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Sample records for kupffer cell blockade

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

    SciTech Connect

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

    1990-05-01

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

  2. Kupffer Cell Metabolism and Function

    PubMed Central

    Nguyen-Lefebvre, Anh Thu; Horuzsko, Anatolij

    2015-01-01

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

  3. Kupffer Cells in the Liver

    PubMed Central

    Dixon, Laura J.; Barnes, Mark; Tang, Hui; Pritchard, Michele T.; Nagy, Laura E.

    2016-01-01

    Kupffer cells are a critical component of the mononuclear phagocytic system and are central to both the hepatic and systemic response to pathogens. Kupffer cells are reemerging as critical mediators of both liver injury and repair. Kupffer cells exhibit a tremendous plasticity; depending on the local metabolic and immune environment, then can express a range of polarized phenotypes, from the proinflammatory M1 phenotype to the alternative/M2 phenotype. Multiple M2 phenotypes can be distinguished, each involved in the resolution of inflammation and wound healing. Here, we have provided an update on recent research that has contributed to the developing delineation of the contribution of Kupffer cells to different types of liver injury, with an emphasis on alcoholic and nonalcoholic liver diseases. These recent advances in our understanding of Kupffer cell function and regulation will likely provide new insights into the potential for therapeutic manipulation of Kupffer cells to promote the resolution of inflammation and enhance wound healing in liver disease. PMID:23720329

  4. 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.

  5. Characterisation of Kupffer cells in some Amphibia

    PubMed Central

    CORSARO, CONCETTA; SCALIA, MARINA; LEOTTA, NICOLA; MONDIO, FILIPPO; SICHEL, GIOVANNI

    2000-01-01

    A study on the Kupffer cells (KCs) of Amphibia was undertaken in order to compare these cells with those of endothermic animals. Liver tissue and isolated and cultured KCs were studied by light microscopy and by transmission and scanning electron microscopy. We have shown that amphibian KCs can be divided into 2 principal types: ‘small’ and ‘large’. Both cell types possess the distinctive KC morphology. They show nonspecific esterase activity, weak endogenous peroxidase activity in the nuclear envelope and in the rough endoplasmic reticulum, and the ability to engulf naturally present cell debris or experimentally administered zymosan or latex particles. The principal difference between the small and the large cells consists in the substantial quantity of inclusion bodies that exist only in the latter. We conclude that amphibian KCs, apart from their ability to build melanosomes and synthesise melanins, are very similar to mammalian KCs. PMID:10739021

  6. Characterization of Kupffer cells in livers of developing mice

    PubMed Central

    2011-01-01

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

  7. Perfluorochemical emulsions decrease Kupffer cell phagocytosis

    SciTech Connect

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

    1991-07-01

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

  8. 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. Copyright © 2013 Wiley Periodicals, Inc.

  9. 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

  10. [Trend of molecular biological studies on the liver sinusoidal cells--Kupffer cells].

    PubMed

    Tsutsui, H; Morisawa, S

    1993-02-01

    The Kupffer cells are sessile macrophages residing in the hepatic sinusoids. The Kupffer cells are well known as the first line of defence against pathogenic agents derived from not-self substances, in some cases even from self proteins. Indeed, Kupffer cells play an important roles in such a defence system: they act as an antigen presenting cells, and also produce various kinds of cytokines and chemical mediators such as prostaglandins, leukotrienes and platelet activating factor. In this article, we introduce these functions of Kupffer cells and discuss the possible pathway of the signal transduction.

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

    PubMed Central

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

    2016-01-01

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

  12. In vivo assessment of phagocytic properties of Kupffer cells

    SciTech Connect

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

    1981-05-01

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

  13. In vivo assessment of phagocytic properties of Kupffer cells

    SciTech Connect

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

    1981-05-01

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

  14. 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.

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

    PubMed

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

    2002-09-01

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

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

    PubMed

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

    2015-10-01

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

  17. 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.

  18. How Inflammation Impinges on NAFLD: A Role for Kupffer Cells.

    PubMed

    Duarte, Nádia; Coelho, Inês C; Patarrão, Rita S; Almeida, Joana I; Penha-Gonçalves, Carlos; Macedo, M Paula

    2015-01-01

    Nonalcoholic fatty liver disease (NAFLD) is rapidly becoming the most prevalent cause of liver disease worldwide and afflicts adults and children as currently associated with obesity and insulin resistance. Even though lately some advances have been made to elucidate the mechanism and causes of the disease much remains unknown about NAFLD. The aim of this paper is to discuss the present knowledge regarding the pathogenesis of the disease aiming at the initial steps of NAFLD development, when inflammation impinges on fat liver deposition. At this stage, the Kupffer cells attain a prominent role. This knowledge becomes subsequently relevant for the development of future diagnostic, prevention, and therapeutic options for the management of NAFLD.

  19. Activated Kupffer cells inhibit insulin sensitivity in obese mice

    PubMed Central

    Tencerova, Michaela; Aouadi, Myriam; Vangala, Pranitha; Nicoloro, Sarah M.; Yawe, Joseph C.; Cohen, Jessica L.; Shen, Yuefei; Garcia-Menendez, Lorena; Pedersen, David J.; Gallagher-Dorval, Karen; Perugini, Richard A.; Gupta, Olga T.; Czech, Michael P.

    2015-01-01

    Obesity promotes insulin resistance associated with liver inflammation, elevated glucose production, and type 2 diabetes. Although insulin resistance is attenuated in genetic mouse models that suppress systemic inflammation, it is not clear whether local resident macrophages in liver, denoted Kupffer cells (KCs), directly contribute to this syndrome. We addressed this question by selectively silencing the expression of the master regulator of inflammation, NF-κB, in KCs in obese mice. We used glucan-encapsulated small interfering RNA particles (GeRPs) that selectively silence gene expression in macrophages in vivo. Following intravenous injections, GeRPs containing siRNA against p65 of the NF-κB complex caused loss of NF-κB p65 expression in KCs without disrupting NF-κB in hepatocytes or macrophages in other tissues. Silencing of NF-κB expression in KCs in obese mice decreased cytokine secretion and improved insulin sensitivity and glucose tolerance without affecting hepatic lipid accumulation. Importantly, GeRPs had no detectable toxic effect. Thus, KCs are key contributors to hepatic insulin resistance in obesity and a potential therapeutic target for metabolic disease.—Tencerova, M., Aouadi, M., Vangala, P., Nicoloro, S. M., Yawe, J. C., Cohen, J. L., Shen, Y., Garcia-Menendez, L., Pedersen, D. J., Gallagher-Dorval, K., Perugini, R. A., Gupta, O. T., Czech, M. P. Activated Kupffer cells inhibit insulin sensitivity in obese mice. PMID:25805830

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2001-05-04

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

  2. 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.

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

    PubMed

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

    1985-01-01

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

  4. 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.

  5. Activation of Kupffer cells and caspase-3 involved in rat hepatocyte apoptosis induced by endotoxin.

    PubMed

    Hamada, E; Nishida, T; Uchiyama, Y; Nakamura, J; Isahara, K; Kazuo, H; Huang, T P; Momoi, T; Ito, T; Matsuda, H

    1999-05-01

    Sepsis and lipopolysaccharides (LPS) cause mild to severe hepatic dysfunction. In this study, Kupffer cell activation, involvement of TNFalpha and caspases downstream of the TNFalpha receptor were examined in hepatocyte apoptosis induced by LPS. In in vivo experiments, male Sprague-Dawley rats were injected intravenously with LPS, and small amounts of the blood and liver were sampled to evaluate apoptosis. Kupffer cells were inactivated by pretreatment with gadolinium chloride for 2 days. In in vitro experiments, hepatocytes and Kupffer cells were separately isolated from rat livers using collagenase perfusion. LPS induced time-dependent and dose-dependent increases in the number of TUNEL-positive cells, which coincided with the apoptotic features of hepatocytes demonstrated by electron microscopy and DNA ladder. Activation of caspase-3-like proteases was observed with an increase in the number of apoptotic hepatocytes. Immunostaining with activated caspase-3-specific antibody showed that caspase-3 was activated only in the cytoplasm of TUNEL-positive hepatocytes. Inactivation of Kupffer cells by gadolinium chloride was concomitantly accompanied by the prevention of caspase-3 activation, hepatocyte apoptosis and liver injury induced by LPS. The co-culture system of hepatocytes and Kupffer cells, but neither cell culture system, individually, showed LPS-induced hepatocyte apoptosis. Kupffer cell-conditioned medium induced hepatocyte apoptosis, whereas addition of anti-TNFalpha antibody to Kupffer cell-conditioned medium did not. Additions of acetyl-DEVD-CHO, acetyl-YVAD-CHO, and acetyl-IETD-CHO to Kupffer cell-conditioned medium decreased the number of apoptotic hepatocytes. These results suggest that the activation of Kupffer cells, TNFalpha and caspases downstream of TNFR1 were involved in hepatocyte apoptosis induced by LPS.

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

    PubMed

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

    1987-12-01

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

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

    PubMed Central

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

    1987-01-01

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

  8. How Inflammation Impinges on NAFLD: A Role for Kupffer Cells

    PubMed Central

    Duarte, Nádia; Coelho, Inês C.; Patarrão, Rita S.; Almeida, Joana I.; Penha-Gonçalves, Carlos; Macedo, M. Paula

    2015-01-01

    Nonalcoholic fatty liver disease (NAFLD) is rapidly becoming the most prevalent cause of liver disease worldwide and afflicts adults and children as currently associated with obesity and insulin resistance. Even though lately some advances have been made to elucidate the mechanism and causes of the disease much remains unknown about NAFLD. The aim of this paper is to discuss the present knowledge regarding the pathogenesis of the disease aiming at the initial steps of NAFLD development, when inflammation impinges on fat liver deposition. At this stage, the Kupffer cells attain a prominent role. This knowledge becomes subsequently relevant for the development of future diagnostic, prevention, and therapeutic options for the management of NAFLD. PMID:26090470

  9. Chlamydia pneumoniae replicates in Kupffer cells in mouse model of liver infection

    PubMed Central

    Marangoni, Antonella; Donati, Manuela; Cavrini, Francesca; Aldini, Rita; Accardo, Silvia; Sambri, Vittorio; Montagnani, Marco; Cevenini, Roberto

    2006-01-01

    AIM: To develop an animal model of liver infection with Chlamydia pneumoniae (C. pneumoniae) in intraperitoneally infected mice for studying the presence of chlamydiae in Kupffer cells and hepatocytes. METHODS: A total of 80 BALB/c mice were inoculated intraperitoneally with C. pneumoniae and sacrificed at various time points after infection. Chlamydiae were looked for in liver homogenates as well as in Kupffer cells and hepatocytes separated by liver perfusion with collagenase. C. pneumoniae was detected by both isolation in LLC-MK2 cells and fluorescence in situ hybridization (FISH). The releasing of TNFA-α by C. pneumoniae in vitro stimulated Kupffer cells was studied by enzyme-linked immunosorbent assay. RESULTS: C. pneumoniae isolation from liver homogenates reached a plateau on d 7 after infection when 6 of 10 animals were positive, then decreased, and became negative by d 20. C. pneumoniae isolation from separated Kupffer cells reached a plateau on d 7 when 5 of 10 animals were positive, and became negative by d 20. The detection of C. pneumoniae in separated Kupffer cells by FISH, confirmed the results obtained by culture. Isolated hepatocytes were always negative. Stimulation of Kupffer cells by alive C. pneumoniae elicited high TNF-α levels. CONCLUSION: A productive infection by C. pneumoniae may take place in Kupffer cells and C. pneumoniae induces a local pro-inflammatory activity. C. pneumoniae is therefore, able to act as antigenic stimulus when localized in the liver. One could speculate that C. pneumoniae infection, involving cells of the innate immunity such as Kupffer cells, could also trigger pathological immune reactions involving the liver, as observed in human patients with primary biliary cirrhosis. PMID:17072977

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

    PubMed Central

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

    2008-01-01

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

  11. 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.

  12. Kupffer cell heterogeneity: functional properties of bone marrow derived and sessile hepatic macrophages.

    PubMed

    Klein, Ingo; Cornejo, Judith C; Polakos, Noelle K; John, Beena; Wuensch, Sherry A; Topham, David J; Pierce, Robert H; Crispe, Ian Nicholas

    2007-12-01

    Kupffer cells form a large intravascular macrophage bed in the liver sinusoids. The differentiation history and diversity of Kupffer cells is disputed; some studies argue that they are derived from blood monocytes, whereas others support a local origin from intrahepatic precursor cells. In the present study, we used both flow cytometry and immunohistochemistry to distinguish 2 subsets of Kupffer cells that were revealed in the context both of bone marrow transplantation and of orthotopic liver transplantation. One subset was radiosensitive and rapidly replaced from hematogenous precursors, whereas the other was relatively radioresistant and long-lived. Both were phagocytic but only the former population was recruited into inflammatory foci in response to CD8(+) T-cell activation. We propose the name "sessile" for the radioresistant Kupffer cells that do not participate in immunoinflammatory reactions. However, we found no evidence that these sessile Kupffer cells arise from immature intrahepatic precursors. Our conclusions resolve a long-standing controversy and explain how different experimental approaches may reveal one or both of these subsets.

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

    SciTech Connect

    Sim, W.L.W.

    1988-01-01

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

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

    PubMed Central

    Rous, Peyton; Beard, J. W.

    1934-01-01

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

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

    PubMed

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

    1988-01-01

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

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

    PubMed Central

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

    2013-01-01

    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 (GdCl3; 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

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

    PubMed

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

    2007-04-01

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

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

    PubMed Central

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

    1984-01-01

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

  19. 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.

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

    PubMed Central

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

    2011-01-01

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

  1. Propofol reduces liver dysfunction caused by tumor necrosis factor-α production in Kupffer cells.

    PubMed

    Li, Jiazheng; Kandatsu, Nobuhisa; Feng, Guo-Gang; Jiang, Jia-Zhen; Huang, Lei; Kinoshita, Hiroyuki; Okada, Shoshiro; Fujiwara, Yoshihiro

    2016-06-01

    The present study, conducted in rats, investigated whether propofol attenuates lipopolysaccharide (LPS)-triggered liver dysfunction via regulation of tumor necrosis factor (TNF)-α production in activated Kupffer cells. Rats received LPS (500 μg/kg) under Urethane™ sedation (1 g/kg) in combination with propofol (5 mg/kg/h) or Intralipid™ from 1 h before to 6 h after LPS administration. Some rats were treated with 10 mg/kg gadolinium chloride (GdCl3) to induce Kupffer cell depletion. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), TNF-α mRNA and protein expression, caspase-3 activation and apoptosis were evaluated in hepatocytes. Immunofluorescence staining revealed expression of the pan-macrophage marker CD68 as well as TNF-α in Kupffer cells. ALT and AST serum levels increased approximately four-fold in LPS-exposed rats compared with Intralipid™-treated rats at 6 h after LPS administration, whereas propofol and GdCl3 reduced the LPS-induced increases. LPS simultaneously augmented TNF-α expression in Kupffer cells, followed by increased caspase-3 activity and apoptosis in hepatocytes. Immunofluorescence staining and immunoblotting assay showed that TNF-α expression in Kupffer cells was inhibited by propofol and GdCl3, resulting in a reduction of caspase-3 activity and apoptosis in LPS-treated rat hepatocytes. Propofol (5 mg/kg/h) attenuated LPS-triggered liver dysfunction via inhibition of TNF-α production in activated Kupffer cells. These results suggest that propofol is capable of inhibiting inflammation-induced liver dysfunction in vivo.

  2. 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

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

    PubMed Central

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

    2016-01-01

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

  4. Distinct development and functions of resident and recruited liver Kupffer cells/macrophages.

    PubMed

    Ikarashi, Masami; Nakashima, Hiroyuki; Kinoshita, Manabu; Sato, Atsushi; Nakashima, Masahiro; Miyazaki, Hiromi; Nishiyama, Kiyoshi; Yamamoto, Junji; Seki, Shuhji

    2013-12-01

    Although mouse liver F4/80(+) Kupffer cells consist of cytokine-producing CD11b(+) cells and phagocytic CD68(+) cells, an undefined CD11b(-) CD68(-) subset (30%) also exists. We herein demonstrate a more fundamental classification by adding CD32 (FcγRII), which covers most liver F4/80(+) cells and the distinct functions of them. Among the F4/80(+) cells, 50%, 40%, and 30% of cells were CD32(+), CD68(+), and CD11b(+), respectively, and one-half of the CD68(+) cells coexpressed CD32. CD68(+) and CD32(+) cells, but not CD11b(+) cells, expressed a phagocytosis-related CRIg. Gy (6) irradiation depleted liver CD11b(+) cells and those in the spleen, bone marrow, and peripheral blood but not liver CD32/CD68(+) cells. Transfer of bone marrow cells into the irradiated mice reconstituted liver CD11b(+) cells. Conversely, clodronate pretreatment depleted only liver CD32/CD68(+) cells but not liver CD11b(+) cells and peripheral blood or spleen CD11b(+) monocytes/macrophages. Moreover, the CD32(+) cells might be precursors of CD68(+) cells, as a large proportion of CD32(+) cells expressed the c-kit (CD117), and CD34 and CD32(+) cells acquired CD68 immediately after bacteria administration. CD32/CD68(+) cells, but not CD11b(+) cells, expressed resident macrophage-specific MerTK and CD64 (FcγRI). Challenge with Staphylococcus aureus or liver metastatic EL-4 tumor cells indicated that the CD68(+) subset is engaged in systemic bactericidal activity, whereas the CD11b(+) subset is pivotal for liver antitumor immunity. Human liver CD14(+) Kupffer cells could also be classified into three similar subsets. These results suggest that liver CD68(+) Kupffer cells and CD11b(+) Kupffer cells/macrophages are developmentally and functionally distinct subsets.

  5. 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.

  6. 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

  7. 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.

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

    PubMed Central

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

    1997-01-01

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

  9. Chitotriosidase gene expression in Kupffer cells from patients with non‐alcoholic fatty liver disease

    PubMed Central

    Malaguarnera, L; Rosa, M Di; Zambito, A M; dell'Ombra, N; Nicoletti, F; Malaguarnera, M

    2006-01-01

    Background and aims Non‐alcoholic steatohepatitis (NASH) is a clinicopathological condition characterised by a necroinflammatory disorder with fatty infiltration of the hepatocytes. The molecular mechanisms involved in the anomalous behaviour of liver cells have only partially been determined. Human chitotriosidase (Chit) is a chitinolytic enzyme mainly produced by activated macrophages. The aim of this study was to investigate the expression of the chitinase‐like gene in Kupffer cells, to determine how chitotriosidase may be implicated in the progression from uncomplicated steatosis to steatohepatitis with progressive fibrosis. Methods 75 subjects were studied: 40 with NASH, 20 with simple steatosis, and 15 normal controls. Kupffer cells obtained from liver biopsies were used to detect CHIT expression, superoxide anion (O2−), lipid peroxidation, and tumour necrosis factor α (TNFα) and ferritin levels. Results CHIT expression differed markedly in livers from normal controls and in those from patients with simple steatosis or non‐alcoholic steatohepatitis. A significant correlation between mRNA CHIT and O2−, lipid peroxidation, TNFα, and ferritin levels was observed in both NASH and simple steatosis. Conclusions Human Kupffer cells in NASH patients overproduce chitotriosidase. At the highest levels of production, this enzyme may play a role in increasing the risk for a poor outcome in steatohepatitis. PMID:16825325

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

    PubMed

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

    2011-05-06

    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 γFe(2)O(3) 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 × 10(7) 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.

  11. 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

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

    PubMed Central

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

    2017-01-01

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

  13. Ultrastructure of Kupffer cells and hepatocytes in the Dubin-Johnson syndrome: A case report

    PubMed Central

    Sobaniec-Lotowska, Maria Elzbieta; Lebensztejn, Dariusz Marek

    2006-01-01

    Ultrastructure of Kupffer cells and hepatocytes in liver bioptate was evaluated in a 17-year-old boy with Dubin–Johnson syndrome (DJS). The liver tissue obtained by needle biopsy was fixed in glutaraldehyde and paraformaldehyde and routinely processed for electron microscopic analysis. The ultrastructural examinations of liver bioptate revealed the accumulation of membrane-bound, electron-dense lysosomal granules within the cytoplasm of hepatocytes, characteristic of DJS. They were located mainly in the vicinity of the biliary pole, and preferentially in the centrilobular region that corresponded to the pigment deposits seen under light microscope. The presence of the granules was accompanied by dilated elements of the granular endoplasmic reticulum and paracrystalline mitochondrial inclusions as well as dilation of the bile canaliculi. The changes in hepatocytes co-existed with marked stimulation and enhanced phagocytic activity of Kupffer cells. This was manifested in the accumulation of pigment deposits within their cytoplasm that corresponded to those observed in hepatocytes. Hyperactive pericentral Kupffer cells which are involved in the response to pigmentary material originating from disintegrated hepatocytes may play an essential role in the development of DJS. PMID:16521235

  14. Ultrastructure of Kupffer cells and hepatocytes in the Dubin-Johnson syndrome: a case report.

    PubMed

    Sobaniec-Lotowska, Maria Elzbieta; Lebensztejn, Dariusz Marek

    2006-02-14

    Ultrastructure of Kupffer cells and hepatocytes in liver bioptate was evaluated in a 17-year-old boy with Dubin-Johnson syndrome (DJS). The liver tissue obtained by needle biopsy was fixed in glutaraldehyde and paraformaldehyde and routinely processed for electron microscopic analysis. The ultrastructural examinations of liver bioptate revealed the accumulation of membrane-bound, electron-dense lysosomal granules within the cytoplasm of hepatocytes, characteristic of DJS. They were located mainly in the vicinity of the biliary pole, and preferentially in the centrilobular region that corresponded to the pigment deposits seen under light microscope. The presence of the granules was accompanied by dilated elements of the granular endoplasmic reticulum and paracrystalline mitochondrial inclusions as well as dilation of the bile canaliculi. The changes in hepatocytes co-existed with marked stimulation and enhanced phagocytic activity of Kupffer cells. This was manifested in the accumulation of pigment deposits within their cytoplasm that corresponded to those observed in hepatocytes. Hyperactive pericentral Kupffer cells which are involved in the response to pigmentary material originating from disintegrated hepatocytes may play an essential role in the development of DJS.

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

    SciTech Connect

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

    2010-03-15

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

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

    PubMed

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

    2015-06-01

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

  17. In vitro activation of murine Kupffer cells by lymphokines or endotoxins to lyse syngeneic tumor cells.

    PubMed Central

    Xu, Z. L.; Bucana, C. D.; Fidler, I. J.

    1984-01-01

    Murine Kupffer cells (RC) were isolated in sufficient number and purity to allow in vitro investigations of their tumoricidal capabilities. The identity of the adherent cells as KCs was established by morphologic, histochemical, and functional criteria. The yield of KCs varied from young (high) to old (low) mice but was not affected by the mouse strain. KCs activated in vitro by either endotoxins (lipopolysaccharide) or lymphokines were rendered highly cytotoxic against syngeneic melanoma or fibrosarcoma target cells. These studies indicate that KCs may indeed play a role in destruction of tumor cells in vivo and thus be important in host defense against developing hepatic cancer metastases. Images Figure 1 Figure 2 Figure 3 PMID:6391188

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

    SciTech Connect

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

    1990-03-01

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

  19. 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

  20. Cell collectivity regulation within migrating cell cluster during Kupffer's vesicle formation in zebrafish.

    PubMed

    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.

  1. Nuclear receptor atlas of female mouse liver parenchymal, endothelial, and Kupffer cells.

    PubMed

    Li, Zhaosha; Kruijt, J Kar; van der Sluis, Ronald J; Van Berkel, Theo J C; Hoekstra, Menno

    2013-04-01

    The liver consists of different cell types that together synchronize crucial roles in liver homeostasis. Since nuclear receptors constitute an important class of drug targets that are involved in a wide variety of physiological processes, we have composed the hepatic cell type-specific expression profile of nuclear receptors to uncover the pharmacological potential of liver-enriched nuclear receptors. Parenchymal liver cells (hepatocytes) and liver endothelial and Kupffer cells were isolated from virgin female C57BL/6 wild-type mice using collagenase perfusion and counterflow centrifugal elutriation. The hepatic expression pattern of 49 nuclear receptors was generated by real-time quantitative PCR using the NUclear Receptor Signaling Atlas (NURSA) program resources. Thirty-six nuclear receptors were expressed in total liver. FXR-α, EAR2, LXR-α, HNF4-α, and CAR were the most abundantly expressed nuclear receptors in liver parenchymal cells. In contrast, NUR77, COUP-TFII, LXR-α/β, FXR-α, and EAR2 were the most highly expressed nuclear receptors in endothelial and Kupffer cells. Interestingly, members of orphan receptor COUP-TF family showed a distinct expression pattern. EAR2 was highly and exclusively expressed in parenchymal cells, while COUP-TFII was moderately and exclusively expressed in endothelial and Kupffer cells. Of interest, the orphan receptor TR4 showed a similar expression pattern as the established lipid sensor PPAR-γ. In conclusion, our study provides the most complete quantitative assessment of the nuclear receptor distribution in liver reported to date. Our gene expression catalog suggests that orphan nuclear receptors such as COUP-TFII, EAR2, and TR4 may be of significant importance as novel targets for pharmaceutical interventions in liver.

  2. Induction of ischemic tolerance in rat liver via reduced nicotinamide adenine dinucleotide phosphate oxidase in Kupffer cells.

    PubMed

    Tejima, Kazuaki; Arai, Masahiro; Ikeda, Hitoshi; Tomiya, Tomoaki; Yanase, Mikio; Inoue, Yukiko; Nishikawa, Takako; Watanabe, Naoko; Ohtomo, Natsuko; Omata, Masao; Fujiwara, Kenji

    2007-10-14

    To elucidate the mechanisms of hepatocyte preconditioning by H2O2 to better understand the pathophysiology of ischemic preconditioning. The in vitro effect of H2O2 pretreatment was investigated in rat isolated hepatocytes subjected to anoxia/reoxygenation. Cell viability was assessed with propidium iodide fluorometry. In other experiments, rat livers were excised and subjected to warm ischemia/reperfusion in an isolated perfused liver system to determine leakage of liver enzymes. Preconditioning was performed by H2O2 perfusion, or by stopping the perfusion for 10 min followed by 10 min of reperfusion. To inhibit Kupffer cell function or reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, gadolinium chloride was injected prior to liver excision, or diphenyleneiodonium, an inhibitor of NADPH oxidase, was added to the perfusate, respectively. Histological detection of oxygen radical formation in Kupffer cells was performed by perfusion with nitro blue tetrazolium. Anoxia/reoxygenation decreased hepatocyte viability compared to the controls. Pretreatment with H2O2 did not improve such hepatocyte injury. In liver perfusion experiments, however, H2O2 preconditioning reduced warm ischemia/reperfusion injury, which was reversed by inhibition of Kupffer cell function or NADPH oxidase. Histological examination revealed that H2O2 preconditioning induced oxygen radical formation in Kupffer cells. NADPH oxidase inhibition also reversed hepatoprotection by ischemic preconditioning. H2O2 preconditioning protects hepatocytes against warm ischemia/reperfusion injury via NADPH oxidase in Kupffer cells, and not directly. NADPH oxidase also mediates hepatoprotection by ischemic preconditioning.

  3. Kupffer cells are central in the removal of nanoparticles from the organism.

    PubMed

    Sadauskas, Evaldas; Wallin, Håkan; Stoltenberg, Meredin; Vogel, Ulla; Doering, Peter; Larsen, Agnete; Danscher, Gorm

    2007-10-19

    The study aims at revealing the fate of nanoparticles administered intravenously and intraperitoneally to adult female mice, some of which were pregnant. Gold nanoparticles were chosen as a model because these particles have been found to be chemically inert and at the same time are easily traced by autometallography (AMG) at both ultrastructural and light microscopic levels. Gold nanoparticles were injected intravenously (IV) or intraperitoneally (IP) and traced after 1, 4 or 24 hours. For IV injections 2 and 40 nm particles were used; for IP injections 40 nm particles only. The injected nanoparticles were found in macrophages only, and at moderate exposure primarily in the Kupffer cells in the liver. IV injections resulted in a rapid accumulation/clustering of nanoparticles in these liver macrophages, while the uptake in spleen macrophages was moderate. IP injections were followed by a delayed uptake in the liver and included a moderate uptake in macrophages located in mesenteric lymph nodes, spleen and small intestine. Ultrastructurally, the AMG silver enhanced nanocrystals were found in lysosome-like organelles of the Kupffer cells and other macrophages wherever located.Accumulations of gold nanoparticles were not found in any other organs analysed, i.e. kidneys, brain, lungs, adrenals, ovaries, placenta, and fetal liver, and the control animals were all void of AMG staining. Our results suggest that: (1) inert gold nanoparticles do not penetrate cell membranes by non-endocytotic mechanisms, but are rather taken up by endocytosis; (2) gold nanoparticles, independent of size, are taken up primarily by Kupffer cells in the liver and secondarily by macrophages in other places; (3) gold nanoparticles do not seem to penetrate the placenta barrier; (4) the blood-brain barrier seems to protect the central nervous system from gold nanoparticles; (5) 2 nanometer gold particles seem to be removed not only by endocytosis by macrophages, and we hypothesize that part of

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    1987-02-01

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

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

    PubMed

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

    2013-06-28

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

  7. Initiation of remote hepatic injury in the rat: interactions between Kupffer cells, tumor necrosis factor-alpha, and microvascular perfusion.

    PubMed

    Brock, R W; Lawlor, D K; Harris, K A; Potter, R F

    1999-07-01

    Severe trauma may initiate a systemic inflammatory response, which in turn may result in remote organ injury. After limb ischemia/reperfusion (I/R), intravital fluorescence microscopy was applied to the livers of normotensive rats to investigate the initiation of remote injury to the liver. Additionally, we determined whether Kupffer cell activation and tumor necrosis factor-alpha (TNF-alpha) were involved, via perfusion deficits, in such injury. TNF-alpha, measured by immunoassay, peaked at 30 minutes of reperfusion, but returned to baseline within 60 minutes. Limb I/R resulted in significant increases to global hepatocellular injury measured by alanine transaminase (ALT) and lethal hepatocyte injury as seen with intravital fluorescence microscopy. Although the number of perfused sinusoids went unchanged, a significantly augmented perfusion heterogeneity was measured. After 1.5 hours of reperfusion, both TNF-alpha and Kupffer cells were shown to contribute to global hepatocellular injury (e.g., ALT). After 3 hours, TNF-alpha was no longer essential for this injury, suggesting that some other mechanism(s) activated Kupffer cells and initiated hepatocellular injury. Using propidium iodide and fluorescence microscopy, we found that both TNF-alpha and Kupffer cell activation were necessary to drive hepatocytes toward lethal injury. No additional benefits were observed with a combination of TNF-alpha inhibition and Kupffer cell suppression. These results not only implicate both Kupffer cells and TNF-alpha in the initiation of remote hepatic injury, but suggest that sinusoidal perfusion deficits are not essential for the initiation of such injury. Other mechanism(s) are likely involved in the pathogenesis of remote hepatic parenchymal injury.

  8. [Comparison of efficiency and cytotoxicity of different transfection reagents in transfecting RIP140-siRNA into Kupffer cells].

    PubMed

    Li, Ji; Liu, Zuojin

    2015-12-01

    To compare the efficiency and cytotoxicity of different transfection reagents used in transfection of RIP140-siRNA into Kupffer cells to optimize the transfection conditions. Kupffer cells were transfected with RIP140-siRNA labeled with GFP as the reporter gene using lipofectamine 2000, Roche reagent (X-treme GENE siRNA Transfection Reagent) and puro screening lentivirus (1.0×10(8) TU/mL) as the transfection reagents. The transfection effect was observed under a fluorescent inverted microscope, and laser scanning confocal microscopy was used to analyze RIP140 expression in trasnfected Kupffer cells. Flow cytometry was performed to detect cell apoptosis, and CCK-8 test was used to evaluate the cell proliferation inhibition. RT-RCR and Western blotting were performed to detect the expressions of RIP140 mRNA and protein in the trasnfected cells. Puro screening lentivirus yielded the highest cell transfection efficiency, which exceeded 90%, followed by Roche reagent and then by lipofectamine 2000. Flow cytometry and CCK-8 test showed that the cytotoxicity was the mildest with Roche reagent, moderate with lentivirus, and severe with lipofectamine 2000. The cells trasnfected with lentivirus showed a significantly lower RIP140 expression than cells trasnfected with lipofectamine 2000 and Roche reagent (P<0.05). In Kupffer cells, lentivirus-mediated transfection, as compared with the other two trasnfection reagents, can achieve good transfection efficiency with a relativelty low cytotoxicity, and allows for better controllability and stability of the trasnfectiion conditions.

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    1991-09-01

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

  11. Human recombinant apolipoprotein E redirects lipopolysaccharide from Kupffer cells to liver parenchymal cells in rats In vivo.

    PubMed Central

    Rensen, P C; Oosten, M; Bilt, E; Eck, M; Kuiper, J; Berkel, T J

    1997-01-01

    Chylomicrons have been shown to protect mice and rats against a lethal dose of lipopolysaccharide and may serve as a therapeutic means to protect against endotoxemia. However, the requisite of isolation from human lymph hampers pharmaceutical application. Recently, we developed recombinant chylomicrons from commercially available lipids and human recombinant apolipoprotein E. The current study explored the effectiveness of these apoE-enriched emulsions in redirecting LPS from Kupffer cells to liver parenchymal cells. Upon injection into rats, 125I-LPS rapidly and specifically associated with the liver (64.3+/-3.1% of the injected dose) and spleen (4.1+/-0.7%). The uptake of LPS by the spleen was four- to fivefold reduced upon incubation with the apoE-enriched emulsion or free apoE (P < 0.0001), but not with emulsion alone or Lipofundin. Within the liver, 125I-LPS mainly associated with Kupffer cells. The uptake by Kupffer cells was eight- to ninefold reduced by the apoE-enriched emulsion or apoE alone (P < 0.01), and a 19.6-fold increased uptake ratio by liver parenchymal cells over Kupffer cells was observed. The emulsion without apoE had no effect on the in vivo kinetics of LPS. LPS interacted selectively with the apoE moiety of the recombinant chylomicron. Emulsion-associated and free apoE bound approximately two molecules of LPS, possibly by its exposed hydrophilic domain involving arginine residues. We anticipate that the protecting effect of endogenous chylomicrons against LPS-induced endotoxemia may result from the apoE moiety and that human recombinant apoE may serve as a therapeuticum to protect against endotoxemia. PMID:9153287

  12. Pancreatic elastase induces liver injury by activating cytokine production within Kupffer cells via nuclear factor-Kappa B.

    PubMed

    Murr, Michel M; Yang, Jun; Fier, Adam; Kaylor, Pam; Mastorides, Stephen; Norman, James G

    2002-01-01

    Liver injury is a manifestation of the systemic inflammatory response during acute pancreatitis. We have demonstrated that elastase induces macrophage tumor necrosis factor (TNF) production in distant organs, thus mimicking pancreatitis-associated organ injury. The aim of this study was to determine the mechanism by which elastase induces hepatic cytokine production. Rat livers (n = 40) were perfused with elastase +/- gadolinium (Gd) to inhibit Kupffer cells. Liver parenchymal enzymes and TNF were measured in the effluent. In vitro, rat hepatocytes or Kupffer cells were treated with elastase (1 U/ml) +/- Gd (0.5 mg/ml) or pyrrolidine dithiocarbamate (PDTC; 0.5 mg/ml). TNF protein, TNF messenger RNA, and NF-kappa B activation were determined. In vivo, Gd blunted the elastase-induced TNF production and decreased AST, ALT, LDH, and nonviable cells (propidium iodide) (P < or= 0.03 vs. elastase). In vitro, elastase induced TNF production from Kupffer cells (P < 0.001 vs. control) but not from hepatocytes. Gd or PDTC significantly attenuated the elastase-induced TNF production (P < 0.001). Elastase-induced overexpression of TNF messengerRNA and activation of NF-kappa B was attenuated by Gd. Pancreatic elastase induces a pattern of liver injury similar to that seen during acute pancreatitis by activating cytokine production and gene expression within Kupffer cells via NF-kappa B. Gd exhibits a protective effect against elastase-induced liver injury by inhibiting activation of NF-kappa B.

  13. Hexokinase II Binding to Mitochondria Is Necessary for Kupffer Cell Activation and Is Potentiated by Ethanol Exposure*

    PubMed Central

    Shulga, Nataly; Pastorino, John G.

    2014-01-01

    Ethanol exposure promotes the development of steatohepatitis, which can progress to end stage liver disease. Kupffer cells have been documented to play a key role in the genesis and progression of alcoholic liver disease with ethanol exposure enhancing Kupffer cell activation. In the present study, we identified the binding of hexokinase II to the mitochondria as a requirement for LPS-induced activation of Kupffer cells and its potentiation by ethanol. LPS and ethanol exposure induced a reduction in sirtuin-3 activity. In turn, the decline of sirtuin-3 activity led to the activation of cyclophilin-D, which mediated an increased binding of hexokinase II to the mitochondria. Suppression of cyclophilin-D expression or enforced detachment of hexokinase II from the mitochondria abrogated the LPS- and ethanol-induced stimulation of Kupffer cells, preventing NADPH oxidase and inflammasome activation. Moreover, activation of AMP-activated protein kinase restored sirtuin-3 activity, thereby preventing LPS and ethanol from stimulating the binding of hexokinase II to the mitochondria and precluding NADPH oxidase and inflammasome activation. PMID:25096575

  14. 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

  15. High plasma levels of arginine and liver arginase in Kupffer-cell-depleted rats after partial hepatectomy.

    PubMed

    Prins, H A; Meijer, C; Nijveldt, R J; Wiezer, M J; van Leeuwen, P A

    2000-03-01

    The remnant liver after partial hepatectomy releases arginase into the plasma, which is a reliable indicator of hepatocellular damage. Little information is available on how this release affects arginine plasma levels. We hypothesized that Kupffer cells after partial hepatectomy may prevent further hepatocellular damage, contributing to lower arginase release. The aim of the study was to evaluate the role of Kupffer cells in plasma arginase activity and arginine plasma levels after partial hepatectomy. Wag/Rij rats (n=72, 250-275 g) were randomly assigned to receive 1 ml liposome-encapsulated dichloromethylene-diphosphonate in order to eliminate Kupffer cells (DMDP, n=24), 1 ml liposome encapsulated-phosphate buffered saline (PBS, n=24) or 1 ml NaCl 0.9% (NaCl, n=24) intravenously. Forty-eight hours later, all rats had a two-third liver resection. Rats were killed at 0, 24, 48 and 96 h after partial hepatectomy. Arginase plasma activity was higher in the DMDP-treated group compared to NaCl and PBS (both p<0.01, p<0.05, p<0.01 and p<0.05 for 0, 24, 48 and 96 h after partial hepatectomy respectively). Arginine plasma levels increased, but were lower in the DMDP group compared to NaCl and PBS (both p<0.05, 24 h after hepatectomy). The study showed that Kupffer cell depletion results in a higher arginase release from the remnant liver after partial hepatectomy, indicating a hepatocellular protective function of Kupffer cells. Despite this arginase release, arginine plasma levels were increased after partial hepatectomy.

  16. Inhibition of nitric oxide production reverses diabetes-induced Kupffer cell activation and Klebsiella pneumonia liver translocation.

    PubMed

    Lin, Shu-Han; Chung, Pei-Hsuan; Wu, Ying-Ying; Fung, Chang-Phone; Hsu, Ching-Mei; Chen, Lee-Wei

    2017-01-01

    Klebsiella pneumoniae (KP) is the most common pathogen of pyogenic liver abscess in East and Southeast Asia and diabetes mellitus (DM) is a major risk factor. The effect and mechanism of diabetes on KP liver abscess was examined in streptozotocin-induced diabetic mice and Akita mice (C57BL/6J-Ins2Akita). KP translocation to liver and plasma alaine transaminase levels were increased and liver clearance of KP was decreased in DM mice. Diabetic mice exhibited overgrowth of Enterococcus as well as E.coli and decreased lactobacilli/bifidas growth in intestine, increased intestinal iNOS protein and nitrite levels in portal vein, and increased IL-1β and TNF-α expression of Kupffer cells. Fructooligosaccharides (FOS) or dead L. salivarius (dLac) supplementation reversed diabetes-induced enteric dysbiosis, NO levels in portal vein, and KP translocation to liver. L-NAME treatment decreased intestinal iNOS protein expression as well as Kupffer cell activation and increased liver clearance of KP in DM mice. Dead E.coli (2×108 CFU/ml) feeding for one week induced iNOS and TLR4 expression of intestine in germ-free (GF) mice. Dead bacteria feeding induced IL-1β and TNF-α expression of Kupffer cells in GF mice but not in GF TLR4-/- mice. In conclusion, balance of intestinal microflora is important for preventing intestinal iNOS expression, Kupffer cell activation, and KP liver translocation in diabetes. Reversal of diabetes-induced enteric dysbiosis with FOS or dead L. salivarius decreases diabetes-induced intestinal iNOS expression and KP liver translocation. Diabetes induces Kupffer cell activation and KP liver translocation through enteric dysbiosis and nitric oxide production.

  17. Inhibition of nitric oxide production reverses diabetes-induced Kupffer cell activation and Klebsiella pneumonia liver translocation

    PubMed Central

    Wu, Ying-Ying; Fung, Chang-Phone; Hsu, Ching-Mei

    2017-01-01

    Klebsiella pneumoniae (KP) is the most common pathogen of pyogenic liver abscess in East and Southeast Asia and diabetes mellitus (DM) is a major risk factor. The effect and mechanism of diabetes on KP liver abscess was examined in streptozotocin-induced diabetic mice and Akita mice (C57BL/6J-Ins2Akita). KP translocation to liver and plasma alaine transaminase levels were increased and liver clearance of KP was decreased in DM mice. Diabetic mice exhibited overgrowth of Enterococcus as well as E.coli and decreased lactobacilli/bifidas growth in intestine, increased intestinal iNOS protein and nitrite levels in portal vein, and increased IL-1β and TNF-α expression of Kupffer cells. Fructooligosaccharides (FOS) or dead L. salivarius (dLac) supplementation reversed diabetes-induced enteric dysbiosis, NO levels in portal vein, and KP translocation to liver. L-NAME treatment decreased intestinal iNOS protein expression as well as Kupffer cell activation and increased liver clearance of KP in DM mice. Dead E.coli (2×108 CFU/ml) feeding for one week induced iNOS and TLR4 expression of intestine in germ-free (GF) mice. Dead bacteria feeding induced IL-1β and TNF-α expression of Kupffer cells in GF mice but not in GF TLR4-/- mice. In conclusion, balance of intestinal microflora is important for preventing intestinal iNOS expression, Kupffer cell activation, and KP liver translocation in diabetes. Reversal of diabetes-induced enteric dysbiosis with FOS or dead L. salivarius decreases diabetes-induced intestinal iNOS expression and KP liver translocation. Diabetes induces Kupffer cell activation and KP liver translocation through enteric dysbiosis and nitric oxide production. PMID:28493939

  18. 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

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

    PubMed

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

    1995-01-01

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

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

    PubMed Central

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

    1989-01-01

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

  1. 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.

  2. 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

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

    PubMed Central

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

    2007-01-01

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

  4. Contribution of nitric oxide synthase to luminol-dependent chemiluminescence generated by phorbol-ester-activated Kupffer cells.

    PubMed Central

    Wang, J F; Komarov, P; Sies, H; de Groot, H

    1991-01-01

    Phorbol 12-myristate 13-acetate-induced luminol chemiluminescence in rat Kupffer cells was doubled by the addition of L-arginine and significantly (up to 70%) inhibited by NG-nitro-L-arginine and NG-monomethyl-L-arginine, competitive inhibitors of L-arginine-dependent nitric oxide (NO) formation. The release of superoxide anion (O2-) by NADPH oxidase was neither affected by L-arginine nor by the inhibitors. Only very slight luminol chemiluminescence was detectable in lipopolysaccharide-pretreated Kupffer cells, a condition in which significant amounts of NO were formed but no O2-. In a cell-free system, significant luminol chemiluminescence only occurred when both authentic NO and the O2-/H2O2- generating system xanthine/xanthine oxidase were present. The results indicate that luminol chemiluminescence in phorbol-ester-activated Kupffer cells largely depends on L-arginine metabolism by NO synthase, requiring the concurrent formation of NO and O2-/H2O2. PMID:1718262

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

    PubMed Central

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

    1982-01-01

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

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

    PubMed

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

    1982-05-15

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

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

    PubMed Central

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

    2016-01-01

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

  8. 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

  9. Kupffer cells facilitate the acute effects of leptin on hepatic lipid metabolism.

    PubMed

    Metlakunta, Anantha; Huang, Wan; Stefanovic-Racic, Maja; Dedousis, Nikolaos; Sipula, Ian; O'Doherty, Robert M

    2017-01-01

    Leptin has potent effects on lipid metabolism in a number of peripheral tissues. In liver, an acute leptin infusion (~120 min) stimulates hepatic fatty acid oxidation (~30%) and reduces triglycerides (TG, ~40%), effects that are dependent on phosphoinositol-3-kinase (PI3K) activity. In the current study we addressed the hypothesis that leptin actions on liver-resident immune cells are required for these metabolic effects. Myeloid cell-specific deletion of the leptin receptor (ObR) in mice or depletion of liver Kupffer cells (KC) in rats in vivo prevented the acute effects of leptin on liver lipid metabolism, while the metabolic effects of leptin were maintained in mice lacking ObR in hepatocytes. Notably, liver TG were elevated in both lean and obese myeloid cell ObR, but the degree of obesity and insulin resistance induced by a high-fat diet was similar to control mice. In isolated primary hepatocytes (HEP), leptin had no effects on HEP lipid metabolism and only weakly stimulated PI3K. However, the coculture of KC with HEP restored leptin action on HEP fatty acid metabolism and stimulation of HEP PI3K. Notably, leptin stimulated the release from KC of a number of cytokines. However, the exposure of HEP to these cytokines individually [granulocyte macrophage colony-stimulating factor, IL-1α, IL-1β, IL-6, IL-10, and IL-18] or in combination had no effects on HEP lipid metabolism. Together, these data demonstrate a role for liver mononuclear cells in the regulation of liver lipid metabolism by leptin. Copyright © 2017 the American Physiological Society.

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

    PubMed

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

    2001-10-01

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

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

    PubMed

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

    2000-09-01

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

  12. 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.

  13. Production of reactive oxygen species and expression of inducible nitric oxide synthase in rat isolated Kupffer cells stimulated by Leptospira interrogans and Borrelia burgdorferi

    PubMed Central

    Marangoni, Antonella; Accardo, Silvia; Aldini, Rita; Guardigli, Massimo; Cavrini, Francesca; Sambri, Vittorio; Montagnani, Marco; Roda, Aldo; Cevenini, Roberto

    2006-01-01

    AIM: To evaluate the production of reactive oxygen species (ROS) and the expression of inducible nitric oxide synthase (iNOS) in rat isolated Kupffer cells (KCs) stimulated by Leptospira interrogans and Borrelia burgdorferi. METHODS: Rat Kupffer cells were separated by perfusion of the liver with 0.05% collagenase, and purified by Percoll gradients. Purified Kupffer cells were tested in vitro with alive L.interogans and B. burgdorferi preparations. The production of ROS was determined by chemiluminescence, whereas iNOS protein expression was evaluated by Western blot assay using anti-iNOS antibodies. RESULTS: B. burgdorferi and to a less extent L. interrogans induced ROS production with a peak 35 min after infection. The chemiluminescence signal progressively diminished and was undetectable by 180 min of incubation. Leptospirae and borreliae induced an increased iNOS expression in Kupffer cells that peaked at 6 hours and was still evident 22 h after infection. CONCLUSION: Both genera of spirochetes induced ROS and iNOS production in rat Kupffer cells. Since the cause of liver damage both in leptospiral as well as in borrelial infections are still unknown, we suggest that leptospira and borrelia damage of the liver can be initially mediated by oxygen radicals, and is then maintained at least in part by nitric oxide. PMID:16718791

  14. MicroRNA-155 Deficiency in Kupffer Cells Ameliorates Liver Ischemia-Reperfusion Injury in Mice.

    PubMed

    Li, Yakun; Ma, Dongxia; Wang, Zhimin; Yang, Jun

    2017-07-01

    MicroRNA-155 (miR-155) is known to be involved in autoimmune diseases, inflammation, and transplantation. However, its role in a warm hepatic ischemia-reperfusion (IR) model has not been fully elucidated. Partial hepatic IR was performed in wild-type and miR-155-deficient mice treated with or without GdCl3, and then the serum transaminase concentration and histology were analyzed. Kupffer cells (KCs) were isolated from the liver after IR, and immunohistochemistry was used to evaluate activation and polarization. In addition, the mRNA concentrations of various inflammatory cytokines were measured. Macrophages were obtained from the abdominal cavity and challenged with or without lipopolysaccharide to determine the influence of miR-155 deficiency on macrophage polarization in vitro. Furthermore, we used in vitro coculture assays to determine the effect of miR-155 deficiency on hepatocyte apoptosis induced directly by KCs. miR-155 deficiency ameliorated liver IR injury, and inhibition of KCs by GdCl3 abolished this protective effect. miR-155 deficiency decreased CD80, CD86, and major histocompatibility complex class II expression in KCs after IR and tipped the M1/M2 balance toward an anti-inflammatory profile, where proinflammatory cytokine secretion was suppressed and IL-10 was enhanced. In addition, hepatocyte apoptosis was reduced in coculture with miR-155-deficient KCs in vitro. miR-155 deficiency plays an effective role in attenuating liver IR injury likely by regulating the activation and inflammatory response, as well as modifying the polarization of KCs.

  15. 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

  16. Kupffer cell activation by ambient air particulate matter exposure may exacerbate non-alcoholic fatty liver disease.

    PubMed

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

    2009-12-01

    Owing 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 microm (PM(2.5)) is a risk factor for arteriosclerosis and lung disease, but its effect on NAFLD is unknown. PM(2.5) induces pulmonary dysfunction via Toll-like receptor (TLR) activation on alveolar macrophages. TLR 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 PM(2.5) exposure is a significant risk factor for the progression of NAFLD. Thus, following exposure of male C57BL/6 mice fed high fat chow (HFC) to concentrated air particulate matter (CAPs) or filtered air for 6 weeks, progression of NAFLD was evaluated by standardized histological assessment of hepatic inflammation and fibrosis. In mice fed HFC, 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 scores of (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). In conclusion, ambient PM(2.5) exposure may be a significant risk factor for NAFLD progression.

  17. 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

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

    SciTech Connect

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

    2016-01-08

    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. - Highlights: • We constructed Large-Pore Mesoporous Silica Nanospheres (LPMSNs). • LPMSNs adsorbed high quantity of plasmid. • Low cytotoxicity of LPMSNs to Kupffer cells. • LPMSNs delivered plasmid into Kupffer cells.

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  1. Cholera Toxin-Sensitive GTP-Binding Protein-Coupled Activation of Augmenter of Liver Regeneration (ALR) Receptor and Its Function in Rat Kupffer Cells

    PubMed Central

    Gandhi, Chandrashekhar R.; Murase, Noriko; Starzl, Thomas E.

    2010-01-01

    Mitogenic effect of augmenter of liver regeneration (ALR), a protein produced and released by hepatocytes, on hepatocytes in vivo but not in vitro suggests that the effect is mediated by nonparenchymal cells. Since mediators produced by Kupffer cells are implicated in hepatic regeneration, we investigated receptor for ALR and its functions in rat Kupffer cells. Kupffer cells were isolated from rat liver by enzymatic digestion and centrifugal elutriation. Radioligand ([125I] ALR) receptor binding, ALR-induced GTP/G-protein association, and nitric oxide (NO), tumor necrosis factor (TNF)-α, and interleukin-6 (IL-6) synthesis were determined. High-affinity receptor for ALR, belonging to the G-protein family, with Kd of 1.25 ± 0.18 nM and Bmax of 0.26 ± 0.02 fmol/μg DNA was identified. ALR stimulated NO, TNF-α, and IL-6 synthesis via cholera toxin-sensitive G-protein, as well as p38-MAPK activity and nuclear translocation of NFκB. While inhibitor of NFκB (MG132) inhibited ALR-induced NO synthesis, MG132 and p38-MAPK inhibitor (SB203580) abrogated ALR-induced TNF-α and IL-6 synthesis. ALR also prevented the release of mediator(s) from Kupffer cells that cause inhibition of DNA synthesis in hepatocytes. Administration of ALR to 40% partially hepatectomized rats increased expression of TNF-α, IL-6, and inducible nitric oxide synthase (iNOS) and caused augmentation of hepatic regeneration. These results demonstrate specific G-protein coupled binding of ALR and its function in Kupffer cells and suggest that mediators produced by ALR-stimulated Kupffer cells may elicit physiologically important effects on hepatocytes. PMID:19859909

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

    SciTech Connect

    Naito, M.; Takahashi, K. )

    1991-05-01

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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2017-03-16

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

  5. 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

  6. 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.

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

    PubMed

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

    2013-10-01

    Acute liver failure (ALF) is a highly complex syndrome characterized by devastating activation of early activation of Kupffer cells (KCs) has been implicated in the pathogenesis of ALF. However, the factors regulating KC early activation are virtually unexplored. The aim of present study was to determine the role of the intracellular high-mobility group box 1 (HMGB1) in modulating the early activation of KCs during ALF. The intravenous injection of Concanavalin A (Con A) was used to establish a mouse model of ALF. The dynamic pro-inflammatory properties and MHC II expression of KCs were measured by qRT-PCR and flow cytometry. HMGB1 expression in KCs was measured by qRT-PCR and Western blotting. The immunofluorescence was implemented to determine the relocation of HMGB1 in KCs, and the siRNA against HMGB1 was utilized to assess the impact of HMGB1 on KC pro-inflammatory properties. The peak of pro-inflammatory cytokines production and MHC II expression in KCs appeared at the early stage of ALF. The up-regulation of HMGB1 expression and the translocation of HMGB1 in KCs were in parallel with the early activation of KCs. The blockade of intracellular HMGB1 expression caused by siRNA significantly inhibited the production of KC-derived pro-inflammatory cytokines, and led to a down-regulation of MAP kinase activation in KCs. The self-derived HMGB1 is an "early alarmin" of KC activation during Con A-induced ALF. HMGB1 might be a potential target for cell-specific strategy in ALF. Copyright © 2013 Elsevier GmbH. All rights reserved.

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

    PubMed Central

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

    2016-01-01

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

  9. 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.

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

    PubMed

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

    2017-04-01

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

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

    PubMed

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

    2013-08-01

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

  12. 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

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

    PubMed

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

    2013-03-01

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

  14. 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

  15. Glassy droplet inclusions within the cytoplasm of Kupffer cells: A novel ultrastructural feature for the diagnosis of pediatric autoimmune hepatitis.

    PubMed

    Lotowska, Joanna Maria; Sobaniec-Lotowska, Maria Elzbieta; Daniluk, Urszula; Lebensztejn, Dariusz Marek

    2017-08-01

    Since Kupffer cells/macrophages (KCs/MPs) may be involved in the pathogenesis of autoimmune hepatitis (AIH), this pioneer study was undertaken to evaluate KCs/MPs in pediatric AIH in transmission-electron microscope. Ultrastructural analyses were performed using liver biopsies from 14 children with clinicopathologically diagnosed AIH. In all AIH children, ultrastructural findings revealed changes in the cells lining sinusoidal vessels, especially KCs/MPs and endothelial cells. KCs/MPs showed increased phagocytic activity and damaged mitochondria, frequently with accompanying intense fibrosis. In 10/14 AIH patients, the cytoplasm of sinusoidal KCs/MPs contained characteristic glassy droplet inclusions. They were round, sharply circumscribed, and contained homogeneous material and distinct translucent fields. Their ultrastructure was identical with the Russel bodies of plasma cells, which were also found in liver biopsies in the same patients. Ultrastructural identification of characteristic cytoplasmic droplets with glassy appearance in KCs/MPs, never before described in AIH, provides a useful novel morphological feature in the diagnosis of this disease. Copyright © 2017 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

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

    PubMed

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

    1997-02-15

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

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

    PubMed

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

    2013-02-15

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

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed Central

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

    2017-01-01

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

  20. Early changes of graft function, cytokines and superoxide dismutase serum levels after donor liver denervation and Kupffer cell depletion in a rat-to-rat liver transplantation model.

    PubMed

    Zhu, Hong; Marco, Catena; Gianfranco, Ferla

    2009-04-01

    Hepatic reperfusion injury may cause acute inflammatory damage, producing significant organ dysfunction, and is an important problem in liver transplantation. This experiment aimed to study early changes of hepatic function after donor liver denervation and Kupffer cell depletion in rat-to-rat liver transplantation and to evaluate the effect of pre-treatment on liver reperfusion injury. Donor rats were divided into four groups: control group; group G was pre-treated with gadolinium chloride (G), an inhibitor of Kupffer cells; group H with hexamethonium (H), a sympathetic ganglionic blocking agent; and group HG, with combined H and G pre-treatment. Under the same conditions, serum alanine aminotransferase (ALT), arterial ketone body ratio (AKBR), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and superoxide dismutase (SOD) of recipient rats were assessed at 4, 8, 16 and 24 hours after liver transplantation. Histological studies of the grafts were compared. HG pre-treatment significantly decreased ALT, TNF-alpha, and IL-6 levels, increased AKBR and SOD levels, and demonstrated less pathological damage at 8, 16 and 24 hours compared with the control group. Similar trends were also found in the other groups (G and H). However, the differences among them were not significant at 4 post-operative hours. Donor denervation and Kupffer cell depletion had preventive effect on liver reperfusion injury. HG pre-treatment is a feasible and reproducible method to protect grafts from reperfusion injury.

  1. Inflammatory Monocytes Recruited to the Liver within 24 Hours after Virus-Induced Inflammation Resemble Kupffer Cells but Are Functionally Distinct

    PubMed Central

    Movita, Dowty; Biesta, Paula; Kreefft, Kim; Haagmans, Bart; Zuniga, Elina; Herschke, Florence; De Jonghe, Sandra; Janssen, Harry L. A.; Gama, Lucio; Boonstra, Andre

    2015-01-01

    ABSTRACT Due to a scarcity of immunocompetent animal models for viral hepatitis, little is known about the early innate immune responses in the liver. In various hepatotoxic models, both pro- and anti-inflammatory activities of recruited monocytes have been described. In this study, we compared the effect of liver inflammation induced by the Toll-like receptor 4 ligand lipopolysaccharide (LPS) with that of a persistent virus, lymphocytic choriomeningitis virus (LCMV) clone 13, on early innate intrahepatic immune responses in mice. LCMV infection induces a remarkable influx of inflammatory monocytes in the liver within 24 h, accompanied by increased transcript levels of several proinflammatory cytokines and chemokines in whole liver. Importantly, while a single LPS injection results in similar recruitment of inflammatory monocytes to the liver, the functional properties of the infiltrating cells are dramatically different in response to LPS versus LCMV infection. In fact, intrahepatic inflammatory monocytes are skewed toward a secretory phenotype with impaired phagocytosis in LCMV-induced liver inflammation but exhibit increased endocytic capacity after LPS challenge. In contrast, F4/80high-Kupffer cells retain their steady-state endocytic functions upon LCMV infection. Strikingly, the gene expression levels of inflammatory monocytes dramatically change upon LCMV exposure and resemble those of Kupffer cells. Since inflammatory monocytes outnumber Kupffer cells 24 h after LCMV infection, it is highly likely that inflammatory monocytes contribute to the intrahepatic inflammatory response during the early phase of infection. Our findings are instrumental in understanding the early immunological events during virus-induced liver disease and point toward inflammatory monocytes as potential target cells for future treatment options in viral hepatitis. IMPORTANCE Insights into how the immune system deals with hepatitis B virus (HBV) and HCV are scarce due to the lack of

  2. 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.

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

    PubMed Central

    2012-01-01

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

  4. PD-1 blockade expands intratumoral T memory cells

    PubMed Central

    Ribas, Antoni; Shin, Daniel Sanghoon; Zaretsky, Jesse; Frederiksen, Juliet; Cornish, Andrew; Avramis, Earl; Seja, Elizabeth; Kivork, Christine; Siebert, Janet; Kaplan-Lefko, Paula; Wang, Xiaoyan; Chmielowski, Bartosz; Glaspy, John A.; Tumeh, Paul C.; Chodon, Thinle; Pe’er, Dana; Comin-Anduix, Begoña

    2016-01-01

    Tumor responses to PD-1 blockade therapy are mediated by T cells, which we characterized in 102 tumor biopsies obtained from 53 patients treated with pembrolizumab, an antibody to PD-1. Biopsies were dissociated and single cell infiltrates were analyzed by multicolor flow cytometry using two computational approaches to resolve the leukocyte phenotypes at the single cell level. There was a statistically significant increase in the frequency of T cells in patients who responded to therapy. The frequency of intratumoral B cells and monocytic myeloid-derived suppressor cells (moMDSCs) significantly increased in patients’ biopsies taken on treatment. The percentage of cells with a T regulatory phenotype, monocytes, and NK cells did not change while on PD-1 blockade therapy. CD8+ T memory cells were the most prominent phenotype that expanded intratumorally on therapy. However, the frequency of CD4+ T effector memory cells significantly decreased on treatment, whereas CD4+ T effector cells significantly increased in nonresponding tumors on therapy. In peripheral blood, an unusual population of blood cells expressing CD56 were detected in two patients with regressing melanoma. In conclusion, PD-1 blockade increases the frequency of T cells, B cells, and MDSCs in tumors, with the CD8+ T effector memory subset being the major T-cell phenotype expanded in patients with a response to therapy. PMID:26787823

  5. Nobiletin ameliorates ischemia-reperfusion injury by suppressing the function of Kupffer cells after liver transplantation in rats.

    PubMed

    Wu, Yakun; Zhang, Wenfeng; Li, Min; Cao, Ding; Yang, Xiaoli; Gong, Jianping

    2017-03-05

    This study aims to explore the protective effects of nobiletin against hepatic ischemia-reperfusion (IR) injury after liver transplantation. Kupffer cells (KCs) were activated and co-cultured with different concentration of nobiletin for 24h in vitro, inflammatory products and activity of TLR4/NF-κB signaling pathway were detected. Sprague-Dawley rats were selected and underwent orthotopic liver transplantation. Donors were injected intravenously with nobiletin (50mg/kg) or saline solution, once a day for 1 week before the surgery. Recipients were randomly paired and sacrificed at the indicated time points (3, 6, and 24h after the surgery), the graft liver tissues and blood samples were collected for analysis. Hepatic function, inflammatory mediators, apoptosis of hepatocytes, histological changes, KCs and CD4+ T-lymphocyte infiltration were assessed. Results showed nobiletin dose-dependently suppressed the expression of inflammatory mediators and the activity of TLR4/NF-κB signaling pathway in activated KCs. Furthermore, nobiletin alleviated liver damage induced by IR in vivo, significantly decreased the serum levels of alanine aminotransferase, aspartate transaminase, inflammatory cytokines and alleviated the histopathology changes. Moreover, liver in the nobiletin treated group exhibited less KCs and CD4+ lymphocyte infiltration and lower hepatocyte apoptosis after operation. In addition, activity of TLR4/NF-κB signaling pathway in KCs was also suppressed, consistent with the results in vitro. Collectively, Nobiletin can ameliorate IR injury after liver transplantation and may be a promising new strategy to protect against liver IR injury.

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

    PubMed

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

    2017-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  8. 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

  9. 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

  10. The role of Kupffer cells in the morphogenesis of nonalcoholic steatohepatitis - ultrastructural findings. The first report in pediatric patients.

    PubMed

    Lotowska, Joanna Maria; Sobaniec-Lotowska, Maria Elzbieta; Lebensztejn, Dariusz Marek

    2013-03-01

    Until now studies concerning the involvement of hepatic nonparenchymal cells (NPCs), particularly Kupffer cells/macrophages (KCs/MPs), in the pathogenesis of human nonalcoholic steatohepatitis (NASH) have been limited to adult patients; there are no similar reports referring to children. This study aimed to explore, based on ultrastructural analysis, the role of KCs/MPs in the morphogenesis of nonalcoholic steatohepatitis (NASH) in children. Ultrastructural investigations of KCs were conducted on liver bioptates obtained from 10 children, aged 2-14 years, with clinicopathologically diagnosed NASH. Bioptatic material was fixed in solution of paraformaldehyde and glutaraldehyde in cacodylate buffer, routinely processed for transmission-electron microscopic analysis and examined using an Opton EM microscope. The current ultrastructural study revealed within the hepatic sinusoids the presence of numerous enlarged KCs with increased phagocytic activity, which reduced or blocked vascular lumen. Interestingly, the activated KCs not only contained primary and secondary lysosomes, altered mitochondria, and well-developed Golgi apparatus, but also absorbed fragments of erythrocytes. Such macrophages were frequently seen very close to the transformed hepatic stellate cells (T-HSCs) and progenitor/oval cells. Intensive fibrosis was observed in the vicinity of activated KCs/MPs. Bundles of collagen fibers were seen directly adhering to these cells and to other NPCs, especially T-HSCs. KCs are involved in the morphogenesis and development of pediatric NASH. Engulfment of erythrocytes by hepatic macrophages may lead to the accumulation of iron derived from hemoglobin in liver and play a role in triggering the generation of oxidative stress in the disease course.

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

    PubMed

    Marcos, Ricardo; Correia-Gomes, Carla

    2016-12-01

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

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

    PubMed

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

    2014-08-01

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

  13. MicroRNA 181b-3p and its target importin α5 regulate toll-like receptor 4 signaling in Kupffer cells and liver injury in mice in response to ethanol.

    PubMed

    Saikia, Paramananda; Bellos, Damien; McMullen, Megan R; Pollard, Katherine A; de la Motte, Carol; Nagy, Laura E

    2017-08-01

    Increased inflammatory signaling by Kupffer cells contributes to alcoholic liver disease (ALD). Here we investigated the impact of small, specific-sized hyaluronic acid of 35 kD (HA35) on ethanol-induced sensitization of Kupffer cells, as well as ethanol-induced liver injury in mice. Unbiased analysis of microRNA (miRNA) expression in Kupffer cells identified miRNAs regulated by both ethanol and HA35. Toll-like receptor 4 (TLR4)-mediated signaling was assessed in primary cultures of Kupffer cells from ethanol- and pair-fed rats after treatment with HA35. Female C57BL6/J mice were fed ethanol or pair-fed control diets and treated or not with HA35. TLR4 signaling was increased in Kupffer cells by ethanol; this sensitization was normalized by ex vivo treatment with HA35. Next generation sequencing of Kupffer cell miRNA identified miRNA 181b-3p (miR181b-3p) as sensitive to both ethanol and HA35. Importin α5, a protein involved in p65 translocation to the nucleus, was identified as a target of miR181b-3p; importin α5 protein was increased in Kupffer cells from ethanol-fed rats, but decreased by HA35 treatment. Overexpression of miR181b-3p decreased importin α5 expression and normalized lipopolysaccharide-stimulated tumor necrosis factor α expression in Kupffer cells from ethanol-fed rats. In a mouse model of ALD, ethanol feeding decreased miR181b-3p in liver and increased expression of importin α5 in nonparenchymal cells. Treatment with HA35 normalized these changes and also protected mice from ethanol-induced liver and intestinal injury. miR181b-3p is dynamically regulated in Kupffer cells and mouse liver in response to ethanol and treatment with HA35. miR181b-3p modulates expression of importin α5 and sensitivity of TLR4-mediated signaling. This study identifies a miR181b-3p-importin α5 axis in regulating inflammatory signaling pathways in hepatic macrophages. (Hepatology 2017;66:602-615). © 2017 by the American Association for the Study of Liver Diseases.

  14. Defective function of the mononuclear phagocytic system in rats with chronic nephritis. Evidence of a decreased degradation of IgG aggregates by Kupffer cells.

    PubMed Central

    Herrero-Beaumont, G; Egido, J; Sancho, J; González, E; Castañeda, S; Escanero, J F

    1988-01-01

    In a model of chronic nephritis induced by daily injections of ovalbumin (OVA), the fate of stable, well-defined heat-aggregated rat IgG (A-IgG) was examined by comparing it to that observed in normal rats of the same age. Following i.v. injection of traced amounts of 125I-A-IgG 0.5 hr after the administration of OVA (Group I), rats with nephritis showed delayed blood clearance (t1/2 = 4.6 +/- 2.3 min) compared to the control group (t1/2 = 2.4 +/- 0.8 min). At this time, the following data were also obtained from rats with nephritis: (i) sections of liver, examined by immunofluorescence, showed rat IgG in Kupffer cells reflecting the presence of immune complexes (IC), as was demonstrated in blood by the Raji cell assay; and (ii) rosette formation of IgG-sensitized Affigel 701 beads with Kupffer cells demonstrated decreased Fc receptor-ligand binding in comparison to those of control rats (percentage rosettes: 18.3 +/- 7.2 versus 40.9 +/- 7.9; P less than 0.01). To assess the dynamics of the mononuclear phagocytic system (MPS) in the uptake of immune complexes, in another set of experiments the A-IgG was administered 18 hr after the injection of OVA (Group II). Although a delayed blood clearance of A-IgG was also observed (t1/2 = 4.1 +/- 1.2 min) there were neither circulating IgG-immune complexes nor IgG deposits in the liver, and the percentage of rosette-forming Kupffer cells was increased (75.6 +/- 7.4%). To explain the apparently discordant results between the t1/2 and percentages of rosette-forming Kupffer cells in Group II, binding assays (at 4 degrees and 37 degrees), endocytosis, and catabolism of labelled A-IgG for Kupffer cells were studied. The binding studies at 4 degrees showed that the number of receptor sites per cell was similar in rats with nephritis (6.5 +/- 3.3 X 10(4] and control rats (7.0 +/- 3.1 X 10(4]. However, in rats with nephritis, the affinity constant Ka was significantly lower (0.86 +/- 0.4/M X 10(8] than in control rats (37.4 +/- 13

  15. PI3K inhibition protects mice from NAFLD by down-regulating CMKLR1 and NLRP3 in Kupffer cells.

    PubMed

    Zhang, Wenfeng; Liu, Yan; Wu, Mingbing; Zhu, Xiwen; Wang, Tao; He, Kun; Li, Peizhi; Wu, Xiaoling

    2017-09-13

    Inflammation induced by high-fat diet (HFD) is of critical importance in the development of hepatic steatosis. The role of chemerin in the progress of nonalcoholic fatty liver disease (NAFLD) remains controversial. Here, we have evaluated the effects and mechanism of chemerin on insulin sensitivity and inflammation. An inhibitor (wortmannin) and agonist (insulin-like growth factor 1 (IGF-1) of phosphatidyl inositol 3-kinase (PI3K) were applied to Kupffer cells (KCs) after treatment with a concentration gradient of chemerin in vitro. Mice were subjected to both HFD and intra-peritoneal injections of wortmannin and IGF-1 for 12 weeks. Levels of cytokines were evaluated by enzyme-linked immunosorbent assays and the mRNA and protein levels in the KCs were tested by quantitative real-time polymerase chain reaction and western blotting, respectively. Our data suggested that levels of interleukin 1β (IL-1β) and IL-18 in the KCs and mice treated with wortmannin were significantly lower than that of IGF-1. Consistently, the expression of chemokine-like receptor 1 (CMKLR1) and nucleotide oligomerization domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) was significantly lower in the KCs and mice treated with wortmannin than those treated with IGF-1. Consistently, liver function, insulin resistance, and hepatic steatosis were much more severe in mice treated with IGF-1 than those treated with wortmannin. In conclusions, PI3K inhibition attenuates hepatic steatosis and KC-mediated inflammation via down-regulation of CMKLR1 and NLRP3 in HFD mice.

  16. Kupffer cell depletion by CI2MDP-liposomes alters hepatic cytokine expression and delays liver regeneration after partial hepatectomy.

    PubMed

    Meijer, C; Wiezer, M J; Diehl, A M; Schouten, H J; Schouten, H J; Meijer, S; van Rooijen, N; van Lambalgen, A A; Dijkstra, C D; van Leeuwen, P A

    2000-02-01

    Although Kupffer cells (KCs) are capable of producing important growth-stimulating cytokines, their role in liver regeneration following partial hepatectomy (PH) remains poorly understood. In the present study liver regeneration was studied after KC-depletion by intravenous administration of liposome-encapsulated dichloromethylene-diphosphonate (C12MDP), a method known to physically eliminate KCs. Furthermore, splenectomy was performed one week prior to PH to exclude the effect of C12MDP-liposomes on macrophage populations in the spleen. KC-depletion was confirmed in cryostat liver sections stained with the monoclonal antibody ED2, a marker for resident tissue macrophages. Forty-eight hours after PH, the cumulative hepatocyte DNA synthesis, as determined in liver sections by the hepatocyte bromodeoxyuridine labeling index, was significantly decreased in KC-depleted rats when compared to control-rats. The weight of the remnant liver, expressed as a percentage of the initial liver weight, was significantly less at 96 h after PH in KC-depleted rats. KC-depletion abolished the hepatic interleukin-6 (IL-6) and interleukin-10 (IL-10) mRNA synthesis and decreased hepatic expression of tumor necrosis factor-alpha (TNF-alpha), hepatocyte growth factor (HGF) and transforming growth factor-beta1(TGF-beta1) mRNA after PH, as was assessed by reverse-transcriptase polymerase chain reaction (RT-PCR). Moreover, at 4 h after PH the systemic release of IL-6 was significantly decreased in KC-depleted rats. We conclude that KCs are important for hepatocyte regeneration after PH. Delayed liver regeneration in KC-depleted rats can be explained, at least in part, by an imbalanced hepatic cytokine expression, thereby suppressing important growth-stimulating cytokines.

  17. Tattoo Pigments Are Observed in the Kupffer Cells of the Liver Indicating Blood-Borne Distribution of Tattoo Ink.

    PubMed

    Sepehri, Mitra; Sejersen, Tobias; Qvortrup, Klaus; Lerche, Catharina M; Serup, Jørgen

    2017-01-01

    Tattoo pigments are deposited in the skin and known to distribute to regional lymph nodes. Tattoo pigments are small particles and may be hypothesized to reach the blood stream and become distributed to peripheral organs. This has not been studied in the past. The aim of the study was to trace tattoo pigments in internal organs in mice extensively tattooed with 2 different tattoo ink products. Three groups of mice were studied, i.e., 10 tattooed black, 10 tattooed red, and 5 untreated controls. They were tattooed on the entire back with commercial tattoo inks, black and red. Mice were sacrificed after 1 year. Samples were isolated from tattooed skin, lymph nodes, liver, spleen, kidney, and lung. Samples were examined for deposits of tattoo pigments by light microscopy and transmission electron microscopy (TEM). TEM identified intracellular tattoo pigments in the skin and in lymph nodes. TEM in both groups of tattooed mice showed tattoo pigment deposits in the Kupffer cells in the liver, which is a new observation. TEM detected no pigment in other internal organs. Light microscopy showed dense pigment in the skin and in lymph nodes but not in internal organs. The study demonstrated black and red tattoo pigment deposits in the liver; thus, tattoo pigment distributed from the tattooed skin via the blood stream to this important organ of detoxification. The finding adds a new dimension to tattoo pigment distribution in the body, i.e., as observed via the blood in addition to the lymphatic pathway. © 2017 S. Karger AG, Basel.

  18. Differences in the involvement of prostanoids from Kupffer cells in the mediation of anaphylatoxin C5a-, zymosan-, and lipopolysaccharide-dependent hepatic glucose output and flow reduction.

    PubMed

    Pestel, Sabine; Schlaf, Gerald; Götze, Otto; Jungermann, Kurt; Schieferdecker, Henrike L

    2003-12-01

    Various inflammatory stimuli such as anaphylatoxin C5a, zymosan, and lipopolysaccharides (LPSs) have been reported both to enhance glucose output in the perfused rat liver and to induce prostanoid (ie, prostaglandin and thromboxane) release from Kupffer cells, the resident liver macrophages. Because prostanoids can enhance glucose output from hepatocytes, it was the aim of this study to compare the possible roles of prostanoids released after C5a, zymosan, and LPS in the mediation of hepatic glucose output. In perfused livers both C5a and zymosan immediately enhanced glucose output, reduced flow, and induced prostanoid overflow into the hepatic vein, but with different quantities and kinetics. Only the C5a-induced but not the zymosan-induced effects were abrogated by inhibitors of prostanoid signaling as the prostanoid synthesis inhibitor indomethacin and the thromboxane receptor antagonist daltroban. In contrast to C5a and zymosan, LPS had no effect on glucose output, flow rate, or prostanoid overflow. In isolated Kupffer cells, C5a and zymosan induced maximal release of prostaglandins D(2) and E(2) and of thromboxane A(2) within a period of 0 to 2 minutes and 5 to 15 minutes, respectively. In pulse-chase experiments, maximal prostanoid release was already observed after 2 minutes of continuous stimulation with C5a, but only after 10 to 15 minutes of continuous stimulation with zymosan. LPS-dependent prostanoid release was not seen before 1 hour. Thus, even though C5a, zymosan, and LPS induced prostanoid release from Kupffer cells, only C5a quickly regulated hepatic glucose metabolism in a prostanoid-dependent manner (due to the kinetics and quantities of prostanoids released).

  19. Interleukin-1 blockade in refractory giant cell arteritis.

    PubMed

    Ly, Kim-Heang; Stirnemann, Jérôme; Liozon, Eric; Michel, Marc; Fain, Olivier; Fauchais, Anne-Laure

    2014-01-01

    Giant cell arteritis is a primary large-vessel vasculitis characterized by an arterial wall inflammation associated with intimal hyperplasia leading to arterial occlusion. Glucocorticoids remain the mainstay of giant cell arteritis treatment. However, relapses and glucocorticoid-related complications are frequent and therapeutic options for refractory giant cell arteritis are quite limited. Like tumor necrosis factor-α and interleukin-6, interleukin-1β is also highly expressed in inflamed arterial walls of patients with giant cell arteritis and may contribute in the pathogenesis of this disease. We report treatment of three cases of refractory giant cell arteritis successfully treated with anakinra, an interleukin-1 blockade therapy. Anakinra was effective for all patients, yielding improvement in their inflammation biomarkers and/or in their symptoms, as well as a disappearance of arterial inflammation in PET/CT for two of them. Copyright © 2013. Published by Elsevier SAS.

  20. 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

  1. CTLA4 blockade broadens the peripheral T cell receptor repertoire

    PubMed Central

    Robert, Lidia; Tsoi, Jennifer; Wang, Xiaoyan; Emerson, Ryan; Homet, Blanca; Chodon, Thinle; Mok, Stephen; Huang, Rong Rong; Cochran, Alistair J.; Comin-Anduix, Begonya; Koya, Richard C.; Graeber, Thomas G.; Robins, Harlan; Ribas, Antoni

    2014-01-01

    Purpose To evaluate the immunomodulatory effects of CTLA-4 blockade with tremelimumab in peripheral blood mononuclear cells (PBMC). Experimental Design We used next generation sequencing to study the complementarity determining region 3 (CDR3) from the rearranged T cell receptor (TCR) variable beta (V-beta) in PBMC of 21 patients, at baseline and 30–60 days after receiving tremelimumab. Results After receiving tremelimumab there was a median of 30% increase in unique productive sequences of TCR V-beta CDR3 in 19 out of 21 patients, and a median decrease of 30% in only 2 out of 21 patients. These changes were significant for richness (p=0.01) and for Shannon index diversity (p=0.04). In comparison, serially collected PBMC from four healthy donors did not show a significant change in TCR V-beta CDR3 diversity over one year. There was a significant difference in the total unique productive TCR V-beta CDR3 sequences between patients experiencing toxicity with tremelimumab compared to patients without toxicity (p=0.05). No relevant differences were noted between clinical responders and non-responders. Conclusions CTLA4 blockade with tremelimumab diversifies the peripheral T cell pool, representing a pharmacodynamic effect of how this class of antibodies modulates the human immune system. PMID:24583799

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

    PubMed Central

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

    2011-01-01

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

  3. Interleukin 1 beta markedly stimulates nitric oxide formation in the absence of other cytokines or lipopolysaccharide in primary cultured rat hepatocytes but not in Kupffer cells.

    PubMed

    Kitade, H; Sakitani, K; Inoue, K; Masu, Y; Kawada, N; Hiramatsu, Y; Kamiyama, Y; Okumura, T; Ito, S

    1996-04-01

    To investigate whether a single inflammatory cytokine could stimulate nitric oxide formation in the absence of other cytokines or lipopolysaccharide (LPS), NO was measured by the redox chemiluminescence method in primary cultured rat hepatocytes and in rat Kupffer cells. Interleukin (IL) 1 beta, but neither IL-6, tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), nor LPS stimulated NO formation in a dose-dependent manner and induced half-maximal effects at 30 pmol/L. Maximal stimulation was achieved at 12 to 16 hours after the addition of 1I nmol/L of IL-1 beta, and was 50- to 60-fold above basal levels in rat hepatocytes. The combined effect of these cytokines with LPS or IFN-gamma on NO formation was also examined. Neither LPS nor IFN-gamma affected the IL-1 beta-induced NO formation. TNF-alpha, however, stimulated IL-1 beta-induced NO formation, while IL-6 inhibited it, although independently these cytokines had no effect on NO formation. None of the cytokines tested stimulated NO formation in cultured rat Kupffer cells. In hepatocytes, the NO formation induced by IL-l beta was blocked by both the NO synthase (NOS) inhibitor NG-monomethyl-L-arginine (L-NMMA) and by IL-1 receptor antagonist (IL-1ra). Furthermore, IL-1 beta markedly increased NOS activity, and this increase in activity was accompanied by the expression of inducible NOS (iNOS) messenger RNA (mRNA). This study clearly demonstrated that IL-1 beta markedly stimulates NO formation in hepatocytes, in the absence of other cytokines or LPS.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-10-04

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

  6. Blockade of mast cell activation reduces cutaneous scar formation.

    PubMed

    Chen, Lin; Schrementi, Megan E; Ranzer, Matthew J; Wilgus, Traci A; DiPietro, Luisa A

    2014-01-01

    Damage to the skin initiates a cascade of well-orchestrated events that ultimately leads to repair of the wound. The inflammatory response is key to wound healing both through preventing infection and stimulating proliferation and remodeling of the skin. Mast cells within the tissue are one of the first immune cells to respond to trauma, and upon activation they release pro-inflammatory molecules to initiate recruitment of leukocytes and promote a vascular response in the tissue. Additionally, mast cells stimulate collagen synthesis by dermal fibroblasts, suggesting they may also influence scar formation. To examine the contribution of mast cells in tissue repair, we determined the effects the mast cell inhibitor, disodium cromoglycate (DSCG), on several parameters of dermal repair including, inflammation, re-epithelialization, collagen fiber organization, collagen ultrastructure, scar width and wound breaking strength. Mice treated with DSCG had significantly reduced levels of the inflammatory cytokines IL-1α, IL-1β, and CXCL1. Although DSCG treatment reduced the production of inflammatory mediators, the rate of re-epithelialization was not affected. Compared to control, inhibition of mast cell activity caused a significant decrease in scar width along with accelerated collagen re-organization. Despite the reduced scar width, DSCG treatment did not affect the breaking strength of the healed tissue. Tryptase β1 exclusively produced by mast cells was found to increase significantly in the course of wound healing. However, DSCG treatment did not change its level in the wounds. These results indicate that blockade of mast cell activation reduces scar formation and inflammation without further weakening the healed wound.

  7. Blockade of Mast Cell Activation Reduces Cutaneous Scar Formation

    PubMed Central

    Ranzer, Matthew J.; Wilgus, Traci A.; DiPietro, Luisa A.

    2014-01-01

    Damage to the skin initiates a cascade of well-orchestrated events that ultimately leads to repair of the wound. The inflammatory response is key to wound healing both through preventing infection and stimulating proliferation and remodeling of the skin. Mast cells within the tissue are one of the first immune cells to respond to trauma, and upon activation they release pro-inflammatory molecules to initiate recruitment of leukocytes and promote a vascular response in the tissue. Additionally, mast cells stimulate collagen synthesis by dermal fibroblasts, suggesting they may also influence scar formation. To examine the contribution of mast cells in tissue repair, we determined the effects the mast cell inhibitor, disodium cromoglycate (DSCG), on several parameters of dermal repair including, inflammation, re-epithelialization, collagen fiber organization, collagen ultrastructure, scar width and wound breaking strength. Mice treated with DSCG had significantly reduced levels of the inflammatory cytokines IL-1α, IL-1β, and CXCL1. Although DSCG treatment reduced the production of inflammatory mediators, the rate of re-epithelialization was not affected. Compared to control, inhibition of mast cell activity caused a significant decrease in scar width along with accelerated collagen re-organization. Despite the reduced scar width, DSCG treatment did not affect the breaking strength of the healed tissue. Tryptase β1 exclusively produced by mast cells was found to increase significantly in the course of wound healing. However, DSCG treatment did not change its level in the wounds. These results indicate that blockade of mast cell activation reduces scar formation and inflammation without further weakening the healed wound. PMID:24465509

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

    SciTech Connect

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

    2007-12-15

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

  9. Piroxicam and meloxicam ameliorate hepatic oxidative stress and protein carbonylation in Kupffer and sinusoidal endothelial cells promoted by ischemia-reperfusion injury.

    PubMed

    Montalvo-Javé, Eduardo E; Ortega-Salgado, José A; Castell, Andrés; Carrasco-Daza, Daniel; Jay, David; Gleason, Roberto; Muñoz, Eduardo; Montalvo-Arenas, César; Hernández-Muñoz, Rolando; Piña, Enrique

    2011-05-01

    The present study was aimed to assess the effect of protein carbonylation (PC) in hepatic cells and effects of nonsteroidal anti-inflammatory drugs (NSAIDs) on indicators of tissue damage induced by liver ischemia-reperfusion injury (LIRI). Warm ischemia was performed by partial vascular occlusion during 90 min in Wistar rats. In serum, we determined the catalytic activity of Alanine Aminotransferase, Aspartate Aminotransferase, Lacticate Dehydrogenase, and Ornithine Carbamoyltransferase. In liver samples, we studied cellular alterations by means of histologic studies, lipid peroxidation, PC by immunohistochemistry, apoptosis and reactive oxygen species in bile by electron paramagnetic resonance. Based on PC data, sinusoidal endothelial cells (SEC) and Kupffer cells (KC) were the first to exhibit LIRI-associated oxidative damage and prior to parenchymal cells. Administration of piroxicam or meloxicam during the pre-ischemic period produced a highly significant decrease in all studied injury indicators. No significant differences were revealed between the protective action of the two drugs. The data shown here suggest the potential use of NSAIDs such as piroxicam or meloxicam in minimizing ischemic event-caused damage in liver. We also propose that PC may be employed as an adequate tool to assess tissue damage after oxidative stress. © 2011 The Authors. Transplant International © 2011 European Society for Organ Transplantation.

  10. 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

  11. 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.

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

    PubMed

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

    2015-01-01

    Kupffer cells (KCs) are the first line of defense in the liver against pathogens, yet several microbes successfully target the liver, bypass immune surveillance, and effectively develop in this tissue. Our current, albeit poor, understanding of KC-pathogen interactions has been largely achieved through the study of primary cells, requiring isolation from large numbers of animals. To facilitate the study of KC biology, an immortalized rat KC line 1, RKC1, was developed. We performed a comparative global proteomic analysis of RKC1 and primary rat KCs (PRKC) to characterize their respective responses to lipopolysaccharide-mediated immune stimulation. We identified patent differences in the proteomic response profile of RKC1 and PRKC to lipopolysaccharide. We observed that PRKC upregulated more immune function pathways and exhibited marked changes in cellular morphology following stimulation. We consequently analyzed the cytoskeletal signaling pathways of these cells in light of the fact that macrophages are known to induce cytoskeletal changes in response to pathogens. Our findings suggest that KCs respond differently to inflammatory stimulus than do monocyte-derived macrophages, and such data may provide insight into how pathogens, such as the malaria parasite, may have evolved mechanisms of liver entry through KCs without detection. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. 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.

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

    SciTech Connect

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

    1986-03-05

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

  15. Immune Checkpoint Blockade and Hematopoietic Stem Cell Transplant.

    PubMed

    Merryman, Reid W; Armand, Philippe

    2017-02-01

    Allogeneic hematopoietic stem cell transplant (HSCT) relies primarily upon graft-versus-tumor activity for cancer eradication. Relapse remains the principal cause of treatment failure after HSCT, implying frequent immune escape, which in at least some cases, appears to be mediated by increased expression of inhibitory immune checkpoints. In an attempt to restore anti-tumor immunity, checkpoint blockade therapy (CBT) targeting PD-1 and CLTA-4 has been used in conjunction with both allogeneic and autologous HSCT. Clinical experience in this setting is limited to several small clinical trials and case series, but together they suggest that treatment with CBT can effectively amplify anti-tumor immune responses. However, intrinsic to its mechanism is also the risk that CBT in the HSCT setting may also cause significant immune toxicity. Fatal immune-related adverse events and graft-versus-host disease have been observed, but in most cases, immune side effects appear to be reversible with steroids and CBT discontinuation. As clinical investigation continues, improved understanding of immune checkpoint biology will be critical to optimize safe and efficacious treatment strategies.

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

    PubMed Central

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

    2014-01-01

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

  17. 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.

  18. Cholesterol-lowering drugs cause dissolution of cholesterol crystals and disperse Kupffer cell crown-like structures during resolution of NASH

    PubMed Central

    Ioannou, George N.; Van Rooyen, Derrick M.; Savard, Christopher; Haigh, W. Geoffrey; Yeh, Matthew M.; Teoh, Narci C.; Farrell, Geoffrey C.

    2015-01-01

    Cholesterol crystals form within hepatocyte lipid droplets in human and experimental nonalcoholic steatohepatitis (NASH) and are the focus of crown-like structures (CLSs) of activated Kupffer cells (KCs). Obese, diabetic Alms1 mutant (foz/foz) mice were a fed high-fat (23%) diet containing 0.2% cholesterol for 16 weeks and then assigned to four intervention groups for 8 weeks: a) vehicle control, b) ezetimibe (5 mg/kg/day), c) atorvastatin (20 mg/kg/day), or d) ezetimibe and atorvastatin. Livers of vehicle-treated mice developed fibrosing NASH with abundant cholesterol crystallization within lipid droplets calculated to extend over 3.3% (SD, 2.2%) of liver surface area. Hepatocyte lipid droplets with prominent cholesterol crystallization were surrounded by TNFα-positive (activated) KCs forming CLSs (≥3 per high-power field). KCs that formed CLSs stained positive for NLRP3, implicating activation of the NLRP3 inflammasome in response to cholesterol crystals. In contrast, foz/foz mice treated with ezetimibe and atorvastatin showed near-complete resolution of cholesterol crystals [0.01% (SD, 0.02%) of surface area] and CLSs (0 per high-power field), with amelioration of fibrotic NASH. Ezetimibe or atorvastatin alone had intermediate effects on cholesterol crystallization, CLSs, and NASH. These findings are consistent with a causative link between exposure of hepatocytes and KCs to cholesterol crystals and with the development of NASH possibly mediated by NLRP3 activation. PMID:25520429

  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. 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

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

    PubMed Central

    2015-01-01

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

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

    PubMed

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

    1998-11-01

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

  3. Effects of combined alcohol and anti-HIV drugs on cellular stress responses in primary hepatocytes and hepatic stellate and kupffer cells.

    PubMed

    Hu, Jay; Han, Hui; Lau, Mo Yin; Lee, Harrison; MacVeigh-Aloni, Michelle; Ji, Cheng

    2015-01-01

    Certain anti-HIV drugs alone or in combination are often associated with liver damages, which are frequently worsened by alcohol consumption. We previously found an endoplasmic reticulum (ER) stress mechanism for the drug- and alcohol-induced hepatic injuries in animal models and in vitro hepatocytes. However, it is unknown whether anti-HIV drugs and alcohol induce similar cellular stress responses and injuries in liver nonparenchymal cells. Primary mouse hepatocytes (PMH), kupffer cells (KC), and hepatocellular stellate cells (HSC) were freshly isolated from mouse liver and treated with DMSO, stress-inducing pharmaceutical agents, alcohol alone, or in combination with antiviral ritonavir (RIT), lopinavir (LOP), or efavirenz (EFV). Expression of cellular stress markers, protein colocalization, and cell death were analyzed with immunoblotting, immunocytochemistry, and positive double staining with Sytox green and Hoechst blue, respectively. Expression of the ER stress markers of BiP, CHOP, and SERCA and the autophagy marker LC3 was significantly changed in PMH in response to combined alcohol, RIT, and LOP, which was companied by increased cell death compared with control. In contrast, although pharmaceutical agents induced ER stress and cell death, no significant ER stress or cell death was found in KC treated with alcohol, RIT, LOP, and EFV singly or in combination. In HSC, alcohol, RIT, LOP, or EFV induced BiP, but not CHOP, SERCA, or cell death compared with vehicle control. Further in PMH, RIT and LOP or in combination with alcohol-induced dose-dependent inhibition of β-actin. Inhibition of β-actin by RIT and LOP was companied with an inhibited nuclear expression of the antioxidant response regulator Nrf2 and reduced GST downstream of Nrf2. Ascorbic acid treatment reduced the alcohol-, RIT-, and LOP-induced cell death. The data suggest for the first time that sensitivities of hepatocytes and nonparenchymal cells to alcohol and anti-HIV drugs in vitro are

  4. Influence of copper deficiency on binding and uptake of apolipoprotein E-free high density lipoproteins (APO E-free HDL) by isolated rat liver parenchymal and Kupffer cells

    SciTech Connect

    Lei, K.Y.; Hendriks, H.F.J.; Brouwer, A.; Bock, I.; De Ruiter, C.S.J.; Knook, D.L. )

    1989-02-01

    Weanling male Sprague-Dawley rats were assigned to two dietary treatments; copper-deficient (CD, 0.7 ppm) and adequate (CA, 7.0 ppm). After 8 weeks, plasma apo E-free HDL were isolated by a combination of ultracentrifugation, gel filtration, and heparin-Sepharose affinity chromatography. Liver parenchymal (P) and Kupffer (K) cells were obtained by collagenase perfusion and purified by elutriation. Freshly isolated cells were then incubated with {sup 125}I-apo E-free HDL, either from the same treatment group or in a crossover design, in order to establish if treatment differences were associated with cells and/or lipoproteins. Binding studies performed at 4{degree}C with increasing apo E-free HDL concentration indicated an increase in specific binding in P cells from CD rats. In cell association studies at 37{degree}C, P cells from CD rats demonstrated increased in trypsin releasable (surface-bound) and total cell-associated apo E-free HDL. In contrast, K cells from CD rats demonstrated a reduction in apo E-free HDL uptake (internalized). These findings confirm data obtained from cultured P cells and indicated that copper deficiency may exert different effects on lipoprotein metabolism in rat liver parenchymal and Kupffer cells.

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

    PubMed Central

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

    1991-01-01

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

  6. CAR T-cell intrinsic PD-1 checkpoint blockade: A two-in-one approach for solid tumor immunotherapy.

    PubMed

    Chen, Nan; Morello, Aurore; Tano, Zachary; Adusumilli, Prasad S

    2017-01-01

    PD-L1/2 expression in solid tumors inhibits chimeric antigen receptor (CAR) T-cell efficacy. A PD-1 dominant negative receptor expressed in CAR T cells provides cell-intrinsic checkpoint blockade and augments antitumor efficacy. A combinatorial immunotherapeutic strategy of combining CAR T cells with checkpoint blockade is a promising treatment approach for solid tumors.

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

    PubMed

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

    2017-02-01

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

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

    PubMed

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

    1993-02-01

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

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

    PubMed Central

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

    2015-01-01

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

  10. High-Mobility Group Box-1 Induces Proinflammatory Cytokines Production of Kupffer Cells through TLRs-Dependent Signaling Pathway after Burn Injury

    PubMed Central

    Chen, Xu-Lin; Sun, Li; Guo, Feng; Wang, Fei; Liu, Sheng; Liang, Xun; Wang, Ren-Su; Wang, Yong-Jie; Sun, Ye-Xiang

    2012-01-01

    Kupffer cells (KCs) were a significant source of cytokine release during the early stage of severe burns. High mobility group box protein 1 (HMGB1) was recently identified as a new type of proinflammatory cytokine. The ability of HMGB1 to generate inflammatory responses after burn trauma has not been well characterized. KCs were isolated from sham animals and rats with a 30% full-thickness burn, and then were stimulated with increasing concentrations of HMGB1. The levels of Tumor necrosis factor (TNF)-α and interleukin (IL)-1β in culture supernatant were measured by enzyme-linked immunosorbent assay. Northern blot analysis was performed to detect the expressions of TNF-α and IL-1β mRNAs. The activities of p38 MAPK and JNK (by Western blot analysis) as well as NF-κB (by EMSA) in KCs were also examined. As a result, HMGB1 in vitro upregulated expressions of TNF-α and IL-1β of KCs in a dose-dependent manner, and HMGB1 promoted KCs from burn rats to produce significantly more TNF-α and IL-1β proteins than those from sham animals. After harvested from burn rats, KCs were pre-incubated with anti-TLR2 or anti-TLR4 antibody prior to HMGB1 administration. HMGB1 exposure not only significantly increased expressions of TNF-α and IL-1β mRNAs in KCs from burn rats, but also enhanced activities of p38 MAPK, JNK and NF-κB. However, these upregulation events were all reduced by pre-incubation with anti-TLR2 or anti-TLR4 antibody. These results indicate that HMGB1 induces proinflammatory cytokines production of KCs after sever burn injury, and this process might be largely dependent on TLRs-dependent MAPKs/NF-κB signal pathway. PMID:23209806

  11. Inhibition by prostaglandin E(2) of anaphylatoxin C5a- but not zymosan-induced prostanoid release from rat Kupffer cells.

    PubMed

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

    2002-04-01

    The proinflammatory anaphylatoxin C5a induces the release of prostanoids, ie, prostaglandins (PG) and thromboxane (TX), from the resident liver macrophages (Kupffer cells [KC]). Because KC themselves express prostanoid receptors, prostanoids--besides having paracrine functions--might regulate their own release in an autocrine loop. So far, such a possible feedback regulation has not been investigated systematically, probably because of methodological difficulties to measure newly synthesized prostanoids in the presence of added prostanoids. Here, after prelabeling of phospholipids with [(14)C]arachidonate, cellularly formed [(14)C]prostanoids were determined in the presence of added unlabelled prostanoids by thin layer chromatography. In cultured KC, recombinant rat C5a (rrC5a) rapidly increased PGD(2), PGE(2), and TXA(2) release, which was strongly reduced by PGE(2), but neither by PGD(2) nor by the TXA(2) analog U46619. The inhibitory effect of PGE(2) was mimicked by cAMP, indicating that the G(s)-coupled PGE(2) receptors type 2 or 4 were involved. Zymosan also enhanced prostanoid release from KC, but with slightly slower kinetics; this action was neither inhibited by PGE(2) nor by cAMP. Also in perfused rat livers, rrC5a enhanced prostanoid release from KC as shown by prostanoid overflow and thereby indirectly increased glucose output from hepatocytes. Again, PGE(2), but not PGD(2), inhibited rrC5a-elicited prostanoid overflow. This resulted in a complete inhibition of rrC5a-induced, prostanoid-mediated glucose output. Thus, PGE(2) can inhibit specifically the C5a-induced prostanoid release from KC via a feedback mechanism and thereby limit prostanoid-mediated hepatocellular defense reactions, eg, glucose release.

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

    PubMed Central

    2012-01-01

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

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

    PubMed

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

    2017-03-09

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

  14. Regulation of myeloid cells by activated T cells determines the efficacy of PD-1 blockade.

    PubMed

    Eissler, Nina; Mao, Yumeng; Brodin, David; Reuterswärd, Philippa; Andersson Svahn, Helene; Johnsen, John Inge; Kiessling, Rolf; Kogner, Per

    2016-01-01

    Removal of immuno-suppression has been reported to enhance antitumor immunity primed by checkpoint inhibitors. Although PD-1 blockade failed to control tumor growth in a transgenic murine neuroblastoma model, concurrent inhibition of colony stimulating factor 1 receptor (CSF-1R) by BLZ945 reprogrammed suppressive myeloid cells and significantly enhanced therapeutic effects. Microarray analysis of tumor tissues identified a significant increase of T-cell infiltration guided by myeloid cell-derived chemokines CXCL9, 10, and 11. Blocking the responsible chemokine receptor CXCR3 hampered T-cell infiltration and reduced antitumor efficacy of the combination therapy. Multivariate analysis of 59 immune-cell parameters in tumors and spleens detected the correlation between PD-L1-expressing myeloid cells and tumor burden. In vitro, anti-PD-1 antibody Nivolumab in combination with BLZ945 increased the activation of primary human T and NK cells. Importantly, we revealed a previously uncharacterized pathway, in which T cells secreted M-CSF upon PD-1 blockade, leading to enhanced suppressive capacity of monocytes by upregulation of PD-L1 and purinergic enzymes. In multiple datasets of neuroblastoma patients, gene expression of CD73 correlated strongly with myeloid cell markers CD163 and CSF-1R in neuroblastoma tumors, and associated with worse survival in high-risk patients. Altogether, our data reveal the dual role of activated T cells on myeloid cell functions and provide a rationale for the combination therapy of anti-PD-1 antibody with CSF-1R inhibitor.

  15. Sodium permeability and myocardial resistance to cell swelling during metabolic blockade.

    PubMed

    Pine, M B; Kahne, D; Jaski, B; Apstein, C S; Thorp, K; Abelmann, W H

    1980-07-01

    The role of cell membrane permeability to sodium in cell volume regulation during inhibition of the sodium-potassium exchange pump with ouabain and during total metabolic blockade was evaluated in sections of guinea pig renal cortex, ventricle, and atrium incubated in Krebs-Henseleit solution. In all tissues, 2 and 3 h of ouabain and metabolic blockade resulted in similar marked losses of potassium and parallel continuous reductions in resting membrane potentials. Only metabolic blockade of renal cortex increased cell water, chloride, and total monovalent cations (potassium plus sodium) significantly. Compared to ouabain, metabolic blockade markedly increased the rate of cellular washout of 24Na+ from renal cortex (t 1/2 reduced by 47%), which was significantly greater than reductions in t 1/2 from ventricle (16%) and atrium (15%). Thus, inhibition of sodium-potassium exchange pump activity was not sufficient to produce cell swelling unless associated with marked increases in cell membrane permeability to sodium, in which case sodium influx exceeded potassium loss and substantial increases in monovalent cations, chloride, and water occurred.

  16. Regulation of myeloid cells by activated T cells determines the efficacy of PD-1 blockade

    PubMed Central

    Eissler, Nina; Mao, Yumeng; Brodin, David; Andersson Svahn, Helene; Johnsen, John Inge

    2016-01-01

    ABSTRACT Removal of immuno-suppression has been reported to enhance antitumor immunity primed by checkpoint inhibitors. Although PD-1 blockade failed to control tumor growth in a transgenic murine neuroblastoma model, concurrent inhibition of colony stimulating factor 1 receptor (CSF-1R) by BLZ945 reprogrammed suppressive myeloid cells and significantly enhanced therapeutic effects. Microarray analysis of tumor tissues identified a significant increase of T-cell infiltration guided by myeloid cell-derived chemokines CXCL9, 10, and 11. Blocking the responsible chemokine receptor CXCR3 hampered T-cell infiltration and reduced antitumor efficacy of the combination therapy. Multivariate analysis of 59 immune-cell parameters in tumors and spleens detected the correlation between PD-L1-expressing myeloid cells and tumor burden. In vitro, anti-PD-1 antibody Nivolumab in combination with BLZ945 increased the activation of primary human T and NK cells. Importantly, we revealed a previously uncharacterized pathway, in which T cells secreted M-CSF upon PD-1 blockade, leading to enhanced suppressive capacity of monocytes by upregulation of PD-L1 and purinergic enzymes. In multiple datasets of neuroblastoma patients, gene expression of CD73 correlated strongly with myeloid cell markers CD163 and CSF-1R in neuroblastoma tumors, and associated with worse survival in high-risk patients. Altogether, our data reveal the dual role of activated T cells on myeloid cell functions and provide a rationale for the combination therapy of anti-PD-1 antibody with CSF-1R inhibitor. PMID:28123870

  17. IL-7 receptor blockade following T cell depletion promotes long-term allograft survival

    PubMed Central

    Mai, Hoa-Le; Boeffard, Françoise; Longis, Julie; Danger, Richard; Martinet, Bernard; Haspot, Fabienne; Vanhove, Bernard; Brouard, Sophie; Soulillou, Jean-Paul

    2014-01-01

    T cell depletion is commonly used in organ transplantation for immunosuppression; however, a restoration of T cell homeostasis following depletion leads to increased memory T cells, which may promote transplant rejection. The cytokine IL-7 is important for controlling lymphopoiesis under both normal and lymphopenic conditions. Here, we investigated whether blocking IL-7 signaling with a mAb that targets IL-7 receptor α (IL-7Rα) alone or following T cell depletion confers an advantage for allograft survival in murine transplant models. We found that IL-7R blockade alone induced indefinite pancreatic islet allograft survival if anti–IL-7R treatment was started 3 weeks before graft. IL-7R blockade following anti-CD4– and anti-CD8–mediated T cell depletion markedly prolonged skin allograft survival. Furthermore, IL-7 inhibition in combination with T cell depletion synergized with either CTLA-4Ig administration or suboptimal doses of tacrolimus to induce long-term skin graft acceptance in this stringent transplant model. Together, these therapies inhibited T cell reconstitution, decreased memory T cell numbers, increased the relative frequency of Tregs, and abrogated both cellular and humoral alloimmune responses. Our data suggest that IL-7R blockade following T cell depletion has potential as a robust, immunosuppressive therapy in transplantation. PMID:24569454

  18. Blockade of constitutively activated ERK signaling enhances cytotoxicity of microtubule-destabilizing agents in tumor cells.

    PubMed

    Tanimura, Susumu; Uchiyama, Aya; Watanabe, Kazushi; Yasunaga, Masahiro; Inada, Yoshiyuki; Kawabata, Takumi; Iwashita, Ken-Ichi; Noda, Sinji; Ozaki, Kei-Ichi; Kohno, Michiaki

    2009-01-16

    The extracellular signal-regulated kinase (ERK) signaling pathway is constitutively activated in many human tumor cell types. Given the cytoprotective role of this pathway, we examined whether its specific blockade might sensitize human tumor cells to the induction of apoptosis by various anticancer drugs. Although blockade of ERK signaling alone did not induce substantial cell death, it resulted in marked and selective enhancement of the induction of apoptosis by microtubule-destabilizing agents in tumor cells in which the ERK pathway is constitutively activated. The synergistic activation of c-Jun NH(2)-terminal kinase by the combination of an ERK pathway inhibitor and a microtubule-destabilizing agent appeared to be responsible, at least in part, for this effect. These results suggest that administration of the combination of an ERK pathway inhibitor and a microtubule-destabilizing agent is a potential chemotherapeutic strategy for the treatment of tumor cells with constitutive activation of the ERK pathway.

  19. Blockade of the Programmed Death-1 Pathway Restores Sarcoidosis CD4+ T-Cell Proliferative Capacity

    PubMed Central

    Braun, Nicole A.; Celada, Lindsay J.; Herazo-Maya, Jose D.; Abraham, Susamma; Shaginurova, Guzel; Sevin, Carla M.; Grutters, Jan; Culver, Daniel A.; Dworski, Ryszard; Sheller, James; Massion, Pierre P.; Polosukhin, Vasiliy V.; Johnson, Joyce E.; Kaminski, Naftali; Wilkes, David S.; Oswald-Richter, Kyra A.

    2014-01-01

    Rationale: Effective therapeutic interventions for chronic, idiopathic lung diseases remain elusive. Normalized T-cell function is an important contributor to spontaneous resolution of pulmonary sarcoidosis. Up-regulation of inhibitor receptors, such as programmed death-1 (PD-1) and its ligand, PD-L1, are important inhibitors of T-cell function. Objectives: To determine the effects of PD-1 pathway blockade on sarcoidosis CD4+ T-cell proliferative capacity. Methods: Gene expression profiles of sarcoidosis and healthy control peripheral blood mononuclear cells were analyzed at baseline and follow-up. Flow cytometry was used to measure ex vivo expression of PD-1 and PD-L1 on systemic and bronchoalveolar lavage–derived cells of subjects with sarcoidosis and control subjects, as well as the effects of PD-1 pathway blockade on cellular proliferation after T-cell receptor stimulation. Immunohistochemistry analysis for PD-1/PD-L1 expression was conducted on sarcoidosis, malignant, and healthy control lung specimens. Measurements and Main Results: Microarray analysis demonstrates longitudinal increase in PDCD1 gene expression in sarcoidosis peripheral blood mononuclear cells. Immunohistochemistry analysis revealed increased PD-L1 expression within sarcoidosis granulomas and lung malignancy, but this was absent in healthy lungs. Increased numbers of sarcoidosis PD-1+ CD4+ T cells are present systemically, compared with healthy control subjects (P < 0.0001). Lymphocytes with reduced proliferative capacity exhibited increased proliferation with PD-1 pathway blockade. Longitudinal analysis of subjects with sarcoidosis revealed reduced PD-1+ CD4+ T cells with spontaneous clinical resolution but not with disease progression. Conclusions: Analogous to the effects in other chronic lung diseases, these findings demonstrate that the PD-1 pathway is an important contributor to sarcoidosis CD4+ T-cell proliferative capacity and clinical outcome. Blockade of the PD-1 pathway may be a

  20. Challenges and opportunities for checkpoint blockade in T-cell lymphoproliferative disorders.

    PubMed

    Phillips, Tycel; Devata, Sumana; Wilcox, Ryan A

    2016-01-01

    The T-cell lymphoproliferative disorders are a heterogeneous group of non-Hodgkin's lymphomas (NHL) for which current therapeutic strategies are inadequate, as most patients afflicted with these NHL will succumb to disease progression within 2 years of diagnosis. Appreciation of the genetic and immunologic landscape of these aggressive NHL, including PD-L1 (B7-H1, CD274) expression by malignant T cells and within the tumor microenvironment, provides a strong rationale for therapeutic targeting this immune checkpoint. While further studies are needed, the available data suggests that responses with PD-1 checkpoint blockade alone will unlikely approach those achieved in other lymphoproliferative disorders. Herein, we review the unique challenges posed by the T-cell lymphoproliferative disorders and discuss potential strategies to optimize checkpoint blockade in these T-cell derived malignancies.

  1. Blockade of the ERK pathway markedly sensitizes tumor cells to HDAC inhibitor-induced cell death

    SciTech Connect

    Ozaki, Kei-ichi; Minoda, Ai; Kishikawa, Futaba; Kohno, Michiaki . E-mail: kohnom@net.nagasaki-u.ac.jp

    2006-01-27

    Constitutive activation of the extracellular signal-regulated kinase (ERK) pathway is associated with the neoplastic phenotype of a large number of human tumor cells. Although specific blockade of the ERK pathway by treating such tumor cells with potent mitogen-activated protein kinase/ERK kinase (MEK) inhibitors completely suppresses their proliferation, it by itself shows only a modest effect on the induction of apoptotic cell death. However, these MEK inhibitors markedly enhance the efficacy of histone deacetylase (HDAC) inhibitors to induce apoptotic cell death: such an enhanced cell death is observed only in tumor cells in which the ERK pathway is constitutively activated. Co-administration of MEK inhibitor markedly sensitizes tumor cells to HDAC inhibitor-induced generation of reactive oxygen species, which appears to mediate the enhanced cell death induced by the combination of these agents. These results suggest that the combination of MEK inhibitors and HDAC inhibitors provides an efficient chemotherapeutic strategy for the treatment of tumor cells in which the ERK pathway is constitutively activated.

  2. Opiate receptor blockade on human granulosa cells inhibits VEGF release.

    PubMed

    Lunger, Fabian; Vehmas, Anni P; Fürnrohr, Barbara G; Sopper, Sieghart; Wildt, Ludwig; Seeber, Beata

    2016-03-01

    The objectives of this study were to determine whether the main opioid receptor (OPRM1) is present on human granulosa cells and if exogenous opiates and their antagonists can influence granulosa cell vascular endothelial growth factor (VEGF) production via OPRM1. Granulosa cells were isolated from women undergoing oocyte retrieval for IVF. Complementary to the primary cells, experiments were conducted using COV434, a well-characterized human granulosa cell line. Identification and localization of opiate receptor subtypes was carried out using Western blot and flow cytometry. The effect of opiate antagonist on granulosa cell VEGF secretion was assessed by enzyme-linked immunosorbent assay. For the first time, the presence of OPRM1 on human granulosa cells is reported. Blocking of opiate signalling using naloxone, a specific OPRM1 antagonist, significantly reduced granulosa cell-derived VEGF levels in both COV434 and granulosa-luteal cells (P < 0.01). The presence of opiate receptors and opiate signalling in granulosa cells suggest a possible role in VEGF production. Targeting this signalling pathway could prove promising as a new clinical option in the prevention and treatment of ovarian hyperstimulation syndrome.

  3. TLR-2 is upregulated and mobilized to the hepatocyte plasma membrane in the space of Disse and to the Kupffer cells TLR-4 dependently during acute endotoxemia in mice.

    PubMed

    Ojaniemi, Marja; Liljeroos, Mari; Harju, Kirsi; Sormunen, Raija; Vuolteenaho, Reetta; Hallman, Mikko

    2006-02-15

    Membrane components of bacteria and fungi are recognized by Toll-like receptors (TLRs) which, when activated, induce several inflammatory mediators important in the host defense. As the liver is constantly exposed to ingested bacteria, hepatic TLRs must be broadly responsive and highly regulated to prevent uncontrolled inflammatory activation. Although several hepatic cells express microbe recognition molecules and inflammatory mediators in vitro, the regulation and cellular localization of these proteins in vivo remain uncertain. The expression and regulation of TLR-2 and TLR-4, and the cytokine expression patterns were evaluated in mouse tissues using a model of acute inflammation induced by intraperitoneal injection of LPS. Five hours after intraperitoneal LPS, induction of TLR-4 was evident in lung, while the low hepatic TLR-4 expression was non-inducible. TLR-2 mRNA and protein were induced both in lung and liver TLR-4 dependently. However, IL-1alpha also contributed to this induction, and IL-1R1 antibody attenuated the TLR-2 increase. Immunoelectron microscopy showed accumulation of cytoplasmic TLR-2 to vesicles near the hepatocyte plasma membrane in the space of Disse, to the sinusoidal endothelium and to the Kupffer cells. NF-kappaB activation was clear in Kupffer cells and hepatocytes during LPS-challenge, suggesting these cells to be the main source of in vivo cytokine production. Hepatic cytokine response to LPS was remarkably rapid in liver, whereas lung responded less acutely. Secondary inflammatory challenge attenuated the TLR-2 response. The innate immune system of the liver is rapidly and transiently activated during endotoxemia by mechanism involving both TLR-4 and TLR-2.

  4. PD1 blockade enhances cytotoxicity of in vitro expanded natural killer cells towards myeloma cells

    PubMed Central

    Guo, Yanan; Feng, Xiaoli; Jiang, Yang; Shi, Xiaoyun; Xing, Xiangling; Liu, Xiaoli; Li, Nailin; Fadeel, Bengt; Zheng, Chengyun

    2016-01-01

    Aiming for an adoptive natural killer (NK) cell therapy, we have developed a novel protocol to expand NK cells from peripheral blood. With this protocol using anti-human CD16 antibody and interleukin (IL)-2, NK (CD3−CD56+) cells could be expanded about 4000-fold with over 70% purity during a 21-day culture. The expanded NK (exNK) cells were shown to be highly cytotoxic to multiple myeloma (MM) cells (RPMI8226) at low NK-target cell ratios. Furthermore, NK cells expanded in the presence of a blocking antibody (exNK+PD1-blockage) against programmed cell death protein-1 (PD1), a key counteracting molecule for NK and T cell activity, demonstrated more potent cytolytic activity against the RPMI8226 than the exNK cells without PD1 blocking. In parallel, the exNK cells showed significantly higher expression of NK activation receptors NKG2D, NKp44 and NKp30. In a murine model of MM, transfusion of exNK cells, exNK+PD1-blockage, and exNK plus intratumor injection of anti-PD-L2 antibody (exNK+PD-L2 blockage) all significantly suppressed tumor growth and prolonged survival of the myeloma mice. Importantly, exNK+PD1-blockage presented more efficient therapeutic effects. Our results suggest that the NK cell expansion protocol with PD1 blockade presented in this study has considerable potential for the clinical application of allo- and auto-NK cell-based therapies against malignancies. PMID:27356741

  5. Blockade of maitotoxin-induced oncotic cell death reveals zeiosis

    PubMed Central

    Estacion, Mark; Schilling, William P

    2002-01-01

    Background Maitotoxin (MTX) initiates cell death by sequentially activating 1) Ca2+ influx via non-selective cation channels, 2) uptake of vital dyes via formation of large pores, and 3) release of lactate dehydrogenase, an indication of cell lysis. MTX also causes formation of membrane blebs, which dramatically dilate during the cytolysis phase. To determine the role of phospholipase C (PLC) in the cell death cascade, U73122, a specific inhibitor of PLC, and U73343, an inactive analog, were examined on MTX-induced responses in bovine aortic endothelial cells. Results Addition of either U73122 or U73343, prior to MTX, produced a concentration-dependent inhibition of the cell death cascade (IC50 ≈ 1.9 and 0.66 μM, respectively) suggesting that the effect of these agents was independent of PLC. Addition of U73343 shortly after MTX, prevented or attenuated the effects of the toxin, but addition at later times had little or no effect. Time-lapse videomicroscopy showed that U73343 dramatically altered the blebbing profile of MTX-treated cells. Specifically, U73343 blocked bleb dilation and converted the initial blebbing event into "zeiosis", a type of membrane blebbing commonly associated with apoptosis. Cells challenged with MTX and rescued by subsequent addition of U73343, showed enhanced caspase-3 activity 48 hr after the initial insult, consistent with activation of the apoptotic program. Conclusions Within minutes of MTX addition, endothelial cells die by oncosis. Rescue by addition of U73343 shortly after MTX showed that a small percentage of cells are destined to die by oncosis, but that a larger percentage survive; cells that survive the initial insult exhibit zeiosis and may ultimately die by apoptotic mechanisms. PMID:11825342

  6. Grid cell spatial tuning reduced following systemic muscarinic receptor blockade

    PubMed Central

    Newman, Ehren L.; Climer, Jason R.; Hasselmo, Michael E.

    2014-01-01

    Grid cells of the medial entorhinal cortex exhibit a periodic and stable pattern of spatial tuning that may reflect the output of a path integration system. This grid pattern has been hypothesized to serve as a spatial coordinate system for navigation and memory function. The mechanisms underlying the generation of this characteristic tuning pattern remain poorly understood. Systemic administration of the muscarinic antagonist scopolamine flattens the typically positive correlation between running speed and entorhinal theta frequency in rats. The loss of this neural correlate of velocity, an important signal for the calculation of path integration, raises the question of what influence scopolamine has on the grid cell tuning as a read out of the path integration system. To test this, the spatial tuning properties of grid cells were compared before and after systemic administration of scopolamine as rats completed laps on a circle track for food rewards. The results show that the spatial tuning of the grid cells was reduced following scopolamine administration. The tuning of head direction cells, in contrast, was not reduced by scopolamine. This is the first report to demonstrate a link between cholinergic function and grid cell tuning. This work suggests that the loss of tuning in the grid cell network may underlie the navigational disorientation observed in Alzheimer's patients and elderly individuals with reduced cholinergic tone. PMID:24493379

  7. Restoration of HBV-specific CD8+ T cell function by PD-1 blockade in inactive carrier patients is linked to T cell differentiation.

    PubMed

    Bengsch, Bertram; Martin, Bianca; Thimme, Robert

    2014-12-01

    The upregulation of several inhibitory signalling pathways by exhausted HBV-specific CD8+ T cells in chronic infection is thought to contribute to viral persistence. Blockade of inhibitory receptors to reinvigorate exhausted T cell function is a promising novel therapeutic approach. However, little information is available regarding the relative contribution of individual inhibitory pathways to HBV-specific CD8+ T cell failure and the impact of inhibitory receptor blockade on restoration of T cell function in chronic HBV. 98 HLA-A2+ chronically infected patients were analysed ex vivo for HBV-specific CD8+ T cell responses, the expression of multiple inhibitory receptors and T cell differentiation markers. The effects of inhibitory receptor blockade targeting PD-1, 2B4, Tim-3, CTLA-4, and BTLA were assessed in vitro. In our cohort, ex vivo HBV-specific CD8+ T cell responses were identified preferentially in HBeAg patients with low ALT and low viral load (inactive carriers). We observed a clear hierarchy of inhibitory receptor expression dominated by PD-1. The response to inhibitory receptor blockade was heterogeneous. Compared to the blockade of other inhibitory receptors, blockade of the PD-1 pathway resulted in the strongest increase in function. Of note, a positive effect of PD-1 blockade was linked to intermediate T cell differentiation. Despite the expression of multiple inhibitory receptors by HBV-specific CD8+ T cells, expression and response to blockade was dominated by PD-1. However, PD-1 expression did not predict response to blockade. Rather, response to blockade was associated with intermediate T cell differentiation. These findings have important implications for our understanding of inhibitory receptor blockade as a novel therapeutic strategy. Copyright © 2014. Published by Elsevier B.V.

  8. Blockade of Glutamine Synthetase Enhances Inflammatory Response in Microglial Cells

    PubMed Central

    Palmieri, Erika M.; Menga, Alessio; Lebrun, Aurore; Hooper, Douglas C.; Butterfield, D. Allan

    2017-01-01

    Abstract Aims: Microglial cells are brain-resident macrophages engaged in surveillance and maintained in a constant state of relative inactivity. However, their involvement in autoimmune diseases indicates that in pathological conditions microglia gain an inflammatory phenotype. The mechanisms underlying this change in the microglial phenotype are still unclear. Since metabolism is an important modulator of immune cell function, we focused our attention on glutamine synthetase (GS), a modulator of the response to lipopolysaccharide (LPS) activation in other cell types, which is expressed by microglia. Results: GS inhibition enhances release of inflammatory mediators of LPS-activated microglia in vitro, leading to perturbation of the redox balance and decreased viability of cocultured neurons. GS inhibition also decreases insulin-mediated glucose uptake in microglia. In vivo, microglia-specific GS ablation enhances expression of inflammatory markers upon LPS treatment. In the spinal cords from experimental autoimmune encephalomyelitis (EAE), GS expression levels and glutamine/glutamate ratios are reduced. Innovation: Recently, metabolism has been highlighted as mediator of immune cell function through the discovery of mechanisms that (behind these metabolic changes) modulate the inflammatory response. The present study shows for the first time a metabolic mechanism mediating microglial response to a proinflammatory stimulus, pointing to GS activity as a master modulator of immune cell function and thus unraveling a potential therapeutic target. Conclusions: Our study highlights a new role of GS in modulating immune response in microglia, providing insights into the pathogenic mechanisms associated with inflammation and new strategies of therapeutic intervention. Antioxid. Redox Signal. 26, 351–363. PMID:27758118

  9. [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

  10. Structure-activity relationship between carboxylic acids and T cell cycle blockade.

    PubMed

    Gilbert, Kathleen M; DeLoose, Annick; Valentine, Jimmie L; Fifer, E Kim

    2006-04-04

    This study was designed to examine the potential structure-activity relationship between carboxylic acids, histone acetylation and T cell cycle blockade. Toward this goal a series of structural homologues of the short-chain carboxylic acid n-butyrate were studied for their ability to block the IL-2-stimulated proliferation of cloned CD4+ T cells. The carboxylic acids were also tested for their ability to inhibit histone deacetylation. In addition, Western blotting was used to examine the relative capacity of the carboxlic acids to upregulate the cyclin kinase-dependent inhibitor p21cip1 in T cells. As shown earlier n-butyrate effectively inhibited histone deacetylation. The increased acetylation induced by n-butyrate was associated with the upregulation of the cyclin-dependent kinase inhibitor p21cip1 and the cell cycle blockade of CD4+ T cells. Of the other carboxylic acids studied, the short chain acids, C3-C5, without branching were the best inhibitors of histone deacetylase. This inhibition correlated with increased expression of the cell cycle blocker p21cip1, and the associated suppression of CD4+ T cell proliferation. The branched-chain carboxylic acids tested were ineffective in all the assays. These results underline the relationship between the ability of a carboxylic acid to inhibit histone deacetylation, and their ability to block T cell proliferation, and suggests that branching inhibits these effects.

  11. Evolution of Neoantigen Landscape during Immune Checkpoint Blockade in Non-Small Cell Lung Cancer.

    PubMed

    Anagnostou, Valsamo; Smith, Kellie N; Forde, Patrick M; Niknafs, Noushin; Bhattacharya, Rohit; White, James; Zhang, Theresa; Adleff, Vilmos; Phallen, Jillian; Wali, Neha; Hruban, Carolyn; Guthrie, Violeta B; Rodgers, Kristen; Naidoo, Jarushka; Kang, Hyunseok; Sharfman, William; Georgiades, Christos; Verde, Franco; Illei, Peter; Li, Qing Kay; Gabrielson, Edward; Brock, Malcolm V; Zahnow, Cynthia A; Baylin, Stephen B; Scharpf, Robert B; Brahmer, Julie R; Karchin, Rachel; Pardoll, Drew M; Velculescu, Victor E

    2017-03-01

    Immune checkpoint inhibitors have shown significant therapeutic responses against tumors containing increased mutation-associated neoantigen load. We have examined the evolving landscape of tumor neoantigens during the emergence of acquired resistance in patients with non-small cell lung cancer after initial response to immune checkpoint blockade with anti-PD-1 or anti-PD-1/anti-CTLA-4 antibodies. Analyses of matched pretreatment and resistant tumors identified genomic changes resulting in loss of 7 to 18 putative mutation-associated neoantigens in resistant clones. Peptides generated from the eliminated neoantigens elicited clonal T-cell expansion in autologous T-cell cultures, suggesting that they generated functional immune responses. Neoantigen loss occurred through elimination of tumor subclones or through deletion of chromosomal regions containing truncal alterations, and was associated with changes in T-cell receptor clonality. These analyses provide insight into the dynamics of mutational landscapes during immune checkpoint blockade and have implications for the development of immune therapies that target tumor neoantigens.Significance: Acquired resistance to immune checkpoint therapy is being recognized more commonly. This work demonstrates for the first time that acquired resistance to immune checkpoint blockade can arise in association with the evolving landscape of mutations, some of which encode tumor neoantigens recognizable by T cells. These observations imply that widening the breadth of neoantigen reactivity may mitigate the development of acquired resistance. Cancer Discov; 7(3); 264-76. ©2017 AACR.See related commentary by Yang, p. 250This article is highlighted in the In This Issue feature, p. 235.

  12. TNF Blockade Maintains an IL-10+ Phenotype in Human Effector CD4+ and CD8+ T Cells

    PubMed Central

    Roberts, Ceri A.; Durham, Lucy E.; Fleskens, Veerle; Evans, Hayley G.; Taams, Leonie S.

    2017-01-01

    CD4+ and CD8+ effector T cell subpopulations can display regulatory potential characterized by expression of the prototypically anti-inflammatory cytokine IL-10. However, the underlying cellular mechanisms that regulate expression of IL-10 in different T cell subpopulations are not yet fully elucidated. We recently showed that TNF inhibitors (TNFi) promote IL-10 expression in human CD4+ T cells, including IL-17+ CD4+ T cells. Here, we further characterized the regulation of IL-10 expression via blockade of TNF signaling or other cytokine/co-stimulatory pathways, in human T cell subpopulations. Addition of the TNFi drug adalimumab to anti-CD3-stimulated human CD4+ T cell/monocyte cocultures led to increased percentages of IL-10+ cells in pro-inflammatory IL-17+, IFNγ+, TNFα+, GM-CSF+, and IL-4+ CD4+ T cell subpopulations. Conversely, exogenous TNFα strongly decreased IL-10+ cell frequencies. TNF blockade also regulated IL-10 expression in CD4+ T cells upon antigenic stimulation. Using time course experiments in whole peripheral blood mononuclear cell (PBMC) cultures, we show that TNF blockade maintained, rather than increased, IL-10+ cell frequencies in both CD4+ and CD8+ T cells following in vitro stimulation in a dose- and time-dependent manner. Blockade of IL-17, IFNγ, IL-6R, or CD80/CD86-mediated co-stimulation did not significantly regulate IL-10 expression within CD4+ or CD8+ T cell subpopulations. We show that TNF blockade acts directly on effector CD4+ T cells, in the absence of monocytes or CD4+ CD25highCD127low regulatory T cells and independently of IL-27, resulting in higher IL-10+ frequencies after 3 days in culture. IL-10/IL-10R blockade reduced the frequency of IL-10-expressing cells both in the presence and absence of TNF blockade. Addition of recombinant IL-10 alone was insufficient to drive an increase in IL-10+ CD4+ T cell frequencies in 3-day CD4+ T cell/monocyte cocultures, but resulted in increased IL-10 expression at later time points in

  13. Human CAR T cells with cell-intrinsic PD-1 checkpoint blockade resist tumor-mediated inhibition

    PubMed Central

    Cherkassky, Leonid; Morello, Aurore; Villena-Vargas, Jonathan; Feng, Yang; Dimitrov, Dimiter S.; Jones, David R.; Sadelain, Michel; Adusumilli, Prasad S.

    2016-01-01

    Following immune attack, solid tumors upregulate coinhibitory ligands that bind to inhibitory receptors on T cells. This adaptive resistance compromises the efficacy of chimeric antigen receptor (CAR) T cell therapies, which redirect T cells to solid tumors. Here, we investigated whether programmed death-1–mediated (PD-1–mediated) T cell exhaustion affects mesothelin-targeted CAR T cells and explored cell-intrinsic strategies to overcome inhibition of CAR T cells. Using an orthotopic mouse model of pleural mesothelioma, we determined that relatively high doses of both CD28- and 4-1BB–based second-generation CAR T cells achieved tumor eradication. CAR-mediated CD28 and 4-1BB costimulation resulted in similar levels of T cell persistence in animals treated with low T cell doses; however, PD-1 upregulation within the tumor microenvironment inhibited T cell function. At lower doses, 4-1BB CAR T cells retained their cytotoxic and cytokine secretion functions longer than CD28 CAR T cells. The prolonged function of 4-1BB CAR T cells correlated with improved survival. PD-1/PD-1 ligand [PD-L1] pathway interference, through PD-1 antibody checkpoint blockade, cell-intrinsic PD-1 shRNA blockade, or a PD-1 dominant negative receptor, restored the effector function of CD28 CAR T cells. These findings provide mechanistic insights into human CAR T cell exhaustion in solid tumors and suggest that PD-1/PD-L1 blockade may be an effective strategy for improving the potency of CAR T cell therapies. PMID:27454297

  14. Human CAR T cells with cell-intrinsic PD-1 checkpoint blockade resist tumor-mediated inhibition.

    PubMed

    Cherkassky, Leonid; Morello, Aurore; Villena-Vargas, Jonathan; Feng, Yang; Dimitrov, Dimiter S; Jones, David R; Sadelain, Michel; Adusumilli, Prasad S

    2016-08-01

    Following immune attack, solid tumors upregulate coinhibitory ligands that bind to inhibitory receptors on T cells. This adaptive resistance compromises the efficacy of chimeric antigen receptor (CAR) T cell therapies, which redirect T cells to solid tumors. Here, we investigated whether programmed death-1-mediated (PD-1-mediated) T cell exhaustion affects mesothelin-targeted CAR T cells and explored cell-intrinsic strategies to overcome inhibition of CAR T cells. Using an orthotopic mouse model of pleural mesothelioma, we determined that relatively high doses of both CD28- and 4-1BB-based second-generation CAR T cells achieved tumor eradication. CAR-mediated CD28 and 4-1BB costimulation resulted in similar levels of T cell persistence in animals treated with low T cell doses; however, PD-1 upregulation within the tumor microenvironment inhibited T cell function. At lower doses, 4-1BB CAR T cells retained their cytotoxic and cytokine secretion functions longer than CD28 CAR T cells. The prolonged function of 4-1BB CAR T cells correlated with improved survival. PD-1/PD-1 ligand [PD-L1] pathway interference, through PD-1 antibody checkpoint blockade, cell-intrinsic PD-1 shRNA blockade, or a PD-1 dominant negative receptor, restored the effector function of CD28 CAR T cells. These findings provide mechanistic insights into human CAR T cell exhaustion in solid tumors and suggest that PD-1/PD-L1 blockade may be an effective strategy for improving the potency of CAR T cell therapies.

  15. Modulation of Cell Signaling Networks after CTLA4 Blockade in Patients with Metastatic Melanoma

    PubMed Central

    Comin-Anduix, Begoña; Sazegar, Hooman; Chodon, Thinle; Matsunaga, Douglas; Jalil, Jason; von Euw, Erika; Escuin-Ordinas, Helena; Balderas, Robert; Chmielowski, Bartosz; Gomez-Navarro, Jesus; Koya, Richard C.; Ribas, Antoni

    2010-01-01

    Background The effects on cell signalling networks upon blockade of cytotoxic T lymphocyte-associated antigen-4 (CTLA4) using the monoclonal antibody tremelimumab were studied in peripheral blood mononuclear cell (PBMC) samples from patients with metastatic melanoma. Methodology/Principal Findings Intracellular flow cytometry was used to detect phosphorylated (p) signaling molecules downstream of the T cell receptor (TCR) and cytokine receptors. PBMC from tremelimumab-treated patients were characterized by increase in pp38, pSTAT1 and pSTAT3, and decrease in pLck, pERK1/2 and pSTAT5 levels. These changes were noted in CD4 and CD8 T lymphocytes but also in CD14 monocytes. A divergent pattern of phosphorylation of Zap70, LAT, Akt and STAT6 was noted in patients with or without an objective tumor response. Conclusions/Significance The administration of the CTLA4-blocking antibody tremelimumab to patients with metastatic melanoma influences signaling networks downstream of the TCR and cytokine receptors both in T cells and monocytes. The strong modulation of signaling networks in monocytes suggests that this cell subset may be involved in clinical responses to CTLA4 blockade. Clinical Trial Registration clinicaltrials.gov; Registration numbers NCT00090896 and NCT00471887 PMID:20856802

  16. T cell costimulation blockade blunts pressure overload-induced heart failure

    PubMed Central

    Kallikourdis, Marinos; Martini, Elisa; Carullo, Pierluigi; Sardi, Claudia; Roselli, Giuliana; Greco, Carolina M.; Vignali, Debora; Riva, Federica; Ormbostad Berre, Anne Marie; Stølen, Tomas O.; Fumero, Andrea; Faggian, Giuseppe; Di Pasquale, Elisa; Elia, Leonardo; Rumio, Cristiano; Catalucci, Daniele; Papait, Roberto; Condorelli, Gianluigi

    2017-01-01

    Heart failure (HF) is a leading cause of mortality. Inflammation is implicated in HF, yet clinical trials targeting pro-inflammatory cytokines in HF were unsuccessful, possibly due to redundant functions of individual cytokines. Searching for better cardiac inflammation targets, here we link T cells with HF development in a mouse model of pathological cardiac hypertrophy and in human HF patients. T cell costimulation blockade, through FDA-approved rheumatoid arthritis drug abatacept, leads to highly significant delay in progression and decreased severity of cardiac dysfunction in the mouse HF model. The therapeutic effect occurs via inhibition of activation and cardiac infiltration of T cells and macrophages, leading to reduced cardiomyocyte death. Abatacept treatment also induces production of anti-inflammatory cytokine interleukin-10 (IL-10). IL-10-deficient mice are refractive to treatment, while protection could be rescued by transfer of IL-10-sufficient B cells. These results suggest that T cell costimulation blockade might be therapeutically exploited to treat HF. PMID:28262700

  17. Antibody blockade of the Cripto CFC domain suppresses tumor cell growth in vivo.

    PubMed

    Adkins, Heather B; Bianco, Caterina; Schiffer, Susan G; Rayhorn, Paul; Zafari, Mohammad; Cheung, Anne E; Orozco, Olivia; Olson, Dian; De Luca, Antonella; Chen, Ling Ling; Miatkowski, Konrad; Benjamin, Chris; Normanno, Nicola; Williams, Kevin P; Jarpe, Matthew; LePage, Doreen; Salomon, David; Sanicola, Michele

    2003-08-01

    Cripto, a cell surface-associated protein belonging to the EGF-CFC family of growth factor-like molecules, is overexpressed in many human solid tumors, including 70-80% of breast and colon tumors, yet how it promotes cell transformation is unclear. During embryogenesis, Cripto complexes with Alk4 via its unique cysteine-rich CFC domain to facilitate signaling by the TGF-beta ligand Nodal. We report, for the first time to our knowledge, that Cripto can directly bind to another TGF-beta ligand, Activin B, and that Cripto overexpression blocks Activin B growth inhibition of breast cancer cells. This result suggests a novel mechanism for antagonizing Activin signaling that could promote tumorigenesis by deregulating growth homeostasis. We show that an anti-CFC domain antibody, A8.G3.5, both disrupts Cripto-Nodal signaling and reverses Cripto blockade of Activin B-induced growth suppression by blocking Cripto's association with either Alk4 or Activin B. In two xenograft models, testicular and colon cancer, A8.G3.5 inhibited tumor cell growth by up to 70%. Both Nodal and Activin B expression was found in the xenograft tumor, suggesting that either ligand could be promoting tumorigenesis. These data validate that functional blockade of Cripto inhibits tumor growth and highlight antibodies that block Cripto signaling mediated through its CFC domain as an important class of antibodies for further therapeutic development.

  18. Antibody blockade of the Cripto CFC domain suppresses tumor cell growth in vivo

    PubMed Central

    Adkins, Heather B.; Bianco, Caterina; Schiffer, Susan G.; Rayhorn, Paul; Zafari, Mohammad; Cheung, Anne E.; Orozco, Olivia; Olson, Dian; De Luca, Antonella; Chen, Ling Ling; Miatkowski, Konrad; Benjamin, Chris; Normanno, Nicola; Williams, Kevin P.; Jarpe, Matthew; LePage, Doreen; Salomon, David; Sanicola, Michele

    2003-01-01

    Cripto, a cell surface–associated protein belonging to the EGF-CFC family of growth factor–like molecules, is overexpressed in many human solid tumors, including 70–80% of breast and colon tumors, yet how it promotes cell transformation is unclear. During embryogenesis, Cripto complexes with Alk4 via its unique cysteine-rich CFC domain to facilitate signaling by the TGF-β ligand Nodal. We report, for the first time to our knowledge, that Cripto can directly bind to another TGF-β ligand, Activin B, and that Cripto overexpression blocks Activin B growth inhibition of breast cancer cells. This result suggests a novel mechanism for antagonizing Activin signaling that could promote tumorigenesis by deregulating growth homeostasis. We show that an anti–CFC domain antibody, A8.G3.5, both disrupts Cripto-Nodal signaling and reverses Cripto blockade of Activin B–induced growth suppression by blocking Cripto’s association with either Alk4 or Activin B. In two xenograft models, testicular and colon cancer, A8.G3.5 inhibited tumor cell growth by up to 70%. Both Nodal and Activin B expression was found in the xenograft tumor, suggesting that either ligand could be promoting tumorigenesis. These data validate that functional blockade of Cripto inhibits tumor growth and highlight antibodies that block Cripto signaling mediated through its CFC domain as an important class of antibodies for further therapeutic development. PMID:12925698

  19. Biomimetic biodegradable artificial antigen presenting cells synergize with PD-1 blockade to treat melanoma.

    PubMed

    Kosmides, A K; Meyer, R A; Hickey, J W; Aje, K; Cheung, K N; Green, J J; Schneck, J P

    2017-02-01

    Biomimetic materials that target the immune system and generate an anti-tumor responses hold promise in augmenting cancer immunotherapy. These synthetic materials can be engineered and optimized for their biodegradability, physical parameters such as shape and size, and controlled release of immune-modulators. As these new platforms enter the playing field, it is imperative to understand their interaction with existing immunotherapies since single-targeted approaches have limited efficacy. Here, we investigate the synergy between a PLGA-based artificial antigen presenting cell (aAPC) and a checkpoint blockade molecule, anti-PD1 monoclonal antibody (mAb). The combination of antigen-specific aAPC-based activation and anti-PD-1 mAb checkpoint blockade induced the greatest IFN-γ secretion by CD8+ T cells in vitro. Combination treatment also acted synergistically in an in vivo murine melanoma model to result in delayed tumor growth and extended survival, while either treatment alone had no effect. This was shown mechanistically to be due to decreased PD-1 expression and increased antigen-specific proliferation of CD8+ T cells within the tumor microenvironment and spleen. Thus, biomaterial-based therapy can synergize with other immunotherapies and motivates the translation of biomimetic combinatorial treatments.

  20. Interleukin-1 blockade overcomes erlotinib resistance in head and neck squamous cell carcinoma.

    PubMed

    Stanam, Aditya; Gibson-Corley, Katherine N; Love-Homan, Laurie; Ihejirika, Nnamdi; Simons, Andrean L

    2016-11-15

    Erlotinib has demonstrated poor clinical response rates for head and neck squamous cell carcinoma (HNSCC) to date and the majority of respondents acquire resistance to erlotinib relatively quickly. To elucidate novel pathways involved in erlotinib resistance, we compared the gene expression profiles of erlotinib-resistant (ER) vs. erlotinib-sensitive (ES) HNSCC cell lines. Enrichment analysis of microarray data revealed a deregulation of the IL-1 signaling pathway in ER versus ES-HNSCC cells. Gene expression of interleukin-1 alpha (IL1A) and interleukin-1 beta (IL1B) were significantly upregulated by > 2 fold in ER-SQ20B and ER-CAL 27 cells compared to their respective ES-cells. Secretion of the IL-1 receptor antagonist (IL-1RA) was significantly reduced in ER-cells compared to ES-cells. Blockade of IL-1 signaling using a recombinant IL-1R antagonist (anakinra) was able to inhibit the growth of ER-SQ20B and ER-CAL 27 but not ES-SQ20B and ES-CAL 27 xenografts as a single agent and in combination with erlotinib. ER-SQ20B xenografts treated with anakinra ± erlotinib were found to be less vascularized than ER-SQ20B xenografts treated with water or erlotinib. Mice bearing ER-SQ20B xenografts had significantly lesser circulating levels of G-CSF and IL-1β when treated with anakinra ± erlotinib compared to those treated with water or erlotinib alone. Furthermore, augmented mRNA levels of IL1A or interleukin-1 receptor accessory protein (IL1RAP) were associated with shortened survival in HNSCC patients. Altogether, blockade of the IL-1 pathway using anakinra overcame erlotinib resistance in HNSCC xenografts and may represent a novel strategy to overcome EGFR inhibitor resistance for treatment of HNSCC patients.

  1. Interleukin-1 blockade overcomes erlotinib resistance in head and neck squamous cell carcinoma

    PubMed Central

    Stanam, Aditya; Gibson-Corley, Katherine N.; Love-Homan, Laurie; Ihejirika, Nnamdi; Simons, Andrean L.

    2016-01-01

    Erlotinib has demonstrated poor clinical response rates for head and neck squamous cell carcinoma (HNSCC) to date and the majority of respondents acquire resistance to erlotinib relatively quickly. To elucidate novel pathways involved in erlotinib resistance, we compared the gene expression profiles of erlotinib-resistant (ER) vs. erlotinib-sensitive (ES) HNSCC cell lines. Enrichment analysis of microarray data revealed a deregulation of the IL-1 signaling pathway in ER versus ES-HNSCC cells. Gene expression of interleukin-1 alpha (IL1A) and interleukin-1 beta (IL1B) were significantly upregulated by > 2 fold in ER-SQ20B and ER-CAL 27 cells compared to their respective ES-cells. Secretion of the IL-1 receptor antagonist (IL-1RA) was significantly reduced in ER-cells compared to ES-cells. Blockade of IL-1 signaling using a recombinant IL-1R antagonist (anakinra) was able to inhibit the growth of ER-SQ20B and ER-CAL 27 but not ES-SQ20B and ES-CAL 27 xenografts as a single agent and in combination with erlotinib. ER-SQ20B xenografts treated with anakinra ± erlotinib were found to be less vascularized than ER-SQ20B xenografts treated with water or erlotinib. Mice bearing ER-SQ20B xenografts had significantly lesser circulating levels of G-CSF and IL-1β when treated with anakinra ± erlotinib compared to those treated with water or erlotinib alone. Furthermore, augmented mRNA levels of IL1A or interleukin-1 receptor accessory protein (IL1RAP) were associated with shortened survival in HNSCC patients. Altogether, blockade of the IL-1 pathway using anakinra overcame erlotinib resistance in HNSCC xenografts and may represent a novel strategy to overcome EGFR inhibitor resistance for treatment of HNSCC patients. PMID:27738319

  2. Precision medicine in immune checkpoint blockade therapy for non-small cell lung cancer.

    PubMed

    Liu, Xiaoming; Cho, William C

    2017-12-01

    Immune checkpoint blockade therapy by targeting the programmed death protein 1/programmed death ligand 1 (PD-L1) axis using antibodies has yielded promising clinical responses in patients with non-small cell lung cancer (NSCLC). However, owing to the dynamic expression of PD-L1, degree of mutational/neoantigen load, intratumoral heterogeneity, infiltrated immune cells of tumor microenvironment of NSCLC, the response rates to these agents are limited, despite several companion diagnostic assays by detecting PD-L1 in tumor cells have been introduced into clinical practice. Therefore, in this era of precision medicine, there is an urgent need for predictive biomarkers to identify NSCLC patients likely to benefit from this novel therapy.

  3. TNF-α blockade induces IL-10 expression in human CD4+ T cells

    NASA Astrophysics Data System (ADS)

    Evans, Hayley G.; Roostalu, Urmas; Walter, Gina J.; Gullick, Nicola J.; Frederiksen, Klaus S.; Roberts, Ceri A.; Sumner, Jonathan; Baeten, Dominique L.; Gerwien, Jens G.; Cope, Andrew P.; Geissmann, Frederic; Kirkham, Bruce W.; Taams, Leonie S.

    2014-02-01

    IL-17+ CD4+ T (Th17) cells contribute to the pathogenesis of several human inflammatory diseases. Here we demonstrate that TNF inhibitor (TNFi) drugs induce the anti-inflammatory cytokine IL-10 in CD4+ T cells including IL-17+ CD4+ T cells. TNFi-mediated induction of IL-10 in IL-17+ CD4+ T cells is Treg-/Foxp3-independent, requires IL-10 and is overcome by IL-1β. TNFi-exposed IL-17+ CD4+ T cells are molecularly and functionally distinct, with a unique gene signature characterized by expression of IL10 and IKZF3 (encoding Aiolos). We show that Aiolos binds conserved regions in the IL10 locus in IL-17+ CD4+ T cells. Furthermore, IKZF3 and IL10 expression levels correlate in primary CD4+ T cells and Aiolos overexpression is sufficient to drive IL10 in these cells. Our data demonstrate that TNF-α blockade induces IL-10 in CD4+ T cells including Th17 cells and suggest a role for the transcription factor Aiolos in the regulation of IL-10 in CD4+ T cells.

  4. Tetrandrine induces lipid accumulation through blockade of autophagy in a hepatic stellate cell line

    SciTech Connect

    Miyamae, Yusaku; Nishito, Yukina; Nakai, Naomi; Nagumo, Yoko; Usui, Takeo; Masuda, Seiji; Kambe, Taiho; Nagao, Masaya

    2016-08-12

    Macroautophagy, or autophagy, is a cellular response in which unnecessary cytoplasmic components, including lipids and organelles, are self-degraded. Recent studies closely related autophagy to activation of hepatic stellate cells (HSCs), a process critical in the pathogenesis of liver fibrosis. During HSC activation, cytoplasmic lipid droplets (LDs) are degraded as autophagic cargo, and then cells express fibrogenic genes. Thus, inhibition of autophagy in HSCs is a potential therapeutic approach for attenuating liver fibrosis. We found that tetrandrine, a bisbenzylisoquinoline alkaloid isolated from Stephania tetrandra, induced lipid accumulation, a phenotype associated with quiescent HSCs, through blockade of autophagy in the rat-derived HSC line HSC-T6. Tetrandrine inhibited autophagic flux without affecting lysosomal function. A phenotypic comparison using siRNA knockdown suggested that tetrandrine may target regulators, involved in fusion between autophagosomes and lysosomes (e.g., syntaxin 17). Moreover, perilipin 1, an LD-coated protein, co-localized specifically with LC3, a marker protein for autophagosomes, in tetrandrine-treated HSC-T6 cells. This suggests a potential role for perilipin 1 in autophagy-mediated LD degradation in HSCs. Our results identified tetrandrine as a potential tool for prevention and treatment of HSC activation. - Highlights: • Autophagy is closely related to lipid degradation in hepatic stellate cells. • Tetrandrine (Tet) causes lipid accumulation via blockade of autophagy in HSC-T6 cells. • Tet blocked autophagy without affecting lysosomal function unlike bafilomycin A{sub 1}. • Perilipin 1 was specifically co-localized with LC3 in Tet-treated cells. • Perilipin 1 may play potential roles in autophagy-mediated lipid degradation.

  5. Dual HER2 Blockade in Non-Small Cell Lung Cancer Harboring a HER2 Mutation.

    PubMed

    Mar, Nataliya; Vredenburgh, James J

    2015-10-01

    Identification of targetable oncogenic mutations in non-small cell lung cancer (NSCLC) has been a major advance in cancer treatment. Laboratory techniques to assess human epidermal growth factor receptor 2 (HER2) positivity in NSCLC include immunohistochemistry (IHC) for protein overexpression, fluorescent in situ hybridization (FISH) for gene amplification, and next generation sequencing (NGS) for HER2 gene mutations. These tests have a controversial prognostic and predictive value, with an emerging association between HER2 gene mutations and treatment response to HER2 targeted therapy. We present a case of a woman with metastatic lung adenocarcinoma with HER2 positivity assessed by IHC and FISH, as well as a high gene copy number noted on NGS. She was observed to have significant disease progression following standard first-line platinum doublet chemotherapy. She was started on dual HER2 blockade in the second-line setting, which yielded a great response in the liver with stable disease elsewhere. To our knowledge, this is the first report describing successful use of dual HER2 blockade in metastatic HER2 positive NSCLC. We also review common laboratory techniques for determining HER2 positivity in NSCLC and their clinical applications.

  6. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade

    PubMed Central

    McGranahan, Nicholas; Furness, Andrew J. S.; Rosenthal, Rachel; Ramskov, Sofie; Lyngaa, Rikke; Saini, Sunil Kumar; Jamal-Hanjani, Mariam; Wilson, Gareth A.; Birkbak, Nicolai J.; Hiley, Crispin T.; Watkins, Thomas B. K.; Shafi, Seema; Murugaesu, Nirupa; Mitter, Richard; Akarca, Ayse U.; Linares, Joseph; Marafioti, Teresa; Henry, Jake Y.; Van Allen, Eliezer M.; Miao, Diana; Schilling, Bastian; Schadendorf, Dirk; Garraway, Levi A.; Makarov, Vladimir; Rizvi, Naiyer A.; Snyder, Alexandra; Hellmann, Matthew D.; Merghoub, Taha; Wolchok, Jedd D.; Shukla, Sachet A.; Wu, Catherine J.; Peggs, Karl S.; Chan, Timothy A.; Hadrup, Sine R.; Quezada, Sergio A.; Swanton, Charles

    2016-01-01

    As tumors grow, they acquire mutations, some of which create neoantigens that influence the response of patients to immune checkpoint inhibitors. We explored the impact of neoantigen intratumor heterogeneity (ITH) on antitumor immunity. Through integrated analysis of ITH and neoantigen burden, we demonstrate a relationship between clonal neoantigen burden and overall survival in primary lung adenocarcinomas. CD8+ tumor-infiltrating lymphocytes reactive to clonal neoantigens were identified in early-stage non–small cell lung cancer and expressed high levels of PD-1. Sensitivity to PD-1 and CTLA-4 blockade in patients with advanced NSCLC and melanoma was enhanced in tumors enriched for clonal neoantigens. T cells recognizing clonal neoantigens were detectable in patients with durable clinical benefit. Cytotoxic chemotherapy–induced subclonal neoantigens, contributing to an increased mutational load, were enriched in certain poor responders. These data suggest that neoantigen heterogeneity may influence immune surveillance and support therapeutic developments targeting clonal neoantigens. PMID:26940869

  7. Blockade of glucagon signaling prevents or reverses diabetes onset only if residual β-cells persist

    PubMed Central

    Damond, Nicolas; Thorel, Fabrizio; Moyers, Julie S; Charron, Maureen J; Vuguin, Patricia M; Powers, Alvin C; Herrera, Pedro L

    2016-01-01

    Glucagon secretion dysregulation in diabetes fosters hyperglycemia. Recent studies report that mice lacking glucagon receptor (Gcgr-/-) do not develop diabetes following streptozotocin (STZ)-mediated ablation of insulin-producing β-cells. Here, we show that diabetes prevention in STZ-treated Gcgr-/- animals requires remnant insulin action originating from spared residual β-cells: these mice indeed became hyperglycemic after insulin receptor blockade. Accordingly, Gcgr-/- mice developed hyperglycemia after induction of a more complete, diphtheria toxin (DT)-induced β-cell loss, a situation of near-absolute insulin deficiency similar to type 1 diabetes. In addition, glucagon deficiency did not impair the natural capacity of α-cells to reprogram into insulin production after extreme β-cell loss. α-to-β-cell conversion was improved in Gcgr-/- mice as a consequence of α-cell hyperplasia. Collectively, these results indicate that glucagon antagonism could i) be a useful adjuvant therapy in diabetes only when residual insulin action persists, and ii) help devising future β-cell regeneration therapies relying upon α-cell reprogramming. DOI: http://dx.doi.org/10.7554/eLife.13828.001 PMID:27092792

  8. Immune checkpoint blockade in small cell lung cancer: is there a light at the end of the tunnel?

    PubMed Central

    Paglialunga, Luca; Salih, Zena; Ricciuti, Biagio; Califano, Raffaele

    2016-01-01

    Small cell lung cancer (SCLC) is a very aggressive disease, characterised by rapid growth, high response rates to both chemotherapy and radiotherapy and subsequent development of treatment resistance in the vast majority of patients. In the past 30 years, little progress has been made in systemic treatments and the established management paradigm of platinum-based chemotherapy has reached an efficacy plateau. Several clinical trials have investigated targeted therapies, without producing clinically significant benefits. Recently presented early phase clinical trials with immune checkpoint inhibitors (blockade of the cytotoxic T-lymphocyte antigen-4 (CTLA-4) and blockade of the programmed cell death-1 (PD-1) receptor) have shown promising results. In this review, we present the emerging evidence on immune checkpoint blockade for SCLC. PMID:27843619

  9. Beta-blockade prevents hematopoietic progenitor cell suppression after hemorrhagic shock.

    PubMed

    Elhassan, Ihab O; Hannoush, Edward J; Sifri, Ziad C; Jones, Eyone; Alzate, Walter D; Rameshwar, Pranela; Livingston, David H; Mohr, Alicia M

    2011-08-01

    Severe injury is accompanied by sympathetic stimulation that induces bone marrow (BM) dysfunction by both suppression of hematopoietic progenitor cell (HPC) growth and loss of cells via HPC mobilization to the peripheral circulation and sites of injury. Previous work demonstrated that beta-blockade (BB) given prior to tissue injury both reduces HPC mobilization and restores HPC colony growth within the BM. This study examined the effect and timing of BB on BM function in a hemorrhagic shock (HS) model. Male Sprague-Dawley rats underwent HS via blood withdrawal, maintaining the mean arterial blood pressure at 30-40 mm Hg for 45 min, after which the extracted blood was reinfused. Propranolol (10 mg/kg) was given either prior to or immediately after HS. Blood pressure, heart rate, BM cellularity, and death were recorded. Bone marrow HPC growth was assessed by counting colony-forming unit-granulocyte-, erythrocyte-, monocyte-, megakaryocyte (CFU-GEMM), burst-forming unit-erythroid (BFU-E), and colony-forming unit-erythroid (CFU-E) cells. Administration of BB prior to injury restored HPC growth to that of naïve animals (CFU-GEMM 59 ± 11 vs. 61 ± 4, BFU-E 68 ± 9 vs. 73 ± 3, and CFU-E 81 ± 35 vs. 78 ± 14 colonies/plate). Beta-blockade given after HS increased the growth of CFU-GEMM, BFU-E, and CFU-E significantly and improved BM cellularity compared with HS alone. The mortality rate was not increased in the groups receiving BB. Administration of propranolol either prior to injury or immediately after resuscitation significantly reduced post-shock BM suppression. After HS, BB may improve BM cellularity by decreasing HPC mobilization. Therefore, the early use of BB post-injury may play an important role in attenuating the BM dysfunction accompanying HS.

  10. Receptor-selective retinoids inhibit the growth of normal and malignant breast cells by inducing G1 cell cycle blockade.

    PubMed

    Wu, Kendall; DuPré, Elizabeth; Kim, Heetae; Tin-U, Caesar K; Bissonnette, Reid P; Lamph, William W; Brown, Powel H

    2006-03-01

    Despite advances in treatment, breast cancer continues to be the second leading cause of cancer mortality in women. Statistics suggest that while focus on treatment should continue, chemopreventive approaches should also be pursued. Previous studies have demonstrated that naturally occurring retinoids such as 9-cis retinoic acid (9cRA) can prevent breast cancer in animal models. However, these studies have also shown that these compounds are too toxic for general use. Work from our laboratory showed that an RXR-selective retinoid LGD1069 prevented tumor development in animal models of cancer with reduced toxicity as compared to an RAR-selective retinoid TTNPB. In the present study, we investigated the mechanisms by which receptor-selective retinoids inhibit the growth of normal and malignant breast cells. Our results demonstrate that the synthetic retinoids tested are as effective as 9cRA in suppressing the growth of normal human mammary epithelial cells (HMECs) and estrogen receptor-positive (ER-positive) breast cancer cells. Although the receptor-selective retinoids induce minimal amounts of apoptosis in T47D breast cancer cells, the predominant factor that leads to growth arrest is G1 cell cycle blockade. Our data indicate that this blockade results from the downregulation of Cyclin D1 and Cyclin D3, which in turn causes Rb hypophosphorylation. Non-toxic retinoids that are potent inducers of cell cycle arrest may be particularly useful for the prevention of breast cancer.

  11. Synergistic reversal of intrahepatic HCV-specific CD8 T cell exhaustion by combined PD-1/CTLA-4 blockade.

    PubMed

    Nakamoto, Nobuhiro; Cho, Hyosun; Shaked, Abraham; Olthoff, Kim; Valiga, Mary E; Kaminski, Mary; Gostick, Emma; Price, David A; Freeman, Gordon J; Wherry, E John; Chang, Kyong-Mi

    2009-02-01

    Viral persistence is associated with hierarchical antiviral CD8 T cell exhaustion with increased programmed death-1 (PD-1) expression. In HCV persistence, HCV-specific CD8 T cells from the liver (the site of viral replication) display increased PD-1 expression and a profound functional impairment that is not reversed by PD-1 blockade alone. Here, we report that the inhibitory receptor cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is preferentially upregulated in PD-1(+) T cells from the liver but not blood of chronically HCV-infected patients. PD-1/CTLA-4 co-expression in intrahepatic T cells was associated with a profound HCV-specific effector dysfunction that was synergistically reversed by combined PD-1/CTLA-4 blockade in vitro, but not by blocking PD-1 or CTLA-4 alone. A similar effect was observed in circulating HCV-specific CD8 T cells with increased PD-1/CTLA-4 co-expression during acute hepatitis C. The functional response to combined blockade was directly associated with CTLA-4 expression, lost with CD28-depletion and CD4-independent (including CD4(+)FoxP3(+) Tregs). We conclude that PD-1 and CTLA-4 pathways both contribute to virus-specific T cell exhaustion at the site of viral replication by a redundant mechanism that requires combined PD-1/CTLA-4 blockade to reverse. These findings provide new insights into the mechanisms of virus-specific T cell dysfunction, and suggest that the synergistic effect by combined inhibitory receptor blockade might have a therapeutic application against chronic viral infection in vivo, provided that it does not induce autoimmunity.

  12. Effect of cholecystokinin-2 receptor blockade on rat stomach ECL cells. A histochemical, electron-microscopic and chemical study.

    PubMed

    Chen, D; Zhao, C M; Norlén, P; Björkqvist, M; Ding, X Q; Kitano, M; Håkanson, R

    2000-01-01

    The ECL cells in the oxyntic mucosa of rat stomach produce histamine and chromogranin A-derived peptides such as pancreastatin. The cells respond to gastrin via cholecystokinin-2 (CCK2) receptors. A CCK2 receptor blockade was induced by treatment (for up to 8 weeks) with two receptor antagonists, YM022 and YF476. Changes in ECL-cell morphology were examined by immunocytochemistry and electron microscopy, while changes in ECL cell-related biochemical parameters were monitored by measuring serum pancreastatin and oxyntic mucosal pancreastatin, and histamine concentrations, and histidine decarboxylase (HDC) activity. The CCK2 receptor blockade reduced the ECL-cell density only marginally, if at all, but transformed the ECL cells from slender, elongated cells with prominent projections to small, spherical cells without projections. The Golgi complex and the rough endoplasmic reticulum were diminished. Secretory vesicles were greatly reduced in volume density in the trans Golgi area. Circulating pancreastatin concentration and oxyntic mucosal HDC activity were lowered within a few hours. Oxyntic mucosal histamine and pancreastatin concentrations were reduced only gradually. The CCK2 receptor blockade was found to prevent the effects of omeprazole-evoked hypergastrinaemia on the ECL-cell activity and density. In conclusion, gastrin, acting on CCK2 receptors, is needed to maintain the shape, size and activity of the ECL cells, but not for maintaining the ECL-cell population.

  13. Blockade of bovine PD-1 increases T cell function and inhibits bovine leukemia virus expression in B cells in vitro.

    PubMed

    Ikebuchi, Ryoyo; Konnai, Satoru; Okagawa, Tomohiro; Yokoyama, Kazumasa; Nakajima, Chie; Suzuki, Yasuhiko; Murata, Shiro; Ohashi, Kazuhiko

    2013-07-22

    Programmed death-1 (PD-1) is a known immunoinhibitory receptor that contributes to immune evasion of various tumor cells and pathogens causing chronic infection, such as bovine leukemia virus (BLV) infection. First, in this study, to establish a method for the expression and functional analysis of bovine PD-1, hybridomas producing monoclonal antibodies (mAb) specific for bovine PD-1 were established. Treatment with these anti-PD-1 mAb enhanced interferon-gamma (IFN-γ) production of bovine peripheral blood mononuclear cells (PBMC). Next, to examine whether PD-1 blockade by anti-PD-1 mAb could upregulate the immune reaction during chronic infection, the expression and functional analysis of PD-1 in PBMC isolated from BLV-infected cattle with or without lymphoma were performed using anti-PD-1 mAb. The frequencies of both PD-1+ CD4+ T cells in blood and lymph node and PD-1+ CD8+ T cells in lymph node were higher in BLV-infected cattle with lymphoma than those without lymphoma or control uninfected cattle. PD-1 blockade enhanced IFN-γ production and proliferation and reduced BLV-gp51 expression and B-cell activation in PBMC from BLV-infected cattle in response to BLV-gp51 peptide mixture. These data show that anti-bovine PD-1 mAb could provide a new therapy to control BLV infection via upregulation of immune response.

  14. Blockade of Deubiquitylating Enzyme USP1 Inhibits DNA Repair and Triggers Apoptosis in Multiple Myeloma Cells.

    PubMed

    Das, Deepika Sharma; Das, Abhishek; Ray, Arghya; Song, Yan; Samur, Mehmet Kemal; Munshi, Nikhil C; Chauhan, Dharminder; Anderson, Kenneth C

    2017-08-01

    Purpose: The ubiquitin proteasome pathway is a validated therapeutic target in multiple myeloma. Deubiquitylating enzyme USP1 participates in DNA damage response and cellular differentiation pathways. To date, the role of USP1 in multiple myeloma biology is not defined. In the present study, we investigated the functional significance of USP1 in multiple myeloma using genetic and biochemical approaches.Experimental Design: To investigate the role of USP1 in myeloma, we utilized USP1 inhibitor SJB3-019A (SJB) for studies in myeloma cell lines and patient multiple myeloma cells.Results: USP1-siRNA knockdown decreases multiple myeloma cell viability. USP1 inhibitor SJB selectively blocks USP1 enzymatic activity without blocking other DUBs. SJB also decreases the viability of multiple myeloma cell lines and patient tumor cells, inhibits bone marrow plasmacytoid dendritic cell-induced multiple myeloma cell growth, and overcomes bortezomib resistance. SJB triggers apoptosis in multiple myeloma cells via activation of caspase-3, caspase-8, and caspase-9. Moreover, SJB degrades USP1 and downstream inhibitor of DNA-binding proteins as well as inhibits DNA repair via blockade of Fanconi anemia pathway and homologous recombination. SJB also downregulates multiple myeloma stem cell renewal/survival-associated proteins Notch-1, Notch-2, SOX-4, and SOX-2. Moreover, SJB induced generation of more mature and differentiated plasma cells. Combination of SJB and HDACi ACY-1215, bortezomib, lenalidomide, or pomalidomide triggers synergistic cytotoxicity.Conclusions: Our preclinical studies provide the framework for clinical evaluation of USP1 inhibitors, alone or in combination, as a potential novel multiple myeloma therapy. Clin Cancer Res; 23(15); 4280-9. ©2017 AACR. ©2017 American Association for Cancer Research.

  15. Blockade of voltage-gated sodium channels inhibits invasion of endocrine-resistant breast cancer cells.

    PubMed

    Mohammed, Fatima H; Khajah, Maitham A; Yang, Ming; Brackenbury, William J; Luqmani, Yunus A

    2016-01-01

    Voltage-gated Na+ channels (VGSCs) are membrane proteins which are normally expressed in excitable cells but have also been detected in cancer cells, where they are thought to be involved in malignancy progression. In this study we examined the ion current and expression profile of VGSC (Nav1.5) in estrogen receptor (ER)-positive (MCF-7) and silenced (pII) breast cancer cells and its possible influence on their proliferation, motility and invasion. VGSC currents were analysed by whole cell patch clamp recording. Nav1.5 expression and localization, in response to EGF stimulation, was examined by western blotting and immunofluorescence respectively. Cell invasion (under-agarose and Matrigel assays), motility (wound healing assay) and proliferation (MTT assay) were assessed in pII cells in response to VGSC blockers, phenytoin (PHT) and tetrodotoxin (TTX), or by siRNA knockdown of Nav1.5. The effect of PHT and TTX on modulating EGF-induced phosphorylation of Akt and ERK1/2 was determined by western blotting. Total matrix metalloproteinase (MMP) was determined using a fluorometric-based activity assay. The level of various human proteases was detected by using proteome profiler array kit. VGSC currents were detected in pII cells, but were absent in MCF-7. Nav1.5 showed cytoplasmic and perinuclear expression in both MCF-7 and pII cells, with enhanced expression upon EGF stimulation. Treatment of pII cells with PHT, TTX or siRNA significantly reduced invasion towards serum components and EGF, in part through reduction of P-ERK1/2 and proteases such as cathepsin E, kallikrein-10 and MMP-7, as well as total MMP activity. At high concentrations, PHT inhibited motility while TTX reduced cell proliferation. Pharmacological or genetic blockade of Nav1.5 may serve as a potential anti-metastatic therapy for breast cancer.

  16. Blockade of TGF-beta enhances tumor vaccine efficacy mediated by CD8(+) T cells.

    PubMed

    Takaku, Shun; Terabe, Masaki; Ambrosino, Elena; Peng, Judy; Lonning, Scott; McPherson, John M; Berzofsky, Jay A

    2010-04-01

    Though TGF-beta inhibition enhances antitumor immunity mediated by CD8(+) T cells in several tumor models, it is not always sufficient for rejection of tumors. In this study, to maximize the antitumor effect of TGF-beta blockade, we tested the effect of anti-TGF-beta combined with an irradiated tumor vaccine in a subcutaneous CT26 colon carcinoma tumor model. The irradiated tumor cell vaccine alone in prophylactic setting significantly delayed tumor growth, whereas anti-TGF-beta antibodies alone did not show any antitumor effect. However, tumor growth was inhibited significantly more in vaccinated mice treated with anti-TGF-beta antibodies compared to vaccinated mice without anti-TGF-beta, suggesting that anti-TGF-beta synergistically enhanced irradiated tumor vaccine efficacy. CD8(+) T-cell depletion completely abrogated the vaccine efficacy, and so protection required CD8(+) T cells. Depletion of CD25(+) T regulatory cells led to the almost complete rejection of tumors without the vaccine, whereas anti-TGF-beta did not change the number of CD25(+) T regulatory cells in unvaccinated and vaccinated mice. Though the abrogation of CD1d-restricted NKT cells, which have been reported to induce TGF-beta production by MDSC through an IL-13-IL-4R-STAT6 pathway, partially enhanced antitumor immunity regardless of vaccination, abrogation of the NKT cell-IL-13-IL-4R-STAT-6 immunoregulatory pathway did not enhance vaccine efficacy. Taken together, these data indicated that anti-TGF-beta enhances efficacy of a prophylactic vaccine in normal individuals despite their not having the elevated TGF-beta levels found in patients with cancer and that the effect is not dependent on TGF-beta solely from CD4(+)CD25(+) T regulatory cells or the NKT cell-IL-13-IL-4R-STAT-6 immunoregulatory pathway.

  17. Pathogen Stimulation History Impacts Donor-Specific CD8(+) T Cell Susceptibility to Costimulation/Integrin Blockade-Based Therapy.

    PubMed

    Badell, I R; Kitchens, W H; Wagener, M E; Lukacher, A E; Larsen, C P; Ford, M L

    2015-12-01

    Recent studies have shown that the quantity of donor-reactive memory T cells is an important factor in determining the relative heterologous immunity barrier posed during transplantation. Here, we hypothesized that the quality of T cell memory also potently influences the response to costimulation blockade-based immunosuppression. Using a murine skin graft model of CD8(+) memory T cell-mediated costimulation blockade resistance, we elicited donor-reactive memory T cells using three distinct types of pathogen infections. Strikingly, we observed differential efficacy of a costimulation and integrin blockade regimen based on the type of pathogen used to elicit the donor-reactive memory T cell response. Intriguingly, the most immunosuppression-sensitive memory T cell populations were composed primarily of central memory cells that possessed greater recall potential, exhibited a less differentiated phenotype, and contained more multi-cytokine producers. These data, therefore, demonstrate that the memory T cell barrier is dependent on the specific type of pathogen infection via which the donor-reactive memory T cells are elicited, and suggest that the immune stimulation history of a given transplant patient may profoundly influence the relative barrier posed by heterologous immunity during transplantation.

  18. Pathogen stimulation history impacts donor-specific CD8+ T cell susceptibility to costimulation/integrin blockade-based therapy

    PubMed Central

    Badell, IR; Kitchens, WH; Wagener, ME; Lukacher, AE; Larsen, CP; Ford, ML

    2017-01-01

    Recent studies have shown that the quantity of donor-reactive memory T cells is an important factor in determining the relative heterologous immunity barrier posed during transplantation. Here, we hypothesized that the quality of T cell memory also potently influences the response to costimulation blockade-based immunosuppression. Using a murine skin graft model of CD8+ memory T cell-mediated costimulation blockade resistance, we elicited donor-reactive memory T cells using three distinct types of pathogen infections. Strikingly, we observed differential efficacy of a costimulation and integrin blockade regimen based on the type of pathogen used to elicit the donor-reactive memory T cell response. Intriguingly, the most immunosuppression-sensitive memory T cell populations were composed primarily of central memory cells that possessed greater recall potential, exhibited a less differentiated phenotype, and contained more multi-cytokine producers. These data therefore demonstrate that the memory T cell barrier is dependent on the specific type of pathogen infection via which the donor-reactive memory T cells are elicited, and suggest that the immune stimulation history of a given transplant patient may profoundly influence the relative barrier posed by heterologous immunity during transplantation. PMID:26228897

  19. Hyperlipidemia Alters Regulatory T Cell Function and Promotes Resistance to Tolerance Induction Through Costimulatory Molecule Blockade

    PubMed Central

    Bagley, J.; Yuan, J.; Chandrakar, A.; Iacomini, J.

    2016-01-01

    Recent work from our laboratory has shown that hyperlipidemia promotes accelerated rejection of vascularized cardiac allografts in mice by inducing anti-donor Th17 reactivity and production of IL-17. Here, we show that hyperlipidemia also affects FoxP3+ regulatory T cells (Tregs). Hyperlipidemia promotes the development of Tregs that express low levels of CD25. Hyperlipidemia also promotes a decrease in central Tregs and an increase in effector Tregs that appears to account for the increase in the frequency of CD25low Tregs. Alterations in Treg subsets also appear to lead to alterations in Treg function. The ability of FoxP3+, CD25high, CD4+ Tregs from hyperlipidemic mice to inhibit proliferation of effector T cells stimulated with anti-CD3 and CD28 was reduced when compared with Tregs from control mice. Regulatory T cells isolated from hyperlipidemic recipients exhibit increased activation of Akt, and a reduction in Bim levels that permits the expansion of FoxP3+CD25lowCD4+ T cells. Hyperlipidemic mice were also resistant to tolerance induction using costimulatory molecule blockade consisting of anti-CD154 and CTLA4Ig, a strategy that requires Tregs. Together, our data suggest that hyperlipidemia profoundly affects Treg subsets and function as well as the ability to induce tolerance. PMID:26079467

  20. T Cell-Mediated Rejection of Human CD34(+) Cells Is Prevented by Costimulatory Blockade in a Xenograft Model.

    PubMed

    Oh, Annie L; Mahmud, Dolores; Nicolini, Benedetta; Mahmud, Nadim; Senyuk, Vitalyi; Patel, Pritesh R; Bonetti, Elisa; Arpinati, Mario; Ferrara, James L M; Rondelli, Damiano

    2017-08-14

    A xenograft model of stem cell rejection was developed by co-transplantating human CD34(+) and allogeneic CD3(+) T cells into NOD-scid ɣ-chain(null) mice. T cells caused graft failure when transplanted at any CD34/CD3 ratio between 1:50 and 1:.1. Kinetics experiments showed that 2 weeks after transplantation CD34(+) cells engrafted the marrow and T cells expanded in the spleen. Then, at 4 weeks only memory T cells populated both sites and rejected CD34(+) cells. Blockade of T cell costimulation was tested by injecting the mice with abatacept (CTLA4-IgG1) from day -1 to +27 (group A), from day -1 to +13 (group B), or from day +14 to +28 (group C). On day +56 groups B and C had rejected the graft, whereas in group A graft failure was completely prevented, although with lower stem cell engraftment than in controls (P = .03). Retransplantation of group A mice with same CD34(+) cells obtained a complete reconstitution of human myeloid and B cell lineages and excluded latent alloreactivity. In this first xenograft model of stem cell rejection we showed that transplantation of HLA mismatched CD34(+) cells may be facilitated by treatment with abatacept and late stem cell boost. Copyright © 2017 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

  1. Ex Vivo Costimulatory Blockade to Generate Regulatory T Cells From Patients Awaiting Kidney Transplantation.

    PubMed

    Guinan, E C; Cole, G A; Wylie, W H; Kelner, R H; Janec, K J; Yuan, H; Oppatt, J; Brennan, L L; Turka, L A; Markmann, J

    2016-07-01

    Short-term outcomes of kidney transplantation have improved dramatically, but chronic rejection and regimen-related toxicity continue to compromise overall patient outcomes. Development of regulatory T cells (Tregs) as a means to decrease alloresponsiveness and limit the need for pharmacologic immunosuppression is an active area of preclinical and clinical investigation. Nevertheless, the immunomodulatory effects of end-stage renal disease on the efficacy of various strategies to generate and expand recipient Tregs for kidney transplantation are incompletely characterized. In this study, we show that Tregs can be successfully generated from either freshly isolated or previously cryopreserved uremic recipient (responder) and healthy donor (stimulator) peripheral blood mononuclear cells using the strategy of ex vivo costimulatory blockade with belatacept during mixed lymphocyte culture. Moreover, these Tregs maintain a CD3(+) CD4(+) CD25(+) CD127(lo) surface phenotype, high levels of intracellular FOXP3 and significant demethylation of the FOXP3 Treg-specific demethylation region on allorestimulation with donor stimulator cells. These data support evaluation of this simple, brief Treg production strategy in clinical trials of mismatched kidney transplantation.

  2. In vivo blockade of neural activity alters dendritic development of neonatal CA1 pyramidal cells.

    PubMed

    Groc, Laurent; Petanjek, Zdravko; Gustafsson, Bengt; Ben-Ari, Yehezkel; Hanse, Eric; Khazipov, Roustem

    2002-11-01

    During development, neural activity has been proposed to promote neuronal growth. During the first postnatal week, the hippocampus is characterized by an oscillating neural network activity and a rapid neuronal growth. In the present study we tested in vivo, by injecting tetanus toxin into the hippocampus of P1 rats, whether this neural activity indeed promotes growth of pyramidal cells. We have previously shown that tetanus toxin injection leads to a strong reduction in the frequency of spontaneous GABA and glutamatergic synaptic currents, and to a complete blockade of the early neural network activity during the first postnatal week. Morphology of neurobiotin-filled CA1 pyramidal cells was analyzed at the end of the first postnatal week (P6-10). In activity-reduced neurons, the total length of basal dendritic tree was three times less than control. The number, but not the length, of basal dendritic branches was affected. The growth impairment was restricted to the basal dendrites. The apical dendrite, the axons, or the soma grew normally during activity deprivation. Thus, the in vivo neural activity in the neonate hippocampus seems to promote neuronal growth by initiating novel branches.

  3. Blockade of FLT4 suppresses metastasis of melanoma cells by impaired lymphatic vessels.

    PubMed

    Lee, Ji Yoon; Hong, Seok-Ho; Shin, Minsang; Heo, Hye-Ryeon; Jang, In Ho

    2016-09-16

    The metastatic spread of tumor cells via lymphatic vessels affects the relapse of tumor patients. New lymphatic vessel formation, including lymphangiogenesis, is promoted in the tumor environment. The lymphangiogenic factor VEGF-C can mediate lymphatic vessel formation and induce tumor metastasis by binding with FLT4. In melanoma, metastasis via lymphatics such as lymph nodes is one of the main predictors of poor outcome. Thus, we investigated whether blockade of FLT4 can reduce metastasis via the suppression of lymphatic capillaries. Proliferative lymphatic capillaries in melanoma were estimated by immunohistochemistry using FLT4 antibody after the injection of the FLT4 antagonist MAZ51. The numbers of tumor modules in metastasised lungs were calculated by gross examination and lymphatic related factors were examined by qRT-PCR. MAZ51 injection resulted in the suppression of tumor size and module number and the inhibition of proliferative lymphatic vessels in the intratumoral region in the lung and proliferating melanoma cells in the lung compared to those of untreated groups. Additionally, high FLT4 and TNF-alpha were detected in melanoma-induced tissue, while lymphatic markers such as VEGF-C, FLT4 and Prox-1 were significantly decreased in MAZ51 treated groups, implying that anti-lymphangiogenesis by MAZ51 may provide a potential strategy to prevent tumor metastasis in melanoma and high number of lymphatic capillaries could be used diagnosis for severe metastasis. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. NK cell killing of AML and ALL blasts by Killer-Immunoglobulin Receptor (KIR) negative NK cells after NKG2A and LIR-1 blockade

    PubMed Central

    Godal, Robert; Bachanova, Veronika; Gleason, Michelle; McCullar, Valarie; Yun, Gong H.; Cooley, Sarah; Verneris, Michael R.; McGlave, Philip B.; Miller, Jeffrey S.

    2010-01-01

    Although NK cell alloreactivity has been dominated by studies of KIR, we hypothesized that NKG2A and LIR-1, present on 53±13% and 36±18% of normal NK cells, plays a role in NK cell killing of primary leukemia targets. KIR− cells, which comprise nearly half of the circulating NK cell population, exhibited tolerance to primary leukemia targets, suggesting signaling through other inhibitory receptors. Both AML and ALL targets could be rendered susceptible to lysis by fresh resting KIR− NK cells when inhibitory receptor-MHC class I interactions were blocked by pan-HLA antibodies demonstrating that these cells were functionally competent. Blockade of a single inhibitory receptor resulted in slight increases in killing, while combined LIR-1 and NKG2A blockade consistently resulted in increased NK cell cytotoxicity. Dual blockade of NKG2A and LIR-1 led to significant killing of targets by resting KIR− NK cells showing that this population is not hyporesponsive. Together these results suggest that alloreactivity of a significant fraction of KIR− NK cells is determined by NKG2A and LIR-1. Thus strategies to interrupt NKG2A and LIR-1 in combination with anti-KIR blockade hold promise for exploiting NK cell therapy in acute leukemia. PMID:20139023

  5. In vitro TNF blockade enhances ex vivo expansion of regulatory T cells in patients with immune thrombocytopenia

    PubMed Central

    Zhong, Hui; Bussel, James; Yazdanbakhsh, Karina

    2017-01-01

    Summary Tumour necrosis factor-α (TNF) is an inflammatory cytokine that is elevated in a number of autoimmune diseases including immune thrombocytopenia (ITP), a bleeding disorder characterized by low platelet counts. In vitro TNF blockade increases expansion of the regulatory T cell (Treg) IKZF2 (also termed Helios) subset in T cell-monocyte cocultures from healthy donors, but its role on proliferative responses of Tregs in ITP patients, who have altered immunoregulatory compartment, remains unclear. TNF in CD4+ T cells from patients with chronic ITP were elevated and negatively correlated with peripheral Treg frequencies, suggesting a possible inhibitory effect of TNF on ITP Tregs. In vitro antibody neutralization with anti-TNF in T cell-monocyte cocultures resulted in a robust expansion of pre-existing ITP Tregs, higher than in healthy controls. Similar to the effects of anti-TNF antibodies, TNF blockade with antibodies against TNFRSF1B (anti-TNFRSF1B, previously termed anti-TNFRII) almost doubled ITP Treg expansion whereas neutralization with anti-TNFRSF1A (anti-TNFRI) antibodies had no effect on proliferative responses of Tregs. In addition, TNFRSF1B levels on ITP Tregs were significantly elevated, which may explain the increased susceptibility of patient Tregs to the actions of TNF blockade. Altogether, these data raise the possibility that TNF blockers, through their ability to increase Treg proliferation, may be efficacious in ITP patients. PMID:25252160

  6. Ethanol and arachidonic acid synergize to activate Kupffer cells and modulate the fibrogenic response via tumor necrosis factor alpha, reduced glutathione, and transforming growth factor beta-dependent mechanisms.

    PubMed

    Cubero, Francisco Javier; Nieto, Natalia

    2008-12-01

    Because of the contribution of ethanol and polyunsaturated fatty acids (PUFAs) to alcoholic liver disease, we investigated whether chronic ethanol administration and arachidonic acid (AA) could synergistically mediate Kupffer cell (KC) activation and modulate the stellate cell (HSC) fibrogenic response. (1) the effects of ethanol and AA on KC and HSC were as follows: Cell proliferation, lipid peroxidation, H(2)O(2), O(2).(-), nicotinamide adenine dinucleotide phosphate reduced form (NADPH) oxidase activity, and tumor necrosis factor alpha (TNF-alpha) were higher in KC(ethanol) than in KC(control), and were enhanced by AA; HSC(ethanol) proliferated faster, increased collagen, and showed higher GSH than HSC(control), with modest effects by AA. (2) AA effects on the control co-culture: We previously reported the ability of KC to induce a pro-fibrogenic response in HSC via reactive oxygen species (ROS)-dependent mechanisms; we now show that AA further increases cell proliferation and collagen in the control co-culture. The latter was prevented by vitamin E (an antioxidant) and by diphenyleneiodonium (a NADPH oxidase inhibitor). (3) Ethanol effects on the co-cultures: Co-culture with KC(control) or KC(ethanol) induced HSC(control) and HSC(ethanol) proliferation; however, the pro-fibrogenic response in HSC(ethanol) was suppressed because of up-regulation of TNF-alpha and GSH, which was prevented by a TNF-alpha neutralizing antibody (Ab) and by L-buthionine-sulfoximine, a GSH-depleting agent. (4) Ethanol plus AA effects on the co-cultures: AA lowered TNF-alpha in the HSC(control) co-cultures, allowing for enhanced collagen deposition; furthermore, AA restored the pro-fibrogenic response in the HSC(ethanol) co-cultures by counteracting the up-regulation of TNF-alpha and GSH with a significant increase in GSSG and in pro-fibrogenic transforming growth factor beta (TGF-beta). These results unveil synergism between ethanol and AA to the mechanism whereby KC mediate ECM

  7. Functional blockade of α5β1 integrin induces scattering and genomic landscape remodeling of hepatic progenitor cells

    PubMed Central

    2010-01-01

    Background Cell scattering is a physiological process executed by stem and progenitor cells during embryonic liver development and postnatal organ regeneration. Here, we investigated the genomic events occurring during this process induced by functional blockade of α5β1 integrin in liver progenitor cells. Results Cells treated with a specific antibody against α5β1 integrin exhibited cell spreading and scattering, over-expression of liver stem/progenitor cell markers and activation of the ERK1/2 and p38 MAPKs signaling cascades, in a similar manner to the process triggered by HGF/SF1 stimulation. Gene expression profiling revealed marked transcriptional changes of genes involved in cell adhesion and migration, as well as genes encoding chromatin remodeling factors. These responses were accompanied by conspicuous spatial reorganization of centromeres, while integrin genes conserved their spatial positioning in the interphase nucleus. Conclusion Collectively, our results demonstrate that α5β1 integrin functional blockade induces cell migration of hepatic progenitor cells, and that this involves a dramatic remodeling of the nuclear landscape. PMID:20958983

  8. Candida-elicited murine Th17 cells express high Ctla-4 compared with Th1 cells and are resistant to costimulation blockade.

    PubMed

    Krummey, Scott M; Floyd, Tamara L; Liu, Danya; Wagener, Maylene E; Song, Mingqing; Ford, Mandy L

    2014-03-01

    Effector and memory T cells may cross-react with allogeneic Ags to mediate graft rejection. Whereas the costimulation properties of Th1 cells are well studied, relatively little is known about the costimulation requirements of microbe-elicited Th17 cells. The costimulation blocker CTLA-4 Ig has been ineffective in the treatment of several Th17-driven autoimmune diseases and is associated with severe acute rejection following renal transplantation, leading us to investigate whether Th17 cells play a role in CD28/CTLA-4 blockade-resistant alloreactivity. We established an Ag-specific model in which Th1 and Th17 cells were elicited via Mycobacterium tuberculosis and Candida albicans immunization, respectively. C. albicans immunization elicited a higher frequency of Th17 cells and conferred resistance to costimulation blockade following transplantation. Compared with the M. tuberculosis group, C. albicans-elicited Th17 cells contained a higher frequency of IL-17(+)IFN-γ(+) producers and a lower frequency of IL-10(+) and IL-10(+)IL-17(+) cells. Importantly, Th17 cells differentially regulated the CD28/CTLA-4 pathway, expressing similarly high CD28 but significantly greater amounts of CTLA-4 compared with Th1 cells. Ex vivo blockade experiments demonstrated that Th17 cells are more sensitive to CTLA-4 coinhibition and therefore less susceptible to CTLA-4 Ig. These novel insights into the differential regulation of CTLA-4 coinhibition on CD4(+) T cells have implications for the immunomodulation of pathologic T cell responses during transplantation and autoimmunity.

  9. Local blockade of epithelial PDL-1 in the airways enhances T cell function and viral clearance during influenza virus infection.

    PubMed

    McNally, Beth; Ye, Fang; Willette, Meredith; Flaño, Emilio

    2013-12-01

    In order to maintain the gas exchange function of the lung following influenza virus infection, a delicate orchestration of positive and negative regulatory pathways must be maintained to attain viral eradication while minimizing local inflammation. The programmed death receptor 1 ligand/programmed death receptor 1 (PDL-1/PD-1) pathway plays an important immunoregulatory role, particularly in the context of T cell function. Here, we have shown that influenza virus infection of primary airway epithelial cells strongly enhances PDL-1 expression and does so in an alpha interferon receptor (IFNAR) signaling-dependent manner. PD-1 is expressed primarily on effector T cells in the lung, compared to effector memory and central memory cells, and shortly after influenza virus infection, an increased number of PD-1(+) T cells are recruited to the airways. Using in vitro cocultures of airway epithelial cells and T cells and in vivo models of influenza virus infection, we have demonstrated that blockade of airway epithelial PDL-1 improves CD8 T cell function, defined by increased production of gamma interferon (IFN-γ) and granzyme B and expression of CD107ab. Furthermore, PDL-1 blockade in the airways served to accelerate influenza virus clearance and enhance infection recovery. Our findings suggest that local manipulation of the PDL-1/PD-1 axis in the airways may represent a therapeutic alternative during acute influenza virus infection.

  10. Brief report: Blockade of Notch signaling in muscle stem cells causes muscular dystrophic phenotype and impaired muscle regeneration.

    PubMed

    Lin, Shuibin; Shen, Huangxuan; Jin, Baofeng; Gu, Yumei; Chen, Zirong; Cao, Chunxia; Hu, Chengbin; Keller, Charles; Pear, Warren S; Wu, Lizi

    2013-04-01

    Muscular dystrophies are a group of devastating diseases characterized by progressive muscle weakness and degeneration, with etiologies including muscle gene mutations and regenerative defects of muscle stem cells. Notch signaling is critical for skeletal myogenesis and has important roles in maintaining the muscle stem cell pool and preventing premature muscle differentiation. To investigate the functional impact of Notch signaling blockade in muscle stem cells, we developed a conditional knock-in mouse model in which endogenous Notch signaling is specifically blocked in muscle stem cell compartment. Mice with Notch signaling inhibition in muscle stem cells showed several muscular dystrophic features and impaired muscle regeneration. Analyses of satellite cells and isolated primary myoblasts revealed that Notch signaling blockade in muscle stem cells caused reduced activation and proliferation of satellite cells but enhanced differentiation of myoblasts. Our data thus indicate that Notch signaling controls processes that are critical to regeneration in muscular dystrophy, suggesting that Notch inhibitor therapies could have potential side effects on muscle functions. Copyright © 2013 AlphaMed Press.

  11. Blockade of pathological retinal ganglion cell hyperactivity improves optogenetically evoked light responses in rd1 mice

    PubMed Central

    Barrett, John M.; Degenaar, Patrick; Sernagor, Evelyne

    2015-01-01

    Retinitis pigmentosa (RP) is a progressive retinal dystrophy that causes visual impairment and eventual blindness. Retinal prostheses are the best currently available vision-restoring treatment for RP, but only restore crude vision. One possible contributing factor to the poor quality of vision achieved with prosthetic devices is the pathological retinal ganglion cell (RGC) hyperactivity that occurs in photoreceptor dystrophic disorders. Gap junction blockade with meclofenamic acid (MFA) was recently shown to diminish RGC hyperactivity and improve the signal-to-noise ratio (SNR) of RGC responses to light flashes and electrical stimulation in the rd10 mouse model of RP. We sought to extend these results to spatiotemporally patterned optogenetic stimulation in the faster-degenerating rd1 model and compare the effectiveness of a number of drugs known to disrupt rd1 hyperactivity. We crossed rd1 mice with a transgenic mouse line expressing the light-sensitive cation channel channelrhodopsin2 (ChR2) in RGCs, allowing them to be stimulated directly using high-intensity blue light. We used 60-channel ITO multielectrode arrays to record ChR2-mediated RGC responses from wholemount, ex-vivo retinas to full-field and patterned stimuli before and after application of MFA, 18-β-glycyrrhetinic acid (18BGA, another gap junction blocker) or flupirtine (Flu, a Kv7 potassium channel opener). All three drugs decreased spontaneous RGC firing, but 18BGA and Flu also decreased the sensitivity of RGCs to optogenetic stimulation. Nevertheless, all three drugs improved the SNR of ChR2-mediated responses. MFA also made it easier to discern motion direction of a moving bar from RGC population responses. Our results support the hypothesis that reduction of pathological RGC spontaneous activity characteristic in retinal degenerative disorders may improve the quality of visual responses in retinal prostheses and they provide insights into how best to achieve this for optogenetic prostheses

  12. Receptor for advanced glycation end product blockade enhances the chemotherapeutic effect of cisplatin in tongue squamous cell carcinoma by reducing autophagy and modulating the Wnt pathway.

    PubMed

    Zhao, Ziming; Wang, Hongyu; Zhang, Liao; Mei, Xifan; Hu, Jing; Huang, Keqiang

    2017-02-01

    Tongue squamous cell carcinoma (TSCC) is one of the most severe types of cancer with poor outcomes. Cisplatin is used widely to treat cancer cells, but many patients develop acquired drug resistance. The receptor for advanced glycation end products (RAGE) is expressed widely in TSCC and associated with drug-induced chemotherapy resistance. However, the effect of RAGE and cisplatin on Tca-8113 cells remains unknown. We assayed the combined use of RAGE blockade and cisplatin effect on Tca-8113 cells' viability by MTT and apoptosis rate of Tca-8113 cells on RAGE blockade+cisplatin treatment; cisplatin alone; or RAGE blockade alone by flow cytometry. We observed the expressions of autophagy-related proteins beclin1, LC3II, p62; Wnt signaling-related proteins β-catenin, GSK3β, WNT5A, ROR-2; and apoptosis-related protein cleaved caspase-3, bcl-2-associated X proteins using western blot. We determined WNT5A and beclin1 expression on Tca-8113 cells by immunofluorescence. We further observed autophagy vacuoles by monodansylcadaverine staining. We found that RAGE blockade and cisplatin significantly decreased cell viability and increased the cell apoptosis rate compared with cisplatin alone. Furthermore, RAGE blockade suppressed the canonical Wnt pathway proteins β-catenin and GSK-3β, but upregulated noncanonical WNT5A and receptor ROR-2. We show that RAGE blockade suppressed the levels of autophagy-related protein LC3II/I, beclin1, accelerated degradation of autophagy for the increasing p62 expression, and increased cell apoptosis for the increasing expressions of cleaved caspase-3 and bcl-2-associated X proteins. We observed the location of WNT5A and beclin1 expressions on cells by immunofluorescence and their trends were consistent with western blotting. Taken together, our findings suggested that RAGE blockade+cisplatin improved chemotherapeutic effects by reducing autophagy and regulating Wnt/β-catenin to suppress the progression of TSCC.

  13. Blockade of CD47 ameliorates autoimmune inflammation in CNS by suppressing IL-1-triggered infiltration of pathogenic Th17 cells.

    PubMed

    Gao, Qiangguo; Zhang, Yi; Han, Chaofeng; Hu, Xiang; Zhang, Hua; Xu, Xiongfei; Tian, Jun; Liu, Yiqi; Ding, Yuanyuan; Liu, Juan; Wang, Chunmei; Guo, Zhenhong; Yang, Yongguang; Cao, Xuetao

    2016-05-01

    The migration of Th17 cells into central nervous system (CNS) tissue is the key pathogenic step in experimental autoimmune encephalomyelitis (EAE) model. However, the mechanism underlying the pathogenic Th17 cell migration remains elusive. Here we report that blockade of CD47 with CD47-Fc fusion protein is effective in preventing and curing EAE by impairing infiltration of Th17 cells into CNS. However, CD47 deficiency does not directly impair the migration of Th17 cells. Mechanistic studies showed that CD47 deficiency inhibited degradation of inducible nitric oxide synthase (iNOS) in proteasome of macrophages by Src activation and led to the increased nitric oxide (NO) production. Then NO suppressed inflammasome activation-induced IL-1β production. This lower IL-1β reduces the expression of IL-1R1 and migration-related chemokine receptors on CD47(-/-) Th17 cells, inhibiting the ability of Th17 cells to infiltrate into the CNS of CD47(-/-) mice and therefore suppressing EAE development. In vivo administration of exogenous IL-1β indeed promoted the infiltration CD47(-/-) Th17 cells into CNS and antagonized the protective role of CD47 deficiency in EAE pathogenesis. Our results demonstrate a potential preventive and therapeutic application of CD47 blockade in controlling EAE development.

  14. PD-1 blockade for relapsed lymphoma post-allogeneic hematopoietic cell transplant: high response rate but frequent GVHD.

    PubMed

    Haverkos, Bradley M; Abbott, Diana; Hamadani, Mehdi; Armand, Philippe; Flowers, Mary E; Merryman, Reid; Kamdar, Manali; Kanate, Abraham Sebastian; Saad, Ayman; Mehta, Amitkumar; Ganguly, Siddhartha; Fenske, Timothy S; Hari, Parameswaran; Lowsky, Robert; Andritsos, Leslie; Jagasia, Madan; Bashey, Asad; Brown, Stacey; Bachanova, Veronika; Stephens, Deborah; Mineishi, Shin; Nakamura, Ryotaro; Chen, Yi-Bin; Blazar, Bruce R; Gutman, Jonathan; Devine, Steven M

    2017-07-13

    Given the limited treatment options for relapsed lymphoma post-allogeneic hematopoietic cell transplantation (post-allo-HCT) and the success of programmed death 1 (PD-1) blockade in classical Hodgkin lymphoma (cHL) patients, anti-PD-1 monoclonal antibodies (mAbs) are increasingly being used off-label after allo-HCT. To characterize the safety and efficacy of PD-1 blockade in this setting, we conducted a multicenter retrospective analysis of 31 lymphoma patients receiving anti-PD-1 mAbs for relapse post-allo-HCT. Twenty-nine (94%) patients had cHL and 27 had ≥1 salvage therapy post-allo-HCT and prior to anti-PD-1 treatment. Median follow-up was 428 days (range, 133-833) after the first dose of anti-PD-1. Overall response rate was 77% (15 complete responses and 8 partial responses) in 30 evaluable patients. At last follow-up, 11 of 31 patients progressed and 21 of 31 (68%) remain alive, with 8 (26%) deaths related to new-onset graft-versus-host disease (GVHD) after anti-PD-1. Seventeen (55%) patients developed treatment-emergent GVHD after initiation of anti-PD-1 (6 acute, 4 overlap, and 7 chronic), with onset after a median of 1, 2, and 2 doses, respectively. GVHD severity was grade III-IV acute or severe chronic in 9 patients. Only 2 of these 17 patients achieved complete response to GVHD treatment, and 14 of 17 required ≥2 systemic therapies. In conclusion, PD-1 blockade in relapsed cHL allo-HCT patients appears to be highly efficacious but frequently complicated by rapid onset of severe and treatment-refractory GVHD. PD-1 blockade post-allo-HCT should be studied further but cannot be recommended for routine use outside of a clinical trial. © 2017 by The American Society of Hematology.

  15. Combination of Id2 Knockdown Whole Tumor Cells and Checkpoint Blockade: A Potent Vaccine Strategy in a Mouse Neuroblastoma Model.

    PubMed

    Chakrabarti, Lina; Morgan, Clifford; Sandler, Anthony D

    2015-01-01

    Tumor vaccines have held much promise, but to date have demonstrated little clinical success. This lack of success is conceivably due to poor tumor antigen presentation combined with immuno-suppressive mechanisms exploited by the tumor itself. Knock down of Inhibitor of differentiation protein 2 (Id2-kd) in mouse neuroblastoma whole tumor cells rendered these cells immunogenic. Id2-kd neuroblastoma (Neuro2a) cells (Id2-kd N2a) failed to grow in most immune competent mice and these mice subsequently developed immunity against further wild-type Neuro2a tumor cell challenge. Id2-kd N2a cells grew aggressively in immune-compromised hosts, thereby establishing the immunogenicity of these cells. Therapeutic vaccination with Id2-kd N2a cells alone suppressed tumor growth even in established neuroblastoma tumors and when used in combination with immune checkpoint blockade eradicated large established tumors. Mechanistically, immune cell depletion studies demonstrated that while CD8+ T cells are critical for antitumor immunity, CD4+ T cells are also required to induce a sustained long-lasting helper effect. An increase in number of CD8+ T-cells and enhanced production of interferon gamma (IFNγ) was observed in tumor antigen stimulated splenocytes of vaccinated mice. More importantly, a massive influx of cytotoxic CD8+ T-cells infiltrated the shrinking tumor following combined immunotherapy. These findings show that down regulation of Id2 induced tumor cell immunity and in combination with checkpoint blockade produced a novel, potent, T-cell mediated tumor vaccine strategy.

  16. Immunosuppressive Myeloid Cells' Blockade in the Glioma Microenvironment Enhances the Efficacy of Immune-Stimulatory Gene Therapy.

    PubMed

    Kamran, Neha; Kadiyala, Padma; Saxena, Meghna; Candolfi, Marianela; Li, Youping; Moreno-Ayala, Mariela A; Raja, Nicholas; Shah, Diana; Lowenstein, Pedro R; Castro, Maria G

    2017-01-04

    Survival of glioma (GBM) patients treated with the current standard of care remains dismal. Immunotherapeutic approaches that harness the cytotoxic and memory potential of the host immune system have shown great benefit in other cancers. GBMs have developed multiple strategies, including the accumulation of myeloid-derived suppressor cells (MDSCs) to induce immunosuppression. It is therefore imperative to develop multipronged approaches when aiming to generate a robust anti-tumor immune response. Herein, we tested whether combining MDSC depletion or checkpoint blockade would augment the efficacy of immune-stimulatory herpes simplex type-I thymidine kinase (TK) plus Fms-like tyrosine kinase ligand (Flt3L)-mediated immune stimulatory gene therapy. Our results show that MDSCs constitute >40% of the tumor-infiltrating immune cells. These cells express IL-4Rα, inducible nitric oxide synthase (iNOS), arginase, programmed death ligand 1 (PDL1), and CD80, molecules that are critically involved in antigen-specific T cell suppression. Depletion of MDSCs strongly enhanced the TK/Flt3L gene therapy-induced tumor-specific CD8 T cell response, which lead to increased median survival and percentage of long-term survivors. Also, combining PDL1 or CTLA-4 immune checkpoint blockade greatly improved the efficacy of TK/Flt3L gene therapy. Our results, therefore, indicate that blocking MDSC-mediated immunosuppression holds great promise for increasing the efficacy of gene therapy-mediated immunotherapies for GBM.

  17. PD-1 Blockade Can Restore Functions of T-Cells in Epstein-Barr Virus-Positive Diffuse Large B-Cell Lymphoma In Vitro.

    PubMed

    Quan, Lina; Chen, Xue; Liu, Aichun; Zhang, Yan; Guo, Xiuchen; Yan, Shujie; Liu, Yue

    2015-01-01

    Epstein-Barr virus-positive diffuse large B-cell lymphoma (EBV+DLBCL) is an aggressive malignancy that is largely resistant to current therapeutic regimens, and is an attractive target for immune-based therapies. Anti-programmed death-1 (PD-1) antibodies showed encouraging anti-tumor effects in both preclinical models and advanced solid and hematological malignancies, but its efficacy against EBV+DLBCL is unknown. Herein, we performed experiments using co-culture system with T cells and lymphoma cell lines including EBV+DLBCL and EBV-DLBCL [including germinal center B-cell like (GCB)-DLBCL and non-GCB-DLBCL] in vitro. We show that lymphoma cells augmented the expression of PD-1 on T cells, decreased the proliferation of T cells, and altered the secretion of multiple cytokines. However, through PD-1 blockade, these functions could be largely restored. Notbaly, the effect of PD-1 blockade on antitumor immunity was more effective in EBV+DLBCL than that in EBV-DLBCL in vitro. These results suggest that T-cell exhaustion and immune escape in microenvironment is one of the mechanisms underlying DLBCL; and PD-1 blockade could present as a efficacious immunotherapeutic treatment for EBV+DLBCL.

  18. PD-1 Blockade Can Restore Functions of T-Cells in Epstein-Barr Virus-Positive Diffuse Large B-Cell Lymphoma In Vitro

    PubMed Central

    Quan, Lina; Chen, Xue; Liu, Aichun; Zhang, Yan; Guo, Xiuchen; Yan, Shujie; Liu, Yue

    2015-01-01

    Epstein–Barr virus-positive diffuse large B-cell lymphoma (EBV+DLBCL) is an aggressive malignancy that is largely resistant to current therapeutic regimens, and is an attractive target for immune-based therapies. Anti-programmed death-1 (PD-1) antibodies showed encouraging anti-tumor effects in both preclinical models and advanced solid and hematological malignancies, but its efficacy against EBV+DLBCL is unknown. Herein, we performed experiments using co-culture system with T cells and lymphoma cell lines including EBV+DLBCL and EBV-DLBCL [including germinal center B-cell like (GCB)-DLBCL and non-GCB-DLBCL] in vitro. We show that lymphoma cells augmented the expression of PD-1 on T cells, decreased the proliferation of T cells, and altered the secretion of multiple cytokines. However, through PD-1 blockade, these functions could be largely restored. Notbaly, the effect of PD-1 blockade on antitumor immunity was more effective in EBV+DLBCL than that in EBV-DLBCL in vitro. These results suggest that T-cell exhaustion and immune escape in microenvironment is one of the mechanisms underlying DLBCL; and PD-1 blockade could present as a efficacious immunotherapeutic treatment for EBV+DLBCL. PMID:26361042

  19. Activation and Two Modes of Blockade by Strontium of Ca2+-activated K+ Channels in Goldfish Saccular Hair Cells

    PubMed Central

    Sugihara, Izumi

    1998-01-01

    Effects of internal Sr2+ on the activity of large-conductance Ca2+-activated K+ channels were studied in inside-out membrane patches from goldfish saccular hair cells. Sr2+ was approximately one-fourth as potent as Ca2+ in activating these channels. Although the Hill coefficient for Sr2+ was smaller than that for Ca2+, maximum open-state probability, voltage dependence, steady state gating kinetics, and time courses of activation and deactivation of the channel were very similar under the presence of equipotent concentrations of Ca2+ and Sr2+. This suggests that voltage-dependent activation is partially independent of the ligand. Internal Sr2+ at higher concentrations (>100 μM) produced fast and slow blockade both concentration and voltage dependently. The reduction in single-channel amplitude (fast blockade) could be fitted with a modified Woodhull equation that incorporated the Hill coefficient. The dissociation constant at 0 mV, the Hill coefficient, and zd (a product of the charge of the blocking ion and the fraction of the voltage difference at the binding site from the inside) in this equation were 58–209 mM, 0.69–0.75, 0.45–0.51, respectively (n = 4). Long shut events (slow blockade) produced by Sr2+ lasted ∼10–200 ms and could be fitted with single-exponential curves (time constant, τl−s) in shut-time histograms. Durations of burst events, periods intercalated by long shut events, could also be fitted with single exponentials (time constant, τb). A significant decrease in τb and no large changes in τl−s were observed with increased Sr2+ concentration and voltage. These findings on slow blockade could be approximated by a model in which single Sr2+ ions bind to a blocking site within the channel pore beyond the energy barrier from the inside, as proposed for Ba2+ blockade. The dissociation constant at 0 mV and zd in the Woodhull equation for this model were 36–150 mM and 1–1.8, respectively (n = 3). PMID:9450948

  20. Dual blockade of HER2 in HER2-overexpressing tumor cells does not completely eliminate HER3 function

    PubMed Central

    Garrett, Joan T.; Sutton, Cammie R.; Kuba, María Gabriela; Cook, Rebecca S .; Arteaga, Carlos L.

    2012-01-01

    Purpose Dual blockade of HER2 with trastuzumab with lapatinib or with pertuzumab is a superior treatment approach compared to single agent HER2 inhibitors. However, many HER2-overexpressing breast cancers still escape from this combinatorial approach. Inhibition of HER2 and downstream phosphatidylinositol-3 kinase (PI3K)/AKT causes a transcriptional and post-translational upregulation of HER3 which, in turn, counteracts the antitumor action of the HER2-directed therapies. We hypothesized that suppression of HER3 would synergize with dual blockade of HER2 in breast cancer cells sensitive and refractory to HER2 antagonists. Experimental Design Inhibition of HER2/HER3 in HER2+ breast cancer cell lines was evaluated by western blot. We analyzed drug-induced apoptosis and 2- and 3-dimensional growth in vitro. Growth inhibition of PI3K was examined in vivo in xenografts treated with combinations of trastuzumab, lapatinib, and the HER3 neutralizing monoclonal antibody U3-1287. Results Treatment with U3-1287 blocked the upregulation of total and phosphorylated HER3 that followed treatment with lapatinib and trastuzumab and, in turn, enhanced the anti-tumor action of the combination against trastuzumab-sensitive and -resistant cells. Mice bearing HER2+ xenografts treated with lapatinib, trastuzumab, and U3-1287 exhibited fewer recurrences and better survival compared to mice treated with lapatinib and trastuzumab. Conclusions Dual blockade of HER2 with trastuzumab and lapatinib does not eliminate the compensatory upregulation of HER3. Therapeutic inhibitors of HER3 should be considered as part of multi-drug combinations aimed at completely and rapidly disabling the HER2 network in HER2-overexpressing breast cancers. PMID:23224399

  1. Human Dendritic Cells Mitigate NK-Cell Dysfunction Mediated by Nonselective JAK1/2 Blockade.

    PubMed

    Curran, Shane A; Shyer, Justin A; St Angelo, Erin T; Talbot, Lillian R; Sharma, Sneh; Chung, David J; Heller, Glenn; Hsu, Katharine C; Betts, Brian C; Young, James W

    2017-01-01

    Janus kinase (JAK) inhibitors have achieved positive responses in myeloproliferative neoplasms, but at the expense of decreased natural killer (NK) cell numbers and compromised function. Selective JAK2 inhibition may also have a role in preventing and treating graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. Although JAK inhibitors can impair monocyte-derived dendritic cell (moDC) activation and function and suppress effector T-cell responses, the effects on NK cells and the relevant mechanisms remain undefined. Using common γc cytokines and distinct human dendritic cell (DC) subtypes, we compared the effects of a JAK2-specific (TG101348) with a less selective JAK1/2 (ruxolitinib) inhibitor on NK-cell activation and function. Ruxolitinib treatment completely blocked IL2, IL15, and DC-mediated STAT5 phosphorylation, along with the capacity of NK cells to secrete IFNγ or lyse NK cell-sensitive targets. Only NK-cell proliferation stimulated by moDCs resisted ruxolitinib treatment. In contrast, TG101348 treatment of stimulated NK cells resulted in far less functional compromise. TG101348 completely inhibited only soluble IL15-mediated STAT5 phosphorylation, which Langerhans-type DCs (LCs), presenting membrane-bound IL15 in trans, could salvage. These results demonstrate that ruxolitinib's nonselective inhibition of JAK1/2 results in profound NK-cell dysfunction by blocking downstream pSTAT5, hence providing a persuasive rationale for the development of selective JAK2 inhibitors for immunotherapeutic applications. Cancer Immunol Res; 5(1); 52-60. ©2016 AACR.

  2. CDK4/6 or MAPK blockade enhances efficacy of EGFR inhibition in oesophageal squamous cell carcinoma

    PubMed Central

    Zhou, Jin; Wu, Zhong; Wong, Gabrielle; Pectasides, Eirini; Nagaraja, Ankur; Stachler, Matthew; Zhang, Haikuo; Chen, Ting; Zhang, Haisheng; Liu, Jie Bin; Xu, Xinsen; Sicinska, Ewa; Sanchez-Vega, Francisco; Rustgi, Anil K.; Diehl, J. Alan; Wong, Kwok-Kin; Bass, Adam J.

    2017-01-01

    Oesophageal squamous cell carcinoma is a deadly disease where systemic therapy has relied upon empiric chemotherapy despite the presence of genomic alterations pointing to candidate therapeutic targets, including recurrent amplification of the gene encoding receptor tyrosine kinase epidermal growth factor receptor (EGFR). Here, we demonstrate that EGFR-targeting small-molecule inhibitors have efficacy in EGFR-amplified oesophageal squamous cell carcinoma (ESCC), but may become quickly ineffective. Resistance can occur following the emergence of epithelial–mesenchymal transition and by reactivation of the mitogen-activated protein kinase (MAPK) pathway following EGFR blockade. We demonstrate that blockade of this rebound activation with MEK (mitogen-activated protein kinase kinase) inhibition enhances EGFR inhibitor-induced apoptosis and cell cycle arrest, and delays resistance to EGFR monotherapy. Furthermore, genomic profiling shows that cell cycle regulators are altered in the majority of EGFR-amplified tumours and a combination of cyclin-dependent kinase 4/6 (CDK4/6) and EGFR inhibitors prevents the emergence of resistance in vitro and in vivo. These data suggest that upfront combination strategies targeting EGFR amplification, guided by adaptive pathway reactivation or by co-occurring genomic alterations, should be tested clinically. PMID:28059068

  3. Blockade of DNA methylation enhances the therapeutic effect of gefitinib in non-small cell lung cancer cells.

    PubMed

    Li, Xiao-You; Wu, Jian-Zhong; Cao, Hai-Xia; Ma, Rong; Wu, Jian-Qiu; Zhong, Yue-Jiao; Feng, Ji-Feng

    2013-05-01

    The sensitivity of lung cancer to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) has been found to be associated with mutations in the tyrosine kinase domain of EGFR. However, not all mutations are sensitive to gefitinib. While CpG island methylation in the promoter region of the EGFR gene and transcriptional silencing are common in solid tumors, the role of the EGFR gene promoter methylation in affecting resistance to TKIs in non-small cell lung cancer (NSCLC) remains unknown. In this study, we examined the correlation between EGFR gene promoter methylation and the therapeutic effect of gefitinib in NSCLC cells. Three NSCLC cell lines with different EGFR mutation statuses and levels of sensitivity to EGFR-TKIs were used in this study: H1650 (del E746-A750), H1299 (wild-type EGFR) and PC-9 (del E746-A750). Cells were treated with gefitinib or 5-aza-2'-deoxy cytidine (5-aza-CdR), a methylation inhibitor, alone or in combination. Subsequently, the methylation status of the EGFR gene promoter was examined by methylation-specific PCR (MSP). Cell survival and apoptosis assays were performed using the Cell Counting Kit-8 (CCK-8) and flow cytometry. In addition, western blot analysis and quantitative real-time PCR were used to examine the expression levels of EGFR protein and mRNA. Our study showed that the promoter region of the EGFR gene in PC-9 cells was unmethylated, and that the cells were sensitive to gefitinib. By contrast, the promoter region of the EGFR gene in the H1650 and H1299 cells was methylated, and the cells were resistant to gefitinib. Of note, the combination treatment with 5-aza-CdR and gefitinib further enhanced the growth inhibitory effects and led to the induction of apoptosis, while a significant reduction in the expression of EGFR protein and mRNA was observed in the H1650 and H1299 cells. These results suggest that blockade of DNA methylation may enhance the antitumor effects of EGFR-TKIs and gefitinib in NSCLC cells

  4. Human Prostate Tumor Antigen-Specific CD8+ Regulatory T Cells are Inhibited by CTLA-4 or IL-35 Blockade

    PubMed Central

    Olson, Brian M.; Jankowska-Gan, Ewa; Becker, Jordan T.; Vignali, Dario A.A.; Burlingham, William J.; McNeel, Douglas G.

    2012-01-01

    Regulatory T cells play important roles in cancer development and progression by limiting the generation of innate and adaptive anti-tumor immunity. We hypothesized that in addition to natural CD4+CD25+ Tregs and myeloid-derived suppressor cells, tumor antigen-specific regulatory T cells interfere with the detection of anti-tumor immunity following immunotherapy. Using samples from prostate cancer patients immunized with a DNA vaccine encoding prostatic acid phosphatase (PAP) and a trans-vivo delayed type hypersensitivity (tvDTH) assay, we found that the detection of PAP-specific effector responses following immunization was prevented by the activity of PAP-specific regulatory cells. These regulatory cells were CD8+CTLA-4+, and their suppression was relieved by blockade of CTLA-4, but not IL-10 or TGF-β. Moreover, antigen-specific CD8+ regulatory T cells were detected prior to immunization in the absence of PAP-specific effector responses. These PAP-specific CD8+CTLA-4+ suppressor T cells expressed IL-35, which was decreased following blockade of CTLA-4, and inhibition of either CTLA-4 or IL-35 reversed PAP-specific suppression of tvDTH response. PAP-specific CD8+CTLA-4+ T cells also suppressed T-cell proliferation in an IL-35-dependent, contact-independent fashion. Taken together, these findings suggest a novel population of CD8+CTLA-4+ IL-35-secreting tumor antigen-specific regulatory T cells arise spontaneously in some prostate cancer patients, persist during immunization, and can prevent the detection of antigen-specific effector responses by an IL-35-dependent mechanism. PMID:23152566

  5. Vaccination with Dendritic Cell Myeloma Fusions in Conjunction With Stem Cell Transplantation and PD1 Blockade

    DTIC Science & Technology

    2010-05-01

    glycol. Mean expression of PDL- 1 was 97% on DCs (n=5, Figure 1), and 45% on patient derived myeloma cells (n=6, Figure 1). In addition, mean... expression of PDL-1 was 90% on DC/ myeloma fusions (n=2, Figure 2), suggesting this pathway may provide an inhibitory signal that blunts fusion mediated...immunologic response. Expression of PD-1 on T cells isolated from patients with multiple myeloma : In infectious disease models, upregulation of T

  6. Resistance of human glioblastoma multiforme cells to growth factor inhibitors is overcome by blockade of inhibitor of apoptosis proteins

    PubMed Central

    Ziegler, David S.; Wright, Renee D.; Kesari, Santosh; Lemieux, Madeleine E.; Tran, Mary A.; Jain, Monish; Zawel, Leigh; Kung, Andrew L.

    2008-01-01

    Multiple receptor tyrosine kinases (RTKs), including PDGFR, have been validated as therapeutic targets in glioblastoma multiforme (GBM), yet inhibitors of RTKs have had limited clinical success. As various antiapoptotic mechanisms render GBM cells resistant to chemo- and radiotherapy, we hypothesized that these antiapoptotic mechanisms also confer resistance to RTK inhibition. We found that in vitro inhibition of PDGFR in human GBM cells initiated the intrinsic pathway of apoptosis, as evidenced by mitochondrial outer membrane permeabilization, but downstream caspase activation was blocked by inhibitor of apoptosis proteins (IAPs). Consistent with this, inhibition of PDGFR combined with small molecule inactivation of IAPs induced apoptosis in human GBM cells in vitro and had synergistic antitumor effects in orthotopic mouse models of GBM and in primary human GBM neurospheres. These results demonstrate that concomitant inhibition of IAPs can overcome resistance to RTK inhibitors in human malignant GBM cells, and suggest that blockade of IAPs has the potential to improve treatment outcomes in patients with GBM. PMID:18677408

  7. [Effect of blockade of NF-kappaB signaling pathway on cell apoptosis in cutaneous squamous cell carcinoma].

    PubMed

    Xia, Yong-hua; Liu, Dong; Zhang, Cai-feng; Fu, Dan-dan; Li, Min; Li, Zhan-guo; Tian, Zhong-wei

    2011-04-18

    To evaluate the siRNA-mediated inhibitory effect of nuclear factor-kappaB (NF-kappaB) p65 on expression of p65, and explore the effect of blockade of NF-kappaB signal pathway on cell apoptosis in cutaneous squamous cell carcinoma (cutaneous SCC). Cutaneous SCC cell line SCL-1 cells were transfected with 50 nmol/L p65 siRNA. The expression level of p65 mRNA was measured using RT-PCR method at 0, 24, 48 and 72 h . Expressions of p65, bcl-2 and bax proteins were determined using Western blotting. Activities of caspase-3/9 was detected by Caspase-Glo®-3/7, 8 and 9 kit. Finally, cell apoptosis was detected using flow cytometry. The expression level of p65 mRNA in Cutaneous SCC SCL-1 cells was obviously down-regulated 48 h after transfection with p65 siRNA, and a significant difference was detected, as compared with 0 h after (0.23 ± 0.10 vs. 0.66 ± 0.05, P<0.05). The protein levels of p65 and bcl-2 decreased, and the bax protein level and activities of caspase-3/9 increased after transfection with p65 siRNA at h 48 . Further, the results of flow cytometry demonstrated that p65 siRNA could induce apoptosis of SCL-1 cells, and cell apoptosis ratio (20.28% ± 1.87%) in p65 siRNA group was significantly higher than that in the untreated group and control siRNA group (9.13% ± 1.51% and 9.37% ± 1.38%, respectively, F=47.532, P<0.01). p65 siRNA can block NF-kappaB signal pathway, down-regulate expression of bcl-2, elevate the bax level and increase the activities of caspase-3/9, suggesting that NF-kappaB signal pathway may be a key molecular target for therapy of cutaneous SCC.

  8. Monoclonal antibody that inhibits infection of HeLa and rhabdomyosarcoma cells by selected enteroviruses through receptor blockade

    SciTech Connect

    Crowell, R.L.; Field, A.K.; Schleif, W.A.; Long, W.L.; Colonno, R.J.; Mapoles, J.E.; Emini, E. A.

    1986-02-01

    BALB/c mice were immunized with HeLa cells, and their spleen cells were fused with myeloma cells to produce hybridomas. Initial screening of culture fluids from 800 fusion products in a cell protection assay against coxsackievirus B3 (CB3) and the CB3-RD virus variant yielded five presumptive monoclonal antibodies with three specificities: (i) protection against CB3 on HeLa, (ii) protection against CB3-RD on rhabdomyosarcoma (RD) cells, and (iii) protection against both viruses on the respective cells. Only one of the monoclonal antibodies (with dual specificity) survived two subclonings and was studied in detail. The antibody was determined to have an immunoglobulin G2a isotype and protected cells by blockade of cellular receptors, since attachment of (/sup 35/S)methionine-labeled CB3 was inhibited by greater than 90%. The monoclonal antibody protected HeLa cells against infection by CB1, CB3, CB5, echovirus 6, and coxsackievirus A21 and RD cells against CB1-RD, CB3-RD, and CB5-Rd virus variants. The monoclonal antibody did not protect either cell type against 16 other immunotypes of picornaviruses. The monoclonal antibody produced only positive fluorescence on those cells which were protected against infection, and /sup 125/I-labeled antibody confirmed the specific binding to HeLa and RD cells. The results suggest that this monoclonal antibody possesses some of the receptor specificity of the group B coxsackieviruses.

  9. Predictive Efficacy Biomarkers of Programmed Cell Death 1/Programmed Cell Death 1 Ligand Blockade Therapy.

    PubMed

    Fang, Xiao-Na; Fu, Li-Wu

    2016-01-01

    Inhibitors of immune check-point molecule, programmed cell death 1 (PD-1) and its ligand, programmed cell death ligand 1 (PD-L1) have attracted much attention in cancer immunotherapy recently due to their durable antitumor effects in various malignances, especially the advanced ones. Unfortunately, only a fraction of patients with advanced tumors could benefit from anti-PD-1/PD-L1 therapy, while others still worsened. The key to this point is that there are no efficient biomarkers for screening anti-PD-1/PD-L1-sensitive patients. In this review, we aim at summarizing the latest advances of anti-PD-1/PDL1 immunotherapy and the potential predictive efficacy biomarkers to provide evidences for identifying anti-PD-1/PDL1- sensitive patients. The present article also includes the patent review coverage on this topic.

  10. NK cells and CD8+ T cells cooperate to improve therapeutic responses in melanoma treated with interleukin-2 (IL-2) and CTLA-4 blockade.

    PubMed

    Kohlhapp, Frederick J; Broucek, Joseph R; Hughes, Tasha; Huelsmann, Erica J; Lusciks, Jevgenijs; Zayas, Janet P; Dolubizno, Hubert; Fleetwood, Vidyaratna A; Grin, Alisa; Hill, Graham E; Poshepny, Joseph L; Nabatiyan, Arman; Ruby, Carl E; Snook, Joshua D; Rudra, Jai S; Schenkel, Jason M; Masopust, David; Zloza, Andrew; Kaufman, Howard L

    2015-01-01

    Melanoma is one of the few types of cancer with an increasing annual incidence. While a number of immunotherapies for melanoma have been associated with significant clinical benefit, including high-dose IL-2 and cytotoxic T lymphocyte antigen 4 (CTLA-4) blockade, clinical response to either of these single agents has been limited to 11-20% of treated patients. Therefore, in this study, we sought to test the hypothesis that the combination of IL-2 and CTLA-4 blockade could mediate a more profound therapeutic response. Here, B6 mice were challenged with poorly immunogenic B16 melanoma on day 0, and treated with CTLA-4 blocking antibody (100 μg/mouse) on days 3, 6, and 9, and IL-2 (100,000 units) twice daily on days 4-8, or both. A highly significant synergistic effect that delayed tumor growth and prolonged survival was demonstrated with the combination immunotherapy compared to either monotherapy alone. The therapeutic effect of combination immunotherapy was dependent on both CD8+ T and NK cells and co-depletion of these subsets (but not either one alone) abrogated the therapeutic effect. CTLA-4 blockade increased immune cell infiltration (including CD8+ T cells and NK cells) in the tumor and IL-2 reduced the proportion of highly differentiated/exhausted tumor-infiltrating NK cells. These results have implications for the design of clinical trials in patients with metastatic melanoma and provide new insights into how the immune system may be mediating anti-tumor activity with combination IL-2 and CTLA-4 blockade in melanoma.

  11. Blockade of programmed death-1/programmed death ligand pathway enhances the antitumor immunity of human invariant natural killer T cells.

    PubMed

    Kamata, Toshiko; Suzuki, Akane; Mise, Naoko; Ihara, Fumie; Takami, Mariko; Makita, Yuji; Horinaka, Atsushi; Harada, Kazuaki; Kunii, Naoki; Yoshida, Shigetoshi; Yoshino, Ichiro; Nakayama, Toshinori; Motohashi, Shinichiro

    2016-12-01

    The role of invariant natural killer T (iNKT) cells in antitumor immunity has been studied extensively, and clinical trials in patients with advanced cancer have revealed a prolonged survival in some cases. In recent years, humanized blocking antibodies against co-stimulatory molecules such as PD-1 have been developed. The enhancement of T cell function is reported to improve antitumor immunity, leading to positive clinical effects. However, there are limited data on the role of PD-1/programmed death ligand (PDL) molecules in human iNKT cells. In this study, we investigated the interaction between PD-1 on iNKT cells and PDL on antigen-presenting cells (APCs) in the context of iNKT cell stimulation. The blockade of PDL1 at the time of stimulation resulted in increased release of helper T cell (Th) 1 cytokines from iNKT cells, leading to the activation of NK cells. The direct antitumor function of iNKT cells was also enhanced after stimulation with anti-PDL1 antibody-treated APCs. According to these results, we conclude that the co-administration of anti-PDL1 antibody and alpha-galactosylceramide (αGalCer)-pulsed APCs enhances iNKT cell-mediated antitumor immunity.

  12. GD2-specific CAR T Cells Undergo Potent Activation and Deletion Following Antigen Encounter but can be Protected From Activation-induced Cell Death by PD-1 Blockade

    PubMed Central

    Gargett, Tessa; Yu, Wenbo; Dotti, Gianpietro; Yvon, Eric S; Christo, Susan N; Hayball, John D; Lewis, Ian D; Brenner, Malcolm K; Brown, Michael P

    2016-01-01

    Chimeric antigen receptor (CAR) T cells have shown great promise in the treatment of hematologic malignancies but more variable results in the treatment of solid tumors and the persistence and expansion of CAR T cells within patients has been identified as a key correlate of antitumor efficacy. Lack of immunological “space”, functional exhaustion, and deletion have all been proposed as mechanisms that hamper CAR T-cell persistence. Here we describe the events following activation of third-generation CAR T cells specific for GD2. CAR T cells had highly potent immediate effector functions without evidence of functional exhaustion in vitro, although reduced cytokine production reversible by PD-1 blockade was observed after longer-term culture. Significant activation-induced cell death (AICD) of CAR T cells was observed after repeated antigen stimulation, and PD-1 blockade enhanced both CAR T-cell survival and promoted killing of PD-L1+ tumor cell lines. Finally, we assessed CAR T-cell persistence in patients enrolled in the CARPETS phase 1 clinical trial of GD2-specific CAR T cells in the treatment of metastatic melanoma. Together, these data suggest that deletion also occurs in vivo and that PD-1-targeted combination therapy approaches may be useful to augment CAR T-cell efficacy and persistence in patients. PMID:27019998

  13. GD2-specific CAR T Cells Undergo Potent Activation and Deletion Following Antigen Encounter but can be Protected From Activation-induced Cell Death by PD-1 Blockade.

    PubMed

    Gargett, Tessa; Yu, Wenbo; Dotti, Gianpietro; Yvon, Eric S; Christo, Susan N; Hayball, John D; Lewis, Ian D; Brenner, Malcolm K; Brown, Michael P

    2016-06-01

    Chimeric antigen receptor (CAR) T cells have shown great promise in the treatment of hematologic malignancies but more variable results in the treatment of solid tumors and the persistence and expansion of CAR T cells within patients has been identified as a key correlate of antitumor efficacy. Lack of immunological "space", functional exhaustion, and deletion have all been proposed as mechanisms that hamper CAR T-cell persistence. Here we describe the events following activation of third-generation CAR T cells specific for GD2. CAR T cells had highly potent immediate effector functions without evidence of functional exhaustion in vitro, although reduced cytokine production reversible by PD-1 blockade was observed after longer-term culture. Significant activation-induced cell death (AICD) of CAR T cells was observed after repeated antigen stimulation, and PD-1 blockade enhanced both CAR T-cell survival and promoted killing of PD-L1(+) tumor cell lines. Finally, we assessed CAR T-cell persistence in patients enrolled in the CARPETS phase 1 clinical trial of GD2-specific CAR T cells in the treatment of metastatic melanoma. Together, these data suggest that deletion also occurs in vivo and that PD-1-targeted combination therapy approaches may be useful to augment CAR T-cell efficacy and persistence in patients.

  14. Suppression of experimental myasthenia gravis, a B cell-mediated autoimmune disease, by blockade of IL-18.

    PubMed

    Im, S H; Barchan, D; Maiti, P K; Raveh, L; Souroujon, M C; Fuchs, S

    2001-10-01

    Interleukin-18 (IL-18) is a pleiotropic proinflammatory cytokine that plays an important role in interferon gamma (IFN-gamma) production and IL-12-driven Th1 phenotype polarization. Increased expression of IL-18 has been observed in several autoimmune diseases. In this study we have analyzed the role of IL-18 in an antibody-mediated autoimmune disease and elucidated the mechanisms involved in disease suppression mediated by blockade of IL-18, using experimental autoimmune myasthenia gravis (EAMG) as a model. EAMG is a T cell-regulated, antibody-mediated autoimmune disease in which the nicotinic acetylcholine receptor (AChR) is the major autoantigen. Th1- and Th2-type responses are both implicated in EAMG development. We show that treatment by anti-IL-18 during ongoing EAMG suppresses disease progression. The protective effect can be adoptively transferred to naive recipients and is mediated by increased levels of the immunosuppressive Th3-type cytokine TGF-beta and decreased AChR-specific Th1-type cellular responses. Suppression of EAMG is accompanied by down-regulation of the costimulatory factor CD40L and up-regulation of CTLA-4, a key negative immunomodulator. Our results suggest that IL-18 blockade may potentially be applied for immunointervention in myasthenia gravis.

  15. Enhanced T-cell immunity to osteosarcoma through antibody blockade of PD-1/PD-L1 interactions.

    PubMed

    Lussier, Danielle M; O'Neill, Lauren; Nieves, Lizbeth M; McAfee, Megan S; Holechek, Susan A; Collins, Andrea W; Dickman, Paul; Jacobsen, Jeffrey; Hingorani, Pooja; Blattman, Joseph N

    2015-04-01

    Osteosarcoma is the most common bone cancer in children and adolescents. Although 70% of patients with localized disease are cured with chemotherapy and surgical resection, patients with metastatic osteosarcoma are typically refractory to treatment. Numerous lines of evidence suggest that cytotoxic T lymphocytes (CTLs) limit the development of metastatic osteosarcoma. We have investigated the role of PD-1, an inhibitory TNFR family protein expressed on CTLs, in limiting the efficacy of immune-mediated control of metastatic osteosarcoma. We show that human metastatic, but not primary, osteosarcoma tumors express a ligand for PD-1 (PD-L1) and that tumor-infiltrating CTLs express PD-1, suggesting this pathway may limit CTLs control of metastatic osteosarcoma in patients. PD-L1 is also expressed on the K7M2 osteosarcoma tumor cell line that establishes metastases in mice, and PD-1 is expressed on tumor-infiltrating CTLs during disease progression. Blockade of PD-1/PD-L1 interactions dramatically improves the function of osteosarcoma-reactive CTLs in vitro and in vivo, and results in decreased tumor burden and increased survival in the K7M2 mouse model of metastatic osteosarcoma. Our results suggest that blockade of PD-1/PD-L1 interactions in patients with metastatic osteosarcoma should be pursued as a therapeutic strategy.

  16. Eradication of metastatic mouse cancers resistant to immune checkpoint blockade by suppression of myeloid-derived cells.

    PubMed

    Kim, KiBem; Skora, Andrew D; Li, Zhaobo; Liu, Qiang; Tam, Ada J; Blosser, Richard L; Diaz, Luis A; Papadopoulos, Nickolas; Kinzler, Kenneth W; Vogelstein, Bert; Zhou, Shibin

    2014-08-12

    Impressive responses have been observed in patients treated with checkpoint inhibitory anti-programmed cell death-1 (PD-1) or anti-cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) antibodies. However, immunotherapy against poorly immunogenic cancers remains a challenge. Here we report that treatment with both anti-PD-1 and anti-CTLA-4 antibodies was unable to eradicate large, modestly immunogenic CT26 tumors or metastatic 4T1 tumors. Cotreatment with epigenetic-modulating drugs and checkpoint inhibitors markedly improved treatment outcomes, curing more than 80% of the tumor-bearing mice. Functional studies revealed that the primary targets of the epigenetic modulators were myeloid-derived suppressor cells (MDSCs). A PI3K inhibitor that reduced circulating MDSCs also eradicated 4T1 tumors in 80% of the mice when combined with immune checkpoint inhibitors. Thus, cancers resistant to immune checkpoint blockade can be cured by eliminating MDSCs.

  17. Blockade of CD26-mediated T cell costimulation with soluble caveolin-1-Ig fusion protein induces anergy in CD4{sup +}T cells

    SciTech Connect

    Ohnuma, Kei; Uchiyama, Masahiko; Hatano, Ryo; Takasawa, Wataru; Endo, Yuko; Dang, Nam H.; Morimoto, Chikao

    2009-08-21

    CD26 binds to caveolin-1 in antigen-presenting cells (APC), and that ligation of CD26 by caveolin-1 induces T cell proliferation in a TCR/CD3-dependent manner. We report herein the effects of CD26-caveolin-1 costimulatory blockade by fusion protein caveolin-1-Ig (Cav-Ig). Soluble Cav-Ig inhibits T cell proliferation and cytokine production in response to recall antigen, or allogeneic APC. Our data hence suggest that blocking of CD26-associated signaling by soluble Cav-Ig may be an effective approach as immunosuppressive therapy.

  18. The antimalarial amodiaquine causes autophagic-lysosomal and proliferative blockade sensitizing human melanoma cells to starvation- and chemotherapy-induced cell death.

    PubMed

    Qiao, Shuxi; Tao, Shasha; Rojo de la Vega, Montserrat; Park, Sophia L; Vonderfecht, Amanda A; Jacobs, Suesan L; Zhang, Donna D; Wondrak, Georg T

    2013-12-01

    Pharmacological inhibition of autophagic-lysosomal function has recently emerged as a promising strategy for chemotherapeutic intervention targeting cancer cells. Repurposing approved and abandoned non-oncological drugs is an alternative approach to the identification and development of anticancer therapeutics, and antimalarials that target autophagic-lysosomal functions have recently attracted considerable attention as candidates for oncological repurposing. Since cumulative research suggests that dependence on autophagy represents a specific vulnerability of malignant melanoma cells, we screened a focused compound library of antimalarials for antimelanoma activity. Here we report for the first time that amodiaquine (AQ), a clinical 4-aminoquinoline antimalarial with unexplored cancer-directed chemotherapeutic potential, causes autophagic-lysosomal and proliferative blockade in melanoma cells that surpasses that of its parent compound chloroquine. Monitoring an established set of protein markers (LAMP1, LC3-II, SQSTM1) and cell ultrastructural changes detected by electron microscopy, we observed that AQ treatment caused autophagic-lysosomal blockade in malignant A375 melanoma cells, a finding substantiated by detection of rapid inactivation of lysosomal cathepsins (CTSB, CTSL, CTSD). AQ-treatment was associated with early induction of energy crisis (ATP depletion) and sensitized melanoma cells to either starvation- or chemotherapeutic agent-induced cell death. AQ displayed potent antiproliferative effects, and gene expression array analysis revealed changes at the mRNA (CDKN1A, E2F1) and protein level (TP53, CDKN1A, CCND1, phospho-RB1 [Ser 780]/[Ser 807/811], E2F1) consistent with the observed proliferative blockade in S-phase. Taken together, our data suggest that the clinical antimalarial AQ is a promising candidate for repurposing efforts that aim at targeting autophagic-lysosomal function and proliferative control in malignant melanoma cells.

  19. Synergistic mobilization of hemopoietic progenitor cells using concurrent beta1 and beta2 integrin blockade or beta2-deficient mice.

    PubMed

    Papayannopoulou, T; Priestley, G V; Nakamoto, B; Zafiropoulos, V; Scott, L M; Harlan, J M

    2001-03-01

    The hierarchy of cytoadhesion molecules involved in hematopoietic/stem progenitor cell mobilization has not yet been delineated. Previous studies have suggested an important role for alpha4beta1 integrin in this process. To test whether mobilization involves dynamic interactions of alpha4beta1 with other integrins on hematopoietic cells, especially the beta2 integrins, mice and primates were treated with anti-beta1 or anti-beta2 antibodies alone or in combination. A single injection of anti-alpha4beta1 antibody elicited reproducible mobilization in contrast to other antibodies, and 3 injections yielded higher mobilization efficiency than each of the other antibodies. When the anti-beta2 (anti-CD11a or anti-CD18) or anti-alpha5/beta1 integrin antibody was combined with anti-alpha4, an augmentation in mobilization was seen that was either additive or synergistic, depending on the potency of the antibody used. Synergy between anti-alpha4 and anti-CD18 (beta(2)) antibody blockade was seen in primates and confirmed in anti-alpha4-treated CD18-deficient mice. In the latter, there was a 49-fold increase in mobilization with anti-alpha4, much higher than in littermate control animals, in CD18 hypomorphic mice, or in other strains of mice tested. Data from both the antibody blockade and gene-targeted mice suggest that the cooperativity of alpha4beta1 with beta2 integrins becomes evident when they are concurrently inhibited. It is unclear whether this cooperativity is exerted at the stage of reversible adhesion versus migration, and enhancement of and whether the 2 integrins work in a sequential or parallel manner. Whatever the mechanism, the data provide a novel example of beta1 and beta2 integrin crosstalk in stem/progenitor cell mobilization.

  20. Effects of epidermal growth factor receptor blockade on ependymoma stem cells in vitro and in orthotopic mouse models.

    PubMed

    Servidei, Tiziana; Meco, Daniela; Trivieri, Nadia; Patriarca, Valentina; Vellone, Valerio Gaetano; Zannoni, Gian Franco; Lamorte, Giuseppe; Pallini, Roberto; Riccardi, Riccardo

    2012-09-01

    Some lines of evidence suggest that tumors, including ependymoma, might arise from a subpopulation of cells, termed cancer stem cells (CSCs), with self-renewal and tumor-initiation properties. Given the strict dependence of CSCs on epidermal growth factor (EGF) through EGF receptor (EGFR), we investigated the effects of EGFR inhibitors in ependymoma-stem cells (SCs) in vitro and in orthotopic mouse models. We established two ependymoma-SC lines from two recurrent pediatric ependymoma. Both lines expressed markers of radial glia--the candidate SCs of ependymoma--and showed renewal ability, multipotency, and tumorigenicity after orthotopic implantation, despite markedly different expression of CD133 (94 vs. 6%). High phosphorylated-EGFR/EGFR ratio was detected, which decreased after differentiation. EGFR inhibitors (gefitinib and AEE788) reduced clonogenicity, proliferation and survival of ependymoma-SC lines dose-dependently, and blocked EGF-induced activation of EGFR, Akt and extracellular signal-regulated kinase 1/2. Overall, AEE788 was more effective than gefitinib. EGFR blockade as well as differentiation strongly reduced CD133 expression. However, ex vivo treatment with AEE788 did not impair orthotopic tumor engraftment, whereas ex vivo differentiation did, suggesting that CD133 does not absolutely segregate for tumorigenicity in ependymoma-SCs. Orally administered AEE788 prolonged survival of mice bearing ependymoma-SC-driven orthotopic xenografts from 56 to 63 days, close to statistical significance (log-rank p=0.06). Our study describes for the first time EGFR signaling in ependymoma-SCs and the effects of EGFR blockade in complementary in vitro and in vivo systems. The experimental models we developed can be used to further investigate the activity of EGFR inhibitors or other antineoplastic agents in this tumor.

  1. Blockade of Hedgehog Signaling Synergistically Increases Sensitivity to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Non-Small-Cell Lung Cancer Cell Lines

    PubMed Central

    Bai, Xiao-Yan; Zhang, Xu-Chao; Yang, Su-Qing; An, She-Juan; Chen, Zhi-Hong; Su, Jian; Xie, Zhi; Gou, Lan-Ying; Wu, Yi-Long

    2016-01-01

    Aberrant activation of the hedgehog (Hh) signaling pathway has been implicated in the epithelial-to-mesenchymal transition (EMT) and cancer stem-like cell (CSC) maintenance; both processes can result in tumor progression and treatment resistance in several types of human cancer. Hh cooperates with the epidermal growth factor receptor (EGFR) signaling pathway in embryogenesis. We found that the Hh signaling pathway was silenced in EGFR-TKI-sensitive non-small-cell lung cancer (NSCLC) cells, while it was inappropriately activated in EGFR-TKI-resistant NSCLC cells, accompanied by EMT induction and ABCG2 overexpression. Upregulation of Hh signaling through extrinsic SHH exposure downregulated E-cadherin expression and elevated Snail and ABCG2 expression, resulting in gefitinib tolerance (P < 0.001) in EGFR-TKI-sensitive cells. Blockade of the Hh signaling pathway using the SMO antagonist SANT-1 restored E-cadherin expression and downregulate Snail and ABCG2 in EGFR-TKI-resistant cells. A combination of SANT-1 and gefitinib markedly inhibited tumorigenesis and proliferation in EGFR-TKI-resistant cells (P < 0.001). These findings indicate that hyperactivity of Hh signaling resulted in EGFR-TKI resistance, by EMT introduction and ABCG2 upregulation, and blockade of Hh signaling synergistically increased sensitivity to EGFR-TKIs in primary and secondary resistant NSCLC cells. E-cadherin expression may be a potential biomarker of the suitability of the combined application of an Hh inhibitor and EGFR-TKIs in EGFR-TKI-resistant NSCLCs. PMID:26943330

  2. Reversion of immune tolerance in advanced malignancy: modulation of myeloid-derived suppressor cell development by blockade of stem-cell factor function.

    PubMed

    Pan, Ping-Ying; Wang, George X; Yin, Bingjiao; Ozao, Junko; Ku, Teresa; Divino, Celia M; Chen, Shu-Hsia

    2008-01-01

    Tumor growth induced a significant increase of myeloid-derived suppressor cells (MDSCs) in the tumor-bearing host. In our previous study, we showed that MDSCs induced tumor-specific T-cell tolerance and the development of T regulatory cells (Tregs). Tumor-derived factors have been implicated in the accumulation of MDSCs. We hypothesize that reduction of MDSC accumulation in tumor-bearing hosts, through the blockade of tumor factors, can prevent T-cell anergy and Treg development and thereby improve immune therapy for the treatment of advanced tumors. Several tumor-derived factors were identified by gene array analysis. Among the candidate factors, stem- cell factor (SCF) is expressed by various human and murine carcinomas and was selected for further study. Mice bearing tumor cells with SCF siRNA knockdown exhibited significantly reduced MDSC expansion and restored proliferative responses of tumor-infiltrating T cells. More importantly, blockade of SCF receptor (ckit)-SCF interaction by anti-ckit prevented tumor-specific T-cell anergy, Treg development, and tumor angiogenesis. Furthermore, the prevention of MDSC accumulation in conjunction with immune activation therapy showed synergistic therapeutic effect when treating mice bearing large tumors. This information supports the notion that modulation of MDSC development may be required to achieve effective immune-enhancing therapy for the treatment of advanced tumors.

  3. Blockade of Fas signaling in breast cancer cells suppresses tumor growth and metastasis via disruption of Fas signaling-initiated cancer-related inflammation.

    PubMed

    Liu, Qiuyan; Tan, Qinchun; Zheng, Yuanyuan; Chen, Kun; Qian, Cheng; Li, Nan; Wang, Qingqing; Cao, Xuetao

    2014-04-18

    Mechanisms for cancer-related inflammation remain to be fully elucidated. Non-apoptotic functions of Fas signaling have been proposed to play an important role in promoting tumor progression. It has yet to be determined if targeting Fas signaling can control tumor progression through suppression of cancer-related inflammation. In the current study we found that breast cancer cells with constitutive Fas expression were resistant to apoptosis induction by agonistic anti-Fas antibody (Jo2) ligation or Fas ligand cross-linking. Higher expression of Fas in human breast cancer tissue has been significantly correlated with poorer prognosis in breast cancer patients. To determine whether blockade of Fas signaling in breast cancer could suppress tumor progression, we prepared an orthotopic xenograft mouse model with mammary cancer cells 4T1 and found that blockade of Fas signaling in 4T1 cancer cells markedly reduced tumor growth, inhibited tumor metastasis in vivo, and prolonged survival of tumor-bearing mice. Mechanistically, blockade of Fas signaling in cancer cells significantly decreased systemic or local recruitment of myeloid derived suppressor cells (MDSCs) in vivo. Furthermore, blockade of Fas signaling markedly reduced IL-6, prostaglandin E2 production from breast cancer cells by impairing p-p38, and activity of the NFκB pathway. In addition, administration of a COX-2 inhibitor and anti-IL-6 antibody significantly reduced MDSC accumulation in vivo. Therefore, blockade of Fas signaling can suppress breast cancer progression by inhibiting proinflammatory cytokine production and MDSC accumulation, indicating that Fas signaling-initiated cancer-related inflammation in breast cancer cells may be a potential target for treatment of breast cancer.

  4. Influence of Blockade of beta-Adrenoreceptors and Acute Stress on Antibody Formation, Delayed Type of Hypersensitivity, Phagocytic Cell Activity in Local Immune Response.

    PubMed

    Shilov, Juri I.; Gein, Sergei V.; Chereshnev, Valery A.

    2001-10-01

    In the experiments on male Wistar rats a study was made on the effect of acute 6-h immobilization stress on antibody formation, delayed type hypersensitivity (DTH), functional activity of phagocytes in the case of a local form of immune response to sheep red blood cells at the background of beta-adrenergic receptor blockade. It was established that immobilization stress resulted in substantial inhibition of the expressibility of immune inflammation in the case of DTH, cancellation of an increase of the phagocytic activity of macrophages of the regional lymph node and the level of antibodies. The blockade of the beta-adrenergic receptors with propranolol antagonized with these effects of stress. After termination of the immobilization, activation of neutrophil phagocytosis was detected, this being related to an increase of neutrophil immigration from the bone marrow. Eosinophilic phagocytosis at the early period of stress was inhibited, the blockade of beta-adrenergic receptors canceled this effect.

  5. Blockade of vascular endothelial growth factor receptors by tivozanib has potential anti-tumour effects on human glioblastoma cells

    PubMed Central

    Momeny, Majid; Moghaddaskho, Farima; Gortany, Narges K.; Yousefi, Hassan; Sabourinejad, Zahra; Zarrinrad, Ghazaleh; Mirshahvaladi, Shahab; Eyvani, Haniyeh; Barghi, Farinaz; Ahmadinia, Leila; Ghazi-Khansari, Mahmoud; Dehpour, Ahmad R.; Amanpour, Saeid; Tavangar, Seyyed M.; Dardaei, Leila; Emami, Amir H.; Alimoghaddam, Kamran; Ghavamzadeh, Ardeshir; Ghaffari, Seyed H.

    2017-01-01

    Glioblastoma (GBM) remains one of the most fatal human malignancies due to its high angiogenic and infiltrative capacities. Even with optimal therapy including surgery, radiotherapy and temozolomide, it is essentially incurable. GBM is among the most neovascularised neoplasms and its malignant progression associates with striking neovascularisation, evidenced by vasoproliferation and endothelial cell hyperplasia. Targeting the pro-angiogenic pathways is therefore a promising anti-glioma strategy. Here we show that tivozanib, a pan-inhibitor of vascular endothelial growth factor (VEGF) receptors, inhibited proliferation of GBM cells through a G2/M cell cycle arrest via inhibition of polo-like kinase 1 (PLK1) signalling pathway and down-modulation of Aurora kinases A and B, cyclin B1 and CDC25C. Moreover, tivozanib decreased adhesive potential of these cells through reduction of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Tivozanib diminished GBM cell invasion through impairing the proteolytic cascade of cathepsin B/urokinase-type plasminogen activator (uPA)/matrix metalloproteinase-2 (MMP-2). Combination of tivozanib with EGFR small molecule inhibitor gefitinib synergistically increased sensitivity to gefitinib. Altogether, these findings suggest that VEGFR blockade by tivozanib has potential anti-glioma effects in vitro. Further in vivo studies are warranted to explore the anti-tumour activity of tivozanib in combinatorial approaches in GBM. PMID:28287096

  6. Gefitinib induces lung cancer cell autophagy and apoptosis via blockade of the PI3K/AKT/mTOR pathway

    PubMed Central

    ZHAO, ZHONG-QUAN; YU, ZHONG-YANG; LI, JIE; OUYANG, XUE-NONG

    2016-01-01

    Gefitinib is a selective inhibitor of the tyrosine kinase epidermal growth factor receptor, which inhibits tumor pathogenesis, metastasis and angiogenesis, as well as promoting apoptosis. Therefore, gefitinib presents an effective drug for the targeted therapy of lung cancer. However, the underlying mechanisms by which gefitinib induces lung cancer cell death remain unclear. To investigate the effects of gefitinib on lung cancer cells and the mechanism of such, the present study analyzed the effect of gefitinib on the autophagy, apoptosis and proliferation of the A549 and A549-gefitinib-resistant (GR) cell lines GR. The regulation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/ mammalian target of rapamycin (mTOR) pathway was also investigated. Acridine orange staining revealed that gefitinib induced autophagy of A549 cells but not A549-GR cells. In addition, gefitinib promoted apoptosis and inhibited proliferation of A549 cells but not A549-GR cells. Furthermore, western blot analysis demonstrated that gefitinib treatment led to the downregulation of PI3K, AKT, pAKT, mTOR and phosphorylated-mTOR protein expression in A549 cells but not A549-GR cells. LY294002 blocked the PI3K/AKT/mTOR pathway and induced autophagy and apoptosis of A549 cells, however, no synergistic effect was observed following combined treatment with gefitinib and LY294002. In conclusion, the results of the present study indicate that gefitinib promotes autophagy and apoptosis of lung cancer cells via blockade of the PI3K/AKT/mTOR pathway, which leads to lung cancer cell death. PMID:27347100

  7. Effects of the combined blockade of EGFR and ErbB-2 on signal transduction and regulation of cell cycle regulatory proteins in breast cancer cells.

    PubMed

    D'Alessio, Amelia; De Luca, Antonella; Maiello, Monica R; Lamura, Luana; Rachiglio, Anna Maria; Napolitano, Maria; Gallo, Marianna; Normanno, Nicola

    2010-09-01

    Treatment of breast cancer cells with a combination of the EGFR-tyrosine kinase inhibitor (EGFR-TKI) gefitinib and the anti-ErbB-2 monoclonal antibody trastuzumab results in a synergistic antitumor effect. In this study, we addressed the mechanisms involved in this phenomenon. The activation of signaling pathways and the expression of cell cycle regulatory proteins were studied in SK-Br-3 and BT-474 breast cancer cells, following treatment with EGFR and/or ErbB-2 inhibitors. Treatment with the gefitinib/trastuzumab combination produced, as compared with a single agent, a more prolonged blockade of AKT and MAPK activation, a more pronounced accumulation of cells in the G0/G1 phase of the cell cycle, a more significant increase in the levels of p27(kip1) and of hypophosphorylated pRb2, and a decrease in the levels of Cyclin D1 and survivin. Similar findings were observed with the EGFR/ErbB-2 inhibitor lapatinib. Gefitinib, trastuzumab, and their combination increased the stability of p27(kip1), with the combination showing the highest effects. Blockade of both receptors with gefitinib/trastuzumab or lapatinib induced a significant increase in the levels of p27(kip1) mRNA and in the nuclear levels of the p27(kip1) transcription factor FKHRL-1. Inhibition of PI3K signaling also produced a significant raise in p27(kip1) mRNA. Finally, down-modulation of FKHRL-1 with siRNAs prevented the lapatinib-induced increase of p27(kip1) mRNA. The synergism deriving from EGFR and ErbB-2 blockade is mediated by several different alterations in the activation of signaling proteins and in the expression of cell cycle regulatory proteins, including transcriptional and posttranscriptional regulation of p27(kip1) expression.

  8. ZLM-7 exhibits anti-angiogenic effects via impaired endothelial cell function and blockade of VEGF/VEGFR-2 signaling.

    PubMed

    Su, Min; Huang, Jingjia; Li, Jijia; Qin, Xiyuan; Tang, Xiaoning; Jin, Fang; Chen, Shali; Jiang, Chuanming; Zou, Zizheng; Peng, Kunjian; Nuruzzaman, Mohammed; Zhang, Jianting; Luo, Junli; Liu, Suyou; Luo, Zhiyong

    2016-04-05

    Inhibition of angiogenesis is a promising therapeutic strategy against cancer. In this study, we reported that ZLM-7, a combretastain A-4 (CA-4) derivative, exhibited anti-angiogenic activity in vitro and in vivo. In vitro, ZLM-7 induced microtubule cytoskeletal disassembly. It decreased VEGF-induced proliferation, migration, invasion and tube formation in endothelial cells, which are critical steps in angiogenesis. In vivo, ZLM-7 significantly inhibited neovascularization in a chicken chorioallantoic membrane (CAM) model and reduced the microvessel density in tumor tissues of MCF-7 xenograft mouse model. ZLM-7 also displayed comparable antiangiogenic and anti-tumor activities associated with the lead compound CA-4, but exhibited lower toxicity compared with CA-4. The anti-angiogenic effect of ZLM-7 was exerted via blockade of VEGF/VEGFR-2 signaling. ZLM-7 treatment suppressed the expression and secretion of VEGF in endothelial cells and MCF-7 cells under hypoxia. Further, ZLM-7 suppressed the VEGF-induced phosphorylation of VEGFR-2 and its downstream signaling mediators including activated AKT, MEK and ERK in endothelial cells. Overall, these results demonstrate that ZLM-7 exhibits anti-angiogenic activities by impairing endothelial cell function and blocking VEGF/VEGFR-2 signaling, suggesting that ZLM-7 might be a potential angiogenesis inhibitor.

  9. Blockade of Rho-associated protein kinase (ROCK) inhibits the contractility and invasion potential of cancer stem like cells

    PubMed Central

    Mohanty, Sagarajit; Das, Alakesh; Das, Sreya; Kumar, Sushant; Sen, Shamik; Purwar, Rahul

    2017-01-01

    Recent studies have implicated the roles of cancer stem like cells (CSCs) in cancer metastasis. However, very limited knowledge exists at the molecular and cellular level to target CSCs for prevention of cancer metastasis. In this study, we examined the roles of contractile dynamics of CSCs in cell invasion and delineated the underlying molecular mechanisms of their distinct cell invasion potential. Using de-adhesion assay and atomic force microscopy, we show that CSCs derived from melanoma and breast cancer cell lines exhibit increased contractility compared to non-CSCs across all tumor types. In addition, CSCs possess increased ECM remodeling capacity as quantified by collagen degradation assay. More importantly, pharmacological blockade of Rho-associated protein kinase completely abolished the contractility and collagen degradation capacity of both CSCs and non-CSCs. In conclusion, our study demonstrates the importance of cell contractility in regulating invasiveness of CSCs and suggests that pharmacological targeting of ROCK pathway represents a novel strategy for targeting both CSCs and bulk population for the treatment of cancer metastasis. PMID:28199964

  10. Blockade of PD-1 Signaling Enhances Th2 Cell Responses and Aggravates Liver Immunopathology in Mice with Schistosomiasis japonica

    PubMed Central

    Zhou, Sha; Jin, Xin; Li, Yalin; Li, Wei; Chen, Xiaojun; Xu, Lei; Zhu, Jifeng; Xu, Zhipeng; Zhang, Yang; Liu, Feng; Su, Chuan

    2016-01-01

    Background More than 220 million people worldwide are chronically infected with schistosomes, causing severe disease or even death. The major pathological damage occurring in schistosomiasis is attributable to the granulomatous inflammatory response and liver fibrosis induced by schistosome eggs. The inflammatory response is tightly controlled and parallels immunosuppressive regulation, constantly maintaining immune homeostasis and limiting excessive immunopathologic damage in important host organs. It is well known that the activation of programmed death 1 (PD-1) signaling causes a significant suppression of T cell function. However, the roles of PD-1 signaling in modulating CD4+ T cell responses and immunopathology during schistosome infection, have yet to be defined. Methodology/Principal Findings Here, we show that PD-1 is upregulated in CD4+ T cells in Schistosoma japonicum (S. japonicum)-infected patients. We also show the upregulation of PD-1 expression in CD4+ T cells in the spleens, mesenteric lymph nodes, and livers of mice with S. japonicum infection. Finally, we found that the blockade of PD-1 signaling enhanced CD4+ T helper 2 (Th2) cell responses and led to more severe liver immunopathology in mice with S. japonicum infection, without a reduction of egg production or deposition in the host liver. Conclusions/Significance Overall, our study suggests that PD-1 signaling is specifically induced to control Th2-associated inflammatory responses during schistosome infection and is beneficial to the development of PD-1-based control of liver immunopathology. PMID:27792733

  11. Blockade of Rho-associated protein kinase (ROCK) inhibits the contractility and invasion potential of cancer stem like cells.

    PubMed

    Srinivasan, Srisathya; Ashok, Vandhana; Mohanty, Sagarajit; Das, Alakesh; Das, Sreya; Kumar, Sushant; Sen, Shamik; Purwar, Rahul

    2017-02-10

    Recent studies have implicated the roles of cancer stem like cells (CSCs) in cancer metastasis. However, very limited knowledge exists at the molecular and cellular level to target CSCs for prevention of cancer metastasis. In this study, we examined the roles of contractile dynamics of CSCs in cell invasion and delineated the underlying molecular mechanisms of their distinct cell invasion potential. Using de-adhesion assay and atomic force microscopy, we show that CSCs derived from melanoma and breast cancer cell lines exhibit increased contractility compared to non-CSCs across all tumor types. In addition, CSCs possess increased ECM remodeling capacity as quantified by collagen degradation assay. More importantly, pharmacological blockade of Rho-associated protein kinase completely abolished the contractility and collagen degradation capacity of both CSCs and non-CSCs. In conclusion, our study demonstrates the importance of cell contractility in regulating invasiveness of CSCs and suggests that pharmacological targeting of ROCK pathway represents a novel strategy for targeting both CSCs and bulk population for the treatment of cancer metastasis.

  12. The Effect of Therapeutic Blockades of Dust Particles-Induced Ca2+ Signaling and Proinflammatory Cytokine IL-8 in Human Bronchial Epithelial Cells

    PubMed Central

    Yoon, Ju Hee; Jeong, Sung Hwan; Hong, Jeong Hee

    2015-01-01

    Bronchial epithelial cells are the first barrier of defense against respiratory pathogens. Dust particles as extracellular stimuli are associated with inflammatory reactions after inhalation. It has been reported that dust particles induce intracellular Ca2+ signal, which subsequently increases cytokines production such as interleukin- (IL-) 8. However, the study of therapeutic blockades of Ca2+ signaling induced by dust particles in human bronchial epithelial cells is poorly understood. We investigated how to modulate dust particles-induced Ca2+ signaling and proinflammatory cytokine IL-8 expression. Bronchial epithelial BEAS-2B cells were exposed to PM10 dust particles and subsequent mediated intracellular Ca2+ signaling and reactive oxygen species signal. Our results show that exposure to several inhibitors of Ca2+ pathway attenuated the PM10-induced Ca2+ response and subsequent IL-8 mRNA expression. PM10-mediated Ca2+ signal and IL-8 expression were attenuated by several pharmacological blockades such as antioxidants, IP3-PLC blockers, and TRPM2 inhibitors. Our results show that blockades of PLC or TRPM2 reduced both of PM10-mediated Ca2+ signal and IL-8 expression, suggesting that treatment with these blockades should be considered for potential therapeutic trials in pulmonary epithelium for inflammation caused by environmental events such as seasonal dust storm. PMID:26640326

  13. Increased Tim-3 expression in peripheral NK cells predicts a poorer prognosis and Tim-3 blockade improves NK cell-mediated cytotoxicity in human lung adenocarcinoma.

    PubMed

    Xu, Liyun; Huang, Yanyan; Tan, Linlin; Yu, Wei; Chen, Dongdong; Lu, ChangChang; He, Jianying; Wu, Guoqing; Liu, Xiaoguang; Zhang, Yongkui

    2015-12-01

    T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) has been shown to play an important role in mediating NK-cell function in human diseases. However, the relationship between Tim-3 expression in natural killer (NK) cells and human lung adenocarcinoma remains unclear. We therefore investigated the expression of Tim-3 in NK cells and explored the effect of Tim-3 blockade on NK cell-mediated activity in human lung adenocarcinoma. Upregulated expression of Tim-3 on CD3-CD56+ cells (P<0.05) and CD3-CD56(dim) cells (P<0.05) of patients with lung adenocarcinoma was detected by flow cytometry. Moreover, Tim-3 expression in CD3-CD56+ NK cells was higher in patients with lung adenocarcinoma with lymph node metastasis (LNM) (P<0.05) or with tumor stage T3-T4 (P<0.05). Tim-3 expression in CD56(dim) NK-cell subset was higher in patients with tumor size ≥3cm (P<0.05), or LNM (P<0.05) or with tumor stage T3-T4 (P<0.05). Further analysis showed that higher expressions of Tim-3 on both CD3-CD56+ NK cells and CD56(dim) NK-cell subset were independently correlated with shorter overall survival of patients with lung adenocarcinoma (log-rank test, P=0.0418, 0.0406, respectively). Importantly, blockade of Tim-3 signaling with anti-Tim-3 antibodies resulted in the increased cytotoxicity and IFN-γ production of peripheral NK cells from patients with lung adenocarcinoma. Our data indicate that Tim-3 expression in NK cells can function as a prognostic biomarker in human lung adenocarcinoma and support that Tim-3 could be a new target for an immunotherapeutic strategy.

  14. Loss of AXIN1 drives acquired resistance to WNT pathway blockade in colorectal cancer cells carrying RSPO3 fusions.

    PubMed

    Picco, Gabriele; Petti, Consalvo; Centonze, Alessia; Torchiaro, Erica; Crisafulli, Giovanni; Novara, Luca; Acquaviva, Andrea; Bardelli, Alberto; Medico, Enzo

    2017-03-01

    In colorectal cancer (CRC), WNT pathway activation by genetic rearrangements of RSPO3 is emerging as a promising target. However, its low prevalence severely limits availability of preclinical models for in-depth characterization. Using a pipeline designed to suppress stroma-derived signal, we find that RSPO3 "outlier" expression in CRC samples highlights translocation and fusion transcript expression. Outlier search in 151 CRC cell lines identified VACO6 and SNU1411 cells as carriers of, respectively, a canonical PTPRK(e1)-RSPO3(e2) fusion and a novel PTPRK(e13)-RSPO3(e2) fusion. Both lines displayed marked in vitro and in vivo sensitivity to WNT blockade by the porcupine inhibitor LGK974, associated with transcriptional and morphological evidence of WNT pathway suppression. Long-term treatment of VACO6 cells with LGK974 led to the emergence of a resistant population carrying two frameshift deletions of the WNT pathway inhibitor AXIN1, with consequent protein loss. Suppression of AXIN1 in parental VACO6 cells by RNA interference conferred marked resistance to LGK974. These results provide the first mechanism of secondary resistance to WNT pathway inhibition.

  15. Beta-adrenergic receptor blockade during exercise decreases intestinal lymphocyte apoptosis but not cell loss in mice.

    PubMed

    Marra, S; Hoffman-Goetz, L

    2004-07-01

    Catecholamines induce apoptosis in various lymphoid populations. This process can occur with both alpha- and beta-adrenoreceptors. Heavy exercise increases plasma catecholamine concentrations, and is also a cause of lymphocyte apoptosis, a possible explanation for postexercise lymphocytopenia. The purpose of this study was to examine the effects of adrenoreceptor antagonism on exercise-induced decreases and apoptosis of intestinal lymphocytes. Mice received an intraperitoneal injection of phentolamine (a nonselective alpha-blocker), nadolol (a nonselective beta-blocker), or saline (vehicle) prior to an exhaustive bout of exercise. Total intestinal lymphocyte numbers, percent and number of CD3+ lymphocytes, and cell viability were assessed. Neither alpha- nor beta-antagonism prevented exercise-induced cell loss in the intestine; however, pretreatment with nadolol significantly reduced the number of apoptotic and necrotic cells. Phentolamine administration appeared to increase the incidence of cell death among intestinal lymphocytes. Both drugs decreased the percentage of CD3+ intestinal lymphocytes. Our study suggests that catecholamines are not responsible for postexercise lymphocytopenia, but beta-adrenoceptor blockade may confer protection against exercise-induced apoptosis of intestinal lymphocytes.

  16. Implication of combined PD-L1/PD-1 blockade with cytokine-induced killer cells as a synergistic immunotherapy for gastrointestinal cancer

    PubMed Central

    Geng, Ruixuan; Ge, Xiaoxiao; Tang, Wenbo; Chang, Jinjia; Wu, Zheng; Liu, Xinyang; Lin, Ying; Zhang, Zhe; Li, Jin

    2016-01-01

    Cytokine-induced killer (CIK) cells represent a realistic approach in cancer immunotherapy with confirmed survival benefits in the context of metastatic solid tumors. However, therapeutic effects are limited to a fraction of patients. In this study, immune-resistance elements and ideal combination therapies were explored. Initially, phenotypic analysis was performed to document CD3, CD56, NKG2D, DNAM-1, PD-L1, PD-1, CTLA-4, TIM-3, 2B4, and LAG-3 on CIK cells. Upon engagement of CIK cells with the tumor cells, expression of PD-1 on CIK cells and PD-L1 on both cells were up-regulated. Over-expression of PD-L1 levels on tumor cells via lentiviral transduction inhibited tumoricidal activity of CIK cells, and neutralizing of PD-L1/PD-1 signaling axis could enhance their tumor-killing effect. Conversely, blockade of NKG2D, a major activating receptor of CIK cells, largely caused dysfunction of CIK cells. Functional study showed an increase of NKG2D levels along with PD-L1/PD-1 blockade in the presence of other immune effector molecule secretion. Additionally, combined therapy of CIK infusion and PD-L1/PD-1 blockade caused a delay of in vivo tumor growth and exhibited a survival advantage over untreated mice. These results provide a preclinical proof-of-concept for simultaneous PD-L1/PD-1 pathways blockade along with CIK infusion as a novel immunotherapy for unresectable cancers. PMID:26871284

  17. Effects of blockade of endogenous Gi signaling in Tie2-expressing cells on bone formation in a mouse model of heterotopic ossification.

    PubMed

    Wang, Liping; Carroll, Dylan O'; Liu, Xuhui; Roth, Theresa; Kim, Hubert; Halloran, Bernard; Nissenson, Robert A

    2015-08-01

    Available evidence indicates that some Tie2-expressing (Tie2(+) ) cells serve as multipotent progenitors that have robust BMP-dependent osteogenic activity and mediate heterotopic ossification (HO). Since signaling through the G protein Gi is required for cell motility, we hypothesized that blockade of endogenous Gi signaling in Tie2(+) cell populations would prevent HO formation. Blockade of Gi signaling in Tie2(+) cells was accomplished in transgenic mice with expression of pertussis toxin (PTX) under the control of the Tie2 promoter (Tie2(+) /PTX(+) ). Bone formation within HOs was evaluated 2 weeks after BMP injection. Expression of PTX in Tie2(+) cells significantly reduced the bone volume (BV) of HOs in male and female mice. Orthotopic bones were assessed at the distal femur and expression of PTX significantly increased trabecular bone fractional volume and bone formation rate in females only. In adult Tie2(+) /GFP(+) mice, GFP(+) cells appeared both inside and at the surfaces of bone tissue within HOs and in orthotopic bones. In summary, blockade of Gi signaling in Tie2(+) cells reduced the accrual of HOs and stimulated osteogenesis in orthotopic bones. Targeting of Gi protein coupled receptors in Tie2(+) cells may be a novel therapeutic strategy in states of abnormal bone formation such as osteoporosis and HO. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  18. Immune Toxicities Elicted by CTLA-4 Blockade in Cancer Patients Are Associated with Early Diversification of the T-cell Repertoire.

    PubMed

    Oh, David Y; Cham, Jason; Zhang, Li; Fong, Grant; Kwek, Serena S; Klinger, Mark; Faham, Malek; Fong, Lawrence

    2017-03-15

    While immune checkpoint blockade elicits efficacious responses in many patients with cancer, it also produces a diverse and unpredictable number of immune-related adverse events (IRAE). Mechanisms driving IRAEs are generally unknown. Because CTLA-4 blockade leads to proliferation of circulating T cells, we examined in this study whether ipilimumab treatment leads to clonal expansion of tissue-reactive T cells. Rather than narrowing the T-cell repertoire to a limited number of clones, ipilimumab induced greater diversification in the T-cell repertoire in IRAE patients compared with patients without IRAEs. Specifically, ipilimumab triggered increases in the numbers of clonotypes, including newly detected clones and a decline in overall T-cell clonality. Initial broadening in the repertoire occurred within 2 weeks of treatment, preceding IRAE onset. IRAE patients exhibited greater diversity of CD4(+) and CD8(+) T cells, but showed no differences in regulatory T-cell numbers relative to patients without IRAEs. Prostate-specific antigen responses to ipilimumab were also associated with increased T-cell diversity. Our results show how rapid diversification in the immune repertoire immediately after checkpoint blockade can be both detrimental and beneficial for patients with cancer. Cancer Res; 77(6); 1322-30. ©2016 AACR. ©2016 American Association for Cancer Research.

  19. Intratumoral injection of a CpG oligonucleotide reverts resistance to PD-1 blockade by expanding multifunctional CD8+ T cells.

    PubMed

    Wang, Shu; Campos, Jose; Gallotta, Marilena; Gong, Mei; Crain, Chad; Naik, Edwina; Coffman, Robert L; Guiducci, Cristiana

    2016-11-15

    Despite the impressive rates of clinical response to programmed death 1 (PD-1) blockade in multiple cancers, the majority of patients still fail to respond to this therapy. The CT26 tumor in mice showed similar heterogeneity, with most tumors unaffected by anti-PD-1. As in humans, response of CT26 to anti-PD-1 correlated with increased T- and B-cell infiltration and IFN expression. We show that intratumoral injection of a highly interferogenic TLR9 agonist, SD-101, in anti-PD-1 nonresponders led to a complete, durable rejection of essentially all injected tumors and a majority of uninjected, distant-site tumors. Therapeutic efficacy of the combination was also observed with the TSA mammary adenocarcinoma and MCA38 colon carcinoma tumor models that show little response to PD-1 blockade alone. Intratumoral SD-101 substantially increased leukocyte infiltration and IFN-regulated gene expression, and its activity was dependent on CD8(+) T cells and type I IFN signaling. Anti-PD-1 plus intratumoral SD-101 promoted infiltration of activated, proliferating CD8(+) T cells and led to a synergistic increase in total and tumor antigen-specific CD8(+) T cells expressing both IFN-γ and TNF-α. Additionally, PD-1 blockade could alter the CpG-mediated differentiation of tumor-specific CD8(+) T cells into CD127(low)KLRG1(high) short-lived effector cells, preferentially expanding the CD127(high)KLRG1(low) long-lived memory precursors. Tumor control and intratumoral T-cell proliferation in response to the combined treatment is independent of T-cell trafficking from secondary lymphoid organs. These findings suggest that a CpG oligonucleotide given intratumorally may increase the response of cancer patients to PD-1 blockade, increasing the quantity and the quality of tumor-specific CD8(+) T cells.

  20. Dicoumarol relieves serum withdrawal-induced G0/1 blockade in HL-60 cells through a superoxide-dependent mechanism.

    PubMed

    Bello, Rosario I; Gómez-Díaz, Consuelo; López-Lluch, Guillermo; Forthoffer, Nathalie; Córdoba-Pedregosa, María C; Navas, Plácido; Villalba, José M

    2005-06-01

    This work was set to study how dicoumarol affects the cell cycle in human myeloid leukemia HL-60 cells. Cells were accumulated in G0/1 after serum deprivation. However, when cells were treated with 5 microM dicoumarol in serum-free medium, a significant increment in the number of cells in S-phase was observed. Inhibition of G0/1 blockade was confirmed by the increase of thymidine incorporation, the phosphorylation of retinoblastoma protein, and the promotion of cell growth in long-term treatments in the absence of serum. Dicoumarol treatment increased superoxide levels, but did not affect peroxide. Increase of cellular superoxide was essential for inhibition of G0/1 blockade, since scavenging this reactive species with a cell-permeable form of SOD and the SOD mimetics 2-amino-3,5-dibromo-N-[trans-4-hydroxycyclohexyl]benzylamine (ambroxol, 100 microM) and copper[II]diisopropyl salicylate (CuDIPS, 10 microM) completely abolished the effect of dicoumarol. However, N-acetyl-cysteine, overexpression of Bcl-2 or a cell-permeable form of catalase were not effective. 5-Methoxy-1,2-dimethyl-3-[(4-nitrophenol)methyl]-indole-4,7-dione (ES936), a mechanism-based irreversible inhibitor of NAD(P)H:quinone oxidoreductase 1 (NQO1), did not promote S phase entry, and dicoumarol still inhibited G0/1 blockade in the presence of ES936. We demonstrate that dicoumarol inhibits the normal blockade in G0/1 in HL-60 cells through a mechanism involving superoxide, but this effect is not dependent solely on the inhibition of the NQO1 catalytic activity. Our results send a precautionary message about use of dicoumarol to elucidate cellular processes involving oxidoreductases.

  1. Intratumoral injection of a CpG oligonucleotide reverts resistance to PD-1 blockade by expanding multifunctional CD8+ T cells

    PubMed Central

    Wang, Shu; Campos, Jose; Gallotta, Marilena; Gong, Mei; Crain, Chad; Naik, Edwina; Coffman, Robert L.; Guiducci, Cristiana

    2016-01-01

    Despite the impressive rates of clinical response to programmed death 1 (PD-1) blockade in multiple cancers, the majority of patients still fail to respond to this therapy. The CT26 tumor in mice showed similar heterogeneity, with most tumors unaffected by anti–PD-1. As in humans, response of CT26 to anti–PD-1 correlated with increased T- and B-cell infiltration and IFN expression. We show that intratumoral injection of a highly interferogenic TLR9 agonist, SD-101, in anti–PD-1 nonresponders led to a complete, durable rejection of essentially all injected tumors and a majority of uninjected, distant-site tumors. Therapeutic efficacy of the combination was also observed with the TSA mammary adenocarcinoma and MCA38 colon carcinoma tumor models that show little response to PD-1 blockade alone. Intratumoral SD-101 substantially increased leukocyte infiltration and IFN-regulated gene expression, and its activity was dependent on CD8+ T cells and type I IFN signaling. Anti–PD-1 plus intratumoral SD-101 promoted infiltration of activated, proliferating CD8+ T cells and led to a synergistic increase in total and tumor antigen-specific CD8+ T cells expressing both IFN-γ and TNF-α. Additionally, PD-1 blockade could alter the CpG-mediated differentiation of tumor-specific CD8+ T cells into CD127lowKLRG1high short-lived effector cells, preferentially expanding the CD127highKLRG1low long-lived memory precursors. Tumor control and intratumoral T-cell proliferation in response to the combined treatment is independent of T-cell trafficking from secondary lymphoid organs. These findings suggest that a CpG oligonucleotide given intratumorally may increase the response of cancer patients to PD-1 blockade, increasing the quantity and the quality of tumor-specific CD8+ T cells. PMID:27799536

  2. Immunotherapy of murine retrovirus-induced acquired immunodeficiency by CD4 T regulatory cell depletion and PD-1 blockade.

    PubMed

    Li, Wen; Green, William R

    2011-12-01

    LP-BM5 retrovirus induces a complex disease featuring an acquired immunodeficiency syndrome termed murine AIDS (MAIDS) in susceptible strains of mice, such as C57BL/6 (B6). CD4 T helper effector cells are required for MAIDS induction and progression of viral pathogenesis. CD8 T cells are not needed for viral pathogenesis, but rather, are essential for protection from disease in resistant strains, such as BALB/c. We have discovered an immunodominant cytolytic T lymphocyte (CTL) epitope encoded in a previously unrecognized LP-BM5 retroviral alternative (+1 nucleotide [nt]) gag translational open reading frame. CTLs specific for this cryptic gag epitope are the basis of protection from LP-BM5-induced immunodeficiency in BALB/c mice, and the inability of B6 mice to mount an anti-gag CTL response appears critical to the initiation and progression of LP-BM5-induced MAIDS. However, uninfected B6 mice primed by LP-BM5-induced tumors can generate CTL responses to an LP-BM5 retrovirus infection-associated epitope(s) that is especially prevalent on such MAIDS tumor cells, indicating the potential to mount a protective CD8 T-cell response. Here, we utilized this LP-BM5 retrovirus-induced disease system to test whether modulation of normal immune down-regulatory mechanisms can alter retroviral pathogenesis. Thus, following in vivo depletion of CD4 T regulatory (Treg) cells and/or selective interruption of PD-1 negative signaling in the CD8 T-cell compartment, retroviral pathogenesis was significantly decreased, with the combined treatment of CD4 Treg cell depletion and PD-1 blockade working in a synergistic fashion to substantially reduce the induction of MAIDS.

  3. CTLA-4 blockade following relapse of malignancy after allogeneic stem cell transplantation is associated with T cell activation but not with increased levels of T regulatory cells.

    PubMed

    Zhou, Jiehua; Bashey, Asad; Zhong, Ruikun; Corringham, Sue; Messer, Karen; Pu, Minya; Ma, Wenxue; Chut, Theresa; Soiffer, Robert; Mitrovich, Rachel C; Lowy, Israel; Ball, Edward D

    2011-05-01

    Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is a key negative regulator of T cell activation and proliferation. Ipilimumab is a human monoclonal antibody that specifically blocks the binding of CTLA-4 to its ligand. To test the hypothesis that blockade of CTLA-4 by ipilimumab could augment graft-versus-malignancy (GVM) effects without a significant impact on graft-versus-host disease (GVHD), we conducted a phase I clinical trial of ipilimumab infusion in patients with relapsed malignancy following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Here, we report the analysis of peripheral blood T lymphocyte reconstitution, T regulatory cell (Treg) expression, and T cell activation markers after a single dose of ipilimumab in 29 patients. Peripheral blood samples were collected from all patients before and after ipilimumab infusion. Lymphocyte immunophenotyes, including levels of CD4(+)CD25(high) cells and T cell activation markers, were analyzed in all cases. Levels of CD4(+)CD25(high)Foxp3(+) cells and intracellular CTLA-4 in CD4(+) T cells also were evaluated in the last 11 cases. We found lower baseline levels of CD4(+) and CD45RO(+) T cells in patients compared with normal controls. More than 50% of the patients had abnormally low lymphocyte counts (CD4 or/and CD8 T cells), and some had no circulating B lymphocytes. The percentages of both CD4(+)CD25(high) and CD4(+)CD25(high)Foxp3(+) T cells were significantly higher in patients before ipilimumab infusion than in healthy donors. Twenty of 29 patients exhibited an elevated level of CD4(+)CD25(low) activated T cells at baseline, compared with only 3 of 26 healthy donors. Both CD4(+) and CD8(+) T lymphocyte counts were significantly increased after ipilimumab infusion. There was no consistent change in absolute lymphocyte count or in the number of T cells expressing the activation marker CD69. However, increases in CD4(+)CD25(low) T cells were seen in 20 of 29 patients and increases in CD4

  4. Blockade of STAT3 in T Cells Inhibits Germinal Center Reactions against Intranasal Allergens.

    PubMed

    Choi, Garam; Chung, Yeonseok

    2016-05-01

    Understanding the developmental mechanisms of humoral immunity against intranasal antigens is essential for the development of therapeutic approaches against air-borne pathogens as well as allergen-induced pulmonary inflammation. Follicular helper T (Tfh) cells expressing CXCR5 are required for humoral immunity by providing IL-21 and ICOS costimulation to activated B cells. However, the regulation of Tfh cell responses against intranasal antigens remains unclear. Here, we found that the generation of Tfh cells and germinal center B cells in the bronchial lymph node against intranasal proteinase antigens was independent of TGF-β. In contrast, administration of STAT3 inhibitor STA-21 suppressed the generation of Tfh cells and germinal center B cells. Compared with wild-type OT-II T cells, STAT3-deficient OT-II T cells transferred into recipients lacking T cells not only showed significantly reduced frequency Tfh cells, but also induced diminished IgG as well as IgE specific for the intranasal antigens. Cotransfer study of wild-type OT-II and STAT3-deficient OT-II T cells revealed that the latter failed to differentiate into Tfh cells. These findings demonstrate that T cell-intrinsic STAT3 is required for the generation of Tfh cells to intranasal antigens and that targeting STAT3 might be an effective approach to ameliorate antibody-mediated pathology in the lung.

  5. Short-term EGFR blockade enhances immune-mediated cytotoxicity of EGFR mutant lung cancer cells: rationale for combination therapies

    PubMed Central

    Dominguez, Charli; Tsang, Kwong-Yok; Palena, Claudia

    2016-01-01

    The epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) erlotinib has been approved for years as a first-line therapy for patients harboring EGFR-sensitizing mutations. With the promising implementation of immunotherapeutic strategies for the treatment of lung cancer, there is a growing interest in developing combinatorial therapies that could utilize immune approaches in the context of conventional or targeted therapies. Tumor cells are known to evade immune attack by multiple strategies, including undergoing phenotypic plasticity via a process designated as the epithelial–mesenchymal transition (EMT). As signaling through EGFR is a major inducer of EMT in epithelial cells, we have investigated the effect of EGFR inhibition with erlotinib on tumor phenotype and susceptibility to immune attack. Our data shows that short-term exposure of tumor cells to low-dose erlotinib modulates tumor plasticity and immune-mediated cytotoxicity in lung cancer cells harboring a sensitizing EGFR mutation, leading to a remarkable enhancement of tumor lysis mediated by innate NK cells and antigen-specific T cells. This effect positively correlated with the ability of short-term EGFR blockade to modulate tumor phenotype towards a more epithelial one, as well as to increase susceptibility to caspase-mediated apoptosis. The effect, however, was lost when erlotinib was utilized for long periods of time in vitro or in vivo, which resulted in gain of mesenchymal features and decreased (rather than increased) tumor lysis in response to immune effector mechanisms. Our data provides rationale for potential combinations of erlotinib and immunotherapies for the treatment of lung carcinomas in the early setting, before the establishment of tumor relapse with long-term EGFR inhibition. PMID:27685624

  6. Myeloid-Derived Suppressor Cells Associated With Disease Progression in Primary HIV Infection: PD-L1 Blockade Attenuates Inhibition.

    PubMed

    Zhang, Zi-Ning; Yi, Nan; Zhang, Tong-Wei; Zhang, Le-Le; Wu, Xian; Liu, Mei; Fu, Ya-Jing; He, Si-Jia; Jiang, Yong-Jun; Ding, Hai-Bo; Chu, Zhen-Xing; Shang, Hong

    2017-10-01

    Events occurring during the initial phase of human immunodeficiency virus (HIV) infection are intriguing because of their dramatic impact on the subsequent course of the disease. In particular, the relationship between myeloid-derived suppressor cells (MDSCs) and HIV pathogenesis in primary infection remains unknown and the mechanism of MDSCs in HIV infection are incompletely defined. The frequency of MDSC expression in patients with primary HIV infection (PHI) and chronic HIV infection was measured, and the association with disease progression was studied. Programmed death-ligand 1 (PD-L1) and galectin-9 (Gal-9) expression on MDSCs was measured and in vitro blocking experiments were performed to study the role of PD-L1 in MDSCs' inhibition. We found increased levels of HLA-DRCD14CD33CD11b granulocytic(G)-MDSCs in PHI individuals compared with normal controls, which correlated with viral loads and was negatively related to CD4 T-cell levels. When cocultured with purified G-MDSCs, both proliferation and interferon-γ secretion by T cell receptor (TCR)-stimulated CD8 T cells from HIV-infected patients were significantly inhibited. We also demonstrated that PD-L1, but not Gal-9, expression on HLA-DRCD14CD33CD11b cells increased during HIV infection. The suppressive activity of G-MDSCs from HIV-infected patients was attenuated by PD-L1 blockade. We found a significant increase in G-MDSCs in PHI patients that was related to disease progression and PD-L1 was used by MDSCs to inhibit CD8 T cells in HIV infection. Our data improve the understanding of HIV pathogenesis in PHI.

  7. Epigallocatechin gallate (EGCG) attenuates high glucose-induced insulin signaling blockade in human hepG2 hepatoma cells.

    PubMed

    Lin, Chih-Li; Lin, Jen-Kun

    2008-08-01

    Insulin resistance is the primary characteristic of type 2 diabetes which as a result of insulin signaling defects. It has been suggested that the tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) displays some antidiabetic effects, but the mechanism for EGCG insulin-enhancing effects is incompletely understood. In the present study, the investigations of EGCG on insulin signaling are performed in insulin-responsive human HepG2 cells cotreated with high glucose. We found that the high glucose condition causes significant increasing Ser307 phosphorylation of insulin receptor substrate-1 (IRS-1), leading to reduce insulin-stimulated phosphorylation of Akt. As the results, the insulin metabolic effects of glycogen synthesis and glucose uptake are inhibited by high glucose. However, the treatment of EGCG improves insulin-stimulated downsignaling by reducing IRS-1 Ser307 phosphorylation. Furthermore, we also demonstrated these EGCG effects are essential depends on the 5'-AMP-activated protein kinase (AMPK) activation. Together, our data suggest a putative link between high glucose and insulin resistance in HepG2 cells, and the EGCG treatment attenuates insulin signaling blockade by reducing IRS-1 Ser307 phosphorylation through the AMPK activation pathway.

  8. Critical role for CD8 T cells in allograft acceptance induced by DST and CD40/CD154 costimulatory blockade.

    PubMed

    Gao, Donghong; Lunsford, Keri E; Eiring, Anna M; Bumgardner, Ginny L

    2004-07-01

    Donor-specific transfusion (DST) and CD40/CD154 costimulation blockade is a powerful immunosuppressive strategy which prolongs survival of many allografts. The efficacy of DST and anti-CD154 mAb for prolongation of hepatocellular allograft survival was only realized in C57BL/6 mice that have both CD4- and CD8-dependent pathways available (median survival time, MST, 82 days). Hepatocyte rejection in CD8 KO mice which is CD4-dependent was not suppressed by DST and anti-CD154 mAb treatment (MST, 7 days); unexpectedly DST abrogated the beneficial effects of anti-CD154 mAb for suppression of hepatocyte rejection (MST, 42 days) and on donor-reactive alloantibody production. Hepatocyte rejection in CD4 KO mice which is CD8-dependent was suppressed by treatment with DST and anti-CD154 mAb therapy (MST, 35 days) but did not differ significantly from immunotherapy with anti-CD154 mAb alone (MST, 32 days). Induction of hepatocellular allograft acceptance by DST and anti-CD154 mAb immunotherapy was dependent on host CD8(+) T cells, as demonstrated by CD8 depletion studies in C57BL/6 mice (MST, 14 days) and CD8 reconstitution of CD8 KO mice (MST, 56 days). These studies demonstrate that both CD4(+) and CD8(+) T-cell subsets contribute to induction of hepatocellular allograft acceptance by this immunotherapeutic strategy.

  9. Combined blockade of angiotensin II and prorenin receptors ameliorates podocytic apoptosis induced by IgA-activated mesangial cells.

    PubMed

    Leung, Joseph C K; Chan, Loretta Y Y; Saleem, M A; Mathieson, P W; Tang, Sydney C W; Lai, Kar Neng

    2015-07-01

    Glomerulo-podocytic communication plays an important role in the podocytic injury in IgA nephropathy (IgAN). In this study, we examine the role of podocytic angiotensin II receptor subtype 1 (AT1R) and prorenin receptor (PRR) in podocytic apoptosis in IgAN. Polymeric IgA (pIgA) was isolated from patients with IgAN and healthy controls. Conditioned media were prepared from growth arrested human mesangial cells (HMC) incubated with pIgA from patients with IgAN (IgA-HMC media) or healthy controls (Ctl-HMC media). A human podocyte cell line was used as a model to examine the regulation of the expression of AT1R, PRR, TNF-α and CTGF by IgA-HMC media. Podocytic nephrin expression, annexin V binding and caspase 3 activity were used as the functional readout of podocytic apoptosis. IgA-HMC media had no effect on AngII release by podocytes. IgA-HMC media significantly up-regulated the expression of AT1R and PRR, down-regulated nephrin expression and induced apoptosis in podocytes. Mono-blockade of AT1R, PRR, TNF-α or CTGF partially reduced podocytic apoptosis. IgA-HMC media activated NFκB, notch1 and HEY1 expression by podocytes and dual blockade of AT1R with PRR, or anti-TNF-α with anti-CTGF, effectively rescued the podocytic apoptosis induced by IgA-HMC media. Our data suggests that pIgA-activated HMC up-regulates the expression of AT1R and PRR expression by podocytes and the associated activation of NFκB and notch signalling pathways play an essential role in the podocytic apoptosis induced by glomerulo-podocytic communication in IgAN. Simultaneously targeting the AT1R and PRR could be a potential therapeutic option to reduce the podocytic injury in IgAN.

  10. Selective Blockade of Human Natural Killer Cells by a Monoclonal Antibody

    NASA Astrophysics Data System (ADS)

    Newman, Walter

    1982-06-01

    A murine monoclonal antibody, 13.1, which blocks human natural killer (NK) cell-mediated lysis, has been developed. Hybridoma 13.1 was derived by fusion of NS-1 cells with spleen cells from mice immunized with an enriched population of NK cells. Supernatants of growing hybridomas were screened for their ability to block NK cell-mediated lysis of K562 targets. Antibody 13.1 is an IgG1 with a single light chain type and it does not fix complement. The 13.1 antigen is expressed on all peripheral blood mononuclear cells, with an antigen density approximately 1/30th that of HLA antigen heavy chain. Pretreatment and washing experiments revealed that inhibition of cytotoxicity occurred at the effector cell level only. Significant blocking was achieved with nanogram quantities of antibody and was not due to toxic effects on NK cells. Likewise, controls with other antibodies of the same subclass demonstrated that blocking was not a consequence of mere binding to NK cells. When a panel of 17 NK cell-susceptible targets was tested, the lysis of only 5 of these was blocked, namely K562, HL-60, KG-1, Daudi, and HEL, a human erythroleukemic cell line. The lysis of 12 human B cell and T cell line targets was not inhibited. In addition to the demonstration that the 13.1 antigen is a crucial cell surface structure involved in NK lysis, a heterogeneity of target cell recognition has been revealed that argues for the proposition that individual NK cells have multiple recognitive capabilities.

  11. Blockade of CTLA-4 on CD4+CD25+ regulatory T cells abrogates their function in vivo.

    PubMed

    Read, Simon; Greenwald, Rebecca; Izcue, Ana; Robinson, Nicholas; Mandelbrot, Didier; Francisco, Loise; Sharpe, Arlene H; Powrie, Fiona

    2006-10-01

    Naturally occurring CD4+ regulatory T cells (T(R)) that express CD25 and the transcription factor FoxP3 play a key role in immune homeostasis, preventing immune pathological responses to self and foreign Ags. CTLA-4 is expressed by a high percentage of these cells, and is often considered as a marker for T(R) in experimental and clinical analysis. However, it has not yet been proven that CTLA-4 has a direct role in T(R) function. In this study, using a T cell-mediated colitis model, we demonstrate that anti-CTLA-4 mAb treatment inhibits T(R) function in vivo via direct effects on CTLA-4-expressing T(R), and not via hyperactivation of colitogenic effector T cells. Although anti-CTLA-4 mAb treatment completely inhibits T(R) function, it does not reduce T(R) numbers or their homing to the GALT, suggesting the Ab mediates its function by blockade of a signal required for T(R) activity. In contrast to the striking effect of the Ab, CTLA-4-deficient mice can produce functional T(R), suggesting that under some circumstances other immune regulatory mechanisms, including the production of IL-10, are able to compensate for the loss of the CTLA-4-mediated pathway. This study provides direct evidence that CTLA-4 has a specific, nonredundant role in the function of normal T(R). This role has to be taken into account when targeting CTLA-4 for therapeutic purposes, as such a strategy will not only boost effector T cell responses, but might also break T(R)-mediated self-tolerance.

  12. In vitro VLA-4 blockade results in an impaired NK cell-mediated immune surveillance against melanoma.

    PubMed

    Gandoglia, Ilaria; Ivaldi, Federico; Carrega, Paolo; Armentani, Eric; Ferlazzo, Guido; Mancardi, Gianluigi; Kerlero de Rosbo, Nicole; Uccelli, Antonio; Laroni, Alice

    2017-01-01

    cells in the lower chamber, and that it was significantly reduced in presence of NTZ. Our results show that upon exposure to NTZ both cytolytic activity and migration toward melanoma cells were affected, suggesting that binding of NTZ to NK cells affects pathways involved in these NK-cell functions. We analyzed the expression of CD49d on NK cells from MS patients treated with NTZ and observed that it decreases with time of treatment. These data suggest that blockade of VLA-4 on NK-cell surface alters some key functions involved in the immune surveillance toward melanoma by NK cells and may provide a mechanistic explanation for the reported occurrence of melanoma in MS patients treated with NTZ.

  13. Blockade of MUC1 expression by glycerol guaiacolate inhibits proliferation of human breast cancer cells.

    PubMed

    Smith, J S; Colon, J; Madero-Visbal, R; Isley, B; Konduri, S D; Baker, C H

    2010-10-01

    We sought to determine whether administration of glycerol guaiacolate at an optimal biological dose inhibits human breast cancer cell growth. Human breast cancer MCF-7 and ZR-75-1 cells were treated with glycerol guaiacolate and the therapeutic efficacy and biological activity of this drug was investigated on breast cancer cell growth. MCF-7 cells were injected into the mammary fat pad of overectamized female athymic nude mice. Ten days later, animals were treated with daily intraperitoneal injections of glycerol guaiacolate for six weeks. Tumor size and volume was monitored and immunohistochemistry analysis on MUC1, p21 and ki-67 was performed. Glycerol guaiacolate decreased breast cancer cell growth in a dose-dependent manner, decreased cell migration, and caused G1 cell cycle arrest. Our results demonstrate that glycerol guaiacolate inhibits MUC1 protein and mRNA expression levels and significantly increased p21 expression in human breast cancer cells as well as induced PARP cleavage. Similarly, glycerol guaiacolate inhibited breast tumor growth in vivo as well as enhanced p21 expression and decreased breast tumor cell proliferation (ki-67 expression). Collectively, our results demonstrate that glycerol guaiacolate decreased MUC1 expression and enhanced cell growth inhibition by inducing p21 expression in breast cancer cells. These findings suggest that glycerol guaiacolate may provide a novel and effective approach for the treatment of human breast cancer.

  14. Blockade of TNF-α signaling benefits cancer therapy by suppressing effector regulatory T cell expansion.

    PubMed

    Chang, Li-Yuan; Lin, Yung-Chang; Chiang, Jy-Ming; Mahalingam, Jayashri; Su, Shih-Huan; Huang, Ching-Tai; Chen, Wei-Ting; Huang, Chien-Hao; Jeng, Wen-Juei; Chen, Yi-Cheng; Lin, Shi-Ming; Sheen, I-Shyan; Lin, Chun-Yen

    2015-10-01

    Effector but not naive regulatory T cells (Treg cells) can accumulate in the peripheral blood as well as the tumor microenvironment, expand during tumor progression and be one of the main suppressors for antitumor immunity. However, the underlying mechanisms for effector Treg cell expansion in tumor are still unknown. We demonstrate that effector Treg cell-mediated suppression of antitumor CD8(+) T cells is tumor-nonspecific. Furthermore, TNFR2 expression is increased in these Treg cells by Affymetrix chip analysis which was confirmed by monoclonal antibody staining in both hepatocellular carcinoma (HCC) and colorectal cancer (CRC) patients and murine models. Correspondingly, increased levels of TNF-α in both tissue and serum were also demonstrated. Interestingly, TNF-α could not only expand effector Treg cells through TNFR2 signaling, but also enhanced their suppressive activity against antitumor immunity of CD8(+) T cells. Furthermore, targeting TNFR2 signaling with a TNF-α inhibitor could selectively reduce rapid resurgence of effector Treg cells after cyclophosphamide-induced lymphodepletion and markedly inhibit the growth of established tumors. Herein, we propose a novel mechanism in which TNF-α could promote tumor-associated effector Treg cell expansion and suggest a new cancer immunotherapy strategy using TNF-α inhibitors to reduce effector Treg cells expansion after cyclophosphamide-induced lymphodepletion.

  15. Conditional and specific NF-kappaB blockade protects pancreatic beta cells from diabetogenic agents.

    PubMed

    Eldor, R; Yeffet, A; Baum, K; Doviner, V; Amar, D; Ben-Neriah, Y; Christofori, G; Peled, A; Carel, J C; Boitard, C; Klein, T; Serup, P; Eizirik, D L; Melloul, D

    2006-03-28

    Type 1 diabetes is characterized by the infiltration of inflammatory cells into pancreatic islets of Langerhans, followed by the selective and progressive destruction of insulin-secreting beta cells. Islet-infiltrating leukocytes secrete cytokines such as IL-1beta and IFN-gamma, which contribute to beta cell death. In vitro evidence suggests that cytokine-induced activation of the transcription factor NF-kappaB is an important component of the signal triggering beta cell apoptosis. To study the in vivo role of NF-kappaB in beta cell death, we generated a transgenic mouse line expressing a degradation-resistant NF-kappaB protein inhibitor (DeltaNIkappaBalpha), acting specifically in beta cells, in an inducible and reversible manner, by using the tet-on regulation system. In vitro, islets expressing the DeltaNIkappaBalpha protein were resistant to the deleterious effects of IL-1beta and IFN-gamma, as assessed by reduced NO production and beta-cell apoptosis. This effect was even more striking in vivo, where nearly complete protection against multiple low-dose streptozocin-induced diabetes was observed, with reduced intraislet lymphocytic infiltration. Our results show in vivo that beta cell-specific activation of NF-kappaB is a key event in the progressive loss of beta cells in diabetes. Inhibition of this process could be a potential effective strategy for beta-cell protection.

  16. GnRH Episodic Secretion Is Altered by Pharmacological Blockade of Gap Junctions: Possible Involvement of Glial Cells.

    PubMed

    Pinet-Charvet, Caroline; Geller, Sarah; Desroziers, Elodie; Ottogalli, Monique; Lomet, Didier; Georgelin, Christine; Tillet, Yves; Franceschini, Isabelle; Vaudin, Pascal; Duittoz, Anne

    2016-01-01

    Episodic release of GnRH is essential for reproductive function. In vitro studies have established that this episodic release is an endogenous property of GnRH neurons and that GnRH secretory pulses are associated with synchronization of GnRH neuron activity. The cellular mechanisms by which GnRH neurons synchronize remain largely unknown. There is no clear evidence of physical coupling of GnRH neurons through gap junctions to explain episodic synchronization. However, coupling of glial cells through gap junctions has been shown to regulate neuron activity in their microenvironment. The present study investigated whether glial cell communication through gap junctions plays a role in GnRH neuron activity and secretion in the mouse. Our findings show that Glial Fibrillary Acidic Protein-expressing glial cells located in the median eminence in close vicinity to GnRH fibers expressed Gja1 encoding connexin-43. To study the impact of glial-gap junction coupling on GnRH neuron activity, an in vitro model of primary cultures from mouse embryo nasal placodes was used. In this model, GnRH neurons possess a glial microenvironment and were able to release GnRH in an episodic manner. Our findings show that in vitro glial cells forming the microenvironment of GnRH neurons expressed connexin-43 and displayed functional gap junctions. Pharmacological blockade of the gap junctions with 50 μM 18-α-glycyrrhetinic acid decreased GnRH secretion by reducing pulse frequency and amplitude, suppressed neuronal synchronization and drastically reduced spontaneous electrical activity, all these effects were reversed upon 18-α-glycyrrhetinic acid washout.

  17. Reinvigorating Exhausted T Cells by Blockade of the PD-1 Pathway

    PubMed Central

    Lee, Junghwa; Ahn, Eunseon; Kissick, Haydn T.; Ahmed, Rafi

    2017-01-01

    T-cell exhaustion due to persistent antigen stimulation is a key feature of chronic viral infections and cancer. Programmed cell death-1 (PD-1) is a major regulator of T-cell exhaustion, and blocking the PD-1 pathway restores T-cell function and improves pathogen control and tumor eradication. Immunotherapy targeting the PD-1 inhibitory receptor pathway has demonstrated significant antitumor activity. Recently, antibodies blocking PD-1 have been approved for use in cancer patients. In this review, we summarize the role of the PD-1 pathway in chronic infection and cancer and the therapeutic potential of PD-1-directed immunotherapy in patients with chronic infection or cancer.

  18. Therapeutic CD94/NKG2A blockade improves natural killer cell dysfunction in chronic lymphocytic leukemia

    PubMed Central

    McWilliams, Emily M.; Mele, Jennifer M.; Cheney, Carolyn; Timmerman, Elizabeth A.; Fiazuddin, Faraz; Strattan, Ethan J.; Mo, Xiaokui; Byrd, John C.; Muthusamy, Natarajan; Awan, Farrukh T.

    2016-01-01

    ABSTRACT Natural killer (NK)-cell count is predictive of chronic lymphoid leukemia (CLL) disease progression and their dysfunction is well documented, but the etiology of this is currently lacking. CLL cells have been shown to over-express HLA-E, the natural ligand for NKG2A expressed on NK-cells that generates a distinct negative signal relative to direct NK-cell cytotoxicity in other disease models. Utilizing a novel anti-NKG2A monoclonal blocking antibody (mAb), monalizumab, we explored the in vitro preclinical activity of targeting the NKG2A receptor, and the NKG2A/HLA-E interaction as a mechanism of tumor evasion in CLL patients. Our work confirmed overexpression of HLA-E on CLL B-cells and demonstrated NKG2A expression on CD56+/16+ NK-cells from CLL patients. We also demonstrate that blocking NKG2A on CLL NK-cells was sufficient to restore direct cytotoxicity ability of NK-cells against HLA-E-expressing targets without impacting NK-cell mediated antibody-dependent cellular cytotoxicity. Additionally, we proved the specificity of monalizumab in blocking NKG2A through Fc-blocking mechanisms. This paper provides justification for the potential clinical utility of monalizumab in the treatment of patients with CLL. PMID:27853650

  19. Therapeutic CD94/NKG2A blockade improves natural killer cell dysfunction in chronic lymphocytic leukemia.

    PubMed

    McWilliams, Emily M; Mele, Jennifer M; Cheney, Carolyn; Timmerman, Elizabeth A; Fiazuddin, Faraz; Strattan, Ethan J; Mo, Xiaokui; Byrd, John C; Muthusamy, Natarajan; Awan, Farrukh T

    2016-01-01

    Natural killer (NK)-cell count is predictive of chronic lymphoid leukemia (CLL) disease progression and their dysfunction is well documented, but the etiology of this is currently lacking. CLL cells have been shown to over-express HLA-E, the natural ligand for NKG2A expressed on NK-cells that generates a distinct negative signal relative to direct NK-cell cytotoxicity in other disease models. Utilizing a novel anti-NKG2A monoclonal blocking antibody (mAb), monalizumab, we explored the in vitro preclinical activity of targeting the NKG2A receptor, and the NKG2A/HLA-E interaction as a mechanism of tumor evasion in CLL patients. Our work confirmed overexpression of HLA-E on CLL B-cells and demonstrated NKG2A expression on CD56+/16+ NK-cells from CLL patients. We also demonstrate that blocking NKG2A on CLL NK-cells was sufficient to restore direct cytotoxicity ability of NK-cells against HLA-E-expressing targets without impacting NK-cell mediated antibody-dependent cellular cytotoxicity. Additionally, we proved the specificity of monalizumab in blocking NKG2A through Fc-blocking mechanisms. This paper provides justification for the potential clinical utility of monalizumab in the treatment of patients with CLL.

  20. Interleukin-6 blockade attenuates lung cancer tissue construction integrated by cancer stem cells.

    PubMed

    Ogawa, Hiroyuki; Koyanagi-Aoi, Michiyo; Otani, Kyoko; Zen, Yoh; Maniwa, Yoshimasa; Aoi, Takashi

    2017-09-26

    In the present study, we successfully generated lung cancer stem cell (CSC)-like cells by introducing a small set of transcription factors into a lung cancer cell line. In addition to properties that are conventionally referred to as CSC properties, the lung induced CSCs exhibited the ability to form lung cancer-like tissues in vitro with vascular cells and mesenchymal stem cells, which showed structures and immunohistological patterns that were similar to human lung cancer tissues. We named them "lung cancer organoids". We found that interleukin-6 (IL-6), which was expressed in the lung induced CSCs, facilitates the formation of lung cancer organoids via the conversion of mesenchymal stem cells into alpha-smooth muscle actin (αSMA)-positive cells. Interestingly, the combination of anti-IL-6 antibody and cisplatin could destroy the lung cancer organoids, while cisplatin alone could not. Furthermore, IL-6 mRNA-positive cancer cells were found in clinical lung cancer samples. These results suggest that IL-6 could be a novel therapeutic target in lung cancer.

  1. Selective blockade of T-type Ca2+ channels suppresses human breast cancer cell proliferation.

    PubMed

    Taylor, James T; Huang, Luping; Pottle, Jonathan E; Liu, Kai; Yang, Yali; Zeng, Xiangbin; Keyser, Brian M; Agrawal, Krishna C; Hansen, J Bondo; Li, Ming

    2008-08-18

    We have measured the expression of T-type Ca2+ channel mRNA in breast cancer cell lines (MCF-7 (ERalpha+) using Western blot and quantitative real-time PCR (Q-RT-PCR). These results revealed that the MCF-7 cells express both alpha1G and alpha1H isoforms of T-type Ca2+ channels. In order to further clarify the role of T-type Ca2+ channels in proliferation, we tested the effects of a selective T-type Ca2+ channel inhibitor NNC-55-0396 on cellular proliferation. MCF-7 (ERalpha+) cellular proliferation was inhibited by the compound. In contrast, NNC-55-0396 at same concentration had no effect on the proliferation of MCF-10A cells, a non-cancer breast epithelial cell line. We also found that message expression of the T-type Ca2+ channels were only expressed in rapidly growing non-confluent cells but not in the cytostatic confluent cells. Knocking down the expression of T-type Ca2+ channels with siRNA targeting both alpha1G and alpha1H resulted in growth inhibition as much as 45%+/-5.0 in MCF-7 cells as compared to controls. In conclusion, our results suggest that T-type Ca2+ channel antagonism/silencing may reduce cellular proliferation in mitogenic breast cells.

  2. Correction of abnormal B-cell subset distribution by interleukin-6 receptor blockade in polymyalgia rheumatica.

    PubMed

    Carvajal Alegria, Guillermo; Devauchelle-Pensec, Valérie; Renaudineau, Yves; Saraux, Alain; Pers, Jacques-Olivier; Cornec, Divi

    2017-08-01

    The aim was to study lymphocyte subsets and circulating cytokines at diagnosis of PMR and after tocilizumab monotherapy. Eighteen untreated patients with PMR were included in a prospective study and received 3-monthly tocilizumab infusions without glucocorticoids. Lymphocyte subset distribution was assessed by flow cytometry and serum cytokines were assayed by a 34-cytokine array and ELISA, at baseline and during follow-up. Baseline data were also compared with age- and sex-matched controls. At baseline, total lymphocytes, T-cell subsets and NK cell counts were similar in patients and controls, but patients had significantly lower B-cell counts attributable to lower transitional, naïve and post-switch memory B-cell subsets. Circulating B-cell counts were positively correlated with the PMR activity score (PMR-AS) in untreated active patients at baseline, but subsequently increased to normal values while disease activity was controlled after tocilizumab therapy. Among serum cytokines, IL-6 showed the largest concentration difference between patients and controls, and the serum IL-6 concentration was correlated with baseline PMR-AS. The effects of tocilizumab on serum IL-6 concentration were heterogeneous, and the patients whose serum IL-6 decreased after tocilizumab therapy exhibited a significant increase in circulating B-cell counts. In patients with PMR, B-cell lymphopenia and abnormal B-cell subset distribution are associated with disease activity and IL-6 concentration, and both are corrected by the IL-6 antagonist tocilizumab.

  3. Mutational landscape determines sensitivity to PD-1 blockade in non–small cell lung cancer

    PubMed Central

    Rizvi, Naiyer A.; Hellmann, Matthew D.; Snyder, Alexandra; Kvistborg, Pia; Makarov, Vladimir; Havel, Jonathan J.; Lee, William; Yuan, Jianda; Wong, Phillip; Ho, Teresa S.; Miller, Martin L.; Rekhtman, Natasha; Moreira, Andre L.; Ibrahim, Fawzia; Bruggeman, Cameron; Gasmi, Billel; Zappasodi, Roberta; Maeda, Yuka; Sander, Chris; Garon, Edward B.; Merghoub, Taha; Wolchok, Jedd D.; Schumacher, Ton N.; Chan, Timothy A.

    2016-01-01

    Immune checkpoint inhibitors, which unleash a patient’s own T cells to kill tumors, are revolutionizing cancer treatment. To unravel the genomic determinants of response to this therapy, we used whole-exome sequencing of non–small cell lung cancers treated with pembrolizumab, an antibody targeting programmed cell death-1 (PD-1). In two independent cohorts, higher nonsynonymous mutation burden in tumors was associated with improved objective response, durable clinical benefit, and progression-free survival. Efficacy also correlated with the molecular smoking signature, higher neoantigen burden, and DNA repair pathway mutations; each factor was also associated with mutation burden. In one responder, neoantigen-specific CD8+ T cell responses paralleled tumor regression, suggesting that anti–PD-1 therapy enhances neoantigen-specific T cell reactivity. Our results suggest that the genomic landscape of lung cancers shapes response to anti–PD-1 therapy. PMID:25765070

  4. BK K+ channel blockade inhibits radiation-induced migration/brain infiltration of glioblastoma cells

    PubMed Central

    Klumpp, Lukas; Haehl, Erik; Schilbach, Karin; Lukowski, Robert; Kühnle, Matthias; Bernhardt, Günther; Buschauer, Armin; Zips, Daniel; Ruth, Peter; Huber, Stephan M.

    2016-01-01

    Infiltration of the brain by glioblastoma cells reportedly requires Ca2+ signals and BK K+ channels that program and drive glioblastoma cell migration, respectively. Ionizing radiation (IR) has been shown to induce expression of the chemokine SDF-1, to alter the Ca2+ signaling, and to stimulate cell migration of glioblastoma cells. Here, we quantified fractionated IR-induced migration/brain infiltration of human glioblastoma cells in vitro and in an orthotopic mouse model and analyzed the role of SDF-1/CXCR4 signaling and BK channels. To this end, the radiation-induced migratory phenotypes of human T98G and far-red fluorescent U-87MG-Katushka glioblastoma cells were characterized by mRNA and protein expression, fura-2 Ca2+ imaging, BK patch-clamp recording and transfilter migration assay. In addition, U-87MG-Katushka cells were grown to solid glioblastomas in the right hemispheres of immunocompromised mice, fractionated irradiated (6 MV photons) with 5 × 0 or 5 × 2 Gy, and SDF-1, CXCR4, and BK protein expression by the tumor as well as glioblastoma brain infiltration was analyzed in dependence on BK channel targeting by systemic paxilline application concomitant to IR. As a result, IR stimulated SDF-1 signaling and induced migration of glioblastoma cells in vitro and in vivo. Importantly, paxilline blocked IR-induced migration in vivo. Collectively, our data demonstrate that fractionated IR of glioblastoma stimulates and BK K+ channel targeting mitigates migration and brain infiltration of glioblastoma cells in vivo. This suggests that BK channel targeting might represent a novel approach to overcome radiation-induced spreading of malignant brain tumors during radiotherapy. PMID:26893360

  5. Chromatin structural changes in synchronized cells blocked in early S phase by sequential use of isoleucine deprivation and hydroxyurea blockade

    SciTech Connect

    D'Anna, J.A.; Prentice, D.A.

    1983-01-01

    The authors have investigated the loss of histone H1 from chromatin and the structure of chromatin from Chinese hamster (line CHO) cells blocked in early S phase by sequential use of isoleucine deprivation G/sub 1/ block and 1 mM hydroxyurea (HU) blockade. Measurements of H1 content in the cell and histone turnover indicate that H1 is lost from the cell and that there is negligible replacement synthesis of H1 during the period of the S-phase block. As H1 is lost, chromatin appears to undergo structural change. After 10 h of HU block, the new deoxyribonucleic acid (DNA) and a portion of the old DNA have measured nucleosome repeat lengths (37/sup 0/C digestion). By 24 h of HU block, nearly all of the chromatin has assumed a pseudoimmature conformation in which the nucleosome cores appear to be more closely packed along the DNA chain, but the new DNA is slightly more resistant than old DNA to attack by micrococcal nuclease. Electrophoretic analysis of nucleoprotein particles produced by micrococcal nuclease digestion of nuclei indicates that: the distribution of mononucleosome species changes during Hu block and some mononucleosome species appear to be enriched in normally minor proteins which may determine the electrophoretic mobility of the nucleoprotein particles in agarose-acrylamide gels. The results raise the possibility that: during the early stages of replication (or prior to the passage of the replication fork), H1 is dissociated from initiated replicons and H1 does not reassociate in a concerted fashion with the H1-depleted chromatin until the replication fork has passed and, perhaps, a substantial portion of the replicon has been replicated.

  6. Angiotensin type 1 receptor resistance to blockade in the opossum proximal tubule cell due to variations in the binding pocket.

    PubMed

    Nistala, Ravi; Andresen, Bradley T; Pulakat, Lakshmi; Meuth, Alex; Sinak, Catherine; Mandavia, Chirag; Thekkumkara, Thomas; Speth, Robert C; Whaley-Connell, Adam; Sowers, James R

    2013-04-15

    Blockade of the angiotensin (ANG) II receptor type 1 (AT(1)R) with angiotensin receptor blockers (ARBs) is widely used in the treatment of hypertension. However, ARBs are variably effective in reducing blood pressure, likely due, in part, to polymorphisms in the ARB binding pocket of the AT(1)R. Therefore, we need a better understanding of variations/polymorphisms that alter binding of ARBs in heterogeneous patient populations. The opossum proximal tubule cell (OKP) line is commonly used in research to evaluate renal sodium handling and therefore blood pressure. Investigating this issue, we found natural sequence variations in the opossum AT(1)R paralleling those observed in the human AT(1)R. Therefore, we posited that these sequence variations may explain ARB resistance. We demonstrate that OKP cells express AT(1)R mRNA, bind (125)I-labeled ANG II, and exhibit ANG II-induced phosphorylation of Jak2. However, Jak2 phosphorylation is not inhibited by five different ARBs commonly used to treat hypertension. Additionally, nonradioactive ANG II competes (125)I-ANG II efficiently, whereas a 10-fold molar excess of olmesartan and the ANG II receptor type 2 blocker PD-123319 is unable to block (125)I-ANG II binding. In contrast, ANG II binding to OKP cells stably expressing rat AT(1A)Rs, which have a conserved AT(1)R-binding pocket with human AT(1)R, is efficiently inhibited by olmesartan. A novel observation was that resistance to ARB binding to opossum AT(1)Rs correlates with variations from the human receptor at positions 108, 163, 192, and 198 within the ARB-binding pocket. These observations highlight the potential utility of evaluating AT(1)R polymorphisms within the ARB-binding pocket in various hypertensive populations.

  7. Angiotensin type 1 receptor resistance to blockade in the opossum proximal tubule cell due to variations in the binding pocket

    PubMed Central

    Nistala, Ravi; Andresen, Bradley T.; Pulakat, Lakshmi; Meuth, Alex; Sinak, Catherine; Mandavia, Chirag; Thekkumkara, Thomas; Speth, Robert C.; Whaley-Connell, Adam

    2013-01-01

    Blockade of the angiotensin (ANG) II receptor type 1 (AT1R) with angiotensin receptor blockers (ARBs) is widely used in the treatment of hypertension. However, ARBs are variably effective in reducing blood pressure, likely due, in part, to polymorphisms in the ARB binding pocket of the AT1R. Therefore, we need a better understanding of variations/polymorphisms that alter binding of ARBs in heterogeneous patient populations. The opossum proximal tubule cell (OKP) line is commonly used in research to evaluate renal sodium handling and therefore blood pressure. Investigating this issue, we found natural sequence variations in the opossum AT1R paralleling those observed in the human AT1R. Therefore, we posited that these sequence variations may explain ARB resistance. We demonstrate that OKP cells express AT1R mRNA, bind 125I-labeled ANG II, and exhibit ANG II-induced phosphorylation of Jak2. However, Jak2 phosphorylation is not inhibited by five different ARBs commonly used to treat hypertension. Additionally, nonradioactive ANG II competes 125I-ANG II efficiently, whereas a 10-fold molar excess of olmesartan and the ANG II receptor type 2 blocker PD-123319 is unable to block 125I-ANG II binding. In contrast, ANG II binding to OKP cells stably expressing rat AT1ARs, which have a conserved AT1R-binding pocket with human AT1R, is efficiently inhibited by olmesartan. A novel observation was that resistance to ARB binding to opossum AT1Rs correlates with variations from the human receptor at positions 108, 163, 192, and 198 within the ARB-binding pocket. These observations highlight the potential utility of evaluating AT1R polymorphisms within the ARB-binding pocket in various hypertensive populations. PMID:23389452

  8. Growth inhibition of non-small cell lung cancer cells by AP-1 blockade using a cJun dominant-negative mutant

    PubMed Central

    Shimizu, Y; Kinoshita, I; Kikuchi, J; Yamazaki, K; Nishimura, M; Birrer, M J; Dosaka-Akita, H

    2008-01-01

    cJun, a major constituent of AP-1 transcription factor transducing multiple mitogen growth signals, is frequently overexpressed in non-small cell lung cancers (NSCLCs). The purpose of this study is to determine the effects of AP-1 blockade on the growth of NSCLC cells using a cJun dominant-negative mutant, TAM67. Transiently transfected TAM67 inhibited AP-1 transcriptional activity in NSCLC cell lines, NCI-H1299 (H1299), A549 and NCI-H520 (H520). The colony-forming efficiency of H1299 and A549 was reduced by TAM67, while that of H520 was not. To elucidate the effects of TAM67 on the growth of H1299, we established H1299 clone cells that expressed TAM67 under the control of a doxycycline-inducible promoter. In the H1299 clone cells, the induced TAM67 inhibited anchorage-dependent growth by promoting G1 cell-cycle block, but not by apoptosis. The induced TAM67 decreased the expression of a cell-cycle regulatory protein, cyclin A. TAM67 also inhibited anchorage-independent growth of these cells. Furthermore, TAM67 reduced growth of established xenograft tumours from these cells in nude mice. These results suggest that AP-1 plays an essential role in the growth of at least some of NSCLC cells. PMID:18283312

  9. CD28 family of receptors on T cells in chronic HBV infection: Expression characteristics, clinical significance and correlations with PD-1 blockade

    PubMed Central

    Tang, Zong-Sheng; Hao, You-Hua; Zhang, E-Juan; Xu, Chun-Li; Zhou, Yun; Zheng, Xin; Yang, Dong-Liang

    2016-01-01

    The aim of the present study was to investigate the overall clinical expression characteristics of the cluster of differentiation (CD)28 family receptors [CD28, inducible T-cell co-stimulator, programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 and B- and T-lymphocyte attenuator] on T cells in patients with chronic hepatitis B (CHB), analyze the correlations among these receptors and the clinical parameters, and to investigate the effects of PD-1 blockade on the receptor expression profiles, T-cell function and other biological effects. The expression characteristics of the CD28 family of receptors, the effects of PD-1 blockade on the receptor expression profiles and the levels of interferon (IFN)-γ were investigated in the T cells of patients with CHB. In addition, the transcription factor, T-box 21 (T-bet) and GATA binding protein 3 (GATA-3) mRNA expression levels were investigated in the peripheral blood mononuclear cells (PBMCs) of patients with CHB. The expression levels of the CD28 family receptors in the T cells of patients with CHB demonstrated distinct characteristics, for example levels of PD-1 and CTLA-4 on CD4 T cells and ICOS, PD-1, and BTLA on CD8 T cells were increased in cells from patients with CHB compared with those from the healthy individuals. A significant positive correlation was demonstrated among the serum HBV DNA titers and the levels of PD-1 on CD8+ T cells with the highest expression of PD-1 corresponding to viral levels >106 IU/ml. A significant positive correlation was observed between the serum HBV DNA titers and the expression levels of BTLA on CD8+ T cells with the highest expression of BTLA corresponding to viral levels >106 IU/ml. PD-1 blockade altered the expression profiles of CD28 family receptors in the T cells of patients with CHB, partly enhanced T cell function and increased the ratio of T-bet/GATA-3 mRNA in PBMCs. Thus, CD28 family receptors are potential clinical indicators for the rapid

  10. Prevention of Breast Cancer Cell Transformation by Blockade of the AP-1 Transcription Factor

    DTIC Science & Technology

    2001-10-01

    TX; M. Ahotupa, MCA Re- ity shift assays and a reporter gene sys- Another possible mechanism for search Laboratory, Department of Physiology , Uni- tem...formation. Biochim Biophys Acta 1991;1072: (34) Glock GE, McLean P. Further studies on the (50) Blask DE, Wilson ST, Zalatan F. Physiological 129-57...deprivation in pheochromocytoma cells (49). The mechanism by and differentiation of HCI I mammary epithelial cells. Mol. Endocrinol., 9: 14-23. which c-Jun

  11. Overcoming resistance to checkpoint blockade therapy by targeting PI3Kγ in myeloid cells.

    PubMed

    De Henau, Olivier; Rausch, Matthew; Winkler, David; Campesato, Luis Felipe; Liu, Cailian; Cymerman, Daniel Hirschhorn; Budhu, Sadna; Ghosh, Arnab; Pink, Melissa; Tchaicha, Jeremy; Douglas, Mark; Tibbitts, Thomas; Sharma, Sujata; Proctor, Jennifer; Kosmider, Nicole; White, Kerry; Stern, Howard; Soglia, John; Adams, Julian; Palombella, Vito J; McGovern, Karen; Kutok, Jeffery L; Wolchok, Jedd D; Merghoub, Taha

    2016-11-17

    Recent clinical trials using immunotherapy have demonstrated its potential to control cancer by disinhibiting the immune system. Immune checkpoint blocking (ICB) antibodies against cytotoxic-T-lymphocyte-associated protein 4 or programmed cell death protein 1/programmed death-ligand 1 have displayed durable clinical responses in various cancers. Although these new immunotherapies have had a notable effect on cancer treatment, multiple mechanisms of immune resistance exist in tumours. Among the key mechanisms, myeloid cells have a major role in limiting effective tumour immunity. Growing evidence suggests that high infiltration of immune-suppressive myeloid cells correlates with poor prognosis and ICB resistance. These observations suggest a need for a precision medicine approach in which the design of the immunotherapeutic combination is modified on the basis of the tumour immune landscape to overcome such resistance mechanisms. Here we employ a pre-clinical mouse model system and show that resistance to ICB is directly mediated by the suppressive activity of infiltrating myeloid cells in various tumours. Furthermore, selective pharmacologic targeting of the gamma isoform of phosphoinositide 3-kinase (PI3Kγ), highly expressed in myeloid cells, restores sensitivity to ICB. We demonstrate that targeting PI3Kγ with a selective inhibitor, currently being evaluated in a phase 1 clinical trial (NCT02637531), can reshape the tumour immune microenvironment and promote cytotoxic-T-cell-mediated tumour regression without targeting cancer cells directly. Our results introduce opportunities for new combination strategies using a selective small molecule PI3Kγ inhibitor, such as IPI-549, to overcome resistance to ICB in patients with high levels of suppressive myeloid cell infiltration in tumours.

  12. Calcium channel blockade reduces mechanical strain-induced extracellular matrix gene response in lamina cribrosa cells.

    PubMed

    Quill, B; Irnaten, M; Docherty, N G; McElnea, E M; Wallace, D M; Clark, A F; O'Brien, C J

    2015-07-01

    This study examines the effect of the L-type calcium channel blocker verapamil on mechanical strain-induced extracellular matrix genes in optic nerve head lamina cribrosa (LC) cells. Changes in LC cell intracellular calcium [Ca(2+)]i following hypotonic cell membrane stretch were measured with the fluorescent probe fura-2/AM. Fluorescence intensity was measured, after labelling, by calcium (Ca2+) imaging confocal microscopy. Confluent human LC cell cultures were serum starved for 24 h prior to exposure to cyclical mechanical strain (1 Hz, 15%) for 24 h in the presence or absence of verapamil (10 mm). Transforming growth factor-β 1 (TGF-β1), collagen 6A3 (COL6A3) and chondroitin sulfate proteoglycan 2 (CSPG2) mRNA expression levels were assessed by quantitative RT-PCR. Hypotonic cell membrane stretch of LC cells from normal donors significantly increased [Ca2+]i (p<0.05). Exposure to cyclical mechanical strain (15% strain) produced a statistically significant increase in the three matrix genes that were examined (TGF-β1, COL6A3 and CSPG2). This response in both cyclical and mechanical stretch was significantly reduced by pretreating LC cells with the L-type calcium channel blocker verapamil (p<0.05). This study provides evidence of a novel mechanotransduction pathway linking mechanical strain, cation channel function and the induction of LC cell matrix gene transcription. This highlights the potential involvement of calcium influx in the activation of matrix remodelling responses in the optic nerve head and supports the rationale that calcium channel blockers may attenuate disease progression in glaucoma. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  13. Glucocorticoid receptor blockade inhibits brain cell addition and aggressive signaling in electric fish, Apteronotus leptorhynchus.

    PubMed

    Dunlap, Kent D; Jashari, Denisa; Pappas, Kristina M

    2011-08-01

    When animals are under stress, glucocorticoids commonly inhibit adult neurogenesis by acting through glucocorticoid receptors (GRs). However, in some cases, conditions that elevate glucocorticoids promote adult neurogenesis, and the role of glucocorticoid receptors in these circumstances is not well understood. We examined the involvement of GRs in social enhancement of brain cell addition and aggressive signaling in electric fish, Apteronotus leptorhynchus. In this species, long-term social interaction simultaneously elevates plasma cortisol, enhances brain cell addition and increases production of aggressive electrocommunication signals ("chirps"). We implanted isolated and paired fish with capsules containing nothing (controls) or the GR antagonist, RU486, recorded chirp production and locomotion for 7d, and measured the density of newborn cells in the periventricular zone. Compared to isolated controls, paired controls showed elevated chirping in two phases: much higher chirp rates in the first 5h and moderately higher nocturnal rates thereafter. Treating paired fish with RU486 reduced chirp rates in both phases to those of isolated fish, demonstrating that GR activation is crucial for socially induced chirping. Neither RU486 nor social interaction affected locomotion. RU486 treatment to paired fish had a partial effect on cell addition: paired RU486 fish had less cell addition than paired control fish but more than isolated fish. This suggests that cortisol activation of GRs contributes to social enhancement of cell addition but works in parallel with another GR-independent mechanism. RU486 also reduced cell addition in isolated fish, indicating that GRs participate in the regulation of cell addition even when cortisol levels are low.

  14. In Vitro Apoptotic Effects of Farnesyltransferase blockade in Acute Myeloid Leukemia Cells

    PubMed Central

    Giudice, V; Ricci, P; Marino, L; Rocco, M; Villani, G; Langella, M; Manente, L; Seneca, E; Ferrara, I; Pezzullo, L; Serio, B; Selleri, C

    2016-01-01

    Farnesyltransferase inhibitors (FTIs) are a class of oral anti-cancer drugs currently tested in phase I-II clinical trials for treatment of hematological malignancies. The in vitro effects of various FTIs (alpha-hydroxyfarnesylphosphonic acid, manumycin-A and SCH66336) were tested on CD34+ KG1a cell line and in primary acute myeloid leukemia (AML) cells from 64 patients. By cell viability and clonogeneic methylcellulose assays, FTIs showed a significant inhibitory activity in CD34+ KG1a and primary bone marrow (BM) leukemic cells from 56% of AML patients. FTIs also induced activation of caspase-3 and Fas-independent apoptosis, confirmed by the finding that inhibition of caspase-8 was not associated with the rescue of FTI-treated cells. We concluded that other cellular events induced by FTIs may trigger activation of caspase-3 and subsequent apoptosis, but the expression of proapoptotic molecules, as Bcl-2 and Bcl-XL, and antiapoptotic, as Bcl-X(s), were not modified by FTIs. By contrast, expression of inducible nitric oxide synthase (iNOS) was increased in FTI-treated AML cells. Our results suggest a very complex mechanism of action of FTIs that require more studies for a better clinical use of the drugs alone or in combination in the treatment of hematological malignancies. PMID:27896224

  15. Blockade of GLUT1 by WZB117 resensitizes breast cancer cells to adriamycin.

    PubMed

    Chen, Qing; Meng, Ya-Qiu; Xu, Xiao-Fan; Gu, Jun

    2017-09-01

    The tolerance to adriamycin of cancer as a common and stubborn obstacle occurred during curing breast cancer patients needs to be overcome. In the present study, we explored whether inhibiting the glucose transporter 1 (GLUT1) could restore the activity of adriamycin in breast cancer cell line MCF-7 resistant to adriamycin and the possible underlying mechanisms. Adriamycin-resistant cell line MCF-7/ADR was selected stepwise from the parental MCF-7 cells and the level of GLUT1 was measured. Then, the MCF-7/ADR cells were incubated with adriamycin, WZB117 (a specific GLUT1 inhibitor), or both. The viability, proliferation and apoptosis of cells and the level of glucose and lactate were measured, respectively. Finally, the cytosolic and mitochondrial proteins were isolated and the activity of the adenosine monophosphate-activated protein kinase (AMPK)/phosphorylated AMPK, mammalian target of rapamycin (mTOR)/phosphorylated mTOR, and apoptotic-related protein BCL-2-associated X protein (BAX), Bcl-2 was assayed by western blot. We found that WZB117 resensitized MCF-7/ADR to adriamycin and increased BAX translocated to mitochondria, which through activation of AMPK and inhibition of mTOR in a high probability. Inhibition of the GLUT1 could partially restore the antineoplastic effects of adriamycin in the adriamycin-resistant MCF-7 cell line possibly through activating the AMPK, downregulating the mTOR pathway, and increasing the BAX translocation to mitochondria.

  16. IL-1R signaling enables bystander cells to overcome bacterial blockade of host protein synthesis

    PubMed Central

    Copenhaver, Alan M.; Casson, Cierra N.; Nguyen, Hieu T.; Duda, Matthew M.; Shin, Sunny

    2015-01-01

    The innate immune system is critical for host defense against microbial pathogens, yet many pathogens express virulence factors that impair immune function. Here, we used the bacterial pathogen Legionella pneumophila to understand how the immune system successfully overcomes pathogen subversion mechanisms. L. pneumophila replicates within macrophages by using a type IV secretion system to translocate bacterial effectors into the host cell cytosol. As a consequence of effector delivery, host protein synthesis is blocked at several steps, including translation initiation and elongation. Despite this translation block, infected cells robustly produce proinflammatory cytokines, but the basis for this is poorly understood. By using a reporter system that specifically discriminates between infected and uninfected cells within a population, we demonstrate here that infected macrophages produced IL-1α and IL-1β, but were poor producers of IL-6, TNF, and IL-12, which are critical mediators of host protection. Uninfected bystander cells robustly produced IL-6, TNF, and IL-12, and this bystander response required IL-1 receptor (IL-1R) signaling during early pulmonary infection. Our data demonstrate functional heterogeneity in production of critical protective cytokines and suggest that collaboration between infected and uninfected cells enables the immune system to bypass pathogen-mediated translation inhibition to generate an effective immune response. PMID:26034289

  17. IL-1R signaling enables bystander cells to overcome bacterial blockade of host protein synthesis.

    PubMed

    Copenhaver, Alan M; Casson, Cierra N; Nguyen, Hieu T; Duda, Matthew M; Shin, Sunny

    2015-06-16

    The innate immune system is critical for host defense against microbial pathogens, yet many pathogens express virulence factors that impair immune function. Here, we used the bacterial pathogen Legionella pneumophila to understand how the immune system successfully overcomes pathogen subversion mechanisms. L. pneumophila replicates within macrophages by using a type IV secretion system to translocate bacterial effectors into the host cell cytosol. As a consequence of effector delivery, host protein synthesis is blocked at several steps, including translation initiation and elongation. Despite this translation block, infected cells robustly produce proinflammatory cytokines, but the basis for this is poorly understood. By using a reporter system that specifically discriminates between infected and uninfected cells within a population, we demonstrate here that infected macrophages produced IL-1α and IL-1β, but were poor producers of IL-6, TNF, and IL-12, which are critical mediators of host protection. Uninfected bystander cells robustly produced IL-6, TNF, and IL-12, and this bystander response required IL-1 receptor (IL-1R) signaling during early pulmonary infection. Our data demonstrate functional heterogeneity in production of critical protective cytokines and suggest that collaboration between infected and uninfected cells enables the immune system to bypass pathogen-mediated translation inhibition to generate an effective immune response.

  18. Combined blockade of signalling pathways shows marked anti-tumour potential in phaeochromocytoma cell lines

    PubMed Central

    Nölting, Svenja; Garcia, Edwin; Alusi, Ghassan; Giubellino, Alessio; Pacak, Karel; Korbonits, Márta; Grossman, Ashley B

    2016-01-01

    Currently, there is no completely effective therapy available for metastatic phaeochromocytomas (PCCs) and paragangliomas. In this study, we explore new molecular targeted therapies for these tumours, using one more benign (mouse phaeochromocytoma cell (MPC)) and one more malignant (mouse tumour tissue (MTT)) mouse PCC cell line –both generated from heterozygous neurofibromin 1 knockout mice. Several PCC-promoting gene mutations have been associated with aberrant activation of PI3K/AKT, mTORC1 and RAS/RAF/ERK signalling. We therefore investigated different agents that interfere specifically with these pathways, including antagonism of the IGF1 receptor by NVP-AEW541. We found that NVP-AEW541 significantly reduced MPC and MTT cell viability at relatively high doses but led to a compensatory up-regulation of ERK and mTORC1 signalling at suboptimal doses while PI3K/AKT inhibition remained stable. We subsequently investigated the effect of the dual PI3K/mTORC1/2 inhibitor NVP-BEZ235, which led to a significant decrease of MPC and MTT cell viability at doses down to 50 nM but again increased ERK signalling. Accordingly, we next examined the combination of NVP-BEZ235 with the established agent lovastatin, as this has been described to inhibit ERK signalling. Lovastatin alone significantly reduced MPC and MTT cell viability at therapeutically relevant doses and inhibited both ERK and AKT signalling, but increased mTORC1/p70S6K signalling. Combination treatment with NVP-BEZ235 and lovastatin showed a significant additive effect in MPC and MTT cells and resulted in inhibition of both AKT and mTORC1/p70S6K signalling without ERK up-regulation. Simultaneous inhibition of PI3K/AKT, mTORC1/2 and ERK signalling suggests a novel therapeutic approach for malignant PCCs. PMID:22715163

  19. Dendritic Cell Vaccination Combined with CTLA4 Blockade in Patients with Metastatic Melanoma

    PubMed Central

    Ribas, Antoni; Comin-Anduix, Begoña; Chmielowski, Bartosz; Jalil, Jason; de la Rocha, Pilar; McCannel, Tara A.; Ochoa, Maria Teresa; Seja, Elizabeth; Villanueva, Arturo; Oseguera, Denise K.; Straatsma, Bradley R.; Cochran, Alistair J.; Glaspy, John A.; Hui, Liu; Marincola, Francesco M.; Wang, Ena; Economou, James S.; Gomez-Navarro, Jesus

    2009-01-01

    Purpose Tumor antigen-loaded dendritic cells (DC) are believed to activate antitumor immunity by stimulating T cells, and cytotoxic T lymphocyte-associated antigen 4 (CTLA4)-blocking antibodies should release a key negative regulatory pathway on T cells. The combination was tested in a phase 1 clinical trial in patients with advanced melanoma. Experimental Design Autologous DC were pulsed with MART-126-35 peptide and administered with a dose escalation of the CTLA4 blocking antibody tremelimumab. Sixteen patients were accrued to 5 dose levels. Primary endpoints were safety and immune effects; clinical efficacy was a secondary endpoint. Results Dose-limiting toxicities (DLTs) of grade 3 diarrhea and grade 2 hypophysitis developed in 2 out of 3 patients receiving tremelimumab at 10 mg/kg monthly. Four patients had an objective tumor response, two partial responses (PR) and two complete responses (CR), all melanoma-free between 2 and 4 years after study initiation. There was no difference in immune monitoring results between patients with an objective tumor response and those without a response. Exploratory gene expression analysis suggested that immune-related gene signatures, in particular for B cell function, may be important in predicting response. Conclusion The combination of MART-1 peptide-pulsed DC and tremelimumab results in objective and durable tumor responses at the higher range of the expected response rate with either agent alone. PMID:19789309

  20. Mitochondrial activation chemicals synergize with surface receptor PD-1 blockade for T cell-dependent antitumor activity.

    PubMed

    Chamoto, Kenji; Chowdhury, Partha S; Kumar, Alok; Sonomura, Kazuhiro; Matsuda, Fumihiko; Fagarasan, Sidonia; Honjo, Tasuku

    2017-01-31

    Although immunotherapy by PD-1 blockade has dramatically improved the survival rate of cancer patients, further improvement in efficacy is required to reduce the fraction of less sensitive patients. In mouse models of PD-1 blockade therapy, we found that tumor-reactive cytotoxic T lymphocytes (CTLs) in draining lymph nodes (DLNs) carry increased mitochondrial mass and more reactive oxygen species (ROS). We show that ROS generation by ROS precursors or indirectly by mitochondrial uncouplers synergized the tumoricidal activity of PD-1 blockade by expansion of effector/memory CTLs in DLNs and within the tumor. These CTLs carry not only the activation of mechanistic target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) but also an increment of their downstream transcription factors such as PPAR-gamma coactivator 1α (PGC-1α) and T-bet. Furthermore, direct activators of mTOR, AMPK, or PGC-1α also synergized the PD-1 blockade therapy whereas none of above-mentioned chemicals alone had any effects on tumor growth. These findings will pave a way to developing novel combinatorial therapies with PD-1 blockade.

  1. Mitochondrial activation chemicals synergize with surface receptor PD-1 blockade for T cell-dependent antitumor activity

    PubMed Central

    Chamoto, Kenji; Chowdhury, Partha S.; Kumar, Alok; Sonomura, Kazuhiro; Matsuda, Fumihiko; Fagarasan, Sidonia; Honjo, Tasuku

    2017-01-01

    Although immunotherapy by PD-1 blockade has dramatically improved the survival rate of cancer patients, further improvement in efficacy is required to reduce the fraction of less sensitive patients. In mouse models of PD-1 blockade therapy, we found that tumor-reactive cytotoxic T lymphocytes (CTLs) in draining lymph nodes (DLNs) carry increased mitochondrial mass and more reactive oxygen species (ROS). We show that ROS generation by ROS precursors or indirectly by mitochondrial uncouplers synergized the tumoricidal activity of PD-1 blockade by expansion of effector/memory CTLs in DLNs and within the tumor. These CTLs carry not only the activation of mechanistic target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) but also an increment of their downstream transcription factors such as PPAR-gamma coactivator 1α (PGC-1α) and T-bet. Furthermore, direct activators of mTOR, AMPK, or PGC-1α also synergized the PD-1 blockade therapy whereas none of above-mentioned chemicals alone had any effects on tumor growth. These findings will pave a way to developing novel combinatorial therapies with PD-1 blockade. PMID:28096382

  2. Structure-Guided Blockade of CSF1R Kinase in Tenosynovial Giant-Cell Tumor.

    PubMed

    Tap, William D; Wainberg, Zev A; Anthony, Stephen P; Ibrahim, Prabha N; Zhang, Chao; Healey, John H; Chmielowski, Bartosz; Staddon, Arthur P; Cohn, Allen Lee; Shapiro, Geoffrey I; Keedy, Vicki L; Singh, Arun S; Puzanov, Igor; Kwak, Eunice L; Wagner, Andrew J; Von Hoff, Daniel D; Weiss, Glen J; Ramanathan, Ramesh K; Zhang, Jiazhong; Habets, Gaston; Zhang, Ying; Burton, Elizabeth A; Visor, Gary; Sanftner, Laura; Severson, Paul; Nguyen, Hoa; Kim, Marie J; Marimuthu, Adhirai; Tsang, Garson; Shellooe, Rafe; Gee, Carolyn; West, Brian L; Hirth, Peter; Nolop, Keith; van de Rijn, Matt; Hsu, Henry H; Peterfy, Charles; Lin, Paul S; Tong-Starksen, Sandra; Bollag, Gideon

    2015-07-30

    Expression of the colony-stimulating factor 1 (CSF1) gene is elevated in most tenosynovial giant-cell tumors. This observation has led to the discovery and clinical development of therapy targeting the CSF1 receptor (CSF1R). Using x-ray co-crystallography to guide our drug-discovery research, we generated a potent, selective CSF1R inhibitor, PLX3397, that traps the kinase in the autoinhibited conformation. We then conducted a multicenter, phase 1 trial in two parts to analyze this compound. In the first part, we evaluated escalations in the dose of PLX3397 that was administered orally in patients with solid tumors (dose-escalation study). In the second part, we evaluated PLX3397 at the chosen phase 2 dose in an extension cohort of patients with tenosynovial giant-cell tumors (extension study). Pharmacokinetic and tumor responses in the enrolled patients were assessed, and CSF1 in situ hybridization was performed to confirm the mechanism of action of PLX3397 and that the pattern of CSF1 expression was consistent with the pathological features of tenosynovial giant-cell tumor. A total of 41 patients were enrolled in the dose-escalation study, and an additional 23 patients were enrolled in the extension study. The chosen phase 2 dose of PLX3397 was 1000 mg per day. In the extension study, 12 patients with tenosynovial giant-cell tumors had a partial response and 7 patients had stable disease. Responses usually occurred within the first 4 months of treatment, and the median duration of response exceeded 8 months. The most common adverse events included fatigue, change in hair color, nausea, dysgeusia, and periorbital edema; adverse events rarely led to discontinuation of treatment. Treatment of tenosynovial giant-cell tumors with PLX3397 resulted in a prolonged regression in tumor volume in most patients. (Funded by Plexxikon; ClinicalTrials.gov number, NCT01004861.).

  3. Blockade of interleukin-6 receptor suppresses the proliferation of H460 lung cancer stem cells.

    PubMed

    Yi, Hee; Cho, Hee-Jung; Cho, Soo-Min; Jo, Kyul; Park, Jin-A; Kim, Na-Hyun; Amidon, Gordon L; Kim, Jin-Suk; Shin, Ho-Chul

    2012-07-01

    IL-6/6R signaling is closely associated with tumor growth and poor prognosis. Although there is evidence that interleukin-6 receptor (IL-6R)-mediated signaling promotes the growth and malignancy of cancer, the role of IL-6R in cancer stem cells (CSCs) is poorly defined. This study investigated the role of IL-6R in the proliferation of CSCs. Sphere-forming cells were isolated from the H460 non-small cell lung cancer (NSCLC) cell line and identified as CSCs using confocal microscopy, RT-PCR and WST-1 assay. The H460 spheres demonstrated the typical characteristics of CSCs, including CD133 expression, upregulation of Nanog, self-renewal, and drug resistance to methotrexate (MTX) and fluorouracil (5-FU). The release of IL-6R and its ligand, IL-6, were quantitatively determined and compared between CSCs and non-CSCs. The concentration of soluble IL-6R (sIL-6R) was remarkably high in CSCs compared to that in non-CSCs. Furthermore, significant upregulation of the IL-6R gene was also observed in the CSCs. The growth of CSCs was significantly inhibited by transfection with IL-6R small-interfering RNA (siRNA), as well as with the IL-6R monoclonal antibody (mAb). In addition, blocking both IL-6R and IL-6 using siRNA or mAbs intensified the inhibition of CSC proliferation. These findings indicate that IL-6R is present in CSCs and has an important role in the proliferation of CSCs in the H460 lung cancer cell line. Therefore, we suggest that IL-6R is both a viable target for the development of CSC-directed lung cancer therapeutics and a potential CSC marker in NSCLC.

  4. Prevention of Breast Cancer Cell Transformation by Blockade of the AP-1 Transcription Factor

    DTIC Science & Technology

    1998-10-01

    Project (0704-0188), Washington, DC 20503. 1. AGENCY USE ONLY (Leave blank) 2 . REPORT DATE 3. REPORT TYPE AND DATES COVERED October 1998 Annual (1...cell lines are necessary reagents to address studies proposed in specific aim 2 and specific aim 3 for months 25-48. In the following years of...LIMITATION OF ABSTRAC OF REPORT OF THIS PAGE OF ABSTRACT Unclassified Unclassified 21 Unclassified Unlimited NSN 7540-01-280-5500 Standard Form 298 (Rev. 2

  5. CK2 blockade causes MPNST cell apoptosis and promotes degradation of β-catenin.

    PubMed

    Kendall, Jed J; Chaney, Katherine E; Patel, Ami V; Rizvi, Tilat A; Largaespada, David A; Ratner, Nancy

    2016-08-16

    Malignant peripheral nerve sheath tumors (MPNSTs) are soft tissue sarcomas that are a major cause of mortality of Neurofibromatosis type 1 (NF1) patients. MPNST patients have few therapeutic options available and only complete surgical resection can be curative. MPNST formation and survival are dependent on activated β-catenin signaling. The goal of this study was to determine if inhibition of the CK2 enzyme can be therapeutically exploited in MPNSTs, given CK2's role in mainta ining oncogenic phenotypes including stabilization of β-catenin. We found that CK2α is over-expressed in MPNSTs and is critical for maintaining cell survival, as the CK2 inhibitor, CX-4945 (Silmitasertib), and shRNA targeting CK2α each significantly reduce MPNST cell viability. These effects were preceded by loss of critical signaling pathways in MPNSTs, including destabilization of β-catenin and TCF8. CX-4945 administration in vivo slowed tumor growth and extends survival time. We conclude that CK2 inhibition is a promising approach to blocking β-catenin in MPNST cells, although combinatorial therapies may be required for maximal efficacy.

  6. CK2 blockade causes MPNST cell apoptosis and promotes degradation of β-catenin

    PubMed Central

    Kendall, Jed J.; Chaney, Katherine E.; Patel, Ami V.; Rizvi, Tilat A.; Largaespada, David A.; Ratner, Nancy

    2016-01-01

    Malignant peripheral nerve sheath tumors (MPNSTs) are soft tissue sarcomas that are a major cause of mortality of Neurofibromatosis type 1 (NF1) patients. MPNST patients have few therapeutic options available and only complete surgical resection can be curative. MPNST formation and survival are dependent on activated β-catenin signaling. The goal of this study was to determine if inhibition of the CK2 enzyme can be therapeutically exploited in MPNSTs, given CK2's role in mainta ining oncogenic phenotypes including stabilization of β-catenin. We found that CK2α is over-expressed in MPNSTs and is critical for maintaining cell survival, as the CK2 inhibitor, CX-4945 (Silmitasertib), and shRNA targeting CK2α each significantly reduce MPNST cell viability. These effects were preceded by loss of critical signaling pathways in MPNSTs, including destabilization of β-catenin and TCF8. CX-4945 administration in vivo slowed tumor growth and extends survival time. We conclude that CK2 inhibition is a promising approach to blocking β-catenin in MPNST cells, although combinatorial therapies may be required for maximal efficacy. PMID:27448963

  7. Bepridil (CERM-1978) blockade of action potentials in cultured rat aortic smooth muscle cells.

    PubMed

    Mras, S; Sperelakis, N

    1981-04-24

    Reaggregate cultures (primary) were prepared from enzyme-dispersed vascular smooth muscle (VSM) cells from rat aortas. The cultures were incubated for 7-10 days, and then studied by the intracellular microelectrode technique. The cells were electrically quiescent (mean resting potential of --47 mV), and extracellular electrical stimulation usually did not elicit a membrane response. Addition of 10 mM tetraethylammonium rapidly induced excitability, allowing the VSM cells to fire Ca2+-dependent action potentials in response to electrical stimulation. The electrical responses often had two components, an initial spike and a later plateau-like component. The action potential spikes had a mean amplitude of 22 mV but occasionally were overshooting; the plateaus had a mean duration (at 50% repolarization) of 3.8 sec. A new anti-anginal agent, bepridil (10(-8)-10(-5) M), depressed the amplitude and duration of the plateau and blocked the spike component of the action potential in a dose-dependent fashion without affecting the resting potential. This finding is consistent with the view that bepridil acts as a Ca2+-antagonistic agent to prevent the generation of the action potentials, and this action can explain its antianginal properties.

  8. VEGF blockade enables oncolytic cancer virotherapy in part by modulating intratumoral myeloid cells.

    PubMed

    Currier, Mark A; Eshun, Francis K; Sholl, Allyson; Chernoguz, Artur; Crawford, Kelly; Divanovic, Senad; Boon, Louis; Goins, William F; Frischer, Jason S; Collins, Margaret H; Leddon, Jennifer L; Baird, William H; Haseley, Amy; Streby, Keri A; Wang, Pin-Yi; Hendrickson, Brett W; Brekken, Rolf A; Kaur, Balveen; Hildeman, David; Cripe, Timothy P

    2013-05-01

    Understanding the host response to oncolytic viruses is important to maximize their antitumor efficacy. Despite robust cytotoxicity and high virus production of an oncolytic herpes simplex virus (oHSV) in cultured human sarcoma cells, intratumoral (ITu) virus injection resulted in only mild antitumor effects in some xenograft models, prompting us to characterize the host inflammatory response. Virotherapy induced an acute neutrophilic infiltrate, a relative decrease of ITu macrophages, and a myeloid cell-dependent upregulation of host-derived vascular endothelial growth factor (VEGF). Anti-VEGF antibodies, bevacizumab and r84, the latter of which binds VEGF and selectively inhibits binding to VEGF receptor-2 (VEGFR2) but not VEGFR1, enhanced the antitumor effects of virotherapy, in part due to decreased angiogenesis but not increased virus production. Neither antibody affected neutrophilic infiltration but both partially mitigated virus-induced depletion of macrophages. Enhancement of virotherapy-mediated antitumor effects by anti-VEGF antibodies could largely be recapitulated by systemic depletion of CD11b(+) cells. These data suggest the combined effect of oHSV virotherapy and anti-VEGF antibodies is in part due to modulation of a host inflammatory reaction to virus. Our data provide strong preclinical support for combined oHSV and anti-VEGF antibody therapy and suggest that understanding and counteracting the innate host response may help enable the full antitumor potential of oncolytic virotherapy.

  9. Blockade of PAR1 signaling with cell-penetrating pepducins inhibits Akt-survival pathways in breast cancer cells and suppresses tumor survival and metastasis

    PubMed Central

    Yang, Eric; Boire, Adrienne; Agarwal, Anika; Nguyen, Nga; O'Callaghan, Katie; Tu, Powen; Kuliopulos, Athan; Covic, Lidija

    2009-01-01

    Protease-activated receptor 1 (PAR1) is a G protein-coupled receptor that is not expressed in normal breast epithelia, but is up-regulated in invasive breast carcinomas. In the present study, we found that matrix metalloprotease-1 (MMP-1) robustly activates the PAR1-Akt survival pathway in breast carcinoma cells. This process is blocked by a cell-penetrating lipopeptide ‘pepducin’, P1pal-7, which is a potent inhibitor of cell viability in breast carcinoma cells expressing PAR1. Both a MMP-1 inhibitor and P1pal-7 significantly promote apoptosis in breast tumor xenografts and inhibit metastasis to the lungs by up to 88%. Dual therapy with P1pal-7 and taxotere inhibits the growth of MDA-MB-231 xenografts by 95%. Consistently, biochemical analysis of xenograft tumors treated with P1pal-7 or MMP-1 inhibitor demonstrated attenuated Akt activity. Ectopic expression of constitutively active Akt rescues breast cancer cells from the synergistic cytotoxicity of P1pal-7 and taxotere, suggesting that Akt is a critical component of PAR1-dependent cancer cell viability. Together, these findings indicate that blockade of MMP1-PAR1 signaling may provide a benefit beyond treatment with taxotere alone in advanced, metastatic breast cancer. PMID:19622769

  10. Degron protease blockade sensor to image epigenetic histone protein methylation in cells and living animals.

    PubMed

    Sekar, Thillai V; Foygel, Kira; Devulapally, Rammohan; Paulmurugan, Ramasamy

    2015-01-16

    Lysine methylation of histone H3 and H4 has been identified as a promising therapeutic target in treating various cellular diseases. The availability of an in vivo assay that enables rapid screening and preclinical evaluation of drugs that potentially target this cellular process will significantly expedite the pace of drug development. This study is the first to report the development of a real-time molecular imaging biosensor (a fusion protein, [FLuc2]-[Suv39h1]-[(G4S)3]-[H3-K9]-[cODC]) that can detect and monitor the methylation status of a specific histone lysine methylation mark (H3-K9) in live animals. The sensitivity of this sensor was assessed in various cell lines, in response to down-regulation of methyltransferase EHMT2 by specific siRNA, and in nude mice with lysine replacement mutants. In vivo imaging in response to a combination of methyltransferase inhibitors BIX01294 and Chaetocin in mice reveals the potential of this sensor for preclinical drug evaluation. This biosensor thus has demonstrated its utility in the detection of H3-K9 methylations in vivo and potential value in preclinical drug development.

  11. Selective blockade and recovery of cell surface alpha 2-adrenergic receptors in human erythroleukemia (HEL) cells. Studies with the irreversible antagonist benextramine

    SciTech Connect

    McKernan, R.M.; Strickland, W.R.; Insel, P.A.

    1988-01-01

    alpha 2-Adrenergic receptors are present on human erythroleukemia (HEL) cells, both on the cell surface and in a sequestered compartment. In the current study we show that benextramine, a hydrophilic irreversible antagonist, can be used to investigate alpha 2-adrenergic receptor compartmentation in these cells. In membranes prepared from HEL cells, benextramine competed for all alpha 2-adrenergic receptors ( (/sup 3/H)yohimbine sites). In intact cells, at 4 degrees, benextramine exhibited a biphasic competition curve for alpha 2-adrenergic receptors, with EC50 values of approximately 10 microM and greater than 1 mM for the high and low affinity components, respectively. We propose that the alpha 2-adrenergic receptors preferentially blocked by benextramine are those on the surface of the cell, whereas those with low affinity are sequestered receptors because: 1) only epinephrine-accessible sites are removed by prior treatment of cells with benextramine, 2) a preparation enriched with surface membranes is also enriched in receptors with a high affinity for benextramine; and 3) after blockade of cell surface receptors (54 +/- 6% of total sites, n = 7) by benextramine, the ability of the alpha 2-adrenergic agonists epinephrine and UK-14,304 to inhibit forskolin-stimulated cAMP accumulation is lost. The latter result implies that only cell surface and not sequestered receptors are functionally coupled to adenylate cyclase. The return of receptors from the sequestered compartment to the cell surface and the recovery of alpha 2-adrenergic receptor function were measured after HEL cells were treated with benextramine (50 microM for 1 hr at 4 degrees). The recovery of receptor binding (t1/2 = 25 min) was somewhat slower than the recovery of function (t1/2 approximately 8 min).

  12. Targeting myeloid-derived suppressor cells with colony stimulating factor-1 receptor blockade can reverse immune resistance to immunotherapy in indoleamine 2,3-dioxygenase-expressing tumors

    PubMed Central

    Holmgaard, Rikke B.; Zamarin, Dmitriy; Lesokhin, Alexander; Merghoub, Taha; Wolchok, Jedd D.

    2016-01-01

    Tumor indoleamine 2,3-dioxygenase (IDO) promotes immunosuppression by direct action on effector T cells and Tregs and through recruitment, expansion and activation of myeloid-derived suppressor cells (MDSCs). Targeting of MDSCs is clinically being explored as a therapeutic strategy, though optimal targeting strategies and biomarkers predictive of response are presently unknown. Maturation and tumor recruitment of MDSCs are dependent on signaling through the receptor tyrosine kinase CSF-1R on myeloid cells. Here, we show that MDSCs are the critical cell population in IDO-expressing B16 tumors in mediating accelerated tumor outgrowth and resistance to immunotherapy. Using a clinically relevant drug, we show that inhibition of CSF-1R signaling can functionally block tumor-infiltrating MDSCs and enhance anti-tumor T cell responses. Furthermore, inhibition of CSF-1R sensitizes IDO-expressing tumors to immunotherapy with T cell checkpoint blockade, and combination of CSF-1R blockade with IDO inhibitors potently elicits tumor regression. These findings provide evidence for a critical and functional role for MDSCs on the in vivo outcome of IDO-expressing tumors. PMID:27211548

  13. Residual Neuromuscular Blockade.

    PubMed

    Plummer-Roberts, Anna L; Trost, Christina; Collins, Shawn; Hewer, Ian

    2016-02-01

    This article provides an update on residual neuromuscular blockade for nurse anesthetists. The neuromuscular junction, pharmacology for producing and reversing neuromuscular blockade, monitoring sites and methods, and patient implications relating to incomplete reversal of neuromuscular blockade are reviewed. Overall recommendations include using multiple settings when employing a peripheral nerve stimulator for monitoring return of neuromuscular function and administering pharmacologic reversal when the train-of-four ratio is below 0.9.

  14. Expression of PD-L1 on Canine Tumor Cells and Enhancement of IFN-γ Production from Tumor-Infiltrating Cells by PD-L1 Blockade

    PubMed Central

    Maekawa, Naoya; Konnai, Satoru; Ikebuchi, Ryoyo; Okagawa, Tomohiro; Adachi, Mami; Takagi, Satoshi; Kagawa, Yumiko; Nakajima, Chie; Suzuki, Yasuhiko; Murata, Shiro; Ohashi, Kazuhiko

    2014-01-01

    Programmed death 1 (PD-1), an immunoinhibitory receptor, and programmed death ligand 1 (PD-L1), its ligand, together induce the “exhausted” status in antigen-specific lymphocytes and are thus involved in the immune evasion of tumor cells. In this study, canine PD-1 and PD-L1 were molecularly characterized, and their potential as therapeutic targets for canine tumors was discussed. The canine PD-1 and PD-L1 genes were conserved among canine breeds. Based on the sequence information obtained, the recombinant canine PD-1 and PD-L1 proteins were constructed; they were confirmed to bind each other. Antibovine PD-L1 monoclonal antibody effectively blocked the binding of recombinant PD-1 with PD-L1–expressing cells in a dose-dependent manner. Canine melanoma, mastocytoma, renal cell carcinoma, and other types of tumors examined expressed PD-L1, whereas some did not. Interestingly, anti-PD-L1 antibody treatment enhanced IFN-γ production from tumor-infiltrating cells. These results showed that the canine PD-1/PD-L1 pathway is also associated with T-cell exhaustion in canine tumors and that its blockade with antibody could be a new therapeutic strategy for canine tumors. Further investigations are needed to confirm the ability of anti-PD-L1 antibody to reactivate canine antitumor immunity in vivo, and its therapeutic potential has to be further discussed. PMID:24915569

  15. Expression of PD-L1 on canine tumor cells and enhancement of IFN-γ production from tumor-infiltrating cells by PD-L1 blockade.

    PubMed

    Maekawa, Naoya; Konnai, Satoru; Ikebuchi, Ryoyo; Okagawa, Tomohiro; Adachi, Mami; Takagi, Satoshi; Kagawa, Yumiko; Nakajima, Chie; Suzuki, Yasuhiko; Murata, Shiro; Ohashi, Kazuhiko

    2014-01-01

    Programmed death 1 (PD-1), an immunoinhibitory receptor, and programmed death ligand 1 (PD-L1), its ligand, together induce the "exhausted" status in antigen-specific lymphocytes and are thus involved in the immune evasion of tumor cells. In this study, canine PD-1 and PD-L1 were molecularly characterized, and their potential as therapeutic targets for canine tumors was discussed. The canine PD-1 and PD-L1 genes were conserved among canine breeds. Based on the sequence information obtained, the recombinant canine PD-1 and PD-L1 proteins were constructed; they were confirmed to bind each other. Antibovine PD-L1 monoclonal antibody effectively blocked the binding of recombinant PD-1 with PD-L1-expressing cells in a dose-dependent manner. Canine melanoma, mastocytoma, renal cell carcinoma, and other types of tumors examined expressed PD-L1, whereas some did not. Interestingly, anti-PD-L1 antibody treatment enhanced IFN-γ production from tumor-infiltrating cells. These results showed that the canine PD-1/PD-L1 pathway is also associated with T-cell exhaustion in canine tumors and that its blockade with antibody could be a new therapeutic strategy for canine tumors. Further investigations are needed to confirm the ability of anti-PD-L1 antibody to reactivate canine antitumor immunity in vivo, and its therapeutic potential has to be further discussed.

  16. Opioid blockade effect on insulin beta-cells secretory patterns in polycystic ovary syndrome. Oral glucose load versus intravenous glucagon bolus.

    PubMed

    Ciampelli, M; Fulghesu, A M; Guido, M; Murgia, F; Muzj, G; Belosi, C; Fortini, A; Cento, R; Lanzone, A

    1998-01-01

    In order to evaluate the involvement of endogenous opiates in the insulin disorders of polycystic ovary syndrome (PCOs) a total of 25 PCOs women and 11 normo-ovulatory controls were studied by comparing the effect of a chronic opioid blockade on beta-cells responsiveness to oral glucose load and to intravenous glucagon bolus. Each patient, studied on follicular phase, underwent to oral glucose tolerance test (OGTT), and, 2 days later, to a glucagon intravenous bolus (1 mg); these tests were then repeated after 6 weeks of naltrexone treatment (50 mg orally). Naltrexone treatment did not modify the insulin secretory patterns of control subjects, whereas the same therapy significantly reduced, in hyperinsulinemic PCOs women, the beta-cell hyperresponsiveness both to oral glucose load and to intravenous glucagon (p < 0.05 and p < 0.01, respectively), even if with different mean percent decrease (32% OGTT vs. 45% glucagon, p < 0.05). Moreover, normoinsulinemic PCOs patients showed a slight, but not significantly increase in the beta-cells response to OGTT after opioid blockade, whereas, in the same situation, the insulin release after glucagon bolus was significantly reduced (p < 0.01). Chronic opioid blockade did not modify gonadotropins, steroids and SHBG levels in either group. Our data show that naltrexone treatment is able to reduce the beta-cell response to a direct intravenous secretagogue stimulus in all PCOs patients, while only in hyperinsulinemic PCOs subjects the same treatment is effective in reducing the exaggerated insulin secretion after oral glucose load. The reason for such a discrepancy could be ascribed to a different effect of opioids on first- and second-phase insulin secretion, or, alternatively, to an involvement of other secretagogue factors, such as glucoincretins.

  17. CSF1/CSF1R blockade reprograms tumor-infiltrating macrophages and improves response to T-cell checkpoint immunotherapy in pancreatic cancer models.

    PubMed

    Zhu, Yu; Knolhoff, Brett L; Meyer, Melissa A; Nywening, Timothy M; West, Brian L; Luo, Jingqin; Wang-Gillam, Andrea; Goedegebuure, S Peter; Linehan, David C; DeNardo, David G

    2014-09-15

    Cancer immunotherapy generally offers limited clinical benefit without coordinated strategies to mitigate the immunosuppressive nature of the tumor microenvironment. Critical drivers of immune escape in the tumor microenvironment include tumor-associated macrophages and myeloid-derived suppressor cells, which not only mediate immune suppression, but also promote metastatic dissemination and impart resistance to cytotoxic therapies. Thus, strategies to ablate the effects of these myeloid cell populations may offer great therapeutic potential. In this report, we demonstrate in a mouse model of pancreatic ductal adenocarcinoma (PDAC) that inhibiting signaling by the myeloid growth factor receptor CSF1R can functionally reprogram macrophage responses that enhance antigen presentation and productive antitumor T-cell responses. Investigations of this response revealed that CSF1R blockade also upregulated T-cell checkpoint molecules, including PDL1 and CTLA4, thereby restraining beneficial therapeutic effects. We found that PD1 and CTLA4 antagonists showed limited efficacy as single agents to restrain PDAC growth, but that combining these agents with CSF1R blockade potently elicited tumor regressions, even in larger established tumors. Taken together, our findings provide a rationale to reprogram immunosuppressive myeloid cell populations in the tumor microenvironment under conditions that can significantly empower the therapeutic effects of checkpoint-based immunotherapeutics. ©2014 American Association for Cancer Research.

  18. CSF1/CSF1R Blockade Reprograms Tumor-Infiltrating Macrophages and Improves Response to T Cell Checkpoint Immunotherapy in Pancreatic Cancer Models

    PubMed Central

    Zhu, Yu; Knolhoff, Brett L.; Meyer, Melissa A.; Nywening, Timothy M.; West, Brian L.; Luo, Jingqin; Wang-Gillam, Andrea; Goedegebuure, S Peter; Linehan, David C.; DeNardo, David G.

    2014-01-01

    Cancer immunotherapy generally offers limited clinical benefit without coordinated strategies to mitigate the immunosuppressive nature of the tumor microenvironment. Critical drivers of immune escape in the tumor microenvironment include tumor-associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC), which not only mediate immune suppression but also promote metastatic dissemination and impart resistance to cytotoxic therapies. Thus, strategies to ablate the effects of these myeloid cell populations may offer great therapeutic potential. In this report, we demonstrate in a mouse model of pancreatic ductal adenocarcinoma (PDAC) that inhibiting signaling by the myeloid growth factor receptor CSF1R can functionally reprogram macrophage responses that enhance antigen presentation and productive anti-tumor T cell responses. Investigations of this response revealed that CSF1R blockade also upregulated T cell checkpoint molecules, including PDL1 and CTLA4, thereby restraining beneficial therapeutic effects. We found that PD1 and CTLA4 antagonists showed limited efficacy as single agents to restrain PDAC growth, but that that combining these agents with CSF1R blockade potently elicited tumor regressions, even in larger established tumors. Taken together, our findings provide a rationale to reprogram immunosuppressive myeloid cell populations in the tumor microenvironment under conditions that can significantly empower the therapeutic effects of checkpoint-based immunotherapeutics. PMID:25082815

  19. PD-1 blockade attenuates immunosuppressive myeloid cells due to inhibition of CD47/SIRPα axis in HPV negative head and neck squamous cell carcinoma.

    PubMed

    Yu, Guang-Tao; Bu, Lin-Lin; Huang, Cong-Fa; Zhang, Wen-Feng; Chen, Wan-Jun; Gutkind, J Silvio; Kulkarni, Ashok B; Sun, Zhi-Jun

    2015-12-08

    Myeloid-derived suppressor cells (MDSCs) and tumor associated macrophages (TAMs) play key roles in the tumor immune suppressive network and tumor progression. However, precise roles of programmed death-1 (PD-1) in immunological functions of MDSCs and TAMs in head and neck squamous cell carcinoma (HNSCC) have not been clearly elucidated. In the present study, we show that PD-1 and PD-L1 levels were significantly higher in human HNSCC specimen than in normal oral mucosa. MDSCs and TAMs were characterized in mice and human HNSCC specimen, correlated well with PD-1 and PD-L1 expression. αPD-1 treatment was well tolerated and significantly reduced tumor growth in the HNSCC mouse model along with significant reduction in MDSCs and TAMs in immune organs and tumors. Molecular analysis suggests a reduction in the CD47/SIRPα pathway by PD-1 blockade, which regulates MDSCs, TAMs, dendritic cell as well as effector T cells. Hence, these data identify that PD-1/PD-L1 axis is significantly increased in human and mouse HNSCC. Adoptive αPD-1 immunotherapy may provide a novel therapeutic approach to modulate the micro- and macro-environment in HNSCC.

  20. Therapeutic PD-1 pathway blockade augments with other modalities of immunotherapy T-cell function to prevent immune decline in ovarian cancer.

    PubMed

    Duraiswamy, Jaikumar; Freeman, Gordon J; Coukos, George

    2013-12-01

    The tumor microenvironment mediates induction of the immunosuppressive programmed cell death-1 (PD-1) pathway, and targeted interventions against this pathway can help restore antitumor immunity. To gain insight into these responses, we studied the interaction between PD-1 expressed on T cells and its ligands (PD-1:PD-L1, PD-1:PD-L2, and PD-L1:B7.1), expressed on other cells in the tumor microenvironment, using a syngeneic orthotopic mouse model of epithelial ovarian cancer (ID8). Exhaustion of tumor-infiltrating lymphocytes (TIL) correlated with expression of PD-1 ligands by tumor cells and tumor-derived myeloid cells, including tumor-associated macrophages (TAM), dendritic cells, and myeloid-derived suppressor cells (MDSC). When combined with GVAX or FVAX vaccination (consisting of irradiated ID8 cells expressing granulocyte macrophage colony-stimulating factor or FLT3 ligand) and costimulation by agonistic α-4-1BB or TLR 9 ligand, antibody-mediated blockade of PD-1 or PD-L1 triggered rejection of ID8 tumors in 75% of tumor-bearing mice. This therapeutic effect was associated with increased proliferation and function of tumor antigen-specific effector CD8(+) T cells, inhibition of suppressive regulatory T cells (Treg) and MDSC, upregulation of effector T-cell signaling molecules, and generation of T memory precursor cells. Overall, PD-1/PD-L1 blockade enhanced the amplitude of tumor immunity by reprogramming suppressive and stimulatory signals that yielded more powerful cancer control.

  1. Time-specific blockade of PDGFR with Imatinib (Glivec®) causes cataract and disruption of lens fiber cells in neonatal mice.

    PubMed

    Zhou, Yin-Pin; He, Yang-Tao; Chen, Cheng-Li; Ji, Jun; Niu, Jian-Qin; Wang, Han-Zhi; Li, Shi-Feng; Huang, Lan; Mei, Feng

    2011-03-01

    This study aimed at investigating the response of lens epithelial cells in postnatal mice to Imatinib (Glivec®, a potent inhibitor of platelet-derived growth factor receptor (PDGFR)) treatment. Mouse eyes were sampled 10 days after administration of Imatinib (0.5 mg·g(-1)·day(-1)) for 3 days, at either 7, 14, or 21 days postpartum. Structural changes of lens were revealed by routine H.E. staining. Levels of proliferation and apoptosis were revealed by BrdU incorporation and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, respectively, and immunofluorescent staining with anti-PDGFRα antibody was carried out on the sections of eyeball. PDGFRα and p-PDGFRαprotein levels were evaluated by Western blot. Our results indicated that administration of Imatinib led to blockade of PDGFR signaling. Formation of cataracts was found only in those mice where treatment started from 7 days postpartum (P7), but was not observed in those samples from P14 nor P21. Fiber cells were disorganized in cataract lens core as observed histologically, and migration of epithelial cells was also inhibited. No apoptosis was detected with the TUNEL method. Our results indicated blockade of PDGFR at the neonatal stage (P7) would lead to cataracts and lens fiber cells disorganization, suggesting that PDGFR signaling plays a time-specific and crucial role in the postnatal development of lens in the mouse, and also may provide a new approach to produce a congenital cataract animal model.

  2. OX40- and CD27-mediated co-stimulation synergize with anti-PD-L1 blockade by forcing exhausted CD8+ T cells to exit quiescence

    PubMed Central

    Rogel, Anne; Edwards, Noha; Zhang, Lei; Sivakumaran, Shivajanani; Ghorashian, Sara; Carpenter, Ben; Bennett, Clare; Freeman, Gordon J.; Sykes, Megan; Croft, Michael; Al-Shamkhani, Aymen; Chakraverty, Ronjon

    2014-01-01

    Exhaustion of chronically stimulated CD8+ T cells is a significant obstacle to immune control of chronic infections or tumors. Although co-inhibitory checkpoint blockade with anti-programmed death-ligand 1 (PD-L1) antibody can restore functions to exhausted T cell populations, recovery is often incomplete and dependent upon the pool size of a quiescent T-bethigh subset that express lower levels of PD-1. In a model where unhelped, HY-specific CD8+ T cells gradually lose function following transfer to male BMT recipients, we have explored the effect of shifting the balance away from co-inhibition and toward co-stimulation by combining anti-PD-L1 with agonistic antibodies to the tumor-necrosis factor receptor superfamily members, OX40 and CD27. Several weeks following T cell transfer, both agonistic antibodies but especially anti-CD27 demonstrated synergy with anti-PD-L1 by enhancing CD8+ T cell proliferation and effector cytokine generation. Anti-CD27 and anti-PD-L1 synergised by downregulating the expression of multiple quiescence-related genes concomitant with a reduced frequency of T-bethigh cells within the exhausted population. However, in the presence of persistent antigen, the CD8+ T cell response was not sustained and the overall size of the effector cytokine-producing pool eventually contracted to levels below that of controls. Thus, CD27-mediated co-stimulation can synergize with co-inhibitory checkpoint blockade to switch off molecular programs for quiescence in exhausted T cell populations but at the expense of losing precursor cells required to maintain a response. PMID:25404365

  3. AP-1 blockade in breast cancer cells causes cell cycle arrest by suppressing G1 cyclin expression and reducing cyclin-dependent kinase activity.

    PubMed

    Liu, Yongmin; Lu, Chunhua; Shen, Qiang; Munoz-Medellin, Debbie; Kim, Heetae; Brown, Powel H

    2004-10-28

    The AP-1 transcription factor is a central component of signal transduction pathways in many cells, although the exact role of AP-1 in controlling cell growth and malignant transformation is unknown. We have previously shown that AP-1 complexes are activated by peptide and steroid growth factors in both normal and malignant breast cells, and that blocking AP-1 by overexpressing a dominant-negative form of cJun (cJun-DN, TAM67) inhibits breast cancer cell growth both in vivo and in vitro. We hypothesized that TAM67 inhibits cell growth by altering the expression of cell cycle regulatory proteins, thus causing a cell cycle block. In the present study, we used clones of MCF7 breast cancer cells that express TAM67 under the control of an inducible promoter. First, we determined the effect of AP-1 blockade on cell growth, then we performed 3H-thymidine incorporation and flow cytometry assays to investigate whether TAM67 inhibits the cell cycle. We observed that in the presence of serum TAM67 inhibited cell growth and caused a block in the G1 phase of the cell cycle. Next, we performed Western-blotting and CDK kinase assays to determine the effects of TAM67 on retinoblastoma (Rb) phosphorylation, the expression of cell cycle regulatory proteins, and CDK activity. We discovered that TAM67 inhibited Rb phosphorylation and reduced E2F activity. We also found that TAM67 decreased the expression of D and E cyclins, reduced CDK2 and CDK4 activity, and increased the CDK inhibitor p27. The studies of gene expression at the RNA level showed that TAM67 decreased cyclin Ds mRNA expression. Our study suggests that in the presence of serum, TAM67 inhibits breast cancer growth predominantly by inducing inhibitors of cyclin-dependent kinases (such as p27) and by reducing the expression of the cyclins involved in transitioning from G1 into S phase of the cell cycle. These studies lay the foundation for future attempt to develop new agents for the treatment and prevention of breast

  4. Blockade of dendritic cell development by bacterial fermentation products butyrate and propionate through a transporter (Slc5a8)-dependent inhibition of histone deacetylases.

    PubMed

    Singh, Nagendra; Thangaraju, Muthusamy; Prasad, Puttur D; Martin, Pamela M; Lambert, Nevin A; Boettger, Thomas; Offermanns, Stefan; Ganapathy, Vadivel

    2010-09-03

    Mammalian colon harbors trillions of bacteria, yet there is no undue inflammatory response by the host against these bacteria under normal conditions. The bacterial fermentation products acetate, propionate, and butyrate are believed, at least in part, to be responsible for these immunosuppressive effects. Dendritic cells play an essential role in presentation of antigens to T lymphocytes and initiation of adaptive immune responses. Here we report that butyrate and propionate block the generation of dendritic cells from bone marrow stem cells, without affecting the generation of granulocytes. This effect is dependent on the Na(+)-coupled monocarboxylate transporter Slc5a8, which transports butyrate and propionate into cells, and on the ability of these two bacterial metabolites to inhibit histone deacetylases. Acetate, which is also a substrate for Slc5a8 but not an inhibitor of histone deacetylases, does not affect dendritic cell development, indicating the essential role of histone deacetylase inhibition in the process. The blockade of dendritic cell development by butyrate and propionate is associated with decreased expression of the transcription factors PU.1 and RelB. Butyrate also elicits its biologic effects through its ability to activate the G-protein-coupled receptor Gpr109a, but this mechanism is not involved in butyrate-induced blockade of dendritic cell development. The participation of Slc5a8 and the non-involvement of Gpr109a in butyrate effects have been substantiated using bone marrow cells obtained from Slc5a8(-/-) and Gpr109a(-/-) mice. These findings uncover an important mechanism underlying the anti-inflammatory functions of the bacterial fermentation products butyrate and propionate.

  5. Blockade of Dendritic Cell Development by Bacterial Fermentation Products Butyrate and Propionate through a Transporter (Slc5a8)-dependent Inhibition of Histone Deacetylases

    PubMed Central

    Singh, Nagendra; Thangaraju, Muthusamy; Prasad, Puttur D.; Martin, Pamela M.; Lambert, Nevin A.; Boettger, Thomas; Offermanns, Stefan; Ganapathy, Vadivel

    2010-01-01

    Mammalian colon harbors trillions of bacteria, yet there is no undue inflammatory response by the host against these bacteria under normal conditions. The bacterial fermentation products acetate, propionate, and butyrate are believed, at least in part, to be responsible for these immunosuppressive effects. Dendritic cells play an essential role in presentation of antigens to T lymphocytes and initiation of adaptive immune responses. Here we report that butyrate and propionate block the generation of dendritic cells from bone marrow stem cells, without affecting the generation of granulocytes. This effect is dependent on the Na+-coupled monocarboxylate transporter Slc5a8, which transports butyrate and propionate into cells, and on the ability of these two bacterial metabolites to inhibit histone deacetylases. Acetate, which is also a substrate for Slc5a8 but not an inhibitor of histone deacetylases, does not affect dendritic cell development, indicating the essential role of histone deacetylase inhibition in the process. The blockade of dendritic cell development by butyrate and propionate is associated with decreased expression of the transcription factors PU.1 and RelB. Butyrate also elicits its biologic effects through its ability to activate the G-protein-coupled receptor Gpr109a, but this mechanism is not involved in butyrate-induced blockade of dendritic cell development. The participation of Slc5a8 and the non-involvement of Gpr109a in butyrate effects have been substantiated using bone marrow cells obtained from Slc5a8−/− and Gpr109a−/− mice. These findings uncover an important mechanism underlying the anti-inflammatory functions of the bacterial fermentation products butyrate and propionate. PMID:20601425

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

    PubMed

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

    2007-10-15

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

  7. Blockade of GpIIb/IIIa inhibits the release of vascular endothelial growth factor (VEGF) from tumor cell-activated platelets and experimental metastasis.

    PubMed

    Amirkhosravi, A; Amaya, M; Siddiqui, F; Biggerstaff, J P; Meyer, T V; Francis, J L

    1999-01-01

    Evidence that platelets play a role in tumor metastasis includes the observation of circulating tumor cell-platelet aggregates and the anti-metastatic effect of thrombocytopenia and anti-platelet drugs. Platelets have recently been shown to contain vascular endothelial growth factor (VEGF) which is released during clotting. We therefore studied the effects of (1) tumor cell-platelet adherence and tumor cell TF activity on platelet VEGF release; and (2) the effects of GpIIb/IIIa blockade on tumor cell-induced platelet VEGF release, tumor cell-induced thrombocytopenia and experimental metastasis. Adherent A375 human melanoma cells (TF+) and KG1 myeloid leukemia (TF-) cells were cultured in RPMI containing 10% fetal bovine serum. Platelet-rich plasma was obtained from normal citrated whole blood and the presence of VEGF (34 and 44 kDa isoforms) confirmed by immunoblotting. Platelet-rich plasma with or without anti-GpIIb/IIIa (Abciximab) was added to A375 monolayers and supernatant VEGF measured by ELISA. Tumor cell-induced platelet activation and release were determined by CD62P expression and serotonin release respectively. In vitro, tumor cell-platelet adherence was evaluated by flow cytometry. In vivo, thrombocytopenia and lung seeding were assessed 30 min and 18 days, respectively, after i.v. injection of Lewis Lung carcinoma (LL2) cells into control or murine 7E3 F(ab')(2) (6 mg/ kg) athymic rats. Maximal in vitro platelet activation (72% serotonin release) occurred 30 min after adding platelets to tumor cells. At this time, 87% of the A375 cells had adhered to platelets. Abciximab significantly (P<0.05) reduced platelet adherence to tumor cells as evidenced by flow cytometry. Incubation of A375 cells with platelets induced VEGF release in a time-dependent manner. This release was significantly inhibited by Abciximab (81% at 30 min; P<0.05). In the presence of fibrinogen and FII, VEGF release induced by A375 (TF+) cells was significantly higher than that induced

  8. T-Cell Immunoglobulin- and Mucin-Domain-Containing Molecule 3 Signaling Blockade Improves Cell-Mediated Immunity Against Malaria.

    PubMed

    Hou, Nan; Zou, Yang; Piao, Xianyu; Liu, Shuai; Wang, Lei; Li, Shanshan; Chen, Qijun

    2016-11-15

    Cell-mediated immune responses play important roles in immune protection against Plasmodium infection. However, impaired immunity, such as lymphocyte exhaustion, is a common phenomenon in malaria. T-cell immunoglobulin- and mucin-domain-containing molecule 3 (Tim-3) is an important regulatory molecule in cell-mediated immunity and has been implicated in malaria. In this study, it was found that Tim-3 expression on key populations of lymphocytes was significantly increased in both Plasmodium falciparum-infected patients and Plasmodium berghei ANKA (PbANKA)-infected C57BL/6 mice. Upregulation of Tim-3 led to lymphocyte exhaustion, while blocking Tim-3 signaling with an anti-Tim-3 antibody restored lymphocyte activity in Plasmodium infections. Further, anti-Tim-3 treatment accelerated the parasite clearance and relieved the symptoms of cerebral malaria in PbANKA-infected mice. In conclusion, Tim-3 on immune cells negatively regulates cell-mediated immunity against Plasmodium infection, and blocking Tim-3 signaling enhances sterile immunity and may play a protective role during malarial parasite infections. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  9. Blockade of NFκB activity by Sunitinib increases cell death in Bortezomib-treated endometrial carcinoma cells.

    PubMed

    Sorolla, Anabel; Yeramian, Andrée; Valls, Joan; Dolcet, Xavier; Bergadà, Laura; Llombart-Cussac, Antoni; Martí, Rosa Maria; Matias-Guiu, Xavier

    2012-10-01

    Endometrial carcinoma is one of the most common malignancies in the female genital tract, usually treated by surgery and radiotherapy. Chemotherapy is used when endometrial carcinoma is associated with widespread metastasis or when the tumor recurs after radiation therapy. In the present study, we demonstrate that the tyrosine kinase receptor inhibitor Sunitinib reduces cell viability, proliferation, clonogenicity and induces apoptotic cell death in endometrial carcinoma cell lines, which is not due to its action through the most known targets like VEGFR, nor through EGFR as demonstrated in this work. Interestingly, Sunitinib reduces NFκB transcriptional activity either at basal level or activation by EGF or TNF-α. We observed that Sunitinib was able to inhibit the Bortezomib-induced NFκB transcriptional activity which correlates with a decrease of the phosphorylated levels of IKKα and β, p65 and IκBα. We evaluated the nature of the interaction between Sunitinib and Bortezomib by the dose effect method and identified a synergistic effect (combination index < 1). Analogously, silencing of p65 expression by lentiviral-mediated short-hairpin RNA delivery in Bortezomib treated cells leads to a strongly increased sensitivity to Bortezomib apoptotic cell death. Altogether our results suggest that the combination of Sunitinib and Bortezomib could be considered a promising treatment for endometrial carcinoma after failure of surgery and radiation. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  10. Blockade of T-cell activation by dithiocarbamates involves novel mechanisms of inhibition of nuclear factor of activated T cells.

    PubMed Central

    Martínez-Martínez, S; Gómez del Arco, P; Armesilla, A L; Aramburu, J; Luo, C; Rao, A; Redondo, J M

    1997-01-01

    Dithiocarbamates (DTCs) have recently been reported as powerful inhibitors of NF-kappaB activation in a number of cell types. Given the role of this transcription factor in the regulation of gene expression in the inflammatory response, NF-kappaB inhibitors have been suggested as potential therapeutic drugs for inflammatory diseases. We show here that DTCs inhibited both interleukin 2 (IL-2) synthesis and membrane expression of antigens which are induced during T-cell activation. This inhibition, which occurred with a parallel activation of c-Jun transactivating functions and expression, was reflected by transfection experiments at the IL-2 promoter level, and involved not only the inhibition of NF-kappaB-driven reporter activation but also that of nuclear factor of activated T cells (NFAT). Accordingly, electrophoretic mobility shift assays (EMSAs) indicated that pyrrolidine DTC (PDTC) prevented NF-kappaB, and NFAT DNA-binding activity in T cells stimulated with either phorbol myristate acetate plus ionophore or antibodies against the CD3-T-cell receptor complex and simultaneously activated the binding of AP-1. Furthermore, PDTC differentially targeted both NFATp and NFATc family members, inhibiting the transactivation functions of NFATp and mRNA induction of NFATc. Strikingly, Western blotting and immunocytochemical experiments indicated that PDTC promoted a transient and rapid shuttling of NFATp and NFATc, leading to their accelerated export from the nucleus of activated T cells. We propose that the activation of an NFAT kinase by PDTC could be responsible for the rapid shuttling of the NFAT, therefore transiently converting the sustained transactivation of this transcription factor that occurs during lymphocyte activation, and show that c-Jun NH2-terminal kinase (JNK) can act by directly phosphorylating NFATp. In addition, the combined inhibitory effects on NFAT and NF-KB support a potential use of DTCs as immunosuppressants. PMID:9343406

  11. Klf8 regulates left-right asymmetric patterning through modulation of Kupffer's vesicle morphogenesis and spaw expression.

    PubMed

    Lin, Che-Yi; Tsai, Ming-Yuan; Liu, Yu-Hsiu; Lu, Yu-Fen; Chen, Yi-Chung; Lai, Yun-Ren; Liao, Hsin-Chi; Lien, Huang-Wei; Yang, Chung-Hsiang; Huang, Chang-Jen; Hwang, Sheng-Ping L

    2017-07-17

    Although vertebrates are bilaterally symmetric organisms, their internal organs are distributed asymmetrically along a left-right axis. Disruption of left-right axis asymmetric patterning often occurs in human genetic disorders. In zebrafish embryos, Kupffer's vesicle, like the mouse node, breaks symmetry by inducing asymmetric expression of the Nodal-related gene, spaw, in the left lateral plate mesoderm (LPM). Spaw then stimulates transcription of itself and downstream genes, including lft1, lft2, and pitx2, specifically in the left side of the diencephalon, heart and LPM. This developmental step is essential to establish subsequent asymmetric organ positioning. In this study, we evaluated the role of krüppel-like factor 8 (klf8) in regulating left-right asymmetric patterning in zebrafish embryos. Zebrafish klf8 expression was disrupted by both morpholino antisense oligomer-mediated knockdown and a CRISPR-Cas9 system. Whole-mount in situ hybridization was conducted to evaluate gene expression patterns of Nodal signalling components and the positions of heart and visceral organs. Dorsal forerunner cell number was evaluated in Tg(sox17:gfp) embryos and the length and number of cilia in Kupffer's vesicle were analyzed by immunocytochemistry using an acetylated tubulin antibody. Heart jogging, looping and visceral organ positioning were all defective in zebrafish klf8 morphants. At the 18-22 s stages, klf8 morphants showed reduced expression of genes encoding Nodal signalling components (spaw, lft1, lft2, and pitx2) in the left LPM, diencephalon, and heart. Co-injection of klf8 mRNA with klf8 morpholino partially rescued spaw expression. Furthermore, klf8 but not klf8△zf overexpressing embryos showed dysregulated bilateral expression of Nodal signalling components at late somite stages. At the 10s stage, klf8 morphants exhibited reductions in length and number of cilia in Kupffer's vesicle, while at 75% epiboly, fewer dorsal forerunner cells were observed

  12. IL-10R blockade during chronic schistosomiasis mansoni results in the loss of B cells from the liver and the development of severe pulmonary disease.

    PubMed

    Fairfax, Keke C; Amiel, Eyal; King, Irah L; Freitas, Tori C; Mohrs, Markus; Pearce, Edward J

    2012-01-01

    In schistosomiasis patients, parasite eggs trapped in hepatic sinusoids become foci for CD4+ T cell-orchestrated granulomatous cellular infiltrates. Since the immune response is unable to clear the infection, the liver is subjected to ongoing cycles of focal inflammation and healing that lead to vascular obstruction and tissue fibrosis. This is mitigated by regulatory mechanisms that develop over time and which minimize the inflammatory response to newly deposited eggs. Exploring changes in the hepatic inflammatory infiltrate over time in infected mice, we found an accumulation of schistosome egg antigen-specific IgG1-secreting plasma cells during chronic infection. This population was significantly diminished by blockade of the receptor for IL-10, a cytokine implicated in plasma cell development. Strikingly, IL-10R blockade precipitated the development of portal hypertension and the accumulation of parasite eggs in the lungs and heart. This did not reflect more aggressive Th2 cell responsiveness, increased hepatic fibrosis, or the emergence of Th1 or Th17 responses. Rather, a role for antibody in the prevention of severe disease was suggested by the finding that pulmonary involvement was also apparent in mice unable to secrete class switched antibody. A major effect of anti-IL-10R treatment was the loss of a myeloid population that stained positively for surface IgG1, and which exhibited characteristics of regulatory/anti-inflammatory macrophages. This finding suggests that antibody may promote protective effects within the liver through local interactions with macrophages. In summary, our data describe a role for IL-10-dependent B cell responses in the regulation of tissue damage during a chronic helminth infection.

  13. PD-L1 is a novel direct target of HIF-1α, and its blockade under hypoxia enhanced MDSC-mediated T cell activation.

    PubMed

    Noman, Muhammad Zaeem; Desantis, Giacomo; Janji, Bassam; Hasmim, Meriem; Karray, Saoussen; Dessen, Philippe; Bronte, Vincenzo; Chouaib, Salem

    2014-05-05

    Tumor-infiltrating myeloid cells such as myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) form an important component of the hypoxic tumor microenvironment. Here, we investigated the influence of hypoxia on immune checkpoint receptors (programmed death [PD]-1 and CTLA-4) and their respective ligands (PD-1 ligand 1 [PD-L1], PD-L2, CD80, and CD86) on MDSCs. We demonstrate that MDSCs at the tumor site show a differential expression of PD-L1 as compared with MDSCs from peripheral lymphoid organ (spleen). Hypoxia caused a rapid, dramatic, and selective up-regulation of PD-L1 on splenic MDSCs in tumor-bearing mice. This was not limited to MDSCs, as hypoxia also significantly increased the expression of PD-L1 on macrophages, dendritic cells, and tumor cells. Furthermore, PD-L1 up-regulation under hypoxia was dependent on hypoxia-inducible factor-1α (HIF-1α) but not HIF-2α. Chromatin immunoprecipitation and luciferase reporter assay revealed direct binding of HIF-1α to a transcriptionally active hypoxia-response element (HRE) in the PD-L1 proximal promoter. Blockade of PD-L1 under hypoxia enhanced MDSC-mediated T cell activation and was accompanied by the down-regulation of MDSCs IL-6 and IL-10. Finally, neutralizing antibodies against IL-10 under hypoxia significantly abrogated the suppressive activity of MDSCs. Simultaneous blockade of PD-L1 along with inhibition of HIF-1α may thus represent a novel approach for cancer immunotherapy.

  14. Blockade of Notch signaling promotes acetaminophen-induced liver injury.

    PubMed

    Jiang, Longfeng; Ke, Michael; Yue, Shi; Xiao, Wen; Yan, Youde; Deng, Xiaozhao; Ying, Qi-Long; Li, Jun; Ke, Bibo

    2017-03-13

    Liver injury after experimental acetaminophen treatment is mediated both by direct hepatocyte injury through a P450-generated toxic metabolite and indirectly by activated liver Kupffer cells and neutrophils. This study was designed to investigate the role of Notch signaling in the regulation of innate immune responses in acetaminophen (APAP)-induced liver injury. Using a mouse model of APAP-induced liver injury, wild-type (WT) and toll-like receptor 4 knockout (TLR4 KO) mice were injected intraperitoneally with APAP or PBS. Some animals were injected with γ-secretase inhibitor DAPT or DMSO vehicle. For the in vitro study, bone marrow-derived macrophages (BMMs) were transfected with Notch1 siRNA, TLR4 siRNA, and non-specific (NS) siRNA and stimulated with LPS. Indeed, paracetamol/acetaminophen-induced liver damage was worse after Notch blockade with DAPT in wild-type mice, which was accompanied by significantly increased ALT levels, diminished hairy and enhancer of split-1 (Hes1), and phosphorylated Stat3 and Akt but enhanced high mobility group box 1 (HMGB1), TLR4, NF-κB, and NLRP3 activation after APAP challenge. Mice receiving DAPT increased macrophage and neutrophil accumulation and hepatocellular apoptosis. However, TLR4 KO mice that received DAPT reduced APAP-induced liver damage and NF-κB, NLRP3, and cleaved caspase-1 activation. BMMs transfected with Notch1 siRNA reduced Hes1 and phosphorylated Stat3 and Akt but augmented HMGB1, TLR4, NF-κB, and NLRP3. Furthermore, TLR4 siRNA knockdown resulted in decreased NF-κB and NLRP3 and cleaved caspase-1 and IL-1β levels following LPS stimulation. These results demonstrate that Notch signaling regulates innate NLRP3 inflammasome activation through regulation of HMGB1/TLR4/NF-κB activation in APAP-induced liver injury. Our novel findings underscore the critical role of the Notch1-Hes1 signaling cascade in the regulation of innate immunity in APAP-triggered liver inflammation. This might imply a novel therapeutic

  15. Blockade of Indoleamine 2,3-Dioxygenase Reduces Mortality from Peritonitis and Sepsis in Mice by Regulating Functions of CD11b+ Peritoneal Cells

    PubMed Central

    Osawa, Yosuke; Ito, Hiroyasu; Ohtaki, Hirofumi; Ando, Tatsuya; Takamatsu, Manabu; Hara, Akira; Saito, Kuniaki; Seishima, Mitsuru

    2014-01-01

    Indoleamine 2,3-dioxygenase-1 (Ido), which catalyzes the first and limiting step of tryptophan catabolism, has been implicated in immune tolerance. However, the roles of Ido in systemic bacterial infection are complicated and remain controversial. To explore this issue, we examined the roles of Ido in bacterial peritonitis and sepsis after cecal ligation and puncture (CLP) in mice by using the Ido inhibitor 1-methyl-d,l-tryptophan (1-MT), by comparing Ido+/+ and Ido−/− mice, or by using chimeric mice in which Ido in the bone marrow-derived cells was deficient. Ido expression in the peritoneal CD11b+ cells and its metabolite l-kynurenine in the serum were increased after CLP. 1-MT treatment or Ido deficiency, especially in bone marrow-derived cells, reduced mortality after CLP. Compared to Ido+/+ mice, Ido−/− mice showed increased recruitment of neutrophils and mononuclear cells into the peritoneal cavity and a decreased bacterial count in the blood accompanied by increased CXCL-2 and CXCL-1 mRNA in the peritoneal cells. Ido has an inhibitory effect on LPS-induced CXCL-2 and CXCL-1 production in cultured peritoneal cells. These findings indicate that inhibition of Ido reduces mortality from peritonitis and sepsis after CLP via recruitment of neutrophils and mononuclear cells by chemokine production in peritoneal CD11b+ cells. Thus, blockade of Ido plays a beneficial role in host protection during bacterial peritonitis and sepsis. PMID:25114116

  16. Co-Stimulatory Blockade of the CD28/CD80-86/CTLA-4 Balance in Transplantation: Impact on Memory T Cells?

    PubMed Central

    Ville, Simon; Poirier, Nicolas; Blancho, Gilles; Vanhove, Bernard

    2015-01-01

    CD28 and CTLA-4 are prototypal co-stimulatory and co-inhibitory cell surface signaling molecules interacting with CD80/86, known to be critical for immune response initiation and regulation, respectively. Initial “bench-to-beside” translation, two decades ago, resulted in the development of CTLA4-Ig, a biologic that targets CD80/86 and prevents T-cell costimulation. In spite of its proven effectiveness in inhibiting allo-immune responses, particularly in murine models, clinical experience in kidney transplantation with belatacept (high-affinity CTLA4-Ig molecule) reveals a high incidence of acute, cell-mediated rejection. Originally, the etiology of belatacept-resistant graft rejection was thought to be heterologous immunity, i.e., the cross-reactivity of the pool of memory T cells from pathogen-specific immune responses with alloantigens. Recently, the standard view that memory T cells arise from effector cells after clonal contraction has been challenged by a “developmental” model, in which less differentiated memory T cells generate effector cells. This review delineates how this shift in paradigm, given the differences in co-stimulatory and co-inhibitory signal depending on the maturation stage, could profoundly affect our understanding of the CD28/CD80-86/CTLA-4 blockade and highlights the potential advantages of selectively targeting CD28, instead of CD80/86, to control post-transplant immune responses. PMID:26322044

  17. Intraluminal Blockade of Cell-Surface CD74 and Glucose Regulated Protein 78 Prevents Substance P-Induced Bladder Inflammatory Changes in the Rat

    PubMed Central

    Vera, Pedro L.; Wang, Xihai; Bucala, Richard J.; Meyer-Siegler, Katherine L.

    2009-01-01

    Background Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine constitutively expressed by urothelial cells. During inflammatory stimuli, MIF is released into the lumen complexed to other proteins and these complexes can bind to urothelial cell-surface receptors to activate signaling pathways. Since MIF is complexed to α1-inhibitor III (A1-I3; a member of the α2-macroglubulin family) and glucose regulated protein 78 (GRP78) is a receptor for A1-I3 the goals of this study were to determine if substance P elicits urothelial cell-surface expression of GRP78 and to assess the functional role of CD74 (receptor for MIF) or GRP78 in substance P-induced bladder inflammatory changes. Methodology/Principal Findings Anesthetized male Sprague-Dawley rats received either saline or substance P (s.c.), bladders were collected 1 hour after treatment and processed for histology or protein/mRNA. The expression of GRP78 at urothelial cell-surface was determined by performing in vivo biotinylation of urothelial cell-surface proteins. Finally, in order to determine the effects of receptor blockade on substance P-induced MIF release and inflammatory changes, rats received either intraluminal antibodies to CD74, GRP78, both, or non-specific IgG (as a control). GRP78 and MIF immunostaining was simultaneously visualized in umbrella cells only after substance P treatment. Immunoprecipitation studies showed GRP78-MIF complexes increased after substance P while in vivo biotinylation confirmed substance P-induced GRP78 cell-surface expression in urothelial cells. Intraluminal blockade of CD74 and/or GRP78 prevented substance P-induced changes, including bladder edema, intraluminal MIF release by urothelial cells and production of inflammatory cytokines by urothelial cells. Conclusions/Significance GRP78 is expressed on the surface of urothelial cells after substance P treatment where it can bind MIF complexes. Blocking CD74 (receptor for MIF) and/or GRP78 prevented

  18. T-cell epitope strength in WAP-T mouse mammary carcinomas is an important determinant in PD1/PD-L1 immune checkpoint blockade therapy

    PubMed Central

    Bruns, Michael; Wanger, Jara; Schumacher, Udo; Deppert, Wolfgang

    2016-01-01

    Using the SV40 transgenic WAP-T/WAP-TNP mouse models for mammary carcinomas, we compared the response to immune checkpoint blockade therapy in tumor mice expressing either SV40 T-antigen containing the LCMV NP-epitope (T-AgNP in WAP-TNP mice), or the unmodified T-antigen (T-Ag in WAP-T mice). Specifically, we asked, whether the presence of the highly immunogenic NP-epitope in T-AgNP influences this response in comparison to the weakly immunogenic T-cell epitopes of T-Ag in WAP-T tumor mice. Treatment of WAP-TNP tumor mice with either anti-PD1 or anti-PD-L1 antibodies led to tumor regression, with anti-PD-L1 treatment being more effective. However, tumors had fully re-appeared after 21 days, indicating that CTL exhaustion had been rapidly re-established. Surprisingly, the same treatment applied to WAP-T tumor mice resulted in a significantly prolonged period of tumor regression. We provide evidence that in contrast to the weak antigenic stimuli exerted by T-cell epitopes of T-Ag, the strong antigenic stimulus of the NP-epitope in T-AgNP has a dual effect: (i) a rapid generation of active NP-specific CTLs, accompanied (ii) by accelerated CTL exhaustion. Our data support the hypothesis that the immunogenicity of tumor antigen T-cell epitopes strongly influences the success of immune checkpoint blockade therapy. PMID:27579535

  19. Tasquinimod modulates tumor-infiltrating myeloid cells and improves the antitumor immune response to PD-L1 blockade in bladder cancer.

    PubMed

    Nakhlé, Jessica; Pierron, Valérie; Bauchet, Anne-Laure; Plas, Pascale; Thiongane, Amath; Meyer-Losic, Florence; Schmidlin, Fabien

    2016-06-01

    The infiltration of myeloid cells helps tumors to overcome immune surveillance and imparts resistance to cancer immunotherapy. Thus, strategies to modulate the effects of these immune cells may offer a potential therapeutic benefit. We report here that tasquinimod, a novel immunotherapy which targets S100A9 signaling, reduces the immunosuppressive properties of myeloid cells in preclinical models of bladder cancer (BCa). As single anticancer agent, tasquinimod treatment was effective in preventing early stage tumor growth, but did not achieve a clear antitumor effect in advanced tumors. Investigations of this response revealed that tasquinimod induces an increase in the expression of a negative regulator of T cell activation, Programmed-death-ligand 1 (PD-L1). This markedly weakens its antitumor immunity, yet provokes an "inflamed" milieu rendering tumors more prone to T cell-mediated immune attack by PD-L1 blockade. Interestingly, the combination of tasquinimod with an Anti-PD-L1 antibody enhanced the antitumor immune response in bladder tumors. This combination synergistically modulated tumor-infiltrating myeloid cells, thereby strongly affecting proliferation and activation of effector T cells. Together, our data provide insight into the rational combination of therapies that activate both innate and adaptive immune system, such as the association of S100A9-targeting agents with immune checkpoints inhibitors, to improve the response to cancer immunotherapeutic agents in BCa.

  20. Overexpression of CD85j in TNBC patients inhibits Cetuximab-mediated NK-cell ADCC but can be restored with CD85j functional blockade.

    PubMed

    Roberti, María P; Juliá, Estefanía P; Rocca, Yamila S; Amat, Mora; Bravo, Alicia I; Loza, José; Coló, Federico; Loza, Carlos M; Fabiano, Verónica; Maino, Mercedes; Podhorzer, Ariel; Fainboim, Leonardo; Barrio, María M; Mordoh, José; Levy, Estrella M

    2015-05-01

    Clinical studies suggest that triple negative breast cancer (TNBC) patients with epidermal growth factor receptor (EGFR)-expressing tumors could benefit from therapy with Cetuximab, which targets EGFR. NK cells are the primary effectors of antibody (Ab)-dependent cell-mediated cytotoxicity (ADCC) and thus play a role in Ab-based therapies. We have previously described diminished levels of Cetuximab-mediated ADCC in vitro in patients with advanced breast cancer. Here, we investigated the potential causes of this NK-cell functional deficiency. We characterized NK-cell activating/inhibitory receptors in the peripheral blood of breast cancer patients and found CD85j inhibitory receptor overexpression. The capacity of NK cells to perform Cetuximab-triggered ADCC against TNBC cells correlated inversely with CD85j expression, even in the presence of the stimulatory cytokines IL-2 or IL-15. Hence, patients expressing high levels of CD85j had an impaired ability to lyse TNBC cells in the presence of Cetuximab. We also found that CD85j overexpression was associated with HLA-I and soluble HLA-G expression by tumors. A CD85j functional blockade with a CD85j antagonist Ab restored ADCC levels in breast cancer patients and reverted this negative effect. Our data suggest that strategies that overcome the hurdles of immune activation could improve Cetuximab clinical efficacy.

  1. Tasquinimod modulates tumor-infiltrating myeloid cells and improves the antitumor immune response to PD-L1 blockade in bladder cancer

    PubMed Central

    Nakhlé, Jessica; Pierron, Valérie; Bauchet, Anne-Laure; Plas, Pascale; Thiongane, Amath; Meyer-Losic, Florence; Schmidlin, Fabien

    2016-01-01

    ABSTRACT The infiltration of myeloid cells helps tumors to overcome immune surveillance and imparts resistance to cancer immunotherapy. Thus, strategies to modulate the effects of these immune cells may offer a potential therapeutic benefit. We report here that tasquinimod, a novel immunotherapy which targets S100A9 signaling, reduces the immunosuppressive properties of myeloid cells in preclinical models of bladder cancer (BCa). As single anticancer agent, tasquinimod treatment was effective in preventing early stage tumor growth, but did not achieve a clear antitumor effect in advanced tumors. Investigations of this response revealed that tasquinimod induces an increase in the expression of a negative regulator of T cell activation, Programmed-death-ligand 1 (PD-L1). This markedly weakens its antitumor immunity, yet provokes an “inflamed” milieu rendering tumors more prone to T cell-mediated immune attack by PD-L1 blockade. Interestingly, the combination of tasquinimod with an Anti-PD-L1 antibody enhanced the antitumor immune response in bladder tumors. This combination synergistically modulated tumor-infiltrating myeloid cells, thereby strongly affecting proliferation and activation of effector T cells. Together, our data provide insight into the rational combination of therapies that activate both innate and adaptive immune system, such as the association of S100A9-targeting agents with immune checkpoints inhibitors, to improve the response to cancer immunotherapeutic agents in BCa. PMID:27471612

  2. Induction of regulatory cells and control of cellular but not vascular rejection by costimulation blockade in hamster-to-rat heart xenotransplantation.

    PubMed

    Séveno, Céline; Coulon, Flora; Haspot, Fabienne; Mérieau, Emmanuel; Renaudin, Karine; Martinet, Bernard; Vanhove, Bernard

    2007-01-01

    In heart allograft in the rat, a sustained costimulation blockade with CTLA4Ig prevents alloreactive T-cell activation and promotes a long-term graft survival through the action of tolerogeneic dendritic cells. It is unclear whether similar mechanisms might occur after xenotransplantation. To test that hypothesis, we have analyzed the action of CTLA4Ig in a model of CD4(+)T cell-mediated xenograft rejection. Hamster hearts were transplanted into LEW.1A rats receiving an accommodation-inducing treatment consisting of a short course administration of LF15-0195 and a daily administration of cyclosporine A (CSA). To achieve long-term delivery of CTLA4Ig, an intravenous administration of an adenovirus vector coding for mouse CTLA4Ig (Ad-CTLA4Ig) was added to the accommodation induction protocol. On day 40 post-transplantation, rejection was induced by CSA withdrawal. In other xenograft recipients, CD28/B7 costimulation was inhibited at that time only by injections of CTLA4Ig or anti-CD28 antibodies. Graft survival, immunohistology, as well as development of antibodies and regulatory cells were examined. Xenografts survived 6 days after CSA withdrawal in controls and were rejected, as previously described, through the action of CD4(+) xenoreactive T cells. Interfering with CD28/B7 costimulation inhibited this xenoreactive T cell response and delayed rejection to day 10. In recipients that had received Ad-CTLA4Ig, survival was prolonged to day 19 and this was accompanied by the appearance of regulatory cells exhibiting non-donor-specific suppressive activity dependent on IL-2, NO, and IDO. These regulatory cells were different from those previously identified after Ad-CTLA4Ig administration in heart allograft in the rat. In these recipients, rejection occurred as a consequence of an evoked anti-donor IgM response and complement activation and not of a cellular rejection as complement inhibition with cobra venom factor further prolonged xenograft survival. CD28/B7 blockade

  3. Disabling immune tolerance by programmed death-1 blockade with pidilizumab after autologous hematopoietic stem-cell transplantation for diffuse large B-cell lymphoma: results of an international phase II trial.

    PubMed

    Armand, Philippe; Nagler, Arnon; Weller, Edie A; Devine, Steven M; Avigan, David E; Chen, Yi-Bin; Kaminski, Mark S; Holland, H Kent; Winter, Jane N; Mason, James R; Fay, Joseph W; Rizzieri, David A; Hosing, Chitra M; Ball, Edward D; Uberti, Joseph P; Lazarus, Hillard M; Mapara, Markus Y; Gregory, Stephanie A; Timmerman, John M; Andorsky, David; Or, Reuven; Waller, Edmund K; Rotem-Yehudar, Rinat; Gordon, Leo I

    2013-11-20

    The Programmed Death-1 (PD-1) immune checkpoint pathway may be usurped by tumors, including diffuse large B-cell lymphoma (DLBCL), to evade immune surveillance. The reconstituting immune landscape after autologous hematopoietic stem-cell transplantation (AHSCT) may be particularly favorable for breaking immune tolerance through PD-1 blockade. We conducted an international phase II study of pidilizumab, an anti-PD-1 monoclonal antibody, in patients with DLBCL undergoing AHSCT, with correlative studies of lymphocyte subsets. Patients received three doses of pidilizumab beginning 1 to 3 months after AHSCT. Sixty-six eligible patients were treated. Toxicity was mild. At 16 months after the first treatment, progression-free survival (PFS) was 0.72 (90% CI, 0.60 to 0.82), meeting the primary end point. Among the 24 high-risk patients who remained positive on positron emission tomography after salvage chemotherapy, the 16-month PFS was 0.70 (90% CI, 0.51 to 0.82). Among the 35 patients with measurable disease after AHSCT, the overall response rate after pidilizumab treatment was 51%. Treatment was associated with increases in circulating lymphocyte subsets including PD-L1E-bearing lymphocytes, suggesting an on-target in vivo effect of pidilizumab. This is the first demonstration of clinical activity of PD-1 blockade in DLBCL. Given these results, PD-1 blockade after AHSCT using pidilizumab may represent a promising therapeutic strategy in this disease.

  4. Disabling Immune Tolerance by Programmed Death-1 Blockade With Pidilizumab After Autologous Hematopoietic Stem-Cell Transplantation for Diffuse Large B-Cell Lymphoma: Results of an International Phase II Trial

    PubMed Central

    Armand, Philippe; Nagler, Arnon; Weller, Edie A.; Devine, Steven M.; Avigan, David E.; Chen, Yi-Bin; Kaminski, Mark S.; Holland, H. Kent; Winter, Jane N.; Mason, James R.; Fay, Joseph W.; Rizzieri, David A.; Hosing, Chitra M.; Ball, Edward D.; Uberti, Joseph P.; Lazarus, Hillard M.; Mapara, Markus Y.; Gregory, Stephanie A.; Timmerman, John M.; Andorsky, David; Or, Reuven; Waller, Edmund K.; Rotem-Yehudar, Rinat; Gordon, Leo I.

    2013-01-01

    Purpose The Programmed Death-1 (PD-1) immune checkpoint pathway may be usurped by tumors, including diffuse large B-cell lymphoma (DLBCL), to evade immune surveillance. The reconstituting immune landscape after autologous hematopoietic stem-cell transplantation (AHSCT) may be particularly favorable for breaking immune tolerance through PD-1 blockade. Patients and Methods We conducted an international phase II study of pidilizumab, an anti–PD-1 monoclonal antibody, in patients with DLBCL undergoing AHSCT, with correlative studies of lymphocyte subsets. Patients received three doses of pidilizumab beginning 1 to 3 months after AHSCT. Results Sixty-six eligible patients were treated. Toxicity was mild. At 16 months after the first treatment, progression-free survival (PFS) was 0.72 (90% CI, 0.60 to 0.82), meeting the primary end point. Among the 24 high-risk patients who remained positive on positron emission tomography after salvage chemotherapy, the 16-month PFS was 0.70 (90% CI, 0.51 to 0.82). Among the 35 patients with measurable disease after AHSCT, the overall response rate after pidilizumab treatment was 51%. Treatment was associated with increases in circulating lymphocyte subsets including PD-L1E–bearing lymphocytes, suggesting an on-target in vivo effect of pidilizumab. Conclusion This is the first demonstration of clinical activity of PD-1 blockade in DLBCL. Given these results, PD-1 blockade after AHSCT using pidilizumab may represent a promising therapeutic strategy in this disease. PMID:24127452

  5. In vivo blockade of α1-adrenergic receptors mitigates stress-disturbed cAMP and cGMP signaling in Leydig cells.

    PubMed

    Stojkov, Natasa J; Baburski, Aleksandar Z; Bjelic, Maja M; Sokanovic, Srdjan J; Mihajlovic, Aleksandar I; Drljaca, Dragana M; Janjic, Marija M; Kostic, Tatjana S; Andric, Silvana A

    2014-01-01

    The molecular mechanism of stress-associated reproductive dysfunction is complex and largely unknown. This study was designed to systematically analyze molecular effects of systemic in vivo blockade of α1-adrenergic receptors (α1-ADRs) on stress-induced disturbance of cAMP/cGMP signaling in testosterone-producing Leydig cells using the following parameters (i) level of circulating stress hormones, LH and testosterone; (ii) level of main molecular markers of Leydig cell functionality (testosterone, Insl3, cAMP); (iii) expression of cAMP signaling (cAMP 'producers'/'effectors'/'removers') and (iv) expression of NO-cGMP signaling (NO-cGMP 'producers'/'effectors'/'removers'). The results showed that oral administration of α1-ADR blocker before stress increased cGMP and diminished stress-reduced cAMP production in Leydig cells. In the same cells, stress-induced effects on cAMP/cGMP signaling pathways elements were changed. Sustained in vivo α1-ADR blockade completely abolished stress-increased transcription of most abundantly expressed phosphodiesterase that remove cAMP (Pde4b) and potentiated stress-increased expression of PRKA, the main stimulator of Leydig cell steroidogenesis. In the same Leydig cells, stress-decreased NOS3 expression was abolished, while stress-increased GUCY1 (cGMP 'producer') and PRKG1 (cGMP 'effector') were potentiated. It is possible that all molecules mentioned could contribute, at least in part, in recovery of Leydig cell testosterone production. Presented data provide new role of α1-ADRs in stress-triggered disturbance of cAMP/cGMP signaling, and new molecular insights into the relationship between stress and mammalian reproduction. Regardless of whether the effects of α1-blocker + stress are direct or indirect, the results are important in terms of human reproductive health and the wide use of α1-ADR antagonists, alone or in combination, to treat post-traumatic stress disorders, hypertension, benign prostatic hyperplasia symptoms and

  6. Self-Sustained Resistance to Suppression of CD8+ Teff Cells at the Site of Autoimmune Inflammation Can Be Reversed by Tumor Necrosis Factor and Interferon-γ Blockade.

    PubMed

    Petrelli, Alessandra; Wehrens, Ellen J; Scholman, Rianne C; Prakken, Berent J; Vastert, Sebastian J; van Wijk, Femke

    2016-01-01

    Resistance of Teff cells to Treg cell-mediated suppression contributes to the breakdown of peripheral tolerance in the inflamed joints of patients with juvenile idiopathic arthritis (JIA). However, unanswered questions are whether this resistant phenotype is self-sustained and whether CD8+ and CD4+ Teff cells share the same mechanism of resistance to suppression. We undertook this study to investigate intrinsic resistance of CD8+ Teff cells to suppression and to determine how this can be targeted therapeutically. CD8+ or CD4+ Teff cells were cultured with or without antigen-presenting cells (APCs) in Treg cell-dependent and -independent suppression assays. Synovial fluid (SF)-derived Teff cells were crosscultured with peripheral blood (PB) Treg cells from JIA patients or healthy controls. Tumor necrosis factor (TNF) or interferon-γ (IFNγ) blocking agents were used to restore Teff cell responsiveness to suppression. Suppression of cell proliferation and cytokine production in CD8+ Teff cells from the SF of JIA patients was severely impaired compared to that in CD8+ Teff cells from the PB of JIA patients, regardless of the presence of APCs and CD4+ Teff cells. Similar to CD4+ Teff cells, impaired suppression of CD8+ Teff cells was shown to be an intrinsic feature of this cell population. While TNF blockade restored both CD8+ and CD4+ Teff cell susceptibility to suppression, autocrine release of IFNγ selectively sustained CD8+ Teff cell resistance, which could be relieved by IFNγ blockade. Unlike CD4+ Teff cells, resistance of CD8+ Teff cells to suppression at the site of autoimmune inflammation is maintained by autocrine release of IFNγ, and blockade of IFNγ restores CD8+ Teff cell responsiveness to suppression. These findings indicate a potential therapeutic value of blocking IFNγ to restore immune regulation in JIA. © 2016, American College of Rheumatology.

  7. Enhancement of Muscle T Regulatory Cells and Improvement of Muscular Dystrophic Process in mdx Mice by Blockade of Extracellular ATP/P2X Axis.

    PubMed

    Gazzerro, Elisabetta; Baldassari, Simona; Assereto, Stefania; Fruscione, Floriana; Pistorio, Angela; Panicucci, Chiara; Volpi, Stefano; Perruzza, Lisa; Fiorillo, Chiara; Minetti, Carlo; Traggiai, Elisabetta; Grassi, Fabio; Bruno, Claudio

    2015-12-01

    Infiltration of immune cells and chronic inflammation substantially affect skeletal and cardiac muscle degeneration in Duchenne muscular dystrophy. In the immune system, extracellular adenosine triphosphate (ATP) released by dying cells is sensed as a danger associated molecular pattern through P2 purinergic receptors. Specifically, the P2X7 subtype has a prominent role in regulating immune system physiology and contributes to inflammasome activation also in muscle cells. Here, we show that in vivo blockade of the extracellular ATP/P2X purinergic signaling pathway by periodate-oxidized ATP delayed the progression of the dystrophic phenotype and dampened the local inflammatory response in mdx mice, a spontaneous mouse model of dystrophin deficiency. Reduced infiltration of leukocytes and macrophages and decreased expression of IL-6 were revealed in the muscles of periodate-oxidized ATP-treated mdx mice. Concomitantly, an increase in Foxp3(+) immunosuppressive regulatory T cells was observed and correlated with enhanced myofiber regeneration. Moreover, we detected reduced concentrations of profibrotic cytokines, including transforming growth factor-β and connective tissue growth factor, in muscles of periodate-oxidized ATP-treated mdx mice. The improvement of inflammatory features was associated with increased strength and reduced necrosis, thus suggesting that pharmacologic purinergic antagonism altering the adaptive immune component in the muscle infiltrates might represent a promising therapeutic approach in Duchenne muscular dystrophy.

  8. GM-CSF signalling blockade and chemotherapeutic agents act in concert to inhibit the function of myeloid-derived suppressor cells in vitro

    PubMed Central

    Gargett, Tessa; Christo, Susan N; Hercus, Timothy R; Abbas, Nazim; Singhal, Nimit; Lopez, Angel F; Brown, Michael P

    2016-01-01

    Immune evasion is a recently defined hallmark of cancer, and immunotherapeutic approaches that stimulate an immune response to tumours are gaining recognition. However tumours may evade the immune response and resist immune-targeted treatment by promoting an immune-suppressive environment and stimulating the differentiation or recruitment of immunosuppressive cells. Myeloid-derived suppressor cells (MDSC) have been identified in a range of cancers and are often associated with tumour progression and poor patient outcomes. Pancreatic cancer in particular supports MDSC differentiation via the secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF), and MDSC are believed to contribute to the profoundly immune-suppressive microenvironment present in pancreatic tumours. MDSC-targeted therapies that deplete or inhibit this cell population have been proposed as a way to shift the balance in favour of a tumour-clearing immune response. In this study, we have modelled MDSC differentiation and function in vitro and this has provided us with the opportunity to test a range of potential MDSC-targeted therapies to identify candidates for further investigation. Using in vitro modelling we show here that the combination of GM-CSF-signalling blockade and gemcitabine suppresses both the MDSC phenotype and the inhibition of T-cell function by MDSC. PMID:28090321

  9. Pharmacological blockade of either cannabinoid CB1 or CB2 receptors prevents both cocaine-induced conditioned locomotion and cocaine-induced reduction of cell proliferation in the hippocampus of adult male rat.

    PubMed

    Blanco-Calvo, Eduardo; Rivera, Patricia; Arrabal, Sergio; Vargas, Antonio; Pavón, Francisco Javier; Serrano, Antonia; Castilla-Ortega, Estela; Galeano, Pablo; Rubio, Leticia; Suárez, Juan; Rodriguez de Fonseca, Fernando

    2014-01-01

    Addiction to major drugs of abuse, such as cocaine, has recently been linked to alterations in adult neurogenesis in the hippocampus. The endogenous cannabinoid system modulates this proliferative response as demonstrated by the finding that pharmacological activation/blockade of cannabinoid CB1 and CB2 receptors not only modulates neurogenesis but also modulates cell death in the brain. In the present study, we evaluated whether the endogenous cannabinoid system affects cocaine-induced alterations in cell proliferation. To this end, we examined whether pharmacological blockade of either CB1 (Rimonabant, 3 mg/kg) or CB2 receptors (AM630, 3 mg/kg) would affect cell proliferation [the cells were labeled with 5-bromo-2'-deoxyuridine (BrdU)] in the subventricular zone (SVZ) of the lateral ventricle and the dentate subgranular zone (SGZ). Additionally, we measured cell apoptosis (as monitored by the expression of cleaved caspase-3) and glial activation [by analyzing the expression of glial fibrillary acidic protein (GFAP) and Iba-1] in the striatum and hippocampus during acute and repeated (4 days) cocaine administration (20 mg/kg). The results showed that acute cocaine exposure decreased the number of BrdU-immunoreactive (ir) cells in the SVZ and SGZ. In contrast, repeated cocaine exposure reduced the number of BrdU-ir cells only in the SVZ. Both acute and repeated cocaine exposure increased the number of cleaved caspase-3-, GFAP- and Iba1-ir cells in the hippocampus, and this effect was counteracted by AM630 or Rimonabant, which increased the number of BrdU-, GFAP-, and Iba1-ir cells in the hippocampus. These results indicate that the changes in neurogenic, apoptotic and gliotic processes that were produced by repeated cocaine administration were normalized by pharmacological blockade of CB1 and CB2. The restorative effects of cannabinoid receptor blockade on hippocampal cell proliferation were associated with the prevention of the induction of conditioned locomotion

  10. Ca2+ signals mediated by bradykinin type 2 receptors in normal pancreatic stellate cells can be inhibited by specific Ca2+ channel blockade

    PubMed Central

    Gryshchenko, Oleksiy; Gerasimenko, Julia V.

    2015-01-01

    Key points Bradykinin may play a role in the autodigestive disease acute pancreatitis, but little is known about its pancreatic actions.In this study, we have investigated bradykinin‐elicited Ca2+ signal generation in normal mouse pancreatic lobules.We found complete separation of Ca2+ signalling between pancreatic acinar (PACs) and stellate cells (PSCs). Pathophysiologically relevant bradykinin concentrations consistently evoked Ca2+ signals, via B2 receptors, in PSCs but never in neighbouring PACs, whereas cholecystokinin, consistently evoking Ca2+ signals in PACs, never elicited Ca2+ signals in PSCs.The bradykinin‐elicited Ca2+ signals were due to initial Ca2+ release from inositol trisphosphate‐sensitive stores followed by Ca2+ entry through Ca2+ release‐activated channels (CRACs). The Ca2+ entry phase was effectively inhibited by a CRAC blocker.B2 receptor blockade reduced the extent of PAC necrosis evoked by pancreatitis‐promoting agents and we therefore conclude that bradykinin plays a role in acute pancreatitis via specific actions on PSCs. Abstract Normal pancreatic stellate cells (PSCs) are regarded as quiescent, only to become activated in chronic pancreatitis and pancreatic cancer. However, we now report that these cells in their normal microenvironment are far from quiescent, but are capable of generating substantial Ca2+ signals. We have compared Ca2+ signalling in PSCs and their better studied neighbouring acinar cells (PACs) and found complete separation of Ca2+ signalling in even closely neighbouring PACs and PSCs. Bradykinin (BK), at concentrations corresponding to the slightly elevated plasma BK levels that have been shown to occur in the auto‐digestive disease acute pancreatitis in vivo, consistently elicited substantial Ca2+ signals in PSCs, but never in neighbouring PACs, whereas the physiological PAC stimulant cholecystokinin failed to evoke Ca2+ signals in PSCs. The BK‐induced Ca2+ signals were mediated by B2 receptors and B2

  11. Ca(2+) signals mediated by bradykinin type 2 receptors in normal pancreatic stellate cells can be inhibited by specific Ca(2+) channel blockade.

    PubMed

    Gryshchenko, Oleksiy; Gerasimenko, Julia V; Gerasimenko, Oleg V; Petersen, Ole H

    2016-01-15

    Bradykinin may play a role in the autodigestive disease acute pancreatitis, but little is known about its pancreatic actions. In this study, we have investigated bradykinin-elicited Ca(2+) signal generation in normal mouse pancreatic lobules. We found complete separation of Ca(2+) signalling between pancreatic acinar (PACs) and stellate cells (PSCs). Pathophysiologically relevant bradykinin concentrations consistently evoked Ca(2+) signals, via B2 receptors, in PSCs but never in neighbouring PACs, whereas cholecystokinin, consistently evoking Ca(2+) signals in PACs, never elicited Ca(2+) signals in PSCs. The bradykinin-elicited Ca(2+) signals were due to initial Ca(2+) release from inositol trisphosphate-sensitive stores followed by Ca(2+) entry through Ca(2+) release-activated channels (CRACs). The Ca(2+) entry phase was effectively inhibited by a CRAC blocker. B2 receptor blockade reduced the extent of PAC necrosis evoked by pancreatitis-promoting agents and we therefore conclude that bradykinin plays a role in acute pancreatitis via specific actions on PSCs. Normal pancreatic stellate cells (PSCs) are regarded as quiescent, only to become activated in chronic pancreatitis and pancreatic cancer. However, we now report that these cells in their normal microenvironment are far from quiescent, but are capable of generating substantial Ca(2+) signals. We have compared Ca(2+) signalling in PSCs and their better studied neighbouring acinar cells (PACs) and found complete separation of Ca(2+) signalling in even closely neighbouring PACs and PSCs. Bradykinin (BK), at concentrations corresponding to the slightly elevated plasma BK levels that have been shown to occur in the auto-digestive disease acute pancreatitis in vivo, consistently elicited substantial Ca(2+) signals in PSCs, but never in neighbouring PACs, whereas the physiological PAC stimulant cholecystokinin failed to evoke Ca(2+) signals in PSCs. The BK-induced Ca(2+) signals were mediated by B2 receptors and B2

  12. Cocoa flavonoids attenuate high glucose-induced insulin signalling blockade and modulate glucose uptake and production in human HepG2 cells.

    PubMed

    Cordero-Herrera, Isabel; Martín, María Ángeles; Goya, Luis; Ramos, Sonia

    2014-02-01

    Insulin resistance is the primary characteristic of type 2 diabetes. Cocoa and its main flavanol, (-)-epicatechin (EC), display some antidiabetic effects, but the mechanisms for their preventive activities related to glucose metabolism and insulin signalling in the liver remain largely unknown. In the present work, the preventive effect of EC and a cocoa polyphenolic extract (CPE) on insulin signalling and on both glucose production and uptake are studied in insulin-responsive human HepG2 cells treated with high glucose. Pre-treatment of cells with EC or CPE reverted decreased tyrosine-phosphorylated and total levels of IR, IRS-1 and -2 triggered by high glucose. EC and CPE pre-treatment also prevented the inactivation of the PI3K/AKT pathway and AMPK, as well as the diminution of GLUT-2 levels induced by high glucose. Furthermore, pre-treatment of cells with EC and CPE avoided the increase in PEPCK levels and the diminished glucose uptake provoked by high glucose, returning enhanced levels of glucose production and decreased glycogen content to control values. These findings suggest that EC and CPE improved insulin sensitivity of HepG2 treated with high glucose, preventing or delaying a potential hepatic dysfunction through the attenuation of the insulin signalling blockade and the modulation of glucose uptake and production.

  13. Blockade of CCR7 leads to decreased dendritic cell migration to draining lymph nodes and promotes graft survival in low-risk corneal transplantation.

    PubMed

    Hos, D; Dörrie, J; Schaft, N; Bock, F; Notara, M; Kruse, F E; Krautwald, S; Cursiefen, C; Bachmann, B O

    2016-05-01

    The chemokine receptor CCR7 is essential for migration of mature dendritic cells (DCs) to the regional lymph nodes, and it has been shown that blocking of CCR7 improves graft survival after high-risk corneal transplantation in vascularized recipient corneas. However, it is so far unknown whether blocking of CCR7 reduces migration of DCs from the avascular cornea to the draining lymph nodes and whether this leads to improved graft survival also in the low-risk setting of corneal transplantation, which accounts for the majority of perforating transplantations performed. Therefore, in this study, pellets containing Freund's adjuvant and bovine serum albumin (BSA) conjugated to Alexa488 fluorescent dye were implanted into the corneal stroma of BALB/c mice to analyze antigen uptake by corneal DCs and their migration to the regional lymph nodes. After pellet implantation, mice were either treated by local administration of a CCR7 blocking fusion protein that consisted of CCL19 fused to the Fc part of human IgG1 or a control-IgG. In vivo fluorescence microscopy showed uptake of Alexa488-conjugated BSA by corneal DCs within 8 h. Furthermore, analysis of single cell suspensions of draining lymph nodes prepared after 48 h revealed that 2.1 ± 0.3% of CD11c(+) cells were also Alexa488(+). Importantly, DC migration was significantly reduced after topical administration of CCL19-IgG (1.2 ± 0.2%; p < 0.05). To test the effect of CCR7 blockade on graft rejection after allogeneic low-risk keratoplasty, corneal transplantations were performed using C57BL/6-mice as donors and BALB/c-mice as recipients. Treatment mice received two intraperitoneal loading doses of CCL19-IgG prior to transplantation, followed by local treatment with CCL19-IgG containing eye drops for the first two weeks after transplantation. Control mice received same amounts of control-IgG. Kaplan-Meier survival analysis showed that in the CCL19-IgG treated group, 76% of the grafts survived through the end

  14. PD1 blockade with pembrolizumab is highly effective in relapsed or refractory NK/T-cell lymphoma failing l-asparaginase.

    PubMed

    Kwong, Yok-Lam; Chan, Thomas S Y; Tan, Daryl; Kim, Seok Jin; Poon, Li-Mei; Mow, Benjamin; Khong, Pek-Lan; Loong, Florence; Au-Yeung, Rex; Iqbal, Jabed; Phipps, Colin; Tse, Eric

    2017-04-27

    Natural killer (NK)/T-cell lymphomas failing L-asparaginse regimens have no known salvage and are almost invariably fatal. Seven male patients with NK/T-cell lymphoma (median age, 49 years; range, 31-68 years) for whom a median of 2 (range, 1-5) regimens (including l-asparaginase regimens and allogeneic hematopoietic stem-cell transplantation [HSCT] in 2 cases) failed were treated with the anti-programmed death 1 (PD1) antibody pembrolizumab. All patients responded, according to various clinical, radiologic (positron emission tomography), morphologic, and molecular (circulating Epstein-Barr virus [EBV] DNA) criteria. Two patients achieved complete response (CR) in all parameters. Three patients achieved clinical and radiologic CRs, with two having molecular remission (undetectable EBV DNA) but minimal EBV-encoded RNA-positive cells in lesions comprising predominantly CD3(+)CD4(+) and CD3(+)CD8(+) T cells (which ultimately disappeared, suggesting they represented pseudoprogression) and one having detectable EBV DNA despite morphologic CR. Two patients achieved partial response (PR). After a median of 7 (range, 2-13) cycles of pembrolizumab and a follow-up of a median of 6 (range, 2-10) months, all five CR patients were still in remission. The only adverse event was grade 2 skin graft-versus-host disease in one patient with previous allogeneic HSCT. Expression of the PD1 ligand was strong in 4 patients (3 achieving CR) and weak in 1 (achieving PR). PD1 blockade with pembrolizumab was a potent strategy for NK/T-cell lymphomas failing l-asparaginase regimens. © 2017 by The American Society of Hematology.

  15. Loss of oxidative defense and potential blockade of satellite cell maturation in the skeletal muscle of patients with cancer but not in the healthy elderly

    PubMed Central

    Brzeszczyńska, Joanna; Johns, Neil; Schilb, Alain; Degen, Simone; Degen, Martin; Langen, Ramon; Schols, Annemie; Glass, David J; Roubenoff, Ronenn; Greig, Carolyn A.

    2016-01-01

    Muscle wasting in old age or cancer may result from failed myofiber regeneration and/or accelerated atrophy. This study aimed to determine from transcriptomic analysis of human muscle the integrity of the cellular stress response system in relation to satellite cell differentiation or apoptosis in patients with cancer (weight-stable (CWS) or weight-losing (CWL)) or healthy elderly (HE) when compared with healthy middle-aged controls (HMA). 28 patients with cancer (CWS: 18 and CWL: 10), HE: 21 and HMA: 20 underwent biopsy of quadriceps muscle. The expression of transcription factors for muscle regeneration (Pax3, Pax7 and MyoD) was increased in CWS and HE compared with HMA (p<0.001). In contrast, the expression of the late myogenic differentiation marker MyoG was reduced in CWS and CWL but increased in HE (p<0.0001). Bax was significantly increased in CWS, CWL and HE (P<0.0001). Expression of the oxidative defense genes SOD2, GCLM, and Nrf2 was decreased in CWS and CWL but increased in HE (p<0.0001). There is evidence for blockade of satellite cell maturation, upregulation of apoptosis and reduced oxidative defense in the muscle of cancer patients. In the healthy elderly the potential for differentiation and oxidative defense is maintained. PMID:27454226

  16. Loss of oxidative defense and potential blockade of satellite cell maturation in the skeletal muscle of patients with cancer but not in the healthy elderly.

    PubMed

    Brzeszczyńska, Joanna; Johns, Neil; Schilb, Alain; Degen, Simone; Degen, Martin; Langen, Ramon; Schols, Annemie; Glass, David J; Roubenoff, Ronenn; Greig, Carolyn A; Jacobi, Carsten; Fearon, Kenneth Ch; Ross, James A

    2016-08-01

    Muscle wasting in old age or cancer may result from failed myofiber regeneration and/or accelerated atrophy. This study aimed to determine from transcriptomic analysis of human muscle the integrity of the cellular stress response system in relation to satellite cell differentiation or apoptosis in patients with cancer (weight-stable (CWS) or weight-losing (CWL)) or healthy elderly (HE) when compared with healthy middle-aged controls (HMA). 28 patients with cancer (CWS: 18 and CWL: 10), HE: 21 and HMA: 20 underwent biopsy of quadriceps muscle. The expression of transcription factors for muscle regeneration (Pax3, Pax7 and MyoD) was increased in CWS and HE compared with HMA (p≤0.001). In contrast, the expression of the late myogenic differentiation marker MyoG was reduced in CWS and CWL but increased in HE (p≤0.0001). Bax was significantly increased in CWS, CWL and HE (p≤0.0001). Expression of the oxidative defense genes SOD2, GCLM, and Nrf2 was decreased in CWS and CWL but increased in HE (p≤0.0001). There is evidence for blockade of satellite cell maturation, upregulation of apoptosis and reduced oxidative defense in the muscle of cancer patients. In the healthy elderly the potential for differentiation and oxidative defense is maintained.

  17. Sprouty2 Suppresses Epithelial-Mesenchymal Transition of Human Lens Epithelial Cells through Blockade of Smad2 and ERK1/2 Pathways

    PubMed Central

    Chen, Chuan; Chen, Xiaoyun; Qin, Yingyan; Qu, Bo; Luo, Lixia; Lin, Haotian; Wu, Mingxing; Chen, Weirong; Liu, Yizhi

    2016-01-01

    Transforming growth factor β (TGFβ)-induced epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) plays a key role in the pathogenesis of anterior subcapsular cataract (ASC) and capsule opacification. In mouse lens, Sprouty2 (Spry2) has a negative regulatory role on TGFβ signaling. However, the regulation of Spry2 during ASC development and how Spry2 modulates TGFβ signaling pathway in human LECs have not been characterized. Here, we demonstrate that Spry2 expression level is decreased in anterior capsule LECs of ASC patients. Spry2 negatively regulates TGFβ2-induced EMT and migration of LECs through inhibition of Smad2 and ERK1/2 phosphorylation. Also, blockade of Smad2 or ERK1/2 activation suppresses EMT caused by Spry2 downregulation. Collectively, our results for the first time show in human LECs that Spry2 has an inhibitory role in TGFβ signaling pathway. Our findings in human lens tissue and epithelial cells suggest that Spry2 may become a novel therapeutic target for the prevention and treatment of ASC and capsule opacification. PMID:27415760

  18. Testing chemotherapeutic agents in the feather follicle identifies a selective blockade of cell proliferation and a key role for sonic hedgehog signaling in chemotherapy-induced tissue damage.

    PubMed

    Xie, Guojiang; Wang, Hangwei; Yan, Zhipeng; Cai, Linyan; Zhou, Guixuan; He, Wanzhong; Paus, Ralf; Yue, Zhicao

    2015-03-01

    Chemotherapeutic agents induce complex tissue responses in vivo and damage normal organ functions. Here we use the feather follicle to investigate details of this damage response. We show that cyclophosphamide treatment, which causes chemotherapy-induced alopecia in mice and man, induces distinct defects in feather formation: feather branching is transiently and reversibly disrupted, thus leaving a morphological record of the impact of chemotherapeutic agents, whereas the rachis (feather axis) remains unperturbed. Similar defects are observed in feathers treated with 5-fluorouracil or taxol but not with doxorubicin or arabinofuranosyl cytidine (Ara-C). Selective blockade of cell proliferation was seen in the feather branching area, along with a downregulation of sonic hedgehog (Shh) transcription, but not in the equally proliferative rachis. Local delivery of the Shh inhibitor, cyclopamine, or Shh silencing both recapitulated this effect. In mouse hair follicles, those chemotherapeutic agents that disrupted feather formation also downregulated Shh gene expression and induced hair loss, whereas doxorubicin or Ara-C did not. Our results reveal a mechanism through which chemotherapeutic agents damage rapidly proliferating epithelial tissue, namely via the cell population-specific, Shh-dependent inhibition of proliferation. This mechanism may be targeted by future strategies to manage chemotherapy-induced tissue damage.

  19. Brief Report: Blockade of TANK-Binding Kinase 1/IKKɛ Inhibits Mutant Stimulator of Interferon Genes (STING)-Mediated Inflammatory Responses in Human Peripheral Blood Mononuclear Cells.

    PubMed

    Frémond, Marie-Louise; Uggenti, Carolina; Van Eyck, Lien; Melki, Isabelle; Bondet, Vincent; Kitabayashi, Naoki; Hertel, Christina; Hayday, Adrian; Neven, Bénédicte; Rose, Yoann; Duffy, Darragh; Crow, Yanick J; Rodero, Mathieu P

    2017-07-01

    Gain-of-function mutations in TMEM173, encoding the stimulator of interferon (IFN) genes (STING) protein, underlie a novel type I interferonopathy that is minimally responsive to conventional immunosuppressive therapies and associated with high frequency of childhood morbidity and mortality. STING gain-of-function causes constitutive oversecretion of IFN. This study was undertaken to determine the effects of a TANK-binding kinase 1 (TBK-1)/IKKɛ inhibitor (BX795) on secretion and signaling of IFN in primary peripheral blood mononuclear cells (PBMCs) from patients with mutations in STING. PBMCs from 4 patients with STING-associated disease were treated with BX795. The effect of BX795 on IFN pathways was assessed by Western blotting and an IFNβ reporter assay, as well as by quantification of IFNα in cell lysates, staining for STAT-1 phosphorylation, and measurement of IFN-stimulated gene (ISG) messenger RNA (mRNA) expression. Treatment of PBMCs with BX795 inhibited the phosphorylation of IFN regulatory factor 3 and IFNβ promoter activity induced in HEK 293T cells by cyclic GMP-AMP or by genetic activation of STING. In vitro exposure to BX795 inhibited IFNα production in PBMCs of patients with STING-associated disease without affecting cell survival. In addition, BX795 decreased STAT-1 phosphorylation and ISG mRNA expression independent of IFNα blockade. These findings demonstrate the effect of BX795 on reducing type I IFN production and IFN signaling in cells from patients with gain-of-function mutations in STING. A combined inhibition of TBK-1 and IKKɛ therefore holds potential for the treatment of patients carrying STING mutations, and may also be relevant in other type I interferonopathies. © 2017, American College of Rheumatology.

  20. [Cancer immunotherapy by immuno-checkpoint blockade].

    PubMed

    Kawakami, Yutaka

    2015-10-01

    As cancer immunotherapies utilizing anti-tumor T-cell responses, immuno-checkpoint blockade and adoptive T-cell immunotherapy have recently achieved durable responses even in advanced cancer patients with metastases. Administration of antibodies on the T-cell surface, CTLA-4 and PD-1 (or PD-1 ligand PD-L1), resulted in tumor regression of not only melanoma and renal cell cancer which were known to be relatively sensitive to immunotherapy, but also various malignancies including lung, bladder, ovarian, gastric, and head and neck cancers, as well as hematological malignancies such as Hodgkin and B-cell malignant lymphomas. These findings have changed the status of immunotherapy in the development of cancer treatments. Currently, development of combinations employing cancer immunotherapy with immuno-checkpoint blockade, as well as personalized cancer immunotherapy based on the evaluation of pretreatment immune status, are in progress.

  1. Rapid blockade of telomerase activity and tumor cell growth by the DPL lipofection of ribbon antisense to hTR.

    PubMed

    Bajpai, Arun K; Park, Jeong-Hoh; Moon, Ik-Jae; Kang, Hyungu; Lee, Yun-Han; Doh, Kyung-Oh; Suh, Seong-Il; Chang, Byeong-Churl; Park, Jong-Gu

    2005-09-29

    Ribbon antisense (RiAS) to the hTR RNA, a component of the telomerase complex, was employed to inhibit telomerase activity and cancer cell growth. The antisense molecule, hTR-RiAS, combined with enhanced cellular uptake was shown to effectively inhibit telomerase activity and cause rapid cell death in various cancer cell lines. When cancer cells were treated with hTR-RiAS, the level of hTR RNA was reduced by more than 90% accompanied with reduction in telomerase activity. When checked for cancer cell viability, cancer cell lines treated with hTR-RiAS using DNA+Peptide+Lipid complex showed 70-80% growth inhibition in 3 days. The reduced cell viability was due to apoptosis as the percentage of cells exhibiting the sub-G0 arrest and DNA fragmentation increased after antisense treatment. Further, when subcutaneous tumors of a colon cancer cell line (SW480) were treated intratumorally with hTR-RiAS, tumor growth was markedly suppressed with almost total ablation of hTR RNA in the tumor tissue. Cells in the tumor tissue were also found to undergo apoptosis after hTR-RiAS treatment. These results suggest that hTR-RiAS is an effective anticancer reagent, with a potential for broad efficacy to diverse malignant tumors.

  2. A randomized controlled clinical trial on the impact of CCR5 blockade with maraviroc in early infection on T-cell dynamics

    PubMed Central

    Karris, Maile Y.; Umlauf, Anya; Vaida, Florin; Richman, Douglas; Little, Susan; Smith, Davey

    2016-01-01

    Abstract Background: Initiation of antiretroviral therapy (ART) in early HIV infection demonstrates clinical benefits including enhanced CD4+ T-lymphocyte recovery and minimization of the latent HIV reservoir. Whether ART intensification with CCR5 blockade provides additional benefits is unknown. Trial design : This randomized controlled trial evaluated the impact of maraviroc (MVC) intensification in persons starting ART in acute and early HIV (AEH, within 3 months of estimated date of infection). Methods: Twenty persons in AEH in San Diego underwent double-blind randomization to receive either standard of care (SOC) ART or SOC + MVC to evaluate the hypothesis that early CCR5 blockage with a CCR5-containing ART regimen may provide immunologic and clinical benefit. The primary outcome of this study was the difference from baseline to week 48 in the proportion of CCR5+ CD4+ memory T cells. Blood was drawn at baseline and weeks 12, 24, and 48 to evaluate CCR5+ CD4+ and CD8+ T-cell dynamics using multicolor flow cytometry. Results: MVC intensification (n = 10) did not significantly alter CCR5+ T-cell dynamics at week 48 of study compared to SOC (n = 9) in this fully recruited study (mean 1.12 vs 0.63, t = 0.36, df = 16, P = 0.727). Exploratory analyses of additional T-cell subsets suggest that MVC intensification in AEH trended to early greater increases in naïve and activated and proliferating CD4+ T cells (P = 0.11, 0.19), and greater decreases in senescent memory CD4+ T cells (P = 0.06), but these differences did not remain by week 48. CD8+ T-cell evaluations did demonstrate trends to differences at week 48 with slower increases in naïve cells and slower decreases in activated memory cells (P = 0.16, 0.09). There were no reported harms or significantly different adverse events. Conclusions: We did observe a few trends, but did not find compelling evidence that MVC intensification during AEH significantly impacts CD4+ and CD8+ T-cell

  3. Blockade of TRPA1 with HC-030031 attenuates visceral nociception by a mechanism independent of inflammatory resident cells, nitric oxide and the opioid system.

    PubMed

    Pereira, L M S; Lima-Júnior, R C P; Bem, A X C; Teixeira, C G; Grassi, L S; Medeiros, R P; Marques-Neto, R D; Callado, R B; Aragão, K S; Wong, D V T; Vale, M L; Brito, G A C; Ribeiro, R A

    2013-02-01

    Some studies have shown a somatic nociceptive response due to the activation of transient receptor potential A1 channels (TRPA1), which is modulated by the TRPA1 antagonist HC-030031. However, a few studies report the role of TRPA1 in visceral pain. Therefore, we investigated the participation of TRPA1 in visceral nociception and the involvement of nitric oxide, the opioid system and resident cells in the modulation of these channels. Mice were treated with vehicle or HC-030031 (18.75-300 mg/kg) before ifosfamide (400 mg/kg), 0.75% mustard oil (50 μL/colon), acetic acid 0.6% (10 mL/kg), zymosan (1 mg/cavity) or misoprostol (1 μg/cavity) injection. Visceral nociception was assessed through the electronic von Frey test or the writhing response. Ifosfamide-administered mice were euthanized for bladder analysis. The involvement of nitric oxide and the opioid system were investigated in mice injected with ifosfamide and mustard oil, respectively. The participation of resident peritoneal cells in acetic acid-, zymosan- or misoprostol-induced nociception was also evaluated. HC-030031 failed to protect animals against ifosfamide-induced bladder injury (p > 0.05). However, a marked antinociceptive effect against ifosfamide, mustard oil, acetic acid, zymosan and misoprostol was observed (p < 0.05). Neither L-arginine (600 mg/kg) nor naloxone (2 mg/kg) could reverse the antinociceptive effect of HC-030031. The reduction of the peritoneal cell population inhibited the acetic acid and zymosan-related writhes without interfering with the misoprostol effect. Our findings suggest that the blockade of TRPA1 attenuates visceral nociception by a mechanism independent of the modulation of resident cells, nitric oxide and opioid pathways. © 2012 European Federation of International Association for the Study of Pain Chapters.

  4. Therapeutic blockade of CD54 attenuates pulmonary barrier damage in T cell-induced acute lung injury.

    PubMed

    Svedova, Julia; Ménoret, Antoine; Mittal, Payal; Ryan, Joseph M; Buturla, James A; Vella, Anthony T

    2017-07-01

    Acute respiratory distress syndrome (ARDS) is a serious, often fatal condition without available pharmacotherapy. Although the role of innate cells in ARDS has been studied extensively, emerging evidence suggests that T cells may be involved in disease etiology. Staphylococcus aureus enterotoxins are potent T-cell mitogens capable of triggering life-threatening shock. We demonstrate that 2 days after inhalation of S. aureus enterotoxin A, mice developed T cell-mediated increases in vascular permeability, as well as expression of injury markers and caspases in the lung. Pulmonary endothelial cells underwent sequential phenotypic changes marked by rapid activation coinciding with inflammatory events secondary to T-cell priming, followed by reductions in endothelial cell number juxtaposing simultaneous T-cell expansion and cytotoxic differentiation. Although initial T-cell activation influenced the extent of lung injury, CD54 (ICAM-1) blocking antibody administered well after enterotoxin exposure substantially attenuated pulmonary barrier damage. Thus CD54-targeted therapy may be a promising candidate for further exploration into its potential utility in treating ARDS patients. Copyright © 2017 the American Physiological Society.

  5. Chemical and genetic blockade of HDACs enhances osteogenic differentiation of human adipose tissue-derived stem cells by oppositely affecting osteogenic and adipogenic transcription factors

    SciTech Connect

    Maroni, Paola; Brini, Anna Teresa; Arrigoni, Elena; Girolamo, Laura de; Niada, Stefania; Matteucci, Emanuela; Bendinelli, Paola; Desiderio, Maria Alfonsina

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer Acetylation affected hASCs osteodifferentiation through Runx2-PPAR{gamma}. Black-Right-Pointing-Pointer HDACs knocking-down favoured the commitment effect of osteogenic medium. Black-Right-Pointing-Pointer HDACs silencing early activated Runx2 and ALP. Black-Right-Pointing-Pointer PPAR{gamma} reduction and calcium/collagen deposition occurred later. Black-Right-Pointing-Pointer Runx2/PPAR{gamma} target genes were modulated in line with HDACs role in osteo-commitment. -- Abstract: The human adipose-tissue derived stem/stromal cells (hASCs) are an interesting source for bone-tissue engineering applications. Our aim was to clarify in hASCs the role of acetylation in the control of Runt-related transcription factor 2 (Runx2) and Peroxisome proliferator activated receptor (PPAR) {gamma}. These key osteogenic and adipogenic transcription factors are oppositely involved in osteo-differentiation. The hASCs, committed or not towards bone lineage with osteoinductive medium, were exposed to HDACs chemical blockade with Trichostatin A (TSA) or were genetically silenced for HDACs. Alkaline phosphatase (ALP) and collagen/calcium deposition, considered as early and late osteogenic markers, were evaluated concomitantly as index of osteo-differentiation. TSA pretreatment, useful experimental protocol to analyse pan-HDAC-chemical inhibition, and switch to osteogenic medium induced early-osteoblast maturation gene Runx2, while transiently decreased PPAR{gamma} and scarcely affected late-differentiation markers. Time-dependent effects were observed after knocking-down of HDAC1 and 3: Runx2 and ALP underwent early activation, followed by late-osteogenic markers increase and by PPAR{gamma}/ALP activity diminutions mostly after HDAC3 silencing. HDAC1 and 3 genetic blockade increased and decreased Runx2 and PPAR{gamma} target genes, respectively. Noteworthy, HDACs knocking-down favoured the commitment effect of osteogenic medium. Our results reveal

  6. Double Blockade of Glioma Cell Proliferation and Migration by Temozolomide Conjugated with NPPB, a Chloride Channel Blocker.

    PubMed

    Park, Miri; Song, Chiman; Yoon, Hojong; Choi, Kee-Hyun

    2016-03-16

    Glioblastoma is the most common and aggressive primary malignant brain tumor. Temozolomide (TMZ), a chemotherapeutic agent combined with radiation therapy, is used as a standard treatment. The infiltrative nature of glioblastoma, however, interrupts effective treatment with TMZ and increases the tendency to relapse. Voltage-gated chloride channels have been identified as crucial regulators of glioma cell migration and invasion by mediating cell shape and volume change. Accordingly, chloride current inhibition by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), a chloride channel blocker, suppresses cell movement by diminishing the osmotic cell volume regulation. In this study, we developed a novel compound, TMZ conjugated with NPPB (TMZ-NPPB), as a potential anticancer drug. TMZ-NPPB blocked chloride currents in U373MG, a severely invasive human glioma cell line, and suppressed migration and invasion of U373MG cells. Moreover, TMZ-NPPB exhibited DNA modification activity similar to that of TMZ, and surprisingly showed remarkably enhanced cytotoxicity relative to TMZ by inducing apoptotic cell death via DNA damage. These findings indicate that TMZ-NPPB has a dual function in blocking both proliferation and migration of human glioma cells, thereby suggesting its potential to overcome challenges in current glioblastoma therapy.

  7. CXCL13 Blockade Disrupts B Lymphocyte Organization in Tertiary Lymphoid Structures without Altering B Cell Receptor Bias or Preventing Diabetes in Nonobese Diabetic Mice

    PubMed Central

    Henry, Rachel A.; Kendall, Peggy L.

    2013-01-01

    Lymphocytes that invade nonlymphoid tissues often organize into follicle-like structures known as tertiary lymphoid organs (TLOs). These structures resemble those found in spleen or lymph nodes, but their function is unknown. TLOs are recognized in many autoimmune diseases, including the NOD mouse model of type 1 diabetes. In some cases, TLOs have been associated with the B lymphocyte chemoattractant, CXCL13. Studies presented in this article show that CXCL13 is present in inflamed islets of NOD mice. Ab blockade of this chemokine unraveled B lymphocyte organization in islet TLOs, without reducing their proportion in the islets. These chaotic milieus contained B lymphocytes with the same distinct repertoire of B cell receptors as those found in mice with well-organized structures. Somatic hypermutation, associated with T–B interactions, was not impaired in these disorganized insulitis lesions. Finally, loss of B lymphocyte organization in islets did not provide disease protection. Thus, B lymphocytes infiltrating islets in NOD mice do not require the morphology of secondary lymphoid tissues to support their role in disease. PMID:20574003

  8. Antibodies targeting BTLA or TIM-3 enhance HIV-1 specific T cell responses in combination with PD-1 blockade.

    PubMed

    Grabmeier-Pfistershammer, Katharina; Stecher, Carmen; Zettl, Markus; Rosskopf, Sandra; Rieger, Armin; Zlabinger, Gerhard J; Steinberger, Peter

    2017-09-04

    Persistent stimulation with antigens derived from viruses that establish chronic infections or tumour antigens results in the exhaustion of T cells. Coinhibitory receptors like PD-1 and CTLA-4 function as immune checkpoints on exhausted T cells. Blocking these molecules with antibodies improve immunity to cancer cells. Immune checkpoint inhibitors targeting other coinhibitory receptors might have a similar role in improving T cell function and thus also utility in cancer therapy. Using HIV-specific T cells as a model for exhaustion we have evaluated the capacity of antibodies targeting TIM-3, BTLA, CD160, LAG-3 and CTLA-4 alone or in combination with a PD-1 antibody to enhance proliferation and cytokine production in response to Gag and Nef peptides. Antibodies targeting BTLA and TIM-3 enhanced CD8 T cell proliferation. Moreover, our results indicate that blocking BTLA and TIM-3 in combination with PD-1 might be especially effective in enhancing responses of exhausted human T cells. Copyright © 2017. Published by Elsevier Inc.

  9. Nebivolol exerts beneficial effects on endothelial function, early endothelial progenitor cells, myocardial neovascularization, and left ventricular dysfunction early after myocardial infarction beyond conventional β1-blockade.

    PubMed

    Sorrentino, Sajoscha A; Doerries, Carola; Manes, Costantina; Speer, Thimoteus; Dessy, Chantal; Lobysheva, Irina; Mohmand, Wazma; Akbar, Razma; Bahlmann, Ferdinand; Besler, Christian; Schaefer, Arnd; Hilfiker-Kleiner, Denise; Lüscher, Thomas F; Balligand, Jean-Luc; Drexler, Helmut; Landmesser, Ulf

    2011-02-01

    The aim of this study was to investigate whether nebivolol has added effects on left ventricular (LV) dysfunction and remodeling early after myocardial infarction (MI) beyond its β₁-receptor-blocking properties. Nebivolol is a third-generation selective β₁-adrenoreceptor antagonist that stimulates endothelial cell nitric oxide (NO) production and prevents vascular reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation. Both endothelial NO synthase-derived NO production and NADPH oxidase activation are critical modulators of LV dysfunction early after MI. Mice with extensive anterior MI (n = 90) were randomized to treatment with nebivolol (10 mg/kg/day), metoprolol-succinate (20 mg/kg/day), or placebo for 30 days starting on day 1 after surgery. Infarct size was similar among the groups. Both β₁-adrenergic receptor antagonists caused a similar decrease in heart rate. Nebivolol therapy improved endothelium-dependent vasorelaxation and increased early endothelial progenitor cells 4 weeks after MI compared with metoprolol and placebo. Nebivolol, but not metoprolol, inhibited cardiac NADPH oxidase activation after MI, as detected by electron spin resonance spectroscopy analysis. Importantly, nebivolol, but not metoprolol, improved LV dysfunction 4 weeks after MI (LV ejection fraction: nebivolol vs. metoprolol vs. placebo: 32 ± 4% vs. 17 ± 6% vs. 19 ± 4%; nebivolol vs. metoprolol: p < 0.05) and was associated with improved survival 4 weeks post-MI compared with placebo. Nebivolol had a significantly more pronounced inhibitory effect on cardiomyocyte hypertrophy after MI compared with metoprolol. Nebivolol improves LV dysfunction and survival early after MI likely beyond the effects provided by conventional β₁-receptor blockade. Nebivolol induced effects on NO-mediated endothelial function, early endothelial progenitor cells and inhibition of myocardial NADPH oxidase likely contribute to these beneficial effects of nebivolol early

  10. Synthetic tambjamine analogues induce mitochondrial swelling and lysosomal dysfunction leading to autophagy blockade and necrotic cell death in lung cancer.

    PubMed

    Rodilla, Ananda M; Korrodi-Gregório, Luís; Hernando, Elsa; Manuel-Manresa, Pilar; Quesada, Roberto; Pérez-Tomás, Ricardo; Soto-Cerrato, Vanessa

    2017-02-15

    Current pharmacological treatments for lung cancer show very poor clinical outcomes, therefore, the development of novel anticancer agents with innovative mechanisms of action is urgently needed. Cancer cells have a reversed pH gradient compared to normal cells, which favours cancer progression by promoting proliferation, metabolic adaptation and evasion of apoptosis. In this regard, the use of ionophores to modulate intracellular pH appears as a promising new therapeutic strategy. Indeed, there is a growing body of evidence supporting ionophores as novel antitumour drugs. Despite this, little is known about the implications of pH deregulation and homeostasis imbalance triggered by ionophores at the cellular level. In this work, we deeply analyse for the first time the anticancer effects of tambjamine analogues, a group of highly effective anion selective ionophores, at the cellular and molecular levels. First, their effects on cell viability were determined in several lung cancer cell lines and patient-derived cancer stem cells, demonstrating their potent cytotoxic effects. Then, we have characterized the induced lysosomal deacidification, as well as, the massive cytoplasmic vacuolization observed after treatment with these compounds, which is consistent with mitochondrial swelling. Finally, the activation of several proteins involved in stress response, autophagy and apoptosis was also detected, although they were not significantly responsible for the cell death induced. Altogether, these evidences suggest that tambjamine analogues provoke an imbalance in cellular ion homeostasis that triggers mitochondrial dysfunction and lysosomal deacidification leading to a potent cytotoxic effect through necrosis in lung cancer cell lines and cancer stem cells.

  11. Targeting mantle cell lymphoma metabolism and survival through simultaneous blockade of mTOR and nuclear transporter exportin-1

    PubMed Central

    Sekihara, Kazumasa; Saitoh, Kaori; Han, Lina; Ciurea, Stefan; Yamamoto, Shinichi; Kikkawa, Mika; Kazuno, Saiko; Taka, Hikari; Kaga, Naoko; Arai, Hajime; Miida, Takashi; Andreeff, Michael; Konopleva, Marina; Tabe, Yoko

    2017-01-01

    Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma with poor prognosis, characterized by aberrant expression of growth-regulating and oncogenic effectors and requiring novel anticancer strategies. The nuclear transporter exportin-1 (XPO1) is highly expressed in MCL and is associated with its pathogenesis. mTOR signaling, a central regulator of cell metabolism, is frequently activated in MCL and is also an important therapeutic target in this cancer. This study investigated the antitumor effects and molecular/metabolic changes induced by the combination of the small-molecule selective inhibitor XPO1 inhibitor KPT-185 and the dual mTORC1/2 kinase inhibitor AZD-2014 on MCL cells. AZD-2014 enhanced the KPT-185–induced inhibition of cell growth and repression of cell viability. The combination of KPT-185 and AZD-2014 downregulated c-Myc and heat shock factor 1 (HSF1) with its target heat shock protein 70 (HSP70). As a consequence, the combination caused repression of ribosomal biogenesis demonstrated by iTRAQ proteomic analyses. Metabolite assay by CETOF-MS showed that AZD-2014 enhanced the KPT-185–induced repression of MCL cellular energy metabolism through the TCA (Krebs) cycle, and further repressed KPT-185–caused upregulation of glycolysis. Thus the simultaneous inhibition of XPO1 and mTOR signaling is a novel and promising strategy targeting prosurvival metabolism in MCL. PMID:28388555

  12. Basal cell carcinoma: PD-L1/PD-1 checkpoint expression and tumor regression after PD-1 blockade.

    PubMed

    Lipson, Evan J; Lilo, Mohammed T; Ogurtsova, Aleksandra; Esandrio, Jessica; Xu, Haiying; Brothers, Patricia; Schollenberger, Megan; Sharfman, William H; Taube, Janis M

    2017-01-01

    Monoclonal antibodies that block immune regulatory proteins such as programmed death-1 (PD-1) have demonstrated remarkable efficacy in controlling the growth of multiple tumor types. Unresectable or metastatic basal cell carcinoma, however, has largely gone untested. Because PD-Ligand-1 (PD-L1) expression in other tumor types has been associated with response to anti-PD-1, we investigated the expression of PD-L1 and its association with PD-1 expression in the basal cell carcinoma tumor microenvironment. Among 40 basal cell carcinoma specimens, 9/40 (22%) demonstrated PD-L1 expression on tumor cells, and 33/40 (82%) demonstrated PD-L1 expression on tumor-infiltrating lymphocytes and associated macrophages. PD-L1 was observed in close geographic association to PD-1+ tumor infiltrating lymphocytes. Additionally, we present, here, the first report of an objective anti-tumor response to pembrolizumab (anti-PD-1) in a patient with metastatic PD-L1 (+) basal cell carcinoma, whose disease had previously progressed through hedgehog pathway-directed therapy. The patient remains in a partial response 14 months after initiation of therapy. Taken together, our findings provide a rationale for testing anti-PD-1 therapy in patients with advanced basal cell carcinoma, either as initial treatment or after acquired resistance to hedgehog pathway inhibition.

  13. Compound C induces protective autophagy in cancer cells through AMPK inhibition-independent blockade of Akt/mTOR pathway.

    PubMed

    Vucicevic, Ljubica; Misirkic, Maja; Janjetovic, Kristina; Vilimanovich, Urosh; Sudar, Emina; Isenovic, Esma; Prica, Marko; Harhaji-Trajkovic, Ljubica; Kravic-Stevovic, Tamara; Bumbasirevic, Vladimir; Trajkovic, Vladimir

    2011-01-01

    In the present study, we report that compound C, an inhibitor of a key intracellular energy sensor AMP-activated protein kinase (AMPK), can induce autophagy in cancer cells. The induction of autophagy in U251 human glioma cell line was demonstrated by acridine orange staining of intracellular acidic vesicles, Beclin 1 induction, p62 decrease and conversion of LC3-I to autophagosome-associated LC3-II in the presence of proteolysis inhibitors. The presence of autophagosome-like vesicles was confirmed by transmission electron microscopy. Compound C-mediated inhibition of AMPK and raptor in U251 cells was associated with paradoxical decrease in phosphorylation of AMPK/raptor-repressed mTOR, a major negative regulator of autophagy, and its downstream target p70S6K. The phosphorylation of an mTOR activator Akt and the PI3K-activating kinase Src was also impaired in compound C-treated cells. The siRNA-mediated AMPK silencing did not reduce the activity of the Akt/mTOR/p70S6K pathway and AMPK activators metformin and AIC AR failed to block compound C-induced autophagy. Autophagy inhibitors bafilomycin and chloroquine significantly increased the cytotoxicity of compound C towards U251 cells, as confirmed by increase in lactate dehydrogenase release, DNA fragmentation and caspase-3 activation. Similar effects of compound C were also observed in C6 rat glioma, L929 mouse fibrosarcoma and B16 mouse melanoma cell lines. Since compound C has previously been reported to suppress AMPK-dependent autophagy in different cell types, our findings suggest that the effects of compound C on autophagy might be dose-, cell type- and/or context-dependent. By demonstrating the ability of compound C to induce autophagic response in cancer cells via AMPK inhibition-independent downregulation of Akt/mTOR pathway, our results warrant caution when using compound C to inhibit AMPK-dependent cellular responses, but also support further exploration of compound C and related molecules as potential

  14. Post-Therapeutic Relapse of Psoriasis after CD11a Blockade Is Associated with T Cells and Inflammatory Myeloid DCs

    PubMed Central

    Johnson-Huang, Leanne M.; Pensabene, Cara A.; Shah, Kejal R.; Pierson, Katherine C.; Kikuchi, Toyoko; Lentini, Tim; Gilleaudeau, Patricia; Sullivan-Whalen, Mary; Cueto, Inna; Khatcherian, Artemis; Hyder, Luke A.; Suárez-Fariñas, Mayte; Krueger, James G.; Lowes, Michelle A.

    2012-01-01

    To understand the development of new psoriasis lesions, we studied a group of moderate-to-severe psoriasis patients who experienced a relapse after ceasing efalizumab (anti-CD11a, Raptiva, Genentech). There were increased CD3+ T cells, neutrophils, CD11c+ and CD83+ myeloid dendritic cells (DCs), but no increase in CD1c+ resident myeloid DCs. In relapsed lesions, there were many CD11c+CD1c−, inflammatory myeloid DCs identified by TNFSF10/TRAIL, TNF, and iNOS. CD11c+ cells in relapsed lesions co-expressed CD14 and CD16 in situ. Efalizumab induced an improvement in many psoriasis genes, and during relapse, the majority of these genes reversed back to a lesional state. Gene Set Enrichment Analysis (GSEA) of the transcriptome of relapsed tissue showed that many of the gene sets known to be present in psoriasis were also highly enriched in relapse. Hence, on ceasing efalizumab, T cells and myeloid cells rapidly enter the skin to cause classic psoriasis. Trial registration Clinicaltrials.gov NCT00115076 PMID:22348003

  15. Lysosomal dysfunction and autophagy blockade contribute to IMB-6G-induced apoptosis in pancreatic cancer cells

    PubMed Central

    Liu, Lu; Zhang, Na; Dou, Yueying; Mao, Genxiang; Bi, Chongwen; Pang, Weiqiang; Liu, Xiaojia; Song, Danqing; Deng, Hongbin

    2017-01-01

    Targeting the autophagic pathway is currently regarded as an attractive strategy for cancer drug discovery. Our previous work showed that IMB-6G is a novel N-substituted sophoridinic acid derivative with potent cytotoxicity against tumor cells, yet the effect of IMB-6G on autophagy and pancreatic cancer cell death remains unknown. Here, we show that IMB-6G inhibits the growth of MiaPaCa-2 and HupT-3 pancreatic cancer cells and induces caspase-mediated apoptosis, which is correlated with an accumulation of autophagic vacuoles. IMB-6G promotes autophagosome accumulation from the early stage of treatment but blocks autophagic flux in the degradation stage, mainly through attenuation of lysosomal cathepsin activity in pancreatic cancer cells. Moreover, IMB-6G triggers lysosomal membrane permeabilization (LMP), followed by cathepsin B/CTSB and cathepsin D/CTSD release from lysosomes into the cytoplasm. Inhibition of autophagosome formation with siRNA against autophagy protein 5 (Atg5) attenuates IMB-6G-induced LMP and apoptosis. Furthermore, cathepsin inhibitors relieve IMB-6G-induced apoptosis as well. Altogether, our findings demonstrate that IMB-6G is a novel autophagy inhibitor, which induces autophagy-dependent apoptosis through autophagosomal-cathepsin axis in pancreatic cancer cells and indicate the potential value of IMB-6G as a novel antitumor drug candidate. PMID:28139733

  16. Adrenomedullin blockade suppresses sunitinib-resistant renal cell carcinoma growth by targeting the ERK/MAPK pathway

    PubMed Central

    Meng, Qingsong; Zhang, Ming; Wang, Xin; Jia, Jianghua; Yang, Shuwen; Qu, Changbao; Li, Wei; Wang, Dongbin

    2016-01-01

    Purpose To evaluate the mechanisms underlying sunitinib resistance in RCC and to identify targets that may be used to overcome this resistance. Results Reanalysis of transcriptome microarray datasets (GSE64052 and GSE76068) showed that adrenomedullin expression was increased in sunitinib-resistant tumors. And adrenomedullin expression was increased in sunitinib-resistant tumor xenografts, accompanied by upregulation of phospho-ERK levels. However, blocking adrenomedullin inhibited sunitinib-resistant tumor growth. Treatment of RCC cells with sunitinib and ADM22-52 was superior to monotherapy with either agent. Additionally, adrenomedullin upregulated cAMP and activated the ERK/MAPK pathway, promoting cell proliferation, while knockdown of adrenomedullin inhibited RCC cell growth and invasion in vitro. Materials and methods We searched the Gene Expression Omnibus (GEO) database to find data regarding sunitinib-resistant RCC. These data were subsequently reanalyzed to identify targets that contribute to sunitinib resistance, and adrenomedullin upregulation was found to mediate sunitinib resistance in RCC. Then, we created an RCC mouse xenograft model. Mice were treated with sunitinib, an adrenomedullin receptor antagonist (ADM22-52), a MEK inhibitor (PD98059) and different combinations of these three drugs to investigate their effects on tumor growth. RCC cells (786-0) were cultured in vitro and treated with an ADM22-52 or PD98059 to determine whether adrenomedullin activates the ERK/MAPK pathway. Adrenomedullin was knocked down in 786-0 cells via siRNA, and the effects of this knockdown on cell were subsequently investigated. Conclusions Adrenomedullin plays an important role in RCC resistance to sunitinib treatment. The combination of sunitinib and an adrenomedullin receptor antagonist may result in better outcomes in advanced RCC patients. PMID:27556517

  17. Blockade of Tumor Cell TGF-Betas: A Strategy to Reverse Antiestrogen Resistance in Human Breast Cancer

    DTIC Science & Technology

    2002-01-01

    junctions and the acquisition of spindle cell morphology. LY294002, an inhibitor of the pl 10 catalytic subunit of P13K, and a dominant- negative...kinase- phorylation of Smad2 in HaCaT cells was not altered by inactive version of Akt in which Lys179 in the catalytic dn Akt. domain has been mutated...that result in LY294002, a small molecule inhibitor of the p110 catalytic growth inhibition may be independent from those involved subunit of P13K

  18. Embryonic GABA(B) receptor blockade alters cell migration, adult hypothalamic structure, and anxiety- and depression-like behaviors sex specifically in mice.

    PubMed

    Stratton, Matthew S; Staros, Michelle; Budefeld, Tomaz; Searcy, Brian T; Nash, Connor; Eitel, Chad; Carbone, David; Handa, Robert J; Majdic, Gregor; Tobet, Stuart A

    2014-01-01

    Neurons of the paraventricular nucleus of the hypothalamus (PVN) regulate the hypothalamic- pituitary-adrenal (HPA) axis and the autonomic nervous system. Females lacking functional GABA(B) receptors because of a genetic disruption of the R1 subunit have altered cellular characteristics in and around the PVN at birth. The genetic disruption precluded appropriate assessments of physiology or behavior in adulthood. The current study was conducted to test the long term impact of a temporally restricting pharmacological blockade of the GABA(B) receptor to a 7-day critical period (E11-E17) during embryonic development. Experiments tested the role of GABA(B) receptor signaling in fetal development of the PVN and later adult capacities for adult stress related behaviors and physiology. In organotypic slices containing fetal PVN, there was a female specific, 52% increase in cell movement speeds with GABA(B) receptor antagonist treatment that was consistent with a sex-dependent lateral displacement of cells in vivo following 7 days of fetal exposure to GABA(B) receptor antagonist. Anxiety-like and depression-like behaviors, open-field activity, and HPA mediated responses to restraint stress were measured in adult offspring of mothers treated with GABA(B) receptor antagonist. Embryonic exposure to GABA(B) receptor antagonist resulted in reduced HPA axis activation following restraint stress and reduced depression-like behaviors. There was also increased anxiety-like behavior selectively in females and hyperactivity in males. A sex dependent response to disruptions of GABA(B) receptor signaling was identified for PVN formation and key aspects of physiology and behavior. These changes correspond to sex specific prevalence in similar human disorders, namely anxiety disorders and hyperactivity.

  19. Embryonic GABAB Receptor Blockade Alters Cell Migration, Adult Hypothalamic Structure, and Anxiety- and Depression-Like Behaviors Sex Specifically in Mice

    PubMed Central

    Stratton, Matthew S.; Staros, Michelle; Budefeld, Tomaz; Searcy, Brian T.; Nash, Connor; Eitel, Chad; Carbone, David; Handa, Robert J.; Majdic, Gregor; Tobet, Stuart A.

    2014-01-01

    Neurons of the paraventricular nucleus of the hypothalamus (PVN) regulate the hypothalamic- pituitary-adrenal (HPA) axis and the autonomic nervous system. Females lacking functional GABAB receptors because of a genetic disruption of the R1 subunit have altered cellular characteristics in and around the PVN at birth. The genetic disruption precluded appropriate assessments of physiology or behavior in adulthood. The current study was conducted to test the long term impact of a temporally restricting pharmacological blockade of the GABAB receptor to a 7-day critical period (E11–E17) during embryonic development. Experiments tested the role of GABAB receptor signaling in fetal development of the PVN and later adult capacities for adult stress related behaviors and physiology. In organotypic slices containing fetal PVN, there was a female specific, 52% increase in cell movement speeds with GABAB receptor antagonist treatment that was consistent with a sex-dependent lateral displacement of cells in vivo following 7 days of fetal exposure to GABAB receptor antagonist. Anxiety-like and depression-like behaviors, open-field activity, and HPA mediated responses to restraint stress were measured in adult offspring of mothers treated with GABAB receptor antagonist. Embryonic exposure to GABAB receptor antagonist resulted in reduced HPA axis activation following restraint stress and reduced depression-like behaviors. There was also increased anxiety-like behavior selectively in females and hyperactivity in males. A sex dependent response to disruptions of GABAB receptor signaling was identified for PVN formation and key aspects of physiology and behavior. These changes correspond to sex specific prevalence in similar human disorders, namely anxiety disorders and hyperactivity. PMID:25162235

  20. Protection by imidazol(ine) drugs and agmatine of glutamate-induced neurotoxicity in cultured cerebellar granule cells through blockade of NMDA receptor

    PubMed Central

    Olmos, Gabriel; DeGregorio-Rocasolano, Nuria; Regalado, M Paz; Gasull, Teresa; Boronat, M Assumpció; Trullas, Ramón; Villarroel, Alvaro; Lerma, Juan; García-Sevilla, Jesús A

    1999-01-01

    neuroprotective against glutamate-induced necrotic neuronal cell death in vitro and that this effect is mediated through NMDA receptor blockade by interacting with a site located within the NMDA channel pore. PMID:10455281

  1. Kaempferol Inhibits Pancreatic Cancer Cell Growth and Migration through the Blockade of EGFR-Related Pathway In Vitro.

    PubMed

    Lee, Jungwhoi; Kim, Jae Hoon

    2016-01-01

    Pancreatic cancer is one of the most appalling cancers with a pessimistic prognosis. Despite many therapies, there has been no improvement of survival rates. In this study, we assessed the anti-cancer effects of kaempferol, a well known flavonoid having functional bio-activity against various malignant tumors. Kaempferol had anti-cancer effects on Miapaca-2, Panc-1, and SNU-213 human pancreatic cancer cells. In a dose-dependent manner, kaempferol decreased viability of these pancreatic cancer cells by increasing apoptosis. In particular, kaempferol effectively inhibited the migratory activity of human pancreatic cancer cells at relatively low dosages without any toxicity. The anti-cancer effect of kaempferol was mediated by inhibition of EGFR related Src, ERK1/2, and AKT pathways. These results collectively indicate that kaempferol, a phytochemical ingredient reported to have anti-viability and anti-oxidant properties, can act as a safety anti-migration reagent in human pancreatic cancer cells, which provide the rationale for further investigation of kaempferol as a strong candidate for the potential clinical trial of malignant pancreatic cancers.

  2. Kaempferol Inhibits Pancreatic Cancer Cell Growth and Migration through the Blockade of EGFR-Related Pathway In Vitro

    PubMed Central

    Lee, Jungwhoi; Kim, Jae Hoon

    2016-01-01

    Pancreatic cancer is one of the most appalling canc