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Sample records for complex antigens inhibition

  1. Brucella abortus Inhibits Major Histocompatibility Complex Class II Expression and Antigen Processing through Interleukin-6 Secretion via Toll-Like Receptor 2▿

    PubMed Central

    Barrionuevo, Paula; Cassataro, Juliana; Delpino, M. Victoria; Zwerdling, Astrid; Pasquevich, Karina A.; Samartino, Clara García; Wallach, Jorge C.; Fossati, Carlos A.; Giambartolomei, Guillermo H.

    2008-01-01

    The strategies that allow Brucella abortus to survive inside macrophages for prolonged periods and to avoid the immunological surveillance of major histocompatibility complex class II (MHC-II)-restricted gamma interferon (IFN-γ)-producing CD4+ T lymphocytes are poorly understood. We report here that infection of THP-1 cells with B. abortus inhibited expression of MHC-II molecules and antigen (Ag) processing. Heat-killed B. abortus (HKBA) also induced both these phenomena, indicating the independence of bacterial viability and involvement of a structural component of the bacterium. Accordingly, outer membrane protein 19 (Omp19), a prototypical B. abortus lipoprotein, inhibited both MHC-II expression and Ag processing to the same extent as HKBA. Moreover, a synthetic lipohexapeptide that mimics the structure of the protein lipid moiety also inhibited MHC-II expression, indicating that any Brucella lipoprotein could down-modulate MHC-II expression and Ag processing. Inhibition of MHC-II expression and Ag processing by either HKBA or lipidated Omp19 (L-Omp19) depended on Toll-like receptor 2 and was mediated by interleukin-6. HKBA or L-Omp19 also inhibited MHC-II expression and Ag processing of human monocytes. In addition, exposure to the synthetic lipohexapeptide inhibited Ag-specific T-cell proliferation and IFN-γ production of peripheral blood mononuclear cells from Brucella-infected patients. Together, these results indicate that there is a mechanism by which B. abortus may prevent recognition by T cells to evade host immunity and establish a chronic infection. PMID:17984211

  2. Cyclosporine inhibits macrophage-mediated antigen presentation

    SciTech Connect

    Ziegler, H.K.; Palay, D.; Wentworth, P.; Cluff, C.

    1986-03-01

    The influence of cyclosporine on antigen-specific, macrophage-dependent T cell activation was analyzed in vitro. Murine T cell activation by antigens derived from Listeria monocytogenes was monitored by the production of interleukin-2. Pretreatment (2 hrs., 37/sup 0/C) of macrophages with cyclosporine resulted in a population of macrophages with a markedly diminished capacity to support the activation of T lymphocytes. When cyclosporine-pretreated macrophages were added to cultures of antigen and untreated T cells, the dose of cyclosporine which produced 50% inhibition was 1.5 ..mu..g/ml. Appropriate control experiments indicated that cyclosporine was indeed inhibiting at the macrophage level. The addition of interleukin-1 or indomethacin to the cultures did not alter the inhibitory effect of cyclosporine. Under conditions which produced >90% inhibition of antigen presentation, macrophage surface Ia expression was not altered, and the uptake and catabolism of radiolabelled antigen was normal. Thus, cyclosporine inhibits antigen presentation by a mechanism which appears unrelated to changes in Il-1 elaboration, prostaglandin production, Ia expression, or antigen uptake and catabolism.

  3. Lipase Processing of Complex Lipid Antigens.

    PubMed

    Sander, Peter; Becker, Katja; Molin, Michael Dal

    2016-09-22

    Mycobacterium tuberculosis synthesizes a wide variety of complex lipids that can serve as antigens in immune recognition of the bacterium. In this issue of Cell Chemical Biology, Gilleron et al. (2016) identify key enzymes essential for lipid antigen processing, which is required for CD1b-restricted T cell activation. PMID:27662250

  4. Mechanism of inhibition of Mycobacterium tuberculosis antigen 85 by ebselen

    PubMed Central

    Favrot, Lorenza; Grzegorzewicz, Anna E.; Lajiness, Daniel H.; Marvin, Rachel K.; Boucau, Julie; Isailovic, Dragan; Jackson, Mary; Ronning, Donald R.

    2014-01-01

    The increasing prevalence of drug-resistant tuberculosis highlights the need for identifying new antitubercular drugs that can treat these infections. The antigen 85 (Ag85) complex has emerged as an intriguing mycobacterial drug target due to its central role in synthesizing major components of the inner and outer leaflets of the mycobacterial outer membrane. Here we identify ebselen as a potent inhibitor of the Mycobacterium tuberculosis Ag85 complex. Mass spectrometry data show that ebselen binds covalently to a cysteine residue (C209) located near the Ag85C active site. The crystal structure of Ag85C in the presence of ebselen shows that C209 modification restructures the active site, thereby disrupting the hydrogen-bonded network within the active site that is essential for enzymatic activity. C209 mutations display marked decreases in enzymatic activity. These data suggest that compounds using this mechanism of action will strongly inhibit the Ag85 complex and minimize the selection of drug resistance. PMID:24193546

  5. Inhibition by chloroquine of the class II major histocompatibility complex-restricted presentation of endogenous antigens varies according to the cellular origin of the antigen-presenting cells, the nature of the T-cell epitope, and the responding T cell.

    PubMed Central

    Lombard-Platlet, S; Bertolino, P; Deng, H; Gerlier, D; Rabourdin-Combe, C

    1993-01-01

    Chloroquine treatment of antigen-presenting cells (APC) was explored as a tool to investigate the processing pathway for major histocompatibility complex (MHC) class II-restricted presentation of the endogenous secreted hen egg lysozyme (HEL) and transmembrane measles virus haemagglutinin (HA). A 72-hr pretreatment of the APC with 25 microM chloroquine blocked the presentation of the HEL(52-61) T-cell epitope generated from endogenous HEL to the I-Ak-restricted 3A9 T-cell hybridoma by MHC class II-transfected L cells expressing the invariant chain (Ii). The presentation of exogenously added HEL peptides was not affected. Under the same conditions, no inhibition of the presentation of HEL(106-116) to the I-Ed-restricted G28 high-avidity T-cell hybridoma, nor of HA when synthesized by L cells, was observed. When B-lymphoid APC were used, inhibition was observed in every case with a low number of B APC pretreated for 48 hr with chloroquine prior to the T-cell stimulation test. Moreover, addition of chloroquine to untreated B APC during the T-cell stimulation assay was sufficient to inhibit completely the presentation of HEL(106-116) to the B10.D24.42 low avidity T-cell hybridoma. Altogether these studies suggest that an apparent resistance of endogenous Ag presentation to chloroquine inhibition may not necessarily indicate the existence of a non-endosomal pathway but may be due to the nature of the T-cell epitope, to the use of 'non-professional' APC such as L cells, to the use of T cells of high avidity, and to high amounts of pre-existing MHC class II-peptide complexes expressed by the APC. We demonstrate here that, at least in conventional APC such as B cells, class II-restricted presentation of both endogenous secreted HEL and transmembrane HA involves an endosomal pathway. PMID:7508420

  6. Crystal structure of the jacalin-T-antigen complex and a comparative study of lectin-T-antigen complexes.

    PubMed

    Jeyaprakash, A Arockia; Geetha Rani, P; Banuprakash Reddy, G; Banumathi, S; Betzel, C; Sekar, K; Surolia, A; Vijayan, M

    2002-08-23

    Thomsen-Friedenreich antigen (Galbeta1-3GalNAc), generally known as T-antigen, is expressed in more than 85% of human carcinomas. Therefore, proteins which specifically bind T-antigen have potential diagnostic value. Jacalin, a lectin from jack fruit (Artocarpus integrifolia) seeds, is a tetramer of molecular mass 66kDa. It is one of the very few proteins which are known to bind T-antigen. The crystal structure of the jacalin-T-antigen complex has been determined at 1.62A resolution. The interactions of the disaccharide at the binding site are predominantly through the GalNAc moiety, with Gal interacting only through water molecules. They include a hydrogen bond between the anomeric oxygen of GalNAc and the pi electrons of an aromatic side-chain. Several intermolecular interactions involving the bound carbohydrate contribute to the stability of the crystal structure. The present structure, along with that of the Me-alpha-Gal complex, provides a reasonable qualitative explanation for the known affinities of jacalin to different carbohydrate ligands and a plausible model of the binding of the lectin to T-antigen O-linked to seryl or threonyl residues. Including the present one, the structures of five lectin-T-antigen complexes are available. GalNAc occupies the primary binding site in three of them, while Gal occupies the site in two. The choice appears to be related to the ability of the lectin to bind sialylated sugars. In either case, most of the lectin-disaccharide interactions are at the primary binding site. The conformation of T-antigen in the five complexes is nearly the same.

  7. Recovery of a cell surface fetal antigen from circulating immune complexes of melanoma patients.

    PubMed

    Wong, J H; Aguero, B; Gupta, R K; Morton, D L

    1988-01-01

    A well-characterized 69.5 x 10(3) dalton glycoprotein fetal antigen (FA), isolated from the spent culture medium of a melanoma cell line, UCLA-SO-14 (M14), was utilized to characterize the antigen component of circulating immune complexes (CIC) from melanoma patients. Ten serum samples from five patients with stage II melanoma at 1 and 4 months prior to the clinical detection of recurrent disease were selected for study. The CIC were dissociated with low pH and ultrafiltered through a 100 x 10(3) dalton exclusion limit membrane. The low pH treatment resulted in an increase in antibody titer in eight of ten serum samples. The antibody activity in membrane immunofluorescence was quantitatively inhibited by the filtered antigen fraction and purified FA, suggesting the presence of anti-FA antibodies in the treated serum, which possibly were complexed with FA in the untreated sample. As determined by competitive inhibition in an enzyme-linked immunosorbent assay, the filtrate (antigen fraction) contained an antigen that was immunologically similar to FA. These results clearly demonstrate that FA, expressed on the cell surface of melanoma cells, is present in CIC of selected melanoma patients.

  8. A synthetic random basic copolymer with promiscuous binding to class II major histocompatibility complex molecules inhibits T-cell proliferative responses to major and minor histocompatibility antigens in vitro and confers the capacity to prevent murine graft-versus-host disease in vivo.

    PubMed Central

    Schlegel, P G; Aharoni, R; Chen, Y; Chen, J; Teitelbaum, D; Arnon, R; Sela, M; Chao, N J

    1996-01-01

    Graft-versus-host disease (GVHD) is a T-cell-mediated disease of transplanted donor T cells recognizing host alloantigens. Data presented in this report show, to our knowledge, for the first time that a synthetic copolymer of the amino acids L-Glu, L-Lys, L-Ala, and L-Tyr (molecular ratio, 1.9:6.0:4.7:1.0; Mr, 6000-8500) [corrected], termed GLAT, with promiscuous binding to multiple major histocompatibility complex class II alleles is capable of preventing lethal GVHD in the B10.D2 --> BALB/c model (both H-2d) across minor histocompatibility barriers. Administration of GLAT over a limited time after transplant significantly reduced the incidence, onset, and severity of disease. GLAT also improved long-term survival from lethal GVHD: 14/25 (56%) of experimental mice survived > 140 days after transplant compared to 2/26 of saline-treated or to 1/10 of hen egg lysozyme-treated control mice (P < 0.01). Long-term survivors were documented to be fully chimeric by PCR analysis of a polymorphic microsatellite region in the interleukin 1beta gene. In vitro, GLAT inhibited the mixed lymphocyte culture in a dose-dependent fashion across a variety of major barriers tested. Furthermore, GLAT inhibited the response of nylon wool-enriched T cells to syngeneic antigen-presenting cells presenting minor histocompatibility antigens. Prepulsing of the antigen-presenting cells with GLAT reduced the proliferative response, suggesting that GLAT inhibits antigen presentation. Images Fig. 1 Fig. 2 Fig. 4 PMID:8643529

  9. Enhanced Direct Major Histocompatibility Complex Class I Self-Antigen Presentation Induced by Chlamydia Infection

    PubMed Central

    Cram, Erik D.; Simmons, Ryan S.; Palmer, Amy L.; Hildebrand, William H.; Rockey, Daniel D.

    2015-01-01

    The direct major histocompatibility complex (MHC) class I antigen presentation pathway ensures intracellular peptides are displayed at the cellular surface for recognition of infected or transformed cells by CD8+ cytotoxic T lymphocytes. Chlamydia spp. are obligate intracellular bacteria and, as such, should be targeted by CD8+ T cells. It is likely that Chlamydia spp. have evolved mechanisms to avoid the CD8+ killer T cell responses by interfering with MHC class I antigen presentation. Using a model system of self-peptide presentation which allows for posttranslational control of the model protein's stability, we tested the ability of various Chlamydia species to alter direct MHC class I antigen presentation. Infection of the JY lymphoblastoid cell line limited the accumulation of a model host protein and increased presentation of the model-protein-derived peptides. Enhanced self-peptide presentation was detected only when presentation was restricted to defective ribosomal products, or DRiPs, and total MHC class I levels remained unaltered. Skewed antigen presentation was dependent on a bacterial synthesized component, as evidenced by reversal of the observed phenotype upon preventing bacterial transcription, translation, and the inhibition of bacterial lipooligosaccharide synthesis. These data suggest that Chlamydia spp. have evolved to alter the host antigen presentation machinery to favor presentation of defective and rapidly degraded forms of self-antigen, possibly as a mechanism to diminish the presentation of peptides derived from bacterial proteins. PMID:26597986

  10. The Structure and Function of the Rh antigen Complex

    PubMed Central

    Westhoff, Connie M.

    2007-01-01

    The Rh system is one of the most important and complex blood group systems because of the large number of antigens and the serious complications for the fetus of a woman sensitized by transfusion or pregnancy. Major advances in our understanding of the Rh system have occurred with the cloning of the genes and with functional evidence that the Rh blood group proteins belong to an ancient family of membrane proteins involved in ammonia transport. The arrangement and configuration of the genes at the RH locus promotes genetic exchange, generating new antigens. Importantly, RH genetic testing can now be applied to clinical transfusion medicine and prenatal practice. This includes testing for RHD zygosity, confirmation or resolution of D antigen status, and detection of altered RHD and RHCE genes in individuals at risk for producing antibodies to high incidence Rh antigens, particularly sickle cell disease patients. The Rh proteins form a core complex that is critical to the structure of the erythrocyte membrane, and may play a physiologically role in the sequestration of blood ammonia. The Rh family of proteins now includes non-erythroid Rh homologs present in many other tissues, and comparative genomics reveals Rh homologs in all domains of life. PMID:17198846

  11. Immune complexes that contain HIV antigens activate peripheral blood T cells.

    PubMed

    Korolevskaya, L B; Shmagel, K V; Saidakova, E V; Shmagel, N G; Chereshnev, V A

    2016-07-01

    Uninfected donor T cells were treated in vitro by model immune complexes that contained either HIV or hepatitis C virus (HCV) antigens. Unlike HCV antigen-containing complexes, the immune complexes that contained HIV antigens have been shown to activate peripheral blood T cells of uninfected donors under in vitro conditions. Both the antiviral antibodies and HIV antigen were involved in the activation process. The unique properties of the immune complexes formed by HIV antigens and antiviral antibodies are believed to result from the virus-specific antibody properties and molecular conformation of the antigen-antibody complex. PMID:27595830

  12. Neisseria lactamica antigens complexed with a novel cationic adjuvant

    PubMed Central

    Gaspar, Emanuelle B.; Rosetti, Andreza S.; Lincopan, Nilton; De Gaspari, Elizabeth

    2013-01-01

    Colonization of the nasopharynx by non-pathogenic Neisseria species, including N. lactamica, has been suggested to lead to the acquisition of natural immunity against Neisseria meningitidis in young children. The aim of this study was to identify a model complex of antigens and adjuvant for immunological preparation against N. meningitidis B, based on cross reactivity with N. lactamica outer membrane vesicles (OMV) antigens and the (DDA-BF) adjuvant. Complexes of 25 µg of OMV in 0.1 mM of DDA-BF were colloidally stable, exhibiting a mean diameter and charge optimal for antigen presentation. Immunogenicity tests for these complexes were performed in mice. A single dose of OMV/DDA-BF was sufficient to induce a (DTH) response, while the same result was achieved only after two doses of OMV/alum. In addition, to achieve total IgG levels that are similar to a single immunization with OMV/DDA-BF, it was necessary to give the mice a second dose of OMV/alum. Moreover, the antibodies induced from a single immunization with OMV/DDA-BF had an intermediate avidity, but antibodies with a similar avidity were only induced by OMV/alum after two immunizations. The use of this novel cationic adjuvant for the first time with a N. lactamica OMV preparation revealed good potential for future vaccine design. PMID:23296384

  13. Sodium ethylmercurithiosalicylate (Merthiolate) inhibition of antigen-antibody interactions in radioimmunoassay systems

    SciTech Connect

    Mori, M.; Wilber, J.F.

    1984-02-01

    Sodium ethylmercurithiosalicylate (Merthiolate), used for preservation of buffer solutions, was found to inhibit the antigen-antibody binding reactions in radioimmunoassay systems in a noncompetitive manner. This effect is attributable to mercury ion per se. 10 references. 2 figures.

  14. Effective Inhibition of Kb- and Db-Restricted Antigen Presentation in Primary Macrophages by Murine Cytomegalovirus

    PubMed Central

    LoPiccolo, Diane M.; Gold, Marielle C.; Kavanagh, Daniel G.; Wagner, Markus; Koszinowski, Ulrich H.; Hill, Ann B.

    2003-01-01

    Macrophages play an important role in murine cytomegalovirus (MCMV) infection in vivo, both in disseminating infection and in harboring latent virus. MCMV encodes three immune evasion genes (m4, m6, and m152) that interfere with the ability of cytotoxic T cells (CTL) to detect virus-infected fibroblasts, but the efficacy of immune evasion in macrophages has been controversial. Here we show that MCMV immune evasion genes function in H-2b primary bone marrow macrophages (BMMφ) in the same way that they do in fibroblasts. Metabolic labeling experiments showed that class I is retained in the endoplasmic reticulum by MCMV infection and associates with m4/gp34 to a similar extent in fibroblasts and BMMφ. We tested a series of Kb- and Db-restricted CTL clones specific for MCMV early genes against a panel of MCMV wild-type virus and mutants lacking m152, m4, or m6. MCMV immune evasion genes effectively inhibited antigen presentation. m152 appeared sufficient to abolish Db-restricted presentation in infected macrophages, as has been previously observed in infected fibroblasts. However, for inhibition of recognition of infected macrophages by Kb-restricted CTL, m4, m6, and m152 were all required. The contribution of m4 to inhibition of recognition appeared much more important in macrophages than in fibroblasts. Thus, MCMV immune evasion genes function effectively in primary macrophages to prevent CTL recognition of early antigens and show the same pattern of major histocompatibility complex class I allele discrimination as is seen in fibroblasts. Furthermore, for inhibition of Kb-restricted presentation, a strong synergistic effect was noted among m152, m4, and m6. PMID:12477835

  15. Leucocyte migration inhibition test with two gastric antigens in pernicious anaemia and in simple atrophic gastritis.

    PubMed

    Fixa, B; Komárková, O; Nozicka, Z

    1979-02-01

    Leucocyte migration inhibition test was used for evaluation of cell-mediated immunity in patients with pernicious anaemia (PA) and simple atrophic gastritis (SAG). As antigens microsomal antigen from gastric mucosa of swine foetus and relatively pure hog intrinsic factor (IF) were used. Significant differences were found between PA and SAG with microsomal antigen, but not with IF. It was concluded, that the microsomal antigen might be more active than IF. This observation could contribute to explane the higher incidence of parietal cell antibody than that of IF antibody in PA patients.

  16. Efficient major histocompatibility complex class I presentation of exogenous antigen upon phagocytosis by macrophages.

    PubMed Central

    Kovacsovics-Bankowski, M; Clark, K; Benacerraf, B; Rock, K L

    1993-01-01

    Antigens in extracellular fluids can be processed and presented with major histocompatibility complex (MHC) class I molecules by a subset of antigen presenting cells (APCs). Chicken egg ovalbumin (Ova) linked to beads was presented with MHC class I molecules by these cells up to 10(4)-fold more efficiently than soluble Ova. This enhanced presentation was observed with covalently or noncovalently linked Ova and with beads of different compositions. A key parameter in the activity of these conjugates was the size of the beads. The APC that is responsible for this form of presentation is a macrophage. These cells internalize the antigen constructs through phagocytosis, since cytochalasin B inhibited presentation. Processing of the antigen and association with MHC class I molecules appears to occur intracellularly as presentation was observed under conditions where there was no detectable release of peptides into the extracellular fluids. When injected in vivo in C57BL/6 mice, Ova-beads, but not soluble Ova, primed CD4- CD8+ cytotoxic T lymphocytes (CTLs). Similar results were obtained in BALB/c mice immunized with beta-galactosidase-beads. The implications of these findings for development of nonliving vaccines that stimulate CTL immunity are discussed. PMID:8506338

  17. IL-35 inhibits HBV antigen-specific IFN-γ-producing CTLs in vitro.

    PubMed

    Li, Xuefen; Tian, Li; Dong, Yuejiao; Zhu, Qiaoyun; Wang, Yiyin; Han, Wenzheng; Liu, Xia; Ni, Qin; Chen, Yu; Li, Lanjuan

    2015-09-01

    Interleukin (IL)-35 is an inhibitory cytokine consisting of IL-12A and Epstein-Barr virus-induced gene 3 (Ebi3) and is required by regulatory T-cells (Tregs) for maximal activity. During chronic hepatitis B virus (HBV) infection, Tregs have immunosuppressive effects on HBV-specific T helper (Th) cells, yet little is known about the complex regulation of Tregs and their contribution to the inadequate immune system response to the virus. In the present study, we investigated whether IL-35 is involved in HBV-related cellular immune responses. Cluster of differentiation (CD)4(+) T-cells from peripheral blood were derived from healthy volunteers, resolved HBV individuals and chronic active hepatitis B patients and stimulated with CD3/28-conjugated beads. We analysed mRNA and protein levels of IL-35 and assessed the inhibitory effect of IL-35 on HBV core antigen-specific cytotoxic T lymphocytes (CTLs), dendritic cells (DCs) and effector T-cells (Teffs). Correlation analyses between liver inflammation and HBV DNA load were conducted. Results show that chronic HBV patients harbour significantly higher levels of Ebi3 mRNA and protein in CD4(+) T-cells compared with healthy volunteers and resolved HBV individuals. IL-35 suppressed the proliferation of HBV antigen-specific CTLs and interferon (IFN)-γ production in vitro. Ex vivo, IL-35 decreased the proliferation of CD4(+)CD45RA(+) naïve T-cells, especially in CD4(+)CD25(-)CD45RA(+) naïve Teffs. IL-35 inhibited the expansion of CD11c(+) DCs. Our data indicate that IL-35 is highly expressed in chronic HBV CD4(+) T-cells and plays an important role in the inhibition of the cellular immune response in chronic HBV.

  18. Inhibition of immune opsonin-independent phagocytosis by antibody to a pulmonary macrophage cell surface antigen

    SciTech Connect

    Parod, R.J.; Godleski, J.J.; Brain J.D.

    1986-03-15

    Unlike other hamster phagoycytes, hamster pulmonary macrophages (PM) avidly ingest albumin-coated latex particles in the absence of serum. They also possess a highly specific cell surface antigen. To evaluate the relationship between these two characteristics, PM were incubated with mouse monoclonal antibody directed against the PM antigen. After unbound antibody was removed, the amount of bound antibody and the phagocytic capability of PM were measured by flow cytometry and fluorescence microscopy. Maximum antibody binding produced a 25% inhibition of ingestion. Particle attachment was not affected. This effect was antigen specific, since neither a nonspecific mouse myeloma protein of the same subclass nor a mouse antibody that bound to another hamster surface antigen had any effect on binding or ingestion. If antigen-specific F(ab')/sub 2/ fragments were introduced both before and during the period of phagocytosis, the inhibition of particle ingestion approached 100%. Particle binding increased at low F(ab')/sub 2/ concentrations but declined at higher concentrations. Because calcium may play a role in the ingestion process, the effect of antibody on /sup 45/Ca uptake was evaluated. It was observed that antigen-specific F(ab')/sub 2/ fragments stimulated /sup 45/Ca uptake, whereas control antibodies did not. These results suggest that the antigen reacting with the anti-hamster PM monoclonal antibody is involved in immune opsonin-independent phagocytosis and that calcium participates in this phagocytic process.

  19. Inhibition of T-cell antigen receptor-mediated transmembrane signaling by protein kinase C activation.

    PubMed Central

    Abraham, R T; Ho, S N; Barna, T J; Rusovick, K M; McKean, D J

    1988-01-01

    The murine T-lymphoma cell line LBRM-33 is known to require synergistic signals delivered through the antigen receptor (Ti-CD3) complex, together with interleukin 1 (IL-1), for activation of IL-2 gene expression and IL-2 production. Although 12-O-tetradecanoylphorbol-13-acetate (TPA) was capable of replacing IL-1 as an activating stimulus under certain conditions, biologic studies indicated that TPA failed to synergize with Ti-CD3-dependent stimuli under conditions in which IL-1 was clearly active. Acute exposure to TPA and other active phorbol esters resulted in a concentration-dependent inhibition of the increases in phosphoinositide hydrolysis and intracellular free Ca2+ concentration stimulated by phytohemagglutinin or anti-Ti antibodies. TPA treatment induced no direct alteration of phospholipase C enzymatic activities in LBRM-33 cells. In contrast, both Ti-CD3 cross-linkage and TPA rapidly stimulated the phosphorylation of identical CD3 complex polypeptides, presumably via activation of protein kinase C. Exposure of LBRM-33 cells to TPA resulted in a time-dependent, partial down-regulation of surface Ti-CD3 expression. Thus, TPA treatment inhibited the responsiveness of LBRM-33 cells to Ti-CD3-dependent stimuli by inducing an early desensitization of Ti-CD3 receptors, followed by a decrease in membrane receptor expression. These studies indicate that phorbol esters deliver bidirectional signals that both inhibit Ti-CD3-dependent phosphoinositide hydrolysis and augment IL-2 production in LBRM-33 cells. Images PMID:2977423

  20. Pathophysiology of Antigen 85 in Patients with Active Tuberculosis: Antigen 85 Circulates as Complexes with Fibronectin and Immunoglobulin G

    PubMed Central

    Bentley-Hibbert, Stuart I.; Quan, Xin; Newman, Thomas; Huygen, Kris; Godfrey, Henry P.

    1999-01-01

    Antigen 85 (Ag85) complex proteins are major secretory products of Mycobacterium tuberculosis and induce strong cellular and humoral immune responses in infected experimental animals and human beings. We have previously shown that nanogram doses of these 30- to 32-kDa fibronectin-binding proteins inhibit local expression of delayed hypersensitivity by a T-cell fibronectin-dependent mechanism. Circulating levels of Ag85 might be expected to be elevated in patients with active tuberculosis and possibly to play a role in systemic anergy in these patients. To test this hypothesis, Ag85 was measured in serum and urine by a monoclonal antibody-based dot immunobinding assay in 56 patients and controls with known skin test reactivity. Median serum Ag85 levels were 50- to 150-fold higher in patients with active tuberculosis than in patients with active M. avium-intracellulare disease or other nontuberculous pulmonary disease or in healthy controls (P < 0.001). The median and range of serum Ag85 in patients with active tuberculosis was not significantly different between skin test-positive and -negative subjects. Patients with active M. avium disease could be distinguished from those with disease due to M. tuberculosis by monoclonal anti-Ag85 antibodies of appropriate specificities. No increases in urinary Ag85 were detected in any patient, regardless of the Ag85 level in serum. Chromatographic analysis and immunoprecipitation studies of serum revealed that Ag85 existed in the serum of these patients complexed to either fibronectin or immunoglobulin G (IgG). Uncomplexed circulating Ag85 was demonstrable in serum from fewer than 20% of patients with active tuberculosis. In patients with active tuberculosis, Ag85 is therefore likely to circulate primarily as complexes with plasma fibronectin and IgG rather than in unbound form. The existence of Ag85 complexes with plasma proteins would account for its lack of urinary clearance. PMID:9916062

  1. Mapping Epitopes on a Protein Antigen by the Proteolysis of Antigen-Antibody Complexes

    NASA Astrophysics Data System (ADS)

    Jemmerson, Ronald; Paterson, Yvonne

    1986-05-01

    A monoclonal antibody bound to a protein antigen decreases the rate of proteolytic cleavage of the antigen, having the greatest effect on those regions involved in antibody contact. Thus, an epitope can be identified by the ability of the antibody to protect one region of the antigen more than others from proteolysis. By means of this approach, two distinct epitopes, both conformationally well-ordered, were characterized on horse cytochrome c.

  2. Vaccinia Virus A35R Inhibits MHC Class II Antigen Presentation

    PubMed Central

    Rehm, Kristina E.; Connor, Ramsey F.; Jones, Gwendolyn J.B.; Yimbu, Kenneth; Roper, Rachel L.

    2009-01-01

    The Vaccinia virus gene A35R (Copenhagen designation) is highly conserved in mammalian-tropic poxviruses and is an important virulence factor, but its function was unknown. We show herein that A35 does not affect viral infectivity, apoptosis induction, or replication; however, we found that A35 significantly inhibited MHC class II-restricted antigen presentation, immune priming of T lymphocytes, and subsequent chemokine and cytokine synthesis. A35 localized to endosomes and reduced the amount of a model antigenic peptide displayed in the cleft of class II MHC. In addition, A35 decreased VV specific T cell responses in vivo. Thus, this is the first report identifying a function for the A35 protein in virulence as well as the first report identifying a VV gene that inhibits peptide antigen presentation. PMID:19954808

  3. Phospholipase treatment of accessory cells that have been exposed to antigen selectively inhibits antigen-specific Ia-restricted, but not allospecific, stimulation of T lymphocytes.

    PubMed Central

    Falo, L D; Benacerraf, B; Rock, K L

    1986-01-01

    The corecognition of antigen and class II major histocompatibility complex (MHC) molecules (Ia molecules) by the T-cell receptor is a cell surface event. Before antigen is recognized, it must be taken up, processed, and displayed on the surface of an Ia-bearing accessory cell (antigen-presenting cell, APC). The exact nature of antigen processing and the subsequent associations of antigen with the APC plasma membrane, Ia molecules, and/or the T-cell receptor are not well defined. To further analyze these events, we have characterized the processing and presentation of the soluble polypeptide antigen bovine insulin. We found that this antigen requires APC-dependent processing, as evidenced by the inability of metabolically inactivated APCs to present native antigen to antigen plus Ia-specific T-T hybridomas. The ability of the same APCs to present antigen after uptake and processing showed that this antigen subsequently becomes stably associated with the APC plasma membrane. To characterize the basis for this association, we analyzed its sensitivity to enzymatic digestion. APCs exposed to antigen, treated with phospholipase A2, and then immediately fixed lost the ability to stimulate bovine insulin plus I-Ad-specific hybridomas. In contrast, the ability of these same APCs to stimulate I-Ad allospecific hybridomas was unaffected. This effect of phospholipase is not mimicked by the broadly active protease Pronase, nor is there evidence for contaminating proteases in the phospholipase preparation. These results suggest that one consequence of antigen processing may be an antigen-lipid association that contributes to the anchoring of antigen to the APC membrane. The implications of this model are discussed. PMID:3529095

  4. Antigen-specific suppression of anti-influenza antibody production in man. Possible role of a membrane-antigen complex.

    PubMed

    McCaughan, G W; Brown, M H; Basten, A

    1985-03-01

    E rosette-forming (E+) cells from human secondary lymphoid tissue were incubated with high dose influenza A virus (Mem-Bel) in an attempt to generate suppressor T cells. Suppression was assayed by transferring the antigen-pulsed E+ cells into effector cultures consisting of E+ and E- cells stimulated with immunogenic amounts of either the inducing virus Mem-Bel) or the non-cross-reacting influenza B virus (B/HK). The transfer resulted in marked inhibition of IgG, IgA and IgM antibody production to Mem-Bel but not to the control antigen, B/HK virus. The suppressive effect was specific at the level of induction as well as expression since E+ cells exposed to high dose Mem-Bel could provide help to an effector culture containing E- cells and optimal dose of B/HK virus. However, metabolically active cells did not appear to be required for suppression. Thus, it could be elicited (a) after only 15 min incubation of E+ cells with high-dose virus and (b) by E+ cells exposed to irradiation, incubated in the presence of metabolic inhibitors, or disrupted by repeated freeze thawing. In contrast, treatment of E+ cells with pronase reversed the suppressive effect. Interestingly, virus heated to 70 degree C failed to induced suppression, while retailing the ability to elicit a normal helper response. Suppression induced by exposure to standard amounts of high-dose antigen was mediated by T cells of both helper/inducer (Leu-3a+) and suppressor/cytotoxic subsets (Leu-2a+), but not by B cells. Two groups of observations pointed to the B cell as the target of suppression. First, suppression could still be transferred to effector cultures in which helper T cells had been replaced by T cell-replacing factor or suppressor T cells removed by irradiation. Second, significant inhibition of antibody production was obtained when the transfer of antigen-pulsed E+ cells was delayed for up to 120 h after initiation of the effector culture. Taken together the results suggest that suppression in

  5. Phosphorothioate oligonucleotides inhibit the intrinsic tenase complex.

    PubMed

    Sheehan, J P; Lan, H C

    1998-09-01

    concentrations in a chromogenic assay. This activity was oligonucleotide sequence-independent but required the phosphorothioate backbone, suggesting that inhibition of intrinsic tenase is a general property of this class of oligonucleotides. These results are relevant to both the therapeutic use of phosphorothioate oligonucleotides and the potential design of inhibitors of the intrinsic tenase complex, a novel target for anticoagulation.

  6. Restricted diversity of antigen binding residues of antibodies revealed by computational alanine scanning of 227 antibody-antigen complexes.

    PubMed

    Robin, Gautier; Sato, Yoshiteru; Desplancq, Dominique; Rochel, Natacha; Weiss, Etienne; Martineau, Pierre

    2014-11-11

    Antibody molecules are able to recognize any antigen with high affinity and specificity. To get insight into the molecular diversity at the source of this functional diversity, we compiled and analyzed a non-redundant aligned collection of 227 structures of antibody-antigen complexes. Free energy of binding of all the residue side chains was quantified by computational alanine scanning, allowing the first large-scale quantitative description of antibody paratopes. This demonstrated that as few as 8 residues among 30 key positions are sufficient to explain 80% of the binding free energy in most complexes. At these positions, the residue distribution is not only different from that of other surface residues but also dependent on the role played by the side chain in the interaction, residues participating in the binding energy being mainly aromatic residues, and Gly or Ser otherwise. To question the generality of these binding characteristics, we isolated an antibody fragment by phage display using a biased synthetic repertoire with only two diversified complementarity-determining regions and solved its structure in complex with its antigen. Despite this restricted diversity, the structure demonstrated that all complementarity-determining regions were involved in the interaction with the antigen and that the rules derived from the natural antibody repertoire apply to this synthetic binder, thus demonstrating the robustness and universality of our results.

  7. Serum proteases alter the antigenicity of peptides presented by class I major histocompatibility complex molecules.

    PubMed Central

    Falo, L D; Colarusso, L J; Benacerraf, B; Rock, K L

    1992-01-01

    Any effect of serum on the antigenicity of peptides is potentially relevant to their use as immunogens in vivo. Here we demonstrate that serum contains distinct proteases that can increase or decrease the antigenicity of peptides. By using a functional assay, we show that a serum component other than beta 2-microglobulin enhances the presentation of ovalbumin peptides produced by cyanogen bromide cleavage. Three features of this serum activity implicate proteolysis: it is temperature dependent, it results in increased antigenicity in a low molecular weight peptide fraction, and it is inhibited by the protease inhibitor leupeptin. Conversely, presentation of the synthetic peptide OVA-(257-264) is inhibited by serum. This inhibition is unaffected by leupeptin but is blocked by bestatin, a protease inhibitor with distinct substrate specificities. Implications for peptide-based vaccine design and immunotherapy are discussed. PMID:1518868

  8. Human Antibodies to a Mr 155,000 Plasmodium falciparum Antigen Efficiently Inhibit Merozoite Invasion

    NASA Astrophysics Data System (ADS)

    Wahlin, Birgitta; Wahlgren, Mats; Perlmann, Hedvig; Berzins, Klavs; Bjorkman, Anders; Patarroyo, Manuel E.; Perlmann, Peter

    1984-12-01

    IgG from a donor clinically immune to Plasmodium falciparum malaria strongly inhibited reinvasion in vitro of human erythrocytes by the parasite. When added to monolayers of glutaraldehyde-fixed and air-dried erythrocytes infected with the parasite, this IgG also displayed a characteristic immunofluorescence restricted to the surface of infected erythrocytes. Elution of the IgG adsorbed to such monolayers gave an antibody fraction that was 40 times more efficient in the reinvasion inhibition assay (50% inhibition titer, <1 μ g/ml) than the original IgG preparation. The major antibody in this eluate was directed against a parasite-derived antigen of Mr 155,000 (Pf 155) deposited by the parasite in the erythrocyte membrane in the course of invasion. A detailed study of IgG fractions from 11 donors with acute P. falciparum malaria or clinical immunity revealed the existence of an excellent correlation between their capacities to stain the surface of infected erythrocytes, their titers in reinvasion inhibition, and the presence of antibodies to Pf 155 as detected by immunoblotting. No such correlations were seen when the IgG fractions were analyzed for immunofluorescence of intracellular parasites or for the presence of antibodies to other parasite antigens as detected by immunoprecipitation of [35S]methionine-labeled and NaDodSO4/PAGE-separated parasite extracts. The results suggest that Pf 155 has an important role in the process of erythrocyte infection and that host antibodies to this antigen may efficiently interfere with this process.

  9. Interleukin-10 inhibits antigen-induced cellular recruitment into the airways of sensitized mice.

    PubMed Central

    Zuany-Amorim, C; Hailé, S; Leduc, D; Dumarey, C; Huerre, M; Vargaftig, B B; Pretolani, M

    1995-01-01

    This report examines the effect of recombinant murine (rm) IL-10 on antigen-induced cellular recruitment into the airways of sensitized Balb/c mice. The intranasal instillation of 10 micrograms ovalbumin induced an early (6-24 h) increase in the number of neutrophils, and a late rise (24-96 h) in that of eosinophils in the bronchoalveolar lavage (BAL) fluid and bronchial tissue. A single intranasal instillation of 0.01-0.1 microgram of rmIL-10, administered concurrently with ovalbumin, but not 1 or 3 h thereafter, dose-dependently inhibited both airway neutrophilia and eosinophilia. This phenomenon was suppressed by treating the sensitized mice with 1 mg/mouse of a neutralizing anti-IL-10 mAb, which increased significantly ovalbumin-induced neutrophil and eosinophil accumulation in the BAL fluid. These results suggest that antigen stimulation may trigger the in vivo generation of IL-10, which, in turn, participates in the leukocyte infiltration into the airways. rmIL-10 also reduced TNF-alpha release in the BAL fluid observed 1 and 3 h after antigen challenge. Furthermore, the intranasal instillation of an anti-TNF-alpha antiserum to sensitized mice markedly reduced ovalbumin-induced neutrophil and eosinophil accumulation in the BAL fluid. These findings indicate that leukocyte infiltration into the airways of antigen-challenged mice is regulated by IL-10. Furthermore, inhibition of TNF-alpha production by rmIL-10 suggests that allergic airway inflammation and TNF-alpha formation are parallel events in this model. Images PMID:7769104

  10. Mutations which affect the inhibition of protein phosphatase 2A by simian virus 40 small-t antigen in vitro decrease viral transformation.

    PubMed Central

    Mungre, S; Enderle, K; Turk, B; Porrás, A; Wu, Y Q; Mumby, M C; Rundell, K

    1994-01-01

    Three independent point mutations within residues 97 to 103 of the simian virus 40-small-t antigen (small-t) greatly reduced the ability of purified small-t to inhibit protein phosphatase 2A in vitro. These mutations affected the interaction of small-t antigen with the protein phosphatase 2A A subunit translated in vitro, and a peptide from the region identified by these mutations released the A subunit from immune complexes. When introduced into virus, the mutations eliminated the ability of small-t to enhance viral transformation of growth-arrested rat F111 cells. In contrast, the mutant small-t antigens were unimpaired in the transactivation of the adenovirus E2 promoter, an activity which was reduced by a double mutation in small-t residues 43 and 45. Images PMID:8107228

  11. Aldehyde-mannan antigen complexes target the MHC class I antigen-presentation pathway.

    PubMed

    Apostolopoulos, V; Pietersz, G A; Gordon, S; Martinez-Pomares, L; McKenzie, I F

    2000-06-01

    Antigens such as MUC1 coupled to oxidized mannan lead to rapid and efficient MHC class I presentation to CD8+ cells and a preferential T1 response; after reduction there is class II presentation and a T2 immune response. We now show that the selective advantage of the oxidized mannan-MUC1 is due to the presence of aldehydes and not Schiff bases, and that oxidized mannan-MUC1 binds to the mannose and not scavenger receptors and is internalized and presented by MHC class I molecules 1,000 times more efficiently than when reduced. After internalization there is rapid access to the class I pathway via endosomes but not lysosomes, proteasomal processing and transport to the endoplasmic reticulum, Golgi apparatus and cell surface. Aldehydes cause rapid entry into the class I pathway, and can therefore direct the subsequent immune response.

  12. A novel T cell receptor single-chain signaling complex mediates antigen-specific T cell activity and tumor control

    PubMed Central

    Stone, Jennifer D.; Harris, Daniel T.; Soto, Carolina M.; Chervin, Adam S.; Aggen, David H.; Roy, Edward J.; Kranz, David M.

    2014-01-01

    Adoptive transfer of genetically modified T cells to treat cancer has shown promise in several clinical trials. Two main strategies have been applied to redirect T cells against cancer: 1) introduction of a full-length T cell receptor (TCR) specific for a tumor-associated peptide-MHC, or 2) introduction of a chimeric antigen receptor (CAR), including an antibody fragment specific for a tumor cell surface antigen, linked intracellularly to T cell signaling domains. Each strategy has advantages and disadvantages for clinical applications. Here, we present data on the in vitro and in vivo effectiveness of a single-chain signaling receptor incorporating a TCR variable fragment as the targeting element (referred to as TCR-SCS). This receptor contained a single-chain TCR (Vβ-linker-Vα) from a high-affinity TCR called m33, linked to the intracellular signaling domains of CD28 and CD3ζ. This format avoided mispairing with endogenous TCR chains, and mediated specific T cell activity when expressed in either CD4 or CD8 T cells. TCR-SCS-transduced CD8-negative cells showed an intriguing sensitivity, compared to full-length TCRs, to higher densities of less stable pepMHC targets. T cells that expressed this peptide-specific receptor persisted in vivo, and exhibited polyfunctional responses. Growth of metastatic antigen-positive tumors was significantly inhibited by T cells that expressed this receptor, and tumor cells that escaped were antigen loss variants. TCR-SCS receptors represent an alternative targeting receptor strategy that combines the advantages of single-chain expression, avoidance of TCR chain mispairing, and targeting of intracellular antigens presented in complex with MHC proteins. PMID:25082071

  13. Antigen processing by epidermal Langerhans cells correlates with the level of biosynthesis of major histocompatibility complex class II molecules and expression of invariant chain

    PubMed Central

    1990-01-01

    Two prior studies with a small number of T cell lines have shown that the presentation of native protein antigens by epidermal Langerhans cells (LC) is regulated. When freshly isolated, LC are efficient antigen-presenting cells (APC), but after a period of culture LC are inefficient or even inactive. The deficit in culture seems to be a selective loss in antigen processing, since cultured LC are otherwise rich in major histocompatibility complex (MHC) class II products and are active APC for alloantigens and mitogens, which do not require processing. We have extended the analysis by studying presentation to bulk populations of primed lymph node and a T-T hybrid. Only freshly isolated LC can be pulsed with the protein antigens myoglobin and conalbumin, but once pulsed, antigen is retained in an immunogenic form for at least 2 d. The acquisition of antigen, presumably as MHC-peptide complexes, is inhibited if the fresh LC are exposed to foreign protein in the presence of chloroquine or cycloheximide. The latter, in contrast, improves the efficacy of antigen pulsing in anti-Ig- stimulated B blasts. In additional studies of mechanism, we noted that both fresh and cultured LC endocytose similar amounts of an antigen, rhodamineovalbumin, into perinuclear granules. However, freshly isolated LC synthesize high levels class II MHC molecules and express higher amounts of the class II-associated invariant chain. Fresh LC are at least 5-10 times more active than many other cells types in the level of biosynthesis of MHC class II products. These findings provide a physiologic model in which newly synthesized MHC class II molecules appear to be the principal vehicle for effective antigen processing by APC of the dendritic cell lineage. Another APC, the B lymphoblast, does not appear to require newly synthesized MHC class II molecules for presentation. PMID:2121888

  14. Evaluation of the Secretor Status of ABO Blood Group Antigens in Saliva among Southern Rajasthan Population Using Absorption Inhibition Method

    PubMed Central

    Khajuria, Nidhi; Mamta; Ramesh, Gayathri

    2016-01-01

    Introduction The ABO blood group system was the significant element for forensic serological examination of blood and body fluids in the past before the wide adaptation of DNA typing. A significant proportion of individuals (80%) are secretors, meaning that antigens present in the blood are also found in other body fluids such as saliva. Absorption inhibition is one such method that works by reducing strength of an antiserum based on type and amount of antigen present in the stains. Aim To check the efficacy of identifying the blood group antigens in saliva and to know the secretor status using absorption inhibition method among southern Rajasthan population. Materials and Methods Blood and saliva samples were collected from 80 individuals comprising 20 individuals in each blood group. The absorption inhibition method was used to determine the blood group antigens in the saliva and then the results were correlated with the blood group of the collected blood sample. The compiled data was statistically analysed using chi-square test. Results Blood groups A & O revealed 100% secretor status for both males and females. While blood groups B and AB revealed 95% secretor status. Conclusion Secretor status evaluation of the ABO blood group antigen in saliva using absorption inhibition method can be a useful tool in forensic examination. PMID:27042574

  15. Antibody recognition of an immunogenic influenza hemagglutinin-human leukocyte antigen class II complex

    PubMed Central

    1991-01-01

    The A/Japan/57 influenza hemagglutin (HA) peptide HA 128-145, when bound by human histocompatibility leukocyte antigen-DRw11 cells, is recognized by the human CD4+ T cell clone V1. A rabbit antiserum has been raised against HA 128-145 which recognizes not only the free peptide, but also the HA 128-145/DRw11 complex on a solid matrix, in solution, or on the surface of viable cells. The detection of these complexes on viable cells was shown to be class II specific, DRw11 restricted, and commensurate with the level of DRw11 expression. The identity of DRw11 as the cell surface molecule binding HA 128-145 was confirmed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and tryptic peptide mapping. Using this antiserum HA 128-145/DRw11 complexes could be detected on the cell surface as soon as 30 min after the peptide was added, and increased up to 24 h. Dissociation kinetics showed these complexes were long-lived, with a half-life of approximately 14 h. This anti-HA peptide antiserum represents the first direct means of studying antigenic peptide-human leukocyte antigen class II complexes on the surface of living cells without the addition of a non-amino acid moiety to the peptide. The properties of this antiserum thus provide the potential to study naturally processed antigenic peptides as well as the mechanism of processing itself in a physiologically relevant system. PMID:2056278

  16. mTOR inhibition improves antitumor effects of vaccination with antigen-encoding RNA.

    PubMed

    Diken, Mustafa; Kreiter, Sebastian; Vascotto, Fulvia; Selmi, Abderraouf; Attig, Sebastian; Diekmann, Jan; Huber, Christoph; Türeci, Özlem; Sahin, Ugur

    2013-12-01

    Vaccination with in vitro transcribed RNA encoding tumor antigens is an emerging approach in cancer immunotherapy. Attempting to further improve RNA vaccine efficacy, we have explored combining RNA with immunomodulators such as rapamycin. Rapamycin, the inhibitor of mTOR, was used originally for immunosuppression. Recent reports in mouse systems, however, suggest that mTOR inhibition may enhance the formation and differentiation of the memory CD8(+) T-cell pool. Because memory T-cell formation is critical to the outcome of vaccination approaches, we studied the impact of rapamycin on the in vivo primed RNA vaccine-induced immune response using the chicken ovalbumin-expressing B16 melanoma model in C57BL/6 mice. Our data show that treatment with rapamycin at the effector-to-memory transition phase skews the vaccine-induced immune response toward the formation of a quantitatively and qualitatively superior memory pool and results in a better recall response. Tumor-infiltrating immune cells from these mice display a favorable ratio of effector versus suppressor cell populations. Survival of mice treated with the combined regimen of RNA vaccination with rapamycin is significantly longer (91.5 days) than that in the control groups receiving only one of these compounds (32 and 46 days, respectively). Our findings indicate that rapamycin enhances therapeutic efficacy of antigen-specific CD8(+) T cells induced by RNA vaccination, and we propose further clinical exploration of rapamycin as a component of immunotherapeutic regimens. PMID:24778131

  17. HIV infection of monocytes inhibits the T-lymphocyte proliferative response to recall antigens, via production of eicosanoids.

    PubMed Central

    Foley, P; Kazazi, F; Biti, R; Sorrell, T C; Cunningham, A L

    1992-01-01

    Human monocytes infected in vitro with human immunodeficiency virus (HIV) soon after adherence to plastic substrate demonstrated a significantly decreased ability to restimulate autologous immune T-lymphocyte proliferation after exposure to soluble (tetanus toxoid) and particulate [herpes simplex virus (HSV)] antigen. Incubation with the cyclo-oxygenase inhibitor, indomethacin (2-5 microM), prevented inhibition of antigen-stimulated lymphocyte proliferation. The inhibitory activity was identified in ultrafiltrates containing the low molecular weight fraction (less than 3000 MW) of supernatants from HIV-infected monocyte cultures. This activity was significantly and markedly reduced in similar ultrafiltrates prepared from indomethacin-treated cultures. Increased concentrations of prostaglandin E2 (PGE2) were detected in ultrafiltrates from HIV-infected monocyte cultures compared with uninfected cultures and cultures preincubated with indomethacin. Ultrafiltrates were inhibitory when added during the presentation of antigen to T lymphocytes but not when removed from monocyte cultures prior to the addition of lymphocytes. In addition, ultrafiltrates inhibited antigen-stimulated lymphocyte proliferation and PHA-induced lymphocyte proliferation to the same extent. These data indicate that cyclo-oxygenase products of arachidonic acid, including PGE2, are produced in excess by HIV-infected monocytes and that PGE2 and perhaps other cyclo-oxygenase products are implicated in the inhibition of antigen-stimulated lymphocyte proliferation via a direct effect on T lymphocytes. PMID:1572689

  18. Lysophosphatidic Acid (LPA) Receptor 5 Inhibits B Cell Antigen Receptor Signaling and Antibody Response1

    PubMed Central

    Shotts, Kristin; Donovan, Erin E.; Strauch, Pamela; Pujanauski, Lindsey M.; Victorino, Francisco; Al-Shami, Amin; Fujiwara, Yuko; Tigyi, Gabor; Oravecz, Tamas; Pelanda, Roberta; Torres, Raul M.

    2014-01-01

    Lysophospholipids have emerged as biologically important chemoattractants capable of directing lymphocyte development, trafficking and localization. Lysophosphatidic acid (LPA) is a major lysophospholipid found systemically and whose levels are elevated in certain pathological settings such as cancer and infections. Here, we demonstrate that BCR signal transduction by mature murine B cells is inhibited upon LPA engagement of the LPA5 (GPR92) receptor via a Gα12/13 – Arhgef1 pathway. The inhibition of BCR signaling by LPA5 manifests by impaired intracellular calcium store release and most likely by interfering with inositol 1,4,5-trisphosphate receptor activity. We further show that LPA5 also limits antigen-specific induction of CD69 and CD86 expression and that LPA5-deficient B cells display enhanced antibody responses. Thus, these data show that LPA5 negatively regulates BCR signaling, B cell activation and immune response. Our findings extend the influence of lysophospholipids on immune function and suggest that alterations in LPA levels likely influence adaptive humoral immunity. PMID:24890721

  19. Small molecule and peptide-mediated inhibition of Epstein-Barr virus nuclear antigen 1 dimerization

    SciTech Connect

    Kim, Sun Young; Song, Kyung-A; Kieff, Elliott; Kang, Myung-Soo

    2012-07-27

    Highlights: Black-Right-Pointing-Pointer Evidence that targeting EBNA1 dimer, an EBV onco-antigen, can be achievable. Black-Right-Pointing-Pointer A small molecule and a peptide as EBNA1 dimerization inhibitors identified. Black-Right-Pointing-Pointer Both inhibitors associated with EBNA1 and blocked EBNA1 DNA binding activity. Black-Right-Pointing-Pointer Also, prevented its dimerization, and repressed viral gene transcription. -- Abstract: Latent Epstein-Barr virus (EBV) infection is associated with human B cell lymphomas and certain carcinomas. EBV episome persistence, replication, and gene expression are dependent on EBV-encoded nuclear antigen 1 (EBNA1)'s DNA binding domain (DBD)/dimerization domain (DD)-mediated sequence-specific DNA binding activity. Homodimerization of EBNA1 is essential for EBNA1 DNA binding and transactivation. In this study, we characterized a novel small molecule EBNA1 inhibitor EiK1, screened from the previous high throughput screening (HTS). The EiK1 compound specifically inhibited the EBNA1-dependent, OriP-enhanced transcription, but not EBNA1-independent transcription. A Surface Plasmon Resonance Biacore assay revealed that EiK1 associates with EBNA1 amino acid 459-607 DBD/DD. Consistent with the SPR data, in vitro gel shift assays showed that EiK1 suppressed the activity of EBNA1 binding to the cognate familial repeats (FR) sequence, but not control RBP-J{kappa} binding to the J{kappa} site. Subsequently, a cross-linker-mediated in vitro multimerization assay and EBNA1 homodimerization-dependent yeast two-hybrid assay showed that EiK1 significantly inhibited EBNA1 dimerization. In an attempt to identify more highly specific peptide inhibitors, small peptides encompassing the EBNA1 DBD/DD were screened for inhibition of EBNA1 DBD-mediated DNA binding function. The small peptide P85, covering EBNA1 a.a. 560-574, significantly blocked EBNA1 DNA binding activity in vitro, prevented dimerization in vitro and in vivo, associated with

  20. Expression of conformationally constrained adhesion peptide in an antibody CDR loop and inhibition of natural killer cell cytotoxic activity by an antibody antigenized with the RGD motif.

    PubMed Central

    Zanetti, M; Filaci, G; Lee, R H; del Guercio, P; Rossi, F; Bacchetta, R; Stevenson, F; Barnaba, V; Billetta, R

    1993-01-01

    We report that an antibody engineered to express three Arg-Gly-Asp (RGD) repeats in the third complementarity-determining region of the heavy chain (antigenized antibody) efficiently inhibits the lysis of human erythroleukemia K-562 cells by natural killer (NK) cells. Synthetic peptides containing RGD did not inhibit. Inhibition was specific for the (RGD)3-containing loop and required simultaneous occupancy of the Fc receptor (CD16) on effector cells. The antigenized antibody inhibited other forms of cytotoxicity mediated by NK cells but not cytotoxicity mediated by major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL). A three-dimensional model of the engineered antibody loop shows the structure and physicochemical characteristics probably required for the ligand activity. The results indicate that an RGD motif is involved in the productive interaction between NK and target cells. Moreover, they show that peptide expression in the hypervariable loops of an antibody molecule is an efficient procedure for stabilizing oligopeptides within a limited spectrum of tertiary structures. This is a new approach towards imparting ligand properties to antibody molecules and can be used to study the biological function and specificity of short peptide motifs, including those involved in cell adhesion. Images PMID:8223447

  1. Complex Minigene Library Vaccination for Discovery of Pre-Erythrocytic Plasmodium T Cell Antigens

    PubMed Central

    Stone, Brad C.; Kas, Arnold; Billman, Zachary P.; Fuller, Deborah H.; Fuller, James T.; Shendure, Jay; Murphy, Sean C.

    2016-01-01

    Development of a subunit vaccine targeting liver-stage Plasmodium parasites requires the identification of antigens capable of inducing protective T cell responses. However, traditional methods of antigen identification are incapable of evaluating T cell responses against large numbers of proteins expressed by these parasites. This bottleneck has limited development of subunit vaccines against Plasmodium and other complex intracellular pathogens. To address this bottleneck, we are developing a synthetic minigene technology for multi-antigen DNA vaccines. In an initial test of this approach, pools of long (150 bp) antigen-encoding oligonucleotides were synthesized and recombined into vectors by ligation-independent cloning to produce two DNA minigene library vaccines. Each vaccine encoded peptides derived from 36 (vaccine 1) and 53 (vaccine 2) secreted or transmembrane pre-erythrocytic P. yoelii proteins. BALB/cj mice were vaccinated three times with a single vaccine by biolistic particle delivery (gene gun) and screened for interferon-γ-producing T cell responses by ELISPOT. Library vaccination induced responses against four novel antigens. Naïve mice exposed to radiation-attenuated sporozoites mounted a response against only one of the four novel targets (PyMDH, malate dehydrogenase). The response to PyMDH could not be recalled by additional homologous sporozoite immunizations but could be partially recalled by heterologous cross-species sporozoite exposure. Vaccination against the dominant PyMDH epitope by DNA priming and recombinant Listeria boosting did not protect against sporozoite challenge. Improvements in library design and delivery, combined with methods promoting an increase in screening sensitivity, may enable complex minigene screening to serve as a high-throughput system for discovery of novel T cell antigens. PMID:27070430

  2. The lectin Siglec-G inhibits dendritic cell cross-presentation by impairing MHC class I-peptide complex formation.

    PubMed

    Ding, Yuanyuan; Guo, Zhenhong; Liu, Yiqi; Li, Xia; Zhang, Qian; Xu, Xiongfei; Gu, Yan; Zhang, Yi; Zhao, Dezhi; Cao, Xuetao

    2016-10-01

    CD8α(+) dendritic cells (DCs) are specialized at cross-presenting extracellular antigens on major histocompatibility complex (MHC) class I molecules to initiate cytotoxic T lymphocyte (CTL) responses; however, details of the mechanisms that regulate cross-presentation remain unknown. We found lower expression of the lectin family member Siglec-G in CD8α(+) DCs, and Siglec-G deficient (Siglecg(-/-)) mice generated more antigen-specific CTLs to inhibit intracellular bacterial infection and tumor growth. MHC class I-peptide complexes were more abundant on Siglecg(-/-) CD8α(+) DCs than on Siglecg(+/+) CD8α(+) DCs. Mechanistically, phagosome-expressed Siglec-G recruited the phosphatase SHP-1, which dephosphorylated the NADPH oxidase component p47(phox) and inhibited the activation of NOX2 on phagosomes. This resulted in excessive hydrolysis of exogenous antigens, which led to diminished formation of MHC class I-peptide complexes for cross-presentation. Therefore, Siglec-G inhibited DC cross-presentation by impairing such complex formation, and our results add insight into the regulation of cross-presentation in adaptive immunity. PMID:27548433

  3. Mycobacterial antigen 85 complex (Ag85) as a target for ficolins and mannose-binding lectin.

    PubMed

    Świerzko, Anna S; Bartłomiejczyk, Marcin A; Brzostek, Anna; Łukasiewicz, Jolanta; Michalski, Mateusz; Dziadek, Jarosław; Cedzyński, Maciej

    2016-06-01

    The pattern recognition molecules (PRMs) able to activate complement via the lectin pathway are suspected to be involved in the interaction between pathogenic Mycobacteria and the host immune response. Recently, we have found strong interactions between 25 and 35kDa mycobacterial cell fractions and mannose-binding lectin (MBL) and ficolins. Here we demonstrate that two biologically important mycobacterial structures, mannosylated lipoarabinomannan (ManLAM) and the antigen 85 (Ag85) complex, induce activation of the lectin pathway of complement. The strong interaction of recombinant MBL with purified ManLAM was confirmed, but no binding of recombinant ficolins (ficolin-1, -2, -3) with this structure was observed. Interestingly, all PRMs tested reacted with the mycobacterial antigen 85 (Ag85) complex. Based on the use of specific inhibitors (mannan for MBL, acetylated bovine serum albumin for ficolin-1 and -2, Hafnia alvei PCM 1200 lipopolysaccharide for ficolin-3), we concluded that carbohydrate-recognition (MBL) and fibrinogen-like domains (ficolins) were involved in these interactions. Our results indicate that the mycobacterial antigen 85 complex is a target for ficolins and MBL. Furthermore, those PRMs also bound to fibronectin and therefore might influence the Ag85 complex-dependent interaction of Mycobacterium with the extracellular matrix. PMID:27141819

  4. Herpes Simplex Virus 1 Glycoprotein B and US3 Collaborate To Inhibit CD1d Antigen Presentation and NKT Cell Function ▿

    PubMed Central

    Rao, Ping; Pham, Hong Thanh; Kulkarni, Arpita; Yang, Yang; Liu, Xueqiao; Knipe, David M.; Cresswell, Peter; Yuan, Weiming

    2011-01-01

    Herpes simplex viruses (HSVs) are prevalent human pathogens that establish latency in human neuronal cells and efficiently evade the immune system. It has been a major medical challenge to eradicate them and, despite intensive efforts, an effective vaccine is not available. We previously showed that upon infection of antigen-presenting cells, HSV type 1 (HSV-1) rapidly and efficiently downregulates the major histocompatibility complex class I-like antigen-presenting molecule, CD1d, and potently inhibits its recognition by CD1d-restricted natural killer T (NKT) cells. It suppresses CD1d expression primarily by inhibiting its recycling to the cell surface after endocytosis. We identify here the viral glycoprotein B (gB) as the predominant CD1d-interacting protein. gB initiates the interaction with CD1d in the endoplasmic reticulum and stably associates with it throughout CD1d trafficking. However, an additional HSV-1 component, the serine-threonine kinase US3, is required for optimal CD1d downregulation. US3 expression in infected cells leads to gB enrichment in the trans-Golgi network (TGN) and enhances the relocalization of both gB and CD1d to this compartment, suggesting that following internalization CD1d is translocated from the endocytic pathway to the TGN by its association with gB. Importantly, both US3 and gB are required for efficient inhibition of CD1d antigen presentation and NKT cell activation. In summary, our results suggest that HSV-1 uses gB and US3 to rapidly inhibit NKT cell function in the initial antiviral response. PMID:21653669

  5. Experimental studies on the inhibition effects of 1000 Chinese medicinal herbs on the surface antigen of hepatitis B virus.

    PubMed

    Zheng, M; Zheng, Y

    1992-09-01

    The reverse passive hemagglutination inhibition test was used in the screening of 1000 Chinese materia medica for inhibitors of hepatitis B virus surface antigen. The herbal drugs were commercially available and the surface antigen was from sera of hepatitis B patients. 127 effective drugs were obtained from the survey of which 28 were highly inhibitory (8:1), 35 were moderately effective (4:1), and 64 mildly effective (2:1). Further experiments with varying dosages of the drug, dosages of HBsAg and duration of contact showed 10 drugs to be of optimal effect. PMID:1453758

  6. Identification of Small Molecule Proliferating Cell Nuclear Antigen (PCNA) Inhibitor That Disrupts Interactions with PIP-box Proteins and Inhibits DNA Replication*

    PubMed Central

    Punchihewa, Chandanamali; Inoue, Akira; Hishiki, Asami; Fujikawa, Yoshihiro; Connelly, Michele; Evison, Benjamin; Shao, Youming; Heath, Richard; Kuraoka, Isao; Rodrigues, Patrick; Hashimoto, Hiroshi; Kawanishi, Masanobu; Sato, Mamoru; Yagi, Takashi; Fujii, Naoaki

    2012-01-01

    We have discovered that 3,3′,5-triiodothyronine (T3) inhibits binding of a PIP-box sequence peptide to proliferating cell nuclear antigen (PCNA) protein by competing for the same binding site, as evidenced by the co-crystal structure of the PCNA-T3 complex at 2.1 Å resolution. Based on this observation, we have designed a novel, non-peptide small molecule PCNA inhibitor, T2 amino alcohol (T2AA), a T3 derivative that lacks thyroid hormone activity. T2AA inhibited interaction of PCNA/PIP-box peptide with an IC50 of ∼1 μm and also PCNA and full-length p21 protein, the tightest PCNA ligand protein known to date. T2AA abolished interaction of PCNA and DNA polymerase δ in cellular chromatin. De novo DNA synthesis was inhibited by T2AA, and the cells were arrested in S-phase. T2AA inhibited growth of cancer cells with induction of early apoptosis. Concurrently, Chk1 and RPA32 in the chromatin are phosphorylated, suggesting that T2AA causes DNA replication stress by stalling DNA replication forks. T2AA significantly inhibited translesion DNA synthesis on a cisplatin-cross-linked template in cells. When cells were treated with a combination of cisplatin and T2AA, a significant increase in phospho(Ser139)histone H2AX induction and cell growth inhibition was observed. PMID:22383522

  7. Concurrent feline immune-complex nephritis. Tubular antigen-positive and renal amyloidosis.

    PubMed

    Saegusa, S; Shimizu, F; Nagase, M; Kasegawa, A

    1979-08-01

    We describe tubular antigen-positive immune-complex nephritis in a case of feline renal amyloidosis. Amyloid deposition was observed in mesangial area, and thickening of capillary walls was shown in the majority of the glomeruli. This case was also characterized with typical fluorescent granular depositions of cat IgG and C3 along the glomerular capillary walls as seen in human membranous glomerulonephritis. The fluorescent pattern of tubular antigen was identical with that of IgG and C3. Electron micrograph showed the thickening and irregularity of glomerular basement membranes, fusion of foot processes, and deposits of electron-dense or sometimes translucent materials, mostly in the intramembranous location. The causal sequence of the coincidental deposition of amyloid and immune complexes is discussed. PMID:157110

  8. Recognition of Major Histocompatibility Complex Antigens on Cultured Human Biliary Epithelial Cells by Alloreactive Lymphocytes

    PubMed Central

    Saidman, Susan L.; Duquesnoy, Rene J.; Zeevi, Adriana; Fung, John J.; Starzl, Thomas E.; Demetris, A. Jake

    2010-01-01

    We have developed an in vitro system to study the interactions between biliary epithelium and lymphocytes using cultured human biliary epithelial cells. No class II antigens were detected by immunoperoxidase staining of the normal biliary epithelial cells, but alloactivated lymphocyte culture supernatants were able to induce class II expression. The activity of the supernatants was blocked with an anti-γ-interferon monoclonal antibody. In addition, recombinant human γ-interferon alone induced the expression of class II antigens and increased the intensity of class I staining of cultured biliary epithelial cells. Biliary epithelial cell–induced proliferation of alloreactive T lymphocytes demonstrated that the major histocompatibility complex molecules carry functional lymphocyte-activating determinants. The recognition of major histocompatibility complex determinants was confirmed by monoclonal antibody–blocking studies and by stimulation of an alloreactive T-cell clone. However, the biliary epithelial cells were much less potent stimulators than arterial endothelial cells tested in the same assay system. PMID:1704868

  9. Selective inhibition of deactivated mitochondrial complex I by biguanides.

    PubMed

    Matsuzaki, Satoshi; Humphries, Kenneth M

    2015-03-24

    Biguanides are widely used antihyperglycemic agents for diabetes mellitus and prediabetes treatment. Complex I is the rate-limiting step of the mitochondrial electron transport chain (ETC), a major source of mitochondrial free radical production, and a known target of biguanides. Complex I has two reversible conformational states, active and de-active. The deactivated state is promoted in the absence of substrates but is rapidly and fully reversed to the active state in the presence of NADH. The objective of this study was to determine the relative sensitivity of active/de-active complex I to biguanide-mediated inhibition and resulting superoxide radical (O₂(•⁻)) production. Using isolated rat heart mitochondria, we show that deactivation of complex I sensitizes it to metformin and phenformin (4- and 3-fold, respectively), but not to other known complex I inhibitors, such as rotenone. Mitochondrial O₂(•⁻) production by deactivated complex I was measured fluorescently by NADH-dependent 2-hydroxyethidium formation at alkaline pH to impede reactivation. Superoxide production was 260.4% higher than in active complex I at pH 9.4. However, phenformin treatment of de-active complex I decreased O₂(•⁻) production by 14.9%, while rotenone increased production by 42.9%. Mitochondria isolated from rat hearts subjected to cardiac ischemia, a condition known to induce complex I deactivation, were sensitized to phenformin-mediated complex I inhibition. This supports the idea that the effects of biguanides are likely to be influenced by the complex I state in vivo. These results demonstrate that the complex I active and de-active states are a determinant in biguanide-mediated inhibition.

  10. Complement fixing hepatitis B core antigen immune complexes in the liver of patients with HBs antigen positive chronic disease.

    PubMed Central

    Rizzetto, M; Bonino, F; Crivelli, O; Canese, M G; Verme, G

    1976-01-01

    One hundred and fifty-two biopsies from serologically HBsAg positive and negative patients with liver disease were studied in immunofluorescence: for the presence of the surface (HBs) and the core (HBc) antigenic determinants foeterminants of the hepatitis B virus, of immunoglobulins and complement (C) deposits, and for the capacity to fix human C. Circumstantial evidence is presented suggesting that HBc immune-complexes are a relevant feature in the establishment and progression of chronic HBSAg liver disease. C fixation by liver cells was shown in all HBC positive patients with chronic hepatitis; an active form was present in every case, except two with a persistent hepatitis, an inverse ratio of HBc to C binding fluorescence being noted between active chronic hepatitis and cirrhotic patients. HBc without C fixation was observed in only three patients in the incubation phase of infectious hepatitis. IgG deposits were often found in HBc containing, C fixing nuclei. No C binding or IgG deposits were observed in acute self-limited type B hepatitis, in serologically positive patients with normal liver or minimal histological lesions, with and without HBs cytoplasmic fluorescence in their biopsy, or in serologically negative individuals. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:1001973

  11. Sperm-related antigens, antibodies, and circulating immune complexes in sera of recently vasectomized men.

    PubMed Central

    Witkin, S S; Zelikovsky, G; Bongiovanni, A M; Geller, N; Good, R A; Day, N K

    1982-01-01

    Sera from 35 men were collected before and at timed intervals subsequent to vasectomy and examined for the presence of (a) antibody reactive with human spermatozoa, (b) sperm-related antigen, and (c) circulating immune complexes (CIC). Fewer than 10% of the men examined were ever positive for antisperm antibodies. However, sperm-related antigens were elevated in the sera of 18, 18, and 26% of the mean at 2 wk, 2 mo, and 4 mo postvasectomy, respectively. CIC were detected in the sera of some vasectomized men by three different assays. The CIC in patients' sera were precipitated with polyethylene glycol, dissociated, and the individual CIC components identified by an enzyme-linked immunosorbent assay. Most, but not all, of the CIC contained antigen reactive with antisperm immunoglobulin (Ig)G and some also contained complement components C3 and/or Clq. IgA was identified in some of the CIC positive for IgG and sperm antigen and two men had IgM-containing CIC. Analysis of the CIC by sucrose gradient centrifugation revealed them to be heterogeneous in size. PMID:7085887

  12. Structure of the Malaria Antigen AMA1 in Complex with a Growth-Inhibitory Antibody

    PubMed Central

    Bai, Tao; Kim, Hanna; Anders, Robin F; Foley, Michael; Batchelor, Adrian H

    2007-01-01

    Identifying functionally critical regions of the malaria antigen AMA1 (apical membrane antigen 1) is necessary to understand the significance of the polymorphisms within this antigen for vaccine development. The crystal structure of AMA1 in complex with the Fab fragment of inhibitory monoclonal antibody 1F9 reveals that 1F9 binds to the AMA1 solvent-exposed hydrophobic trough, confirming its importance. 1F9 uses the heavy and light chain complementarity-determining regions (CDRs) to wrap around the polymorphic loops adjacent to the trough, but uses a ridge of framework residues to bind to the hydrophobic trough. The resulting 1F9-AMA1–combined buried surface of 2,470 Å2 is considerably larger than previously reported Fab–antigen interfaces. Mutations of polymorphic AMA1 residues within the 1F9 epitope disrupt 1F9 binding and dramatically reduce the binding of affinity-purified human antibodies. Moreover, 1F9 binding to AMA1 is competed by naturally acquired human antibodies, confirming that the 1F9 epitope is a frequent target of immunological attack. PMID:17907804

  13. Investigation of vesicle-capsular plague antigen complex formation by elastic laser radiation scattering

    NASA Astrophysics Data System (ADS)

    Guseva, N. P.; Maximova, Irina S.; Romanov, Sergey V.; Shubochkin, L. P.; Tatarintsev, Sergey N.

    1991-05-01

    Recently a great deal of attention has been given to the investigation artificial lipid liposomes, due to their application as "containers" for directed transport of biologically active compounds into particular cells, organs and tissues for prophylaxis and therapy of infectious diseases. The use of traditional methods of liposome investigation, such as sedimentation, electrophoresis and chromatography is impeded by low liposome resistivity to different deformations. In conjunction with this, optical methods of laser light scattering are promising as they allow nondisturbing, precise and quick investigations. This paper describes the investigation of vesicle systems prepared from egg lecithin of Serva Corporation and their complexes with the capsular antigen of the plague microbe. The capsular antigen Fl was obtained from EV plague microbe grown at 37° C on Huttinger agar. Fl was isolated by gel-filtration on ASA-22 followed by freeze drying of the preparation. Angular dependences of polarized radiation scattering were measured for several liposome suspension samples in a saline solution before and after the interaction with the plague microbe capsular antigen. The aim of the investigation was to analyze the nature of mutual antigen arrangement in a liposome and to develop methods for measuring its inclusion percentage.

  14. Engineering an intracellular pathway for major histocompatibility complex class II presentation of antigens.

    PubMed Central

    Wu, T C; Guarnieri, F G; Staveley-O'Carroll, K F; Viscidi, R P; Levitsky, H I; Hedrick, L; Cho, K R; August, J T; Pardoll, D M

    1995-01-01

    The presentation of antigenic peptides by major histocompatibility complex (MHC) class II molecules to CD4+ T cells is critical to the function of the immune system. In this study, we have utilized the sorting signal of the lysosomal-associated membrane protein LAMP-1 to target a model antigen, human papillomavirus 16 E7 (HPV-16 E7), into the endosomal and lysosomal compartments. The LAMP-1 sorting signal reroutes the antigen into the MHC class II processing pathway, resulting in enhanced presentation to CD4+ cells in vitro. In vivo immunization experiments in mice demonstrated that vaccinia containing the chimeric E7/LAMP-1 gene generated greater E7-specific lymphoproliferative activity, antibody titers, and cytotoxic T-lymphocyte activities than vaccinia containing the wild-type HPV-16 E7 gene. These results suggest that specific targeting of an antigen to the endosomal and lysosomal compartments enhances MHC class II presentation and vaccine potency. Images Fig. 2 Fig. 3 PMID:8524826

  15. Comparison of peptide-major histocompatibility complex tetramers and dextramers for the identification of antigen-specific T cells.

    PubMed

    Dolton, G; Lissina, A; Skowera, A; Ladell, K; Tungatt, K; Jones, E; Kronenberg-Versteeg, D; Akpovwa, H; Pentier, J M; Holland, C J; Godkin, A J; Cole, D K; Neller, M A; Miles, J J; Price, D A; Peakman, M; Sewell, A K

    2014-07-01

    Fluorochrome-conjugated peptide-major histocompatibility complex (pMHC) multimers are widely used for flow cytometric visualization of antigen-specific T cells. The most common multimers, streptavidin-biotin-based 'tetramers', can be manufactured readily in the laboratory. Unfortunately, there are large differences between the threshold of T cell receptor (TCR) affinity required to capture pMHC tetramers from solution and that which is required for T cell activation. This disparity means that tetramers sometimes fail to stain antigen-specific T cells within a sample, an issue that is particularly problematic when staining tumour-specific, autoimmune or MHC class II-restricted T cells, which often display TCRs of low affinity for pMHC. Here, we compared optimized staining with tetramers and dextramers (dextran-based multimers), with the latter carrying greater numbers of both pMHC and fluorochrome per molecule. Most notably, we find that: (i) dextramers stain more brightly than tetramers; (ii) dextramers outperform tetramers when TCR-pMHC affinity is low; (iii) dextramers outperform tetramers with pMHC class II reagents where there is an absence of co-receptor stabilization; and (iv) dextramer sensitivity is enhanced further by specific protein kinase inhibition. Dextramers are compatible with current state-of-the-art flow cytometry platforms and will probably find particular utility in the fields of autoimmunity and cancer immunology. PMID:24673376

  16. Nitric oxide (NO) inhibits antigen-stimulated increases in vasoconstriction and glycogenolysis in perfused livers derived from sensitized rats

    SciTech Connect

    Hines, K.L.; Bates, J.N.; Fisher, R.A. )

    1991-03-11

    Recent studies in the authors laboratory demonstrated that infusion of antigen into perfused livers from sensitized rats produces increases in hepatic portal pressure, increases in hepatic glucose output and decreases in hepatic oxygen consumption. In the present study, effects of NO on these hepatic responses to antigen challenge were investigated. Infusion of NO into perfused livers from sensitized rats attenuated ovalbumin induced increases in hepatic portal pressure and glucose output approximately 85% and 90%, respectively, and abolished ovalbumin-induced decreases in hepatic oxygen consumption. The duration of ovalbumin-stimulated increases in hepatic portal pressure was reduced nearly 90% by NO. Similarly, infusion of NO into perfused livers from sensitized rats inhibited increases in hepatic portal pressure and glucose output in response to platelet-activating factor (PAF) nearly 80 and 90%, respectively. In contrast, NO inhibited completely hepatic vasoconstriction in response to phenylephrine without altering glycogenolytic responses to this {alpha}-adrenergic agonist. These results provide evidence for regulatory effects of NO on hemodynamic and glycogenolytic responses to antigen in perfused livers from sensitized rats. These observations support previous findings which suggest that hepatic responses to sensitizing antigen may be mediated by PAF or other autacoid mediators which stimulate glycogenolysis in liver by indirect mechanisms involving hepatic vasoconstriction.

  17. Ethanol Metabolism Alters Major Histocompatibility Complex Class I-Restricted Antigen Presentation In Liver Cells

    PubMed Central

    Osna, Natalia A.; White, Ronda L.; Thiele, Geoffrey M.; Donohue, Terrence M.

    2009-01-01

    The proteasome is a major enzyme that cleaves proteins for antigen presentation. Cleaved peptides traffic to the cell surface, where they are presented in the context of MHC class I. Recognition of these complexes by cytotoxic T lymphocytes is crucial for elimination of cells bearing “non-self” proteins. Our previous studies revealed that ethanol suppresses proteasome function in ethanol-metabolizing liver cells. We hypothesized that proteasome suppression reduces the hydrolysis of antigenic peptides, thereby decreasing the presentation of the peptide-MHC class I-complexes on the cell surface. To test this, we used the mouse hepatocyte cell line (CYP2E1/ADH-transfected HepB5 cells) or primary mouse hepatocytes, both derived from livers of C57Bl/6 mice, which present the ovalbumin peptide, SIINFEKL, complexed with H2Kb. To induce H2Kb expression, HepB5 cells were treated with interferon gamma (IFNγ) and then exposed to ethanol. In these cells, ethanol metabolism decreased not only proteasome activity, but also hydrolysis of the C-extended peptide, SIINFEKL-TE and the presentation of SIINFEKL-H2Kb complexes measured after the delivery of SIINFEKL-TE to cytoplasm. The suppressive effects of ethanol were, in part, attributed to ethanol-elicited impairment of IFNγ signaling. However, in primary hepatocytes, even in the absence of IFNγ, we observed a similar decline in proteasome activity and antigen presentation after ethanol exposure. We conclude that proteasome function is directly suppressed by ethanol metabolism and indirectly, by preventing the activating effects of IFNγ. Ethanol-elicited reduction in proteasome activity contributes to the suppression of SIINFEKL-H2Kb presentation on the surface of liver cells. Immune response to viral antigens plays a crucial role in the pathogenesis of hepatitis C or B viral infections (HCV and HBV, respectively). Professional antigen-presenting cells (dendritic cells and macrophages) are responsible for priming the

  18. Peptide-dependent Conformational Fluctuation Determines the Stability of the Human Leukocyte Antigen Class I Complex*

    PubMed Central

    Yanaka, Saeko; Ueno, Takamasa; Shi, Yi; Qi, Jianxun; Gao, George F.; Tsumoto, Kouhei; Sugase, Kenji

    2014-01-01

    In immune-mediated control of pathogens, human leukocyte antigen (HLA) class I presents various antigenic peptides to CD8+ T-cells. Long-lived peptide presentation is important for efficient antigen-specific T-cell activation. Presentation time depends on the peptide sequence and the stability of the peptide-HLA complex (pHLA). However, the determinant of peptide-dependent pHLA stability remains elusive. Here, to reveal the pHLA stabilization mechanism, we examined the crystal structures of an HLA class I allomorph in complex with HIV-derived peptides and evaluated site-specific conformational fluctuations using NMR. Although the crystal structures of various pHLAs were almost identical independent of the peptides, fluctuation analyses identified a peptide-dependent minor state that would be more tightly packed toward the peptide. The minor population correlated well with the thermostability and cell surface presentation of pHLA, indicating that this newly identified minor state is important for stabilizing the pHLA and facilitating T-cell recognition. PMID:25028510

  19. Stability of free and complexed human immunodeficiency virus type 1 antigen at 4 degrees C and at room temperature.

    PubMed Central

    Griffith, B P; Garner, R B; Chacko, T M

    1995-01-01

    Free and immune-complex-dissociated (ICD) human immunodeficiency virus type 1 (HIV-1) antigenemias in serum specimens stored at room temperature (RT) and 4 degrees C for 1 to 35 days were evaluated. At all time points examined, there was no significant loss in detectable levels of ICD HIV-1 antigen at either RT or 4 degrees C. Free HIV-1 antigen was not stable in serum samples stored at RT for more than 2 days but was stable in samples stored at 4 degrees C for up to 4 days. Loss of free antigen occurred more rapidly in samples with high antigen content at baseline. Use of the ICD antigen assay allowed accurate quantitation of antigen in samples stored at RT or 4 degrees C for as long as 1 month. PMID:7615753

  20. Pseudomonas aeruginosa Cif protein enhances the ubiquitination and proteasomal degradation of the transporter associated with antigen processing (TAP) and reduces major histocompatibility complex (MHC) class I antigen presentation.

    PubMed

    Bomberger, Jennifer M; Ely, Kenneth H; Bangia, Naveen; Ye, Siying; Green, Kathy A; Green, William R; Enelow, Richard I; Stanton, Bruce A

    2014-01-01

    Cif (PA2934), a bacterial virulence factor secreted in outer membrane vesicles by Pseudomonas aeruginosa, increases the ubiquitination and lysosomal degradation of some, but not all, plasma membrane ATP-binding cassette transporters (ABC), including the cystic fibrosis transmembrane conductance regulator and P-glycoprotein. The goal of this study was to determine whether Cif enhances the ubiquitination and degradation of the transporter associated with antigen processing (TAP1 and TAP2), members of the ABC transporter family that play an essential role in antigen presentation and intracellular pathogen clearance. Cif selectively increased the amount of ubiquitinated TAP1 and increased its degradation in the proteasome of human airway epithelial cells. This effect of Cif was mediated by reducing USP10 deubiquitinating activity, resulting in increased polyubiquitination and proteasomal degradation of TAP1. The reduction in TAP1 abundance decreased peptide antigen translocation into the endoplasmic reticulum, an effect that resulted in reduced antigen available to MHC class I molecules for presentation at the plasma membrane of airway epithelial cells and recognition by CD8(+) T cells. Cif is the first bacterial factor identified that inhibits TAP function and MHC class I antigen presentation.

  1. Cinacalcet inhibits neuroblastoma tumor growth and upregulates cancer-testis antigens

    PubMed Central

    Casalà, Carla; Briansó, Ferran; Castrejón, Nerea; Rodríguez, Eva; Suñol, Mariona; Carcaboso, Angel M.; Lavarino, Cinzia; Mora, Jaume; de Torres, Carmen

    2016-01-01

    The calcium–sensing receptor is a G protein-coupled receptor that exerts cell-type specific functions in numerous tissues and some cancers. We have previously reported that this receptor exhibits tumor suppressor properties in neuroblastoma. We have now assessed cinacalcet, an allosteric activator of the CaSR approved for clinical use, as targeted therapy for this developmental tumor using neuroblastoma cell lines and patient-derived xenografts (PDX) with different MYCN and TP53 status. In vitro, acute exposure to cinacalcet induced endoplasmic reticulum stress coupled to apoptosis via ATF4-CHOP-TRB3 in CaSR-positive, MYCN-amplified cells. Both phenotypes were partially abrogated by phospholipase C inhibitor U73122. Prolonged in vitro treatment also promoted dose- and time-dependent apoptosis in CaSR-positive, MYCN-amplified cells and, irrespective of MYCN status, differentiation in surviving cells. Cinacalcet significantly inhibited tumor growth in MYCN-amplified xenografts and reduced that of MYCN-non amplified PDX. Morphology assessment showed fibrosis in MYCN-amplified xenografts exposed to the drug. Microarrays analyses revealed up-regulation of cancer-testis antigens (CTAs) in cinacalcet-treated MYCN-amplified tumors. These were predominantly CTAs encoded by genes mapping on chromosome X, which are the most immunogenic. Other modulated genes upon prolonged exposure to cinacalcet were involved in differentiation, cell cycle exit, microenvironment remodeling and calcium signaling pathways. CTAs were up-regulated in PDX and in vitro models as well. Moreover, progressive increase of CaSR expression upon cinacalcet treatment was seen both in vitro and in vivo. In summary, cinacalcet reduces neuroblastoma tumor growth and up-regulates CTAs. This effect represents a therapeutic opportunity and provides surrogate circulating markers of neuroblastoma response to this treatment. PMID:26893368

  2. A novel inhibition ELISA for the detection and monitoring of Penicillium marneffei antigen in human serum.

    PubMed

    Prakit, K; Nosanchuk, J D; Pruksaphon, K; Vanittanakom, N; Youngchim, S

    2016-04-01

    The thermally dimorphic fungus Penicillium marneffei is a causative agent of penicilliosis marneffei, a disease considered to be an acquired immune deficiency syndrome (AIDS)-defining illness in Southeast Asia and southern China. We have developed an inhibition enzyme-linked immunosorbent assay (inh-ELISA) incorporating the yeast phase specific mannoprotein-binding monoclonal antibody 4D1 for the detection of P. marneffei infection. In our sample set, the test detected antigenemia in all 45 (100 %) patients with P. marneffei, with a mean antigen concentration of 4.32 μg/ml. No cross-reactivity in this assay was found using serum from 44 additional patients with other fungal infections, such as Aspergillus fumigatus, Cryptococcus neoformans, and Candida albicans, as well as 44 patients with bacterial infections, such as Mycobacterium tuberculosis and Streptococcus suis. Additionally, no reactivity occurred using serum from 31 human immunodeficiency virus (HIV)-infected patients without a history of fungal infections and 113 healthy controls residing in endemic areas. To investigate the potential of the inh-ELISA for disease monitoring, we followed the reduction in antigenemia in six patients who clinically responded to itraconazole and P. marneffei was no longer isolated from their blood or tissues. In contrast, we correlated increased concentrations of antigenemia in patients with relapsed P. marneffei infection with the progression of their clinical symptoms and the isolation of P. marneffei from their clinical specimens. In summary, the P. marneffei inh-ELISA is a promising new assay for the rapid diagnosis of P. marneffei, as well as a tool for evaluating clinical response and clearance of the fungus during treatment. PMID:26838686

  3. Immunostimulatory complexes containing Eimeria tenella antigens and low toxicity plant saponins induce antibody response and provide protection from challenge in broiler chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Immunostimulating complexes (ISCOMs) are unique multimolecular structures formed by encapsulating antigens, lipids and triterpene saponins and are one of the most successful antigen delivery systems for microbial antigens. In the current study, both the route of administration and the antigen conce...

  4. Major histocompatibility complex markers and red cell antibodies to the Rh (D) antigen. Absence of association.

    PubMed

    Kruskall, M S; Yunis, E J; Watson, A; Awdeh, Z; Alper, C A

    1990-01-01

    Between 20 and 35 percent of Rh(D) antigen-negative individuals do not develop antibodies to D even after multiple transfusions of Rh-positive red cells. To evaluate the possibility that antibody production after exposure to the D antigen was related to a major histocompatibility complex immune response gene, analysis of the HLA genotypes of 38 Rh-sensitized women and their families was performed. No significant deviations were found in the frequency of any individual HLA class I, II, or III allele or of any extended haplotype (fixed allelic combinations of HLA-B, HLA-DR, and the complement components BF, C2, C4A, and C4B). Type 1 errors due to the extreme allelic polymorphism of the HLA system, as well as the ethnic variation in patient groups, may have contributed to HLA allele-antibody responder relationships reported in earlier studies.

  5. Posttranscriptional Inhibition of Class I Major Histocompatibility Complex Presentation on Hepatocytes and Lymphoid Cells in Chronic Woodchuck Hepatitis Virus Infection

    PubMed Central

    Michalak, Tomasz I.; Hodgson, Paul D.; Churchill, Norma D.

    2000-01-01

    Woodchuck hepatitis virus (WHV), similar to human hepatitis B virus, causes acute liver inflammation that can progress to chronic hepatitis and hepatocellular carcinoma. WHV also invades cells of the host lymphatic system, where it persists for life. We report here that acute and chronic hepadnavirus hepatitis is characterized by a profound difference in the expression of class I major histocompatibility complex (MHC) molecules on the surface of infected hepatocytes and, notably, lymphoid cells. While acute WHV infection is accompanied by the enhanced hepatocyte surface presentation of class I MHC antigen and upregulated transcription of the relevant hepatic genes, inhibition of class I antigen display on liver cells is a uniform hallmark of chronic WHV infection. This inhibition in chronic hepatitis occurs despite augmented (as in acute infection) expression of hepatic genes for class I MHC heavy chain, β2-microglobulin, and transporters associated with antigen processing (TAP1 and TAP2). Further, the class I antigen inhibition is not related to the histological severity of hepatocellular injury, the extent of lymphocytic infiltrations, the level of intrahepatic gamma interferon induction, or the hepatic WHV load. Importantly, the antigen expression is also inhibited on organ lymphoid cells of chronically infected hosts. The results obtained in this study demonstrate that the defective presentation of class I MHC molecules on cells supporting persistent WHV replication is due to viral posttranscriptional interference. This event may diminish the susceptibility of infected hepatocytes to virus-specific T-cell-mediated elimination, hinder virus clearance, and deregulate the class I MHC-dependent functions of the host immune system. This multifarious effect could be critical for perpetuation of liver damage and evasion of the antiviral immunological surveillance in chronic infection and therefore could be supportive of hepadnavirus persistence. PMID:10775584

  6. A Cronobacter turicensis O1 Antigen-Specific Monoclonal Antibody Inhibits Bacterial Motility and Entry into Epithelial Cells

    PubMed Central

    Lehner, Angelika; Dietrich, Richard; Kleinsteuber, Ina; Canals, Rocío; Zurfluh, Katrin; Weiner, Kerstin; Märtlbauer, Erwin

    2014-01-01

    Cronobacter turicensis is an opportunistic foodborne pathogen that can cause a rare but sometimes lethal infection in neonates. Little is known about the virulence mechanisms and intracellular lifestyle of this pathogen. In this study, we developed an IgG monoclonal antibody (MAb; MAb 2G4) that specifically recognizes the O1 antigen of C. turicensis cells. The antilipopolysaccharide antibody bound predominantly monovalently to the O antigen and reduced bacterial growth without causing cell agglutination. Furthermore, binding of the antibody to the O1 antigen of C. turicensis cells caused a significant reduction of the membrane potential which is required to energize flagellar rotation, accompanied by a decreased flagellum-based motility. These results indicate that binding of IgG to the O antigen of C. turicensis causes a direct antimicrobial effect. In addition, this feature of the antibody enabled new insight into the pathogenicity of C. turicensis. In a tissue culture infection model, pretreatment of C. turicensis with MAb 2G4 showed no difference in adhesion to human epithelial cells, whereas invasion of bacteria into Caco-2 cells was significantly inhibited. PMID:25534937

  7. Analysis of tuberculous meningitis cases by an immunoblotting assay based on a mycobacterial antigen complex.

    PubMed Central

    Zou, Y L; Van Antwerpen, M P; Shi, G Q; Chen, Q X; Sindic, C J; Cocito, C

    1994-01-01

    Tuberculous meningitis cases were analyzed by an immunoblotting test based on Mycobacterium bovis BCG antigen complex A60. Anti-A60 immunoglobulin G (IgG) in cerebrospinal fluid (CSF) allowed early diagnosis, and concentrations decreased after recovery. In primary meningitis forms, anti-A60 IgGs were intrathecally synthesized and specific oligoclonal IgGs were present in CSF. In meningeal complications of pulmonary tuberculosis, there were matching titers of anti-A60 IgG in blood and CSF (mirror pattern). Correlation between CSF-restricted patterns and CSF pleocytosis was shown. Images PMID:7496976

  8. Parasite Manipulation of the Invariant Chain and the Peptide Editor H2-DM Affects Major Histocompatibility Complex Class II Antigen Presentation during Toxoplasma gondii Infection.

    PubMed

    Leroux, Louis-Philippe; Nishi, Manami; El-Hage, Sandy; Fox, Barbara A; Bzik, David J; Dzierszinski, Florence S

    2015-10-01

    Toxoplasma gondii is an obligate intracellular protozoan parasite. This apicomplexan is the causative agent of toxoplasmosis, a leading cause of central nervous system disease in AIDS. It has long been known that T. gondii interferes with major histocompatibility complex class II (MHC-II) antigen presentation to attenuate CD4(+) T cell responses and establish persisting infections. Transcriptional downregulation of MHC-II genes by T. gondii was previously established, but the precise mechanisms inhibiting MHC-II function are currently unknown. Here, we show that, in addition to transcriptional regulation of MHC-II, the parasite modulates the expression of key components of the MHC-II antigen presentation pathway, namely, the MHC-II-associated invariant chain (Ii or CD74) and the peptide editor H2-DM, in professional antigen-presenting cells (pAPCs). Genetic deletion of CD74 restored the ability of infected dendritic cells to present a parasite antigen in the context of MHC-II in vitro. CD74 mRNA and protein levels were, surprisingly, elevated in infected cells, whereas MHC-II and H2-DM expression was inhibited. CD74 accumulated mainly in the endoplasmic reticulum (ER), and this phenotype required live parasites, but not active replication. Finally, we compared the impacts of genetic deletion of CD74 and H2-DM genes on parasite dissemination toward lymphoid organs in mice, as well as activation of CD4(+) T cells and interferon gamma (IFN-γ) levels during acute infection. Cyst burdens and survival during the chronic phase of infection were also evaluated in wild-type and knockout mice. These results highlight the fact that the infection is influenced by multiple levels of parasite manipulation of the MHC-II antigen presentation pathway. PMID:26195549

  9. Immune response to Neospora caninum native antigens formulated with immune stimulating complexes in calves.

    PubMed

    Moore, D P; Echaide, I; Verna, A E; Leunda, M R; Cano, A; Pereyra, S; Zamorano, P I; Odeón, A C; Campero, C M

    2011-02-10

    The aim of this study was to compare the immune responses to live Neospora caninum tachyzoites and N. caninum native antigens formulated with immune stimulating complexes matrix (ISCOM-matrix) in calves. Fifteen calves were used in this study: 3 were intravenously inoculated with 1 × 10(8) live tachyzoites (Group A), 3 were inoculated twice with N. caninum native antigens formulated with ISCOMs (Group B); 3 with N. caninum native antigens in phosphate-buffered saline (PBS) (Group C); 3 received ISCOM-matrix (ISCOMs without antigen) (Group D) and 3 were negative controls receiving PBS (Group E). The last four groups were inoculated subcutaneously. The specific total IgG and its subtypes were analyzed by an indirect enzyme-linked immunosorbent assays (ELISAs) and by Western blot. IFN-γ levels in plasma was quantified using a commercial kit. All calves were challenged intravenously with 1 × 10(8) live tachyzoites at week 11 after receiving the first dose. Parasitemia was assessed in plasma samples by semi-nested PCR. Neospora-specific antibodies were detected in animals from Groups A and B in the week 2 after inoculation. The ELISA OD values were higher in Group B compared with Group A from weeks 6 to 11 (P<0.05). Analysis of the subisotype specific antibodies in experimentally infected calves revealed a predominant IgG(2) response; however, a predominant IgG(1) response was observed in animals inoculated with N. caninum native antigens formulated with ISCOM-matrix. Control calves remained seronegative until challenge infection. The pattern of bands by Western blot was similar when testing sera from animals in Groups A and B. The levels of IFN-γ production after respective immunization schedules were similar between Groups A and B. Neospora-DNA was detected in plasma samples shortly after intravenous challenge in calves from all groups including those receiving the experimental vaccine formulation. The duration of the parasitemia was similar in all groups. PMID

  10. Effective expansion of forkhead box P3⁺ regulatory T cells via early secreted antigenic target 6 and antigen 85 complex B from Mycobacterium tuberculosis.

    PubMed

    Wu, Ying-E; Du, Zhong-Ren; Cai, Ying-Mu; Peng, Wen-Guang; Zheng, Gao-Zhe; Zheng, Geng-Long; Wu, Li-Biao; Li, Ke

    2015-04-01

    The expansion of CD4+ CD25+ forkhead box (FOX)P3+ regulatory T (Treg) cells has been observed in patients with Mycobacterium (M.) tuberculosis; however, the mechanism of expansion remains to be elucidated. The aim of the present study was to examine the role of the early secreted antigenic target 6(ESAT‑6) and antigen 85 complex B (Ag85B) from M. tuberculosis on Treg cell expansion. To investigate the sensitivity of peripheral blood cultures to the M. tuberculosis ESAT‑6 and Ag85B antigens, the proportion of circulating CD4+ CD25+ FOXP3+ Treg cells was determined using flow cytometry and the levels of FOXP3 mRNA were determined using reverse transcription quantitative polymerase chain reaction. The mRNA levels of FOXP3 and the proportion of circulating CD4+ CD25+ FOXP3+ Treg cells were increased in multiplicitous drug‑resistant tuberculosis patients compared with those in healthy controls and patients with latent tuberculosis (TB) infection (LTBI) (P<0.001). The mycobacterial antigens ESAT‑6 and Ag85B increased the expansion of the CD4+ CD25+ FOXP3+ Treg cells and the mRNA levels of FOXP3 in healthy controls and LTBI patients compared with the effect of Bacillus Calmette‑Guerin (P<0.05). Additionally, the mRNA levels of FOXP3 were elevated in the LTBI patients following stimulations with the mycobacterial antigens (P=0.012). Therefore, the M. tuberculosis antigens ESAT‑6 and Ag85B induced CD4+ CD25+ FOXP3+ Treg‑cell expansion, particularly in patients with LTBI. These findings indicated that CD4+ CD25+ FOXP3+ Treg cells may have a primary role in the failure of the host immune system to eradicate M. tuberculosis.

  11. Inhibition of Mitochondrial Complex II by the Anticancer Agent Lonidamine.

    PubMed

    Guo, Lili; Shestov, Alexander A; Worth, Andrew J; Nath, Kavindra; Nelson, David S; Leeper, Dennis B; Glickson, Jerry D; Blair, Ian A

    2016-01-01

    The antitumor agent lonidamine (LND; 1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid) is known to interfere with energy-yielding processes in cancer cells. However, the effect of LND on central energy metabolism has never been fully characterized. In this study, we report that a significant amount of succinate is accumulated in LND-treated cells. LND inhibits the formation of fumarate and malate and suppresses succinate-induced respiration of isolated mitochondria. Utilizing biochemical assays, we determined that LND inhibits the succinate-ubiquinone reductase activity of respiratory complex II without fully blocking succinate dehydrogenase activity. LND also induces cellular reactive oxygen species through complex II, which reduced the viability of the DB-1 melanoma cell line. The ability of LND to promote cell death was potentiated by its suppression of the pentose phosphate pathway, which resulted in inhibition of NADPH and glutathione generation. Using stable isotope tracers in combination with isotopologue analysis, we showed that LND increased glutaminolysis but decreased reductive carboxylation of glutamine-derived α-ketoglutarate. Our findings on the previously uncharacterized effects of LND may provide potential combinational therapeutic approaches for targeting cancer metabolism. PMID:26521302

  12. Inhibition of Mitochondrial Complex II by the Anticancer Agent Lonidamine*

    PubMed Central

    Guo, Lili; Shestov, Alexander A.; Worth, Andrew J.; Nath, Kavindra; Nelson, David S.; Leeper, Dennis B.; Glickson, Jerry D.; Blair, Ian A.

    2016-01-01

    The antitumor agent lonidamine (LND; 1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid) is known to interfere with energy-yielding processes in cancer cells. However, the effect of LND on central energy metabolism has never been fully characterized. In this study, we report that a significant amount of succinate is accumulated in LND-treated cells. LND inhibits the formation of fumarate and malate and suppresses succinate-induced respiration of isolated mitochondria. Utilizing biochemical assays, we determined that LND inhibits the succinate-ubiquinone reductase activity of respiratory complex II without fully blocking succinate dehydrogenase activity. LND also induces cellular reactive oxygen species through complex II, which reduced the viability of the DB-1 melanoma cell line. The ability of LND to promote cell death was potentiated by its suppression of the pentose phosphate pathway, which resulted in inhibition of NADPH and glutathione generation. Using stable isotope tracers in combination with isotopologue analysis, we showed that LND increased glutaminolysis but decreased reductive carboxylation of glutamine-derived α-ketoglutarate. Our findings on the previously uncharacterized effects of LND may provide potential combinational therapeutic approaches for targeting cancer metabolism. PMID:26521302

  13. Antigen/IgG immune complex-primed mucosal mast cells mediate antigen-specific activation of co-cultured T cells

    PubMed Central

    Ding, Jie; Fang, Yu; Xiang, Zou

    2015-01-01

    Mast cells are proposed to be one of the targets for mucosal vaccine adjuvants. We previously demonstrated that mucosal adjuvants containing IgG immune complexes could activate connective tissue mast cells enhancing immune responses. Here we suggest that mucosal mast cells (MMC) may also contribute to augmentation of antigen-specific immune responses following treatment with antigens complexed with IgG. We demonstrated that both bone marrow-derived cultured MMC and tissue resident MMC incorporated ovalbumin (OVA) at a greater level in the presence of anti-OVA IgG. Co-culture of OVA/IgG-pulsed bone marrow-derived MMC with splenocytes from OT-II mice promoted OVA-specific activation and proliferation of T cells, a process known as cross-presentation. Furthermore, bone marrow-derived cultured MMC underwent apoptosis following treatment with IgG immune complexes, a feature that has been described as favouring phagocytosis of mast cells by professional antigen-presenting cells. PMID:25196548

  14. Labor Inhibits Placental Mechanistic Target of Rapamycin Complex 1 Signaling

    PubMed Central

    LAGER, Susanne; AYE, Irving L.M.H.; GACCIOLI, Francesca; RAMIREZ, Vanessa I.; JANSSON, Thomas; POWELL, Theresa L.

    2014-01-01

    Introduction Labor induces a myriad of changes in placental gene expression. These changes may represent a physiological adaptation inhibiting placental cellular processes associated with a high demand for oxygen and energy (e.g., protein synthesis and active transport) thereby promoting oxygen and glucose transfer to the fetus. We hypothesized that mechanistic target of rapamycin complex 1 (mTORC1) signaling, a positive regulator of trophoblast protein synthesis and amino acid transport, is inhibited by labor. Methods Placental tissue was collected from healthy, term pregnancies (n=15 no-labor; n=12 labor). Activation of Caspase-1, IRS1/Akt, STAT, mTOR, and inflammatory signaling pathways was determined by Western blot. NFκB p65 and PPARγ DNA binding activity was measured in isolated nuclei. Results Labor increased Caspase-1 activation and mTOR complex 2 signaling, as measured by phosphorylation of Akt (S473). However, mTORC1 signaling was inhibited in response to labor as evidenced by decreased phosphorylation of mTOR (S2448) and 4EBP1 (T37/46 and T70). Labor also decreased NFκB and PPARγ DNA binding activity, while having no effect on IRS1 or STAT signaling pathway. Discussion and conclusion Several placental signaling pathways are affected by labor, which has implications for experimental design in studies of placental signaling. Inhibition of placental mTORC1 signaling in response to labor may serve to down-regulate protein synthesis and amino acid transport, processes that account for a large share of placental oxygen and glucose consumption. We speculate that this response preserves glucose and oxygen for transfer to the fetus during the stressful events of labor. PMID:25454472

  15. Thalidomide inhibits granulocyte responses in healthy humans after ex vivo stimulation with bacterial antigens.

    PubMed

    Juffermans, N P; Verbon, A; Schultz, M J; Hack, C E; van Deventer, S J; Speelman, P; van der Poll, T

    2001-05-01

    Ingestion of thalidomide was associated with a reduction in the upregulation of the granulocyte activation marker CD11b and a reduced capacity to release elastase and lactoferrin after stimulation with lipopolysaccharide or lipoteichoic acid. A single oral dose of thalidomide attenuates neutrophil activation upon ex vivo stimulation with bacterial antigens.

  16. INTERACTION OF THE RHEUMATOID FACTOR WITH ANTIGEN-ANTIBODY COMPLEXES AND AGGREGATED GAMMA GLOBULIN

    PubMed Central

    Edelman, G. M.; Kunkel, H. G.; Franklin, E. C.

    1958-01-01

    The effect of highly purified rheumatoid factor on the precipitin reactions of various antigen-antibody systems was determined. The amount of nitrogen precipitated was increased over a broad range when the factor was added to ovalbumin, human albumin, or human gamma globulin, and the corresponding rabbit antibodies. In the zone of antigen excess, soluble antigen-antibody complexes were precipitated by rheumatoid factor. Soluble aggregates of human and rabbit gamma globulin, produced by heating at 63°C., treatment with urea plus mercaptoethanol or treatment with guanidine, also precipitated with rheumatoid factor. Ultracentrifugal analysis of dissolved specific precipitates showed the presence of aggregated gamma globulin. The sedimentation rate of reactive aggregates was greater than 20 S, and concentrated preparations free of the non-reactive 7 S gamma globulin could be prepared by various procedures of zone centrifugation. These aggregates showed a high inhibitory capacity in the sensitized sheep cell agglutination reaction. Solid gamma globulin, prepared by heat denaturation, also selectively adsorbed the rheumatoid factor, and removed or decreased the activity in the various precipitation and agglutination reactions. Elution of highly purified active preparations from the solid gamma globulin could be carried out with urea or acid buffers. Evidence for interaction between rheumatoid factor and low molecular weight gamma globulin without precipitation, was also obtained. This interaction appears to occur in the circulation of patients with rheumatoid arthritis. The question of whether the rheumatoid factor represents an antibody to gamma globulin was discussed. Points of similarity to the behavior of complement also were cited. PMID:13549644

  17. Crystal structure of human prostate-specific antigen in a sandwich antibody complex.

    PubMed

    Stura, Enrico A; Muller, Bruno H; Bossus, Marc; Michel, Sandrine; Jolivet-Reynaud, Colette; Ducancel, Frédéric

    2011-12-01

    Human prostate-specific antigen (PSA or human kallikrein-related peptidase 3) present in small quantities in the sera of healthy men becomes elevated in prostate cancer (PCa) and other prostate disorders. The ability to identify the free PSA fraction associated with PCa could increase the reliability of the PSA diagnostic test. Here we present the crystal structure of human PSA from seminal fluid in a sandwich complex with two monoclonal antibodies (mAbs). MAb 5D5A5 captures total PSA with exceptionally high affinity, and mAb 5D3D11 selectively discriminates between free PSA subforms that are more abundant in sera from patients with PCa. Although the antigen is not of seric origin, several insights into cancer diagnosis can be discerned from this complex. MAb 5D3D11 recognizes a PSA conformation different from that previously reported. Interacting with the kallikrein loop, the PSA N-linked glycan attached to asparagine 61 is an uncommonly complex sialated triantennary chain. O-linked glycosylation is observed at threonine 125. The description of how PSA subforms in prostatic fluid can be discriminated using pairs of antibodies is a first step in the design of new strategies that are capable of real discrimination among PSA subforms, which will lead to the formulation of more reliable diagnostic tests. In a companion article [Muller, B. H., Savatier, A., L'Hostis, G., Costa, N., Bossus, M., Michel, S., et al. (2011). In vitro affinity maturation of an anti-PSA antibody for prostate cancer diagnostic assay. J. Mol. Biol.], we describe engineering efforts to improve the affinity of mAb 5D3D11, a first step towards such goal. PMID:22037582

  18. Synthesis of biocompatible nanoparticle drug complexes for inhibition of mycobacteria

    NASA Astrophysics Data System (ADS)

    Bhave, Tejashree; Ghoderao, Prachi; Sanghavi, Sonali; Babrekar, Harshada; Bhoraskar, S. V.; Ganesan, V.; Kulkarni, Anjali

    2013-12-01

    Tuberculosis (TB) is one of the most critical infectious diseases affecting the world today. Current TB treatment involves six months long daily administration of four oral doses of antibiotics. Due to severe side effects and the long treatment, a patient's adherence is low and this results in relapse of symptoms causing an alarming increase in the prevalence of multi-drug resistant (MDR) TB. Hence, it is imperative to develop a new drug delivery technology wherein these effects can be reduced. Rifampicin (RIF) is one of the widely used anti-tubercular drugs (ATD). The present study discusses the development of biocompatible nanoparticle-RIF complexes with superior inhibitory activity against both Mycobacterium smegmatis (M. smegmatis) and Mycobacterium tuberculosis (M. tuberculosis). Iron oxide nanoparticles (NPs) synthesized by gas phase condensation and NP-RIF complexes were tested against M. smegmatis SN2 strain as well as M. tuberculosis H37Rv laboratory strain. These complexes showed significantly better inhibition of M. smegmatis SN2 strain at a much lower effective concentration (27.5 μg ml-1) as compared to neat RIF (125 μg ml-1). Similarly M. tuberculosis H37Rv laboratory strain was susceptible to both nanoparticle-RIF complex and neat RIF at a minimum inhibitory concentration of 0.22 and 1 μg ml-1, respectively. Further studies are underway to determine the efficacy of NPs-RIF complexes in clinical isolates of M. tuberculosis as well as MDR isolates.

  19. Downregulation of major histocompatibility complex antigens in invading glioma cells: stealth invasion of the brain.

    PubMed

    Zagzag, David; Salnikow, Konstantin; Chiriboga, Luis; Yee, Herman; Lan, Li; Ali, M Aktar; Garcia, Roberto; Demaria, Sandra; Newcomb, Elizabeth W

    2005-03-01

    Invasion into surrounding brain tissue is a fundamental feature of gliomas and the major reason for treatment failure. The process of brain invasion in gliomas is not well understood. Differences in gene expression and/or gene products between invading and noninvading glioma cells may identify potential targets for new therapies. To look for genes associated with glioma invasion, we first employed Affymetrix microarray Genechip technology to identify genes differentially expressed in migrating glioma cells in vitro and in invading glioma cells in vivo using laser capture microdissection. We observed upregulation of a variety of genes, previously reported to be linked to glioma cell migration and invasion. Remarkably, major histocompatiblity complex (MHC) class I and II genes were significantly downregulated in migrating cells in vitro and in invading cells in vivo. Decreased MHC expression was confirmed in migrating glioma cells in vitro using RT-PCR and in invading glioma cells in vivo by immunohistochemical staining of human and murine glioblastomas for beta2 microglobulin, a marker of MHC class I protein expression. To the best of our knowledge, this report is the first to describe the downregulation of MHC class I and II antigens in migrating and invading glioma cells, in vitro and in vivo, respectively. These results suggest that the very process of tumor invasion is associated with decreased expression of MHC antigens allowing glioma cells to invade the surrounding brain in a 'stealth'-like manner.

  20. Influence of ions on the antigen-antibody complex formation as measured by radioimmunoassay.

    PubMed

    Vader, H L; Geuskens, L M; Vink, C L

    1977-10-15

    In this study, using radioimmunoassay techniques, we found that ions at concentrations in the order of 0.1 molar influence the antigen-antibody complex formation. The angiotensin I/anti-angiotensin I reaction was studied in detail. Particularly bivalent cations and anions with a strong chaotropic effect (SCN-, I- and ClO4-) were found to influence strongly the specific immunological reaction. However, NO3- had also a remarkably strong influence. We found that the equilibrium constant, rather than the number of binding sites of the antibody, is influenced by the ions. It should be borne in mind that relatively high concentrations of electrolyte (as compared with the concentrations of antigen and antibody) show this effect. Consequently, this effect is of less practical importance for routine radioimmunoassay than is, for example, the effect of pH. However, this phenomenon shows that the radioimmunoassay technique might be valuable not only for quantization of very low hormone concentrations in biological fluids, but has also important potential applications in physical and protein chemistry. Particularly, the high sensitivity of this technique and the possibility of studying a homogeneous reaction system might give it advantages over other techniques.

  1. Yeast surface display of a noncovalent MHC class II heterodimer complexed with antigenic peptide.

    PubMed

    Boder, Eric T; Bill, Jerome R; Nields, Andrew W; Marrack, Philippa C; Kappler, John W

    2005-11-20

    Microbial protein display technologies have enabled directed molecular evolution of binding and stability properties in numerous protein systems. In particular, dramatic improvements to antibody binding affinity and kinetics have been accomplished using these tools in recent years. Examples of successful application of display technologies to other immunological proteins have been limited to date. Herein, we describe the expression of human class II major histocompatibility complex allele (MHCII) HLA-DR4 on the surface of Saccharomyces cerevisiae as a noncovalently associated heterodimer. The yeast-displayed MHCII is fully native as assessed by binding of conformationally specific monoclonal antibodies; failure of antibodies specific for empty HLA-DR4 to bind yeast-displayed protein indicates antigenic peptide is bound. This report represents the first example of a noncovalent protein dimer displayed on yeast and of successful display of wild-type MHCII. Results further point to the potential for using yeast surface display for engineering and analyzing the antigen binding properties of MHCII.

  2. Cancer-testis antigens and immunotherapy in the light of cancer complexity.

    PubMed

    Grizzi, F; Mirandola, L; Qehajaj, D; Cobos, E; Figueroa, J A; Chiriva-Internati, M

    2015-03-01

    The ability of immunotherapy to evoke successful antitumor immune responses has been well documented over the past decade. Despite abundant preclinical data, it is only with the recent approval by the Food and Drug Administration (FDA) of the drugs such as sipuleucel-T and ipilimumab that immunotherapy is finally being recognized as a viable alternative to traditional therapies for treatment of various cancers. Despite the ability of immunotherapy to elicit successful antitumor immune responses, its efficacy is hindered by several factors. Among these are the paucity of tumor-associated antigens (TAA) that can be used as effective targets and the systemic toxicities that often lead to treatment interruption. Indeed, such adverse effects, which can be immunological and/or parenchymal, can be particularly severe and even fatal to some patients. A family of TAA called cancer-testis antigens (CTA) has been identified and their encoding genes have been extensively investigated. CTA expression has been demonstrated in a variety of human cancer tissues, and at least 19 CTA have been found to elicit humoral and/or cellular immune responses in cancer patients. Here we discuss how CTA and immunotherapy will most likely play a major role in the cure of cancer in the light of cancer complexity.

  3. Sialic acid-modified antigens impose tolerance via inhibition of T-cell proliferation and de novo induction of regulatory T cells

    PubMed Central

    Perdicchio, Maurizio; Ilarregui, Juan M.; Verstege, Marleen I.; Cornelissen, Lenneke A. M.; Schetters, Sjoerd T. T.; Engels, Steef; Ambrosini, Martino; Kalay, Hakan; Veninga, Henrike; den Haan, Joke M. M.; van Berkel, Lisette A.; Samsom, Janneke N.; Crocker, Paul R.; Sparwasser, Tim; Berod, Luciana; Garcia-Vallejo, Juan J.; van Kooyk, Yvette; Unger, Wendy W. J.

    2016-01-01

    Sialic acids are negatively charged nine-carbon carboxylated monosaccharides that often cap glycans on glycosylated proteins and lipids. Because of their strategic location at the cell surface, sialic acids contribute to interactions that are critical for immune homeostasis via interactions with sialic acid-binding Ig-type lectins (siglecs). In particular, these interactions may be of importance in cases where sialic acids may be overexpressed, such as on certain pathogens and tumors. We now demonstrate that modification of antigens with sialic acids (Sia-antigens) regulates the generation of antigen-specific regulatory T (Treg) cells via dendritic cells (DCs). Additionally, DCs that take up Sia-antigen prevent formation of effector CD4+ and CD8+ T cells. Importantly, the regulatory properties endowed on DCs upon Sia-antigen uptake are antigen-specific: only T cells responsive to the sialylated antigen become tolerized. In vivo, injection of Sia-antigen–loaded DCs increased de novo Treg-cell numbers and dampened effector T-cell expansion and IFN-γ production. The dual tolerogenic features that Sia-antigen imposed on DCs are Siglec-E–mediated and maintained under inflammatory conditions. Moreover, loading DCs with Sia-antigens not only inhibited the function of in vitro–established Th1 and Th17 effector T cells but also significantly dampened ex vivo myelin-reactive T cells, present in the circulation of mice with experimental autoimmune encephalomyelitis. These data indicate that sialic acid-modified antigens instruct DCs in an antigen-specific tolerogenic programming, enhancing Treg cells and reducing the generation and propagation of inflammatory T cells. Our data suggest that sialylation of antigens provides an attractive way to induce antigen-specific immune tolerance. PMID:26941238

  4. CD4 binding to major histocompatibility complex class II antigens induces LFA-1-dependent and -independent homotypic adhesion of B lymphocytes.

    PubMed

    Kansas, G S; Cambier, J C; Tedder, T F

    1992-01-01

    T helper cells recognize processed antigen (Ag) in the context of major histocompatibility complex (MHC) class II antigens present on the surface of B cells and other Ag-presenting cells. This interaction is mediated through the T cell receptor complex with associate recognition of class II molecules by the CD4 molecule. In this study, the binding of a soluble recombinant CD4/Ig heavy chain fusion protein (CD4-gamma 3) or monoclonal antibody (mAb) to class II antigens on human B cells was shown to induce rapid and specific homotypic adhesion of B cells and most B lymphoblastoid cell lines. mAb reactive with CD4 inhibited CD4-gamma 3-induced adhesion and a mutant B lymphoblastoid cell line deficient in class II antigens failed to respond. Induction of homotypic adhesion was dependent on energy metabolism and a functional cytoskeleton, and class II+ pre-B cells did not exhibit adhesion in response to these stimuli, suggesting that cross-linking of class II molecules generated a transmembrane signal and did not simply aggregate cells. In addition, MHC class II-induced adhesion was Fc receptor independent, as 15 mAb of different Ig isotypes reactive with HLA-D or HLA-DQ gene products induced adhesion. Anti-class II mAb and CD4-gamma 3 were able to induce adhesion at concentrations as low as 10 ng/ml and 100 ng/ml, respectively. Suboptimal stimulation of B cell lines through HLA-D antigens induced homotypic adhesion that was dependent on the activation of LFA-1 (CD11a/CD18), and which could be blocked by specific mAb. However, at greater signal strengths, adhesion was not blocked by mAb against the known adhesion receptors, suggesting the induction of a novel adhesion pathway. Consistent with this, homotypic adhesion induced by engagement of MHC class II antigens was observed with LFA-1-deficient B cell lines, and was independent of CD49d or CD18 expression. Thus, the direct engagement of B cell class II antigens by CD4 is likely to generate transmembrane signals which

  5. Major histocompatibility complex class II antigens are required for both cytokine production and proliferation induced by mercuric chloride in vitro.

    PubMed

    Hu, H; Möller, G; Abedi-Valugerdi, M

    1997-10-01

    Autoimmune diseases induced by mercuric chloride are genetically determined, at least one gene being major histocompatibility complex (MHC)-linked. Previously, we showed that in vitro mercury stimulation induced a high proliferative response in lymphocytes from susceptible mice (high-responders) and that the proliferative response could be restored in lymphocytes from low-responders by pretreating the cells with mercury. We also found that the continuous presence of mercury induced IL-2 and IFN-gamma production, while pretreatment with mercury induced IL-4 production. In this study, we showed that anti-MHC class II monoclonal antibodies blocked both the mercury-induced proliferative responses in lymphocytes from high-responders and the restored proliferative responses in low-responders. In addition, anti-MHC class II antibodies also inhibited the mercury-induced IL-2, IFN-gamma and IL-4 cytokine production in vitro. The results demonstrate that MHC class II antigens directly participate in mercury-induced cytokine production and cell activation, and are required at the onset of the initiation.

  6. Selection of peptide inhibitors of interactions involved in complex protein assemblies: association of the core and surface antigens of hepatitis B virus.

    PubMed Central

    Dyson, M R; Murray, K

    1995-01-01

    As an example for studies of contacts involved in complex biological systems, peptide ligands that bind to the core antigen of hepatitis B virus (HBcAg) have been selected from a random hexapeptide library displayed on filamentous phage. Affinity-purified phage bearing aa sequence LLGRMK, or some related sequences, bound full-length or truncated HBcAg but did not bind denatured HBcAg. The long (L), but not the short (S), hepatitis B virus envelope polypeptide, when synthesized in an in vitro system, bound firmly to HBcAg, indicating that interaction between HBcAg and the pre-S region of the L polypeptide is critical for virus morphogenesis. This interaction was inhibited by peptide ALLGRMKG, suggesting that this and related small molecules may inhibit viral assembly. Images Fig. 2 PMID:7892246

  7. Inhibition of Escherichia coli respiratory complex I by Zn(2+).

    PubMed

    Schulte, Marius; Mattay, Dinah; Kriegel, Sebastien; Hellwig, Petra; Friedrich, Thorsten

    2014-10-14

    The energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, couples NADH oxidation and quinone reduction with the translocation of protons across the membrane. Complex I exhibits a unique L shape with a peripheral arm extending in the aqueous phase and a membrane arm embedded in the lipid bilayer. Both arms have a length of ∼180 Å. The electron transfer reaction is catalyzed by a series of cofactors in the peripheral arm, while the membrane arm catalyzes proton translocation. We used the inhibition of complex I by zinc to shed light on the coupling of the two processes, which is not yet understood. Enzyme kinetics revealed the presence of two high-affinity binding sites for Zn(2+) that are attributed to the proton translocation pathways in the membrane arm. Electrochemically induced Fourier transform infrared difference spectroscopy demonstrated that zinc binding involves at least two protonated acidic residues. Electron paramagnetic resonance spectroscopy showed that one of the cofactors is only partially reduced by NADH in the presence of Zn(2+). We conclude that blocking the proton channels in the membrane arm leads to a partial block of the electron transfer in the peripheral arm, indicating the long-range coupling between both processes.

  8. Characterization of anti-P monoclonal antibodies directed against the ribosomal protein–RNA complex antigen and produced using Murphy Roths large autoimmune-prone mice

    PubMed Central

    Sato, H; Onozuka, M; Hagiya, A; Hoshino, S; Narita, I; Uchiumi, T

    2015-01-01

    Autoantibodies, including anti-ribosomal P proteins (anti-P), are thought to be produced by an antigen-driven immune response in systemic lupus erythematosus (SLE). To test this hypothesis, we reconstituted the ribosomal antigenic complex in vitro using human P0, phosphorylated P1 and P2 and a 28S rRNA fragment covering the P0 binding site, and immunized Murphy Roths large (MRL)/lrp lupus mice with this complex without any added adjuvant to generate anti-P antibodies. Using hybridoma technology, we subsequently obtained 34 clones, each producing an anti-P monoclonal antibody (mAb) that recognized the conserved C-terminal tail sequence common to all three P proteins. We also obtained two P0-specific monoclonal antibodies, but no antibody specific to P1, P2 or rRNA fragment. Two types of mAbs were found among these anti-P antibodies: one type (e.g. 9D5) reacted more strongly with the phosphorylated P1 and P2 than that with their non-phosphorylated forms, whereas the other type (e.g. 4H11) reacted equally with both phosphorylated and non-phosphorylated forms of P1/P2. Both 9D5 and 4H11 inhibited the ribosome/eukaryotic elongation factor-2 (eEF-2)-coupled guanosine triphosphate (GTP)ase activity. However, preincubation with a synthetic peptide corresponding to the C-terminal sequence common to all three P proteins, but not the peptide that lacked the last three C-terminal amino acids, mostly prevented the mAb-induced inhibition of GTPase activity. Thus, at least two types of anti-P were produced preferentially following the immunization of MRL mice with the reconstituted antigenic complex. Presence of multiple copies of the C-termini, particularly that of the last three C-terminal amino acid residues, in the antigenic complex appears to contribute to the immunogenic stimulus. PMID:25255895

  9. Cytokines Regulate Proteolysis in Major Histocompatibility Complex Class II–Dependent Antigen Presentation by Dendritic Cells

    PubMed Central

    Fiebiger, Edda; Meraner, Paul; Weber, Ekkehard; Fang, I-Fei; Stingl, Georg; Ploegh, Hidde; Maurer, Dieter

    2001-01-01

    Endo/lysosomal proteases control two key events in antigen (Ag) presentation: the degradation of protein Ag and the generation of peptide-receptive major histocompatibility complex (MHC) class II molecules. Here we show that the proinflammatory cytokines tumor necrosis factor α and interleukin (IL)-1β rapidly increase the activity of cathepsin (cat) S and catB in human dendritic cells (DCs). As a consequence, a wave of MHC class II sodium dodecyl sulfate stable dimer formation ensues in a catS-dependent fashion. In contrast, the antiinflammatory cytokine IL-10 renders DCs incapable of upregulating catS and catB activity and in fact, attenuates the level of both enzymes. Suppressed catS and catB activity delays MHC class II sodium dodecyl sulfate stable dimer formation and impairs Ag degradation. In DCs exposed to tetanus toxoid, IL-10 accordingly reduces the number of MHC class II–peptide complexes accessible to tetanus toxoid–specific T cell receptors, as analyzed by measuring T cell receptor downregulation in Ag-specific T cell clones. Thus, the control of protease activity by pro- and antiinflammatory cytokines is an essential feature of the Ag presentation properties of DCs. PMID:11304549

  10. Accurate structure prediction of peptide–MHC complexes for identifying highly immunogenic antigens

    SciTech Connect

    Park, Min-Sun; Park, Sung Yong; Miller, Keith R.; Collins, Edward J.; Lee, Ha Youn

    2013-11-01

    Designing an optimal HIV-1 vaccine faces the challenge of identifying antigens that induce a broad immune capacity. One factor to control the breadth of T cell responses is the surface morphology of a peptide–MHC complex. Here, we present an in silico protocol for predicting peptide–MHC structure. A robust signature of a conformational transition was identified during all-atom molecular dynamics, which results in a model with high accuracy. A large test set was used in constructing our protocol and we went another step further using a blind test with a wild-type peptide and two highly immunogenic mutants, which predicted substantial conformational changes in both mutants. The center residues at position five of the analogs were configured to be accessible to solvent, forming a prominent surface, while the residue of the wild-type peptide was to point laterally toward the side of the binding cleft. We then experimentally determined the structures of the blind test set, using high resolution of X-ray crystallography, which verified predicted conformational changes. Our observation strongly supports a positive association of the surface morphology of a peptide–MHC complex to its immunogenicity. Our study offers the prospect of enhancing immunogenicity of vaccines by identifying MHC binding immunogens.

  11. Identification of small molecules that inhibit the interaction of TEM8 with anthrax protective antigen using a FRET assay.

    PubMed

    Cryan, Lorna M; Habeshian, Kaiane A; Caldwell, Thomas P; Morris, Meredith T; Ackroyd, P Christine; Christensen, Kenneth A; Rogers, Michael S

    2013-07-01

    Tumor marker endothelial 8 (TEM8) is a receptor for the protective antigen (PA) component of anthrax toxin. TEM8 is upregulated on endothelial cells lining the blood vessels within tumors, compared with normal blood vessels. A number of studies have demonstrated a pivotal role for TEM8 in developmental and tumor angiogenesis. We have also shown that targeting the anthrax receptors with a mutated form of PA inhibits angiogenesis and tumor formation in vivo. Here we describe the development and testing of a high-throughput fluorescence resonance energy transfer assay to identify molecules that strongly inhibit the interaction of PA and TEM8. The assay we describe is sensitive and robust, with a Z' value of 0.8. A preliminary screen of 2310 known bioactive library compounds identified ebselen and thimerosal as inhibitors of the TEM8-PA interaction. These molecules each contain a cysteine-reactive transition metal, and complementary studies indicate that their inhibition of interaction is due to modification of a cysteine residue in the TEM8 extracellular domain. This is the first demonstration of a high-throughput screening assay that identifies inhibitors of TEM8, with potential application for antianthrax and antiangiogenic diseases.

  12. Pharmacologic IKK/NF-κB inhibition causes antigen presenting cells to undergo TNFα dependent ROS-mediated programmed cell death

    NASA Astrophysics Data System (ADS)

    Tilstra, Jeremy S.; Gaddy, Daniel F.; Zhao, Jing; Davé, Shaival H.; Niedernhofer, Laura J.; Plevy, Scott E.; Robbins, Paul D.

    2014-01-01

    Monocyte-derived antigen presenting cells (APC) are central mediators of the innate and adaptive immune response in inflammatory diseases. As such, APC are appropriate targets for therapeutic intervention to ameliorate certain diseases. APC differentiation, activation and functions are regulated by the NF-κB family of transcription factors. Herein, we examined the effect of NF-κB inhibition, via suppression of the IκB Kinase (IKK) complex, on APC function. Murine bone marrow-derived macrophages and dendritic cells (DC), as well as macrophage and DC lines, underwent rapid programmed cell death (PCD) after treatment with several IKK/NF-κB inhibitors through a TNFα-dependent mechanism. PCD was induced proximally by reactive oxygen species (ROS) formation, which causes a loss of mitochondrial membrane potential and activation of a caspase signaling cascade. NF-κB-inhibition-induced PCD of APC may be a key mechanism through which therapeutic targeting of NF-κB reduces inflammatory pathologies.

  13. Heat-solubilized curry spice curcumin inhibits antibody-antigen interaction in in vitro studies: A possible therapy to alleviate autoimmune disorders

    PubMed Central

    Kurien, Biji T.; D'Souza, Anil; Scofield, R. Hal

    2010-01-01

    Chronic and complex autoimmune diseases, currently treated palliatively with immunosuppressives, require multi-targeted therapy for greater effectiveness. The naturally occurring polyphenol curcumin has emerged as a powerful “nutraceutical” that interacts with multiple targets to regress diseases safely and inexpensively. Upto 8 g/day of curcumin for 18 months was non-toxic to humans. However, curcumin's utility is limited by its aqueous insolubility. We have demonstrated a heat-mediated 12-fold increase in curcumin's aqueous solubility. Here we show by, SDS-PAGE and SPR, that heat-solubilized curcumin binds to proteins. Based on this binding we hypothesized that heat-solubilized curcumin or turmeric would prevent autoantibody targeting of cognate autoantigens. Heat-solubilized curcumin/turmeric significantly decreased binding of autoantibodies from Sjögren's syndrome (SS) (up to 43/70 % respectively) and SLE (up to 52/70 % respectively) patients as well as an animal model of SS (up to 50/60 % respectively) to their cognate antigens. However, inhibition was not specific to autoimmunity. Heat-solubilized curcumin/turmeric also inhibited binding of polyclonal anti-spectrin to spectrin (50/56 % respectively). Thus, we suggest that the multifaceted heat-solubilized curcumin can ameliorate autoimmune disorders. In addition, the non-toxic curcumin could serve as a new protein stain in SDS-PAGE even though it is less sensitive than the Coomassie system which involves toxic chemicals. PMID:20146265

  14. Maternal inhibition of hepatitis B surface antigen gene expression in transgenic mice correlates with de novo methylation.

    PubMed

    Hadchouel, M; Farza, H; Simon, D; Tiollais, P; Pourcel, C

    Differential modifications of the genome during gametogenesis result in a functional difference between the paternal and maternal genomes at the moment of fertilization. A possible cause of this imprinting is the methylation of DNA. The insertion of foreign DNA into transgenic mice allows the tagging of regions that are differentially methylated during gametogenesis. We describe here a transgenic mouse strain in which the expression of the hepatitis B surface antigen gene is irreversibly repressed following its passage through the female germ line. This inhibition is accompanied by the methylation of all the HpaII and HhaI sites within the foreign gene, which we have shown to be integrated into a site on chromosome 13. The irreversibility reported here contrasts with what is found with other transgenic mice sequences which are reversibly methylated after passage through the male or female germ line, though in both cases methylation appears to be important in the imprinting process.

  15. Carcinoembryonic antigen promotes colorectal cancer progression by targeting adherens junction complexes

    SciTech Connect

    Bajenova, Olga; Chaika, Nina; Tolkunova, Elena; Davydov-Sinitsyn, Alexander; Gapon, Svetlana; Thomas, Peter; O’Brien, Stephen

    2014-06-10

    Oncomarkers play important roles in the detection and management of human malignancies. Carcinoembryonic antigen (CEA, CEACAM5) and epithelial cadherin (E-cadherin) are considered as independent tumor markers in monitoring metastatic colorectal cancer. They are both expressed by cancer cells and can be detected in the blood serum. We investigated the effect of CEA production by MIP101 colorectal carcinoma cell lines on E-cadherin adherens junction (AJ) protein complexes. No direct interaction between E-cadherin and CEA was detected; however, the functional relationships between E-cadherin and its AJ partners: α-, β- and p120 catenins were impaired. We discovered a novel interaction between CEA and beta-catenin protein in the CEA producing cells. It is shown in the current study that CEA overexpression alters the splicing of p120 catenin and triggers the release of soluble E-cadherin. The influence of CEA production by colorectal cancer cells on the function of E-cadherin junction complexes may explain the link between the elevated levels of CEA and the increase in soluble E-cadherin during the progression of colorectal cancer. - Highlights: • Elevated level of CEA increases the release of soluble E-cadherin during the progression of colorectal cancer. • CEA over-expression alters the binding preferences between E-cadherin and its partners: α-, β- and p120 catenins in adherens junction complexes. • CEA produced by colorectal cancer cells interacts with beta-catenin protein. • CEA over-expression triggers the increase in nuclear beta-catenin. • CEA over-expression alters the splicing of p120 catenin protein.

  16. Transcriptional activation by simian virus 40 large T antigen: interactions with multiple components of the transcription complex.

    PubMed Central

    Gruda, M C; Zabolotny, J M; Xiao, J H; Davidson, I; Alwine, J C

    1993-01-01

    Simian virus 40 (SV40) large T antigen is a potent transcriptional activator of both viral and cellular promoters. Within the SV40 late promoter, a specific upstream element necessary for T-antigen transcriptional activation is the binding site for transcription-enhancing factor 1 (TEF-1). The promoter structure necessary for T-antigen-mediated transcriptional activation appears to be simple. For example, a promoter consisting of upstream TEF-1 binding sites (or other factor-binding sites) and a downstream TATA or initiator element is efficiently activated. It has been demonstrated that transcriptional activation by T antigen does not require direct binding to the DNA; thus, the most direct effect that T antigen could have on these simple promoters would be through protein-protein interactions with either upstream-bound transcription factors, the basal transcription complex, or both. To determine whether such interactions occur, full-length T antigen or segments of it was fused to the glutathione-binding site (GST fusions) or to the Gal4 DNA-binding domain (amino acids 1 to 147) (Gal4 fusions). With the GST fusions, it was found that TEF-1 and the TATA-binding protein (TBP) bound different regions of T antigen. A GST fusion containing amino acids 5 to 172 (region T1) efficiently bound TBP. TEF-1 bound neither region T1 nor a region between amino acids 168 and 373 (region T2); however, it bound efficiently to the combined region (T5) containing amino acids 5 to 383.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:8423815

  17. Crystal Structure of Plasmodium knowlesi Apical Membrane Antigen 1 and Its Complex with an Invasion-Inhibitory Monoclonal Antibody

    PubMed Central

    van der Eijk, Marjolein; Thomas, Alan W.; Singh, Balbir; Kocken, Clemens H. M.

    2015-01-01

    The malaria parasite Plasmodium knowlesi, previously associated only with infection of macaques, is now known to infect humans as well and has become a significant public health problem in Southeast Asia. This species should therefore be targeted in vaccine and therapeutic strategies against human malaria. Apical Membrane Antigen 1 (AMA1), which plays a role in Plasmodium merozoite invasion of the erythrocyte, is currently being pursued in human vaccine trials against P. falciparum. Recent vaccine trials in macaques using the P. knowlesi orthologue PkAMA1 have shown that it protects against infection by this parasite species and thus should be developed for human vaccination as well. Here, we present the crystal structure of Domains 1 and 2 of the PkAMA1 ectodomain, and of its complex with the invasion-inhibitory monoclonal antibody R31C2. The Domain 2 (D2) loop, which is displaced upon binding the Rhoptry Neck Protein 2 (RON2) receptor, makes significant contacts with the antibody. R31C2 inhibits binding of the Rhoptry Neck Protein 2 (RON2) receptor by steric blocking of the hydrophobic groove and by preventing the displacement of the D2 loop which is essential for exposing the complete binding site on AMA1. R31C2 recognizes a non-polymorphic epitope and should thus be cross-strain reactive. PkAMA1 is much less polymorphic than the P. falciparum and P. vivax orthologues. Unlike these two latter species, there are no polymorphic sites close to the RON2-binding site of PkAMA1, suggesting that P. knowlesi has not developed a mechanism of immune escape from the host’s humoral response to AMA1. PMID:25886591

  18. [Basic and clinical evaluation of an immunoradiometric competitive inhibition assay for 2----6 sialyl Lewis a antigen--1. Evaluation of assay conditions and normal values].

    PubMed

    Imura, H; Takahashi, T; Matsuda, T; Yoshida, O; Ohkura, H; Seitetsu, Y; Seino, Y; Ishii, M; Kuwabara, M; Ariyoshi, Y

    1989-06-01

    We describe an immunoradiometric competitive inhibition assay of the serum levels of the 2----6 sialyl Lewisa antigen, using "SLA 2-6 Otsuka" kits. The assay required only duplicate 50-microliters samples, and the concentration of 2----6 sialyl Lewisa antigen in serum was determined by reference to a standard curve ranging from 0 to 160 arbitrary U/ml. The intra- and inter-assays reproducibilities were good and analytical recovery of antigen were excellent. The serum levels of the antigen were highly dependent on the Lewis blood types of the tested individuals; i.e., the levels of the antigen in the sera of the Lewisa-b- individuals were significantly lower than those of the antigen obtained with the Lewisa+b- and Lewisa-b+ individuals. The cut-off value (42 U/ml) was obtained as mean + 2SD, which was carefully calculated from the antigen levels in sera of the non-Lewisa-b- individuals.

  19. Antigen Transfer from Exosomes to Dendritic Cells as an Explanation for the Immune Enhancement Seen by IgE Immune Complexes

    PubMed Central

    Henningsson, Frida; Heyman, Birgitta; Conrad, Daniel H.

    2014-01-01

    IgE antigen complexes induce increased specific T cell proliferation and increased specific IgG production. Immediately after immunization, CD23+ B cells capture IgE antigen complexes, transport them to the spleen where, via unknown mechanisms, dendritic cells capture the antigen and present it to T cells. CD23, the low affinity IgE receptor, binds IgE antigen complexes and internalizes them. In this study, we show that these complexes are processed onto B-cell derived exosomes (bexosomes) in a CD23 dependent manner. The bexosomes carry CD23, IgE and MHC II and stimulate antigen specific T-cell proliferation in vitro. When IgE antigen complex stimulated bexosomes are incubated with dendritic cells, dendritic cells induce specific T-cell proliferation in vivo, similar to IgE antigen complexes. This suggests that bexosomes can provide the essential transfer mechanism for IgE antigen complexes from B cells to dendritic cells. PMID:25330118

  20. Inhibition of host immune response in colorectal cancer: Human leukocyte antigen-G and beyond

    PubMed Central

    Garziera, Marica; Toffoli, Giuseppe

    2014-01-01

    Colorectal cancer (CRC) is one of the most diffuse cancers worldwide and is still a clinical burden. Increasing evidences associate CRC clinical outcome to immune contexture represented by adaptive immune cells. Their type, density and location are summarized in the Immune Score that has been shown to improve prognostic prediction of CRC patients. The non-classical MHC class I human leukocyte antigen-G (HLA-G), is a crucial tumor-driven immune escape molecule involved in immune tolerance. HLA-G and soluble counterparts are able to exert inhibitory functions by direct interactions with inhibitory receptors present on both innate cells such as natural killer cells, and adaptive immune cells as cytotoxic T and B lymphocytes. HLA-G may play a prominent role in CRC strategies to avoid host immunosurveillance. This review highlights the current knowledge on HLA-G contribution in CRC, in related inflammatory diseases and in other type of cancers and disorders. HLA-G genetic setting (specific haplotypes, genotypes and alleles frequencies) and association with circulating/soluble profiles was highlighted. HLA G prognostic and predictive value in CRC was investigated in order to define a novel prognostic immune biomarker in CRC. PMID:24744572

  1. Outcome of limbic encephalitis with VGKC-complex antibodies: relation to antigenic specificity.

    PubMed

    Malter, M P; Frisch, C; Schoene-Bake, J C; Helmstaedter, C; Wandinger, K P; Stoecker, W; Urbach, H; Surges, R; Elger, C E; Vincent, A V; Bien, C G

    2014-09-01

    In limbic encephalitis (LE) with antibodies (Abs) to the voltage-gated potassium channel complex (VGKC), the Abs are mainly directed to the VGKC-complex proteins, leucine-rich, glioma inactivated 1 protein (LGI1) or contactin-associated protein-like 2 (CASPR-2) or neither. Here, we relate the outcomes of VGKC-LE patients to the presence of Abs to LGI1, CASPR-2 or neither antigen (LGI1/CASPR-2-Ab(-)). Clinical, neuropsychology and MRI data were obtained from patient records for all LE patients from the Bonn Epilepsy Centre positive for VGKC-Abs by radioimmunoprecipitation assay between 2002 and 2011. Eighteen VGKC-LE patients were identified: nine patients (50 %) had LGI1-Abs, three (16 %) had CASPR-2-Abs; and six (33 %) were negative for both LGI1- and CASPR-2-Abs. At first assessment, the groups did not differ clinically or radiologically, but faciobrachial dystonic seizures were only observed in two LGI1-Ab(+) patients. All patients received monthly intravenous methylprednisolone (MP) pulses. At the most recent follow up (median 26 months), thirteen (72 %) were seizure-free, and seizure-freedom rates did not differ between the Ab groups. Hippocampal atrophy had developed in 7/9 LGI1-Ab(+) patients, but in none of the CASPR-2-Ab(+) or LGI/CASPR-2-Ab(-) patients (p = 0.003). While all subgroups improved, memory scores only normalized in six patients (33 %) and LGI1-Ab(+) patients were left with significantly poorer memory than the other two subgroups. Most VGKC-LE patients become seizure-free with pulsed monthly MP, but memory outcome is less favourable. Hippocampal atrophy and poor memory recovery is common in patients with LGI1-Abs and suggests permanent functional damage. More intense immunotherapies could improve outcomes in LGI1-Ab(+)-LE.

  2. Structure-based non-canonical amino acid design to covalently crosslink an antibody–antigen complex

    PubMed Central

    Xu, Jianqing; Tack, Drew; Hughes, Randall A.; Ellington, Andrew D.; Gray, Jeffrey J.

    2014-01-01

    Engineering antibodies to utilize non-canonical amino acids (NCAA) should greatly expand the utility of an already important biological reagent. In particular, introducing crosslinking reagents into antibody complementarity determining regions (CDRs) should provide a means to covalently crosslink residues at the antibody–antigen interface. Unfortunately, finding the optimum position for crosslinking two proteins is often a matter of iterative guessing, even when the interface is known in atomic detail. Computer-aided antibody design can potentially greatly restrict the number of variants that must be explored in order to identify successful crosslinking sites. We have therefore used Rosetta to guide the introduction of an oxidizable crosslinking NCAA, l-3,4-dihydroxyphenylalanine (l-DOPA), into the CDRs of the anti-protective antigen scFv antibody M18, and have measured crosslinking to its cognate antigen, domain 4 of the anthrax protective antigen. Computed crosslinking distance, solvent accessibility, and interface energetics were three factors considered that could impact the efficiency of l-DOPA-mediated crosslinking. In the end, 10 variants were synthesized, and crosslinking efficiencies were generally 10% or higher, with the best variant crosslinking to 52% of the available antigen. The results suggest that computational analysis can be used in a pipeline for engineering crosslinking antibodies. The rules learned from l-DOPA crosslinking of antibodies may also be generalizable to the formation of other crosslinked interfaces and complexes. PMID:23680795

  3. Effects of messenger RNA structure and other translational control mechanisms on major histocompatibility complex-I mediated antigen presentation

    PubMed Central

    Murat, Pierre; Tellam, Judy

    2015-01-01

    Effective T-cell surveillance of antigen-presenting cells is dependent on the expression of an array of antigenic peptides bound to major histocompatibility complex (MHC) class I (MHC-I) or class II (MHC-II) molecules. Pathogens co-evolving with their hosts exploit crucial translational regulatory mechanisms in order to evade host immune recognition and thereby sustain their infection. Evasion strategies that downregulate viral protein synthesis and thereby restrict antigen presentation to cytotoxic T-cells through the endogenous MHC-I pathway have been implicated in the pathogenesis of viral-associated malignancies. An understanding of the mechanisms by which messenger RNA (mRNA) structure modulates both viral mRNA translation and the antigen processing machinery to escape immune surveillance, will stimulate the development of alternative therapeutic strategies focused on RNA-directed drugs designed to enhance immune responses against infected cells. In this review, we discuss regulatory aspects of the MHC-I pathway and summarize current knowledge of the role attributed by mRNA structure and other translational regulatory mechanisms in immune evasion. In particular we highlight the impact of recently identified G-quadruplex structures within virally encoded transcripts as unique regulatory signals for translational control and antigen presentation. WIREs RNA 2015, 6:157–171. doi: 10.1002/wrna.1262 PMID:25264139

  4. Allo-antigen stimulated CD8+ T-cells suppress NF-κB and Ets-1 DNA binding activity, and inhibit phosphorylated NF-κB p65 nuclear localization in CD4+ T-cells.

    PubMed

    Nagashima, Ryuichi; Kawakami, Fumitaka; Takahashi, Shinichiro; Obata, Fumiya; Kubo, Makoto

    2014-08-01

    CD8+ T-cells of asymptomatic HIV-1 carriers (AC) suppress human immunodeficiency virus type 1 (HIV-1) replication in a class I major histocompatibility complex (MHC-I)-restricted and -unrestricted manner. In order to investigate the mechanism of MHC-I-unrestricted CD8+ T-cell-mediated HIV-1 suppression, we previously established allo-antigen stimulated CD8+T-cells from HIV-1-uninfected donors. These allo-antigen stimulated CD8+ T-cells suppressed HIV-1 replication in acutely infected autologous CD4+ T-cells when directly co-cultured. To elucidate the mechanism of HIV-1 replication suppression, we analyzed DNA-binding activity and phosphorylation of transcriptional factors associated with HIV-1 replication by electrophoresis mobility shift assay and Western blotting. When CD4+ T-cells were cultured with allo-antigen stimulated CD8+ T-cells, the reduction of NF-κB and Ets-1 DNA-binding activity was observed. Nuclear localization of NF-κB p65 and Ets-1 was suppressed in CD4+ T-cells. Although NF-κB p65 and Ets-1 are known to be regulated by protein kinase A (PKA), no difference was observed in the expression and phosphorylation of the PKA catalytic subunit in CD4+ T-cells cultured with PHA-treated CD8+ T-cells or allo-antigen stimulated CD8+ T-cells. Cyclic AMP is also known to enter through gap junctions, but the suppression of HIV-1 replication mediated by allo-antigen stimulated CD8+ T-cells was not affected by the gap junction inhibitor. The nuclear transport of phosphorylated NF-κB p65 (Ser276) was inhibited only in CD4+ T-cells cultured with allo-antigen stimulated CD8+ T-cells. Our results indicate that allo-antigen stimulated CD8+ T-cells suppress the transcriptional activity of NF-κB p65 or Ets-1 in an antigen-nonspecific manner, and inhibit the nuclear transport of phosphorylated NF-κB p65 (Ser276).

  5. Pectinesterase Inhibitor from Jelly Fig (Ficus awkeotsang Makino) Achene Inhibits Surface Antigen Expression by Human Hepatitis B Virus

    PubMed Central

    Huang, Yu-Chuen; Jiang, Chii-Ming; Chen, Yu-Jen; Chen, Yu-Yawn

    2013-01-01

    Pectinesterase inhibitor (PEI) isolated from jelly fig (Ficus awkeotsang Makino) is an edible component of a popular drink consumed in Asia. Hepatitis B virus (HBV) infection is prevalent in Asia, and current treatments for HBV infection need improvement. This study aimed to evaluate the effect of PEI on the surface antigen expression by HBV (HBsAg). Human hepatoma cell lines Hep3B and Huh7 served as in vitro models for assessing the cytotoxicity and HBsAg expression. A culture of primary hepatocytes cultured from mice served as the normal counterpart. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. HBsAg expression was evaluated by measuring HBsAg secretion into the culture medium using an enzyme-linked immunosorbent assay. The results showed that PEI did not affect the viability of the human hepatoma cell lines or primary mouse hepatocytes. PEI inhibited the expression of HBsAg in hepatoma cell lines harboring endogenous (Hep3B) and integrated (Huh7) HBV genomes in a concentration- and time-dependent manner, thus implicating a universal activity against HBV gene expression. In conclusion, it suggests that PEI from jelly fig inhibits the expression of human HBsAg in host cells without toxic effects on normal primary hepatocytes. PMID:24302965

  6. Innocuous IFNγ induced by adjuvant-free antigen restores normoglycemia in NOD mice through inhibition of IL-17 production

    PubMed Central

    Jain, Renu; Tartar, Danielle M.; Gregg, Randal K.; Divekar, Rohit D.; Bell, J. Jeremiah; Lee, Hyun-Hee; Yu, Ping; Ellis, Jason S.; Hoeman, Christine M.; Franklin, Craig L.; Zaghouani, Habib

    2008-01-01

    The role of Th17 cells in type I diabetes (TID) remains largely unknown. Glutamic acid decarboxylase (GAD) sequence 206–220 (designated GAD2) represents a late-stage epitope, but GAD2-specific T cell receptor transgenic T cells producing interferon γ (IFNγ) protect against passive TID. Because IFNγ is known to inhibit Th17 cells, effective presentation of GAD2 peptide under noninflammatory conditions may protect against TID at advanced disease stages. To test this premise, GAD2 was genetically incorporated into an immunoglobulin (Ig) molecule to magnify tolerance, and the resulting Ig-GAD2 was tested against TID at different stages of the disease. The findings indicated that Ig-GAD2 could not prevent TID at the preinsulitis phase, but delayed TID at the insulitis stage. More importantly, Ig-GAD2 sustained both clearance of pancreatic cell infiltration and β-cell division and restored normoglycemia when given to hyperglycemic mice at the prediabetic stage. This was dependent on the induction of splenic IFNγ that inhibited interleukin (IL)-17 production. In fact, neutralization of IFNγ led to a significant increase in the frequency of Th17 cells, and the treatment became nonprotective. Thus, IFNγ induced by an adjuvant free antigen, contrary to its usual inflammatory function, restores normoglycemia, most likely by localized bystander suppression of pathogenic IL-17–producing cells. PMID:18195074

  7. Pectinesterase Inhibitor from Jelly Fig (Ficus awkeotsang Makino) Achene Inhibits Surface Antigen Expression by Human Hepatitis B Virus.

    PubMed

    Huang, Yu-Chuen; Jiang, Chii-Ming; Chen, Yu-Jen; Chen, Yu-Yawn

    2013-01-01

    Pectinesterase inhibitor (PEI) isolated from jelly fig (Ficus awkeotsang Makino) is an edible component of a popular drink consumed in Asia. Hepatitis B virus (HBV) infection is prevalent in Asia, and current treatments for HBV infection need improvement. This study aimed to evaluate the effect of PEI on the surface antigen expression by HBV (HBsAg). Human hepatoma cell lines Hep3B and Huh7 served as in vitro models for assessing the cytotoxicity and HBsAg expression. A culture of primary hepatocytes cultured from mice served as the normal counterpart. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. HBsAg expression was evaluated by measuring HBsAg secretion into the culture medium using an enzyme-linked immunosorbent assay. The results showed that PEI did not affect the viability of the human hepatoma cell lines or primary mouse hepatocytes. PEI inhibited the expression of HBsAg in hepatoma cell lines harboring endogenous (Hep3B) and integrated (Huh7) HBV genomes in a concentration- and time-dependent manner, thus implicating a universal activity against HBV gene expression. In conclusion, it suggests that PEI from jelly fig inhibits the expression of human HBsAg in host cells without toxic effects on normal primary hepatocytes. PMID:24302965

  8. Cancer-testis antigen lymphocyte antigen 6 complex locus K is a serologic biomarker and a therapeutic target for lung and esophageal carcinomas.

    PubMed

    Ishikawa, Nobuhisa; Takano, Atsushi; Yasui, Wataru; Inai, Kouki; Nishimura, Hitoshi; Ito, Hiroyuki; Miyagi, Yohei; Nakayama, Haruhiko; Fujita, Masahiro; Hosokawa, Masao; Tsuchiya, Eiju; Kohno, Nobuoki; Nakamura, Yusuke; Daigo, Yataro

    2007-12-15

    Gene expression profile analyses of non-small cell lung carcinomas (NSCLC) and esophageal squamous cell carcinomas (ESCC) revealed that lymphocyte antigen 6 complex locus K (LY6K) was specifically expressed in testis and transactivated in a majority of NSCLCs and ESCCs. Immunohistochemical staining using 406 NSCLC and 265 ESCC specimens confirmed that LY6K overexpression was associated with poor prognosis for patients with NSCLC (P = 0.0003), as well as ESCC (P = 0.0278), and multivariate analysis confirmed its independent prognostic value for NSCLC (P = 0.0035). We established an ELISA to measure serum LY6K and found that the proportion of the serum LY6K-positive cases was 38 of 112 (33.9%) NSCLC and 26 of 81 (32.1%) ESCC, whereas only 3 of 74 (4.1%) healthy volunteers were falsely diagnosed. In most cases, there was no correlation between serum LY6K and conventional tumor markers of carcinoembryonic antigen (CEA) and cytokeratin 19-fragment (CYFRA 21-1) values. A combined ELISA for both LY6K and CEA classified 64.7% of lung adenocarcinoma patients as positive, and the use of both LY6K and CYFRA 21-1 increased sensitivity in the detection of lung squamous cell carcinomas and ESCCs up to 70.4% and 52.5%, respectively, whereas the false positive rate was 6.8% to 9.5%. In addition, knocked down of LY6K expression with small interfering RNAs resulted in growth suppression of the lung and esophageal cancer cells. Our data imply that a cancer-testis antigen, LY6K, should be useful as a new type of tumor biomarker and probably as a target for the development of new molecular therapies for cancer treatment.

  9. Human hepatitis B viral e antigen and its precursor P20 inhibit T lymphocyte proliferation

    SciTech Connect

    Purvina, Maija; Hoste, Astrid; Rossignol, Jean-Michel; Lagaudriere-Gesbert, Cecile

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer P20, precursor of the HBeAg, interacts with the cellular protein gC1qR. Black-Right-Pointing-Pointer HBeAg and P20 bind to T cell surface and inhibit mitogen-induced T cell division. Black-Right-Pointing-Pointer HBeAg and P20 inhibition of T cell proliferation is gC1qR and IL-1RAcP-independent. -- Abstract: The hepatitis B virus (HBV) Precore protein is processed through the secretory pathway directly as HBeAg or with the generation of an intermediate (P20). Precore gene has been shown to be implicated in viral persistence, but the functions of HBeAg and its precursors have not been fully elucidated. We show that the secreted proteins HBeAg and P20 interact with T cell surface and alter Kit-225 and primary T cells proliferation, a process which may facilitate the establishment of HBV persistence. Our data indicate that the N-terminal end of Precore is important for these inhibitory effects and exclude that they are dependent on the association of HBeAg and P20 with two characterized cell surface ligands, the Interleukin-1 Receptor Accessory Protein and gC1qR (present study).

  10. BRAF and MEK inhibition variably affect GD2-specific chimeric antigen receptor (CAR) T-cell function in vitro.

    PubMed

    Gargett, Tessa; Fraser, Cara K; Dotti, Gianpietro; Yvon, Eric S; Brown, Michael P

    2015-01-01

    Cancer immunotherapy has long been used in the treatment of metastatic melanoma, and an anti-CTLA-4 monoclonal antibody treatment has recently been approved by the US Food and Drug Administration. Targeted therapies such as small molecule kinase inhibitors targeting deregulated mitogen-activated protein kinase (MAPK) signaling have markedly improved melanoma control in up to 50% of metastatic disease patients and have likewise been recently approved. Combination therapies for melanoma have been proposed as a way to exploit the high-level but short-term responses associated with kinase inhibitor therapies and the low-level but longer-term responses associated with immunotherapy. Cancer immunotherapy now includes adoptive transfer of autologous tumor-specific chimeric antigen receptor (CAR) T cells and this mode of therapy is a candidate for combination with small molecule drugs. This paper describes CART cells that target GD2-expressing melanoma cells and investigates the effects of approved MAPK pathway-targeted therapies for melanoma [vemurafenib (Vem), dabrafenib (Dab), and trametinib (Tram)] on the viability, activation, proliferation, and cytotoxic T lymphocyte activity of these CAR T cells, as well as on normal peripheral blood mononuclear cells. We report that, although all these drugs lead to inhibition of stimulated T cells at high concentrations in vitro, only Vem inhibited T cells at concentrations equivalent to reported plasma concentrations in treated patients. Although the combination of Dab and Tram also resulted in inhibition of T-cell effector functions at some therapeutic concentrations, Dab itself had little adverse effect on CAR T-cell function. These findings may have implications for novel therapeutic combinations of adoptive CAR T-cell immunotherapy and MAPK pathway inhibitors.

  11. A prostate-specific antigen-dependent fusion polypeptide inhibits growth of prostate cancer cells in vitro and in vivo

    PubMed Central

    Zhang, Xiang; Ma, Yueyun; Wei, Hua; Li, Bin; Xiao, Fengjing; Yang, Jing; Yue, Qiaohong; Yang, Angang; Hao, Xiaoke

    2016-01-01

    Polypeptide APP8 is a prostate-specific antigen (PSA)-activated prodrug that was designed to synergize the effects of the Bcl-2 homology domain 3 (BH3) peptide, K237 and the DG2 peptide. The aim of this study is to evaluate its biodistribution and anticancer effect in vitro and in vivo. In this study, APP8 and each component peptide were synthesized. The biodistribution was identified using con-focal microscopyin both PSA+ cell line and PSA- cell line in vitro. Then cell cycle, MTT and in-cell western blot were accessed to analyze the effect mechanisms. Finally, xenografts were used to confirm the anticancer effect in vivo. Here, it was shown that APP8 was hydrolyzed and BH3 was released into the nucleus, while K237 and DG2 were located predominantly in the cytoplasm, only in LNCaP cells (PSA+), but not PC3 cells (PSA-). K237 and DG2 could induce cell apoptosis through decreasing the phosphorylation of ERK-2 and Flk-1. APP8 also caused the death of LNCaP cells, and was predominantly dependent on BH3 in vitro. In addition, It was noted that as the tumor grew in vivo, APP8 could inhibit the tumor volume to 77.3%, mainly depending on K237 and DG2 via inhibition of the growth of vascular endothelial cells. Our results suggested that APP8 could promote prostate cancer cell death and stop prostate cancer growth via synergizing apoptosis induction of tumor cell and inhibition of the growth of vascular endothelial cells. It provides a novel candidate prodrug for specific therapy of prostate cancer. PMID:27293998

  12. Chimeric antigen receptor T cells targeting HERV-K inhibit breast cancer and its metastasis through downregulation of Ras

    PubMed Central

    Zhou, Fuling; Krishnamurthy, Janani; Wei, Yongchang; Li, Ming; Hunt, Kelly; Johanning, Gary L; Cooper, Laurence JN; Wang-Johanning, Feng

    2015-01-01

    We have previously reported that human endogenous retrovirus-K (HERV-K) envelope (env) protein is a tumor-associated antigen (TAA) for cancer vaccines, and that its antibodies (mAbs) possess antitumor activity against cancer. In this study, a chimeric antigen receptor (CAR) specific for HERV-K env protein (K-CAR) was generated using anti-HERV-K mAb. K-CAR T cells from peripheral blood mononuclear cells (PBMCs) of 9 breast cancer (BC) patients and 12 normal donors were able to inhibit growth of, and to exhibit significant cytotoxicity toward, BC cells but not MCF-10A normal breast cells. The antitumor effects in cancer cells were significantly reduced when control T cells were used, or the expression of HERV-K was knocked down by an shRNA. Secretion of multiple cytokines, including IFNγ, TNF-α, and IL-2, was significantly enhanced in culture media of BC cells treated with K-CARs. Significantly reduced tumor growth and tumor weight was observed in xenograft models bearing MDA-MB-231 or MDA-MB-435.eB1 BC cells. Importantly, the K-CAR prevented tumor metastasis to other organs. Furthermore, downregulation of HERV-K expression in tumors of mice treated with K-CAR correlated with upregulation of p53 and downregulation of MDM2 and p-ERK. Importantly, the expression of HERV-K env protein in metastatic tumor tissues treated with K-CAR T cells correlated with the expression of Ras. Our results indicate that HERV-K env protein is an oncoprotein and may play an important role in tumorigenesis related to p53 and Ras signaling pathways. Anti-HERV-K treatment, including K-CAR treatment, shows potential for immunotherapy of BC. PMID:26451325

  13. Lipopeptides: a novel antigen repertoire presented by major histocompatibility complex class I molecules.

    PubMed

    Morita, Daisuke; Sugita, Masahiko

    2016-10-01

    Post-translationally modified peptides, such as those containing either phosphorylated or O-glycosylated serine/threonine residues, may be presented to cytotoxic T lymphocytes (CTLs) by MHC class I molecules. Most of these modified peptides are captured in the MHC class I groove in a similar manner to that for unmodified peptides. N-Myristoylated 5-mer lipopeptides have recently been identified as a novel chemical class of MHC class I-presented antigens. The rhesus classical MHC class I allele, Mamu-B*098, was found to be capable of binding N-myristoylated lipopeptides and presenting them to CTLs. A high-resolution X-ray crystallographic analysis of the Mamu-B*098:lipopeptide complex revealed that the myristic group as well as conserved C-terminal serine residue of the lipopeptide ligand functioned as anchors, whereas the short stretch of three amino acid residues located in the middle of the lipopeptides was only exposed externally with the potential to interact directly with specific T-cell receptors. Therefore, the modes of lipopeptide-ligand interactions with MHC class I and with T-cell receptors are novel and fundamentally distinct from that for MHC class I-presented peptides. Another lipopeptide-presenting MHC class I allele has now been identified, leading us to the prediction that MHC class I molecules may be separated on a functional basis into two groups: one presenting long peptides and the other presenting short lipopeptides. Since the N-myristoylation of viral proteins is often linked to pathogenesis, CTLs capable of sensing N-myristoylation may serve to control pathogenic viruses, raising the possibility for the development of a new type of lipopeptide vaccine. PMID:27402593

  14. Carcinoembryonic antigen-related cell adhesion molecule-1 regulates granulopoiesis by inhibition of granulocyte colony-stimulating factor receptor.

    PubMed

    Pan, Hao; Shively, John E

    2010-10-29

    Although carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is an activation marker for neutrophils and delays neutrophil apoptosis, the role of CEACAM1 in granulopoiesis and neutrophil-dependent host immune responses has not been investigated. CEACAM1 expression correlated with granulocytic differentiation, and Ceacam1(-/-) mice developed neutrophilia because of loss of the Src-homology-phosphatase-1 (SHP-1)-dependent inhibition of granulocyte colony-stimulating factor receptor (G-CSFR) signal transducer and activator of transcription (Stat3) pathway provided by CEACAM1. Moreover, Ceacam1(-/-) mice were hypersensitive to Listeria Monocytogenes (LM) infection with an accelerated mortality. Reintroduction of CEACAM1 into Ceacam1(-/-) bone marrow restored normal granulopoiesis and host sensitivity to LM infection, while mutation of its immunoreceptor tyrosine-based inhibitory motifs (ITIMs) abrogated this restoration. shRNA-mediated reduction of Stat3 amounts rescued normal granulopoiesis, attenuating host sensitivity to LM infection in Ceacam1(-/-) mice. Thus, CEACAM1 acted as a coinhibitory receptor for G-CSFR regulating granulopoiesis and host innate immune response to bacterial infections.

  15. Serosurvey of West Nile virus and other flaviviruses of the Japanese encephalitis antigenic complex in birds from Andalusia, southern Spain.

    PubMed

    García-Bocanegra, Ignacio; Busquets, Núria; Napp, Sebastián; Alba, Ana; Zorrilla, Irene; Villalba, Rubén; Arenas, Antonio

    2011-08-01

    Flaviviruses of the Japanese encephalitis virus (JEV) antigenic complex, including West Nile virus (WNV), are recognized as emerging and reemerging pathogens. Circulation of flaviviruses has been recently detected in different mosquito and vertebrate species in several European countries. A serosurvey study was carried out to evaluate the circulation of WNV and other flaviviruses of the JEV antigenic complex in different wild bird species in Spain between 2006 and 2009. Seropositiviy against JEV using a competitive enzyme-linked immunosorbent assay was found in common coot, Montagu's Harrier, black kite, black vulture, Bonelli's eagle, Spanish imperial eagle, Egyptian vulture, and Eurasian spoonbill. Seropositivity to JEV antigenic complex viruses was significantly higher in samples collected during autumn compared with animals sampled during summer. Significantly higher seroprevalence was also observed in 2007 compared with 2009, whereas there were no significant differences in seropositivity among taxonomic levels, migratory versus resident behavior, body size (large vs. medium), or habitats (free-ranging vs. captivity). Neutralizing antibodies against WNV were detected in common coot and Spanish imperial eagle using a virus-neutralization test. Oral shedding of WNV was not detected in any of the Spanish imperial eagles, Egyptian vultures, Eurasian Spoonbills, Lammergeiers, and the Black vultures analyzed by means of real-time reverse transcription-polymerase chain reaction. The results indicate that WNV and others flaviviruses of the JEV antigenic group circulated in migratory and resident wild bird species in Spain between 2007 and 2008. Further studies are necessary to determine the precise role that each of these wild bird species, some of them cataloged as "near threatened," "vulnerable," or "endangered," play in the epidemiology of those viruses. PMID:21142954

  16. Latex bead-based artificial antigen-presenting cells induce tumor-specific CTL responses in the native T-cell repertoires and inhibit tumor growth.

    PubMed

    Shen, Chuanlai; Cheng, Kai; Miao, Shenwei; Wang, Wei; He, Yong; Meng, Fanyan; Zhang, Jianqiong

    2013-02-01

    Cell-free artificial antigen-presenting cells (aAPCs) were generated by coupling H-2K(b)/TRP2 tetramers together with anti-CD28 and anti-4-1BB antibodies onto cell-sized latex beads and injected intravenously and subcutaneously into naïve mice and antigen-primed mice (B6, H-2K(b)). Vigorous tumor antigen-specific CTL responses in the native T-cell repertoire in each mouse model were elicited as evaluated by measuring surface CD69 and CD25, intracellular IFN-γ, tetramer staining and cytolysis of melanoma cells. Furthermore, the aAPCs efficiently inhibited subcutaneous tumor growth and markedly delayed tumor progression in tumor-bearing mice. These data suggest that bead-based aAPCs represent a potential strategy for the active immunotherapy of cancers or persistent infections. PMID:23328744

  17. T cell receptor interaction with peptide/major histocompatibility complex (MHC) and superantigen/MHC ligands is dominated by antigen

    PubMed Central

    1993-01-01

    While recent evidence strongly suggests that the third complementarity determining regions (CDR3s) of T cell receptors (TCRs) directly contact antigenic peptides bound to major histocompatibility complex (MHC) molecules, the nature of other TCR contact(s) is less clear. Here we probe the extent to which different antigens can affect this interaction by comparing the responses of T cells bearing structurally related TCRs to cytochrome c peptides and staphylococcal enterotoxin A (SEA) presented by 13 mutant antigen-presenting cell (APC) lines. Each APC expresses a class II MHC molecule (I-Ek) with a single substitution of an amino acid residue predicted to be located on the MHC alpha helices and to point "up" towards the TCR. We find that very limited changes (even a single amino acid) in either a CDR3 loop of the TCR or in a contact residue of the antigenic peptide can have a profound effect on relatively distant TCR/MHC interactions. The extent of these effects can be as great as that observed between T cells bearing entirely different TCRs and recognizing different peptides. We also find that superantigen presentation entails a distinct mode of TCR/MHC interaction compared with peptide presentation. These data suggest that TCR/MHC contacts can be made in a variety of ways between the same TCR and MHC, with the final configuration apparently dominated by the antigen. These observations suggest a molecular basis for recent reports in which either peptide analogues or superantigens trigger distinct pathways of T cell activation. PMID:8393480

  18. Immunoblot analysis for serodiagnosis of tuberculosis using a 45/47-kilodalton antigen complex of Mycobacterium tuberculosis.

    PubMed Central

    Diagbouga, S; Fumoux, F; Zoubga, A; Sanou, P T; Marchal, G

    1997-01-01

    We evaluated the immunoglobulin G (IgG) antibody response to the 45/47-kDa secreted protein of Mycobacterium tuberculosis by immunoblot assay, to assess its potential value for serological diagnosis. Control subjects consisted of healthy volunteers with negative or positive tuberculin skin tests. Most (>98%) scored negative in an immunoblot test when the sera were analyzed at a 1:400 dilution. Approximately 40% of sera (diluted 1 in 400) from tuberculous patients (positive smears) recognized the antigen complex. The sensitivity of the test for patients suffering from extrapulmonary tuberculosis was similar to that for patients suffering from pulmonary tuberculosis but who had negative smears. The frequency of positive reactions among the patients suffering from other pulmonary diseases was similar to that among the control subjects. In tuberculous patients infected with human immunodeficiency virus, the sensitivity of the immunoblot test was significantly lower. Thus, this test based on an antigen complex used in an immunoblot assay to detect the presence of IgG antibody has a specificity of 98% and a sensitivity of 40%. The simultaneous use of different purified antigens, selected at the same high specificity level, may improve the sensitivity of such an assay. PMID:9144373

  19. The unravelling of the complex pattern of tyrosinase inhibition

    PubMed Central

    Deri, Batel; Kanteev, Margarita; Goldfeder, Mor; Lecina, Daniel; Guallar, Victor; Adir, Noam; Fishman, Ayelet

    2016-01-01

    Tyrosinases are responsible for melanin formation in all life domains. Tyrosinase inhibitors are used for the prevention of severe skin diseases, in skin-whitening creams and to avoid fruit browning, however continued use of many such inhibitors is considered unsafe. In this study we provide conclusive evidence of the inhibition mechanism of two well studied tyrosinase inhibitors, KA (kojic acid) and HQ (hydroquinone), which are extensively used in hyperpigmentation treatment. KA is reported in the literature with contradicting inhibition mechanisms, while HQ is described as both a tyrosinase inhibitor and a substrate. By visualization of KA and HQ in the active site of TyrBm crystals, together with molecular modeling, binding constant analysis and kinetic experiments, we have elucidated their mechanisms of inhibition, which was ambiguous for both inhibitors. We confirm that while KA acts as a mixed inhibitor, HQ can act both as a TyrBm substrate and as an inhibitor. PMID:27725765

  20. Inhibition of antigen-induced proliferation of T cells from radiation-induced bone marrow chimeras by a monoclonal antibody directed against an Ia determinant on the antigen-presenting cell.

    PubMed Central

    Longo, D L; Schwartz, R H

    1981-01-01

    Chimeric B10.A T cells that had matured in a (B10.A X B10.Q)F1 environment acquired the ability to respond to poly(Glu56Lys35Phe9) (GL pi), an antigen to which the B10.A mouse is a nonresponder. The response of the chimeric B10.A T cells was initiated by GL phi on responder B10.Q antigen-presenting cells (APC) but not by GL phi on nonresponder B10.A APC. Similarly, chimeric B10.Q T cells that had matured in a (B10.A X B10.Q)F1 environment acquired the ability to respond to poly(Glu60Ala30Tyr10) (GAT) when the antigen was presented on responder B10.A APC, but not when GAT was presented on nonresponder B10.Q APC. No syngeneic haplotype preference was observed for either antigen. These interactions between H-2 nonidentical T cells and APC were inhibited by anti-H-2 antisera and a monoclonal anti-Ia antibody directed against the APC but not by such antibodies when they were directed against the T cell. These data suggest that, when they develop in a responder chimeric environment, genotypic nonresponder T cells become responders by acquiring receptors that allow them to recognize responder I region products on the surface of APC. Furthermore, the data demonstrate that the site of action of the blocking effects of the anti-Ia antibodies is the APC, thus providing strong evidence in support of the idea that Ia antigens on APC are the Ir gene products. PMID:6165995

  1. Peptide-β2-microglobulin-major histocompatibility complex expressing cells are potent antigen-presenting cells that can generate specific T cells

    PubMed Central

    Obermann, Sonja; Petrykowska, Susanne; Manns, Michael P; Korangy, Firouzeh; Greten, Tim F

    2007-01-01

    Adoptive T-cell therapy represents a promising therapeutic approach for the treatment of cancer. Successful adoptive immunotherapy depends on the ex vivo priming and expansion of antigen-specific T cells. However, the in vitro generation of adequate numbers of functional antigen-specific T cell remains a major obstacle. It is important to develop efficient and reproducible methods to generate high numbers of antigen-specific T cells for adoptive T-cell transfer. We have developed a new artificial antigen-presenting cell (aAPC) by transfection of major histocompatibility (MHC) class I negative Daudi cells with a peptide-β2-microglobulin–MHC fusion construct (single-chain aAPC) ensuring presentation of the peptide–MHC complex of interest. Using this artificial antigen-presenting cell, we could generate up to 9·2 × 108 antigen-specific cytotoxic CD8+ T cells from 10 ml blood. In vitro generated T cells lysed endogenously presented antigens. Direct comparison of the single-chain aAPC with autologous monocyte-derived dendritic cells demonstrated that these cells were equally efficient in stimulation of T cells. Finally, we were able to generate antigen-specific T cell lines from perpheral blood mononuclear cells of patients receiving cytotoxic chemotherapy. The use of single-chain aAPC represent a promising option for the generation of antigen-specific CD8+ T cells, which could be used for adoptive T-cell therapy. PMID:17472719

  2. Peptide-beta2-microglobulin-major histocompatibility complex expressing cells are potent antigen-presenting cells that can generate specific T cells.

    PubMed

    Obermann, Sonja; Petrykowska, Susanne; Manns, Michael P; Korangy, Firouzeh; Greten, Tim F

    2007-09-01

    Adoptive T-cell therapy represents a promising therapeutic approach for the treatment of cancer. Successful adoptive immunotherapy depends on the ex vivo priming and expansion of antigen-specific T cells. However, the in vitro generation of adequate numbers of functional antigen-specific T cell remains a major obstacle. It is important to develop efficient and reproducible methods to generate high numbers of antigen-specific T cells for adoptive T-cell transfer. We have developed a new artificial antigen-presenting cell (aAPC) by transfection of major histocompatibility (MHC) class I negative Daudi cells with a peptide-beta2-microglobulin-MHC fusion construct (single-chain aAPC) ensuring presentation of the peptide-MHC complex of interest. Using this artificial antigen-presenting cell, we could generate up to 9.2 x 10(8) antigen-specific cytotoxic CD8(+) T cells from 10 ml blood. In vitro generated T cells lysed endogenously presented antigens. Direct comparison of the single-chain aAPC with autologous monocyte-derived dendritic cells demonstrated that these cells were equally efficient in stimulation of T cells. Finally, we were able to generate antigen-specific T cell lines from perpheral blood mononuclear cells of patients receiving cytotoxic chemotherapy. The use of single-chain aAPC represent a promising option for the generation of antigen-specific CD8(+) T cells, which could be used for adoptive T-cell therapy.

  3. [Internal inhibition in rats after destruction of amygdaloid complex nuclei].

    PubMed

    Bogach, P G; Makarchuk, N E; Chaĭchenko, G M

    1981-01-01

    In experiments on male albino rats bilateral lesions of the basolateral amygdala (BLA) impaired extinction and differentiation of the food-procuring conditioned reflexes where as bilateral corticomedial (CMA) lesions had no effect on them. The BLA damage caused a significant impairment of the aversive stimuli discrimination but had no effect on the extinction of the conditioned avoidance reflex in a shuttle-box. The CMA lesions in both hemispheres led to attenuation of aversive stimuli discrimination and delay of avoidance extinction. The data obtained testify to the BLA dominant role in internal inhibition of the alimentary conditioned reflexes and the CMA dominant role in internal inhibition of avoidance conditioning and furthermore to the importance of BLA in the evaluation of the emotional significance and biological validity of the stimuli (reward or punishment). PMID:7303899

  4. Human Leukocyte Antigen (HLA) Peptides Derived from Tumor Antigens Induced by Inhibition of DNA Methylation for Development of Drug-facilitated Immunotherapy.

    PubMed

    Shraibman, Bracha; Kadosh, Dganit Melamed; Barnea, Eilon; Admon, Arie

    2016-09-01

    Treatment of cancer cells with anticancer drugs often fails to achieve complete remission. Yet, such drug treatments may induce alteration in the tumor's gene expression patterns, including those of Cancer/Testis Antigens (CTA). The degradation products of such antigens can be presented as HLA peptides on the surface of the tumor cells and be developed into anticancer immunotherapeutics. For example, the DNA methyl transferase inhibitor, 5-aza-2'-deoxycytidine (Decitabine) has limited antitumor efficacy, yet it induces the expression of many genes, including CTAs that are normally silenced in the healthy adult tissues. In this study, the presentation of many new HLA peptides derived from CTAs and induced by Decitabine was demonstrated in three human Glioblastoma cell lines. Such presentation of CTA-derived HLA peptides can be exploited for development of new treatment modalities, combining drug treatment with anti-CTA targeted immunotherapy. The Decitabine-induced HLA peptidomes include many CTAs that are not normally detected in healthy tissues or in cancer cells, unless treated with the drug. In addition, the study included large-scale analyses of the simultaneous effects of Decitabine on the transcriptomes, proteomes and HLA peptidomes of the human Glioblastoma cells. It demonstrates the poor correlations between these three levels of gene expression, both in their total levels and in their response to the drug. The proteomics and HLA peptidomics data are available via ProteomeXchange with identifier PXD003790 and the transcriptomics data are available via GEO with identifier GSE80137.

  5. Synthetic antigens reveal dynamics of BCR endocytosis during inhibitory signaling.

    PubMed

    Courtney, Adam H; Bennett, Nitasha R; Zwick, Daniel B; Hudon, Jonathan; Kiessling, Laura L

    2014-01-17

    B cells detect foreign antigens through their B cell antigen receptor (BCR). The BCR, when engaged by antigen, initiates a signaling cascade. Concurrent with signaling is endocytosis of the BCR complex, which acts to downregulate signaling and facilitate uptake of antigen for processing and display on the cell surface. The relationship between signaling and BCR endocytosis is poorly defined. Here, we explore the interplay between BCR endocytosis and antigens that either promote or inhibit B cell activation. Specifically, synthetic antigens were generated that engage the BCR alone or both the BCR and the inhibitory co-receptor CD22. The lectin CD22, a member of the Siglec family, binds sialic acid-containing glycoconjugates found on host tissues, inhibiting BCR signaling to prevent erroneous B cell activation. At low concentrations, antigens that can cocluster the BCR and CD22 promote rapid BCR endocytosis; whereas, slower endocytosis occurs with antigens that bind only the BCR. At higher antigen concentrations, rapid BCR endocytosis occurs upon treatment with either stimulatory or inhibitory antigens. Endocytosis of the BCR, in response to synthetic antigens, results in its entry into early endocytic compartments. Although the CD22-binding antigens fail to activate key regulators of antigen presentation (e.g., Syk), they also promote BCR endocytosis, indicating that inhibitory antigens can be internalized. Together, our observations support a functional role for BCR endocytosis in downregulating BCR signaling. The reduction of cell surface BCR levels in the absence of B cell activation should raise the threshold for BCR subsequent activation. The ability of the activating synthetic antigens to trigger both signaling and entry of the BCR into early endosomes suggests strategies for targeted antigen delivery.

  6. Successive Administration of Streptococcus Type 5 Group A Antigens and S. typhimurium Antigenic Complex Corrects Elevation of Serum Cytokine Concentration and Number of Bone Marrow Stromal Pluripotent Cells in CBA Mice Induced by Each Antigen Separately.

    PubMed

    Gorskaya, Yu F; Danilova, T A; Grabko, V I; Nesterenko, V G

    2015-12-01

    Administration of bacterial antigens to CBA mice induced an increase in serum concentration of virtually all cytokines with a peak in 4 h after administration of S. typhimurium antigens and in 7 h after administration of streptococcus antigens. In 20 h, cytokine concentrations returned to the control level or were slightly below it. In 4 h after administration of S. typhimurium antigens preceded 3 h before by administration of streptococcus antigens, we observed a significant decrease in serum concentrations of IFN-γ, IL-10, GM-CSF, IL-12, and TNF-α, in comparison with injection S. typhimurium antigens alone and IL-5, IL-10, GM-CSF, and TNF-α in comparison with injection of streptococcus antigens alone; the concentrations of IL-2 and IFN-γ, in contrast, increased by 1.5 times in this case. In 20 h after administration of S. typhimurium antigens, the number of multipotential stromal cells (MSC) in the bone marrow and their cloning efficiency (ECF-MSC) increased by 4.8 and 4.4 times, respectively, in comparison with the control, while after administration of streptococcus antigens by 2.6 and 2.4 times, respectively. In 20 h after administration of S. typhimurium antigens preceded 3 h before by administration of streptococcus antigens, these parameters increased by 3.2 and 2.9 times, respectively, in comparison with the control, i.e. the observed increase in the level of MSC count and ECF-MSC is more consistent with the response of the stromal tissue to streptococcus antigens. Thus, successive administration of two bacterial antigens corrected both serum cytokine profiles and MSC response to administration of each antigen separately, which indicates changeability of the stromal tissue in response to changes in the immune response.

  7. Structures of synthetic O-antigen fragments from serotype 2a Shigella flexneri in complex with a protective monoclonal antibody.

    PubMed

    Vulliez-Le Normand, B; Saul, F A; Phalipon, A; Bélot, F; Guerreiro, C; Mulard, L A; Bentley, G A

    2008-07-22

    The anti-LPS IgG mAb F22-4, raised against Shigella flexneri serotype 2a bacteria, protects against homologous, but not heterologous, challenge in an experimental animal model. We report the crystal structures of complexes formed between Fab F22-4 and two synthetic oligosaccharides, a decasaccharide and a pentadecasaccharide that were previously shown to be both immunogenic and antigenic mimics of the S. flexneri serotype 2a O-antigen. F22-4 binds to an epitope contained within two consecutive 2a serotype pentasaccharide repeat units (RU). Six sugar residues from a contiguous nine-residue segment make direct contacts with the antibody, including the nonreducing rhamnose and both branching glucosyl residues from the two RUs. The glucosyl residue, whose position of attachment to the tetrasaccharide backbone of the RU defines the serotype 2a O-antigen, is critical for recognition by F22-4. Although the complete decasaccharide is visible in the electron density maps, the last four pentadecasaccharide residues from the reducing end, which do not contact the antibody, could not be traced. Although considerable mobility in the free oligosaccharides can thus be expected, the conformational similarity between the individual RUs, both within and between the two complexes, suggests that short-range transient ordering to a helical conformation might occur in solution. Although the observed epitope includes the terminal nonreducing residue, binding to internal epitopes within the polysaccharide chain is not precluded. Our results have implications for vaccine development because they suggest that a minimum of two RUs of synthetic serotype 2a oligosaccharide is required for optimal mimicry of O-Ag epitopes.

  8. Antigen nature and complexity influence human antibody light chain usage and specificity.

    PubMed

    Smith, Kenneth; Shah, Hemangi; Muther, Jennifer J; Duke, Angie L; Haley, Kathleen; James, Judith A

    2016-05-27

    Human antibodies consist of a heavy chain and one of two possible light chains, kappa (κ) or lambda (λ). Here we tested how these two possible light chains influence the overall antibody response to polysaccharide and protein antigens by measuring light chain usage in human monoclonal antibodies from antibody secreting cells obtained following vaccination with Pneumovax23. Remarkably, we found that individuals displayed restricted light chain usage to certain serotypes and that lambda antibodies have different specificities and modes of cross-reactivity than kappa antibodies. Thus, at both the monoclonal (7 kappa, no lambda) and serum levels (145μg/mL kappa, 2.82μg/mL lambda), antibodies to cell wall polysaccharide were nearly always kappa. The pneumococcal reference serum 007sp was analyzed for light chain usage to 12 pneumococcal serotypes for which it is well characterized. Similar to results at the monoclonal level, certain serotypes tended to favor one of the light chains (14 and 19A, lambda; 6A and 23F, kappa). We also explored differences in light chain usage at the serum level to a variety of antigens. We examined serum antibodies to diphtheria toxin mutant CRM197 and Epstein-Barr virus protein EBNA-1. These responses tended to be kappa dominant (average kappa-to-lambda ratios of 4.52 and 9.72 respectively). Responses to the influenza vaccine were more balanced with kappa-to-lambda ratio averages having slight strain variations: seasonal H1N1, 1.1; H3N2, 0.96; B, 0.91. We conclude that antigens with limited epitopes tend to produce antibodies with restricted light chain usage and that in most individuals, antibodies with lambda light chains have specificities different and complementary to kappa-containing antibodies.

  9. Antigen nature and complexity influence human antibody light chain usage and specificity.

    PubMed

    Smith, Kenneth; Shah, Hemangi; Muther, Jennifer J; Duke, Angie L; Haley, Kathleen; James, Judith A

    2016-05-27

    Human antibodies consist of a heavy chain and one of two possible light chains, kappa (κ) or lambda (λ). Here we tested how these two possible light chains influence the overall antibody response to polysaccharide and protein antigens by measuring light chain usage in human monoclonal antibodies from antibody secreting cells obtained following vaccination with Pneumovax23. Remarkably, we found that individuals displayed restricted light chain usage to certain serotypes and that lambda antibodies have different specificities and modes of cross-reactivity than kappa antibodies. Thus, at both the monoclonal (7 kappa, no lambda) and serum levels (145μg/mL kappa, 2.82μg/mL lambda), antibodies to cell wall polysaccharide were nearly always kappa. The pneumococcal reference serum 007sp was analyzed for light chain usage to 12 pneumococcal serotypes for which it is well characterized. Similar to results at the monoclonal level, certain serotypes tended to favor one of the light chains (14 and 19A, lambda; 6A and 23F, kappa). We also explored differences in light chain usage at the serum level to a variety of antigens. We examined serum antibodies to diphtheria toxin mutant CRM197 and Epstein-Barr virus protein EBNA-1. These responses tended to be kappa dominant (average kappa-to-lambda ratios of 4.52 and 9.72 respectively). Responses to the influenza vaccine were more balanced with kappa-to-lambda ratio averages having slight strain variations: seasonal H1N1, 1.1; H3N2, 0.96; B, 0.91. We conclude that antigens with limited epitopes tend to produce antibodies with restricted light chain usage and that in most individuals, antibodies with lambda light chains have specificities different and complementary to kappa-containing antibodies. PMID:27113164

  10. Aguacate virus, a new antigenic complex of the genus Phlebovirus (family Bunyaviridae)

    PubMed Central

    Travassos da Rosa, Amelia; Savji, Nazir; Sze, Wilson; Wick, Ivan; Guzman, Hilda; Hutchison, Stephen; Tesh, Robert; Lipkin, W. Ian

    2011-01-01

    Genomic and antigenic characterization of Aguacate virus, a tentative species of the genus Phlebovirus, and three other unclassified viruses, Armero virus, Durania virus and Ixcanal virus, demonstrate a close relationship to one another. They are distinct from the other nine recognized species within the genus Phlebovirus. We propose to designate them as a new (tenth) serogroup or species (Aguacate virus) within the genus. The four viruses were all isolated from phlebotomine sandflies (Lutzomyia sp.) collected in Central and South America. Aguacate virus appears to be a natural reassortant and serves as one more example of the high frequency of reassortment in this genus. PMID:21325481

  11. Aguacate virus, a new antigenic complex of the genus Phlebovirus (family Bunyaviridae).

    PubMed

    Palacios, Gustavo; da Rosa, Amelia Travassos; Savji, Nazir; Sze, Wilson; Wick, Ivan; Guzman, Hilda; Hutchison, Stephen; Tesh, Robert; Lipkin, W Ian

    2011-06-01

    Genomic and antigenic characterization of Aguacate virus, a tentative species of the genus Phlebovirus, and three other unclassified viruses, Armero virus, Durania virus and Ixcanal virus, demonstrate a close relationship to one another. They are distinct from the other nine recognized species within the genus Phlebovirus. We propose to designate them as a new (tenth) serogroup or species (Aguacate virus) within the genus. The four viruses were all isolated from phlebotomine sandflies (Lutzomyia sp.) collected in Central and South America. Aguacate virus appears to be a natural reassortant and serves as one more example of the high frequency of reassortment in this genus.

  12. Preparation and diagnostic utility of a hemagglutination inhibition test antigen derived from the baculovirus-expressed hemagglutinin-neuraminidase protein gene of Newcastle disease virus.

    PubMed

    Choi, Kang-Seuk; Kye, Soo-Jeong; Jeon, Woo-Jin; Park, Mi-Ja; Kim, Saeromi; Seul, Hee-Jung; Kwon, Jun-Hun

    2013-01-01

    A recombinant hemagglutinin-neuraminidase (rHN) protein from Newcastle disease virus (NDV) with hemagglutination (HA) activity was expressed in Spodoptera frugiperda cells using a baculovirus expression system. The rHN protein extracted from infected cells was used as an antigen in a hemagglutination inhibition (HI) test for the detection and titration of NDV-specific antibodies present in chicken sera. The rHN antigen produced high HA titers of 2(13) per 25 μL, which were similar to those of the NDV antigen produced using chicken eggs, and it remained stable without significant loss of the HA activity for at least 12 weeks at 4°C. The rHN-based HI assay specifically detected NDV antibodies, but not the sera of other avian pathogens, with a specificity and sensitivity of 100% and 98.0%, respectively, in known positive and negative chicken sera (n = 430). Compared with an NDV-based HI assay, the rHN-based HI assay had a relative sensitivity and specificity of 96.1% and 95.5%, respectively, when applied to field chicken sera. The HI titers of the rHN-based HI assay were highly correlated with those in an NDV-based HI assay (r = 0.927). Overall, these results indicate that rHN protein provides a useful alternative to NDV antigen in HI assays.

  13. Specific inhibition of in vitro Candida-induced lymphocyte proliferation by polysaccharidic antigens present in the serum of patients with chronic mucocutaneous candidiasis.

    PubMed Central

    Fischer, A; Ballet, J J; Griscelli, C

    1978-01-01

    A specific inhibitory activity of in vitro proliferative responses of normal human lymphocytes to Candida metabolic antigen was found in the serum of 6 out of 23 children with chronic mucocutaneous candidiasis. In each of the six patients, the presence of an inhibitory activity was associated with Candida-specific cellular defects, characterized by a negative-skin test and a lack of in vitro lymphocyte proliferation. The presence of a circulating inhibitor was detected during relapses of the disease and disappeared under antifungal therapy. This inhibitory effect was not associated with any toxicity on tested lymphocytes. The factor was shown to be nondialysable, thermostable, nonprecipitable with ammonium sulfate and absorbable on anti-Candida antibodies or concanavalin A-coupled agarose columns. Altogether, these results suggest that the inhibitory factor is not an immunoglobulin, but rather a polysaccharidic antigen of Candida albicans. An inhibition of Candida-induced proliferative response of normal human lymphocytes was also obtained by addition of polysacharide antigens or purified mannans from C. albicans to cultures. Candida polysaccharidic antigens appeared, therefore, to be involved in specific depression of cellular functions observed in chronic candidiasis. PMID:361754

  14. Genomic polymorphism, recombination, and linkage disequilibrium in human major histocompatibility complex-encoded antigen-processing genes

    SciTech Connect

    van Endert, P.M.; Lopez, M.T.; Patel, S.D.; McDevitt, H.O. ); Monaco, J.J. )

    1992-12-01

    Recently, two subunits of a large cytosolic protease and two putative peptide transporter proteins were found to be encoded by genes within the class II region of the major histocompatibility complex (MHC). These genes have been suggested to be involved in the processing of antigenic proteins for presentation by MHC class I molecules. Because of the high degree of polymorphism in MHC genes, and previous evidence for both functional and polypeptide sequence polymorphism in the proteins encoded by the antigen-processing genes, we tested DNA from 27 consanguineous human cell lines for genomic polymorphism by restriction fragment length polymorphism (RFLP) analysis. These studies demonstrate a strong linkage disequilibrium between TAP1 and LMP2 RFLPs. Moreover, RFLPs, as well as a polymorphic stop codon in the telomeric TAP2 gene, appear to be in linkage disequilibrium with HLA-DR alleles and RFLPs in the HLA-DO gene. A high rate of recombination, however, seems to occur in the center of the complex, between the TAP1 and TAP2 genes.

  15. Structure of a Major Antigenic Site on the Respiratory Syncytial Virus Fusion Glycoprotein in Complex with Neutralizing Antibody 101F

    SciTech Connect

    McLellan, Jason S.; Chen, Man; Chang, Jung-San; Yang, Yongping; Kim, Albert; Graham, Barney S.; Kwong, Peter D.

    2010-11-19

    Respiratory syncytial virus (RSV) is a major cause of pneumonia and bronchiolitis in infants and elderly people. Currently there is no effective vaccine against RSV, but passive prophylaxis with neutralizing antibodies reduces hospitalizations. To investigate the mechanism of antibody-mediated RSV neutralization, we undertook structure-function studies of monoclonal antibody 101F, which binds a linear epitope in the RSV fusion glycoprotein. Crystal structures of the 101F antigen-binding fragment in complex with peptides from the fusion glycoprotein defined both the extent of the linear epitope and the interactions of residues that are mutated in antibody escape variants. The structure allowed for modeling of 101F in complex with trimers of the fusion glycoprotein, and the resulting models suggested that 101F may contact additional surfaces located outside the linear epitope. This hypothesis was supported by surface plasmon resonance experiments that demonstrated 101F bound the peptide epitope {approx}16,000-fold more weakly than the fusion glycoprotein. The modeling also showed no substantial clashes between 101F and the fusion glycoprotein in either the pre- or postfusion state, and cell-based assays indicated that 101F neutralization was not associated with blocking virus attachment. Collectively, these results provide a structural basis for RSV neutralization by antibodies that target a major antigenic site on the fusion glycoprotein.

  16. Immunostimulatory complexes containing Eimeria tenella antigens and low toxicity plant saponins induce antibody response and provide protection from challenge in broiler chickens.

    PubMed

    Berezin, V E; Bogoyavlenskyi, A P; Khudiakova, S S; Alexuk, P G; Omirtaeva, E S; Zaitceva, I A; Tustikbaeva, G B; Barfield, R C; Fetterer, R H

    2010-01-20

    Immunostimulating complexes (ISCOMs) are unique multimolecular structures formed by encapsulating antigens, lipids and triterpene saponins and are one of the most successful antigen delivery systems for microbial antigens. In the current study, both the route of administration and the antigen concentration of ISCOMs, containing Eimeria tenella antigens and saponins from native plants, were evaluated in their ability to stimulate humoral immunity and to protect chickens against a challenge infection with E. tenella. Broiler chickens were immunized with ISCOM preparations containing E. tenella antigens and the purified saponins Gg6, Ah6 and Gp7 isolated from Glycyrrhiza glabra, Aesculus hippocastanum and Gipsophila paniculata, respectively. The effects of the route of administration, dose of antigen and type of saponin used for construction of ISCOMs were evaluated for ability to stimulate serum IgG and IgM and to protect chickens against a homologous challenge. A single intranasal immunization was the most effective route for administering ISCOMs although the in ovo route was also quite effective. Dose titration experiments demonstrated efficacy after single immunization with various ISCOM doses but maximum effects were observed when ISCOMs contain 5-10mug antigen. Immunization of birds by any of the three routes with E. tenella antigens alone or antigens mixed with alum hydroxide adjuvant resulted in lower serum antibody and reduced protection to challenge relative to immunization with ISCOMs. Overall the results of this study confirm that significant immunostimulation and protection to challenge are achieved by immunization of chickens with ISCOMs containing purified saponins and native E. tenella antigens and suggest that ISCOMs may be successfully used to develop a safe and effective vaccine for prevention of avian coccidiosis.

  17. Contribution of dopamine to mitochondrial complex I inhibition and dopaminergic deficits caused by methylenedioxymethamphetamine in mice.

    PubMed

    Barros-Miñones, L; Goñi-Allo, B; Suquia, V; Beitia, G; Aguirre, N; Puerta, E

    2015-06-01

    Methylenedioxymethamphetamine (MDMA) causes a persistent loss of dopaminergic cell bodies in the substantia nigra of mice. Current evidence indicates that MDMA-induced neurotoxicity is mediated by oxidative stress probably due to the inhibition of mitochondrial complex I activity. In this study we investigated the contribution of dopamine (DA) to such effects. For this, we modulated the dopaminergic system of mice at the synthesis, uptake or metabolism levels. Striatal mitochondrial complex I activity was decreased 1 h after MDMA; an effect not observed in the striatum of DA depleted mice or in the hippocampus, a dopamine spare region. The DA precursor, L-dopa, caused a significant reduction of mitochondrial complex I activity by itself and exacerbated the dopaminergic deficits when combined with systemic MDMA. By contrast, no damage was observed when L-dopa was combined with intrastriatal injections of MDMA. On the other hand, dopamine uptake blockade using GBR 12909, inhibited both, the acute inhibition of complex I activity and the long-term dopaminergic toxicity caused by MDMA. Moreover, the inhibition of DA metabolism with the monoamine oxidase (MAO) inhibitor, pargyline, afforded a significant protection against MDMA-induced complex I inhibition and neurotoxicity. Taken together, these findings point to the formation of hydrogen peroxide subsequent to DA metabolism by MAO, rather than a direct DA-mediated mitochondrial complex I inhibition, and the contribution of a peripheral metabolite of MDMA, as the key steps in the chain of biochemical events leading to DA neurotoxicity caused by MDMA in mice.

  18. Cloning, sequencing, and expression of the apa gene coding for the Mycobacterium tuberculosis 45/47-kilodalton secreted antigen complex.

    PubMed

    Laqueyrerie, A; Militzer, P; Romain, F; Eiglmeier, K; Cole, S; Marchal, G

    1995-10-01

    Effective protection against a virulent challenge with Mycobacterium tuberculosis is induced mainly by previous immunization with living attenuated mycobacteria, and it has been hypothesized that secreted proteins serve as major targets in the specific immune response. To identify and purify molecules present in culture medium filtrate which are dominant antigens during effective vaccination, a two-step selection procedure was used to select antigens able to interact with T lymphocytes and/or antibodies induced by immunization with living bacteria and to counterselect antigens interacting with the immune effectors induced by immunization with dead bacteria. A Mycobacterium bovis BCG 45/47-kDa antigen complex, present in BCG culture filtrate, has been previously identified and isolated (F. Romain, A. Laqueyrerie, P. Militzer, P. Pescher, P. Chavarot, M. Lagranderie, G. Auregan, M. Gheorghiu, and G. Marchal, Infect. Immun. 61:742-750, 1993). Since the cognate antibodies recognize the very same antigens present in M. tuberculosis culture medium filtrates, a project was undertaken to clone, express, and sequence the corresponding gene of M. tuberculosis. An M. tuberculosis shuttle cosmid library was transferred in Mycobacterium smegmatis and screened with a competitive enzyme-linked immunosorbent assay to detect the clones expressing the proteins. A clone containing a 40-kb DNA insert was selected, and by means of subcloning in Escherichia coli, a 2-kb fragment that coded for the molecules was identified. An open reading frame in the 2,061-nucleotide sequence codes for a secreted protein with a consensus signal peptide of 39 amino acids and a predicted molecular mass of 28,779 Da. The gene was referred to as apa because of the high percentages of proline (21.7%) and alanine (19%) in the purified protein. Southern hybridization analysis of digested total genomic DNA from M. tuberculosis (reference strains H37Rv and H37Ra) indicated that the apa gene was present as a

  19. Serotype O:8 isolates in the Yersinia pseudotuberculosis complex have different O-antigen gene clusters and produce various forms of rough LPS.

    PubMed

    Kenyon, Johanna J; Duda, Katarzyna A; De Felice, Antonia; Cunneen, Monica M; Molinaro, Antonio; Laitinen, Juha; Skurnik, Mikael; Holst, Otto; Reeves, Peter R; De Castro, Cristina

    2016-04-01

    In Yersinia pseudotuberculosis complex, the O-antigen of LPS is used for the serological characterization of strains, and 21 serotypes have been identified to date. The O-antigen biosynthesis gene cluster and corresponding O-antigen structure have been described for 18, leaving O:8, O:13 and O:14 unresolved. In this study, two O:8 isolates were examined. The O-antigen gene cluster sequence of strain 151 was near identical to serotype O:4a, though a frame-shift mutation was found in ddhD, while No. 6 was different to 151 and carried the O:1b gene cluster. Structural analysis revealed that No. 6 produced a deeply truncated LPS, suggesting a mutation within the waaF gene. Both ddhD and waaF were cloned and expressed in 151 and No. 6 strains, respectively, and it appeared that expression of ddhD gene in strain 151 restored the O-antigen on LPS, while waaF in No. 6 resulted in an LPS truncated less severely but still without the O-antigen, suggesting that other mutations occurred in this strain. Thus, both O:8 isolates were found to be spontaneous O-antigen-negative mutants derived from other validated serotypes, and we propose to remove this serotype from the O-serotyping scheme, as the O:8 serological specificity is not based on the O-antigen.

  20. Loss of T Cell Antigen Recognition Arising from Changes in Peptide and Major Histocompatibility Complex Protein Flexibility: Implications for Vaccine Design

    SciTech Connect

    Insaidoo, Francis K.; Borbulevych, Oleg Y.; Hossain, Moushumi; Santhanagopolan, Sujatha M.; Baxter, Tiffany K.; Baker, Brian M.

    2012-05-08

    Modification of the primary anchor positions of antigenic peptides to improve binding to major histocompatibility complex (MHC) proteins is a commonly used strategy for engineering peptide-based vaccine candidates. However, such peptide modifications do not always improve antigenicity, complicating efforts to design effective vaccines for cancer and infectious disease. Here we investigated the MART-1{sub 27-35} tumor antigen, for which anchor modification (replacement of the position two alanine with leucine) dramatically reduces or ablates antigenicity with a wide range of T cell clones despite significantly improving peptide binding to MHC. We found that anchor modification in the MART-1{sub 27-35} antigen enhances the flexibility of both the peptide and the HLA-A*0201 molecule. Although the resulting entropic effects contribute to the improved binding of the peptide to MHC, they also negatively impact T cell receptor binding to the peptide {center_dot} MHC complex. These results help explain how the 'anchor-fixing' strategy fails to improve antigenicity in this case, and more generally, may be relevant for understanding the high specificity characteristic of the T cell repertoire. In addition to impacting vaccine design, modulation of peptide and MHC flexibility through changes to antigenic peptides may present an evolutionary strategy for the escape of pathogens from immune destruction.

  1. Identification of an elaborate complex mediating postsynaptic inhibition.

    PubMed

    Uezu, Akiyoshi; Kanak, Daniel J; Bradshaw, Tyler W A; Soderblom, Erik J; Catavero, Christina M; Burette, Alain C; Weinberg, Richard J; Soderling, Scott H

    2016-09-01

    Inhibitory synapses dampen neuronal activity through postsynaptic hyperpolarization. The composition of the inhibitory postsynapse and the mechanistic basis of its regulation, however, remain poorly understood. We used an in vivo chemico-genetic proximity-labeling approach to discover inhibitory postsynaptic proteins. Quantitative mass spectrometry not only recapitulated known inhibitory postsynaptic proteins but also revealed a large network of new proteins, many of which are either implicated in neurodevelopmental disorders or are of unknown function. Clustered regularly interspaced short palindromic repeats (CRISPR) depletion of one of these previously uncharacterized proteins, InSyn1, led to decreased postsynaptic inhibitory sites, reduced the frequency of miniature inhibitory currents, and increased excitability in the hippocampus. Our findings uncover a rich and functionally diverse assemblage of previously unknown proteins that regulate postsynaptic inhibition and might contribute to developmental brain disorders. PMID:27609886

  2. Latex-protein complexes from an acute phase recombinant antigen of Toxoplasma gondii for the diagnosis of recently acquired toxoplasmosis.

    PubMed

    Peretti, Leandro E; Gonzalez, Verónica D G; Marcipar, Iván S; Gugliotta, Luis M

    2014-08-01

    The synthesis and characterization of latex-protein complexes (LPC), from the acute phase recombinant antigen P35 (P35Ag) of Toxoplasma gondii and "core-shell" carboxylated or polystyrene (PS) latexes (of different sizes and charge densities) are considered, with the aim of producing immunoagglutination reagents able to detect recently acquired toxoplasmosis. Physical adsorption (PA) and chemical coupling (CC) of P35Ag onto latex particles at different pH were investigated. Greater amounts of adsorbed protein were obtained on PS latexes than on carboxylated latexes, indicating that hydrophobic forces govern the interactions between the protein and the particle surface. In the CC experiments, the highest amount of bound protein was obtained at pH 6, near the isoelectric point of the protein (IP=6.27). At this pH, it decreased both the repulsion between particle surface and protein, and the repulsion between neighboring molecules. The LPC were characterized and the antigenicity of the P35Ag protein coupled on the particles surface was evaluated by Enzyme-Linked ImmunoSorbent Assay (ELISA). Results from ELISA showed that the P35Ag coupled to the latex particles surface was not affected during the particles sensitization by PA and CC and the produced LPC were able to recognize specific anti-P35Ag antibodies present in the acute phase of the disease.

  3. Schistosoma japonicum egg antigen up-regulates fibrogenesis and inhibits proliferation in primary hepatic stellate cells in a concentration-dependent manner

    PubMed Central

    Liu, Ping; Wang, Mi; Lu, Xiao-Dan; Zhang, Shu-Juan; Tang, Wang-Xian

    2013-01-01

    AIM: To investigate the effects of different concentrations of Schistosoma japonicum (S. japonicum) egg antigen on fibrogenesis and apoptosis in primary hepatic stellate cells (HSCs). METHODS: A mouse model of schistosomiasis-associated liver fibrosis (SSLF) was established by infecting mice with schistosomal cercaria via the abdomen. HSCs were isolated from SSLF mice by discontinuous density gradient centrifugation, and their identity was confirmed by immunofluorescence double staining of α-smooth muscle actin (α-SMA) and desmin. The growth inhibitory effect and 50% inhibitory concentration (IC50) of S. japonicum egg antigen for primary HSCs (24 h) were determined using a cell counting kit-8 (CCK-8) assay. The expression levels of α-SMA, matrix metalloproteinase-9 (MMOL/LP-9) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in HSCs in response to different concentrations of S. japonicum egg antigen were detected by Western blotting and real-time reverse transcription-polymerase chain reaction. The levels of phospho-P38 (P-P38), phospho-Jun N-terminal kinase (P-JNK) and phospho-Akt (P-AKT) in HSCs were detected by Western blotting. RESULTS: An SSLF mouse model was established, and primary HSCs were successfully isolated and cultured. S. japonicum egg antigen inhibited HSC proliferation in a concentration-dependent manner. The IC50 of the S. japonicum egg antigen was 244.53 ± 35.26 μg/mL. S. japonicum egg antigen enhanced α-SMA expression at both the mRNA and protein levels and enhanced TIMP-1 expression at the mRNA level in HSCs (P < 0.05), whereas the expression of MMOL/LP-9 was attenuated at both the mRNA and protein levels in a concentration-dependent manner (P < 0.05). A high concentration of S. japonicum egg antigen enhanced P-P38, P-JNK and P-AKT activation (P < 0.05). The changes in α-SMA and MMOL/LP-9 expression induced by S. japonicum egg antigen were closely correlated with P-P38 and P-JNK activation (P < 0.05). The attenuation of MMOL/LP-9

  4. Inhibition of the purified 20S proteasome by non-heme iron complexes

    PubMed Central

    Prakash, Jai; Schmitt, Sara M.; Dou, Q. Ping; Kodanko, Jeremy J.

    2013-01-01

    Polypyridyl pentadentate ligands N4Py (1) and Bn-TPEN (2), along with their respective iron complexes, have been investigated for their ability to inhibit the purified 20S proteasome. Results demonstrated that the iron complexes of both ligands are potent inhibitors of the 20S proteasome (IC50 = 9.2 μM for [FeII(OH2)(N4Py)]2+ (3) and 4.0 μM for [FeII(OH2)(Bn-TPEN)]2+ (4)). Control experiments showed that ligand 1 or FeII alone showed no inhibition, whereas 2 was moderately active (IC50 = 96 μM), suggesting that iron, when bound to these ligands, plays a key role in proteasome inhibition. Results from time-dependent inactivation studies suggest different modes of action for the iron complexes. Time-dependent decay of proteasome activity was observed upon incubation in the presence of 4, which accelerated in the presence of DTT, suggesting reductive activation of O2 and oxidation of the 20S proteasome as a mode of action. In contrast, loss of 20S proteasome activity was not observed with 3 over time, suggesting inhibition through direct binding of the iron complex to the enzyme. Inhibition of the 20S proteasome by 4 was not blocked by reactive oxygen species scavengers, consistent with a unique oxidant being responsible for the time-dependent inhibition observed. PMID:22170477

  5. Inhibition of Beta-Amyloid Fibrillation by Luminescent Iridium(III) Complex Probes

    PubMed Central

    Lu, Lihua; Zhong, Hai-Jing; Wang, Modi; Ho, See-Lok; Li, Hung-Wing; Leung, Chung-Hang; Ma, Dik-Lung

    2015-01-01

    We report herein the application of kinetically inert luminescent iridium(III) complexes as dual inhibitors and probes of beta-amyloid fibrillogenesis. These iridium(III) complexes inhibited Aβ1–40 peptide aggregation in vitro, and protected against Aβ-induced cytotoxicity in neuronal cells. Furthermore, the complexes differentiated between the aggregated and unaggregated forms of Aβ1–40 peptide on the basis of their emission response. PMID:26419607

  6. Heparin inhibits the intrinsic tenase complex by interacting with an exosite on factor IXa.

    PubMed

    Sheehan, John P; Kobbervig, Catherine E; Kirkpatrick, Heidi M

    2003-09-30

    The specific molecular target for direct heparin inhibition of factor X activation by intrinsic tenase (factor IXa-factor VIIIa) was investigated. Comparison of size-fractionated oligosaccharides demonstrated that an octasaccharide was sufficient to inhibit intrinsic tenase. Substitution of soluble dihexanoic phosphatidylserine (C6PS) for phospholipid (PL) vesicles demonstrated that inhibition by low-molecular weight heparin (LMWH) was independent of factor IXa-factor VIIIa membrane assembly. LMWH also inhibited factor X activation by the factor IXa-PL complex via a distinct mechanism that required longer oligosaccharides and was independent of substrate concentrations. The apparent affinity of LMWH for the factor IXa-PL complex was higher in the absence of factor VIIIa, suggesting that the cofactor adversely affected the interaction of heparin with factor IXa-phospholipid. LMWH did not interact directly with the active site, as it failed to inhibit chromogenic substrate cleavage by the factor IXa-PL complex. LMWH induced a modest decrease in factor IXa-factor VIIIa affinity [K(D(app))] on PL vesicles that did not account for the inhibition. In contrast, LMWH caused a substantial reduction in factor IXa-factor VIIIa affinity in the presence of C6PS that fully accounted for the inhibition. Factor IXa bound LMWH with significantly higher affinity than factor X by competition solution affinity analysis, and the K(D(app)) for the factor IXa-LMWH complex agreed with the K(I) for inhibition of the factor IXa-PL complex by LMWH. Thus, LMWH binds to an exosite on factor IXa that antagonizes cofactor activity without disrupting factor IXa-factor VIIIa assembly on the PL surface. This exosite may contribute to the clinical efficacy of heparin and represents a novel target for antithrombotic therapy.

  7. Definition of epitopes and antigens recognized by vaccinia specific immune responses: their conservation in variola virus sequences, and use as a model system to study complex pathogens.

    PubMed

    Sette, Alessandro; Grey, Howard; Oseroff, Carla; Peters, Bjoern; Moutaftsi, Magdalini; Crotty, Shane; Assarsson, Erika; Greenbaum, Jay; Kim, Yohan; Kolla, Ravi; Tscharke, David; Koelle, David; Johnson, R Paul; Blum, Janice; Head, Steven; Sidney, John

    2009-12-30

    In the last few years, a wealth of information has become available relating to the targets of vaccinia virus (VACV)-specific CD4(+) T cell, CD8(+) T cell and antibody responses. Due to the large size of its genome, encoding more than 200 different proteins, VACV represents a useful model system to study immunity to complex pathogens. Our data demonstrate that both cellular and humoral responses target a large number of antigens and epitopes. This broad spectrum of targets is detected in both mice and humans. CD4(+) T cell responses target late and structural antigens, while CD8(+) T cells preferentially recognize early antigens. While both CD4(+) and CD8(+) T cell responses target different types of antigens, the antigens recognized by T(H) cells are highly correlated with those recognized by antibody responses. We further show that protein abundance and antibody recognition can be used to predict antigens recognized by CD4(+) T cell responses, while early expression at the mRNA level predicts antigens targeted by CD8(+) T cells. Finally, we find that the vast majority of VACV epitopes are conserved in variola virus (VARV), thus suggesting that the epitopes defined herein also have relevance for the efficacy of VACV as a smallpox vaccine.

  8. Effect of mitochondrial complex I inhibition on Fe-S cluster protein activity

    SciTech Connect

    Mena, Natalia P.; Bulteau, Anne Laure; Salazar, Julio; Hirsch, Etienne C.; Nunez, Marco T.

    2011-06-03

    Highlights: {yields} Mitochondrial complex I inhibition resulted in decreased activity of Fe-S containing enzymes mitochondrial aconitase and cytoplasmic aconitase and xanthine oxidase. {yields} Complex I inhibition resulted in the loss of Fe-S clusters in cytoplasmic aconitase and of glutamine phosphoribosyl pyrophosphate amidotransferase. {yields} Consistent with loss of cytoplasmic aconitase activity, an increase in iron regulatory protein 1 activity was found. {yields} Complex I inhibition resulted in an increase in the labile cytoplasmic iron pool. -- Abstract: Iron-sulfur (Fe-S) clusters are small inorganic cofactors formed by tetrahedral coordination of iron atoms with sulfur groups. Present in numerous proteins, these clusters are involved in key biological processes such as electron transfer, metabolic and regulatory processes, DNA synthesis and repair and protein structure stabilization. Fe-S clusters are synthesized mainly in the mitochondrion, where they are directly incorporated into mitochondrial Fe-S cluster-containing proteins or exported for cytoplasmic and nuclear cluster-protein assembly. In this study, we tested the hypothesis that inhibition of mitochondrial complex I by rotenone decreases Fe-S cluster synthesis and cluster content and activity of Fe-S cluster-containing enzymes. Inhibition of complex I resulted in decreased activity of three Fe-S cluster-containing enzymes: mitochondrial and cytosolic aconitases and xanthine oxidase. In addition, the Fe-S cluster content of glutamine phosphoribosyl pyrophosphate amidotransferase and mitochondrial aconitase was dramatically decreased. The reduction in cytosolic aconitase activity was associated with an increase in iron regulatory protein (IRP) mRNA binding activity and with an increase in the cytoplasmic labile iron pool. Since IRP activity post-transcriptionally regulates the expression of iron import proteins, Fe-S cluster inhibition may result in a false iron deficiency signal. Given that

  9. Complex formation with nucleic acids and aptamers alters the antigenic properties of platelet factor 4

    PubMed Central

    Jaax, Miriam E.; Krauel, Krystin; Marschall, Thomas; Brandt, Sven; Gansler, Julia; Fürll, Birgitt; Appel, Bettina; Fischer, Silvia; Block, Stephan; Helm, Christiane A.; Müller, Sabine; Preissner, Klaus T.

    2013-01-01

    The tight electrostatic binding of the chemokine platelet factor 4 (PF4) to polyanions induces heparin-induced thrombocytopenia, a prothrombotic adverse drug reaction caused by immunoglobulin G directed against PF4/polyanion complexes. This study demonstrates that nucleic acids, including aptamers, also bind to PF4 and enhance PF4 binding to platelets. Systematic assessment of RNA and DNA constructs, as well as 4 aptamers of different lengths and secondary structures, revealed that increasing length and double-stranded segments of nucleic acids augment complex formation with PF4, while single nucleotides or single-stranded polyA or polyC constructs do not. Aptamers were shown by circular dichroism spectroscopy to induce structural changes in PF4 that resemble those induced by heparin. Moreover, heparin-induced anti-human–PF4/heparin antibodies cross-reacted with human PF4/nucleic acid and PF4/aptamer complexes, as shown by an enzyme immunoassay and a functional platelet activation assay. Finally, administration of PF4/44mer–DNA protein C aptamer complexes in mice induced anti–PF4/aptamer antibodies, which cross-reacted with murine PF4/heparin complexes. These data indicate that the formation of anti-PF4/heparin antibodies in postoperative patients may be augmented by PF4/nucleic acid complexes. Moreover, administration of therapeutic aptamers has the potential to induce anti-PF4/polyanion antibodies and a prothrombotic diathesis. PMID:23673861

  10. Phosphorothioate oligonucleotides inhibit the intrinsic tenase complex by an allosteric mechanism.

    PubMed

    Sheehan, J P; Phan, T M

    2001-04-24

    Phosphorothioate oligonucleotides (PS ODNs) prolong the activated partial thromboplastin time in human plasma by inhibition of intrinsic tenase (factor IXa-factor VIIIa) activity. This inhibition was characterized using ISIS 2302, a 20-mer antisense PS ODN. ISIS 2302 demonstrated hyperbolic, mixed-type inhibition of factor X activation by the intrinsic tenase complex. The decrease in V(max(app)) was analyzed by examining complex assembly, cofactor stability, and protease catalysis. ISIS 2302 did not inhibit factor X activation by the factor IXa-phospholipid complex, or significantly affect factor VIII-phospholipid affinity. Inhibitory concentrations of ISIS 2302 modestly decreased the affinity of factor IXa-factor VIIIa binding in the presence of phospholipid (K(D) = 11.5 vs 4.8 nM). This effect was insufficient to explain the reduction in V(max(app)). ISIS 2302 did not affect the in vitro half-life of factor VIIIa, suggesting it did not destabilize cofactor activity. In the presence of 30% ethylene glycol, the level of factor X activation by the factor IXa-phospholipid complex increased 3-fold, and the level of chromogenic substrate cleavage by factor IXa increased more than 50-fold. ISIS 2302 demonstrated partial inhibition of factor X activation by the factor IXa-phospholipid complex, and chromogenic substrate cleavage by factor IXa, only in the presence of ethylene glycol. Like the intact enzyme complex, ISIS 2302 demonstrated hyperbolic, mixed-type inhibition of chromogenic substrate cleavage by factor IXa (K(I) = 88 nM). Equilibrium binding studies with fluorescein-labeled ISIS 2302 demonstrated a similar affinity (K(D) = 92 nM) for the PS ODN-factor IX interaction. These results suggest that PS ODNs bind to an exosite on factor IXa, modulating catalytic activity of the intrinsic tenase complex.

  11. [Genetic diversity based on swine leukocyte antigen complex mi-crosatellites(SLA-MS) in five pig populations].

    PubMed

    Yu, Hui; Liu, Rong-Hui; Li, Hua; Zuo, Qi-Zhen; Li, Yan; Wu, Zhen-Fang

    2012-11-01

    The genetic diversity of swine leukocyte antigen complex (SLA) was studied among Guangdong local pigs, Huanan wild boars (S.s. chirodontus) and introduced pigs, which aimed at providing a theoretical foundation for further pig anti-disease resistance breeding. Pietrain pigs, Duroc pigs, Large black-white pigs, Lantang pigs, and Huanan wild boars were genotyped by employing 18 microsatellites in swine leukocyte antigen complex (SLA-MS). The result showed that the average diversity in SLA II was higher (He=0.628, PIC=0.581) than that in SLA I (He=0.530, PIC=0.474) and in SLA III (He=0.526, PIC=0.458). The molecular diversity indices (MDI) of Huanan wild boars was the highest(0.716), followed by Lantang pigs (0.614), Large black-white pigs (0.559), Pietrain pigs (0.550) and Duroc pigs (0.507). As a whole, the genetic diversity of Huanan wild boars was the highest over Guangdong native pigs and introduced pigs. Large black-white pigs and Duroc pigs had ever happened a severe bottleneck by comparison with the Garza-Williamson index (GWI) in Huanan wild boar. From the genetic distance, one clade was that Lantang pigs were first clustered with Huanan wild boar, and then grouped together with Large black-white pigs; another clade was that Pietrain pigs were independently clustered with Duroc pigs in the NJ tree. The results would establish the foundation for pig conservation of germplasm resource, disease resistance breeding, and multiplicative strains.

  12. Plasma membrane associated, virus-specific polypeptides required for the formation of target antigen complexes recognized by virus-specific cytotoxic T lymphocytes

    SciTech Connect

    Domber, E.A.

    1986-01-01

    These studies were undertaken to define some of the poxvirus-specific target antigens which are synthesized in infected cells and recognized by vaccinia virus-specific CTLs (VV-CTLs). Since vaccinia virus infected, unmanipulated target cells express numerous virus-specific antigens on the plasma membrane, attempts were made to manipulate expression of the poxvirus genome after infection so that one or a few defined virus-specified antigens were expressed on the surface of infected cells. In vitro (/sup 51/Cr)-release assays determined that viral DNA synthesis and expression of late viral proteins were not necessary to form a target cell which was fully competent for lysis by VV-CTLs. Under the conditions employed in these experiments, 90-120 minutes of viral protein synthesis were necessary to produce a competent cell for lysis by VV-CTLs. In order to further inhibit the expression of early viral proteins in infected cells, partially UV-inactivated vaccinia virus was employed to infect target cells. It was determined that L-cells infected with virus preparations which had been UV-irradiated for 90 seconds were fully competent for lysis by VV-CTLs. Cells infected with 90 second UV-irr virus expressed 3 predominant, plasma membrane associated antigens of 36-37K, 27-28K, and 19-17K. These 3 viral antigens represent the predominant membrane-associated viral antigens available for interaction with class I, major histocompatibility antigens and hence are potential target antigens for VV-CTLs.

  13. Targeting metabolic flexibility by simultaneously inhibiting respiratory complex I and lactate generation retards melanoma progression.

    PubMed

    Chaube, Balkrishna; Malvi, Parmanand; Singh, Shivendra Vikram; Mohammad, Naoshad; Meena, Avtar Singh; Bhat, Manoj Kumar

    2015-11-10

    Melanoma is a largely incurable skin malignancy owing to the underlying molecular and metabolic heterogeneity confounded by the development of resistance. Cancer cells have metabolic flexibility in choosing either oxidative phosphorylation (OXPHOS) or glycolysis for ATP generation depending upon the nutrient availability in tumor microenvironment. In this study, we investigated the involvement of respiratory complex I and lactate dehydrogenase (LDH) in melanoma progression. We show that inhibition of complex I by metformin promotes melanoma growth in mice via elevating lactate and VEGF levels. In contrast, it leads to the growth arrest in vitro because of enhanced extracellular acidification as a result of increased glycolysis. Inhibition of LDH or lactate generation causes decrease in glycolysis with concomitant growth arrest both in vitro and in vivo. Blocking lactate generation in metformin-treated melanoma cells results in diminished cell proliferation and tumor progression in mice. Interestingly, inhibition of either LDH or complex I alone does not induce apoptosis, whereas inhibiting both together causes depletion in cellular ATP pool resulting in metabolic catastrophe induced apoptosis. Overall, our study suggests that LDH and complex I play distinct roles in regulating glycolysis and cell proliferation. Inhibition of these two augments synthetic lethality in melanoma.

  14. HD-03/ES: A Herbal Medicine Inhibits Hepatitis B Surface Antigen Secretion in Transfected Human Hepatocarcinoma PLC/PRF/5 Cells.

    PubMed

    Varma, Sandeep R; Sundaram, R; Gopumadhavan, S; Vidyashankar, Satyakumar; Patki, Pralhad S

    2013-01-01

    HD-03/ES is a herbal formulation used for the treatment of hepatitis B. However, the molecular mechanism involved in the antihepatitis B (HBV) activity of this drug has not been studied using in vitro models. The effect of HD-03/ES on hepatitis B surface antigen (HBsAg) secretion and its gene expression was studied in transfected human hepatocarcinoma PLC/PRF/5 cells. The anti-HBV activity was tested based on the inhibition of HBsAg secretion into the culture media, as detected by HBsAg-specific antibody-mediated enzyme assay (ELISA) at concentrations ranging from 125 to 1000  μ g/mL. The effect of HD-03/ES on HBsAg gene expression was analyzed using semiquantitative multiplex RT-PCR by employing specific primers. The results showed that HD-03/ES suppressed HBsAg production with an IC50 of 380  μ g/mL in PLC/PRF/5 cells for a period of 24 h. HD-03/ES downregulated HBsAg gene expression in PLC/PRF/5 cells. In conclusion, HD-03/ES exhibits strong anti-HBV properties by inhibiting the secretion of hepatitis B surface antigen in PLC/PRF/5 cells, and this action is targeted at the transcription level. Thus, HD-03/ES could be beneficial in the treatment of acute and chronic hepatitis B infections.

  15. HD-03/ES: A Herbal Medicine Inhibits Hepatitis B Surface Antigen Secretion in Transfected Human Hepatocarcinoma PLC/PRF/5 Cells

    PubMed Central

    Varma, Sandeep R.; Sundaram, R.; Gopumadhavan, S.; Vidyashankar, Satyakumar; Patki, Pralhad S.

    2013-01-01

    HD-03/ES is a herbal formulation used for the treatment of hepatitis B. However, the molecular mechanism involved in the antihepatitis B (HBV) activity of this drug has not been studied using in vitro models. The effect of HD-03/ES on hepatitis B surface antigen (HBsAg) secretion and its gene expression was studied in transfected human hepatocarcinoma PLC/PRF/5 cells. The anti-HBV activity was tested based on the inhibition of HBsAg secretion into the culture media, as detected by HBsAg-specific antibody-mediated enzyme assay (ELISA) at concentrations ranging from 125 to 1000 μg/mL. The effect of HD-03/ES on HBsAg gene expression was analyzed using semiquantitative multiplex RT-PCR by employing specific primers. The results showed that HD-03/ES suppressed HBsAg production with an IC50 of 380 μg/mL in PLC/PRF/5 cells for a period of 24 h. HD-03/ES downregulated HBsAg gene expression in PLC/PRF/5 cells. In conclusion, HD-03/ES exhibits strong anti-HBV properties by inhibiting the secretion of hepatitis B surface antigen in PLC/PRF/5 cells, and this action is targeted at the transcription level. Thus, HD-03/ES could be beneficial in the treatment of acute and chronic hepatitis B infections. PMID:23691296

  16. Envelope protein complexes of Mycobacterium avium subsp. paratuberculosis and their antigenicity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s disease, a chronic enteric disease of ruminant animals. In the present study, blue native PAGE electrophoresis and 2D SDS-PAGE were used to separate MAP envelope protein complexes, followed by mass spectrometry (MS) ...

  17. Identification of the p53 protein domain involved in formation of the simian virus 40 large T-antigen-p53 protein complex.

    PubMed Central

    Tan, T H; Wallis, J; Levine, A J

    1986-01-01

    An expression vector utilizing the enhancer and promoter region of the simian virus 40 (SV40) DNA regulating a murine p53 cDNA clone was constructed. The vector produced murine p53 protein in monkey cells identified by five different monoclonal antibodies, three of which were specific for the murine form of p53. The murine p53 produced in monkey cells formed an oligomeric protein complex with the SV40 large tumor antigen. A large number of deletion mutations, in-frame linker insertion mutations, and linker insertion mutations resulting in a frameshift mutation were constructed in the cDNA coding portion of the p53 protein expression vector. The wild-type and mutant p53 cDNA vectors were expressed in monkey cells producing the SV40 large T antigen. The conformation and levels of p53 protein and its ability to form protein complexes with the SV40 T antigen were determined by using five different monoclonal antibodies with quite distinct epitope recognition sites. Insertion mutations between amino acid residues 123 and 215 (of a total of 390 amino acids) eliminated the ability of murine p53 to bind to the SV40 large T antigen. Deletion (at amino acids 11 through 33) and insertion mutations (amino acids 222 through 344) located on either side of this T-antigen-binding protein domain produced a murine p53 protein that bound to the SV40 large T antigen. The same five insertion mutations that failed to bind with the SV40 large T antigen also failed to react with a specific monoclonal antibody, PAb246. In contrast, six additional deletion and insertion mutations that produced p53 protein that did bind with T antigen were each recognized by PAb246. The proposed epitope for PAb246 has been mapped adjacent (amino acids 88 through 109) to the T-antigen-binding domain (amino acids 123 through 215) localized by the mutations mapped in this study. Finally, some insertion mutations that produced a protein that failed to bind to the SV40 T antigen appeared to have an enhanced

  18. Injury and differentiation following inhibition of mitochondrial respiratory chain complex IV in rat oligodendrocytes

    PubMed Central

    Ziabreva, Iryna; Campbell, Graham; Rist, Julia; Zambonin, Jessica; Rorbach, Joanna; Wydro, Mateusz M; Lassmann, Hans; Franklin, Robin J M; Mahad, Don

    2010-01-01

    Oligodendrocyte lineage cells are susceptible to a variety of insults including hypoxia, excitotoxicity, and reactive oxygen species. Demyelination is a well-recognized feature of several CNS disorders including multiple sclerosis, white matter strokes, progressive multifocal leukoencephalopathy, and disorders due to mitochondrial DNA mutations. Although mitochondria have been implicated in the demise of oligodendrocyte lineage cells, the consequences of mitochondrial respiratory chain defects have not been examined. We determine the in vitro impact of established inhibitors of mitochondrial respiratory chain complex IV or cytochrome c oxidase on oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes as well as on differentiation capacity of OPCs from P0 rat. Injury to mature oligodendrocytes following complex IV inhibition was significantly greater than to OPCs, judged by cell detachment and mitochondrial membrane potential (MMP) changes, although viability of cells that remained attached was not compromised. Active mitochondria were abundant in processes of differentiated oligodendrocytes and MMP was significantly greater in differentiated oligodendrocytes than OPCs. MMP dissipated following complex IV inhibition in oligodendrocytes. Furthermore, complex IV inhibition impaired process formation within oligodendrocyte lineage cells. Injury to and impaired process formation of oligodendrocytes following complex IV inhibition has potentially important implications for the pathogenesis and repair of CNS myelin disorders. © 2010 Wiley-Liss, Inc. PMID:20665559

  19. The 2.5 Å Structure of CD1c in Complex with a Mycobacterial Lipid Reveals an Open Groove Ideally Suited for Diverse Antigen Presentation

    SciTech Connect

    Scharf, Louise; Li, Nan-Sheng; Hawk, Andrew J.; Garzón, Diana; Zhang, Tejia; Fox, Lisa M.; Kazen, Allison R.; Shah, Sneha; Haddadian, Esmael J.; Gumperz, Jenny E.; Saghatelian, Alan; Faraldo-Gómez, José D.; Meredith, Stephen C.; Piccirilli, Joseph A.; Adams, Erin J.

    2011-08-24

    CD1 molecules function to present lipid-based antigens to T cells. Here we present the crystal structure of CD1c at 2.5 {angstrom} resolution, in complex with the pathogenic Mycobacterium tuberculosis antigen mannosyl-{beta}1-phosphomycoketide (MPM). CD1c accommodated MPM's methylated alkyl chain exclusively in the A pocket, aided by a unique exit portal underneath the {alpha}1 helix. Most striking was an open F pocket architecture lacking the closed cavity structure of other CD1 molecules, reminiscent of peptide binding grooves of classical major histocompatibility complex molecules. This feature, combined with tryptophan-fluorescence quenching during loading of a dodecameric lipopeptide antigen, provides a compelling model by which both the lipid and peptide moieties of the lipopeptide are involved in CD1c presentation of lipopeptides.

  20. Functional effects of the antigen glatiramer acetate are complex and tightly associated with its composition.

    PubMed

    Hasson, Tal; Kolitz, Sarah; Towfic, Fadi; Laifenfeld, Daphna; Bakshi, Shlomo; Beriozkin, Olga; Shacham-Abramson, Maya; Timan, Bracha; Fowler, Kevin D; Birnberg, Tal; Konya, Attila; Komlosh, Arthur; Ladkani, David; Hayden, Michael R; Zeskind, Benjamin; Grossman, Iris

    2016-01-15

    Glatiramer acetate (Copaxone®; GA) is a non-biological complex drug for multiple sclerosis. GA modulated thousands of genes in genome-wide expression studies conducted in THP-1 cells and mouse splenocytes. Comparing GA with differently-manufactured glatiramoid Polimunol (Synthon) in mice yielded hundreds of differentially expressed probesets, including biologically-relevant genes (e.g. Il18, adj p<9e-6) and pathways. In human monocytes, 700+ probesets differed between Polimunol and GA, enriching for 130+ pathways including response to lipopolysaccharide (adj. p<0.006). Key differences were confirmed by qRT-PCR (splenocytes) or proteomics (THP-1). These studies demonstrate the complexity of GA's mechanisms of action, and may help inform therapeutic equivalence assessment. PMID:26711576

  1. A model of motor inhibition for a complex skill: baseball batting.

    PubMed

    Gray, Rob

    2009-06-01

    The ability to inhibit an ongoing action in response to a signal from the environment is important for many perceptual-motor actions. This paper examines a particular example of this behavior: attempting to inhibit or "check" a swing in baseball batting. A model of motor inhibition in batting is proposed. In the model there are three different inhibition signals (out of range launch angle, early expected-actual trajectory discrepancy, and late expected-actual trajectory discrepancy) resulting in four possible response outcomes for the batter's swing (full swing, inhibited swing, partial response, or interrupted swing). The predictions of the model were compared with the actual batting performance of 20 baseball players using a high-fidelity batting simulator. The proportions of the different response outcomes could be explained by the inhibition model for 17/20 of the batters in the study. These findings suggest that models of motor inhibition developed for simple, discrete tasks can be applied to complex, multistage behaviors. This batting inhibition model could be used to provide a quantitative measure of a player's bat control for training and player-screening purposes. (PsycINFO Database Record (c) 2009 APA, all rights reserved). PMID:19586249

  2. A model of motor inhibition for a complex skill: baseball batting.

    PubMed

    Gray, Rob

    2009-06-01

    The ability to inhibit an ongoing action in response to a signal from the environment is important for many perceptual-motor actions. This paper examines a particular example of this behavior: attempting to inhibit or "check" a swing in baseball batting. A model of motor inhibition in batting is proposed. In the model there are three different inhibition signals (out of range launch angle, early expected-actual trajectory discrepancy, and late expected-actual trajectory discrepancy) resulting in four possible response outcomes for the batter's swing (full swing, inhibited swing, partial response, or interrupted swing). The predictions of the model were compared with the actual batting performance of 20 baseball players using a high-fidelity batting simulator. The proportions of the different response outcomes could be explained by the inhibition model for 17/20 of the batters in the study. These findings suggest that models of motor inhibition developed for simple, discrete tasks can be applied to complex, multistage behaviors. This batting inhibition model could be used to provide a quantitative measure of a player's bat control for training and player-screening purposes. (PsycINFO Database Record (c) 2009 APA, all rights reserved).

  3. Immunization with antigenic peptides complexed with β-glucan induces potent cytotoxic T-lymphocyte activity in combination with CpG-ODNs.

    PubMed

    Mochizuki, Shinichi; Morishita, Hiromi; Kobiyama, Kouji; Aoshi, Taiki; Ishii, Ken J; Sakurai, Kazuo

    2015-12-28

    The induction of antigen-specific immune responses requires immunization with not only antigens, but also adjuvants. CpG oligonucleotides (CpG-ODNs) are well-known ligands for Toll-like receptor 9 and a potent adjuvant that induces both Th1-type humoral and cellular immune responses including cytotoxic T-lymphocyte responses. We previously demonstrated that β-glucan schizophyllan (SPG) can form complexes with CpG-ODNs with attached dA40 (CpG-dA/SPG), which can accumulate in macrophages in the draining inguinal lymph nodes and induce strong immune responses by co-administration of antigenic proteins, namely ovalbumin (OVA). Immunization with antigenic peptides, OVA257-264, did not induce these antigen-specific immune responses even in combination with CpG-dA/SPG, indicating that peptides require a carrier to antigen presenting cells. In this study, we prepared conjugates comprising OVA257-264 and dA40, and made complexes with SPG. Immunization with OVA257-264-dA/SPG induced peptide-specific immune responses in combination with CpG-dA regardless of complexation with SPG both in vitro and in vivo. When splenocytes from immunized mice were incubated with E.G7-OVA tumor model cells presenting OVA peptides, the number of cells drastically decreased after 24h. Furthermore, mice pre-immunized with OVA257-264-dA/SPG and CpG-ODNs exhibited a long delay in tumor growth after tumor inoculation. Therefore, these peptide-dA/SPG and CpG-dA/SPG complexes could be used as a potent vaccine for the treatment of cancers and infectious diseases. PMID:26562685

  4. Endothelial nitric oxide synthase regulates N-Ras activation on the Golgi complex of antigen-stimulated T cells

    PubMed Central

    Ibiza, Sales; Pérez-Rodríguez, Andrea; Ortega, Ángel; Martínez-Ruiz, Antonio; Barreiro, Olga; García-Domínguez, Carlota A.; Víctor, Víctor M.; Esplugues, Juan V.; Rojas, José M.; Sánchez-Madrid, Francisco; Serrador, Juan M.

    2008-01-01

    Ras/ERK signaling plays an important role in T cell activation and development. We recently reported that endothelial nitric oxide synthase (eNOS)-derived NO regulates T cell receptor (TCR)-dependent ERK activation by a cGMP-independent mechanism. Here, we explore the mechanisms through which eNOS exerts this regulation. We have found that eNOS-derived NO positively regulates Ras/ERK activation in T cells stimulated with antigen on antigen-presenting cells (APCs). Intracellular activation of N-, H-, and K-Ras was monitored with fluorescent probes in T cells stably transfected with eNOS-GFP or its G2A point mutant, which is defective in activity and cellular localization. Using this system, we demonstrate that eNOS selectively activates N-Ras but not K-Ras on the Golgi complex of T cells engaged with APC, even though Ras isoforms are activated in response to NO from donors. We further show that activation of N-Ras involves eNOS-dependent S-nitrosylation on Cys118, suggesting that upon TCR engagement, eNOS-derived NO directly activates N-Ras on the Golgi. Moreover, wild-type but not C118S N-Ras increased TCR-dependent apoptosis, suggesting that S-nitrosylation of Cys118 contributes to activation-induced T cell death. Our data define a signaling mechanism for the regulation of the Ras/ERK pathway based on the eNOS-dependent differential activation of N-Ras and K-Ras at specific cell compartments. PMID:18641128

  5. Delta-like 1/Fetal Antigen-1 (Dlk1/FA1) Is a Novel Regulator of Chondrogenic Cell Differentiation via Inhibition of the Akt Kinase-dependent Pathway*

    PubMed Central

    Chen, Li; Qanie, Diyako; Jafari, Abbas; Taipaleenmaki, Hanna; Jensen, Charlotte H.; Säämänen, Anna-Marja; Sanz, Maria Luisa Nueda; Laborda, Jorge; Abdallah, Basem M.; Kassem, Moustapha

    2011-01-01

    Delta-like 1 (Dlk1, also known as fetal antigen-1, FA1) is a member of Notch/Delta family that inhibits adipocyte and osteoblast differentiation; however, its role in chondrogenesis is still not clear. Thus, we overexpressed Dlk1/FA1 in mouse embryonic ATDC5 cells and tested its effects on chondrogenic differentiation. Dlk1/FA1 inhibited insulin-induced chondrogenic differentiation as evidenced by reduction of cartilage nodule formation and gene expression of aggrecan, collagen Type II and X. Similar effects were obtained either by using Dlk1/FA1-conditioned medium or by addition of a purified, secreted, form of Dlk1 (FA1) directly to the induction medium. The inhibitory effects of Dlk1/FA1 were dose-dependent and occurred irrespective of the chondrogenic differentiation stage: proliferation, differentiation, maturation, or hypertrophic conversion. Overexpression or addition of the Dlk1/FA1 protein to the medium strongly inhibited the activation of Akt, but not the ERK1/2, or p38 MAPK pathways, and the inhibition of Akt by Dlk1/FA1 was mediated through PI3K activation. Interestingly, inhibition of fibronectin expression by siRNA rescued the Dlk1/FA1-mediated inhibition of Akt, suggesting interaction of Dlk1/FA1 and fibronectin in chondrogenic cells. Our results identify Dlk1/FA1 as a novel regulator of chondrogenesis and suggest Dlk1/FA1 acts as an inhibitor of the PI3K/Akt pathways that leads to its inhibitory effects on chondrogenesis. PMID:21724852

  6. Association of the bovine leukocyte antigen major histocompatibility complex class II DRB3*4401 allele with host resistance to the Lone Star Tick, Amblyomma americanum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The MHC of cattle, known as the bovine leukocyte antigen (BoLA) complex, plays an integral role in disease and parasite susceptibility, and immune responsiveness of the host. While susceptibility to tick infestation in cattle is believed to be heritable, genes that may be responsible for the manife...

  7. The Impact of Mitochondrial Complex Inhibition on mESC Differentiation

    EPA Science Inventory

    The Impact of Mitochondrial Complex Inhibition on mESC Differentiation JE Royland, SH Warren, S Jeffay, MR Hoopes, HP Nichols, ES Hunter U.S. Environmental Protection Agency, Integrated Systems Toxicology Division, Research Triangle Park, NC The importance of mitochondrial funct...

  8. Eliminating Inhibition of Return by Changing Salient Nonspatial Attributes in a Complex Environment

    ERIC Educational Resources Information Center

    Hu, Frank K.; Samuel, Arthur G.; Chan, Agnes S.

    2011-01-01

    Inhibition of return (IOR) occurs when a target is preceded by an irrelevant stimulus (cue) at the same location: Target detection is slowed, relative to uncued locations. In the present study, we used relatively complex displays to examine the effect of repetition of nonspatial attributes. For both color and shape, attribute repetition produced a…

  9. Mechanisms of cell death pathway activation following drug-induced inhibition of mitochondrial complex I

    PubMed Central

    Imaizumi, Naoki; Kwang Lee, Kang; Zhang, Carmen; Boelsterli, Urs A.

    2015-01-01

    Respiratory complex I inhibition by drugs and other chemicals has been implicated as a frequent mode of mitochondria-mediated cell injury. However, the exact mechanisms leading to the activation of cell death pathways are incompletely understood. This study was designed to explore the relative contributions to cell injury of three distinct consequences of complex I inhibition, i.e., impairment of ATP biosynthesis, increased formation of superoxide and, hence, peroxynitrite, and inhibition of the mitochondrial protein deacetylase, Sirt3, due to imbalance of the NADH/NAD+ ratio. We used the antiviral drug efavirenz (EFV) to model drug-induced complex I inhibition. Exposure of cultured mouse hepatocytes to EFV resulted in a rapid onset of cell injury, featuring a no-effect level at 30 µM EFV and submaximal effects at 50 µM EFV. EFV caused a concentration-dependent decrease in cellular ATP levels. Furthermore, EFV resulted in increased formation of peroxynitrite and oxidation of mitochondrial protein thiols, including cyclophilin D (CypD). This was prevented by the superoxide scavenger, Fe-TCP, or the peroxynitrite decomposition catalyst, Fe-TMPyP. Both ferroporphyrins completely protected from EFV-induced cell injury, suggesting that peroxynitrite contributed to the cell injury. Finally, EFV increased the NADH/NAD+ ratio, inhibited Sirt3 activity, and led to hyperacetylated lysine residues, including those in CypD. However, hepatocytes isolated from Sirt3-null mice were protected against 40 µM EFV as compared to their wild-type controls. In conclusion, these data are compatible with the concept that chemical inhibition of complex I activates multiple pathways leading to cell injury; among these, peroxynitrite formation may be the most critical. PMID:25625582

  10. Crystal structure of tarocystatin-papain complex: implications for the inhibition property of group-2 phytocystatins.

    PubMed

    Chu, Ming-Hung; Liu, Kai-Lun; Wu, Hsin-Yi; Yeh, Kai-Wun; Cheng, Yi-Sheng

    2011-08-01

    Tarocystatin (CeCPI) from taro (Colocasia esculenta cv. Kaohsiung no. 1), a group-2 phytocystatin, shares a conserved N-terminal cystatin domain (NtD) with other phytocystatins but contains a C-terminal cystatin-like extension (CtE). The structure of the tarocystatin-papain complex and the domain interaction between NtD and CtE in tarocystatin have not been determined. We resolved the crystal structure of the phytocystatin-papain complex at resolution 2.03 Å. Surprisingly, the structure of the NtD-papain complex in a stoichiometry of 1:1 could be built, with no CtE observed. Only two remnant residues of CtE could be built in the structure of the CtE-papain complex. Therefore, CtE is easily digested by papain. To further characterize the interaction between NtD and CtE, three segments of tarocystatin, including the full-length (FL), NtD and CtE, were used to analyze the domain-domain interaction and the inhibition ability. The results from glutaraldehyde cross-linking and yeast two-hybrid assay indicated the existence of an intrinsic flexibility in the region linking NtD and CtE for most tarocystatin molecules. In the inhibition activity assay, the glutathione-S-transferase (GST)-fused FL showed the highest inhibition ability without residual peptidase activity, and GST-NtD and FL showed almost the same inhibition ability, which was higher than with NtD alone. On the basis of the structures, the linker flexibility and inhibition activity of tarocystatins, we propose that the overhangs from the cystatin domain may enhance the inhibition ability of the cystatin domain against papain.

  11. Crystal structure of tarocystatin-papain complex: implications for the inhibition property of group-2 phytocystatins.

    PubMed

    Chu, Ming-Hung; Liu, Kai-Lun; Wu, Hsin-Yi; Yeh, Kai-Wun; Cheng, Yi-Sheng

    2011-08-01

    Tarocystatin (CeCPI) from taro (Colocasia esculenta cv. Kaohsiung no. 1), a group-2 phytocystatin, shares a conserved N-terminal cystatin domain (NtD) with other phytocystatins but contains a C-terminal cystatin-like extension (CtE). The structure of the tarocystatin-papain complex and the domain interaction between NtD and CtE in tarocystatin have not been determined. We resolved the crystal structure of the phytocystatin-papain complex at resolution 2.03 Å. Surprisingly, the structure of the NtD-papain complex in a stoichiometry of 1:1 could be built, with no CtE observed. Only two remnant residues of CtE could be built in the structure of the CtE-papain complex. Therefore, CtE is easily digested by papain. To further characterize the interaction between NtD and CtE, three segments of tarocystatin, including the full-length (FL), NtD and CtE, were used to analyze the domain-domain interaction and the inhibition ability. The results from glutaraldehyde cross-linking and yeast two-hybrid assay indicated the existence of an intrinsic flexibility in the region linking NtD and CtE for most tarocystatin molecules. In the inhibition activity assay, the glutathione-S-transferase (GST)-fused FL showed the highest inhibition ability without residual peptidase activity, and GST-NtD and FL showed almost the same inhibition ability, which was higher than with NtD alone. On the basis of the structures, the linker flexibility and inhibition activity of tarocystatins, we propose that the overhangs from the cystatin domain may enhance the inhibition ability of the cystatin domain against papain. PMID:21416241

  12. Structural mimicry of O-antigen by a peptide revealed in a complex with an antibody raised against Shigella flexneri serotype 2a.

    PubMed

    Theillet, François-Xavier; Saul, Frederick A; Vulliez-Le Normand, Brigitte; Hoos, Sylviane; Felici, Franco; Weintraub, Andrej; Mulard, Laurence A; Phalipon, Armelle; Delepierre, Muriel; Bentley, Graham A

    2009-05-15

    The use of carbohydrate-mimicking peptides to induce immune responses against surface polysaccharides of pathogenic bacteria offers a novel approach to vaccine development. Factors governing antigenic and immunogenic mimicry, however, are complex and poorly understood. We have addressed this question using the anti-lipopolysaccharide monoclonal antibody F22-4, which was raised against Shigella flexneri serotype 2a and shown to protect against homologous infection in a mouse model. In a previous crystallographic study, we described F22-4 in complex with two synthetic fragments of the O-antigen, the serotype-specific saccharide moiety of lipopolysaccharide. Here, we present a crystallographic and NMR study of the interaction of F22-4 with a dodecapeptide selected by phage display using the monoclonal antibody. Like the synthetic decasaccharide, the peptide binds to F22-4 with micromolar affinity. Although the peptide and decasaccharide use very similar regions of the antigen-binding site, indicating good antigenic mimicry, immunogenic mimicry by the peptide was not observed. The F22-4-antigen interaction is significantly more hydrophobic with the peptide than with oligosaccharides; nonetheless, all hydrogen bonds formed between the peptide and F22-4 have equivalents in the oligosaccharide complex. Two bridging water molecules are also in common, adding to partial structural mimicry. Whereas the bound peptide is entirely helical, its structure in solution, as shown by NMR, is helical in the central region only. Moreover, docking the NMR structure into the antigen-binding site shows that steric hindrance would occur, revealing poor complementarity between the major solution conformation and the antibody that could contribute to the absence of immunogenic mimicry.

  13. A Small Molecule Inhibitor of Monoubiquitinated Proliferating Cell Nuclear Antigen (PCNA) Inhibits Repair of Interstrand DNA Cross-link, Enhances DNA Double Strand Break, and Sensitizes Cancer Cells to Cisplatin*

    PubMed Central

    Inoue, Akira; Kikuchi, Sotaro; Hishiki, Asami; Shao, Youming; Heath, Richard; Evison, Benjamin J.; Actis, Marcelo; Canman, Christine E.; Hashimoto, Hiroshi; Fujii, Naoaki

    2014-01-01

    Small molecule inhibitors of proliferating cell nuclear antigen (PCNA)/PCNA interacting protein box (PIP-Box) interactions, including T2 amino alcohol (T2AA), inhibit translesion DNA synthesis. The crystal structure of PCNA in complex with T2AA revealed that T2AA bound to the surface adjacent to the subunit interface of the homotrimer of PCNA in addition to the PIP-box binding cavity. Because this site is close to Lys-164, which is monoubiquitinated by RAD18, we postulated that T2AA would affect monoubiquitinated PCNA interactions. Binding of monoubiquitinated PCNA and a purified pol η fragment containing the UBZ and PIP-box was inhibited by T2AA in vitro. T2AA decreased PCNA/pol η and PCNA/REV1 chromatin colocalization but did not inhibit PCNA monoubiquitination, suggesting that T2AA hinders interactions of pol η and REV1 with monoubiquitinated PCNA. Interstrand DNA cross-links (ICLs) are repaired by mechanisms using translesion DNA synthesis that is regulated by monoubiquitinated PCNA. T2AA significantly delayed reactivation of a reporter plasmid containing an ICL. Neutral comet analysis of cells receiving T2AA in addition to cisplatin revealed that T2AA significantly enhanced formation of DNA double strand breaks (DSBs) by cisplatin. T2AA promoted colocalized foci formation of phospho-ATM and 53BP1 and up-regulated phospho-BRCA1 in cisplatin-treated cells, suggesting that T2AA increases DSBs. When cells were treated by cisplatin and T2AA, their clonogenic survival was significantly less than that of those treated by cisplatin only. These findings show that the inhibitors of monoubiquitinated PCNA chemosensitize cells by inhibiting repair of ICLs and DSBs. PMID:24474685

  14. CD8+ TCR repertoire formation is guided primarily by the peptide component of the antigenic complex.

    PubMed

    Koning, Dan; Costa, Ana I; Hoof, Ilka; Miles, John J; Nanlohy, Nening M; Ladell, Kristin; Matthews, Katherine K; Venturi, Vanessa; Schellens, Ingrid M M; Borghans, Jose A M; Kesmir, Can; Price, David A; van Baarle, Debbie

    2013-02-01

    CD8(+) T cells recognize infected or dysregulated cells via the clonotypically expressed αβ TCR, which engages Ag in the form of peptide bound to MHC class I (MHC I) on the target cell surface. Previous studies have indicated that a diverse Ag-specific TCR repertoire can be beneficial to the host, yet the determinants of clonotypic diversity are poorly defined. To better understand the factors that govern TCR repertoire formation, we conducted a comprehensive clonotypic analysis of CD8(+) T cell populations directed against epitopes derived from EBV and CMV. Neither pathogen source nor the restricting MHC I molecule were linked with TCR diversity; indeed, both HLA-A and HLA-B molecules were observed to interact with an overlapping repertoire of expressed TRBV genes. Peptide specificity, however, markedly impacted TCR diversity. In addition, distinct peptides sharing HLA restriction and viral origin mobilized TCR repertoires with distinct patterns of TRBV gene usage. Notably, no relationship was observed between immunodominance and TCR diversity. These findings provide new insights into the forces that shape the Ag-specific TCR repertoire in vivo and highlight a determinative role for the peptide component of the peptide-MHC I complex on the molecular frontline of CD8(+) T cell-mediated immune surveillance.

  15. A Role For Mitochondria In Antigen Processing And Presentation.

    PubMed

    Bonifaz, Lc; Cervantes-Silva, Mp; Ontiveros-Dotor, E; López-Villegas, Eo; Sánchez-García, Fj

    2014-09-23

    Immune synapse formation is critical for T lymphocyte activation, and mitochondria have a role in this process, by localizing close to the immune synapse, regulating intracellular calcium concentration, and providing locally required ATP. The interaction between antigen presenting cells (APCs) and T lymphocytes is a two-way signaling process. However, the role of mitochondria in antigen presenting cells during this process remains unknown. For APCs to be able to activate T lymphocytes, they must first engage in an antigen-uptake, -processing, and -presentation process. Here we show that HEL-loaded B lymphocytes, as a type of APCs, undergo a small but significant mitochondrial depolarization by 1-2 h following antigen exposure thus suggesting an increase in their metabolic demands. Inhibition of ATP synthase (oligomycin) or mitochondrial Ca(2+) uniporter (MCU) (Ruthenium red) had no effect on antigen uptake. Therefore, antigen processing and antigen presentation were further analyzed. Oligomycin treatment reduced the amount of specific MHC-peptide complexes but not total MHC II on the cell membrane of B lymphocytes which correlated with a decrease in antigen presentation. However, oligomycin also reduced antigen presentation by B lymphocytes that endogenously express HEL and by B lymphocytes loaded with the HEL48-62 peptide, although to a lesser extent. ATP synthase inhibition and MCU inhibition had a clear inhibitory effect on antigen processing (DQ-OVA). Taking together these results suggest that ATP synthase and MCU are relevant for antigen processing and presentation. Finally, APCs mitochondria were found to re-organize towards the APC-T immune synapse. This article is protected by copyright. All rights reserved.

  16. Tumor cells in multiple myeloma patients inhibit myeloma-reactive T cells through carcinoembryonic antigen-related cell adhesion molecule-6.

    PubMed

    Witzens-Harig, Mathias; Hose, Dirk; Jünger, Simone; Pfirschke, Christina; Khandelwal, Nisit; Umansky, Ludmila; Seckinger, Anja; Conrad, Heinke; Brackertz, Bettina; Rème, Thierry; Gueckel, Brigitte; Meißner, Tobias; Hundemer, Michael; Ho, Anthony D; Rossi, Jean-Francois; Neben, Kai; Bernhard, Helga; Goldschmidt, Hartmut; Klein, Bernard; Beckhove, Philipp

    2013-05-30

    Although functionally competent cytotoxic, T cells are frequently observed in malignant diseases, they possess little ability to react against tumor cells. This phenomenon is particularly apparent in multiple myeloma. We here demonstrate that cytotoxic T cells reacted against myeloma antigens when presented by autologous dendritic cells, but not by myeloma cells. We further show by gene expression profiling and flow cytometry that, similar to many other malignant tumors, freshly isolated myeloma cells expressed several carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) at varying proportions. Binding and crosslinking of CEACAM-6 by cytotoxic T cells inhibited their activation and resulted in T-cell unresponsiveness. Blocking of CEACAM-6 on the surface of myeloma cells by specific monoclonal antibodies or CEACAM-6 gene knock down by short interfering RNA restored T-cell reactivity against malignant plasma cells. These findings suggest that CEACAM-6 plays an important role in the regulation of CD8+ T-cell responses against multiple myeloma; therefore, therapeutic targeting of CEACAM-6 may be a promising strategy to improve myeloma immunotherapy.

  17. Complex Living Conditions Impair Behavioral Inhibition but Improve Attention in Rats.

    PubMed

    van der Veen, Rixt; Kentrop, Jiska; van der Tas, Liza; Loi, Manila; van IJzendoorn, Marinus H; Bakermans-Kranenburg, Marian J; Joëls, Marian

    2015-01-01

    Rapid adaptation to changes, while maintaining a certain level of behavioral inhibition is an important feature in every day functioning. How environmental context and challenges in life can impact on the development of this quality is still unknown. In the present study, we examined the effect of a complex rearing environment during adolescence on attention and behavioral inhibition in adult male rats. We also tested whether these effects were affected by an adverse early life challenge, maternal deprivation (MD). We found that animals that were raised in large, two floor Marlau(TM) cages, together with 10 conspecifics, showed improved attention, but impaired behavioral inhibition in the 5-choice serial reaction time task. The early life challenge of 24 h MD on postnatal day 3 led to a decline in bodyweight during adolescence, but did not by itself influence responses in the 5-choice task in adulthood, nor did it moderate the effects of complex housing. Our data suggest that a complex rearing environment leads to a faster adaptation to changes in the environment, but at the cost of lower behavioral inhibition. PMID:26733839

  18. Complex Living Conditions Impair Behavioral Inhibition but Improve Attention in Rats

    PubMed Central

    van der Veen, Rixt; Kentrop, Jiska; van der Tas, Liza; Loi, Manila; van IJzendoorn, Marinus H.; Bakermans-Kranenburg, Marian J.; Joëls, Marian

    2015-01-01

    Rapid adaptation to changes, while maintaining a certain level of behavioral inhibition is an important feature in every day functioning. How environmental context and challenges in life can impact on the development of this quality is still unknown. In the present study, we examined the effect of a complex rearing environment during adolescence on attention and behavioral inhibition in adult male rats. We also tested whether these effects were affected by an adverse early life challenge, maternal deprivation (MD). We found that animals that were raised in large, two floor MarlauTM cages, together with 10 conspecifics, showed improved attention, but impaired behavioral inhibition in the 5-choice serial reaction time task. The early life challenge of 24 h MD on postnatal day 3 led to a decline in bodyweight during adolescence, but did not by itself influence responses in the 5-choice task in adulthood, nor did it moderate the effects of complex housing. Our data suggest that a complex rearing environment leads to a faster adaptation to changes in the environment, but at the cost of lower behavioral inhibition. PMID:26733839

  19. The TF-antigen binding lectin from Sclerotium rolfsii inhibits growth of human colon cancer cells by inducing apoptosis in vitro and suppresses tumor growth in vivo.

    PubMed

    Inamdar, Shashikala R; Savanur, Mohammed Azharuddin; Eligar, Sachin M; Chachadi, Vishwanath B; Nagre, Nagaraja N; Chen, Chen; Barclays, Monica; Ingle, Aravind; Mahajan, Praveen; Borges, Anita; Shastry, Padma; Kalraiya, Rajiv D; Swamy, Bale M; Rhodes, Jonathan M; Yu, Lu-Gang

    2012-09-01

    Glycan array analysis of Sclerotium rolfsii lectin (SRL) revealed its exquisite binding specificity to the oncofetal Thomsen-Friedenreich (Galβ1-3GalNAcα-O-Ser/Thr, T or TF) antigen and its derivatives. This study shows that SRL strongly inhibits the growth of human colon cancer HT29 and DLD-1 cells by binding to cell surface glycans and induction of apoptosis through both the caspase-8 and -9 mediated signaling. SRL showed no or very weak binding to normal human colon tissues but strong binding to cancerous and metastatic tissues. Intratumor injection of SRL at subtoxic concentrations in NOD-SCID mice bearing HT29 xenografts resulted in total tumor regression in 9 days and no subsequent tumor recurrence. As the increased expression of TF-associated glycans is commonly seen in human cancers, SRL has the potential to be developed as a therapeutic agent for cancer. PMID:22653662

  20. Complexes of hepatitis B surface antigen and immunoglobulin M in the sera of patients with hepatitis B virus infection.

    PubMed Central

    Palla, M; Rizzi, R; Toti, M; Almi, P; Rizzetto, M; Bonino, F; Purcell, R

    1983-01-01

    Hepatitis B surface antigen (HBsAg) bound to immunoglobulin M (IgM) was detected in sera of HBsAg carriers by a radioimmunoassay based on selective absorption of the immunoglobulin on a solid phase coated with antiserum to human IgM. Isopycnic banding and rate-zonal sedimentation have shown that the reaction is related to particulate forms of the HBsAg complexed with IgM. The binding of IgM possibly occurred because of a selective affinity of these molecules to the surface of HBsAg particles. HBsAg/IgM was found transiently in 24 of 25 (96%) patients with acute self-limited hepatitis B and persistently in 6 of 25 patients whose acute hepatitis B progressed to chronicity. It was also found in 20 of 39 (51%) chronic HBsAg carriers with inactive and asymptomatic infection. The HBsAg/IgM phenomenon is not dependent on replication of hepatitis B virions; its persistence in patients with acute hepatitis B may provide complementary evidence of transition of the infection to chronicity. PMID:6309673

  1. Tumor-Targeted Human T Cells Expressing CD28-Based Chimeric Antigen Receptors Circumvent CTLA-4 Inhibition

    PubMed Central

    Condomines, Maud; Arnason, Jon; Benjamin, Reuben; Gunset, Gertrude; Plotkin, Jason; Sadelain, Michel

    2015-01-01

    Adoptive T cell therapy represents a promising treatment for cancer. Human T cells engineered to express a chimeric antigen receptor (CAR) recognize and kill tumor cells in a MHC-unrestricted manner and persist in vivo when the CAR includes a CD28 costimulatory domain. However, the intensity of the CAR-mediated CD28 activation signal and its regulation by the CTLA-4 checkpoint are unknown. We investigated whether T cells expressing an anti-CD19, CD3 zeta and CD28-based CAR (19-28z) displayed the same proliferation and anti-tumor abilities than T cells expressing a CD3 zeta-based CAR (19z1) costimulated through the CD80/CD28, ligand/receptor pathway. Repeated in vitro antigen-specific stimulations indicated that 19-28z+ T cells secreted higher levels of Th1 cytokines and showed enhanced proliferation compared to those of 19z1+ or 19z1-CD80+ T cells. In an aggressive pre-B cell leukemia model, mice treated with 19-28z+ T cells had 10-fold reduced tumor progression compared to those treated with 19z1+ or 19z1-CD80+ T cells. shRNA-mediated CTLA-4 down-regulation in 19z1-CD80+ T cells significantly increased their in vivo expansion and anti-tumor properties, but had no effect in 19-28z+ T cells. Our results establish that CTLA-4 down-regulation may benefit human adoptive T cell therapy and demonstrate that CAR design can elude negative checkpoints to better sustain T cell function. PMID:26110267

  2. Liposome-Antigen-Nucleic Acid Complexes Protect Mice from Lethal Challenge with Western and Eastern Equine Encephalitis Viruses

    PubMed Central

    Phillips, Aaron T.; Schountz, Tony; Toth, Ann M.; Rico, Amber B.; Jarvis, Donald L.; Powers, Ann M.

    2014-01-01

    Alphaviruses are mosquito-borne viruses that cause significant disease in animals and humans. Western equine encephalitis virus (WEEV) and eastern equine encephalitis virus (EEEV), two New World alphaviruses, can cause fatal encephalitis, and EEEV is a select agent of concern in biodefense. However, we have no antiviral therapies against alphaviral disease, and current vaccine strategies target only a single alphavirus species. In an effort to develop new tools for a broader response to outbreaks, we designed and tested a novel alphavirus vaccine comprised of cationic lipid nucleic acid complexes (CLNCs) and the ectodomain of WEEV E1 protein (E1ecto). Interestingly, we found that the CLNC component, alone, had therapeutic efficacy, as it increased survival of CD-1 mice following lethal WEEV infection. Immunization with the CLNC-WEEV E1ecto mixture (lipid-antigen-nucleic acid complexes [LANACs]) using a prime-boost regimen provided 100% protection in mice challenged with WEEV subcutaneously, intranasally, or via mosquito. Mice immunized with LANACs mounted a strong humoral immune response but did not produce neutralizing antibodies. Passive transfer of serum from LANAC E1ecto-immunized mice to nonimmune CD-1 mice conferred protection against WEEV challenge, indicating that antibody is sufficient for protection. In addition, the LANAC E1ecto immunization protocol significantly increased survival of mice following intranasal or subcutaneous challenge with EEEV. In summary, our LANAC formulation has therapeutic potential and is an effective vaccine strategy that offers protection against two distinct species of alphavirus irrespective of the route of infection. We discuss plausible mechanisms as well the potential utility of our LANAC formulation as a pan-alphavirus vaccine. PMID:24257615

  3. Validation of nanodiamond-extracted CFP-10 antigen as a biomarker in clinical isolates of Mycobacterium tuberculosis complex in broth culture media.

    PubMed

    Soo, Po-Chi; Horng, Yu-Tze; Chen, Ai-Ti; Yang, Shih-Chieh; Chang, Kai-Chih; Lee, Jen-Jyh; Peng, Wen-Ping

    2015-09-01

    With detonation nanodiamonds (DNDs) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS), we previously identified early secreted cell filtrate protein 10 (CFP-10) as a candidate Mycobacterium tuberculosis complex (MTC) biomarker. The performance of the CFP-10 biomarker was initially evaluated in relatively small mycobacterial samples (n = 42 samples) in our previous study. In this study, we conducted DND MALDI-TOF MS experiments to investigate the specificity and sensitivity of the MTC biomarker with 312 MTC and 52 nontuberculous mycobacteria (NTM) clinical samples. The frequency and intensity of the acquired CFP-10 mass-to-charge (m/z) peaks were checked with a program to validate that the singly and doubly charged CFP-10 antigen can be treated as a MTC biomarker. We confirmed that by detecting the singly charged species of CFP-10 antigen, the sensitivity and the specificity of MTC samples could reach 97.4% and 100% and no CFP-10 biomarker could be found in NTM samples. This indicates with CFP-10 biomarker it is easy to distinguish MTC from NTM. Besides, the observed intensity ratio of singly and doubly charged species of CFP-10 antigen was 3.3 ± 2.6 and the CFP-10 antigen could maintain good signal intensity for a week. Our results suggest that, with the DND MALDI-TOF mass spectrometry approach, CFP-10 antigen can be used as an early diagnosis biomarker in clinical practice. PMID:26071665

  4. Biguanides inhibit complex I, II and IV of rat liver mitochondria and modify their functional properties.

    PubMed

    Drahota, Z; Palenickova, E; Endlicher, R; Milerova, M; Brejchova, J; Vosahlikova, M; Svoboda, P; Kazdova, L; Kalous, M; Cervinkova, Z; Cahova, M

    2014-01-01

    In this study, we focused on an analysis of biguanides effects on mitochondrial enzyme activities, mitochondrial membrane potential and membrane permeability transition pore function. We used phenformin, which is more efficient than metformin, and evaluated its effect on rat liver mitochondria and isolated hepatocytes. In contrast to previously published data, we found that phenformin, after a 5 min pre-incubation, dose-dependently inhibits not only mitochondrial complex I but also complex II and IV activity in isolated mitochondria. The enzymes complexes inhibition is paralleled by the decreased respiratory control index and mitochondrial membrane potential. Direct measurements of mitochondrial swelling revealed that phenformin increases the resistance of the permeability transition pore to Ca(2+) ions. Our data might be in agreement with the hypothesis of Schäfer (1976) that binding of biguanides to membrane phospholipids alters membrane properties in a non-specific manner and, subsequently, different enzyme activities are modified via lipid phase. However, our measurements of anisotropy of fluorescence of hydrophobic membrane probe diphenylhexatriene have not shown a measurable effect of membrane fluidity with the 1 mM concentration of phenformin that strongly inhibited complex I activity. Our data therefore suggest that biguanides could be considered as agents with high efficacy but low specifity.

  5. Mitochondrial Complex 1 Inhibition Increases 4-Repeat Isoform Tau by SRSF2 Upregulation

    PubMed Central

    De Andrade, Anderson; Höglinger, Günter

    2014-01-01

    Progressive Supranuclear Palsy (PSP) is a neurodegenerative disorder characterised by intracellular aggregation of the microtubule-associated protein tau. The tau protein exists in 6 predominant isoforms. Depending on alternative splicing of exon 10, three of these isoforms have four microtubule-binding repeat domains (4R), whilst the others only have three (3R). In PSP there is an excess of the 4R tau isoforms, which are thought to contribute significantly to the pathological process. The cause of this 4R increase is so far unknown. Several lines of evidence link mitochondrial complex I inhibition to the pathogenesis of PSP. We demonstrate here for the first time that annonacin and MPP+, two prototypical mitochondrial complex I inhibitors, increase the 4R isoforms of tau in human neurons. We show that the splicing factor SRSF2 is necessary to increase 4R tau with complex I inhibition. We also found SRSF2, as well as another tau splicing factor, TRA2B, to be increased in brains of PSP patients. Thereby, we provide new evidence that mitochondrial complex I inhibition may contribute as an upstream event to the pathogenesis of PSP and suggest that splicing factors may represent an attractive therapeutic target to intervene in the disease process. PMID:25402454

  6. Transformation with Oncogenic Ras and the Simian Virus 40 T Antigens Induces Caspase-Dependent Sensitivity to Fatty Acid Biosynthetic Inhibition

    PubMed Central

    Xu, Shihao; Spencer, Cody M.

    2015-01-01

    ABSTRACT Oncogenesis is frequently accompanied by the activation of specific metabolic pathways. One such pathway is fatty acid biosynthesis, whose induction is observed upon transformation of a wide variety of cell types. Here, we explored how defined oncogenic alleles, specifically the simian virus 40 (SV40) T antigens and oncogenic Ras12V, affect fatty acid metabolism. Our results indicate that SV40/Ras12V-mediated transformation of fibroblasts induces fatty acid biosynthesis in the absence of significant changes in the concentration of fatty acid biosynthetic enzymes. This oncogene-induced activation of fatty acid biosynthesis was found to be mammalian target of rapamycin (mTOR) dependent, as it was attenuated by rapamycin treatment. Furthermore, SV40/Ras12V-mediated transformation induced sensitivity to treatment with fatty acid biosynthetic inhibitors. Pharmaceutical inhibition of acetyl-coenzyme A (CoA) carboxylase (ACC), a key fatty acid biosynthetic enzyme, induced caspase-dependent cell death in oncogene-transduced cells. In contrast, isogenic nontransformed cells were resistant to fatty acid biosynthetic inhibition. This oncogene-induced sensitivity to fatty acid biosynthetic inhibition was independent of the cells' growth rates and could be attenuated by supplementing the medium with unsaturated fatty acids. Both the activation of fatty acid biosynthesis and the sensitivity to fatty acid biosynthetic inhibition could be conveyed to nontransformed breast epithelial cells through transduction with oncogenic Ras12V. Similar to what was observed in the transformed fibroblasts, the Ras12V-induced sensitivity to fatty acid biosynthetic inhibition was independent of the proliferative status and could be attenuated by supplementing the medium with unsaturated fatty acids. Combined, our results indicate that specific oncogenic alleles can directly confer sensitivity to inhibitors of fatty acid biosynthesis. IMPORTANCE Viral oncoproteins and cellular mutations

  7. Complex I and complex III inhibition specifically increase cytosolic hydrogen peroxide levels without inducing oxidative stress in HEK293 cells

    PubMed Central

    Forkink, Marleen; Basit, Farhan; Teixeira, José; Swarts, Herman G.; Koopman, Werner J.H.; Willems, Peter H.G.M.

    2015-01-01

    Inhibitor studies with isolated mitochondria demonstrated that complex I (CI) and III (CIII) of the electron transport chain (ETC) can act as relevant sources of mitochondrial reactive oxygen species (ROS). Here we studied ROS generation and oxidative stress induction during chronic (24 h) inhibition of CI and CIII using rotenone (ROT) and antimycin A (AA), respectively, in intact HEK293 cells. Both inhibitors stimulated oxidation of the ROS sensor hydroethidine (HEt) and increased mitochondrial NAD(P)H levels without major effects on cell viability. Integrated analysis of cells stably expressing cytosolic- or mitochondria-targeted variants of the reporter molecules HyPer (H2O2-sensitive and pH-sensitive) and SypHer (H2O2-insensitive and pH-sensitive), revealed that CI- and CIII inhibition increased cytosolic but not mitochondrial H2O2 levels. Total and mitochondria-specific lipid peroxidation was not increased in the inhibited cells as reported by the C11-BODIPY581/591 and MitoPerOx biosensors. Also expression of the superoxide-detoxifying enzymes CuZnSOD (cytosolic) and MnSOD (mitochondrial) was not affected. Oxyblot analysis revealed that protein carbonylation was not stimulated by CI and CIII inhibition. Our findings suggest that chronic inhibition of CI and CIII: (i) increases the levels of HEt-oxidizing ROS and (ii) specifically elevates cytosolic but not mitochondrial H2O2 levels, (iii) does not induce oxidative stress or substantial cell death. We conclude that the increased ROS levels are below the stress-inducing level and might play a role in redox signaling. PMID:26516986

  8. Janus kinase-2 inhibition induces durable tolerance to alloantigen by human dendritic cell–stimulated T cells yet preserves immunity to recall antigen

    PubMed Central

    Betts, Brian C.; Abdel-Wahab, Omar; Curran, Shane A.; St Angelo, Erin T.; Koppikar, Priya; Heller, Glenn; Levine, Ross L.

    2011-01-01

    Janus kinase-2 (JAK2) conveys receptor-binding signals by several inflammatory cytokines, including IL-6, via phosphorylation of signal transducer and activator of transcription 3 (STAT3). We demonstrate that selective JAK2 inhibition by TG101348 during initial encounters between human T cells and allogeneic monocyte-derived dendritic cells induces durable, profound, and specific T-cell tolerance upon reexposure to the same alloantigens. Subsequent responses by nonalloreactive T cells to stimulation de novo by a pathogenic nominal antigen remain intact. TG101348 also suppresses primed T-cell responses when present only during alloantigen restimulation. TG101348 ablates IL-6/JAK2–mediated phosphorylation of STAT3, but has no off-target effects on IL-2 or IL-15/JAK3/pSTAT5-dependent signaling, which sustain the responses of regulatory T cells (Tregs) and other effector T cells. JAK2 inhibition preserves Treg numbers and thereby enhances the ratio of CD4+ Tregs to CD8+CD25+ effector T cells in favor of Tregs. JAK2 inhibition also reduces the production of IL-6 and TNF-α in allogeneic MLRs, impairing the activation of central and effector memory T cells as well as the expansion of responder Th1 and Th17 cells. While we have reported the limitations of isolated IL-6R-α inhibition on dendritic cell–stimulated alloreactivity, we demonstrate here that JAK2 represents a relevant biologic target for controlling GVHD or allograft rejection without broader immune impairment. PMID:21917753

  9. Statin-Induced Myopathy Is Associated with Mitochondrial Complex III Inhibition.

    PubMed

    Schirris, Tom J J; Renkema, G Herma; Ritschel, Tina; Voermans, Nicol C; Bilos, Albert; van Engelen, Baziel G M; Brandt, Ulrich; Koopman, Werner J H; Beyrath, Julien D; Rodenburg, Richard J; Willems, Peter H G M; Smeitink, Jan A M; Russel, Frans G M

    2015-09-01

    Cholesterol-lowering statins effectively reduce the risk of major cardiovascular events. Myopathy is the most important adverse effect, but its underlying mechanism remains enigmatic. In C2C12 myoblasts, several statin lactones reduced respiratory capacity and appeared to be strong inhibitors of mitochondrial complex III (CIII) activity, up to 84% inhibition. The lactones were in general three times more potent inducers of cytotoxicity than their corresponding acid forms. The Qo binding site of CIII was identified as off-target of the statin lactones. These findings could be confirmed in muscle tissue of patients suffering from statin-induced myopathies, in which CIII enzyme activity was reduced by 18%. Respiratory inhibition in C2C12 myoblasts could be attenuated by convergent electron flow into CIII, restoring respiration up to 89% of control. In conclusion, CIII inhibition was identified as a potential off-target mechanism associated with statin-induced myopathies.

  10. Deep Brain Stimulation: More Complex than the Inhibition of Cells and Excitation of Fibers.

    PubMed

    Florence, Gerson; Sameshima, Koichi; Fonoff, Erich T; Hamani, Clement

    2016-08-01

    High-frequency deep brain stimulation (DBS) is an effective treatment for some movement disorders. Though mechanisms underlying DBS are still unclear, commonly accepted theories include a "functional inhibition" of neuronal cell bodies and the excitation of axonal projections near the electrodes. It is becoming clear, however, that the paradoxical dissociation "local inhibition" and "distant excitation" is far more complex than initially thought. Despite an initial increase in neuronal activity following stimulation, cells are often unable to maintain normal ionic concentrations, particularly those of sodium and potassium. Based on currently available evidence, we proposed an alternative hypothesis. Increased extracellular concentrations of potassium during DBS may change the dynamics of both cells and axons, contributing not only to the intermittent excitation and inhibition of these elements but also to interrupt abnormal pathological activity. In this article, we review mechanisms through which high extracellular potassium may mediate some of the effects of DBS.

  11. The CD4 and CD8 antigens are coupled to a protein-tyrosine kinase (p56lck) that phosphorylates the CD3 complex.

    PubMed Central

    Barber, E K; Dasgupta, J D; Schlossman, S F; Trevillyan, J M; Rudd, C E

    1989-01-01

    Many mammalian receptors have been found to regulate cell growth by virtue of a protein-tyrosine kinase domain in their cytoplasmic tail. We recently described an association of the CD4 antigen with a T-cell-specific protein-tyrosine kinase (p56lck; formerly termed pp58lck; EC 2.7.1.112). This interaction represents a potential mechanism by which T-cell growth may be regulated and offers a model by which other members of the src family (products of c-src, c-yes, c-fgr, etc.) may interact with mammalian growth factor receptors. As in the case of the CD4 antigen, the CD8 antigen appears to serve as a receptor for nonpolymorphic regions of products of the major histocompatibility complex and has been implicated in the regulation of T-cell growth. In this study, we reveal that the human CD8 antigen is also associated with the T-cell-specific protein-tyrosine kinase (p56lck). The associated p56lck kinase was detected by use of both in vitro and in vivo labeling regimes using an antiserum to the C terminus of p56lck. Two-dimensional nonequilibrium pH-gradient gel electrophoresis and sodium dodecyl sulfate/polyacrylamide gel electrophoresis demonstrated the similarity of p56lck to the protein-tyrosine kinase associated with the CD4 antigen. The catalytic activity of p56lck was revealed by the autophosphorylation of the 55- to 60-kDa kinase and the occasional labeling of a 35-kDa protein. Last, we demonstrate directly that members of the CD3 complex, including the gamma, delta, and epsilon chains, as well as a putative zeta subunit, can be phosphorylated at tyrosine residues by the CD4/CD8.p56lck complex. Images PMID:2470098

  12. CD4 ligands inhibit the formation of multifunctional transduction complexes involved in T cell activation.

    PubMed

    Jabado, N; Pallier, A; Le Deist, F; Bernard, F; Fischer, A; Hivroz, C

    1997-01-01

    Ligands binding to the CD4 molecule can inhibit TCR-mediated T cell activation. We have previously reported that transcription factors regulating the expression of the IL-2 gene, NF-AT, NF-kappaB, and AP-1, are targets of this inhibitory effect in an in vitro model using peripheral human CD4+ T cells activated by a CD3 mAb. Two T cell activation pathways involved in the regulation of these transcription factors, calcium flux and the p21ras pathway, were investigated as potential targets. Binding of HIV envelope glycoprotein gp160/gp120 or a CD4 mAb to the CD4+ T cells, prior to TCR/CD3 activation, inhibited the intracellular calcium elevation. This event strongly suggested an inhibition of PLCgamma1 activity. Tyrosine phosphorylation of PLCgamma1, induced by CD3 activation, was not affected, but its association with tyrosine-phosphorylated proteins, including a 62-kDa protein, was disrupted. This PLCgamma1-associated p62 was found to be immunoreactive to p62-Sam68 Abs. The activation-induced phosphorylation of two p21ras effectors, Raf-1 and Erk2, was inhibited by the CD4 ligands, indirectly pointing to inhibition of the p21ras activation pathway. In addition, we demonstrate that TCR activation of normal CD4+ T cells induced the formation of p120GAP and PLCgamma1-containing complexes. These complexes also contain other unidentified proteins. CD4 ligand binding induced a defective formation of these transduction complexes. This may result in inefficient signaling, partially accounting for the inhibitory effects of the CD4 ligands on both p21ras and calcium-activation pathways.

  13. Metformin inhibits mitochondrial complex I of cancer cells to reduce tumorigenesis.

    PubMed

    Wheaton, William W; Weinberg, Samuel E; Hamanaka, Robert B; Soberanes, Saul; Sullivan, Lucas B; Anso, Elena; Glasauer, Andrea; Dufour, Eric; Mutlu, Gokhan M; Budigner, Gr Scott; Chandel, Navdeep S

    2014-05-13

    Recent epidemiological and laboratory-based studies suggest that the anti-diabetic drug metformin prevents cancer progression. How metformin diminishes tumor growth is not fully understood. In this study, we report that in human cancer cells, metformin inhibits mitochondrial complex I (NADH dehydrogenase) activity and cellular respiration. Metformin inhibited cellular proliferation in the presence of glucose, but induced cell death upon glucose deprivation, indicating that cancer cells rely exclusively on glycolysis for survival in the presence of metformin. Metformin also reduced hypoxic activation of hypoxia-inducible factor 1 (HIF-1). All of these effects of metformin were reversed when the metformin-resistant Saccharomyces cerevisiae NADH dehydrogenase NDI1 was overexpressed. In vivo, the administration of metformin to mice inhibited the growth of control human cancer cells but not those expressing NDI1. Thus, we have demonstrated that metformin's inhibitory effects on cancer progression are cancer cell autonomous and depend on its ability to inhibit mitochondrial complex I.DOI: http://dx.doi.org/10.7554/eLife.02242.001.

  14. Inhibiting Smooth Muscle Cell Proliferation via Immobilization of Heparin/Fibronectin Complexes on Titanium Surfaces.

    PubMed

    Li, Gui Cai; Xu, Qi Fei; Yang, Ping

    2015-05-01

    The aim of this study was to investigate the inhibitory effect of heparin/fibronectin (Hep/Fn) complexes on neointimal hyperplasia following endovascular intervention. Hep/Fn complexes were immobilized onto titanium (Ti) surfaces, with subsequent X-ray photoelectron spectroscopy (XPS), Toluidine Blue O (TBO) and immunohistochemistry methods were used to characterize surface properties. Smooth muscle cell (SMC) cultures were used to evaluate the effect of Hep/Fn complexes on SMC proliferation. Results showed that Hep/Fn complexes successfully immobilized onto Ti surfaces and resulted in an inhibition of SMC proliferation. This study suggests that Hep/Fn surface-immobilized biomaterials develop as a new generation of biomaterials to prevent neointimal hyperplasia, particularly for use in cardiovascular implants. PMID:26055566

  15. Boron inhibits the proliferating cell nuclear antigen index, molybdenum containing proteins and ameliorates oxidative stress in hepatocellular carcinoma.

    PubMed

    Zafar, Hina; Ali, Shakir

    2013-01-15

    Hepatocellular carcinoma (HCC) is a common malignancy and the main cause of mortality in patients with chronic liver diseases. This study reports the inhibitory effect of boron on HCC induced in rats by administering thioacetamide (TAA) (0.03%) in drinking water for 400days. Boron (4mg/kg body weight) was administered orally after induction of carcinoma. Treatment was continued for 122days, and cell proliferation, histology and biochemistry of treated and control group of rats were studied. Proliferating cell nuclear antigen (PCNA), and [(3)H]-thymidine incorporation, which increased in rats exposed to carcinogen, significantly decreased after boron treatment. PCNA index decreased from 80 in HCC rats to 32 after boron treatment. In the control group, it was 20. Boron caused a dose-dependent decrease in carcinogen-induced [(3)H]-thymidine uptake by the rat hepatocyte. It could partially reverse the activity of selected biochemical indicators of hepatic damage, oxidative stress, selenium and serum retinol, which are depleted in liver cancer, and improved overall health of animal. The study implicates the elevated levels of mammalian molybdenum Fe-S containing flavin hydroxylases, which increase the free radical production and oxidative stress, consequently causing increased hepatic cell proliferation in HCC, and reports boron to ameliorate these changes in liver cancer.

  16. Inhibition of human amylin fibril formation by insulin-mimetic vanadium complexes.

    PubMed

    He, Lei; Wang, Xuesong; Zhao, Cong; Zhu, Dengsen; Du, Weihong

    2014-05-01

    The toxicity of amyloid-forming proteins can be linked to many degenerative and systemic diseases. Human islet amyloid polypeptide (hIAPP, amylin) has been associated with type II diabetes. Methods for efficient inhibition of amyloid fibril formation are highly clinically important. This study demonstrated the significant inhibitory effects of six vanadium complexes on hIAPP aggregation. Vanadium complexes, such as bis(maltolato)-oxovanadium (BMOV), have been used as insulin-mimetic agents for the treatment of diabetes for many years. Different biophysical methods were applied to investigate the interaction between V complexes and hIAPP. The results indicated that the selected compounds affected the peptide aggregation by different action modes and protected the cells from the cytotoxicity induced by hIAPP. Both the high binding affinity and the ligand spatial effect on inhibiting hIAPP aggregation are significant. Although some of these compounds undergo biotransformation under the conditions of the experiments, and the active species are not identified, it is understood that the effect results from a particular compound and its conversion products. Importantly, our work provided information on the effects of the selected V complexes on hIAPP and demonstrated multiple levels of effects of V complexes against amyloid-related diseases.

  17. Complex I inhibition in the visual pathway induces disorganization of the node of Ranvier.

    PubMed

    Marella, Mathieu; Patki, Gaurav; Matsuno-Yagi, Akemi; Yagi, Takao

    2013-10-01

    Mitochondrial defects can have significant consequences on many aspects of neuronal physiology. In particular, deficiencies in the first enzyme complex of the mitochondrial respiratory chain (complex I) are considered to be involved in a number of human neurodegenerative diseases. The current work highlights a tight correlation between the inhibition of complex I and the state of axonal myelination of the optic nerve. Exposing the visual pathway of rats to rotenone, a complex I inhibitor, resulted in disorganization of the node of Ranvier. The structure and function of the node depend on specific cell adhesion molecules, among others, CASPR (contactin associated protein) and contactin. CASPR and contactin are both on the axonal surfaces and need to be associated to be able to anchor their myelin counterpart. Here we show that inhibition of mitochondrial complex I by rotenone in rats induces reactive oxygen species, disrupts the interaction of CASPR and contactin couple, and thus damages the organization and function of the node of Ranvier. Demyelination of the optic nerve occurs as a consequence which is accompanied by a loss of vision. The physiological impairment could be reversed by introducing an alternative NADH dehydrogenase to the mitochondria of the visual system. The restoration of the nodal structure was specifically correlated with visual recovery in the treated animal. PMID:23816754

  18. Complex I inhibition in the visual pathway induces disorganization of the node of Ranvier

    PubMed Central

    Marella, Mathieu; Patki, Gaurav; Matsuno-Yagi, Akemi; Yagi, Takao

    2013-01-01

    Mitochondrial defects can have significant consequences on many aspects of neuronal physiology. In particular, deficiencies in the first enzyme complex of the mitochondrial respiratory chain (complex I) are considered to be involved in a number of human neurodegenerative diseases. The current work highlights a tight correlation between the inhibition of complex I and the state of axonal myelination of the optic nerve. Exposing the visual pathway of rats to rotenone, a complex I inhibitor, resulted in disorganization of the node of Ranvier. The structure and function of the node depends on specific cell adhesion molecules, among others, CASPR (contactin associated protein) and contactin. CASPR and contactin are both on the axonal surface and need to be associated to be able to anchor their myelin counter part. Here we show that inhibition of mitochondrial complex I by rotenone in rats induces reactive oxygen species, disrupts the interaction of CASPR and contactin couple, and thus damages the organization and function of the node of Ranvier. Demyelination of the optic nerve occurs as a consequence which is accompanied by a loss of vision. The physiological impairment could be reversed by introducing an alternative NADH dehydrogenase to the mitochondria of the visual system. The restoration of the nodal structure was specifically correlated with visual recovery in the treated animal. PMID:23816754

  19. Stat6-Dependent Inhibition of Mincle Expression in Mouse and Human Antigen-Presenting Cells by the Th2 Cytokine IL-4

    PubMed Central

    Hupfer, Thomas; Schick, Judith; Jozefowski, Katrin; Voehringer, David; Ostrop, Jenny; Lang, Roland

    2016-01-01

    The C-type lectin receptors (CLRs) Mincle, Mcl, and Dectin-2 bind mycobacterial and fungal cell wall glycolipids and carbohydrates. Recently, we described that expression of these CLR is downregulated during differentiation of human monocytes to dendritic cells (DC) in the presence of GM-CSF and IL-4. Here, we demonstrate that the Th2 cytokine IL-4 specifically inhibits expression of Mincle, Mcl, and Dectin-2 in human antigen-presenting cells (APC). This inhibitory effect of IL-4 was observed across species, as murine macrophages and DC treated with IL-4 also downregulated these receptors. IL-4 blocked upregulation of Mincle and Mcl mRNA expression and cell surface protein by murine macrophages in response to the Mincle ligand Trehalose-6,6-dibehenate (TDB), whereas the TLR4 ligand LPS overcame inhibition by IL-4. Functionally, downregulation of Mincle expression by IL-4 was accompanied by reduced cytokine production upon stimulation with TDB. These inhibitory effects of IL-4 were dependent on the transcription factor Stat6. Together, our results show that the key Th2 cytokine IL-4 exerts a negative effect on the expression of Mincle and other Dectin-2 cluster CLR in mouse and human macrophages and DC, which may render these sentinel cells less vigilant for sensing mycobacterial and fungal ligands. PMID:27790218

  20. Rapamycin (sirolimus) inhibits proliferating cell nuclear antigen expression and blocks cell cycle in the G1 phase in human keratinocyte stem cells.

    PubMed Central

    Javier, A. F.; Bata-Csorgo, Z.; Ellis, C. N.; Kang, S.; Voorhees, J. J.; Cooper, K. D.

    1997-01-01

    Because the immunosuppressant rapamycin (sirolimus) blocks T cell proliferation in G1 phase, it has been proposed as a potential treatment for psoriasis, a skin disease characterized by T cell activation and keratinocyte stem cell hyperproliferation. To determine another potentially important mechanism through which rapamycin can act as an antipsoriatic agent, we tested its direct effect on keratinocyte stem cell proliferation in vitro as well as in vivo. In vivo cell cycle quiescent (G0 phase) stem cell keratinocytes in primary culture sequentially express de novo cyclin D1 and proliferating cell nuclear antigen (PCNA), prior to S phase entry, and upregulate beta1 integrin. Rapamycin inhibited the growth of keratinocytes that were leaving quiescence as well as those already in cell cycle without affecting cell viability. Although beta1 integrin(bright) expression was not affected, the number of beta1 integrin(bright) cells entering S/G2/M was significantly lowered by rapamycin. Cells treated with rapamycin exhibited decreased PCNA expression while cyclin D1 expression, which precedes PCNA expression in the cell cycle, was not affected. We found similar effects on stem cell keratinocytes in patients with psoriasis treated systemically with rapamycin. Because PCNA is required for cell cycle progression from G1 to S phase, our data indicate that inhibition of PCNA protein synthesis may be an important regulatory element in the ability of rapamycin to exert a G1 block. PMID:9151781

  1. The effect of stable macromolecular complexes of ionic polyphosphazene on HIV Gag antigen and on activation of human dendritic cells and presentation to T-cells.

    PubMed

    Palmer, Christine D; Ninković, Jana; Prokopowicz, Zofia M; Mancuso, Christy J; Marin, Alexander; Andrianov, Alexander K; Dowling, David J; Levy, Ofer

    2014-10-01

    Neonates and infants are susceptible to infection due to distinct immune responses in early life. Therefore, development of vaccine formulation and delivery systems capable of activating human newborn leukocytes is of global health importance. Poly[di(carboxylatophenoxy)phosphazene] (PCPP) belongs to a family of ionic synthetic polyphosphazene polyelectrolyte compounds that can form non-covalent interactions with protein antigens and demonstrate adjuvant activity in animals and in human clinical trials. However, little is known about their ability to activate human immune cells. In this study, we characterized the effects of PCPP alone or in combination with a model antigen (recombinant HIV-Gag (Gag)), on the maturation, activation and antigen presentation by human adult and newborn dendritic cells (DCs) in vitro. PCPP treatment induced DC activation as assessed by upregulation of co-stimulatory molecules and cytokine production. Studies benchmarking PCPP to Alum, the most commonly used vaccine adjuvant, demonstrated that both triggered cell death and release of danger signals in adult and newborn DCs. When complexed with Gag antigen, PCPP maintained its immunostimulatory characteristics while permitting internalization and presentation of Gag by DCs to HIV-Gag-specific CD4(+) T cell clones. The PCPP vaccine formulation outlined here has intrinsic adjuvant activity, can facilitate effective delivery of antigen to DCs, and may be advantageous for induction of beneficial T cell-mediated immunity. Moreover, polyphosphazenes can further reduce cost of vaccine production and distribution through their dose-sparing and antigen-stabilizing properties, thus potentially eliminating the need for cold chain distribution. PMID:25023392

  2. Evolutionary genetics and vector adaptation of recombinant viruses of the western equine encephalitis antigenic complex provides new insights into alphavirus diversity and host switching

    PubMed Central

    Allison, Andrew B.; Stallknecht, David E.; Holmes, Edward C.

    2014-01-01

    Western equine encephalitis virus (WEEV), Highlands J virus (HJV), and Fort Morgan virus (FMV) are the sole representatives of the WEE antigenic complex of the genus Alphavirus, family Togaviridae, that are endemic to North America. All three viruses have their ancestry in a recombination event involving eastern equine encephalitis virus (EEEV) and a Sindbis (SIN)-like virus that gave rise to a chimeric alphavirus that subsequently diversified into the present-day WEEV, HJV, and FMV. Here, we present a comparative analysis of the genetic, ecological, and evolutionary relationships among these recombinant-origin viruses, including the description of a nsP4 polymerase mutation in FMV that allows it to circumvent the host range barrier to Asian tiger mosquito cells, a vector species that is normally refractory to infection. Notably, we also provide evidence that the recombination event that gave rise to these three WEEV antigenic complex viruses may have occurred in North America. PMID:25463613

  3. Evolutionary genetics and vector adaptation of recombinant viruses of the western equine encephalitis antigenic complex provides new insights into alphavirus diversity and host switching.

    PubMed

    Allison, Andrew B; Stallknecht, David E; Holmes, Edward C

    2015-01-01

    Western equine encephalitis virus (WEEV), Highlands J virus (HJV), and Fort Morgan virus (FMV) are the sole representatives of the WEE antigenic complex of the genus Alphavirus, family Togaviridae, that are endemic to North America. All three viruses have their ancestry in a recombination event involving eastern equine encephalitis virus (EEEV) and a Sindbis (SIN)-like virus that gave rise to a chimeric alphavirus that subsequently diversified into the present-day WEEV, HJV, and FMV. Here, we present a comparative analysis of the genetic, ecological, and evolutionary relationships among these recombinant-origin viruses, including the description of a nsP4 polymerase mutation in FMV that allows it to circumvent the host range barrier to Asian tiger mosquito cells, a vector species that is normally refractory to infection. Notably, we also provide evidence that the recombination event that gave rise to these three WEEV antigenic complex viruses may have occurred in North America.

  4. A role for mitochondria in antigen processing and presentation

    PubMed Central

    Bonifaz, Laura C; Cervantes-Silva, Mariana P; Ontiveros-Dotor, Elizabeth; López-Villegas, Edgar O; Sánchez-García, F Javier

    2015-01-01

    Immune synapse formation is critical for T-lymphocyte activation, and mitochondria have a role in this process, by localizing close to the immune synapse, regulating intracellular calcium concentration, and providing locally required ATP. The interaction between antigen-presenting cells (APCs) and T lymphocytes is a two-way signalling process. However, the role of mitochondria in APCs during this process remains unknown. For APCs to be able to activate T lymphocytes, they must first engage in an antigen-uptake, -processing and -presentation process. Here we show that hen egg white lysozyme (HEL) -loaded B lymphocytes, as a type of APC, undergo a small but significant mitochondrial depolarization by 1–2 hr following antigen exposure, suggesting an increase in their metabolic demands. Inhibition of ATP synthase (oligomycin) or mitochondrial Ca2+ uniporter (MCU) (Ruthenium red) had no effect on antigen uptake. Therefore, antigen processing and antigen presentation were further analysed. Oligomycin treatment reduced the amount of specific MHC–peptide complexes but not total MHC II on the cell membrane of B lymphocytes, which correlated with a decrease in antigen presentation. However, oligomycin also reduced antigen presentation by B lymphocytes, which endogenously express HEL and by B lymphocytes loaded with the HEL48–62 peptide, although to a lesser extent. ATP synthase inhibition and MCU inhibition had a clear inhibitory effect on antigen processing (DQ-OVA). Taken together these results suggest that ATP synthase and MCU are relevant for antigen processing and presentation. Finally, APC mitochondria were found to re-organize towards the APC–T immune synapse. PMID:25251370

  5. Tumor-associated antigen/IL-21-transduced dendritic cell vaccines enhance immunity and inhibit immunosuppressive cells in metastatic melanoma.

    PubMed

    Aravindaram, K; Wang, P-H; Yin, S-Y; Yang, N-S

    2014-05-01

    Dendritic cell (DC)-based vaccine approaches are being actively evaluated for developing immunotherapeutic agents against cancers. In this study, we investigated the use of engineered DCs expressing transgenic tumor-associated antigen hgp100 and the regulatory cytokine interleukin-21, namely DC-hgp100/mIL-21, as a therapeutic vaccine against melanoma. Tumor-bearing mice were injected intratumorally with transgenic DCs followed by three booster injections. Transgenic DC-hgp100/mIL-21 showed significant reduction in primary tumor growth and metastasis compared with DC-hgp100 alone and DC-mIL-21 alone. In vivo depletion of specific immune cell types (CD8(+) T, CD4(+) T and Natural killer (NK)-1.1(+) cells) effectively blocked the protective effect of this combinational vaccine. In adoptive transfer experiments, a survival rate of nearly 90% was observed at 60 days post-tumor inoculation for the combinational vaccine group. In contrast, all mice in the DC-hgp100 and DC-mIL-21-only groups died within 43-46 days after tumor challenge. Considerably increased levels of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, granulocyte macrophage colony-stimulating factor (GM-CSF) and cytotoxic T lymphocytes (CTLs) were detected with the combination vaccine group compared with other individual treatment groups. In comparison with the DC-hgp100 or mIL-21 groups, the combinational DC-hgp100/mIL-21 vaccine also drastically suppressed the myeloid-derived suppressor cells (MDSCs) and T-regulatory (Treg) cell populations. Our findings suggest that a combinational DC- and gene-based hgp100 and mIL-21 vaccine therapy strategy warrants further evaluation as a clinically relevant cancer vaccine approach for human melanoma patients.

  6. Enhancement of antigen-presenting ability of B lymphoma cells by partial inhibition of protein synthesis through inducing B7-1 expression.

    PubMed Central

    Aoi, T; Nakano, H; Tanaka, Y; Kakiuchi, T

    1997-01-01

    During the investigation of the role of protein synthesis in antigen-presenting cell (APC) function of A20-HL B lymphoma cells, we found that partial inhibition of protein synthesis enhanced their APC function. The treatment of A20-HL cells with 0.313-2.5 microM emetine, an irreversible inhibitor of protein synthesis, decreased protein synthesis by 60-70%, and enhanced their APC function to stimulate I-Ad/OVA323-339-specific T cells to produce interleukin-2 in response to ovalbumin (OVA). The emetine-treated and paraformaldehyde-fixed A20-HL cells required only 20 nM OVA323-339 peptide to stimulate the T cells, whereas those untreated and fixed required 200 nM peptide. This enhancement of APC function was mostly because of the induction of B7-1 expression on A20-HL cells by the emetine treatment, since B7-1 molecules were detected on the emetine-treated A20-HL cells, but only negligibly, if at all, on the untreated cells, and an anti-B7-1 monoclonal antibody, 1G10, inhibited the enhanced APC function of the emetine-treated A20-HL cells. The emetine-treatment also increased B7-1 mRNA expression in A20-HL cells, suggesting that the induction of B7-1 expression was due to the increase in the accumulation of mRNA and the translation with residual ability to synthesize protein. Thus, partial inhibition of protein synthesis in A20-HL cells increases B7-1 mRNA accumulation and its expression on the cell surface, which results in the enhancement of their APC function. Images Figure 5 PMID:9227319

  7. Inhibiting oral intoxication of botulinum neurotoxin A complex by carbohydrate receptor mimics.

    PubMed

    Lee, Kwangkook; Lam, Kwok-Ho; Kruel, Anna-Magdalena; Mahrhold, Stefan; Perry, Kay; Cheng, Luisa W; Rummel, Andreas; Jin, Rongsheng

    2015-12-01

    Botulinum neurotoxins (BoNTs) cause the disease botulism manifested by flaccid paralysis that could be fatal to humans and animals. Oral ingestion of the toxin with contaminated food is one of the most common routes for botulism. BoNT assembles with several auxiliary proteins to survive in the gastrointestinal tract and is subsequently transported through the intestinal epithelium into the general circulation. Several hemagglutinin proteins form a multi-protein complex (HA complex) that recognizes host glycans on the intestinal epithelial cell surface to facilitate BoNT absorption. Blocking carbohydrate binding to the HA complex could significantly inhibit the oral toxicity of BoNT. Here, we identify lactulose, a galactose-containing non-digestible sugar commonly used to treat constipation, as a prototype inhibitor against oral BoNT/A intoxication. As revealed by a crystal structure, lactulose binds to the HA complex at the same site where the host galactose-containing carbohydrate receptors bind. In vitro assays using intestinal Caco-2 cells demonstrated that lactulose inhibits HA from compromising the integrity of the epithelial cell monolayers and blocks the internalization of HA. Furthermore, co-administration of lactulose significantly protected mice against BoNT/A oral intoxication in vivo. Taken together, these data encourage the development of carbohydrate receptor mimics as a therapeutic intervention to prevent BoNT oral intoxication.

  8. Differential susceptibility of mitochondrial complex II to inhibition by oxaloacetate in brain and heart.

    PubMed

    Stepanova, Anna; Shurubor, Yevgeniya; Valsecchi, Federica; Manfredi, Giovanni; Galkin, Alexander

    2016-09-01

    Mitochondrial Complex II is a key mitochondrial enzyme connecting the tricarboxylic acid (TCA) cycle and the electron transport chain. Studies of complex II are clinically important since new roles for this enzyme have recently emerged in cell signalling, cancer biology, immune response and neurodegeneration. Oxaloacetate (OAA) is an intermediate of the TCA cycle and at the same time is an inhibitor of complex II with high affinity (Kd~10(-8)M). Whether or not OAA inhibition of complex II is a physiologically relevant process is a significant, but still controversial topic. We found that complex II from mouse heart and brain tissue has similar affinity to OAA and that only a fraction of the enzyme in isolated mitochondrial membranes (30.2±6.0% and 56.4±5.6% in the heart and brain, respectively) is in the free, active form. Since OAA could bind to complex II during isolation, we established a novel approach to deplete OAA in the homogenates at the early stages of isolation. In heart, this treatment significantly increased the fraction of free enzyme, indicating that OAA binds to complex II during isolation. In brain the OAA-depleting system did not significantly change the amount of free enzyme, indicating that a large fraction of complex II is already in the OAA-bound inactive form. Furthermore, short-term ischemia resulted in a dramatic decline of OAA in tissues, but it did not change the amount of free complex II. Our data show that in brain OAA is an endogenous effector of complex II, potentially capable of modulating the activity of the enzyme.

  9. Differential susceptibility of mitochondrial complex II to inhibition by oxaloacetate in brain and heart.

    PubMed

    Stepanova, Anna; Shurubor, Yevgeniya; Valsecchi, Federica; Manfredi, Giovanni; Galkin, Alexander

    2016-09-01

    Mitochondrial Complex II is a key mitochondrial enzyme connecting the tricarboxylic acid (TCA) cycle and the electron transport chain. Studies of complex II are clinically important since new roles for this enzyme have recently emerged in cell signalling, cancer biology, immune response and neurodegeneration. Oxaloacetate (OAA) is an intermediate of the TCA cycle and at the same time is an inhibitor of complex II with high affinity (Kd~10(-8)M). Whether or not OAA inhibition of complex II is a physiologically relevant process is a significant, but still controversial topic. We found that complex II from mouse heart and brain tissue has similar affinity to OAA and that only a fraction of the enzyme in isolated mitochondrial membranes (30.2±6.0% and 56.4±5.6% in the heart and brain, respectively) is in the free, active form. Since OAA could bind to complex II during isolation, we established a novel approach to deplete OAA in the homogenates at the early stages of isolation. In heart, this treatment significantly increased the fraction of free enzyme, indicating that OAA binds to complex II during isolation. In brain the OAA-depleting system did not significantly change the amount of free enzyme, indicating that a large fraction of complex II is already in the OAA-bound inactive form. Furthermore, short-term ischemia resulted in a dramatic decline of OAA in tissues, but it did not change the amount of free complex II. Our data show that in brain OAA is an endogenous effector of complex II, potentially capable of modulating the activity of the enzyme. PMID:27287543

  10. cAMP prevents TNF-induced apoptosis through inhibiting DISC complex formation in rat hepatocytes

    SciTech Connect

    Bhattacharjee, Rajesh; Xiang, Wenpei; Wang, Yinna; Zhang, Xiaoying

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer cAMP blocks cell death induced by TNF and actinomycin D in cultured hepatocytes. Black-Right-Pointing-Pointer cAMP blocks NF-{kappa}B activation induced by TNF and actinomycin D. Black-Right-Pointing-Pointer cAMP blocks DISC formation following TNF and actinomycin D exposure. Black-Right-Pointing-Pointer cAMP blocks TNF signaling at a proximal step. -- Abstract: Tumor necrosis factor {alpha} (TNF) is a pleiotropic proinflammatory cytokine that plays a role in immunity and the control of cell proliferation, cell differentiation, and apoptosis. The pleiotropic nature of TNF is due to the formation of different signaling complexes upon the binding of TNF to its receptor, TNF receptor type 1 (TNFR1). TNF induces apoptosis in various mammalian cells when the cells are co-treated with a transcription inhibitor like actinomycin D (ActD). When TNFR1 is activated, it recruits an adaptor protein, TNF receptor-associated protein with death domain (TRADD), through its cytoplasmic death effector domain (DED). TRADD, in turn, recruits other signaling proteins, including TNF receptor-associated protein 2 (TRAF2) and receptor-associated protein kinase (RIPK) 1, to form a complex. Subsequently, this complex combines with FADD and procaspase-8, converts into a death-inducing signaling complex (DISC) to induce apoptosis. Cyclic AMP (cAMP) is a second messenger that regulates various cellular processes such as cell proliferation, gene expression, and apoptosis. cAMP analogues are reported to act as anti-apoptotic agents in various cell types, including hepatocytes. We found that a cAMP analogue, dibutyryl cAMP (db-cAMP), inhibits TNF + ActD-induced apoptosis in rat hepatocytes. The protein kinase A (PKA) inhibitor KT-5720 reverses this inhibitory effect of cAMP on apoptosis. Cytoprotection by cAMP involves down-regulation of various apoptotic signal regulators like TRADD and FADD and inhibition of caspase-8 and caspase-3 cleavage. We also found

  11. Large hepatitis delta antigen in packaging and replication inhibition: role of the carboxyl-terminal 19 amino acids and amino-terminal sequences.

    PubMed

    Lee, C Z; Chen, P J; Chen, D S

    1995-09-01

    Hepatitis delta virus (HDV) encodes two proteins, the small delta antigen (SHDAg) and large delta antigen (LHDAg). The latter is identical to the former except for the presence of additional 19 amino acids at the C terminus. While SHDAg is required for HDV replication, LHDAg inhibits replication and, together with hepatitis B surface antigen (HBsAg), is required for the assembly of HDV. The last 19 C-terminal amino acids of LHDAg are essential for HDV assembly. Most of LHDAg (amino acids 19 to 146 and 163 to 195) had been shown to be dispensable for packaging with HBsAg. To discern whether the last 19 C-terminal amino acids solely constitute the signal for packaging with HBsAg, we constructed two LHDAg deletion mutants and tested their abilities to be packaged with HBsAg in cotransfection experiments. We found that deletion of amino acids 2 to 21 and 142 to 165 did not affect LHDAg packaging. This result suggested that only the last 19 C-terminal amino acids of LHDAg are required for packaging. We further constructed two plasmids which expressed c-H-ras with or without additional 19 C-terminal amino acids identical to those in LHDAg. Only c-H-ras with additional 19 amino acids could be cosecreted with HBsAg in the cotransfection experiment. This result confirmed that the C-terminal 19 amino acids are the packaging signal for HBsAg. We also tested the trans activation activity and trans-dominant inhibitory activity of the deletion mutants of SHDAg and LHDAg, respectively. In contrast to deletion of amino acids 142 to 165, deletion of amino acids 2 to 21 impaired the trans-dominant inhibitory activity of LHDAg. Deletion of amino acids 2 to 21 and 142 to 165 did not affect the trans activation activity of SHDAg. This result suggested that a functional domain which is important for the trans-dominant inhibitory activity of LHDAg exists in the amino terminus of HDAg. PMID:7636976

  12. Evidence for a conformational change in a class II major histocompatibility complex molecule occurring in the same pH range where antigen binding is enhanced

    PubMed Central

    1996-01-01

    Many class II histocompatibility complex molecules bind antigenic peptides optimally at low pH, consistent with their exposure to antigen in acidic endosomal compartments. While it has been suggested that a partially unfolded state serves as an intermediate involved in peptide binding, very little evidence for such a state has been obtained. In this report, we show that the murine class II molecule IE becomes increasingly less stable to sodium dodecyl sulfate-induced dissociation since the pH is decreased in the same range that enhances antigenic peptide binding. Furthermore, at mildly acidic pH levels, IEk binds the fluorescent dye 1-anilino-naphthalene-8-sulfonic acid (ANS), a probe for exposed nonpolar sites in proteins, suggesting that protonation produces a molten globule-like state. The association of IEk with a single high-affinity peptide had only a small effect in these two assays, indicating that the changes that occur are distal to the peptide-binding groove. Circular dichroism analysis shows that a pH shift from neutral to mildly acidic pH causes subtle changes in the environment of aromatic residues but does not grossly disrupt the secondary structure of IEk. We propose a model in which perturbations in interdomain contacts outside the peptide-binding domain of IEk occur at acidic pH, producing a partially unfolded state that facilitates optimal antigen binding. PMID:8551214

  13. Molecular components of the B cell antigen receptor complex of class IgD differ partly from those of IgM.

    PubMed Central

    Wienands, J; Hombach, J; Radbruch, A; Riesterer, C; Reth, M

    1990-01-01

    Two classes of immunoglobulin, IgM and IgD, are present as antigen receptors on the surface of mature B lymphocytes. We show here that IgD molecules are noncovalently associated in the B cell membrane with a heterodimer consisting of two proteins of 35 kd (IgD-alpha) and 39 kd (Ig-beta), respectively. The two novel proteins are not found in the IgD-expressing myeloma J558L delta m, which fails to bring IgD antigen receptor onto the cell surface. In a surface IgD positive variant line of this myeloma, however, membrane-bound IgD molecules are associated with the heterodimer, suggesting that the formation of an antigen receptor complex is required for surface IgD expression. We further demonstrate that the IgD-associated heterodimer differs partly from that of the IgM antigen receptor and that its binding to the heavy chain only requires the presence of the last constant domain and the transmembrane part of the delta m chain. Images Fig. 3. Fig. 5. Fig. 6. Fig. 7. PMID:2303036

  14. Eliminating inhibition of return by changing salient nonspatial attributes in a complex environment.

    PubMed

    Hu, Frank K; Samuel, Arthur G; Chan, Agnes S

    2011-02-01

    Inhibition of return (IOR) occurs when a target is preceded by an irrelevant stimulus (cue) at the same location: Target detection is slowed, relative to uncued locations. In the present study, we used relatively complex displays to examine the effect of repetition of nonspatial attributes. For both color and shape, attribute repetition produced a robust inhibitory effect that followed a time course similar to that for location-based IOR. However, the effect only occurred when the target shared both the feature (i.e., color or shape) and location with the cue; this constraint implicates a primary role for location. The data are consistent with the idea that the system integrates consecutive stimuli into a single object file when attributes repeat, hindering detection of the second stimulus. The results are also consistent with an interpretation of IOR as a form of habituation, with greater habituation occurring with increasing featural overlap of a repeated stimulus. Critically, both of these interpretations bring the IOR effect within more general approaches to attention and perception, rather than requiring a specialized process with a limited function. In this view, there is no process specifically designed to inhibit return, suggesting that IOR may be the wrong framing of inhibitory repetition effects. Instead, we suggest that repetition of stimulus properties can interfere with the ability to focus attention on the aspects of a complex display that are needed to detect the occurrence of the target stimulus; this is a failure of activation, not an inhibition of processing.

  15. Methionine oxidation of amyloid peptides by peroxovanadium complexes: inhibition of fibril formation through a distinct mechanism.

    PubMed

    He, Lei; Wang, Xuesong; Zhu, Dengsen; Zhao, Cong; Du, Weihong

    2015-12-01

    Fibril formation of amyloid peptides is linked to a number of pathological states. The prion protein (PrP) and amyloid-β (Aβ) are two remarkable examples that are correlated with prion disorders and Alzheimer's disease, respectively. Metal complexes, such as those formed by platinum and ruthenium compounds, can act as inhibitors against peptide aggregation primarily through metal coordination. This study revealed the inhibitory effect of two peroxovanadium complexes, (NH4)[VO(O2)2(bipy)]·4H2O (1) and (NH4)[VO(O2)2(phen)]·2H2O (2), on amyloid fibril formation of PrP106-126 and Aβ1-42via site-specific oxidation of methionine residues, besides direct binding of the complexes with the peptides. Complexes 1 and 2 showed higher anti-amyloidogenic activity on PrP106-126 aggregation than on Aβ1-42, though their regulation on the cytotoxicity induced by the two peptides could not be differentiated. The action efficacy may be attributed to the different molecular structures of the vanadium complex and the peptide sequence. Results reflected that methionine oxidation may be a crucial action mode in inhibiting amyloid fibril formation. This study offers a possible application value for peroxovanadium complexes against amyloid proteins. PMID:26444976

  16. Methionine oxidation of amyloid peptides by peroxovanadium complexes: inhibition of fibril formation through a distinct mechanism.

    PubMed

    He, Lei; Wang, Xuesong; Zhu, Dengsen; Zhao, Cong; Du, Weihong

    2015-12-01

    Fibril formation of amyloid peptides is linked to a number of pathological states. The prion protein (PrP) and amyloid-β (Aβ) are two remarkable examples that are correlated with prion disorders and Alzheimer's disease, respectively. Metal complexes, such as those formed by platinum and ruthenium compounds, can act as inhibitors against peptide aggregation primarily through metal coordination. This study revealed the inhibitory effect of two peroxovanadium complexes, (NH4)[VO(O2)2(bipy)]·4H2O (1) and (NH4)[VO(O2)2(phen)]·2H2O (2), on amyloid fibril formation of PrP106-126 and Aβ1-42via site-specific oxidation of methionine residues, besides direct binding of the complexes with the peptides. Complexes 1 and 2 showed higher anti-amyloidogenic activity on PrP106-126 aggregation than on Aβ1-42, though their regulation on the cytotoxicity induced by the two peptides could not be differentiated. The action efficacy may be attributed to the different molecular structures of the vanadium complex and the peptide sequence. Results reflected that methionine oxidation may be a crucial action mode in inhibiting amyloid fibril formation. This study offers a possible application value for peroxovanadium complexes against amyloid proteins.

  17. Transcutaneous antigen delivery system

    PubMed Central

    Lee, Mi-Young; Shin, Meong-Cheol; Yang, Victor C.

    2013-01-01

    Transcutaneous immunization refers to the topical application of antigens onto the epidermis. Transcutaneous immunization targeting the Langerhans cells of the skin has received much attention due to its safe, needle-free, and noninvasive antigen delivery. The skin has important immunological functions with unique roles for antigen-presenting cells such as epidermal Langerhans cells and dermal dendritic cells. In recent years, novel vaccine delivery strategies have continually been developed; however, transcutaneous immunization has not yet been fully exploited due to the penetration barrier represented by the stratum corneum, which inhibits the transport of antigens and adjuvants. Herein we review recent achievements in transcutaneous immunization, focusing on the various strategies for the enhancement of antigen delivery and vaccination efficacy. [BMB Reports 2013; 46(1): 17-24] PMID:23351379

  18. Severe Acute Respiratory Syndrome Coronavirus ORF7a Inhibits Bone Marrow Stromal Antigen 2 Virion Tethering through a Novel Mechanism of Glycosylation Interference

    PubMed Central

    Taylor, Justin K.; Coleman, Christopher M.; Postel, Sandra; Sisk, Jeanne M.; Bernbaum, John G.; Venkataraman, Thiagarajan; Sundberg, Eric J.

    2015-01-01

    ABSTRACT Severe acute respiratory syndrome (SARS) emerged in November 2002 as a case of atypical pneumonia in China, and the causative agent of SARS was identified to be a novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV). Bone marrow stromal antigen 2 (BST-2; also known as CD317 or tetherin) was initially identified to be a pre-B-cell growth promoter, but it also inhibits the release of virions of the retrovirus human immunodeficiency virus type 1 (HIV-1) by tethering budding virions to the host cell membrane. Further work has shown that BST-2 restricts the release of many other viruses, including the human coronavirus 229E (hCoV-229E), and the genomes of many of these viruses encode BST-2 antagonists to overcome BST-2 restriction. Given the previous studies on BST-2, we aimed to determine if BST-2 has the ability to restrict SARS-CoV and if the SARS-CoV genome encodes any proteins that modulate BST-2's antiviral function. Through an in vitro screen, we identified four potential BST-2 modulators encoded by the SARS-CoV genome: the papain-like protease (PLPro), nonstructural protein 1 (nsp1), ORF6, and ORF7a. As the function of ORF7a in SARS-CoV replication was previously unknown, we focused our study on ORF7a. We found that BST-2 does restrict SARS-CoV, but the loss of ORF7a leads to a much greater restriction, confirming the role of ORF7a as an inhibitor of BST-2. We further characterized the mechanism of BST-2 inhibition by ORF7a and found that ORF7a localization changes when BST-2 is overexpressed and ORF7a binds directly to BST-2. Finally, we also show that SARS-CoV ORF7a blocks the restriction activity of BST-2 by blocking the glycosylation of BST-2. IMPORTANCE The severe acute respiratory syndrome coronavirus (SARS-CoV) emerged from zoonotic sources in 2002 and caused over 8,000 infections and 800 deaths in 37 countries around the world. Identifying host factors that regulate SARS-CoV pathogenesis is critical to understanding how

  19. Hydroxychloroquine protects melanocytes from autoantibody-induced injury by reducing the binding of antigen-antibody complexes.

    PubMed

    Li, Dong-Guang; Hu, Wen-Zhi; Ma, Hui-Jun; Liu, Wen; Yang, Qing-Qi; Zhao, Guang

    2016-08-01

    . HCQ also significantly decreased the effects of ADCC and CDC that were mediated by GV IgG. The present study provides evidence that HCQ dissociates autoantibody-antigen complexes on the surface of HMCs and reverses ADCC and CDC activity in vitro. Thus, in addition to its effectiveness as an antimalarial therapeutic agent, HCQ may also be a promising potential treatment for patients with vitiligo. PMID:27277530

  20. Brugia malayi Antigen (BmA) Inhibits HIV-1 Trans-Infection but Neither BmA nor ES-62 Alter HIV-1 Infectivity of DC Induced CD4+ Th-Cells.

    PubMed

    Mouser, Emily E I M; Pollakis, Georgios; Yazdanbakhsh, Maria; Harnett, William; de Jong, Esther C; Paxton, William A

    2016-01-01

    One of the hallmarks of HIV-1 disease is the association of heightened CD4+ T-cell activation with HIV-1 replication. Parasitic helminths including filarial nematodes have evolved numerous and complex mechanisms to skew, dampen and evade human immune responses suggesting that HIV-1 infection may be modulated in co-infected individuals. Here we studied the effects of two filarial nematode products, adult worm antigen from Brugia malayi (BmA) and excretory-secretory product 62 (ES-62) from Acanthocheilonema viteae on HIV-1 infection in vitro. Neither BmA nor ES-62 influenced HIV-1 replication in CD4+ enriched T-cells, with either a CCR5- or CXCR4-using virus. BmA, but not ES-62, had the capacity to bind the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) thereby inhibiting HIV-1 trans-infection of CD4+ enriched T-cells. As for their effect on DCs, neither BmA nor ES-62 could enhance or inhibit DC maturation as determined by CD83, CD86 and HLA-DR expression, or the production of IL-6, IL-10, IL-12 and TNF-α. As expected, due to the unaltered DC phenotype, no differences were found in CD4+ T helper (Th) cell phenotypes induced by DCs treated with either BmA or ES-62. Moreover, the HIV-1 susceptibility of the Th-cell populations induced by BmA or ES-62 exposed DCs was unaffected for both CCR5- and CXCR4-using HIV-1 viruses. In conclusion, although BmA has the potential capacity to interfere with HIV-1 transmission or initial viral dissemination through preventing the virus from interacting with DCs, no differences in the Th-cell polarizing capacity of DCs exposed to BmA or ES-62 were observed. Neither antigenic source demonstrated beneficial or detrimental effects on the HIV-1 susceptibility of CD4+ Th-cells induced by exposed DCs. PMID:26808476

  1. Brugia malayi Antigen (BmA) Inhibits HIV-1 Trans-Infection but Neither BmA nor ES-62 Alter HIV-1 Infectivity of DC Induced CD4+ Th-Cells

    PubMed Central

    Mouser, Emily E. I. M.; Pollakis, Georgios; Yazdanbakhsh, Maria; Harnett, William

    2016-01-01

    One of the hallmarks of HIV-1 disease is the association of heightened CD4+ T-cell activation with HIV-1 replication. Parasitic helminths including filarial nematodes have evolved numerous and complex mechanisms to skew, dampen and evade human immune responses suggesting that HIV-1 infection may be modulated in co-infected individuals. Here we studied the effects of two filarial nematode products, adult worm antigen from Brugia malayi (BmA) and excretory-secretory product 62 (ES-62) from Acanthocheilonema viteae on HIV-1 infection in vitro. Neither BmA nor ES-62 influenced HIV-1 replication in CD4+ enriched T-cells, with either a CCR5- or CXCR4-using virus. BmA, but not ES-62, had the capacity to bind the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) thereby inhibiting HIV-1 trans-infection of CD4+ enriched T-cells. As for their effect on DCs, neither BmA nor ES-62 could enhance or inhibit DC maturation as determined by CD83, CD86 and HLA-DR expression, or the production of IL-6, IL-10, IL-12 and TNF-α. As expected, due to the unaltered DC phenotype, no differences were found in CD4+ T helper (Th) cell phenotypes induced by DCs treated with either BmA or ES-62. Moreover, the HIV-1 susceptibility of the Th-cell populations induced by BmA or ES-62 exposed DCs was unaffected for both CCR5- and CXCR4-using HIV-1 viruses. In conclusion, although BmA has the potential capacity to interfere with HIV-1 transmission or initial viral dissemination through preventing the virus from interacting with DCs, no differences in the Th-cell polarizing capacity of DCs exposed to BmA or ES-62 were observed. Neither antigenic source demonstrated beneficial or detrimental effects on the HIV-1 susceptibility of CD4+ Th-cells induced by exposed DCs. PMID:26808476

  2. Rho-kinase inhibition prevents proteinuria in immune-complex-mediated antipodocyte nephritis.

    PubMed

    Meyer-Schwesinger, Catherine; Dehde, Silke; Sachs, Marlies; Mathey, Sabrina; Arefi, Kazem; Gatzemeier, Stefan; Balabanov, Stefan; Becker, Jan U; Thaiss, Friedrich; Meyer, Tobias N

    2012-10-01

    Podocyte foot process retraction is a hallmark of proteinuric glomerulonephritis. Cytoskeletal rearrangement causes a redistribution of slit membrane proteins from the glomerular filtration barrier towards the cell body. However, the underlying signaling mechanisms are presently unknown. Recently, we have developed a new experimental model of immune-mediated podocyte injury in mice, the antipodocyte nephritis (APN). Podocytes were targeted with a polyclonal antipodocyte antibody causing massive proteinuria around day 10. Rho-kinases play a central role in the organization of the actin cytoskeleton of podocytes. We therefore investigated whether inhibition of Rho-kinases would prevent podocyte disruption. C57/BL6 mice received antipodocyte serum with or without daily treatment with the specific Rho-kinase inhibitor HA-1077 (5 mg/kg). Immunoblot analysis demonstrated activation of Rho-kinase in glomeruli of antipodocyte serum-treated mice, which was prevented by HA-1077. Increased Rho-kinase activity was localized to podocytes in APN mice by immunostainings against the phosphorylated forms of Rho-kinase substrates. Rho-kinase inhibition significantly reduced podocyte loss from the glomerular tuft. Periodic acid staining demonstrated less podocyte hypertrophy in Rho-kinase-inhibited APN mice, despite similar amounts of immune complex deposition. Electron microscopy revealed reduced foot process effacement compared with untreated APN mice. Internalization of the podocyte slit membrane proteins nephrin and synaptopodin was prevented by Rho-kinase inhibition. Functionally, Rho-kinase inhibition significantly reduced proteinuria without influencing blood pressure. In rats with passive Heymann nephritis and human kidney biopsies from patients with membranous nephropathy, Rho-kinase was activated in podocytes. Together, these data suggest that increased Rho-kinase activity in the podocyte may be a mechanism for in vivo podocyte foot process retraction.

  3. Human prostate tumor antigen-specific CD8+ regulatory T cells are inhibited by CTLA-4 or IL-35 blockade.

    PubMed

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

    2012-12-15

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

  4. [Evaluation of penicillin expandase mutants and complex substrate inhibition characteristics at high concentrations of penicillin G].

    PubMed

    Wu, Linjun; Fan, Keqiang; Ji, Junjie; Yang, Keqian

    2015-12-01

    Penicillin expandase, also known as deacetoxycephalosporin C synthase (DAOCS), is an essential enzyme involved in cephalosporin C biosynthesis. To evaluate the catalytic behaviors of penicillin expandase under high penicillin G concentration and to identify mutants suitable for industrial applications, the specific activities of wild-type DAOCS and several mutants with increased activities toward penicillin G were determined by HPLC under high penicillin G concentrations. Their specific activity profiles were compared with theoretical predictions by different catalytic dynamics models. We evaluated the specific activities of wild-type DAOCS and previous reported high-activity mutants H4, H5, H6 and H7 at concentrations ranging from 5.6 to 500 mmol/L penicillin G. The specific activities of wild-type DAOCS and mutant H4 increased as penicillin G concentration increased, but decreased when concentrations of substrate go above 200 mmol/L. Other mutants H5, H6 and H7 showed more complex behaviors under high concentration of penicillin G. Among all tested enzymes, mutant H6 showed the highest activity when concentration of penicillin G is above 100 mmol/L. Our results revealed that the substrate inhibition to wild-type DAOCS' by penicillin G is noncompetitive. Other DAOCS mutants showed more complex trends in their specific activities at high concentration of penicillin G (>100 mmol/L), indicating more complex substrate inhibition mechanism might exist. The substrate inhibition and activity of DAOCS mutants at high penicillin G concentration provide important insight to help select proper mutants for industrial application. PMID:27093832

  5. The Herbal Medicine KIOM-MA128 Inhibits the Antigen/IgE-Mediated Allergic Response in Vitro and in Vivo.

    PubMed

    Park, Kwang Il; Kim, Dong Gun; Yoo, Jae Myung; Ma, Jin Yeul

    2016-01-01

    KIOM-MA128, a novel herbal medicine, has been reported to exert some beneficial effects on various biological events, such as atopic dermatitis, inflammation and cancer. The aim of this study is to investigate how KIOM-MA128 regulates the allergic response. We measured the activity of β-hexosaminidase and the levels of allergic mediators in the conditioned media of antigen/IgE (Ag/IgE)-activated RBL-2H3 mast cells. We examined the levels of proteins associated with both the FcεRI and arachidonate cascades. Finally, we established the passive cutaneous anaphylaxis (PCA) model in mice to confirm the anti-allergic effects of KIOM-MA128 in vivo. KIOM-MA128 dose-dependently inhibited degranulation and the production of the allergic mediators described above, with no significant cytotoxicity. In the arachidonate cascade, KIOM-MA128 significantly reduced both cytosolic phospholipase A₂ (cPLA₂) phosphorylation and cyclooxygenase-2 (COX-2) expression. Moreover, in the FcεRI cascade, KIOM-MA128 not only inhibited activation of LYN, FYN and SYK, known as the rate-limiting proteins of the FcεRI cascade, but also suppressed the phosphorylation of ERK, p38 and JNK, which is related to cytokine expression. Finally, 50 to 100 mg/kg KIOM-MA128 significantly attenuated the Ag/IgE-induced PCA reaction in mice. These findings provide novel information and improve our understanding of the anti-allergic effects of KIOM-MA128 on allergic diseases. PMID:27527133

  6. A mathematical model for dynamic simulation of anaerobic digestion of complex substrates: Focusing on ammonia inhibition

    SciTech Connect

    Angelidaki, I.; Ellegaard, L.; Ahring, B.K. )

    1993-06-20

    A mathematical model for anaerobic degradation of complex organic material, such as manure, has been developed. The model includes an enzymatic hydrolytic step and four bacterial steps and involves 12 chemical compounds. The model focuses on ammonia inhibition and includes a detailed description of pH and temperature characteristics in order to accurately simulate free ammonia concentration. Free ammonia and acetate constitute the primary modulating factors in the model. The model has been applied for the simulation of digestion of cattle manure in continuously stirred tank reactors (CSTRs), and results compare favorably with experimental data.

  7. Vanadium(IV) complexes inhibit adhesion, migration and colony formation of UMR106 osteosarcoma cells.

    PubMed

    Molinuevo, María S; Cortizo, Ana M; Etcheverry, Susana B

    2008-04-01

    Vanadium is a trace element widely distributed in the environment. In vertebrates it is mainly stored in bone tissue. The unique cellular environment in the bone and the variety of interactions that mediate cancer metastasis determine that certain types of cancer, such as breast and prostate cancer, preferentially metastize in the skeleton. Since this effect usually signifies serious morbidity and grave prognosis there is an increasing interest in the development of new treatments for this pathology. The present work shows that vanadium complexes can inhibit some parameters related to cancer metastasis such as cell adhesion, migration and clonogenicity. We have also investigated the role of protein kinase A in these processes.

  8. Inhibition of influenza virus infection with chitosan-sialyloligosaccharides ionic complex.

    PubMed

    Cheng, Shuihong; Zhao, Huiqin; Xu, Yaozu; Yang, Yawei; Lv, Xun; Wu, Peixing; Li, Xuebing

    2014-07-17

    With the recent emergence of drug-resistant influenza viruses, effective means of preventing and treating these contagious pathogens have become imperative. The binding receptors of influenza virus are sialyloligosaccharides (SOS), which are present on the surfaces of host cells, and are therefore attractive targets for antiviral development. We report the preparation and identification of a novel influenza virus entry inhibitor, designated chitosan-SOS complex (CS complex). The CS complex was formed through noncovalent adsorption between cationic chitosan and anionic SOS, the latter derived from bovine colostrum. The preparation was accomplished in gram quantities from chitosan and bovine colostrum oligosaccharides by a one-step dialysis process. The inhibitory activity of the complex against influenza virus infection was determined by cytotoxicity inhibition assay (IC50=42 μM). This simple preparation, combined with efficient anti-infective activity and the rich natural availability of chitosan and SOS, highlights the potential of the CS complex as a safe, practical agent for influenza prevention and control. PMID:24702928

  9. The Small Molecule IMR-1 Inhibits the Notch Transcriptional Activation Complex to Suppress Tumorigenesis.

    PubMed

    Astudillo, Luisana; Da Silva, Thiago G; Wang, Zhiqiang; Han, Xiaoqing; Jin, Ke; VanWye, Jeffrey; Zhu, Xiaoxia; Weaver, Kelly; Oashi, Taiji; Lopes, Pedro E M; Orton, Darren; Neitzel, Leif R; Lee, Ethan; Landgraf, Ralf; Robbins, David J; MacKerell, Alexander D; Capobianco, Anthony J

    2016-06-15

    In many cancers, aberrant Notch activity has been demonstrated to play a role in the initiation and maintenance of the neoplastic phenotype and in cancer stem cells, which may allude to its additional involvement in metastasis and resistance to therapy. Therefore, Notch is an exceedingly attractive therapeutic target in cancer, but the full range of potential targets within the pathway has been underexplored. To date, there are no small-molecule inhibitors that directly target the intracellular Notch pathway or the assembly of the transcriptional activation complex. Here, we describe an in vitro assay that quantitatively measures the assembly of the Notch transcriptional complex on DNA. Integrating this approach with computer-aided drug design, we explored potential ligand-binding sites and screened for compounds that could disrupt the assembly of the Notch transcriptional activation complex. We identified a small-molecule inhibitor, termed Inhibitor of Mastermind Recruitment-1 (IMR-1), that disrupted the recruitment of Mastermind-like 1 to the Notch transcriptional activation complex on chromatin, thereby attenuating Notch target gene transcription. Furthermore, IMR-1 inhibited the growth of Notch-dependent cell lines and significantly abrogated the growth of patient-derived tumor xenografts. Taken together, our findings suggest that a novel class of Notch inhibitors targeting the transcriptional activation complex may represent a new paradigm for Notch-based anticancer therapeutics, warranting further preclinical characterization. Cancer Res; 76(12); 3593-603. ©2016 AACR. PMID:27197169

  10. High-resolution structure of HLA-A*0201 in complex with a tumour-specific antigenic peptide encoded by the MAGE-A4 gene.

    PubMed

    Hillig, R C; Coulie, P G; Stroobant, V; Saenger, W; Ziegler, A; Hülsmeyer, M

    2001-07-27

    The heterotrimeric complex of the human major histocompatibity complex (MHC) molecule HLA-A*0201, beta2-microglobulin and the decameric peptide GVYDGREHTV derived from the melanoma antigen (MAGE-A4 protein has been determined by X-ray crystallography at 1.4 A resolution. MAGE-A4 belongs to a family of genes that are specifically expressed in a variety of tumours. MAGE-A4-derived peptides are presented by MHC molecules at the cell surface to cytotoxic T-lymphocytes. As the HLA-A*0201:MAGE-A4 complex occurs only on tumour cells, it is considered to be an appropriate target for immunotherapy. The structure presented here reveals potential epitopes specific to the complex and indicates which peptide residues could be recognised by T-cell receptors. In addition, as the structure could be refined anisotropically, it was possible to describe the movements of the bound peptide in more detail.

  11. Walnut polyphenol metabolites, urolithins A and B, inhibit the expression of the prostate-specific antigen and the androgen receptor in prostate cancer cells.

    PubMed

    Sánchez-González, Claudia; Ciudad, Carlos J; Noé, Véronique; Izquierdo-Pulido, Maria

    2014-11-01

    Walnuts have been gathering attention for their health-promoting properties. They are rich in polyphenols, mainly ellagitannins (ETs) that after consumption are hydrolyzed to release ellagic acid (EA). EA is further metabolized by microbiota to form urolithins, such as A and B, which are absorbed. ETs, EA and urolithins have shown to slow the proliferation and growth of different types of cancer cells but the mechanisms remain unclear. We investigate the role of urolithins in the regulatory mechanisms in prostate cancer, specifically those related to the androgen receptor (AR), which have been linked to the development of this type of cancer. In our study, urolithins down-regulated the mRNA and protein levels of both prostate specific antigen (PSA) and AR in LNCaP cells. The luciferase assay performed with a construct containing three androgen response elements (AREs) showed that urolithins inhibit AR-mediated PSA expression at the transcriptional level. Electrophoretic mobility shift assays revealed that urolithins decreased AR binding to its consensus response element. Additionally, urolithins induced apoptosis in LNCaP cells, and this effect correlated with a decrease in Bcl-2 protein levels. In summary, urolithins attenuate the function of the AR by repressing its expression, causing a down-regulation of PSA levels and inducing apoptosis. Our results suggest that a diet rich in ET-containing foods, such as walnuts, could contribute to the prevention of prostate cancer.

  12. Application of a recombinant Fab fragment from a phage display library for sensitive detection of a target antigen by an inhibition ELISA system.

    PubMed

    Itoh, K; Suzuki, K; Ishiwata, S; Tezuka, T; Mizugaki, M; Suzuki, T

    1999-02-01

    We have found that the recombinant Fab (rFab) produced by phage display system was detectable for a target antigen more sensitive than the parental monoclonal antibody (MoAb). The Fab phage display library was constructed from hybridoma cells producing APU-6 MoAb specific for a modified nucleoside, pseudouridine that have been studied as a urinary marker for malignancy. Fab-displayed phage clones were screened by a direct ELISA, and the single positive clone was finally obtained. Although the reaction pattern of rFab against pseudouridine and uridine was almost identical to that of MoAb, detection sensitivity of rFab was approximately 30 times higher than that of MoAb. Since the sensitivity of rFab was almost identical to that of Fab fragment prepared by papain digestion of MoAb, the increased sensitivity is considered to be the nature of Fab fragment. The sensitivity of established assay system was sufficient for quantitative determination of serum pseudouridine levels in healthy individuals and cancer patients. This procedure may be applicable for improvement of detection sensitivity of a MoAb-based inhibition ELISA system for drugs or low molecular weight compounds.

  13. Deviating the level of proliferating cell nuclear antigen in Trypanosoma brucei elicits distinct mechanisms for inhibiting proliferation and cell cycle progression.

    PubMed

    Valenciano, Ana L; Ramsey, Aaron C; Mackey, Zachary B

    2015-01-01

    The DNA replication machinery is spatially and temporally coordinated in all cells to reproduce a single exact copy of the genome per division, but its regulation in the protozoan parasite Trypanosoma brucei is not well characterized. We characterized the effects of altering the levels of proliferating cell nuclear antigen, a key component of the DNA replication machinery, in bloodstream form T. brucei. This study demonstrated that tight regulation of TbPCNA levels was critical for normal proliferation and DNA replication in the parasite. Depleting TbPCNA mRNA reduced proliferation, severely diminished DNA replication, arrested the synthesis of new DNA and caused the parasites to accumulated in G2/M. Attenuating the parasite by downregulating TbPCNA caused it to become hypersensitive to hydroxyurea. Overexpressing TbPCNA in T. brucei arrested proliferation, inhibited DNA replication and prevented the parasite from exiting G2/M. These results indicate that distinct mechanisms of cell cycle arrest are associated with upregulating or downregulating TbPCNA. The findings of this study validate deregulating intra-parasite levels of TbPCNA as a potential strategy for therapeutically exploiting this target in bloodstream form T. brucei.

  14. The major histocompatibility complex-restricted antigen receptor on T cells. I. Isolation with a monoclonal antibody

    PubMed Central

    1983-01-01

    An antibody-secreting B cell hybridoma, KJ1-26.1, has been prepared from mice immunized with the T cell hybridoma DO-11.10, which recognizes chicken ovalbumin in association with I-Ad (cOVA/I-Ad). KJ1- 26.1 blocks I-restricted antigen recognition by DO-11.10 and a subclone of this T cell hybridoma, DO-11.10.24, which has the same specificity for cOVA/I-Ad as its parent. KJ1-26.1 does not block I-restricted antigen recognition by any other T cell hybridoma tested, including a number of T cell hybridomas closely related to DO-11.10, with similar, but not identical, specificities for antigen/I. Moreover, KJ1-26.1 binds to DO-11.10 and DO-11.10.24, but not to any other T cell hybridomas tested, including three subclones of DO-11.10 that have lost the ability to recognize cOVA/I-Ad. Thus, in every regard KJ1-26.1 appears to be binding to all or part of the receptors for antigen/I on the T cell hybridoma DO-11.10. KJ1-26.1 appears to bind to approximately 15,000 molecules/cell on the surface of DO-11.10. The antibody precipitates an 80,000 dimer from the cells, which on reduction migrates as 40-44,000 monomers. The receptor(s) for antigen/I on DO-11.10 therefore includes molecules with these properties. PMID:6601175

  15. Murine aortic smooth muscle cells acquire, though fail to present exogenous protein antigens on major histocompatibility complex class II molecules.

    PubMed

    Maddaluno, Marcella; MacRitchie, Neil; Grassia, Gianluca; Ialenti, Armando; Butcher, John P; Garside, Paul; Brewer, James M; Maffia, Pasquale

    2014-01-01

    In the present study aortic murine smooth muscle cell (SMC) antigen presentation capacity was evaluated using the Eα-GFP/Y-Ae system to visualize antigen uptake through a GFP tag and tracking of Eα peptide/MHCII presentation using the Y-Ae Ab. Stimulation with IFN-γ (100 ng/mL) for 72 h caused a significant (P < 0.01) increase in the percentage of MHC class II positive SMCs, compared with unstimulated cells. Treatment with Eα-GFP (100 μg/mL) for 48 h induced a significant (P < 0.05) increase in the percentage of GFP positive SMCs while it did not affect the percentage of Y-Ae positive cells, being indicative of antigen uptake without its presentation in the context of MHC class II. After IFN-γ-stimulation, ovalbumin- (OVA, 1 mg/mL) or OVA323-339 peptide-(0.5 μg/mL) treated SMCs failed to induce OT-II CD4(+) T cell activation/proliferation; this was also accompanied by a lack of expression of key costimulatory molecules (OX40L, CD40, CD70, and CD86) on SMCs. Finally, OVA-treated SMCs failed to induce DO11.10-GFP hybridoma activation, a process independent of costimulation. Our results demonstrate that while murine primary aortic SMCs express MHC class II and can acquire exogenous antigens, they fail to activate T cells through a failure in antigen presentation and a lack of costimulatory molecule expression.

  16. Inhibition of experimental lung metastasis by aerosol delivery of PEI-p53 complexes.

    PubMed

    Gautam, A; Densmore, C L; Waldrep, J C

    2000-10-01

    Mutations in the p53 tumor suppressor gene and the pathways mediated by the p53 protein are common in many human cancers. Replacement of functional p53 by gene therapy is a potential way of combating these cancers and the associated drug resistance and tumor growth. Aerosol delivery of genes is a noninvasive way of targeting genes to the lung for gene therapy. Here we demonstrate, using a murine melanoma lung metastasis model, that aerosol delivery of polyethyleneimine-p53 (PEI-p53) complexes inhibits the growth of lung metastasis. A significantly reduced number of visible foci were observed in C57BL/6 mice injected with B16-F10 melanoma and treated with PEI-p53 complexes by aerosol for 3 weeks at twice a week. Fifty percent of the mice in the PEI-p53-treated group exhibited no visible tumor foci. There was a significant reduction in the lung weights of p53-treated mice (P < 0.01) compared to control groups. The tumor burden was also significantly lower (P < 0.001) in mice treated with PEI-p53 complexes. No extrapulmonary metastasis was observed in the groups treated with PEI-p53 complexes compared to 50% of the mice in control groups, which showed metastasis to lymph nodes in the neck or abdomen. Treatment with PEI-p53 aerosol also led to about a 50% increase in the mean length of survival of the mice injected with B16-F10 cells. These data suggest that delivery of the p53 gene by aerosol using PEI as the gene delivery vector can inhibit the growth of lung metastasis. PMID:11020346

  17. Inhibition of heparin/protamine complex-induced complement activation by Compstatin in baboons.

    PubMed

    Soulika, A M; Khan, M M; Hattori, T; Bowen, F W; Richardson, B A; Hack, C E; Sahu, A; Edmunds, L H; Lambris, J D

    2000-09-01

    Complement activation products are major components of the inflammatory response induced by cardiac surgery and cardiopulmonary bypass which contribute to postoperative organ dysfunction, fluid accumulation, and morbidity. Activation of the complement system occurs during extracorporeal circulation, during reperfusion of ischemic tissue, and after the formation of heparin-protamine complexes. In this study we examine the efficacy of Compstatin, a recently discovered peptide inhibitor of complement, in preventing heparin/protamine-induced complement activation in baboons. The study was performed in baboons because Compstatin binds to baboon C3 and is resistant to proteolytic cleavage in baboon blood (similar to humans); Compstatin inhibits only the activation of primates' complement system. After testing various doses and administration regimens, Compstatin produced complete inhibition at a total dose of 21 mg/kg when given as a combination of bolus injection and infusion. Compstatin completely inhibited in vivo heparin/protamine-induced complement activation without adverse effects on heart rate or systemic arterial, central venous, and pulmonary arterial pressures. This study indicates that Compstatin is a safe and effective complement inhibitor that has the potential to prevent complement activation during and after clinical cardiac surgery. Furthermore, Compstatin can serve as the prototype for designing an orally administrated drug.

  18. Pyrrolidine dithiocarbamate-zinc(II) and -copper(II) complexes induce apoptosis in tumor cells by inhibiting the proteasomal activity☆

    PubMed Central

    Milacic, Vesna; Chen, Di; Giovagnini, Lorena; Diez, Alejandro; Fregona, Dolores; Dou, Q. Ping

    2013-01-01

    Zinc and copper are trace elements essential for proper folding, stabilization and catalytic activity of many metalloenzymes in living organisms. However, disturbed zinc and copper homeostasis is reported in many types of cancer. We have previously demonstrated that copper complexes induced proteasome inhibition and apoptosis in cultured human cancer cells. In the current study we hypothesized that zinc complexes could also inhibit the proteasomal chymotrypsin-like activity responsible for subsequent apoptosis induction. We first showed that zinc(II) chloride was able to inhibit the chymotrypsin-like activity of a purified 20S proteasome with an IC50 value of 13.8 μM, which was less potent than copper(II) chloride (IC50 5.3 μM). We then compared the potencies of a pyrrolidine dithiocarbamate (PyDT)-zinc(II) complex and a PyDT-copper(II) complex to inhibit cellular proteasomal activity, suppress proliferation and induce apoptosis in various human breast and prostate cancer cell lines. Consistently, zinc complex was less potent than copper complex in inhibiting the proteasome and inducing apoptosis. Additionally, zinc and copper complexes appear to use somewhat different mechanisms to kill tumor cells. Zinc complexes were able to activate calpain-, but not caspase-3-dependent pathway, while copper complexes were able to induce activation of both proteases. Furthermore, the potencies of these PyDT-metal complexes depend on the nature of metals and also on the ratio of PyDT to the metal ion within the complex, which probably affects their stability and availability for interacting with and inhibiting the proteasome in tumor cells. PMID:18501397

  19. Pyrrolidine dithiocarbamate-zinc(II) and -copper(II) complexes induce apoptosis in tumor cells by inhibiting the proteasomal activity

    SciTech Connect

    Milacic, Vesna; Chen Di; Giovagnini, Lorena; Diez, Alejandro; Fregona, Dolores; Dou, Q. Ping

    2008-08-15

    Zinc and copper are trace elements essential for proper folding, stabilization and catalytic activity of many metalloenzymes in living organisms. However, disturbed zinc and copper homeostasis is reported in many types of cancer. We have previously demonstrated that copper complexes induced proteasome inhibition and apoptosis in cultured human cancer cells. In the current study we hypothesized that zinc complexes could also inhibit the proteasomal chymotrypsin-like activity responsible for subsequent apoptosis induction. We first showed that zinc(II) chloride was able to inhibit the chymotrypsin-like activity of a purified 20S proteasome with an IC{sub 50} value of 13.8 {mu}M, which was less potent than copper(II) chloride (IC{sub 50} 5.3 {mu}M). We then compared the potencies of a pyrrolidine dithiocarbamate (PyDT)-zinc(II) complex and a PyDT-copper(II) complex to inhibit cellular proteasomal activity, suppress proliferation and induce apoptosis in various human breast and prostate cancer cell lines. Consistently, zinc complex was less potent than copper complex in inhibiting the proteasome and inducing apoptosis. Additionally, zinc and copper complexes appear to use somewhat different mechanisms to kill tumor cells. Zinc complexes were able to activate calpain-, but not caspase-3-dependent pathway, while copper complexes were able to induce activation of both proteases. Furthermore, the potencies of these PyDT-metal complexes depend on the nature of metals and also on the ratio of PyDT to the metal ion within the complex, which probably affects their stability and availability for interacting with and inhibiting the proteasome in tumor cells.

  20. Pyrrolidine dithiocarbamate-zinc(II) and -copper(II) complexes induce apoptosis in tumor cells by inhibiting the proteasomal activity.

    PubMed

    Milacic, Vesna; Chen, Di; Giovagnini, Lorena; Diez, Alejandro; Fregona, Dolores; Dou, Q Ping

    2008-08-15

    Zinc and copper are trace elements essential for proper folding, stabilization and catalytic activity of many metalloenzymes in living organisms. However, disturbed zinc and copper homeostasis is reported in many types of cancer. We have previously demonstrated that copper complexes induced proteasome inhibition and apoptosis in cultured human cancer cells. In the current study we hypothesized that zinc complexes could also inhibit the proteasomal chymotrypsin-like activity responsible for subsequent apoptosis induction. We first showed that zinc(II) chloride was able to inhibit the chymotrypsin-like activity of a purified 20S proteasome with an IC(50) value of 13.8 microM, which was less potent than copper(II) chloride (IC(50) 5.3 microM). We then compared the potencies of a pyrrolidine dithiocarbamate (PyDT)-zinc(II) complex and a PyDT-copper(II) complex to inhibit cellular proteasomal activity, suppress proliferation and induce apoptosis in various human breast and prostate cancer cell lines. Consistently, zinc complex was less potent than copper complex in inhibiting the proteasome and inducing apoptosis. Additionally, zinc and copper complexes appear to use somewhat different mechanisms to kill tumor cells. Zinc complexes were able to activate calpain-, but not caspase-3-dependent pathway, while copper complexes were able to induce activation of both proteases. Furthermore, the potencies of these PyDT-metal complexes depend on the nature of metals and also on the ratio of PyDT to the metal ion within the complex, which probably affects their stability and availability for interacting with and inhibiting the proteasome in tumor cells. PMID:18501397

  1. HIV-1 reverse transcriptase complex with DNA and nevirapine reveals nonnucleoside inhibition mechanism

    PubMed Central

    Das, Kalyan; Martinez, Sergio E.; Bauman, Joseph D.; Arnold, Eddy

    2012-01-01

    Combinations of nucleoside and nonnucleoside inhibitors (NNRTIs) of HIV-1 reverse transcriptase (RT) are widely used in anti-AIDS therapies. Five NNRTIs including nevirapine are clinical drugs; however, the molecular mechanism of inhibition by NNRTIs is not clear. We determined the crystal structures of RT–DNA–nevirapine, RT–DNA, and RT–DNA–AZT-triphosphate complexes at 2.85, 2.70, and 2.80 Å, respectively. The RT–DNA complex in the crystal could bind nevirapine or AZT-triphosphate; however, not both. Binding of nevirapine led to opening of the NNRTI-binding pocket. The pocket formation caused shifting of the 3’-end of DNA primer by ~5.5 Å away from its polymerase active site position. Nucleic acid interactions with fingers and palm subdomains were reduced, the dNTP-binding pocket was distorted, and the thumb opened up. The structures elucidate complementary roles of nucleoside and nonnucleoside inhibitors in inhibiting RT. PMID:22266819

  2. Mutant Cockayne syndrome group B protein inhibits repair of DNA topoisomerase I-DNA covalent complex.

    PubMed

    Horibata, Katsuyoshi; Saijo, Masafumi; Bay, Mui N; Lan, Li; Kuraoka, Isao; Brooks, Philip J; Honma, Masamitsu; Nohmi, Takehiko; Yasui, Akira; Tanaka, Kiyoji

    2011-01-01

    Two UV-sensitive syndrome patients who have mild photosensitivity without detectable somatic abnormalities lack detectable Cockayne syndrome group B (CSB) protein because of a homozygous null mutation in the CSB gene. In contrast, mutant CSB proteins are produced in CS-B patients with the severe somatic abnormalities of Cockayne syndrome and photosensitivity. It is known that the piggyBac transposable element derived 3 is integrated within the CSB intron 5, and that CSB-piggyBac transposable element derived 3 fusion (CPFP) mRNA is produced by alternative splicing. We found that CPFP or truncated CSB protein derived from CPFP mRNA was stably produced in CS-B patients, and that wild-type CSB, CPFP, and truncated CSB protein interacted with DNA topoisomerase I. We also found that CPFP inhibited repair of a camptothecin-induced topoisomerase I-DNA covalent complex. The inhibition was suppressed by the presence of wild-type CSB, consistent with the autosomal recessive inheritance of Cockayne syndrome. These results suggested that reduced repair of a DNA topoisomerase I-DNA covalent complex because of truncated CSB proteins is involved in the pathogenesis of CS-B. PMID:21143350

  3. Molecular Modeling Analysis of the Inhibition of Mitochondrial Cytochrome BC1 Complex Activity by Tocol Derivatives

    NASA Astrophysics Data System (ADS)

    Singh, Awantika; Hauer-Jensen, Martin; Compadre, Cesar M.; Kumar, K. Sree

    2011-06-01

    The biological functions of vitamin E related compounds have been of interest in biomedical research for several decades. Among those compounds, α-, β-, δ-, and γ-tocopherols and their oxidation products, α-, β-, δ-, γ-tocopherylquinone and their analogs α-TQo, γ-TQo, TMC20 and TMC40 were recently shown to inhibit the mitochondrial cytochrome bc1 complex. In this investigation the effects of the structural variation on the inhibition of the mitochondrial cytochrome bc1 complex were analyzed using Comparative Molecular Field Analysis (CoMFA). CoMFA performed using steric and electrostatic molecular fields produced a very good correlation. The best CoMFA models were obtained using the manual alignment of 12 compounds with 5 components (q2 = 0.589, SPRESS = 0.515, r2 = 0.992, s = 0.068 and F value = 156.520). The resulting contour maps produced by the best CoMFA model were helpful in identifying the structural features required for the biological activity of compounds under study. These results would be helpful for predicting the activity of new compounds, and they could be used for guiding the design, synthesis and development of new and more effective agents.

  4. Analysis of the inhibition of PAI-1 by metal theaflavin complexes and their degradation products.

    PubMed

    Jankun, Jerzy; Kondray, Victor; Skrzypczak-Jankun, Ewa

    2013-05-01

    The inhibition of elements of the plasminogen activator system [urokinase (uPA), tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1)] plays an important role in human diseases. PAI-1 is overexpressed in obesity and diabetes, and the inhibition of this protein has been postulated to alleviate the symptoms of both disorders. We found that two theaflavins (TFs) from black tea inhibit PAI-1 and we suggest that the beneficial effects of drinking tea may be associated with the suppression of PAI-1 activity by theaflavins. Epidemiological studies are controversial; some studies show the beneficial effects of drinking black tea on obesity and diabetes, while others do not. TFs, a family of compounds that can comprise up to 40% the dry weight of black tea, are responsible for the characteristic color, and they are known to chelate metals. We hypothesized that the content/variety of metals present in drinking water may be one of the reasons for such controversies in the population studies. TFs are excellent chelating compounds by trapping metals into complexes; thus, the quality of water used for tea preparation may influence changes in the formation of new products according to TF affinity for different metals, as well as their high molecular weight oxidation products. Our modeling and docking studies suggest that TF/metal complexes have similar affinity to PAI-1 as native TFs. However, analyses using liquid chromatography-mass spectroscopy (LC-MS) revealed the presence of TF degradation products in tea brewed using water containing metal salts. These can further form high molecular weight oxidation products. Thus, metals present in tea could diminish the beneficial effects of black tea by reducing TF concentration via metal-induced degradation and precipitation.

  5. Inhibition of Phosphate Uptake in Corn Roots by Aluminum-Fluoride Complexes1

    PubMed Central

    Façanha, Arnoldo Rocha; Okorokova-Façanha, Anna L.

    2002-01-01

    F forms stable complexes with Al at conditions found in the soil. Fluoroaluminate complexes (AlFx) have been widely described as effective analogs of inorganic phosphate (Pi) in Pi-binding sites of several proteins. In this work, we explored the possibility that the phytotoxicity of AlFx reflects their activity as Pi analogs. For this purpose, 32P-labeled phosphate uptake by excised roots and plasma membrane H+-ATPase activity were investigated in an Al-tolerant variety of maize (Zea mays L. var. dwarf hybrid), either treated or not with AlFx. In vitro, AlFx competitively inhibited the rate of root phosphate uptake as well as the H+-ATPase activity. Conversely, pretreatment of seedlings with AlFx in vivo promoted no effect on the H+-ATPase activity, whereas a biphasic effect on Pi uptake by roots was observed. Although the initial rate of phosphate uptake by roots was inhibited by AlFx pretreatment, this situation changed over the following minutes as the rate of uptake increased and a pronounced stimulation in subsequent 32Pi uptake was observed. This kinetic behavior suggests a reversible and competitive inhibition of the phosphate transporter by fluoroaluminates. The stimulation of root 32Pi uptake induced by AlFx pretreatment was tentatively interpreted as a phosphate starvation response. This report places AlF3 and AlF4− among Al-phytotoxic species and suggests a mechanism of action where the accumulation of Pi-mimicking fluoroaluminates in the soil may affect the phosphate absorption by plants. The biochemical, physiological, and environmental significance of these findings is discussed. PMID:12177489

  6. The use of sucrose-acetone-extracted Rift Valley fever virus antigen derived from cell culture in an indirect enzyme-linked immunosorbent assay and haemagglutination-inhibition test.

    PubMed

    Paweska, J T; Barnard, B J; Williams, R

    1995-12-01

    A sucrose-acetone-extracted, Madin-Darby-bovine-kidney (MDBK)-derived Rift Valley fever virus (RVFV) antigen was tested both in an indirect ELISA and a haemagglutination-inhibition test for its ability to detect serum antibodies to RVFV. Optimal conditions for antigen concentration, serum and conjugate dilutions for the ELISA were established by checkerboard titration. The specificity and sensitivity of ELISA were determined by the use of paired pre- and post-vaccination sheep-serum samples. Compared with the virus neutralization test, the overall ELISA specificity and sensitivity were 97.4 and 97.3%, respectively. There was a 100% correlation between the results obtained in haemagglutination-inhibition tests with a RVFV sucrose-acetone-extracted antigen derived from hamster liver, and from MDBK cells. A total of 10 582 field-serum samples (84 cattle, 3,659 sheep, 6,839 goats) collected in 1994-1995 from animals of unknown vaccination status in different regions of South Africa were tested with ELISA for antibodies against RVFV. There were no seropositive cattle, 0.16% seropositive sheep and 0.12% seropositive goats. This study demonstrates the potential diagnostic application of cell-culture-derived, sucrose-acetone-extracted RVFV antigen in an indirect ELISA and HI test.

  7. [Complex immunochemical analysis of the proteinogram and the system of soluble leukocytic antigens in children with chronic and recurrent infections].

    PubMed

    Petrunin, D D; Khakhalin, L N; Porkhovatyĭ, S Ia; Olefirenko, G A

    1985-09-01

    The immunochemical study of the blood sera of children with chronic and relapsing infections has shown an increase in the content of alpha 2-macroglobulin and alpha 1-antitrypsin in the absence of significant changes in the concentration of immunoglobulins and complement components. The immunochemical analysis of the system of soluble leukocytic antigens (SLA) has revealed a decrease in the level of SLA-1 simultaneously with the presence of redundant amounts of SLA-5 and SLA-8.

  8. Chronic administration of dexamethasone results in Fc receptor up-regulation and inhibition of class I antigen expression on macrophages from MRL/lpr autoimmune mice.

    PubMed Central

    Zuckerman, S H; Evans, G F; Bryan, N

    1997-01-01

    The MRL/lpr mouse develops, after approximately 8 weeks of age, a severe autoimmune syndrome with many features resembling human systemic lupus erythematosus, including autoantibodies against DNA and basement membranes resulting in immune complexes, vasculitis, and multiorgan disease. While this murine model of lupus has been used for the identification of therapeutics with potential efficacy in human autoimmune disease, the long-term impact of chronic immunosuppressive therapy on macrophage function in this paradigm is not understood. To this end, MRL/lpr mice were treated prophylactically with dexamethasone at 0.01, 0.1, and 1 mg/kg of body weight for 20 weeks or were allowed to develop autoimmune disease and, at 15 weeks of age, treated therapeutically with 1-mg/kg dexamethasone for 8 additional weeks. Analysis of surface antigens on resident peritoneal macrophages demonstrated a progressive loss in class I expression with a concomitant increase in Fc receptor expression. Neither phagocytosis nor CD11b expression was modulated with chronic steroid treatment. Furthermore, dexamethasone treatment was associated with a reduction in anti-DNA antibodies and total immunoglobulin G and yet an elevation in serum cholesterol due to an increase in high-density lipoproteins. Therefore, the MRL/lpr mouse serves not only as a small-animal model of autoimmune disease but also as one in which the negative and positive sequelae associated with chronic immunosuppression can be further understood. PMID:9302207

  9. Analysis of antigen presentation by metabolically inactive accessory cells and their isolated membranes.

    PubMed Central

    Falo, L D; Sullivan, K; Benacerraf, B; Mescher, M F; Rock, K L

    1985-01-01

    Several amino acid copolymers are potent immunogens under the control of major histocompatibility complex (MHC)-encoded Ir genes. We have further characterized their accessory-cell-dependent, MHC-restricted presentation to T lymphocytes. We initially characterized their processing requirements by investigating the ability of paraformaldehyde-fixed antigen-presenting cells (APC) to present these copolymers. Fixed APC can present poly(Glu56Lys35Phe9) and poly(Glu60Ala30Tyr10) provided that they have been incubated with antigen prior to fixation. The inability of these same fixed preparations to present soluble antigen indicates a fixation-sensitive antigen-processing step. In contrast, the antigens poly(Glu55Lys35Leu10) and poly(Glu55Lys35Tyr10) can be presented by APC fixed before antigen exposure. This differential requirement for antigen processing was exploited to analyze the events of antigen presentation in two related systems. First, the ability of isolated APC membranes to process and present antigen was assessed. APC membranes can present the antigens poly(GluLysLeu) and poly(GluLysTyr) in a specific and MHC-restricted manner. However, the isolated membranes fail to present either poly(GluLysPhe) or poly(GluAlaTyr), suggesting that such preparations can present but not process antigen. Second, the distinct properties of the various copolymers were used with fixed APC to test the effects of antigen processing on the phenomenon of antigen competition. APC that had processed poly(GluLysPhe) or poly(GluAlaTyr) were subsequently fixed and used to present antigen in the presence or absence of various antagonists. Under these conditions, poly(GluLysLeu) and poly(Glu50Tyr50) could effect specific inhibition, clearly indicating that antigen competition occurs distal to and does not require antigen processing. In contrast, native antigen with an absolute processing requirement is not capable of competing with preprocessed antigen on fixed APC. Taken together, these

  10. Inhibition of BK virus replication in human kidney cells by BK virus large tumor antigen-specific shRNA delivered by JC virus-like particles.

    PubMed

    Lin, Mien-Chun; Wang, Meilin; Fang, Chiung-Yao; Chen, Pei-Lain; Shen, Cheng-Huang; Chang, Deching

    2014-03-01

    Polyomavirus-associated nephropathy (PVAN) due to lytic infection by the BK polyomavirus (BKPyV) remains an important cause of allograft dysfunction and graft loss in renal transplant recipients. PVAN is commonly treated by reducing the dosage of immunosuppressive drugs and adding adjuvant antiviral agents, but the outcomes have been less than satisfactory. The BKPyV early protein large tumor antigen (LT) is indispensable for viral genome replication and viral late protein expression. Therefore, suppressing LT expression may be a way to inhibit BKPyV replication without harming the host human kidney cells. Previous studies have shown that JC polyomavirus (JCPyV) virus-like particles (VLPs), which have tropism for the human kidney, can package and transfer exogenous genes into human kidney cells for expression. In this study, we constructed an expression plasmid for a BKPyV LT-specific shRNA (shLT) and used JCPyV VLPs as a delivery vehicle to transduce the shLT plasmid into BKPyV-infected human kidney cells. The expression of BKPyV early (LT) and late (VP1) proteins was examined after transduction by immunofluorescence microscopy and Western blotting. We found that transduction with the shLT plasmid decreased the proportions of BKPyV LT- and VP1-expressing cells by 73% and 82%, respectively, relative to control. The viral genomes were also decreased by 56%. These results point to the promising possibility of developing shLT-transducing JCPyV VLPs as a specific anti-BKPyV approach for PVAN treatment.

  11. Vanadium limits the expression of proliferating cell nuclear antigen and inhibits early DNA damage during diethylnitrosamine-induced hepatocellular preneoplasia in rats.

    PubMed

    Chakraborty, Tridib; Chatterjee, Amrita; Dhachinamoorthi, Duraisami; Srivastawa, Sunil; Panayappan, Lakshmanan; Chatterjee, Malay

    2006-10-01

    Previous studies from our laboratory have shown that vanadium stabilizes xenobiotic metabolizing enzymes and antioxidant status and suppresses DNA-protein crosslinks during chemically-induced hepatocarcinogenesis in rats. In the present study, we have further investigated the in vivo antitumor potential of this micronutrient by determining the effect of 0.5 ppm vanadium in drinking water on biomarkers for the early stages of hepatocarcinogenesis; the biomarkers included gamma-glutamyl transpeptidase (GGT)-positive foci and glycogen-storage foci, in situ expression of proliferating cell nuclear antigen (PCNA), and genotoxic DNA damage assessed by the alkaline Comet assay. Histomorphometry also was assessed during the study. Hepatocarcinogenesis was induced by treating 4-week-old male Sprague-Dawley rats with a single, necrogenic, intraperitoneal (i.p.) injection of 200 mg/kg body weight diethylnitrosamine (DEN). Compared to the carcinogen control, vanadium administration over the 32 weeks of the experiment reduced the relative liver weight by 30%, the incidence of nodules by 69.34%, the total number and multiplicity of nodules by 80.77%, and remodeled the hepatocellular premalignant architecture towards a normal phenotype. Moreover, long-term vanadium treatment reduced the development of GGT foci by 76.2% (P < 0.001), decreased periodic acid-Schiff's reactivity by 59.49% (P < 0.01), and decreased PCNA expression, with the concomitant reduction in PCNA immunolabeling index by 93.36% (P < 0.001). Finally, vanadium inhibited early DNA damage (DNA strand-breaks) in DEN-treated rat hepatocytes as expressed in the Comet assay by a 60.04% reduction in the length:width value of DNA mass (P < 0.01) and a 51.54% reduction in the tail length of the DNA comets (P < 0.001). Our results indicate that continuous supplementation with 0.5 ppm vanadium suppresses hepatocellular neoplastic transformation in rats. PMID:16878318

  12. A Novel Prostate-Specific Membrane-Antigen (PSMA) Targeted Micelle-Encapsulating Wogonin Inhibits Prostate Cancer Cell Proliferation via Inducing Intrinsic Apoptotic Pathway

    PubMed Central

    Zhang, Hailong; Liu, Xiaogang; Wu, Fengbo; Qin, Feifei; Feng, Ping; Xu, Ting; Li, Xiang; Yang, Li

    2016-01-01

    Prostate cancer (PCa) is a malignant tumor for which there are no effective treatment strategies. In this study, we developed a targeted strategy for prostate-specific membrane-antigen (PSMA)-positive PCa in vitro based on 2-(3-((S)-5-amino-1-carboxypentyl)ureido) pentanedioic acid (ACUPA) modified polyethylene glycol (PEG)-Cholesterol micelles containing wogonin (WOG), which was named ACUPA-M-WOG. ACUPA-M-WOG was conventionally prepared using a self-assembling method, which produced stable particle size and ζ potential. Moreover, ACUPA-M-WOG showed good drug encapsulating capacity and drug release profiles. Fluorescence activated cell sorting (FACS) results suggested that ACUPA modified PEG-Cholesterol micelles could effectively enhance the drug uptake on PSMA(+) PCa cells, and the cytotoxicity of ACUPA-M-WOG was stronger than other controls according to in vitro cellular proliferation and apoptosis assays, separately through methyl thiazolyl tetrazolium (MTT) and Annexin V/Propidium Iodide (PI) staining. Finally, the molecular mechanisms of ACUPA-M-WOG’s effects on human PSMA(+) PCa were investigated, and were mainly the intrinsic or extrinsic apoptosis signaling pathways. The Western blot results suggested that ACUPA-M-WOG could enhance the WOG-induced apoptosis, which was mainly via the intrinsic signaling pathway rather than the extrinsic signaling pathway. In conclusion, ACUPA-M-WOG was successfully developed for WOG-selective delivery to PSMA(+) PCa cells and had stronger inhibition than free drugs, which might make it an effective strategy for PSMA(+) PCa. PMID:27196894

  13. A Novel Prostate-Specific Membrane-Antigen (PSMA) Targeted Micelle-Encapsulating Wogonin Inhibits Prostate Cancer Cell Proliferation via Inducing Intrinsic Apoptotic Pathway.

    PubMed

    Zhang, Hailong; Liu, Xiaogang; Wu, Fengbo; Qin, Feifei; Feng, Ping; Xu, Ting; Li, Xiang; Yang, Li

    2016-01-01

    Prostate cancer (PCa) is a malignant tumor for which there are no effective treatment strategies. In this study, we developed a targeted strategy for prostate-specific membrane-antigen (PSMA)-positive PCa in vitro based on 2-(3-((S)-5-amino-1-carboxypentyl)ureido) pentanedioic acid (ACUPA) modified polyethylene glycol (PEG)-Cholesterol micelles containing wogonin (WOG), which was named ACUPA-M-WOG. ACUPA-M-WOG was conventionally prepared using a self-assembling method, which produced stable particle size and ζ potential. Moreover, ACUPA-M-WOG showed good drug encapsulating capacity and drug release profiles. Fluorescence activated cell sorting (FACS) results suggested that ACUPA modified PEG-Cholesterol micelles could effectively enhance the drug uptake on PSMA(+) PCa cells, and the cytotoxicity of ACUPA-M-WOG was stronger than other controls according to in vitro cellular proliferation and apoptosis assays, separately through methyl thiazolyl tetrazolium (MTT) and Annexin V/Propidium Iodide (PI) staining. Finally, the molecular mechanisms of ACUPA-M-WOG's effects on human PSMA(+) PCa were investigated, and were mainly the intrinsic or extrinsic apoptosis signaling pathways. The Western blot results suggested that ACUPA-M-WOG could enhance the WOG-induced apoptosis, which was mainly via the intrinsic signaling pathway rather than the extrinsic signaling pathway. In conclusion, ACUPA-M-WOG was successfully developed for WOG-selective delivery to PSMA(+) PCa cells and had stronger inhibition than free drugs, which might make it an effective strategy for PSMA(+) PCa. PMID:27196894

  14. Proteasome inhibition blocks NF-κB and ERK1/2 pathways, restores antigen expression and sensitizes resistant human melanoma to TCR-engineered CTLs

    PubMed Central

    Jazirehi, Ali R.; Economou, James S.

    2012-01-01

    Adoptive cell transfer (ACT) of ex vivo engineered autologous lymphocytes encoding high-affinity MART-1/HLA-A*0201-specific T-cell receptor (TCR) α/β chains (F5 CTL), densely infiltrate into sites of metastatic disease, mediating dramatic but partial clinical responses in melanoma patients. We hypothesized that MART-1 down-modulation in addition to aberrant apoptotic/survival signaling could confer resistance to death signals delivered by transgenic CTLs. To explore this hypothesis, we established an in vitro model of resistant (R) lines from MART-1+/HLA-A*0201+ F5 CTL-sensitive parental (P) lines under serial F5 CTL-selective pressure. We have recently reported that several melanoma R lines, while retaining MART-1 expression, exhibited constitutive NF-κB activation and over-expression of NF-κB-dependent resistance factors. Another established melanoma cell line M244, otherwise sensitive to F5 CTL, yielded R lines after serial F5 CTL selective pressure which had both reduced MART-1 expression levels, thus, could not be recognized, and were resistant to CTL-delivered apoptotic death signals. The proteasome inhibitor bortezomib blocked NF-κB activity, decreased phopspho-ERK1/2, increased phospho-JNK levels, reduced expression of resistance-factors, restored MART-1 expression to sufficient levels, which in combination allowed M244R lines be sensitized to F5 CTL-killing. These findings suggest that proteasome inhibition in immune resistant tumors can restore proapoptotic signaling and improve tumor antigen expression. PMID:22532603

  15. BET bromodomain inhibition releases the Mediator complex from select cis-regulatory elements

    PubMed Central

    Bhagwat, Anand S.; Roe, Jae-Seok; Mok, Beverly A.; Hohmann, Anja F.; Shi, Junwei; Vakoc, Christopher R.

    2016-01-01

    The bromodomain and extraterminal (BET) protein BRD4 can physically interact with the Mediator complex, but the relevance of this association to the therapeutic effects of BET inhibitors in cancer is unclear. Here, we show that BET inhibition causes a rapid release of Mediator from a subset of cis-regulatory elements in the genome of acute myeloid leukemia (AML) cells. These sites of Mediator eviction were highly correlated with transcriptional suppression of neighboring genes, which are enriched for targets of the transcription factor MYB and for functions related to leukemogenesis. An shRNA screen of Mediator in AML cells identified the MED12, MED13, MED23, and MED24 subunits as performing a similar regulatory function to BRD4 in this context, including a shared role in sustaining a block in myeloid maturation. These findings suggest that the interaction between BRD4 and Mediator has functional importance for gene-specific transcriptional activation and for AML maintenance. PMID:27068464

  16. Tannins: thermostable pigments which complex dietary proteins and inhibit digestive enzymes.

    PubMed

    Carmona, A

    1996-12-01

    The presence of antinutritional factors in legume seeds and other vegetables has been considered as an expression of the chemical warfare of plants against their predators. As a consequence, the nutritional utilization of these foods has only been possible through the use of a variety of treatments (cooking, fermentation, germination) which increase nutrient bioavailability. Nonetheless, some factors are not destroyed by effect of seed processing, among which stand a family of polymeric polyphenols called tannins. These pigments have the ability to complex and precipitate proteins and inhibit digestive enzymes. This paper describes what has been accomplished in regards to the selection of an appropriate solvent to extract bean polyphenols, the assessment of the most commonly used assay procedures, the purification of bean tannins and the evaluation of their interaction with proteins and digestive enzymes, responsible for their antinutritional effect.

  17. Human CRB2 Inhibits γ-Secretase Cleavage of Amyloid Precursor Protein by Binding to the Presenilin Complex*

    PubMed Central

    Mitsuishi, Yachiyo; Hasegawa, Hiroshi; Matsuo, Akinori; Araki, Wataru; Suzuki, Toshiharu; Tagami, Shinji; Okochi, Masayasu; Takeda, Masatoshi; Roepman, Ronald; Nishimura, Masaki

    2010-01-01

    Drosophila Crumbs has been reported to attenuate Notch signaling by inhibition of γ-secretase cleavage at the wing margins. γ-Secretase is an intramembrane protease that is responsible for the generation of amyloid-β (Aβ) peptides from the β-amyloid precursor protein (APP). Here, we re-examined γ-secretase inhibition by human CRB2, which is the most abundant Crumbs ortholog in the brain. Transfected CRB2 inhibited proteolytic production of Aβ and APP intracellular domains from APP C-terminal fragments in HEK293 and SH-SY5Y cells. Conversely, knockdown of endogenous CRB2 increased γ-secretase cleavage products in SH-SY5Y cells. CRB2 inhibition of γ-cleavage was also detected in cell-free assays. CRB2 interacted with the γ-secretase complex, but was not a competitive substrate for γ-cleavage. The transmembrane domain of CRB2 was indispensable for inhibition of Aβ generation and mediated CRB2 binding with the γ-secretase complex. In addition, the cytoplasmic domain appeared to play a supportive role in γ-secretase inhibition, whereas mutational disruption of the two protein-binding motifs involved in the formation of cell adhesion complexes did not affect γ-secretase inhibition. Co-overexpression of presenilin-1 or APH-1 abrogated γ-secretase inhibition probably through prevention of the incorporation of CRB2 into the γ-secretase complex. Our results suggest that CRB2 functions as an inhibitory binding protein that is involved in the formation of a mature but inactive pool of the γ-secretase complex. PMID:20299451

  18. Delayed inhibition in cortical receptive fields and the discrimination of complex stimuli.

    PubMed

    Narayan, Rajiv; Ergün, Ayla; Sen, Kamal

    2005-10-01

    Although auditory cortex is thought to play an important role in processing complex natural sounds such as speech and animal vocalizations, the specific functional roles of cortical receptive fields (RFs) remain unclear. Here, we study the relationship between a behaviorally important function: the discrimination of natural sounds and the structure of cortical RFs. We examine this problem in the model system of songbirds, using a computational approach. First, we constructed model neurons based on the spectral temporal RF (STRF), a widely used description of auditory cortical RFs. We focused on delayed inhibitory STRFs, a class of STRFs experimentally observed in primary auditory cortex (ACx) and its analog in songbirds (field L), which consist of an excitatory subregion and a delayed inhibitory subregion cotuned to a characteristic frequency. We quantified the discrimination of birdsongs by model neurons, examining both the dynamics and temporal resolution of discrimination, using a recently proposed spike distance metric (SDM). We found that single model neurons with delayed inhibitory STRFs can discriminate accurately between songs. Discrimination improves dramatically when the temporal structure of the neural response at fine timescales is considered. When we compared discrimination by model neurons with and without the inhibitory subregion, we found that the presence of the inhibitory subregion can improve discrimination. Finally, we modeled a cortical microcircuit with delayed synaptic inhibition, a candidate mechanism underlying delayed inhibitory STRFs, and showed that blocking inhibition in this model circuit degrades discrimination.

  19. LINE-1 retroelements complexed and inhibited by activation induced cytidine deaminase.

    PubMed

    Metzner, Mirjam; Jäck, Hans-Martin; Wabl, Matthias

    2012-01-01

    LINE-1 (abbreviated L1) is a major class of retroelements in humans and mice. If unrestricted, retroelements accumulate in the cytoplasm and insert their DNA into the host genome, with the potential to cause autoimmune disease and cancer. Retroviruses and other retroelements are inhibited by proteins of the APOBEC family, of which activation-induced cytidine deaminase (AID) is a member. Although AID is mainly known for being a DNA mutator shaping the antibody repertoire in B lymphocytes, we found that AID also restricts de novo L1 integrations in B- and non-B-cell lines. It does so by decreasing the protein level of open reading frame 1 (ORF1) of both exogenous and endogenous L1. In activated B lymphocytes, AID deficiency increased L1 mRNA 1.6-fold and murine leukemia virus (MLV) mRNA 2.7-fold. In cell lines and activated B lymphocytes, AID forms cytoplasmic high-molecular-mass complexes with L1 mRNA, which may contribute to L1 restriction. Because AID-deficient activated B lymphocytes do not express ORF1 protein, we suggest that ORF1 protein expression is inhibited by additional restriction factors in these cells. The greater increase in MLV compared to L1 mRNA in AID-deficient activated B lymphocytes may indicate less strict surveillance of retrovirus. PMID:23133680

  20. Therapeutic Blockade of Immune Complex-Mediated Glomerulonephritis by Highly Selective Inhibition of Bruton's Tyrosine Kinase.

    PubMed

    Chalmers, Samantha A; Doerner, Jessica; Bosanac, Todd; Khalil, Sara; Smith, Dustin; Harcken, Christian; Dimock, Janice; Der, Evan; Herlitz, Leal; Webb, Deborah; Seccareccia, Elise; Feng, Di; Fine, Jay S; Ramanujam, Meera; Klein, Elliott; Putterman, Chaim

    2016-01-01

    Lupus nephritis (LN) is a potentially dangerous end organ pathology that affects upwards of 60% of lupus patients. Bruton's tyrosine kinase (BTK) is important for B cell development, Fc receptor signaling, and macrophage polarization. In this study, we investigated the effects of a novel, highly selective and potent BTK inhibitor, BI-BTK-1, in an inducible model of LN in which mice receive nephrotoxic serum (NTS) containing anti-glomerular antibodies. Mice were treated once daily with vehicle alone or BI-BTK-1, either prophylactically or therapeutically. When compared with control treated mice, NTS-challenged mice treated prophylactically with BI-BTK-1 exhibited significantly attenuated kidney disease, which was dose dependent. BI-BTK-1 treatment resulted in decreased infiltrating IBA-1+ cells, as well as C3 deposition within the kidney. RT-PCR on whole kidney RNA and serum profiling indicated that BTK inhibition significantly decreased levels of LN-relevant inflammatory cytokines and chemokines. Renal RNA expression profiling by RNA-seq revealed that BI-BTK-1 dramatically modulated pathways related to inflammation and glomerular injury. Importantly, when administered therapeutically, BI-BTK-1 reversed established proteinuria and improved renal histopathology. Our results highlight the important role for BTK in the pathogenesis of immune complex-mediated nephritis, and BTK inhibition as a promising therapeutic target for LN. PMID:27192942

  1. Delayed inhibition in cortical receptive fields and the discrimination of complex stimuli.

    PubMed

    Narayan, Rajiv; Ergün, Ayla; Sen, Kamal

    2005-10-01

    Although auditory cortex is thought to play an important role in processing complex natural sounds such as speech and animal vocalizations, the specific functional roles of cortical receptive fields (RFs) remain unclear. Here, we study the relationship between a behaviorally important function: the discrimination of natural sounds and the structure of cortical RFs. We examine this problem in the model system of songbirds, using a computational approach. First, we constructed model neurons based on the spectral temporal RF (STRF), a widely used description of auditory cortical RFs. We focused on delayed inhibitory STRFs, a class of STRFs experimentally observed in primary auditory cortex (ACx) and its analog in songbirds (field L), which consist of an excitatory subregion and a delayed inhibitory subregion cotuned to a characteristic frequency. We quantified the discrimination of birdsongs by model neurons, examining both the dynamics and temporal resolution of discrimination, using a recently proposed spike distance metric (SDM). We found that single model neurons with delayed inhibitory STRFs can discriminate accurately between songs. Discrimination improves dramatically when the temporal structure of the neural response at fine timescales is considered. When we compared discrimination by model neurons with and without the inhibitory subregion, we found that the presence of the inhibitory subregion can improve discrimination. Finally, we modeled a cortical microcircuit with delayed synaptic inhibition, a candidate mechanism underlying delayed inhibitory STRFs, and showed that blocking inhibition in this model circuit degrades discrimination. PMID:15917327

  2. Polyion complex micellar nanoparticles for integrated fluorometric detection and bacteria inhibition in aqueous media.

    PubMed

    Li, Yamin; Hu, Xianglong; Tian, Sidan; Li, Yang; Zhang, Guoqing; Zhang, Guoying; Liu, Shiyong

    2014-02-01

    The development of portable and inexpensive detection methods can significantly contribute to the prevention of water-borne infectious diseases caused by pathogenic bacteria. Here we designed a nanosystem capable of both bacterial detection and inhibition, where polyion complex (PIC) micelles are constructed from negatively-charged tetraphenylethylene (TPE) sulfonate derivatives, which exhibit the aggregation-induced emission (AIE) feature, and cationic diblock copolymers, poly(ethylene oxide)-b-quaternized poly(2-(dimethylamino)ethyl methacrylate) (PEO-b-PQDMA). Upon contacting with bacteria, the PIC nanosystem disintegrates presumably due to competitive binding of polycation blocks with negatively-charged bacterial surfaces. This process is accompanied by a conspicuous quenching of TPE fluorescence emission, serving as a real-time module for microbial detection. Furthermore, the sharp decrease in CFU is indicative of prominent anti-microbial activities. Thus, PIC micelles possess dual functions of fluorometric detection and inhibition for bacteria in aqueous media. By tuning the charge density of TPE sulfonate derivatives and chain length of cationic PQDMA blocks, optimal performance against Gram-negative Escherichia coli has been achieved with a detection limit of 5.5 × 10(4) CFU/mL and minimum inhibitory concentration (MIC) of 19.7 μg/mL. Tests against Gram-positive Staphylococcus aureus were also conducted to demonstrate versatility of the nanosystem.

  3. A Simple Proteomics-Based Approach to Identification of Immunodominant Antigens from a Complex Pathogen: Application to the CD4 T Cell Response against Human Herpesvirus 6B.

    PubMed

    Becerra-Artiles, Aniuska; Dominguez-Amorocho, Omar; Stern, Lawrence J; Calvo-Calle, J Mauricio

    2015-01-01

    Most of humanity is chronically infected with human herpesvirus 6 (HHV-6), with viral replication controlled at least in part by a poorly characterized CD4 T cell response. Identification of viral epitopes recognized by CD4 T cells is complicated by the large size of the herpesvirus genome and a low frequency of circulating T cells responding to the virus. Here, we present an alternative to classical epitope mapping approaches used to identify major targets of the T cell response to a complex pathogen like HHV-6B. In the approach presented here, extracellular virus preparations or virus-infected cells are fractionated by SDS-PAGE, and eluted fractions are used as source of antigens to study cytokine responses in direct ex vivo T cell activation studies. Fractions inducing significant cytokine responses are analyzed by mass spectrometry to identify viral proteins, and a subset of peptides from these proteins corresponding to predicted HLA-DR binders is tested for IFN-γ production in seropositive donors with diverse HLA haplotypes. Ten HHV-6B viral proteins were identified as immunodominant antigens. The epitope-specific response to HHV-6B virus was complex and variable between individuals. We identified 107 peptides, each recognized by at least one donor, with each donor having a distinctive footprint. Fourteen peptides showed responses in the majority of donors. Responses to these epitopes were validated using in vitro expanded cells and naturally expressed viral proteins. Predicted peptide binding affinities for the eight HLA-DRB1 alleles investigated here correlated only modestly with the observed CD4 T cell responses. Overall, the response to the virus was dominated by peptides from the major capsid protein U57 and major antigenic protein U11, but responses to other proteins including glycoprotein H (U48) and tegument proteins U54 and U14 also were observed. These results provide a means to follow and potentially modulate the CD4 T-cell immune response to HHV-6

  4. 1,10-Phenanthroline promotes copper complexes into tumor cells and induces apoptosis by inhibiting the proteasome activity.

    PubMed

    Zhang, Zhen; Bi, Caifeng; Schmitt, Sara M; Fan, Yuhua; Dong, Lili; Zuo, Jian; Dou, Q Ping

    2012-12-01

    Indole-3-acetic acid and indole-3-propionic acid, two potent natural plant growth hormones, have attracted attention as promising prodrugs in cancer therapy. Copper is known to be a cofactor essential for tumor angiogenesis. We have previously reported that taurine, L-glutamine, and quinoline-2-carboxaldehyde Schiff base copper complexes inhibit cell proliferation and proteasome activity in human cancer cells. In the current study, we synthesized two types of copper complexes, dinuclear complexes and ternary complexes, to investigate whether a certain structure could easily carry copper into cancer cells and consequently inhibit tumor proteasome activity and induce apoptosis. We observed that ternary complexes binding with 1,10-phenanthroline are more potent proteasome inhibitors and apoptosis inducers than dinuclear complexes in PC-3 human prostate cancer cells. Furthermore, the ternary complexes potently inhibit proteasome activity before induction of apoptosis in MDA-MB-231 human breast cancer cells, but not in nontumorigenic MCF-10A cells. Our results suggest that copper complexes binding with 1,10-phenanthroline as the third ligand could serve as potent, selective proteasome inhibitors and apoptosis inducers in tumor cells, and that the ternary complexes may be good potential anticancer drugs.

  5. 1,10-Phenanthroline promotes copper complexes into tumor cells and induces apoptosis by inhibiting the proteasome activity.

    PubMed

    Zhang, Zhen; Bi, Caifeng; Schmitt, Sara M; Fan, Yuhua; Dong, Lili; Zuo, Jian; Dou, Q Ping

    2012-12-01

    Indole-3-acetic acid and indole-3-propionic acid, two potent natural plant growth hormones, have attracted attention as promising prodrugs in cancer therapy. Copper is known to be a cofactor essential for tumor angiogenesis. We have previously reported that taurine, L-glutamine, and quinoline-2-carboxaldehyde Schiff base copper complexes inhibit cell proliferation and proteasome activity in human cancer cells. In the current study, we synthesized two types of copper complexes, dinuclear complexes and ternary complexes, to investigate whether a certain structure could easily carry copper into cancer cells and consequently inhibit tumor proteasome activity and induce apoptosis. We observed that ternary complexes binding with 1,10-phenanthroline are more potent proteasome inhibitors and apoptosis inducers than dinuclear complexes in PC-3 human prostate cancer cells. Furthermore, the ternary complexes potently inhibit proteasome activity before induction of apoptosis in MDA-MB-231 human breast cancer cells, but not in nontumorigenic MCF-10A cells. Our results suggest that copper complexes binding with 1,10-phenanthroline as the third ligand could serve as potent, selective proteasome inhibitors and apoptosis inducers in tumor cells, and that the ternary complexes may be good potential anticancer drugs. PMID:23053530

  6. Molecular and biological interaction between major histocompatibility complex class I antigens and luteinizing hormone receptors or beta-adrenergic receptors triggers cellular response in mice.

    PubMed Central

    Solano, A R; Cremaschi, G; Sánchez, M L; Borda, E; Sterin-Borda, L; Podestá, E J

    1988-01-01

    Purified IgG from BALB/c mouse anti-C3H serum exerts positive inotropic and chronotropic effects in C3H mouse atria and induces testosterone synthesis in C3H mouse Leydig cells. The effect depends on IgG concentration and can be abolished by beta-adrenergic-receptor and luteinizing hormone-receptor antagonists. IgG interferes with the binding of dihydroalprenolol and luteinizing hormone. Monoclonal antibodies against major histocompatibility complex class I antigens were active on the Leydig cells of C3H and BALB/c mice. There was a parallelism between the effect of each individual monoclonal antibody with specificity for a particular haplotype and the response of the target cell from the strains carrying such haplotypes. These antibodies could precipitate the soluble luteinizing hormone-receptor complex. The results suggested that bound hormone triggers the association of major histocompatibility class I antigen with the receptor, thereby activating the respective target cells. PMID:2839829

  7. Activation of human monocytes by streptococcal rhamnose glucose polymers is mediated by CD14 antigen, and mannan binding protein inhibits TNF-alpha release.

    PubMed

    Soell, M; Lett, E; Holveck, F; Schöller, M; Wachsmann, D; Klein, J P

    1995-01-15

    The present work was initiated to define mechanisms that account for the binding on human monocytes of streptococcal cell wall polysaccharides formed by rhamnose glucose polymers (RGPs), and subsequent stimulatory activities. We show here that RGPs bind to and stimulate human monocytes to produce TNF-alpha in a dose-dependent manner. To detect cell surface RGPs binding proteins, intact monocytes were biotinylated before lysis with Nonidet P-40 and solubilized proteins were incubated with RGPs Affi-Prep beads. One major membrane protein of 55 kDa was specifically detected and identified as CD14 because it reacted with anti-CD14 mAbs. Furthermore, anti-CD14 mAbs were able to perform a dose-dependent inhibition of RGPs binding, and suppressed TNF-alpha release from RGPs-stimulated monocytes. Moreover, we demonstrated that RGPs also bind to CD11b; however, this binding is not implicated in synthesis of TNF-alpha. Interestingly, RGPs binding to monocytes was enhanced by human normal serum (HNS) whereas HNS inhibits the TNF-alpha-stimulating activity of RGPs. Western blotting analysis of HNS proteins purified on RGPs Affi-prep beads revealed three specific bands of 75, 55, and 32 kDa reactive with anti-C3 Abs, anti-CD14 mAbs (TUK4), and anti-human mannan binding protein (hMBP)-derived peptide IgG, respectively. These results suggest that C3, soluble CD14, and hMBP form complexes that are probably active in enhancing the binding of RGPs to monocytes. Additional studies have shown that hMBP that recognizes RGPs prevents, unlike the LPS binding protein, TNF-alpha release by inhibiting the binding of RGPs to CD14 Ag. By incubating cells with a constant amount of RGPs-hMBP complexes in the presence or absence of increasing concentrations of C1q, we also demonstrated that C1q receptor mediates the binding and probably the uptake of RGPs-hMBP complexes by human monocytes. PMID:7529289

  8. Effect of oxygen on activation state of complex I and lack of oxaloacetate inhibition of complex II in Langendorff perfused rat heart.

    PubMed

    Maklashina, Elena; Kotlyar, Alexander B; Karliner, Joel S; Cecchini, Gary

    2004-01-01

    Two main entry points for electrons into the mitochondrial respiratory chain are NADH:ubiquinone oxidoreductase (complex I) and succinate:ubiquinone oxidoreductase (complex II). Metabolic regulation of these two respiratory complexes is not understood in detail. It has been suggested that the Krebs cycle metabolic intermediate oxaloacetate (OAA) inhibits complex II in vivo, whereas complex I undergoes a reversible active/de-active transition. In normoxic and anoxic hearts it has been shown that the proportion of complex I in the active and de-active states is different suggesting a possible mode of regulation of the enzyme by oxygen concentration. In the current studies rapid isolation of mitochondrial membranes in a state that preserves the activity of both complex I and complex II has been achieved using Langendorff perfused rat hearts. The findings indicate that the state of activation of complex I is controlled by the oxygen saturation in the perfusate. In addition, these studies show that complex II is fully active in the mitochondrion and not inhibited by OAA regardless of the oxygen concentration.

  9. The role of enzymatic activity in inhibition of the extrinsic tenase complex by phospholipase A2 isoenzymes from Naja nigricollis venom.

    PubMed

    Kini, R M; Evans, H J

    1995-12-01

    Three phospholipase A2 isoenzymes from Naja nigricollis venom inhibit the extrinsic tenase complex. We examined the role of their enzymatic activity in this inhibition by studying the effects of native and His-modified enzymes. Only CM-IV of the His-modified, catalytically inactive proteins showed significant inhibition of the activity of the complex. This indicates that strongly anticoagulant CM-IV inhibits the complex by both enzymatic and nonenzymatic mechanisms, whereas the weakly anticoagulant isoenzymes, CM-I and CM-II, inhibit primarily by catalytic degradation of phospholipids. This indicates a functional difference in the mode of inhibition between strongly and weakly anticoagulant phospholipase A2 enzymes.

  10. Efficient Targeting of Protein Antigen to the Dendritic Cell Receptor DEC-205 in the Steady State Leads to Antigen Presentation on Major Histocompatibility Complex Class I Products and Peripheral CD8+ T Cell Tolerance

    PubMed Central

    Bonifaz, Laura; Bonnyay, David; Mahnke, Karsten; Rivera, Miguel; Nussenzweig, Michel C.; Steinman, Ralph M.

    2002-01-01

    To identify endocytic receptors that allow dendritic cells (DCs) to capture and present antigens on major histocompatibility complex (MHC) class I products in vivo, we evaluated DEC-205, which is abundant on DCs in lymphoid tissues. Ovalbumin (OVA) protein, when chemically coupled to monoclonal αDEC-205 antibody, was presented by CD11c+ lymph node DCs, but not by CD11c− cells, to OVA-specific, CD4+ and CD8+ T cells. Receptor-mediated presentation was at least 400 times more efficient than unconjugated OVA and, for MHC class I, the DCs had to express transporter of antigenic peptides (TAP) transporters. When αDEC-205:OVA was injected subcutaneously, OVA protein was identified over a 4–48 h period in DCs, primarily in the lymph nodes draining the injection site. In vivo, the OVA protein was selectively presented by DCs to TCR transgenic CD8+ cells, again at least 400 times more effectively than soluble OVA and in a TAP-dependent fashion. Targeting of αDEC-205:OVA to DCs in the steady state initially induced 4–7 cycles of T cell division, but the T cells were then deleted and the mice became specifically unresponsive to rechallenge with OVA in complete Freund's adjuvant. In contrast, simultaneous delivery of a DC maturation stimulus via CD40, together with αDEC-205:OVA, induced strong immunity. The CD8+ T cells responding in the presence of agonistic αCD40 antibody produced large amounts of interleukin 2 and interferon γ, acquired cytolytic function in vivo, emigrated in large numbers to the lung, and responded vigorously to OVA rechallenge. Therefore, DEC-205 provides an efficient receptor-based mechanism for DCs to process proteins for MHC class I presentation in vivo, leading to tolerance in the steady state and immunity after DC maturation. PMID:12486105

  11. Efficient targeting of protein antigen to the dendritic cell receptor DEC-205 in the steady state leads to antigen presentation on major histocompatibility complex class I products and peripheral CD8+ T cell tolerance.

    PubMed

    Bonifaz, Laura; Bonnyay, David; Mahnke, Karsten; Rivera, Miguel; Nussenzweig, Michel C; Steinman, Ralph M

    2002-12-16

    To identify endocytic receptors that allow dendritic cells (DCs) to capture and present antigens on major histocompatibility complex (MHC) class I products in vivo, we evaluated DEC-205, which is abundant on DCs in lymphoid tissues. Ovalbumin (OVA) protein, when chemically coupled to monoclonal alphaDEC-205 antibody, was presented by CD11c+ lymph node DCs, but not by CD11c- cells, to OVA-specific, CD4+ and CD8+ T cells. Receptor-mediated presentation was at least 400 times more efficient than unconjugated OVA and, for MHC class I, the DCs had to express transporter of antigenic peptides (TAP) transporters. When alphaDEC-205:OVA was injected subcutaneously, OVA protein was identified over a 4-48 h period in DCs, primarily in the lymph nodes draining the injection site. In vivo, the OVA protein was selectively presented by DCs to TCR transgenic CD8+ cells, again at least 400 times more effectively than soluble OVA and in a TAP-dependent fashion. Targeting of alphaDEC-205:OVA to DCs in the steady state initially induced 4-7 cycles of T cell division, but the T cells were then deleted and the mice became specifically unresponsive to rechallenge with OVA in complete Freund's adjuvant. In contrast, simultaneous delivery of a DC maturation stimulus via CD40, together with alphaDEC-205:OVA, induced strong immunity. The CD8+ T cells responding in the presence of agonistic alphaCD40 antibody produced large amounts of interleukin 2 and interferon gamma, acquired cytolytic function in vivo, emigrated in large numbers to the lung, and responded vigorously to OVA rechallenge. Therefore, DEC-205 provides an efficient receptor-based mechanism for DCs to process proteins for MHC class I presentation in vivo, leading to tolerance in the steady state and immunity after DC maturation.

  12. Epitope mapping of 7S cashew antigen in complex with antibody by solution-phase H/D exchange monitored by FT-ICR mass spectrometry.

    PubMed

    Guan, Xiaoyan; Noble, Kyle A; Tao, Yeqing; Roux, Kenneth H; Sathe, Shridhar K; Young, Nicolas L; Marshall, Alan G

    2015-06-01

    The potential epitope of a recombinant food allergen protein, cashew Ana o 1, reactive to monoclonal antibody, mAb 2G4, has been mapped by solution-phase amide backbone H/D exchange (HDX) monitored by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Purified mAb 2G4 was incubated with recombinant Ana o 1 (rAna o 1) to form antigen:monoclonal antibody (Ag:mAb) complexes. Complexed and uncomplexed (free) rAna o 1 were then subjected to HDX-MS analysis. Five regions protected from H/D exchange upon mAb binding are identified as potential conformational epitope-contributing segments. PMID:26169135

  13. Inhibition of atherosclerosis-promoting microRNAs via targeted polyelectrolyte complex micelles

    PubMed Central

    Kuo, Cheng-Hsiang; Leon, Lorraine; Chung, Eun Ji; Huang, Ru-Ting; Sontag, Timothy J.; Reardon, Catherine A.; Getz, Godfrey S.; Tirrell, Matthew; Fang, Yun

    2015-01-01

    Polyelectrolyte complex micelles have great potential as gene delivery vehicles because of their ability to encapsulate charged nucleic acids forming a core by neutralizing their charge, while simultaneously protecting the nucleic acids from non-specific interactions and enzymatic degradation. Furthermore, to enhance specificity and transfection efficiency, polyelectrolyte complex micelles can be modified to include targeting capabilities. Here, we describe the design of targeted polyelectrolyte complex micelles containing inhibitors against dys-regulated microRNAs (miRNAs) that promote atherosclerosis, a leading cause of human mortality and morbidity. Inhibition of dys-regulated miRNAs in diseased cells associated with atherosclerosis has resulted in therapeutic efficacy in animal models and has been proposed to treat human diseases. However, the non-specific targeting of microRNA inhibitors via systemic delivery has remained an issue that may cause unwanted side effects. For this reason, we incorporated two different peptide sequences to our miRNA inhibitor containing polyelectrolyte complex micelles. One of the peptides (Arginine-Glutamic Acid-Lysine-Alanine or REKA) was used in another micellar system that demonstrated lesion-specific targeting in a mouse model of atherosclerosis. The other peptide (Valine-Histidine-Proline-Lysine-Glutamine-Histidine-Arginine or VHPKQHR) was identified via phage display and targets vascular endothelial cells through the vascular cell adhesion molecule-1 (VCAM-1). In this study we have tested the in vitro efficacy and efficiency of lesion- and cell-specific delivery of microRNA inhibitors to the cells associated with atherosclerotic lesions via peptide-targeted polyelectrolyte complex micelles. Our results show that REKA-containing micelles (fibrin-targeting) and VHPKQHR-containing micelles (VCAM-1 targeting) can be used to carry and deliver microRNA inhibitors into macrophages and human endothelial cells, respectively

  14. A surface antigen influenza vaccine. 2. Pyrogenicity and antigenicity.

    PubMed Central

    Brady, M. I.; Furminger, I. G.

    1976-01-01

    Conventional influenza vaccine containing whole virus particles purified on a zonal centrifuge is pyrogenic and can cause systemic and local adverse side effects. An improved vaccine was therefore prepared which contained only the surface antigens of the virus adsorbed to aluminium hydroxide. The antigenicity of this vaccine was compared with conventional vaccine in chickens. Both vaccines induced similar titres of serum haemagglutination-inhibition and neuraminidase inhibition antibody. The dose response curves, however, were different. The surface antigens at vaccine strength without aluminium hydroxide were of negligible pyrogenicity in rabbits. PMID:1068196

  15. Endosidin2 targets conserved exocyst complex subunit EXO70 to inhibit exocytosis.

    PubMed

    Zhang, Chunhua; Brown, Michelle Q; van de Ven, Wilhelmina; Zhang, Zhi-Min; Wu, Bin; Young, Michael C; Synek, Lukáš; Borchardt, Dan; Harrison, Reed; Pan, Songqin; Luo, Nan; Huang, Yu-Ming M; Ghang, Yoo-Jin; Ung, Nolan; Li, Ruixi; Isley, Jonathan; Morikis, Dimitrios; Song, Jikui; Guo, Wei; Hooley, Richard J; Chang, Chia-En A; Yang, Zhenbiao; Zarsky, Viktor; Muday, Gloria K; Hicks, Glenn R; Raikhel, Natasha V

    2016-01-01

    The exocyst complex regulates the last steps of exocytosis, which is essential to organisms across kingdoms. In humans, its dysfunction is correlated with several significant diseases, such as diabetes and cancer progression. Investigation of the dynamic regulation of the evolutionarily conserved exocyst-related processes using mutants in genetically tractable organisms such as Arabidopsis thaliana is limited by the lethality or the severity of phenotypes. We discovered that the small molecule Endosidin2 (ES2) binds to the EXO70 (exocyst component of 70 kDa) subunit of the exocyst complex, resulting in inhibition of exocytosis and endosomal recycling in both plant and human cells and enhancement of plant vacuolar trafficking. An EXO70 protein with a C-terminal truncation results in dominant ES2 resistance, uncovering possible distinct regulatory roles for the N terminus of the protein. This study not only provides a valuable tool in studying exocytosis regulation but also offers a potentially new target for drugs aimed at addressing human disease. PMID:26607451

  16. EGCG inhibit chemical reactivity of iron through forming an Ngal–EGCG–iron complex

    PubMed Central

    Bao, Guan-Hu; Xu, Jie; Hu, Feng-Lin; Wan, Xiao-Chun; Deng, Shi-Xian

    2015-01-01

    Accumulated evidence indicates that the interconversion of iron between ferric (Fe3+) and ferrous (Fe2+) can be realized through interaction with reactive oxygen species in the Fenton and Haber–Weiss reactions and thereby physiologically effects redox cycling. The imbalance of iron and ROS may eventually cause tissue damage such as renal proximal tubule injury and necrosis. Many approaches were exploited to ameliorate the oxidative stress caused by the imbalance. (−)-Epigallocatechin-3-gallate, the most active and most abundant catechin in tea, was found to be involved in the protection of a spectrum of renal injuries caused by oxidative stress. Most of studies suggested that EGCG works as an antioxidant. In this paper, Multivariate analysis of the LC–MS data of tea extracts and binding assays showed that the tea polyphenol EGCG can form stable complex with iron through the protein Ngal, a biomarker of acute kidney injury. UV–Vis and Luminescence spectrum methods showed that Ngal can inhibit the chemical reactivity of iron and EGCG through forming an Ngal–EGCG–iron complex. In thinking of the interaction of iron and ROS, we proposed that EGCG may work as both antioxidant and Ngal binding siderphore in protection of kidney from injuries. PMID:24158698

  17. Endosidin2 targets conserved exocyst complex subunit EXO70 to inhibit exocytosis

    PubMed Central

    Zhang, Chunhua; Brown, Michelle Q.; van de Ven, Wilhelmina; Zhang, Zhi-Min; Wu, Bin; Young, Michael C.; Synek, Lukáš; Borchardt, Dan; Harrison, Reed; Pan, Songqin; Luo, Nan; Huang, Yu-ming M.; Ghang, Yoo-Jin; Ung, Nolan; Li, Ruixi; Isley, Jonathan; Morikis, Dimitrios; Song, Jikui; Guo, Wei; Hooley, Richard J.; Chang, Chia-en A.; Yang, Zhenbiao; Zarsky, Viktor; Muday, Gloria K.; Hicks, Glenn R.; Raikhel, Natasha V.

    2016-01-01

    The exocyst complex regulates the last steps of exocytosis, which is essential to organisms across kingdoms. In humans, its dysfunction is correlated with several significant diseases, such as diabetes and cancer progression. Investigation of the dynamic regulation of the evolutionarily conserved exocyst-related processes using mutants in genetically tractable organisms such as Arabidopsis thaliana is limited by the lethality or the severity of phenotypes. We discovered that the small molecule Endosidin2 (ES2) binds to the EXO70 (exocyst component of 70 kDa) subunit of the exocyst complex, resulting in inhibition of exocytosis and endosomal recycling in both plant and human cells and enhancement of plant vacuolar trafficking. An EXO70 protein with a C-terminal truncation results in dominant ES2 resistance, uncovering possible distinct regulatory roles for the N terminus of the protein. This study not only provides a valuable tool in studying exocytosis regulation but also offers a potentially new target for drugs aimed at addressing human disease. PMID:26607451

  18. Astrocyte-Specific Overexpression of Nrf2 Protects Striatal Neurons from Mitochondrial Complex II Inhibition

    PubMed Central

    Calkins, Marcus J.; Vargas, Marcelo R.; Johnson, Delinda A.; Johnson, Jeffrey A.

    2010-01-01

    Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor that is known to regulate a variety of cytoprotective genes through the antioxidant response element (ARE). This endogenous response is one of the major pathways by which cells are protected from xenobiotic or innate oxidative insults. Furthermore, in neural systems, astrocyte-specific activation of Nrf2 is known to protect neurons. In previous work, our laboratory found that Nrf2 protects from intrastriatal injections of the mitochondrial complex II inhibitor malonate. Here, we extend these results to show that multiple methods of astrocyte-specific Nrf2 overexpression provide protection from neurotoxicity in vivo. GFAP-Nrf2 transgenic mice are significantly more resistant to malonate lesioning. This outcome is associated with an increased basal resistance, but more so, an enhanced Nrf2 response to lesioning that attenuated the ensuing neurotoxicity. Furthermore, striatal transplantation of neuroprogenitor cells overexpressing Nrf2 that differentiate into astrocytes after grafting also significantly reduced malonate toxicity. Overall, these data establish that enhanced astrocytic Nrf2 response and Nrf2 preconditioning are both sufficient to protect from acute lesions from mitochondrial complex II inhibition. PMID:20211941

  19. Crystallization and preliminary X-ray crystallographic characterization of a public CMV-specific TCR in complex with its cognate antigen.

    PubMed

    Reiser, Jean Baptiste; Legoux, François; Machillot, Paul; Debeaupuis, Emilie; Le Moullac-Vaydie, Béatrice; Chouquet, Anne; Saulquin, Xavier; Bonneville, Marc; Housset, Dominique

    2009-11-01

    The T-cell response to human cytomegalovirus is characterized by a dramatic reduction of clonal diversity in patients undergoing chronic inflammation or immunodepression. In order to check whether all the selected high-avidity T-cell clones recognize the immunodominant pp65 peptide antigen pp65(495-503) (NLVPMVATV) presented by the major histocompatibility complex (MHC) molecule HLA-A2 in a similar manner, several public high-affinity T-cell receptors (TCRs) specific for the pp65(495-503)-HLA-A2 complex have been investigated. Expression, purification and crystallization were performed and preliminary crystallographic data were collected to 4.7 angstrom resolution for the RA15 TCR in complex with the pp65(495-503)-HLA-A2 complex. Comparison of the RA15-pp65(495-503)-HLA-A2 complex molecular-replacement solution with the structure of another high-affinity pp65(495-503)-HLA-A2-specific TCR, RA14, shows a shared docking mode, indicating that the clonal focusing could be accompanied by the selection of a most favoured peptide-readout mode. However, the position of the RA15 V beta domain is significantly shifted, suggesting a different interatomic interaction network. PMID:19923740

  20. An S6:S18 complex inhibits translation of E. coli rpsF

    PubMed Central

    Babina, Arianne M.; Soo, Mark W.; Fu, Yang; Meyer, Michelle M.

    2015-01-01

    More than half of the ribosomal protein operons in Escherichia coli are regulated by structures within the mRNA transcripts that interact with specific ribosomal proteins to inhibit further protein expression. This regulation is accomplished using a variety of mechanisms and the RNA structures responsible for regulation are often not conserved across bacterial phyla. A widely conserved mRNA structure preceding the ribosomal protein operon containing rpsF and rpsR (encoding S6 and S18) was recently identified through comparative genomics. Examples of this RNA from both E. coli and Bacillus subtilis were shown to interact in vitro with an S6:S18 complex. In this work, we demonstrate that in E. coli, this RNA structure regulates gene expression in response to the S6:S18 complex. β-galactosidase activity from a lacZ reporter translationally fused to the 5′ UTR and first nine codons of E. coli rpsF is reduced fourfold by overexpression of a genomic fragment encoding both S6 and S18 but not by overexpression of either protein individually. Mutations to the mRNA structure, as well as to the RNA-binding site of S18 and the S6–S18 interaction surfaces of S6 and S18, are sufficient to derepress β-galactosidase activity, indicating that the S6:S18 complex is the biologically active effector. Measurement of transcript levels shows that although reporter levels do not change upon protein overexpression, levels of the native transcript are reduced fourfold, suggesting that the mRNA regulator prevents translation and this effect is amplified on the native transcript by other mechanisms. PMID:26447183

  1. Mechanism of Origin DNA Recognition and Assembly of an Initiator-Helicase Complex by SV40 Large Tumor Antigen

    PubMed Central

    Chang, Y. Paul; Xu, Meng; Machado, Ana Carolina Dantas; Yu, Xian Jessica; Rohs, Remo; Chen, Xiaojiang S.

    2013-01-01

    SUMMARY The DNA tumor virus Simian virus 40 (SV40) is a model system for studying eukaryotic replication. SV40 large tumor antigen (LTag) is the initiator/helicase that is essential for genome replication. LTag recognizes and assembles at the viral replication origin. We determined the structure of two multidomain LTag subunits bound to origin DNA. The structure reveals that the origin binding domains (OBDs) and Zn and AAA+ domains are involved in origin recognition and assembly. Notably, the OBDs recognize the origin in an unexpected manner. The histidine residues of the AAA+ domains insert into a narrow minor groove region with enhanced negative electrostatic potential. Computational analysis indicates that this region is intrinsically narrow, demonstrating the role of DNA shape readout in origin recognition. Our results provide important insights into the assembly of the LTag initiator/ helicase at the replication origin and suggest that histidine contacts with the minor groove serve as a mechanism of DNA shape readout. PMID:23545501

  2. Inhibition of Viability, Proliferation, Cytokines Secretion, Surface Antigen Expression, and Adipogenic and Osteogenic Differentiation of Adipose-Derived Stem Cells by Seven-Day Exposure to 0.5 T Static Magnetic Fields.

    PubMed

    Wang, Jian; Xiang, Bo; Deng, Jixian; Freed, Darren H; Arora, Rakesh C; Tian, Ganghong

    2016-01-01

    After seven-day exposure to 0.5-Tesla Static Magnetic Field (SMF), Adipose-derived Stem Cells (ASCs) and those labeled by superparamagnetic iron oxide (SPIO) nanoparticles were examined for viability by methyl thiazol tetrazolium (MTT) assay, proliferation by cell counting and bromodeoxyuridine (BrdU) incorporation, DNA integrity by single cell gel electrophoresis, surface antigen by flow cytometry analysis, and the expression of cytokines and genetic markers by reverse transcription-PCR and underwent adipogenic and osteogenic differentiation assessed by quantifying related specific genes expression. The SMF slightly reduced cell viability and proliferation and inhibited the expression of CD49d, CD54, and CD73 but did not damage DNA integrity. The SMF slightly downregulated the expression of cytokines including Vascular Endothelial Growth Factor (VEGF), Insulin-like Growth Factor-1 (IGF-1), Transforming Growth Factor Beta 1 (TGF-β1), genetic markers comprising Stem Cell Antigen-1 (Sca1), Octamer-4 (Oct-4), ATP-binding Cassette Subfamily B Member 1 (ABCB1), adipogenic marker genes containing Lipoprotein Lipase (LPL), Peroxisome Proliferator-Activated Receptor Gamma (PPAR-γ), and osteogenic marker genes including Secreted Phosphor-protein 1 (SPP1) and Osterix (OSX). Exposure to 0.5 T SMF for seven days inhibited viability, proliferation, surface antigen expression, cytokine secretion, stem cell genetic marker expression, and adipogenic and osteogenic differentiation but did not affect the DNA integrity in ASCs with or without SPIO labeling. PMID:26880984

  3. Inhibition of Viability, Proliferation, Cytokines Secretion, Surface Antigen Expression, and Adipogenic and Osteogenic Differentiation of Adipose-Derived Stem Cells by Seven-Day Exposure to 0.5 T Static Magnetic Fields

    PubMed Central

    Wang, Jian; Xiang, Bo; Deng, Jixian; Freed, Darren H.; Arora, Rakesh C.; Tian, Ganghong

    2016-01-01

    After seven-day exposure to 0.5-Tesla Static Magnetic Field (SMF), Adipose-derived Stem Cells (ASCs) and those labeled by superparamagnetic iron oxide (SPIO) nanoparticles were examined for viability by methyl thiazol tetrazolium (MTT) assay, proliferation by cell counting and bromodeoxyuridine (BrdU) incorporation, DNA integrity by single cell gel electrophoresis, surface antigen by flow cytometry analysis, and the expression of cytokines and genetic markers by reverse transcription-PCR and underwent adipogenic and osteogenic differentiation assessed by quantifying related specific genes expression. The SMF slightly reduced cell viability and proliferation and inhibited the expression of CD49d, CD54, and CD73 but did not damage DNA integrity. The SMF slightly downregulated the expression of cytokines including Vascular Endothelial Growth Factor (VEGF), Insulin-like Growth Factor-1 (IGF-1), Transforming Growth Factor Beta 1 (TGF-β1), genetic markers comprising Stem Cell Antigen-1 (Sca1), Octamer-4 (Oct-4), ATP-binding Cassette Subfamily B Member 1 (ABCB1), adipogenic marker genes containing Lipoprotein Lipase (LPL), Peroxisome Proliferator-Activated Receptor Gamma (PPAR-γ), and osteogenic marker genes including Secreted Phosphor-protein 1 (SPP1) and Osterix (OSX). Exposure to 0.5 T SMF for seven days inhibited viability, proliferation, surface antigen expression, cytokine secretion, stem cell genetic marker expression, and adipogenic and osteogenic differentiation but did not affect the DNA integrity in ASCs with or without SPIO labeling. PMID:26880984

  4. A Recombinant Antibody with the Antigen-Specific, Major Histocompatibility Complex-Restricted Specificity of T Cells

    NASA Astrophysics Data System (ADS)

    Andersen, Peter S.; Stryhn, Anette; Hansen, Bjarke E.; Fugger, Lars; Engberg, Jan; Buus, Soren

    1996-03-01

    Specific recognition of peptide/major histocompatibility complex (MHC) molecule complexes by the T-cell receptor is a key reaction in the specific immune response. Antibodies against peptide/MHC complexes would therefore be valuable tools in studying MHC function and T-cell recognition and might lead to novel approaches in immunotherapy. However, it has proven difficult to generate antibodies with the specificity of T cells by conventional hybridoma techniques. Here we report that the phage display technology is a feasible alternative to generate antibodies recognizing specific, predetermined peptide/MHC complexes.

  5. Affinity improvement of a therapeutic antibody by structure-based computational design: generation of electrostatic interactions in the transition state stabilizes the antibody-antigen complex.

    PubMed

    Kiyoshi, Masato; Caaveiro, Jose M M; Miura, Eri; Nagatoishi, Satoru; Nakakido, Makoto; Soga, Shinji; Shirai, Hiroki; Kawabata, Shigeki; Tsumoto, Kouhei

    2014-01-01

    The optimization of antibodies is a desirable goal towards the development of better therapeutic strategies. The antibody 11K2 was previously developed as a therapeutic tool for inflammatory diseases, and displays very high affinity (4.6 pM) for its antigen the chemokine MCP-1 (monocyte chemo-attractant protein-1). We have employed a virtual library of mutations of 11K2 to identify antibody variants of potentially higher affinity, and to establish benchmarks in the engineering of a mature therapeutic antibody. The most promising candidates identified in the virtual screening were examined by surface plasmon resonance to validate the computational predictions, and to characterize their binding affinity and key thermodynamic properties in detail. Only mutations in the light-chain of the antibody are effective at enhancing its affinity for the antigen in vitro, suggesting that the interaction surface of the heavy-chain (dominated by the hot-spot residue Phe101) is not amenable to optimization. The single-mutation with the highest affinity is L-N31R (4.6-fold higher affinity than wild-type antibody). Importantly, all the single-mutations showing increase affinity incorporate a charged residue (Arg, Asp, or Glu). The characterization of the relevant thermodynamic parameters clarifies the energetic mechanism. Essentially, the formation of new electrostatic interactions early in the binding reaction coordinate (transition state or earlier) benefits the durability of the antibody-antigen complex. The combination of in silico calculations and thermodynamic analysis is an effective strategy to improve the affinity of a matured therapeutic antibody. PMID:24475232

  6. The structure of the antigen-binding groove of major histocompatibility complex class I molecules determines specific selection of self-peptides.

    PubMed Central

    van Bleek, G M; Nathenson, S G

    1991-01-01

    We have examined the effect of diversity in the antigen-binding groove of the Kb, Db, Kbm1, and Kbm8 major histocompatibility complex (MHC) class I molecules on the set of self-peptides they present on the cell surface, by using a procedure we recently developed in our laboratory to isolate endogenously processed peptides bound to MHC class I molecules. We found that such naturally processed peptides are 7-10 amino acids long. A major motif of tyrosine and phenylalanine residues at positions three and five was found for peptides binding to Kb. The availability of Kb mutant molecules Kbm1 and Kbm8, each with localized clustered changes in the antigen-binding cleft, allowed us to probe the effect of such small alterations on peptide selection. We found that such changes in different regions in the antigen-binding groove exert an absolute effect by changing subsets of self-peptides bound to these MHC molecules. In the Kbm1 mutant, the binding of the characteristic major set of Kb-associated peptides with tyrosine at position three or both positions three and five is abrogated, although this MHC molecule still binds peptides with tyrosine at position seven; the latter peptides also bind to Kb. Kbm8 shares the major Tyr-3, Tyr-5 peptide set that binds to Kb but does not bind the peptides with tyrosine at position seven. Thus differences in binding selectivity in Kbm1 and Kbm8 appear to be the major determinant for the observed alterations in in vivo immune responses. PMID:1763019

  7. Lipid peroxidation causes endosomal antigen release for cross-presentation

    PubMed Central

    Dingjan, Ilse; Verboogen, Daniëlle RJ; Paardekooper, Laurent M; Revelo, Natalia H; Sittig, Simone P; Visser, Linda J; Mollard, Gabriele Fischer von; Henriet, Stefanie SV; Figdor, Carl G; ter Beest, Martin; van den Bogaart, Geert

    2016-01-01

    Dendritic cells (DCs) present foreign antigen in major histocompatibility complex (MHC) class I molecules to cytotoxic T cells in a process called cross-presentation. An important step in this process is the release of antigen from the lumen of endosomes into the cytosol, but the mechanism of this step is still unclear. In this study, we show that reactive oxygen species (ROS) produced by the NADPH-oxidase complex NOX2 cause lipid peroxidation, a membrane disrupting chain-reaction, which in turn results in antigen leakage from endosomes. Antigen leakage and cross-presentation were inhibited by blocking ROS production or scavenging radicals and induced when using a ROS-generating photosensitizer. Endosomal antigen release was impaired in DCs from chronic granulomatous disease (CGD) patients with dysfunctional NOX2. Thus, NOX2 induces antigen release from endosomes for cross-presentation by direct oxidation of endosomal lipids. This constitutes a new cellular function for ROS in regulating immune responses against pathogens and cancer. PMID:26907999

  8. Cytotoxic and Nitric Oxide Inhibition Activities of Propolis Extract along with Microencapsulation by Complex Coacervation.

    PubMed

    Onbas, Rabia; Kazan, Aslihan; Nalbantsoy, Ayse; Yesil-Celiktas, Ozlem

    2016-09-01

    In this study, cytotoxicity of ethanol extract of propolis (EEP) originating from Sivas, Turkey was screened against several cancer cell lines, namely PC-3, U87MG, A-549, mPANC96, CaCo-2, MCF-7, HeLa, MDA-MB-231 and a non-tumor cell line HEK293 by MTT assay. The inhibition levels of inducible nitric oxide synthase (iNOS) were also determined by using RAW 264.7 macrophage cells following lipopolysaccharide (LPS) treatment. EEP exhibited significant cytotoxic nitric oxide inhibition activities with an IC50 value of 0.1 ± 0.1 μg/ml indicating a high potential as an anti-inflammatory agent. In spite of these promising results and the fact that propolis is a highly nutritive substance, its low solubility and bitter taste limit the applications as a natural supplement. Encapsulation might serve as a good strategy in order to overcome these problems. Complex coacervation was applied where the main focus was on surfactant type, polymer ratio (alginate:gelatin), stirring rate and concentration of core material. The mean particle size of unloaded microparticles were 22.62 μm obtained with gelatin:alginate ratio of 1:1 at a stirring rate of 1400 rpm with 2 ml of 1 % (w/v) sodium carboxymethyl cellulose (Na-CMC), whereas addition of EEP at a concentration of 100 mg/ml increased the mean particle size to 36.44 μm and yielded an encapsulation efficiency of 98.77 %. The cytotoxicities of EEP loaded microparticles were also assessed both on MCF-7 and MDA-MB-231 where similar results were achieved as free EEP which can enhance the possible use of propolis extract in the industry as a natural supplement. PMID:27380456

  9. Xanthohumol induces generation of reactive oxygen species and triggers apoptosis through inhibition of mitochondrial electron transfer chain complex I.

    PubMed

    Zhang, Bo; Chu, Wei; Wei, Peng; Liu, Ying; Wei, Taotao

    2015-12-01

    Xanthohumol is a prenylflavonoid extracted from hops (Humulus lupulus). It possesses anti-cancer and anti-inflammatory activities in vitro and in vivo, and offers therapeutic benefits for treatment of metabolic syndromes. However, the precise mechanisms underlying its pharmacological effects remain to be elucidated, together with its cellular target. Here, we provide evidence that xanthohumol directly interacts with the mitochondrial electron transfer chain complex I (NADH dehydrogenase), inhibits the oxidative phosphorylation, triggers the production of reactive oxygen species, and induces apoptosis. In addition, we show that as a result of the inhibition of the mitochondrial oxidative phosphorylation, xanthohumol exposure causes a rapid decrease of mitochondrial transmembrane potential. Furthermore, we showed that xanthohumol up-regulates the glycolytic capacity in cells, and thus compensates cellular ATP generation. Dissection of the multiple steps of aerobic respiration by extracellular flux assays revealed that xanthohumol specifically inhibits the activity of mitochondrial complex I, but had little effect on that of complex II, III and IV. Inhibition of complex I by xanthohumol caused the overproduction of reactive oxygen species, which are responsible for the induction of apoptosis in cancer cells. We also found that isoxanthohumol, the structural isomer of xanthohumol, is inactive to cells, suggesting that the reactive 2-hydroxyl group of xanthohumol is crucial for its targeting to the mitochondrial complex I. Together, the remodeling of cell metabolism revealed here has therapeutic potential for the use of xanthohumol.

  10. EBV Nuclear Antigen 3C Mediates Regulation of E2F6 to Inhibit E2F1 Transcription and Promote Cell Proliferation.

    PubMed

    Pei, Yonggang; Banerjee, Shuvomoy; Sun, Zhiguo; Jha, Hem Chandra; Saha, Abhik; Robertson, Erle S

    2016-08-01

    Epstein-Barr virus (EBV) is considered a ubiquitous herpesvirus with the ability to cause latent infection in humans worldwide. EBV-association is evidently linked to different types of human malignancies, mainly of epithelial and lymphoid origin. Of interest is the EBV nuclear antigen 3C (EBNA3C) which is critical for EBV-mediated immortalization. Recently, EBNA3C was shown to bind the E2F1 transcription regulator. The E2F transcription factors have crucial roles in various cellular functions, including cell cycle, DNA replication, DNA repair, cell mitosis, and cell fate. Specifically, E2F6, one of the unique E2F family members, is known to be a pRb-independent transcription repressor of E2F-target genes. In our current study, we explore the role of EBNA3C in regulating E2F6 activities. We observed that EBNA3C plays an important role in inducing E2F6 expression in LCLs. Our study also shows that EBNA3C physically interacts with E2F6 at its amino and carboxy terminal domains and they form a protein complex in human cells. In addition, EBNA3C stabilizes the E2F6 protein and is co-localized in the nucleus. We also demonstrated that both EBNA3C and E2F6 contribute to reduction in E2F1 transcriptional activity. Moreover, E2F1 forms a protein complex with EBNA3C and E2F6, and EBNA3C competes with E2F1 for E2F6 binding. E2F6 is also recruited by EBNA3C to the E2F1 promoter, which is critical for EBNA3C-mediated cell proliferation. These results demonstrate a critical role for E2F family members in EBV-induced malignancies, and provide new insights for targeting E2F transcription factors in EBV-associated cancers as potential therapeutic intervention strategies. PMID:27548379

  11. EBV Nuclear Antigen 3C Mediates Regulation of E2F6 to Inhibit E2F1 Transcription and Promote Cell Proliferation.

    PubMed

    Pei, Yonggang; Banerjee, Shuvomoy; Sun, Zhiguo; Jha, Hem Chandra; Saha, Abhik; Robertson, Erle S

    2016-08-01

    Epstein-Barr virus (EBV) is considered a ubiquitous herpesvirus with the ability to cause latent infection in humans worldwide. EBV-association is evidently linked to different types of human malignancies, mainly of epithelial and lymphoid origin. Of interest is the EBV nuclear antigen 3C (EBNA3C) which is critical for EBV-mediated immortalization. Recently, EBNA3C was shown to bind the E2F1 transcription regulator. The E2F transcription factors have crucial roles in various cellular functions, including cell cycle, DNA replication, DNA repair, cell mitosis, and cell fate. Specifically, E2F6, one of the unique E2F family members, is known to be a pRb-independent transcription repressor of E2F-target genes. In our current study, we explore the role of EBNA3C in regulating E2F6 activities. We observed that EBNA3C plays an important role in inducing E2F6 expression in LCLs. Our study also shows that EBNA3C physically interacts with E2F6 at its amino and carboxy terminal domains and they form a protein complex in human cells. In addition, EBNA3C stabilizes the E2F6 protein and is co-localized in the nucleus. We also demonstrated that both EBNA3C and E2F6 contribute to reduction in E2F1 transcriptional activity. Moreover, E2F1 forms a protein complex with EBNA3C and E2F6, and EBNA3C competes with E2F1 for E2F6 binding. E2F6 is also recruited by EBNA3C to the E2F1 promoter, which is critical for EBNA3C-mediated cell proliferation. These results demonstrate a critical role for E2F family members in EBV-induced malignancies, and provide new insights for targeting E2F transcription factors in EBV-associated cancers as potential therapeutic intervention strategies.

  12. EBV Nuclear Antigen 3C Mediates Regulation of E2F6 to Inhibit E2F1 Transcription and Promote Cell Proliferation

    PubMed Central

    Sun, Zhiguo; Jha, Hem Chandra; Saha, Abhik; Robertson, Erle S.

    2016-01-01

    Epstein–Barr virus (EBV) is considered a ubiquitous herpesvirus with the ability to cause latent infection in humans worldwide. EBV-association is evidently linked to different types of human malignancies, mainly of epithelial and lymphoid origin. Of interest is the EBV nuclear antigen 3C (EBNA3C) which is critical for EBV-mediated immortalization. Recently, EBNA3C was shown to bind the E2F1 transcription regulator. The E2F transcription factors have crucial roles in various cellular functions, including cell cycle, DNA replication, DNA repair, cell mitosis, and cell fate. Specifically, E2F6, one of the unique E2F family members, is known to be a pRb-independent transcription repressor of E2F-target genes. In our current study, we explore the role of EBNA3C in regulating E2F6 activities. We observed that EBNA3C plays an important role in inducing E2F6 expression in LCLs. Our study also shows that EBNA3C physically interacts with E2F6 at its amino and carboxy terminal domains and they form a protein complex in human cells. In addition, EBNA3C stabilizes the E2F6 protein and is co-localized in the nucleus. We also demonstrated that both EBNA3C and E2F6 contribute to reduction in E2F1 transcriptional activity. Moreover, E2F1 forms a protein complex with EBNA3C and E2F6, and EBNA3C competes with E2F1 for E2F6 binding. E2F6 is also recruited by EBNA3C to the E2F1 promoter, which is critical for EBNA3C-mediated cell proliferation. These results demonstrate a critical role for E2F family members in EBV-induced malignancies, and provide new insights for targeting E2F transcription factors in EBV-associated cancers as potential therapeutic intervention strategies. PMID:27548379

  13. Diagnosis of tuberculosis in an Indian population by an indirect ELISA protocol based on detection of Antigen 85 complex: a prospective cohort study

    PubMed Central

    Kashyap, Rajpal S; Rajan, Anju N; Ramteke, Sonali S; Agrawal, Vijay S; Kelkar, Sanjivani S; Purohit, Hemant J; Taori, Girdhar M; Daginawala, Hatim F

    2007-01-01

    Background Diagnosis of tuberculosis (TB) remains problematic despite many new advanced diagnostic methods. A reliable and rapid diagnostic test, which could be performed in any standard pathology laboratory, would help to obtain definitive early diagnoses of TB. In the present study we describe a prospective evaluation for demonstrating Antigen (Ag) 85 complex in the sera from TB patients. Methods Indirect ELISA, employing monoclonal antibodies (mAb) against the purified Ag 85 complex, was used to demonstrate Ag 85 complex in sera from TB patients. Serum samples were obtained from 197 different groups of patients: confirmed TB {n = 24}, clinically diagnosed TB {n = 104}, disease controls {n = 49} and healthy controls {n = 20}. Receiver operating curve (ROC) was used to calculate the cut off value and comparison between TB and non-TB groups were done by the chi-square test. Results The indirect ELISA method, using an mAb against Ag 85 complex, yielded 82% sensitivity (95% confidence interval [CI] 67 to 93%) and 86% specificity (95% CI, 57 to 98%) for the diagnosis of TB. The serum positivities for Ag 85 complex in cases of confirmed and clinically diagnosed TB patients were 96% (23/24) and 79% (82/104) respectively, while the positivity for patients in the non-tuberculosis group was 14% (10/69). Conclusion The detection of Ag 85 complex in sera from TB patients by indirect ELISA using mAb against purified Ag 85 complex gives a reliable diagnosis and can be used to develop an immunodiagnostic assay with increased sensitivity and specificity. PMID:17620147

  14. Pharmacological and small interference RNA-mediated inhibition of breast cancer-associated fatty acid synthase (oncogenic antigen-519) synergistically enhances Taxol (paclitaxel)-induced cytotoxicity.

    PubMed

    Menendez, Javier A; Vellon, Luciano; Colomer, Ramon; Lupu, Ruth

    2005-05-20

    The relationship between breast cancer-associated fatty acid synthase (FAS; oncogenic antigen-519) and chemotherapy-induced cell damage has not been studied. We examined the ability of C75, a synthetic slow-binding inhibitor of FAS activity, to modulate the cytotoxic activity of the microtubule-interfering agent Taxol (paclitaxel) in SK-Br3, MDA-MB-231, MCF-7 and multidrug-resistant MDR-1 (P-Glycoprotein)-overexpressing MCF-7/AdrR breast cancer cells. When the combination of C75 with Taxol in either concurrent (C75 + Taxol 24 hr) or sequential (C75 24 hr --> Taxol 24 hr) schedules were tested for synergism, addition or antagonism using the isobologram and the median-effect plot analyses, co-exposure of C75 and Taxol mostly demonstrated synergistic effects, whereas sequential exposure to C75 followed by Taxol mainly showed additive or antagonistic interactions. Because the nature of the cytotoxic interactions was definitely schedule-dependent in MCF-7 cells, we next evaluated the effects of C75 on Taxol-induced apoptosis as well as Taxol-activated cell death and cell survival-signaling pathways in this breast cancer cell model. An ELISA for histone-associated DNA fragments demonstrated that C75 and Taxol co-exposure caused a synergistic enhancement of apoptotic cell death, whereas C75 pre-treatment did not enhance the apoptosis-inducing activity of Taxol. Co-exposure to C75 and Taxol induced a remarkable nuclear accumulation of activated p38 mitogen-activated protein kinase (p38 MAPK), which was accompanied by a synergistic nuclear accumulation of the p53 tumor-suppressor protein that was phosphorylated at Ser46, a p38 MAPK-regulated pro-apoptotic modification of p53. As single agents, FAS blocker C75 and Taxol induced a significant stimulation of the proliferation and cell survival mitogen-activated protein kinase extracellular signal-regulated kinase (ERK1/ERK2 MAPK) activity, whereas, in combination, they interfered with ERK1/ERK2 activation. Moreover, the

  15. Major histocompatibility complex class II dextramers: New tools for the detection of antigen-specific, CD4 T cells in basic and clinical research

    PubMed Central

    Massilamany, Chandirasegaran; Krishnan, Bharathi; Reddy, Jay

    2015-01-01

    The advent of major histocompatibility complex (MHC) tetramer technology has been a major contribution to T cell immunology, because tetramer reagents permit detection of antigen-specific T cells at the single-cell level in heterogeneous populations by flow cytometry. However, unlike MHC class I tetramers, the utility of MHC class II tetramers has been less frequently reported. MHC class II tetramers can be used successfully to enumerate the frequencies of antigen-specific CD4 T cells in cells activated in vitro, but their use for ex vivo analyses continues to be a problem, due in part to their activation dependency for binding with T cells. To circumvent this problem, we recently reported the creation of a new generation of reagents called MHC class II dextramers, which were found to be superior to their counterparts. In this review, we discuss the utility of class II dextramers vis-a-vis tetramers, with respect to their specificity and sensitivity, including potential applications and limitations. PMID:26207337

  16. A possible functional necklace formed by placental antigen X-P2-immunoreactive and intensely acetylcholinesterase-reactive (PAX/IAE) glomerular complexes in the rat olfactory bulb.

    PubMed

    Shinoda, K; Ohtsuki, T; Nagano, M; Okumura, T

    1993-07-30

    The relationship between placental antigen X-P2 (PAX)-immunoreactive glomeruli and intensely acetylcholinesterase-reactive (IAE) patchy regions was evaluated by comparison of neighboring cryostat sections of the rat olfactory bulb. Both groups of distribution show similar necklace patterns. Each IAE region consists of heterologous glomerulus-like structures with variable acetylcholinesterase reactivity: strongly and less-reactive (IAE-S and IAE-L) structures. The PAX-immunoreactive glomeruli were detected as parts of the IAE-L portions. Three heterologous PAX/IAE glomeruli or glomerulus-like structures (IAE-S, IAE-L/PAX and IAE-L/non-PAX structures) locally form a distinct glomerular complex, the 'PAX/IAE glomerular complex'. At the caudal end of the main olfactory bulb, nine to sixteen such complexes occur at intervals and form a circumferential 'necklace'. Since one of them corresponds to the 'modified glomerular complex' involved in rat suckling behavior, the entire 'necklace' may be associated with processing olfactory stimuli eliciting or suppressing the suckling response.

  17. A Model of Motor Inhibition for a Complex Skill: Baseball Batting

    ERIC Educational Resources Information Center

    Gray, Rob

    2009-01-01

    The ability to inhibit an ongoing action in response to a signal from the environment is important for many perceptual-motor actions. This paper examines a particular example of this behavior: attempting to inhibit or "check" a swing in baseball batting. A model of motor inhibition in batting is proposed. In the model there are three different…

  18. Tracking global changes induced in the CD4 T-cell receptor repertoire by immunization with a complex antigen using short stretches of CDR3 protein sequence

    PubMed Central

    Thomas, Niclas; Best, Katharine; Cinelli, Mattia; Reich-Zeliger, Shlomit; Gal, Hilah; Shifrut, Eric; Madi, Asaf; Friedman, Nir; Shawe-Taylor, John; Chain, Benny

    2014-01-01

    Motivation: The clonal theory of adaptive immunity proposes that immunological responses are encoded by increases in the frequency of lymphocytes carrying antigen-specific receptors. In this study, we measure the frequency of different T-cell receptors (TcR) in CD4 + T cell populations of mice immunized with a complex antigen, killed Mycobacterium tuberculosis, using high throughput parallel sequencing of the TcRβ chain. Our initial hypothesis that immunization would induce repertoire convergence proved to be incorrect, and therefore an alternative approach was developed that allows accurate stratification of TcR repertoires and provides novel insights into the nature of CD4 + T-cell receptor recognition. Results: To track the changes induced by immunization within this heterogeneous repertoire, the sequence data were classified by counting the frequency of different clusters of short (3 or 4) continuous stretches of amino acids within the antigen binding complementarity determining region 3 (CDR3) repertoire of different mice. Both unsupervised (hierarchical clustering) and supervised (support vector machine) analyses of these different distributions of sequence clusters differentiated between immunized and unimmunized mice with 100% efficiency. The CD4 + TcR repertoires of mice 5 and 14 days postimmunization were clearly different from that of unimmunized mice but were not distinguishable from each other. However, the repertoires of mice 60 days postimmunization were distinct both from naive mice and the day 5/14 animals. Our results reinforce the remarkable diversity of the TcR repertoire, resulting in many diverse private TcRs contributing to the T-cell response even in genetically identical mice responding to the same antigen. However, specific motifs defined by short stretches of amino acids within the CDR3 region may determine TcR specificity and define a new approach to TcR sequence classification. Availability and implementation: The analysis was

  19. Native MS and ECD Characterization of a Fab-Antigen Complex May Facilitate Crystallization for X-ray Diffraction

    NASA Astrophysics Data System (ADS)

    Zhang, Ying; Cui, Weidong; Wecksler, Aaron T.; Zhang, Hao; Molina, Patricia; Deperalta, Galahad; Gross, Michael L.

    2016-07-01

    Native mass spectrometry (MS) and top-down electron-capture dissociation (ECD) combine as a powerful approach for characterizing large proteins and protein assemblies. Here, we report their use to study an antibody Fab (Fab-1)-VEGF complex in its near-native state. Native ESI with analysis by FTICR mass spectrometry confirms that VEGF is a dimer in solution and that its complex with Fab-1 has a binding stoichiometry of 2:2. Applying combinations of collisionally activated dissociation (CAD), ECD, and infrared multiphoton dissociation (IRMPD) allows identification of flexible regions of the complex, potentially serving as a guide for crystallization and X-ray diffraction analysis.

  20. Native MS and ECD Characterization of a Fab-Antigen Complex May Facilitate Crystallization for X-ray Diffraction.

    PubMed

    Zhang, Ying; Cui, Weidong; Wecksler, Aaron T; Zhang, Hao; Molina, Patricia; Deperalta, Galahad; Gross, Michael L

    2016-07-01

    Native mass spectrometry (MS) and top-down electron-capture dissociation (ECD) combine as a powerful approach for characterizing large proteins and protein assemblies. Here, we report their use to study an antibody Fab (Fab-1)-VEGF complex in its near-native state. Native ESI with analysis by FTICR mass spectrometry confirms that VEGF is a dimer in solution and that its complex with Fab-1 has a binding stoichiometry of 2:2. Applying combinations of collisionally activated dissociation (CAD), ECD, and infrared multiphoton dissociation (IRMPD) allows identification of flexible regions of the complex, potentially serving as a guide for crystallization and X-ray diffraction analysis. Graphical Abstract ᅟ.

  1. Inhibition of the STAT3 Protein by a Dinuclear Macrocyclic Complex.

    PubMed

    Mesquita, Lígia M; Herrera, Federico; Esteves, Catarina V; Lamosa, Pedro; André, Vânia; Mateus, Pedro; Delgado, Rita

    2016-04-01

    A new diethylenetriamine-derived macrocycle bearing 2-methylpyridyl arms and containing m-xylyl spacers, L, was prepared, and its dinuclear copper(II) and zinc(II) complexes were used as receptors for the recognition in aqueous solution of a phosphorylated peptide derived from a sequence of the STAT3 protein. A detailed study of the acid-base behavior of L and of its complexation properties as well as of the association of the phosphorylated peptide to the receptor was carried out by potentiometry in aqueous solution at 298.2 K and I = 0.10 M in KNO3. The data revealed that the receptor forms stable associations with several protonated forms of the substrate, with constant values ranging from 3.32 to 4.25 log units. The affinity of the receptor for the phosphorylated substrate studied is higher at a pH value where the receptor is mainly in the [Cu2L](4+) form and the pY residue of the substrate is in the dianionic form (pH 6.55). These results, also supported by (31)P NMR studies, showed that the phosphopeptide is bound through the phosphoryl group in a bridging mode. Additionally, the receptor inhibited binding between active (phosphorylated) STAT3 and its target DNA sequence in a dose-dependent manner (IC50 63 ± 3.4 μM) in human nuclear extracts in vitro. Treatment of whole cells with the inhibitor revealed that it is bioactive in living cells and has oncostatic properties that could be interesting for the fight against cancer and other pathologies involving the STAT3 protein. PMID:26999534

  2. Depolymerized holothurian glycosaminoglycan and heparin inhibit the intrinsic tenase complex by a common antithrombin-independent mechanism.

    PubMed

    Sheehan, John P; Walke, Erik N

    2006-05-15

    Depolymerized holothurian glycosaminoglycan (DHG) is a fucosylated chrondroitin sulfate that possesses antithrombin-independent antithrombotic properties and inhibits factor X activation by the intrinsic tenase complex (factor IXa-factor VIIIa). The mechanism and molecular target for intrinsic tenase inhibition were determined and compared with inhibition by low-molecular-weight heparin (LMWH). DHG inhibited factor X activation in a noncompetitive manner (reduced V(max(app))), with 50-fold higher apparent affinity than LMWH. DHG did not affect factor VIIIa half-life or chromogenic substrate cleavage by factor IXa-phospholipid but reduced the affinity of factor IXa for factor VIIIa. DHG competed factor IXa binding to immobilized LMWH with an EC(50) 35-fold lower than soluble LWMH. Analysis of intrinsic tenase inhibition, employing factor IXa with mutations in the heparin-binding exosite, demonstrated that relative affinity (K(i)) for DHG was as follows: wild type > K241A > H92A > R170A > > R233A, with partial rather than complete inhibition of the mutants. This rank order for DHG potency correlated with the effect of these mutations on factor IXa-LMWH affinity and the potency of LMWH for intrinsic tenase. DHG also accelerated decay of the intact intrinsic tenase complex. Thus, DHG binds to an exosite on factor IXa that overlaps with the binding sites for LMWH and factor VIIIa, disrupting critical factor IXa-factor VIIIa interactions.

  3. [Basic and clinical evaluation of an immunoradiometric competitive inhibition assay for 2----6 sialyl Lewis a antigen--2. Evaluation of clinical significance].

    PubMed

    Imura, H; Takahashi, T; Matsuda, T; Yoshida, O; Ohkura, H; Seitetsu, Y; Seino, Y; Ishii, M; Kuwabara, M; Ariyoshi, Y

    1989-06-01

    The clinical significance of serum 2----6 sialyl Lewisa antigen was evaluated using "SLA 2----6 Otsuka" kits. Results indicated that the antigen was frequently elevated in sera obtained from patients with various cancers, including pancreas (73%), liver (67%), bilialy tract (66%), uterus (35%), and stomach (33%). Among other tumor markers examined, CA 19-9 (2----3 sialyl Lewisa) had a very similar cancer spectrum as 2----6 sialyl Lewisa. In the sera of patients with malignant disorders of digestive and respiratory organs, including stomach, intestine, pancreas, biliary tract and lung, the serum levels of CA19-9 tended to be higher than those of 2----6 sialyl Lewisa, usually exceeded those of CA19-9. This suggests that the 2----6 sialylation of Lewisa antigen is equally observed in malignant and non-malignant diseases, while the 2----3 sialylation is relatively specific to cancers. As a result, the ratio of the two antigens, CA 19-9: 2----6 sialyl Lewisa antigen ratio, exceeded 2.0 in most of the sera obtained from patients with malignancy, whereas the ratio was below 2.0 in most of patients with corresponding non-malignant diseases of those organs. The determination of the ratio may be clinically useful in the differential diagnosis of the malignant and non-malignant diseases in those organs.

  4. Immunoregulatory effects of covalent antigen-antibody complexes. IV. Priming and tolerance in T-dependent responses.

    PubMed Central

    Tite, J P; Morrison, C A; Taylor, R B

    1982-01-01

    Stable, covalently bonded, monomeric complexes of rabbit anti-NAP (4-azido-2-nitrophenyl) antibodies and NAP-bovine pancreatic ribonuclease (RNase), when injected into mice, prime the subsequent response to a soluble challenge of RNase. This effect is shown to be dependent on an intact Fc portion of the rabbit antibody and not simply due to foreign determinants recognized on the latter. A study of the kinetics of elimination of radioiodinated complexes from the serum indicates that the generation of a primary anti-rabbit IgG response and subsequent clearance of the complex leads to priming of the anti-RNase response. If mice are previously rendered tolerant to rabbit IgG or the complexes are ultracentrifuged, the priming to RNase is often abolished and tolerance may be induced. PMID:6179858

  5. Deletion of naïve T cells recognizing the minor histocompatibility antigen HY with toxin-coupled peptide-MHC class I tetramers inhibits cognate CTL responses and alters immunodominance.

    PubMed

    Hess, Sabrina M; Young, Ellen F; Miller, Keith R; Vincent, Benjamin G; Buntzman, Adam S; Collins, Edward J; Frelinger, Jeffrey A; Hess, Paul R

    2013-12-01

    Alloreactive T-cell responses directed against minor histocompatibility (H) antigens, which arise from diverse genetic disparities between donor and recipient outside the MHC, are an important cause of rejection of MHC-matched grafts. Because clinically significant responses appear to be directed at only a few antigens, the selective deletion of naïve T cells recognizing donor-specific, immunodominant minor H antigens in recipients before transplantation may be a useful tolerogenic strategy. We have previously demonstrated that peptide-MHC class I tetramers coupled to a toxin can efficiently eliminate specific TCR-transgenic T cells in vivo. Here, using the minor histocompatibility antigen HY as a model, we investigated whether toxic tetramers could inhibit the subsequent priming of the two H2-D(b)-restricted, immunodominant T-cell responses by deleting precursor CTL. Immunization of female mice with male bone marrow elicited robust CTL activity against the Uty and Smcy epitopes, with Uty constituting the major response. As hypothesized, toxic tetramer administration prior to immunization increased survival of cognate peptide-pulsed cells in an in vivo CTL assay, and reduced the frequency of corresponding T cells. However, tetramer-mediated decreases in either T-cell population magnified CTL responses against the non-targeted epitope, suggesting that D(b)-Uty(+) and D(b)-Smcy(+) T cells compete for a limited common resource during priming. Toxic tetramers conceivably could be used in combination to dissect manipulate CD8(+) T-cell immunodominance hierarchies, and to prevent the induction of donor-specific, minor H antigen CTL responses in allotransplantation.

  6. Deletion of naïve T cells recognizing the minor histocompatibility antigen HY with toxin-coupled peptide-MHC class I tetramers inhibits cognate CTL responses and alters immunodominance

    PubMed Central

    Hess, Sabrina M.; Young, Ellen F.; Miller, Keith R.; Vincent, Benjamin G.; Buntzman, Adam S.; Collins, Edward J.; Frelinger, Jeffrey A.; Hess, Paul R.

    2013-01-01

    Alloreactive T-cell responses directed against minor histocompatibility (H) antigens, which arise from diverse genetic disparities between donor and recipient outside the MHC, are an important cause of rejection of MHC-matched grafts. Because clinically significant responses appear to be directed at only a few antigens, the selective deletion of naïve T cells recognizing donor-specific, immunodominant minor H antigens in recipients before transplantation may be a useful tolerogenic strategy. We have previously demonstrated that peptide-MHC class I tetramers coupled to a toxin can efficiently eliminate specific TCR-transgenic T cells in vivo. Here, using the minor histocompatibility antigen HY as a model, we investigated whether toxic tetramers could inhibit the subsequent priming of the two H2-Db-restricted, immunodominant T-cell responses by deleting precursor CTL. Immunization of female mice with male bone marrow elicited robust CTL activity against the Uty and Smcy epitopes, with Uty constituting the major response. As hypothesized, toxic tetramer administration prior to immunization increased survival of cognate peptide-pulsed cells in an in vivo CTL assay, and reduced the frequency of corresponding T cells. However, tetramer-mediated decreases in either T-cell population magnified CTL responses against the non-targeted epitope, suggesting that Db-Uty+ and Db-Smcy+ T cells compete for a limited common resource during priming. Toxic tetramers conceivably could be used in combination to dissect or manipulate CD8+ T-cell immunodominance hierarchies, and to prevent the induction of donor-specific, minor H antigen CTL responses in allotransplantation. PMID:24161680

  7. Mutant p53 inhibits miRNA biogenesis by interfering with the microprocessor complex.

    PubMed

    Garibaldi, F; Falcone, E; Trisciuoglio, D; Colombo, T; Lisek, K; Walerych, D; Del Sal, G; Paci, P; Bossi, G; Piaggio, G; Gurtner, A

    2016-07-21

    Downregulation of microRNAs (miRNAs) is commonly observed in cancers and promotes tumorigenesis suggesting that miRNAs may function as tumor suppressors. However, the mechanism through which miRNAs are regulated in cancer, and the connection between oncogenes and miRNA biogenesis remain poorly understood. The TP53 tumor-suppressor gene is mutated in half of human cancers resulting in an oncogene with gain-of-function activities. Here we demonstrate that mutant p53 (mutp53) oncoproteins modulate the biogenesis of a subset of miRNAs in cancer cells inhibiting their post-transcriptional maturation. Interestingly, among these miRNAs several are also downregulated in human tumors. By confocal, co-immunoprecipitation and RNA-chromatin immunoprecipitation experiments, we show that endogenous mutp53 binds and sequesters RNA helicases p72/82 from the microprocessor complex, interfering with Drosha-pri-miRNAs association. In agreement with this, the overexpression of p72 leads to an increase of mature miRNAs levels. Moreover, functional experiments demonstrate the oncosuppressive role of mutp53-dependent miRNAs (miR-517a, -519a, -218, -105). Our study highlights a previously undescribed mechanism by which mutp53 interferes with Drosha-p72/82 association leading, at least in part, to miRNA deregulation observed in cancer.

  8. Trypanosoma cruzi Infection Modulates In Vivo Expression of Major Histocompatibility Complex Class II Molecules on Antigen-Presenting Cells and T-Cell Stimulatory Activity of Dendritic Cells in a Strain-Dependent Manner

    PubMed Central

    Alba Soto, Catalina D.; Mirkin, Gerardo A.; Solana, Maria E.; González Cappa, Stella M.

    2003-01-01

    A striking feature of Chagas' disease is the diversity of clinical presentations. Such variability may be due to the heterogeneity among Trypanosoma cruzi isolates or to the host immune response. Employing two strains which differ in their virulence, we investigated the effect of in vivo infection on professional antigen-presenting cells (APC). Acute infection with the virulent RA strain downregulated the expression of major histocompatibility complex (MHC) class II on splenic dendritic cells (DC) and inhibited its induction on peritoneal macrophages and splenic B cells. It also impaired the ability of DC to prime allogeneic T cells and to form homotypic clusters, suggesting a low maturation state of these cells. In contrast, the low-virulence K98 strain maintained the expression of MHC class II on DC or stimulated it on peritoneal macrophages and B cells and preserved DC's T-cell priming capacity and homotypic clustering. DC from RA-infected mice elicited a lower activation of T. cruzi-specific T-cell proliferation than those from K98-infected mice. APC from RA-infected mice that reached the chronic phase of infection restored MHC class II levels to those found in K98-infected mice and upregulated costimulatory molecules expression, suggesting that the immunosuppression caused by this strain is only transient. Taken together, the results indicate that in vivo infection with T. cruzi modulates APC functionality and that this is accomplished in a strain-dependent manner. PMID:12595432

  9. Class II major histocompatibility complex mutant mice to study the germ-line bias of T-cell antigen receptors.

    PubMed

    Silberman, Daniel; Krovi, Sai Harsha; Tuttle, Kathryn D; Crooks, James; Reisdorph, Richard; White, Janice; Gross, James; Matsuda, Jennifer L; Gapin, Laurent; Marrack, Philippa; Kappler, John W

    2016-09-20

    The interaction of αβ T-cell antigen receptors (TCRs) with peptides bound to MHC molecules lies at the center of adaptive immunity. Whether TCRs have evolved to react with MHC or, instead, processes in the thymus involving coreceptors and other molecules select MHC-specific TCRs de novo from a random repertoire is a longstanding immunological question. Here, using nuclease-targeted mutagenesis, we address this question in vivo by generating three independent lines of knockin mice with single-amino acid mutations of conserved class II MHC amino acids that often are involved in interactions with the germ-line-encoded portions of TCRs. Although the TCR repertoire generated in these mutants is similar in size and diversity to that in WT mice, the evolutionary bias of TCRs for MHC is suggested by a shift and preferential use of some TCR subfamilies over others in mice expressing the mutant class II MHCs. Furthermore, T cells educated on these mutant MHC molecules are alloreactive to each other and to WT cells, and vice versa, suggesting strong functional differences among these repertoires. Taken together, these results highlight both the flexibility of thymic selection and the evolutionary bias of TCRs for MHC. PMID:27588903

  10. Class II major histocompatibility complex mutant mice to study the germ-line bias of T-cell antigen receptors

    PubMed Central

    Silberman, Daniel; Krovi, Sai Harsha; Tuttle, Kathryn D.; Crooks, James; Reisdorph, Richard; White, Janice; Gross, James; Matsuda, Jennifer L.; Gapin, Laurent; Marrack, Philippa; Kappler, John W.

    2016-01-01

    The interaction of αβ T-cell antigen receptors (TCRs) with peptides bound to MHC molecules lies at the center of adaptive immunity. Whether TCRs have evolved to react with MHC or, instead, processes in the thymus involving coreceptors and other molecules select MHC-specific TCRs de novo from a random repertoire is a longstanding immunological question. Here, using nuclease-targeted mutagenesis, we address this question in vivo by generating three independent lines of knockin mice with single-amino acid mutations of conserved class II MHC amino acids that often are involved in interactions with the germ-line–encoded portions of TCRs. Although the TCR repertoire generated in these mutants is similar in size and diversity to that in WT mice, the evolutionary bias of TCRs for MHC is suggested by a shift and preferential use of some TCR subfamilies over others in mice expressing the mutant class II MHCs. Furthermore, T cells educated on these mutant MHC molecules are alloreactive to each other and to WT cells, and vice versa, suggesting strong functional differences among these repertoires. Taken together, these results highlight both the flexibility of thymic selection and the evolutionary bias of TCRs for MHC. PMID:27588903

  11. Antigen nonspecific effect of major histocompatibility complex haplotype on autoantibody levels in systemic lupus erythematosus-prone lpr mice.

    PubMed Central

    Cohen, P L; Creech, E; Nakul-Aquaronne, D; McDaniel, R; Ackler, S; Rapoport, R G; Sobel, E S; Eisenberg, R A

    1993-01-01

    MHC-linked genes strongly influence susceptibility to autoimmune diseases and also regulate responses to exogenous antigens. To begin to understand the mechanism of this MHC effect on disease, we have investigated MHC-congenic mouse strains that develop spontaneous autoimmunity because of the lpr gene. C57BL6/lpr (B6/lpr) mice (H-2b) are known to have substantial levels of autoantibodies to chromatin, single stranded DNA (ssDNA3), and IgG of different murine subclasses (rheumatoid factor). We have crossed the H-2d and the H-2bm12 (la mutant) haplotypes onto the B6/lpr background. Surprisingly, levels of all the autoantibodies were markedly lower in B6/lpr.H-2d, but levels in B6/lpr.H-2bm12 were no different from those in B6/lpr mice. The downregulating influence of the H-2d allele was dominant, and there was no effect on autoantibody fine specificities. The genetics of the H-2d effect and its diffuse influence on multiple autoantibody specificities, in addition to the lack of effect of the bm12 mutation, which modifies the peptide-binding groove of I-A, together raise the question of whether MHC-linked genes other than classical (IR) genes may be responsible for MHC disease associations in this model. Images PMID:7685774

  12. Class II major histocompatibility complex mutant mice to study the germ-line bias of T-cell antigen receptors.

    PubMed

    Silberman, Daniel; Krovi, Sai Harsha; Tuttle, Kathryn D; Crooks, James; Reisdorph, Richard; White, Janice; Gross, James; Matsuda, Jennifer L; Gapin, Laurent; Marrack, Philippa; Kappler, John W

    2016-09-20

    The interaction of αβ T-cell antigen receptors (TCRs) with peptides bound to MHC molecules lies at the center of adaptive immunity. Whether TCRs have evolved to react with MHC or, instead, processes in the thymus involving coreceptors and other molecules select MHC-specific TCRs de novo from a random repertoire is a longstanding immunological question. Here, using nuclease-targeted mutagenesis, we address this question in vivo by generating three independent lines of knockin mice with single-amino acid mutations of conserved class II MHC amino acids that often are involved in interactions with the germ-line-encoded portions of TCRs. Although the TCR repertoire generated in these mutants is similar in size and diversity to that in WT mice, the evolutionary bias of TCRs for MHC is suggested by a shift and preferential use of some TCR subfamilies over others in mice expressing the mutant class II MHCs. Furthermore, T cells educated on these mutant MHC molecules are alloreactive to each other and to WT cells, and vice versa, suggesting strong functional differences among these repertoires. Taken together, these results highlight both the flexibility of thymic selection and the evolutionary bias of TCRs for MHC.

  13. A Novel Mechanism for Adenylyl Cyclase Inhibition from the Crystal Structure of its Complex with Catechol Estrogen

    SciTech Connect

    Steegborn,C.; Litvin, T.; Hess, K.; Capper, A.; Taussig, R.; Buck, J.; Levin, L.; Wu, H.

    2005-01-01

    Catechol estrogens are steroid metabolites that elicit physiological responses through binding to a variety of cellular targets. We show here that catechol estrogens directly inhibit soluble adenylyl cyclases and the abundant trans-membrane adenylyl cyclases. Catechol estrogen inhibition is non-competitive with respect to the substrate ATP, and we solved the crystal structure of a catechol estrogen bound to a soluble adenylyl cyclase from Spirulina platensis in complex with a substrate analog. The catechol estrogen is bound to a newly identified, conserved hydrophobic patch near the active center but distinct from the ATP-binding cleft. Inhibitor binding leads to a chelating interaction between the catechol estrogen hydroxyl groups and the catalytic magnesium ion, distorting the active site and trapping the enzyme substrate complex in a non-productive conformation. This novel inhibition mechanism likely applies to other adenylyl cyclase inhibitors, and the identified ligand-binding site has important implications for the development of specific adenylyl cyclase inhibitors.

  14. Adeno-Associated Virus Serotype 1 (AAV1)- and AAV5-Antibody Complex Structures Reveal Evolutionary Commonalities in Parvovirus Antigenic Reactivity

    PubMed Central

    Tseng, Yu-Shan; Gurda, Brittney L.; Chipman, Paul; McKenna, Robert; Afione, Sandra; Chiorini, John A.; Muzyczka, Nicholas; Olson, Norman H.; Baker, Timothy S.; Kleinschmidt, Jürgen

    2014-01-01

    ABSTRACT The clinical utility of the adeno-associated virus (AAV) gene delivery system has been validated by the regulatory approval of an AAV serotype 1 (AAV1) vector for the treatment of lipoprotein lipase deficiency. However, neutralization from preexisting antibodies is detrimental to AAV transduction efficiency. Hence, mapping of AAV antigenic sites and engineering of neutralization-escaping vectors are important for improving clinical efficacy. We report the structures of four AAV-monoclonal antibody fragment complexes, AAV1-ADK1a, AAV1-ADK1b, AAV5-ADK5a, and AAV5-ADK5b, determined by cryo-electron microscopy and image reconstruction to a resolution of ∼11 to 12 Å. Pseudoatomic modeling mapped the ADK1a epitope to the protrusions surrounding the icosahedral 3-fold axis and the ADK1b and ADK5a epitopes, which overlap, to the wall between depressions at the 2- and 5-fold axes (2/5-fold wall), and the ADK5b epitope spans both the 5-fold axis-facing wall of the 3-fold protrusion and portions of the 2/5-fold wall of the capsid. Combined with the six antigenic sites previously elucidated for different AAV serotypes through structural approaches, including AAV1 and AAV5, this study identified two common AAV epitopes: one on the 3-fold protrusions and one on the 2/5-fold wall. These epitopes coincide with regions with the highest sequence and structure diversity between AAV serotypes and correspond to regions determining receptor recognition and transduction phenotypes. Significantly, these locations overlap the two dominant epitopes reported for autonomous parvoviruses. Thus, rather than the amino acid sequence alone, the antigenic sites of parvoviruses appear to be dictated by structural features evolved to enable specific infectious functions. IMPORTANCE The adeno-associated viruses (AAVs) are promising vectors for in vivo therapeutic gene delivery, with more than 20 years of intense research now realized in a number of successful human clinical trials that

  15. Desensitization in vitro: the role of T-suppressor cells, T-suppressor factor and T-acceptor cells in the inhibition of the passive transfer of contact sensitivity to picryl chloride by exposure to antigen in vitro

    PubMed Central

    Zembala, M.; Asherson, G. L.; Colizzi, V.; Watkins, Madeleine C.

    1982-01-01

    This paper investigates desensitization in vitro, e.g. the inhibition of the transfer of contact sensitivity to picryl chloride by incubation of the passive transfer population with picrylated spleen cells. It asks whether desensitization is based on the same T-suppressor circuit which is responsible for the inhibition of passive transfer by antigen-specific T-suppressor factor (TsF). In this circuit, the T-suppressor cell which acts at the efferent stage (Ts-eff) makes TsF. This TsF depresses contact sensitivity indirectly by arming a T-acceptor cell (Tacc). The armed Tacc, when exposed to antigen (picrylated spleen cells), liberates a non-specific inhibitor which blocks the transfer of contact sensitivity. The three elements of this T-suppressor circuit occur in nylon wool-purified T cells prepared from the lymph nodes and spleens of mice four days after immunization with picryl chloride. This population transfers contact sensitivity and can be desensitized in vitro. It contains Ts-eff which can be isolated by panning (adherence) on picrylated albumin and detected by their ability to inhibit passive transfer. The 24 hr supernatant of cultures of these cells contains TsF. Finally the population contains Tacc which appear in the spleen 2 days after immunization and virtually disappear by 10 days. Further experiments demonstrated that the Ts-eff and the Tacc were not merely present but actually required for desensitization in vitro. Immune cells depleted of both Ts-eff (by panning on picrylated albumin) and Tacc (by arming with anti-oxazolone TsF and panning on oxazolonated albumin) cannot be desensitized. To restore desensitization both Ts-eff and Tacc must be added back. The Ts-eff were characterized as cyclophosphamide resistant, adult thymectomy sensitive cells (Cyr, ATxs), which adhered to antigen and were produced only by specific immunization. The Tacc were characterized as Cys, ATxs cells which adhered to antigen only after arming with antigen-specific T

  16. Structures of MART-126/27-35Peptide/HLA-A2 Complexes Reveal a Remarkable Disconnect between Antigen Structural Homology and T Cell Recognition

    SciTech Connect

    Borbulevych, Oleg Y; Insaidoo, Francis K; Baxter, Tiffany K; Powell, Jr., Daniel J.; Johnson, Laura A; Restifo, Nicholas P; Baker, Brian M

    2008-09-17

    Small structural changes in peptides presented by major histocompatibility complex (MHC) molecules often result in large changes in immunogenicity, supporting the notion that T cell receptors are exquisitely sensitive to antigen structure. Yet there are striking examples of TCR recognition of structurally dissimilar ligands. The resulting unpredictability of how T cells will respond to different or modified antigens impacts both our understanding of the physical bases for TCR specificity as well as efforts to engineer peptides for immunomodulation. In cancer immunotherapy, epitopes and variants derived from the MART-1/Melan-A protein are widely used as clinical vaccines. Two overlapping epitopes spanning amino acid residues 26 through 35 are of particular interest: numerous clinical studies have been performed using variants of the MART-1 26-35 decamer, although only the 27-35 nonamer has been found on the surface of targeted melanoma cells. Here, we show that the 26-35 and 27-35 peptides adopt strikingly different conformations when bound to HLA-A2. Nevertheless, clonally distinct MART-1{sub 26/27-35}-reactive T cells show broad cross-reactivity towards these ligands. Simultaneously, however, many of the cross-reactive T cells remain unable to recognize anchor-modified variants with very subtle structural differences. These dichotomous observations challenge our thinking about how structural information on unligated peptide/MHC complexes should be best used when addressing questions of TCR specificity. Our findings also indicate that caution is warranted in the design of immunotherapeutics based on the MART-1 26/27-35 epitopes, as neither cross-reactivity nor selectivity is predictable based on the analysis of the structures alone.

  17. Structures of a pan-specific antagonist antibody complexed to different isoforms of TGFβ reveal structural plasticity of antibody-antigen interactions.

    PubMed

    Moulin, Aaron; Mathieu, Magali; Lawrence, Catherine; Bigelow, Russell; Levine, Mark; Hamel, Christine; Marquette, Jean-Piere; Le Parc, Josiane; Loux, Christophe; Ferrari, Paul; Capdevila, Cecile; Dumas, Jacques; Dumas, Bruno; Rak, Alexey; Bird, Julie; Qiu, Huawei; Pan, Clark Q; Edmunds, Tim; Wei, Ronnie R

    2014-12-01

    Various important biological pathways are modulated by TGFβ isoforms; as such they are potential targets for therapeutic intervention. Fresolimumab, also known as GC1008, is a pan-TGFβ neutralizing antibody that has been tested clinically for several indications including an ongoing trial for focal segmental glomerulosclerosis. The structure of the antigen-binding fragment of fresolimumab (GC1008 Fab) in complex with TGFβ3 has been reported previously, but the structural capacity of fresolimumab to accommodate tight interactions with TGFβ1 and TGFβ2 was insufficiently understood. We report the crystal structure of the single-chain variable fragment of fresolimumab (GC1008 scFv) in complex with target TGFβ1 to a resolution of 3.00 Å and the crystal structure of GC1008 Fab in complex with TGFβ2 to 2.83 Å. The structures provide further insight into the details of TGFβ recognition by fresolimumab, give a clear indication of the determinants of fresolimumab pan-specificity and provide potential starting points for the development of isoform-specific antibodies using a fresolimumab scaffold.

  18. Isolation and characterization of a membrane protein from rat erythrocytes which inhibits lysis by the membrane attack complex of rat complement.

    PubMed Central

    Hughes, T R; Piddlesden, S J; Williams, J D; Harrison, R A; Morgan, B P

    1992-01-01

    The membrane attack complex (MAC) of complement in humans is regulated by several membrane-bound proteins; however, no such proteins have so far been described in other species. Here we report the isolation and characterization of a rat erythrocyte membrane glycoprotein of molecular mass 21 kDa which inserts into cell membranes and is a potent inhibitor of the rat MAC. This protein, here called rat inhibitory protein (RIP), was first partially purified by column chromatography from a butanol extract of rat erythrocyte membranes. Monoclonal antibodies (Mabs) were raised against RIP and used for its affinity purification. Affinity-purified RIP was shown to inhibit in a dose-dependent manner the cobra venom factor (CVF)-mediated 'reactive' lysis of guinea pig erythrocytes by rat complement. Conversely, the anti-RIP MAbs 6D1 and TH9 were shown to markedly enhance the CVF-mediated lysis of rat erythrocytes by rat complement. RIP acted late in the assembly of the MAC (at or after the C5b-8 stage) and was releasable from the membranes of rat erythrocytes by phosphatidylinositol-specific phospholipase C. These features, together with its size, deglycosylation pattern and N-terminal amino acid sequence, lead us to conclude that RIP is the rat homologue of the human MAC-inhibitory protein CD59 antigen. Images Fig. 1. Fig. 3. PMID:1376109

  19. Complex preimplantation genetic diagnosis for beta-thalassaemia, sideroblastic anaemia, and human leukocyte antigen (HLA)-typing.

    PubMed

    Kakourou, Georgia; Vrettou, Christina; Kattamis, Antonis; Destouni, Aspasia; Poulou, Myrto; Moutafi, Maria; Kokkali, Georgia; Pantos, Konstantinos; Davies, Stephen; Kitsiou-Tzeli, Sophia; Kanavakis, Emmanuel; Traeger-Synodinos, Joanne

    2016-01-01

    Preimplantation genetic diagnosis (PGD) to select histocompatible siblings to facilitate curative haematopoeitic stem-cell transplantation (HSCT) is now an acceptable option in the absence of an available human leukocyte antigen (HLA) compatible donor. We describe a case where the couple who requested HLA-PGD, were both carriers of two serious haematological diseases, beta-thalassaemia and sideroblastic anaemia. Their daughter, affected with sideroblastic anaemia, was programmed to have HSCT. A multiplex-fluorescent-touchdown-PCR protocol was optimized for the simultaneous amplification of: the two HBB-gene mutated regions (c.118C> T, c.25-26delAA), four short tandem repeats (STRs) in chr11p15.5 linked to the HBB gene, the SLC25A38 gene mutation (c.726C > T), two STRs in chr3p22.1 linked to the SLC25A38 gene, plus eleven informative STRs for HLA-haplotyping (chr6p22.1-21.3). This was followed by real-time nested PCR and high-resolution melting analysis (HRMA) for the detection of HBB and SLC25A38 gene mutations, as well as the analysis of all STRs on an automatic genetic analyzer (sequencer). The couple completed four clinical in vitro fertilization (IVF)/PGD cycles. At least one matched unaffected embryo was identified and transferred in each cycle. A twin pregnancy was established in the fourth PGD cycle and genotyping results at all loci were confirmed by prenatal diagnosis. Two healthy baby girls were delivered at week 38 of pregnancy. The need to exclude two familial disorders for HLA-PGD is rarely encountered. The methodological approach described here is fast, accurate, clinically-validated, and of relatively low cost. PMID:26636621

  20. Polyphosphates form antigenic complexes with platelet factor 4 (PF4) and enhance PF4-binding to bacteria.

    PubMed

    Brandt, Sven; Krauel, Krystin; Jaax, Miriam; Renné, Thomas; Helm, Christiane A; Hammerschmidt, Sven; Delcea, Mihaela; Greinacher, Andreas

    2015-11-25

    Short chain polyphosphates (polyP) are pro-coagulant and pro-inflammatory platelet released inorganic polymers. The platelet chemokine platelet factor 4 (PF4) binds to lipid A on bacteria, inducing an antibody mediated host defense mechanism, which can be misdirected against PF4/heparin complexes leading to the adverse drug reaction heparin-induced thrombocytopenia (HIT). Here, we demonstrate that PF4 complex formation with soluble short chain polyP contributes to host defense mechanisms. Circular dichroism spectroscopy and isothermal titration calorimetry revealed that PF4 changed its structure upon binding to polyP in a similar way as seen in PF4/heparin complexes. Consequently, PF4/polyP complexes exposed neoepitopes to which human anti-PF4/heparin antibodies bound. PolyP enhanced binding of PF4 to Escherichia coli, hereby facilitating bacterial opsonisation and, in the presence of human anti-PF4/polyanion antibodies, phagocytosis. Our study indicates a role of polyP in enhancing PF4-mediated defense mechanisms of innate immunity.

  1. Structures and contribution to the antigenicity of oligosaccharides of Japanese cedar (Cryptomeria japonica) pollen allergen Cry j I: relationship between the structures and antigenic epitopes of plant N-linked complex-type glycans.

    PubMed

    Ogawa, H; Hijikata, A; Amano, M; Kojima, K; Fukushima, H; Ishizuka, I; Kurihara, Y; Matsumoto, I

    1996-08-01

    The oligosaccharide structures of Cry j I, a major allergenic glycoprotein of Cryptomeria japonica (Japanese cedar, sugi), were analysed by 400 MHz 1H-NMR and two-dimensional sugar mapping analyses. The four major fractions comprised a series of biantennary complex type N-linked oligosaccharides that share a fucose/xylose-containing core and glucosamine branches including a novel structure with a nongalactosylated fucosylglucosamine branch. Rabbit polyclonal anti-Cry j I IgG antibodies cross-reacted with three different plant glycoproteins having the same or shorter N-linked oligosaccharides as Cry j I. ELISA and ELISA inhibition studies with intact glycoproteins, glycopeptides and peptides indicated that both anti-Cry j I IgGs and anti-Sophora japonica bark lectin II (B-SJA-II) IgGs included oligosaccharide-specific antibodies with different specificities, and that the epitopic structures against anti-Cry j I IgGs include a branch containing alpha 1-6 linked fucose and a core containing fucose/xylose, while those against anti-B-SJA-II IgGs include nonreducing terminal mannose residues. The cross-reactivities of human allergic sera to miraculin and Clerodendron Trichotomum lectin (CTA) were low, and inhibition studies suggested that the oligosaccharides on Cry j I contribute little or only conformationally to the reactivity of specific IgE antibodies. PMID:8872112

  2. Presentation of hepatocellular antigens

    PubMed Central

    Grakoui, Arash; Crispe, Ian Nicholas

    2016-01-01

    The liver is an organ in which antigen-specific T-cell responses manifest a bias toward immune tolerance. This is clearly seen in the rejection of allogeneic liver transplants, and multiple other phenomena suggest that this effect is more general. These include tolerance toward antigens introduced via the portal vein, immune failure to several hepatotropic viruses, the lack of natural liver-stage immunity to malaria parasites, and the frequent metastasis of cancers to the liver. Here we review the mechanisms by which T cells engage with hepatocellular antigens, the context in which such encounters occur, and the mechanisms that act to suppress a full T-cell response. While many mechanisms play a role, we will argue that two important processes are the constraints on the cross-presentation of hepatocellular antigens, and the induction of negative feedback inhibition driven by interferons. The constant exposure of the liver to microbial products from the intestine may drive innate immunity, rendering the local environment unfavorable for specific T-cell responses through this mechanism. Nevertheless, tolerance toward hepatocellular antigens is not monolithic and under specific circumstances allows both effective immunity and immunopathology. PMID:26924525

  3. Characterization of antigen association with accessory cells: specific removal of processed antigens from the cell surface by phospholipases.

    PubMed Central

    Falo, L D; Haber, S I; Herrmann, S; Benacerraf, B; Rock, K L

    1987-01-01

    To characterize the basis for the cell surface association of processed antigen with the antigen-presenting cell (APC) we analyzed its sensitivity to enzymatic digestion. Antigen-exposed APC that are treated with phospholipase and then immediately fixed lose their ability to stimulate antigen-plus-Ia-specific T-T hybridomas. This effect is seen with highly purified phospholipase A2 and phospholipase C. In addition it is observed with three distinct antigens--ovalbumin, bovine insulin, and poly(LGlu56LLys35LPhe9) [(GluLysPhe)n]. The effect of phospholipases is highly specific. Identically treated APC are equivalent to controls in their ability to stimulate alloreactive hybridomas specific for precisely the same Ia molecule that is corecognized by antigen-plus-Ia-specific hybrids. Furthermore, the antigen-presenting function of enzyme-treated, fixed APC can be reconstituted by the addition of exogenous in vitro processed or "processing independent" antigens. In parallel studies 125I-labeled avidin was shown to specifically bind to APC that were previously exposed and allowed to process biotin-insulin. Biotin-insulin-exposed APC that are pretreated with phospholipase bind significantly less 125I-labeled avidin than do untreated, exposed APC. Identical enzyme treatment does not reduce the binding of avidin to a biotinylated antibody already bound to class II major histocompatibility complex molecules of APC. At least some of the biotin-insulin surface sites are immunologically relevant, because the presentation of processed biotin-insulin by fixed APC is blocked by avidin. This effect is specific. Avidin binding to biotin-insulin-exposed APC does not inhibit allospecific stimulation nor the presentation of unconjugated insulin. These studies demonstrate that phospholipase effectively removes processed cell surface antigen. PMID:3467371

  4. The structure of a furin-antibody complex explains non-competitive inhibition by steric exclusion of substrate conformers

    PubMed Central

    Dahms, Sven O.; Creemers, John W. M.; Schaub, Yvonne; Bourenkov, Gleb P.; Zögg, Thomas; Brandstetter, Hans; Than, Manuel E.

    2016-01-01

    Proprotein Convertases (PCs) represent highly selective serine proteases that activate their substrates upon proteolytic cleavage. Their inhibition is a promising strategy for the treatment of cancer and infectious diseases. Inhibitory camelid antibodies were developed, targeting the prototypical PC furin. Kinetic analyses of them revealed an enigmatic non-competitive mechanism, affecting the inhibition of large proprotein-like but not small peptidic substrates. Here we present the crystal structures of furin in complex with the antibody Nb14 and of free Nb14 at resolutions of 2.0 Å and 2.3 Å, respectively. Nb14 binds at a site distant to the substrate binding pocket to the P-domain of furin. Interestingly, no major conformational changes were observed upon complex formation, neither for the protease nor for the antibody. Inhibition of furin by Nb14 is instead explained by steric exclusion of specific substrate conformers, explaining why Nb14 inhibits the processing of bulky protein substrates but not of small peptide substrates. This mode of action was further supported by modelling studies with the ternary factor X-furin-antibody complex and a mutation that disrupted the interaction interface between furin and the antibody. The observed binding mode of Nb14 suggests a novel approach for the development of highly specific antibody-based proprotein convertase inhibitors. PMID:27670069

  5. Heterometallic titanium-gold complexes inhibit renal cancer cells in vitro and in vivo‡

    PubMed Central

    Sadhukha, Tanmoy; Prabha, Swayam; Sanaú, Mercedes; Rotenberg, Susan A.

    2016-01-01

    Following recent work on heterometallic titanocene-gold complexes as potential chemotherapeutics for renal cancer, we report here on the synthesis, characterization and stability studies of new titanocene complexes containing a methyl group and a carboxylate ligand (mba = S-C6H4-COO−) bound to gold(I)-phosphane fragments through a thiolate group ([(η-C5H5)2TiMe(μ-mba)Au(PR3)]. The compounds are more stable in physiological media than those previously reported and are highly cytotoxic against human cancer renal cell lines. We describe here preliminary mechanistic data involving studies on the interaction of selected compounds with plasmid (pBR322) DNA used as a model nucleic acid, and with selected protein kinases from a panel of 35 protein kinases having oncological interest. Preliminary mechanistic studies in Caki-1 renal cells indicate that the cytotoxic and anti-migration effects of the most active compound 5 ([(η-C5H5)2TiMe(μ-mba)Au(PPh3)] involve inhibition of thioredoxin reductase and loss of expression of protein kinases that drive cell migration (AKT, p90-RSK, and MAPKAPK3). The co-localization of both titanium and gold metals (1:1 ratio) in Caki-1 renal cells was demonstrated for 5 indicating the robustness of the heterometallic compound in vitro. Two compounds were selected for further in vivo studies on mice based on their selectivity in vitro against renal cancer cell lines when compared to non-tumorigenic human kidney cell lines (HEK-293T and RPTC) and the favourable preliminary toxicity profile in C57BL/6 mice. Evaluation of Caki-1 xenografts in NOD.CB17-Prkdc SCID/J mice showed an impressive tumor reduction (67%) after treatment for 28 days (3 mg/kg/every other day) with heterometallic compound 5 as compared with the previously described [(η-C5H5)2Ti{OC(O)-4-C6H4-P(Ph2)AuCI}2] 3 which was non-inhibitory. These findings indicate that structural modifications on the ligand scaffold affect the in vivo efficacy of this class of compounds. PMID

  6. Effect of oil overlay on inhibition potential of roscovitine in sheep cumulus-oocyte complexes.

    PubMed

    Crocomo, L F; Marques Filho, W C; Ulian, C M V; Branchini, N S; Silva, D T; Ackermann, C L; Landim-Alvarenga, F C; Bicudo, S D

    2015-06-01

    Inhibitors of cyclin-dependent kinases, as roscovitine, have been used to prevent the spontaneous resumption of meiosis in vitro and to improve the oocyte developmental competence. In this study, the interference of oil overlay on the reversible arrest capacity of roscovitine in sheep oocytes as well as its effects on cumulus expansion was evaluated. For this, cumulus-oocyte complexes (COCs) were cultured for 20 h in TCM 199 with 10% foetal bovine serum (Control) containing 75 μm roscovitine (Rosco). Subsequently, they were in vitro matured (IVM) for further 18 h in inhibitor-free medium with LH and FSH. The culture was performed in Petri dishes under mineral oil (+) or in 96 well plates without oil overlay (-) at 38.5°C and 5% CO2 . At 20 and 38 h, the cumulus expansion and nuclear maturation were evaluated under stereomicroscope and by Hoechst 33342 staining, respectively. No group presented cumulus expansion at 20 h. After additional culture with gonadotrophins, a significant rate of COCs from both Control groups (+/-) exhibited total expansion while in both Rosco groups (+/-) the partial expansion prevailed. Among the oocytes treated with roscovitine, 65.2% were kept at GV in the absence of oil overlay while 40.6% of them reached MII under oil cover (p < 0.05). This meiotic arrest was reversible, and proper meiosis progression also occurred in the Control groups (+/-). So, the culture system without oil overlay improved the meiotic inhibition promoted by roscovitine without affecting the cumulus expansion rate or the subsequent meiosis progression.

  7. The pro-oxidant chromium(VI) inhibits mitochondrial complex I, complex II, and aconitase in the bronchial epithelium: EPR markers for Fe-S proteins

    PubMed Central

    Myers, Charles R.; Antholine, William E.; Myers, Judith M.

    2010-01-01

    Hexavalent chromium [Cr(VI)] compounds (e.g. chromates) are strong oxidants that readily enter cells where they are reduced to reactive Cr species that also facilitate reactive oxygen species (ROS) generation. Recent studies demonstrated inhibition and oxidation of the thioredoxin system, with greater effects on mitochondrial thioredoxin (Trx2). This implies that Cr(VI)-induced oxidant stress may be especially directed at the mitochondria. Examination of other redox-sensitive mitochondrial functions showed that Cr(VI) treatments that cause Trx2 oxidation in human bronchial epithelial cells also result in pronounced and irreversible inhibition of aconitase, a TCA cycle enzyme that has an iron-sulfur (Fe-S) center that is labile with respect to certain oxidants. The activities of electron transport complexes I and II were also inhibited, whereas complex III was not. Electron paramagnetic resonance (EPR) studies of samples at liquid helium temperature (10 K) showed a strong signal at g = 1.94 that is consistent with the inhibition of electron flow through complexes I and/or II. A signal at g = 2.02 was also observed which is consistent with oxidation of the Fe-S center of aconitase. The g = 1.94 signal was particularly intense and remained after extracellular Cr(VI) was removed, whereas the g = 2.02 signal declined in intensity after Cr(VI) was removed. A similar inhibition of these activities and analogous EPR findings were noted in bovine airways treated ex vivo with Cr(VI). Overall, the data support the hypothesis that Cr(VI) exposure has deleterious effects on a number of redox-sensitive core mitochondrial proteins. The g = 1.94 signal could prove to be an important biomarker for oxidative damage resulting from Cr(VI) exposure. The EPR spectra simultaneously showed signals for Cr(V) and Cr(III) which verify Cr(VI) exposure and its intracellular reductive activation. PMID:20883776

  8. Antigens and allergic asthma

    SciTech Connect

    Reed, C.E.; Swanson, M.C.

    1987-06-01

    There are few reliable epidemiologic data on the overall frequency and importance of allergy. We describe a practical method for quantifying the concentration of both amorphous and morphologically defined antigens in the air. A high volume air sampler is used to collect airborne particles and has a facility to separate samples into different particle sizes. Samples are tested for allergenic activity by radioallergosorbent test inhibition assay. Preliminary findings from studies of community wide, amorphous and common household allergens are reported.

  9. Efficient convergent synthesis of bi-, tri-, and tetra-antennary complex type N-glycans and their HIV-1 antigenicity.

    PubMed

    Shivatare, Sachin S; Chang, Shih-Huang; Tsai, Tsung-I; Ren, Chien-Tai; Chuang, Hong-Yang; Hsu, Li; Lin, Chih-Wei; Li, Shiou-Ting; Wu, Chung-Yi; Wong, Chi-Huey

    2013-10-16

    The structural diversity of glycoproteins often comes from post-translational glycosylation with heterogeneous N-glycans. Understanding the complexity of glycans related to various biochemical processes demands a well-defined synthetic sugar library. We report herein a unified convergent strategy for the rapid production of bi-, tri-, and tetra-antennary complex type N-glycans with and without terminal N-acetylneuraminic acid residues connected via the α-2,6 or α-2,3 linkages. Moreover, using sialyltransferases to install sialic acid can minimize synthetic steps through the use of shared intermediates to simplify the complicated procedures associated with conventional sialic acid chemistry. Furthermore, these synthetic complex oligosaccharides were compiled to create a glycan array for the profiling of HIV-1 broadly neutralizing antibodies PG9 and PG16 that were isolated from HIV infected donors. From the study of antibody PG16, we identified potential natural and unnatural glycan ligands, which may facilitate the design of carbohydrate-based immunogens and hasten the HIV vaccine development.

  10. Bm-CPI-2, a cystatin from Brugia malayi nematode parasites, differs from Caenorhabditis elegans cystatins in a specific site mediating inhibition of the antigen-processing enzyme AEP.

    PubMed

    Murray, Janice; Manoury, Bénédicte; Balic, Adam; Watts, Colin; Maizels, Rick M

    2005-02-01

    The filarial parasite Brugia malayi survives for many years in the human lymphatic system. One immune evasion mechanism employed by Brugia is thought to be the release of cysteine protease inhibitors (cystatins), and we have previously shown that the recombinant cystatin Bm-CPI-2 interferes with protease-dependent antigen processing in the MHC class II antigen presentation pathway. Analogy with vertebrate cystatins suggested that Bm-CPI-2 is bi-functional, with one face of the protein blocking papain-like proteases, and the other able to inhibit legumains such as asparaginyl endopeptidase (AEP). Site-directed mutagenesis was carried out on Bm-CPI-2 at Asn-77, the residue on which AEP inhibition is dependent in vertebrate homologues. Two mutations at this site (to Asp and Lys) showed 10-fold diminished and ablated activity respectively, in assays of AEP inhibition, while blocking of papain-like proteases was reduced by only a small degree. Comparison of the B. malayi cystatins with two homologues encoded by the free-living model organism, Caenorhabditis elegans, suggested that while the papain site may be intact, the AEP site would not be functional. This supposition was tested with recombinant C. elegans proteins, Ce-CPI-1 (K08B4.6) and Ce-CPI-2 (R01B10.1), both of which block cathepsins and neither of which possess the ability to block AEP. Thus, Brugia CPI-2 may have convergently evolved to inhibit an enzyme important only in the mammalian environment.

  11. T−B+NK+ severe combined immunodeficiency caused by complete deficiency of the CD3ζ subunit of the T-cell antigen receptor complex

    PubMed Central

    Lauritsen, Jens Peter H.; Cooney, Myriah; Parrott, Roberta E.; Sajaroff, Elisa O.; Win, Chan M.; Keller, Michael D.; Carpenter, Jeffery H.; Carabana, Juan; Krangel, Michael S.; Sarzotti, Marcella; Zhong, Xiao-Ping; Wiest, David L.; Buckley, Rebecca H.

    2007-01-01

    CD3ζ is a subunit of the T-cell antigen receptor (TCR) complex required for its assembly and surface expression that also plays an important role in TCR-mediated signal transduction. We report here a patient with T−B+NK+ severe combined immunodeficiency (SCID) who was homozygous for a single C insertion following nucleotide 411 in exon 7 of the CD3ζ gene. The few T cells present contained no detectable CD3ζ protein, expressed low levels of cell surface CD3ε, and were nonfunctional. CD4+CD8−CD3εlow, CD4−CD8+CD3εlow, and CD4−CD8−CD3εlow cells were detected in the periphery, and the patient also exhibited an unusual population of CD56−CD16+ NK cells with diminished cytolytic activity. Additional studies demonstrated that retrovirally transduced patient mutant CD3ζ cDNA failed to rescue assembly of nascent complete TCR complexes or surface TCR expression in CD3ζ-deficient MA5.8 murine T-cell hybridoma cells. Nascent transduced mutant CD3ζ protein was also not detected in metabolically labeled MA5.8 cells, suggesting that it was unstable and rapidly degraded. Taken together, these findings provide the first demonstration that complete CD3ζ deficiency in humans can cause SCID by preventing normal TCR assembly and surface expression. PMID:17170122

  12. Activation of CD8-dependent cytotoxic T lymphocyte adhesion and degranulation by peptide class I antigen complexes.

    PubMed

    Kane, K P; Mescher, M F

    1993-06-01

    Activation of CTL requires engagement of both the TCR and the CD8 coreceptor. Immobilized class I proteins and in vitro-formed peptide class I Ag complexes have been used to examine the relative contributions of TCR and CD8 to the adhesion and response of cloned, class I-restricted CTL. The extent of degranulation was found to be directly proportional to the concentration of peptide used to pulse class I, suggesting that activation is a direct function of TCR occupancy level. In contrast, activation of degranulation as a function of the amount of class I on the surface displayed a marked threshold density dependence. Essentially the same density dependence was found for the response of CTL to fluid phase anti-TCR mAb and non-Ag class I, indicating that CD8-class I interaction must exceed a threshold before effective cosignaling can occur. Adhesion and degranulation of CTL was minimal in response to in vitro peptide-class I complexes prepared at a class I density below the threshold. However, the same density of peptide class I initiated both adhesion and response if additional non-Ag class I was coimmobilized on the same surface at levels above threshold. Thus, when surface levels of peptide class I complex are low, as is likely to be the case under physiologic conditions, the level of TCR occupancy achieved is, by itself, insufficient to mediate cell adhesion or activate degranulation. The results demonstrate, however, that low TCR occupancy is sufficient to provide the signal to prime CD8. Provided that the surface density of class I is sufficiently high, CD8 then mediates strong adhesion and provides the costimulatory signal(s) to activate response.

  13. Salivary Antigen-5/CAP Family Members Are Cu2+-dependent Antioxidant Enzymes That Scavenge O2⨪ and Inhibit Collagen-induced Platelet Aggregation and Neutrophil Oxidative Burst*

    PubMed Central

    Assumpção, Teresa C. F.; Ma, Dongying; Schwarz, Alexandra; Reiter, Karine; Santana, Jaime M.; Andersen, John F.; Ribeiro, José M. C.; Nardone, Glenn; Yu, Lee L.; Francischetti, Ivo M. B.

    2013-01-01

    The function of the antigen-5/CAP family of proteins found in the salivary gland of bloodsucking animals has remained elusive for decades. Antigen-5 members from the hematophagous insects Dipetalogaster maxima (DMAV) and Triatoma infestans (TIAV) were expressed and discovered to attenuate platelet aggregation, ATP secretion, and thromboxane A2 generation by low doses of collagen (<1 μg/ml) but no other agonists. DMAV did not interact with collagen, glycoprotein VI, or integrin α2β1. This inhibitory profile resembles the effects of antioxidants Cu,Zn-superoxide dismutase (Cu,Zn-SOD) in platelet function. Accordingly, DMAV was found to inhibit cytochrome c reduction by O2⨪ generated by the xanthine/xanthine oxidase, implying that it exhibits antioxidant activity. Moreover, our results demonstrate that DMAV blunts the luminescence signal of O2⨪ generated by phorbol 12-myristate 13-acetate-stimulated neutrophils. Mechanistically, inductively coupled plasma mass spectrometry and fluorescence spectroscopy revealed that DMAV, like Cu,Zn-SOD, interacts with Cu2+, which provides redox potential for catalytic removal of O2⨪. Notably, surface plasmon resonance experiments (BIAcore) determined that DMAV binds sulfated glycosaminoglycans (e.g. heparin, KD ∼100 nmol/liter), as reported for extracellular SOD. Finally, fractions of the salivary gland of D. maxima with native DMAV contain Cu2+ and display metal-dependent antioxidant properties. Antigen-5/CAP emerges as novel family of Cu2+-dependent antioxidant enzymes that inhibit neutrophil oxidative burst and negatively modulate platelet aggregation by a unique salivary mechanism. PMID:23564450

  14. Domain interactions control complex formation and polymerase specificity in the biosynthesis of the Escherichia coli O9a antigen.

    PubMed

    Liston, Sean D; Clarke, Bradley R; Greenfield, Laura K; Richards, Michele R; Lowary, Todd L; Whitfield, Chris

    2015-01-01

    The Escherichia coli O9a O-polysaccharide (O-PS) is a prototype for bacterial glycan synthesis and export by an ATP-binding cassette transporter-dependent pathway. The O9a O-PS possesses a tetrasaccharide repeat unit comprising two α-(1→2)- and two α-(1→3)-linked mannose residues and is extended on a polyisoprenoid lipid carrier by the action of a polymerase (WbdA) containing two glycosyltransferase active sites. The N-terminal domain of WbdA possesses α-(1→2)-mannosyltransferase activity, and we demonstrate in this study that the C-terminal domain is an α-(1→3)-mannosyltransferase. Previous studies established that the size of the O9a polysaccharide is determined by the chain-terminating dual kinase/methyltransferase (WbdD) that is tethered to the membrane and recruits WbdA into an active enzyme complex by protein-protein interactions. Here, we used bacterial two-hybrid analysis to identify a surface-exposed α-helix in the C-terminal mannosyltransferase domain of WbdA as the site of interaction with WbdD. However, the C-terminal domain was unable to interact with WbdD in the absence of its N-terminal partner. Through deletion analysis, we demonstrated that the α-(1→2)-mannosyltransferase activity of the N-terminal domain is regulated by the activity of the C-terminal α-(1→3)-mannosyltransferase. In mutants where the C-terminal catalytic site was deleted but the WbdD-interaction site remained, the N-terminal mannosyltransferase became an unrestricted polymerase, creating a novel polymer comprising only α-(1→2)-linked mannose residues. The WbdD protein therefore orchestrates critical localization and coordination of activities involved in chain extension and termination. Complex domain interactions are needed to position the polymerase components appropriately for assembly into a functional complex located at the cytoplasmic membrane. PMID:25422321

  15. Electrochemical Study on the Inhibition Effect of Phenanthroline and Its Cobalt Complex as Corrosion Inhibitors for Mild Steel

    NASA Astrophysics Data System (ADS)

    Liu, Xia; Okafor, Peter C.; Jiang, Bin; Hu, Hongxiang; Zheng, Yugui

    2015-09-01

    The corrosion inhibition effect of phenanthroline (Phen) and its cobalt complex (CoPhen) on the corrosion of carbon steel in sulphuric acid solutions was studied using potentiodynamic polarization and electrochemical impedance spectroscopy techniques at 20, 30, and 40 °C. Scanning electron microscopy techniques were used to characterize the mild steel surface. The results indicate that the compounds inhibit the corrosion of mild steel in H2SO4 solutions through a predominant physical adsorption following the Langmuir adsorption isotherm. Polarization curves suggest that the complex and ligand are mixed-type inhibitors. The efficiency of the inhibitors is concentration- and temperature-dependent and follows the trend CoPhen > Phen.

  16. HIV-1 Vpr Protein Inhibits Telomerase Activity via the EDD-DDB1-VPRBP E3 Ligase Complex*

    PubMed Central

    Wang, Xin; Singh, Shailbala; Jung, Hae-Yun; Yang, Guojun; Jun, Sohee; Sastry, K. Jagannadha; Park, Jae-Il

    2013-01-01

    Viral pathogens utilize host cell machinery for their benefits. Herein, we identify that HIV-1 Vpr (viral protein R) negatively modulates telomerase activity. Telomerase enables stem and cancer cells to evade cell senescence by adding telomeric sequences to the ends of chromosomes. We found that Vpr inhibited telomerase activity by down-regulating TERT protein, a catalytic subunit of telomerase. As a molecular adaptor, Vpr enhanced the interaction between TERT and the VPRBP substrate receptor of the DYRK2-associated EDD-DDB1-VPRBP E3 ligase complex, resulting in increased ubiquitination of TERT. In contrast, the Vpr mutant identified in HIV-1-infected long-term nonprogressors failed to promote TERT destabilization. Our results suggest that Vpr inhibits telomerase activity by hijacking the host E3 ligase complex, and we propose the novel molecular mechanism of telomerase deregulation in possibly HIV-1 pathogenesis. PMID:23612978

  17. A Small Molecule Inhibitor of Polycomb Repressive Complex 1 Inhibits Ubiquitin Signaling at DNA Double-strand Breaks*

    PubMed Central

    Ismail, Ismail Hassan; McDonald, Darin; Strickfaden, Hilmar; Xu, Zhizhong; Hendzel, Michael J.

    2013-01-01

    Polycomb-repressive complex 1 (PRC1)-mediated histone ubiquitylation plays an important role in aberrant gene silencing in human cancers and is a potential target for cancer therapy. Here we show that 2-pyridine-3-yl-methylene-indan-1,3-dione (PRT4165) is a potent inhibitor of PRC1-mediated H2A ubiquitylation in vivo and in vitro. The drug also inhibits the accumulation of all detectable ubiquitin at sites of DNA double-strand breaks (DSBs), the retention of several DNA damage response proteins in foci that form around DSBs, and the repair of the DSBs. In vitro E3 ubiquitin ligase activity assays revealed that PRT4165 inhibits both RNF2 and RING 1A, which are partially redundant paralogues that together account for the E3 ubiquitin ligase activity found in PRC1 complexes, but not RNF8 nor RNF168. Because ubiquitylation is completely inhibited despite the efficient recruitment of RNF8 to DSBs, our results suggest that PRC1-mediated monoubiquitylation is required for subsequent RNF8- and/or RNF168-mediated polyubiquitylation. Our results demonstrate the unique feature of PRT4165 as a novel chromatin-remodeling compound and provide a new tool for the inhibition of ubiquitylation signaling at DNA double-strand breaks. PMID:23902761

  18. Concepts and applications for influenza antigenic cartography

    PubMed Central

    Cai, Zhipeng; Zhang, Tong; Wan, Xiu-Feng

    2011-01-01

    Influenza antigenic cartography projects influenza antigens into a two or three dimensional map based on immunological datasets, such as hemagglutination inhibition and microneutralization assays. A robust antigenic cartography can facilitate influenza vaccine strain selection since the antigenic map can simplify data interpretation through intuitive antigenic map. However, antigenic cartography construction is not trivial due to the challenging features embedded in the immunological data, such as data incompleteness, high noises, and low reactors. To overcome these challenges, we developed a computational method, temporal Matrix Completion-Multidimensional Scaling (MC-MDS), by adapting the low rank MC concept from the movie recommendation system in Netflix and the MDS method from geographic cartography construction. The application on H3N2 and 2009 pandemic H1N1 influenza A viruses demonstrates that temporal MC-MDS is effective and efficient in constructing influenza antigenic cartography. The web sever is available at http://sysbio.cvm.msstate.edu/AntigenMap. PMID:21761589

  19. The PD-1/PD-L1 complex resembles the antigen-binding Fv domains of antibodies and T cell receptors

    SciTech Connect

    Lin, David Yin-wei; Tanaka, Yoshimasa; Iwasaki, Masashi; Gittis, Apostolos G.; Su, Hua-Poo; Mikami, Bunzo; Okazaki, Taku; Honjo, Tasuku; Minato, Nagahiro; Garboczi, David N.

    2008-07-29

    Signaling through the programmed death 1 (PD-1) inhibitory receptor upon binding its ligand, PD-L1, suppresses immune responses against autoantigens and tumors and plays an important role in the maintenance of peripheral immune tolerance. Release from PD-1 inhibitory signaling revives 'exhausted' virus-specific T cells in chronic viral infections. Here we present the crystal structure of murine PD-1 in complex with human PD-L1. PD-1 and PD-L1 interact through the conserved front and side of their Ig variable (IgV) domains, as do the IgV domains of antibodies and T cell receptors. This places the loops at the ends of the IgV domains on the same side of the PD-1/PD-L1 complex, forming a surface that is similar to the antigen-binding surface of antibodies and T cell receptors. Mapping conserved residues allowed the identification of residues that are important in forming the PD-1/PD-L1 interface. Based on the structure, we show that some reported loss-of-binding mutations involve the PD-1/PD-L1 interaction but that others compromise protein folding. The PD-1/PD-L1 interaction described here may be blocked by antibodies or by designed small-molecule drugs to lower inhibitory signaling that results in a stronger immune response. The immune receptor-like loops offer a new surface for further study and potentially the design of molecules that would affect PD-1/PD-L1 complex formation and thereby modulate the immune response.

  20. Complex inhibition of tyrosinase by thiol-composed Cu2+ chelators: a clue for designing whitening agents.

    PubMed

    Park, Yong-Doo; Lyou, You-Jeong; Hahn, Hwa-Sun; Hahn, Myong-Joon; Yang, Jun-Mo

    2006-10-01

    The inhibition of tyrosinase has attracted considerable attention for potential medicinal and cosmetic applications, as well as in agriculture. This study investigated the inhibition effects of thiol-associated Cu(2+) chelators and deduced a strategy for designing and/or selecting tyrosinase inhibitors. Among the several compounds tested, dithioglycerine (DTGC) was selected for further experiments on the inhibition kinetics on tyrosinase. Different types of tyrosinases derived from mushroom and from the transient overexpression in HEK293 cells were tested individually. The results showed that DTGC significantly inhibited human tyrosinase in a complex manner (slope-parabolic mixed-type inhibition), which was comparable to mushroom tyrosinase. The affinity of DTGC affinity to human tyrosinase was evaluated by setting up a K(i slope) equation. The results suggest that a Cu(2+) chelator modified with thiol groups has potential as a whitening agent. In addition, a strategy for designing and/or selecting tyrosinase inhibitors that target the active enzyme site was also suggested. PMID:16928136

  1. Pathways of Antigen Processing

    PubMed Central

    Blum, Janice S.; Wearsch, Pamela A.; Cresswell, Peter

    2014-01-01

    T cell recognition of antigen presenting cells depends on their expression of a spectrum of peptides bound to Major Histocompatibility Complex class I (MHC-I) and class II (MHC-II) molecules. Conversion of antigens from pathogens or transformed cells into MHC-I and MHC-II-bound peptides is critical for mounting protective T cell responses, and similar processing of self proteins is necessary to establish and maintain tolerance. Cells use a variety of mechanisms to acquire protein antigens, from translation in the cytosol to variations on the theme of endocytosis, and to degrade them once acquired. In this review we highlight the aspects of MHC-I and MHC-II biosynthesis and assembly that have evolved to intersect these pathways and sample the peptides that are produced. PMID:23298205

  2. Small molecule binding sites on the Ras:SOS complex can be exploited for inhibition of Ras activation.

    PubMed

    Winter, Jon J G; Anderson, Malcolm; Blades, Kevin; Brassington, Claire; Breeze, Alexander L; Chresta, Christine; Embrey, Kevin; Fairley, Gary; Faulder, Paul; Finlay, M Raymond V; Kettle, Jason G; Nowak, Thorsten; Overman, Ross; Patel, S Joe; Perkins, Paula; Spadola, Loredana; Tart, Jonathan; Tucker, Julie A; Wrigley, Gail

    2015-03-12

    Constitutively active mutant KRas displays a reduced rate of GTP hydrolysis via both intrinsic and GTPase-activating protein-catalyzed mechanisms, resulting in the perpetual activation of Ras pathways. We describe a fragment screening campaign using X-ray crystallography that led to the discovery of three fragment binding sites on the Ras:SOS complex. The identification of tool compounds binding at each of these sites allowed exploration of two new approaches to Ras pathway inhibition by stabilizing or covalently modifying the Ras:SOS complex to prevent the reloading of Ras with GTP. Initially, we identified ligands that bound reversibly to the Ras:SOS complex in two distinct sites, but these compounds were not sufficiently potent inhibitors to validate our stabilization hypothesis. We conclude by demonstrating that covalent modification of Cys118 on Ras leads to a novel mechanism of inhibition of the SOS-mediated interaction between Ras and Raf and is effective at inhibiting the exchange of labeled GDP in both mutant (G12C and G12V) and wild type Ras.

  3. Non-covalent pomegranate (Punica granatum) hydrolyzable tannin-protein complexes modulate antigen uptake, processing and presentation by a T-cell hybridoma line co-cultured with murine peritoneal macrophages.

    PubMed

    Madrigal-Carballo, Sergio; Haas, Linda; Vestling, Martha; Krueger, Christian G; Reed, Jess D

    2016-12-01

    In this work we characterize the interaction of pomegranate hydrolyzable tannins (HT) with hen egg-white lysozyme (HEL) and determine the effects of non-covalent tannin-protein complexes on macrophage endocytosis, processing and presentation of antigen. We isolated HT from pomegranate and complex to HEL, the resulting non-covalent tannin-protein complex was characterized by gel electrophoresis and MALDI-TOF MS. Finally, cell culture studies and confocal microscopy imaging were conducted on the non-covalent pomegranate HT-HEL protein complexes to evaluate its effect on macrophage antigen uptake, processing and presentation to T-cell hybridomas. Our results indicate that non-covalent pomegranate HT-HEL protein complexes modulate uptake, processing and antigen presentation by mouse peritoneal macrophages. After 4 h of pre-incubation, only trace amounts of IL-2 were detected in the co-cultures treated with HEL alone, whereas a non-covalent pomegranate HT-HEL complex had already reached maximum IL-2 expression. Pomegranate HT may increase rate of endocytose of HEL and subsequent expression of IL-2 by the T-cell hybridomas. PMID:27406472

  4. From ligand to complexes. Part 2. Remarks on human immunodeficiency virus type 1 integrase inhibition by beta-diketo acid metal complexes.

    PubMed

    Bacchi, Alessia; Biemmi, Mariano; Carcelli, Mauro; Carta, Fabrizio; Compari, Carlotta; Fisicaro, Emilia; Rogolino, Dominga; Sechi, Mario; Sippel, Martin; Sotriffer, Christoph A; Sanchez, Tino W; Neamati, Nouri

    2008-11-27

    Previously, we synthesized a series of beta-diketo acid metal complexes as novel HIV-1 integrase (IN) inhibitors (J. Med. Chem. 2006, 46, 4248-4260). Herein, a further extension of this study is reported. First, detailed docking studies were performed in order to investigate the mode of binding in the active site of the free ligands and of their metal complexes. Second, a series of potentiometric measurements were conducted for two diketo acids chosen as model ligands, with Mn(2+) and Ca(2+), in order to outline a speciation model. Third, we designed and synthesized a new set of complexes with different stoichiometries and tested them in an in vitro assay specific for IN. Finally, we obtained the first X-ray structure of a metal complex with HIV-1 IN inhibition activity. Analysis of these results supports the hypothesis that the diketo acids could act as complexes and form complexes with the metal ions on the active site of the enzyme.

  5. Crystal structures of human group-VIIA phospholipase A2 inhibited by organophosphorus nerve agents exhibit non-aged complexes.

    PubMed

    Samanta, Uttamkumar; Kirby, Stephen D; Srinivasan, Prabhavathi; Cerasoli, Douglas M; Bahnson, Brian J

    2009-08-15

    The enzyme group-VIIA phospholipase A2 (gVIIA-PLA2) is bound to lipoproteins in human blood and hydrolyzes the ester bond at the sn-2 position of phospholipid substrates with a short sn-2 chain. The enzyme belongs to a serine hydrolase superfamily of enzymes, which react with organophosphorus (OP) nerve agents. OPs ultimately exert their toxicity by inhibiting human acetycholinesterase at nerve synapses, but may additionally have detrimental effects through inhibition of other serine hydrolases. We have solved the crystal structures of gVIIA-PLA2 following inhibition with the OPs diisopropylfluorophosphate, sarin, soman and tabun. The sarin and soman complexes displayed a racemic mix of P(R) and P(S) stereoisomers at the P-chiral center. The tabun complex displayed only the P(R) stereoisomer in the crystal. In all cases, the crystal structures contained intact OP adducts that had not aged. Aging refers to a secondary process OP complexes can go through, which dealkylates the nerve agent adduct and results in a form that is highly resistant to either spontaneous or oxime-mediated reactivation. Non-aged OP complexes of the enzyme were corroborated by trypsin digest and matrix-assisted laser desorption ionization mass spectrometry of OP-enzyme complexes. The lack of stereoselectivity of sarin reaction was confirmed by gas chromatography/mass spectrometry using a chiral column to separate and quantitate the unbound stereoisomers of sarin following incubation with enzyme. The structural details and characterization of nascent reactivity of several toxic nerve agents is discussed with a long-term goal of developing gVIIA-PLA2 as a catalytic bioscavenger of OP nerve agents. PMID:19394314

  6. A New Way Forward in Cancer Drug Discovery: Inhibiting the SWI/SNF Chromatin Remodelling Complex.

    PubMed

    Zinzalla, Giovanna

    2016-04-15

    Mutations in subunits of the SWI/SNF chromatin remodelling complex are found in 20 % of human cancers. At face value, this would appear to indicate that this multiprotein complex is a potent tumour suppressor. However, it has recently emerged that some mutations in the SWI/SNF complex can have a gain-of-function effect and that in other tumours, such as pancreatic cancer, leukaemia, and breast cancer, the wild-type complex is used to drive cancer. Thus, paradoxically, this "tumour suppressor" has become an attractive target for developing anticancer agents. The SWI/SNF complex makes several protein-protein interactions both within the complex and with a wide range of transcription factors, and targeting these protein-protein interactions is emerging as the best approach to modulating the activity of the complex selectively.

  7. The inhibition of antigen-presenting activity of dendritic cells resulting from UV irradiation of murine skin is restored by in vitro photorepair of cyclobutane pyrimidine dimers.

    PubMed

    Vink, A A; Moodycliffe, A M; Shreedhar, V; Ullrich, S E; Roza, L; Yarosh, D B; Kripke, M L

    1997-05-13

    Exposing skin to UVB (280-320 nm) radiation suppresses contact hypersensitivity by a mechanism that involves an alteration in the activity of cutaneous antigen-presenting cells (APC). UV-induced DNA damage appears to be an important molecular trigger for this effect. The specific target cells in the skin that sustain DNA damage relevant to the immunosuppressive effect have yet to be identified. We tested the hypothesis that UV-induced DNA damage in the cutaneous APC was responsible for their impaired ability to present antigen after in vivo UV irradiation. Cutaneous APC were collected from the draining lymph nodes of UVB-irradiated, hapten-sensitized mice and incubated in vitro with liposomes containing a photolyase (Photosomes; Applied Genetics, Freeport, NY), which, upon absorption of photoreactivating light, splits UV-induced cyclobutane pyrimidine dimers. Photosome treatment followed by photoreactivating light reduced the number of dimer-containing APC, restored the in vivo antigen-presenting activity of the draining lymph node cells, and blocked the induction of suppressor T cells. Neither Photosomes nor photoreactivating light alone, nor photoreactivating light given before Photosomes, restored APC activity, and Photosome treatment did not reverse the impairment of APC function when isopsoralen plus UVA (320-400 nm) radiation was used instead of UVB. These controls indicate that the restoration of APC function matched the requirements of Photosome-mediated DNA repair for dimers and post-treatment photoreactivating light. These results provide compelling evidence that it is UV-induced DNA damage in cutaneous APC that leads to reduced immune function. PMID:9144224

  8. ATP is required for the release of the anaphase-promoting complex/cyclosome from inhibition by the mitotic checkpoint

    PubMed Central

    Miniowitz-Shemtov, Shirly; Teichner, Adar; Sitry-Shevah, Danielle; Hershko, Avram

    2010-01-01

    The mitotic (or spindle assembly) checkpoint system ensures accurate segregation of chromosomes by delaying anaphase until all chromosomes are correctly attached to the mitotic spindle. This system acts by inhibiting the activity of the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase to target securin for degradation. APC/C is inhibited by a mitotic checkpoint complex (MCC) composed of BubR1, Bub3, Mad2, and Cdc20. The molecular mechanisms of the inactivation of the mitotic checkpoint, including the release of APC/C from inhibition, remain obscure. It has been reported that polyubiquitylation by the APC/C is required for the inactivation of the mitotic checkpoint [Reddy SK, Rape M, Margansky WA, Kirschner MW (2007) Nature, 446:921–924]. We confirmed the involvement of polyubiquitylation, but found that another process, which requires ATP cleavage at the β–γ position (as opposed to α–β bond scission involved in ubiquitylation), is essential for the release of APC/C from checkpoint inhibition. ATP (β–γ) cleavage is required both for the dissociation of MCC components from APC/C and for the disassembly of free MCC, whereas polyubiquitylation is involved only in the former process. We find that the requirement for ATP (β–γ) cleavage is not due to the involvement of the 26S proteasome and that the phenomena observed are not due to sustained activity of protein kinase Cdk1/cyclin B, caused by inhibition of the degradation of cyclin B. Thus, some other energy-consuming process is needed for the inactivation of the mitotic checkpoint. PMID:20212161

  9. Crystal Structures of GII.10 and GII.12 Norovirus Protruding Domains in Complex with Histo-Blood Group Antigens Reveal Details for a Potential Site of Vulnerability

    SciTech Connect

    Hansman, Grant S.; Biertümpfel, Christian; Georgiev, Ivelin; McLellan, Jason S.; Chen, Lei; Zhou, Tongqing; Katayama, Kazuhiko; Kwong, Peter D.

    2011-10-10

    Noroviruses are the dominant cause of outbreaks of gastroenteritis worldwide, and interactions with human histo-blood group antigens (HBGAs) are thought to play a critical role in their entry mechanism. Structures of noroviruses from genogroups GI and GII in complex with HBGAs, however, reveal different modes of interaction. To gain insight into norovirus recognition of HBGAs, we determined crystal structures of norovirus protruding domains from two rarely detected GII genotypes, GII.10 and GII.12, alone and in complex with a panel of HBGAs, and analyzed structure-function implications related to conservation of the HBGA binding pocket. The GII.10- and GII.12-apo structures as well as the previously solved GII.4-apo structure resembled each other more closely than the GI.1-derived structure, and all three GII structures showed similar modes of HBGA recognition. The primary GII norovirus-HBGA interaction involved six hydrogen bonds between a terminal {alpha}fucose1-2 of the HBGAs and a dimeric capsid interface, which was composed of elements from two protruding subdomains. Norovirus interactions with other saccharide units of the HBGAs were variable and involved fewer hydrogen bonds. Sequence analysis revealed a site of GII norovirus sequence conservation to reside under the critical {alpha}fucose1-2 and to be one of the few patches of conserved residues on the outer virion-capsid surface. The site was smaller than that involved in full HBGA recognition, a consequence of variable recognition of peripheral saccharides. Despite this evasion tactic, the HBGA site of viral vulnerability may provide a viable target for small molecule- and antibody-mediated neutralization of GII norovirus.

  10. Epstein-Barr virus nuclear antigen 3A partially coincides with EBNA3C genome-wide and is tethered to DNA through BATF complexes.

    PubMed

    Schmidt, Stefanie C S; Jiang, Sizun; Zhou, Hufeng; Willox, Bradford; Holthaus, Amy M; Kharchenko, Peter V; Johannsen, Eric C; Kieff, Elliott; Zhao, Bo

    2015-01-13

    Epstein-Barr Virus (EBV) conversion of B-lymphocytes to Lymphoblastoid Cell Lines (LCLs) requires four EBV nuclear antigen (EBNA) oncoproteins: EBNA2, EBNALP, EBNA3A, and EBNA3C. EBNA2 and EBNALP associate with EBV and cell enhancers, up-regulate the EBNA promoter, MYC, and EBV Latent infection Membrane Proteins (LMPs), which up-regulate BCL2 to protect EBV-infected B-cells from MYC proliferation-induced cell death. LCL proliferation induces p16(INK4A) and p14(ARF)-mediated cell senescence. EBNA3A and EBNA3C jointly suppress p16(INK4A) and p14(ARF), enabling continuous cell proliferation. Analyses of the EBNA3A human genome-wide ChIP-seq landscape revealed 37% of 10,000 EBNA3A sites to be at strong enhancers; 28% to be at weak enhancers; 4.4% to be at active promoters; and 6.9% to be at weak and poised promoters. EBNA3A colocalized with BATF-IRF4, ETS-IRF4, RUNX3, and other B-cell Transcription Factors (TFs). EBNA3A sites clustered into seven unique groups, with differing B-cell TFs and epigenetic marks. EBNA3A coincidence with BATF-IRF4 or RUNX3 was associated with stronger EBNA3A ChIP-Seq signals. EBNA3A was at MYC, CDKN2A/B, CCND2, CXCL9/10, and BCL2, together with RUNX3, BATF, IRF4, and SPI1. ChIP-re-ChIP revealed complexes of EBNA3A on DNA with BATF. These data strongly support a model in which EBNA3A is tethered to DNA through a BATF-containing protein complexes to enable continuous cell proliferation.

  11. Roles of inhibition in creating complex auditory responses in the inferior colliculus: facilitated combination-sensitive neurons.

    PubMed

    Nataraj, Kiran; Wenstrup, Jeffrey J

    2005-06-01

    We studied roles of inhibition on temporally sensitive facilitation in combination-sensitive neurons from the mustached bat's inferior colliculus (IC). In these integrative neurons, excitatory responses to best frequency (BF) tones are enhanced by much lower frequency signals presented in a specific temporal relationship. Most facilitated neurons (76%) showed inhibition at delays earlier than or later than the delays causing facilitation. The timing of inhibition at earlier delays was closely related to the best delay of facilitation, but the inhibition had little influence on the duration or strength of the facilitatory interaction. Local iontophoretic application of antagonists to receptors for glycine (strychnine, STRY) and gamma-aminobutyric acid (GABA) (bicuculline, BIC) showed that STRY abolished facilitation in 96% of tested units, but BIC eliminated facilitation in only 28%. This suggests that facilitatory interactions are created in IC and reveals a differential role for these neurotransmitters. The facilitation may be created by coincidence of a postinhibitory rebound excitation activated by the low-frequency signal with the BF-evoked excitation. Unlike facilitation, inhibition at earlier delays was not eliminated by application of antagonists, suggesting an origin in lower brain stem nuclei. However, inhibition at delays later than facilitation, like facilitation itself, appears to originate within IC and to be more dependent on glycinergic than GABAergic mechanisms. Facilitatory and inhibitory interactions displayed by these combination-sensitive neurons encode information within sonar echoes and social vocalizations. The results indicate that these complex response properties arise through a series of neural interactions in the auditory brain stem and midbrain.

  12. Functional study of hepatitis delta virus large antigen in packaging and replication inhibition: role of the amino-terminal leucine zipper.

    PubMed

    Chen, P J; Chang, F L; Wang, C J; Lin, C J; Sung, S Y; Chen, D S

    1992-05-01

    The large hepatitis delta antigen (HDAg) has been found to be essential for the assembly of the hepatitis delta virion. Furthermore, in a cotransfection experiment, the large HDAg itself, without the hepatitis delta virus (HDV) genome and small HDAg, could be packaged into hepatitis B surface antigen (HBsAg) particles. By deletion analysis, it was shown that the amino-terminal leucine zipper domain was dispensable for packaging. The large HDAg could also help in copackaging of the small HDAg into HBsAg particles without the need for HDV RNA. This process was probably mediated through direct interaction of the two HDAgs as a mutated large HDAg whose leucine zipper domain was deleted such that it could not help in copackaging of the small HDAg. This mutated large HDAg did not suppress HDV replication, suggesting that this effect is probably also via protein interaction. These results indicated that functional domains of the large HDAg responsible for packaging with HBsAg particles and for the trans-negative effect on HDV replication can be separated. PMID:1560529

  13. Selective inhibition of tumor growth by clonal NK cells expressing an ErbB2/HER2-specific chimeric antigen receptor.

    PubMed

    Schönfeld, Kurt; Sahm, Christiane; Zhang, Congcong; Naundorf, Sonja; Brendel, Christian; Odendahl, Marcus; Nowakowska, Paulina; Bönig, Halvard; Köhl, Ulrike; Kloess, Stephan; Köhler, Sylvia; Holtgreve-Grez, Heidi; Jauch, Anna; Schmidt, Manfred; Schubert, Ralf; Kühlcke, Klaus; Seifried, Erhard; Klingemann, Hans G; Rieger, Michael A; Tonn, Torsten; Grez, Manuel; Wels, Winfried S

    2015-02-01

    Natural killer (NK) cells are an important effector cell type for adoptive cancer immunotherapy. Similar to T cells, NK cells can be modified to express chimeric antigen receptors (CARs) to enhance antitumor activity, but experience with CAR-engineered NK cells and their clinical development is still limited. Here, we redirected continuously expanding and clinically usable established human NK-92 cells to the tumor-associated ErbB2 (HER2) antigen. Following GMP-compliant procedures, we generated a stable clonal cell line expressing a humanized CAR based on ErbB2-specific antibody FRP5 harboring CD28 and CD3ζ signaling domains (CAR 5.28.z). These NK-92/5.28.z cells efficiently lysed ErbB2-expressing tumor cells in vitro and exhibited serial target cell killing. Specific recognition of tumor cells and antitumor activity were retained in vivo, resulting in selective enrichment of NK-92/5.28.z cells in orthotopic breast carcinoma xenografts, and reduction of pulmonary metastasis in a renal cell carcinoma model, respectively. γ-irradiation as a potential safety measure for clinical application prevented NK cell replication, while antitumor activity was preserved. Our data demonstrate that it is feasible to engineer CAR-expressing NK cells as a clonal, molecularly and functionally well-defined and continuously expandable cell therapeutic agent, and suggest NK-92/5.28.z cells as a promising candidate for use in adoptive cancer immunotherapy. PMID:25373520

  14. Interaction between the CD8 coreceptor and major histocompatibility complex class I stabilizes T cell receptor-antigen complexes at the cell surface.

    PubMed

    Wooldridge, Linda; van den Berg, Hugo A; Glick, Meir; Gostick, Emma; Laugel, Bruno; Hutchinson, Sarah L; Milicic, Anita; Brenchley, Jason M; Douek, Daniel C; Price, David A; Sewell, Andrew K

    2005-07-29

    The off-rate (k(off)) of the T cell receptor (TCR)/peptide-major histocompatibility complex class I (pMHCI) interaction, and hence its half-life, is the principal kinetic feature that determines the biological outcome of TCR ligation. However, it is unclear whether the CD8 coreceptor, which binds pMHCI at a distinct site, influences this parameter. Although biophysical studies with soluble proteins show that TCR and CD8 do not bind cooperatively to pMHCI, accumulating evidence suggests that TCR associates with CD8 on the T cell surface. Here, we titrated and quantified the contribution of CD8 to TCR/pMHCI dissociation in membrane-constrained interactions using a panel of engineered pMHCI mutants that retain faithful TCR interactions but exhibit a spectrum of affinities for CD8 of >1,000-fold. Data modeling generates a "stabilization factor" that preferentially increases the predicted TCR triggering rate for low affinity pMHCI ligands, thereby suggesting an important role for CD8 in the phenomenon of T cell cross-reactivity.

  15. Inhibition of lipoprotein-associated phospholipase A2 reduces complex coronary atherosclerotic plaque development

    PubMed Central

    Wilensky, Robert L; Shi, Yi; Mohler, Emile R; Hamamdzic, Damir; Burgert, Mark E; Li, Jun; Postle, Anthony; Fenning, Robert S; Bollinger, James G; Hoffman, Bryan E; Pelchovitz, Daniel J; Yang, Jisheng; Mirabile, Rosanna C; Webb, Christine L; Zhang, LeFeng; Zhang, Ping; Gelb, Michael H; Walker, Max C; Zalewski, Andrew; Macphee, Colin H

    2010-01-01

    Increased lipoprotein-associated phospholipase A2 (Lp-PLA2) activity is associated with increased risk of cardiac events, but it is not known whether Lp-PLA2 is a causative agent. Here we show that selective inhibition of Lp-PLA2 with darapladib reduced development of advanced coronary atherosclerosis in diabetic and hypercholesterolemic swine. Darapladib markedly inhibited plasma and lesion Lp-PLA2 activity and reduced lesion lysophosphatidylcholine content. Analysis of coronary gene expression showed that darapladib exerted a general anti-inflammatory action, substantially reducing the expression of 24 genes associated with macrophage and T lymphocyte functioning. Darapladib treatment resulted in a considerable decrease in plaque area and, notably, a markedly reduced necrotic core area and reduced medial destruction, resulting in fewer lesions with an unstable phenotype. These data show that selective inhibition of Lp-PLA2 inhibits progression to advanced coronary atherosclerotic lesions and confirms a crucial role of vascular inflammation independent from hypercholesterolemia in the development of lesions implicated in the pathogenesis of myocardial infarction and stroke. PMID:18806801

  16. Induction of tumor cell apoptosis by taurine Schiff base copper complex is associated the with inhibition of proteasomal activity

    PubMed Central

    ZHANG, XIA; BI, CAIFENG; FAN, YUHUA; CUI, QIUZHI; CHEN, DI; XIAO, YAN; DOU, Q. PING

    2013-01-01

    Schiff bases have been intensively investigated due to their antibacterial and antitumor properties. Copper is a cofactor essential for the tumor angiogenesis processes, whereas other transition metals are not. Consistently, high serum or tissue levels of copper were found in many types of human cancer including breast, prostate, colon, lung, and brain, supporting the idea that copper could be used as a novel selective target for cancer therapies. In the current study we hypothesize that a synthetic taurine Schiff base copper complex (Compound 1) could suppress tumor cell growth via the direct inhibition of proteasome activity. Compound 1 potently inhibits the activity of purified 20S and 26S proteasome in human breast cancer MDA-MB-231 and leukemia Jurkat T cells. Inhibition of tumor cellular proteasomal activity by Compound 1 results in the accumulation of ubiquitinated protein and the proteasome target proteins p27 and Bax, followed by the induction of apoptosis. Our results strongly suggest that taurine Schiff base copper complexes, as potent proteasome inhibitors, have great potential to be developed into novel anticancer drugs. PMID:18949390

  17. Inhibition of nuclear factor kappaB proteins-platinated DNA interactions correlates with cytotoxic effectiveness of the platinum complexes

    PubMed Central

    Brabec, Viktor; Kasparkova, Jana; Kostrhunova, Hana; Farrell, Nicholas P.

    2016-01-01

    Nuclear DNA is the target responsible for anticancer activity of platinum anticancer drugs. Their activity is mediated by altered signals related to programmed cell death and the activation of various signaling pathways. An example is activation of nuclear factor kappaB (NF-κB). Binding of NF-κB proteins to their consensus sequences in DNA (κB sites) is the key biochemical activity responsible for the biological functions of NF-κB. Using gel-mobility-shift assays and surface plasmon resonance spectroscopy we examined the interactions of NF-κB proteins with oligodeoxyribonucleotide duplexes containing κB site damaged by DNA adducts of three platinum complexes. These complexes markedly differed in their toxic effects in tumor cells and comprised highly cytotoxic trinuclear platinum(II) complex BBR3464, less cytotoxic conventional cisplatin and ineffective transplatin. The results indicate that structurally different DNA adducts of these platinum complexes exhibit a different efficiency to affect the affinity of the platinated DNA (κB sites) to NF-κB proteins. Our results support the hypothesis that structural perturbations induced in DNA by platinum(II) complexes correlate with their higher efficiency to inhibit binding of NF-κB proteins to their κB sites and cytotoxicity as well. However, the full generalization of this hypothesis will require to evaluate a larger series of platinum(II) complexes. PMID:27574114

  18. Inhibition of nuclear factor kappaB proteins-platinated DNA interactions correlates with cytotoxic effectiveness of the platinum complexes.

    PubMed

    Brabec, Viktor; Kasparkova, Jana; Kostrhunova, Hana; Farrell, Nicholas P

    2016-01-01

    Nuclear DNA is the target responsible for anticancer activity of platinum anticancer drugs. Their activity is mediated by altered signals related to programmed cell death and the activation of various signaling pathways. An example is activation of nuclear factor kappaB (NF-κB). Binding of NF-κB proteins to their consensus sequences in DNA (κB sites) is the key biochemical activity responsible for the biological functions of NF-κB. Using gel-mobility-shift assays and surface plasmon resonance spectroscopy we examined the interactions of NF-κB proteins with oligodeoxyribonucleotide duplexes containing κB site damaged by DNA adducts of three platinum complexes. These complexes markedly differed in their toxic effects in tumor cells and comprised highly cytotoxic trinuclear platinum(II) complex BBR3464, less cytotoxic conventional cisplatin and ineffective transplatin. The results indicate that structurally different DNA adducts of these platinum complexes exhibit a different efficiency to affect the affinity of the platinated DNA (κB sites) to NF-κB proteins. Our results support the hypothesis that structural perturbations induced in DNA by platinum(II) complexes correlate with their higher efficiency to inhibit binding of NF-κB proteins to their κB sites and cytotoxicity as well. However, the full generalization of this hypothesis will require to evaluate a larger series of platinum(II) complexes. PMID:27574114

  19. Radioimmunoassays of hidden viral antigens

    SciTech Connect

    Neurath, A.R.; Strick, N.; Baker, L.; Krugman, S.

    1982-07-01

    Antigens corresponding to infectious agents may be present in biological specimens only in a cryptic form bound to antibodies and, thus, may elude detection. We describe a solid-phase technique for separation of antigens from antibodies. Immune complexes are precipitated from serum by polyethylene glycol, dissociated with NaSCN, and adsorbed onto nitrocellulose or polystyrene supports. Antigens remain topographically separated from antibodies after removal of NaSCN and can be detected with radiolabeled antibodies. Genomes from viruses immobilized on nitrocellulose can be identified by nucleic acid hybridization. Nanogram quantities of sequestered hepatitis B surface and core antigens and picogram amounts of hepatitis B virus DNA were detected. Antibody-bound adenovirus, herpesvirus, and measles virus antigens were discerned by the procedure.

  20. Competitive binding inhibition enzyme-linked immunosorbent assay that uses the secreted aspartyl proteinase of Candida albicans as an antigenic marker for diagnosis of disseminated candidiasis.

    PubMed

    Morrison, Christine J; Hurst, Steven F; Reiss, Errol

    2003-09-01

    The secreted aspartyl proteinases (Saps) of Candida albicans have been implicated as virulence factors associated with adherence and tissue invasion. The potential use of proteinases as markers of invasive candidiasis led us to develop a competitive binding inhibition enzyme-linked immunosorbent assay (ELISA) to detect Sap in clinical specimens. Daily serum and urine specimens were collected from rabbits that had been immunosuppressed with cyclophosphamide and cortisone acetate and infected intravenously with 10(7) C. albicans blastoconidia. Disseminated infection was confirmed by organ culture and histopathology. Although ELISA inhibition was observed when serum specimens from these rabbits were used, more significant inhibition, which correlated with disease progression, occurred when urine specimens were used. Urine collected as early as 1 day after infection resulted in significant ELISA inhibition (mean inhibition +/- standard error [SE] compared with preinfection control urine, 15.7% +/- 2.7% [P < 0.01]), and inhibition increased on days 2 through 5 (29.4% +/- 4.8% to 44.5% +/- 3.5% [P < 0.001]). Urine specimens from immunosuppressed rabbits infected intravenously with Candida tropicalis, Candida parapsilosis, Candida krusei, Cryptococcus neoformans, Aspergillus fumigatus, or Staphylococcus aureus were negative in the assay despite culture-proven dissemination. Nonimmunosuppressed rabbits receiving oral tetracycline and gentamicin treatment were given 2 x 10(8) C. albicans blastoconidia orally or intraurethrally to establish colonization of the gastrointestinal tract or bladder, respectively, without systemic dissemination; urine specimens from these rabbits also gave negative ELISA results. Dissemination to the kidney and spleen occurred in one rabbit challenged by intragastric inoculation, and urine from this rabbit demonstrated significant inhibition in the ELISA (mean inhibition +/- SE by day 3 after infection, 32.9% +/- 2.7% [P < 0.001]). The overall

  1. Enzyme Mechanism and Slow-Onset Inhibition of Plasmodium falciparum Enoyl-Acyl Carrier Protein Reductase by an Inorganic Complex

    PubMed Central

    de Medeiros, Patrícia Soares de Maria; Ducati, Rodrigo Gay; Basso, Luiz Augusto; Santos, Diógenes Santiago; da Silva, Luiz Hildebrando Pereira

    2011-01-01

    Malaria continues to be a major cause of children's morbidity and mortality worldwide, causing nearly one million deaths annually. The human malaria parasite, Plasmodium falciparum, synthesizes fatty acids employing the Type II fatty acid biosynthesis system (FAS II), unlike humans that rely on the Type I (FAS I) pathway. The FAS II system elongates acyl fatty acid precursors of the cell membrane in Plasmodium. Enoyl reductase (ENR) enzyme is a member of the FAS II system. Here we present steady-state kinetics, pre-steady-state kinetics, and equilibrium fluorescence spectroscopy data that allowed proposal of P. falciparum ENR (PfENR) enzyme mechanism. Moreover, building on previous results, the present study also evaluates the PfENR inhibition by the pentacyano(isoniazid)ferrateII compound. This inorganic complex represents a new class of lead compounds for the development of antimalarial agents focused on the inhibition of PfENR. PMID:21603269

  2. Enzyme Mechanism and Slow-Onset Inhibition of Plasmodium falciparum Enoyl-Acyl Carrier Protein Reductase by an Inorganic Complex.

    PubMed

    de Medeiros, Patrícia Soares de Maria; Ducati, Rodrigo Gay; Basso, Luiz Augusto; Santos, Diógenes Santiago; da Silva, Luiz Hildebrando Pereira

    2011-01-01

    Malaria continues to be a major cause of children's morbidity and mortality worldwide, causing nearly one million deaths annually. The human malaria parasite, Plasmodium falciparum, synthesizes fatty acids employing the Type II fatty acid biosynthesis system (FAS II), unlike humans that rely on the Type I (FAS I) pathway. The FAS II system elongates acyl fatty acid precursors of the cell membrane in Plasmodium. Enoyl reductase (ENR) enzyme is a member of the FAS II system. Here we present steady-state kinetics, pre-steady-state kinetics, and equilibrium fluorescence spectroscopy data that allowed proposal of P. falciparum ENR (PfENR) enzyme mechanism. Moreover, building on previous results, the present study also evaluates the PfENR inhibition by the pentacyano(isoniazid)ferrateII compound. This inorganic complex represents a new class of lead compounds for the development of antimalarial agents focused on the inhibition of PfENR.

  3. Antigenic variation in ciliates: antigen structure, function, expression.

    PubMed

    Simon, Martin C; Schmidt, Helmut J

    2007-01-01

    In the past decades, the major focus of antigen variation research has been on parasitic protists. However, antigenic variation occurs also in free-living protists. The antigenic systems of the ciliates Paramecium and Tetrahymena have been studied for more than 100 yr. In spite of different life strategies and distant phylogenetic relationships of free-living ciliates and parasitic protists, their antigenic systems have features in common, such as the presence of repeated protein motifs and multigene families. The function of variable surface antigens in free-living ciliates is still unknown. Up to now no detailed monitoring of antigen expression in free-living ciliates in natural habitats has been performed. Unlike stochastic switching in parasites, antigen expression in ciliates can be directed, e.g. by temperature, which holds great advantages for research on the expression mechanism. Regulated expression of surface antigens occurs in an exclusive way and the responsible mechanism is complex, involving both transcriptional and post-transcriptional features. The involvement of homology-dependent effects has been proposed several times but has not been proved yet.

  4. Structural basis of oncogenic histone H3K27M inhibition of human polycomb repressive complex 2

    PubMed Central

    Justin, Neil; Zhang, Ying; Tarricone, Cataldo; Martin, Stephen R.; Chen, Shuyang; Underwood, Elizabeth; De Marco, Valeria; Haire, Lesley F.; Walker, Philip A.; Reinberg, Danny; Wilson, Jon R.; Gamblin, Steven J.

    2016-01-01

    Polycomb repressive complex 2 (PRC2) silences gene expression through trimethylation of K27 of histone H3 (H3K27me3) via its catalytic SET domain. A missense mutation in the substrate of PRC2, histone H3K27M, is associated with certain pediatric brain cancers and is linked to a global decrease of H3K27me3 in the affected cells thought to be mediated by inhibition of PRC2 activity. We present here the crystal structure of human PRC2 in complex with the inhibitory H3K27M peptide bound to the active site of the SET domain, with the methionine residue located in the pocket that normally accommodates the target lysine residue. The structure and binding studies suggest a mechanism for the oncogenic inhibition of H3K27M. The structure also reveals how binding of repressive marks, like H3K27me3, to the EED subunit of the complex leads to enhancement of the catalytic efficiency of the SET domain and thus the propagation of this repressive histone modification. PMID:27121947

  5. Streptococcal inhibitor of complement (SIC) inhibits the membrane attack complex by preventing uptake of C567 onto cell membranes.

    PubMed

    Fernie-King, B A; Seilly, D J; Willers, C; Würzner, R; Davies, A; Lachmann, P J

    2001-07-01

    Streptococcal inhibitor of complement (SIC) was first described in 1996 as a putative inhibitor of the membrane attack complex of complement (MAC). SIC is a 31 000 MW protein secreted in large quantities by the virulent Streptococcus pyogenes strains M1 and M57, and is encoded by a gene which is extremely variable. In order to study further the interactions of SIC with the MAC, we have made a recombinant form of SIC (rSIC) in Escherichia coli and purified native M1 SIC which was used to raise a polyclonal antibody. SIC prevented reactive lysis of guinea pig erythrocytes by the MAC at a stage prior to C5b67 complexes binding to cell membranes, presumably by blocking the transiently expressed membrane insertion site on C7. The ability of SIC and clusterin (another putative fluid phase complement inhibitor) to inhibit complement lysis was compared, and found to be equally efficient. In parallel, by enzyme-linked immunosorbent assay both SIC and rSIC bound strongly to C5b67 and C5b678 complexes and to a lesser extent C5b-9, but only weakly to individual complement components. The implications of these data for virulence of SIC-positive streptococci are discussed, in light of the fact that Gram-positive organisms are already protected against complement lysis by the presence of their peptidoglycan cell walls. We speculate that MAC inhibition may not be the sole function of SIC.

  6. Inhibiting complex IL-17A and IL-17RA interactions with a linear peptide

    PubMed Central

    Liu, Shenping; Desharnais, Joel; Sahasrabudhe, Parag V.; Jin, Ping; Li, Wei; Oates, Bryan D.; Shanker, Suman; Banker, Mary Ellen; Chrunyk, Boris A.; Song, Xi; Feng, Xidong; Griffor, Matt; Jimenez, Judith; Chen, Gang; Tumelty, David; Bhat, Abhijit; Bradshaw, Curt W.; Woodnutt, Gary; Lappe, Rodney W.; Thorarensen, Atli; Qiu, Xiayang; Withka, Jane M.; Wood, Lauren D.

    2016-01-01

    IL-17A is a pro-inflammatory cytokine that has been implicated in autoimmune and inflammatory diseases. Monoclonal antibodies inhibiting IL-17A signaling have demonstrated remarkable efficacy, but an oral therapy is still lacking. A high affinity IL-17A peptide antagonist (HAP) of 15 residues was identified through phage-display screening followed by saturation mutagenesis optimization and amino acid substitutions. HAP binds specifically to IL-17A and inhibits the interaction of the cytokine with its receptor, IL-17RA. Tested in primary human cells, HAP blocked the production of multiple inflammatory cytokines. Crystal structure studies revealed that two HAP molecules bind to one IL-17A dimer symmetrically. The N-terminal portions of HAP form a β-strand that inserts between two IL-17A monomers while the C-terminal section forms an α helix that directly blocks IL-17RA from binding to the same region of IL-17A. This mode of inhibition suggests opportunities for developing peptide antagonists against this challenging target. PMID:27184415

  7. Interaction between limulus amoebocyte lysate and soluble antigens from Pseudomonas aeruginosa and Staphylococcus aureus studied by quantitative immunoelectrophoresis.

    PubMed Central

    Baek, L; Høiby, N; Hertz, J B; Espersen, F

    1985-01-01

    To investigate the interaction of Limulus amoebocyte lysate (LAL) with gram-negative bacteria, soluble antigens from sonicated Pseudomonas aeruginosa were studied by various crossed-immunoelectrophoresis methods before and after reaction with LAL. Of 64 possible, at least 7 antigens were affected, as indicated by precipitin pattern, after the reaction with LAL. The precipitates corresponding to lipopolysaccharide and Pseudomonas "common antigen" disappeared. This reaction was inhibited when LAL was pretreated with lipopolysaccharide or by heating. Several of the reacting antigens have been shown to cross-react with many other strains of both gram-negative and gram-positive bacteria. Soluble antigens from a protein A-deficient strain of Staphylococcus aureus were also studied. LAL reacted with at least four of these antigens, including the teichoic acid complex. It is concluded that LAL is highly reactive with lipopolysaccharide, but it can react with other antigens from gram-negative and gram-positive bacteria as well. It is suggested that LAL interacts with biologically important antigens from the bacterial membrane. It is proposed that the reactivity and specificity of LAL for various microbial antigens can be studied by immunoelectrophoretic techniques. Images PMID:3928680

  8. Determinant selection of major histocompatibility complex class I- restricted antigenic peptides is explained by class I-peptide affinity and is strongly influenced by nondominant anchor residues

    PubMed Central

    1994-01-01

    The contribution of major histocompatibility complex (MHC) class I- peptide affinity to immunodominance of particular peptide antigens (Ags) in the class I-restricted cytotoxic T lymphocyte (CTL) response is not clearly established. Therefore, we have compared the H-2Kb- restricted binding and presentation of the immunodominant ovalbumin (OVA)257-264 (SIINFEKL) determinant to that of a subdominant OVA determinant OVA55-62 (KVVRFDKL). Immunodominance of OVA257-264 was not attributable to the specific T cell repertoire but correlated instead with more efficient Ag presentation. This enhanced Ag presentation could be accounted for by the higher affinity of Kb/OVA257-264 compared with Kb/OVA55-62 despite the presence of a conserved Kb-binding motif in both peptides. Kinetic binding studies using purified soluble H-2Kb molecules (Kbs) and biosensor techniques indicated that the Kon for association of OVA257-264-C6 and Kbs at 25 degrees C was integral of 10- fold faster (5.9 x 10(3) M-1 s-1 versus 6.5 x 10(2) M-1 s-1), and the Koff approximately twofold slower (9.1 x 10(-6) s-1 versus 1.6 x 10(-5) s-1), than the rate constants for interaction of OVA55-62-C6 and Kbs. The association of these peptides with Kb was significantly influenced by multiple residues at presumed nonanchor sites within the peptide sequence. The contribution of each peptide residue to Kb-binding was dependent upon the sequence context and the summed contributions were not additive. Thus the affinity of MHC class I-peptide binding is a critical factor controlling presentation of peptide Ag and immunodominance in the class I-restricted CTL response. PMID:7523572

  9. Up-regulation of lymphocyte antigen 6 complex expression in side-population cells derived from a human trophoblast cell line HTR-8/SVneo.

    PubMed

    Inagaki, Tetsunori; Kusunoki, Soshi; Tabu, Kouichi; Okabe, Hitomi; Yamada, Izumi; Taga, Tetsuya; Matsumoto, Akemi; Makino, Shintaro; Takeda, Satoru; Kato, Kiyoko

    2016-01-01

    The continual proliferation and differentiation of trophoblasts are critical for the maintenance of pregnancy. It is well known that the tissue stem cells are associated with the development of tissues and pathologies. It has been demonstrated that side-population (SP) cells identified by fluorescence-activated cell sorting (FACS) are enriched with stem cells. The SP cells in HTR-8/SVneo cells derived from human primary trophoblast cells were isolated by FACS. HTR-8/SVneo-SP cell cultures generated both SP and non-SP (NSP) subpopulations. In contrast, NSP cell cultures produced NSP cells and failed to produce SP cells. These SP cells showed self-renewal capability by serial colony-forming assay. Microarray expression analysis using a set of HTR-8/SVneo-SP and -NSP cells revealed that SP cells overexpressed several stemness genes including caudal type homeobox2 (CDX2) and bone morphogenic proteins (BMPs), and lymphocyte antigen 6 complex locus D (LY6D) gene was the most highly up-regulated in HTR-8/SVneo-SP cells. LY6D gene reduced its expression in the course of a 7-day cultivation in differentiation medium. SP cells tended to reduce its fraction by treatment of LY6D siRNA indicating that LY6D had potential to maintain cell proliferation of HTR-8/SVneo-SP cells. On ontology analysis, epithelial-mesenchymal transition (EMT) pathway was involved in the up-regulated genes on microarray analysis. HTR-SVneo-SP cells showed enhanced migration. This is the first report that LY6D was important for the maintenance of HTR-8/SVneo-SP cells. EMT was associated with the phenotype of these SP cells.

  10. Evaluation of squamous cell carcinoma antigen-immunoglobulin M complex (SCCA-IGM) and alpha-L-fucosidase (AFU) as novel diagnostic biomarkers for hepatocellular carcinoma.

    PubMed

    Mossad, Nehad A; Mahmoud, Enas H; Osman, Enas A; Mahmoud, Sherif H; Shousha, Hend I

    2014-11-01

    Hepatocellular carcinoma (HCC) surveillance lacks a reliable biomarker. Alpha-fetoprotein (AFP) is the most widely used. However, not all HCCs secrete AFP. AFP may be elevated with cirrhosis in the absence of HCC. Serum alpha-L-fucosidase (AFU) and squamous cell carcinoma antigen-immunoglobulin M complex (SCCA-IgM) were found to be useful markers in diagnosing HCC. SCCA-IgM and AFU were assessed by ELISA technique; AFP was measured by enzyme chemiluminescence in serum of 40 patients with HCC, 30 patients with liver cirrhosis, and 20 healthy control participants to compare their accuracy in early diagnosis of HCC. Serum SCCA-IgM and AFU levels were significantly elevated in HCC group compared to cirrhotic group (P value<0.001 and <0.001, respectively). Receiver operating characteristic curve showed the optimal cutoff value for SCCA-IgM was 233 AU/ml with sensitivity 87.5% and specificity 66% and for AFU was 25 U/L with sensitivity 87.5% and specificity 98%. AFP cutoff value was 48 ng/mL with sensitivity of 70% and specificity of 53.3%. The simultaneous determination of AFP and SCCA-IgM activity increased the sensitivity to 92.5% and specificity to 62.1%. There were positive significant correlations between SCCA-IgM and each of AFU (r=0.296, P=0.005) and AFP (r=0.284, P=0.007) and no correlation between AFP and AFU. All markers did not correlate with the tumor size or affected by the Child score. The significant difference between SCCA-IgM and AFU levels among HCC and cirrhotic patients suggests their use as potential diagnostic tools and allows identifying a new group of HCC patients even in the absence of elevated AFP. PMID:25129443

  11. Biopsy Quantitative Patohistology and Seral Values of Prostate Specific Antigen-Alpha (1) Antichymotrypsine Complex in Prediction of Adverse Pathology Findings after Radical Prostatectomy.

    PubMed

    Tomasković, Igor; Milicić, Valerija; Tomić, Miroslav; Ruzić, Boris; Ulamec, Monika

    2015-09-01

    In this prospective study we examined the utility of parameters obtained on prostate needle biopsy and prostate specific antigen-alpha(1)-antichymotripsine complex (PSA-ACT) to predict adverse pathologic findings after radical prostatectomy. 45 consecutive patients assigned for radical prostatectomy due to clinically localized prostate cancer were included in the study. Prostate biopsy parameters such as number of positive cores, the greatest percentage of tumor in the positive cores, Gleason score, perineural invasion, unilaterality or bilaterality of the tumor were recorded. PSA-ACT was determined using sandwich immunoassay chemiluminiscent method (Bayer, Tarrytown, New York). We analyzed relationship of preoperative PSA, PSA-ACTand quantitative biopsy parameters with final pathology after prostatectomy. Adverse findings were considered when extracapsular extension of cancer (pT3) was noted. Postoperatively, 29 (64.4%) patients were diagnosed with pT2 disease and 16 (35.6%) with pT3 disease. There was a significant difference in localized vs. locally advanced disease in number of positive biopsy cores (p<0.001), greatest percentage of tumor in the core (p=0.008), localization of the tumor (p=0.003) and perineural invasion (p=0.004). Logistic regression was used to develop a model on the multivariate level. It included number of positive cores and PSA-ACT and was significant on our cohort with the reliability of 82.22%. The combination of PSA-ACT and a large scale of biopsy parameters could be used in prediction of adverse pathologic findings after radical prostatectomy. Clinical decisions and patients counselling could be influenced by these predictors but further confirmation on a larger population is necessary. PMID:26898067

  12. Structural analysis of the human interferon gamma receptor: a small segment of the intracellular domain is specifically required for class I major histocompatibility complex antigen induction and antiviral activity.

    PubMed Central

    Cook, J R; Jung, V; Schwartz, B; Wang, P; Pestka, S

    1992-01-01

    Mutations of the human interferon gamma (IFN-gamma) receptor intracellular domain have permitted us to define a restricted region of that domain as necessary for both induction of class I major histocompatibility complex antigen by IFN-gamma and protection against encephalomyocarditis virus. This region consists of five amino acids (YDKPH), all of which are conserved in the human and murine receptors. Tyr-457 and His-461 are essential for activity. Approximately 80% of the amino acids of the intracellular domain of the receptor is not required for major histocompatibility complex class I antigen induction or for antiviral protection against encephalomyocarditis virus. The observation that there was no protection by IFN-gamma against vesiculostomatitis virus indicates that other factors, in addition to chromosome 21 accessory factor(s), are required to generate the full complement of transduction signals from the human IFN-gamma receptor. Images PMID:1454813

  13. Serum Albumin Binding Inhibits Nuclear Uptake of Luminescent Metal-Complex-Based DNA Imaging Probes.

    PubMed

    Wragg, Ashley; Gill, Martin R; McKenzie, Luke; Glover, Caroline; Mowll, Rachel; Weinstein, Julia A; Su, Xiaodi; Smythe, Carl; Thomas, Jim A

    2015-08-10

    The DNA binding and cellular localization properties of a new luminescent heterobimetallic Ir(III) Ru(II) tetrapyridophenazine complex are reported. Surprisingly, in standard cell media, in which its tetracationic, isostructural Ru(II) Ru(II) analogue is localized in the nucleus, the new tricationic complex is poorly taken up by live cells and demonstrates no nuclear staining. Consequent cell-free studies reveal that the Ir(III) Ru(II) complex binds bovine serum albumin, BSA, in Sudlow's Site I with a similar increase in emission and binding affinity to that observed with DNA. Contrastingly, in serum-free conditions the complex is rapidly internalized by live cells, where it localizes in cell nuclei and functions as a DNA imaging agent. The absence of serum proteins also greatly alters the cytotoxicity of the complex, where high levels of oncosis/necrosis are observed due to this enhanced uptake. This suggests that simply increasing the lipophilicity of a DNA imaging probe to enhance cellular uptake can be counterproductive as, due to increased binding to serum albumin protein, this strategy can actually disrupt nuclear targeting.

  14. Shigella effector IpaB-induced cholesterol relocation disrupts the Golgi complex and recycling network to inhibit host cell secretion.

    PubMed

    Mounier, Joëlle; Boncompain, Gaëlle; Senerovic, Lidija; Lagache, Thibault; Chrétien, Fabrice; Perez, Franck; Kolbe, Michael; Olivo-Marin, Jean-Christophe; Sansonetti, Philippe J; Sauvonnet, Nathalie

    2012-09-13

    Shigella infection causes destruction of the human colonic epithelial barrier. The Golgi network and recycling endosomes are essential for maintaining epithelial barrier function. Here we show that Shigella epithelial invasion induces fragmentation of the Golgi complex with consequent inhibition of both secretion and retrograde transport in the infected host cell. Shigella induces tubulation of the Rab11-positive compartment, thereby affecting cell surface receptor recycling. The molecular process underlying the observed damage to the Golgi complex and receptor recycling is a massive redistribution of plasma membrane cholesterol to the sites of Shigella entry. IpaB, a virulence factor of Shigella that is known to bind cholesterol, is necessary and sufficient to induce Golgi fragmentation and reorganization of the recycling compartment. Shigella infection-induced Golgi disorganization was also observed in vivo, suggesting that this mechanism affecting the sorting of cell surface molecules likely contributes to host epithelial barrier disruption associated with Shigella pathogenesis.

  15. BAFF/APRIL Inhibition Decreases Selection of Naive but Not Antigen-Induced Autoreactive B Cells in Murine Systemic Lupus Erythematosus

    PubMed Central

    Huang, Weiqing; Moisini, Ioana; Bethunaickan, Ramalingam; Sahu, Ranjit; Akerman, Meredith; Eilat, Dan; Lesser, Martin; Davidson, Anne

    2011-01-01

    BAFF inhibition is a new B cell-directed therapeutic strategy for autoimmune disease. Our purpose was to analyze the effect of BAFF/APRIL availability on the naive and Ag-activated B cell repertoires in systemic lupus erythematosus, using the autoreactive germline D42 H chain (glD42H) site-directed transgenic NZB/W mouse. In this article, we show that the naive Vκ repertoire in both young and diseased glD42H NZB/W mice is dominated by five L chains that confer no or low-affinity polyreactivity. In contrast, glD42H B cells expressing L chains that confer high-affinity autoreactivity are mostly deleted before the mature B cell stage, but are positively selected and expanded in the germinal centers (GCs) as the mice age. Of these, the most abundant is VκRF (Vκ16-104*01), which is expressed by almost all IgG anti-DNA hybridomas derived from the glD42H mouse. Competition with nonautoreactive B cells or BAFF/APRIL inhibition significantly inhibited selection of glD42H B cells at the late transitional stage, with only subtle effects on the glD42H-associated L chain repertoire. However, glD42H/VκRF-encoded B cells were still vastly overrepresented in the GC, and serum IgG anti-DNA Abs arose with only a slight delay. Thus, although BAFF/APRIL inhibition increases the stringency of negative selection of the naive autoreactive B cell repertoire in NZB/W mice, it does not correct the major breach in B cell tolerance that occurs at the GC checkpoint. PMID:22102726

  16. A monoclonal antibody against the nuclear pore complex inhibits nucleocytoplasmic transport of protein and RNA in vivo

    PubMed Central

    1988-01-01

    A monoclonal antibody that reacts with proteins in the nuclear pore complex of rat liver (Snow, C. M., A. Senior, and L. Gerace. 1987. J. Cell Biol. 104:1143-1156) has been shown to cross react with similar components in Xenopus oocytes, as determined by immunofluorescence microscopy and immunoblotting. We have microinjected the antibody into oocytes to study the possible role of these polypeptides in nucleocytoplasmic transport. The antibody inhibits import of a large nuclear protein, nucleoplasmin, in a time- and concentration-dependent manner. It also inhibits export of 5S ribosomal RNA and mature tRNA, but has no effect on transcription or intranuclear tRNA processing. The antibody does not affect the rate of diffusion into the nucleus of two small proteins, myoglobin and ovalbumin, indicating that antibody binding does not result in occlusion of the channel for diffusion. This suggests that inhibition of protein and RNA transport occurs by binding of the antibody at or near components of the pore that participate in mediated transport. PMID:2459127

  17. Dopaminergic inhibition by G9a/Glp complex on tyrosine hydroxylase in nerve injury-induced hypersensitivity

    PubMed Central

    Wang, Nan; Shen, Xiaofeng; Bao, Senzhu; Feng, Shan-Wu; Wang, Wei; Liu, Yusheng; Wang, Yiquan; Wang, Xian; Guo, Xirong; Shen, Rong; Wu, Haibo; Lei, Liming; Wang, Fuzhou

    2016-01-01

    The neural balance between facilitation and inhibition determines the final tendency of central sensitization. Nerve injury-induced hypersensitivity was considered as the results from the enhanced ascending facilitation and the diminished descending inhibition. The role of dopaminergic transmission in the descending inhibition has been well documented, but its underlying molecular mechanisms are unclear. Previous studies demonstrated that the lysine dimethyltransferase G9a/G9a-like protein (Glp) complex plays a critical role in cocaine-induced central plasticity, and given cocaine’s role in the nerve system is relied on its function on dopamine system, we herein proposed that the reduced inhibition of dopaminergic transmission was from the downregulation of tyrosine hydroxylase expression by G9a/Glp complex through methylating its gene Th. After approval by the Animal Care and Use Committee, C57BL/6 mice were used for pain behavior using von Frey after spared nerve injury, and Th CpG islands methylation was measured using bisulfite sequencing at different nerve areas. The inhibitor of G9a/Glp, BIX 01294, was administered intraventricularly daily with bolus injection. The protein levels of G9a, Glp, and tyrosine hydroxylase were measured with immunoblotting. Dopamine levels were detected using high-performance liquid chromatography. The expression of G9a but not Glp was upregulated in ventral tegmental area at post-injury day 4 till day 49 (the last day of the behavioral test). Correspondingly, the Th CpG methylation is increased, but the tyrosine hydroxylase expression was downregulated and the dopamine level was decreased. After the intracerebroventriclar injection of BIX 01294 since the post-injury days 7 and 14 for consecutive three days, three weeks, and six weeks, the expression of tyrosine hydroxylase was upregulated with a significant decrease in Th methylation and increase in dopamine level. Moreover, the pain after G9a/Glp inhibitor was attenuated

  18. Dopaminergic inhibition by G9a/Glp complex on tyrosine hydroxylase in nerve injury-induced hypersensitivity.

    PubMed

    Wang, Nan; Shen, Xiaofeng; Bao, Senzhu; Feng, Shan-Wu; Wang, Wei; Liu, Yusheng; Wang, Yiquan; Wang, Xian; Guo, Xirong; Shen, Rong; Wu, Haibo; Lei, Liming; Xu, Shiqin; Wang, Fuzhou

    2016-01-01

    The neural balance between facilitation and inhibition determines the final tendency of central sensitization. Nerve injury-induced hypersensitivity was considered as the results from the enhanced ascending facilitation and the diminished descending inhibition. The role of dopaminergic transmission in the descending inhibition has been well documented, but its underlying molecular mechanisms are unclear. Previous studies demonstrated that the lysine dimethyltransferase G9a/G9a-like protein (Glp) complex plays a critical role in cocaine-induced central plasticity, and given cocaine's role in the nerve system is relied on its function on dopamine system, we herein proposed that the reduced inhibition of dopaminergic transmission was from the downregulation of tyrosine hydroxylase expression by G9a/Glp complex through methylating its gene Th After approval by the Animal Care and Use Committee, C57BL/6 mice were used for pain behavior using von Frey after spared nerve injury, and Th CpG islands methylation was measured using bisulfite sequencing at different nerve areas. The inhibitor of G9a/Glp, BIX 01294, was administered intraventricularly daily with bolus injection. The protein levels of G9a, Glp, and tyrosine hydroxylase were measured with immunoblotting. Dopamine levels were detected using high-performance liquid chromatography. The expression of G9a but not Glp was upregulated in ventral tegmental area at post-injury day 4 till day 49 (the last day of the behavioral test). Correspondingly, the Th CpG methylation is increased, but the tyrosine hydroxylase expression was downregulated and the dopamine level was decreased. After the intracerebroventriclar injection of BIX 01294 since the post-injury days 7 and 14 for consecutive three days, three weeks, and six weeks, the expression of tyrosine hydroxylase was upregulated with a significant decrease in Th methylation and increase in dopamine level. Moreover, the pain after G9a/Glp inhibitor was attenuated

  19. Identification of the antigen content of electroimmunoprecipitates.

    PubMed

    Beyer, N Helena; Heegaard, Niels H H

    2013-01-01

    Polyclonal antibodies including purified antibody fractions and animal or human antisera may react with unknown antigens or antigens other than their main specificity in reactions that are best visualized by gel electroimmunoprecipitation methods, e.g., when analyzing complex antigen mixtures. The great advantage of gel immunoprecipitation approaches is that each immunoprecipitate contains antigen in a pure form and that the precipitate is separated by position, shape, and size from other precipitates in the complex patterns of crossed immunoelectrophoresis. The identification of the antigen content of such immunoprecipitates is important but challenging because of the very stable, high affinity complex formation leading to precipitation in the gels. Here, we present detailed step-by-step recipes for identifying the antigen content of electroimmunoprecipitates.

  20. Studies on Inhibition of Respiratory Cytochrome bc1 Complex by the Fungicide Pyrimorph Suggest a Novel Inhibitory Mechanism

    PubMed Central

    Xiao, Yu-Mei; Esser, Lothar; Zhou, Fei; Li, Chang; Zhou, Yi-Hui; Yu, Chang-An; Qin, Zhao-Hai; Xia, Di

    2014-01-01

    The respiratory chain cytochrome bc1 complex (cyt bc1) is a major target of numerous antibiotics and fungicides. All cyt bc1 inhibitors act on either the ubiquinol oxidation (QP) or ubiquinone reduction (QN) site. The primary cause of resistance to bc1 inhibitors is target site mutations, creating a need for novel agents that act on alternative sites within the cyt bc1 to overcome resistance. Pyrimorph, a synthetic fungicide, inhibits the growth of a broad range of plant pathogenic fungi, though little is known concerning its mechanism of action. In this study, using isolated mitochondria from pathogenic fungus Phytophthora capsici, we show that pyrimorph blocks mitochondrial electron transport by affecting the function of cyt bc1. Indeed, pyrimorph inhibits the activities of both purified 11-subunit mitochondrial and 4-subunit bacterial bc1 with IC50 values of 85.0 μM and 69.2 μM, respectively, indicating that it targets the essential subunits of cyt bc1 complexes. Using an array of biochemical and spectral methods, we show that pyrimorph acts on an area near the QP site and falls into the category of a mixed-type, noncompetitive inhibitor with respect to the substrate ubiquinol. In silico molecular docking of pyrimorph to cyt b from mammalian and bacterial sources also suggests that pyrimorph binds in the vicinity of the quinol oxidation site. PMID:24699450

  1. Studies on inhibition of respiratory cytochrome bc1 complex by the fungicide pyrimorph suggest a novel inhibitory mechanism.

    PubMed

    Xiao, Yu-Mei; Esser, Lothar; Zhou, Fei; Li, Chang; Zhou, Yi-Hui; Yu, Chang-An; Qin, Zhao-Hai; Xia, Di

    2014-01-01

    The respiratory chain cytochrome bc1 complex (cyt bc1) is a major target of numerous antibiotics and fungicides. All cyt bc1 inhibitors act on either the ubiquinol oxidation (QP) or ubiquinone reduction (QN) site. The primary cause of resistance to bc1 inhibitors is target site mutations, creating a need for novel agents that act on alternative sites within the cyt bc1 to overcome resistance. Pyrimorph, a synthetic fungicide, inhibits the growth of a broad range of plant pathogenic fungi, though little is known concerning its mechanism of action. In this study, using isolated mitochondria from pathogenic fungus Phytophthora capsici, we show that pyrimorph blocks mitochondrial electron transport by affecting the function of cyt bc1. Indeed, pyrimorph inhibits the activities of both purified 11-subunit mitochondrial and 4-subunit bacterial bc1 with IC50 values of 85.0 μM and 69.2 μM, respectively, indicating that it targets the essential subunits of cyt bc1 complexes. Using an array of biochemical and spectral methods, we show that pyrimorph acts on an area near the QP site and falls into the category of a mixed-type, noncompetitive inhibitor with respect to the substrate ubiquinol. In silico molecular docking of pyrimorph to cyt b from mammalian and bacterial sources also suggests that pyrimorph binds in the vicinity of the quinol oxidation site.

  2. FRK inhibits migration and invasion of human glioma cells by promoting N-cadherin/β-catenin complex formation.

    PubMed

    Shi, Qiong; Song, Xu; Wang, Jun; Gu, Jia; Zhang, Weijian; Hu, Jinxia; Zhou, Xiuping; Yu, Rutong

    2015-01-01

    Fyn-related kinase (FRK), a member of Src-related tyrosine kinases, is recently reported to function as a potent tumor suppressor in several cancer types. Our previous study has also shown that FRK over-expression inhibited the migration and invasion of glioma cells. However, the mechanism of FRK effect on glioma cell migration and invasion, a feature of human malignant gliomas, is still not clear. In this study, we found that FRK over-expression increased the protein level of N-cadherin, but not E-cadherin. Meanwhile, FRK over-expression promoted β-catenin translocation to the plasma membrane, where it formed complex with N-cadherin, while decreased β-catenin level in the nuclear fraction. In addition, down-regulation of N-cadherin by siRNA promoted the migration and invasion of glioma U251 and U87 cells and abolished the inhibitory effect of FRK on glioma cell migration and invasion. In summary, these results indicate that FRK inhibits migration and invasion of human glioma cells by promoting N-cadherin/β-catenin complex formation.

  3. Metabolites from invasive pests inhibit mitochondrial complex II: A potential strategy for the treatment of human ovarian carcinoma?

    PubMed

    Ferramosca, Alessandra; Conte, Annalea; Guerra, Flora; Felline, Serena; Rimoli, Maria Grazia; Mollo, Ernesto; Zara, Vincenzo; Terlizzi, Antonio

    2016-05-13

    The red pigment caulerpin, a secondary metabolite from the marine invasive green algae Caulerpa cylindracea can be accumulated and transferred along the trophic chain, with detrimental consequences on biodiversity and ecosystem functioning. Despite increasing research efforts to understand how caulerpin modifies fish physiology, little is known on the effects of algal metabolites on mammalian cells. Here we report for the first time the mitochondrial targeting activity of both caulerpin, and its closely related derivative caulerpinic acid, by using as experimental model rat liver mitochondria, a system in which bioenergetics mechanisms are not altered. Mitochondrial function was tested by polarographic and spectrophotometric methods. Both compounds were found to selectively inhibit respiratory complex II activity, while complexes I, III, and IV remained functional. These results led us to hypothesize that both algal metabolites could be used as antitumor agents in cell lines with defects in mitochondrial complex I. Ovarian cancer cisplatin-resistant cells are a good example of cell lines with a defective complex I function on which these molecules seem to have a toxic effect on proliferation. This provided novel insight toward the potential use of metabolites from invasive Caulerpa species for the treatment of human ovarian carcinoma cisplatin-resistant cells. PMID:27091429

  4. An early pseudorabies virus protein down-regulates porcine MHC class I expression by inhibition of transporter associated with antigen processing (TAP).

    PubMed

    Ambagala, A P; Hinkley, S; Srikumaran, S

    2000-01-01

    The objectives of this study were to identify the mechanism(s) of pseudorabies virus (PrV)-induced down-regulation of porcine class I molecules and the viral protein(s) responsible for the effect. The ability of PrV to interfere with the peptide transport activity of TAP was determined by an in vitro transport assay. In this assay, porcine kidney (PK-15) cells were permeabilized with streptolysin-O and incubated with a library of 125I-labeled peptides having consensus motifs for glycosylation in the endoplasmic reticulum (ER). The efficiency of transport of peptides from the cytosol into the ER was determined by adsorbing the ER-glycosylated peptides onto Con A-coupled Sepharose beads. Dose-dependent inhibition of TAP activity was observed in PrV-infected PK-15 cells. This inhibition, which occurred as early as 2 h postinfection (h.p.i.), reached the maximum level by 6 h.p.i., indicating that TAP inhibition is one of the mechanisms by which PrV down-regulates porcine class I molecules. Infection of cells with PrV in the presence of metabolic inhibitors revealed that cycloheximide a protein synthesis inhibitor, but not phosphonoacetic acid a herpesvirus DNA synthesis inhibitor, could restore the cell surface expression of class I molecules, indicating that late proteins are not responsible for the down-regulation. Infection in the presence of cycloheximide followed by actinomycin-D, which results in accumulation of the immediate-early protein, failed to down-regulate class I, indicating that one or more early proteins are responsible for the down-regulation of class I molecules.

  5. Antigen-specific inhibition of CD8+ T cell response by immature myeloid cells in cancer is mediated by reactive oxygen species.

    PubMed

    Kusmartsev, Sergei; Nefedova, Yulia; Yoder, Daniel; Gabrilovich, Dmitry I

    2004-01-15

    Tumor growth is associated with the accumulation of immature myeloid cells (ImC), which in mice are characterized by the expression of Gr-1 and CD11b markers. These cells suppress Ag-specific CD8+ T cells via direct cell-cell contact. However, the mechanism of immunosuppressive activity of tumor-derived ImC remains unclear. In this study we analyzed the function of ImC isolated from tumor-free control and tumor-bearing mice. Only ImC isolated from tumor-bearing mice, not those from their control counterparts, were able to inhibit the Ag-specific response of CD8+ T cells. ImC obtained from tumor-bearing mice had significantly higher levels of reactive oxygen species (ROS) than ImC isolated from tumor-free animals. Accumulation of H2O2, but not superoxide or NO, was a major contributor to this increased pool of ROS. It appears that arginase activity played an important role in H2O2 accumulation in these cells. Inhibition of ROS in ImC completely abrogated the inhibitory effect of these cells on T cells, indicating that ImC generated in tumor-bearing hosts suppress the CD8+ T cell response via production of ROS. Interaction of ImC with Ag-specific T cells in the presence of specific Ags resulted in a significant increase in ROS production compared with control Ags. That increase was independent of IFN-gamma production by T cells, but was mediated by integrins CD11b, CD18, and CD29. Blocking of these integrins with specific Abs abrogated ROS production and ImC-mediated suppression of CD8+ T cell responses. This study demonstrates a new mechanism of Ag-specific T cell inhibition mediated by ROS produced by ImCs in cancer.

  6. Water-Soluble Coenzyme Q10 Inhibits Nuclear Translocation of Apoptosis Inducing Factor and Cell Death Caused by Mitochondrial Complex I Inhibition

    PubMed Central

    Li, Haining; Chen, Guisheng; Ma, Wanrui; Li, Ping-An Andy

    2014-01-01

    The objectives of the study were to explore the mechanism of rotenone-induced cell damage and to examine the protective effects of water-soluble Coenzyme Q10 (CoQ10) on the toxic effects of rotenone. Murine hippocampal HT22 cells were cultured with mitochondrial complex I inhibitor rotenone. Water-soluble CoQ10 was added to the culture media 3 h prior to the rotenone incubation. Cell viability was determined by alamar blue, reactive oxygen species (ROS) production by dihydroethidine (DHE) and mitochondrial membrane potential by tetramethyl rhodamine methyl ester (TMRM). Cytochrome c, caspase-9 and apoptosis-inducing factor (AIF) were measured using Western blotting after 24 h rotenone incubation. Rotenone caused more than 50% of cell death, increased ROS production, AIF nuclear translocation and reduction in mitochondrial membrane potential, but failed to cause mitochondrial cytochrome c release and caspase-9 activation. Pretreatment with water-soluble CoQ10 enhanced cell viability, decreased ROS production, maintained mitochondrial membrane potential and prevented AIF nuclear translocation. The results suggest that rotenone activates a mitochondria-initiated, caspase-independent cell death pathway. Water-soluble CoQ10 reduces ROS accumulation, prevents the fall of mitochondrial membrane potential, and inhibits AIF translocation and subsequent cell death. PMID:25089873

  7. Chemopreventive properties of phytosterols and maslinic acid extracted from Coleus tuberosus in inhibiting the expression of EBV early-antigen in Raji cells.

    PubMed

    Mooi, Lim Yang; Wahab, Norhanom Abdul; Lajis, Nordin Haji; Ali, Abdul Manaf

    2010-05-01

    Bioassay-guided fractionation of a MeOH extract of tubers of Coleus tuberosus afforded the active anti-tumor-promoting compounds identified as the triterpenoid 2alpha,3beta-dihydroxyolean-12-en-28-oic acid (maslinic acid; CT2) and a phytosterol mixture (CT1). CT1 consists of stigmasterol (32%), beta-sitosterol (40.3%), and campesterol (27.7%) as determined by capillary gas chromatography. CT1 and CT2 showed very strong anti-tumor-promoting activities at IC(50) 0.7 microg/ml and 0.1 microg/ml, respectively, in a convenient, short-term in vitro assay, i.e., the inhibition of Epstein-Barr virus (EBV) activation induced by phorbol 12-myristate 13-acetate (PMA) and sodium butyrate. We report for the first time the anti-tumor-promoting activity of 2alpha,3beta-dihydroxyolean-12-en-28-oic acid and show that a mixture of stigmasterol, beta-sitosterol, and campesterol is more potent than the individual components in inhibiting tumor-promoting activity.

  8. The role of protein kinase C in transmembrane signaling by the T cell antigen receptor complex. Effects of stimulation with soluble or immobilized CD3 antibodies.

    PubMed

    Manger, B; Weiss, A; Imboden, J; Laing, T; Stobo, J D

    1987-10-15

    Phorbol esters, such as phorbol myristate acetate (PMA), are known to be potent co-stimulants with calcium ionophores for activation of T lymphocytes. The most extensively studied intracellular effect of PMA is its ability to activate the cytoplasmic enzyme protein kinase C (pkC). Herein, we examined the role of pkC activation during T cell activation. During physiologic activation, this enzyme is activated by diacylglycerol which is generated through the hydrolysis of polyphosphoinositides. Therefore, we studied the activation of T lymphocytes induced by a synthetic diacylglycerol, dioctanoylglycerol. In contrast to PMA, this compound can be metabolized in T cells and presumably more closely mimics physiologic activation of pkC. Dioctanoylglycerol together with reagents that induce increases in intracellular free Ca2+ concentration, Ca2+ ionophores, or anti-cluster designation (CD)3 monoclonal antibodies (mAb) were able to induce interleukin 2 receptor expression and proliferation of T lymphocytes. Previous studies have demonstrated that the stimulation of T cells via the CD3/T cell antigen receptor complex by mAb against CD3 leads to an increase in cytoplasmic free Ca2+ and to an activation of pkC. Paradoxically, however, soluble CD3 antibodies do not cause proliferation of resting purified T cells. Inasmuch as immobilization of CD3 mAb has been shown to influence the agonist properties of such antibodies, we compared the ability of soluble and immobilized CD3 mAb to activate pkC. We demonstrated herein that soluble CD3 mAb cause only a very transient activation of pkC in the T cell leukemic line Jurkat. This pkC activation is markedly prolonged when Jurkat cells are stimulated with immobilized rather than soluble CD3 antibodies. These studies suggest that activation of pkC plays a major role in T cell activation and that the activation of pkC is influenced by the form in which CD3 mAb is presented to T cells.

  9. Helminth Antigens Enable CpG-Activated Dendritic Cells to Inhibit the Symptoms of Collagen-induced Arthritis through Foxp3+ Regulatory T Cells

    PubMed Central

    Carranza, Franco; Falcón, Cristian Roberto; Nuñez, Nicolás; Knubel, Carolina; Correa, Silvia Graciela; Bianco, Ismael; Maccioni, Mariana; Fretes, Ricardo; Triquell, María Fernanda; Motrán, Claudia Cristina; Cervi, Laura

    2012-01-01

    Dendritic cells (DC) have the potential to control the outcome of autoimmunity by modulating the immune response. In this study, we tested the ability of Fasciola hepatica total extract (TE) to induce tolerogenic properties in CpG-ODN (CpG) maturated DC, to then evaluate the therapeutic potential of these cells to diminish the inflammatory response in collagen induced arthritis (CIA). DBA/1J mice were injected with TE plus CpG treated DC (T/C-DC) pulsed with bovine collagen II (CII) between two immunizations with CII and clinical scores CIA were determined. The levels of CII-specific IgG2 and IgG1 in sera, the histological analyses in the joints, the cytokine profile in the draining lymph node (DLN) cells and in the joints, and the number, and functionality of CD4+CD25+Foxp3+ T cells (Treg) were evaluated. Vaccination of mice with CII pulsed T/C-DC diminished the severity and incidence of CIA symptoms and the production of the inflammatory cytokine, while induced the production of anti-inflammatory cytokines. The therapeutic effect was mediated by Treg cells, since the adoptive transfer of CD4+CD25+ T cells, inhibited the inflammatory symptoms in CIA. The in vitro blockage of TGF-β in cultures of DLN cells plus CII pulsed T/C-DC inhibited the expansion of Treg cells. Vaccination with CII pulsed T/C-DC seems to be a very efficient approach to diminish exacerbated immune response in CIA, by inducing the development of Treg cells, and it is therefore an interesting candidate for a cell-based therapy for rheumatoid arthritis (RA). PMID:22848374

  10. Substrate complexes of human dipeptidyl peptidase III reveal the mechanism of enzyme inhibition

    PubMed Central

    Kumar, Prashant; Reithofer, Viktoria; Reisinger, Manuel; Wallner, Silvia; Pavkov-Keller, Tea; Macheroux, Peter; Gruber, Karl

    2016-01-01

    Human dipeptidyl-peptidase III (hDPP III) is a zinc-dependent hydrolase cleaving dipeptides off the N-termini of various bioactive peptides. Thus, the enzyme is likely involved in a number of physiological processes such as nociception and is also implicated in several forms of cancer. We present high-resolution crystal structures of hDPP III in complex with opioid peptides (Met-and Leu-enkephalin, endomorphin-2) as well as with angiotensin-II and the peptide inhibitor IVYPW. These structures confirm the previously reported large conformational change of the enzyme upon ligand binding and show that the structure of the closed conformation is independent of the nature of the bound peptide. The overall peptide-binding mode is also conserved ensuring the correct positioning of the scissile peptide bond with respect to the catalytic zinc ion. The structure of the angiotensin-II complex shows, how longer peptides are accommodated in the binding cleft of hDPP III. Differences in the binding modes allow a distinction between real substrates and inhibitory peptides or “slow” substrates. The latter displace a zinc bound water molecule necessitating the energetically much less favoured anhydride mechanism as opposed to the favoured promoted-water mechanism. The structural data also form the necessary framework for the design of specific hDPP III inhibitors. PMID:27025154

  11. Interaction of NCOR/SMRT Repressor Complexes with Papillomavirus E8^E2C Proteins Inhibits Viral Replication.

    PubMed

    Dreer, Marcel; Fertey, Jasmin; van de Poel, Saskia; Straub, Elke; Madlung, Johannes; Macek, Boris; Iftner, Thomas; Stubenrauch, Frank

    2016-04-01

    Infections with high-risk human papillomaviruses (HR-HPV) such as HPV16 and 31 can lead to ano-genital and oropharyngeal cancers and HPV types from the beta genus have been implicated in the development of non-melanoma skin cancer. HPV replicate as nuclear extrachromosomal plasmids at low copy numbers in undifferentiated cells. HPV16 and 31 mutants have indicated that these viruses express an E8^E2C protein which negatively regulates genome replication. E8^E2C shares the DNA-binding and dimerization domain (E2C) with the essential viral replication activator E2 and the E8 domain replaces the replication/transcription activation domain of E2. The HR-HPV E8 domain is required for inhibiting viral transcription and the replication of the viral origin mediated by viral E1 and E2 proteins. We show now that E8^E2C also limits replication of HPV1, a mu-PV and HPV8, a beta-PV, in normal human keratinocytes. Proteomic analyses identified all NCoR/SMRT corepressor complex components (HDAC3, GPS2, NCoR, SMRT, TBL1 and TBLR1) as co-precipitating host cell proteins for HPV16 and 31 E8^E2C proteins. Co-immunoprecipitation and co-localization experiments revealed that NCoR/SMRT components interact with HPV1, 8, 16 and 31 E8^E2C proteins in an E8-dependent manner. SiRNA knock-down experiments confirm that NCoR/SMRT components are critical for both the inhibition of transcription and HPV origin replication by E8^E2C proteins. Furthermore, a dominant-negative NCoR fragment activates transcription and replication only from HPV16 and 31 wt but not from mutant genomes encoding NCoR/SMRT-binding deficient E8^E2C proteins. In summary, our data suggest that the repressive function of E8^E2C is highly conserved among HPV and that it is mediated by an E8-dependent interaction with NCoR/SMRT complexes. Our data also indicate for the first time that NCoR/SMRT complexes not only are involved in inhibiting cellular and viral transcription but also in controlling the replication of HPV origins

  12. Expression of Ley antigen in human immunodeficiency virus-infected human T cell lines and in peripheral lymphocytes of patients with acquired immune deficiency syndrome (AIDS) and AIDS-related complex (ARC)

    PubMed Central

    1988-01-01

    Ley determinant (Fuc alpha 1----2Gal beta 1----4[Fuc alpha 1---- 3]GlcNAc beta 1----R) defined by mAb BM-1 is highly expressed in human immunodeficiency virus (HIV)-infected T cell lines and in CD3+ peripheral mature T cells of patients with acquired immune deficiency syndrome (AIDS) or with AIDS-related complex (ARC). Ley expression increased greatly in the CD3+ population in the advanced stage of AIDS when the CD4+ population decreased greatly. Six other carbohydrate antigens tested by their respective mAbs were not detected in these same cells. None of the carbohydrate antigens tested by the seven mAbs used in this study were found in noninfected T cell lines and in normal peripheral blood lymphocytes. PMID:3258005

  13. Complex effects of inhibiting hepatic apolipoprotein B100 synthesis in humans

    PubMed Central

    Reyes-Soffer, Gissette; Moon, Byoung; Hernandez-Ono, Antonio; Dionizovik-Dimanovski, Marija; Jimenez, Jhonsua; Obunike, Joseph; Thomas, Tiffany; Ngai, Colleen; Fontanez, Nelson; Donovan, Daniel S.; Karmally, Wahida; Holleran, Stephen; Ramakrishnan, Rajasekhar; Mittleman, Robert S.; Ginsberg, Henry N.

    2016-01-01

    Mipomersen is a 20mer antisense oligonucleotide (ASO) that inhibits apolipoprotein B (apoB) synthesis; its low-density lipoprotein (LDL)–lowering effects should therefore result from reduced secretion of very-low-density lipoprotein (VLDL). We enrolled 17 healthy volunteers who received placebo injections weekly for 3 weeks followed by mipomersen weekly for 7 to 9 weeks. Stable isotopes were used after each treatment to determine fractional catabolic rates and production rates of apoB in VLDL, IDL (intermediate-density lipoprotein), and LDL, and of triglycerides in VLDL. Mipomersen significantly reduced apoB in VLDL, IDL, and LDL, which was associated with increases in fractional catabolic rates of VLDL and LDL apoB and reductions in production rates of IDL and LDL apoB. Unexpectedly, the production rates of VLDL apoB and VLDL triglycerides were unaffected. Small interfering RNA–mediated knockdown of apoB expression in human liver cells demonstrated preservation of apoB secretion across a range of apoB synthesis. Titrated ASO knockdown of apoB mRNA in chow-fed mice preserved both apoB and triglyceride secretion. In contrast, titrated ASO knockdown of apoB mRNA in high-fat–fed mice resulted in stepwise reductions in both apoB and triglyceride secretion. Mipomersen lowered all apoB lipoproteins without reducing the production rate of either VLDL apoB or triglyceride. Our human data are consistent with longstanding models of posttranscriptional and posttranslational regulation of apoB secretion and are supported by in vitro and in vivo experiments. Targeting apoB synthesis may lower levels of apoB lipoproteins without necessarily reducing VLDL secretion, thereby lowering the risk of steatosis associated with this therapeutic strategy. PMID:26819195

  14. Lack of association between Behçet's disease and major histocompatibility complex class II antigens in an ethnically diverse North American Caucasoid patient group.

    PubMed

    Moore, S B; O'Duffy, J D

    1986-08-01

    A group of 25 North American Caucasoid patients with well defined Behcet's disease were serologically typed for HLA-DR and DQw antigens. No significant associations were seen when results were compared with a group of 73 normal Caucasoid controls tested concomitantly. PMID:3772926

  15. The effects of serial skin testing with purified protein derivative on the level and quality of antibodies to complex and defined antigens in Mycobacterium bovis-infected cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several serologic tests designed to detect antibodies to immunodominant Mycobacterium bovis antigens have recently emerged as ancillary tests for the detection of bovine tuberculosis in cattle, particularly when applied after injection of purified protein derivative (PPD) for skin test that signific...

  16. Borrelia burgdorferi BBK32 Inhibits the Classical Pathway by Blocking Activation of the C1 Complement Complex.

    PubMed

    Garcia, Brandon L; Zhi, Hui; Wager, Beau; Höök, Magnus; Skare, Jon T

    2016-01-01

    Pathogens that traffic in blood, lymphatics, or interstitial fluids must adopt strategies to evade innate immune defenses, notably the complement system. Through recruitment of host regulators of complement to their surface, many pathogens are able to escape complement-mediated attack. The Lyme disease spirochete, Borrelia burgdorferi, produces a number of surface proteins that bind to factor H related molecules, which function as the dominant negative regulator of the alternative pathway of complement. Relatively less is known about how B. burgdorferi evades the classical pathway of complement despite the observation that some sensu lato strains are sensitive to classical pathway activation. Here we report that the borrelial lipoprotein BBK32 potently and specifically inhibits the classical pathway by binding with high affinity to the initiating C1 complex of complement. In addition, B. burgdorferi cells that produce BBK32 on their surface bind to both C1 and C1r and a serum sensitive derivative of B. burgdorferi is protected from killing via the classical pathway in a BBK32-dependent manner. Subsequent biochemical and biophysical approaches localized the anti-complement activity of BBK32 to its globular C-terminal domain. Mechanistic studies reveal that BBK32 acts by entrapping C1 in its zymogen form by binding and inhibiting the C1 subcomponent, C1r, which serves as the initiating serine protease of the classical pathway. To our knowledge this is the first report of a spirochetal protein acting as a direct inhibitor of the classical pathway and is the only example of a biomolecule capable of specifically and noncovalently inhibiting C1/C1r. By identifying a unique mode of complement evasion this study greatly enhances our understanding of how pathogens subvert and potentially manipulate host innate immune systems.

  17. Borrelia burgdorferi BBK32 Inhibits the Classical Pathway by Blocking Activation of the C1 Complement Complex

    PubMed Central

    Wager, Beau; Höök, Magnus; Skare, Jon T.

    2016-01-01

    Pathogens that traffic in blood, lymphatics, or interstitial fluids must adopt strategies to evade innate immune defenses, notably the complement system. Through recruitment of host regulators of complement to their surface, many pathogens are able to escape complement-mediated attack. The Lyme disease spirochete, Borrelia burgdorferi, produces a number of surface proteins that bind to factor H related molecules, which function as the dominant negative regulator of the alternative pathway of complement. Relatively less is known about how B. burgdorferi evades the classical pathway of complement despite the observation that some sensu lato strains are sensitive to classical pathway activation. Here we report that the borrelial lipoprotein BBK32 potently and specifically inhibits the classical pathway by binding with high affinity to the initiating C1 complex of complement. In addition, B. burgdorferi cells that produce BBK32 on their surface bind to both C1 and C1r and a serum sensitive derivative of B. burgdorferi is protected from killing via the classical pathway in a BBK32-dependent manner. Subsequent biochemical and biophysical approaches localized the anti-complement activity of BBK32 to its globular C-terminal domain. Mechanistic studies reveal that BBK32 acts by entrapping C1 in its zymogen form by binding and inhibiting the C1 subcomponent, C1r, which serves as the initiating serine protease of the classical pathway. To our knowledge this is the first report of a spirochetal protein acting as a direct inhibitor of the classical pathway and is the only example of a biomolecule capable of specifically and noncovalently inhibiting C1/C1r. By identifying a unique mode of complement evasion this study greatly enhances our understanding of how pathogens subvert and potentially manipulate host innate immune systems. PMID:26808924

  18. Antigen Processing and Presentation Mechanisms in Myeloid Cells.

    PubMed

    Roche, Paul A; Cresswell, Peter

    2016-06-01

    Unlike B cells, CD8-positive and CD4-positive T cells of the adaptive immune system do not recognize intact foreign proteins but instead recognize polypeptide fragments of potential antigens. These antigenic peptides are expressed on the surface of antigen presenting cells bound to MHC class I and MHC class II proteins. Here, we review the basics of antigen acquisition by antigen presenting cells, antigen proteolysis into polypeptide fragments, antigenic peptide binding to MHC proteins, and surface display of both MHC class I-peptide and MHC class II-peptide complexes.

  19. Differential inhibition of PDKs by phenylbutyrate and enhancement of pyruvate dehydrogenase complex activity by combination with dichloroacetate.

    PubMed

    Ferriero, Rosa; Iannuzzi, Clara; Manco, Giuseppe; Brunetti-Pierri, Nicola

    2015-09-01

    Pyruvate dehydrogenase complex (PDHC) is a key enzyme in metabolism linking glycolysis to tricarboxylic acid cycle and its activity is tightly regulated by phosphorylation catalyzed by four pyruvate dehydrogenase kinase (PDK) isoforms. PDKs are pharmacological targets for several human diseases including cancer, diabetes, obesity, heart failure, and inherited PDHC deficiency. We investigated the inhibitory activity of phenylbutyrate toward PDKs and found that PDK isoforms 1-to-3 are inhibited whereas PDK4 is unaffected. Moreover, docking studies revealed putative binding sites of phenylbutyrate on PDK2 and 3 that are located on different sites compared to dichloroacetate (DCA), a previously known PDK inhibitor. Based on these findings, we showed both in cells and in mice that phenylbutyrate combined to DCA results in greater increase of PDHC activity compared to each drug alone. These results suggest that therapeutic efficacy can be enhanced by combination of drugs increasing PDHC enzyme activity. PMID:25601413

  20. Tetrathiomolybdate inhibits mitochondrial complex IV and mediates degradation of hypoxia-inducible factor-1α in cancer cells.

    PubMed

    Kim, Kyu Kwang; Abelman, Sarah; Yano, Naohiro; Ribeiro, Jennifer R; Singh, Rakesh K; Tipping, Marla; Moore, Richard G

    2015-01-01

    Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that triggers adaptive responses upon low oxygen conditions and plays a crucial role in cancer metabolism and therapy resistance. Tetrathiomolybdate (TM), a therapy option for copper overload disorder, has also been shown to be capable of limiting tumor angiogenesis, although its underlying mechanism remains unclear. Using ovarian and endometrial cancer cell lines, we observed that TM downregulates HIF-1α protein levels and HIF-transcriptional targets involved in tumor angiogenesis and glycolysis, but did not affect HIF-1α protein synthesis. TM-mediated HIF-1α downregulation was suppressed when HIF-prolyl hydroxylase activity was pharmacologically inhibited using deferoxamine or dimethyloxaloylglycine, and also when the oxygen-dependent degradation domains of HIF-1α, which are responsible for the interaction with HIF-prolyl hydroxylase, were deleted. These findings suggest that TM causes HIF-1α downregulation in a HIF-prolyl hydroxylase-dependent manner. Our studies showed that TM inhibits the activity of the copper-dependent mitochondrial complex IV and reduces mitochondrial respiration, thereby possibly increasing oxygen availability, which is crucial for HIF-prolyl hydroxylase activity. Pimonidazole staining also showed that TM elevates oxygen tension in hypoxic cells. Our studies provide mechanistic evidence for TM-mediated HIF-1α regulation and suggest its therapeutic potential as a method of blocking angiogenesis in ovarian and endometrial tumors.

  1. Inhibition of Human Steroid 5-Reductase (AKR1D1) by Finasteride and Structure of the Enzyme-Inhibitor Complex

    SciTech Connect

    Drury, J.; Di Costanzo, L; Penning, T; Christianson, D

    2009-01-01

    The {Delta}{sup 4}-3-ketosteroid functionality is present in nearly all steroid hormones apart from estrogens. The first step in functionalization of the A-ring is mediated in humans by steroid 5{alpha}- or 5{beta}-reductase. Finasteride is a mechanism-based inactivator of 5{alpha}-reductase type 2 with subnanomolar affinity and is widely used as a therapeutic for the treatment of benign prostatic hyperplasia. It is also used for androgen deprivation in hormone-dependent prostate carcinoma, and it has been examined as a chemopreventive agent in prostate cancer. The effect of finasteride on steroid 5{beta}-reductase (AKR1D1) has not been previously reported. We show that finasteride competitively inhibits AKR1D1 with low micromolar affinity but does not act as a mechanism-based inactivator. The structure of the AKR1D1 {center_dot} NADP{sup +} {center_dot} finasteride complex determined at 1.7 {angstrom} resolution shows that it is not possible for NADPH to reduce the {Delta}{sup 1-2}-ene of finasteride because the cofactor and steroid are not proximal to each other. The C3-ketone of finasteride accepts hydrogen bonds from the catalytic residues Tyr-58 and Glu-120 in the active site of AKR1D1, providing an explanation for the competitive inhibition observed. This is the first reported structure of finasteride bound to an enzyme involved in steroid hormone metabolism.

  2. Tetrathiomolybdate inhibits mitochondrial complex IV and mediates degradation of hypoxia-inducible factor-1α in cancer cells

    PubMed Central

    Kwang Kim, Kyu; Abelman, Sarah; Yano, Naohiro; Ribeiro, Jennifer R.; Singh, Rakesh K.; Tipping, Marla; Moore, Richard G.

    2015-01-01

    Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that triggers adaptive responses upon low oxygen conditions and plays a crucial role in cancer metabolism and therapy resistance. Tetrathiomolybdate (TM), a therapy option for copper overload disorder, has also been shown to be capable of limiting tumor angiogenesis, although its underlying mechanism remains unclear. Using ovarian and endometrial cancer cell lines, we observed that TM downregulates HIF-1α protein levels and HIF-transcriptional targets involved in tumor angiogenesis and glycolysis, but did not affect HIF-1α protein synthesis. TM-mediated HIF-1α downregulation was suppressed when HIF-prolyl hydroxylase activity was pharmacologically inhibited using deferoxamine or dimethyloxaloylglycine, and also when the oxygen-dependent degradation domains of HIF-1α, which are responsible for the interaction with HIF-prolyl hydroxylase, were deleted. These findings suggest that TM causes HIF-1α downregulation in a HIF-prolyl hydroxylase-dependent manner. Our studies showed that TM inhibits the activity of the copper-dependent mitochondrial complex IV and reduces mitochondrial respiration, thereby possibly increasing oxygen availability, which is crucial for HIF-prolyl hydroxylase activity. Pimonidazole staining also showed that TM elevates oxygen tension in hypoxic cells. Our studies provide mechanistic evidence for TM-mediated HIF-1α regulation and suggest its therapeutic potential as a method of blocking angiogenesis in ovarian and endometrial tumors. PMID:26469226

  3. Therapeutic Blockade of Immune Complex-Mediated Glomerulonephritis by Highly Selective Inhibition of Bruton’s Tyrosine Kinase

    PubMed Central

    Chalmers, Samantha A.; Doerner, Jessica; Bosanac, Todd; Khalil, Sara; Smith, Dustin; Harcken, Christian; Dimock, Janice; Der, Evan; Herlitz, Leal; Webb, Deborah; Seccareccia, Elise; Feng, Di; Fine, Jay S.; Ramanujam, Meera; Klein, Elliott; Putterman, Chaim

    2016-01-01

    Lupus nephritis (LN) is a potentially dangerous end organ pathology that affects upwards of 60% of lupus patients. Bruton’s tyrosine kinase (BTK) is important for B cell development, Fc receptor signaling, and macrophage polarization. In this study, we investigated the effects of a novel, highly selective and potent BTK inhibitor, BI-BTK-1, in an inducible model of LN in which mice receive nephrotoxic serum (NTS) containing anti-glomerular antibodies. Mice were treated once daily with vehicle alone or BI-BTK-1, either prophylactically or therapeutically. When compared with control treated mice, NTS-challenged mice treated prophylactically with BI-BTK-1 exhibited significantly attenuated kidney disease, which was dose dependent. BI-BTK-1 treatment resulted in decreased infiltrating IBA-1+ cells, as well as C3 deposition within the kidney. RT-PCR on whole kidney RNA and serum profiling indicated that BTK inhibition significantly decreased levels of LN-relevant inflammatory cytokines and chemokines. Renal RNA expression profiling by RNA-seq revealed that BI-BTK-1 dramatically modulated pathways related to inflammation and glomerular injury. Importantly, when administered therapeutically, BI-BTK-1 reversed established proteinuria and improved renal histopathology. Our results highlight the important role for BTK in the pathogenesis of immune complex-mediated nephritis, and BTK inhibition as a promising therapeutic target for LN. PMID:27192942

  4. Inhibition of Tumor Proteasome Activity by Gold Dithiocarbamato Complexes via both Redox-Dependent and –Independent Processes

    PubMed Central

    Milacic, Vesna; Ronconi, Luca; Fan, Yuhua; Bi, Caifeng; Fregona, Dolores; Dou, Q Ping

    2013-01-01

    We have previously reported on a gold(III) complex, namely [AuBr2(DMDT)] (N,N-dimethyldithiocarbamate) showing potent in vitro and in vivo growth inhibitory activities toward human cancer cells and identifying the cellular proteasome as one of the major targets. However, the importance of the oxidation state of the gold center and the involved mechanism of action has yet to be established. Here we show that both gold(III)- and gold(I)-dithiocarbamato species, namely [AuBr2(ESDT)] (AUL12) and [Au(ESDT)]2 (AUL15), could inhibit the chymotrypsin-like activity of purified 20S proteasome and 26S proteasome in human breast cancer MDA-MB-231 cells, resulting in accumulation of ubiquitinated proteins and proteasome target proteins, and induction of cell death, but at significantly different levels. Gold(I) and gold(III) compounds-mediated proteasome inhibition and cell death induction were completely reversed by the addition of a reducing agent, dithiothreitol or N-acetyl-l-cysteine, suggesting the involvement of redox processes. Furthermore, treatment of MDA-MB-231 cells with gold(III) compound (AUL12), but not the gold(I) analogue (AUL15), resulted in the production of significant level of reactive oxygen species. Our study provides strong evidence that the cellular proteasome is an imporant target of both gold(I) and gold(III) dithiocarbamates, but distinct cellular mechanisms of action are responsible for their different overall effect. PMID:19911377

  5. Salicylic Acid Inhibits the Replication of Tomato bushy stunt virus by Directly Targeting a Host Component in the Replication Complex.

    PubMed

    Tian, Miaoying; Sasvari, Zsuzsanna; Gonzalez, Paulina Alatriste; Friso, Giulia; Rowland, Elden; Liu, Xiao-Min; van Wijk, Klaas J; Nagy, Peter D; Klessig, Daniel F

    2015-04-01

    Although the plant hormone salicylic acid (SA) plays a central role in signaling resistance to viral infection, the underlying mechanisms are only partially understood. Identification and characterization of SA's direct targets have been shown to be an effective strategy for dissecting the complex SA-mediated defense signaling network. In search of additional SA targets, we previously developed two sensitive approaches that utilize SA analogs in conjunction with either a photoaffinity labeling technique or surface plasmon resonance-based technology to identify and evaluate candidate SA-binding proteins (SABPs) from Arabidopsis. Using these approaches, we have now identified several members of the Arabidopsis glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein family, including two chloroplast-localized and two cytosolic isoforms, as SABPs. Cytosolic GAPDH is a well-known glycolytic enzyme; it also is an important host factor involved in the replication of Tomato bushy stunt virus (TBSV), a single-stranded RNA virus. Using a yeast cell-free extract, an in vivo yeast replication system, and plant protoplasts, we demonstrate that SA inhibits TBSV replication. SA does so by inhibiting the binding of cytosolic GAPDH to the negative (-)RNA strand of TBSV. Thus, this study reveals a novel molecular mechanism through which SA regulates virus replication. PMID:25584724

  6. Mondo complexes regulate TFEB via TOR inhibition to promote longevity in response to gonadal signals

    PubMed Central

    Nakamura, Shuhei; Karalay, Özlem; Jäger, Philipp S.; Horikawa, Makoto; Klein, Corinna; Nakamura, Kayo; Latza, Christian; Templer, Sven E.; Dieterich, Christoph; Antebi, Adam

    2016-01-01

    Germline removal provokes longevity in several species and shifts resources towards survival and repair. Several Caenorhabditis elegans transcription factors regulate longevity arising from germline removal; yet, how they work together is unknown. Here we identify a Myc-like HLH transcription factor network comprised of Mondo/Max-like complex (MML-1/MXL-2) to be required for longevity induced by germline removal, as well as by reduced TOR, insulin/IGF signalling and mitochondrial function. Germline removal increases MML-1 nuclear accumulation and activity. Surprisingly, MML-1 regulates nuclear localization and activity of HLH-30/TFEB, a convergent regulator of autophagy, lysosome biogenesis and longevity, by downregulating TOR signalling via LARS-1/leucyl-transfer RNA synthase. HLH-30 also upregulates MML-1 upon germline removal. Mammalian MondoA/B and TFEB show similar mutual regulation. MML-1/MXL-2 and HLH-30 transcriptomes show both shared and preferential outputs including MDL-1/MAD-like HLH factor required for longevity. These studies reveal how an extensive interdependent HLH transcription factor network distributes responsibility and mutually enforces states geared towards reproduction or survival. PMID:27001890

  7. Mondo complexes regulate TFEB via TOR inhibition to promote longevity in response to gonadal signals.

    PubMed

    Nakamura, Shuhei; Karalay, Özlem; Jäger, Philipp S; Horikawa, Makoto; Klein, Corinna; Nakamura, Kayo; Latza, Christian; Templer, Sven E; Dieterich, Christoph; Antebi, Adam

    2016-01-01

    Germline removal provokes longevity in several species and shifts resources towards survival and repair. Several Caenorhabditis elegans transcription factors regulate longevity arising from germline removal; yet, how they work together is unknown. Here we identify a Myc-like HLH transcription factor network comprised of Mondo/Max-like complex (MML-1/MXL-2) to be required for longevity induced by germline removal, as well as by reduced TOR, insulin/IGF signalling and mitochondrial function. Germline removal increases MML-1 nuclear accumulation and activity. Surprisingly, MML-1 regulates nuclear localization and activity of HLH-30/TFEB, a convergent regulator of autophagy, lysosome biogenesis and longevity, by downregulating TOR signalling via LARS-1/leucyl-transfer RNA synthase. HLH-30 also upregulates MML-1 upon germline removal. Mammalian MondoA/B and TFEB show similar mutual regulation. MML-1/MXL-2 and HLH-30 transcriptomes show both shared and preferential outputs including MDL-1/MAD-like HLH factor required for longevity. These studies reveal how an extensive interdependent HLH transcription factor network distributes responsibility and mutually enforces states geared towards reproduction or survival. PMID:27001890

  8. Inhibition of the Tcf/beta-catenin complex increases apoptosis and impairs adrenocortical tumor cell proliferation and adrenal steroidogenesis

    PubMed Central

    Leal, Letícia F.; Bueno, Ana Carolina; Gomes, Débora C.; Abduch, Rafael; de Castro, Margaret; Antonini, Sonir R.

    2015-01-01

    Background To date, there is no effective therapy for patients with advanced/metastatic adrenocortical cancer (ACC). The activation of the Wnt/beta-catenin signaling is frequent in ACC and this pathway is a promising therapeutic target. Aim To investigate the effects of the inhibition of the Wnt/beta-catenin in ACC cells. Methods Adrenal (NCI-H295 and Y1) and non-adrenal (HeLa) cell lines were treated with PNU-74654 (5–200 μM) for 24–96 h to assess cell viability (MTS-based assay), apoptosis (Annexin V), expression/localization of beta-catenin (qPCR, immunofluorescence, immunocytochemistry and western blot), expression of beta-catenin target genes (qPCR and western blot), and adrenal steroidogenesis (radioimmunoassay, qPCR and western blot). Results In NCI-H295 cells, PNU-74654 significantly decreased cell proliferation 96 h after treatment, increased early and late apoptosis, decreased nuclear beta-catenin accumulation, impaired CTNNB1/beta-catenin expression and increased beta-catenin target genes 48 h after treatment. No effects were observed on HeLa cells. In NCI-H295 cells, PNU-74654 decreased cortisol, testosterone and androstenedione secretion 24 and 48 h after treatment. Additionally, in NCI-H295 cells, PNU-74654 decreased SF1 and CYP21A2 mRNA expression as well as the protein levels of STAR and aldosterone synthase 48 h after treatment. In Y1 cells, PNU-74654 impaired corticosterone secretion 24 h after treatment but did not decrease cell viability. Conclusions Blocking the Tcf/beta-catenin complex inhibits the Wnt/beta-catenin signaling in adrenocortical tumor cells triggering increased apoptosis, decreased cell viability and impairment of adrenal steroidogenesis. These promising findings pave the way for further experiments inhibiting the Wnt/beta-catenin pathway in pre-clinical models of ACC. The inhibition of this pathway may become a promising adjuvant therapy for patients with ACC. PMID:26515592

  9. Antioxidant-mediated reversal of oxidative damage in mouse modeling of complex I inhibition.

    PubMed

    Parameshwaran, Kodeeswaran; Irwin, Michael H; Steliou, Kosta; Suppiramaniam, Vishnu; Pinkert, Carl A

    2015-03-01

    Mitochondrial dysfunction is a key component of various aging-related pathologies of the brain that result in dementia. As such, it provides an important avenue in development of therapeutic interventions for a host of neurological disorders. A requirement for functional mitochondrial respiratory chain complex I (CI), to accomplish the normal physiological processes regulating memory, seems intuitive. In the present study, a synthetic lipoylcarnitine antioxidant (PMX-500FI; 100 mg/kg/day po) was administered to female ICR mice (3-4-month old) that were subsequently treated with the mitochondrial CI inhibitor, rotenone (400 mg/kg/day). After 1 week, rotenone-induced impairment of neuronal function was evaluated in the hippocampus, a brain region that is involved in regulating memory formation. Electrophysiological recordings in live brain slices showed that long-term potentiation (LTP) was reduced by rotenone exposure (P < 0.05) while pretreatment with PMX-500FI maintained LTP similar to control levels (P > 0.05). Potentiation during theta burst stimulation (TBS) was similar among treatment groups (P > 0.05); however, neurotransmitter release, which increased in control mice after TBS, was lower in rotenone treated mice (P < 0.05), and was accompanied by reduced basal synaptic transmission (P < 0.05), increased proapoptotic signaling and decreased extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylation (P < 0.05). For each of these determinations, pretreatment with PMX-500FI alleviated the harmful effects of rotenone. These results illustrate that treatment with antioxidant PMX-500FI is protective against rotenone-induced impairment of neuronal bioenergetics in the mouse hippocampus, in regard to both excitatory synaptic physiology and proapoptotic signaling. The protective effect of PMX-500FI against rotenone-induced disruption of cellular bioenergetics may have important therapeutic implications for treating aging-related dementia

  10. Rationally designed inhibitor targeting antigen-trimming aminopeptidases enhances antigen presentation and cytotoxic T-cell responses.

    PubMed

    Zervoudi, Efthalia; Saridakis, Emmanuel; Birtley, James R; Seregin, Sergey S; Reeves, Emma; Kokkala, Paraskevi; Aldhamen, Yasser A; Amalfitano, Andrea; Mavridis, Irene M; James, Edward; Georgiadis, Dimitris; Stratikos, Efstratios

    2013-12-01

    Intracellular aminopeptidases endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2), and as well as insulin-regulated aminopeptidase (IRAP) process antigenic epitope precursors for loading onto MHC class I molecules and regulate the adaptive immune response. Their activity greatly affects the antigenic peptide repertoire presented to cytotoxic T lymphocytes and as a result can regulate cytotoxic cellular responses contributing to autoimmunity or immune evasion by viruses and cancer cells. Therefore, pharmacological regulation of their activity is a promising avenue for modulating the adaptive immune response with possible applications in controlling autoimmunity, in boosting immune responses to pathogens, and in cancer immunotherapy. In this study we exploited recent structural and biochemical analysis of ERAP1 and ERAP2 to design and develop phosphinic pseudopeptide transition state analogs that can inhibit this family of enzymes with nM affinity. X-ray crystallographic analysis of one such inhibitor in complex with ERAP2 validated our design, revealing a canonical mode of binding in the active site of the enzyme, and highlighted the importance of the S2' pocket for achieving inhibitor potency. Antigen processing and presentation assays in HeLa and murine colon carcinoma (CT26) cells showed that these inhibitors induce increased cell-surface antigen presentation of transfected and endogenous antigens and enhance cytotoxic T-cell responses, indicating that these enzymes primarily destroy epitopes in those systems. This class of inhibitors constitutes a promising tool for controlling the cellular adaptive immune response in humans by modulating the antigen processing and presentation pathway.

  11. Ia-antigen-T-cell interactions for a thymus-independent antigen composed of D amino acids.

    PubMed Central

    Zisman, E; Dayan, M; Sela, M; Mozes, E

    1993-01-01

    Synthetic polypeptide antigens of L amino acids, although bearing repeating sequences, are thymus-dependent (L-TD), whereas the same polymers composed of D amino acids are thymus-independent (D-TI), probably due to a slower rate of metabolism. Yet we found that lymph-node cells of BALB/c mice immunized with D-TI proliferate in response to it in vitro. To follow T-cell activation by D-TI, we established T-cell hybridomas to D-TI and to its analog composed of L isomers, L-TD, for comparison. The T-cell hybridomas express membrane alpha/beta T-cell receptors and secrete interleukin 2 upon stimulation with the respective antigen. In addition, D-TI-specific hybridomas are stimulated, to a lesser extent, by the L-TD antigen, whereas only some L-TD-specific hybridomas recognize D-TI. Moreover, biotinylated analogs of D-TI and L-TD bind to splenic antigen-presenting cells (APCs) from BALB/c mice. Binding is inhibited by an excess of nonbiotinylated L-TD, and by an excess of a peptide comprising residues 259-271 of the human acetylcholine receptor alpha subunit, which binds to I-Ad and I-Ed molecules without prior processing. Analysis of APC lysates following incubation of the APCs with biotinylated D-TI and L-TD reveals that the biotinylated antigen moiety is associated with Ia molecules. D-TI and L-TD bind to Ia molecules on intact APCs with similar KD values, 5 x 10(-8) M and 3 x 10(-8) M, respectively. However, D-TI has faster kinetics of binding than L-TD, probably due to different processing requirements. Hence, we have demonstrated a major histocompatibility complex class II-mediated T-cell response to a thymus-independent antigen. Images PMID:8381541

  12. Selective inhibition of the prothrombinase complex: factor Va alters macromolecular recognition of a tick anticoagulant peptide mutant by factor Xa.

    PubMed

    Betz, A; Vlasuk, G P; Bergum, P W; Krishnaswamy, S

    1997-01-01

    The prothrombinase complex assembles through reversible interactions between the protease, factor Xa, the cofactor, factor Va, and acidic phospholipid membranes in the presence of calcium ions. Changes in macromolecular recognition by factor Xa which may result from its interaction with factor Va in the prothrombinase complex have been probed using a recombinant derivative of tick anticoagulant peptide where Arg3 has been replaced with Ala (R3A-TAP). In contrast to the wild type inhibitor, R3A-TAP was a weak competitive inhibitor of factor Xa (Ki = 794 nM). The inhibition of the prothrombinase complex by R3A-TAP was characterized by slow, tight-binding kinetics with an increased affinity of approximately 4000-fold (Ki* = 0.195 nM) relative to that of solution-phase factor Xa. Stopped-flow measurements using p-aminobenzamidine (PAB) demonstrated that the reaction between solution-phase factor Xa and R3A-TAP could be adequately described by a single reversible step with rate constants that were consistent with equilibrium binding measurements. The rate-limiting bimolecular combination of R3A-TAP and factor Xa was competitive with PAB binding of the protease. In contrast, the reaction of R3A-TAP with prothrombinase measured using PAB yielded biphasic stopped-flow traces, indicating a multistep pathway for the reaction of the inhibitor with the enzyme complex. The kinetic measurements were consistent with the initial formation of a ternary complex between R3A-TAP, prothrombinase, and PAB followed by two unimolecular steps which lead to PAB dissociation from the enzyme. In this case, prior occupation of the active site by PAB had no effect on the bimolecular reaction between R3A-TAP and prothrombinase. Thus, the interaction of factor Xa with factor Va on the membrane surface alters recognition of R3A-TAP by the protease, leading to changes in the thermodynamics as well as in the observed kinetic mechanism for the reaction. Therefore, a single amino acid substitution in

  13. Id2 complexes with the SNAG domain of Snai1 inhibiting Snai1-mediated repression of integrin β4.

    PubMed

    Chang, Cheng; Yang, Xiaofang; Pursell, Bryan; Mercurio, Arthur M

    2013-10-01

    The epithelial-mesenchymal transition (EMT) is a fundamental process that underlies development and cancer. Although the EMT involves alterations in the expression of specific integrins that mediate stable adhesion to the basement membrane, such as α6β4, the mechanisms involved are poorly understood. Here, we report that Snai1 inhibits β4 transcription by increasing repressive histone modification (trimethylation of histone H3 at K27 [H3K27Me3]). Surprisingly, Snai1 is expressed and localized in the nucleus in epithelial cells, but it does not repress β4. We resolved this paradox by discovering that Id2 complexes with the SNAG domain of Snai1 on the β4 promoter and constrains the repressive function of Snai1. Disruption of the complex by depleting Id2 resulted in Snai1-mediated β4 repression with a concomitant increase in H3K27Me3 modification on the β4 promoter. These findings establish a novel function for Id2 in regulating Snai1 that has significant implications for the regulation of epithelial gene expression.

  14. Inhibition of constitutive signal transducer and activator of transcription 3 activation by novel platinum complexes with potent antitumor activity.

    PubMed

    Turkson, James; Zhang, Shumin; Palmer, Jay; Kay, Heidi; Stanko, Joseph; Mora, Linda B; Sebti, Said; Yu, Hua; Jove, Richard

    2004-12-01

    DNA-alkylating agents that are platinum complexes induce apoptotic responses and have wide application in cancer therapy. The potential for platinum compounds to modulate signal transduction events that contribute to their therapeutic outcome has not been extensively examined. Among the signal transducer and activator of transcription (STAT) proteins, Stat3 activity is frequently up-regulated in many human tumors. Various lines of evidence have established a causal role for aberrant Stat3 activity in malignant transformation and provided validation for its targeting in the development of small-molecule inhibitors as novel cancer therapeutics. We report here that platinum-containing compounds disrupt Stat3 signaling and suppress its biological functions. The novel platinum (IV) compounds, CPA-1, CPA-7, and platinum (IV) tetrachloride block Stat3 activity in vitro at low micromolar concentrations. In malignant cells that harbor constitutively activated Stat3, CPA-1, CPA-7, and platinum (IV) tetrachloride inhibit cell growth and induce apoptosis in a manner that reflects the attenuation of persistent Stat3 activity. By contrast, cells that do not contain persistent Stat3 activity are marginally affected or are not affected by these compounds. Moreover, CPA-7 induces the regression of mouse CT26 colon tumor, which correlates with the abrogation of persistent Stat3 activity in tumors. Thus, the modulation of oncogenic signal transduction pathways, such as Stat3, may be one of the key molecular mechanisms for the antitumor effects of platinum (IV)-containing complexes.

  15. Inhibition of the FACT Complex Reduces Transcription from the Human Cytomegalovirus Major Immediate Early Promoter in Models of Lytic and Latent Replication.

    PubMed

    O'Connor, Christine M; Nukui, Masatoshi; Gurova, Katerina V; Murphy, Eain A

    2016-04-01

    The successful colonization of the majority of the population by human cytomegalovirus is a direct result of the virus's ability to establish and, more specifically, reactivate from latency. The underlying cellular factors involved in viral reactivation remain unknown. Here, we show that the host complexfacilitateschromatintranscription (FACT) binds to the major immediate early promoter (MIEP) and that inhibition of this complex reduces MIEP transactivation, thus inhibiting viral reactivation. PMID:26865717

  16. Protein C inhibits endocytosis of thrombin-thrombomodulin complexes in A549 lung cancer cells and human umbilical vein endothelial cells

    SciTech Connect

    Maruyama, I.; Majerus, P.W.

    1987-05-01

    We investigated the effect of protein C on the endocytosis of thrombin-thrombomodulin complexes. We previously showed that exposure of umbilical vein endothelial cells to thrombin stimulated the internalization and degradation of thrombin. A similar internalization was stimulated by a monoclonal antithrombomodulin antibody. We have repeated these studies in the presence of protein C and found that endocytosis of /sup 125/I-thrombin-thrombomodulin complexes, but not /sup 125/I-antithrombomodulin-thrombomodulin complexes, is inhibited. Activated protein C did not inhibit endocytosis of thrombin-thrombomodulin complexes. Protein C inhibited both internalization and degradation of /sup 125/I-thrombin and diisopropylphosphoryl (DIP) /sup 125/I-thrombin in human lung cancer cells (A549). These effects were observed at protein C concentrations found in human plasma. Protein S had no effect on the inhibition of endocytosis of thrombin-thrombomodulin complexes by protein C. We propose that protein C may regulate the rate of endocytosis of thrombin-thrombomodulin complexes in vivo and thereby control the capacity for endothelium to activate protein C.

  17. Icariin inhibits oxidized low-density lipoprotein-induced proliferation of vascular smooth muscle cells by suppressing activation of extracellular signal-regulated kinase 1/2 and expression of proliferating cell nuclear antigen.

    PubMed

    Hu, Yanwu; Liu, Kai; Yan, Mengtong; Zhang, Yang; Wang, Yadi; Ren, Liqun

    2016-03-01

    Icariin, a flavonoid isolated from the traditional Chinese herbal medicine Epimedium brevicornum Maxim, has been shown to possess anti-inflammatory, anti‑oxidant and anti-atherosclerotic activities in vivo and in vitro. The aim of the present study was to investigate the effects of icariin on oxidized low‑density lipoprotein (ox-LDL)-induced proliferation of vascular smooth muscle cells (VSMCs) and the possible underlying mechanism. VSMCs were cultured and pre‑treated with various concentrations of icariin (0, 10, 20 or 40 µm) prior to stimulation by ox‑LDL (50 µg/ml). Cell proliferation was evaluated by an MTT assay. Flow cytometry was used to study the influence of icariin on the cell cycle. Proliferating cell nuclear antigen (PCNA) expression and phosphorylation levels of extracellular signal-regulated kinase (ERK)1/2 were detected by western blot analysis. The results indicated that icariin significantly inhibited ox‑LDL‑induced proliferation of VSMCs and phosphorylation of ERK1/2. Furthermore, icariin also blocked the ox‑LDL‑induced cell‑cycle progression at G1/S‑interphase and downregulated the expression of PCNA in VSMCs. In conclusion, the present study indicated for the first time that icariin reduced the amount of ox‑LDL‑induced proliferation of VSMCs through suppression of PCNA expression and inactivation of ERK1/2.

  18. Cancer vaccine--Antigenics.

    PubMed

    2002-01-01

    purified complexes of tumour-derived HSPs linked to tumour antigen peptides. When these HSPPC are readministered to a patient following surgery or biopsy of the tumour, the antigenic tumour peptides are expressed on the surface of potent antigen-presenting cells of the immune system, such as macrophages and dendritic cells. This stimulates a much more powerful anti-tumour immune response than that generated by expression of the same antigens by the tumour cell. Thus, Antigenics autologous HSP technology is attractive because it is highly specific for individual patients and circumvents the need for identification of specific antigens for individual cancers (i.e. it does not require definition of the antigenic epitopes on cancer cells) and it overcomes the immune tolerance associated with various tumours. Oncophage is manufactured in a 10-hour process from surgically resected autologous tumour. A minimum of 1-3g of tumour tissue is required to produce enough Oncophage for a course of treatment. The major limiting factor for producing Oncophage from a particular cancer is the ability to purify HSP from that cancer. From clinical studies to date, Antigenics has been able to produce HSP from 100, 98, 90, 71 and 30% of colorectal carcinoma, renal cell carcinoma, melanoma, gastric cancer and pancreatic cancer tumours, respectively. The low success rate with pancreatic cancers is because of the high concentration of proteases in that tissue type. HSPs are a family of highly conserved proteins present in the cells of all organisms. They function as molecular chaperones, assisting the correct folding of polypeptides and aiding intracellular protein transport. In addition, HSPs associate with a broad range of peptides derived from intracellular protein degradation, including antigenic peptides produced in tumour cells. Antigenics has exclusively licensed worldwide rights to its HSP immunotherapeutic complexes from Mount Sinai School of Medicine and Fordham University in the USA. On

  19. Properties of glycolipid-enriched membrane rafts in antigen presentation.

    PubMed

    Rodgers, William; Smith, Kenneth

    2005-01-01

    Presentation of antigen to T cells represents one of the central events in the engagement of the immune system toward the defense of the host against pathogens. Accordingly, understanding the mechanisms by which antigen presentation occurs is critical toward our understanding the properties of host defense against foreign antigen, as well as insight into other features of the immune system, such as autoimmune disease. The entire antigen-presentation event is complex, and many features of it remain poorly understood. However, recent studies have provided evidence showing that glycolipid-enriched membrane rafts are important for efficient antigen presentation; the studies suggest that one such function of rafts is trafficking of antigen-MHC II complexes to the presentation site on the surface of the antigen-presenting cell. Here, we present a critical discussion of rafts and their proposed functions in antigen presentation. Emerging topics of rafts and antigen presentation that warrant further investigation are also highlighted.

  20. The production and crystallization of the human leukocyte antigen class II molecules HLA-DQ2 and HLA-DQ8 complexed with deamidated gliadin peptides implicated in coeliac disease

    SciTech Connect

    Henderson, Kate N.; Reid, Hugh H.; Borg, Natalie A.; Broughton, Sophie E.; Huyton, Trevor; Anderson, Robert P.; McCluskey, James; Rossjohn, Jamie

    2007-12-01

    The production and crystallization of human leukocyte antigen class II molecules HLA-DQ2 and HLA-DQ8 in complex with deamidated gliadin peptides is reported. Crystals of HLA-DQ2{sup PQPELPYPQ} diffracted to 3.9 Å, while the HLA-DQ8{sup EGSFQPSQE} crystals diffracted to 2.1 Å, allowing structure determination by molecular replacement. The major histocompatibility complex (MHC) class II molecules HLA-DQ2 and HLA-DQ8 are key risk factors in coeliac disease, as they bind deamidated gluten peptides that are subsequently recognized by CD4{sup +} T cells. Here, the production and crystallization of both HLA-DQ2 and HLA-DQ8 in complex with the deamidated gliadin peptides DQ2 α-I (PQPELPYPQ) and DQ8 α-I (EGSFQPSQE), respectively, are reported.

  1. Carbon monoxide impairs mitochondria-dependent endosomal maturation and antigen presentation in dendritic cells.

    PubMed

    Riquelme, Sebastián A; Pogu, Julien; Anegon, Ignacio; Bueno, Susan M; Kalergis, Alexis M

    2015-12-01

    Heme-oxygenase 1 (HO-1) prevents T cell-mediated inflammatory disease by producing carbon monoxide (CO) and impairing DC immunogenicity. However, the cellular mechanisms causing this inhibition are unknown. Here, we show that CO impairs mitochondrial function in DCs by reducing both the mitochondrial membrane potential and ATP production, and resembling the effect of a nonlethal dose of a classical mitochondria uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Moreover, both CO and CCCP reduced cargo transport, endosome-to-lysosome fusion, and antigen processing, dampening the production of peptide-MHC complexes on the surface of DCs. As a result, the inhibition of naive CD4(+) T-cell priming was observed. Furthermore, mitochondrial dysfunction in DCs also significantly reduced CD8(+) T cell-dependent type 1 diabetes onset in vivo. These results showed for the first time that CO interferes with T-cell priming by blocking an unknown mitochondria-dependent antigen-processing pathway in mature DC. Interestingly, other immune functions in DCs such as antigen capture, cytokine secretion, costimulation, and cell survival relied on glycolysis, suggesting that oxidative phosphorylation might only play a key role for the maturation of antigen-containing endosomes. In conclusion, CO produced by HO-1 impairs antigen-dependent inflammation by regulating DC immunogenicity by a mitochondria-dependent mechanism. PMID:26461179

  2. Carbon monoxide impairs mitochondria-dependent endosomal maturation and antigen presentation in dendritic cells.

    PubMed

    Riquelme, Sebastián A; Pogu, Julien; Anegon, Ignacio; Bueno, Susan M; Kalergis, Alexis M

    2015-12-01

    Heme-oxygenase 1 (HO-1) prevents T cell-mediated inflammatory disease by producing carbon monoxide (CO) and impairing DC immunogenicity. However, the cellular mechanisms causing this inhibition are unknown. Here, we show that CO impairs mitochondrial function in DCs by reducing both the mitochondrial membrane potential and ATP production, and resembling the effect of a nonlethal dose of a classical mitochondria uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Moreover, both CO and CCCP reduced cargo transport, endosome-to-lysosome fusion, and antigen processing, dampening the production of peptide-MHC complexes on the surface of DCs. As a result, the inhibition of naive CD4(+) T-cell priming was observed. Furthermore, mitochondrial dysfunction in DCs also significantly reduced CD8(+) T cell-dependent type 1 diabetes onset in vivo. These results showed for the first time that CO interferes with T-cell priming by blocking an unknown mitochondria-dependent antigen-processing pathway in mature DC. Interestingly, other immune functions in DCs such as antigen capture, cytokine secretion, costimulation, and cell survival relied on glycolysis, suggesting that oxidative phosphorylation might only play a key role for the maturation of antigen-containing endosomes. In conclusion, CO produced by HO-1 impairs antigen-dependent inflammation by regulating DC immunogenicity by a mitochondria-dependent mechanism.

  3. Overexpression of Mxi1 inhibits the induction of the human ornithine decarboxylase gene by the Myc/Max protein complex.

    PubMed

    Wu, S; Peña, A; Korcz, A; Soprano, D R; Soprano, K J

    1996-02-01

    We have previously shown that the Myc/Max protein complex plays a role in the growth-associated expression of the human ornithine decarboxylase gene. Mxi1 and Mad, novel Max-associated proteins have been identified and shown to form heterodimers with Max which bind efficiently to the Myc/Max consensus recognition sequence, CACGTG, in vitro. However, formation of Max/Mxi1 or Max/Mad heterodimers results in a reduction in Myc/Max dependent transcriptional activation of reporter plasmid constructs containing the consensus element. In light of the evidence that ODC is transcriptionally regulated in vitro and in vivo by the Myc/Max protein complex and the potential role of Mxi1 and Mad as antagonists of Myc transactivation activity, we set out to determine if one of these Max associated proteins, Mxi1, could affect the regulation of ODC expression by Myc/Max and if this regulation was correlated to growth status. Our results show that overexpression of Mxi1 does in fact inhibit ODC gene expression in a dose-dependent manner both in vivo and in vitro. In addition, evidence is presented which shows that levels of Mxi1 are up-regulated during long term quiescence and down-regulated following growth stimulation by serum. These results suggest that alterations in the levels of Max-associated proteins such as Mxi1 can modulate critical levels of functional Myc/Max protein complexes. This can alter transcriptional transactivation of Myc-regulated targets and as a consequence affect levels of genes essential for initiation and/or maintenance of growth.

  4. Small structural changes on a hydroquinone scaffold determine the complex I inhibition or uncoupling of tumoral oxidative phosphorylation.

    PubMed

    Urra, Félix A; Córdova-Delgado, Miguel; Lapier, Michel; Orellana-Manzano, Andrea; Acevedo-Arévalo, Luis; Pessoa-Mahana, Hernán; González-Vivanco, Jaime M; Martínez-Cifuentes, Maximiliano; Ramírez-Rodríguez, Oney; Millas-Vargas, Juan Pablo; Weiss-López, Boris; Pavani, Mario; Ferreira, Jorge; Araya-Maturana, Ramiro

    2016-01-15

    Mitochondria participate in several distinctiveness of cancer cell, being a promising target for the design of anti-cancer compounds. Previously, we described that ortho-carbonyl hydroquinone scaffold 14 inhibits the complex I-dependent respiration with selective anti-proliferative effect on mouse mammary adenocarcinoma TA3/Ha cancer cells; however, the structural requirements of this hydroquinone scaffold to affect the oxidative phosphorylation (OXPHOS) of cancer cells have not been studied in detail. Here, we characterize the mitochondrial metabolism of TA3/Ha cancer cells, which exhibit a high oxidative metabolism, and evaluate the effect of small structural changes of the hydroquinone scaffold 14 on the respiration of this cell line. Our results indicate that these structural changes modify the effect on OXPHOS, obtaining compounds with three alternative actions: inhibitors of complex I-dependent respiration, uncoupler of OXPHOS and compounds with both actions. To confirm this, the effect of a bicyclic hydroquinone (9) was evaluated in isolated mitochondria. Hydroquinone 9 increased mitochondrial respiration in state 4o without effects on the ADP-stimulated respiration (state 3ADP), decreasing the complexes I and II-dependent respiratory control ratio. The effect on mitochondrial respiration was reversed by 6-ketocholestanol addition, indicating that this hydroquinone is a protonophoric uncoupling agent. In intact TA3/Ha cells, hydroquinone 9 caused mitochondrial depolarization, decreasing intracellular ATP and NAD(P)H levels and GSH/GSSG ratio, and slightly increasing the ROS levels. Moreover, it exhibited selective NAD(P)H availability-dependent anti-proliferative effect on cancer cells. Therefore, our results indicate that the ortho-carbonyl hydroquinone scaffold offers the possibility to design compounds with specific actions on OXPHOS of cancer cells. PMID:26712467

  5. Small structural changes on a hydroquinone scaffold determine the complex I inhibition or uncoupling of tumoral oxidative phosphorylation.

    PubMed

    Urra, Félix A; Córdova-Delgado, Miguel; Lapier, Michel; Orellana-Manzano, Andrea; Acevedo-Arévalo, Luis; Pessoa-Mahana, Hernán; González-Vivanco, Jaime M; Martínez-Cifuentes, Maximiliano; Ramírez-Rodríguez, Oney; Millas-Vargas, Juan Pablo; Weiss-López, Boris; Pavani, Mario; Ferreira, Jorge; Araya-Maturana, Ramiro

    2016-01-15

    Mitochondria participate in several distinctiveness of cancer cell, being a promising target for the design of anti-cancer compounds. Previously, we described that ortho-carbonyl hydroquinone scaffold 14 inhibits the complex I-dependent respiration with selective anti-proliferative effect on mouse mammary adenocarcinoma TA3/Ha cancer cells; however, the structural requirements of this hydroquinone scaffold to affect the oxidative phosphorylation (OXPHOS) of cancer cells have not been studied in detail. Here, we characterize the mitochondrial metabolism of TA3/Ha cancer cells, which exhibit a high oxidative metabolism, and evaluate the effect of small structural changes of the hydroquinone scaffold 14 on the respiration of this cell line. Our results indicate that these structural changes modify the effect on OXPHOS, obtaining compounds with three alternative actions: inhibitors of complex I-dependent respiration, uncoupler of OXPHOS and compounds with both actions. To confirm this, the effect of a bicyclic hydroquinone (9) was evaluated in isolated mitochondria. Hydroquinone 9 increased mitochondrial respiration in state 4o without effects on the ADP-stimulated respiration (state 3ADP), decreasing the complexes I and II-dependent respiratory control ratio. The effect on mitochondrial respiration was reversed by 6-ketocholestanol addition, indicating that this hydroquinone is a protonophoric uncoupling agent. In intact TA3/Ha cells, hydroquinone 9 caused mitochondrial depolarization, decreasing intracellular ATP and NAD(P)H levels and GSH/GSSG ratio, and slightly increasing the ROS levels. Moreover, it exhibited selective NAD(P)H availability-dependent anti-proliferative effect on cancer cells. Therefore, our results indicate that the ortho-carbonyl hydroquinone scaffold offers the possibility to design compounds with specific actions on OXPHOS of cancer cells.

  6. Immune complex glomerulonephritis in experimental kala-azar. II: Detection and characterization of parasite antigens and antibodies eluted from kidneys of Leishmania donovani-infected hamsters.

    PubMed Central

    Sartori, A; Roque-Barreira, M C; Coe, J; Campos-Neto, A

    1992-01-01

    In a previous report analysing kidney sections by immunofluorescence we showed that hamsters infected with L. donovani develop a glomerulonephritis (GN) associated with deposition of hamster immunoglobulins and parasite antigens in the glomeruli. In this study we characterize these immune components eluted from the kidneys. The eluted immunoglobulins showed specificity for L. donovani antigens and hamster immunoglobulins (rheumatoid factor-like activity). The four isotypes IgG1, IgG2, IgA and IgM were detected. Several L. donovani antigens were detected in the renal eluates by Western blot and immunoprecipitation using 125I-labelled eluates. Proteins with mol. wt of 134, 82, 52, 31, and 26 kD were detected by Western blot and proteins with 134, 110, 93, 89 and 48 kD were detected by immunoprecipitation. With the exception of the 134 kD protein which was recognized by both rabbit anti-promastigote and rabbit anti-amastigote sera all the others were recognized only by the anti-amastigote serum. The 134 kD protein was the only one isolated from the kidneys of infected hamster immunocomplexed with IgG and was the only one detected in a promastigote lysate using IgG from L. donovani-infected hamsters. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:1544224

  7. Integrating influenza antigenic dynamics with molecular evolution

    PubMed Central

    Bedford, Trevor; Suchard, Marc A; Lemey, Philippe; Dudas, Gytis; Gregory, Victoria; Hay, Alan J; McCauley, John W; Russell, Colin A; Smith, Derek J; Rambaut, Andrew

    2014-01-01

    Influenza viruses undergo continual antigenic evolution allowing mutant viruses to evade host immunity acquired to previous virus strains. Antigenic phenotype is often assessed through pairwise measurement of cross-reactivity between influenza strains using the hemagglutination inhibition (HI) assay. Here, we extend previous approaches to antigenic cartography, and simultaneously characterize antigenic and genetic evolution by modeling the diffusion of antigenic phenotype over a shared virus phylogeny. Using HI data from influenza lineages A/H3N2, A/H1N1, B/Victoria and B/Yamagata, we determine patterns of antigenic drift across viral lineages, showing that A/H3N2 evolves faster and in a more punctuated fashion than other influenza lineages. We also show that year-to-year antigenic drift appears to drive incidence patterns within each influenza lineage. This work makes possible substantial future advances in investigating the dynamics of influenza and other antigenically-variable pathogens by providing a model that intimately combines molecular and antigenic evolution. DOI: http://dx.doi.org/10.7554/eLife.01914.001 PMID:24497547

  8. Synthesis, structure and urease inhibition studies of Schiff base copper(II) complexes with planar four-coordinate copper(II) centers.

    PubMed

    Dong, Xiongwei; Guo, Taolian; Li, Yuguang; Cui, Yongming; Wang, Qiang

    2013-10-01

    Seven new Schiff base copper(II) complexes with planar four-coordinate copper(II) centers have been synthesized and structurally characterized. The solid state structures of complexes 1, 3, 4, 5, 6 and 7 present a square-planar coordination geometry at the metal centers while complex 2 shows a slightly distorted square-planar geometry. Density functional theory calculations have been performed to evaluate the electronic structure of copper(II) complex 7. Inhibition of jack bean urease by copper(II) complexes 1-7 have also been investigated, and potent inhibitory activities with IC50 range of 2.60-17.00μM have been observed for these mononuclear copper(II) complexes. A docking analysis using a DOCK program was conducted to explain the urease inhibitory activity of the copper(II) complexes and the structure-activity relationships were further discussed.

  9. Antigen-specific suppression of cultured lymphocytes from patients with neurocysticercosis

    PubMed Central

    Bueno, E C; Vaz, A J; Machado, L R; Livramento, J A; Ávila, S L M; Ferreira, A W

    2001-01-01

    The biological parasite–host interactions involved in neurocysticercosis (NC) are of a complex nature. A lymphoproliferation assay was performed using mononuclear cells from 11 patients with NC, who were classified according to the alterations obtained by imaging examinations. Antigen extracts from the membrane and/or scolex of Taenia solium and from the vesicular fluid of Taenia crassiceps were used. Mononuclear cells from patients with NC showed antigen-specific suppression when compared with a control group. The patients presenting calcified cysts showed higher suppression when compared with patients in the active phase of disease. The antigen in the vesicular fluid of T. crassiceps seems to play a suppressor role in vitro, completely inhibiting cell proliferation induced by the mitogens phytohaemagglutinin, concanavalin A and pokeweed mitogen. PMID:11703375

  10. The transcription factor TFEB acts as a molecular switch that regulates exogenous antigen-presentation pathways.

    PubMed

    Samie, Mohammad; Cresswell, Peter

    2015-07-01

    Dendritic cells (DCs) can initiate immune responses by presenting exogenous antigens to T cells via both major histocompatibility complex (MHC) class I pathways and MHC class II pathways. Lysosomal activity has an important role in modulating the balance between these two pathways. The transcription factor TFEB regulates lysosomal function by inducing lysosomal activation. Here we report that TFEB expression inhibited the presentation of exogenous antigen by MHC class I while enhancing presentation via MHC class II. TFEB promoted phagosomal acidification and protein degradation. Furthermore, we found that the activation of TFEB was regulated during DC maturation and that phagosomal acidification was impaired in DCs in which the gene encoding TFEB was silenced. Our data indicate that TFEB is a key participant in the differential regulation of the presentation of exogenous antigens by DCs.

  11. Interaction of the transcription factor TFIID with simian virus 40 (SV40) large T antigen interferes with replication of SV40 DNA in vitro.

    PubMed

    Herbig, U; Weisshart, K; Taneja, P; Fanning, E

    1999-02-01

    Simian virus 40 (SV40) large tumor (T) antigen is the major regulatory protein that directs the course of viral infection, primarily by interacting with host cell proteins and modulating their functions. Initiation of viral DNA replication requires specific interactions of T antigen bound to the viral origin of DNA replication with cellular replication proteins. Transcription factors are thought to stimulate initiation of viral DNA replication, but the mechanism of stimulation is poorly understood. Since the transcription factor TATA-binding protein (TBP) binds to sequences within the origin of replication and interacts specifically with T antigen, we examined whether TBP complexes stimulate SV40 DNA replication in vitro. On the contrary, we found that depletion of TBP complexes from human cell extracts increased their ability to support viral DNA replication, and readdition of TBP complexes to the depleted extracts diminished their activity. We have mapped the sites of interaction between the proteins to residues 181 to 205 of T antigen and 184 to 220 of TBP. Titration of fusion proteins containing either of these peptides into undepleted cell extracts stimulated their replication activity, suggesting that they prevented the T antigen-TBP interaction that interfered with replication activity. TBP complexes also interfered with origin DNA unwinding by purified T antigen, and addition of either the T antigen or the TBP fusion peptide relieved the inhibition. These results suggest that TBP complexes associate with a T-antigen surface that is also required for origin DNA unwinding and viral DNA replication. We speculate that competition among cellular proteins for T antigen may play a role in regulating the course of viral infection.

  12. The gallium(III)-salicylidene acylhydrazide complex shows synergistic anti-biofilm effect and inhibits toxin production by Pseudomonas aeruginosa.

    PubMed

    Rzhepishevska, Olena; Hakobyan, Shoghik; Ekstrand-Hammarström, Barbro; Nygren, Yvonne; Karlsson, Torbjörn; Bucht, Anders; Elofsson, Mikael; Boily, Jean-François; Ramstedt, Madeleine

    2014-09-01

    Bacterial biofilms cause a range of problems in many areas and especially in health care. Biofilms are difficult to eradicate with traditional antibiotics and consequently there is a need for alternative ways to prevent and/or remove bacterial biofilms. Furthermore, the emergence of antibiotic resistance in bacteria creates a challenge to find new types of antibiotics with a lower evolutionary pressure for resistance development. One route to develop such drugs is to target the so called virulence factors, i.e. bacterial systems used when bacteria infect a host cell. This study investigates synergy effects between Ga(III) ions, previously reported to suppress biofilm formation and growth in bacteria, and salicylidene acylhydrazides (hydrazones) that have been proposed as antivirulence drugs targeting the type three secretion system used by several Gram-negative pathogens, including Pseudomonas aerugionosa, during bacterial infection of host cells. A library of hydrazones was screened for: Fe(III) binding, enhanced anti-biofilm effect with Ga(III) on P. aeruginosa, and low cytotoxicity to mammalian cells. The metal coordination for the most promising ligand, 2-Oxo-2-[N-(2,4,6-trihydroxy-benzylidene)-hydrazino]-acetamide (ME0163) with Ga(III) was investigated using extended X-ray absorption fine structure spectroscopy as well as density functional theory. The results showed that Ga(III) chelates the hydrazone with 5- and 6-membered chelating rings, and that the Ga(III)-ME0163 complex enhanced the antibiofilm effect of Ga(III) while suppressing the type three secretion system in P. aeruginosa. The latter effect was not observed for the hydrazone alone and was similar for Ga(III)-citrate and Ga(III)-ME0163 complexes, indicating that the inhibition of virulence was caused by Ga(III).

  13. Tiny T antigen: an autonomous polyomavirus T antigen amino-terminal domain.

    PubMed Central

    Riley, M I; Yoo, W; Mda, N Y; Folk, W R

    1997-01-01

    Three mRNAs from the murine polyomavirus early region encode the three well-characterized tumor antigens. We report the existence of a fourth alternatively spliced mRNA which encodes a fourth tumor antigen, tiny T antigen, which comprises the amino-terminal domain common to all of the T antigens but is extended by six unique amino acid residues. The amount of tiny T antigen in infected cells is small because of its short half-life. Tiny T antigen stimulates the ATPase activity of Hsc70, most likely because of its DnaJ-like motif. The common amino-terminal domain may interface with chaperone complexes to assist the T antigens in carrying out their diverse functions of replication, transcription, and transformation in the appropriate cellular compartments. PMID:9223500

  14. Preliminary X-ray diffraction analysis of crystals from the recombinantly expressed human major histocompatibility antigen HLA-B*2704 in complex with a viral peptide and with a self-peptide

    SciTech Connect

    Loll, Bernhard; Biesiadka, Jacek; Saenger, Wolfram

    2005-10-01

    Crystallization of HLA-B*2704 in complex with two peptides. The product of the human leukocyte antigen (HLA) gene HLA-B*2704 differs from that of the prototypical subtype HLA-B*2705 by three amino acids at heavy-chain residues 77 (Ser instead of Asp), 152 (Glu instead of Val) and 211 (Gly instead of Ala). In contrast to the ubiquitous HLA-B*2705 subtype, HLA-B*2704 occurs only in orientals. Both subtypes are strongly associated with spondyloarthropathies and the peptides presented by these subtypes are suspected to play a role in disease pathogenesis. HLA-B*2704 was crystallized in complex with a viral peptide and with a self-peptide using the hanging-drop vapour-diffusion method with PEG as a precipitant. Both crystals belong to space group P2{sub 1}2{sub 1}2{sub 1}. Data sets were collected to 1.60 Å (complex with the self-peptide pVIPR) or to 1.90 Å (complex with the viral peptide pLMP2) resolution using synchrotron radiation. With HLA-B*2705 complexed with pVIPR as a search model, unambiguous molecular-replacement solutions were found for the complexes of HLA-B*2704 with both peptides.

  15. Tobacco rattle virus 16K silencing suppressor binds ARGONAUTE 4 and inhibits formation of RNA silencing complexes.

    PubMed

    Fernández-Calvino, Lourdes; Martínez-Priego, Llúcia; Szabo, Edit Z; Guzmán-Benito, Irene; González, Inmaculada; Canto, Tomás; Lakatos, Lóránt; Llave, César

    2016-01-01

    The cysteine-rich 16K protein of tobacco rattle virus (TRV), the type member of the genus Tobravirus, is known to suppress RNA silencing. However, the mechanism of action of the 16K suppressor is not well understood. In this study, we used a GFP-based sensor strategy and an Agrobacterium-mediated transient assay in Nicotiana benthamiana to show that 16K was unable to inhibit the activity of existing small interfering RNA (siRNA)- and microRNA (miRNA)-programmed RNA-induced silencing effector complexes (RISCs). In contrast, 16K efficiently interfered with de novo formation of miRNA- and siRNA-guided RISCs, thus preventing cleavage of target RNA. Interestingly, we found that transiently expressed endogenous miR399 and miR172 directed sequence-specific silencing of complementary sequences of viral origin. 16K failed to bind small RNAs, although it interacted with ARGONAUTE 4, as revealed by bimolecular fluorescence complementation and immunoprecipitation assays. Site-directed mutagenesis demonstrated that highly conserved cysteine residues within the N-terminal and central regions of the 16K protein are required for protein stability and/or RNA silencing suppression. PMID:26498945

  16. OLA1 regulates protein synthesis and integrated stress response by inhibiting eIF2 ternary complex formation

    PubMed Central

    Chen, Huarong; Song, Renduo; Wang, Guohui; Ding, Zonghui; Yang, Chunying; Zhang, Jiawei; Zeng, Zihua; Rubio, Valentina; Wang, Luchang; Zu, Nancy; Weiskoff, Amanda M.; Minze, Laurie J.; Jeyabal, Prince V.S.; Mansour, Oula C.; Bai, Li; Merrick, William C.; Zheng, Shu; Shi, Zheng-Zheng

    2015-01-01

    Translation is a fundamental cellular process, and its dysregulation can contribute to human diseases such as cancer. During translation initiation the eukaryotic initiation factor 2 (eIF2) forms a ternary complex (TC) with GTP and the initiator methionyl-tRNA (tRNAi), mediating ribosomal recruitment of tRNAi. Limiting TC availability is a central mechanism for triggering the integrated stress response (ISR), which suppresses global translation in response to various cellular stresses, but induces specific proteins such as ATF4. This study shows that OLA1, a member of the ancient Obg family of GTPases, is an eIF2-regulatory protein that inhibits protein synthesis and promotes ISR by binding eIF2, hydrolyzing GTP, and interfering with TC formation. OLA1 thus represents a novel mechanism of translational control affecting de novo TC formation, different from the traditional model in which phosphorylation of eIF2α blocks the regeneration of TC. Depletion of OLA1 caused a hypoactive ISR and greater survival in stressed cells. In vivo, OLA1-knockdown rendered cancer cells deficient in ISR and the downstream proapoptotic effector, CHOP, promoting tumor growth and metastasis. Our work suggests that OLA1 is a novel translational GTPase and plays a suppressive role in translation and cell survival, as well as cancer growth and progression. PMID:26283179

  17. Curcumin Prevents Formation of Polyglutamine Aggregates by Inhibiting Vps36, a Component of the ESCRT-II Complex

    PubMed Central

    Verma, Meenakshi; Sharma, Abhishek; Naidu, Swarna; Bhadra, Ankan Kumar; Kukreti, Ritushree; Taneja, Vibha

    2012-01-01

    Small molecules with antioxidative properties have been implicated in amyloid disorders. Curcumin is the active ingredient present in turmeric and known for several biological and medicinal effects. Adequate evidence substantiates the importance of curcumin in Alzheimer's disease and recent evidence suggests its role in Prion and Parkinson's disease. However, contradictory effects have been suggested for Huntington's disease. This difference provided a compelling reason to investigate the effect of curcumin on glutamine-rich (Q-rich) and non-glutamine-rich (non Q-rich) amyloid aggregates in the well established yeast model system. Curcumin significantly inhibited the formation of htt72Q-GFP (a Q-rich) and Het-s-GFP (a non Q-rich) aggregates in yeast. We show that curcumin prevents htt72Q-GFP aggregation by down regulating Vps36, a component of the ESCRT-II (Endosomal sorting complex required for transport). Moreover, curcumin disrupted the htt72Q-GFP aggregates that were pre-formed in yeast and cured the yeast prion, [PSI+]. PMID:22880132

  18. Titanocene–Gold Complexes Containing N-Heterocyclic Carbene Ligands Inhibit Growth of Prostate, Renal, and Colon Cancers in Vitro

    PubMed Central

    2016-01-01

    We report on the synthesis, characterization, and stability studies of new titanocene complexes containing a methyl group and a carboxylate ligand (mba = −OC(O)-p-C6H4-S−) bound to gold(I)–N-heterocyclic carbene fragments through the thiolate group: [(η5-C5H5)2TiMe(μ-mba)Au(NHC)]. The cytotoxicities of the heterometallic compounds along with those of novel monometallic gold–N-heterocyclic carbene precursors [(NHC)Au(mbaH)] have been evaluated against renal, prostate, colon, and breast cancer cell lines. The highest activity and selectivity and a synergistic effect of the resulting heterometallic species was found for the prostate and colon cancer cell lines. The colocalization of both titanium and gold metals (1:1 ratio) in PC3 prostate cancer cells was demonstrated for the selected compound 5a, indicating the robustness of the heterometallic compound in vitro. We describe here preliminary mechanistic data involving studies on the interaction of selected mono- and bimetallic compounds with plasmid (pBR322) used as a model nucleic acid and the inhibition of thioredoxin reductase in PC3 prostate cancer cells. The heterometallic compounds, which are highly apoptotic, exhibit strong antimigratory effects on the prostate cancer cell line PC3. PMID:27182101

  19. Structure of the complex of F-actin and DNGR-1, a C-type lectin receptor involved in dendritic cell crosspresentation of dead cell-associated antigens

    PubMed Central

    Iborra, Salvador; Yamada, Yurika; Huotari, Jatta; Schulz, Oliver; Ahrens, Susan; Kjær, Svend; Way, Michael; Sancho, David; Namba, Keiichi; Reis e Sousa, Caetano

    2016-01-01

    Summary DNGR-1 is a C-type lectin receptor that binds F-actin exposed by dying cells and facilitates cross-presentation of dead cell-associated antigens by dendritic cells. Here we present the structure of DNGR-1 bound to F-actin at 7.7 Å resolution. Unusually for F-actin binding proteins, the DNGR-1 ligand binding domain contacts three actin subunits helically arranged in the actin filament, bridging over two protofilaments, as well as two neighboring actin subunits along one protofilament. Mutation of residues predicted to mediate ligand binding led to loss of DNGR-1-dependent cross-presentation of dead cell-associated antigens, formally demonstrating that the latter depends on F-actin recognition. Notably, DNGR-1 has relatively modest affinity for F-actin but multivalent interactions allow a marked increase in binding strength. Our findings shed light on modes of actin binding by cellular proteins and reveal how extracellular detection of cytoskeletal components by dedicated receptors allows immune monitoring of loss of cellular integrity. PMID:25979418

  20. A mouse aminopeptidase N is a marker for antigen-presenting cells and appears to be co-expressed with major histocompatibility complex class II molecules.

    PubMed

    Hansen, A S; Norén, O; Sjöström, H; Werdelin, O

    1993-09-01

    To analyze the expression of mouse aminopeptidase N (APN) on the cells of the immune system a panel of rat monoclonal antibodies against mouse intestinal APN was generated. These antibodies were used to affinity purify functional mouse APN from both intestine and kidney, and by flow cytometry to examine the APN expression of the cells of the mouse immune system. An APN closely related, perhaps identical, to the intestinal APN was expressed on a subpopulation of spleen cells and stimulated peritoneal exudate cells, primarily representing antigen-presenting cells, such as B cells, macrophages, dendritic cells, and veiled cells. In contrast this APN expression could not be detected on thymocytes or spleen T cells. As a corollary, APN was expressed on monocyte, macrophage, and B lymphoma cell lines, but not on T hybridoma or thymoma cell lines. The expression of APN showed a striking correlation with the MHC class II expression in all the cell populations studied. This apparent co-expression suggests a role for APN in antigen processing.

  1. A trans-acting major histocompatibility complex-linked gene whose alleles determine gain and loss changes in the antigenic structure of a classical class I molecule

    PubMed Central

    1989-01-01

    The RT1.A locus of the rat MHC encodes the H chain of the single classical class I molecule of this species. One of the alleles of this polymorphic locus, RT1.Aa, is present in several laboratory inbred, congenic, and MHC recombinant rat strains. Studies of the RT1.Aa class I molecule from a number of these strains as a target for CTL show that its antigenicity, both as an alloantigen and a restricting element, is subject to gain and loss alterations by the action of a gene mapping in the MHC to the right of RT1.A. This locus is apparently present in two allelic forms (one possibly a null allele) corresponding to the presence or absence of a dominant transacting modifier, and has been named class I modification, or cim. The antigenic change brought about by cim is scarcely detectable serologically but highly immunogenic for CTL. Biochemical investigations show that cim affects the post- translational modification of RT1.Aa. PMID:2475574

  2. Cellular and computational studies of proteasome inhibition and apoptosis induction in human cancer cells by amino acid Schiff base–copper complexes

    PubMed Central

    Zuo, Jian; Bi, Caifeng; Fan, Yuhua; Buac, Daniela; Nardon, Chiara; Daniel, Kenyon G.; Dou, Q. Ping

    2013-01-01

    Proliferation and apoptosis pathways are tightly regulated in a cell by the ubiquitin–proteasome system (UPS) and alterations in the UPS may result in cellular transformation or other pathological conditions. Indeed, the proteasome is often found to be overactive in cancer cells. It has also been found that cancer cells are more sensitive to proteasome inhibition than normal cells, and therefore proteasome inhibitors are pursued as antitumor drugs. The use of the proteasome inhibitor Bortezomib for treatment of multiple myeloma and mantle cell lymphoma has proved this principle. Recent studies have suggested that copper complexes can inhibit proteasome activity and induce apoptosis in some human cancer cells. However, the involved molecular mechanism is unknown. In this study, we investigated the biological activities of four amino acid Schiff base–copper(II) complexes by using human breast (MDA-MB-231 and MCF-7) and prostate (PC-3) cancer cells. The complexes C1 and C3, but not their counterparts C2 and C4, inhibit the chymotrypsin-like activity of purified 20S proteasome and human cancer cellular 26S proteasome, cause accumulation of proteasome target proteins Bax and IκB-α, and induce growth inhibition and apoptosis in concentration- and time-dependent manners. Docking analysis shows that C1, but not C2 has hydrophobic, pi–pi, pi–cation and hydrogen bond interactions with the proteasomal chymotrypsin-like pocket and could stably fit into the S3 region, leading to specific inhibition. Our study has identified the mechanism of action of these copper complexes on inhibiting tumor cell proteasome and suggested their great potential as novel anticancer agents. PMID:23142973

  3. Purification, crystallization and preliminary X-ray diffraction analysis of the human major histocompatibility antigen HLA-B*2703 complexed with a viral peptide and with a self-peptide

    SciTech Connect

    Loll, Bernhard; Biesiadka, Jacek; Saenger, Wolfram

    2005-04-01

    The product of the human leukocyte antigen (HLA) gene HLA-B*2703 differs from that of the prototypical subtype HLA-B*2705 by a single amino acid at heavy-chain residue 59 that is involved in anchoring the peptide N-terminus within the A pocket of the molecule. Two B*2703–peptide complexes were crystallized using the hanging-drop vapour-diffusion method using PEG 8000 as a precipitant. A pocket of the molecule, two HLA-B*2703–peptide complexes were crystallized and data sets were collected to high resolution using synchrotron radiation. The product of the human leukocyte antigen (HLA) gene HLA-B*2703 differs from that of the prototypical subtype HLA-B*2705 by a single amino acid at heavy-chain residue 59 that is involved in anchoring the peptide N-terminus within the A pocket of the molecule. Two B*2703–peptide complexes were crystallized using the hanging-drop vapour-diffusion method using PEG 8000 as a precipitant. The crystals belong to space group P2{sub 1} (pVIPR peptide) or P2{sub 1}2{sub 1}2{sub 1} (pLMP2 peptide). Data sets were collected to 1.55 Å (B*2703–pVIPR) or 2.0 Å (B*2703–pLMP2) resolution using synchrotron radiation. With B*2705–pVIPR as a search model, a clear molecular-replacement solution was found for both B*2703 complexes.

  4. Oxidation of defined antigens allows protein unfolding and increases both proteolytic processing and exposes peptide epitopes which are recognized by specific T cells.

    PubMed Central

    Carrasco-Marín, E; Paz-Miguel, J E; López-Mato, P; Alvarez-Domínguez, C; Leyva-Cobián, F

    1998-01-01

    The participation of oxidative mechanisms in major histocompatibility complex (MHC) class II-restricted antigen presentation was studied in vitro. In general, antigen processing is inhibited when peritoneal macrophages (MO) are incubated with scavengers of reactive oxygen intermediates (ROI): mannitol (an.OH scavenger), dimethylurea (DMTU, which reacts with H2O2 and HOCl) and NCO-700 (an epoxysuccinic acid derivative which inhibits oxidant production by activated phagocytes and can scavenge reactive oxygen species in both NaOCl and hypoxanthine (XOD) systems). However, neither rotenone and antimycins (inhibitors of O-2 production at the NADH dehydrogenase and ubiquinone-cytochrome b regions, respectively) nor aminoguanidine (an inducible nitric oxide synthase inhibitor) impaired antigen presentation, thus indirectly discarding the participation of mitochondrial oxidation and reactive nitrogen intermediates (RNI) in antigen processing. ROI scavengers do not inhibit the MHC class II-restricted presentation of antigens that need processing but have their disulphide bonds reduced. It can be shown that oxidation of protein antigens (either by chlorination or performic acid treatment) allow protein unfolding and enhance both processing and exposure of immunogenic epitopes to specific T cells. PMID:9824492

  5. Structural and Functional Studies of a Bothropic Myotoxin Complexed to Rosmarinic Acid: New Insights into Lys49-PLA2 Inhibition

    PubMed Central

    dos Santos, Juliana I.; Cardoso, Fábio F.; Soares, Andreimar M.; dal Pai Silva, Maeli; Gallacci, Márcia; Fontes, Marcos R. M.

    2011-01-01

    Snakebite envenoming is an important public health problem in many tropical and subtropical countries, and is considered a neglected tropical disease by the World Health Organization. Most severe cases are inflicted by species of the families Elapidae and Viperidae, and lead to a number of systemic and local effects in the victim. One of the main problems regarding viperidic accidents is prominent local tissue damage whose pathogenesis is complex and involves the combined actions of a variety of venom components. Phospholipases A2 (PLA2s) are the most abundant muscle-damaging components of these venoms. Herein, we report functional and structural studies of PrTX-I, a Lys49-PLA2 from Bothops pirajai snake venom, and the influence of rosmarinic acid (RA) upon this toxin's activities. RA is a known active component of some plant extracts and has been reported as presenting anti-myotoxic properties related to bothopic envenomation. The myotoxic activity of Lys49-PLA2s is well established in the literature and although no in vivo neurotoxicity has been observed among these toxins, in vitro neuromuscular blockade has been reported for some of these proteins. Our in vitro studies show that RA drastically reduces both the muscle damage and the neuromuscular blockade exerted by PrTX-I on mice neuromuscular preparations (by ∼80% and ∼90%, respectively). These results support the hypothesis that the two effects are closely related and lead us to suggest that they are consequences of the muscle membrane-destabilizing activity of the Lys49-PLA2. Although the C-terminal region of these proteins has been reported to comprise the myotoxic site, we demonstrate by X-ray crystallographic studies that RA interacts with PrTX-I in a different region. Consequently, a new mode of Lys49-PLA2 inhibition is proposed. Comparison of our results with others in the literature suggests possible new ways to inhibit bothropic snake venom myotoxins and improve serum therapy. PMID:22205953

  6. Surface antigens of smooth brucellae.

    PubMed

    Diaz, R; Jones, L M; Leong, D; Wilson, J B

    1968-10-01

    Surface antigens of smooth brucellae were extracted by ether-water, phenol-water, trichloroacetic acid, and saline and examined by immunoelectrophoresis and gel diffusion with antisera from infected and immunized rabbits. Ether-water extracts of Brucella melitensis contained a lipopolysaccharide protein component, which was specific for the surface of smooth brucellae and was correlated with the M agglutinogen of Wilson and Miles, a polysaccharide protein component devoid of lipid which was not restricted to the surface of smooth brucellae and was not correlated with the smooth agglutinogen (component 1), and several protein components which were associated with internal antigens of rough and smooth brucellae. Immunoelectrophoretic analysis of ether-water extracts of B. abortus revealed only two components, a lipopolysaccharide protein component, which was correlated with the A agglutinogen, and component 1. Component 1 from B. melitensis and B. abortus showed identity in gel diffusion tests, whereas component M from B. melitensis and component A from B. abortus showed partial identity with unabsorbed antisera and no cross-reactions with monospecific sera. Attempts to prepare monospecific sera directly by immunization of rabbits with cell walls or ether-water extracts were unsuccessful. Absorption of antisera with heavy fraction of ether-water extracts did not always result in monospecific sera. It was concluded (as has been described before) that the A and M antigens are present on a single antigenic complex, in different proportions depending upon the species and biotype, and that this component is a lipopolysaccharide protein complex of high molecular weight that diffuses poorly through agar gel. Components 1, A, and M were also demonstrated in trichloroacetic acid and phenol-water extracts. With all extracts, B. melitensis antigen showed greater diffusibility in agar than B. abortus antigens. After mild acid hydrolysis, B. abortus ether-water extract was able

  7. Induction of the Epstein-Barr virus latent membrane protein 2 antigen-specific cytotoxic T lymphocytes using human leukocyte antigen tetramer-based artificial antigen-presenting cells.

    PubMed

    Lu, Xiao-Ling; Liang, Zhi-Hui; Zhang, Cai-E; Lu, Sheng-Jun; Weng, Xiu-Fang; Wu, Xiong-Wen

    2006-03-01

    Cytotoxic T lymphocytes (CTLs) specific for the Epstein-Barr virus (EBV) latent membrane protein 2 (LMP2) antigen are important reagents for the treatment of some EBV-associated malignancies, such as EBV-positive Hodgkin's disease and nasopharyngeal carcinoma. However, the therapeutic amount of CTLs is often hampered by the limited supply of antigen-presenting cells. To address this issue, an artificial antigen-presenting cell (aAPC) was made by coating a human leukocyte antigen (HLA)-pLMP2 tetrameric complex, anti-CD28 antibody and CD54 molecule to a cell-sized latex bead, which provided the dual signals required for T cell activation. By co-culture of the HLA-A2-LMP2 bearing aAPC and peripheral blood mononuclear cells from HLA-A2 positive healthy donors, LMP2 antigen-specific CTLs were induced and expanded in vitro. The specificity of the aAPC-induced CTLs was demonstrated by both HLA-A2-LMP2 tetramer staining and cytotoxicity against HLA-A2-LMP2 bearing T2 cell, the cytotoxicity was inhibited by the anti-HLA class I antibody (W6/32). These results showed that LMP2 antigen-specific CTLs could be induced and expanded in vitro by the HLA-A2-LMP2-bearing aAPC. Thus, aAPCs coated with an HLA-pLMP2 complex, anti-CD28 and CD54 might be promising tools for the enrichment of LMP2-specific CTLs for adoptive immunotherapy. PMID:16518539

  8. Tumor-associated antigens in gynecologic cancer.

    PubMed

    DiSaia, P J

    1975-12-01

    antigenic determinants of these two gynecologic cancers. It would appear that the mediated (lymphocyte) effect is considerably more cytotoxic and definitive than the humoral factors measured. In addition, the allogenic experiments would suggest strongly that indeed (at least with regard to these two cancers) histologically similar cancers from the same organ share common antigenic determinants. The identification and isolation of these tumor-associated antigens appears complex. The complexity is increased when one studies patients afflicted with these cancers for plasma carcinoembryonic antigens. This antigen, which was thought to be specific for adenocarcinoma of the colon, is found in the blood of a significant number of patients with adenocarcinoma of the ovary and squamous cell carcinoma of the cervix.

  9. A computational framework for influenza antigenic cartography.

    PubMed

    Cai, Zhipeng; Zhang, Tong; Wan, Xiu-Feng

    2010-10-07

    Influenza viruses have been responsible for large losses of lives around the world and continue to present a great public health challenge. Antigenic characterization based on hemagglutination inhibition (HI) assay is one of the routine procedures for influenza vaccine strain selection. However, HI assay is only a crude experiment reflecting the antigenic correlations among testing antigens (viruses) and reference antisera (antibodies). Moreover, antigenic characterization is usually based on more than one HI dataset. The combination of multiple datasets results in an incomplete HI matrix with many unobserved entries. This paper proposes a new computational framework for constructing an influenza antigenic cartography from this incomplete matrix, which we refer to as Matrix Completion-Multidimensional Scaling (MC-MDS). In this approach, we first reconstruct the HI matrices with viruses and antibodies using low-rank matrix completion, and then generate the two-dimensional antigenic cartography using multidimensional scaling. Moreover, for influenza HI tables with herd immunity effect (such as those from Human influenza viruses), we propose a temporal model to reduce the inherent temporal bias of HI tables caused by herd immunity. By applying our method in HI datasets containing H3N2 influenza A viruses isolated from 1968 to 2003, we identified eleven clusters of antigenic variants, representing all major antigenic drift events in these 36 years. Our results showed that both the completed HI matrix and the antigenic cartography obtained via MC-MDS are useful in identifying influenza antigenic variants and thus can be used to facilitate influenza vaccine strain selection. The webserver is available at http://sysbio.cvm.msstate.edu/AntigenMap.

  10. Mechanism of follicular trapping: similarities and differences in trapping of antibody-complexed antigens and carbon particles in the follicles of the spleen

    SciTech Connect

    Groeneveld, P.H.; Eikelenboom, P.; van Rooijen, N.

    1983-02-01

    Both immune complexes and carbon particles were trapped in spleen follicles soon after intravenous injection. The localization pattern of carbon particles and immune complexes were identical 24 hr after injection. Since there is no reason to believe that lymphocytes are involved in the transport of carbon particles from the marginal zone towards the follicle centers, these results indicate that follicular trapping is based on a purely mechanical process. Pretreatment with endotoxin completely prevented the trapping of immune complexes but not carbon particles. Endotoxin administered after the injection of immune complexes caused the rapid removal of trapped complexes from the follicles. However, the effect of endotoxin on trapped carbon particles was less pronounced. Apart from a mechanical trapping of diffusing compounds in the follicular web, a distinct phase is suggested in which immune complexes are fixed to and retained on the surface of the follicular dendritic cells.

  11. The intracellular pathway for the presentation of vitamin B-related antigens by the antigen-presenting molecule MR1.

    PubMed

    McWilliam, Hamish E G; Eckle, Sidonia B G; Theodossis, Alex; Liu, Ligong; Chen, Zhenjun; Wubben, Jacinta M; Fairlie, David P; Strugnell, Richard A; Mintern, Justine D; McCluskey, James; Rossjohn, Jamie; Villadangos, Jose A

    2016-05-01

    The antigen-presenting molecule MR1 presents vitamin B-related antigens (VitB antigens) to mucosal-associated invariant T (MAIT) cells through an uncharacterized pathway. We show that MR1, unlike other antigen-presenting molecules, does not constitutively present self-ligands. In the steady state it accumulates in a ligand-receptive conformation within the endoplasmic reticulum. VitB antigens reach this location and form a Schiff base with MR1, triggering a 'molecular switch' that allows MR1-VitB antigen complexes to traffic to the plasma membrane. These complexes are endocytosed with kinetics independent of the affinity of the MR1-ligand interaction and are degraded intracellularly, although some MR1 molecules acquire new ligands during passage through endosomes and recycle back to the surface. MR1 antigen presentation is characterized by a rapid 'off-on-off' mechanism that is strictly dependent on antigen availability. PMID:27043408

  12. Effect of herpes simplex virus types 1 and 2 on surface expression of class I major histocompatibility complex antigens on infected cells.

    PubMed Central

    Jennings, S R; Rice, P L; Kloszewski, E D; Anderson, R W; Thompson, D L; Tevethia, S S

    1985-01-01

    Cytotoxic T lymphocytes (CTL) generated in C57BL/6 (H-2b) mice in response to infection with the serologically distinct herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) were cross-reactive against target cells infected with either serotype. However, HSV-2-infected cells were shown to be much less susceptible to CTL-mediated lysis, and analysis through the use of HSV-1 X HSV-2 intertypic recombinants mapped the reduced susceptibility to a region contained within 0.82 to 1.00 map units of the HSV-2 genome. The study reported here was undertaken to determine the possible reasons for the reduced susceptibility of HSV-2-infected cells to lysis by CTL. Competition for the specific lysis of labeled HSV-1-infected cells by either HSV-1- or HSV-2-infected, unlabeled inhibitor cells and frequency analysis of the CTL precursor able to recognize HSV-1- and HSV-2-infected cells suggested that the reduced susceptibility of HSV-2-infected cells to lysis could be explained, at least in part, by reduced levels of target cell recognition. A determination of the surface expression of the critical elements involved in target cell recognition by CTL following infection with HSV-1 or HSV-2 revealed that all the major HSV-specific glycoprotein species were expressed. Infection with both HSV-1 and HSV-2 caused a reduction in the expression of the class I H-2 antigens. However, this reduction was much greater following infection with HSV-2. This suggested that one important factor contributing to reduced lysis of HSV-2-infected cells may be the altered or reduced expression of the class I H-2 self-antigens. PMID:2999432

  13. Establishment of the reversible peptide-major histocompatibility complex (pMHC) class I Histamer technology: tool for visualization and selection of functionally active antigen-specific CD8(+) T lymphocytes.

    PubMed

    Tischer, Sabine; Kaireit, Till; Figueiredo, Constança; Hiller, Oliver; Maecker-Kolhoff, Britta; Geyeregger, Renè; Immenschuh, Stephan; Blasczyk, Rainer; Eiz-Vesper, Britta

    2012-09-01

    Multimers of soluble peptide-major histocompatibilty complex (pMHC) molecules are used in both basic and clinical immunology. They allow the specific visualization and isolation of antigen-specific T cells from ex vivo samples. Adoptive transfer of antigen-specific T cells sorted by pMHC multimers is an effective strategy for treatment of patients with malignancies or infectious diseases after transplantation. We developed a new reversible pMHC multimer called 'Histamer' to enable the specific detection and isolation of antiviral T cells from peripheral blood. HLA-A*02:01/CMVpp65 (495-503) Histamer (A02/CMV Histamer) was generated by coupling 6xHis-tagged pMHC molecules onto cobalt-based magnetic beads. The specificity of the Histamer was evaluated by flow cytometry. Sorting of antiviral CD8(+) cytotoxic T lymphocytes (CTLs) was performed by magnetic cell separation, followed by the monomerization of the Histamer after addition of the competitor L-histidine. Sorted T cells were analyzed for phenotype and function. The reversible pMHC Histamer proved to be highly specific and sensitive. CMV-specific T cells of up to 99.6% purity were isolated using the Histamer technology. Rapid and complete disassembly of the T-cell surface-bound A02/CMV Histamer followed by the subsequent dissociation of the pMHC monomers from CD8(+) CTL receptors was achieved using 100 mM L-histidine. The function of CMV-specific T cells enriched by Histamer staining did not differ from CTLs induced by standard T-cell assays. This reversible T-cell staining procedure preserves the functionality of antigen-specific T cells and can be adapted to good manufacturing practice conditions. The pMHC Histamer technology offers full flexibility and fulfills all requirements to generate clinical-grade T lymphocytes. PMID:22740564

  14. Recruitment of trimeric proliferating cell nuclear antigen by G1-phase cyclin-dependent kinases following DNA damage with platinum-based antitumour agents

    PubMed Central

    He, G; Kuang, J; Koomen, J; Kobayashi, R; Khokhar, A R; Siddik, Z H

    2013-01-01

    Background: In cycling tumour cells, the binary cyclin-dependent kinase Cdk4/cyclin D or Cdk2/cyclin E complex is inhibited by p21 following DNA damage to induce G1 cell-cycle arrest. However, it is not known whether other proteins are also recruited within Cdk complexes, or their role, and this was investigated. Methods: Ovarian A2780 tumour cells were exposed to the platinum-based antitumour agent 1R,2R-diaminocyclohexane(trans-diacetato)(dichloro)platinum(IV) (DAP), which preferentially induces G1 arrest in a p21-dependent manner. The Cdk complexes were analysed by gel filtration chromatography, immunoblot and mass spectrometry. Results: The active forms of Cdk4 and Cdk2 complexes in control tumour cells have a molecular size of ∼140 kDa, which increased to ∼290 kDa when inhibited following G1 checkpoint activation by DAP. Proteomic analysis identified Cdk, cyclin, p21 and proliferating cell nuclear antigen (PCNA) in the inhibited complex, and biochemical studies provided unequivocal evidence that the increase in ∼150 kDa of the inhibited complex is consistent with p21-dependent recruitment of PCNA as a trimer, likely bound to three molecules of p21. Although p21 alone was sufficient to inhibit the Cdk complex, PCNA was critical for stabilising p21. Conclusion: G1 Cdk complexes inhibited by p21 also recruit PCNA, which inhibits degradation and, thereby, prolongs act