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Sample records for human fc gamma

  1. A novel human immunoglobulin Fc gamma Fc epsilon bifunctional fusion protein inhibits Fc epsilon RI-mediated degranulation.

    Science.gov (United States)

    Zhu, Daocheng; Kepley, Christopher L; Zhang, Min; Zhang, Ke; Saxon, Andrew

    2002-05-01

    Human mast cells and basophils that express the high-affinity immunoglobulin E (IgE) receptor, Fc epsilon receptor 1 (Fc epsilon RI), have key roles in allergic diseases. Fc epsilon RI cross-linking stimulates the release of allergic mediators. Mast cells and basophils co-express Fc gamma RIIb, a low affinity receptor containing an immunoreceptor tyrosine-based inhibitory motif and whose co-aggregation with Fc epsilon RI can block Fc epsilon RI-mediated reactivity. Here we designed, expressed and tested the human basophil and mast-cell inhibitory function of a novel chimeric fusion protein, whose structure is gamma Hinge-CH gamma 2-CH gamma 3-15aa linker-CH epsilon 2-CH epsilon 3-CH epsilon 4. This Fc gamma Fc epsilon fusion protein was expressed as the predicted 140-kappa D dimer that reacted with anti-human epsilon- and gamma-chain specific antibodies. Fc gamma Fc epsilon bound to both human Fc epsilon RI and Fc gamma RII. It also showed dose- and time-dependent inhibition of antigen-driven IgE-mediated histamine release from fresh human basophils sensitized with IgE directed against NIP (4-hydroxy-3-iodo-5-nitrophenylacetyl). This was associated with altered Syk signaling. The fusion protein also showed increased inhibition of human anti-NP (4-hydroxy-3-nitrophenylacetyl) and anti-dansyl IgE-mediated passive cutaneous anaphylaxis in transgenic mice expressing human Fc epsilon RI alpha. Our results show that this chimeric protein is able to form complexes with both Fc epsilon RI and Fc gamma RII, and inhibit mast-cell and basophil function. This approach, using a Fc gamma Fc epsilon fusion protein to co-aggregate Fc epsilon RI with a receptor containing an immunoreceptor tyrosine-based inhibition motif, has therapeutic potential in IgE- and Fc epsilon RI-mediated diseases.

  2. Gamma interferon augments Fc gamma receptor-mediated dengue virus infection of human monocytic cells.

    OpenAIRE

    Kontny, U.; Kurane, I; Ennis, F A

    1988-01-01

    It has been reported that anti-dengue antibodies at subneutralizing concentrations augment dengue virus infection of monocytic cells. This is due to the increased uptake of dengue virus in the form of virus-antibody complexes by cells via Fc gamma receptors. We analyzed the effects of recombinant human gamma interferon (rIFN-gamma) on dengue virus infection of human monocytic cells. U937 cells, a human monocytic cell line, were infected with dengue virus in the form of virus-antibody complexe...

  3. Structural Basis for Fc[gamma]RIIa Recognition of Human IgG and Formation of Inflammatory Signaling Complexes

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    Ramsland, Paul A.; Farrugia, William; Bradford, Tessa M.; Sardjono, Caroline Tan; Esparon, Sandra; Trist, Halina M.; Powell, Maree S.; Tan, Peck Szee; Cendron, Angela C.; Wines, Bruce D.; Scott, Andrew M.; Hogarth, P. Mark (Burnet); (Monash); (LICR); (Melbourne)

    2011-09-20

    The interaction of Abs with their specific FcRs is of primary importance in host immune effector systems involved in infection and inflammation, and are the target for immune evasion by pathogens. Fc{gamma}RIIa is a unique and the most widespread activating FcR in humans that through avid binding of immune complexes potently triggers inflammation. Polymorphisms of Fc{gamma}RIIa (high responder/low responder [HR/LR]) are linked to susceptibility to infections, autoimmune diseases, and the efficacy of therapeutic Abs. In this article, we define the three-dimensional structure of the complex between the HR (arginine, R134) allele of Fc{gamma}RIIa (Fc{gamma}RIIa-HR) and the Fc region of a humanized IgG1 Ab, hu3S193. The structure suggests how the HR/LR polymorphism may influence Fc{gamma}RIIa interactions with different IgG subclasses and glycoforms. In addition, mutagenesis defined the basis of the epitopes detected by FcR blocking mAbs specific for Fc{gamma}RIIa (IV.3), Fc{gamma}RIIb (X63-21), and a pan Fc{gamma}RII Ab (8.7). The epitopes detected by these Abs are distinct, but all overlap with residues defined by crystallography to contact IgG. Finally, crystal structures of LR (histidine, H134) allele of Fc{gamma}RIIa and Fc{gamma}RIIa-HR reveal two distinct receptor dimers that may represent quaternary states on the cell surface. A model is presented whereby a dimer of Fc{gamma}RIIa-HR binds Ag-Ab complexes in an arrangement that possibly occurs on the cell membrane as part of a larger signaling assembly.

  4. Genetic variation in human Fc gamma receptors: Functional consequences of polymorphisms and copy number variation

    NARCIS (Netherlands)

    van der Heijden, J.

    2014-01-01

    Fc gamma receptors (FcγRs) are receptors for immunoglobulin G (IgG), the most abundant of five classes of antibodies. They are expressed on almost all immune cells and mediate a range of cellular functions, such as phagocytosis, antibody-dependent cellular cytotoxicity, activation of the NADPH-oxida

  5. Type I (CD64) and type II (CD32) Fc gamma receptor-mediated phagocytosis by human blood dendritic cells.

    Science.gov (United States)

    Fanger, N A; Wardwell, K; Shen, L; Tedder, T F; Guyre, P M

    1996-07-15

    Three classes of Fc receptors for IgG, Fc gamma RI (CD64), Fc gamma RII (CD32), and Fc gamma RIII (CD16), are expressed on blood leukocytes. Although Fc gamma R are important phagocytic receptors on phagocytes, most reports suggest that dendritic cells lack Fc gamma R-mediated phagocytosis and express significant levels of only CD32. We now report that phagocytically active forms of both CD64 and CD32 are expressed significantly on at least one subset of human blood dendritic cells. Countercurrent elutriation and magnetic bead selection were used to rapidly enrich subsets of blood dendritic cells (CD33brightCD14-HLA-DRbrightCD83-) and monocytes (CD33brightCD14brightHLA-DRdimCD83-). Upon culture for 2 days, dendritic cells became CD83-positive and markedly increased HLA-DR expression, whereas monocytes did not express CD83 and exhibited reduced levels of HLA-DR. Constitutive CD64 expression was identified on this circulating dendritic cell population, but at a lower level than on monocytes. CD64 expression by dendritic cells and monocytes did not decrease during 2 days in culture, and was up-regulated on both cell types following incubation with IFN-gamma. Freshly isolated blood dendritic cells performed CD64- and CD32-mediated phagocytosis, although at a lower level than monocytes. Dendritic cells generated by culture of adherent mononuclear cells in granulocyte-macrophage CSF and IL-4 also up-regulated CD64 following IFN-gamma stimulation, and mediated CD64-dependent phagocytosis. These results indicate that both CD64 and CD32 expressed on blood dendritic cells may play a role in uptake of foreign particles and macromolecules through a phagocytic mechanism before trafficking to T cell-reactive areas.

  6. Fc gamma RI blockade and modulation for immunotherapy.

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    Wallace, P K; Keler, T; Guyre, P M; Fanger, M W

    1997-01-01

    Splenectomy and corticosteroids are the treatment of choice for patients with immune thrombocytopenic purpura (ITP). However, for the 10%-15% of patients who do not respond to conventional therapy, high-dose i.v. IgG can induce life-saving transient responses. The benefits of i.v. IgG have been attributed to Fc receptor blockade; however, the involvement of the individual Fc receptors for IgG (Fc gamma R) in ITP remain to be more completely defined. Recently a mAb, designated mAb H22, which recognizes an epitope on Fc gamma RI (CD64) outside the ligand-binding domain, was humanized. Because mAb H22 is a human IgG1 and Fc gamma RI has a high affinity for human IgG1 antibodies, we predicted that mAb H22 would bind to the Fc gamma RI ligand-binding site through its Fc domain and to its external Fc gamma RI epitope through both Fab domains. These studies demonstrate that mAb H22 blocked Fc gamma RI-mediated phagocytosis of opsonized red blood cells more effectively than an irrelevant IgG. Moreover, cross-linking Fc gamma RI with mAb H22 down-modulated Fc gamma RI expression on monocytes, an effect seen within 2 h.

  7. Fc gamma receptor IIIB (Fc gamma RIIIB) polymorphisms are associated with clinical malaria in Ghanaian children

    DEFF Research Database (Denmark)

    Adu, Bright; Dodoo, Daniel; Adukpo, Selorme;

    2012-01-01

    Plasmodium falciparum malaria kills nearly a million people annually. Over 90% of these deaths occur in children under five years of age in sub-Saharan Africa. A neutrophil mediated mechanism, the antibody dependent respiratory burst (ADRB), was recently shown to correlate with protection from...... clinical malaria. Human neutrophils constitutively express Fc gamma receptor-Fc¿RIIA and Fc¿RIIIB by which they interact with immunoglobulin (Ig) G (IgG)-subclass antibodies. Polymorphisms in exon 4 of FCGR2A and exon 3 of FCGR3B genes encoding Fc¿RIIA and Fc¿RIIIB respectively have been described to alter...... malaria and provides justification for further functional characterization of variants of the classical Fc¿RIIIB allotypes. This would be crucial to the improvement of neutrophil mediated functional assays such as the ADRB assay aimed at assessing the functionality of antibodies induced by candidate...

  8. A novel association of Fc receptor gamma-chain with glycoprotein VI and their co-expression as a collagen receptor in human platelets.

    Science.gov (United States)

    Tsuji, M; Ezumi, Y; Arai, M; Takayama, H

    1997-09-19

    The mechanism by which occupancy of collagen receptors is coupled to platelet activation has been uncertain. Our group previously demonstrated that glycoprotein (GP) VI, an uncharacterized platelet membrane protein, is specifically required for collagen-platelet interaction leading to activation of protein-tyrosine kinase Syk. Since collagen stimulation of platelets has recently been found to induce tyrosine phosphorylation of Fc receptor (FcR) gamma-chain, a signal-generating subunit of FcR, we further investigated the relationships between FcR gamma-chain and GPVI in human platelets. Our present study revealed the following. FcR gamma-chain was physically and stably associated with GPVI in human platelets; both FcR gamma-chain and GPVI were proportionally absent in GPVI-deficient platelets; GPVI cross-linking or collagen stimulation of platelets resulted in tyrosine phosphorylation of GPVI-associated FcR gamma-chain accompanied by Syk association and activation. These findings strongly suggest that the associated complex of GPVI and FcR gamma-chain is a collagen receptor featuring the signaling through immune receptors.

  9. Physical and functional association of the Src family kinases Fyn and Lyn with the collagen receptor glycoprotein VI-Fc receptor gamma chain complex on human platelets.

    Science.gov (United States)

    Ezumi, Y; Shindoh, K; Tsuji, M; Takayama, H

    1998-07-20

    We have previously shown that uncharacterized glycoprotein VI (GPVI), which is constitutively associated and coexpressed with Fc receptor gamma chain (FcRgamma) in human platelets, is essential for collagen-stimulated tyrosine phosphorylation of FcRgamma, Syk, and phospholipase Cgamma2 (PLCgamma2), leading to platelet activation. Here we investigated involvement of the Src family in the proximal signals through the GPVI-FcRgamma complex, using the snake venom convulxin from Crotalus durissus terrificus, which specifically recognizes GPVI and activates platelets through cross-linking GPVI. Convulxin-coupled beads precipitated the GPVI-FcRgamma complex from platelet lysates. Collagen and convulxin induced tyrosine phosphorylation of FcRgamma, Syk, and PLCgamma2 and recruited tyrosine-phosphorylated Syk to the GPVI-FcRgamma complex. Using coprecipitation methods with convulxin-coupled beads and antibodies against FcRgamma and the Src family, we showed that Fyn and Lyn, but not Yes, Src, Fgr, Hck, and Lck, were physically associated with the GPVI-FcRgamma complex irrespective of stimulation. Furthermore, Fyn was rapidly activated by collagen or cross-linking GPVI. The Src family-specific inhibitor PP1 dose-dependently inhibited collagen- or convulxin-induced tyrosine phosphorylation of proteins including FcRgamma, Syk, and PLCgamma2, accompanied by a loss of aggregation and ATP release reaction. These results indicate that the Src family plays a critical role in platelet activation via the collagen receptor GPVI-FcRgamma complex.

  10. Characterization of expression, cytokine regulation, and effector function of the high affinity IgG receptor Fc gamma RI (CD64) expressed on human blood dendritic cells.

    Science.gov (United States)

    Fanger, N A; Voigtlaender, D; Liu, C; Swink, S; Wardwell, K; Fisher, J; Graziano, R F; Pfefferkorn, L C; Guyre, P M

    1997-04-01

    The mechanisms responsible for efficient sequestration of Ag by cells of the dendritic cell (DC) lineage remain incompletely characterized. One pathway, internalization of Ag-IgG complexes via CD32 (the type II IgG FcR, Fc gamma RII) enhances Ag presentation 100-fold over noncomplexed Ag. Blood leukocytes differentially express two additional IgG FcR, Fc gamma RI (CD64) and Fc gamma RIII (CD16), which may also participate in leukocyte functions such as phagocytosis, Ab-dependent cellular cytotoxicity (ADCC), release of oxygen intermediates, and enhancement of Ag presentation. A phagocytically active form of CD64 was recently demonstrated on human blood DC, but complete functional potential of CD64 on the DC lineage remains undefined. Therefore, highly purified human blood DC (CD33(2)+, CD14-, CD11c2+, HLA-DR3+, CD64+ (CD83+ after overnight culture)) and monocytes (CD33(2)+, CD14(3)+, CD11c2+, HLA-DR+, CD64(2)+, CD83-) were compared for cytokine modulation and effector functions of CD64. Both DC and monocyte CD64 expression was increased by IFN-gamma and IL-10, but while monocyte CD64 was decreased by IL-4, DC CD64 remained unchanged. FcR-mediated functional differences were also evident between the DC and the monocytes. Monocytes generated robust Fc gamma R-dependent superoxide anion release and ADCC activity, while DC failed to release reactive oxygen intermediates and demonstrated minimal ADCC activity, despite apparently normal expression of the gamma-chain subunit and the signaling molecule Syk. In contrast, DC were more efficient than monocytes with respect to T cell activation when Ag was targeted specifically to CD64. These new findings suggest a previously unappreciated potential for CD64 to shape the immune response by dramatically increasing the efficiency with which DC sequester Ag prior to achieving full T cell stimulatory potential.

  11. Soluble Fc gamma R (sFc gamma R): detection in biological fluids and production of a murine recombinant sFc gamma R biologically active in vitro and in vivo.

    Science.gov (United States)

    Sautès, C; Teillaud, C; Mazières, N; Tartour, E; Bouchard, C; Galinha, A; Jourde, M; Spagnoli, R; Fridman, W H

    1992-08-01

    Soluble forms of receptors for the Fc portion of IgG (sFc gamma R) were detected in biological fluids from mice and humans. In mouse bearing tumors, circulating amounts of sFc gamma R increased concurrently with tumor growth. Tumors secreting IgG2a, IgG2b or IgG3 led to a 5- to 10-fold increase in serum sFc gamma R levels whereas tumors secreting IgG1, IgGA or other types of tumors (non Ig B cell tumors, T cell lymphoma and a melanoma) increased 2- to 3-fold the levels of circulating sFc gamma R. In the human, sFc gamma R were also detected in whole unstimulated saliva. Levels of sFc gamma RII and of sFc gamma RIII were variable and did not seem to depend on the dental status of the individuals. Finally, a murine recombinant sFc gamma R (rsFc gamma R) composed of the two extracellular domains of Fc gamma RII was produced by culture of transfected L cells in bioreactors. The purified rsFc gamma R was found to inhibit antibody production in vitro in anti-SRBC responses and by cultures of small B cells stimulated by anti-IgM antibodies in the presence of IL-4 and IL-5. Moreover, the i.p. injection of this material into adult mice immunized with SRBC led to a decrease of IgG antibody production by splenocytes, as measured by a hemolytic plaque assay, and in serum, as measured by antigen-specific ELISA.

  12. Cytotoxicity mediated by human Fc receptors for IgG.

    Science.gov (United States)

    Fanger, M W; Shen, L; Graziano, R F; Guyre, P M

    1989-03-01

    The Fc receptors for IgG(Fc gamma R) play a major role in the removal of antibody-coated infectious agents and may be important molecules for triggering cytotoxicity of tumor cells; they may also serve as an entry for infection of Fc gamma R-bearing cells by viral (including HIV and Dengue), and perhaps other infectious agents. Although central to immune defense, an understanding of the role of these Fc gamma R in cytotoxicity has been complicated in part by the presence of several biochemically distinct types of receptor that have different distributions, specificities, affinities and modes of activation for killing. The development of monoclonal antibodies specific for Fc gamma R on human leukocytes has established the existence of three distinct Fc gamma R and furthermore has helped clarify the function of each of these receptors. In this review, Michael Fanger and colleagues discuss the use of Fc gamma R-specific mAb and the hybridoma cell lines that produce them in examining the ability of each of these unique receptors to mediate killing of tumor and red cell targets. In particular, the use of self-directed hybridoma cells as a model of tumor-cell killing and of bi-specific antibodies to link target cells to effector cells through the different Fc gamma R is discussed. The results of these studies suggest that the ability of a given Fc gamma R to trigger killing is sometimes dependent on the type of Fc gamma R, but is also markedly influenced by the type of target cell and by the nature and state of activation of the effector cell.

  13. A Case of Neonatal Neutropenia Due to Anti-Fc Gamma Receptor IIIb Isoantibodies Treated with Recombinant Human Granulocyte Colony Stimulating Factor

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    Maja Tomicic

    2009-01-01

    Full Text Available Alloimmunization to granulocyte-specific antigens can occur during pregnancy. Maternal antibodies of IgG class can cross the placenta to result in alloimmune neonatal neutropenia. Antibodies to human neutrophil antigens anti-HNA-1a, HNA-1b, and HNA-2a have been most commonly reported to cause alloimmune neonatal neutropenia. Isoantibodies to Fc gamma RIIIb (CD16 if mother is a HNA-null phenotype are rarely involved in neonatal neutropenia. We report on a case of severe neutropenia (440 neutrophils/μL due to anti-Fc gamma RIIIb (CD16 isoimmunization. On day 14 severe omphalitis developed, which was treated for 7 days by an antibiotic (ceftriaxone in a dose of 80 mg/kg/d according to umbilical swab finding. Omphalitis persisted for 10 days in spite of antibiotic therapy and only resolved upon the introduction of rhG-CSF therapy. Therapy with rh-GCSF proved efficient and led to neutrophil count increase to 1970/μL and cure of omphalitis. However, therapeutic effect on granulocyte count was of transient nature, as granulocyte count fell to 760 n/μL on day 4 of therapy discontinuation. Neutropenia persisted for 2 months. The newborn was discharged from the hospital on day 26 with normal clinical status with clinical and laboratory control examinations at 2-week intervals. No additional infections were observed during the course of neutropenia.

  14. Signalling through neutrophil Fc gamma RIII, Fc gamma RII, and CD59 is not impaired in active rheumatoid arthritis.

    OpenAIRE

    1996-01-01

    OBJECTIVE: To compare neutrophil Fc receptor (Fc gamma R) and CD59 signalling responses in normal healthy subjects and patients with active rheumatoid arthritis (RA). METHODS: Intracellular free calcium concentrations were measured in neutrophils loaded with the fluorescent calcium indicator fura-2, using a spectrofluorimeter. RESULTS: Basal intracellular calcium ion concentrations were similar in both groups when no primary antibody, CD59, or CD32 (Fc gamma RIII) antibody was added. When CD1...

  15. Structural recognition and functional activation of Fc[gamma]R by innate pentraxins

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    Lu, Jinghua; Marnell, Lorraine L.; Marjon, Kristopher D.; Mold, Carolyn; Du Clos, Terry W.; Sun, Peter D. (UNM); (NIH)

    2009-10-05

    Pentraxins are a family of ancient innate immune mediators conserved throughout evolution. The classical pentraxins include serum amyloid P component (SAP) and C-reactive protein, which are two of the acute-phase proteins synthesized in response to infection. Both recognize microbial pathogens and activate the classical complement pathway through C1q. More recently, members of the pentraxin family were found to interact with cell-surface Fc{gamma} receptors (Fc{gamma}R) and activate leukocyte-mediated phagocytosis. Here we describe the structural mechanism for pentraxin's binding to Fc{gamma}R and its functional activation of Fc{gamma}R-mediated phagocytosis and cytokine secretion. The complex structure between human SAP and Fc{gamma}RIIa reveals a diagonally bound receptor on each SAP pentamer with both D1 and D2 domains of the receptor contacting the ridge helices from two SAP subunits. The 1:1 stoichiometry between SAP and Fc{gamma}RIIa infers the requirement for multivalent pathogen binding for receptor aggregation. Mutational and binding studies show that pentraxins are diverse in their binding specificity for Fc{gamma}R isoforms but conserved in their recognition structure. The shared binding site for SAP and IgG results in competition for Fc{gamma}R binding and the inhibition of immune-complex-mediated phagocytosis by soluble pentraxins. These results establish antibody-like functions for pentraxins in the Fc{gamma}R pathway, suggest an evolutionary overlap between the innate and adaptive immune systems, and have new therapeutic implications for autoimmune diseases.

  16. A novel human immunoglobulin Fcγ–Fcε bifunctional fusion protein inhibits FcεRI-mediated degranulation

    OpenAIRE

    Zhu, Daocheng; Kepley, Christopher L.; Zhang, Min; Zhang, Ke; Saxon, Andrew

    2002-01-01

    Human mast cells and basophils that express the high-affinity immunoglobulin E (IgE) receptor, Fcε receptor 1 (FcεRI), have key roles in allergic diseases. FcεRI cross-linking stimulates the release of allergic mediators1. Mast cells and basophils co-express FcγRIIb, a low affinity receptor containing an immunoreceptor tyrosine-based inhibitory motif and whose co-aggregation with FcεRI can block FcεRI-mediated reactivity2–4. Here we designed, expressed and tested the human basophil and mast-c...

  17. Identification of residues within the extracellular domain 1 of bovine Fc gamma 2R essential for binding bovine IgG2.

    Science.gov (United States)

    Morton, H C; Howard, C J; Storset, A K; Brandtzaeg, P

    2001-12-21

    Neutrophils and monocytes in cattle express a novel class of immunoglobulin Fc receptor, specific for bovine IgG2 (bIgG2), termed bFc gamma 2R. In cows, the ability of neutrophils to kill immunoglobulin-opsonized microorganisms appears to depend largely on this subclass, whose interaction with bFc gamma 2R initiates the killing process. bFc gamma 2R is a transmembrane glycoprotein consisting of two extracellular immunoglobulin-like domains, followed by a 19-amino acid membrane-spanning region and a short cytoplasmic tail. Although related to other mammalian Fc gamma Rs, bFc gamma 2R belongs to a novel gene family that includes the human killer cell inhibitory receptor and Fc alpha RI (CD89) proteins. We have shown previously (Morton, H. C., van Zandbergen, G., van Kooten, C., Howard, C. J., van de Winkel, J. G., and Brandtzaeg, P. (1999) J. Exp. Med. 189, 1715-1722) that like these proteins (and unlike other Fc gamma Rs), bFc gamma 2R binds bIgG2 via the membrane-distal extracellular domain 1 (EC1). In this present study, we introduced mutations into the predicted loop regions of the EC1 domain and assayed the resulting bFc gamma 2R mutants for their ability to bind bIgG2. Our results indicated that the bIgG2 binding site lies within the predicted F-G loop region of the EC1 domain. Furthermore, single amino acid mutational analysis of this region identified Phe-82 and Trp-87 as being critical for bIgG2 binding.

  18. Role of Fc Gamma Receptors in Triggering Host Cell Activation and Cytokine Release by Borrelia burgdorferi

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    Talkington, Jeffrey; Nickell, Steven P.

    2001-01-01

    Borrelia burgdorferi, the spirochetal bacterium that causes human Lyme disease, encodes numerous lipoproteins which have the capacity to trigger the release of proinflammatory cytokines from a variety of host cell types, and it is generally believed that these cytokines contribute to the disease process in vivo. We previously reported that low-passage-number infectious B. burgdorferi spirochetes express a novel lipidation-independent activity which induces secretion of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) by the mouse MC/9 mast cell line. Using RNase protection assays, we determined that mast cells exposed in vitro to low-passage-number, but not high-passage-number, B. burgdorferi spirochetes show increased expression of additional mRNAs representing several chemokines, including macrophage-inflammatory protein 1α (MIP-1α), MIP-1β, and TCA3, as well as the proinflammatory cytokine interleukin-6. Furthermore, mast cell TNF-α secretion can be inhibited by the phosphatidylinositol 3-kinase inhibitor wortmannin and also by preincubation with purified mouse immunoglobulin G1 (IgG1) and IgG2a, but not mouse IgG3, and by a mouse Fc gamma receptor II and III (FcγRII/III)-specific rat monoclonal antibody, suggesting the likely involvement of host FcγRIII in B. burgdorferi-mediated signaling. A role for passively adsorbed rabbit or bovine IgG or serum components in B. burgdorferi-mediated FcγR signaling was excluded in control experiments. These studies confirm that low-passage-number B. burgdorferi spirochetes express a novel activity which upregulates the expression of a variety of host cell chemokine and cytokine genes, and they also establish a novel antibody-independent role for FcγRs in transduction of activation signals by bacterial products. PMID:11119532

  19. Intravenous immunoglobulin ameliorates ITP via activating Fc gamma receptors on dendritic cells.

    Science.gov (United States)

    Siragam, Vinayakumar; Crow, Andrew R; Brinc, Davor; Song, Seng; Freedman, John; Lazarus, Alan H

    2006-06-01

    Despite a more than 20-year experience of therapeutic benefit, the relevant molecular and cellular targets of intravenous immunoglobulin (IVIg) in autoimmune disease remain unclear. Contrary to the prevailing theories of IVIg action in autoimmunity, we show that IVIg drives signaling through activating Fc gamma receptors (Fc gammaR) in the amelioration of mouse immune thrombocytopenic purpura (ITP). The actual administration of IVIg was unnecessary because as few as 10(5) IVIg-treated cells could, upon adoptive transfer, ameliorate ITP. IVIg did not interact with the inhibitory Fc gammaRIIB on the initiator cell, although Fc gammaRIIB does have a role in the late phase of IVIg action. Notably, only IVIg-treated CD11c+ dendritic cells could mediate these effects. We hypothesize that IVIg forms soluble immune complexes in vivo that prime dendritic-cell regulatory activity. In conclusion, the clinical effects of IVIg in ameliorating ITP seem to involve the acute interaction of IVIg with activating Fc gammaR on dendritic cells.

  20. Fc gamma receptor polymorphisms and periodontal status : a prospective follow-up study

    NARCIS (Netherlands)

    Wolf, D. L.; Neiderud, A. M.; Hinckley, K.; Dahlen, G.; van de Winkel, J. G. J.; Papapanou, P. N.

    2006-01-01

    Aims: The aims of this study were to assess: (i) the distribution of Fc gamma receptor polymorphisms among patients with chronic periodontitis ("cases") and control subjects with no/minimal loss of periodontal tissue support in a Caucasian population; (ii) whether these polymorphisms can serve as se

  1. Altered polymorphonuclear leukocyte Fc gamma R expression contributes to decreased candicidal activity during intraabdominal sepsis

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    Simms, H.H.; D' Amico, R.; Monfils, P.; Burchard, K.W. (Rhode Island Hospital, Providence (USA))

    1991-03-01

    We investigated the effects of untreated intraabdominal sepsis on polymorphonuclear leukocyte (PMN) candicidal activity. Two groups of swine were studied. Group I (n=6) underwent sham laparotomy, group II (n=7) underwent cecal ligation and incision. Untreated intraabdominal sepsis resulted in a progressive decrease in PMN candicidal activity. Concomitant rosetting and phagocytosis assays demonstrated a decrease in both the attachment and phagocytosis of Candida albicans opsonized with both normal and septic swine serum by PMNs in group II. Iodine 125-labeled swine immunoglobulin G (IgG) and fluorescein isothioalanate (FITC)-labeled swine IgG were used to investigate Fc gamma receptor ligand interactions. Scatchard analyses demonstrated a progressive decline in both the binding affinity constant and number of IgG molecules bound per PMN. Stimulation of the oxidative burst markedly reduced 125I-labeled IgG binding in both group I and group II, with a greater decrement being seen in animals with intraabdominal sepsis. Further, in group II, PMN recycling of the Fc gamma receptor to the cell surface after generation of the oxidative burst was reduced by postoperative day 4. Binding of monoclonal antibodies to Fc gamma receptor II, but not Fc gamma receptor I/III markedly reduced intracellular candicidal activity. Immunofluorescence studies revealed a homogeneous pattern of FITC-IgG uptake by nearly all group I PMNs, whereas by postoperative day 8 a substantial number of PMNs from group II failed to internalize the FITC-IgG. These studies suggest that untreated intraabdominal sepsis reduces PMN candicidal activity and that this is due, in part, to altered PMN Fc gamma receptor ligand interactions.

  2. Fc gamma receptor CD64 modulates the inhibitory activity of infliximab.

    Directory of Open Access Journals (Sweden)

    Kacper A Wojtal

    Full Text Available BACKGROUND: Tumor necrosis factor (TNF is an important cytokine in the pathogenesis of inflammatory bowel disease (IBD. Anti-TNF antibodies have been successfully implemented in IBD therapy, however their efficacies differ among IBD patients. Here we investigate the influence of CD64 Fc receptor on the inhibitory activity of anti-TNFs in cells of intestinal wall. METHODS: Intestinal cell lines, monocytes/macrophages and peripheral blood mononuclear cells (PBMCs were used as models. The efficacies of adalimumab, infliximab and certolizumab-pegol were assessed by RT-PCR for target genes. Protein levels and localizations were examined by Western blotting and immunofluorescence. Antibody fragments were obtained by proteolytic digestion, immunoprecipitation and protein chip analysis. Knock-down of specific gene expression was performed using siRNAs. RESULTS: Infliximab had limited efficacy towards soluble TNF in cell types expressing Fc gamma receptor CD64. Both adalimumab and infliximab had lower efficacies in PBMCs of IBD patients, which express elevated levels of CD64. Infliximab-TNF complexes were more potent in activating CD64 in THP-1 cells than adalimumab, which was accompanied by distinct phospho-tyrosine signals. Blocking Fc parts and isolation of Fab fragments of infliximab improved its efficacy. IFN-γ-induced expression of CD64 correlated with a loss of efficacy of infliximab, whereas reduction of CD64 expression by either siRNA or PMA treatment improved inhibitory activity of this drug. Colonic mRNA expression levels of CD64 and other Fc gamma receptors were significantly increased in the inflamed tissues of infliximab non-responders. CONCLUSIONS: CD64 modulates the efficacy of infliximab both in vitro and ex vivo, whereas the presence of this receptor has no impact on the inhibitory activity of certolizumab-pegol, which lacks Fc fragment. These data could be helpful in both predicting and evaluating the outcome of anti-TNF therapy in

  3. Transfusion-induced, Fc gamma-receptor-blocking antibodies: spectrum of cellular reactivity.

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    Forwell, M A; Peel, M G; Froebel, K S; Belch, J J; MacSween, R N; Sandilands, G P

    1986-06-01

    In this study we have shown that transfusion-induced Fc gamma R-blocking antibodies have the capacity to react with various cell types which are known to possess this receptor i.e., lymphocytes (T and B cells), polymorphs and platelets. In contrast we were unable to demonstrate any reactivity with K (or NK) lymphocytes or with monocytes. The spectrum of cellular reactivity exhibited by these antibodies suggests that their effect on the immune system may be complex.

  4. Imaging and measuring the biophysical properties of Fc gamma receptors on single macrophages using atomic force microscopy

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    Li, Mi [State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Sciences, Shenyang 110016 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Liu, Lianqing, E-mail: lqliu@sia.cn [State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Sciences, Shenyang 110016 (China); Xi, Ning [Department of Mechanical and Biomedical Engineering, City University of Hong Kong, Hong Kong (China); Wang, Yuechao [State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Sciences, Shenyang 110016 (China); Xiao, Xiubin [Department of Lymphoma, Affiliated Hospital of Military Medical Academy of Sciences, Beijing 100071 (China); Zhang, Weijing, E-mail: zhangwj3072@163.com [Department of Lymphoma, Affiliated Hospital of Military Medical Academy of Sciences, Beijing 100071 (China)

    2013-09-06

    Highlights: •Nanoscale cellular ultra-structures of macrophages were observed. •The binding affinities of FcγRs were measured directly on macrophages. •The nanoscale distributions of FcγRs were mapped on macrophages. -- Abstract: Fc gamma receptors (FcγR), widely expressed on effector cells (e.g., NK cells, macrophages), play an important role in clinical cancer immunotherapy. The binding of FcγRs to the Fc portions of antibodies that are attached to the target cells can activate the antibody-dependent cell-mediated cytotoxicity (ADCC) killing mechanism which leads to the lysis of target cells. In this work, we used atomic force microscopy (AFM) to observe the cellular ultra-structures and measure the biophysical properties (affinity and distribution) of FcγRs on single macrophages in aqueous environments. AFM imaging was used to obtain the topographies of macrophages, revealing the nanoscale cellular fine structures. For molecular interaction recognition, antibody molecules were attached onto AFM tips via a heterobifunctional polyethylene glycol (PEG) crosslinker. With AFM single-molecule force spectroscopy, the binding affinities of FcγRs were quantitatively measured on single macrophages. Adhesion force mapping method was used to localize the FcγRs, revealing the nanoscale distribution of FcγRs on local areas of macrophages. The experimental results can improve our understanding of FcγRs on macrophages; the established approach will facilitate further research on physiological activities involved in antibody-based immunotherapy.

  5. Certolizumab pegol does not bind the neonatal Fc receptor (FcRn): Consequences for FcRn-mediated in vitro transcytosis and ex vivo human placental transfer.

    Science.gov (United States)

    Porter, Charlene; Armstrong-Fisher, Sylvia; Kopotsha, Tim; Smith, Bryan; Baker, Terry; Kevorkian, Lara; Nesbitt, Andrew

    2016-08-01

    Antibodies to tumor necrosis factor (anti-TNF) are used to treat inflammatory diseases, which often affect women of childbearing age. The active transfer of these antibodies across the placenta by binding of the Fc-region to the neonatal Fc receptor (FcRn) may result in adverse fetal or neonatal effects. In contrast to other anti-TNFs, certolizumab pegol lacks an Fc-region. The objective of this study was to determine whether the structure of certolizumab pegol limits active placental transfer. Binding affinities of certolizumab pegol, infliximab, adalimumab and etanercept to human FcRn and FcRn-mediated transcytosis were determined using in vitro assays. Human placentas were perfused ex vivo to measure transfer of certolizumab pegol and positive control anti-D IgG from the maternal to fetal circulation. FcRn binding affinity (KD) was 132nM, 225nM and 1500nM for infliximab, adalimumab and etanercept, respectively. There was no measurable certolizumab pegol binding affinity, similar to that of the negative control. FcRn-mediated transcytosis across a cell layer (mean±SD; n=3) was 249.6±25.0 (infliximab), 159.0±20.2 (adalimumab) and 81.3±13.1ng/mL (etanercept). Certolizumab pegol transcytosis (3.2±3.4ng/mL) was less than the negative control antibody (5.9±4.6ng/mL). No measurable transfer of certolizumab pegol from the maternal to the fetal circulation was observed in 5 out of 6 placentas that demonstrated positive-control IgG transport in the ex vivo perfusion model. Together these results support the hypothesis that the unique structure of certolizumab pegol limits its transfer through the placenta to the fetus and may be responsible for previously reported differences in transfer of other anti-TNFs from mother to fetus.

  6. Neuronal Fc gamma receptor I as a novel mediator for IgG immune complex-induced peripheral sensitization

    Institute of Scientific and Technical Information of China (English)

    Lintao Qu

    2012-01-01

    Chronic pain often accompanies immune-related diseases with an elevated level of IgG immune complex (IgG-IC) in the serum and/or the affected tissues though the underlying mechanisms are largely unknown. Fc gamma receptors (FcγRs), known as the receptors for the Fc domain of immunoglobulin G (IgG), are typically expressed on immune cells. A general consensus is that the activation of FcγRs by IgG-IC in such immune cells induces the release of proinflammatory cytokines from the immune cells, which may contribute to the IgG-IC-mediated peripheral sensitization. In addition to the immune cells, recent studies have revealed that FcγRI, but not FcγRII and FcγRIII, is also expressed in a subpopulation of primary sensory neurons. Moreover, IgG-IC directly excites the primary sensory neurons through neuronal FcγRI. These findings indicate that neuronal FcγRI provides a novel direct linkage between immunoglobulin and primary sensory neurons, which may be a novel target for the treatment of pain in the immune-related disorders. In this review, we summarize the expression pattern, functions, and the associated cellular signaling of FcγRs in the primary sensory neurons.

  7. Characterization of upFc, a fragment of human immunoglobulin G1 produced by pepsin in urea.

    Science.gov (United States)

    Parr, D M; Hofmann, T; Connell, G E

    1976-09-01

    The digestion of human IgG1/K myeloma proteins with pepsin in the presence of 8 M-urea produces fragments that differ from those produced by aqueous peptic digestion, and from other characteristic immunoglobulin fragments. Fb'2, the larger urea/pepsin fragment, was previously shown to consist of the constant regions of the light chains, and the CH1 domains and hinge regions of the heavy chains. The smaller fragment, upFc, has now been characterized. After reduction, three peptides were released from fragment upFc. Amino acid sequencing, N- and C-terminal determinations and amino acid compositions have enabled these peptides to be identified as residues Ile-253 to Leu-306, residues Thr-307 to Asp-376 and residues Thr-411 to Gly-446 of the heavy chain. Fragment upFc therefore contains the entire Fc region, beginning at residue Ile-253, except for a 34-residue section from within the CH3-domain disulphide loop. Peptic digestion of IgG1/K proteins in 8M-urea therefore provides a method for isolating from gamma1 heavy chains five homogeneous peptides in good yield, which account for almost the entire constant region. Characterization of fragments Fb'2 and upFc has shown that the action of pepsin in urea is entirely different from that of aqueous pepsin. Two gamma1 heavy chains have been shown to differ in sequence at three positions from the sequence reported for protein Eu.

  8. A critical review of the role of Fc gamma receptor polymorphisms in the response to monoclonal antibodies in cancer

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    Mellor James D

    2013-01-01

    Full Text Available Abstract Antibody-dependent cellular cytotoxicity (ADCC is a major mechanism of action of therapeutic monoclonal antibodies (mAbs such as cetuximab, rituximab and trastuzumab. Fc gamma receptors (FcgR on human white blood cells are an integral part of the ADCC pathway. Differential response to therapeutic mAbs has been reported to correlate with specific polymorphisms in two of these genes: FCGR2A (H131R and FCGR3A (V158F. These polymorphisms are associated with differential affinity of the receptors for mAbs. This review critically examines the current evidence for genotyping the corresponding single nucleotide polymorphisms (SNPs to predict response to mAbs in patients with cancer.

  9. X-ray Crystal Structures of Monomeric and Dimeric Peptide Inhibitors in Complex with the Human Neonatal Fc Receptor, FcRn

    Energy Technology Data Exchange (ETDEWEB)

    Mezo, Adam R.; Sridhar, Vandana; Badger, John; Sakorafas, Paul; Nienaber, Vicki (Zenobia); (Biogen)

    2010-10-28

    The neonatal Fc receptor, FcRn, is responsible for the long half-life of IgG molecules in vivo and is a potential therapeutic target for the treatment of autoimmune diseases. A family of peptides comprising the consensus motif GHFGGXY, where X is preferably a hydrophobic amino acid, was shown previously to inhibit the human IgG:human FcRn protein-protein interaction (Mezo, A. R., McDonnell, K. A., Tan Hehir, C. A., Low, S. C., Palombella, V. J., Stattel, J. M., Kamphaus, G. D., Fraley, C., Zhang, Y., Dumont, J. A., and Bitonti, A. J. (2008) Proc. Natl. Acad. Sci. U.S.A., 105, 2337-2342). Herein, the x-ray crystal structure of a representative monomeric peptide in complex with human FcRn was solved to 2.6 {angstrom} resolution. The structure shows that the peptide binds to human FcRn at the same general binding site as does the Fc domain of IgG. The data correlate well with structure-activity relationship data relating to how the peptide family binds to human FcRn. In addition, the x-ray crystal structure of a representative dimeric peptide in complex with human FcRn shows how the bivalent ligand can bridge two FcRn molecules, which may be relevant to the mechanism by which the dimeric peptides inhibit FcRn and increase IgG catabolism in vivo. Modeling of the peptide:FcRn structure as compared with available structural data on Fc and FcRn suggest that the His-6 and Phe-7 (peptide) partially mimic the interaction of His-310 and Ile-253 (Fc) in binding to FcRn, but using a different backbone topology.

  10. Fully human monoclonal antibody inhibitors of the neonatal Fc receptor (FcRn reduce circulating IgG in nonhuman primates

    Directory of Open Access Journals (Sweden)

    Andrew E Nixon

    2015-04-01

    Full Text Available The therapeutic management of antibody mediated autoimmune disease typically involves immunosuppressant and immunomodulatory strategies. However, perturbing the fundamental role of the neonatal Fc receptor (FcRn in salvaging IgG from lysosomal degradation provides a novel approach – depleting the body of pathogenic immunoglobulin by preventing IgG binding to FcRn and thereby increasing the rate of IgG catabolism. Herein, we describe the discovery and preclinical evaluation of fully human monoclonal IgG antibody inhibitors of FcRn. Using phage display, we identified several potent inhibitors of human FcRn in which binding to FcRn is pH independent, with over 1000-fold higher affinity for human FcRn than human IgG-Fc at pH 7.4. FcRn antagonism in vivo using a human-FcRn knock-in transgenic mouse model caused enhanced catabolism of exogenously administered human IgG. In non-human primates we observed reductions in endogenous circulating IgG of > 60% with no changes in albumin, IgM, or IgA. FcRn antagonism did not disrupt the ability of non-human primates to mount IgM/IgG primary and secondary immune responses. Interestingly, the therapeutic anti-FcRn antibodies had a short serum half-life but caused a prolonged reduction in IgG levels. This may be explained by the high affinity of the antibodies to FcRn at both acidic and neutral pH. These results provide important preclinical proof of concept data in support of FcRn antagonism as a novel approach to the treatment of antibody mediated autoimmune diseases.

  11. Attenuated atherosclerotic lesions in apoe-fc gamma-chain-deficient hyperlipidemic mouse model is associated with inhibition of Th17 cells and promotion of regulatory T cells

    Science.gov (United States)

    Though the presence of antioxidized low-density lipoprotein IgG is well documented in clinical and animal studies, the role for Fc gamma Rs to the progression of atherosclerosis has not been studied in detail. In the current study, we investigated the role for activating Fc gamma R in the progressio...

  12. Importance of neonatal FcR in regulating the serum half-life of therapeutic proteins containing the Fc domain of human IgG1: a comparative study of the affinity of monoclonal antibodies and Fc-fusion proteins to human neonatal FcR.

    Science.gov (United States)

    Suzuki, Takuo; Ishii-Watabe, Akiko; Tada, Minoru; Kobayashi, Tetsu; Kanayasu-Toyoda, Toshie; Kawanishi, Toru; Yamaguchi, Teruhide

    2010-02-15

    The neonatal FcR (FcRn) binds to the Fc domain of IgG at acidic pH in the endosome and protects IgG from degradation, thereby contributing to the long serum half-life of IgG. To date, more than 20 mAb products and 5 Fc-fusion protein products have received marketing authorization approval in the United States, the European Union, or Japan. Many of these therapeutic proteins have the Fc domain of human IgG1; however, the serum half-lives differ in each protein. To elucidate the role of FcRn in the pharmacokinetics of Fc domain-containing therapeutic proteins, we evaluated the affinity of the clinically used human, humanized, chimeric, or mouse mAbs and Fc-fusion proteins to recombinant human FcRn by surface plasmon resonance analysis. The affinities of these therapeutic proteins to FcRn were found to be closely correlated with the serum half-lives reported from clinical studies, suggesting the important role of FcRn in regulating their serum half-lives. The relatively short serum half-life of Fc-fusion proteins was thought to arise from the low affinity to FcRn. The existence of some mAbs having high affinity to FcRn and a short serum half-life, however, suggested the involvement of other critical factor(s) in determining the serum half-life of such Abs. We further investigated the reason for the relatively low affinity of Fc-fusion proteins to FcRn and suggested the possibility that the receptor domain of Fc-fusion protein influences the structural environment of the FcRn binding region but not of the FcgammaRI binding region of the Fc domain.

  13. Association of variation in Fc gamma receptor 3B gene copy number with rheumatoid arthritis in Caucasian samples

    NARCIS (Netherlands)

    McKinney, Cushla; Fanciulli, Manuela; Merriman, Marilyn E.; Phipps-Green, Amanda; Alizadeh, Behrooz Z.; Koeleman, Bobby P. C.; Dalbeth, Nicola; Gow, Peter J.; Harrison, Andrew A.; Highton, John; Jones, Peter B.; Stamp, Lisa K.; Steer, Sophia; Barrera, Pilar; Coenen, Marieke J. H.; Franke, Barbara; van Riel, Piet L. C. M.; Vyse, Tim J.; Aitman, Tim J.; Radstake, Timothy R. D. J.; Merriman, Tony R.

    2010-01-01

    Objective There is increasing evidence that variation in gene copy number (CN) influences clinical phenotype. The low-affinity Fc gamma receptor 3B (FCGR3B) located in the FCGR gene cluster is a CN polymorphic gene involved in the recruitment to sites of inflammation and activation of polymorphonucl

  14. Therapeutic effect of AdCMVCD/5-FC system and metabolism of 5-FC in the treatment of human tongue squamous cell carcinoma

    Institute of Scientific and Technical Information of China (English)

    王安训; 黄洪章; 李苏

    2003-01-01

    Objective To investigate the therapeutic effect and metabolism of 5-fluorocytosine (5-FC ) in human tongue squamous carcinoma cells after treatment with adenovirus-medi ated cytosine deaminase (AdCMVCD)/5-FC system. Methods Human tongue squamous carcinoma cells (Tca8113 cell line) and its xenografts in BALB/c nude mice were treated with AdCMVCD/5-FC system. The killing effect in vitro and bystander effect were detected by microculture tetrazolium (MTT) assay . Tumor inhibition effect and histopathological changes were observed in vivo. High-performance liquid chromatography (HPLC) was performed to determine the metabolism of 5-FC in vitro and in vivo. Results AdCMVCD/5-FC system had strong killing effect and bystander effect on Tca8113 cells. Both condition media and cell extracts showed two peaks identified as 5- FC and 5-fluorouracil (5-FU) by HPLC and a time-dependent generation of 5-FU and concomitant time-dependent decreases of 5-FC. Compared to the control groups, mice treated with AdCMVCD/5-FC system demonstrated significant tumor regr ession (P<0.001); the tumor doubling time prolonged and inhibition rate was 92.62%. There were substantial tumor necrotic areas and infiltrative lymphocy tes around necrotic areas in the AdCMVCD/5-FC treated group under light microscope. There was a significantly low concentration of 5-FC and high concentratio n of 5-FU in tumor tissue, but only 5-FC was found in blood. Conclusion AdCMVCD/5-FC suicide gene system had significant in vitro and in vivo anti-tumor effect on human tongue squamous cell carcinomadue to convert 5-FC into 5-F U.

  15. FcRn expression, ligands binding properties and its regulation in human immune cells and hepatocytes

    OpenAIRE

    2007-01-01

    ABSTRACT Expression and diverse functions of MHC class I related neonatal Fc receptor in different tissues is continually reported. To contribute to the understanding of how the receptor functions according to cell type, we investigated the expression and ligands binding properties of FcRn in human immune cells and hepatocytes. Here, we report that heterodimeric FcRn is expressed in these cells as evidenced by RT-PCR, Western immunoblottting and flow cytometry. The receptor expression i...

  16. Human cytomegalovirus Fcγ binding proteins gp34 and gp68 antagonize Fcγ receptors I, II and III.

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    Eugenia Corrales-Aguilar

    2014-05-01

    Full Text Available Human cytomegalovirus (HCMV establishes lifelong infection with recurrent episodes of virus production and shedding despite the presence of adaptive immunological memory responses including HCMV immune immunoglobulin G (IgG. Very little is known how HCMV evades from humoral and cellular IgG-dependent immune responses, the latter being executed by cells expressing surface receptors for the Fc domain of IgG (FcγRs. Remarkably, HCMV expresses the RL11-encoded gp34 and UL119-118-encoded gp68 type I transmembrane glycoproteins which bind Fcγ with nanomolar affinity. Using a newly developed FcγR activation assay, we tested if the HCMV-encoded Fcγ binding proteins (HCMV FcγRs interfere with individual host FcγRs. In absence of gp34 or/and gp68, HCMV elicited a much stronger activation of FcγRIIIA/CD16, FcγRIIA/CD32A and FcγRI/CD64 by polyclonal HCMV-immune IgG as compared to wildtype HCMV. gp34 and gp68 co-expression culminates in the late phase of HCMV replication coinciding with the emergence of surface HCMV antigens triggering FcγRIII/CD16 responses by polyclonal HCMV-immune IgG. The gp34- and gp68-dependent inhibition of HCMV immune IgG was fully reproduced when testing the activation of primary human NK cells. Their broad antagonistic function towards FcγRIIIA, FcγRIIA and FcγRI activation was also recapitulated in a gain-of-function approach based on humanized monoclonal antibodies (trastuzumab, rituximab and isotypes of different IgG subclasses. Surface immune-precipitation showed that both HCMV-encoded Fcγ binding proteins have the capacity to bind trastuzumab antibody-HER2 antigen complexes demonstrating simultaneous linkage of immune IgG with antigen and the HCMV inhibitors on the plasma membrane. Our studies reveal a novel strategy by which viral FcγRs can compete for immune complexes against various Fc receptors on immune cells, dampening their activation and antiviral immunity.

  17. A strategy for bacterial production of a soluble functional human neonatal Fc receptor.

    Science.gov (United States)

    Andersen, Jan Terje; Justesen, Sune; Berntzen, Gøril; Michaelsen, Terje E; Lauvrak, Vigdis; Fleckenstein, Burkhard; Buus, Søren; Sandlie, Inger

    2008-02-29

    The major histocompatibility complex (MHC) class I related receptor, the neonatal Fc receptor (FcRn), rescues immunoglobulin G (IgG) and albumin from lysosomal degradation by recycling in endothelial cells. FcRn also contributes to passive immunity by mediating transport of IgG from mother to fetus (human) or newborn (rodents), and may translocate IgG over mucosal surfaces. FcRn interacts with the Fc-region of IgG and domain III of albumin with binding at pH 6.0 and release at pH 7.4. Knowledge of these interactions has facilitated design of recombinant proteins with altered serum half-lives and/or altered biodistribution. To generate further research in this field, there is a great need for large amounts of soluble human FcRn (shFcRn) for in vitro interaction studies. In this report, we describe a novel laboratory scale production of functional shFcRn in Escherichia coli (E. coli) at milligram level. Truncated wild type hFcRn heavy chains were expressed, extracted, purified from inclusion bodies under denaturing non-reducing conditions, and subsequently refolded in the presence of human beta(2)-microglobulin (hbeta(2)m). The secondary structural elements of refolded heterodimeric shFcRn were correctly formed as demonstrated by circular dichroism (CD). Furthermore, functional and stringent pH dependent binding to IgG and human serum albumin were demonstrated by ELISA and surface plasmon resonance (SPR). This method may be easily adapted for the expression of large amounts of other FcRn species and MHC class I related molecules.

  18. Genetic variation of the Fc gamma receptor 3B gene and association with rheumatoid arthritis.

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    Rute B Marques

    Full Text Available BACKGROUND: Fc gamma receptors (FcγRs play a crucial role in immunity by linking IgG antibody-mediated responses with cellular effector and regulatory functions. Genetic variants in these receptors have been previously identified as risk factors for several chronic inflammatory conditions. The present study aimed to investigate the presence of copy number variations (CNVs in the FCGR3B gene and its potential association with the autoimmune disease rheumatoid arthritis (RA. METHODOLOGY/PRINCIPAL FINDINGS: CNV of the FCGR3B gene was studied using Multiplex Ligation Dependent Probe Amplification (MLPA in 518 Dutch RA patients and 304 healthy controls. Surprisingly, three independent MLPA probes targeting the FCGR3B promoter measured different CNV frequencies, with probe#1 and #2 measuring 0 to 5 gene copies and probe#3 showing little evidence of CNV. Quantitative-PCR correlated with the copy number results from MLPA probe#2, which detected low copy number (1 copy in 6.7% and high copy number (≥3 copies in 9.4% of the control population. No significant difference was observed between RA patients and the healthy controls, neither in the low copy nor the high copy number groups (p-values = 0.36 and 0.71, respectively. Sequencing of the FCGR3B promoter region revealed an insertion/deletion (indel that explained the disparate CNV results of MLPA probe#1. Finally, a non-significant trend was found between the novel -256A>TG indel and RA (40.7% in healthy controls versus 35.9% in RA patients; P = 0.08. CONCLUSIONS/SIGNIFICANCE: The current study highlights the complexity and poor characterization of the FCGR3B gene sequence, indicating that the design and interpretation of genotyping assays based on specific probe sequences must be performed with caution. Nonetheless, we confirmed the presence of CNV and identified novel polymorphisms in the FCGR3B gene in the Dutch population. Although no association was found between RA and FCGR3B CNV, the

  19. The Fc and not CD4 Receptor Mediates Antibody Enhancement of HIV Infection in Human Cells

    Science.gov (United States)

    Homsy, Jacques; Meyer, Mia; Tateno, Masatoshi; Clarkson, Sarah; Levy, Jay A.

    1989-06-01

    Antibodies that enhance human immunodeficiency virus (HIV) infectivity have been found in the blood of infected individuals and in infected or immunized animals. These findings raise serious concern for the development of a safe vaccine against acquired immunodeficiency syndrome. To address the in vivo relevance and mechanism of this phenomenon, antibody-dependent enhancement of HIV infectivity in peripheral blood macrophages, lymphocytes, and human fibroblastoid cells was studied. Neither Leu3a, a monoclonal antibody directed against the CD4 receptor, nor soluble recombinant CD4 even at high concentrations prevented this enhancement. The addition of monoclonal antibody to the Fc receptor III (anti-FcRIII), but not of antibodies that react with FcRI or FcRII, inhibited HIV type 1 and HIV type 2 enhancement in peripheral blood macrophages. Although enhancement of HIV infection in CD4+ lymphocytes could not be blocked by anti-FcRIII, it was inhibited by the addition of human immunoglobulin G aggregates. The results indicate that the FcRIII receptor on human macrophages and possibly another Fc receptor on human CD4+ lymphocytes mediate antibody-dependent enhancement of HIV infectivity and that this phenomenon proceeds through a mechanism independent of the CD4 protein.

  20. Increased platelet expression of FcGammaRIIa and its potential impact on platelet reactivity in patients with end stage renal disease

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    Sobel Burton E

    2007-06-01

    Full Text Available Abstract Background Increased platelet reactivity has been implicated in cardiovascular disease – the major cause of death in patients with end stage renal disease (ESRD. FcGammaRIIA is a component of glycoprotein VI and Ib-IX-V that mediate activation of platelets by collagen and von Willebrand factor. To determine whether expression of FcGammaRIIA impacts platelet reactivity we quantified its expression and platelet reactivity in 33 patients with ESRD who were undergoing hemodialysis. Methods Blood samples were obtained from patients immediately before hemodialysis and before administration of heparin. Platelet expression of FcGammaRIIA and the activation of platelets in response to low concentrations of convulxin (1 ng/ml, selected to mimic effects of collagen, thrombin (1 nM, adenosine diphosphate (ADP, 0.2 uM, or platelet activating factor (PAF, 1 nM were determined with the use of flow cytometry in samples of whole blood anticoagulated with corn trypsin inhibitor (a specific inhibitor of Factor XIIa. Results Patients were stratified with respect to the median expression of FcGammaRIIA. Patients with high platelet expression of FcGammaRIIA exhibited 3-fold greater platelet reactivity compared with that in those with low expression in response to convulxin (p Conclusion Increased platelet reactivity in response to low concentrations of diverse agonists is associated with high expression of FcGammaRIIA and may contribute to an increased risk of thrombosis in patients with ESRD.

  1. Constitutive and functional association of the platelet collagen receptor glycoprotein VI-Fc receptor gamma-chain complex with membrane rafts.

    Science.gov (United States)

    Ezumi, Yasuharu; Kodama, Kumi; Uchiyama, Takashi; Takayama, Hiroshi

    2002-05-01

    The platelet collagen receptor glycoprotein (GP) VI-Fc receptor gamma-chain (FcRgamma) complex transduces signals in an immunoreceptorlike manner. We examined a role for the Triton X-100-insoluble membrane rafts in GPVI-FcRgamma complex signaling. Methyl-beta-cyclodextrin (MbetaCD)-induced disruption of the membrane rafts inhibited not only platelet aggregation and secretion but also tyrosine phosphorylation of signaling molecules on stimulation through the GPVI-FcRgamma complex. The GPVI-FcRgamma complex was constitutively associated with membrane rafts wherein the Src family kinases and LAT were also present. Their association was not affected by the complex engagement but was highly sensitive to MbetaCD treatment. Thus, we provide the first evidence that the GPVI-FcRgamma complex is constitutively and functionally associated with membrane rafts.

  2. Improvement of the method of obtaining human IgA Fc-fragments

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    O. Y. Galkin

    2015-02-01

    Full Text Available To address a number of fundamental and applied problems in immunology, molecular and cellular biology and biotechnology it is necessary to obtain Fc-fragments of immunoglobulins. Fc-fragments may be used for studying of the effector functions of antibodies which are mediated by these areas. They are often used as an immunogen to produce anti-specie (based on so-called secondary antibody conjugate in the development of serological tests for diagnostics (predominantly such conjugate based on monoclonal antibodies. The work is aimed to develop improved methods of obtaining and allocation of Fc-fragments of human IgA. To achieve this objective, optimization of hydrolysis of IgA with subsequent purification of Fс-fragments have been carried out. Improved method of obtaining Fc-fragments of IgA provides: papain hydrolysis of immunoglobulin in the environment of nitrogen for 4 h, allowing to achieve maximum output of Fc-fragments without their further degradation: isolation and purification of Fc-fragments of human IgA by one-stage gel filtration on sephadex G-100; control of purity of the target product by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate and Ouchterlony immunodiffusion. Enzymatic hydrolysis was carried out at the optimal temperature of papain (37 °C. As the oxygen in the air may have inhibitory effect on enzymatic hydrolysis reaction, the reaction mixture was incubated in the nitrogen atmosphere to prevent inactivation of papain. To reduce the incident degradation of immunoglobulin molecules, papain hydrolysis was carried out without using an enzyme activator (cysteine. Usage of the proposed scheme allows obtaining Fc-fragments of human IgA of high purity. Outcome of Fc-fragments after all stages of purification was about 18% of the initial amount of IgA in the preparation. Molecular weight from Fc-fragments of human IgA was equal to approximately 70 kDa.

  3. (Some cellular mechanisms influencing the transcription of human endogenous retrovirus, HERV-Fc1.

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    Magdalena Janina Laska

    Full Text Available DNA methylation and histone acetylation are epigenetic modifications that act as regulators of gene expression. DNA methylation is considered an important mechanism for silencing of retroelements in the mammalian genome. However, the methylation of human endogenous retroviruses (HERVs is not well investigated. The aim of this study was to investigate the transcriptional potential of HERV-Fc1 proviral 5'LTR in more detail, and examined the specific influence of CpG methylation on this LTR in number of cell lines. Specifically, the role of demethylating chemicals e.g. 5-aza-2' deoxycytidine and Trichostatin-A, in inducing or reactivating expression of HERV-Fc1 specific sequences and the mechanisms were investigated. In our present study, 5-aza-dC is shown to be a powerful inducer of HERV-Fc1, and at the same time it strongly inhibits methylation of DNA. Treatment with this demethylating agent 5-aza-dC, results in significantly increased levels of HERV-Fc1 expression in cells previously not expressing HERV-Fc1, or with a very low expression level. The extent of expression of HERV-Fc1 RNAs precisely correlates with the apparent extent of demethylation of the related DNA sequences. In conclusion, the results suggest that inhibition of DNA methylation/histone deacetylase can interfere with gene silencing mechanisms affecting HERV-Fc1 expression in human cells.

  4. Immunoglobulin Fc gamma receptor promotes immunoglobulin uptake, immunoglobulin-mediated calcium increase, and neurotransmitter release in motor neurons

    Science.gov (United States)

    Mohamed, Habib A.; Mosier, Dennis R.; Zou, Ling L.; Siklos, Laszlo; Alexianu, Maria E.; Engelhardt, Jozsef I.; Beers, David R.; Le, Wei-dong; Appel, Stanley H.

    2002-01-01

    Receptors for the Fc portion of immunoglobulin G (IgG; FcgammaRs) facilitate IgG uptake by effector cells as well as cellular responses initiated by IgG binding. In earlier studies, we demonstrated that amyotrophic lateral sclerosis (ALS) patient IgG can be taken up by motor neuron terminals and transported retrogradely to the cell body and can alter the function of neuromuscular synapses, such as increasing intracellular calcium and spontaneous transmitter release from motor axon terminals after passive transfer. In the present study, we examined whether FcgammaR-mediated processes can contribute to these effects of ALS patient immunoglobulins. F(ab')(2) fragments (which lack the Fc portion) of ALS patient IgG were not taken up by motor axon terminals and were not retrogradely transported. Furthermore, in a genetically modified mouse lacking the gamma subunit of the FcR, the uptake of whole ALS IgG and its ability to enhance intracellular calcium and acetylcholine release were markedly attenuated. These data suggest that FcgammaRs appear to participate in IgG uptake into motor neurons as well as IgG-mediated increases in intracellular calcium and acetylcholine release from motor axon terminals. Copyright 2002 Wiley-Liss, Inc.

  5. Colocalization of Fc gamma RI-targeted antigen with class I MHC: implications for antigen processing.

    Science.gov (United States)

    Guyre, C A; Barreda, M E; Swink, S L; Fanger, M W

    2001-02-15

    The high-affinity receptor for IgG (CD64 or FcgammaRI) is constitutively expressed exclusively on professional APCs (monocytes, macrophages, and dendritic cells). When Ag is targeted specifically to FcgammaRI, Ag presentation is markedly enhanced, although the mechanism of this enhancement is unknown. In an effort to elucidate the pathways involved in FcgammaRI targeting, we developed a model targeted Ag using enhanced green fluorescent protein (eGFP). This molecule, wH22xeGFP, consists of the entire humanized anti-FcgammaRI mAb H22 with eGFP genetically fused to the C-terminal end of each CH3 domain. wH22xeGFP binds within the ligand-binding region by its Fc end, as well as outside the ligand-binding region by its Fab ends, thereby cross-linking FcgammaRI. Confocal microscopy studies revealed that wH22xeGFP was rapidly internalized by the high-FcgammaRI-expressing cell line U937 10.6, but did not associate with intracellular proteins Rab4, Rab5a, or Lamp-1, suggesting that the targeted fusion protein was not localized in early endosomes, recycling vesicles, or lysosomes. Interestingly, wH22xeGFP was found colocalized with intracellular MHC class I, suggesting that FcgammaRI-targeted Ags may converge upon a class I processing pathway. These data are in agreement with studies in the mouse showing that FcgammaRI targeting can lead to Ag-specific activation of cytotoxic T cells. Data obtained from these studies should lead to a better understanding of how Ags targeted to FcgammaRI are processed and under what conditions they lead to presentation of antigenic peptides in MHC class I, as a foundation for the use of FcgammaRI-targeted Ags as vaccines.

  6. Fc functional antibodies in humans with severe H7N9 and seasonal influenza

    Science.gov (United States)

    Vanderven, Hillary A.; Liu, Lu; Ana-Sosa-Batiz, Fernanda; Nguyen, Thi H.O.; Wan, Yanmin; Hogarth, P. Mark; Tilmanis, Danielle; Parsons, Matthew S.; Hurt, Aeron C.; Davenport, Miles P.; Kotsimbos, Tom; Cheng, Allen C.; Kedzierska, Katherine; Zhang, Xiaoyan; Xu, Jianqing; Kent, Stephen J.

    2017-01-01

    BACKGROUND. Both seasonal and novel avian influenza viruses can result in severe infections requiring hospitalization. Anti-influenza antibodies (Abs) with Fc-mediated effector functions, such as Ab-dependent cellular cytotoxicity (ADCC), are of growing interest in control of influenza but have not previously been studied during severe human infections. As such, the objective of this study was to examine Fc-mediated Ab functions in humans hospitalized with influenza infection. METHODS. Serum Ab response was studied in subjects hospitalized with either pandemic H7N9 avian influenza virus in China (n = 18) or circulating seasonal influenza viruses in Melbourne, Australia (n = 16). Recombinant soluble Fc receptor dimer ELISAs, natural killer (NK) cell activation assays, and Ab-dependent killing assays with influenza-infected target cells were used to assess the Fc functionality of anti-influenza hemagglutinin (HA) Abs during severe human influenza infection. RESULTS. We found that the peak generation of Fc functional HA Abs preceded that of neutralizing Abs for both severe H7N9 and seasonal influenza infections. Subjects who succumbed to complications of H7N9 infection demonstrated reduced HA-specific Fc receptor–binding Abs (in magnitude and breadth) immediately prior to death compared with those who survived. Subjects who recovered from H7N9 and severe seasonal influenza infections demonstrated increased Fc receptor–binding Abs not only against the homologous infecting strain but against HAs from different influenza A subtypes. CONCLUSION. Collectively, survivors of severe influenza infection rapidly generate a functional Ab response capable of mediating ADCC against divergent influenza viruses. Broadly binding HA Abs with Fc-mediated functions may be a useful component of protective immunity to severe influenza infection. FUNDING. The National Health and Medical Research Council ([NHMRC] grants 1023294, 1041832, and 1071916), the Australian Department of Health

  7. Association of Fc gamma-receptors IIa, IIIa, and IIIb genetic polymorphism with susceptibility to chronic periodontitis in South Indian population

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    Veenu Madaan Hans

    2015-01-01

    Full Text Available Background and Objective: Fc gamma receptors (FcγRs are the members of the immunoglobulin superfamily and may play a role in the pathogenesis of periodontitis. Genetic variation in these receptors and its link with various forms of periodontitis is being studied in different populations. The aim of the present study is to determine whether specific FcγRIIa, FcγRIIIa, and FcγRIIIb alleles and/or genotypes are associated with risk for susceptibility to generalized chronic periodontitis (GCP in South Indian population. Materials and Methods: The study population consisted of 120 South Indian subjects; 60 with GCP and 60 periodontally healthy. Deoxyribonucleic acid (DNA was extracted from samples collected by scrapping buccal epithelium. FcγRIIa and FcγRIIIa genotyping were performed by polymerase chain reaction (PCR amplification of DNA with allele-specific primers followed by allele-specific restriction digestion of the products. However, FcγRIIIb genotyping was done by allele-specific PCR. Results: No significant difference in the distribution of FcγRIIa H/R and FcγRIIIa NA1/NA2 genotypes or their respective alleles was observed in GCP patients and healthy subjects. For FcγRIIIa F/V genetic polymorphism, the homozygous V/V genotype and V allele were significantly overrepresented in GCP patients while F/F genotype and F allele in controls. Conclusion: The present study demonstrates that FcγRIIIa V/V genotype, as well as V allele, could be a possible risk factor for chronic periodontitis in South Indian population.

  8. Protection of Mice from Lethal Endotoxemia by Chimeric Human BPI-Fcγ1 Gene Delivery

    Institute of Scientific and Technical Information of China (English)

    Chen Li; Jing Li; Zhe Lv; Xinghua Guo; Qinghua Chen; Qingli Kong; Yunqing An

    2006-01-01

    To evaluate the potentiality of applying gene therapy to endotoxemia in high-risk patients, we investigated the effects of transferring an adeno-associated virus serotype 2 (AAV2)-mediated BPI-Fcγ1 gene on protecting mice from challenge of lethal endotoxin. The chimeric BPI-Fcγ1 gene consists of two parts, one encods functional N-terminus (1 to 199 amino acidic residues) of human BPI, which is a bactericidal/permeability-increasing protein,and the other encodes Fc segment of human immunoglobulin G1 (Fcγ1). Our results indicated that the target protein could be expressed and secreted into the serum of the gene-transferred mice. After lethal endotoxin challenge, the levels of endotoxin and TNF-α in the gene-transferred mice were decreased. The survival rate of the BPI-Fcγ1 gene-transferred mice was markedly increased. Our data suggest that AAV2-mediated chimeric BPI-Fcγ1 gene delivery can potentially be used clinically for the protection and treatment of endotoxemia and endotoxic shock in high-risk individuals.

  9. Controlled release of human growth hormone fused with a human hybrid Fc fragment through a nanoporous polymer membrane

    Science.gov (United States)

    Kim, Eung-Sam; Jang, Do Soo; Yang, Seung Yun; Lee, Mi Nam; Jin, Kyeong Sik; Cha, Hyung Jin; Kim, Jin Kon; Sung, Young Chul; Choi, Kwan Yong

    2013-05-01

    Nanotechnology has been applied to the development of more effective and compatible drug delivery systems for therapeutic proteins. Human growth hormone (hGH) was fused with a hybrid Fc fragment containing partial Fc domains of human IgD and IgG4 to produce a long-acting fusion protein. The fusion protein, hGH-hyFc, resulted in the increase of the hydrodynamic diameter (ca. 11 nm) compared with the diameter (ca. 5 nm) of the recombinant hGH. A diblock copolymer membrane with nanopores (average diameter of 14.3 nm) exhibited a constant release rate of hGH-hyFc. The hGH-hyFc protein released in a controlled manner for one month was found to trigger the phosphorylation of Janus kinase 2 (JAK2) in human B lymphocyte and to exhibit an almost identical circular dichroism spectrum to that of the original hGH-hyFc, suggesting that the released fusion protein should maintain the functional and structural integrity of hGH. Thus, the nanoporous release device could be a potential delivery system for the long-term controlled release of therapeutic proteins fused with the hybrid Fc fragment.Nanotechnology has been applied to the development of more effective and compatible drug delivery systems for therapeutic proteins. Human growth hormone (hGH) was fused with a hybrid Fc fragment containing partial Fc domains of human IgD and IgG4 to produce a long-acting fusion protein. The fusion protein, hGH-hyFc, resulted in the increase of the hydrodynamic diameter (ca. 11 nm) compared with the diameter (ca. 5 nm) of the recombinant hGH. A diblock copolymer membrane with nanopores (average diameter of 14.3 nm) exhibited a constant release rate of hGH-hyFc. The hGH-hyFc protein released in a controlled manner for one month was found to trigger the phosphorylation of Janus kinase 2 (JAK2) in human B lymphocyte and to exhibit an almost identical circular dichroism spectrum to that of the original hGH-hyFc, suggesting that the released fusion protein should maintain the functional and

  10. A soluble form of the high affinity IgE receptor, Fc-epsilon-RI, circulates in human serum.

    Directory of Open Access Journals (Sweden)

    Eleonora Dehlink

    Full Text Available Soluble IgE receptors are potential in vivo modulators of IgE-mediated immune responses and are thus important for our basic understanding of allergic responses. We here characterize a novel soluble version of the IgE-binding alpha-chain of Fc-epsilon-RI (sFcεRI, the high affinity receptor for IgE. sFcεRI immunoprecipitates as a protein of ∼40 kDa and contains an intact IgE-binding site. In human serum, sFcεRI is found as a soluble free IgE receptor as well as a complex with IgE. Using a newly established ELISA, we show that serum sFcεRI levels correlate with serum IgE in patients with elevated IgE. We also show that serum of individuals with normal IgE levels can be found to contain high levels of sFcεRI. After IgE-antigen-mediated crosslinking of surface FcεRI, we detect sFcεRI in the exosome-depleted, soluble fraction of cell culture supernatants. We further show that sFcεRI can block binding of IgE to FcεRI expressed at the cell surface. In summary, we here describe the alpha-chain of FcεRI as a circulating soluble IgE receptor isoform in human serum.

  11. Pharmacokinetics, Pharmacodynamics, and Efficacy of a Novel Long-Acting Human Growth Hormone: Fc Fusion Protein.

    Science.gov (United States)

    Kim, Su Jin; Kwak, Hyun-Hee; Cho, Sung Yoon; Sohn, Young Bae; Park, Sung Won; Huh, Rimm; Kim, Jinsup; Ko, Ah-Ra; Jin, Dong-Kyu

    2015-10-05

    The current recombinant human growth hormone (rhGH) therapy requires daily subcutaneous (sc) injections, which results in poor patient compliance, especially in young children. To reduce the dosing frequency, we generated a chimeric protein of rhGH and the Fc-domain of immunoglobulin G (IgG) (rhGH-Fc). The pharmacokinetics and pharmacodynamics of sc-injected rhGH-Fc were assessed in male Sprague-Dawley rats and hypophysectomized rats, respectively. A single sc injection of rhGH-Fc at a dose of 0.2 mg/kg slowly reached a Cmax of 16.80 ng/mL and remained for 7 days with a half-life of 51.1 h. Conversely, a single sc injection of rhGH 0.2 mg/kg rapidly reached a Cmax of 46.88 ng/mL and declined with a half-life of 0.55 h to baseline values in 4 h. In the efficacy study, the sc-injected rhGH-Fc induced rapid weight gain and tibial width growth at a dose of 240 μg/animal. The effect of two injections of rhGH-Fc separated by 1 week was comparable to that of the same dose of 14 daily injections of rhGH. The rhGH-Fc is a novel candidate for long-acting rhGH therapy with more convenient weekly administration, as it reduces glomerular filtration and receptor-mediated clearance while allowing for the rapid reversal of potential adverse events.

  12. Phagocytosis via complement or Fc-gamma receptors is compromised in monocytes from type 2 diabetes patients with chronic hyperglycemia.

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    Blanca I Restrepo

    Full Text Available Type 2 diabetes patients (DM2 have a higher risk of tuberculosis (TB that may be attributed to functional defects in their mononuclear phagocytes given the critical role of these cells in Mycobacterium tuberculosis containment. Our previous findings suggest that monocytes from DM2 have reduced association with serum-opsonized M. tuberculosis. To determine if this alteration is due to defects in phagocytosis via complement or Fc-gamma receptors (FcγRs, in this study we evaluated the uptake of sheep red blood cells coated with IgG or complement, respectively, by monocytes from individuals with and without DM2. We found that chronic hyperglycemia was significantly associated with reduced phagocytosis via either receptor by univariable and multivariable analyses. This defect was independent of host serum opsonins and flow cytometry data indicated this was not attributed to reduced expression of these phagocytic receptors on DM2 monocytes. The positive correlation between both pathways (R = 0.64; p = 0.003 indicate that monocytes from individuals with chronic hyperglycemia have a defect in the two predominant phagocytic pathways of these cells. Given that phagocytosis is linked to activation of effector mechanisms for bacterial killing, it is likely that this defect is one factor contributing to the higher susceptibility of DM2 patients to pathogens like M. tuberculosis.

  13. Rab20 regulates phagosome maturation in RAW264 macrophages during Fc gamma receptor-mediated phagocytosis.

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    Youhei Egami

    Full Text Available Rab20, a member of the Rab GTPase family, is known to be involved in membrane trafficking, however its implication in FcγR-mediated phagocytosis is unclear. We examined the spatiotemporal localization of Rab20 during phagocytosis of IgG-opsonized erythrocytes (IgG-Es in RAW264 macrophages. By the live-cell imaging of fluorescent protein-fused Rab20, it was shown that Rab20 was transiently associated with the phagosomal membranes. During the early stage of phagosome formation, Rab20 was not localized on the membranes of phagocytic cups, but was gradually recruited to the newly formed phagosomes. Although Rab20 was colocalized with Rab5 to some extent, the association of Rab20 with the phagosomes persisted even after the loss of Rab5 from the phagosomal membranes. Then, Rab20 was colocalized with Rab7 and Lamp1, late endosomal/lysosomal markers, on the internalized phagosomes. Moreover, our analysis of Rab20 mutant expression revealed that the maturation of phagosomes was significantly delayed in cells expressing the GDP-bound mutant Rab20-T19N. These data suggest that Rab20 is an important component of phagosome and regulates the phagosome maturation during FcγR-mediated phagocytosis.

  14. (Some) cellular mechanisms influencing the transcription of human endogenous retrovirus, HERV-Fc1

    DEFF Research Database (Denmark)

    Laska, Magdalena Janina; Nissen, Kari Konstantin; Nexø, Bjørn Andersen

    2013-01-01

    DNA methylation and histone acetylation are epigenetic modifications that act as regulators of gene expression. DNA methylation is considered an important mechanism for silencing of retroelements in the mammalian genome. However, the methylation of human endogenous retroviruses (HERVs) is not well...... investigated. The aim of this study was to investigate the transcriptional potential of HERV-Fc1 proviral 5'LTR in more detail, and examined the specific influence of CpG methylation on this LTR in number of cell lines. Specifically, the role of demethylating chemicals e.g. 5-aza-2' deoxycytidine...... and Trichostatin-A, in inducing or reactivating expression of HERV-Fc1 specific sequences and the mechanisms were investigated. In our present study, 5-aza-dC is shown to be a powerful inducer of HERV-Fc1, and at the same time it strongly inhibits methylation of DNA. Treatment with this demethylating agent 5-aza...

  15. Human Endogenous Retrovirus HERV-Fc1 Association with Multiple Sclerosis Susceptibility: A Meta-Analysis

    Science.gov (United States)

    García-Montojo, Marta; Alcina, Antonio; Fedetz, María; Alloza, Iraide; Astobiza, Ianire; Leyva, Laura; Fernández, Oscar; Izquierdo, Guillermo; Antigüedad, Alfredo; Arroyo, Rafael; Álvarez-Lafuente, Roberto; Vandenbroeck, Koen; Matesanz, Fuencisla; Urcelay, Elena

    2014-01-01

    Background Human endogenous retroviruses (HERVs) are repetitive sequences derived from ancestral germ-line infections by exogenous retroviruses and different HERV families have been integrated in the genome. HERV-Fc1 in chromosome X has been previously associated with multiple sclerosis (MS) in Northern European populations. Additionally, HERV-Fc1 RNA levels of expression have been found increased in plasma of MS patients with active disease. Considering the North-South latitude gradient in MS prevalence, we aimed to evaluate the role of HERV-Fc1on MS risk in three independent Spanish cohorts. Methods A single nucleotide polymorphism near HERV-Fc1, rs391745, was genotyped by Taqman chemistry in a total of 2473 MS patients and 3031 ethnically matched controls, consecutively recruited from: Northern (569 patients and 980 controls), Central (883 patients and 692 controls) and Southern (1021 patients and 1359 controls) Spain. Our results were pooled in a meta-analysis with previously published data. Results Significant associations of the HERV-Fc1 polymorphism with MS were observed in two Spanish cohorts and the combined meta-analysis with previous data yielded a significant association [rs391745 C-allele carriers: pM-H = 0.0005; ORM-H (95% CI) = 1.27 (1.11–1.45)]. Concordantly to previous findings, when the analysis was restricted to relapsing remitting and secondary progressive MS samples, a slight enhancement in the strength of the association was observed [pM-H = 0.0003, ORM-H (95% CI) = 1.32 (1.14–1.53)]. Conclusion Association of the HERV-Fc1 polymorphism rs391745 with bout-onset MS susceptibility was confirmed in Southern European cohorts. PMID:24594754

  16. Human endogenous retrovirus HERV-Fc1 association with multiple sclerosis susceptibility: a meta-analysis.

    Directory of Open Access Journals (Sweden)

    Belén de la Hera

    Full Text Available BACKGROUND: Human endogenous retroviruses (HERVs are repetitive sequences derived from ancestral germ-line infections by exogenous retroviruses and different HERV families have been integrated in the genome. HERV-Fc1 in chromosome X has been previously associated with multiple sclerosis (MS in Northern European populations. Additionally, HERV-Fc1 RNA levels of expression have been found increased in plasma of MS patients with active disease. Considering the North-South latitude gradient in MS prevalence, we aimed to evaluate the role of HERV-Fc1on MS risk in three independent Spanish cohorts. METHODS: A single nucleotide polymorphism near HERV-Fc1, rs391745, was genotyped by Taqman chemistry in a total of 2473 MS patients and 3031 ethnically matched controls, consecutively recruited from: Northern (569 patients and 980 controls, Central (883 patients and 692 controls and Southern (1021 patients and 1359 controls Spain. Our results were pooled in a meta-analysis with previously published data. RESULTS: Significant associations of the HERV-Fc1 polymorphism with MS were observed in two Spanish cohorts and the combined meta-analysis with previous data yielded a significant association [rs391745 C-allele carriers: pM-H = 0.0005; ORM-H (95% CI = 1.27 (1.11-1.45]. Concordantly to previous findings, when the analysis was restricted to relapsing remitting and secondary progressive MS samples, a slight enhancement in the strength of the association was observed [pM-H = 0.0003, ORM-H (95% CI = 1.32 (1.14-1.53]. CONCLUSION: Association of the HERV-Fc1 polymorphism rs391745 with bout-onset MS susceptibility was confirmed in Southern European cohorts.

  17. Detection of EPO-Fc fusion protein in human blood: screening and confirmation protocols for sports drug testing.

    Science.gov (United States)

    Reichel, Christian; Thevis, Mario

    2012-11-01

    The neonatal Fc receptor (FcRn) has been under investigation for several years as a pharmaceutical drug target. Clinical studies have shown that fusion proteins consisting of human recombinant erythropoietin (rhEPO) and the Fc-part of IgG can be transported after pulmonary administration via FcRn across the airway epithelium to the blood stream. So far, no clinically approved pharmaceutical formulation of EPO-Fc is available. Since various forms of recombinant erythropoietins have been frequently misused by athletes as performance-enhancing agents, EPO-Fc might play a similar role in sports in the future. In order to investigate the detectability of EPO-Fc in human blood, different strategies were tested and developed. Only two of them fulfilled the necessary requirements regarding sensitivity and specificity. A rapid protocol useful for screening purposes first enriches EPO-Fc from human serum via high capacity protein A beads and subsequently detects EPO-Fc in the eluate with a commercial EPO ELISA kit. The limit of detection (LOD) of the method is about 5 pg (45 amol) EPO-Fc and is independent of the serum volume used. For screening and/or confirmation purposes a second protocol was evaluated, which consists of a fast EPO immunopurification step followed by sodium dodecyl sulfate or sarcosyl polyacrylamide gel electrophoresis (SDS-PAGE, SAR-PAGE) and Western double-blotting with chemiluminescence detection - a method already established in routine EPO anti-doping control. The latter strategy allows the detection of EPO-Fc in serum together with all other recombinant erythropoietins and with an identical LOD (5 pg/45 amol) as for the rapid screening protocol.

  18. Fc gamma receptors regulate immune activation and susceptibility during Mycobacterium tuberculosis infection.

    Science.gov (United States)

    Maglione, Paul J; Xu, Jiayong; Casadevall, Arturo; Chan, John

    2008-03-01

    The critical role of cellular immunity during tuberculosis (TB) has been extensively studied, but the impact of Abs upon this infection remains poorly defined. Previously, we demonstrated that B cells are required for optimal protection in Mycobacterium tuberculosis-infected mice. FcgammaR modulate immunity by engaging Igs produced by B cells. We report that C57BL/6 mice deficient in inhibitory FcgammaRIIB (RIIB-/-) manifested enhanced mycobacterial containment and diminished immunopathology compared with wild-type controls. These findings corresponded with enhanced pulmonary Th1 responses, evidenced by increased IFN-gamma-producing CD4+ T cells, and elevated expression of MHC class II and costimulatory molecules B7-1 and B7-2 in the lungs. Upon M. tuberculosis infection and immune complex engagement, RIIB-/- macrophages produced more of the p40 component of the Th1-promoting cytokine IL-12. These data strongly suggest that FcgammaRIIB engagement can dampen the TB Th1 response by attenuating IL-12p40 production or activation of APCs. Conversely, C57BL/6 mice lacking the gamma-chain shared by activating FcgammaR had enhanced susceptibility and exacerbated immunopathology upon M. tuberculosis challenge, associated with increased production of the immunosuppressive cytokine IL-10. Thus, engagement of distinct FcgammaR can divergently affect cytokine production and susceptibility during M. tuberculosis infection.

  19. Fc gamma receptor polymorphisms in systemic lupus erythematosus and their correlation with the clinical severity of the disease

    Directory of Open Access Journals (Sweden)

    Pradhan Vandana

    2008-01-01

    Full Text Available Receptors for the Fc domains of IgG (Fc γ R play a critical role in linking humoral and cellular immune responses. The various Fc γ R genes may contribute to differences in infectious and immune related diseases in various ethnic populations. Polymorphisms of Fc γ R mainly Fc γ R IIA, IIB, IIIA, IIIB have been identified as genetic factors influencing susceptibility to disease or disease course of a prototype autoimmune disease like Systemic Lupus Erythematosus (SLE. Activated and inhibitory Fc γ Rs seem to play an important role in the pathogenesis of SLE, in initiation of autoimmunity, the subsequent development of inflammatory lesions and finally immune clearance mechanisms. This review focuses on the role of Fc γ R polymorphism and their association with clinical manifestations and initiation of autoantibody production, inflammatory handling of immune complexes and disease development in SLE patients.

  20. Novel human IgG1 and IgG4 Fc-engineered antibodies with completely abolished immune effector functions.

    Science.gov (United States)

    Schlothauer, Tilman; Herter, Sylvia; Koller, Claudia Ferrara; Grau-Richards, Sandra; Steinhart, Virginie; Spick, Christian; Kubbies, Manfred; Klein, Christian; Umaña, Pablo; Mössner, Ekkehard

    2016-10-01

    Recombinant human IgG antibodies (hIgGs) completely devoid of binding to Fcγ receptors (FcγRs) and complement protein C1q, and thus with abolished immune effector functions, are of use for various therapeutic applications in order to reduce FcγR activation and Fc-mediated toxicity. Fc engineering approaches described to date only partially achieve this goal or employ a large number of mutations, which may increase the risk of anti-drug antibody generation. We describe here two new, engineered hIgG Fc domains, hIgG1-P329G LALA and hIgG4-P329G SPLE, with completely abolished FcγR and C1q interactions, containing a limited number of mutations and with unaffected FcRn interactions and Fc stability. Both 'effector-silent' Fc variants are based on a novel Fc mutation, P329G that disrupts the formation of a proline sandwich motif with the FcγRs. As this motif is present in the interface of all IgG Fc/FcγR complexes, its disruption can be applied to all human and most of the other mammalian IgG subclasses in order to create effector silent IgG molecules.

  1. Evidence for non-random distribution of Fc gamma receptor genotype combinations

    NARCIS (Netherlands)

    van der Pol, WL; Jansen, MD; Sluiter, WJ; van de Sluis, B; Leppers-van de Straat, FGJ; Kobayashi, T; Westendorp, RGJ; Huizinga, TWJ; van de Winkel, JGJ

    2003-01-01

    Human IgG receptors (FcgammaR) display considerable heterogeneity, and are crucial immune response modulating molecules. FcgammaRIIA, FcgammaRIIIA, and FcgammaRIIIB display functional biallelic polymorphisms. FcgammaR polymorphisms have been found associated with susceptibility to infectious and aut

  2. Fcγ receptor antigen targeting potentiates cross-presentation by human blood and lymphoid tissue BDCA-3+ dendritic cells.

    Science.gov (United States)

    Flinsenberg, Thijs W H; Compeer, Ewoud B; Koning, Dan; Klein, Mark; Amelung, Femke J; van Baarle, Debbie; Boelens, Jaap Jan; Boes, Marianne

    2012-12-20

    The reactivation of human cytomegalovirus (HCMV) poses a serious health threat to immune compromised individuals. As a treatment strategy, dendritic cell (DC) vaccination trials are ongoing. Recent work suggests that BDCA-3(+) (CD141(+)) subset DCs may be particularly effective in DC vaccination trials. BDCA-3(+) DCs had however been mostly characterized for their ability to cross-present antigen from necrotic cells. We here describe our study of human BDCA-3(+) DCs in elicitation of HCMV-specific CD8(+) T-cell clones. We show that Fcgamma-receptor (FcγR) antigen targeting facilitates antigen cross-presentation in several DC subsets, including BDCA-3(+) DCs. FcγR antigen targeting stimulates antigen uptake by BDCA-1(+) rather than BDCA-3(+) DCs. Conversely, BDCA-3(+) DCs and not BDCA-1(+) DCs show improved cross-presentation by FcγR targeting, as measured by induced release of IFNγ and TNF by antigen-specific CD8(+) T cells. FcγR-facilitated cross-presentation requires antigen processing in both an acidic endosomal compartment and by the proteasome, and did not induce substantial DC maturation. FcγRII is the most abundantly expressed FcγR on both BDCA-1(+) and BDCA-3(+) DCs. Furthermore we show that BDCA-3(+) DCs express relatively more stimulatory FcγRIIa than inhibitory FcγRIIb in comparison with BDCA-1(+) DCs. These studies support the exploration of FcγR antigen targeting to BDCA-3(+) DCs for human vaccination purposes.

  3. Characterization of Aggregation Propensity of a Human Fc-Fusion Protein Therapeutic by Hydrogen/Deuterium Exchange Mass Spectrometry

    Science.gov (United States)

    Huang, Richard Y.-C.; Iacob, Roxana E.; Krystek, Stanley R.; Jin, Mi; Wei, Hui; Tao, Li; Das, Tapan K.; Tymiak, Adrienne A.; Engen, John R.; Chen, Guodong

    2017-05-01

    Aggregation of protein therapeutics has long been a concern across different stages of manufacturing processes in the biopharmaceutical industry. It is often indicative of aberrant protein therapeutic higher-order structure. In this study, the aggregation propensity of a human Fc-fusion protein therapeutic was characterized. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) was applied to examine the conformational dynamics of dimers collected from a bioreactor. HDX-MS data combined with spatial aggregation propensity calculations revealed a potential aggregation interface in the Fc domain. This study provides a general strategy for the characterization of the aggregation propensity of Fc-fusion proteins at the molecular level.

  4. Computational modeling of the Fc αRI receptor binding in the Fc α domain of the human antibody IgA: Normal Modes Analysis (NMA) study

    Science.gov (United States)

    Jayasinghe, Manori; Posgai, Monica; Tonddast-Navaei, Sam; Ibrahim, George; Stan, George; Herr, Andrew; George Stan Group Collaboration; Herr's Group Team

    2014-03-01

    Fc αRI receptor binding in the Fc α domain of the antibody IgA triggers immune effector responses such as phagocytosis and antibody-dependent cell-mediated cytotoxicity in eukaryotic cells. Fc α is a dimer of heavy chains of the IgA antibody and each Fc α heavy chain which consisted of two immunoglobulin constant domains, CH2 and CH3, can bind one Fc αRI molecule at the CH2-CH3 interface forming a 2:1 stoichiometry. Experimental evidences confirmed that Fc αRI binding to the Fc α CH2-CH3 junction altered the kinetics of HAA lectin binding at the distant IgA1 hinge. Our focus in this research was to understand the conformational changes and the network of residues which co-ordinate the receptor binding dynamics of the Fc α dimer complex. Structure-based elastic network modeling was used to compute normal modes of distinct Fc α configurations. Asymmetric and un-liganded Fc α configurations were obtained from the high resolution crystal structure of Fc α-Fc αRI 2:1 symmetric complex of PDB ID 1OW0. Our findings confirmed that Fc αRI binding, either in asymmetric or symmetric complex with Fc α, propagated long-range conformational changes across the Fc domains, potentially also impacting the distant IgA1 hinge.

  5. [Expression of human IL-35-IgG4 (Fc) fusion protein in CHO/DG44 cells].

    Science.gov (United States)

    Tang, Jing; Gao, Wenda; Zhang, Qing; Zhang, Dawei; Chen, Yang; He, Bo; Liu, Quansheng

    2009-01-01

    We constructed the eukaryotic expression vector of human IL-35-IgG4 (Fc)-pOptiVEC-TOPO by gene recombination technique and expressed the fusion protein human IL-35-IgG4 (Fc) in CHO/DG44 cells. The two components of the newly discovered cytokine human IL-35, EBI3 and IL-12p35, were amplified by PCR from the cDNA library derived from the KG-I cells after LPS induction. The two PCR-amplified cDNA fragments of human IL-35 were linked by over-lapping PCR and then cloned into the IgG4 (Fc)-pOptiVEC-TOPO vector. The constructed plasmid with the recombinant cDNA IL-35-IgG4 (Fc) was verified by restriction enzyme digestion analysis, PCR and DNA sequencing. The verified plasmid with the recombinant cDNA was transfected into CHO/DG44 cells using Lipofectamine 2000. The success of the transfection was examined and confirmed by RT-PCR. After selection in alpha-MEM (-) medium, the IL-35-Ig G4 (Fc) positive CHO/DG44 clones were chosen and the media from these positive clones were collected to be used to purify the fusion protein. The positive CHO/DG44 clones were further cultured in increasing concentrations of MTX and the expression levels of the fusion protein IL-35-Ig G4 (Fc) were repetitively induced by MTX-induced gene amplification. The IL-35-IgG4 (Fc) fusion protein was purified from the media collected from the positive CHO/DG44 clones by protein G affinity chromatography and then identified by SDS-PAGE and Western blotting. The results showed that one protein band was found to match well with the predicted relative molecular mass of human IL-35-IgG4 (Fc) and this protein could specifically bind to anti-human IgG4 (Fc) monoclonal antibody. In conclusion, our study successfully established an IL-35-IgG4 (Fc) positive DG44 cell line which could stably express IL-35-IgG4 (Fc) fusion protein.

  6. Bacillus anthracis peptidoglycan activates human platelets through FcγRII and complement

    Science.gov (United States)

    Sun, Dawei; Popescu, Narcis I.; Raisley, Brent; Keshari, Ravi S.; Dale, George L.; Lupu, Florea

    2013-01-01

    Platelet activation frequently accompanies sepsis and contributes to the sepsis-associated vascular leakage and coagulation dysfunction. Our previous work has implicated peptidoglycan (PGN) as an agent causing systemic inflammation in gram-positive sepsis. We used flow cytometry and fluorescent microscopy to define the effects of PGN on the activation of human platelets. PGN induced platelet aggregation, expression of the activated form of integrin αIIbβ3, and exposure of phosphatidylserine (PS). These changes were dependent on immunoglobulin G and were attenuated by the Fcγ receptor IIa–blocking antibody IV.3, suggesting they are mediated by PGN–anti-PGN immune complexes signaling through Fcγ receptor IIa. PS exposure was not blocked by IV.3 but was sensitive to inhibitors of complement activation. PGN was a potent activator of the complement cascade in human plasma and caused deposition of C5b-9 on the platelet surface. Platelets with exposed PS had greatly accelerated prothrombinase activity. We conclude that PGN derived from gram-positive bacteria is a potent platelet agonist when complexed with anti-PGN antibody and could contribute to the coagulation dysfunction accompanying gram-positive infections. PMID:23733338

  7. Bruton’s Tyrosine Kinase Mediates FcγRIIa/Toll-Like Receptor–4 Receptor Crosstalk in Human Neutrophils

    Science.gov (United States)

    Krupa, Agnieszka; Fudala, Rafal; Florence, Jon M.; Tucker, Torry; Allen, Timothy C.; Standiford, Theodore J.; Luchowski, Rafal; Fol, Marek; Rahman, Moshiur; Gryczynski, Zygmunt; Gryczynski, Ignacy

    2013-01-01

    Previous observations by our laboratory indicate that the presence of anti–IL-8 autoantibody:IL-8 immune complexes in lung fluids from patients with acute lung injury/acute respiratory distress syndrome (ALI/ARDS) comprises an important prognostic indicator in the development and ultimate outcome of ALI/ARDS. We also showed that these complexes display proinflammatory activity toward neutrophils through the engagement of FcγRIIa receptors. Because sepsis is one of the most common risk factors for ALI/ARDS, the initial goal of our present study involved investigating the effects of LPS on the expression of FcγRIIa receptors in neutrophils. Our results indicate that LPS triggers an increase in the expression of FcγRIIa on the neutrophil surface, which leads to shortening of the molecular distance between FcγRIIa and Toll-like receptor–4 (TLR4). When such neutrophils are stimulated with anti–IL-8:IL-8 complexes, the TLR4 cascade becomes activated via the engagement of FcγRIIa. The underlying molecular mechanism has been subsequently examined and involves Bruton’s tyrosine kinase (Btk). In conclusion, our study reveals the existence of Btk-dependent molecular cooperation between FcγRIIa and TLR4 signaling cascades in LPS-“primed” human neutrophils. Furthermore, we used fluorescence lifetime imaging to study the interactions between TLR4 and FcγRIIa in human alveolar neutrophils from patients with ALI/ARDS. The results from these experiments confirm the existence of the molecular cooperation between TLR4 and FcγRIIa. PMID:23239500

  8. A strategy for bacterial production of a soluble functional human neonatal Fc receptor

    DEFF Research Database (Denmark)

    Andersen, Jan Terje; Justesen, Sune; Berntzen, Gøril

    2008-01-01

    The major histocompatibility complex (MHC) class I related receptor, the neonatal Fc receptor (FcRn), rescues immunoglobulin G (IgG) and albumin from lysosomal degradation by recycling in endothelial cells. FcRn also contributes to passive immunity by mediating transport of IgG from mother to fetus...

  9. Fc-Gamma Receptor 3B Copy Number Variation Is Not a Risk Factor for Behçet’s Disease

    Directory of Open Access Journals (Sweden)

    Rachel Black

    2012-01-01

    Full Text Available Behçet’s disease (BD is an immune-mediated systemic vasculitis associated with HLAB51. Other gene associations are likely and may provide further insight into the pathogenesis of this disease. Fc-gamma receptors play an important role in regulating immune function. Copy number variation (CNV of the Fc-gamma receptor 3B (FCGR3B gene is associated with other inflammatory conditions and may also play a role in BD. The aim of this study was to determine whether CNV of the FCGR3B gene is associated with BD or its clinical features. FCGR3B copy number was determined for 187 Iranian patients and 178 ethnicity-matched controls using quantitative real-time PCR. The genotype frequencies were comparable in both BD patients and controls. The odds ratio for low copy number (2CN was 0.75 (=0.50. There was no association found between high or low CN of the FCGR3B gene and BD or its clinical features in this Iranian population. We are the first to report this finding which, when looked at in the context of other genetic studies, gives us further insight into the complex pathogenesis of BD.

  10. Quantitation of Fc gamma RII mRNA in platelets and megakaryoblastic cell lines by a new method of in situ hybridization.

    Science.gov (United States)

    Markovic, B; Wu, Z H; Chesterman, C N; Chong, B H

    1994-06-03

    We have developed a highly sensitive and quantitative, non-isotopic method of in situ hybridization in which the level of probe binding to intracellular mRNA is determined using an ELISA based detection method. Highly purified cell preparations or cells from a cultured cell line are centrifuged into 96 well microtiter plates. The cells are fixed with formalin and pre-treated with Triton X-100 and Nonidet P40 before photobiotin labeled cDNA probes are applied. The biotin from the hybridization is detected using multiple applications of streptavidin and biotinylated alkaline phosphatase and then visualized by the p-NPP (p-nitrophenyl phosphate) conversion method. We have determined a number of the optimal parameters in the procedure including the effects of cell numbers per well, development times and standardization of data using ubiquitous beta-actin mRNA and poly-A+ RNA expression as controls. We have used the technique to study the level of expression of FcgR mRNA in platelets and precursors. We found that platelets and megakaryoblastic cell lines only express mRNA for Fc gamma RII. The presence of the Fc gamma RII molecules was confirmed by complementary studies using immunohistochemistry with specific monoclonal antibodies IV.3 and KB61.

  11. Physical and Functional Association of the Src Family Kinases Fyn and Lyn with the Collagen Receptor Glycoprotein VI-Fc Receptor γ Chain Complex on Human Platelets

    Science.gov (United States)

    Ezumi, Yasuharu; Shindoh, Keisuke; Tsuji, Masaaki; Takayama, Hiroshi

    1998-01-01

    We have previously shown that uncharacterized glycoprotein VI (GPVI), which is constitutively associated and coexpressed with Fc receptor γ chain (FcRγ) in human platelets, is essential for collagen-stimulated tyrosine phosphorylation of FcRγ, Syk, and phospholipase Cγ2 (PLCγ2), leading to platelet activation. Here we investigated involvement of the Src family in the proximal signals through the GPVI–FcRγ complex, using the snake venom convulxin from Crotalus durissus terrificus, which specifically recognizes GPVI and activates platelets through cross-linking GPVI. Convulxin-coupled beads precipitated the GPVI–FcRγ complex from platelet lysates. Collagen and convulxin induced tyrosine phosphorylation of FcRγ, Syk, and PLCγ2 and recruited tyrosine-phosphorylated Syk to the GPVI–FcRγ complex. Using coprecipitation methods with convulxin-coupled beads and antibodies against FcRγ and the Src family, we showed that Fyn and Lyn, but not Yes, Src, Fgr, Hck, and Lck, were physically associated with the GPVI–FcRγ complex irrespective of stimulation. Furthermore, Fyn was rapidly activated by collagen or cross-linking GPVI. The Src family–specific inhibitor PP1 dose-dependently inhibited collagen- or convulxin-induced tyrosine phosphorylation of proteins including FcRγ, Syk, and PLCγ2, accompanied by a loss of aggregation and ATP release reaction. These results indicate that the Src family plays a critical role in platelet activation via the collagen receptor GPVI–FcRγ complex. PMID:9670039

  12. Human gamma oscillations during slow wave sleep.

    Directory of Open Access Journals (Sweden)

    Mario Valderrama

    Full Text Available Neocortical local field potentials have shown that gamma oscillations occur spontaneously during slow-wave sleep (SWS. At the macroscopic EEG level in the human brain, no evidences were reported so far. In this study, by using simultaneous scalp and intracranial EEG recordings in 20 epileptic subjects, we examined gamma oscillations in cerebral cortex during SWS. We report that gamma oscillations in low (30-50 Hz and high (60-120 Hz frequency bands recurrently emerged in all investigated regions and their amplitudes coincided with specific phases of the cortical slow wave. In most of the cases, multiple oscillatory bursts in different frequency bands from 30 to 120 Hz were correlated with positive peaks of scalp slow waves ("IN-phase" pattern, confirming previous animal findings. In addition, we report another gamma pattern that appears preferentially during the negative phase of the slow wave ("ANTI-phase" pattern. This new pattern presented dominant peaks in the high gamma range and was preferentially expressed in the temporal cortex. Finally, we found that the spatial coherence between cortical sites exhibiting gamma activities was local and fell off quickly when computed between distant sites. Overall, these results provide the first human evidences that gamma oscillations can be observed in macroscopic EEG recordings during sleep. They support the concept that these high-frequency activities might be associated with phasic increases of neural activity during slow oscillations. Such patterned activity in the sleeping brain could play a role in off-line processing of cortical networks.

  13. Postneonatal Mortality and Liver Changes in Cloned Pigs Associated with Human Tumor Necrosis Factor Receptor I-Fc and Human Heme Oxygenase-1 Overexpression

    Directory of Open Access Journals (Sweden)

    Geon A. Kim

    2017-01-01

    Full Text Available Soluble human tumor necrosis factor (shTNFRI-Fc and human heme oxygenase 1 (hHO-1 are key regulators for protection against oxidative and inflammatory injury for xenotransplantation. Somatic cells with more than 10 copy numbers of shTNFRI-Fc and hHO-1 were employed in somatic cell nuclear transfer to generate cloned pigs, thereby resulting in seven cloned piglets. However, produced piglets were all dead within 24 hours after birth. Obviously, postnatal death with liver apoptosis was reported in the higher copy number of shTNFRI-Fc and hHO-1 piglets. In liver, the transcript levels of ferritin heavy chain, light chain, transferrin, and inducible nitric oxide synthase were significantly highly expressed compared to those of lower copy number of shTNFRI-Fc and hHO-1 piglets (P<0.05. Also, H2O2 contents were increased, and superoxide dismutase was significantly lower in the higher copy number of shTNFRI-Fc and hHO-1 piglets (P<0.05. These results indicate that TNFRI-Fc and hHO-1 overexpression may apparently induce free iron in the liver and exert oxidative stress by enhancing reactive oxygen species production and block normal postneonatal liver metabolism.

  14. Perturbation of thermal unfolding and aggregation of human IgG1 Fc fragment by Hofmeister anions.

    Science.gov (United States)

    Zhang-van Enk, Jian; Mason, Bruce D; Yu, Lei; Zhang, Le; Hamouda, Wael; Huang, Gang; Liu, Dingjiang; Remmele, Richard L; Zhang, Jifeng

    2013-02-04

    The thermal unfolding and subsequent aggregation of the unglycosylated Fc fragment of a human IgG1 antibody (Fc) were studied in the salt solutions of Na(2)SO(4), KF, KCl and KSCN at pH 4.8 and 7.2 below and at its pI of 7.2, respectively, using differential scanning calorimetry (DSC), far ultraviolet circular dichroism (far-UV CD), size exclusion chromatography (SE-HPLC) and light scattering. First, our experimental results demonstrated that the thermal unfolding of the C(H)2 domain of the Fc was sufficient to induce aggregation. Second, at both pH conditions, the anions (except F(-)) destabilized the C(H)2 domain where the effectiveness of SO(4)(2-) > SCN(-) > Cl(-) > F(-) was more apparent at pH 4.8. In addition, the thermal stability of the C(H)2 domain was less sensitive to the change in salt concentration at pH 7.2 than at pH 4.8. Third, at pH 4.8 when the Fc had a net positive charge, the anions accelerated the aggregation reaction with SO(4)(2-) > SCN(-) > Cl(-) > F(-) in effectiveness. But these anions slowed down the aggregation kinetics at pH 7.2 with similar effectiveness when the Fc was net charge neutral. We hypothesize that the effectiveness of the anion on destabilizing the C(H)2 domain could be attributed to its ability to perturb the free energy for both of the native and unfolded states. The effect of the anions on the kinetics of the aggregation reaction could be interpreted based on the modulation of the electrostatic protein-protein interactions by the anions.

  15. Structural insights into the interaction of human IgG1 with FcγRI: no direct role of glycans in binding

    Energy Technology Data Exchange (ETDEWEB)

    Oganesyan, Vaheh, E-mail: oganesyanv@medimmune.com; Mazor, Yariv; Yang, Chunning; Cook, Kimberly E.; Woods, Robert M. [MedImmune LLC, 1 MedImmune Way, Gaithersburg, MD 20878 (United States); Ferguson, Andrew [AstraZeneca Pharmaceuticals, 35 Gatehouse Drive, Mailstop E3, Waltham, MA 02451 (United States); Bowen, Michael A.; Martin, Tom; Zhu, Jie; Wu, Herren; Dall’Acqua, William F., E-mail: oganesyanv@medimmune.com [MedImmune LLC, 1 MedImmune Way, Gaithersburg, MD 20878 (United States)

    2015-10-31

    In an effort to identify the critical structural features responsible for the high-affinity interaction of IgG1 Fc with FcγRI, the structure of the corresponding complex was solved at a resolution of 2.4 Å. The three-dimensional structure of a human IgG1 Fc fragment bound to wild-type human FcγRI is reported. The structure of the corresponding complex was solved at a resolution of 2.4 Å using molecular replacement; this is the highest resolution achieved for an unmutated FcγRI molecule. This study highlights the critical structural and functional role played by the second extracellular subdomain of FcγRI. It also explains the long-known major energetic contribution of the Fc ‘LLGG’ motif at positions 234–237, and particularly of Leu235, via a ‘lock-and-key’ mechanism. Finally, a previously held belief is corrected and a differing view is offered on the recently proposed direct role of Fc carbohydrates in the corresponding interaction. Structural evidence is provided that such glycan-related effects are strictly indirect.

  16. Human epidermal Langerhans cells express the high affinity receptor for immunoglobulin E (Fc epsilon RI)

    OpenAIRE

    1992-01-01

    It has been suggested that epidermal Langerhans cells (LC) bearing immunoglobulin E (IgE) may be involved in the genesis of atopic disease. The identity of the IgE receptor(s) on LC remained unclear, although it represents a crucial point in understanding cellular events linked to the binding of allergens to LC via IgE. In this report, we demonstrate that epidermal LC express the high affinity receptor for the Fc fragment of IgE (Fc epsilon RI) which has, so far, only been described on mast c...

  17. Production and characterization of soluble human TNFRI-Fc and human HO-1(HMOX1) transgenic pigs by using the F2A peptide.

    Science.gov (United States)

    Park, Sol Ji; Cho, Bumrae; Koo, Ok Jae; Kim, Hwajung; Kang, Jung Taek; Hurh, Sunghoon; Kim, Su Jin; Yeom, Hye Jung; Moon, Joonho; Lee, Eun Mi; Choi, Ji Yei; Hong, Ju Ho; Jang, Goo; Hwang, Joing-Ik; Yang, Jaeseok; Lee, Byeong Chun; Ahn, Curie

    2014-06-01

    Generation of transgenic pigs for xenotransplantation is one of the most promising technologies for resolving organ shortages. Human heme oxygenase-1 (hHO-1/HMOX1) can protect transplanted organs by its strong anti-oxidative, anti-apoptotic, and anti-inflammatory effects. Soluble human TNFRI-Fc (shTNFRI-Fc) can inhibit the binding of human TNF-α (hTNF-α) to TNF receptors on porcine cells, and thereby, prevent hTNF-α-mediated inflammation and apoptosis. Herein, we successfully generated shTNFRI-Fc-F2A-HA-hHO-1 transgenic (TG) pigs expressing both shTNFRI-Fc and hemagglutinin-tagged-human heme oxygenase-1 (HA-hHO-1) by using an F2A self-cleaving peptide. shTNFRI-Fc and HA-hHO-1 transgenes containing the F2A peptide were constructed under the control of the CAG promoter. Transgene insertion and copy number in the genome of transgenic pigs was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. Expressions of shTNFRI-Fc and HA-hHO-1 in TG pigs were confirmed using PCR, RT-PCR, western blot, ELISA, and immunohistochemistry. shTNFRI-Fc and HA-hHO-1 were expressed in various organs, including the heart, lung, and spleen. ELISA assays detected shTNFRI-Fc in the sera of TG pigs. For functional analysis, fibroblasts isolated from a shTNFRI-Fc-F2A-HA-hHO-1 TG pig (i.e., #14; 1 × 10(5) cells) were cultured with hTNF-α (20 ng/mL) and cycloheximide (10 μg/mL). The viability of shTNFRI-Fc-F2A-HA-hHO-1 TG pig fibroblasts was significantly higher than that of the wild type (wild type vs. shTNFRI-Fc-F2A-HA-hHO-1 TG at 24 h, 31.6 ± 3.2 vs. 60.4 ± 8.3 %, respectively; p hHO-1 TG pig fibroblasts was lower than that of the wild type pig fibroblasts (wild type vs. shTNFRI-Fc-F2A-HA-hHO-1 TG at 12 h, 812,452 ± 113,078 RLU vs. 88,240 ± 10,438 RLU, respectively; p hHO-1 TG pigs generated by the F2A self-cleaving peptide express both shTNFRI-Fc and HA-hHO-1 molecules, which provides protection against oxidative and inflammatory injury

  18. Inhibition of th17 cells and promotion of tregs in fc gamma chain-deficient mice contributes to the attenuated atherosclerotic lesions

    Science.gov (United States)

    The presence of anti-oxLDL IgG is well documented in clinical and animal studies. However, the role for Fc Rs to the progression of atherosclerosis has not been studied in detail. In the present study, we investigated the role for activating Fc R in the progression of atherosclerosis using apoE-Fc -...

  19. Human FcRn can mediate the transport across intestinal mucosal barrier and prolong the half-life of rabbit IgG in vivo

    Directory of Open Access Journals (Sweden)

    Guangchang Pang

    2015-06-01

    Full Text Available FcRn (neonatal Fc receptor plays an important role in IgG transportation, antigen presentation and signal transmission. In this study, the complement fixation test and flow cytometry test were performed to verify whether the heterologous antibody could be transmitted to the serum or leukocyte with FcγR (Fc gamma receptor across the intestinal mucosa. The results showed that rabbit anti-bovine IgG could be detected in both the serum and the leukocytes, which indicated that the heterologous antibody could transport across the intestinal mucosa to enter the blood and be effectively delivered to the leukocytes with FcγR. In addition, the results also showed that the rabbit anti-bovine IgG still could be detected in the leukocyte group (P=0.044<0.05 after 21 days. It indicated that the rabbit IgG could exist in the body for a long term (up to 21 days after being transported to the cells containing FcγR.

  20. Low-affinity FcγR interactions can decide the fate of novel human IgG-sensitised red blood cells and platelets.

    Science.gov (United States)

    Armour, Kathryn L; Smith, Cheryl S; Turner, Craig P; Kirton, Christopher M; Wilkes, Anthony M; Hadley, Andrew G; Ghevaert, Cedric; Williamson, Lorna M; Clark, Michael R

    2014-03-01

    G1Δnab is a mutant human IgG1 constant region with a lower ability to interact with FcγR than the natural IgG constant regions. Radiolabelled RBCs and platelets sensitised with specific G1Δnab Abs were cleared more slowly from human circulation than IgG1-sensitised counterparts. However, non-destructive splenic retention of G1Δnab-coated RBCs required investigation and plasma radioactivities now suggest this also occurred for platelets sensitised with an IgG1/G1Δnab mixture. In vitro assays with human cells showed that G1Δnab-sensitised RBCs did not cause FcγRI-mediated monocyte activation, FcγRIIIa-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) or macrophage phagocytosis although they did adhere to macrophages. Thus, FcγRII was implicated in the adhesion despite the Δnab mutation reducing the already low-affinity binding to this receptor class. Additional contacts via P-selectin enhance the interaction of sensitised platelets with monocytes and this system provided evidence of FcγRII-dependent activation by G1Δnab. These results emphasise the physiological relevance of low-affinity interactions: It appears that FcγRII interactions of G1Δnab allowed splenic retention of G1Δnab-coated RBCs with inhibitory FcγRIIb binding preventing RBC destruction and that FcγRIIb engagement by G1Δnab on IgG1/G1Δnab-sensitised platelets overcame activation by IgG1. Considering therapeutic blocking Abs, G1Δnab offers lower FcγR binding and a greater bias towards inhibition than IgG2 and IgG4 constant regions.

  1. Fc gamma receptor IIIb polymorphism and systemic lupus erythematosus: association with disease susceptibility and identification of a novel FCGR3B*01 variant.

    Science.gov (United States)

    Santos, V C; Grecco, M; Pereira, K M C; Terzian, C C N; Andrade, L E C; Silva, N P

    2016-10-01

    The objective of this study was to evaluate the association between Fc gamma receptor IIIb polymorphism and susceptibility to systemic lupus erythematosus and clinical traits of the disease. Genomic DNA was obtained from 303 consecutive systemic lupus erythematosus patients and 300 healthy blood donors from the southeastern region of Brazil. The polymorphic region of the FCGR3B gene was sequenced and the alleles FCGR3B*01, FCGR3B*02 and FCGR3B*03 were analyzed. The FCGR3B*01 allele was more frequent in systemic lupus erythematosus patients (43.1%) while the FCGR3B*02 allele prevailed among controls (63.7%) (P = 0.001). The FCGR3B*03 allele was found equally in both groups. The FCGR3B*01/*01 (20.7%) and FCGR3B*01/*02 (41.1%) genotypes were more frequent among systemic lupus erythematosus patients (P = 0.028 and P = 0.012, respectively) while the FCGR3B*02/*02 genotype was more frequent in controls (45.5%) (P systemic lupus erythematosus was associated with the FCGR3B*01 allele, as well as with the FCGR3B*01/*01 and FCGR3B*01/*02 genotypes. No association was found between FCGR3B genotypes and clinical manifestations, disease severity or the presence of autoantibodies. © The Author(s) 2016.

  2. An Fc Gamma Receptor-Mediated Upregulation of the Production of Interleukin 10 by Intravenous Immunoglobulin in Bone-Marrow-Derived Mouse Dendritic Cells Stimulated with Lipopolysaccharide In Vitro

    Directory of Open Access Journals (Sweden)

    Akihiro Fujii

    2013-01-01

    Full Text Available Intravenous immunoglobulin (IVIG, a highly purified immunoglobulin fraction prepared from pooled plasma of several thousand donors, increased anti-inflammatory cytokine IL-10 production, while decreased proinflammatory cytokine IL-12p70 production in bone-marrow-derived mouse dendritic cells (BMDCs stimulated with lipopolysaccharide (LPS. The changes of cytokine production were confirmed with the transcription levels of these cytokines. To study the mechanisms of this bidirectional effect, we investigated changes of intracellular molecules in the LPS-induced signaling pathway and observed that IVIG upregulated ERK1/2 phosphorylation while downregulated p38 MAPK phosphorylation. Using chemical inhibitors specific to protein kinases involved in activation of Fc gamma receptors (FcγRs, which mediate IgG signals, we found that hyperphosphorylation of ERK1/2 and Syk phosphorylation occurred after stimulation of BMDC with LPS and IVIG, and the increasing effect on IL-10 production was abolished by these inhibitors. Furthermore, an antibody specific to FcγRI, one of FcγRs involved in immune activation, inhibited IVIG-induced increases in IL-10 production, but not IL-12p70 decreases, whereas the anti-IL-10 antibody restored the decrease in IL-12p70 induced by IVIG. These findings suggest that IVIG induced the upregulation of IL-10 production through FcγRI activation, and IL-10 was indispensable to the suppressing effect of IVIG on the production of IL-12p70 in LPS-stimulated BMDC.

  3. Postneonatal Mortality and Liver Changes in Cloned Pigs Associated with Human Tumor Necrosis Factor Receptor I-Fc and Human Heme Oxygenase-1 Overexpression.

    Science.gov (United States)

    Kim, Geon A; Jin, Jun-Xue; Lee, Sanghoon; Taweechaipaisankul, Anukul; Oh, Hyun Ju; Hwang, Joing-Ik; Ahn, Curie; Saadeldin, Islam M; Lee, Byeong Chun

    2017-01-01

    Soluble human tumor necrosis factor (shTNFRI-Fc) and human heme oxygenase 1 (hHO-1) are key regulators for protection against oxidative and inflammatory injury for xenotransplantation. Somatic cells with more than 10 copy numbers of shTNFRI-Fc and hHO-1 were employed in somatic cell nuclear transfer to generate cloned pigs, thereby resulting in seven cloned piglets. However, produced piglets were all dead within 24 hours after birth. Obviously, postnatal death with liver apoptosis was reported in the higher copy number of shTNFRI-Fc and hHO-1 piglets. In liver, the transcript levels of ferritin heavy chain, light chain, transferrin, and inducible nitric oxide synthase were significantly highly expressed compared to those of lower copy number of shTNFRI-Fc and hHO-1 piglets (P hHO-1 piglets (P hHO-1 overexpression may apparently induce free iron in the liver and exert oxidative stress by enhancing reactive oxygen species production and block normal postneonatal liver metabolism.

  4. A recombinant mimetics of the HIV-1 gp41 prehairpin fusion intermediate fused with human IgG Fc fragment elicits neutralizing antibody response in the vaccinated mice

    Energy Technology Data Exchange (ETDEWEB)

    Qi, Zhi; Pan, Chungen; Lu, Hong; Shui, Yuan [Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10065 (United States); Li, Lin [Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10065 (United States); School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, Guangdong 510515 (China); Li, Xiaojuan; Xu, Xueqing; Liu, Shuwen [School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, Guangdong 510515 (China); Jiang, Shibo, E-mail: sjiang@nybloodcenter.org [Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10065 (United States); School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, Guangdong 510515 (China)

    2010-07-30

    Research highlights: {yields} One recombinant mimetics of gp41 prehairpin fusion intermediate (PFI) consisting of gp41 N46 sequence, foldon and IgG Fc, designated N46FdFc, was expressed. {yields} N46FdFc-induced antibodies in mice that neutralized HIV-1 infection, inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. {yields} These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines. -- Abstract: HIV-1 gp41 prehairpin fusion intermediate (PFI) composed of three N-terminal heptad repeats (NHR) plays a crucial role in viral fusion and entry and represents an attractive target for anti-HIV therapeutics (e.g., enfuvirtide) and vaccines. In present study, we constructed and expressed two recombinant gp41 PFI mimetics, designated N46Fd and N46FdFc. N46Fd consists of N46 (residues 536-581) in gp41 NHR and foldon (Fd), a trimerization motif. N46FdFc is composed of N46Fd fused with human IgG Fc fragment as an immunoenhancer. We immunized mice with N46 peptide, N46Fd and N46FdFc, respectively, and found that only N46FdFc elicited neutralizing antibody response in mice against infection by HIV-1 strains IIIB (clade B, X4), 92US657 (clade B, R5), and 94UG103 (clade A, X4R5). Anti-N46FdFc antibodies inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines.

  5. Fc gamma receptor gene polymorphisms in Japanese patients with systemic lupus erythematosus - Contribution of FCGR2B to genetic susceptibility

    NARCIS (Netherlands)

    Kyogoku, C; Dijstelbloem, HM; Tsuchiya, N; Hatta, Y; Kato, H; Yamaguchi, A; Fukazawa, T; Jansen, MD; Hashimoto, H; van de Winkel, JGJ; Kallenberg, CGM; Tokunaga, K

    Objective. Human low-affinity Fcgamma receptors (FcgammaR) constitute a clustered gene family located on chromosome 1q23, that consists of FcgammaRIIA, IIB, IIC, IIIA, and IIIB genes. FcgammaRIIB is unique in its ability to transmit inhibitory signals, and recent animal studies demonstrated a role

  6. Ligand binding and antigenic properties of a human neonatal Fc receptor with mutation of two unpaired cysteine residues

    DEFF Research Database (Denmark)

    Andersen, Jan T; Justesen, Sune; Fleckenstein, Burkhard

    2008-01-01

    The neonatal Fc receptor (FcRn) is a major histocompatibility complex class I-related molecule that regulates the half-life of IgG and albumin. In addition, FcRn directs the transport of IgG across both mucosal epithelium and placenta and also enhances phagocytosis in neutrophils. This new knowle...

  7. Is Fc gamma receptor IIA (FcγRIIA) polymorphism associated with clinical malaria and Plasmodium falciparum specific antibody levels in children from Burkina Faso?

    DEFF Research Database (Denmark)

    Cherif, Mariama K; Sanou, Guillaume S; Bougouma, Edith C;

    2015-01-01

    In the present study, the influences of FcγRIIA polymorphism on susceptibility to malaria and antibody responses to Plasmodium falciparum antigens were analyzed in children. We recruited 96 healthy children between 3 and 10 years at the beginning of the high transmission season and we followed up...

  8. Differential sensitivity of Chironomus and human hemoglobin to gamma radiation

    Energy Technology Data Exchange (ETDEWEB)

    Gaikwad, Pallavi S. [Stress Biology Research Laboratory, Department of Zoology, Savitribai Phule University, Pune, 411007 (India); Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai, 400085 (India); Panicker, Lata [Solid State Physics Division, Bhabha Atomic Research Centre, Trombay, Mumbai, 400085 (India); Mohole, Madhura; Sawant, Sangeeta [Bioinformatics Center, Savitribai Phule Pune University, Pune, 411007 (India); Mukhopadhyaya, Rita [Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai, 400085 (India); Nath, Bimalendu B., E-mail: bbnath@gmail.com [Stress Biology Research Laboratory, Department of Zoology, Savitribai Phule University, Pune, 411007 (India)

    2016-08-05

    Chironomus ramosus is known to tolerate high doses of gamma radiation exposure. Larvae of this insect possess more than 95% of hemoglobin (Hb) in its circulatory hemolymph. This is a comparative study to see effect of gamma radiation on Hb of Chironomus and humans, two evolutionarily diverse organisms one having extracellular and the other intracellular Hb respectively. Stability and integrity of Chironomus and human Hb to gamma radiation was compared using biophysical techniques like Dynamic Light Scattering (DLS), UV-visible spectroscopy, fluorescence spectrometry and CD spectroscopy after exposure of whole larvae, larval hemolymph, human peripheral blood, purified Chironomus and human Hb. Sequence- and structure-based bioinformatics methods were used to analyze the sequence and structural similarities or differences in the heme pockets of respective Hbs. Resistivity of Chironomus Hb to gamma radiation is remarkably higher than human Hb. Human Hb exhibited loss of heme iron at a relatively low dose of gamma radiation exposure as compared to Chironomus Hb. Unlike human Hb, the heme pocket of Chironomus Hb is rich in aromatic amino acids. Higher hydophobicity around heme pocket confers stability of Chironomus Hb compared to human Hb. Previously reported gamma radiation tolerance of Chironomus can be largely attributed to its evolutionarily ancient form of extracellular Hb as evident from the present study. -- Highlights: •Comparison of radiation tolerant Chironomus Hb and radiation sensitive Human Hb. •Amino acid composition of midge and human heme confer differential hydrophobicity. •Heme pocket of evolutionarily ancient midge Hb provide gamma radiation resistivity.

  9. Structure of the human G gamma-A gamma-delta-beta-globin gene locus

    NARCIS (Netherlands)

    Bernards, R.A.; Little, P.F.R.; Annison, F.; Williamson, R.; Flavell, R.A.

    1979-01-01

    We have constructed a physical map of the human G gamma-, A gamma-, delta-, and beta-globin genes. The previously described maps of the fetal and adult beta-like globin genes have been linked to one another by identification of a DNA fragment, generated by BamHI, that contains part of each of the A

  10. Electrophoretic separation of A gamma and G gamma human globin chains in Nonidet P-40.

    Science.gov (United States)

    Guerrasio, A; Saglio, G; Mazza, U; Pich, P; Camaschella, C; Ricco, G; Gianazza, E; Righetti, P G

    1979-11-15

    Electrophoresis in cellulose acetate in the presence of 3% Nonidet P-40 can resolve two neutral genetic variants, A gamma and G gamma human fetal globin chains. The ratio of these two chains, determined by densitometry of the electrophoretic strips, is in excellent agreement with the Gly-Ala ratio obtained by chemical analysis of the cyanogen bromide fragment gamma CB3. It is suggested that the detergent binds preferentially to the hydrophobic amino acid segment 133-141 in the A gamma chain, thus masking either a Lys or an Arg residue at the two extremes.

  11. Fusion of Na-ASP-2 with human immunoglobulin Fcγ abrogates histamine release from basophils sensitized with anti-Na-ASP-2 IgE.

    Science.gov (United States)

    Zhan, Bin; Santiago, H; Keegan, B; Gillespie, P; Xue, J; Bethony, J; de Oliveira, L M; Jiang, D; Diemert, D; Xiao, S-H; Jones, K; Feng, X; Hotez, P J; Bottazzi, M E

    2012-01-01

    Na-ASP-2 is a major protein secreted by infective third-stage larvae (L3) of the human hookworm Necator americanus upon host entry. It was chosen as a lead vaccine candidate for its ability to elicit protective immune responses. However, clinical development of this antigen as a recombinant vaccine was halted because it caused allergic reactions among some of human volunteers previously infected with N. americanus. To prevent IgE-mediated allergic reactions induced by Na-ASP-2 but keep its immunogenicity as a vaccine antigen, we designed and tested a genetically engineered fusion protein, Fcγ/Na-ASP-2, composed of full-length Na-ASP-2 and truncated human IgG Fcγ1 that targets the negative signalling receptor FcγRIIb expressed on pro-allergic cells. The chimeric recombinant Fcγ/Na-ASP-2 protein was expressed in Pichia pastoris and shared the similar antigenicity as native Na-ASP-2. Compared to Na-ASP-2, the chimeric fusion protein efficiently reduced the release of histamine in human basophils sensitized with anti-Na-ASP-2 IgE obtained from individuals living in a hookworm-endemic area. In dogs infected with canine hookworm, Fcγ/Na-ASP-2 resulted in significantly reduced immediate-type skin reactivity when injected intradermally compared with Na-ASP-2. Hamsters vaccinated with Fcγ/Na-ASP-2 formulated with Alhydrogel(®) produced specific IgG that recognized Na-ASP-2 and elicited similar protection level against N. americanus L3 challenge as native Na-ASP-2. © 2012 Blackwell Publishing Ltd.

  12. Efficient production and purification of extracellular domain of human FGFR-Fc fusion proteins from Chinese hamster ovary cells.

    Science.gov (United States)

    Sokolowska-Wedzina, Aleksandra; Borek, Aleksandra; Chudzian, Julia; Jakimowicz, Piotr; Zakrzewska, Malgorzata; Otlewski, Jacek

    2014-07-01

    The family of fibroblast growth factor receptors (FGFRs) plays an important role in cell growth, survival, differentiation and angiogenesis. The three immunoglobulin-like extracellular domains of FGFR (D1, D2, and D3) are critical for ligand binding and specificity towards fibroblast growth factor and heparan sulfate. Fibroblast growth factor receptors are overexpressed in a wide variety of tumors, such as breast, bladder, and prostate cancer, and therefore they are attractive targets for different types of anticancer therapies. In this study, we have cloned, expressed in CHO cells and purified Fc-fused extracellular domains of different types of FGFRs (ECD_FGFR1a-Fc, ECD_FGFR1b-Fc, ECD_FGFR2a-Fc, ECD_FGFR2b-Fc, ECD_FGFR3a-Fc, ECD_FGFR3b-Fc, ECD_FGFR4a-Fc, ECD_FGFR4b-Fc), which could be used as molecular targets for the selection of specific antibodies. The fusion proteins were analyzed using gel electrophoresis, Western blotting and mass spectrometry. To facilitate their full characterization, the fusion proteins were deglycosylated using PNGase F enzyme. With an optimized transient transfection protocol and purification procedure we were able to express the proteins at a high level and purify them to homogeneity.

  13. Prokaryotic expression of recombinant human p75NTR-Fc fusion protein and its effect on the neurite outgrowth of dorsal root ganglia neuron

    Institute of Scientific and Technical Information of China (English)

    Zhu Feng; Wang Yongtang; Lu Xiumin; Zeng Lin; Wu Yamin

    2009-01-01

    Objective: To clone, express, and identify the extracellular domain gene of human p75 neurotrophin receptor with IgG-Fc (hp75NTR-Fc) in prokaryotic expression system, and investigate the effect of the recombinant protein on dorsal root ganglia (DRG) neuron neurites. Methods: The hp75NTR-Fc coding sequence was amplified from pcDNA-hp75NTR-Fc by polymerase chain reaction (PCR) and subcloned into vector pET30a (+), in which hp75NTR-Fc expression was controlled under the T7 promoter. The recombinant vectors were amplified in E. coli DH5a and identified by PCR, enzyme digestion and sequencing, and then transformed into E. coli BL21 (DE3). The expression product was analyzed with SDS-PAGE and Western blot. Then after the recombinant protein purified with Protein A affinity chromatograph, and renaturated with dialysis, respectively, the effect of the recombinant protein on DRG neuron neuritis was further investigated. Results: The results of PCR, enzyme digestion, and sequencing demonstrated the success of inserting the hp75NTR-Fc fragment into vector pET30a (+). SDS-PAGE and Western blot showed a positive protein band with molecular weight about 50 kD in the expression product, which is accordant with the interest protein, and this band could be specifically recognized by rabbit anti-NGFRp75 antibody. The purified infusion protein following dialysis could promote neurite outgrowth of DRG neurons cultured with myelin-associated glycoprotein (MAG). Conclusion: The hp75NTR-Fc coding sequence was subcloned into the expression vector pET30a (+) correctly and expressed successfully in the prokaryotic expression system. The infusion protein could promote neurite outgrowth of DRG neurons cultured with MAG.

  14. Characterization of the promoter of the human gene encoding the high-affinity IgG receptor: Transcriptional induction by. gamma. -interferon is mediated through common DNA response elements

    Energy Technology Data Exchange (ETDEWEB)

    Pearse, R.N.; Feinman, R.; Ravetch, J.V. (DeWitt Wallace Research Lab., New York, NY (United States))

    1991-12-15

    Expression of the high-affinity receptor for IgG (Fc{sub {gamma}}RI) is restricted to cells of myeloid lineage and is induced by {gamma}-interferon (IFN-{gamma}) but not by IFN-{alpha}/{beta}. The organization of the human Fc{sub {gamma}}RI gene has been determined and the DNA elements governing its cell type-restricted transcription and IFN-{gamma} induction are reported here. A 39-nucleotide sequence (IFN-{gamma} response region, or GRR) is defined that is both necessary and sufficient for IFN-{gamma} inducibility. Sequence analysis of the GRR reveals the presence of promoter elements initially defined for the major histocompatibility complex class 2 genes: i.e., X, H, and {gamma}-IRE sequences. Comparison of a number of genes whose expression is induced selectively by IFN-{gamma} indicated that the presence of these elements is a general feature of IFN-{gamma}-responsive genes. The studies suggest that the combination of X, H, and {gamma}-IRE elements is a common motif in the pathway of transcriptional induction by this lymphokine.

  15. Differential sensitivity of Chironomus and human hemoglobin to gamma radiation.

    Science.gov (United States)

    Gaikwad, Pallavi S; Panicker, Lata; Mohole, Madhura; Sawant, Sangeeta; Mukhopadhyaya, Rita; Nath, Bimalendu B

    2016-08-05

    Chironomus ramosus is known to tolerate high doses of gamma radiation exposure. Larvae of this insect possess more than 95% of hemoglobin (Hb) in its circulatory hemolymph. This is a comparative study to see effect of gamma radiation on Hb of Chironomus and humans, two evolutionarily diverse organisms one having extracellular and the other intracellular Hb respectively. Stability and integrity of Chironomus and human Hb to gamma radiation was compared using biophysical techniques like Dynamic Light Scattering (DLS), UV-visible spectroscopy, fluorescence spectrometry and CD spectroscopy after exposure of whole larvae, larval hemolymph, human peripheral blood, purified Chironomus and human Hb. Sequence- and structure-based bioinformatics methods were used to analyze the sequence and structural similarities or differences in the heme pockets of respective Hbs. Resistivity of Chironomus Hb to gamma radiation is remarkably higher than human Hb. Human Hb exhibited loss of heme iron at a relatively low dose of gamma radiation exposure as compared to Chironomus Hb. Unlike human Hb, the heme pocket of Chironomus Hb is rich in aromatic amino acids. Higher hydophobicity around heme pocket confers stability of Chironomus Hb compared to human Hb. Previously reported gamma radiation tolerance of Chironomus can be largely attributed to its evolutionarily ancient form of extracellular Hb as evident from the present study.

  16. Peptide mimetics of immunoglobulin A (IgA) and FcαRI block IgA-induced human neutrophil activation and migration.

    Science.gov (United States)

    Heineke, Marieke H; van der Steen, Lydia P E; Korthouwer, Rianne M; Hage, J Joris; Langedijk, Johannes P M; Benschop, Joris J; Bakema, Jantine E; Slootstra, Jerry W; van Egmond, Marjolein

    2017-07-24

    The cross-linking of the IgA Fc receptor (FcαRI) by IgA induces release of the chemoattractant LTB4, thereby recruiting neutrophils in a positive feedback loop. IgA autoantibodies of patients with autoimmune blistering skin diseases therefore induce massive recruitment of neutrophils, resulting in severe tissue damage. To interfere with neutrophil mobilization and reduce disease morbidity, we developed a panel of specific peptides mimicking either IgA or FcαRI sequences. CLIPS technology was used to stabilize three-dimensional structures and to increase peptides' half-life. IgA and FcαRI peptides reduced phagocytosis of IgA-coated beads, as well as IgA-induced ROS production and neutrophil migration in in vitro and ex vivo (human skin) experiments. Since topical application would be the preferential route of administration, Cetomacrogol cream containing an IgA CLIPS peptide was developed. In the presence of a skin permeation enhancer, peptides in this cream were shown to penetrate the skin, while not diffusing systemically. Finally, epitope mapping was used to discover sequences important for binding between IgA and FcαRI. In conclusion, a cream containing IgA or FcαRI peptide mimetics, which block IgA-induced neutrophil activation and migration in the skin may have therapeutic potential for patients with IgA-mediated blistering skin diseases. © 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Association of low-affinity FC gamma receptor 3B (FCGR3B) copy number variation with rheumatoid arthritis in Caucasian subjects

    NARCIS (Netherlands)

    Merriman, T.R.; Fanciulli, M.; Merriman, M.E.; Alizadeh, B.Z.; Koeleman, B.P.C.; Dalbeth, N.; Gow, P.; Harrison, A.A.; Highton, J.; Jones, P.B.; Stamp, L.K.; Steer, S.; Barrera, P.; Coenen, M.J.H.; Franke, B.; Vyse, T.; Aitman, T.; Radstake, T.; McKinney, C.

    2009-01-01

    Aim: There is increasing evidence that gene copy-number variation influences phenotypic variation. The low-affinity Fc receptor 3B (FCGR3B) is a copy-number polymorphic gene involved in the recruitment to sites of inflammation and activation of polymorphonuclear neutrophils (PMN). Given the importan

  18. Copy number variation of Fc gamma receptor genes in HIV-infected and HIV-tuberculosis co-infected individuals in sub-Saharan Africa.

    Directory of Open Access Journals (Sweden)

    Lee R Machado

    Full Text Available AIDS, caused by the retrovirus HIV, remains the largest cause of morbidity in sub-Saharan Africa yet almost all genetic studies have focused on cohorts from Western countries. HIV shows high co-morbidity with tuberculosis (TB, as HIV stimulates the reactivation of latent tuberculosis (TB. Recent clinical trials suggest that an effective anti-HIV response correlates with non-neutralising antibodies. Given that Fcγ receptors are critical in mediating the non-neutralising effects of antibodies, analysis of the extensive variation at Fcγ receptor genes is important. Single nucleotide variation and copy number variation (CNV of Fcγ receptor genes affects the expression profile, activatory/inhibitory balance, and IgG affinity of the Fcγ receptor repertoire of each individual. In this study we investigated whether CNV of FCGR2C, FCGR3A and FCGR3B as well as the HNA1 allotype of FCGR3B is associated with HIV load, response to highly-active antiretroviral therapy (HAART and co-infection with TB. We confirmed an effect of TB-co-infection status on HIV load and response to HAART, but no conclusive effect of the genetic variants we tested. We observed a small effect, in Ethiopians, of FCGR3B copy number, where deletion was more frequent in HIV-TB co-infected patients than those infected with HIV alone.

  19. Receptor modulation by Fc gamma RI-specific fusion proteins is dependent on receptor number and modified by IgG.

    Science.gov (United States)

    Guyre, C A; Keler, T; Swink, S L; Vitale, L A; Graziano, R F; Fanger, M W

    2001-12-01

    The high-affinity IgG receptor, FcgammaRI (CD64), is constitutively expressed exclusively on professional APCs. Human FcgammaRI binds monomeric IgG with high affinity and is, therefore, saturated in vivo. The binding of IgG to FcgammaRI causes receptor recycling, while Abs that cross-link FcgammaRI cause rapid down-modulation of surface FcgammaRI. Because studies performed in the absence of ligand may not be representative of FcgammaRI modulation in vivo, we investigated the ability of FcgammaRI-cross-linking Abs and non-cross-linking derivatives to modulate FcgammaRI in the presence and absence of ligand. In the absence of ligand mAb H22 and wH22xeGFP, an enhanced green fluorescent protein (eGFP)-labeled fusion protein of H22, cross-linked and rapidly down-modulated surface FcgammaRI on the human myeloid cell line, U937, and its high FcgammaRI-expressing subclone, 10.6. This effect was dependent on the concentration of fusion protein and the level of FcgammaRI expression and correlated with internalization of both wH22xeGFP and FcgammaRI, itself, as assessed by confocal microscopy. A single-chain Fv version, sFv22xeGFP, which does not cross-link FcgammaRI, was unable to modulate FcgammaRI in the absence of IgG. However, if ligand was present, treatment with either monovalent or cross-linking fusion protein led to intracellular receptor accumulation. These findings suggest at least two alternate mechanisms of internalization that are influenced by ligand and demonstrate the physiologic potential of FcgammaRI to transport a large antigenic load into APCs for processing. These studies may lead to the development of better FcgammaRI-targeted vaccines, as well as therapies to down-modulate FcR involved in autoimmune diseases.

  20. Contribution of Fcγ receptors to human respiratory syncytial virus pathogenesis and the impairment of T-cell activation by dendritic cells.

    Science.gov (United States)

    Gómez, Roberto S; Ramirez, Bruno A; Céspedes, Pablo F; Cautivo, Kelly M; Riquelme, Sebastián A; Prado, Carolina E; González, Pablo A; Kalergis, Alexis M

    2016-01-01

    Human respiratory syncytial virus (hRSV) is the leading cause of infant hospitalization related to respiratory disease. Infection with hRSV produces abundant infiltration of immune cells into the airways, which combined with an exacerbated pro-inflammatory immune response can lead to significant damage to the lungs. Human RSV re-infection is extremely frequent, suggesting that this virus may have evolved molecular mechanisms that interfere with host adaptive immunity. Infection with hRSV can be reduced by administering a humanized neutralizing antibody against the virus fusion protein in high-risk infants. Although neutralizing antibodies against hRSV effectively block the infection of airway epithelial cells, here we show that both, bone marrow-derived dendritic cells (DCs) and lung DCs undergo infection with IgG-coated virus (hRSV-IC), albeit abortive. Yet, this is enough to negatively modulate DC function. We observed that such a process is mediated by Fcγ receptors (FcγRs) expressed on the surface of DCs. Remarkably, we also observed that in the absence of hRSV-specific antibodies FcγRIII knockout mice displayed significantly less cellular infiltration in the lungs after hRSV infection, compared with wild-type mice, suggesting a potentially harmful, IgG-independent role for this receptor in hRSV disease. Our findings support the notion that FcγRs can contribute significantly to the modulation of DC function by hRSV and hRSV-IC. Further, we provide evidence for an involvement of FcγRIII in the development of hRSV pathogenesis.

  1. New insights in the structure and biology of the high affinity receptor for IgE (Fc epsilon RI) on human epidermal Langerhans cells.

    Science.gov (United States)

    Bieber, T; Kraft, S; Jürgens, M; Strobel, I; Haberstok, J; Tomov, H; Regele, D; de la Salle, H; Wollenberg, A; Hanau, D

    1996-10-01

    The recent structural and functional analysis of the high affinity receptor for IgE (Fc epsilon RI) expressed on human epidermal Langerhans cells (LC) revealed new aspects of the biology of this structure. In contrast to basophils and mast cells where this receptor seems to be expressed constitutively at a constant level, the expression of Fc epsilon RI on LC varies on the donor and the inflammatory environment of the cells and lacks the classical beta-chain. This also implies functional differences most probably related to the expression level. Although the signalling pathway seems to be similar to that of basophils or mast cells, LC from individuals with atopic dermatitis are fully activated by receptor ligation while LC from normal individuals fail to exhibit calcium mobilization under the same conditions. Finally, LC from normal and atopic individuals use Fc epsilon RI to maximize antigen uptake via specific IgE and subsequent presentation to T cells. Thus, Fc epsilon RI expressed on LC differs in terms of structure and function from that expressed on effector cells of anaphylaxis.

  2. Increment in Drug Loading on an Antibody-Drug Conjugate Increases Its Binding to the Human Neonatal Fc Receptor in Vitro.

    Science.gov (United States)

    Brachet, Guillaume; Respaud, Renaud; Arnoult, Christophe; Henriquet, Corinne; Dhommée, Christine; Viaud-Massuard, Marie-Claude; Heuze-Vourc'h, Nathalie; Joubert, Nicolas; Pugnière, Martine; Gouilleux-Gruart, Valérie

    2016-04-04

    Antibody-drug conjugates, such as brentuximab vedotin (BTXv), are an innovative category of monoclonal antibodies. BTXv is bioconjugated via the chemical reduction of cysteine residues involved in disulfide bonds. Species of BTXv containing zero, two, four, six, or eight vedotin molecules per antibody coexist in the stock solution. We investigated the influence of drug loading on the binding of the antibody to FcRn, a major determinant of antibody pharmacokinetics in humans. We developed a hydrophobic interaction chromatography (HIC) method for separating the different species present in the stock solution of BTXv, and we purified and characterized the collected species before use. We assessed the binding of these different species to FcRn in a cellular assay based on flow cytometry and surface plasmon resonance. HIC separated the different species of BTXv and allowed their collection at adequate levels of purity. Physicochemical characterization showed that species with higher levels of drug loading tended to form more aggregates. FcRn binding assays showed that the most conjugated species, particularly those with saturated loading, interacted more strongly than unconjugated BTXv with the FcRn.

  3. Humanized mAb H22 binds the human high affinity Fc receptor for IgG (FcgammaRI), blocks phagocytosis, and modulates receptor expression.

    Science.gov (United States)

    Wallace, P K; Keler, T; Coleman, K; Fisher, J; Vitale, L; Graziano, R F; Guyre, P M; Fanger, M W

    1997-10-01

    About 10-15% of patients with immune thrombocytopenic purpura (ITP) cannot be controlled by corticosteroid therapy and splenectomy. For these patients treatment with high-dose IVIgG induces partial or complete responses. The clinical benefits of IVIgG could be due to blockade of Fc receptors for IgG (FcgammaR), because several model systems clearly show that functional FcgammaR are essential for establishment of ITP and related diseases. However, the specific contributions of the three individual classes of FcgammaR remain to be more completely defined. Recently monoclonal antibody (mAb) H22, which recognizes an epitope on FcgammaRI (CD64) outside the ligand binding domain, was humanized by grafting its complementarity determining regions onto human IgG1 constant domains. Because FcgammaRI has a high affinity for human IgG1 antibodies, we predicted mAb H22 would also bind to FcgammaRI through its Fc domain and block FcgammaRI-mediated phagocytosis. These studies demonstrate that mAb H22 blocked phagocytosis of opsonized red blood cells 1000 times more effectively than an irrelevant IgG. Moreover, cross-linking FcgammaRI with mAb H22 rapidly down-modulated FcgammaRI expression on monocytes without affecting other surface antigens. We conclude that because mAb H22 is a humanized mAb that blocks the FcgammaRI ligand binding domain and down-modulates FcgammaRI expression, it is a particularly good candidate for evaluating the role of FcgammaRI in patients with ITP.

  4. Lin- CD34hi CD117int/hi FcεRI+ cells in human blood constitute a rare population of mast cell progenitors.

    Science.gov (United States)

    Dahlin, Joakim S; Malinovschi, Andrei; Öhrvik, Helena; Sandelin, Martin; Janson, Christer; Alving, Kjell; Hallgren, Jenny

    2016-01-28

    Mast cells are rare tissue-resident immune cells that are involved in allergic reactions, and their numbers are increased in the lungs of asthmatics. Murine lung mast cells arise from committed bone marrow-derived progenitors that enter the blood circulation, migrate through the pulmonary endothelium, and mature in the tissue. In humans, mast cells can be cultured from multipotent CD34(+) progenitor cells. However, a population of distinct precursor cells that give rise to mast cells has remained undiscovered. To our knowledge, this is the first report of human lineage-negative (Lin(-)) CD34(hi) CD117(int/hi) FcεRI(+) progenitor cells, which represented only 0.0053% of the isolated blood cells in healthy individuals. These cells expressed integrin β7 and developed a mast cell-like phenotype, although with a slow cell division capacity in vitro. Isolated Lin(-) CD34(hi) CD117(int/hi) FcεRI(+) blood cells had an immature mast cell-like appearance and expressed high levels of many mast cell-related genes as compared with human blood basophils in whole-transcriptome microarray analyses. Furthermore, serglycin, tryptase, and carboxypeptidase A messenger RNA transcripts were detected by quantitative reverse transcription-polymerase chain reaction. Altogether, we propose that the Lin(-) CD34(hi) CD117(int/hi) FcεRI(+) blood cells are closely related to human tissue mast cells and likely constitute an immediate precursor population, which can give rise to predominantly mast cells. Furthermore, asthmatics with reduced lung function had a higher frequency of Lin(-) CD34(hi) CD117(int/hi) FcεRI(+) blood mast cell progenitors than asthmatics with normal lung function.

  5. Stimulus Dependence of Gamma Oscillations in Human Visual Cortex.

    Science.gov (United States)

    Hermes, D; Miller, K J; Wandell, B A; Winawer, J

    2015-09-01

    A striking feature of some field potential recordings in visual cortex is a rhythmic oscillation within the gamma band (30-80 Hz). These oscillations have been proposed to underlie computations in perception, attention, and information transmission. Recent studies of cortical field potentials, including human electrocorticography (ECoG), have emphasized another signal within the gamma band, a nonoscillatory, broadband signal, spanning 80-200 Hz. It remains unclear under what conditions gamma oscillations are elicited in visual cortex, whether they are necessary and ubiquitous in visual encoding, and what relationship they have to nonoscillatory, broadband field potentials. We demonstrate that ECoG responses in human visual cortex (V1/V2/V3) can include robust narrowband gamma oscillations, and that these oscillations are reliably elicited by some spatial contrast patterns (luminance gratings) but not by others (noise patterns and many natural images). The gamma oscillations can be conspicuous and robust, but because they are absent for many stimuli, which observers can see and recognize, the oscillations are not necessary for seeing. In contrast, all visual stimuli induced broadband spectral changes in ECoG responses. Asynchronous neural signals in visual cortex, reflected in the broadband ECoG response, can support transmission of information for perception and recognition in the absence of pronounced gamma oscillations.

  6. Subthreshold desensitization of human basophils re-capitulates the loss of Syk and FcεRI expression characterized by other methods of desensitization.

    Science.gov (United States)

    MacGlashan, D

    2012-07-01

    Clinical desensitization of patients to drugs involves progressive exposure to escalating doses of drug over a period of 24 h. In prior studies, this method was re-capitulated in vitro to also demonstrate loss of mast cell or basophil responsiveness. However, most signalling studies of human basophils have identified changes in signalling by using other methods of inducing cellular desensitization. This study examined two well-described endpoints of basophil desensitization, loss of syk or FcεRI expression, under conditions of subthreshold desensitization. The loss of FcεRI and syk was examined in human basophils. It was shown that both loss of syk and FcεRI/IgE occurred during an escalating series of stimulation (anti-IgE Ab) and that expression loss occurred despite the presence of little histamine release. If basophils were first cultured for 3 days in 10 ng/mL IL-3, the concentration-dependence of histamine release shifted to 100-fold lower concentrations of stimulus. However, loss of syk did not show any change in its EC50 while loss of FcεRI also shifted 100-fold. From the perspective of early signal element activation, the marked shift in the EC50 for histamine release was not accompanied by similar shifts in the EC50s for several signalling elements. The EC50s for phospho-Src, phospho-SHIP1, phospho-Syk, or phospho-Cbl did not change while the EC50s for phospho-Erk and the cytosolic calcium response did shift 100-fold. These studies show that under normal conditions, subthreshold desensitization leads to loss of two critical signalling molecules (FcεRI and syk) but under at least one condition, treatment with IL-3, it is possible to markedly blunt the loss of syk, but not FcεRI, while executing a proper subthreshold titration. These data also suggest that IL-3 modifies only the sensitivity of signalling elements that are downstream of syk activation. © 2012 Blackwell Publishing Ltd.

  7. Fc gamma receptor IIa-H131R polymorphism and malaria susceptibility in sympatric ethnic groups, Fulani and Dogon of Mali.

    Science.gov (United States)

    Maiga, B; Dolo, A; Touré, O; Dara, V; Tapily, A; Campino, S; Sepulveda, N; Corran, P; Rockett, K; Clark, T G; Blomberg, M Troye; Doumbo, O K

    2014-01-01

    It has been previously shown that there are some interethnic differences in susceptibility to malaria between two sympatric ethnic groups of Mali, the Fulani and the Dogon. The lower susceptibility to Plasmodium falciparum malaria seen in the Fulani has not been fully explained by genetic polymorphisms previously known to be associated with malaria resistance, including haemoglobin S (HbS), haemoglobin C (HbC), alpha-thalassaemia and glucose-6-phosphate dehydrogenase (G6PD) deficiency. Given the observed differences in the distribution of FcγRIIa allotypes among different ethnic groups and with malaria susceptibility that have been reported, we analysed the rs1801274-R131H polymorphism in the FcγRIIa gene in a study of Dogon and Fulani in Mali (n = 939). We confirm that the Fulani have less parasite densities, less parasite prevalence, more spleen enlargement and higher levels of total IgG antibodies (anti-CSP, anti-AMA1, anti-MSP1 and anti-MSP2) and more total IgE (P Dogon ethnic group. Furthermore, the Fulani exhibit higher frequencies of the blood group O (56.5%) compared with the Dogon (43.5%) (P Dogon 0.341, P < 0.0001), which was associated with greater total IgE production (P = 0.004). Our findings show that the FcγRIIa polymorphism might have an implication in the relative protection seen in the Fulani tribe, with confirmatory studies required in other malaria endemic settings. © 2013 The Authors. Scandinavian Journal of Immunology published by John Wiley & Sons Ltd on behalf of Scandanavian Society of Immunology (SSI).

  8. Importance of the Side Chain at Position 296 of Antibody Fc in Interactions with FcγRIIIa and Other Fcγ Receptors.

    Directory of Open Access Journals (Sweden)

    Yuya Isoda

    Full Text Available Antibody-dependent cellular cytotoxicity (ADCC is an important effector function determining the clinical efficacy of therapeutic antibodies. Core fucose removal from N-glycans on the Fc portion of immunoglobulin G (IgG improves the binding affinity for Fcγ receptor IIIa (FcγRIIIa and dramatically enhances ADCC. Our previous structural analyses revealed that Tyr-296 of IgG1-Fc plays a critical role in the interaction with FcγRIIIa, particularly in the enhanced FcγRIIIa binding of nonfucosylated IgG1. However, the importance of the Tyr-296 residue in the antibody in the interaction with various Fcγ receptors has not yet been elucidated. To further clarify the biological importance of this residue, we established comprehensive Tyr-296 mutants as fucosylated and nonfucosylated anti-CD20 IgG1s rituximab variants and examined their binding to recombinant soluble human Fcγ receptors: shFcγRI, shFcγRIIa, shFcγRIIIa, and shFcγRIIIb. Some of the mutations affected the binding of antibody to not only shFcγRIIIa but also shFcγRIIa and shFcγRIIIb, suggesting that the Tyr-296 residue in the antibody was also involved in interactions with FcγRIIa and FcγRIIIb. For FcγRIIIa binding, almost all Tyr-296 variants showed lower binding affinities than the wild-type antibody, irrespective of their core fucosylation, particularly in Y296K and Y296P. Notably, only the Y296W mutant showed improved binding to FcγRIIIa. The 3.00 Å-resolution crystal structure of the nonfucosylated Y296W mutant in complex with shFcγRIIIa harboring two N-glycans revealed that the Tyr-to-Trp substitution increased the number of potential contact atoms in the complex, thus improving the binding of the antibody to shFcγRIIIa. The nonfucosylated Y296W mutant retained high ADCC activity, relative to the nonfucosylated wild-type IgG1, and showed greater binding affinity for FcγRIIa. Our data may improve our understanding of the biological importance of human IgG1-Fc Tyr-296

  9. Expression of HERV-Fc1, a human endogenous retrovirus, is increased in patients with active Multiple Sclerosis

    DEFF Research Database (Denmark)

    Laska, Magdalena Janina; Brudek, Tomasz; Nissen, Kari Konstantin

    2012-01-01

    been detected as transcripts and proteins in the central nervous system (CNS) and peripheral blood, frequently in the context of neuroinflammation. HERV-Fc1, which belongs to the HERV-H/F family, has received our attention largely because of the genetic association with MS. We studied the expression...

  10. Modification of the Fc Region of a Human Anti-oncostatin M Monoclonal Antibody for Higher Affinity to FcRn Receptor and Extension of Half-life in Cynomolgus Monkeys.

    Science.gov (United States)

    Nnane, Ivo P; Han, Chao; Jiao, Qun; Tam, Susan H; Davis, Hugh M; Xu, Zhenhua

    2017-01-28

    The purpose of this study was to evaluate the pharmacokinetics (PK) of anti-oncostatin M (OSM) IgG1 monoclonal antibodies, CNTO 1119 and its Fc variant (CNTO 8212), which incorporates the LS(Xtend) mutation to extend terminal half-life (T1/2 ), after a single intravenous (IV) or subcutaneous (SC) administration in cynomolgus monkeys, and to predict human PK. In study 1, single doses of CNTO 1119 and CNTO 8212 were administered IV or SC at 3 mg/kg to cynomolgus monkeys (n = 3 per group). In study 2, single doses of CNTO 8212 were administered IV at 1, 5 or 20 mg/kg, or SC at 5 mg/kg to cynomolgus monkeys (n = 5 per group). Serial blood samples were collected for assessment of serum concentrations of CNTO 1119 and/or CNTO 8212. A two-compartment population PK model with first-order elimination was utilized to simultaneously describe the serum concentrations of CNTO 1119 and CNTO 8212 over time after IV and SC administration in cynomolgus monkeys. The typical population PK parameter estimates for CNTO 1119 in cynomolgus monkeys were clearance (CL) = 2.81 mL/day/kg, volume of distribution of central compartment (V1 ) = 31.3 mL/kg, volume of distribution of peripheral compartment (V2 ) = 23.3 mL/kg, absolute bioavailability (F) = 0.84 and T1/2 = 13.4 days. In comparison, the typical population PK parameter estimates for CNTO 8212 in cynomolgus monkeys were CL = 1.41 mL/day/kg, V1 = 39.8 mL/kg, V2 = 32.6 mL/kg, F = 0.75 and T1/2 = 35.7 days. The mean CL of CNTO 8212 was ~50% lower compared with that for CNTO 1119 in cynomolgus monkeys. The overall volume of distribution (V1 +V2 ) for CNTO 8212 was about 32% larger compared with that for CNTO 1119, but generally similar to the vascular volume in cynomolgus monkeys. The T1/2 of CNTO 8212 was significantly (p monkeys. Thus, the modification of the Fc portion of an anti-OSM IgG1 mAb for higher FcRn binding affinity resulted in lower systemic clearance and a longer terminal half-life in cynomolgus monkeys. CNTO 8212

  11. Human Eosinophils Express the High Affinity IgE Receptor, FcεRI, in Bullous Pemphigoid

    Science.gov (United States)

    Messingham, Kelly N.; Holahan, Heather M.; Frydman, Alexandra S.; Fullenkamp, Colleen; Srikantha, Rupasree; Fairley, Janet A.

    2014-01-01

    Bullous pemphigoid (BP) is an autoimmune blistering disease mediated by autoantibodies targeting BP180 (type XVII collagen). Patient sera and tissues typically have IgG and IgE autoantibodies and elevated eosinophil numbers. Although the pathogenicity of the IgE autoantibodies is established in BP, their contribution to the disease process is not well understood. Our aims were two-fold: 1) To establish the clinical relationships between total and BP180-specific IgE, eosinophilia and other markers of disease activity; and 2) To determine if eosinophils from BP patients express the high affinity IgE receptor, FcεRI, as a potential mechanism of action for IgE in BP. Our analysis of 48 untreated BP patients revealed a correlation between BP180 IgG and both BP180 IgE and peripheral eosinophil count. Additionally, we established a correlation between total IgE concentration and both BP180 IgE levels and eosinophil count. When only sera from patients (n = 16) with total IgE≥400 IU/ml were analyzed, BP180 IgG levels correlated with disease severity, BP230 IgG, total circulating IgE and BP180 IgE. Finally, peripheral eosinophil count correlated more strongly with levels of BP180 IgE then with BP180 IgG. Next, eosinophil FcεRI expression was investigated in the blood and skin using several methods. Peripheral eosinophils from BP patients expressed mRNA for all three chains (α, β and γ) of the FcεRI. Surface expression of the FcεRIα was confirmed on both peripheral and tissue eosinophils from most BP patients by immunostaining. Furthermore, using a proximity ligation assay, interaction of the α- and β-chains of the FcεRI was observed in some biopsy specimens, suggesting tissue expression of the trimeric receptor form in some patients. These studies provide clinical support for the relevance of IgE in BP disease and provide one mechanism of action of these antibodies, via binding to the FcεRI on eosinophils. PMID:25255430

  12. Human eosinophils express the high affinity IgE receptor, FcεRI, in bullous pemphigoid.

    Directory of Open Access Journals (Sweden)

    Kelly N Messingham

    Full Text Available Bullous pemphigoid (BP is an autoimmune blistering disease mediated by autoantibodies targeting BP180 (type XVII collagen. Patient sera and tissues typically have IgG and IgE autoantibodies and elevated eosinophil numbers. Although the pathogenicity of the IgE autoantibodies is established in BP, their contribution to the disease process is not well understood. Our aims were two-fold: 1 To establish the clinical relationships between total and BP180-specific IgE, eosinophilia and other markers of disease activity; and 2 To determine if eosinophils from BP patients express the high affinity IgE receptor, FcεRI, as a potential mechanism of action for IgE in BP. Our analysis of 48 untreated BP patients revealed a correlation between BP180 IgG and both BP180 IgE and peripheral eosinophil count. Additionally, we established a correlation between total IgE concentration and both BP180 IgE levels and eosinophil count. When only sera from patients (n = 16 with total IgE ≥ 400 IU/ml were analyzed, BP180 IgG levels correlated with disease severity, BP230 IgG, total circulating IgE and BP180 IgE. Finally, peripheral eosinophil count correlated more strongly with levels of BP180 IgE then with BP180 IgG. Next, eosinophil FcεRI expression was investigated in the blood and skin using several methods. Peripheral eosinophils from BP patients expressed mRNA for all three chains (α, β and γ of the FcεRI. Surface expression of the FcεRIα was confirmed on both peripheral and tissue eosinophils from most BP patients by immunostaining. Furthermore, using a proximity ligation assay, interaction of the α- and β-chains of the FcεRI was observed in some biopsy specimens, suggesting tissue expression of the trimeric receptor form in some patients. These studies provide clinical support for the relevance of IgE in BP disease and provide one mechanism of action of these antibodies, via binding to the FcεRI on eosinophils.

  13. CTLA4Fcε, a novel soluble fusion protein that binds B7 molecules and the IgE receptors, and reduces human in vitro soluble CD23 production and lymphocyte proliferation.

    Science.gov (United States)

    Perez-Witzke, Daniel; Miranda-García, María Auxiliadora; Suárez, Nuris; Becerra, Raquel; Duque, Kharelys; Porras, Verónica; Fuenmayor, Jaheli; Montano, Ramon Fernando

    2016-05-01

    Immunoglobulin E-mediated allergy and certain autoimmune diseases are characterized by the presence of a T helper type 2 (Th2) immune response and allergen-specific or self-reactive IgE. Soluble CD23 (sCD23) is a B-cell factor that fosters IgE class-switching and synthesis, suggesting that sCD23 may be a therapeutic target for these pathologies. We produced a recombinant protein, CTLA4Fcε, by fusing the ectodomain of the immunoregulatory molecule cytotoxic T-lymphocyte antigen 4 (CTLA-4) with a fragment of the IgE H-chain constant region. In SDS-PAGE/inmunoblot analyses, CTLA4Fcε appeared as a 70,000 MW polypeptide that forms homodimers. Flow cytometry showed that CTLA4Fcε binds to IgE receptors FcεRI and FcεRII/CD23, as well as to CTLA-4 counter-receptors CD80 and CD86. Binding of CTLA4Fcε to FcεRII/CD23 appeared stronger than that of IgE. Since the cells used to study CD23 binding express CD80 and CD86, simultaneous binding of CTLA4Fcε to CD23 and CD80/CD86 seems to occur and would explain this difference. As measured by a human CD23-specific ELISA, CTLA4Fcε - but not IgE - induced a concentration-dependent reduction of sCD23 in culture supernatants of RPMI-8866 cells. Our results suggest that the simultaneous binding of CTLA4Fcɛ to CD23-CD80/CD86 may cause the formation of multi-molecular complexes that are either internalized or pose a steric hindrance to enzymatic proteolysis, so blocking sCD23 generation. CTLA4Fcε caused a concentration-dependent reduction of lymphocyte proliferation in human peripheral blood mononuclear cell samples stimulated in vitro with concanavalin A. The ability to bind IgE receptors on effector cells, to regulate the production of sCD23 and to inhibit lymphocyte proliferation suggests that CTLA4Fcɛ has immunomodulatory properties on human Th2 responses.

  14. Posttranscriptional control of human gamma interferon gene expression in transfected mouse fibroblasts.

    OpenAIRE

    1986-01-01

    Human gamma interferon genomic DNA was introduced into NIH 3T3 fibroblasts by calcium phosphate precipitation and was not expressed in these cells at the cytoplasmic mRNA or protein level. Treatment of the transfected cells with cycloheximide (1 microgram/ml) induced the accumulation of cytoplasmic gamma interferon mRNA and biologically active human gamma interferon. Analysis of the nuclear enriched RNA from untreated cells indicated that human gamma interferon mRNA was present, suggesting th...

  15. Homology models of human gamma-crystallins: structural study of the extensive charge network in gamma-crystallins.

    Science.gov (United States)

    Salim, Asmat; Zaidi, Zafar H

    2003-01-17

    The lens is composed of highly stable and long-lived proteins, the crystallins which are divided into alpha-, beta-, and gamma-crystallins. Human gamma-crystallins belong to the betagamma superfamily. A large number of gamma-crystallins have been sequenced and have been found to share remarkable sequence homology with each other. Some of the gamma-crystallins from various sources have also been elucidated structurally by X-ray crystallographic or NMR spectroscopic experiments. Their three-dimensional structures are also similar having consisted of two domains each possessing two Greek key motifs. In this study we have constructed the comparative or homology models of the four major human gamma-crystallins, gammaA-,gammaB-, gammaC-, and gammaD-crystallins and studied the charge network in these crystallins. Despite an overall structural similarity between these crystallins, differences in the ion pair formation do exist which is partly due to the differences in their primary sequence and partly due to the structural orientation of the neighboring amino acids. In this study, we present an elaborate analysis of these charged interactions and their formation or loss with respect to the structural changes.

  16. Identification of human IgG1 variant with enhanced FcRn binding and without increased binding to rheumatoid factor autoantibody

    Science.gov (United States)

    Maeda, Atsuhiko; Iwayanagi, Yuki; Haraya, Kenta; Tachibana, Tatsuhiko; Nakamura, Genki; Nambu, Takeru; Esaki, Keiko; Hattori, Kunihiro; Igawa, Tomoyuki

    2017-01-01

    ABSTRACT Various studies have demonstrated that Fc engineering to enhance neonatal Fc receptor (FcRn) binding is effective for elongating half-life or increasing cellular uptake of IgG. A previous study has shown that a N434H mutation to enhance FcRn binding resulted in increased binding to rheumatoid factor (RF) autoantibody, which is not desirable for therapeutic use in autoimmune disease. In this study, we first showed that all the existing Fc variants with enhanced FcRn binding also show increased RF binding, and then identified specific mutations that could be introduced to those Fc variants to reduce the RF binding. Furthermore, we generated novel Fc variants that do not increase RF binding and show half-lives of 45 d in cynomolgus monkey, which is longer than those of previously reported Fc variants. In addition, we generated novel Fc variants with antigen sweeping activity that do not increase RF binding. We expect that these novel Fc variants will be useful as antibody therapeutics against autoimmune diseases. PMID:28387635

  17. Robust expression of the human neonatal Fc receptor in a truncated soluble form and as a full-length membrane-bound protein in fusion with eGFP.

    Directory of Open Access Journals (Sweden)

    Johan Seijsing

    Full Text Available Studies on the neonatal Fc receptor (FcRn have revealed a multitude of important functions in mammals, including protection of IgG and serum albumin (SA from lysosomal degradation. The pharmacokinetic behavior of therapeutic antibodies, IgG-Fc- and SA-containing drugs is therefore influenced by their interaction with FcRn. Pre-clinical development of such drugs is facilitated if their interaction with FcRn can be studied in vitro. For this reason we have developed a robust system for production of the soluble extracellular domain of human FcRn as well as the full-length receptor as fusion to green fluorescent protein, taking advantage of a lentivirus-based gene delivery system where stable over-expressing cells are easily and rapidly generated. Production of the extracellular domain in multiple-layered culture flasks, followed by affinity purification using immobilized IgG, resulted in capture of milligram amounts of soluble receptor per liter cell culture with retained IgG binding. The receptor was further characterized by SDS-PAGE, western blotting, circular dichroism spectroscopy, ELISA, surface plasmon resonance and a temperature stability assay showing a functional and stable protein of high purity. The full-length receptor was found to be successfully over-expressed in a membrane-bound form with retained pH-dependent IgG- and SA-binding.

  18. A Monosaccharide Residue Is Sufficient to Maintain Mouse and Human IgG Subclass Activity and Directs IgG Effector Functions to Cellular Fc Receptors

    Directory of Open Access Journals (Sweden)

    Daniela Kao

    2015-12-01

    Full Text Available Immunoglobulin G (IgG glycosylation modulates antibody activity and represents a major source of heterogeneity within antibody preparations. Depending on their glycosylation pattern, individual IgG glycovariants present in recombinant antibody preparations may trigger effects ranging from enhanced pro-inflammatory activity to increased anti-inflammatory activity. In contrast, reduction of IgG glycosylation beyond the central mannose core is generally believed to result in impaired IgG activity. However, this study reveals that a mono- or disaccharide structure consisting of one N-acetylglucosamine with or without a branching fucose residue is sufficient to retain the activity of the most active human and mouse IgG subclasses in vivo and further directs antibody activity to cellular Fcγ receptors. Notably, the activity of minimally glycosylated antibodies is not predicted by in vitro assays based on a monomeric antibody-Fcγ-receptor interaction analysis, whereas in vitro assay systems using immune complexes are more suitable to predict IgG activity in vivo.

  19. Fc-fusion mimetics

    OpenAIRE

    2016-01-01

    The Fc-fusion mimetic RpR 2 was prepared by disulfide bridging conjugation using a PEG in the place of the Fc. RpR 2 displayed higher affinity for VEGF than aflibercept caused primarily by a slower dissociation rate, which can prolong a drug at its site of action. RpRs have considerable potential for development as stable, organ specific therapeutics.

  20. Computer simulations of human interferon gamma mutated forms

    Science.gov (United States)

    Lilkova, E.; Litov, L.; Petkov, P.; Petkov, P.; Markov, S.; Ilieva, N.

    2010-01-01

    In the general framework of the computer-aided drug design, the method of molecular-dynamics simulations is applied for investigation of the human interferon-gamma (hIFN-γ) binding to its two known ligands (its extracellular receptor and the heparin-derived oligosaccharides). A study of 100 mutated hIFN-γ forms is presented, the mutations encompassing residues 86-88. The structural changes are investigated by comparing the lengths of the α-helices, in which these residues are included, in the native hIFN-γ molecule and in the mutated forms. The most intriguing cases are examined in detail.

  1. The long elusive IgM Fc receptor, FcμR.

    Science.gov (United States)

    Kubagawa, Hiromi; Oka, Satoshi; Kubagawa, Yoshiki; Torii, Ikuko; Takayama, Eiji; Kang, Dong-Won; Jones, Dewitt; Nishida, Naonori; Miyawaki, Toshio; Bertoli, Luigi F; Sanders, Sheila K; Honjo, Kazuhito

    2014-07-01

    IgM exists as both a monomer on the surface of B cells and a pentamer secreted by plasma cells. Both pre-immune "natural" and antigen-induced "immune" IgM antibodies are important for protective immunity and for immune regulation of autoimmune processes by recognizing pathogens and self-antigens. Effector proteins interacting with the Fc portion of IgM, such as complement and complement receptors, have thus far been proposed but fail to fully account for the IgM-mediated protection and regulation. A major reason for this deficit in our understanding of IgM function seems to be lack of data on a long elusive Fc receptor for IgM (FcμR). We have recently identified a bona fide FcμR in both humans and mice. In this article we briefly review what we have learned so far about FcμR.

  2. Human Antibodies Can Cross Guinea Pig Placenta and Bind Its Neonatal Fc Receptor: Implications for Studying Immune Prophylaxis and Therapy during Pregnancy

    Directory of Open Access Journals (Sweden)

    Evi Budo Struble

    2012-01-01

    Full Text Available Despite increased use of monoclonal and polyclonal antibody therapies, including during pregnancy, there is little data on appropriate animal models that could humanely be used to understand determinants of protection and to evaluate safety of these biologics in the mother and the developing fetus. Here, we demonstrate that pregnant guinea pigs can transport human IgG transplacentally at the end of pregnancy. We also observe that human IgG binds to an engineered soluble variant of the guinea pig neonatal Fc receptor in vitro in a manner similar to that demonstrated for the human variant, suggesting that this transplacental transport mirrors the receptor-based mechanism seen in humans. Using an intravenous antihepatitis B-specific immune globulin preparation as an example, we show that this transport results in neutralizing activity in the mother and the newborn that would potentially be prophylactic against hepatitis B viral infection. These preliminary data lay the groundwork for introducing pregnant guinea pigs as an appropriate model for the evaluation of antibody therapies and advancing the health of women and neonates.

  3. FcGammaRIIa polymorphism and anti-malaria specific IgG and IgG subclass responses in populations differing in susceptibility to malaria in Burkina Faso

    DEFF Research Database (Denmark)

    Cherif, Mariama K; Sanou, Guillaume S; Maiga, Boubakar;

    2012-01-01

    Fc¿RIIa is known to be polymorphic; and certain variants err associated with differt susceptibilities to malaria. Studies involving the Fulani ethnic group reported an ethnic difference in Fc¿RIIa-R131H genotype frequencies between the Fulani and other sympatric groups. No previous studies have a...

  4. Introduction of human gamma 1 immunoglobulin genes into fertilized mouse eggs.

    Science.gov (United States)

    Yamamura, K; Kikutani, H; Takahashi, N; Taga, T; Akira, S; Kawai, K; Fukuchi, K; Kumahara, Y; Honjo, T; Kishimoto, T

    1984-08-01

    A rearranged human gamma 1 immunoglobulin gene was introduced into fertilized mouse eggs. The phage Ch4A-VCE-gamma 1 was constructed by ligating an EcoRI and BglII fragment of pBR322-CESSV(CE-1) containing the VDJ region with an EcoRI and BamHI fragment of Ch4A-HIg gamma 1-10 containing the gamma 1 constant region. About 200 copies of Ch4A-VCE-gamma 1 genes were introduced into fertilized mouse eggs. Of 489 eggs injected with these genes, 319 survived and were transferred to oviducts of foster mothers. Thirtyeight mice were born and were screened for the presence of human gamma 1 immunoglobulin genes by Southern blot hybridization. Five of these 38 mice had integrated human gamma 1 immunoglobulin genes. None of the human gamma 1 copies in each mouse had undergone deletions or rearrangements as judged by the Southern blotting patterns for several restriction enzymes. Human gamma 1 gene was present in several different tissues. All the mice tested so far transmit the human gamma 1 gene to a fraction of their offspring in an autosomal dominant manner. Spleen cells from transgenic mice were analyzed for immunoglobulin production by reverse plaque assay or immunofluorescence staining of cytoplasmic immunoglobulin, but synthesis and secretion of human gamma 1 chains could not be detected. No human gamma 1 immunoglobulin mRNA was detected in the liver and spleen of a transgenic mouse. The presence of the human gamma 1 immunoglobulin gene appeared to have no effect on the expression of endogenous mouse immunoglobulin genes.

  5. NEW ROLES FOR FC RECEPTORS IN NEURODEGENERATION-THE IMPACT ON IMMUNOTHERAPY FOR ALZHEIMER’S DISEASE

    Directory of Open Access Journals (Sweden)

    James P. Fuller

    2014-08-01

    Full Text Available There are an estimated 18 million Alzheimer’s disease (AD sufferers worldwide and with no disease modifying treatment currently available, development of new therapies represents an enormous unmet clinical need. AD is characterised by episodic memory loss followed by severe cognitive decline and is associated with many neuropathological changes. AD is characterised by deposits of amyloid beta (Aβ, neurofibrillary tangles, and neuroinflammation. Active immunisation or passive immunisation against Aβ leads to the clearance of deposits in transgenic mice expressing human Aβ. This clearance is associated with reversal of associated cognitive deficits, but these results have failed to translate to humans, with both active and passive immunotherapy failing to improve memory loss. One explanation for these observations is that certain anti-Aβ antibodies mediate damage to the cerebral vasculature limiting the top dose and potentially reducing efficacy. Fc gamma receptors (Fcγ are a family of immunoglobulin like receptors which bind to the Fc portion of IgG, and mediate the response of effector cells to immune complexes. Data from both mouse and human studies suggest that cross-linking Fc receptors by therapeutic antibodies and the subsequent pro-inflammatory response mediates the vascular side effects seen following immunotherapy. Increasing evidence is emerging that Fc receptor expression on CNS resident cells, including microglia and neurons, is increased during aging and functionally involved in the pathogenesis of age-related neurodegenerative diseases. We propose that increased expression and ligation of Fc receptors in the CNS, either by endogenous IgG or therapeutic antibodies, has the potential to induce vascular damage and exacerbate neurodegeneration. To produce safe and effective immunotherapies for AD and other neurodegenerative diseases it will be vital to understand the role of Fc receptors in the healthy and diseased brain.

  6. Assessment of three human FcεRI-transfected RBL cell-lines for identifying IgE induced degranulation utilizing peanut-allergic patient sera and peanut protein extract

    NARCIS (Netherlands)

    Ladics, G.S.; Bilsen, J.H.M. van; Brouwer, H.M.H.; Vogel, L.; Vieths, S.; Knippels, L.M.J.

    2008-01-01

    Specific IgE sera screening studies are employed to investigate protein cross-reactivity. Such nonfunctional immunochemical methods cannot measure the biological activity of proteins. Therefore, an assay using RBL cells transfected with human FcεRI was developed. Our objective was to evaluate the de

  7. Quantitative evaluation of fucose reducing effects in a humanized antibody on Fcγ receptor binding and antibody-dependent cell-mediated cytotoxicity activities.

    Science.gov (United States)

    Chung, Shan; Quarmby, Valerie; Gao, Xiaoying; Ying, Yong; Lin, Linda; Reed, Chae; Fong, Chris; Lau, Wendy; Qiu, Zhihua J; Shen, Amy; Vanderlaan, Martin; Song, An

    2012-01-01

    The presence or absence of core fucose in the Fc region N-linked glycans of antibodies affects their binding affinity toward FcγRIIIa as well as their antibody-dependent cell-mediated cytotoxicity (ADCC) activity. However, the quantitative nature of this structure-function relationship remains unclear. In this study, the in vitro biological activity of an afucosylated anti-CD20 antibody was fully characterized. Further, the effect of fucose reduction on Fc effector functions was quantitatively evaluated using the afucosylated antibody, its "regular" fucosylated counterpart and a series of mixtures containing varying proportions of "regular" and afucosylated materials. Compared with the "regular" fucosylated antibody, the afucosylated antibody demonstrated similar binding interactions with the target antigen (CD20), C1q and FcγRIa, moderate increases in binding to FcγRIIa and IIb, and substantially increased binding to FcγRIIIa. The afucosylated antibodies also showed comparable complement-dependent cytotoxicity activity but markedly increased ADCC activity. Based on EC 50 values derived from dose-response curves, our results indicate that the amount of afucosylated glycan in antibody samples correlate with both FcγRIIIa binding activity and ADCC activity in a linear fashion. Furthermore, the extent of ADCC enhancement due to fucose depletion was not affected by the FcγRIIIa genotype of the effector cells.

  8. Fcγ receptors and ligands and cardiovascular disease.

    Science.gov (United States)

    Tanigaki, Keiji; Sundgren, Nathan; Khera, Amit; Vongpatanasin, Wanpen; Mineo, Chieko; Shaul, Philip W

    2015-01-16

    Fcγ receptors (FcγRs) classically modulate intracellular signaling on binding of the Fc region of IgG in immune response cells. How FcγR and their ligands affect cardiovascular health and disease has been interrogated recently in both preclinical and clinical studies. The stimulation of activating FcγR in endothelial cells, vascular smooth muscle cells, and monocytes/macrophages causes a variety of cellular responses that may contribute to vascular disease pathogenesis. Stimulation of the lone inhibitory FγcR, FcγRIIB, also has adverse consequences in endothelial cells, antagonizing NO production and reparative mechanisms. In preclinical disease models, activating FcγRs promote atherosclerosis, whereas FcγRIIB is protective, and activating FcγRs also enhance thrombotic and nonthrombotic vascular occlusion. The FcγR ligand C-reactive protein (CRP) has undergone intense study. Although in rodents CRP does not affect atherosclerosis, it causes hypertension and insulin resistance and worsens myocardial infarction. Massive data have accumulated indicating an association between increases in circulating CRP and coronary heart disease in humans. However, Mendelian randomization studies reveal that CRP is not likely a disease mediator. CRP genetics and hypertension warrant further investigation. To date, studies of genetic variants of activating FcγRs are insufficient to implicate the receptors in coronary heart disease pathogenesis in humans. However, a link between FcγRIIB and human hypertension may be emerging. Further knowledge of the vascular biology of FcγR and their ligands will potentially enhance our understanding of cardiovascular disorders, particularly in patients whose greater predisposition for disease is not explained by traditional risk factors, such as individuals with autoimmune disorders.

  9. Fc Receptor-Mediated Activities of Env-Specific Human Monoclonal Antibodies Generated from Volunteers Receiving the DNA Prime-Protein Boost HIV Vaccine DP6-001.

    Science.gov (United States)

    Costa, Matthew R; Pollara, Justin; Edwards, Regina Whitney; Seaman, Michael S; Gorny, Miroslaw K; Montefiori, David C; Liao, Hua-Xin; Ferrari, Guido; Lu, Shan; Wang, Shixia

    2016-11-15

    HIV-1 is able to elicit broadly potent neutralizing antibodies in a very small subset of individuals only after several years of infection, and therefore, vaccines that elicit these types of antibodies have been difficult to design. The RV144 trial showed that moderate protection is possible and that this protection may correlate with antibody-dependent cellular cytotoxicity (ADCC) activity. Our previous studies demonstrated that in an HIV vaccine phase I trial, the DP6-001 trial, a polyvalent Env DNA prime-protein boost formulation could elicit potent and broadly reactive, gp120-specific antibodies with positive neutralization activities. Here we report on the production and analysis of HIV-1 Env-specific human monoclonal antibodies (hMAbs) isolated from vaccinees in the DP6-001 trial. For this initial report, 13 hMAbs from four vaccinees in the DP6-001 trial showed broad binding to gp120 proteins of diverse subtypes both autologous and heterologous to vaccine immunogens. Equally cross-reactive Fc receptor-mediated functional activities, including ADCC and antibody-dependent cellular phagocytosis (ADCP) activities, were present with both immune sera and isolated MAbs, confirming the induction of nonneutralizing functional hMAbs by the DNA prime-protein boost vaccination. Elicitation of broadly reactive hMAbs by vaccination in healthy human volunteers confirms the value of the polyvalent formulation in this HIV vaccine design. The roles of Fc receptor-mediated protective antibody responses are gaining more attention due to their potential contribution to the low-level protection against HIV-1 infection that they provided in the RV144 trial. At the same time, information about hMabs from other human HIV vaccine studies is very limited. In the current study, both immune sera and monoclonal antibodies from vaccinated humans showed not only high-level ADCC and ADCP activities but also cross-subtype ADCC and ADCP activities when a polyvalent DNA prime-protein boost

  10. Peroxisome proliferator-activated receptor gamma overexpression suppresses proliferation of human colon cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Tsukahara, Tamotsu, E-mail: ttamotsu@shinshu-u.ac.jp [Department of Integrative Physiology and Bio-System Control, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan); Haniu, Hisao [Department of Orthopaedic Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621 (Japan)

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer We examined the correlation between PPAR{gamma} expression and cell proliferation. Black-Right-Pointing-Pointer PPAR{gamma} overexpression reduces cell viability. Black-Right-Pointing-Pointer We show the synergistic effect of cell growth inhibition by a PPAR{gamma} agonist. -- Abstract: Peroxisome proliferator-activated receptor gamma (PPAR{gamma}) plays an important role in the differentiation of intestinal cells and tissues. Our previous reports indicate that PPAR{gamma} is expressed at considerable levels in human colon cancer cells. This suggests that PPAR{gamma} expression may be an important factor for cell growth regulation in colon cancer. In this study, we investigated PPAR{gamma} expression in 4 human colon cancer cell lines, HT-29, LOVO, DLD-1, and Caco-2. Real-time polymerase chain reaction (PCR) and Western blot analysis revealed that the relative levels of PPAR{gamma} mRNA and protein in these cells were in the order HT-29 > LOVO > Caco-2 > DLD-1. We also found that PPAR{gamma} overexpression promoted cell growth inhibition in PPAR{gamma} lower-expressing cell lines (Caco-2 and DLD-1), but not in higher-expressing cells (HT-29 and LOVO). We observed a correlation between the level of PPAR{gamma} expression and the cells' sensitivity for proliferation.

  11. 新生儿Fc受体在人肾小球肾炎及大鼠肾炎模型中的表达%Expression of neonatal Fc receptor on human nephritis and rat nephritis models

    Institute of Scientific and Technical Information of China (English)

    冯松涛; 甘华磊; 孙建永; 蒋涛; 刘宝利; 赵仲华; 郭慕依; 张志刚

    2012-01-01

    Objective To study the expression of neonatal Fc receptor in podocytes in human nephritis and immune-induced rat nephritis models:anti-Thy1.1 nephritis and Heymann nephritis.Methods Thirty-nine cases of renal biopsies were enrolled from September 2009 to February 2010,including 8 cases of minimal change disease, 4 cases of focal segmental glomerulosclerosis, 9 cases of membranous nephropathy,12 cases of IgA nephropathy and 6 cases of lupus nephritis.Five normal kidney tissue samples adjacent to renal clear-cell carcinoma were served as normal controls.Laser capture microdissection and realtime RT-PCR were used to assess the expression level of FcRn mRNA in glomeruli of various glomerulonephritides,and immunohistochemistry (IHC) of FcRn by SuperVision method was performed.In addition,rat models of mesangial proliferative nephritis (anti-Thyl.1 nephritis) and passive membranous nephropathy (Heymann nephritis ) were established and FcRn was examined in renal tissues by IHC.Results The FcRn mRNA level in lupus nephritis was statistically higher than that of normal controls ( P < 0.05 ).FcRn protein expression by IHC was seen in lupus nephritis (6/6),membranous nephropathy (6/9) and IgA nephropathy (7/12),significantly higher than that of normal controls (0/5),P < 0.05.Minimal change disease and focal segmental glomerular sclerosis showed minimal or none expression of FcRn (1/8,0/4 respectively) and not statistically difference from that of normal controls. Furthermore,FcRn expression in podocytes was detected in rat anti-Thy1.1 (3/5) and Heymann nephritis models (2/7) but was not detected in normal controls. Conclusions Expression of FcRn in podocytes was up-regulated in immune-induced human nephritis and rat nephritis models of anti-Thyl.1 nephritis and Heymann nephritis.FcRn may play a role in the development of immune-induced glomerulonephritis.%目的 观察人肾小球肾炎及大鼠肾炎动物模型中,足细胞新生儿Fc受体(FcRn)的表达.方法 (1)

  12. Formation of gamma-glutamylpropargylglycylglycine from propargylglycine in human blood and erythrocytes.

    Directory of Open Access Journals (Sweden)

    Nagamine N

    1999-02-01

    Full Text Available Gamma-Glutamylpropargylglycylglycine (gamma-Glu-PPG-Gly was isolated as a metabolite of propargylglycine (2-amino-4-pentynoic acid, a natural and synthetic inhibitor of cystathionine gamma-lyase from human blood incubated with D,L-propargylglycine in the presence of L-glutamate and glycine, and identified by fast-atom-bombardment mass spectrometry, indicating that human blood can metabolize propargylglycine to gamma-Glu-PPG-Gly. When whole blood was incubated with 2 mM D,L-propargylglycine in the presence of 10 mM L-glutamate and 10 mM glycine at 37 degrees C for 16h, 0.094+/-0.013 micromol of gamma-Glu-PPG-Gly was formed per ml of whole blood. When erythrocytes were incubated under the same conditions for 16h, 0.323+/-0.060 micromol of gamma-Glu-PPG-Gly was formed per ml of erythrocytes, suggesting a large contribution of erythrocytes to gamma-Glu-PPG-Gly formation in whole blood. The apparent Km value of gamma-Glu-PPG-Gly formation in human erythrocytes for D,L-propargylglycine was 0.32 mM. The observed rate of gamma-Glu-PPG-Gly formation and the Km value for D,L-propargylglycine suggest that metabolism of propargylglycine to gamma-Glu-PPG-Gly can play a definite biological role in human subjects who are loaded with propargylglycine.

  13. Aggregated IgG inhibits the differentiation of human fibrocytes.

    Science.gov (United States)

    Pilling, Darrell; Tucker, Nancy M; Gomer, Richard H

    2006-06-01

    Fibrocytes are fibroblast-like cells, which appear to participate in wound healing and are present in pathological lesions associated with asthma, pulmonary fibrosis, and scleroderma. Fibrocytes differentiate from CD14+ peripheral blood monocytes, and the presence of serum delays this process dramatically. We previously purified the factor in serum, which inhibits fibrocyte differentiation, and identified it as serum amyloid P (SAP). As SAP binds to Fc receptors for immunoglobulin G (IgG; Fc gammaRs), Fc gammaR activation may be an inhibitory signal for fibrocyte differentiation. Fc gammaR are activated by aggregated IgG, and we find aggregated but not monomeric, human IgG inhibits human fibrocyte differentiation. Monoclonal antibodies that bind to Fc gammaRI (CD64) or Fc gammaRII (CD32) also inhibit fibrocyte differentiation. Aggregated IgG lacking Fc domains or aggregated IgA, IgE, or IgM do not inhibit fibrocyte differentiation. Incubation of monocytes with SAP or aggregated IgG inhibited fibrocyte differentiation. Using inhibitors of protein kinase enzymes, we show that Syk- and Src-related tyrosine kinases participate in the inhibition of fibrocyte differentiation. These observations suggest that fibrocyte differentiation can occur in situations where SAP and aggregated IgG levels are low, such as the resolution phase of inflammation.

  14. FcγRI mediates serum amyloid P inhibition of fibrocyte differentiation.

    Science.gov (United States)

    Crawford, Jeffrey R; Pilling, Darrell; Gomer, Richard H

    2012-10-01

    Fibrotic diseases, such as cardiac and pulmonary fibrosis, have a poor prognosis with no FDA approved therapies. Monocyte-derived, fibroblast-like cells, called fibrocytes, participate in the formation of fibrotic lesions. The conserved pentraxin protein SAP inhibits fibrocyte differentiation in cell culture, and injections of SAP significantly reduce fibrosis in several animal models. SAP binds to the receptors for the Fc portion of IgG (FcγR) and has been crystallized bound to FcγRIIa (CD32a). The in vivo activity of SAP appears to be dependent on the FcRγ. We find that mutagenesis of the residues critical for SAP binding to FcγRIIa only moderately decreases the ability of SAP to inhibit fibrocyte differentiation. In murine cells, deletion of FcRγ or FcγRI (CD64) significantly reduced sensitivity to SAP. Deletion of the combination of FcγRIIb, FcγRIIIa, and FcγRIV did not significantly affect sensitivity to SAP, whereas deletion of just the inhibitory receptor FcγRIIb (CD32b) increased sensitivity to SAP. In human cells, siRNA-mediated reduction of FcRγ or FcγRI levels significantly decreased sensitivity to SAP, whereas reduction of FcγRIIb levels increased sensitivity to SAP. These observations suggest that SAP, at least in part, uses FcγRI and FcRγ to inhibit fibrocyte differentiation.

  15. Liposomes containing NY‑ESO‑1/tetanus toxoid and adjuvant peptides targeted to human dendritic cells via the Fc receptor for cancer vaccines.

    Science.gov (United States)

    Cruz, Luis J; Rueda, Felix; Simón, Lorena; Cordobilla, Begoña; Albericio, Fernando; Domingo, Joan C

    2014-04-01

    To improve the immunological response against tumors, a vaccine based on nanoliposomes targeted to the Fcg-receptor was developed to enhance the immunogenicity of tumor-associated antigens (TAAs). Using human dendritic cells in vitro, a fragment of the TAA NY-ESO-1 combined with a T-helper peptide from the tetanus toxoid encapsulated in nanoliposomes was evaluated. In addition, peptides Palm-IL-1 and MAP-IFN-g were coadministered as adjuvants to enhance the immunological response. Coadministration of Palm-IL-1 or MAP-IFN-g peptide adjuvants and the hybrid NY-ESO-1-tetanus toxoid (soluble or encapsulated in nanoliposomes without targeting) increased immunogenicity. However, the most potent immunological response was obtained when the peptide adjuvants were encapsulated in liposomes targeted to human dendritic cells via the Fc receptor. This targeted vaccine strategy is a promising tool to activate and deliver antigens to dendritic cells, thus improving immunotherapeutic response in situations in which the immune system is frequently compromised, as in advanced cancers.

  16. The Etiology of Multiple Sclerosis: Genetic Evidence for the Involvement of the Human Endogenous Retrovirus HERV-Fc1

    DEFF Research Database (Denmark)

    Nexø, Bjørn Andersen; Christensen, Tove; Frederiksen, Jette;

    2011-01-01

    We have investigated the role of human endogenous retroviruses in multiple sclerosis by analyzing the DNA of patients and controls in 4 cohorts for associations between multiple sclerosis and polymorphisms near viral restriction genes or near endogenous retroviral loci with one or more intact...

  17. Gamma oscillations in human primary somatosensory cortex reflect pain perception.

    Directory of Open Access Journals (Sweden)

    Joachim Gross

    2007-05-01

    Full Text Available Successful behavior requires selection and preferred processing of relevant sensory information. The cortical representation of relevant sensory information has been related to neuronal oscillations in the gamma frequency band. Pain is of invariably high behavioral relevance and, thus, nociceptive stimuli receive preferred processing. Here, by using magnetoencephalography, we show that selective nociceptive stimuli induce gamma oscillations between 60 and 95 Hz in primary somatosensory cortex. Amplitudes of pain-induced gamma oscillations vary with objective stimulus intensity and subjective pain intensity. However, around pain threshold, perceived stimuli yielded stronger gamma oscillations than unperceived stimuli of equal stimulus intensity. These results show that pain induces gamma oscillations in primary somatosensory cortex that are particularly related to the subjective perception of pain. Our findings support the hypothesis that gamma oscillations are related to the internal representation of behaviorally relevant stimuli that should receive preferred processing.

  18. Monomeric Immunoglobulin A from Plasma Inhibits Human Th17 Responses In Vitro Independent of FcαRI and DC-SIGN

    Science.gov (United States)

    Saha, Chaitrali; Das, Mrinmoy; Patil, Veerupaxagouda; Stephen-Victor, Emmanuel; Sharma, Meenu; Wymann, Sandra; Jordi, Monika; Vonarburg, Cédric; Kaveri, Srini V.; Bayry, Jagadeesh

    2017-01-01

    Circulating immunoglobulins including immunoglobulin G (IgG) and IgM play a critical role in the immune homeostasis by modulating functions of immune cells. These functions are mediated in part by natural antibodies. However, despite being second most abundant antibody in the circulation, the immunoregulatory function of IgA is relatively unexplored. As Th17 cells are the key mediators of a variety of autoimmune, inflammatory, and allergic diseases, we investigated the ability of monomeric IgA (mIgA) isolated from pooled plasma of healthy donors to modulate human Th17 cells. We show that mIgA inhibits differentiation and amplification of human Th17 cells and the production of their effector cytokine IL-17A. mIgA also suppresses IFN-γ responses under these experimental conditions. Suppressive effect of mIgA on Th17 responses is associated with reciprocal expansion of FoxP3-positive regulatory T cells. The effect of mIgA on Th17 cells is dependent on F(ab′)2 fragments and independent of FcαRI (CD89) and DC-SIGN. Mechanistically, the modulatory effect of mIgA on Th17 cells implicates suppression of phosphorylation of signal transducer and activator of transcription 3. Furthermore, mIgA binds to CD4+ T cells and recognizes in a dose-dependent manner the receptors for cytokines (IL-6Rα and IL-1RI) that mediate Th17 responses. Our findings thus reveal novel anti-inflammatory functions of IgA and suggest potential therapeutic utility of mIgA in autoimmune and inflammatory diseases that implicate Th17 cells. PMID:28352269

  19. Developmental- and differentiation-specific patterns of human gamma- and beta-globin promoter DNA methylation.

    Science.gov (United States)

    Mabaera, Rodwell; Richardson, Christine A; Johnson, Kristin; Hsu, Mei; Fiering, Steven; Lowrey, Christopher H

    2007-08-15

    The mechanisms underlying the human fetal-to-adult beta-globin gene switch remain to be determined. While there is substantial experimental evidence to suggest that promoter DNA methylation is involved in this process, most data come from studies in nonhuman systems. We have evaluated human gamma- and beta-globin promoter methylation in primary human fetal liver (FL) and adult bone marrow (ABM) erythroid cells. Our results show that, in general, promoter methylation and gene expression are inversely related. However, CpGs at -162 of the gamma promoter and -126 of the beta promoter are hypomethylated in ABM and FL, respectively. We also studied gamma-globin promoter methylation during in vitro differentiation of erythroid cells. The gamma promoters are initially hypermethylated in CD34(+) cells. The upstream gamma promoter CpGs become hypomethylated during the preerythroid phase of differentiation and are then remethylated later, during erythropoiesis. The period of promoter hypomethylation correlates with transient gamma-globin gene expression and may explain the previously observed fetal hemoglobin production that occurs during early adult erythropoiesis. These results provide the first comprehensive survey of developmental changes in human gamma- and beta-globin promoter methylation and support the hypothesis that promoter methylation plays a role in human beta-globin locus gene switching.

  20. Expression Profile of Human Fc Receptor-Like 1, 2, and 4 Molecules in Peripheral Blood Mononuclear Cells of Patients with Hashimoto's Thyroiditis and Graves' Disease.

    Science.gov (United States)

    Rostamzadeh, D; Dabbaghmanesh, M H; Shabani, M; Hosseini, A; Amirghofran, Z

    2015-08-01

    Recently identified Fc receptor-like (FCRL) molecules are new members of the immunoglobulin superfamily dominantly expressed by B cells. Although FCRL expression patterns have been studied in normal and malignant cells, their biological functions and roles remain to be clearly identified in humans. Research has particularly focused on FCRL gene polymorphisms in autoimmune diseases, however, their involvement in the pathogenesis of autoimmune diseases is an interesting field for investigation. In the present study, we have investigated the gene expression profiles of FCRL1, 2, and 4 in 2 common thyroid diseases, Hashimoto's thyroiditis (HT) and Graves' disease (GD). FCRL1, 2, and 4 expressions were determined in peripheral blood samples of 55 HT patients, 40 GD patients and equal numbers of normal subjects by quantitative real-time PCR. Our results showed downregulation of FCRL1 and upregulation of FCRL2 transcripts in both HT and GD groups compared to healthy counterparts. Overexpression of FCRL4 was observed only in GD patients compared to controls. A significant correlation was observed between all FCRL gene expression levels in HT patients. Only FCRL2 and 4 had a correlation in GD patients. In addition, FCRL1, 2, and 4 gene expressions showed no correlations with the level of anti-thyroid peroxidase antibody (anti-TPO) or anti-thyroglobulin (anti-Tg) antibody from patients' sera. In conclusion, expressions of activating or inhibitory FCRL1, 2, and 4 showed significant alterations in HT and GD patients compared to healthy subjects. © Georg Thieme Verlag KG Stuttgart · New York.

  1. Gamma oscillations correlate with working memory load in humans

    National Research Council Canada - National Science Library

    Howard, Marc W; Rizzuto, Daniel S; Caplan, Jeremy B; Madsen, Joseph R; Lisman, John; Aschenbrenner-Scheibe, Richard; Schulze-Bonhage, Andreas; Kahana, Michael J

    2003-01-01

    .... Spectral analyses revealed that, in both patients, gamma (30-60 Hz) oscillations increased approximately linearly with memory load, tracking closely with memory load over the course of the trial...

  2. Developing the IVIG biomimetic, hexa-Fc, for drug and vaccine applications.

    Science.gov (United States)

    Czajkowsky, Daniel M; Andersen, Jan Terje; Fuchs, Anja; Wilson, Timothy J; Mekhaiel, David; Colonna, Marco; He, Jianfeng; Shao, Zhifeng; Mitchell, Daniel A; Wu, Gang; Dell, Anne; Haslam, Stuart; Lloyd, Katy A; Moore, Shona C; Sandlie, Inger; Blundell, Patricia A; Pleass, Richard J

    2015-04-27

    The remarkable clinical success of Fc-fusion proteins has driven intense investigation for even more potent replacements. Using quality-by-design (QbD) approaches, we generated hexameric-Fc (hexa-Fc), a ~20 nm oligomeric Fc-based scaffold that we here show binds low-affinity inhibitory receptors (FcRL5, FcγRIIb, and DC-SIGN) with high avidity and specificity, whilst eliminating significant clinical limitations of monomeric Fc-fusions for vaccine and/or cancer therapies, in particular their poor ability to activate complement. Mass spectroscopy of hexa-Fc reveals high-mannose, low-sialic acid content, suggesting that interactions with these receptors are influenced by the mannose-containing Fc. Molecular dynamics (MD) simulations provides insight into the mechanisms of hexa-Fc interaction with these receptors and reveals an unexpected orientation of high-mannose glycans on the human Fc that provides greater accessibility to potential binding partners. Finally, we show that this biosynthetic nanoparticle can be engineered to enhance interactions with the human neonatal Fc receptor (FcRn) without loss of the oligomeric structure, a crucial modification for these molecules in therapy and/or vaccine strategies where a long plasma half-life is critical.

  3. Beta- and gamma-range human lower limb corticomuscular coherence

    Directory of Open Access Journals (Sweden)

    Joseph T Gwin

    2012-09-01

    Full Text Available Coherence between electroencephalography (EEG recorded on the scalp above the motor cortex and electromyography (EMG recorded on the skin of the limbs is thought to reflect corticospinal coupling between motor cortex and muscle motor units. Beta-range (13-30 Hz corticomuscular coherence has been extensively documented during static force output while gamma-range (31-45 Hz coherence has been linked to dynamic force output. However, the explanation for this beta-to-gamma coherence shift remains unclear. We recorded 264-channel EEG and 8-channel lower limb electromyography (EMG while 8 healthy subjects performed isometric and isotonic, knee and ankle exercises. Adaptive mixture independent component analysis (AMICA parsed EEG into models of underlying source signals. We computed magnitude squared coherence between electrocortical source signals and EMG. Significant coherence between contralateral motor cortex electrocortical signals and lower limb EMG was observed in the beta- and gamma-range for all exercise types. Gamma-range coherence was significantly greater for isotonic exercises than for isometric exercises. We conclude that active muscle movement modulates the speed of corticospinal oscillations. Specifically, isotonic contractions shift corticospinal oscillations towards the gamma-range while isometric contractions favor beta-range oscillations. Prior research has suggested that tasks requiring increased integration of visual and somatosensory information may shift corticomuscular coherence to the gamma-range. The isometric and isotonic tasks studied here likely required similar amounts of visual and somatosensory integration. This suggests that muscle dynamics, including the amount and type of proprioception, may play a role in the beta-to-gamma shift.

  4. Glucagon-like Peptide 1 Conjugated to Recombinant Human Serum Albumin Variants with Modified Neonatal Fc Receptor Binding Properties. Impact on Molecular Structure and Half-Life

    DEFF Research Database (Denmark)

    Bukrinski, Jens T.; Sønderby, Pernille; Antunes, Filipa

    2017-01-01

    cells of blood vessels, which rescues circulating HSA from lysosomal degradation. We have conjugated GLP-1 to C34 of native sequence recombinant HSA (rHSA) and two rHSA variants; one with increased and one with decreased binding affinity to hFcRn. We have investigated the impact of conjugation on Fc......Rn binding affinities, GLP-1 potency and pharmacokinetics, combined with the solution structure of the rHSA variants and GLP-1 albumin conjugates. The solution structures, determined by small angle X-ray scattering, show the GLP-1 pointing away from the surface of rHSA. Combining the solution structures...... with the available structural information on the FcRn and GLP-1 receptor (GLP-1R) obtained from X-ray crystallography, we can explain the observed in-vitro and in-vivo behaviour. We conclude that the conjugation of GLP-1 to rHSA does not affect the interaction between rHSA and FcRn, while the observed decrease...

  5. Analysis of FcR non-binding anti-CD3 mAb in humanized mice identifies novel human gut tropic cells with regulatory function that are found in patients

    Science.gov (United States)

    Waldron-Lynch, Frank; Henegariu, Octavian; Deng, Songyan; Preston-Hurlburt, Paula; Tooley, James; Flavell, Richard; Herold, Kevan C.

    2014-01-01

    The development and optimization of immune therapies in patients has been hampered by the lack of preclinical models in which their effects on human immune cells can be studied. As a result, observations that have been made in preclinical studies have suggested mechanisms of drug action in murine models that may not be confirmed in clinical studies. We have utilized a humanized mouse reconstituted with human hematopoetic stem cells to circumvent these limitations. We have studied the effects of teplizumab in this model, a Fc receptor non-binding humanized monoclonal anti-CD3 antibody that has been used to treat patients with Type 1 diabetes mellitus. A novel mechanism of action was identified where human gut tropic CCR6+ T cells leave the circulation and secondary lymph organs and migrate to the small intestine. They become producers of IL-10 which can be detected in the peripheral circulation. Blockade of migration of T cells to the small intestine by natalizumab abolishes the treatment effects of teplizumab. Direct translation of these findings was possible in patients with Type 1 diabetes treated with teplizumab since we found there is increased expression of IL-10 by CD4+CD25highCCR6+FoxP3 cells when they emerge into the peripheral circulation. These findings demonstrate that humanized mice may be used to identify novel immunologic mechanisms that occur in patients treated with immune modulators. PMID:22277969

  6. Characterization of the ligand binding site of the bovine IgA Fc receptor (bFc alpha R).

    Science.gov (United States)

    Morton, H Craig; Pleass, Richard J; Woof, Jenny M; Brandtzaeg, Per

    2004-12-24

    Recently, we identified a bovine IgA Fc receptor (bFc alpha R), which shows high homology to the human myeloid Fc alpha R, CD89. IgA binding has previously been shown to depend on several specific residues located in the B-C and F-G loops of the membrane-distal extracellular domain 1 of CD89. To compare the ligand binding properties of these two Fc alpha Rs, we have mapped the IgA binding site of bFc alpha R. We show that, in common with CD89, Tyr-35 in the B-C loop is essential for IgA binding. However, in contrast to earlier observations on CD89, mutation of residues in the F-G loop did not significantly inhibit IgA binding.

  7. [Gamma interferon induced in human leukocytes by phytohemagglutinin: its production and biological characteristics].

    Science.gov (United States)

    Danielescu, G; Maniu, H; Georgescu, T; Cajal, N

    1988-01-01

    Human gamma type interferon (IFN) preparations were obtained through phytohemagglutinin stimulation of leukocytes from the peripheral blood. Biological value of these preparations varied between 160 u and 800 u/ml, depending on leukocyte incubation medium, culture system and inductor conservation. The rising of the antiviral activity through association between gamma (3 u) and alpha (27 u) interferons was revealed by the virus quantity reduction (in this case the vesicular stomatitis virus was used) during a 24-hour multiplication cycle. The protection ensured by the mixture of the two types of interferon was about ten times higher than the additive effect of the two preparations. Study of the antiproliferative activity of a gamma interferon preparation was conducted on two human cell lines of tumoral origin (T-10 from a glioblastoma, and HEp-2) and revealed the difficulties to quantify precisely this property of the crude gamma interferon preparations.

  8. SIMPLE EXTRACTION OF GAMMA-HYDROXYBUTYRATE IN HUMAN WHOLE BLOOD BY HEADSPACE SOLID-PHASE MICROEXTRACTION

    OpenAIRE

    2001-01-01

    We have developed a simple method for the extraction of gamma-hydroxybutyrate (GHB) in human whole blood using headspace solid-phase microextraction (SPME). The procedure involves the conversion of GHB to gamma-butyrolactone (GBL) with acid catalysis; gamma-valerolactone (GVL) was used as internal standard (IS). After heating a vial containing a whole blood sample with GHB and IS at 80℃ for 5 min in the presence of H3PO4 solution, a Carboxen/polydimethylsiloxane -coated fiber was exposed to t...

  9. Sialylation of IgG Fc domain impairs complement-dependent cytotoxicity.

    Science.gov (United States)

    Quast, Isaak; Keller, Christian W; Maurer, Michael A; Giddens, John P; Tackenberg, Björn; Wang, Lai-Xi; Münz, Christian; Nimmerjahn, Falk; Dalakas, Marinos C; Lünemann, Jan D

    2015-11-01

    IgG molecules exert both pro- and antiinflammatory effector functions based on the composition of the fragment crystallizable (Fc) domain glycan. Sialylated IgG Fc domains have antiinflammatory properties that are attributed to their ability to increase the activation threshold of innate effector cells to immune complexes by stimulating the upregulation of the inhibitory Fcγ receptor IIB (FcγRIIB). Here, we report that IgG Fc sialylation of human monoclonal IgG1 molecules impairs their efficacy to induce complement-mediated cytotoxicity (CDC). Fc sialylation of a CD20-targeting antibody had no impact on antibody-dependent cellular cytotoxicity and did not change the affinity of the antibody for activating Fcγ receptors. In contrast, the presence of sialic acid abrogated the increased binding of C1q to Fc-galactosylated IgG1 and resulted in decreased levels of C3b deposition on the cell surface. Similar to monoclonal antibodies, sialic acid inhibited the increased C1q binding to galactosylated Fc fragments in human polyclonal IgG. In sera derived from patients with chronic inflammatory demyelinating polyneuropathy, an autoimmune disease of the peripheral nervous system in which humoral immune responses mediate tissue damage, induction of IgG Fc sialylation was associated with clinical disease remission. Thus, impairment of CDC represents an FcγR-independent mechanism by which Fc-sialylated glycovariants might limit proinflammatory IgG effector functions.

  10. Investigating the Interaction between the Neonatal Fc Receptor and Monoclonal Antibody Variants by Hydrogen/Deuterium Exchange Mass Spectrometry

    DEFF Research Database (Denmark)

    Jensen, Pernille Foged; Larraillet, Vincent; Schlothauer, Tilman

    2015-01-01

    to map sites perturbed by binding on both partners of the IgG-FcRn complex. Several regions in the antibody Fc region and the FcRn were protected from exchange upon complex formation, in good agreement with previous crystallographic studies of FcRn in complex with the Fc fragment. Interestingly, we found......The recycling of immunoglobulins by the neonatal Fc receptor (FcRn) is of crucial importance in the maintenance of antibody levels in plasma and is responsible for the long half-lives of endogenous and recombinant monoclonal antibodies. From a therapeutic point of view there is great interest...... in understanding and modulating the IgG-FcRn interaction to optimize antibody pharmacokinetics and ultimately improve efficacy and safety. Here we studied the interaction between a full-length human IgG1 and human FcRn via hydrogen/deuterium exchange mass spectrometry and targeted electron transfer dissociation...

  11. Single-domain antibody-based and linker-free bispecific antibodies targeting FcγRIII induce potent antitumor activity without recruiting regulatory T cells.

    Science.gov (United States)

    Rozan, Caroline; Cornillon, Amélie; Pétiard, Corinne; Chartier, Martine; Behar, Ghislaine; Boix, Charlotte; Kerfelec, Brigitte; Robert, Bruno; Pèlegrin, André; Chames, Patrick; Teillaud, Jean-Luc; Baty, Daniel

    2013-08-01

    Antibody-dependent cell-mediated cytotoxicity, one of the most prominent modes of action of antitumor antibodies, suffers from important limitations due to the need for optimal interactions with Fcγ receptors. In this work, we report the design of a new bispecific antibody format, compact and linker-free, based on the use of llama single-domain antibodies that are capable of circumventing most of these limitations. This bispecific antibody format was created by fusing single-domain antibodies directed against the carcinoembryonic antigen and the activating FcγRIIIa receptor to human Cκ and CH1 immunoglobulin G1 domains, acting as a natural dimerization motif. In vitro and in vivo characterization of these Fab-like bispecific molecules revealed favorable features for further development as a therapeutic molecule. They are easy to produce in Escherichia coli, very stable, and elicit potent lysis of tumor cells by human natural killer cells at picomolar concentrations. Unlike conventional antibodies, they do not engage inhibitory FcγRIIb receptor, do not compete with serum immunoglobulins G for receptor binding, and their cytotoxic activity is independent of Fc glycosylation and FcγRIIIa polymorphism. As opposed to anti-CD3 bispecific antitumor antibodies, they do not engage regulatory T cells as these latter cells do not express FcγRIII. Studies in nonobese diabetic/severe combined immunodeficient gamma mice xenografted with carcinoembryonic antigen-positive tumor cells showed that Fab-like bispecific molecules in the presence of human peripheral blood mononuclear cells significantly slow down tumor growth. This new compact, linker-free bispecific antibody format offers a promising approach for optimizing antibody-based therapies.

  12. Interferon gamma response region in the promoter of the human DPA gene.

    OpenAIRE

    1990-01-01

    The interferon gamma (IFN-gamma) response region of the human class II major histocompatibility complex gene, DPA, has been localized to a 52-base-pair (bp) DNA fragment in the proximal promotor at -107 to -55 bp after transfection into HeLa cells of a series of 5', 3', and gap deletion mutants linked to a reporter gene, human growth hormone, as well as of synthetic oligonucleotides fused to the heterologous promoter thymidine kinase. The 52-mer sequence contains the X and Y box elements cons...

  13. In vitro and in vivo effect of 5-FC combined gene therapy with TNF-α and CD suicide gene on human laryngeal carcinoma cell line Hep-2.

    Science.gov (United States)

    Chai, Li-Ping; Wang, Zhang-Feng; Liang, Wei-Ying; Chen, Lei; Chen, Dan; Wang, An-Xun; Zhang, Zhao-Qiang

    2013-01-01

    This study was aimed to investigate the effect of combined cancer gene therapy with exogenous tumor necrosis factor-alpha (TNF-α) and cytosine deaminase (CD) suicide gene on laryngeal carcinoma cell line Hep-2 in vitro and in vivo. Transfection of the recombinant eukaryotic vectors of pcDNA3.1 (+) containing TNF-α and/or CD into Hep-2 cells resulted in expression of TNF-α and/or CD gene in vitro. The significant increase in apoptotic Hep-2 cells and decrease of Hep-2 cell proliferation were observed using 5-FC treatment combined with TNF-a expression by CD/5-FC suicide system. Moreover, bystander effect was also observed in the TNF-α and CD gene co-expression group. Laryngeal squamous cell carcinoma (LSCC) mice model was established by using BALB/c mice which different transfected Hep-2 cells with pcDNA3.1 (+) containing TNF-α and/or CD were applied subcutaneously. So these mice are divided into four groups, namely, (1)Hep-2/TIC group; (2)Hep-2/CD group; (3)Hep-2/TNF-α group; (4)Hep-2/0 group. At day 29 after cell inoculation, volume of grafted tumor had significant difference between each two of them (P<0.05). These results showed that the products of combined CD and TNF-α genes inhibited the growth of transplanted LSCC in mice model. So by our observed parameters and many others results, we hypothesized that 5-FC combined gene therapy with TNF-αand CD suicide gene should be an effective treatment on Laryngeal carcinoma.

  14. Induction of ceruloplasmin synthesis by IFN-gamma in human monocytic cells

    Science.gov (United States)

    Mazumder, B.; Mukhopadhyay, C. K.; Prok, A.; Cathcart, M. K.; Fox, P. L.

    1997-01-01

    Ceruloplasmin is a 132-kDa glycoprotein abundant in human plasma. It has multiple in vitro activities, including copper transport, lipid pro- and antioxidant activity, and oxidation of ferrous ion and aromatic amines; however, its physiologic role is uncertain. Although ceruloplasmin is synthesized primarily by the liver in adult humans, production by cells of monocytic origin has been reported. We here show that IFN-gamma is a potent inducer of ceruloplasmin synthesis by monocytic cells. Activation of human monoblastic leukemia U937 cells with IFN-gamma increased the production of ceruloplasmin by at least 20-fold. The identity of the protein was confirmed by plasmin fingerprinting. IFN-gamma also increased ceruloplasmin mRNA. Induction followed a 2- to 4-h lag and was partially blocked by cycloheximide, indicating a requirement for newly synthesized factors. Ceruloplasmin induction in monocytic cells was agonist specific, as IL-1, IL-4, IL-6, IFN-alpha, IFN-beta, TNF-alpha, and LPS were completely ineffective. The induction was also cell type specific, as IFN-gamma did not induce ceruloplasmin synthesis in endothelial or smooth muscle cells. In contrast, IFN-gamma was stimulatory in other monocytic cells, including THP-1 cells and human peripheral blood monocytes, and also in HepG2 cells. Ceruloplasmin secreted by IFN-gamma-stimulated U937 cells had ferroxidase activity and was, in fact, the only secreted protein with this activity. Monocytic cell-derived ceruloplasmin may contribute to defense responses via its ferroxidase activity, which may drive iron homeostasis in a direction unfavorable to invasive organisms.

  15. Femtosecond fluorescence spectra of tryptophan in human gamma-crystallin mutants: site-dependent ultrafast quenching.

    Science.gov (United States)

    Xu, Jianhua; Chen, Jiejin; Toptygin, Dmitri; Tcherkasskaya, Olga; Callis, Patrik; King, Jonathan; Brand, Ludwig; Knutson, Jay R

    2009-11-25

    The eye lens Crystallin proteins are subject to UV irradiation throughout life, and the photochemistry of damage proceeds through the excited state; thus, their tryptophan (Trp) fluorescence lifetimes are physiologically important properties. The time-resolved fluorescence spectra of single Trps in human gammaD- and gammaS-Crystallins have been measured with both an upconversion spectrophotofluorometer on the 300 fs to 100 ps time scale, and a time correlated single photon counting apparatus on the 100 ps to 10 ns time scale, respectively. Three Trps in each wild type protein were replaced by phenylalanine, leading to single-Trp mutants: W68-only and W156-only of HgammaD- and W72-only and W162-only of HgammaS-Crystallin. These proteins exhibit similar ultrafast signatures: positive definite decay associated spectra (DAS) for 50-65 ps decay constants that indicate dominance of fast, heterogeneous quenching. The quenched population (judged by amplitude) of this DAS differs among mutants. Trps 68, 156 in human gammaD- and Trp72 in human gammaS-Crystallin are buried, but water can reach amide oxygen and ring HE1 atoms through narrow channels. QM-MM simulations of quenching by electron transfer predict heterogeneous decay times from 50-500 ps that agree with our experimental results. Further analysis of apparent radiative lifetimes allow us to deduce that substantial subpopulations of Trp are fully quenched in even faster (sub-300 fs) processes for several of the mutants. The quenching of Trp fluorescence of human gammaD- and gammaS-Crystallin may protect them from ambient light induced photo damage.

  16. A recombinant vaccine of H5N1 HA1 fused with foldon and human IgG Fc induced complete cross-clade protection against divergent H5N1 viruses.

    Directory of Open Access Journals (Sweden)

    Lanying Du

    Full Text Available Development of effective vaccines to prevent influenza, particularly highly pathogenic avian influenza (HPAI caused by influenza A virus (IAV subtype H5N1, is a challenging goal. In this study, we designed and constructed two recombinant influenza vaccine candidates by fusing hemagglutinin 1 (HA1 fragment of A/Anhui/1/2005(H5N1 to either Fc of human IgG (HA1-Fc or foldon plus Fc (HA1-Fdc, and evaluated their immune responses and cross-protection against divergent strains of H5N1 virus. Results showed that these two recombinant vaccines induced strong immune responses in the vaccinated mice, which specifically reacted with HA1 proteins and an inactivated heterologous H5N1 virus. Both proteins were able to cross-neutralize infections by one homologous strain (clade 2.3 and four heterologous strains belonging to clades 0, 1, and 2.2 of H5N1 pseudoviruses as well as three heterologous strains (clades 0, 1, and 2.3.4 of H5N1 live virus. Importantly, immunization with these two vaccine candidates, especially HA1-Fdc, provided complete cross-clade protection against high-dose lethal challenge of different strains of H5N1 virus covering clade 0, 1, and 2.3.4 in the tested mouse model. This study suggests that the recombinant fusion proteins, particularly HA1-Fdc, could be developed into an efficacious universal H5N1 influenza vaccine, providing cross-protection against infections by divergent strains of highly pathogenic H5N1 virus.

  17. Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies

    Science.gov (United States)

    Schlothauer, Tilman; Rueger, Petra; Stracke, Jan Olaf; Hertenberger, Hubert; Fingas, Felix; Kling, Lothar; Emrich, Thomas; Drabner, Georg; Seeber, Stefan; Auer, Johannes; Koch, Stefan; Papadimitriou, Apollon

    2013-01-01

    The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG antibodies in vivo. Analysis of the interaction between FcRn and IgG in vitro might provide insight into the structural and functional integrity of therapeutic IgG that may affect pharmacokinetics (PK) in vivo. We developed a standardized pH gradient FcRn affinity liquid chromatography method with conditions closely resembling the physiological mechanism of interaction between IgG and FcRn. This method allows the separation of molecular IgG isoforms, degradation products and engineered molecules based on their affinity to FcRn. Human FcRn was immobilized on the column and a linear pH gradient from pH 5.5 to 8.8 was applied. FcRn chromatography was used in comparison to surface plasmon resonance to characterize different monoclonal IgG preparations, e.g., oxidized or aggregated species. Wild-type and engineered IgGs were compared in vitro by FcRn chromatography and in vivo by PK studies in huFcRn transgenic mice. Analytical FcRn chromatography allows differentiation of IgG samples and variants by peak pattern and retention time profile. The method can distinguish: 1) IgGs with different Fabs, 2) oxidized from native IgG, 3) aggregates from monomer and 4) antibodies with mutations in the Fc part from wild-type IgGs. Changes in the FcRn chromatographic behavior of mutant IgGs relative to the wild-type IgG correlate to changes in the PK profile in the FcRn transgenic mice. These results demonstrate that FcRn affinity chromatography is a useful new method for the assessment of IgG integrity. PMID:23765230

  18. Past makes future: role of pFC in prediction.

    Science.gov (United States)

    Fuster, Joaquín M; Bressler, Steven L

    2015-04-01

    The pFC enables the essential human capacities for predicting future events and preadapting to them. These capacities rest on both the structure and dynamics of the human pFC. Structurally, pFC, together with posterior association cortex, is at the highest hierarchical level of cortical organization, harboring neural networks that represent complex goal-directed actions. Dynamically, pFC is at the highest level of the perception-action cycle, the circular processing loop through the cortex that interfaces the organism with the environment in the pursuit of goals. In its predictive and preadaptive roles, pFC supports cognitive functions that are critical for the temporal organization of future behavior, including planning, attentional set, working memory, decision-making, and error monitoring. These functions have a common future perspective and are dynamically intertwined in goal-directed action. They all utilize the same neural infrastructure: a vast array of widely distributed, overlapping, and interactive cortical networks of personal memory and semantic knowledge, named cognits, which are formed by synaptic reinforcement in learning and memory acquisition. From this cortex-wide reservoir of memory and knowledge, pFC generates purposeful, goal-directed actions that are preadapted to predicted future events.

  19. Inhibition of growth of Toxoplasma gondii in cultured fibroblasts by human recombinant gamma interferon.

    Science.gov (United States)

    Pfefferkorn, E R; Guyre, P M

    1984-01-01

    The growth of Toxoplasma gondii in cultured human fibroblasts was inhibited by recombinant human gamma interferon at concentrations of 8 to 16 U/ml. The interferon was titrated by observing a total inhibition of parasite plaque formation 7 days after infection. Inhibition of the growth of T. gondii in the early days after infection was measured by marked reductions in the incorporation of radioactive uracil, a precursor that can only be used by the parasites. This assay showed that when cells were pretreated with gamma interferon for 1 day and then infected, inhibition of T. gondii growth could be readily detected 1 or 2 days after infection. When the pretreatment was omitted and parasites and gamma interferon were added at the same time, no inhibition of parasite growth could be detected 1 day later, although it was apparent after 2 days. Cultures from which the gamma interferon had been removed by washing after a 1-day treatment showed inhibition of T. gondii growth. Gamma interferon had no effect on the viability of extracellular parasites, but it did inhibit the synthesis of host cell RNA and protein by ca. 50% 3 days after treatment. This degree of inhibition is unlikely, of itself, to compromise the growth of T. gondii. Recombinant alpha and beta interferons had no effect on the growth of T. gondii. Images PMID:6425215

  20. Alterations in interferon-gamma and nitric oxide levels in human echinococcosis.

    Science.gov (United States)

    Ait Aissa, S; Amri, M; Bouteldja, R; Wietzerbin, J; Touil-Boukoffa, C

    2006-05-15

    Human cystic hydatid disease is characterized by the long-term coexistence of Echinococcus granulosus and its host without effective rejection of the parasite. This parasitic helminth infection currently constitutes a major health problem in Algeria. We investigated interferon-gamma (IFN-gamma) and nitrite (NO2-) production in PBMC culture 2 supernatants from Algerian patients (n = 35), stimulated by a major antigen (antigen 5). Nitrite was also observed in 74 sera and 28 cyst fluids of patients carrying cysts in different locations. In addition, we report the detection of Nitric Oxide Synthase-2 (NOS2) in liver biopsies of patients (n = 8) by an immunochemical method using human NOS2 antibody. In vivo nitrite levels in host sera and cyst biological fluid point to a tight relation between host response and macro-parasite effects. Our in vitro results indicate a correlation between nitrite and IFN-gamma production in PBMC culture supernatants. Furthermore, by immunohistochemistry NOS2 expression was observed in hepatocytes and Küpffer cells from hydatid patients. Collectively, our data imply NO production in host defense against the extracellular parasite, probably in response to an IFN-gamma activating signal. Concomitant enhanced levels of IFN-gamma and nitrite represent useful indicators of the clinical aggressiveness of hydatidosis.

  1. Distinguishing mechanisms of gamma frequency oscillations in human current source signals using a computational model of a laminar neocortical network

    Directory of Open Access Journals (Sweden)

    Shane eLee

    2013-12-01

    Full Text Available Gamma frequency rhythms have been implicated in numerous studies for their role in healthy and abnormal brain function. The frequency band has been described to encompass as broad a range as 30–150 Hz. Crucial to understanding the role of gamma in brain function is an identification of the underlying neural mechanisms, which is particularly difficult in the absence of invasive recordings in macroscopic human signals such as those from magnetoencephalography (MEG and electroencephalography (EEG. Here, we studied features of current dipole (CD signals from two distinct mechanisms of gamma generation, using a computational model of a laminar cortical circuit designed specifically to simulate CDs in a biophysically principled manner (Jones et al., 2007; Jones et al., 2009. We simulated spiking pyramidal interneuronal gamma (PING whose period is regulated by the decay time constant of GABAA-mediated synaptic inhibition and also subthreshold gamma driven by gamma-periodic exogenous excitatory synaptic drive. Our model predicts distinguishable CD features created by spiking PING compared to subthreshold driven gamma that can help to disambiguate mechanisms of gamma oscillations in human signals. We found that gamma rhythms in neocortical layer 5 can obscure a simultaneous, independent gamma in layer 2/3. Further, we arrived at a novel interpretation of the origin of high gamma frequency rhythms (100–150 Hz, showing that they emerged from a specific temporal feature of CDs associated with single cycles of PING activity and did not reflect a separate rhythmic process. Last we show that the emergence of observable subthreshold gamma required highly coherent exogenous drive. Our results are the first to demonstrate features of gamma oscillations in human current source signals that distinguish cellular and circuit level mechanisms of these rhythms and may help guide understanding of their functional role.

  2. MOUSE ANTIBODY RESPONSE FOLLOWING REPETITIVE INJECTIONS OF GAMMA-IRRADIATED HUMAN PLACENTA COLLAGENA

    Institute of Scientific and Technical Information of China (English)

    刘秉慈; MelvinSpira; 许增禄

    1994-01-01

    Injectable bovine collagen has been used clinically for years.But both the necessity of repeated injections to maintain corrections and the question of adverse allergic reactions developing from the use of a xenogenic collagen have been an area of serious concern.To overoome these adyerse effects,we have developed injectable collagen preparations from human placenta.Gamma irradiation was used for sterilization and crosslinking of the collagen.We observed the mouse immune respose to gamma-irradiated human placenta soluble and insoluble collagen follow-ing multiple injections.After six injections of these materials,no total IgG level increase was found,nor was anti-body specifically directed against human collagen found.Mouse antibody levels were also observed following Zyderm Ⅱ and Zyplast repetitive injections and follow-ing repetitive implantations of coated vicryl and chromic gut.No humoral immune response was found in this het-erologous type system.

  3. Scanning electron microscopic assessment on surface morphology of preserved human amniotic membrane after gamma sterilisation.

    Science.gov (United States)

    Ab Hamid, Suzina Sheikh; Zahari, Nor Kamalia; Yusof, Norimah; Hassan, Asnah

    2014-03-01

    Human amniotic membrane that has been processed and sterilised by gamma irradiation is widely used as a biological dressing in surgical applications. The morphological structure of human amniotic membrane was studied under scanning electron microscopy (SEM) to assess effects of gamma radiation on human amniotic membrane following different preservation methods. The amniotic membrane was preserved by either air drying or submerged in glycerol before gamma irradiated at 15, 25 and 35 kGy. Fresh human amniotic membrane, neither preserved nor irradiated was used as the control. The surface morphology of glycerol preserved amnion was found comparable to the fresh amniotic membrane. The cells of the glycerol preserved was beautifully arranged, homogonous in size and tended to round up. The cell structure in the air dried preserved amnion seemed to be flattened and dehydrated. The effects of dehydration on intercellular channels and the microvilli on the cell surface were clearly seen at higher magnifications (10,000×). SEM revealed that the changes of the cell morphology of the glycerol preserved amnion were visible at 35 kGy while the air dried already changed at 25 kGy. Glycerol preservation method is recommended for human amniotic membrane as the cell morphological structure is maintained and radiation doses lower than 25 kGy for sterilization did not affect the appearance of the preserved amnion.

  4. Gelam Honey Protects against Gamma-Irradiation Damage to Antioxidant Enzymes in Human Diploid Fibroblasts

    OpenAIRE

    Suzana Makpol; Tengku Ahbrizal Farizal Tengku Ahmad; Yasmin Anum Mohd Yusof; Nor Fadilah Rajab; Khairuddin Abdul Rahim; Zakiah Jubri

    2013-01-01

    The present study was designed to determine the radioprotective effects of Malaysian Gelam honey on gene expression and enzyme activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) of human diploid fibroblasts (HDFs) subjected to gamma-irradiation. Six groups of HDFs were studied: untreated control, irradiated HDFs, Gelam honey-treated HDFs and HDF treated with Gelam honey pre-, during- and post-irradiation. HDFs were treated with 6 mg/mL of sterilized Gelam ...

  5. Dose-Response Curve of Chromosome Aberrations in Human Lymphocytes Induced by Gamma-Rays

    Directory of Open Access Journals (Sweden)

    Y. Lusiyanti

    2013-12-01

    Full Text Available Chromosome aberration is a biomarker to predict the level of cell damage caused by exposure to ionizing radiation on human body. Dicentric chromosome is a specific chromosome aberration caused by ionizing radiation and is used as a gold standard biodosimetry of individuals over exposed to ionizing radiation. In radiation accident the dicentric assays has been applied as biological dosimetry to estimate radiation absorbed dose and also to confirm the radiation dose received to radiation workers.The purpose of this study was to generate a dose response curve of chromosome aberration (dicentric in human lymphocyte induced by gamma radiation. Peripheral blood samples from three non smoking healthy volunteers aged between 25-48 years old with informed consent were irradiated with dose between 0.1-4.0 Gy and a control using gamma teletherapy source. The culture procedure was conducted following the IAEA standard procedures with slight modifications. Analysis of dose-response curves used was LQ model Y = a + αD + βD2. The result showed that α and β values of the curve obtained were 0.018 ± 0.006 and 0.013 ± 0.002, respectively. Dose response calibration curve for dicentric chromosome aberrations in human lymphocytes induced by gamma-radiation fitted to linear quadratic model. In order to apply the dose response curve of chromosome aberration disentric for biodosimetry, this standar curve still need to be validated.

  6. Gelam Honey Protects against Gamma-Irradiation Damage to Antioxidant Enzymes in Human Diploid Fibroblasts

    Directory of Open Access Journals (Sweden)

    Suzana Makpol

    2013-02-01

    Full Text Available The present study was designed to determine the radioprotective effects of Malaysian Gelam honey on gene expression and enzyme activity of superoxide dismutase (SOD, catalase (CAT and glutathione peroxidase (GPx of human diploid fibroblasts (HDFs subjected to gamma-irradiation. Six groups of HDFs were studied: untreated control, irradiated HDFs, Gelam honey-treated HDFs and HDF treated with Gelam honey pre-, during- and post-irradiation. HDFs were treated with 6 mg/mL of sterilized Gelam honey (w/v for 24 h and exposed to 1 Gray (Gy of gamma rays at the dose rate of 0.25 Gy/min. Gamma-irradiation was shown to down-regulate SOD1, SOD2, CAT and GPx1 gene expressions (p < 0.05. Conversely, HDFs treated with Gelam honey alone showed up-regulation of all genes studied. Similarly, SOD, CAT and GPx enzyme activities in HDFs decreased with gamma-irradiation and increased when cells were treated with Gelam honey (p < 0.05. Furthermore, of the three different stages of study treatment, pre-treatment with Gelam honey caused up-regulation of SOD1, SOD2 and CAT genes expression and increased the activity of SOD and CAT. As a conclusion, Gelam honey modulates the expression of antioxidant enzymes at gene and protein levels in irradiated HDFs indicating its potential as a radioprotectant agent.

  7. The human glia maturation factor-gamma gene: genomic structure and mutation analysis in gliomas with chromosome 19q loss.

    Science.gov (United States)

    Peters, N; Smith, J S; Tachibana, I; Lee, H K; Pohl, U; Portier, B P; Louis, D N; Jenkins, R B

    1999-09-01

    Human glia maturation factor-gamma (hGMF-gamma) is a recently identified gene that may be involved in glial differentiation, neural regeneration, and inhibition of tumor cell proliferation. The gene maps to the long arm of chromosome 19 at band q13.2, a region that is frequently deleted in human malignant gliomas and is thus suspected to harbor a glioma tumor suppressor gene. Given the putative role of hGMF-gamma in cell differentiation and proliferation and its localization to chromosome 19q13, this gene is an interesting candidate for the chromosome 19q glioma tumor suppressor gene. To evaluate this possibility, we determined the genomic structure of human hGMF-gamma and performed mutation screening in a series of 41 gliomas with and without allelic loss of chromosome 19q. Mutations were not detected, which suggests that hGMF-gamma is not the chromosome 19q glioma suppressor gene. However, the elucidation of the genomic structure of hGMF-gamma may prove useful in future investigations of hGMF-gamma in the normal adult and developing human nervous system.

  8. Distribution of FcγR gene polymorphisms among two sympatric populations in Mali: differing allele frequencies, associations with malariometric indices and implications for genetic susceptibility to malaria.

    Science.gov (United States)

    Cherif, Mariama; Amoako-Sakyi, Daniel; Dolo, Amagana; Pearson, Jan-Olov; Gyan, Ben; Obiri-Yeboah, Dorcas; Nebie, Issa; Sirima, Sodiomon B; Doumbo, Ogobara; Troye-Blomberg, Marita; Bakary, Maiga

    2016-01-19

    Genetic polymorphisms in the complex gene cluster encoding human Fc-gamma receptors (FcγRs) may influence malaria susceptibility and pathogenesis. Studying genetic susceptibility to malaria is ideal among sympatric populations because the distribution of polymorphic genes among such populations can help in the identification malaria candidate genes. This study determined the distribution of three FcyRs single nucleotide polymorphisms (SNPs) (FcγRIIB-rs1050519, FcγRIIC-rs3933769 and FcγRIIIA-rs396991) among sympatric Fulani and Dogon children with uncomplicated malaria. The association of these SNPs with clinical, malariometric and immunological indices was also tested. This study involved 242 Fulani and Dogon volunteers from Mali age under 15 years. All SNPs were genotyped with predesigned TaqMan(®) SNP Genotyping Assays. Genotypic and allelic distribution of SNPs was compared across ethnic groups using the Fisher exact test. Variations in clinical, malariometric and immunologic indices between groups were tested with Kruskal-Wallis H, Mann-Whitney U test and Fisher exact test where appropriate. The study confirmed known malariometric and immunologic differences between sympatric Fulani and non-Fulani tribes. Parasite density was lower in the Fulani than the Dogon (p Dogon (p Dogon (p = 0.0043). The difference in the mutant allele frequency of FcγRIIB (rs1050519) between the two ethnic groups was however not statistically significant (p = 0.064). The mutant allele of rs396991 was associated with high malaria-specific IgG1 and IgG3 in the entire study population and Dogon tribe, p = 0.023 and 0.015, respectively. Parasite burden was lower in carriers of the FcγRIIC (rs3933769) mutant allele than non-carriers in the entire study population (p Dogon indirectly suggest that these SNPs may influence malaria susceptibility and pathogenesis in the study population. The high frequency of the FcγRIIC (rs3933769) mutant allele in the Fulani and its

  9. Fcγ receptor expression on splenic macrophages in adult immune thrombocytopenia

    NARCIS (Netherlands)

    Audia, S; Santegoets, K; Laarhoven, A G; Vidarsson, G; Facy, O; Ortega-Deballon, P; Samson, M; Janikashvili, N; Saas, P; Bonnotte, B; Radstake, T R

    2017-01-01

    Splenic macrophages play a key role in immune thrombocytopenia (ITP) pathogenesis by clearing opsonized platelets. Fcγ receptors (FcγR) participate in this phenomenon, but their expression on splenic macrophages and their modulation by treatment have scarcely been studied in human ITP. We aimed to

  10. Effect of gamma radiation and endodontic treatment on mechanical properties of human and bovine root dentin

    Energy Technology Data Exchange (ETDEWEB)

    Novais, Veridiana Resende; Soares, Priscilla Barbosa Ferreira; Guimaraes, Carlla Martins; Schliebe, Lais Rani Sales Oliveira; Braga, Stella Sueli Lourenco; Soares, Carlos Jose, E-mail: carlosjsoares@ufu.br [Universidade Federal de Uberlandia (UFU), MG (Brazil)

    2016-11-15

    This study evaluated the effect of gamma radiation and endodontic treatment on the microhardness and flexural strength of human and bovine root dentin. Forty single rooted human teeth and forty bovine incisor teeth were collected, cleaned and stored in distilled water at 4 °C. The human and bovine teeth were divided into 4 groups (n=10) resulting from the combination of two study factors: first, regarding the endodontic treatment in 2 levels: with or without endodontic treatment; and second, radiotherapy in two levels: with or without radiotherapy by 60 Gy of Co-60 gamma radiation fractioned into 2 Gy daily doses five days per week. Each tooth was longitudinally sectioned in two parts; one-half was used for the three-point bending test and the other for the Knoop hardness test (KHN). Data were analyzed by 3-way ANOVA and Tukey HSD test (α=0.05). No significant difference was found for flexural strength values. The human dentin had significantly higher KHN than the bovine. The endodontic treatment and radiotherapy resulted in significantly lower KHN irrespective of tooth origin. The results indicated that the radiotherapy had deleterious effects on the microhardness of human and bovine dentin and this effect is increased by the interaction with endodontic therapy. The endodontic treatment adds additional negative effect on the mechanical properties of radiated tooth dentin; the restorative protocols should be designed taking into account this effect. (author)

  11. Parametric variation of gamma frequency and power with luminance contrast: A comparative study of human MEG and monkey LFP and spike responses.

    Science.gov (United States)

    Hadjipapas, A; Lowet, E; Roberts, M J; Peter, A; De Weerd, P

    2015-05-15

    Gamma oscillations contribute significantly to the manner in which neural activity is bound into functional assemblies. The mechanisms that underlie the human gamma response, however, are poorly understood. Previous computational models of gamma rely heavily on the results of invasive recordings in animals, and it is difficult to assess whether these models hold in humans. Computational models of gamma predict specific changes in gamma spectral response with increased excitatory drive. Hence, differences and commonalities between spikes, LFPs and MEG in the spectral responses to changes in excitatory drive can lead to a refinement of existing gamma models. We compared gamma spectral responses to varying contrasts in a monkey dataset acquired previously (Roberts et al., 2013) with spectral responses to similar contrast variations in a new human MEG dataset. We found parametric frequency shifts with increasing contrast in human MEG at the single-subject and the single-trial level, analogous to those observed in the monkey. Additionally, we observed parametric modulations of spectral asymmetry, consistent across spikes, LFP and MEG. However, while gamma power scaled linearly with contrast in MEG, it saturated at high contrasts in both the LFP and spiking data. Thus, while gamma frequency changes to varying contrasts were comparable across spikes, LFP and MEG, gamma power changes were not. This indicates that gamma frequency may be a more stable parameter across scales of measurements and species than gamma power. The comparative approach undertaken here represents a fruitful path towards a better understanding of gamma oscillations in the human brain.

  12. Integrity of Proteins in Human Saliva after Sterilization by Gamma Irradiation▿

    Science.gov (United States)

    Ruhl, Stefan; Berlenbach, Pereshia; Langenfelder, Sabine; Hörl, Dagmar; Lehn, Norbert; Hiller, Karl-Anton; Schmalz, Gottfried; Durchschlag, Helmut

    2011-01-01

    Microbial contamination of whole human saliva is unwanted for certain in vitro applications, e.g., when utilizing it as a growth substratum for biofilm experiments. The aim of this investigation was to test gamma irradiation for its suitability to sterilize saliva and to investigate the treatment's influence on the composition and integrity of salivary proteins in comparison to filter sterilization. For inhibition of bacterial growth by gamma irradiation, a sterility assurance level of 10−6 was determined to be reached at a dose of 3.5 kGy. At this dose, the integrity of proteins, as measured by fluorescence, circular dichroism, and gel electrophoretic banding pattern, and the enzymatic activities of salivary amylase and lysozyme were virtually unchanged. Filtration reduced the total protein concentration to about half of its original value and decreased lysozyme activity to about 10%. It can be concluded that irradiation is suitable for sterilizing whole saliva in its native form. PMID:21148692

  13. Cell death is induced by ciglitazone, a peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonist, independently of PPAR{gamma} in human glioma cells

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Myoung Woo; Kim, Dae Seong; Kim, Hye Ryung; Kim, Hye Jin; Yang, Jin Mo; Ryu, Somi; Noh, Yoo Hun; Lee, Soo Hyun; Son, Meong Hi; Jung, Hye Lim; Yoo, Keon Hee [Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of); Koo, Hong Hoe, E-mail: hhkoo@skku.edu [Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of); Sung, Ki Woong, E-mail: kwsped@skku.edu [Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of)

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Greater than 30 {mu}M ciglitazone induces cell death in glioma cells. Black-Right-Pointing-Pointer Cell death by ciglitazone is independent of PPAR{gamma} in glioma cells. Black-Right-Pointing-Pointer CGZ induces cell death by the loss of MMP via decreased Akt. -- Abstract: Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) regulates multiple signaling pathways, and its agonists induce apoptosis in various cancer cells. However, their role in cell death is unclear. In this study, the relationship between ciglitazone (CGZ) and PPAR{gamma} in CGZ-induced cell death was examined. At concentrations of greater than 30 {mu}M, CGZ, a synthetic PPAR{gamma} agonist, activated caspase-3 and induced apoptosis in T98G cells. Treatment of T98G cells with less than 30 {mu}M CGZ effectively induced cell death after pretreatment with 30 {mu}M of the PPAR{gamma} antagonist GW9662, although GW9662 alone did not induce cell death. This cell death was also observed when cells were co-treated with CGZ and GW9662, but was not observed when cells were treated with CGZ prior to GW9662. In cells in which PPAR{gamma} was down-regulated cells by siRNA, lower concentrations of CGZ (<30 {mu}M) were sufficient to induce cell death, although higher concentrations of CGZ ( Greater-Than-Or-Slanted-Equal-To 30 {mu}M) were required to induce cell death in control T98G cells, indicating that CGZ effectively induces cell death in T98G cells independently of PPAR{gamma}. Treatment with GW9662 followed by CGZ resulted in a down-regulation of Akt activity and the loss of mitochondrial membrane potential (MMP), which was accompanied by a decrease in Bcl-2 expression and an increase in Bid cleavage. These data suggest that CGZ is capable of inducing apoptotic cell death independently of PPAR{gamma} in glioma cells, by down-regulating Akt activity and inducing MMP collapse.

  14. Modulating immunogenicity of factor IX by fusion to an immunoglobulin Fc domain: a study using a hemophilia B mouse model.

    Science.gov (United States)

    Levin, D; Lagassé, H A D; Burch, E; Strome, S; Tan, S; Jiang, H; Sauna, Z E; Golding, B

    2017-04-01

    Essentials Fc-fusion increases a therapeutic's half-life, but FcγR interactions may impact immunogenicity. Species-specific Fc-FcγR interactions allow for mechanistic in vivo studies using mouse models. Fc fusion modulates the immune response to factor IX in hemophilia B mice by eliciting Th1 bias. This model could inform future studies of IgE-associated anaphylaxis in hemophilia B patients. Background Fc fusion is a platform technology used to increase the circulating half-life of protein and peptide therapeutics. However, there are potential immunological consequences with this approach, such as changes in the molecule's immunogenicity as well as possible interactions with a repertoire of Fc receptors (FcR) that can modulate immune responses. Objectives/Methods Using a mouse hemophilia B (HB) model, we compared the immune responses to infusions of recombinant human factor IX (hFIX) and hFIX fused to mouse IgG2a-Fc (hFIX-mFc). The mFc was employed to allow species-specific Fc-FcγR interactions. Results Although treatment with hFIX-mFc altered the early development of anti-FIX IgG, no significant differences in anti-FIX antibody titers were observed at the end of the treatment regimen (5 weeks) or upon anamnestic response (5 months). However, treatment with hFIX-mFc elicited higher FIX-neutralizing antibody levels and resulted in reduced IgE titers compared with the hFIX-treated group. Additionally, differences in plasma cytokine levels and in vitro CD4(+) T-cell responses suggest that whereas hFIX treatment triggered a Th2-biased immune response, hFIX-mFc treatment induced Th1-biased CD4(+) T cells. We also show that hFIX-mFc bound to soluble FcγRs and engaged with FcγRs on different cell types, which may impact antigen presentation. Conclusions These studies provide a model system to study how Fc-fusion proteins may affect immune mechanisms. We used this model to demonstrate a plausible mechanism by which Fc fusion may modulate the IgE response to hFIX. This

  15. Interferon gamma blocks the growth of Toxoplasma gondii in human fibroblasts by inducing the host cells to degrade tryptophan.

    Science.gov (United States)

    Pfefferkorn, E R

    1984-01-01

    Treatment of human fibroblasts with human recombinant gamma interferon blocked the growth of Toxoplasma gondii, an obligate intracellular protozoan parasite. Growth of the parasite was measured by a plaque assay 7 days after infection or by the incorporation of [3H]uracil 1 or 2 days after infection. The antitoxoplasma activity induced in the host cells by gamma interferon was strongly dependent upon the tryptophan concentration of the medium. Progressively higher minimal inhibitory concentrations of gamma interferon were observed as the tryptophan concentration in the culture medium was increased. Treatment with gamma interferon did not make the cells impermeable to tryptophan. The kinetics of [3H]tryptophan uptake into the acid-soluble pools of control and gamma interferon-treated cultures were identical during the first 48 sec. Thereafter uptake of [3H]tryptophan into the acid-soluble pool of control fibroblasts reached the expected plateau after 96 sec. In contrast, uptake of [3H]tryptophan continued for at least 12 min in the gamma interferon-treated cultures. At that time, the acid-soluble pool of the gamma interferon-treated cultures contained 8 times the radioactivity of the control cultures. This continued accumulation was the result of rapid intracellular degradation of [3H]tryptophan into kynurenine and N-formylkynurenine that leaked slowly from the cells. These two metabolites were also recovered from the medium of cultures treated for 1 or 2 days with gamma interferon. Human recombinant alpha and beta interferons, which have no antitoxoplasma activity, did not induce any detectable degradation of tryptophan. Several hypotheses are presented to explain how the intracellular degradation of tryptophan induced by gamma interferon could restrict the growth of an obligate intracellular parasite. Images PMID:6422465

  16. Human gamma interferon and tumor necrosis factor exert a synergistic blockade on the replication of herpes simplex virus.

    Science.gov (United States)

    Feduchi, E; Alonso, M A; Carrasco, L

    1989-03-01

    The replication of herpes simplex virus type 1 (HSV-1) is not inhibited in either HeLa or HEp-2 cells treated with human alpha interferon (HuIFN-alpha), particularly when high multiplicities of infection are used. However, HuIFN-gamma partially inhibits HSV-1 translation in HEp-2 cells infected at low multiplicities. Under these conditions, the transcription of genes alpha 22, TK, and gamma 0 is greatly diminished. The combined addition of human tumor necrosis factor (TNF) and HuIFN-gamma to HEp-2 cells exerts a synergistic inhibition of HSV-1 translation. Cells treated with both cytokines continue synthesizing cellular proteins, even 20 h after HSV-1 infection. As little as 10 U of IFN-gamma per ml blocked HSV-1 DNA replication, provided that TNF was also present in the medium. Analyses of HSV-1 gene transcription suggest that the action of both TNF and IFN-gamma blocked a step that comes at or prior to early HSV-1 gene expression. This early step in HSV-1 replication inhibited by TNF and IFN-gamma occurs after virus attachment and entry into cells, since the internalization of radioactive HSV-1 virion particles was not blocked by the presence of the two cytokines. Therefore, we conclude that the synergistic action of TNF plus IFN-gamma affects a step in HSV-1 replication that comes after virus entry but before or at the transcription of immediate-early genes.

  17. Association between Fcγ receptor IIA, IIIA and IIIB genetic polymorphisms and susceptibility to severe malaria anemia in children in western Kenya.

    Science.gov (United States)

    Munde, Elly O; Okeyo, Winnie A; Raballah, Evans; Anyona, Samuel B; Were, Tom; Ong'echa, John M; Perkins, Douglas J; Ouma, Collins

    2017-04-20

    Naturally-acquired immunity to Plasmodium falciparum malaria develops after several episodes of infection. Fc gamma receptors (FcγRs) bind to immunoglobulin G (IgG) antibodies and mediate phagocytosis of opsonized microbes, thereby, linking humoral and cellular immunity. FcγR polymorphisms influence binding affinity to IgGs and consequently, can influence clinical malaria outcomes. Specifically, variations in FcγRIIA -131Arg/His, FcγRIIIA-176F/V and FcγRIIIB-NA1/NA2 modulate immune responses through altered binding preferences to IgGs and immune complexes. Differential binding, in turn, changes ability of immune cells to respond to infection through production of inflammatory mediators during P. falciparum infection. We determined the association between haplotypes of FcγRIIA-131Arg/His, FcγRIIIA-176F/V and FcγRIIIB-NA1/NA2 variants and severe malarial anemia (SMA; hemoglobin malaria in a holoendemic transmission region of western Kenya. FcγRIIA-131Arg/His and FcγRIIIA-176F/V genotypes were determined using TaqMan® SNP genotyping, while FcγRIIIBNA1/NA2 genotypes were determined using restriction fragment length polymorphism. Hematological and parasitological indices were measured in all study participants. Carriage of FcγRIIA-131Arg/FcγRIIIA-176F/FcγRIIIBNA2 haplotype was associated with susceptibility to SMA (OR = 1.70; 95% CI; 1.02-2.93; P = 0.036), while the FcγRIIA-131His/ FcγRIIIA-176F/ FcγRIIIB NA1 haplotype was marginally associated with enhanced susceptibility to SMA (OR: 1.80, 95% CI; 0.98-3.30, P = 0.057) and higher levels of parasitemia (P = 0.009). Individual genotypes of FcγRIIA-131Arg/His, FcγRIIIA-176F/V and FcγRIIIB-NA1/NA2 were not associated with susceptibility to SMA. The study revealed that haplotypes of FcγRs are important in conditioning susceptibility to SMA in immune-naive children from P. falciparum holoendemic region of western Kenya.

  18. High-level expression of a chemically synthesized gene for human interferon-gamma using a prokaryotic expression vector.

    OpenAIRE

    1984-01-01

    A chemically synthesized gene for human interferon-gamma has been cloned into a prokaryotic expression vector under the regulation of a synthetic constitutive transcriptional-translational control unit that contains a strong bacteriophage T5 early promoter and a strong ribosome-binding site. Cells harboring the recombinant plasmid express high levels (4 X 10(9) units per liter of culture) of antiviral activity specific for interferon-gamma. Analysis of total cell lysates on NaDodSO4/polyacryl...

  19. HAL/S-FC compiler system specifications

    Science.gov (United States)

    1976-01-01

    This document specifies the informational interfaces within the HAL/S-FC compiler, and between the compiler and the external environment. This Compiler System Specification is for the HAL/S-FC compiler and its associated run time facilities which implement the full HAL/S language. The HAL/S-FC compiler is designed to operate stand-alone on any compatible IBM 360/370 computer and within the Software Development Laboratory (SDL) at NASA/JSC, Houston, Texas.

  20. A prominent lack of IgG1-Fc fucosylation of platelet alloantibodies in pregnancy.

    Science.gov (United States)

    Kapur, Rick; Kustiawan, Iwan; Vestrheim, Anne; Koeleman, Carolien A M; Visser, Remco; Einarsdottir, Helga K; Porcelijn, Leendert; Jackson, Dave; Kumpel, Belinda; Deelder, André M; Blank, Dennis; Skogen, Björn; Killie, Mette Kjaer; Michaelsen, Terje E; de Haas, Masja; Rispens, Theo; van der Schoot, C Ellen; Wuhrer, Manfred; Vidarsson, Gestur

    2014-01-23

    Immunoglobulin G (IgG) formed during pregnancy against human platelet antigens (HPAs) of the fetus mediates fetal or neonatal alloimmune thrombocytopenia (FNAIT). Because antibody titer or isotype does not strictly correlate with disease severity, we investigated by mass spectrometry variations in the glycosylation at Asn297 in the IgG Fc because the composition of this glycan can be highly variable, affecting binding to phagocyte IgG-Fc receptors (FcγR). We found markedly decreased levels of core fucosylation of anti-HPA-1a-specific IgG1 from FNAIT patients (n = 48), but not in total serum IgG1. Antibodies with a low amount of fucose displayed higher binding affinity to FcγRIIIa and FcγRIIIb, but not to FcγRIIa, compared with antibodies with a high amount of Fc fucose. Consequently, these antibodies with a low amount of Fc fucose showed enhanced phagocytosis of platelets using FcγRIIIb(+) polymorphonuclear cells or FcγRIIIa(+) monocytes as effector cells, but not with FcγRIIIa(-) monocytes. In addition, the degree of anti-HPA-1a fucosylation correlated positively with the neonatal platelet counts in FNAIT, and negatively to the clinical disease severity. In contrast to the FNAIT patients, no changes in core fucosylation were observed for anti-HLA antibodies in refractory thrombocytopenia (post platelet transfusion), indicating that the level of fucosylation may be antigen dependent and/or related to the immune milieu defined by pregnancy.

  1. NFκB induces overexpression of bovine FcRn: a novel mechanism that further contributes to the enhanced immune response in genetically modified animals carrying extra copies of FcRn.

    Science.gov (United States)

    Cervenak, Judit; Doleschall, Márton; Bender, Balázs; Mayer, Balázs; Schneider, Zita; Doleschall, Zoltán; Zhao, Yaofeng; Bősze, Zsuzsanna; Hammarström, Lennart; Oster, Wolfgang; Kacskovics, Imre

    2013-01-01

    Among the many functions of the neonatal Fc receptor (FcRn) for IgG, it binds to IgG-opsonized antigen complexes and propagates their traffic into lysosomes where antigen processing occurs. We previously reported that transgenic (Tg) mice and rabbits that carry multiple copies and overexpress FcRn have augmented humoral immune responses. Nuclear factor-kappa B (NFκB) is a critical molecule in the signaling cascade in the immune response. NFκB induces human FcRn expression and our previous in silico analysis suggested NFκB binding sites in the promoter region of the bovine (b) FcRn α-chain gene (FCGRT). Here, we report the identification of three NFκB transcription binding sites in the promoter region of this gene using luciferase reporter gene technology, electromobility shift assay and supershift analysis. Stimulation of primary bovine endothelial cells with the Toll-like receptor-4 ligand lipopolysaccharide (LPS), which mediates its effect via NFκB, resulted in rapid upregulation of the bFcRn expression and a control gene, bovine E-selectin. This rapid bFcRn gene induction was also observed in the spleen of bFcRn Tg mice treated with intraperitoneally injected LPS, analyzed by northern blot analysis. Finally, NFκB-mediated bFcRn upregulation was confirmed at the protein level in macrophages isolated from the bFcRn Tg mice using flow cytometry with a newly developed FcRn specific monoclonal antibody that does not cross-react with the mouse FcRn. We conclude that NFκB regulates bFcRn expression and thus optimizes its functions, e.g., in the professional antigen presenting cells, and contributes to the much augmented humoral immune response in the bFcRn Tg mice.

  2. Distinct Fcγ receptors mediate the effect of serum amyloid p on neutrophil adhesion and fibrocyte differentiation.

    Science.gov (United States)

    Cox, Nehemiah; Pilling, Darrell; Gomer, Richard H

    2014-08-15

    The plasma protein serum amyloid P (SAP) reduces neutrophil adhesion, inhibits the differentiation of monocytes into fibroblast-like cells called fibrocytes, and promotes phagocytosis of cell debris by macrophages. Together, these effects of SAP reduce key aspects of inflammation and fibrosis, and SAP injections improve lung function in pulmonary fibrosis patients. SAP functions are mediated, in part, by FcγRs, but the contribution of each FcγR is not fully understood. We found that aa Q55 and E126 in human SAP affect human fibrocyte differentiation and SAP binding to FcγRI. E126, K130, and Q128 affect neutrophil adhesion and SAP affinity for FcγRIIa. Q128 also affects phagocytosis by macrophages and SAP affinity for FcγRI. All the identified functionally significant amino acids in SAP form a binding site that is distinct from the previously described SAP-FcγRIIa binding site. Blocking FcγRI with an IgG-blocking Ab reduces the SAP effect on fibrocyte differentiation, and ligating FcγRIIa with Abs reduces neutrophil adhesion. Together, these results suggest that SAP binds to FcγRI on monocytes to inhibit fibrocyte differentiation, and binds to FcγRIIa on neutrophils to reduce neutrophil adhesion.

  3. Characterization of commercial laminin preparations from human placenta in comparison to recombinant laminins 2 (alpha2beta1gamma1), 8 (alpha4beta1gamma1), 10 (alpha5beta1gamma1).

    Science.gov (United States)

    Wondimu, Zenebech; Gorfu, Gezahegn; Kawataki, Tomoyuki; Smirnov, Sergei; Yurchenco, Peter; Tryggvason, Karl; Patarroyo, Manuel

    2006-03-01

    Laminins, a family of large heterotrimeric (alphabetagamma) proteins, are major components of basement membranes implicated in a variety of cellular functions. Different commercial laminin preparations isolated from human placenta have been widely used in functional studies but their molecular properties are poorly known. In the present study, we characterized several of these preparations by ELISA, silver staining and Western blotting, in comparison to mouse laminin 1 (alpha1beta1gamma1), and recombinant human laminins 2 (alpha2beta1gamma1), 8 (alpha4beta1gamma1) and 10 (alpha5beta1gamma1). The cell migration-promoting activity of different batches was also tested. The placenta laminin preparations differed from one another and consisted of highly fragmented proteins, a mixture of laminin isoforms, and/or contaminating fibronectin. Major functional differences between batches were also observed, reflecting molecular heterogeneity. Previous data obtained in functional studies using these preparations need to be interpreted with caution and may require revision, and future functional studies demand prior molecular characterization of the laminins, particularly their alpha-chain.

  4. Phospholipase C-gamma1 is required for subculture-induced terminal differentiation of normal human oral keratinocytes.

    Science.gov (United States)

    Oh, Ju-Eun; Kook, Joong-Ki; Park, Kyung-Hee; Lee, Gene; Seo, Byoung-Moo; Min, Byung-Moo

    2003-04-01

    Serial subculture of primary normal human oral keratinocytes (NHOKs) to the post-mitotic stage induces terminal differentiation, which is in part linked to elevated levels of phospholipase C (PLC)-gamma1. Therefore, PLC-gamma1 may be involved in the signal transduction system that leads to the calcium regulation of subculture-induced keratinocyte differentiation. To test this hypothesis, the expression of PLC-gamma1 in primary NHOKs was blocked by transfecting cells with the antisense PLC-gamma1 cDNA construct. These cells demonstrated dramatic reductions in PLC-gamma1 protein and in the differentiation markers involucrin and transglutaminase following calcium exposure and an increase (15-20%) in in vitro life span versus empty vector-transfected cells. In addition, we established the ability of antisense PLC-gamma1 to block the serial subculture-induced rise in intracellular calcium. Similar observations were made following treatment with the specific PLC inhibitor U73122. These results indicate that the terminal differentiation of NHOKs by serial subculture is associated with PLC-gamma1, which mediates calcium regulation by mobilizing intracellular calcium.

  5. Peroxisome proliferator-activated receptor- gamma expression in human malignant and normal brain, breast and prostate-derived cells.

    Science.gov (United States)

    Nwankwo, J O; Robbins, M E

    2001-01-01

    The constitutive and gamma -linolenic acid (GLA)-induced expression of peroxisome proliferator-activated receptor gamma (PPAR gamma) immunoreactive protein in a panel of human malignant brain (U87MG, T98G); breast (MCF-7, MB MDA-231, MB MDA 435) and prostate (ALVA, DU-145, LNCaP, PC3) cell lines have been compared with those for their normal cell counterparts, the human normal astrocyte (NHA), mammary epithelial (HMEC) and prostate epithelial (PrEC) cells, respectively. Constitutive levels of expression for PPAR gamma protein were significantly higher in the malignant cell lines relative to their normal cells. GLA supplementation did not affect the protein expression in malignant cells but caused 6- and 3-fold increases in normal breast and prostate cells, respectively. Since activation of PPAR gamma protein in some human malignant cell lines has been demonstrated to induce tumour cell death, these findings signal the need to exploit the significantly elevated expression of this protein in the therapy of human cancer. Copyright 2001 Harcourt Publishers Ltd.

  6. HIV-1 envelope gp41 peptides promote migration of human Fc epsilon RI+ cells and inhibit IL-13 synthesis through interaction with formyl peptide receptors.

    Science.gov (United States)

    de Paulis, Amato; Florio, Giovanni; Prevete, Nella; Triggiani, Massimo; Fiorentino, Isabella; Genovese, Arturo; Marone, Gianni

    2002-10-15

    We evaluated the effects of synthetic peptides (2017, 2019, 2020, 2021, 2023, 2027, 2029, 2030, 2031, and 2035) encompassing the structure of HIV-1(MN) envelope gp41 on both chemotaxis of human basophils and the release of preformed mediators (histamine) and of cytokines (IL-13). Peptides 2019 and 2021 were potent basophil chemoattractants, whereas the other peptides examined were ineffective. Preincubation of basophils with FMLP or gp41 2019 resulted in complete desensitization to a subsequent challenge with homologous stimulus. Incubation of basophils with low concentration (5 x 10(-7) M) of FMLP, which binds with high affinity to N-formyl peptide receptor (FPR), but not to FPR-like 1, did not affect the chemotactic response to a heterologous stimulus (gp41 2019). In contrast, a high concentration (10(-4) M) of FMLP, which binds also to FPR-like 1, significantly reduced the chemotactic response to gp41 2019. The FPR antagonist cyclosporin H inhibited chemotaxis induced by FMLP, but not by gp41 2019. None of these peptides singly induced the release of histamine or cytokines (IL-4 and IL-13) from basophils. However, low concentrations of peptides 2019 and 2021 (10(-8)-10(-6) M) inhibited histamine release from basophils challenged with FMLP but not the secretion caused by anti-IgE and gp120. Preincubation of basophils with peptides 2019 and 2021 inhibited the expression of both IL-13 mRNA, and the FMLP-induced release of IL-13 from basophils. These data highlight the complexity of the interactions between viral and bacterial peptides with FPR subtypes on human basophils.

  7. Early and Late Chromosome Damages in Human Lymphocytes Induced by Gamma Rays and Fe Ions

    Science.gov (United States)

    Sunagawa, Mayumi; Zhang, Ye; Yeshitla, Samrawit; Kadhim, Munira; Wilson, Bobby; Wu, Honglu

    2014-01-01

    Chromosomal translocations and inversions are considered stable, and cells containing these types of chromosome aberrations can survive multiple cell divisions. An efficient method to detect an inversion is multi-color banding fluorescent in situ hybridization (mBAND) which allows identification of both inter- and intrachromosome aberrations simultaneously. Post irradiation, chromosome aberrations may also arise after multiple cell divisions as a result of genomic instability. To investigate the stable or late-arising chromosome aberrations induced after radiation exposure, we exposed human lymphocytes to gamma rays and Fe ions ex vivo, and cultured the cells for multiple generations. Chromosome aberrations were analyzed in cells collected at first mitosis and at several time intervals during the culture period post irradiation. With gamma irradiation, about half of the damages observed at first mitosis remained after 7 day- and 14 day- culture, suggesting the transmissibility of damages to the surviving progeny. Detailed analysis of chromosome break ends participating in exchanges revealed a greater fraction of break ends involved in intrachromosome aberrations in the 7- and 14-day samples in comparison to the fraction at first mitosis. In particular, simple inversions were found at 7 and 14 days, but not at the first mitosis, suggesting that some of the aberrations might be formed days post irradiation. In contrast, at the doses that produced similar frequencies of gamma-induced chromosome aberrations as observed at first mitosis, a significantly lower yield of aberrations remained at the same population doublings after Fe ion exposure. At these equitoxic doses, more complex type aberrations were observed for Fe ions, indicating that Fe ion-induced initial chromosome damages are more severe and may lead to cell death. Comparison between low and high doses of Fe ion irradiation in the induction of late damages will also be discussed.

  8. A Novel Factor H-Fc Chimeric Immunotherapeutic Molecule against Neisseria gonorrhoeae.

    Science.gov (United States)

    Shaughnessy, Jutamas; Gulati, Sunita; Agarwal, Sarika; Unemo, Magnus; Ohnishi, Makoto; Su, Xia-Hong; Monks, Brian G; Visintin, Alberto; Madico, Guillermo; Lewis, Lisa A; Golenbock, Douglas T; Reed, George W; Rice, Peter A; Ram, Sanjay

    2016-02-15

    Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhea, has developed resistance to almost every conventional antibiotic. There is an urgent need to develop novel therapies against gonorrhea. Many pathogens, including N. gonorrhoeae, bind the complement inhibitor factor H (FH) to evade complement-dependent killing. Sialylation of gonococcal lipooligosaccharide, as occurs in vivo, augments binding of human FH through its domains 18-20 (FH18-20). We explored the use of fusing FH18-20 with IgG Fc (FH18-20/Fc) to create a novel anti-infective immunotherapeutic. FH18-20 also binds to select host glycosaminoglycans to limit unwanted complement activation on host cells. To identify mutation(s) in FH18-20 that eliminated complement activation on host cells, yet maintained binding to N. gonorrhoeae, we created four mutations in domains 19 or 20 described in atypical hemolytic uremic syndrome that prevented binding of mutated fH to human erythrocytes. One of the mutant proteins (D to G at position 1119 in domain 19; FHD1119G/Fc) facilitated complement-dependent killing of gonococci similar to unmodified FH18-20/Fc but, unlike FH18-20/Fc, did not lyse human erythrocytes. FHD1119G/Fc bound to all (100%) of 15 sialylated clinical N. gonorrhoeae isolates tested (including three contemporary ceftriaxone-resistant strains), mediated complement-dependent killing of 10 of 15 (67%) strains, and enhanced C3 deposition (≥10-fold above baseline levels) on each of the five isolates not directly killed by complement. FHD1119G/Fc facilitated opsonophagocytic killing of a serum-resistant strain by human polymorphonuclear neutrophils. FHD1119G/Fc administered intravaginally significantly reduced the duration and burden of gonococcal infection in the mouse vaginal colonization model. FHD1119G/Fc represents a novel immunotherapeutic against multidrug-resistant N. gonorrhoeae.

  9. Immunoglobulin superantigen protein L induces IL-4 and IL-13 secretion from human Fc epsilon RI+ cells through interaction with the kappa light chains of IgE.

    Science.gov (United States)

    Genovese, Arturo; Borgia, Guglielmo; Björck, Lars; Petraroli, Angelica; de Paulis, Amato; Piazza, Marcello; Marone, Gianni

    2003-02-15

    Peptostreptococcus magnus protein L is a multidomain bacterial surface protein that correlates with virulence. It consists of up to five homologous Ig-binding domains (B1-B5) that interact with the variable domain of Ig kappa L chains. Intact protein L stimulates the synthesis and the release of IL-4 and IL-13 from human basophils in vitro. A protein L fragment covering the Ig-binding domains B1-B4 also induced IL-4 and IL-13 release from basophils. There was an excellent correlation (r(s) = 0.82; p ADZ (kappa chains) blocked both anti-IgE- and protein L-induced secretion. Cyclosporin A, but not cyclosporin H, inhibited protein L-induced release of IL-4 and IL-13 from basophils. Thus, protein L acts as a bacterial Ig superantigen to induce the synthesis and release of IL-4 and IL-13 from basophils by interacting with kappa L chains of the IgE isotype.

  10. Purification and characterization of biologically active human recombinant 37 kDa soluble CD23 (sFc epsilon RII) expressed in insect cells.

    Science.gov (United States)

    Graber, P; Jansen, K; Pochon, S; Shields, J; Aubonney, N; Turcatti, G; Bonnefoy, J Y

    1992-05-18

    Human recombinant soluble 37 kDa CD23 has been expressed in insect cells and secreted into the culture medium using the IL-2 leader sequence. The 37 kDa CD23 was purified 600-fold to homogeneity by monoclonal antibody affinity chromatography and gel filtration. The pure protein is monomeric, glycosylated, depleted of one N terminal amino acid and contains four disulphide bonds. It degrades into smaller fragments of 33, 29 and 25 kDa if purified in the absence of protease inhibitors. The same pattern of proteolytic fragments is observed when the pure preparation is incubated at room temperature for 3 weeks. Physical characterization of the 37 kDa CD23 by circular dichroism indicates that the protein contains mainly beta sheet and 20% of alpha helical structures. Specific binding of IgE to natural CD23 (low affinity IgE receptor) was inhibited by purified recombinant 37 kDa CD23. Moreover, purified recombinant 37kDa CD23 and interleukin-1 promoted the survival of germinal centre B cells.

  11. Fcγ receptor IIb strongly regulates Fcγ receptor-facilitated T cell activation by dendritic cells

    NARCIS (Netherlands)

    N. van Montfoort (Nadine); P.A.C. 't Hoen (Peter); S.M. Mangsbo (Sara); M. Camps (Marcel); P. Boross (Peter); C.J.M. Melief (Cornelis); F. Ossendorp (Ferry); J.S. Verbeek (Sjef)

    2012-01-01

    textabstractFcγR ligation by Ag-Ab immune complexes (IC) not only mediates effective Ag uptake, but also strongly initiates dendritic cell (DC) maturation, a requirement for effective T cell activation. Besides the activating FcγRI, FcγRIII, and FcγRIV, the inhibitory FcγRIIb is expressed on DCs. It

  12. Dicty_cDB: FC-AV24 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AV24 (Link to dictyBase) - - - Contig-U16482-1 FC-AV24E (Li...nk to Original site) - - - - - - FC-AV24E 591 Show FC-AV24 Library FC (Link to library) Clone ID FC-AV24 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AV/FC-AV24Q.Seq.d/ Representative seq. ID FC-AV...24E (Link to Original site) Representative DNA sequence >FC-AV24 (FC-AV24Q) /CSM/FC/FC-AV/FC-AV24Q.Seq....RFWYFLSKIVKMKKSTGEIL NVTEIFEDKPQKVKNFGVFIRYNSRSGTHNIYKEYRDLTRCGAVSQMYDEMASRHSARES SIHIIDIKEIAASLTRRANTKQFHDS

  13. Dicty_cDB: FC-AI21 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AI21 (Link to dictyBase) - - - Contig-U16254-1 FC-AI21Z (Li...nk to Original site) - - FC-AI21Z 696 - - - - Show FC-AI21 Library FC (Link to library) Clone ID FC-AI21 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI21Q.Seq.d/ Representative seq. ID FC-AI...21Z (Link to Original site) Representative DNA sequence >FC-AI21 (FC-AI21Q) /CSM/FC/FC-AI/FC-AI21Q.Seq....YPGYMYTDLSTIYERAGRIQGRNGSITQI PILTMPNDDITHPIPDLTGYITEGQIFIDRQINNRQIYPPINVLPSLSRLMKSAI

  14. Dicty_cDB: FC-AI05 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AI05 (Link to dictyBase) - - - Contig-U15516-1 FC-AI05E (Li...nk to Original site) - - - - - - FC-AI05E 1189 Show FC-AI05 Library FC (Link to library) Clone ID FC-AI05 (L...//dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI05Q.Seq.d/ Representative seq. ID FC-AI...05E (Link to Original site) Representative DNA sequence >FC-AI05 (FC-AI05Q) /CSM/FC/FC-AI/FC-AI05Q.Seq...KIVGEASLKNKGKMSRVLAAKAALSARFD ALCEVSDTSYGIAYKGAVDRRAAAIEGREVRKSLNAVKPEKSGNVAKYDHTKSATTNTTR DVATKSSKESSIKQEKQ

  15. Dicty_cDB: FC-AI11 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AI11 (Link to dictyBase) - - - Contig-U15122-1 FC-AI11E (Li...nk to Original site) - - - - - - FC-AI11E 1040 Show FC-AI11 Library FC (Link to library) Clone ID FC-AI11 (L...//dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI11Q.Seq.d/ Representative seq. ID FC-AI...11E (Link to Original site) Representative DNA sequence >FC-AI11 (FC-AI11Q) /CSM/FC/FC-AI/FC-AI11Q.Seq...VLSPEIKKGSWDEAEEELLFQLVDKHGQSWKNVAIEIKTRTDIQCRYQYFKAI MSRQTEWNQLEDDILTKKIKLMTQNNEKISFQQVSKHLARAKTTKIPRTALECK

  16. Dicty_cDB: FC-AI04 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AI04 (Link to dictyBase) - - - Contig-U15121-1 FC-AI04E (Li...nk to Original site) - - - - - - FC-AI04E 772 Show FC-AI04 Library FC (Link to library) Clone ID FC-AI04 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI04Q.Seq.d/ Representative seq. ID FC-AI...04E (Link to Original site) Representative DNA sequence >FC-AI04 (FC-AI04Q) /CSM/FC/FC-AI/FC-AI04Q.Seq....qvnkhqqvvtktvsd vlvphqvhnqvfphipqqmtlvnkhqpvvtktvsdvlvphqvhnqvfphtpqlkiqvylq vfqvvvvtiisai

  17. Dicty_cDB: FC-AI10 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AI10 (Link to dictyBase) - - - Contig-U16270-1 FC-AI10F (Li...nk to Original site) FC-AI10F 405 - - - - - - Show FC-AI10 Library FC (Link to library) Clone ID FC-AI10 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI10Q.Seq.d/ Representative seq. ID FC-AI...10F (Link to Original site) Representative DNA sequence >FC-AI10 (FC-AI10Q) /CSM/FC/FC-AI/FC-AI10Q.Seq.... sequence RKKRKSDYTSFSTYIHKLLKQITPPTNAKSNEKGDRKFTISSKAMSVMNSFVHDIFDRIA TEASGLAKKKKRQTLHSRDIQVAVRIILTGELAXHAI

  18. Dicty_cDB: FC-AI23 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AI23 (Link to dictyBase) - - - Contig-U15308-1 FC-AI23Z (Li...nk to Original site) - - FC-AI23Z 603 - - - - Show FC-AI23 Library FC (Link to library) Clone ID FC-AI23 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI23Q.Seq.d/ Representative seq. ID FC-AI...23Z (Link to Original site) Representative DNA sequence >FC-AI23 (FC-AI23Q) /CSM/FC/FC-AI/FC-AI23Q.Seq....LNTLAKKNEQVVEGEILAKQLTGVTAEELSEFKACFSHFDKDN DNKLNRLEFSSCLKSIGDELTEEQLNQVISKIDTDGNGTISFEEFIDYMVSSRKGTDSVE STKAAFKVMAEDKDFITEAQIRAAI

  19. Gamma-aminobutyric acid promotes human hepatocellular carcinoma growth through overexpressed gamma-aminobutyric acid A receptor α3 subunit

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    AIM: To investigate the expression pattern of gamma-aminobutyric acid A (GABAA) receptors in hepatocellular carcinoma (HCC) and indicate the relationship among gamma-aminobutyric acid (GABA), gamrna-aminobutyric acid A receptor α3 subunit (GABRA3) and HCC.METHODS: HCC cell line Chang, HepG2, normal liver cell line L-02 and 8 samples of HCC tissues and paired non-cancerous tissues were analyzed with semiquantitative polymerase chain reaction (PCR) for the expression of GABAA receptors. HepG2 cells were treated with gamma-aminobutyric acid (GABA) at serial concentrations (0, 1, 10, 20, 40 and 60 μmol/L), and their proliferating abilities were analyzed with the 3-(4, 5-methylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, cell doubling time test, colon formation assay, cell cycle analysis and tumor planted in nude mice. Small interfering RNA was used for knocking down the endogenous GABRA3 in HepG2. oliferating abilities of these cells treated with or without GABA were analyzed.RESULTS: We identified the overexpression of GABRA3 in HCC cells. Knockdown of endogenous GABRA3 expression in HepG2 attenuated HCC cell growth, suggesting its role in HCC cell viability. We determined the in vitro and in vivo effect of GABA in the proliferation of GABRA3-positive cell lines, and found that GABA increased HCC growth in a dose-dependent manner. Notably, the addition of GABA into the cell culture medium promoted the proliferation of GABRA3-expressing HepG2 cells, but not GABRA3-knockdown HepG2 cells. This means that GABA stimulates HepG2 cell growth through GABRA3. CONCLUSION: GABA and GABRA3 play important roles in HCC development and progression and can be a promising molecular target for the development of new diagnostic and therapeutic strategies for HCC.

  20. Expression of human vascular endothelial growth factor receptor-Fc in DG44 cells and biological activity of expressed product%人血管内皮生长因子受体-Fc在DG44细胞中的表达及其生物活性

    Institute of Scientific and Technical Information of China (English)

    胡伟伟; 范清林; 尼钢钢; 张玲; 黄辉; 宋礼华

    2013-01-01

    Objective To express human vascular endothelial growth factor receptor (VEGFR)-Fc in Chinese hamster ovary(CHO) DG44 cells and determine its biological activity.Methods The sequence VEGFR1D2-VEGFR2D3 encoding the second and the third human immunoglobulin domains of VEGFR2 was synthesized,and fused with human IgG1Fc by overlap PCR to generate VEGFR-Fc fusion gene,which was then subcloned into eukaryotic expression plasmid pD2.The constructed recombinant plasmid pD2-VEGFR-Fc was transfected to DG44 cells in mediation of FreeStyleTM MAX Reagent and OptiPROTM SFM,and a cell line stably expressing VEGFR-Fc protein was screened by MTX pressure screening.The expressed VEGFR-Fc in cell culture supernatant was determined by SDS-PAGE,Western blot and ELISA,then purified through HiTrapTM ProteinA FF column,and determined for activity by using microscopy and endothelial ECV304 cell model.Results The length of PCR product of VEGFR-Fc gene was 1 377 bp.Restriction analysis and sequencing proved that recombinant plasmid pD2-VEGFR-Fc was constructed correctly.VEGFR-Fc protein was detected in culture supernatant of DG44 cells transfected with plasmid pD2-VEGFR-Fc,of which the expression level was 0.5 g / L.After purification by HiTrapTM ProteinA FF column chromatography,the foreign protein in cell culture supernatant was removed effectively.Purified VEGFR-Fc showed specific binding to VEGF and inhibited the growth of ECV304 cells.Conclusion The VEGFR-Fc with biological activity was successfully expressed in DG44 cells,which laid a foundation of further study on its role in angiogenesis inhibition and anticancer therapy.%目的 在中国仓鼠卵巢细胞DG44中表达人血管内皮生长因子受体(vascular endothelial growth factor receptor,VEGFR)-Fc,并检测其生物活性.方法 化学合成人VEGFR1的第2免疫球蛋白结构域(VEGFR1D2)基因和人VEGFR2的第3免疫球蛋白结构域(VEGFR2D3)基因,通过重叠PCR(Overlap PCR)将VEGFR1D2-VEGFR2D3和人IgG1Fc拼接形

  1. 1. cap alpha. ,25-Dihydroxyvitamin D/sub 3/ inhibits. gamma. -interferon synthesis by normal human peripheral blood lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Reichel, H.; Koeffler, H.P.; Tobler, A.; Norman, A.W.

    1987-05-01

    1..cap alpha..,25-Dihydroxyvitamin D/sub 3/ (1,25-(OH)/sub 2/D/sub 3/), the biologically active metabolite of vitamin D/sub 3/, inhibited synthesis of ..gamma..-interferon (IFN-..gamma..) by phytohemagglutinin-activated peripheral blood lymphocytes (PBLs). A significant reduction of IFN-..gamma.. protein levels in PBL culture medium was achieved with a physiologic 1,25-(OH)/sub 2/D/sub 3/ concentration, 1,25-(OH)/sub 2/D/sub 3/ also inhibited accumulation of IFN-..gamma.. mRNA in activated PBLs in a dose-dependent fashion. The ability of 1,25-(OH)/sub 2/D/sub 3/ to modulate IFN-..gamma.. protein synthesis was unaltered in the presence of high concentrations of recombinant human interleukin 2. The suppression of IFN-..gamma.. synthesis by PBLs was specific for 1,25-(OH)/sub 2/D/sub 3/; the potencies of other vitamin D/sub 3/ metabolites were correlated with their affinities for the cellular 1,25-(OH)/sub 2/D/sub 3/ receptor. The time course of 1,25-(OH)/sub 2/D/sub 3/ receptor expression in phytohemagglutinin-activated PBLs was correlated with the time course of 1,25-(OH)/sub 2/D/sub 3/-mediated inhibition of IFN-..gamma.. synthesis. Finally, the authors examined the effects of 1,25-(OH)/sub 2/D/sub 3/ on the constitutive IFN-..gamma.. production by two human T-lymphocyte lines transformed by human T-lymphotropic virus type I. The cell lines were established from a normal donor (cell line S-LB1) and from a patient with vitamin D-dependent rickets type 2 (cell line Ab-VDR). IFN-..gamma.. synthesis by S-LB1 cells was inhibited in a dose-dependent fashion by 1,25-(OH)/sub 2/D/sub 3/, whereas IFN-..gamma.. synthesis by Ab-VDR cells was not altered by 1,25-(OH)/sub 2/D/sub 3/. The data presented in this study provide evidence for a role of 1,25-(OH)/sub 2/D/sub 3/ in immunoregulation.

  2. Engineering of Immunoglobulin Fc Heterodimers Using Yeast Surface-Displayed Combinatorial Fc Library Screening.

    Directory of Open Access Journals (Sweden)

    Hye-Ji Choi

    Full Text Available Immunoglobulin Fc heterodimers, which are useful scaffolds for the generation of bispecific antibodies, have been mostly generated through structure-based rational design methods that introduce asymmetric mutations into the CH3 homodimeric interface to favor heterodimeric Fc formation. Here, we report an approach to generate heterodimeric Fc variants through directed evolution combined with yeast surface display. We developed a combinatorial heterodimeric Fc library display system by mating two haploid yeast cell lines, one haploid cell line displayed an Fc chain library (displayed FcCH3A with mutations in one CH3 domain (CH3A on the yeast cell surface, and the other cell line secreted an Fc chain library (secreted FcCH3B with mutations in the other CH3 domain (CH3B. In the mated cells, secreted FcCH3B is displayed on the cell surface through heterodimerization with the displayed FcCH3A, the detection of which enabled us to screen the library for heterodimeric Fc variants. We constructed combinatorial heterodimeric Fc libraries with simultaneous mutations in the homodimer-favoring electrostatic interaction pairs K370-E357/S364 or D399-K392/K409 at the CH3 domain interface. High-throughput screening of the libraries using flow cytometry yielded heterodimeric Fc variants with heterodimer-favoring CH3 domain interface mutation pairs, some of them showed high heterodimerization yields (~80-90% with previously unidentified CH3 domain interface mutation pairs, such as hydrogen bonds and cation-π interactions. Our study provides a new approach for engineering Fc heterodimers that could be used to engineer other heterodimeric protein-protein interactions through directed evolution combined with yeast surface display.

  3. Engineering of Immunoglobulin Fc Heterodimers Using Yeast Surface-Displayed Combinatorial Fc Library Screening.

    Science.gov (United States)

    Choi, Hye-Ji; Kim, Ye-Jin; Choi, Dong-Ki; Kim, Yong-Sung

    2015-01-01

    Immunoglobulin Fc heterodimers, which are useful scaffolds for the generation of bispecific antibodies, have been mostly generated through structure-based rational design methods that introduce asymmetric mutations into the CH3 homodimeric interface to favor heterodimeric Fc formation. Here, we report an approach to generate heterodimeric Fc variants through directed evolution combined with yeast surface display. We developed a combinatorial heterodimeric Fc library display system by mating two haploid yeast cell lines, one haploid cell line displayed an Fc chain library (displayed FcCH3A) with mutations in one CH3 domain (CH3A) on the yeast cell surface, and the other cell line secreted an Fc chain library (secreted FcCH3B) with mutations in the other CH3 domain (CH3B). In the mated cells, secreted FcCH3B is displayed on the cell surface through heterodimerization with the displayed FcCH3A, the detection of which enabled us to screen the library for heterodimeric Fc variants. We constructed combinatorial heterodimeric Fc libraries with simultaneous mutations in the homodimer-favoring electrostatic interaction pairs K370-E357/S364 or D399-K392/K409 at the CH3 domain interface. High-throughput screening of the libraries using flow cytometry yielded heterodimeric Fc variants with heterodimer-favoring CH3 domain interface mutation pairs, some of them showed high heterodimerization yields (~80-90%) with previously unidentified CH3 domain interface mutation pairs, such as hydrogen bonds and cation-π interactions. Our study provides a new approach for engineering Fc heterodimers that could be used to engineer other heterodimeric protein-protein interactions through directed evolution combined with yeast surface display.

  4. Q{sub {gamma}-H2AX}, an analysis method for partial-body radiation exposure using {gamma}-H2AX in non-human primate lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Redon, Christophe E., E-mail: redonc@mail.nih.gov [NIH, NCI, CCR, Laboratory of Molecular Pharmacology, Bethesda, MD 20892 (United States); Nakamura, Asako J.; Gouliaeva, Ksenia [NIH, NCI, CCR, Laboratory of Molecular Pharmacology, Bethesda, MD 20892 (United States); Rahman, Arifur; Blakely, William F. [Armed Forces Radiobiology Research Institute, Uniformed Services University, Bethesda, MD 20889-5603 (United States); Bonner, William M. [NIH, NCI, CCR, Laboratory of Molecular Pharmacology, Bethesda, MD 20892 (United States)

    2011-09-15

    We previously used the {gamma}-H2AX assay as a biodosimeter for total-body irradiation (TBI) exposure ({gamma}-rays) in a rhesus macaque (Macaca mulatta) model. Utilizing peripheral blood lymphocytes and plucked hairs, we obtained statistically significant {gamma}-H2AX responses days after total-body exposure to 1-8.5 Gy ({sup 60}Co {gamma}-rays at 55 cGy min{sup -1}). Here, we introduce a partial-body exposure analysis method, Q{sub {gamma}-H2AX}, which is based on the number of {gamma}-H2AX foci per damaged cells as evident by having one or more {gamma}-H2AX foci per cell. Results from the rhesus monkey - TBI study were used to establish Q{sub {gamma}-H2AX} dose-response calibration curves to assess acute partial-body exposures. {gamma}-H2AX foci were detected in plucked hairs for several days after in vivo irradiation demonstrating this assay's utility for dose assessment in various body regions. The quantitation of {gamma}-H2AX may provide a robust biodosimeter for analyzing partial-body exposures to ionizing radiation in humans.

  5. Fc gamma receptor polymorphisms in relation to periodontitis

    NARCIS (Netherlands)

    Loos, BG; Leppers-Van de Straat, FGJ; Van de Winkel, JGJ; Van der Velden, U

    Objectives: Evidence suggests functional relevance for polymorphisms in Fcgamma R in relation to inflammatory and infectious diseases. The present aim was to investigate genetic polymorphisms in three Fcgamma R in relation to susceptibility and severity of periodontitis. Material and Methods: The

  6. Fc gamma receptor polymorphisms in relation to periodontitis

    NARCIS (Netherlands)

    Loos, BG; Leppers-Van de Straat, FGJ; Van de Winkel, JGJ; Van der Velden, U

    2003-01-01

    Objectives: Evidence suggests functional relevance for polymorphisms in Fcgamma R in relation to inflammatory and infectious diseases. The present aim was to investigate genetic polymorphisms in three Fcgamma R in relation to susceptibility and severity of periodontitis. Material and Methods: The st

  7. Toll-like receptor 2 ligands regulate monocyte Fcγ receptor expression and function.

    Science.gov (United States)

    Shah, Prexy; Fatehchand, Kavin; Patel, Hemal; Fang, Huiqing; Justiniano, Steven E; Mo, Xiaokui; Jarjoura, David; Tridandapani, Susheela; Butchar, Jonathan P

    2013-04-26

    Fcγ receptor (FcγR) clustering on monocytes/macrophages results in phagocytosis and inflammatory cytokine production, which serve to eliminate antibody-opsonized targets and activate neighboring immune cells. Toll-like receptor 2 (TLR2), which recognizes a range of both bacterial and fungal components, elicits strong proinflammatory responses in these cells when stimulated by ligands, either natural or synthetic. Thus, we explored the possibility that TLR2 agonists could strengthen FcγR activity within the context of antibody therapy. Human peripheral blood monocytes treated with the TLR2 agonist Pam2CSK4 showed significantly enhanced FcγR-mediated cytokine production as well as phagocytic ability. An examination of the molecular mechanism behind this enhancement revealed increased expression of both FcγRIIa and the common γ subunit following Pam2CSK4 treatment. Interestingly however, expression of the inhibitory receptor FcγRIIb was also modestly increased. Further investigation revealed that Pam2CSK4 also dramatically decreased the expression of SHIP, the major mediator of FcγRIIb inhibitory activity. Using a murine Her2/neu solid tumor model of antibody therapy, we found that Pam2CSK4 significantly enhanced the ability of anti-Her2 antibody to reduce the rate of tumor growth. To verify that the FcγR enhancement was not unique to the diacylated Pam2CSK4, we also tested Pam3CSK4, a related triacylated TLR2 agonist. Results showed significant enhancement in FcγR function and expression. Taken together, these findings indicate that TLR2 activation can positively modulate FcγR and suggest that TLR2 agonists should be considered for testing as adjuvants for antitumor antibody therapy.

  8. Toll-like Receptor 2 Ligands Regulate Monocyte Fcγ Receptor Expression and Function*

    Science.gov (United States)

    Shah, Prexy; Fatehchand, Kavin; Patel, Hemal; Fang, Huiqing; Justiniano, Steven E.; Mo, Xiaokui; Jarjoura, David; Tridandapani, Susheela; Butchar, Jonathan P.

    2013-01-01

    Fcγ receptor (FcγR) clustering on monocytes/macrophages results in phagocytosis and inflammatory cytokine production, which serve to eliminate antibody-opsonized targets and activate neighboring immune cells. Toll-like receptor 2 (TLR2), which recognizes a range of both bacterial and fungal components, elicits strong proinflammatory responses in these cells when stimulated by ligands, either natural or synthetic. Thus, we explored the possibility that TLR2 agonists could strengthen FcγR activity within the context of antibody therapy. Human peripheral blood monocytes treated with the TLR2 agonist Pam2CSK4 showed significantly enhanced FcγR-mediated cytokine production as well as phagocytic ability. An examination of the molecular mechanism behind this enhancement revealed increased expression of both FcγRIIa and the common γ subunit following Pam2CSK4 treatment. Interestingly however, expression of the inhibitory receptor FcγRIIb was also modestly increased. Further investigation revealed that Pam2CSK4 also dramatically decreased the expression of SHIP, the major mediator of FcγRIIb inhibitory activity. Using a murine Her2/neu solid tumor model of antibody therapy, we found that Pam2CSK4 significantly enhanced the ability of anti-Her2 antibody to reduce the rate of tumor growth. To verify that the FcγR enhancement was not unique to the diacylated Pam2CSK4, we also tested Pam3CSK4, a related triacylated TLR2 agonist. Results showed significant enhancement in FcγR function and expression. Taken together, these findings indicate that TLR2 activation can positively modulate FcγR and suggest that TLR2 agonists should be considered for testing as adjuvants for antitumor antibody therapy. PMID:23504312

  9. Effects of sleep deprivation on gamma oscillation of waking human EEG

    Institute of Scientific and Technical Information of China (English)

    Ning Li; Yan Wang; Mingshi Wang; Haiying Liu

    2008-01-01

    This study compares and evaluates the effect of sleep deprivation (SD) on human brain cognition by analyzing the recorded EEG data under normal and 24 h sleep deprived states.EEG auditory event-related potentials were collected from 14 healthy volunteers,and the statistical values of wavelet-transformed EEG in gamma band were decomposed by parallel factor analysis (PARAFAC) to identify where the differences appeared in the time,frequency and spatial domains.The results showed that the changes of brain states caused by SD appeared around 40 Hz at about 400 ms after stimulation on prefrontal and frontal lobes.Negative effects of SD on neuronal activity and oscillation were observed,The analysis of the EEG data by the wavelet transform and PARAFAC can be an integrated way to estimate the change of brain states in the three domains.

  10. Zebrafish mast cells possess an FcɛRI-like receptor and participate in innate and adaptive immune responses.

    Science.gov (United States)

    Da'as, Sahar; Teh, Evelyn M; Dobson, J Tristan; Nasrallah, Gheyath K; McBride, Eileen R; Wang, Hao; Neuberg, Donna S; Marshall, Jean S; Lin, Tong-Jun; Berman, Jason N

    2011-01-01

    We previously identified a zebrafish mast cell (MC) lineage and now aim to determine if these cells function analogously in innate and adaptive immunity like their mammalian counterparts. Intraperitoneal (IP) injection of compound 48/80 or live Aeromonas salmonicida resulted in significant MC degranulation evident histologically and by increased plasma tryptase compared with saline-injected controls (p=0.0006, 0.005, respectively). Pre-treatment with ketotifen abrogated these responses (p=0.0004, 0.005, respectively). Cross-reactivity was observed in zebrafish to anti-human high-affinity IgE receptor gamma (FcɛRIγ) and IgE heavy chain-directed antibodies. Whole mount in situ hybridization on 7-day embryos demonstrated co-localization of cpa5, a MC-specific marker, with myd88, a toll-like receptor adaptor, and zebrafish FcɛRI subunit homologs. Zebrafish injected IP with matched dinitrophenyl-sensitized mouse (anti-DNP) IgE and DNP-BSA or trinitrophenyl-sensitized mouse (anti-TNP) IgE and TNP-BSA demonstrated increased plasma tryptase compared with mismatched controls (p=0.03, 0.010, respectively). These results confirm functional conservation and validate the zebrafish model as an in vivo screening tool for novel MC modulating agents. Copyright © 2010 Elsevier Ltd. All rights reserved.

  11. IFN-gamma is not induced through increased plasma concentrations of interleukin-12/interleukin-18 during human endotoxemia.

    NARCIS (Netherlands)

    Dorresteijn, M.J.; Pickkers, P.; Netea, M.G.; Hoeven, J.G. van der

    2005-01-01

    Endotoxin administration to animals and humans is an accepted experimental model of Gram-negative sepsis, and endotoxin is believed to play a major role in triggering the activation of cytokines. In septic patients, the IL-12/IL-18/IFN-gamma axis is activated and correlates with mortality. Our aim w

  12. Fractionation of rat IgG subclasses and screening for IgG Fc-binding to bacteria.

    Science.gov (United States)

    Nilsson, R; Myhre, E; Kronvall, G; Sjögren, H O

    1982-01-01

    The four IgG subclasses of the rat, IgGl, IgG2a, IgG2b and IgG2c, were purified from normal serum by a combination of protein A-affinity chromatography and DEAE-cellulose chromatography. Purified, radiolabelled preparations of IgG were tested for binding to Gram-positive bacteria representing five different Fc-receptor (FcR) types. Distinct rat subclass-specific Fc-binding was noted to bacterial species belonging to different Fc-receptor types. Staphylococcus aureus (FcR I) strains bind IgGl and IgG2c as shown by others. Group C and G Streptococci (FcR III) bind all four subclasses of rat IgG. Streptococcus zooepidemicus strains (FcR V) also bind all four subclases but only to a lower degree. Human group A Streptococci (FcR II) and bovine group G Streptococci (FcR IV) do not bind any of the rat IgG subclasses. Elution studies on two strains. Staphylococcus aureus, Cowan I, and human group G Streptococcus, G 148, showed that both thiocyanate and pH-elution might be useful for the fractionation of IgG subclasses bound to bacterial cells. The present work indicates the possible use of bacterial cells as solid-phase absorbents in immunological studies of rat IgG.

  13. Secretion of interferon gamma from human immune cells is altered by exposure to tributyltin and dibutyltin.

    Science.gov (United States)

    Lawrence, Shanieek; Reid, Jacqueline; Whalen, Margaret

    2015-05-01

    Tributyltin (TBT) and dibutyltin (DBT) are widespread environmental contaminants found in food, beverages, and human blood samples. Both of these butyltins (BTs) interfere with the ability of human natural killer (NK) cells to lyse target cells and alter secretion of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) from human immune cells in vitro. The capacity of BTs to interfere with secretion of other pro-inflammatory cytokines has not been examined. Interferon gamma (IFNγ) is a modulator of adaptive and innate immune responses, playing an important role in overall immune competence. This study shows that both TBT and DBT alter secretion of IFNγ from human immune cells. Peripheral blood cell preparations that were increasingly reconstituted were used to determine if exposures to either TBT or DBT affected IFNγ secretion and how the makeup of the cell preparation influenced that effect. IFNγ secretion was examined after 24 h, 48 h, and 6 day exposures to TBT (200 - 2.5 nM) and DBT (5 - 0.05 µM) in highly enriched human NK cells, a monocyte-depleted preparation of PBMCs, and monocyte-containing PBMCs. Both BTs altered IFNγ secretion from immune cells at most of the conditions tested (either increasing or decreasing secretion). However, there was significant variability among donors as to the concentrations and time points that showed changes as well as the baseline secretion of IFNγ. The majority of donors showed an increase in IFNγ secretion in response to at least one concentration of TBT or DBT at a minimum of one length of exposure.

  14. IFN-gamma is produced by polymorphonuclear neutrophils in human uterine endometrium and by cultured peripheral blood polymorphonuclear neutrophils.

    Science.gov (United States)

    Yeaman, G R; Collins, J E; Currie, J K; Guyre, P M; Wira, C R; Fanger, M W

    1998-05-15

    Cytokines present in the human uterus play an important role both in modulating immune responses to infectious challenge and in the establishment and maintenance of pregnancy. In particular, successful implantation and pregnancy is thought to require the establishment of a Th2 environment, while Th1 cytokines are associated with pregnancy loss and infertility. On the other hand, a Th1 response appears to be required for the resolution of acute infection. Using novel confocal microscopic analysis of fresh sections of human tissue, we have investigated the production of IFN-gamma, a Th1 cytokine, in human endometria. Extracellular IFN-gamma, mostly associated with matrix components, was located immediately beneath the luminal epithelium and along the glandular epithelium proximal to the lumen. As evidenced by intracellular staining, IFN-gamma is produced by both stromal cells and intraepithelial lymphocytes through all stages of the menstrual cycle. Surprisingly, the stromal cell containing intracellular IFN-gamma was identified as a polymorphonuclear neutrophil on the basis of its reactivity with a panel of mAbs and its nuclear morphology. We further found that polymorphonuclear neutrophils isolated from normal donors produce IFN-gamma in response to stimulation with LPS, IL-12, and TNF-alpha. Taken together, these findings suggest that polymorphonuclear neutrophils are capable of producing IFN-gamma both in vitro and in vivo, indicating that their role in shaping immune responses may be more extensive than previously thought. Furthermore, these studies strongly suggest that polymorphonuclear neutrophils play an important role in determining immune responsiveness within the female reproductive tract.

  15. Review of the expression of peroxisome proliferator-activated receptors alpha (PPAR alpha), beta (PPAR beta), and gamma (PPAR gamma) in rodent and human development.

    Science.gov (United States)

    Abbott, Barbara D

    2009-06-01

    The peroxisome proliferator-activated receptors (PPAR) belong to the nuclear hormone receptor superfamily and there are three primary subtypes, PPARalpha, beta, and gamma. These receptors regulate important physiological processes that impact lipid homeostasis, inflammation, adipogenesis, reproduction, wound healing, and carcinogenesis. These nuclear receptors have important roles in reproduction and development and their expression may influence the responses of an embryo exposed to PPAR agonists. PPARs are relevant to the study of the biological effects of the perfluorinated alkyl acids as these compounds, including perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), activate PPARalpha. Exposure of the rodent to PFOA or PFOS during gestation results in neonatal deaths, developmental delay and growth deficits. Studies in PPARalpha knockout mice demonstrate that the developmental effects of PFOA, but not PFOS, depend on expression of PPARalpha. This review provides an overview of PPARalpha, beta, and gamma protein and mRNA expression during mouse, rat, and human development. The review presents the results from many published studies and the information is organized by organ system and collated to show patterns of expression at comparable developmental stages for human, mouse, and rat. The features of the PPAR nuclear receptor family are introduced and what is known or inferred about their roles in development is discussed relative to insights from genetically modified mice and studies in the adult.

  16. IgG Fc Fragment as a Scaffold for Development of Targeted Therapeutics.

    Science.gov (United States)

    Wozniak-Knopp, Gordana; Stadlmayr, Gerhard; Rueker, Florian

    2016-11-14

    Monoclonal antibodies and Fc fusion proteins have been successful therapeutics in the field of cancer and immune disease. As their pharmacological activity is dependent on the Fc fragment governing their effector functions and long in vivo half-life, the extensive engineering of the Fc for altered binding of its natural ligands that enable these properties has delivered molecules optimized for their therapeutic effect. Recently, the IgG1 Fc region itself and its subunits monomeric Fc fragment, CH2 and monomeric CH3 domain, have been engineered into scaffolds with favorable biophysical properties and a high potential for de novo antigen recognition. A dimeric Fc fragment with an antigen binding site has proven suitable for evaluation in animal models and will soon be entering human trials. Such modified constant domains can easily be incorporated into an antibody or fused with antibody domains of a second specificity. The small size of the Fc and its subunits that enhances their tissue penetration, as well as the unique topology of their binding sites that allows novel modes of contact with the antigen, are attractive features that prompt their development into promising candidate therapeutics.

  17. Fc-fragment removal allows the EPO-Fc fusion protein to be detected in blood samples by IEF-PAGE.

    Science.gov (United States)

    Postnikov, Pavel; Krotov, Grigory; Mesonzhnik, Natalia; Efimova, Yulia; Rodchenkov, Grigory

    2015-01-01

    EPO-Fc proteins have been under investigation as a potential drug for treating anaemia and have shown larger half-life values than other erythropoiesis-stimulating agents (ESAs). Sodium dodecyl sulfate/sodium N-lauroylsarcosinate polyacrylamide gel electrophoresis (SDS/SAR-PAGE) methods and subsequent immunoblotting are used for routine anti-doping analysis. This paper reports that EPO-Fc fusion proteins can be detected in serum samples by isoelectric focusing-polyacrylamide gel electrophoresis (IEF-PAGE) in carrier ampholyte-based gels with a pH 2-6 gradient after removing the Fc part via site-specific IdeS protease cleavage. The IdeS-digested EPO-Fc protein yields three fragments: two Fc fragments and one dimeric EPO-hinge fragment. After IEF-PAGE was followed by double Western blotting with chemiluminescent detection, the dimeric EPO-hinge fragment showed a unique isoelectric pattern, which differed from those of any other currently known analogue of EPO. We observed that the removal of the Fc fragment from EPO-Fc reduced the apparent molecular weight of entire fusion protein and increased its electrophoretic mobility. As a result, the band for the EPO-hinge fragment was located in a region between the rEPO and NESP standards, at which lower amounts of serum proteins are present. Simple and selective protocols for determining the EPO-Fc protein in human serum were developed to extend the methodological anti-doping arsenal. This protocol has been characterized. The limit of detection (LOD) of the IEF-PAGE method was 20 pg, and that of SDS/SAR-PAGE was 15 pg.

  18. Peroxisome proliferator-activated receptor-gamma suppresses cyclooxygenase-2 expression in human prostate cells.

    Science.gov (United States)

    Sabichi, Anita L; Subbarayan, Vemparala; Llansa, Norma; Lippman, Scott M; Menter, David G

    2004-11-01

    Recent studies have found that cyclooxygenase-2 (COX-2) protein expression was low and inducible with cytokines in prostate cancer cells (in the absence of serum) and that, in contrast, COX-2 expression was high in normal prostate epithelial cells (EC). Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) was expressed at high levels in the prostate cancer cell line PC-3 but not in ECs. In contrast to previous findings by others, PPAR-gamma ligands did not induce PPAR-gamma expression in EC or PC-3. The present study examined the relationship between PPAR-gamma and COX-2 expression patterns in EC and PC-3 in the presence and absence of serum and/or the PPAR-gamma agonist 15-deoxy-Delta12,14-prostaglandin J(2) (15d-PGJ(2)). We also evaluated the effects that the forced expression of PPAR-gamma1 and PPAR-gamma2 had on COX-2 in ECs. We found that expression of PPAR-gamma and COX-2 protein was inversely correlated in ECs and PC-3. Low COX-2 expression in PC-3 was up-regulated by serum, and 15d-PGJ(2) blocked serum-induced COX-2 expression and activity in a dose-dependent manner. 15d-PGJ(2) had no effect on COX-2 expression in ECs or PPAR-gamma expression in either cell type. However, forced expression of PPAR-gamma1 or PPAR-gamma2 in ECs suppressed the high level of endogenous COX-2. This effect was not isoform specific and was augmented by 15d-PGJ(2). The present study showed that PPAR-gamma activation can be an important regulator of COX-2 in prostate cells and may be an important target for prostate cancer chemoprevention.

  19. Dicty_cDB: FC-BS10 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-BS10 (Link to dictyBase) - - - Contig-U16283-1 FC-BS10Z (Li...nk to Original site) - - FC-BS10Z 720 - - - - Show FC-BS10 Library FC (Link to library) Clone ID FC-BS10 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-BS/FC-BS10Q.Seq.d/ Representative seq. ID FC-BS10...Z (Link to Original site) Representative DNA sequence >FC-BS10 (FC-BS10Q) /CSM/FC/FC-BS/FC-BS10Q.Seq....E Sequences producing significant alignments: (bits) Value FC-BS10 (FC-BS10Q) /CSM/FC/FC-BS/FC-BS10Q.Seq.d/

  20. Dicty_cDB: FC-BS11 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-BS11 (Link to dictyBase) - - - Contig-U16517-1 FC-BS11Z (Li...nk to Original site) - - FC-BS11Z 683 - - - - Show FC-BS11 Library FC (Link to library) Clone ID FC-BS11 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-BS/FC-BS11Q.Seq.d/ Representative seq. ID FC-BS1...1Z (Link to Original site) Representative DNA sequence >FC-BS11 (FC-BS11Q) /CSM/FC/FC-BS/FC-BS11Q.Seq....s producing significant alignments: (bits) Value FC-BS11 (FC-BS11Q) /CSM/FC/FC-BS/FC-BS1

  1. Dicty_cDB: FC-BS16 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-BS16 (Link to dictyBase) - - - Contig-U15105-1 FC-BS16E (Li...nk to Original site) - - - - - - FC-BS16E 671 Show FC-BS16 Library FC (Link to library) Clone ID FC-BS16 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-BS/FC-BS16Q.Seq.d/ Representative seq. ID FC-BS1...6E (Link to Original site) Representative DNA sequence >FC-BS16 (FC-BS16Q) /CSM/FC/FC-BS/FC-BS16Q.Seq....VSH7-A/VSH720Q.Seq.d/ 1241 0.0 VSE854 (VSE854Q) /CSM/VS/VSE8-C/VSE854Q.Seq.d/ 1241 0.0 FC-BS16 (FC-BS16Q) /CSM/FC/FC-BS/FC-BS1

  2. Dicty_cDB: FC-BS19 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-BS19 (Link to dictyBase) - - - Contig-U16583-1 FC-BS19E (Li...nk to Original site) - - - - - - FC-BS19E 604 Show FC-BS19 Library FC (Link to library) Clone ID FC-BS19 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-BS/FC-BS19Q.Seq.d/ Representative seq. ID FC-BS1...9E (Link to Original site) Representative DNA sequence >FC-BS19 (FC-BS19Q) /CSM/FC/FC-BS/FC-BS19Q.Seq....E Sequences producing significant alignments: (bits) Value FC-BS19 (FC-BS19Q) /CSM/FC/FC-BS/FC-BS19Q.Seq.d/

  3. Dicty_cDB: FC-AL16 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AL16 (Link to dictyBase) - - - Contig-U15130-1 FC-AL16E (Li...nk to Original site) - - - - - - FC-AL16E 648 Show FC-AL16 Library FC (Link to library) Clone ID FC-AL16 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AL/FC-AL16Q.Seq.d/ Representative seq. ID FC-AL1...6E (Link to Original site) Representative DNA sequence >FC-AL16 (FC-AL16Q) /CSM/FC/FC-AL/FC-AL16Q.Seq....ences producing significant alignments: (bits) Value FC-AL16 (FC-AL16Q) /CSM/FC/FC-AL/FC-AL16Q.Seq.d/ 1241 0

  4. Dicty_cDB: FC-AI13 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AI13 (Link to dictyBase) - - - Contig-U16263-1 FC-AI13F (Li...nk to Original site) FC-AI13F 507 - - - - - - Show FC-AI13 Library FC (Link to library) Clone ID FC-AI13 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI13Q.Seq.d/ Representative seq. ID FC-AI...13F (Link to Original site) Representative DNA sequence >FC-AI13 (FC-AI13Q) /CSM/FC/FC-AI/FC-AI13Q.Seq....nificant alignments: (bits) Value FC-AI13 (FC-AI13Q) /CSM/FC/FC-AI/FC-AI13Q.Seq.d/ 963 0.0 VSA730 (VSA730Q)

  5. Dicty_cDB: FC-AI01 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AI01 (Link to dictyBase) - - - Contig-U15592-1 FC-AI01E (Li...nk to Original site) - - - - - - FC-AI01E 307 Show FC-AI01 Library FC (Link to library) Clone ID FC-AI01 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI01Q.Seq.d/ Representative seq. ID FC-AI...01E (Link to Original site) Representative DNA sequence >FC-AI01 (FC-AI01Q) /CSM/FC/FC-AI/FC-AI01Q.Seq....uences producing significant alignments: (bits) Value FC-AI01 (FC-AI01Q) /CSM/FC/FC-AI/FC-AI01Q.Seq.d/ 68 8e

  6. Chemoenzymatic Fc Glycosylation via Engineered Aldehyde Tags

    OpenAIRE

    2014-01-01

    Glycoproteins with chemically defined glycosylation sites and structures are important biopharmaceutical targets and critical tools for glycobiology. One approach toward constructing such molecules involves chemical glycosylation of aldehyde-tagged proteins. Here, we report the installation of a genetically encoded aldehyde tag at the internal glycosylation site of the crystallizable fragment (Fc) of IgG1. We replaced the natural Fc N-glycosylation sequon with a five amino-acid sequence that ...

  7. Two essential regulatory elements in the human interferon gamma promoter confer activation specific expression in T cells.

    Science.gov (United States)

    Penix, L; Weaver, W M; Pang, Y; Young, H A; Wilson, C B

    1993-11-01

    Like interleukin 2 (IL-2), interferon gamma (IFN-gamma) is an early response gene in T cells and both are prototypical T helper cell type 1 (Th-1) lymphokines. Yet IL-2 and IFN-gamma production are independently regulated, as demonstrated by their differential expression in certain T cell subsets, suggesting that the regulatory elements in these two genes must differ. To explore this possibility, the 5' flank of the human IFN-gamma gene was analyzed. Expression of IFN-gamma promoter-driven beta-galactosidase reporter constructs containing 538 bp of 5' flank was similar to that by constructs driven by the IL-2 promoter in activated Jurkat T cells; expression nearly as great was observed with the construct containing only 108 bp of IFN-gamma 5' flank. These IFN-gamma promoter constructs faithfully mirrored expression of the endogenous gene, in that expression required activation both with ionomycin and PMA, was inhibited by cyclosporin A, and was not observed in U937 or THP-1 cells. The region between -108 and -40 bp in the IFN-gamma promoter was required for promoter function and contained two elements that are conserved across species. Deletion of 10 bp within either element reduced promoter function by 70%, whereas deletions in nonconserved portions of this region had little effect on promoter function. The distal conserved element (-96 to -80 bp) contained a consensus GATA motif and a potential regulatory motif found in the promoter regions of the GM-CSF and macrophage inflammatory protein (MIP) genes. Factors binding to this element, including GATA-3, were found in Jurkat nuclear extracts by electromobility shift assays and two of the three complexes observed were altered in response to activation. One or both of these motifs are present in the 5' flank of multiple, other lymphokine genes, including IL-3, IL-4, IL-5, and GM-CSF, but neither is present in the promoter of the IL-2 gene. The proximal conserved element (-73 to -48 bp) shares homology with the NFIL-2

  8. Effect of gamma irradiation on the wear behaviour of human tooth enamel

    Science.gov (United States)

    Qing, Ping; Huang, Shengbin; Gao, Shanshan; Qian, Linmao; Yu, Haiyang

    2015-06-01

    Radiotherapy is a frequently used treatment for oral cancer. Extensive research has been conducted to detect the mechanical properties of dental hard tissues after irradiation at the macroscale. However, little is known about the influence of irradiation on the tribological properties of enamel at the micro- or nanoscale. Therefore, this study aimed to investigate the effect of gamma irradiation on the wear behaviour of human tooth enamel in relation to prism orientation. Nanoscratch tests, surface profilometer and scanning electron microscope (SEM) analysis were used to evaluate the friction behaviour of enamel slabs before and after treatment with identical irradiation procedures. X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) were performed to analyse the changes in crystallography and chemical composition induced by irradiation. Surface microhardness (SMH) alteration was also evaluated. The results showed that irradiation resulted in different scratch morphologies, friction coefficients and remnant depth and width at different loads. An inferior nanoscratch resistance was observed independent of prism orientation. Moreover, the variation of wear behaviours was closely related to changes in the crystallography, chemical composition and SMH of the enamel. Together, these measures indicated that irradiation had a direct deleterious effect on the wear behaviour of human tooth enamel.

  9. Interaction of 2'-deoxyguanosine triphosphate analogue inhibitors of HIV reverse transcriptase with human mitochondrial DNA polymerase gamma.

    Science.gov (United States)

    Ray, Adrian S; Feng, Joy Y; Murakami, Eisuke; Chu, Chung K; Schinazi, Raymond F; Anderson, Karen S

    2007-01-01

    Mitochondrial toxicity is a limiting factor in the use of some nucleoside reverse transcriptase inhibitors of HIV. To further understand the impact of structural features on the incorporation and exonuclease removal of nucleoside monophosphate (MP) analogues by human mitochondrial DNA polymerase (pol gamma), transient kinetic studies were done with analogues of 2'-deoxyguanosine triphosphate. The kinetic parameters for the incorporation and removal of carbovir (CBV)-MP, dioxolane guanosine (DXG)-MP and 2',3'-dideoxy-2',3'-didehydroguanosine (d4G)-MP were studied with pol gamma holoenzyme. The importance of the ribose oxygen in incorporation by pol gamma was illustrated by an approximate 3,000-fold decrease in the incorporation efficiency of an analogue lacking the ribose oxygen (CBV-TP) relative to those containing a ribose oxygen (DXG-TP and d4G-TP). As a result, a comparison with previous data for the incorporation by HIV reverse transcriptase showed CBV-TP to be approximately 800-8,000-fold more selective for its antiviral target over pol gamma relative to the other guanosine analogues. However, DXG-TP and d4G-TP were found to be much more selective than previously reported values for mitochondrial toxic nucleoside analogues. Structural modelling based on sequence homology with other polymerase A family members suggests that an interaction between the ribose oxygen and arginine 853 in pol gamma may play a critical role in causing this differential incorporation. Exonuclease removal of a chain-terminating CBV-MP was also found to be more efficient by pol gamma. These results help to further elucidate the structure activity relationships for pol gamma and should aid in the design of more selective antiviral agents.

  10. Dose-response relationship of {gamma}-H2AX foci induction in human lymphocytes after X-rays exposure

    Energy Technology Data Exchange (ETDEWEB)

    Mandina, Tania [Centro de Proteccion e Higiene de las Radiaciones, Calle 20 No. 4113 e/41y 47 Miramar, AP 6195 C. Habana (Cuba); Roch-Lefevre, Sandrine H.; Voisin, Pascale [Institut de Radioprotection et de Surete Nucleaire (IRSN), DRPH, SRBE, LDB, BP17, 92262 Fontenay-aux-Roses (France); Gonzalez, Jorge E.; Lamadrid, Ana I.; Romero, Ivonne [Centro de Proteccion e Higiene de las Radiaciones, Calle 20 No. 4113 e/41y 47 Miramar, AP 6195 C. Habana (Cuba); Garcia, Omar, E-mail: omar@cphr.edu.cu [Centro de Proteccion e Higiene de las Radiaciones, Calle 20 No. 4113 e/41y 47 Miramar, AP 6195 C. Habana (Cuba); Voisin, Philippe; Roy, Laurence [Institut de Radioprotection et de Surete Nucleaire (IRSN), DRPH, SRBE, LDB, BP17, 92262 Fontenay-aux-Roses (France)

    2011-09-15

    Biological dosimeters are recommended for dose estimation in case of human overexposure to ionising radiation. Rapid measurement of {gamma}-H2AX foci as a marker of DNA double-strand breaks (DSB) induction has been recently tested with this purpose. Here we reported a dose-response relationship after X-ray irradiation at different times post-exposure. Blood samples were obtained from several healthy donors and exposed to doses between 0 and 2 Gy. After irradiation, blood samples were incubated at 37 deg. C during 0.5 h, 5 h, and 8 h. Scoring of cells and {gamma}-H2AX foci was performed by software. The dose-response curves for different incubation times were as follows: Y{sub (0.5h)} = 11.66D + 0.15 (R{sup 2} = 0.99), Y{sub (5h)} = 2.44D + 0.15 (R{sup 2} = 0.99), Y{sub (8h)} = 1.57D + 0.22 (R{sup 2} = 0.99). At 0.5 h post-exposure, the dose-response relationship for X-irradiated lymphocytes was similar to the one obtained after gamma-irradiation using the same protocol. On the other hand, the results were not similar after 8 h due to different kinetics after gamma- and X-irradiation. Our results confirm the possibilities of using {gamma}-H2AX foci method for dose estimation in a period from 0.5 h up to 8 h post X-irradiation and support the hypothesis of differences in {gamma}-H2AX foci kinetics after gamma- and X-irradiation in vitro.

  11. Inhibition of tumor growth and metastasis by ATF-Fc, an engineered antibody targeting urokinase receptor.

    Science.gov (United States)

    Hu, Xian-Wen; Duan, Hai-Feng; Gao, Li-Hua; Pan, Shu-Yuan; Li, Yong-Mei; Xi, Yongyi; Zhao, Su-Rong; Yin, Liang; Li, Jin-Feng; Chen, Hui-Peng; Wu, Chu-Tse

    2008-05-01

    Urokinase (uPA) and its receptor (uPAR) play an important role in tumor growth and metastasis, and overexpression of these molecules is strongly correlated with poor prognosis in a variety of malignant tumors. In this study, ATF-Fc, an antibody-like molecule comprising the amino-terminal fragment of human uPA (ATF) linked to the Fc fragment of human IgG1 via a flexible linker was developed. Its antitumor activities were evaluated in vitro and in vivo. The results showed that ATF-Fc had obvious cytotoxic effect on several types of tumor cells, which is dependent on cellular expression of uPAR and its Fc fragment. Treatment with ATF-Fc caused a significant suppression on tumor growth and metastasis of xenograft human tumors (MCF-7 breast cancer and BGC-823 gastric cancer) in athymic nude mice. Furthermore, we demonstrated that ATF-Fc had an anti-angiogenesis activity both in vitro and in vivo. In conclusion, we provided a novel therapeutic antibody-like molecule in the management of a variety of solid tumors by disrupting the uPA/uPAR interaction.

  12. FC-NIRS: A Functional Connectivity Analysis Tool for Near-Infrared Spectroscopy Data

    Directory of Open Access Journals (Sweden)

    Jingping Xu

    2015-01-01

    Full Text Available Functional near-infrared spectroscopy (fNIRS, a promising noninvasive imaging technique, has recently become an increasingly popular tool in resting-state brain functional connectivity (FC studies. However, the corresponding software packages for FC analysis are still lacking. To facilitate fNIRS-based human functional connectome studies, we developed a MATLAB software package called “functional connectivity analysis tool for near-infrared spectroscopy data” (FC-NIRS. This package includes the main functions of fNIRS data preprocessing, quality control, FC calculation, and network analysis. Because this software has a friendly graphical user interface (GUI, FC-NIRS allows researchers to perform data analysis in an easy, flexible, and quick way. Furthermore, FC-NIRS can accomplish batch processing during data processing and analysis, thereby greatly reducing the time cost of addressing a large number of datasets. Extensive experimental results using real human brain imaging confirm the viability of the toolbox. This novel toolbox is expected to substantially facilitate fNIRS-data-based human functional connectome studies.

  13. Sound identification in human auditory cortex: Differential contribution of local field potentials and high gamma power as revealed by direct intracranial recordings.

    Science.gov (United States)

    Nourski, Kirill V; Steinschneider, Mitchell; Rhone, Ariane E; Oya, Hiroyuki; Kawasaki, Hiroto; Howard, Matthew A; McMurray, Bob

    2015-09-01

    High gamma power has become the principal means of assessing auditory cortical activation in human intracranial studies, albeit at the expense of low frequency local field potentials (LFPs). It is unclear whether limiting analyses to high gamma impedes ability of clarifying auditory cortical organization. We compared the two measures obtained from posterolateral superior temporal gyrus (PLST) and evaluated their relative utility in sound categorization. Subjects were neurosurgical patients undergoing invasive monitoring for medically refractory epilepsy. Stimuli (consonant-vowel syllables varying in voicing and place of articulation and control tones) elicited robust evoked potentials and high gamma activity on PLST. LFPs had greater across-subject variability, yet yielded higher classification accuracy, relative to high gamma power. Classification was enhanced by including temporal detail of LFPs and combining LFP and high gamma. We conclude that future studies should consider utilizing both LFP and high gamma when investigating the functional organization of human auditory cortex. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. CONSTRUCTION, EXPRESSION AND BIOLOGICAL ASSESSMENT OF BPI23-Fcγ 1 RECOMBINANT PROTEIN PROKARYOTIC EXPRESSION VECTOR

    Institute of Scientific and Technical Information of China (English)

    安云庆; 管远志; 柯岩; 杨贵贞

    2002-01-01

    Objective. To construct pBV-BPI600-Fcγ 1700 recombinant expression vector, to transform it into Escherichia coli DH5α , and to induce the expression of BPI23-Fcγ 1 anti-bacterial recombinant protein. Methods. Genes coding for BPI23 and Fcγ 1 were amplified by RT-PCR from mRNA extracted from HL-60 cell and normal human leukocytes; recombinant cloning vector and recombinant expression vector were then constructed. pBV-BPI600-Fcγ 1700 recombinant expression vector was transformed into the competent Escherichia coli DH5α and BPI23-Fcγ 1 recombinant protein was expressed by a temperature-induced method. Results. (1) Expected amplified products BPI600bp and Fcγ 1700bp were obtained by RT-PCR method. (2) pUC18-BPI180, pUC18-BPI420 and pUC18-Fcγ 1700 recombinant cloning vectors were successfully constructed, and sequences were identical with the reported ones. (3) pBV-BPI600-Fcγ 1700 recombinant expression vector was successfully constructed, and the enzyme digestion analysis showed an expected result. (4) The expression level of BPI23-Fcγ 1 recombinant protein accounted for 20% of total bacterial proteins. (5) The renatured BPI23-Fcγ 1 recombinant protein showed bacteriocidal activity and biological function of complement fixation, and opsonization. Conclusion. pBV-BPI600-Fcγ 1700 recombinant expression vector was successfully constructed, and BPI23-Fcγ 1 recombinant protein with double biological activity of BPI and IgGFc was expressed in Escherichia coli.

  15. CONSTRUCTION,EXPRESSION AND BIOLOGICAL ASSESSMENT OF BPI23—Fcγ1 RECOMBINANT PROTEIN PROKARYOTIC EXPRESSION VECTOR

    Institute of Scientific and Technical Information of China (English)

    安云庆; 管远志; 等

    2002-01-01

    Objective:To construct pBV-BPI600-Fcγ1700 recombinant expression vector,to transform it into Escherichia coli DH5α,and to induce the expression of BPI23-Fcγ1 anti-bacterial recombinant protein.Methods:Genes coding for BPI23 and Fcγ1 were amplified by RT-PCR from mRNA extracted from Hl-60 cell and normal human leukocytes;recombinant cloning vector and recombinant expression vector were then constructed.pBV-BPI600-Fcγ1700 recombinant expression vector was transformed into the competent Escherichia coli DH5α and BPI23-Fcγ1 recombinant protein was expressed by a temperature-induced method.Results:(1)Expected amplified products BPI600bp and Fcγ1700bp were obtained by RT-PCR method.(2)pUC18-BPI180,pUC18-BPI420 and pUC18-Fcγ1700 recombinant cloning vectors were successfully constructed, and sequences were identical with the reported ones.(3)pBV-BPI600-Fcγ1700 recombinant expression vector was successfully constructed,and the enzyme digestion analysis showed an expected result.(4)The expression level of BPI23-Fcγ1 recombinant protein accounted for 20% of total bacterial proteins.(5)The renatured BPI23-Fcγ1 recombinant protein showed bacteriocidal activity and biological function of complement fixation,and opsonization.Conclusion:pBV-BPI600-Fcγ1700 recombinant expression vector was successfully constructed,and BPI23-Fcγ1 recombinant protein with double biological activity of BPI and IgGFc was expressed in Escherichia coli.

  16. pH-dependent binding engineering reveals an FcRn affinity threshold that governs IgG recycling.

    Science.gov (United States)

    Borrok, M Jack; Wu, Yanli; Beyaz, Nurten; Yu, Xiang-Qing; Oganesyan, Vaheh; Dall'Acqua, William F; Tsui, Ping

    2015-02-13

    The Fc domain of IgG has been the target of multiple mutational studies aimed at altering the pH-dependent IgG/FcRn interaction to modulate IgG pharmacokinetics. These studies have yielded antibody variants with disparate pharmacokinetic characteristics, ranging from extended in vivo half-life to those exhibiting extremely rapid clearance. To better understand pH-dependent binding parameters that govern these outcomes and limit FcRn-mediated half-life extension, we generated a panel of novel Fc variants with high affinity binding at acidic pH that vary in pH 7.4 affinities and assessed pharmacokinetic outcomes. Pharmacokinetic studies in human FcRn transgenic mice and cynomolgus monkeys showed that multiple variants with increased FcRn affinities at acidic pH exhibited extended serum half-lives relative to the parental IgG. Importantly, the results reveal an underappreciated affinity threshold of neutral pH binding that determines IgG recycling efficiency. Variants with pH 7.4 FcRn affinities below this threshold recycle efficiently and can exhibit increased serum persistence. Increasing neutral pH FcRn affinity beyond this threshold reduced serum persistence by offsetting the benefits of increased pH 6.0 binding. Ultra-high affinity binding to FcRn at both acidic and neutral pH leads to rapid serum clearance.

  17. Downmodulation of Vaccine-Induced Immunity and Protection against the Intracellular Bacterium Francisella tularensis by the Inhibitory Receptor FcγRIIB

    Directory of Open Access Journals (Sweden)

    Brian J. Franz

    2015-01-01

    Full Text Available Fc gamma receptor IIB (FcγRIIB is the only Fc gamma receptor (FcγR which negatively regulates the immune response, when engaged by antigen- (Ag- antibody (Ab complexes. Thus, the generation of Ag-specific IgG in response to infection or immunization has the potential to downmodulate immune protection against infection. Therefore, we sought to determine the impact of FcγRIIB on immune protection against Francisella tularensis (Ft, a Category A biothreat agent. We utilized inactivated Ft (iFt as an immunogen. Naïve and iFt-immunized FcγRIIB knockout (KO or wildtype (WT mice were challenged with Ft-live vaccine strain (LVS. While no significant difference in survival between naïve FcγRIIB KO versus WT mice was observed, iFt-immunized FcγRIIB KO mice were significantly better protected than iFt-immunized WT mice. Ft-specific IgA in serum and bronchial alveolar lavage, as well as IFN-γ, IL-10, and TNF-α production by splenocytes harvested from iFt-immunized FcγRIIB KO, were also significantly elevated. In addition, iFt-immunized FcγRIIB KO mice exhibited a reduction in proinflammatory cytokine levels in vivo at 5 days after challenge, which correlates with increased survival following Ft-LVS challenge in published studies. Thus, these studies demonstrate for the first time the ability of FcγRIIB to regulate vaccine-induced IgA production and downmodulate immunity and protection. The immune mechanisms behind the above observations and their potential impact on vaccine development are discussed.

  18. Glia maturation factor gamma regulates the migration and adherence of human T lymphocytes

    Directory of Open Access Journals (Sweden)

    Lippert Dustin ND

    2012-04-01

    Full Text Available Abstract Background Lymphocyte migration and chemotaxis are essential for effective immune surveillance. A critical aspect of migration is cell polarization and the extension of pseudopodia in the direction of movement. However, our knowledge of the underlying molecular mechanisms responsible for these events is incomplete. Proteomic analysis of the isolated leading edges of CXCL12 stimulated human T cell lines was used to identify glia maturation factor gamma (GMFG as a component of the pseudopodia. This protein is predominantly expressed in hematopoietic cells and it has been shown to regulate cytoskeletal branching. The present studies were undertaken to examine the role of GMFG in lymphocyte migration. Results Microscopic analysis of migrating T-cells demonstrated that GMFG was distributed along the axis of movement with enrichment in the leading edge and behind the nucleus of these cells. Inhibition of GMFG expression in T cell lines and IL-2 dependent human peripheral blood T cells with shRNAmir reduced cellular basal and chemokine induced migration responses. The failure of the cells with reduced GMFG to migrate was associated with an apparent inability to detach from the substrates that they were moving on. It was also noted that these cells had an increased adherence to extracellular matrix proteins such as fibronectin. These changes in adherence were associated with altered patterns of β1 integrin expression and increased levels of activated integrins as detected with the activation specific antibody HUTS4. GMFG loss was also shown to increase the expression of the β2 integrin LFA-1 and to increase the adhesion of these cells to ICAM-1. Conclusions The present studies demonstrate that GMFG is a component of human T cell pseudopodia required for migration. The reduction in migration and increased adherence properties associated with inhibition of GMFG expression suggest that GMFG activity influences the regulation of integrin mediated

  19. Selection of IgG variants with increased FcRn binding using random and directed mutagenesis: impact on effector functions

    Directory of Open Access Journals (Sweden)

    Céline eMonnet

    2015-02-01

    Full Text Available Despite the reasonably long half-life of IgGs, market pressure for higher patient convenience while conserving efficacy continues to drive IgG half-life improvement. IgG half-life is dependent on the neonatal Fc receptor FcRn, which amongst other functions, protects IgG from catabolism. FcRn binds the Fc domain of IgG at an acidic pH ensuring that endocytosed IgG will not be degraded in lysosomal compartments and will then be released into the bloodstream. Consistent with this mechanism of action, several Fc engineered IgG with increased FcRn affinity and conserved pH-dependency were designed and resulted in longer half-life in vivo in human FcRn transgenic mice (hFcRn, cynomolgus monkeys and recently in healthy humans. These IgG variants were usually obtained by in silico approaches or directed mutagenesis in the FcRn binding site. Using random mutagenesis, combined with a pH-dependent phage display selection process, we isolated IgG variants with improved FcRn-binding which exhibited longer in vivo half-life in hFcRn mice. Interestingly, many mutations enhancing Fc/FcRn interaction were located at a distance from the FcRn binding site validating our random molecular approach. Directed mutagenesis was then applied to generate new variants to further characterize our IgG variants and the effect of the mutations selected. Since these mutations are distributed over the whole Fc sequence, binding to other Fc effectors, such as complement C1q and FcgRs, was dramatically modified, even by mutations distant from these effectors’ binding sites. Hence, we obtained numerous IgG variants with increased FcRn binding and different binding patterns to other Fc effectors, including variants without any effector function, providing distinct fit-for-purpose Fc molecules. We therefore provide evidence that half-life and effector functions should be optimized simultaneously as mutations can have unexpected effects on all Fc receptors that are critical for Ig

  20. Structural and neurochemical correlates of individual differences in gamma frequency oscillations in human visual cortex.

    Science.gov (United States)

    Robson, Siân E; Muthukumarawswamy, Suresh D; John Evans, C; Shaw, Alexander; Brealy, Jennifer; Davis, Brittany; McNamara, Grainne; Perry, Gavin; Singh, Krish D

    2015-10-01

    Neuronal oscillations in the gamma frequency range play an important role in stimulus processing in the brain. The frequency of these oscillations can vary widely between participants and is strongly genetically determined, but the cause of this variability is not understood. Previous studies have reported correlations between individual differences in gamma frequency and the concentration of the inhibitory neurotransmitter, gamma-aminobutyric acid (GABA), as well as with age and primary visual cortex (V1) area and thickness. This study assessed the relationships between all of these variables in the same group of participants. There were no significant correlations between gamma frequency and GABA+ concentration, V1 area or V1 thickness, although the relationship with GABA+/Cr approached significance. Considering age as a covariate further reduced the strength of all correlations and, in an additional dataset with a larger age range, gamma frequency was strongly inversely correlated with age but not V1 thickness or area, suggesting that age modulates gamma frequency via an additional, as yet unknown, mechanism. Consistent with other recent studies, these findings do not demonstrate a clear relationship between gamma frequency and GABA+ concentration. Further investigation of additional variables and the interactions between them will be necessary in order to more accurately determine predictors of the frequency of gamma oscillations. © 2015 Anatomical Society.

  1. Isolation of novel human cDNA (hGMF-gamma) homologous to Glia Maturation Factor-beta gene.

    Science.gov (United States)

    Asai, K; Fujita, K; Yamamoto, M; Hotta, T; Morikawa, M; Kokubo, M; Moriyama, A; Kato, T

    1998-03-13

    A novel full-length human cDNA homologous to Glia Maturation Factor-beta (GMF-beta) gene was isolated. Sequence analysis of the entire cDNA revealed an open reading frame of 426 nucleotides with a deduced protein sequence of 142 amino acid residues. The deduced amino acid sequences of its putative product is highly homologous to human GMF-beta (82% identity) and named for GMF-gamma. Northern blot analysis indicated that a message of 0.9 kb long, but not 4.1 kb of GMF-beta, is predominantly expressed in human lung, heart, and placenta.

  2. Comparison of techniques for morphologic evaluation of glycerol-preserved human skim subjected to gamma radiation

    Energy Technology Data Exchange (ETDEWEB)

    Bringel, Fabiana de A. [Faculty of Humanities, Economic and Health Sciences of Araguaina ITPAC (FAHESA/ITPAC/TO) Araguaina, TO (Brazil); Isaac, Cesar [Faculty of Medicine, University of Sao Paulo (FMUSP/SP) Sao Paulo, SP (Brazil); Herson, Marisa R., E-mail: marisah@vifm.org [Tissue Bank of Victoria, Victoria (Australia); Freitas, Anderson Z. de; Martinho Junior, Antonio C.; Mathor, Monica B., E-mail: azanardi@ipen.br, E-mail: mathor@ipen.br [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil). Radiation Technology Centre; Oliveira, Sergio F. de [Institute of Biomedical Sciences, University of Sao Paulo (ICB-USP/SP), SP (Brazil)

    2011-07-01

    Extensive skin lesions expose the body to damaging agents, which makes spontaneous regeneration difficult and, in many cases, leads patient to death. In such cases, if there are no donating areas for auto graft, allografts can be used. In this type of graft, tissue is processed in tissue banks, where it can be subjected to radiosterilization. According to in vitro studies, gamma radiation, in doses higher than 25 kGy, causes breakdown of collagen I fibrils in the skin preserved in glycerol at 85% and this change influences fibroblast migration and deposition of new collagen. In order to assess if the alterations observed in vitro, would compromise in vivo use, transplants of human tissue, irradiated or not, were performed in Nude mice. After the surgery the skins of the mice was subjected to macroscopic analysis on the 3{sup rd}, 7{sup th}, 21{sup st} and 90{sup th} days; optical coherence tomography on the 90{sup th} day and histological assay on the 3{sup rd}, 7{sup th}, 21{sup st} days to compare the results of the repair process among the techniques, considering that the OCT allows in vivo and not destructive morphological analysis. According to the results obtained through OCT it was possible to observe a more organized repair process in the animals which received irradiated grafts (25 and 50 kGy) if compared to unirradiated grafts. It was not possible to observe such phenomena through macroscopic or histological evaluation. (author)

  3. Multiscale Aspects of Generation of High-Gamma Activity during Seizures in Human Neocortex123

    Science.gov (United States)

    Marcuccilli, Charles J.; Ben-Mabrouk, Faiza; Lew, Sean M.; Goodman, Robert R.; McKhann, Guy M.; Frim, David M.; Kohrman, Michael H.; Schevon, Catherine A.; van Drongelen, Wim

    2016-01-01

    High-gamma (HG; 80-150 Hz) activity in macroscopic clinical records is considered a marker for critical brain regions involved in seizure initiation; it is correlated with pathological multiunit firing during neocortical seizures in the seizure core, an area identified by correlated multiunit spiking and low frequency seizure activity. However, the effects of the spatiotemporal dynamics of seizure on HG power generation are not well understood. Here, we studied HG generation and propagation, using a three-step, multiscale signal analysis and modeling approach. First, we analyzed concurrent neuronal and microscopic network HG activity in neocortical slices from seven intractable epilepsy patients. We found HG activity in these networks, especially when neurons displayed paroxysmal depolarization shifts and network activity was highly synchronized. Second, we examined HG activity acquired with microelectrode arrays recorded during human seizures (n = 8). We confirmed the presence of synchronized HG power across microelectrode records and the macroscale, both specifically associated with the core region of the seizure. Third, we used volume conduction-based modeling to relate HG activity and network synchrony at different network scales. We showed that local HG oscillations require high levels of synchrony to cross scales, and that this requirement is met at the microscopic scale, but not within macroscopic networks. Instead, we present evidence that HG power at the macroscale may result from harmonics of ongoing seizure activity. Ictal HG power marks the seizure core, but the generating mechanism can differ across spatial scales. PMID:27257623

  4. Deficient recovery from potentially lethal damage in some gamma-irradiated human fibroblast cell strains

    Energy Technology Data Exchange (ETDEWEB)

    Arlett, C.F.; Priestley, A. (Medical Research Council, Brighton (UK). Cell Mutation Unit)

    1984-01-01

    The repair of potentially lethal damage following treatment with gamma radiation was investigated in human fibroblasts held in a non-cycling state by maintenance in a medium containing 0.5% foetal calf serum. Normal cells were found to be competent in the repair of PLD. Ataxia-telangiectasia cells were deficient as was a heterozygote suggesting that a failure to repair PLD may make it possible to detect such heterozygotes. Fibroblasts from Huntington's disease patients were either slightly or no more sensitive than cells from normal individuals. Cultures from two individuals in the former class showed limited capacity to repair PLD but cells from the latter class were as competent as normals. Thus assays of radiosensitivity where conditions allow for the repair of PLD may maximise small differences in sensitivity. Cells taken from three patients suffering from Basal Cell Naevus Syndrome were also shown to be defective in the repair of PLD. The existence of such a defect may be related to the increased frequency of basal cell cancer observed in exposed fields following irradiation of such individuals.

  5. Effect of a topical treatment in organotypic culture of human breast skin after exposure to gamma-rays

    Directory of Open Access Journals (Sweden)

    N Gagliano

    2009-08-01

    Full Text Available The early radiation of epidermal reactions can lead to healing of the lesion or radiation necrosis. There is no general agreement for either the prevention and/or treatment of radiation skin response, also as little is known about the immediate phases of this phenomenon. We investigated the early effects exerted by Healing and Wound Emulsion (HWE on human skin response after ionizing radiation. Epidermal morphology, Heat Shock Protein (HSP 70, and Transforming Growth Factor-b1 (TGF-b1 gene expression were investigated in organotypic human skin cultures undergoing a double dose of gamma-rays (2 Gy. HSP70 gene expression tended to be induced in the HWE group 6 hours after cream administration and was significantly up-regulated after 48 hours, when epidermal morphological alterations were evident. TGF- b1 seems not affected in cream treated samples. HWE may stimulate skin to mount an early defensive response against damage induced by gamma rays.

  6. Effects of gamma-ray-induced free radicals on the metal content and amino acid composition of human metallothionein-1

    Indian Academy of Sciences (India)

    Lieven Goossens

    2011-06-01

    Metallothioneins (MTs), a low-mass class of metalloproteins, are characterized by a high thiolate sulphur and metal content. MTs are involved in metal homeostasis and heavy metal detoxification, and are efficient scavengers of free radicals. This article describes zinc release from human MT-1 and modification of its amino acid composition when subjected to free radicals generated during gamma ray radiolysis. The effect of gamma ray radiolysis of untreated and metal-depleted human MT-1 was tested under multiple aerobic and anaerobic conditions at increasing irradiation doses. Under all conditions, a rapid increase of serine in the early stages of irradiation was observed. Irradiation for longer times led to cysteic acid formation, except under argon atmosphere. Several other amino acid concentrations gradually decreased. Formation of limited amounts of hydroxyproline, hydroxylysine and ornithine as well as some less common derivatives such as cystathionine occurred as side-effects.

  7. IL-1 and Tumor Necrosis Factor-Alpha Each Up-Regulate Both the Expression of IFN-Gamma Receptors and Enhance IFN-Gamma-Induced HLA-DR expression on Human Monocytes and a Human Monocytic Cell Line (THP-1),

    Science.gov (United States)

    1993-02-01

    Demoi stration and partial characterization DR antigen expression in ,itro bi, lymphokines and recoin of the interferon gamma receptor on human...independent pathwkay tit Ma’- class 1f induction in human islet cells by interferon - gamma rophage activation, defined in the SCID mouse. lmnnun~ol

  8. Cytotoxic activities of amentoflavone against human breast and cervical cancers are mediated by increasing of PTEN expression levels due to peroxisomes proliferate-activated receptor {gamma} activation

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Eunjung; Shin, Soyoung; Lee, Jeeyoung; Lee, So Jung; Kim, Jinkyoung; Yoon, Doyoung; Kim, Yangmee [Konkuk Univ., Seoul (Korea, Republic of); Woo, Eunrhan [Chosun Univ., Gwangju (Korea, Republic of)

    2012-07-15

    Human peroxisomes proliferate-activated receptor gamma (hPPAR{gamma}) has been implicated in numerous pathologies, including obesity, diabetes, and cancer. Previously, we verified that amentoflavone is an activator of hPPAR{gamma} and probed the molecular basis of its action. In this study, we investigated the mechanism of action of amentoflavone in cancer cells and demonstrated that amentoflavone showed strong cytotoxicity against MCF-7 and HeLa cancer cell lines. We showed that hPPAR{gamma} expression in MCF-7 and HeLa cells is specifically stimulated by amentoflavone, and suggested that amentoflavone-induced cytotoxic activities are mediated by activation of hPPAR{gamma} in these two cancer cell lines. Moreover, amentoflavone increased PTEN levels in these two cancer cell lines, indicating that the cytotoxic activities of amentoflavone are mediated by increasing of PTEN expression levels due to hPPAR{gamma} activation.

  9. Dicty_cDB: FC-BS13 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-BS13 (Link to dictyBase) - - - Contig-U15818-1 FC-BS13P (Li...nk to Original site) FC-BS13F 636 FC-BS13Z 565 FC-BS13P 1201 - - Show FC-BS13 Library FC (Link to library) Clone ID FC-BS1...nal site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-BS/FC-BS13Q.Seq.d/ Repr...esentative seq. ID FC-BS13P (Link to Original site) Representative DNA sequence >FC-BS13 (FC-BS13Q) /CSM/FC/FC-BS/FC-BS1...tiii*skr*rnn**ekiiifwkiint *idinqkkkk Homology vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value FC-BS1

  10. Adsorption of biopolymers human serum albumin and human gamma globulin to well-defined surfaces of self-assembled monolayers

    Science.gov (United States)

    Cregger, Tricia Ann

    The tenacity with which the blood proteins Human Serum Albumin (HSA) and Human Gamma Globulin (HGG) adsorb to a surface modified with a monomolecular coating varies with the packing of the alkyl chains in the coating. The adsorption of proteins onto well-defined surfaces of self-assembled monolayers (SAMs) was studied with X-ray reflectometry (XR), neutron reflectometry (NR), optical reflectometry, and total internal reflection fluorescence (TIRF). NR and XR was used to study adsorption in the absence of flow, while optical reflectometry and TIRF were used to probe the adsorption under flow conditions. In particular, competitive adsorption measurements of binary solutions of HSA, HGG and Fibrinogen (FIB) were performed with TIRE The properties of the surface were varied by altering the alkyl chains' packing density and the chain end functionality of the SAMs. The depth profiles of protein concentration near the adsorbing surface measured by NR were dependent upon the chain packing density in the case of HSA. The concentration depth profile of HGG was unaltered by varying chain packing density. Measurements performed under flow using optical reflectometry showed a different behavior: the surface excess of adsorbed HSA was relatively independent of the surface packing, while the surface excess of HGG depended on the packing density of the SAM. The tenacity with which the proteins adsorbed to different functionalized surfaces was determined by attempting to remove the protein using a strong surfactant, sodium dodecyl sulfate (SDS). Ex situ XR measurements suggested that both HSA and HGG adsorb more tenaciously to a less densely-packed monolayer, almost independent of surface functionality. Two exceptions were a less densely-packed vinyl-terminated monolayer and a less densely-packed bromine-terminated monolayer, from which HSA could not be removed at all.

  11. Hypoxia upregulates Bcl-2 expression and suppresses interferon-gamma induced antiangiogenic activity in human tumor derived endothelial cells.

    LENUS (Irish Health Repository)

    Wang, Jiang Huai

    2012-02-03

    BACKGROUND: Hypoxia in solid tumors potentially stimulates angiogenesis by promoting vascular endothelial growth factor (VEGF) production and upregulating VEGF receptor expression. However, it is unknown whether hypoxia can modulate the effect of anti-angiogenic treatment on tumor-derived endothelium. METHODS: Human tumor-derived endothelial cells (HTDEC) were freshly isolated from surgically removed human colorectal tumors by collagenase\\/DNase digestion and Percol gradient sedimentation. Cell proliferation was assessed by measuring BrdU incorporation, and capillary tube formation was measured using Matrigel. Cell apoptosis was assessed by flow cytometry and ELISA, and Bcl-2 expression was detected by Western blot analysis. RESULTS: Under aerobic culture conditions (5% CO2 plus 21% O2) HTDEC expressed less Bcl-2 and were more susceptible to IFN-gamma-induced apoptosis with significant reductions in both cell proliferation and capillary tube formation, when compared with normal human macrovascular and microvascular EC. Following exposure of HTDEC to hypoxia (5% CO2 plus 2% O2), IFN-gamma-induced cell apoptosis, and antiangiogenic activity (i.e. an inhibition in cell proliferation and capillary tube formation) in HTDEC were markedly attenuated. This finding correlated with hypoxia-induced upregulation of Bcl-2 expression in HTDEC. CONCLUSIONS: These results indicate that hypoxia can protect HTDEC against IFN-gamma-mediated cell death and antiangiogenic activity, and suggest that improvement of tumor oxygenation may potentiate the efficacy of anti-cancer therapies specifically targeting the inhibition of tumor angiogenesis.

  12. Multistep process of neoplastic transformation of normal human fibroblasts by 60Co gamma rays and Harvey sarcoma viruses

    Energy Technology Data Exchange (ETDEWEB)

    Namba, M.; Nishitani, K.; Fukushima, F.; Kimoto, T.; Nose, K.

    1986-03-15

    As reported previously (Namba et al., 1985), normal human fibroblasts were transformed by 60Co gamma-ray irradiation into immortal cells with abnormal karyotypes. These transformed cells (KMST-6), however, showed a low cloning efficiency in soft agar and no transplantability. However, upon treatment with Harvey murine sarcoma virus (Ha-MSV), the cells acquired elevated clonability in soft agar and transplantability in nude mice. Ha-MSV alone, however, did not convert normal human fibroblasts into either immortal or tumorigenic cells. The Ha-MSV-transformed KMST-6 cells showed an enhanced expression of the ras oncogene, but normal and 60Co gamma-ray-transformed cells did not. Our current data suggest that gamma rays worked against normal human cells as an initiator, giving rise to chromosome aberrations and immortality, and that Ha-MSV, probably through its ras oncogene, played a role in the progression of the malignant cell population to a more malignant one showing enhanced colony formation in soft agar and tumorigenicity in nude mice.

  13. Evaluate an impact of incident alpha particle and gamma ray on human blood components: A comparison study

    Energy Technology Data Exchange (ETDEWEB)

    Ismail, Asaad H.; Yaba, Sardar P.; Ismail, Haider J. [Medical Physics Research Group, Physics Department, Education College, Salahaddin University-Erbil, Iraqi Kurdistan (Iraq)

    2015-07-01

    An impact of alpha and gamma irradiation on human blood components have been evaluated and compared for healthy blood samples (male and females). Irradiation dose and time of irradiation calibrated and considered as a main comparison factors. Density of blood components measured for each in vitro irradiation before and after irradiation for males and females. Survey radiation dosimeter (Inspector Exp) and nuclear track detectors type CR-39 used to evaluate exposure dose rate and incident density of alpha particles, respectively. Experiment results verified that the irradiation of blood makes ionizing of blood components, either alpha or gamma irradiation dose, and the impacts of ionizing radiation were relativity for WBC, RBC, and PLT. Limited irradiation doses of 1-5 μSv/hr considered as a low radiation dose of alpha and gamma radiation sources ({sup 226}Ra, and {sup 137}Cs). Density of alpha particles accumulated on the blood surface was 34 (alpha particle/cm{sup 2}) for selected dose of incident alpha particle. Optimum value of irradiation dose and time of irradiation were 5 μSv/hr and 4 second for males and females. On the other hands, the values of irradiation dose and time of irradiation were 2.1 μSv/hr and 2 second for males and females for gamma irradiation. Thus, present results demonstrated that densities of RBC and WBC cells are capable of inducing reproduction in vitro for both type of irradiation. (authors)

  14. Neonatal Fc receptor expression in dendritic cells mediates protective immunity against colorectal cancer.

    Science.gov (United States)

    Baker, Kristi; Rath, Timo; Flak, Magdalena B; Arthur, Janelle C; Chen, Zhangguo; Glickman, Jonathan N; Zlobec, Inti; Karamitopoulou, Eva; Stachler, Matthew D; Odze, Robert D; Lencer, Wayne I; Jobin, Christian; Blumberg, Richard S

    2013-12-12

    Cancers arising in mucosal tissues account for a disproportionately large fraction of malignancies. Immunoglobulin G (IgG) and the neonatal Fc receptor for IgG (FcRn) have an important function in the mucosal immune system that we have now shown extends to the induction of CD8(+) T cell-mediated antitumor immunity. We demonstrate that FcRn within dendritic cells (DCs) was critical for homeostatic activation of mucosal CD8(+) T cells that drove protection against the development of colorectal cancers and lung metastases. FcRn-mediated tumor protection was driven by DCs activation of endogenous tumor-reactive CD8(+) T cells via the cross-presentation of IgG complexed antigens (IgG IC), as well as the induction of cytotoxicity-promoting cytokine secretion, particularly interleukin-12, both of which were independently triggered by the FcRn-IgG IC interaction in murine and human DCs. FcRn thus has a primary role within mucosal tissues in activating local immune responses that are critical for priming efficient anti-tumor immunosurveillance.

  15. Dose-rate effects for apoptosis and micronucleus formation in gamma-irradiated human lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Boreham, D.R.; Dolling, J.-A.; Maves, S.R. [Atomic Energy of Canada Limited, Chalk River, Ontario (Canada); Siwarungsun, N. [Chulalongkorn Univ., Bangkok (Thailand); Mitchel, R.E.J. [Atomic Energy of Canada Limited, Chalk River, Ontario (Canada)

    2000-07-01

    We have compared dose-rate effects for {gamma}-radiation-induced apoptosis and micronucleus formation in human lymphocytes. Long-term assessment of individual radiation-induced apoptosis showed little intraindividual variation but significant interindividual variation. The effectiveness of radiation exposure to cause apoptosis or micronucleus formation was reduced by low-dose-rate exposures, but the reduction was apparent at different dose rates for these two end points. Micronucleus formation showed a dose-rate effect when the dose rate was lowered to 0.29 cGy/min, but there was no accompanying cell cycle delay. A further increase in the dose-rate effect was seen at 0.15 cGy/min, but was now accompanied by cell cycle delay. There was no dose-rate effect for the induction of apoptosis until the dose rate was reduced to 0.15 cGy/min, indicating that the mechanisms or signals for processing radiation-induced lesions for these two end points must be different at least in part. There appear to be two mechanisms that contribute to the dose-rate effect for micronucleus formation. One of these does not affect binucleate cell frequency and occurs at dose rates higher than that required to produce a dose-rate effect for apoptosis, and one affects binucleate cell frequency, induced only at the very low dose rate which coincidentally produces a dose-rate effect for apoptosis. Since the dose rate at which cells showed reduced apoptosis as well as a further reduction in micronucleus formation was very low, we conclude that the processing of the radiation-induced lesions that induce apoptosis, and some micronuclei, is very slow in quiescent and PHA-stimulated lymphocytes, respectively. (author)

  16. Revisiting Field Capacity (FC: variation of definition of FC and its estimation from pedotransfer functions

    Directory of Open Access Journals (Sweden)

    Theophilo Benedicto Ottoni Filho

    2014-12-01

    Full Text Available Taking into account the nature of the hydrological processes involved in in situ measurement of Field Capacity (FC, this study proposes a variation of the definition of FC aiming not only at minimizing the inadequacies of its determination, but also at maintaining its original, practical meaning. Analysis of FC data for 22 Brazilian soils and additional FC data from the literature, all measured according to the proposed definition, which is based on a 48-h drainage time after infiltration by shallow ponding, indicates a weak dependency on the amount of infiltrated water, antecedent moisture level, soil morphology, and the level of the groundwater table, but a strong dependency on basic soil properties. The dependence on basic soil properties allowed determination of FC of the 22 soil profiles by pedotransfer functions (PTFs using the input variables usually adopted in prediction of soil water retention. Among the input variables, soil moisture content θ (6 kPa had the greatest impact. Indeed, a linear PTF based only on it resulted in an FC with a root mean squared residue less than 0.04 m³ m-3 for most soils individually. Such a PTF proved to be a better FC predictor than the traditional method of using moisture content at an arbitrary suction. Our FC data were compatible with an equivalent and broader USA database found in the literature, mainly for medium-texture soil samples. One reason for differences between FCs of the two data sets of fine-textured soils is due to their different drainage times. Thus, a standardized procedure for in situ determination of FC is recommended.

  17. IL-2R{gamma} gene microdeletion demonstrates that canine X-linked severe combined immunodeficiency is a homologue of the human disease

    Energy Technology Data Exchange (ETDEWEB)

    Henthorn, P.S.; Fimiani, V.M.; Patterson, D.F. [Univ. of Pennsylvania School of Veterinary Medicine, Philadelphia, PA (United States)] [and others

    1994-09-01

    X-linked severe combined immunodeficiency (SCID) is characterized by profound defects in cellular and humoral immunity and, in humans, is associated with mutations in the gene for the {gamma} chain of the IL-2 receptor (IL-2R{gamma}). We have examined this gene in a colony of dogs established from a single X-linked SCID carrier female. Affected dogs have a 4-bp deletion in the first exon of the IL-2R{gamma} gene, which precludes the production of a functional protein, demonstrating that the canine disease is a true homologue of human X-linked SCID. 37 refs., 3 figs.

  18. Evaluation of FcεRl-binding serum IgE in patients with ocular allergic diseases

    Directory of Open Access Journals (Sweden)

    Satoru Matsumoto

    1999-01-01

    Full Text Available We evaluated high-affinity receptor for IgE (FcεRI- binding serum IgE in patients with atopic keratoconjunctivitis (AKC; n=31 and with seasonal allergic conjunctivitis (SAC; n=13 by enzyme-linked immunosorbent assay (ELISA using a recombinant soluble form of the human FcεRIα ectodomain (soluble α. The quantities of FcεRI-binding IgE are compared with those of total IgE measured by a conventional sandwich ELISA. Both of the quantities of FcεRI-binding and total IgE in AKC were significantly larger than those in SAC (P<0.001. In contrast, the proportion of FcεRI- binding IgE (FcεRI-binding IgE/total IgE; % in SAC was significantly larger than that in AKC (P <0.001, although significant reverse correlation was observed between the proportion of FcεRI-binding IgE and total IgE in both AKC and SAC. Significantly, a higher proportion of FcεRI-binding IgE in SAC than that in AKC may reflect the differences in pathologic states of AKC and SAC that are caused by a disparity in immune responses in these diseases.

  19. Dicty_cDB: FC-BS14 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-BS14 (Link to dictyBase) - - - Contig-U16399-1 FC-BS14E (Li...nk to Original site) - - - - - - FC-BS14E 534 Show FC-BS14 Library FC (Link to library) Clone ID FC-BS14 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-BS/FC-BS14Q.Seq.d/ Representative seq. ID FC-BS1...4E (Link to Original site) Representative DNA sequence >FC-BS14 (FC-BS14Q) /CSM/FC/FC-BS/FC-BS14Q.Seq.... vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value FC-BS14 (FC-BS1

  20. Dicty_cDB: FC-AV04 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AV04 (Link to dictyBase) - - - Contig-U15991-1 FC-AV04P (Li...nk to Original site) FC-AV04F 307 FC-AV04Z 363 FC-AV04P 670 - - Show FC-AV04 Library FC (Link to library) Clone ID FC-AV...al site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AV/FC-AV04Q.Seq.d/ Representative seq. ID FC-AV...04P (Link to Original site) Representative DNA sequence >FC-AV04 (FC-AV04Q) /CSM/FC/FC-AV/FC-AV...B: ifnftskekkk*nnsfmfsvvtlffnfilfyffifnyffnyffisfffpiqiiiifnyfl fylffls*ipk*lki*av*dyfsnf*l**c*cslrnrkit**--

  1. Dicty_cDB: FC-AS12 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AS12 (Link to dictyBase) - - - Contig-U15984-1 FC-AS12P (Li...nk to Original site) FC-AS12F 486 FC-AS12Z 445 FC-AS12P 931 - - Show FC-AS12 Library FC (Link to library) Clone ID FC-AS...12 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U15984-1 Origin...al site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AS/FC-AS12Q.Seq.d/ Representative seq. ID FC-AS...12P (Link to Original site) Representative DNA sequence >FC-AS12 (FC-AS12Q) /CSM/FC/FC-AS/FC-AS

  2. Dicty_cDB: FC-AS17 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AS17 (Link to dictyBase) - - - Contig-U15985-1 FC-AS17P (Li...nk to Original site) FC-AS17F 390 FC-AS17Z 158 FC-AS17P 548 - - Show FC-AS17 Library FC (Link to library) Clone ID FC-AS...17 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U15985-1 Origin...al site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AS/FC-AS17Q.Seq.d/ Representative seq. ID FC-AS...17P (Link to Original site) Representative DNA sequence >FC-AS17 (FC-AS17Q) /CSM/FC/FC-AS/FC-AS

  3. Dicty_cDB: FC-AS20 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AS20 (Link to dictyBase) - - - Contig-U15987-1 FC-AS20P (Li...nk to Original site) FC-AS20F 620 FC-AS20Z 287 FC-AS20P 907 - - Show FC-AS20 Library FC (Link to library) Clone ID FC-AS...20 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U15987-1 Origin...al site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AS/FC-AS20Q.Seq.d/ Representative seq. ID FC-AS...20P (Link to Original site) Representative DNA sequence >FC-AS20 (FC-AS20Q) /CSM/FC/FC-AS/FC-AS

  4. Dicty_cDB: FC-AI18 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AI18 (Link to dictyBase) - - - Contig-U15590-1 FC-AI18P (Li...nk to Original site) FC-AI18F 621 FC-AI18Z 703 FC-AI18P 1324 - - Show FC-AI18 Library FC (Link to library) Clone ID FC-AI...nal site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI18Q.Seq.d/ Representative seq. ID FC-AI...18P (Link to Original site) Representative DNA sequence >FC-AI18 (FC-AI18Q) /CSM/FC/FC-AI/FC-AI...AVWPLIPGYERA DGEKQYPVAAMLCNFTKPTPTTPSLLTHDEVVTFFHEFGHVMHNMSTKVHYSMFSGTSVE RDFVECPSQLFEFWCWNKDVLVNKLSGHXKDHSKKLPTDLVERMIAAKNLNVAI

  5. Dicty_cDB: FC-AI17 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AI17 (Link to dictyBase) - - - Contig-U15690-1 FC-AI17P (Li...nk to Original site) FC-AI17F 587 FC-AI17Z 626 FC-AI17P 1212 - - Show FC-AI17 Library FC (Link to library) Clone ID FC-AI...nal site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI17Q.Seq.d/ Representative seq. ID FC-AI...17P (Link to Original site) Representative DNA sequence >FC-AI17 (FC-AI17Q) /CSM/FC/FC-AI/FC-AI...slated Amino Acid sequence ANIATVGDFLKADTVVPKMIITYNKRKQGTDYLKAVIGPILSNVIKQELNLELKPNLVYA AIISEQEIRTGEKSTLDRNV

  6. Dicty_cDB: FC-AI03 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AI03 (Link to dictyBase) - - - Contig-U15833-1 FC-AI03P (Li...nk to Original site) FC-AI03F 690 FC-AI03Z 651 FC-AI03P 1341 - - Show FC-AI03 Library FC (Link to library) Clone ID FC-AI...nal site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI03Q.Seq.d/ Representative seq. ID FC-AI...03P (Link to Original site) Representative DNA sequence >FC-AI03 (FC-AI03Q) /CSM/FC/FC-AI/FC-AI...li*l*ivtlskv*qswyqvfcslmrftcwi*nv fht*ivhwsqhwhql*flqpivaiv*skapitifnlhmaslwif*ivl*sfvpfhiitmk sfkfspfvpqlki

  7. Dicty_cDB: FC-AI02 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AI02 (Link to dictyBase) - - - Contig-U16513-1 FC-AI02E (Li...nk to Original site) - - - - - - FC-AI02E 535 Show FC-AI02 Library FC (Link to library) Clone ID FC-AI02 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI02Q.Seq.d/ Representative seq. ID FC-AI...02E (Link to Original site) Representative DNA sequence >FC-AI02 (FC-AI02Q) /CSM/FC/FC-AI/FC-AI02Q.Seq....ogy vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value FC-AI02 (FC-AI

  8. Melanoma cells treated with GGTI and IFN-gamma allow murine vaccination and enhance cytotoxic response against human melanoma cells.

    Directory of Open Access Journals (Sweden)

    Guillaume Sarrabayrouse

    Full Text Available BACKGROUND: Suboptimal activation of T lymphocytes by melanoma cells is often due to the defective expression of class I major histocompatibility antigens (MHC-I and costimulatory molecules. We have previously shown that geranylgeranyl transferase inhibition (done with GGTI-298 stimulates anti-melanoma immune response through MHC-I and costimulatory molecule expression in the B16F10 murine model [1]. METHODOLOGY/PRINCIPAL FINDINGS: In this study, it is shown that vaccination with mIFN-gand GGTI-298 pretreated B16F10 cells induces a protection against untreated tumor growth and pulmonary metastases implantation. Furthermore, using a human melanoma model (LB1319-MEL, we demonstrated that in vitro treatment with hIFN-gamma and GGTI-298 led to the up regulation of MHC-I and a costimulatory molecule CD86 and down regulation of an inhibitory molecule PD-1L. Co-culture experiments with peripheral blood mononuclear cells (PBMC revealed that modifications induced by hIFN-gamma and GGTI-298 on the selected melanoma cells, enables the stimulation of lymphocytes from HLA compatible healthy donors. Indeed, as compared with untreated melanoma cells, pretreatment with hIFN-gamma and GGTI-298 together rendered the melanoma cells more efficient at inducing the: i activation of CD8 T lymphocytes (CD8+/CD69+; ii proliferation of tumor-specific CD8 T cells (MelanA-MART1/TCR+; iii secretion of hIFN-gamma; and iv anti-melanoma specific cytotoxic cells. CONCLUSIONS/SIGNIFICANCE: These data indicate that pharmacological treatment of melanoma cell lines with IFN-gamma and GGTI-298 stimulates their immunogenicity and could be a novel approach to produce tumor cells suitable for vaccination and for stimulation of anti-melanoma effector cells.

  9. Probiotics and commensals reverse TNF-alpha- and IFN-gamma-induced dysfunction in human intestinal epithelial cells.

    Science.gov (United States)

    Resta-Lenert, Silvia; Barrett, Kim E

    2006-03-01

    Commensal bacteria are crucial for the development of the mucosal immune system. Probiotics are commensals with special characteristics and may protect mucosal surfaces against pathogens. Pathogens cause significant phenotypic alterations in infected epithelial cells, and probiotics reverse these deleterious responses. We hypothesized that probiotics and/or commensals may also reverse epithelial damage produced by cytokines. Human intestinal epithelial cells were exposed basolaterally to interferon (IFN)-gamma (10(3) U/mL) or tumor necrosis factor (TNF)-alpha (10 ng/mL) for up to 48 hours and assessed for ion transport, transepithelial resistance (TER), and epithelial permeability in the presence or absence of probiotics (Streptococcus thermophilus [ST] and Lactobacillus acidophilus [LA]), or the commensal, Bacteroides thetaiotaomicron (BT). Agonist-stimulated chloride secretion was inhibited by IFN-gamma, an effect prevented by ST/LA or BT. The ability of ST/LA or BT to restore Cl(-) secretion was blocked by inhibitors of p38 MAPK, ERK1, 2, and PI3K. The cystic fibrosis transmembrane conductance regulator (CFTR) and the NKCC1 cotransporter were down-regulated by IFN-gamma, and ST/LA pretreatment reversed this effect. Both TNF-alpha and IFN-gamma significantly reduced TER and increased epithelial permeability, effects prevented by ST/LA or BT. A Janus kinase (JAK) inhibitor synergistically potentiated effects of ST/LA or BT on TER and permeability, but p38, ERK1, 2, or PI3K inhibition did not. Finally, only probiotic-treated epithelial cells exposed to cytokines showed reduced activation of SOCS3 and STAT1,3. Deleterious effects of TNF-alpha and IFN-gamma on epithelial function are prevented by probiotic, and to a lesser extent, commensal pretreatment. These data extend the spectrum of effects of such bacteria on intestinal epithelial function and may justify their use in inflammatory disorders.

  10. Dose-response calibration curves of {sup 137}Cs gamma rays for dicentric chromosome aberrations in human lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Jo, Wol Soon; Oh, Su Jung; Jeong, Soo Kyun; Yang, Kwang Mo [Dept. of Research center, Dong Nam Institute of Radiological and Medical Sciences, Busan (Korea, Republic of); Jeong, Min Ho [Dept. of Microbiology, Dong A University College of Medicine, Busan (Korea, Republic of)

    2012-11-15

    Recently, the increased threat of radiologically industrial accident such as radiation nondestructive inspection or destruction of nuclear accident by natural disaster such as Fukushima accident requires a greater capacity for cytogenetic biodosimetry, which is critical for clinical triage of potentially thousands of radiation-exposed individuals. Dicentric chromosome aberration analysis is the conventional means of assessing radiation exposure. Dose–response calibration curves for {sup 13}'7Cs gamma rays have been established for unstable chromosome aberrations in human peripheral blood lymphocytes in many laboratories of international biodosimetry network. In this study, therefore, we established dose– response calibration curves of our laboratory for {sup 137}Cs gamma raysaccording to the IAEA protocols for conducting the dicentric chromosome assay We established in vitro dose–response calibration curves for dicentric chromosome aberrations in human lymphocytes for{sup 13}'7Cs gamma rays in the 0 to 5 Gy range, using the maximum likelihood linear-quadratic model, Y = c+αD+βD2. The estimated coefficients of the fitted curves were within the 95% confidence intervals (CIs) and the curve fitting of dose–effect relationship data indicated a good fit to the linear-quadratic model. Hence, meaningful dose estimation from unknown sample can be determined accurately by using our laboratory’s calibration curve according to standard protocol.

  11. Antitumour effects on human colorectal carcinomas cells by stable silencing of phospholipase C-gamma 1 with lentivirus-delivered siRNA

    Institute of Scientific and Technical Information of China (English)

    TAN Li; XIAO Bing-xiang; ZENG Wei-sen; LIN Jun; ZOU Zhi-peng; XU Ai-min; LUO Shen-qiu

    2007-01-01

    Background In most colorectal carcinomas, the level of phospholipase C (PLC)-gamma 1 expression is greatly elevated. Increased expression of PLC-gamma 1 may play an important role in colon carcinogenesis, but the mechanism is not well known. The aim of this study was to evaluate the role of PLC-gamma 1 in colon carcinogenesis by using recombinant lentivirus that stably suppressed the PLC-gamma 1 expression in human colorectal carcinoma LoVo cells.Methods Recombinant lentivirus producing PLC-gamma 1 siRNA were prepared. After LoVo cells were transduced by each lentivirus, stably transduced cells were selected by Blasticidin. The protein and mRNA expression of PLC-gamma 1 were examined by Western-blot and reverse transcription-polymerase chain reaction (RT-PCR) analysis, and the effects of the lentivirus on the cell adhesion, migration and apoptosis were analyzed.Results Stable LoVo cell line deficient in PLC-gamma 1, was established. Notably, PLC-gamma 1 was silenced without affecting the levels of other subtypes of PLC so that the role of PLC-gamma 1 in colon carcinogenesis could be examined.Silencing of endogenous PLC-gamma 1 resulted in efficient inhibition of the adhesion and migration of LoVo cells in vitro and a great increase of 5-fluorouracil induced apoptosis (30%-40%) of LoVo cells.Conclusions PLC-gamma 1 may play an important role in metastasis and anti-apoptosis in human colorectal carcinomas.

  12. CREB, ATF, and AP-1 transcription factors regulate IFN-gamma secretion by human T cells in response to mycobacterial antigen.

    Science.gov (United States)

    Samten, Buka; Townsend, James C; Weis, Steven E; Bhoumik, Anindita; Klucar, Peter; Shams, Homayoun; Barnes, Peter F

    2008-08-01

    IFN-gamma production by T cells is pivotal for defense against many pathogens, and the proximal promoter of IFN-gamma, -73 to -48 bp upstream of the transcription start site, is essential for its expression. However, transcriptional regulation mechanisms through this promoter in primary human cells remain unclear. We studied the effects of cAMP response element binding protein/activating transcription factor (CREB/ATF) and AP-1 transcription factors on the proximal promoter of IFN-gamma in human T cells stimulated with Mycobacterium tuberculosis. Using EMSA, supershift assays, and promoter pulldown assays, we demonstrated that CREB, ATF-2, and c-Jun, but not cyclic AMP response element modulator, ATF-1, or c-Fos, bind to the proximal promoter of IFN-gamma upon stimulation, and coimmunoprecipitation indicated the possibility of interaction among these transcription factors. Chromatin immunoprecipitation confirmed the recruitment of these transcription factors to the IFN-gamma proximal promoter in live Ag-activated T cells. Inhibition of ATF-2 activity in T cells with a dominant-negative ATF-2 peptide or with small interfering RNA markedly reduced the expression of IFN-gamma and decreased the expression of CREB and c-Jun. These findings suggest that CREB, ATF-2, and c-Jun are recruited to the IFN-gamma proximal promoter and that they up-regulate IFN-gamma transcription in response to microbial Ag. Additionally, ATF-2 controls expression of CREB and c-Jun during T cell activation.

  13. Purification and characterisation of recombinant human eukaryotic elongation factor 1 gamma.

    Science.gov (United States)

    Achilonu, Ikechukwu; Siganunu, Thendo P; Dirr, Heini W

    2014-07-01

    The eukaryotic elongation factor 1 gamma (eEF1γ) is a multi-domain protein, which consist of a glutathione transferase (GST)-like N-terminus domain. In association with α, β and δ subunits, eEF1γ forms part of the eukaryotic elongation factor complex, which is mainly involved in protein biosynthesis. The N-terminus GST domain of eEF1γ interacts with the β subunit. eEF1γ subunit is over-expressed in human carcinoma. The role of human eEF1γ (heEF1γ) is poorly understood. A successful purification of recombinant heEF1γ is the first step towards determining unknown properties of the protein, including putative GST-like activities and the structure of the protein. This paper describes the over-expression, purification and characterisation of recombinant full-length, and the N- and C-terminus domains of heEF1γ. All three recombinant heEF1γ constructs over-expressed in the soluble Escherichia coli cell fraction and were purified to homogeneity. Secondary structure analysis indicates that the heEF1γ constructs have high α-helical structural character. The full-length and N-terminus domain are dimeric, while the C-terminus is monomeric. Both full-length and N-terminus domain interact with 8-anilino-1-naphthalene sulfonate (ANS) with KD=70.0 (±5.7) μM and with reduced glutathione (GSH). Glutathione sulfonate displaced ANS bound to hydrophobic binding sites in the recombinant N-terminus domain. Using the standard GSH-1-chloro-2,4-dinitrobenzene conjugation assay, the N-domain showed some enzyme activity (0.03μmolmin(-1) mg(-1) protein), while the full-length heEF1γ did not catalyse the GSH-CDNB conjugation. Consequently, we hypothesize the presence of a presumed GST-like active site structure in the heEF1γ, which comprises a glutathione binding site and a hydrophobic substrate binding site.

  14. Rapid polyclonal desensitization with antibodies to IgE and FcεRIα.

    Science.gov (United States)

    Khodoun, Marat V; Kucuk, Zeynep Yesim; Strait, Richard T; Krishnamurthy, Durga; Janek, Kevin; Lewkowich, Ian; Morris, Suzanne C; Finkelman, Fred D

    2013-06-01

    Rapid desensitization, a procedure in which persons allergic to an antigen are treated at short intervals with increasing doses of that antigen until they tolerate a large dose, is an effective, but risky, way to induce temporary tolerance. We wanted to determine whether this approach can be adapted to suppress all IgE-mediated allergies in mice by injecting serially increasing doses of monoclonal antibodies (mAbs) to IgE or FcεRIα. Active and passive models of antigen- and anti-IgE mAb-induced IgE-mediated anaphylaxis were used. Mice were desensitized with serially increasing doses of anti-IgE mAb, anti-FcεRIα mAb, or antigen. Development of shock (hypothermia), histamine and mast cell protease release, cytokine secretion, calcium flux, and changes in cell number and FcεRI and IgE expression were evaluated. Rapid desensitization with anti-IgE mAb suppressed IgE-mediated immediate hypersensitivity; however, some mice developed mild anaphylaxis during desensitization. Rapid desensitization with anti-FcεRIα mAb that only binds FcεRI that is not occupied by IgE suppressed both active and passive IgE-mediated anaphylaxis without inducing disease. It quickly, but temporarily, suppressed IgE-mediated anaphylaxis by decreasing mast cell signaling through FcεRI, then slowly induced longer lasting mast cell unresponsiveness by removing membrane FcεRI. Rapid desensitization with anti-FcεRIα mAb was safer and longer lasting than rapid desensitization with antigen. A rapid desensitization approach with anti-FcεRIα mAb safely desensitizes mice to IgE-mediated anaphylaxis by inducing mast cell anergy and later removing all mast cell IgE. Rapid desensitization with an anti-human FcεRIα mAb may be able to prevent human IgE-mediated anaphylaxis. Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  15. Simultaneous Targeting of FcγRs and FcαRI Enhances Tumor Cell Killing

    NARCIS (Netherlands)

    Brandsma, Arianne M; Ten Broeke, Toine; Nederend, Maaike; Meulenbroek, Laura A P M; van Tetering, Geert; Meyer, Saskia; Jansen, J H Marco; Beltrán Buitrago, M Alejandra; Nagelkerke, Sietse Q; Németh, István; Ubink, Ruud; Rouwendal, Gerard; Lohse, Stefan; Valerius, Thomas; Leusen, Jeanette H W; Boross, Peter

    2015-01-01

    Efficacy of anticancer monoclonal antibodies (mAb) is limited by the exhaustion of effector mechanisms. IgG mAbs mediate cellular effector functions through FcγRs expressed on effector cells. IgA mAbs can also induce efficient tumor killing both in vitro and in vivo. IgA mAbs recruit FcαRI-expressin

  16. Biodistribution of [sup 125]I labeled monoclonal antibody against gamma seminoprotein in the nude mice bearing human benign prostatic hyperplasia xenografts

    Energy Technology Data Exchange (ETDEWEB)

    Fujino, Awato (Kitasato Univ., Sagamihara, Kanagawa (Japan). School of Medicine)

    1993-07-01

    The biodistribution of [sup 125]I labeled monoclonal antibody against gamma seminoprotein ([gamma]-Sm) in the nude mice bearing human benign prostatic hyperplasia xenograft was evaluated by whole body autoradiography and by counting of radioactivity in organs. The monoclonal antibody (murine Ig G[sub 1], K) to [gamma]-Sm which was established in this institute and its F(ab')[sub 2] fragment were radioiodinated using Iodogen method. The autoradiograms demonstrated specific uptake of [sup 125]I-intact Ig G as well as [sup 125]I-F(ab')[sub 2] within the prostatic adenoma xenografts 4 days after intravenous administrations. Radioactivity in the xenograft was relatively higher than those in the liver, kidney or lung. These results suggest that radio-labeled monoclonal antibody against [gamma]-Sm might be applicable for radioimmunodetection of prostatic tumors which produce [gamma]-Sm. (author).

  17. Organization of human interferon gamma-heparin complexes from solution properties and hydrodynamics.

    Science.gov (United States)

    Perez Sanchez, Horacio; Tatarenko, Karine; Nigen, Michael; Pavlov, Georges; Imberty, Anne; Lortat-Jacob, Hugues; Garcia de la Torre, Jose; Ebel, Christine

    2006-11-01

    Heparan sulfate (HS) recognizes a variety of proteins, one of which is the pleiotropic cytokine IFN-gamma, and as such modulates many biological processes. IFN-gamma is a homodimer with a well-defined core and two flexible C-termini that constitute HS binding domains. We show here using molecular modeling that an extended IFN-gamma structure overlaps a HS fragment of 16 disaccharides (16 nm). Since a 21-24-disaccharide HS fragment was experimentally defined as the minimum size that interacts with IFN-gamma [Lortat-Jacob, H., Turnbull, J. E., and Grimaud, J. A. (1995) Biochem. J. 310 (Part 2), 497-505], this raises the question of the complexe organization. We combine analytical ultracentrifugation, size exclusion chromatography, and hydrodynamic bead modeling to characterize the complexes formed in solution with heparin oligosaccharides. For oligosaccharides of 14 and 20 nm, two types of complexes are formed with one IFN-gamma and one or two heparin molecules. Complexes consisting of two IFN-gamma and one or two heparin molecules are present for a fragment of 25 nm and aggregates for a fragment of 35 nm. The complexes are rather compact and can be formed without major conformational changes of the partners. The complex pattern of interaction is related to the size of the partners and their multiple binding possibilities. These various possibilities suggest networks of interactions at the crowded surface of the cells. Hydrodynamic methods used here proved to be very efficient tools for describing protein-HS complexes that, due to the intrinsic heterogeneity and flexibility of the partners, are otherwise very difficult to analyze.

  18. Impact of head modeling and sensor types in localizing human gamma-band oscillations.

    Science.gov (United States)

    Mideksa, K G; Hoogenboom, N; Hellriegel, H; Krause, H; Schnitzler, A; Deuschl, G; Raethjen, J; Heute, U; Muthuraman, M

    2014-01-01

    An effective mechanism in neuronal communication is oscillatory neuronal synchronization. The neuronal gamma-band (30-100 Hz) synchronization is associated with attention which is induced by a certain visual stimuli. Numerous studies have shown that the gamma-band activity is observed in the visual cortex. However, impact of different head modeling techniques and sensor types to localize gamma-band activity have not yet been reported. To do this, the brain activity was recorded using 306 magnetoencephalography (MEG) sensors, consisting of 102 magnetometers and 102 pairs of planar gradiometers (one measuring the derivative of the magnetic field along the latitude and the other along the longitude), and the data were analyzed with respect to time, frequency, and location of the strongest response. The spherical head models with a single-shell and overlapping spheres (local sphere) have been used as a forward model for calculating the external magnetic fields generated from the gamma-band activity. For each sensor type, the subject-specific frequency range of the gamma-band activity was obtained from the spectral analysis. The identified frequency range of interest with the highest gamma-band activity is then localized using a spatial-filtering technique known as dynamic imaging of coherent sources (DICS). The source analysis for all the subjects revealed that the gradiometer sensors which measure the derivative along the longitude, showed sources close to the visual cortex (cuneus) as compared to the other gradiometer sensors which measure the derivative along the latitude. However, using the magnetometer sensors, it was not possible to localize the sources in the region of interest. When comparing the two head models, the local-sphere model helps in localizing the source more focally as compared to the single-shell head model.

  19. Transepithelial Transport of Fc -Targeted Nanoparticles by the Neonatal Fc Receptor for Oral Delivery

    Science.gov (United States)

    Pridgen, Eric M.; Alexis, Frank; Kuo, Timothy T.; Levy-Nissenbaum, Etgar; Karnik, Rohit; Blumberg, Richard S.; Langer, Robert; Farokhzad, Omid C.

    2014-01-01

    Nanoparticles are poised to have a tremendous impact on the treatment of many diseases, but their broad application is limited because currently they can only be administered by parenteral methods. Oral administration of nanoparticles is preferred but remains a challenge because transport across the intestinal epithelium is limited. Here, we show that nanoparticles targeted to the neonatal Fc receptor (FcRn), which is known to mediate the transport of IgG antibodies across epithelial barriers, are efficiently transported across the intestinal epithelium using both in vitro and in vivo models. In mice, orally administered FcRn-targeted nanoparticles crossed the intestinal epithelium and reached systemic circulation with a mean absorption efficiency of 13.7%*h compared with only 1.2%*h for non-targeted nanoparticles. In addition, targeted nanoparticles containing insulin as a model nanoparticle-based therapy for diabetes were orally administered at a clinically relevant insulin dose of 1.1 U/kg and elicited a prolonged hypoglycemic response in wild-type mice. This effect was abolished in FcRn knockout mice, indicating the enhanced nanoparticle transport was due specifically to FcRn. FcRn-targeted nanoparticles may have a major impact on the treatment of many diseases by enabling drugs currently limited by low bioavailability to be efficiently delivered though oral administration. PMID:24285486

  20. Crosstalk between the peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) and the vitamin D receptor (VDR) in human breast cancer cells: PPAR{gamma} binds to VDR and inhibits 1{alpha},25-dihydroxyvitamin D{sub 3} mediated transactivation

    Energy Technology Data Exchange (ETDEWEB)

    Alimirah, Fatouma; Peng, Xinjian; Yuan, Liang; Mehta, Rajeshwari R. [Cancer Biology Division, IIT Research Institute, 10 West 35th Street, Chicago, IL 60616 (United States); Knethen, Andreas von [Institute of Biochemistry, Johann Wolfgang Goethe University, Frankfurt (Germany); Choubey, Divaker [Department of Environmental Health, University of Cincinnati, 3223 Eden Avenue, P.O. Box 670056, Cincinnati, OH 45267 (United States); Mehta, Rajendra G., E-mail: rmehta@iitri.org [Cancer Biology Division, IIT Research Institute, 10 West 35th Street, Chicago, IL 60616 (United States)

    2012-11-15

    Heterodimerization and cross-talk between nuclear hormone receptors often occurs. For example, estrogen receptor alpha (ER{alpha}) physically binds to peroxisome proliferator-activated receptor gamma (PPAR{gamma}) and inhibits its transcriptional activity. The interaction between PPAR{gamma} and the vitamin D receptor (VDR) however, is unknown. Here, we elucidate the molecular mechanisms linking PPAR{gamma} and VDR signaling, and for the first time we show that PPAR{gamma} physically associates with VDR in human breast cancer cells. We found that overexpression of PPAR{gamma} decreased 1{alpha},25-dihydroxyvitamin D{sub 3} (1,25D{sub 3}) mediated transcriptional activity of the vitamin D target gene, CYP24A1, by 49% and the activity of VDRE-luc, a vitamin D responsive reporter, by 75% in T47D human breast cancer cells. Deletion mutation experiments illustrated that helices 1 and 4 of PPAR{gamma}'s hinge and ligand binding domains, respectively, governed this suppressive function. Additionally, abrogation of PPAR{gamma}'s AF2 domain attenuated its repressive action on 1,25D{sub 3} transactivation, indicating that this domain is integral in inhibiting VDR signaling. PPAR{gamma} was also found to compete with VDR for their binding partner retinoid X receptor alpha (RXR{alpha}). Overexpression of RXR{alpha} blocked PPAR{gamma}'s suppressive effect on 1,25D{sub 3} action, enhancing VDR signaling. In conclusion, these observations uncover molecular mechanisms connecting the PPAR{gamma} and VDR pathways. -- Highlights: PPAR{gamma}'s role on 1{alpha},25-dihydroxyvitamin D{sub 3} transcriptional activity is examined. Black-Right-Pointing-Pointer PPAR{gamma} physically binds to VDR and inhibits 1{alpha},25-dihydroxyvitamin D{sub 3} action. Black-Right-Pointing-Pointer PPAR{gamma}'s hinge and ligand binding domains are important for this inhibitory effect. Black-Right-Pointing-Pointer PPAR{gamma} competes with VDR for the availability of their binding

  1. Neonatal Fc receptors discriminates and monitors the pathway of native and modified immunoglobulin G in placental endothelial cells.

    Science.gov (United States)

    Radulescu, Luminita; Antohe, Felicia; Jinga, Victor; Ghetie, Victor; Simionescu, Maya

    2004-06-01

    In the placenta, immunoglobulin G (IgG) is selectively transported from mother to fetus by a highly regulated transcellular mechanism aimed to achieve fetal humoral immunity. We questioned the role of neonatal Fc receptors (FcRn) in the traffic of IgG in human placental endothelial cells (HPEC). Cells were cultured in a double-chamber system and further exposed to IgG or Fc or to diethylpyrocarbonate-modified IgG or Fc in which the receptor recognition domain of the molecule (CH2-CH3) was altered. We provide evidence that the native IgG/Fc probes are transcytosed or recycled by HPEC, whereas the probes with the altered receptor recognition domain (which do not bind to FcRn) massively accumulate into the endocytic/lysosomal compartments. The results indicate that FcRn distinguishes between the intact and modified IgG and control their cellular traffic: native IgG is salvaged and released out of the cells, whereas modified IgG is retained and sorted to a degradative pathway. The data advance the understanding of the basic mechanism for IgG traffic in human endothelial cells, which may be exploited for the specific transport of antibodies in various immune disorders.

  2. IgG receptor FcγRIIB plays a key role in obesity-induced hypertension.

    Science.gov (United States)

    Sundgren, Nathan C; Vongpatanasin, Wanpen; Boggan, Brigid-Meghan D; Tanigaki, Keiji; Yuhanna, Ivan S; Chambliss, Ken L; Mineo, Chieko; Shaul, Philip W

    2015-02-01

    There is a well-recognized association between obesity, inflammation, and hypertension. Why obesity causes hypertension is poorly understood. We previously demonstrated using a C-reactive protein (CRP) transgenic mouse that CRP induces hypertension that is related to NO deficiency. Our prior work in cultured endothelial cells identified the Fcγ receptor IIB (FcγRIIB) as the receptor for CRP whereby it antagonizes endothelial NO synthase. Recognizing known associations between CRP and obesity and hypertension in humans, in the present study we tested the hypothesis that FcγRIIB plays a role in obesity-induced hypertension in mice. Using radiotelemetry, we first demonstrated that the hypertension observed in transgenic mouse-CRP is mediated by the receptor, indicating that FcγRIIB is capable of modifying blood pressure. We then discovered in a model of diet-induced obesity yielding equal adiposity in all study groups that whereas FcγRIIB(+/+) mice developed obesity-induced hypertension, FcγRIIB(-/-) mice were fully protected. Levels of CRP, the related pentraxin serum amyloid P component which is the CRP-equivalent in mice, and total IgG were unaltered by diet-induced obesity; FcγRIIB expression in endothelium was also unchanged. However, whereas IgG isolated from chow-fed mice had no effect, IgG from high-fat diet-fed mice inhibited endothelial NO synthase in cultured endothelial cells, and this was an FcγRIIB-dependent process. Thus, we have identified a novel role for FcγRIIB in the pathogenesis of obesity-induced hypertension, independent of processes regulating adiposity, and it may entail an IgG-induced attenuation of endothelial NO synthase function. Approaches targeting FcγRIIB may potentially offer new means to treat hypertension in obese individuals. © 2014 American Heart Association, Inc.

  3. Fc-epsilon-RI, the high affinity IgE-receptor, is robustly expressed in the upper gastrointestinal tract and modulated by mucosal inflammation.

    Directory of Open Access Journals (Sweden)

    Christina Bannert

    Full Text Available BACKGROUND: The role of the high affinity IgE receptor, FcεRI, in IgE-mediated immune responses of the gastrointestinal (GI mucosa is poorly understood. Currently, a detailed characterization of FcεRI expression throughout the human gut is lacking. The aim of this study was to define the expression pattern of FcεRI in the GI tract. METHODS/PRINCIPAL FINDINGS: We compared FcεRI expression in children with gastritis/esophagitis (n = 10, celiac disease (n = 10, inflammatory bowel disease (IBD (n = 9, and normal mucosa (n = 5. The α-subunit of FcεRI (FcεRIα, detected by immunohistochemistry, was found on cells infiltrating the mucosa of the esophagus, the stomach, and the duodenum, but was rarely detected in more distal sections of the GI tract. Accordingly, quantitative RT-PCR analysis on esophagus, stomach, duodenum, colon, and rectum biopsies revealed that FcεRIα and -β expression levels decreased towards the distal intestine. mRNA transcripts of the common Fc-receptor-γ chain were present in the entire GI mucosa. Double-immunofluorescence staining of esophageal specimens confirmed that FcεRIα was expressed on intraepithelial mast cells and Langerhans cells. The mRNA expression levels of the α, β, and γ subunits of FcεRI did not correlate with total serum IgE but were associated with mucosal inflammation. CONCLUSION/SIGNIFICANCE: Our data define the upper GI tract as the main site for IgE-mediated immune activation via FcεRI. Tissue mRNA levels of FcεRIα are regulated by inflammatory conditions rather than serum IgE, indicating that FcεRI might also play a role in pathologies other than allergy.

  4. IgG-Fc-glycosylation in immune-mediated cytopenias

    NARCIS (Netherlands)

    Sonneveld, M.E.

    2017-01-01

    Antibody Fc-glycosylation affects allo- and autoimmunity towards blood cells. Low Fc-fucosylation increases IgG-binding affinity to FcγRIIIa/b, thereby contributing to enhanced cell breakdown and hence, a worse clinical outcome. In this thesis we contribute to the development of new diagnostic assay

  5. Transgenic rabbits that overexpress the neonatal Fc receptor (FcRn generate higher quantities and improved qualities of anti-thymocyte globulin (ATG.

    Directory of Open Access Journals (Sweden)

    Mária Baranyi

    Full Text Available Immune suppression with rabbit anti-thymocyte globulin (rATG is a well-established therapeutic concept for preventing host rejection of transplanted organs and graft versus host disease. Increasing the efficiency of rATG production by reducing the number of animals would be highly beneficial to lower cost and to improve quality standards. We have developed transgenic (Tg mice and rabbits that overexpress the neonatal Fc receptor (FcRn and have shown an augmented humoral immune response in these animals. To test whether our FcRn Tg rabbits produced rATG more efficiently, we immunized them and their New Zealand White controls with live Jurkat cells. By day 21 after immunization, Tg animals produced significantly, 1.5 times higher amount of total IgG compared to their wt littermates. Also, the binding efficiency of Tg sera to Jurkat cells and their complement-mediated cytotoxicity was significantly higher. The purified Tg IgG preparation contained 2.6 the amount of Jurkat specific IgG as the wt preparation analyzed by complement-mediated lysis, suggesting greater antigen-specific B cell activation in the Tg rabbits. To test this hypothesis, immunization with ovalbumin and human α1-antitrypsin was performed, resulting in significantly greater numbers of antigen-specific B-cells in the FcRn Tg rabbits as compared with wt controls. The shift towards significantly larger populations of antigen-specific B cells relative to the non-specific B cell pool is further corroborated by our previous findings in FcRn Tg mice. Consequently, our FcRn Tg rabbits have the potential to offer substantial qualitative and quantitative improvements for the production of rATG and other polyclonal or monoclonal antibodies.

  6. Inhibition of human lung cancer cell growth by the peroxisome proliferator-activated receptor-gamma agonists through induction of apoptosis.

    Science.gov (United States)

    Tsubouchi, Y; Sano, H; Kawahito, Y; Mukai, S; Yamada, R; Kohno, M; Inoue, K; Hla, T; Kondo, M

    2000-04-13

    Peroxisome proliferator-activated receptors (PPARs), members of the nuclear hormone receptors superfamily, have an important regulatory role in adipogenesis and inflammation. PPAR-gamma ligands induce terminal differentiation and growth inhibition of human breast cancer cells and prostatic cancer cells. In this study, we demonstrated that PPAR-gamma, but not PPAR-alpha, was expressed in human lung cancer cell lines by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. We also found that the synthetic PPAR-gamma agonist thiazolidinedione compounds (troglitazone) and the endogenous PPAR-gamma ligand, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), inhibited the growth of human lung cancer cells through the induction of apoptosis. However, PPAR-alpha agonist (bezafibrate) and other prostanoids (PGE(2), PGF(2alpha)) did not induce apoptosis. These findings suggest that PPAR-gamma may play an important role in the pathogenesis of lung cancer and that PPAR-gamma agonist may be useful therapeutic agents in the treatment of human lung cancer. Copyright 2000 Academic Press.

  7. 重组人可溶性PDGFRβ/Fc在昆虫细胞Sf9中的表达%Expression of recombinant human soluble platelet-derived growth factor receptor Beta/Fc chimera in insect cell Sf9

    Institute of Scientific and Technical Information of China (English)

    谢秋玲; 刘兰; 刘秀贵; 张玲; 徐丽慧; 洪岸

    2009-01-01

    [目的]利用昆虫细胞Bac-to-Bac杆状病毒表达系统表达血小板源性生长因子受体β (PDGFRβ)链膜外区与人IgG Fc片段的可溶性受体融合蛋白sPDGFRβ/Fc,并检测重组蛋白的特异性和生物活性.[方法]采用Bac-to-Bac系统,构建重组转移质粒pFastbac-sPDGFRβ/Fc,转化到含穿梭载体Bacmid的感受态细胞DH10Bac中,使目的基因与杆状病毒基因组DNA发生位点特异性重组,获得重组病毒DNA,将其通过脂质体转染昆虫细胞Sf9获得重组病毒.将该重组病毒感染Sf9无血清细胞系,在Sf9细胞中表达sPDGFRβ/Fc,对表达产物进行Western blotting检测和Protein A亲合层析纯化,并进一步通过MTT法检测获得的重组蛋白生物学活性.[结果]重组病毒感染Sf9细胞后,经Western blotting分析,能检测到一条分子量约为97 kDa的特异性条带,与目的蛋白大小相符.通过Protein A亲和层析,获得了纯度达75%以上,表达量为1 μg/mL细胞培养上清的重组融合蛋白,MTT结果显示该重组融合蛋白sPDGFRβ/Fc具有抑制PDGF刺激的Balb/c 3T3细胞增殖的能力.[结论]具有生物活性的重组可溶性受体融合蛋白sPDGFRβ/Fc可在昆虫细胞中成功地得到表达.

  8. Bispecific-armed, interferon gamma-primed macrophage-mediated phagocytosis of malignant non-Hodgkin's lymphoma.

    Science.gov (United States)

    Ely, P; Wallace, P K; Givan, A L; Graziano, R F; Guyre, P M; Fanger, M W

    1996-05-01

    To show that macrophages can be effectively targeted against malignant B cells, bispecific antibodies (BsAb) were constructed from two antibodies having specificity for the high-affinity Fc receptor for IgG (Fc gamma RI/CD64) and the B-cell differentiation antigens CD19 and CD37. Using a flow cytometry-based assay and confocal imaging, we show that these constructs mediated significant phagocytosis of B lymphocytes by macrophages that could be enhanced with interferon gamma (IFN gamma) and IFN gamma in combination with macrophage colony-stimulating factor. BsAb-dependent phagocytosis was triggered through Fc gamma RI and could be blocked only by using F(ab')2 fragments from the parent molecule or by cross-linking Fc gamma RI. BsAb-dependent phagocytosis was not blocked by antibodies to the other Fc receptors, Fc gamma RII and Fc gamma RIII. Because these antibody constructs bind to an epitope outside the Fc gamma RI ligand binding site, we show that autologous serum, polyclonal IgG, and monomeric IgG1 did not block BsAb-dependent phagocytosis, whereas autologous serum and the IgG fractions blocked parent molecule monoclonal antibody-dependent phagocytosis due to the avid binding of monomeric IgG to Fc gamma RI. Finally, BsAb-mediated phagocytosis was effective against the malignant B cells of patients with mantle cell lymphoma, prolymphocytic leukemia, and chronic lymphocytic leukemia. Based on these studies, we propose that BsAbs may provide an effective means of immunomodulation for patients with B-cell malignancies.

  9. DNA repair in modeled microgravity: Double strand break rejoining activity in human lymphocytes irradiated with {gamma}-rays

    Energy Technology Data Exchange (ETDEWEB)

    Mognato, Maddalena, E-mail: maddalena.mognato@unipd.it [Dipartimento di Biologia, Universita di Padova, via U. Bassi 58 B, 35121 Padova (Italy); Girardi, Cristina; Fabris, Sonia [Dipartimento di Biologia, Universita di Padova, via U. Bassi 58 B, 35121 Padova (Italy); Celotti, Lucia [Dipartimento di Biologia, Universita di Padova, via U. Bassi 58 B, 35121 Padova (Italy); Laboratori Nazionali di Legnaro, INFN, Padova (Italy)

    2009-04-26

    Cell response to ionising radiation depends, besides on genetic and physiological features of the biological systems, on environmental conditions occurring during DNA repair. Many data showed that microgravity, experienced by astronauts during space flights or modeled on Earth, causes apoptosis, cytoskeletal alteration, cell growth inhibition, increased frequency of mutations and chromosome aberrations. In this study, we analysed the progression of the rejoining of double strand breaks (DSBs) in human peripheral blood lymphocytes (PBLs) irradiated with {gamma}-rays and incubated in static condition (1g) or in modeled microgravity (MMG). {gamma}-H2AX foci formation and disappearance, monitored during the repair incubation, showed that the kinetics of DSBs rejoining was different in the two gravity conditions. The fraction of foci-positive cells decreased slower in MMG than in 1g at 6 and 24 h after irradiation (P < 0.01) and the mean number of {gamma}-H2AX foci per nucleus was significantly higher in MMG than in 1g at the same time-points (P < 0.001). In the same samples we determined apoptotic level and the rate of DSB rejoining during post-irradiation incubation. A significant induction of apoptosis was observed in MMG at 24 h after irradiation (P < 0.001), whereas at shorter times the level of apoptosis was slightly higher in MMG respect to 1g. In accordance with the kinetics of {gamma}-H2AX foci, the slower rejoining of radiation-induced DSBs in MMG was observed by DNA fragmentation analyses during the repair incubation; the data of pulsed-field gel electrophoresis assay showed that the fraction of DNA released in the gel was significantly higher in PBL incubated in MMG after irradiation with respect to cells maintained in 1g. Our results provide evidences that MMG incubation during DNA repair delayed the rate of radiation-induced DSB rejoining, and increased, as a consequence, the genotoxic effects of ionising radiation.

  10. Generation of new peptide-Fc fusion proteins that mediate antibody-dependent cellular cytotoxicity against different types of cancer cells

    Directory of Open Access Journals (Sweden)

    Mouldy Sioud

    2015-01-01

    Full Text Available Antibody-dependent cellular cytotoxicity (ADCC, a key effector function for the clinical effectiveness of monoclonal antibodies, is triggered by the engagement of the antibody Fc domain with the Fcγ receptors expressed by innate immune cells such as natural killer (NK cells and macrophages. Here, we fused cancer cell-binding peptides to the Fc domain of human IgG1 to engineer novel peptide-Fc fusion proteins with ADCC activity. The designed fusion proteins were expressed in human embryonic kidney 293T cells, followed by purification and characterization by western blots. One of the engineered variants (WN-Fc, bound with high affinity to a wide range of solid tumor cell lines (e.g., colon, lung, prostate, skin, ovarian, and mammary tumors. Treatment of cancer cells with the engineered peptide-Fc fusions in the presence of effector NK cells potentially enhanced cytotoxicity, degranulation, and interferon-γ production by NK cells when compared to cells treated with the Fc control. The presence of competing peptides inhibited NK cell activation. Furthermore, a bispecific peptide-Fc fusion protein activated NK cells against HER-1- and/or HER-2-expressing cancer cells. Collectively, the engineered peptide-Fc fusions constitute a new promising strategy to recruit and activate NK cells against tumor cells, a primary goal of cancer immunotherapy.

  11. Reciprocal regulation of activating and inhibitory Fcγ receptors by TLR7/8 activation: Implications for tumor immunotherapy

    Science.gov (United States)

    Butchar, Jonathan P.; Mehta, Payal; Justiniano, Steven E.; Guenterberg, Kristan D.; Kondadasula, Sri-Vidya; Mo, Xiaokui; Chemudupati, Mahesh; Kanneganti, Thirumala-Devi; Amer, Amal; Muthusamy, Natarajan; Jarjoura, David; Marsh, Clay B.; Carson, William E.; Byrd, John C.; Tridandapani, Susheela

    2010-01-01

    Purpose Activation of Toll-like Receptors (TLR) 7 and 8 by engineered agonists has been shown to aid in combating viruses and tumors. Here, we wished to test the effect of TLR7/8 activation on monocyte Fcγ receptor (FcγR) function, as they are critical mediators of antibody therapy. Experimental Design The effect of the TLR7/8 agonist R-848 on cytokine production and antibody-dependent cellular cytotoxicity (ADCC) by human peripheral blood monocytes (PBM) was tested. Affymetrix microarrays were done to examine genomewide transcriptional responses of monocytes to R-848, and Western blots were done to measure protein levels of FcγR. Murine bone marrow-derived macrophages (BMM) from wild-type and knockout mice were examined to determine the downstream pathway involved with regulating FcγR expression. The efficacy of R-848 as an adjuvant for antibody therapy was tested using a CT26-HER2/neu solid tumor model. Results Overnight incubation with R-848 increased FcγR-mediated cytokine production and ADCC in human PBM. Expression of FcγRI, FcγRIIa and the common γ-subunit was increased. Surprisingly, expression of the inhibitory FcγRIIb was almost completely abolished. In BMM, this required TLR7 and MyD88, as R-848 did not increase expression of the γ-subunit in TLR7−/− nor MyD88−/− cells. In a mouse solid tumor model, R-848 treatment superadditively enhanced the effects of antitumor antibody. Conclusions These results demonstrate an as-yet undiscovered regulatory and functional link between the TLR7/8 and FcγR pathways. This suggests that TLR7/8 agonists may be especially beneficial during antibody therapy. PMID:20332325

  12. The High Affinity IgE Receptor (FcεRI as a Target for Anti-allergic Agents

    Directory of Open Access Journals (Sweden)

    Kyoko Takahashi

    2005-01-01

    Full Text Available Prevention of the effector cell activation via high affinity IgE receptor (FcεRI is thought to be a straightforward strategy for suppressing the allergic reaction. Among the numerous methods to prevent the activation through FcεRI, three versions are described in this article. The first and second ideas involve inhibition of binding between FcεRI and IgE with a soluble form of the FceRI α chain and a humanized antibody directed against the a chain, respectively. Both of these paths involve suppression the histamine release from human peripheral blood basophils in vitro. They also inhibited the allergic reaction in vivo. The soluble α inhibited the anaphylactic reaction in rodents and the Fab fragments of the humanized anti-FcεRI α chain antibody suppressed the dermal response in rhesus monkeys. The third idea involves repression of FcεRI expression by suppressing the transcription of the genes encoding the subunits of FceRI. Although no plausible candidate molecule for actualizing this idea can be identified at present, further analyses of the transcriptional regulatory mechanisms in the human FcεRI α and β chain genes will lead to the discovery of novel targets for developing anti-allergic agents.

  13. Dicty_cDB: FC-BS17 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-BS17 (Link to dictyBase) - - - Contig-U16491-1 FC-BS17Z (Li...nk to Original site) - - FC-BS17Z 725 - - - - Show FC-BS17 Library FC (Link to library) Clone ID FC-BS17 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-BS/FC-BS17Q.Seq.d/ Representative seq. ID FC-BS1...7Z (Link to Original site) Representative DNA sequence >FC-BS17 (FC-BS17Q) /CSM/FC/FC-BS/FC-BS17Q.Seq....-cDNA Score E Sequences producing significant alignments: (bits) Value VSF675 (VSF675Q) /CSM/VS/VSF6-D/VSF67

  14. Dicty_cDB: FC-AV01 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AV01 (Link to dictyBase) - - - Contig-U16311-1 FC-AV01Z (Li...nk to Original site) - - FC-AV01Z 643 - - - - Show FC-AV01 Library FC (Link to library) Clone ID FC-AV01 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AV/FC-AV01Q.Seq.d/ Representative seq. ID FC-AV...01Z (Link to Original site) Representative DNA sequence >FC-AV01 (FC-AV01Q) /CSM/FC/FC-AV/FC-AV01Q.Seq....ed Amino Acid sequence ---SGSHGGSQSQSAGSDSQSAGSESSQSESGSQSQSESGSQSQSQSGSQSFSGSLYSGS YSGSQSGSQSGNSGAAVKQTGAGS

  15. Dicty_cDB: FC-AS10 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AS10 (Link to dictyBase) - - - Contig-U16538-1 FC-AS10Z (Li...nk to Original site) - - FC-AS10Z 549 - - - - Show FC-AS10 Library FC (Link to library) Clone ID FC-AS10 (Link to dictyBase) Atlas... ID - NBRP ID - dictyBase ID - Link to Contig Contig-U16538-1 Original site URL http:/.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AS/FC-AS10Q.Seq.d/ Representative seq. ID FC-AS...10Z (Link to Original site) Representative DNA sequence >FC-AS10 (FC-AS10Q) /CSM/FC/FC-AS/FC-AS10Q.Seq.

  16. Dicty_cDB: FC-AS15 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AS15 (Link to dictyBase) - - - Contig-U16090-1 FC-AS15E (Li...nk to Original site) - - - - - - FC-AS15E 616 Show FC-AS15 Library FC (Link to library) Clone ID FC-AS15 (Link to dictyBase) Atlas... ID - NBRP ID - dictyBase ID - Link to Contig Contig-U16090-1 Original site URL http:/.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AS/FC-AS15Q.Seq.d/ Representative seq. ID FC-AS...15E (Link to Original site) Representative DNA sequence >FC-AS15 (FC-AS15Q) /CSM/FC/FC-AS/FC-AS15Q.Seq.

  17. Dicty_cDB: FC-AS03 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AS03 (Link to dictyBase) - - - - FC-AS03Z (Link to Original site) - - FC-AS...03Z 540 - - - - Show FC-AS03 Library FC (Link to library) Clone ID FC-AS03 (Link to dictyBase) Atlas... ID - NBRP ID - dictyBase ID - Link to Contig - Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AS/FC-AS...03Q.Seq.d/ Representative seq. ID FC-AS03Z (Link to Original s...ite) Representative DNA sequence >FC-AS03 (FC-AS03Q) /CSM/FC/FC-AS/FC-AS03Q.Seq.d/ XXXXXXXXXXACAAAAGATTACAAT

  18. Dicty_cDB: FC-AS07 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AS07 (Link to dictyBase) - - - Contig-U14940-1 FC-AS07E (Li...nk to Original site) - - - - - - FC-AS07E 730 Show FC-AS07 Library FC (Link to library) Clone ID FC-AS07 (Link to dictyBase) Atlas... ID - NBRP ID - dictyBase ID - Link to Contig Contig-U14940-1 Original site URL http:/.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AS/FC-AS07Q.Seq.d/ Representative seq. ID FC-AS...07E (Link to Original site) Representative DNA sequence >FC-AS07 (FC-AS07Q) /CSM/FC/FC-AS/FC-AS07Q.Seq.

  19. Dicty_cDB: FC-AS06 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AS06 (Link to dictyBase) - - - Contig-U15075-1 FC-AS06Z (Li...nk to Original site) - - FC-AS06Z 367 - - - - Show FC-AS06 Library FC (Link to library) Clone ID FC-AS06 (Link to dictyBase) Atlas... ID - NBRP ID - dictyBase ID - Link to Contig Contig-U15075-1 Original site URL http:/.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AS/FC-AS06Q.Seq.d/ Representative seq. ID FC-AS...06Z (Link to Original site) Representative DNA sequence >FC-AS06 (FC-AS06Q) /CSM/FC/FC-AS/FC-AS06Q.Seq.

  20. Dicty_cDB: FC-AS08 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AS08 (Link to dictyBase) - - - Contig-U16491-1 FC-AS08Z (Li...nk to Original site) - - FC-AS08Z 572 - - - - Show FC-AS08 Library FC (Link to library) Clone ID FC-AS08 (Link to dictyBase) Atlas... ID - NBRP ID - dictyBase ID - Link to Contig Contig-U16491-1 Original site URL http:/.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AS/FC-AS08Q.Seq.d/ Representative seq. ID FC-AS...08Z (Link to Original site) Representative DNA sequence >FC-AS08 (FC-AS08Q) /CSM/FC/FC-AS/FC-AS08Q.Seq.

  1. Dicty_cDB: FC-AS01 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AS01 (Link to dictyBase) - - - Contig-U16172-1 FC-AS01Z (Li...nk to Original site) - - FC-AS01Z 583 - - - - Show FC-AS01 Library FC (Link to library) Clone ID FC-AS01 (Link to dictyBase) Atlas... ID - NBRP ID - dictyBase ID - Link to Contig Contig-U16172-1 Original site URL http:/.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AS/FC-AS01Q.Seq.d/ Representative seq. ID FC-AS...01Z (Link to Original site) Representative DNA sequence >FC-AS01 (FC-AS01Q) /CSM/FC/FC-AS/FC-AS01Q.Seq.

  2. Dicty_cDB: FC-AS23 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AS23 (Link to dictyBase) - - - Contig-U15550-1 FC-AS23Z (Li...nk to Original site) - - FC-AS23Z 519 - - - - Show FC-AS23 Library FC (Link to library) Clone ID FC-AS23 (Link to dictyBase) Atlas... ID - NBRP ID - dictyBase ID - Link to Contig Contig-U15550-1 Original site URL http:/.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AS/FC-AS23Q.Seq.d/ Representative seq. ID FC-AS...23Z (Link to Original site) Representative DNA sequence >FC-AS23 (FC-AS23Q) /CSM/FC/FC-AS/FC-AS23Q.Seq.

  3. Dicty_cDB: FC-AS24 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AS24 (Link to dictyBase) - - - Contig-U15127-1 FC-AS24Z (Li...nk to Original site) - - FC-AS24Z 454 - - - - Show FC-AS24 Library FC (Link to library) Clone ID FC-AS24 (Link to dictyBase) Atlas... ID - NBRP ID - dictyBase ID - Link to Contig Contig-U15127-1 Original site URL http:/.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AS/FC-AS24Q.Seq.d/ Representative seq. ID FC-AS...24Z (Link to Original site) Representative DNA sequence >FC-AS24 (FC-AS24Q) /CSM/FC/FC-AS/FC-AS24Q.Seq.

  4. Dicty_cDB: FC-BL11 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-BL11 (Link to dictyBase) - - - Contig-U15198-1 FC-BL11Z (Li...nk to Original site) - - FC-BL11Z 671 - - - - Show FC-BL11 Library FC (Link to library) Clone ID FC-BL11 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-BL/FC-BL11Q.Seq.d/ Representative seq. ID FC-BL1...1Z (Link to Original site) Representative DNA sequence >FC-BL11 (FC-BL11Q) /CSM/FC/FC-BL/FC-BL11Q.Seq....ALDNSCSLVDGTEDVYQQIFYSPSFQ*in*ifeitikkkkk Homology vs CSM-cDNA Score E Sequences producing significant align

  5. Dicty_cDB: FC-BF11 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-BF11 (Link to dictyBase) - - - Contig-U16455-1 FC-BF11Z (Li...nk to Original site) - - FC-BF11Z 718 - - - - Show FC-BF11 Library FC (Link to library) Clone ID FC-BF11 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-BF/FC-BF11Q.Seq.d/ Representative seq. ID FC-BF1...1Z (Link to Original site) Representative DNA sequence >FC-BF11 (FC-BF11Q) /CSM/FC/FC-BF/FC-BF11Q.Seq....: (bits) Value N X55973 |X55973.1 D. discoideum EF1-I gene for elongation factor 1 alpha. 1292 0.0 1 X55972

  6. Dicty_cDB: FC-AI15 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AI15 (Link to dictyBase) - G01730 DDB0214993 Contig-U15123-1 FC-AI...15E (Link to Original site) - - - - - - FC-AI15E 856 Show FC-AI15 Library FC (Link to library) Clone ID FC-AI...3-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI15Q.Se...q.d/ Representative seq. ID FC-AI15E (Link to Original site) Representative DNA sequence >FC-AI15 (FC-AI15Q) /CSM/FC/FC-AI/FC-AI...AAAAAAAATA sequence update 1996.12.24 Translated Amino Acid sequence kt*riyi*KMMIKYITIAILFIASLVKADLQFSLCPTCV

  7. Dicty_cDB: FC-AI06 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AI06 (Link to dictyBase) - - - Contig-U16465-1 FC-AI06E (Li...nk to Original site) - - - - - - FC-AI06E 1138 Show FC-AI06 Library FC (Link to library) Clone ID FC-AI06 (L...//dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI06Q.Seq.d/ Representative seq. ID FC-AI...06E (Link to Original site) Representative DNA sequence >FC-AI06 (FC-AI06Q) /CSM/FC/FC-AI/FC-AI06Q.Seq...FGRGIDIERVNVVINYDMAESADTYLHRVGRAGRFGTK GLAISFVPSKEDPVLEQVQSKFVVSIKELVATPDPSTYMSG*kkkkkkkknlfvlksikk k*kkk*in

  8. Dicty_cDB: FC-AI09 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AI09 (Link to dictyBase) - - - Contig-U16149-1 FC-AI09Z (Li...nk to Original site) - - FC-AI09Z 591 - - - - Show FC-AI09 Library FC (Link to library) Clone ID FC-AI09 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI09Q.Seq.d/ Representative seq. ID FC-AI...09Z (Link to Original site) Representative DNA sequence >FC-AI09 (FC-AI09Q) /CSM/FC/FC-AI/FC-AI09Q.Seq....*tkl ik*ilifykiknnkkkkkk Frame B: ---gt*kvpeflailfkrmasrsvlwy*rcltkakkglkapqtltik

  9. Dicty_cDB: FC-AI19 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AI19 (Link to dictyBase) - - - Contig-U15115-1 FC-AI19Z (Li...nk to Original site) - - FC-AI19Z 661 - - - - Show FC-AI19 Library FC (Link to library) Clone ID FC-AI19 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI19Q.Seq.d/ Representative seq. ID FC-AI...19Z (Link to Original site) Representative DNA sequence >FC-AI19 (FC-AI19Q) /CSM/FC/FC-AI/FC-AI19Q.Seq....lmrqswvkkiesi*lvl krrkkkknnkkkkkkkkkkklfn*lvnkkn*ik*kkllcnqkk Frame B: ---*ekaieilsklfsin*kfn**ysiiigkkstkyq

  10. Dicty_cDB: FC-AI14 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AI14 (Link to dictyBase) - - - Contig-U16280-1 FC-AI14Z (Li...nk to Original site) - - FC-AI14Z 671 - - - - Show FC-AI14 Library FC (Link to library) Clone ID FC-AI14 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI14Q.Seq.d/ Representative seq. ID FC-AI...14Z (Link to Original site) Representative DNA sequence >FC-AI14 (FC-AI14Q) /CSM/FC/FC-AI/FC-AI14Q.Seq....nqrllv*lvvlskklqllnsnqsfkfkkvq rmkknsvkntkn*rfvllt*nlkslkrmpksknsptkliifilkly Frame B: ---lkdl*krtphl*stcfhhptlcssrrfclwslnai

  11. Dicty_cDB: FC-AI12 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AI12 (Link to dictyBase) - - - Contig-U15484-1 FC-AI12Z (Li...nk to Original site) - - FC-AI12Z 614 - - - - Show FC-AI12 Library FC (Link to library) Clone ID FC-AI12 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI12Q.Seq.d/ Representative seq. ID FC-AI...12Z (Link to Original site) Representative DNA sequence >FC-AI12 (FC-AI12Q) /CSM/FC/FC-AI/FC-AI12Q.Seq....EKIVRRI ELLDGITCYRNEKAKDEIVLTGNSLELLSQSCATIQLRSAIKYKDVRKFLDGIYVSERNV LESN*in*riys

  12. Dicty_cDB: FC-AI24 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AI24 (Link to dictyBase) - - - - FC-AI24Z (Link to Original site) - - FC-AI...24Z 693 - - - - Show FC-AI24 Library FC (Link to library) Clone ID FC-AI24 (Link to dictyBas...e) Atlas ID - NBRP ID - dictyBase ID - Link to Contig - Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI...24Q.Seq.d/ Representative seq. ID FC-AI24Z (Link to Original s...ite) Representative DNA sequence >FC-AI24 (FC-AI24Q) /CSM/FC/FC-AI/FC-AI24Q.Seq.d/ XXXXXXXXXXAAATTAGAAAACAAA

  13. Dicty_cDB: FC-BC24 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-BC24 (Link to dictyBase) - - - Contig-U15479-1 FC-BC24Z (Li...nk to Original site) - - FC-BC24Z 578 - - - - Show FC-BC24 Library FC (Link to library) Clone ID FC-BC24 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-BC/FC-BC24Q.Seq.d/ Representative seq. ID FC-BC2...4Z (Link to Original site) Representative DNA sequence >FC-BC24 (FC-BC24Q) /CSM/FC/FC-BC/FC-BC24Q.Seq....m mRNA for ribosomal acidic phosphoprotein P0. 1068 0.0 2 CD681220 |CD681220.1 tac23d06.y1 Hydra EST -IV Hyd

  14. Dicty_cDB: FC-IC1118 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC1118 (Link to dictyBase) - - - - FC-IC1118E (Link to Original site) FC-IC... to library) Clone ID FC-IC1118 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig - Ori...re E Sequences producing significant alignments: (bits) Value FC-IC1118 (FC-IC1118Q) /CSM/FC-IC/FC-IC...%: extracellular, including cell wall 4.0 %: cytoskeletal 4.0 %: vacuolar >> prediction for FC-IC1118 is cyt 5' end seq. ID FC-IC...1118F 398 FC-IC1118Z 400 FC-IC1118P 778 FC-IC1118E 390 Show FC-IC1118 Library FC-IC (Link

  15. Dicty_cDB: FC-IC0790 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC0790 (Link to dictyBase) - - - Contig-U14940-1 FC-IC07... (Link to library) Clone ID FC-IC0790 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link t...o Contig Contig-U14940-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC...y vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value FC-IC0790 (FC-IC0790Q) /CSM/FC-IC/FC-IC...90E (Link to Original site) FC-IC0790F 305 FC-IC0790Z 305 FC-IC0790P 590 FC-IC0790E 295 Show FC-IC0790 Library FC-IC

  16. Dicty_cDB: FC-IC1261 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC1261 (Link to dictyBase) - - - - FC-IC1261E (Link to Original site) FC-IC... to library) Clone ID FC-IC1261 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig - Ori...SM-cDNA Score E Sequences producing significant alignments: (bits) Value FC-IC1760 (FC-IC1760Q) /CSM/FC-IC/FC-IC...etal 4.0 %: Golgi 4.0 %: cytoplasmic >> prediction for FC-IC1261 is end 5' end seq. ID FC-IC...1261F 255 FC-IC1261Z 439 FC-IC1261P 674 FC-IC1261E 429 Show FC-IC1261 Library FC-IC (Link

  17. Dicty_cDB: FC-IC0142 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC0142 (Link to dictyBase) - G24282 DDB0231822 Contig-U03313-1 FC-IC...tig Contig-U03313-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC...E Sequences producing significant alignments: (bits) Value FC-IC0142 (FC-IC0142Q) /CSM/FC-IC/FC-IC0142Q.Seq....les of secretory system >> prediction for FC-IC0142 is cyt 5' end seq. ID FC-IC0142F 5' end seq. >FC-IC...0142F (Link to Original site) FC-IC0142F 280 - - - - - - Show FC-IC0142 Library FC-IC (Link to library) Clone ID FC-IC

  18. Gamma watermarking

    Science.gov (United States)

    Ishikawa, Muriel Y.; Wood, Lowell L.; Lougheed, Ronald W.; Moody, Kenton J.; Wang, Tzu-Fang

    2004-05-25

    A covert, gamma-ray "signature" is used as a "watermark" for property identification. This new watermarking technology is based on a unique steganographic or "hidden writing" digital signature, implemented in tiny quantities of gamma-ray-emitting radioisotopic material combinations, generally covertly emplaced on or within an object. This digital signature may be readily recovered at distant future times, by placing a sensitive, high energy-resolution gamma-ray detecting instrument reasonably precisely over the location of the watermark, which location may be known only to the object's owner; however, the signature is concealed from all ordinary detection means because its exceedingly low level of activity is obscured by the natural radiation background (including the gamma radiation naturally emanating from the object itself, from cosmic radiation and material surroundings, from human bodies, etc.). The "watermark" is used in object-tagging for establishing object identity, history or ownership. It thus may serve as an aid to law enforcement officials in identifying stolen property and prosecuting theft thereof. Highly effective, potentially very low cost identification-on demand of items of most all types is thus made possible.

  19. Gamma watermarking

    Energy Technology Data Exchange (ETDEWEB)

    Ishikawa, Muriel Y.; Wood, Lowell L.; Lougheed, Ronald W.; Moody, Kenton J.; Wang, Tzu-Fang

    2004-05-25

    A covert, gamma-ray "signature" is used as a "watermark" for property identification. This new watermarking technology is based on a unique steganographic or "hidden writing" digital signature, implemented in tiny quantities of gamma-ray-emitting radioisotopic material combinations, generally covertly emplaced on or within an object. This digital signature may be readily recovered at distant future times, by placing a sensitive, high energy-resolution gamma-ray detecting instrument reasonably precisely over the location of the watermark, which location may be known only to the object's owner; however, the signature is concealed from all ordinary detection means because its exceedingly low level of activity is obscured by the natural radiation background (including the gamma radiation naturally emanating from the object itself, from cosmic radiation and material surroundings, from human bodies, etc.). The "watermark" is used in object-tagging for establishing object identity, history or ownership. It thus may serve as an aid to law enforcement officials in identifying stolen property and prosecuting theft thereof. Highly effective, potentially very low cost identification-on demand of items of most all types is thus made possible.

  20. Characterization of the rabbit neonatal Fc receptor (FcRn) and analyzing the immunophenotype of the transgenic rabbits that overexpresses FcRn.

    Science.gov (United States)

    Catunda Lemos, Ana Paula; Cervenak, Judit; Bender, Balázs; Hoffmann, Orsolya Ivett; Baranyi, Mária; Kerekes, Andrea; Farkas, Anita; Bosze, Zsuzsanna; Hiripi, László; Kacskovics, Imre

    2012-01-01

    The neonatal Fc receptor (FcRn) regulates IgG and albumin homeostasis, mediates maternal IgG transport, takes an active role in phagocytosis, and delivers antigen for presentation. We have previously shown that overexpression of FcRn in transgenic mice significantly improves the humoral immune response. Because rabbits are an important source of polyclonal and monoclonal antibodies, adaptation of our FcRn overexpression technology in this species would bring significant advantages. We cloned the full length cDNA of the rabbit FcRn alpha-chain and found that it is similar to its orthologous analyzed so far. The rabbit FcRn - IgG contact residues are highly conserved, and based on this we predicted pH dependent interaction, which we confirmed by analyzing the pH dependent binding of FcRn to rabbit IgG using yolk sac lysates of rabbit fetuses by Western blot. Using immunohistochemistry, we detected strong FcRn staining in the endodermal cells of the rabbit yolk sac membrane, while the placental trophoblast cells and amnion showed no FcRn staining. Then, using BAC transgenesis we generated transgenic rabbits carrying and overexpressing a 110 kb rabbit genomic fragment encoding the FcRn. These transgenic rabbits--having one extra copy of the FcRn when hemizygous and two extra copies when homozygous--showed improved IgG protection and an augmented humoral immune response when immunized with a variety of different antigens. Our results in these transgenic rabbits demonstrate an increased immune response, similar to what we described in mice, indicating that FcRn overexpression brings significant advantages for the production of polyclonal and monoclonal antibodies.

  1. Genetic association of multiple sclerosis with the marker rs391745 near the endogenous retroviral locus HERV-Fc1: analysis of disease subtypes

    DEFF Research Database (Denmark)

    Hansen, Bettina; Oturai, Annette Bang; Harbo, Hanne F;

    2011-01-01

    We have previously described the occurrence of multiple sclerosis (MS) to be associated with human endogenous retroviruses, specifically the X-linked viral locus HERV-Fc1. The aim of this study was to investigate a possible association of the HERV-Fc1 locus with subtypes of MS. MS patients are ge...

  2. Fcγ Receptor Heterogeneity in Leukocyte Functional Responses

    Science.gov (United States)

    Rosales, Carlos

    2017-01-01

    Antibodies participate in defense of the organism from all types of pathogens, including viruses, bacteria, fungi, and protozoa. IgG antibodies recognize their associated antigen via their two Fab portions and are in turn recognized though their Fc portion by specific Fcγ receptors (FcγRs) on the membrane of immune cells. Multiple types and polymorphic variants of FcγR exist. These receptors are expressed in many cells types and are also redundant in inducing cell responses. Crosslinking of FcγR on the surface of leukocytes activates several effector functions aimed toward the destruction of pathogens and the induction of an inflammatory response. In the past few years, new evidence on how the particular IgG subclass and the glycosylation pattern of the antibody modulate the IgG–FcγR interaction has been presented. Despite these advances, our knowledge of what particular effector function is activated in a certain cell and in response to a specific type of FcγR remains very limited today. On one hand, each immune cell could be programmed to perform a particular cell function after FcγR crosslinking. On the other, each FcγR could activate a particular signaling pathway leading to a unique cell response. In this review, I describe the main types of FcγRs and our current view of how particular FcγRs activate various signaling pathways to promote unique leukocyte functions. PMID:28373871

  3. REGULATION OF Fc RECEPTOR ENDOCYTIC TRAFFICKING BY UBIQUITINATION

    Directory of Open Access Journals (Sweden)

    Rosa eMolfetta

    2014-09-01

    Full Text Available Most immune cells, particularly phagocytes, express various receptors for the Fc-portion of the different immunoglobulin isotypes (Fc receptors, FcRs. By binding to the antibody, they provide a link between the adaptive immune system and the powerful effector functions triggered by innate immune cells such as mast cells, neutrophils, macrophages, and NK cells. Upon ligation of the immune complexes, the downstream signalling pathways initiated by the different receptors are quite similar for different FcR classes leading to the secretion of preformed and de novo synthesized pro-inflammatory mediators. FcR engagement also promotes negative signals through the combined action of several molecules that limit the extent and duration of positive signalling. To this regard, ligand-induced ubiquitination of Fc receptors for IgE (FcεR and IgG (FcγR has become recognized as a key modification that generates signals for the internalization and/or delivery of engaged receptor complexes to lysosomes or cytoplasmic proteasomes for degradation, providing negative-feedback regulation of Fc receptor activity.In this review, we discuss recent advances in our understanding of the molecular mechanisms that ensure the clearance of engaged Fcε and Fcγ receptor complexes from the cell surface with an emphasis given to the cooperation between the ubiquitin pathway and endosomal adaptors including the endosomal sorting complex required for transport (ESCRT in controlling receptor internalization and sorting along the endocytic compartments.

  4. FC-normal and extended stratified logic program

    Institute of Scientific and Technical Information of China (English)

    许道云; 丁德成

    2002-01-01

    This paper investigates the consistency property of FC-normal logic program and presentsan equivalent deciding condition whether a logic program P is an FC-normal program. The decidingcondition describes the characterizations of FC-normal program. By the Petri-net presentation ofa logic program, the characterizations of stratification of FC-normal program are investigated. Thestratification of FC-normal program motivates us to introduce a new kind of stratification, extendedstratification, over logic program. It is shown that an extended (locally) stratified logic program isan FC-normal program. Thus, an extended (locally) stratified logic program has at least one stablemodel. Finally, we have presented algorithms about computation of consistency property and a fewequivalent deciding methods of the finite FC-normal program.

  5. PCC-ring induction in human lymphocytes exposed to gamma and neutron irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Lamadrid B, A.I.; Garcia L, O. [CPHR, Calle 20 No. 4113 e/41 y 47, Playa, La Habana 11300 (Cuba); Delbos, M.; Voisin, P.; Roy, L. [Institut de Radioprotection et de Surete Nucleaire, BP 17, 92262 Fontenay-aux-Roses (France)]. e-mail: ana@cphr.edu.cu

    2006-07-01

    Dose-effect curves for dose assessment in Gamma and neutron overexposures to high doses are presented in this paper for the first time in literature. The relationships were obtained by plotting the Premature Chromosome Condensation -rings (PCC-R) frequencies in PCC Iymphocytes obtained by chemical induction with Calyculin A in vitro, with radiation doses between 5 to 25 Gy. For the elaboration of these curves 9 676 PCC cells in Gl G2 and M stages were analyzed. The results were fitted to a lineal quadratic model in Gamma irradiation. For neutron irradiation the data was fitted to a lineal quadratic model up to 10 Gy and then a markedly cell cycle arrest and saturation was observed. These curves are of particular interest for victims exposed to doses exceeding 5 Gy where it is always very difficult to estimate a dose using the conventional technique. (Author)

  6. Retargeting T cells for HER2-positive tumor killing by a bispecific Fv-Fc antibody.

    Directory of Open Access Journals (Sweden)

    Lei Wang

    Full Text Available To exploit the biological and pharmacological properties of immunoglobulin constant domain Fc fragment and increase the killing efficacy of T cells, a single chain variable fragment specific to CD3 was fused with Fcab (Fc antigen binding, a mutant Fc fragment with specificity against Human epidermal growth factor receptor 2 (HER2 developed by F-star. The bispecific fusion named as FcabCD3 was expressed by transient transfection in HEK-293T cells and purified by affinity chromatography. Specific cytolytic activity of retargeted T cells to kill HER2 positive SKBR3 cell line was evaluated in vitro. FcabCD3 was able to retarget T cells to kill both Herceptin insensitive Colo205-luc cell line and HER2 low expression MDA-MB-231-luc cell line. Furthermore, FcabCD3 was effective in eliminating the Colo205 tumor established on BALB/c nu/nu mice.

  7. Incorporation and distribution of dihomo-gamma-linolenic acid, arachidonic acid, and eicosapentaenoic acid in cultured human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Punnonen, K.; Puustinen, T.; Jansen, C.T.

    1986-02-01

    Human keratinocytes in culture were labelled with /sup 14/C-dihomo-gamma-linolenic acid, /sup 14/C-arachidonic acid or /sup 14/C-eicosapentaenoic acid. All three eicosanoid precursor fatty acids were effectively incorporated into the cells. In phospholipids most of the radioactivity was recovered, in neutral lipids a substantial amount, and as free unesterified fatty acids only a minor amount. Most of the radioactivity was found in phosphatidylethanolamine which was also the major phospholipid as measured by phosphorous assay. The incorporation of dihomo-gamma-linolenic acid and arachidonic acid into lipid subfractions was essentially similar. Eicosapentaenoic acid was, however, much less effectively incorporated into phosphatidylinositol + phosphatidylserine and, correspondingly, more effectively into triacylglycerols as compared to the two other precursor fatty acids. Once incorporated, the distribution of all three precursor fatty acids was relatively stable, and only minor amounts of fatty acids were released into the culture medium during short term culture (two days). Our study demonstrates that eicosanoid precursor fatty acids are avidly taken up by human keratinocytes and esterified into membrane lipids. The clinical implication of this finding is that dietary manipulations might be employed to cause changes in the fatty acid composition of keratinocytes.

  8. Homophilic interaction of NTBA, a member of the CD2 molecular family: induction of cytotoxicity and cytokine release in human NK cells.

    Science.gov (United States)

    Falco, Michela; Marcenaro, Emanuela; Romeo, Elisa; Bellora, Francesca; Marras, Daniele; Vély, Frédéric; Ferracci, Géraldine; Moretta, Lorenzo; Moretta, Alessandro; Bottino, Cristina

    2004-06-01

    NK-T-B antigen (NTBA) is a CD2 family member that functions as a coreceptor in human NK cell activation. Several receptor/ligand interactions occur between different members of this molecular family. In this study, in order to identify the natural ligand of NTBA, we produced a chimeric protein formed by the NTBA extracellular region fused with the Fc portion of human IgG1 (termed NTBA-Fc*). NTBA-Fc* specifically binds to NTBA cell transfectants but not to cells transfected with other CD2 family members including CD2, CD48, CD84, CD150, CD229, and CD244. Moreover, NTBA-Fc* also binds to NTBA(+) but not to NTBA(-) T cell lines. Enzyme-linked immunosorbent assays, plasmon resonance analysis, as well as NTBA-Fc*-mediated down-regulation of NTBA surface expression further confirmed the occurrence of NTBA/NTBA homophilic interaction. Functionally, in NK cells, NTBA-Fc* promoted a strong production of IFN-gamma and TNF-alpha. Moreover, NTBA-transfected targets displayed increased susceptibility to NK-mediated killing as compared to untransfected cells and this effect was specifically inhibited by anti-NTBA mAb. Altogether our data indicate that NTBA is characterized by self recognition.

  9. Close linkage of the mouse and human CD3. gamma. - and delta-chain genes suggests that their transcription is controlled by common regulatory elements

    Energy Technology Data Exchange (ETDEWEB)

    Saito, H.; Koyama, T.; Georgopoulos, K.; Clevers, H.; Haser, W.G.; LeBien, T.; Tonegawa, S.; Terhorst, C.

    1987-12-01

    Antigen receptors on the T-cell surface are noncovalently associated with at least four invariant polypeptide chains, CD3-..gamma.., -delta, -epsilon, and -zeta. The mouse CD3-..gamma.. gene, consisting of seven exons, was found to be highly homologous to the CD3-..gamma.. described earlier. Both the high level of sequence homology and the exon/intron organization indicate that the CD3-..gamma.. and -delta genes arose by gene duplication. Surprisingly, murine and human genomic DNA clones could be isolated that contained elements of both the CD3-..gamma.. and CD3-delta genes. In fact, the putative transcription start site of the mouse CD3-..gamma.. gene is less than 1.4 kilobases from the transcription initiation site of the mouse CD3-delta gene. Common elements that regulate the divergent transcription of the two genes are therefore proposed to be located in the intervening 1.4-kilobase DNA segment. This might contribute to the coordinate expression of the CD3-..gamma.. and -delta genes during intrathymic maturation of T lymphocytes.

  10. Dicty_cDB: FC-AS18 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AS18 (Link to dictyBase) - G03233 DDB0231612 Contig-U15937-1 FC-AS... to library) Clone ID FC-AS18 (Link to dictyBase) Atlas ID - NBRP ID G03233 dictyBase ID DDB0231612 Link to ...18P (Link to Original site) FC-AS18F 508 FC-AS18Z 542 FC-AS18P 1050 - - Show FC-AS18 Library FC (Link...Contig Contig-U15937-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AS/FC-AS...18Q.Seq.d/ Representative seq. ID FC-AS18P (Link to Original site) Representative DNA sequence >FC-AS18 (FC-AS

  11. Dicty_cDB: FC-AS21 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AS21 (Link to dictyBase) - - - Contig-U15326-1 FC-AS21P (Li... %: nuclear 12.0 %: cytoplasmic 4.0 %: cytoskeletal >> prediction for FC-AS21 is mit 5' end seq. ID FC-AS...nk to Original site) FC-AS21F 506 FC-AS21Z 184 FC-AS21P 690 - - Show FC-AS21 Library FC (Link to library) Clone ID FC-AS...21 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U15326-1 Origin...al site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AS/FC-AS21Q.Seq.d/ Representative seq. ID FC-AS

  12. Dicty_cDB: FC-AI22 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AI22 (Link to dictyBase) - - - Contig-U15369-1 | Contig-U15732-1 FC-AI...22P (Link to Original site) FC-AI22F 583 FC-AI22Z 683 FC-AI22P 1266 - - Show FC-AI22 Library FC (...Link to library) Clone ID FC-AI22 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Con...tig-U15369-1 | Contig-U15732-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI...22Q.Seq.d/ Representative seq. ID FC-AI22P (Link to Original site) Representative DNA sequence >FC-AI22 (FC-AI

  13. Dicty_cDB: FC-AI07 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AI07 (Link to dictyBase) - - - Contig-U15296-1 | Contig-U15756-1 FC-AI...07P (Link to Original site) FC-AI07F 580 FC-AI07Z 723 FC-AI07P 1303 - - Show FC-AI07 Library FC (...Link to library) Clone ID FC-AI07 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Con...tig-U15296-1 | Contig-U15756-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI...07Q.Seq.d/ Representative seq. ID FC-AI07P (Link to Original site) Representative DNA sequence >FC-AI07 (FC-AI

  14. Dicty_cDB: FC-AI08 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AI08 (Link to dictyBase) - G01729 DDB0233148 Contig-U14939-1 FC-AI...08P (Link to Original site) FC-AI08F 654 FC-AI08Z 563 FC-AI08P 1217 - - Show FC-AI08 Library FC (Link... to library) Clone ID FC-AI08 (Link to dictyBase) Atlas ID - NBRP ID G01729 dictyBase ID DDB0233148 Link to ...Contig Contig-U14939-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI...08Q.Seq.d/ Representative seq. ID FC-AI08P (Link to Original site) Representative DNA sequence >FC-AI08 (FC-AI

  15. Dicty_cDB: FC-IC0282 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC0282 (Link to dictyBase) - - - Contig-U16527-1 - (Link to Original site) FC-IC...(Link to library) Clone ID FC-IC0282 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig ...Contig-U16527-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC...ogy vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value FC-IC1760 (FC-IC1760Q) /CSM/FC-IC/FC-IC...0282F 255 FC-IC0282Z 429 FC-IC0282P 673 FC-IC0282E 429 Show FC-IC0282 Library FC-IC

  16. Dicty_cDB: FC-IC0374 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC0374 (Link to dictyBase) - - - Contig-U12354-1 FC-IC03... (Link to library) Clone ID FC-IC0374 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link t...o Contig Contig-U12354-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC...les of secretory system >> prediction for FC-IC0374 is end 5' end seq. ID FC-IC0374F 5' end seq. >FC-IC...74E (Link to Original site) FC-IC0374F 223 FC-IC0374Z 324 FC-IC0374P 547 FC-IC0374E 324 Show FC-IC0374 Library FC-IC

  17. Dicty_cDB: FC-IC1268 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC1268 (Link to dictyBase) - - - Contig-U16527-1 FC-IC12...ink to library) Clone ID FC-IC1268 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Co...ntig-U16527-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC...lpy*iw Homology vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value FC-IC1760 (FC-IC1760Q) /CSM/FC-IC/FC-IC...68P (Link to Original site) FC-IC1268F 231 FC-IC1268Z 250 FC-IC1268P 461 - - Show FC-IC1268 Library FC-IC (L

  18. Development and applications of AlphaScreen-based FcRn binding assay to characterize monoclonal antibodies.

    Science.gov (United States)

    Wu, Qiang; Lee, Ho Young; Wong, Pin Yee; Jiang, Guoying; Gazzano-Santoro, Hélène

    2015-05-01

    IgG antibodies are important pharmaceutical molecules that successfully treat a variety of human diseases. The neonatal Fc receptor (FcRn) interacts with IgG Fc in the CH2-CH3 domain and plays a key role in IgG antibody homeostasis and affects its pharmacokinetic properties. An in vitro FcRn binding assay could be a highly valuable complementary tool to assess IgG antibody pharmacokinetics in IgG engineering and screening during the early optimization stage. In addition, it could be useful in biological characterization studies for antibody minor variants, process optimization, and comparability study at later stages of antibody development. Here we developed a homogeneous AlphaScreen-based FcRn assay to assess the binding of FcRn to IgG antibody in vitro. The assay is found to be accurate, precise, specific, and simple: donor beads loaded with FcRn and acceptor beads loaded with IgG1 mAb1 are mixed together with sample IgG at various dilutions and incubated for 1h before acquiring data with a fluorescence reader. This assay can run up to four samples per plate in 2h, which is time and cost effective compared with other FcRn binding methods such as cell-based fluorescent-activated cell scan and surface plasma resonance. Our data demonstrated that this assay is suitable for assessing the FcRn binding in vitro and provides a platform approach that can be readily applied to various antibodies. Copyright © 2015. Published by Elsevier B.V.

  19. Ebolavirus Glycoprotein Fc Fusion Protein Protects Guinea Pigs against Lethal Challenge.

    Science.gov (United States)

    Konduru, Krishnamurthy; Shurtleff, Amy C; Bradfute, Steven B; Nakamura, Siham; Bavari, Sina; Kaplan, Gerardo

    2016-01-01

    Ebola virus (EBOV), a member of the Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health. Currently, there are no licensed vaccines or therapeutics to prevent and treat EBOV infection. Several vaccines based on the EBOV glycoprotein (GP) are under development, including vectored, virus-like particles, and protein-based subunit vaccines. We previously demonstrated that a subunit vaccine containing the extracellular domain of the Ebola ebolavirus (EBOV) GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc) protected mice against EBOV lethal challenge. Here, we show that the EBOVgp-Fc vaccine formulated with QS-21, alum, or polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) adjuvants induced strong humoral immune responses in guinea pigs. The vaccinated animals developed anti-GP total antibody titers of approximately 105-106 and neutralizing antibody titers of approximately 103 as assessed by a BSL-2 neutralization assay based on vesicular stomatitis virus (VSV) pseudotypes. The poly-ICLC formulated EBOVgp-Fc vaccine protected all the guinea pigs against EBOV lethal challenge performed under BSL-4 conditions whereas the same vaccine formulated with QS-21 or alum only induced partial protection. Vaccination with a mucin-deleted EBOVgp-Fc construct formulated with QS-21 adjuvant did not have a significant effect in anti-GP antibody levels and protection against EBOV lethal challenge compared to the full-length GP construct. The bulk of the humoral response induced by the EBOVgp-Fc vaccine was directed against epitopes outside the EBOV mucin region. Our findings indicate that different adjuvants can eliciting varying levels of protection against lethal EBOV challenge in guinea pigs vaccinated with EBOVgp-Fc, and suggest that levels of total anti-GP antibodies elicit by protein-based GP subunit vaccines do not correlate with protection. Our data further support

  20. Site-selective conjugation of an anticoagulant aptamer to recombinant albumins and maintenance of neonatal Fc receptor binding

    Science.gov (United States)

    Schmøkel, Julie; Voldum, Anders; Tsakiridou, Georgia; Kuhlmann, Matthias; Cameron, Jason; Sørensen, Esben S.; Wengel, Jesper; Howard, Kenneth A.

    2017-05-01

    Aptamers are an attractive molecular medicine that offers high target specificity. Nucleic acid-based aptamers, however, are prone to nuclease degradation and rapid renal excretion that require blood circulatory half-life extension enabling technologies. The long circulatory half-life, predominately facilitated by engagement with the cellular recycling neonatal Fc receptor (FcRn), and ligand transport properties of albumin promote it as an attractive candidate to improve the pharmacokinetic profile of aptamers. This study investigates the effect of Cys34 site-selective covalent attachment of a factor IXa anticoagulant aptamer on aptamer functionality and human FcRn (hFcRn) engagement using recombinant human albumin (rHA) of either a wild type (WT) or an engineered human FcRn high binding variant (HB). Albumin-aptamer conjugates, connected covalently through a heterobifunctional succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate linker, were successfully prepared and purified by high performance liquid chromatography as confirmed by gel electrophoresis band-shift analysis and matrix-assisted laser desorption/ionization time of flight. Minimal reduction (∼25%) in activity of WT-linked aptamer to that of aptamer alone was found using an anticoagulant activity assay measuring temporal levels of activated partial thrombin. Covalent albumin-aptamer conjugation, however, substantially compromized binding to hFcRn, to 10% affinity of that of non-conjugated WT, determined by biolayer interferometry. Binding could be rescued by aptamer conjugation to recombinant albumin engineered for higher FcRn affinity (HB) that exhibited an 8-fold affinity compared to WT alone. This work describes a novel albumin-based aptamer delivery system whose hFcRn binding can be increased using a HB engineered albumin.

  1. Homology modeling of human alpha 1 beta 2 gamma 2 and house fly beta 3 GABA receptor channels and Surflex-docking of fipronil.

    Science.gov (United States)

    Cheng, Jin; Ju, Xiu-Lian; Chen, Xiang-Yang; Liu, Gen-Yan

    2009-09-01

    To further explore the mechanism of selective binding of the representative gamma-aminobutyric acid receptors (GABARs) noncompetitive antagonist (NCA) fipronil to insect over mammalian GABARs, three-dimensional models of human alpha 1 beta 2 gamma 2 and house fly beta 3 GABAR were generated by homology modeling, using the cryo-electron microscopy structure of the nicotinic acetylcholine receptor (nAChR) of Torpedo marmorata as a template. Fipronil was docked into the putative binding site of the human alpha 1 beta 2 gamma 2 and house fly beta 3 receptors by Surflex-docking, and the calculated docking energies are in agreement with experimental results. The GABA receptor antagonist fipronil exhibited higher potency with house fly beta 3 GABAR than with human alpha 1 beta 2 gamma 2 GABAR. Furthermore, analyses of Surflex-docking suggest that the H-bond interaction of fipronil with Ala2 and Thr6 in the second transmembrane segment (TM2) of these GABARs plays a relatively important role in ligand selective binding. The different subunit assemblies of human alpha 1 beta 2 gamma 2 and house fly beta 3 GABARs may result in differential selectivity for fipronil.

  2. Effects of gamma-radiation on cell growth, cycle arrest, death, and superoxide dismutase expression by DU 145 human prostate cancer cells

    Directory of Open Access Journals (Sweden)

    Vucic V.

    2006-01-01

    Full Text Available Gamma-irradiation (gamma-IR is extensively used in the treatment of hormone-resistant prostate carcinoma. The objective of the present study was to investigate the effects of 60Co gamma-IR on the growth, cell cycle arrest and cell death of the human prostate cancer cell line DU 145. The viability of DU 145 cells was measured by the Trypan blue exclusion assay and the 3(4,5-dimethylthiazol-2-yl-2,5,diphenyltetrazolium bromide test. Bromodeoxyuridine incorporation was used for the determination of cell proliferation. Cell cycle arrest and cell death were analyzed by flow cytometry. Superoxide dismutase (SOD, specifically CuZnSOD and MnSOD protein expression, after 10 Gy gamma-IR, was determined by Western immunoblotting analysis. gamma-IR treatment had a significant (P < 0.001 antiproliferative and cytotoxic effect on DU 145 cells. Both effects were time and dose dependent. Also, the dose of gamma-IR which inhibited DNA synthesis and cell proliferation by 50% was 9.7 Gy. Furthermore, gamma-IR induced cell cycle arrest in the G2/M phase and the percentage of cells in the G2/M phase was increased from 15% (control to 49% (IR cells, with a nonsignificant induction of apoptosis. Treatment with 10 Gy gamma-IR for 24, 48, and 72 h stimulated CuZnSOD and MnSOD protein expression in a time-dependent manner, approximately by 3- to 3.5-fold. These data suggest that CuZnSOD and MnSOD enzymes may play an important role in the gamma-IR-induced changes in DU 145 cell growth, cell cycle arrest and cell death.

  3. Effects of gamma-radiation on cell growth, cycle arrest, death, and superoxide dismutase expression by DU 145 human prostate cancer cells

    Directory of Open Access Journals (Sweden)

    V. Vucic

    2006-02-01

    Full Text Available Gamma-irradiation (gamma-IR is extensively used in the treatment of hormone-resistant prostate carcinoma. The objective of the present study was to investigate the effects of 60Co gamma-IR on the growth, cell cycle arrest and cell death of the human prostate cancer cell line DU 145. The viability of DU 145 cells was measured by the Trypan blue exclusion assay and the 3(4,5-dimethylthiazol-2-yl-2,5,diphenyltetrazolium bromide test. Bromodeoxyuridine incorporation was used for the determination of cell proliferation. Cell cycle arrest and cell death were analyzed by flow cytometry. Superoxide dismutase (SOD, specifically CuZnSOD and MnSOD protein expression, after 10 Gy gamma-IR, was determined by Western immunoblotting analysis. gamma-IR treatment had a significant (P < 0.001 antiproliferative and cytotoxic effect on DU 145 cells. Both effects were time and dose dependent. Also, the dose of gamma-IR which inhibited DNA synthesis and cell proliferation by 50% was 9.7 Gy. Furthermore, gamma-IR induced cell cycle arrest in the G2/M phase and the percentage of cells in the G2/M phase was increased from 15% (control to 49% (IR cells, with a nonsignificant induction of apoptosis. Treatment with 10 Gy gamma-IR for 24, 48, and 72 h stimulated CuZnSOD and MnSOD protein expression in a time-dependent manner, approximately by 3- to 3.5-fold. These data suggest that CuZnSOD and MnSOD enzymes may play an important role in the gamma-IR-induced changes in DU 145 cell growth, cell cycle arrest and cell death.

  4. mBAND Analysis of Late Chromosome Aberrations in Human Lymphocytes Induced by Gamma Rays and Fe Ions

    Science.gov (United States)

    Sunagawa, Mayumi; Zhang, Ye; Yeshitla, Samrawit; Kadhim, Munira; Wilson, Bobby; Wu, Honglu

    2014-01-01

    Chromosomal translocations and inversions are considered stable, and cells containing these types of chromosome aberrations can survive multiple cell divisions. An efficient method to detect an inversion is multi-color banding fluorescent in situ hybridization (mBAND) which allows identification of both inter- and intrachromosome aberrations simultaneously. Post irradiation, chromosome aberrations may also arise after multiple cell divisions as a result of genomic instability. To investigate the stable or late-arising chromosome aberrations induced after radiation exposure, we exposed human lymphocytes to gamma rays and Fe ions ex vivo, and cultured the cells for multiple generations. Chromosome aberrations were analyzed in cells collected at first mitosis and at several time intervals during the culture period post irradiation. With gamma irradiation, about half of the damages observed at first mitosis remained after 7 day- and 14 day- culture, suggesting the transmissibility of damages to the surviving progeny. Detailed analysis of chromosome break ends participating in exchanges revealed a greater fraction of break ends involved in intrachromosome aberrations in the 7- and 14-day samples in comparison to the fraction at first mitosis. In particular, simple inversions were found at 7 and 14 days, but not at the first mitosis, suggesting that some of the aberrations might be formed days post irradiation. In contrast, at the doses that produced similar frequencies of gamma-induced chromosome aberrations as observed at first mitosis, a significantly lower yield of aberrations remained at the same population doublings after Fe ion exposure. At these equitoxic doses, more complex type aberrations were observed for Fe ions, indicating that Fe ion-induced initial chromosome damages are more severe and may lead to cell death. Comparison between low and high doses of Fe ion irradiation in the induction of late damages will also be discussed.

  5. Break Point Distribution on Chromosome 3 of Human Epithelial Cells exposed to Gamma Rays, Neutrons and Fe Ions

    Science.gov (United States)

    Hada, M.; Saganti, P. B.; Gersey, B.; Wilkins, R.; Cucinotta, F. A.; Wu, H.

    2007-01-01

    Most of the reported studies of break point distribution on the damaged chromosomes from radiation exposure were carried out with the G-banding technique or determined based on the relative length of the broken chromosomal fragments. However, these techniques lack the accuracy in comparison with the later developed multicolor banding in situ hybridization (mBAND) technique that is generally used for analysis of intrachromosomal aberrations such as inversions. Using mBAND, we studied chromosome aberrations in human epithelial cells exposed in vitro to both low or high dose rate gamma rays in Houston, low dose rate secondary neutrons at Los Alamos National Laboratory and high dose rate 600 MeV/u Fe ions at NASA Space Radiation Laboratory. Detailed analysis of the inversion type revealed that all of the three radiation types induced a low incidence of simple inversions. Half of the inversions observed after neutron or Fe ion exposure, and the majority of inversions in gamma-irradiated samples were accompanied by other types of intrachromosomal aberrations. In addition, neutrons and Fe ions induced a significant fraction of inversions that involved complex rearrangements of both inter- and intrachromosome exchanges. We further compared the distribution of break point on chromosome 3 for the three radiation types. The break points were found to be randomly distributed on chromosome 3 after neutrons or Fe ions exposure, whereas non-random distribution with clustering break points was observed for gamma-rays. The break point distribution may serve as a potential fingerprint of high-LET radiation exposure.

  6. Persistence of gamma-H2AX and 53BP1 foci in proliferating and nonproliferating human mammary epithelial cells after exposure to gamma-rays or iron ions

    Energy Technology Data Exchange (ETDEWEB)

    Groesser, Torsten; Chang, Hang; Fontenay, Gerald; Chen, James; Costes, Sylvain V.; Barcellos-Hoff, Mary Helen; Parvin, Bahram; Rydberg, Bjorn

    2010-12-22

    To investigate {gamma}-H2AX (phosphorylated histone H2AX) and 53BP1 (tumour protein 53 binding protein No. 1) foci formation and removal in proliferating and non-proliferating human mammary epithelial cells (HMEC) after exposure to sparsely and densely ionizing radiation under different cell culture conditions. HMEC cells were grown either as monolayers (2D) or in extracellular matrix to allow the formation of acinar structures in vitro (3D). Foci numbers were quantified by image analysis at various time points after exposure. Our results reveal that in non-proliferating cells under 2D and 3D cell culture conditions, iron-ion induced {gamma}-H2AX foci were still present at 72 h after exposure, although 53BP1 foci returned to control levels at 48 h. In contrast in proliferating HMEC, both {gamma}-H2AX and 53BP1 foci decreased to control levels during the 24-48 h time interval after irradiation under 2D conditions. Foci numbers decreased faster after {gamma}-ray irradiation and returned to control levels by 12 h regardless of marker, cell proliferation status, and cell culture condition. Conclusions: The disappearance of radiation induced {gamma}-H2AX and 53BP1 foci in HMEC have different dynamics that depend on radiation quality and proliferation status. Notably, the general patterns do not depend on the cell culture condition (2D versus 3D). We speculate that the persistent {gamma}-H2AX foci in iron-ion irradiated non-proliferating cells could be due to limited availability of double strand break (DSB) repair pathways in G0/G1-phase, or that repair of complex DSB requires replication or chromatin remodeling.

  7. Roles of interferon-gamma and its target genes in schizophrenia: Proteomics-based reverse genetics from mouse to human.

    Science.gov (United States)

    Kim, Hak-Jae; Eom, Chi-Yong; Kwon, Joseph; Joo, Jaesoon; Lee, Sujeong; Nah, Seong-Su; Kim, Il-Chul; Jang, Ik-Soon; Chung, Young-Ho; Kim, Seung Il; Chung, Joo-Ho; Choi, Jong-Soon

    2012-06-01

    A decreased production of interferon gamma (IFNG) has been observed in acute schizophrenia. In order to explore the possible relationship between IFNG and schizophrenia, we attempted to analyze the differentially expressed proteins in the brains of interferon-gamma knockout (Ifng-KO) mice. Five upregulated and five downregulated proteins were identified with 2D gels and MALDI-TOF/TOF MS analyses in Ifng-KO mouse brain. Of the identified proteins, we focused on creatine kinase brain (CKB) and triose phosphate isomerase 1 (TPI1). Consistent with the proteomic data, reverse transcriptase-mediated PCR, immunoblotting, and immunohistochemistry analyses confirmed that the levels of gene expressions of Ckb and Tpi1 were downregulated and upregulated, respectively. When we analyzed the genetic polymorphisms of the single nucleotide polymorphisms (SNPs) of their human orthologous genes in a Korean population, the promoter SNPs of CKB and TPI1 were weakly associated with schizophrenia. In addition, IFNG polymorphisms were associated with schizophrenia. These results suggest that IFNG and proteins affected by IFNG may play a role in the pathogenesis of schizophrenia.

  8. Analysis of eight genes modulating interferon gamma and human genetic susceptibility to tuberculosis: a case-control association study

    Directory of Open Access Journals (Sweden)

    Hoal Eileen G

    2010-06-01

    Full Text Available Abstract Background Interferon gamma is a major macrophage-activating cytokine during infection with Mycobacterium tuberculosis, the causative pathogen of tuberculosis, and its role has been well established in animal models and in humans. This cytokine is produced by activated T helper 1 cells, which can best deal with intracellular pathogens such as M. tuberculosis. Based on the hypothesis that genes which regulate interferon gamma may influence tuberculosis susceptibility, we investigated polymorphisms in eight candidate genes. Methods Fifty-four polymorphisms in eight candidate genes were genotyped in over 800 tuberculosis cases and healthy controls in a population-based case-control association study in a South African population. Genotyping methods used included the SNPlex Genotyping System™, capillary electrophoresis of fluorescently labelled PCR products, TaqMan® SNP genotyping assays or the amplification mutation refraction system. Single polymorphisms as well as haplotypes of the variants were tested for association with TB using statistical analyses. Results A haplotype in interleukin 12B was nominally associated with tuberculosis (p = 0.02, but after permutation testing, done to assess the significance for the entire analysis, this was not globally significant. In addition a novel allele was found for the interleukin 12B D5S2941 microsatellite. Conclusions This study highlights the importance of using larger sample sizes when attempting validation of previously reported genetic associations. Initial studies may be false positives or may propose a stronger genetic effect than subsequently found to be the case.

  9. FC vehicle hybridisation: an affordable solution for an energy-efficient FC powered drive train

    Science.gov (United States)

    Pede, G.; Iacobazzi, A.; Passerini, S.; Bobbio, A.; Botto, G.

    Fuel cells (FCs) have potential as clean and efficient energy sources for automotive applications without sacrifice in performance or driving range. However, the complete FC system must operate as efficiently as possible over the range of driving conditions that may be encountered while maintaining a low cost. To achieve this target, a storage unit can be introduced in the FC system to reduce the size of the fuel cell that is the most expensive component. This "hybrid" concept would not only reduce the drive train total cost but it also allow the recover of the braking energy and the operation at the voltage-current point of maximum efficiency for the FC system. Pro-and-cons of the "full-power" versus the "hybrid" configuration are shown in this work. The "Hybridisation rate" or "Hybridisation degree", a parameter expressed by the relationship between two installed powers, the generation power and the traction power, is also introduced and it is demonstrated that for each category of hybrid vehicles there is an optimal value of hybridisation degree. The storage systems considered are based on high power batteries or ultra capacitors (UCs) or a combination of them. A preliminary design of a sport utility vehicle (SUV) using a combined storage system and a FC energy source (called Triple Hybrid), is proposed. Finally, the experience of the Italian industry in this field is also reviewed.

  10. Construction of lentiviral vector of mFVII/Fc fusion gene and the research of its expression in human bone mesenchymal stem cells%mFVII/Fc融合基因慢病毒载体的构建及在hBMSCs中表达的研究

    Institute of Scientific and Technical Information of China (English)

    刘胜来; 陈捷; 王共先; 汪泱; 汪新辉; 余刚; 薛金雄; 熊礼生

    2011-01-01

    目的 构建人凝血因子VII(FVII)与免疫球蛋白Fc片段融合基因(mFVII/Fc)的慢病毒表达载体,并检测其在人骨髓间充质干细胞(hBMSCs)中的表达情况,获取mFVII/Fc稳定表达的干细胞载体.方法 体外克隆人凝血因子VII,用点突变技术在基因水平将341部位Lys突变为Ala后,利用DNA连接酶与免疫球蛋白IgG1 Fc片段的基因融合,经酶切整合,测序鉴定后转染人胚肾293T细胞包装为重组mFVII/Fc慢病毒载体,鉴定载体构建成功后,测定慢病毒滴度.确定转染第三代hBMSCs的最佳MOI值后批量转染,荧光显微镜下观察GFP的表达情况,RT-PCR、ELISA分别在不同时间点对mFVII/Fc的mRNA及蛋白表达进行相对定量分析.结果 所构建融合基因与GenBank ID,AF272774对比,除同义变异外完全相符;包装的慢病毒滴度为2×108TU/ml;hBM-SCs鉴定结果为CD+29(98.08%)、CD+44(97.63%)、CD34(0.31%)、CD+45(0.58%);转染hBMSCs 72 h后的效率为(84±3)%,经RT-PCR及ELISA证实转染mFVII/Fc基因的hBMSCs有大量mRNA转录和蛋白表达水平.结论 实验成功获得了融合基因的hBMSCs稳定遗传表达载体,为靶向前列腺癌组织因子研究及肿瘤基因治疗研究提供了实验依据.%Objective To construct a recombinant Ientiviral vector of mFVII/Fc and investigate its transfective efficiency into human bone mesenchymal stem cells (hBMSCs),and to detect the expression of mFVII/Fc fusion gene in vitro. Methods Coagulation factor VII (FVII) was cloned in vitro,with a point mutation from Lys to Ala in the position of 341 in the gene level.The cDNA fragments of mutational FVII (mFVII) and those of IgG1Fc were fused together with DNA ligase.After digestion,integration and sequencing,the fusion DNA was identified and transfected human embryonic kidney 293T cell packaging for re-mFVII/Fc lentiviral vector.After successful identification of vectors,detect the Ientiviral titer determination,bulk transfer after the determination of best MOI

  11. Fc fusion as a platform technology: potential for modulating immunogenicity.

    Science.gov (United States)

    Levin, Ditza; Golding, Basil; Strome, Scott E; Sauna, Zuben E

    2015-01-01

    The platform technology of fragment crystallizable (Fc) fusion, in which the Fc region of an antibody is genetically linked to an active protein drug, is among the most successful of a new generation of bioengineering strategies. Immunogenicity is a critical safety concern in the development of any protein therapeutic. While the therapeutic goal of generating Fc-fusion proteins has been to extend half-life, there is a critical mass of literature from immunology indicating that appropriate design of the Fc component has the potential to engage the immune system for product-specific outcomes. In the context of Fc-fusion therapeutics, a review of progress in understanding Fc biology suggests the prospect of engineering products that have an extended half-life and are able to modulate the immune system.

  12. Dicty_cDB: FC-AS13 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AS13 (Link to dictyBase) - - - Contig-U15149-1 FC-AS13P (Li...racellular, including cell wall 32.0 %: plasma membrane 20.0 %: endoplasmic reticulum 12.0 %: Golgi >> prediction for FC-AS...nk to Original site) FC-AS13F 390 FC-AS13Z 534 FC-AS13P 924 - - Show FC-AS13 Library FC (Link to library) Clone ID FC-AS...13 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U15149-1 Origin...al site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AS/FC-AS13Q.Seq.d/ Representative seq. ID FC-AS

  13. Dicty_cDB: FC-AS19 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AS19 (Link to dictyBase) - - - Contig-U15986-1 FC-AS19P (Li...00 m_ : 1.00 76.0 %: nuclear 12.0 %: cytoplasmic 8.0 %: mitochondrial 4.0 %: cytoskeletal >> prediction for FC-AS...nk to Original site) FC-AS19F 136 FC-AS19Z 295 FC-AS19P 431 - - Show FC-AS19 Library FC (Link to library) Clone ID FC-AS...19 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U15986-1 Origin...al site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AS/FC-AS19Q.Seq.d/ Representative seq. ID FC-AS

  14. Dicty_cDB: FC-IC0618 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC0618 (Link to dictyBase) - - - Contig-U07877-1 FC-IC06...ink to library) Clone ID FC-IC0618 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Co...ntig-U07877-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC...les of secretory system >> prediction for FC-IC0618 is cyt 5' end seq. ID FC-IC0618F 5' end seq. >FC-IC...18P (Link to Original site) FC-IC0618F 583 FC-IC0618Z 169 FC-IC0618P 732 - - Show FC-IC0618 Library FC-IC (L

  15. Dicty_cDB: FC-IC1558 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC1558 (Link to dictyBase) - - - Contig-U15578-1 FC-IC15...ink to library) Clone ID FC-IC1558 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Co...ntig-U15578-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC...y system 4.0 %: peroxisomal >> prediction for FC-IC1558 is cyt 5' end seq. ID FC-IC1558F 5' end seq. >FC-IC1...58P (Link to Original site) FC-IC1558F 205 FC-IC1558Z 298 FC-IC1558P 483 - - Show FC-IC1558 Library FC-IC (L

  16. Dicty_cDB: FC-IC1004 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC1004 (Link to dictyBase) - G24305 DDB0232093 Contig-U13941-1 FC-IC... (Link to library) Clone ID FC-IC1004 (Link to dictyBase) Atlas ID - NBRP ID G24305 dic...mrvimmwlinkl*i*slkvvhylllvw*hslracv Homology vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value FC-IC... 4.0 %: mitochondrial >> prediction for FC-IC1004 is cyt 5' end seq. ID FC-IC1004F 5' end seq. >FC-IC...1004E (Link to Original site) FC-IC1004F 187 FC-IC1004Z 187 FC-IC1004P 354 FC-IC1004E 177 Show FC-IC1004 Library FC-IC

  17. Dicty_cDB: FC-IC1504 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC1504 (Link to dictyBase) - - - Contig-U07877-1 FC-IC15... (Link to library) Clone ID FC-IC1504 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link ...to Contig Contig-U07877-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC...0104Q) /CSM/FC-IC/FC-IC0104Q.Seq.d/ 533 e-151 own update 2001.11.19 Homology vs DNA Score E Sequences producing signific...04E (Link to Original site) FC-IC1504F 581 FC-IC1504Z 583 FC-IC1504P 1144 FC-IC1504E 573 Show FC-IC1504 Library FC-IC

  18. Dicty_cDB: FC-IC0422 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC0422 (Link to dictyBase) - - - Contig-U16233-1 FC-IC04...22Z (Link to Original site) - - FC-IC0422Z 549 - - - - Show FC-IC0422 Library FC-IC (Link to library) Clone ID FC-IC... vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value FC-IC0422 (FC-IC0422Q) /CSM/FC-IC/FC-IC...0422 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U16233-1 Original site URL http://dic...tycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC0422Q.Seq.d/ Representative seq. ID FC-IC

  19. Dicty_cDB: FC-IC1498 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC1498 (Link to dictyBase) - - - Contig-U16527-1 FC-IC14... (Link to library) Clone ID FC-IC1498 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link t...o Contig Contig-U16527-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC...xiyldilxlrnhxk Homology vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value FC-IC1760 (FC-IC...98E (Link to Original site) FC-IC1498F 438 FC-IC1498Z 439 FC-IC1498P 857 FC-IC1498E 429 Show FC-IC1498 Library FC-IC

  20. Dicty_cDB: FC-IC1278 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC1278 (Link to dictyBase) - - - Contig-U12354-1 FC-IC12... (Link to library) Clone ID FC-IC1278 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link t...o Contig Contig-U12354-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC... 16.0 %: nuclear 8.0 %: cytoskeletal 8.0 %: plasma membrane >> prediction for FC-IC1278 is cyt 5' end seq. ID FC-IC...78E (Link to Original site) FC-IC1278F 334 FC-IC1278Z 334 FC-IC1278P 648 FC-IC1278E 324 Show FC-IC1278 Library FC-IC

  1. Dicty_cDB: FC-IC0929 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC0929 (Link to dictyBase) - - - Contig-U16527-1 FC-IC09...ink to library) Clone ID FC-IC0929 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Co...ntig-U16527-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC...les of secretory system >> prediction for FC-IC0929 is end 5' end seq. ID FC-IC0929F 5' end seq. >FC-IC...29P (Link to Original site) FC-IC0929F 313 FC-IC0929Z 320 FC-IC0929P 613 - - Show FC-IC0929 Library FC-IC (L

  2. Dicty_cDB: FC-IC1321 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC1321 (Link to dictyBase) - - - Contig-U15335-1 FC-IC13... (Link to library) Clone ID FC-IC1321 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link t...o Contig Contig-U15335-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC...0 %: mitochondrial 4.0 %: vacuolar 4.0 %: endoplasmic reticulum >> prediction for FC-IC1321 is cyt 5' end seq. ID FC-IC...21E (Link to Original site) FC-IC1321F 194 FC-IC1321Z 194 FC-IC1321P 368 FC-IC1321E 184 Show FC-IC1321 Library FC-IC

  3. Dicty_cDB: FC-IC0181 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC0181 (Link to dictyBase) - - - Contig-U03326-1 FC-IC01...81F (Link to Original site) FC-IC0181F 553 - - - - - - Show FC-IC0181 Library FC-IC (Link to library) Clone ID FC-IC... Homology vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value FC-IC0181 (FC-IC0181Q) /CSM/FC-IC/FC-IC...ulum >> prediction for FC-IC0181 is cyt 5' end seq. ID FC-IC0181F 5' end seq. >FC-IC...0181 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U03326-1 Original site URL http://dic

  4. Dicty_cDB: FC-IC0175 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC0175 (Link to dictyBase) - - - Contig-U13957-1 FC-IC01...75F (Link to Original site) FC-IC0175F 212 - - - - - - Show FC-IC0175 Library FC-IC (Link to library) Clone ID FC-IC...e E Sequences producing significant alignments: (bits) Value FC-IC0175 (FC-IC0175Q) /CSM/FC-IC/FC-IC0175Q.Se...letal 4.0 %: vacuolar >> prediction for FC-IC0175 is cyt 5' end seq. ID FC-IC0175F 5' end seq. >FC-IC0175F.S...0175 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U13957-1 Original site URL http://dic

  5. Dicty_cDB: FC-IC0169 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC0169 (Link to dictyBase) - - - Contig-U15064-1 FC-IC01...69F (Link to Original site) FC-IC0169F 288 - - - - - - Show FC-IC0169 Library FC-IC (Link to library) Clone ID FC-IC...0169 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U15064-1 Original site URL http://dic...tycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC0169Q.Seq.d/ Representative seq. ID FC-IC...0169F (Link to Original site) Representative DNA sequence >FC-IC0169 (FC-IC0169Q) /CSM/FC-IC/FC-IC

  6. Chlamydia trachomatis Is Resistant to Inclusion Ubiquitination and Associated Host Defense in Gamma Interferon-Primed Human Epithelial Cells

    Science.gov (United States)

    Haldar, Arun K.; Piro, Anthony S.; Finethy, Ryan; Espenschied, Scott T.; Brown, Hannah E.; Giebel, Amanda M.; Frickel, Eva-Maria; Nelson, David E.

    2016-01-01

    ABSTRACT The cytokine gamma interferon (IFN-γ) induces cell-autonomous immunity to combat infections with intracellular pathogens, such as the bacterium Chlamydia trachomatis. The present study demonstrates that IFN-γ-primed human cells ubiquitinate and eliminate intracellular Chlamydia-containing vacuoles, so-called inclusions. We previously described how IFN-γ-inducible immunity-related GTPases (IRGs) employ ubiquitin systems to mark inclusions for destruction in mouse cells and, furthermore, showed that the rodent pathogen Chlamydia muridarum blocks ubiquitination of its inclusions by interfering with mouse IRG function. Here, we report that ubiquitination of inclusions in human cells is independent of IRG and thus distinct from the murine pathway. We show that C. muridarum is susceptible to inclusion ubiquitination in human cells, while the closely related human pathogen C. trachomatis is resistant. C. muridarum, but not C. trachomatis, inclusions attract several markers of cell-autonomous immunity, including the ubiquitin-binding protein p62, the ubiquitin-like protein LC3, and guanylate-binding protein 1. Consequently, we find that IFN-γ priming of human epithelial cells triggers the elimination of C. muridarum, but not C. trachomatis, inclusions. This newly described defense pathway is independent of indole-2,3-dioxygenase, a known IFN-γ-inducible anti-Chlamydia resistance factor. Collectively, our observations indicate that C. trachomatis evolved mechanisms to avoid a human-specific, ubiquitin-mediated response as part of its unique adaptation to its human host. PMID:27965446

  7. [Eukaryotic Expression and Immunogenic Research of Recombination Ebola Virus Membrane Protein Gp-Fc].

    Science.gov (United States)

    Zhang, Xiaoguang; Yang, Ren; Wang, Jiao; Wang, Xuan; Hou, Mieling; An, Lina; Zhu, Ying; Cao, Yuxi; Zeng, Yi

    2016-01-01

    We used 293 cells to express the recombinant membrane protein of the Ebola virus. Then, the immunogenicity of the recombinant protein was studied by immunized BALB/c mice. According to the codon use frequency of humans, the gene encoding the extracellular domain of the Ebola virus membrane protein was optimized, synthesized, and inserted into the eukaryotic expression plasmid pXG-Fc to construct the human IgG Fc and Ebola GP fusion protein expression plasmid pXG-modGP-Fc. To achieve expression, the fusion protein expression vector was transfected into high-density 293 cells using transient transfection technology. The recombinant protein was purified by protein A affinity chromatography. BALB/c mice were immunized with the purified fusion protein, and serum antibody titers evaluated by an indirect enzyme-linked immunosorbent assay (ELISA). Purification and analyses of the protein revealed that the eukaryotic expression vector could express the recombinant protein GP-Fc effectively, and that the recombinant protein in the supernatant of the cell culture was present as a dimer. After immunization with the purified recombinant protein, a high titer of antigen-specific IgG could be detected in the serum of immunized mice by indirect ELISA, showing that the recombinant protein had good immunogenicity. These data suggest that we obtained a recombinant protein with good immunogenicity. Our study is the basis for development of a vaccine against the Ebola virus and for screening of monoclonal antibodies.

  8. [The macrophage disappearance reaction in guinea pigs sensitized with bovine gamma globulin or human scrum albumin (author's transl)].

    Science.gov (United States)

    Schimke, R; Bernstein, B; Ambrosius, H

    1977-01-01

    The macrophage disappearance reaction (MDR) is a suitable test for detection of cell mediated immunity against bovine gamma globulin (BGG) and human serum albumin (HSA) in guinea pigs. The MDR is a technical simple, good manipulable, and quantifiable test. The optimal test conditions for the antigens BGC and HSA are the following: Peritoneal exudat cells (PEC) were stimulated with paraffin oil. On the 5th day after receiving oil the animals were injected with 80 microgram BGG or 30 microgram HSA i.p. 5 hours later the PEC were harvested and counted. With the MDR it is possible to detect differences with respect to degree of cell-mediated immunity. Supernatants of sensitized lymphocytes produces the MDR too.

  9. The expression of Fcγ receptors in Hashimoto's thyroiditis.

    Science.gov (United States)

    Liu, Yalei; Liu, Mingming; Zhang, Yang; Qu, Chenxue; Lu, Guizhi; Huang, Youyuan; Zhang, Hong; Yu, Nan; Yuan, Shanshan; Gao, Ying; Gao, Yanming; Guo, Xiaohui

    2015-03-01

    The pathophysiological mechanism underlying Hashimoto's thyroiditis (HT) is still unclear. Thyroglobulin antibody (TgAb) and thyroid peroxidase antibody (TPOAb) are diagnostic hallmarks of HT. These IgG antibodies regulate the balance of immunologic tolerance and autoimmunity via Fcγ receptors (FcγRs). The aim of our study was to investigate the role of FcγRs in the pathogenesis of HT. The percentage of peripheral blood mononuclear cells (PBMCs) from HT patients bearing FcγRII was significantly lower than that seen in healthy donors, and the mean fluorescence intensity (MFI) value of FcγRII on PBMCs from HT patients was significantly higher. The percentage of PBMCs positive for FcγRIII also was significantly higher in HT patients, and the percentage of B cells bearing FcγRIIB in HT patients was significantly lower than that seen in healthy donors. Our study therefore provides evidence for FcγRs, especially FcγRIIB, being involved in the pathogenesis of HT.

  10. CONSTRUCTION AND IDENTIFICATION OF THE RECOMBINANT OF THE AAV VECTOR AND HUMAN INTERFERON-GAMMA

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    To construct and identify further a recombinant of Adeno-associated virus and interferon-gamma for gene therapy, the full-length IFN-γcDNA containing signal peptide was amplified by PCR, and then cloned into the pUC18. After screening, the fragment from the positive clone was then subcloned into pwpl9. After the correct recombinant was identified by digestion with SacI and BamHI, it was transfected into lympho- cyte cell line H9 mediated by calcium phosphate, and the expression of IFN-γ was detected by RT-PCR and ELISA. The result showed that the IFN-y were expressed in the H9 cells transfected with pwp/IFN-y. The so constructed recombinant plasmid pwpl9/IFN-y containing the full-length IFN-y gene was expressed in mam- malian cells.

  11. Inhibitory Mechanism of Inteferon-gamma on Human Fibroblasts from Tenon's Capsule

    Institute of Scientific and Technical Information of China (English)

    韩波; 胡义珍; 熊新春

    2004-01-01

    Summary: The inhibitory mechanism of interferon-gamma (IFN-γ) on the fibroblasts from Tenon's capsule was studied. By using immunohistochemical SP method and pathological image system, the inhibitory effects of IFN-γ on the expression of transforming growth factor beta receptor I in the in vitro cultured fibroblasts from Tenon's capsule were quantitatively analyzed. The results showed that IFN-γ could reduce the expression of transforming growth factor beta receptor I in the fibroblasts with the following dose-effect relationship: Y= 1937.5-134.2 Igx (r= -0. 971, P<0.01).It was concluded that IFN-γ could inhibit the expression of transforming growth factor beta receptor I in the fibroblasts from Tenon's capsule. The modulation of the transforming growth factor beta receptor I expression by IFN-γ may be beneficial to the alleviation of the hyperplasia of scar after trabeculectomy.

  12. Sparse Spectro-Temporal Receptive Fields Based on Multi-Unit and High-Gamma Responses in Human Auditory Cortex.

    Directory of Open Access Journals (Sweden)

    Rick L Jenison

    Full Text Available Spectro-Temporal Receptive Fields (STRFs were estimated from both multi-unit sorted clusters and high-gamma power responses in human auditory cortex. Intracranial electrophysiological recordings were used to measure responses to a random chord sequence of Gammatone stimuli. Traditional methods for estimating STRFs from single-unit recordings, such as spike-triggered-averages, tend to be noisy and are less robust to other response signals such as local field potentials. We present an extension to recently advanced methods for estimating STRFs from generalized linear models (GLM. A new variant of regression using regularization that penalizes non-zero coefficients is described, which results in a sparse solution. The frequency-time structure of the STRF tends toward grouping in different areas of frequency-time and we demonstrate that group sparsity-inducing penalties applied to GLM estimates of STRFs reduces the background noise while preserving the complex internal structure. The contribution of local spiking activity to the high-gamma power signal was factored out of the STRF using the GLM method, and this contribution was significant in 85 percent of the cases. Although the GLM methods have been used to estimate STRFs in animals, this study examines the detailed structure directly from auditory cortex in the awake human brain. We used this approach to identify an abrupt change in the best frequency of estimated STRFs along posteromedial-to-anterolateral recording locations along the long axis of Heschl's gyrus. This change correlates well with a proposed transition from core to non-core auditory fields previously identified using the temporal response properties of Heschl's gyrus recordings elicited by click-train stimuli.

  13. Induction of micronuclei in human and mouse lymphocytes irradiated with gamma radiation and effect of panax ginseng C. A. Meyer

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sung Ho; Oh, Heon; Lee, Song Eun [Chonnam National Univ., Kwangju (Korea, Republic of); Lee, Yun Sil; Kim, Tae Hwan [Korea Cancer Center Hospital, Seoul (Korea, Republic of); Jeong, Kyu Sik [Korea Research Institute of Bioscience and Biotechnology, Taejon (Korea, Republic of); Ryu, Si Yun [Chungnam National Univ., Taejon (Korea, Republic of)

    1997-09-01

    The frequencies of {gamma}-ray-induced micronuclei (MN) in Cytokinesis-Blocked (CB) lymphocytes at several doses were measured in three donors of human and C57BL/6 mice. Measurements performed after irradiation showed a dose-related increases in MN frequency in each of the donors studied. The relative sensitivity of mouse in Spleen Lymphocytes (SLs) compared with human Peripheral Blood Lymphocytes (PBLs) was estimated by best fitting linear-quadratic model based on the radiation-induced MN data over the range from 0 cGy to 400 cGy. In the case of MN frequency with 0.2 per CB cell, the relative sensitivity of mouse SLs was 1.67. Compared with the radiation-induced MN formation in the PBLs of human, the SLs of mouse were more radiosensitive. Using this MN assay with human PBLs and mouse SLs, studies were performed to determine whether the water fraction of ginseng (Panax ginseng C.A.Meyer)against radiation-induced MN in human PBLs after in vitro irradiation (3Gy) and in SLs of C57BL/6 mice after in vivo irradiation (3Gy). The frequency of MN in human PBLs was reduced by water fraction of ginseng (0.5mg/ml of medium) both pre-and post treatment (p<0.01) in vitro. In addition, the frequency of MN in mouse SLs was also reduced by pretreatment of ginseng (2mg/ml of drinking water for 7 days) in vivo.

  14. Dual role of Fcγ receptors in host defense and disease in Borrelia burgdorferi-infected mice

    Directory of Open Access Journals (Sweden)

    Alexia Anne Belperron

    2014-06-01

    Full Text Available Arthritis in mice infected with the Lyme disease spirochete, Borrelia burgdorferi, results from the influx of innate immune cells responding to the pathogen in the joint and is influenced in part by mouse genetics. Production of inflammatory cytokines by innate immune cells in vitro is largely mediated by Toll-like receptor (TLR interaction with Borrelia lipoproteins, yet surprisingly mice deficient in TLR2 or the TLR signaling molecule MyD88 still develop arthritis comparable to that seen in wild type mice after B. burgdorferi infection. These findings suggest that other, MyD88-independent inflammatory pathways can contribute to arthritis expression. Clearance of B. burgdorferi is dependent on the production of specific antibody and phagocytosis of the organism. As Fc receptors (FcγR are important for IgG-mediated clearance of immune complexes and opsonized particles by phagocytes, we examined the role that FcγR play in host defense and disease in B. burgdorferi-infected mice. B. burgdorferi-infected mice deficient in the Fc receptor common gamma chain (FcεRγ-/- mice harbored ~10 fold more spirochetes than similarly infected wild type mice, and this was associated with a transient increase in arthritis severity. While the elevated pathogen burdens seen in B. burgdorferi-infected MyD88-/- mice were not affected by concomitant deficiency in FcγR, arthritis was reduced in FcεRγ-/-MyD88-/- mice in comparison to wild type or single knockout mice. Gene expression analysis from infected joints demonstrated that absence of both MyD88 and FcγR lowers mRNA levels of proteins involved in inflammation, including Cxcl1 (KC, Xcr1 (Gpr5, IL-1beta, and C reactive protein. Taken together, our results demonstrate a role for FcγR-mediated immunity in limiting pathogen burden and arthritis in mice during the acute phase of B. burgdorferi infection, and further suggest that this pathway contributes to the arthritis that develops in B. burgdorferi

  15. Peroxisome proliferator-activated receptor gamma-mediated up-regulation of syndecan-1 by n-3 fatty acids promotes apoptosis of human breast cancer cells.

    Science.gov (United States)

    Sun, Haiguo; Berquin, Isabelle M; Owens, Rick T; O'Flaherty, Joseph T; Edwards, Iris J

    2008-04-15

    Diets enriched in n-3 polyunsaturated fatty acids (n-3 PUFA) may protect against breast cancer but biochemical mechanisms are unclear. Our studies showed that the n-3 fatty acid docosahexaenoic acid (DHA) up-regulated syndecan-1 (SDC-1) in human breast cancer cells, and we tested the hypothesis that DHA-mediated up-regulation of SDC-1 induces apoptosis. DHA was delivered to MCF-7 cells by n-3 PUFA-enriched low-density lipoproteins (LDL) or by albumin in the presence or absence of SDC-1 small interfering RNA. The n-3 PUFA induced apoptosis, which was blocked by SDC-1 silencing. We also confirmed that SDC-1 up-regulation and apoptosis promotion by n-3 PUFA was mediated by peroxisome proliferator-activated receptor gamma (PPAR gamma). Using a luciferase gene driven by either a PPAR response element or a DR-1 site present in the SDC-1 promoter, reporter activities were enhanced by n-3 LDL, DHA, and PPAR gamma agonist, whereas activity of a luciferase gene placed downstream of a mutant DR-1 site was unresponsive. Cotransfection with dominant-negative PPAR gamma DNA eliminated the increase in luciferase activity. These data provide strong evidence that SDC-1 is a molecular target of n-3 PUFA in human breast cancer cells through activation of PPAR gamma and that n-3 PUFA-induced apoptosis is mediated by SDC-1. This provides a novel mechanism for the chemopreventive effects of n-3 PUFA in breast cancer.

  16. Noninvasively decoding the contents of visual working memory in the human prefrontal cortex within high-gamma oscillatory patterns.

    Science.gov (United States)

    Polanía, Rafael; Paulus, Walter; Nitsche, Michael A

    2012-02-01

    The temporal maintenance and subsequent retrieval of information that no longer exists in the environment is called working memory. It is believed that this type of memory is controlled by the persistent activity of neuronal populations, including the prefrontal, temporal, and parietal cortex. For a long time, it has been controversially discussed whether, in working memory, the PFC stores past sensory events or, instead, its activation is an extramnemonic source of top-down control over posterior regions. Recent animal studies suggest that specific information about the contents of working memory can be decoded from population activity in prefrontal areas. However, it has not been shown whether the contents of working memory during the delay periods can be decoded from EEG recordings in the human brain. We show that by analyzing the nonlinear dynamics of EEG oscillatory patterns it is possible to noninvasively decode with high accuracy, during encoding and maintenance periods, the contents of visual working memory information within high-gamma oscillations in the human PFC. These results are thus in favor of an active storage function of the human PFC in working memory; this, without ruling out the role of PFC in top-down processes. The ability to noninvasively decode the contents of working memory is promising in applications such as brain computer interfaces, together with computation of value function during planning and decision making processes.

  17. Histamine and histamine-receptor antagonists modify gene expression and biosynthesis of interferon gamma in peripheral human blood mononuclear cells and in CD19-depleted cell subsets

    NARCIS (Netherlands)

    Horváth, B V; Szalai, C; Mándi, Y; László, V; Radvány, Z; Darvas, Z; Falus, A

    1999-01-01

    The effect of histamine and histamine antagonists was examined on gene expression and biosynthesis of bacterial endotoxin (LPS) induced interferon gamma (IFNgamma) both in human peripheral mononuclear cells (PMBC) and in T-cell enriched fractions. We found, that histamine inhibited the LPS induced t

  18. A randomized controlled study of juvenile idiopathic arthritis treated with recombinant human Ⅱ tumor necrosis factor-Fc function protein%重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白治疗幼年特发性关节炎的随机对照研究

    Institute of Scientific and Technical Information of China (English)

    曾萍; 谢颖; 唐盈; 李丰; 曾华松

    2012-01-01

    及合并巨噬细胞活化综合征的全身型JIA患儿可以考虑使用rhTNFR:Fc.%Objective Through the application of recombinant human Ⅱ tumor necrosis factor-Fc function protein (rhTNFR:Fc) in the treatment of juvenile idiopathic arthritis (JIA) with randomized control study,clinical characteristic and clinical effect were summarized.Methods According to the randomized controlled principle,124 patients with JIA were divided into control group and treatment group.The basic treatment in two groups were one antirheumatic slow-acting drug,nonsteroidal drug,adrenal cortical hormone.There were no significant differences between clinical type and basic treatment in two groups (P > 0.05).Sixty-two patients of JIA treated with rhTNFR:Fc by subcutaneous injection.The doses was 0.8mg /kg per week.There were 17 cases of oligoarthritis,15 cases of polyarthritis,30 cases of systemic arthritis in the treatment group and control group respectively.The basic antirheumatic drugs,nonsteroidal anti-inflamatory drugs ( NSAIDs),adrenal cortex hormone were allowed to continued.Clinical evaluation index included ACR Pedi 30,ACR Pedi 50 and ACR Pedi 70.The adverse drug reactions were recorded.Results The remission rate of ACR Pedi 30,50,70 in 2 weeks,one month,three monthes and six monthes were different in types of JIA patients in the treatment group ( P < 0.05 ).The remission rate of systemic arthritis was lower than the other two groups of arthritis ( P < 0.05 ).Only 44% ACR Pedi 50 remission was achieved after three monthes medication in systemic arthritis and 41.7% ACR Pedi 50,29.2% ACR Pedi 70 were achieved after six monthes.The remission rate in the types of oligoarthritis and polyarthritis at different time points (2 weeks,one month,three monthes,six monthes) of ACR Pedi 30,50,70 were similar.After six monthes,more than 80% reached ACR Pedi 50 remission,more than half of patients reached ACR Pedi 70 remission.Three cases of macrophage activation syndrome in

  19. Site-selective conjugation of an anticoagulant aptamer to recombinant albumins and maintenance of neonatal Fc receptor binding

    DEFF Research Database (Denmark)

    Schmøkel, Julie; Voldum, Anders; Tsakiridou, Georgia

    2017-01-01

    Aptamers are an attractive molecular medicine that offers high target specificity. Nucleic acid-based aptamers, however, are prone to nuclease degradation and rapid renal excretion that require blood circulatory half-life extension enabling technologies. The long circulatory half...... of a factor IXa anticoagulant aptamer on aptamer functionality and human FcRn (hFcRn) engagement using recombinant human albumin (rHA) of either a wild type (WT) or an engineered human FcRn high binding variant (HB). Albumin-aptamer conjugates, connected covalently through a heterobifunctional succinimidyl 4......-(N-maleimidomethyl)cyclohexane-1-carboxylate linker, were successfully prepared and purified by high performance liquid chromatography as confirmed by gel electrophoresis band-shift analysis and matrix-assisted laser desorption/ionization time of flight. Minimal reduction (∼25%) in activity of WT...

  20. Dicty_cDB: FC-IC1367 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC1367 (Link to dictyBase) - - - - FC-IC1367E (Link to Original site) FC-IC... to library) Clone ID FC-IC1367 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig - Ori...cytoskeletal 8.0 %: mitochondrial 4.0 %: vacuolar 4.0 %: endoplasmic reticulum >> prediction for FC-IC1367 i...1367F 271 FC-IC1367Z 271 FC-IC1367P 522 FC-IC1367E 261 Show FC-IC1367 Library FC-IC (Link...ginal site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC1367Q.Seq.d/ Rep

  1. Dicty_cDB: FC-IC1728 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC1728 (Link to dictyBase) - - - - - (Link to Original site) - - FC-IC...1728Z 373 - - - - Show FC-IC1728 Library FC-IC (Link to library) Clone ID FC-IC1728 (Link to dic...tyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig - Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC...vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value FC-IC1785 (FC-IC1785Q) /CSM/FC-IC/FC-IC...6.0 %: plasma membrane 12.0 %: mitochondrial 4.0 %: cytoskeletal 4.0 %: Golgi 4.0 %: cytoplasmic >> prediction for FC-IC

  2. Dicty_cDB: FC-IC1667 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC1667 (Link to dictyBase) - - - Contig-U16382-1 FC-IC16... (Link to library) Clone ID FC-IC1667 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link t...o Contig Contig-U16382-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC...y vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value FC-IC1667 (FC-IC1667Q) /CSM/FC-IC/FC-IC...%: mitochondrial 8.0 %: nuclear >> prediction for FC-IC1667 is mit 5' end seq. ID FC-IC1667F 5' end seq. >FC-IC

  3. Dicty_cDB: FC-IC0107 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC0107 (Link to dictyBase) - - - - FC-IC0107F (Link to Original site) FC-IC...0107F 430 - - - - - - Show FC-IC0107 Library FC-IC (Link to library) Clone ID FC-IC0107 (Link to dic...tyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig - Original site URL http://dictycdb.bi...ore E Sequences producing significant alignments: (bits) Value FC-IC0107 (FC-IC0107Q) /CSM/FC-IC/FC-IC0107Q.Seq.d/ 258 6e-68 FC-IC...asma membrane 12.0 %: mitochondrial 4.0 %: cytoskeletal 4.0 %: Golgi 4.0 %: cytoplasmic >> prediction for FC-IC

  4. Dicty_cDB: FC-IC1423 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC1423 (Link to dictyBase) - - - Contig-U16122-1 FC-IC14... (Link to library) Clone ID FC-IC1423 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link t...o Contig Contig-U16122-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC...mitochondrial 4.0 %: cytoskeletal 4.0 %: Golgi 4.0 %: vesicles of secretory system >> prediction for FC-IC14...23E (Link to Original site) FC-IC1423F 250 FC-IC1423Z 254 FC-IC1423P 484 FC-IC1423E 244 Show FC-IC1423 Library FC-IC

  5. Dicty_cDB: FC-IC1251 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC1251 (Link to dictyBase) - - - Contig-U08307-1 FC-IC12...Link to library) Clone ID FC-IC1251 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig C...ontig-U08307-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC... 4.0 %: plasma membrane >> prediction for FC-IC1251 is mit 5' end seq. ID FC-IC...51P (Link to Original site) FC-IC1251F 648 FC-IC1251Z 650 FC-IC1251P 1278 - - Show FC-IC1251 Library FC-IC (

  6. Dicty_cDB: FC-IC1707 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC1707 (Link to dictyBase) - - - Contig-U13402-1 FC-IC17... (Link to library) Clone ID FC-IC1707 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link ...to Contig Contig-U13402-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC...tracellular, including cell wall 8.0 %: endoplasmic reticulum 4.0 %: cytoskeletal 4.0 %: peroxisomal >> prediction for FC-IC...07E (Link to Original site) FC-IC1707F 532 FC-IC1707Z 533 FC-IC1707P 1045 FC-IC1707E 523 Show FC-IC1707 Library FC-IC

  7. Dicty_cDB: FC-IC0095 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC0095 (Link to dictyBase) - G24277 DDB0231825 Contig-U03300-1 FC-IC...tig Contig-U03300-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC... vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value FC-IC0095 (FC-IC0095Q) /CSM/FC-IC/FC-IC...0095F (Link to Original site) FC-IC0095F 288 - - - - - - Show FC-IC0095 Library FC-IC (Link to library) Clone ID FC-IC...0095 (Link to dictyBase) Atlas ID - NBRP ID G24277 dictyBase ID DDB0231825 Link to Con

  8. Dicty_cDB: FC-IC0932 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC0932 (Link to dictyBase) - - - - - (Link to Original site) - - FC-IC...0932Z 239 - - - - Show FC-IC0932 Library FC-IC (Link to library) Clone ID FC-IC0932 (Link to dic...tyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig - Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC... 8.0 %: extracellular, including cell wall 4.0 %: cytoskeletal >> prediction for FC-IC0932 is mit 5' ...te) Representative DNA sequence >FC-IC0932 (FC-IC0932Q) /CSM/FC-IC/FC-IC0932Q.Seq.d/ XXXXXXXXXXTCACCAATGTTGA

  9. Dicty_cDB: FC-IC1144 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC1144 (Link to dictyBase) - - - Contig-U15215-1 FC-IC11...Link to library) Clone ID FC-IC1144 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig C...ontig-U15215-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC...tochondrial 32.0 %: nuclear 16.0 %: cytoplasmic 4.0 %: cytoskeletal >> prediction for FC-IC1144 is mit 5' end seq. ID FC-IC...44P (Link to Original site) FC-IC1144F 715 FC-IC1144Z 681 FC-IC1144P 1376 - - Show FC-IC1144 Library FC-IC (

  10. Dicty_cDB: FC-IC1678 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC1678 (Link to dictyBase) - - - Contig-U16583-1 FC-IC16... (Link to library) Clone ID FC-IC1678 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link t...o Contig Contig-U16583-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC...s Homology vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value FC-IC1678 (FC-IC1678Q) /CSM/FC-IC/FC-IC... 8.0 %: cytoskeletal 8.0 %: mitochondrial >> prediction for FC-IC1678 is nuc 5' end seq. ID FC-IC1678F 5' end seq. >FC-IC

  11. Dicty_cDB: FC-IC0522 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC0522 (Link to dictyBase) - G24294 DDB0169466 Contig-U03376-1 FC-IC... (Link to library) Clone ID FC-IC0522 (Link to dictyBase) Atlas ID - NBRP ID G24294 dic...tnfski*ki rniyklsislc Homology vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value FC-IC0522 (FC-IC... 12.0 %: cytoskeletal 8.0 %: mitochondrial 4.0 %: vacuolar >> prediction for FC-IC...0522E (Link to Original site) FC-IC0522F 215 FC-IC0522Z 215 FC-IC0522P 430 FC-IC0522E 215 Show FC-IC0522 Library FC-IC

  12. Dicty_cDB: FC-IC0335 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC0335 (Link to dictyBase) - - - Contig-U08330-1 FC-IC03... (Link to library) Clone ID FC-IC0335 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link t...o Contig Contig-U08330-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC...0 m_ : 1.00 44.0 %: cytoplasmic 40.0 %: nuclear 12.0 %: mitochondrial 4.0 %: plasma membrane >> prediction for FC-IC...35E (Link to Original site) FC-IC0335F 270 FC-IC0335Z 270 FC-IC0335P 540 FC-IC0335E 270 Show FC-IC0335 Library FC-IC

  13. Dicty_cDB: FC-IC1095 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC1095 (Link to dictyBase) - - - Contig-U10067-1 FC-IC10... (Link to library) Clone ID FC-IC1095 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link t...o Contig Contig-U10067-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC...CSM-cDNA Score E Sequences producing significant alignments: (bits) Value FC-IC1156 (FC-IC...%: cytoskeletal 12.0 %: mitochondrial 4.0 %: vacuolar 4.0 %: Golgi >> prediction for FC-IC

  14. CF750-A33scFv-Fc-Based Optical Imaging of Subcutaneous and Orthotopic Xenografts of GPA33-Positive Colorectal Cancer in Mice

    Directory of Open Access Journals (Sweden)

    Danfeng Wei

    2015-01-01

    Full Text Available Antibody-based imaging agents are attractive as adjuvant diagnostic tools for solid tumors. GPA33 is highly expressed in most human colorectal cancers and has been verified as a diagnostic and therapeutic target. Here, we built an A33scFv-Fc antibody against GPA33 by fusing A33scFv to the Fc fragment of human IgG1 antibodies. The A33scFv-Fc specifically binds GPA33-positive colorectal cancer cells and tumor tissues. After the intravenous injection of mice bearing subcutaneous GPA33-positive LS174T tumor grafts with near-infrared fluorescence probe CF750-labeled A33scFv-Fc (CF750-A33scFv-Fc, high contrast images of the tumor grafts could be kinetically documented within 24 h using an optical imaging system. However, GPA33-negative SMMC7721 tumor grafts could not be visualized by injecting the same amount of CF750-A33scFv-Fc. Moreover, in subcutaneous LS174T tumor-bearing mice, tissue scanning revealed that the CF750-A33scFv-Fc accumulated in the tumor grafts, other than the kidney and liver. In mice with orthotopic tumor transplantations, excrescent LS174T tumor tissues in the colon were successfully removed under guidance by CF750-A33scFv-Fc-based optical imaging. These results indicate that CF750-A33scFv-Fc can target GPA33, suggesting the potential of CF750-A33scFv-Fc as an imaging agent for the diagnosis of colorectal cancer.

  15. Fc receptor inside-out signaling and possible impact on antibody therapy

    NARCIS (Netherlands)

    Brandsma, Arianne M; Jacobino, Shamir R; Meyer, Saskia; ten Broeke, Toine; Leusen, Jeanette H W

    2015-01-01

    Fc receptors (FcR) are expressed on immune cells and bind to the Fc tail of antibodies. This interaction is essential for FcR-mediated signaling and triggering of cellular effector functions. FcR activation is tightly regulated to prevent immune responses by non-antigen bound antibodies or in the ab

  16. Temporal evolution of gamma activity in human cortex during an overt and covert word repetition task

    Directory of Open Access Journals (Sweden)

    Eric eLeuthardt

    2012-05-01

    Full Text Available Different models for cortical processing of speech have been proposed. Classically, the regions participating in language were thought to be modular with a linear sequence of activations. More recently, modern theoretical models have posited a more hierarchical and distributed interaction of anatomic areas for the various stages of speech processing. Traditional imaging techniques can only define the location or time of cortical activation, which impedes the further validation and refinement of these models. In this study, we take advantage of recordings from the surface of the brain (electrocorticography ECoG, which can accurately detect the location and timing of cortical activations, to study the time course of ECoG gamma modulations during an overt and covert word repetition task for different cortical areas. For overt word production, our results show substantial perisylvian cortical activations early in the perceptual phase of the task that were maintained through word articulation. However, this broad activation is attenuated during the expressive phase of covert word repetition. Across the different repetition tasks, the utilization of the different cortical sites within the perisylvian region varied dependent on which stimulus was provided (auditory or visual cue and whether the word was to be spoken or imagined. Taken together, the data supports current models of speech that were defined with functional imaging. Moreover, this study demonstrates that the broad perisylvian speech network variably utilizes these cortical resources based on the nature of the cognitive task.

  17. ON COLLECTIVELY FIXED POINT THEOREMS ON FC-SPACES

    Institute of Scientific and Technical Information of China (English)

    Yongjie Piao

    2010-01-01

    Based on a KKM type theorem on FC-space,some new fixed point theorems for Fan-Browder type are established,and then some collectively fixed point theorems for a family of Φ-maps defined on product space of FC-spaees are given.These results generalize and improve many corresponding results.

  18. Effect of gamma radiation on the expression of mRNA growth factors in glycerol cryopreserved human amniotic membrane.

    Science.gov (United States)

    Yatim, Rusidah Mat; Kannan, Thirumulu Ponnuraj; Ab Hamid, Suzina Sheikh

    2016-12-01

    Human amniotic membrane (HAM) due to its high biocompatibility, low immunogenicity, anti-microbial, anti-viral properties as well as the presence of growth factors has been used in various clinical applications. The growth factors play an important role in wound healing. The current study aimed to explore the effect of 15 kGy gamma radiation dose on selected growth factors and receptors mRNA present in HAM. Eight growth factors, namely, EGF, HGF, KGF, TGF-α, TGF-β1, TGF-β2, TGF-β3 and bFGF and two growth factor receptors, HGFR and KGFR were evaluated in this study. The total RNA was extracted and converted to complimentary DNA using commercial kits. Subsequently, the mRNA expressions of these growth factors were evaluated using real-time PCR and the results were statistically analyzed using REST-MCS software. This study confirmed the presence of these mRNA growth factors and receptors in fresh, glycerol cryopreserved and irradiated glycerol cryopreserved HAM. In glycerol cryopreserved HAM, the results showed up-regulation of HGF and bFGF and down-regulation of EGF, HGFR, KGF, KGFR, TGF-α, TGF-β1, TGF-β2 and TGF-β3 relative to the fresh HAM which acted as the control, whereas in irradiated glycerol cryopreserved HAM, the results showed up-regulation of EGF, HGF, KGF, KGFR, TGF-β1, TGF-β2 and TGF-β3 and down-regulation of HGFR, TGF-α and bFGF relative to the glycerol cryopreserved HAM which acted as the control. However, these mRNA expressions did not show any statistical significant difference compared to the control groups. This study concluded that a dose of 15 kGy of gamma radiation did not affect the mRNA expression for the growth factors' and receptors' in the glycerol cryopreserved HAM.

  19. Essential involvement of cross-talk between IFN-gamma and TNF-alpha in CXCL10 production in human THP-1 monocytes.

    Science.gov (United States)

    Qi, Xu-Feng; Kim, Dong-Heui; Yoon, Yang-Suk; Jin, Dan; Huang, Xue-Zhu; Li, Jian-Hong; Deung, Young-Kun; Lee, Kyu-Jae

    2009-09-01

    Interferon (IFN)-gamma-induced protein 10 (IP-10/CXCL10), a CXC chemokine, has been documented in several inflammatory and autoimmune disorders including atopic dermatitis and bronchial asthma. Although CXCL10 could be induced by IFN-gamma depending on cell type, the mechanisms regulating CXCL10 production following treatment with combination of IFN-gamma and TNF-alpha have not been adequately elucidated in human monocytes. In this study, we showed that TNF-alpha had more potential than IFN-gamma to induce CXCL10 production in THP-1 monocytes. Furthermore, IFN-gamma synergistically enhanced the production of CXCL10 in parallel with the activation of NF-kappaB in TNF-alpha-stimulated THP-1 cells. Blockage of STAT1 or NF-kappaB suppressed CXCL10 production. JAKs inhibitors suppressed IFN-gamma plus TNF-alpha-induced production of CXCL10 in parallel with activation of STAT1 and NF-kappaB, while ERK inhibitor suppressed production of CXCL10 as well as activation of NF-kappaB, but not that of STAT1. IFN-gamma-induced phosphorylation of JAK1 and JAK2, whereas TNF-alpha induced phosphorylation of ERK1/2. Interestingly, IFN-gamma alone had no effect on phosphorylation and degradation of IkappaB-alpha, whereas it significantly promoted TNF-alpha-induced phosphorylation and degradation of IkappaB-alpha. These results suggest that TNF-alpha induces CXCL10 production by activating NF-kappaB through ERK and that IFN-gamma induces CXCL10 production by increasing the activation of STAT1 through JAKs pathways. Of note, TNF-alpha-induced NF-kappaB may be the primary pathway contributing to CXCL10 production in THP-1 cells. IFN-gamma potentiates TNF-alpha-induced CXCL10 production in THP-1 cells by increasing the activation of STAT1 and NF-kappaB through JAK1 and JAK2.

  20. Transcription of human resistin gene involves an interaction of Sp1 with peroxisome proliferator-activating receptor gamma (PPARgamma.

    Directory of Open Access Journals (Sweden)

    Anil K Singh

    Full Text Available BACKGROUND: Resistin is a cysteine rich protein, mainly expressed and secreted by circulating human mononuclear cells. While several factors responsible for transcription of mouse resistin gene have been identified, not much is known about the factors responsible for the differential expression of human resistin. METHODOLOGY/PRINCIPAL FINDING: We show that the minimal promoter of human resistin lies within approximately 80 bp sequence upstream of the transcriptional start site (-240 whereas binding sites for cRel, CCAAT enhancer binding protein alpha (C/EBP-alpha, activating transcription factor 2 (ATF-2 and activator protein 1 (AP-1 transcription factors, important for induced expression, are present within sequences up to -619. Specificity Protein 1(Sp1 binding site (-276 to -295 is also present and an interaction of Sp1 with peroxisome proliferator activating receptor gamma (PPARgamma is necessary for constitutive expression in U937 cells. Indeed co-immunoprecipitation assay demonstrated a direct physical interaction of Sp1 with PPARgamma in whole cell extracts of U937 cells. Phorbol myristate acetate (PMA upregulated the expression of resistin mRNA in U937 cells by increasing the recruitment of Sp1, ATF-2 and PPARgamma on the resistin gene promoter. Furthermore, PMA stimulation of U937 cells resulted in the disruption of Sp1 and PPARgamma interaction. Chromatin immunoprecipitation (ChIP assay confirmed the recruitment of transcription factors phospho ATF-2, Sp1, Sp3, PPARgamma, chromatin modifier histone deacetylase 1 (HDAC1 and the acetylated form of histone H3 but not cRel, C/EBP-alpha and phospho c-Jun during resistin gene transcription. CONCLUSION: Our findings suggest a complex interplay of Sp1 and PPARgamma along with other transcription factors that drives the expression of resistin in human monocytic U937 cells.

  1. Diethyldithiocarbamate inhibits iNOS expression in human lens epithelial cells stimulated by IFN-gamma and LPS

    Institute of Scientific and Technical Information of China (English)

    De-xin LI; Si-ling WANG; Yoshimasa ITO; Jing-hai ZHANG; Chun-fu WU

    2005-01-01

    Aim: To investigate the biological activity of human lens epithelial cells (HLEC) in producing inducible nitric oxide synthase (iNOS) and nitric oxide (NO), and to assesse the effect of diethyldithiocarbamate (DDC) on iNOS mRNA levels and expression of NOS. Methods: The human lens epithelial cell line SRA 01/04 was used in this experiment. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were used to detect, respectively, iNOS mRNA expression and protein production. Results: A costimulation by interferon gamma (IFN-γ) and lipopolysaccharide (LPS) was necessary for iNOS expression in HLEC. The expression of iNOS was significantly reduced in a dosedependent manner by adding DDC from 10 μmol/L to 1 mmol/L. Conclusion:The expression of iNOS in HLEC needs co-stimulation with IFN-γ and LPS and it is inhibited by DDC.

  2. Validation of a simple gas chromatographic-mass spectrometric method for the determination of gamma-butyrolactone in human plasma.

    Science.gov (United States)

    Fukui, Yousuke; Matsusima, Eiji; Muramoto, Kouichi; Nagai, Nobutaka; Ohama, Koso; Yamashita, Kazumasa

    2003-02-25

    A gas chromatographic-mass spectrometric (GC-MS) method is described for the determination of human plasma levels of gamma-butyrolactone (GBL) is described. The method is sensitive and simple. The plasma sample spiked with the internal standard was extracted by dichloromethane (CH(2)Cl(2)) in acidic conditions, and the concentrated organic layer was injected into GC-MS. Because of endogenous GBL in human plasma, the method used a standard calibration curve. The calibration curve was linear from 10 to 1000 ng/ml. The method has been validated for accuracy and precision with the relative error and C.V. for intra- and inter-day within 10%. GBL-spiked plasma samples stored at -80 degrees C were stable for a 3-month period. The stability of plasma samples after three cycles of freezing and thawing and of prepared samples on an autosampler for 48 h were demonstrated. Plasma concentrations of GBL before and after administration of UFT were 24.3+/-14.2 and 84.9+/-22.4 ng/ml, respectively.

  3. Anti-leukemia activity of in vitro-expanded human gamma delta T cells in a xenogeneic Ph+ leukemia model.

    Directory of Open Access Journals (Sweden)

    Gabrielle M Siegers

    Full Text Available Gamma delta T cells (GDTc lyse a variety of hematological and solid tumour cells in vitro and in vivo, and are thus promising candidates for cellular immunotherapy. We have developed a protocol to expand human GDTc in vitro, yielding highly cytotoxic Vgamma9/Vdelta2 CD27/CD45RA double negative effector memory cells. These cells express CD16, CD45RO, CD56, CD95 and NKG2D. Flow cytometric, clonogenic, and chromium release assays confirmed their specific cytotoxicity against Ph(+ cell lines in vitro. We have generated a fluorescent and bioluminescent Ph(+ cell line, EM-2eGFPluc, and established a novel xenogeneic leukemia model. Intravenous injection of EM-2eGFPluc into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG mice resulted in significant dose-dependent bone marrow engraftment; lower levels engrafted in blood, lung, liver and spleen. In vitro-expanded human GDTc injected intraperitoneally were found at higher levels in blood and organs compared to those injected intravenously; GDTc survived at least 33 days post-injection. In therapy experiments, we documented decreased bone marrow leukemia burden in mice treated with GDTc. Live GDTc were found in spleen and bone marrow at endpoint, suggesting the potential usefulness of this therapy.

  4. Simvastatin Sodium Salt and Fluvastatin Interact with Human Gap Junction Gamma-3 Protein.

    Science.gov (United States)

    Marsh, Andrew; Casey-Green, Katherine; Probert, Fay; Withall, David; Mitchell, Daniel A; Dilly, Suzanne J; James, Sean; Dimitri, Wade; Ladwa, Sweta R; Taylor, Paul C; Singer, Donald R J

    2016-01-01

    Finding pleiomorphic targets for drugs allows new indications or warnings for treatment to be identified. As test of concept, we applied a new chemical genomics approach to uncover additional targets for the widely prescribed lipid-lowering pro-drug simvastatin. We used mRNA extracted from internal mammary artery from patients undergoing coronary artery surgery to prepare a viral cardiovascular protein library, using T7 bacteriophage. We then studied interactions of clones of the bacteriophage, each expressing a different cardiovascular polypeptide, with surface-bound simvastatin in 96-well plates. To maximise likelihood of identifying meaningful interactions between simvastatin and vascular peptides, we used a validated photo-immobilisation method to apply a series of different chemical linkers to bind simvastatin so as to present multiple orientations of its constituent components to potential targets. Three rounds of biopanning identified consistent interaction with the clone expressing part of the gene GJC3, which maps to Homo sapiens chromosome 7, and codes for gap junction gamma-3 protein, also known as connexin 30.2/31.3 (mouse connexin Cx29). Further analysis indicated the binding site to be for the N-terminal domain putatively 'regulating' connexin hemichannel and gap junction pores. Using immunohistochemistry we found connexin 30.2/31.3 to be present in samples of artery similar to those used to prepare the bacteriophage library. Surface plasmon resonance revealed that a 25 amino acid synthetic peptide representing the discovered N-terminus did not interact with simvastatin lactone, but did bind to the hydrolysed HMG CoA inhibitor, simvastatin acid. This interaction was also seen for fluvastatin. The gap junction blockers carbenoxolone and flufenamic acid also interacted with the same peptide providing insight into potential site of binding. These findings raise key questions about the functional significance of GJC3 transcripts in the vasculature and

  5. Simvastatin Sodium Salt and Fluvastatin Interact with Human Gap Junction Gamma-3 Protein.

    Directory of Open Access Journals (Sweden)

    Andrew Marsh

    Full Text Available Finding pleiomorphic targets for drugs allows new indications or warnings for treatment to be identified. As test of concept, we applied a new chemical genomics approach to uncover additional targets for the widely prescribed lipid-lowering pro-drug simvastatin. We used mRNA extracted from internal mammary artery from patients undergoing coronary artery surgery to prepare a viral cardiovascular protein library, using T7 bacteriophage. We then studied interactions of clones of the bacteriophage, each expressing a different cardiovascular polypeptide, with surface-bound simvastatin in 96-well plates. To maximise likelihood of identifying meaningful interactions between simvastatin and vascular peptides, we used a validated photo-immobilisation method to apply a series of different chemical linkers to bind simvastatin so as to present multiple orientations of its constituent components to potential targets. Three rounds of biopanning identified consistent interaction with the clone expressing part of the gene GJC3, which maps to Homo sapiens chromosome 7, and codes for gap junction gamma-3 protein, also known as connexin 30.2/31.3 (mouse connexin Cx29. Further analysis indicated the binding site to be for the N-terminal domain putatively 'regulating' connexin hemichannel and gap junction pores. Using immunohistochemistry we found connexin 30.2/31.3 to be present in samples of artery similar to those used to prepare the bacteriophage library. Surface plasmon resonance revealed that a 25 amino acid synthetic peptide representing the discovered N-terminus did not interact with simvastatin lactone, but did bind to the hydrolysed HMG CoA inhibitor, simvastatin acid. This interaction was also seen for fluvastatin. The gap junction blockers carbenoxolone and flufenamic acid also interacted with the same peptide providing insight into potential site of binding. These findings raise key questions about the functional significance of GJC3 transcripts in the

  6. Des-Gamma-Carboxy Prothrombin (DCP Antagonizes the Effects of Gefitinib on Human Hepatocellular Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Yu-Sheng Zhang

    2015-01-01

    Full Text Available Background/Aims: Des-gamma-carboxy prothrombin (DCP, an aberrant prothrombin produced by hepatocellular carcinoma (HCC cells, is known as a marker for HCC. Recent studies indicated that high levels of DCP are associated with the malignant potential of HCC. In this study, we aimed to investigate the association of DCP with gefitinib treatment failure in HCC and whether DCP counteracts gefitinib-induced growth inhibition and apoptosis of HCC. Methods: The experiments were performed in HCC cell lines HepG2 and PLC/PRF/5. The effects of gefitinib on HCC in the presence or absence of DCP were evaluated by the 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT assay. Apoptotic cells were identified by Annexin V-FITC/PI staining. Western blotting was performed to analyze the expressions of molecules related to the apoptotic caspase-dependent pathway and epidermal growth factor receptor (EGFR pathway. Results: Gefitinib inhibited HCC cell proliferation and induced apoptosis in HCC cells. The effects of gefitinib on HCC cells were antagonized by DCP. In the presence of DCP, HCC cells were resistant to the gefitinib-induced inhibition of proliferation and stimulation of apoptosis. DCP prevented the activation of the apoptotic caspase-dependent pathway induced by gefitinib. These antagonistic effects of DCP also arose from its ability to up-regulate EGFR, c-Met and hepatocyte growth factor (HGF in HCC cells. Conclusion: DCP antagonized gefitinib-induced HCC cell growth inhibition by counteracting apoptosis and up-regulating the EGFR pathway. High levels of DCP might thus lead to low response rates or possibly no response to gefitinib in patients with HCC.

  7. Induction of genomic instability in TK6 human lymphoblasts exposed to 137Cs gamma radiation: comparison to the induction by exposure to accelerated 56Fe particles

    Science.gov (United States)

    Evans, Helen H.; Horng, Min-Fen; Ricanati, Marlene; Diaz-Insua, M.; Jordan, Robert; Schwartz, Jeffrey L.

    2003-01-01

    The induction of genomic instability in TK6 human lymphoblasts by exposure to (137)Cs gamma radiation was investigated by measuring the frequency and characteristics of unstable clones isolated approximately 36 generations after exposure. Clones surviving irradiation and control clones were analyzed for 17 characteristics including chromosomal aberrations, growth defects, alterations in response to a second irradiation, and mutant frequencies at the thymidine kinase and Na(+)/K(+) ATPase loci. Putative unstable clones were defined as those that exhibited a significant alteration in one or more characteristics compared to the controls. The frequency and characteristics of the unstable clones were compared in clones exposed to (137)Cs gamma rays or (56)Fe particles. The majority of the unstable clones isolated after exposure to either gamma rays or (56)Fe particles exhibited chromosomal instability. Alterations in growth characteristics, radiation response and mutant frequencies occurred much less often than cytogenetic alterations in these unstable clones. The frequency and complexity of the unstable clones were greater after exposure to (56)Fe particles than to gamma rays. Unstable clones that survived 36 generations after exposure to gamma rays exhibited increases in the incidence of dicentric chromosomes but not of chromatid breaks, whereas unstable clones that survived 36 generations after exposure to (56)Fe particles exhibited increases in both chromatid and chromosome aberrations.

  8. IgG Binding Characteristics of Rhesus Macaque FcγR.

    Science.gov (United States)

    Chan, Ying N; Boesch, Austin W; Osei-Owusu, Nana Y; Emileh, Ali; Crowley, Andrew R; Cocklin, Sarah L; Finstad, Samantha L; Linde, Caitlyn H; Howell, Rebecca A; Zentner, Isaac; Cocklin, Simon; Miles, Adam R; Eckman, Joshua W; Alter, Galit; Schmitz, Joern E; Ackerman, Margaret E

    2016-10-01

    Indian rhesus macaques (Macaca mulatta) are routinely used in preclinical studies to evaluate therapeutic Abs and candidate vaccines. The efficacy of these interventions in many cases is known to rely heavily on the ability of Abs to interact with a set of Ab FcγR expressed on innate immune cells. Yet, despite their presumed functional importance, M. mulatta Ab receptors are largely uncharacterized, posing a fundamental limit to ensuring accurate interpretation and translation of results from studies in this model. In this article, we describe the binding characteristics of the most prevalent allotypic variants of M. mulatta FcγR for binding to both human and M. mulatta IgG of varying subclasses. The resulting determination of the affinity, specificity, and glycan sensitivity of these receptors promises to be useful in designing and evaluating studies of candidate vaccines and therapeutic Abs in this key animal model and exposes significant evolutionary divergence between humans and macaques.

  9. EEG beta suppression and low gamma modulation are different elements of human upright walking

    NARCIS (Netherlands)

    Seeber, M.; Scherer, R.; Wagner, J.; Solis Escalante, T.; Müller-Putz, G.R.

    2014-01-01

    Cortical involvement during upright walking is not well-studied in humans. We analyzed non-invasive electroencephalographic (EEG) recordings from able-bodied volunteers who participated in a robot-assisted gait-training experiment. To enable functional neuroimaging during walking, we applied source

  10. Identification and characterization of human neuronal voltage-gated calcium channel gamma 3 subunit gene

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    By homologous expressed sequence tag (EST) searching,one EST (GenBank: W29095) was obtained,which shows 75% identity in 435 bp overlap with the coding sequence of mouse Cacng2 gene. A 1 545 bp cDNA fragment was obtained from the nested polymerase chain reaction (PCR) and rapid applification of cDNA end (RACE) reaction in the human brain prefrontal cortex cDNA library and the human brain Ready cDNA with the primers designed on W29095. The fragment contained a 948-bp open reading frame (ORF) encoding 315 amino acids,and was named CACNG3. As it was identical to a BAC clone (GenBank: AC004125) from chromosome 16p12-p13.1,the CACNG3 gene was mapped to human chromosome 16p12-p13.1,and the coding region was composed of 4 exons. Reverse transcription PCR (RT-PCR) analysis showed that the CACNG3 gene expressed in human adult brain and fetal brain. Single strand comformation polymorphism (SSCP) analysis was performed in 3 pedigrees with autosomal recessive retinitis pigmentosa,8 pedigrees with autosomal recessive retinitis pigmentosa accompanied by deafness and 2 pedigrees with epilepsy,but no mutation was detected.

  11. Predominant alpha2/beta2/gamma3 AMPK activation during exercise in human skeletal muscle

    DEFF Research Database (Denmark)

    Birk, Jesper Bratz; Wojtaszewski, Jørgen

    2006-01-01

    5'AMP-activated protein kinase (AMPK) is a key regulator of cellular metabolism and is regulated in muscle during exercise. We have previously established that only three of 12 possible AMPK a/ß/¿-heterotrimers are present in human skeletal muscle. Previous studies describe discrepancies between ...

  12. Human keratinocytes produce the complement inhibitor factor H: synthesis is regulated by interferon-gamma.

    NARCIS (Netherlands)

    Timar, K.K.; Pasch, M.C.; Bosch, N.H. van den; Jarva, H.; Junnikkala, S.; Meri, S.; Bos, J.D.; Asghar, S.S.

    2006-01-01

    Locally synthesized complement is believed to play an important role in host defense and inflammation at organ level. In the epidermis, keratinocytes have so far been shown to synthesize two complement components, C3 and factor B. Here, we studied the synthesis of factor H by human keratinocytes. We

  13. Gamma irradiation does not induce detectable changes in DNA methylation directly following exposure of human cells.

    Directory of Open Access Journals (Sweden)

    Christoph Lahtz

    Full Text Available Environmental chemicals and radiation have often been implicated in producing alterations of the epigenome thus potentially contributing to cancer and other diseases. Ionizing radiation, released during accidents at nuclear power plants or after atomic bomb explosions, is a potentially serious health threat for the exposed human population. This type of high-energy radiation causes DNA damage including single- and double-strand breaks and induces chromosomal rearrangements and mutations, but it is not known if ionizing radiation directly induces changes in the epigenome of irradiated cells. We treated normal human fibroblasts and normal human bronchial epithelial cells with different doses of γ-radiation emitted from a cesium 137 ((137Cs radiation source. After a seven-day recovery period, we analyzed global DNA methylation patterns in the irradiated and control cells using the methylated-CpG island recovery assay (MIRA in combination with high-resolution microarrays. Bioinformatics analysis revealed only a small number of potential methylation changes with low fold-difference ratios in the irradiated cells. These minor methylation differences seen on the microarrays could not be verified by COBRA (combined bisulfite restriction analysis or bisulfite sequencing of selected target loci. Our study shows that acute γ-radiation treatment of two types of human cells had no appreciable direct effect on DNA cytosine methylation patterns in exposed cells.

  14. Macrophage Polarization Modulates FcγR- and CD13-Mediated Phagocytosis and Reactive Oxygen Species Production, Independently of Receptor Membrane Expression

    Science.gov (United States)

    Mendoza-Coronel, Elizabeth; Ortega, Enrique

    2017-01-01

    In response to microenvironmental cues, macrophages undergo a profound phenotypic transformation acquiring distinct activation phenotypes ranging from pro-inflammatory (M1) to anti-inflammatory (M2). To study how activation phenotype influences phagocytosis and production of reactive oxygen species (ROS) mediated by receptors for IgG antibodies (Fcγ receptors) and by CD13, human monocyte-derived macrophages were polarized to distinct phenotypes using IFN-γ (Mϕ-IFN-γ), IL-4 (Mϕ-IL-4), or IL-10 (Mϕ-IL-10). Phenotypically, Mϕ-IFN-γ were characterized as CD14+CD80+CD86+ cells, Mϕ-IL-4 as CD209highCD206+CD11b+CD14low, and Mϕ-IL-10 as CD16+CD163+ cells. Compared to non-polarized macrophages, FcγRI expression increased in Mϕ-IFN-γ and Mϕ-IL-10 and FcγRIII expression increased in Mϕ-IL-10. None of the polarizing cytokines modified FcγRII or CD13 expression. Functionally, we found that cytokine-mediated activation significantly and distinctively affected FcγR- and CD13-mediated phagocytosis and ROS generation. Compared to non-polarized macrophages, FcγRI-, FcγRII-, and CD13-mediated phagocytosis was significantly increased in Mϕ-IL-10 and decreased in Mϕ-IFN-γ, although both cytokines significantly upregulated FcγRI expression. IL-10 also increased phagocytosis of Escherichia coli, showing that the effect of IL-10 on macrophage phagocytosis is not specific for a particular receptor. Interestingly, Mϕ-IL-4, which showed poor FcγR- and CD13-mediated phagocytosis, showed very high phagocytosis of E. coli and zymosan. Coupled with phagocytosis, macrophages produce ROS that contribute to microbial killing. As expected, Mϕ-IFN-γ showed significant production of ROS after FcγRI-, FcγRII-, or CD13-mediated phagocytosis. Unexpectedly, we found that Mϕ-IL-10 can also produce ROS after simultaneous stimulation through several phagocytic receptors, as coaggregation of FcγRI/FcγRII/CD13 induced a belated but significant ROS production. Together, these

  15. Dicty_cDB: FC-AS11 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AS11 (Link to dictyBase) - - - - FC-AS11Z (Link to Original site) - - FC-AS...11Z 593 - - - - Show FC-AS11 Library FC (Link to library) Clone ID FC-AS11 (Link to dictyBase) Atlas... ID - NBRP ID - dictyBase ID - Link to Contig - Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AS/FC-AS...ytoskeletal 4.0 %: vacuolar 4.0 %: plasma membrane 4.0 %: peroxisomal >> prediction for FC-AS11 is cyt 5' en...11Q.Seq.d/ Representative seq. ID FC-AS11Z (Link to Original s

  16. Dicty_cDB: FC-AS09 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AS09 (Link to dictyBase) - G24051 DDB0191911 Contig-U15148-1 FC-AS...mic 4.0 %: mitochondrial 4.0 %: plasma membrane >> prediction for FC-AS09 is nuc 5' end seq. ID FC-AS...09F (Link to Original site) FC-AS09F 448 - - - - - - Show FC-AS09 Library FC (Link to library) Clone ID FC-AS...09 (Link to dictyBase) Atlas ID - NBRP ID G24051 dictyBase ID DDB0191911 Link to Contig Contig-U1514...8-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AS/FC-AS09Q.Se

  17. Dicty_cDB: FC-IC0564 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC0564 (Link to dictyBase) - - - Contig-U16036-1 FC-IC05...ink to library) Clone ID FC-IC0564 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Co...ntig-U16036-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC... Score E Sequences producing significant alignments: (bits) Value FC-IC0564 (FC-IC0564Q) /CSM/FC-IC/FC-IC056...lar, including cell wall 4.0 %: cytoskeletal 4.0 %: vacuolar >> prediction for FC-IC0564 is cyt 5' end seq. ID FC-IC

  18. Dicty_cDB: FC-IC0216 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC0216 (Link to dictyBase) - G24287 DDB0201591 Contig-U0... (Link to library) Clone ID FC-IC0216 (Link to dictyBase) Atlas ID - NBRP ID G24287 dictyBase I...D DDB0201591 Link to Contig Contig-U03331-1 Original site URL http://dictycdb.bio... 4.0 %: mitochondrial 4.0 %: peroxisomal >> prediction for FC-IC0216 is nuc 5' end seq. ID FC-IC0216F 5' end seq. >FC-IC...3331-1 - (Link to Original site) FC-IC0216F 315 FC-IC0216Z 315 FC-IC0216P 630 FC-IC0216E 315 Show FC-IC0216 Library FC-IC

  19. Dicty_cDB: FC-IC0364 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC0364 (Link to dictyBase) - - - Contig-U01120-1 - (Link to Original site) FC-IC...(Link to library) Clone ID FC-IC0364 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig ...Contig-U01120-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC...re E Sequences producing significant alignments: (bits) Value FC-IC0364 (FC-IC0364Q) /CSM/FC-IC/FC-IC0364Q.S...24.0 %: nuclear 8.0 %: cytoplasmic 4.0 %: cytoskeletal >> prediction for FC-IC0364 is mit 5' end seq. ID FC-IC

  20. Dicty_cDB: FC-IC0491 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC0491 (Link to dictyBase) - - - Contig-U12373-1 FC-IC04... (Link to library) Clone ID FC-IC0491 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link ...to Contig Contig-U12373-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC...omology vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value FC-IC1285 (FC-IC1285Q) /CSM/FC-IC/FC-IC...ear 12.0 %: cytoskeletal 4.0 %: mitochondrial 4.0 %: peroxisomal >> prediction for FC-IC0491 is cyt 5' end seq. ID FC-IC

  1. Dicty_cDB: FC-IC1222 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC1222 (Link to dictyBase) - - - Contig-U11840-1 FC-IC12...ink to library) Clone ID FC-IC1222 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Co...ntig-U11840-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC...equences producing significant alignments: (bits) Value FC-IC1222 (FC-IC1222Q) /CSM/FC-IC/FC-IC1222Q.Seq.d/ ...%: Golgi 4.0 %: plasma membrane 4.0 %: vesicles of secretory system >> prediction for FC-IC1222 is end 5' end seq. ID FC-IC

  2. Dicty_cDB: FC-IC1569 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC1569 (Link to dictyBase) - - - Contig-U16026-1 FC-IC15... (Link to library) Clone ID FC-IC1569 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link t...o Contig Contig-U16026-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC...vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value FC-IC1569 (FC-IC1569Q) /CSM/FC-IC/FC-IC...les of secretory system >> prediction for FC-IC1569 is end 5' end seq. ID FC-IC

  3. Dicty_cDB: FC-IC1756 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC1756 (Link to dictyBase) - - - Contig-U16026-1 FC-IC17...ink to library) Clone ID FC-IC1756 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Co...ntig-U16026-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC...isomal 4.0 %: extracellular, including cell wall 4.0 %: vacuolar 4.0 %: vesicles of secretory system >> prediction for FC-IC...56P (Link to Original site) FC-IC1756F 156 FC-IC1756Z 284 FC-IC1756P 420 - - Show FC-IC1756 Library FC-IC (L

  4. Dicty_cDB: FC-IC1753 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC1753 (Link to dictyBase) - - - - - (Link to Original site) - - FC-IC...1753Z 284 - - - - Show FC-IC1753 Library FC-IC (Link to library) Clone ID FC-IC1753 (Link to dic...tyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig - Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC...te) Representative DNA sequence >FC-IC1753 (FC-IC1753Q) /CSM/FC-IC/FC-IC1753Q.Seq.d/ XXXXXXXXXXTGTGTTTTGGAGT...vllvlgwlelvllvle*lelvwlvlvwlelv*lelgllfl eykeehp*ihllgnmnmklqiyldilslrnhmk Homology vs CSM-cDNA Score E Sequences producing signific

  5. Dicty_cDB: FC-IC0417 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC0417 (Link to dictyBase) - - - Contig-U03353-1 FC-IC04...17F (Link to Original site) FC-IC0417F 285 - - - - - - Show FC-IC0417 Library FC-IC (Link to library) Clone ID FC-IC... %: cytoskeletal 4.0 %: vacuolar >> prediction for FC-IC0417 is nuc 5' end seq. ID FC-IC...0417 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U03353-1 Original site URL http://dic...tycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC0417Q.Seq.d/ Representative seq. ID FC-IC

  6. LyGDI, a novel SHIP-interacting protein, is a negative regulator of FcγR-mediated phagocytosis.

    Science.gov (United States)

    Mehta, Payal; Wavreille, Anne-Sophie; Justiniano, Steven E; Marsh, Rachel L; Yu, Jianhua; Burry, Richard W; Jarjoura, David; Eubank, Timothy; Caligiuri, Michael A; Butchar, Jonathan P; Tridandapani, Susheela

    2011-01-01

    SHIP and SHIP-2 are inositol phosphatases that regulate FcγR-mediated phagocytosis through catalytic as well as non-catalytic mechanisms. In this study we have used two-dimensional fluorescence difference gel electrophoresis (DIGE) analysis to identify downstream signaling proteins that uniquely associate with SHIP or SHIP-2 upon FcγR clustering in human monocytes. We identified LyGDI as a binding partner of SHIP, associating inducibly with the SHIP/Grb2/Shc complex. Immunodepletion and competition experiments with recombinant SHIP domains revealed that Grb2 and the proline-rich domain of SHIP were necessary for SHIP-LyGDI association. Functional studies in primary human monocytes showed that LyGDI sequesters Rac in the cytosol, preventing it from localizing to the membrane. Consistent with this, suppression of LyGDI expression resulted in significantly enhanced FcγR-mediated phagocytosis.

  7. LyGDI, a novel SHIP-interacting protein, is a negative regulator of FcγR-mediated phagocytosis.

    Directory of Open Access Journals (Sweden)

    Payal Mehta

    Full Text Available SHIP and SHIP-2 are inositol phosphatases that regulate FcγR-mediated phagocytosis through catalytic as well as non-catalytic mechanisms. In this study we have used two-dimensional fluorescence difference gel electrophoresis (DIGE analysis to identify downstream signaling proteins that uniquely associate with SHIP or SHIP-2 upon FcγR clustering in human monocytes. We identified LyGDI as a binding partner of SHIP, associating inducibly with the SHIP/Grb2/Shc complex. Immunodepletion and competition experiments with recombinant SHIP domains revealed that Grb2 and the proline-rich domain of SHIP were necessary for SHIP-LyGDI association. Functional studies in primary human monocytes showed that LyGDI sequesters Rac in the cytosol, preventing it from localizing to the membrane. Consistent with this, suppression of LyGDI expression resulted in significantly enhanced FcγR-mediated phagocytosis.

  8. Antibody with an engineered Fc region as a therapeutic agent against dengue virus infection.

    Science.gov (United States)

    Ramadhany, Ririn; Hirai, Itaru; Sasaki, Tadahiro; Ono, Ken-ichiro; Ramasoota, Pongrama; Ikuta, Kazuyoshi; Kurosu, Takeshi

    2015-12-01

    Antibody-dependent enhancement (ADE) of dengue virus (DENV) infectivity is thought to play a crucial role in severe dengue disease. It occurs when pre-existing sub-neutralizing anti-DENV antibody (Ab) produced from a primary infection encounters a DENV serotype different from that of the initial infection and forms immune complexes, which enable the efficient infection of Fcγ receptor-bearing cells. However, the exact role played by Abs during a secondary infection of patients remains unknown. We previously obtained a broadly cross-reactive neutralizing IgG1 human monoclonal anti-DENV envelope (E) Ab (HuMAb) D23-1G7C2-IgG1 from a DENV-infected patient; however, D23-1G7C2-IgG1 had ADE activity. With the aim of being able to reduce the ADE activity, we exchanged the Fc region of D23-1G7C2 to generate Abs bearing each of the three other IgG subclasses (IgG2-4). In addition, N297A, a mutation known to reduce the affinity of the IgG1 Fc region for Fcγ receptors, was introduced into D23-1G7C2-IgG1. Swapping D23-1G7C2-IgG1 to IgG2 or IgG4 subclasses reduced ADE activity in FcγRI and FcγRII-bearing THP-1 cells. By contrast, in FcγRII-bearing K562 cells, the change to IgG2 increased ADE activity. Introducing the N297A mutation into D23-1G7C2-IgG1 resulted in a marked reduction in ADE activity in both cell types. Compared to D23-1G7C2-IgG1, D23-1G7C2-IgG1-N297A was less protective in IFN-α/β/γ receptor knockout mice infected with a lethal dose of recombinant chimeric DENV, carrying prME of DENV-2 in Japanese encephalitis virus (80% vs. 40% survival, respectively). These observations provide valuable information regarding the use of recombinant Abs as therapeutics.

  9. Significance of soluble Fc epsilon receptor II (sFc epsilon RII/CD23) in serum and possible application of sFc epsilon RII for the prevention of allergic reactions.

    Science.gov (United States)

    Suemura, M; Kikutani, H; Sugiyama, K; Uchibayashi, N; Aitani, M; Kuritani, T; Barsumian, E L; Yamatodani, A; Kishimoto, T

    1991-01-01

    The significance of sFc epsilon RII in IgE-mediated allergic disease was examined. sFc epsilon RII in serum was found to decrease following clinical improvement, suggesting sFc epsilon RII in serum may be an indicator of allergic diseases. Significant proportions of sFc epsilon RII in serum were present as complexes with IgE in normals as well as in atopic patients, and these complexes were more prominent in the former than in the latter group. From these observations, attempts were made to inhibit IgE-mediated allergic reactions in vitro employing recombinant sFc epsilon RII. sFc epsilon RII inhibited IgE-binding as well as IgE-mediated release of chemical mediators from Fc epsilon RI and Fc epsilon RII expressing cells. These results show the functional significance of sFc epsilon RII in the negative regulation of IgE-mediated allergic reactions.

  10. IgG2 Fc structure and the dynamic features of the IgG CH2-CH3 interface.

    Science.gov (United States)

    Teplyakov, Alexey; Zhao, Yonghong; Malia, Thomas J; Obmolova, Galina; Gilliland, Gary L

    2013-11-01

    The analyses of two human IgG2 Fc structures, determined in different crystal forms, and the comparison with IgG1 Fc structures reveals molecular features that are involved in accommodating and stabilizing structural conformations. In the IgG2 Fc structures relative positions of the CH2 domains with respect to the CH3 domains vary significantly from those observed for IgG1 Fc structures in similar unit cells. The analysis reveals that the movement of the CH2 domain in all of the Fc structures results from a pivoting around a highly conserved ball-and-socket-like joint in which the CH2 L251 side chain (the ball) interacts with a pocket (the socket) formed by CH3 M428, H429, E430, and H435. Despite the change in positioning of the CH2 and CH3 domains, conserved hydrogen bonds and electrostatic interactions are retained, stabilizing the Fc domain interface. In the high resolution IgG2 and IgG1 Fc structures the position and number of water molecules, and water networks bridging the two domains differ significantly because of the difference in positions of CH2 relative to CH3. At the domain interface, only CH2 T339 in IgG2 differs from alanine found in IgG1 and IgG4. This residue's side chain influences the water structure at the interface by interacting either directly or through a bridging water molecule with D376 in the CH3 BC loop. Thus, the analyses of the IgG2 Fc structures and their comparisons with IgG1 Fc structures reveals similar, but distinctly different dynamic CH2-CH3 interfaces that can accommodate a wide range of CH2-CH3 conformations, that in conjunction with the amino acid residues in the hinge region, may influence FcγR effector function profiles. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. Defining the Binding Region in Factor H to Develop a Therapeutic Factor H-Fc Fusion Protein against Non-Typeable Haemophilus influenzae.

    Science.gov (United States)

    Wong, Sandy M; Shaughnessy, Jutamas; Ram, Sanjay; Akerley, Brian J

    2016-01-01

    Non-typeable Haemophilus influenzae (NTHi) cause a range of illnesses including otitis media, sinusitis, and exacerbation of chronic obstructive pulmonary disease, infections that contribute to the problem of antibiotic resistance and are themselves often intractable to standard antibiotic treatment regimens. We investigated a strategy to exploit binding of the complement inhibitor Factor H (FH) to NTHi as a functional target for an immunotherapeutic containing the NTHi binding domain of FH fused to the Fc domain of IgG1. Chimeric proteins containing the regions that most FH-binding bacteria use to engage human FH, domains 6 and 7 (FH6,7/Fc) and/or 18 through 20 (FH18-20/Fc), were evaluated for binding to NTHi. FH6,7/Fc bound strongly to each of seven NTHi clinical isolates tested and efficiently promoted complement-mediated killing by normal human serum. FH18-20/Fc bound weakly to three of the strains but did not promote complement dependent killing. Outer-membrane protein P5 has been implicated in FH binding by NTHi, and FH6,7/Fc binding was greatly diminished in five of seven P5 deficient isogenic mutant strains tested, implicating an alternative FH binding protein in some strains. Binding of FH18-20/Fc was decreased in the P5 mutant of one strain. A murine model was used to evaluate potential therapeutic application of FH6,7/Fc. FH6,7/Fc efficiently promoted binding of C3 to NTHi exposed to mouse serum, and intranasal delivery of FH6,7/Fc resulted in significantly enhanced clearance of NTHi from the lung. Moreover, a P5 deficient mutant was attenuated for survival in the lung model, suggesting that escape mutants lacking P5 would be less likely to replace strains susceptible to FH6,7/Fc. These results provide evidence for the potential utility of FH6,7/Fc as a therapeutic against NTHi lung infection. FH binding is a common property of many respiratory tract pathogens and FH/Fc chimeras may represent promising alternative or adjunctive therapeutics against such

  12. Defining the binding region in factor H to develop a therapeutic factor H-Fc fusion protein against nontypeable Haemophilus influenzae

    Directory of Open Access Journals (Sweden)

    Sandy M. Wong

    2016-04-01

    Full Text Available Nontypeable Haemophilus influenzae (NTHi cause a range of illnesses including otitis media, sinusitis, and exacerbation of chronic obstructive pulmonary disease, infections that contribute to the problem of antibiotic resistance and are themselves often intractable to standard antibiotic treatment regimens. We investigated a strategy to exploit binding of the complement inhibitor Factor H (FH to NTHi as a functional target for an immunotherapeutic containing the NTHi binding domain of FH fused to the Fc domain of IgG1. Chimeric proteins containing the regions that most FH-binding bacteria use to engage human FH, domains 6 and 7 (FH6,7/Fc and/or 18 through 20 (FH18-20/Fc, were evaluated for binding to NTHi. FH6,7/Fc bound strongly to each of seven NTHi clinical isolates tested and efficiently promoted complement-mediated killing by normal human serum. FH18-20/Fc bound weakly to three of the strains but did not promote complement dependent killing. Outer-membrane protein P5 has been implicated in FH binding by NTHi, and FH6,7/Fc binding was greatly diminished in five of seven P5 deficient isogenic mutant strains tested, implicating an alternative FH binding protein in some strains. Binding of FH18-20/Fc was decreased in the P5 mutant of one strain. A murine model was used to evaluate potential therapeutic application of FH6,7/Fc. FH6,7/Fc efficiently promoted binding of C3 to NTHi exposed to mouse serum, and intranasal delivery of FH6,7/Fc resulted in significantly enhanced clearance of NTHi from the lung. Moreover, a P5 deficient mutant was attenuated for survival in the lung model, suggesting that escape mutants lacking P5 would be less likely to replace strains susceptible to FH6,7/Fc. These results provide evidence for the potential utility of FH6,7/Fc as a therapeutic against NTHi lung infection. FH binding is a common property of many respiratory tract pathogens and FH/Fc chimeras may represent promising alternative or adjunctive

  13. Short-Latency Median-Nerve Somatosensory-Evoked Potentials and Induced Gamma-Oscillations in Humans

    Science.gov (United States)

    Fukuda, Miho; Nishida, Masaaki; Juhasz, Csaba; Muzik, Otto; Sood, Sandeep; Chugani, Harry T.; Asano, Eishi

    2008-01-01

    Recent studies have suggested that cortical gamma-oscillations are tightly linked with various forms of physiological activity. In the present study, the dynamic changes of intracranially recorded median-nerve somatosensory-evoked potentials (SEPs) and somatosensory-induced gamma-oscillations were animated on a three-dimensional MR image, and the…

  14. Dicty_cDB: FC-IC1182 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC1182 (Link to dictyBase) - - - Contig-U07877-1 FC-IC11...Link to library) Clone ID FC-IC1182 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig C...ontig-U07877-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC...kl*s*swfrcctnsycwsffksfncsw **psycmwtlan* Homology vs CSM-cDNA Score E Sequences producing significant align...les of secretory system >> prediction for FC-IC1182 is cyt 5' end seq. ID FC-IC

  15. Dicty_cDB: FC-IC1622 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC1622 (Link to dictyBase) - - - Contig-U16527-1 FC-IC16... (Link to library) Clone ID FC-IC1622 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link t...o Contig Contig-U16527-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC...k tnntssnhpktnntssnhsktnhtssnysktnntspnntspninsysrldfipnsnkrsr tmrimlgic**cst*isl...ghllvvqhlnlvtllnm Homology vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value FC-IC1760 (FC-IC

  16. Unlike PPAR{gamma}, PPAR{alpha} or PPAR{beta}/{delta} activation does not promote human monocyte differentiation toward alternative macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Bouhlel, Mohamed Amine [Univ Lille Nord de France, F-59000 Lille (France); Inserm U545, F-59000 Lille (France); UDSL, F-59000 Lille (France); Institut Pasteur de Lille, F-59019 Lille (France); Brozek, John [Genfit, Loos (France); Derudas, Bruno [Univ Lille Nord de France, F-59000 Lille (France); Inserm U545, F-59000 Lille (France); UDSL, F-59000 Lille (France); Institut Pasteur de Lille, F-59019 Lille (France); Zawadzki, Christophe; Jude, Brigitte [Inserm ERI-9 and Equipe d' Accueil 2693, IFR114, Universite de Lille, Lille (France); Staels, Bart, E-mail: bart.staels@pasteur-lille.fr [Univ Lille Nord de France, F-59000 Lille (France); Inserm U545, F-59000 Lille (France); UDSL, F-59000 Lille (France); Institut Pasteur de Lille, F-59019 Lille (France); Chinetti-Gbaguidi, Giulia [Univ Lille Nord de France, F-59000 Lille (France); Inserm U545, F-59000 Lille (France); UDSL, F-59000 Lille (France); Institut Pasteur de Lille, F-59019 Lille (France)

    2009-08-28

    Macrophages adapt their response to micro-environmental signals. While Th1 cytokines promote pro-inflammatory M1 macrophages, Th2 cytokines promote an 'alternative' anti-inflammatory M2 macrophage phenotype. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors expressed in macrophages where they control the inflammatory response. It has been shown that PPAR{gamma} promotes the differentiation of monocytes into anti-inflammatory M2 macrophages in humans and mice, while a role for PPAR{beta}/{delta} in this process has been reported only in mice and no data are available for PPAR{alpha}. Here, we show that in contrast to PPAR{gamma}, expression of PPAR{alpha} and PPAR{beta}/{delta} overall does not correlate with the expression of M2 markers in human atherosclerotic lesions, whereas a positive correlation with genes of lipid metabolism exists. Moreover, unlike PPAR{gamma}, PPAR{alpha} or PPAR{beta}/{delta} activation does not influence human monocyte differentiation into M2 macrophages in vitro. Thus, PPAR{alpha} and PPAR{beta}/{delta} do not appear to modulate the alternative differentiation of human macrophages.

  17. Functional characterization of an scFv-Fc antibody that immunotherapeutically targets the common cancer cell surface proteoglycan CSPG4.

    Science.gov (United States)

    Wang, Xinhui; Katayama, Akihiro; Wang, Yangyang; Yu, Ling; Favoino, Elvira; Sakakura, Koichi; Favole, Alessandra; Tsuchikawa, Takahiro; Silver, Susan; Watkins, Simon C; Kageshita, Toshiro; Ferrone, Soldano

    2011-12-15

    Cell surface chondroitin sulfate proteoglycan 4 (CSPG4) is an attractive target for antibody-based cancer immunotherapy because of its role in tumor cell biology, its high expression on malignant cells including cancer-initiating cells, and its restricted distribution in normal tissues. The clinical use of CSPG4 has been hampered by the lack of a CSPG4-specific chimeric, humanized, or fully human monoclonal antibody. To overcome this limitation, we generated a CSPG4-specific fully human single-chain antibody termed scFv-FcC21 and characterized its specificity and antitumor activity. Viable CSPG4(+) melanoma cells were used in a screen of a human scFv phage display library that included CDR3 engineered to optimize antibody binding sites. The scFv antibody isolated was then recombinantly engineered with a human immunoglobulin G1 Fc region to construct the fully human antibody scFv-FcC21, which recognized tumors of neuroectodermal origin, various types of carcinomas, mesotheliomas, and sarcomas as well as myeloid leukemias. scFv-FcC21 inhibited in vitro growth and migration of tumor cells and in vivo growth of human tumor xenografts. These effects were mediated by inhibition of the activation of extracellular signal-regulated kinase and focal adhesion kinase signaling pathways that are critical for tumor cell growth and migration, respectively. Our findings define the CSPG4-specific fully human scFv-FcC21 antibody as a candidate therapeutic agent to target the many types of tumors that express CSPG4.

  18. Gamma Knife

    Science.gov (United States)

    ... Resources Professions Site Index A-Z Gamma Knife Gamma Knife® is a radiation therapy that uses computerized treatment planning software to help ... sparing surrounding tissue. If you're scheduled for radiation therapy using Gamma Knife®, a treatment team consisting of a radiation ...

  19. Gamma Knife

    Science.gov (United States)

    ... News Physician Resources Professions Site Index A-Z Gamma Knife Gamma Knife® is a radiation therapy that uses computerized treatment ... If you're scheduled for radiation therapy using Gamma Knife®, a treatment team consisting of a radiation oncologist, ...

  20. Generalized constrained multiobjective games in locally FC-uniform spaces

    Institute of Scientific and Technical Information of China (English)

    DING Xie-ping; LEE Chin-san; YAO Jen-chih

    2008-01-01

    A new class of generalized constrained multiobjective games is introduced and studied in locally FC-uniform spaces without convexity structure where the number of players may be finite or infinite and all payoff functions get their values in an infinite-dimensional space.By using a Himmelberg type fixed point theorem in locally FC-uniform spaces due to author,some existence theorems of weak Pareto equilibria for the generalized constrained multiobjective games are established in locally FC-uniform spaces.These theorems improve,unify and generalize the corresponding results in recent literatares.

  1. The Tat protein of human immunodeficiency virus-1 enhances hepatitis C virus replication through interferon gamma-inducible protein-10

    Directory of Open Access Journals (Sweden)

    Qu Jing

    2012-04-01

    Full Text Available Abstract Background Co-infection with human immunodeficiency virus-1 (HIV-1 and hepatitis C virus (HCV is associated with faster progression of liver disease and an increase in HCV persistence. However, the mechanism by which HIV-1 accelerates the progression of HCV liver disease remains unknown. Results HIV-1/HCV co-infection is associated with increased expression of interferon gamma-induced protein-10 (IP-10 mRNA in peripheral blood mononuclear cells (PBMCs. HCV RNA levels were higher in PBMCs of patients with HIV-1/HCV co-infection than in patients with HCV mono-infection. HIV-1 Tat and IP-10 activated HCV replication in a time-dependent manner, and HIV-1 Tat induced IP-10 production. In addition, the effect of HIV-1 Tat on HCV replication was blocked by anti-IP-10 monoclonal antibody, demonstrating that the effect of HIV-1 Tat on HCV replication depends on IP-10. Taken together, these results suggest that HIV-1 Tat protein activates HCV replication by upregulating IP-10 production. Conclusions HIV-1/HCV co-infection is associated with increased expression of IP-10 mRNA and replication of HCV RNA. Furthermore, both HIV-1 Tat and IP-10 activate HCV replication. HIV-1 Tat activates HCV replication by upregulating IP-10 production. These results expand our understanding of HIV-1 in HCV replication and the mechanism involved in the regulation of HCV replication mediated by HIV-1 during co-infection.

  2. Energetics of Glutathione Binding to Human Eukaryotic Elongation Factor 1 Gamma: Isothermal Titration Calorimetry and Molecular Dynamics Studies.

    Science.gov (United States)

    Tshabalala, Thabiso N; Tomescu, Mihai-Silviu; Prior, Allan; Balakrishnan, Vijayakumar; Sayed, Yasien; Dirr, Heini W; Achilonu, Ikechukwu

    2016-12-01

    The energetics of ligand binding to human eukaryotic elongation factor 1 gamma (heEF1γ) was investigated using reduced glutathione (GSH), oxidised glutathione (GSSG), glutathione sulfonate and S-hexylglutathione as ligands. The experiments were conducted using isothermal titration calorimetry, and the findings were supported using computational studies. The data show that the binding of these ligands to heEF1γ is enthalpically favourable and entropically driven (except for the binding of GSSG). The full length heEF1γ binds GSSG with lower affinity (K d = 115 μM), with more hydrogen-bond contacts (ΔH = -73.8 kJ/mol) and unfavourable entropy (-TΔS = 51.7 kJ/mol) compared to the glutathione transferase-like N-terminus domain of heEF1γ, which did not show preference to any specific ligand. Computational free binding energy calculations from the 10 ligand poses show that GSSG and GSH consistently bind heEF1γ, and that both ligands bind at the same site with a folded bioactive conformation. This study reveals the possibility that heEF1γ is a glutathione-binding protein.

  3. Interferon-gamma improves impaired dentinogenic and immunosuppressive functions of irreversible pulpitis-derived human dental pulp stem cells.

    Science.gov (United States)

    Sonoda, Soichiro; Yamaza, Haruyoshi; Ma, Lan; Tanaka, Yosuke; Tomoda, Erika; Aijima, Reona; Nonaka, Kazuaki; Kukita, Toshio; Shi, Songtao; Nishimura, Fusanori; Yamaza, Takayoshi

    2016-01-18

    Clinically, irreversible pulpitis is treated by the complete removal of pulp tissue followed by replacement with artificial materials. There is considered to be a high potential for autologous transplantation of human dental pulp stem cells (DPSCs) in endodontic treatment. The usefulness of DPSCs isolated from healthy teeth is limited. However, DPSCs isolated from diseased teeth with irreversible pulpitis (IP-DPSCs) are considered to be suitable for dentin/pulp regeneration. In this study, we examined the stem cell potency of IP-DPSCs. In comparison with healthy DPSCs, IP-DPSCs expressed lower colony-forming capacity, population-doubling rate, cell proliferation, multipotency, in vivo dentin regeneration, and immunosuppressive activity, suggesting that intact IP-DPSCs may be inadequate for dentin/pulp regeneration. Therefore, we attempted to improve the impaired in vivo dentin regeneration and in vitro immunosuppressive functions of IP-DPSCs to enable dentin/pulp regeneration. Interferon gamma (IFN-γ) treatment enhanced in vivo dentin regeneration and in vitro T cell suppression of IP-DPSCs, whereas treatment with tumor necrosis factor alpha did not. Therefore, these findings suggest that IFN-γ may be a feasible modulator to improve the functions of impaired IP-DPSCs, suggesting that autologous transplantation of IFN-γ-accelerated IP-DPSCs might be a promising new therapeutic strategy for dentin/pulp tissue engineering in future endodontic treatment.

  4. Shifting Phases for Patchy Particles - Effect of mutagenesis and chemical modification on the phase diagram of human gamma D crystallin

    Science.gov (United States)

    McManus, Jennifer J.; James, Susan; McNamara, Ruth; Quinn, Michelle

    2014-03-01

    Single mutations in human gamma D crystallin (HGD), a protein found in the eye lens are associated with several childhood cataracts. Phase diagrams for several of these protein mutants have been measured and reveal that phase boundaries are shifted compared with the native protein, leading to condensation of protein in a physiologically relevant regime. Using HGD as a model protein, we have constructed phase diagrams for double mutants of the protein, incorporating two single amino acid substitutions for which phase diagrams are already known. In doing so, the characteristics of each of the single mutations are maintained but both are now present in the same protein particle. While these proteins are not of interest physiologically, this strategy allows the controlled synthesis of nano-scale patchy particles in which features associated with a known phase behavior can be included. It can also provide a strategy for the controlled crystallisation of proteins. Phase boundaries also change after the chemical modification of the protein, through the covalent attachment of fluorescent labels, for example, and this will also be discussed. The authors acknowledge Science Foundation Ireland Stokes Lectureship and Grant 11/RFP.1/PHY/3165. The authors also acknowledge the Irish Research Council and the John and Pat Hume Scholarship.

  5. Two mutational hotspots in the interleukin-2 receptor {gamma} chain gene causing human X-linked severe combined immunodeficiency

    Energy Technology Data Exchange (ETDEWEB)

    Pepper, A.E.; Puck, J.M. [National Institutes of Health, Bethesda, MD (United States); Buckley, R.H. [and others

    1995-09-01

    Human severe combined immunodeficiency (SCID), a syndrome of profoundly impaired cellular and humoral immunity, is most commonly caused by mutations in the X-linked gene for interleukin-2 (IL-2) receptor {gamma} chain (IL2RG). For mutational analysis of IL2RG in males with SCID, SSCP screening was followed by DNA sequencing. Of 40 IL2RG mutations found in unrelated SCID patients, 6 were point mutations at the CpG dinucleotide at cDNA 690-691, encoding amino acid R226. This residue lies in the extracellular domain of the protein in a region not previously recognized to be significantly conserved in the cytokine receptor gene family, 11 amino acids upstream from the highly conserved WSXWS motif. Three additional instances of mutation at another CpG dinucleotide at cDNA 879 produced a premature termination signal in the intracellular domain of IL2RG, resulting in loss of the SH2-homologous intracellular domain known to be essential for signaling from the IL-2 receptor complex. Mutations at these two hotspots constitute >20% of the X-linked SCID mutations found by our group and a similar proportion of all reported IL2RG mutations. 41 refs., 5 figs., 1 tab.

  6. Gamma radiation at a human relevant low dose rate is genotoxic in mice

    Science.gov (United States)

    Graupner, Anne; Eide, Dag M.; Instanes, Christine; Andersen, Jill M.; Brede, Dag A.; Dertinger, Stephen D.; Lind, Ole C.; Brandt-Kjelsen, Anicke; Bjerke, Hans; Salbu, Brit; Oughton, Deborah; Brunborg, Gunnar; Olsen, Ann K.

    2016-09-01

    Even today, 70 years after Hiroshima and accidents like in Chernobyl and Fukushima, we still have limited knowledge about the health effects of low dose rate (LDR) radiation. Despite their human relevance after occupational and accidental exposure, only few animal studies on the genotoxic effects of chronic LDR radiation have been performed. Selenium (Se) is involved in oxidative stress defence, protecting DNA and other biomolecules from reactive oxygen species (ROS). It is hypothesised that Se deficiency, as it occurs in several parts of the world, may aggravate harmful effects of ROS-inducing stressors such as ionising radiation. We performed a study in the newly established LDR-facility Figaro on the combined effects of Se deprivation and LDR γ exposure in DNA repair knockout mice (Ogg1-/-) and control animals (Ogg1+/-). Genotoxic effects were seen after continuous radiation (1.4 mGy/h) for 45 days. Chromosomal damage (micronucleus), phenotypic mutations (Pig-a gene mutation of RBCCD24-) and DNA lesions (single strand breaks/alkali labile sites) were significantly increased in blood cells of irradiated animals, covering three types of genotoxic activity. This study demonstrates that chronic LDR γ radiation is genotoxic in an exposure scenario realistic for humans, supporting the hypothesis that even LDR γ radiation may induce cancer.

  7. The use of gamma-irradiation and ultraviolet-irradiation in the preparation of human melanoma cells for use in autologous whole-cell vaccines

    Directory of Open Access Journals (Sweden)

    Denlinger Chadrick E

    2008-12-01

    Full Text Available Abstract Background Human cancer vaccines incorporating autologous tumor cells carry a risk of implantation and subsequent metastasis of viable tumor cells into the patient who is being treated. Despite the fact that the melanoma cell preparations used in a recent vaccine trial (Mel37 were gamma-irradiated (200 Gy, approximately 25% of the preparations failed quality control release criteria which required that the irradiated cells incorporate 3H-thymidine at no more than 5% the level seen in the non-irradiated cells. We have, therefore, investigated ultraviolet (UV-irradiation as a possible adjunct to, or replacement for gamma-irradiation. Methods Melanoma cells were gamma- and/or UV-irradiated. 3H-thymidine uptake was used to assess proliferation of the treated and untreated cells. Caspase-3 activity and DNA fragmentation were measured as indicators of apoptosis. Immunohistochemistry and Western blot analysis was used to assess antigen expression. Results UV-irradiation, either alone or in combination with gamma-irradiation, proved to be extremely effective in controlling the proliferation of melanoma cells. In contrast to gamma-irradiation, UV-irradiation was also capable of inducing significant levels of apoptosis. UV-irradiation, but not gamma-irradiation, was associated with the loss of tyrosinase expression. Neither form of radiation affected the expression of gp100, MART-1/MelanA, or S100. Conclusion These results indicate that UV-irradiation may increase the safety of autologous melanoma vaccines, although it may do so at the expense of altering the antigenic profile of the irradiated tumor cells.

  8. Structure and dynamics of IgE-receptor interactions: FcεRI and CD23/FcεRII.

    Science.gov (United States)

    Sutton, Brian J; Davies, Anna M

    2015-11-01

    Immunoglobulin E (IgE) is well known for its role in allergic disease, the manifestations of which are mediated through its two Fc receptors, FcεRI and CD23 (FcεRII). IgE and its interactions with these receptors are therefore potential targets for therapeutic intervention, and exciting progress has been made in this direction. Furthermore, recent structural studies of IgE-Fc, the two receptors, and of their complexes, have revealed a remarkable degree of plasticity at the IgE-CD23 interface and an even more remarkable degree of dynamic flexibility within the IgE molecule. Indeed, there is allosteric communication between the two receptor-binding sites, which we now know are located at some distance from each other in IgE-Fc (at opposite ends of the Cε3 domain). The conformational changes associated with FcεRI and CD23 binding to IgE-Fc ensure that their interactions are mutually incompatible, and it may be that this functional imperative has driven IgE to evolve such a dynamic structure. Appreciation of these new structural data has revised our view of IgE structure, shed light on the co-evolution of antibodies and their receptors, and may open up new therapeutic opportunities.

  9. Crystal structure of the HSV-1 Fc receptor bound to Fc reveals a mechanism for antibody bipolar bridging.

    Directory of Open Access Journals (Sweden)

    Elizabeth R Sprague

    2006-06-01

    Full Text Available Herpes simplex virus type-1 expresses a heterodimeric Fc receptor, gE-gI, on the surfaces of virions and infected cells that binds the Fc region of host immunoglobulin G and is implicated in the cell-to-cell spread of virus. gE-gI binds immunoglobulin G at the basic pH of the cell surface and releases it at the acidic pH of lysosomes, consistent with a role in facilitating the degradation of antiviral antibodies. Here we identify the C-terminal domain of the gE ectodomain (CgE as the minimal Fc-binding domain and present a 1.78-angstroms CgE structure. A 5-angstroms gE-gI/Fc crystal structure, which was independently verified by a theoretical prediction method, reveals that CgE binds Fc at the C(H2-C(H3 interface, the binding site for several mammalian and bacterial Fc-binding proteins. The structure identifies interface histidines that may confer pH-dependent binding and regions of CgE implicated in cell-to-cell spread of virus. The ternary organization of the gE-gI/Fc complex is compatible with antibody bipolar bridging, which can interfere with the antiviral immune response.

  10. Ubiquitin/proteasome pathway regulates levels of retinoic acid receptor gamma and retinoid X receptor alpha in human keratinocytes.

    Science.gov (United States)

    Boudjelal, M; Wang, Z; Voorhees, J J; Fisher, G J

    2000-04-15

    Repeated exposure of human skin to solar UV radiation leads to premature aging (photoaging) and skin cancer. UV-induced skin damage can be ameliorated by all-trans retinoic acid treatment. The actions of retinoic acid in skin keratinocytes are mediated primarily by nuclear retinoic acid receptor gamma (RARgamma) and retinoid X receptor alpha (RXRalpha). We found that exposure of cultured primary human keratinocytes to UV irradiation (30 mJ/cm2) substantially reduced (50-90%) RARgamma and RXRalpha mRNA and protein within 8 h. The rates of disappearance of RARgamma and RXRalpha proteins after UV exposure or treatment with the protein synthesis inhibitor cycloheximide were similar. UV irradiation did not increase the rate of breakdown of RARgamma or RXRalpha but rather reduced their rate of synthesis. The addition of proteasome inhibitors MG132 and LLvL, but not the lysosomal inhibitor E64, prevented loss of RARgamma and RXRalpha proteins after exposure of keratinocytes to either UV radiation or cycloheximide. Soluble extracts from nonirradiated or UV-irradiated keratinocytes possessed similar levels of proteasome activity that degraded RARgamma and RXRalpha proteins in vitro. Furthermore, RARgamma and RXRalpha were polyubiquitinated in intact cells. RXRalpha was found to contain two proline, glutamate/aspartate, serine, and threonine (PEST) motifs, which confer rapid turnover of many short-lived regulatory proteins that are degraded by the ubiquitin/proteasome pathway. However, the PEST motifs in RXRalpha did not function to regulate its stability, because deletion of the PEST motifs individually or together did not alter ubiquitination or proteasome-mediated degradation of RXRalpha. These results demonstrate that loss of RARgamma and RXRalpha proteins after UV irradiation results from degradation via the ubiquitin/proteasome pathway. Taken together, the data here indicate that ubiquitin/proteasome-mediated breakdown is an important mechanism regulating the levels of

  11. Characterizing low dose and dose rate effects in rodent and human neural stem cells exposed to proton and gamma irradiation

    Directory of Open Access Journals (Sweden)

    Bertrand P. Tseng

    2013-01-01

    Full Text Available Past work has shown that exposure to gamma rays and protons elicit a persistent oxidative stress in rodent and human neural stem cells (hNSCs. We have now adapted these studies to more realistic exposure scenarios in space, using lower doses and dose rates of these radiation modalities, to further elucidate the role of radiation-induced oxidative stress in these cells. Rodent neural stem and precursor cells grown as neurospheres and human neural stem cells grown as monolayers were subjected to acute and multi-dosing paradigms at differing dose rates and analyzed for changes in reactive oxygen species (ROS, reactive nitrogen species (RNS, nitric oxide and superoxide for 2 days after irradiation. While acute exposures led to significant changes in both cell types, hNSCs in particular, exhibited marked and significant elevations in radiation-induced oxidative stress. Elevated oxidative stress was more significant in hNSCs as opposed to their rodent counterparts, and hNSCs were significantly more sensitive to low dose exposures in terms of survival. Combinations of protons and γ-rays delivered as lower priming or higher challenge doses elicited radioadaptive changes that were associated with improved survival, but in general, only under conditions where the levels of reactive species were suppressed compared to cells irradiated acutely. Protective radioadaptive effects on survival were eliminated in the presence of the antioxidant N-acetylcysteine, suggesting further that radiation-induced oxidative stress could activate pro-survival signaling pathways that were sensitive to redox state. Data corroborates much of our past work and shows that low dose and dose rate exposures elicit significant changes in oxidative stress that have functional consequences on survival.

  12. The contribution of cell surface FcRn in monoclonal antibody serum uptake from the intestine in suckling rat pups

    Directory of Open Access Journals (Sweden)

    Philip R Cooper

    2014-10-01

    Full Text Available The neonatal Fc Receptor (FcRn in intestinal epithelium is the primary mechanism for transfer of maternal immunoglobulin G (IgG from suckled milk to serum; but the factors contributing to the rapid uptake of IgG are poorly understood. These studies help to determine the contribution of cell-surface FcRn in IgG uptake in 2-week old rat pups by varying local pH and binding conditions. Variants of a human wild-type IgG monoclonal antibody (mAb WT were assessed for binding affinity (KD to rat (rFcRn at pH6.0 and subsequent off-rate at pH7.4 (1/s by Surface Plasmon Resonance. Selected mAbs were administered intra-intestinally in isofluorane-anesthetized 2-week rat pups. Full-length mAb in serum was quantified by immunoassay, (rFcRn mRNA expression by RT-PCR, and mAb epithelial localization was visualized by immunohistochemistry. After duodenal administration, serum levels of mAb variants correlated with their rFcRn off-rate at pH7.4, but not their affinity at pH6.0. The greatest serum levels of IgG were measured when mAb was administered in the duodenum where rFcRn mRNA expression is greatest, and was increased further by duodenal administration in pH6.0 buffer. More intense human IgG immunostaining was detected in epithelium than the same variant administered at higher pH. These data suggest an increased contribution for cell-surface receptor. We conclude that, in the neonate duodenum, receptor off-rates are as important as affinities for FcRn mediated uptake, and cell surface binding of IgG to rFcRn plays contributes to IgG uptake alongside pinocytosis; both of which responsible for increased IgG uptake.

  13. In Silico Prediction of Human Pathogenicity in the gamma-Proteobacteria

    DEFF Research Database (Denmark)

    Andreatta, Massimo; Nielsen, Morten; Aarestrup, Frank Møller

    2010-01-01

    strongly needed to mine the fast growing number of genome sequences and assess in a rapid and reliable way the pathogenicity of novel bacteria. Methodology/Principal Findings: We describe a new in silico method for the prediction of bacterial pathogenicity, based on the identification in microbial genomes...... to be able to separate pathogenic organisms from non-pathogenic ones. Using traditional experimental methods for this purpose can be very costly and time-consuming, and also uncertain since animal models are not always good predictors for pathogenicity in humans. Bioinformatics-based methods are therefore....... A set of protein families that differentiate pathogenic and non-pathogenic strains were identified, including families of yet uncharacterized proteins that are suggested to be involved in bacterial pathogenicity....

  14. Effects of Gamma Irradiation on Bacterial Microflora Associated with Human Amniotic Membrane

    Directory of Open Access Journals (Sweden)

    Fahmida Binte Atique

    2013-01-01

    Full Text Available Human amniotic membrane is considered a promising allograft material for the treatment of ocular surface reconstruction, burns, and other skin defects. In order to avoid the transmission of any diseases, grafts should be perfectly sterile. Twenty-five amniotic sacs were collected to determine the microbiological quality of human amniotic membrane, to analyze the radiation sensitivity pattern of the microorganism, and to detect the radiation decimal reduction dose (D10 values. All the samples were found to be contaminated, and the bioburden was ranged from 3.4×102 to 1.2×105 cfu/g. Initially, a total fifty bacterial isolates were characterized according to their cultural, morphological, and biochemical characteristics and then tested for the radiation sensitivity in an incremental series of radiation doses from 1 to 10 KGy. The results depict gradual decline in bioburden with incline of radiation doses. Staphylococcus spp. were the most frequently isolated bacterial contaminant in tissue samples (44%. The D10 values of the bacterial isolates were ranged from 0.6 to 1.27 KGy. Streptococcus spp. were found to be the highest radioresistant strain with the radiation sterilization dose (RSD of 11.4 KGy for a bioburden level of 1000. To compare the differences, D10 values were also calculated by graphical evaluations of the data with two of the representative isolates of each bacterial species which showed no significant variations. Findings of this study indicate that lower radiation dose is quite satisfactory for the sterilization of amniotic membrane grafts. Therefore, these findings would be helpful to predict the efficacy of radiation doses for the processing of amniotic membrane for various purposes.

  15. Effects of Gamma Irradiation on Bacterial Microflora Associated with Human Amniotic Membrane

    Science.gov (United States)

    Binte Atique, Fahmida; Ahmed, Kazi Tahsin; Asaduzzaman, S. M.; Hasan, Kazi Nadim

    2013-01-01

    Human amniotic membrane is considered a promising allograft material for the treatment of ocular surface reconstruction, burns, and other skin defects. In order to avoid the transmission of any diseases, grafts should be perfectly sterile. Twenty-five amniotic sacs were collected to determine the microbiological quality of human amniotic membrane, to analyze the radiation sensitivity pattern of the microorganism, and to detect the radiation decimal reduction dose (D10) values. All the samples were found to be contaminated, and the bioburden was ranged from 3.4 × 102 to 1.2 × 105 cfu/g. Initially, a total fifty bacterial isolates were characterized according to their cultural, morphological, and biochemical characteristics and then tested for the radiation sensitivity in an incremental series of radiation doses from 1 to 10 KGy. The results depict gradual decline in bioburden with incline of radiation doses. Staphylococcus spp. were the most frequently isolated bacterial contaminant in tissue samples (44%). The D10 values of the bacterial isolates were ranged from 0.6 to 1.27 KGy. Streptococcus spp. were found to be the highest radioresistant strain with the radiation sterilization dose (RSD) of 11.4 KGy for a bioburden level of 1000. To compare the differences, D10 values were also calculated by graphical evaluations of the data with two of the representative isolates of each bacterial species which showed no significant variations. Findings of this study indicate that lower radiation dose is quite satisfactory for the sterilization of amniotic membrane grafts. Therefore, these findings would be helpful to predict the efficacy of radiation doses for the processing of amniotic membrane for various purposes. PMID:24063009

  16. mBAND analysis of chromosome aberrations in human epithelial cells induced by gamma-rays and secondary neutrons of low dose rate.

    Science.gov (United States)

    Hada, M; Gersey, B; Saganti, P B; Wilkins, R; Cucinotta, F A; Wu, H

    2010-08-14

    Human risks from chronic exposures to both low- and high-LET radiation are of intensive research interest in recent years. In the present study, human epithelial cells were exposed in vitro to gamma-rays at a dose rate of 17 mGy/h or secondary neutrons of 25 mGy/h. The secondary neutrons have a broad energy spectrum that simulates the Earth's atmosphere at high altitude, as well as the environment inside spacecrafts like the Russian MIR station and the International Space Station (ISS). Chromosome aberrations in the exposed cells were analyzed using the multicolor banding in situ hybridization (mBAND) technique with chromosome 3 painted in 23 colored bands that allows identification of both inter- and intrachromosome exchanges including inversions. Comparison of present dose responses between gamma-rays and neutron irradiations for the fraction of cells with damaged chromosome 3 yielded a relative biological effectiveness (RBE) value of 26+/-4 for the secondary neutrons. Our results also revealed that secondary neutrons of low dose rate induced a higher fraction of intrachromosome exchanges than gamma-rays, but the fractions of inversions observed between these two radiation types were indistinguishable. Similar to the previous findings after acute radiation exposures, most of the inversions observed in the present study were accompanied by other aberrations. The fractions of complex type aberrations and of unrejoined chromosomal breakages were also found to be higher in the neutron-exposed cells than after gamma-rays. We further analyzed the location of the breaks involved in chromosome aberrations along chromosome 3, and observed hot spots after gamma-ray, but not neutron, exposures.

  17. Dicty_cDB: FC-AS02 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC (Link to library) FC-AS02 (Link to dictyBase) - - - Contig-U16382-1 FC-AS02Z (Li...nk to Original site) - - FC-AS02Z 561 - - - - Show FC-AS02 Library FC (Link to library) Clone ID FC-AS02 (Link to dictyBase) Atlas....00 m3a: 0.00 m3b: 0.00 m_ : 1.00 44.0 %: cytoplasmic 44.0 %: nuclear 8.0 %: cytoskeletal 4.0 %: mitochondri... ID - NBRP ID - dictyBase ID - Link to Contig Contig-U16382-1 Original site URL http:/.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AS/FC-AS02Q.Seq.d/ Representative seq. ID FC-AS

  18. Dicty_cDB: FC-IC0430 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC0430 (Link to dictyBase) - - - Contig-U16271-1 FC-IC04... (Link to library) Clone ID FC-IC0430 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link t...o Contig Contig-U16271-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC...ology vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value FC-IC0430 (FC-IC...l 4.0 %: mitochondrial 4.0 %: endoplasmic reticulum >> prediction for FC-IC0430 is cyt 5' end seq. ID FC-IC0430F 5' end seq. >FC-IC

  19. Dicty_cDB: FC-IC0649 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC0649 (Link to dictyBase) - - - Contig-U16566-1 FC-IC06...ink to library) Clone ID FC-IC0649 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Co...ntig-U16566-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC... Score E Sequences producing significant alignments: (bits) Value FC-IC0649 (FC-IC0649Q) /CSM/FC-IC/FC-IC064...ory system 4.0 %: cytoskeletal 4.0 %: vacuolar 4.0 %: Golgi 4.0 %: mitochondrial >> prediction for FC-IC0649

  20. Dicty_cDB: FC-IC0993 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC0993 (Link to dictyBase) - G24303 DDB0220699 Contig-U08379-1 FC-IC... (Link to library) Clone ID FC-IC0993 (Link to dictyBase) Atlas ID - NBRP ID G24303 dic...gi 4.0 %: plasma membrane 4.0 %: vesicles of secretory system 4.0 %: extracellular, including cell wall >> prediction for FC-IC...0993E (Link to Original site) FC-IC0993F 526 FC-IC0993Z 526 FC-IC0993P 1032 FC-IC0993E 516 Show FC-IC0993 Library FC-IC...tyBase ID DDB0220699 Link to Contig Contig-U08379-1 Original site URL http://dic

  1. Dicty_cDB: FC-IC0027 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC0027 (Link to dictyBase) - - - Contig-U08312-1 FC-IC00...27F (Link to Original site) FC-IC0027F 578 - - - - - - Show FC-IC0027 Library FC-IC (Link to library) Clone ID FC-IC...0005Q) /CSM/FC-IC/FC-IC0005Q.Seq.d/ 531 e-150 own update 2001.11.18 Homology vs DNA Score E Sequences producing signific...les of secretory system 4.0 %: endoplasmic reticulum >> prediction for FC-IC0027 is nuc 5' end seq. ID FC-IC...0027 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U08312-1 Original site URL http://dic

  2. Dicty_cDB: FC-IC0758 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC0758 (Link to dictyBase) - - - Contig-U07877-1 FC-IC07...ink to library) Clone ID FC-IC0758 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Co...ntig-U07877-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC...cldr Homology vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value FC-IC0758 (FC-IC0758Q) /CSM/FC-IC/FC-IC...0 %: mitochondrial 4.0 %: plasma membrane 4.0 %: vesicles of secretory system >> prediction for FC-IC

  3. Dicty_cDB: FC-IC1688 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC1688 (Link to dictyBase) - - - Contig-U16382-1 - (Link... to Original site) - - FC-IC1688Z 288 - - - - Show FC-IC1688 Library FC-IC (Link to library) Clone ID FC-IC1688 (Link to dic...IV Homology vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value FC-IC1688 (FC-IC...:FC-AG0... 414 e-112 1 ( AU275195 ) Dictyostelium discoideum gamete cDNA clone:FC-IC1... 414 e-112 1 ( AU272515 ) Dic...tyostelium discoideum gamete cDNA clone:FC-IC1... 414 e-112 1 ( AU272386 ) Dic

  4. Dicty_cDB: FC-IC1440 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC1440 (Link to dictyBase) - - - Contig-U15453-1 FC-IC14... (Link to library) Clone ID FC-IC1440 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link t...o Contig Contig-U15453-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC...re E Sequences producing significant alignments: (bits) Value FC-IC1440 (FC-IC1440Q) /CSM/FC-IC/FC-IC...clear 36.0 %: cytoplasmic 8.0 %: cytoskeletal 8.0 %: mitochondrial 4.0 %: vacuolar >> prediction for FC-IC14

  5. Dicty_cDB: FC-IC0501 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC0501 (Link to dictyBase) - - - Contig-U16382-1 FC-IC05... (Link to library) Clone ID FC-IC0501 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link t...o Contig Contig-U16382-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC...y vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value FC-IC0501 (FC-IC...les of secretory system >> prediction for FC-IC0501 is nuc 5' end seq. ID FC-IC0501F 5' end seq. >FC-IC

  6. Dicty_cDB: FC-IC0954 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC0954 (Link to dictyBase) - - - Contig-U16382-1 FC-IC09... (Link to library) Clone ID FC-IC0954 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link t...o Contig Contig-U16382-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC...SM-cDNA Score E Sequences producing significant alignments: (bits) Value FC-IC0954 (FC-IC0954Q) /CSM/FC-IC/FC-IC.../FC-IC1696Q.Seq.d/ 761 0.0 own update 2004.12.25 Homology vs DNA Score E Sequences producing signific

  7. Dicty_cDB: FC-IC1149 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC1149 (Link to dictyBase) - - - Contig-U16271-1 - (Link to Original site) FC-IC...1149F 272 - - - - - - Show FC-IC1149 Library FC-IC (Link to library) Clone ID FC-IC1149 (Link to dic...tyostelium discoideum gamete cDNA clone:FC-IC1... 519 e-143 1 ( AU272187 ) Dictyostelium discoideu...m gamete cDNA clone:FC-IC1... 519 e-143 1 ( AU272185 ) Dictyostelium discoideum gamete cDNA clone:FC-IC1... ...elium discoideum gamete cDNA clone:FC-IC1... 517 e-143 1 ( AU275192 ) Dictyostelium discoideum gamete cDNA clone:FC-IC

  8. Dicty_cDB: FC-IC1236 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC1236 (Link to dictyBase) - - - Contig-U12406-1 - (Link... to Original site) - - FC-IC1236Z 497 - - - - Show FC-IC1236 Library FC-IC (Link to library) Clone ID FC-IC1236 (Link to dic...xxxlvxslcfxw Homology vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value FC-IC1236 (FC-IC1236Q) /CSM/FC-IC...tyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U12406-1 Original site URL http://dic...tycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC1236Q.Seq.d/ Representative se

  9. Dicty_cDB: FC-IC0067 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC0067 (Link to dictyBase) - - - Contig-U16382-1 FC-IC00...67F (Link to Original site) FC-IC0067F 267 - - - - - - Show FC-IC0067 Library FC-IC (Link to library) Clone ID FC-IC...tochondrial 8.0 %: Golgi 4.0 %: vesicles of secretory system 4.0 %: endoplasmic reticulum >> prediction for FC-IC...0067 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U16382-1 Original site URL http://dic...tycdb.biol.tsukuba.ac.jp/CSM/FC-IC/FC-IC0067Q.Seq.d/ Representative seq. ID FC-IC

  10. Dicty_cDB: FC-IC0162 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC0162 (Link to dictyBase) - - - Contig-U10099-1 FC-IC01...62F (Link to Original site) FC-IC0162F 358 - - - - - - Show FC-IC0162 Library FC-IC (Link to library) Clone ID FC-IC...DGPDDEALPVVDGPDDEALP-- - Homology vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value FC-IC0162 (FC-IC...%: cytoskeletal 4.0 %: Golgi 4.0 %: cytoplasmic >> prediction for FC-IC0162 is end 5' end seq. ID FC-IC...0162 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U10099-1 Original site URL http://dic

  11. Dicty_cDB: FC-IC1178 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available FC-IC (Link to library) FC-IC1178 (Link to dictyBase) - - - Contig-U16026-1 - (Link... to Original site) - - FC-IC1178Z 442 - - - - Show FC-IC1178 Library FC-IC (Link to library) Clone ID FC-IC1178 (Link to dic... vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value FC-IC1178 (FC-IC1178Q) /CSM/FC-IC/FC-IC...les of secretory system >> prediction for FC-IC1178 is end 5' end seq. ID - 5' end seq. - Length ...tyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U16026-1 Original site URL http://dic

  12. Purification of chimeric heavy chain monoclonal antibody EG2-hFc using hydrophobic interaction membrane chromatography: an alternative to protein-A affinity chromatography.

    Science.gov (United States)

    Sadavarte, Rahul; Spearman, Maureen; Okun, Natalie; Butler, Michael; Ghosh, Raja

    2014-06-01

    Heavy chain monoclonal antibodies are being considered as alternative to whole-IgG monoclonal antibodies for certain niche applications. Protein-A chromatography which is widely used for purifying IgG monoclonal antibodies is also used for purifying heavy chain monoclonal antibodies as these molecules possess fully functional Fc regions. However, the acidic conditions used to elute bound antibody may sometimes also leach protein-A, which is immunotoxic. Low pH conditions also tend to make the mAb molecules unstable and prone to aggregation. Moreover, protein-A affinity chromatography does not remove aggregates already present in the feed. Hydrophobic interaction membrane chromatography (or HIMC) has already been studied as an alternative to protein-A chromatography for purifying whole-IgG monoclonal antibodies. This paper describes the use of HIMC for capturing a humanized chimeric heavy chain monoclonal antibody (EG2-hFC). Binding and eluting conditions were suitably optimized using pure EG2-hFC. Based on this, an HIMC method was developed for capture of EG2-hFC directly from cell culture supernatant. The EG2-hFc purity obtained in this single-step process was high. The glycan profiles of protein-A and HIMC purified monoclonal antibody samples were similar, clearly demonstrating that both techniques captured similarly glycosylated population of EG2-hFc. Moreover, this technique was able to resolve aggregates from monomeric form of the EG2-hFc.

  13. Modulation of the allergen-induced human IgE response in Hu-SCID mice: inhibitory effect of human recombinant IFN-gamma and allergen-derived lipopeptide.

    Science.gov (United States)

    Duez, C; Gras-Masse, H; Hammad, H; Akoum, H; Didierlaurent, A; André, C; Tonnel, A B; Pestel, J

    2001-01-01

    We have previously established a model to study the in vivo human IgE response using humanized SCID mice. Allergic SCID mice were obtained following intraperitoneal injection with mononuclear cells from Dermatophagoides pteronyssinus (Dpt)-sensitive patients, and sensitization by Dpt allergen intraperitoneal injection (immunization) or Dpt aerosol (inhalation). Human serum IgE was measured in allergic SCID mice after administration of human recombinant IFN-gamma or the lipopeptide LP 52-71 (derived from peptide p52-71 from Der p 1, Dpt major allergen, coupled to a lipophilic moiety), during the immunization or the inhalation phase. IFN-gamma inhibited human IgE production when given at the time of immunization, but not during inhalation. This effect was long-lasting as Dpt aerosol, given one month after immunization and IFN-gamma administration, failed to increase IgE levels. Unlike Dpt or p52-71, LP 52-71 failed to induce human IgE production at day 14 and 21 after its injection, but did inhibit the development of the IgE response after a secondary Dpt-challenge. Moreover, LP 52-71 administration 14 days after Dpt inhalation decreased IgE levels, in contrast to peptide 52-71, which increased IgE levels. Thus, taken together these results indicate that the development of the human IgE response in allergic SCID mice can be modulated by modified allergen and a Th1 cytokine.

  14. The high affinity IgE receptor (FcεRI) expression and function in airway smooth muscle.

    Science.gov (United States)

    Redhu, Naresh Singh; Gounni, Abdelilah S

    2013-02-01

    The airway smooth muscle (ASM) is no longer considered as merely a contractile apparatus and passive recipient of growth factors, neurotransmitters and inflammatory mediators signal but a critical player in the perpetuation and modulation of airway inflammation and remodeling. In recent years, a molecular link between ASM and IgE has been established through Fc epsilon receptors (FcεRs) in modulating the phenotype and function of these cells. Particularly, the expression of high affinity IgE receptor (FcεRI) has been noted in primary human ASM cells in vitro and in vivo within bronchial biopsies of allergic asthmatic subjects. The activation of FcεRI on ASM cells suggests a critical yet almost completely ignored network which may modulate ASM cell function in allergic asthma. This review is intended to provide a historical perspective of IgE effects on ASM and highlights the recent updates in the expression and function of FcεRI, and to present future perspectives of activation of this pathway in ASM cells.

  15. EEG beta suppression and low gamma modulation are different elements of human upright walking.

    Science.gov (United States)

    Seeber, Martin; Scherer, Reinhold; Wagner, Johanna; Solis-Escalante, Teodoro; Müller-Putz, Gernot R

    2014-01-01

    Cortical involvement during upright walking is not well-studied in humans. We analyzed non-invasive electroencephalographic (EEG) recordings from able-bodied volunteers who participated in a robot-assisted gait-training experiment. To enable functional neuroimaging during walking, we applied source modeling to high-density (120 channels) EEG recordings using individual anatomy reconstructed from structural magnetic resonance imaging scans. First, we analyzed amplitude differences between the conditions, walking and upright standing. Second, we investigated amplitude modulations related to the gait phase. During active walking upper μ (10-12 Hz) and β (18-30 Hz) oscillations were suppressed [event-related desynchronization (ERD)] compared to upright standing. Significant β ERD activity was located focally in central sensorimotor areas for 9/10 subjects. Additionally, we found that low γ (24-40 Hz) amplitudes were modulated related to the gait phase. Because there is a certain frequency band overlap between sustained β ERD and gait phase related modulations in the low γ range, these two phenomena are superimposed. Thus, we observe gait phase related amplitude modulations at a certain ERD level. We conclude that sustained μ and β ERD reflect a movement related state change of cortical excitability while gait phase related modulations in the low γ represent the motion sequence timing during gait. Interestingly, the center frequencies of sustained β ERD and gait phase modulated amplitudes were identified to be different. They may therefore be caused by different neuronal rhythms, which should be taken under consideration in future studies.

  16. Up-Regulation of CCR5 and CXCR4 Expression on Human Monocytes by Interferon Gamma

    Institute of Scientific and Technical Information of China (English)

    陆韵; 刘祖强; 陈应华

    2003-01-01

    Chemokine receptors, mainly CCR5 and CXCR4, have been proved to be the important coreceptors in HIV-1 entry.HIV-1 disease progression is, in general, characterized by an initial predominance of CCR5 using macrophage tropic, non-syncytium-inducing (NSI) isolates, switching later to CXCR4 using T-cell tropic, syncytium-inducing (SI) isolates.How this shift occurs and how the shift can be controlled are still unclear.Since patients with rapid decline of T cell counts have constantly high levels of IFN-γ in the sera and lymphoid nodes, we investigated the influence of this cytokine on the expression of the HIV-1 coreceptors CCR5 and CXCR4 on the cell surfaces of human monocytic cell line U937 and promonocyte NB4.IFN-γ could intensively enhance the expression of both, while a low level of CCR5 expression was detected in two cell lines before stimulation.The results of semiquantitative RT-PCR also confirm the up-regulation.As the newly generated X4-strains have been demonstrated to be insensitive to chemokine in some reports, IFN-γ may play an important role in selecting CXCR4-used strains.

  17. GENERALIZED VECTOR VARIATIONAL-TYPE INEQUALITIES IN FC-SPACES

    Institute of Scientific and Technical Information of China (English)

    FANG Min; DING Xie-ping

    2006-01-01

    A class of generalized vector variational-type inequality problems (GVVTIP) are studied in FC-spaces, which includes the most of vector equilibrium problems, vector variational inequality problems, generalized vector equilibrium problems and generalized vector variational inequality problem as special cases. By using F-KKM theorem,some new existence results for GVVTIP are established in noncompact FC-space. As consequences, some recent known results in literature are obtained under much weaker assumption.

  18. Effects of tumor necrosis factor-alpha and interferon-gamma on expressions of matrix metalloproteinase-2 and -9 in human bladder cancer cells.

    Science.gov (United States)

    Shin, K Y; Moon, H S; Park, H Y; Lee, T Y; Woo, Y N; Kim, H J; Lee, S J; Kong, G

    2000-10-31

    We have investigated the effects of tumor necrosis factor-alpha (TNF-alpha) and interferon (INF-gamma), the potent Bacillus Calmette-Guerin (BCG)-induced cytokines on the production of MMP-2, MMP-9, TIMP-1, TIMP-2 and MT1-MMP in high grade human bladder cancer cell lines, T-24, J-82 and HT-1376 cell lines. MMP-2 expression and activity were decreased in T-24 cells treated with both cytokines in a dose dependent manner. However, J-82 cells treated with TNF-alpha and INF-gamma revealed dose dependent increases of MMP-9 expression and activity with similar baseline expression and activity of MMP-2. HT-1376 cells after exposure to TNF-alpha only enhanced the expression and activity of MMP-9. These results indicate that TNF-alpha and INF-gamma could regulate the production of MMP-2 or MMP-9 on bladder cancer cells and their patterns of regulation are cell specific. Furthermore, this diverse response of bladder cancer cells to TNF-alpha and INF-gamma suggests that BCG immunotherapy may enhance the invasiveness of bladder cancer in certain conditions with induction of MMPs.

  19. EEG beta suppression and low gamma modulation are different elements of human upright walking

    Directory of Open Access Journals (Sweden)

    Martin eSeeber

    2014-07-01

    Full Text Available Cortical involvement during upright walking is not well-studied in humans. We analyzed non-invasive electroencephalographic (EEG recordings from able-bodied volunteers who participated in a robot-assisted gait-training experiment. To enable functional neuroimaging during walking, we applied source modeling to high-density (120 channels EEG recordings using individual anatomy reconstructed from structural magnetic resonance imaging (MRI scans. First, we analyzed amplitude differences between the conditions, walking and upright standing. Second, we investigated amplitude modulations related to the gait phase. During active walking upper µ (10-12Hz and β (18-30Hz oscillations were suppressed (event-related desynchronization, ERD compared to upright standing. Significant β ERD activity was located focally in central sensorimotor areas for 9/10 subjects. Additionally, we found that low γ (24-40Hz amplitudes were modulated related to the gait phase. Because there is a certain frequency band overlap between sustained β ERD and gait phase related modulations in the low γ range, these two phenomena are superimposed. Thus, we observe gait phase related amplitude modulations at a certain ERD level. We conclude that sustained µ and β ERD reflect a movement related state change of cortical excitability while gait phase related modulations in the low γ represent the motion sequence timing during gait. Interestingly, the center frequencies of sustained β ERD and gait phase modulated amplitudes were identified to be different. They may therefore be caused by different neuronal rhythms, which should be taken under consideration in future studies.

  20. Factor structure of the human gamma band oscillatory response to visual (contrast) stimulation.

    Science.gov (United States)

    Carozzo, Simone; De Carli, Fabrizio; Beelke, Manolo; Saturno, Moreno; Garbarino, Sergio; Martello, Cristina; Sannita, Walter G

    2004-07-01

    Visual contrast stimulation evokes in man an oscillatory mass response at approximately 20.0-35.0 Hz, consistent with stimulus-dependent synchronous oscillations in multiunit animal recordings from visual cortex, but shorter in duration and phase-locked to stimulus. A factor analysis was applied to characterize the signal structure under stimulus conditions inducing an oscillatory response and to identify possible subcomponents in normal volunteers. Contrast stimuli were gratings with a sinusoidal luminance profile (9.0 degrees; 5.0 cycle/degree; 80% contrast; reversal 1.06 Hz). The amplitude spectrum of the signal was computed by Discrete Fourier Transform (DFT) and the oscillatory response was separated from the corresponding visually evoked potential (VEP) by DFT high-pass filter at 19.0 Hz. Nine consecutive waves were identified in all subjects (60 volunteers), with amplitudes/latencies consistent with normative studies. A factor analysis was computed 1- in the frequency domain, on the amplitude values of the signal components (2 Hz resolution), and 2- in the time domain, on the latencies/amplitudes of the averaged VEP and oscillatory responses. (1) Two non-overlapping factors accounted for the approximately 2-20.0 and approximately 20.0-40.0 Hz signal components, with separation of the approximately 20.0-35.0 Hz oscillatory response from low frequency VEPs. (2) Two factors on latencies and one factor on amplitudes (independent of each other and from those of VEPs) accounted for the average approximately 20.0-35.0 Hz oscillatory response. The factor structure further indicates an oscillatory structure and some independence from conventional VEPs of the human oscillatory response to contrast, with a separation between the oscillatory response early and late waves possibly reflecting functional differences.

  1. Consistency Property of Finite FC-Normal Logic Programs

    Institute of Scientific and Technical Information of China (English)

    Yi-Song Wang; Ming-Yi Zhang; Yu-Ping Shen

    2007-01-01

    Marek's forward-chaining construction is one of the important techniques for investigating the non-monotonic reasoning. By introduction of consistency property over a logic program, they proposed a class of logic programs, FC-normal programs, each of which has at least one stable model. However, it is not clear how to choose one appropriate consistency property for deciding whether or not a logic program is FC-normal. In this paper, we firstly discover that, for any finite logic program ∏, there exists the least consistency property LCon(∏) over ∏, which just depends on ∏ itself, such that, ∏ is FC-normal if and only if ∏ is FC-normal with respect to (w.r.t.) LCon(∏). Actually, in order to determine the FC-normality of a logic program, it is sufficient to check the monotonic closed sets in LCon(∏) for all non-monotonic rules, that is LFC(∏). Secondly, we present an algorithm for computing LFC(∏). Finally, we reveal that the brave reasoning task and cautious reasoning task for FC-normal logic programs are of the same difficulty as that of normal logic programs.

  2. Alpha, beta and gamma electrocorticographic rhythms in somatosensory, motor, premotor and prefrontal cortical areas differ in movement execution and observation in humans.

    Science.gov (United States)

    Babiloni, Claudio; Del Percio, Claudio; Vecchio, Fabrizio; Sebastiano, Fabio; Di Gennaro, Giancarlo; Quarato, Pier P; Morace, Roberta; Pavone, Luigi; Soricelli, Andrea; Noce, Giuseppe; Esposito, Vincenzo; Rossini, Paolo Maria; Gallese, Vittorio; Mirabella, Giovanni

    2016-01-01

    In the present study, we tested the hypothesis that both movement execution and observation induce parallel modulations of alpha, beta, and gamma electrocorticographic (ECoG) rhythms in primary somatosensory (Brodmann area 1-2, BA1-2), primary motor (BA4), ventral premotor (BA6), and prefrontal (BA44 and BA45, part of putative human mirror neuron system underlying the understanding of actions of other people) areas. ECoG activity was recorded in drug-resistant epileptic patients during the execution of actions to reach and grasp common objects according to their affordances, as well as during the observation of the same actions performed by an experimenter. Both action execution and observation induced a desynchronization of alpha and beta rhythms in BA1-2, BA4, BA6, BA44 and BA45, which was generally higher in amplitude during the former than the latter condition. Action execution also induced a major synchronization of gamma rhythms in BA4 and BA6, again more during the execution of an action than during its observation. Human primary sensorimotor, premotor, and prefrontal areas do generate alpha, beta, and gamma rhythms and differently modulate them during action execution and observation. Gamma rhythms of motor areas are especially involved in action execution. Oscillatory activity of neural populations in sensorimotor, premotor and prefrontal (part of human mirror neuron system) areas represents and distinguishes own actions from those of other people. This methodological approach might be used for a neurophysiological diagnostic imaging of social cognition in epileptic patients. Copyright © 2015 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.

  3. Broadly Neutralizing Hemagglutinin Stalk-Specific Antibodies Induce Potent Phagocytosis of Immune Complexes by Neutrophils in an Fc-Dependent Manner.

    Science.gov (United States)

    Mullarkey, Caitlin E; Bailey, Mark J; Golubeva, Diana A; Tan, Gene S; Nachbagauer, Raffael; He, Wenqian; Novakowski, Kyle E; Bowdish, Dawn M; Miller, Matthew S; Palese, Peter

    2016-10-04

    Broadly neutralizing antibodies that recognize the conserved hemagglutinin (HA) stalk have emerged as exciting new biotherapeutic tools to combat seasonal and pandemic influenza viruses. Our general understanding of the mechanisms by which stalk-specific antibodies achieve protection is rapidly evolving. It has recently been demonstrated that broadly neutralizing HA stalk-specific IgG antibodies require Fc-Fcγ receptor (FcγR) interactions for optimal protection in vivo Here we examine the neutrophil effector functions induced by stalk-specific antibodies. As the most abundant subset of blood leukocytes, neutrophils represent a critical innate effector cell population and serve an instrumental role in orchestrating downstream adaptive responses to influenza virus infection. Yet, the interplay of HA stalk-specific IgG, Fc-FcγR engagement, and neutrophils has remained largely uncharacterized. Using an in vitro assay to detect the production of reactive oxygen species (ROS), we show that human and mouse monoclonal HA stalk-specific IgG antibodies are able to induce the production of ROS by neutrophils, while HA head-specific antibodies do not. Furthermore, our results indicate that the production of ROS is dependent on Fc receptor (FcR) engagement and phagocytosis. We went on to assess the ability of monoclonal HA stalk-specific IgA antibodies to induce ROS. Consistent with our findings for monoclonal IgGs, only HA stalk-specific IgA antibodies elicited ROS production by neutrophils. This induction is dependent on the engagement of FcαR1. Taken together, our findings describe a novel FcR-dependent effector function induced by HA stalk-specific IgG and IgA antibodies, and importantly, our studies shed light on the mechanisms by which HA stalk-specific antibodies achieve protection.

  4. Broadly Neutralizing Hemagglutinin Stalk-Specific Antibodies Induce Potent Phagocytosis of Immune Complexes by Neutrophils in an Fc-Dependent Manner

    Directory of Open Access Journals (Sweden)

    Caitlin E. Mullarkey

    2016-10-01

    Full Text Available Broadly neutralizing antibodies that recognize the conserved hemagglutinin (HA stalk have emerged as exciting new biotherapeutic tools to combat seasonal and pandemic influenza viruses. Our general understanding of the mechanisms by which stalk-specific antibodies achieve protection is rapidly evolving. It has recently been demonstrated that broadly neutralizing HA stalk-specific IgG antibodies require Fc-Fcγ receptor (FcγR interactions for optimal protection in vivo. Here we examine the neutrophil effector functions induced by stalk-specific antibodies. As the most abundant subset of blood leukocytes, neutrophils represent a critical innate effector cell population and serve an instrumental role in orchestrating downstream adaptive responses to influenza virus infection. Yet, the interplay of HA stalk-specific IgG, Fc-FcγR engagement, and neutrophils has remained largely uncharacterized. Using an in vitro assay to detect the production of reactive oxygen species (ROS, we show that human and mouse monoclonal HA stalk-specific IgG antibodies are able to induce the production of ROS by neutrophils, while HA head-specific antibodies do not. Furthermore, our results indicate that the production of ROS is dependent on Fc receptor (FcR engagement and phagocytosis. We went on to assess the ability of monoclonal HA stalk-specific IgA antibodies to induce ROS. Consistent with our findings for monoclonal IgGs, only HA stalk-specific IgA antibodies elicited ROS production by neutrophils. This induction is dependent on the engagement of FcαR1. Taken together, our findings describe a novel FcR-dependent effector function induced by HA stalk-specific IgG and IgA antibodies, and importantly, our studies shed light on the mechanisms by which HA stalk-specific antibodies achieve protection.

  5. Interferon-gamma increased epithelial barrier function via upregulating claudin-7 expression in human submandibular gland duct epithelium.

    Science.gov (United States)

    Abe, Ayumi; Takano, Kenichi; Kojima, Takashi; Nomura, Kazuaki; Kakuki, Takuya; Kaneko, Yakuto; Yamamoto, Motohisa; Takahashi, Hiroki; Himi, Tetsuo

    2016-06-01

    Tight junctions (TJs) are necessary for salivary gland function and may se