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Sample records for high-affinity il-2 receptors

  1. Role of alpha chain-IL-2 complex in the formation of the ternary complex of IL-2 and high-affinity IL-2 receptor.

    PubMed

    Kamio, M; Uchiyama, T; Arima, N; Itoh, K; Ishikawa, T; Hori, T; Uchino, H

    1990-01-01

    Using anti-Tac (anti-alpha chain) and 2R-B (anti-beta chain) antibodies, we studied the roles of IL-2 receptor subunits (alpha and beta chains) in the formation of IL-2 and high-affinity IL-2 receptor complex, which is the initial event of IL-2 induced T cell growth. High-affinity IL-2 binding which was undetectable in the presence of 2R-B antibody at 4 degrees C became fully detectable when examined at 37 degrees C, which explained the lack of inhibition by 2R-B antibody of IL-2-induced proliferation of the cells expressing high-affinity IL-2 receptor. We further studied the mechanism of the 'reappearance' of high-affinity IL-2 binding in the presence of 2R-B antibody. The addition of IL-2 to the cells preincubated with radiolabeled or fluorescence-labeled 2R-B antibody resulted in a marked decrease in the antibody bound to the cells expressing high-affinity IL-2 receptor at 37 degrees C. This decrease was blocked by the presence of anti-Tac antibody, which inhibited IL-2 binding to alpha chain, but not by 7G7/B6 antibody, which recognized a non-IL-2 binding site of its chain. Furthermore, the decrease in cell-bound 2R-B antibody was not due to the internalization of beta chain-2R-B antibody complex, because the amount of cell-bound Mik-beta3 antibody recognizing a non-IL-2 binding epitope of beta chain remained unchanged, nor to the inhibition by simple competitive binding of IL-2 molecules to beta chain as judged from comparative studies of competitive binding inhibition. Taking these data together, the reappearance of high-affinity IL-2 binding was considered to be caused by the replacement of 2R-B antibody at the IL-2 binding site of beta chain by alpha chain-mediated IL-2, and it was strongly suggested that alpha chain-IL-2 complex has a key role in the formation of the ternary complex of IL-2 and high-affinity IL-2 receptor. alpha chain may function as a dimension converter of IL-2 to effectively deliver IL-2 molecules to a relatively small number of beta

  2. Expression of low-, intermediate-, and high-affinity IL-2 receptors on B cell lines derived from patients with undifferentiated lymphoma of Burkitt's and non-Burkitt's types

    SciTech Connect

    Benjamin, D.; Rosolen, A.; Wormsley, S.B.; DeBault, L.E.; Colamonici, O.R. )

    1990-08-01

    IL-2 receptors on T cells exist in at least three forms which differ in their ligand-binding affinity. The low-affinity IL-2 receptor (IL-2R) consists of the 55-kDa Tac protein (p55 alpha), the intermediate-affinity site corresponds to the 70-kDa molecule (p70 beta), and the high-affinity IL-2R consists of a noncovalent heterodimeric structure involving both p55 alpha and p70 beta. We studied 24 B cell lines (8 EBV-negative and 16 EBV-positive) for IL-2R expression in the presence or absence of the tumor promoter, teleocidin. 125I-IL-2 radioreceptor binding assays and crosslinking studies demonstrated the sole expression of p55 alpha in EBV-negative cell lines only, whereas p55 alpha present in EBV-positive cell lines was always associated with p70 beta to construct high-affinity IL-2R. p70 beta was not detected in any of the EBV-negative cell lines, but was expressed on most of the EBV-positive cell lines (13 of 16). Our data also indicate that the expression of p55 alpha and p70 beta by radiolabeling correlates with their expression in flow cytometry, and that a large excess of p55 alpha is required to construct high-affinity IL-2R. Coexpression of p55 alpha and p70 beta on human B cells contributed to constructing high-affinity IL-2R hybrid complex as shown by rapid association rate contributed by p55 alpha and slow dissociation rate by p70 beta; teleocidin's ability to induce p55 alpha on cell lines which express p70 beta only, resulting in appearance of high-affinity IL-2R; and blocking p55 alpha by anti-Tac mAb in cell lines which constitutively express high-affinity IL-2R eliminated both high- and low-affinity components. The existence of low, intermediate, and high IL-2R on human B cells bears important future implications for understanding the mechanism of IL-2 signaling and the role of IL-2 in B cell activation, proliferation, and differentiation.

  3. Curcumin blocks interleukin (IL)-2 signaling in T-lymphocytes by inhibiting IL-2 synthesis, CD25 expression, and IL-2 receptor signaling

    SciTech Connect

    Forward, Nicholas A.; Conrad, David M.; Power Coombs, Melanie R.; Doucette, Carolyn D.; Furlong, Suzanne J.; Lin, Tong-Jun; Hoskin, David W.

    2011-04-22

    Highlights: {yields} Curcumin inhibits CD4{sup +} T-lymphocyte proliferation. {yields} Curcumin inhibits interleukin-2 (IL-2) synthesis and CD25 expression by CD4{sup +} T-lymphocytes. {yields} Curcumin interferes with IL-2 receptor signaling by inhibiting JAK3 and STAT5 phosphorylation. {yields} IL-2-dependent regulatory T-lymphocyte function and Foxp3 expression is downregulated by curcumin. -- Abstract: Curcumin (diferulomethane) is the principal curcuminoid in the spice tumeric and a potent inhibitor of activation-induced T-lymphocyte proliferation; however, the molecular basis of this immunosuppressive effect has not been well studied. Here we show that micromolar concentrations of curcumin inhibited DNA synthesis by mouse CD4{sup +} T-lymphocytes, as well as interleukin-2 (IL-2) and CD25 ({alpha} chain of the high affinity IL-2 receptor) expression in response to antibody-mediated cross-linking of CD3 and CD28. Curcumin acted downstream of protein kinase C activation and intracellular Ca{sup 2+} release to inhibit I{kappa}B phosphorylation, which is required for nuclear translocation of the transcription factor NF{kappa}B. In addition, IL-2-dependent DNA synthesis by mouse CTLL-2 cells, but not constitutive CD25 expression, was impaired in the presence of curcumin, which demonstrated an inhibitory effect on IL-2 receptor (IL-2R) signaling. IL-2-induced phosphorylation of STAT5A and JAK3, but not JAK1, was diminished in the presence of curcumin, indicating inhibition of critical proximal events in IL-2R signaling. In line with the inhibitory action of curcumin on IL-2R signaling, pretreatment of CD4{sup +}CD25{sup +} regulatory T-cells with curcumin downregulated suppressor function, as well as forkhead box p3 (Foxp3) expression. We conclude that curcumin inhibits IL-2 signaling by reducing available IL-2 and high affinity IL-2R, as well as interfering with IL-2R signaling.

  4. Interleukin 2 (IL2) PE40 is cytotoxic to cells displaying either the p55 or p70 subunit of the IL2 receptor.

    PubMed

    Lorberboum-Galski, H; Kozak, R W; Waldmann, T A; Bailon, P; FitzGerald, D J; Pastan, I

    1988-12-15

    IL2-PE40 is a chimeric protein composed of human interleukin 2 (IL2) genetically fused to the amino terminus of a modified form of pseudomonas exotoxin (PE). Internalization of IL2 via the individual p55 and p70 subunits of the IL2 receptor was studied using IL2-PE40 on several mouse and human cell lines expressing either the p55, the p70, or both IL2 receptor subunits. Internalization was assessed by measuring inhibition of protein synthesis caused by the toxin moiety of IL2-PE40. The results demonstrate that IL2 internalization is mediated by either the p55 receptor subunit or by the p70 subunit but is much more efficient when high affinity receptors composed of both subunits are present. IL2-PE40 is a powerful reagent for studying IL2 receptor interactions and for analyzing pathways of the immune response and its regulation. PMID:3143716

  5. Association of intercellular adhesion molecule 1 with the multichain high-affinity interleukin 2 receptor.

    PubMed Central

    Burton, J; Goldman, C K; Rao, P; Moos, M; Waldmann, T A

    1990-01-01

    Previously, using flow cytometric resonance energy transfer and lateral diffusion measurements, we demonstrated that a 95-kDa protein identified by two monoclonal antibodies (OKT27 and OKT27b) interacts physically with the 55-kDa alpha protein of the high-affinity interleukin 2 (IL-2) receptor. In the present study, this 95-kDa protein (p95) was purified and amino acid sequence data were obtained that showed strong homology to the human intercellular adhesion molecule 1 (ICAM-1). The identity of the p95 protein with ICAM-1 was confirmed by sequential immunoprecipitations using OKT27 and an antibody, WEHI-CAM-1, that is directed toward ICAM-1. We confirmed the physical proximity of p95/ICAM-1 to the IL-2 receptor alpha subunit by demonstrating that radiolabeled IL-2 could be cross-linked to this protein expressed on activated T cells. In functional studies, the antibodies OKT27 and OKT27b inhibited T-cell proliferative responses to OKT3, to soluble antigen, and to heterologous cells (mixed lymphocyte reaction). However, these antibodies did not inhibit IL-2-induced proliferation of an IL-2-dependent T-cell line. Taken together with our previous observations, the present studies suggest that ICAM-1 is in proximity and interacts physically with the high-affinity IL-2 receptor. The association of ICAM-1 with the IL-2 receptor may facilitate the paracrine IL-2-mediated stimulation of T cells expressing IL-2 receptors by augmenting homotypic T-T-cell interaction, by receptor-directed focusing of IL-2 release by helper T cells, and by focusing IL-2 receptors of the physically linked cells to the site of lymphocyte function-associated antigen 1-ICAM-1-IL-2 receptor interaction. Images PMID:1976256

  6. Only high-affinity receptors for interleukin 2 mediate internalization of ligand

    SciTech Connect

    Weissman, A.M.; Harford, J.B.; Svetlik, P.B.; Leonard, W.L.; Depper, J.M.; Waldmann, T.A.; Greene, W.C.; Klausner, R.D.

    1986-03-01

    Interleukin 2 (IL-2) receptors are expressed on activated T cells and in select T-cell leukemias. Recently, it has been demonstrated that at least two classes of receptor for IL-2 exist with markedly different affinities for ligand. All known biological actions of IL-2 have been correlated with occupancy of high-affinity sites; the function of the low-affinity sites remains unknown. Receptor-mediated endocytosis is the primary means of internalization of cell-surface receptors and their ligands. The internalization of IL-2 bound to high- and low-affinity receptor sites was studied in a human T-cell lymphotrophic virus type 1 (HTLV-1)-infected human T-cell leukemia cell line and in a cloned murine cytotoxic T-cell line (CTLL). Internalization of IL-2 occurred only when bound to high-affinity sites. In addition, an anti-receptor antibody (anti-Tac), which binds equally well to high- and low-affinity sites, demonstrated no detectable internalization. The implications of these findings as they relate to IL-2 receptor structure and function are discussed.

  7. IL-2 regulation of soluble IL-2 receptor levels following thermal injury.

    PubMed Central

    Teodorczyk-Injeyan, J A; Sparkes, B G; Lalani, S; Peters, W J; Mills, G B

    1992-01-01

    In the immunosuppressed burn patient serum levels of both IL-2 and a soluble form of IL-2 receptor alpha (sIL-2R alpha) are significantly elevated. Strikingly, the production of these markers by the in vitro activated patients' cells is decreased. This study examines the role of IL-2 in the decreased production of the sIL-2R alpha in vitro in patients with major burns (n = 18, 30 to greater than 70% total body surface area). Peripheral blood mononuclear cell (PBMC) cultures from patients with highly elevated serum sIL-2R alpha, and from healthy controls (n = 12) were activated with concanavalin A (Con A) at initiation. In patients' cultures mitogen-induced increments of sIL-2R alpha levels were significantly lower. There was a significant negative correlation (r = 0.64, P less than 0.001) between a high serum sIL-2R alpha level and a decreased lectin-induced sIL-2R alpha release in vitro. Low levels of sIL-2R alpha in patients' samples were not normalized by increasing the number of T lymphocytes. Also exogenous rIL-1 was without effect, whereas rIL-3 increased sIL-2R alpha release in some cultures. However, sIL-2R alpha levels were significantly increased in patients' cultures by (i) addition of exogenous IL-2; (ii) removal of adherent cells; (iii) addition of cyclooxygenase inhibitor, indomethacin; (iv) bypassing cell surface activation by the combination of the calcium ionophore A23187 and the phorbol ester 12-o-tetradecanoyl acetate. The cyclic AMP-elevating drug, forskolin, abrogated the ability of exogenous IL-2 to increase sIL-2R alpha production. Thus, in the burn patient, the reduced in vitro sIL-2R alpha release appears to relate to abnormalities in IL-2 production and action mediated through its functional surface receptor. Elevated levels of sIL-2R alpha in vivo may, therefore, reflect systemic activation of T lymphocytes in response to biologically active IL-2. PMID:1382903

  8. Intermediate- and high-affinity interleukin-2 receptors expressed in an IL-4-dependent T-cell line induce different signals.

    PubMed Central

    Rebollo, A; Silva, A

    1993-01-01

    In order to understand the respective roles of IL-2R alpha and IL-2R beta subunits in transmission of the interleukin-2 (IL-2)-mediated growth signals, we have established two IL-4-dependent murine T-cell clones stably expressing the human IL-2R beta chain and three clones stably expressing the human IL-2R alpha chain. Whereas parental LD8 cells (which express only the murine IL-2R beta chain) do not proliferate in response to IL-2, cell lines stably expressing human IL-2R beta or the chimeric IL-2R alpha beta complex proliferate in response to IL-2. Stably transfected cells expressing the chimeric high-affinity receptor (human IL-2R alpha and murine IL-2R beta) expressed de novo endogenous murine IL-2R alpha when cultured in the presence of IL-2 but not IL-4. Both chimeric and endogenous receptors are functional in response to IL-2, since only addition of both anti-human and anti-murine IL-2R alpha monoclonal antibodies (mAb) inhibited IL-2-induced proliferation. Taken together, our results strongly suggest that human and murine IL-2R beta molecules are different since interaction of IL-2 with human p70 IL-2R is sufficient for transduction of proliferative signals in the absence of p55 IL-2R or, alternatively, that over-expression of the IL-2R beta chain renders cells responsive to IL-2. In addition, IL-2 stimulation of T cells through different forms of IL-2R results in the induction of distinct cellular responses. PMID:8262551

  9. Gangliosides as high affinity receptors for tetanus neurotoxin.

    PubMed

    Chen, Chen; Fu, Zhuji; Kim, Jung-Ja P; Barbieri, Joseph T; Baldwin, Michael R

    2009-09-25

    Tetanus neurotoxin (TeNT) is an exotoxin produced by Clostridium tetani that causes paralytic death to hundreds of thousands of humans annually. TeNT cleaves vesicle-associated membrane protein-2, which inhibits neurotransmitter release in the central nervous system to elicit spastic paralysis, but the molecular basis for TeNT entry into neurons remains unclear. TeNT is a approximately 150-kDa protein that has AB structure-function properties; the A domain is a zinc metalloprotease, and the B domain encodes a translocation domain and C-terminal receptor-binding domain (HCR/T). Earlier studies showed that HCR/T bound gangliosides via two carbohydrate-binding sites, termed the lactose-binding site (the "W" pocket) and the sialic acid-binding site (the "R" pocket). Here we report that TeNT high affinity binding to neurons is mediated solely by gangliosides. Glycan array and solid phase binding analyses identified gangliosides that bound exclusively to either the W pocket or the R pocket of TeNT; GM1a bound to the W pocket, and GD3 bound to the R pocket. Using these gangliosides and mutated forms of HCR/T that lacked one or both carbohydrate-binding pocket, gangliosides binding to both of the W and R pockets were shown to be necessary for high affinity binding to neuronal and non-neuronal cells. The crystal structure of a ternary complex of HCR/T with sugar components of two gangliosides bound to the W and R supported the binding of gangliosides to both carbohydrate pockets. These data show that gangliosides are functional dual receptors for TeNT. PMID:19602728

  10. The expression of functional IL-2 receptor on activated macrophages depends on the stimulus applied.

    PubMed Central

    Valitutti, S; Carbone, A; Castellino, F; Maggiano, N; Ricci, R; Larocca, L M; Musiani, P

    1989-01-01

    Human peripheral blood monocytes (Mo) synthesize prostaglandin E2 (PGE2) when activated with lipopolysaccharide (LPS). This production is strongly enhanced by the addition of supernatant from phytohaemagglutinin (PHA)-activated T cells. To evaluate the factor(s) responsible for this enhancement we studied the effect of several cytokines on the PGE2 metabolism. Recombinant interleukin-1 (IL-1) or recombinant IL-2 strongly enhanced PGE2 synthesis in LPS-stimulated Mo cultures, whereas recombinant interferon-gamma (IFN-gamma) partially inhibited its production. To see whether the effect of IL-2 on Mo was due to the presence of IL-2 receptor (IL-2R) on the cell surface, flow cytometric analysis and electron microscopy were used to investigate IL-2R expression in unstimulated and stimulated Mo. Stimulated, but not resting, Mo displayed the p55 IL-2R chain on their cellular surface and associated with the polyribosomes of the rough endoplasmic reticulum in the cytoplasm. This finding strongly suggested that the p55 chain of the IL-2R was synthesized by activated Mo. To confirm this result, 125I-labelled IL-2 was bound under high- and low-affinity conditions and cross-linked to Mo cultured in the presence of LPS, IFN-gamma or IL-1. The cross-linked 125I-IL-2/IL-2R complexes were analysed by SDS-PAGE. Mo cultured with LPS, IFN-gamma and IL-1 expressed the p55 protein detected by low-affinity cross-linking, whereas only LPS-stimulated Mo displayed a barely detectable band with an apparent MW of 70,000 under high-affinity binding conditions. In addition, stimulated Mo were found capable of producing the soluble form of IL-2R. Finally, LPS-activated Mo only responded to the addition of IL-2 by an increase in PGE2 production, suggesting that the function of IL-2R on activated Mo is linked to the stimulus applied. Images Figure 2 Figure 3 PMID:2661416

  11. Early immune response and regulation of IL-2 receptor subunits

    NASA Technical Reports Server (NTRS)

    Hughes-Fulford, Millie; Sugano, Eiko; Schopper, Thomas; Li, Chai-Fei; Boonyaratanakornkit, J. B.; Cogoli, Augusto

    2005-01-01

    Affymetrix oligonucleotide arrays were used to monitor expression of 8796 genes and probe sets in activated T-cells; analysis revealed that 217 genes were significantly upregulated within 4 h. Induced genes included transcription factors, cytokines and their receptor genes. Analysis by semi-quantitative RT-PCR confirmed the significant induction of IL-2, IL-2R(gamma) and IL-2R(alpha). Forty-eight of the 217 induced genes are known to or predicted to be regulated by a CRE promoter/enhancer. We found that T-cell activation caused a significant increase in CREB phosphorylation furthermore, inhibition of the PKC pathway by GF109203 reduced CREB activation by 50% and inhibition of the PKA pathway caused a total block of CREB phosphorylation and significantly reduced IFN(gamma), IL-2 and IL-2R(alpha) gene expression by approximately 40% (p<0.001). PKC(theta) plays a major role in T-cell activation: inhibition of PKC significantly reduced the expression of IFN(gamma), IL-2 and IL-2R(alpha). Since PKC blocked activation of CREB, we studied potential cross-talk between the PKC and the PKA/MAPK pathways, PMA-stimulated Jurkat cells were studied with specific signal pathway inhibitors. Extracellular signal-regulated kinase-2 (ERK2) pathway was found to be significantly activated greater than seven-fold within 30 min; however, there was little activation of ERK-1 and no activation of JNK or p38 MAPK. Inhibition of the PKA pathway, but not the PKC pathway, resulted in inhibition of ERK1/2 activation at all time points, inhibition of MEK1 and 2 significantly blocked expression of IL-2 and IL-2R(alpha). Gene expression of IL-2R(alpha) and IFN(gamma) was dependent on PKA in S49 wt cells but not in kin- mutants. Using gel shift analysis, we found that forskolin activation of T-cells resulted in activation of AP1 sites; this increase in nuclear extract AP1 was significantly blocked by MEK1 inhibitor U0126. Taken together, these results suggest that the PKA in addition to PKC and

  12. Reduced secondary cytokine induction by BAY 50-4798, a high-affinity receptor-specific interleukin-2 analog.

    PubMed

    Steppan, Sonja; Eckart, Michael R; Bajsarowicz, Krystyna; Sternberg, Lawrence R; Greve, Jeffrey M; Cassell, Delanie J

    2006-03-01

    Recombinant interleukin-2 (IL-2) (aldesleukin, Proleukin, Chiron, Emeryville, CA) is approved for treatment of cancer patients and under investigation in HIV-infected individuals. However, treatment with aldesleukin is associated with toxicity, which may be due to its elicitation of inflammatory mediators from cells that express the intermediate-affinity IL-2 receptor. BAY 50-4798, a novel IL-2 analog, is a selective agonist for the high-affinity receptor. It induces the proliferation of activated T cells with a potency similar to that of aldesleukin but has reduced activity on cells expressing the intermediate-affinity receptor. In the current study, we compared cytokine responses elicited in peripheral blood mononuclear cell (PBMC) cultures stimulated with BAY 50-4798 or aldesleukin. BAY 50-4798 induced approximately 5-fold lower mean levels of endogenous IL-2 than aldesleukin, and at least 50% lower levels of proinflammatory cytokines, such as tumor necrosis fctor-alpha (TNF-alpha), IL-1beta, IL-6, and interferon-gamma (IFN-gamma). Furthermore, statistically significant reductions in the levels of IL-5, IL-8, IL-10, IL-13, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were observed in response to BAY 50-4798. These findings increase our understanding of the biologic action of BAY 50-4798 and suggest a mechanism by which it may exhibit better safety than aldesleukin in humans. PMID:16542139

  13. OX48, a monoclonal antibody against a 70,000 MW rat activation antigen expressed by T cells bearing the high-affinity interleukin-2 receptor.

    PubMed Central

    Somoza, C; Fernández-Ruiz, E; Rebollo, A; Sanz, E; Ramírez, F; Silva, A

    1990-01-01

    The monoclonal antibody (mAb) OX48 recognizes a 70,000 MW cell-surface protein present in a small percentage of activated rat T cells and in CD8+ rat x BW5147 interleukin-2 (IL-2)-dependent T-cell hybridomas, but not in resting spleen cells or in IL-2-independent T-cell hybrids. OX48 antibody added simultaneously with concanavalin A (Con A) to resting spleen cells inhibits the cell proliferation and reduces the IL-2 production. However, addition of IL-2 does not restore the mitogenic response. Growth of rat blast T cells or IL-2-dependent hybrids is not affected by the OX48 antibody. There is a close correlation between the expression of high-affinity IL-2 receptors (IL-2R) and the OX48 antigen in T-cell hybridomas. In spite of this striking correlation, OX48 mAb does not inhibit the binding of 125I-IL-2 to the IL-2-dependent hybrids, and is unable to immunoprecipitate any of the proteins chemically cross-linked to 125I-IL-2. Therefore, the OX48 molecule represents a new rat activation antigen, undefined in other species, and probably involved in the early steps of T-cell activation. Images Figure 5 Figure 7 PMID:2373518

  14. High-affinity binding of (/sup 3/H)acetylcholine to muscarinic cholinergic receptors

    SciTech Connect

    Kellar, K.J.; Martino, A.M.; Hall, D.P. Jr.; Schwartz, R.D.; Taylor, R.L.

    1985-06-01

    High-affinity binding of (/sup 3/H)acetylcholine to muscarinic cholinergic sites in rat CNS and peripheral tissues was measured in the presence of cytisin, which occupies nicotinic cholinergic receptors. The muscarinic sites were characterized with regard to binding kinetics, pharmacology, anatomical distribution, and regulation by guanyl nucleotides. These binding sites have characteristics of high-affinity muscarinic cholinergic receptors with a Kd of approximately 30 nM. Most of the muscarinic agonist and antagonist drugs tested have high affinity for the (/sup 3/H)acetylcholine binding site, but pirenzepine, an antagonist which is selective for M-1 receptors, has relatively low affinity. The ratio of high-affinity (/sup 3/H)acetylcholine binding sites to total muscarinic binding sites labeled by (/sup 3/H)quinuclidinyl benzilate varies from 9 to 90% in different tissues, with the highest ratios in the pons, medulla, and heart atrium. In the presence of guanyl nucleotides, (/sup 3/H) acetylcholine binding is decreased, but the extent of decrease varies from 40 to 90% in different tissues, with the largest decreases being found in the pons, medulla, cerebellum, and heart atrium. The results indicate that (/sup 3/H)acetylcholine binds to high-affinity M-1 and M-2 muscarinic receptors, and they suggest that most M-2 sites have high affinity for acetylcholine but that only a small fraction of M-1 sites have such high affinity.

  15. Effect of IL-2-Bax, a novel interleukin-2-receptor-targeted chimeric protein, on bleomycin lung injury1

    PubMed Central

    Segel, Michael J; Aqeilan, Rami; Zilka, Keren; Lorberboum-Galski, Haya; Wallach-Dayan, Shulamit B; Conner, Michael W; Christensen, Thomas G; Breuer, Raphael

    2005-01-01

    The role of lymphocytes in the pathogenesis of lung fibrosis is not clear, but the weight of the evidence supports a pro-fibrotic effect for lymphocytes. The high-affinity interleukin-2 receptor (haIL-2R) is expressed on activated, but not quiescent, T lymphocytes. This selective expression of haIL-2R provides the basis for therapeutic strategies that target IL-2R-expressing cells. We hypothesized that elimination of activated lymphocytes by IL-2R-targeted chimeric proteins might ameliorate lung fibrosis. We investigated the effects of IL-2-Bax, a novel apoptosis-inducing IL-2R-targeted chimeric protein, on bleomycin-induced lung injury in mice. Treatment groups included (i) a single intratracheal instillation of bleomycin and twice-daily intraperitoneal injections of IL-2-Bax; (ii) intratracheal bleomycin and intraperitoneal IL-2-PE664Glu, an older-generation chimeric protein; (iii) intratracheal bleomycin/intraperitoneal PBS; (iv) intratracheal saline/intraperitoneal PBS. Lung injury was evaluated 14 days after intratracheal instillation by cell count in bronchoalveolar lavage (BAL) fluid, semi-quantitative and quantitative histomorphological measurements and by biochemical analysis of lung hydroxyproline. Bleomycin induced a BAL lymphocytosis that was significantly attenuated by IL-2-Bax and IL-2-PE664Glu. However, morphometric parameters and lung hydroxyproline were unaffected by the chimeric proteins. These results show that IL-2-Bax reduces the lymphocytic infiltration of the lungs in response to bleomycin, but this effect is not accompanied by a decrease in lung fibrosis. PMID:16191100

  16. Neuroprotective effects of high affinity sigma 1 receptor selective compounds

    PubMed Central

    Luedtke, Robert R.; Perez, Evelyn; Yang, Shao-Hua; Liu, Ran; Vangveravong, Suwanna; Tu, Zhude; Mach, Robert H.; Simpkins, James W.

    2014-01-01

    We previously reported that the antipsychotic drug haloperidol, a multifunctional D2-like dopamine and sigma receptor subtype antagonist, has neuroprotective properties. In this study we further examined the association between neuroprotection and receptor antagonism by evaluating a panel of novel compounds with varying affinity at sigma and D2-like dopamine receptors. These compounds were evaluated using an in vitro cytotoxicity assay that utilizes a hippocampal-derived cell line, HT-22, in the presence or absence of varying concentrations (5 to 20 mM) of glutamate. While haloperidol was found to be a potent neuroprotective agent in this in vitro cell assay, the prototypic sigma 1 receptor agonist (+)-pentazocine was found not to be neuroprotective. Subsequently, the potency for the neuroprotection of HT-22 cells was evaluated for a) three SV series indoles which have nMolar affinity at D2-like receptors but varying affinity at sigma 1 receptor and b) two benzyl phenylacetamides sigma 1 receptor selective compounds which bind with low affinity at D2-like receptors but have nMolar affinity for the sigma 1 receptor. We observed that cytoprotection correlated with the affinity of the compounds for sigma 1 receptors. Based upon results from the HT-22 cell-based in vitro assay, two phenylacetamides, LS-127 and LS-137, were further evaluated in vivo using a transient middle cerebral artery occlusion (t-MCAO) model of stroke. At a dose of 100 µg/kg, both LS-127 and LS-137 attenuated infarct volume by approximately 50%. These studies provide further evidence that sigma 1 receptor selective compounds can provide neuroprotection in cytotoxic situations. These results also demonstrate that sigma 1 receptor selective benzyl phenylacetamides are candidate pharmacotherapeutic agents that could be used to minimize neuronal death after a stroke or head trauma. PMID:22285434

  17. Evidence for enhanced interleukin 2 (IL-2) secretion and IL-2 receptor presentation by synovial fluid lymphocytes in rheumatoid arthritis.

    PubMed Central

    Lemm, G; Warnatz, H

    1986-01-01

    Synovial fluid lymphocytes (SFL) and peripheral blood lymphocytes (PBL) from patients with rheumatoid arthritis (RA) and reactive oligoarthritis were investigated for activated T cells (Ia+SIg-), IL-2 receptor bearing cells (Tac+) and IL-2 production in vivo and in vitro. In contrast to negative results with blood, the synovial fluid of the arthritic joints contains considerable amounts of IL-2 activity (median: 11.8 mu/ml), elevated proportions of Ia+SIg- activated T cells (median: 12.5%) and of IL-2 receptor bearing cells (median: 2.5%). In vitro, after stimulation with several Concanavalin A (Con A) doses, SFL develop proportions of IL-2 receptor cells comparable to PBL. Furthermore, they produce higher values of IL-2 activity than comparable PBL cultures. The proportions of Ia+SIg- activated T cells increase only moderately after Con A stimulation compared to in vivo data, indicating different activated T cell subsets in the synovial fluid (Ia+SIg-, Tac+). The findings are discussed as an expression of an acute hyperactivation of lymphocytes in an inflamed joint. PMID:3089651

  18. GHB receptor targets in the CNS: focus on high-affinity binding sites.

    PubMed

    Bay, Tina; Eghorn, Laura F; Klein, Anders B; Wellendorph, Petrine

    2014-01-15

    γ-Hydroxybutyric acid (GHB) is an endogenous compound in the mammalian brain with both low- and high-affinity receptor targets. GHB is used clinically in the treatment of symptoms of narcolepsy and alcoholism, but also illicitly abused as the recreational drug Fantasy. Major pharmacological effects of exogenous GHB are mediated by GABA subtype B (GABAB) receptors that bind GHB with low affinity. The existence of GHB high-affinity binding sites has been known for more than three decades, but the uncovering of their molecular identity has only recently begun. This has been prompted by the generation of molecular tools to selectively study high-affinity sites. These include both genetically modified GABAB knock-out mice and engineered selective GHB ligands. Recently, certain GABA subtype A (GABAA) receptor subtypes emerged as high-affinity GHB binding sites and potential physiological mediators of GHB effects. In this research update, a description of the various reported receptors for GHB is provided, including GABAB receptors, certain GABAA receptor subtypes and other reported GHB receptors. The main focus will thus be on the high-affinity binding targets for GHB and their potential functional roles in the mammalian brain. PMID:24269284

  19. Rational design of high affinity tachykinin NK1 receptor antagonists.

    PubMed

    Boyle, S; Guard, S; Higginbottom, M; Horwell, D C; Howson, W; McKnight, A T; Martin, K; Pritchard, M C; O'Toole, J; Raphy, J

    1994-05-01

    The rational design of a non-peptide tachykinin NK1 receptor antagonist, [(2-benzofuran)-CH2OCO]-(R)-alpha-MeTrp-(S)-NHCH(CH3)P h (28, PD 154075) is described. Compound 28 has a Ki = 9 and 0.35 nM for the NK1 receptor binding site in guinea-pig cerebral cortex membranes and human IM9, cells respectively (using [125I] Bolton-Hunter-SP as the radioligand). It is a potent antagonist in vitro where it antagonises the contractions mediated by SPOMe in the guinea-pig ileum (KB = 0.3 nM). Compound 28 is active in vivo in the guinea-pig plasma extravasation model, where it is able to block the SPOMe-induced protein plasma extravasation (monitored by Evans Blue) in the bladder with an ID50 of 0.02 mg kg-1 iv. PMID:7922147

  20. High-affinity benzodiazepine receptor ligands among benzodiazepines and betacarbolines with different intrinsic activity

    SciTech Connect

    Yliniemelae, A.; Gynther, J. ); Konschin, H.; Tylli, H. ); Rouvinen, J. )

    1989-01-01

    Structural and electrostatic features of diazepam, flumazenil, and methyl betacarboline-3-carboxylate (BCCM) have been investigated using the molecular superimposition method. These high-affinity benzodiazepine (BZ) receptor ligands are structurally unrelated and they have different intrinsic activity. These ligands are superimposed in such a way that common structural and electrostatic features essential for the high receptor binding affinity overlap. In addition to this binding pharmacophore, there are roughly three separate binding zones in the BZ receptor, one for each class of ligands. The intrinsic activity of BZ receptor ligands depends on the molecular structures and the way the ligand approaches the receptor.

  1. Putative M2 muscarinic receptors of rat heart have high affinity for organophosphorus anticholinesterases.

    PubMed

    Silveira, C L; Eldefrawi, A T; Eldefrawi, M E

    1990-05-01

    The M2 subtype of muscarinic receptor is predominant in heart, and such receptors were reported to be located in muscles as well as in presynaptic cholinergic and adrenergic nerve terminals. Muscarinic receptors of rat heart were identified by the high affinity binding of the agonist (+)-[3H]cis-methyldioxolane ([3H]CD), which has been used to label a high affinity population of M2 receptors. A single population of sites (KD 2.74 nM; Bmax of 82 fmol/mg protein) was detected and [3H]CD binding was sensitive to the M2 antagonist himbacine but much less so to pirenzepine, the M1 antagonist. These cardiac receptors had different sensitivities to NiCl2 and N-ethylmaleimide from brain muscarinic receptors, that were also labeled with [3H]CD and considered to be of the M2 subtype. Up to 70% of the [3H]CD-labeled cardiac receptors had high affinities for several organophosphate (OP) anticholinesterases. [3H]CD binding was inhibited by the nerve agents soman, VX, sarin, and tabun, with K0.5 values of 0.8, 2, 20, and 50 nM, respectively. It was also inhibited by echothiophate and paraoxon with K0.5 values of 100 and 300 nM, respectively. The apparent competitive nature of inhibition of [3H]CD binding by both sarin and paraoxon suggests that the OPs bind to the acetylcholine binding site of the muscarinic receptor. Other OP insecticides had lower potencies, inhibiting less than 50% of 5 nM [3H]CD binding by 1 microM of EPN, coumaphos, dioxathion, dichlorvos, or chlorpyriphos. There was poor correlation between the potencies of the OPs in reversibly inhibiting [3H]CD binding, and their anticholinesterase activities and toxicities. Acetylcholinesterases are the primary targets for these OP compounds because of the irreversible nature of their inhibition, which results in building of acetylcholine concentrations that activate muscarinic and nicotinic receptors and desensitize them, thereby inhibiting respiration. Nevertheless, the high affinities that cardiac muscarinic

  2. Purification of high affinity benzodiazepine receptor binding site fragments from rat brain

    SciTech Connect

    Klotz, K.L.

    1984-01-01

    In central nervous system benzodiazepine recognition sites occur on neuronal cell surfaces as one member of a multireceptor complex, including recognition sites for benzodiazepines, gamma aminobutyric acid (GABA), barbiturates and a chloride ionophore. During photoaffinity labelling, the benzodiazepine agonist, /sup 3/H-flunitrazepam, is irreversibly bound to central benzodiazepine high affinity recognition sites in the presence of ultraviolet light. In these studies a /sup 3/H-flunitrazepam radiolabel was used to track the isolation and purification of high affinity agonist binding site fragments from membrane-bound benzodiazepine receptor in rat brain. The authors present a method for limited proteolysis of /sup 3/H-flunitrazepam photoaffinity labeled rat brain membranes, generating photolabeled benzodiazepine receptor fragments containing the agonist binding site. Using trypsin chymotrypsin A/sub 4/, or a combination of these two proteases, they have demonstrated the extent and time course for partial digestion of benzodiazepine receptor, yielding photolabeled receptor binding site fragments. These photolabeled receptor fragments have been further purified on the basis of size, using ultrafiltration, gel permeation chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as well as on the basis of hydrophobicity, using a high performance liquid chromatography (HPLC) precolumn, several HPLC elution schemes, and two different HPLC column types. Using these procedures, they have purified three photolabeled benzodiazepine receptor fragments containing the agonist binding site which appear to have a molecular weight of less than 2000 daltons each.

  3. Cloning of a type I cytokine receptor most related to the IL-2 receptor β chain

    PubMed Central

    Ozaki, Katsutoshi; Kikly, Kristine; Michalovich, David; Young, Peter R.; Leonard, Warren J.

    2000-01-01

    We have identified a type I cytokine receptor, which we have termed novel interleukin receptor (NILR), that is most related to the IL-2 receptor β chain (IL-2Rβ) and physically adjacent to the IL-4 receptor α chain gene on chromosome 16. NILR mRNA is most highly expressed in thymus and spleen, and is induced by phytohemagglutinin in human peripheral blood mononuclear cells. NILR protein was detected on human T cell lymphotropic virus type I-transformed T cell lines, Raji B cells, and YT natural killer-like cells. Artificial homodimerization of the NILR cytoplasmic domain confers proliferation to Ba/F3 murine pro-B cells but not to 32D myeloid progenitor cells or CTLL-2 murine helper T cells. In these latter cells, heterodimerization of IL-2Rβ and the common cytokine receptor γ chain (γc) cytoplasmic domains allows potent proliferation, whereas such heterodimerization of NILR with γc does not. This finding suggests that NILR has signaling potential but that a full understanding of its signaling partner(s) is not yet clear. Like IL-2Rβ, NILR associates with Jak1 and mediates Stat5 activation. PMID:11016959

  4. Sensitivity of binding of high-affinity dopamine receptor radioligands to increased synaptic dopamine.

    PubMed

    Gatley, S J; Gifford, A N; Carroll, F I; Volkow, N D

    2000-12-15

    PET and SPECT studies have documented that D2 radioligands of moderate affinity, but not radioligands of high affinity, are sensitive to pharmacological challenges that alter synaptic dopamine levels. The objective of this work was to determine whether the brain kinetics of high-affinity radioligands for dopamine D1 ([(3)H]SCH 23390) and D2 ([(123)I]epidepride) receptors were altered by a prolonged elevation of synaptic dopamine induced by the potent cocaine analog RTI-55. Mice were injected intravenously with radioligands either 30 min after or 4 h before intraperitoneal administration of RTI-55 (2 mg/kg). In separate experiments, the pharmacological effects of RTI-55 were assessed biochemically by measuring uptake of dopamine in synaptosomes prepared from RTI-treated mice and behaviorally by monitoring locomotor activity. Consistent with the expected elevation of synaptic dopamine, RTI-55 induced a long-lasting decrement in dopamine uptake measured ex vivo, and a prolonged increase in locomotor activity. RTI-55 injected prior to the radioligands induced a significant (P < 0.05) increase in striatal concentration of [(123)I]epidepride at 15 min, relative to saline-treated controls, but there were no differences between the two groups at later time-points. For [(3)H]SCH 23390, both initial striatal uptake and subsequent clearance were slightly increased by preadministration of RTI-55. Administration of RTI-55 4 h after the radioligands (i.e., when it was presumed that a state of near equilibrium binding of the radioligands had been reached), was associated with a significant reduction of striatal radioactivity for both radiotracers. Our results are consistent with increased competition between dopamine and radioligand for binding to both D1 and D2 receptors after treatment with RTI-55. We suggest that the magnitude of the competition is reduced by failure of the receptor binding of high-affinity radioligands to rapidly attain equilibrium. PMID:11044896

  5. High-Affinity Fc Receptor Expression Indicates Relative Immaturity in Human Monocytes.

    PubMed

    Clanchy, Felix I L

    2016-05-01

    Within monocyte heterogeneity, subsets represent discrete, well-characterized phenotypes. Although many studies have highlighted differences between subsets, there is evidence that subpopulations represent contiguous stages in a maturational series. As CD14(hi)CD64(hi) monocytes have higher proliferative potential than CD14(hi)CD64(lo) monocytes, the surface marker profile on 4 subsets defined by CD14 and CD64 was measured. The profiles were compared to that of subsets defined by the high-affinity IgE receptor (FcɛRIα), CD16, and CD14; further differences in size, granularity, and buoyancy were measured in subsets delineated by these markers. There was a positive correlation between proliferative monocyte (PM) prevalence and CD64 expression on the classical monocyte subset, and also between PM prevalence and circulating FcɛRIα(+) monocytes. The expression of CD64, the high-affinity IgG receptor, on canonical human monocyte subsets was determined before and after short-term culture, and in response to interleukin (IL)-6, IL-10, macrophage colony-stimulating factor, granulocyte/macrophage colony-stimulating factor and interferon-γ; the influence of these cytokines on monocyte subset transition was also measured. The loss of FcɛRIα expression preceded an increase in CD16 expression in whole blood cultures. These data indicate that high-affinity Fc receptors are expressed on less mature monocytes and that FcɛRIα(+) monocytes are developmentally antecedent to the canonical classical and intermediate monocyte subsets. PMID:26714112

  6. Association study of schizophrenia and IL-2 receptor {beta} chain gene

    SciTech Connect

    Nimgaonkar, V.L.; Yang, Z.W.; Zhang, X.R.; Brar, J.S.

    1995-10-09

    A case-control association study was conducted in Caucasian patients with schizophrenia (DSM-III-R, n = 42) and unaffected controls (n = 47) matched for ethnicity and area of residence. Serum interleukin-2 receptor (IL-2R) concentrations, as well as a dinucleotide repeat polymorphism in the IL-2RP chain gene, were examined in both groups. No significant differences in IL-2R concentrations or in the distribution of the polymorphism were noted. This study does not support an association between schizophrenia and the IL-2RP gene locus, contrary to the suggestive evidence from linkage analysis in multicase families. 17 refs., 2 tabs.

  7. Increased production of interleukin-2 and IL-2 receptor in primary IgA nephropathy.

    PubMed

    Schena, F P; Mastrolitti, G; Jirillo, E; Munno, I; Pellegrino, N; Fracasso, A R; Aventaggiato, L

    1989-03-01

    The production of interleukin-2 (IL-2) by peripheral blood mononuclear cells (PBMC) in 13 patients with IgA nephropathy (IgAN) and 9 patients with chronic glomerulonephritis was investigated. Moreover, the distribution of IL-2 receptor (IL-2R) expression was studied in the purified T cell population versus the non-T cell population of IgAN patients. The results show a spontaneous significant production of IL-2 in cultures of PBMC from patients with IgAN (P less than 0.025) that increased after PHA stimulation. IgAN patients also had a significantly higher expression of IL-2R on the surface of PBMC than did patients with chronic glomerulonephritis (P less than 0.05). IL-2R was usually detected on unstimulated purified T cells that expressed the activation DR antigen. Moreover, a high number of DR helper T cells was associated to a reduced number of suppressor T cells (OKT8+M1+). These findings suggest that the increased production of IL-2 in patients with IgAN may be responsible for the increased activity of helper T cells. The high number of IL-2R expressed by freshly separated PBMC implies an in vivo continuous stimulation of these cells, and this finding is in agreement with the demonstrated spontaneous hyperproduction of IL-2. Moreover, the low number of suppressor T cells may contribute to the overactivity of helper T cells bearing IL-2R in IgAN patients. PMID:2785227

  8. Collagens are functional, high affinity ligands for the inhibitory immune receptor LAIR-1

    PubMed Central

    Lebbink, Robert Jan; de Ruiter, Talitha; Adelmeijer, Jelle; Brenkman, Arjan B.; van Helvoort, Joop M.; Koch, Manuel; Farndale, Richard W.; Lisman, Ton; Sonnenberg, Arnoud; Lenting, Peter J.; Meyaard, Linde

    2006-01-01

    Collagens are the most abundant proteins in the human body, important in maintenance of tissue structure and hemostasis. Here we report that collagens are high affinity ligands for the broadly expressed inhibitory leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1). The interaction is dependent on the conserved Gly-Pro-Hyp collagen repeats. Antibody cross-linking of LAIR-1 is known to inhibit immune cell function in vitro. We now show that collagens are functional ligands for LAIR-1 and directly inhibit immune cell activation in vitro. Thus far, all documented ligands for immune inhibitory receptors are membrane molecules, implying a regulatory role in cell–cell interaction. Our data reveal a novel mechanism of peripheral immune regulation by inhibitory immune receptors binding to extracellular matrix collagens. PMID:16754721

  9. Synthetic Receptors for the High-Affinity Recognition of O-GlcNAc Derivatives.

    PubMed

    Rios, Pablo; Carter, Tom S; Mooibroek, Tiddo J; Crump, Matthew P; Lisbjerg, Micke; Pittelkow, Michael; Supekar, Nitin T; Boons, Geert-Jan; Davis, Anthony P

    2016-03-01

    The combination of a pyrenyl tetraamine with an isophthaloyl spacer has led to two new water-soluble carbohydrate receptors ("synthetic lectins"). Both systems show outstanding affinities for derivatives of N-acetylglucosamine (GlcNAc) in aqueous solution. One receptor binds the methyl glycoside GlcNAc-β-OMe with Ka ≈20,000 m(-1), whereas the other one binds an O-GlcNAcylated peptide with Ka ≈70,000 m(-1). These values substantially exceed those usually measured for GlcNAc-binding lectins. Slow exchange on the NMR timescale enabled structural determinations for several complexes. As expected, the carbohydrate units are sandwiched between the pyrenes, with the alkoxy and NHAc groups emerging at the sides. The high affinity of the GlcNAcyl-peptide complex can be explained by extra-cavity interactions, raising the possibility of a family of complementary receptors for O-GlcNAc in different contexts. PMID:26822115

  10. Characterization of specific high affinity receptors for human tumor necrosis factor on mouse fibroblasts

    SciTech Connect

    Hass, P.E.; Hotchkiss, A.; Mohler, M.; Aggarwal, B.B.

    1985-10-05

    Mouse L-929 fibroblasts, an established line of cells, are very sensitive to lysis by human lymphotoxin (hTNF-beta). Specific binding of a highly purified preparation of hTNF-beta to these cells was examined. Recombinant DNA-derived hTNF-beta was radiolabeled with (TH)propionyl succinimidate at the lysine residues of the molecule to a specific activity of 200 microCi/nmol of protein. (TH)hTNF-beta was purified by high performance gel permeation chromatography and the major fraction was found to be monomeric by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The labeled hTNF-beta was fully active in causing lysis of L-929 fibroblasts and bound specifically to high affinity binding sites on these cells. Scatchard analysis of the binding data revealed the presence of a single class of high affinity receptors with an apparent Kd of 6.7 X 10(-11) M and a capacity of 3200 binding sites/cell. Unlabeled recombinant DNA-derived hTNF-beta was found to be approximately 5-fold more effective competitive inhibitor of binding than the natural hTNF-beta. The binding of hTNF-beta to these mouse fibroblasts was also correlated with the ultimate cell lysis. Neutralizing polyclonal antibodies to hTNF-beta efficiently inhibited the binding of (TH)hTNF-beta to the cells. The authors conclude that the specific high affinity binding site is the receptor for hTNF-beta and may be involved in lysis of cells.

  11. Solubilization and reconstitution of the D-1 dopamine receptor: potentiation of the agonist high-affinity state of the receptor

    SciTech Connect

    Sidhu, A.

    1988-11-29

    The D-1 dopamine receptor was extracted from rat striatal membranes with sodium cholate and NaCl in the presence of a specific agonist and phospholipids. The soluble receptor then was reconstituted into phospholipid vesicles by further addition of phospholipids prior to detergent removal. Of the total membrane receptors, up to 48% were extracted and 36% were reconstituted into phospholipid vesicles. Yields were greatly reduced if the agonist was omitted or replaced with an antagonist. The solubilized and reconstituted D-1 receptors retained the pharmacological properties of the membrane-bound receptors, including the ability to discriminate between active and inactive enantiomers of specific agonists and antagonists. In this regard, the affinity of the reconstituted receptors for the D-1 specific antagonist /sup 125/I SCH 23982 was similar to that of the membrane-bound receptors with a Kd of 1.5 nM. Both the soluble and reconstituted forms of the D-1 receptor exhibited two affinity states for the D-1 specific agonist SKandF R-38393. In contrast to the low proportion of the receptors that had a high affinity for the agonists in striatal membranes (less than 6%), there was a dramatic increase following solubilization (22%) and reconstitution (40%). Similar results were obtained by using dopamine; the proportion of high-affinity sites increased from 4% (membrane-bound) to 48% (reconstituted) of the total receptor population. These high-affinity sites were coupled to G proteins, as guanyl nucleotides completely abolished them. Addition of guanyl nucleotides prior to solubilization or to reconstitution, however, had no effect on the subsequent yield of the reconstituted receptors.

  12. Further characterization of the subunits of the receptor with high affinity for immunoglobulin E

    SciTech Connect

    Alcaraz, G.; Kinet, J.P.; Liu, T.Y.; Metzger, H.

    1987-05-05

    The ..cap alpha.., ..beta.., ..gamma.. subunits of the receptor with high affinity for immunoglobulin E were isolated and their compositions assessed by direct amino acid analysis and by incorporation of radioactive precursors. The compositions show no unusual features other than a rather high content of tryptophan in the ..cap alpha.. chain as assessed from the incorporation studies. The results combined with future sequence data will permit unambiguous determination of the multiplicity of the chains in the receptor. Chymotryptic peptide maps of the extrinsically iodinated subunits show several similar peptides, particularly for ..cap alpha.. and ..beta... However, these putative homologies were not apparent when tryptic maps of the biosynthetically ((/sup 3/H)leucine) labeled subunits were analyzed.

  13. Rational development of high-affinity T-cell receptor-like antibodies.

    PubMed

    Stewart-Jones, Guillaume; Wadle, Andreas; Hombach, Anja; Shenderov, Eugene; Held, Gerhard; Fischer, Eliane; Kleber, Sascha; Nuber, Natko; Stenner-Liewen, Frank; Bauer, Stefan; McMichael, Andrew; Knuth, Alexander; Abken, Hinrich; Hombach, Andreas A; Cerundolo, Vincenzo; Jones, E Yvonne; Renner, Christoph

    2009-04-01

    T-cell interaction with a target cell is a key event in the adaptive immune response and primarily driven by T-cell receptor (TCR) recognition of peptide-MHC (pMHC) complexes. TCR avidity for a given pMHC is determined by number of MHC molecules, availability of coreceptors, and TCR affinity for MHC or peptide, respectively, with peptide recognition being the most important factor to confer target specificity. Here we present high-resolution crystal structures of 2 Fab antibodies in complex with the immunodominant NY-ESO-1(157-165) peptide analogue (SLLMWITQV) presented by HLA-A*0201 and compare them with a TCR recognizing the same pMHC. Binding to the central methionine-tryptophan peptide motif and orientation of binding were almost identical for Fabs and TCR. As the MW "peg" dominates the contacts between Fab and peptide, we estimated the contributions of individual amino acids between the Fab and peptide to provide the rational basis for a peptide-focused second-generation, high-affinity antibody library. The final Fab candidate achieved better peptide binding by 2 light-chain mutations, giving a 20-fold affinity improvement to 2-4 nM, exceeding the affinity of the TCR by 1,000-fold. The high-affinity Fab when grafted as recombinant TCR on T cells conferred specific killing of HLA-A*0201/NY-ESO-1(157-165) target cells. In summary, we prove that affinity maturation of antibodies mimicking a TCR is possible and provide a strategy for engineering high-affinity antibodies that can be used in targeting specific pMHC complexes for diagnostic and therapeutic purposes. PMID:19307587

  14. Rational development of high-affinity T-cell receptor-like antibodies

    PubMed Central

    Stewart-Jones, Guillaume; Wadle, Andreas; Hombach, Anja; Shenderov, Eugene; Held, Gerhard; Fischer, Eliane; Kleber, Sascha; Nuber, Natko; Stenner-Liewen, Frank; Bauer, Stefan; McMichael, Andrew; Knuth, Alexander; Abken, Hinrich; Hombach, Andreas A.; Cerundolo, Vincenzo; Jones, E. Yvonne; Renner, Christoph

    2009-01-01

    T-cell interaction with a target cell is a key event in the adaptive immune response and primarily driven by T-cell receptor (TCR) recognition of peptide-MHC (pMHC) complexes. TCR avidity for a given pMHC is determined by number of MHC molecules, availability of coreceptors, and TCR affinity for MHC or peptide, respectively, with peptide recognition being the most important factor to confer target specificity. Here we present high-resolution crystal structures of 2 Fab antibodies in complex with the immunodominant NY-ESO-1157–165 peptide analogue (SLLMWITQV) presented by HLA-A*0201 and compare them with a TCR recognizing the same pMHC. Binding to the central methionine-tryptophan peptide motif and orientation of binding were almost identical for Fabs and TCR. As the MW “peg” dominates the contacts between Fab and peptide, we estimated the contributions of individual amino acids between the Fab and peptide to provide the rational basis for a peptide-focused second-generation, high-affinity antibody library. The final Fab candidate achieved better peptide binding by 2 light-chain mutations, giving a 20-fold affinity improvement to 2–4 nM, exceeding the affinity of the TCR by 1,000-fold. The high-affinity Fab when grafted as recombinant TCR on T cells conferred specific killing of HLA-A*0201/NY-ESO-1157–165 target cells. In summary, we prove that affinity maturation of antibodies mimicking a TCR is possible and provide a strategy for engineering high-affinity antibodies that can be used in targeting specific pMHC complexes for diagnostic and therapeutic purposes. PMID:19307587

  15. Role of the High Affinity Immunoglobulin E Receptor in Bacterial Translocation and Intestinal Inflammation

    PubMed Central

    Dombrowicz, David; Nutten, Sophie; Desreumaux, Pierre; Neut, Christel; Torpier, Gérard; Peeters, Marc; Colombel, Jean-Frédéric; Capron, Monique

    2001-01-01

    A role for immunoglobulin E and its high affinity receptor (FcεRI) in the control of bacterial pathogenicity and intestinal inflammation has been suggested, but relevant animal models are lacking. Here we compare transgenic mice expressing a humanized FcεRI (hFcεRI), with a cell distribution similar to that in humans, to FcεRI-deficient animals. In hFcεRI transgenic mice, levels of colonic interleukin 4 were higher, the composition of fecal flora was greatly modified, and bacterial translocation towards mesenteric lymph nodes was increased. In hFcεRI transgenic mice, 2,4,6-tri-nitrobenzenesulfonic acid (TNBS)-induced colitis was also more pronounced, whereas FcεRI-deficient animals were protected from colitis, demonstrating that FcεRI can affect the onset of intestinal inflammation. PMID:11136818

  16. Glycan-based high-affinity ligands for toxins and pathogen receptors.

    PubMed

    Kulkarni, Ashish A; Weiss, Alison A; Iyer, Suri S

    2010-03-01

    Glycans decorate over 95% of the mammalian cell surface in the form of glycolipids and glycoproteins. Several toxins and pathogens bind to these glycans to enter the cells. Understanding the fundamentals of the complex interplay between microbial pathogens and their glycan receptors at the molecular level could lead to the development of novel therapeutics and diagnostics. Using Shiga toxin and influenza virus as examples, we describe the complex biological interface between host glycans and these infectious agents, and recent strategies to develop glycan-based high-affinity ligands. These molecules are expected to ultimately be incorporated into diagnostics and therapeutics, and can be used as probes to study important biological processes. Additionally, by focusing on the specific glycans that microbial pathogens target, we can begin to decipher the "glycocode" and how these glycans participate in normal and aberrant cellular communication. PMID:20135686

  17. N-Alkyl Ammonium Resorcinarene Salts as High-Affinity Tetravalent Chloride Receptors.

    PubMed

    Beyeh, N Kodiah; Pan, Fangfang; Bhowmik, Sandip; Mäkelä, Toni; Ras, Robin H A; Rissanen, Kari

    2016-01-22

    N-Alkyl ammonium resorcinarene salts (NARYs, Y=triflate, picrate, nitrate, trifluoroacetates and NARBr) as tetravalent receptors, are shown to have a strong affinity for chlorides. The high affinity for chlorides was confirmed from a multitude of exchange experiments in solution (NMR and UV/Vis), gas phase (mass spectrometry), and solid-state (X-ray crystallography). A new tetra-iodide resorcinarene salt (NARI) was isolated and fully characterized from exchange experiments in the solid-state. Competition experiments with a known monovalent bis-urea receptor (5) with strong affinity for chloride, reveals these receptors to have a much higher affinity for the first two chlorides, a similar affinity as 5 for the third chloride, and lower affinity for the fourth chloride. The receptors affinity toward chloride follows the trend K1 ≫K2 ≫K3 ≈5>K4, with Ka =5011 m(-1) for 5 in 9:1 CDCl3/[D6]DMSO. PMID:26671730

  18. Effects of histamine type-2 receptor antagonists on indomethacin and IL-2 immunotherapy of metastasis.

    PubMed

    Saarloos, M N; Khoo, N K; Lala, P K

    1993-05-01

    Histamine type-2 receptor antagonists (H-2RA) have been used chronically to prevent dyspepsia in cancer patients subjected to immunotherapy with chronic indomethacin (Indo) and intermittent IL-2 in our cancer centre. We tested the effects of these agents during immunotherapy of C3H/HeJ mice transplanted s.c. with 5 x 10(5) C3L5 mammary adenocarcinoma cells. Tumor-transplanted mice were divided into groups receiving: (1) Indo (14 micrograms/ml); (2) H-2RA, i.e. (a) ranitidine at 28.6 micrograms/ml (Ran-lo) or 143 micrograms/ml (Ran-hi), or (b) famotidine (Fam) at 4.3 micrograms/ml, or (c) cimetidine (Cim) at 107 micrograms/ml, all in the drinking water on days 5-24; (3) IL-2 (1.5 x 10(3) Cetus U i.p. every 8 h on days 10-14 and 20-24); (4) combinations of H-2RA + Indo; or (5) combinations of H-2RA + Indo + IL-2. Animals were killed on day 24 for examination of primary s.c. tumor growth, secondary lung metastasis and splenocyte cytotoxicity against YAC-1 lymphoma cells (51Cr release assay). Results revealed: (1) primary tumor growth was reduced in mice treated with Fam + Indo, Indo + IL-2 and any of the H-2RA + Indo + IL-2 (no difference observed within the last two groups); (2) lung metastases decreased in mice treated with IL-2 alone, Indo + IL-2, and Indo + IL-2 + Ran-hi; (3) splenic cytotoxicity was suppressed in tumor-bearing controls, with partial restoration seen in Ran (both doses), Ran-lo + Indo, Ran-lo + Indo + IL-2, and Cim + Indo + IL-2 treated groups. Nearly complete restoration was seen in Cim, Cim + Indo, Indo + IL-2, Ran-hi + Indo + IL-2, and Fam + Indo + IL-2 groups. Thus, addition of H-2RA did not alter the overall therapeutic efficacy of the standard Indo + IL-2 tumor immunotherapy. PMID:8097142

  19. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

    SciTech Connect

    Wang, Deng-Liang; Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan; Yang, Hai-Tao; Wang, Jiang-Jie; Yao, Pei-Sen; Pan, Ru-Jun; Yang, Chaoyong James; Kang, De-Zhi

    2014-10-31

    Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K{sub d} 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K{sub d} 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.

  20. Binding characteristics of [3H]14-methoxymetopon, a high affinity mu-opioid receptor agonist.

    PubMed

    Spetea, Mariana; Tóth, Fanni; Schütz, Johannes; Otvös, Ferenc; Tóth, Géza; Benyhe, Sandor; Borsodi, Anna; Schmidhammer, Helmut

    2003-07-01

    The highly potent micro -opioid receptor agonist 14-methoxymetopon (4,5alpha-epoxy-3-hydroxy-14beta-methoxy-5beta,17-dimethylmorphinan-6-one) was prepared in tritium labelled form by a catalytic dehalogenation method resulting in a specific radioactivity of 15.9 Ci/mmol. Opioid binding characteristics of [3H]14-methoxymetopon were determined using radioligand binding assay in rat brain membranes. [3H]14-Methoxymetopon specifically labelled a single class of opioid sites with affinity in low subnanomolar range (Ki = 0.43 nm) and maximal number of binding sites of 314 fmol/mg protein. Binding of [3H]14-methoxymetopon was inhibited by ligands selective for the micro -opioid receptor with high potency, while selective kappa-opioids and delta-opioids were weaker inhibitors. 14-Methoxymetopon increased guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]GTPgammaS) binding with an EC50 of 70.9 nm, thus, providing evidence for the agonist character of this ligand. The increase of [35S]GTPgammaS binding was inhibited by naloxone and selective micro -opioid antagonists, indicating a micro -opioid receptor-mediated action. [3H]14-Methoxymetopon is one of the few nonpeptide mu-opioid receptor agonists available in radiolabelled form up to now. Due to its high affinity and selectivity, high stability and extremely low nonspecific binding (<10%), this radioligand would be an important and useful tool in probing mu-opioid receptor mechanisms, as well as to promote a further understanding of the opioid system at the cellular and molecular level. PMID:12887410

  1. Endocytosis of lutropin by Leydig cells through a pathway distinct from the high-affinity receptor.

    PubMed

    Bozon, V; Pajot-Augy, E; Vignon, X; Salesse, R

    1998-08-25

    In porcine Leydig cells in primary culture, 95% of the internalization of [125I]porcine lutropin ([125I]pLH, which bears sulfated GalNAc) could not be ascribed to the high-affinity LH receptor (LHR). In contrast, >40% of [125I]human choriogonadotropin (hCG, with sialylated sugar chains) uptake was performed by the LHR itself. When the LHR was down-regulated by excess unlabeled hormone, the LHR-independent incorporation of [125I]pLH could be inhibited in a dose-dependent fashion by sulfated polysaccharides such as fucoidan or chondroitin-(4 or 6)-sulfate, but not by other polyanionic compounds, nor by sulfated chondroitin disaccharides. Endocytosis occurred through a clathrin-dependent pathway and was inhibited by low temperature, endocytosis inhibitors, increased ionic strength, or by EDTA and dithiothreitol. Taken together, these results suggest that a Leydig cell membrane protein (possibly a lectin, or a glycosaminoglycan receptor) could perform specific LH clearance in the testis via recognition of its sulfated sugars. PMID:9806348

  2. Molecular cloning of a second subunit of the receptor for human granulocyte - macrophage colony-stimulating factor (GM-CSF): Reconstitution of a high-affinity GM-CSF receptor

    SciTech Connect

    Hayashida, Kazuhiro; Kitamura, Toshio; Gorman, D.M.; Miyajima, Atsushi ); Arai, Kenichi; Yokota, Takashi )

    1990-12-01

    Using the mouse interleukin 3 (IL-3) receptor cDNA as a probe, the authors obtained a monologous cDNA (KH97) from a cDNA library of a human hemopoietic cell line, TF-1. The protein encoded by the KH97 cDNA has 56% amino acid sequence identity with the mouse IL-3 receptor and retains features common to the family of cytokine receptors. Fibroblasts transfected with the KH97 cDNA expressed a protein of 120 kDa but did not bind any human cytokines, including IL-3 and granulocyte - macrophage colony-stimulating factor (GM-CSF). Interestingly, cotransfection of cDNAs for KH97 and the low-affinity human GM-CSF receptor in fibroblasts resulted in formation of a high-affinity receptor for GM-CSF. The dissociation rate of GM-CSF from the reconstituted high-affinity receptor was slower than that from the low-affinity site, whereas the association rate was unchanged. Cross-linking of {sup 125}I-labeled GM-CSF to fibroblasts cotransfected with both cDNAs revealed the same cross-linking patterns as in TF-1 cells - i.e., two major proteins of 80 and 120 kDa which correspond to the low-affinity GM-CSF receptor and the KH97 protein, respectively. These results indicate that the high-affinity GM-CSF receptor is composed of at least two components in a manner analogous to the IL-2 receptor. They therefore propose to designate the low-affinity GM-CSF receptor and the KH97 protein as the {alpha} and {beta} subunits of the GM-CSF receptor, respectively.

  3. G Protein-Coupled Receptors Directly Bind Filamin A with High Affinity and Promote Filamin Phosphorylation

    PubMed Central

    2015-01-01

    Although interaction of a few G protein-coupled receptors (GPCRs) with Filamin A, a key actin cross-linking and biomechanical signal transducer protein, has been observed, a comprehensive structure–function analysis of this interaction is lacking. Through a systematic sequence-based analysis, we found that a conserved filamin binding motif is present in the cytoplasmic domains of >20% of the 824 GPCRs encoded in the human genome. Direct high-affinity interaction of filamin binding motif peptides of select GPCRs with the Ig domain of Filamin A was confirmed by nuclear magnetic resonance spectroscopy and isothermal titration calorimetric experiments. Engagement of the filamin binding motif with the Filamin A Ig domain induced the phosphorylation of filamin by protein kinase A in vitro. In transfected cells, agonist activation as well as constitutive activation of representative GPCRs dramatically elicited recruitment and phosphorylation of cellular Filamin A, a phenomenon long known to be crucial for regulating the structure and dynamics of the cytoskeleton. Our data suggest a molecular mechanism for direct GPCR–cytoskeleton coupling via filamin. Until now, GPCR signaling to the cytoskeleton was predominantly thought to be indirect, through canonical G protein-mediated signaling cascades involving GTPases, adenylyl cyclases, phospholipases, ion channels, and protein kinases. We propose that the GPCR-induced filamin phosphorylation pathway is a conserved, novel biochemical signaling paradigm. PMID:26460884

  4. Alterations of cortical pyramidal neurons in mice lacking high-affinity nicotinic receptors

    PubMed Central

    Ballesteros-Yáñez, Inmaculada; Benavides-Piccione, Ruth; Bourgeois, Jean-Pierre; Changeux, Jean-Pierre; DeFelipe, Javier

    2010-01-01

    The neuronal nicotinic acetylcholine receptors (nAChRs) are allosteric membrane proteins involved in multiple cognitive processes, including attention, learning, and memory. The most abundant form of heterooligomeric nAChRs in the brain contains the β2- and α4- subunits and binds nicotinic agonists with high affinity. In the present study, we investigated in the mouse the consequences of the deletion of one of the nAChR components: the β2-subunit (β2−/−) on the microanatomy of cortical pyramidal cells. Using an intracellular injection method, complete basal dendritic arbors of 650 layer III pyramidal neurons were sampled from seven cortical fields, including primary sensory, motor, and associational areas, in both β2−/− and WT animals. We observed that the pyramidal cell phenotype shows significant quantitative differences among different cortical areas in mutant and WT mice. In WT mice, the density of dendritic spines was rather similar in all cortical fields, except in the prelimbic/infralimbic cortex, where it was significantly higher. In the absence of the β2-subunit, the most significant reduction in the density of spines took place in this high-order associational field. Our data suggest that the β2-subunit is involved in the dendritic morphogenesis of pyramidal neurons and, in particular, in the circuits that contribute to the high-order functional connectivity of the cerebral cortex. PMID:20534523

  5. Comparison of three high affinity SPECT radiotracers for the dopamine D2 receptor.

    PubMed

    al-Tikriti, M S; Baldwin, R M; Zea-Ponce, Y; Sybirska, E; Zoghbi, S S; Laruelle, M; Malison, R T; Kung, H F; Kessler, R M; Charney, D S

    1994-02-01

    The regional brain distribution and pharmacological specificity of three high affinity tracers for the dopamine (DA) D2 receptor: [123I]IBF, [123I]epidepride, and [123I]2'-ISP were assessed by SPECT imaging of non-human primates. The ratios of striatal-to-occipital activities at the time of peak striatal uptake were 2.2, 6.3 and 1.7, respectively. From the peak striatal activities, washout rates were 33, 4 and 16%/h for [123I]IBF, [123I]epidepride and [123I]2'-ISP, respectively. The reversibility of the striatal uptake of all three agents was demonstrated by the rapid displacement induced by the dopamine D2 selective antipsychotic agent raclopride, which increased washout rates to 96, 58 and 43%/h. The administration of d-amphetamine, which induces release of dopamine, had no noticeable effect on [123I]epidepride but increased the washout rate of [123I]IBF. These results suggest that, among these three agents, [123I]epidepride is the superior tracer for in vivo displacement studies because of its slow washout and high target-to-background ratios. However, for tracer kinetic modeling, [123I]IBF may be the superior agent because of its early time of peak uptake and its higher target-to-background ratios than [123I]2'-ISP. PMID:9234281

  6. High-affinity receptors for peptides of the bombesin family in Swiss 3T3 cells

    SciTech Connect

    Zachary, I.; Rozengurt, E.

    1985-11-01

    Gastrin-releasing peptide (GRP) labeled with /sup 125/I at tyrosine-15 (/sup 125/I-GRP) binds to intact quiescent Swiss 3T3 cells in a specific and saturable manner. Scatchard analysis indicates the presence of a single class of high-affinity binding sites of Kd = 0.5 X 10(-9) M and a value for the number of sites per cell of about 100,000. /sup 125/I-GRP binding was not inhibited by other mitogens for these cells, and cell lines that are mitogenically unresponsive to GRP do not exhibit specific GRP binding. Structure-activity relationships show a close parallel between the ability of a range of GRP-related peptides to both inhibit GRP binding and to stimulate mitogenesis. Further, GRP binding is selectively blocked in a competitive fashion by a novel bombesin antagonist, (D-Arg1, D-Pro2, D-Trp7,9, Leu11) substance P. In addition, this compound selectively inhibits GRP and bombesin-induced mitogenesis. These results demonstrate that the mitogenic response of Swiss 3T3 cells to peptides of the bombesin family is mediated by a class of receptors distinct from those of other mitogens for these cells.

  7. High-affinity cannabinoid binding site in brain: A possible marijuana receptor

    SciTech Connect

    Nye, J.S.

    1988-01-01

    The mechanism by which delta{sup 9} tetrahydrocannabinol (delta{sup 9}THC), the major psychoactive component of marijuana or hashish, produces its potent psychological and physiological effects is unknown. To find receptor binding sites for THC, we designed a water-soluble analog for use as a radioligand. 5{prime}-Trimethylammonium-delta{sup 8}THC (TMA) is a positively charged analog of delta-{sup 8}THC modified on the 5{prime} carbon, a portion of the molecule not important for its psychoactivity. We have studied the binding of ({sup 3}H)-5{prime}-trimethylammonium-delta-{sup 8}THC (({sup 3}H)TMA) to rat neuronal membranes. ({sup 3}H)TMA binds saturably and reversibly to brain membranes with high affinity to apparently one class of sites. Highest binding site density occurs in brain, but several peripheral organs also display specific binding. Detergent solubilizes the sites without affecting their pharmacologial properties. Molecular sieve chromatography reveals a bimodal peak of ({sup 3}H)TMA binding activity of approximately 60,000 daltons apparent molecular weight.

  8. Characterization of the Staphylococcal enterotoxin A: Vβ receptor interaction using human receptor fragments engineered for high affinity.

    PubMed

    Sharma, P; Postel, S; Sundberg, E J; Kranz, D M

    2013-12-01

    Staphylococcal food poisoning is a gastrointestinal disorder caused by the consumption of food containing Staphylococcal enterotoxins. Staphylococcal enterotoxin A (SEA) is the most common enterotoxin recovered from food poisoning outbreaks in the USA. In addition to its enteric activity, SEA also acts as a potent superantigen through stimulation of T cells, although less is known about its interactions than the superantigens SEB, SEC and toxic shock syndrome toxin-1. To understand more about SEA:receptor interactions, and to develop toxin-detection systems for use in food testing, we engineered various SEA-binding receptor mutants. The extracellular domain of the receptor, a variable region of the beta chain (Vβ22) of the T-cell receptor, was engineered for stability as a soluble protein and for high affinity, using yeast-display technology. The highest affinity mutant was shown to bind SEA with a Kd value of 4 nM. This was a 25 000-fold improvement in affinity compared with the wild-type receptor, which bound to SEA with low affinity (Kd value of 100 µM), similar to other superantigen:Vβ interactions. The SEA:Vβ interface was centered around residues within the complementarity determining region 2 loop. The engineered receptor was specific for SEA, in that it did not bind to two other closely related enterotoxins SEE or SED, providing information on the SEA residues possibly involved in the interaction. The specificity and affinity of these high-affinity Vβ proteins also provide useful agents for the design of more sensitive and specific systems for SEA detection. PMID:24167300

  9. Synthesis and characterization of a high affinity radioiodinated probe for the alpha 2-adrenergic receptor

    SciTech Connect

    Lanier, S.M.; Hess, H.J.; Grodski, A.; Graham, R.M.; Homcy, C.J.

    1986-03-01

    The availability of radioiodinated probes has facilitated the localization and molecular characterization of cell membrane receptors for hormones and neurotransmitters. However, such probes are not available for the study of the alpha 2-adrenergic receptor. This report describes the synthesis and characterization of functionalized derivatives of the selective alpha 2-adrenergic antagonists, rauwolscine and yohimbine, which can be radiolabeled to high specific activity with 125I. Following demethylation of rauwolscine or yohimbine, the resultant carboxylic acid derivatives were reacted with 4-aminophenethylamine to yield the respective 4-aminophenethyl carboxamides, 17 alpha-hydroxy-20 alpha-yohimban-16 beta-(N-4-amino-phenethyl)carboxamide (rau-pAPC) and 17 alpha-hydroxy-20 beta-yohimban-16 alpha-(N-4-aminophenethyl)carboxamide. In competitive inhibition studies using rat renal membranes and the radioligand (3H)rauwolscine, rau-pAPC (Ki = 11 +/- 1 nM) exhibited a 14-fold greater affinity than the corresponding yohimbine derivative (Ki = 136 +/- 45 nM). The higher affinity compound, rau-pAPC, was radioiodinated by the chloramine T method, and the product, 125I-rau-pAPC (17 alpha-hydroxy-20 alpha-yohimban-16 beta-(N-4-amino-3 -(125I)iodophenethyl)carboxamide), was purified by reverse phase HPLC to high specific activity (2175 Ci/mmol) and its binding characteristics were investigated in rat kidney membranes. Specific binding of 125I-rau-pAPC was saturable and of high affinity as determined by Scatchard analysis (KD = 1.8 +/- 0.3 nM) or from kinetic studies (KD = k2/k1 = 0.056 +/- 0.013 min-1)/4.3 +/- 0.2 X 10(7) M-1 min-1 = 1.3 +/- 0.3 nM).

  10. Production of a High-affinity Monoclonal Antibody Reactive with Folate Receptors Alpha and Beta.

    PubMed

    Nagai, Taku; Furusho, Yuko; Li, Hua; Hasui, Kazuhisa; Matsukita, Sumika; Sueyoshi, Kazunobu; Yanagi, Masakazu; Hatae, Masaki; Takao, Sonshin; Matsuyama, Takami

    2015-06-01

    Folate receptors α (FRα) and β (FRβ) are two isoforms of the cell surface glycoprotein that binds folate. The expression of FRα is rare in normal cells and elevated in cancer cells. Thus, FRα-based tumor-targeted therapy has been a focus area of laboratory research and clinical trials. Recently, it was shown that a significant fraction of tumor-associated macrophages expresses FRβ and that these cells can enhance tumor growth. Although FRα and FRβ share 70% identity in their deduced amino acid sequence, a monoclonal antibody (MAb) reactive with both receptors has not been developed. A MAb that can target both FRα-expressing cancer cells and FRβ-expressing tumor-associated macrophages may provide a more potent therapeutic tool for cancer than individual anti-FRα or anti-FRβ MAbs. In this study, we developed a MAb that recognizes both FRα and FRβ (anti-FRαβ). The anti-FRαβ specifically stained trophoblasts and macrophages from human placenta, synovial macrophages from rheumatoid arthritis patient, liver macrophages from cynomolgus monkey and common marmoset, and cancer cells and tumor-associated macrophages from ovary and lung carcinomas. Surface plasmon resonance showed that the anti-FRαβ bound to soluble forms of the FRα and FRβ proteins with high affinity (KD=6.26×10(-9) M and 4.33×10(-9) M, respectively). In vitro functional analysis of the anti-FRαβ showed that this MAb mediates complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity, and antibody-dependent cellular phagocytosis of FRα-expressing and FRβ-expressing cell lines. The anti-FRαβ MAb is a promising therapeutic candidate for cancers in which macrophages promote tumor progression. PMID:26090596

  11. The molecular basis for high affinity of a universal ligand for human bombesin receptor (BnR) family members

    PubMed Central

    Uehara, Hirotsugu; Hocart, Simon J.; González, Nieves; Mantey, Samuel A.; Nakagawa, Tomoo; Katsuno, Tatsuro; Coy, David H.; Jensen, Robert T.

    2012-01-01

    There is increased interest in the Bn-receptor family because they are frequently over/ectopically-expressed by tumors and thus useful as targets for imaging or receptor-targeted-cytotoxicity. The synthetic Bn-analog,[D-Tyr6,β-Ala11,Phe13,Nle14]Bn(6-14)[Univ.Lig] has the unique property of having high affinity for all three human BNRs(GRPR,NMBR,BRS-3), and thus could be especially useful for this approach. However, the molecular basis of this property is unclear and is the subject of this study. To accomplish this, site-directed mutagenesis was used after identifying potentially important amino acids using sequence homology analysis of all BnRs with high affinity for Univ.Lig compared to the Cholecystokinin-receptor(CCKAR), which has low affinity. Using various criteria 74 amino acids were identified and 101 mutations made in GRPR by changing each to those of CCKAR or to alanine. 22 GRPR mutations showed a significant decrease in affinity for Univ.Lig(>2-fold) with 2 in EC2[ D97N,G112V], 1 in UTM6[Y284A], 2 in EC4[R287N,H300S] showing >10-fold decrease in Univ.Lig affinity. Additional mutations were made to explore the molecular basis for these changes. Our results show that high affinity for Univ.Lig by human Bn-receptors requires positively charged amino acids in extracellular (EC)-domain 4 and to a lesser extent EC2 and EC3 suggesting charge-charge interactions may be particularly important for determining the general high affinity of this ligand. Furthermore, transmembrane amino acids particularly in UTM6 are important contributing both charge-charge interactions as well as interaction with a tyrosine residue in close proximity suggesting possible receptor-peptide cation-pi or H–bonding interactions are also important for determining its high affinity. PMID:22828605

  12. Soluble interleukin-2 receptors (sIL-2R) in Hodgkin's disease: outcome and clinical implications.

    PubMed Central

    Viviani, S.; Camerini, E.; Bonfante, V.; Santoro, A.; Balzarotti, M.; Fornier, M.; Devizzi, L.; Verderio, P.; Valagussa, P.; Bonadonna, G.

    1998-01-01

    The aim of this study was to assess the prognostic role of soluble interleukin-2 receptors (sIL-2R) in Hodgkin's disease (HD) both in the achievement of complete remission (CR) and in predicting disease relapse. Between August 1988 and June 1993 sIL-2R serum levels were measured in 174 untreated patients; in 137 of them evaluation was repeated at the end of treatment and in 132 also during the follow-up. Baseline sIL-2R levels (mean+/-standard error) were significantly higher in patients than in 65 healthy control subjects (1842+/-129 U ml(-1) vs 420+/-10 U ml(-10, P< 0.0001). At the end of treatment 135 out of 137 evaluated patients achieved complete response (CR) and their mean sIL-2R serum levels were significantly lower than those at diagnosis (635+/-19 U ml(-1) vs 1795+/-122 U ml(-1), P=0.0001). After a median follow-up of 5 years, sIL-2R remained low in 114 patients in continuous CR, while they increased in 9 out of 12 patients (75%) who relapsed. However, a temporary increase was also observed in six patients (5%) still in CR. Treatment outcome in terms of freedom from progression was linearly related to sIL-2R levels. Our study confirms that patients with untreated HD have increased baseline levels of sIL-2R compared with healthy subjects and that their pretreatment values may be an indication of disease outcome similar to other conventional prognostic factors, such as number of involved sites, presence of B symptoms and extranodal extent. PMID:9528846

  13. Early Signs of Pathological Cognitive Aging in Mice Lacking High-Affinity Nicotinic Receptors.

    PubMed

    Konsolaki, Eleni; Tsakanikas, Panagiotis; Polissidis, Alexia V; Stamatakis, Antonios; Skaliora, Irini

    2016-01-01

    In order to address pathological cognitive decline effectively, it is critical to adopt early preventive measures in individuals considered at risk. It is therefore essential to develop approaches that identify such individuals before the onset of irreversible dementia. A deficient cholinergic system has been consistently implicated as one of the main factors associated with a heightened vulnerability to the aging process. In the present study we used mice lacking high affinity nicotinic receptors (β2-/-), which have been proposed as an animal model of accelerated/premature cognitive aging. Our aim was to identify behavioral signs that could serve as indicators or predictors of impending cognitive decline. We used test batteries in order to assess cognitive functions and additional tasks to investigate spontaneous behaviors, such as species-specific activities and exploration/locomotion in a novel environment. Our data confirm the hypothesis that β2-/- animals exhibit age-related cognitive impairments in spatial learning. In addition, they document age-related deficits in other areas, such as recognition memory, burrowing and nesting building, thereby extending the validity of this animal model for the study of pathological aging. Finally, our data reveal deficits in spontaneous behavior and habituation processes that precede the onset of cognitive decline and could therefore be useful as a non-invasive behavioral screen for identifying animals at risk. To our knowledge, this is the first study to perform an extensive behavioral assessment of an animal model of premature cognitive aging, and our results suggest that β2-nAChR dependent cognitive deterioration progressively evolves from initial subtle behavioral changes to global dementia due to the combined effect of the neuropathology and aging. PMID:27199738

  14. Early Signs of Pathological Cognitive Aging in Mice Lacking High-Affinity Nicotinic Receptors

    PubMed Central

    Konsolaki, Eleni; Tsakanikas, Panagiotis; Polissidis, Alexia V.; Stamatakis, Antonios; Skaliora, Irini

    2016-01-01

    In order to address pathological cognitive decline effectively, it is critical to adopt early preventive measures in individuals considered at risk. It is therefore essential to develop approaches that identify such individuals before the onset of irreversible dementia. A deficient cholinergic system has been consistently implicated as one of the main factors associated with a heightened vulnerability to the aging process. In the present study we used mice lacking high affinity nicotinic receptors (β2-/-), which have been proposed as an animal model of accelerated/premature cognitive aging. Our aim was to identify behavioral signs that could serve as indicators or predictors of impending cognitive decline. We used test batteries in order to assess cognitive functions and additional tasks to investigate spontaneous behaviors, such as species-specific activities and exploration/locomotion in a novel environment. Our data confirm the hypothesis that β2-/- animals exhibit age-related cognitive impairments in spatial learning. In addition, they document age-related deficits in other areas, such as recognition memory, burrowing and nesting building, thereby extending the validity of this animal model for the study of pathological aging. Finally, our data reveal deficits in spontaneous behavior and habituation processes that precede the onset of cognitive decline and could therefore be useful as a non-invasive behavioral screen for identifying animals at risk. To our knowledge, this is the first study to perform an extensive behavioral assessment of an animal model of premature cognitive aging, and our results suggest that β2-nAChR dependent cognitive deterioration progressively evolves from initial subtle behavioral changes to global dementia due to the combined effect of the neuropathology and aging. PMID:27199738

  15. The marine phycotoxin gymnodimine targets muscular and neuronal nicotinic acetylcholine receptor subtypes with high affinity.

    PubMed

    Kharrat, Riadh; Servent, Denis; Girard, Emmanuelle; Ouanounou, Gilles; Amar, Muriel; Marrouchi, Riadh; Benoit, Evelyne; Molgó, Jordi

    2008-11-01

    Gymnodimines (GYMs) are phycotoxins exhibiting unusual structural features including a spirocyclic imine ring system and a trisubstituted tetrahydrofuran embedded within a 16-membered macrocycle. The toxic potential and the mechanism of action of GYM-A, highly purified from contaminated clams, have been assessed. GYM-A in isolated mouse phrenic hemidiaphragm preparations produced a concentration- and time-dependent block of twitch responses evoked by nerve stimulation, without affecting directly elicited muscle twitches, suggesting that it may block the muscle nicotinic acetylcholine (ACh) receptor (nAChR). This was confirmed by the blockade of miniature endplate potentials and the recording of subthreshold endplate potentials in GYM-A paralyzed frog and mouse isolated neuromuscular preparations. Patch-clamp recordings in Xenopus skeletal myocytes revealed that nicotinic currents evoked by constant iontophoretical ACh pulses were blocked by GYM-A in a reversible manner. GYM-A also blocked, in a voltage-independent manner, homomeric human alpha7 nAChR expressed in Xenopus oocytes. Competition-binding assays confirmed that GYM-A is a powerful ligand interacting with muscle-type nAChR, heteropentameric alpha3beta2, alpha4beta2, and chimeric alpha7-5HT(3) neuronal nAChRs. Our data show for the first time that GYM-A broadly targets nAChRs with high affinity explaining the basis of its neurotoxicity, and also pave the way for designing specific tests for accurate GYM-A detection in shellfish samples. PMID:18990115

  16. Selectively Promiscuous Opioid Ligands: Discovery of High Affinity/Low Efficacy Opioid Ligands with Substantial Nociceptin Opioid Peptide Receptor Affinity

    PubMed Central

    2015-01-01

    Emerging clinical and preclinical evidence suggests that a compound displaying high affinity for μ, κ, and δ opioid (MOP, KOP, and DOP) receptors and antagonist activity at each, coupled with moderate affinity and efficacy at nociceptin opioid peptide (NOP) receptors will have utility as a relapse prevention agent for multiple types of drug abuse. Members of the orvinol family of opioid ligands have the desired affinity profile but have typically displayed substantial efficacy at MOP and or KOP receptors. In this study it is shown that a phenyl ring analogue (1d) of buprenorphine displays the desired profile in vitro with high, nonselective affinity for the MOP, KOP, and DOP receptors coupled with moderate affinity for NOP receptors. In vivo, 1d lacked any opioid agonist activity and was an antagonist of both the MOP receptor agonist morphine and the KOP receptor agonist ethylketocyclazocine, confirming the desired opioid receptor profile in vivo. PMID:24761755

  17. Biphasic regulation of development of the high-affinity saxitoxin receptor by innervation in rat skeletal muscle

    SciTech Connect

    Sherman, S.J.; Catterall, W.A.

    1982-11-01

    Specific binding of /sup 3/H-saxitoxin (STX) was used to quantitate the density of voltage-sensitive sodium channels in developing rat skeletal muscle. In adult triceps surae, a single class of sites with a KD . 2.9 nM and a density of 21 fmol/mg wet wt was detected. The density of these high-affinity sites increased from 2.0 fmol/mg wet wt to the adult value in linear fashion during days 2-25 after birth. Denervation of the triceps surae at day 11 or 17 reduced final saxitoxin receptor site density to 10.4 or 9.2 fmol/mg wet wt, respectively, without changing KD. Denervation of the triceps surae at day 5 did not alter the subsequent development of saxitoxin receptor sites during days 5-9 and accelerated the increase of saxitoxin receptor sites during days 9-13. After day 13, saxitoxin receptor development abruptly ceased and the density of saxitoxin receptor sites declined to 11 fmol/wg wet wt. These results show that the regulation of high-affinity saxitoxin receptor site density by innervation is biphasic. During the first phase, which is independent of continuing innervation, the saxitoxin receptor density increases to 47-57% of the adult level. After day 11, the second phase of development, which is dependent on continuing innervation, gives rise to the adult density of saxitoxin receptors.

  18. Molecular basis for high affinity and selectivity of peptide antagonist, Bantag-1, for the orphan BB3 receptor.

    PubMed

    Nakamura, Taichi; Ramos-Álvarez, Irene; Iordanskaia, Tatiana; Moreno, Paola; Mantey, Samuel A; Jensen, R T

    2016-09-01

    Bombesin-receptor-subtype-3 (BB3 receptor) is a G-protein-coupled-orphan-receptor classified in the mammalian Bombesin-family because of high homology to gastrin-releasing peptide (BB2 receptor)/neuromedin-B receptors (BB1 receptor). There is increased interest in BB3 receptor because studies primarily from knockout-mice suggest it plays roles in energy/glucose metabolism, insulin-secretion, as well as motility and tumor-growth. Investigations into its roles in physiological/pathophysiological processes are limited because of lack of selective ligands. Recently, a selective, peptide-antagonist, Bantag-1, was described. However, because BB3 receptor has low-affinity for all natural, Bn-related peptides, little is known of the molecular basis of its high-affinity/selectivity. This was systematically investigated in this study for Bantag-1 using a chimeric-approach making both Bantag-1 loss-/gain-of-affinity-chimeras, by exchanging extracellular (EC) domains of BB3/BB2 receptor, and using site-directed-mutagenesis. Receptors were transiently expressed and affinities determined by binding studies. Bantag-1 had >5000-fold selectivity for BB3 receptor over BB2/BB1 receptors and substitution of the first EC-domain (EC1) in loss-/gain-of affinity-chimeras greatly affected affinity. Mutagenesis of each amino acid difference in EC1 between BB3 receptor/BB2 receptor showed replacement of His(107) in BB3 receptor by Lys(107) (H107K-BB3 receptor-mutant) from BB2 receptor, decreased affinity 60-fold, and three replacements [H107K, E11D, G112R] decreased affinity 500-fold. Mutagenesis in EC1's surrounding transmembrane-regions (TMs) demonstrated TM2 differences were not important, but R127Q in TM3 alone decreased affinity 400-fold. Additional mutants in EC1/TM3 explored the molecular basis for these changes demonstrated in EC1, particularly important is the presence of aromatic-interactions by His(107), rather than hydrogen-bonding or charge-charge interactions, for determining

  19. A biophysical approach to IL-2 and IL-15 receptor function: localization, conformation and interactions.

    PubMed

    Bodnár, Andrea; Nizsalóczki, Eniko; Mocsár, Gábor; Szalóki, Nikoletta; Waldmann, Thomas A; Damjanovich, Sándor; Vámosi, György

    2008-03-15

    Interleukin-2 and interleukin-15 (IL-2, IL-15) are key participants in T and NK cell activation and function. Sharing the beta and gamma receptor subunits results in several common functions: e.g. the promotion of T cell proliferation. On the other hand, due to their distinct alpha receptor subunits, they also play opposing roles in immune processes such as activation induced cell death and immunological memory. Divergence of signaling pathways must ensue already at the plasma membrane where the cytokines interact with their receptors. Therefore understanding molecular details of receptor organization and mapping interactions with other membrane proteins that might influence receptor conformation and function, are of key importance. Biophysical/advanced microscopic methods (fluorescence resonance energy transfer (FRET), fluorescence crosscorrelation spectroscopy (FCCS), near-field scanning optical microscopy (NSOM), X-ray crystallography, surface plasmon resonance, NMR spectroscopy) have been instrumental in clarifying the details of receptor structure and organization from the atomic level to the assembly and dynamics of supramolecular clusters. In this short review some important contributions shaping our current view of IL-2 and IL-15 receptors are presented. PMID:18280585

  20. Characterization of tetanus toxin binding to rat brain membranes. Evidence for a high-affinity proteinase-sensitive receptor.

    PubMed Central

    Pierce, E J; Davison, M D; Parton, R G; Habig, W H; Critchley, D R

    1986-01-01

    Binding of 125I-labelled tetanus toxin to rat brain membranes in 25 mM-Tris/acetate, pH 6.0, was saturable and there was a single class of high-affinity site (KD 0.26-1.14 nM) present in high abundance (Bmax. 0.9-1.89 nmol/mg). The sites were largely resistant to proteolysis and heating but were markedly sensitive to neuraminidase. Trisialogangliosides were effective inhibitors of toxin binding (IC50 10 nM) and trisialogangliosides inserted into membranes lacking a toxin receptor were able to bind toxin with high affinity (KD 2.6 nM). The results are consistent with previous studies and the hypothesis that di- and trisialogangliosides act as the primary receptor for tetanus toxin under these conditions. In contrast, when toxin binding was assayed in Krebs-Ringer buffer, pH 7.4, binding was greatly reduced, was non-saturable and competition binding studies showed evidence for a small number of high-affinity sites (KD 0.42 nM, Bmax. 0.90 pmol/mg) and a larger number of low-affinity sites (KD 146 nM, Bmax. 179 pmol/mg). Treatment of membranes with proteinases, heat, and neuraminidase markedly reduced binding. Trisialogangliosides were poor inhibitors of toxin binding (IC50 11.0 microM), and trisialogangliosides inserted into membranes bound toxin with low affinity. The results suggest that in physiological buffers tetanus toxin binds with high affinity to a protein receptor, and that gangliosides represent only a low-affinity site. Images Fig. 5. PMID:3539106

  1. Inter-residue coupling contributes to high-affinity subtype-selective binding of α-bungarotoxin to nicotinic receptors

    PubMed Central

    Sine, Steven M.; Huang, Sun; Li, Shu-Xing; daCOSTA, Corrie J. B.; Chen, Lin

    2014-01-01

    The crystal structure of a pentameric α7 ligand-binding domain chimaera with bound α-btx (α-bungarotoxin) showed that of the five conserved aromatic residues in α7, only Tyr184 in loop C of the ligand-binding site was required for high-affinity binding. To determine whether the contribution of Tyr184 depends on local residues, we generated mutations in an α7/5HT3A (5-hydroxytryptamine type 3A) receptor chimaera, individually and in pairs, and measured 125I-labelled α-btx binding. The results show that mutations of individual residues near Tyr184 do not affect α-btx affinity, but pairwise mutations decrease affinity in an energetically coupled manner. Kinetic measurements show that the affinity decreases arise through increases in the α-btx dissociation rate with little change in the association rate. Replacing loop C in α7 with loop C from the α-btx-insensitive α2 or α3 subunits abolishes high-affinity α-btx binding, but preserves acetylcholine-elicited single channel currents. However, in both the α2 and α3 construct, mutating either residue that flanks Tyr184 to its α7 counterpart restores high-affinity α-btx binding. Analogously, in α7, mutating both residues that flank Tyr184 to the α2 or α3 counterparts abolishes high-affinity α-btx binding. Thus interaction between Tyr184 and local residues contributes to high-affinity subtype-selective α-btx binding. PMID:23802200

  2. Point mutation increases a form of the NK1 receptor with high affinity for neurokinin A and B and septide

    PubMed Central

    Ciucci, Alessandra; Palma, Carla; Manzini, Stefano; Werge, Thomas M

    1998-01-01

    The binding modalities of substance P and neurokinin A on the wild type and Gly166 to-Cys mutant NK1 receptors expressed on CHO cells were investigated in homologous and heterologous binding experiments using both radiolabelled substance P and neurokinin A.On the wild type NK1 receptor NKA displaces radiolabelled substance P with very low apparent affinity, despite its high-affinity binding constant (determined in homologous binding experiments). The Gly166 to-Cys substitution in the NK1 tachykinin receptor greatly enhances the apparent affinity of neurokinin A in competition for radiolabelled substance P, but it does not change the binding constant of neurokinin A. The mutation, thereby, eliminates the discrepancy between the low apparent affinity and the high binding constant of neurokinin A.On the wild type receptor the binding capacity of neurokinin A is significantly smaller than that of substance P. In contrast, the two tachykinins bind to approximately the same number of sites on the mutant receptor.Simultaneous mass action law analysis of binding data in which multiple radioligands were employed in parallel demonstrated that a one-site model was unable to accommodate all the experimental data, whereas a two-site model provided a dramatically better description.These two receptor-sites display equally high affinity for substance P, while neurokinin A strongly discriminates between a high and a low affinity component. The binding affinities of neurokinin A are not affected by the mutation, which instead specifically alters the distribution between receptor sites in favour of a high affinity neurokinin A binding form.The low apparent affinity and binding capacity of neurokinin A on the wild type receptor results from neurokinin A binding with high affinity only to a fraction of the sites labelled by substance P. The mutation increases the proportion of this site, and consequently enhances the apparent affinity and binding capacity of neurokinin A.The binding

  3. Current concepts. I. High affinity receptors for bombesin/GRP-like peptides on human small cell lung cancer

    SciTech Connect

    Moody, T.W.; Carney, D.N.; Cuttitta, F.; Quattrocchi, K.; Minna, J.D.

    1985-07-15

    The binding of a radiolabeled bombesin analogue to human small cell lung cancer (SCLC) cell lines was investigated. (/sup 125/I-Tyr/sup 4/)bombesin bound with high affinity (Kd = 0.5 nM) to a single class of sites (2000/cell) using SCLC line NCI-H446. Binding was reversible, saturable and specific. The pharmacology of binding was investigated, using NCI-H466 and SCLC line NCI-H345. Bombesin and structurally related peptides, such as gastrin releasing peptide (GRP), but not other peptides, such as substance P or vasopressin, inhibited high affinity (/sup 125/I-Tyr/sup 4/)BN binding activity. Finally, the putative receptor, a 78,000 dalton polypeptide, was identified by purifying radiolabeled cell lysates on bombesin or GRP affinity resins and then displaying the bound polypeptides on sodium dodecylsulfate polyacrylamide gels. Because SCLC both produces bombesin/GRP-like peptides and contains high affinity receptors for these peptides, they may function as important autocrine regulatory factors for human SCLC. 31 references, 6 figures, 2 tables.

  4. Activation of the IL-2 Receptor in Podocytes: A Potential Mechanism for Podocyte Injury in Idiopathic Nephrotic Syndrome?

    PubMed Central

    Zea, Arnold H.; Stewart, Tyrus; Ascani, Jeannine; Tate, David J.; Finkel-Jimenez, Beatriz; Wilk, Anna; Reiss, Krzysztof; Smoyer, William E.; Aviles, Diego H.

    2016-01-01

    The renal podocyte plays an important role in maintaining the structural integrity of the glomerular basement membrane. We have previously reported that patients with idiopathic nephrotic syndrome (INS) have increased IL-2 production. We hypothesized that podocytes express an IL-2 receptor (IL-2R) and signaling through this receptor can result in podocyte injury. To confirm the presence of the IL-2R, we tested a conditionally immortalized murine podocyte cell line by flow cytometry, qPCR, and Western blot. To test for the presence of the IL-2R in vivo, immunohistochemical staining was performed on human renal biopsies in children with FSGS and control. Podocytes were stimulated with IL-2 in vitro, to study signaling events via the JAK/STAT pathway. The results showed that stimulation with IL-2 resulted in increased mRNA and protein expression of STAT 5a, phosphorylated STAT 5, JAK 3, and phosphorylated JAK 3. We then investigated for signs of cellular injury and the data showed that pro-apoptotic markers Bax and cFLIP were significantly increased following IL-2 exposure, whereas LC3 II was decreased. Furthermore, mitochondrial depolarization and apoptosis were both significantly increased following activation of the IL-2R. We used a paracellular permeability assay to monitor the structural integrity of a podocyte monolayer following IL-2 exposure. The results showed that podocytes exposed to IL-2 have increased albumin leakage across the monolayer. We conclude that murine podocytes express the IL-2R, and that activation through the IL-2R results in podocyte injury. PMID:27389192

  5. Two high-affinity ligand binding states of uterine estrogen receptor distinguished by modulation of hydrophobic environment

    SciTech Connect

    Hutchens, T.W.; Li, C.M.; Zamah, N.M.; Besch, P.K.

    1987-02-10

    The steroid binding function of soluble (cytosolic) estrogen receptors from calf uteri was evaluated under conditions known to modify the extent of hydrophobic interaction with receptor-associated proteins. Receptor preparations were equilibrated into 6 M urea buffers and control buffers by chromatography through small columns of Sephadex G-25 or by dialysis at 0.6 /sup 0/C. Equilibrium dissociation constants (K/sub d/) and binding capacities (n) of experimental and control receptor preparations were determined by 13-point Scatchard analyses using concentrations of 17..beta..-(/sup 3/H)estradiol from 0.05 to 10 nM. Nonspecific binding was determined at each concentration by parallel incubations with a 200-fold molar excess of the receptor-specific competitor diethylstilbestrol. The control receptor population was consistently found to be a single class of binding sites with a high affinity for estradiol which was unaffected by G-25 chromatography, by dialysis, by dilution, or by the presence of 0.4 M KCl. However, equilibration into 6 M urea induced a discrete (10-fold) reduction in receptor affinity to reveal a second, thermodynamically stable, high-affinity binding state. The presence of 0.4 M KCl did not significantly influence the discrete change in receptor affinity induced by urea. The effects of urea on both receptor affinity and binding capacity were reversible, suggesting a lack of covalent modification. These results demonstrate nonenzymatic means by which not only the binding capacity but also the affinity of receptor for estradiol can be reversibly controlled, suggesting that high concentrations of urea might be more effectively utilized during the physicochemical characterization and purification of steroid receptor proteins.

  6. Soluble T cell receptor Vβ domains engineered for high-affinity binding to staphylococcal or streptococcal superantigens.

    PubMed

    Sharma, Preeti; Wang, Ningyan; Kranz, David M

    2014-02-01

    Staphylococcus aureus and group A Streptococcus secrete a collection of toxins called superantigens (SAgs), so-called because they stimulate a large fraction of an individual's T cells. One consequence of this hyperactivity is massive cytokine release leading to severe tissue inflammation and, in some cases, systemic organ failure and death. The molecular basis of action involves the binding of the SAg to both a T cell receptor (TCR) on a T cell and a class II product of the major histocompatibility complex (MHC) on an antigen presenting cell. This cross-linking leads to aggregation of the TCR complex and signaling. A common feature of SAgs is that they bind with relatively low affinity to the variable region (V) of the beta chain of the TCR. Despite this low affinity binding, SAgs are very potent, as each T cell requires only a small fraction of their receptors to be bound in order to trigger cytokine release. To develop high-affinity agents that could neutralize the activity of SAgs, and facilitate the development of detection assays, soluble forms of the Vβ regions have been engineered to affinities that are up to 3 million-fold higher for the SAg. Over the past decade, six different Vβ regions against SAgs from S. aureus (SEA, SEB, SEC3, TSST-1) or S. pyogenes (SpeA and SpeC) have been engineered for high-affinity using yeast display and directed evolution. Here we review the engineering of these high-affinity Vβ proteins, structural features of the six different SAgs and the Vβ proteins, and the specific properties of the engineered Vβ regions that confer high-affinity and specificity for their SAg ligands. PMID:24476714

  7. Localization of the binding site for the human high-affinity Fc receptor on IgG.

    PubMed

    Duncan, A R; Woof, J M; Partridge, L J; Burton, D R; Winter, G

    1988-04-01

    A major pathway in the clearance of pathogens involves the coating of the pathogen with specific antibodies, and the binding of the antibody Fc region to cell receptors. This can trigger engulfment of the pathogen by phagocytes or lysis by killer cells. By oligonucleotide site-directed mutagenesis we have engineered a single amino acid change in a mouse IgG2b antibody (Glu 235----Leu) which now enables the antibody to bind to the FcRI (high affinity) receptor on human monocytes with a 100-fold improvement in affinity. This indicates that Leu 235 is a major determinant in the binding of antibody to FcRI and that the receptor may interact directly with the region linking the CH2 domain to the hinge. Tailoring the affinity of antibodies for cell receptors could help dissect their role in clearing pathogen. PMID:2965792

  8. Temperature dependence of high-affinity CCK receptor binding and CCK internalization in rat pancreatic acini

    SciTech Connect

    Williams, J.A.; Bailey, A.C.; Roach, E. Univ. of California, San Francisco )

    1988-04-01

    {sup 125}I-labeled cholecystokinin (CCK) binding and internalization were studied as a function of temperatures in isolated rat pancreatic acini. At 37{degree}C, acini readily bound and degraded {sup 125}I-CCK. When labeled hormone binding was inhibited by increasing amounts of unlabeled CCK, competition-inhibition curves were biphasic, consistent with both high- (K{sub d}, 18 pM) and low-affinity (K{sub d}, 13 nM) binding sites. At 4{degree}C, acini bound only one-third as much {sup 125}I-CCK and degradation was essentially abolished. At 4{degree}C, CCK competition curves were consistent with a single class of low-affinity binding sites (K{sub d}, 19 nM). Internalization of {sup 125}I-CCK was evaluated by three washing procedures utilizing acid, base, and trypsin. All were shown to remove membrane-bound {sup 125}I-CCK, and this finding was validated for trypsin by electron microscope autotradiography. When internalization of {sup 125}I-CCK was evaluated as a function of the medium concentration of CCK, both high- and low-affinity components were observed. These results suggest that high-affinity CCK binding and CCK internalization are separate temperature-sensitive processes. Moreover, internalization is not uniquely associated with high-affinity binding.

  9. Essential biphasic role for JAK3 catalytic activity in IL-2 receptor signaling.

    PubMed

    Smith, Geoffrey A; Uchida, Kenji; Weiss, Arthur; Taunton, Jack

    2016-05-01

    To drive lymphocyte proliferation and differentiation, common γ-chain (γc) cytokine receptors require hours to days of sustained stimulation. JAK1 and JAK3 kinases are found together in all γc-receptor complexes, but how their respective catalytic activities contribute to signaling over time is not known. Here we dissect the temporal requirements for JAK3 kinase activity with a selective covalent inhibitor (JAK3i). By monitoring phosphorylation of the transcription factor STAT5 over 20 h in CD4(+) T cells stimulated with interleukin 2 (IL-2), we document a second wave of signaling that is much more sensitive to JAK3i than the first wave. Selective inhibition of this second wave is sufficient to block cyclin expression and entry to S phase. An inhibitor-resistant JAK3 mutant (C905S) rescued all effects of JAK3i in isolated T cells and in mice. Our chemical genetic toolkit elucidates a biphasic requirement for JAK3 kinase activity in IL-2-driven T cell proliferation and will find broad utility in studies of γc-receptor signaling. PMID:27018889

  10. Solubilization of high affinity corticotropin-releasing factor receptors from rat brain: Characterization of an active digitonin-solubilized receptor complex

    SciTech Connect

    Grigoriadis, D.E.; Zaczek, R.; Pearsall, D.M.; De Souza, E.B. )

    1989-12-01

    The binding characteristics of CRF receptors in rat frontal cerebral cortex membranes solubilized in 1% digitonin were determined. The binding of (125I)Tyro-ovine CRF ((125I)oCRF) to solubilized membrane proteins was dependent on incubation time, temperature, and protein concentration, was saturable and of high affinity, and was absent in boiled tissue. The solubilized receptors retained their high affinity for (125I) oCRF in the solubilized state, exhibiting a dissociation constant (KD) of approximately 200 pM, as determined by direct binding saturation isotherms. Solubilized CRF receptors maintained the rank order of potencies for various related and unrelated CRF peptides characteristic of the membrane CRF receptor: rat/human CRF congruent to ovine CRF congruent to Nle21,38-rat CRF greater than alpha-helical oCRF-(9-41) greater than oCRF-(7-41) much greater than vasoactive intestinal peptide, arginine vasopressin, or the substance-P antagonist. Furthermore, the absolute potencies (Ki values) for the various CRF-related peptides in solubilized receptors were almost identical to those observed in the membrane preparations, indicating that the CRF receptor retained its high affinity binding capacity in the digitonin-solubilized state. Chemical affinity cross-linking of digitonin-solubilized rat cortical membrane proteins revealed a specifically labeled protein with an apparent mol wt of 58,000 which was similar to the labeled protein in native membrane homogenates. Although solubilized CRF receptors retained their high affinity for agonists, their sensitivity for guanine nucleotide was lost. Size exclusion chromatography substantiated these results, demonstrating that in the presence or absence of guanine nucleotides, (125I)oCRF labeled the same size receptor complex.

  11. Trifluoromethoxyl Substituted Phenylethylene Diamines as High Affinity σ Receptor Ligands with Potent Anti-Cocaine Actions

    PubMed Central

    Smith, Trudy A.; Yang, Xiaowen; Wu, Huifang; Pouw, Buddy; Matsumoto, Rae R.; Coop, Andrew

    2012-01-01

    The phenylethylene diamines are a class of σ receptor ligands with excellent selectivity over other biological systems and with anti-cocaine actions that involve antagonism of σ1 receptors. In order to increase the potency of the aromatic methoxyl substituted analogues, trifluoromethoxyl groups were introduced to prevent metabolic demethylation. The para-substituted trifluoromethoxyl substituted analogues were shown to have increased σ receptor affinity and represent the most potent anti-cocaine phenylethylene diamines yet described. PMID:18461921

  12. Progress toward a high-affinity allosteric enhancer at muscarinic M1 receptors.

    PubMed

    Lazareno, Sebastian; Popham, Angela; Birdsall, Nigel J M

    2003-01-01

    Loss of forebrain acetylcholine is an early neurochemical lesion in Alzheimer's disease (AD). As muscarinic acetylcholine receptors are involved in memory and cognition, a muscarinic agonist could therefore provide a "replacement therapy" in this disease. However, muscarinic receptors occur throughout the CNS and the periphery. A selective locus of action of a muscarinic agonist is therefore crucial in order to avoid intolerable side effects. The five subtypes of muscarinic receptors, M1-M5, have distinct regional distributions with M2 and M3 receptors mediating most of the peripheral effects. M1 receptors are the major receptor subtype in the cortex and hippocampus-the two brain regions most associated with memory and cognition. This localization has led to a, so far unsuccessful, search for a truly M1-selective muscarinic agonist. However, acetylcholinesterase inhibitors, such as donepezil (Aricept), which potentiate cholinergic neurotransmission, do have a therapeutic role in the management of AD and so the M1 receptor remains a viable therapeutic target. Our approach is to develop muscarinic allosteric enhancers-compounds that bind to the receptor at an "allosteric" site, which is distinct from the "primary" site to which ACh binds, and which enhance ACh affinity (or efficacy). Having discovered that a commercially available compound, WIN 62577, is an allosteric enhancer with micromolar potency at M3 receptors, we report here some results of a chemical synthesis project to develop this hit. Modification of WIN 62577 has led to compounds with over 1000-fold increased affinity but, so far, none of these extremely potent compounds are allosteric enhancers. PMID:14501021

  13. A role for the intermediate affinity IL-2R in the protection against glucocorticoid-induced apoptosis.

    PubMed Central

    Rebollo, A; Pitton, C; García, A; Gómez, J; Silva, A

    1995-01-01

    Recent work has shown that T lymphocytes undergo apoptosis upon treatment with the glucocorticoid analogue dexamethasone. These cells can be protected from the effect of dexamethasone by interleukin-2 (IL-2) or IL-4. We were interested in analysing whether a transfected cell dependent on three different lymphokines could be protected by them from the effect of dexamethasone. In addition, we took advantage of our cellular system, in which we expressed intermediate- or high-affinity IL-2R independently, to analyse the role of these receptors in the protection from glucocorticoid-induced apoptosis. In this report we show that IL-2 rescues murine T cells expressing exogenous intermediate- (TS1 beta) or high-affinity (TS1 alpha beta) IL-2 receptor (IL-2R) from dexamethasone-induced apoptosis. This result suggests that intermediate-affinity IL-2R alone can replace high-affinity IL-2R for the protection from the effect of dexamethasone. In addition, IL-4 and IL-9 are rescue-factors, as well as IL-2, of glucocorticoid-treated TS1 beta and TS1 alpha beta cells. Our data suggest that the presence of the alpha-chain of the IL-2R is not required for rescue by IL-2 from the effect of dexamethasone. In addition, we show that proliferation is not required for preventing glucocorticoid-induced apoptosis. This result implies a new role for the intermediate-affinity IL-2R. Images Figure 4 Figure 7 PMID:7751021

  14. Anticancer immunotherapy by CTLA-4 blockade: obligatory contribution of IL-2 receptors and negative prognostic impact of soluble CD25

    PubMed Central

    Hannani, Dalil; Vétizou, Marie; Enot, David; Rusakiewicz, Sylvie; Chaput, Nathalie; Klatzmann, David; Desbois, Melanie; Jacquelot, Nicolas; Vimond, Nadège; Chouaib, Salem; Mateus, Christine; Allison, James P; Ribas, Antoni; Wolchok, Jedd D; Yuan, Jianda; Wong, Philip; Postow, Michael; Mackiewicz, Andrzej; Mackiewicz, Jacek; Schadendorff, Dirk; Jaeger, Dirk; Korman, Alan J; Bahjat, Keith; Maio, Michele; Calabro, Luana; Teng, Michele WL; Smyth, Mark J; Eggermont, Alexander; Robert, Caroline; Kroemer, Guido; Zitvogel, Laurence

    2015-01-01

    The cytotoxic T lymphocyte antigen-4 (CTLA-4)-blocking antibody ipilimumab induces immune-mediated long-term control of metastatic melanoma in a fraction of patients. Although ipilimumab undoubtedly exerts its therapeutic effects via immunostimulation, thus far clinically useful, immunologically relevant biomarkers that predict treatment efficiency have been elusive. Here, we show that neutralization of IL-2 or blocking the α and β subunits of the IL-2 receptor (CD25 and CD122, respectively) abolished the antitumor effects and the accompanying improvement of the ratio of intratumoral T effector versus regulatory cells (Tregs), which were otherwise induced by CTLA-4 blockade in preclinical mouse models. CTLA-4 blockade led to the reduction of a suppressive CD4+ T cell subset expressing Lag3, ICOS, IL-10 and Egr2 with a concomitant rise in IL-2-producing effector cells that lost FoxP3 expression and accumulated in regressing tumors. While recombinant IL-2 improved the therapeutic efficacy of CTLA-4 blockade, the decoy IL-2 receptor α (IL-2Rα, sCD25) inhibited the anticancer effects of CTLA-4 blockade. In 262 metastatic melanoma patients receiving ipilimumab, baseline serum concentrations of sCD25 represented an independent indicator of overall survival, with high levels predicting resistance to therapy. Altogether, these results unravel a role for IL-2 and IL-2 receptors in the anticancer activity of CTLA-4 blockade. Importantly, our study provides the first immunologically relevant biomarker, namely elevated serum sCD25, that predicts resistance to CTLA-4 blockade in patients with melanoma. PMID:25582080

  15. Anticancer immunotherapy by CTLA-4 blockade: obligatory contribution of IL-2 receptors and negative prognostic impact of soluble CD25.

    PubMed

    Hannani, Dalil; Vétizou, Marie; Enot, David; Rusakiewicz, Sylvie; Chaput, Nathalie; Klatzmann, David; Desbois, Melanie; Jacquelot, Nicolas; Vimond, Nadège; Chouaib, Salem; Mateus, Christine; Allison, James P; Ribas, Antoni; Wolchok, Jedd D; Yuan, Jianda; Wong, Philip; Postow, Michael; Mackiewicz, Andrzej; Mackiewicz, Jacek; Schadendorff, Dirk; Jaeger, Dirk; Zörnig, Inka; Hassel, Jessica; Korman, Alan J; Bahjat, Keith; Maio, Michele; Calabro, Luana; Teng, Michele Wl; Smyth, Mark J; Eggermont, Alexander; Robert, Caroline; Kroemer, Guido; Zitvogel, Laurence

    2015-02-01

    The cytotoxic T lymphocyte antigen-4 (CTLA-4)-blocking antibody ipilimumab induces immune-mediated long-term control of metastatic melanoma in a fraction of patients. Although ipilimumab undoubtedly exerts its therapeutic effects via immunostimulation, thus far clinically useful, immunologically relevant biomarkers that predict treatment efficiency have been elusive. Here, we show that neutralization of IL-2 or blocking the α and β subunits of the IL-2 receptor (CD25 and CD122, respectively) abolished the antitumor effects and the accompanying improvement of the ratio of intratumoral T effector versus regulatory cells (Tregs), which were otherwise induced by CTLA-4 blockade in preclinical mouse models. CTLA-4 blockade led to the reduction of a suppressive CD4(+) T cell subset expressing Lag3, ICOS, IL-10 and Egr2 with a concomitant rise in IL-2-producing effector cells that lost FoxP3 expression and accumulated in regressing tumors. While recombinant IL-2 improved the therapeutic efficacy of CTLA-4 blockade, the decoy IL-2 receptor α (IL-2Rα, sCD25) inhibited the anticancer effects of CTLA-4 blockade. In 262 metastatic melanoma patients receiving ipilimumab, baseline serum concentrations of sCD25 represented an independent indicator of overall survival, with high levels predicting resistance to therapy. Altogether, these results unravel a role for IL-2 and IL-2 receptors in the anticancer activity of CTLA-4 blockade. Importantly, our study provides the first immunologically relevant biomarker, namely elevated serum sCD25, that predicts resistance to CTLA-4 blockade in patients with melanoma. PMID:25582080

  16. Towards a high-affinity allosteric enhancer at muscarinic M1 receptors.

    PubMed

    Lazareno, Sebastian; Popham, Angela; Birdsall, Nigel J M

    2002-01-01

    Loss of forebrain acetylcholine (ACh) is an early neurochemical lesion in Alzheimer's Disease (AD), and muscarinic receptors for ACh are involved in memory and cognition, so a muscarinic agonist could provide 'replacement therapy' in this disease. Muscarinic receptors, which couple to G-proteins, occur throughout the CNS, and in the periphery they mediate the responses of the parasympathetic nervous system, so selectivity is crucial. The five subtypes of muscarinic receptor, M1-M5, have a distinct regional distribution, with M2 and M3 mediating most of the peripheral effects, M2 predominating in hindbrain areas, and M1 predominating in the cortex and hippocampus--the brain regions most associated with memory and cognition, which has lead to a search for a truly M1-selective muscarinic agonist. That search has so far been unsuccessful, but acetylcholinesterase inhibitors such as donepezil (Aricept), which potentiate cholinergic neurotransmission, have a therapeutic role in the management of AD; so the M1 receptor remains a therapeutic target. Our approach is to develop allosteric enhancers--compounds which bind to the receptor at an 'allosteric' site which is distinct from the 'primary' site to which the endogenous ligand binds, and which enhance the affinity (or efficacy) of the endogenous ligand. We have developed radioligand binding assays and analyses for the detection and quantitatitation of allosteric interactions of a test agent with labelled and unlabelled 'primary' ligands, and we report here some results of the initial phase of a chemical synthesis project to develop potent and selective allosteric enhancers at muscarinic M1 receptors. PMID:12212769

  17. Molecular docking approaches in identification of High affinity inhibitors of Human SMO receptor

    PubMed Central

    Akare, Uday Raj; Bandaru, Srinivas; Shaheen, Uzma; Singh, Pramod Kumar; Tiwari, Geet; Singare, Paramanand; Nayarisseri, Anuraj; Banerjee, Tushar

    2014-01-01

    Inappropriate activation of the Hh signaling pathway has been implicated in the development of several types of cancers including prostate, lung, pancreas, breast, brain and skin. Present study identified the binding affinities of eight established inhibitors viz., Cyclopamine, Saridegib, Itraconazole, LDE-225, TAK-441, BMS-833923 (XL139), PF-04449913 and Vismodegib targeting SMO receptor - a candidate protein involved in hedgehog pathway and sought to identify the best amongst the established inhibitors through by molecular docking. Exelxis® BMS 833923 (XL 139) demonstrated superior binding affinity aided by MolDock scoring docking algorithm. Further BMS 833923 (XL 139) was evaluated for pharmacophoric features which revealed appreciable ligand receptor interactions. PMID:25670876

  18. Human and rat mast cell high-affinity immunoglobulin E receptors: Characterization of putative. alpha. -chain gene products

    SciTech Connect

    Shimizu, Akira; Benfey, P.N.; Leder, P. ); Tepler, I. Brigham and Women's Hospital, Boston, MA ); Berenstein, E.H.; Siraganian, R.P. )

    1988-03-01

    The authors have cloned and determined the entire nucleotide sequence of cDNAs corresponding to the putative {alpha} subunits of the human and rat mast cell high-affinity IgE receptors. Both human and rat cDNAs encode an NH{sub 2}-terminal signal peptide, two immunoglobulin-like extracellular domains (encoded by discrete exons), a hydrophobic transmembrane region, and a positively charged cytoplasmic tail. The human and rat {alpha} subunits share an overall homology with one another and the immunoglobulin gene family, suggesting that they arose from a common ancestral gene and continue to share structural homology with their ligands. In addition, the rat gene is transcribed into at least three distinct forms, each of which yields a somewhat different coding sequence.

  19. The M2 selective antagonist AF-DX 116 shows high affinity for muscarine receptors in bovine tracheal membranes.

    PubMed

    Roffel, A F; in't Hout, W G; de Zeeuw, R A; Zaagsma, J

    1987-05-01

    We have characterized the muscarine receptors in bovine tracheal and left ventricular membranes using 3H-dexetimide/pirenzepine and 3H-dexetimide/AF-DX 116 competition studies. Pirenzepine exhibited low (M2) affinity binding to both preparations; Kd was 590 nM in left ventricle and 463 nM in trachea. AF-DX 116 exhibited high (M2) affinity binding to left ventricle (Kd = 95.6 nM); in tracheal membranes it bound with high (M2) affinity (Kd = 40.7 nM) to 74% of the receptors and with low (M3) affinity (Kd = 2.26 microM) to 26% of the receptors. It is concluded that bovine tracheal muscle membranes contain a heterogeneous population of muscarine binding sites, the majority having M2 (heart) subtype characteristics and being located on the smooth muscle membranes; a minority having M3 (exocrine gland) subtype characteristics and presumed to be located in submucosal glands. This is the first report of high affinity binding of AF-DX 116 to non-cardiac peripheral muscarine receptors. PMID:3614390

  20. A high-affinity receptor for urokinase plasminogen activator on human keratinocytes: characterization and potential modulation during migration.

    PubMed Central

    McNeill, H; Jensen, P J

    1990-01-01

    Low passage cultures of normal human keratinocytes produce several components of the plasminogen activator/plasmin proteolytic cascade, including urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), and two specific inhibitors. Studies here presented demonstrate that these cells also contain a high-affinity (Kd = 3 x 10(-10) M) plasma membrane-binding site for uPA. High molecular weight uPA, either as the single-chain precursor or two-chain activated form, bound to the receptor; however, low molecular weight (33 kD) uPA, tPA, or epidermal growth factor did not compete for binding, demonstrating specificity. Acid treatment, which removed endogenous uPA from the receptor, was required to detect maximal binding (45,000 sites per cell). To investigate the possibility that the uPA receptor on keratinocytes may be involved in epithelial migration during wound repair, cultures were wounded and allowed to migrate into the wounded site. Binding sites for uPA were localized by autoradiographic analysis of 125I-uPA binding as well as by immunocytochemical studies using anti-uPA IgG. With both techniques uPA binding sites were detected selectively on the plasma membrane of cells at the leading edge of the migrating epithelial sheet. This localization pattern suggests that uPA receptor expression on keratinocytes may be coupled to cell migration during cutaneous wounding. Images PMID:1965151

  1. High affinity dopamine D2 receptor radioligands. 1. Regional rat brain distribution of iodinated benzamides.

    PubMed

    Kessler, R M; Ansari, M S; de Paulis, T; Schmidt, D E; Clanton, J A; Smith, H E; Manning, R G; Gillespie, D; Ebert, M H

    1991-08-01

    Five 125I-labeled substituted benzamides, which are close structural analogues of (S)-sulpiride, eticlopride, and isoremoxipride, were evaluated for their selective in vivo uptake into dopamine D2 receptor rich tissue of the rat brain. "Iodopride" (KD 0.88 nM), an iodine substituted benzamide structurally related to sulpiride, displayed a maximal striatum: cerebellar uptake ratio of 7.6. Demonstration of saturation of the receptor with [125I]iodopride in striatum required uptake in frontal cortex to be used, rather than cerebellar uptake, to define nonspecific binding. Two other ligands structurally related to eticlopride, "iclopride" (KD 0.23 nM) and "itopride" (KD 0.16 nM), displayed maximal striatal: cerebellar uptake ratios of 9.8 and 3.3, respectively. The most potent ligands, "epidepride" (KD 0.057 nM) and "ioxipride" (KD 0.070 nM) showed striatal:cerebellar uptake ratios of 234 and 65, respectively. The observed uptake ratios correlated poorly with the affinity constants for the dopamine D2 receptor alone, but were highly correlated (r = 0.92) with the product of the receptor dissociation constant (KD) and the apparent lipophilicity (kw), as determined by reverse-phase HPLC at pH 7.5. Total striatal uptake also appeared dependent on lipophilicity, with maximal uptake occurring for ligands having log kw 2.4-2.8. PMID:1831229

  2. High affinity dopamine D2 receptor radioligands. 1. Regional rat brain distribution of iodinated benzamides

    SciTech Connect

    Kessler, R.M.; Ansari, M.S.; de Paulis, T.; Schmidt, D.E.; Clanton, J.A.; Smith, H.E.; Manning, R.G.; Gillespie, D.; Ebert, M.H. )

    1991-08-01

    Five 125I-labeled substituted benzamides, which are close structural analogues of (S)-sulpiride, eticlopride, and isoremoxipride, were evaluated for their selective in vivo uptake into dopamine D2 receptor rich tissue of the rat brain. Iodopride (KD 0.88 nM), an iodine substituted benzamide structurally related to sulpiride, displayed a maximal striatum: cerebellar uptake ratio of 7.6. Demonstration of saturation of the receptor with (125I)iodopride in striatum required uptake in frontal cortex to be used, rather than cerebellar uptake, to define nonspecific binding. Two other ligands structurally related to eticlopride, iclopride (KD 0.23 nM) and itopride (KD 0.16 nM), displayed maximal striatal: cerebellar uptake ratios of 9.8 and 3.3, respectively. The most potent ligands, epidepride (KD 0.057 nM) and ioxipride (KD 0.070 nM) showed striatal:cerebellar uptake ratios of 234 and 65, respectively. The observed uptake ratios correlated poorly with the affinity constants for the dopamine D2 receptor alone, but were highly correlated (r = 0.92) with the product of the receptor dissociation constant (KD) and the apparent lipophilicity (kw), as determined by reverse-phase HPLC at pH 7.5. Total striatal uptake also appeared dependent on lipophilicity, with maximal uptake occurring for ligands having log kw 2.4-2.8.

  3. Bodilisant—A Novel Fluorescent, Highly Affine Histamine H3 Receptor Ligand

    PubMed Central

    2012-01-01

    A piperidine-based lead structure for the human histamine H3 receptor (hH3R) was coupled with the BODIPY fluorophore and resulted in a strong green fluorescent (quantum yield, 0.92) hH3R ligand with affinity in the nanomolar concentration range (Ki hH3R = 6.51 ± 3.31 nM), named Bodilisant. Screening for affinities at histamine and dopamine receptor subtypes showed high hH3R preference. Bodilisant was used for visualization of hH3R in hH3R overexpressing HEK-293 cells with fluorescence confocal laser scanning microscopy. In addition, in native human brain tissues, Bodilisant showed clear and displaceable images of labeled hH3R. PMID:24900647

  4. Micro-structured peptide surfaces for the detection of high-affinity peptide-receptor interactions in living cells.

    PubMed

    Lipp, Anna-Maria; Ji, Bozhi; Hager, Roland; Haas, Sandra; Schweiggl, Simone; Sonnleitner, Alois; Haselgrübler, Thomas

    2015-12-15

    Peptide ligands have great potential as selective agents for diagnostic imaging and therapeutic targeting of human cancers. A number of high-throughput assays for screening potential candidate peptides have been developed. Although these screening assays are indispensable for the identification of peptide ligands at a large scale, it is crucial to validate peptide binding and selectivity for targeted receptors in a live-cell context. For testing high-affinity peptide-receptor interactions in the plasma membrane of living cells, we developed cell-resistant, micro-structured glass surfaces with high-density and high-contrast peptide features. Cell adhesion and recruitment of fluorescent receptors to micro-patterned peptides in the live-cell membrane were evaluated by reflection interference contrast (RIC) and total internal reflection (TIRF) microscopy, respectively. To demonstrate both the specificity and modularity of the assay, co-patterning of fluorescent receptors with three different immobilized micro-structured ligands was shown: first, interaction of green fluorescent protein (GFP)-tagged epidermal growth factor (EGF) receptor expressed in Jurkat cells with immobilized EGF was detected and quantified. Second, using Jurkat cells, we demonstrated specific interaction of yellow fluorescent protein (YFP)-tagged β3 integrin with c(RGDfK) peptide. Third, we identified indirect recruitment of GFP-tagged α5 integrin to an 11-mer peptide. In summary, our results show that the developed micro-structured surfaces are a useful tool for the validation and quantification of peptide-receptor interactions in their natural cellular environment. PMID:26210593

  5. ZK91587: a novel synthetic antimineralocorticoid displays high affinity for corticosterone (type I) receptors in the rat hippocampus

    SciTech Connect

    Sutanto, W.; de Kloet, E.R.

    1988-01-01

    In vitro cytosol binding assays have shown the properties of binding of a novel steroid, ZK91587 (15..beta.., 16..beta..b-methylene-mexrenone) in the brain of rats. Scatchard and Woolf analyses of the binding data reveal the binding of (/sup 3/H) ZK91587 to the total hippocampal coritcosteroid receptor sites with high affinity, and low capacity. When 100-fold excess RU28362 was included simultaneously with (/sup 3/H) ZK91587, the labelled steroid binds with the same affinity and capacity. Relative binding affinities (RBA) of various steroids for the Type I or Type II corticosteroid receptor in these animals are: Type I: ZK91587 = corticosterone (B) > cortisol (F); Type II: B > F >>> ZK91587. In the binding kinetic study, ZK91587 has a high association rate of binding in the rat. The steroid dissociates following a one slope pattern, indicating, the present data demonstrate that in the rat hippocampus, ZK91587 binds specifically to the Type I (corticosterone-preferring/mineralocorticoid-like receptor.

  6. Progress in the discovery of selective, high affinity A(2B) adenosine receptor antagonists as clinical candidates.

    PubMed

    Kalla, Rao V; Zablocki, Jeff

    2009-03-01

    The selective, high affinity A(2B) adenosine receptor (AdoR) antagonists that were synthesized by several research groups should aid in determining the role of the A(2B) AdoR in inflammatory diseases like asthma or rheumatoid arthritis (RA) and angiogenic diseases like diabetic retinopathy or cancer. CV Therapeutics scientists discovered the selective, high affinity A(2B) AdoR antagonist 10, a 8-(4-pyrazolyl)-xanthine derivative [CVT-6883, K(i)(hA(2B)) = 22 nM; K(i)(hA(1)) = 1,940 nM; K(i)(hA(2A)) = 3,280; and K(i)(hA(3)) = 1,070 nM] that has favorable pharmacokinetic (PK) properties (t (1/2) = 4 h and F > 35% rat). Compound 10 demonstrated functional antagonism at the A(2B) AdoR (K(B) = 6 nM) and efficacy in a mouse model of asthma. In two phase 1 clinical trials, CVT-6883 was found to be safe, well tolerated, and suitable for once daily dosing. A second compound 20, 8-(5-pyrazolyl)-xanthine, has been nominated for development from Baraldi's group in conjunction with King Pharmaceuticals that has favorable A(2B) AdoR affinity and selectivity [K(i)(hA(2B)) = 5.5 nM; K(i)(hA(1)) > 1,000 nM; K(i)(hA(2A)) > 1,000; and K(i)(hA(3)) > 1,000 nM], and it has been demonstrated to be a functional antagonist. A third compound 32, a 2-aminopyrimidine, from the Almirall group has high A(2B) AdoR affinity and selectivity [K(i)(hA(2B)) = 17 nM; K(i)(hA(1)) > 1,000 nM; K(i)(hA(2A)) > 2,500; and K(i)(hA(3)) > 1,000 nM], and 32 has been moved into preclinical safety testing. Since three highly selective, high affinity A(2B) AdoR antagonists have been nominated for development with 10 (CVT-6883) being the furthest along in the development process, the role of the A(2B) AdoR in various disease states will soon be established. PMID:18568423

  7. AGIA Tag System Based on a High Affinity Rabbit Monoclonal Antibody against Human Dopamine Receptor D1 for Protein Analysis

    PubMed Central

    Yano, Tomoya; Takeda, Hiroyuki; Uematsu, Atsushi; Yamanaka, Satoshi; Nomura, Shunsuke; Nemoto, Keiichirou; Iwasaki, Takahiro; Takahashi, Hirotaka; Sawasaki, Tatsuya

    2016-01-01

    Polypeptide tag technology is widely used for protein detection and affinity purification. It consists of two fundamental elements: a peptide sequence and a binder which specifically binds to the peptide tag. In many tag systems, antibodies have been used as binder due to their high affinity and specificity. Recently, we obtained clone Ra48, a high-affinity rabbit monoclonal antibody (mAb) against dopamine receptor D1 (DRD1). Here, we report a novel tag system composed of Ra48 antibody and its epitope sequence. Using a deletion assay, we identified EEAAGIARP in the C-terminal region of DRD1 as the minimal epitope of Ra48 mAb, and we named this sequence the “AGIA” tag, based on its central sequence. The tag sequence does not include the four amino acids, Ser, Thr, Tyr, or Lys, which are susceptible to post-translational modification. We demonstrated performance of this new tag system in biochemical and cell biology applications. SPR analysis demonstrated that the affinity of the Ra48 mAb to the AGIA tag was 4.90 × 10−9 M. AGIA tag showed remarkably high sensitivity and specificity in immunoblotting. A number of AGIA-fused proteins overexpressed in animal and plant cells were detected by anti-AGIA antibody in immunoblotting and immunostaining with low background, and were immunoprecipitated efficiently. Furthermore, a single amino acid substitution of the second Glu to Asp (AGIA/E2D) enabled competitive dissociation of AGIA/E2D-tagged protein by adding wild-type AGIA peptide. It enabled one-step purification of AGIA/E2D-tagged recombinant proteins by peptide competition under physiological conditions. The sensitivity and specificity of the AGIA system makes it suitable for use in multiple methods for protein analysis. PMID:27271343

  8. AGIA Tag System Based on a High Affinity Rabbit Monoclonal Antibody against Human Dopamine Receptor D1 for Protein Analysis.

    PubMed

    Yano, Tomoya; Takeda, Hiroyuki; Uematsu, Atsushi; Yamanaka, Satoshi; Nomura, Shunsuke; Nemoto, Keiichirou; Iwasaki, Takahiro; Takahashi, Hirotaka; Sawasaki, Tatsuya

    2016-01-01

    Polypeptide tag technology is widely used for protein detection and affinity purification. It consists of two fundamental elements: a peptide sequence and a binder which specifically binds to the peptide tag. In many tag systems, antibodies have been used as binder due to their high affinity and specificity. Recently, we obtained clone Ra48, a high-affinity rabbit monoclonal antibody (mAb) against dopamine receptor D1 (DRD1). Here, we report a novel tag system composed of Ra48 antibody and its epitope sequence. Using a deletion assay, we identified EEAAGIARP in the C-terminal region of DRD1 as the minimal epitope of Ra48 mAb, and we named this sequence the "AGIA" tag, based on its central sequence. The tag sequence does not include the four amino acids, Ser, Thr, Tyr, or Lys, which are susceptible to post-translational modification. We demonstrated performance of this new tag system in biochemical and cell biology applications. SPR analysis demonstrated that the affinity of the Ra48 mAb to the AGIA tag was 4.90 × 10-9 M. AGIA tag showed remarkably high sensitivity and specificity in immunoblotting. A number of AGIA-fused proteins overexpressed in animal and plant cells were detected by anti-AGIA antibody in immunoblotting and immunostaining with low background, and were immunoprecipitated efficiently. Furthermore, a single amino acid substitution of the second Glu to Asp (AGIA/E2D) enabled competitive dissociation of AGIA/E2D-tagged protein by adding wild-type AGIA peptide. It enabled one-step purification of AGIA/E2D-tagged recombinant proteins by peptide competition under physiological conditions. The sensitivity and specificity of the AGIA system makes it suitable for use in multiple methods for protein analysis. PMID:27271343

  9. Behavioral interactions between ethanol and imidazodiazepines with high affinities for benzodiazepine receptors

    SciTech Connect

    Lister, R.G.

    1988-01-01

    The intrinsic effect of two imidazodiazepines RO 15-3505 and RO 17-1812 on the behavior of mice in a holeboard test were investigated. The interactions of these two drugs with ethanol were also studied. RO 15-3505 failed to significantly alter either exploratory head-dipping or locomotor activity when administered alone but doses of 0.75 and 1.5 mg/kg reversed the reduction in the number of head-dips caused by ethanol and partially reversed ethanol's locomotor stimulant action. In contrast, RO 17-1812 increased locomotor activity when administered alone, and enhanced the reduction in exploration caused by ethanol. Neither RO 15-3505 nor RO 17-1812 altered blood alcohol concentrations suggesting a pharmacodynamic basis for these interactions. The results suggest that in the holeboard test the interactions of imidazodiazepines with ethanol are related to the nature of their interaction with benzodiazepine receptors, inverse agonists antagonising and agonists enhancing ethanol's effects on exploration.

  10. NMR and computational evidence that high-affinity bradykinin receptor antagonists adopt C-terminal beta-turns.

    PubMed

    Kyle, D J; Blake, P R; Smithwick, D; Green, L M; Martin, J A; Sinsko, J A; Summers, M F

    1993-05-14

    Three tetrapeptides were prepared, each corresponding to the four C-terminal amino acid residues of highly potent, second-generation bradykinin receptor antagonists. The tetrapeptides are (IA) Ser-D-Phe-Oic-Arg, (IIA) Ser-D-Tic-Oic-Arg, and (IIIA) Ser-D-Hype(trans-propyl)-Oic-Arg. Solution conformations for each were determined by incorporating interproton distance restraints, determined by 2D NMR experiments performed in water at neutral pH, into a series of distance geometry/simulated annealing model building calculations. Similarly, systematic conformational analyses were performed for each using molecular mechanics calculations. Both the NMR-derived structures, as well as the calculated structures, are shown to adopt a beta-turn as the primary conformation. Excellent agreement between the predicted structures and the NMR-derived structures is demonstrated. Aside from being the first examples of linear tetrapeptides reported to be ordered in aqueous solvent, the results presented support the hypothesis that high-affinity bradykinin receptor antagonists must adopt C-terminal beta-turn conformations. PMID:8388469

  11. The Analysis of the Human High Affinity IgE Receptor FceRIa from Multiple Crystal Forms

    SciTech Connect

    Garman, S.C.; Sechi, S.; Kinet, J.-P.; Jardetzky, T.S.

    2010-03-05

    We have solved the structure of the human high affinity IgE receptor, Fc{var_epsilon}RI{alpha}, in six different crystal forms, showing the structure in 15 different chemical environments. This database of structures shows no change in the overall shape of the molecule, as the angle between domains 1 and 2 (D1 and D2) varies little across the ensemble. However, the receptor has local conformational variability in the C' strand of D2 and in the BC loop of D1. In every crystal form, a residue inserts between tryptophan residues 87 and 110, mimicking the position of a proline from the IgE ligand. The different crystal forms reveal a distribution of carbohydrates lining the front and back surfaces of the structure. An analysis of crystal contacts in the different forms indicates regions where the molecule interacts with other proteins, and reveals a potential new binding site distal to the IgE binding site. The results of this study point to new directions for the design of molecules to inhibit the interaction of Fc{var_epsilon}RI{alpha} with its natural ligand and thus to prevent a primary step in the allergic response.

  12. Development of indazolylpyrimidine derivatives as high-affine EphB4 receptor ligands and potential PET radiotracers.

    PubMed

    Ebert, Kristin; Wiemer, Jens; Caballero, Julio; Köckerling, Martin; Steinbach, Jörg; Pietzsch, Jens; Mamat, Constantin

    2015-09-01

    Due to their essential role in the pathogenesis of cancer, members of the Eph (erythropoietin-producing hepatoma cell line-A2) receptor tyrosine kinase family represent promising candidates for molecular imaging. Thus, the development and preparation of novel radiotracers for the noninvasive imaging of the EphB4 receptor via positron emission tomography (PET) is described. First in silico investigations with the indazolylpyrimidine lead compound which is known to be highly affine to EphB4 were executed to identify favorable labeling positions for an introduction of fluorine-18 to retain the affinity. Based on this, reference compounds as well as precursors were developed and labeled with carbon-11 and fluorine-18, respectively. For this purpose, a protecting group strategy essentially had to be generated to prevent unwanted methylation and to enable the introduction of fluorine-18. Further, a convenient radiolabeling strategy using [(11)C]methyl iodide was established which afforded the isotopically labeled radiotracer in 30-35% RCY (d.c.) which is identical with the original inhibitor molecule. A spiro ammonium precursor was prepared for radiolabeling with fluorine-18. Unfortunately, the labeling did not lead to the desired (18)F-radiotracer under the chosen conditions. PMID:26189032

  13. Human Adenosine A2A Receptor Binds Calmodulin with High Affinity in a Calcium-Dependent Manner

    PubMed Central

    Piirainen, Henni; Hellman, Maarit; Tossavainen, Helena; Permi, Perttu; Kursula, Petri; Jaakola, Veli-Pekka

    2015-01-01

    Understanding how ligands bind to G-protein-coupled receptors and how binding changes receptor structure to affect signaling is critical for developing a complete picture of the signal transduction process. The adenosine A2A receptor (A2AR) is a particularly interesting example, as it has an exceptionally long intracellular carboxyl terminus, which is predicted to be mainly disordered. Experimental data on the structure of the A2AR C-terminus is lacking, because published structures of A2AR do not include the C-terminus. Calmodulin has been reported to bind to the A2AR C-terminus, with a possible binding site on helix 8, next to the membrane. The biological meaning of the interaction as well as its calcium dependence, thermodynamic parameters, and organization of the proteins in the complex are unclear. Here, we characterized the structure of the A2AR C-terminus and the A2AR C-terminus-calmodulin complex using different biophysical methods, including native gel and analytical gel filtration, isothermal titration calorimetry, NMR spectroscopy, and small-angle X-ray scattering. We found that the C-terminus is disordered and flexible, and it binds with high affinity (Kd = 98 nM) to calmodulin without major conformational changes in the domain. Calmodulin binds to helix 8 of the A2AR in a calcium-dependent manner that can displace binding of A2AR to lipid vesicles. We also predicted and classified putative calmodulin-binding sites in a larger group of G-protein-coupled receptors. PMID:25692595

  14. IL-18-induced expression of high-affinity IL-2R on murine NK cells is essential for NK-cell IFN-γ production during murine Plasmodium yoelii infection.

    PubMed

    Stegmann, Kerstin A; De Souza, J Brian; Riley, Eleanor M

    2015-12-01

    Early production of pro-inflammatory cytokines, including IFN-γ, is essential for control of blood-stage malaria infections. We have shown that IFN-γ production can be induced among human natural killer (NK) cells by coculture with Plasmodium falciparum infected erythrocytes, but the importance of this response is unclear. To further explore the role of NK cells during malaria infection, we have characterized the NK-cell response of C57BL/6 mice during lethal (PyYM) or nonlethal (Py17XNL) P. yoelii infection. Ex vivo flow cytometry revealed that NK cells are activated within 24 h of Py17XNL blood-stage infection, expressing CD25 and producing IFN-γ; this response was blunted and delayed during PyYM infection. CD25 expression and IFN-γ production were highly correlated, suggesting a causal relationship between the two responses. Subsequent in vitro experiments revealed that IL-18 signaling is essential for induction of CD25 and synergizes with IL-12 to enhance CD25 expression on splenic NK cells. In accordance with this, Py17XNL-infected erythrocytes induced NK-cell CD25 expression and IFN-γ production in a manner that is completely IL-18- and partially IL-12-dependent, and IFN-γ production is enhanced by IL-2. These data suggest that IL-2 signaling via CD25 amplifies IL-18- and IL-12-mediated NK-cell activation during malaria infection. PMID:26420375

  15. Mutual enhancement of IL-2 and IL-7 on DNA vaccine immunogenicity mainly involves regulations on their receptor expression and receptor-expressing lymphocyte generation.

    PubMed

    Zhang, Yonghong; Liang, Shuang; Li, Xiujin; Wang, Liyue; Zhang, Jianlou; Xu, Jian; Huo, Shanshan; Cao, Xuebin; Zhong, Zhenyu; Zhong, Fei

    2015-07-01

    Our previous study showed that IL-2 and IL-7 could mutually enhance the immunogenicity of canine parvovirus VP2 DNA vaccine, although the underlying mechanism remained unknown. Here, we used the OVA gene as a DNA vaccine in a mouse model to test their enhancement on DNA vaccine immunogenicity and to explore the molecular mechanism. Results showed that both IL-2 and IL-7 genes significantly increased the immunogenicity of OVA DNA vaccine in mice. Co-administration of IL-2 and IL-7 genes with OVA DNA significantly increased OVA-specific antibody titers, T cell proliferation and IFN-γ production compared with IL-2 or IL-7 alone, confirming that IL-2 and IL-7 mutually enhanced DNA vaccine immunogenicity. Mechanistically, we have shown that IL-2 significantly stimulated generation of IL-7 receptor-expressing lymphocytes, and that IL-7 significantly induced IL-2 receptor expression. These results contribute to an explanation of the mechanism of the mutual effects of IL-2 and IL-7 on enhancing DNA vaccine immunogenicity and provided a basis for further investigation on their mutual effects on adjuvant activity and immune regulation. PMID:26055295

  16. Triaryl-Substituted Schiff Bases Are High-Affinity Subtype-Selective Ligands for the Estrogen Receptor

    PubMed Central

    2015-01-01

    We have explored the isoelectronic replacement of the C=C double bond found at the core of many nonsteroidal estrogen ligands with a simple Schiff base (C=N). Di- and triaryl-substituted imine derivatives were conveniently prepared by the condensation of benzophenones with various anilines without the need for phenolic hydroxy protection. Most of these imines demonstrated high affinity for the estrogen receptors, which, in some cases exceeded that of estradiol. In cell-based assays, these imines profiled as ERα agonists but as ERβ antagonists, showing preferential reliance on the N-terminal activation function (AF1), which is more active in ERα. X-ray analysis revealed that the triaryl-imines distort the ligand-binding pocket in a new way: by controlling the separation of helices 3 and 11, which appears to alter the C-terminal AF2 surface that binds transcriptional coactivators. This work suggests that C=N for C=C substitution might be more widely considered as a general strategy for preparing drug analogues. PMID:24708493

  17. Clathrin-independent endocytosis used by the IL-2 receptor is regulated by Rac1, Pak1 and Pak2

    PubMed Central

    Grassart, Alexandre; Dujeancourt, Annick; Lazarow, Paul B; Dautry-Varsat, Alice; Sauvonnet, Nathalie

    2008-01-01

    There are several endocytic pathways, which are either dependent on or independent of clathrin. This study focuses on a poorly characterized mechanism—clathrin- and caveolae-independent endocytosis—used by the interleukin-2 receptor β (IL-2Rβ). We address the question of its regulation in comparison with the clathrin-dependent pathway. First, we show that Ras-related C3 botulinum toxin substrate 1 (Rac1) is specifically required for IL-2Rβ entry, and we identify p21-activated kinases (Paks) as downstream targets. By RNA interference, we show that Pak1 and Pak2 are both necessary for IL-2Rβ uptake, in contrast to the clathrin-dependent route. We observe that cortactin, a partner of actin and dynamin—two essential endocytic factors—is required for IL-2Rβ uptake. Furthermore, we find that cortactin acts downstream from Paks, suggesting control of its function by these kinases. Thus, we describe a cascade composed of Rac1, Paks and cortactin specifically regulating IL-2Rβ internalization. This study indicates Paks as the first specific regulators of the clathrin-independent endocytosis pathway. PMID:18344974

  18. Clathrin-independent endocytosis used by the IL-2 receptor is regulated by Rac1, Pak1 and Pak2.

    PubMed

    Grassart, Alexandre; Dujeancourt, Annick; Lazarow, Paul B; Dautry-Varsat, Alice; Sauvonnet, Nathalie

    2008-04-01

    There are several endocytic pathways, which are either dependent on or independent of clathrin. This study focuses on a poorly characterized mechanism-clathrin- and caveolae-independent endocytosis-used by the interleukin-2 receptor beta (IL-2R beta). We address the question of its regulation in comparison with the clathrin-dependent pathway. First, we show that Ras-related C3 botulinum toxin substrate 1 (Rac1) is specifically required for IL-2R beta entry, and we identify p21-activated kinases (Paks) as downstream targets. By RNA interference, we show that Pak1 and Pak2 are both necessary for IL-2R beta uptake, in contrast to the clathrin-dependent route. We observe that cortactin, a partner of actin and dynamin-two essential endocytic factors-is required for IL-2R beta uptake. Furthermore, we find that cortactin acts downstream from Paks, suggesting control of its function by these kinases. Thus, we describe a cascade composed of Rac1, Paks and cortactin specifically regulating IL-2R beta internalization. This study indicates Paks as the first specific regulators of the clathrin-independent endocytosis pathway. PMID:18344974

  19. Molecular cloning of a type A chicken corticotropin-releasing factor receptor with high affinity for urotensin I.

    PubMed

    Yu, J; Xie, L Y; Abou-Samra, A B

    1996-01-01

    The hypothalamic-pituitary-adrenal axis is an essential physiological system in many species. CRF, the major neuropeptide regulating ACTH secretion, is highly conserved in its primary sequence. Evolutionary conservation of the CRF sequence suggests that the CRF receptor (CRF-R) complementary DNA and examined its properties. The avian CRF-R complementary DNA encodes a 420-amino acid protein that is 87-88% identical to those of human, rat, and mouse. Most sequence divergence occurs in the putative signal peptide and the extracellular amino-terminus of the receptor. Five additional amino acids are inserted in the amino-terminus of the cCRF-R. When expressed in COS-7 cells, the cCRF-R binds the CRF and urotensin I radioligands with high affinities. Urotensin I competes for binding to the chicken CRF-R, expressed in COS-7 cells, with an apparent affinity 20 times higher than that of CRF. Both urotensin I and sauvagine were more effective in stimulating cAMP accumulation in COS-7 cells transfected with the cCRF-R than CRF. The effects of CRF and urotensin I on inositol phosphate accumulation were also tested. Urotensin I was an effective as CRF in stimulating inositol phosphate accumulation in COS-7 cells transfected with the cCRF-R. These data suggest that the sequence of the CRF-R is highly conserved from avian to mammalian species and that, despite its high sequence homology to the type A mammalian CRF-R, the ligand binding properties of cCRF-R are similar to those of the type B CRF-R i.e. a higher affinity for urotensin I than for CRF. PMID:8536612

  20. Multiple mode of binding of phencyclidines: high affinity association between phencyclidine receptors in rat brain and a monovalent ion-sensitive polypeptide

    SciTech Connect

    Haring, R.; Kloog, Y.; Harshak-Felixbrodt, N.A.; Sokolovsky, M.

    1987-01-30

    Two populations of phencyclidine (PCP) binding sites are shown to exist in the rat brain: a high-affinity monovalent ion-sensitive site (Kd of 10-14 nM for (/sup 3/H)TCP, (/sup 3/H)N-(1-(2-thienyl)cyclohexyl)piperidine), which exists in both the frontal cortex and the hippocampus, and a lower affinity site (Kd of 80-130 nM for (/sup 3/H)TCP) which is found in the hippocampus but not in the frontal cortex. The nature of the interactions between the ion-binding sites and the high affinity PCP receptors depend on both ligand structure (PCP or TCP) and the ion involved (K or Na). The high-affinity sites are associated with an Mr 90,000 polypeptide whose labeling by (/sup 3/H)azido phencyclidine is selectively inhibited by monovalent ions.

  1. New Molecular and Cellular Mechanisms of Tolerance: Tolerogenic Actions of IL-2.

    PubMed

    Pérol, Louis; Piaggio, Eliane

    2016-01-01

    Interleukin-2 (IL-2) is an old molecule with brand new functions. Indeed, IL-2 has been first described as a T-cell growth factor but recent data pointed out that its main function in vivo is the maintenance of immune tolerance. Mechanistically, IL-2 is essential for the development and function of CD4(+) Foxp3(+) regulatory T cells (Treg cells) that are essential players in the control of immune responded to self, tumors, microbes and grafts. Treg cells are exquisitely sensitive to IL-2 due to their constitutive expression of the high affinity IL-2 receptor (IL-2R) and the new paradigm suggests that low-doses of IL-2 could selectively boost Treg cells in vivo. Consequently, a growing body of clinical research is aiming at using IL-2 at low doses as a tolerogenic drug to boost endogenous Treg cells in patients suffering from autoimmune or inflammatory conditions. In this manuscript, we briefly review IL-2/IL-2R biology and the role of IL-2 in the development, maintenance, and function of Treg cells; and also its effects on other immune cell populations such as CD4(+) T helper cells and CD8(+) memory T cells. Then, focusing on type 1 diabetes, we review the preclinical studies and clinical trials supporting the use of low-doses IL-2 as a tolerogenic immunotherapy. Finally, we discuss the limitations and future directions for IL-2 based immunotherapy. PMID:26530792

  2. Placenta-specific Expression of the Interleukin-2 (IL-2) Receptor β Subunit from an Endogenous Retroviral Promoter*

    PubMed Central

    Cohen, Carla J.; Rebollo, Rita; Babovic, Sonja; Dai, Elizabeth L.; Robinson, Wendy P.; Mager, Dixie L.

    2011-01-01

    The long terminal repeat (LTR) sequences of endogenous retroviruses and retroelements contain promoter elements and are known to form chimeric transcripts with nearby cellular genes. Here we show that an LTR of the THE1D retroelement family has been domesticated as an alternative promoter of human IL2RB, the gene encoding the β subunit of the IL-2 receptor. The LTR promoter confers expression specifically in the placental trophoblast as opposed to its native transcription in the hematopoietic system. Rather than sequence-specific determinants, DNA methylation was found to regulate transcription initiation and splicing efficiency in a tissue-specific manner. Furthermore, we detected the cytoplasmic signaling domain of the IL-2Rβ protein in the placenta, suggesting that IL-2Rβ undergoes preferential proteolytic cleavage in this tissue. These findings implicate novel functions for this cytokine receptor subunit in the villous trophoblast and reveal an intriguing example of ancient LTR exaptation to drive tissue-specific gene expression. PMID:21865161

  3. BAY 50-4798, a novel, high-affinity receptor-specific recombinant interleukin-2 analog, induces dose-dependent increases in CD25 expression and proliferation among unstimulated, human peripheral blood mononuclear cells in vitro.

    PubMed

    Matthews, Lynn; Chapman, Sherita; Ramchandani, Meena S; Lane, H Clifford; Davey, Richard T; Sereti, Irini

    2004-12-01

    Interleukin-2 administration induces CD4 T cell expansion in HIV-infected patients, however, toxicity can limit dosing. BAY 50-4798 is a recombinant IL-2 analog with >1000-fold specificity for the high-affinity IL-2 receptor. The effects of this compound on unstimulated human PBMC were evaluated. PBMC from HIV(-) and HIV(+) donors were cultured in vitro with incremental doses of BAY 50-4798 or aldesleukin. CD25 expression and proliferation were evaluated with flow cytometry. Cytokine levels were measured by ELISA in culture supernatants. BAY 50-4798 induced dose-dependent increases in CD25 expression and proliferation of T cells, NK, and B cells and showed selectivity for CD4 T cells expressing CD25. Induction of pro-inflammatory cytokines was also dose-dependent and was observed at the concentrations of BAY 50-4798 with the highest biologic activity. These data suggest that BAY 50-4798 can induce proliferation of unstimulated T cells but loss of T cell selectivity and induction of pro-inflammatory cytokines occur at concentrations exerting the highest biologic activity. PMID:15507389

  4. [125I]AT-1012, a New High Affinity Radioligand for the α3β4 Nicotinic Acetylcholine Receptors

    PubMed Central

    Wu, Jinhua; Perry, David C.; Bupp, James E.; Jiang, Faming; Polgar, Willma E.; Toll, Lawrence; Zaveri, Nurulain T.

    2013-01-01

    Recent genetic and pharmacological studies have implicated the α3, β4 and a5 subunits of the nicotinic acetylcholine receptor (nAChR) in dependence to nicotine and other abused drugs and nicotine withdrawal. The α3β4* nAChR subtype has been shown to co-assemble with the α5 or β3 nAChR subunits, and is found mainly in the autonomic ganglia and select brain regions. It has been difficult to study the α3β4 nAChR because there have been no selective nonpeptidic ligands available to independently examine its pharmacology. We recently reported the synthesis of a [125I]-radiolabeled analog of a high affinity, selective small-molecule α3β4 nAChR ligand, AT-1012. We report here the vitro characterization of this radioligand in receptor binding and in vitro autoradiographic studies targeting the α3β4* nAChR. Binding of [125I]AT-1012 was characterized at the rat α3β4- and α4β2 nAChR transfected into HEK cells as well as at the human α3β4α5 nAChR in HEK cells. Binding affinity of [125I]AT-1012 at the rat α3β4 nAChR was 1.4 nM, with a Bmax of 10.3 pmol/mg protein, similar to what was determined using [3H]epibatidine. Saturation isotherms suggested that [125I]AT-1012 binds to a single site on the α3β4 nAChR. Similar high binding affinity was also observed for [125I]AT-1012 at human α3β4α5 nAChR in a human α3β4aα nAChR transfected cell line. [125I]AT-1012 did not bind with high affinity to membranes from α4β2 nAChR-transfected HEK cells, and [3H]epibatidine binding studies showed that AT-1012 had over 100-fold binding selectivity for the α3β4 over α4β2 nAChR. Ki values determined for known nAChR compounds using [125I]AT-1012 as radioligand were comparable to those obtained with [3H]epibatidine. [125I]AT-1012 was also used to label the α3β4 nAChR in rat brain slices in vitro using autoradiography which showed highly localized binding of the radioligand in brain regions consistent with the discreet localization of the α3β4 nAChR. We

  5. The High Affinity IgE Receptor FcεRI Is Expressed by Human Intestinal Epithelial Cells

    PubMed Central

    Starkl, Philipp; Bevins, Charles L.; Scheiner, Otto; Boltz-Nitulescu, George; Wrba, Fritz; Jensen-Jarolim, Erika

    2010-01-01

    Background IgE antibodies play a paramount role in the pathogenesis of various intestinal disorders. To gain insights in IgE-mediated pathophysiology of the gut, we investigated the expression of the high affinity IgE receptor FcεRI in human intestinal epithelium. Methodology/Principal Findings FcεRI α-chain, as detected by immunohistochemistry, was positive in epithelial cells for eight of eleven (8/11) specimens from colon cancer patients and 5/11 patients with inflammation of the enteric mucosa. The FcεRIα positive epithelial cells co-expressed FcεRIγ, whereas with one exception, none of the samples was positive for the β-chain in the epithelial layer. The functionality of FcεRI was confirmed in situ by human IgE binding. In experiments with human intestinal tumor cell lines, subconfluent Caco-2/TC7 and HCT-8 cells were found to express the α- and γ-chains of FcεRI and to bind IgE, whereas confluent cells were negative for γ-chains. Conclusions/Significance Our data provide the first evidence that the components of a functional FcεRI are in vitro expressed by the human intestinal epithelial cells depending on differentiation and, more importantly, in situ in epithelia of patients with colon cancer or gastrointestinal inflammations. Thus, a contribution of FcεRI either to immunosurveillance or pathophysiology of the intestinal epithelium is suggested. PMID:20126404

  6. Development of BODIPY FL Vindoline as a Novel and High-Affinity Pregnane X Receptor Fluorescent Probe

    PubMed Central

    2015-01-01

    The pregnane X receptor (PXR) regulates the metabolism and excretion of xenobiotics and endobiotics by regulating the expression of drug-metabolizing enzymes and transporters. The unique structure of PXR allows it to bind many drugs and drug leads, possibly causing undesired drug–drug interactions. Therefore, it is crucial to evaluate whether chemicals or drugs bind to PXR. Fluorescence-based assays are preferred because of their sensitivity and nonradioactive nature. On the basis of our previously characterized 4 (BODIPY FL vinblastine), a high-affinity PXR probe, we developed 20 (BODIPY FL vindoline) and showed that it is a novel and potent PXR fluorescent probe with Kd of 256 nM in a time-resolved fluorescence resonance energy transfer (TR-FRET) binding assay with PXR. By using 20 (BODIPY FL vindoline) in the PXR TR-FRET assay, we obtained a more than 7-fold signal-to-background ratio and high signal stability (signal was stable for at least 120 min, and Z′-factor > 0.85 from 30 to 240 min). The assay can tolerate DMSO up to 2%. This assay has been used to evaluate a panel of PXR ligands for their PXR-binding affinities. The performance of 20 (BODIPY FL vindoline) in the PXR TR-FRET assay makes it an ideal PXR fluorescent probe, and the newly developed PXR TR-FRET assay with 20 (BODIPY FL vindoline) as a fluorescent probe is suitable for high-throughput screening to identify PXR-binding ligands. PMID:25133934

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

    PubMed Central

    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

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

    PubMed

    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

  9. Synthesis, structure-affinity relationships, and radiolabeling of selective high-affinity 5-HT4 receptor ligands as prospective imaging probes for positron emission tomography.

    PubMed

    Xu, Rong; Hong, Jinsoo; Morse, Cheryl L; Pike, Victor W

    2010-10-14

    In a search for high-affinity receptor ligands that might serve for development as radioligands for the imaging of brain 5-HT(4) receptors in vivo with positron emission tomography (PET), structural modifications were made to the high-affinity 5-HT(4) antagonist (1-butylpiperidin-4-yl)methyl 8-amino-7-iodo-2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (1, SB 207710). These modifications were made mainly on the aryl side of the ester bond to permit possible rapid labeling of the carboxylic acid component with a positron emitter, either carbon-11 (t(1/2) = 20.4 min) or fluorine-18 (t(1/2) = 109.7 min), and included (i) replacement of the iodine atom with a small substituent such as nitrile, methyl, or fluoro, (ii) methylation of the 8-amino group, (iii) opening of the dioxan ring, and (iv) alteration of the length of the N-alkyl goup. High-affinity ligands were discovered for recombinant human 5-HT(4) receptors with amenability to labeling with a positron emitter and potential for development as imaging probes. The ring-opened radioligand, (([methoxy-(11)C]1-butylpiperidin-4-yl)methyl 4-amino-3-methoxybenzoate; [(11)C]13), showed an especially favorable array of properties for future evaluation as a PET radioligand for brain 5-HT(4) receptors. PMID:20812727

  10. Synthesis, Structure-affinity Relationships and Radiolabeling of Selective High-affinity 5-HT4 Receptor Ligands as Prospective Imaging Probes for PET

    PubMed Central

    Xu, Rong; Hong, Jinsoo; Morse, Cheryl L.; Pike, Victor W.

    2010-01-01

    In a search for high-affinity receptor ligands that might serve for development as radioligands for the imaging of brain 5-HT4 receptors in vivo with positron emission tomography (PET), structural modifications were made to the high-affinity 5-HT4 antagonist, (1-butylpiperidin-4-yl)methyl 8-amino-7-iodo-2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate (1, SB 207710). These modifications were made mainly on the aryl side of the ester bond to permit possible rapid labeling of the carboxylic acid component with a positron-emitter, either carbon-11 (t1/2 = 20.4 min) or fluorine-18 (t1/2 = 109.7 min), and included, i) replacement of the iodine atom with a small substituent such as nitrile, methyl or fluoro, ii) methylation of the 8-amino group, iii) opening of the dioxan ring, and iv) alteration of the length of the N-alkyl goup. High-affinity ligands were discovered for recombinant human 5-HT4 receptors with amenability to labeling with a positron-emitter and potential for development as imaging probes. The ring-opened radioligand, (([methoxy-11C]1-butylpiperidin-4-yl)methyl 4-amino-3-methoxybenzoate; [11C]13), showed an especially favorable array of properties for future evaluation as a PET radioligand for brain 5-HT4 receptors. PMID:20812727

  11. Photoaffinity labeling of mammalian. cap alpha. /sub 1/-adrenergic receptors: identification of the ligand binding subunit with a high affinity radioiodinated probe. [Rats, guinea pigs, rabbits

    SciTech Connect

    Leeb-Lundberg, L.M.F.; Dickinson, K.E.J.; Heald, S.L.; Wikberg, J.E.S.; Hagen, P.O.; DeBernardis, J.F.; Winn, M.; Arendsen, D.L.; Lefkowitz, R.J.; Caron, M.G.

    1984-02-01

    A description is given of the synthesised and characterization of a novel high affinity radioiodinated ..cap alpha../sub 1/-adrenergic receptor photoaffinity probe, 4-amino-6,7-dimethoxy-2-(4-(5-(4-azido-3-(/sup 125/I)iodophenyl)pentanoyl)-1-piperazinyl) quinazoline. In the absence of light, this ligand binds with high affinity (K/sub d/ = 130 pm) in a reverisble and saturable manner to sites in rat hepatic plasma membranes. The binding is stereoselective and competitively inhibited by adrenergic agonists and antagonists with an ..cap alpha../sub 1/-adrenergic specificity. Upon photolysis, this ligand incorporates irreversibly into plasma membranes prepared from several mammalian tissues including rat liver, rat, guinea pig, and rabbit spleen, rabbit lung, and rabbit aorta vascular smooth muscle cells, also with typical ..cap alpha../sub 1/-adrenergic specificity. Autoradiograms of such membrane samples subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveal a major specifically labeled polypeptide at M/sub 4/ = 78,000-85,000, depending on the tissue used, in addition to some lower molecular weight peptides. Protease inhibitors, in particular EDTA, a metalloprotease inhibitor, dramatically increases the predominance of the M/sub r/ = 78,000-85,000 polypeptide while attenuating the labeling of the lower molecular weight bands. This new high affinity radioiodinated photoaffinity probe should be of great value for the molecular characterization of the ..cap alpha../sub 1/-adrenergic receptor.

  12. Characterization of a Pachymedusa dacnicolor–Sauvagine Analog as a New High-Affinity Radioligand for Corticotropin-Releasing Factor Receptor Studies

    PubMed Central

    Perrin, Marilyn H.; Tan, Laura A.; Vaughan, Joan M.; Lewis, Kathy A.; Donaldson, Cynthia J.; Miller, Charleen; Erchegyi, Judit; Rivier, Jean E.

    2015-01-01

    The corticotropin-releasing factor (CRF) peptide family comprises the mammalian peptides CRF and the urocortins as well as frog skin sauvagine and fish urophyseal urotensin. Advances in understanding the roles of the CRF ligand family and associated receptors have often relied on radioreceptor assays using labeled CRF ligands. These assays depend on stable, high-affinity CRF analogs that can be labeled, purified, and chemically characterized. Analogs of several of the native peptides have been used in this context, most prominently including sauvagine from the frog Phyllomedusa sauvageii (PS-Svg). Because each of these affords both advantages and disadvantages, new analogs with superior properties would be welcome. We find that a sauvagine-like peptide recently isolated from a different frog species, Pachymedusa dacnicolor (PD-Svg), is a high-affinity agonist whose radioiodinated analog, [125ITyr0-Glu1, Nle17]-PD-Svg, exhibits improved biochemical properties over those of earlier iodinated agonists. Specifically, the PD-Svg radioligand binds both CRF receptors with comparably high affinity as its PS-Svg counterpart, but detects a greater number of sites on both type 1 and type 2 receptors. PD-Svg is also ∼10 times more potent at stimulating cAMP accumulation in cells expressing the native receptors. Autoradiographic localization using the PD-Svg radioligand shows robust specific binding to rodent brain and peripheral tissues that identifies consensus CRF receptor–expressing sites in a greater number and/or with greater sensitivity than its PS-Svg counterpart. We suggest that labeled analogs of PD-Svg may be useful tools for biochemical, structural, pharmacological, and anatomic studies of CRF receptors. PMID:25736419

  13. Structural basis for binding of human IgG1 to its high-affinity human receptor FcγRI

    PubMed Central

    Kiyoshi, Masato; Caaveiro, Jose M.M.; Kawai, Takeaki; Tashiro, Shinya; Ide, Teruhiko; Asaoka, Yoshiharu; Hatayama, Kouta; Tsumoto, Kouhei

    2015-01-01

    Cell-surface Fcγ receptors mediate innate and adaptive immune responses. Human Fcγ receptor I (hFcγRI) binds IgGs with high affinity and is the only Fcγ receptor that can effectively capture monomeric IgGs. However, the molecular basis of hFcγRI's interaction with Fc has not been determined, limiting our understanding of this major immune receptor. Here we report the crystal structure of a complex between hFcγRI and human Fc, at 1.80 Å resolution, revealing an unique hydrophobic pocket at the surface of hFcγRI perfectly suited for residue Leu235 of Fc, which explains the high affinity of this complex. Structural, kinetic and thermodynamic data demonstrate that the binding mechanism is governed by a combination of non-covalent interactions, bridging water molecules and the dynamic features of Fc. In addition, the hinge region of hFcγRI-bound Fc adopts a straight conformation, potentially orienting the Fab moiety. These findings will stimulate the development of novel therapeutic strategies involving hFcγRI. PMID:25925696

  14. The Ketamine Analogue Methoxetamine and 3- and 4-Methoxy Analogues of Phencyclidine Are High Affinity and Selective Ligands for the Glutamate NMDA Receptor

    PubMed Central

    Roth, Bryan L.; Gibbons, Simon; Arunotayanun, Warunya; Huang, Xi-Ping; Setola, Vincent; Treble, Ric; Iversen, Les

    2013-01-01

    In this paper we determined the pharmacological profiles of novel ketamine and phencyclidine analogues currently used as ‘designer drugs’ and compared them to the parent substances via the resources of the National Institute of Mental Health Psychoactive Drug Screening Program. The ketamine analogues methoxetamine ((RS)-2-(ethylamino)-2-(3-methoxyphenyl)cyclohexanone) and 3-MeO-PCE (N-ethyl-1-(3-methoxyphenyl)cyclohexanamine) and the 3- and 4-methoxy analogues of phencyclidine, (1-[1-(3-methoxyphenyl)cyclohexyl]piperidine and 1-[1-(4-methoxyphenyl)cyclohexyl]piperidine), were all high affinity ligands for the PCP-site on the glutamate NMDA receptor. In addition methoxetamine and PCP and its analogues displayed appreciable affinities for the serotonin transporter, whilst the PCP analogues exhibited high affinities for sigma receptors. Antagonism of the NMDA receptor is thought to be the key pharmacological feature underlying the actions of dissociative anaesthetics. The novel ketamine and PCP analogues had significant affinities for the NMDA receptor in radioligand binding assays, which may explain their psychotomimetic effects in human users. Additional actions on other targets could be important for delineating side-effects. PMID:23527166

  15. Establishment of a novel high-affinity IgE receptor-positive canine mast cell line with wild-type c-kit receptors

    SciTech Connect

    Amagai, Yosuke; Tanaka, Akane; Ohmori, Keitaro; Matsuda, Hiroshi

    2008-02-15

    Much is known regarding participations of mast cells with innate and acquired immunity by secreting various cytokines and chemical mediators. However, details of mast cell biology still remain unclear. In this study, we successfully established a novel growth factor-independent mast cell line (MPT-1) derived from canine mast cell tumor. MPT-1 cells manifested factor-independent proliferation as floating cells containing a large amount of histamine, as well as chymase-like dog mast cell protease 3, in cytosolic granules. Particularly, MPT-1 cells expressed high-affinity IgE receptors (Fc{epsilon}RI) and wild-type c-kit receptors. Degranulation of MPT-1 cells was induced not only by stimulation with calcium ionophore but also by cross-linkage of the surface IgE. Given that MPT-1 is the first mast cell line with Fc{epsilon}RI which has no c-kit mutations, MPT-1 cells may provide great contribution for investigation of IgE-mediated activation mechanisms of mast cells, leading to development of effective treatment for allergic disorders.

  16. Analysis of D2 dopamine receptor occupancy with quantitative SPET using the high-affinity ligand [123I]epidepride: resolving conflicting findings.

    PubMed

    Erlandsson, Kjell; Bressan, Rodrigo A; Mulligan, Rachel S; Ell, Peter J; Cunningham, Vincent J; Pilowsky, Lyn S

    2003-07-01

    Recent studies of limbic cortical dopamine D(2) receptor occupancy by clozapine using high-affinity PET and SPET radioligands have produced conflicting findings. It has been suggested that these divergent findings are due to between-study differences in the method used to estimate D(2) receptor-binding potential. We compared different methods for estimating striatal and temporal cortical D(2) receptor occupancy with high-affinity tracers. In vivo experimental SPET data, obtained with [(123)I]epidepride were analysed with reference tissue kinetic modeling and with the ratio method, applied to data corresponding to short (60 min) and long (240 min) acquisition times. Dopamine D(2) receptor occupancy by the atypical antipsychotic drug risperidone was evaluated. Simulation experiments were also performed, comparing occupancy values obtained for different receptor densities in relation to different data acquisition times. The simulation results revealed that previously published data regarding errors in occupancy estimation by analysis of time activity data acquired for 60 min cannot be extrapolated to studies performed over 240 min. The ratio method provided accurate temporal cortical D(2) receptor occupancy values when applied to data from a late time period, but underestimated the occupancy with earlier data. In striatum, both the late data ratio method and reference tissue kinetic modeling using all data underestimated D(2) receptor occupancy. However, more accurate analyses of striatal D(2) occupancy still showed selective limbic/cortical occupancy by risperidone. Our results substantiate the previous [(123)I]epidepride findings of high temporal cortical occupancy by other atypical antipsychotic drugs and suggest that a potential source of conflicting findings might be short scanning times imposed by [(11)C]FLB 457, leading to underestimation of temporal cortical D(2) receptor occupancy by this method. PMID:12880845

  17. Michael Acceptor Approach to the Design of New Salvinorin A-based High Affinity Ligands for the Kappa-Opioid Receptor

    PubMed Central

    Polepally, Prabhakar R.; Huben, Krzysztof; Vardy, Eyal; Setola, Vincent; Mosier, Philip D.; Roth, Bryan L.; Zjawiony, Jordan K.

    2014-01-01

    The neoclerodane diterpenoid salvinorin A is a major secondary metabolite isolated from the psychoactive plant Salvia divinorum. Salvinorin A has been shown to have high affinity and selectivity for the κ-opioid receptor (KOR). To study the ligand–receptor interactions that occur between salvinorin A and the KOR, a new series of salvinorin A derivatives bearing potentially reactive Michael acceptor functional groups at C-2 was synthesized and used to probe the salvinorin A binding site. The κ-, δ-, and μ-opioid receptor (KOR, DOR and MOR, respectively) binding affinities and KOR efficacies were measured for the new compounds. Although none showed wash-resistant irreversible binding, most of them showed high affinity for the KOR, and some exhibited dual affinity to KOR and MOR. Molecular modeling techniques based on the recently-determined crystal structure of the KOR combined with results from mutagenesis studies, competitive binding, functional assays and structure–activity relationships, and previous salvinorin A–KOR interaction models were used to identify putative interaction modes of the new compounds with the KOR and MOR. PMID:25193297

  18. Distinction between high-affinity (/sup 3/H)phencyclidine binding sites and muscarinic receptors in guinea-pig ileum muscle

    SciTech Connect

    El-Fakahany, E.E.; Triggle, D.J.; Eldefrawi, A.T.; Eldefrawi, M.E.

    1984-05-01

    (/sup 3/H)Phencyclidine ((/sup 3/H)PCP) binding was studied in guinea-pig ileum muscle membranes. Specific binding of (/sup 3/H)PCP was time dependent, reversible and saturable, with an equilibrium dissociation constant of 154 nM and maximum binding of 12.9 pmol/mg of protein at pH 9. Its pH dependency suggests that the un-ionized PCP is the pharmacologically active form. The binding site was on a protein which was sensitive to heat, proteolytic enzymes and the carboxylic group reagent dicyclohexylcarbodiimide, but insensitive to phospholipase A and C, concanavalin A, dithiothreitol and N-ethylmaleimide. Specific (/sup 3/H)PCP binding was displaced effectively by several PCP analogs and Ca/sup + +/ channel antagonists including verapamil, to which these sites had a high affinity. These high-affinity PCP-binding sites were found at a much higher concentration in the same membrane preparation than muscarinic receptor sites identified by their specific binding of (/sup 3/H)quinuclidinyl benzilate. PCP bound to both sites, but with a lower affinity to the muscarinic receptor sites. The PCP and muscarinic receptor sites differed in their sensitivities to pH and drug specifities.

  19. Dopamine inhibition of anterior pituitary adenylate cyclase is mediated through the high-affinity state of the D/sub 2/ receptor

    SciTech Connect

    Borgundvaag, B.; George, S.R.

    1985-07-29

    The diterpinoid forskolin stimulated adenylate cyclase activity (measured by conversion of (/sup 3/H)-ATP to (/sup 3/H)-cAMP) in anterior pituitary from male and female rats. Inhibition of stimulated adenylate cyclase activity by potent dopaminergic agonists was demonstrable only in female anterior pituitary. The inhibition of adenylate cyclase activity displayed a typically dopaminergic rank order of agonist potencies and could be completely reversed by a specific dopamine receptor antagonist. The IC/sub 50/ values of dopamine agonist inhibition of adenylate cyclase activity correlated with equal molarity with the dissociation constant of the high-affinity dopamine agonist-detected receptor binding site and with the IC/sub 50/ values for inhibition of prolactin secretion. These findings support the hypothesis that it is the high-affinity form of the D/sub 2/ dopamine receptor in anterior pituitary which is responsible for mediating the dopaminergic function of attenuating adenylate cyclase activity. 12 references, 4 figures, 1 table.

  20. Nicotine ameliorates NMDA receptor antagonist-induced deficits in contextual fear conditioning through high-affinity nicotinic acetylcholine receptors in the hippocampus.

    PubMed

    André, Jessica M; Leach, Prescott T; Gould, Thomas J

    2011-03-01

    NMDA glutamate receptors (NMDARs) and nicotinic acetylcholine receptors (nAChRs) are both involved in learning and synaptic plasticity. Increasing evidence suggests processes mediated by these receptors may interact to modulate learning; however, little is known about the neural substrates involved in these interactive processes. The present studies investigated the effects of nicotine on MK-801 hydrogen maleate (MK-801) and DL-2-Amino-5-phosphonovaleric acid (APV)-induced disruption of contextual fear conditioning in male C57BL/6J mice, using direct drug infusion and selective nAChR antagonists to define the brain regions and the nAChR subtypes involved. Mice treated with MK-801 showed a deficit in contextual fear conditioning that was ameliorated by nicotine. Direct drug infusion demonstrated that the NMDAR antagonists disrupted hippocampal function and that nicotine acted in the dorsal hippocampus to ameliorate the deficit in learning. The high-affinity nAChR antagonist Dihydro-β-erythroidine hydrobromide (DhβE) blocked the effects of nicotine on MK-801-induced deficits while the α7 nAChR antagonist methyllycaconitine citrate salt hydrate (MLA) did not. These results suggest that NMDARs and nAChRs may mediate similar hippocampal processes involved in contextual fear conditioning. Furthermore, these results may have implications for developing effective therapeutics for the cognitive deficits associated with schizophrenia because a large subset of patients with schizophrenia exhibit cognitive deficits that may be related to NMDAR dysfunction and smoke at much higher rates than the healthy population, which may be an attempt to ameliorate cognitive deficits. PMID:21167848

  1. Synthesis and in vitro autoradiographic evaluation of a novel high-affinity radioiodinated ligand for imaging brain cannabinoid subtype-1 receptors.

    PubMed

    Donohue, Sean R; Varnäs, Katarina; Jia, Zhisheng; Gulyás, Balázs; Pike, Victor W; Halldin, Christer

    2009-11-01

    There is strong interest to study the involvement of brain cannabinoid subtype-1 (CB1) receptors in neuropsychiatric disorders with single photon emission computed tomography (SPECT) and a suitable radioligand. Here we report the synthesis of a novel high-affinity radioiodinated CB1 receptor ligand ([125I]8, [125I]1-(2-iodophenyl)-4-cyano-5-(4-methoxyphenyl)-N-(piperidin-1-yl)-1H-pyrazole-3-carboxylate, [125I]SD7015). By autoradiography in vitro, [125I]8 showed selective binding to CB1 receptors on human brain postmortem cryosections and now merits labeling with iodine-123 for further evaluation as a SPECT radioligand in non-human primate. PMID:19767206

  2. Potentials and pitfalls using high affinity radioligands in PET and SPET determinations on regional drug induced D2 receptor occupancy--a simulation study based on experimental data.

    PubMed

    Olsson, H; Farde, L

    2001-10-01

    The D2 dopamine receptor density ranges from 0.2 to 40 nM among human brain regions. For high density regions radioligands like [(11)C]raclopride provide accurate and reliable estimates of the receptor density. In research on neuropsychiatric disorders there is, however, a growing need for quantitative approaches that accurately measure D2 dopamine receptor occupancy induced by drugs or endogenous dopamine in regions with low receptor density. The new high affinity radioligands [(11)C]FLB 457 and [(123)I]epidepride have been shown to provide a signal for extrasriatal D2 dopamine receptor populations in the human brain in vivo. Initial observations indicate, however, that the time required to reach equilibrium is dependent on receptor density. Ratio analyses may thus not be readily used for comparisons among different brain regions. The aim of the present simulation study was to examine commonly used approaches for calculation of drug induced D2 dopamine receptor occupancy among regions with widely different receptor density. The input functions and the rate constants of [(11)C]FLB 457 and the reference ligand [(11)C]raclopride were first used in a simulation estimating the effect of receptor density on equilibrium time. In a second step we examined how errors produced by inaccurate determination of the binding potential parameter propagate to calculations of drug induced receptor occupancy. The simulations showed a marked effect of receptor density on equilibrium time for [(11)C]FLB 457, but not for [(11)C]raclopride. For [(11)C]FLB 457, a receptor density above about 7 nM caused the time of equilibrium to fall beyond time of data acquisition (1 h). The use of preequilibrium data caused the peak equilibrium and the end time ratio approaches but not the simplified reference tissue model (SRTM) approach to underestimate the binding potential and thus also the drug occupancy calculated for high-density regions. The study supports the use of ratio and SRTM analyses in

  3. Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcɛRI.

    PubMed

    Sabban, Sari; Ye, Hongtu; Helm, Birgit

    2013-05-15

    The binding of immunoglobulin E (IgE) to its high-affinity receptor (FcɛRI) is the central protein interaction in IgE-mediated allergic reactions. The cross-linking of the IgE/FcɛRI complex, through cognate allergens, on the surface of mast cells and basophil cells results in mediator release, and thus leads to the symptoms of type I hypersensitivity responses in mammals. To develop a baseline value for subsequent equine anti-allergy drug and vaccine research, the interaction of equine IgE with its high-affinity FcɛRI receptor was investigated following the cloning and expression of equine IgE with specificity for NIP-HSA (4-hydroxy-5-iodo-3-nitrophenylacetic acid conjugated to human serum albumin). Receptor recognition and effector functions were assessed in Rat Basophil Leukemia (RBL-2H3.1) cells transfected with the α chain of equine and canine FcɛRI. Results obtained showed that the equine FcɛRI receptor recognizes both equine and canine IgE and supports similar β-hexosaminidase release levels from RBL cells transfected with equine FcɛRI, peaking at 36.68% at 100ngml(-1) antigen and 32.00% at 100ngml(-1) antigen respectively. Furthermore, the binding kinetics of the equine IgE to the equine FcɛRI receptor and the canine IgE to the same receptor was measured to be KA=6.33×10(9)M(-1) and KA=1.84×10(9)M(-1) respectively. This research established basic reagents and vitro assay systems to underpin the development of rational therapeutic intervention strategies to combat equine allergic manifestations. PMID:23485176

  4. Inflammation Enhances IL-2 Driven Differentiation of Cytolytic CD4 T Cells

    PubMed Central

    Workman, Aspen M.; Jacobs, Ashley K.; Vogel, Alexander J.; Condon, Shirley; Brown, Deborah M.

    2014-01-01

    Cytolytic CD4 T cells (CD4 CTL) have been identified in vivo in response to viral infections; however, the factors necessary for driving the cytolytic phenotype have not been fully elucidated. Our previously published work suggests IL-2 may be the master regulator of perforin-mediated cytotoxicity in CD4 effectors. To further dissect the role of IL-2 in CD4 CTL generation, T cell receptor transgenic mice deficient in the ability to produce IL-2 or the high affinity IL-2 receptor (IL-2Rα, CD25) were used. Increasing concentrations of IL-2 were necessary to drive perforin (Prf) expression and maximal cytotoxicity. Granzyme B (GrB) expression and killing correlated with STAT5 activation and CD25 expression in vitro, suggesting that signaling through the high affinity IL-2R is critical for full cytotoxicity. IL-2 signaling was also necessary in vivo for inducing the Th1 phenotype and IFN-γ expression in CD4 T cells during influenza A (IAV) infection. In addition, GrB expression, as measured by mean fluorescent intensity, was decreased in CD25 deficient cells; however, the frequency of CD4 cells expressing GrB was unchanged. Similarly, analysis of cytolytic markers such as CD107a/b and Eomesodermin indicate high IL-2Rα expression is not necessary to drive the CD4 CTL phenotype during IAV infection. Thus, inflammatory signals induced by viral infection may overcome the need for strong IL-2 signals in driving cytotoxicity in CD4 cells. PMID:24586481

  5. Competing feedback loops shape IL-2 signaling between helper and regulatory T lymphocytes in cellular microenvironments

    PubMed Central

    Busse, Dorothea; de la Rosa, Maurus; Hobiger, Kirstin; Thurley, Kevin; Flossdorf, Michael; Scheffold, Alexander; Höfer, Thomas

    2010-01-01

    Cytokines are pleiotropic and readily diffusible messenger molecules, raising the question of how their action can be confined to specific target cells. The T cell cytokine interleukin-2 (IL-2) is essential for the homeostasis of regulatory T (Treg) cells that suppress (auto)immunity and stimulates immune responses mediated by conventional T cells. We combined mathematical modeling and experiments to dissect the dynamics of the IL-2 signaling network that links the prototypical IL-2 producers, conventional T helper (Th) cells, and Treg cells. We show how the IL-2-induced upregulation of high-affinity IL-2 receptors (IL-2R) establishes a positive feedback loop of IL-2 signaling. This feedback mediates a digital switch for the proliferation of Th cells and functions as an analog amplifier for the IL-2 uptake capacity of Treg cells. Unlike other positive feedbacks in cell signaling that augment signal propagation, the IL-2/IL-2R loop enhances the capture of the signal molecule and its degradation. Thus Treg and Th cells can compete for IL-2 and restrict its range of action through efficient cellular uptake. Depending on activation status and spatial localization of the cells, IL-2 may be consumed exclusively by Treg or Th cells, or be shared between them. In particular, a Treg cell can deprive a stimulated Th cell of its IL-2, but only when the cells are located in close proximity, within a few tens of micrometers. The present findings explain how IL-2 can play two disctinct roles in immune regulation and point to a hitherto largely unexplored spatiotemporal complexity of cytokine signaling. PMID:20133667

  6. The novel VIP-like hypothalamic polypeptide PACAP interacts with high affinity receptors in the human neuroblastoma cell line NB-OK

    SciTech Connect

    Cauvin, A.; Buscail, L.; Gourlet, P.; De Neef, P.; Gossen, D.; Arimura, A.; Miyata, A.; Coy, D.H.; Robberecht, P.; Christophe, J. )

    1990-07-01

    We investigated the ability of two forms of Pituitary Adenylate Cyclase Activating Polypeptide (PACAP-38, the 38 amino acid peptide isolated from ovine hypothalamus, and PACAP-27, a shorter N-terminal (1-27) amidated version) to interact with specific receptors in membranes from the human neuroblastoma cell line NB-OK. ({sup 125}I)PACAP-27 bound rapidly and specifically to one class of high affinity sites (Kd 0.5 nM). VIP inhibited ({sup 125}I)PACAP-27 binding 300- to 1000-fold less potently than PACAP-27 and PACAP-38. One microM PHI prevented tracer binding only partially and secretin, glucagon and GRF(1-29)NH2 were ineffective in this respect. PACAP-27 and PACAP-38 stimulated adenylate cyclase activity dose dependently and with similar efficacy (Kact 0.2-0.3 nM), this activation being compatible with the occupancy of specific high affinity PACAP receptor. VIP was markedly less potent and less efficient on this enzyme than PACAP. Chemical cross-linking of ({sup 125}I)PACAP-27 followed by SDS-PAGE and autoradiography revealed specific cross-linking with a 68 kDa protein.

  7. Combined Stimulation of IL-2 and 4-1BB Receptors Augments the Antitumor Activity of E7 DNA Vaccines by Increasing Ag-Specific CTL Responses

    PubMed Central

    Kim, Ha; Kwon, Byungsuk; Sin, Jeong-Im

    2013-01-01

    Human papillomavirus (HPV) infection is a major cause of cervical cancer. Here, we investigate whether concurrent therapy using HPV E7 DNA vaccines (pE7) plus IL-2 vs. IL-15 cDNA and anti-4-1BB Abs might augment antitumor activity against established tumors. IL-2 cDNA was slightly better than IL-15 cDNA as a pE7 adjuvant. Co-delivery of pE7+IL-2 cDNA increased tumor cure rates from 7% to 27%, whereas co-delivery of pE7+IL-2 cDNA with anti-4-1BB Abs increased tumor cure rates from 27% to 67% and elicited long-term memory responses. This increased activity was concomitant with increased induction of Ag-specific CTL activity and IFN-γ responses, but not with Ag-specific IgG production. Moreover, the combined stimulation of IL-2 and 4-1BB receptors with rIL-2 and anti-4-1BB Abs resulted in enhanced production of IFN-γ from Ag-specific CD8+ T cells. However, this effect was abolished by treatment with anti-IL-2 Abs and 4-1BB-Fc, suggesting that the observed effect was IL-2- and anti-4-1BB Ab-specific. A similar result was also obtained for Ag-specific CTL activity. Thus, these studies demonstrate that combined stimulation through the IL-2 and 4-1BB receptors augments the Ag-specific CD8+ CTL responses induced by pE7, increasing tumor cure rates and long-term antitumor immune memory. These findings may have implications for the design of DNA-based therapeutic vaccines against cancer. PMID:24391824

  8. Combined stimulation of IL-2 and 4-1BB receptors augments the antitumor activity of E7 DNA vaccines by increasing Ag-specific CTL responses.

    PubMed

    Kim, Ha; Kwon, Byungsuk; Sin, Jeong-Im

    2013-01-01

    Human papillomavirus (HPV) infection is a major cause of cervical cancer. Here, we investigate whether concurrent therapy using HPV E7 DNA vaccines (pE7) plus IL-2 vs. IL-15 cDNA and anti-4-1BB Abs might augment antitumor activity against established tumors. IL-2 cDNA was slightly better than IL-15 cDNA as a pE7 adjuvant. Co-delivery of pE7+IL-2 cDNA increased tumor cure rates from 7% to 27%, whereas co-delivery of pE7+IL-2 cDNA with anti-4-1BB Abs increased tumor cure rates from 27% to 67% and elicited long-term memory responses. This increased activity was concomitant with increased induction of Ag-specific CTL activity and IFN-γ responses, but not with Ag-specific IgG production. Moreover, the combined stimulation of IL-2 and 4-1BB receptors with rIL-2 and anti-4-1BB Abs resulted in enhanced production of IFN-γ from Ag-specific CD8+ T cells. However, this effect was abolished by treatment with anti-IL-2 Abs and 4-1BB-Fc, suggesting that the observed effect was IL-2- and anti-4-1BB Ab-specific. A similar result was also obtained for Ag-specific CTL activity. Thus, these studies demonstrate that combined stimulation through the IL-2 and 4-1BB receptors augments the Ag-specific CD8+ CTL responses induced by pE7, increasing tumor cure rates and long-term antitumor immune memory. These findings may have implications for the design of DNA-based therapeutic vaccines against cancer. PMID:24391824

  9. P2X7 receptor activation downmodulates Na(+)-dependent high-affinity GABA and glutamate transport into rat brain cortex synaptosomes.

    PubMed

    Barros-Barbosa, A R; Lobo, M G; Ferreirinha, F; Correia-de-Sá, P; Cordeiro, J M

    2015-10-15

    Sodium-dependent high-affinity amino-acid transporters play crucial roles in terminating synaptic transmission in the central nervous system (CNS). However, there is lack of information about the mechanisms underlying the regulation of amino-acid transport by fast-acting neuromodulators, like ATP. Here, we investigated whether activation of the ATP-sensitive P2X7 receptor modulates Na(+)-dependent high-affinity γ-aminobutyric acid (GABA) and glutamate uptake into nerve terminals (synaptosomes) of the rat cerebral cortex. Radiolabeled neurotransmitter accumulation was evaluated by liquid scintillation spectrometry. The cell-permeant sodium-selective fluorescent indicator, SBFI-AM, was used to estimate Na(+) influx across plasma membrane. 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP, 3-300 μM), a prototypic P2X7 receptor agonist, concentration-dependently decreased [(3)H]GABA (14%) and [(14)C]glutamate (24%) uptake; BzATP decreased transport maximum velocity (Vmax) without affecting the Michaelis constant (Km) values. The selective P2X7 receptor antagonist, A-438079 (3 μM), prevented inhibition of [(3)H]GABA and [(14)C]glutamate uptake by BzATP (100 μM). The inhibitory effect of BzATP coincided with its ability to increase intracellular Na(+) and was mimicked by Na(+) ionophores, like gramicidin and monensin. Increases in intracellular Na(+) (with veratridine or ouabain) or substitution of extracellular Na(+) by N-methyl-D-glucamine (NMDG)(+) all decreased [(3)H]GABA and [(14)C]glutamate uptake and attenuated BzATP effects. Uptake inhibition by BzATP (100 μM) was also attenuated by calmidazolium, which selectively inhibits Na(+) currents through the P2X7 receptor pore. In conclusion, disruption of the Na(+) gradient by P2X7 receptor activation downmodulates high-affinity GABA and glutamate uptake into rat cortical synaptosomes. Interference with amino-acid transport efficacy may constitute a novel target for therapeutic management of cortical excitability. PMID

  10. Human metabolites of synthetic cannabinoids JWH-018 and JWH-073 bind with high affinity and act as potent agonists at cannabinoid type-2 receptors

    SciTech Connect

    Rajasekaran, Maheswari; Brents, Lisa K.; Franks, Lirit N.; Moran, Jeffery H.; Prather, Paul L.

    2013-06-01

    K2 or Spice is an emerging drug of abuse that contains synthetic cannabinoids, including JWH-018 and JWH-073. Recent reports indicate that monohydroxylated metabolites of JWH-018 and JWH-073 retain high affinity and activity at cannabinoid type-1 receptors (CB{sub 1}Rs), potentially contributing to the enhanced toxicity of K2 compared to marijuana. Since the parent compounds also bind to cannabinoid type-2 receptors (CB{sub 2}Rs), this study investigated the affinity and intrinsic activity of JWH-018, JWH-073 and several monohydroxylated metabolites at human CB{sub 2}Rs (hCB{sub 2}Rs). The affinity of cannabinoids for hCB{sub 2}Rs was determined by competition binding studies employing CHO-hCB{sub 2} membranes. Intrinsic activity of compounds was assessed by G-protein activation and adenylyl cyclase (AC)-inhibition in CHO-hCB{sub 2} cells. JWH-073, JWH-018 and several of their human metabolites exhibit nanomolar affinity and act as potent agonists at hCB{sub 2}Rs. Furthermore, a major omega hydroxyl metabolite of JWH-073 (JWH-073-M5) binds to CB{sub 2}Rs with 10-fold less affinity than the parent molecule, but unexpectedly, is equipotent in regulating AC-activity when compared to the parent molecule. Finally, when compared to CP-55,940 and Δ{sup 9}-tetrahydrocannabinol (Δ{sup 9}-THC), JWH-018, JWH-018-M5 and JWH-073-M5 require significantly less CB{sub 2}R occupancy to produce similar levels of AC-inhibition, indicating that these compounds may more efficiently couple CB{sub 2}Rs to AC than the well characterized cannabinoid agonists examined. These results indicate that JWH-018, JWH-073 and several major human metabolites of these compounds exhibit high affinity and demonstrate distinctive signaling properties at CB{sub 2}Rs. Therefore, future studies examining pharmacological and toxicological properties of synthetic cannabinoids present in K2 products should consider potential actions of these drugs at both CB{sub 1} and CB{sub 2}Rs. - Highlights: • JWH-018

  11. (I-125) 17. cap alpha. -Iodovinyl 11. beta. -methoxyestradiol: in vivo and in vitro properties of a high-affinity estrogen-receptor radiopharmaceutical

    SciTech Connect

    Jagoda, E.M.; Gibson, R.E.; Goodgold, H.; Ferreira, N.; Francis, B.E.; Reba, R.C.; Rzeszotarski, W.J.; Eckelman, W.C.

    1984-04-01

    17 ..cap alpha..-(/sup 125/I)Iodovinyl 11 ..beta..-methoxyestradiol ((I-125)MIVE/sub 2/) has been prepared with high specific activity (155-2000 Ci/mmol) and a high affinity for the estrogen receptor. In vivo distribution studies using immature rats result in high levels of activity in the uterus (20-30% dose/g) with uterus-to-plasma ratios on the order of 68 to 100. Peak activity in the uterus is obtained between 2 and 4 hr, and by 6 hr 50% of the activity has washed out. The radioactive labeling of MIVE/sub 2/ is sufficiently rapid so that (I-123)MIVE/sub 2/ has been synthesized and is currently in clinical trials. These results suggest that MIVE/sub 2/ would be an excellent agent for the study of estrogen receptors in vivo and in vitro.

  12. Does thalidomide affect IL-2 response and production?

    PubMed

    Fernandez, L P; Schlegel, P G; Baker, J; Chen, Y; Chao, N J

    1995-08-01

    The exact mechanism of immunosuppression by thalidomide is poorly understood. A common denominator in the pathogenesis of graft-vs.-host disease, graft rejection, reactional lepromatous leprosy, and autoimmune disorders modulated by thalidomide is the activation of T lymphocytes culminating in the synthesis of interleukin-2 (IL-2), the expression of high-affinity IL-2 receptors, and the induction of proliferation. We investigated the effect of thalidomide on the production of IL-2 by the human leukemia cell line Jurkat through induction of IL-2 gene enhancer activity and through the presence of IL-2 in supernatants. beta-galactosidase activity, encoded by a reporter lac z construct and controlled by a transcription factor in thalidomide-treated PMA- and ionomycin-stimulated Jurkat cells, was similar (97 +/- 1.33%; p > 0.1) to non-thalidomide-treated controls at all drug concentrations tested. IL-2 enhancer-driven beta-galactose activity of thalidomide-treated and stimulated cells was also similar to that of untreated controls (p > 0.2). The IL-2 production of activated nontransfected Jurkat cells was gauged by using the IL-2-dependent cell line HT-2 as a readout and by ELISA. Jurkat cells were subcloned by limiting dilution. Bulk cultures and three subclones (J.5.2.5., J.5.2.9., and J.5.3.8.) were assayed at 6, 12, and 24 hours after PHA/PMA-induced stimulation. No inhibitory effect on the IL-2 production by thalidomide could be detected at any of the drug concentrations tested (5-30 micrograms/mL), whereas 10 to 100 ng/mL of cyclosporine inhibited the IL-2 production by 95 to 100%. In addition, we observed neither inhibition of IL-2-dependent proliferation of HT-2 nor inhibition of PHA-induced proliferation of peripheral mononuclear cells by thalidomide at all drug concentrations used (5-30 micrograms/mL). These results do not support the possibility of a modulatory effect on the immune response by thalidomide via IL-2 production and IL-2 response. PMID:7635184

  13. High-affinity cholecystokinin type A receptor/cytosolic phospholipase A2 pathways mediate Ca2+ oscillations via a positive feedback regulation by calmodulin kinase in pancreatic acini.

    PubMed

    Lankisch, T O; Nozu, F; Owyang, C; Tsunoda, Y

    1999-09-01

    In rat pancreatic acini, we previously demonstrated that depending on the agonist used, activation of cholecystokinin type A (CCKA) receptor (CCK-AR) results in the differential involvement of the cytosolic phospholipase A2 (cPLA2), phospholipase Cbeta1 (PLCbeta1) and Src/protein tyrosine kinase (PTK) pathways. The high-affinity CCK-AR appears to be coupled to the Gbeta/cPLA2/arachidonic acid (AA) cascade in mediating Ca2+ oscillations. The low-affinity CCK-AR is coupled to both the Galphaq/11/PLCbeta1/inositol 1,4,5-trisphosphate (IP3) to evoke intracellular Ca2+ release and the Src/PTK pathway to mediate extracellular Ca2+ influx. The objectives of this study were to provide evidence that cPLA2 is present in pancreatic acini and to evaluate the possibility that its activation results in Ca2+ oscillations and amylase secretion. Furthermore, we investigated the mechanism of Ca2+ oscillations mediated by the high-affinity CCK-AR. In rat pancreatic acini, immunoprecipitation studies using an anti-cPLA2 monoclonal antibody, demonstrated a cPLA2 band at the location of 110 kDa. A selective inhibitor of cPLA2, AACOCF3 (100 microM), inhibited production of AA metabolites, Ca2+ oscillations and amylase secretion elicited by the high-affinity CCK-AR agonist, CCK-OPE (10-1000 nM). In addition, through the repetitive release of intracellular Ca2+, CCK-OPE enhanced phosphotransferase activities of Ca2+/calmodulin-dependent protein kinase type IV (CaMK IV), which were inhibited by AACOCF3. The CaMK inhibitor, K252-a (1-3 microM), also abolished basal and CCK-OPE-stimulated CaMK IV activities. The CaM inhibitor, W-7 (100 microM), and K252-a inhibited Ca2+ oscillations and amylase secretion evoked by CCK-OPE without affecting the AA formation. Therefore, it appears that Ca2+ oscillations elicited by the high-affinity CCK-AR/Gbeta/cPLA2/AA pathway activate CaMK IV. Activated CaMK, in turn, regulates Ca2+ oscillations through a positive feedback mechanism to mediate pancreatic

  14. Identification of specific calcitonin-like receptor residues important for calcitonin gene-related peptide high affinity binding

    PubMed Central

    Banerjee, Sugato; Evanson, Janel; Harris, Erik; Lowe, Stephen L; Thomasson, Kathryn A; Porter, James E

    2006-01-01

    Background Calcitonin gene-related peptide (CGRP) is a vasoactive neuropeptide whose biological activity has potential therapeutic value for many vascular related diseases. CGRP is a 37 amino acid neuropeptide that signals through a G protein-coupled receptor belonging to the secretin receptor family. Previous studies on the calcitonin-like receptor (CLR), which requires co-expression of the receptor-activity-modifying protein-1 (RAMP1) to function as a CGRP receptor, have shown an 18 amino acid N-terminus sequence important for binding CGRP. Moreover, several investigations have recognized the C-terminal amidated phenylalanine (F37) of CGRP as essential for docking to the mature receptor. Therefore, we hypothesize that hydrophobic amino acids within the previously characterized 18 amino acid CLR N-terminus domain are important binding contacts for the C-terminal phenylalaninamide of CGRP. Results Two leucine residues within this previously characterized CLR N-terminus domain, when mutated to alanine and expressed on HEK293T cells stably transfected with RAMP1, demonstrated a significantly decreased binding affinity for CGRP compared to wild type receptor. Additional decreases in binding affinity for CGRP were not found when both leucine mutations were expressed in the same CLR construct. Decreased binding characteristic of these leucine mutant receptors was observed for all CGRP ligands tested that contained the necessary amidated phenylalanine at their C-terminus. However, there was no difference in the potency of CGRP to increase cAMP production by these leucine mutant receptors when compared to wild type CLR, consistent with the notion that the neuropeptide C-terminal F37 is important for docking but not activation of the receptor. This observation was conserved when modified CGRP ligands lacking the amidated F37 demonstrated similar potencies to generate cAMP at both wild type and mutant CLRs. Furthermore, these modified CGRP ligands displayed a significant

  15. Poly(Ethylene Glycol) as a Scaffold for High-Affinity Open-Channel Blockers of the Mouse Nicotinic Acetylcholine Receptor

    PubMed Central

    Lin, Wan-Chen; Licht, Stuart

    2014-01-01

    High-affinity blockers for an ion channel often have complex molecular structures that are synthetically challenging and/or laborious. Here we show that high-affinity blockers for the mouse nicotinic acetylcholine receptor (AChR) can be prepared from a structurally simple material, poly(ethylene glycol) (PEG). The PEG-based blockers (PQ1–5), comprised of a flexible octa(ethylene glycol) scaffold and two terminal quaternary ammonium groups, exert low- to sub-micromolar affinities for the open AChR pore (measured via single-channel analysis of AChRs expressed in human embryonic kidney cells). PQ1–5 are comparable in pore-binding affinity to the strongest AChR open-channel blockers previously reported, which have complex molecular structures. These results suggest a general approach for designing potent open-channel blockers from a structurally flexible polymer. This design strategy involves simple synthetic procedures and does not require detailed information about the structure of an ion-channel pore. PMID:25386750

  16. LYR3, a high-affinity LCO-binding protein of Medicago truncatula, interacts with LYK3, a key symbiotic receptor.

    PubMed

    Fliegmann, Judith; Jauneau, Alain; Pichereaux, Carole; Rosenberg, Charles; Gasciolli, Virginie; Timmers, Antonius C J; Burlet-Schiltz, Odile; Cullimore, Julie; Bono, Jean-Jacques

    2016-05-01

    LYR3, LYK3, and NFP are lysin motif-containing receptor-like kinases (LysM-RLKs) from Medicago truncatula, involved in perception of symbiotic lipo-chitooligosaccharide (LCO) signals. Here, we show that LYR3, a high-affinity LCO-binding protein, physically interacts with LYK3, a key player regulating symbiotic interactions. In vitro, LYR3 is phosphorylated by the active kinase domain of LYK3. Fluorescence lifetime imaging/Förster resonance energy transfer (FLIM/FRET) experiments in tobacco protoplasts show that the interaction between LYR3 and LYK3 at the plasma membrane is disrupted or inhibited by addition of LCOs. Moreover, LYR3 attenuates the cell death response, provoked by coexpression of NFP and LYK3 in tobacco leaves. PMID:27129432

  17. A solid-phase combinatorial approach for indoloquinolizidine-peptides with high affinity at D(1) and D(2) dopamine receptors.

    PubMed

    Molero, Anabel; Vendrell, Marc; Bonaventura, Jordi; Zachmann, Julian; López, Laura; Pardo, Leonardo; Lluis, Carme; Cortés, Antoni; Albericio, Fernando; Casadó, Vicent; Royo, Miriam

    2015-06-01

    Ligands acting at multiple dopamine receptors hold potential as therapeutic agents for a number of neurodegenerative disorders. Specifically, compounds able to bind at D1R and D2R with high affinity could restore the effects of dopamine depletion and enhance motor activation on degenerated nigrostriatal dopaminergic systems. We have directed our research towards the synthesis and characterisation of heterocycle-peptide hybrids based on the indolo[2,3-a]quinolizidine core. This privileged structure is a water-soluble and synthetically accessible scaffold with affinity for diverse GPCRs. Herein we have prepared a solid-phase combinatorial library of 80 indoloquinolizidine-peptides to identify compounds with enhanced binding affinity at D2R, a receptor that is crucial to re-establish activity on dopamine-depleted degenerated GABAergic neurons. We applied computational tools and high-throughput screening assays to identify 9a{1,3,3} as a ligand for dopamine receptors with nanomolar affinity and agonist activity at D2R. Our results validate the application of indoloquinolizidine-peptide combinatorial libraries to fine-tune the pharmacological profiles of multiple ligands at D1 and D2 dopamine receptors. PMID:25969169

  18. EP 171: a high affinity thromboxane A2-mimetic, the actions of which are slowly reversed by receptor blockade.

    PubMed Central

    Jones, R. L.; Wilson, N. H.; Lawrence, R. A.

    1989-01-01

    1. Replacement of the four-carbon omega-terminus in 9,11-endoxy-10a-homo prostaglandin H2 with a p-fluorophenoxy group produces a compound (EP 171) with very high agonist potency at TP-receptors. 2. On six isolated smooth muscle preparations EP 171 was 33-167 times more potent as a TP-receptor agonist than U-46619 (11,9-epoxymethano PGH2); EC50 values ranged from 45 to 138 pM. The actions of EP 171 were difficult to study because of their slow onset and offset. For example, on the guinea-pig trachea the time required for 50% reversal of EP 171-induced contractions during washout was about 3 h. 3. On the pig pulmonary artery, a more rapidly responding preparation, it was possible to show that the TP-receptor antagonist EP 092 blocked the contractile actions of EP 171 and U-46619 to similar extents: pA2 = 8.09 and 8.15 respectively. 4. EP 171 was also a very potent activator of human blood platelets, being about 90 times more potent than U-46619. Both shape change (0.1 nM) and aggregation (1 nM) were slow in onset, a profile not previously observed for a thromboxane A2-mimetic. 5. When potencies at TP-, EP1-(guinea-pig fundus) and FP-(dog iris sphincter) receptors were compared, EP 171 showed a higher specificity as a TP-receptor agonist than either STA2 or U-46619. These studies also showed that contrary to earlier reports, the guinea-pig fundus does contain TP-receptors mediating muscle contraction. However, the maximal response due to activation of TP-receptors was only about 35% of the PGE2 maximum. 6. Established responses to EP 171 were slowly reversed following addition of a high concentration of a TP-receptor antagonist (EP 092, GR 32191 or BM 13177). Faster reversals of three less potent 16-p-halophenoxy prostanoids and U-46619 were obtained. Half-times for offset (and onset) of agonist action appeared to correlate with potency rather than with lipophilicity. 7. Competition between the agonists and a radio iodinated PTA2 derivative ([125I]-PTA-OH) for

  19. Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcεRI.

    PubMed

    Sabban, Sari; Ye, Hongtu; Helm, Birgit

    2014-01-01

    The interaction of IgE with its high-affinity Fc receptor (FcεRI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and FcεRI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine FcεRIα was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine α-chain formed a functional receptor complex with the endogenous rat β- and γ-chains. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine FcεRIα transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using β-hexosaminidase release as a readout. Mediator release peaked at 36.68% ± 4.88% at 100 ng ml(-1) of antigen. This assay was modified from previous assays used to study human and canine allergic responses. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease. PMID:25406512

  20. Purified murine granulocyte/macrophage progenitor cells express a high-affinity receptor for recombinant murine granulocyte/macrophage colony-stimulating factor

    SciTech Connect

    Williams, D.E.; Bicknell, D.C.; Park, L.S.; Straneva, J.E.; Cooper, S.; Broxmeyer, H.E.

    1988-01-01

    Purified recombinant murine granulocyte/macrophage colony-stimulating factor (GM-CSF) was labeled with /sup 125/I and used to examine the GM-CSF receptor on unfractionated normal murine bone marrow cells, casein-induced peritoneal exudate cells, and highly purified murine granulocyte/macrophage progenitor cells (CFU-GM). CFU-GM were isolated from cyclophosphamide-treated mice by Ficoll-Hypaque density centrifugation followed by counterflow centrifugal elutriation. The resulting population had a cloning efficiency of 62-99% in cultures containing conditioned medium from pokeweed mitogen-stimulated spleen cells and 55-86% in the presence of a plateau concentration of purified recombinant murine GM-CSF. Equilibrium binding studies with /sup 125/I-labeled GM-CSF showed that normal bone marrow cells, casein-induced peritoneal exudate cells, and purified CFU-GM had a single class of high-affinity receptor. Affinity crosslinking studies demonstrated that /sup 125/I-labeled GM-CSF bound specifically to two species of M/sub r/ 180,000 and 70,000 on CFU-GM, normal bone marrow cells, and peritoneal exudate cells. The M/sub r/ 70,000 species is thought to be a proteolytic fragment of the intact M/sub r/ 180,000 receptor. The present studies indicate that the GM-CSF receptor expressed on CFU-GM and mature myeloid cells are structurally similar. In addition, the number of GM-CSF receptors on CFU-GM is twice the average number of receptors on casein-induced mature myeloid cells, suggesting that receptor number may decrease as CFU-GM mature.

  1. Impaired NK-mediated regulation of T-cell activity in multiple sclerosis is reconstituted by IL-2 receptor modulation.

    PubMed

    Gross, Catharina C; Schulte-Mecklenbeck, Andreas; Rünzi, Anna; Kuhlmann, Tanja; Posevitz-Fejfár, Anita; Schwab, Nicholas; Schneider-Hohendorf, Tilman; Herich, Sebastian; Held, Kathrin; Konjević, Matea; Hartwig, Marvin; Dornmair, Klaus; Hohlfeld, Reinhard; Ziemssen, Tjalf; Klotz, Luisa; Meuth, Sven G; Wiendl, Heinz

    2016-05-24

    Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS) resulting from a breakdown in peripheral immune tolerance. Although a beneficial role of natural killer (NK)-cell immune-regulatory function has been proposed, it still needs to be elucidated whether NK cells are functionally impaired as part of the disease. We observed NK cells in active MS lesions in close proximity to T cells. In accordance with a higher migratory capacity across the blood-brain barrier, CD56(bright) NK cells represent the major intrathecal NK-cell subset in both MS patients and healthy individuals. Investigating the peripheral blood and cerebrospinal fluid of MS patients treated with natalizumab revealed that transmigration of this subset depends on the α4β1 integrin very late antigen (VLA)-4. Although no MS-related changes in the migratory capacity of NK cells were observed, NK cells derived from patients with MS exhibit a reduced cytolytic activity in response to antigen-activated CD4(+) T cells. Defective NK-mediated immune regulation in MS is mainly attributable to a CD4(+) T-cell evasion caused by an impaired DNAX accessory molecule (DNAM)-1/CD155 interaction. Both the expression of the activating NK-cell receptor DNAM-1, a genetic alteration consistently found in MS-association studies, and up-regulation of the receptor's ligand CD155 on CD4(+) T cells are reduced in MS. Therapeutic immune modulation of IL-2 receptor restores impaired immune regulation in MS by increasing the proportion of CD155-expressing CD4(+) T cells and the cytolytic activity of NK cells. PMID:27162345

  2. Visualizing Microtubule-Dependent Vasopressin Type 2 Receptor Trafficking Using a New High-Affinity Fluorescent Vasopressin Ligand

    PubMed Central

    Chen, Sylvia; Webber, Matthew J.; Vilardaga, Jean-Pierre; Khatri, Ashok; Brown, Dennis; Ausiello, Dennis A.; Lin, Herbert Y.

    2011-01-01

    The vasopressin receptor type 2 (V2R) is the major target of vasopressin (VP) in renal epithelial cells. Although it is known that VP induces V2R internalization, accumulation in the perinuclear area, and degradation, the V2R intracellular trafficking pathways remain elusive. We visualized this process by developing a new fluorescent VP analog tagged by tetramethylrhodamine (TMR)-[Lys-(PEG)2-Suc-TMR8]VP or (VPTMR). This ligand is fully functional as revealed by its high binding affinity toward V2R [(Kd) =157 ± 52 nm] and ability to increase intracellular cAMP 32-fold. VPTMR induced V2R internalization in LLC-PK1 cells expressing either a FLAG-tagged receptor (FLAG-V2R) or V2R C-terminally tagged with green fluorescent protein (GFP) (V2R-GFP). After internalization, VPTMR and V2R-GFP colocalized in the perinuclear area, suggesting that the hormone and receptor traffic along the same pathway. VPTMR and V2R colocalized initially with the early endosome markers EEA1 and Rab5, and later with the recycling and late endosome markers Rab11 and Rab25. Epifluorescence microscopy of LLC-PK1 cells expressing GFP-tagged microtubules (MT) showed that VPTMR-containing vesicles travel along the MT network, and even remain attached to MT during the metaphase and anaphase of mitosis. Colchicine, a MT-depolymerizing agent, abolished perinuclear accumulation of VPTMR, and Western blot analysis showed that VP-induced V2R-GFP degradation is markedly retarded, but not abolished, by colchicine (10 μM). We conclude that the new VPTMR ligand is suitable for dissecting V2R and VP internalization and trafficking in cells, and that V2R trafficking and down-regulation is an MT-dependent mechanism. PMID:21828182

  3. PET studies with low and high affinity dopamine D2 receptor radioligands: Effects of 4-hydroxybutyrate (4HB)

    SciTech Connect

    Gatley, S.J.; Fowler, J.S.; Dewey, S.

    1994-05-01

    D2 radioligands of varying affinities have been developed as PET and SPECT radiotracers, but no consensus has been reached on the abilities of these tracers to quantify D2 receptor concentrations in vivo. Amongst other differences, competition of the radioligand with endogenous DA is expected to depend on affinity for the D2 receptor, so that changes in DA might confound estimates of Bmax. We examined the uptake and kinetics if C-11 raclopride (RAC; Kd = 1.2 nM) and C-11 N-methylspiperone (NMS); Kd = 75 pM in baboon striatum after pretreatment with 4HB (200 mg/Kg, i/v) which inhibits DA release by nigrostriatal nerve terminals. While 4HB diminished uptake (%ID/g) of NMS, it prolonged tissue retention of RAC, confirming previous observations in rodent models. Logan (for RAC) and Patlak (for NMS) plots gave changes of +24% and -20%, respectively, between control and 4HB treated animals. Since decreased competition with DA should increase uptake of NMS as well as RAC the paradoxical decrease in NMS uptake could be due to a second synaptic effect of DA, such as a decrease in agonist mediated internalization of NMS. Alternatively, it could result from an independent effect of 4HB, perhaps related to this drug`s ability to induce anesthesia and to depress cerebral glucose utilization. Although previous work in the rat suggests that 4HB does not alter brain blood flow, we found O-15 water that baboon striatal blood flow was decreased 22% and 42% at 30 and 60 minutes, respectively, after 4HB. Smaller changes were seen in cerebellar blood flow. Though a 4HB induced decrease in blood flow does not rule out a DA mediated alteration in D2 receptor Bmax or Kd for NMS, or other factor, it is unnecessary to invoke this to account for our results.

  4. Characterization of radioiodinated TISCH: a high-affinity and selective ligand for mapping CNS D1 dopamine receptor.

    PubMed

    Billings, J J; Kung, M P; Chumpradit, S; Mozley, D; Alavi, A; Kung, H F

    1992-01-01

    In developing CNS D1 dopamine receptor-imaging agents with improved specificity and longer brain retention, an iodinated D1 ligand was synthesized. In vitro and in vivo radiolabeling studies of a new iodinated benzazepine, TISCH [7-chloro-8-hydroxy-1-(3'-iodophenyl)-3-methyl-2,3,4,5-tetrahydro-1H-3- benzazepine], an analog of SCH 23390 (7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepin e), were investigated. After an intravenous injection, the R(+) isomer of TISCH showed high brain uptake in rats (2.20 and 0.57% dose per whole brain at 2 and 60 min, respectively). The striatum/cerebellum ratio increased progressively with time (12 at 60 min). Ex vivo autoradiography of rat brain sections, after intravenous injection of R(+)-[125I]TISCH, displayed the highest uptake in striatum and substantia nigra, regions known to have a high concentration of D1 receptors, whereas the S(-) isomer displayed no specific uptake. Furthermore, the specific uptake can be blocked by pretreatment with SCH 23390. In vitro binding studies using the rat striatum tissue preparation showed high specific and low nonspecific bindings (KD = 0.21 +/- 0.03 nM). The rank order of potency exhibiting high specificity to the D1 receptor was SCH 23390 greater than (+/-)-TISCH greater than (+)-butaclamol = (+/-)-FISCH [7-chloro-8-hydroxy-1-(4'-iodophenyl)-3-methyl-2,3,4,5-tetrahydro-1 H-3-benzazepine] much greater than WB4101 = spiperone greater than dopamine, serotonin, (+/-)-propranolol, and naloxone. Imaging studies in a monkey with the resolved isomer, R(+)-[123I]TISCH, demonstrated a high uptake in the basal ganglia and prolonged retention. The preliminary data suggest that R(+)-TISCH is selective for the CNS D1 receptor and is potentially useful for in vivo and in vitro pharmacological studies. When labeled with iodine-123, it may be suitable for noninvasive imaging in humans. PMID:1530770

  5. Quantitative Contribution of IL2Rγ to the Dynamic Formation of IL2-IL2R Complexes

    PubMed Central

    Ponce, Luis F.; García-Martínez, Karina; León, Kalet

    2016-01-01

    Interleukin-2 (IL2) is a growth factor for several immune cells and its function depends on its binding to IL2Rs in the cell membrane. The most accepted model for the assembling of IL2-IL2R complexes in the cell membrane is the Affinity Conversion Model (ACM). This model postulates that IL2R receptor association is sequential and dependent on ligand binding. Most likely free IL2 binds first to IL2Rα, and then this complex binds to IL2Rβ, and finally to IL2Rγ (γc). However, in previous mathematical models representing this process, the binding of γc has not been taken into account. In this work, the quantitative contribution of the number of IL2Rγ chain to the IL2-IL2R apparent binding affinity and signaling is studied. A mathematical model of the affinity conversion process including the γ chain in the dynamic, has been formulated. The model was calibrated by fitting it to experimental data, specifically, Scatchard plots obtained using human cell lines. This paper demonstrates how the model correctly explains available experimental observations. It was estimated, for the first time, the value of the kinetic coefficients of IL2-IL2R complexes interaction in the cell membrane. Moreover, the number of IL2R components in different cell lines was also estimated. It was obtained a variable distribution in the number of IL2R components depending on the cell type and the activation state. Of most significance, the study predicts that not only the number of IL2Rα and IL2Rβ, but also the number of γc determine the capacity of the cell to capture and retain IL2 in signalling complexes. Moreover, it is also showed that different cells might use different pathways to bind IL2 as consequence of its IL2R components distribution in the membrane. PMID:27195783

  6. Localized low-level re-expression of high-affinity mesolimbic nicotinic acetylcholine receptors restores nicotine-induced locomotion but not place conditioning.

    PubMed

    Mineur, Y S; Brunzell, D H; Grady, S R; Lindstrom, J M; McIntosh, J M; Marks, M J; King, S L; Picciotto, M R

    2009-04-01

    High-affinity, beta2-subunit-containing (beta2*) nicotinic acetylcholine receptors (nAChRs) are essential for nicotine reinforcement; however, these nAChRs are found on both gamma-aminobutyric acid (GABA) and dopaminergic (DA) neurons in the ventral tegmental area (VTA) and also on terminals of glutamatergic and cholinergic neurons projecting from the pedunculopontine tegmental area and the laterodorsal tegmental nucleus. Thus, systemic nicotine administration stimulates many different neuronal subtypes in various brain nuclei. To identify neurons in which nAChRs must be expressed to mediate effects of systemic nicotine, we investigated responses in mice with low-level, localized expression of beta2* nAChRs in the midbrain/VTA. Nicotine-induced GABA and DA release were partially rescued in striatal synaptosomes from transgenic mice compared with tissue from beta2 knockout mice. Nicotine-induced locomotor activation, but not place preference, was rescued in mice with low-level VTA expression, suggesting that low-level expression of beta2* nAChRs in DA neurons is not sufficient to support nicotine reward. In contrast to control mice, transgenic mice with low-level beta2* nAChR expression in the VTA showed no increase in overall levels of cyclic AMP response element-binding protein (CREB) but did show an increase in CREB phosphorylation in response to exposure to a nicotine-paired chamber. Thus, CREB activation in the absence of regulation of total CREB levels during place preference testing was not sufficient to support nicotine place preference in beta2 trangenic mice. This suggests that partial activation of high-affinity nAChRs in VTA might block the rewarding effects of nicotine, providing a potential mechanism for the ability of nicotinic partial agonists to aid in smoking cessation. PMID:19077117

  7. Roles of the NMDA Receptor and EAAC1 Transporter in the Modulation of Extracellular Glutamate by Low and High Affinity AMPA Receptors in the Cerebellum in Vivo: Differential Alteration in Chronic Hyperammonemia.

    PubMed

    Cabrera-Pastor, Andrea; Taoro, Lucas; Llansola, Marta; Felipo, Vicente

    2015-12-16

    The roles of high- and low-affinity AMPA receptors in modulating extracellular glutamate in the cerebellum remain unclear. Altered glutamatergic neurotransmission is involved in neurological alterations in hyperammonemia, which differently affects high- and low-affinity AMPA receptors. The aims were to assess by in vivo microdialysis (a) the effects of high- and low-affinity AMPA receptor activation on extracellular glutamate in the cerebellum; (b) whether chronic hyperammonemia alters extracellular glutamate modulation by high- and/or low-affinity AMPA receptors; and (c) the contribution of NMDA receptors and EAAC1 transporter to AMPA-induced changes in extracellular glutamate. In control rats, high affinity receptor activation does not affect extracellular glutamate but increases glutamate if NMDA receptors are blocked. Low affinity AMPA receptor activation increases transiently extracellular glutamate followed by reduction below basal levels and return to basal values. The reduction is associated with transient increased membrane expression of EAAC1 and is prevented by blocking NMDA receptors. Blocking NMDA receptors with MK-801 induces a transient increase in extracellular glutamate which is associated with reduced membrane expression of EAAC1 followed by increased membrane expression of the glutamate transporter GLT-1. Chronic hyperammonemia does not affect responses to activation of low affinity AMPA receptors. Activation of high affinity AMPA receptors increases extracellular glutamate in hyperammonemic rats by an NMDA receptor-dependent mechanism. In conclusion, these results show that there is a tightly controlled interplay between AMPA and NMDA receptors and an EAAC1 transporter in controlling extracellular glutamate. Hyperammonemia alters high- but not low-affinity AMPA receptors. PMID:26428532

  8. Differential effects of chronic hyperammonemia on modulation of the glutamate-nitric oxide-cGMP pathway by metabotropic glutamate receptor 5 and low and high affinity AMPA receptors in cerebellum in vivo.

    PubMed

    Cabrera-Pastor, Andrea; Llansola, Marta; Reznikov, Vitaliy; Boix, Jordi; Felipo, Vicente

    2012-07-01

    Previous studies show that chronic hyperammonemia impairs learning ability of rats by impairing the glutamate-nitric oxide (NO)-cyclic guanosine mono-phosphate (cGMP) pathway in cerebellum. Three types of glutamate receptors cooperate in modulating the NO-cGMP pathway: metabotropic glutamate receptor 5 (mGluR5), (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-d-aspartic acid (NMDA) receptors. The aim of this work was to assess whether hyperammonemia alters the modulation of this pathway by mGluR5 and AMPA receptors in cerebellum in vivo. The results support that in control rats: (1) low AMPA concentrations (0.1mM) activate nearly completely Ca(2+)-permeable (glutamate receptor subunit 2 (GluR2)-lacking) AMPA receptors and the NO-cGMP pathway; (2) higher AMPA concentrations (0.3 mM) also activate Ca(2+)-impermeable (GluR2-containing) AMPA receptors, leading to activation of NMDA receptors and of NO-cGMP pathway. Moreover, the data support that chronic hyperammonemia: (1) reduces glutamate release and activation of the glutamate-NO-cGMP pathway by activation of mGluR5; (2) strongly reduces the direct activation by AMPA receptors of the NO-cGMP pathway, likely due to reduced entry of Ca(2+) through GluR2-lacking, high affinity AMPA receptors; (3) strongly increases the indirect activation of the NO-cGMP pathway by high affinity AMPA receptors, likely due to increased entry of Na(+) through GluR2-lacking AMPA receptors and NMDA receptors activation; (4) reduces the indirect activation of the NO-cGMP pathway by low affinity AMPA receptors, likely due to reduced activation of NMDA receptors. PMID:22521775

  9. Insecticidal 3-benzamido-N-phenylbenzamides specifically bind with high affinity to a novel allosteric site in housefly GABA receptors.

    PubMed

    Ozoe, Yoshihisa; Kita, Tomo; Ozoe, Fumiyo; Nakao, Toshifumi; Sato, Kazuyuki; Hirase, Kangetsu

    2013-11-01

    γ-Aminobutyric acid (GABA) receptors (GABARs) are an important target for existing insecticides such as fiproles. These insecticides act as noncompetitive antagonists (channel blockers) for insect GABARs by binding to a site within the intrinsic channel of the GABAR. Recently, a novel class of insecticides, 3-benzamido-N-phenylbenzamides (BPBs), was shown to inhibit GABARs by binding to a site distinct from the site for fiproles. We examined the binding site of BPBs in the adult housefly by means of radioligand-binding and electrophysiological experiments. 3-Benzamido-N-(2,6-dimethyl-4-perfluoroisopropylphenyl)-2-fluorobenzamide (BPB 1) (the N-demethyl BPB) was a partial, but potent, inhibitor of [(3)H]4'-ethynyl-4-n-propylbicycloorthobenzoate (GABA channel blocker) binding to housefly head membranes, whereas the 3-(N-methyl)benzamido congener (the N-methyl BPB) had low or little activity. A total of 15 BPB analogs were tested for their abilities to inhibit [(3)H]BPB 1 binding to the head membranes. The N-demethyl analogs, known to be highly effective insecticides, potently inhibited the [(3)H]BPB 1 binding, but the N-methyl analogs did not even though they, too, are considered highly effective. [(3)H]BPB 1 equally bound to the head membranes from wild-type and dieldrin-resistant (rdl mutant) houseflies. GABA allosterically inhibited [(3)H]BPB 1 binding. By contrast, channel blocker-type antagonists enhanced [(3)H]BPB 1 binding to housefly head membranes by increasing the affinity of BPB 1. Antiparasitic macrolides, such as ivermectin B1a, were potent inhibitors of [(3)H]BPB 1 binding. BPB 1 inhibited GABA-induced currents in housefly GABARs expressed in Xenopus oocytes, whereas it failed to inhibit l-glutamate-induced currents in inhibitory l-glutamate receptors. Overall, these findings indicate that BPBs act at a novel allosteric site that is different from the site for channel blocker-type antagonists and that is probably overlapped with the site for macrolides

  10. IL-1 binds to high affinity receptors on human osteosarcoma cells and potentiates prostaglandin E2 stimulation of cAMP production

    SciTech Connect

    Rodan, S.B.; Wesolowski, G.; Chin, J.; Limjuco, G.A.; Schmidt, J.A.; Rodan, G.A. )

    1990-08-15

    IL-1 is a potent bone resorbing agent. Its mechanism of action is unknown, but the presence of osteoblasts was shown to be necessary for IL-1 stimulation of bone resorption by isolated osteoclasts. This study examines the presence of IL-1R and IL-1 effects in osteoblastic cells from a clonal human osteosarcoma cell line, Saos-2/B-10. We found that the binding affinity and the number of binding sites increases substantially during the postconfluent stage. Scatchard and curve-fitting analysis revealed one class of high affinity binding sites, with Kd/Ki's of 40 +/- 17 pM (mean +/- SD) for IL-1 alpha (n = 5) and 9 +/- 7 pM for IL-1 beta (n = 5) and 2916 +/- 2438 (n = 6) receptors/cell. Incubation of the cells with 125I-IL-1 alpha (100 pM) at 4 degrees C, followed by incubation at 37 degrees C up to 4 h, revealed internalization of receptor-bound IL-1 alpha. Chemical cross-linking studies showed that the IL-1R in Saos-2/B-10 cells had a molecular mass of approximately 80 kDa. To assess the biologic effect of IL-1 in Saos-2/B-10 cells, we determined PGE2 content and adenylate cyclase activity. Although IL-1 had no effect on PGE2 synthesis, both IL-1 alpha and IL-1 beta enhanced PGE2 stimulation of adenylate cyclase two- to four-fold in a dose-dependent manner. The half-maximal effect for IL-1 alpha was seen at 8 to 10 pM and for IL-1 beta at 0.6 to 1.8 pM. IL-1 did not enhance basal adenylate cyclase or stimulation by parathyroid hormone, isoproterenol, or forskolin. IL-1 enhancement of PGE2-stimulated adenylate cyclase was detected between 1 to 2 h, was maximal at 4 to 5 h, was not prevented by cycloheximide treatment, and was seen in membranes from IL-1 pretreated cells. These data show effects of IL-1 on a human osteoblast-like cell line that are mediated by high affinity receptors. These IL-1 effects could contribute to the biologic action of IL-1 on bone.

  11. (3H) 5,7-dichlorokynurenic acid, a high affinity ligand for the NMDA receptor glycine regulatory site

    SciTech Connect

    Hurt, S.D.; Baron, B.M. )

    1991-01-01

    The NMDA subtype of glutamate receptors is allosterically linked to a strychnine-insensitive glycine regulatory site. Kynurenic acid and its halogenated derivatives are non-competitive NMDA antagonists acting at the glycine site. The authors have prepared (3H) 5,7-dichlorokyrurenic acid (DCKA) as an antagonist radioligand and have characterized its binding. 3-Bromo-5,7-DCKA was catalytically dehalogenated in the presence of tritium gas and HPLC purified to yield (3H) 5,7-DCKA with a specific activity of 17.6 Ci/mmol. (3H) 5,7-DCKA bound to rat brain synaptosomes with a Kd of 69 {plus minus} 23 nM and Bmax = 14.5 {plus minus} 3.2 pmoles/mg protein. Binding was 65-70% specific at 10 nM (3H) 5,7-DCKA. This ligand is thus more selective and has higher affinity than (3H) glycine, in addition to being an antagonist.

  12. Nucleotide binding by the widespread high-affinity cyclic di-GMP receptor MshEN domain.

    PubMed

    Wang, Yu-Chuan; Chin, Ko-Hsin; Tu, Zhi-Le; He, Jin; Jones, Christopher J; Sanchez, David Zamorano; Yildiz, Fitnat H; Galperin, Michael Y; Chou, Shan-Ho

    2016-01-01

    C-di-GMP is a bacterial second messenger regulating various cellular functions. Many bacteria contain c-di-GMP-metabolizing enzymes but lack known c-di-GMP receptors. Recently, two MshE-type ATPases associated with bacterial type II secretion system and type IV pilus formation were shown to specifically bind c-di-GMP. Here we report crystal structure of the MshE N-terminal domain (MshEN1-145) from Vibrio cholerae in complex with c-di-GMP at a 1.37 Å resolution. This structure reveals a unique c-di-GMP-binding mode, featuring a tandem array of two highly conserved binding motifs, each comprising a 24-residue sequence RLGxx(L/V/I)(L/V/I)xxG(L/V/I)(L/V/I)xxxxLxxxLxxQ that binds half of the c-di-GMP molecule, primarily through hydrophobic interactions. Mutating these highly conserved residues markedly reduces c-di-GMP binding and biofilm formation by V. cholerae. This c-di-GMP-binding motif is present in diverse bacterial proteins exhibiting binding affinities ranging from 0.5 μM to as low as 14 nM. The MshEN domain contains the longest nucleotide-binding motif reported to date. PMID:27578558

  13. Syntheses of 2-amino and 2-halothiazole derivatives as high-affinity metabotropic glutamate receptor subtype 5 ligands and potential radioligands for in vivo imaging.

    PubMed

    Siméon, Fabrice G; Wendahl, Matthew T; Pike, Victor W

    2011-02-10

    The structure of the potent selective mGlu(5) ligand, SP203 (1, 3-fluoro-5-[[2-(fluoromethyl)thiazol-4-yl]ethynyl]benzonitrile), was modified by replacing the 2-fluoromethyl substituent with an amino or halo substituent and by variation of substituents in the distal aromatic ring to provide a series of new high-affinity mGlu(5) ligands. In this series, among the most potent ligands obtained, the 2-chloro-thiazoles 7a and 7b and the 2-fluorothiazole 10b showed subnanomolar mGlu(5) affinity. 10b also displayed >10000-fold selectivity over all other metabotropic receptor subtypes plus a wide range of other receptors and binding sites. The 2-fluorothiazoles 10a and 10b were labeled using [(18)F]fluoride ion (t(1/2) = 109.7 min) in moderately high radiochemical yield to provide potential radioligands that may resist troublesome radiodefluorination during the imaging of brain mGlu(5) with position emission tomography. The iodo compound 9b has nanomolar affinity for mGlu(5) and may also serve as a lead to a potential (123)I-labeled ligand for imaging brain mGlu(5) with single photon emission computed tomography. PMID:21207959

  14. Selection of a high-affinity WW domain against the extracellular region of VEGF receptor isoform-2 from a combinatorial library using CIS display.

    PubMed

    Patel, Seema; Mathonet, Pascale; Jaulent, Agnes M; Ullman, Christopher G

    2013-04-01

    WW domains are small β-sheet motifs that are involved in intracellular signalling through the recognition of proline-rich or phosphorylated linear peptide sequences. Here, we describe modification of this motif to provide a framework for engineering the side chains exposed on its concave surface. This non-natural scaffold incorporates an additional tryptophan, has a shorter loop 1 and supports modification of 25% of the natural protein to form a novel affinity reagent. We demonstrate the utility of this structure by selecting a high-affinity binder to the extracellular region of human vascular endothelial growth factor receptor isoform 2 (VEGFR-2) from a library of modifications, using a cell-free molecular display platform, CIS display. The isolate has low nanomolar affinity to VEGFR-2 and inhibits binding of human VEGF to its receptor with nanomolar activity. The structure is amenable to cyclisation to improve its proteolytic stability and has advantages over larger protein scaffolds in that it can be synthesised chemically to high yields offering potential for therapeutic and non-therapeutic applications. PMID:23378640

  15. High Affinity Dopamine D3 Receptor (D3R)-Selective Antagonists Attenuate Heroin Self-Administration in Wild-Type but not D3R Knockout Mice.

    PubMed

    Boateng, Comfort A; Bakare, Oluyomi M; Zhan, Jia; Banala, Ashwini K; Burzynski, Caitlin; Pommier, Elie; Keck, Thomas M; Donthamsetti, Prashant; Javitch, Jonathan A; Rais, Rana; Slusher, Barbara S; Xi, Zheng-Xiong; Newman, Amy Hauck

    2015-08-13

    The dopamine D3 receptor (D3R) is a promising target for the development of pharmacotherapeutics to treat substance use disorders. Several D3R-selective antagonists are effective in animal models of drug abuse, especially in models of relapse. Nevertheless, poor bioavailability, metabolic instability, and/or predicted toxicity have impeded success in translating these drug candidates to clinical use. Herein, we report a series of D3R-selective 4-phenylpiperazines with improved metabolic stability. A subset of these compounds was evaluated for D3R functional efficacy and off-target binding at selected 5-HT receptor subtypes, where significant overlap in SAR with D3R has been observed. Several high affinity D3R antagonists, including compounds 16 (Ki = 0.12 nM) and 32 (Ki = 0.35 nM), showed improved metabolic stability compared to the parent compound, PG648 (6). Notably, 16 and the classic D3R antagonist SB277011A (2) were effective in reducing self-administration of heroin in wild-type but not D3R knockout mice. PMID:26203768

  16. Syntheses of 2-Amino and 2-Halothiazole Derivatives as High-Affinity Metabotropic Glutamate Receptor Subtype 5 Ligands and Potential Radioligands for In Vivo Imaging

    PubMed Central

    Siméon, Fabrice G; Wendahl, Matthew T.; Pike, Victor W.

    2011-01-01

    The structure of the potent selective mGlu5 ligand, SP203 (1, 3-fluoro-5-[[2-(fluoromethyl)thiazol-4-yl]ethynyl]benzonitrile), was modified by replacing the 2-fluoromethyl substituent with an amino or halo substituent and by variation of substituents in the distal aromatic ring to provide a series of new high-affinity mGlu5 ligands. In this series, among the most potent ligands obtained, the 2-chloro-thiazoles 7a and 7b and the 2-fluorothiazole 10b showed sub-nanomolar mGlu5 affinity. 10b also displayed >10,000-fold selectivity over all other metabotropic receptor subtypes plus a wide range of other receptors and binding sites. The 2-fluorothiazoles 10a and 10b were labeled using [18F]fluoride ion (t1/2 = 109.7 min) in moderately high radiochemical yield to provide potential radioligands that may resist troublesome radiodefluorination during the imaging of brain mGlu5 with position emission tomography. The iodo compound 9b has nanomolar affinity for mGlu5 and may also serve as a lead to a potential 123I-labeled ligand for imaging brain mGlu5 with single photon emission computed tomography. PMID:21207959

  17. Certain photooxidized derivatives of tryptophan bind with very high affinity to the Ah receptor and are likely to be endogenous signal substances

    SciTech Connect

    Rannug, A.; Rannug, U.; Rosenkranz, H.S.; Winqvist, L.; Westerholm, R.; Agurell, E.; Grafstroem, A.K.

    1987-11-15

    The purpose of the present study was to determine whether ultraviolet light (UV) irradiation of amino acids produces compounds with affinity for the Ah receptor. Aqueous solutions of L-tryptophan were exposed to radiation from an unfiltered high-pressure mercury lamp. The photoproducts formed were solvent-extracted or concentrated on Sep-Pak C18 cartridges. The concentrated extracts or eluants were treated for their ability to compete with /sup 3/H-labeled 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Binding was assayed in liver cytosolic preparations from Sprague-Dawley rats using a technique based on hydroxylapatite separation. Photoproducts with receptor affinity were formed in a time-dependent manner. Histidine and tryptamine also gave products upon UV irradiation that competed with TCDD. Commercial tryptophan, at least aged, contained trace amounts of impurities with receptor affinity. Analysis by TLC and high-pressure liquid chromatography of the photo-products of tryptophan showed a minimum of three different binding compounds. Two of the products were studied in greater detail. One of them, showing UV absorbance and yellow fluorescence, gave a molecular ion (M+) of 284 and the other gave M+ 312 but showed little UV absorption and fluorescence. The concentration, based on mass spectrometry quantifications, of the two compounds that displaced more than 50% of TCDD was found to be extremely low, giving Kd values of 0.44 nM (M+ 312) and 0.07 nM (M+ 284). The existence of high affinity receptors for oxidized amino acids is postulated and their possible role in the proliferative cellular responses to TCDD and tryptophan is discussed briefly.

  18. A Bg1I polymorphism for the interleukin-2 receptor gene (IL2R) on chromosome 10

    SciTech Connect

    Ardinger, R.H.; Murray, J.C. )

    1988-08-25

    pIL2R2 is a cDNA for IL2R cloned into the EcoRI site of pBR322 and encoding from the 5{prime} flanking region to exon 7. Bg1I identifies a two allele polymorphism with 17 kb and 16 kb alleles. Co-dominant inheritance was demonstrated in 6 CEPH families. SpeI shows a possible RFLP with I12R2.

  19. A systemic defect in Toll-like receptor 4 signaling increases lipopolysaccharide-induced suppression of IL-2-dependent T-cell proliferation in COPD.

    PubMed

    Knobloch, Jürgen; Chikosi, Sarah-Jane; Yanik, Sarah; Rupp, Jan; Jungck, David; Koch, Andrea

    2016-01-01

    The susceptibility to bacterial infections is increased in chronic obstructive pulmonary disease (COPD). This promotes exacerbations. IL-2 triggers CD4(+)/Th1-cell proliferation, which is important for infection defense. Bacterial endotoxin (LPS) activates MyD88/IRAK and TRIF/IKKε/TBK1 pathways via Toll-like receptor-4 (TLR4) in Th1 cells. Systemic defects in TLR pathways in CD4(+)/Th1 cells cause an impairment of IL-2-dependent immune responses to bacterial infections in COPD. Peripheral blood CD4(+) T cells of never smokers, smokers without COPD, and smokers with COPD (each n = 10) were ex vivo activated towards Th1 and stimulated with LPS. IL-2, MyD88, and TRIF expression, and cell proliferation was analyzed by ELISA, quantitative RT-PCR, and bromodeoxyuridine (BrdU) and trypan blue staining comparative among the cohorts. IL-2 release from activated T cells was increased in COPD vs. smokers and never smokers. LPS reduced IL-2 expression and T-cell proliferation. These effects were increased in COPD vs. never smokers and inversely correlated with FEV1 (%predicted). The MyD88/TRIF ratio was decreased in Th1 cells of COPD. The suppression of IL-2 by LPS was abolished by MyD88/IRAK blockade in never smokers but by TRIF/IKKε/TBK1 blockade in COPD. Moxifloxacin restored IL-2 expression and T-cell proliferation in the presence of LPS by blocking p38 MAPK. The increased IL-2 release from Th1 cells in COPD might contribute to airway inflammation in disease exacerbations. A switch from MyD88/IRAK to TRIF/IKKε/TBK1 signaling amplifies the suppression of IL-2-dependent proliferation of CD4(+) T cells by LPS in COPD. This molecular pathology is of systemic origin, might impair adaptive immune responses, and could explain the increased susceptibility to bacterial infections in COPD. Targeting TLR4-downstream signaling, for example, with moxifloxacin, might reduce exacerbation rates. PMID:26498252

  20. Neuronal acetylcholine receptors in Drosophila: the ARD protein is a component of a high-affinity alpha-bungarotoxin binding complex.

    PubMed Central

    Schloss, P; Hermans-Borgmeyer, I; Betz, H; Gundelfinger, E D

    1988-01-01

    The ard gene of Drosophila melanogaster encodes a structural homologue of vertebrate nicotinic acetylcholine receptors (AChR) and is expressed exclusively in nervous tissue. To study the nature of the ARD protein, antibodies were raised against fusion constructs containing two regions of this polypeptide. One segment is putatively extracellular (amino acids 65-212), the other domain is exposed to the cytoplasm (amino acids 305-444). The ARD antisera obtained served to investigate the physical relationship between the ARD protein and alpha-bungarotoxin (alpha-Btx) binding sites occurring in Drosophila. Two different high-affinity binding sites for [125I]alpha-Btx, a highly potent antagonist of vertebrate muscle AChR, were detected in fly head membranes. Equilibrium binding and kinetic studies revealed Kd values of approximately 0.1 nM (site 1) and approximately 4 nM (site 2). The estimated maximal binding (Bmax) was approximately 240 and 1080 fmol/mg protein respectively. Both sites exhibited a nicotinic-cholinergic pharmacology. Immunoprecipitation experiments with the ARD antisera indicated that the ARD protein is associated with the [125I]alpha-Btx binding site 1 only. These data support the previously postulated hypothesis that the ARD protein is part of an alpha-Btx binding neuronal AChR of Drosophila. Furthermore, they indicate heterogeneity in nicotinic-cholinergic binding sites in the insect nervous system. PMID:3141150

  1. High-affinity NGF receptor in the rat spinal cord during acute and chronic phases of experimental autoimmune encephalomyelitis: a possible functional significance.

    PubMed

    Oderfeld-Nowak, B; Zaremba, M; Lipkowski, A W; Kwiatkowska-Patzer, B; Triaca, V; Aloe, L

    2003-03-01

    The biological effects of Nerve Growth Factor (NGF) are primarily mediated via its high affinity receptor-TrkA. In the present study, we examined the effect of experimental autoimmune encephalomyelitis (EAE) upon the expression of TrkA in neuronal and non-neuronal cells of the spinal cord of Lewis rats during the acute (14 days postimmunization) and chronic (12 months postimmunization) phases of the disease. In the normal spinal cord, both of mature and aged rats, we found TrkA immunoreaction (TrkA-IR) in the motoneurons of the Rexed lamina IX and in both oligo- and astroglia cells. In the acute phase of the disease, we found a reduction of TrkA immunoreactivity in motoneurons and its up-regulation in oligodendroglia, mainly in the white matter. We also confirmed our previous findings concerning the up-regulation of TrkA-IR in astroglia. Both neuronal and non-neuronal changes of TrkA immunoreactivity had a transient character: they were not seen in the chronic phase of the disease. Our results suggest that both neuronal and glial TrkA expression changes depend on inflammation. Moreover, our data indicate that, during the acute phase of EAE, the glial cells become more receptive to NGF, pointing to glia as an important target for pharmacological manipulations, particularly for exogenously administered NGF. PMID:12825322

  2. Analysis of the conformation and thermal stability of the high-affinity IgE Fc receptor β chain polymorphic proteins.

    PubMed

    Terada, Tomoyoshi; Takahashi, Teppei; Arikawa, Hajime; Era, Seiichi

    2016-07-01

    The high-affinity IgE Fc receptor (FcεRI) β chain acts as a signal amplifier through the immunoreceptor tyrosine-based activation motif in its C-terminal intracellular region. Polymorphisms in FcεRI β have been linked to atopy, asthma, and allergies. We investigated the secondary structure, conformation, and thermal stability of FcεRI β polymorphic (β-L172I, β-L174V, and β-E228G) proteins. Polymorphisms did not affect the secondary structure and conformation of FcεRI β. However, we calculated Gibbs free energy of unfolding (ΔGunf) and significant differences were observed in ΔGunf values between the wild-type FcεRI β (β-WT) and β-E228G. These results suggested that β-E228G affected the thermal stability of FcεRI β. The role of β-E228G in biological functions and its involvement in allergic reactions have not yet been elucidated in detail; therefore, differences in the thermal stability of β-E228G may affect the function of FcεRI β. PMID:26940508

  3. Defect in the membrane expression of high affinity 72-kD Fc gamma receptors on phagocytic cells in four healthy subjects.

    PubMed Central

    Ceuppens, J L; Baroja, M L; Van Vaeck, F; Anderson, C L

    1988-01-01

    Three different receptors for the Fc portion of IgG (FcR) have been characterized on human leukocytes. We have identified four healthy members of one family, whose blood phagocytic cells lack functional 72 kD high-affinity FcRI. Their monocytes were unable to bind the Fc portion of mouse (m)-IgG2a and of monomeric human IgG, and they were unreactive with two anti-FcRI monoclonal antibodies. Thus, FcRI is either absent, expressed at very low density, or is so structurally altered as to be unable to bind both its ligand and the anti-FcRI antibodies. The failure to bind the Fc portion of mIgG2a underlies the previously reported inability of these monocytes to support T cell mitogenesis on OKT3 stimulation. FcRI was not inducible upon incubation of their monocytes or neutrophils in gamma interferon. However, their monocytes were able to bind aggregated human IgG, and to phagocytose IgG-coated particles in vitro. Both functions could be blocked with a monoclonal antibody to the 40-kD low-affinity FcRII and therefore apparently were mediated exclusively through FcRII. This also demonstrates that FcRII can mediate phagocytosis independently. Despite the FcRI defect, these subjects had no circulating immune complexes, no evidence of autoimmune pathology and no increased susceptibility to infections. PMID:2969920

  4. A Human Platelet Receptor Protein Microarray Identifies the High Affinity Immunoglobulin E Receptor Subunit α (FcεR1α) as an Activating Platelet Endothelium Aggregation Receptor 1 (PEAR1) Ligand*

    PubMed Central

    Sun, Yi; Vandenbriele, Christophe; Kauskot, Alexandre; Verhamme, Peter; Hoylaerts, Marc F.; Wright, Gavin J.

    2015-01-01

    Genome-wide association studies to identify loci responsible for platelet function and cardiovascular disease susceptibility have repeatedly identified polymorphisms linked to a gene encoding platelet endothelium aggregation receptor 1 (PEAR1), an “orphan” cell surface receptor that is activated to stabilize platelet aggregates. To investigate how PEAR1 signaling is initiated, we sought to identify its extracellular ligand by creating a protein microarray representing the secretome and receptor repertoire of the human platelet. Using an avid soluble recombinant PEAR1 protein and a systematic screening assay designed to detect extracellular interactions, we identified the high affinity immunoglobulin E (IgE) receptor subunit α (FcεR1α) as a PEAR1 ligand. FcεR1α and PEAR1 directly interacted through their membrane-proximal Ig-like and 13th epidermal growth factor domains with a relatively strong affinity (KD ∼ 30 nm). Precomplexing FcεR1α with IgE potently inhibited the FcεR1α-PEAR1 interaction, and this was relieved by the anti-IgE therapeutic omalizumab. Oligomerized FcεR1α potentiated platelet aggregation and led to PEAR1 phosphorylation, an effect that was also inhibited by IgE. These findings demonstrate how a protein microarray resource can be used to gain important insight into the function of platelet receptors and provide a mechanistic basis for the initiation of PEAR1 signaling in platelet aggregation. PMID:25713122

  5. High-affinity binding of [3H]estradiol-17 beta by an estrogen receptor in the liver of the turtle.

    PubMed

    Ho, S M; Fehrer, S; Yu, M; Liang, L C; Press, D

    1988-06-01

    Specific [3H]estradiol-17 beta ([3H]E2) binding activity (EBA) with characteristics of an estrogen receptor (ER) was demonstrated in cytosols and nuclear extracts of the female turtle, Chrysemys picta. Three different receptor assays (dextran-coated charcoal assay, hydroxylapatite batch procedure, and DNA-cellulose chromatography) were evaluated in terms of their applicability in analyzing large numbers of samples. For the measurement of cytosolic EBA, the hydroxylapatite batch procedure was found to be the most reliable assay. On the other hand, the dextran-coated charcoal assay was found to be the most appropriate method for the measurement of nuclear EBA. Turtle hepatic EBA binds [3H]E2 with high affinity (cytosolic, 17.4 +/- 2.8 X 10(9) M-1; nuclear, 17.7 +/- 1.9 X 10(9) M-1), limited capacity (cytosolic, 133.7 +/- 4.6 fmol/g tissue; nuclear, 81.1 +/- 9.0 fmol/g tissue), and strict steroid specificity. The EBA bound natural estrogens (E2, estrone, estriol) as well as the nonsteroidal estrogen, diethylstilbestrol, but exhibited little affinity for androgens, progesterone, or corticosterone. The turtle hepatic EBA resembled mammalian and avian ERs in terms of binding characteristics; however, unlike mammalian and avian ERs it was shown to be heat-labile. Incubation at 30 degrees caused rapid loss of [3H]E2 binding activity in both cytosolic and nuclear fractions. The exchange between [3H]E2 and the endogenously bound estrogen was slow at 4 and 15 degrees, but the exchange process was facilitated in the presence of the chaotropic salt, NaSCN. Establishment of quantitation methods for both cytosolic and nuclear forms of EBA will enable future investigation of the mechanism and regulation of estrogen action in the liver of this turtle species. PMID:3417113

  6. Chemically Modified DNA Aptamers Bind Interleukin-6 with High Affinity and Inhibit Signaling by Blocking Its Interaction with Interleukin-6 Receptor

    PubMed Central

    Gupta, Shashi; Hirota, Masao; Waugh, Sheela M.; Murakami, Ikuo; Suzuki, Tomoki; Muraguchi, Masahiro; Shibamori, Masafumi; Ishikawa, Yuichi; Jarvis, Thale C.; Carter, Jeffrey D.; Zhang, Chi; Gawande, Bharat; Vrkljan, Michael; Janjic, Nebojsa; Schneider, Daniel J.

    2014-01-01

    Interleukin-6 (IL-6) is a pleiotropic cytokine that regulates immune and inflammatory responses, and its overproduction is a hallmark of inflammatory diseases. Inhibition of IL-6 signaling with the anti-IL-6 receptor antibody tocilizumab has provided some clinical benefit to patients; however, direct cytokine inhibition may be a more effective option. We used the systematic evolution of ligands by exponential enrichment (SELEX) process to discover slow off-rate modified aptamers (SOMAmers) with hydrophobic base modifications that inhibit IL-6 signaling in vitro. Two classes of IL-6 SOMAmers were isolated from modified DNA libraries containing 40 random positions and either 5-(N-benzylcarboxamide)-2′-deoxyuridine (Bn-dU) or 5-[N-(1-naphthylmethyl)carboxamide]-2′-deoxyuridine (Nap-dU) replacing dT. These modifications facilitate the high affinity binding interaction with IL-6 and provide resistance against degradation by serum endonucleases. Post-SELEX optimization of one Bn-dU and one Nap-dU SOMAmer led to improvements in IL-6 binding (10-fold) and inhibition activity (greater than 20-fold), resulting in lead SOMAmers with sub-nanomolar affinity (Kd = 0.2 nm) and potency (IC50 = 0.2 nm). Although similar in inhibition properties, the two SOMAmers have unique sequences and different ortholog specificities. Furthermore, these SOMAmers were stable in human serum in vitro for more than 48 h. Both SOMAmers prevented IL-6 signaling by blocking the interaction of IL-6 with its receptor and inhibited the proliferation of tumor cells in vitro as effectively as tocilizumab. This new class of IL-6 inhibitor may be an effective therapeutic alternative for patients suffering from inflammatory diseases. PMID:24415766

  7. High-affinity binding of (/sup 3/H)estradiol-17 beta by an estrogen receptor in the liver of the turtle

    SciTech Connect

    Ho, S.M.; Fehrer, S.; Yu, M.; Liang, L.C.; Press, D.

    1988-06-01

    Specific (3H)estradiol-17 beta ((3H)E2) binding activity (EBA) with characteristics of an estrogen receptor (ER) was demonstrated in cytosols and nuclear extracts of the female turtle, Chrysemys picta. Three different receptor assays (dextran-coated charcoal assay, hydroxylapatite batch procedure, and DNA-cellulose chromatography) were evaluated in terms of their applicability in analyzing large numbers of samples. For the measurement of cytosolic EBA, the hydroxylapatite batch procedure was found to be the most reliable assay. On the other hand, the dextran-coated charcoal assay was found to be the most appropriate method for the measurement of nuclear EBA. Turtle hepatic EBA binds (3H)E2 with high affinity (cytosolic, 17.4 +/- 2.8 X 10(9) M-1; nuclear, 17.7 +/- 1.9 X 10(9) M-1), limited capacity (cytosolic, 133.7 +/- 4.6 fmol/g tissue; nuclear, 81.1 +/- 9.0 fmol/g tissue), and strict steroid specificity. The EBA bound natural estrogens (E2, estrone, estriol) as well as the nonsteroidal estrogen, diethylstilbestrol, but exhibited little affinity for androgens, progesterone, or corticosterone. The turtle hepatic EBA resembled mammalian and avian ERs in terms of binding characteristics; however, unlike mammalian and avian ERs it was shown to be heat-labile. Incubation at 30 degrees caused rapid loss of (3H)E2 binding activity in both cytosolic and nuclear fractions. The exchange between (3H)E2 and the endogenously bound estrogen was slow at 4 and 15 degrees, but the exchange process was facilitated in the presence of the chaotropic salt, NaSCN. Establishment of quantitation methods for both cytosolic and nuclear forms of EBA will enable future investigation of the mechanism and regulation of estrogen action in the liver of this turtle species.

  8. Detection of high affinity receptor sites for IL 1. beta. on a human B lymphoblastoid line which fail to recognize IL 1. cap alpha

    SciTech Connect

    Chin, J.; Cameron, P.; Sigal, N.H.; Schmidt, J.A.

    1986-03-05

    A large number of EBV-transformed human B lines were screened for their ability to bind human pI 6.8 IL 1 (IL 1..beta..) which was labeled to high specific radioactivity with Bolton-Hunter reagent. One of these, designated 2C2, bound (/sup 125/)I-IL 1 in a saturable dose-dependent fashion. Scatchard analysis of direct binding data obtained at equilibrium suggested a single family of receptor sites, at approx. 10,000 sites per cell, with a K/sub d/ = 1.5 +/- 0.2 (+SD) nM. Competition experiments with cold pI 6.8 IL 1 gave a K/sub i/ = 1.0 +/- 0.3 nM. No competition was seen with a 20-fold molar excess of human IL 2, human gamma-INF, or the pI 5.2 and pI 5.4 species of human IL 1. These anionic species of IL 1 have recently been purified to homogeneity by us from monocyte culture supernatants. Amino acid sequence analysis of the pI 5.4 species demonstrates that it is encoded by the recently reported IL 1..cap alpha.. cDNA. Cross linking of pI 6.8 (/sup 125/)I-IL 1 to intact 2C2 cells with increasing amounts of cross linker revealed a single band with a MW congruent to 80,000. Cross-linking was totally abolished by excess unlabeled pI 6.8 IL 1 but not by excess pI 5.4 IL 1. These results show that the receptor for IL 1..beta.. on 2C2 cells is highly specific for one species of human IL 1 and raises the possibility that IL 1..cap alpha.. and IL 1..beta.., though very similar in their biological properties, have separate receptor sites.

  9. Genotypes of NK cell KIR Receptors, Their Ligands, and Fcγ Receptors in the Response of Neuroblastoma Patients to Hu14.18-IL2 Immunotherapy

    PubMed Central

    Delgado, DC; Hank, JA; Kolesar, J; Lorentzen, D; Gan, J; Seo, S; Kim, KM; Shusterman, S; Gillies, SD; Reisfeld, RA; Yang, R; Gadbaw, B; DeSantes, KD; London, WB; Seeger, RC; Maris, JM; Sondel, PM

    2010-01-01

    Response to immunocytokine (IC) therapy is dependent on natural killer (NK) cells in murine neuroblastoma (NBL) models. Furthermore, killer immunoglobulin-like receptor (KIR) / KIR-ligand mismatch is associated with improved outcome to autologous stem cell transplant for NBL. Additionally, clinical anti-tumor response to monoclonal antibodies has been associated with specific polymorphic-FcγR alleles. Relapsed/refractory NBL patients received the hu14.18-IL2 IC (humanized anti-GD2 monoclonal antibody linked to human IL2) in a Children’s Oncology Group (COG) Phase II trial. In this report, these patients were genotyped for KIR, HLA and FcR alleles to determine if KIR receptor-ligand mismatch or specific FcγR alleles were associated with anti-tumor response. Thirty-eight of 39 patients enrolled had DNA available for analysis; 24 were found to have autologous KIR/KIR -ligand mismatch; 14 were matched. Seven of 24 mismatched patients experienced either complete response or improvement of their disease after IC therapy. There was no response or comparable improvement of disease in patients who were matched. Thus KIR/KIR-ligand mismatch was associated with response/improvement to IC (p = 0.03). There was a trend towards patients with the FcγR2A 131-H/H genotype showing a higher response rate compared to other FcγR2A genotypes (p = 0.06). These analyses indicate that response or improvement of relapsed/refractory neuroblastoma patients after IC treatment is associated with autologous KIR/KIR-ligand mismatch, consistent with a role for NK cells in this clinical response. PMID:20935224

  10. Loss of high-affinity prostacyclin receptors in platelets and the lack of prostaglandin-induced inhibition of platelet-stimulated thrombin generation in subjects with spinal cord injury.

    PubMed Central

    Kahn, N N; Bauman, W A; Sinha, A K

    1996-01-01

    Coronary artery disease is a leading cause of death in individuals with chronic spinal cord injury (SCI). However, platelets of those with SCI (n = 30) showed neither increased aggregation nor resistance to the antiaggregatory effects of prostacyclin when compared with normal controls (n = 30). Prostanoid-induced cAMP synthesis was similar in both groups. In contrast, prostacyclin, which completely inhibited the platelet-stimulated thrombin generation in normal controls, failed to do so in those with SCI. Scatchard analysis of the binding of [3H]prostaglandin E1, used as a prostacyclin receptor probe, showed the presence of one high-affinity (Kd1 = 8.11 +/- 2.80 nM; n1 = 172 +/- 32 sites per cell) and one low-affinity (Kd2 = 1.01 +/- 0.3 microM; n2 = 1772 +/- 226 sites per cell) prostacyclin receptor in normal platelets. In contrast, the same analysis in subjects with SCI showed significant loss (P < 0.001) of high-affinity receptor sites (Kd1 = 6.34 +/- 1.91 nM; n1 = 43 +/- 10 sites per cell) with no significant change in the low affinity-receptors (Kd2 = 1.22 +/- 0.23; n2 = 1820 +/- 421). Treatment of these platelets with insulin, which has been demonstrated to restore both of the high- and low-affinity prostaglandin receptor numbers to within normal ranges in coronary artery disease, increased high-affinity receptor numbers and restored the prostacyclin effect on thrombin generation. These results demonstrate that the loss of the inhibitory effect of prostacyclin on the stimulation of thrombin generation was due to the loss of platelet high-affinity prostanoid receptors, which may contribute to atherogenesis in individuals with chronic SCI. PMID:8552614

  11. High Affinity Binding of the Receptor-associated Protein D1D2 Domains with the Low Density Lipoprotein Receptor-related Protein (LRP1) Involves Bivalent Complex Formation: CRITICAL ROLES OF LYSINES 60 AND 191.

    PubMed

    Prasad, Joni M; Young, Patricia A; Strickland, Dudley K

    2016-08-26

    The LDL receptor-related protein 1 (LRP1) is a large endocytic receptor that binds and mediates the endocytosis of numerous structurally diverse ligands. Currently, the basis for ligand recognition by LRP1 is not well understood. LRP1 requires a molecular chaperone, termed the receptor-associated protein (RAP), to escort the newly synthesized receptor from the endoplasmic reticulum to the Golgi. RAP is a three-domain protein that contains the following two high affinity binding sites for LRP1: one is located within domains 1 and 2, and one is located in its third domain. Studies on the interaction of the RAP third domain with LRP1 reveal critical contributions by lysine 256 and lysine 270 for this interaction. From these studies, a model for ligand recognition by this class of receptors has been proposed. Here, we employed surface plasmon resonance to investigate the binding of RAP D1D2 to LRP1. Our results reveal that the high affinity of D1D2 for LRP1 results from avidity effects mediated by the simultaneous interactions of lysine 60 in D1 and lysine 191 in D2 with sites on LRP1 to form a bivalent D1D2-LRP1 complex. When lysine 60 and 191 are both mutated to alanine, the binding of D1D2 to LRP1 is ablated. Our data also reveal that D1D2 is able to bind to a second distinct site on LRP1 to form a monovalent complex. The studies confirm the canonical model for ligand recognition by this class of receptors, which is initiated by pairs of lysine residues that dock into acidic pockets on the receptor. PMID:27402839

  12. The discovery of epidepride and its analogs as high-affinity radioligands for imaging extrastriatal dopamine D(2) receptors in human brain.

    PubMed

    de Paulis, Tomas

    2003-01-01

    [(123)I]Epidepride, [(18)F]fallypride, and [(76)Br]isoremoxipride (FLB-457) and their corresponding [(11)C]labeled derivatives belong to a class of high-affinity radioligands for SPECT or PET imaging of dopamine D(2) receptors in the human brain. In contrast to previously used imaging agents, these ligands are capable of identifying extrastriatal dopamine D(2) receptors. The design of these substituted benzamides derive its origin from the atypical antipsychotic agent, remoxipride. Starting in the late 1970's, halogenated analogs of (S)-sulpiride were evaluated in binding assays and behavioral studies, leading to the discovery of remoxipride. Remoxipride was 10 times weaker than sulpiride in vitro but 50 times more potent in vivo. Search for a putative active metabolite of remoxipride led to the discovery of raclopride and eticlopride, the former becoming a useful radioligand as tritium or carbon-11 labeled form for receptor binding and PET studies, respectively. In the US, the mono-iodine analog of raclopride, [(123)I]iodobenzamide (IBZM), was found to have moderate putamen-to-cerebellum ratio in rat and human brain. Continued search for metabolites of remoxipride led to the discovery of its 3,6-dihydroxy derivative, NCQ-344, with an extremely potent in vivo activity in the rat. SAR studies of the metabolites of remoxipride led to the discovery of the 3-methoxy isomer, isoremoxipride (FLB-457) and its corresponding 6-hydroxy analog, FLB-463, both having affinities for the dopamine D(2) receptor in the 20-30 pM range. Later, the 5-[(123)I]iodo analog of FLB-463, [(123)I]ioxipride ([(123)I]NCQ-298), became a potential SPECT imaging agent. In the mean time, the deshydroxy analog of IBZM, [(125)I]iodopride, showed binding potential in the rat similar to [(125)I]IBZM. Epidepride was designed by combining the structure of isoremoxipride with that of iodopride. In 1988, epidepride was independently prepared and radiolabeled in three separate laboratories in Stockholm

  13. Ethanol Inhibits High-Affinity Immunoglobulin E Receptor (FcεRI) Signaling in Mast Cells by Suppressing the Function of FcεRI-Cholesterol Signalosome

    PubMed Central

    Draberova, Lubica; Paulenda, Tomas; Halova, Ivana; Potuckova, Lucie; Bugajev, Viktor; Bambouskova, Monika; Tumova, Magda; Draber, Petr

    2015-01-01

    Ethanol has multiple effects on biochemical events in a variety of cell types, including the high-affinity immunoglobulin E receptor (FcεRI) signaling in antigen-activated mast cells. However, the underlying molecular mechanism remains unknown. To get better understanding of the effect of ethanol on FcεRI-mediated signaling we examined the effect of short-term treatment with non-toxic concentrations of ethanol on FcεRI signaling events in mouse bone marrow-derived mast cells. We found that 15 min exposure to ethanol inhibited antigen-induced degranulation, calcium mobilization, expression of proinflammatory cytokine genes (tumor necrosis factor-α, interleukin-6, and interleukin-13), and formation of reactive oxygen species in a dose-dependent manner. Removal of cellular cholesterol with methyl-β-cyclodextrin had a similar effect and potentiated some of the inhibitory effects of ethanol. In contrast, exposure of the cells to cholesterol-saturated methyl-β-cyclodextrin abolished in part the inhibitory effect of ethanol on calcium response and production of reactive oxygen species, supporting lipid-centric theories of ethanol action on the earliest stages of mast cell signaling. Further studies showed that exposure to ethanol and/or removal of cholesterol inhibited early FcεRI activation events, including tyrosine phosphorylation of the FcεRI β and γ subunits, SYK kinases, LAT adaptor protein, phospholipase Cγ, STAT5, and AKT and internalization of aggregated FcεRI. Interestingly, ethanol alone, and particularly in combination with methyl-β-cyclodextrin, enhanced phosphorylation of negative regulatory tyrosine 507 of LYN kinase. Finally, we found that ethanol reduced passive cutaneous anaphylactic reaction in mice, suggesting that ethanol also inhibits FcεRI signaling under in vivo conditions. The combined data indicate that ethanol interferes with early antigen-induced signaling events in mast cells by suppressing the function of Fc

  14. Ethanol Inhibits High-Affinity Immunoglobulin E Receptor (FcεRI) Signaling in Mast Cells by Suppressing the Function of FcεRI-Cholesterol Signalosome.

    PubMed

    Draberova, Lubica; Paulenda, Tomas; Halova, Ivana; Potuckova, Lucie; Bugajev, Viktor; Bambouskova, Monika; Tumova, Magda; Draber, Petr

    2015-01-01

    Ethanol has multiple effects on biochemical events in a variety of cell types, including the high-affinity immunoglobulin E receptor (FcεRI) signaling in antigen-activated mast cells. However, the underlying molecular mechanism remains unknown. To get better understanding of the effect of ethanol on FcεRI-mediated signaling we examined the effect of short-term treatment with non-toxic concentrations of ethanol on FcεRI signaling events in mouse bone marrow-derived mast cells. We found that 15 min exposure to ethanol inhibited antigen-induced degranulation, calcium mobilization, expression of proinflammatory cytokine genes (tumor necrosis factor-α, interleukin-6, and interleukin-13), and formation of reactive oxygen species in a dose-dependent manner. Removal of cellular cholesterol with methyl-β-cyclodextrin had a similar effect and potentiated some of the inhibitory effects of ethanol. In contrast, exposure of the cells to cholesterol-saturated methyl-β-cyclodextrin abolished in part the inhibitory effect of ethanol on calcium response and production of reactive oxygen species, supporting lipid-centric theories of ethanol action on the earliest stages of mast cell signaling. Further studies showed that exposure to ethanol and/or removal of cholesterol inhibited early FcεRI activation events, including tyrosine phosphorylation of the FcεRI β and γ subunits, SYK kinases, LAT adaptor protein, phospholipase Cγ, STAT5, and AKT and internalization of aggregated FcεRI. Interestingly, ethanol alone, and particularly in combination with methyl-β-cyclodextrin, enhanced phosphorylation of negative regulatory tyrosine 507 of LYN kinase. Finally, we found that ethanol reduced passive cutaneous anaphylactic reaction in mice, suggesting that ethanol also inhibits FcεRI signaling under in vivo conditions. The combined data indicate that ethanol interferes with early antigen-induced signaling events in mast cells by suppressing the function of Fc

  15. Pulmonary Administration of GW0742, a High-Affinity Peroxisome Proliferator-Activated Receptor Agonist, Repairs Collapsed Alveoli in an Elastase-Induced Mouse Model of Emphysema.

    PubMed

    Ozawa, Chihiro; Horiguchi, Michiko; Akita, Tomomi; Oiso, Yuki; Abe, Kaori; Motomura, Tomoki; Yamashita, Chikamasa

    2016-01-01

    Pulmonary emphysema is a disease in which lung alveoli are irreversibly damaged, thus compromising lung function. Our previous study revealed that all-trans-retinoic acid (ATRA) induces the differentiation of human lung alveolar epithelial type 2 progenitor cells and repairs the alveoli of emphysema model mice. ATRA also reportedly has the ability to activate peroxisome proliferator-activated receptor (PPAR) β/δ. A selective PPARβ/δ ligand has been reported to induce the differentiation of human keratinocytes during wound repair. Here, we demonstrate that treatment using a high-affinity PPARβ/δ agonist, GW0742, reverses the lung tissue damage induced by elastase in emphysema-model mice and improves respiratory function. Mice treated with elastase, which collapsed their alveoli, were then treated with either 10% dimethyl sulfoxide (DMSO) in saline (control group) or GW0742 (1.0 mg/kg twice a week) by pulmonary administration. Treatment with GW0742 for 2 weeks increased the in vivo expression of surfactant proteins A and D, which are known alveolar type II epithelial cell markers. GW0742 treatment also shortened the average distance between alveolar walls in the lungs of emphysema model mice, compared with a control group treated with 10% DMSO in saline. Treatment with GW0742 for 3 weeks also improved tissue elastance (cm H2O/mL), as well as the ratio of the forced expiratory volume in the first 0.05 s to the forced vital capacity (FEV 0.05/FVC). In each of these experiments, GW0742 treatment reversed the damage caused by elastase. In conclusion, PPARβ/δ agonists are potential therapeutic agents for pulmonary emphysema. PMID:27150147

  16. Mapping the binding pocket of a novel, high-affinity, slow dissociating tachykinin NK3 receptor antagonist: biochemical and electrophysiological characterization.

    PubMed

    Malherbe, Pari; Knoflach, Frédéric; Marcuz, Anne; Bohnert, Claudia; Weber, Michael; Knust, Henner; Ratni, Hasane; Spooren, Will; Ballard, Theresa M; Bissantz, Caterina

    2014-11-01

    The NK3 receptor is a GPCR that is prominently expressed in limbic areas of the brain, many of which have been implicated in schizophrenia. Phase II clinical trials in schizophrenia with two selective NK3 antagonists (osanetant and talnetant) have demonstrated significant improvement in positive symptoms. The objective of this study was to characterize the properties of a novel dual NK2/NK3 antagonist, RO5328673. [(3)H]RO5328673 bound to a single saturable site on hNK2, hNK3 and gpNK3 with high-affinity. RO5328673 acted as an insurmountable antagonist at both human and guinea-pig NK3 receptors in the [(3)H]IP accumulation assay. In binding kinetic analyses, [(3)H]RO5328673 had fast association and dissociation rates at hNK2 while it had a fast association rate and a remarkably slow dissociation rate at gp and hNK3. In electrophysiological recordings of gp SNpc, RO5328673 inhibited the senktide-induced potentiation of spontaneous activity of dopaminergic neurons with an insurmountable mechanism of action. RO5328673 exhibited in-vivo activity in gerbils, robustly reversing the senktide-induced locomotor activity. The TM2 residue gpNK3-A114(2.58) (threonine in all other species) was identified as the critical residue for the RO5328673's slower dissociation kinetics and stronger insurmountable mode of antagonism in the guinea-pig as compared to hNK3-T139(2.58). Using site-directed mutagenesis, [(3)H]RO5328673 binding and rhodopsin-based modeling, the important molecular determinants of the RO5328673-binding pocket of hNK3 were determined. A comparison of the RO5328673-binding pocket with that of osanetant showed that two antagonists have similar contact sides on hNK3 binding crevice except for three mutations V95L(1.42), Y247W(5.38), V255I(5.46), which behaved differently between interacting modes of two antagonists in hNK3. PMID:25107588

  17. Diphtheria toxin-based bivalent human IL-2 fusion toxin with improved efficacy for targeting human CD25(+) cells.

    PubMed

    Peraino, Jaclyn Stromp; Zhang, Huiping; Rajasekera, Priyani V; Wei, Min; Madsen, Joren C; Sachs, David H; Huang, Christene A; Wang, Zhirui

    2014-03-01

    Regulatory T cells (Treg) constitute a major inhibitory cell population which suppresses immune responses. Thus, Treg have proven to be key players in the induction of transplantation tolerance, protection from autoimmune disease and prevention of the development of effective anti-tumor immune reactions. Treg express high levels of the high affinity interleukin-2 receptor (IL-2R) consisting of IL-2Rα (CD25) together with IL-2Rβ (CD122) and the common γ-chain (CD132). An effective reagent capable of depleting Treg in vivo would facilitate better cancer treatment and allow mechanistic studies of the role of Treg in transplantation tolerance and the development of autoimmune disease. In this study, we have developed a novel bivalent human IL-2 fusion toxin along with an Ontak®-like monovalent human IL-2 fusion toxin and compared the functional ability of these reagents in vitro. Here we show that genetically linking two human IL-2 domains in tandem, thereby generating a bivalent fusion toxin, results in significantly improved capacity in targeting human CD25(+) cells in vitro. Binding analysis by flow cytometry showed that the bivalent human IL-2 fusion toxin has notably increased affinity for human CD25(+) cells. In vitro functional analysis demonstrated that the bivalent isoform has an increased potency of approximately 2 logs in inhibiting cellular proliferation and protein synthesis in human CD25(+) cells compared to the monovalent human IL-2 fusion toxin. Additionally, we performed two inhibition assays in order to verify that the fusion toxins target the cells specifically through binding of the human IL-2 domain of the fusion toxin to the human IL-2 receptor on the cell surface. These results demonstrated that 1) both monovalent and bivalent human IL-2 fusion toxins are capable of blocking the binding of biotinylated human IL-2 to human CD25 by flow cytometry; and 2) human IL-2 blocked the fusion toxins from inhibiting protein synthesis and cellular

  18. Identification of contact and respiratory sensitizers according to IL-4 receptor α expression and IL-2 production

    SciTech Connect

    Goutet, Michèle Pépin, Elsa; Langonné, Isabelle; Huguet, Nelly; Ban, Masarin

    2012-04-15

    Identification of allergenic chemicals is an important occupational safety issue. While several methods exist to identify contact sensitizers, there is currently no validated model to predict the potential of chemicals to act as respiratory sensitizers. Previously, we reported that cytometry analysis of the local immune responses induced in mice dermally exposed to the respiratory sensitizer trimellitic anhydride (TMA 10%) and contact sensitizer dinitrochlorobenzene (DNCB 1%) could identify divergent expression of several immune parameters. The present study confirms, first, that IgE-positive B cells, MHC class II molecules, interleukin (IL)-2, IL-4 and IL-4Rα can differentiate the allergic reactions caused by high doses of strong respiratory (TMA, phthalic anhydride and toluene diisocyanate) and contact sensitizers (DNCB, dinitrofluorobenzene and oxazolone). The second part of the study was designed to test the robustness of these markers when classing the weakly immunogenic chemicals most often encountered. Six respiratory allergens, including TMA (2.5%), five contact allergens, including DNCB (0.25%), and two irritants were compared at doses of equivalent immunogenicity. The results indicated that IL-4Rα and IL-2 can be reliably used to discriminate sensitizers. Respiratory sensitizers induced markedly higher IL-4Rα levels than contact allergens, while irritants had no effect on this parameter. Inversely, contact allergens tended to induce higher percentages of IL-2{sup +}CD8{sup +} cells than respiratory allergens. In contrast, the markers MHC-II, IgE and IL-4 were not able to classify chemicals with low immunogenic potential. In conclusion, IL-4Rα and IL-2 have the potential to be used in classifying a variety of chemical allergens. -- Highlights: ► Identification of chemical allergens is an important occupational safety issue. ► There is currently no model to predict the potential of chemicals to induce asthma. ► We analyze immune responses induced

  19. /sup 125/I-BW-A844U, an antagonist radioligand with high affinity and selectivity for adenosine A1 receptors, and /sup 125/I-azido-BW-A844U, a photoaffinity label

    SciTech Connect

    Patel, A.; Craig, R.H.; Daluge, S.M.; Linden, J.

    1988-06-01

    3-(4-Amino)phenethyl-1-propyl-8-cyclopentylxanthine (BW-A844U) has been synthesized and shown to bind with high affinity to adenosine A1 receptors of bovine brain membranes (KD = 0.23 nM). This compound is highly selective for A1 receptors; the KI for binding to A2 receptors of human platelet membranes is 2.0 microM (A2/A1 ratio = 8700). Radioiodination of the 3-aminophenethyl group resulted in 125I-BW-A844U, a radioligand that retains high affinity for A1 receptors in bovine brain membranes (KD = 0.14 nM) and to 3-((3-cholamidopropyl)-dimethylammonio)-1-propane sulfonate-solubilized receptors (KD = 0.34 nM). Specific binding of 125I-BW-A844U represented greater than 90% of the total binding at the KD. From the association constant (K1 = 5.0 X 10(8) M-1min-1) and the dissociation constant (K-1 = 0.064 min-1), the kinetic KD (K-1/K1) in membranes was calculated to be 0.13 nM. NaCl (1 M) had little effect on the binding affinity of 125I-BW-A844U, in contrast to the large effect of salt on the binding affinity of acidic antagonist radioligands. 8-Sulfophenyltheophylline inhibited radioligand binding with a Hill coefficient of 1.0, indicative of a single affinity binding state for the antagonist. By comparison, two distinct agonist affinity states of A1 receptors for the agonist (R)-phenylisopropyladenosine could be resolved, a high affinity state (62%, KH = 74 pM) and a low affinity state (KL = 3.83 nM). The addition of 0.1 mM guanylylimidodiphosphate converted all receptors to the low affinity state. Addition of NaCl (0.5 M) decreased the fraction of receptors in the high affinity state and increased both KH and KL, suggesting that NaCl alters coupling of receptors to G proteins and influences the conformation of the receptor polypeptide, whether or not the receptor is coupled to a G protein.

  20. Celecoxib offsets the negative renal influences of cyclosporine via modulation of the TGF-β1/IL-2/COX-2/endothelin ET{sub B} receptor cascade

    SciTech Connect

    El-Gowelli, Hanan M.; Helmy, Maged W.; Ali, Rabab M.; El-Mas, Mahmoud M.

    2014-03-01

    Endothelin (ET) signaling provokes nephrotoxicity induced by the immunosuppressant drug cyclosporine A (CSA). We tested the hypotheses that (i): celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, counterbalances renal derangements caused by CSA in rats and (ii) the COX-2/endothelin ET{sub B} receptor signaling mediates the CSA-celecoxib interaction. Ten-day treatment with CSA (20 mg/kg/day) significantly increased biochemical indices of renal function (serum urea, creatinine), inflammation (interleukin-2, IL-2) and fibrosis (transforming growth factor-β{sub 1}, TGF-β{sub 1}). Histologically, CSA caused renal tubular atrophy along with interstitial fibrosis. These detrimental renal effects of CSA were largely reduced in rats treated concurrently with celecoxib (10 mg/kg/day). We also report that cortical glomerular and medullary tubular protein expressions of COX-2 and ET{sub B} receptors were reduced by CSA and restored to near-control values in rats treated simultaneously with celecoxib. The importance of ET{sub B} receptors in renal control and in the CSA-celecoxib interaction was further verified by the findings (i) most of the adverse biochemical, inflammatory, and histopathological profiles of CSA were replicated in rats treated with the endothelin ET{sub B} receptor antagonist BQ788 (0.1 mg/kg/day, 10 days), and (ii) the BQ788 effects, like those of CSA, were alleviated in rats treated concurrently with celecoxib. Together, the data suggest that the facilitation of the interplay between the TGF-β1/IL-2/COX-2 pathway and the endothelin ET{sub B} receptors constitutes the cellular mechanism by which celecoxib ameliorates the nephrotoxic manifestations of CSA in rats. - Highlights: • Celecoxib abolishes nephrotoxic manifestations of CSA in rats. • Blockade of ETB receptors by BQ788 mimicked the nephrotoxic effects of CSA. • CSA or BQ788 reduces renal protein expression of COX-2 and endothelin ETB receptors. • Enhanced TGFβ1/IL-2/COX2/ETB

  1. High affinity dopamine D2 receptor radioligands. 2. [125I]epidepride, a potent and specific radioligand for the characterization of striatal and extrastriatal dopamine D2 receptors.

    PubMed

    Kessler, R M; Ansari, M S; Schmidt, D E; de Paulis, T; Clanton, J A; Innis, R; al-Tikriti, M; Manning, R G; Gillespie, D

    1991-01-01

    Epidepride, (S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-iodo-2,3-dimethoxybenzamide+ ++, the iodine analogue of isoremoxipride (FLB 457), was found to be a very potent dopamine D2 receptor antagonist. Optimal in vitro binding required incubation at 25 degrees C for 4 h at pH 7.4 in a buffer containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2 and 1 mM MgCl2. Scatchard analysis of in vitro binding to striatal, medial frontal cortical, hippocampal and cerebellar membranes revealed a KD of 24 pM in all regions, with Bmax's of 36.7, 1.04, 0.85, and 0.37 pmol/g tissue, respectively. The Hill coefficients ranged from 0.91-1.00 in all four regions. The IC50's for inhibition of [125I]epidepride binding to striatal, medial frontal cortical, and hippocampal membranes for SCH 23390, SKF 83566, serotonin, ketanserin, mianserin, naloxone, QNB, prasozin, clonidine, alprenolol, and norepinephrine ranged from 1 microM to greater than 10 microM. Partial displacement of [125I]epidepride by nanomolar concentrations of clonidine was noted in the frontal cortex and hippocampus, but not in the striatum. Scatchard analysis of epidepride binding to alpha 2 noradrenergic receptors in the frontal cortex and hippocampus revealed an apparent KD of 9 nM. At an epidepride concentration equal to the KD for the D2 receptor, i.e. 25 pM, no striatal alpha 2 binding was seen and only 7% of the specific epidepride binding in the cortex or hippocampus was due to binding at the alpha 2 site. Correlation of inhibition of [3H]spiperone and [125I]epidepride binding to striatal membranes by a variety of D2 ligands revealed a correlation coefficient of 0.99, indicating that epidepride labels a D2 site. In vitro autoradiography revealed high densities of receptor binding in layers V and VI of prefrontal and cingulate cortices as well as in striatum. In vivo rat brain uptake revealed a hippocampal:cerebellar and frontal cortical:cerebellar ratio of 2.2:1 which fell to 1.1:1 following haloperidol pretreatment. These

  2. Discovery of high affinity anti-ricin antibodies by B cell receptor sequencing and by yeast display of combinatorial VH:VL libraries from immunized animals.

    PubMed

    Wang, Bo; Lee, Chang-Han; Johnson, Erik L; Kluwe, Christien A; Cunningham, Josephine C; Tanno, Hidetaka; Crooks, Richard M; Georgiou, George; Ellington, Andrew D

    2016-01-01

    Ricin is a toxin that could potentially be used as a bioweapon. We identified anti-ricin A chain antibodies by sequencing the antibody repertoire from immunized mice and by selecting high affinity antibodies using yeast surface display. These methods led to the isolation of multiple antibodies with high (sub-nanomolar) affinity. Interestingly, the antibodies identified by the 2 independent approaches are from the same clonal lineages, indicating for the first time that yeast surface display can identify native antibodies. The new antibodies represent well-characterized reagents for biodefense diagnostics and therapeutics development. PMID:27224530

  3. Further characterization of a high affinity thyrotropin binding site on the rat thyrotropin receptor which is an epitope for blocking antibodies from idiopathic myxedema patients but not thyroid stimulating antibodies from Graves' patients.

    PubMed

    Kosugi, S; Ban, T; Akamizu, T; Kohn, L D

    1991-10-31

    Cysteine 390 of the rat thyrotropin (TSH) receptor, when mutated to serine, results in a receptor with a reduced ability of TSH to bind and increase cAMP levels but a preserved ability of thyroid stimulating autoantibodies (TSAbs) from hyperthyroid Graves' patients to increase cAMP levels. The ability of receptor autoantibodies from hypothyroid patients with idiopathic myxedema to inhibit the TSAb activity which is preserved is, however, like TSH binding, significantly reduced. Cysteine 390, together with tyrosine 385, thus appears to be an important determinant in a high affinity TSH binding site which is an epitope for receptor autoantibodies which block TSH or TSAb action and cause hypothyroidism rather than TSAbs which increase cAMP levels and are associated with hyperthyroidism. Threonine 388 and aspartic acid 403 may contribute to this ligand interaction site. PMID:1719963

  4. Development of a diphtheria toxin-based recombinant porcine IL-2 fusion toxin for depleting porcine CD25+ cells.

    PubMed

    Peraino, Jaclyn Stromp; Schenk, Marian; Li, Guoying; Zhang, Huiping; Farkash, Evan A; Sachs, David H; Huang, Christene A; Duran-Struuck, Raimon; Wang, Zhirui

    2013-12-15

    Regulatory T cells (Tregs) have been widely recognized as crucial players in controlling immune responses. Because their major role is to ensure that the immune system is not over reactive, Tregs have been the focus of multiple research studies including those investigating transplantation tolerance, autoimmunity and cancer treatment. On their surface Tregs constitutively express CD25, a high affinity receptor for the cytokine interleukin-2 (IL-2). The reagents constructed in this study were generated by genetically linking porcine IL-2 to the truncated diphtheria toxin (DT390). This reagent functions by first binding to the cell surface via the porcine IL-2/porcine CD25 interaction then the DT390 domain facilitates internalization followed by inhibition of protein synthesis resulting in cell death. Four versions of the porcine IL-2 fusion toxin were designed in an interest to find the most effective isoform: 1) monovalent glycosylated porcine IL-2 fusion toxin (Gly); 2) monovalent non-N-glycosylated porcine IL-2 fusion toxin (NonGly); 3) bivalent glycosylated porcine IL-2 fusion toxin (Bi-Gly); 4) bivalent non-N-glycosylated porcine IL-2 fusion toxin (Bi-NonGly). Using a porcine CD25(+) B cell lymphoma cell line (LCL13271) in vitro analysis of the fusion toxins' ability to inhibit protein synthesis demonstrated that the Bi-NonGly fusion toxin is the most efficient reagent. These in vitro results are consistent with binding affinity as the Bi-NonGly fusion toxin binds strongest to CD25 on the same LCL13271 cells. The Bi-Gly fusion toxin significantly prolonged the survival (p=0.028) of tumor-bearing NOD/SCID IL-2 receptor γ(-/-) (NSG) mice injected with LCL13271 cells compared with untreated controls. This recombinant protein has great potential to function as a useful tool for in vivo depletion of porcine CD25(+) cells for studying immune regulation. PMID:24055128

  5. Development of a Diphtheria Toxin-Based Recombinant Porcine IL-2 Fusion Toxin for Depleting Porcine CD25+ Cells

    PubMed Central

    Peraino, Jaclyn Stromp; Schenk, Marian; Li, Guoying; Zhang, Huiping; Farkash, Evan A.; Sachs, David H.; Huang, Christene A.; Duran-Struuck, Raimon; Wang, Zhirui

    2013-01-01

    Regulatory T cells (Tregs) have been widely recognized as crucial players in controlling immune responses. Because their major role is to ensure that the immune system is not over reactive, Tregs have been the focus of multiple research studies including those investigating transplantation tolerance, autoimmunity and cancer treatment. On their surface Tregs constitutively express CD25, a high affinity receptor for the cytokine interleukin-2 (IL-2). The reagents constructed in this study were generated by genetically linking porcine IL-2 to the truncated diphtheria toxin (DT390). This reagent functions by first binding to the cell surface via the porcine IL-2/porcine CD25 interaction then the DT390 domain facilitates internalization followed by inhibition of protein synthesis resulting in cell death. Four versions of the porcine IL-2 fusion toxin were designed in an interest to find the most effective isoform: 1) monovalent glycosylated porcine IL-2 fusion toxin (Gly); 2) monovalent non-N-glycosylated porcine IL-2 fusion toxin (NonGly); 3) bivalent glycosylated porcine IL-2 fusion toxin (Bi-Gly); 4) bivalent non-N-glycosylated porcine IL-2 fusion toxin (Bi-NonGly). Using a porcine CD25+ B cell lymphoma cell line (LCL13271) in vitro analysis of the fusion toxins’ ability to inhibit protein synthesis demonstrated that the Bi-NonGly fusion toxin is the most efficient reagent. These in vitro results are consistent with binding affinity as the Bi-NonGly fusion toxin binds strongest to CD25 on the same LCL13271 cells. The Bi-Gly fusion toxin significantly prolonged the survival (p=0.028) of tumor-bearing NOD/SCID IL-2 receptor γ−/− (NSG) mice injected with LCL13271 cells compared with untreated controls. This recombinant protein has great potential to function as a useful tool for in vivo depletion of porcine CD25+ cells for studying immune regulation. PMID:24055128

  6. Regulation of the human interleukin-2/interleukin-2 receptor system: A role for immunosuppression

    SciTech Connect

    Kaempfer, R.

    1994-12-31

    The strength of the cellular immune response is regulated to a large extent by the amount of interleukin-2 (IL-2) produced in response to a stimulus. The ability of lymphocytes and other cells to respond to IL-2 depends upon the expression of cell surface IL-2 receptors. Formation of a high-affinity IL-2 receptor is regulated primarily through induction of its {alpha} subunit, IL-2R{alpha}. Once formed, the IL-2R{alpha} chain turns over rapidly, rendering expression of high-affinity IL-2 receptors during the immune response dependent upon continuous activity of the IL-2R{alpha} gene. The induced expression of both human IL-2 and IL-2R{alpha} chains is sensitive to cell-mediated suppression by CD8 cells; depletion of CD8 cells leads to extensive superinduction. This coupled suppression of IL-2 and IL-2R{alpha} genes greatly increases the extend of control, and strongly limits the strength, of the signal transduced by this ligand/receptor system during an immune response. 29 refs., 3 figs.

  7. Interactions of dopaminergic agonists and antagonists with dopaminergic D3 binding sites in rat striatum. Evidence that (/sup 3/H)dopamine can label a high affinity agonist-binding state of the D1 dopamine receptor

    SciTech Connect

    Leff, S.E.; Creese, I.

    1985-02-01

    The interactions of dopaminergic agonists and antagonists with /sup 3/H-agonist labeled D3 dopaminergic binding sites of rat striatum have been characterized by radioligand-binding techniques. When the binding of (/sup 3/H)dopamine and (/sup 3/H)apomorphine to D2 dopamine receptors is blocked by the inclusion of D2 selective concentrations of unlabeled spiroperidol or domperidone, these ligands appear to label selectively the previously termed D3 binding site. Antagonist/(/sup 3/H)dopamine competition curves are of uniformly steep slope (nH . 1.0), suggesting the presence of a single D3 binding site. The relative potencies of antagonists to inhibit D3 specific (/sup 3/H)dopamine binding are significantly correlated with their potencies to block D1 dopamine receptors as measured by the inhibition of both dopamine-stimulated adenylate cyclase and (/sup 3/H)flupentixol-binding activities. The affinities of agonists to inhibit D3 specific (/sup 3/H)dopamine binding are also correlated with estimates of these agonists affinities for the high affinity binding component of agonist/(/sup 3/H)flupentixol competition curves. Both D3 specific (/sup 3/H) dopamine binding and the high affinity agonist-binding component of dopamine/(/sup 3/H)flupentixol competition curves show a similar sensitivity to guanine nucleotides. Taken together, these data strongly suggest that the D3 binding site is related to a high affinity agonist-binding state of the D1 dopamine receptor.

  8. Structure of the high-affinity binding site for noncompetitive blockers of the acetylcholine receptor: (/sup 3/H)chlorpromazine labels homologous residues in the. beta. and delta chains

    SciTech Connect

    Giraudat, J.; Dennis, M.; Heidmann, T.; Haumont, P.Y.; Lederer, F.; Changeux, J.P.

    1987-05-05

    The membrane-bound acetylcholine receptor from Torpedo marmorata was photolabeled by the noncompetitive channel blocker (/sup 3/H)chlorpromazine under equilibrium conditions in the presence of the agonist carbamoylcholine. The amount of radioactivity incorporated into all subunits was reduced by addition of phencyclidine, a specific ligand for the high-affinity site for noncompetitive blockers. The labeled ..beta.. chain was purified and digested with trypsin or CNBr, and the resulting fragments were fractionated by high-performance liquid chromatography. Sequence analysis resulted in the identification of Ser-254 and Leu-257 as residues labeled by (/sup 3/H)chlorpromazine in a phencyclidine-sensitive manner. These residues are located in the hydrophobic and potentially transmembrane segment M II of the ..beta.. chain, a region homologous to that containing the chlorpromazine-labeled Ser-262 in the delta chain. These results show that homologous regions of different receptor subunits contribute to the unique high-affinity site for noncompetitive blockers, a finding consistent with the location of this site on the axis of symmetry of the receptor molecule.

  9. Crystallization and preliminary X-ray structural studies of a high-affinity CD8αα co-receptor to pMHC

    SciTech Connect

    Cole, David K.; Rizkallah, Pierre J.; Sami, Malkit; Lissin, Nikolai M.; Gao, Feng; Bell, John I.; Boulter, Jonathan M.; Glick, Meir; Vuidepot, Anne-Lise; Jakobsen, Bent K.; Gao, George F.

    2005-03-01

    A high-affinity mutant CD8 (haCD8) has been developed with the aim of developing a therapeutic immunosuppressor. In order to fully understand the nature of the haCD8 interaction, this protein was crystallized using the sitting-drop vapour-diffusion method. The class I CD8 positive T-cell response is involved in a number of conditions in which artificial down-regulation and control would be therapeutically beneficial. Such conditions include a number of autoimmune diseases and graft rejection in transplant patients. Although the CD8 T-cell response is dominated by the TCR–pMHC interaction, activation of T cells is in most cases also dependent on a number of associated signalling molecules. Previous work has demonstrated the ability of one such molecule (CD8) to act as an antagonist to T-cell activation if added in soluble form. Therefore, a high-affinity mutant CD8 (haCD8) has been developed with the aim of developing a therapeutic immunosuppressor. In order to fully understand the nature of the haCD8 interaction, this protein was crystallized using the sitting-drop vapour-diffusion method. Single haCD8 crystals were cryocooled and used for data collection. These crystals belonged to space group P6{sub 4}22 (assumed by similarity to the wild type), with unit-cell parameters a = 101.08, c = 56.54 Å. V{sub M} calculations indicated one molecule per asymmetric unit. A 2 Å data set was collected and the structure is currently being determined using molecular replacement.

  10. [76Br]BMK-152, a non-peptide analogue, with high affinity and low non-specific binding for the Corticotropin-Releasing Factor Type 1 Receptor (CRF1 receptor)

    PubMed Central

    Jagoda, Elaine M.; Lang, Lixin; McCullough, Karen; Contoreggi, Carlo; Kim, B. Moon; Ma, Ying; Rice, Kenner C.; Szajek, Lawrence P; Eckelman, William C.; Kiesewetter, Dale O.

    2013-01-01

    Corticotropin-releasing factor (CRF), a neuropeptide, regulates endocrine and autonomic responses to stress through G-protein coupled receptors, CRF1 or CRF2. A PET ligand able to monitor changes in CRF1 receptor occupancy in vivo would aid in understanding the pathophysiology of stress related diseases as well as in the clinical development of non-peptide antagonists with therapeutic value. We have radiolabeled the CRF1 receptor ligand, BMK-152 ([8-(4-bromo-2,6-dimethoxyphenyl)-2,7-dimethylpyrazolo[1,5-α][1,3,5]triazin-4-yl]-N,N-bis-(2-methoxyethyl)amine; ClogP= 2.6), at both the 3 and 4 position with [76Br]. Using in vitro autoradiography saturation studies the 4-[76Br]BMK-152 exhibited high affinity binding to both rat (Kd = 0.23 ± 0.07 nM; n=3) and monkey frontal cortex (Kd = 0.31 ± 0.08 nM; n=3) consistent with CRF1 receptor regional distribution whereas with the 3-[76Br]BMK-152, the Kd's could not be determined due to high non-specific binding. In vitro autoradiography competition studies using [125I]Tyr0-o-CRF confirmed that 3-Br-BMK-152 (Ki = 24.4 ± 4.9 nM; n=3) had lower affinity (70 fold) than 4-Br-BMK-152 (Ki = 0.35 ± 0.07 nM; n=3) in monkey frontal cortex and similiar studies using [125I]Sauvagine confirmed CRF1 receptor selectivity. In vivo studies with P-glycoprotein (PGP) knockout mice (KO) and their wildtype littermates (WT) showed that the brain uptake of 3-[76Br]BMK/4-[76Br]BMK was increased < 2 fold in KO vs WT indicating that 3-[76Br]BMK-152/4-[76Br]BMK was not a Pgp substrate. Rat brain uptakes of 4-[76Br] BMK-152 from ex vivo autoradiography studies showed regional localization consistent with known published CRF1 receptor distribution and potential as a PET ligand for in vivo imaging of CRF1 receptors. PMID:21308801

  11. Sodium-dependent isomerization of dopamine D-2 receptors characterized using [125I]epidepride, a high-affinity substituted benzamide ligand.

    PubMed

    Neve, K A; Henningsen, R A; Kinzie, J M; De Paulis, T; Schmidt, D E; Kessler, R M; Janowsky, A

    1990-03-01

    We have characterized the in vitro binding of a new ligand, [125I]epidepride, and used this substituted benzamide to assess the sensitivity of dopamine D-2 receptors to sodium. Both direct and indirect binding studies with [125I]epidepride and unlabeled epidepride, respectively, demonstrated that the affinity of D-2 receptors for the ligand was decreased from 20 to 30 pM in the presence of sodium to 350 to 500 pM in the absence of sodium. The density of binding sites for [125I]epidepride was identical in the presence and absence of NaCl. The time courses for association of [125I]epidepride to and dissociation from D-2 receptors in the presence of sodium were not consistent with simple bimolecular reactions, suggesting the possibility of a sodium-dependent ligand-induced receptor isomerization. Thus, dissociation of [125I]epidepride was biphasic in the presence of sodium, but monophasic in the absence of sodium. The rank order of potency for inhibition of [125I]epidepride binding by drugs was identical in rat striatum and cells expressing a D-2 receptor cDNA, and similar to the previously described pharmacological profile of D-2 receptors labeled by [3H]spiperone. [125I]Epidepride bound to two classes of binding sites in rat medial prefrontal cortex. One class, present at a density of 10 fmol/mg of protein and with a Kd value of approximately 40 pM, was pharmacologically indistinguishable from D-2 receptors in striatum and transfected cells. The pharmacological profile of the second class of sites was similar to that of alpha-2 adrenergic receptors. [125I]Epidepride had 50- to 100-fold lower affinity (approximately 2 nM) for alpha-2 receptors than for D-2 receptors.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2138666

  12. High Affinity Agonists of the Neuropeptide Y (NPY) Y4 Receptor Derived from the C-Terminal Pentapeptide of Human Pancreatic Polypeptide (hPP): Synthesis, Stereochemical Discrimination, and Radiolabeling.

    PubMed

    Kuhn, Kilian K; Ertl, Thomas; Dukorn, Stefanie; Keller, Max; Bernhardt, Günther; Reiser, Oliver; Buschauer, Armin

    2016-07-14

    The diastereomeric mixture of d/l-2,7-diaminooctanedioyl-bis(YRLRY-NH2) (BVD-74D, 2) was described in the literature as a high affinity Y4 receptor agonist. Here we report on the synthesis and pharmacological characterization of the pure diastereomers (2R,7R)- and (2S,7S)-2 and a series of homo- and heterodimeric analogues in which octanedioic acid was used as an achiral linker. To investigate the role of the Arg residues, one or two arginines were replaced by Ala. Moreover, N(ω)-(6-aminohexylaminocarbonyl)Arg was introduced as an arginine replacement (17). (2R,7R)-2 was superior to (2S,7S)-2 in binding and functional cellular assays and equipotent with 17. [(3)H]Propionylation of one amino group in the linker of (2R,7R)-2 or at the primary amino group in 17 resulted in high affinity Y4R radioligands ([(3)H]-(2R,7R)-10, [(3)H]18) with subnanomolar Kd values. PMID:27223253

  13. 12-HETER1/GPR31, a high-affinity 12(S)-hydroxyeicosatetraenoic acid receptor, is significantly up-regulated in prostate cancer and plays a critical role in prostate cancer progression.

    PubMed

    Honn, Kenneth V; Guo, Yande; Cai, Yinlong; Lee, Menq-Jer; Dyson, Gregory; Zhang, Wenliang; Tucker, Stephanie C

    2016-06-01

    Previously we identified and deorphaned G-protein-coupled receptor 31 (GPR31) as the high-affinity 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] receptor (12-HETER1). Here we have determined its distribution in prostate cancer tissue and its role in prostate tumorigenesis using in vitro and in vivo assays. Data-mining studies strongly suggest that 12-HETER1 expression positively correlates with the aggressiveness and progression of prostate tumors. This was corroborated with real-time PCR analysis of human prostate tumor tissue arrays that revealed the expression of 12-HETER1 positively correlates with the clinical stages of prostate cancers and Gleason scores. Immunohistochemistry analysis also proved that the expression of 12-HETER1 is positively correlated with the grades of prostate cancer. Knockdown of 12-HETER1 in prostate cancer cells markedly reduced colony formation and inhibited tumor growth in animals. To discover the regulatory factors, 5 candidate 12-HETER1 promoter cis elements were assayed as luciferase reporter fusions in Chinese hamster ovary (CHO) cells, where the putative cis element required for gene regulation was mapped 2 kb upstream of the 12-HETER1 transcriptional start site. The data implicate 12-HETER1 in a critical new role in the regulation of prostate cancer progression and offer a novel alternative target for therapeutic intervention.-Honn, K. V., Guo, Y., Cai, Y., Lee, M.-J., Dyson, G., Zhang, W., Tucker, S. C. 12-HETER1/GPR31, a high-affinity 12(S)-hydroxyeicosatetraenoic acid receptor, is significantly up-regulated in prostate cancer and plays a critical role in prostate cancer progression. PMID:26965684

  14. Affinities and densities of high-affinity (/sup 3/H)muscimol (GABA-A) binding sites and of central benzodiazepine receptors are unchanged in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy

    SciTech Connect

    Butterworth, R.F.; Lavoie, J.; Giguere, J.F.; Pomier-Layrargues, G.

    1988-09-01

    The integrity of GABA-A receptors and of central benzodiazepine receptors was evaluated in membrane preparations from prefrontal cortex and caudate nuclei obtained at autopsy from nine cirrhotic patients who died in hepatic coma and an equal number of age-matched control subjects. Histopathological studies revealed Alzheimer Type II astrocytosis in all cases in the cirrhotic group; controls were free from neurological, psychiatric or hepatic diseases. Binding to GABA-A receptors was studied using (/sup 3/H)muscimol as radioligand. The integrity of central benzodiazepine receptors was evaluated using (/sup 3/H)flunitrazepam and (/sup 3/H)Ro15-1788. Data from saturation binding assays was analyzed by Scatchard plot. No modifications of either affinities (Kd) or densities (Bmax) of (/sup 3/H)muscimol of central benzodiazepine binding sites were observed. These findings do not support recent suggestions that alterations of either high-affinity GABA or benzodiazepine receptors play a significant role in the pathogenesis of hepatic encephalopathy.

  15. Cell-autonomous role of TGFβ and IL-2 receptors in CD4+ and CD8+ inducible regulatory T-cell generation during GVHD

    PubMed Central

    Sawamukai, Norifumi; Satake, Atsushi; Schmidt, Amanda M.; Lamborn, Ian T.; Ojha, Priti; Tanaka, Yoshiya

    2012-01-01

    FoxP3+ regulatory T cells (Tregs) suppress GVHD while preserving graft-versus-tumor effects, making them an attractive target for GVHD therapy. The donor-derived Treg pool can potentially be derived from the expansion of preexisting natural Tregs (nTregs) or from de novo generation of inducible Tregs (iTregs) from donor Tconvs in the transplantation recipient. Using an MHC-mismatched model of acute GVHD, in the present study we found that the Treg pool was comprised equally of donor-derived nTregs and iTregs. Experiments using various combinations of T cells from wild-type and FoxP3-deficient mice suggested that both preexisting donor nTregs and the generation of iTregs in the recipient mice contribute to protection against GVHD. Surprisingly, CD8+FoxP3+ T cells represented approximately 70% of the iTreg pool. These CD8+FoxP3+ T cells shared phenotypic markers with their CD4+ counterparts and displayed suppressive activity, suggesting that they were bona fide iTregs. Both CD4+ and CD8+ Tregs appeared to be protective against GVHD-induced lethality and required IL-2 and TGFβ receptor expression for their generation. These data illustrate the complex makeup of the donor-derived FoxP3+ Treg pool in allogeneic recipients and their potential role in protection against GVHD. PMID:22496155

  16. Cell-autonomous role of TGFβ and IL-2 receptors in CD4+ and CD8+ inducible regulatory T-cell generation during GVHD.

    PubMed

    Sawamukai, Norifumi; Satake, Atsushi; Schmidt, Amanda M; Lamborn, Ian T; Ojha, Priti; Tanaka, Yoshiya; Kambayashi, Taku

    2012-06-01

    FoxP3(+) regulatory T cells (Tregs) suppress GVHD while preserving graft-versus-tumor effects, making them an attractive target for GVHD therapy. The donor-derived Treg pool can potentially be derived from the expansion of preexisting natural Tregs (nTregs) or from de novo generation of inducible Tregs (iTregs) from donor Tconvs in the transplantation recipient. Using an MHC-mismatched model of acute GVHD, in the present study we found that the Treg pool was comprised equally of donor-derived nTregs and iTregs. Experiments using various combinations of T cells from wild-type and FoxP3-deficient mice suggested that both preexisting donor nTregs and the generation of iTregs in the recipient mice contribute to protection against GVHD. Surprisingly, CD8(+)FoxP3(+) T cells represented approximately 70% of the iTreg pool. These CD8(+)FoxP3(+) T cells shared phenotypic markers with their CD4(+) counterparts and displayed suppressive activity, suggesting that they were bona fide iTregs. Both CD4(+) and CD8(+) Tregs appeared to be protective against GVHD-induced lethality and required IL-2 and TGFβ receptor expression for their generation. These data illustrate the complex makeup of the donor-derived FoxP3(+) Treg pool in allogeneic recipients and their potential role in protection against GVHD. PMID:22496155

  17. Benzodiazepine receptor ligands. 8: synthesis and pharmacological evaluation of new pyrazolo[5,1-c] [1,2,4]benzotriazine 5-oxide 3- and 8-disubstituted: high affinity ligands endowed with inverse-agonist pharmacological efficacy.

    PubMed

    Guerrini, Gabriella; Costanzo, Annarella; Ciciani, Giovanna; Bruni, Fabrizio; Selleri, Silvia; Costagli, Camilla; Besnard, François; Costa, Barbara; Martini, Claudia; De Siena, Gaetano; Malmberg-Aiello, Petra

    2006-02-01

    The synthesis and the binding study of new 3-arylesters and 3-heteroarylpyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide 8-substituted are reported. The nature of these substituents (in terms of lipophilic and electronic features) seems to influence the binding affinity. High-affinity ligands were studied in mice in vivo for their pharmacological effects, considering six potential benzodiazepine actions: anxiolytic-like effects, muscle relaxant effects, motor coordination, anticonvulsant action, spontaneous motor activity, and ethanol-potentiating action. Compounds 4d and 6d showed an inverse-agonist profile. These compounds were evaluated also for their binding at benzodiazepine site on GABAA receptor complex (GABAA/BzR complex) subtype to evaluate their subtype selectivity. PMID:16214350

  18. Crystal Structure of Human Interferon-[lamda]1 in Complex with Its High-Affinity Receptor Interferon-[lamda]R1

    SciTech Connect

    Miknis, Zachary; Magracheva, Eugenia; Li, Wei; Zdanov, Alexander; Kotenko, Sergei V.; Wlodawer, Alexander

    2010-12-01

    Interferon (IFN)-{lambda}1 [also known as interleukin (IL)-29] belongs to the recently discovered group of type III IFNs. All type III IFNs initiate signaling processes through formation of specific heterodimeric receptor complexes consisting of IFN-{lambda}R1 and IL-10R2. We have determined the structure of human IFN-{lambda}1 complexed with human IFN-{lambda}R1, a receptor unique to type III IFNs. The overall structure of IFN-{lambda}1 is topologically similar to the structure of IL-10 and other members of the IL-10 family of cytokines. IFN-{lambda}R1 consists of two distinct domains having fibronectin type III topology. The ligand-receptor interface includes helix A, loop AB, and helix F on the IFN site, as well as loops primarily from the N-terminal domain and inter-domain hinge region of IFN-{lambda}R1. Composition and architecture of the interface that includes only a few direct hydrogen bonds support an idea that long-range ionic interactions between ligand and receptor govern the process of initial recognition of the molecules while hydrophobic interactions finalize it.

  19. SH2 domains prevent tyrosine dephosphorylation of the EGF receptor: identification of Tyr992 as the high-affinity binding site for SH2 domains of phospholipase C gamma.

    PubMed Central

    Rotin, D; Margolis, B; Mohammadi, M; Daly, R J; Daum, G; Li, N; Fischer, E H; Burgess, W H; Ullrich, A; Schlessinger, J

    1992-01-01

    Several cytoplasmic tyrosine kinases contain a conserved, non-catalytic stretch of approximately 100 amino acids called the src homology 2 (SH2) domain, and a region of approximately 50 amino acids called the SH3 domain. SH2/SH3 domains are also found in several other proteins, including phospholipase C-gamma (PLC gamma). Recent studies indicate that SH2 domains promote association between autophosphorylated growth factor receptors such as the epidermal growth factor (EGF) receptor and signal transducing molecules such as PLC gamma. Because SH2 domains bind specifically to protein sequences containing phosphotyrosine, we examined their capacity to prevent tyrosine dephosphorylation of the EGF and other receptors with tyrosine kinase activity. For this purpose, various SH2/SH3 constructs of PLC gamma were expressed in Escherichia coli as glutathione-S-transferase fusion proteins. Our results show that purified SH2 domains of PLC gamma are able to prevent tyrosine dephosphorylation of the EGF receptor and other receptors with tyrosine activity. The inhibition of tyrosine dephosphorylation paralleled the capacity of various SH2-containing constructs to bind to the EGF receptor, suggesting that the tyrosine phosphatase and the SH2 domain compete for the same tyrosine phosphorylation sites in the carboxy-terminal tail of the EGF receptor. Analysis of the phosphorylation sites protected from dephosphorylation by PLC gamma-SH2 revealed substantial inhibition of dephosphorylation of Tyr992 at 1 microM SH2. This indicates that Tyr992 and its flanking sequence is the high-affinity binding site for SH2 domains of PLC gamma.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:1537335

  20. Investigations on the 4-quinolone-3-carboxylic acid motif. 6. Synthesis and pharmacological evaluation of 7-substituted quinolone-3-carboxamide derivatives as high affinity ligands for cannabinoid receptors.

    PubMed

    Pasquini, Serena; De Rosa, Maria; Ligresti, Alessia; Mugnaini, Claudia; Brizzi, Antonella; Caradonna, Nicola P; Cascio, Maria Grazia; Bolognini, Daniele; Pertwee, Roger G; Di Marzo, Vincenzo; Corelli, Federico

    2012-12-01

    Within our studies on structure-activity relationships of 4-quinolone-3-carboxamides as cannabinoid ligands, a new series of compounds characterized by a fluoro or phenylthio group at 7-position and different substituents at N1 and carboxamide nitrogen were synthesized and evaluated for their binding ability to cannabinoid type 1 (CB1) and type 2 (CB2) receptors. Most of the compounds showed affinity for one or both cannabinoid receptors at nanomolar concentration, with K(i)(CB1) and K(i)(CB2) values ranging from 2.45 to >10,000 nM and from 0.09 to 957 nM, respectively. The N-(3,4-dichlorobenzyl)amide derivatives 27 and 40 displayed relatively low affinity, but high selectivity towards the CB1 receptor. Compounds 4 and 40, a CB2 and a CB1 ligand, respectively, behaved as partial agonists in the [(35)S]GTPγS assay. They showed very low permeability through (MDCK-MDR1) cells and might, therefore, represent possible lead structures for further optimization in the search for cannabinoid ligands unable to cross the blood-brain barrier. PMID:23085772

  1. Influence of in vitro IL-2 or IL-15 alone or in combination with Hsp-70-derived 14-mer peptide (TKD) on the expression of NK cell activatory and inhibitory receptors.

    PubMed

    Hromadnikova, Ilona; Pirkova, Petra; Sedlackova, Lucie

    2013-01-01

    NK cells represent a potential tool for adoptive immunotherapy against tumors. Membrane-bound Hsp70 acts as a tumor-specific marker enhancing NK cell activity. Using flow cytometry the effect of in vitro stimulation with IL-2 or IL-15 alone or in combination with Hsp70-derived 14-mer peptide (TKD) on cell surface expression of NK activatory receptors (CD16, NKG2D, NKG2C, NKp46, NKp44, NKp30, KIR2DL4, DNAM-1, and LAMP1) and NK inhibitory receptors (NKG2A, KIR2DL2/L3, LIR1/ILT-2, and NKR-P1A) in healthy individuals was studied. Results were expressed as the percentage of receptor expressing cells and the amount of receptor expressed by CD3(-)CD56(+) cellular population. CD94, NKG2D, NKp44, NKp30, KIR2DL4, DNAM-1, LAMP1, NKG2A, and NKR-P1A were upregulated after the stimulation with IL-2 or IL-15 alone or in combination with TKD. KIR2DL2/L3 was upregulated only by IL-15 and IL-15/TKD. Concurrently, an increase in a number of NK cells positive for CD94, NKp44, NKp30, KIR2DL4, and LAMP1 was observed. IL-15 and IL-15/TKD caused also cell number rise positive for KIR2DL2/L3 and NKR-P1A. Cell number positive for NKG2C and NKG2A was increased only by IL-2 and IL-2/TKD. The diverse effect of IL-2 or IL-15 w or w/o TKD on cell surface expression was observed in CD16, NKp46, and LIR1/ILT-2. PMID:23476104

  2. Biophysical Fragment Screening of the β1-Adrenergic Receptor: Identification of High Affinity Arylpiperazine Leads Using Structure-Based Drug Design

    PubMed Central

    2013-01-01

    Biophysical fragment screening of a thermostabilized β1-adrenergic receptor (β1AR) using surface plasmon resonance (SPR) enabled the identification of moderate affinity, high ligand efficiency (LE) arylpiperazine hits 7 and 8. Subsequent hit to lead follow-up confirmed the activity of the chemotype, and a structure-based design approach using protein–ligand crystal structures of the β1AR resulted in the identification of several fragments that bound with higher affinity, including indole 19 and quinoline 20. In the first example of GPCR crystallography with ligands derived from fragment screening, structures of the stabilized β1AR complexed with 19 and 20 were determined at resolutions of 2.8 and 2.7 Å, respectively. PMID:23517028

  3. Structure-Guided Design of a High-Affinity Platelet Integrin αIIbβ3 Receptor Antagonist That Disrupts Mg2+ Binding to the MIDAS

    PubMed Central

    Zhu, Jieqing; Choi, Won-Seok; McCoy, Joshua G.; Negri, Ana; Zhu, Jianghai; Naini, Sarasija; Li, Jihong; Shen, Min; Huang, Wenwei; Bougie, Daniel; Rasmussen, Mark; Aster, Richard; Thomas, Craig J.; Filizola, Marta; Springer, Timothy A.; Coller, Barry S.

    2012-01-01

    An integrin found on platelets, αIIbβ3 mediates platelet aggregation, and αIIbβ3 antagonists are effective antithrombotic agents in the clinic. Ligands bind to integrins in part by coordinating a magnesium ion (Mg2+) located in the β subunit metal ion–dependent adhesion site (MIDAS). Drugs patterned on the integrin ligand sequence Arg-Gly-Asp have a basic moiety that binds the αIIb subunit and a carboxyl group that coordinates the MIDAS Mg2+ in the β3 subunits. They induce conformational changes in the β3 subunit that may have negative consequences such as exposing previously hidden epitopes and inducing the active conformation of the receptor. We recently reported an inhibitor of αIIbβ3 (RUC-1) that binds exclusively to the αIIb subunit; here, we report the structure-based design and synthesis of RUC-2, a RUC-1 derivative with a ~100-fold higher affinity. RUC-2 does not induce major conformational changes in β3 as judged by monoclonal antibody binding, light scattering, gel chromatography, electron microscopy, and a receptor priming assay. X-ray crystallography of the RUC-2–IIbβ3 headpiece complex in 1 mM calcium ion (Ca2+)/5 mM Mg2+ at 2.6 Å revealed that RUC-2 binds to αIIb the way RUC-1 does, but in addition, it binds to the β3 MIDAS residue glutamic acid 220, thus displacing Mg2+ from the MIDAS. When the Mg2+ concentration was increased to 20 mM, however, Mg2+ was identified in the MIDAS and RUC-2 was absent. RUC-2′s ability to inhibit ligand binding and platelet aggregation was diminished by increasing the Mg2+ concentration. Thus, RUC-2 inhibits ligand binding by a mechanism different from that of all other αIIbβ3 antagonists and may offer advantages as a therapeutic agent. PMID:22422993

  4. Discovery of 4-(phenyl)thio-1H-pyrazole derivatives as agonists of GPR109A, a high affinity niacin receptor.

    PubMed

    Kim, Hyeon Young; Jadhav, Vithal B; Jeong, Dae Young; Park, Woo Kyu; Song, Jong-Hwan; Lee, Sunkyung; Cho, Heeyeong

    2015-06-01

    Even though nicotinic acid (niacin) appears to have beneficial effects on human lipid profiles, niacin-induced cutaneous vasodilatation called flushing limits its remedy to patient. GPR109A is activated by niacin and mediates the anti-lipolytic effects. Based on the hypothesis that β-arrestin signaling mediates niacin-induced flushing, but not its anti-lipolytic effect, we tried to find GPR109A agonists which selectively elicit Gi-protein-biased signaling devoid of β-arrestin internalization using a β-lactamase assay. We identified a 4-(phenyl)thio-1H-pyrazole as a novel scaffold for GPR109A agonist in a high throughput screen, which has no carboxylic acid moiety known to be important for binding. While 1-nicotinoyl derivatives (5a-g, 6a-e) induced β-arrestin recruitment, 1-(pyrazin-2-oyl) derivatives were found to play as G-protein-biased agonists without GPR109A receptor internalization. The activity of compound 5a (EC50 = 45 nM) was similar to niacin (EC50 = 52 nM) and MK-6892 (EC50 = 74 nM) on calcium mobilization assay, but its activity at 10 μM on β-arrestin recruitment were around two and five times weaker than niacin and MK-6892, respectively. The development of G-protein biased GPR109A ligands over β-arrestin pathway is attainable and might be important in differentiation of pharmacological efficacy. PMID:25599616

  5. Role of berberine in anti-bacterial as a high-affinity LPS antagonist binding to TLR4/MD-2 receptor

    PubMed Central

    2014-01-01

    Background Berberine is an isoquinoline alkaloid mainly extracted from Rhizoma Coptidis and has been shown to possess a potent inhibitory activity against bacterial. However, the role of berberine in anti-bacterial action has not been extensively studied. Methods The animal model was established to investigate the effects of berberine on bacterial and LPS infection. Docking analysis, Molecular dynamics simulations and Real-time RT-PCR analysis was adopted to investigate the molecular mechanism. Results Treatment with 40 mg/kg berberine significantly increased the survival rate of mice challenged with Salmonella typhimurium (LT2), but berberine show no effects in bacteriostasis. Further study indicated that treatment with 0.20 g/kg berberine markedly increased the survival rate of mice challenged with 2 EU/ml bacterial endotoxin (LPS) and postpone the death time of the dead mice. Moreover, pretreatment with 0.05 g/kg berberine significantly lower the increasing temperature of rabbits challenged with LPS. The studies of molecular mechanism demonstrated that Berberine was able to bind to the TLR4/MD-2 receptor, and presented higher affinity in comparison with LPS. Furthermore, berberine could significantly suppressed the increasing expression of NF-κB, IL-6, TNFα, and IFNβ in the RAW264.7 challenged with LPS. Conclusion Berberine can act as a LPS antagonist and block the LPS/TLR4 signaling from the sourse, resulting in the anti-bacterial action. PMID:24602493

  6. Open conformers of HLA-F are high-affinity ligands of the activating NK-cell receptor KIR3DS1.

    PubMed

    Garcia-Beltran, Wilfredo F; Hölzemer, Angelique; Martrus, Gloria; Chung, Amy W; Pacheco, Yovana; Simoneau, Camille R; Rucevic, Marijana; Lamothe-Molina, Pedro A; Pertel, Thomas; Kim, Tae-Eun; Dugan, Haley; Alter, Galit; Dechanet-Merville, Julie; Jost, Stephanie; Carrington, Mary; Altfeld, Marcus

    2016-09-01

    The activating natural killer (NK)-cell receptor KIR3DS1 has been linked to the outcome of various human diseases, including delayed progression of disease caused by human immunodeficiency virus type 1 (HIV-1), yet a ligand that would account for its biological effects has remained unknown. We screened 100 HLA class I proteins and found that KIR3DS1 bound to HLA-F, a result we confirmed biochemically and functionally. Primary human KIR3DS1(+) NK cells degranulated and produced antiviral cytokines after encountering HLA-F and inhibited HIV-1 replication in vitro. Activation of CD4(+) T cells triggered the transcription and surface expression of HLA-F mRNA and HLA-F protein, respectively, and induced binding of KIR3DS1. HIV-1 infection further increased the transcription of HLA-F mRNA but decreased the binding of KIR3DS1, indicative of a mechanism for evading recognition by KIR3DS1(+) NK cells. Thus, we have established HLA-F as a ligand of KIR3DS1 and have demonstrated cell-context-dependent expression of HLA-F that might explain the widespread influence of KIR3DS1 in human disease. PMID:27455421

  7. Synthesis and pharmacological characterization of [125I]MRS1898, a high-affinity, selective radioligand for the rat A3 adenosine receptor

    PubMed Central

    Gao, Zhan-Guo; Teng, Bao; Wu, Haitao; Joshi, Bhalchandra V.; Griffiths, Gary L.

    2008-01-01

    A known selective agonist of the A3 adenosine receptors (AR), MRS1898 [(1′R,2′R,3′S,4′R,5′S)-4-{2-chloro-6-[(3-iodophenylmethyl)amino]purin-9-yl}-1-(methylaminocarbonyl)bicyclo[3.1.0]hexane-2,3-diol], was synthesized in radioactive form and characterized pharmacologically. This agonist ligand series, based on nucleoside analogues containing a rigid, bicyclic ring system in place of the ribose moiety, was selected for radiolabeling due to its high A3AR affinity across species, with nanomolar binding at both rat and human A3ARs. The radioiodination of MRS1898 on its N6–3-iodobenzyl substituent was accomplished in 76% radiochemical yield by iododestannylation of a 3-(trimethylstannyl)benzyl precursor. [125I]MRS1898 bound to the rat A3AR with a Kd value of 0.17 ± 0.04 nM and a Bmax value of 0.66 ± 0.15 pmol/mg protein. The competition binding profiles for other agonists and antagonists obtained with this radioligand are similar to those previously obtained with other radioligands. The advantages of [125I]MRS1898 compared with previously used radioligands are primarily its high selectivity and affinity for the rat A3AR and also its facile synthesis and radiochemical stability; however, a relatively high level of nonspecific binding presents a limitation. Thus, we have introduced the first selective radioligand for the rat A3AR. PMID:18528782

  8. Synthesis and resolution of (+-)-7-chloro-8-hydroxy-1-(3'-iodophenyl)-3-methyl-2,3,4,5-tetrahydro- 1H-3- benzazepine (TISCH): A high affinity and selective iodinated ligand for CNS D1 dopamine receptor

    SciTech Connect

    Chumpradit, S.; Kung, M.P.; Billings, J.J.; Kung, H.F. )

    1991-03-01

    The synthesis and resolution of (+-)-7-chloro-8-hydroxy-1-(3'-iodophenyl)-3-methyl-2,3,4,5-tetrahydro-1 H-3- benzazepine, (+/-)-TISCH (8) has been achieved by resolution of intermediate 4, the O-methoxyl, 3'-bromo derivative, as the diastereomeric camphor sulfonate salt. The final products, R-(+)-8 and S-(-)-8, were prepared by treatment of R-(+)- or S-(-)-7, the 3'-tributyltin intermediates, with iodine in chloroform, followed by O-demethylation. By using HPLC with a chiral column, the optical purity (greater than 99%) of the intermediates and the final compounds was determined. Radioiodination was achieved by an iodo-destannylation reaction with sodium (125I)iodide and hydrogen peroxide. As expected, the R-(+)-(125I)-8 (the active isomer) displayed high affinity and selectivity to the CNS D-1 receptor in rat striatum tissue preparation (Kd = 0.205 nM). The rank order of potency was as follows: SCH-23390 (1a) greater than (+/-)-8 greater than (+)-butaclamol greater than spiperone, WB4101 greater than dopamine, 5-HT. After an iv injection, the R-(+)-(125I)-8 penetrated the blood-brain barrier with ease and displayed specific regional distribution corresponding to the D-1 receptor density, while the S-(-)-(125I)-8 showed no specific uptake. The data suggest that the ligand may be useful as a pharmacological tool for characterizing the D-1 dopamine receptor. When labeled with I-123, this ligand is a potential agent for in vivo imaging of CNS D-1 dopamine receptor.

  9. (D-Pen2,4 prime -125I-Phe4,D-Pen5)enkephalin: A selective high affinity radioligand for delta opioid receptors with exceptional specific activity

    SciTech Connect

    Knapp, R.J.; Sharma, S.D.; Toth, G.; Duong, M.T.; Fang, L.; Bogert, C.L.; Weber, S.J.; Hunt, M.; Davis, T.P.; Wamsley, J.K. )

    1991-09-01

    (D-Pen2,4{prime}-125I-Phe4,D-Pen5)enkephalin ((125I)DPDPE) is a highly selective radioligand for the delta opioid receptor with a specific activity (2200 Ci/mmol) that is over 50-fold greater than that of tritium-labeled DPDPE analogs. (125I)DPDPE binds to a single site in rat brain membranes with an equilibrium dissociation constant (Kd) value of 421 {plus minus} 67 pM and a receptor density (Bmax) value of 36.4 {plus minus} 2.7 fmol/mg protein. The high affinity of this site for delta opioid receptor ligands and its low affinity for mu or kappa receptor-selective ligands are consistent with its being a delta opioid receptor. The distribution of these sites in rat brain, observed by receptor autoradiography, is also consistent with that of delta opioid receptors. Association and dissociation binding kinetics of 1.0 nM (125I) DPDPE are monophasic at 25 degrees C. The association rate (k + 1 = 5.80 {plus minus} 0.88 {times} 10(7) M-1 min-1) is about 20- and 7-fold greater than that measured for 1.0 nM (3H) DPDPE and 0.8 nM (3H) (D-Pen2,4{prime}-Cl-Phe4, D-Pen5)enkephalin, respectively. The dissociation rate of (125I)DPDPE (0.917 {plus minus} 0.117 {times} 10(-2) min-1) measured at 1.0 nM is about 3-fold faster than is observed for either of the other DPDPE analogs. The rapid binding kinetics of (125I)DPDPE is advantageous because binding equilibrium is achieved with much shorter incubation times than are required for other cyclic enkephalin analogs. This, in addition to its much higher specific activity, makes (125I)DPDPE a valuable new radioligand for studies of delta opioid receptors.

  10. IL2RA/CD25 Gene Polymorphisms: Uneven Association with Multiple Sclerosis (MS) and Type 1 Diabetes (T1D)

    PubMed Central

    Alcina, Antonio; Fedetz, María; Ndagire, Dorothy; Fernández, Oscar; Leyva, Laura; Guerrero, Miguel; Abad-Grau, María M.; Arnal, Carmen; Delgado, Concepción; Lucas, Miguel; Izquierdo, Guillermo; Matesanz, Fuencisla

    2009-01-01

    Background IL-2 receptor (IL2R) alpha is the specific component of the high affinity IL2R system involved in the immune response and in the control of autoimmunity. Methods and Results Here we perform a replication and fine mapping of the IL2RA gene region analyzing 3 SNPs previously associated with multiple sclerosis (MS) and 5 SNPs associated with type 1 diabetes (T1D) in a collection of 798 MS patients and 927 matched Caucasian controls from the south of Spain. We observed association with MS in 6 of 8 SNPs. The rs1570538, at the 3′- UTR extreme of the gene, previously reported to have a weak association with MS, is replicated here (P = 0.032). The most associated T1D SNP (rs41295061) was not associated with MS in the present study. However, the rs35285258, belonging to another independent group of SNPs associated with T1D, showed the maximal association in this study but different risk allele. We replicated the association of only one (rs2104286) of the two IL2RA SNPs identified in the recently performed genome-wide association study of MS. Conclusions These findings confirm and extend the association of this gene with MS and reveal a genetic heterogeneity of the associated polymorphisms and risk alleles between MS and T1D suggesting different immunopathological roles of IL2RA in these two diseases. PMID:19125193

  11. Systemic radioimmunotherapy using a monoclonal antibody, anti-Tac directed toward the alpha subunit of the IL-2 receptor armed with the alpha-emitting radionuclides (212)Bi or (211)At.

    PubMed

    Wesley, Jon N; McGee, Edwin C; Garmestani, Kayhan; Brechbiel, Martin W; Yordanov, Alexander T; Wu, Chuanchu; Gansow, Otto A; Eckelman, William C; Bacher, John D; Flynn, Michael; Goldman, Carolyn K; MacLin, Melvin; Schwartz, Uwe P; Jackson-White, Terri; Phillip, Celeste M; Decker, Jean; Waldmann, Thomas A

    2004-04-01

    To exploit the fact that IL-2 receptors are expressed by T-cells responding to foreign antigens but not by resting T-cells, humanized anti-Tac (HAT) armed with alpha-emitting radionuclides (212)Bi and (211)At was evaluated in a cynomolgus cardiac allograft model. Control graft survival was 8.2+/- 0.5 days compared with 14.0+/-1.3 days (p<0.01) survival for monkeys treated with (212)Bi labeled HAT and 26.7+/-2.4 days survival (p<0.001 versus controls) with (211)At labeled HAT. Thus, (211)At labeled HAT may have application in organ transplantation and in treatment of IL-2 receptor expressing T-cell leukemia. PMID:15028248

  12. Immunosuppressive effect of the anti-IL-2-receptor monoclonal antibody, AMT-13, on organ-cultured fetal pancreas allograft survival

    SciTech Connect

    Burkhardt, K.; Loughnan, M.S.; Diamantstein, T.; Mandel, T.E.

    1988-11-01

    Recently, prolongation of cardiac allograft survival in mice was reported using a rat anti-IL-2R mAb (AMT-13). However, its immunosuppressive action in vivo, alone and in combination with other immunosuppressants, and its effect on other organ transplants has not been extensively studied. We grafted cultured fetal pancreas from CBA (H-2k) donors to Balb/c (H-2d) mice. Recipients were treated with 10 consecutive daily injections each of 20 micrograms AMT-13 only, or with an additional mild immunosuppression of 350 rads irradiation. Control groups received rat immunoglobulin or 350 rads irradiation. Graft survival and the phenotype of infiltrating cells were assessed histologically and immunocytochemically on days 12, 17, and 21, and soluble IL-2R levels were measured in the serum with a quantitative ELISA in all recipients. Two of five grafts in the AMT-13-treated group had islets on day 12 posttransplantation despite lymphocytic infiltration in all grafts, while at this time all grafts of rat Ig treated control mice were completely rejected with only scar tissue and a few lymphocytes remaining. Additional immunosuppression with 350 rads irradiation had a marked additive effect with AMT-13. Soluble IL-2R levels in the serum of untreated recipients were not elevated compared with normal serum levels, but recipients injected with AMT-13 had multifold increased soluble IL-2R levels. The percentage of IL-2R+ cells in the grafts of AMT-13-treated animals was either normal (less than 5%) or increased (20%) in the additionally irradiated mice, providing strong evidence that the immunosuppressive effect of AMT-13 is not due to a depletion of activated IL-2R+ lymphocytes.

  13. (/sup 3/H)leukotriene B/sub 4/ binding to the guinea pig spleen membranes: a rich tissue source for a high affinity leukotriene B/sub 4/ receptor site

    SciTech Connect

    Cheng, J.B.; Kohi, F.; Townley, R.G.

    1986-03-05

    To select a tissue rich for the high affinity leukotriene (LT)B/sub 4/ receptor site, they compared binding of 1 nM (/sup 3/H)LTB/sub 4/ (180 Ci/mmol) to the crude membrane preparations of guinea pig spleen, thymus, lung, uterus, bladder, brain, adrenal gland, small intestine, liver, kidney and heart. They found that the membrane preparations from spleen contained the highest binding activity per mg protein. They characterized the LTB/sub 4/ binding to the spleen preparation in detail. LTB/sub 4/ binding was rapid, reversible, stereoselective and saturable. The data from equilibrium experiments showed a linear Scatchard plot with a K/sub d/ of 1.6 nM and a binding site density of 259 fmol/mg prot. The rank order of agents competing for spleen (/sup 3/H)LTB/sub 4/ binding at 25/sup 0/C was: LTB/sub 4/ (K/sub i/ = 2.8 nM) > 20-OH-LTB/sub 4/ (23 nM) > LTA/sub 4/ (48 nM) > LTA/sub 4/ methyl ester (0.13 ..mu..M) > 20-COOH-LTB/sub 4/ (> 6.6 ..mu..M) greater than or equal to arachidonic acid (0.15 mM) similarly ordered FPL-55,712 (0.11 mM). At 4/sup 0/C, LTB/sub 4/ (2.3 nM) competed at least 10x more effectively than 20-OH-LTB/sub 4/ (29 nM) and 20-COOH-LTB/sub 4/ (> 6.6 ..mu..M). HPLC analysis indicated that incubation of 84 ng LTB/sub 4/ with the spleen membrane at 25/sup 0/C did not result in the formation of 20-OH-LTB/sub 4/ (< 1 ng) in the filtrate. They conclude that guinea pig spleen contains a rich tissue source of high affinity (/sup 3/H)LTB/sub 4/ receptor binding sites.

  14. The High Affinity Binding Site on Plasminogen Activator Inhibitor-1 (PAI-1) for the Low Density Lipoprotein Receptor-related Protein (LRP1) Is Composed of Four Basic Residues.

    PubMed

    Gettins, Peter G W; Dolmer, Klavs

    2016-01-01

    Plasminogen activator inhibitor 1 (PAI-1) is a serpin inhibitor of the plasminogen activators urokinase-type plasminogen activator (uPA) and tissue plasminogen activator, which binds tightly to the clearance and signaling receptor low density lipoprotein receptor-related protein 1 (LRP1) in both proteinase-complexed and uncomplexed forms. Binding sites for PAI-1 within LRP1 have been localized to CR clusters II and IV. Within cluster II, there is a strong preference for the triple CR domain fragment CR456. Previous mutagenesis studies to identify the binding site on PAI-1 for LRP1 have given conflicting results or implied small binding contributions incompatible with the high affinity PAI-1/LRP1 interaction. Using a highly sensitive solution fluorescence assay, we have examined binding of CR456 to arginine and lysine variants of PAI-1 and definitively identified the binding site as composed of four basic residues, Lys-69, Arg-76, Lys-80, and Lys-88. These are highly conserved among mammalian PAI-1s. Individual mutations result in a 13-800-fold increase in Kd values. We present evidence that binding involves engagement of CR4 by Lys-88, CR5 by Arg-76 and Lys-80, and CR6 by Lys-69, with the strongest interactions to CR5 and CR6. Collectively, the individual binding contributions account quantitatively for the overall PAI-1/LRP1 affinity. We propose that the greater efficiency of PAI-1·uPA complex binding and clearance by LRP1, compared with PAI-1 alone, is due solely to simultaneous binding of the uPA moiety in the complex to its receptor, thereby making binding of the PAI-1 moiety to LRP1 a two-dimensional surface-localized association. PMID:26555266

  15. 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

    SciTech Connect

    Pearse, R.N.; Feinman, R.; Ravetch, J.V. )

    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.

  16. Effects of Cudrania tricuspidata Fruit Extract and Its Active Compound, 5,7,3',4'-Tetrahydroxy-6,8-diprenylisoflavone, on the High-Affinity IgE Receptor-Mediated Activation of Syk in Mast Cells.

    PubMed

    Lee, Taehun; Kwon, Jaeyoung; Lee, Dongho; Mar, Woongchon

    2015-06-10

    Cudrania tricuspidata fruit extract contains a rich source of prenylated flavonoids with potential antiatherosclerotic, hepatoprotective, and anti-inflammatory properties. However, the effect of C. tricuspidata fruit extracts and its active compounds on the high-affinity IgE receptor (FcεRI)-mediated signaling remains unknown. In the present study, the effect of methanol extract from the fruits of C. tricuspidata (MFC) and its active compound, 5,7,3',4'-tetrahydroxy-6,8-diprenylisoflavone (THDPI), on FcεRI-mediated signaling in mast cells was investigated. MFC and THDPI suppressed mast cell degranulation and Ca(2+) influx. MFC also interfered with IgE-FcεRI interaction and decreased FcεRIβ mRNA expression in mast cells. Furthermore, MFC and THDPI inhibited the phosphorylation of Syk, LAT, and PLCγ and F-actin redistribution. These results indicate that MFC and its active compound, THDPI, inhibit mast cell activation through the inhibition of FcεRI-mediated Syk activation, suggesting a therapeutic potential for controlling mast cell activation in inflammatory and/or allergic processes. PMID:25989241

  17. Synthesis and Structure-Affinity Relationships of Selective High-Affinity 5-HT4 Receptor Antagonists: Application to the Design of New Potential Single Photon Emission Computed Tomography (SPECT) Tracers

    PubMed Central

    Dubost, Emmanuelle; Dumas, Noé; Fossey, Christine; Magnelli, Rosa; Butt-Gueulle, Sabrina; Ballandonne, Céline; Caignard, Daniel H.; Dulin, Fabienne; de-Oliveira Santos, Jana Sopkova; Millet, Philippe; Charnay, Yves; Rault, Sylvain; Cailly, Thomas; Fabis, Frederic

    2012-01-01

    The work described herein aims at finding new potential ligands for the brain imaging of 5-HT4 receptors using single-photon emission computed tomography (SPECT). Starting from the non-substituted phenanthridine compound 4a exhibiting a Ki value of 51 nM on 5-HT4R, we explored structure-affinity in this series. We found that substitution in position 4 of the tricycle with a fluorine atom gave the best result. Introduction of an additional nitrogen atom inside the tricyclic framework led to increase both the affinity and the selectivity for 5-HT4R suggesting the design of the antagonist 4v exhibiting a high affinity of 0.04 nM. Several iodinated analogues were then synthesized as potential SPECT tracers. The iodinated compound 11d was able to displace the reference radioiodinated 5-HT4R antagonist (1-butylpiperidin-4-yl)methyl-8-amino-7-iodo[123I]-2,3-dihydrobenzo[b][1,4]dioxine-5-carboxylate ([123I]1, [123I]SB 207710) both in vitro and in vivo in brain. Compound 11d was radiolabeled with [125I]iodine, providing a potential SPECT candidate for brain imaging of 5-HT4R. PMID:23102207

  18. A novel intronic splice site deletion of the IL-2 receptor common gamma chain results in expression of a dysfunctional protein and T-cell-positive X-linked Severe combined immunodeficiency.

    PubMed

    Gray, P E A; Logan, G J; Alexander, I E; Poulton, S; Roscioli, T; Ziegler, J

    2015-02-01

    X-linked severe combined immunodeficiency is caused by mutations in the IL-2 receptor common gamma chain and classically presents in the first 6 months of life with predisposition to bacterial, viral and fungal infections. In most instances, affected individuals are lymphopenic with near complete absence of T cells and NK cells. We report a boy who presented at 12 months of age with Pneumocystis jiroveci pneumonia and a family history consistent with X-linked recessive inheritance. He had a normal lymphocyte count including the presence of T cells and a broad T-cell-receptor diversity, as well as normal surface expression of the common gamma chain (CD132) protein. He however had profound hypogammaglobulinaemia, and IL-2-induced STAT5 phosphorylation was absent. Sequencing of IL-2RG demonstrated a 12-base pair intronic deletion close to the canonical splice site of exon 5, which resulted in a variety of truncated IL2RG mRNA species. A review of the literature identified 4 other patients with T-cell-positive X-SCID, with the current patient being the first associated with an mRNA splicing defect. This case raises the question of how a dysfunctional protein incapable of mediating STAT5 phosphorylation might nonetheless support T-cell development. Possible explanations are that STAT5-mediated signal transduction may be less relevant to IL7-receptor-mediated T-cell development than are other IL7R-induced intracellular transduction pathways or that a low level of STAT5 phosphorylation, undetectable in the laboratory, may be sufficient to support some T-cell development. PMID:25443657

  19. Therapeutic enhancement of IL-2 through molecular design.

    PubMed

    Cassell, Delanie J; Choudhri, Shurjeel; Humphrey, Rachel; Martell, Robert E; Reynolds, Theresa; Shanafelt, Armen B

    2002-01-01

    A recombinant human IL-2 analog (rIL-2, Proleukin) is currently being evaluated for clinical benefit in HIV infected patients. It is approved for therapy of patients with metastatic melanoma and renal cell carcinoma. Treatment of cancer patients with rIL-2 results in durable responses but is associated with life-threatening toxicity, which limits its use to patients in relatively good health. Antitumor efficacy associated with rIL-2 therapy are hypothesized to be mediated by distinct types of cells that express structurally different forms of the IL-2 receptor. This hypothesis suggests that it might be possible to engineer an IL-2 variant addressing the risks associated with the therapeutic use of IL-2. In this article, we review the clinical experience with IL-2 and its analogs, the evidence that different IL-2 receptors may dissociate efficacy and toxicity, and describe the generation of a novel IL-2 variant with the potential for a superior therapeutic index. PMID:12369861

  20. Involvement of the high-affinity receptor for IgG (Fc gamma RI; CD64) in enhanced tumor cell cytotoxicity of neutrophils during granulocyte colony-stimulating factor therapy.

    PubMed

    Valerius, T; Repp, R; de Wit, T P; Berthold, S; Platzer, E; Kalden, J R; Gramatzki, M; van de Winkel, J G

    1993-08-01

    Three different classes of Fc receptors for IgG (Fc gamma R) are currently distinguished in humans, of which polymorphonuclear phagocytes (PMN) normally express both low-affinity receptor classes--Fc gamma RII (CD32) and Fc gamma RIII (CD16). During therapy with granulocyte colony-stimulating factor (G-CSF), neutrophils from patients with various malignancies and different hematologic disorders were found to additionally express high levels of the receptor with high affinity for IgG (Fc gamma RI; CD64). For these patients, the relative fluorescence intensity (rFI) for Fc gamma RI was 5.3 (range, 1.7 to 10.3; n = 19), compared with 1.0 (range, 1.0 to 1.1; n = 8) for healthy donors. The expression of Fc gamma RI during G-CSF therapy could be confirmed by using a panel of six CD64-specific antibodies, and by showing mRNA for Fc gamma RI. So far, three genes for Fc gamma RI have been identified, encoding four distinct transcription products. By reverse transcriptase-polymerase chain reaction technology, transcripts for both membrane-associated isoforms (hFc gamma RIa and hFc gamma RIb2) could be detected. The functional activity of Fc gamma RI on PMN during G-CSF therapy was shown by measuring binding of monomeric human IgG and antibody-dependent cellular cytotoxicity (ADCC). Thus, Fc gamma RI-positive neutrophils displayed enhanced ADCC activity to glioma (A1207), squamous cell (A431), and ovarian (SK-ov3) carcinoma cell lines. The involvement of Fc gamma RI in this increased cytotoxic activity was shown by blocking Fc gamma receptors with monoclonal antibodies, and by using F(ab')2 x F(ab')2-bispecific antibodies with specificities against tumor-related antigens and Fc gamma RI, resulting in solely Fc gamma RI-mediated cytotoxicity. Therapeutically, this additional Fc receptor on PMN may increase the efficacy of experimental antibody therapy. PMID:7687898

  1. Two cis-DNA elements involved in myeloid-cell-specific expression and gamma interferon (IFN-gamma) activation of the human high-affinity Fc gamma receptor gene: a novel IFN regulatory mechanism.

    PubMed Central

    Perez, C; Wietzerbin, J; Benech, P D

    1993-01-01

    The human high-affinity receptor for the constant region of immunoglobulin G (human Fc gamma R1) is encoded by two mRNAs induced selectively by gamma interferon (IFN-gamma) and expressed in cells of myeloid lineage. The cis-DNA element (GRR) previously found to confer IFN-gamma responsiveness to this gene acts as an inducible enhancer and is the target of an IFN-gamma-activated factor(s) (GIRE-BP) in cells of different origins. Although the GRR motif is not related to the DNA elements involved in the regulation of other IFN-stimulated genes, GIRE-BP binding depends on the IFN-gamma-dependent activation of the 91-kDa protein known to be one of the factors of a transcriptional complex activated by IFN-alpha. Deletions of the Fc gamma R1 promoter allowed us to identify a 25-bp element, downstream from the GRR motif, conferring cell-type-specific expression. This element, called MATE (myeloid activating transcription element), is the DNA target for constitutive factors forming two complexes, MATE-BP1 and MATE-BP2. In accordance with the functional analysis, MATE-BP binding activities were detected in extracts prepared from myeloid cell lines such as THP-1, HL-60, and U-937 but not in HeLa cell extracts. The MATE motif is present not only in the promoter of other Fc receptor genes but also in several promoters of genes whose expression is restricted to monocytic cells. Our results suggest that human Fc gamma R1 gene expression in myeloid cells is initiated by the interaction of IFN-gamma-activated factors with cell-type-specific factors through their binding to the GRR and MATE motifs. Images PMID:8455606

  2. High affinity binding of [3H]propionyl-[Met(O2)11]substance P(7-11), a tritiated septide-like peptide, in Chinese hamster ovary cells expressing human neurokinin-1 receptors and in rat submandibular glands.

    PubMed

    Sagan, S; Beaujouan, J C; Torrens, Y; Saffroy, M; Chassaing, G; Glowinski, J; Lavielle, S

    1997-07-01

    Propionyl-[Met(O2)11]substance P(7-11) [ALIE-124 or propionyl-[Met(O2)11]SP(7-11)] has been designed as a septide-like ligand adequate for tritiation and, therefore, adequate for binding studies. In Chinese hamster ovary (CHO) cells expressing human tachykinin neurokinin (NK)-1 receptors, ALIE-124 displaced [3H][Pro9]substance P (SP) from its binding site at micromolar concentrations. However, ALIE-124 stimulated phosphatidylinositol hydrolysis, as previously shown for septide-like peptides. With [3H]ALIE-124 (95 Ci/mmol), we have been able to reveal a high affinity binding site in CHO cells (Kd = 6.6 +/- 1.0 nM), with a low maximal binding capacity. [3H]ALIE-124 specific maximal binding represented only 15-20% of that observed with [3H][Pro9]SP in CHO cells. Septide-like peptides, including septide and NKA, were potent competitors (in the nanomolar range) of [3H]ALIE-124 specific binding site. Interestingly, SP and [Pro9]SP were also potent competitors, with 10-fold greater potency for sites labeled with [3H]ALIE-124 than for sites labeled with [3H][Pro9]SP. The NK-1 antagonist RP 67580 also showed a higher potency for [3H]ALIE-124 than for [3H][Pro9]SP-specific binding sites. NKB and [Lys5,methyl-Leu9,Nle10]NKA(4-10) displaced [3H]ALIE-124 binding but with lower potency, whereas senktide had no affinity. The existence of [3H]ALIE-124 specific binding sites was also demonstrated in rat submandibular gland. In this tissue, [3H]ALIE-124 specific maximal binding was higher, reaching 40-50% of that achieved with [3H][Pro9]SP. PMID:9224821

  3. Tracking human multiple myeloma xenografts in NOD-Rag-1/IL-2 receptor gamma chain-null mice with the novel biomarker AKAP-4

    PubMed Central

    2011-01-01

    Background Multiple myeloma (MM) is a fatal malignancy ranking second in prevalence among hematological tumors. Continuous efforts are being made to develop innovative and more effective treatments. The preclinical evaluation of new therapies relies on the use of murine models of the disease. Methods Here we describe a new MM animal model in NOD-Rag1null IL2rgnull (NRG) mice that supports the engraftment of cell lines and primary MM cells that can be tracked with the tumor antigen, AKAP-4. Results Human MM cell lines, U266 and H929, and primary MM cells were successfully engrafted in NRG mice after intravenous administration, and were found in the bone marrow, blood and spleen of tumor-challenged animals. The AKAP-4 expression pattern was similar to that of known MM markers, such as paraproteins, CD38 and CD45. Conclusions We developed for the first time a murine model allowing for the growth of both MM cell lines and primary cells in multifocal sites, thus mimicking the disease seen in patients. Additionally, we validated the use of AKAP-4 antigen to track tumor growth in vivo and to specifically identify MM cells in mouse tissues. We expect that our model will significantly improve the pre-clinical evaluation of new anti-myeloma therapies. PMID:21923911

  4. NKG2D is a Key Receptor for Recognition of Bladder Cancer Cells by IL-2-Activated NK Cells and BCG Promotes NK Cell Activation

    PubMed Central

    García-Cuesta, Eva María; López-Cobo, Sheila; Álvarez-Maestro, Mario; Esteso, Gloria; Romera-Cárdenas, Gema; Rey, Mercedes; Cassady-Cain, Robin L.; Linares, Ana; Valés-Gómez, Alejandro; Reyburn, Hugh Thomson; Martínez-Piñeiro, Luis; Valés-Gómez, Mar

    2015-01-01

    Intravesical instillation of bacillus Calmette–Guérin (BCG) is used to treat superficial bladder cancer, either papillary tumors (after transurethral resection) or high-grade flat carcinomas (carcinoma in situ), reducing recurrence in about 70% of patients. Initially, BCG was proposed to work through an inflammatory response, mediated by phagocytic uptake of mycobacterial antigens and cytokine release. More recently, other immune effectors such as monocytes, natural killer (NK), and NKT cells have been suggested to play a role in this immune response. Here, we provide a comprehensive study of multiple bladder cancer cell lines as putative targets for immune cells and evaluated their recognition by NK cells in the presence and absence of BCG. We describe that different bladder cancer cells can express multiple activating and inhibitory ligands for NK cells. Recognition of bladder cancer cells depended mainly on NKG2D, with a contribution from NKp46. Surprisingly, exposure to BCG did not affect the immune phenotype of bladder cells nor increased NK cell recognition of purified IL-2-activated cell lines. However, NK cells were activated efficiently when BCG was included in mixed lymphocyte cultures, suggesting that NK activation after mycobacteria treatment requires the collaboration of various immune cells. We also analyzed the percentage of NK cells in peripheral blood of a cohort of bladder cancer patients treated with BCG. The total numbers of NK cells did not vary during treatment, indicating that a more detailed study of NK cell activation in the tumor site will be required to evaluate the response in each patient. PMID:26106390

  5. The IL-2/IL-2R system: from basic science to therapeutic applications to enhance immune regulation

    PubMed Central

    Bayer, Allison L.; Pugliese, Alberto

    2014-01-01

    IL-2 plays a critical role in the normal function of the immune system. A trophic factor for lymphocytes, IL-2 is required for mounting and sustaining adaptive T cell responses; however, IL-2 is also critical for immune regulation via its effects on regulatory T cells (Treg cells). Over the years, we have contributed to the understanding of the biology of IL-2 and its signaling through the IL-2 receptor and helped define the key role played by IL-2 in Treg development and function. Our data show that Treg cells have a heightened sensitivity to IL-2, which may create a therapeutic window to promote immune regulation by selective stimulation of Treg cells. We are now developing new efforts to translate this knowledge to the clinical arena, through our focused interest in Type 1 diabetes as a prototypic autoimmune disease. Specifically, we aim at developing IL-2-based therapeutic regimens and incorporate means to enhance antigen-specific Treg responses, for improved and more selective regulation of islet autoimmunity. In parallel, we are pursuing studies in preclinical models of autoimmunity and transplantation to define critical factors for successful adoptive Treg therapy and develop clinically applicable therapeutic protocols. PMID:24214027

  6. Pleiotropic Effects of IL-2 on Cancer: Its Role in Cervical Cancer

    PubMed Central

    Valle-Mendiola, Arturo; Gutiérrez-Hoya, Adriana; Lagunas-Cruz, María del Carmen; Weiss-Steider, Benny; Soto-Cruz, Isabel

    2016-01-01

    IL-2 receptor (IL-2R) signalling is critical for normal lymphocyte proliferation, but its role in cervical cancer is not fully understood. The receptor is composed of three chains: IL-2α, IL-2β, and IL-2γ. Intracellular signalling is initiated by ligand-induced heterodimerization of the IL-2β and IL-2γ chains, resulting in the activation of multiple intracellular kinases. Recently, IL-2R was shown to be expressed on nonhaematopoietic cells, especially on several types of tumour cells. However, the function of this receptor on malignant cells has not been clearly defined. The expression of IL-2R and the production of IL-2 in cervical cancer cells have been documented as well as expression of molecules of the JAK-STAT pathway. In the current review we have highlighted the differences in the responses of molecules downstream from the IL-2R in normal lymphocytes and tumour cells that could explain the presence of tumour cells in an environment in which cytotoxic lymphocytes also exist and compete and also the effect of different concentrations of IL-2 that could activate effector cells of the immune system cells, which favour the elimination of tumour cells, or concentrations that may promote a regulatory microenvironment in which tumour cells can easily grow. PMID:27293315

  7. Failure of low-dose recombinant human IL-2 to support the survival of virus-specific CTL clones infused into severe combined immunodeficient foals: lack of correlation between in vitro activity and in vivo efficacy.

    PubMed

    Mealey, Robert H; Littke, Matt H; Leib, Steven R; Davis, William C; McGuire, Travis C

    2008-01-15

    Although CTL are important for control of lentiviruses, including equine infectious anemia virus (EIAV), it is not known if CTL can limit lentiviral replication in the absence of CD4 help and neutralizing antibody. Adoptive transfer of EIAV-specific CTL clones into severe combined immunodeficient (SCID) foals could resolve this issue, but it is not known whether exogenous IL-2 administration is sufficient to support the engraftment and proliferation of CTL clones infused into immunodeficient horses. To address this question we adoptively transferred EIAV Rev-specific CTL clones into four EIAV-challenged SCID foals, concurrent with low-dose aldesleukin (180,000U/m2), a modified recombinant human IL-2 (rhuIL-2) product. The dose was calculated based on the specific activity on equine PBMC in vitro, and resulted in plasma concentrations considered sufficient to saturate high affinity IL-2 receptors in humans. Despite specific activity on equine PBMC that was equivalent to recombinant equine IL-2 and another form of rhuIL-2, aldesleukin did not support the engraftment and expansion of infused CTL clones, and control of viral load and clinical disease did not occur. It was concluded that survival of Rev-specific CTL clones infused into EIAV-challenged SCID foals was not enhanced by aldesleukin at the doses used in this study, and that in vitro specific activity did not correlate with in vivo efficacy. Successful adoptive immunotherapy with CTL clones in immunodeficient horses will likely require higher doses of rhuIL-2, co-infusion of CD4+ T lymphocytes, or administration of equine IL-2. PMID:17727961

  8. High affinity dopamine D2 receptor radioligands. 2. ( sup 125 I)epidepride, a potent and specific radioligand for the characterization of striatal and extrastriatal dopamine D2 receptors

    SciTech Connect

    Kessler, R.M.; Ansari, M.S.; Schmidt, D.E.; de Paulis, T.; Clanton, J.A.; Manning, R.G.; Gillespie, D. ); Innis, R.; Al-Tikriti, M. )

    1991-01-01

    Epidepride, (S)-N-((1-ethyl-2-pyrrolidinyl)methyl)-5-iodo-2,3-dimethoxybenzamide, the iodine analogue of isoremoxipride (FLB 457), was found to be a very potent dopamine D2 receptor antagonist. Optimal in vitro binding required incubation at 25C for 4 h at pH 7.4 in a buffer containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl{sub 2} and 1 nM MgCl{sub 2}. Scatchard analysis of in vitro binding to striatal, medical frontal cortical, hippocampal and cerebellar membranes revealed a K{sub D} of 24 pM in all regions, with Bmax's of 36.7, 1.04, 0.85, and 0.37 pmol/g tissue, respectively. The Hill coefficients ranged from 0.91-1.00 in all four regions. The IC{sub 50}'s for inhibition of ({sup 125}I)epidepride binding to striatal, medial frontal cortical, and hippocampal membranes for SCH 23390, SKF 83566, serotonin, ketanserin, mianserin, naloxone, QNB, prasozin, clonidine, alprenolol, and norepinephrine ranged from 1 {mu}M to >10 {mu}M. Partial displacement of ({sup 125}I)epidepride by nanomolar concentrations of clonidine was noted in the frontal cortex and hippocampus, but not in the striatum. Scatchard analysis of epidepride binding to {alpha}{sub 2} noradrenergic receptors in the frontal cortex and hippocampus revealed an apparent K{sub D} of 9 nM. At an epidepride concentration equal to the K{sub D} for the D2 receptor, i.e., 25 pM, no striatal {alpha}{sub 2} binding was seen and only 7% of the specific epidepride binding in the cortex or hippocampus was due to binding at the {alpha}{sub 2} site. Correlation of inhibition of ({sup 3}H)spiperone and ({sup 125}I)epidepride binding to striatal membranes by a variety of D2 ligands revealed a correlation coefficient of 0.99, indicating that epidepride labels a D2 site.

  9. CD4+CD25+Foxp3+ T regulatory cells, Th1 (CCR5, IL-2, IFN-γ) and Th2 (CCR4, IL-4, Il-13) type chemokine receptors and intracellular cytokines in children with common variable immunodeficiency.

    PubMed

    Kutukculer, Necil; Azarsiz, Elif; Aksu, Guzide; Karaca, Neslihan Edeer

    2016-06-01

    Common variable immunodeficiency (CVID) is a heterogeneous group of primary antibody deficiencies characterized by decreased serum immunoglobulin G along with a decrease in serum IgA and/or IgM, defective specific antibody production, and recurrent bacterial infections. Abnormal lymphocyte trafficking, dysregulated cellular responses to chemokines, and uncontrolled T cell polarization may be involved in the pathogenesis and may help to understand the clinical complications. We evaluated T helper cell subsets (chemokine receptors CCR4, CCR5, and CCR7), expressions on T lymphocytes, intracellular cytokines - IL-2, IL-4, IL-13, IFN- γ-on CD4(+) T cells, and expression of CD4(+)CD25(+)Foxp3(+) regulatory T cells of 20 CVID patients and 26 healthy controls. Autoimmune clinical findings and other complications were also determined. Percentages and absolute numbers of CD4(+)CD25(+) Foxp3(+) cells did not show any significant difference between CVID cases and healthy controls nor between severe and moderate disease patients. The only significant difference regarding Th1 and Th2 type intracellular cytokines was the decreased absolute numbers of CD3(+)CD4(+)IL4(+) cells in CVID cases. There were some findings about T helper cell type dominance in CVID patients such as positive correlation between hepatomegaly and high IL-2 and IFN-γ in CD3(+)CD4(+) cells and very high expression of CCR5 (Th1) on CD3(+)CD4(+) cells in patients with granuloma. Th1 (CCR5) and Th2 (CCR4) type chemokine receptors did not show any dominance in CVID cases. However, frequencies of CCR7 expressing CD3(+) T cells, CD3(+)CD4(+) T helper cells and CD3(+)CD8(+) T cytotoxic cells were significantly lower in severe CVID patients. In addition, presence of autoimmune clinical findings was negatively correlated with CCR7(+) cells. As CCR7 is a key mediator balancing immunity and tolerance in the immune system, the abnormality of this mediator may contribute to the profound immune dysregulation seen in CVID

  10. Localization of a type 1 diabetes locus in the IL2RA/CD25 region by use of tag single-nucleotide polymorphisms.

    PubMed

    Vella, Adrian; Cooper, Jason D; Lowe, Christopher E; Walker, Neil; Nutland, Sarah; Widmer, Barry; Jones, Richard; Ring, Susan M; McArdle, Wendy; Pembrey, Marcus E; Strachan, David P; Dunger, David B; Twells, Rebecca C J; Clayton, David G; Todd, John A

    2005-05-01

    As part of an ongoing search for genes associated with type 1 diabetes (T1D), a common autoimmune disease, we tested the biological candidate gene IL2RA (CD25), which encodes a subunit (IL-2R alpha) of the high-affinity interleukin-2 (IL-2) receptor complex. We employed a tag single-nucleotide polymorphism (tag SNP) approach in large T1D sample collections consisting of 7,457 cases and controls and 725 multiplex families. Tag SNPs were analyzed using a multilocus test to provide a regional test for association. We found strong statistical evidence in the case-control collection (P=6.5x10(-8)) for a T1D locus in the CD25 region of chromosome 10p15 and replicated the association in the family collection (P=7.3x10(-3); combined P=1.3x10(-10)). These results illustrate the utility of tag SNPs in a chromosome-regional test of disease association and justify future fine mapping of the causal variant in the region. PMID:15776395

  11. [Biological properties and therapeutic use of interleukin 2 (IL-2)].

    PubMed

    Robak, T

    1995-01-01

    A cytokine produced by the subpopulation of activated helper lymphocytes T has been called interleukin-2 (IL-2). The obtaining of recombinant cytokine has facilitated the study of its biological properties and its application in the treatment of certain neoplastic and infectious diseases. IL-2 affects the target cells by means of a receptor of great affinity consisting of three independent chains: alpha, beta, gamma. The cytokine is the most important growth factor of lymphocytes T, conditioning their clonal expansion. Antigen stimulation is the condition for the expression of IL-2 does not, however, affect resting lymphocytes T. The expression of the receptor for this cytokine on NK cells is, however, continuous in character but only a very small percentage of these cells has receptors of great affinity. IL-2 plays a great role in adoptive immunotherapy consisting in intravenous administration of cells with cytotoxic properties. Cells obtained from peripheral blood and grown in vitro are called LAK cells (lymphocyte activated killer cells), while cells obtained from neoplasms and grown in similar conditions are named TIL cells (tumor infiltrated lymphocytes). LAK and TIL cells reveal a similar antineoplastic activity in vivo. At present, however, recombinant IL-2 alone is used more often, either intravenously or subcutaneously. The cytokine is effective in the treatment of patients with disseminate cancer of the kidney and melanoma, and in adjuvant therapy of acute myeloid leukemia. Attempts have been made to apply it in the treatment of AIDS and leprosy. The toxic effect of IL-2 depends on the dose and the mode of administration. In the majority of patients parainfluenza symptoms appear. Most undesirable effects are connected with multisystemic syndrome of capillary vessels hyperpermeability leading to the increased fluid retention into extravascular spaces, oedema, hypotonia and oliguria. PMID:8657637

  12. Complex high affinity interactions occur between MHCI and superantigens

    NASA Technical Reports Server (NTRS)

    Chapes, S. K.; Herpich, A. R.; Spooner, B. S. (Principal Investigator)

    1998-01-01

    Staphylococcal enterotoxins A and C1 (SEA or SEC1) bound to major histocompatibility-I (MHCI) molecules with high affinity (binding constants ranging from 1.1 microM to 79 nM). SEA and SEC1 directly bound MHCI molecules that had been captured by monoclonal antibodies specific for H-2Kk, H-2Dk, or both. In addition, MHCI-specific antibodies inhibited the binding of SEC1 to LM929 cells and SEA competitively inhibited SEC1 binding; indicating that the superantigens bound to MHCI on the cell surface. The affinity and number of superantigen binding sites differed depending on whether MHCI was expressed in the membrane of LM929 cells or whether it was captured. These data support the hypothesis that MHCI molecules can serve as superantigen receptors.

  13. Conserved IL-2Rγc Signaling Mediates Lymphopoiesis in Zebrafish.

    PubMed

    Sertori, Robert; Liongue, Clifford; Basheer, Faiza; Lewis, Kanako L; Rasighaemi, Parisa; de Coninck, Dennis; Traver, David; Ward, Alister C

    2016-01-01

    The IL-2 receptor γ common (IL-2Rγc) chain is the shared subunit of the receptors for the IL-2 family of cytokines, which mediate signaling through JAK3 and various downstream pathways to regulate lymphopoiesis. Inactivating mutations in human IL-2Rγc result in SCID, a primary immunodeficiency characterized by greatly reduced numbers of lymphocytes. This study used bioinformatics, expression analysis, gene ablation, and specific pharmacologic inhibitors to investigate the function of two putative zebrafish IL-2Rγc paralogs, il-2rγc.a and il-2rγc.b, and downstream signaling components during early lymphopoiesis. Expression of il-2rγc.a commenced at 16 h post fertilization (hpf) and rose steadily from 4-6 d postfertilization (dpf) in the developing thymus, with il-2rγc.a expression also confirmed in adult T and B lymphocytes. Transcripts of il-2rγc.b were first observed from 8 hpf, but waned from 16 hpf before reaching maximal expression at 6 dpf, but this was not evident in the thymus. Knockdown of il-2rγc.a, but not il-2rγc.b, substantially reduced embryonic lymphopoiesis without affecting other aspects of hematopoiesis. Specific targeting of zebrafish Jak3 exerted a similar effect on lymphopoiesis, whereas ablation of zebrafish Stat5.1 and pharmacologic inhibition of PI3K and MEK also produced significant but smaller effects. Ablation of il-2rγc.a was further demonstrated to lead to an absence of mature T cells, but not B cells in juvenile fish. These results indicate that conserved IL-2Rγc signaling via JAK3 plays a key role during early zebrafish lymphopoiesis, which can be potentially targeted to generate a zebrafish model of human SCID. PMID:26590317

  14. Cloning of murine interferon gamma receptor cDNA: expression in human cells mediates high-affinity binding but is not sufficient to confer sensitivity to murine interferon gamma.

    PubMed Central

    Hemmi, S; Peghini, P; Metzler, M; Merlin, G; Dembic, Z; Aguet, M

    1989-01-01

    A full-length cDNA encoding the murine interferon gamma (IFN-gamma) receptor was isolated from a lambda gt11 library using a human IFN-gamma receptor cDNA probe. The deduced amino acid sequence of the murine IFN-gamma receptor shows approximately 53% homology to its human counterpart but no homology to other known proteins. Murine IFN-gamma receptor cDNA was expressed in human HEp-2 cells, which do not bind murine IFN-gamma and are insensitive to its action. Transfectants displayed the same binding properties as mouse cells. The biological responsiveness of such transfectants to various biological effects of both human and murine IFN-gamma was investigated, including modulation of major histocompatibility complex class I and class II antigen expression, inhibition of cell growth, and antiviral activity. Like parental HEp-2 cells, these transfectants responded only to human, but not to murine, IFN-gamma. Inversely, mouse L929 cells transfected with human IFN-gamma receptor cDNA were insensitive to human IFN-gamma. These results confirm and extend previous findings, suggesting that species-specific cofactors are needed for IFN-gamma-mediated signal transduction. Images PMID:2532365

  15. Polymorphisms in the IL2, IL2RA and IL2RB genes in multiple sclerosis risk

    PubMed Central

    Cavanillas, María L; Alcina, Antonio; Núñez, Concepción; de las Heras, Virginia; Fernández-Arquero, Miguel; Bartolomé, Manuel; de la Concha, Emilio G; Fernández, Oscar; Arroyo, Rafael; Matesanz, Fuencisla; Urcelay, Elena

    2010-01-01

    Interleukin (IL)-2/IL-2R signalling promotes proliferation and survival of activated T cells and has an essential non-redundant role in the production of regulatory T cells. Associations with different autoimmune diseases of polymorphisms in a linkage disequilibrium block in which the IL2/IL21 genes map (4q27), and also in genes encoding the IL2RA and IL2RB subunits (located in 10p15 and 22q13, respectively), were identified through genome-wide studies. Polymorphisms in these three genes were studied in 430 multiple sclerosis (MS) patients and in 550 ethnically matched controls from Madrid (Spain). Replication and meta-analysis with results from an independent cohort of 771 MS patients and 759 controls from Andalucía (Spain) confirmed the association of polymorphisms in the IL2RA gene (PMantel–Haenszel, odds ratio (OR)M–H (95% confidence interval, CI) for rs2104286: 0.0001, 0.75 (0.65–0.87); for rs11594656/rs35285258: 0.004, 1.19 (1.06–1.34); for rs41295061: 0.03, 0.77 (0.60–0.98)); showed a trend for association of the IL2/IL21 rs6822844 (PM–H=0.07, ORM–H (95% CI)=0.86 (0.73–1.01)), but did not corroborate the association for IL2RB. Regression analyses of the combined Spanish cohort revealed the independence of two IL2RA association signals: rs2104286 and rs11594656/rs35285258. The relevant role of the IL2RA gene on MS susceptibility adds support to its common effect on autoimmune risk and the suggestive association of IL2/IL21 warrants further investigation. PMID:20179739

  16. Human IL-2 mutein with higher antitumor efficacy than wild type IL-2.

    PubMed

    Carmenate, Tania; Pacios, Anabel; Enamorado, Michel; Moreno, Ernesto; Garcia-Martínez, Karina; Fuente, Dasha; León, Kalet

    2013-06-15

    IL-2 has been used for the treatment of melanoma and renal cell carcinoma, but this therapy has limited efficacy and severe toxicity. Currently, it is assumed that part of the limited efficacy is due to the IL-2-driven preferential expansion of regulatory T cells, which dampen the antitumor immunity. In this study, we characterize a human IL-2 mutant with higher antitumor efficacy and lower toxicity than wild type human IL-2 (wtIL-2). The mutant differs from wtIL-2 by four mutations at the interface with the α subunit of IL-2R. The IL-2 mutant induces in vitro proliferation of CD8(+)CD44(hi) and NK1.1 cells as efficiently as does wtIL-2, but it shows a reduced capacity to induce proliferation of CD4(+)Foxp3(+) regulatory T cells. The IL-2 mutant shows a higher antimetastatic effect than does wtIL-2 in several transplantable tumor models: the experimental metastasis model of MB16F0 melanoma and the experimental and spontaneous metastasis models for the mouse pulmonary carcinoma 3LL-D1222. Relevantly, the IL-2 mutant also exhibits lower lung and liver toxicity than does wtIL-2 when used at high doses in mice. In silico simulations, using a calibrated mathematical model, predict that the properties of IL-2 mutein are a consequence of the reduction, of at least two orders of magnitude, in its affinity for the α subunit of IL-2R (CD25). The human IL-2 mutant described in the present work could be a good candidate for improving cancer therapy based on IL-2. PMID:23677467

  17. Analysis of IL-2-like factor in lymphocyte culture supernatant of olive flounder, Paralichthys oliveaceus

    NASA Astrophysics Data System (ADS)

    Wu, Riqin; Zhang, Peijun; Li, Jun; Xu, Yongli

    2005-03-01

    To study immune mechanism of fish lymphocyte we performed a proliferation assay and ELISA using monoclonal antibody against human IL-2. The result showed that an interleukin-2 (IL-2)-like factor was detected in the supernatant of plant haemoglutinin (PHA)-stimulated lymphocyte culture from peripheral blood, spleen and head kidney of olive flounder, Paralichthys olivaceus. The quantities of IL-2-like factor in the supernatant from different lymphoid tissues were quite different. The IL-2 like factor in the supernatant from cultured head kidney lymphocytes was much higher than those of peripheral blood lymphocytes and spleen lymphocytes ( P<0.01). The IL-2 activity was found in either mouse thymocyte proliferation assay or flounder head kidney lymphocyte proliferation assay and shown to have obvious enhancing effect on proliferation of the above two types of cell. The recombinant human IL-2, (rhIL-2) was able to stimulate flounder thymocyte proliferation and used to detect the IL-2 receptor (IL-2R) on the surface of flounder lymphocyte. The cross-reaction between the lymphocytes of flounder peripheral blood and CD25(IL-2R) was detected with flow cytometry and shown that the percentage of CD25-positive cell in peripheral blood was 7.74±0.67%.

  18. High affinity dopamine D2 receptor radioligands. 3. [123I] and [125I]epidepride: in vivo studies in rhesus monkey brain and comparison with in vitro pharmacokinetics in rat brain.

    PubMed

    Kessler, R M; Votaw, J R; Schmidt, D E; Ansari, M S; Holdeman, K P; de Paulis, T; Clanton, J A; Pfeffer, R; Manning, R G; Ebert, M H

    1993-01-01

    Studies of [123I]epidepride uptake in rhesus monkey brain were performed using single photon tomography. Striatal uptake peaked at 0.85% of administered dose/g at 107 min post-injection, then declined slowly to 0.70% of administered dose/g at 6 h. Striatal:posterior brain ratios rose from 2 at 25 min to 6.8 at 105 min, to 15 at 4 h and to 58 at 6.4 h. [123I]Epidepride was displaced by haloperidol (0.1 and 1 mg/kg) with a half-life of washout of 55 min. Little displacement of [123I]epidepride was observed following administration of 1 or 2 mg/kg d-amphetamine, respectively, indicating [123I]epidepride is not easily displaced by endogenous dopamine. In vitro equilibrium binding studies using rat striatum revealed a KD of 46 pM and Bmax of 33 pmol/g tissue at 37 degrees C, while at 25 degrees C the KD was 25 pM and the Bmax 32 pmol/g tissue. In vitro kinetic analysis of association and dissociation curves revealed a half-life for receptor dissociation at 37 degrees C of 15 min and 79-90 min at 25 degrees C. Allowing for the temperature difference, there is good correspondence between in vivo and in vitro dissociation kinetics at 25 degrees C. Increasing in vitro incubation temperature from 25 to 37 degrees C caused a 6-fold increase in the dissociation rate, suggesting that there is a change in binding kinetics at the dopamine D2 receptor at 37 degrees C compared to in vivo binding. The results of this study indicate that [123I]epidepride is an excellent radioligand for SPECT studies of the dopamine D2 receptor in man. PMID:8321085

  19. N(ω)-Carbamoylation of the Argininamide Moiety: An Avenue to Insurmountable NPY Y1 Receptor Antagonists and a Radiolabeled Selective High-Affinity Molecular Tool ([(3)H]UR-MK299) with Extended Residence Time.

    PubMed

    Keller, Max; Weiss, Stefan; Hutzler, Christoph; Kuhn, Kilian K; Mollereau, Catherine; Dukorn, Stefanie; Schindler, Lisa; Bernhardt, Günther; König, Burkhard; Buschauer, Armin

    2015-11-25

    Analogues of the argininamide-type NPY Y1 receptor (Y1R) antagonist BIBP3226, bearing carbamoyl moieties at the guanidine group, revealed subnanomolar Ki values and caused depression of the maximal response to NPY (calcium assay) by up to 90% in a concentration- and time-dependent manner, suggesting insurmountable antagonism. To gain insight into the mechanism of binding of the synthesized compounds, a tritiated antagonist, (R)-N(α)-diphenylacetyl-N(ω)-[2-([2,3-(3)H]propionylamino)ethyl]aminocarbonyl-(4-hydroxybenzyl)arginin-amide ([(3)H]UR-MK299, [(3)H]38), was prepared. [(3)H]38 revealed a dissociation constant in the picomolar range (Kd 0.044 nM, SK-N-MC cells) and very high Y1R selectivity. Apart from superior affinity, a considerably lower target off-rate (t1/2 95 min) was characteristic of [(3)H]38 compared to that of the higher homologue containing a tetramethylene instead of an ethylene spacer (t1/2 3 min, Kd 2.0 nM). Y1R binding of [(3)H]38 was fully reversible and fully displaceable by nonpeptide antagonists and the agonist pNPY. Therefore, the insurmountable antagonism observed in the functional assay has to be attributed to the extended target-residence time, a phenomenon of relevance in drug research beyond the NPY receptor field. PMID:26466164

  20. Assignment of the gene (EPLG2) encoding a high-affinity binding protein for the receptor tyrosine kinase elk to a 200-kilobasepair region in human chromosome Xq12

    SciTech Connect

    Fletcher, F.A.; Beckmann, M.P.; Lyman, S.D.

    1995-01-01

    Elk is a member of the eph family of receptor tyrosine kinases. Elk is expressed only in the brain and testes of the developing and adult rat, and the interaction of elk with its ligand(s) has been suggested to play a role in the development or maintenance of the nervous system. The mouse gene Eplg2 encodes a potential elk ligand that is highly conserved among rat, mouse, and human. Eplg2 has been mapped to the central portion of the mouse X chromosome, tightly linked to the androgen receptor (Ar) locus. Linkage conservation between the mouse and the human X chromosomes suggested that the human homologue (EPLG2) would map near human AR, in the interval Xq11-q12. In the present study, we have confirmed this prediction and have localized EPLG2 to a 200-kb interval in Xq12 by somatic cell hybrid analysis, two-color fluorescence in situ hybridization (FISH), and yeast artificial chromosome (YAC) hybridization. 12 refs., 1 fig.

  1. High affinity dopamine D2 receptor radioligands. 3. [[sup 123]I] and [[sup 125]I]epidepride: In vivo studies in rhesus monkey brain and comparison with in vitro pharmacokinetics in rat brain

    SciTech Connect

    Kessler, R.M.; Votaw, J.R.; Schmidt, D.E.; Ansari, M.S.; Holdeman, K.P.; Paulis, T. de; Clanton, J.A.; Pfeffer, R.; Manning, R.G.; Ebert, M.H. )

    1993-01-01

    Studies of [[sup 123]I]epidepride uptake in rhesus monkey brain were performed using single photon tomography. Striatal uptake peaked at 0.85% of administered dose/g at 107 min post-injection, then declined slowly to 0.70% of administered dose/g at 6 h. Striatal:posterior brain ratios rose from 2 at 25 min to 6.8 at 105 min, to 15 at 4 h and to 58 at 6.4 h. [[sup 123]I]Epidepride was displaced by haloperidol (0.1 and 1 mg/kg) with a half-life of washout of 55 min. Little displacement of [[sup 123]I]epidepride was observed following administration of 1 or 2 mg/kg d-amphetamine, respectively, indicating [[sup 123]I]epidepride is not easily displaced by endogenous dopamine. In vitro equilibrium binding studies with [[sup 125]I]epidepride using rat striatum revealed a K[sub D] of 46 pM and B[sub max] of 33 pmol/g tissue at 37[degrees]C, while at 25[degrees]C the K[sub D] was 25 pM and the B[sub max] 32 pmol/g tissue. In vitro kinetic analysis of association and dissociation curves revealed a half-life for receptor dissociation at 37[degrees]C of 15 min and 79--90 min at 25[degrees]C. Allowing for the temperature difference, there is good correspondence between in vivo and in vitro dissociation kinetics at 25[degrees]C. Increasing in vitro incubation temperature from 25 to 37[degrees]C caused a 6-fold increase in the dissociation rate, suggesting that there is a change in binding kinetics at the dopamine D2 receptor at 37[degrees]C compared to in vivo binding. The results of this study indicate that [[sup 123]I]epidepride is an excellent radioligand for SPECT studies of the dopamine D2 receptor in man. 34 refs., 4 figs.

  2. Influence of In Vitro IL-2 or IL-15 Alone or in Combination with Hsp 70 Derived 14-Mer Peptide (TKD) on the Expression of NK Cell Activatory and Inhibitory Receptors on Peripheral Blood T Cells, B Cells and NKT Cells.

    PubMed

    Hromadnikova, Ilona; Li, Shuang; Kotlabova, Katerina; Dickinson, Anne M

    2016-01-01

    Previous studies from Multhoff and colleagues reported that plasma membrane Hsp70 acts as a tumour-specific recognition structure for activated NK cells, and that the incubation of NK cells with Hsp70 and/or a 14-mer peptide derived from the N-terminal sequence of Hsp70 (TKDNNLLGRFELSG, TKD, aa 450-463) plus a low dose of IL-2 triggers NK cell proliferation and migration, and their capacity to kill cancer cells expressing membrane Hsp70. Herein, we have used flow cytometry to determine the influence of in vitro stimulation of peripheral blood mononuclear cells from healthy individuals with IL-2 or IL-15, either alone or in combination with TKD peptide on the cell surface expression of CD94, NK cell activatory receptors (CD16, NK2D, NKG2C, NKp30, NKp44, NKp46, NKp80, KIR2DL4, DNAM-1 and LAMP1) and NK cell inhibitory receptors (NKG2A, KIR2DL2/L3, LIR1/ILT-2 and NKR-P1A) by CD3+CD56+ (NKT), CD3+CD4+, CD3+CD8+ and CD19+ populations. NKG2D, DNAM-1, LAMP1 and NKR-P1A expression was upregulated after the stimulation with IL-2 or IL-15 alone or in combination with TKD in NKT, CD8+ T cells and B cells. CD94 was upregulated in NKT and CD8+ T cells. Concurrently, an increase in a number of CD8+ T cells expressing LIR1/ILT-2 and CD4+ T cells positive for NKR-P1A was observed. The proportion of CD8+ T cells that expressed NKG2D was higher after IL-2/TKD treatment, when compared with IL-2 treatment alone. In comparison with IL-15 alone, IL-15/TKD treatment increased the proportion of NKT cells that were positive for CD94, LAMP1 and NKRP-1A. The more potent effect of IL-15/TKD on cell surface expression of NKG2D, LIR1/ILT-2 and NKRP-1A was observed in B cells compared with IL-15 alone. However, this increase was not of statistical significance. IL-2/TKD induced significant upregulation of LAMP1 in CD8+ T cells compared with IL-2 alone. Besides NK cells, other immunocompetent cells present within the fraction of peripheral blood mononuclear cells were influenced by the treatment

  3. Influence of In Vitro IL-2 or IL-15 Alone or in Combination with Hsp 70 Derived 14-Mer Peptide (TKD) on the Expression of NK Cell Activatory and Inhibitory Receptors on Peripheral Blood T Cells, B Cells and NKT Cells

    PubMed Central

    Hromadnikova, Ilona; Li, Shuang; Kotlabova, Katerina; Dickinson, Anne M.

    2016-01-01

    Previous studies from Multhoff and colleagues reported that plasma membrane Hsp70 acts as a tumour-specific recognition structure for activated NK cells, and that the incubation of NK cells with Hsp70 and/or a 14-mer peptide derived from the N-terminal sequence of Hsp70 (TKDNNLLGRFELSG, TKD, aa 450–463) plus a low dose of IL-2 triggers NK cell proliferation and migration, and their capacity to kill cancer cells expressing membrane Hsp70. Herein, we have used flow cytometry to determine the influence of in vitro stimulation of peripheral blood mononuclear cells from healthy individuals with IL-2 or IL-15, either alone or in combination with TKD peptide on the cell surface expression of CD94, NK cell activatory receptors (CD16, NK2D, NKG2C, NKp30, NKp44, NKp46, NKp80, KIR2DL4, DNAM-1 and LAMP1) and NK cell inhibitory receptors (NKG2A, KIR2DL2/L3, LIR1/ILT-2 and NKR-P1A) by CD3+CD56+ (NKT), CD3+CD4+, CD3+CD8+ and CD19+ populations. NKG2D, DNAM-1, LAMP1 and NKR-P1A expression was upregulated after the stimulation with IL-2 or IL-15 alone or in combination with TKD in NKT, CD8+ T cells and B cells. CD94 was upregulated in NKT and CD8+ T cells. Concurrently, an increase in a number of CD8+ T cells expressing LIR1/ILT-2 and CD4+ T cells positive for NKR-P1A was observed. The proportion of CD8+ T cells that expressed NKG2D was higher after IL-2/TKD treatment, when compared with IL-2 treatment alone. In comparison with IL-15 alone, IL-15/TKD treatment increased the proportion of NKT cells that were positive for CD94, LAMP1 and NKRP-1A. The more potent effect of IL-15/TKD on cell surface expression of NKG2D, LIR1/ILT-2 and NKRP-1A was observed in B cells compared with IL-15 alone. However, this increase was not of statistical significance. IL-2/TKD induced significant upregulation of LAMP1 in CD8+ T cells compared with IL-2 alone. Besides NK cells, other immunocompetent cells present within the fraction of peripheral blood mononuclear cells were influenced by the treatment

  4. First-In-Human Study Demonstrating Tumor-Angiogenesis by PET/CT Imaging with 68Ga-NODAGA-THERANOST, a High-Affinity Peptidomimetic for αvβ3 Integrin Receptor Targeting

    PubMed Central

    Baum, Richard P.; Kulkarni, Harshad R.; Müller, Dirk; Danthi, Narasimhan; Kim, Young-Seung; Brechbiel, Martin W.

    2015-01-01

    Abstract 68Ga-NODAGA-THERANOST™ is an αvβ3 integrin antagonist and the first radiolabeled peptidomimetic to reach clinical development for targeting integrin receptors. In this first-in-human study, the feasibility of integrin receptor peptidomimetic positron emission tomography/computed tomography (PET/CT) imaging was confirmed in patients with non-small-cell lung cancer and breast cancer. Methods: Patients underwent PET/CT imaging with 68Ga NODAGA-THERANOST. PET images were analyzed qualitatively and quantitatively and compared to 2-deoxy-2-(18F) fluoro-d-glucose (18F-FDG) findings. Images were obtained 60 minutes postinjection of 300–500 MBq of 68Ga-NODAGA-THERANOST. Results: 68Ga-NODAGA-THERANOST revealed high tumor-to-background ratios (SUVmax=4.8) and uptake at neoangiogenesis sites. Reconstructed fused images distinguished cancers with high malignancy potential and enabled enhanced bone metastasis detection. 18F-FDG-positive lung and lymph node metastases did not show uptake, indicating the absence of neovascularization. Conclusions: 68Ga-NODAGA-THERANOST was found to be safe and effective, exhibiting in this study rapid blood clearance, stability, rapid renal excretion, favorable biodistribution and PK/PD, low irradiation burden (μSv/MBq/μg), and convenient radiolabeling. This radioligand might enable theranostics, that is, a combination of diagnostics followed by the appropriate therapeutics, namely antiangiogenic therapy, image-guided presurgical assessment, treatment response evaluation, prediction of pathologic response, neoadjuvant-peptidomimetic-radiochemotherapy, and personalized medicine strategies. Further clinical trials evaluating 68Ga-NODAGA-THERANOST are warranted. PMID:25945808

  5. Role of IL-2 in cancer immunotherapy.

    PubMed

    Jiang, Tao; Zhou, Caicun; Ren, Shengxiang

    2016-06-01

    Interleukin-2 (IL-2) is one of the key cytokines with pleiotropic effects on immune system. It has been approved for the treatment of metastatic renal cell carcinoma and metastatic melanoma. Recent progress has been made in our understanding of IL-2 in regulating lymphocytes that has led to exciting new directions for cancer immunotherapy. While improved IL-2 formulations might be used as monotherapies, their combination with other anticancer immunotherapies, such as adoptive cell transfer regimens, antigen-specific vaccination, and blockade of immune checkpoint inhibitory molecules, for example cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and programmed death 1 (PD-1) mono-antibodies, would held the promise of treating metastatic cancer. Despite the comprehensive studies of IL-2 on immune system have established the application of IL-2 for cancer immunotherapy, a number of poignant obstacles remain for future research. In the present review, we will focus on the key biological features of IL-2, current applications, limitations, and future directions of IL-2 in cancer immunotherapy. PMID:27471638

  6. Structure-guided design of a high-affinity platelet integrin αIIbβ3 receptor antagonist that disrupts Mg²⁺ binding to the MIDAS.

    PubMed

    Zhu, Jieqing; Choi, Won-Seok; McCoy, Joshua G; Negri, Ana; Zhu, Jianghai; Naini, Sarasija; Li, Jihong; Shen, Min; Huang, Wenwei; Bougie, Daniel; Rasmussen, Mark; Aster, Richard; Thomas, Craig J; Filizola, Marta; Springer, Timothy A; Coller, Barry S

    2012-03-14

    An integrin found on platelets, α(IIb)β(3) mediates platelet aggregation, and α(IIb)β(3) antagonists are effective antithrombotic agents in the clinic. Ligands bind to integrins in part by coordinating a magnesium ion (Mg(2+)) located in the β subunit metal ion-dependent adhesion site (MIDAS). Drugs patterned on the integrin ligand sequence Arg-Gly-Asp have a basic moiety that binds the α(IIb) subunit and a carboxyl group that coordinates the MIDAS Mg(2+) in the β(3) subunits. They induce conformational changes in the β(3) subunit that may have negative consequences such as exposing previously hidden epitopes and inducing the active conformation of the receptor. We recently reported an inhibitor of α(IIb)β(3) (RUC-1) that binds exclusively to the α(IIb) subunit; here, we report the structure-based design and synthesis of RUC-2, a RUC-1 derivative with a ~100-fold higher affinity. RUC-2 does not induce major conformational changes in β(3) as judged by monoclonal antibody binding, light scattering, gel chromatography, electron microscopy, and a receptor priming assay. X-ray crystallography of the RUC-2-α(IIb)β(3) headpiece complex in 1 mM calcium ion (Ca(2+))/5 mM Mg(2+) at 2.6 Å revealed that RUC-2 binds to α(IIb) the way RUC-1 does, but in addition, it binds to the β(3) MIDAS residue glutamic acid 220, thus displacing Mg(2+) from the MIDAS. When the Mg(2+) concentration was increased to 20 mM, however, Mg(2+) was identified in the MIDAS and RUC-2 was absent. RUC-2's ability to inhibit ligand binding and platelet aggregation was diminished by increasing the Mg(2+) concentration. Thus, RUC-2 inhibits ligand binding by a mechanism different from that of all other α(IIb)β(3) antagonists and may offer advantages as a therapeutic agent. PMID:22422993

  7. Synthesis, structure activity relationship, radiolabeling and preclinical evaluation of high affinity ligands for the ion channel of the N-methyl-d-aspartate receptor as potential imaging probes for positron emission tomography.

    PubMed

    Klein, Pieter J; Christiaans, Johannes A M; Metaxas, Athanasios; Schuit, Robert C; Lammertsma, Adriaan A; van Berckel, Bart N M; Windhorst, Albert D

    2015-03-01

    The N-methyl-d-aspartate receptor (NMDAr) is involved in many neurological and psychiatric disorders including Alzheimer's disease and schizophrenia. Currently, it is not possible to assess NMDAr availability in vivo. The purpose of this study was to develop a positron emission tomography (PET) ligand for the NMDAr ion channel. A series of di- and tri-N-substituted diarylguanidines was synthesized. In addition, in vitro binding affinity for the NMDAr ion channel in rat forebrain membrane fractions was assessed. Compounds 10, 11 and 32 were radiolabeled with either carbon-11 or fluorine-18. Ligands [(11)C]10 and [(18)F]32 were evaluated ex vivo in B6C3 mice. Biodistribution studies showed higher uptake of [(11)C]10 and [(18)F]32 in forebrain regions compared with cerebellum. In addition, for [(11)C]10 54% and for [(18)F]32 70% of activity in the brain at 60min was due to intact tracer. Pre-treatment with MK-801 (0.6mg·kg(-1), ip) slightly decreased uptake in NMDAr-specific regions for [(18)F]32, but not for [(11)C]10. As such [(18)F]32 has the best characteristics as a PET tracer for the ion channel of the NMDAr. PMID:25648682

  8. A comparative autoradiography study in post mortem whole hemisphere human brain slices taken from Alzheimer patients and age-matched controls using two radiolabelled DAA1106 analogues with high affinity to the peripheral benzodiazepine receptor (PBR) system.

    PubMed

    Gulyás, Balázs; Makkai, Boglárka; Kása, Péter; Gulya, Károly; Bakota, Lidia; Várszegi, Szilvia; Beliczai, Zsuzsa; Andersson, Jan; Csiba, László; Thiele, Andrea; Dyrks, Thomas; Suhara, Tetsua; Suzuki, Kazutoshi; Higuchi, Makato; Halldin, Christer

    2009-01-01

    The binding of two radiolabelled analogues (N-(5-[125I]Iodo-2-phenoxyphenyl)-N-(2,5-dimethoxybenzyl)acetamide ([125I]desfluoro-DAA1106) and N-(5-[125I]Fluoro-2-phenoxyphenyl)-N-(2-[125I]Iodo-5-methoxybenzyl)acetamide ([125I]desmethoxy-DAA1106) of the peripheral benzodiazepine receptor (PBR) (or TSPO, 18kDa translocator protein) ligand DAA1106 was examined by in vitro autoradiography on human post mortem whole hemisphere brain slices obtained from Alzheimer's disease (AD) patients and age-matched controls. Both [(125)I]desfluoro-IDAA1106 and [(125)I]desmethoxy-IDAA1106 were effectively binding to various brain structures. The binding could be blocked by the unlabelled ligand as well as by other PBR specific ligands. With both radiolabelled compounds, the binding showed regional inhomogeneity and the specific binding values proved to be the highest in the hippocampus, temporal and parietal cortex, the basal ganglia and thalamus in the AD brains. Compared with age-matched control brains, specific binding in several brain structures (temporal and parietal lobes, thalamus and white matter) in Alzheimer brains was significantly higher, indicating that the radioligands can effectively label-activated microglia and the up-regulated PBR/TSPO system in AD. Complementary immunohistochemical studies demonstrated reactive microglia activation in the AD brain tissue and indicated that increased ligand binding coincides with increased regional microglia activation due to neuroinflammation. These investigations yield further support to the PBR/TSPO binding capacity of DAA1106 in human brain tissue, demonstrate the effective usefulness of its radio-iodinated analogues as imaging biomarkers in post mortem human studies, and indicate that its radiolabelled analogues, labelled with short half-time bioisotopes, can serve as prospective in vivo imaging biomarkers of activated microglia and the up-regulated PBR/TSPO system in the human brain. PMID:18984021

  9. A high-affinity inositol 1,3,4,5-tetrakisphosphate receptor protein from brain is specifically labelled by a newly synthesized photoaffinity analogue, N-(4-azidosalicyl)aminoethanol(1)-1-phospho-D-myo-inositol 3,4,5-trisphosphate.

    PubMed Central

    Reiser, G; Schäfer, R; Donié, F; Hülser, E; Nehls-Sahabandu, M; Mayr, G W

    1991-01-01

    A photolabile arylazido analogue of Ins(1,3,4,5)P4 selectively substituted at the 1-phosphate group was synthesized by coupling 2-aminoethanol(1)-1-phospho-D-myo-inositol 4,5-bisphosphate with N-hydroxysuccinimidyl-4-azidosalicylic acid [Schäfer, Nehls-Sahabandu, Grabowsky, Dehlinger-Kremer, Schulz & Mayr (1990) Biochem. J. 272, 817-825] and subsequently phosphorylating the product by bovine brain Ins(1,4,5)P3 3-kinase. The product, N-(4-azidosalicyl)-aminoethanol(1)-1-phospho-D-myo-inositol 3,4,5-trisphosphate [AsaIns(1,3,4,5)P4] was radioiodinated and purified by anion-exchange chromatography. AsaIns(1,3,4,5)P4 bound to a high-affinity Ins(1,3,4,5)P4 receptor from pig cerebellum with an affinity only 3-fold lower than that of Ins(1,3,4,5)P4. Photoirradiation of 125I-AsaIns(1,3,4,5)P4 in the presence of the receptor preparation revealed that the radioactive label was specifically associated with a protein band of apparent molecular mass 42 kDa, which Donié & Reiser [(1991) Biochem. J. 275, 453-457] had previously tentatively assigned to the Ins(1,3,4,5)P4 receptor protein. The radioactive label was displaced from the receptor when the binding reaction with 125I-AsaIns(1,3,4,5)P4 was carried out in the presence of 5 microM-Ins(1,3,4,5)P4. Images Fig. 3. PMID:1660714

  10. Detection of Waterborne Viruses Using High Affinity Molecularly Imprinted Polymers.

    PubMed

    Altintas, Zeynep; Gittens, Micah; Guerreiro, Antonio; Thompson, Katy-Anne; Walker, Jimmy; Piletsky, Sergey; Tothill, Ibtisam E

    2015-07-01

    Molecularly imprinted polymers (MIPs) are artificial receptor ligands which can recognize and specifically bind to a target molecule. They are more resistant to chemical and biological damage and inactivation than antibodies. Therefore, target specific-MIP nanoparticles are aimed to develop and implemented to biosensors for the detection of biological toxic agents such as viruses, bacteria, and fungi toxins that cause many diseases and death due to the environmental contamination. For the first time, a molecularly imprinted polymer (MIP) targeting the bacteriophage MS2 as the template was investigated using a novel solid-phase synthesis method to obtain the artificial affinity ligand for the detection and removal of waterborne viruses through optical-based sensors. A high affinity between the artificial ligand and the target was found, and a regenerative MIP-based virus detection assay was successfully developed using a new surface plasmon resonance (SPR)-biosensor which provides an alternative technology for the specific detection and removal of waterborne viruses that lead to high disease and death rates all over the world. PMID:26008649

  11. In-vitro characterization of novel and functional regulatory SNPs in the promoter region of IL2 and IL2R alpha in a Gabonese population

    PubMed Central

    2012-01-01

    Background The selection pressure imposed by the parasite has a functional consequence on the immune genes, leading to altered immune function in which regulatory T cells (Tregs) induced by parasites during infectious challenges modulate or thwart T effector cell mechanism. Methods We identified and investigated regulatory polymorphisms in the immune gene IL2 and its receptor IL2R alpha (also known as CD25) in Gabonese individuals exposed to plentiful parasitic infections. Results We identified two reported variants each for IL2 and its receptor IL2R alpha gene loci. Also identified were two novel variants, -83 /-84 CT deletions (ss410961576) for IL2 and -409C/T (ss410961577) for IL2R alpha. We further validated all identified promoter variants for their allelic gene expression using transient transfection assays. Three promoter variants of the IL2 locus revealed no significant expression of the reporter gene. The identified novel variant (ss410961577C/T) of the IL2R alpha revealed a significant higher expression of the reporter gene in comparison to the major allele (P<0.05). In addition, the rs12722616C/T variant of the IL2R alpha locus altered the transcription factor binding site TBP (TATA box binding protein) and C/EBP beta (CCAAT/enhancer binding protein beta) that are believed to regulate the Treg function. Conclusions The identification and validation of such regulatory polymorphisms in the immune genes may provide a basis for future studies on parasite susceptibility in a population where T cell functions are compromised. PMID:23217119

  12. Solution assembly of cytokine receptor ectodomain complexes

    SciTech Connect

    Wu, Zining; Ciardelli, T.L.; Johnson, K.W.

    1995-09-01

    For the majority of single transmembrane-spanning cell surface receptors, signal transmission across the lipid bilayer barrier involves several discrete components of molecular recognition. The interaction between ligand and the extracellular segment of its cognate receptor (ectodomain) initiates either homomeric or heteromeric association of receptor subunits. Specific recognition among these subunits may then occur between ectodomain regions, within the membrane by interhelical contact or inside the cell between cytoplasmic domains. Any or all of these interactions may contribute to the stability of the signaling complex. It is the characteristics of ligand binding by the ectodomains of these receptors that controls the heteromeric or homomeric nature and the stoichiometry of the complex. Cytokines and their receptors belong to a growing family of macromolecular systems that exhibit these functional features and share many structural similarities as well. Interleukin-2 is a multifunctional cytokine that represents, perhaps, the most complex example to date of ligand recognition among the hematopoietin receptor family. It is the cooperative binding of IL-2 by all three proteins on the surface of activated T-lymphocytes, however, that ultimately results in crosslinking of the {beta}- and {gamma}-subunits and signaling via association of their cytoplasmic domains. Although the high-affinity IL-2R functions as a heterotrimer, heterodimers of the receptor subunits are also physiologically important. The {alpha}/{beta} heterodimer or {open_quotes}pseudo-high affinity{close_quotes} receptor captures IL-2 as a preformed cell surface complex while the {beta}/{gamma} intermediate affinity site exists, in the absence of the {alpha} subunit, on the majority of natural killer cells. We have begun to study stable complexes of cytokine receptor ectodomains of defined composition and that mimic the ligand binding characteristics of the equivalent cell surface receptor sites.

  13. Calcium channel antagonists. Omega-conotoxin defines a new high affinity site.

    PubMed

    Cruz, L J; Olivera, B M

    1986-05-15

    The omega-conotoxins, a class of Ca2+ channel antagonists from fish-hunting marine snails, have recently been described (Olivera, B. M., McIntosh, J. M., Zeikus, R., Gray, W. R., Varga, J., Rivier, J., de Santos, V., and Cruz, L. J. (1985) Science, 230, 1338-1343). One of these peptide neurotoxins, omega-conotoxin GVIA, was radiolabeled with iodine, and the 125I-labeled toxin was shown to bind specifically to high affinity sites on chick brain synaptosomes. The toxin-receptor complex was extremely stable; addition of an excess of unlabeled toxin did not cause significant displacement of the labeled toxin after 2 h. Binding competition data suggest that omega-conotoxin defines a new high affinity receptor site affecting voltage-activated Ca2+ channels, distinct from both the verapamil and dihydropyridine target sites. PMID:2939072

  14. "DAKLI": a multipurpose ligand with high affinity and selectivity for dynorphin (kappa opioid) binding sites.

    PubMed Central

    Goldstein, A; Nestor, J J; Naidu, A; Newman, S R

    1988-01-01

    We describe a synthetic ligand, "DAKLI" (Dynorphin A-analogue Kappa LIgand), related to the opioid peptide dynorphin A. A single reactive amino group at the extended carboxyl terminus permits various reporter groups to be attached, such as 125I-labeled Bolton-Hunter reagent, fluorescein isothiocyanate, or biotin. These derivatives have high affinity and selectivity for the dynorphin (kappa opioid) receptor. An incidental finding is that untreated guinea pig brain membranes have saturable avidin binding sites. PMID:2902630

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

    SciTech Connect

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

    1990-11-01

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

  16. Reconstitution of high-affinity opioid agonist binding in brain membranes

    SciTech Connect

    Remmers, A.E.; Medzihradsky, F. )

    1991-03-15

    In synaptosomal membranes from rat brain cortex, the {mu} selective agonist ({sup 3}H)dihydromorphine in the absence of sodium, and the nonselective antagonist ({sup 3}H)naltrexone in the presence of sodium, bound to two populations of opioid receptor sites with K{sub d} values of 0.69 and 8.7 nM for dihydromorphine, and 0.34 and 5.5 nM for naltrexone. The addition of 5 {mu}M guanosine 5{prime}-({gamma}-thio)triphosphate (GTP({gamma}S)) strongly reduced high-affinity agonist but not antagonist binding. Exposure of the membranes to high pH reduced the number of GTP({gamma}-{sup 35}S) binding sites by 90% and low K{sub m}, opioid-sensitive GTPase activity by 95%. In these membranes, high-affinity agonist binding was abolished and modulation of residual binding by GTP({gamma}S) was diminished. Alkali treatment of the glioma cell membranes prior to fusion inhibited most of the low K{sub m} GTPase activity and prevented the reconstitution of agonist binding. The results show that high-affinity opioid agonist binding reflects the ligand-occupied receptor - guanine nucleotide binding protein complex.

  17. IL-2/IL-2 Ab Therapy Induces Target Organ NK Cells that Inhibit CNS Inflammation

    PubMed Central

    Hao, Junwei; Campagnolo, Denise; Liu, Ruolan; Piao, Wenhua; Shi, Samuel; Hu, Baoyong; Xiang, Rong; Zhou, Qinghua; Vollmer, Timothy; Van Kaer, Luc; La Cava, Antonio; Shi, Fu-Dong

    2010-01-01

    Objective The role of natural killer (NK) cells in regulating multiple sclerosis (MS) is not well understood. Additional studies with NK cells might provide insight into the mechanism of action of MS therapies such as daclizumab, an antibody against the IL-2R α-chain, which induces expansion of CD56bright NK cells. Methods In a relapsing-remitting form of the experimental autoimmune encephalomyelitis (EAE) model of MS induced in SJL mice, we expanded NK cells with IL-2 coupled with an anti-IL-2 mAb and evaluated the effects of these NK cells on EAE. Further, we investigated the effect of the human version of IL-2/IL-2 mAb on NK cells from MS patients and its effect on CNS inflammation and pathology in a human-mouse chimera model and assessed the underlying mechanisms. Results IL-2/IL-2 mAb dramatically expands NK cells both in the peripheral lymphoid organs and in the central nervous system (CNS), and attenuates CNS inflammation and neurological deficits. Disease protection is conferred by CNS-resident NK cells. Importantly, the human version of IL-2/IL-2 mAb restored the defective CD56+ NK cells from MS patients in a human-mouse chimera model. Both the CD56bright and CD56dim subpopulations were required to attenuate disease in this model. Interpretation These findings unveil the immunotherapeutic potential of NK cells, which can act as critical suppressor cells in target organs of autoimmunity. These results also have implications to better understand the mechanism of action of daclizumab in MS. PMID:21425186

  18. Functional NK cell repertoires are maintained through IL-2Rα and FasL

    PubMed Central

    Felices, Martin; Lenvik, Todd R.; Ankarlo, Dave E.M.; Foley, Bree; Curtsinger, Julie; Luo, Xianghua; Blazar, Bruce R.; Anderson, Stephen K.; Miller, Jeffrey S.

    2014-01-01

    Acquisition of a functional natural killer (NK) cell repertoire, known as education or licensing, is a complex process mediated through inhibitory receptors that recognize self. We found that NK cells containing self-killer immunoglobulin-like receptors (KIR) for cognate HLA-ligand in vivo were less susceptible to apoptosis. In vitro IL-15 withdrawal showed that uneducated NK cells upregulated Bim and Fas. Conversely, educated NK cells upregulated FasL under these conditions. Induction of cell death and Bim expression on uneducated cells correlated with increased IL-2Rα expression. Overexpression and knockdown studies showed that higher IL-2Rα limits NK cell survival in a novel manner that is independent from the role of IL-2 in activation induced cell death (AICD). To study the role of FasL in induction of IL-2Rαhi NK cell death, a co-culture assay with FasL blocking antibodies was used. IL-15 withdrawal led to FasL dependent killing of IL-2Rαhi NK cells by more educated IL-2Rαlo NK cells. Finally, CMV reactivation induces a potent long-lasting population of licensed NK cells with enhanced survival. These findings show education dependent NK cell survival advantages and killing of uneducated NK result in the maintenance of a functional repertoire, which may be manipulated to exploit NK cells for cancer immunotherapy. PMID:24634493

  19. PD-L1 blockade synergizes with IL-2 therapy in reinvigorating exhausted T cells

    PubMed Central

    West, Erin E.; Jin, Hyun-Tak; Rasheed, Ata-Ur; Penaloza-MacMaster, Pablo; Ha, Sang-Jun; Tan, Wendy G.; Youngblood, Ben; Freeman, Gordon J.; Smith, Kendall A.; Ahmed, Rafi

    2013-01-01

    The inhibitory receptor programmed cell death 1 (PD-1) plays a major role in functional exhaustion of T cells during chronic infections and cancer, and recent clinical data suggest that blockade of the PD-1 pathway is an effective immunotherapy in treating certain cancers. Thus, it is important to define combinatorial approaches that increase the efficacy of PD-1 blockade. To address this issue, we examined the effect of IL-2 and PD-1 ligand 1 (PD-L1) blockade in the mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection. We found that low-dose IL-2 administration alone enhanced CD8+ T cell responses in chronically infected mice. IL-2 treatment also decreased inhibitory receptor levels on virus-specific CD8+ T cells and increased expression of CD127 and CD44, resulting in a phenotype resembling that of memory T cells. Surprisingly, IL-2 therapy had only a minimal effect on reducing viral load. However, combining IL-2 treatment with blockade of the PD-1 inhibitory pathway had striking synergistic effects in enhancing virus-specific CD8+ T cell responses and decreasing viral load. Interestingly, this reduction in viral load occurred despite increased numbers of Tregs. These results suggest that combined IL-2 therapy and PD-L1 blockade merits consideration as a regimen for treating human chronic infections and cancer. PMID:23676462

  20. Dendritic cell derived IL-2 inhibits survival of terminally mature cells via an autocrine signaling pathway.

    PubMed

    Balachander, Akhila; Nabti, Sabrina; Sobota, Radoslaw M; Foo, Shihui; Zolezzi, Francesca; Lee, Bernett T K; Poidinger, Michael; Ricciardi-Castagnoli, Paola

    2015-05-01

    DCs are crucial for sensing pathogens and triggering immune response. Upon activation by pathogen-associated molecular pattern (PAMP) ligands, GM-CSF myeloid DCs (GM-DCs) secrete several cytokines, including IL-2. DC IL-2 has been shown to be important for innate and adaptive immune responses; however, IL-2 importance in DC physiology has never been demonstrated. Here, we show that autocrine IL-2 signaling is functional in murine GM-DCs in an early time window after PAMPs stimulation. IL-2 signaling selectively activates the JAK/STAT5 pathway by assembling holo-receptor complexes at the cell surface. Using the sensitivity of targeted mass spectrometry, we show conclusively that GM-DCs express CD122, the IL-2 receptor β-chain, at steady state. In myeloid DCs, this cytokine pathway inhibits survival of PAMP-matured GM-DCs which is crucial for maintaining immune tolerance and preventing autoimmunity. Our findings suggest that immune regulation by this novel autocrine signaling pathway can potentially be used in DC immunotherapy. PMID:25652593

  1. Evaluation of the IL2/IL21, IL2RA and IL2RB genetic variants influence on the endogenous non-anterior uveitis genetic predisposition

    PubMed Central

    2013-01-01

    Background Recently, different genetic variants located within the IL2/IL21 genetic region as well as within both IL2RA and IL2RB loci have been associated to multiple autoimmune disorders. We aimed to investigate for the first time the potential influence of the IL2/IL21, IL2RA and IL2RB most associated polymorphisms with autoimmunity on the endogenous non-anterior uveitis genetic predisposition. Methods A total of 196 patients with endogenous non-anterior uveitis and 760 healthy controls, all of them from Caucasian population, were included in the current study. The IL2/IL21 (rs2069762, rs6822844 and rs907715), IL2RA (2104286, rs11594656 and rs12722495) and IL2RB (rs743777) genetic variants were genotyped using TaqMan® allelic discrimination assays. Results A statistically significant difference was found for the rs6822844 (IL2/IL21 region) minor allele frequency in the group of uveitis patients compared with controls (P-value=0.02, OR=0.64 CI 95%=0.43-0.94) although the significance was lost after multiple testing correction. Furthermore, no evidence of association with uveitis was detected for the analyzed genetic variants of the IL2RA or IL2RB loci. Conclusion Our results indicate that analyzed IL2/IL21, IL2RA and IL2RB polymorphisms do not seem to play a significant role on the non-anterior uveitis genetic predisposition although further studies are needed in order to clear up the influence of these loci on the non-anterior uveitis susceptibility. PMID:23676143

  2. Clinical Significance of sIL-2R Levels in B-Cell Lymphomas

    PubMed Central

    Yoshida, Noriaki; Oda, Miyo; Kuroda, Yoshiaki; Katayama, Yuta; Okikawa, Yoshiko; Masunari, Taro; Fujiwara, Megumu; Nishisaka, Takashi; Sasaki, Naomi; Sadahira, Yoshito; Mihara, Keichiro; Asaoku, Hideki; Matsui, Hirotaka; Seto, Masao; Kimura, Akiro; Arihiro, Koji; Sakai, Akira

    2013-01-01

    Elevated soluble interleukin-2 receptor (sIL-2R) in sera is observed in patients with malignant lymphoma (ML). Therefore, sIL-2R is commonly used as a diagnostic and prognostic marker for ML, but the mechanisms responsible for the increase in sIL-2R levels in patients with B-cell lymphomas have not yet been elucidated. We first hypothesized that lymphoma cells expressing IL-2R and some proteinases such as matrix metalloproteinases (MMPs) in the tumor microenvironment can give rise to increased sIL-2R in sera. However, flow cytometric studies revealed that few lymphoma cells expressed IL-2R α chain (CD25) in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL), and most CD25-expressing cells in the tumor were T-cells. Distinct correlations between CD25 expression on B-lymphoma cells and sIL-2R levels were not observed. We then confirmed that MMP-9 plays an important role in producing sIL-2R in functional studies. Immunohistochemical (IHC) analysis also revealed that MMP-9 is mainly derived from tumor-associated macrophages (TAMs). We therefore evaluated the number of CD68 and CD163 positive macrophages in the tumor microenvironment using IHC analysis. A positive correlation between the levels of sIL-2R in sera and the numbers of CD68 positive macrophages in the tumor microenvironment was confirmed in FL and extranodal DLBCL. These results may be useful in understanding the pathophysiology of B-cell lymphomas. PMID:24236041

  3. High-affinity binding sites involved in the import of porin into mitochondria.

    PubMed Central

    Pfaller, R; Neupert, W

    1987-01-01

    The specific recognition by mitochondria of the precursor of porin and the insertion into the outer membrane were studied with a radiolabeled water-soluble form of porin derived from the mature protein. High-affinity binding sites had a number of 5-10 pmol/mg mitochondrial protein and a ka of 1-5 X 10(8) M-1. Binding was abolished after trypsin pretreatment of mitochondria indicating that binding sites were of protein-aceous nature. Specifically bound porin could be extracted at alkaline pH but not by high salt and was protected against low concentrations of proteinase K. It could be chased to a highly protease resistant form corresponding to mature porin. High-affinity binding sites could be extracted from mitochondria with detergent and reconstituted in asolectin-ergosterol liposomes. Water-soluble porin competed for the specific binding and import of the precursor of the ADP/ATP carrier, an inner membrane protein. We suggest that (i) binding of precursors to proteinaceous receptors serves as an initial step for recognition, (ii) the receptor for porin may also be involved in the import of precursors of inner membrane proteins, and (iii) interaction with the receptor triggers partial insertion of the precursor into the outer membrane. Images Fig. 4. PMID:2960520

  4. Selective high affinity polydentate ligands and methods of making such

    SciTech Connect

    DeNardo, Sally; DeNardo, Gerald; Balhorn, Rodney

    2010-02-16

    This invention provides novel polydentate selective high affinity ligands (SHALs) that can be used in a variety of applications in a manner analogous to the use of antibodies. SHALs typically comprise a multiplicity of ligands that each bind different region son the target molecule. The ligands are joined directly or through a linker thereby forming a polydentate moiety that typically binds the target molecule with high selectivity and avidity.

  5. Green tea EGCG suppresses T cell proliferation through impairment of IL-2/IL-2 receptor signaling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Studies have suggested a benefit of consuming green tea in promoting general health and reducing the risk of certain diseases. However, little is known about the effect of green tea on immune function. In this study we determined the effect of epigallocatechin-3-gallate (EGCG), the major active comp...

  6. Conditional IL-2 Gene Deletion: Consequences for T Cell Proliferation

    PubMed Central

    Popmihajlov, Zoran; Xu, Dong; Morgan, Heather; Milligan, Zoie; Smith, Kendall A.

    2012-01-01

    To explore the role of interleukin-2 (IL-2) in T cell proliferation, and to circumvent the IL-2 deficiency autoimmune syndrome of conventional il2 gene deletion, mice were created to allow conditional il2 gene deletion when treated with the estrogen analog, tamoxifen (TAM) as adults. Splenocytes from four different mouse strains, C57Bl/6 wild type (WT), conventional IL-2(−/−), TAM-treated Cre recombinase-negative (Cre−)/IL2fl/fl, and Cre recombinase-positive (Cre+)/IL2fl/fl, were activated with anti-CD3 and anti-CD28, and monitored for CD4+ and CD8+ T cell lymphocyte blastogenesis, aerobic glycolysis, BrdU incorporation into newly synthesized DNA, and CFSE dye dilution to monitor cell division. IL-2 production was monitored by quantitative ELISA and multiple additional cytokines were monitored by quantitative protein-bead arrays. Splenocytes from conventional IL-2(−/−) and TAM-treated Cre+ mice resulted in undetectable IL-2 production by ELISA, so that both strains were IL-2-deficient. As monitored by flow cytometry, activated CD4+ and CD8+ T cells from WT, Cre+, and Cre− mice all underwent blastogenesis, whereas far fewer cells from conventional IL-2(−/−) mice did so. By comparison, only cells from IL-2 sufficient WT and Cre− mice switched to aerobic glycolysis as evidenced by a drop in media pH. Blastogenesis was mirrored by BrdU incorporation and CFSE dye dilution by CD4+ and CD8+ T cells from WT, Cre+, and Cre− mice, which were all equivalent, while proliferation of cells from conventional IL-2(−/−) mice was compromised. Splenocytes from IL-2 deficient conventional IL-2(−/−) mice produced low or undetectable other γc-chain cytokines (IL-4, IL-7, IL-9, IL-13, IL-15, and IL-21), whereas production of these γc-chain cytokines from IL-2-deficient conditional IL-2(−/−) Cre+ mice were comparable with WT and Cre− mice. These results indicate that CD4+ and CD8+ T cell blastogenesis cannot be attributable to IL-2 alone, but a switch

  7. Specific high-affinity binding of high density lipoproteins to cultured human skin fibroblasts and arterial smooth muscle cells.

    PubMed

    Biesbroeck, R; Oram, J F; Albers, J J; Bierman, E L

    1983-03-01

    Binding of human high density lipoproteins (HDL, d = 1.063-1.21) to cultured human fibroblasts and human arterial smooth muscle cells was studied using HDL subjected to heparin-agarose affinity chromatography to remove apoprotein (apo) E and B. Saturation curves for binding of apo E-free 125I-HDL showed at least two components: low-affinity nonsaturable binding and high-affinity binding that saturated at approximately 20 micrograms HDL protein/ml. Scatchard analysis of high-affinity binding of apo E-free 125I-HDL to normal fibroblasts yielded plots that were significantly linear, indicative of a single class of binding sites. Saturation curves for binding of both 125I-HDL3 (d = 1.125-1.21) and apo E-free 125I-HDL to low density lipoprotein (LDL) receptor-negative fibroblasts also showed high-affinity binding that yielded linear Scatchard plots. On a total protein basis, HDL2 (d = 1.063-1.10), HDL3 and very high density lipoproteins (VHDL, d = 1.21-1.25) competed as effectively as apo E-free HDL for binding of apo E-free 125I-HDL to normal fibroblasts. Also, HDL2, HDL3, and VHDL competed similarly for binding of 125I-HDL3 to LDL receptor-negative fibroblasts. In contrast, LDL was a weak competitor for HDL binding. These results indicate that both human fibroblasts and arterial smooth muscle cells possess specific high affinity HDL binding sites. As indicated by enhanced LDL binding and degradation and increased sterol synthesis, apo E-free HDL3 promoted cholesterol efflux from fibroblasts. These effects also saturated at HDL3 concentrations of 20 micrograms/ml, suggesting that promotion of cholesterol efflux by HDL is mediated by binding to the high-affinity cell surface sites. PMID:6826722

  8. 01-ERD-111 - The Development of Synthetic High Affinity Ligands

    SciTech Connect

    Perkins, J; Balhorn, R; Cosman, M; Lightstone, F; Zeller, L

    2004-02-05

    The aim of this project was to develop Synthetic High-Affinity Ligands (SHALs), which bind with high affinity and specificity to proteins of interest for national security and cancer therapy applications. The aim of producing synthetic ligands for sensory devices as an alternative to antibody-based detection assays and therapeutic agents is to overcome the drawbacks associated with antibody-based in next-generation sensors and systems. The focus area of the project was the chemical synthesis of the SHALs. The project concentrated on two different protein targets. (a) The C fragment of tetanus and botulinum toxin, potential biowarfare agents. A SHAL for tetanus or botulinum toxin would be incorporated into a sensory device for the toxins. (b) HLA-DR10, a protein found in high abundance on the surface of Non-Hodgkins Lymphoma. A SHAL specific to a tumor marker, labeled with a radionuclide, would enable the targeted delivery of radiation therapy to metastatic disease. The technical approach used to develop a SHAL for each protein target will be described in more detail below. However, in general, the development of a SHAL requires a combination of computational modeling techniques, modern nuclear magnetic resonance spectroscopy (NMR) and synthetic chemistry.

  9. Multipurpose ligand, DAKLI (Dynorphin A-analogue Kappa LIgand), with high affinity and selectivity for dynorphin (. kappa. opioid) binding sites

    SciTech Connect

    Goldstein, A.; Nestor, J.J. Jr.; Naidu, A.; Newman, S.R. )

    1988-10-01

    The authors describe a synthetic ligand, DALKI (Dynorphin A-analogue Kappa LIgand), related to the opioid peptide dynorphin A. A single reactive amino group at the extended carboxyl terminus permits various reporter groups to be attached, such as {sup 125}I-labeled Bolton-Hunter reagent, fluorescein isothiocyanate, or biotin. These derivatives have high affinity and selectivity for the dynorphin ({kappa} opioid) receptor. An incidental finding is that untreated guinea pig brain membranes have saturable avidin binding sites.

  10. IL-2: a two-faced master regulator of autoimmunity.

    PubMed

    Sharma, Rahul; Fu, Shu Man; Ju, Shyr-Te

    2011-03-01

    CD4(+) T-cell (Th) cytokines provide important regulatory and effector functions of T-cells. Among them, IL-2 plays a unique role. IL-2 is required for the generation and maintenance of regulatory T-cells (Treg) to provide lifelong protection from autoimmune disease. Whether IL-2 is also required for autoimmune disease development is less clear as Il2(-/)(-) mice themselves spontaneously develop multi-organ inflammation (MOI). In this communication, we discuss evidence that support the thesis that IL-2 is required for the development of autoimmune response, although some aspects of autoimmune response are not regulated by IL-2. Potential IL-2-dependent mechanisms operating at specific stages of the inflammation process are presented. The interplays among Treg, IL-2, autoimmune response and adaptive immunity are discussed. Overall, available information indicates that IL-2 is a two-faced master regulator of autoimmunity: one to prevent autoimmunity while the other promotes autoimmune response. The latter is an unfortunate consequence of IL-2 function that is used to promote the adaptive immune response against foreign antigens and pathogens. PMID:21282039

  11. High affinity binding of (/sup 3/H)cocaine to rat liver microsomes

    SciTech Connect

    El-Maghrabi, E.A.; Calligaro, D.O.; Eldefrawi, M.E.

    1988-01-01

    )/sup 3/H)cocaine bound reversible, with high affinity and stereospecificity to rat liver microsomes. Little binding was detected in the lysosomal, mitochondrial and nuclear fractions. The binding kinetics were slow and the kinetically calculated K/sub D/ was 2 nM. Induction of mixed function oxidases by phenobarbital did not produce significant change in (/sup 3/H)cocaine binding. On the other hand, chronic administration of cocaine reduced (/sup 3/H)cocaine binding drastically. Neither treatment affected the affinity of the liver binding protein for cocaine. Microsomes from mouse and human livers had less cocaine-binding protein and lower affinity for cocaine than those from rat liver. Binding of (/sup 3/H)cocaine to rat liver microsomes was insensitive to monovalent cations and > 10 fold less sensitive to biogenic amines than the cocaine receptor in rat striatum. However, the liver protein had higher affinity for cocaine and metabolites except for norcocaine. Amine uptake inhibitors displaced (/sup 3/H)cocaine binding to liver with a different rank order of potency than their displacement of (/sup 3/H)cocaine binding to striatum. This high affinity (/sup 3/H)cocaine binding protein in liver is not likely to be monooxygenase, but may have a role in cocaine-induced hepatotoxicity

  12. Rac1 Protein Regulates Glycogen Phosphorylase Activation and Controls Interleukin (IL)-2-dependent T Cell Proliferation*

    PubMed Central

    Arrizabalaga, Onetsine; Lacerda, Hadriano M.; Zubiaga, Ana M.; Zugaza, José L.

    2012-01-01

    Small GTPases of the Rho family have been implicated in important cellular processes such as cell migration and adhesion, protein secretion, and/or gene transcription. In the lymphoid system, these GTPases participate in the signaling cascades that are activated after engagement of antigen receptors. However, little is known about the role that Rho GTPases play in IL-2-mediated responses. Here, we show that IL-2 induces Rac1 activation in Kit 225 T cells. We identified by mass spectrometry the muscle isoform of glycogen phosphorylase (PYGM) as a novel Rac1 effector molecule in IL-2-stimulated cells. The interaction between the active form of Rac1 (Rac1-GTP) and PYGM was established directly through a domain comprising amino acids 191–270 of PYGM that exhibits significant homology with the Rac binding domain of PAK1. The integrity of this region was crucial for PYGM activation. Importantly, IL-2-dependent cellular proliferation was inhibited upon blocking both the activation of Rac1 and the activity of PYGM. These results reveal a new role for Rac1 in cell signaling, showing that this GTPase triggers T cell proliferation upon IL-2 stimulation by associating with PYGM and modulating its enzymatic activity. PMID:22337875

  13. Mutation analysis of IL2RG in human X-linked severe combined immunodeficiency.

    PubMed

    Puck, J M; Pepper, A E; Henthorn, P S; Candotti, F; Isakov, J; Whitwam, T; Conley, M E; Fischer, R E; Rosenblatt, H M; Small, T N; Buckley, R H

    1997-03-15

    Severe combined immunodeficiency (SCID) is a syndrome of profoundly impaired cellular and humoral immunity. In humans, SCID is most commonly caused by mutations in the X-linked gene IL2RG, which encodes the common gamma chain, gamma c, of the leukocyte receptors for interleukin-2 and multiple other cytokines. To investigate the frequency and variety of IL2RG mutations that cause SCID, we analyzed DNA, RNA, and B-cell lines from a total of 103 unrelated SCID-affected males and their relatives using a combination of molecular and immunologic techniques. Sixty-two different mutations spanning all eight IL2RG exons were found in 87 cases, making possible correlations between mutation type and functional consequences. Although skewed maternal X chromosome inactivation, single-strand conformation polymorphism, mRNA expression, and cell surface staining with anti-gamma c antibodies were all helpful in establishing IL2RG defects as the cause of SCID, only dideoxy fingerprinting and DNA sequence determination each detected 100% of the IL2RG mutations in our series. Abnormal gamma c chains may be expressed in the lymphocytes of as many as two thirds of patients with X-linked SCID. Specific mutation diagnosis thus remains technically challenging, but it is important for genetic counseling and perhaps for helping to select appropriate subjects for retroviral gene therapy trials, This is a US government work. There are no restrictions on its use. PMID:9058718

  14. Solubilization and characterization of high-affinity [3H]serotonin binding sites from bovine cortical membranes.

    PubMed Central

    VandenBerg, S R; Allgren, R L; Todd, R D; Ciaranello, R D

    1983-01-01

    High-affinity [3H]serotonin binding activity has been solubilized from bovine cerebral cortical membranes by using Triton X-100, Tween-80, and octyl-beta-D-glucopyranoside. This mixture of detergents solubilizes the high-affinity [3H]serotonin binding activity present in crude membrane preparations with retention of 75-90% specific binding. The detergent mixture was chosen because it can easily be removed from the solubilized fraction by dialysis and polystyrene bead adsorption, thus permitting further purification and isolation of the binding sites. Saturation analysis reveals multiple components of high-affinity [3H]serotonin binding. In crude bovine cortical membranes, at least two binding components are present. A higher-affinity binding component, as defined from curvilinear Scatchard plots, has a Kd for [3H]serotonin of 1-3 nM, whereas a lower-affinity component has a Kd of 10-20 nM. In the solubilized preparation, only a single class of binding sites is apparent, with a Kd of 50-100 nM. Removal of detergents by dialysis and polystyrene bead adsorption results in restoration of the curvilinear Scatchard plot with apparent Kds similar to those observed in crude membrane preparations and with increased Bmax values for each component. [3H]Serotonin binding activity in the solubilized preparation is stable to Sephacryl S-300 column chromatography and to glycerol gradient sedimentation. Saturation analysis of the peak binding fractions from both these procedures once again yields curvilinear Scatchard plots, indicating that the multiple high-affinity binding components are preserved and migrate together. The molecular weight, Stokes radius, and frictional coefficient of the binding site(s) have been calculated. After detergent removal the solubilized material shows many of the characteristics usually attributed to S1 receptors, such as high affinity for [3H]serotonin and its analogs and low affinity for serotonin antagonists. PMID:6574495

  15. High-affinity uptake of noradrenaline in postsynaptic neurones.

    PubMed Central

    al-Damluji, S.; Krsmanovic, L. Z.; Catt, K. J.

    1993-01-01

    1. Neurotransmitters released from nerve endings are inactivated by re-uptake into the presynaptic nerve terminals and possibly into neighbouring glial cells. While analysing the functional properties of alpha 1-adrenoceptors in the hypothalamus, we observed a high-affinity uptake process for noradrenaline in postsynaptic peptidergic neurones. 2. In primary hypothalamic cell cultures and in a hypothalamic neuronal cell line, [3H]-prazosin bound with high affinity and was displaced by unlabelled prazosin in concentrations of 10(-10) to 10(-7) M. However, at concentrations of unlabelled prazosin above 10(-7) M, there was a paradoxical increase in apparent [3H]-prazosin binding. 3. Methoxamine, an alpha 1-adrenoceptor ligand that is not subject to significant neuronal uptake, displaced [3H]-prazosin but did not cause the paradoxical increase in the apparent binding of [3H]-prazosin. Cooling the cells to 4 degrees C reduced the total amount of prazosin associated with the cells; under these conditions, methoxamine almost completely inhibited [3H]-prazosin binding to the cells. 4. In the presence of desipramine (DMI), unlabelled prazosin displaced [3H]-prazosin as before, but no paradoxical increase in apparent binding was seen above 10(-7) M. 5. The paradoxical increase of [3H]-prazosin binding was not observed in membrane preparations of hypothalamic neurones. These findings indicated that the paradoxical increase in apparent [3H]-prazosin binding was due to a cellular uptake process that becomes evident at high concentrations of the ligand. 6. DMI (10(-5) M) had no effect on the specific binding of [3H]-prazosin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8358534

  16. Autophagy is required for IL-2-mediated fibroblast growth

    SciTech Connect

    Kang, Rui; Tang, Daolin; Lotze, Michael T.; Zeh III, Herbert J.

    2013-02-15

    Autophagy is an evolutionarily conserved pathway responsible for delivery of cytoplasmic material into the lysosomal degradation pathway to enable vesicular exocytosis. Interleukin (IL)-2 is produced by T-cells and its activity is important for immunoregulation. Fibroblasts are an immune competent cell type, playing a critical role in wound healing, chronic inflammation, and tumor development. Although autophagy plays an important role in each of these processes, whether it regulates IL-2 activity in fibroblasts is unknown. Here, we show that autophagy is required for IL-2-induced cell growth in fibroblasts. IL-2 significantly induced autophagy in mouse embryonic fibroblasts (MEFs) and primary lung fibroblasts. Autophagy inhibitors (e.g., 3-methylamphetamine and bafilomycin A1) or knockdown of ATG5 and beclin 1 blocked clinical grade IL-2-induced autophagy. Moreover, IL-2 induced HMGB1 cytoplasmic translocation in MEFs and promoted interaction between HMGB1 and beclin1, which is required for autophagy induction. Pharmacological and genetic inhibition of autophagy inhibited IL-2-induced cell proliferation and enhanced IL-2-induced apoptosis. These findings suggest that autophagy is an important pro-survival regulator for IL-2-induced cell growth in fibroblasts.

  17. Structure-based Design of Peptides with High Affinity and Specificity to HER2 Positive Tumors

    PubMed Central

    Geng, Lingling; Wang, Zihua; Yang, Xiaoliang; Li, Dan; Lian, Wenxi; Xiang, Zhichu; Wang, Weizhi; Bu, Xiangli; Lai, Wenjia; Hu, Zhiyuan; Fang, Qiaojun

    2015-01-01

    To identify peptides with high affinity and specificity against human epidermal growth factor receptor 2 (HER2), a series of peptides were designed based on the structure of HER2 and its Z(HER2:342) affibody. By using a combination protocol of molecular dynamics modeling, MM/GBSA binding free energy calculations, and binding free energy decomposition analysis, two novel peptides with 27 residues, pep27 and pep27-24M, were successfully obtained. Immunocytochemistry and flow cytometry analysis verified that both peptides can specifically bind to the extracellular domain of HER2 protein at cellular level. The Surface Plasmon Resonance imaging (SPRi) analysis showed that dissociation constants (KD) of these two peptides were around 300 nmol/L. Furthermore, fluorescence imaging of peptides against nude mice xenografted with SKBR3 cells indicated that both peptides have strong affinity and high specificity to HER2 positive tumors. PMID:26284145

  18. Structure-based Design of Peptides with High Affinity and Specificity to HER2 Positive Tumors.

    PubMed

    Geng, Lingling; Wang, Zihua; Yang, Xiaoliang; Li, Dan; Lian, Wenxi; Xiang, Zhichu; Wang, Weizhi; Bu, Xiangli; Lai, Wenjia; Hu, Zhiyuan; Fang, Qiaojun

    2015-01-01

    To identify peptides with high affinity and specificity against human epidermal growth factor receptor 2 (HER2), a series of peptides were designed based on the structure of HER2 and its Z(HER2:342) affibody. By using a combination protocol of molecular dynamics modeling, MM/GBSA binding free energy calculations, and binding free energy decomposition analysis, two novel peptides with 27 residues, pep27 and pep27-24M, were successfully obtained. Immunocytochemistry and flow cytometry analysis verified that both peptides can specifically bind to the extracellular domain of HER2 protein at cellular level. The Surface Plasmon Resonance imaging (SPRi) analysis showed that dissociation constants (K D) of these two peptides were around 300 nmol/L. Furthermore, fluorescence imaging of peptides against nude mice xenografted with SKBR3 cells indicated that both peptides have strong affinity and high specificity to HER2 positive tumors. PMID:26284145

  19. Interleukin-2 (IL-2, Proleukin) and immune function.

    PubMed

    2003-01-01

    IL-2 is an immune-based therapy that results in dramatic increases in CD4+ cell counts when used in conjunction with anti-HIV therapy. Although IL-2 has been discussed in previous issues of PI Perspective, new information warrants a further look at the product. PMID:12647677

  20. Local therapy of cancer with free IL-2

    PubMed Central

    Jacobs, John J. L.; Battermann, Jan J.; Hordijk, Gerrit Jan; Krastev, Zachary; Moiseeva, Ekaterina V.; Stewart, Rachel J. E.; Ziekman, Paul G. P. M.; Koten, Jan Willem

    2008-01-01

    This is a position paper about the therapeutic effects of locally applied free IL-2 in the treatment of cancer. Local therapy: IL-2 therapy of cancer was originally introduced as a systemic therapy. This therapy led to about 20% objective responses. Systemic therapy however was very toxic due to the vascular leakage syndrome. Nevertheless, this treatment was a break-through in cancer immunotherapy and stimulated some interesting questions: Supposing that the mechanism of IL-2 treatment is both proliferation and tumoricidal activity of the tumor infiltrating cells, then locally applied IL-2 should result in a much higher local IL-2 concentration than systemic IL-2 application. Consequently a greater beneficial effect could be expected after local IL-2 application (peritumoral = juxtatumoral, intratumoral, intra-arterial, intracavitary, or intratracheal = inhalation). Free IL-2: Many groups have tried to prepare a more effective IL-2 formulation than free IL-2. Examples are slow release systems, insertion of the IL-2 gene into a tumor cell causing prolonged IL-2 release. However, logistically free IL-2 is much easier to apply; hence we concentrated in this review and in most of our experiments on the use of free IL-2. Local therapy with free IL-2 may be effective against transplanted tumors in experimental animals, and against various spontaneous carcinomas, sarcomas, and melanoma in veterinary and human cancer patients. It may induce rejection of very large, metastasized tumor loads, for instance advanced clinical tumors. The effects of even a single IL-2 application may be impressive. Not each tumor or tumor type is sensitive to local IL-2 application. For instance transplanted EL4 lymphoma or TLX9 lymphoma were not sensitive in our hands. Also the extent of sensitivity differs: In Bovine Ocular Squamous Cell Carcinoma (BOSCC) often a complete regression is obtained, whereas with the Bovine Vulval Papilloma and Carcinoma Complex (BVPCC) mainly stable disease is

  1. Differential expression of proteins in naïve and IL-2 stimulated primary human NK cells identified by global proteomic analysis

    PubMed Central

    Ma, Di; Felder, Mildred; Scarlett, Cameron O.; Patankar, Manish S.; Li, Lingjun

    2015-01-01

    Natural killer (NK) cells efficiently cytolyse tumors and virally infected cells. Despite the important role that interleukin (IL)-2 plays in stimulating the proliferation of NK cells and increasing NK cell activity, little is known about the alterations in the global NK cell proteome following IL-2 activation. To compare the proteomes of naïve and IL-2-activated primary NK cells and identify key cellular pathways involved in IL-2 signaling, we isolated proteins from naïve and IL-2-activated NK cells from healthy donors, the proteins were trypsinized and the resulting peptides were analyzed by 2D LC ESI-MS/MS followed by label-free quantification. In total, more than 2000 proteins were identified from naïve and IL-2-activated NK cells where 383 proteins were found to be differentially expressed following IL-2 activation. Functional annotation of IL-2 regulated proteins revealed potential targets for future investigation of IL-2 signaling in human primary NK cells. A pathway analysis was performed and revealed several pathways that were not previously known to be involved in IL-2 response, including ubiquitin proteasome pathway, integrin signaling pathway, platelet derived growth factor (PDGF) signaling pathway, epidermal growth factor receptor (EGFR) signaling pathway and Wnt signaling pathway. PMID:23806757

  2. Solubilization and partial characterization of a microsomal high affinity GTPase

    SciTech Connect

    Nicchitta, C.; Williamson, J.R.

    1987-05-01

    Isolated rat liver microsomes release sequestered Ca/sup 2 +/ following addition of GTP. In contrast to permeabilized cells, GTP dependent microsomal Ca/sup 2 +/ release requires low concentrations of polyethylene glycol (PEG). They have identified a microsomal, PEG-sensitive high affinity GTPase which shares a number of characteristics with the GTP-dependent Ca/sup 2 +/ release system. To aid in further characterization of this activity they have initiated studies on the solubilization and purification of the microsomal GTPases. When microsomes are solubilized under the following conditions (150 mM NaCl, 5 mg protein/ml, 1% Triton X-114) PEG sensitive GTPase activity selectively partitions into the detergent rich phase of the Triton X-114 extract. As observed in intact microsomal membranes the Triton X-114 soluble GTPase is maximally stimulated by 3% PEG. Half maximal stimulation is observed at 1% PEG. PEG increases the Vmax of this activity; no effects on Km were observed. The Km for GTP of the detergent soluble GTPase is 5 ..mu..M. This GTPase is sensitive to inhibition by sulfhydryl reagents. PEG-sensitive GTPase activity was completely inhibited in the presence of 25 ..mu..M p-hydroxymercuribenzoate (PHMB); half maximal inhibition was observed at 5 ..mu..M. Labeling of the Triton X-114 extract with the photosensitive compound (/sup 32/P) 8-azido GTP indicated the presence of two prominent GTP binding proteins of approximate molecular weights 17 and 54 kD.

  3. The common γ-chain cytokine receptor: tricks-and-treats for T cells.

    PubMed

    Waickman, Adam T; Park, Joo-Young; Park, Jung-Hyun

    2016-01-01

    Originally identified as the third subunit of the high-affinity IL-2 receptor complex, the common γ-chain (γc) also acts as a non-redundant receptor subunit for a series of other cytokines, collectively known as γc family cytokines. γc plays essential roles in T cell development and differentiation, so that understanding the molecular basis of its signaling and regulation is a critical issue in T cell immunology. Unlike most other cytokine receptors, γc is thought to be constitutively expressed and limited in its function to the assembly of high-affinity cytokine receptors. Surprisingly, recent studies reported a series of findings that unseat γc as a simple housekeeping gene, and unveiled γc as a new regulatory molecule in T cell activation and differentiation. Cytokine-independent binding of γc to other cytokine receptor subunits suggested a pre-association model of γc with proprietary cytokine receptors. Also, identification of a γc splice isoform revealed expression of soluble γc proteins (sγc). sγc directly interacted with surface IL-2Rβ to suppress IL-2 signaling and to promote pro-inflammatory Th17 cell differentiation. As a result, endogenously produced sγc exacerbated autoimmune inflammatory disease, while the removal of endogenous sγc significantly ameliorated disease outcome. These data provide new insights into the role of both membrane and soluble γc in cytokine signaling, and open new venues to interfere and modulate γc signaling during immune activation. These unexpected discoveries further underscore the perspective that γc biology remains largely uncharted territory that invites further exploration. PMID:26468051

  4. The Conundrum of the High-Affinity NGF Binding Site Formation Unveiled?

    PubMed Central

    Covaceuszach, Sonia; Konarev, Petr V.; Cassetta, Alberto; Paoletti, Francesca; Svergun, Dmitri I.; Lamba, Doriano; Cattaneo, Antonino

    2015-01-01

    The homodimer NGF (nerve growth factor) exerts its neuronal activity upon binding to either or both distinct transmembrane receptors TrkA and p75NTR. Functionally relevant interactions between NGF and these receptors have been proposed, on the basis of binding and signaling experiments. Namely, a ternary TrkA/NGF/p75NTR complex is assumed to be crucial for the formation of the so-called high-affinity NGF binding sites. However, the existence, on the cell surface, of direct extracellular interactions is still a matter of controversy. Here, supported by a small-angle x-ray scattering solution study of human NGF, we propose that it is the oligomerization state of the secreted NGF that may drive the formation of the ternary heterocomplex. Our data demonstrate the occurrence in solution of a concentration-dependent distribution of dimers and dimer of dimers. A head-to-head molecular assembly configuration of the NGF dimer of dimers has been validated. Overall, these findings prompted us to suggest a new, to our knowledge, model for the transient ternary heterocomplex, i.e., a TrkA/NGF/p75NTR ligand/receptors molecular assembly with a (2:4:2) stoichiometry. This model would neatly solve the problem posed by the unconventional orientation of p75NTR with respect to TrkA, as being found in the crystal structures of the TrkA/NGF and p75NTR/NGF complexes. PMID:25650935

  5. EBI2 augments Tfh cell fate by promoting interaction with IL-2-quenching dendritic cells.

    PubMed

    Li, Jianhua; Lu, Erick; Yi, Tangsheng; Cyster, Jason G

    2016-05-01

    T follicular helper (Tfh) cells are a subset of T cells carrying the CD4 antigen; they are important in supporting plasma cell and germinal centre responses. The initial induction of Tfh cell properties occurs within the first few days after activation by antigen recognition on dendritic cells, although how dendritic cells promote this cell-fate decision is not fully understood. Moreover, although Tfh cells are uniquely defined by expression of the follicle-homing receptor CXCR5 (refs 1, 2), the guidance receptor promoting the earlier localization of activated T cells at the interface of the B-cell follicle and T zone has been unclear. Here we show that the G-protein-coupled receptor EBI2 (GPR183) and its ligand 7α,25-dihydroxycholesterol mediate positioning of activated CD4 T cells at the interface of the follicle and T zone. In this location they interact with activated dendritic cells and are exposed to Tfh-cell-promoting inducible co-stimulator (ICOS) ligand. Interleukin-2 (IL-2) is a cytokine that has multiple influences on T-cell fate, including negative regulation of Tfh cell differentiation. We demonstrate that activated dendritic cells in the outer T zone further augment Tfh cell differentiation by producing membrane and soluble forms of CD25, the IL-2 receptor α-chain, and quenching T-cell-derived IL-2. Mice lacking EBI2 in T cells or CD25 in dendritic cells have reduced Tfh cells and mount defective T-cell-dependent plasma cell and germinal centre responses. These findings demonstrate that distinct niches within the lymphoid organ T zone support distinct cell fate decisions, and they establish a function for dendritic-cell-derived CD25 in controlling IL-2 availability and T-cell differentiation. PMID:27147029

  6. Fast and high-affinity binding of B-lymphotropic papovavirus to human B-lymphoma cell lines.

    PubMed Central

    Herrmann, M; Oppenländer, M; Pawlita, M

    1995-01-01

    Binding of B-lymphotropic papovavirus (LPV) to host cells differing in susceptibility to viral infection was determined by a newly established, direct, nonradioactive virus binding assay, which allows quantitative description of the binding characteristics by receptor saturation and Scatchard analysis. LPV binding to the highly susceptible human B-lymphoma cell line BJA-B K88 is specific, saturable, and noncooperative. Binding occurs very fast, with an association rate constant (k1) of 6.7 x 10(7) M-1s-1, and is of high affinity, with a dissociation constant (Kd) of 2.9 x 10(-12) M; and the virus-receptor complex is stable, with a half life of 70 min. The binding affinities of receptors on four other highly, moderately, or weakly susceptible human B-lymphoma cell lines were similar, with up to twofold variation around a mean Kd value of 3 x 10(-12) M, suggesting the presence of the same LPV receptor on all of these cell lines. This view is further supported by the finding that in all cases a terminal sialic acid is necessary for LPV binding. Tunicamycin has been shown to drastically induce LPV susceptibility and LPV binding in weakly and moderately susceptible B-lymphoma cell lines (O.T. Keppler, M. Herrmann, M. Oppenländer, W. Meschede, and M. Pawlita, J. Virol. 68:6933-6939, 1994). The hypothesis that the constitutively expressed and tunicamycin-induced LPV receptors are identical is strengthened by our finding that both receptor types displayed the same high affinity. LPV susceptibility of different B-lymphoma cell lines was correlated with receptor number but not with receptor affinity. The numbers of receptors per cell on highly and moderately susceptible cell lines ranged from 2,000 to 400 and were directly proportional to LPV susceptibility. This indicates that the number of high-affinity receptors per cell is a key regulating factor for the LPV host range. PMID:7474091

  7. High affinity IgM(+) memory B cells are generated through a germinal center-dependent pathway.

    PubMed

    Hara, Yasushi; Tashiro, Yasuyuki; Murakami, Akikazu; Nishimura, Miyuki; Shimizu, Takeyuki; Kubo, Masato; Burrows, Peter D; Azuma, Takachika

    2015-12-01

    During a T cell-dependent immune response, B cells undergo clonal expansion and selection and the induction of isotype switching and somatic hypermutation (SHM). Although somatically mutated IgM(+) memory B cells have been reported, it has not been established whether they are really high affinity B cells. We tracked (4-hydroxy-3-nitrophenyl) acetyl hapten-specific GC B cells from normal immunized mice based on affinity of their B cell receptor (BCR) and performed BCR sequence analysis. SHM was evident by day 7 postimmunization and increased with time, such that high affinity IgM(+) as well as IgG(+) memory B cells continued to be generated up to day 42. In contrast, class-switch recombination (CSR) was almost completed by day 7 and then the ratio of IgG1(+)/IgM(+) GC B cells remained unchanged. Together these findings suggest that IgM(+) B cells undergo SHM in the GC to generate high affinity IgM(+) memory cells and that this process continues even after CSR is accomplished. PMID:26514429

  8. [Peritumoral administered IL-2-induced tumor regression in melanoma. Pilot study].

    PubMed

    Gutwald, J; Groth, W; Mahrle, G

    1994-08-01

    Since the immune system plays an important role in the rejection of tumours, current tumour therapy includes immunostimulation. This can be done by interleukin 2 (IL-2), which activates T and killer cells and induces lysis of the tumour. Because the intravenous application of IL-2 may have serious side effects, we treated two patients with peritumoral injections in a pilot study. Both patients suffered from multiple cutaneous metastases of melanoma. A total of 31 and 39 x 10(6) IU recombinant IL-2 (Proleukin) respectively was injected in increasing concentrations in one metastasis of each patient. Histologically, almost complete necrosis of the tumour was induced. In comparison with the control specimen, the T-cell-rich infiltrate increased intra- and, in particular, peritumorally. While the ratio of helper to suppressor T cells remained unchanged, the proportions of NK cells, monocytes/macrophages and IL-2-receptor-bearing cells increased. This reaction was restricted to treated metastases. No clinical side effects or laboratory changes were registered apart from local erythema and swelling. PMID:7960753

  9. A novel high affinity human monoclonal antibody to mesothelin

    PubMed Central

    Ho, Mitchell; Feng, Mingqian; Fisher, Robert J.; Rader, Christoph; Pastan, Ira

    2010-01-01

    Mesothelin is a glycosylphosphatidylinisotol-anchored glycoprotein that is highly expressed on the cell surface of mesothelioma, ovarian cancer and other malignant tumors. The interaction between mesothelin and CA125 (also called MUC16) may facilitate the implantation and metastasis of tumors in the peritoneal cavity. A desirable therapeutic agent involves finding a fully human monoclonal antibody (mAb) that binds to mesothelin or CA125 and inhibits their interaction. Here we report the identification of a novel human mAb to mesothelin. HN1, a human single chain Fv specific for mesothelin, was isolated from a naïve human scFv phage display library. To investigate HN1 as a potential therapeutic, we generated a fully human IgG with the γ 1 heavy chain and the κ light chain, and an immuntoxin by fusing the HN1 scFv to a truncated Pseudomonas exotoxin A. The HN1 IgG kills cancer cells with very strong antibody-dependent cell-mediated cytotoxicity. HN1 binds a conformation-sensitive epitope in human mesothelin with high affinity (KD = 3 nM). The HN1 epitope is different from that of SS1, a mouse Fv used to develop therapeutic antibodies that are currently in clinical trials. HN1 binds to cell surface-associated mesothelin on human mesothelioma, ovarian cancer, lung adenocarcinoma and pancreatic cancer cells. In addition, HN1 can functionally block the interaction of mesothelin and CA125 on cancer cells. Most importantly, because the HN1 immuntoxin kills mesothelin-expressing cancer cells with high cytotoxic activity, we believe that it has significant potential for mesothelin-expressing cancer treatment and diagnosis. PMID:20635390

  10. IL2Rβ-dependent signals drive terminal exhaustion and suppress memory development during chronic viral infection.

    PubMed

    Beltra, Jean-Christophe; Bourbonnais, Sara; Bédard, Nathalie; Charpentier, Tania; Boulangé, Moana; Michaud, Eva; Boufaied, Ines; Bruneau, Julie; Shoukry, Naglaa H; Lamarre, Alain; Decaluwe, Hélène

    2016-09-13

    Exhaustion of CD8(+) T cells severely impedes the adaptive immune response to chronic viral infections. Despite major advances in our understanding of the molecular regulation of exhaustion, the cytokines that directly control this process during chronicity remain unknown. We demonstrate a direct impact of IL-2 and IL-15, two common gamma-chain-dependent cytokines, on CD8(+) T-cell exhaustion. Common to both cytokine receptors, the IL-2 receptor β (IL2Rβ) chain is selectively maintained on CD8(+) T cells during chronic lymphocytic choriomeningitis virus and hepatitis C virus infections. Its expression correlates with exhaustion severity and identifies terminally exhausted CD8(+) T cells both in mice and humans. Genetic ablation of the IL2Rβ chain on CD8(+) T cells restrains inhibitory receptor induction, in particular 2B4 and Tim-3; precludes terminal differentiation of highly defective PD-1(hi) effectors; and rescues memory T-cell development and responsiveness to IL-7-dependent signals. Together, we ascribe a previously unexpected role to IL-2 and IL-15 as instigators of CD8(+) T-cell exhaustion during chronic viral infection. PMID:27573835

  11. Intrathymic radioresistant stem cells follow an IL-2/IL-2R pathway during thymic regeneration after sublethal irradiation

    SciTech Connect

    Zuniga-Pfluecker, J.C.K.; Kruisbeek, A.M. )

    1990-05-15

    Sublethally irradiated mice undergo thymic regeneration which follows a phenotypic pattern of events similar to that observed during normal fetal development. Thymic regeneration after irradiation is the product of a limited pool of intrathymic radioresistant stem cells undergoing simultaneous differentiation. We show that in this model of T cell development, thymic regeneration follows a pathway in which the IL-2R is transiently expressed on CD4-/CD8- cells. IL-2R expression occurred during the exponential growth period of thymic regeneration, and IL-2R blocking prevented this explosive growth. Flow cytometry analysis revealed that the IL-2R blockade affected primarily the development of the immature CD3-/CD4-/CD8- (triple negative) cells and their ability to generate CD3+/CD4+/CD8+ or CD3+/CD4+/CD8- and CD3+/CD4-/CD8+ thymocytes. Thus, our findings demonstrate that blocking of the IL-2R resulted in an arrest in proliferation and differentiation by intrathymic radioresistant stem cells, indicating that the IL-2/IL-2R pathway is necessary for the expansion of immature triple negative T cells.

  12. In vivo Expansion of Naïve CD4+CD25high FOXP3+ Regulatory T Cells in Patients with Colorectal Carcinoma after IL-2 Administration

    PubMed Central

    Beyer, Marc; Schumak, Beatrix; Weihrauch, Martin R.; Andres, Bettina; Giese, Thomas; Endl, Elmar; Knolle, Percy A.; Classen, Sabine; Limmer, Andreas; Schultze, Joachim L.

    2012-01-01

    Regulatory T cells (Treg cells) are increased in context of malignancies and their expansion can be correlated with higher disease burden and decreased survival. Initially, interleukin 2 (IL-2) has been used as T-cell growth factor in clinical vaccination trials. In murine models, however, a role of IL-2 in development, differentiation, homeostasis, and function of Treg cells was established. In IL-2 treated cancer patients a further Treg-cell expansion was described, yet, the mechanism of expansion is still elusive. Here we report that functional Treg cells of a naïve phenotype - as determined by CCR7 and CD45RA expression - are significantly expanded in colorectal cancer patients. Treatment of 15 UICC stage IV colorectal cancer patients with IL-2 in a phase I/II peptide vaccination trial further enlarges the already increased naïve Treg-cell pool. Higher frequencies of T-cell receptor excision circles in naïve Treg cells indicate IL-2 dependent thymic generation of naïve Treg cells as a mechanism leading to increased frequencies of Treg cells post IL-2 treatment in cancer patients. This finding could be confirmed in naïve murine Treg cells after IL-2 administration. These results point to a more complex regulation of Treg cells in context of IL-2 administration. Future strategies therefore might aim at combining IL-2 therapy with novel strategies to circumvent expansion and differentiation of naïve Treg cells. PMID:22276195

  13. A 45-amino acid scaffold mined from the Protein Data Bank for high affinity ligand engineering

    PubMed Central

    Kruziki, Max A.; Bhatnagar, Sumit; Woldring, Daniel R.; Duong, Vandon T.; Hackel, Benjamin J.

    2015-01-01

    Summary Small protein ligands can provide superior physiological distribution versus antibodies and improved stability, production, and specific conjugation. Systematic evaluation of the Protein Data Bank identified a scaffold to push the limits of small size and robust evolution of stable, high-affinity ligands: 45-residue T7 phage gene 2 protein (Gp2) contains an α-helix opposite a β-sheet with two adjacent loops amenable to mutation. De novo ligand discovery from 108 mutants and directed evolution towards four targets yielded target-specific binders with affinities as strong as 200 ±100 pM, Tm’s from 65 ±3 °C to 80 ±1 °C, and retained activity after thermal denaturation. For cancer targeting, a Gp2 domain for epidermal growth factor receptor was evolved with 18 ±8 nM affinity, receptor-specific binding, and high thermal stability with refolding. The efficiency of evolving new binding function and the size, affinity, specificity, and stability of evolved domains render Gp2 a uniquely effective ligand scaffold. PMID:26165154

  14. High Affinity Binding of Indium and Ruthenium Ions by Gastrins

    PubMed Central

    Baldwin, Graham S.; George, Graham N.; Pushie, M. Jake

    2015-01-01

    The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated forms of the hormone. Since gastrins act as growth factors in gastrointestinal cancers, and as peptides labelled with Ga and In isotopes are increasingly used for cancer diagnosis, the ability of gastrins to bind other metal ions was investigated systematically by absorption spectroscopy. The coordination structures of the complexes were characterized by extended X-ray absorption fine structure (EXAFS) spectroscopy. Changes in the absorption of gastrin in the presence of increasing concentrations of Ga3+ were fitted by a 2 site model with dissociation constants (Kd) of 3.3 x 10−7 and 1.1 x 10−6 M. Although the absorption of gastrin did not change upon the addition of In3+ ions, the changes in absorbance on Fe3+ ion binding in the presence of indium ions were fitted by a 2 site model with Kd values for In3+ of 6.5 x 10−15 and 1.7 x 10−7 M. Similar results were obtained with Ru3+ ions, although the Kd values for Ru3+ of 2.6 x 10−13 and 1.2 x 10−5 M were slightly larger than observed for In3+. The structures determined by EXAFS all had metal:gastrin stoichiometries of 2:1 but, while the metal ions in the Fe, Ga and In complexes were bridged by a carboxylate and an oxygen with a metal-metal separation of 3.0–3.3 Å, the Ru complex clearly demonstrated a short range Ru—Ru separation, which was significantly shorter, at 2.4 Å, indicative of a metal-metal bond. We conclude that gastrin selectively binds two In3+ or Ru3+ ions, and that the affinity of the first site for In3+ or Ru3+ ions is higher than for ferric ions. Some of the metal ion-gastrin complexes may be useful for cancer diagnosis and therapy. PMID:26457677

  15. Diphtheria toxin-based recombinant murine IL-2 fusion toxin for depleting murine regulatory T cells in vivo.

    PubMed

    Wei, Min; Marino, Jose; Trowell, Aaron; Zhang, Huiping; Stromp Peraino, Jaclyn; Rajasekera, Priyani V; Madsen, Joren C; Sachs, David H; Huang, Christene A; Benichou, Gilles; Wang, Zhirui

    2014-09-01

    Regulatory T cells (Tregs) are a subpopulation of CD4(+) T cells which suppress immune responses of effector cells and are known to play a very important role in protection against autoimmune disease development, induction of transplantation tolerance and suppression of effective immune response against tumor cells. An effective in vivo Treg depletion agent would facilitate Treg-associated studies across many research areas. In this study, we have developed diphtheria toxin-based monovalent and bivalent murine IL-2 fusion toxins for depleting murine IL-2 receptor positive cells including CD25(+) Treg in vivo. Their potencies were assessed by in vitro protein synthesis inhibition and cell proliferation inhibition assays using a murine CD25(+) CTLL-2 cell line. Surprisingly, in contrast to our previously developed recombinant fusion toxins, the monovalent isoform (DT390-mIL-2) was approximately 4-fold more potent than its bivalent counterpart (DT390-bi-mIL-2). Binding analysis by flow cytometry demonstrated that the monovalent isoform bound stronger than the bivalent version. In vivo Treg depletion with the monovalent murine IL-2 fusion toxin was performed using C57BL/6J (B6) mice. Spleen Treg were significantly depleted with a maximum reduction of ∼70% and detectable as early as 12 h after the last injection. The spleen Treg numbers were reduced until Day 3 and returned to control levels by Day 7. We believe that this monovalent murine IL-2 fusion toxin will be an effective in vivo murine Treg depleter. PMID:25147093

  16. Biphasic competition between opiates and enkephalins: does it indicate the existence of a common high affinity (mu-1) binding site

    SciTech Connect

    Sarne, Y.; Kenner, A.

    1987-08-03

    Displacement from brain membranes of labeled opiates by low concentrations of enkephalins and of labeled enkephalins by low concentrations of opiates has been previously explained by the existance of a common high affinity site termed mu-1. An alternative interpretation of the same results is that the trough seen in the low concentration zone of the displacement curves represents cross binding of mu and delta opioid ligands to delta and mu receptors, respectively. In three sets of experiments with brain membranes, the size of the trough is shown to be dependent on the labeled ligand used: The ratio between the size of troughs seen with (TH)D-Ala, D-Leu enkephalin and with (TH)morphine varies with experimental conditions (storage of membranes at 4C for 72h), with ratio of mu:delta receptors (e.g. in thalamus and cortex which are enriched in mu and delta sites, respectively) and with pretreatment of membranes with naloxonazine. These results cannot be explained by a common high affinity site, but rather by binding of (TH)D-Ala, D-Leu enkephalin to mu and of (TH)morphine to delta opioid receptors. 17 references, 3 figures.

  17. High-Affinity Self-Reactive Human Antibodies by Design and Selection: Targeting the Integrin Ligand Binding Site

    NASA Astrophysics Data System (ADS)

    Barbas, Carlos F., III; Languino, Lucia R.; Smith, Jeffrey W.

    1993-11-01

    A strategy for the design and selection of human antibodies that bind receptors is described. We have demonstrated the validity of the approach by producing semisynthetic human antibodies that bind integrins α_vβ_3 and αIIbβ_3 with high affinity (10-10 M). The selected antibodies mimic the integrins' natural ligands as demonstrated by their ability to compete with these ligands and Arg-Gly-Asp (RGD)-containing peptides for binding to the integrins. Furthermore, the antibodies bind in a cation-dependent fashion and are functional in cell adhesion assays. Antibodies that are high-affinity inhibitors of cell adhesion receptors should be of use in assessing receptor function and dissecting mechanisms of adhesion. Semisynthetic human antibodies that target integrins are potential therapeutic agents for the treatment of a number of diseases including thrombosis and metastasis. Furthermore, antibodies that are optimized to bind by a single complementarity determining region may be important lead compounds for the design of small molecule pharmaceuticals.

  18. Phase I clinical trial combining imatinib mesylate and IL-2

    PubMed Central

    Chaput, Nathalie; Flament, Caroline; Locher, Clara; Desbois, Mélanie; Rey, Annie; Rusakiewicz, Sylvie; Poirier-Colame, Vichnou; Pautier, Patricia; Le Cesne, Axel; Soria, Jean-Charles; Paci, Angelo; Rosenzwajg, Michelle; Klatzmann, David; Eggermont, Alexander; Robert, Caroline; Zitvogel, Laurence

    2013-01-01

    We performed a Phase I clinical trial from October 2007 to October 2009, enrolling patients affected by refractory solid tumors, to determine the maximum tolerated dose (MTD) of interleukin (IL)-2 combined with low dose cyclophosphamide (CTX) and imatinib mesylate (IM). In a companion paper published in this issue of OncoImmunology, we show that the MTD of IL-2 is 6 MIU/day for 5 consecutive days, and that IL-2 increases the impregnation of both IM and of its main metabolite, CGP74588. Among the secondary objectives, we wanted to determine immunological markers that might be associated with progression-free survival (PFS) and/or overall survival (OS). The combination therapy markedly reduced the absolute counts of B, CD4+ T and CD8+ T cells in a manner that was proportional to IL-2 dose. There was a slight (less than 2-fold) increase in the proportion of regulatory T cells (Tregs) among CD4+ T cells in response to IM plus IL-2. The natural killer (NK)-cell compartment was activated, exhibiting a significant upregulation of HLA-DR, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and CD56. The abundance of HLA-DR+ NK cells after one course of combination therapy positively correlated with both PFS and OS. The IL-2-induced rise of the CD4+:CD8+ T-cell ratio calculated after the first cycle of treatment was also positively associated with OS. Overall, the combination of IM and IL-2 promoted the rapid expansion of HLA-DR+ NK cells and increased the CD4+:CD8+ T-cell ratio, both being associated with clinical benefits. This combinatorial regimen warrants further investigation in Phase II clinical trials, possibly in patients affected by gastrointestinal stromal tumors, a setting in which T and NK cells may play an important therapeutic role. PMID:23525357

  19. Differential effects of Denileukin Diftitox IL-2 immunotoxin on NK and regulatory T cells in non-human primates

    PubMed Central

    Yamada, Yohei; Aoyama, Akihiro; Tocco, Georges; Boskovic, Svjetlan; Nadazdin, Ognjenka; Alessandrini, Alessandro; Madsen, Joren C.; Cosimi, A. Benedict; Benichou, Gilles; Kawai, Tatsuo

    2012-01-01

    Denileukin Diftitox (DD), a fusion protein comprised of IL-2 and diphtheria toxin was initially expected to enhance anti-tumor immunity by selectively eliminating regulatory T cells (Tregs) displaying the high affinity IL-2R (α-β-γ trimers). While DD has been shown to deplete some Tregs in primates, its effects on NK cells (CD16+CD8+NKG2A+CD3−), which constitutively express the intermediate affinity IL-2R (β-γ dimers) and play a critical role in anti-tumor immunity, are still unknown. To address this question, cynomolgus monkeys were injected intravenously with two different doses of DD (8 or 18 μg/Kg). This treatment resulted in a rapid but short-term reduction in detectable peripheral blood resting Tregs (R-Tregs: CD4+CD45RA+Foxp3+) and a transient increase in the number of activated Tregs (A-Tregs: CD4+CD45RA−Foxp3high) followed by their partial depletion (50–60%). On the other hand, all NK cells were deleted immediately and durably after DD administration. This difference was not due to a higher binding or internalization of DD by NK cells as compared to Tregs. Co-administration of DD with IL-15, which binds to IL-2Rβ-γ, abrogated DD-induced NK cell deletion in vitro and in vivo while it did not affect Tregs elimination. Taken together, these results show that DD exerts a potent cytotoxic effect on NK cells, a phenomenon which might impair its anti-tumoral properties. However, co-administration of IL-15 with DD could alleviate this problem by selectively protecting potentially oncolytic NK cells while allowing the depletion of immunosuppressive regulatory T cells in cancer patients. PMID:22586034

  20. Differential effects of denileukin diftitox IL-2 immunotoxin on NK and regulatory T cells in nonhuman primates.

    PubMed

    Yamada, Yohei; Aoyama, Akihiro; Tocco, Georges; Boskovic, Svjetlan; Nadazdin, Ognjenka; Alessandrini, Alessandro; Madsen, Joren C; Cosimi, A Benedict; Benichou, Gilles; Kawai, Tatsuo

    2012-06-15

    Denileukin diftitox (DD), a fusion protein comprising IL-2 and diphtheria toxin, was initially expected to enhance antitumor immunity by selectively eliminating regulatory T cells (Tregs) displaying the high-affinity IL-2R (α-β-γ trimers). Although DD was shown to deplete some Tregs in primates, its effects on NK cells (CD16(+)CD8(+)NKG2A(+)CD3(-)), which constitutively express the intermediate-affinity IL-2R (β-γ dimers) and play a critical role in antitumor immunity, are still unknown. To address this question, cynomolgus monkeys were injected i.v. with two doses of DD (8 or 18 μg/kg). This treatment resulted in a rapid, but short-term, reduction in detectable peripheral blood resting Tregs (CD4(+)CD45RA(+)Foxp3(+)) and a transient increase in the number of activated Tregs (CD4(+)CD45RA(-)Foxp3(high)), followed by their partial depletion (50-60%). In contrast, all NK cells were deleted immediately and durably after DD administration. This difference was not due to a higher binding or internalization of DD by NK cells compared with Tregs. Coadministration of DD with IL-15, which binds to IL-2Rβ-γ, abrogated DD-induced NK cell deletion in vitro and in vivo, whereas it did not affect Treg elimination. Taken together, these results show that DD exerts a potent cytotoxic effect on NK cells, a phenomenon that might impair its antitumoral properties. However, coadministration of IL-15 with DD could alleviate this problem by selectively protecting potentially oncolytic NK cells, while allowing the depletion of immunosuppressive Tregs in cancer patients. PMID:22586034

  1. Photoaffinity labelling of high affinity dopamine binding proteins

    SciTech Connect

    Ross, G.M.; McCarry, B.E.; Mishra, R.K.

    1986-03-01

    A photoactive analogue of the dopamine agonist 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronapthalene (ADTN) has been synthesized and used to photoaffinity label dopamine binding proteins prepared from bovine caudate nucleus. N-(3-)N'-4-azidobenzamidol)-aminopropyl)-aminopropyl)-ADTN (AzB-AP-ADTN) was incubated with caudate membranes and irradiated with UV light. Membranes were then repeatedly washed by centrifugation to remove excess photolabel. A binding assay, using (/sup 3/H)-SCH 23390 (a D/sub 1/ specific antagonist), was then performed to evaluate the loss of receptor density in the photolyzed preparation. AzB-AP-ADTN irreversibly blocked (/sup 3/H)-SCH 23390 binding in a dose-dependent manner. Scatchard analysis revealed a decrease in the B/sub max/, with no significant change in the K/sub d/, of (/sup 3/H)-SCH 23390 binding. Compounds which compete for D/sub 1/ receptor binding (such as dopamine, SKF 38393 or apomorphine), proteted the SCH 23390 binding site from inactivation. This data would suggest that the novel photoaffinity ligand, AzB-AP-ADTN, can covalently label the D/sub 1/ (adenylate cyclase linked) dopamine receptor.

  2. Monochromatic multicomponent fluorescence sedimentation velocity for the study of high-affinity protein interactions

    PubMed Central

    Zhao, Huaying; Fu, Yan; Glasser, Carla; Andrade Alba, Eric J; Mayer, Mark L; Patterson, George; Schuck, Peter

    2016-01-01

    The dynamic assembly of multi-protein complexes underlies fundamental processes in cell biology. A mechanistic understanding of assemblies requires accurate measurement of their stoichiometry, affinity and cooperativity, and frequently consideration of multiple co-existing complexes. Sedimentation velocity analytical ultracentrifugation equipped with fluorescence detection (FDS-SV) allows the characterization of protein complexes free in solution with high size resolution, at concentrations in the nanomolar and picomolar range. Here, we extend the capabilities of FDS-SV with a single excitation wavelength from single-component to multi-component detection using photoswitchable fluorescent proteins (psFPs). We exploit their characteristic quantum yield of photo-switching to imprint spatio-temporal modulations onto the sedimentation signal that reveal different psFP-tagged protein components in the mixture. This novel approach facilitates studies of heterogeneous multi-protein complexes at orders of magnitude lower concentrations and for higher-affinity systems than previously possible. Using this technique we studied high-affinity interactions between the amino-terminal domains of GluA2 and GluA3 AMPA receptors. DOI: http://dx.doi.org/10.7554/eLife.17812.001 PMID:27436096

  3. Identification of high affinity folate binding proteins in human erythrocyte membranes.

    PubMed

    Antony, A C; Kincade, R S; Verma, R S; Krishnan, S R

    1987-09-01

    Mature human erythrocyte membranes contained specific, high affinity (Kd 3.3 X 10(-11) M) folate binding moieties. Folate binding was pH, time- and temperature-dependent, saturable, and was much greater for pteroylmonoglutamate and 5-methyltetrahydrofolate than 5-formyltetrahydrofolate and amethopterin. On detergent solubilization of membranes, two peaks of specific folate binding with Mr greater than or equal to 200,000 and 160,000 were identified on Sephacryl S-200 gel filtration chromatography in Triton X-100, and this corresponded to two similar peaks of immunoprecipitated material when solubilized iodinated membranes were probed with anti-human placental folate receptor antiserum. Age-dependent separation of erythrocytes by Stractan density gradients revealed a sevenfold greater folate binding capacity in membranes purified from younger compared with aged erythrocytes. Since this difference was not reflected in proportionately higher immunoreactive folate binding protein, (as determined by a specific radioimmunoassay for these proteins), or differences in affinity in younger than aged cells, these findings indicate that erythrocyte folate binding proteins become progressively nonfunctional at the onset of red cell aging. PMID:3624486

  4. Monochromatic multicomponent fluorescence sedimentation velocity for the study of high-affinity protein interactions.

    PubMed

    Zhao, Huaying; Fu, Yan; Glasser, Carla; Andrade Alba, Eric J; Mayer, Mark L; Patterson, George; Schuck, Peter

    2016-01-01

    The dynamic assembly of multi-protein complexes underlies fundamental processes in cell biology. A mechanistic understanding of assemblies requires accurate measurement of their stoichiometry, affinity and cooperativity, and frequently consideration of multiple co-existing complexes. Sedimentation velocity analytical ultracentrifugation equipped with fluorescence detection (FDS-SV) allows the characterization of protein complexes free in solution with high size resolution, at concentrations in the nanomolar and picomolar range. Here, we extend the capabilities of FDS-SV with a single excitation wavelength from single-component to multi-component detection using photoswitchable fluorescent proteins (psFPs). We exploit their characteristic quantum yield of photo-switching to imprint spatio-temporal modulations onto the sedimentation signal that reveal different psFP-tagged protein components in the mixture. This novel approach facilitates studies of heterogeneous multi-protein complexes at orders of magnitude lower concentrations and for higher-affinity systems than previously possible. Using this technique we studied high-affinity interactions between the amino-terminal domains of GluA2 and GluA3 AMPA receptors. PMID:27436096

  5. The presence of high-affinity, low-capacity estradiol-17β binding in rainbow trout scale indicates a possible endocrine route for the regulation of scale resorption

    USGS Publications Warehouse

    Persson, Petra; Shrimpton, J.M.; McCormick, S.D.; Bjornsson, Bjorn Thrandur

    2000-01-01

    High-affinity, low-capacity estradiol-17β (E2) binding is present in rainbow trout scale. The Kd and Bmax of the scale E2 binding are similar to those of the liver E2 receptor (Kd is 1.6 ± 0.1 and 1.4 ± 0.1 nM, and Bmax is 9.1 ± 1.2 and 23.1 ± 2.2 fmol x mg protein-1, for scale and liver, respectively), but different from those of the high-affinity, low-capacity E2 binding in plasma (Kd is 4.0 ± 0.4 nM and Bmax is 625.4 ± 63.1 fmol x mg protein-1). The E2 binding in scale was displaced by testosterone, but not by diethylstilbestrol. Hence, the ligand binding specificity is different from that of the previously characterized liver E2 receptor, where E2 is displaced by diethylstilbestrol, but not by testosterone. The putative scale E2 receptor thus appears to bind both E2 and testosterone, and it is proposed that the increased scale resorption observed during sexual maturation in both sexes of several salmonid species may be mediated by this receptor. No high-affinity, low-capacity E2 binding could be detected in rainbow trout gill or skin.

  6. Single-cell quantification of IL-2 response by effector and regulatory T cells reveals critical plasticity in immune response

    PubMed Central

    Feinerman, Ofer; Jentsch, Garrit; Tkach, Karen E; Coward, Jesse W; Hathorn, Matthew M; Sneddon, Michael W; Emonet, Thierry; Smith, Kendall A; Altan-Bonnet, Grégoire

    2010-01-01

    Understanding how the immune system decides between tolerance and activation by antigens requires addressing cytokine regulation as a highly dynamic process. We quantified the dynamics of interleukin-2 (IL-2) signaling in a population of T cells during an immune response by combining in silico modeling and single-cell measurements in vitro. We demonstrate that IL-2 receptor expression levels vary widely among T cells creating a large variability in the ability of the individual cells to consume, produce and participate in IL-2 signaling within the population. Our model reveals that at the population level, these heterogeneous cells are engaged in a tug-of-war for IL-2 between regulatory (Treg) and effector (Teff) T cells, whereby access to IL-2 can either increase the survival of Teff cells or the suppressive capacity of Treg cells. This tug-of-war is the mechanism enforcing, at the systems level, a core function of Treg cells, namely the specific suppression of survival signals for weakly activated Teff cells but not for strongly activated cells. Our integrated model yields quantitative, experimentally validated predictions for the manipulation of Treg suppression. PMID:21119631

  7. Nonresponsive lymphocytes from bone marrow transplant patients proliferate in vitro following exposure to rIL-2

    SciTech Connect

    Hank, J.A.; Sondel, P.M.; Flynn, B.; Hong, R.; Kohler, P.C.

    1986-03-01

    In vitro T-cell responses are depressed following bone marrow transplantation (BMT). Normal proliferative responses to mitogens, alloantigens and soluble antigens do not return for many months. Despite poor T-cell responses following EMT, peripheral blood lymphocytes respond readily to human rIL-2 as measured by /sup 3/H-thymidine incorporation. Lymphocytes from 8 patients have been studied following BMT. Six received whole marrow and 2 received marrow depleted of T-cells. While these patients were still unable to respond to mitogens, soluble antigens or in MLC following engraftment, their responses to rIL-2 were remarkable. These responses were often greater than the responses of the patients own cryopreserved remission cells, donor cells, or lymphocytes from healthy controls to either rIL-2 or antigens. Whether these IL-2 responsive cells are lymphocytes that have been activated in vivo by alloantigens, and thus express their IL-2 receptor, or are lymphokine activated killer (LAK) precursors, or are a primitive regenerating cell remains to be determined. Preliminary results indicate that these cells mediate nonspecific cytotoxicity.

  8. The effect of peripheral blood lymphocyte stimulation on zeta chain expression and IL-2 production in Hodgkin's disease

    PubMed Central

    Frydecka, I; Boćko, D; Kosmaczewska, A; Ciszak, L; Morilla, R

    2001-01-01

    It has been reported that peripheral blood T cells and NK cells express reduced levels of the T-cell receptor signal-transducing zeta chain in Hodgkin's disease (HD). The zeta chain has emerged as a key subunit of the T-cell antigen receptor, which plays a central role in the signal-transducing events leading to T and NK-cell activation. We were interested in determining whether the low zeta chain expression in HD could be corrected by anti-CD3, anti-CD3-rIL-2 ex vivo stimulation. Zeta chain expression was analysed by dual immunofluorescence on permeabilized cells before and after 72 hours of culture. The IL-2 concentration in the culture supernatants was measured by ELISA. Zeta chain was significantly reduced on unstimulated CD4+, CD8+ and CD56+ cells from patients in active disease compared with normal subjects. In patients in complete remission, the values were normal except for CD8+ cells, on which zeta expression remained significantly reduced. Stimulation with anti-CD3 did not change zeta expression. Co-stimulation with rIL-2 increased but did not normalize the proportions of CD4+/zeta+, CD8+/zeta+and CD56+/zeta+cells and IL-2 production in active disease. Stimulation of cells from patients in clinical remission with anti-CD3+rIL-2 increased the proportion of CD8+zeta+cells and normalized IL-2 production levels. Considering the pivotal role of CD3-zeta in immune response, our data suggest that successful immunotherapy approaches in active HD should consider inclusion of other potent cytokines, as well as genetically engineered tumour vaccines. © 2001 Cancer Research Campaign www.bjcancer.com PMID:11355944

  9. Identifying high-affinity aptamer ligands with defined cross-reactivity using high-throughput guided systematic evolution of ligands by exponential enrichment

    PubMed Central

    Levay, Agata; Brenneman, Randall; Hoinka, Jan; Sant, David; Cardone, Marco; Trinchieri, Giorgio; Przytycka, Teresa M.; Berezhnoy, Alexey

    2015-01-01

    Oligonucleotide aptamers represent a novel platform for creating ligands with desired specificity, and they offer many potentially significant advantages over monoclonal antibodies in terms of feasibility, cost, and clinical applicability. However, the isolation of high-affinity aptamer ligands from random oligonucleotide pools has been challenging. Although high-throughput sequencing (HTS) promises to significantly facilitate systematic evolution of ligands by exponential enrichment (SELEX) analysis, the enormous datasets generated in the process pose new challenges for identifying those rare, high-affinity aptamers present in a given pool. We show that emulsion PCR preserves library diversity, preventing the loss of rare high-affinity aptamers that are difficult to amplify. We also demonstrate the importance of using reference targets to eliminate binding candidates with reduced specificity. Using a combination of bioinformatics and functional analyses, we show that the rate of amplification is more predictive than prevalence with respect to binding affinity and that the mutational landscape within a cluster of related aptamers can guide the identification of high-affinity aptamer ligands. Finally, we demonstrate the power of this selection process for identifying cross-species aptamers that can bind human receptors and cross-react with their murine orthologs. PMID:26007661

  10. Reconstitution of high affinity. cap alpha. /sub 2/ adrenergic agonist binding by fusion with a pertussis toxin substrate

    SciTech Connect

    Kim, M.H.; Neubig, R.R.

    1986-03-05

    High affinity ..cap alpha../sub 2/ adrenergic agonist binding is thought to occur via a coupling of the ..cap alpha../sub 2/ receptor with N/sub i/, the inhibitory guanyl nucleotide binding protein. Human platelet membranes pretreated at pH 11.5 exhibit a selective inactivation of agonist binding and N/sub i/. To further study the mechanism of agonist binding, alkali treated membranes (ATM) were mixed with membranes pretreated with 10 ..mu..M phenoxybenzamine to block ..cap alpha../sub 2/ receptors (POB-M). The combined membrane pellet was incubated in 50% polyethylene glycol (PEG) to promote membrane-membrane fusion and assayed for binding to the ..cap alpha../sub 2/ agonist (/sup 3/H)UK 14,304 (UK) and the antagonist (/sup 3/H) yohimbine. PEG treatment resulted in a 2-4 fold enhancement of UK binding whereas yohimbine binding was unchanged. No enhancement of UK binding was observed in the absence of PEG treatment. The reconstitution was dependent on the addition of POB-M. They found that a 1:1 ratio of POB-M:ATM was optimal. Reconstituted binding was inhibited by GppNHp. Fusion of rat C6 glioma cell membranes, which do not contain ..cap alpha../sub 2/ receptors, also enhanced agonist binding to ATM. Fusion of C6 membranes from cells treated with pertussis toxin did not enhance (/sup 3/H) UK binding. These data show that a pertussis toxin sensitive membrane component, possibly N/sub i/, can reconstitute high affinity ..cap alpha../sub 2/ agonist binding.

  11. CM156, a high affinity sigma ligand, attenuates the stimulant and neurotoxic effects of methamphetamine in mice

    PubMed Central

    Kaushal, Nidhi; Seminerio, Michael J.; Shaikh, Jamaluddin; Medina, Mark A.; Mesangeau, Christophe; Wilson, Lisa L.; McCurdy, Christopher R.; Matsumoto, Rae R.

    2012-01-01

    Methamphetamine (METH) is a highly addictive psychostimulant drug of abuse. Low and high dose administration of METH leads to locomotor stimulation, and dopaminergic and serotonergic neurotoxicity, respectively. The behavioral stimulant and neurotoxic effects of METH can contribute to addiction and other neuropsychiatric disorders, thus necessitating the identification of potential pharmacotherapeutics against these effects produced by METH. METH binds to σ receptors at physiologically relevant concentrations. Also, σ receptors are present on and can modulate dopaminergic and serotonergic neurons. Therefore, σ receptors provide a viable target for the development of pharmacotherapeutics against the adverse effects of METH. In the present study, CM156, a σ receptor ligand with high affinity and selectivity for σ receptors over 80 other non-σ binding sites, was evaluated against METH-induced stimulant, hyperthermic, and neurotoxic effects. Pretreatment of male, Swiss Webster mice with CM156 dose dependently attenuated the locomotor stimulation, hyperthermia, striatal dopamine and serotonin depletions, and striatal dopamine and serotonin transporter reductions produced by METH, without significant effects of CM156 on its own. These results demonstrate the ability of a highly selective σ ligand to mitigate the effects of METH. PMID:21762711

  12. Heterogenetiy of glucocorticoid binders: a high affinity triamcinolone acetonide binder in bovine serum.

    PubMed

    Do, Y; Feldman, D

    1980-11-01

    While investigating glucocorticoid-binding proteins in bovine tissues, a new binder was found in fresh bovine serum which exhibited high affinity for certain synthetic glucocorticoids. This serum binder was characterized using [3H]triamcinolone acetonide (TA) as the ligand. On sucrose gradients, the [3H]TA peak sedimented at 8S, which was easily distinguishable from the [3H]cortisol-transcortin peak at 4S. Unlike the tissue receptor, which showed ionically dependent transformation from 8S in equilibrium or formed from 4S, the serum binder sedimented at 8S in both hypo- and hypertonic gradients. Binding properties were evaluated employing sephadex G-50 chromatography to separate bound from free steroid. Scatchard analysis of specific [3H]TA binding data revealed a straight line. The apparent equilibrium dissociation constant (Kd) was 7.8 +/- 0.7 X 10(-8) M, and the binding capacity was 772 +/- 70 fmol/mg serum protein. Hormonal specificity was determined by a competitive binding assay and revealed the following sequence: TA (100%) > betamethasone (47%) > triamcinolone (33%) > dexamethasone (2%) = cortisol = progesterone; aldosterone, estradiol, and testosterone exhibited negligible competitive activity. The serum binder was very stable, withstanding heating to 37 C for 60 min and long term storage in the frozen state. However, binding was significantly destroyed by trypsin. The binder was absent from fresh samples of chicken, mouse, rat, rabbit, dog, monkey, and human sera and frozen horse and porcine sera, but was clearly present in commercially available frozen calf, fetal calf, and lamb sera. At this time, we are unable to define the function of this binder, although its existence in both ovine and bovine sera suggests a possible role in ruminants. However, since bovine serum is routinely employed in tissue culture studies, the presence of this glucocorticoid binder might significantly influence many experiments. PMID:6775927

  13. Insulin Regulates the Activity of the High-Affinity Choline Transporter CHT.

    PubMed

    Fishwick, Katherine J; Rylett, R Jane

    2015-01-01

    Studies in humans and animal models show that neuronal insulin resistance increases the risk of developing Alzheimer's Disease (AD), and that insulin treatment may promote memory function. Cholinergic neurons play a critical role in cognitive and attentional processing and their dysfunction early in AD pathology may promote the progression of AD pathology. Synthesis and release of the neurotransmitter acetylcholine (ACh) is closely linked to the activity of the high-affinity choline transporter protein (CHT), but the impact of insulin receptor signaling and neuronal insulin resistance on these aspects of cholinergic function are unknown. In this study, we used differentiated SH-SY5Y cells stably-expressing CHT proteins to study the effect of insulin signaling on CHT activity and function. We find that choline uptake activity measured after acute addition of 20 nM insulin is significantly lower in cells that were grown for 24 h in media containing insulin compared to cells grown in the absence of insulin. This coincides with loss of ability to increase phospho-Protein Kinase B (PKB)/Akt levels in response to acute insulin stimulation in the chronic insulin-treated cells. Inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3-kinase) in cells significantly lowers phospho-PKB/Akt levels and decreases choline uptake activity. We show total internal reflection microscopy (TIRF) imaging of the dynamic movement of CHT proteins in live cells in response to depolarization and drug treatments. These data show that acute exposure of depolarized cells to insulin is coupled to transiently increased levels of CHT proteins at the cell surface, and that this is attenuated by chronic insulin exposure. Moreover, prolonged inhibition of PI3-kinase results in enhanced levels of CHT proteins at the cell surface by decreasing their rate of internalization. PMID:26161852

  14. High-affinity σ1 protein agonist reduces clinical and pathological signs of experimental autoimmune encephalomyelitis

    PubMed Central

    Oxombre, B; Lee-Chang, C; Duhamel, A; Toussaint, M; Giroux, M; Donnier-Maréchal, M; Carato, P; Lefranc, D; Zéphir, H; Prin, L; Melnyk, P; Vermersch, P

    2015-01-01

    Background and Purpose Selective agonists of the sigma-1 receptor (σ1 protein) are generally reported to protect against neuronal damage and modulate oligodendrocyte differentiation. Human and rodent lymphocytes possess saturable, high-affinity binding sites for compounds binding to the σ1 protein and potential immunomodulatory properties have been described for σ1 protein ligands. Experimental autoimmune encephalomyelitis (EAE) is recognized as a valuable model of the inflammatory aspects of multiple sclerosis (MS). Here, we have assessed the role of a σ1 protein agonist, containing the tetrahydroisoquinoline-hydantoin structure, in EAE. Experimental Approach EAE was induced in SJL/J female mice by active immunization with myelin proteolipid protein (PLP)139–151 peptide. The σ1 protein agonist was injected i.p. at the time of immunization (day 0). Disease severity was assessed clinically and by histopathological evaluation of the CNS. Phenotyping of B-cell subsets and regulatory T-cells were performed by flow cytometry in spleen and cervical lymph nodes. Key Results Prophylactic treatment of EAE mice with the σ1 protein agonist prevented mononuclear cell accumulation and demyelination in brain and spinal cord and increased T2 B-cells and regulatory T-cells, resulting in an overall reduction in the clinical progression of EAE. Conclusions and Implications This σ1 protein agonist, containing the tetrahydroisoquinoline-hydantoin structure, decreased the magnitude of inflammation in EAE. This effect was associated with increased proportions of B-cell subsets and regulatory T-cells with potential immunoregulatory functions. Targeting of the σ1 protein might thus provide new therapeutic opportunities in MS. PMID:25521311

  15. Insulin Regulates the Activity of the High-Affinity Choline Transporter CHT

    PubMed Central

    Fishwick, Katherine J.; Rylett, R. Jane

    2015-01-01

    Studies in humans and animal models show that neuronal insulin resistance increases the risk of developing Alzheimer’s Disease (AD), and that insulin treatment may promote memory function. Cholinergic neurons play a critical role in cognitive and attentional processing and their dysfunction early in AD pathology may promote the progression of AD pathology. Synthesis and release of the neurotransmitter acetylcholine (ACh) is closely linked to the activity of the high-affinity choline transporter protein (CHT), but the impact of insulin receptor signaling and neuronal insulin resistance on these aspects of cholinergic function are unknown. In this study, we used differentiated SH-SY5Y cells stably-expressing CHT proteins to study the effect of insulin signaling on CHT activity and function. We find that choline uptake activity measured after acute addition of 20 nM insulin is significantly lower in cells that were grown for 24 h in media containing insulin compared to cells grown in the absence of insulin. This coincides with loss of ability to increase phospho-Protein Kinase B (PKB)/Akt levels in response to acute insulin stimulation in the chronic insulin-treated cells. Inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3-kinase) in cells significantly lowers phospho-PKB/Akt levels and decreases choline uptake activity. We show total internal reflection microscopy (TIRF) imaging of the dynamic movement of CHT proteins in live cells in response to depolarization and drug treatments. These data show that acute exposure of depolarized cells to insulin is coupled to transiently increased levels of CHT proteins at the cell surface, and that this is attenuated by chronic insulin exposure. Moreover, prolonged inhibition of PI3-kinase results in enhanced levels of CHT proteins at the cell surface by decreasing their rate of internalization. PMID:26161852

  16. Synthesis and biological activity of a novel series of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate inhibitors of purine biosynthesis with selectivity for high affinity folate receptors and the proton-coupled folate transporter over the reduced folate carrier for cellular entry†

    PubMed Central

    Wang, Lei; Cherian, Christina; Desmoulin, Sita Kugel; Polin, Lisa; Deng, Yijun; Wu, Jianmei; Hou, Zhanjun; White, Kathryn; Kushner, Juiwanna; Matherly, Larry H.; Gangjee, Aleem

    2010-01-01

    2-Amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidines with a thienoyl side chain and 4-6 carbon bridge lengths (compounds 1-3) were synthesized as substrates for folate receptors (FRs) and the proton-coupled folate transporter (PCFT). Conversion of acetylene carboxylic acids to α-bromomethylketones and condensation with 2,4-diamino-6-hydroxypyrimidine afforded the 6-substituted pyrrolo[2,3-d]pyrimidines. Sonogashira coupling with (S)-2-[(5-bromo-thiophene-2-carbonyl)-amino]-pentanedioic acid diethyl ester, followed by hydrogenation and saponification, afforded 1-3. Compounds 1 and 2 potently inhibited KB and IGROV1 human tumor cells that express FRα, reduced folate carrier (RFC), and PCFT. The analogs were selective for FR- and PCFT over RFC. Glycinamide ribonucleotide formyltransferase was the principal cellular target. In SCID mice with KB tumors, 1 was highly active against both early (3.5 log kill, 1/5 cures) and advanced (3.7 log kill, 4/5 complete remissions) stage tumors. Our results demonstrate potent in vitro and in vivo antitumor activity for 1 due to selective transport by FRs and PCFT over RFC. PMID:20085328

  17. Corticotropin-releasing factor antagonist blocks microwave-induced decreases in high-affinity choline uptake in the rat brain

    SciTech Connect

    Lai, H.; Carino, M.A.; Horita, A.; Guy, A.W. )

    1990-10-01

    Acute (45-min) irradiation with pulsed low-level microwaves (2450-MHz, 2 microseconds pulses at 500 pps, average power density of 1 mW/cm2, whole-body average specific absorption rate of 0.6 W/kg) decreased sodium-dependent high-affinity choline uptake (HACU) activity in the frontal cortex and hippocampus of the rat. These effects were blocked by pretreating the animals before exposure with intracerebroventricular injection of the specific corticotropin-releasing factor (CRF) receptor antagonist, alpha-helical-CRF9-41 (25 micrograms). Similar injection of the antagonist had no significant effect on HACU in the brain of the sham-exposed rats. These data suggest that low-level microwave irradiation activates CRF in the brain, which in turn causes the changes in central HACU.

  18. Characterization of high affinity (/sup 3/H)triazolam binding in rat brain

    SciTech Connect

    Earle, M.; Concas, A.; Yamamura, H.I.

    1986-03-01

    The hypnotic Triazolam (TZ), a triazolo (1,4)-benzodiazepine, displays a short physiological half life and has been used for the treatment of insomnia related to anxiety states. Specific binding properties of this recently tritiated TZ were characterized. The authors major objectives were the direct measurement of the temperature dependence and the GABA effect on (/sup 3/H)TZ binding. Saturation studies showed a shift to lower affinity at 37/sup 0/C (K/sub d/ = 0.25 +/- 0.01 nM at O/sup 0/C; K/sub d/ = 1.46 +/- 0.03 nM at 37/sup 0/C) while the B/sub max/ values remained unchanged (1003 +/- 37 fmoles/mg prot. at 0/sup 0/C and 1001 +/- 43 fmoles/mg prot. at 37/sup 0/C). Inhibition studies showed that (/sup 3/H)TZ binding displayed no GABA shift at 0/sup 0/C(K/sub i/ 0.37 +/- 0.03 nM/- GABA and K/sub i/ = 0.55 +/- 0.13 nM/+GABA) but a nearly two-fold shift was apparent at 37/sup 0/C (K/sub i/ = 2.92 +/- 0.2 nM/-GABA; K/sub i/ = 1.37 +/- 0.11 mM/+GABA). These results were also confirmed by saturation studies in the presence or absence of GABA showing a shift to higher affinity in the presence of GABA only at 37/sup 0/C. In Ro 15-1788/(/sup 3/H)TZ competition experiments the presence of GABA did not affect the inhibitory potency of Ro 15-1788 on (/sup 3/H)TZ binding at both temperatures. In conclusion (/sup 3/H)TZ binding showed an extremely high affinity for benzodiazepine receptors. In contrast to reported literature, the findings suggest that TZ interacts with benzodiazepine receptors similar to other benzodiazepine agonists.

  19. INVOLVEMENT OF KRAS G12A MUTATION IN THE IL-2-INDEPENDENT GROWTH OF A HUMAN T-LGL LEUKEMIA CELL LINE, PLT-2

    PubMed Central

    MIZUTANI, NAOKI; ITO, HIROMI; HAGIWARA, KAZUMI; KOBAYASHI, MISA; HOSHIKAWA, ASUKA; NISHIDA, YAYOI; TAKAGI, AKIRA; KOJIMA, TETSUHITO; SUZUKI, MOTOSHI; OSAWA, YOSUKE; OHNISHI, KAZUNORI; DAIBATA, MASANORI; MURATE, TAKASHI

    2012-01-01

    ABSTRACT Cytokine-dependent cell lines have been used to analyze the cytokine-induced cellular signaling and the mechanism of oncogenesis. In the current study, we analyzed MOTN-1 and PLT-2 cell lines established from different stages of a T-cell large granular lymphocyte leukemia patient (Daibata et al. 2004). MOTN-1 is IL-2-dependent derived from the chronic phase, whereas IL-2-independent PLT-2 is from the aggressive and terminal stage. They shared considerable chromosome abnormalities and the pattern of T-cell receptor rearrangement, presuming that the cytokine independence of PLT-2 was due to the additive genetic abnormality. Besides IL-2, IL-15 supported MOTN-1 cell growth, because these receptors share β- and γ-subunits. IL-2 activated ERK, AKT and STAT pathway of MOTN-1. STAT3 pathway of PLT-2 was also activated by IL-2, suggesting intact IL-2 induces signal transduction of PLT-2. However, ERK1/2 but not AKT, was continuously activated in PLT-2, consistent with the increased Ras-activity of PLT-2. Sequence analysis revealed KRAS G12A mutation but not NRAS and HRAS mutation of PLT-2 but not MOTN-1. Another signaling molecule affecting Ras-signaling pathway, SHP2, which has been frequently mutated in juvenile myelomonocytic leukemia (JMML), did not show mutation. Moreover, MEK inhibitor, PD98059, as well as farnesylation inhibitor inhibited PLT-2 cell growth. Using NIH3T3 and MOTN-1, ERK activation, increased cell proliferation and survival by KRAS G12A were shown, suggesting the important role of KRAS G12A in IL-2-independent growth of PLT-2. Taken together, KRAS G12A is important for IL-2-independent growth of PLT-2 cells and suggests the possibility of involvement of KRAS mutation with disease progression. PMID:23092099

  20. IL-21 signalling via STAT3 primes human naive B cells to respond to IL-2 to enhance their differentiation into plasmablasts.

    PubMed

    Berglund, Lucinda J; Avery, Danielle T; Ma, Cindy S; Moens, Leen; Deenick, Elissa K; Bustamante, Jacinta; Boisson-Dupuis, Stephanie; Wong, Melanie; Adelstein, Stephen; Arkwright, Peter D; Bacchetta, Rosa; Bezrodnik, Liliana; Dadi, Harjit; Roifman, Chaim M; Fulcher, David A; Ziegler, John B; Smart, Joanne M; Kobayashi, Masao; Picard, Capucine; Durandy, Anne; Cook, Matthew C; Casanova, Jean-Laurent; Uzel, Gulbu; Tangye, Stuart G

    2013-12-01

    B-cell responses are guided by the integration of signals through the B-cell receptor (BCR), CD40, and cytokine receptors. The common γ chain (γc)-binding cytokine interleukin (IL)-21 drives humoral immune responses via STAT3-dependent induction of transcription factors required for plasma cell generation. We investigated additional mechanisms by which IL-21/STAT3 signaling modulates human B-cell responses by studying patients with STAT3 mutations. IL-21 strongly induced CD25 (IL-2Rα) in normal, but not STAT3-deficient, CD40L-stimulated naïve B cells. Chromatin immunoprecipitation confirmed IL2RA as a direct target of STAT3. IL-21-induced CD25 expression was also impaired on B cells from patients with IL2RG or IL21R mutations, confirming a requirement for intact IL-21R signaling in this process. IL-2 increased plasmablast generation and immunoglobulin secretion from normal, but not CD25-deficient, naïve B cells stimulated with CD40L/IL-21. IL-2 and IL-21 were produced by T follicular helper cells, and neutralizing both cytokines abolished the B-cell helper capacity of these cells. Our results demonstrate that IL-21, via STAT3, sensitizes B cells to the stimulatory effects of IL-2. Thus, IL-2 may play an adjunctive role in IL-21-induced B-cell differentiation. Lack of this secondary effect of IL-21 may amplify the humoral immunodeficiency in patients with mutations in STAT3, IL2RG, or IL21R due to impaired responsiveness to IL-21. PMID:24159173

  1. A β-hairpin structure in a 13-mer peptide that binds α-bungarotoxin with high affinity and neutralizes its toxicity

    PubMed Central

    Scherf, Tali; Kasher, Roni; Balass, Moshe; Fridkin, Mati; Fuchs, Sara; Katchalski-Katzir, Ephraim

    2001-01-01

    Snake-venom α-bungarotoxin is a member of the α-neurotoxin family that binds with very high affinity to the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction. The structure of the complex between α-bungarotoxin and a 13-mer peptide (WRYYESSLEPYPD) that binds the toxin with high affinity, thus inhibiting its interactions with AChR with an IC50 of 2 nM, has been solved by 1H-NMR spectroscopy. The bound peptide folds into a β-hairpin structure created by two antiparallel β-strands, which combine with the already existing triple-stranded β-sheet of the toxin to form a five-stranded intermolecular, antiparallel β-sheet. Peptide residues Y3P, E5P, and L8P have the highest intermolecular contact area, indicating their importance in the binding of α-bungarotoxin; W1P, R2P, and Y4P also contribute significantly to the binding. A large number of characteristic hydrogen bonds and electrostatic and hydrophobic interactions are observed in the complex. The high-affinity peptide exhibits inhibitory potency that is better than any known peptide derived from AChR, and is equal to that of the whole α-subunit of AChR. The high degree of sequence similarity between the peptide and various types of AChRs implies that the binding mode found within the complex might possibly mimic the receptor binding to the toxin. The design of the high-affinity peptide was based on our previous findings: (i) the detection of a lead peptide (MRYYESSLKSYPD) that binds α-bungarotoxin, using a phage-display peptide library, (ii) the information about the three-dimensional structure of α-bungarotoxin/lead-peptide complex, and (iii) the amino acid sequence analysis of different AChRs. PMID:11381118

  2. Architecture of high-affinity unnatural-base DNA aptamers toward pharmaceutical applications

    PubMed Central

    Matsunaga, Ken-ichiro; Kimoto, Michiko; Hanson, Charlotte; Sanford, Michael; Young, Howard A.; Hirao, Ichiro

    2015-01-01

    We present a remodeling method for high-affinity unnatural-base DNA aptamers to augment their thermal stability and nuclease resistance, for use as drug candidates targeting specific proteins. Introducing a unique mini-hairpin DNA provides robust stability to unnatural-base DNA aptamers generated by SELEX using genetic alphabet expansion, without reducing their high affinity. By this method, >80% of the remodeled DNA aptamer targeting interferon-γ (KD of 33 pM) survived in human serum at 37 °C after 3 days under our experimental conditions, and sustainably inhibited the biological activity of interferon-γ. PMID:26690672

  3. Architecture of high-affinity unnatural-base DNA aptamers toward pharmaceutical applications.

    PubMed

    Matsunaga, Ken-ichiro; Kimoto, Michiko; Hanson, Charlotte; Sanford, Michael; Young, Howard A; Hirao, Ichiro

    2015-01-01

    We present a remodeling method for high-affinity unnatural-base DNA aptamers to augment their thermal stability and nuclease resistance, for use as drug candidates targeting specific proteins. Introducing a unique mini-hairpin DNA provides robust stability to unnatural-base DNA aptamers generated by SELEX using genetic alphabet expansion, without reducing their high affinity. By this method, >80% of the remodeled DNA aptamer targeting interferon-γ (KD of 33 pM) survived in human serum at 37 °C after 3 days under our experimental conditions, and sustainably inhibited the biological activity of interferon-γ. PMID:26690672

  4. In vitro ozone exposure inhibits mitogen-induced lymphocyte proliferation and IL-2 production

    SciTech Connect

    Becker, S.; Jordan, R.L.; Orlando, G.S.; Koren, H.S.

    1989-01-01

    Human blood mononuclear cells were exposed to ozone in vitro and thereafter analyzed for competence in mitogen-induced proliferation as well as IL-1 and IL-2 production. Proliferative responses induced by phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM) were all depressed in lymphocytes exposed to an ozone concentration of 1 ppm for 4-6 h. The response to PWM was most sensitive to the ozone effect (38% suppression); responses to Con A and PHA were suppressed to a lesser extent, 23% and 18%, respectively, and were not significantly different from each other. PWM responses were affected at an ozone concentration as low as 0.1 ppm; however, no suppression of Con A-induced proliferation was seen below 0.18 ppm or of PHA-induced proliferation below 0.5 ppm. When lymphocytes and monocytes were exposed separately to ozone and then mixed back with control air-exposed monocytes or lymphocytes, both cell types appeared to be affected and the functional defects caused by the pollutant were additive. Monocyte IL-1 production induced by endotoxin was not affected by ozone exposure, while surface expression of HLA-DR on exposed monocytes was reduced by 40% 24 h after exposure. Moreover, lymphocytes exposed to ozone produced 46% less IL-2 while expressing similar surface density of IL-2 receptors. Taken together, these results show that exposure to ozone has distinct adverse effects on lymphocytes and monocytes, both of which are important in local immune defenses in the lung.

  5. Failure of the three compartment model to describe the pharmacokinetics in brain of a high affinity substituted benzamide.

    PubMed

    Votaw, J R; Kessler, R M; de Paulis, T

    1993-11-01

    The applicability of using the standard 3-compartment model to describe the neuropharmacokinetics of a high affinity substituted benzamide was investigated. We performed the following experiments using the [18F]-5-(3-fluoropropyl) analog of epidepride ([18F]5-FPrEpid), a potent dopamine D2 receptor antagonist: constant left ventricular infusion, first-pass clearance, varying ligand specific activity, and displacing bound ligand with varying amounts of unlabelled ligand. Taken together, the information from these experiments rigorously tests the standard 3-compartment model. The obtained data and predictions from the model of the kinetic behavior of the ligand are inconsistent. The measured and model predicted dissociation rate (measured koff = 0.065 min-1, model prediction koff = 0.007 min-1) and the equilibrium dissociation constant (measured KD = 0.14 nM, model prediction KD = 2.2 nM) differ by an order of magnitude. Furthermore, the model cannot be used to accurately estimate the receptor density. We postulate that the synapse geometry and physical relationship between receptors are necessary components of a model that describes the pharmacokinetics of [18F]5-FPrEpid. PMID:8278896

  6. The presence of high-affinity, low-capacity estradiol-17β binding in rainbow trout scale indicates a possible endocrine route for the regulation of scale resorption

    USGS Publications Warehouse

    Persson, Petra; Shrimpton, J. Mark; McCormick, Stephen D.; Bjornsson, Bjorn Thrandur

    2000-01-01

    High-affinity, low-capacity estradiol-17β (E2) binding is present in rainbow trout scale. The Kd and Bmax of the scale E2 binding are similar to those of the liver E2 receptor (Kd is 1.6 ± 0.1 and 1.4 ± 0.1 nM, and Bmax is 9.1 ± 1.2 and 23.1 ± 2.2 fmol × mg protein-1, for scale and liver, respectively), but different from those of the high-affinity, low-capacity E2 binding in plasma (Kd is 4.0 ± 0.4 nM and Bmax is 625.4 ± 63.1 fmol × mg protein−1). The E2 binding in scale was displaced by testosterone, but not by diethylstilbestrol. Hence, the ligand binding specificity is different from that of the previously characterized liver E2 receptor, where E2 is displaced by diethylstilbestrol, but not by testosterone. The putative scale E2 receptor thus appears to bind both E2 and testosterone, and it is proposed that the increased scale resorption observed during sexual maturation in both sexes of several salmonid species may be mediated by this receptor. No high-affinity, low-capacity E2 binding could be detected in rainbow trout gill or skin.

  7. High-affinity immobilization of proteins using biotin- and GST-based coupling strategies.

    PubMed

    Hutsell, Stephanie Q; Kimple, Randall J; Siderovski, David P; Willard, Francis S; Kimple, Adam J

    2010-01-01

    Surface plasmon resonance (SPR) is a highly sensitive method for the detection of molecular interactions. One interacting partner is immobilized on the sensor chip surface while the other is injected across the sensor surface. This chapter focuses on high-affinity immobilization of protein substrates for affinity and kinetic analyses using biotin/streptavidin interaction and GST/anti-GST-antibody interaction. PMID:20217614

  8. SORPTION OF LEAD ON A HIGH AFFINITY OXIDE: MACROSCOPIC AND MICROSCOPIC STUDIES

    EPA Science Inventory

    Sorption of lead (Pb) was investigated on an innovative metal oxide compound using macroscopic and microscopic techniques. The objective of this study was to elucidate the sorption mechanism of Pb on the high-affinity engineered oxide with time at pH 6 employing batch methods an...

  9. SORPTION OF LEAD ON A HIGH AFFINITY OXIDE: MACROSCOPIC AND MICROSCOPIC STUDIES (ABSTRACT)

    EPA Science Inventory

    Sorption of lead (Pb) was investigated on an innovative metal oxide compound using macroscopic and microscopic techniques. The objective of this study was to elucidate the sorption mechanism of Pb on the high-affinity engineered oxide with time at pH 6 employing batch methods an...

  10. Substantial high-affinity methanotroph populations in Andisols effect high rates of atmospheric methane oxidation.

    PubMed

    Maxfield, Pete J; Hornibrook, Ed R C; Evershed, Richard P

    2009-10-01

    Methanotrophic bacteria in soils derived from volcanic ash (Andisols) were characterized via time series (13) C-phospholipid fatty acid (PLFA) labelling. Three Andisols were incubated under 2 ppmv (13) CH4 for up to 18 weeks, thus enabling high-affinity methanotrophs to be selectively characterized and quantified. PLFA profiles from all soils were broadly similar, but the magnitude of the high-affinity methanotrophic populations determined through (13) C-PLFA-stable isotope probing displayed sizeable differences. Substantial incorporation of (13) C indicated very large high-affinity methanotrophic populations in two of the soils. Such high values are far in excess (10×) of those observed for a range of mineral soils incubated under similar conditions (Bull et al., 2000; Maxfield et al., 2006; 2008a, b). Two of the three Andisols studied also displayed high but variable CH4 oxidation rates ranging from 0.03 to 1.58 nmol CH4 g(-1) d.wt. h(-1) . These findings suggest that Andisols, a previously unstudied soil class with respect to high-affinity methanotrophic bacteria, may oxidize significant amounts of atmospheric methane despite their low areal coverage globally. PMID:23765899

  11. 4,5-Disubstituted oxazolidinones: High affinity molecular effectors of RNA function.

    PubMed

    Anupam, Rajaneesh; Nayek, Abhijit; Green, Nicholas J; Grundy, Frank J; Henkin, Tina M; Means, John A; Bergmeier, Stephen C; Hines, Jennifer V

    2008-06-15

    The T box transcription antitermination system is a riboswitch found primarily in Gram-positive bacteria which monitors the aminoacylation of the cognate tRNA and regulates a variety of amino acid-related genes. Novel 4,5-disubstituted oxazolidinones were identified as high affinity RNA molecular effectors that modulate the transcription antitermination function of the T box riboswitch. PMID:18502126

  12. High affinity immobilization of proteins using biotin- and GST-based coupling strategies

    PubMed Central

    Hutsell, Stephanie Q.; Kimple, Randall J.; Siderovski, David P.; Willard, Francis S.; Kimple, Adam J.

    2011-01-01

    Surface Plasmon Resonance (SPR) is a highly sensitive method for the detection of molecular interactions. One interacting partner is immobilized on the sensor chip surface while the other is injected across the sensor surface. This chapter focuses on high affinity immobilization of protein substrates for affinity and kinetic analyses using biotin/streptavidin interaction and GST/anti-GST-antibody interaction. PMID:20217614

  13. High Affinity Iron Permease is Required for Virulence of Rhizopus oryzae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rhizopus oryzae is the most common cause of mucormycosis. Clinical and animal model data clearly demonstrate that the presence of elevated available serum iron predisposes the host to develop mucormycosis. The high affinity iron permease gene (rFTR1) is required for R. oryzae iron transport in iro...

  14. Radioligand binding assays for high affinity binders in the presence of endogenous ligands

    SciTech Connect

    White, H.B. III; McGahan, T.

    1986-05-01

    Endogenous ligands complicate radioligand-binding assays of high-affinity binding proteins by obscuring binding sites or by diluting the labeled ligand. They have developed a mathematical model for such systems where structurally identical radioligand and endogenous ligand can be equilibrated on the binding site and bound radioligand measured. A double-reciprocal plot of bound radioligand, *L/sub B/, versus sample volume, V, yields a straight line. Introduction of scaling factors for sample dilution, F, and total radioligand available, *L/sub T/, produces a plot in which the x-intercept yields the endogenous ligand concentration, (L/sub T/); the slope is the reciprocal of the binding protein concentration, (P/sub T/)/sup -1/; and the y-intercept is the fractional saturation of the high-affinity binder, L/sub T//P/sub T/. This type of analysis has been applied to the assay of high-affinity biotin-binding proteins in egg yolk. Its use led to the detection of a second biotin-binding protein which is heat labile. The conceptual approach can be applied to the assay of other high-affinity binders.

  15. High affinity group III mGluRs regulate mossy fiber input to CA3 interneurons

    PubMed Central

    Cosgrove, Kathleen E.; Meriney, Stephen D.; Barrionuevo, Germán

    2010-01-01

    Stratum lacunosum-moleculare interneurons (L-Mi) in hippocampal area CA3 target the apical dendrite of pyramidal cells providing feedforward inhibition. Here we report that selective activation of group III metabotropic glutamate receptors (mGluRs) 4/8 with L-(+)-2-amino-4-phosphnobytyric acid (L-AP4; 10 μM) decreased the probability of glutamate release from the mossy fiber (MF) terminals synapsing onto L-Mi. Consistent with this interpretation, application of L-AP4 in the presence of 3 mM strontium decreased the frequency of asynchronous MF EPSCs in L-Mi. Furthermore, the dose response curve showed that L-AP4 at 400 μM produced no further decrease in MF EPSC amplitude compared to 20 μM L-AP4, indicating the lack of mGluRs 7 at these MF terminals. We also found that one mechanism of mGluRs 4/8-mediated inhibition of release is linked to N-type voltage gated calcium channels at MF terminals. Application of the group III mGluR antagonist MSOP (100 μM) demonstrated that mGluRs 4/8 are neither tonically active nor activated by low and moderate frequencies of activity. However, trains of stimuli to the MF at 20 and 40Hz delivered during the application of MSOP revealed a relief of inhibition of transmitter release and an increase in the overall probability of action potential firing in the postsynaptic L-Mi. Interestingly, the time to first action potential was significantly shorter in the presence of MSOP, indicating that mGluR 4/8 activation delays L-Mi firing in response to MF activity. Taken together, our data demonstrate that the timing and probability of action potentials in L-Mi evoked by MF synaptic input is regulated by the activation of presynaptic high affinity group III mGluRs. PMID:20824730

  16. High Affinity Binders to EphA2 Isolated from Abdurin Scaffold Libraries; Characterization, Binding and Tumor Targeting

    PubMed Central

    Ullman, Christopher; Mathonet, Pascale; Oleksy, Arkadiusz; Diamandakis, Agata; Tomei, Licia; Demartis, Anna; Nardi, Chiara; Sambucini, Sonia; Missineo, Antonino; Alt, Karen; Hagemeyer, Christoph E.; Harris, Matt; Hedt, Amos; Weis, Roland; Gehlsen, Kurt R.

    2015-01-01

    Abdurins are a novel antibody-like scaffold derived from the engineering of a single isolated CH2 domain of human IgG. Previous studies established the prolonged serum half-life of Abdurins, the result of a retained FcRn binding motif. Here we present data on the construction of large, diverse, phage-display and cell-free DNA display libraries and the isolation of high affinity binders to the cancer target, membrane-bound ephrin receptor tyrosine kinase class A2 (EphA2). Antigen binding regions were created by designing combinatorial libraries into the structural loops and Abdurins were selected using phage display methods. Initial binders were reformatted into new maturation libraries and low nanomolar binders were isolated using cell-free DNA display, CIS display. Further characterization confirmed binding of the Abdurins to both human and murine EphA2 proteins and exclusively to cell lines that expressed EphA2, followed by rapid internalization. Two different EphA2 binders were labeled with 64Cu, using a bifunctional MeCOSar chelator, and administered to mice bearing tumors from transplanted human prostate cancer cells, followed by PET/CT imaging. The anti-EphA2 Abdurins localized in the tumors as early as 4 hours after injection and continued to accumulate up to 48 hours when the imaging was completed. These data demonstrate the ability to isolate high affinity binders from the engineered Abdurin scaffold, which retain a long serum half-life, and specifically target tumors in a xenograft model. PMID:26313909

  17. Immunotherapy of cancer using local administration of lymphoid cells transformed by IL-2 cDNA and constitutively producing IL-2.

    PubMed

    Bubeník, J; Símová, J; Jandlová, T

    1990-02-01

    Peritumoral administration of X63-m-IL-2 cells transformed by IL-2 cDNA and constitutively producing large quantities of recombinant IL-2 mediated regression of X63-Ag8.653 plasmocytoma and MC14 sarcoma transplanted in syngeneic mice. Injections of the IL-2-producing cells containing an inserted, modified IL-2 gene effectively inhibited tumour growth also in allogeneic recipients. Activation of murine spleen cells in vitro by co-cultivation with X63-m-IL-2 cells gave rise to lymphokine-activated killer (LAK) cell populations cytotoxic for the X63-Ag8.653 plasmocytoma and MC14 sarcoma. The results suggest that peritumoral administration of lymphoid cells transformed with IL-2 cDNA and constitutively producing IL-2 in the immediate tumour vicinity is sufficient for the effective activation of local IL-2-dependent tumour defence mechanisms and, therefore, can be considered a novel, genetic approach to the immunotherapy of cancer. PMID:2347603

  18. Further characterization of the low and high affinity binding components of the thyrotropin receptor

    SciTech Connect

    McQuade, R.; Thomas, C.G. Jr.; Nayfeh, S.N.

    1986-05-29

    Following cross-linking with disuccinimdiyl suberate and analysis by SDS-PAGE and autoradiography, both the high- and low-affinity TSH binding components exhibited two similar /sup 125/I-TSH-labeled bands, with Mr values of 80,000 and 68,000. IgG fractions from patients with Graves' disease inhibited /sup 125/I-TSH binding to both components, while normal IgG had no effect. Although not entirely conclusive, these results suggest that the high- and low-affinity components share similar subunit composition and antigenic determinants.

  19. Neuroendocrine changes in colon of mice with a disrupted IL-2 gene.

    PubMed

    Qian, B F; El-Salhy, M; Melgar, S; Hammarström, M L; Danielsson, A

    2000-06-01

    Neuroendocrine peptides have a variety of physiological functions in the gastrointestinal tract. This study was carried out to investigate the impact of IL-2 deficiency on the neuroendocrine system in normal colon, and the neuroendocrine changes during colonic inflammation. Mice with homozygous disrupted IL-2 gene (IL-2-/-) spontaneously developed a bowel disease with similarities to human ulcerative colitis. Different types of colonic endocrine cells and myenteric nerves were analysed in the IL-2-/- mice using immunomorphometry. The neuropeptide contents in the colonic tissues were determined by radioimmunoassay. Age-matched healthy IL-2+/- and IL-2+/+ mice served as controls and the colonic IL-2 levels were compared between these two groups of mice by ELISA. Our data showed that less than half the amount of IL-2 was synthesized in the colon of IL-2+/- mice compared with the IL-2+/+ wild-type mice. Two major differences in the neuroendocrine colon were found between the mice with an intact and disrupted IL-2 gene. One was age-related. The frequencies of various endocrine cells and myenteric nerves increased with age in the IL-2+/+ mice. However, no such increases were seen in the mice with a disrupted IL-2 gene. Instead, the volume densities of enteroglucagon, serotonin cells and substance P (SP), vasoactive intestinal polypeptide (VIP) and total myenteric nerves were lower in the older IL-2+/- and IL-2-/- mice compared with the wild type. The other was disease-related. Polypeptide YY (PYY) cells and tissue levels of PYY, SP and VIP were significantly decreased in the IL-2-/- mice during the course of bowel inflammation compared with the healthy IL-2+/- and IL-2+/+ controls. These findings indicate that colonic neuroendocrine alterations did occur in the mice with a disrupted IL-2 gene and diminished local IL-2 level, suggesting a role of IL-2 in the regulation of the neuroendocrine system and a prevalent interaction between the immune and neuroendocrine systems

  20. The shared and contrasting roles of interleukin-2 (IL-2) and IL-15 in the life and death of normal and neoplastic lymphocytes: implications for cancer therapy

    PubMed Central

    Waldmann, Thomas A.

    2015-01-01

    Interleukin-2 (IL2) and IL15, members of the 4α-helix bundle family of cytokines, play pivotal roles in the control of the life and death of lymphocytes. Although their heterotrimeric receptors have two receptor subunits in common these two cytokines have contrasting roles in adaptive immune responses. The unique role of IL2 through maintenance of fitness of regulatory T cells (Treg) and activation-induced cell death (AICD) is the elimination of self-reactive T cells to prevent autoimmunity. In contrast to IL2, IL15 is dedicated to the prolonged maintenance of memory T-cell responses to invading pathogens. Blockade of IL2 and IL15 using monoclonal antibodies has been reported to be of value in the treatment of patients with leukemia, autoimmune disorders and in the prevention of allograft rejection. IL2 has been approved by the FDA for the treatment of patients with malignant renal cell cancer and metastatic malignant melanoma. Clinical trials involving recombinant human IL15 given by bolus infusions have been completed, and by subcutaneous and continuous intravenous infusions are underway in patients with metastatic malignancy. Furthermore, clinical trials are being initiated that employ the combination of IL15 with IL15Rα+/− IgFc. PMID:25736261

  1. High-affinity FRβ-specific CAR T cells eradicate AML and normal myeloid lineage without HSC toxicity.

    PubMed

    Lynn, R C; Feng, Y; Schutsky, K; Poussin, M; Kalota, A; Dimitrov, D S; Powell, D J

    2016-06-01

    Acute myeloid leukemia (AML) is an aggressive malignancy, and development of new treatments to prolong remissions is warranted. Chimeric antigen receptor (CAR) T-cell therapies appear promising but on-target, off-tumor recognition of antigen in healthy tissues remains a concern. Here we isolated a high-affinity (HA) folate receptor beta (FRβ)-specific single-chain variable fragment (2.48 nm KD) for optimization of FRβ-redirected CAR T-cell therapy for AML. T cells stably expressing the HA-FRβ CAR exhibited greatly enhanced antitumor activity against FRβ(+) AML in vitro and in vivo compared with a low-affinity FRβ CAR (54.3 nm KD). Using the HA-FRβ immunoglobulin G, FRβ expression was detectable in myeloid-lineage hematopoietic cells; however, expression in CD34(+) hematopoietic stem cells (HSCs) was nearly undetectable. Accordingly, HA-FRβ CAR T cells lysed mature CD14(+) monocytes, while HSC colony formation was unaffected. Because of the potential for elimination of mature myeloid lineage, mRNA CAR electroporation for transient CAR expression was evaluated. mRNA-electroporated HA-FRβ CAR T cells retained effective antitumor activity in vitro and in vivo. Together, our results highlight the importance of antibody affinity in target protein detection and CAR development and suggest that transient delivery of potent HA-FRβ CAR T cells is highly effective against AML and reduces the risk for long-term myeloid toxicity. PMID:26898190

  2. Structural determinants in phycotoxins and AChBP conferring high affinity binding and nicotinic AChR antagonism

    PubMed Central

    Bourne, Yves; Radić, Zoran; Aráoz, Rómulo; Talley, Todd T.; Benoit, Evelyne; Servent, Denis; Taylor, Palmer; Molgó, Jordi; Marchot, Pascale

    2010-01-01

    Spirolide and gymnodimine macrocyclic imine phycotoxins belong to an emerging class of chemical agents associated with marine algal blooms and shellfish toxicity. Analysis of 13-desmethyl spirolide C and gymnodimine A by binding and voltage-clamp recordings on muscle-type α12βγδ and neuronal α3β2 and α4β2 nicotinic acetylcholine receptors reveals subnanomolar affinities, potent antagonism, and limited subtype selectivity. Their binding to acetylcholine-binding proteins (AChBP), as soluble receptor surrogates, exhibits picomolar affinities governed by diffusion-limited association and slow dissociation, accounting for apparent irreversibility. Crystal structures of the phycotoxins bound to Aplysia-AChBP (≈2.4Å) show toxins neatly imbedded within the nest of ar-omatic side chains contributed by loops C and F on opposing faces of the subunit interface, and which in physiological conditions accommodates acetylcholine. The structures also point to three major features: (i) the sequence-conserved loop C envelops the bound toxins to maximize surface complementarity; (ii) hydrogen bonding of the protonated imine nitrogen in the toxins with the carbonyl oxygen of loop C Trp147 tethers the toxin core centered within the pocket; and (iii) the spirolide bis-spiroacetal or gymnodimine tetrahydrofuran and their common cyclohexene-butyrolactone further anchor the toxins in apical and membrane directions, along the subunit interface. In contrast, the se-quence-variable loop F only sparingly contributes contact points to preserve the broad receptor subtype recognition unique to phycotoxins compared with other nicotinic antagonists. These data offer unique means for detecting spiroimine toxins in shellfish and identify distinctive ligands, functional determinants and binding regions for the design of new drugs able to target several receptor subtypes with high affinity. PMID:20224036

  3. Structural determinants in phycotoxins and AChBP conferring high affinity binding and nicotinic AChR antagonism.

    PubMed

    Bourne, Yves; Radic, Zoran; Aráoz, Rómulo; Talley, Todd T; Benoit, Evelyne; Servent, Denis; Taylor, Palmer; Molgó, Jordi; Marchot, Pascale

    2010-03-30

    Spirolide and gymnodimine macrocyclic imine phycotoxins belong to an emerging class of chemical agents associated with marine algal blooms and shellfish toxicity. Analysis of 13-desmethyl spirolide C and gymnodimine A by binding and voltage-clamp recordings on muscle-type alpha1(2)betagammadelta and neuronal alpha3beta2 and alpha4beta2 nicotinic acetylcholine receptors reveals subnanomolar affinities, potent antagonism, and limited subtype selectivity. Their binding to acetylcholine-binding proteins (AChBP), as soluble receptor surrogates, exhibits picomolar affinities governed by diffusion-limited association and slow dissociation, accounting for apparent irreversibility. Crystal structures of the phycotoxins bound to Aplysia-AChBP ( approximately 2.4A) show toxins neatly imbedded within the nest of ar-omatic side chains contributed by loops C and F on opposing faces of the subunit interface, and which in physiological conditions accommodates acetylcholine. The structures also point to three major features: (i) the sequence-conserved loop C envelops the bound toxins to maximize surface complementarity; (ii) hydrogen bonding of the protonated imine nitrogen in the toxins with the carbonyl oxygen of loop C Trp147 tethers the toxin core centered within the pocket; and (iii) the spirolide bis-spiroacetal or gymnodimine tetrahydrofuran and their common cyclohexene-butyrolactone further anchor the toxins in apical and membrane directions, along the subunit interface. In contrast, the se-quence-variable loop F only sparingly contributes contact points to preserve the broad receptor subtype recognition unique to phycotoxins compared with other nicotinic antagonists. These data offer unique means for detecting spiroimine toxins in shellfish and identify distinctive ligands, functional determinants and binding regions for the design of new drugs able to target several receptor subtypes with high affinity. PMID:20224036

  4. An amino acid residue in the second extracellular loop determines the agonist-dependent tolerance property of the human D3 dopamine receptor.

    PubMed

    Gil-Mast, Sara; Kortagere, Sandhya; Kota, Kokila; Kuzhikandathil, Eldo V

    2013-06-19

    The D3 dopamine receptor is a therapeutic target for treating various nervous system disorders such as schizophrenia, Parkinson's disease, depression, and addictive behaviors. The crystal structure of the D3 receptor bound to an antagonist was recently described; however, the structural features that contribute to agonist-induced conformational changes and signaling properties are not well understood. We have previously described the conformation-dependent tolerance and slow response termination (SRT) signaling properties of the D3 receptor and identified the C147 residue in the second intracellular loop (IL2) of the D3 receptor as important for the tolerance property. Interestingly, while IL2 and the C147 residue, in particular, were important for dopamine- and quinpirole-induced tolerance, this residue did not affect the severe tolerance induced by the high affinity, D3 receptor-selective agonist, PD128907. Here, we used D2/D3 receptor chimeras and site-specific D3 receptor mutants to identify another residue, D187, in the second extracellular loop (EC2) of the human D3 receptor that mediates the tolerance property induced by PD128907, quinpirole, pramipexole, and dopamine. Molecular dynamics simulations confirmed the distinct conformation adopted by D3 receptor during tolerance and suggested that in the tolerant D3 receptor the D187 residue in EC2 forms a salt bridge with the H354 residue in EC3. Indeed, site-directed mutation of the H354 residue resulted in loss of PD1287907-induced tolerance. The mapping of specific amino acid residues that contribute to agonist-dependent conformation changes and D3 receptor signaling properties refines the agonist-bound D3 receptor pharmacophore model which will help develop novel D3 receptor agonists. PMID:23477444

  5. Inhibition of Gαs/cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4+ T Helper Cells

    PubMed Central

    Hynes, Thomas R.; Yost, Evan A.; Yost, Stacy M.; Hartle, Cassandra M.; Ott, Braden J.

    2015-01-01

    Background: The role of cAMP in regulating T cell activation and function has been controversial. cAMP is generally known as an immunosuppressant, but it is also required for generating optimal immune responses. As the effect of cAMP is likely to depend on its cellular context, the current study investigated whether the mechanism of activation of Gαs and adenylyl cyclase influences their effect on T cell receptor (TCR)-stimulated interleukin-2 (IL-2) mRNA levels. Methods: The effect of blocking Gs-coupled receptor (GsPCR)-mediated Gs activation on TCR-stimulated IL-2 mRNA levels in CD4+ T cells was compared with that of knocking down Gαs expression or inhibiting adenylyl cyclase activity. The effect of knocking down Gαs expression on TCR-stimulated cAMP accumulation was compared with that of blocking GsPCR signaling. Results: ZM-241385, an antagonist to the Gs-coupled A2A adenosine receptor (A2AR), enhanced TCR-stimulated IL-2 mRNA levels in primary human CD4+ T helper cells and in Jurkat T cells. A dominant negative Gαs construct, GαsDN3, also enhanced TCR-stimulated IL-2 mRNA levels. Similar to GsPCR antagonists, GαsDN3 blocked GsPCR-dependent activation of both Gαs and Gβγ. In contrast, Gαs siRNA and 2’,5’-dideoxyadenosine (ddA), an adenylyl cyclase inhibitor, decreased TCR-stimulated IL-2 mRNA levels. Gαs siRNA, but not GαsDN3, decreased TCR-stimulated cAMP synthesis. Potentiation of IL-2 mRNA levels by ZM-241385 required at least two days of TCR stimulation, and addition of ddA after three days of TCR stimulation enhanced IL-2 mRNA levels. Conclusions: GsPCRs play an inhibitory role in the regulation of TCR-stimulated IL-2 mRNA levels whereas Gαs and cAMP can play a stimulatory one. Additionally, TCR-dependent activation of Gαs does not appear to involve GsPCRs. These results suggest that the context of Gαs/cAMP activation and the stage of T cell activation and differentiation determine the effect on TCR-stimulated IL-2 mRNA levels. PMID

  6. Effect of A-63162 on lymphocyte proliferation, IL-2 production, mRNA for Il-2 and LTB sub 4 production from human mononuclear cells

    SciTech Connect

    Atluru, D.; Atluru, S. ); Woloschak, G.E. )

    1991-03-15

    Lipoxygenase metabolites of arachidonic acid have diversified effects on human lymphocytes. In the present investigation, the authors measured the effects of A-63162, a specific 5-lipoxygenase inhibitor on lymphocyte proliferation, IL-2 production, mRNA for IL-2 and LTB{sub 4} synthesis from peripheral blood human mononuclear cells. A-63162 inhibited the {sup 3}H-thymidine incorporation from PHA or PMA plus A23187 stimulated cultures. The synthesis of IL-2 was also inhibited from PHA plus PMA or PMA plus A23187 stimulated cultures. Further, they found A-63162 also inhibited the accumulation of IL-2 mRNA. And, A-63162 at that above concentration also inhibited the LTB{sub 4} synthesis from A23187 stimulated cultures. The results suggest that endogenous LTB{sub 4} may play an important role in regulating IL-2 production at the message level.

  7. Three Recombinant Engineered Antibodies against Recombinant Tags with High Affinity and Specificity

    PubMed Central

    Zhao, Hongyu; Shen, Ao; Xiang, Yang K.; Corey, David P.

    2016-01-01

    We describe three recombinant engineered antibodies against three recombinant epitope tags, constructed with divalent binding arms to recognize divalent epitopes and so achieve high affinity and specificity. In two versions, an epitope is inserted in tandem into a protein of interest, and a homodimeric antibody is constructed by fusing a high-affinity epitope-binding domain to a human or mouse Fc domain. In a third, a heterodimeric antibody is constructed by fusing two different epitope-binding domains which target two different binding sites in GFP, to polarized Fc fragments. These antibody/epitope pairs have affinities in the low picomolar range and are useful tools for many antibody-based applications. PMID:26943906

  8. RNA mango aptamer-fluorophore: a bright, high-affinity complex for RNA labeling and tracking.

    PubMed

    Dolgosheina, Elena V; Jeng, Sunny C Y; Panchapakesan, Shanker Shyam S; Cojocaru, Razvan; Chen, Patrick S K; Wilson, Peter D; Hawkins, Nancy; Wiggins, Paul A; Unrau, Peter J

    2014-10-17

    Because RNA lacks strong intrinsic fluorescence, it has proven challenging to track RNA molecules in real time. To address this problem and to allow the purification of fluorescently tagged RNA complexes, we have selected a high affinity RNA aptamer called RNA Mango. This aptamer binds a series of thiazole orange (fluorophore) derivatives with nanomolar affinity, while increasing fluorophore fluorescence by up to 1,100-fold. Visualization of RNA Mango by single-molecule fluorescence microscopy, together with injection and imaging of RNA Mango/fluorophore complex in C. elegans gonads demonstrates the potential for live-cell RNA imaging with this system. By inserting RNA Mango into a stem loop of the bacterial 6S RNA and biotinylating the fluorophore, we demonstrate that the aptamer can be used to simultaneously fluorescently label and purify biologically important RNAs. The high affinity and fluorescent properties of RNA Mango are therefore expected to simplify the study of RNA complexes. PMID:25101481

  9. Novel Ubiquitin-derived High Affinity Binding Proteins with Tumor Targeting Properties*

    PubMed Central

    Lorey, Susan; Fiedler, Erik; Kunert, Anja; Nerkamp, Jörg; Lange, Christian; Fiedler, Markus; Bosse-Doenecke, Eva; Meysing, Maren; Gloser, Manja; Rundfeldt, Chris; Rauchhaus, Una; Hänssgen, Ilka; Göttler, Thomas; Steuernagel, Arnd; Fiedler, Ulrike; Haupts, Ulrich

    2014-01-01

    Targeting effector molecules to tumor cells is a promising mode of action for cancer therapy and diagnostics. Binding proteins with high affinity and specificity for a tumor target that carry effector molecules such as toxins, cytokines, or radiolabels to their intended site of action are required for these applications. In order to yield high tumor accumulation while maintaining low levels in healthy tissues and blood, the half-life of such conjugates needs to be in an optimal range. Scaffold-based binding molecules are small proteins with high affinity and short systemic circulation. Due to their low molecular complexity, they are well suited for combination with effector molecules as well as half-life extension technologies yielding therapeutics with half-lives adapted to the specific therapy. We have identified ubiquitin as an ideal scaffold protein due to its outstanding biophysical and biochemical properties. Based on a dimeric ubiquitin library, high affinity and specific binding molecules, so-called Affilin® molecules, have been selected against the extradomain B of fibronectin, a target almost exclusively expressed in tumor tissues. Extradomain B-binding molecules feature high thermal and serum stability as well as strong in vitro target binding and in vivo tumor accumulation. Application of several half-life extension technologies results in molecules of largely unaffected affinity but significantly prolonged in vivo half-life and tumor retention. Our results demonstrate the utility of ubiquitin as a scaffold for the generation of high affinity binders in a modular fashion, which can be combined with effector molecules and half-life extension technologies. PMID:24474690

  10. High-affinity triplex-forming oligonucleotide target sequences in mammalian genomes.

    PubMed

    Wu, Qi; Gaddis, Sara S; MacLeod, Michael C; Walborg, Earl F; Thames, Howard D; DiGiovanni, John; Vasquez, Karen M

    2007-01-01

    Site-specific recognition of duplex DNA by triplex-forming oligonucleotides (TFOs) provides a promising approach to manipulate mammalian genomes. A prerequisite for successful gene targeting using this approach is that the targeted gene must contain specific, high-affinity TFO target sequences (TTS). To date, TTS have been identified and characterized in only approximately 37 human or rodent genes, limiting the application of triplex-directed gene targeting. We searched the complete human and mouse genomes using an algorithm designed to identify high-affinity TTS. The resulting data set contains 1.9 million potential TTS for each species. We found that 97.8% of known human and 95.2% of known mouse genes have at least one potential high-affinity TTS in the promoter and/or transcribed gene regions. Importantly, 86.5% of known human and 83% of the known mouse genes have at least one TTS that is unique to that gene. Thus, it is possible to target the majority of human and mouse genes with specific TFOs. We found substantially more potential TTS in the promoter sequences than in the transcribed gene sequences or intergenic sequences in both genomes. We selected 12 mouse genes and 2 human genes critical for cell signaling, proliferation, and/or carcinogenesis, identified potential TTS in each, and determined TFO binding affinities to these sites in vitro. We identified at least one high-affinity, specific TFO binding site within each of these genes. Using this information, many genes involved in mammalian cell proliferation and carcinogenesis can now be targeted. PMID:17013831

  11. Low and high affinity dopamine transporter inhibitors block dopamine uptake within 5 sec of intravenous injection

    PubMed Central

    Yorgason, Jordan T.; Jones, Sara R.; España, Rodrigo A.

    2011-01-01

    Extensive evidence suggests that the reinforcing effects of cocaine involve inhibition of dopamine transporters (DAT) and subsequent increases in dopamine (DA) levels in the striatum. We have previously reported that cocaine inhibits the DAT within 4–5 sec of intravenous injection, matching the temporal profile of the behavioral and subjective effects of cocaine. Intravenous injection of GBR-12909, a high affinity, long-acting DAT inhibitor, also inhibits DA uptake within 5 sec. Given that high affinity, long-acting drugs are considered to have relatively low abuse potential, we found it intriguing that GBR-12909 had an onset profile similar to that of cocaine. To further explore the onset kinetics of both low and high affinity DAT inhibitors, we examined the effects of intravenous cocaine (1.5 mg/kg), methylphenidate (1.5 mg/kg), nomifensine (1.5 mg/kg), GBR-12909 (1.5 mg/kg), PTT (0.5 mg/kg), and WF23 (0.5 mg/kg) on electrically-evoked DA release and uptake in the nucleus accumbens core. Results indicate that all of the DAT inhibitors significantly inhibited DA uptake within 5 sec of injection. However, the timing of peak uptake inhibition varied greatly between the low and high affinity uptake inhibitors. Uptake inhibition following cocaine, methylphenidate, and nomifensine peaked 30 sec following injection. In contrast, peak effects for GBR-12909, PTT, and WF23 occurred between 20 and 60 min following injection. These observations suggest that the initial onset for intravenous DAT inhibitors is extremely rapid and does not appear to be dictated by a drug’s affinity. PMID:21402130

  12. Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging

    PubMed Central

    Maute, Roy L.; Gordon, Sydney R.; Mayer, Aaron T.; McCracken, Melissa N.; Natarajan, Arutselvan; Ring, Nan Guo; Kimura, Richard; Tsai, Jonathan M.; Manglik, Aashish; Kruse, Andrew C.; Gambhir, Sanjiv S.; Weissman, Irving L.; Ring, Aaron M.

    2015-01-01

    Signaling through the immune checkpoint programmed cell death protein-1 (PD-1) enables tumor progression by dampening antitumor immune responses. Therapeutic blockade of the signaling axis between PD-1 and its ligand programmed cell death ligand-1 (PD-L1) with monoclonal antibodies has shown remarkable clinical success in the treatment of cancer. However, antibodies have inherent limitations that can curtail their efficacy in this setting, including poor tissue/tumor penetrance and detrimental Fc-effector functions that deplete immune cells. To determine if PD-1:PD-L1–directed immunotherapy could be improved with smaller, nonantibody therapeutics, we used directed evolution by yeast-surface display to engineer the PD-1 ectodomain as a high-affinity (110 pM) competitive antagonist of PD-L1. In contrast to anti–PD-L1 monoclonal antibodies, high-affinity PD-1 demonstrated superior tumor penetration without inducing depletion of peripheral effector T cells. Consistent with these advantages, in syngeneic CT26 tumor models, high-affinity PD-1 was effective in treating both small (50 mm3) and large tumors (150 mm3), whereas the activity of anti–PD-L1 antibodies was completely abrogated against large tumors. Furthermore, we found that high-affinity PD-1 could be radiolabeled and applied as a PET imaging tracer to efficiently distinguish between PD-L1–positive and PD-L1–negative tumors in living mice, providing an alternative to invasive biopsy and histological analysis. These results thus highlight the favorable pharmacology of small, nonantibody therapeutics for enhanced cancer immunotherapy and immune diagnostics. PMID:26604307

  13. Selective high-affinity polydentate ligands and methods of making such

    DOEpatents

    DeNardo, Sally; DeNardo, Gerald; Balhorn, Rodney

    2013-09-17

    This invention provides polydentate selective high affinity ligands (SHALs) that can be used in a variety of applications in a manner analogous to the use of antibodies. SHALs typically comprise a multiplicity of ligands that each binds different regions on the target molecule. The ligands are joined directly or through a linker thereby forming a polydentate moiety that typically binds the target molecule with high selectivity and avidity.

  14. EF5 Is the High-Affinity Mg(2+) Site in ALG-2.

    PubMed

    Tanner, John J; Frey, Benjamin B; Pemberton, Travis; Henzl, Michael T

    2016-09-13

    The penta-EF-hand (PEF) protein ALG-2 (apoptosis-linked gene 2) has been implicated in several important physiological processes, including endoplasmic reticulum-Golgi vesicular transport and endosomal biogenesis/transport. ALG-2 was recently shown to harbor a metal ion-binding site with a high affinity for Mg(2+) and a low affinity for Ca(2+). We herein present the X-ray structure of Mg(2+)-bound ALG-2des23(wt). Although the C(α) trace is nearly indistinguishable from that of the Ca(2+)-free protein, the orientation of the C-terminal helix differs in the two structures. Consistent with that observation, replacement of the +x ligand in EF5, D169, with alanine eliminates high-affinity Mg(2+) binding. It also eliminates the low-affinity Ca(2+) site and lowers the affinity of the remaining Ca(2+)-binding sites, EF3 and EF1. The coordination environment in EF5 approaches ideal Mg(2+) octahedral geometry. The ligand array, consisting of three carboxylates (+x, +y, +z), a backbone carbonyl (-y), and two water molecules (-x, -z), may offer a recipe for a high-affinity, high-selectivity Mg(2+)-binding site. Sequence data for other PEF proteins indicate that select calpain large subunits, notably CAPN1 and CAPN8, may also possess a high-affinity Mg(2+)-binding site. In Mg(2+)-bound ALG-2, the carbonyl of F188 and the C-terminal carboxylate of V191 interact with the ε-ammonium group of K137 in the opposing subunit, suggesting that Mg(2+) binding could have an impact on dimerization. Interestingly, EF1 and EF3 are also occupied in the crystal, despite having modest affinity for Mg(2+). The results of a calorimetry-based analysis indicate that their Mg(2+) binding constants are 2 orders of magnitude lower than that determined for EF5. PMID:27541325

  15. Development of a high-affinity GABA uptake system in embryonic amphibian spinal neurons.

    PubMed

    Lamborghini, J E; Iles, A

    1985-11-01

    High-affinity uptake systems for amino acid neurotransmitter precursors have been highly correlated with the use of the particular amino acid or its derivative as a transmitter. We have found interneurons in the Xenopus embryo spinal cord which accumulate GABA by a high-affinity uptake system. They originate near the end of gastrulation and their ability to accumulate GABA first appears at the early tail bud stage. By position and appearance they are comparable to some of the embryonic interneurons described by A. Roberts and J. D. W. Clarke (1982, Phil. Trans. R. Soc. London Ser. B 296, 195-212). GABA-accumulating neurons also develop in dissociated cell cultures made from the presumptive spinal cord of neural plate stage Xenopus embryos. GABA accumulation in cultured neurons, as in cells in vivo, occurs via a high-affinity uptake system; GABA-accumulating cells have the same time of origin as the cells in vivo, and the ability to accumulate GABA in the population of cultured neurons appears at a time equivalent to that observed in intact sibling embryos. Thus it seems likely that the population of GABA-accumulating neurons developing in cell culture corresponds to the GABA-accumulating interneurons in vivo. The development of these neurons in dissociated cell cultures permits perturbation experiments that would be difficult to perform in vivo. We have examined the development of high-affinity GABA uptake in conditions that permit no electrical impulse activity in the cultures. The onset and extent of development of GABA accumulation in the neuronal population are normal under these conditions. PMID:3932109

  16. Brain immune interactions and air pollution: macrophage inhibitory factor (MIF), prion cellular protein (PrPC), Interleukin-6 (IL-6), interleukin 1 receptor antagonist (IL-1Ra), and interleukin-2 (IL-2) in cerebrospinal fluid and MIF in serum differentiate urban children exposed to severe vs. low air pollution

    PubMed Central

    Calderón-Garcidueñas, Lilian; Cross, Janet V.; Franco-Lira, Maricela; Aragón-Flores, Mariana; Kavanaugh, Michael; Torres-Jardón, Ricardo; Chao, Chih-kai; Thompson, Charles; Chang, Jing; Zhu, Hongtu; D'Angiulli, Amedeo

    2013-01-01

    Mexico City Metropolitan Area children chronically exposed to high concentrations of air pollutants exhibit an early brain imbalance in genes involved in oxidative stress, inflammation, innate and adaptive immune responses along with accumulation of misfolded proteins observed in the early stages of Alzheimer and Parkinson's diseases. A complex modulation of serum cytokines and chemokines influences children's brain structural and gray/white matter volumetric responses to air pollution. The search for biomarkers associating systemic and CNS inflammation to brain growth and cognitive deficits in the short term and neurodegeneration in the long-term is our principal aim. We explored and compared a profile of cytokines, chemokines (Multiplexing LASER Bead Technology) and Cellular prion protein (PrPC) in normal cerebro-spinal-fluid (CSF) of urban children with high vs. low air pollution exposures. PrPC and macrophage inhibitory factor (MIF) were also measured in serum. Samples from 139 children ages 11.91 ± 4.2 years were measured. Highly exposed children exhibited significant increases in CSF MIF (p = 0.002), IL6 (p = 0.006), IL1ra (p = 0.014), IL-2 (p = 0.04), and PrPC (p = 0.039) vs. controls. MIF serum concentrations were higher in exposed children (p = 0.009). Our results suggest CSF as a MIF, IL6, IL1Ra, IL-2, and PrPC compartment that can possibly differentiate air pollution exposures in children. MIF, a key neuro-immune mediator, is a potential biomarker bridge to identify children with CNS inflammation. Fine tuning of immune-to-brain communication is crucial to neural networks appropriate functioning, thus the short and long term effects of systemic inflammation and dysregulated neural immune responses are of deep concern for millions of exposed children. Defining the linkage and the health consequences of the brain / immune system interactions in the developing brain chronically exposed to air pollutants ought to be of pressing importance for public health

  17. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates.

    PubMed

    Noy-Porat, Tal; Rosenfeld, Ronit; Ariel, Naomi; Epstein, Eyal; Alcalay, Ron; Zvi, Anat; Kronman, Chanoch; Ordentlich, Arie; Mazor, Ohad

    2016-01-01

    Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit) was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (koff < 1 × 10(-7) s(-1)) that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication. PMID:26950154

  18. High affinity binding of (/sup 3/H)neurotensin of rat uterus

    SciTech Connect

    Pettibone, D.J.; Totaro, J.A.

    1987-11-01

    (/sup 3/H)Neurotensin (NT) was found to bind specifically and with high affinity to crude membranes prepared from rat uterus. Scatchard analysis of saturation binding studies indicated that (/sup 3/H)NT apparently binds to two sites (high affinity Kd 0.5 nM; low affinity Kd 9 nM) with the density of high affinity sites (41 fmoles/mg prot.) being about one-third that of the low affinity sites (100 fmoles/mg prot.). In competition studies, NT and various fragments inhibited (/sup 3/H)NT binding with the following potencies (approximately IC50): NT 8-13 (0.4 nM), NT 1-13 (4 nM), NT 9-13 (130 nM), NT 1-11, NT 1-8 (greater than 100 microM). Quantitatively similar results were obtained using brain tissue. These findings raise the possibility of a role for NT in uterine function.

  19. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates

    PubMed Central

    Noy-Porat, Tal; Rosenfeld, Ronit; Ariel, Naomi; Epstein, Eyal; Alcalay, Ron; Zvi, Anat; Kronman, Chanoch; Ordentlich, Arie; Mazor, Ohad

    2016-01-01

    Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit) was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (koff < 1 × 10−7 s−1) that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication. PMID:26950154

  20. ELISA-mimic screen for synthetic polymer nanoparticles with high affinity to target proteins.

    PubMed

    Yonamine, Yusuke; Hoshino, Yu; Shea, Kenneth J

    2012-09-10

    Synthetic polymer nanoparticles (NPs) that display high affinity to protein targets have significant potential for medical and biotechnological applications as protein capture agents or functional replacements of antibodies ("plastic antibodies"). In this study, we modified an immunological assay (enzyme-linked immunosorbent assay: ELISA) into a high-throughput screening method to select nanoparticles with high affinity to target proteins. Histone and fibrinogen were chosen as target proteins to demonstrate this concept. The selection process utilized a biotinylated NP library constructed with combinations of functional monomers. The screen identified NPs with distinctive functional group compositions that exhibited high affinity to either histone or fibrinogen. The variation of protein affinity with changes in the nature and amount of functional groups in the NP provided chemical insight into the principle determinants of protein-NP binding. The NP affinity was semiquantified using the ELISA-mimic assay by varying the NP concentrations. The screening results were found to correlate with solution-based assay results. This screening system utilizing a biotinylated NP is a general approach to optimize functional monomer compositions and can be used to rapidly search for synthetic polymers with high (or low) affinity for target biological macromolecules. PMID:22813352

  1. Transmembrane-truncated alphavbeta3 integrin retains high affinity for ligand binding: evidence for an 'inside-out' suppressor?

    PubMed Central

    Mehta, R J; Diefenbach, B; Brown, A; Cullen, E; Jonczyk, A; Güssow, D; Luckenbach, G A; Goodman, S L

    1998-01-01

    The molecular mechanisms of alphavbeta3 integrin affinity regulation have important biological implications in tumour development, wound repair and angiogenesis. We expressed, purified and characterized recombinant forms of human alphavbeta3 (r-alphavbeta3) and compared the activation state of these with alphavbeta3 in its cellular environment. The ligand specificity and selectivity of recombinant full-length and double transmembrane truncations of r-alphavbeta3 cloned in BacPAK6 vectors and expressed in Sf9 and High Five insect cells were compared with those of native placental alphavbeta3 and the receptor in situ on the cell surface. r-alphavbeta3 integrins were purified by affinity chromatography from detergent extracts of cells (full-length), and from the culture medium of cells expressing double-truncated r-alphavbeta3. r-alphavbeta3 had the same epitopes, ligand-binding specificities, bivalent cation requirements and susceptibility to RGD-containing peptides as native alphavbeta3. On M21-L4 melanoma cells, alphavbeta3 mediated binding to vitronectin, but not to fibrinogen unless activated with Mn2+. Non-activated alphaIIbbeta3 integrin as control in M21-L-IIb cells had the opposite profile, mediating binding to fibrinogen, but not to vitronectin unless activated with Mn2+. Thus these receptors had moderate to low ligand affinity. In marked contrast, purified alphavbeta3 receptors, with or without transmembrane and cytoplasmic domains, were constitutively of high affinity and able to bind strongly to vitronectin, fibronectin and fibrinogen under physiological conditions. Our data suggest that, in contrast with the positive regulation of alphaIIbbeta3 in situ, intracellular controls lower the affinity of alphavbeta3, and the cytoplasmic domains may act as a target for negative regulators of alphavbeta3 activity. PMID:9480902

  2. Antitumor effect of murine dendritic and tumor cells transduced with IL-2 gene.

    PubMed

    Wojas-Turek, Justyna; Pajtasz-Piasecka, Elżbieta; Rossowska, Joanna; Piasecki, Egbert; Duś, Danuta

    2012-01-01

    Interleukin (IL-) 2 acts on a number of types of immune cells promoting their effector functions. To replace systemic administration of recombinant form of this cytokine, various genetically modified cells have been used indifferent preclinical models for tumor growth inhibition. In this study, dendritic or tumor cells transduced with retroviral vector carrying IL-2 gene (JAWS II/IL-2, X63/IL-2, MC38/IL-2 cells) alone or combined with tumor antigen-stimulated dendritic cells (JAWS II/TAg) were exploited to treat colon carcinoma MC38-bearing mice. After the peritumoral injection of vaccine cells, the tumor growth delay and the increase in the number of tumor infiltrating CD4⁺ and CD8⁺ T lymphocytes were noted. A considerable increase in CD4⁺ cell influx into tumor tissue was observed when JAWS II/IL-2 cells or JAWS II/TAg with syngeneic MC38/IL-2 cells were applied. The increase in intensity of CD8⁺ cell infiltration was associated with immune reaction triggered by the same combination of applied cells or JAWS II/TAg with allogeneic X63/IL-2 cells. The effect observed in vivo was accompanied by MC38/0 cell specific cytotoxic activity of spleen cells in vitro. Thus, the application of vaccines, including IL-2-secreting cells of various origins, was able to induce different antitumor responses polarized by exogenous IL-2 and the encountered tumor antigen. PMID:23042272

  3. IL-2 / α-IL-2 Complex Treatment Cannot Be Substituted for the Adoptive Transfer of Regulatory T cells to Promote Bone Marrow Engraftment

    PubMed Central

    Mahr, Benedikt; Unger, Lukas; Hock, Karin; Pilat, Nina; Baranyi, Ulrike; Schwarz, Christoph; Maschke, Svenja; Farkas, Andreas Michael; Wekerle, Thomas

    2016-01-01

    Cell therapy with recipient Tregs achieves engraftment of allogeneic bone marrow (BM) without the need for cytoreductive conditioning (i.e., without irradiation or cytotoxic drugs). Thereby mixed chimerism and transplantation tolerance are established in recipients conditioned solely with costimulation blockade and rapamycin. However, clinical translation would be substantially facilitated if Treg-stimulating pharmaceutical agents could be used instead of individualized cell therapy. Recently, it was shown that interleukin-2 (IL-2) complexed with a monoclonal antibody (mAb) (clone JES6-1A12) against IL-2 (IL-2 complexes) potently expands and activates Tregs in vivo. Therefore, we investigated whether IL-2 complexes can replace Treg therapy in a costimulation blockade-based and irradiation-free BM transplantation (BMT) model. Unexpectedly, the administration of IL-2 complexes at the time of BMT (instead of Tregs) failed to induce BM engraftment in non-irradiated recipients (0/6 with IL-2 complexes vs. 3/4 with Tregs, p<0.05). Adding IL-2 complexes to an otherwise effective regimen involving recipient irradiation (1Gy) but no Treg transfer indeed actively triggered donor BM rejection at higher doses (0/8 with IL-2 complexes vs. 9/11 without, p<0.01) and had no detectable effect at two lower doses (3/5 vs. 9/11, p>0.05). CD8 T cells and NK cells of IL-2 complex-treated naïve mice showed an enhanced proliferative response towards donor antigens in vitro despite the marked expansion of Tregs. However, IL-2 complexes also expanded conventional CD4 T cells, CD8 T cells, NK cells, NKT cells and notably even B cells, albeit to a lesser extent. Notably, IL-2 complex expanded Tregs featured less potent suppressive functions than in vitro activated Tregs in terms of T cell suppression in vitro and BM engraftment in vivo. In conclusion, these data suggest that IL-2 complexes are less effective than recipient Tregs in promoting BM engraftment and in contrast actually trigger BM

  4. IL-2/α-IL-2 Complex Treatment Cannot Be Substituted for the Adoptive Transfer of Regulatory T cells to Promote Bone Marrow Engraftment.

    PubMed

    Mahr, Benedikt; Unger, Lukas; Hock, Karin; Pilat, Nina; Baranyi, Ulrike; Schwarz, Christoph; Maschke, Svenja; Farkas, Andreas Michael; Wekerle, Thomas

    2016-01-01

    Cell therapy with recipient Tregs achieves engraftment of allogeneic bone marrow (BM) without the need for cytoreductive conditioning (i.e., without irradiation or cytotoxic drugs). Thereby mixed chimerism and transplantation tolerance are established in recipients conditioned solely with costimulation blockade and rapamycin. However, clinical translation would be substantially facilitated if Treg-stimulating pharmaceutical agents could be used instead of individualized cell therapy. Recently, it was shown that interleukin-2 (IL-2) complexed with a monoclonal antibody (mAb) (clone JES6-1A12) against IL-2 (IL-2 complexes) potently expands and activates Tregs in vivo. Therefore, we investigated whether IL-2 complexes can replace Treg therapy in a costimulation blockade-based and irradiation-free BM transplantation (BMT) model. Unexpectedly, the administration of IL-2 complexes at the time of BMT (instead of Tregs) failed to induce BM engraftment in non-irradiated recipients (0/6 with IL-2 complexes vs. 3/4 with Tregs, p<0.05). Adding IL-2 complexes to an otherwise effective regimen involving recipient irradiation (1Gy) but no Treg transfer indeed actively triggered donor BM rejection at higher doses (0/8 with IL-2 complexes vs. 9/11 without, p<0.01) and had no detectable effect at two lower doses (3/5 vs. 9/11, p>0.05). CD8 T cells and NK cells of IL-2 complex-treated naïve mice showed an enhanced proliferative response towards donor antigens in vitro despite the marked expansion of Tregs. However, IL-2 complexes also expanded conventional CD4 T cells, CD8 T cells, NK cells, NKT cells and notably even B cells, albeit to a lesser extent. Notably, IL-2 complex expanded Tregs featured less potent suppressive functions than in vitro activated Tregs in terms of T cell suppression in vitro and BM engraftment in vivo. In conclusion, these data suggest that IL-2 complexes are less effective than recipient Tregs in promoting BM engraftment and in contrast actually trigger BM

  5. Identification and affinity of very high affinity binding sites for the phenylalkylamine series of Ca/sup +/ channel blockers in the Drosophila nervous system

    SciTech Connect

    Pauron, D.; Qar, J.; Barhanin, J.; Fournier, D.; Cuany, A.; Pralavorio, M.; Berge, J.B.; Lazdunski, M.

    1987-10-06

    The interaction of putative Ca/sup 2 +/ channels of Drosophila head membranes with molecules of the phenylalkylamine series was studied from binding experiments using (-)-(/sup 3/H)D888 and (+/-)-(/sup 3/H)verapamil. These ligands recognize a single class of very high affinity binding sites. The most potent molecule in the phenylalkylamine series was (-)-verapamil with a K/sub d/ value as exceptional low as 4.7 pM. Molecules in the benzothiazepine and diphenylbutylpiperidine series of Ca/sup 2 +/ channel blockers as well as bepridil inhibited (-)-(/sup 3/H)D888 binding in a competitive way with K/sub d/ values between 12 and 190 nM, suggesting a close correlation, as in the mammalian system, between these receptor sites and those recognizing phenylalkylamines. A tritiated (arylazido)phenylalkylamine with high affinity for the Drosophila head membranes, phenylalkylamine receptor was used in photoaffinity experiments. A protein of M/sub r/ 135,000 +/- 5000 was specifically labeled after ultraviolet irradiation.

  6. Large-scale genetic fine mapping and genotype-phenotype associations implicate polymorphism in the IL2RA region in type 1 diabetes.

    PubMed

    Lowe, Christopher E; Cooper, Jason D; Brusko, Todd; Walker, Neil M; Smyth, Deborah J; Bailey, Rebecca; Bourget, Kirsi; Plagnol, Vincent; Field, Sarah; Atkinson, Mark; Clayton, David G; Wicker, Linda S; Todd, John A

    2007-09-01

    Genome-wide association studies are now identifying disease-associated chromosome regions. However, even after convincing replication, the localization of the causal variant(s) requires comprehensive resequencing, extensive genotyping and statistical analyses in large sample sets leading to targeted functional studies. Here, we have localized the type 1 diabetes (T1D) association in the interleukin 2 receptor alpha (IL2RA) gene region to two independent groups of SNPs, spanning overlapping regions of 14 and 40 kb, encompassing IL2RA intron 1 and the 5' regions of IL2RA and RBM17 (odds ratio = 2.04, 95% confidence interval = 1.70-2.45; P = 1.92 x 10(-28); control frequency = 0.635). Furthermore, we have associated IL2RA T1D susceptibility genotypes with lower circulating levels of the biomarker, soluble IL-2RA (P = 6.28 x 10(-28)), suggesting that an inherited lower immune responsiveness predisposes to T1D. PMID:17676041

  7. Inhibition of CD25 (IL-2R alpha) expression and T-cell proliferation by polyclonal anti-thymocyte globulins.

    PubMed Central

    Bonnefoy-Berard, N; Verrier, B; Vincent, C; Revillard, J P

    1992-01-01

    Anti-lymphocyte and anti-thymocyte globulins (ATG) are currently used as immunosuppressive agents in organ transplantation. Their administration in vivo may induce not only lymphocyte depletion but also functional effects which were investigated in the present study. In vitro ATG inhibited T-cell proliferation induced by monocyte-dependent T-cell mitogens, like CD3 antibodies, phytohaemagglutinin (PHA) and concanavalin A (Con A), by monocyte-independent mitogens, like CD2 antibodies, or by protein kinase C activators (phorbol esters) associated with a calcium ionophore. The inhibitory effect of ATG was therefore not solely accounted for by a suppression of co-stimulatory signals delivered by monocytes, but rather implied a direct action on T cells. Addition of recombinant human interleukin-2 (rIL-2) did not overcome the inhibition. Suppression of T-cell proliferation by ATG was characterized by normal RNA synthesis and IL-2 secretion contrasting with markedly reduced expression of the CD25 protein [p55, the alpha-chain of interleukin-2 receptor (IL-2R)] both in cytoplasm and on T-cell membrane, as well as a decreased secretion of interferon-gamma (IFN-gamma). Northern blot analysis revealed increased levels of CD25 and IFN-gamma mRNA, suggesting a post-transcriptional inhibition of these molecules, whereas IL-2 mRNA levels were unchanged. These data demonstrate that inhibition of T-cell proliferation by ATG can be attributed primarily to a post-transcriptional defect of CD25 expression, implying a novel mechanism different from those described with other immunosuppressive agents. Blocking of T-cell proliferation in the late G1 phase of the cell cycle may contribute to the immunosuppressive activity of ATG in prophylactic treatment of allograft rejection. Images Figure 2 PMID:1398765

  8. Shark Attack: high affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation.

    PubMed

    Zielonka, Stefan; Weber, Niklas; Becker, Stefan; Doerner, Achim; Christmann, Andreas; Christmann, Christine; Uth, Christina; Fritz, Janine; Schäfer, Elena; Steinmann, Björn; Empting, Martin; Ockelmann, Pia; Lierz, Michael; Kolmar, Harald

    2014-12-10

    A novel method for stepwise in vitro affinity maturation of antigen-specific shark vNAR domains is described that exclusively relies on semi-synthetic repertoires derived from non-immunized sharks. Target-specific molecules were selected from a CDR3-randomized bamboo shark (Chiloscyllium plagiosum) vNAR library using yeast surface display as platform technology. Various antigen-binding vNAR domains were easily isolated by screening against several therapeutically relevant antigens, including the epithelial cell adhesion molecule (EpCAM), the Ephrin type-A receptor 2 (EphA2), and the human serine protease HTRA1. Affinity maturation was demonstrated for EpCAM and HTRA1 by diversifying CDR1 of target-enriched populations which allowed for the rapid selection of nanomolar binders. EpCAM-specific vNAR molecules were produced as soluble proteins and more extensively characterized via thermal shift assays and biolayer interferometry. Essentially, we demonstrate that high-affinity binders can be generated in vitro without largely compromising the desirable high thermostability of the vNAR scaffold. PMID:24862193

  9. Elongated fibrillar structure of a streptococcal adhesin assembled by the high-affinity association of [alpha]- and PPII-helices

    SciTech Connect

    Larson, Matthew R.; Rajashankar, Kanagalaghatta R.; Patel, Manisha H.; Robinette, Rebekah A.; Crowley, Paula J.; Michalek, Suzanne; Brady, L. Jeannine; Deivanayagam, Champion

    2010-08-18

    Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein adhesin that interacts with salivary components within the salivary pellicle. AgI/II contributes to virulence and has been studied as an immunological and structural target, but a fundamental understanding of its underlying architecture has been lacking. Here we report a high-resolution (1.8 {angstrom}) crystal structure of the A{sub 3}VP{sub 1} fragment of S. mutans AgI/II that demonstrates a unique fibrillar form (155 {angstrom}) through the interaction of two noncontiguous regions in the primary sequence. The A{sub 3} repeat of the alanine-rich domain adopts an extended {alpha}-helix that intertwines with the P{sub 1} repeat polyproline type II (PPII) helix to form a highly extended stalk-like structure heretofore unseen in prokaryotic or eukaryotic protein structures. Velocity sedimentation studies indicate that full-length AgI/II that contains three A/P repeats extends over 50 nanometers in length. Isothermal titration calorimetry revealed that the high-affinity association between the A{sub 3} and P{sub 1} helices is enthalpically driven. Two distinct binding sites on AgI/II to the host receptor salivary agglutinin (SAG) were identified by surface plasmon resonance (SPR). The current crystal structure reveals that AgI/II family proteins are extended fibrillar structures with the number of alanine- and proline-rich repeats determining their length.

  10. ESCRT-III-Associated Protein ALIX Mediates High-Affinity Phosphate Transporter Trafficking to Maintain Phosphate Homeostasis in Arabidopsis

    PubMed Central

    Cardona-López, Ximena; Cuyas, Laura; Marín, Elena; Irigoyen, María Luisa; Gil, Erica; Puga, María Isabel; Bligny, Richard; Nussaume, Laurent; Geldner, Niko; Paz-Ares, Javier

    2015-01-01

    Prior to the release of their cargoes into the vacuolar lumen, sorting endosomes mature into multivesicular bodies (MVBs) through the action of ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT (ESCRT) protein complexes. MVB-mediated sorting of high-affinity phosphate transporters (PHT1) to the vacuole limits their plasma membrane levels under phosphate-sufficient conditions, a process that allows plants to maintain phosphate homeostasis. Here, we describe ALIX, a cytosolic protein that associates with MVB by interacting with ESCRT-III subunit SNF7 and mediates PHT1;1 trafficking to the vacuole in Arabidopsis thaliana. We show that the partial loss-of-function mutant alix-1 displays reduced vacuolar degradation of PHT1;1. ALIX derivatives containing the alix-1 mutation showed reduced interaction with SNF7, providing a simple molecular explanation for impaired cargo trafficking in alix-1 mutants. In fact, the alix-1 mutation also hampered vacuolar sorting of the brassinosteroid receptor BRI1. We also show that alix-1 displays altered vacuole morphogenesis, implying a new role for ALIX proteins in vacuolar biogenesis, likely acting as part of ESCRT-III complexes. In line with a presumed broad target spectrum, the alix-1 mutation is pleiotropic, leading to reduced plant growth and late flowering, with stronger alix mutations being lethal, indicating that ALIX participates in diverse processes in plants essential for their life. PMID:26342016

  11. Elongated fibrillar structure of a streptococcal adhesin assembled by the high-affinity association of α- and PPII-helices

    PubMed Central

    Larson, Matthew R.; Rajashankar, Kanagalaghatta R.; Patel, Manisha H.; Robinette, Rebekah A.; Crowley, Paula J.; Michalek, Suzanne; Brady, L. Jeannine; Deivanayagam, Champion

    2010-01-01

    Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein adhesin that interacts with salivary components within the salivary pellicle. AgI/II contributes to virulence and has been studied as an immunological and structural target, but a fundamental understanding of its underlying architecture has been lacking. Here we report a high-resolution (1.8 Å) crystal structure of the A3VP1 fragment of S. mutans AgI/II that demonstrates a unique fibrillar form (155 Å) through the interaction of two noncontiguous regions in the primary sequence. The A3 repeat of the alanine-rich domain adopts an extended α-helix that intertwines with the P1 repeat polyproline type II (PPII) helix to form a highly extended stalk-like structure heretofore unseen in prokaryotic or eukaryotic protein structures. Velocity sedimentation studies indicate that full-length AgI/II that contains three A/P repeats extends over 50 nanometers in length. Isothermal titration calorimetry revealed that the high-affinity association between the A3 and P1 helices is enthalpically driven. Two distinct binding sites on AgI/II to the host receptor salivary agglutinin (SAG) were identified by surface plasmon resonance (SPR). The current crystal structure reveals that AgI/II family proteins are extended fibrillar structures with the number of alanine- and proline-rich repeats determining their length. PMID:20231452

  12. High-affinity choline uptake and acetylcholine-metabolizing enzymes in CNS white matter. A quantitative study.

    PubMed

    Hassel, Bjørnar; Solyga, Volker; Lossius, Andreas

    2008-12-01

    The presence of nicotinic and muscarinic receptors suggests the occurrence of cholinergic neurotransmission in white matter; however no quantitative information exists on acetylcholine formation and breakdown in white matter. We compared white structures of pig brain (fimbria, corpus callosum, pyramidal tracts, and occipital white matter) to gray structures (temporal, parietal and cerebellar cortices, hippocampus, and caudate) and found that sodium-dependent, high-affinity choline uptake in white structures was 25-31% of that in hippocampus. White matter choline acetyltransferase activity was 10-50% of the hippocampal value; the highest activity was found in fimbria. Acetylcholine esterase activity in white structures was 20-25% of that in hippocampus. The caudate, which is rich in cholinergic interneurons, gave values for all three parameters that were 2.8-4 times higher than in hippocampus. The results suggest a certain capacity for cholinergic neurotransmission in central nervous white matter. The white matter activity of pyruvate dehydrogenase, which provides acetyl-CoA for acetylcholine synthesis, ranged between 33 and 50% of the hippocampal activity; the activity in the caudate was similar to that in hippocampus and the other gray structures, which was true also for other enzymes of glucose metabolism: hexokinase, phosphoglucomutase, and glucose-6-phosphate dehydrogenase. Acetylcholine esterase activity in white matter was inhibited by the nerve agent soman, which may help explain the reported deleterious effect of soman on white matter. Further, this finding suggests that acetylcholine esterase inhibitors used in Alzheimer's disease may have an effect in white matter. PMID:18674580

  13. Challenges and developing solutions for increasing the benefits of IL-2 treatment in tumor therapy

    PubMed Central

    Skrombolas, Denise; Frelinger, John G.

    2014-01-01

    Summary Interleukin-2 (IL-2) is a cytokine with pleiotropic effects on the immune system. Systemic IL-2 treatment has produced durable responses in melanoma and renal cancer patients, but unfortunately this is effective only in a fraction of patients. Moreover, IL-2 treatment also engenders serious side effects, which limit its clinical utility. It is now appreciated that IL-2 not only stimulates NK and effector T cells but also has a critical role in the generation and maintenance of regulatory T cells, which act to dampen immune responses. Thus, successful immunotherapy of cancers using IL-2 has to address two fundamentally important issues: (1) how to limit side effects yet be active where it is needed, and (2) how to preferentially activate effector T cells while limiting the stimulation of Tregs. Strategies are now being developed to address these critical obstacles that may lead to a renaissance of IL-2 therapy. PMID:24410537

  14. Analysis of the high-affinity iron uptake system at the Chlamydomonas reinhardtii plasma membrane.

    PubMed

    Terzulli, Alaina; Kosman, Daniel J

    2010-05-01

    Multicopper ferroxidases play a vital role in iron metabolism in bacteria, fungi, algae, and mammals. Saccharomyces cerevisiae utilizes a channeling mechanism to couple the ferroxidase activity of Fet3p to Fe(3+) transport into the cell by Ftr1p. In contrast, the mechanisms by which mammals couple the ferroxidase reaction to iron trafficking is unclear. The human ferroxidases ceruloplasmin and hephaestin are twice the size of Fet3p and interact with proteins that are not expressed in fungi. Chlamydomonas FOX1 is a homolog of the human ferroxidases but likely supports iron uptake in a manner similar to that of yeast, since Chlamydomonas reinhardtii expresses a ferric iron permease homolog, FTR1. The results presented support this hypothesis. We show that FOX1 is trafficked to the plasma membrane and is oriented with its multicopper oxidase/ferroxidase domain in the exocytoplasmic space. Our analysis of FTR1 indicates its topology is similar to that of S. cerevisiae Ftr1p, with a potential exocytoplasmic iron channeling motif and two potential iron permeation motifs in membrane-spanning regions. We demonstrate that high-affinity iron uptake is dependent on FOX1 and the copper status of the cell. Kinetic inhibition of high-affinity iron uptake by a ferric iron chelator does not reflect the strength of the chelator, supporting a ferric iron channeling mechanism for high-affinity iron uptake in Chlamydomonas. Last, recombinant FOX1 (rFOX1) has been isolated in a partially holo form that exhibits the UV-visible absorbance spectrum of a multicopper oxidase and the catalytic activity of a ferroxidase. PMID:20348389

  15. Phase I trial with recombinant interleukin-2 (rIL-2): immune activation by rIL-2 alone or following pretreatment with recombinant interferon-gamma.

    PubMed Central

    Farace, F; Mathiot, C; Brandely, M; Tursz, T; Dorval, T; Pouillart, P; Triebel, F; Hercend, T; Fridman, W H

    1990-01-01

    Alterations of immunological parameters were analysed in patients with advanced malignancies during a phase I trial with rIL-2. Five-day infusions of rIL-2 at doses from 1 x 10(6) to 24 x 10(6) biological response modifiers program (BRMP) U/m2 per day were given to 29 patients, with a minimum of three patients per dose. The dose of 24 x 10(6) U/m2 per day was the maximal tolerated dose (MTD). Immunological parameters were analyzed at days 0, 8 and 11 of the rIL-2 courses. Following a leucopenia during rIL-2 infusion, a lymphocytosis was found in all patients except one. The lymphocytosis peaked at day 8 and was detected at doses of rIL-2 as low as 1 x 10(6) U/m2 per day, reaching a plateau at a dose of 16 x 10(6) U/m2 per day. Although all lymphocyte subsets were increased in patients receiving rIL-2, some patients had predominant T cells (CD3+, NKH1(CD56)-), others had predominant natural killer (NK) cells (CD3-, NKH1 (CD56)+), and yet others showed a mixed profile. A strong induction of cells cytotoxic for K562 targets was found in all patients at days 8 and 11. Eighteen patients received, 1 month later, a second treatment in which infusion of rIL-2 was preceded by a course of 5 days infusion of 2 x 10(6) U/m2 per day recombinant interferon-gamma (rIFN-gamma). The infusion of rIFN-gamma prior to rIL-2 had no effect on the rIL-2-induced alterations of immunological parameters. Taken together, our results suggest that immune stimulation by rIL-2 occurs even at low doses and is maximal at a dose below the MTD; and that pretreatment with low-dose rIFN-gamma does not modify the immune stimulation by rIL-2. PMID:2122928

  16. Influence of macrophages on HSV-1 induced IL 2 production by human lymphocytes

    SciTech Connect

    Clouse, K.A.; Orosz, C.G.; Sheridan, J.F.

    1986-03-05

    Previous work has demonstrated that human peripheral blood mononuclear cells (PBMC) from HSV-1 seropositive individuals produce interleukin 2 (IL 2) following stimulation in vitro with uv-inactivated herpes simplex virus type 1 (HSV antigen). This study investigated the accessory macrophage (MO) and monokine requirements for IL 2 production by enriched T lymphocytes from HSV-1 seropositive individuals. Following removal of accessory MO populations, enriched T lymphocytes did not secrete IL 2 in response to HSV antigen. However, IL 2 production was restored by the addition of autologous, ..gamma..-irradiated (5000R) MO. HSV antigen-pulsed MO also induced IL 2 production by enriched T lymphocytes. Furthermore, when HSV-pulsed macrophages were treated with paraformaldehyde they no longer caused T lymphocytes to produce IL 2 unless exogenous monokines were provided. Neither exogenous monokines nor purified human IL 1 could support HSV antigen induced IL 2 production in the absence of MO. These studies demonstrated that MO are required for HSV-induced IL 2 production by T lymphocytes from HSV-1 seropositive individuals. Furthermore, these MO appear to provide two functions required for IL 2 production: viral antigen display and monokine production.

  17. [Persistent inflammatory reaction at the injection site of Il-2 with lymphoma-like inflammatory infiltrates].

    PubMed

    Assmann, K; Nashan, D; Grabbe, S; Luger, T A; Metze, D

    2002-08-01

    Interleukin-2 (Il-2) is widely used for treatment of carcinomas, leukemia, and melanoma. Adverse drug effects of Il-2 include various systemic and generalized cutaneous drug reactions. In the following we report on a persistent inflammatory reaction at the injection site of Il-2. A patient received chemoimmuno-therapy for treatment of metastasizing melanoma, including subcutaneous application of recombinant interleukin-2 (Proleukin((R))). Within a few days reddish nodules developed at the injection sites that persisted after cessation of Il-2 injections for two years until final lethal outcome. Histologic examination revealed a lobular panniculitis expressing atypical lymphocytes and multinucleated histiocytes. PMID:12221472

  18. Microwave-assisted Organic Synthesis of a High-affinity Pyrazolo-pyrimidinyl TSPO Ligand

    PubMed Central

    Tang, Dewei; Buck, Jason R.; Hight, Matthew R.; Manning, H. Charles

    2010-01-01

    We herein report a dramatically improved total synthesis of the high-affinity translocator protein (TSPO) ligand DPA-714, featuring microwave-assisted organic synthesis (MAOS). Compared with previously described approaches, our novel MAOS method dramatically reduces overall reaction time without adversely effecting reaction yields. We envision that the described MAOS protocol may be suitably applied to high-throughput, diversity-oriented synthesis of novel compounds based on the pyrazolo-pyrimidinyl scaffold. Such an approach could accelerate the development of focused libraries of novel TSPO ligands with potential for future development as molecular imaging and therapeutic agents. PMID:20689673

  19. Studies on the nature of the high-affinity trialkyltin binding site of rat liver mitochondria.

    PubMed Central

    Dawson, A P; Farrow, B G; Selwyn, M J

    1982-01-01

    1. The proteolipid fraction isolated from rat liver mitochondria pretreated with [3H]triphenyltin chloride is enriched in triphenyltin compared with the original mitochondria. 2. Part of this [3H]triphenyltin is eluted with a protein of Mr 5000-6000 on Sephadex LH20 chromatography. 2. Mössbauer spectra of the proteolipid fraction treated with 119Sn-enriched triethyltin chloride show a doublet which corresponds closely with that assigned previously [Farrow & Dawson (1978) Eur. J. Biochem. 86. 85-95] to the absorption of triethyltin bound to the high-affinity binding site of the mitochondrial ATPase. PMID:7082305

  20. Designing and optimizing library selection strategies for generating high-affinity antibodies.

    PubMed

    Hoogenboom, H R

    1997-02-01

    Since its invention at the beginning of the 1990s, antibody phage display has revolutionized the generation of monoclonal antibodies and their engineering. It is now possible to create antibodies binding to any chosen target antigen without the use of laboratory animals or hybridomas, in a system that completely by-passes the immune system. Making antibodies from single-pot phage libraries, and improving their affinity up to the picomolar range if necessary, has never appeared easier. In this review, a variety of phage library-based strategies for the isolation of high-affinity antibodies are presented. PMID:9081300

  1. High affinity FRβ-specific CAR T cells eradicate AML and normal yeloid lineage without HSC toxicity

    PubMed Central

    Lynn, Rachel C; Feng, Yang; Schutsky, Keith; Poussin, Mathilde; Kalota, Anna; Dimitrov, Dimiter S; Powell, Daniel J

    2016-01-01

    Acute myeloid leukemia (AML) is an aggressive malignancy, and development of new treatments to prolong remissions is warranted. Chimeric antigen receptor (CAR) T-cell therapies appear promising but on-target, off-tumor recognition of antigen in healthy tissues remains a concern. Here, we isolated a high affinity (HA) folate receptor beta (FRβ)-specific scFv (2.48nM KD) for optimization of FRβ-redirected CAR T-cell therapy for AML. T-cells stably expressing the HA-FRβ CAR exhibited greatly enhanced antitumor activity against FRβ+ AML in vitro and in vivo compared to a low affinity (LA) FRβ CAR (54.3nM KD). Using the HA-FRβ IgG, FRβ expression was detectable in myeloid-lineage hematopoietic cells; however, expression in CD34+ hematopoietic stem cells (HSCs) was nearly undetectable. Accordingly, HA-FRβ CAR T-cells lysed mature CD14+ monocytes, while HSC colony formation was unaffected. Because of the potential for elimination of mature myeloid lineage, mRNA CAR electroporation for transient CAR expression was evaluated. mRNA-electroporated HA-FRβ CAR T-cells retained effective anti-tumor activity in vitro and in vivo. Together, our results highlight the importance of antibody affinity in target protein detection and CAR development and suggest that transient delivery of potent HA-FRβ CAR T-cells is highly effective against AML and reduces the risk for long-term myeloid toxicity. PMID:26898190

  2. The HXT2 gene of Saccharomyces cerevisiae is required for high-affinity glucose transport.

    PubMed Central

    Kruckeberg, A L; Bisson, L F

    1990-01-01

    The HXT2 gene of the yeast Saccharomyces cerevisiae was identified on the basis of its ability to complement the defect in glucose transport of a snf3 mutant when present on the multicopy plasmid pSC2. Analysis of the DNA sequence of HXT2 revealed an open reading frame of 541 codons, capable of encoding a protein of Mr 59,840. The predicted protein displayed high sequence and structural homology to a large family of procaryotic and eucaryotic sugar transporters. These proteins have 12 highly hydrophobic regions that could form transmembrane domains; the spacing of these putative transmembrane domains is also highly conserved. Several amino acid motifs characteristic of this sugar transporter family are also present in the HXT2 protein. An hxt2 null mutant strain lacked a significant component of high-affinity glucose transport when under derepressing (low-glucose) conditions. However, the hxt2 null mutation did not incur a major growth defect on glucose-containing media. Genetic and biochemical analyses suggest that wild-type levels of high-affinity glucose transport require the products of both the HXT2 and SNF3 genes; these genes are not linked. Low-stringency Southern blot analysis revealed a number of other sequences that cross-hybridize with HXT2, suggesting that S. cerevisiae possesses a large family of sugar transporter genes. Images PMID:2233722

  3. AtKUP1: an Arabidopsis gene encoding high-affinity potassium transport activity.

    PubMed Central

    Kim, E J; Kwak, J M; Uozumi, N; Schroeder, J I

    1998-01-01

    Because plants grow under many different types of soil and environmental conditions, we investigated the hypothesis that multiple pathways for K+ uptake exist in plants. We have identified a new family of potassium transporters from Arabidopsis by searching for homologous sequences among the expressed sequence tags of the GenBank database. The deduced amino acid sequences of AtKUP (for Arabidopsis thaliana K+ uptake transporter) cDNAs are highly homologous to the non-plant Kup and HAK1 potassium transporters from Escherichia coli and Schwanniomyces occidentalis, respectively. Interestingly, AtKUP1 and AtKUP2 are able to complement the potassium transport deficiency of an E. coli triple mutant. In addition, transgenic Arabidopsis suspension cells overexpressing AtKUP1 showed increased Rb+ uptake at micromolar concentrations with an apparent K(m) of approximately 22 microM, indicating that AtKUP1 encodes a high-affinity potassium uptake activity in vivo. A small, low-affinity Rb+ uptake component was also detected in AtKUP1-expressing cells. RNA gel blot analysis showed that the various members of the AtKUP family have distinct patterns of expression, with AtKUP3 transcript levels being strongly induced by K+ starvation. It is proposed that plants contain multiple potassium transporters for high-affinity uptake and that the AtKUP family may provide important components of high- and low-affinity K+ nutrition and uptake into various plant cell types. PMID:9477571

  4. Rapid high-affinity transport of a chemotherapeutic amino acid across the blood-brain barrier.

    PubMed

    Takada, Y; Vistica, D T; Greig, N H; Purdon, D; Rapoport, S I; Smith, Q R

    1992-04-15

    The therapeutic efficacy of many anticancer drugs against intracerebral tumors is limited by poor uptake into the central nervous system. One way to enhance brain delivery is to design agents that are transported into the brain by the saturable nutrient carriers of the blood-brain barrier. In this paper, we describe a nitrogen mustard amino acid, DL-2-amino-7-bis[(2-chloroethyl)amino/bd-1,2,3,4-tetrahydro-2-napthoi c acid, that is taken up into brain with high affinity by the large neutral amino acid carrier of the blood-brain barrier. Brain transport of DL-2-amino-7-bis[(2-chloroethyl)aminol-1,2,3,4-tetrahydro-2-naphth oic acid in the rat was found to be rapid (cerebrovascular permeability-surface area product approximately 2 x 10(-2) ml/s/g), saturable and inhibitable by large neutral amino acids. Maximal influx rate (Vmax) and half-saturation (Km) constants equaled 0.26 nmol/min/g and 0.19 microM, respectively, in the parietal cortex. Regional brain uptake of acid exceeded that of the clinical analogue, melphalan, by greater than 20-fold. The results demonstrate that drug modification to produce high-affinity ligands for the cerebrovascular nutrient carriers is a viable means to enhance drug delivery to brain for the treatment of brain tumors and other central nervous system disorders. PMID:1559223

  5. Genetic evidence of a high-affinity cyanuric acid transport system in Pseudomonas sp. ADP.

    PubMed

    Platero, Ana I; Santero, Eduardo; Govantes, Fernando

    2014-03-01

    The Pseudomonas sp. ADP plasmid pADP-1 encodes the activities involved in the hydrolytic degradation of the s-triazine herbicide atrazine. Here, we explore the presence of a specific transport system for the central intermediate of the atrazine utilization pathway, cyanuric acid, in Pseudomonas sp. ADP. Growth in fed-batch cultures containing limiting cyanuric acid concentrations is consistent with high-affinity transport of this substrate. Acquisition of the ability to grow at low cyanuric acid concentrations upon conjugal transfer of pADP1 to the nondegrading host Pseudomonas putida KT2442 suggests that all activities required for this phenotype are encoded in this plasmid. Co-expression of the pADP1-borne atzDEF and atzTUVW genes, encoding the cyanuric acid utilization pathway and the subunits of an ABC-type solute transport system, in P. putida KT2442 was sufficient to promote growth at cyanuric acid concentrations as low as 50 μM in batch culture. Taken together, our results strongly suggest that the atzTUVW gene products are involved in high-affinity transport of cyanuric acid. PMID:24484197

  6. High affinity sorption domains in soil are blocked by polar soil organic matter components.

    PubMed

    Mitchell, Perry J; Simpson, Myrna J

    2013-01-01

    Reported correlations between organic contaminant sorption affinity and soil organic matter (OM) structure vary widely, suggesting the importance of OM physical conformation and accessibility. Batch equilibration experiments were used to examine the sorption affinity of bisphenol A, atrazine, and diuron to five soils of varying OM composition. (13)C cross-polarization magic angle spinning NMR spectroscopy was used to characterize the organic carbon chemistry of the soil samples. High sorption by a soil low in O-alkyl components suggested that these structures may block high affinity sorption sites in soil OM. As such, soil samples were subjected to acid hydrolysis, and NMR results showed a decrease in the O-alkyl carbon signal intensity for all soils. Subsequent sorption experiments revealed that organic carbon-normalized distribution coefficient (K(OC)) values increased for all three contaminants. Before hydrolysis, K(OC) values correlated positively with soil aromatic carbon content and negatively with polar soil O-alkyl carbon content. While these correlations were weaker after hydrolysis, the correlation between K(OC) values and soil alkyl carbon content improved. This study suggests that hydrolyzable O-alkyl soil OM components may block high affinity sorption sites and further highlights the importance of OM physical conformation and accessibility with respect to sorption processes. PMID:23206246

  7. A high affinity monoclonal antibody recognizing the light chain of human coagulating factor VII.

    PubMed

    Sarial, Sheila; Asadi, Farzad; Jeddi-Tehrani, Mahmood; Hadavi, Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Taghizadeh-Jahed, Masoud; Shokri, Fazel; Rabbani, Hodjattallah

    2012-12-01

    Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII. PMID:23244324

  8. Screening of high-affinity scFvs from a ribosome displayed library using BIAcore biosensor.

    PubMed

    Yuan, Qing; Wang, Zhongkang; Nian, Siji; Yin, Youping; Chen, Gang; Xia, Yuxian

    2009-02-01

    An experimental protocol was developed to screen high-affinity single-chain Fv antibody fragments (scFvs) from a Xanthomonas axonopodis pv. citri (Xac) immunized ribosome display library using BIAcore biosensor. The screening methods involved immobilizing antigen [lipopolysaccharides (LPS) of Xac] on sensor chip HPA and then unpurified expression products of scFvs flowing over the immobilized sensor chip. The affinity-improved scFvs were selected based on dissociation rate constants (k (d)). Thirty-five enzyme-linked immunosorbent assay-positive scFvs were analyzed by BIAcore, and three of those (scFv A1, B2, and C5) with lower k (d) were screened. To demonstrate the accuracy of the screening method, the three scFvs were expressed in Escherichia coli HB2151 and purified. The purified scFvs were subsequently further identified according to association rate and affinity constants. The results showed that the three scFvs (A1, B2, and C5) had high affinity for LPS of Xac (3.51 x 10(-11), 1.13 x 10(-10), 5.06 x 10(-10) M, respectively). Furthermore, the scFv B2 was highly specific for LPS of Xac and had no any cross-reactions with bovine serum albumin and LPS from Xac-related bacteria. This provided evidence that the information from the BIAcore screening assay could be accurate. PMID:18574567

  9. Cationic polymer brush-modified cellulose nanocrystals for high-affinity virus binding.

    PubMed

    Rosilo, Henna; McKee, Jason R; Kontturi, Eero; Koho, Tiia; Hytönen, Vesa P; Ikkala, Olli; Kostiainen, Mauri A

    2014-10-21

    Surfaces capable of high-affinity binding of biomolecules are required in several biotechnological applications, such as purification, transfection, and sensing. Therein, the rod-shaped, colloidal cellulose nanocrystals (CNCs) are appealing due to their large surface area available for functionalization. In order to exploit electrostatic binding, their intrinsically anionic surfaces have to be cationized as biological supramolecules are predominantly anionic. Here we present a facile way to prepare cationic CNCs by surface-initiated atom-transfer radical polymerization of poly(N,N-dimethylaminoethyl methacrylate) and subsequent quaternization of the polymer pendant amino groups. The cationic polymer brush-modified CNCs maintained excellent dispersibility and colloidal stability in water and showed a ζ-potential of +38 mV. Dynamic light scattering and electron microscopy showed that the modified CNCs electrostatically bind cowpea chlorotic mottle virus and norovirus-like particles with high affinity. Addition of only a few weight percent of the modified CNCs in water dispersions sufficed to fully bind the virus capsids to form micrometer-sized assemblies. This enabled the concentration and extraction of the virus particles from solution by low-speed centrifugation. These results show the feasibility of the modified CNCs in virus binding and concentrating, and pave the way for their use as transduction enhancers for viral delivery applications. PMID:25171730

  10. The human organic cation transporter OCT1 mediates high affinity uptake of the anticancer drug daunorubicin.

    PubMed

    Andreev, Emil; Brosseau, Nicolas; Carmona, Euridice; Mes-Masson, Anne-Marie; Ramotar, Dindial

    2016-01-01

    Anthracyclines such as daunorubicin are anticancer agents that are transported into cells, and exert cytotoxicity by blocking DNA metabolism. Although there is evidence for active uptake of anthracyclines into cells, the specific transporter involved in this process has not been identified. Using the high-grade serous ovarian cancer cell line TOV2223G, we show that OCT1 mediated the high affinity (Km ~ 5 μM) uptake of daunorubicin into the cells, and that micromolar amounts of choline completely abolished the drug entry. OCT1 downregulation by shRNA impaired daunorubicin uptake into the TOV2223G cells, and these cells were significantly more resistant to the drug in comparison to the control shRNA. Transfection of HEK293T cells, which accommodated the ectopic expression of OCT1, with a plasmid expressing OCT1-EYFP showed that the transporter was predominantly localized to the plasma membrane. These transfected cells exhibited an increase in the uptake of daunorubicin in comparison to control cells transfected with an empty EYFP vector. Furthermore, a variant of OCT1, OCT1-D474C-EYFP, failed to enhance daunorubicin uptake. This is the first report demonstrating that human OCT1 is involved in the high affinity transport of anthracyclines. We postulate that OCT1 defects may contribute to the resistance of cancer cells treated with anthracyclines. PMID:26861753

  11. Choline uptake in Agrobacterium tumefaciens by the high-affinity ChoXWV transporter.

    PubMed

    Aktas, Meriyem; Jost, Kathinka A; Fritz, Christiane; Narberhaus, Franz

    2011-10-01

    Agrobacterium tumefaciens is a facultative phytopathogen that causes crown gall disease. For successful plant transformation A. tumefaciens requires the membrane lipid phosphatidylcholine (PC), which is produced via the methylation and the PC synthase (Pcs) pathways. The latter route is dependent on choline. Although choline uptake has been demonstrated in A. tumefaciens, the responsible transporter(s) remained elusive. In this study, we identified the first choline transport system in A. tumefaciens. The ABC-type choline transporter is encoded by the chromosomally located choXWV operon (ChoX, binding protein; ChoW, permease; and ChoV, ATPase). The Cho system is not critical for growth and PC synthesis. However, [14C]choline uptake is severely reduced in A. tumefaciens choX mutants. Recombinant ChoX is able to bind choline with high affinity (equilibrium dissociation constant [KD] of ≈2 μM). Since other quaternary amines are bound by ChoX with much lower affinities (acetylcholine, KD of ≈80 μM; betaine, KD of ≈470 μM), the ChoXWV system functions as a high-affinity transporter with a preference for choline. Two tryptophan residues (W40 and W87) located in the predicted ligand-binding pocket are essential for choline binding. The structural model of ChoX built on Sinorhizobium meliloti ChoX resembles the typical structure of substrate binding proteins with a so-called "Venus flytrap mechanism" of substrate binding. PMID:21803998

  12. Eosinophil cationic protein high-affinity binding to bacteria-wall lipopolysaccharides and peptidoglycans.

    PubMed

    Torrent, Marc; Navarro, Susanna; Moussaoui, Mohammed; Nogués, M Victòria; Boix, Ester

    2008-03-18

    The eosinophil cationic protein (ECP) is an eosinophil-secreted RNase involved in the immune host defense, with a cytotoxic activity against a wide range of pathogens. The protein displays antimicrobial activity against both Gram-negative and Gram-positive strains. The protein can destabilize lipid bilayers, although the action at the membrane level can only partially account for its bactericidal activity. We have now shown that ECP can bind with high affinity to the bacteria-wall components. We have analyzed its specific association to lipopolysaccharides (LPSs), its lipid A component, and peptidoglycans (PGNs). ECP high-affinity binding capacity to LPSs and lipid A has been analyzed by a fluorescent displacement assay, and the corresponding dissociation constants were calculated using the protein labeled with a fluorophor. The protein also binds in vivo to bacteria cells. Ultrastructural analysis of cell bacteria wall and morphology have been visualized by scanning and transmission electron microscopy in both Escherichia coli and Staphylococcus aureus strains. The protein damages the bacteria surface and induces the cell population aggregation on E. coli cultures. Although both bacteria strain cells retain their shape and no cell lysis is patent, the protein can induce in E. coli the outer membrane detachment. ECP also activates the cytoplasmic membrane depolarization in both strains. Moreover, the depolarization activity on E. coli does not require any pretreatment to overcome the outer membrane barrier. The protein binding to the bacteria-wall surface would represent a first encounter step key in its antimicrobial mechanism of action. PMID:18293932

  13. The human organic cation transporter OCT1 mediates high affinity uptake of the anticancer drug daunorubicin

    PubMed Central

    Andreev, Emil; Brosseau, Nicolas; Carmona, Euridice; Mes-Masson, Anne-Marie; Ramotar, Dindial

    2016-01-01

    Anthracyclines such as daunorubicin are anticancer agents that are transported into cells, and exert cytotoxicity by blocking DNA metabolism. Although there is evidence for active uptake of anthracyclines into cells, the specific transporter involved in this process has not been identified. Using the high-grade serous ovarian cancer cell line TOV2223G, we show that OCT1 mediated the high affinity (Km ~ 5 μM) uptake of daunorubicin into the cells, and that micromolar amounts of choline completely abolished the drug entry. OCT1 downregulation by shRNA impaired daunorubicin uptake into the TOV2223G cells, and these cells were significantly more resistant to the drug in comparison to the control shRNA. Transfection of HEK293T cells, which accommodated the ectopic expression of OCT1, with a plasmid expressing OCT1-EYFP showed that the transporter was predominantly localized to the plasma membrane. These transfected cells exhibited an increase in the uptake of daunorubicin in comparison to control cells transfected with an empty EYFP vector. Furthermore, a variant of OCT1, OCT1-D474C-EYFP, failed to enhance daunorubicin uptake. This is the first report demonstrating that human OCT1 is involved in the high affinity transport of anthracyclines. We postulate that OCT1 defects may contribute to the resistance of cancer cells treated with anthracyclines. PMID:26861753

  14. A molecular recognizing system of serotonin in rat fetal axonal growth cones: uptake and high affinity binding.

    PubMed

    Mercado, R; Hernández, J

    1992-09-18

    Axonal growth cone particles (AGCP) isolated from prenatal and postnatal rat brain had different high-affinity 5-HT uptake characteristics. In postnatal AGCP the uptake behaves as in the adult rat brain, while in the prenatal AGCP the uptake characteristics seem to be in a transitional stage. Also in prenatal AGCP we observed specific, high-affinity 5-HT binding sites. These results support the idea of an important role for 5-HT during axogenesis. PMID:1424085

  15. Quantitative and qualitative effects of N10-methylfolate on high-affinity folate binding in human leukocytes.

    PubMed

    Holm, J; Hansen, S I; Lyngbye, J

    1984-01-01

    N10-methylfolate acted as a potent competitive inhibitor of high-affinity [3H] folate binding in human leukocytes, while methotrexate had no effect. Furthermore, folate binding changed into a non-cooperative type in the presence of N10-methylfolate. Hence, in qualitative and quantitative respects, the substrate specificity characteristics of leukocyte folate binding resemble those of other high-affinity folate binding systems. PMID:6500843

  16. IL-2 therapy promotes suppressive ICOS+ Treg expansion in melanoma patients

    PubMed Central

    Sim, Geok Choo; Martin-Orozco, Natalia; Jin, Lei; Yang, Yan; Wu, Sheng; Washington, Edwina; Sanders, Deborah; Lacey, Carol; Wang, Yijun; Vence, Luis; Hwu, Patrick; Radvanyi, Laszlo

    2013-01-01

    High-dose (HD) IL-2 therapy in patients with cancer increases the general population of Tregs, which are positive for CD4, CD25, and the Treg-specific marker Foxp3. It is unknown whether specific subsets of Tregs are activated and expanded during HD IL-2 therapy or whether activation of any particular Treg subset correlates with clinical outcome. Here, we evaluated Treg population subsets that were induced in patients with melanoma following HD IL-2 therapy. We identified a Treg population that was positive for CD4, CD25, Foxp3, and the inducible T cell costimulator (ICOS). This Treg population increased more than any other lymphocyte subset during HD IL-2 therapy and had an activated Treg phenotype, as indicated by high levels of CD39, CD73, and TGF-β. ICOS+ Tregs were the most proliferative lymphocyte population in the blood after IL-2 therapy. Patients with melanoma with enhanced expansion of ICOS+ Tregs in blood following the first cycle of HD IL-2 therapy had worse clinical outcomes than patients with fewer ICOS+ Tregs. However, there was no difference in total Treg expansion between HD IL-2 responders and nonresponders. These data suggest that increased expansion of the ICOS+ Treg population following the first cycle of HD IL-2 therapy may be predictive of clinical outcome. PMID:24292706

  17. PKCα and PKCβ cooperate functionally in CD3-induced de novo IL-2 mRNA transcription.

    PubMed

    Lutz-Nicoladoni, Christina; Christina, Lutz-Nicoladoni; Thuille, Nikolaus; Nikolaus, Thuille; Wachowicz, Katarzyna; Katarzyna, Wachowicz; Gruber, Thomas; Thomas, Gruber; Leitges, Michael; Michael, Leitges; Baier, Gottfried; Gottfried, Baier

    2013-03-01

    The physiological functions of PKCα and PKCθ isotypes downstream of the antigen receptor have been defined in CD3(+) T cells. In contrast, no function of the second conventional PKC member, PKCβ, has been described yet in T cell antigen receptor signalling. To investigate the hypothesis that both conventional PKCα and PKCβ isotypes may have overlapping functions in T cell activation signalling, we generated mice that lacked the genes for both isotypes. We found that PKCα(-/-)/β(-/-) animals are viable, live normal life spans and display normal T cell development. However, these animals possess additive defects in T cell responses in comparison to animals that carry single mutations in these genes. Our studies demonstrate that the activities of PKCα and PKCβ converge to regulate IL-2 cytokine responses in anti-CD3 stimulated primary mouse T cells. Here, we present genetic evidence that PKCα and PKCβ cooperate in IL-2 transcriptional transactivation in primary mouse T cells independently of the actions of PKCθ. PMID:23439007

  18. PKCα and PKCβ cooperate functionally in CD3-induced de novo IL-2 mRNA transcription

    PubMed Central

    Christina, Lutz-Nicoladoni; Nikolaus, Thuille; Katarzyna, Wachowicz; Thomas, Gruber; Michael, Leitges; Gottfried, Baier

    2013-01-01

    The physiological functions of PKCα and PKCθ isotypes downstream of the antigen receptor have been defined in CD3+ T cells. In contrast, no function of the second conventional PKC member, PKCβ, has been described yet in T cell antigen receptor signalling. To investigate the hypothesis that both conventional PKCα and PKCβ isotypes may have overlapping functions in T cell activation signalling, we generated mice that lacked the genes for both isotypes. We found that PKCα−/−/β−/− animals are viable, live normal life spans and display normal T cell development. However, these animals possess additive defects in T cell responses in comparison to animals that carry single mutations in these genes. Our studies demonstrate that the activities of PKCα and PKCβ converge to regulate IL-2 cytokine responses in anti-CD3 stimulated primary mouse T cells. Here, we present genetic evidence that PKCα and PKCβ cooperate in IL-2 transcriptional transactivation in primary mouse T cells independently of the actions of PKCθ. PMID:23439007

  19. Synthesis and receptor binding of N-substituted tropane derivatives. High-affinity ligands for the cocaine receptor

    SciTech Connect

    Milius, R.A.; Saha, J.K.; Madras, B.K.; Neumeyer, J.L. )

    1991-05-01

    The synthesis and pharmacological characterization of a series of N-substituted 3-(4-fluorophenyl)tropane derivatives is reported. The compounds displayed binding characteristics that paralleled those of cocaine, and several had substantially higher affinity at cocaine recognition sites. Conjugate addition of 4-fluorophenyl magnesium bromide to anhydroecgonine methyl ester gave 2 beta-(carbomethoxy)-3 beta-(4-fluorophenyl)tropane (4a, designated CFT, also known as WIN 35,428) after flash chromatography. N demethylation of 4a was effected by Zn/HOAc reduction of the corresponding 2,2,2-trichloroethyl carbamate to give 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)nortropane (5), which was alkylated with allyl bromide to afford the N-allyl analogue, 6. The N-propyl analogue, 7, was prepared by catalytic reduction (Pd/C) of 6. The most potent analogue, 4a, was tritiated at a specific activity of 81.3 Ci/mmol. ({sup 3}H)4a bound rapidly and reversibly to caudate putamen membranes; the two-component binding curve typical of cocaine analogues was observed. Equilibrium was achieved within 2 h and was stable for at least 4 h. High- and low-affinity Kd values observed for ({sup 3}H)4a (4.7 and 60 nM, respectively) were more than 4 times lower than those for ({sup 3}H)cocaine, and the density of binding sites (Bmax = 50 pmol/g, high, and 290 pmol/g, low) for the two drugs were comparable. Nonspecific binding of ({sup 3}H)4a was 5-10% of total binding.

  20. Deep Sequencing-guided Design of a High Affinity Dual Specificity Antibody to Target Two Angiogenic Factors in Neovascular Age-related Macular Degeneration.

    PubMed

    Koenig, Patrick; Lee, Chingwei V; Sanowar, Sarah; Wu, Ping; Stinson, Jeremy; Harris, Seth F; Fuh, Germaine

    2015-09-01

    The development of dual targeting antibodies promises therapies with improved efficacy over mono-specific antibodies. Here, we engineered a Two-in-One VEGF/angiopoietin 2 antibody with dual action Fab (DAF) as a potential therapeutic for neovascular age-related macular degeneration. Crystal structures of the VEGF/angiopoietin 2 DAF in complex with its two antigens showed highly overlapping binding sites. To achieve sufficient affinity of the DAF to block both angiogenic factors, we turned to deep mutational scanning in the complementarity determining regions (CDRs). By mutating all three CDRs of each antibody chain simultaneously, we were able not only to identify affinity improving single mutations but also mutation pairs from different CDRs that synergistically improve both binding functions. Furthermore, insights into the cooperativity between mutations allowed us to identify fold-stabilizing mutations in the CDRs. The data obtained from deep mutational scanning reveal that the majority of the 52 CDR residues are utilized differently for the two antigen binding function and permit, for the first time, the engineering of several DAF variants with sub-nanomolar affinity against two structurally unrelated antigens. The improved variants show similar blocking activity of receptor binding as the high affinity mono-specific antibodies against these two proteins, demonstrating the feasibility of generating a dual specificity binding surface with comparable properties to individual high affinity mono-specific antibodies. PMID:26088137

  1. Immunotherapy of murine sarcomas with interleukin 2. I. Local administration of human recombinant IL-2 preparations.

    PubMed

    Bubeník, J; Indrová, M; Toulcová, A

    1986-01-01

    The immunotherapeutic effect of human recombinant interleukin 2 was examined with a panel of MC-induced murine sarcomas carrying individual tumour-specific transplantation antigens. Repeated peritumoral injections of RIL-2 inhibited growth of five (MC11, MC13, MC14, MC15, MC16) out of six sarcomas in syngeneic mice. The sixth murine sarcoma (MC12) was resistant to the tumour-inhibitory effect of human recombinant IL-2 as well as to the tumour-inhibitory effect of murine and rat lymphoid IL-2 preparations. Since the IL-2-sensitive and IL-2-resistant sarcomas were induced with MC in mice of identical genotype and share most of their characteristics, they represent a useful model for investigation of structural target cell determinants and functional target cell properties responsible for the sensitivity of tumours to the immunotherapeutic effects of IL-2. PMID:3492396

  2. A nitrogen-dependent switch in the high affinity ammonium transport in Medicago truncatula.

    PubMed

    Straub, Daniel; Ludewig, Uwe; Neuhäuser, Benjamin

    2014-11-01

    Ammonium transporters (AMTs) are crucial for the high affinity primary uptake and translocation of ammonium in plants. In the model legume Medicago truncatula, the genomic set of AMT-type ammonium transporters comprises eight members. Only four genes were abundantly expressed in young seedlings, both in roots and shoots. While the expression of all AMTs in the shoot was not affected by the nitrogen availability, the dominating MtAMT1;1 gene was repressed by nitrogen in roots, despite that cellular nitrogen concentrations were far above deficiency levels. A contrasting de-repression by nitrogen was observed for MtAMT1;4 and MtAMT2;1, which were both expressed at intermediate level. Weak expression was found for MtAMT1;2 and MtAMT2;3, while the other AMTs were not detected in young seedlings. When expressed from their endogenous promoters, translational fusion proteins of MtAMT1;1 and MtAMT2;1 with green fluorescent protein were co-localized in the plasma membrane of rhizodermal cells, but also detected in cortical root layers. Both transporter proteins similarly functionally complemented a yeast strain that is deficient in high affinity ammonium transport, both at acidic and neutral pH. The uptake into yeast mediated by these transporters saturated with Km AMT1;1 = 89 µM and Km AMT2;1 = 123 µM, respectively. When expressed in oocytes, MtAMT1;1 mediated much larger (15)N-ammonium uptake than MtAMT2;1, but NH4 (+) currents were only recorded for MtAMT1;1. These currents saturated with a voltage-dependent Km = 90 µM at -80 mV. The cellular localization and regulation of the AMTs suggests that MtAMT1;1 encodes the major high affinity ammonium transporter gene in low nitrogen grown young M. truncatula roots and despite the similar localization and substrate affinity, MtAMT2;1 appears functionally distinct and more important at higher nitrogen supply. PMID:25164101

  3. IL-2 production by intestinal lamina propria cells in normal inflamed and cancer-bearing colons.

    PubMed Central

    Pullman, W E; Doe, W F

    1992-01-01

    Biologically significant levels of IL-2 activity were produced by isolated lamina propria mononuclear cells (LPMC) from normal intestine (n = 12), cancer-bearing colons (n = 35) and inflammatory bowel disease (IBD) affected tissue (n = 12). The levels of IL-2 produced were similar for all three sources of LPMC (normal 252 +/- 48 U/ml, IBD-affected mucosa 197 +/- 42 U/ml and colon cancer 285 +/- 43 U/ml). These levels were significantly greater than those produced by peripheral blood mononuclear cells (20 +/- 5 U/ml, P less than 0.01) on a per cell basis. In mucosa from cancer-bearing colons the amount of IL-2 produced by LPMC was unaffected by the invasiveness of the colon cancer. LPMC IL-2 production was markedly suppressed by drugs used in IBD therapy. 5-Aminosalicylic acid (5-ASA) reduced activity in a dose-dependent fashion. At a dose equivalent to the faecal therapeutic level of 0.5 mg/ml activity, IL-2 production by LPMC was suppressed to 3.4% of controls. Similarly, exposure of LPMC to cyclosporin A (CyA) and hydrocortisone (HC) at therapeutic levels reduced IL-2 activity to less than 1% of controls. The major producers of IL-2 activity were shown to be CD3+ T lymphocytes and those bearing the activation markers IL-2R and TFR. Suppression of mucosal IL-2 production represents an important therapeutic mechanism of drugs used in the management of IBD including HC, 5-ASA and CyA. These results suggest that mucosal T cells produce appreciable levels of IL-2 activity that may be important in maintaining immune homeostasis in the normal intestine, provide anti-neoplastic cytotoxic activity and contribute to the inflammatory events that characterize the mucosal lesions of IBD. PMID:1563100

  4. IL-4 acts as a homeostatic regulator of IL-2-induced TNF and IFN-gamma.

    PubMed Central

    Bello-Fernandez, C; Oblakowski, P; Meager, A; Duncombe, A S; Rill, D M; Hoffbrand, A V; Brenner, M K

    1991-01-01

    Interleukin-4 (IL-4) is a cytokine secreted by interleukin-2 (IL-2)-activated lymphocytes. IL-2-stimulated lymphocytes also secrete two cytokines, tumour necrosis factor (TNF) and gamma-interferon (IFN-gamma), which contribute to effector function and which may themselves recruit fresh, cytokine-secreting effector cells. We have now investigated whether the IL-4 induced is able to homeostatically regulate secretion of the TNF and IFN-gamma. Peripheral blood mononuclear cells or lymphocytes from normal donors and from patients with neoplastic disease were cultured in the presence of IL-2 alone, IL-4 alone or with both cytokines. IL-2 induced high levels of TNF and IFN-gamma secretion in both groups. The addition of recombinant IL-4 to these IL-2-stimulated cultures lead to significant inhibition of IFN-gamma and TNF production. IFN-gamma secretion was reduced by 50-99% in normal donors and by between 11% and 99% in patients (P less than 0.001). TNF levels induced by IL-2 were similarly reduced by IL-4 both in normal donors (P less than 0.003) and in patients (P less than 0.01). These inhibitory effects were produced by IL-4 at doses of IL-2 attainable in vivo. Inhibition appears to represent a homeostatic regulatory mechanism which may limit recruitment of fresh activated killer (AK) cells. When endogenous IL-4 activity in IL-2-activated lymphocytes was blocked by anti-IL-4 antibody, significantly higher levels of IFN-gamma and TNF were secreted (P less than 0.05). Since both TNF and IFN-gamma may contribute to the anti-neoplastic action of IL-2, manipulating the level of IL-4 activity in vivo could augment the benefits of IL-2 immunotherapy. PMID:1901829

  5. IL-2 activated NK cell immunotherapy of three children after haploidentical stem cell transplantation.

    PubMed

    Koehl, Ulrike; Sörensen, Jan; Esser, Ruth; Zimmermann, Stefanie; Grüttner, Hans Peter; Tonn, Torsten; Seidl, Christian; Seifried, Erhard; Klingebiel, Thomas; Schwabe, Dirk

    2004-01-01

    Natural killer (NK) cells are thought to be of benefit in HLA-mismatched hematopoietic transplantation (H-SCT). Therefore, we developed a protocol for clinical-use expansion of highly enriched and IL-2-stimulated NK cells. Purification of unstimulated leukaphereses by a two-step T cell depletion with a final CD56 enrichment procedure leads to a mean purity of 95% CD56(+)CD3- NK cells with a four- to five-log depletion of T cells. So far, three pediatric patients with multiply relapsed acute lymphoblastic leukemia (ALL) or acute myelogenous leukemia (AML) were treated with repeated transfusions post-H-SCT. Directed killer immunoglobulin-like receptor (KIR) mismatches were demonstrated in all three cases. Although all patients showed blast persistence at the time of transplant, they reached complete remission and complete donor chimerism within 1 month post-H-SCT. NK cell therapy was tolerated well without graft-versus-host disease (GvHD) induction or other adverse events. The AML patient died of early relapse on day +80, while the ALL patients died of thrombotic-thrombocytopenic purpura and atypical viral pneumonia on days +45 and +152, respectively. This initial trial showed the feasibility of good manufacturing practice (GMP)-compliant NK cell isolation and expansion for clinical applications. We now launch a clinical phase I trial with activated NK cells post-H-SCT. PMID:15528141

  6. Cationic polymer brush-modified cellulose nanocrystals for high-affinity virus binding

    NASA Astrophysics Data System (ADS)

    Rosilo, Henna; McKee, Jason R.; Kontturi, Eero; Koho, Tiia; Hytönen, Vesa P.; Ikkala, Olli; Kostiainen, Mauri A.

    2014-09-01

    Surfaces capable of high-affinity binding of biomolecules are required in several biotechnological applications, such as purification, transfection, and sensing. Therein, the rod-shaped, colloidal cellulose nanocrystals (CNCs) are appealing due to their large surface area available for functionalization. In order to exploit electrostatic binding, their intrinsically anionic surfaces have to be cationized as biological supramolecules are predominantly anionic. Here we present a facile way to prepare cationic CNCs by surface-initiated atom-transfer radical polymerization of poly(N,N-dimethylaminoethyl methacrylate) and subsequent quaternization of the polymer pendant amino groups. The cationic polymer brush-modified CNCs maintained excellent dispersibility and colloidal stability in water and showed a ζ-potential of +38 mV. Dynamic light scattering and electron microscopy showed that the modified CNCs electrostatically bind cowpea chlorotic mottle virus and norovirus-like particles with high affinity. Addition of only a few weight percent of the modified CNCs in water dispersions sufficed to fully bind the virus capsids to form micrometer-sized assemblies. This enabled the concentration and extraction of the virus particles from solution by low-speed centrifugation. These results show the feasibility of the modified CNCs in virus binding and concentrating, and pave the way for their use as transduction enhancers for viral delivery applications.Surfaces capable of high-affinity binding of biomolecules are required in several biotechnological applications, such as purification, transfection, and sensing. Therein, the rod-shaped, colloidal cellulose nanocrystals (CNCs) are appealing due to their large surface area available for functionalization. In order to exploit electrostatic binding, their intrinsically anionic surfaces have to be cationized as biological supramolecules are predominantly anionic. Here we present a facile way to prepare cationic CNCs by surface

  7. High affinity anti-Internalin B VHH antibody fragments isolated from naturally and artificially immunized repertoires.

    PubMed

    Gene, Robert W; Kumaran, Jyothi; Aroche, Cristina; van Faassen, Henk; Hall, J Christopher; MacKenzie, C Roger; Arbabi-Ghahroudi, Mehdi

    2015-01-01

    The need for rapid and easy technologies for the detection of food-borne and environmental pathogens is essential for safeguarding the health of populations. Furthermore, distribution of tainted food and water can have consequences which can affect whole economies. Antibodies and antibody fragments have been historically used in detection platforms due to their antigen specificity and robust physicochemical properties. In this study, we report the isolation and characterization of antibody fragments from the heavy chain antibody repertoire (VHH) of Camelidae which bind with specificity and high affinity to the Listeria monocytogenes invasin, Internalin B (InlB). To the best of our knowledge, this is the first report of anti-InlB VHHs from camelids. These anti-InlB VHHs were not cross-reactive to the structurally related Listeria invasin Internalin A (InlA) and are potential reagents to be used in the development of detection and medical technologies. PMID:25450000

  8. Experimental conditions can obscure the second high-affinity site in LeuT.

    PubMed

    Quick, Matthias; Shi, Lei; Zehnpfennig, Britta; Weinstein, Harel; Javitch, Jonathan A

    2012-02-01

    Neurotransmitter:Na(+) symporters (NSSs), the targets of antidepressants and psychostimulants, recapture neurotransmitters from the synapse in a Na(+)-dependent symport mechanism. The crystal structure of the NSS homolog LeuT from Aquifex aeolicus revealed one leucine substrate in an occluded, centrally located (S1) binding site next to two Na(+) ions. Computational studies combined with binding and flux experiments identified a second substrate (S2) site and a molecular mechanism of Na(+)-substrate symport that depends upon the allosteric interaction of substrate molecules in the two high-affinity sites. Here we show that the S2 site, which has not yet been identified by crystallographic approaches, can be blocked during preparation of detergent-solubilized LeuT, thereby obscuring its crucial role in Na(+)-coupled symport. This finding points to the need for caution in selecting experimental environments in which the properties and mechanistic features of membrane proteins can be delineated. PMID:22245968

  9. Protein unfolding as a switch from self-recognition to high-affinity client binding.

    PubMed

    Groitl, Bastian; Horowitz, Scott; Makepeace, Karl A T; Petrotchenko, Evgeniy V; Borchers, Christoph H; Reichmann, Dana; Bardwell, James C A; Jakob, Ursula

    2016-01-01

    Stress-specific activation of the chaperone Hsp33 requires the unfolding of a central linker region. This activation mechanism suggests an intriguing functional relationship between the chaperone's own partial unfolding and its ability to bind other partially folded client proteins. However, identifying where Hsp33 binds its clients has remained a major gap in our understanding of Hsp33's working mechanism. By using site-specific Fluorine-19 nuclear magnetic resonance experiments guided by in vivo crosslinking studies, we now reveal that the partial unfolding of Hsp33's linker region facilitates client binding to an amphipathic docking surface on Hsp33. Furthermore, our results provide experimental evidence for the direct involvement of conditionally disordered regions in unfolded protein binding. The observed structural similarities between Hsp33's own metastable linker region and client proteins present a possible model for how Hsp33 uses protein unfolding as a switch from self-recognition to high-affinity client binding. PMID:26787517

  10. An Arabidopsis thaliana high-affinity molybdate transporter required for efficient uptake of molybdate from soil

    PubMed Central

    Tomatsu, Hajime; Takano, Junpei; Takahashi, Hideki; Watanabe-Takahashi, Akiko; Shibagaki, Nakako; Fujiwara, Toru

    2007-01-01

    Molybdenum (Mo) is a trace element essential for living organisms, however no molybdate transporter has been identified in eukaryotes. Here, we report the identification of a molybdate transporter, MOT1, from Arabidopsis thaliana. MOT1 is expressed in both roots and shoots, and the MOT1 protein is localized, in part, to plasma membranes and to vesicles. MOT1 is required for efficient uptake and translocation of molybdate and for normal growth under conditions of limited molybdate supply. Kinetics studies in yeast revealed that the Km value of MOT1 for molybdate is ≈20 nM. Furthermore, Mo uptake by MOT1 in yeast was not affected by coexistent sulfate, and MOT1 did not complement a sulfate transporter-deficient yeast mutant strain. These data confirmed that MOT1 is specific for molybdate and that the high affinity of MOT1 allows plants to obtain scarce Mo from soil. PMID:18003916

  11. Protein unfolding as a switch from self-recognition to high-affinity client binding

    PubMed Central

    Groitl, Bastian; Horowitz, Scott; Makepeace, Karl A. T.; Petrotchenko, Evgeniy V.; Borchers, Christoph H.; Reichmann, Dana; Bardwell, James C. A.; Jakob, Ursula

    2016-01-01

    Stress-specific activation of the chaperone Hsp33 requires the unfolding of a central linker region. This activation mechanism suggests an intriguing functional relationship between the chaperone's own partial unfolding and its ability to bind other partially folded client proteins. However, identifying where Hsp33 binds its clients has remained a major gap in our understanding of Hsp33's working mechanism. By using site-specific Fluorine-19 nuclear magnetic resonance experiments guided by in vivo crosslinking studies, we now reveal that the partial unfolding of Hsp33's linker region facilitates client binding to an amphipathic docking surface on Hsp33. Furthermore, our results provide experimental evidence for the direct involvement of conditionally disordered regions in unfolded protein binding. The observed structural similarities between Hsp33's own metastable linker region and client proteins present a possible model for how Hsp33 uses protein unfolding as a switch from self-recognition to high-affinity client binding. PMID:26787517

  12. AIRE-Deficient Patients Harbor Unique High-Affinity Disease-Ameliorating Autoantibodies.

    PubMed

    Meyer, Steffen; Woodward, Martin; Hertel, Christina; Vlaicu, Philip; Haque, Yasmin; Kärner, Jaanika; Macagno, Annalisa; Onuoha, Shimobi C; Fishman, Dmytro; Peterson, Hedi; Metsküla, Kaja; Uibo, Raivo; Jäntti, Kirsi; Hokynar, Kati; Wolff, Anette S B; Krohn, Kai; Ranki, Annamari; Peterson, Pärt; Kisand, Kai; Hayday, Adrian

    2016-07-28

    APS1/APECED patients are defined by defects in the autoimmune regulator (AIRE) that mediates central T cell tolerance to many self-antigens. AIRE deficiency also affects B cell tolerance, but this is incompletely understood. Here we show that most APS1/APECED patients displayed B cell autoreactivity toward unique sets of approximately 100 self-proteins. Thereby, autoantibodies from 81 patients collectively detected many thousands of human proteins. The loss of B cell tolerance seemingly occurred during antibody affinity maturation, an obligatorily T cell-dependent step. Consistent with this, many APS1/APECED patients harbored extremely high-affinity, neutralizing autoantibodies, particularly against specific cytokines. Such antibodies were biologically active in vitro and in vivo, and those neutralizing type I interferons (IFNs) showed a striking inverse correlation with type I diabetes, not shown by other anti-cytokine antibodies. Thus, naturally occurring human autoantibodies may actively limit disease and be of therapeutic utility. PMID:27426947

  13. Guanine Nucleotide Exchange Factor αPIX Leads to Activation of the Rac 1 GTPase/Glycogen Phosphorylase Pathway in Interleukin (IL)-2-stimulated T Cells

    PubMed Central

    Llavero, Francisco; Urzelai, Bakarne; Osinalde, Nerea; Gálvez, Patricia; Lacerda, Hadriano M.; Parada, Luis A.; Zugaza, José L.

    2015-01-01

    Recently, we have reported that the active form of Rac 1 GTPase binds to the glycogen phosphorylase muscle isoform (PYGM) and modulates its enzymatic activity leading to T cell proliferation. In the lymphoid system, Rac 1 and in general other small GTPases of the Rho family participate in the signaling cascades that are activated after engagement of the T cell antigen receptor. However, little is known about the IL-2-dependent Rac 1 activator molecules. For the first time, a signaling pathway leading to the activation of Rac 1/PYGM in response to IL-2-stimulated T cell proliferation is described. More specifically, αPIX, a known guanine nucleotide exchange factor for the small GTPases of the Rho family, preferentially Rac 1, mediates PYGM activation in Kit 225 T cells stimulated with IL-2. Using directed mutagenesis, phosphorylation of αPIX Rho-GEF serines 225 and 488 is required for activation of the Rac 1/PYGM pathway. IL-2-stimulated serine phosphorylation was corroborated in Kit 225 T cells cultures. A parallel pharmacological and genetic approach identified PKCθ as the serine/threonine kinase responsible for αPIX serine phosphorylation. The phosphorylated state of αPIX was required to activate first Rac 1 and subsequently PYGM. These results demonstrate that the IL-2 receptor activation, among other early events, leads to activation of PKCθ. To activate Rac 1 and consequently PYGM, PKCθ phosphorylates αPIX in T cells. The biological significance of this PKCθ/αPIX/Rac 1 GTPase/PYGM signaling pathway seems to be the control of different cellular responses such as migration and proliferation. PMID:25694429

  14. High affinity binding of [3H]-tyramine in the central nervous system.

    PubMed Central

    Vaccari, A.

    1986-01-01

    Optimum assay conditions for the association of [3H]-para-tyramine [( 3H]-pTA) with rat brain membranes were characterized, and a saturable, reversible, drug-specific, and high affinity binding mechanism for this trace amine was revealed. The binding capacity (Bmax) for [3H]-pTA in the corpus striatum was approximately 30 times higher than that in the cerebellum, with similar dissociation constants (KD). The binding process of [3H]-pTA involved the dopamine system, inasmuch as (a) highest binding capacity was associated with dopamine-rich regions; (b) dopamine and pTA equally displaced specifically bound [3H]-pTA; (c) there was a severe loss in striatal binding capacity for [3H]-pTA and, reportedly, for [3H]-dopamine, following unilateral nigrostriatal lesion; (d) acute in vivo reserpine treatment markedly decreased the density of [3H]-pTA and, reportedly, of [3H]-dopamine binding sites. In competition experiments [3H]-pTA binding sites, though displaying nanomolar affinity for dopamine, revealed micromolar affinities for the dopamine agonists apomorphine and pergolide, and for several dopamine antagonists, while having very high affinity for reserpine, a marker for the catecholamine transporter in synaptic vesicles. The binding process of [3H]-pTA was both energy-dependent (ouabain-sensitive), and ATP-Mg2+-insensitive; furthermore, the potencies of various drugs in competing for [3H]-pTA binding to rat striatal membranes correlated well (r = 0.96) with their reported potencies in inhibiting [3H]-dopamine uptake into striatal synaptosomes. It is concluded that [3H]-pTA binds at a site located on/within synaptic vesicles where it is involved in the transport mechanism of dopamine. PMID:3801770

  15. High affinity ATP/ADP analogues as new tools for studying CFTR gating.

    PubMed

    Zhou, Zhen; Wang, Xiaohui; Li, Min; Sohma, Yoshiro; Zou, Xiaoqin; Hwang, Tzyh-Chang

    2005-12-01

    Previous studies using non-hydrolysable ATP analogues and hydrolysis-deficient cystic fibrosis transmembrane conductance regulator (CFTR) mutants have indicated that ATP hydrolysis precedes channel closing. Our recent data suggest that ATP binding is also important in modulating the closing rate. This latter hypothesis predicts that ATP analogues with higher binding affinities should stabilize the open state more than ATP. Here we explore the possibility of using N6-modified ATP/ADP analogues as high-affinity ligands for CFTR gating, since these analogues have been shown to be more potent than native ATP/ADP in other ATP-binding proteins. Among the three N6-modified ATP analogues tested, N6-(2-phenylethyl)-ATP (P-ATP) was the most potent, with a K(1/2) of 1.6 +/- 0.4 microm (>50-fold more potent than ATP). The maximal open probability (P(o)) in the presence of P-ATP was approximately 30% higher than that of ATP, indicating that P-ATP also has a higher efficacy than ATP. Single-channel kinetic analysis showed that as [P-ATP] was increased, the opening rate increased, whereas the closing rate decreased. The fact that these two kinetic parameters have different sensitivities to changes of [P-ATP] suggests an involvement of two different ATP-binding sites, a high-affinity site modulating channel closing and a low affinity site controlling channel opening. The effect of P-ATP on the stability of open states was more evident when ATP hydrolysis was abolished, either by mutating the nucleotide-binding domain 2 (NBD2) Walker B glutamate (i.e. E1371) or by using the non-hydrolysable ATP analogue AMP-PNP. Similar strategies to develop nucleotide analogues with a modified adenine ring could be valuable for future studies of CFTR gating. PMID:16223764

  16. High affinity ATP/ADP analogues as new tools for studying CFTR gating

    PubMed Central

    Zhou, Zhen; Wang, Xiaohui; Li, Min; Sohma, Yoshiro; Zou, Xiaoqin; Hwang, Tzyh-Chang

    2005-01-01

    Previous studies using non-hydrolysable ATP analogues and hydrolysis-deficient cystic fibrosis transmembrane conductance regulator (CFTR) mutants have indicated that ATP hydrolysis precedes channel closing. Our recent data suggest that ATP binding is also important in modulating the closing rate. This latter hypothesis predicts that ATP analogues with higher binding affinities should stabilize the open state more than ATP. Here we explore the possibility of using N6-modified ATP/ADP analogues as high-affinity ligands for CFTR gating, since these analogues have been shown to be more potent than native ATP/ADP in other ATP-binding proteins. Among the three N6-modified ATP analogues tested, N6-(2-phenylethyl)-ATP (P-ATP) was the most potent, with a K½ of 1.6 ± 0.4 μm (>50-fold more potent than ATP). The maximal open probability (Po) in the presence of P-ATP was ∼30% higher than that of ATP, indicating that P-ATP also has a higher efficacy than ATP. Single-channel kinetic analysis showed that as [P-ATP] was increased, the opening rate increased, whereas the closing rate decreased. The fact that these two kinetic parameters have different sensitivities to changes of [P-ATP] suggests an involvement of two different ATP-binding sites, a high-affinity site modulating channel closing and a low affinity site controlling channel opening. The effect of P-ATP on the stability of open states was more evident when ATP hydrolysis was abolished, either by mutating the nucleotide-binding domain 2 (NBD2) Walker B glutamate (i.e. E1371) or by using the non-hydrolysable ATP analogue AMP-PNP. Similar strategies to develop nucleotide analogues with a modified adenine ring could be valuable for future studies of CFTR gating. PMID:16223764

  17. Concurrent low- and high-affinity sulfate reduction kinetics in marine sediment

    NASA Astrophysics Data System (ADS)

    Harder Tarpgaard, Irene; Røy, Hans; Jørgensen, Bo Barker

    Bacterial sulfate reduction in marine sediments generally occurs in the presence of high millimolar concentrations of sulfate. Published data indicate that low sulfate concentrations may limit sulfate reduction rates below 0.2-2 mM. Yet, high sulfate reduction rates occur in the 1-100 μM range in freshwater sediments and at the sulfate-methane transition in marine sediments. Through a combination of 35S-tracer experiments, including initial velocity experiments and time course experiments, we searched for different sulfate affinities in the mixed community of sulfate reducers in a marine sediment. We supported the radiotracer experiments with a highly sensitive ion chromatographic technique for sulfate with a detection limit of 0.15 μM SO 42- in marine pore water. Our results showed that high and low affinities for sulfate co-occur and that the applied experimental approach may determine the observed apparent half saturation constant, Km. Our experimental and model data both show that sulfate reduction in the studied marine sediment could be explained by two dominating affinities for sulfate: a low affinity with a mean half saturation constant, Km, of 430 μM SO 42- and a high affinity with a mean Km of 2.6 μM SO 42-. The high-affinity sulfate reduction was thermodynamically un-constrained down to <1 μM SO 42-, both in our experiments and under in situ conditions. The reduction of radio-labeled sulfate was partly reversible due to concurrent re-oxidation of sulfide by Fe(III) and possibly due to a reversibility of the enzymatic pathway of sulfate reduction. A literature survey of apparent Km values for sediments and pure cultures is presented and discussed.

  18. Low-Dose IL-2 Induces Regulatory T Cell-Mediated Control of Experimental Food Allergy.

    PubMed

    Bonnet, Benjamin; Vigneron, James; Levacher, Béatrice; Vazquez, Thomas; Pitoiset, Fabien; Brimaud, Faustine; Churlaud, Guillaume; Klatzmann, David; Bellier, Bertrand

    2016-07-01

    Regulatory T cells (Tregs) are pivotal for maintenance of immune self-tolerance and also regulate immune responses to exogenous Ags, including allergens. Both decreased Treg number and function have been reported in allergic patients, offering new therapeutic perspectives. We previously demonstrated that Tregs can be selectively expanded and activated by low doses of IL-2 (ld-IL-2) inducing immunoregulation without immunosuppression and established its protective effect in autoimmune diseases. In this study, we evaluated the ability of ld-IL-2 to control allergy in an experimental model of food allergy. Ld-IL-2 induced Treg expansion and activation that elicited protection against clinical manifestations of food allergy in two mouse models with OVA and peanut. This clinical effect was lost in Treg-depleted mice, demonstrating the major contribution of Tregs in ld-IL-2 efficacy. Mechanistic studies further indicated that protection from allergy could be explained by a Treg-dependent local modification of the Th1/Th2 balance and an inhibition of mast cell recruitment and activation. Preventive and therapeutic effects of ld-IL-2 were observed over a 7-mo-period, highlighting its long-term efficacy. This study demonstrated that ld-IL-2 is efficient to prevent and to treat allergic immune responses, and thus represents a promising therapeutic strategy for managing allergic diseases. PMID:27259854

  19. Generation of antitumor response by IL-2-transduced JAWS II dendritic cells.

    PubMed

    Rossowska, Joanna; Pajtasz-Piasecka, Elżbieta; Ryśnik, Oliwia; Wojas, Justyna; Krawczenko, Agnieszka; Szyda, Anna; Duś, Danuta

    2011-10-01

    Antigen-loaded dendritic cells (DCs) are a promising tool for inducing a tumor-specific immune response. It seems probable that co-administration of those cells together with cytokine-transduced DCs can further increase effectiveness of the antitumor vaccine. The local production of IL-2 by genetically modified DCs may result in alteration of the unfavorable tumor environment causing immune response dysfunction. In the presented study murine DCs of an established JAWS II cell line were transduced with a retroviral vector carrying murine IL-2 gene (JAWS II/IL-2). JAWS II/IL-2 cells demonstrated slightly decreased tumor antigen (TAg) uptake capacities. However, this modification resulted in enhanced ability of the cells to migrate in vivo. The multiple injection of vaccines containing JAWS II/IL-2 cells caused MC38 tumor growth delay and prolonged mice survival. The immunological response was manifested as cytotoxic natural killer (NK) and T cell activation and tumor tissue infiltration by CD8(+) and CD4(+) cells, accompanied by increased IFN-γ production by spleen cells. These observations suggest that repeated peritumoral administration of IL-2-producing dendritic cells can inhibit tumor growth by intensification of CD8(+) and CD4(+) cells' influx into tumor tissue and further activation of the systemic antitumor response. It can be concluded that IL-2 transduced dendritic cells may be used as a potent adjuvant in antitumor immunotherapy. PMID:21676487

  20. Manipulating Memory CD8 T Cell Numbers by Timed Enhancement of IL-2 Signals.

    PubMed

    Kim, Marie T; Kurup, Samarchith P; Starbeck-Miller, Gabriel R; Harty, John T

    2016-09-01

    As a result of the growing burden of tumors and chronic infections, manipulating CD8 T cell responses for clinical use has become an important goal for immunologists. In this article, we show that dendritic cell (DC) immunization coupled with relatively early (days 1-3) or late (days 4-6) administration of enhanced IL-2 signals increase peak effector CD8 T cell numbers, but only early IL-2 signals enhance memory numbers. IL-2 signals delivered at relatively late time points drive terminal differentiation and marked Bim-mediated contraction and do not increase memory T cell numbers. In contrast, early IL-2 signals induce effector cell metabolic profiles that are more conducive to memory formation. Of note, downregulation of CD80 and CD86 was observed on DCs in vivo following early IL-2 treatment. Mechanistically, early IL-2 treatment enhanced CTLA-4 expression on regulatory T cells, and CTLA-4 blockade alongside IL-2 treatment in vivo prevented the decrease in CD80 and CD86, supporting a cell-extrinsic role for CTLA-4 in downregulating B7 ligand expression on DCs. Finally, DC immunization followed by early IL-2 treatment and anti-CTLA-4 blockade resulted in lower memory CD8 T cell numbers compared with the DC+early IL-2 treatment group. These data suggest that curtailed signaling through the B7-CD28 costimulatory axis during CD8 T cell activation limits terminal differentiation and preserves memory CD8 T cell formation; thus, it should be considered in future T cell-vaccination strategies. PMID:27439516

  1. Phase I clinical trial combining imatinib mesylate and IL-2 in refractory cancer patients

    PubMed Central

    Pautier, Patricia; Locher, Clara; Robert, Caroline; Deroussent, Alain; Flament, Caroline; Le Cesne, Axel; Rey, Annie; Bahleda, Ratislav; Ribrag, Vincent; Soria, Jean-Charles; Vassal, Gilles; Eggermont, Alexander; Zitvogel, Laurence; Chaput, Nathalie; Paci, Angelo

    2013-01-01

    Imatinib mesylate (IM) is a small molecule inhibitor of protein tyrosine kinases. In addition to its direct effect on malignant cells, it has been suggested IM may activate of natural killer (NK) cells, hence exerting immunomodulatory functions. In preclinical settings, improved antitumor responses have been observed when IM and interleukin-2 (IL-2), a cytokine that enhances NK cells functions, were combined. The goals of this study were to determine the maximum tolerated dose (MTD) of IL-2 combined with IM at a constant dose of 400 mg, the pharmacokinetics of IM and IL-2, as well as toxicity and clinical efficacy of this immunotherapeutic regimen in patients affected by advanced tumors. The treatment consisted in 50 mg/day cyclophosphamide from 21 d before the initiation of IM throughout the first IM cycle (from D-21 to D14), 400 mg/day IM for 14 d (D1 to D14) combined with escalating doses of IL-2 (3, 6, 9 and 12 MIU/day) from days 10 to 14. This treatment was administered at three week intervals to 17 patients. Common side effects of the combination were mild to moderate, including fever, chills, fatigue, nausea and hepatic enzyme elevation. IL-2 dose level II, 6 MIU/day, was determined as the MTD with the following dose-limiting toxicities: systemic capillary leak syndrome, fatigue and anorexia. Pharmacokinetic studies revealed that the area under the curve and the maximum concentration of IM and its main metabolite CGP74588 increased significantly when IM was concomitantly administered with IL-2. In contrast, IM did not modulate IL-2 pharmacokinetics. No objective responses were observed. The best response obtained was stable disease in 8/17 (median duration: 12 weeks). Finally, IL-2 augmented the impregnation of IM and its metabolite. The combination of IM (400 mg/day) and IL-2 (6 MIU/day) in tumors that express IM targets warrants further investigation. PMID:23525192

  2. A Pilot Study of IL2 in Drug-Resistant Idiopathic Nephrotic Syndrome

    PubMed Central

    Bonanni, Alice; Bertelli, Roberta; Rossi, Roberta; Bruschi, Maurizio; Di Donato, Armando; Ravani, Pietro; Ghiggeri, Gian Marco

    2015-01-01

    Tregs infusion reverts proteinuria and reduces renal lesions in most animal models of nephrotic syndrome (i.e. Buffalo/Mna, Adriamycin, Promycin, LPS). IL2 up-regulates Tregs and may be an alternative to cell-therapy in this setting. To evaluate a potential role of IL2 as Tregs inducer and proteinuria lowering agent in human nephrotic syndrome we treated 5 nephrotic patients with 6 monthly cycles of low-dose IL2 (1x106 U/m2 first month, 1.5x106 U/m2 following months). The study cohort consisted of 5 children (all boys, 11–17 years) resistant to all the available treatments (i.e. steroids, calcineurin inhibitors, mycophenolate, Rituximab). Participants had Focal Segmental Glomerulosclerosis (3 cases) or Minimal Change Nephropathy (2 cases). IL2 was safe in all but one patient who had an acute asthma attack after the first IL2 dose and did not receive further doses. Circulating Tregs were stably increased (>10%) during the whole study period in 2 cases while were only partially modified in the other two children who started with very low levels and partially responded to single IL2 Proteinuria and renal function were not modified by IL2 at any phase of the study. We concluded that low-dose IL2 given in monthly pulses is safe and modifies the levels of circulating Tregs. This drug may not be able to lower proteinuria or affect renal function in children with idiopathic nephrotic syndrome. We were unable to reproduce in humans the effects of IL2 described in rats and mice reducing de facto the interest on this drug in nephrotic syndrome. Trial Registration ClinicalTrials.gov NCT02455908 PMID:26413873

  3. 4-1BB Signaling in Conventional T Cells Drives IL-2 Production That Overcomes CD4+CD25+FoxP3+ T Regulatory Cell Suppression

    PubMed Central

    Barsoumian, Hampartsoum B.

    2016-01-01

    Costimulation with the recombinant SA-4-1BBL agonist of 4-1BB receptor on conventional CD4+ T cells (Tconvs) overcomes the suppression mediated by naturally occurring CD4+CD25+FoxP3+ T regulatory cells (Tregs). The mechanistic basis of this observation has remained largely unknown. Herein we show that Tconvs, but not Tregs, are the direct target of SA-4-1BBL-mediated evasion of Treg suppression. IL-2 produced by Tconvs in response to 4-1BB signaling is both necessary and sufficient for overcoming Treg suppression. Supernatant from Tconvs stimulated with SA-4-1BBL contains high levels of IL-2 and overcomes Treg suppression in ex vivo Tconv:Treg cocultures. Removal of IL-2 from such supernatant restores Treg suppression and repletion of Tconv:Treg cocultures with exogenous recombinant IL-2 overcomes suppression. This study establishes 4-1BB signaling as a key circuit that regulates physical and functional equilibrium between Tregs and Tconvs with important implications for immunotherapy for indications where a fine balance between Tregs and Teffs plays a decisive role. PMID:27049955

  4. A High-Affinity Protein Binder that Blocks the IL-6/STAT3 Signaling Pathway Effectively Suppresses Non–Small Cell Lung Cancer

    PubMed Central

    Lee, Joong-jae; Kim, Hyun Jung; Yang, Chul-Su; Kyeong, Hyun-Ho; Choi, Jung-Min; Hwang, Da-Eun; Yuk, Jae-Min; Park, Keunwan; Kim, Yu Jung; Lee, Seung-Goo; Kim, Dongsup; Jo, Eun-Kyeong; Cheong, Hae-Kap; Kim, Hak-Sung

    2014-01-01

    Interleukin-6 (IL-6) is a multifunctional cytokine that regulates immune responses for host defense and tumorigenic process. Upregulation of IL-6 is known to constitutively phosphorylate signal transducer and activator of transcription 3 (STAT3), leading to activation of multiple oncogene pathways and inflammatory cascade. Here, we present the development of a high-affinity protein binder, termed repebody, which effectively suppresses non–small cell lung cancer in vivo by blocking the IL-6/STAT3 signaling. We selected a repebody that prevents human IL-6 (hIL-6) from binding to its receptor by a competitive immunoassay, and modulated its binding affinity for hIL-6 up to a picomolar range by a modular approach that mimics the combinatorial assembly of diverse modules to form antigen-specific receptors in nature. The resulting repebody was highly specific for hIL-6, effectively inhibiting the STAT3 phosphorylation in a dose- and binding affinity-response manner in vitro. The repebody was shown to have a remarkable suppression effect on the growth of tumors and STAT3 phosphorylation in xenograft mice with non–small cell lung cancer by blocking the hIL-6/STAT3 signaling. Structural analysis of the repebody and IL-6 complex revealed that the repebody binds the site 2a of hIL-6, overlapping a number of epitope residues at site 2a with gp130, and consequently causes a steric hindrance to the formation of IL-6/IL-6Rα complex. Our results suggest that high-affinity repebody targeting the IL-6/STAT3 pathway can be developed as therapeutics for non–small cell lung cancer. PMID:24682171

  5. Itk is required for Th9 differentiation via TCR-mediated induction of IL-2 and IRF4.

    PubMed

    Gomez-Rodriguez, Julio; Meylan, Françoise; Handon, Robin; Hayes, Erika T; Anderson, Stacie M; Kirby, Martha R; Siegel, Richard M; Schwartzberg, Pamela L

    2016-01-01

    Th9 cells produce interleukin (IL)-9, a cytokine implicated in allergic asthma and autoimmunity. Here we show that Itk, a mediator of T cell receptor signalling required for Th2 immune responses and the development of asthma, is a positive regulator of Th9 differentiation. In a model of allergic lung disease, Itk-deficient mice show reduced pulmonary inflammation and IL-9 production by T cells and innate lymphoid type 2 cells (ILC2), despite normal early induction of ILC2s. In vitro, Itk(-/-) CD4(+) T cells do not produce IL-9 and have reduced levels of IRF4 (Interferon Regulator Factor 4), a critical transcription factor for effector T cell function. Both IL-9 and IRF4 expression are rescued by either IL-2 or constitutively active STAT5, but not NFATc1. STAT5 binds the Irf4 promoter, demonstrating one mechanism by which IL-2 rescues weakly activated T cells. Itk inhibition also reduces IL-9 expression by human T cells, implicating ITK as a key regulator of Th9 induction. PMID:26936133

  6. Itk is required for Th9 differentiation via TCR-mediated induction of IL-2 and IRF4

    PubMed Central

    Gomez-Rodriguez, Julio; Meylan, Françoise; Handon, Robin; Hayes, Erika T.; Anderson, Stacie M.; Kirby, Martha R.; Siegel, Richard M.; Schwartzberg, Pamela L.

    2016-01-01

    Th9 cells produce interleukin (IL)-9, a cytokine implicated in allergic asthma and autoimmunity. Here we show that Itk, a mediator of T cell receptor signalling required for Th2 immune responses and the development of asthma, is a positive regulator of Th9 differentiation. In a model of allergic lung disease, Itk-deficient mice show reduced pulmonary inflammation and IL-9 production by T cells and innate lymphoid type 2 cells (ILC2), despite normal early induction of ILC2s. In vitro, Itk−/− CD4+ T cells do not produce IL-9 and have reduced levels of IRF4 (Interferon Regulator Factor 4), a critical transcription factor for effector T cell function. Both IL-9 and IRF4 expression are rescued by either IL-2 or constitutively active STAT5, but not NFATc1. STAT5 binds the Irf4 promoter, demonstrating one mechanism by which IL-2 rescues weakly activated T cells. Itk inhibition also reduces IL-9 expression by human T cells, implicating ITK as a key regulator of Th9 induction. PMID:26936133

  7. Chronic hypoxia in pregnancy affects thymus development in Balb/c mouse offspring via IL2 Signaling.

    PubMed

    Zhang, Xiaopeng; Zhou, Xiuwen; Li, Lingjun; Sun, Miao; Gao, Qingqing; Zhang, Pengjie; Tang, Jiaqi; He, Yu; Zhu, Di; Xu, Zhice

    2016-04-01

    Hypoxia during pregnancy can adversely affect development. This study, addressed the impact of prenatal hypoxia on thymus development in the rodent offspring. Pregnant Balb/c mice were exposed to hypoxia or normoxia during pregnancy, and the thymuses of their offspring were tested. Chronic hypoxia during pregnancy resulted in significantly decreased fetal body weight, with an increased thymus-to-body weight ratio. Histological analysis revealed a smaller cortical zone in the thymus of the offspring exposed to hypoxia. A reduction in the cortical T lymphocyte population corresponded to increased mRNA abundance of caspase 3 (Casp3) and decreased expression of the proliferation marker Ki-67 (Mki67). Differences in T lymphocyte sub-populations in the thymus further indicate that thymus development in offspring was retarded or stagnated by prenatal hypoxia. The abundance of IL2 and its receptor was reduced in the thymus following prenatal hypoxia. This was accompanied by an increase in thymus HIF1A and IKKβ and a decrease in phosphorylated NFKB, MAP2K1, and MAPK1/3 compared to control pregnancies. Together, these results implicate deficiencies in IL2-mediated signaling as one source of prenatal-hypoxia-impaired thymus development. PMID:26918321

  8. Kinetics of calcium dissociation from its high-affinity transport sites on sarcoplasmic reticulum ATPase.

    PubMed

    Orlowski, S; Champeil, P

    1991-01-15

    We investigated the kinetics of calcium dissociation from its high-affinity transport sites on sarcoplasmic reticulum Ca2(+)-ATPase by combining fast filtration with stopped-flow fluorescence measurements. At pH 6 and 20 degrees C, in the absence of potassium and in the presence of 20 mM MgCl2, isotopic exchange of bound calcium exhibited biphasic kinetics, with two phases of equal amplitude, regardless of the initial extent of binding site saturation. The rapidly exchangeable site, whose occupancy by calcium controlled the rate constant of the slow phase, had an apparent affinity for calcium of about 3-6 microM. A similar high affinity was also deduced from measurements of the calcium dependence of the rate constant for ATPase fluorescence changes. This affinity was higher than the overall affinity for calcium deduced from the equilibrium binding measurements (dissociation constant of 15-20 microM); this was consistent with the occurrence of cooperativity (Hill coefficient of 1.6-1.8). The drop in intrinsic fluorescence observed upon chelation of calcium was always slightly faster than the dissociation of calcium itself, although the rates for both this drop in fluorescence and calcium dissociation varied slightly from one preparation to the other. This fluorescence drop was therefore mainly due to dissociation of the bound ions, not to slow transconformation of the ATPase. Dissociation of the two bound calcium ions in a medium containing EGTA exhibited monophasic kinetics in the presence of a calcium ionophore, with a rate constant about half that of the fast phase of isotopic exchange. This particular pattern was observed over a wide range of experimental conditions, including the presence of KCl, dimethyl sulfoxide, 4-nonylphenol, or a nucleotide analogue, at pH 6 or 7, and at various temperatures. The kinetics of calcium dissociation under the above various conditions were not correlated with the ATPase affinity for calcium deduced from equilibrium

  9. New Synthesis and Tritium Labeling of a Selective Ligand for Studying High-affinity γ-Hydroxybutyrate (GHB) Binding Sites

    PubMed Central

    Vogensen, Stine B.; Marek, Aleš; Bay, Tina; Wellendorph, Petrine; Kehler, Jan; Bundgaard, Christoffer; Frølund, Bente; Pedersen, Martin H.F.; Clausen, Rasmus P.

    2013-01-01

    3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [3H]-1 is described. The radiosynthesis employs in situ generated lithium trimethoxyborotritide. Screening of 1 against different CNS targets establishes a high selectivity and we demonstrate in vivo brain penetration. In vitro characterization of [3H]-1 binding shows high specificity to the high-affinity GHB binding sites. PMID:24053696

  10. Preferential loss of IL-2-secreting CD4+ T helper cells in chronic HCV infection.

    PubMed

    Semmo, Nasser; Day, Cheryl L; Ward, Scott M; Lucas, Michaela; Harcourt, Gillian; Loughry, Andrew; Klenerman, Paul

    2005-05-01

    Hepatitis C virus (HCV) becomes persistent in the majority of infected individuals. In doing so, the virus evades host adaptive immune responses, although the mechanisms responsible in this evasion are not clear. Several groups have demonstrated weak or absent HCV-specific CD4+ T cell responses during chronic HCV infection using proliferation assays and, more recently, class II tetramers. However, the functional status of HCV-specific CD4+ T cells in resolved and persistent infection is poorly understood. Using interferon gamma (IFN-gamma) and interleukin 2 (IL-2) enzyme-linked immunospot assays, we analyzed cytokine secretion patterns in chronically infected patients and compared them with those with resolved infection. In the spontaneous resolver group, strong IL-2 secretion in relation to IFN-gamma secretion was observed. However, in the persistently infected group, a consistent and significant loss of IL-2-secreting cells, compared with IFN-gamma-secreting cells, was identified. In vitro addition of IL-2 had a substantial effect in restoring CD4+ T cell activity. In conclusion, failure of IL-2 secretion, as opposed to physical deletion or complete functional unresponsiveness, appears to be an important determinant of the status of CD4+ T cell populations in chronic HCV infection. Loss of IL-2 secretory capacity may lead to disruption of IFN-gamma and proliferative function in vivo-a status that characterizes the cellular immune response in both CD4+ and CD8+ compartments in chronic disease. PMID:15841456

  11. Designing High-Affinity Peptides for Organic Molecules by Explicit Solvent Molecular Dynamics.

    PubMed

    Gladich, Ivan; Rodriguez, Alex; Hong Enriquez, Rolando P; Guida, Filomena; Berti, Federico; Laio, Alessandro

    2015-10-15

    Short peptides offer a cheap alternative to antibodies for developing sensing units in devices for concentration measurement. We here describe a computational procedure that allows designing peptides capable of binding with high affinity a target organic molecule in aqueous or nonstandard solvent environments. The algorithm is based on a stochastic search in the space of the possible sequences of the peptide, and exploits finite temperature molecular dynamics simulations in explicit solvent to check if a proposed mutation improves the binding affinity or not. The procedure automatically produces peptides which form thermally stable complexes with the target. The estimated binding free energy reaches the 13 kcal/mol for Irinotecan anticancer drug, the target considered in this work. These peptides are by construction solvent specific; namely, they recognize the target only in the solvent in which they have been designed. This feature of the algorithm calls for applications in devices in which the peptide-based sensor is required to work in denaturants or under extreme conditions of pressure and temperature. PMID:26398715

  12. Off-rate screening for selection of high-affinity anti-drug antibodies.

    PubMed

    Ylera, Francisco; Harth, Stefan; Waldherr, Dirk; Frisch, Christian; Knappik, Achim

    2013-10-15

    The rapidly increasing number of therapeutic antibodies in clinical development and on the market requires corresponding detection reagents for monitoring the concentration of these drugs in patient samples and as positive controls for measurement of anti-drug antibodies. Phage display of large recombinant antibody libraries has been shown to enable the rapid development of fully human anti-idiotypic antibodies binding specifically to antibody drugs, since the in vitro panning approach allows for incorporation of suitable blockers to drive selection toward the paratope of the drug. A typical bottleneck in antibody generation projects is ranking of the many candidates obtained after panning on the basis of antibody binding strength. Ideally, such method will work without prior labeling of antigens and with crude bacterial lysates. We developed an off-rate screening method of crude Escherichia coli lysates containing monovalent Fab fragments obtained after phage display of the HuCAL PLATINUM® antibody library. We used the antibody drugs trastuzumab and cetuximab as antigen examples. Using the Octet® RED384 label-free sensor instrument we show that antibody off rates can be reliably determined in crude bacterial lysates with high throughput. We also demonstrate that the method can be applied to screening for high-affinity antibodies typically obtained after affinity maturation. PMID:23906643

  13. Hydroxamate Production as a High Affinity Iron Acquisition Mechanism in Paracoccidioides Spp

    PubMed Central

    Silva-Bailão, Mirelle Garcia; Bailão, Elisa Flávia Luiz Cardoso; Lechner, Beatrix Elisabeth; Gauthier, Gregory M.; Lindner, Herbert; Bailão, Alexandre Melo; Haas, Hubertus; de Almeida Soares, Célia Maria

    2014-01-01

    Iron is a micronutrient required by almost all living organisms, including fungi. Although this metal is abundant, its bioavailability is low either in aerobic environments or within mammalian hosts. As a consequence, pathogenic microorganisms evolved high affinity iron acquisition mechanisms which include the production and uptake of siderophores. Here we investigated the utilization of these molecules by species of the Paracoccidioides genus, the causative agents of a systemic mycosis. It was demonstrated that iron starvation induces the expression of Paracoccidioides ortholog genes for siderophore biosynthesis and transport. Reversed-phase HPLC analysis revealed that the fungus produces and secretes coprogen B, which generates dimerumic acid as a breakdown product. Ferricrocin and ferrichrome C were detected in Paracoccidioides as the intracellular produced siderophores. Moreover, the fungus is also able to grow in presence of siderophores as the only iron sources, demonstrating that beyond producing, Paracoccidioides is also able to utilize siderophores for growth, including the xenosiderophore ferrioxamine. Exposure to exogenous ferrioxamine and dimerumic acid increased fungus survival during co-cultivation with macrophages indicating that these molecules play a role during host-pathogen interaction. Furthermore, cross-feeding experiments revealed that Paracoccidioides siderophores promotes growth of Aspergillus nidulans strain unable to produce these iron chelators. Together, these data denote that synthesis and utilization of siderophores is a mechanism used by Paracoccidioides to surpass iron limitation. As iron paucity is found within the host, siderophore production may be related to fungus pathogenicity. PMID:25157575

  14. Characterization of a high affinity cocaine binding site in rat brain

    SciTech Connect

    Calligaro, D.; Eldefrawi, M.

    1986-03-05

    Binding of (/sup 3/H)cocaine to synaptic membranes from whole rat brain was reversible and saturable. Nonlinear regression analysis of binding isotherms indicated two binding affinities: one with k/sub d/ = 16 nM, B/sub max/ = 0.65 pmoles/mg protein and the other with K/sub d/ = 660 nM, B/sub max/ = 5.1 pmoles/mg protein. The high-affinity binding of (/sup 3/H)cocaine was sensitive to the actions of trypsin and chymotrypsin but not carboxypeptidase, and was eliminated by exposure of the membranes to 95/sup 0/C for 5 min. Specific binding at 2 nM was higher at pH 8.8 than at pH 7.0. Binding of (/sup 3/H)cocaine (15 nM) was inhibited by increasing concentrations of Na/sup +/ ions. Several cocaine analogues, neurotransmitter uptake inhibitors and local anesthetics displaced specific (/sup 3/H)cocaine binding at 2 nM with various potencies. The cocaine analogue (-)-norcocaine was the most potent (IC/sub 50/ = 10 nM), while the local anesthetic tetracaine was the least potent in inhibiting (/sup 3/H)cocaine binding. Several biogenic amine uptake inhibitors, including tricyclic antidepressants and phencyclidine, had IC/sub 50/ values below ..mu..M concentrations.