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Sample records for international kupffer cell

  1. Pathogenesis of Kupffer Cells in Cholestatic Liver Injury.

    PubMed

    Sato, Keisaku; Hall, Chad; Glaser, Shannon; Francis, Heather; Meng, Fanyin; Alpini, Gianfranco

    2016-09-01

    Kupffer cells are the resident macrophages in the liver. They are located in hepatic sinusoid, which allows them to remove foreign materials, pathogens, and apoptotic cells efficiently. Activated Kupffer cells secrete various mediators, including cytokines and chemokines, to initiate immune responses, inflammation, or recruitment of other liver cells. Bile duct ligation (BDL) surgery in rodents is often studied as an animal model of cholestatic liver disease, characterized by obstruction of bile flow. BDL mice show altered functional activities of Kupffer cells compared with sham-operated mice, including elevated cytokine secretion and impaired bacterial clearance. Various mediators produced by other liver cells can regulate Kupffer cell activation, which suggest that Kupffer cells orchestrate with other liver cells to relay inflammatory signals and to maintain liver homeostasis during BDL-induced liver injury. Blocking or depletion of Kupffer cells, an approach for the treatment of liver diseases, has shown controversial implications. Procedures in Kupffer cell research have limitations and may produce various results in Kupffer cell research. It is important, however, to reveal underlying mechanisms of activation and functions of Kupffer cells, followed by hepatic inflammation and fibrosis. This review summarizes present Kupffer cell studies in cholestatic liver injury. PMID:27452297

  2. 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.

  3. 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.

  4. Effective delivery of chemotherapeutic nanoparticles by depleting host Kupffer cells.

    PubMed

    Ohara, Yusuke; Oda, Tatsuya; Yamada, Keiichi; Hashimoto, Shinji; Akashi, Yoshimasa; Miyamoto, Ryoichi; Kobayashi, Akihiko; Fukunaga, Kiyoshi; Sasaki, Ryoko; Ohkohchi, Nobuhiro

    2012-11-15

    Although chemotherapeutic nanoparticles would confer various advantages, the majority of administrated nanoparticles are known to be spoiled by the reticuloendothelial system (RES). Intending to more effectively deliver therapeutic nanoparticles to target regions in vivo, host RES, especially Kupffer cells in the liver, have been depleted ahead of drug administration. To demonstrate this hypothesis, clodronate liposomes were preinjected into BALB/c nude mice for depletion of Kupffer cells 2 days before, and pegylated liposomal doxorubicin (Doxil) at the doses of 1.25, 2.5 and 5.0 mg/kg was administered. As a result, doxorubicin accumulation in the liver was decreased from 36 to 26% injected dose/organ by the Kupffer cells depletion, and consequently, the plasma concentration of doxorubicin was significantly enhanced threefold (from 11 to 33 μg/mL) on day 1 at 1.25 mg/kg-dose group. Doxorubicin accumulation in the tumor was increased from 0.78 to 3.0 μg/g-tissue on day 3, and tumor growth inhibition by Doxil was significantly boosted (tumor volumes from 751 to 482 mm(3) on day 24) by the Kupffer cells depletion. In conclusion, Kupffer cells depletion by clodronate liposomes enhanced the plasma concentration and antitumor effects of Doxil, and would be widely applicable for various clinical cancer chemotherapies using nanoparticles. PMID:22362271

  5. 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. PMID:24142864

  6. Alcoholic hepatitis: The pivotal role of Kupffer cells.

    PubMed

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

    2015-11-15

    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

  7. 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

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

    PubMed Central

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

    2013-01-01

    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. PMID:24074569

  9. Contribution of programmed cell death receptor (PD)-1 to Kupffer cell dysfunction in murine polymicrobial sepsis.

    PubMed

    Wang, Fei; Huang, Xin; Chung, Chun-Shiang; Chen, Yaping; Hutchins, Noelle A; Ayala, Alfred

    2016-08-01

    Recent studies suggest that coinhibitory receptors appear to be important in contributing sepsis-induced immunosuppression. Our laboratory reported that mice deficient in programmed cell death receptor (PD)-1 have increased bacterial clearance and improved survival in experimental sepsis induced by cecal ligation and puncture (CLP). In response to infection, the liver clears the blood of bacteria and produces cytokines. Kupffer cells, the resident macrophages in the liver, are strategically situated to perform the above functions. However, it is not known if PD-1 expression on Kupffer cells is altered by septic stimuli, let alone if PD-1 ligation contributes to the altered microbial handling seen. Here we report that PD-1 is significantly upregulated on Kupffer cells during sepsis. PD-1-deficient septic mouse Kupffer cells displayed markedly enhanced phagocytosis and restoration of the expression of major histocompatibility complex II and CD86, but reduced CD80 expression compared with septic wild-type (WT) mouse Kupffer cells. In response to ex vivo LPS stimulation, the cytokine productive capacity of Kupffer cells derived from PD-1-/- CLP mice exhibited a marked, albeit partial, restoration of the release of IL-6, IL-12, IL-1β, monocyte chemoattractant protein-1, and IL-10 compared with septic WT mouse Kupffer cells. In addition, PD-1 gene deficiency decreased LPS-induced apoptosis of septic Kupffer cells, as indicated by decreased levels of cleaved caspase-3 and reduced terminal deoxynucleotidyl transferase dUTP nick end-labeling-positive cells. Exploring the signal pathways involved, we found that, after ex vivo LPS stimulation, septic PD-1-/- mouse Kupffer cells exhibited an increased Akt phosphorylation and a reduced p38 phosphorylation compared with septic WT mouse Kupffer cells. Together, these results indicate that PD-1 appears to play an important role in regulating the development of Kupffer cell dysfunction seen in sepsis. PMID:27288425

  10. 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.

  11. The Role of IL-1 Family Members and Kupffer Cells in Liver Regeneration

    PubMed Central

    Tan, Quanhui; Hu, Jianjun; Yu, Xiaolan; Guan, Wen; Lu, Huili; Yu, Yan; Yu, Yongsheng; Zang, Guoqiang; Tang, Zhenghao

    2016-01-01

    Interleukin-1 (IL-1) family and Kupffer cells are linked with liver regeneration, but their precise roles remain unclear. IL-1 family members are pleiotropic factors with a range of biological roles in liver diseases, inducing hepatitis, cirrhosis, and hepatocellular carcinoma, as well as liver regeneration. Kupffer cells are the main source of IL-1 and IL-1 receptor antagonist (IL-1Ra), the key members of IL-1 family. This systemic review highlights a close association of IL-1 family members and Kupffer cells with liver regeneration, although their specific roles are inconclusive. Moreover, IL-1 members are proposed to induce effects on liver regeneration through Kupffer cells. PMID:27092311

  12. 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.

  13. 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

  14. Kupffer cell activation after no-flow ischemia versus hemorrhagic shock.

    PubMed

    Jaeschke, Hartmut; Farhood, Anwar

    2002-07-15

    Kupffer cell-derived oxidant stress is critical for reperfusion injury after no-flow ischemia. However, the importance of Kupffer cells as source of reactive oxygen formation is unclear in a hemorrhagic shock model. Therefore, we evaluated Kupffer cell activation after 60 or 120 min of hemorrhage and 90 min of resuscitation (HS/RS) in pentobarbital-anesthetized male Fischer rats. Plasma glutathione disulfide (GSSG) as indicator for a vascular oxidant stress showed no significant changes after HS/RS. Plasma ALT activities were only moderately increased (100-200 U/L). Kupffer cells isolated from postischemic livers did not generate more superoxide than cells from sham controls. In contrast, the 10-fold increase of plasma GSSG and the 9-fold higher spontaneous superoxide formation of Kupffer cells after 60 min of hepatic no-flow ischemia followed by 90 min of reperfusion demonstrated the activation of Kupffer cells in this experimental model. Plasma ALT activities (1930 +/- 240 U/L) indicated severe liver injury. These results demonstrate a fundamental difference in the degree of Kupffer cell activation between the two models of warm hepatic ischemia. Our findings suggest that different therapeutic strategies are necessary to ameliorate the initial injury after low flow ischemia (hemorrhage) compared to cold (transplantation) or warm (Pringle maneuver) no-flow ischemia. PMID:12106817

  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. Repopulation of murine Kupffer cells after intravenous administration of liposome-encapsulated dichloromethylene diphosphonate.

    PubMed Central

    Yamamoto, T.; Naito, M.; Moriyama, H.; Umezu, H.; Matsuo, H.; Kiwada, H.; Arakawa, M.

    1996-01-01

    Kupffer cells were selectively eliminated in mice by the intravenous administration of liposome-entrapped dichloromethylene diphosphonate. At 5 days, small peroxidase-negative and acid-phosphatase-weakly-positive macrophages appeared, increased in number, and differentiated into peroxidase- and acid-phosphatase-positive Kupffer cells. Repopulating small macrophages actively proliferated, and the number of Kupffer cells returned to the normal level by day 14. The numbers of macrophage precursors in the liver as detected by the monoclonal antibodies ER-MP20 and ER-MP58 increased after liposome-entrapped dichloromethylene diphosphonate injection. ER-MP58-positive cells proliferated and differentiated into ER-MP20-positive cells and eventually into BM8-positive Kupffer cells in the liver. Bone-marrow-derived ER-MP58-positive cells were also detectable in the liver and differentiated into ER-MP20-positive cells, but they did not become BM8-positive macrophages. Macrophage colony-stimulating factor mRNA expression was enhanced in the liver 1 day after injection. The administration of macrophage colony-stimulating factor did not shorten the period of Kupffer cell depletion but increased the number and the proliferative capacity of repopulating Kupffer cells. These findings implied that repopulating Kupffer cells are derived from a macrophage precursor pool in the liver rather than from bone-marrow-derived monocytes. Local production of macrophage colony-stimulating factor in the liver plays a crucial role in the differentiation, maturation, and proliferation of Kupffer cells. Images Figure 3 Figure 4 Figure 5 Figure 7 Figure 8 Figure 9 Figure 11 Figure 13 Figure 12 Figure 18 PMID:8863675

  17. Bimodal role of Kupffer cells during colorectal cancer liver metastasis.

    PubMed

    Wen, Shu Wen; Ager, Eleanor I; Christophi, Christopher

    2013-07-01

    Kupffer cells (KCs) are resident liver macrophages that play a crucial role in liver homeostasis and in the pathogenesis of liver disease. Evidence suggests KCs have both stimulatory and inhibitory functions during tumor development but the extent of these functions remains to be defined. Using KC depletion studies in an orthotopic murine model of colorectal cancer (CRC) liver metastases we demonstrated the bimodal role of KCs in determining tumor growth. KC depletion with gadolinium chloride before tumor induction was associated with an increased tumor burden during the exponential growth phase. In contrast, KC depletion at the late stage of tumor growth (day 18) decreased liver tumor load compared with non-depleted animals. This suggests KCs exhibit an early inhibitory and a later stimulatory effect. These two opposing functions were associated with changes in iNOS and VEGF expression as well as T-cell infiltration. KC depletion at day 18 increased numbers of CD3 (+) T cells and iNOS-expressing infiltrating cells in the tumor, but decreased the number of VEGF-expressing infiltrating cells. These alterations may be responsible for the observed reduction in tumor burden following depletion of pro-tumor KCs at the late stage of metastatic growth. Taken together, our results indicate that the bimodal role of KC activity in liver tumors may provide the key to timing immunomodulatory intervention for the treatment of CRC liver metastases. PMID:23792646

  18. 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.

  19. [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

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

    PubMed Central

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

    2016-01-01

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

  1. Mammary-carcinoma cells in mouse liver: infiltration of liver tissue and interaction with Kupffer cells.

    PubMed Central

    Roos, E.; Dingemans, K. P.; Van de Pavert, I. V.; Van den Bergh-Weerman, M. A.

    1978-01-01

    Interactions between TA3 mammary-carcinoma cells and liver cells were studied with the electron microscope in mouse livers that had been perfused with a defined medium containing the tumour cells. Infiltration of liver tissue by the TA3 cells proceeded in the following steps. First, numerous small protrusions were extended through endothelial cells and into hepatocytes. Next, some cells had larger processes deeply indenting hepatocytes. Finally a few tumour cells became located outside the blood vessels. Two variant cell lines, TA3/Ha and TA3/St, differing in cell coat and surface charge, did not differ in the extent of infiltration. TA3/Ha cells were often encircled by thin processes of liver macrophages (Kupffer cells). Encircled cells were initially intact, but later some of them degenerated. These observations suggest that TA3/Ha cells were phagocytized by the Kupffer cells. Encirclement appeared to be inhibited after only 30 min, when many cells were still partly surrounded. Encirclement of TA3/St was much less frequent. After injection of tumour cells intra-portally in vivo, similar results were obtained, which demonstrated the validity of the perfused liver model. TA3/Ha cells formed much fewer tumour nodules in the liver than TA3/St cells. Images Fig. 7 Fig. 8 Fig. 9 Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 11 Fig. 12 Fig. 13 Fig. 15 Fig. 16 Fig. 17 Fig. 18 Fig. 19 PMID:687522

  2. Cross-Activating Invariant NKT Cells and Kupffer Cells Suppress Cholestatic Liver Injury in a Mouse Model of Biliary Obstruction

    PubMed Central

    Duwaerts, Caroline C.; Sun, Eric P.; Cheng, Chao-Wen; van Rooijen, Nico; Gregory, Stephen H.

    2013-01-01

    Both Kupffer cells and invariant natural killer T (iNKT) cells suppress neutrophil-dependent liver injury in a mouse model of biliary obstruction. We hypothesize that these roles are interdependent and require iNKT cell-Kupffer cell cross-activation. Female, wild-type and iNKT cell-deficient C57Bl/6 mice were injected with magnetic beads 3 days prior to bile duct ligation (BDL) in order to facilitate subsequent Kupffer cell isolation. On day three post-BDL, the animals were euthanized and the livers dissected. Necrosis was scored; Kupffer cells were isolated and cell surface marker expression (flow cytometry), mRNA expression (qtPCR), nitric oxide (NO.) production (Griess reaction), and protein secretion (cytometric bead-array or ELISAs) were determined. To address the potential role of NO. in suppressing neutrophil accumulation, a group of WT mice received 1400W, a specific inducible nitric oxide synthase (iNOS) inhibitor, prior to BDL. To clarify the mechanisms underlying Kupffer cell-iNKT cell cross-activation, WT animals were administered anti-IFN-γ or anti-lymphocyte function-associated antigen (LFA)-1 antibody prior to BDL. Compared to their WT counterparts, Kupffer cells obtained from BDL iNKT cell-deficient mice expressed lower iNOS mRNA levels, produced less NO., and secreted more neutrophil chemoattractants. Both iNOS inhibition and IFN-γ neutralization increased neutrophil accumulation in the livers of BDL WT mice. Anti-LFA-1 pre-treatment reduced iNKT cell accumulation in these same animals. These data indicate that the LFA-1-dependent cross-activation of iNKT cells and Kupffer cells inhibits neutrophil accumulation and cholestatic liver injury. PMID:24260285

  3. Cross-activating invariant NKT cells and kupffer cells suppress cholestatic liver injury in a mouse model of biliary obstruction.

    PubMed

    Duwaerts, Caroline C; Sun, Eric P; Cheng, Chao-Wen; van Rooijen, Nico; Gregory, Stephen H

    2013-01-01

    Both Kupffer cells and invariant natural killer T (iNKT) cells suppress neutrophil-dependent liver injury in a mouse model of biliary obstruction. We hypothesize that these roles are interdependent and require iNKT cell-Kupffer cell cross-activation. Female, wild-type and iNKT cell-deficient C57Bl/6 mice were injected with magnetic beads 3 days prior to bile duct ligation (BDL) in order to facilitate subsequent Kupffer cell isolation. On day three post-BDL, the animals were euthanized and the livers dissected. Necrosis was scored; Kupffer cells were isolated and cell surface marker expression (flow cytometry), mRNA expression (qtPCR), nitric oxide (NO (.) ) production (Griess reaction), and protein secretion (cytometric bead-array or ELISAs) were determined. To address the potential role of NO (.) in suppressing neutrophil accumulation, a group of WT mice received 1400W, a specific inducible nitric oxide synthase (iNOS) inhibitor, prior to BDL. To clarify the mechanisms underlying Kupffer cell-iNKT cell cross-activation, WT animals were administered anti-IFN-γ or anti-lymphocyte function-associated antigen (LFA)-1 antibody prior to BDL. Compared to their WT counterparts, Kupffer cells obtained from BDL iNKT cell-deficient mice expressed lower iNOS mRNA levels, produced less NO (.) , and secreted more neutrophil chemoattractants. Both iNOS inhibition and IFN-γ neutralization increased neutrophil accumulation in the livers of BDL WT mice. Anti-LFA-1 pre-treatment reduced iNKT cell accumulation in these same animals. These data indicate that the LFA-1-dependent cross-activation of iNKT cells and Kupffer cells inhibits neutrophil accumulation and cholestatic liver injury. PMID:24260285

  4. Colloidal carbon stimulation of Kupffer cells triggers Nrf2 activation in the isolated perfused rat liver.

    PubMed

    Núñez, Bárbara; Vargas, Romina; Castillo, Iván; Videla, Luis A

    2012-06-01

    Activation of transcription factor Nrf2 was investigated in the isolated perfused rat liver infused with 0.5 mg of colloidal carbon (CC)/ml for 5-15 min to stimulated Kupffer cell function. Infusion of CC enhanced liver O(2) consumption over basal levels, with a time-dependent increase in CC-induced O(2) uptake, at constant rates of CC phagocytosis by Kupffer cells, as assessed histologically, and adequate viability conditions of the livers, as shown by the marginal (0.34 %) total sinusoidal lactate dehydrogenase (LDH) efflux over intrahepatic LDH activity. Under these conditions, cytosolic protein levels of Nrf2 (Western blot) and inhibitor of Nrf2 Keap1 progressively declined by CC infusion, those of nuclear Nrf2 increased, leading to enhancement in the nuclear/cytosolic Nrf2 ratios. It is concluded that the respiratory burst of CC-stimulated Kupffer cells triggers Nrf2 activation in the perfused liver, a response that may afford cellular protection under pro-oxidant conditions underlying Kupffer cell stimulation. PMID:22461194

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

    PubMed Central

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

    2015-01-01

    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. PMID:19371622

  6. 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.

  7. 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

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

    PubMed

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

    2015-05-01

    Radiation-induced liver injury remains a clinical problem and data suggest that sinusoidal endothelial cells (SECs) are an important target. The purpose of this study was to determine whether the inhibition of Kupffer cells before exposure would protect SECs from radiation-induced injury. Sprague-Dawley rats were intravenously injected 24 h before irradiation with Kupffer cell inhibitor gadolinium chloride (GdCl3) (10 mg/kg body weight). Three groups of animals were treated: 1. control group (saline and sham irradiation); 2. GdCl3 + 30 Gy radiation group and 3. 30 Gy radiation only group. Specimens were collected at 2, 6, 12, 24 and 48 h after completion of each treatment. Liver tissue was assessed for inflammatory cytokine expression and radiation-induced SEC injury based on serum hyaluronic acid (HA) level, apoptosis and ultrastructural and histological analyses. The results showed that radiation exposure caused apoptosis of SECs, but not hepatocytes. Inflammatory cytokine expression, including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) expression, was significantly attenuated in the GdCl3 + 30 Gy radiation group, compared with the 30 Gy radiation-only group (P < 0.05). The GdCl3 + radiation-treated rats exhibited significantly lower levels of HA and SEC apoptosis than the radiation-treated only rats at early time points, and radiation-induced liver injury was also attenuated. In conclusion, we hypothesize that selective Kupffer cell inhibition by gadolinium chloride was shown to reduce apoptosis in SECs caused by irradiation of the live and protected the liver against radiation-induced injury. PMID:25897555

  9. 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

  10. 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

  11. Kupffer cell-mediated exacerbation of methimazole-induced acute liver injury in rats.

    PubMed

    Akai, Sho; Uematsu, Yasuaki; Tsuneyama, Koichi; Oda, Shingo; Yokoi, Tsuyoshi

    2016-05-01

    Methimazole (MTZ), an anti-thyroid drug, is known to cause liver injury in humans. It has been demonstrated that MTZ-induced liver injury in Balb/c mice is accompanied by T helper (Th) 2 cytokine-mediated immune responses; however, there is little evidence for immune responses associated with MTZ-induced liver injury in rats. To investigate species differences in MTZ-induced liver injury, we administered MTZ with a glutathione biosynthesis inhibitor, L-buthionine-S,R-sulfoximine (BSO), to F344 rats and subsequently observed an increase in plasma alanine aminotransferase (ALT) and high-mobility group box 1 (HMGB1), which are associated with hepatic lesions. The hepatic mRNA expression of innate immune-related genes significantly increased in BSO- and MTZ-treated rats, but the change in Th2-related genes was not much greater than the change observed in the previous mouse study. Moreover, an increase in Kupffer cells and an induction of the phosphorylation of extracellular signal-regulated kinase (ERK)/c-Jun N-terminal kinase (JNK) proteins were accompanied by an increase in Toll-like receptor 4 (TLR4) expression, indicating that Kupffer cell activation occurs through HMGB1-TLR4 signaling. To elucidate the mechanism of liver injury in rats, gadolinium chloride, which inactivates the function of Kupffer cells, was administered before BSO and MTZ administration. The gadolinium chloride treatment significantly suppressed the increased ALT, which was accompanied by decreased hepatic mRNA expression related to innate immune responses and ERK/JNK phosphorylation. In conclusion, Kupffer cell-mediated immune responses are crucial factors for the exacerbation of MTZ-induced liver injury in rats, indicating apparent species differences in the immune-mediated exacerbation of liver injury between mice and rats. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26177832

  12. 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.

  13. Gadolinium chloride, a Kupffer cell inhibitor, attenuates hepatic injury in a rat model of chronic cholestasis.

    PubMed

    Zandieh, Ali; Payabvash, Seyedmehedi; Pasalar, Parvin; Morteza, Afsaneh; Zandieh, Basira; Tavangar, Seyed Mohammad; Dehpour, Ahmad Reza

    2011-11-01

    The aim of the current study was to elucidate the effect of Kupffer cells inhibition on hepatic injury induced by chronic cholestasis. Sprague-Dawley rats underwent bile duct ligation (BDL) or sham operation and were treated with either saline solution or gadolinium chloride (GdCl(3), a specific Kupffer cell inhibitor, 20 mg/kg i.p. daily). Serum and liver samples were collected after 28 days. Direct and total bilirubin concentrations and serum enzyme activities of alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and γ-glutamyl transpeptidase (GGT) increased following BDL (p < 0.01). On the contrary to bilirubin concentrations and AST activity, GdCl(3) partially prevented the elevation in ALP, ALT and GGT enzyme activities (p < 0.05). GdCl(3) alleviated lipid peroxidation (reflected by malondialdehyde [MDA] concentration) and increased the activities of antioxidant enzymes (i.e. catalase and glutathione peroxidase) in liver samples after BDL (p < 0.05). Fibrosis, ductular proliferation and portal inflammation were also scored in liver samples. Among morphological changes appeared following BDL (i.e. marked fibrosis, portal inflammation and ductular proliferation); only ductular proliferation was not alleviated by GdCl(3). Therefore, Kupffer cells inhibition has beneficial effects against the development of hepatic injury induced by chronic cholestasis. PMID:21339256

  14. Cannabinoid CB2 receptors protect against alcoholic liver disease by regulating Kupffer cell polarization in mice.

    PubMed

    Louvet, Alexandre; Teixeira-Clerc, Fatima; Chobert, Marie-Noële; Deveaux, Vanessa; Pavoine, Catherine; Zimmer, Andreas; Pecker, Françoise; Mallat, Ariane; Lotersztajn, Sophie

    2011-10-01

    Activation of Kupffer cells plays a central role in the pathogenesis of alcoholic liver disease. Because cannabinoid CB2 receptors (CB2) display potent anti-inflammatory properties, we investigated their role in the pathogenesis of alcoholic liver disease, focusing on the impact of CB2 on Kupffer cell polarization and the consequences on liver steatosis. Wild-type (WT) mice fed an alcohol diet showed an induction of hepatic classical (M1) and alternative (M2) markers. Cotreatment of alcohol-fed mice with the CB2 agonist, JWH-133, decreased hepatic M1 gene expression without affecting the M2 profile. In keeping with this, genetic ablation of CB2 enhanced hepatic induction of M1 gene signature and blunted the induction of M2 markers. CB2 also modulated alcohol-induced fatty liver, as shown by the reduction of hepatocyte steatosis in JWH-133-treated mice and its enhancement in CB2-/- animals. Studies in isolated Kupffer cells and cultured macrophages further demonstrated that CB2 inhibits M1 polarization and favors the transition to an M2 phenotype. In addition, conditioned-medium experiments showed that preventing M1 polarization in CB2-activated macrophages protects from lipid accumulation in hepatocytes. Heme oxygenase-1 (HO-1) mediated the anti-inflammatory effects of CB2 receptors. Indeed, alcohol-fed mice treated with JWH-133 showed increased hepatic expression of macrophage HO-1, as compared to vehicle-treated counterparts. In keeping with this, JWH-133 induced HO-1 expression in cultured macrophages, and the HO-1 inhibitor, zinc protoporphyrin, blunted the inhibitory effect of JWH-133 on lipopolysaccharide-induced nuclear factor-kappa B activation and M1 polarization. Altogether, these findings demonstrate that CB2 receptors display beneficial effects on alcohol-induced inflammation by regulating M1/M2 balance in Kupffer cells, thereby reducing hepatocyte steatosis via paracrine interactions between Kupffer cells and hepatocytes. These data identify CB2

  15. 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

  16. Toll Like Receptor 4 Dependent Kupffer Cell Activation and Liver Injury in a Novel Mouse Model of Parenteral Nutrition

    PubMed Central

    El Kasmi, Karim C.; Anderson, Aimee L.; Devereaux, Michael W.; Fillon, Sophie A.; Harris, J. Kirk; Lovell, Mark A.; Finegold, Milton J.; Sokol, Ronald J.

    2011-01-01

    Infants with intestinal failure who are parenteral nutrition (PN)-dependent may develop cholestatic liver injury and cirrhosis (PN-associated liver injury: PNALI). The pathogenesis of PNALI remains incompletely understood. We hypothesized that intestinal injury with increased intestinal permeability combined with administration of PN promotes LPS-TLR4 signaling dependent Kupffer cell activation as an early event in the pathogenesis of PNALI. We developed a mouse model in which intestinal injury and increased permeability were induced by oral treatment for 4 days with dextran sulphate sodium (DSS) followed by continuous infusion of soy lipid-based PN solution through a central venous catheter for 7 (PN/DSS7d) and 28 (PN/DSS28d) days. Liver injury and cholestasis were evaluated by serum AST, ALT, bile acids, total bilirubin, and by histology. Purified Kupffer cells were probed for transcription of pro-inflammatory cytokines. PN/DSS7d mice showed elevated portal vein LPS levels, evidence of hepatocyte injury and cholestasis, and increased Kupffer cell expression of IL6, TNFα, and TGFβ. Serological markers of liver injury remained elevated in PN/DSS28d mice associated with focal inflammation, hepatocyte apoptosis, peliosis, and Kupffer cell hypertrophy and hyperplasia. PN infusion without DSS pre-treatment or DSS pre-treatment alone did not result in liver injury or Kupffer cell activation. Suppression of the intestinal microbiota with broad spectrum antibiotics or ablation of TLR4 signaling in TLR4 mutant mice resulted in significantly reduced Kupffer cell activation and markedly attenuated liver injury in PN/DSS7d mice. Conclusion These data suggest that intestinal-derived LPS activates Kupffer cells through TLR4 signaling in early stages of PNALI. PMID:22120983

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

    SciTech Connect

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

    1990-03-01

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

  18. Activation of NLRP3 and AIM2 inflammasomes in Kupffer cells in hepatic ischemia/reperfusion.

    PubMed

    Kim, Hyo-Yeon; Kim, Seok-Joo; Lee, Sun-Mee

    2015-01-01

    Inflammasome activation by danger signals in ischemia/reperfusion (I/R) injury is responsible for the sterile inflammatory response. Signals triggering formation and activation of the inflammasome involve the generation of oxidative stress. The aim of this study was to examine the molecular mechanisms of inflammasome activation and the involvement of reactive oxygen species in hepatic I/R. I/R induced the formation of nucleotide-binding domain leucine-rich repeat containing family pyrin domain containing 3 (NLRP3) and absent in melanoma 2 (AIM2) inflammasomes and the subsequent serum release of interleukin 1β. Pannexin-1 inhibitor and anti-cathepsin B antibody attenuated I/R-induced inflammasome activation and hepatic injury. The expression of the thioredoxin-interacting protein gene and the interaction between NLRP3 and the thioredoxin-interacting protein increased after I/R. Treatment with the antioxidant N-acetylcysteine significantly attenuated protein conversion of interleukin 1β after hepatic I/R. Moreover, pannexin-1 protein expression and cathepsin B release were strongly attenuated by N-acetylcysteine. The depletion of Kupffer cells with gadolinium chloride markedly decreased NLRP3 and AIM2 inflammasome expression and activation of their signaling pathways, and also reduced the level of caspase-1 protein in F4/80-positive cells. Our findings suggest that reactive-oxygen-species-mediated activation of NLRP3 and AIM2 inflammasomes leads to I/R-induced inflammatory responses in which Kupffer cells play a crucial role. PMID:25327779

  19. Liver innate immune cells and insulin resistance: the multiple facets of Kupffer cells.

    PubMed

    Jager, J; Aparicio-Vergara, M; Aouadi, M

    2016-08-01

    Obesity, which affects 600 million adults worldwide, is a major risk factor for type 2 diabetes (T2D) and insulin resistance. Current therapies for these metabolic disorders include weight management by lifestyle intervention or bariatric surgery and pharmacological treatment with the aim of regulating blood glucose. Probably because of their short-term effectiveness, these therapies have not been able to stop the rapidly rising prevalence of T2D over the past decades, highlighting an urgent need to develop new therapeutic strategies. The role of immune cells, such as macrophages, in insulin resistance has been extensively studied. Major advances have been made to elucidate the role of adipose tissue macrophages in these pathogeneses. Recently, anti-inflammatory drugs have been suggested as an alternative treatment for T2D, and clinical trials of these agents are currently ongoing. In addition, results of previous clinical trials using antibodies against inflammatory cytokines, which showed modest effects, are now being rigorously re-evaluated. However, it is still unclear how liver macrophages [termed Kupffer cells (KCs)], which constitute the major source of macrophages in the body, contribute to the development of insulin resistance. In this review, we will discuss the present understanding of the role of liver immune cells in the development of insulin resistance. We will particularly focus on KCs, which could represent an attractive target for the treatment of metabolic diseases. PMID:26864622

  20. 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

  1. 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...

  2. Oxidative products from alcohol metabolism differentially modulate pro-inflammatory cytokine expression in Kupffer cells and hepatocytes.

    PubMed

    Dong, Daoyin; Zhong, Wei; Sun, Qian; Zhang, Wenliang; Sun, Xinguo; Zhou, Zhanxiang

    2016-09-01

    Pro-inflammatory cytokines play a vital role in the pathogenesis of alcoholic steatohepatitis. The present study was to determine the role of alcohol-induced oxidative stress in modulating cytokine production. A rat model of alcohol consumption was used to determine alcohol-induced hepatic cytokine expression. Chronic alcohol exposure caused lipid accumulation, oxidative stress, and inflammation in the livers of Wistar rats. The role of oxidative stress in regulating cell type-specific cytokine production was further dissected in vitro. Lipopolysaccharide (LPS) dose-dependently upregulated TNF-α, MIP-1α, MCP-1, and CINC-1 in Kupffer cells-SV40, whereas TNF-α dose-dependently induced CINC-1, IP-10, and MIP-2 expression in H4IIEC3 hepatoma cells. An additive effect on cytokine production was observed in both Kupffer cells-SV40 and hepatocytes when combined hydrogen peroxide with LPS or TNF-α, respectively, which was associated with NF-κB activation and histone H3 hyper-acetylation. Unexpectedly, an inhibitory effect of 4-hydroxynonenal on cytokine production was revealed in LPS-treated Kupffer cells-SV40. Mechanistic study showed that 4-hydroxynonenal significantly enhanced mRNA degradation of TNF-α, MCP-1, and MIP-1α, and decreased the protein levels of MCP-1 in LPS-stimulated Kupffer cells-SV40 through reducing the phosphorylation of mRNA binding proteins. This study suggests that Kupffer cells and hepatocytes express distinct pro-inflammatory cytokines/chemokines in response to alcohol intoxication, and oxidative products (4-hydroxynonenal) differentially modulate pro-inflammatory cytokine/chemokine production via NF-κB signaling, histone acetylation, and mRNA stability. PMID:27314544

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

    PubMed Central

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

    2016-01-01

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

  4. Arachidonic acid stimulates TNFα production in Kupffer cells via a reactive oxygen species-pERK1/2-Egr1-dependent mechanism.

    PubMed

    Cubero, Francisco Javier; Nieto, Natalia

    2012-07-15

    Kupffer cells are a key source of mediators of alcohol-induced liver damage such as reactive oxygen species, chemokines, growth factors, and eicosanoids. Since diets rich in polyunsaturated fatty acids are a requirement for the development of alcoholic liver disease, we hypothesized that polyunsaturated fatty acids could synergize with ethanol to promote Kupffer cell activation and TNFα production, hence, contributing to liver injury. Primary Kupffer cells from control and from ethanol-fed rats incubated with arachidonic acid showed similar proliferation rates than nontreated cells; however, arachidonic acid induced phenotypic changes, lipid peroxidation, hydroperoxides, and superoxide radical generation. Similar effects occurred in human Kupffer cells. These events were greater in Kupffer cells from ethanol-fed rats, and antioxidants and inhibitors of arachidonic acid metabolism prevented them. Arachidonic acid treatment increased NADPH oxidase activity. Inhibitors of NADPH oxidase and of arachidonic acid metabolism partially prevented the increase in oxidant stress. Upon arachidonic acid stimulation, there was a rapid and sustained increase in TNFα, which was greater in Kupffer cells from ethanol-fed rats than in Kupffer cells from control rats. Arachidonic acid induced ERK1/2 phosphorylation and nuclear translocation of early growth response-1 (Egr1), and ethanol synergized with arachidonic acid to promote this effect. PD98059, a mitogen extracellular kinase 1/2 inhibitor, and curcumin, an Egr1 inhibitor, blocked the arachidonic acid-mediated upregulation of TNFα in Kupffer cells. This study unveils the mechanism whereby arachidonic acid and ethanol increase TNFα production in Kupffer cells, thus contributing to alcoholic liver disease. PMID:22538404

  5. 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

  6. The activation of Epimedium polysaccharide-propolis flavone liposome on Kupffer cells.

    PubMed

    Fan, Yunpeng; Ren, Meimei; Hou, Weifeng; Guo, Chao; Tong, Dewen; Ma, Lin; Zhang, Weimin; He, Mengmeng; Song, Xiaoping

    2015-11-20

    Epimedium polysaccharide-propolis flavone liposome (EPL), a potent immunological pharmaceutical preparation, was investigated for the immunomodulatory activity on Kupffer cells (KCs) in vitro. The results showed that EPL could significantly induce the secretion of chemokines (RANTES and MCP-1), promote the production of nitric oxide and induced nitric oxide synthase, improve the pinocytic and phagocytic activity of KCs, promote the mRNA expression of TNF-α and IL-1β, and enhance the expression of costimulatory molecules (CD11b and CD68) in KCs compared with Epimedium polysaccharide-propolis flavone (EP) at 30-7.5μg/mL. In addition, the abilities of KCs on stimulating lymphocytes proliferation and antigen presenting were significantly enhanced after stimulated with EPL compared with EP. These results suggested that EPL could activate KCs and possessed the stronger immunomodulatory effect, which provided the theoretical basis for further studying the mechanism of EPL on improving the immune response. PMID:26344320

  7. 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

  8. 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.

  9. 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

  10. 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

  11. Kupffer Cells Protect Liver Sinusoidal Endothelial Cells from Fas-Dependent Apoptosis in Sepsis by Down-Regulating gp130

    PubMed Central

    Hutchins, Noelle A.; Chung, Chun-Shiang; Borgerding, Joshua N.; Ayala, Carol A.; Ayala, Alfred

    2014-01-01

    Endothelial cell (EC) dysfunction is a key feature of multiple organ injury, the primary cause of fatality seen in critically ill patients. Although the development of EC dysfunction in the heart and lung is well studied in sepsis, it remains unclear in the liver. Herein, we report that liver sinusoidal ECs (LSECs; defined as CD146+CD45−) exhibit increased intercellular adhesion molecule-1 (CD54) and Fas in response to sepsis induced by cecal ligation and puncture (CLP). By using magnetically enriched LSEC (CD146+) populations, we show evidence of marked apoptosis, with a twofold decline in viable LSECs in CLP animals compared with sham controls. These changes and increased serum alanine aminotransferase levels were all mitigated in septic Fas−/− and Fas ligand−/− animals. Although we previously reported increased numbers of Fas ligand expressing CD8+ T lymphocytes in the septic liver, CD8+ T-cell deficiency did not reverse the onset of LSEC apoptosis/damage. However, Kupffer cell depletion with clodronate liposomes resulted in greater apoptosis and Fas expression after CLP and a decrease in glycoprotein 130 expression on LSECs, suggesting that STAT3 activation may protect these cells from injury. Our results document a critical role for death receptor–mediated LSEC injury and show the first evidence that Kupffer cells are essential to the viability of LSECs, which appears to be mediated through glycoprotein 130 expression in sepsis. PMID:23306157

  12. Iron- and inflammation-induced hepcidin gene expression in mice is not mediated by Kupffer cells in vivo.

    PubMed

    Lou, Dan-Qing; Lesbordes, Jeanne-Claire; Nicolas, Gaël; Viatte, Lydie; Bennoun, Myriam; Van Rooijen, Nico; Kahn, Axel; Renia, Laurent; Vaulont, Sophie

    2005-05-01

    Hepcidin, a recently discovered iron regulatory peptide, is believed to inhibit the release of iron from absorptive enterocytes and macrophages. Liver hepcidin synthesis is induced in vivo by iron stores and inflammation. The molecular basis of the regulation of hepcidin gene expression by these effectors in hepatocytes is currently unknown, although there is strong evidence that indirect mechanisms are involved. The aims of this study were to gain insight into these mechanisms and to determine to what extent other liver cell types are responsible for transducing the signal by which hepcidin expression is regulated in mouse hepatocytes. For this, we depleted Kupffer cells by injection of liposome-encapsulated clodronate and then studied iron- and inflammation-induced hepcidin gene expression. In addition, we directly evaluated the role of the inflammatory cytokine interleukin 6 (IL-6) by using IL-6-deficient mice. Our results show that iron is able to induce hepcidin gene expression independently of Kupffer cells in the liver and circulating IL-6. In contrast, we show that hepcidin gene induction by inflammation is also independent of Kupffer cells, but involves, at least partly, IL-6. In conclusion, these results show that two independent regulatory pathways control hepcidin gene expression and suggest that hepatocytes play a key role in the regulation of hepcidin gene expression by sensing iron and inflammatory signals. PMID:15793843

  13. Characterization of the Inflammasome in Human Kupffer Cells in Response to Synthetic Agonists and Pathogens.

    PubMed

    Zannetti, Claudia; Roblot, Guillaume; Charrier, Emily; Ainouze, Michelle; Tout, Issam; Briat, François; Isorce, Nathalie; Faure-Dupuy, Suzanne; Michelet, Maud; Marotel, Marie; Kati, Semra; Schulz, Thomas F; Rivoire, Michel; Traverse-Glehen, Alexandra; Luangsay, Souphalone; Alatiff, Omran; Henry, Thomas; Walzer, Thierry; Durantel, David; Hasan, Uzma

    2016-07-01

    The liver is the largest gland in the human body and functions as an innate immune organ. Liver macrophages called Kupffer cells (KC) constitute the largest group of macrophages in the human body. Innate immune responses involving KC represent the first line of defense against pathogens in the liver. Human monocyte-derived macrophages have been used to characterize inflammasome responses that lead to the release of the proinflammatory cytokines IL-1β and IL-18, but it has not yet been determined whether human KC contain functional inflammasomes. We show, to our knowledge for the first time, that KC express genes and proteins that make up several different inflammasome complexes. Moreover, activation of KC in response to the absent in melanoma 2 (AIM2) inflammasome led to the production of IL-1β and IL-18, which activated IL-8 transcription and hepatic NK cell activity, respectively. Other inflammasome responses were also activated in response to selected bacteria and viruses. However, hepatitis B virus inhibited the AIM2 inflammasome by reducing the mRNA stability of IFN regulatory factor 7, which regulated AIM2 transcription. These data demonstrate the production of IL-1β and IL-18 in KC, suggesting that KC contain functional inflammasomes that could be important players in the innate immune response following certain infections of the liver. We think our findings could potentially aid therapeutic approaches against chronic liver diseases that activate the inflammasome. PMID:27226092

  14. Gliotoxin causes apoptosis and necrosis of rat Kupffer cells in vitro and in vivo in the absence of oxidative stress: Exacerbation by caspase and serine protease inhibition

    PubMed Central

    Anselmi, Kristin; Stolz, Donna B.; Nalesnik, Michael; Watkins, Simon C.; Kamath, Ravindra; Gandhi, Chandrashekhar R.

    2009-01-01

    Background/Aims A potential application of gliotoxin therapy for liver fibrosis was suggested by its apoptotic effect on fibrogenic activated stellate cells. We investigated if gliotoxin exerts similar effects on hepatic macrophages Kupffer cells. Methods Effects of gliotoxin on Kupffer cells isolated from the normal liver and in vivo following its administration to CCl4–induced cirrhotic rats were studied. Results Gliotoxin caused apoptosis of cultured Kupffer cells, the effect being apparent at 0.3 μM concentration within 1 hour; longer incubation caused necrosis. This effect was associated with mitochondroial cytochrome c release, caspase-3 activation and ATP depletion. Interestingly, inhibition of caspase-3 and serine proteases accelerated and augmented gliotoxin-induced cell death via necrosis. Gliotoxin stimulated nuclear translocation of NFκB, and phosphorylation of p38, ERK1/2 and JNK MAP kinases, but these signaling molecules were not involved in gliotoxin-induced death of Kupffer cells. In vivo administration of gliotoxin to cirrhotic rats caused apoptosis of Kupffer cells, stellate cells and hepatocytes. In control rats, the effect was minimal on the nonparenchymal cells and not apparent on hepatocytes. Conclusions In the fibrotic liver, gliotoxin nonspecifically causes death of hepatic cell types. Modification of gliotoxin molecule may be necessary for selective targeting and elimination of activated stellate cells. PMID:17466404

  15. Kupffer cells and activation of endothelial TLR4 coordinate neutrophil adhesion within liver sinusoids during endotoxemia.

    PubMed

    McDonald, Braedon; Jenne, Craig N; Zhuo, Lisheng; Kimata, Koji; Kubes, Paul

    2013-12-01

    A key pathological feature of the systemic inflammatory response of sepsis/endotoxemia is the accumulation of neutrophils within the microvasculature of organs such as the liver, where they cause tissue damage and vascular dysfunction. There is emerging evidence that the vascular endothelium is critical to the orchestration of inflammatory responses to blood-borne microbes and microbial products in sepsis/endotoxemia. In this study, we aimed to understand the role of endothelium, and specifically endothelial TLR4 activation, in the regulation of neutrophil recruitment to the liver during endotoxemia. Intravital microscopy of bone marrow chimeric mice revealed that TLR4 expression by non-bone marrow-derived cells was required for neutrophil recruitment to the liver during endotoxemia. Furthermore, LPS-induced neutrophil adhesion in liver sinusoids was equivalent between wild-type mice and transgenic mice that express TLR4 only on endothelium (tlr4(-/-)Tie2(tlr4)), revealing that activation of endothelial TLR4 alone was sufficient to initiate neutrophil adhesion. Neutrophil arrest within sinusoids of endotoxemic mice requires adhesive interactions between neutrophil CD44 and endothelial hyaluronan. Intravital immunofluorescence imaging demonstrated that stimulation of endothelial TLR4 alone was sufficient to induce the deposition of serum-derived hyaluronan-associated protein (SHAP) within sinusoids, which was required for CD44/hyaluronan-dependent neutrophil adhesion. In addition to endothelial TLR4 activation, Kupffer cells contribute to neutrophil recruitment via a distinct CD44/HA/SHAP-independent mechanism. This study sheds new light on the control of innate immune activation within the liver vasculature during endotoxemia, revealing a key role for endothelial cells as sentinels in the detection of intravascular infections and coordination of neutrophil recruitment to the liver. PMID:24113769

  16. Histones activate the NLRP3 Inflammasome in Kupffer Cells during Sterile Inflammatory Liver Injury

    PubMed Central

    Huang, Hai; Chen, Hui-Wei; Evankovich, John; Yan, Wei; Rosborough, Brian R.; Nace, Gary W.; Ding, Qing; Loughran, Patricia; Beer-Stolz, Donna; Billiar, Timothy R.; Esmon, Charles T.; Tsung, Allan

    2013-01-01

    Cellular processes that drive sterile inflammatory injury after hepatic ischemia/reperfusion (I/R) injury are not completely understood. Activation of the inflammasome plays a key role in response to invading intracellular pathogens, but mounting evidence suggests it also plays a role in inflammation driven by endogenous danger-associate molecular pattern (DAMP) molecules released after ischemic injury. The nucleotide-binding domain, leucine-rich repeat containing protein 3 (NLRP3) inflammasome is one such process, and the mechanism by which its activation results in damage and inflammatory responses following liver I/R is unknown. Here we report that both NLRP3 and its downstream target Caspase-1 are activated I/R and are essential for hepatic I/R injury as both NLRP3 and Caspase-1 KO mice are protected from injury. Furthermore, inflammasome-mediated injury is dependent on Caspase-1 expression in liver non-parenchymal cells. While upstream signals that activate the inflammasome during ischemic injury are not well characterized, we show that endogenous extracellular histones activate the NLRP3 inflammasome during liver I/R through Toll-like Receptor-9 (TLR9). This occurs through TLR9-dependent generation of reactive oxygen species. This mechanism is operant in resident liver Kupffer cells, which drive innate immune responses after I/R injury by recruiting additional cell types, including neutrophils and inflammatory monocytes. These novel findings illustrate a new mechanism by which extracellular histones and activation of NLRP3 inflammasome contribute to liver damage and activation of innate immunity during sterile inflammation. PMID:23904166

  17. TREM2 governs Kupffer cell activation and explains belr1 genetic resistance to malaria liver stage infection

    PubMed Central

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

    2013-01-01

    Plasmodium liver stage infection is a target of interest for the treatment of and vaccination against malaria. Here we used forward genetics to search for mechanisms underlying natural host resistance to infection and identified triggering receptor expressed on myeloid cells 2 (TREM2) and MHC class II molecules as determinants of Plasmodium berghei liver stage infection in mice. Locus belr1 confers resistance to malaria liver stage infection. The use of newly derived subcongenic mouse lines allowed to map belr1 to a 4-Mb interval on mouse chromosome 17 that contains the Trem2 gene. We show that Trem2 expression in the nonparenchymal liver cells closely correlates with resistance to liver stage infection, implicating TREM2 as a mediator of the belr1 genetic effect. Trem2-deficient mice are more susceptible to liver stage infection than their WT counterparts. We found that Kupffer cells are the principle cells expressing TREM2 in the liver, and that Trem2−/− Kupffer cells display altered functional activation on exposure to P. berghei sporozoites. TREM2 expression in Kupffer cells contributes to the limitation of parasite expansion in isolated hepatocytes in vitro, potentially explaining the increased susceptibility of Trem2−/− mice to liver stage infection. The MHC locus was also found to control liver parasite burden, possibly owing to the expression of MHC class II molecules in hepatocytes. Our findings implicate unexpected Kupffer–hepatocyte cross-talk in the control Plasmodium liver stage infection and demonstrate that TREM2 is involved in host responses against the malaria parasite. PMID:24218563

  18. The renin angiotensin system regulates Kupffer cells in colorectal liver metastases.

    PubMed

    Wen, Shu Wen; Ager, Eleanor I; Neo, Jaclyn; Christophi, Christopher

    2013-08-01

    Blockade of the renin angiotensin system (RAS) can inhibit tumor growth and this may be mediated via undefined immunomodulatory actions. This study investigated the effects of RAS blockade on liver macrophages (Kupffer cells; KCs) in an orthotopic murine model of colorectal cancer (CRC) liver metastases. Here we showed that pharmacological targeting of the RAS [ANG II (31.25 µg/kg/h i.p.), ANG-(1-7) (24 µg/kg/h i.p.) or the ACE inhibitor; captopril (750 mg/kg/d i.p.)] altered endogenous KC numbers in the tumor-bearing liver throughout metastatic growth. Captopril, and to a lesser extent ANG-(1-7), increased KC numbers in the liver but not tumor. KCs were found to express the key RAS components: ACE and AT1R. Treatment with captopril and ANG II increased the number of AT1R-expressing KCs, although total KC numbers were not affected by ANG II. Captopril (0.1 µM) also increased macrophage invasion in vitro. Additionally, captopril was administered with KC depletion before tumor induction (day 0) or at established metastatic growth (day 18) using gadolinium chloride (GdCl 3; 20 mg/kg). Livers were collected at day 21 and quantitative stereology used as a measure of tumor burden. Captopril reduced growth of CRC liver metastases. However, when captopril was combined with early KC depletion (day 0) tumor growth was significantly increased compared with captopril alone. In contrast, late KC depletion (day 18) failed to influence the anti-tumor effects of captopril. The result of these studies suggests that manipulation of the RAS can alter KC numbers and may subsequently influence progression of CRC liver metastases. PMID:23792575

  19. 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. PMID:26319418

  20. 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. PMID:23007174

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

    PubMed Central

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

    2009-01-01

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

  2. 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

  3. Thyroid hormone-induced cytosol-to-nuclear translocation of rat liver Nrf2 is dependent on Kupffer cell functioning.

    PubMed

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

    2012-01-01

    L-3,3',5-triiodothyronine (T(3)) administration upregulates nuclear factor-E2-related factor 2 (Nrf2) in rat liver, which is redox-sensitive transcription factor mediating cytoprotection. In this work, we studied the role of Kupffer cell respiratory burst activity, a process related to reactive oxygen species generation and liver homeostasis, in Nrf2 activation using the macrophage inactivator gadolinium chloride (GdCl(3); 10 mg/kg i.v. 72 h before T(3) [0.1 mg/kg i.p.]) or NADPH oxidase inhibitor apocynin (1.5 mmol/L added to the drinking water for 7 days before T(3)), and determinations were performed 2 h after T(3). T(3) increased nuclear/cytosolic Nrf2 content ratio and levels of heme oxygenase 1 (HO-1), catalytic subunit of glutamate cysteine ligase, and thioredoxin (Western blot) over control values, proteins whose gene transcription is induced by Nrf2. These changes were suppressed by GdCl(3) treatment prior to T(3), an agent-eliciting Kupffer-cell depletion, inhibition of colloidal carbon phagocytosis, and the associated respiratory burst activity, with enhancement in nuclear inhibitor of Nrf2 kelch-like ECH-associated protein 1 (Keap1)/Nrf2 content ratios suggesting Nrf2 degradation. Under these conditions, T(3)-induced tumor necrosis factor-α (TNF-α) response was eliminated by previous GdCl(3) administration. Similar to GdCl(3), apocynin given before T(3) significantly reduced liver Nrf2 activation and HO-1 expression, a NADPH oxidase inhibitor eliciting abolishment of colloidal carbon-induced respiratory burst activity without altering carbon phagocytosis. It is concluded that Kupffer cell functioning is essential for upregulation of liver Nrf2-signaling pathway by T(3). This contention is supported by suppression of the respiratory burst activity of Kupffer cells and the associated reactive oxygen species production by GdCl(3) or apocynin given prior to T(3), thus hindering Nrf2 activation. PMID:22649286

  4. SR-A and SREC-I Are Kupffer and Endothelial Cell Receptors for Helper-dependent Adenoviral Vectors

    PubMed Central

    Piccolo, Pasquale; Vetrini, Francesco; Mithbaokar, Pratibha; Grove, Nathan C; Bertin, Terry; Palmer, Donna; Ng, Philip; Brunetti-Pierri, Nicola

    2013-01-01

    Helper-dependent adenoviral (HDAd) vectors can mediate long-term, high-level transgene expression from transduced hepatocytes with no chronic toxicity. However, a toxic acute response with potentially lethal consequences has hindered their clinical applications. Liver sinusoidal endothelial cells (LSECs) and Kupffer cells are major barriers to efficient hepatocyte transduction. Understanding the mechanisms of adenoviral vector uptake by non-parenchymal cells may allow the development of strategies aimed at overcoming these important barriers and to achieve preferential hepatocyte gene transfer with reduced toxicity. Scavenger receptors on Kupffer cells bind adenoviral particles and remove them from the circulation, thus preventing hepatocyte transduction. In the present study, we show that HDAd particles interact in vitro and in vivo with scavenger receptor-A (SR-A) and with scavenger receptor expressed on endothelial cells-I (SREC-I) and we exploited this knowledge to increase the efficiency of hepatocyte transduction by HDAd vectors in vivo through blocking of SR-A and SREC-I with specific fragments antigen-binding (Fabs). PMID:23358188

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

    SciTech Connect

    Naito, M.; Takahashi, K. )

    1991-05-01

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

  6. 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

  7. 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

  8. 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

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

    PubMed

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

    2016-03-15

    Central insulin action activates hepatic IL-6/STAT3 signaling, which suppresses the gene expression of hepatic gluconeogenic enzymes. The vagus nerve plays an important role in this centrally mediated hepatic response; however, the precise mechanism underlying this brain-liver interaction is unclear. Here, we present our findings that the vagus nerve suppresses hepatic IL-6/STAT3 signaling via α7-nicotinic acetylcholine receptors (α7-nAchR) on Kupffer cells, and that central insulin action activates hepatic IL-6/STAT3 signaling by suppressing vagal activity. Indeed, central insulin-mediated hepatic IL-6/STAT3 activation and gluconeogenic gene suppression were impeded in mice with hepatic vagotomy, pharmacological cholinergic blockade, or α7-nAchR deficiency. In high-fat diet-induced obese and insulin-resistant mice, control of the vagus nerve by central insulin action was disturbed, inducing a persistent increase of inflammatory cytokines. These findings suggest that dysregulation of the α7-nAchR-mediated control of Kupffer cells by central insulin action may affect the pathogenesis of chronic hepatic inflammation in obesity. PMID:26947072

  10. TNFα-Mediated Liver Destruction by Kupffer Cells and Ly6Chi Monocytes during Entamoeba histolytica Infection

    PubMed Central

    Ernst, Thomas; Ittrich, Harald; Jacobs, Thomas; Heeren, Joerg; Tacke, Frank; Tannich, Egbert; Lotter, Hannelore

    2013-01-01

    Amebic liver abscess (ALA) is a focal destruction of liver tissue due to infection by the protozoan parasite Entamoeba histolytica (E. histolytica). Host tissue damage is attributed mainly to parasite pathogenicity factors, but massive early accumulation of mononuclear cells, including neutrophils, inflammatory monocytes and macrophages, at the site of infection raises the question of whether these cells also contribute to tissue damage. Using highly selective depletion strategies and cell-specific knockout mice, the relative contribution of innate immune cell populations to liver destruction during amebic infection was investigated. Neutrophils were not required for amebic infection nor did they appear to be substantially involved in tissue damage. In contrast, Kupffer cells and inflammatory monocytes contributed substantially to liver destruction during ALA, and tissue damage was mediated primarily by TNFα. These data indicate that besides direct antiparasitic drugs, modulating innate immune responses may potentially be beneficial in limiting ALA pathogenesis. PMID:23300453

  11. Kupffer Cell Transplantation in Mice for Elucidating Monocyte/Macrophage Biology and for Potential in Cell or Gene Therapy.

    PubMed

    Merlin, Simone; Bhargava, Kuldeep K; Ranaldo, Gabriella; Zanolini, Diego; Palestro, Christopher J; Santambrogio, Laura; Prat, Maria; Follenzi, Antonia; Gupta, Sanjeev

    2016-03-01

    Kupffer cells (KC) play major roles in immunity and tissue injury or repair. Because recapitulation of KC biology and function within liver will allow superior insights into their functional repertoire, we studied the efficacy of the cell transplantation approach for this purpose. Mouse KC were isolated from donor livers, characterized, and transplanted into syngeneic recipients. To promote cell engraftment through impairments in native KC, recipients were preconditioned with gadolinium chloride. The targeting, fate, and functionality of transplanted cells were evaluated. The findings indicated that transplanted KC engrafted and survived in recipient livers throughout the study period of 3 months. Transplanted KC expressed macrophage functions, including phagocytosis and cytokine expression, with or without genetic modifications using lentiviral vectors. This permitted studies of whether transplanted KC could affect outcomes in the context of acetaminophen hepatotoxicity or hepatic ischemia-reperfusion injury. Transplanted KC exerted beneficial effects in these injury settings. The benefits resulted from cytoprotective factors including vascular endothelial growth factor. In conclusion, transplanted adult KC were successfully targeted and engrafted in the liver with retention of innate immune and tissue repair functions over the long term. This will provide excellent opportunities to address critical aspects in the biogenesis, fate, and function of KC within their native liver microenvironment and to develop the cell and gene therapy potential of KC transplantation. PMID:26773351

  12. Purinergic signaling via P2X7 receptor mediates IL-1β production in Kupffer cells exposed to silica nanoparticle.

    PubMed

    Kojima, Shuji; Negishi, Yusuke; Tsukimoto, Mitsutoshi; Takenouchi, Takato; Kitani, Hiroshi; Takeda, Ken

    2014-07-01

    There is extensive evidence that nanoparticles (NPs) cause adverse effects in multiple organs, including liver, though the mechanisms involved remain to be fully established. Kupffer cells are macrophages resident in the liver, and play important roles in liver inflammation induced by various toxic agents, including lipopolysaccharide (LPS). Interleukin-1 (IL-1) family members IL-1α,β are released from LPS-primed macrophages exposed to NPs, including silica NPs (SNPs), via activation of nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 inflammasomes. Here, we investigated the mechanism of production of IL-1β via activation of inflammasomes in mouse Kupffer cell line KUP5, focusing on the role of purinergic signaling via P2X7 receptor. IL-1β production by LPS-primed KUP5 cells exposed to SNPs was increased dose-dependently, and was greatest in response to SNPs with a diameter of 30 nm (SNP30), as compared with 70-nm and 300-nm SNPs (SNP70 and SNP300). ATP release was also highest in cells exposed to SNP30. Treatment of LPS-primed KUP5 cells with ATP also induced a high level of IL-1β production, similar to that induced by SNP30. IL-1β production was significantly inhibited by apyrase (an ecto-nucleotidase) and A438079 (a P2X7 antagonist/ATP-release inhibitor). Production of reactive oxygen species (ROS) was confirmed in cells exposed to SNP30. In conclusion, ATP released from P2X7 receptor in response to stimulation of KUP5 cells with SNP30 induces ROS production via cell-membrane NADPH oxidase. The ROS causes activation of inflammasomes, leading to caspase-1-dependent processing of IL-1β. PMID:24685903

  13. Dietary omega-3 fatty acids decrease mortality and Kupffer cell prostaglandin E2 production in a rat model of chronic sepsis.

    PubMed

    Barton, R G; Wells, C L; Carlson, A; Singh, R; Sullivan, J J; Cerra, F B

    1991-06-01

    We tested the hypothesis that substitution of omega-3 fat for dietary omega-6 fat would reduce mortality and decrease Kupffer cell prostaglandin E2 (PGE2) production in a rat model of chronic sepsis. Rats were fed via gastrostomy for 12 days with isonitrogenous, isocaloric diets containing 15% of calories as either safflower oil (omega-6) or a 10:1 mixture of menhaden oil (omega-3) and safflower oil. After five days of feeding, animals received an intra-abdominal abscess of defined bacterial content. Survivors were killed on post-laparotomy day 6 in conjunction with liver perfusion and protease liver digestion for Kupffer cell isolation. Kupffer cell PGE2 production was measured by radioimmunoassay after 18 hours of cell culture and again after stimulation with 0 LPS, 10 ng/ml LPS, and 10 micrograms/LPS. Mortality was decreased in menhaden oil-fed animals compared with safflower oil-fed animals (16% vs. 35%). Kupffer cell PGE2 production was decreased in menhaden oil-fed animals at 18 hours (354 +/- 54 vs. 570 +/- 95 pg/0.1 ml; p = 0.09) and after stimulation with 10 micrograms/ml LPS (140 +/- 41 vs. 288 +/- 45 pg/0.1 ml; p = 0.03) compared with safflower oil-fed animals. PMID:2056540

  14. Non-Canonical Wnt Predominates in Activated Rat Hepatic Stellate Cells, Influencing HSC Survival and Paracrine Stimulation of Kupffer Cells

    PubMed Central

    Corbett, Laura; Mann, Jelena; Mann, Derek A.

    2015-01-01

    The Wnt system is highly complex and is comprised of canonical and non-canonical pathways leading to the activation of gene expression. Our aim was to examine changes in the expression of Wnt ligands and regulators during hepatic stellate cell (HSC) transdifferentiation and assess the relative contributions of the canonical and non-canonical Wnt pathways in fibrogenic activated HSC. The expression profile of Wnt ligands and regulators in HSC was not supportive for a major role for β-catenin-dependent canonical Wnt signalling, this verified by inability to induce Topflash reporter activity in HSC even when expressing a constitutive active β-catenin. We detected expression of Wnt5a in activated HSC which can signal via non-canonical mechanisms and showed evidence for non-canonical signalling in these cells involving phosphorylation of Dvl2 and pJNK. Stimulation of HSC or Kupffer cells with Wnt5a regulated HSC apoptosis and expression of TGF-β1 and MCP1 respectively. We were unable to confirm a role for β-catenin-dependent canonical Wnt in HSC and instead propose autocrine and paracrine functions for Wnts expressed by activated HSC via non-canonical pathways. The data warrant detailed investigation of Wnt5a in liver fibrosis. PMID:26566235

  15. 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.

  16. 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

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

    PubMed Central

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

    2016-01-01

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

  18. SiO2 nanoparticle-induced impairment of mitochondrial energy metabolism in hepatocytes directly and through a Kupffer cell-mediated pathway in vitro

    PubMed Central

    Xue, Yang; Chen, Qingqing; Ding, Tingting; Sun, Jiao

    2014-01-01

    The liver has been shown to be a primary target organ for SiO2 nanoparticles in vivo, and may be highly susceptible to damage by these nanoparticles. However, until now, research focusing on the potential toxic effects of SiO2 nanoparticles on mitochondria-associated energy metabolism in hepatocytes has been lacking. In this work, SiO2 nanoparticles 20 nm in diameter were evaluated for their ability to induce dysfunction of mitochondrial energy metabolism. First, a buffalo rat liver (BRL) cell line was directly exposed to SiO2 nanoparticles, which induced cytotoxicity and mitochondrial damage accompanied by decreases in mitochondrial dehydrogenase activity, mitochondrial membrane potential, enzymatic expression in the Krebs cycle, and activity of the mitochondrial respiratory chain complexes I, III and IV. Second, the role of rat-derived Kupffer cells was evaluated. The supernatants from Kupffer cells treated with SiO2 nanoparticles were transferred to stimulate BRL cells. We observed that SiO2 nanoparticles had the ability to activate Kupffer cells, leading to release of tumor necrosis factor-α, nitric oxide, and reactive oxygen species from these cells and subsequently to inhibition of mitochondrial respiratory chain complex I activity in BRL cells. PMID:24959077

  19. 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

  20. P2X7 receptor-NADPH oxidase axis mediates protein radical formation and Kupffer cell activation in carbon tetrachloride-mediated steatohepatitis in obese mice.

    PubMed

    Chatterjee, Saurabh; Rana, Ritu; Corbett, Jean; Kadiiska, Maria B; Goldstein, Joyce; Mason, Ronald P

    2012-05-01

    While some studies show that carbon tetrachloride-mediated metabolic oxidative stress exacerbates steatohepatitic-like lesions in obese mice, the redox mechanisms that trigger the innate immune system and accentuate the inflammatory cascade remain unclear. Here we have explored the role of the purinergic receptor P2X7-NADPH oxidase axis as a primary event in recognizing the heightened release of extracellular ATP from CCl(4)-treated hepatocytes and generating redox-mediated Kupffer cell activation in obese mice. We found that an underlying condition of obesity led to the formation of protein radicals and posttranslational nitration, primarily in Kupffer cells, at 24h post-CCl(4) administration. The free radical-mediated oxidation of cellular macromolecules, which was NADPH oxidase and P2X7 receptor-dependent, correlated well with the release of TNF-α and MCP-2 from Kupffer cells. The Kupffer cells in CCl(4)-treated mice exhibited increased expression of MHC Class II proteins and showed an activated phenotype. Increased expression of MHC Class II was inhibited by the NADPH oxidase inhibitor apocynin , P2X7 receptor antagonist A438709 hydrochloride, and genetic deletions of the NADPH oxidase p47 phox subunit or the P2X7 receptor. The P2X7 receptor acted upstream of NADPH oxidase activation by up-regulating the expression of the p47 phox subunit and p47 phox binding to the membrane subunit, gp91 phox. We conclude that the P2X7 receptor is a primary mediator of oxidative stress-induced exacerbation of inflammatory liver injury in obese mice via NADPH oxidase-dependent mechanisms. PMID:22343416

  1. Re-evaluation of thin layer chromatography as an alternative method for the quantification of prostaglandins from rat Kupffer cells.

    PubMed

    Pestel, Sabine; Jungermann, Kurt; Schieferdecker, Henrike L

    2005-01-01

    In contrast to conventionally used immunoassays, thin layer chromatography (TLC)--by prelabeling of cells with radioactive arachidonic acid (AA)--allows to differentiate between cellularly built and added prostanoids and thus to investigate feedback effects of prostanoids on their own release. PGD2, TXB2 and PGE2 released from zymosan-stimulated Kupffer cells were separated with distinct RF-values, corresponding to those of the pure substances. Quantification of PGD2 and PGE2 gave comparable results with TLC and immunoassays, but measurement in the presence of added prostanoids was only possible with TLC. Moreover TLC was superior to immunoassays in having a longer linear range while being comparably sensitive. Cellularly built TXB2 in its radioactively labeled form was not detectable by TLC. Inhibition of TXB2 release by externally added AA or technical artifacts were excluded, suggesting that the cellular AA-pools used for prostaglandin and thromboxane synthesis differ in their accessibility for added AA. Thus, TLC is a simple, sensitive and precise method for the quantification of cellularly built prostaglandins but not of thromboxane even in the presence of added prostanoids. PMID:15789620

  2. 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

  3. Mainstream cigarette smoke accelerates the progression of nonalcoholic steatohepatitis by modulating Kupffer cell-mediated hepatocellular apoptosis in adolescent mice.

    PubMed

    Park, Surim; Kim, Jong Won; Yun, Hyejin; Choi, Seong-Jin; Lee, Sang-Hyub; Choi, Kyung-Chul; Lim, Chae Woong; Lee, Kyuhong; Kim, Bumseok

    2016-08-10

    Cigarette smoking in adolescents is considered to be a major cause of preventable morbidity and mortality. The purpose of this study is to investigate the role of mainstream cigarette smoke (MSCS) on the progression of nonalcoholic steatohepatitis in adolescents. Three-week-old C57BL/6 mice were fed either a methionine and choline-deficient plus high fat (MCDHF) diet for 6 weeks. Each group was exposed to MSCS (300, 600 ug/L) or fresh air for 2h per day during the first 3 weeks of MCDHF diet feeding. MSCS increased MCDHF diet-induced NASH by increasing serum ALT/AST levels, steatosis, inflammation, and fibrosis. Furthermore, MSCS was associated with the degree of oxidative stress and hepatocellular apoptosis in NASH mice, but not prominent in controls. In vitro, cigarette smoke extract (CSE) activated Kupffer cells (KCs) to release inflammatory cytokines and oxidative stress, which induced hepatocellular apoptosis. In conclusion, MSCS exposure accelerates the progression and severity of NASH by modulating KC-mediated hepatocellular apoptosis. Our results support the regulation of CS in adolescents with steatohepatitis. PMID:27180087

  4. 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

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

    PubMed

    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. Kupffer cells suppress perfluorononanoic acid-induced hepatic peroxisome proliferator-activated receptor α expression by releasing cytokines.

    PubMed

    Fang, Xuemei; Zou, Shanshan; Zhao, Yuanyuan; Cui, Ruina; Zhang, Wei; Hu, Jiayue; Dai, Jiayin

    2012-10-01

    Kupffer cells (KCs) have been demonstrated to play a role in the regulation of intra-hepatic lipid metabolism through the synthesis and secretion of biologically active products. The involvement of KCs in the disturbance of lipid metabolism that induced by perfluorononanoic acid (PFNA), a known agonist of the peroxisome proliferator-activated receptor alpha (PPARα), was investigated in this study. Rats were exposed to PFNA or PFNA combined with gadolinium chloride, an inhibitor of KCs, for 14 days. PFNA exposure dose-dependently increased absolute and relative liver weights, induced triglyceride accumulation, up-regulated the expression of both SERBP-1c and PPARα, and stimulated the release of TNFα and IL-1β. Inactivation of KCs markedly lowered TNFα and IL-1β level, enhanced PFNA-induced expression of PPARα and its target genes, and reduced liver triglyceride levels. In vitro, PFNA-induced expression of PPARα in primary cultured hepatocytes was suppressed by recombinant rat TNFα and IL-1β. However, inhibition of the NF-κB pathway prevented this. Transient transfection and promoter analysis further revealed that these two cytokines and NF-κB were coordinately involved in the suppression of PPARα promoter activity. Our data demonstrate that TNFα and IL-1β released from KCs following PFNA exposure can suppress the expression of PPARα via NF-κB pathway, which partially contribute to the evident accumulation of triglycerides in rat liver. PMID:22648072

  7. 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

  8. 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

  9. 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

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

    PubMed Central

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

    2013-01-01

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

  11. Kupffer Cells Support Hepatitis B Virus-Mediated CD8+ T Cell Exhaustion via Hepatitis B Core Antigen-TLR2 Interactions in Mice.

    PubMed

    Li, Min; Sun, Rui; Xu, Long; Yin, Wenwei; Chen, Yongyan; Zheng, Xiaodong; Lian, Zhexiong; Wei, Haiming; Tian, Zhigang

    2015-10-01

    Hepatitis B virus (HBV) persistence is a fundamental process in chronic HBV infection and a key factor in all related liver diseases; however, the mechanisms have yet to be elucidated. We studied the role of TLR2 in HBV persistence using a well-established HBV-carrier mouse model generated by hydrodynamically injecting a phospho-adeno-associated virus/HBV1.2 plasmid into mice. We found that a genetic deficiency in TLR2 improves HBV elimination, whereas activating TLR2 led to more stable HBV persistence, suggesting that TLR2 activation is critical in HBV persistence. Furthermore, we noted that TLR2 activation could inhibit CD8(+) T cell function, causing the exhaustion phenotype in HBV-carrier mice, because TLR2 deficiency might rescue CD8(+) T cell function in a cellular adoptive experiment. TLR2 expression on Kupffer cells (KCs) was upregulated in HBV-carrier mice, which accounts for HBV persistence, because the difference in anti-HBV immunity between HBV-carrier wild-type and Tlr2(-/-) mice did not exist after KC depletion. In addition, similar to TLR2 deficiency, after KC depletion, CD8(+) T cells were more efficiently activated in HBV-carrier mice, leading to rapid HBV elimination. KCs produced more IL-10 upon TLR2 activation in response to direct hepatitis B core Ag stimulation, and the elevated IL-10 inhibited CD8(+) T cell function in HBV-carrier mice, because IL-10 deficiency or anti-IL-10R treatment resulted in CD8(+) T cells with stronger antiviral function. In conclusion, KCs support liver tolerance by inducing anti-HBV CD8(+) T cell exhaustion via IL-10 production after TLR2 activation by hepatitis B core Ag stimulation. PMID:26304988

  12. Stereological assessment of sexual dimorphism in the rat liver reveals differences in hepatocytes and Kupffer cells but not hepatic stellate cells.

    PubMed

    Marcos, Ricardo; Lopes, Célia; Malhão, Fernanda; Correia-Gomes, Carla; Fonseca, Sónia; Lima, Margarida; Gebhardt, Rolf; Rocha, Eduardo

    2016-06-01

    There is long-standing evidence that male and female rat livers differ in enzyme activity. More recently, differences in gene expression profiling have also been found to exist; however, it is still unclear whether there is morphological expression of male/female differences in the normal liver. Such differences could help to explain features seen at the pathological level, such as the greater regenerative potential generally attributed to the female liver. In this paper, hepatocytes (HEP), Kupffer cells (KC) and hepatic stellate cells (HSC) of male and female rats were examined to investigate hypothesised differences in number, volume and spatial co-localisation of these cell types. Immunohistochemistry and design-based stereology were used to estimate total numbers, numbers per gram and mean cell volumes. The position of HSC within lobules (periportal vs. centrilobular) and their spatial proximity to KC was also assessed. In addition, flow cytometry was used to investigate the liver ploidy. In the case of HEP and KC, differences in the measured cell parameters were observed between male and female specimens; however, no such differences were detected for HSC. Female samples contained a higher number of HEP per gram, with more binucleate cells. The HEP nuclei were smaller in females, which was coincident with more abundant diploid particles in these animals. The female liver also had a greater number of KC per gram, with a lower percentage of KC in the vicinity of HSC compared with males. In this study, we document hitherto unknown morphological sexual dimorphism in the rat liver, namely in HEP and KC. These differences may account for the higher regenerative potential of the female liver and lend weight to the argument for considering the rat liver as a sexually dimorphic organ. PMID:26892301

  13. 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

  14. 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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

    Drug induced liver injury (DILI) is an idiosyncratic adverse drug reaction leading to severe liver damage. Kupffer cells (KC) sense hepatic tissue stress/damage and therefore could be a tool for the estimation of consequent effects associated with DILI. Aim of the present study was to establish a human in vitro liver model for the investigation of immune-mediated signaling in the pathogenesis of DILI. Hepatocytes and KC were isolated from human liver specimens. The isolated KC yield was 1.2 ± 0.9 × 10(6) cells/g liver tissue with a purity of >80%. KC activation was investigated by the measurement of reactive oxygen intermediates (ROI, DCF assay) and cell activity (XTT assay). The initial KC activation levels showed broad donor variability. Additional activation of KC using supernatants of hepatocytes treated with hepatotoxic drugs increased KC activity and led to donor-dependent changes in the formation of ROI compared to KC incubated with supernatants from untreated hepatocytes. Additionally, a compound- and donor-dependent increase in proinflammatory cytokines or in anti-inflammatory cytokines was detected. In conclusion, KC related immune signaling in hepatotoxicity was successfully determined in a newly established in vitro liver model. KC were able to detect hepatocyte stress/damage and to transmit a donor- and compound-dependent immune response via cytokine production. PMID:26491234

  17. 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. PMID:26232728

  18. Kupffer cell depletion attenuates leptin-mediated methoxamine-stimulated portal perfusion pressure and thromboxane A2 release in a rodent model of NASH-cirrhosis.

    PubMed

    Yang, Ying-Ying; Huang, Yi-Tsau; Tsai, Tung-Hu; Hou, Ming-Chih; Lee, Fa-Yauh; Lee, Shou-Dong; Lin, Han-Chieh

    2012-12-01

    Cirrhotic portal hypertension is characterized by increased hepatic oxidative stress, AA (arachidonic acid)-derived TXA(2) (thromboxane A(2)) release and exaggerated hepatic response to the α-adrenergic agonist MTX (methoxamine). Besides promoting hepatic fibrosis, the role of hyperleptinaemia in the modulation of vascular response in NASH (non-alcoholic steatohepatitis) rat livers remains unknown. The aim of the present study was to explore the possible links between hyperleptinaemia and the disarrangement in the hepatic microcirculation. NASH-cirrhosis with hyperleptinaemia was induced in lean rats by feeding with an HF/MCD (high-fat/methionine-choline-deficient) diet. Portal haemodynamics, various substances, protein and mRNA expression and PUFA (polyunsaturated fatty acid) composition were measured. Finally, the effects of leptin pre-infusion on TXA(2) release and concentration-PPP (portal perfusion pressure) curves in response to MTX were evaluated by simultaneously pre-treatment with the Kupffer cell inactivators GdCl(3) (gadolinium chloride) or EC (encapsulated clodronate), the TXS (TXA(2) synthase) inhibitor furegrelate, the TP receptor (TXA(2) receptor) antagonist SQ29548 and the dual TXS/TP receptor antagonist BM567. In HF/MCD+leptin-lean rats, cirrhosis-induced PPP and MTX hyper-responsiveness were associated with increased hepatic TXA(2) production, TBARS (thiobarbituric acid-reacting substances) levels and the AA (arachidonic acid)/n-3 PUFA ratio, and up-regulation of hepatic leptin, FAS (fatty acid synthase), NADPH oxidase subunits, TXS, TP receptor, TGFβ(1) (transforming growth factor β(1)) proteins and mRNAs. Pre-infusion of leptin significantly enhanced MTX-stimulated PPP elevation and TXA(2) release, which were attenuated by GdCl(3) and EC pre-treatment. Concomitantly pre-incubation with BM567, but not furegrelate or SQ29548, significantly abolished the leptin-enhanced MTX-stimulated increase in PPP in NASH-cirrhotic rats. Hyperleptinaemia

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

    PubMed Central

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

    2014-01-01

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

  20. 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

  1. 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

  2. Mercury-selenium relationships in liver of Guiana dolphin: the possible role of Kupffer cells in the detoxification process by tiemannite formation.

    PubMed

    Lailson-Brito, José; Cruz, Renato; 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

  3. 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.

  4. 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

  5. C5a anaphylatoxin as a product of complement activation up-regulates the complement inhibitory factor H in rat Kupffer cells.

    PubMed

    Schlaf, Gerald; Nitzki, Frauke; Heine, Ines; Hardeland, Rüdiger; Schieferdecker, Henrike L; Götze, Otto

    2004-11-01

    The 155-kDa complement regulator factor H (FH) is the predominant soluble regulatory protein of the complement system. It acts as a cofactor for the factor I-mediated conversion of the component C3b to iC3b, competes with factor B for a binding site on C3b and C3(H2O) and promotes the dissociation of the C3bBb complex. The primary site of synthesis is the liver, i.e. FH-specific mRNA and protein were identified in both hepatocytes (HC) and Kupffer cells (KC). Previous studies in rat primary HC and KC had shown that the proinflammatory cytokine IFN-gamma influences the balance between activation and inhibition of the complement system through up-regulation of the inhibitory FH. In this study we show that C5a, as a product of complement activation, stimulates the expression of FH-specific mRNA and protein in KC and thus induces a negative feedback. Quantitative-competitive RT-PCR showed an approximate threefold C5a-induced up-regulation of FH. ELISA analyses revealed a corresponding increase in FH protein in the supernatants of KC. The up-regulation of FH was completely inhibited by the C5a-blocking monoclonal antibody 6-9F. Furthermore, an involvement of LPS and IFN-gamma was excluded, which strongly indicates a direct effect of C5a on the expression of FH in KC. PMID:15376195

  6. 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

  7. Autoregulation by eicosanoids of human Kupffer cell secretory products. A study of interleukin-1, interleukin-6, tumor necrosis factor-alpha, transforming growth factor-beta, and nitric oxide.

    PubMed Central

    Roland, C R; Goss, J A; Mangino, M J; Hafenrichter, D; Flye, M W

    1994-01-01

    OBJECTIVE: Methods employed previously to analyze the secretory behavior of rodent Kupffer cells (KC) were used to examine the human KC's secretory response to lipopolysaccharide (LPS). SUMMARY BACKGROUND DATA: As the resident hepatic macrophage, the KC resides at the interface between the portal and systemic circulations. Consequently, this cell may play an integral role in the immune response to antigens and bacteria in the sinusoid. Study of cytokine production by the KC has relied predominantly on the rat as the source of these cells. Whether human KCs respond similarly to rat KCs after LPS stimulation has been a matter of speculation. METHODS: Kupffer cells obtained from seven human livers were tested under conditions identical to those used to study rat KCs. Kupffer cells rested for 12 hours after isolation were stimulated with LPS (2.5 micrograms/mL). Arginine concentration in the culture medium varied from 0.01 to 1.2 mM. To examine the role of eicosanoids, parallel culture wells received indomethacin (10 microM). Culture supernatants were assayed for interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta), prostaglandin E2 (PGE2), and nitric oxide. RESULTS: Similar to the rat KC, LPS-stimulated human KCs released IL-1, IL-6, TNF-alpha, TGF-beta, and PGE2. However, unlike rat KCs, nitric oxide could not be detected, regardless of whether the human KCs were exposed to LPS, interferon-gamma (INF-gamma), or LPS + IFN-gamma. Similar to rat KCs, indomethacin prevented PGE2 release while significantly upregulating TNF-alpha, IL-1, and IL-6, but not TGF-beta, consistent with an autoregulatory control of eicosanoids over proinflammatory cytokines. As has been shown in the rat, physiologic levels of L-arginine (0.01 mM) significantly enhanced LPS-induced PGE2 secretion relative to the response in medium containing standard L-arginine concentration (1.2 mM); however, unlike the rat KC, the human

  8. [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

  9. 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. PMID:26825450

  10. 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.

  11. 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.

  12. 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

  13. 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

  14. Galactosylated LDL nanoparticles: a novel targeting delivery system to deliver antigen to macrophages and enhance antigen specific T cell responses.

    PubMed

    Wu, Fang; Wuensch, Sherry A; Azadniv, Mitra; Ebrahimkhani, Mohammad R; Crispe, I Nicholas

    2009-01-01

    We aim to define the role of Kupffer cells in intrahepatic antigen presentation, using the selective delivery of antigen to Kupffer cells rather than other populations of liver antigen-presenting cells. To achieve this we developed a novel antigen delivery system that can target antigens to macrophages, based on a galactosylated low-density lipoprotein nanoscale platform. Antigen was delivered via the galactose particle receptor (GPr), internalized, degraded and presented to T cells. The conjugation of fluoresceinated ovalbumin (FLUO-OVA) and lactobionic acid with LDL resulted in a substantially increased uptake of FLUO-OVA by murine macrophage-like ANA1 cells in preference to NIH3T3 cells, and by primary peritoneal macrophages in preference to primary hepatic stellate cells. Such preferential uptake led to enhanced proliferation of OVA specific T cells, showing that the galactosylated LDL nanoscale platform is a successful antigen carrier, targeting antigen to macrophages but not to all categories of antigen presenting cells. This system will allow targeted delivery of antigen to macrophages in the liver and elsewhere, addressing the question of the role of Kupffer cells in liver immunology. It may also be an effective way of delivering drugs or vaccines directly at macrophages. PMID:19637876

  15. Cell morphology and focal adhesion location alters internal cell stress.

    PubMed

    Mullen, C A; Vaughan, T J; Voisin, M C; Brennan, M A; Layrolle, P; McNamara, L M

    2014-12-01

    Extracellular mechanical cues have been shown to have a profound effect on osteogenic cell behaviour. However, it is not known precisely how these cues alter intracellular mechanics to initiate changes in cell behaviour. In this study, a combination of in vitro culture of MC3T3-E1 cells and finite-element modelling was used to investigate the effects of passive differences in substrate stiffness on intracellular mechanics. Cells on collagen-based substrates were classified based on the presence of cell processes and the dimensions of various cellular features were quantified. Focal adhesion (FA) density was quantified from immunohistochemical staining, while cell and substrate stiffnesses were measured using a live-cell atomic force microscope. Computational models of cell morphologies were developed using an applied contraction of the cell body to simulate active cell contraction. The results showed that FA density is directly related to cell morphology, while the effect of substrate stiffness on internal cell tension was modulated by both cell morphology and FA density, as investigated by varying the number of adhesion sites present in each morphological model. We propose that the cells desire to achieve a homeostatic stress state may play a role in osteogenic cell differentiation in response to extracellular mechanical cues. PMID:25297316

  16. Cell morphology and focal adhesion location alters internal cell stress

    PubMed Central

    Mullen, C. A.; Vaughan, T. J.; Voisin, M. C.; Brennan, M. A.; Layrolle, P.; McNamara, L. M.

    2014-01-01

    Extracellular mechanical cues have been shown to have a profound effect on osteogenic cell behaviour. However, it is not known precisely how these cues alter intracellular mechanics to initiate changes in cell behaviour. In this study, a combination of in vitro culture of MC3T3-E1 cells and finite-element modelling was used to investigate the effects of passive differences in substrate stiffness on intracellular mechanics. Cells on collagen-based substrates were classified based on the presence of cell processes and the dimensions of various cellular features were quantified. Focal adhesion (FA) density was quantified from immunohistochemical staining, while cell and substrate stiffnesses were measured using a live-cell atomic force microscope. Computational models of cell morphologies were developed using an applied contraction of the cell body to simulate active cell contraction. The results showed that FA density is directly related to cell morphology, while the effect of substrate stiffness on internal cell tension was modulated by both cell morphology and FA density, as investigated by varying the number of adhesion sites present in each morphological model. We propose that the cells desire to achieve a homeostatic stress state may play a role in osteogenic cell differentiation in response to extracellular mechanical cues. PMID:25297316

  17. 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

  18. 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.

  19. Fuel cell with internal flow control

    DOEpatents

    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.

  20. 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).

  1. International Society for Stem Cell Research

    MedlinePlus

    ... Industry Committee Session RUCDR Humanity in a Dish Stem Cell Engineering Junior Investigator Events Career Panel Meet the ... Scientific Program Confirmed Speakers Support/Exhibit Meeting Supporters Stem Cell Engineering 2014 Program Committee Featured Speakers Deepak Srivastava ...

  2. 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.

  3. 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.

  4. Rapid internalization of the insulin receptor in rat hepatoma cells

    SciTech Connect

    Backer, J.M.; White, M.F.; Kahn, C.R.

    1987-05-01

    The authors have studied the internalization of the insulin receptor (IR) in rat hepatoma cells (Fao). The cells were surface-iodinated at 4C, stimulated with insulin at 37C, and then cooled rapidly, trypsinized at 4C and solubilized. The IR was immunoprecipitated with a specific antibody, and internalization of the IR was assessed by the appearance of trypsin-resistant bands on SDS-PAGE. Insulin induced the internalization of surface receptors with a t 1/2 of 9-10 mins; cells not exposed to insulin internalized less than 20% of the IR during 1 h at 37C. Further experiments demonstrated that the accumulation of trypsin-resistant IR paralleled a loss of receptor from the cell surface. Insulin-stimulated cells were chilled and iodinated at 4C, followed by solubilization, immunoprecipitation and SDS-PAGE; alternatively, insulin-stimulated cells were chilled, surface-bound ligand removed by washing the cells at pH 4.2, and specific ( SVI)insulin binding measured at 4C. Both techniques confirmed the disappearance of IR from the cell surface at rates comparable to the insulin-stimulated internalization described above. The total amount of phosphotyrosine-containing IR, as assessed by immunoprecipitation with an anti-phosphotyrosine antibody, remained constant during this time interval, suggesting that active kinase is translocated into the cell. In summary, the authors data indicate that insulin binding increases the rate of IR internalization of Fao cells. This relocation may facilitate the interaction of the activated tyrosine kinase in the IR with intracellular substrates, thus transmitting the insulin signal to metabolic pathways.

  5. Electrochemical cell having internal short inhibitor

    SciTech Connect

    Hooke, J.W.

    1984-04-24

    An electrochemical cell comprises a spirally wound assembly, the assembly including a negative plate; a porous polyester layer disposed on each major surface of the negative plate; a porous, electrically non-conductive separator disposed on each of the polyester layers; and a positive plate disposed on one of the separators. The cell further includes a housing for enclosing the assembly and an electrolyte such that the electrolyte comes in contact with the plates, polyester layers and separators. The housing includes a pair of external terminals each of which being connected to one of the plates.

  6. 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

  7. 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

  8. 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.

  9. 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

  10. Filamin A Regulates Caveolae Internalization and Trafficking in Endothelial Cells

    PubMed Central

    Sverdlov, Maria; Shinin, Vasily; Place, Aaron T.; Castellon, Maricela

    2009-01-01

    Transcytosis via caveolae is critical for maintaining vascular homeostasis by regulating the tissue delivery of macromolecules, hormones, and lipids. In the present study, we test the hypothesis that interactions between F-actin cross-linking protein filamin A and caveolin-1 facilitate the internalization and trafficking of caveolae. Small interfering RNA-mediated knockdown of filamin A, but not filamin B, reduced the uptake and transcytosis of albumin by ∼35 and 60%, respectively, without altering the actin cytoskeletal structure or cell–cell adherens junctions. Mobility of both intracellular caveolin-1–green fluorescent protein (GFP)-labeled vesicles measured by fluorescence recovery after photobleaching and membrane-associated vesicles measured by total internal reflection-fluorescence microscopy was decreased in cells with reduced filamin A expression. In addition, in melanoma cells that lack filamin A (M2 cells), the majority of caveolin-1-GFP was localized on the plasma membrane, whereas in cells in which filamin A expression was reconstituted (A7 cells and M2 cells transfected with filamin A-RFP), caveolin-1-GFP was concentrated in intracellular vesicles. Filamin A association with caveolin-1 in endothelial cells was confirmed by cofractionation of these proteins in density gradients, as well as by coimmunoprecipitation. Moreover, this interaction was enhanced by Src activation, associated with increased caveolin-1 phosphorylation, and blocked by Src inhibition. Taken together, these data suggest that filamin A association with caveolin-1 promotes caveolae-mediated transport by regulating vesicle internalization, clustering, and trafficking. PMID:19759182

  11. 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)

  12. Fragments of Target Cells are Internalized into Retroviral Envelope Protein-Expressing Cells during Cell-Cell Fusion by Endocytosis

    PubMed Central

    Izumida, Mai; Kamiyama, Haruka; Suematsu, Takashi; Honda, Eri; Koizumi, Yosuke; Yasui, Kiyoshi; Hayashi, Hideki; Ariyoshi, Koya; Kubo, Yoshinao

    2016-01-01

    Retroviruses enter into host cells by fusion between viral and host cell membranes. Retroviral envelope glycoprotein (Env) induces the membrane fusion, and also mediates cell-cell fusion. There are two types of cell-cell fusions induced by the Env protein. Fusion-from-within is induced by fusion between viral fusogenic Env protein-expressing cells and susceptible cells, and virions induce fusion-from-without by fusion between adjacent cells. Although entry of ecotropic murine leukemia virus (E-MLV) requires host cell endocytosis, the involvement of endocytosis in cell fusion is unclear. By fluorescent microscopic analysis of the fusion-from-within, we found that fragments of target cells are internalized into Env-expressing cells. Treatment of the Env-expressing cells with an endocytosis inhibitor more significantly inhibited the cell fusion than that of the target cells, indicating that endocytosis in Env-expressing cells is required for the cell fusion. The endocytosis inhibitor also attenuated the fusion-from-without. Electron microscopic analysis suggested that the membrane fusion resulting in fusion-from-within initiates in endocytic membrane dents. This study shows that two types of the viral cell fusion both require endocytosis, and provides the cascade of fusion-from-within. PMID:26834711

  13. Intersectin Regulates Fission and Internalization of Caveolae in Endothelial Cells

    PubMed Central

    Predescu, Sanda A.; Predescu, Dan N.; Timblin, Barbara K.; Stan, Radu V.; Malik, Asrar B.

    2003-01-01

    Intersectin, a multiple Eps15 homology and Src homology 3 (SH3) domain–containing protein, is a component of the endocytic machinery in neurons and nonneuronal cells. However, its role in endocytosis via caveolae in endothelial cells (ECs) is unclear. We demonstrate herein by coimmunoprecipitation, velocity sedimentation on glycerol gradients, and cross-linking that intersectin is present in ECs in a membrane-associated protein complex containing dynamin and SNAP-23. Electron microscopy (EM) immunogold labeling studies indicated that intersectin associated preferentially with the caveolar necks, and it remained associated with caveolae after their fission from the plasmalemma. A cell-free system depleted of intersectin failed to support caveolae fission from the plasma membrane. A biotin assay used to quantify caveolae internalization and extensive EM morphological analysis of ECs overexpressing wt-intersectin indicated a wide range of morphological changes (i.e., large caveolae clusters marginated at cell periphery and pleiomorphic caveolar necks) as well as impaired caveolae internalization. Biochemical evaluation of caveolae-mediated uptake by ELISA showed a 68.4% inhibition by reference to control. We also showed that intersectin interaction with dynamin was important in regulating the fission and internalization of caveolae. Taken together, the results indicate the crucial role of intersectin in the mechanism of caveolae fission in endothelial cells. PMID:12960435

  14. Mitotic internalization of planar cell polarity proteins preserves tissue polarity.

    PubMed

    Devenport, Danelle; Oristian, Daniel; Heller, Evan; Fuchs, Elaine

    2011-08-01

    Planar cell polarity (PCP) is the collective polarization of cells along the epithelial plane, a process best understood in the terminally differentiated Drosophila wing. Proliferative tissues such as mammalian skin also show PCP, but the mechanisms that preserve tissue polarity during proliferation are not understood. During mitosis, asymmetrically distributed PCP components risk mislocalization or unequal inheritance, which could have profound consequences for the long-range propagation of polarity. Here, we show that when mouse epidermal basal progenitors divide PCP components are selectively internalized into endosomes, which are inherited equally by daughter cells. Following mitosis, PCP proteins are recycled to the cell surface, where asymmetry is re-established by a process reliant on neighbouring PCP. A cytoplasmic dileucine motif governs mitotic internalization of atypical cadherin Celsr1, which recruits Vang2 and Fzd6 to endosomes. Moreover, embryos transgenic for a Celsr1 that cannot mitotically internalize exhibit perturbed hair-follicle angling, a hallmark of defective PCP. This underscores the physiological relevance and importance of this mechanism for regulating polarity during cell division. PMID:21743464

  15. 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.

  16. 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.

  17. 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. PMID:27048816

  18. Internalization of Proteinase 3 Is Concomitant with Endothelial Cell Apoptosis and Internalization of Myeloperoxidase with Generation of Intracellular Oxidants

    PubMed Central

    Yang, Jia Jin; Preston, Gloria A.; Pendergraft, William F.; Segelmark, Mårten; Heeringa, Peter; Hogan, Susan L.; Jennette, J. Charles; Falk, Ronald J.

    2001-01-01

    The important issue addressed by the studies presented here is the mechanism of neutrophil-mediated damage to endothelial and epithelial cells during inflammation. Binding of neutrophil-released granule proteins to endothelial cells may be involved in vascular damage in patients with inflammatory vascular diseases. We have determined whether granule proteins proteinase 3(PR3) and/or myeloperoxidase (MPO) are internalized into endothelial cells, as examined by UV light, confocal, and electron microscopy. Coincident induction of apoptosis and/or the generation of intracellular oxidants were monitored. The results indicate that human endothelial cells (human umbilical vein endothelial cells, human umbilical arterial endothelial cells, human lung microvascular endothelial cells) internalize both PR3 and MPO, which are detected on the cell surface, in the cytoplasm, and possibly nuclear. Epithelial cells (small airway epithelial cells) internalized MPO but not PR3, implying that the mechanism of PR3 internalization may be cell-type specific and different from that of MPO. Internalization of PR3, but not MPO, correlated with activation of apoptosis. Internalization of MPO correlated with an increase in intracellular oxidant radicals. The requirement for the proteolytic activity of PR3 for the induction of apoptosis was examined by generating PR3-truncated fragments that did not contain the components of the catalytic triad. An apoptotic function was localized to the C-terminal portion of PR3. These studies reveal novel mechanisms by which the neutrophil granule proteins PR3 and MPO contribute to tissue injury at sites of inflammation. PMID:11159195

  19. Nonprofessional Phagocytic Cell Receptors Involved in Staphylococcus aureus Internalization

    PubMed Central

    Alva-Murillo, Nayeli; López-Meza, Joel Edmundo

    2014-01-01

    Staphylococcus aureus is a successful human and animal pathogen. The majority of infections caused by this pathogen are life threatening, primarily because S. aureus has developed multiple evasion strategies, possesses intracellular persistence for long periods, and targets the skin and soft tissues. Therefore, it is very important to understand the mechanisms employed by S. aureus to colonize and proliferate in these cells. The aim of this review is to describe the recent discoveries concerning the host receptors of nonprofessional phagocytes involved in S. aureus internalization. Most of the knowledge related to the interaction of S. aureus with its host cells has been described in professional phagocytic cells such as macrophages. Here, we showed that in nonprofessional phagocytes the α5β1 integrin host receptor, chaperons, and the scavenger receptor CD36 are the main receptors employed during S. aureus internalization. The characterization and identification of new bacterial effectors and the host cell receptors involved will undoubtedly lead to new discoveries with beneficial purposes. PMID:24826382

  20. 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

  1. Internalized Chitosan Nanoparticles Persist for Long Time in Cultured Cells

    PubMed Central

    Malatesta, M.; Grecchi, S.; Chiesa, E.; Cisterna, B.; Costanzo, M.; Zancanaro, C.

    2015-01-01

    Chitosan-based nanoparticles (chiNPs) are considered to be potentially good carriers for the sustained intracellular delivery of specific molecules. However, scarce attention has been paid to the long-lasting permanence of these NPs in the intracellular milieu, as well as to their intracellular fate (i.e., distribution, interaction with cell organelles, and degradation) in the long term. In the present study, the presence and subcellular location of FITC-labelled chiNPs were monitored in HeLa cells up to 14 days post-administration using multicolorfluorescence confocal microscopy and diaminobenzidine photo-oxidation at transmission electron microscopy. The main result of the present study is the demonstration that internalized chiNPs persist inside the cell up to two weeks, occurring in both the cytoplasm and nucleus; accordingly, chiNPs are able to pass from mother to daughter cells through several mitotic cycles. The cells did not show increased mortality or structural damage up to 14 days after chiNP exposure. PMID:25820565

  2. Kuppfer Cells Trigger Nonalcoholic Steatohepatitis Development in Diet-induced Mouse Model through Tumor Necrosis Factor-α Production*

    PubMed Central

    Tosello-Trampont, Annie-Carole; Landes, Susan G.; Nguyen, Virginia; Novobrantseva, Tatiana I.; Hahn, Young S.

    2012-01-01

    Nonalcoholic steatohepatitis (NASH), characterized by lipid deposits within hepatocytes (steatosis), is associated with hepatic injury and inflammation and leads to the development of fibrosis, cirrhosis, and hepatocarcinoma. However, the pathogenic mechanism of NASH is not well understood. To determine the role of distinct innate myeloid subsets in the development of NASH, we examined the contribution of liver resident macrophages (i.e. Kupffer cells) and blood-derived monocytes in triggering liver inflammation and hepatic damage. Employing a murine model of NASH, we discovered a previously unappreciated role for TNFα and Kupffer cells in the initiation and progression of NASH. Sequential depletion of Kupffer cells reduced the incidence of liver injury, steatosis, and proinflammatory monocyte infiltration. Furthermore, our data show a differential contribution of Kupffer cells and blood monocytes during the development of NASH; Kupffer cells increased their production of TNFα, followed by infiltration of CD11bintLy6Chi monocytes, 2 and 10 days, respectively, after starting the methionine/choline-deficient (MCD) diet. Importantly, targeted knockdown of TNFα expression in myeloid cells decreased the incidence of NASH development by decreasing steatosis, liver damage, monocyte infiltration, and the production of inflammatory chemokines. Our findings suggest that the increase of TNFα-producing Kupffer cells in the liver is crucial for the early phase of NASH development by promoting blood monocyte infiltration through the production of IP-10 and MCP-1. PMID:23066023

  3. 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

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

    PubMed

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

    2015-01-01

    In autosomal dominant polycystic kidney disease (ADPKD), cyst inflation and continuous enlargement are associated with marked transepithelial ion and fluid secretion into the cyst lumen via cystic fibrosis transmembrane conductance regulator (CFTR). Indeed, the inhibition or degradation of CFTR prevents the fluid accumulation within cysts. The in vivo mechanisms by which the lack of Polycystin-2 leads to CFTR stimulation are an outstanding challenge in ADPKD research and may bring important biomarkers for the disease. However, hampering their study, the available ADPKD in vitro cellular models lack the three-dimensional architecture of renal cysts and the ADPKD mouse models offer limited access for live-imaging experiments in embryonic kidneys. Here, we tested the zebrafish Kupffer's vesicle (KV) as an alternative model-organ. KV is a fluid-filled vesicular organ, lined by epithelial cells that express both CFTR and Polycystin-2 endogenously, being each of them easily knocked-down. Our data on the intracellular distribution of Polycystin-2 support its involvement in the KV fluid-flow induced Ca(2+)-signalling. Mirroring kidney cysts, the KV lumen inflation is dependent on CFTR activity and, as we clearly show, the knockdown of Polycystin-2 results in larger KV lumens through overstimulation of CFTR. In conclusion, we propose the zebrafish KV as a model organ to study the renal cyst inflation. Favouring its use, KV volume can be easily determined by in vivo imaging offering a live readout for screening compounds and genes that may prevent cyst enlargement through CFTR inhibition. PMID:26432887

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

    DOEpatents

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

    2008-10-07

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

  6. 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

  7. A common clathrin-mediated machinery coordinates cell-cell adhesion and bacterial internalization

    PubMed Central

    Bonazzi, Matteo; Kühbacher, Andreas; Toledo-Arana, Alejandro; Mallet, Adeline; Vasudevan, Lavanya; Pizarro-Cerdá, Javier; Brodsky, Frances M.; Cossart, Pascale

    2013-01-01

    Invasive bacterial pathogens often target cellular proteins involved in adhesion as a first event during infection. For example, Listeria monocytogenes uses the bacterial protein InlA to interact with E-cadherin, hijack the host adherens junction machinery, and invade non-phagocytic cells by a clathrin-dependent mechanism. Here we investigate a potential role for clathrin in cell-cell adhesion. We observed that the initial steps of adherens junction formation trigger the phosphorylation of clathrin, and its transient localization at forming cell-cell contacts. Furthermore, we show that clathrin serves as a hub for the recruitment of proteins that are necessary for the actin rearrangements that accompany the maturation of adherens junctions. Using an InlA/E-cadherin chimera, we show that adherent cells expressing the chimera form adherens junctions with cells expressing E-cadherin. To model bacterial invasion, we demonstrate that non-adherent cells expressing the InlA chimera can be internalized by E-cadherin-expressing adherent cells. Together these results reveal that a common clathrin-mediated machinery may regulate internalization and cell adhesion and that the relative mobility of one of the interacting partners plays an important role in the commitment to either one of these processes. PMID:22984946

  8. 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.

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

    NASA Technical Reports Server (NTRS)

    Wittel, deceased, Charles F. (Inventor)

    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.

  10. 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.

  11. 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. PMID:26399046

  12. 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

  13. 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.

  14. 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

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

    PubMed

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

    2006-01-01

    The bacterium Clostridium botulinum type C produces a progenitor toxin (C16S toxin) that binds to O-linked sugar chains terminating with sialic acid on the surface of HT-29 cells prior to internalization [A. Nishikawa, N. Uotsu, H. Arimitsu, J.C. Lee, Y. Miura, Y. Fujinaga, H. Nakada, T. Watanabe, T. Ohyama, Y. Sakano, K. Oguma, Biochem. Biophys. Res. Commun. 319 (2004) 327-333] [21]. Based on this, it was hypothesized that the C16S toxin is internalized via clathrin-coated pits. To examine this possibility, the internalized toxin was observed with a fluorescent antibody using confocal laser-scanning microscopy. The confocal images clearly indicated that the C16S toxin was internalized mainly via clathrin-coated pits and localized in early endosomes. The toxin was colocalized with caveolin-1 which is one of the components of caveolae, however, implying the toxin was also internalized via caveolae. The confocal images also showed that the neurotoxin transported to the endosome was transferred to the Golgi apparatus. However, the non-toxic components were not merged with the Golgi marker protein, TGN38, implying the neurotoxin was dissociated from progenitor toxin in endosomes. These results suggested that the C16S toxin was separated to the neurotoxin and other proteins in endosome and the neurotoxin was further transferred to the Golgi apparatus which is the center for protein sorting. PMID:16413070

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

    SciTech Connect

    Mishchenko, Alexey; Zocco, Alessandro

    2012-12-15

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

  17. 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

  18. 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.

  19. 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

  20. 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.

  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. Relationships between Membrane Binding, Affinity and Cell Internalization Efficacy of a Cell-Penetrating Peptide: Penetratin as a Case Study

    PubMed Central

    Alves, Isabel D.; Bechara, Cherine; Walrant, Astrid; Zaltsman, Yefim; Jiao, Chen-Yu; Sagan, Sandrine

    2011-01-01

    Background Penetratin is a positively charged cell-penetrating peptide (CPP) that has the ability to bind negatively charged membrane components, such as glycosaminoglycans and anionic lipids. Whether this primary interaction of penetratin with these cell surface components implies that the peptide will be further internalized is not clear. Methodology Using mass spectrometry, the amount of internalized and membrane bound penetratin remaining after washings, were quantified in three different cell lines: wild type (WT), glycosaminoglycans- (GAGneg) and sialic acid-deficient (SAneg) cells. Additionally, the affinity and kinetics of the interaction of penetratin to membrane models composed of pure lipids and membrane fragments from the referred cell lines was investigated, as well as the thermodynamics of such interactions using plasmon resonance and calorimetry. Principal Findings Penetratin internalized with the same efficacy in the three cell lines at 1 µM, but was better internalized at 10 µM in SAneg>WT>GAGneg. The heat released by the interaction of penetratin with these cells followed the ranking order of internalization efficiency. Penetratin had an affinity of 10 nM for WT cells and µM for SAneg and GAGneg cells and model membrane of phospholipids. The remaining membrane-bound penetratin after cells washings was similar in WT and GAGneg cells, which suggested that these binding sites relied on membrane phospholipids. The interaction of penetratin with carbohydrates was more superficial and reversible while it was stronger with phospholipids, likely because the peptide can intercalate between the fatty acid chains. Conclusion/Significance These results show that accumulation and high-affinity binding of penetratin at the cell-surface do not reflect the internalization efficacy of the peptide. Altogether, these data further support translocation (membrane phospholipids interaction) as being the internalization pathway used by penetratin at low

  3. Roles of regulated internalization in the polarization of cell surface receptors

    PubMed Central

    Tian, Wei; Cao, Youfang; Ismael, Amber; Stone, David

    2016-01-01

    Cell polarization, the generation of cellular asymmetries, is a fundamental biological process. Polarity of different molecules can arise through several mechanisms. Among these, internalization has been shown to play an important role in the polarization of cell surface receptors. The internalization of cell surface receptors can be upregulated upon ligand binding. Additional regulatory mechanism can downregulate the internalization process. Here we describe a general model, which incorporates these two opposing processes, to study the role of internalization in the establishment of cell polarity. We find that the competition between these two processes is sufficient to induce receptor polarization. Our results show that regulated internalization provides additional regulation on polarization as well. In addition, we discuss applications of our model to the yeast system, which shows the capability and potential of the model. PMID:25570171

  4. 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.

  5. 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

  6. Far infrared multiple-path cell without internal mirrors

    NASA Astrophysics Data System (ADS)

    Marteau, Ph.; Obriot, J.

    1990-11-01

    A mirrorless multiple path cell which can be efficiently used in the FIR is described. The device is based on the White cell and light pipe methods, and requires no setup even after cooling. It is shown how the system can be efficiently operated if certain geometrical conditions are maintained which ensure a uniform light energy distribution inside the cell. The optimization of some physical parameters is discussed, and it is shown that an effective absorption pathlength as long as 4 or 5 m can be obtained with a simple absorption cell which is 10 cm long. The FIR spectrum of CH4 obtained using the cell is presented.

  7. 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.

  8. Internalization and cellular processing of cholecystokinin in rat pancreatic acinar cells

    SciTech Connect

    Izzo, R.S.; Pellecchia, C.; Praissman, M. )

    1988-12-01

    To evaluate the internalization of cholecystokinin, monoiodinated imidoester of cholecystokinin octapeptide ({sup 125}I-(IE)-CCK-8) was bound to dispersed pancreatic acinar cells, and surface-bound and internalized radioligand were differentiated by treating with an acidified glycine buffer. The amount of internalized radioligand was four- and sevenfold greater at 24 and 37{degree}C than at 4{degree}C between 5 and 60 min of association. Specific binding of radioligand to cell surface receptors was not significantly different at these temperatures. Chloroquine, a lysosomotropic agent that blocks intracellular proteolysis, significantly increased the amount of CCK-8 internalized by 18 and 16% at 30 and 60 min of binding, respectively, compared with control. Dithiothreitol (DTT), a sulfhydryl reducing agent, also augmented the amount of CCK-8 radioligand internalized by 25 and 29% at 30 and 60 min, respectively. The effect of chloroquine and DTT on the processing of internalized radioligand was also considered after an initial 60 min of binding of radioligand to acinar cells. After 180 min of processing, the amount of radioligand internalized was significantly greater in the presence of chloroquine compared with controls, whereas the amount of radioligand declined in acinar cells treated with DTT. Internalized and released radioactivity from acinar cells was rebound to pancreatic membrane homogenates to determine the amount of intact radioligand during intracellular processing. Chloroquine significantly increased the amount of intact {sup 125}I-(IE)-CCK-8 radioligand in released and internalized radioactivity while DTT increased the amount of intact radioligand only in internalized samples. This study shows that pancreatic acinar cells rapidly internalize large amounts of CCK-8 and that chloroquine and DTT inhibit intracellular degradation.

  9. 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

  10. 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.

  11. Pretreatment of Epithelial Cells with Rifaximin Alters Bacterial Attachment and Internalization Profiles▿

    PubMed Central

    Brown, Eric L.; Xue, Qiong; Jiang, Zhi-Dong; Xu, Yi; DuPont, Herbert L.

    2010-01-01

    Rifaximin is a poorly absorbed semisynthetic antibiotic derivative of rifampin licensed for use in the treatment of traveler's diarrhea. Rifaximin reduces the symptoms of enteric infection, often without pathogen eradication and with limited effects on intestinal flora. Epithelial cells (HEp-2 [laryngeal], HCT-8 [ileocecal], A549 [lung], and HeLa [cervical]) were pretreated with rifaximin (or control antibiotics) prior to the addition of enteroaggregative Escherichia coli (EAEC). EAEC adherence was significantly reduced following rifaximin pretreatment compared to pretreatment with rifampin or doxycycline for three of the four cell lines tested. The rifaximin-mediated changes to epithelial cells were explored further by testing the attachment and internalization of either Bacillus anthracis or Shigella sonnei into A549 or HeLa cells, respectively. The attachment and internalization of B. anthracis were significantly reduced following rifaximin pretreatment. In contrast, neither the attachment nor the internalization of S. sonnei was affected by rifaximin pretreatment of HeLa cells, suggesting that rifaximin-mediated modulation of host cell physiology affected bacteria utilizing distinct attachment/internalization mechanisms differently. In addition, rifaximin pretreatment of HEp-2 cells led to reduced concentrations of inflammatory cytokines from uninfected cells. The study provides evidence that rifaximin-mediated changes in epithelial cell physiology are associated with changes in bacterial attachment/internalization and reduced inflammatory cytokine release. PMID:19858255

  12. An improved method for differentiating cell-bound from internalized particles by imaging flow cytometry

    PubMed Central

    Smirnov, Asya; Solga, Michael D; Lannigan, Joanne; Criss, Alison K

    2015-01-01

    Recognition, binding, internalization, and elimination of pathogens and cell debris are important functions of professional as well as non-professional phagocytes. However, high-throughput methods for quantifying cell-associated particles and discriminating bound from internalized particles have been lacking. Here we describe a protocol for using imaging flow cytometry to quantify the attached and phagocytosed particles that are associated with a population of cells. Cells were exposed to fluorescent particles, fixed, and exposed to an antibody of a different fluorophore that recognizes the particles. The antibody is added without cell permeabilization, such that the antibody only binds extracellular particles. Cells with and without associated particles were identified by imaging flow cytometry. For each cell with associated particles, a spot count algorithm was employed to quantify the number of extracellular (double fluorescent) and intracellular (single fluorescent) particles per cell, from which the percent particle internalization was determined. The spot count algorithm was empirically validated by examining the fluorescence and phase contrast images acquired by the flow cytometer. We used this protocol to measure binding and internalization of the bacterium Neisseria gonorrhoeae by primary human neutrophils, using different bacterial variants and under different cellular conditions. The results acquired using imaging flow cytometry agreed with findings that were previously obtained using conventional immunofluorescence microscopy. This protocol provides a rapid, powerful method for measuring the association and internalization of any particle by any cell type. PMID:25967947

  13. 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.

  14. 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

  15. Neonatal Fc Receptor Mediates Internalization of Fc in Transfected Human Endothelial Cells

    PubMed Central

    Goebl, Nancy A.; Babbey, Clifford M.; Datta-Mannan, Amita; Witcher, Derrick R.; Wroblewski, Victor J.

    2008-01-01

    The neonatal Fc receptor, FcRn mediates an endocytic salvage pathway that prevents degradation of IgG, thus contributing to the homeostasis of circulating IgG. Based on the low affinity of IgG for FcRn at neutral pH, internalization of IgG by endothelial cells is generally believed to occur via fluid-phase endocytosis. To investigate the role of FcRn in IgG internalization, we used quantitative confocal microscopy to characterize internalization of fluorescent Fc molecules by HULEC-5A lung microvascular endothelia transfected with GFP fusion proteins of human or mouse FcRn. In these studies, cells transfected with FcRn accumulated significantly more intracellular Fc than untransfected cells. Internalization of FcRn-binding forms of Fc was proportional to FcRn expression level, was enriched relative to dextran internalization in proportion to FcRn expression level, and was blocked by incubation with excess unlabeled Fc. Because we were unable to detect either surface expression of FcRn or surface binding of Fc, these results suggest that FcRn-dependent internalization of Fc may occur through sequestration of Fc by FcRn in early endosomes. These studies indicate that FcRn-dependent internalization of IgG may be important not only in cells taking up IgG from an extracellular acidic space, but also in endothelial cells participating in homeostatic regulation of circulating IgG levels. PMID:18843053

  16. 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.

  17. 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.

  18. [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

  19. 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.

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

    SciTech Connect

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

    2016-01-01

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

  1. 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

  2. Comparison of picornaviral IRES-driven internal initiation of translation in cultured cells of different origins.

    PubMed Central

    Borman, A M; Le Mercier, P; Girard, M; Kean, K M

    1997-01-01

    We recently compared the efficiency of six picornaviral internal ribosome entry segments (IRESes) and the hepatitis C virus (HCV) IRES for their ability to drive internal initiation of translationin vitro. Here we present the results of a similar comparison performed in six different cultured cell lines infected with a recombinant vaccinia virus expressing the T7 polymerase and transfected with dicistronic plasmids. The IRESes could be divided into three groups: (i) the cardiovirus and aphthovirus IRESes (and the HCV element) direct internal initiation efficiently in all cell lines tested; (ii) the enterovirus and rhinovirus IRESes are at least equally efficient in several cell lines, but are extremely inefficient in certain cell types; and (iii) the hepatitis A virus IRES is incapable of directing efficient internal initiation in any of the cell lines used (including human hepatocytes). These are the same three groups found when IRESes were classified according to their activitiesin vitro, or according to sequence homologies. In a mouse neuronal cell line, the poliovirus and other type I IRESes were not functional in an artificial bicistronic context. However, infectious poliovirions were produced efficiently after transfection of these cells with a genomic length RNA. Furthermore, activity of the type I IRESes was dramatically increased upon co-expression of the poliovirus 2A proteinase, demonstrating that while IRES efficiency may vary considerably from one cell type to another, at least in some cases viral proteins are capable of overcoming cell-specific translational defects. PMID:9023100

  3. 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

    2014-01-01

    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

  4. 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

  5. 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.

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

    SciTech Connect

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

    1990-01-01

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

  7. 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

  8. 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.

  9. 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

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

    PubMed Central

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

    2010-01-01

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

  11. [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

  12. Experimental simulation of internal short circuit in Li-ion and Li-ion-polymer cells

    NASA Astrophysics Data System (ADS)

    Cai, Wei; Wang, Hsin; Maleki, Hossein; Howard, Jason; Lara-Curzio, Edgar

    A multi-parameter controlled pinch test was developed to study the occurrence of internal short circuits in Li-ion and Li-ion-polymer cells. By tuning the control parameters (i.e., cell voltage as well as pinching area, load, and speed), the pinch test can reproducibly create an internal short between a cell jelly-roll's inner layer electrodes as small as 1-mm wide. This recreates conditions similar to those that may occur during service. In this paper we demonstrate the use of the pinch test as a means to assess design and manufacturing changes in Li-ion-polymer cells on their thermal stability and to identify features or characteristics that lower risk of potential thermal events created by internal short circuits.

  13. 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. PMID:23768638

  14. 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

  15. AFBI assay - Aptamer Fluorescence Binding and Internalization assay for cultured adherent cells.

    PubMed

    Thiel, William H; Giangrande, Paloma H

    2016-07-01

    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

  16. 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

  17. 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

  18. Caveolae internalization repairs wounded cells and muscle fibers

    PubMed Central

    Corrotte, Matthias; Almeida, Patricia E; Tam, Christina; Castro-Gomes, Thiago; Fernandes, Maria Cecilia; Millis, Bryan A; Cortez, Mauro; Miller, Heather; Song, Wenxia; Maugel, Timothy K; Andrews, Norma W

    2013-01-01

    Rapid repair of plasma membrane wounds is critical for cellular survival. Muscle fibers are particularly susceptible to injury, and defective sarcolemma resealing causes muscular dystrophy. Caveolae accumulate in dystrophic muscle fibers and caveolin and cavin mutations cause muscle pathology, but the underlying mechanism is unknown. Here we show that muscle fibers and other cell types repair membrane wounds by a mechanism involving Ca2+-triggered exocytosis of lysosomes, release of acid sphingomyelinase, and rapid lesion removal by caveolar endocytosis. Wounding or exposure to sphingomyelinase triggered endocytosis and intracellular accumulation of caveolar vesicles, which gradually merged into larger compartments. The pore-forming toxin SLO was directly visualized entering cells within caveolar vesicles, and depletion of caveolin inhibited plasma membrane resealing. Our findings directly link lesion removal by caveolar endocytosis to the maintenance of plasma membrane and muscle fiber integrity, providing a mechanistic explanation for the muscle pathology associated with mutations in caveolae proteins. DOI: http://dx.doi.org/10.7554/eLife.00926.001 PMID:24052812

  19. 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

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

    SciTech Connect

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

    1981-11-01

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

  1. 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

  2. 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

  3. Cytoprotective effects of graphene oxide for mammalian cells against internalization of exogenous materials

    NASA Astrophysics Data System (ADS)

    Na, Hee-Kyung; Kim, Mi-Hee; Lee, Jieon; Kim, Young-Kwan; Jang, Hongje; Lee, Kyung Eun; Park, Hyerim; Do Heo, Won; Jeon, Hyesung; Choi, Insung S.; Lee, Younghoon; Min, Dal-Hee

    2013-01-01

    To date, graphene oxide (GO), an oxidized version of graphene, has been utilized in many research areas including bioapplications such as drug delivery and bioanalysis. Unlike other spherical or polygonal nanomaterials, GO exhibits a sheet-like structure, which in itself suggests interesting applications based on its shape. Here we show that GO can protect cells from internalization of toxic hydrophobic molecules, nanoparticles, and nucleic acids such as siRNA and plasmid DNA by interacting with cell surface lipid bilayers without noticeably reducing cell viability. Furthermore, the cytoprotective effect of GO against the internalization of extracellular materials enabled spatial control over gene transfection through region-selective gene delivery only into GO-untreated cells, and not into the GO-treated cells.To date, graphene oxide (GO), an oxidized version of graphene, has been utilized in many research areas including bioapplications such as drug delivery and bioanalysis. Unlike other spherical or polygonal nanomaterials, GO exhibits a sheet-like structure, which in itself suggests interesting applications based on its shape. Here we show that GO can protect cells from internalization of toxic hydrophobic molecules, nanoparticles, and nucleic acids such as siRNA and plasmid DNA by interacting with cell surface lipid bilayers without noticeably reducing cell viability. Furthermore, the cytoprotective effect of GO against the internalization of extracellular materials enabled spatial control over gene transfection through region-selective gene delivery only into GO-untreated cells, and not into the GO-treated cells. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr33800a

  4. 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

  5. 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. PMID:26748311

  6. 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.

  7. 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.

  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.

    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.

  9. 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

  10. 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

  11. 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

  12. 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.

  13. 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

  14. Numerical analysis of molten carbonate fuel cell stack performance: diagnosis of internal conditions using cell voltage profiles

    NASA Astrophysics Data System (ADS)

    Yoshiba, F.; Abe, T.; Watanabe, T.

    A numerical model to diagnose the internal conditions of a molten carbonate fuel cell (MCFC) has been developed to calculate both the temperature and performance of stacks. The performance of the stack is evaluated by applying a `formula for MCFC performance' which has been derived from tests on single small cells with the same active components as the stack. Concerning the separator temperature and the cell performance, calculated results are compared with experimental data acquired during the operation of a 100-kW class stack. Good agreement is obtained. The applied numerical electric circuit model is modified to analyse the voltage distribution within each individual cell. The purpose of the model is to identify the cause of unexpected voltage differences within each cell during operation of a 100-kW class stack. Two causes are identified, namely, increase in the partial internal resistance (IR) and insufficient supply of fuel gas to the cell. The calculated cell voltage distribution and the observed voltage difference for a given cell exhibit similar behaviour.

  15. 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}.

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. On the mechanism of cell internalization of chrysotile fibers: An immunocytochemical and ultrastructural study

    SciTech Connect

    Malorni, W.; Iosi, F.; Falchi, M.; Donelli, G. )

    1990-08-01

    Human breast carcinoma cells (CG5) and human laryngeal carcinoma cells (HEp-2) were exposed to 10 and 50 {mu}ml (about 5 {mu}m) chrysotile asbestos fibers. Morphological and ultrastructural changes were evaluated by means of immunocytochemistry and by scanning and transmission electron microscopy. The authors attention was focused on the mechanisms of cell internalization and on transport of chrysotile fibers. The fibers appeared to penetrate the cell cytoplasm and to be translocated in proximity of the nucleus. Small chrysotile fibers could also be found inside the nucleus of interphase cells. Involvement of the main cytoskeletal components, i.e., microfilaments, intermediate filaments, and microtubules, in the cytotoxicity of chrysotile fibers was also evaluated. Their findings suggest that after fiber penetration, a rearrangement of the cytoskeletal apparatus occurs. It has also been observed that small fibers remain associated with the cytoskeletal framework, which can thus play a role in asbestos intracytoplasmic translocation in epithelial cells. Furthermore, after the cell has completely recovered its morphology, fiber internalization ultimately seems to lead to the formation of giant multinucleated cells. These data could be indicative of an interaction occurring between asbestos fibers and the normal mitotic process.

  1. 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

  2. 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. PMID:25212471

  3. Envelope Glycoprotein Internalization Protects Human and Simian Immunodeficiency Virus-Infected Cells from Antibody-Dependent Cell-Mediated Cytotoxicity

    PubMed Central

    von Bredow, Benjamin; Arias, Juan F.; Heyer, Lisa N.; Gardner, Matthew R.; Farzan, Michael; Rakasz, Eva G.

    2015-01-01

    ABSTRACT The cytoplasmic tails of human and simian immunodeficiency virus (HIV and SIV, respectively) envelope glycoproteins contain a highly conserved, membrane-proximal endocytosis motif that prevents the accumulation of Env on the surface of infected cells prior to virus assembly. Using an assay designed to measure the killing of virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC), we show that substitutions in this motif increase the susceptibility of HIV-1- and SIV-infected cells to ADCC in a manner that directly correlates with elevated Env levels on the surface of virus-infected cells. In the case of HIV-1, this effect is additive with a deletion in vpu recently shown to enhance the susceptibility of HIV-1-infected cells to ADCC as a result of tetherin-mediated retention of budding virions on the cell surface. These results reveal a previously unappreciated role for the membrane-proximal endocytosis motif of gp41 in protecting HIV-1- and SIV-infected cells from antibody responses by regulating the amount of Env present on the cell surface. IMPORTANCE This study reveals an unappreciated role for the membrane-proximal endocytosis motif of gp41 in protecting HIV-1- and SIV-infected cells from elimination by Env-specific antibodies. Thus, strategies designed to interfere with this mechanism of Env internalization may improve the efficacy of antibody-based vaccines and antiretroviral therapies designed to enhance the immunological control of HIV-1 replication in chronically infected individuals. PMID:26269175

  4. Characterization of Ehrlichia risticii binding, internalization, and proliferation in host cells by flow cytometry.

    PubMed Central

    Messick, J B; Rikihisa, Y

    1993-01-01

    The binding, internalization, and proliferation of Ehrlichia risticii in P388D1 cells and equine polymorphonuclear (PMN) leukocytes were studied by immunofluorescent staining and flow cytometric analysis. The binding of ehrlichiae to P388D1 cells at 4 degrees C was dose dependent, and the antigens of bound organisms were susceptible to pronase treatment. Additionally, the binding of ehrlichiae to P388D1 cells was diminished when either P388D1 cells or ehrlichiae were treated with 1% paraformaldehyde for 30 min or 0.25% trypsin for 15 min. These results indicate that the ehrlichial ligand and host cell receptor are likely surface proteins. Following incubation at 37 degrees C, bound E. risticii and/or its antigens were removed with pronase and indirect immunofluorescent staining in the presence of saponin was used to examine intracellular ehrlichiae. Our results indicate that E. risticii was internalized into P388D1 cells within 3 h and proliferated by 48 h of incubation. The microfilament-disrupting agent cytochalasin D and the transglutaminase inhibitor monodansylcadaverine were used to differentiate between phagocytosis (sensitive to cytochalasin) and receptor-mediated endocytosis (sensitive to monodansylcadaverine) of E. risticii by P388D1 cells. In concentrations that produced distinctive morphological changes and inhibited phagocytosis of polystyrene latex beads, cytochalasin D did not suppress the infectivity of E. risticii. Binding, internalization, or proliferation of E. risticii was not affected by cytochalasin D. However, monodansylcadaverine inhibited infection of E. risticii in a dose-dependent manner. The agent did not affect the attachment of ehrlichiae to host cells, but it did suppress internalization and proliferation. These results suggest that E. risticii is internalized by receptor-mediated endocytosis and that productive infection by E. risticii does not depend on phagocytosis by the P388D1 cells. Although E. risticii did not bind to the

  5. Cellular Internalization of Quantum Dots Noncovalently Conjugated with Arginine-Rich Cell-Penetrating Peptides

    PubMed Central

    Liu, Betty R.; Li, Jheng-Fong; Lu, Shu-Wan; Lee, Han-Jung; Huang, Yue-Wern; Shannon, Katie B.; Aronstam, Robert S.

    2010-01-01

    Protein transduction domains comprised of basic amino acid-rich peptides, can efficiently deliver covalently fused macromolecules into cells. Quantum dots (QDs) are luminescent semiconductor nanocrystals that are finding increasing application in biological imaging. Previous studies showed that protein transduction domains mediate the internalization of covalently attached QDs. In this study, we demonstrate that arginine-rich intracellular delivery peptides (cell-penetrating peptides; CPPs), analogs of naturally-occuring protein transduction domains, deliver noncovalently associated QDs into living cells; CPPs dramatically increase the rate and efficiency of cellular uptake of QD probes. The optimal molecular ratio between arginine-rich CPPs and QD cargoes for cellular internalization is approximately 60:1. Upon entry into cells, the QDs are concentrated in the perinuclear region. There is no cytotoxicity following transport of QDs present at concentrations up to 200 nM. The mechanism for arginine-rich CPP/QD complexes to traverse cell membrane appears to involve a combination of internalization pathways. These results provide insight into the mechanism of arginine-rich CPP delivery of noncovalently attached cargoes, and may provide a powerful tool for imaging in vivo. PMID:21137758

  6. 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.

  7. PTH-induced internalization of a type IIa Na/Pi cotransporter in OK-cells.

    PubMed

    Jankowski, M; Biber, J; Murer, H

    1999-10-01

    Regulatory phenomena in brush border membrane sodium/phosphate (Na/Pi) cotransport are directly related to the type IIa Na/Pi-cotransporter and can be analyzed in opossum kidney cells (OK-cells). Parathyroid hormone (PTH) leads to a decreased expression of the type IIa Na/Pi-cotransporter protein at the apical cell surface. To provide evidence for PTH-induced membrane retrieval of the cotransporter protein we labeled OK-cell surface membrane protein NH2-groups with N-hydroxysuccinimide bound via a disulfide bond to biotin (NHS-SS-biotin) prior to or after treatment with PTH. Biotinylated transporters can be detected by streptavidin precipitation and Western blotting using type IIa Na/Pi-cotransporter specific antibodies. To detect only internalized biotinylated transporters biotin located at the cell surface was removed ("stripped") by disulfide bond splitting reagents under reducing conditions. Neither biotinylation per se, nor "stripping" interfered with PTH-induced inhibition of Na/Pi-cotransport activity. The internalization of the transporter was highly increased in response to PTH treatment. The data document that the first step in PTH regulation is internalization of the type IIa Na/Pi-cotransporter protein from the apical membrane. PMID:10555567

  8. SERS and integrative imaging upon internalization of quantum dots into human oral epithelial cells.

    PubMed

    Cepeda-Pérez, Elisa; López-Luke, Tzarara; Plascencia-Villa, Germán; Perez-Mayen, Leonardo; Ceja-Fdez, Andrea; Ponce, Arturo; Vivero-Escoto, Juan; de la Rosa, Elder

    2016-07-01

    CdTe quantum dots (QDs) are widely used in bio-applications due to their size and highly efficient optical properties. However internalization mechanisms thereof for the variety of freshly extracted, not cultivated human cells and their specific molecular interactions remains an open topic for discussion. In this study, we assess the internalization mechanism of CdTe quantum dots (3.3 nm) capped with thioglycolic acid using non cultivated oral epithelial cells obtained from healthy donors. Naked gold nanoparticles (40 nm) were successfully used as nanosensors for surface-enhanced Raman spectroscopy to efficiently identify characteristic Raman peaks, providing new evidence indicating that the first interactions of these QDs with epithelial cells occurred preferentially with aromatic rings and amine groups of amino acid residues and glycans from trans-membrane proteins and cytoskeleton. Using an integrative combination of advanced imaging techniques, including ultra-high resolution SEM, high resolution STEM coupled with EDX spectroscopy together with the results obtained by Raman spectroscopy, it was determined that thioglycolic acid capped CdTe QDs are efficiently internalized into freshly extracted oral epithelial cells only by facilitated diffusion, distributed into cytoplasm and even within the cell nucleus in three minutes. PMID:27120043

  9. Pressure and flow distribution in internal gas manifolds of a fuel-cell stack

    NASA Astrophysics Data System (ADS)

    Koh, Joon-Ho; Seo, Hai-Kyung; Lee, Choong Gon; Yoo, Young-Sung; Lim, Hee Chun

    Gas-flow dynamics in internal gas manifolds of a fuel-cell stack are analyzed to investigate overall pressure variation and flow distribution. Different gas-flow patterns are considered in this analysis. Gas-flow through gas channels of each cell is modeled by means of Darcy's law where permeability should be determined on an experimental basis. Gas-flow in manifolds is modeled from the macroscopic mechanical energy balance with pressure-loss by wall friction and geometrical effects. A systematic algorithm to solve the proposed flow model is suggested to calculate pressure and flow distribution in fuel-cell stacks. Calculation is done for a 100-cell molten carbonate fuel-cell stack with internal manifolds. The results show that the pressure-loss by wall friction is negligible compared with the pressure recovery in inlet manifolds or loss in outlet manifolds due to mass dividing or combining flow at manifold-cell junctions. A more significant effect on manifold pressure possibly arises from the geometrical manifold structure which depends on the manifold size and shape. The geometrical effect is approximated from pressure-loss coefficients of several types of fittings and valves. The overall pressure and flow distribution is significantly affected by the value of the geometrical pressure-loss coefficient. It is also found that the flow in manifolds is mostly turbulent in the 100-cell stack and this way result in an uneven flow distribution when the stack manifold is incorrectly, designed.

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

    NASA Astrophysics Data System (ADS)

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

    2005-01-01

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

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. 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.

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

    PubMed

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

    2016-08-01

    In the chicken embryo, GH gene expression occurs in the neural retina and retinal GH promotes cell survival and induces axonal growth of retinal ganglion cells. Neuroretinal GH is therefore of functional importance before the appearance of somatotrophs and the onset of pituitary GH secretion to the peripheral plasma (at ED15-17). Endocrine actions of pituitary GH in the development and function of the chicken embryo eye are, however, unknown. This possibility has therefore been investigated in ED15 embryos and using the quail neuroretinal derived cell line (QNR/D). During this research, we studied for the first time, the coexistence of exogenous (endocrine) and local GH (autocrine/paracrine) in retinal ganglion cells (RGCs). In ovo systemic injections of Cy3-labeled GH demonstrated that GH in the embryo bloodstream was translocated into the neural retina and internalized into RGC's. Pituitary GH may therefore be functionally involved in retinal development during late embryogenesis. Cy3-labelled GH was similarly internalized into QNR/D cells after its addition into incubation media. The uptake of exogenous GH was by a receptor-mediated mechanism and maximal after 30-60min. The exogenous (endocrine) GH induced STAT5 phosphorylation and increased growth associated protein 43 (GAP43) and SNAP-25 immunoreactivity. Ex ovo intravitreal injections of Cy3-GH in ED12 embryos resulted in GH internalization and STAT5 activation. Interestingly, the CY3-labeled GH accumulated in perinuclear regions of the QNR/D cells, but was not found in the cytoplasm of neurite outgrowths, in which endogenous retinal GH is located. This suggests that exogenous (endocrine) and local (autocrine/paracrine) GH are both involved in retinal function in late embryogenesis but they co-exist in separate intracellular compartments within retinal ganglion cells. PMID:27036926

  18. Plasmid-encoded toxin of enteroaggregative Escherichia coli is internalized by epithelial cells.

    PubMed

    Navarro-García, F; Canizalez-Roman, A; Luna, J; Sears, C; Nataro, J P

    2001-02-01

    We have previously described a 104-kDa protein termed Pet (for plasmid-encoded toxin) secreted by some strains of enteroaggregative Escherichia coli (EAEC). Through an unknown mechanism, this toxin (i) raises transepithelial short-circuit current (Isc) and decreases the electrical resistance of rat jejunum mounted in the Ussing chamber, (ii) causes cytoskeletal alterations in HEp-2 cells and HT29/C1 cells, and (iii) is required for histopathologic effects of EAEC on human intestinal mucosa. Pet is a member of the autotransporter class of secreted proteins and together with Tsh, EspP, EspC, ShMu, and SepA proteins comprises the SPATE subfamily. Here, we show that Pet is internalized by HEp-2 cells and that internalization appears to be required for the induction of cytopathic effects. Evidence supporting Pet internalization includes the facts that (i) the effects of Pet on epithelial cells were inhibited by brefeldin A, which interferes with various steps of intracellular vesicular transport; (ii) immunoblots using anti-Pet antibodies detected Pet in the cytoplasmic fraction of intoxicated HEp-2 cells; (iii) Pet was detected inside HEp-2 cells by confocal microscopy; and (iv) a mutant in the passenger domain cleavage site, which prevents Pet release from the bacterial outer membrane, did not produce cytopathic effects on epithelial cells, whereas the release of mutant Pet from the outer membrane with trypsin yielded active toxin. We have also shown that the Pet serine protease motif is required to produce cytopathic effects but not for Pet secretion. Our results suggest an intracellular mode of action for the Pet protease and are consistent with we our recent report suggesting an intracellular mode of action for Pet. PMID:11160002

  19. 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

  20. 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

  1. Cell-surface nucleolin is involved in lipopolysaccharide internalization and signalling in alveolar macrophages.

    PubMed

    Wang, Yi; Mao, Mei; Xu, Jian-cheng

    2011-07-01

    C23 (nucleolin) shuttling between the nucleus, cytoplasm and cell surface has been implicated in controlling regulatory processes and may play a role in pathogen infection and autoimmune diseases. It has been reported that cell surface-expressed C23 on THP-1 monocytes is involved in the inflammatory response induced by LPS (lipopolysaccharide). This study investigates whether C23 is a membrane receptor for LPS during LPS-induced AMs (alveolar macrophages) activation. First, using immunofluorescence and microscopy, we detected the expression of C23 on the surface of AMs. Second, using LPS affinity columns, we demonstrated that C23 directly binds to LPS. Third, we found that LPS colocalized with C23 on both the cell surface and in the cytoplasm. Finally, knockdown of C23 expression on the cell surface using siRNA (small interfering RNA) led to significant reductions in the internalization of LPS, in LPS-induced NF-κB (nuclear factor κB)-DNA binding and in the protein expression of TNF (tumour necrosis factor)-α and IL-6 (interleukin-6). These findings provide evidence that cell-surface C23 on AMs may serve as a receptor for LPS and are essential for internalization and transport of LPS. Furthermore, C23 participates in the regulation of LPS-induced inflammation of AMs, which indicates that cell-surface C23 is a new and promising therapeutic target for the treatment of bacterial infections. PMID:21309751

  2. 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

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

    PubMed

    Sayyid, Faiz; Kalvala, Sara

    2016-07-01

    Spatial effects such as cell shape have very often been considered negligible in models of cellular pathways, and many existing simulation infrastructures do not take such effects into consideration. Recent experimental results are reversing this judgement by showing that very small spatial variations can make a big difference in the fate of a cell. This is particularly the case when considering eukaryotic cells, which have a complex physical structure and many subtle control mechanisms, but bacteria are also interesting for the huge variation in shape both between species and in different phases of their lifecycle. In this work we perform simulations that measure the effect of three common bacterial shapes on the behaviour of model cellular pathways. To perform these experiments we develop ReDi-Cell, a highly scalable GPGPU cell simulation infrastructure for the modelling of cellular pathways in spatially detailed environments. ReDi-Cell is validated against known-good simulations, prior to its use in new work. We then use ReDi-Cell to conduct novel experiments that demonstrate the effect that three common bacterial shapes (Cocci, Bacilli and Spirilli) have on the behaviour of model cellular pathways. Pathway wavefront shape, pathway concentration gradients, and chemical species distribution are measured in the three different shapes. We also quantify the impact of internal cellular clutter on the same pathways. Through this work we show that variations in the shape or configuration of these common cell shapes alter model cell behaviour. PMID:27262415

  4. 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.

  5. 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

  6. [Change in phospholipid content in platelets, immunocompetent cells and myometrial tissue in patients with internal endometriosis].

    PubMed

    Damirov, M M; Kulakov, V I; Sliusra', N N; Bakuleva, L P; Kargapolov, A V

    1994-01-01

    A method of flow horizontal chromatography has been developed permitting investigation of blood and tissue cellular phospholipids ruling out lipid peroxidation effects on cellular membranes. Phospholipid levels of blood and myometrial tissue cells were measured by this method in 67 patients with histologically verified internal endometriosis. Phospholipid and phosphatidyl inosite levels in platelets and immunocompetent cells of these patients reliably differed from those in healthy women. Phosphatidyl inosite levels of heterotopic endometrial tissue was increased by 1.4 times vs. the norm on an average. In parallel with this, a reliable change of phosphatidyl cholines and inosites levels in endometriosis foci were detected as against their levels in intact tissue. The authors suggest a method for the diagnosis of internal endometriosis by phosphatidyl inosite levels in blood lymphocytes. PMID:8209956

  7. 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.

  8. 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

  9. 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

  10. 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