ratiometric imaging revealed, in addition to membrane-bound species, a significant fraction of free peptide in cytosol showing the characteristic emission from aqueous medium. At higher concentrations of penetratin, mainly peptides bound to cell membrane structures were observed. These observations confirmed the ability of penetratin to enter the cytosol by direct translocation through the cell plasma membrane, in addition to the classical entry by endocytosis. The present probe constitutes thus a powerful tool to study the interaction of peptides with living cells and their internalization mechanisms. PMID:25072870

  4. Capsaicin induces NKCC1 internalization and inhibits chloride secretion in colonic epithelial cells independently of TRPV1.

    PubMed

    Bouyer, Patrice G; Tang, Xu; Weber, Christopher R; Shen, Le; Turner, Jerrold R; Matthews, Jeffrey B

    2013-01-15

    Colonic chloride secretion is regulated via the neurohormonal and immune systems. Exogenous chemicals (e.g., butyrate, propionate) can affect chloride secretion. Capsaicin, the pungent ingredient of the chili peppers, exerts various effects on gastrointestinal function. Capsaicin is known to activate the transient receptor potential vanilloid type 1 (TRPV1), expressed in the mesenteric nervous system. Recent studies have also demonstrated its presence in epithelial cells but its role remains uncertain. Because capsaicin has been reported to inhibit colonic chloride secretion, we tested whether this effect of capsaicin could occur by direct action on epithelial cells. In mouse colon and model T84 human colonic epithelial cells, we found that capsaicin inhibited forskolin-dependent short-circuit current (FSK-I(sc)). Using PCR and Western blot, we demonstrated the presence of TRPV1 in colonic epithelial cells. In T84 cells, TRPV1 localized at the basolateral membrane and in vesicular compartments. In permeabilized monolayers, capsaicin activated apical chloride conductance, had no effect on basolateral potassium conductance, but induced NKCC1 internalization demonstrated by immunocytochemistry and basolateral surface biotinylation. AMG-9810, a potent inhibitor of TRPV1, did not prevent the inhibition of the FSK-I(sc) by capsaicin. Neither resiniferatoxin nor N-oleoyldopamine, two selective agonists of TRPV1, blocked the FSK-I(sc). Conversely capsaicin, resiniferatoxin, and N-oleoyldopamine raised intracellular calcium ([Ca(2+)](i)) in T84 cells and AMG-9810 blocked the rise in [Ca(2+)](i) induced by capsaicin and resiniferatoxin suggesting the presence of a functional TRPV1 channel. We conclude that capsaicin inhibits chloride secretion in part by causing NKCC1 internalization, but by a mechanism that appears to be independent of TRPV1. PMID:23139219

  5. Reduced in vitro T-cell responses induced by glutaraldehyde-modified allergen extracts are caused mainly by retarded internalization of dendritic cells.

    PubMed

    Heydenreich, Bärbel; Bellinghausen, Iris; Lorenz, Steffen; Henmar, Helene; Strand, Dennis; Würtzen, Peter A; Saloga, Joachim

    2012-06-01

    Although allergen-specific immunotherapy is a clinically effective therapy for IgE-mediated allergic diseases, the risk of IgE-mediated adverse effects still exists. For this reason, chemically modified allergoids have been introduced, which may destroy IgE-binding sites while T-cell activation should be retained. The aim of the study was to analyse the differences between intact allergens and differently modified/aggregated allergoids concerning their internalization as well as T-cell and basophil activation. For this purpose human monocyte-derived immature dendritic cells (DC) were incubated with Phleum pratense or Betula verrucosa pollen extract or with the corresponding allergoids, modified with formaldehyde or glutaraldehyde. After an additional maturation process, the antigen-loaded mature DC were co-cultured with autologous CD4(+) T cells. Allergenicity was tested by leukotriene release from basophils. In addition, the uptake of intact allergens and allergoids by immature DC was analysed. The proliferation of, as well as the interleukin-4 (IL-4), IL-10, IL-13 and interferon-γ production by, CD4(+) T cells which had been stimulated with glutaraldehyde allergoid-treated DC was reduced compared with CD4(+) T cells stimulated with intact allergen-treated or formaldehyde allergoid-treated DC. In line with this, glutaraldehyde-modified allergoids were more aggregated and were internalized more slowly. Furthermore, only the allergoids modified with glutaraldehyde induced a decreased leukotriene release by activated basophils. These findings suggest that IgE-reactive epitopes were destroyed more efficiently by modification with glutaraldehyde than with formaldehyde under the conditions chosen for these investigations. Glutaraldehyde-modified allergoids also displayed lower T-cell stimulatory capacity, which is mainly the result of greater modification/aggregation and diminished uptake by DC.

  6. Cholecalciferol (vitamin D) differentially regulates antimicrobial peptide expression in bovine mammary epithelial cells: implications during Staphylococcus aureus internalization.

    PubMed

    Téllez-Pérez, Ana Dolores; Alva-Murillo, Nayeli; Ochoa-Zarzosa, Alejandra; López-Meza, Joel E

    2012-11-01

    Vitamin D has immunomodulatory functions regulating the expression of host defense genes. The aim of this study was to determine the effect of cholecalciferol (vitamin D3) on S. aureus internalization into bovine mammary epithelial cells (bMEC) and antimicrobial peptide (AP) mRNA expression. Cholecalciferol (1-200 nM) did not affect S. aureus growth and bMEC viability; but it reduced bacterial internalization into bMEC (15-74%). Also, bMEC showed a basal expression of all AP genes evaluated, which were induced by S. aureus. Cholecalciferol alone or together with bacteria diminished tracheal antimicrobial peptide (TAP) and bovine neutrophil β-defensin (BNBD) 5 mRNA expression; while alone induced the expression of lingual antimicrobial peptide (LAP), bovine β-defensin 1 (DEFB1) and bovine psoriasin (S100A7), which was inhibited in the presence of S. aureus. This compound (50 nM) increased BNBD10 mRNA expression coinciding with the greatest reduction in S. aureus internalization. Genes of vitamin D pathway (25-hydroxylase and 1 α-hydroxylase) show basal expression, which was induced by cholecalciferol or bacteria. S. aureus induced vitamin D receptor (VDR) mRNA expression, but not in the presence of cholecalciferol. In conclusion, cholecalciferol can reduce S. aureus internalization and differentially regulates AP expression in bMEC. Thus, vitamin D could be an effective innate immunity modulator in mammary gland, which leads to a better defense against bacterial infection.

  7. 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 .

  8. 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.

  9. Intracellular distribution of TM4SF1 and internalization of TM4SF1-antibody complex in vascular endothelial cells.

    PubMed

    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. PMID:26241677

  10. Nitrergic neuromuscular transmission in the mouse internal anal sphincter is accomplished by multiple pathways and postjunctional effector cells

    PubMed Central

    Sotherton, A. G.; Peri, L. E.; Sanders, K. M.; Ward, S. M.; Keef, K. D.

    2014-01-01

    The effector cells and second messengers participating in nitrergic neuromuscular transmission (NMT) were investigated in the mouse internal anal sphincter (IAS). Protein expression of guanylate cyclase (GCα, GCβ) and cyclic GMP-dependent protein kinase I (cGKI) were examined in cryostat sections with dual-labeling immunohistochemical techniques in PDGFRα+ cells, interstitial cells of Cajal (ICC), and smooth muscle cells (SMC). Gene expression levels were determined with quantitative PCR of dispersed cells from Pdgfrαegfp/+, KitcopGFP/+, and smMHCCre-egfp mice sorted with FACS. The relative gene and protein expression levels of GCα and GCβ were PDGFRα+ cells > ICC ≫ SMC. In contrast, cGKI gene expression sequence was SMC = ICC > PDGFRα+ cells whereas cGKI protein expression sequence was neurons > SMC ≫ ICC = PDGFRα+ cells. The functional role of cGKI was investigated in cGKI−/− mice. Relaxation with 8-bromo (8-Br)-cGMP was greatly reduced in cGKI−/− mice whereas responses to sodium nitroprusside (SNP) were partially reduced and forskolin responses were unchanged. A nitrergic relaxation occurred with nerve stimulation (NS, 5 Hz, 60 s) in cGKI+/+ and cGKI−/− mice although there was a small reduction in the cGKI−/− mouse. Nω-nitro-l-arginine (l-NNA) abolished responses during the first 20–30 s of NS in both animals. The GC inhibitor ODQ greatly reduced or abolished SNP and nitrergic NS responses in both animals. These data confirm an essential role for GC in NO-induced relaxation in the IAS. However, the expression of GC and cGKI by all three cell types suggests that each may participate in coordinating muscular responses to NO. The persistence of nitrergic NMT in the cGKI−/− mouse suggests the presence of a significant GC-dependent, cGKI-independent pathway. PMID:25301187

  11. All-In-One: Advanced preparation of Human Parenchymal and Non-Parenchymal Liver Cells

    PubMed Central

    Werner, Melanie; Driftmann, Sabrina; Kleinehr, Kathrin; Kaiser, Gernot M.; Mathé, Zotlan; Treckmann, Juergen-Walter; Paul, Andreas; Skibbe, Kathrin; Timm, Joerg; Canbay, Ali; Gerken, Guido; Schlaak, Joerg F.; Broering, Ruth

    2015-01-01

    Background & Aims Liver cells are key players in innate immunity. Thus, studying primary isolated liver cells is necessary for determining their role in liver physiology and pathophysiology. In particular, the quantity and quality of isolated cells are crucial to their function. Our aim was to isolate a large quantity of high-quality human parenchymal and non-parenchymal cells from a single liver specimen. Methods Hepatocytes, Kupffer cells, liver sinusoidal endothelial cells, and stellate cells were isolated from liver tissues by collagenase perfusion in combination with low-speed centrifugation, density gradient centrifugation, and magnetic-activated cell sorting. The purity and functionality of cultured cell populations were controlled by determining their morphology, discriminative cell marker expression, and functional activity. Results Cell preparation yielded the following cell counts per gram of liver tissue: 2.0±0.4×107 hepatocytes, 1.8±0.5×106 Kupffer cells, 4.3±1.9×105 liver sinusoidal endothelial cells, and 3.2±0.5×105 stellate cells. Hepatocytes were identified by albumin (95.5±1.7%) and exhibited time-dependent activity of cytochrome P450 enzymes. Kupffer cells expressed CD68 (94.5±1.2%) and exhibited phagocytic activity, as determined with 1μm latex beads. Endothelial cells were CD146+ (97.8±1.1%) and exhibited efficient uptake of acetylated low-density lipoprotein. Hepatic stellate cells were identified by the expression of α-smooth muscle actin (97.1±1.5%). These cells further exhibited retinol (vitamin A)-mediated autofluorescence. Conclusions Our isolation procedure for primary parenchymal and non-parenchymal liver cells resulted in cell populations of high purity and quality, with retained physiological functionality in vitro. Thus, this system may provide a valuable tool for determining liver function and disease. PMID:26407160

  12. The business of human embryonic stem cell research and an international analysis of relevant laws.

    PubMed

    De Trizio, Ella; Brennan, Christopher S

    2004-01-01

    Few sciences have held out such therapeutic promise and correspondingly stirred so much controversy in countries throughout the world as the developing science surrounding human embryonic stem cells. Since the first reported development of several lines of human embryonic stem cells in 1988, many governments around the world have attempted to address the thorny ethical issues raised by human embryonic stem cell research by the passage of laws. In some cases these laws have directly regulated governmental funding of the science; in other cases they have created a legal environment that has either encouraged or discouraged both governmental and private funding of the science. This article first differentiates human embryonic stem cells from other types of stem cells and frames the ethical controversy surrounding human embryonic stem cell research, then surveys laws governing human embryonic stem cell research in various scientifically advanced countries located throughout the Pacific Rim, Europe and North America and explains the impact these laws have had on governmental and private funding of human embryonic stem cell research.

  13. 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.

  14. 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.

  15. Differential internalization of amphotericin B--conjugated nanoparticles in human cells and the expression of heat shock protein 70.

    PubMed

    Paulo, Cristiana S O; Lino, Miguel M; Matos, António A; Ferreira, Lino S

    2013-07-01

    Although a variety of nanoparticles (NPs) functionalized with amphotericin B, an antifungal agent widely used in the clinic, have been studied in the last years their cytotoxicity profile remains elusive. Here we show that human endothelial cells take up high amounts of silica nanoparticles (SNPs) conjugated with amphotericin B (AmB) (SNP-AmB) (65.4 ± 12.4 pg of Si per cell) through macropinocytosis while human fibroblasts internalize relatively low amounts (2.3 ± 0.4 pg of Si per cell) because of their low capacity for macropinocytosis. We further show that concentrations of SNP-AmB and SNP up to 400 μg/mL do not substantially affect fibroblasts. In contrast, endothelial cells are sensitive to low concentrations of NPs (above 10 μg/mL), in particular to SNP-AmB. This is because of their capacity to internalize high concentration of NPs and high sensitivity of their membrane to the effects of AmB. Low-moderate concentrations of SNP-AmB (up to 100 μg/mL) induce the production of reactive oxygen species (ROS), LDH release, high expression of pro-inflammatory cytokines and chemokines (IL-8, IL-6, G-CSF, CCL4, IL-1β and CSF2) and high expression of heat shock proteins (HSPs) at gene and protein levels. High concentrations of SNP-AmB (above 100 μg/mL) disturb membrane integrity and kill rapidly human cells (60% after 5 h). This effect is higher in SNP-AmB than in SNP.

  16. S1PR4 Signaling Attenuates ILT 7 Internalization To Limit IFN-α Production by Human Plasmacytoid Dendritic Cells.

    PubMed

    Dillmann, Christina; Ringel, Christian; Ringleb, Julia; Mora, Javier; Olesch, Catherine; Fink, Annika F; Roberts, Edward; Brüne, Bernhard; Weigert, Andreas

    2016-02-15

    Plasmacytoid dendritic cells (pDCs) produce large amounts of type I IFN in response to TLR7/9 ligands. This conveys antiviral effects, activates other immune cells (NK cells, conventional DCs, B, and T cells), and causes the induction and expansion of a strong inflammatory response. pDCs are key players in various type I IFN-driven autoimmune diseases such as systemic lupus erythematosus or psoriasis, but pDCs are also involved in (anti-)tumor immunity. The sphingolipid sphingosine-1-phosphate (S1P) signals through five G-protein-coupled receptors (S1PR1-5) to regulate, among other activities, immune cell migration and activation. The present study shows that S1P stimulation of human, primary pDCs substantially decreases IFN-α production after TLR7/9 activation with different types of CpG oligodeoxynucleotides or tick-borne encephalitis vaccine, which occurred in an S1PR4-dependent manner. Mechanistically, S1PR4 activation preserves the surface expression of the human pDC-specific inhibitory receptor Ig-like transcript 7. We provide novel information that Ig-like transcript 7 is rapidly internalized upon receptor-mediated endocytosis of TLR7/9 ligands to allow high IFN-α production. This is antagonized by S1PR4 signaling, thus decreasing TLR-induced IFN-α secretion. At a functional level, attenuated IFN-α production failed to alter Ag-driven T cell proliferation in pDC-dependent T cell activation assays, but shifted cytokine production of T cells from a Th1 (IFN-γ) to a regulatory (IL-10) profile. In conclusion, S1PR4 agonists block human pDC activation and may therefore be a promising tool to restrict pathogenic IFN-α production. PMID:26783340

  17. Voltage-dependent block by internal Ca2+ ions of inwardly rectifying K+ channels in guinea-pig ventricular cells.

    PubMed Central

    Matsuda, H; Cruz, J dos S

    1993-01-01

    1. The block of the inwardly rectifying K+ channel by intracellular Ca2+ was studied in guinea-pig ventricular cells. 2. Single-channel currents through the inwardly rectifying K+ channel were recorded in the inside-out configuration at 150 mM external and internal K+. Internal Ca2+, at a concentration of 0.4-10 microM, induced subconductance levels with one-third and two-thirds of the unitary amplitude in the outward currents without affecting the inward currents. 3. Occupancy at each sublevel was estimated from the amplitude histogram which showed four equally spaced peaks in the presence of internal Ca2+. At different degrees of blockade, the distribution of the current levels showed a reasonable agreement with the binomial theorem. 4. The outward mean open-channel currents were measured at different Ca2+ concentrations and voltages. The current-voltage relation rectified inwardly in the presence of internal Ca2+ in a concentration-dependent manner. 5. The outward mean open-channel currents were normalized to unitary amplitudes in the absence of Ca2+. The normalized current-Ca2+ concentration curve was fitted by saturation kinetics with a Hill coefficient of 1 at each voltage. The voltage dependence of the dissociation constants gives the value for the fractional electrical distance of the Ca2+ binding site of 0.7. 6. The dwell times in each substrate were distributed exponentially. On the assumption that the inwardly rectifying K+ channel of cardiac cells is composed of three identical conducting subunits and each subunit is blocked by Ca2+ independently, the blocking (mu) and unblocking (lambda) rates were calculated. The value of mu increased with higher Ca2+ concentrations or larger depolarizations, while lambda was independent of Ca2+ and decreased with larger depolarization. 7. It is thus concluded that internal Ca2+ produces a voltage-dependent block of the channel to cause inward rectification although the blocking effect is less potent than that of Mg2

  18. Enhanced light absorption in GaAs solar cells with internal Bragg reflectors

    NASA Astrophysics Data System (ADS)

    Tobin, S. P.; Vernon, S. M.; Sanfacon, M. M.; Mastrovito, A.

    The use of epitaxial multilayer dielectric mirrors (Bragg reflectors) as back-surface reflectors in thin-film GaAs solar cells on GaAs and silicon substrates is investigated. Al0.3Ga0.9As/Al0.85Ga0.15As Bragg reflectors were grown by low-pressure MOCVD on GaAs substrates and shown to exhibit near-ideal optical reflectance and structural perfection. Thin GaAs solar cells grown on Bragg reflectors showed increases in short-circuit current (0.5 to 1.0 mA/sq cm) and efficiency (0.7 percentage points) relative to cells without back reflectors. Efficiencies of 24.7 percent at one sun AM1.5 were measured for GaAs cells only 2 microns thick on Bragg reflectors. In addition to the optical enhancements, Bragg reflectors also appear to improve the defect structure of GaAs-on-Si solar cells. This approach should lead to improved efficiency for GaAs-on-Si solar cells and improved radiation resistance on GaAs cells.

  19. Improved angiogenic cell penetration in vitro and in vivo in collagen scaffolds with internal channels.

    PubMed

    Yahyouche, Asma; Zhidao, Xia; Triffitt, James T; Czernuszka, Jan T; Clover, A J P

    2013-06-01

    Porous scaffolds are limited in volume due to diffusion constraint and delay of vascular network formation. Channels have the potential to speed up cellular penetration. Their effectiveness in improving angiogenic cell penetration was assessed in vitro and in vivo in 3-D collagen scaffolds. In vitro, channelled and non-channelled scaffolds were seeded with vascular smooth muscle cells. Results demonstrated that the scaffolds supported angiogenic cell ingrowth in culture and the channels improved the depth of cell penetration into the scaffold (P < 0.05). The cells reside mainly around and migrate along the channels. In vivo, channels increased cell migration into the scaffolds (P < 0.05) particularly angiogenic cells (P < 0.05) resulting in a clear branched vascular network of microvessels after 2 weeks in the channelled samples which was not apparent in the non-channelled samples. Channels could aid production of tissue engineered constructs by offering the possibility of rapid blood vessel infiltration into collagen scaffolds.

  20. 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.

  1. 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

  2. Detection of DNA Damage by Space Radiation in Human Fibroblast Cells Flown on the International Space Station

    NASA Technical Reports Server (NTRS)

    Wu, Honglu; Lu, Tao; Wong, Michael; Beno, Jonathan; Countryman, Stefanie; Stodieck, Louis; Karouia, Fathi; Zhang, Ye

    2015-01-01

    Although charged particles in space have been detected with radiation detectors on board spacecraft since the early discovery of the Van Allen Belt, reports on effects of direct exposure to space radiation in biological systems have been limited. Measurement of biological effects of space radiation has been difficult due to the low dose and low dose rate nature of the radiation environment, and the difficulty in separating the radiation effects from microgravity and other space environmental factors. In astronauts, only a small number of changes, such as increased chromosome aberrations in lymphocytes and early onset of cataracts, attributed primarily to the exposure to space radiation. In a recent experiment, human fibroblast cells were flown on the International Space Station (ISS). Cells fixed on Days 3 and 14 after reaching orbit were analyzed for phosphorylation of a histone protein H2AX by immunofluorescent staining of cells, which is a widely used marker for DNA double strand breaks. The 3-dimensional gamma-H2AX foci were captured with a laser confocal microscope. Quantitative analysis revealed a small fraction of foci that were larger and displayed a track pattern in the flight samples in comparison to the ground control. Human fibroblast cells were also exposed to low dose rate gamma rays, as well as to protons and Fe ions. Comparison of the pattern and distribution of the foci after gamma ray and charged particle exposure to our flight results confirmed that the foci found in the flown cells were indeed induced by space radiation.

  3. Rare Presentation of Giant Cell Tumor in the Internal Auditory Canal: Case Report and Review of the Literature

    PubMed Central

    Jada, Ajit S.; Shrivastava, Raj K.; Mannan, Abul; Kobets, Andrew; Manolidis, Spiros

    2015-01-01

    Giant cell tumor (GCT) is a benign but locally aggressive bone tumor that usually involves the end of long bones. It is a relatively common neoplasm in patients, constituting 5 to 10% of all benign bone tumors. Approximately 2% of GCTs occur in the craniofacial skeleton with a predilection for the ethmoid, sphenoid, and temporal bones. The skull base location is unique and not commonly described. Hearing loss, headache, tinnitus, and subcutaneous masses are the most commonly reported symptoms in GCTs of the skull base. In this case report we present the first description of a GCT within the internal auditory canal causing cranial neuropathy and review the recent pertinent literature. PMID:26251814

  4. 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

  5. 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.

  6. 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.

  7. Photochemical internalization of tamoxifens transported by a "Trojan-horse" nanoconjugate into breast-cancer cell lines.

    PubMed

    Theodossiou, Theodossis A; Gonçalves, A Ricardo; Yannakopoulou, Konstantina; Skarpen, Ellen; Berg, Kristian

    2015-04-13

    Photochemical internalization (PCI) has shown great promise as a therapeutic alternative for targeted drug delivery by light-harnessed activation. However, it has only been applicable to therapeutic macromolecules or medium-sized molecules. Herein we describe the use of an amphiphilic, water-soluble porphyrin-β-cyclodextrin conjugate (mTHPP-βCD) as a "Trojan horse" to facilitate the endocytosis of CD-guest tamoxifens into breast-cancer cells. Upon irradiation, the porphyrin core of mTHPP-βCD expedited endosomal membrane rupture and tamoxifen release into the cytosol, as documented by confocal microscopy. The sustained complexation of mTHPP-βCD with tamoxifen was corroborated by 2D NMR spectroscopy and FRET studies. Following the application of PCI protocols with 4-hydroxytamoxifen (4-OHT), estrogen-receptor β-positive (Erβ+, but not ERβ-) cell groups exhibited extensive cytotoxicity and/or growth suspension even at 72 h after irradiation. PMID:25663536

  8. Panoramic view of the Fifth International Symposium on Stem Cell Therapy and Applied Cardiovascular Biotechnology, April 2008, Madrid (Spain).

    PubMed

    Villa, Adolfo; Sanz, Ricardo; Fernandez, M Eugenia; Elizaga, Jaime; Ludwig, Indrig; Sanchez, Pedro L; Fernandez-Aviles, Francisco

    2009-03-01

    The Fifth International Symposium on Stem Cell Therapy and Applied Cardiovascular Biotechnology was held on April 24th-25th, 2008, at the Auditorium of the High Council of Scientific Research of Spain (CSIC) in Madrid, as a continuation of a series of yearly meetings, organized in an attempt to encourage translational research in this field and facilitate a positive interaction among experts from several countries, along with industry representatives and journalists. In addition, members of the Task Force of the European Society concerning the clinical investigation of the use of autologous adult stem cells for repair of the heart gathered and discussed an update of the previous consensus, still pending of publication. In this article, we summarize some of the main topics of discussion, the state-of-the-art and latest advances in this field, and new challenges brought up for the near future.

  9. Super-resolution imaging-based single particle tracking reveals dynamics of nanoparticle internalization by live cells.

    PubMed

    Li, Yiming; Shang, Li; Nienhaus, G Ulrich

    2016-04-14

    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. PMID:27001905

  10. 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

  11. 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

  12. 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

  13. Report of the First International Consensus on Standardized Nomenclature of Antinuclear Antibody HEp-2 Cell Patterns 2014–2015

    PubMed Central

    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 antinuclea