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Sample records for antibody potently inhibits

  1. Fully Human Antagonistic Antibodies against CCR4 Potently Inhibit Cell Signaling and Chemotaxis

    PubMed Central

    Géraudie, Solène; Scheffler, Ulrike; Griep, Remko A.; Reiersen, Herald; Duncan, Alexander R.; Kiprijanov, Sergej M.

    2014-01-01

    Background CC chemokine receptor 4 (CCR4) represents a potentially important target for cancer immunotherapy due to its expression on tumor infiltrating immune cells including regulatory T cells (Tregs) and on tumor cells in several cancer types and its role in metastasis. Methodology Using phage display, human antibody library, affinity maturation and a cell-based antibody selection strategy, the antibody variants against human CCR4 were generated. These antibodies effectively competed with ligand binding, were able to block ligand-induced signaling and cell migration, and demonstrated efficient killing of CCR4-positive tumor cells via ADCC and phagocytosis. In a mouse model of human T-cell lymphoma, significant survival benefit was demonstrated for animals treated with the newly selected anti-CCR4 antibodies. Significance For the first time, successful generation of anti- G-protein coupled chemokine receptor (GPCR) antibodies using human non-immune library and phage display on GPCR-expressing cells was demonstrated. The generated anti-CCR4 antibodies possess a dual mode of action (inhibition of ligand-induced signaling and antibody-directed tumor cell killing). The data demonstrate that the anti-tumor activity in vivo is mediated, at least in part, through Fc-receptor dependent effector mechanisms, such as ADCC and phagocytosis. Anti-CC chemokine receptor 4 antibodies inhibiting receptor signaling have potential as immunomodulatory antibodies for cancer. PMID:25080123

  2. Potent Inhibition of Human Immunodeficiency Virus Type 1 Replication by an Intracellular Anti-Rev Single-Chain Antibody

    NASA Astrophysics Data System (ADS)

    Duan, Lingxun; Bagasra, Omar; Laughlin, Mark A.; Oakes, Joseph W.; Pomerantz, Roger J.

    1994-05-01

    Human immunodeficiency virus type 1 (HIV-1) has a complex life cycle, which has made it a difficult target for conventional therapeutic modalities. A single-chain antibody moiety, directed against the HIV-1 regulatory protein Rev, which rescues unspliced viral RNA from the nucleus of infected cells, has now been developed. This anti-Rev single-chain construct (SFv) consists of both light and heavy chain variable regions of an anti-Rev monoclonal antibody, which, when expressed intracellularly within human cells, potently inhibits HIV-1 replication. This intracellular SFv molecule is demonstrated to specifically antagonize Rev function. Thus, intracellular SFv expression, against a retroviral regulatory protein, may be useful as a gene therapeutic approach to combat HIV-1 infections.

  3. A Single-Domain Llama Antibody Potently Inhibits the Enzymatic Activity of Botulinum Neurotoxin by Binding to the Non-Catalytic [alpha]-Exosite Binding Region

    SciTech Connect

    Dong, Jianbo; Thompson, Aaron A.; Fan, Yongfeng; Lou, Jianlong; Conrad, Fraser; Ho, Mengfei; Pires-Alves, Melissa; Wilson, Brenda A.; Stevens, Raymond C.; Marks, James D.

    2010-08-13

    Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K{sub d}) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K{sub d} for BoNT/A Lc of 1.47 x 10{sup -10} M and an IC{sub 50} (50% inhibitory concentration) of 4.7 x 10{sup -10} M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 {angstrom} resolution. The structure reveals that the Aa1 VHH binds in the {alpha}-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc {alpha}-exosite as a target for inhibitor development.

  4. Combination therapy with anti-ErbB3 monoclonal antibodies and EGFR TKIs potently inhibits Non-small Cell Lung Cancer

    PubMed Central

    Noto, Alessia; De Vitis, Claudia; Roscilli, Giuseppe; Fattore, Luigi; Malpicci, Debora; Marra, Emanuele; Luberto, Laura; D'Andrilli, Antonio; Coluccia, Pierpaolo; Giovagnoli, Maria Rosaria; Normanno, Nicola; Ruco, Luigi; Aurisicchio, Luigi; Mancini, Rita; Ciliberto, Gennaro

    2013-01-01

    Personalized therapy of advanced non-small cell lung cancer (NSCLC) has been improved by the introduction of EGFR tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib. EGFR TKIs induce dramatic objective responses and increase survival in patients bearing sensitizing mutations in the EGFR intracytoplasmic tyrosine kinase domain. However, virtually all patients develop resistance, and this is responsible for disease relapse. Hence several efforts are being undertaken to understand the mechanisms of resistance in order to develop combination treatments capable to sensitize resistant cells to EGFR TKIs. Recent studies have suggested that upregulation of another member of the EGFR receptor family, namely ErbB3 is involved in drug resistance, through increased phosphorylation of its intracytoplasmic domain and activation of PI3K/AKT signaling. In this paper we first show, by using a set of malignant pleural effusion derived cell cultures (MPEDCC) from patients with lung adenocarcinoma, that surface ErbB3 expression correlates with increased AKT phosphorylation. Antibodies against ErbB3, namely A3, which we previously demonstrated to induce receptor internalization and degradation, inhibit growth and induce apoptosis only in cells overexpressing surface ErbB3. Furthermore, combination of anti-ErbB3 antibodies with EGFR TKIs synergistically affect cell proliferation in vitro, cause cell cycle arrest, up-regulate p21 expression and inhibit tumor growth in mouse xenografts. Importantly, potentiation of gefitinib by anti-ErbB3 antibodies occurs both in de novo and in ab initio resistant cells. Anti-ErbB3 mAbs strongly synergize also with the dual EGFR and HER2 inhibitor lapatinib. Our results suggest that combination treatment with EGFR TKI and antibodies against ErbB3 should be a promising approach to pursue in the clinic. PMID:23896512

  5. Highly potent anti-human GPVI monoclonal antibodies derived from GPVI knockout mouse immunization.

    PubMed

    Matsumoto, Yutaka; Takizawa, Hisao; Gong, Xiaoqi; Le, Sang; Lockyer, Simon; Okuyama, Keiji; Tanaka, Michinori; Yoshitake, Masuhiro; Tandon, Narendra N; Kambayashi, Junichi

    2007-01-01

    Recent progress in the understanding of thrombus formation has suggested an important role for glycoprotein (GP) VI in this process. To clarify the exact role in detail, it is necessary to use specific, high affinity inhibitory antibodies. However, possibly due to the conserved structure of GPVI among species, it has been difficult to obtain potent antibodies. In this study, we developed highly potent anti-human GPVI monoclonal antibodies using GPVI knockout mice for immunization. Fab fragments of these antibodies, named OM1 and OM2, potently inhibit collagen-induced platelet aggregation. The IC(50) values for OM1 and OM2 are 0.6+/-0.05 and 1.7+/-0.5 microg/mL, respectively, showing potency greater than, or equal to that of abciximab (1.7+/-0.3 microg/mL), an anti-GPIIb/IIIa antibody. Fab fragments of OM1 and OM2 also potently inhibit collagen-induced ATP release, thromboxane A(2) formation, and platelet adhesion to immobilized collagen under static and flow conditions. Interestingly, platelet aggregation induced with collagen-related peptide was potently inhibited by OM2 but not OM1, indicating that OM1 recognizes an epitope that is different from collagen-related peptide-binding site on GPVI. These results suggest that OM1 and OM2 may be useful tools to understand the role of GPVI in thrombus formation. Furthermore, these antibodies have the potential to be developed as a new class of therapeutic tool.

  6. Diversity Against Adversity: How Adaptive Immune System Evolves Potent Antibodies

    NASA Astrophysics Data System (ADS)

    Heo, Muyoung; Zeldovich, Konstantin B.; Shakhnovich, Eugene I.

    2011-07-01

    Adaptive immunity is an amazing mechanism, whereby new protein functions—affinity of antibodies (Immunoglobulins) to new antigens—evolve through mutation and selection in a matter of a few days. Despite numerous experimental studies, the fundamental physical principles underlying immune response are still poorly understood. In considerable departure from past approaches, here, we propose a microscopic multiscale model of adaptive immune response, which consists of three essential players: The host cells, viruses, and B-cells in Germinal Centers (GC). Each moiety carries a genome, which encodes proteins whose stability and interactions are determined from their sequences using laws of Statistical Mechanics, providing an exact relationship between genomic sequences and strength of interactions between pathogens and antibodies and antibodies and host proteins (autoimmunity). We find that evolution of potent antibodies (the process known as Affinity Maturation (AM)) is a delicate balancing act, which has to reconcile the conflicting requirements of protein stability, lack of autoimmunity, and high affinity of antibodies to incoming antigens. This becomes possible only when antibody producing B cells elevate their mutation rates (process known as Somatic Hypermutation (SHM)) to fall into a certain range—not too low to find potency increasing mutations but not too high to destroy stable Immunoglobulins and/or already achieved affinity. Potent antibodies develop through clonal expansion of initial B cells expressing marginally potent antibodies followed by their subsequent affinity maturation through mutation and selection. As a result, in each GC the population of mature potent Immunoglobulins is monoclonal being ancestors of a single cell from initial (germline) pool. We developed a simple analytical theory, which provides further rationale to our findings. The model and theory reveal the molecular factors that determine the efficiency of affinity maturation

  7. Combination of antibody that inhibits ligand-independent HER3 dimerization and a p110α inhibitor potently blocks PI3K signaling and growth of HER2+ breast cancers.

    PubMed

    Garrett, Joan T; Sutton, Cammie R; Kurupi, Richard; Bialucha, Carl Uli; Ettenberg, Seth A; Collins, Scott D; Sheng, Qing; Wallweber, Jerry; Defazio-Eli, Lisa; Arteaga, Carlos L

    2013-10-01

    We examined the effects of LJM716, an HER3 (ERBB3) neutralizing antibody that inhibits ligand-induced and ligand-independent HER3 dimerization, as a single agent and in combination with BYL719, an ATP competitive p110α-specific inhibitor, against HER2-overexpressing breast and gastric cancers. Treatment with LJM716 reduced HER2-HER3 and HER3-p85 dimers, P-HER3 and P-AKT, both in vitro and in vivo. Treatment with LJM716 alone markedly reduced growth of BT474 xenografts. The combination of LJM716/lapatinib/trastuzumab significantly improved survival of mice with BT474 xenografts compared with lapatinib/trastuzumab (P = 0.0012). LJM716 and BYL719 synergistically inhibited growth in a panel of HER2+ and PIK3CA mutant cell lines. The combination also inhibited P-AKT in HER2-overexpressing breast cancer cells and growth of HER2+ NCI-N87 gastric cancer xenografts more potently than LJM716 or BYL719 alone. Trastuzumab-resistant HER2+/PIK3CA mutant MDA453 xenografts regressed completely after 3 weeks of therapy with LJM716 and BYL719, whereas either single agent inhibited growth only partially. Finally, mice with BT474 xenografts treated with trastuzumab/LJM716, trastuzumab/BYL719, LJM716/BYL719, or trastuzumab/LJM716/BYL719 exhibited similar rates of tumor regression after 3 weeks of treatment. Thirty weeks after treatment discontinuation, 14% of mice were treated with trastuzumab/LJM716/BYL719, whereas >80% in all other treatment groups were sacrificed due to a recurrent large tumor burden (P = 0.0066). These data suggest that dual blockade of the HER2 signaling network with an HER3 antibody that inhibits HER2-HER3 dimers in combination with a p110α-specific inhibitor in the absence of a direct HER2 antagonist is an effective treatment approach against HER2-overexpressing cancers.

  8. A truncated receptor-binding domain of MERS-CoV spike protein potently inhibits MERS-CoV infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines.

    PubMed

    Du, Lanying; Kou, Zhihua; Ma, Cuiqing; Tao, Xinrong; Wang, Lili; Zhao, Guangyu; Chen, Yaoqing; Yu, Fei; Tseng, Chien-Te K; Zhou, Yusen; Jiang, Shibo

    2013-01-01

    An emerging respiratory infectious disease with high mortality, Middle East respiratory syndrome (MERS), is caused by a novel coronavirus (MERS-CoV). It was first reported in 2012 in Saudi Arabia and has now spread to eight countries. Development of effective therapeutics and vaccines is crucial to save lives and halt the spread of MERS-CoV. Here, we show that a recombinant protein containing a 212-amino acid fragment (residues 377-588) in the truncated receptor-binding domain (RBD: residues 367-606) of MERS-CoV spike (S) protein fused with human IgG Fc fragment (S377-588-Fc) is highly expressed in the culture supernatant of transfected 293T cells. The purified S377-588-Fc protein efficiently binds to dipeptidyl peptidase 4 (DPP4), the receptor of MERS-CoV, and potently inhibited MERS-CoV infection, suggesting its potential to be further developed as a therapeutic modality for treating MERS-CoV infection and saving the patients' lives. The recombinant S377-588-Fc is able to induce in the vaccinated mice strong MERS-CoV S-specific antibodies, which blocks the binding of RBD to DPP4 receptor and effectively neutralizes MERS-CoV infection. These findings indicate that this truncated RBD protein shows promise for further development as an effective and safe vaccine for the prevention of MERS-CoV infection.

  9. Neutralization mechanism of a highly potent antibody against Zika virus

    PubMed Central

    Zhang, Shuijun; Kostyuchenko, Victor A.; Ng, Thiam-Seng; Lim, Xin-Ni; Ooi, Justin S. G.; Lambert, Sebastian; Tan, Ter Yong; Widman, Douglas G.; Shi, Jian; Baric, Ralph S.; Lok, Shee-Mei

    2016-01-01

    The rapid spread of Zika virus (ZIKV), which causes microcephaly and Guillain-Barré syndrome, signals an urgency to identify therapeutics. Recent efforts to rescreen dengue virus human antibodies for ZIKV cross-neutralization activity showed antibody C10 as one of the most potent. To investigate the ability of the antibody to block fusion, we determined the cryoEM structures of the C10-ZIKV complex at pH levels mimicking the extracellular (pH8.0), early (pH6.5) and late endosomal (pH5.0) environments. The 4.0 Å resolution pH8.0 complex structure shows that the antibody binds to E proteins residues at the intra-dimer interface, and the virus quaternary structure-dependent inter-dimer and inter-raft interfaces. At pH6.5, antibody C10 locks all virus surface E proteins, and at pH5.0, it locks the E protein raft structure, suggesting that it prevents the structural rearrangement of the E proteins during the fusion event—a vital step for infection. This suggests antibody C10 could be a good therapeutic candidate. PMID:27882950

  10. Generation of potent mouse monoclonal antibodies to self-proteins using T-cell epitope "tags".

    PubMed

    Percival-Alwyn, Jennifer L; England, Elizabeth; Kemp, Benjamin; Rapley, Laura; Davis, Nicola H E; McCarthy, Grant R; Majithiya, Jayesh B; Corkill, Dominic J; Welsted, Sarah; Minton, Kevin; Cohen, E Suzanne; Robinson, Matthew J; Dobson, Claire; Wilkinson, Trevor C I; Vaughan, Tristan J; Groves, Maria A T; Tigue, Natalie J

    2015-01-01

    Immunization of mice or rats with a "non-self" protein is a commonly used method to obtain monoclonal antibodies, and relies on the immune system's ability to recognize the immunogen as foreign. Immunization of an antigen with 100% identity to the endogenous protein, however, will not elicit a robust immune response. To develop antibodies to mouse proteins, we focused on the potential for breaking such immune tolerance by genetically fusing two independent T-cell epitope-containing sequences (from tetanus toxin (TT) and diphtheria toxin fragment A (DTA)) to a mouse protein, mouse ST2 (mST2). Wild-type CD1 mice were immunized with three mST2 tagged proteins (Fc, TT and DTA) and the specific serum response was determined. Only in mice immunized with the T-cell epitope-containing antigens were specific mST2 serum responses detected; hybridomas generated from these mice secreted highly sequence-diverse IgGs that were capable of binding mST2 and inhibiting the interaction of mST2 with its ligand, mouse interleukin (IL)-33 (mIL-33). Of the hundreds of antibodies profiled, we identified five potent antibodies that were able to inhibit IL-33 induced IL-6 release in a mast cell assay; notably one such antibody was sufficiently potent to suppress IL-5 release and eosinophilia infiltration in an Alternaria alternata challenge mouse model of asthma. This study demonstrated, for the first time, that T-cell epitope-containing tags have the ability to break tolerance in wild-type mice to 100% conserved proteins, and it provides a compelling argument for the broader use of this approach to generate antibodies against any mouse protein or conserved ortholog.

  11. A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

    PubMed

    Scalley-Kim, Michelle L; Hess, Bruce W; Kelly, Ryan L; Krostag, Anne-Rachel F; Lustig, Kurt H; Marken, John S; Ovendale, Pamela J; Posey, Aaron R; Smolak, Pamela J; Taylor, Janelle D L; Wood, C L; Bienvenue, David L; Probst, Peter; Salmon, Ruth A; Allison, Daniel S; Foy, Teresa M; Raport, Carol J

    2012-01-01

    Chemokines play a key role in leukocyte recruitment during inflammation and are implicated in the pathogenesis of a number of autoimmune diseases. As such, inhibiting chemokine signaling has been of keen interest for the development of therapeutic agents. This endeavor, however, has been hampered due to complexities in the chemokine system. Many chemokines have been shown to signal through multiple receptors and, conversely, most chemokine receptors bind to more than one chemokine. One approach to overcoming this complexity is to develop a single therapeutic agent that binds and inactivates multiple chemokines, similar to an immune evasion strategy utilized by a number of viruses. Here, we describe the development and characterization of a novel therapeutic antibody that targets a subset of human CC chemokines, specifically CCL3, CCL4, and CCL5, involved in chronic inflammatory diseases. Using a sequential immunization approach, followed by humanization and phage display affinity maturation, a therapeutic antibody was developed that displays high binding affinity towards the three targeted chemokines. In vitro, this antibody potently inhibits chemotaxis and chemokine-mediated signaling through CCR1 and CCR5, primary chemokine receptors for the targeted chemokines. Furthermore, we have demonstrated in vivo efficacy of the antibody in a SCID-hu mouse model of skin leukocyte migration, thus confirming its potential as a novel therapeutic chemokine antagonist. We anticipate that this antibody will have broad therapeutic utility in the treatment of a number of autoimmune diseases due to its ability to simultaneously neutralize multiple chemokines implicated in disease pathogenesis.

  12. Biochemical characterization and structure determination of a potent, selective antibody inhibitor of human MMP9.

    PubMed

    Appleby, Todd C; Greenstein, Andrew E; Hung, Magdeleine; Liclican, Albert; Velasquez, Maile; Villaseñor, Armando G; Wang, Ruth; Wong, Melanie H; Liu, Xiaohong; Papalia, Giuseppe A; Schultz, Brian E; Sakowicz, Roman; Smith, Victoria; Kwon, Hyock Joo

    2017-02-24

    Matrix metalloproteinase 9 (MMP9) is a key regulator of the extracellular matrix (ECM), involved in the degradation of various ECM proteins. MMP9 is a member of a large family of proteases that are secreted as inactive zymogens. MMP9 plays a pathological role in a variety of inflammatory and oncology disorders and has long been considered an attractive therapeutic target. GS-5745 is a potent, highly selective humanized monoclonal antibody inhibitor of MMP9 that has shown promise in treating ulcerative colitis and gastric cancer. Here we describe the crystal structure of GS-5745:MMP9 complex and biochemical studies to elucidate the mechanism of GS-5745 inhibition of MMP9. GS-5745 binds MMP9 distal to the active site, near the junction between the prodomain and catalytic domain. GS-5745 inhibits MMP9 by two mechanisms: binding to active MMP9 allosterically inhibits MMP9 activity and binding to pro-MMP9 prevents MMP9 activation.

  13. The heptide repeat 2 and upstream region of TGEV induces potent cross-neutralizing antibodies against group I coronaviruses.

    PubMed

    Shi, Huiling; Wu, Nannan; Wang, Xiaoming; Wang, Tianhou

    2012-10-01

    The coronavirus heptide repeat (HR) region in the spike protein induces neutralizing antibodies that block the postfusion core formation and inhibit virus entry into target cells. The HR2 regions for coronaviruses of the same serogroup share high homology. We found that polyclonal antibodies derived from transmissible gastroenteritis coronavirus HR2 and upstream region were cross-reactive with the S proteins of the same serogroup in western blotting. The polyclonal antibodies also potently cross-neutralized viruses from the same serogroup. This study provides new insight for designing vaccine and therapeutic reagents against coronavirus infections.

  14. Potent neutralizing monoclonal antibodies against Ebola virus infection

    PubMed Central

    Zhang, Qi; Gui, Miao; Niu, Xuefeng; He, Shihua; Wang, Ruoke; Feng, Yupeng; Kroeker, Andrea; Zuo, Yanan; Wang, Hua; Wang, Ying; Li, Jiade; Li, Chufang; Shi, Yi; Shi, Xuanling; Gao, George F.; Xiang, Ye; Qiu, Xiangguo; Chen, Ling; Zhang, Linqi

    2016-01-01

    Ebola virus infections cause a deadly hemorrhagic disease for which no vaccines or therapeutics has received regulatory approval. Here we show isolation of three (Q206, Q314 and Q411) neutralizing monoclonal antibodies (mAbs) against the surface glycoprotein (GP) of Ebola virus identified in West Africa in 2014 through sequential immunization of Chinese rhesus macaques and antigen-specific single B cell sorting. These mAbs demonstrated potent neutralizing activities against both pseudo and live Ebola virus independent of complement. Biochemical, single particle EM, and mutagenesis analysis suggested Q206 and Q411 recognized novel epitopes in the head while Q314 targeted the glycan cap in the GP1 subunit. Q206 and Q411 appeared to influence GP binding to its receptor NPC1. Treatment with these mAbs provided partial but significant protection against disease in a mouse model of Ebola virus infection. These novel mAbs could serve as promising candidates for prophylactic and therapeutic interventions against Ebola virus infection. PMID:27181584

  15. Monochloramine potently inhibits arachidonic acid metabolism in rat platelets.

    PubMed

    Fujimoto, Yohko; Ikeda, Mai; Sakuma, Satoru

    2006-05-26

    In the present study, the effects of hypochlorous acid (HOCl), monochloramine (NH(2)Cl), glutamine-chloramine (Glu-Cl) and taurine-chloramine (Tau-Cl) on the formation of 12-lipoxygenase (LOX) metabolite, 12-HETE, and cyclooxygenase (COX) metabolites, TXB(2), and 12-HHT, from exogenous arachidonic acid (AA) in rat platelets were examined. Rat platelets (4x10(8)/ml) were preincubated with drugs for 5min at 37 degrees C prior to the incubation with AA (40microM) for 2min at 37 degrees C. HOCl (50-250microM) showed an inhibition on the formation of LOX metabolite (12-HETE, 5-67% inhibition) and COX metabolites (TXB(2), 33-73% inhibition; 12-HHT, 27-74% inhibition). Although Tau-Cl and Glu-Cl up to 100microM were without effect on the formation of 12-HETE, TXB(2) and 12-HTT, NH(2)Cl showed a strong inhibition on the formation of all three metabolites (10-100microM NH(2)Cl, 12-HETE, 21-92% inhibition; TXB(2), 58-94% inhibition; 12-HHT, 36-92% inhibition). Methionine reversed a reduction of formation of LOX and COX metabolites induced by NH(2)Cl, and taurine restoring that induced by both NH(2)Cl and HOCl. These results suggest that NH(2)Cl is a more potent inhibitor of COX and LOX pathways in platelets than HOCl, and taurine and methionine can be modulators of NH(2)Cl-induced alterations in the COX and LOX pathways in vivo.

  16. Potent inhibition of tau fibrillization with a multivalent ligand

    SciTech Connect

    Honson, Nicolette S.; Jensen, Jordan R.; Darby, Michael V.; Kuret, Jeff

    2007-11-09

    Small-molecule inhibitors of tau fibrillization are under investigation as tools for interrogating the tau aggregation pathway and as potential therapeutic agents for Alzheimer's disease. Established inhibitors include thiacarbocyanine dyes, which can inhibit recombinant tau fibrillization in the presence of anionic surfactant aggregation inducers. In an effort to increase inhibitory potency, a cyclic bis-thiacarbocyanine molecule containing two thiacarbocyanine moieties was synthesized and characterized with respect to tau fibrillization inhibitory activity by electron microscopy and ligand aggregation state by absorbance spectroscopy. Results showed that the inhibitory activity of the bis-thiacarbocyanine was qualitatively similar to a monomeric cyanine dye, but was more potent with 50% inhibition achieved at {approx}80 nM concentration. At all concentrations tested in aqueous solution, the bis-thiacarbocyanine collapsed to form a closed clamshell structure. However, the presence of tau protein selectively stabilized the open conformation. These results suggest that the inhibitory activity of bis-thiacarbocyanine results from multivalency, and reveal a route to more potent tau aggregation inhibitors.

  17. Potent neutralizing anti-CD1d antibody reduces lung cytokine release in primate asthma model

    PubMed Central

    Nambiar, Jonathan; Clarke, Adam W; Shim, Doris; Mabon, David; Tian, Chen; Windloch, Karolina; Buhmann, Chris; Corazon, Beau; Lindgren, Matilda; Pollard, Matthew; Domagala, Teresa; Poulton, Lynn; Doyle, Anthony G

    2015-01-01

    CD1d is a receptor on antigen-presenting cells involved in triggering cell populations, particularly natural killer T (NKT) cells, to release high levels of cytokines. NKT cells are implicated in asthma pathology and blockade of the CD1d/NKT cell pathway may have therapeutic potential. We developed a potent anti-human CD1d antibody (NIB.2) that possesses high affinity for human and cynomolgus macaque CD1d (KD ∼100 pM) and strong neutralizing activity in human primary cell-based assays (IC50 typically <100 pM). By epitope mapping experiments, we showed that NIB.2 binds to CD1d in close proximity to the interface of CD1d and the Type 1 NKT cell receptor β-chain. Together with data showing that NIB.2 inhibited stimulation via CD1d loaded with different glycolipids, this supports a mechanism whereby NIB.2 inhibits NKT cell activation by inhibiting Type 1 NKT cell receptor β-chain interactions with CD1d, independent of the lipid antigen in the CD1d antigen-binding cleft. The strong in vitro potency of NIB.2 was reflected in vivo in an Ascaris suum cynomolgus macaque asthma model. Compared with vehicle control, NIB.2 treatment significantly reduced bronchoalveolar lavage (BAL) levels of Ascaris-induced cytokines IL-5, IL-8 and IL-1 receptor antagonist, and significantly reduced baseline levels of GM-CSF, IL-6, IL-15, IL-12/23p40, MIP-1α, MIP-1β, and VEGF. At a cellular population level NIB.2 also reduced numbers of BAL lymphocytes and macrophages, and blood eosinophils and basophils. We demonstrate that anti-CD1d antibody blockade of the CD1d/NKT pathway modulates inflammatory parameters in vivo in a primate inflammation model, with therapeutic potential for diseases where the local cytokine milieu is critical. PMID:25751125

  18. Potent neutralizing anti-CD1d antibody reduces lung cytokine release in primate asthma model.

    PubMed

    Nambiar, Jonathan; Clarke, Adam W; Shim, Doris; Mabon, David; Tian, Chen; Windloch, Karolina; Buhmann, Chris; Corazon, Beau; Lindgren, Matilda; Pollard, Matthew; Domagala, Teresa; Poulton, Lynn; Doyle, Anthony G

    2015-01-01

    CD1d is a receptor on antigen-presenting cells involved in triggering cell populations, particularly natural killer T (NKT) cells, to release high levels of cytokines. NKT cells are implicated in asthma pathology and blockade of the CD1d/NKT cell pathway may have therapeutic potential. We developed a potent anti-human CD1d antibody (NIB.2) that possesses high affinity for human and cynomolgus macaque CD1d (KD ∼100 pM) and strong neutralizing activity in human primary cell-based assays (IC50 typically <100 pM). By epitope mapping experiments, we showed that NIB.2 binds to CD1d in close proximity to the interface of CD1d and the Type 1 NKT cell receptor β-chain. Together with data showing that NIB.2 inhibited stimulation via CD1d loaded with different glycolipids, this supports a mechanism whereby NIB.2 inhibits NKT cell activation by inhibiting Type 1 NKT cell receptor β-chain interactions with CD1d, independent of the lipid antigen in the CD1d antigen-binding cleft. The strong in vitro potency of NIB.2 was reflected in vivo in an Ascaris suum cynomolgus macaque asthma model. Compared with vehicle control, NIB.2 treatment significantly reduced bronchoalveolar lavage (BAL) levels of Ascaris-induced cytokines IL-5, IL-8 and IL-1 receptor antagonist, and significantly reduced baseline levels of GM-CSF, IL-6, IL-15, IL-12/23p40, MIP-1α, MIP-1β, and VEGF. At a cellular population level NIB.2 also reduced numbers of BAL lymphocytes and macrophages, and blood eosinophils and basophils. We demonstrate that anti-CD1d antibody blockade of the CD1d/NKT pathway modulates inflammatory parameters in vivo in a primate inflammation model, with therapeutic potential for diseases where the local cytokine milieu is critical.

  19. Ricin-Holotoxin-Based Vaccines: Induction of Potent Ricin-Neutralizing Antibodies.

    PubMed

    Sabo, Tamar; Kronman, Chanoch; Mazor, Ohad

    2016-01-01

    Ricin is one of the most potent and lethal toxins known to which there is no available antidote. Currently, the most promising therapy is based on neutralizing antibodies elicited by active vaccination or given passively. Here, detailed protocols are provided for the production of two ricin holotoxin-based vaccines: monomerized subunit-based vaccine, and a formaldehyde-based ricin toxoid vaccine. Both vaccines were found to be stable with no toxic activity reversion even after long-term storage while eliciting high anti-ricin antibody titers possessing a potent neutralizing activity. The use of these vaccines is highly suitable for both the production of sera that can be used in passive protection experiments and immunization aimed to isolate potent anti-ricin monoclonal antibodies.

  20. Potent neutralization of botulinum neurotoxin by recombinant oligoclonal antibody

    PubMed Central

    Nowakowski, A.; Wang, C.; Powers, D. B.; Amersdorfer, P.; Smith, T. J.; Montgomery, V. A.; Sheridan, R.; Blake, R.; Smith, L. A.; Marks, J. D.

    2002-01-01

    The botulinum neurotoxins (BoNTs) cause the paralytic human disease botulism and are one of the highest-risk threat agents for bioterrorism. To generate a pharmaceutical to prevent or treat botulism, monoclonal antibodies (mAbs) were generated by phage display and evaluated for neutralization of BoNT serotype A (BoNT/A) in vivo. Although no single mAb significantly neutralized toxin, a combination of three mAbs (oligoclonal Ab) neutralized 450,000 50% lethal doses of BoNT/A, a potency 90 times greater than human hyperimmune globulin. The potency of oligoclonal Ab was primarily due to a large increase in functional Ab binding affinity. The results indicate that the potency of the polyclonal humoral immune response can be deconvoluted to a few mAbs binding nonoverlapping epitopes, providing a route to drugs for preventing and treating botulism and diseases caused by other pathogens and biologic threat agents. PMID:12177434

  1. HIV-specific CD4-induced Antibodies Mediate Broad and Potent Antibody-dependent Cellular Cytotoxicity Activity and are Commonly Detected in Plasma from HIV-infected Humans

    PubMed Central

    Williams, Katherine L.; Cortez, Valerie; Dingens, Adam S.; Gach, Johannes S.; Rainwater, Stephanie; Weis, Julie F.; Chen, Xuemin; Spearman, Paul; Forthal, Donald N.; Overbaugh, Julie

    2015-01-01

    HIV-specific antibodies (Abs) can reduce viral burden by blocking new rounds of infection or by destroying infected cells via activation of effector cells through Fc–FcR interaction. This latter process, referred to as antibody-dependent cellular cytotoxicity (ADCC), has been associated with viral control and improved clinical outcome following both HIV and SIV infections. Here we describe an HIV viral-like particle (VLP)-based sorting strategy that led to identification of HIV-specific memory B cells encoding Abs that mediate ADCC from a subtype A-infected Kenyan woman at 914 days post-infection. Using this strategy, 12 HIV-envelope-specific monoclonal antibodies (mAbs) were isolated and three mediated potent ADCC activity when compared to well-characterized ADCC mAbs. The ADCC-mediating Abs also mediated antibody-dependent cell-mediated virus inhibition (ADCVI), which provides a net measure of Fc receptor-triggered effects against replicating virus. Two of the three ADCC-mediating Abs targeted a CD4-induced (CD4i) epitope also bound by the mAb C11; the third antibody targeted the N-terminus of V3. Both CD4i Abs identified here demonstrated strong cross-clade breadth with activity against 10 of 11 envelopes tested, including those from clades A, B, C, A/D and C/D, whereas the V3-specific antibody showed more limited breadth. Variants of these CD4i, C11-like mAbs engineered to interrupt binding to FcγRs inhibited a measurable percentage of the donor's ADCC activity starting as early as 189 days post-infection. C11-like antibodies also accounted for between 18–78% of ADCC activity in 9 chronically infected individuals from the same cohort study. Further, the two CD4i Abs originated from unique B cells, suggesting that antibodies targeting this epitope can be commonly produced. Taken together, these data provide strong evidence that CD4i, C11-like antibodies develop within the first 6 months of infection and they can arise from unique B-cell lineages in the

  2. Crystal structure of HIV-1 primary receptor CD4 in complex with a potent antiviral antibody.

    PubMed

    Freeman, Michael M; Seaman, Michael S; Rits-Volloch, Sophia; Hong, Xinguo; Kao, Chia-Ying; Ho, David D; Chen, Bing

    2010-12-08

    Ibalizumab is a humanized, anti-CD4 monoclonal antibody. It potently blocks HIV-1 infection and targets an epitope in the second domain of CD4 without interfering with immune functions mediated by interaction of CD4 with major histocompatibility complex (MHC) class II molecules. We report here the crystal structure of ibalizumab Fab fragment in complex with the first two domains (D1-D2) of CD4 at 2.2 Å resolution. Ibalizumab grips CD4 primarily by the BC-loop (residues 121-125) of D2, sitting on the opposite side of gp120 and MHC-II binding sites. No major conformational change in CD4 accompanies binding to ibalizumab. Both monovalent and bivalent forms of ibalizumab effectively block viral infection, suggesting that it does not need to crosslink CD4 to exert antiviral activity. While gp120-induced structural rearrangements in CD4 are probably minimal, CD4 structural rigidity is dispensable for ibalizumab inhibition. These results could guide CD4-based immunogen design and lead to a better understanding of HIV-1 entry.

  3. Crystal structure of HIV-1 primary receptor CD4 in complex with a potent antiviral antibody

    PubMed Central

    Freeman, Michael M.; Seaman, Michael S.; Rits-Volloch, Sophia; Hong, Xinguo; Kao, Chia-Ying; Ho, David D.; Chen, Bing

    2010-01-01

    Summary Ibalizumab is a humanized, anti-CD4 monoclonal antibody. It potently blocks HIV-1 infection and targets an epitope in the second domain of CD4 without interfering with immune functions mediated by interaction of CD4 with major histocompatibility complex (MHC) class II molecules. We report here the crystal structure of ibalizumab Fab fragment in complex with the first two domains (D1-D2) of CD4 at 2.2 Å resolution. Ibalizumab grips CD4 primarily by the BC-loop (residues 121-125) of D2, sitting on the opposite side of gp120 and MHC-II binding sites. No major conformational change in CD4 accompanies binding to ibalizumab. Both monovalent and bivalent forms of ibalizumab effectively block viral infection, suggesting that it does not need to crosslink CD4 to exert antiviral activity. While gp120-induced structural rearrangements in CD4 are probably minimal, CD4 structural rigidity is dispensable for ibalizumab inhibition. These results could guide CD4-based immunogen design and lead to a better understanding of HIV-1 entry. PMID:21134642

  4. Affinity-purified respiratory syncytial virus antibodies from intravenous immunoglobulin exert potent antibody-dependent cellular cytotoxicity.

    PubMed

    Gupta, Nimesh; LeGoff, Jerome; Chamat, Soulaima; Mercier-Delarue, Severine; Touzelet, Olivier; Power, Ultan F; Kazatchkine, Michel D; Simon, Francois; Lacroix-Desmazes, Sebastien; Bayry, Jagadeesh; Kaveri, Srinivas V

    2013-01-01

    Mixed infections are one of the major therapeutic challenges, as the current strategies have had limited success. One of the most common and widespread conditions of mixed infection is respiratory syncytial virus-mediated pathology of the respiratory tract in children. There is a dire need for the development of novel therapeutic approaches during mixed infections. Therapeutic intravenous immunoglobulin preparations, obtained from plasma pools of healthy donors have been used in immune deficiencies. This study was thus designed to characterize the functional efficacy of RSV-specific antibodies in IVIg. To explore the functional ability of these affinity-purified RSV-specific antibodies, the antibody-dependent and complement dependent cytotoxicity was determined using peripheral cells of healthy donors. This study demonstrates the existence of highly potent RSV-specific antibodies in IVIg preparations and provides the basis for the use of IVIg as broad-spectrum protective shield to RSV-infected children during mixed infections.

  5. Affinity-Purified Respiratory Syncytial Virus Antibodies from Intravenous Immunoglobulin Exert Potent Antibody-Dependent Cellular Cytotoxicity

    PubMed Central

    Gupta, Nimesh; LeGoff, Jerome; Chamat, Soulaima; Mercier-Delarue, Severine; Touzelet, Olivier; Power, Ultan F.; Kazatchkine, Michel D.; Simon, Francois; Lacroix-Desmazes, Sebastien; Bayry, Jagadeesh; Kaveri, Srinivas V.

    2013-01-01

    Mixed infections are one of the major therapeutic challenges, as the current strategies have had limited success. One of the most common and widespread conditions of mixed infection is respiratory syncytial virus-mediated pathology of the respiratory tract in children. There is a dire need for the development of novel therapeutic approaches during mixed infections. Therapeutic intravenous immunoglobulin preparations, obtained from plasma pools of healthy donors have been used in immune deficiencies. This study was thus designed to characterize the functional efficacy of RSV-specific antibodies in IVIg. To explore the functional ability of these affinity-purified RSV-specific antibodies, the antibody-dependent and complement dependent cytotoxicity was determined using peripheral cells of healthy donors. This study demonstrates the existence of highly potent RSV-specific antibodies in IVIg preparations and provides the basis for the use of IVIg as broad-spectrum protective shield to RSV-infected children during mixed infections. PMID:23894466

  6. Bispecific Antibodies Targeting Different Epitopes on the HIV-1 Envelope Exhibit Broad and Potent Neutralization

    PubMed Central

    Asokan, M.; Rudicell, R. S.; Louder, M.; McKee, K.; O'Dell, S.; Stewart-Jones, G.; Wang, K.; Xu, L.; Chen, X.; Choe, M.; Chuang, G.; Georgiev, I. S.; Joyce, M. G.; Kirys, T.; Ko, S.; Pegu, A.; Shi, W.; Todd, J. P.; Yang, Z.; Bailer, R. T.; Rao, S.; Kwong, P. D.; Nabel, G. J.

    2015-01-01

    ABSTRACT The potency and breadth of the recently isolated neutralizing human monoclonal antibodies to HIV-1 have stimulated interest in their use to prevent or to treat HIV-1 infection. Due to the antigenically diverse nature of the HIV-1 envelope (Env), no single antibody is highly active against all viral strains. While the physical combination of two broadly neutralizing antibodies (bNAbs) can improve coverage against the majority of viruses, the clinical-grade manufacturing and testing of two independent antibody products are time and resource intensive. In this study, we constructed bispecific immunoglobulins (IgGs) composed of independent antigen-binding fragments with a common Fc region. We developed four different bispecific IgG variants that included antibodies targeting four major sites of HIV-1 neutralization. We show that these bispecific IgGs display features of both antibody specificities and, in some cases, display improved coverage over the individual parental antibodies. All four bispecific IgGs neutralized 94% to 97% of antigenically diverse viruses in a panel of 206 HIV-1 strains. Among the bispecific IgGs tested, VRC07 × PG9-16 displayed the most favorable neutralization profile. It was superior in breadth to either of the individual antibodies, neutralizing 97% of viruses with a median 50% inhibitory concentration (IC50) of 0.055 μg/ml. This bispecific IgG also demonstrated in vivo pharmacokinetic parameters comparable to those of the parental bNAbs when administered to rhesus macaques. These results suggest that IgG-based bispecific antibodies are promising candidates for the prevention and treatment of HIV-1 infection in humans. IMPORTANCE To prevent or treat HIV-1 infection, antibodies must potently neutralize nearly all strains of HIV-1. Thus, the physical combination of two or more antibodies may be needed to broaden neutralization coverage and diminish the possibility of viral resistance. A bispecific antibody that has two different

  7. Isolation of potent neutralizing antibodies from a survivor of the 2014 Ebola virus outbreak

    PubMed Central

    Bornholdt, Zachary A.; Turner, Hannah L.; Murin, Charles D.; Li, Wen; Sok, Devin; Souders, Colby A.; Piper, Ashley E.; Goff, Arthur; Shamblin, Joshua D.; Wollen, Suzanne E.; Sprague, Thomas R.; Fusco, Marnie L.; Pommert, Kathleen B.J.; Cavacini, Lisa A.; Smith, Heidi L.; Klempner, Mark; Reimann, Keith A.; Krauland, Eric; Gerngross, Tillman U.; Wittrup, Dane K.; Saphire, Erica Ollmann; Burton, Dennis R.; Glass, Pamela J.; Ward, Andrew B.; Walker, Laura M.

    2016-01-01

    Antibodies targeting the Ebola virus surface glycoprotein (EBOV GP) are implicated in protection against lethal disease, but the characteristics of the human antibody response to EBOV GP remain poorly understood. Here we isolated and characterized 349 GP-specific monoclonal antibodies (mAbs) from the peripheral B cells of a convalescent donor who survived the 2014 EBOV Zaire outbreak. Remarkably, 77% of the mAbs neutralize live EBOV and several mAbs exhibit unprecedented potency. Structures of selected mAbs in complex with GP reveal a site of vulnerability located in the GP stalk region proximal to the viral membrane. Neutralizing antibodies (NAbs) targeting this site show potent therapeutic efficacy against lethal EBOV challenge in mice. The results provide a framework for the design of new EBOV vaccine candidates and immunotherapies. PMID:26912366

  8. A novel bispecific antibody, S-Fab, induces potent cancer cell killing.

    PubMed

    Li, Li; He, Ping; Zhou, Changhua; Jing, Li; Dong, Bin; Chen, Siqi; Zhang, Ning; Liu, Yawei; Miao, Ji; Wang, Zhong; Li, Qing

    2015-01-01

    Bispecific antibodies that engage immune cells to kill cancer cells have been actively studied in cancer immunotherapy. In this study, we present a novel bispecific format, S-Fab, fabricated by linking a single-domain anti-carcinoembryonic antigen VHH to a conventional anti-CD3 Fab. In contrast to most bispecific antibodies, the S-Fab bispecific antibody can be efficiently expressed and purified from bacteria. The purified S-Fab is stable in serum and is able to recruit T cells to drive potent cancer cell killing. In xenograft models, the S-Fab antibody suppresses tumor growth in the presence of human immune cells. Our study suggested that the bispecific S-Fab format can be applied to a wide range of immunotherapies.

  9. ARGX-110, a highly potent antibody targeting CD70, eliminates tumors via both enhanced ADCC and immune checkpoint blockade

    PubMed Central

    Silence, Karen; Dreier, Torsten; Moshir, Mahan; Ulrichts, Peter; Gabriels, Sofie ME; Saunders, Michael; Wajant, Harald; Brouckaert, Peter; Huyghe, Leander; Van Hauwermeiren, Tim; Thibault, Alain; De Haard, Hans J

    2014-01-01

    Overexpression of CD70 has been documented in a variety of solid and hematological tumors, where it is thought to play a role in tumor proliferation and evasion of immune surveillance. Here, we describe ARGX-110, a defucosylated IgG1 monoclonal antibody (mAb) that selectively targets and neutralizes CD70, the ligand of CD27.   ARGX-110 was generated by immunization of outbred llamas. The antibody was germlined to 95% human identity, and its anti-tumor efficacy was tested in several in vitro assays. ARGX-110 binds CD70 with picomolar affinity. In depletion studies, ARGX-110 lyses tumor cells with greater efficacy than its fucosylated version. In addition, ARGX-110 demonstrates strong complement-dependent cytotoxicity and antibody-dependent cellular phagocytosis activity. ARGX-110 inhibits signaling of CD27, which results in blocking of the activation and proliferation of Tregs. In a Raji xenograft model, administration of the fucosylated version of ARGX-110 resulted in a prolonged survival at doses of 0.1 mg/kg and above. The pharmacokinetics of ARGX-110 was tested in cynomolgus monkeys; the calculated half-life is 12 days. In conclusion, ARGX-110 is a potent blocking mAb with a dual mode of action against both CD70-bearing tumor cells and CD70-dependent Tregs. This antibody is now in a Phase 1 study in patients with advanced malignancies expressing CD70 (NCT01813539). PMID:24492296

  10. Structural and pharmacological characterization of novel potent and selective monoclonal antibody antagonists of glucose-dependent insulinotropic polypeptide receptor.

    PubMed

    Ravn, Peter; Madhurantakam, Chaithanya; Kunze, Susan; Matthews, Evelyn; Priest, Claire; O'Brien, Siobhan; Collinson, Andie; Papworth, Monika; Fritsch-Fredin, Maria; Jermutus, Lutz; Benthem, Lambertus; Gruetter, Markus; Jackson, Ronald H

    2013-07-05

    Glucose-dependent insulinotropic polypeptide (GIP) is an endogenous hormonal factor (incretin) that, upon binding to its receptor (GIPr; a class B G-protein-coupled receptor), stimulates insulin secretion by beta cells in the pancreas. There has been a lack of potent inhibitors of the GIPr with prolonged in vivo exposure to support studies on GIP biology. Here we describe the generation of an antagonizing antibody to the GIPr, using phage and ribosome display libraries. Gipg013 is a specific competitive antagonist with equally high potencies to mouse, rat, dog, and human GIP receptors with a Ki of 7 nm for the human GIPr. Gipg013 antagonizes the GIP receptor and inhibits GIP-induced insulin secretion in vitro and in vivo. A crystal structure of Gipg013 Fab in complex with the human GIPr extracellular domain (ECD) shows that the antibody binds through a series of hydrogen bonds from the complementarity-determining regions of Gipg013 Fab to the N-terminal α-helix of GIPr ECD as well as to residues around its highly conserved glucagon receptor subfamily recognition fold. The antibody epitope overlaps with the GIP binding site on the GIPr ECD, ensuring competitive antagonism of the receptor. This well characterized antagonizing antibody to the GIPr will be useful as a tool to further understand the biological roles of GIP.

  11. A potent broad-spectrum protective human monoclonal antibody crosslinking two haemagglutinin monomers of influenza A virus

    PubMed Central

    Wu, Ying; Cho, MyungSam; Shore, David; Song, Manki; Choi, JungAh; Jiang, Tao; Deng, Yong-Qiang; Bourgeois, Melissa; Almli, Lynn; Yang, Hua; Chen, Li-Mei; Shi, Yi; Qi, Jianxu; Li, An; Yi, Kye Sook; Chang, MinSeok; Bae, Jin Soo; Lee, HyunJoo; Shin, JiYoung; Stevens, James; Hong, SeoungSuh; Qin, Cheng-Feng; Gao, George F.; Chang, Shin Jae; Donis, Ruben O.

    2015-01-01

    Effective annual influenza vaccination requires frequent changes in vaccine composition due to both antigenic shift for different subtype hemagglutinins (HAs) and antigenic drift in a particular HA. Here we present a broadly neutralizing human monoclonal antibody with an unusual binding modality. The antibody, designated CT149, was isolated from convalescent patients infected with pandemic H1N1 in 2009. CT149 is found to neutralize all tested group 2 and some group 1 influenza A viruses by inhibiting low pH-induced, HA-mediated membrane fusion. It promotes killing of infected cells by Fc-mediated antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. X-ray crystallographic data reveal that CT149 binds primarily to the fusion domain in HA2, and the light chain is also largely involved in binding. The epitope recognized by this antibody comprises amino-acid residues from two adjacent protomers of HA. This binding characteristic of CT149 will provide more information to support the design of more potent influenza vaccines. PMID:26196962

  12. Crystal structure of human antibody 2909 reveals conserved features of quaternary structure-specific antibodies that potently neutralize HIV-1.

    PubMed

    Changela, Anita; Wu, Xueling; Yang, Yongping; Zhang, Baoshan; Zhu, Jiang; Nardone, Glenn A; O'Dell, Sijy; Pancera, Marie; Gorny, Miroslaw K; Phogat, Sanjay; Robinson, James E; Stamatatos, Leonidas; Zolla-Pazner, Susan; Mascola, John R; Kwong, Peter D

    2011-03-01

    Monoclonal antibody 2909 belongs to a class of potently neutralizing antibodies that recognize quaternary epitopes on HIV-1. Some members of this class, such as 2909, are strain specific, while others, such as antibody PG16, are broadly neutralizing; all, however, recognize a region on the gp120 envelope glycoprotein that includes two loops (V2 and V3) and forms appropriately only in the oligomeric HIV-1 spike (gp120(3)/gp41(3)). Here we present the crystal structure of 2909 and report structure-function analysis with antibody chimeras composed of 2909 and other members of this antibody class. The 2909 structure was dominated by a heavy-chain third-complementarity-determining region (CDR H3) of 21 residues, which comprised 36% of the combining surface and formed a β-hairpin club extending ∼20 Å beyond the rest of the antibody. Sequence analysis and mass spectrometry identified sites of tyrosine sulfation at the middle and top of CDR H3; substitutions with phenylalanine either ablated (middle substitution) or substantially diminished (top substitution) neutralization. Chimeric antibodies composed of heavy and light chains, exchanged between 2909 and other members of the class, indicated a substantial lack of complementation. Comparison of 2909 to PG16 (which is tyrosine sulfated and the only other member of the class for which a structure has previously been reported) showed that both utilize protruding, anionic CDR H3s for recognition. Thus, despite some diversity, members of this class share structural and functional similarities, with conserved features of the CDR H3 subdomain likely reflecting prevalent solutions by the human immune system for recognition of a quaternary site of HIV-1 vulnerability.

  13. Conformation-dependent high-affinity potent ricin-neutralizing monoclonal antibodies.

    PubMed

    Hu, Wei-Gang; Yin, Junfei; Chau, Damon; Hu, Charles Chen; Lillico, Dustin; Yu, Justin; Negrych, Laurel M; Cherwonogrodzky, John W

    2013-01-01

    Ricin is a potential biothreat agent with no approved antidote available for ricin poisoning. The aim of this study was to develop potent antibody-based antiricin antidotes. Four strong ricin resistant hybridoma clones secreting antiricin monoclonal antibodies (mAbs) were developed. All four mAbs are bound to conformational epitopes of ricin toxin B (RTB) with high affinity (KD values from 2.55 to 36.27 nM). RTB not only triggers cellular uptake of ricin, but also facilitates transport of the ricin toxin A (RTA) from the endoplasmic reticulum to the cytosol, where RTA exerts its toxic activity. The four mAbs were found to have potent ricin-neutralizing capacities and synergistic effects among them as determined by an in vitro neutralization assay. In vivo protection assay demonstrated that all four mAbs had strong efficacy against ricin challenges. D9 was found to be exceptionally effective. Intraperitoneal (i.p.) administration of D9, at a dose of 5 μ g, 6 weeks before or 6 hours after an i.p. challenge with 5 × LD50 of ricin was able to protect or rescue 100% of the mice, indicating that mAb D9 is an excellent candidate to be developed as a potent antidote against ricin poisoning for both prophylactic and therapeutic purposes.

  14. Conformation-Dependent High-Affinity Potent Ricin-Neutralizing Monoclonal Antibodies

    PubMed Central

    Hu, Wei-Gang; Yin, Junfei; Chau, Damon; Hu, Charles Chen; Lillico, Dustin; Yu, Justin; Negrych, Laurel M.; Cherwonogrodzky, John W.

    2013-01-01

    Ricin is a potential biothreat agent with no approved antidote available for ricin poisoning. The aim of this study was to develop potent antibody-based antiricin antidotes. Four strong ricin resistant hybridoma clones secreting antiricin monoclonal antibodies (mAbs) were developed. All four mAbs are bound to conformational epitopes of ricin toxin B (RTB) with high affinity (KD values from 2.55 to 36.27 nM). RTB not only triggers cellular uptake of ricin, but also facilitates transport of the ricin toxin A (RTA) from the endoplasmic reticulum to the cytosol, where RTA exerts its toxic activity. The four mAbs were found to have potent ricin-neutralizing capacities and synergistic effects among them as determined by an in vitro neutralization assay. In vivo protection assay demonstrated that all four mAbs had strong efficacy against ricin challenges. D9 was found to be exceptionally effective. Intraperitoneal (i.p.) administration of D9, at a dose of 5 μg, 6 weeks before or 6 hours after an i.p. challenge with 5 × LD50 of ricin was able to protect or rescue 100% of the mice, indicating that mAb D9 is an excellent candidate to be developed as a potent antidote against ricin poisoning for both prophylactic and therapeutic purposes. PMID:23484120

  15. Potent neutralization of VEGF biological activities with a fully human antibody Fab fragment directed against VEGF receptor 2

    SciTech Connect

    Miao, H.-Q. . E-mail: hua-quan.miao@imclone.com; Hu, Kun; Jimenez, Xenia; Navarro, Elizabeth; Zhang, Haifan; Lu Dan; Ludwig, Dale L.; Balderes, Paul; Zhu Zhenping . E-mail: zhenping.zhu@imclone.com

    2006-06-23

    Compelling evidence suggest that vascular endothelial growth factor (VEGF) and its receptors, especially receptor 2 (VEGFR2, or kinase insert domain-containing receptor, KDR), play a critical role in angiogenesis under both physiological and pathological conditions, including cancer and angiogenic retinopathies such as age-related macular degeneration (AMD). To this end, inhibition of angiogenesis with antagonists to either VEGF or KDR has yielded significant therapeutic efficacy both in preclinical studies in animal models and in clinical trials in patients with cancer and AMD. We previously reported the identification of a high affinity, fully human anti-KDR antibody fragment, 1121B Fab, through a highly stringent affinity maturation process with a Fab originally isolated from a naive human antibody phage display library. In this study, we demonstrate that 1121B Fab is able to strongly block KDR/VEGF interaction, resulting in potent inhibition of an array of biological activities of VEGF, including activation of the receptor and its signaling pathway, intracellular calcium mobilization, and migration and proliferation of endothelial cells. Taken together, our data lend strong support to the further development of 1121B Fab fragment as an anti-angiogenesis agent in both cancer and angiogenic retinopathies.

  16. Human monomeric antibody fragments to TRAIL-R1 and TRAIL-R2 that display potent in vitro agonism

    PubMed Central

    Main, Sarah; Newton, Philip; Chodorge, Matthieu; Cadwallader, Karen; Humphreys, Robin; Albert, Vivian; Vaughan, Tristan J; Minter, Ralph R; Edwards, Bryan M

    2009-01-01

    Apoptosis through the TRAIL receptor pathway can be induced via agonistic IgG to either TRAIL-R1 or TRAIL-R2. Here we describe the use of phage display to isolate a substantive panel of fully human anti-TRAIL receptor single chain Fv fragments (scFvs); 234 and 269 different scFvs specific for TRAIL-R1 and TRAIL-R2 respectively. In addition, 134 different scFvs that were cross-reactive for both receptors were isolated. To facilitate screening of all 637 scFvs for potential agonistic activity in vitro, a novel high-throughput surrogate apoptosis assay was developed. Ten TRAIL-R1 specific scFv and 6 TRAIL-R2 specific scFv were shown to inhibit growth of tumor cells in vitro in the absence of any cross-linking agents. These scFv were all highly specific for either TRAIL-R1 or TRAIL-R2, potently inhibited tumor cell proliferation, and were antagonists of TRAIL binding. Moreover, further characterization of TRAIL-R1 agonistic scFv demonstrated significant anti-tumor activity when expressed and purified as a monomeric Fab fragment. Thus, scFv and Fab fragments, in addition to whole IgG, can be agonistic and induce tumor cell death through specific binding to either TRAIL-R1 or TRAIL-R2. These potent agonistic scFv were all isolated directly from the starting phage antibody library and demonstrated significant tumor cell killing properties without any requirement for affinity maturation. Some of these selected scFv have been converted to IgG format and are being studied extensively in clinical trials to investigate their potential utility as human monoclonal antibody therapeutics for the treatment of human cancer. PMID:20068388

  17. On the role of phosphatidylethanolamine in the inhibition of activated protein C activity by antiphospholipid antibodies.

    PubMed Central

    Smirnov, M D; Triplett, D T; Comp, P C; Esmon, N L; Esmon, C T

    1995-01-01

    Phosphatidylethanolamine (PE) is an important membrane component for supporting activated protein C anticoagulant activity but has little influence on prothrombin activation. This difference constitutes a potential mechanism for selective inhibition of the protein C anticoagulant pathway by lupus anticoagulants and/or antiphospholipid antibodies. In this study, we demonstrate that the presence of PE augments lupus anticoagulant activity. In the plasma of some patients with lupus anticoagulants, activated protein C anticoagulant activity is more potently inhibited than prothrombin activation. As a result, in the presence of activated protein C and PE, these patient plasmas clot faster than normal plasma. Patients with minimal lupus anticoagulant activity are identified whose plasma potently inhibits activated protein C anticoagulant activity. This process is also PE dependent. In three patient plasmas, these phenomena are shown to be due to immunoglobulins. The PE requirement in the expression of activated protein C anticoagulant activity and the PE dependence of some antiphospholipid antibodies provide a mechanistic basis for the selective inhibition of the protein C pathway. Inhibition of activated protein C function may be a common mechanism contributing to increased thrombotic risk in certain patients with antiphospholipid antibodies. PMID:7814631

  18. CROSS-REACTIVE AND POTENT NEUTRALIZING ANTIBODY RESPONSES IN HUMAN SURVIVORS OF NATURAL EBOLAVIRUS INFECTION

    PubMed Central

    Flyak, Andrew I.; Shen, Xiaoli; Murin, Charles D.; Turner, Hannah L.; David, Joshua A.; Fusco, Marnie L.; Lampley, Rebecca; Kose, Nurgun; Ilinykh, Philipp A.; Kuzmina, Natalia; Branchizio, Andre; King, Hannah; Brown, Leland; Bryan, Christopher; Davidson, Edgar; Doranz, Benjamin J.; Slaughter, James C.; Sapparapu, Gopal; Klages, Curtis; Ksiazek, Thomas G.; Saphire, Erica Ollmann; Ward, Andrew B.; Bukreyev, Alexander; Crowe, James E.

    2015-01-01

    Summary Recent studies have suggested that antibody-mediated protection against the Ebolaviruses may be achievable, but little is known about whether or not antibodies can confer cross-reactive protection against viruses belonging to diverse Ebolavirus species, such as Ebola virus (EBOV), Sudan virus (SUDV) and Bundibugyo virus (BDBV). We isolated a large panel of human monoclonal antibodies (mAbs) against BDBV glycoprotein (GP) using peripheral blood B cells from survivors of the 2007 BDBV outbreak in Uganda. We determined that a large proportion of mAbs with potent neutralizing activity against BDBV bind to the glycan cap and recognize diverse epitopes within this major antigenic site. We identified several glycan cap-specific mAbs that neutralized multiple ebolaviruses including SUDV, and a cross-reactive mAb that completely protected guinea pigs from the lethal challenge with heterologous EBOV. Our results provide a roadmap to develop a single antibody-based treatment effective against multiple Ebolavirus infections. PMID:26806128

  19. Production of Potent Fully Human Polyclonal Antibodies against Ebola Zaire Virus in Transchromosomal Cattle

    PubMed Central

    Dye, John M.; Wu, Hua; Hooper, Jay W.; Khurana, Surender; Kuehne, Ana I.; Coyle, Elizabeth M.; Ortiz, Ramon A.; Fuentes, Sandra; Herbert, Andrew S.; Golding, Hana; Bakken, Russell A.; Brannan, Jennifer M.; Kwilas, Steve A.; Sullivan, Eddie J.; Luke, Thomas C.; Smith, Gale; Glenn, Gregory; Li, Wenfang; Ye, Ling; Yang, Chinglai; Compans, Richard W.; Tripp, Ralph A.; Jiao, Jin-an

    2016-01-01

    Polyclonal antibodies, derived from humans or hyperimmunized animals, have been used prophylactically or therapeutically as countermeasures for a variety of infectious diseases. SAB Biotherapeutics has successfully developed a transchromosomic (Tc) bovine platform technology that can produce fully human immunoglobulins rapidly, and in substantial quantities, against a variety of disease targets. In this study, two Tc bovines expressing high levels of fully human IgG were hyperimmunized with a recombinant glycoprotein (GP) vaccine consisting of the 2014 Ebola virus (EBOV) Makona isolate. Serum collected from these hyperimmunized Tc bovines contained high titers of human IgG against EBOV GP as determined by GP specific ELISA, surface plasmon resonance (SPR), and virus neutralization assays. Fully human polyclonal antibodies against EBOV were purified and evaluated in a mouse challenge model using mouse adapted Ebola virus (maEBOV). Intraperitoneal administration of the purified anti-EBOV IgG (100 mg/kg) to BALB/c mice one day after lethal challenge with maEBOV resulted in 90% protection; whereas 100% of the control animals succumbed. The results show that hyperimmunization of Tc bovines with EBOV GP can elicit protective and potent neutralizing fully human IgG antibodies rapidly and in commercially viable quantities. PMID:27109916

  20. Production of Potent Fully Human Polyclonal Antibodies against Ebola Zaire Virus in Transchromosomal Cattle.

    PubMed

    Dye, John M; Wu, Hua; Hooper, Jay W; Khurana, Surender; Kuehne, Ana I; Coyle, Elizabeth M; Ortiz, Ramon A; Fuentes, Sandra; Herbert, Andrew S; Golding, Hana; Bakken, Russell A; Brannan, Jennifer M; Kwilas, Steve A; Sullivan, Eddie J; Luke, Thomas C; Smith, Gale; Glenn, Gregory; Li, Wenfang; Ye, Ling; Yang, Chinglai; Compans, Richard W; Tripp, Ralph A; Jiao, Jin-An

    2016-04-25

    Polyclonal antibodies, derived from humans or hyperimmunized animals, have been used prophylactically or therapeutically as countermeasures for a variety of infectious diseases. SAB Biotherapeutics has successfully developed a transchromosomic (Tc) bovine platform technology that can produce fully human immunoglobulins rapidly, and in substantial quantities, against a variety of disease targets. In this study, two Tc bovines expressing high levels of fully human IgG were hyperimmunized with a recombinant glycoprotein (GP) vaccine consisting of the 2014 Ebola virus (EBOV) Makona isolate. Serum collected from these hyperimmunized Tc bovines contained high titers of human IgG against EBOV GP as determined by GP specific ELISA, surface plasmon resonance (SPR), and virus neutralization assays. Fully human polyclonal antibodies against EBOV were purified and evaluated in a mouse challenge model using mouse adapted Ebola virus (maEBOV). Intraperitoneal administration of the purified anti-EBOV IgG (100 mg/kg) to BALB/c mice one day after lethal challenge with maEBOV resulted in 90% protection; whereas 100% of the control animals succumbed. The results show that hyperimmunization of Tc bovines with EBOV GP can elicit protective and potent neutralizing fully human IgG antibodies rapidly and in commercially viable quantities.

  1. Vaccine-Derived Neutralizing Antibodies to the Human Cytomegalovirus gH/gL Pentamer Potently Block Primary Cytotrophoblast Infection

    PubMed Central

    Chiuppesi, Flavia; Wussow, Felix; Johnson, Erica; Bian, Chao; Zhuo, Meng; Rajakumar, Augustine; Barry, Peter A.; Britt, William J.; Chakraborty, Rana

    2015-01-01

    ABSTRACT Human cytomegalovirus (HCMV) elicits neutralizing antibodies (NAb) of various potencies and cell type specificities to prevent HCMV entry into fibroblasts (FB) and epithelial/endothelial cells (EpC/EnC). NAb targeting the major essential envelope glycoprotein complexes gB and gH/gL inhibit both FB and EpC/EnC entry. In contrast to FB infection, HCMV entry into EpC/EnC is additionally blocked by extremely potent NAb to conformational epitopes of the gH/gL/UL128/130/131A pentamer complex (PC). We recently developed a vaccine concept based on coexpression of all five PC subunits by a single modified vaccinia virus Ankara (MVA) vector, termed MVA-PC. Vaccination of mice and rhesus macaques with MVA-PC resulted in a high titer and sustained NAb that blocked EpC/EnC infection and lower-titer NAb that inhibited FB entry. However, antibody function responsible for the neutralizing activity induced by the MVA-PC vaccine is uncharacterized. Here, we demonstrate that MVA-PC elicits NAb with cell type-specific neutralization potency and antigen recognition pattern similar to human NAb targeting conformational and linear epitopes of the UL128/130/131A subunits or gH. In addition, we show that the vaccine-derived PC-specific NAb are significantly more potent than the anti-gH NAb to prevent HCMV spread in EpC and infection of human placental cytotrophoblasts, cell types thought to be of critical importance for HCMV transmission to the fetus. These findings further validate MVA-PC as a clinical vaccine candidate to elicit NAb that resembles those induced during HCMV infection and provide valuable insights into the potency of PC-specific NAb to interfere with HCMV cell-associated spread and infection of key placental cells. IMPORTANCE As a consequence of the leading role of human cytomegalovirus (HCMV) in causing permanent birth defects, developing a vaccine against HCMV has been assigned a major public health priority. We have recently introduced a vaccine strategy based

  2. Transient antibody targeting of CD45RC induces transplant tolerance and potent antigen-specific regulatory T cells

    PubMed Central

    Picarda, Elodie; Bézie, Séverine; Boucault, Laetitia; Autrusseau, Elodie; Kilens, Stéphanie; Martinet, Bernard; Daguin, Véronique; Donnart, Audrey; Charpentier, Eric; Anegon, Ignacio

    2017-01-01

    Rat and human CD4+ and CD8+ Tregs expressing low levels of CD45RC have strong immunoregulatory properties. We describe here that human CD45 isoforms are nonredundant and identify distinct subsets of cells. We show that CD45RC is not expressed by CD4+ and CD8+ Foxp3+ Tregs, while CD45RA/RB/RO are. Transient administration of a monoclonal antibody (mAb) targeting CD45RC in a rat cardiac allotransplantation model induced transplant tolerance associated with inhibition of allogeneic humoral responses but maintained primary and memory responses against cognate antigens. Anti-CD45RC mAb induced rapid death of CD45RChigh T cells through intrinsic cell signaling but preserved and potentiated CD4+ and CD8+ CD45RClow/– Tregs, which are able to adoptively transfer donor-specific tolerance to grafted recipients. Anti-CD45RC treatment results in distinct transcriptional signature of CD4+ and CD8+ CD45RClow/– Tregs. Finally, we demonstrate that anti-human CD45RC treatment inhibited graft-versus-host disease (GVHD) in immune-humanized NSG mice. Thus, short-term anti-CD45RC is a potent therapeutic candidate to induce transplantation tolerance in human. PMID:28194440

  3. Potent single-domain antibodies that arrest respiratory syncytial virus fusion protein in its prefusion state

    PubMed Central

    Rossey, Iebe; Gilman, Morgan S. A.; Kabeche, Stephanie C.; Sedeyn, Koen; Wrapp, Daniel; Kanekiyo, Masaru; Chen, Man; Mas, Vicente; Spitaels, Jan; Melero, José A.; Graham, Barney S.; Schepens, Bert; McLellan, Jason S.; Saelens, Xavier

    2017-01-01

    Human respiratory syncytial virus (RSV) is the main cause of lower respiratory tract infections in young children. The RSV fusion protein (F) is highly conserved and is the only viral membrane protein that is essential for infection. The prefusion conformation of RSV F is considered the most relevant target for antiviral strategies because it is the fusion-competent form of the protein and the primary target of neutralizing activity present in human serum. Here, we describe two llama-derived single-domain antibodies (VHHs) that have potent RSV-neutralizing activity and bind selectively to prefusion RSV F with picomolar affinity. Crystal structures of these VHHs in complex with prefusion F show that they recognize a conserved cavity formed by two F protomers. In addition, the VHHs prevent RSV replication and lung infiltration of inflammatory monocytes and T cells in RSV-challenged mice. These prefusion F-specific VHHs represent promising antiviral agents against RSV. PMID:28194013

  4. Vacuolin-1 potently and reversibly inhibits autophagosome-lysosome fusion by activating RAB5A

    PubMed Central

    Lu, Yingying; Dong, Shichen; Hao, Baixia; Li, Chang; Zhu, Kaiyuan; Guo, Wenjing; Wang, Qian; Cheung, King-Ho; Wong, Connie WM; Wu, Wu-Tian; Markus, Huss; Yue, Jianbo

    2014-01-01

    Autophagy is a catabolic lysosomal degradation process essential for cellular homeostasis and cell survival. Dysfunctional autophagy has been associated with a wide range of human diseases, e.g., cancer and neurodegenerative diseases. A large number of small molecules that modulate autophagy have been widely used to dissect this process and some of them, e.g., chloroquine (CQ), might be ultimately applied to treat a variety of autophagy-associated human diseases. Here we found that vacuolin-1 potently and reversibly inhibited the fusion between autophagosomes and lysosomes in mammalian cells, thereby inducing the accumulation of autophagosomes. Interestingly, vacuolin-1 was less toxic but at least 10-fold more potent in inhibiting autophagy compared with CQ. Vacuolin-1 treatment also blocked the fusion between endosomes and lysosomes, resulting in a defect in general endosomal-lysosomal degradation. Treatment of cells with vacuolin-1 alkalinized lysosomal pH and decreased lysosomal Ca2+ content. Besides marginally inhibiting vacuolar ATPase activity, vacuolin-1 treatment markedly activated RAB5A GTPase activity. Expression of a dominant negative mutant of RAB5A or RAB5A knockdown significantly inhibited vacuolin-1-induced autophagosome-lysosome fusion blockage, whereas expression of a constitutive active form of RAB5A suppressed autophagosome-lysosome fusion. These data suggest that vacuolin-1 activates RAB5A to block autophagosome-lysosome fusion. Vacuolin-1 and its analogs present a novel class of drug that can potently and reversibly modulate autophagy. PMID:25483964

  5. Potent inhibition of native TREK-1 K+ channels by selected dihydropyridine Ca2+ channel antagonists.

    PubMed

    Liu, Haiyan; Enyeart, Judith A; Enyeart, John J

    2007-10-01

    Bovine adrenal zona fasciculata (AZF) cells express bTREK-1 background K+ channels that set the resting membrane potential. Whole-cell and single-channel patch-clamp recording were used to compare five Ca2+ channel antagonists with respect to their potency as inhibitors of native bTREK-1 K+ channels. The dihydropyridine (DHP) Ca2+ channel antagonists amlodipine and niguldipine potently and specifically inhibited bTREK-1 with IC50 values of 0.43 and 0.75 microM, respectively. The other Ca2+ channel antagonists, including the DHP nifedipine, the diphenyldiperazine flunarizine, and the cannabinoid anandamide were less potent, with IC50 values of 8.18, 2.48, and 5.07 microM, respectively. Additional studies with the highly prescribed antihypertensive amlodipine showed that inhibition of bTREK-1 by this agent was voltage-independent and specific. At concentrations that produced near complete block of bTREK-1, amlodipine inhibited voltage-gated Kv1.4 K+ and T-type Ca2+ currents in AZF cells by less than 10%. At the single-channel level, amlodipine reduced bTREK-1 open probability without altering the unitary conductance. The results demonstrate that selected DHP L-type Ca2+ channel antagonists potently inhibit native bTREK-1 K+ channels, whereas other Ca2+ channel antagonists also inhibit bTREK-1 at higher concentrations. Collectively, organic Ca2+ channel antagonists make up the most potent class of TREK-1 inhibitors yet described. Because TREK-1 K+ channels are widely expressed in the central nervous and cardiovascular systems, it is possible that some of the therapeutic or toxic effects of frequently prescribed drugs such as amlodipine may be due to their interaction with TREK-1 K+ rather L-type Ca2+ channels.

  6. A three monoclonal antibody combination potently neutralizes multiple botulinum neurotoxin serotype F subtypes

    PubMed Central

    Fan, Yongfeng; Garcia-Rodriguez, Consuelo; Lou, Jianlong; Wen, Weihua; Conrad, Fraser; Zhai, Wenwu; Smith, Theresa J.; Smith, Leonard A.

    2017-01-01

    Human botulism is primarily caused by botulinum neurotoxin (BoNT) serotypes A, B and E, with around 1% caused by serotype F (BoNT/F). BoNT/F comprises at least seven different subtypes with the amino acid sequence difference between subtypes as high as 36%. The sequence differences present a significant challenge for generating monoclonal antibodies (mAbs) that can bind, detect and neutralize all BoNT/F subtypes. We used repertoire cloning of immune mouse antibody variable (V) regions and yeast display to generate a panel of 33 lead single chain Fv (scFv) mAbs that bound one or more BoNT/F subtypes with a median equilibrium dissociation constant (KD) of 4.06 × 10−9 M. By diversifying the V-regions of the lead mAbs and selecting for cross reactivity we generated five mAbs that bound each of the seven subtypes. Three scFv binding non-overlapping epitopes were converted to IgG that had KD for the different BoNT/F subtypes ranging from 2.2×10−8 M to 1.47×10−12 pM. An equimolar combination of the mAbs was able to potently neutralize BoNT/F1, F2, F4 and F7 in the mouse neutralization assay. The mAbs have potential utility as diagnostics capable of recognizing the known BoNT/F subtypes and could be developed as antitoxins to prevent and treat type F botulism. PMID:28323873

  7. A three monoclonal antibody combination potently neutralizes multiple botulinum neurotoxin serotype F subtypes.

    PubMed

    Fan, Yongfeng; Garcia-Rodriguez, Consuelo; Lou, Jianlong; Wen, Weihua; Conrad, Fraser; Zhai, Wenwu; Smith, Theresa J; Smith, Leonard A; Marks, James D

    2017-01-01

    Human botulism is primarily caused by botulinum neurotoxin (BoNT) serotypes A, B and E, with around 1% caused by serotype F (BoNT/F). BoNT/F comprises at least seven different subtypes with the amino acid sequence difference between subtypes as high as 36%. The sequence differences present a significant challenge for generating monoclonal antibodies (mAbs) that can bind, detect and neutralize all BoNT/F subtypes. We used repertoire cloning of immune mouse antibody variable (V) regions and yeast display to generate a panel of 33 lead single chain Fv (scFv) mAbs that bound one or more BoNT/F subtypes with a median equilibrium dissociation constant (KD) of 4.06 × 10-9 M. By diversifying the V-regions of the lead mAbs and selecting for cross reactivity we generated five mAbs that bound each of the seven subtypes. Three scFv binding non-overlapping epitopes were converted to IgG that had KD for the different BoNT/F subtypes ranging from 2.2×10-8 M to 1.47×10-12 pM. An equimolar combination of the mAbs was able to potently neutralize BoNT/F1, F2, F4 and F7 in the mouse neutralization assay. The mAbs have potential utility as diagnostics capable of recognizing the known BoNT/F subtypes and could be developed as antitoxins to prevent and treat type F botulism.

  8. Potent Selective Inhibition of Monoamine Oxidase A by Alternariol Monomethyl Ether Isolated from Alternaria brassicae.

    PubMed

    Lee, Hyun Woo; Kim, Yeon Ji; Nam, Sang-Jip; Kim, Hoon

    2017-02-28

    Alternariol monomethyl ether (AME), a dibenzopyrone derivative, was isolated from Alternaria brassicae along with altertoxin II (ATX-II). The compounds were tested for the inhibitory activity of monoamine oxidase (MAO), which catalyzes neurotransmitting monoamines. AME was found to be a highly potent and selective inhibitor of human MAO-A with an IC50 value of 1.71 µM; however, it was found to be ineffective for MAO-B inhibition. ATX-II was not effective for the inhibition of either MAO-A or MAO-B. The inhibition of MAO-A using AME was apparently instantaneous. MAO-A activity was almost completely recovered after the dilution of the inhibited enzyme with an excess amount of AME, suggesting AME is a reversible inhibitor. AME showed mixed inhibition for MAO-A in Lineweaver-Burk plots with a Ki value of 0.34 µM. The findings of this study suggest that microbial metabolites and dibenzopyrone could be potent MAO inhibitors. In addition, AME could be a useful lead compound for developing reversible MAO-A inhibitors to treat depression, Parkinson's disease, and Alzheimer's disease.

  9. Rational Design and Characterization of the Novel, Broad and Potent Bispecific HIV-1 Neutralizing Antibody iMabm36

    PubMed Central

    Sun, Ming; Pace, Craig S.; Yao, Xin; Yu, Faye; Padte, Neal N.; Huang, Yaoxing; Seaman, Michael S.; Li, Qihan; Ho, David D.

    2014-01-01

    While broadly neutralizing monoclonal antibodies (bNAbs) have always been considered potential therapeutic options for the prophylactic and treatment of HIV infection, their lack of breadth against all HIV variants has been one of the limiting factors. To provide sufficient neutralization breadth and potency against diverse viruses, including neutralization escape variants, strategies to combine different bNAbs have been explored recently. We rationally designed and engineered a novel bispecific HIV-1 neutralizing antibody (bibNAb), iMabm36, for high potency and breadth against HIV. iMabm36 is composed of the anti-CD4 Ab ibalizumab (iMab) linked to two copies of the single-domain Ab m36 which targets a highly conserved CD4-induced epitope. iMabm36 neutralizes a majority of a large, multi-clade panel of pseudoviruses (96%, n=118) at an IC50 concentration of less than 10 μg/mL, with 83% neutralized at an IC50 concentration of less than 0.1μg/ml. In addition, iMabm36 neutralizes six replication-competent transmitted-founder viruses to 100% inhibition at a concentration of less than 0.1μg/ml in a PBMC-based neutralizing assay. Mechanistically, improved antiviral activity of iMabm36 is dependent on both CD4 binding activity of iMab component and CD4i binding activity of the m36 component. After characterizing viral resistance to iMabm36 neutralization was due to mutations residing in the bridging sheet of gp120, an optimized m36 variant was engineered that, when fused to iMab, improved antiviral activity significantly. Together inter-dependency of this dual mechanism of action enables iMabm36 to potently inhibit HIV-1 entry. These results demonstrate that mechanistic-based design of bibNAbs could generate potential preventive and therapeutic candidates for HIV/AIDS. PMID:24853313

  10. Gossypol enantiomers potently inhibit human placental 3β-hydroxysteroid dehydrogenase 1 and aromatase activities.

    PubMed

    Dong, Yaoyao; Mao, Baiping; Li, Linxi; Guan, Hongguo; Su, Ying; Li, Xiaoheng; Lian, Qingquan; Huang, Ping; Ge, Ren-Shan

    2016-03-01

    Gossypol is a chemical isolated from cotton seeds. It exists as (+) or (-) enantiomer and has been tested for anticancer, abortion-inducing, and male contraception. Progesterone formed from pregnenolone by 3β-hydroxysteroid dehydrogenase 1 (HSD3B1) and estradiol from androgen by aromatase (CYP19A1) are critical for the maintenance of pregnancy or associated with some cancers. In this study we compared the potencies of (+)- and (-)-gossypol enantiomers in the inhibition of HSD3B1 and aromatase activities as well as progesterone and estradiol production in human placental JEG-3 cells. (+) Gossypol showed potent inhibition on human placental HSD3B1 with IC50 value of 2.3 μM, while (-) gossypol weakly inhibited it with IC50 over 100 μM. In contrast, (-) gossypol moderately inhibited CYP19A1 activity with IC50 of 23 μM, while (+) gossypol had no inhibition when the highest concentration (100 μM) was tested. (+) Gossypol enantiomer competitively inhibited HSD3B1 against substrate pregnenolone and showed mixed mode against NAD(+). (-) Gossypol competitively inhibited CYP19A1 against substrate testosterone. Gossypol enantiomers showed different potency related to their inhibition on human HSD3B1 and CYP19A1. Whether gossypol enantiomer is used alone or in combination relies on its application and beneficial effects.

  11. Potent inhibition of cytochrome P450 2B6 by sibutramine in human liver microsomes.

    PubMed

    Bae, Soo Hyeon; Kwon, Min Jo; Choi, Eu Jin; Zheng, Yu Fen; Yoon, Kee Dong; Liu, Kwang-Hyeon; Bae, Soo Kyung

    2013-09-05

    The present study was performed to evaluate the potency and specificity of sibutramine as an inhibitor of the activities of nine human CYP isoforms in liver microsomes. Using a cocktail assay, the effects of sibutramine on specific marker reactions of the nine CYP isoforms were measured in human liver microsomes. Sibutramine showed potent inhibition of CYP2B6-mediated bupropion 6-hydroxylation with an IC50 value of 1.61μM and Ki value of 0.466μM in a competitive manner at microsomal protein concentrations of 0.25mg/ml; this was 3.49-fold more potent than the typical CYP2B6 inhibitor thio-TEPA (Ki=1.59μM). In addition, sibutramine slightly inhibited CYP2C19 activity (Ki=16.6μM, noncompetitive inhibition) and CYP2D6 activity (Ki=15.7μM, noncompetitive inhibition). These observations indicated 35.6- and 33.7-fold decreases in inhibition potency, respectively, compared with that of CYP2B6 by sibutramine. However, no inhibition of CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6, or CYP2E1 activities was observed. In addition, the CYP2B6 inhibitory potential of sibutramine was enhanced at a lower microsomal protein concentration of 0.05mg/ml. After 30min preincubation of human liver microsomes with sibutramine in the presence of NADPH, no shift in IC50 was observed in terms of inhibition of the activities of the nine CYPs, suggesting that sibutramine is not a time-dependent inactivator. These observations suggest that sibutramine is a selective and potent inhibitor of CYP2B6 in vitro, whereas inhibition of other CYPs is substantially lower. These in vitro data support the use of sibutramine as a well-known inhibitor of CYP2B6 for routine screening of P450 reversible inhibition when human liver microsomes are used as the enzyme source.

  12. NCI Researchers Discover Exceptionally Potent Antibodies with Potential for Prophylaxis and Therapy of MERS-Coronavirus Infections | Poster

    Cancer.gov

    By Andrea Frydl, Contributing Writer In a recent article published in the Journal of Virology, Tianlei Ying, Ph.D., Dimiter Dimitrov, Ph.D., and their colleagues in the Laboratory of Experimental Immunology (LEI), Cancer and Inflammation Program, NCI Center for Cancer Research, reported the identification of three human monoclonal antibodies (m336, m337, and m338) that target the part of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) that is responsible for binding to its receptor. These antibodies are exceptionally potent inhibitors of MERS-CoV infection and also provide a basis for creating a future MERS-CoV vaccine.

  13. The Interglomerular Circuit Potently Inhibits Olfactory Bulb Output Neurons by Both Direct and Indirect Pathways

    PubMed Central

    Puche, Adam C.; Shipley, Michael T.

    2016-01-01

    Sensory processing shapes our perception. In mammals, odor information is encoded by combinatorial activity patterns of olfactory bulb (OB) glomeruli. Glomeruli are richly interconnected by short axon cells (SACs), which form the interglomerular circuit (IGC). It is unclear how the IGC impacts OB output to downstream neural circuits. We combined in vitro and in vivo electrophysiology with optogenetics in mice and found the following: (1) the IGC potently and monosynaptically inhibits the OB output neurons mitral/tufted cells (MTCs) by GABA release from SACs: (2) gap junction-mediated electrical coupling is strong for the SAC→MTC synapse, but negligible for the SAC→ETC synapse; (3) brief IGC-mediated inhibition is temporally prolonged by the intrinsic properties of MTCs; and (4) sniff frequency IGC activation in vivo generates persistent MTC inhibition. These findings suggest that the temporal sequence of glomerular activation by sensory input determines which stimulus features are transmitted to downstream olfactory networks and those filtered by lateral inhibition. SIGNIFICANCE STATEMENT Odor identity is encoded by combinatorial patterns of activated glomeruli, the initial signal transformation site of the olfactory system. Lateral circuit processing among activated glomeruli modulates olfactory signal transformation before transmission to higher brain centers. Using a combination of in vitro and in vivo optogenetics, this work demonstrates that interglomerular circuitry produces potent inhibition of olfactory bulb output neurons via direct chemical and electrical synapses as well as by indirect pathways. The direct inhibitory synaptic input engages mitral cell intrinsic membrane properties to generate inhibition that outlasts the initial synaptic action. PMID:27629712

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

    PubMed

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

    2013-08-01

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

  15. JMV641: a potent bombesin receptor antagonist that inhibits Swiss 3T3 cell proliferation.

    PubMed

    Azay, J; Gagne, D; Devin, C; Llinares, M; Fehrentz, J A; Martinez, J

    1996-08-27

    The peptides of the bombesin family are involved in stimulation of mitogenesis in various cell lines, including cancerous cell lines. Bombesin receptor antagonists are of great interest to inhibit this proliferation. We have synthesized a potent bombesin receptor antagonist, e.g., compound JMV641 [H-DPhe-Gln-Trp-Ala-Val-Gly-His-NH-*CH[CH2-CH(CH3)2]-**CHOH- (CH2)3-CH3 [*(S); **92% of (S) isomer], in which a pseudopeptide bond mimicking the transition state analogue replaced the peptide bond between the two C-terminal residues. This compound was highly potent to dose-dependently inhibit binding of 125I-GRP to Swiss 3T3 cells (IC50 = 0.85 +/- 0.15 nM) and bombesin-stimulated Swiss 3T3 proliferation (pA2 = 8.78). However, compound JMV641 can inhibit bombesin-induced AP-1 regulated genes that are nuclear messengers mediating the actions of signal transduction pathways stimulated by growth factors.

  16. Potent inhibition by star fruit of human cytochrome P450 3A (CYP3A) activity.

    PubMed

    Hidaka, Muneaki; Fujita, Ken-ichi; Ogikubo, Tetsuya; Yamasaki, Keishi; Iwakiri, Tomomi; Okumura, Manabu; Kodama, Hirofumi; Arimori, Kazuhiko

    2004-06-01

    There has been very limited information on the capacities of tropical fruits to inhibit human cytochrome P450 3A (CYP3A) activity. Thus, the inhibitory effects of tropical fruits on midazolam 1'-hydroxylase activity of CYP3A in human liver microsomes were evaluated. Eight tropical fruits such as common papaw, dragon fruit, kiwi fruit, mango, passion fruit, pomegranate, rambutan, and star fruit were tested. We also examined the inhibition of CYP3A activity by grapefruit (white) and Valencia orange as controls. The juice of star fruit showed the most potent inhibition of CYP3A. The addition of a star fruit juice (5.0%, v/v) resulted in the almost complete inhibition of midazolam 1'-hydroxylase activity (residual activity of 0.1%). In the case of grape-fruit, the residual activity was 14.7%. The inhibition depended on the amount of fruit juice added to the incubation mixture (0.2-6.0%, v/v). The elongation of the preincubation period of a juice from star fruit (1.25 or 2.5%, v/v) with the microsomal fraction did not alter the CYP3A inhibition, suggesting that the star fruit did not contain a mechanism-based inhibitor. Thus, we discovered filtered extracts of star fruit juice to be inhibitors of human CYP3A activity in vitro.

  17. A single-domain antibody-linked Fab bispecific antibody Her2-S-Fab has potent cytotoxicity against Her2-expressing tumor cells.

    PubMed

    Li, Aifen; Xing, Jieyu; Li, Li; Zhou, Changhua; Dong, Bin; He, Ping; Li, Qing; Wang, Zhong

    2016-12-01

    Her2, which is frequently overexpressed in breast cancer, is one of the most studied tumor-associated antigens for cancer therapy. Anti-HER2 monoclonal antibody, trastuzumab, has achieved significant clinical benefits in metastatic breast cancer. In this study, we describe a novel bispecific antibody Her2-S-Fab targeting Her2 by linking a single domain anti-CD16 VHH to the trastuzumab Fab. The Her2-S-Fab antibody can be efficiently expressed and purified from Escherichia coli, and drive potent cancer cell killing in HER2-overexpressing cancer cells. In xenograft model, the Her2-S-Fab suppresses tumor growth in the presence of human immune cells. Our results suggest that the bispecific Her2-S-Fab may provide a valid alternative to Her2 positive cancer therapy.

  18. Generation of Potent Anti-Vascular Endothelial Growth Factor Neutralizing Antibodies from Mouse Phage Display Library for Cancer Therapy.

    PubMed

    Lai, Yan-Da; Wu, Yen-Yu; Tsai, Yi-Jiue; Tsai, Yi-San; Lin, Yu-Ying; Lai, Szu-Liang; Huang, Chao-Yang; Lok, Ying-Yung; Hu, Chih-Yung; Lai, Jiann-Shiun

    2016-02-05

    Vascular endothelial growth factor (VEGF) is an important stimulator for angiogenesis in solid tumors. Blocking VEGF activity is an effective therapeutic strategy to inhibit tumor growth and metastasis. Avastin, a humanized monoclonal antibody recognizes VEGF, has been approved by the US Food and Drug Administration. To generate potential VEGF-recognizing antibodies with better tumor regression ability than that of Avastin, we have designed a systematic antibody selection plan. From mice immunized with recombinant human VEGF, we generated three phage display libraries, scFv-M13KO7, Fab-M13KO7, and scFv-Hyperphage, in single-chain Fv (scFv) or Fab format, displayed using either M13KO7 helper phage or Hyperphage. Solid-phase and solution-phase selection strategies were then applied to each library, generating six panning combinations. A total of sixty-four antibodies recognizing VEGF were obtained. Based on the results of epitope mapping, binding affinity, and biological functions in tumor inhibition, eight antibodies were chosen to examine their abilities in tumor regression in a mouse xenograft model using human COLO 205 cancer cells. Three of them showed improvement in the inhibition of tumor growth (328%-347% tumor growth ratio (% of Day 0 tumor volume) on Day 21 vs. 435% with Avastin). This finding suggests a potential use of these three antibodies for VEGF-targeted therapy.

  19. Sansanmycin natural product analogues as potent and selective anti-mycobacterials that inhibit lipid I biosynthesis.

    PubMed

    Tran, Anh T; Watson, Emma E; Pujari, Venugopal; Conroy, Trent; Dowman, Luke J; Giltrap, Andrew M; Pang, Angel; Wong, Weng Ruh; Linington, Roger G; Mahapatra, Sebabrata; Saunders, Jessica; Charman, Susan A; West, Nicholas P; Bugg, Timothy D H; Tod, Julie; Dowson, Christopher G; Roper, David I; Crick, Dean C; Britton, Warwick J; Payne, Richard J

    2017-03-01

    Tuberculosis (TB) is responsible for enormous global morbidity and mortality, and current treatment regimens rely on the use of drugs that have been in use for more than 40 years. Owing to widespread resistance to these therapies, new drugs are desperately needed to control the TB disease burden. Herein, we describe the rapid synthesis of analogues of the sansanmycin uridylpeptide natural products that represent promising new TB drug leads. The compounds exhibit potent and selective inhibition of Mycobacterium tuberculosis, the etiological agent of TB, both in vitro and intracellularly. The natural product analogues are nanomolar inhibitors of Mtb phospho-MurNAc-pentapeptide translocase, the enzyme responsible for the synthesis of lipid I in mycobacteria. This work lays the foundation for the development of uridylpeptide natural product analogues as new TB drug candidates that operate through the inhibition of peptidoglycan biosynthesis.

  20. Sansanmycin natural product analogues as potent and selective anti-mycobacterials that inhibit lipid I biosynthesis

    NASA Astrophysics Data System (ADS)

    Tran, Anh T.; Watson, Emma E.; Pujari, Venugopal; Conroy, Trent; Dowman, Luke J.; Giltrap, Andrew M.; Pang, Angel; Wong, Weng Ruh; Linington, Roger G.; Mahapatra, Sebabrata; Saunders, Jessica; Charman, Susan A.; West, Nicholas P.; Bugg, Timothy D. H.; Tod, Julie; Dowson, Christopher G.; Roper, David I.; Crick, Dean C.; Britton, Warwick J.; Payne, Richard J.

    2017-03-01

    Tuberculosis (TB) is responsible for enormous global morbidity and mortality, and current treatment regimens rely on the use of drugs that have been in use for more than 40 years. Owing to widespread resistance to these therapies, new drugs are desperately needed to control the TB disease burden. Herein, we describe the rapid synthesis of analogues of the sansanmycin uridylpeptide natural products that represent promising new TB drug leads. The compounds exhibit potent and selective inhibition of Mycobacterium tuberculosis, the etiological agent of TB, both in vitro and intracellularly. The natural product analogues are nanomolar inhibitors of Mtb phospho-MurNAc-pentapeptide translocase, the enzyme responsible for the synthesis of lipid I in mycobacteria. This work lays the foundation for the development of uridylpeptide natural product analogues as new TB drug candidates that operate through the inhibition of peptidoglycan biosynthesis.

  1. Sansanmycin natural product analogues as potent and selective anti-mycobacterials that inhibit lipid I biosynthesis

    PubMed Central

    Tran, Anh T.; Watson, Emma E.; Pujari, Venugopal; Conroy, Trent; Dowman, Luke J.; Giltrap, Andrew M.; Pang, Angel; Wong, Weng Ruh; Linington, Roger G.; Mahapatra, Sebabrata; Saunders, Jessica; Charman, Susan A.; West, Nicholas P.; Bugg, Timothy D. H.; Tod, Julie; Dowson, Christopher G.; Roper, David I.; Crick, Dean C.; Britton, Warwick J.; Payne, Richard J.

    2017-01-01

    Tuberculosis (TB) is responsible for enormous global morbidity and mortality, and current treatment regimens rely on the use of drugs that have been in use for more than 40 years. Owing to widespread resistance to these therapies, new drugs are desperately needed to control the TB disease burden. Herein, we describe the rapid synthesis of analogues of the sansanmycin uridylpeptide natural products that represent promising new TB drug leads. The compounds exhibit potent and selective inhibition of Mycobacterium tuberculosis, the etiological agent of TB, both in vitro and intracellularly. The natural product analogues are nanomolar inhibitors of Mtb phospho-MurNAc-pentapeptide translocase, the enzyme responsible for the synthesis of lipid I in mycobacteria. This work lays the foundation for the development of uridylpeptide natural product analogues as new TB drug candidates that operate through the inhibition of peptidoglycan biosynthesis. PMID:28248311

  2. Human Antibodies Fix Complement to Inhibit Plasmodium falciparum Invasion of Erythrocytes and Are Associated with Protection against Malaria

    PubMed Central

    Boyle, Michelle J.; Reiling, Linda; Feng, Gaoqian; Langer, Christine; Osier, Faith H.; Aspeling-Jones, Harvey; Cheng, Yik Sheng; Stubbs, Janine; Tetteh, Kevin K.A.; Conway, David J.; McCarthy, James S.; Muller, Ivo; Marsh, Kevin; Anders, Robin F.; Beeson, James G.

    2015-01-01

    Summary Antibodies play major roles in immunity to malaria; however, a limited understanding of mechanisms mediating protection is a major barrier to vaccine development. We have demonstrated that acquired human anti-malarial antibodies promote complement deposition on the merozoite to mediate inhibition of erythrocyte invasion through C1q fixation and activation of the classical complement pathway. Antibody-mediated complement-dependent (Ab-C′) inhibition was the predominant invasion-inhibitory activity of human antibodies; most antibodies were non-inhibitory without complement. Inhibitory activity was mediated predominately via C1q fixation, and merozoite surface proteins 1 and 2 were identified as major targets. Complement fixation by antibodies was very strongly associated with protection from both clinical malaria and high-density parasitemia in a prospective longitudinal study of children. Ab-C′ inhibitory activity could be induced by human immunization with a candidate merozoite surface-protein vaccine. Our findings demonstrate that human anti-malarial antibodies have evolved to function by fixing complement for potent invasion-inhibitory activity and protective immunity. PMID:25786180

  3. Potent and long-lasting inhibition of human P2X2 receptors by copper

    PubMed Central

    Punthambaker, Sukanya; Hume, Richard I.

    2013-01-01

    P2X receptors are ion channels gated by ATP. In rodents these channels are modulated by zinc and copper. Zinc is co-released with neurotransmitter at some synapses and can modulate neuronal activity, but the role of copper in the brain is unclear. Rat P2X2 receptors show potentiation by 2–100 µM zinc or copper in the presence of a submaximal concentration of ATP but are inhibited by zinc or copper at concentrations above 100 µM. In contrast, human P2X2 (hP2X2) receptors show no potentiation and are strongly inhibited by zinc over the range of 2–100 µM. The effect of copper on hP2X2 is of interest because there are human brain disorders in which copper concentration is altered. We found that hP2X2 receptors are potently inhibited by copper (IC50 = 40 nM). ATP responsiveness recovered extremely slowly after copper washout, with full recovery requiring over 1 h. ATP binding facilitated copper binding but not unbinding from this inhibitory site. A mutant receptor in which the first six extracellular cysteines were deleted, C(1–6)S, showed normal copper inhibition, however reducing agents dramatically accelerated recovery from copper inhibition in wild type hP2X2 and the C(1–6)S mutant, indicating that the final two disulfide bonds are required to maintain the high affinity copper binding site. Three histidine residues required for normal zinc inhibition were also required for normal copper inhibition. Humans with untreated Wilson’s disease have excess amounts of copper in the brain. The high copper sensitivity of hP2X2 receptors suggests that they are non-functional in these patients. PMID:24067922

  4. Selective inhibition of the cytochrome P450 isoform by hyperoside and its potent inhibition of CYP2D6.

    PubMed

    Song, Min; Hong, Miri; Lee, Min Young; Jee, Jun-Goo; Lee, You Mie; Bae, Jong-Sup; Jeong, Tae Cheon; Lee, Sangkyu

    2013-09-01

    Hyperoside, quercetin-3-O-galactoside, is a flavonoid isolated from Oenanthe javanica. In the present study, we investigated potential herb-drug inhibitory effects of hyperoside on nine cytochrome P450 (CYP) isoforms in pooled human liver microsomes (HLMs) and human recombinant cDNA expressed CYP using a cocktail probe assay. Hyperoside strongly inhibited CYP2D6-catalyzed dextromethorphan O-demethylation, with IC₅₀ values of 1.2 and 0.81 μM after 0 and 15 min of preincubation, and a Ki value of 2.01 μM in HLMs, respectively. Hyperoside strongly decreased CYP2D6 activity dose-, but not time-, dependently in HLMs. In addition, the Lineweaver-Burk and Secondary plots for the inhibition of CYP2D6 in HLMs fitted a competitive inhibition mode. Furthermore, hyperoside decreased CYP2D6-catalyzed dextromethorphan O-demethylation activity of human recombinant cDNA-expressed CYP2D6, with an IC₅₀ value of 3.87 μM. However, other CYPs were not inhibited significantly by hyperoside. In conclusion, our data demonstrate that hyperoside is a potent selective CYP2D6 inhibitor in HLMs, and suggest that hyperoside might cause herb-drug interactions when co-administrated with CYP2D substrates.

  5. Broad and potent HIV-1 neutralization by a human antibody that binds the gp41-gp120 interface

    SciTech Connect

    Huang, Jinghe; Kang, Byong H.; Pancera, Marie; Lee, Jeong Hyun; Tong, Tommy; Feng, Yu; Imamichi, Hiromi; Georgiev, Ivelin S.; Chuang, Gwo-Yu; Druz, Aliaksandr; Doria-Rose, Nicole A.; Laub, Leo; Sliepen, Kwinten; van Gils, Marit J.; de la Peña, Alba Torrents; Derking, Ronald; Klasse, Per-Johan; Migueles, Stephen A.; Bailer, Robert T.; Alam, Munir; Pugach, Pavel; Haynes, Barton F.; Wyatt, Richard T.; Sanders, Rogier W.; Binley, James M.; Ward, Andrew B.; Mascola, John R.; Kwong, Peter D.; Connors, Mark

    2015-10-15

    The isolation of human monoclonal antibodies is providing important insights into the specificities that underlie broad neutralization of HIV-1 (reviewed in ref. 1). Here we report a broad and extremely potent HIV-specific monoclonal antibody, termed 35O22, which binds a novel HIV-1 envelope glycoprotein (Env) epitope. 35O22 neutralized 62% of 181 pseudoviruses with a half-maximum inhibitory concentration (IC50) <50 μg ml-1. The median IC50 of neutralized viruses was 0.033 μg ml-1, among the most potent thus far described. 35O22 did not bind monomeric forms of Env tested, but did bind the trimeric BG505 SOSIP.664. Mutagenesis and a reconstruction by negative-stain electron microscopy of the Fab in complex with trimer revealed that it bound to a conserved epitope, which stretched across gp120 and gp41. The specificity of 35O22 represents a novel site of vulnerability on HIV Env, which serum analysis indicates to be commonly elicited by natural infection. Binding to this new site of vulnerability may thus be an important complement to current monoclonal-antibody-based approaches to immunotherapies, prophylaxis and vaccine design.

  6. A Humanized Anti-CD22-Onconase Antibody-Drug Conjugate Mediates Highly Potent Destruction of Targeted Tumor Cells

    PubMed Central

    Weber, Tobias; Mavratzas, Athanasios; Kiesgen, Stefan; Haase, Stephanie; Bötticher, Benedikt; Exner, Evelyn; Mier, Walter; Grosse-Hovest, Ludger; Jäger, Dirk; Arndt, Michaela A. E.; Krauss, Jürgen

    2015-01-01

    Antibody-drug conjugates (ADCs) have evolved as a new class of potent cancer therapeutics. We here report on the development of ADCs with specificity for the B-cell lineage specific (surface) antigen CD22 being expressed in the majority of hematological malignancies. As targeting moiety a previously generated humanized anti-CD22 single-chain variable fragment (scFv) derivative from the monoclonal antibody RFB4 was reengineered into a humanized IgG1 antibody format (huRFB4). Onconase (ranpirnase), a clinically active pancreatic-type ribonuclease, was employed as cytotoxic payload moiety. Chemical conjugation via thiol-cleavable disulfide linkage retained full enzymatic activity and full binding affinity of the ADC. Development of sophisticated purification procedures using size exclusion and ion exchange chromatography allowed the separation of immunoconjugate species with stoichiometrically defined number of Onconase cargos. A minimum of two Onconase molecules per IgG was required for achieving significant in vitro cytotoxicity towards lymphoma and leukemia cell lines. Antibody-drug conjugates with an Onconase to antibody ratio of 3 : 1 exhibited an IC50 of 0.08 nM, corresponding to more than 18,400-fold increased cytotoxicity of the ADC when compared with unconjugated Onconase. These results justify further development of this ADC as a promising first-in-class compound for the treatment of CD22-positive malignancies. PMID:26605343

  7. Ley specific antibody with potent anti-tumor activity is internalized and degraded in lysosomes.

    PubMed Central

    Garrigues, J.; Garrigues, U.; Hellström, I.; Hellström, K. E.

    1993-01-01

    BR96 is a monoclonal antibody (MAb) that recognizes many human carcinomas and can kill antigen-positive tumor cells in vitro. Using both gold and radiolabeled MAb, the distribution and cellular processing of BR96 during cytolysis has been determined. After a brief (< 3 minutes) MAb treatment, cells in suspension are stained by the nuclear viability dye propidium iodide. Whole MAb and F(ab')2 fragments are equally cytotoxic; monovalent F(ab) fragments, however, have no effect on dye uptake unless cross-linked with goat anti-mouse IgG. The level of toxicity is dependent on both MAb dose and on cell surface receptor density. Cell contact may regulate receptor expression. BR96 receptors are more abundant on cells migrating into the open areas of a scratch wounded confluent culture than on the adjacent contact-inhibited cells. BR96 can also inhibit the anchorage-independent growth of tumor cells in soft agar showing that its effects on propidium iodide staining are not due to transient changes in membrane permeability. Immunogold electron microscopy reveals that, after a 1-minute treatment, BR96 induces significant infolding of the plasma membrane and that internalized MAb is localized to these structures. Immediately thereafter, large cell surface and intracellular vesicles form, mitochondria are swollen, and membrane integrity is lost. Therefore, BR96 seems to cause morphological changes characteristic of necrosis rather than apoptosis. When bound to adherent carcinoma cells, BR96 is distributed uniformly on the apical surface of cells labeled at 4 C and is enriched at points of cell substratum contact. Upon warming of the cells to 37 C, BR96 localizes in small perinuclear clusters and the cell margin is now devoid of label. Immunogold electron microscopy reveals that BR96 undergoes receptor mediated internalization and is localized within the same coated pits, endosomes, and lysosomes as the transferrin receptor. Quantitative studies using iodinated BR96 show that

  8. Resveratrol analogue 4,4′-dihydroxy-trans-stilbene potently inhibits cancer invasion and metastasis

    PubMed Central

    Savio, Monica; Ferraro, Daniela; Maccario, Cristina; Vaccarone, Rita; Jensen, Lasse D.; Corana, Federica; Mannucci, Barbara; Bianchi, Livia; Cao, Yihai; Stivala, Lucia Anna

    2016-01-01

    We investigated the preventive effects of resveratrol analogue 4,4′-dihydroxy-trans-stilbene (DHS) on cancer invasion and metastasis. Two different in vivo approaches of mouse and zebrafish lung cancer invasion models were employed in our study. The in vitro results showed that DHS displays potent inhibition on anchorage-dependent or -independent cell growth of LLC cells, leading to impairment of the cell cycle progression with reduction of cell numbers arresting at the G1 phase, an evident accumulation of pre-G1 events correlated with apoptotic behaviour. In addition, DHS induces a marked inhibition of LLC cell migration and matrigel invasion. In a murine lung cancer model, tumour volume, cell proliferation, and tumour angiogenesis were significantly inhibited by DHS. Importantly, liver metastatic lesions were significantly reduced in DHS-treated mice. Similarly, DHS significantly inhibits lung cancer cell dissemination, invasion and metastasis in a zebrafish tumour model. These findings demonstrate that DHS could potentially be developed as a novel therapeutic agent for treatment of cancer and metastasis. PMID:26829331

  9. Potent inhibition of human neutrophil activations by bractelactone, a novel chalcone from Fissistigma bracteolatum

    SciTech Connect

    Wu, Yang-Chang; Sureshbabu, Munisamy; Fang, Yao-Ching; Wu, Yi-Hsiu; Lan, Yu-Hsuan; Chang, Fang-Rong; Chang, Ya-Wen; Hwang, Tsong-Long

    2013-02-01

    Fissistigma bracteolatum is widely used in traditional medicine to treat inflammatory diseases. However, its active components and mechanisms of action remain unclear. In this study, (3Z)-6,7-dihydroxy-4-methoxy-3-(phenylmethylidene)-5-(3-phenylpropanoyl) -1-benzofuran-2(3H) (bractelactone), a novel chalcone from F. bracteolatum, showed potent inhibitory effects against superoxide anion (O{sub 2}{sup ·−}) production, elastase release, and CD11b expression in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-induced human neutrophils. However, bractelactone showed only weak inhibition of phorbol myristate acetate-caused O{sub 2}{sup ·−} production. The peak cytosolic calcium concentration ([Ca{sup 2+}]{sub i}) was unaltered by bractelactone in FMLP-induced neutrophils, but the decay time of [Ca{sup 2+}]{sub i} was significantly shortened. In a calcium-free solution, changes in [Ca{sup 2+}]{sub i} caused by the addition of extracellular Ca{sup 2+} were inhibited by bractelactone in FMLP-activated cells. In addition, bractelactone did not alter the phosphorylation of p38 MAPK, ERK, JNK, or AKT or the concentration of cAMP. These results suggest that bractelactone selectively inhibits store-operated calcium entry (SOCE). In agreement with this concept, bractelactone suppressed sustained [Ca{sup 2+}]{sub i} changes in thapsigargin-activated neutrophils. Furthermore, bractelactone did not alter FMLP-induced formation of inositol 1,4,5-triphosphate. Taken together, our results demonstrate that the anti-inflammatory effects of bractelactone, an active ingredient of F. bracteolatum, in human neutrophils are through the selective inhibition of SOCE. Highlights: ► Bractelactone isolated from Fissistigma bracteolatum. ► Bractelactone inhibited FMLP-induced human neutrophil activations. ► Bractelactone had no effect on IP3 formation. ► Bractelactone did not alter MAPKs, AKT, and cAMP pathways. ► Bractelactone inhibited store-operated calcium entry.

  10. Passive transfer of modest titers of potent and broadly neutralizing anti-HIV monoclonal antibodies block SHIV infection in macaques

    PubMed Central

    Shingai, Masashi; Donau, Olivia K.; Plishka, Ronald J.; Buckler-White, Alicia; Mascola, John R.; Nabel, Gary J.; Nason, Martha C.; Montefiori, David; Moldt, Brian; Poignard, Pascal; Diskin, Ron; Bjorkman, Pamela J.; Eckhaus, Michael A.; Klein, Florian; Mouquet, Hugo; Cetrulo Lorenzi, Julio Cesar; Gazumyan, Anna; Burton, Dennis R.; Nussenzweig, Michel C.

    2014-01-01

    It is widely appreciated that effective human vaccines directed against viral pathogens elicit neutralizing antibodies (NAbs). The passive transfer of anti–HIV-1 NAbs conferring sterilizing immunity to macaques has been used to determine the plasma neutralization titers, which must be present at the time of exposure, to prevent acquisition of SIV/HIV chimeric virus (SHIV) infections. We administered five recently isolated potent and broadly acting anti-HIV neutralizing monoclonal antibodies (mAbs) to rhesus macaques and challenged them intrarectally 24 h later with either of two different R5-tropic SHIVs. By combining the results obtained from 60 challenged animals, we determined that the protective neutralization titer in plasma preventing virus infection in 50% of the exposed monkeys was relatively modest (∼1:100) and potentially achievable by vaccination. PMID:25155019

  11. Dual inhibiting OCT4 and AKT potently suppresses the propagation of human cancer cells

    PubMed Central

    Li, Wenxin; Zhou, Yanwen; Zhang, Xiaoqian; Yang, Ying; Dan, Songsong; Su, Tong; She, Shiqi; Dong, Weilai; Zhao, Qingwei; Jia, Jia; Yao, Hangping; Zheng, Min; Kang, Bo; Wang, Ying-Jie

    2017-01-01

    AKT serves as an epigenetic modulator that links epigenetic regulation to cell survival and proliferation while the epigenetic mediator OCT4 critically controls stem cell pluripotency and self-renewal. Emerging evidence indicated their complicated interplays in cancer cells and cancer stem cells (CSCs), and inhibiting either one may activate the other. Thus, in this study, we propose a strategy to targeting both factors simultaneously. Firstly, a combination of an OCT4-specific shRNA and the specific AKT inhibitor Akti-1/2 potently suppressed the propagation of human embryonal carcinoma cells, adherent cancer cells and stem-like cancer cells, establishing the proof-of-concept that dual inhibiting OCT4 and AKT can effectively target various cancer cells. Next, we combined Akti-1/2 with metformin, a widely-prescribed drug for treating type 2 diabetes, which was reported to down-regulate OCT4 expression. The metformin + Akti-1/2 combo significantly altered multiple signaling and epigenetic pathways, induced growth arrest and cell death of adherent and stem-like glioblastoma U87 cells, and attenuated their tumorigenicity in vivo. Taken together, we demonstrate here that simultaneously targeting an epigenetic mediator and an epigenetic modulator, by dual inhibiting OCT4 and AKT, can have significantly improved efficacies over single treatment in suppressing the propagation of CSCs as well as the entire bulk of differentiated cancer cells. PMID:28383051

  12. Potent inhibition of protein tyrosine phosphatases by copper complexes with multi-benzimidazole derivatives.

    PubMed

    Li, Ying; Lu, Liping; Zhu, Miaoli; Wang, Qingming; Yuan, Caixia; Xing, Shu; Fu, Xueqi; Mei, Yuhua

    2011-12-01

    A series of copper complexes with multi-benzimidazole derivatives, including mono- and di-nuclear, were synthesized and characterized by Fourier transform IR spectroscopy, UV-Vis spectroscopy, elemental analysis, electrospray ionization mass spectrometry. The speciation of Cu/NTB in aqueous solution was investigated by potentiometric pH titrations. Their inhibitory effects against human protein tyrosine phosphatase 1B (PTP1B), T-cell protein tyrosine phosphatase (TCPTP), megakaryocyte protein tyrosine phosphatase 2 (PTP-MEG2), srchomology phosphatase 1 (SHP-1) and srchomology phosphatase 2 (SHP-2) were evaluated in vitro. The five copper complexes exhibit potent inhibition against PTP1B, TCPTP and PTP-MEG2 with almost same inhibitory effects with IC(50) at submicro molar level and about tenfold weaker inhibition versus SHP-1, but almost no inhibition against SHP-2. Kinetic analysis indicates that they are reversible competitive inhibitors of PTP1B. Fluorescence study on the interaction between PTP1B and complex 2 or 4 suggests that the complexes bind to PTP1B with the formation of a 1:1 complex. The binding constant are about 1.14 × 10(6) and 1.87 × 10(6) M(-1) at 310 K for 2 and 4, respectively.

  13. Potent inhibition of human immunodeficiency virus by MDL 101028, a novel sulphonic acid polymer.

    PubMed

    Taylor, D L; Brennan, T M; Bridges, C G; Mullins, M J; Tyms, A S; Jackson, R; Cardin, A D

    1995-10-01

    MDL 101028, a novel biphenyl disulphonic acid urea co-polymer was designed and synthesised as a heparin mimetic. This low molecular weight polymer showed potent inhibition of human immunodeficiency virus type 1 (HIV-1) replication in a number of host-cell/virus systems, including primary clinical isolates of the virus cultured in human peripheral blood mononuclear cells (PBMCs). When compared with the heterogeneous polysulphated molecules, heparin and dextran sulphate, this chemically defined compound showed equivalent antiviral activity with 50% inhibitory concentrations (IC50s) in the range 0.27-3.0 micrograms/ml in the host-cell/virus systems tested. MDL 101028 also inhibited the replication of HIV type 2 and the simian immunodeficiency virus (SIV), as well as HIV-1 variants resistant to reverse transcriptase inhibitors. Virus growth was blocked when exposure of T-lymphocytes to MDL 101028 was restricted to the virus absorption stage, or even in whole blood conditions. MDL 101028 did not irreversibly inactivate virions, and in contrast to heparin, did not inhibit the attachment of radiolabelled HIV-1 to CD4+ T-cells. MDL 101028 blocked HIV-induced cell-to-cell fusion and this activity appears to explain the mechanism of its antiviral action. The antiviral evaluation of discrete oligomer molecules of MDL 101028 showed that a polymer chain length of six repeating units had optimal potency. The lack of anticoagulant properties and significant antiviral activity in whole blood may allow the development of MDL 101028 as a treatment of HIV infections.

  14. Antibodies elicited by yeast glycoproteins recognize HIV-1 virions and potently neutralize virions with high mannose N-glycans

    PubMed Central

    Zhang, Hong; Fu, Hu; Luallen, Robert J.; Liu, Bingfen; Lee, Fang-Hua; Doms, Robert W.; Geng, Yu

    2015-01-01

    The glycan shield on the human immunodeficiency virus 1 (HIV-1) envelope (Env) glycoprotein has drawn attention as a target for HIV-1 vaccine design given that an increasing number of potent and broadly neutralizing antibodies (bNAbs) recognize epitopes entirely or partially comprised of high mannose type N-linked glycans. In an attempt to generate immunogens that target the glycan shield of HIV-1, we previously engineered a triple mutant (TM) strain of Saccharomyces cerevisiae that results in exclusive presentation of high mannose type N-glycans, and identified five TM yeast glycoproteins that support strong binding of 2G12, a bNAb that targets a cluster of high mannose glycans on the gp120 subunit of Env. Here, we further analyzed the antigenicity and immunogenicity of these proteins in inducing anti-HIV responses. Our study demonstrated that the 2G12-reactive TM yeast glycoproteins efficiently bound to recently identified bNAbs including PGT125–130 and PGT135 that recognize high mannose glycan-dependent epitopes. Immunization of rabbits with a single TM yeast glycoprotein (Gp38 or Pst1), when conjugated to a promiscuous T-cell epitope peptide and coadministered with a Toll-like receptor 2 agonist, induced glycan-specific HIV-1 Env cross-reactive antibodies. The immune sera bound to both synthetic mannose oligosaccharides and gp120 proteins from a broad range of HIV-1 strains. The purified antibodies recognized and captured virions that contain both complex- and high mannose-type of N-glycans, and potently neutralized virions from different HIV-1 clades but only when the virions were enforced to retain high mannose N-glycans. This study provides insights into the elicitation of anti-carbohydrate, HIV-1 Env-cross reactive antibodies with a heterologous glycoprotein and may have applications in the design and administration of immunogens that target the viral glycan shield for development of an effective HIV-1 vaccine. PMID:26277072

  15. Poliovirus RNA synthesis in vitro: structural elements and antibody inhibition

    SciTech Connect

    Semler, B.L.; Hanecak, R.; Dorner, L.F.; Anderson, C.W.; Wimmer, E.

    1983-01-01

    The poliovirus RNA polymerase complex has been analyzed by immunoautoradiography using antibody probes derived from purified replicase (P3) region viral polypeptides. Antibody preparations made against the polio RNA polymerase, P3-4b, detected a previously unreported cellular protein that copurifies with the RNA polymerase. An IgG fraction purified from rabbit antiserum to polypeptide P3-2, a precursor fo the RNA polymerase, specifically inhibits poliovirus RNA synthesis in vitro. The authors have also immunoprecipitated a 60,000-dalton protein (P3-4a) with antiserum to protein P3-4b and have determined the precise genomic map position of this protein by automated Edman degradation. Protein P3-4a originates by cleavage of the RNA polymerase precursor at a glutamine-glucine amino acid pair not previously reported to be a viral cleavage site.

  16. Alkamides from the fruits of Piper longum and Piper nigrum displaying potent cell adhesion inhibition.

    PubMed

    Lee, Seung Woong; Kim, Young Kook; Kim, Koanhoi; Lee, Hyun Sun; Choi, Jung Ho; Lee, Woo Song; Jun, Chang-Duk; Park, Jee Hun; Lee, Jeong Min; Rho, Mun-Chual

    2008-08-15

    Eight alkamides 1-8 were isolated by bioassay-guided isolation of EtOH extracts of the fruits of Piper longum and Piper nigum (Piperaceae). Their structures were elucidated by spectroscopic analysis ((1)H, (13)C NMR, and ESI-MS) as follows: guineensine (1), retrofracamide C (2), (2E,4Z,8E)-N-[9-(3,4-methylenedioxyphenyl)-2,4,8-nonatrienoyl]piperidine (3), pipernonaline (4), piperrolein B (5), piperchabamide D (6), pellitorin (7), and dehydropipernonaline (8). Their compounds 3-5, 7, and 8 inhibited potently the direct binding between sICAM-1 and LFA-1 of THP-1 cells in a dose-dependent manner, with IC(50) values of 10.7, 8.8, 13.4, 13.5, and 6.0 microg/mL, respectively.

  17. Inhibition of alpha-glucosidase by aqueous extracts of some potent antidiabetic medicinal herbs.

    PubMed

    Onal, Seçil; Timur, Suna; Okutucu, Burcu; Zihnioğlu, Figen

    2005-01-01

    Diabetes mellitus is one of the most prevalant diseases of adults. Agents with alpha-glucosidase inhibitory activity have been useful as oral hypoglycemic drugs for the control of hyperglycemia in patients with type 2; noninsulin-dependent, diabetes mellitus (NIDDM). Investigation of some medicinal herbs: Urtica dioica, Taraxacum officinale, Viscum album, and Myrtus communis with alpha-glucosidase inhibitor activity was conducted to identify a prophylactic effect for diabetes in vitro. All plants showed differing potent alpha-glucosidase inhibitory activity. However, Myrtus communis strongly inhibited the enzyme (IC50 = 38 microg/mL). The inhibitory effect of these plants and some common antidiabetic drugs against the enzyme source (baker's yeast, rabbit liver, and small intestine) were also searched. Approximately all inhibitors used in this study showed quite different inhibitory activities, according to alpha-glucosidase origins. Furthermore, subsequent separation of the active material from Myrtus communis by HPLC showed that only one fraction acted as an a-glucosidase inhibitor.

  18. Glycosynthase Mutants of Endoglycosidase S2 Show Potent Transglycosylation Activity and Remarkably Relaxed Substrate Specificity for Antibody Glycosylation Remodeling.

    PubMed

    Li, Tiezheng; Tong, Xin; Yang, Qiang; Giddens, John P; Wang, Lai-Xi

    2016-08-05

    Glycosylation can exert a profound impact on the structures and biological functions of antibodies. Glycosylation remodeling using the endoglycosidase-catalyzed deglycosylation and transglycosylation approach is emerging as a promising platform to produce homogeneous glycoforms of antibodies, but the broad application of this method will require the availability of highly efficient glycosynthase mutants. We describe in this paper a systematic site-directed mutagenesis of an endoglycosidase from Streptococcus pyogenes of serotype M49 (Endo-S2) and the evaluation of the resulting mutants for their hydrolysis and transglycosylation activities. We found that mutations at the Asp-184 residue gave mutants that demonstrated significantly different properties, some possessed potent transglycosylation activity with diminished hydrolysis activity but others did not, which would be otherwise difficult to predict without the comparative study. In contrast to the previously reported Endo-S mutants that are limited to action on complex type N-glycans, the Endo-S2 glycosynthases described here, including D184M and D184Q, were found to have remarkably relaxed substrate specificity and were capable of transferring three major types (complex, high-mannose, and hybrid type) of N-glycans for antibody glycosylation remodeling. In addition, the Endo-S2 glycosynthase mutants were found to be much more active in general than the Endo-S mutants for transglycosylation. The usefulness of these Endo-S2 glycosynthase mutants was exemplified by an efficient glycosylation remodeling of two therapeutic monoclonal antibodies, rituximab and trastuzumab (Herceptin).

  19. Building a better dynasore: the dyngo compounds potently inhibit dynamin and endocytosis.

    PubMed

    McCluskey, Adam; Daniel, James A; Hadzic, Gordana; Chau, Ngoc; Clayton, Emma L; Mariana, Anna; Whiting, Ainslie; Gorgani, Nick N; Lloyd, Jonathan; Quan, Annie; Moshkanbaryans, Lia; Krishnan, Sai; Perera, Swetha; Chircop, Megan; von Kleist, Lisa; McGeachie, Andrew B; Howes, Mark T; Parton, Robert G; Campbell, Michael; Sakoff, Jennette A; Wang, Xuefeng; Sun, Jian-Yuan; Robertson, Mark J; Deane, Fiona M; Nguyen, Tam H; Meunier, Frederic A; Cousin, Michael A; Robinson, Phillip J

    2013-12-01

    Dynamin GTPase activity increases when it oligomerizes either into helices in the presence of lipid templates or into rings in the presence of SH3 domain proteins. Dynasore is a dynamin inhibitor of moderate potency (IC₅₀ ~ 15 μM in vitro). We show that dynasore binds stoichiometrically to detergents used for in vitro drug screening, drastically reducing its potency (IC₅₀ = 479 μM) and research tool utility. We synthesized a focused set of dihydroxyl and trihydroxyl dynasore analogs called the Dyngo™ compounds, five of which had improved potency, reduced detergent binding and reduced cytotoxicity, conferred by changes in the position and/or number of hydroxyl substituents. The Dyngo compound 4a was the most potent compound, exhibiting a 37-fold improvement in potency over dynasore for liposome-stimulated helical dynamin activity. In contrast, while dynasore about equally inhibited dynamin assembled in its helical or ring states, 4a and 6a exhibited >36-fold reduced activity against rings, suggesting that they can discriminate between helical or ring oligomerization states. 4a and 6a inhibited dynamin-dependent endocytosis of transferrin in multiple cell types (IC₅₀ of 5.7 and 5.8 μM, respectively), at least sixfold more potently than dynasore, but had no effect on dynamin-independent endocytosis of cholera toxin. 4a also reduced synaptic vesicle endocytosis and activity-dependent bulk endocytosis in cultured neurons and synaptosomes. Overall, 4a and 6a are improved and versatile helical dynamin and endocytosis inhibitors in terms of potency, non-specific binding and cytotoxicity. The data further suggest that the ring oligomerization state of dynamin is not required for clathrin-mediated endocytosis.

  20. Structural Basis for Broad and Potent Neutralization of HIV-1 by Antibody VRC01

    SciTech Connect

    Zhou, Tongqing; Georgiev, Ivelin; Wu, Xueling; Yang, Zhi-Yong; Dai, Kaifan; Finzi, Andrés; Kwon, Young Do; Scheid, Johannes F.; Shi, Wei; Xu, Ling; Yang, Yongping; Zhu, Jiang; Nussenzweig, Michel C.; Sodroski, Joseph; Shapiro, Lawrence; Nabel, Gary J.; Mascola, John R.; Kwong, Peter D.

    2010-08-26

    During HIV-1 infection, antibodies are generated against the region of the viral gp120 envelope glycoprotein that binds CD4, the primary receptor for HIV-1. Among these antibodies, VRC01 achieves broad neutralization of diverse viral strains. We determined the crystal structure of VRC01 in complex with a human immunodeficiency virus HIV-1 gp120 core. VRC01 partially mimics CD4 interaction with gp120. A shift from the CD4-defined orientation, however, focuses VRC01 onto the vulnerable site of initial CD4 attachment, allowing it to overcome the glycan and conformational masking that diminishes the neutralization potency of most CD4-binding-site antibodies. To achieve this recognition, VRC01 contacts gp120 mainly through immunoglobulin V-gene regions substantially altered from their genomic precursors. Partial receptor mimicry and extensive affinity maturation thus facilitate neutralization of HIV-1 by natural human antibodies.

  1. Developmental pathway for potent V1V2-directed HIV-neutralizing antibodies.

    PubMed

    Doria-Rose, Nicole A; Schramm, Chaim A; Gorman, Jason; Moore, Penny L; Bhiman, Jinal N; DeKosky, Brandon J; Ernandes, Michael J; Georgiev, Ivelin S; Kim, Helen J; Pancera, Marie; Staupe, Ryan P; Altae-Tran, Han R; Bailer, Robert T; Crooks, Ema T; Cupo, Albert; Druz, Aliaksandr; Garrett, Nigel J; Hoi, Kam H; Kong, Rui; Louder, Mark K; Longo, Nancy S; McKee, Krisha; Nonyane, Molati; O'Dell, Sijy; Roark, Ryan S; Rudicell, Rebecca S; Schmidt, Stephen D; Sheward, Daniel J; Soto, Cinque; Wibmer, Constantinos Kurt; Yang, Yongping; Zhang, Zhenhai; Mullikin, James C; Binley, James M; Sanders, Rogier W; Wilson, Ian A; Moore, John P; Ward, Andrew B; Georgiou, George; Williamson, Carolyn; Abdool Karim, Salim S; Morris, Lynn; Kwong, Peter D; Shapiro, Lawrence; Mascola, John R

    2014-05-01

    Antibodies capable of neutralizing HIV-1 often target variable regions 1 and 2 (V1V2) of the HIV-1 envelope, but the mechanism of their elicitation has been unclear. Here we define the developmental pathway by which such antibodies are generated and acquire the requisite molecular characteristics for neutralization. Twelve somatically related neutralizing antibodies (CAP256-VRC26.01-12) were isolated from donor CAP256 (from the Centre for the AIDS Programme of Research in South Africa (CAPRISA)); each antibody contained the protruding tyrosine-sulphated, anionic antigen-binding loop (complementarity-determining region (CDR) H3) characteristic of this category of antibodies. Their unmutated ancestor emerged between weeks 30-38 post-infection with a 35-residue CDR H3, and neutralized the virus that superinfected this individual 15 weeks after initial infection. Improved neutralization breadth and potency occurred by week 59 with modest affinity maturation, and was preceded by extensive diversification of the virus population. HIV-1 V1V2-directed neutralizing antibodies can thus develop relatively rapidly through initial selection of B cells with a long CDR H3, and limited subsequent somatic hypermutation. These data provide important insights relevant to HIV-1 vaccine development.

  2. Potent in vitro and in vivo activity of an Fc-engineered humanized anti-HM1.24 antibody against multiple myeloma via augmented effector function.

    PubMed

    Tai, Yu-Tzu; Horton, Holly M; Kong, Sun-Young; Pong, Erik; Chen, Hsing; Cemerski, Saso; Bernett, Matthew J; Nguyen, Duc-Hanh T; Karki, Sher; Chu, Seung Y; Lazar, Greg A; Munshi, Nikhil C; Desjarlais, John R; Anderson, Kenneth C; Muchhal, Umesh S

    2012-03-01

    HM1.24, an immunologic target for multiple myeloma (MM) cells, has not been effectively targeted with therapeutic monoclonal antibodies (mAbs). In this study, we investigated in vitro and in vivo anti-MM activities of XmAb5592, a humanized anti-HM1.24 mAb with Fc-domain engineered to significantly enhance FcγR binding and associated immune effector functions. XmAb5592 increased antibody-dependent cellular cytotoxicity (ADCC) several fold relative to the anti-HM1.24 IgG1 analog against both MM cell lines and primary patient myeloma cells. XmAb5592 also augmented antibody dependent cellular phagocytosis (ADCP) by macrophages. Natural killer (NK) cells became more activated by XmAb5592 than the IgG1 analog, evidenced by increased cell surface expression of granzyme B-dependent CD107a and MM cell lysis, even in the presence of bone marrow stromal cells. XmAb5592 potently inhibited tumor growth in mice bearing human MM xenografts via FcγR-dependent mechanisms, and was significantly more effective than the IgG1 analog. Lenalidomide synergistically enhanced in vitro ADCC against MM cells and in vivo tumor inhibition induced by XmAb5592. A single dose of 20 mg/kg XmAb5592 effectively depleted both blood and bone marrow plasma cells in cynomolgus monkeys. These results support clinical development of XmAb5592, both as a monotherapy and in combination with lenalidomide, to improve patient outcome of MM.

  3. Arctigenin, a phenylpropanoid dibenzylbutyrolactone lignan, inhibits MAP kinases and AP-1 activation via potent MKK inhibition: the role in TNF-alpha inhibition.

    PubMed

    Cho, Min Kyung; Jang, Young Pyo; Kim, Young Choong; Kim, Sang Geon

    2004-10-01

    Arctigenin, naturally occurring in Bardanae fructus, Saussurea medusa, Arctium lappa L., Torreya nucifera and Ipomea cairica, is a phenylpropanoid dibenzylbutyrolactone lignan with antioxidant and anti-inflammatory activities. Previously, we showed that arctigenin potently inhibited the induction of nitric oxide synthase (iNOS) by lipopolysaccharide (LPS), which involved suppression of NF-kappaB activation. In the present study, we examined the effects of arctigenin on mitogen-activated protein (MAP) kinase activation in Raw264.7 cells and MAP kinase kinase (MKK) activity. The effect of arctigenin on activator protein-1 (AP-1) activation was also studied in association with tumor necrosis factor-alpha (TNF-alpha) expression. Immunoblot analysis showed that arctigenin inhibited phosphorylation of MAP kinases ERK1/2, p38 kinase and JNK and their activities in Raw264.7 cells treated with LPS. Arctigenin potently inhibited the activity of MKK1 in vitro with the IC(50) value of 1 nM. Gel shift and reporter gene analyses revealed that arctigenin inhibited LPS-inducible AP-1 binding to the AP-1 consensus oligonucleotide and AP-1-mediated reporter gene expression. In view of the potential role of AP-1 in the induction of TNF-alpha, we next examined the inhibitory effects of arctigenin on the expression of TNF-alpha. Arctigenin blocked TNF-alpha production and decreased the level of TNF-alpha mRNA in the cells exposed to LPS. These results showed that arctigenin inhibited activation of MAP kinases including ERK1/2, p38 kinase and JNK through the inhibition of MKK activities, leading to AP-1 inactivation, which might, at least in part, contribute to the inhibition of TNF-alpha production.

  4. A potent anti-dengue human antibody preferentially recognizes the conformation of E protein monomers assembled on the virus surface

    PubMed Central

    Fibriansah, Guntur; Tan, Joanne L; Smith, Scott A; Alwis, Adamberage R; Ng, Thiam-Seng; Kostyuchenko, Victor A; Ibarra, Kristie D; Wang, Jiaqi; Harris, Eva; Silva, Aravinda; Crowe, James E; Lok, Shee-Mei

    2014-01-01

    Dengue virus (DENV), which consists of four serotypes (DENV1-4), infects over 400 million people annually. Previous studies have indicated most human monoclonal antibodies (HMAbs) from dengue patients are cross-reactive and poorly neutralizing. Rare neutralizing HMAbs are usually serotype-specific and bind to quaternary structure-dependent epitopes. We determined the structure of DENV1 complexed with Fab fragments of a highly potent HMAb 1F4 to 6 Å resolution by cryo-EM. Although HMAb 1F4 appeared to bind to virus and not E proteins in ELISAs in the previous study, our structure showed that the epitope is located within an envelope (E) protein monomer, and not across neighboring E proteins. The Fab molecules bind to domain I (DI), and DI-DII hinge of the E protein. We also showed that HMAb 1F4 can neutralize DENV at different stages of viral entry in a cell type and receptor dependent manner. The structure reveals the mechanism by which this potent and specific antibody blocks viral infection. Subject Categories Microbiology, Virology & Host Pathogen Interaction; Immunology PMID:24421336

  5. Structural basis for germ-line gene usage of a potent class of antibodies targeting the CD4-binding site of HIV-1 gp120.

    PubMed

    West, Anthony P; Diskin, Ron; Nussenzweig, Michel C; Bjorkman, Pamela J

    2012-07-24

    A large number of anti-HIV-1 antibodies targeting the CD4-binding site (CD4bs) on the envelope glycoprotein gp120 have recently been reported. These antibodies, typified by VRC01, are remarkable for both their breadth and their potency. Crystal structures have revealed a common mode of binding for several of these antibodies; however, the precise relationship among CD4bs antibodies remains to be defined. Here we analyze existing structural and sequence data, propose a set of signature features for potent VRC01-like (PVL) antibodies, and verify the importance of these features by mutagenesis. The signature features explain why PVL antibodies derive from a single germ-line human V(H) gene segment and why certain gp120 sequences are associated with antibody resistance. Our results bear on vaccine development and structure-based design to improve the potency and breadth of anti-CD4bs antibodies.

  6. A Potent and Broad Neutralizing Antibody Recognizes and Penetrates the HIV Glycan Shield

    SciTech Connect

    Pejchal, Robert; Doores, Katie J.; Walker, Laura M.; Khayat, Reza; Huang, Po-Ssu; Wang, Sheng-Kai; Stanfield, Robyn L.; Julien, Jean-Philippe; Ramos, Alejandra; Crispin, Max; Depetris, Rafael; Katpally, Umesh; Marozsan, Andre; Cupo, Albert; Maloveste, Sebastien; Liu, Yan; McBride, Ryan; Ito, Yukishige; Sanders, Rogier W.; Ogohara, Cassandra; Paulson, James C.; Feizi, Ten; Scanlan, Christopher N.; Wong, Chi-Huey; Moore, John P.; Olson, William C.; Ward, Andrew B.; Poignard, Pascal; Schief, William R.; Burton, Dennis R.; Wilson, Ian A.

    2015-10-15

    The HIV envelope (Env) protein gp120 is protected from antibody recognition by a dense glycan shield. However, several of the recently identified PGT broadly neutralizing antibodies appear to interact directly with the HIV glycan coat. Crystal structures of antigen-binding fragments (Fabs) PGT 127 and 128 with Man{sub 9} at 1.65 and 1.29 angstrom resolution, respectively, and glycan binding data delineate a specific high mannose-binding site. Fab PGT 128 complexed with a fully glycosylated gp120 outer domain at 3.25 angstroms reveals that the antibody penetrates the glycan shield and recognizes two conserved glycans as well as a short {beta}-strand segment of the gp120 V3 loop, accounting for its high binding affinity and broad specificify. Furthermore, our data suggest that the high neutralization potency of PGT 127 and 128 immunoglobulin Gs may be mediated by cross-linking Env trimers on the viral surface.

  7. A potent and broad neutralizing antibody recognizes and penetrates the HIV glycan shield.

    PubMed

    Pejchal, Robert; Doores, Katie J; Walker, Laura M; Khayat, Reza; Huang, Po-Ssu; Wang, Sheng-Kai; Stanfield, Robyn L; Julien, Jean-Philippe; Ramos, Alejandra; Crispin, Max; Depetris, Rafael; Katpally, Umesh; Marozsan, Andre; Cupo, Albert; Maloveste, Sebastien; Liu, Yan; McBride, Ryan; Ito, Yukishige; Sanders, Rogier W; Ogohara, Cassandra; Paulson, James C; Feizi, Ten; Scanlan, Christopher N; Wong, Chi-Huey; Moore, John P; Olson, William C; Ward, Andrew B; Poignard, Pascal; Schief, William R; Burton, Dennis R; Wilson, Ian A

    2011-11-25

    The HIV envelope (Env) protein gp120 is protected from antibody recognition by a dense glycan shield. However, several of the recently identified PGT broadly neutralizing antibodies appear to interact directly with the HIV glycan coat. Crystal structures of antigen-binding fragments (Fabs) PGT 127 and 128 with Man(9) at 1.65 and 1.29 angstrom resolution, respectively, and glycan binding data delineate a specific high mannose-binding site. Fab PGT 128 complexed with a fully glycosylated gp120 outer domain at 3.25 angstroms reveals that the antibody penetrates the glycan shield and recognizes two conserved glycans as well as a short β-strand segment of the gp120 V3 loop, accounting for its high binding affinity and broad specificity. Furthermore, our data suggest that the high neutralization potency of PGT 127 and 128 immunoglobulin Gs may be mediated by cross-linking Env trimers on the viral surface.

  8. Protective monotherapy against lethal Ebola virus infection by a potently neutralizing antibody.

    PubMed

    Corti, Davide; Misasi, John; Mulangu, Sabue; Stanley, Daphne A; Kanekiyo, Masaru; Wollen, Suzanne; Ploquin, Aurélie; Doria-Rose, Nicole A; Staupe, Ryan P; Bailey, Michael; Shi, Wei; Choe, Misook; Marcus, Hadar; Thompson, Emily A; Cagigi, Alberto; Silacci, Chiara; Fernandez-Rodriguez, Blanca; Perez, Laurent; Sallusto, Federica; Vanzetta, Fabrizia; Agatic, Gloria; Cameroni, Elisabetta; Kisalu, Neville; Gordon, Ingelise; Ledgerwood, Julie E; Mascola, John R; Graham, Barney S; Muyembe-Tamfun, Jean-Jacques; Trefry, John C; Lanzavecchia, Antonio; Sullivan, Nancy J

    2016-03-18

    Ebola virus disease in humans is highly lethal, with case fatality rates ranging from 25 to 90%. There is no licensed treatment or vaccine against the virus, underscoring the need for efficacious countermeasures. We ascertained that a human survivor of the 1995 Kikwit Ebola virus disease outbreak maintained circulating antibodies against the Ebola virus surface glycoprotein for more than a decade after infection. From this survivor we isolated monoclonal antibodies (mAbs) that neutralize recent and previous outbreak variants of Ebola virus and mediate antibody-dependent cell-mediated cytotoxicity in vitro. Strikingly, monotherapy with mAb114 protected macaques when given as late as 5 days after challenge. Treatment with a single human mAb suggests that a simplified therapeutic strategy for human Ebola infection may be possible.

  9. A potent and broad neutralizing antibody recognizes and penetrates the HIV glycan shield

    PubMed Central

    Pejchal, Robert; Doores, Katie J.; Walker, Laura M.; Khayat, Reza; Huang, Po-Ssu; Wang, Sheng-Kai; Stanfield, Robyn L.; Julien, Jean-Philippe; Ramos, Alejandra; Crispin, Max; Depetris, Rafael; Katpally, Umesh; Marozsan, Andre; Cupo, Albert; Maloveste, Sebastien; Liu, Yan; McBride, Ryan; Ito, Yukishige; Sanders, Rogier W.; Ogohara, Cassandra; Paulson, James C.; Feizi, Ten; Scanlan, Christopher N.; Wong, Chi-Huey; Moore, John P.; Olson, William C.; Ward, Andrew B.; Poignard, Pascal; Schief, William R.; Burton, Dennis R.; Wilson, Ian A.

    2012-01-01

    The HIV envelope (Env) protein gp120 is protected from antibody recognition by a dense glycan shield. However, several of the recently identified PGT broadly neutralizing antibodies appear to interact directly with the HIV glycan coat. Crystal structures of Fabs PGT 127 and 128 with Man9 at 1.65 and 1.29 Å resolution, respectively, and glycan binding data delineate a specific high mannose binding site. Fab PGT 128 complexed with a fully-glycosylated gp120 outer domain at 3.25 Å reveals that the antibody penetrates the glycan shield and recognizes two conserved glycans as well as a short β-strand segment of the gp120 V3 loop, accounting for its high binding affinity and broad specificify. Furthermore, our data suggest that the high neutralization potency of PGT 127 and 128 IgGs may be mediated by cross-linking Env trimers on the viral surface. PMID:21998254

  10. Rhodanine hydrolysis leads to potent thioenolate mediated metallo-β-lactamase inhibition

    NASA Astrophysics Data System (ADS)

    Brem, Jürgen; van Berkel, Sander S.; Aik, Weishen; Rydzik, Anna M.; Avison, Matthew B.; Pettinati, Ilaria; Umland, Klaus-Daniel; Kawamura, Akane; Spencer, James; Claridge, Timothy D. W.; McDonough, Michael A.; Schofield, Christopher J.

    2014-12-01

    The use of β-lactam antibiotics is compromised by resistance, which is provided by β-lactamases belonging to both metallo (MBL)- and serine (SBL)-β-lactamase subfamilies. The rhodanines are one of very few compound classes that inhibit penicillin-binding proteins (PBPs), SBLs and, as recently reported, MBLs. Here, we describe crystallographic analyses of the mechanism of inhibition of the clinically relevant VIM-2 MBL by a rhodanine, which reveal that the rhodanine ring undergoes hydrolysis to give a thioenolate. The thioenolate is found to bind via di-zinc chelation, mimicking the binding of intermediates in β-lactam hydrolysis. Crystallization of VIM-2 in the presence of the intact rhodanine led to observation of a ternary complex of MBL, a thioenolate fragment and rhodanine. The crystallographic observations are supported by kinetic and biophysical studies, including 19F NMR analyses, which reveal the rhodanine-derived thioenolate to be a potent broad-spectrum MBL inhibitor and a lead structure for the development of new types of clinically useful MBL inhibitors.

  11. Structurally novel steroidal spirooxindole by241 potently inhibits tumor growth mainly through ROS-mediated mechanisms

    PubMed Central

    Shi, Xiao-Jing; Yu, Bin; Wang, Jun-Wei; Qi, Ping-Ping; Tang, Kai; Huang, Xin; Liu, Hong-Min

    2016-01-01

    Cancer cells always have increased ROS levels, thus making them more vulnerable to persistent endogenous oxidative stress. The biochemical difference between cancer and normal cells could be exploited to achieve selective cancer cell killing by exogenous ROS-producing agents. Herein we described a structurally novel steroidal spirooxindole by241 and its anticancer efficacy. By241 exhibited potent inhibition against human cancer cells and less toxic to normal cells. By241 concentration-dependently induced apoptosis of MGC-803 and EC9706 cells, accompanied with the mitochondrial dysfunction and increased ROS levels. NAC can completely restore the decreased cell viability of MGC-803 cells caused by by241, suggesting ROS-mediated mechanisms. The expression levels of proteins involved in the mitochondrion-related pathways were detected, showing increased expression of proapoptotic proteins and decreased expression of anti-apoptotic proteins, and activation of caspases-9/-3, but without activating caspase-8 expression. Pretreatment with Z-VAD-FMK partially rescued by241-induced apoptosis of MGC-803 cells. Additionally, by241 inhibited mTOR, activated p53 and its downstream proteins, cleaved MDM2 and PI3K/AKT as well as NF-κB signaling pathway. In vivo experiments showed that by241 did not have significant acute oral toxicity and exerted good anticancer efficacy against MGC-803 bearing mice models. Therefore, by241 may serve as a lead for further development for cancer therapy. PMID:27527552

  12. Potent inhibition of macrophage migration inhibitory factor (MIF) by myeloperoxidase-dependent oxidation of epicatechins.

    PubMed

    Dickerhof, Nina; Magon, Nicholas J; Tyndall, Joel D A; Kettle, Anthony J; Hampton, Mark B

    2014-09-01

    MIF (macrophage migration inhibitory factor) plays a central role in the promotion and maintenance of the inflammatory response. It is implicated in a number of inflammatory diseases including sepsis, arthritis and colitis, and in diseases with an inflammatory component, such as atherosclerosis, diabetes and cancer. MIF has an unusual N-terminal proline with catalytic activity, and targeting of this residue by small-molecule inhibitors has been shown to interfere with the biological activity of MIF. The objective of the present study was to determine if MIF was susceptible to modification by epicatechins, a group of dietary flavonoids with known anti-inflammatory properties. Epicatechins are substrates for peroxidases including neutrophil-derived MPO (myeloperoxidase). In the present study we show that oxidation of the catechol moiety of epicatechins to an ο-quinone by MPO generates potent MIF inhibitors. Near complete inhibition of MIF by the MPO/H2O2/epicatechin system was achieved at equimolar concentrations of epicatechin and MIF, even in the presence of other MPO substrates. We have characterized the modification introduced by oxidized (-)-epicatechin on MIF by LC-MS (liquid chromatography MS) and found it to occur at the N-terminal proline. We propose that MIF inhibition by oxidized epicatechins contributes to the anti-inflammatory activity of these compounds.

  13. Novel, potent, and selective GABAC antagonists inhibit myopia development and facilitate learning and memory.

    PubMed

    Chebib, Mary; Hinton, Tina; Schmid, Katrina L; Brinkworth, Darren; Qian, Haohua; Matos, Susana; Kim, Hye-Lim; Abdel-Halim, Heba; Kumar, Rohan J; Johnston, Graham A R; Hanrahan, Jane R

    2009-02-01

    This study reports pharmacological and physiological effects of cis- and trans-(3-aminocyclopentanyl)butylphosphinic acid (cis- and trans-3-ACPBPA). These compounds are conformationally restricted analogs of the orally active GABA(B/C) receptor antagonist (3-aminopropyl)-n-butylphosphinic acid (CGP36742 or SGS742). cis-[IC(50)(rho1) = 5.06 microM and IC(50)(rho2) = 11.08 microM; n = 4] and trans-3-ACPMPA [IC(50)(rho1) = 72.58 microM and IC(50)(rho2) = 189.7 microM; n = 4] seem competitive at GABA(C) receptors expressed in Xenopus laevis oocytes, having no effect as agonists (1 mM) but exerting weak antagonist (1 mM) effects on human GABA(A) and GABA(B) receptors. cis-3-ACPBPA was more potent and selective than the trans-compound, being more than 100 times more potent at GABA(C) than GABA(A) or GABA(B) receptors. cis-3-ACPBPA was further evaluated on dissociated rat retinal bipolar cells and dose-dependently inhibited the native GABA(C) receptor (IC(50) = 47 +/- 4.5 microM; n = 6). When applied to the eye as intravitreal injections, cis- and trans-3-ACPBPA prevented experimental myopia development and inhibited the associated vitreous chamber elongation, in a dose-dependent manner in the chick model. Doses only 10 times greater than required to inhibit recombinant GABA(C) receptors caused the antimyopia effects. Using intraperitoneal administration, cis- (30 mg/kg) and trans-3-ACPBPA (100 mg/kg) enhanced learning and memory in male Wistar rats; compared with vehicle there was a significant reduction in time for rats to find the platform in the Morris water maze task (p < 0.05; n = 10). As the physiological effects of cis- and trans-3-ACPBPA are similar to those reported for CGP36742, the memory and refractive effects of CGP36742 may be due in part to its GABA(C) activity.

  14. Proteasome Inhibition Suppresses Dengue Virus Egress in Antibody Dependent Infection

    PubMed Central

    Costa, Vivian V.; Tan, Hwee Cheng; Horrevorts, Sophie; Ooi, Eng Eong

    2015-01-01

    The mosquito-borne dengue virus (DENV) is a cause of significant global health burden, with an estimated 390 million infections occurring annually. However, no licensed vaccine or specific antiviral treatment for dengue is available. DENV interacts with host cell factors to complete its life cycle although this virus-host interplay remains to be fully elucidated. Many studies have identified the ubiquitin proteasome pathway (UPP) to be important for successful DENV production, but how the UPP contributes to DENV life cycle as host factors remains ill defined. We show here that proteasome inhibition decouples infectious virus production from viral RNA replication in antibody-dependent infection of THP-1 cells. Molecular and imaging analyses in β-lactone treated THP-1 cells suggest that proteasome function does not prevent virus assembly but rather DENV egress. Intriguingly, the licensed proteasome inhibitor, bortezomib, is able to inhibit DENV titers at low nanomolar drug concentrations for different strains of all four serotypes of DENV in primary monocytes. Furthermore, bortezomib treatment of DENV-infected mice inhibited the spread of DENV in the spleen as well as the overall pathological changes. Our findings suggest that preventing DENV egress through proteasome inhibition could be a suitable therapeutic strategy against dengue. PMID:26565697

  15. Production of Potent Fully Human Polyclonal Antibodies Against Zaire Ebola Virus in Transchromosomal Cattle

    DTIC Science & Technology

    2016-07-01

    recombinant glycoprotein (GP) vaccine consisting of the 2014 Ebola virus (EBOV)-Makona isolate. Serum collected from these hyperimmunized Tc...countermeasures for EBOV infections in humans. Effective countermeasures that include vaccines , antivirals, and other prophylactic and therapeutic...traditional animal systems used to produce polyclonal antibodies, Tc bovines can be hyperimmunized over a long period of time with vaccines containing strong

  16. SL-01, an oral gemcitabine derivative, inhibited human cancer growth more potently than gemcitabine

    SciTech Connect

    Zhao, Cuirong; Yue, Bin; Liu, Huiping; Sun, Cuicui; Li, Wenbao; Qu, Xianjun

    2012-08-01

    SL-01, an oral gemcitabine derivative, was synthesized by introducing the moiety of 3-(dodecyloxycarbonyl)pyrazine-2-carbonyl at the N4-position on the cytidine ring of gemcitabine. Our goal in this study was to evaluate the efficacy of SL-01 on the growth of human cancers with gemcitabine as control. Experiments were performed on human non-small cell lung cancer NCI-H460 and colon cancer HCT-116 both in vitro and in vivo. In vitro assays, SL-01 significantly inhibited the growth of cancer cells as determined by the 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. Further studies indicated that SL-01 induced the cancer cells to apoptosis showing chromatin condensation and externalization of phosphatidylserine. In in vivo studies, we evaluated the efficacy of SL-01 in nude mice bearing human cancer xenografts. SL-01 effectively delayed the growth of NCI-H460 and HCT-116 without significant loss of body weight. Molecular analysis indicated that the high efficacy of SL-01 was associated with its ability to induce apoptosis as evidenced by increase of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining cells, activation of caspase-9, caspase-3 and cleaved poly ADP-ribose polymerase (PARP) in tumor tissues. SL-01 also increased Bax/Bcl-2 ratio in cancer cells. These biological activities of SL-01 were more potential than that of gemcitabine. Based on these in vitro and in vivo results, SL-01 is proposed as a potent oral anticancer agent that may supplant the use of gemcitabine in the clinic. -- Highlights: ► An oral gemcitabine derivative SL-01 was synthesized. ► The effects of SL-01 were evaluated and its efficacy was compared with gemcitabine. ► The biological activities of SL-01 were more potent than that of gemcitabine. ► SL-01 could replace gemcitabine for clinical use.

  17. Potent and Selective Inhibition of Plasma Membrane Monoamine Transporter by HIV Protease Inhibitors

    PubMed Central

    Duan, Haichuan; Hu, Tao; Foti, Robert S.; Pan, Yongmei; Swaan, Peter W.

    2015-01-01

    Plasma membrane monoamine transporter (PMAT) is a major uptake-2 monoamine transporter that shares extensive substrate and inhibitor overlap with organic cation transporters 1–3 (OCT1–3). Currently, there are no PMAT-specific inhibitors available that can be used in in vitro and in vivo studies to differentiate between PMAT and OCT activities. In this study, we showed that IDT307 (4-(4-(dimethylamino)phenyl)-1-methylpyridinium iodide), a fluorescent analog of 1-methyl-4-phenylpyridinium (MPP+), is a transportable substrate for PMAT and that IDT307-based fluorescence assay can be used to rapidly identify and characterize PMAT inhibitors. Using the fluorescent substrate-based assays, we analyzed the interactions of eight human immunodeficiency virus (HIV) protease inhibitors (PIs) with human PMAT and OCT1–3 in human embryonic kidney 293 (HEK293) cells stably transfected with individual transporters. Our data revealed that PMAT and OCTs exhibit distinct sensitivity and inhibition patterns toward HIV PIs. PMAT is most sensitive to PI inhibition whereas OCT2 and OCT3 are resistant. OCT1 showed an intermediate sensitivity and a distinct inhibition profile from PMAT. Importantly, lopinavir is a potent PMAT inhibitor and exhibited >120 fold selectivity toward PMAT (IC50 = 1.4 ± 0.2 µM) over OCT1 (IC50 = 174 ± 40 µM). Lopinavir has no inhibitory effect on OCT2 or OCT3 at maximal tested concentrations. Lopinavir also exhibited no or much weaker interactions with uptake-1 monoamine transporters. Together, our results reveal that PMAT and OCTs have distinct specificity exemplified by their differential interaction with HIV PIs. Further, we demonstrate that lopinavir can be used as a selective PMAT inhibitor to differentiate PMAT-mediated monoamine and organic cation transport from those mediated by OCT1–3. PMID:26285765

  18. Monoclonal antibody-induced ErbB3 receptor internalization and degradation inhibits growth and migration of human melanoma cells.

    PubMed

    Belleudi, Francesca; Marra, Emanuele; Mazzetta, Francesca; Fattore, Luigi; Giovagnoli, Maria Rosaria; Mancini, Rita; Aurisicchio, Luigi; Torrisi, Maria Rosaria; Ciliberto, Gennaro

    2012-04-01

    Members of the ErbB receptor family are targets of a growing numbers of small molecules and monoclonal antibodies inhibitors currently under development for the treatment of cancer. Although historical efforts have been directed against ErbB1 (EGFR) and ErbB2 (HER2/neu), emerging evidences have pointed to ErbB3 as a key node in the activation of proliferation/survival pathways from the ErbB receptor family and have fueled enthusiasm toward the clinical development of anti-ErbB3 agents. In this study, we have evaluated the potential therapeutic efficacy of a set of three recently generated anti-human ErbB3 monoclonals, A2, A3 and A4, in human primary melanoma cells. We show that in melanoma cells expressing ErbB1, ErbB3 and ErbB4 but not ErbB2 receptor ligands activate the PI3K/AKT pathway, and this leads to increased cell proliferation and migration. While antibodies A3 and A4 are able to potently inhibit ligand-induced signaling, proliferation and migration, antibody A2 is unable to exert this effect. In attempt to understand the mechanism of action and the basis of this different behavior, we demonstrate, through a series of combined approaches, that antibody efficacy strongly correlates with antibody-induced receptor internalization, degradation and inhibition of receptor recycling to the cell surface. Finally, fine epitope mapping studies through a peptide array show that inhibiting vs. non-inhibiting antibodies have a dramatically different mode of binding to the to the receptor extracellular domain. Our study confirms the key role of ErbB3 and points to exploitation of novel combination therapies for treatment of malignant melanoma.

  19. New pyrrole derivatives with potent tubulin polymerization inhibiting activity as anticancer agents including hedgehog-dependent cancer.

    PubMed

    La Regina, Giuseppe; Bai, Ruoli; Coluccia, Antonio; Famiglini, Valeria; Pelliccia, Sveva; Passacantilli, Sara; Mazzoccoli, Carmela; Ruggieri, Vitalba; Sisinni, Lorenza; Bolognesi, Alessio; Rensen, Whilelmina Maria; Miele, Andrea; Nalli, Marianna; Alfonsi, Romina; Di Marcotullio, Lucia; Gulino, Alberto; Brancale, Andrea; Novellino, Ettore; Dondio, Giulio; Vultaggio, Stefania; Varasi, Mario; Mercurio, Ciro; Hamel, Ernest; Lavia, Patrizia; Silvestri, Romano

    2014-08-14

    We synthesized 3-aroyl-1-arylpyrrole (ARAP) derivatives as potential anticancer agents having different substituents at the pendant 1-phenyl ring. Both the 1-phenyl ring and 3-(3,4,5-trimethoxyphenyl)carbonyl moieties were mandatory to achieve potent inhibition of tubulin polymerization, binding of colchicine to tubulin, and cancer cell growth. ARAP 22 showed strong inhibition of the P-glycoprotein-overexpressing NCI-ADR-RES and Messa/Dx5MDR cell lines. Compounds 22 and 27 suppressed in vitro the Hedgehog signaling pathway, strongly reducing luciferase activity in SAG treated NIH3T3 Shh-Light II cells, and inhibited the growth of medulloblastoma D283 cells at nanomolar concentrations. ARAPs 22 and 27 represent a new potent class of tubulin polymerization and cancer cell growth inhibitors with the potential to inhibit the Hedgehog signaling pathway.

  20. New Pyrrole Derivatives with Potent Tubulin Polymerization Inhibiting Activity As Anticancer Agents Including Hedgehog-Dependent Cancer

    PubMed Central

    La Regina, Giuseppe; Bai, Ruoli; Coluccia, Antonio; Famiglini, Valeria; Pelliccia, Sveva; Passacantilli, Sara; Mazzoccoli, Carmela; Ruggieri, Vitalba; Sisinni, Lorenza; Bolognesi, Alessio; Rensen, Whilelmina Maria; Miele, Andrea; Nalli, Marianna; Alfonsi, Romina; Di Marcotullio, Lucia; Gulino, Alberto; Brancale, Andrea; Novellino, Ettore; Dondio, Giulio; Vultaggio, Stefania; Varasi, Mario; Mercurio, Ciro; Hamel, Ernest; Lavia, Patrizia; Silvestri, Romano

    2014-01-01

    We synthesized 3-aroyl-1-arylpyrrole (ARAP) derivatives as potential anticancer agents having different substituents at the pendant 1-phenyl ring. Both the 1-phenyl ring and 3-(3,4,5-trimethoxyphenyl)carbonyl moieties were mandatory to achieve potent inhibition of tubulin polymerization, binding of colchicine to tubulin, and cancer cell growth. ARAP 22 showed strong inhibition of the P-glycoprotein-overexpressing NCI-ADR-RES and Messa/Dx5MDR cell lines. Compounds 22 and 27 suppressed in vitro the Hedgehog signaling pathway, strongly reducing luciferase activity in SAG treated NIH3T3 Shh-Light II cells, and inhibited the growth of medulloblastoma D283 cells at nanomolar concentrations. ARAPs 22 and 27 represent a new potent class of tubulin polymerization and cancer cell growth inhibitors with the potential to inhibit the Hedgehog signaling pathway. PMID:25025991

  1. Delta- and gamma-tocotrienol isomers are potent in inhibiting inflammation and endothelial activation in stimulated human endothelial cells

    PubMed Central

    Muid, Suhaila; Froemming, Gabriele R. Anisah; Rahman, Thuhairah; Ali, A. Manaf; Nawawi, Hapizah M.

    2016-01-01

    Background Tocotrienols (TCTs) are more potent antioxidants than α-tocopherol (TOC). However, the effectiveness and mechanism of the action of TCT isomers as anti-atherosclerotic agents in stimulated human endothelial cells under inflammatory conditions are not well established. Aims 1) To compare the effects of different TCT isomers on inflammation, endothelial activation, and endothelial nitric oxide synthase (eNOS). 2) To identify the two most potent TCT isomers in stimulated human endothelial cells. 3) To investigate the effects of TCT isomers on NFκB activation, and protein and gene expression levels in stimulated human endothelial cells. Methods Human umbilical vein endothelial cells were incubated with various concentrations of TCT isomers or α-TOC (0.3–10 µM), together with lipopolysaccharides for 16 h. Supernatant cells were collected and measured for protein and gene expression of cytokines (interleukin-6, or IL-6; tumor necrosis factor-alpha, or TNF-α), adhesion molecules (intercellular cell adhesion molecule-1, or ICAM-1; vascular cell adhesion molecule-1, or VCAM-1; and e-selectin), eNOS, and NFκB. Results δ-TCT is the most potent TCT isomer in the inhibition of IL-6, ICAM-1, VCAM-1, and NFκB, and it is the second potent in inhibiting e-selectin and eNOS. γ-TCT isomer is the most potent isomer in inhibiting e-selectin and eNOS, and it is the second most potent in inhibiting is IL-6, VCAM-1, and NFκB. For ICAM-1 protein expression, the most potent is δ-TCT followed by α-TCT. α- and β-TCT inhibit IL-6 at the highest concentration (10 µM) but enhance IL-6 at lower concentrations. γ-TCT markedly increases eNOS expression by 8–11-fold at higher concentrations (5–10 µM) but exhibits neutral effects at lower concentrations. Conclusion δ- and γ-TCT are the two most potent TCT isomers in terms of the inhibition of inflammation and endothelial activation whilst enhancing eNOS, possibly mediated via the NFκB pathway. Hence, there is a

  2. Structural and Functional Characterization of Anti-A33 Antibodies Reveal a Potent Cross-Species Orthopoxviruses Neutralizer

    PubMed Central

    Matho, Michael H.; Schlossman, Andrew; Meng, Xiangzhi; Benhnia, Mohammed Rafii-El-Idrissi; Kaever, Thomas; Buller, Mark; Doronin, Konstantin; Parker, Scott; Peters, Bjoern; Crotty, Shane; Xiang, Yan; Zajonc, Dirk M.

    2015-01-01

    Vaccinia virus A33 is an extracellular enveloped virus (EEV)-specific type II membrane glycoprotein that is essential for efficient EEV formation and long-range viral spread within the host. A33 is a target for neutralizing antibody responses against EEV. In this study, we produced seven murine anti-A33 monoclonal antibodies (MAbs) by immunizing mice with live VACV, followed by boosting with the soluble A33 homodimeric ectodomain. Five A33 specific MAbs were capable of neutralizing EEV in the presence of complement. All MAbs bind to conformational epitopes on A33 but not to linear peptides. To identify the epitopes, we have adetermined the crystal structures of three representative neutralizing MAbs in complex with A33. We have further determined the binding kinetics for each of the three antibodies to wild-type A33, as well as to engineered A33 that contained single alanine substitutions within the epitopes of the three crystallized antibodies. While the Fab of both MAbs A2C7 and A20G2 binds to a single A33 subunit, the Fab from MAb A27D7 binds to both A33 subunits simultaneously. A27D7 binding is resistant to single alanine substitutions within the A33 epitope. A27D7 also demonstrated high-affinity binding with recombinant A33 protein that mimics other orthopoxvirus strains in the A27D7 epitope, such as ectromelia, monkeypox, and cowpox virus, suggesting that A27D7 is a potent cross-neutralizer. Finally, we confirmed that A27D7 protects mice against a lethal challenge with ectromelia virus. PMID:26325270

  3. The 1.51-Angstrom structure of the poxvirus L1 protein, a target of potent neutralizing antibodies.

    PubMed

    Su, Hua-Poo; Garman, Scott C; Allison, Timothy J; Fogg, Christiana; Moss, Bernard; Garboczi, David N

    2005-03-22

    Although eradicated from nature more than two decades ago, the threat of smallpox has reemerged because of concerns over its use as a biological weapon. We present the structure of the poxvirus L1 protein, a molecule that is conserved throughout the poxvirus family and is nearly identical in vaccinia virus and in variola virus, which causes smallpox. L1 is a myristoylated envelope protein that is a potent target for neutralizing antibodies and an important component of current experimental vaccines. The L1 structure reveals a hydrophobic cavity located adjacent to its N terminus. The cavity would be capable of shielding the myristate moiety, which is essential for virion assembly. The structure of L1 is a step in the elucidation of molecular mechanisms common to all poxviruses that may stimulate the design of safer vaccines and new antipoxvirus drugs.

  4. Potent inhibition of scrapie prion replication in cultured cells by bis-acridines

    PubMed Central

    May, Barnaby C. H.; Fafarman, Aaron T.; Hong, Septima B.; Rogers, Michael; Deady, Leslie W.; Prusiner, Stanley B.; Cohen, Fred E.

    2003-01-01

    Prion diseases are characterized by an accumulation of PrPSc, a misfolded isoform of the normal cellular prion protein, PrPC. We previously reported the bioactivity of acridine-based compounds against PrPSc replication in scrapie-infected neuroblastoma cells and now report the improved potency of bis-acridine compounds. Bis-acridines are characterized by a dimeric motif, comprising two acridine heterocycles tethered by a linker. A library of bis-(6-chloro-2-methoxy-acridin-9-yl) and bis-(7-chloro-2-methoxy-benzo[b][1,5]naphthyridin-10-yl) analogs was synthesized to explore the effect of structurally diverse linkers on PrPSc replication in scrapie-infected neuroblastoma cells. Structure–activity analysis revealed that linker length and structure are important determinants for inhibition of prion replication in cultured scrapied cells. Three bis-acridine analogs, (6-chloro-2-methoxy-acridin-9-yl)-(3-{4-[3-(6-chloro-2-methoxy-acridin-9-ylamino)-propyl]-piperazin-1-yl}-propyl)-amine, N,N′-bis-(6-chloro-2-methoxy-acridin-9-yl)-1,8-diamino-3,6-dioxaoctane, and (1-{[4-(6-chloro-2-methoxy-acridin-9-ylamino)-butyl]-[3-(6-chloro-2-methoxy-acridin-9-ylamino)-propyl]-carbamoyl}-ethyl)-carbamic acid tert-butyl ester, showed half-maximal inhibition of PrPSc formation at 40, 25, and 30 nM, respectively, and were not cytotoxic to uninfected neuroblastoma cells at concentrations of 500 nM. Our data suggest that bis-acridine analogs may provide a potent alternative to the acridine-based compound quinacrine, which is currently under clinical evaluation for the treatment of prion disease. PMID:12626750

  5. Potent inhibition of feline coronaviruses with peptidyl compounds targeting coronavirus 3C-like protease.

    PubMed

    Kim, Yunjeong; Mandadapu, Sivakoteswara Rao; Groutas, William C; Chang, Kyeong-Ok

    2013-02-01

    Feline coronavirus infection is common among domestic and exotic felid species and usually associated with mild or asymptomatic enteritis; however, feline infectious peritonitis (FIP) is a fatal disease of cats that is caused by systemic infection with a feline infectious peritonitis virus (FIPV), a variant of feline enteric coronavirus (FECV). Currently, there is no specific treatment approved for FIP despite the importance of FIP as the leading infectious cause of death in young cats. During the replication process, coronavirus produces viral polyproteins that are processed into mature proteins by viral proteases, the main protease (3C-like [3CL] protease) and the papain-like protease. Since the cleavages of viral polyproteins are an essential step for virus replication, blockage of viral protease is an attractive target for therapeutic intervention. Previously, we reported the generation of broad-spectrum peptidyl inhibitors against viruses that possess a 3C or 3CL protease. In this study, we further evaluated the antiviral effects of the peptidyl inhibitors against feline coronaviruses, and investigated the interaction between our protease inhibitor and a cathepsin B inhibitor, an entry blocker, against a feline coronavirus in cell culture. Herein we report that our compounds behave as reversible, competitive inhibitors of 3CL protease, potently inhibited the replication of feline coronaviruses (EC(50) in a nanomolar range) and, furthermore, combination of cathepsin B and 3CL protease inhibitors led to a strong synergistic interaction against feline coronaviruses in a cell culture system.

  6. Broad and potent HIV-1 neutralization by a human antibody that binds the gp41-120 interface

    PubMed Central

    Huang, Jinghe; Kang, Byong H.; Pancera, Marie; Lee, Jeong Hyun; Tong, Tommy; Feng, Yu; Georgiev, Ivelin S.; Chuang, Gwo-Yu; Druz, Aliaksandr; Doria-Rose, Nicole A.; Laub, Leo; Sliepen, Kwinten; van Gils, Marit J.; de la Peña, Alba Torrents; Derking, Ronald; Klasse, Per-Johan; Migueles, Stephen A.; Bailer, Robert T.; Alam, Munir; Pugach, Pavel; Haynes, Barton F.; Wyatt, Richard T.; Sanders, Rogier W.; Binley, James M.; Ward, Andrew B.; Mascola, John R.; Kwong, Peter D.; Connors, Mark

    2014-01-01

    The isolation of human monoclonal antibodies (mAbs) is providing important insights regarding the specificities that underlie broad neutralization of HIV-1 (reviewed in1). Here we report a broad and extremely potent HIV-specific mAb, termed 35O22, which binds novel HIV-1 envelope glycoprotein (Env) epitope. 35O22 neutralized 62% of 181 pseudoviruses with an IC50<50 μg/ml. The median IC50 of neutralized viruses was 0.033 μg/ml, among the most potent thus far described. 35O22 did not bind monomeric forms of Env tested, but did bind the trimeric BG505 SOSIP.664. Mutagenesis and a reconstruction by negative-stain electron microscopy of the Fab in complex with trimer revealed it to bind a conserved epitope, which stretched across gp120 and gp41. The specificity of 35O22 represents a novel site of vulnerability on HIV Env, which serum analysis indicates to be commonly elicited by natural infection. Binding to this new site of vulnerability may thus be an important complement to current mAb-based approaches to immunotherapies, prophylaxis, and vaccine design. PMID:25186731

  7. The Antibody Targeting the E314 Peptide of Human Kv1.3 Pore Region Serves as a Novel, Potent and Specific Channel Blocker

    PubMed Central

    Li, Xiao-Wei; Cheng, Long-Xian; Liu, Jin-Ping; Wang, Yan-Fu; Gao, Xiang; Liao, Yu-Hua; Wang, Min; Zeng, Qiu-Tang; Liu, Kun

    2012-01-01

    Selective blockade of Kv1.3 channels in effector memory T (TEM) cells was validated to ameliorate autoimmune or autoimmune-associated diseases. We generated the antibody directed against one peptide of human Kv1.3 (hKv1.3) extracellular loop as a novel and possible Kv1.3 blocker. One peptide of hKv1.3 extracellular loop E3 containing 14 amino acids (E314) was chosen as an antigenic determinant to generate the E314 antibody. The E314 antibody specifically recognized 63.8KD protein stably expressed in hKv1.3-HEK 293 cell lines, whereas it did not recognize or cross-react to human Kv1.1(hKv1.1), Kv1.2(hKv1.2), Kv1.4(hKv1.4), Kv1.5(hKv1.5), KCa3.1(hKCa3.1), HERG, hKCNQ1/hKCNE1, Nav1.5 and Cav1.2 proteins stably expressed in HEK 293 cell lines or in human atrial or ventricular myocytes by Western blotting analysis and immunostaining detection. By the technique of whole-cell patch clamp, the E314 antibody was shown to have a directly inhibitory effect on hKv1.3 currents expressed in HEK 293 or Jurkat T cells and the inhibition showed a concentration-dependence. However, it exerted no significant difference on hKv1.1, hKv1.2, hKv1.4, hKv1.5, hKCa3.1, HERG, hKCNQ1/hKCNE1, L-type Ca2+ or voltage-gated Na+ currents. The present study demonstrates that the antibody targeting the E314 peptide of hKv1.3 pore region could be a novel, potent and specific hKv1.3 blocker without affecting a variety of closely related Kv1 channels, KCa3.1 channels and functional cardiac ion channels underlying central nervous systerm (CNS) disorders or drug-acquired arrhythmias, which is required as a safe clinic-promising channel blocker. PMID:22558454

  8. Synergistic anti-tumor therapy by a comb-like multifunctional antibody nanoarray with exceptionally potent activity

    NASA Astrophysics Data System (ADS)

    Li, Huafei; Sun, Yun; Chen, Di; Zhao, He; Zhao, Mengxin; Zhu, Xiandi; Ke, Changhong; Zhang, Ge; Jiang, Cheng; Zhang, Li; Zhang, Fulei; Wei, Huafeng; Li, Wei

    2015-10-01

    Simultaneously blocking multiple mediators offers new hope for the treatment of complex diseases. However, the curative potential of current combination therapy by chronological administration of separate monoclonal antibodies (mAbs) or multi-specific mAbs is still moderate due to inconvenient manipulation, low cooperative effectors, poor pharmacokinetics and insufficient tumor accumulation. Here, we describe a facile strategy that arms distinct mAbs with cooperative effectors onto a long chain to form a multicomponent comb-like nano mAb. Unlike dissociative parental mAbs, the multifunctional mAb nanoarray (PL-RB) constructed from type I/II anti-CD20 mAbs shows good pharmacokinetics. This PL-RB simultaneously targets distinct epitopes on a single antigen (Ag) and neighboring Ags on different lymphocytes. This unique intra- and intercellular Ag cross-linking endows the multifunctional mAb nanoarray with potent apoptosis activity. The exceptional apoptosis, complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) that are synchronously evoked by the nano PL-RB are further synergistically promoted via enhanced permeability and retention (EPR), which resulted in high intratumor accumulation and excellent anti-lymphoma efficiency.

  9. Epitope Mapping of M36, a Human Antibody Domain with Potent and Broad HIV-1 Inhibitory Activity

    PubMed Central

    Chen, Weizao; Yuan, Xiaohui; Chong, Huihui; Prabakaran, Ponraj; Dimitrov, Dimiter S.; He, Yuxian

    2013-01-01

    M36 is the first member of a novel class of potent HIV-1 entry inhibitors based on human engineered antibody domains (eAds). It exhibits broad inhibitory activity suggesting that its CD4-induced epitope is highly conserved. Here, we describe fine mapping of its epitope by using several approaches. First, a panel of mimotopes was affinity-selected from a random peptide library and potential m36-binding residues were computationally predicted. Second, homology modeling of m36 and molecular docking of m36 onto gp120 revealed potentially important residues in gp120-m36 interactions. Third, the predicted contact residues were verified by site-directed mutagenesis. Taken together, m36 epitope comprising three discontinuous sites including six key gp120 residues (Site C1: Thr123 and Pro124; Site C3: Glu370 and Ile371; Site C4: Met426 and Trp427) were identified. In the 3D structure of gp120, the sites C1 and C4 are located in the bridging sheet and the site C3 is within the β15-α3 excursion, which play essential roles for the receptor- and coreceptor-binding and are major targets of neutralizing antibodies. Based on these results we propose a precise localization of the m36 epitope and suggest a mechanism of its broad inhibitory activity which could help in the development of novel HIV-1 therapeutics based on eAds. PMID:23776690

  10. Simultaneous inhibition of multiple oncogenic miRNAs by a multi-potent microRNA sponge.

    PubMed

    Jung, Jaeyun; Yeom, Chanjoo; Choi, Yeon-Sook; Kim, Sinae; Lee, EunJi; Park, Min Ji; Kang, Sang Wook; Kim, Sung Bae; Chang, Suhwan

    2015-08-21

    The roles of oncogenic miRNAs are widely recognized in many cancers. Inhibition of single miRNA using antagomiR can efficiently knock-down a specific miRNA. However, the effect is transient and often results in subtle phenotype, as there are other miRNAs contribute to tumorigenesis. Here we report a multi-potent miRNA sponge inhibiting multiple miRNAs simultaneously. As a model system, we targeted miR-21, miR-155 and miR-221/222, known as oncogenic miRNAs in multiple tumors including breast and pancreatic cancers. To achieve efficient knockdown, we generated perfect and bulged-matched miRNA binding sites (MBS) and introduced multiple copies of MBS, ranging from one to five, in the multi-potent miRNA sponge. Luciferase reporter assay showed the multi-potent miRNA sponge efficiently inhibited 4 miRNAs in breast and pancreatic cancer cells. Furthermore, a stable and inducible version of the multi-potent miRNA sponge cell line showed the miRNA sponge efficiently reduces the level of 4 target miRNAs and increase target protein level of these oncogenic miRNAs. Finally, we showed the miRNA sponge sensitize cells to cancer drug and attenuate cell migratory activity. Altogether, our study demonstrates the multi-potent miRNA sponge is a useful tool to examine the functional impact of simultaneous inhibition of multiple miRNAs and proposes a therapeutic potential.

  11. XGFR*, a novel affinity-matured bispecific antibody targeting IGF-1R and EGFR with combined signaling inhibition and enhanced immune activation for the treatment of pancreatic cancer

    PubMed Central

    Schanzer, Juergen M.; Wartha, Katharina; Moessner, Ekkehard; Hosse, Ralf J.; Moser, Samuel; Croasdale, Rebecca; Trochanowska, Halina; Shao, Cuiying; Wang, Peng; Shi, Lei; Weinzierl, Tina; Rieder, Natascha; Bacac, Marina; Ries, Carola H.; Kettenberger, Hubert; Schlothauer, Tilman; Friess, Thomas; Umana, Pablo; Klein, Christian

    2016-01-01

    ABSTRACT The epidermal growth factor receptor (EGFR) and the insulin-like growth factor-1 receptor (IGF-1R) play critical roles in tumor growth, providing a strong rationale for the combined inhibition of IGF-1R and EGFR signaling in cancer therapy. We describe the design, affinity maturation, in vitro and in vivo characterization of the bispecific anti-IGF-1R/EGFR antibody XGFR*. XGFR* is based on the bispecific IgG antibody XGFR, which enabled heterodimerization of an IGF-1R binding scFab heavy chain with an EGFR-binding light and heavy chain by the “knobs-into-holes” technology. XGFR* is optimized for monovalent binding of human EGFR and IGF-1R with increased binding affinity for IGF-1R due to affinity maturation and highly improved protein stability to oxidative and thermal stress. It bears an afucosylated Fc-portion for optimal induction of antibody-dependent cell-mediated cytotoxicity (ADCC). Stable Chinese hamster ovary cell clones with production yields of 2–3 g/L were generated, allowing for large scale production of the bispecific antibody. XGFR* potently inhibits EGFR- and IGF-1R-dependent receptor phosphorylation, reduces tumor cell proliferation in cells with heterogeneous levels of IGF-1R and EGFR receptor expression and induces strong ADCC in vitro. A comparison of pancreatic and colorectal cancer lines demonstrated superior responsiveness to XGFR*-mediated signaling and tumor growth inhibition in pancreatic cancers that frequently show a high degree of IGF-1R/EGFR co-expression. XGFR* showed potent anti-tumoral efficacy in the orthotopic MiaPaCa-2 pancreatic xenograft model, resulting in nearly complete tumor growth inhibition with significant number of tumor remissions. In summary, the bispecific anti-IGF-1R/EGFR antibody XGFR* combines potent signaling and tumor growth inhibition with enhanced ADCC induction and represents a clinical development candidate for the treatment of pancreatic cancer. PMID:26984378

  12. Novel neutralizing hedgehog antibody MEDI-5304 exhibits antitumor activity by inhibiting paracrine hedgehog signaling.

    PubMed

    Michaud, Neil R; Wang, Youzhen; McEachern, Kristen A; Jordan, Jerold J; Mazzola, Anne Marie; Hernandez, Axel; Jalla, Sanjoo; Chesebrough, Jon W; Hynes, Mark J; Belmonte, Matthew A; Wang, Lidong; Kang, Jaspal S; Jovanovic, Jelena; Laing, Naomi; Jenkins, David W; Hurt, Elaine; Liang, Meina; Frantz, Christopher; Hollingsworth, Robert E; Simeone, Diane M; Blakey, David C; Bedian, Vahe

    2014-02-01

    The hedgehog pathway has been implicated in the tumorigenesis, tumor progression, and metastasis of numerous human cancers. We generated the first fully human hedgehog antibody MEDI-5304 and characterized its antitumor activity and preclinical toxicology. MEDI-5304 bound sonic hedgehog (SHH) and Indian hedgehog (IHH) with low picomolar affinity and neutralized SHH and IHH activity in cellular mGLI1 reporter assays. The antibody inhibited transcription of hedgehog target genes and osteoblast differentiation of C3H10T1/2 cells. We evaluated the activity of MEDI-5304 in vivo in model systems that allowed us to evaluate two primary hypotheses of hedgehog function in human cancer, paracrine signaling between tumor and stromal cells and cancer stem cell (CSC) self-renewal. MEDI-5304 displayed robust pharmacodynamic effects in stromal cells that translated to antitumor efficacy as a single agent in an HT-29/MEF coimplantation model of paracrine hedgehog signaling. MEDI-5304 also improved responses to carboplatin in the HT-29/MEF model. The antibody, however, had no effect as a single agent or in combination with gemcitabine on the CSC frequency or growth of several primary pancreatic cancer explant models. These findings support the conclusion that hedgehog contributes to tumor biology via paracrine tumor-stromal signaling but not via CSC maintenance or propagation. Finally, the only safety study finding associated with MEDI-5304 was ondontodysplasia in rats. Thus, MEDI-5304 represents a potent dual hedgehog inhibitor suitable for continued development to evaluate efficacy and safety in human patients with tumors harboring elevated levels of SHH or IHH.

  13. Suxiao Jiuxin Pill Induces Potent Relaxation and Inhibition on Contraction in Human Artery and the Mechanism

    PubMed Central

    Bai, Xiao-Yan; Zhang, Ping; Yang, Qin; Liu, Xiao-Cheng; Wang, Jun; Tong, Yong-Ling; Xiong, Song-Jin; Liu, Li-Hua; Wang, Lei; He, Guo-Wei

    2014-01-01

    Suxiao Jiuxin Pill, a compound Chinese traditional medicine with main components of tetramethylpyrazine and borneol, is widely used for antiangina treatment in China but its pharmacological effect on human blood vessels is unknown. We investigated the effect and possible mechanism of SJP in the human internal mammary artery (IMA, n = 78) taken from patients undergoing coronary surgery. SJP caused full relaxation in KCl- (99.4 ± 10.5%, n = 6) and U46619- (99.9 ± 5.6%, n = 6) contracted IMA. Pretreatment of IMA with plasma concentrations of SJP (1 mg/mL), calculated from the plasma concentration of its major component borneol, significantly depressed the maximal contraction to KCl (from 35.8 ± 6.0 mN to 12.6 ± 5.6 mN, P = 0.03) and U46619 (from 19.4 ± 2.9 mN to 5.7 ± 2.4 mN, P = 0.007) while SJP at 10 mg/mL abolished the subsequent contraction. Endothelium denudation and inhibition of eNOS significantly altered the SJP-induced relaxation without changes of eNOS expression. We conclude that SJP has a potent inhibitory effect on the vasoconstriction mediated by a variety of vasoconstrictors in human arteries. The vasorelaxation involves both endothelium-dependent and -independent mechanisms. Thus, the effect of SJP on human arteries demonstrated in this study may prove to be particularly important in vasorelaxing therapy in cardiovascular disease. PMID:24808920

  14. Soy extract is more potent than genistein on tumor growth inhibition.

    PubMed

    Kim, Hyeon-A; Jeong, Kyu-Shik; Kim, Yoo Kyeong

    2008-01-01

    Soybean and soy products have received much attention for their potential heath benefits. Recently it has been reported that the bioactivity of soy products is influenced by the degree of soy processing. This study was conducted to evaluate and compare the influence of diets containing genistein and soy extract on the growth of the estrogen-independent human breast cancer cells, MDA-MB-231, implanted into female Balb/c mice. Four-week-old female athymic nude mice (Balb/c) were acclimatized to an AIN-93G control diet for one week prior to initiating the experimental diets. The animals were placed into three treatment groups, each of which was provided with containing DMSO, genistein (750 microg/g AIN-93G diet) or 0.6% soy extract (containing genistein at 750 microg/g AIN-93G diet) for three weeks from one week prior to the injection of MDA-MB-231 cells (1 x 10(6)/site) and subsequently fed on the AIN-93G control diet until sacrifice. The tumor volumes increased steeply in the control group and the genistein-treated group. However, tumor growth was significantly reduced in the soy extract-treated group compared to the control and genistein-treated groups. Immunohistochemistry of proliferating cell nuclear antigen (PCNA) also revealed that the soy extract treatment effectively reduced cell proliferation of the implanted tumors. In conclusion, soy extract is more potent than genistein in the inhibition of tumor growth, presumably resulting from the synergistic effect of the various bioactive components in the soy extract.

  15. A chalcone with potent inhibiting activity against biofilm formation by nontypeable Haemophilus influenzae.

    PubMed

    Kunthalert, Duangkamol; Baothong, Sudarat; Khetkam, Pichit; Chokchaisiri, Suwadee; Suksamrarn, Apichart

    2014-10-01

    Nontypeable Haemophilus influenzae (NTHi), an important human respiratory pathogen, frequently causes biofilm infections. Currently, resistance of bacteria within the biofilm to conventional antimicrobials poses a major obstacle to effective medical treatment on a global scale. Novel agents that are effective against NTHi biofilm are therefore urgently required. In this study, a series of natural and synthetic chalcones with various chemical substituents were evaluated in vitro for their antibiofilm activities against strong biofilm-forming strains of NTHi. Of the test chalcones, 3-hydroxychalcone (chalcone 8) exhibited the most potent inhibitory activity, its mean minimum biofilm inhibitory concentration (MBIC50 ) being 16 μg/mL (71.35 μM), or approximately sixfold more active than the reference drug, azithromycin (MBIC50 419.68 μM). The inhibitory activity of chalcone 8, which is a chemically modified chalcone, appeared to be superior to those of the natural chalcones tested. Significantly, chalcone 8 inhibited biofilm formation by all studied NTHi strains, indicating that the antibiofilm activities of this compound occur across multiple strong-biofilm forming NTHi isolates of different clinical origins. According to antimicrobial and growth curve assays, chalcone 8 at concentrations that decreased biofilm formation did not affect growth of NTHi, suggesting the biofilm inhibitory effect of chalcone 8 is non-antimicrobial. In terms of structure-activity relationship, the possible substituent on the chalcone backbone required for antibiofilm activity is discussed. These findings indicate that 3-hydroxychalcone (chalcone 8) has powerful antibiofilm activity and suggest the potential application of chalcone 8 as a new therapeutic agent for control of NTHi biofilm-associated infections.

  16. Nanomolecular HLA-DR10 antibody mimics: A potent system for molecular targeted therapy and imaging.

    PubMed

    DeNardo, Gerald L; Natarajan, Arutselvan; Hok, Saphon; Mirick, Gary; DeNardo, Sally J; Corzett, Michele; Sysko, Vladimir; Lehmann, Joerg; Beckett, Laurel; Balhorn, Rod

    2008-12-01

    To mimic the molecular specificity and cell selectivity of monoclonal antibody (mAb) binding while decreasing size, nanomolecules (selective high-affinity ligands; SHALs), based on in silico modeling, have been created to bind to human leukocyte antigen-DR (HLA-DR10), a signaling receptor protein upregulated on the malignant B-lymphocytes of non-Hodgkin's lymphoma and chronic lymphocytic leukemia. SHALs were synthesized with a biotin or DOTA chelate (1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid), using a solid-phase lysine-polyethyleneglycol backbone to link sets of ligands shown previously to bind to HLA-DR10. Using cell-binding and death assays and confocal microscopy, SHAL uptake, residualization, and cytocidal activity were evaluated in HLA-DR10 expressing and nonexpressing live, human lymphoma cell lines. All of the SHALs tested were selective for, and accumulated in, expressing cells. Reflecting binding to HLA-DR10 inside the cells, SHALs having the Ct ligand (3-(2-([3-chloro-5-trifluoromethyl)-2-pyridinyl]oxy)-anilino)-3-oxopropanionic acid) residualized in expressing cells greater than 179 times more than accountable by cell-surface membrane HLA-DR10. Confocal microscopy confirmed the intracellular residualization of these SHALs. Importantly, SHALs with a Ct ligand had direct cytocidal activity, similar in potency to that of Lym-1 mAb and rituximab, selectively for HLA-DR10 expressing lymphoma cells and xenografts. The results show that SHALs containing the Ct ligand residualize intracellularly and have cytocidal effects mediated by HLA-DR10. These SHALs have extraordinary potential as novel molecules for the selective targeting of lymphoma and leukemia for molecular therapy and imaging. Further, these SHALs can be used to transport and residualize cytotoxic agents near critical sites inside these malignant cells.

  17. Halogenated pyrrolopyrimidine analogues of adenosine from marine organisms: pharmacological activities and potent inhibition of adenosine kinase.

    PubMed

    Davies, L P; Jamieson, D D; Baird-Lambert, J A; Kazlauskas, R

    1984-02-01

    Two novel halogenated pyrrolopyrimidine analogues of adenosine, isolated from marine sources, have been examined for pharmacological and biochemical activities. 4-Amino-5-bromo-pyrrolo[2,3-d]pyrimidine, from a sponge of the genus Echinodictyum, had bronchodilator activity at least as potent as theophylline but with a different biochemical profile; unlike theophylline it had no antagonist activity at CNS adenosine receptors and it was quite a potent inhibitor of adenosine uptake and adenosine kinase in brain tissue. 5'-Deoxy-5-iodotubercidin, isolated from the red alga Hypnea valentiae, caused potent muscle relaxation and hypothermia when injected into mice. This compound was a very potent inhibitor of adenosine uptake into rat and guinea-pig brain slices and an extremely potent inhibitor of adenosine kinase from guinea-pig brain and rat brain and liver. Neither of these two pyrrolopyrimidine analogues was a substrate for, or an inhibitor of, adenosine deaminase. Neither compound appeared to have any direct agonist activity on guinea-pig brain adenosine-stimulated adenylate cyclase (A2 adenosine receptors). 5'-Deoxy-5-iodotubercidin is unique in two respects: it appears to be the first naturally-occurring example of a 5'-deoxyribosyl nucleoside and is the first example of a specifically iodinated nucleoside from natural sources. It may be the most potent adenosine kinase inhibitor yet described and, by virtue of its structure, may prove to be the most specific.

  18. Inhibition of CD73 AMP hydrolysis by a therapeutic antibody with a dual, non-competitive mechanism of action

    PubMed Central

    Geoghegan, James C.; Diedrich, Gundo; Lu, Xiaojun; Rosenthal, Kim; Sachsenmeier, Kris F.; Wu, Herren; Dall'Acqua, William F.; Damschroder, Melissa M.

    2016-01-01

    ABSTRACT CD73 (ecto-5′-nucleotidase) has recently been established as a promising immuno-oncology target. Given its role in activating purinergic signaling pathways to elicit immune suppression, antagonizing CD73 (i.e., releasing the brake) offers a complimentary pathway to inducing anti-tumor immune responses. Here, we describe the mechanistic activity of a new clinical therapeutic, MEDI9447, a human monoclonal antibody that non-competitively inhibits CD73 activity. Epitope mapping, structural, and mechanistic studies revealed that MEDI9447 antagonizes CD73 through dual mechanisms of inter-CD73 dimer crosslinking and/or steric blocking that prevent CD73 from adopting a catalytically active conformation. To our knowledge, this is the first report of an antibody that inhibits an enzyme's function through 2 distinct modes of action. These results provide a finely mapped epitope that can be targeted for selective, potent, and non-competitive inhibition of CD73, as well as establish a strategy for inhibiting enzymes that function in both membrane-bound and soluble states. PMID:26854859

  19. Comparison of growth inhibition of a human ovarian adenocarcinoma cell line by free monoclonal antibodies and their corresponding antibody-recombinant ricin A chain immunotoxins.

    PubMed

    Ettenson, D; Sheldon, K; Marks, A; Houston, L L; Baumal, R

    1988-01-01

    Four mouse monoclonal antibodies (mAb) (8C, IgG2a; M2A, IgG2a; M2D, IgG2b; 10B, IgG1) directed against the human ovarian adenocarcinoma cell line HEY were compared for their ability in the free form and as immunotoxins made with recombinant ricin A chain (rRA) to inhibit the growth of HEY cells. For in vitro studies cultured HEY cells were assayed for protein synthesis and plated in agarose to form colonies, and for in vivo studies they were injected intraperitoneally (i.p.) into BALB/c nu/nu (nude) mice at a challenge dose (3 X 10(5) cells) which produced carcinomatosis with ascites, leading to death 30 days following injection. In the free form, mAB 8C was the most potent in inhibiting colony formation in the complement (C)-mediated and ADCC (antibody-dependent cell-mediated cytoxicity) assays in vitro. This mAb was also the only one capable of prolonging survival of mice, both in tumor cell neutralization, and tumor growth inhibition experiments. The four mAb-rRA immunotoxins were effective in inhibiting protein synthesis in vitro in the presence of 10(-7) M monensin. However, in vivo, only 8C-rRA and M2A-rRA were capable of prolonging survival of mice in tumor growth inhibition experiments. Our results suggest that mAb 8C might be useful in the free form and as an 8C-rRA immunotoxin for i.p. immunotherapy of ovarian cancer.

  20. Artemisinin-derived dimer ART-838 potently inhibited human acute leukemias, persisted in vivo, and synergized with antileukemic drugs

    PubMed Central

    Fox, Jennifer M.; Moynihan, James R.; Mott, Bryan T.; Mazzone, Jennifer R.; Anders, Nicole M.; Brown, Patrick A.; Rudek, Michelle A.; Liu, Jun O.; Arav-Boger, Ravit; Posner, Gary H.

    2016-01-01

    Artemisinins, endoperoxide-containing molecules, best known as antimalarials, have potent antineoplastic activity. The established antimalarial, artesunate (AS), and the novel artemisinin-derived trioxane diphenylphosphate dimer 838 (ART-838) inhibited growth of all 23 tested acute leukemia cell lines, reduced cell proliferation and clonogenicity, induced apoptosis, and increased intracellular levels of reactive oxygen species (ROS). ART-838 was 88-fold more potent that AS in vitro, inhibiting all leukemia cell lines at submicromolar concentrations. Both ART-838 and AS cooperated with several established antileukemic drugs and newer kinase inhibitors to inhibit leukemia cell growth. ART-838 had a longer plasma half-life than AS in immunodeficient NOD-SCID-IL2Rgnull (NSG) mice, remaining at effective antileukemic concentrations for >8h. Intermittent cycles of ART-838 inhibited growth of acute leukemia xenografts and primagrafts in NSG mice, at higher potency than AS. Based on these preclinical data, we propose that AS, with its established low toxicity and low cost, and ART-838, with its higher potency and longer persistence in vivo, should be further developed toward integration into antileukemic regimens. PMID:26771236

  1. Chemical or genetic Pin1 inhibition exerts potent anticancer activity against hepatocellular carcinoma by blocking multiple cancer-driving pathways

    PubMed Central

    Liao, Xin-Hua; Zhang, Arina Li; Zheng, Min; Li, Mei-Qing; Chen, Champ Peng; Xu, Huijuan; Chu, Qing-Song; Yang, Dayun; Lu, Wenxian; Tsai, Ting-Fen; Liu, Hekun; Zhou, Xiao Zhen; Lu, Kun Ping

    2017-01-01

    Hepatocellular carcinoma (HCC) is one of the most prevalent and malignant cancers with high inter- and intra-tumor heterogeneity. A central common signaling mechanism in cancer is proline-directed phosphorylation, which is further regulated by the unique proline isomerase Pin1. Pin1 is prevalently overexpressed in human cancers including ~70% of HCC, and promotes tumorigenesis by activating multiple cancer-driving pathways. However, it was challenging to evaluate the significance of targeting Pin1 in cancer treatment until the recent identification of all-trans retinoic acid (ATRA) as a Pin1 inhibitor. Here we systematically investigate functions of Pin1 and its inhibitor ATRA in the development and treatment of HCC. Pin1 knockdown potently inhibited HCC cell proliferation and tumor growth in mice. ATRA-induced Pin1 degradation inhibited the growth of HCC cells, although at a higher IC50 as compared with breast cancer cells, likely due to more active ATRA metabolism in liver cells. Indeed, inhibition of ATRA metabolism enhanced the sensitivity of HCC cells to ATRA. Moreover, slow-releasing ATRA potently and dose-dependently inhibited HCC growth in mice. Finally, chemical or genetic Pin1 ablation blocked multiple cancer-driving pathways simultaneously in HCC cells. Thus, targeting Pin1 offers a promising therapeutic approach to simultaneously stop multiple cancer-driving pathways in HCC. PMID:28262728

  2. Artemisinin-derived dimer ART-838 potently inhibited human acute leukemias, persisted in vivo, and synergized with antileukemic drugs.

    PubMed

    Fox, Jennifer M; Moynihan, James R; Mott, Bryan T; Mazzone, Jennifer R; Anders, Nicole M; Brown, Patrick A; Rudek, Michelle A; Liu, Jun O; Arav-Boger, Ravit; Posner, Gary H; Civin, Curt I; Chen, Xiaochun

    2016-02-09

    Artemisinins, endoperoxide-containing molecules, best known as antimalarials, have potent antineoplastic activity. The established antimalarial, artesunate (AS), and the novel artemisinin-derived trioxane diphenylphosphate dimer 838 (ART-838) inhibited growth of all 23 tested acute leukemia cell lines, reduced cell proliferation and clonogenicity, induced apoptosis, and increased intracellular levels of reactive oxygen species (ROS). ART-838 was 88-fold more potent that AS in vitro, inhibiting all leukemia cell lines at submicromolar concentrations. Both ART-838 and AS cooperated with several established antileukemic drugs and newer kinase inhibitors to inhibit leukemia cell growth. ART-838 had a longer plasma half-life than AS in immunodeficient NOD-SCID-IL2Rgnull (NSG) mice, remaining at effective antileukemic concentrations for >8h. Intermittent cycles of ART-838 inhibited growth of acute leukemia xenografts and primagrafts in NSG mice, at higher potency than AS. Based on these preclinical data, we propose that AS, with its established low toxicity and low cost, and ART-838, with its higher potency and longer persistence in vivo, should be further developed toward integration into antileukemic regimens.

  3. Neutralizing Antibodies Induced by Recombinant Virus-Like Particles of Enterovirus 71 Genotype C4 Inhibit Infection at Pre- and Post-attachment Steps

    PubMed Central

    Ku, Zhiqiang; Ye, Xiaohua; Huang, Xulin; Cai, Yicun; Liu, Qingwei; Li, Yan; Su, Zhiguo; Huang, Zhong

    2013-01-01

    Background Enterovirus 71 (EV71) is a major causative agent of hand, foot and mouth disease, which has been prevalent in Asia–Pacific regions, causing significant morbidity and mortality in young children. Antibodies elicited by experimental EV71 vaccines could neutralize infection in vitro and passively protect animal models from lethal challenge, indicating that neutralizing antibodies play an essential role in protection. However, how neutralizing antibodies inhibit infection in vitro remains unclear. Methods/Findings In the present study, we explored the mechanisms of neutralization by antibodies against EV71 virus-like particles (VLPs). Recombinant VLPs of EV71 genotype C4 were produced in insect cells using baculovirus vectors. Immunization with the VLPs elicited a high-titer, EV71-specific antibody response in mice. Anti-VLP mouse sera potently neutralized EV71 infection in vitro. The neutralizing antibodies in the anti-VLP mouse sera were found to target mainly an extremely conserved epitope (FGEHKQEKDLEYGAC) located at the GH loop of the VP1 protein. The neutralizing anti-VLP antisera were able to inhibit virus binding to target cells efficiently. In addition, post-attachment treatment of virus-bound cells with the anti-VLP antisera also neutralized virus infection, although the antibody concentration required was higher than that of the pre-attachment treatment. Conclusions Collectively, our findings represent a valuable addition to the understanding of mechanisms of EV71 neutralization and have strong implications for EV71 vaccine development. PMID:23451250

  4. Inhibition and Reversal of Microbial Attachment by an Antibody with Parasteric Activity against the FimH Adhesin of Uropathogenic E. coli

    PubMed Central

    Friend, Della; Jalan, Aachal; Gupta, Shivani; Interlandi, Gianluca; Liu, Yan; Tchesnokova, Veronika; Rodriguez, Victoria B.; Sumida, John P.; Strong, Roland K.; Wu, Xue-Ru; Thomas, Wendy E.; Sokurenko, Evgeni V.

    2015-01-01

    Attachment proteins from the surface of eukaryotic cells, bacteria and viruses are critical receptors in cell adhesion or signaling and are primary targets for the development of vaccines and therapeutic antibodies. It is proposed that the ligand-binding pocket in receptor proteins can shift between inactive and active conformations with weak and strong ligand-binding capability, respectively. Here, using monoclonal antibodies against a vaccine target protein - fimbrial adhesin FimH of uropathogenic Escherichia coli, we demonstrate that unusually strong receptor inhibition can be achieved by antibody that binds within the binding pocket and displaces the ligand in a non-competitive way. The non-competitive antibody binds to a loop that interacts with the ligand in the active conformation of the pocket but is shifted away from ligand in the inactive conformation. We refer to this as a parasteric inhibition, where the inhibitor binds adjacent to the ligand in the binding pocket. We showed that the receptor-blocking mechanism of parasteric antibody differs from that of orthosteric inhibition, where the inhibitor replaces the ligand or allosteric inhibition where the inhibitor binds at a site distant from the ligand, and is very potent in blocking bacterial adhesion, dissolving surface-adherent biofilms and protecting mice from urinary bladder infection. PMID:25974133

  5. Potent inhibition of retinoic acid metabolism enzyme(s) by novel azolyl retinoids.

    PubMed

    Njar, V C; Nnane, I P; Brodie, A M

    2000-09-04

    Novel (+/-)-4-azolyl retinoic acid analogues 4, 5, 7 and 8 have been designed and synthesized and have been shown to be powerful inhibitors of hamster microsomal all-trans-retinoic acid 4-hydroxylase enzyme(s). (+/-)-4-(1H-Imidazol-1-yl)retinoic acid (4) is the most potent inhibitor of this enzyme reported to date.

  6. Inhibition of breast cancer resistance protein (ABCG2) in human myeloid dendritic cells induces potent tolerogenic functions during LPS stimulation.

    PubMed

    Jin, Jun-O; Zhang, Wei; Wong, Ka-Wing; Kwak, Minseok; van Driel, Ian R; Yu, Qing

    2014-01-01

    Breast cancer resistance protein (ABCG2), a member of the ATP-binding cassette transporters has been identified as a major determinant of multidrug resistance (MDR) in cancer cells, but ABC transporter inhibition has limited therapeutic value in vivo. In this research, we demonstrated that inhibition of efflux transporters ABCG2 induced the generation of tolerogenic DCs from human peripheral blood myeloid DCs (mDCs). ABCG2 expression was present in mDCs and was further increased by LPS stimulation. Treatment of CD1c+ mDCs with an ABCG2 inhibitor, Ko143, during LPS stimulation caused increased production of IL-10 and decreased production of pro-inflammatory cytokines and decreased expression of CD83 and CD86. Moreover, inhibition of ABCG2 in monocyte-derived DCs (MDDCs) abrogated the up-regulation of co-stimulatory molecules and production of pro-inflammatory cytokines in these cells in response to LPS. Furthermore, CD1c+ mDCs stimulated with LPS plus Ko143 inhibited the proliferation of allogeneic and superantigen-specific syngenic CD4+ T cells and promoted expansion of CD25+FOXP3+ regulatory T (Treg) cells in an IL-10-dependent fashion. These tolerogenic effects of ABCG2 inhibition could be abolished by ERK inhibition. Thus, we demonstrated that inhibition of ABCG2 in LPS-stimulated mDCs can potently induce tolerogenic potentials in these cells, providing crucial new information that could lead to development of better strategies to combat MDR cancer.

  7. Targeted In Vivo Inhibition of Specific Protein–Protein Interactions Using Recombinant Antibodies

    PubMed Central

    Zábrady, Matej; Hrdinová, Vendula; Müller, Bruno; Conrad, Udo; Hejátko, Jan; Janda, Lubomír

    2014-01-01

    With the growing availability of genomic sequence information, there is an increasing need for gene function analysis. Antibody-mediated “silencing” represents an intriguing alternative for the precise inhibition of a particular function of biomolecules. Here, we describe a method for selecting recombinant antibodies with a specific purpose in mind, which is to inhibit intrinsic protein–protein interactions in the cytosol of plant cells. Experimental procedures were designed for conveniently evaluating desired properties of recombinant antibodies in consecutive steps. Our selection method was successfully used to develop a recombinant antibody inhibiting the interaction of ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 3 with such of its upstream interaction partners as the receiver domain of CYTOKININ INDEPENDENT HISTIDINE KINASE 1. The specific down-regulation of the cytokinin signaling pathway in vivo demonstrates the validity of our approach. This selection method can serve as a prototype for developing unique recombinant antibodies able to interfere with virtually any biomolecule in the living cell. PMID:25299686

  8. Potent inhibition of HIV type 1 infection of mononuclear phagocytes by synthetic peptide analogs of HIV type 1 protease substrates.

    PubMed

    Dukes, C S; Matthews, T J; Lambert, D M; Dreyer, G B; Petteway, S R; Weinberg, J B

    1996-06-10

    The HIV-1 genome encodes a protease that is required for viral processing of the precursor polyproteins Pr55gag and Pr160gag-pol. Interference with this process in human lymphocytes inhibits production of infectious virus. We tested the ability of several protease inhibitors to decrease replication of HIV-1BaL in human monocytes and peritoneal macrophages. The compounds tested are oligopeptide analogs of HIV-1 protease substrates in which the scissile dipeptide has been replaced by a hydroxyethylene isostere. The protease inhibitors were added only once, 1 hr prior to inoculation with virus. Every 3-5 days, half the medium was replaced with fresh medium. Inhibition of virus production was assessed by measuring reverse transcriptase (RT) activity in supernatant medium 14 days after infection. The concentration of drug required to inhibit infection by 50% (IC50) in monocytes ranged from 0.17 to 2.99 microM; IC50 values for peritoneal macrophages ranged from 0.21 to 1.9 microM. The IC50 values for these compounds were 1.1- to 10-fold higher when tested in monocytes compared to their inhibitory effect in lymphocytes, although still potently effective in the dosage range that appeared nontoxic to cells. Cell toxicity was seen only at concentrations greater than 10 microM, and varied among the drugs tested. Immunoblot analysis of two of the drugs (SB205700 and SB108922) confirmed inhibition of polyprotein processing. In control cells, 22% of viral protein pr55 was processed to p24 by 24 hr, and 51% was processed by 48 hr. In cells treated with the protease inhibitors (2 microM), Pr55 processing was inhibited 77% at 24 hr and 89% at 48 hr. Thus, these synthetic peptide analogs potently inhibit productive infection of mononuclear phagocytes by HIV-1. Drugs of this class may be useful for the treatment of HIV-1 infection in humans.

  9. Newcastle disease viruses in birds in the Atlantic flyway: isolations, haemagglutination-inhibition and elution-inhibition antibody profiles.

    PubMed

    Graves, I L

    1996-01-01

    The study involved 15 avian species with 5,012 attempts to isolate Newcastle disease viruses (NDV) from their faeces over a three-year period (1977-1979). NDV were isolated from asymptomatic adult Canada geese, nestling Royal terms, a juvenile European Mute swan, and adult Tundra swans on the Eastern flyway. Ring-billed gulls were negative for haemagglutination-inhibition (HI), elution-inhibition (EI) (anti-neuraminidase) antibodies, and NDV despite 3,403 isolation attempts. The EI antibody assay used a strain isolated from a Mute swan. The prevalence of EI antibodies in the swan and geese ranged from 3-41%, while the geometric mean titre (GMT) varied from 20-36. The prevalence of HI antibodies in the swans and geese ranged from 4-62%, while the GMT varied from 16-42. In each of three years (1977-1979), the adult Mute swans had a higher HI antibody prevalence than the juveniles (P < 0.01). Among the Mute swans the HI and EI assays detected serologic conversions and persistent antibodies over the three-year period. The HI and EI assays were effective in showing differences in antibody prevalences in populations of feral birds of different species and age. The EI assay is applicable for population studies of the anti-neuraminidase antibody.

  10. An Anti-Human Lutheran Glycoprotein Phage Antibody Inhibits Cell Migration on Laminin-511: Epitope Mapping of the Antibody

    PubMed Central

    Enomoto-Okawa, Yurie; Maeda, Yuka; Harashima, Nozomi; Sugawara, Yumika; Katagiri, Fumihiko; Hozumi, Kentaro; Hui, Kam Man; Nomizu, Motoyoshi; Ito, Yuji; Kikkawa, Yamato

    2017-01-01

    The Lutheran glycoprotein (Lu), also known as basal cell adhesion molecule (B-CAM), is an Ig superfamily (IgSF) transmembrane receptor for laminin α5. Although Lu is not present in normal hepatocytes, its expression is significantly increased in hepatocellular carcinoma (HCC). In this study, we isolated thirteen phage antibodies to Lu from a phage library of peripheral blood from HCC patients, suggesting that these patients produced autoantibodies against endogenous Lu. To characterize the phage antibodies, we determined the Lu domains they recognize. The extracellular domain of Lu contains five IgSF domains, D1-D2-D3-D4-D5. The epitope of one phage antibody (A7) was localized to the D5 domain. The other phage antibodies recognized the D2 domain, which is also recognized by a function blocking mouse monoclonal antibody. One of the antibodies to D2 (C7) inhibited the binding of Lu to ligand, and it also prevented tumor cell migration on laminin-511 (LM-511). However, the C7 scFv purified from the periplasm fraction of bacteria did not exhibit the inhibitory effects, indicating that the scFv form could not sterically inhibit the binding of Lu to LM-511. We also identified the amino acid residues that form the epitope recognized by the C7 phage antibody. Mutagenesis studies showed that Arg247 is necessary for forming the epitope. The C7 phage antibody and its epitope may be useful for developing drugs to prevent HCC progression and/or metastasis. PMID:28060819

  11. Core-Scaffold-Inspired Asymmetric Synthesis of Polysubstituted Chiral Hexahydropyridazines that Potently Inhibit Breast Cancer Cell Proliferation by Inducing Apoptosis.

    PubMed

    Leng, Hai-Jun; Peng, Fu; Zingales, Sarah; Huang, Wei; Wang, Biao; Zhao, Qian; Zhou, Rui; He, Gu; Peng, Cheng; Han, Bo

    2015-12-07

    The highly enantioselective preparation of pharmacologically interesting hexahydropyridazine derivatives based on a multicomponent cascade reaction is described. This one-pot approach utilizes an organocatalytic Michael reaction followed by intermolecular α-amination and intramolecular hemiaminalization to yield a chiral pyridazine backbone with contiguous stereogenic centers and multiple functional groups in good yield and with high stereoselectivity. Compounds synthesized by this method potently inhibited proliferation of MCF-7 breast cancer cells. Mechanistic studies suggest that compound 5 c exerts these anticancer effects by inducing apoptosis through extracellular signal related kinase (ERK)- and poly(adenosine diphosphate ribose) polymerase (PARP)-regulated pathways, as well as mitochondrial pathways.

  12. Frequent Use of the IgA Isotype in Human B Cells Encoding Potent Norovirus-Specific Monoclonal Antibodies That Block HBGA Binding

    PubMed Central

    Shanker, Sreejesh; Prasad, B. V. Venkataram; Atmar, Robert L.; Estes, Mary K.; Crowe, James E.

    2016-01-01

    Noroviruses (NoV) are the most common cause of non-bacterial acute gastroenteritis and cause local outbreaks of illness, especially in confined situations. Despite being identified four decades ago, the correlates of protection against norovirus gastroenteritis are still being elucidated. Recent studies have shown an association of protection with NoV-specific serum histo-blood group antigen-blocking antibody and with serum IgA in patients vaccinated with NoV VLPs. Here, we describe the isolation and characterization of human monoclonal IgG and IgA antibodies against a GI.I NoV, Norwalk virus (NV). A higher proportion of the IgA antibodies blocked NV VLP binding to glycans than did IgG antibodies. We generated isotype-switched variants of IgG and IgA antibodies to study the effects of the constant domain on blocking and binding activities. The IgA form of antibodies appears to be more potent than the IgG form in blocking norovirus binding to histo-blood group antigens. These studies suggest a unique role for IgA antibodies in protection from NoV infections by blocking attachment to cell receptors. PMID:27355511

  13. The anti-erbB3 antibody MM-121/SAR256212 in combination with trastuzumab exerts potent antitumor activity against trastuzumab-resistant breast cancer cells

    PubMed Central

    2013-01-01

    Background Elevated expression of erbB3 receptor has been reported to induce resistance to therapeutic agents, including trastuzumab in erbB2-overexpressing breast cancer. Our recent studies indicate that erbB3 interacts with both erbB2 and IGF-1 receptor to form a heterotrimeric complex in trastuzumab-resistant breast cancer cells. Herein, we investigate the antitumor activity of MM-121/SAR256212, a fully human anti-erbB3 antibody (Ab), against two erbB2-overexpressing breast cancer cell lines resistant to trastuzumab. Methods MTS-based proliferation assays were used to determine cell viability upon treatment of trastuzumab and/or MM-121/SAR256212. Cell cycle progression was examined by flow cytometric analysis. Western blot analyses were performed to determine the expression and activation of proteins. Tumor xenografts were established by inoculation of the trastuzumab-resistant BT474-HR20 cells into nude mice. The tumor-bearing mice were treated with trastuzumab and/or MM-121/SAR256212 via i.p injection to determine the Abs’ antitumor activity. Immunohistochemical analyses were carried out to study the Abs’ inhibitory effects on tumor cell proliferation and induction of apoptosis in vivo. Results MM-121 significantly enhanced trastuzumab-induced growth inhibition in two sensitive and two resistant breast cancer cell lines. MM-121 in combination with trastuzumab resulted in a dramatic reduction of phosphorylated erbB3 (P-erbB3) and Akt (P-Akt) in the in vitro studies. MM-121 combined with trastuzumab did not induce apoptosis in the trastuzumab-resistant cell lines under our cell culture condition, rather induced cell cycle G1 arrest mainly associated with the upregulation of p27kip1. Interestingly, in the tumor xenograft model established from the trastuzumab-resistant cells, MM-121 in combination with trastuzumab as compared to either agent alone dramatically inhibited tumor growth correlated with a significant reduction of Ki67 staining and increase of

  14. CCR7 expressing mesenchymal stem cells potently inhibit graft-versus-host disease by spoiling the fourth supplemental Billingham's tenet.

    PubMed

    Li, Hong; Jiang, Yan-Ming; Sun, Yan-Feng; Li, Ping; Dang, Rui-Jie; Ning, Hong-Mei; Li, Yu-Hang; Zhang, Ying-Jie; Jiang, Xiao-Xia; Guo, Xi-Min; Wen, Ning; Han, Yan; Mao, Ning; Chen, Hu; Zhang, Yi

    2014-01-01

    The clinical acute graft-versus-host disease (GvHD)-therapy of mesenchymal stem cells (MSCs) is not as satisfactory as expected. Secondary lymphoid organs (SLOs) are the major niches serve to initiate immune responses or induce tolerance. Our previous study showed that CCR7 guide murine MSC line C3H10T1/2 migrating to SLOs. In this study, CCR7 gene was engineered into murine MSCs by lentivirus transfection system (MSCs/CCR7). The immunomodulatory mechanism of MSCs/CCR7 was further investigated. Provoked by inflammatory cytokines, MSCs/CCR7 increased the secretion of nitric oxide and calmed down the T cell immune response in vitro. Immunofluorescent staining results showed that transfused MSCs/CCR7 can migrate to and relocate at the appropriate T cell-rich zones within SLOs in vivo. MSCs/CCR7 displayed enhanced effect in prolonging the survival and alleviating the clinical scores of the GvHD mice than normal MSCs. Owing to the critical relocation sites, MSCs/CCR7 co-infusion potently made the T cells in SLOs more naïve like, thus control T cells trafficking from SLOs to the target organs. Through spoiling the fourth supplemental Billingham's tenet, MSCs/CCR7 potently inhibited the development of GvHD. The study here provides a novel therapeutic strategy of MSCs/CCR7 infusion at a low dosage to give potent immunomodulatory effect for clinical immune disease therapy.

  15. Antibody-mediated inhibition of ricin toxin retrograde transport.

    PubMed

    Yermakova, Anastasiya; Klokk, Tove Irene; Cole, Richard; Sandvig, Kirsten; Mantis, Nicholas J

    2014-04-08

    Ricin is a member of the ubiquitous family of plant and bacterial AB toxins that gain entry into the cytosol of host cells through receptor-mediated endocytosis and retrograde traffic through the trans-Golgi network (TGN) and endoplasmic reticulum (ER). While a few ricin toxin-specific neutralizing monoclonal antibodies (MAbs) have been identified, the mechanisms by which these antibodies prevent toxin-induced cell death are largely unknown. Using immunofluorescence confocal microscopy and a TGN-specific sulfation assay, we demonstrate that 24B11, a MAb against ricin's binding subunit (RTB), associates with ricin in solution or when prebound to cell surfaces and then markedly enhances toxin uptake into host cells. Following endocytosis, however, toxin-antibody complexes failed to reach the TGN; instead, they were shunted to Rab7-positive late endosomes and LAMP-1-positive lysosomes. Monovalent 24B11 Fab fragments also interfered with toxin retrograde transport, indicating that neither cross-linking of membrane glycoproteins/glycolipids nor the recently identified intracellular Fc receptor is required to derail ricin en route to the TGN. Identification of the mechanism(s) by which antibodies like 24B11 neutralize ricin will advance our fundamental understanding of protein trafficking in mammalian cells and may lead to the discovery of new classes of toxin inhibitors and therapeutics for biodefense and emerging infectious diseases. IMPORTANCE Ricin is the prototypic member of the AB family of medically important plant and bacterial toxins that includes cholera and Shiga toxins. Ricin is also a category B biothreat agent. Despite ongoing efforts to develop vaccines and antibody-based therapeutics against ricin, very little is known about the mechanisms by which antibodies neutralize this toxin. In general, it is thought that antibodies simply prevent toxins from attaching to cell surface receptors or promote their clearance through Fc receptor (FcR)-mediated uptake

  16. Insulin action is blocked by a monoclonal antibody that inhibits insulin receptor kinase

    SciTech Connect

    Morgan, D.O.; Ho, L.; Korn, L.J.; Roth, R.A.

    1986-01-01

    Thirty-six monoclonal antibodies to the human insulin receptor were produced. Thirty-four bound the intracellular domain of the receptor ..beta.. subunit, the domain containing the tyrosine-specific kinase activity. Of these 34 antibodies, 33 recognized the rat receptor and 1 was shown to precipitate the receptors from mice, chickens and frogs with high affinity. Another of the antibodies inhibited the kinase activities of the human and frog receptors with equal potencies. This antibody inhibited the kinase activities of these receptors by more than 90%, whereas others had no effect on either kinase activity. Microinjection of the inhibiting antibody into Xenopus oocytes blocked the ability of insulin to stimulate oocyte maturation. In contrast, this inhibiting antibody did not block the ability of progesterone to stimulate the same response. Furthermore, control immunoglobulin and a noninhibiting antibody to the receptor ..beta.. subunit did not block this response to insulin. These results strongly support a role for the tyrosine-specific kinase activity of the insulin receptor in mediating this biological effect of insulin.

  17. Monoclonal Antibodies to the Apical Chloride Channel in Necturus Gallbladder Inhibit the Chloride Conductance

    NASA Astrophysics Data System (ADS)

    Finn, Arthur L.; Tsai, Lih-Min; Falk, Ronald J.

    1989-10-01

    Monoclonal antibodies raised by injecting Necturus gallbladder cells into mice were tested for their ability to inhibit the apical chloride conductance induced by elevation of cellular cAMP. Five of these monoclonal antibodies bound to the apical cells, as shown by indirect immunofluorescence microscopy, and inhibited the chloride conductance; one antibody that bound only to subepithelial smooth muscle, by indirect immunofluorescence microscopy, showed no inhibition of chloride transport. The channel or a closely related molecule is present in the membrane whether or not the pathway is open, since, in addition to inhibiting the conductance of the open channel, the antibody also bound to the membrane in the resting state and prevented subsequent opening of the channel. The antibody was shown to recognize, by ELISA, epitopes from the Necturus gallbladder and small intestine. Finally, by Western blot analysis of Necturus gallbladder homogenates, the antibody was shown to recognize two protein bands of Mr 219,000 and Mr 69,000. This antibody should permit isolation and characterization of this important ion channel.

  18. Insulin Action is Blocked by a Monoclonal Antibody That Inhibits the Insulin Receptor Kinase

    NASA Astrophysics Data System (ADS)

    Morgan, David O.; Ho, Lisa; Korn, Laurence J.; Roth, Richard A.

    1986-01-01

    Thirty-six monoclonal antibodies to the human insulin receptor were produced. Thirty-four bound the intracellular domain of the receptor β subunit, the domain containing the tyrosine-specific kinase activity. Of these 34 antibodies, 33 recognized the rat receptor and 1 was shown to precipitate the receptors from mice, chickens, and frogs with high affinity. Another of the antibodies inhibited the kinase activities of the human and frog receptors with equal potencies. This antibody inhibited the kinase activities of these receptors by more than 90%, whereas others had no effect on either kinase activity. Microinjection of the inhibiting antibody into Xenopus oocytes blocked the ability of insulin to stimulate oocyte maturation. In contrast, this inhibiting antibody did not block the ability of progesterone to stimulate the same response. Furthermore, control immunoglobulin and a noninhibiting antibody to the receptor β subunit did not block this response to insulin. These results strongly support a role for the tyrosine-specific kinase activity of the insulin receptor in mediating this biological effect of insulin.

  19. Potent inhibition of alcohol self-administration in alcohol-preferring rats by a κ-opioid receptor antagonist.

    PubMed

    Cashman, John R; Azar, Marc R

    2014-07-01

    A substituted aryl amide derivative of 6-naltrexamine--17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(4'-trimethylfluoro)benzamido]morphinan-hydrochloride--(compound 5), previously shown to be a potent κ-opioid receptor antagonist, was used to characterize the physicochemical properties and efficacy to decrease alcohol self-administration in alcohol-preferring rats (P-rats) and binge-like P-rats. Previous studies showed that compounds closely related to compound 5 possessed favorable properties regarding penetration of the blood-brain barrier. Pharmacokinetic studies showed that compound 5 had acceptable bioavailability. In contrast to other κ-receptor antagonists, in particular norbinaltorphimine, compound 5 showed favorable drug-like properties. Based on these findings, further studies were done. Safety studies showed that compound 5 was not hepatotoxic at doses 200-fold greater than an efficacious dose. The effects of compound 5 or naltrexone on the hepatotoxicity of thiobenzamide were investigated. In contrast to naltrexone, which exacerbated thiobenzamide-mediated hepatotoxicity, compound 5 was observed to be hepatoprotective. Based on the physicochemical properties of compound 5, the compound was examined in rat animal models of alcohol self-administration. The inhibition of ethanol self-administration by compound 5 in alcohol-dependent and alcohol-nondependent P-rats trained to self-administer a 10% (w/v) ethanol solution, using operant techniques, showed very potent efficacy (i.e., estimated ED50 values of 4-5 μg/kg). In a binge-like P-rat animal model, inhibition of alcohol self-administration by compound 5 had an estimated ED50 value of 8 μg/kg. The results suggest that compound 5 is a potent drug-like κ-opioid receptor antagonist of utility in alcohol cessation medications development.

  20. Monoclonal antibodies against the native urease of Helicobacter pylori: synergistic inhibition of urease activity by monoclonal antibody combinations.

    PubMed Central

    Nagata, K; Mizuta, T; Tonokatu, Y; Fukuda, Y; Okamura, H; Hayashi, T; Shimoyama, T; Tamura, T

    1992-01-01

    Monoclonal antibodies (MAbs) against the native urease of Helicobacter pylori NCTC 11637 were found to clearly inhibit the urease activity. Interestingly, synergistic inhibition by two MAbs recognizing different subunits was also observed. Ten MAbs were produced and classified as two isotypes of the immunoglobulin G (IgG) subclass, IgG1, and IgG2a. Western blot (immunoblot) analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that five MAbs recognized the large subunit and the other five recognized the small subunit of the urease. Among the MAbs, L2 and S2, which recognized the large and the small subunits, respectively, were also able to inhibit the urease activity of clinical isolates from H. pylori-infected patients. The combination of L2 and S2 led to augmented synergistic inhibition. L2, but not S2, could also inhibit the urease activity from Helicobacter mustelae; enzyme-linked immunosorbent assay and Western blot analysis showed that L2 cross-reacted with this urease. These results suggested that the epitope recognized by L2 had a structure common to both Helicobacter species and may be involved in the active site of the urease. In contrast to the MAbs, a polyclonal antibody in sera from mice immunized with H. pylori urease did not have the ability to inhibit H. pylori urease activity. However, the polyclonal antibody retained the ability to abolish the inhibitory action of these MAbs. Moreover, other MAbs which could not inhibit H. pylori urease activity also abolished the inhibitory action. Images PMID:1383158

  1. Antigenic Characterization of the HCMV gH/gL/gO and Pentamer Cell Entry Complexes Reveals Binding Sites for Potently Neutralizing Human Antibodies

    PubMed Central

    Ciferri, Claudio; Chandramouli, Sumana; Leitner, Alexander; Donnarumma, Danilo; Cianfrocco, Michael A.; Gerrein, Rachel; Friedrich, Kristian; Aggarwal, Yukti; Palladino, Giuseppe; Aebersold, Ruedi; Norais, Nathalie; Settembre, Ethan C.; Carfi, Andrea

    2015-01-01

    Human Cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and in fetuses following congenital infection. The glycoprotein complexes gH/gL/gO and gH/gL/UL128/UL130/UL131A (Pentamer) are required for HCMV entry in fibroblasts and endothelial/epithelial cells, respectively, and are targeted by potently neutralizing antibodies in the infected host. Using purified soluble forms of gH/gL/gO and Pentamer as well as a panel of naturally elicited human monoclonal antibodies, we determined the location of key neutralizing epitopes on the gH/gL/gO and Pentamer surfaces. Mass Spectrometry (MS) coupled to Chemical Crosslinking or to Hydrogen Deuterium Exchange was used to define residues that are either in proximity or part of neutralizing epitopes on the glycoprotein complexes. We also determined the molecular architecture of the gH/gL/gO- and Pentamer-antibody complexes by Electron Microscopy (EM) and 3D reconstructions. The EM analysis revealed that the Pentamer specific neutralizing antibodies bind to two opposite surfaces of the complex, suggesting that they may neutralize infection by different mechanisms. Together, our data identify the location of neutralizing antibodies binding sites on the gH/gL/gO and Pentamer complexes and provide a framework for the development of antibodies and vaccines against HCMV. PMID:26485028

  2. A new and potent calmodulin antagonist, HF-2035, which inhibits vascular relaxation induced by nitric oxide synthase.

    PubMed

    Win, N H; Ishikawa, T; Saito, N; Kato, M; Yokokura, H; Watanabe, Y; Iida, Y; Hidaka, H

    1996-03-28

    HF-2035, 2-[N-(2-aminoethyl)-N-(2,4,5-trichlorobenzenesulfonyl)] amino-N-(4-chlorocinnamyl)-N-methylbenzylamine, was synthesized and its effects on calmodulin-dependent enzymes were investigated. HF-2035 inhibited calmodulin kinase I, calmodulin kinase II and myosin light-chain kinase with IC50 values of 1.3 microM, 1.6 microM and 68 microM, respectively. HF-2035 also inhibited the activity of recombinant rat neuronal nitric oxide synthase, one of the calmodulin-dependent enzymes, with a Ki of 0.78 microM. Partially purified nitric oxide synthase of rat brain was also inhibited by HF-2035 with an IC50 of 3.2 microM. Kinetic analysis indicated that this inhibitory effect of HF-2035 was competitive with respect to calmodulin. We examined the effects of HF-2035 on constitutive nitric oxide synthase in a bioassay using vascular strips of rabbit carotid artery with and without endothelium. HF-2035 inhibited acetylcholine- and calcium ionophore, A23187 (6S-[6 alpha (2S*,3S*),8 beta (R*),9 beta, 11 alpha]-5- (methylamino)-2-[[3,9,11-trimethyl-8-[1-methyl-2-oxo-2-(1H-pyrrol-2-yl)- ethyl]-1,7-dioxaspiro[5.5]undec-2-yl]methyl]-4-benzoxazol ecarboxylic acid)-induced relaxation of endothelium-intact strips with an ED50 of 1.5 +/- 0.5 microM and 2.8 +/- 1 microM, respectively. This compound, however, did not inhibit N-nitroso-N-morpholinoaminoacetonitrile (SIN-1A), an exogenous nitric oxide donor, -induced relaxation of endothelium-denuded strips. W-7 (N-(6-aminohexyl)-5-chloro-1- naphthalenesulfonamide) inhibited acetylcholine-induced relaxation with an ED50 of 46 +/- 7 microM, which was 30-fold less potent than HF-2035. HF-2035 was unable to inhibit the activity of the inducible form of nitric oxide synthase in isolated thoracic aorta of rat treated with Escherichia coli lipopolysaccharide. These findings suggest that HF-2035 is a new and potent calmodulin antagonist, and may be used as a mother compound to develop more selective inhibitors of constitutive nitric oxide

  3. Potent Human α-Amylase Inhibition by the β-Defensin-like Protein Helianthamide

    PubMed Central

    2016-01-01

    Selective inhibitors of human pancreatic α-amylase (HPA) are an effective means of controlling blood sugar levels in the management of diabetes. A high-throughput screen of marine natural product extracts led to the identification of a potent (Ki = 10 pM) peptidic HPA inhibitor, helianthamide, from the Caribbean sea anemone Stichodactyla helianthus. Active helianthamide was produced in Escherichia coli via secretion as a barnase fusion protein. X-ray crystallographic analysis of the complex of helianthamide with porcine pancreatic α-amylase revealed that helianthamide adopts a β-defensin fold and binds into and across the amylase active site, utilizing a contiguous YIYH inhibitory motif. Helianthamide represents the first of a novel class of glycosidase inhibitors and provides an unusual example of functional malleability of the β-defensin fold, which is rarely seen outside of its traditional role in antimicrobial peptides. PMID:27066537

  4. Potent Human α-Amylase Inhibition by the β-Defensin-like Protein Helianthamide.

    PubMed

    Tysoe, Christina; Williams, Leslie K; Keyzers, Robert; Nguyen, Nham T; Tarling, Chris; Wicki, Jacqueline; Goddard-Borger, Ethan D; Aguda, Adeleke H; Perry, Suzanne; Foster, Leonard J; Andersen, Raymond J; Brayer, Gary D; Withers, Stephen G

    2016-03-23

    Selective inhibitors of human pancreatic α-amylase (HPA) are an effective means of controlling blood sugar levels in the management of diabetes. A high-throughput screen of marine natural product extracts led to the identification of a potent (Ki = 10 pM) peptidic HPA inhibitor, helianthamide, from the Caribbean sea anemone Stichodactyla helianthus. Active helianthamide was produced in Escherichia coli via secretion as a barnase fusion protein. X-ray crystallographic analysis of the complex of helianthamide with porcine pancreatic α-amylase revealed that helianthamide adopts a β-defensin fold and binds into and across the amylase active site, utilizing a contiguous YIYH inhibitory motif. Helianthamide represents the first of a novel class of glycosidase inhibitors and provides an unusual example of functional malleability of the β-defensin fold, which is rarely seen outside of its traditional role in antimicrobial peptides.

  5. Targeting membrane-bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus.

    PubMed

    Lundin, Anna; Dijkman, Ronald; Bergström, Tomas; Kann, Nina; Adamiak, Beata; Hannoun, Charles; Kindler, Eveline; Jónsdóttir, Hulda R; Muth, Doreen; Kint, Joeri; Forlenza, Maria; Müller, Marcel A; Drosten, Christian; Thiel, Volker; Trybala, Edward

    2014-05-01

    Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs), a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6), a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV), and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections.

  6. 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.

  7. Antibody inhibition of polymorphonuclear phagocytosis. Dissociation of bacterial attachment and bacterial killing.

    PubMed

    Crowley, J P; Valeri, C R

    1980-06-01

    The inhibition of killing of Staphylococcus aureus 502A by PMNs treated with the IgG fraction of serum from a group of patients with demonstrable leukocyte antibodies was investigated. The uptake of opsonized thymidine-labeled S. aureus 502A by PMNs treated with allogeneic antibody was essentially unimpaired, despite significantly decreased killing. The findings were similar to bacteria opsonized by serum complement or bacteria opsonized with specific lapine antibody. An increased proportion of PMN-bound bacteria susceptible to lysis by lysostaphin indicated a reduced rate of translocation of bacteria from the surface of allogeneic antibody-treated PMNs. Antibody did not stimulate the basal oxidative metabolism, but the oxidative metabolism of antibody-treated PMNs during phagocytosis was increased. Although the precise mechanism of inhibition of PMN killing by antibody is uncertain, the data suggest that the impairment of bacterial killing by PMNs treated with allogeneic leukocyte antibody is associated with inefficient translocation of bacteria into phagolysosomes rather than by interference with the binding of bacteria to specific PMN opsonic receptors.

  8. Anti-MUC1 antibody inhibits EGF receptor signaling in cancer cells

    SciTech Connect

    Hisatsune, Akinori; Nakayama, Hideki; Kawasaki, Mitsuru; Horie, Ichiro; Miyata, Takeshi; Isohama, Yoichiro; Kim, Kwang Chul; Katsuki, Hiroshi

    2011-02-18

    Research highlights: {yields} We identified changes in the expression and function of EGFR by anti-MUC1 antibody. {yields} An anti-MUC1 antibody GP1.4 decreased EGFR from cell surface by internalization. {yields} GP1.4 specifically inhibited ERK signaling triggered EGF-EGFR signaling pathway. {yields} Internalization of EGFR was dependent on the presence of MUC1 on cell surface. {yields} GP1.4 significantly inhibited EGF-dependent cancer cell proliferation and migration. -- Abstract: MUC1 is a type I transmembrane glycoprotein aberrantly overexpressed in various cancer cells. High expression of MUC1 is closely associated with cancer progression and metastasis, leading to poor prognosis. We previously reported that MUC1 is internalized by the binding of the anti-MUC1 antibody, from the cell surface to the intracellular region via the macropinocytotic pathway. Since MUC1 is closely associated with ErbBs, such as EGF receptor (EGFR) in cancer cells, we examined the effect of the anti-MUC1 antibody on EGFR trafficking. Our results show that: (1) anti-MUC1 antibody GP1.4, but not another anti-MUC1 antibody C595, triggered the internalization of EGFR in pancreatic cancer cells; (2) internalization of EGFR by GP1.4 resulted in the inhibition of ERK phosphorylation by EGF stimulation, in a MUC1 dependent manner; (3) inhibition of ERK phosphorylation by GP1.4 resulted in the suppression of proliferation and migration of pancreatic cancer cells. We conclude that the internalization of EGFR by anti-MUC1 antibody GP1.4 inhibits the progression of cancer cells via the inhibition of EGFR signaling.

  9. Differential Inhibition of Human Atherosclerotic Plaque–Induced Platelet Activation by Dimeric GPVI-Fc and Anti-GPVI Antibodies

    PubMed Central

    Jamasbi, Janina; Megens, Remco T.A.; Bianchini, Mariaelvy; Münch, Götz; Ungerer, Martin; Faussner, Alexander; Sherman, Shachar; Walker, Adam; Goyal, Pankaj; Jung, Stephanie; Brandl, Richard; Weber, Christian; Lorenz, Reinhard; Farndale, Richard; Elia, Natalie; Siess, Wolfgang

    2015-01-01

    Background Glycoprotein VI (GPVI) is the essential platelet collagen receptor in atherothrombosis, but its inhibition causes only a mild bleeding tendency. Thus, targeting this receptor has selective antithrombotic potential. Objectives This study sought to compare compounds interfering with platelet GPVI–atherosclerotic plaque interaction to improve current antiatherothrombotic therapy. Methods Human atherosclerotic plaque–induced platelet aggregation was measured in anticoagulated blood under static and arterial flow conditions (550/s, 1,100/s, and 1,500/s). Inhibition by dimeric GPVI fragment crystallizable region of IgG (Fc) masking GPVI binding sites on collagen was compared with that of 3 anti-GPVI antibodies: BLO8-1, a human domain antibody; 5C4, a fragment antigen-binding (Fab fragment) of monoclonal rat immunoglobulin G; and m-Fab-F, a human recombinant sFab against GPVI dimers. Results GPVI-Fc reduced plaque-triggered platelet aggregation in static blood by 51%, BLO8-1 by 88%, and 5C4 by 93%. Under arterial flow conditions, BLO8-1 and 5C4 almost completely inhibited platelet aggregation while preserving platelet adhesion on plaque. Inhibition by GPVI-Fc, even at high concentrations, was less marked but increased with shear rate. Advanced optical imaging revealed rapid persistent GPVI-Fc binding to collagen under low and high shear flow, upstream and downstream of plaque fragments. At low shear particularly, platelets adhered in plaque flow niches to GPVI-Fc–free segments of collagen fibers and recruited other platelets onto aggregates via ADP and TxA2 release. Conclusions Anti-GPVI antibodies inhibit atherosclerotic plaque-induced platelet aggregation under static and flow conditions more effectively than GPVI-Fc. However, potent platelet inhibition by GPVI-Fc at a higher shear rate (1,500/s) suggests localized antithrombotic efficacy at denuded or fissured stenotic high-risk lesions without systemic bleeding. The compound-specific differences

  10. Structure and function of broadly reactive antibody PG16 reveal an H3 subdomain that mediates potent neutralization of HIV-1

    SciTech Connect

    Pejchal, Robert; Walker, Laura M.; Stanfield, Robyn L.; Phogat, Sanjay K.; Koff, Wayne C.; Poignard, Pascal; Burton, Dennis R.; Wilson, Ian A.

    2010-11-15

    Development of an effective vaccine against HIV-1 will likely require elicitation of broad and potent neutralizing antibodies against the trimeric surface envelope glycoprotein (Env). Monoclonal antibodies (mAbs) PG9 and PG16 neutralize {approx}80% of HIV-1 isolates across all clades with extraordinary potency and target novel epitopes preferentially expressed on Env trimers. As these neutralization properties are ideal for a vaccine-elicited antibody response to HIV-1, their structural basis was investigated. The crystal structure of the antigen-binding fragment (Fab) of PG16 at 2.5 {angstrom} resolution revealed its unusually long, 28-residue, complementarity determining region (CDR) H3 forms a unique, stable subdomain that towers above the antibody surface. A 7-residue 'specificity loop' on the 'hammerhead' subdomain was identified that, when transplanted from PG16 to PG9 and vice versa, accounted for differences in the fine specificity and neutralization of these two mAbs. The PG16 electron density maps also revealed that a CDR H3 tyrosine was sulfated, which was confirmed for both PG9 (doubly) and PG16 (singly) by mass spectral analysis. We further showed that tyrosine sulfation plays a role in binding and neutralization. An N-linked glycan modification is observed in the variable light chain, but not required for antigen recognition. Further, the crystal structure of the PG9 light chain at 3.0 {angstrom} facilitated homology modeling to support the presence of these unusual features in PG9. Thus, PG9 and PG16 use unique structural features to mediate potent neutralization of HIV-1 that may be of utility in antibody engineering and for high-affinity recognition of a variety of therapeutic targets.

  11. IMGN853, a Folate Receptor-α (FRα)-Targeting Antibody-Drug Conjugate, Exhibits Potent Targeted Antitumor Activity against FRα-Expressing Tumors.

    PubMed

    Ab, Olga; Whiteman, Kathleen R; Bartle, Laura M; Sun, Xiuxia; Singh, Rajeeva; Tavares, Daniel; LaBelle, Alyssa; Payne, Gillian; Lutz, Robert J; Pinkas, Jan; Goldmacher, Victor S; Chittenden, Thomas; Lambert, John M

    2015-07-01

    A majority of ovarian and non-small cell lung adenocarcinoma cancers overexpress folate receptor α (FRα). Here, we report the development of an anti-FRα antibody-drug conjugate (ADC), consisting of a FRα-binding antibody attached to a highly potent maytansinoid that induces cell-cycle arrest and cell death by targeting microtubules. From screening a large panel of anti-FRα monoclonal antibodies, we selected the humanized antibody M9346A as the best antibody for targeted delivery of a maytansinoid payload into FRα-positive cells. We compared M9346A conjugates with various linker/maytansinoid combinations, and found that a conjugate, now denoted as IMGN853, with the N-succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB) linker and N(2')-deacetyl-N(2')-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4) exhibited the most potent antitumor activity in several FRα-expressing xenograft tumor models. The level of expression of FRα on the surface of cells was a major determinant in the sensitivity of tumor cells to the cytotoxic effect of the conjugate. Efficacy studies of IMGN853 in xenografts of ovarian cancer and non-small cell lung cancer cell lines and of a patient tumor-derived xenograft model demonstrated that the ADC was highly active against tumors that expressed FRα at levels similar to those found on a large fraction of ovarian and non-small cell lung cancer patient tumors, as assessed by immunohistochemistry. IMGN853 displayed cytotoxic activity against FRα-negative cells situated near FRα-positive cells (bystander cytotoxic activity), indicating its ability to eradicate tumors with heterogeneous expression of FRα. Together, these findings support the clinical development of IMGN853 as a novel targeted therapy for patients with FRα-expressing tumors.

  12. Production of mouse monoclonal antibodies which inhibit in vitro adherence of Entamoeba histolytica trophozoites.

    PubMed Central

    Ravdin, J I; Petri, W A; Murphy, C F; Smith, R D

    1986-01-01

    Adherence by axenic Entamoeba histolytica trophozoites to mammalian cells is mediated by an N-acetylgalactosamine (GalNAc)-inhibitable adhesin on the surface of the parasite. We isolated 35 hybridoma cell lines producing antibodies to E. histolytica as indicated by ELISA with sonicated amebic protein or by immunofluorescence assay with fixed whole trophozoites. Tissue culture supernatants were further screened for subcloning by the ability to bind to Chinese hamster ovary (CHO) cells which were first exposed to a partially purified soluble preparation of the amebic GalNAc-inhibitable lectin. Eight tissue culture supernatants were positive in this assay. Antibodies from four subcloned cell lines (D3-14, H8-5, I12-2, and I1-21) inhibited amebic adherence to CHO cells (P less than 0.01). Of the original 35 tissue culture supernatants, 3 also inhibited amebic adherence (P less than 0.01; F1, F14, and J10); monoclonal antibodies in these supernatants did not bind to lectin-exposed CHO cells. Three purified monoclonal antibodies (H8-5, I12-2, and I1-21) inhibited amebic adherence at greater than or equal to 2 micrograms/10(4) amebae (P less than 0.05). None of these inhibitory monoclonal antibodies immunoprecipitated with a soluble amebic protein preparation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis under denaturing conditions. Monoclonal antibodies which inhibit in vitro adherence by E. histolytica will be useful in purification of the GalNAc-inhibitable lectin. PMID:2873102

  13. Potent Inhibition of Junín Virus Infection by Interferon in Murine Cells

    PubMed Central

    Huang, Cheng; Walker, Aida G.; Grant, Ashley M.; Kolokoltsova, Olga A.; Yun, Nadezhda E.; Seregin, Alexey V.; Paessler, Slobodan

    2014-01-01

    The new world arenavirus Junín virus (JUNV) is the causative agent of Argentine hemorrhagic fever, a lethal human infectious disease. Adult laboratory mice are generally resistant to peripheral infection by JUNV. The mechanism underlying the mouse resistance to JUNV infection is largely unknown. We have reported that interferon receptor knockout mice succumb to JUNV infection, indicating the critical role of interferon in restricting JUNV infection in mice. Here we report that the pathogenic and vaccine strains of JUNV were highly sensitive to interferon in murine primary cells. Treatment with low concentrations of interferon abrogated viral NP protein expression in murine cells. The replication of both JUNVs was enhanced in IRF3/IRF7 deficient cells. In addition, the vaccine strain of JUNV displayed impaired growth in primary murine cells. Our data suggested a direct and potent role of host interferon response in restricting JUNV replication in mice. The defect in viral growth for vaccine JUNV might also partially explain its attenuation in mice. PMID:24901990

  14. Potent Inhibition of Enterovirus D68 and Human Rhinoviruses by Dipeptidyl Aldehydes and α-Ketoamides

    PubMed Central

    Kim, Yunjeong; Galasiti Kankanamalage, Anushka C.; Damalanka, Vishnu C.; Weerawarna, Pathum M.; Groutas, William C.; Chang, Kyeong-Ok

    2015-01-01

    Enterovirus D68 (EV-D68) is an emerging pathogen responsible for mild to severe respiratory infections that occur mostly in infants, children and teenagers. EV-D68, one of more than 100 non-polio enteroviruses, is acid-labile and biologically similar to human rhinoviruses (HRV) (originally classified as HRV87). However, there is no approved preventive or therapeutic measure against EV-D68, HRV, or other enteroviruses. In this study, we evaluated the antiviral activity of series of dipeptidyl compounds against EV-D68 and HRV strains, and demonstrated that several peptidyl aldehyde and α-ketoamide peptidyl compounds are potent inhibitors of EV-D68 and HRV strains with high in-vitro therapeutic indices (>1000). One of the α-ketoamide compounds is shown to have favorable pharmacokinetics profiles, including a favorable oral bioavailability in rats. Recent successful development of α-ketoamide protease inhibitors against hepatitis C virus suggests these compounds may have a high potential for further optimization and development against emerging EV-D68, as well as HRV. PMID:26658373

  15. Non-proteinogenic amino acids in lacticin 481 analogues result in more potent inhibition of peptidoglycan transglycosylation.

    PubMed

    Knerr, Patrick J; Oman, Trent J; Garcia De Gonzalo, Chantal V; Lupoli, Tania J; Walker, Suzanne; van der Donk, Wilfred A

    2012-11-16

    Lantibiotics are ribosomally synthesized and post-translationally modified peptide natural products that contain the thioether structures lanthionine and methyllanthionine and exert potent antimicrobial activity against Gram-positive bacteria. At present, detailed modes-of-action are only known for a small subset of family members. Lacticin 481, a tricyclic lantibiotic, contains a lipid II binding motif present in related compounds such as mersacidin and nukacin ISK-1. Here, we show that lacticin 481 inhibits PBP1b-catalyzed peptidoglycan formation. Furthermore, we show that changes in potency of analogues of lacticin 481 containing non-proteinogenic amino acids correlate positively with the potency of inhibition of the transglycosylase activity of PBP1b. Thus, lipid II is the likely target of lacticin 481, and use of non-proteinogenic amino acids resulted in stronger inhibition of the target. Additionally, we demonstrate that lacticin 481 does not form pores in the membranes of susceptible bacteria, a common mode-of-action of other lantibiotics.

  16. Characterization of the variable regions of a chimpanzee monoclonal antibody with potent neutralizing activity against HIV-1.

    PubMed

    Vijh-Warrier, S; Murphy, E; Yokoyama, I; Tilley, S A

    1995-10-01

    The variable (V) regions of C108G, a potent neutralizing chimpanzee mAb against a glycan-dependent epitope in the V2 region of HIV-1 gp120, have been characterized for reactivity with human VH and VK family-specific antisera, and their nucleotide sequences have been determined and analysed. To our knowledge, this is the first study characterizing expressed chimpanzee VH and VK genes. Results show that C108G expresses members of the VH3 and VK1 families, the largest VH and VK families in humans, respectively. Nucleotide and amino acid sequence analyses reveal that C108G VH is most homologous to the human VH3 germline gene, hsigdp33 or V3-43, and the human JH4 minigene. The human germline VK1 gene that is most homologous to C108G VK, hsigk1012, was previously observed in unmutated form in a human autoantibody with anti-i red blood cell antigen specificity and in seven human Fabs and a mAb directed against epitopes overlapping the CD4-binding site of HIV-1 gp120. This germline gene was unmutated in three of the human Fabs and was somatically mutated in the other four Fabs and the mAb. In addition, the JK minigene was used in C108G VK, JK2, is apparently over-represented in anti-HIV-1 mAbs/Fabs; this minigene was used in 61% of the anti-gp120 human Fabs recently described and in three other anti-CD4-binding site human mAbs derived by EBV transformation. While the significance of these findings is unclear, they may suggest a bias in VK/JK gene usage and/or network regulation involving an hsigk1012/JK2 idiotope(s) in the antibody response to HIV-1. Both the C108G VH and VK genes showed evidence of somatic mutation and antigen selection that apparently occurred in vivo during chronic exposure to HIV-1 and its antigens. Surprisingly, this somatic mutation was most profound in the CDR3 region of C108G VK; this region shared only 48% nucleotide homology with hsigk1012 contrasted with a homology of 94% over the remainder of these two V gene sequences. Perhaps the most

  17. Platelet-activating factor acetylhydrolase: selective inhibition by potent n-alkyl methylphosphonofluoridates.

    PubMed

    Quistad, Gary B; Fisher, Karl J; Owen, Sarah C; Klintenberg, Rebecka; Casida, John E

    2005-06-01

    Platelet-activating factor (PAF) is a potent endogenous phospholipid modulator of diverse biological activities, including inflammation and shock. PAF levels are primarily regulated by PAF acetylhydrolases (PAF-AHs). These enzymes are candidate secondary targets of organophosphorus (OP) pesticides and related toxicants. Previously known OP inhibitors of other serine hydrolases were tested with PAF-AH from mouse brain and testes of established functional importance compared with the structurally different human plasma enzyme. Several key OP pesticides and their oxon metabolites were very poor inhibitors of mouse brain and human plasma PAF-AH in vitro but moderately active for mouse brain and blood PAF-AH in vivo (e.g., tribufos defoliant and profenofos insecticide, presumably following oxidative bioactivation). OP compounds were then designed for maximum in vitro potency and selectivity for mouse brain PAF-AH vs. acetylcholinesterase (AChE). Lead compounds were found in a series of benzodioxaphosphorin 2-oxides. Ultrahigh potency and selectivity were achieved with n-alkyl methylphosphonofluoridates (long-chain sarin analogs): mouse brain and testes IC50 < or = 5 nM for C(8)-C(18) analogs and 0.1-0.6 nM for C(13) and C(14) compounds; human plasma IC50 < or = 2 nM for C(13)-C(18) analogs. AChE inhibitory potency decreased as chain length increased with maximum brain PAF-AH/AChE selectivity (>3000-fold) for C(13)-C(18) compounds. The toxicity of i.p.-administered PAF (LD50 ca. 0.5 mg/kg) was increased less than 2-fold by pretreatment with tribufos or the C(13)n-alkyl methylphosphonofluoridate. These studies with a mouse model indicate that PAF-AH is not a major secondary target of OP pesticide poisoning. The optimized PAF-AH inhibitors may facilitate investigations on other aspects of PAF metabolism and action.

  18. Overcoming the Constraints of Anti-HIV/CD89 Bispecific Antibodies That Limit Viral Inhibition

    PubMed Central

    Duval, Mark; Posner, Marshall R.

    2016-01-01

    Innovative strategies are necessary to maximize the clinical application of HIV neutralizing antibodies. To this end, bispecific constructs of human antibody F240, reactive with well-conserved gp41 epitope and antibody 14A8, reactive with the IgA receptor (CD89) on effector cells, were constructed. A F240 × 14A8 bispecific single chain variable region (scFv) molecule was constructed by linking two scFvs using a conventional GGGGS linker. Despite immunoreactivity with HIV gp41 and neutrophils, this bispecific scFv failed to inhibit HIV infection. This is in sharp contrast to viral inhibition using a chemical conjugate of the Fab of these two antibodies. Therefore, we constructed two novel Fab-like bispecific antibody molecules centered on fusion of the IgG1 CH1 domain or CH1-hinge domain to the C-terminus of F240scFv and fusion of the kappa chain CL domain to the C-terminus of 14A8scFv. Both Bi-Fab antibodies showed significant ADCVI activity for multiple clade B and clade C isolates by arming the neutrophils to inhibit HIV infection. The approach presented in this study is unique for HIV immunotherapy in that the impetus of neutralization is to arm and mobilize PMN to destroy HIV and HIV infected cells. PMID:27419146

  19. Potent and Selective Peptide-based Inhibition of the G Protein Gαq.

    PubMed

    Charpentier, Thomas H; Waldo, Gary L; Lowery-Gionta, Emily G; Krajewski, Krzysztof; Strahl, Brian D; Kash, Thomas L; Harden, T Kendall; Sondek, John

    2016-12-02

    In contrast to G protein-coupled receptors, for which chemical and peptidic inhibitors have been extensively explored, few compounds are available that directly modulate heterotrimeric G proteins. Active Gαq binds its two major classes of effectors, the phospholipase C (PLC)-β isozymes and Rho guanine nucleotide exchange factors (RhoGEFs) related to Trio, in a strikingly similar fashion: a continuous helix-turn-helix of the effectors engages Gαq within its canonical binding site consisting of a groove formed between switch II and helix α3. This information was exploited to synthesize peptides that bound active Gαq in vitro with affinities similar to full-length effectors and directly competed with effectors for engagement of Gαq A representative peptide was specific for active Gαq because it did not bind inactive Gαq or other classes of active Gα subunits and did not inhibit the activation of PLC-β3 by Gβ1γ2 In contrast, the peptide robustly prevented activation of PLC-β3 or p63RhoGEF by Gαq; it also prevented G protein-coupled receptor-promoted neuronal depolarization downstream of Gαq in the mouse prefrontal cortex. Moreover, a genetically encoded form of this peptide flanked by fluorescent proteins inhibited Gαq-dependent activation of PLC-β3 at least as effectively as a dominant-negative form of full-length PLC-β3. These attributes suggest that related, cell-penetrating peptides should effectively inhibit active Gαq in cells and that these and genetically encoded sequences may find application as molecular probes, drug leads, and biosensors to monitor the spatiotemporal activation of Gαq in cells.

  20. Method for Extracting Viral Hemagglutination-Inhibiting Antibodies from the Nonspecific Inhibitors of Serum

    PubMed Central

    Altemeier, William A.; Mundon, Francis K.; Top, Franklin H.; Russell, Philip K.

    1970-01-01

    Various methods are used to remove nonspecific inhibitors from sera before titering viral hemagglutination-inhibiting antibodies. These methods have several undesirable features; some are tedious and time-consuming, some remove antibody along with nonspecific inhibitors, and different techniques are usually required to remove the nonspecific inhibitors for different viruses. This communication describes a single method that uses diethylaminoethyl-Sephadex to extract the immunoglobulin G antibodies for several viruses from nonspecific inhibitors. The procedure is fast, simple to perform, and removed the nonspecific inhibitors for influenza, Western equine encephalitis, dengue-2, and rubella viruses. Images PMID:5463576

  1. Adamantyl analogues of paracetamol as potent analgesic drugs via inhibition of TRPA1.

    PubMed

    Fresno, Nieves; Pérez-Fernández, Ruth; Goicoechea, Carlos; Alkorta, Ibon; Fernández-Carvajal, Asia; de la Torre-Martínez, Roberto; Quirce, Susana; Ferrer-Montiel, Antonio; Martín, M Isabel; Goya, Pilar; Elguero, José

    2014-01-01

    Paracetamol also known as acetaminophen, is a widely used analgesic and antipyretic agent. We report the synthesis and biological evaluation of adamantyl analogues of paracetamol with important analgesic properties. The mechanism of nociception of compound 6a/b, an analog of paracetamol, is not exerted through direct interaction with cannabinoid receptors, nor by inhibiting COX. It behaves as an interesting selective TRPA1 channel antagonist, which may be responsible for its analgesic properties, whereas it has no effect on the TRPM8 nor TRPV1 channels. The possibility of replacing a phenyl ring by an adamantyl ring opens new avenues in other fields of medicinal chemistry.

  2. Phenoxy herbicides and fibrates potently inhibit the human chemosensory receptor subunit T1R3

    PubMed Central

    Maillet, Emeline L.; Margolskee, Robert F.; Mosinger, Bedrich

    2009-01-01

    We show that phenoxy-auxin herbicides and lipid-lowering fibrates inhibit human but not rodent T1R3. T1R3 as a co-receptor in taste cells responds to sweet compounds and amino-acids; in endocrine cells of gut and pancreas T1R3 contributes to glucose sensing. Thus, certain effects of fibrates in treating hyperlipidemia and type II diabetes may be via actions on T1R3. Likewise, phenoxy-herbicides may have adverse metabolic effects in humans that would have gone undetected in studies on rodents. PMID:19817384

  3. Adamantyl Analogues of Paracetamol as Potent Analgesic Drugs via Inhibition of TRPA1

    PubMed Central

    Fresno, Nieves; Pérez-Fernández, Ruth; Goicoechea, Carlos; Alkorta, Ibon; Fernández-Carvajal, Asia; de la Torre-Martínez, Roberto; Quirce, Susana; Ferrer-Montiel, Antonio; Martín, M. Isabel; Goya, Pilar; Elguero, José

    2014-01-01

    Paracetamol also known as acetaminophen, is a widely used analgesic and antipyretic agent. We report the synthesis and biological evaluation of adamantyl analogues of paracetamol with important analgesic properties. The mechanism of nociception of compound 6a/b, an analog of paracetamol, is not exerted through direct interaction with cannabinoid receptors, nor by inhibiting COX. It behaves as an interesting selective TRPA1 channel antagonist, which may be responsible for its analgesic properties, whereas it has no effect on the TRPM8 nor TRPV1 channels. The possibility of replacing a phenyl ring by an adamantyl ring opens new avenues in other fields of medicinal chemistry. PMID:25438056

  4. In vitro inhibition of Cryptosporidium parvum infection by human monoclonal antibodies.

    PubMed Central

    Elliot, B C; Wisnewski, A V; Johnson, J; Fenwick-Smith, D; Wiest, P; Hamer, D; Kresina, T; Flanigan, T P

    1997-01-01

    Cryptosporidium parvum infection of the small epithelial intestine causes unremitting diarrhea and malabsorption that can lead to chronic and sometimes fatal illness in patients with AIDS. The illness may be ameliorated by passive oral immunoglobulin therapy. The objective of this study was to produce anti-Cryptosporidium human monoclonal antibodies for evaluation as potential therapy. All human monoclonal cell lines that produced C. parvum antibodies were originally generated from the peripheral blood lymphocytes of a human immunodeficiency virus-seronegative woman. She had recovered from C. parvum infection and had a high specific antibody titer. Hybridization of these lymphocytes with a tumor cell line was accomplished by hypo-osmolar electrofusion. Twelve clones were identified by enzyme-linked immunosorbent assay (ELISA) as secreting anti-Cryptosporidium antibodies after the initial hybridization. From the 12 positive clones, two high antibody-secreting clones, 17A and 17B, were maintained in long-term culture. A second hybridization produced two other human monoclonal cell lines, EC5 and BB2. Human monoclonal antibody from the first two cell lines bound to C. parvum sporozoites and oocysts by immunofluorescence. The ability of human monoclonal antibodies to inhibit C. parvum infection in vitro was assessed by using a human enterocyte cell line, HT29.74. The antibodies of the four different human hybridomas inhibited infection by 35 to 68% (P < 0.05) compared to a control irrelevant human monoclonal antibody derived in a similar fashion. Human monoclonal antibodies are candidate molecules for immunotherapy of C. parvum infection. PMID:9284173

  5. A monoclonal antibody targeting ErbB2 domain III inhibits ErbB2 signaling and suppresses the growth of ErbB2-overexpressing breast tumors

    PubMed Central

    Meng, Y; Zheng, L; Yang, Y; Wang, H; Dong, J; Wang, C; Zhang, Y; Yu, X; Wang, L; Xia, T; Zhang, D; Guo, Y; Li, B

    2016-01-01

    The anti-ErbB2 antibodies trastuzumab and pertuzumab in combination have recently been approved for the treatment of patients with ErbB2-positive metastatic breast cancer. Pertuzumab, which binds to ErbB2 near the center of domain II, and trastuzumab, which binds to the juxtamembrane region of ErbB2 domain IV, directly interfere with domain II- and domain IV-mediated heterodimerization contacts, respectively. In this study, we report a novel anti-ErbB2 antibody, 3E10, which binds to an epitope in domain III that appears to be located opposite to the dimerization interfaces in domain II and domain IV of ErbB2. Our data show that the 3E10 antibody inhibits ErbB2 heterodimerization via a mechanism that strikingly differs from trastuzumab and pertuzumab. It could be speculated that the 3E10 antibody may affect ErbB2 heterodimerization by causing major conformational changes of ErbB2. Furthermore, 3E10 provides synergistic inhibition of ErbB2 heterodimerization and signaling in combination with either trastuzumab or pertuzumab. The combination of these three anti-ErbB2 antibodies that have complementary mechanisms of action appears to be an extremely potent ErbB2 heterodimerization blocker. Compared with trastuzumab plus pertuzumab, the combination of trastuzumab, pertuzumab and 3E10 provides a more potent blockade of ErbB2 signaling. Consistent with this, trastuzumab plus pertuzumab plus 3E10 results in greater in vitro and in vivo antitumor activity in ErbB2-overexpressing breast tumor models, suggesting its potential use for treating ErbB2-overexpressing breast cancer. PMID:26999718

  6. A monoclonal antibody targeting ErbB2 domain III inhibits ErbB2 signaling and suppresses the growth of ErbB2-overexpressing breast tumors.

    PubMed

    Meng, Y; Zheng, L; Yang, Y; Wang, H; Dong, J; Wang, C; Zhang, Y; Yu, X; Wang, L; Xia, T; Zhang, D; Guo, Y; Li, B

    2016-03-21

    The anti-ErbB2 antibodies trastuzumab and pertuzumab in combination have recently been approved for the treatment of patients with ErbB2-positive metastatic breast cancer. Pertuzumab, which binds to ErbB2 near the center of domain II, and trastuzumab, which binds to the juxtamembrane region of ErbB2 domain IV, directly interfere with domain II- and domain IV-mediated heterodimerization contacts, respectively. In this study, we report a novel anti-ErbB2 antibody, 3E10, which binds to an epitope in domain III that appears to be located opposite to the dimerization interfaces in domain II and domain IV of ErbB2. Our data show that the 3E10 antibody inhibits ErbB2 heterodimerization via a mechanism that strikingly differs from trastuzumab and pertuzumab. It could be speculated that the 3E10 antibody may affect ErbB2 heterodimerization by causing major conformational changes of ErbB2. Furthermore, 3E10 provides synergistic inhibition of ErbB2 heterodimerization and signaling in combination with either trastuzumab or pertuzumab. The combination of these three anti-ErbB2 antibodies that have complementary mechanisms of action appears to be an extremely potent ErbB2 heterodimerization blocker. Compared with trastuzumab plus pertuzumab, the combination of trastuzumab, pertuzumab and 3E10 provides a more potent blockade of ErbB2 signaling. Consistent with this, trastuzumab plus pertuzumab plus 3E10 results in greater in vitro and in vivo antitumor activity in ErbB2-overexpressing breast tumor models, suggesting its potential use for treating ErbB2-overexpressing breast cancer.

  7. A-62176, a potent topoisomerase inhibitor, inhibits the expression of human epidermal growth factor receptor 2.

    PubMed

    Kim, Hye-Lin; Jeon, Kyung-Hwa; Jun, Kyu-Yeon; Choi, Yongmun; Kim, Dae-Kee; Na, Younghwa; Kwon, Youngjoo

    2012-12-01

    HER2 overexpression is observed in ∼6-35% of all gastric cancers, while co-amplification of topoisomerase IIα occurs in ∼32-90% of all cancers with HER2 amplification. The present study reports that HER2 expression is down-regulated by A-62176, a fluoroquinophenoxazine derivative that we previously demonstrated to inhibit topoisomerase I and IIα. The results suggest that A-62176 inhibits the interaction between the ESX, an ets transcription factor, and its co-activator Sur2, leading to the attenuation of HER2-mediated phosphorylation of MAPK/Akt. A-62176 arrests the cell cycle in the G1 phase via the down-regulation of cyclin D1 and the up-regulation of p27(Kip1) in NCI-N87 gastric cancer cells. The combination of A-62176 with doxorubicin provides a strong synergistic activity. We propose that A-62176 is a dual inhibitor that impairs the expression of HER2 and restrains the activity of topoisomerase IIα. Our results may lead to the rational design of anticancer molecules targeting a subgroup of gastric cancer cells overexpressing both HER2 and topoisomerase IIα.

  8. Isatin-pyrazole benzenesulfonamide hybrids potently inhibit tumor-associated carbonic anhydrase isoforms IX and XII.

    PubMed

    Ibrahim, Hany S; Abou-Seri, Sahar M; Tanc, Muhammet; Elaasser, Mahmoud M; Abdel-Aziz, Hatem A; Supuran, Claudiu T

    2015-10-20

    New series of benzenesulfonamide derivatives incorporating pyrazole and isatin moieties were prepared using celecoxib as lead molecule. Biological evaluation of the target compounds was performed against the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) and more precisely against the human isoforms hCA I, II (cytosolic), IX and XII (transmembrane, tumor-associated enzymes). Most of the tested compounds efficiently inhibited hCA I, II and IX, with KIs of 2.5-102 nM, being more effective than the reference drug acetazolamide. Compounds 11e, 11f, 16e and 16f were found to inhibit hCA XII with Ki of 3.7, 6.5, 5.4 and 7.2 nM, respectively. Compounds 11e and 16e, with 5-NO2 substitution on the isatin ring, were found to be selective inhibitors of hCA IX and hCA XII. Docking studies revealed that the NO2 group of both compounds participate in interactions with Asp132 within the hCA IX active site, and with residues Lys67 and Asp130 in hCA XII, respectively.

  9. Actions of adenosine A1 and A2 receptor antagonists on CFTR antibody-inhibited β-adrenergic mucin secretion response

    PubMed Central

    Pereira, M M C; Lloyd Mills, C; Dormer, R L; McPherson, M A

    1998-01-01

    The cystic fibrosis gene protein, the cystic fibrosis transmembrane conductance regulator (CFTR) acts as a chloride channel and is a key regulator of mucin secretion. The mechanism by which 3-isobutyl-1-methylxanthine (IBMX) corrects the defect in CFTR mediated β-adrenergic stimulation of mucin secretion has not been determined. The present study has investigated the actions of adenosine A1 and A2 receptor antagonists to determine whether ability to stimulate mucin secretion correlates with correction of CFTR antibody inhibited β-adrenergic response and whether excessive cyclic AMP rise is required.CFTR antibodies were introduced into living rat submandibular acini by hypotonic swelling. Following recovery, mucin secretion in response to isoproterenol was measured.The adenosine A1 receptor antagonist, 8 cyclopentyltheophylline (CPT) was a less potent stimulator of mucin secretion than was the A2 receptor antagonist dimethylpropargylxanthine (DMPX). A concentration of CPT close to the Ki for A1 receptor antagonism (10 nM) did not stimulate mucin secretion.DMPX, although a potent stimulator of mucin secretion, did not correct CFTR antibody inhibited mucin secretion.CPT corrected defective CFTR antibody inhibited mucin secretion at a high (1 mM) concentration, suggesting a mechanism other than adenosine receptor antagonism.DMPX potentiated the isoproterenol induced cyclic AMP rise, whereas CPT did not.Correction of the defective CFTR mucin secretion response did not correlate with ability to stimulate mucin secretion and did not require potentiation of β-adrenergic induced increases in cyclic AMP. This affords real promise for the development of a selective drug treatment for cystic fibrosis. PMID:9831904

  10. Antisense oligodeoxynucleotide inhibition as a potent diagnostic tool for gene function in plant biology

    SciTech Connect

    Jansson, Christer; Sun, Chuanxin; Ghebramedhin, Haile; Hoglund, Anna-Stina; Jansson, Christer

    2008-01-15

    Antisense oligodeoxynucleotide (ODN) inhibition emerges as an effective means for probing gene function in plant cells. Employing this method we have established the importance of the SUSIBA2 transcription factor for regulation of starch synthesis in barley endosperm, and arrived at a model for the role of the SUSIBAs in sugar signaling and source-sink commutation during cereal endosperm development. In this addendum we provide additional data demonstrating the suitability of the antisense ODN technology in studies on starch branching enzyme activities in barley leaves. We also comment on the mechanism for ODN uptake in plant cells. Antisense ODNs are short (12-25 nt-long) stretches of single-stranded ODNs that hybridize to the cognate mRNA in a sequence-specific manner, thereby inhibiting gene expression. They are naturally occurring in both prokaryotes and eukaryotes where they partake in gene regulation and defense against viral infection. The mechanisms for antisense ODN inhibition are not fully understood but it is generally considered that the ODN either sterically interferes with translation or promotes transcript degradation by RNase H activation. The earliest indication of the usefulness of antisense ODN technology for the purposes of molecular biology and medical therapy was the demonstration in 1978 that synthetic ODNs complementary to Raos sarcoma virus could inhibit virus replication in tissue cultures of chick embryo fibroblasts. Since then the antisense ODN technology has been widely used in animal sciences and as an important emerging therapeutic approach in clinical medicine. However, antisense ODN inhibition has been an under-exploited strategy for plant tissues, although the prospects for plant cells in suspension cultures to take up single-stranded ODNs was reported over a decade ago. In 2001, two reports from Malho and coworker demonstrated the use of cationic-complexed antisense ODNs to suppress expression of genes encoding pollen

  11. A human antibody to the CD4 binding site of gp120 capable of highly potent but sporadic cross clade neutralization of primary HIV-1.

    PubMed

    Gach, Johannes S; Quendler, Heribert; Tong, Tommy; Narayan, Kristin M; Du, Sean X; Whalen, Robert G; Binley, James M; Forthal, Donald N; Poignard, Pascal; Zwick, Michael B

    2013-01-01

    Primary isolates of HIV-1 resist neutralization by most antibodies to the CD4 binding site (CD4bs) on gp120 due to occlusion of this site on the trimeric spike. We describe 1F7, a human CD4bs monoclonal antibody that was found to be exceptionally potent against the HIV-1 primary isolate JR-FL. However, 1F7 failed to neutralize a patient-matched primary isolate, JR-CSF even though the two isolates differ by <10% in gp120 at the protein level. In an HIV-1 cross clade panel (n = 157), 1F7 exhibited moderate breadth, but occasionally achieved considerable potency. In binding experiments using monomeric gp120s of select resistant isolates and domain-swap chimeras between JR-FL and JR-CSF, recognition by 1F7 was limited by sequence polymorphisms involving at least the C2 region of Env. Putative N-linked glycosylation site (PNGS) mutations, notably at position 197, allowed 1F7 to neutralize JR-CSF potently without improving binding to the cognate, monomeric gp120. In contrast, flow cytometry experiments using the same PNGS mutants revealed that 1F7 binding is enhanced on cognate trimeric Env. BN-PAGE mobility shift experiments revealed that 1F7 is sensitive to the diagnostic mutation D368R in the CD4 binding loop of gp120. Our data on 1F7 reinforce how exquisitely targeted CD4bs antibodies must be to achieve cross neutralization of two closely related primary isolates. High-resolution analyses of trimeric Env that show the orientation of glycans and polymorphic elements of the CD4bs that affect binding to antibodies like 1F7 are desirable to understand how to promote immunogenicity of more conserved elements of the CD4bs.

  12. Potent and Specific Inhibition of Human Immunodeficiency Virus Type 1 Replication by RNA Interference

    PubMed Central

    Coburn, Glen A.; Cullen, Bryan R.

    2002-01-01

    Synthetic small interfering RNAs (siRNAs) have been shown to induce the degradation of specific mRNA targets in human cells by inducing RNA interference (RNAi). Here, we demonstrate that siRNA duplexes targeted against the essential Tat and Rev regulatory proteins encoded by human immunodeficiency virus type 1 (HIV-1) can specifically block Tat and Rev expression and function. More importantly, we show that these same siRNAs can effectively inhibit HIV-1 gene expression and replication in cell cultures, including those of human T-cell lines and primary lymphocytes. These observations demonstrate that RNAi can effectively block virus replication in human cells and raise the possibility that RNAi could provide an important innate protective response, particularly against viruses that express double-stranded RNAs as part of their replication cycle. PMID:12186906

  13. Miniaturized Growth Inhibition Assay to Assess the Anti-blood Stage Activity of Antibodies.

    PubMed

    Duncan, Elizabeth H; Bergmann-Leitner, Elke S

    2015-01-01

    While no immune correlate for blood-stage specific immunity against Plasmodium falciparum malaria has been identified, there is strong evidence that antibodies directed to various malarial antigens play a crucial role. In an effort to evaluate the role of antibodies in inhibiting growth and/or invasion of erythrocytic stages of the malaria parasite it will be necessary to test large sample sets from Phase 2a/b trials as well as epidemiological studies. The major constraints for such analyses are (1) availability of sufficient sample quantities (especially from infants and small children) and (2) the throughput of standard growth inhibition assays. The method described here assesses growth- and invasion inhibition by measuring the metabolic activity and viability of the parasite (by using a parasite lactate dehydrogenase-specific substrate) in a 384-microtiter plate format. This culture method can be extended beyond the described detection system to accommodate other techniques commonly used for growth/invasion-inhibition.

  14. A Novel Sulindac Derivative That Does Not Inhibit Cyclooxygenases but Potently Inhibits Colon Tumor Cell Growth and Induces Apoptosis with Antitumor Activity

    PubMed Central

    Piazza, Gary A.; Keeton, Adam B.; Tinsley, Heather N.; Gary, Bernard D.; Whitt, Jason D.; Mathew, Bini; Thaiparambil, Jose; Coward, Lori; Gorman, Gregory; Li, Yonghe; Sani, Brahma; Hobrath, Judith V.; Maxuitenko, Yulia Y.; Reynolds, Robert C.

    2011-01-01

    Nonsteroidal anti-inflammatory drugs such as sulindac have shown promising antineoplastic activity, although toxicity from cyclooxygenase (COX) inhibition and the suppression of prostaglandin synthesis limits their use for chemoprevention. Previous studies have concluded that the mechanism responsible for their antineoplastic activity may be COX independent. To selectively design out the COX inhibitory activity of sulindac sulfide (SS), in silico modeling studies were done that revealed the crucial role of the carboxylate moiety for COX-1 and COX-2 binding. These studies prompted the synthesis of a series of SS derivatives with carboxylate modifications that were screened for tumor cell growth and COX inhibitory activity. A SS amide (SSA) with a N,N-dimethylethyl amine substitution was found to lack COX-1 and COX-2 inhibitory activity, yet potently inhibit the growth of human colon tumor cell lines, HT-29, SW480, and HCT116 with IC50 values of 2 to 5 µmol/L compared with 73 to 85 µmol/L for SS. The mechanism of growth inhibition involved the suppression of DNA synthesis and apoptosis induction. Oral administration of SSA was well-tolerated in mice and generated plasma levels that exceeded its in vitro IC50 for tumor growth inhibition. In the human HT-29 colon tumor xenograft mouse model, SSA significantly inhibited tumor growth at a dosage of 250 mg/kg. Combined treatment of SSA with the chemotherapeutic drug, Camptosar, caused a more sustained suppression of tumor growth compared with Camptosar treatment alone. These results indicate that SSA has potential safety and efficacy advantages for colon cancer chemoprevention as well as utility for treating malignant disease if combined with chemotherapy. PMID:19470791

  15. A novel sulindac derivative that does not inhibit cyclooxygenases but potently inhibits colon tumor cell growth and induces apoptosis with antitumor activity.

    PubMed

    Piazza, Gary A; Keeton, Adam B; Tinsley, Heather N; Gary, Bernard D; Whitt, Jason D; Mathew, Bini; Thaiparambil, Jose; Coward, Lori; Gorman, Gregory; Li, Yonghe; Sani, Brahma; Hobrath, Judith V; Maxuitenko, Yulia Y; Reynolds, Robert C

    2009-06-01

    Nonsteroidal anti-inflammatory drugs such as sulindac have shown promising antineoplastic activity, although toxicity from cyclooxygenase (COX) inhibition and the suppression of prostaglandin synthesis limits their use for chemoprevention. Previous studies have concluded that the mechanism responsible for their antineoplastic activity may be COX independent. To selectively design out the COX inhibitory activity of sulindac sulfide (SS), in silico modeling studies were done that revealed the crucial role of the carboxylate moiety for COX-1 and COX-2 binding. These studies prompted the synthesis of a series of SS derivatives with carboxylate modifications that were screened for tumor cell growth and COX inhibitory activity. A SS amide (SSA) with a N,N-dimethylethyl amine substitution was found to lack COX-1 and COX-2 inhibitory activity, yet potently inhibit the growth of human colon tumor cell lines, HT-29, SW480, and HCT116 with IC(50) values of 2 to 5 micromol/L compared with 73 to 85 micromol/L for SS. The mechanism of growth inhibition involved the suppression of DNA synthesis and apoptosis induction. Oral administration of SSA was well-tolerated in mice and generated plasma levels that exceeded its in vitro IC(50) for tumor growth inhibition. In the human HT-29 colon tumor xenograft mouse model, SSA significantly inhibited tumor growth at a dosage of 250 mg/kg. Combined treatment of SSA with the chemotherapeutic drug, Camptosar, caused a more sustained suppression of tumor growth compared with Camptosar treatment alone. These results indicate that SSA has potential safety and efficacy advantages for colon cancer chemoprevention as well as utility for treating malignant disease if combined with chemotherapy.

  16. A novel small molecule agent displays potent anti-myeloma activity by inhibiting the JAK2-STAT3 signaling pathway

    PubMed Central

    Zhu, Jingyu; Xu, Yujia; Wang, Siyu; Xu, Xin; Ji, Peng; Yu, Yang; Cao, Biyin; Han, Kunkun; Hou, Tingjun; Xu, Zhuan; Kong, Yan; Jiang, Gaofeng; Tang, Xiaowen; Qiao, Chunhua; Mao, Xinliang

    2016-01-01

    The oncogenic STAT3 signaling pathway is emerging as a promising target for the treatment of multiple myeloma (MM). In the present study, we identified a novel STAT3 inhibitor SC99 in a target-based high throughput screen. SC99 inhibited JAK2-STAT3 activation but had no effects on other transcription factors such as NF-κB, and kinases such as AKT, ERK, and c-Src that are in association with STAT3 signaling pathway. Furthermore, SC99 downregulated the expression of STAT3-modulated genes, including Bcl-2, Bcl-xL, VEGF, cyclin D2, and E2F-1. By inhibiting the STAT3 signaling, SC99 induced MM cell apoptosis which could be partly abolished by the ectopic expression of STAT3. Furthermore, SC99 displayed potent anti-MM activity in two independent MM xenograft models in nude mice. Oral administration of SC99 led to marked decrease of tumor growth within 10 days at a daily dosage of 30 mg/kg, but did not raise toxic effects. Taken together, this study identified a novel oral JAK2/STAT3 inhibitor that could be developed as an anti-myeloma agent. PMID:26814430

  17. Development and Characterization of a Humanized Anti-HER2 Antibody HuA21 with Potent Anti-Tumor Properties in Breast Cancer Cells

    PubMed Central

    Li, Ruilin; Hu, Siyi; Chang, Yan; Zhang, Zhihui; Zha, Zhao; Huang, Hui; Shen, Guodong; Liu, Jing; Song, Lihua; Wei, Wei

    2016-01-01

    Human epidermal growth factor receptor 2 (HER2) is one of the most studied tumor-associated antigens for cancer immunotherapy. An engineered anti-HER-2 chimeric A21 antibody (chA21) is a chimeric antibody targeted to subdomain I of the HER2 extracellular domain. Here, we report the anti-tumor activity of the novel engineered monoclonal antibody humanized chA21 (HuA21) that targets HER2 on the basis of chA21, and we describe the underlying mechanisms. Our results reveal that HuA21 markedly inhibits the proliferation and migration of HER2-overexpressing breast cancer cells and causes enhanced antibody-dependent cell-mediated cytotoxicity potency against HER2-overexpressing tumor cells. In particular, HuA21, but not trastuzumab (Tra), markedly suppresses growth and enhances the internalization of the antibody in Tra-resistant BT-474 breast cancer cells. These characteristics are highly associated with the intrinsic ability of HuA21 to down-regulate HER2 activation and inhibit the extracellular signal-regulated kinase 1/2 (ERK1/2) and protein kinase B (Akt) signaling pathways. Furthermore, the combination of HuA21 with Tra synergistically enhances the anti-tumor effects in vitro and in vivo and inhibits HER2 activation and the ERK1/2 and Akt signaling pathways. Altogether, our results suggest that HuA21 may represent a unique anti-HER2 antibody with potential as a therapeutic candidate alone or in combination with other anti-HER2 reagents in cancer therapy. PMID:27092488

  18. Development and Characterization of a Humanized Anti-HER2 Antibody HuA21 with Potent Anti-Tumor Properties in Breast Cancer Cells.

    PubMed

    Li, Ruilin; Hu, Siyi; Chang, Yan; Zhang, Zhihui; Zha, Zhao; Huang, Hui; Shen, Guodong; Liu, Jing; Song, Lihua; Wei, Wei

    2016-04-15

    Human epidermal growth factor receptor 2 (HER2) is one of the most studied tumor-associated antigens for cancer immunotherapy. An engineered anti-HER-2 chimeric A21 antibody (chA21) is a chimeric antibody targeted to subdomain I of the HER2 extracellular domain. Here, we report the anti-tumor activity of the novel engineered monoclonal antibody humanized chA21 (HuA21) that targets HER2 on the basis of chA21, and we describe the underlying mechanisms. Our results reveal that HuA21 markedly inhibits the proliferation and migration of HER2-overexpressing breast cancer cells and causes enhanced antibody-dependent cell-mediated cytotoxicity potency against HER2-overexpressing tumor cells. In particular, HuA21, but not trastuzumab (Tra), markedly suppresses growth and enhances the internalization of the antibody in Tra-resistant BT-474 breast cancer cells. These characteristics are highly associated with the intrinsic ability of HuA21 to down-regulate HER2 activation and inhibit the extracellular signal-regulated kinase 1/2 (ERK1/2) and protein kinase B (Akt) signaling pathways. Furthermore, the combination of HuA21 with Tra synergistically enhances the anti-tumor effects in vitro and in vivo and inhibits HER2 activation and the ERK1/2 and Akt signaling pathways. Altogether, our results suggest that HuA21 may represent a unique anti-HER2 antibody with potential as a therapeutic candidate alone or in combination with other anti-HER2 reagents in cancer therapy.

  19. Phyto-power dietary supplement potently inhibits dimethylnitrosamine-induced liver fibrosis in rats.

    PubMed

    Lee, Ming-Fen; Tsai, Mei-Ling; Sun, Pei-Pei; Chien, Ling-Lung; Cheng, An-Chin; Ma, Nianhan Jia-Lin; Ho, Chi-Tang; Pan, Min-Hsiung

    2013-02-26

    Curcumin has been extensively studied for its therapeutic effects in a variety of disorders. Fermented soy consumption is associated with a low incidence rate of chronic diseases in many Asian countries. The aim of this study was to investigate the potential underlying mechanisms of the effect of a phyto-power dietary supplement on liver fibrosis. Sprague-Dawley rats were intraperitoneally injected with dimethylnitrosamine (DMN; 10 mg kg(-1)) three times a week for four consecutive weeks. A phyto-power dietary supplement (50 or 100 mg kg(-1)) was administered by oral gavage daily for four weeks. Liver morphology, function, and fibrotic status were examined in DMN induced hepatic fibrogenesis. However, a phyto-power dietary supplement alleviated liver damage as indicated by histopathological examination of the α-smooth muscle actin (α-SMA) and collagen I, accompanied by the concomitant reduction of transforming growth factor-β1 (TGF-β1) and matrix metalloproteinase 2 (MMP2). These data indicate that the phyto-power dietary supplement may inhibit the TGF-β1/Smad signaling and relieve liver damage in experimental fibrosis.

  20. Tafamidis, a potent and selective transthyretin kinetic stabilizer that inhibits the amyloid cascade

    PubMed Central

    Bulawa, Christine E.; Connelly, Stephen; DeVit, Michael; Wang, Lan; Weigel, Charlotte; Fleming, James A.; Packman, Jeff; Powers, Evan T.; Wiseman, R. Luke; Foss, Theodore R.; Wilson, Ian A.; Kelly, Jeffery W.; Labaudinière, Richard

    2012-01-01

    The transthyretin amyloidoses (ATTR) are invariably fatal diseases characterized by progressive neuropathy and/or cardiomyopathy. ATTR are caused by aggregation of transthyretin (TTR), a natively tetrameric protein involved in the transport of thyroxine and the vitamin A–retinol-binding protein complex. Mutations within TTR that cause autosomal dominant forms of disease facilitate tetramer dissociation, monomer misfolding, and aggregation, although wild-type TTR can also form amyloid fibrils in elderly patients. Because tetramer dissociation is the rate-limiting step in TTR amyloidogenesis, targeted therapies have focused on small molecules that kinetically stabilize the tetramer, inhibiting TTR amyloid fibril formation. One such compound, tafamidis meglumine (Fx-1006A), has recently completed Phase II/III trials for the treatment of Transthyretin Type Familial Amyloid Polyneuropathy (TTR-FAP) and demonstrated a slowing of disease progression in patients heterozygous for the V30M TTR mutation. Herein we describe the molecular and structural basis of TTR tetramer stabilization by tafamidis. Tafamidis binds selectively and with negative cooperativity (Kds ∼2 nM and ∼200 nM) to the two normally unoccupied thyroxine-binding sites of the tetramer, and kinetically stabilizes TTR. Patient-derived amyloidogenic variants of TTR, including kinetically and thermodynamically less stable mutants, are also stabilized by tafamidis binding. The crystal structure of tafamidis-bound TTR suggests that binding stabilizes the weaker dimer-dimer interface against dissociation, the rate-limiting step of amyloidogenesis. PMID:22645360

  1. Tafamidis, a potent and selective transthyretin kinetic stabilizer that inhibits the amyloid cascade.

    PubMed

    Bulawa, Christine E; Connelly, Stephen; Devit, Michael; Wang, Lan; Weigel, Charlotte; Fleming, James A; Packman, Jeff; Powers, Evan T; Wiseman, R Luke; Foss, Theodore R; Wilson, Ian A; Kelly, Jeffery W; Labaudinière, Richard

    2012-06-12

    The transthyretin amyloidoses (ATTR) are invariably fatal diseases characterized by progressive neuropathy and/or cardiomyopathy. ATTR are caused by aggregation of transthyretin (TTR), a natively tetrameric protein involved in the transport of thyroxine and the vitamin A-retinol-binding protein complex. Mutations within TTR that cause autosomal dominant forms of disease facilitate tetramer dissociation, monomer misfolding, and aggregation, although wild-type TTR can also form amyloid fibrils in elderly patients. Because tetramer dissociation is the rate-limiting step in TTR amyloidogenesis, targeted therapies have focused on small molecules that kinetically stabilize the tetramer, inhibiting TTR amyloid fibril formation. One such compound, tafamidis meglumine (Fx-1006A), has recently completed Phase II/III trials for the treatment of Transthyretin Type Familial Amyloid Polyneuropathy (TTR-FAP) and demonstrated a slowing of disease progression in patients heterozygous for the V30M TTR mutation. Herein we describe the molecular and structural basis of TTR tetramer stabilization by tafamidis. Tafamidis binds selectively and with negative cooperativity (K(d)s ~2 nM and ~200 nM) to the two normally unoccupied thyroxine-binding sites of the tetramer, and kinetically stabilizes TTR. Patient-derived amyloidogenic variants of TTR, including kinetically and thermodynamically less stable mutants, are also stabilized by tafamidis binding. The crystal structure of tafamidis-bound TTR suggests that binding stabilizes the weaker dimer-dimer interface against dissociation, the rate-limiting step of amyloidogenesis.

  2. Inhibition of triple-negative breast cancer models by combinations of antibodies to EGFR

    PubMed Central

    Ferraro, Daniela A.; Gaborit, Nadège; Maron, Ruth; Cohen-Dvashi, Hadas; Porat, Ziv; Pareja, Fresia; Lavi, Sara; Lindzen, Moshit; Ben-Chetrit, Nir; Sela, Michael; Yarden, Yosef

    2013-01-01

    Breast tumors lacking expression of human epidermal growth factor receptor 2 (HER2) and the estrogen and the progesterone receptors (triple negative; TNBC) are more aggressive than other disease subtypes, and no molecular targeted agents are currently available for their treatment. Because TNBC commonly displays EGF receptor (EGFR) expression, and combinations of monoclonal antibodies to EGFR effectively inhibit other tumor models, we addressed the relevance of this strategy to treatment of TNBC. Unlike a combination of the clinically approved monoclonal antibodies, cetuximab and panitumumab, which displaced each other and displayed no cooperative effects, several other combinations resulted in enhanced inhibition of TNBC’s cell growth both in vitro and in animals. The ability of certain antibody mixtures to remove EGFR from the cell surface and to promote its intracellular degradation correlated with the inhibitory potential. However, unlike EGF-induced sorting of EGFR to lysosomal degradation, the antibody-induced pathway displayed independence from the intrinsic kinase activity and dimer formation ability of EGFR, and it largely avoided the recycling route. In conclusion, although TNBC clinical trials testing EGFR inhibitors reported lack of benefit, our results offer an alternative strategy that combines noncompetitive antibodies to achieve robust degradation of EGFR and tumor inhibition. PMID:23319610

  3. Anti-tissue transglutaminase antibody inhibits apoptotic cell clearance by macrophages in pregnant NOD mice.

    PubMed

    Sóñora, Cecilia; Mourglia-Ettlin, Gustavo; Calo, Guillermina; Hauk, Vanesa; Ramhorst, Rosanna; Hernández, Ana; Leirós, Claudia Pérez

    2014-06-01

    Autoimmunity is a feature of celiac disease (CD) with tissue transglutaminase (tTG) as a major autoantigen. A correlation between gynecological-obstetric disorders in CD patients and the presence of circulating antibodies anti-tTG that inhibited tTG activity was reported. Serum anti-tTG antibodies were detected in a non-obese diabetic (NOD) mouse model of type I insulin-dependent diabetes mellitus and Sjögren's syndrome, two comorbid states with CD. Since pregnancy complications have been described in NOD mice, we evaluated the ability of anti-tTG antibodies to affect the functions of tTG relevant to the normal course of an early pregnancy like extracellular matrix assembling and apoptotic cell phagocytosis by macrophages. Circulating IgG antibodies against tTG were detected in NOD mice with titers that decreased at early pregnancy; interestingly, the in vitro transamidating activity of tTG was reduced by NOD serum samples. Particularly, anti-tTG antibody inhibited apoptotic cell phagocytosis by peritoneal macrophages from pregnant NOD mice that express the enzyme on surface. Evidence provided support for a role for anti-tTG antibodies through reduced transamidating activity and reduced apoptotic cell clearance by the macrophages of pregnant NOD mice.

  4. Diethylstilbestrol can effectively accelerate estradiol-17-O-glucuronidation, while potently inhibiting estradiol-3-O-glucuronidation

    SciTech Connect

    Zhu, Liangliang; Xiao, Ling; Xia, Yangliu; Zhou, Kun; Wang, Huili; Huang, Minyi; Ge, Guangbo; Wu, Yan; Wu, Ganlin; Yang, Ling

    2015-03-01

    This in vitro study investigates the effects of diethylstilbestrol (DES), a widely used toxic synthetic estrogen, on estradiol-3- and 17-O- (E2-3/17-O) glucuronidation, via culturing human liver microsomes (HLMs) or recombinant UDP-glucuronosyltransferases (UGTs) with DES and E2. DES can potently inhibit E2-3-O-glucuronidation in HLM, a probe reaction for UGT1A1. Kinetic assays indicate that the inhibition follows a competitive inhibition mechanism, with the Ki value of 2.1 ± 0.3 μM, which is less than the possible in vivo level. In contrast to the inhibition on E2-3-O-glucuronidation, the acceleration is observed on E2-17-O-glucuronidation in HLM, in which cholestatic E2-17-O-glucuronide is generated. In the presence of DES (0–6.25 μM), K{sub m} values for E2-17-O-glucuronidation are located in the range of 7.2–7.4 μM, while V{sub max} values range from 0.38 to 1.54 nmol/min/mg. The mechanism behind the activation in HLM is further demonstrated by the fact that DES can efficiently elevate the activity of UGT1A4 in catalyzing E2-17-O-glucuronidation. The presence of DES (2 μM) can elevate V{sub max} from 0.016 to 0.81 nmol/min/mg, while lifting K{sub m} in a much lesser extent from 4.4 to 11 μM. Activation of E2-17-O-glucuronidation is well described by a two binding site model, with K{sub A}, α, and β values of 0.077 ± 0.18 μM, 3.3 ± 1.1 and 104 ± 56, respectively. However, diverse effects of DES towards E2-3/17-O-glucuronidation are not observed in liver microsomes from several common experimental animals. In summary, this study issues new potential toxic mechanisms for DES: potently inhibiting the activity of UGT1A1 and powerfully accelerating the formation of cholestatic E2-17-O-glucuronide by UGT1A4. - Highlights: • E2-3-O-glucuronidation in HLM is inhibited when co-incubated with DES. • E2-17-O-glucuronidation in HLM is stimulated when co-incubated with DES. • Acceleration of E2-17-O-glucuronidationin in HLM by DES is via activating the

  5. Potent and Specific Inhibition of Glycosidases by Small Artificial Binding Proteins (Affitins)

    PubMed Central

    Mechaly, Ariel E.; Obal, Gonzalo; Béhar, Ghislaine; Mouratou, Barbara; Oppezzo, Pablo; Alzari, Pedro M.; Pecorari, Frédéric

    2014-01-01

    Glycosidases are associated with various human diseases. The development of efficient and specific inhibitors may provide powerful tools to modulate their activity. However, achieving high selectivity is a major challenge given that glycosidases with different functions can have similar enzymatic mechanisms and active-site architectures. As an alternative approach to small-chemical compounds, proteinaceous inhibitors might provide a better specificity by involving a larger surface area of interaction. We report here the design and characterization of proteinaceous inhibitors that specifically target endoglycosidases representative of the two major mechanistic classes; retaining and inverting glycosidases. These inhibitors consist of artificial affinity proteins, Affitins, selected against the thermophilic CelD from Clostridium thermocellum and lysozyme from hen egg. They were obtained from libraries of Sac7d variants, which involve either the randomization of a surface or the randomization of a surface and an artificially-extended loop. Glycosidase binders exhibited affinities in the nanomolar range with no cross-recognition, with efficient inhibition of lysozyme (Ki = 45 nM) and CelD (Ki = 95 and 111 nM), high expression yields in Escherichia coli, solubility, and thermal stabilities up to 81.1°C. The crystal structures of glycosidase-Affitin complexes validate our library designs. We observed that Affitins prevented substrate access by two modes of binding; covering or penetrating the catalytic site via the extended loop. In addition, Affitins formed salt-bridges with residues essential for enzymatic activity. These results lead us to propose the use of Affitins as versatile selective glycosidase inhibitors and, potentially, as enzymatic inhibitors in general. PMID:24823716

  6. LOCALIZATION, FERTILITY INHIBITION, AND EPITOPE MAPS USING ANTIBODIES TO THE SPERM PROTEIN SP22

    EPA Science Inventory

    LOCALIZATION, FERTILITY INHIBITION, AND EPITOPE MAPS USING ANTIBODIES TO THE SPERM PROTEIN SP22. GR Klinefelter1, JE Welch*1, HDM Moore*2, K Bobseine*1, J Suarez*1 ,N Roberts*1 ,R Zucker *1 1U.S. EPA, NHEERL, Reproductive Toxicology Division, RTP, NC and 2University of Sheffield...

  7. The derivation of a minimum immune titre of rubella haemagglutination-inhibition (HI) antibody*

    PubMed Central

    Bradstreet, C. M. Patricia; Kirkwood, B.; Pattison, J. R.; Tobin, J. O'H.

    1978-01-01

    Ten laboratories collaborated in a study of minimum immune titre (MIT) of rubella haemagglutination-inhibiting (HI) antibody with one laboratory acting as a reference laboratory to provide a uniform basis for comparison of the HI results. The international unitage equivalent to the MIT used by the ten laboratories was found to vary from 24 to 98 units. Testing of the sera by immunofluorescence and by HI after flotation centrifugation indicated that residual non-specific inhibitors may interfere with HI antibody testing to an extent equivalent to 12-15 units. An acceptable MIT would therefore be equivalent to 24-48 units of rubella HI antibody. The single radial haemolysis (SRH) results on the sera indicate that this is a sensitive and specific test for rubella antibody. PMID:366017

  8. Specific inhibition of AGC protein kinases by antibodies against C-terminal epitopes.

    PubMed

    Traincard, François; Giacomoni, Véronique; Veron, Michel

    2004-08-13

    The sequences contributing to the catalytic site of protein kinases are not all comprised within the highly conserved catalytic core. Thus, in mammalian cAMP-dependent protein kinase (PKA), the C-terminal sequence participates in substrate binding. Using synthetic peptides mimicking the FxxF motif present at most C-termini of AGC kinases, we have raised highly specific antibodies which are potent and specific inhibitors of the catalytic activity of the cognate protein kinase. Taking into account the structure of PKA, these results point to the potential of the C-terminal region of protein kinases as a target for designing specific protein kinase inhibitors.

  9. A Novel Antibody against Human Properdin Inhibits the Alternative Complement System and Specifically Detects Properdin from Blood Samples

    PubMed Central

    Pauly, Diana; Nagel, Benedikt M.; Reinders, Jörg; Killian, Tobias; Wulf, Matthias; Ackermann, Susanne; Ehrenstein, Boris; Zipfel, Peter F.; Skerka, Christine; Weber, Bernhard H. F.

    2014-01-01

    The complement system is an essential part of the innate immune system by acting as a first line of defense which is stabilized by properdin, the sole known positive regulator of the alternative complement pathway. Dysregulation of complement can promote a diversity of human inflammatory diseases which are treated by complement inhibitors. Here, we generated a novel blocking monoclonal antibody (mAb) against properdin and devised a new diagnostic assay for this important complement regulator. Mouse mAb 1340 specifically detected native properdin from human samples with high avidity. MAb 1340 inhibited specifically the alternative complement mediated cell lysis within a concentration range of 1–10 µg/mL. Thus, in vitro anti-properdin mAb 1340 was up to fifteen times more efficient in blocking the complement system as compared to anti-C5 or anti-Ba antibodies. Computer-assisted modelling suggested a three-dimensional binding epitope in a properdin-C3(H2O)-clusterin complex to be responsible for the inhibition. Recovery of properdin in a newly established sandwich ELISA using mAb 1340 was determined at 80–125% for blood sample dilutions above 1∶50. Reproducibility assays showed a variation below 25% at dilutions less than 1∶1,000. Systemic properdin concentrations of healthy controls and patients with age-related macular degeneration or rheumatic diseases were all in the range of 13–30 µg/mL and did not reveal significant differences. These initial results encourage further investigation into the functional role of properdin in the development, progression and treatment of diseases related to the alternative complement pathway. Thus, mAb 1340 represents a potent properdin inhibitor suitable for further research to understand the exact mechanisms how properdin activates the complement C3-convertase and to determine quantitative levels of properdin in biological samples. PMID:24797388

  10. A novel antibody against human properdin inhibits the alternative complement system and specifically detects properdin from blood samples.

    PubMed

    Pauly, Diana; Nagel, Benedikt M; Reinders, Jörg; Killian, Tobias; Wulf, Matthias; Ackermann, Susanne; Ehrenstein, Boris; Zipfel, Peter F; Skerka, Christine; Weber, Bernhard H F

    2014-01-01

    The complement system is an essential part of the innate immune system by acting as a first line of defense which is stabilized by properdin, the sole known positive regulator of the alternative complement pathway. Dysregulation of complement can promote a diversity of human inflammatory diseases which are treated by complement inhibitors. Here, we generated a novel blocking monoclonal antibody (mAb) against properdin and devised a new diagnostic assay for this important complement regulator. Mouse mAb 1340 specifically detected native properdin from human samples with high avidity. MAb 1340 inhibited specifically the alternative complement mediated cell lysis within a concentration range of 1-10 µg/mL. Thus, in vitro anti-properdin mAb 1340 was up to fifteen times more efficient in blocking the complement system as compared to anti-C5 or anti-Ba antibodies. Computer-assisted modelling suggested a three-dimensional binding epitope in a properdin-C3(H2O)-clusterin complex to be responsible for the inhibition. Recovery of properdin in a newly established sandwich ELISA using mAb 1340 was determined at 80-125% for blood sample dilutions above 1∶50. Reproducibility assays showed a variation below 25% at dilutions less than 1∶1,000. Systemic properdin concentrations of healthy controls and patients with age-related macular degeneration or rheumatic diseases were all in the range of 13-30 µg/mL and did not reveal significant differences. These initial results encourage further investigation into the functional role of properdin in the development, progression and treatment of diseases related to the alternative complement pathway. Thus, mAb 1340 represents a potent properdin inhibitor suitable for further research to understand the exact mechanisms how properdin activates the complement C3-convertase and to determine quantitative levels of properdin in biological samples.

  11. Junctional and allele-specific residues are critical for MERS-CoV neutralization by an exceptionally potent germline-like antibody

    DOE PAGES

    Ying, Tianlei; Prabakaran, Ponraj; Du, Lanying; ...

    2015-09-15

    The MERS-CoV is an emerging virus, which already infected more than 1,300 humans with high (~36%) mortality. Here, we show that m336, an exceptionally potent human anti-MERS-CoV antibody, is almost germline with only one somatic mutation in the heavy chain. The structure of Fab m336 in complex with the MERS-CoV receptor-binding domain reveals that its IGHV1-69-derived heavy chain provides more than 85% binding surface and that its epitope almost completely overlaps with the receptor-binding site. Analysis of antibodies from 69 healthy humans suggests an important role of the V(D)J recombination-generated junctional and allele-specific residues for achieving high affinity of bindingmore » at such low levels of somatic hypermutation. Our results also have important implications for development of vaccine immunogens based on the newly identified m336 epitope as well as for elucidation of mechanisms of neutralization by m336-like antibodies and their elicitation in vivo.« less

  12. Junctional and allele-specific residues are critical for MERS-CoV neutralization by an exceptionally potent germline-like antibody

    SciTech Connect

    Ying, Tianlei; Prabakaran, Ponraj; Du, Lanying; Shi, Wei; Feng, Yang; Wang, Yanping; Wang, Lingshu; Li, Wei; Jiang, Shibo; Dimitrov, Dimiter S.; Zhou, Tongqing

    2015-09-15

    The MERS-CoV is an emerging virus, which already infected more than 1,300 humans with high (~36%) mortality. Here, we show that m336, an exceptionally potent human anti-MERS-CoV antibody, is almost germline with only one somatic mutation in the heavy chain. The structure of Fab m336 in complex with the MERS-CoV receptor-binding domain reveals that its IGHV1-69-derived heavy chain provides more than 85% binding surface and that its epitope almost completely overlaps with the receptor-binding site. Analysis of antibodies from 69 healthy humans suggests an important role of the V(D)J recombination-generated junctional and allele-specific residues for achieving high affinity of binding at such low levels of somatic hypermutation. Our results also have important implications for development of vaccine immunogens based on the newly identified m336 epitope as well as for elucidation of mechanisms of neutralization by m336-like antibodies and their elicitation in vivo.

  13. Inhibition of HIV replication by pokeweed antiviral protein targeted to CD4+ cells by monoclonal antibodies

    NASA Astrophysics Data System (ADS)

    Zarling, Joyce M.; Moran, Patricia A.; Haffar, Omar; Sias, Joan; Richman, Douglas D.; Spina, Celsa A.; Myers, Dorothea E.; Kuebelbeck, Virginia; Ledbetter, Jeffrey A.; Uckun, Fatih M.

    1990-09-01

    FUNCTIONAL impairment and selective depletion of CD4+ T cells, the hallmark of AIDS, are at least partly caused by human immunodeficiency virus (HIV-1) type 1 binding to the CD4 molecule and infecting CD4+ cells1,2. It may, therefore, be of therapeutic value to target an antiviral agent to CD4+ cells to prevent infection and to inhibit HIV-1 production in patients' CD4+ cells which contain proviral DNA3,4. We report here that HIV-1 replication in normal primary CD4+ T cells can be inhibited by pokeweed antiviral protein, a plant protein of relative molecular mass 30,000 (ref. 5), which inhibits replication of certain plant RNA viruses6-8, and of herpes simplex virus, poliovirus and influenza virus9-11. Targeting pokeweed antiviral protein to CD4+ T cells by conjugating it to monoclonal antibodies reactive with CDS, CD7 or CD4 expressed on CD4+ cells, increased its anti-HIV potency up to 1,000-fold. HIV-1 replication is inhibited at picomolar concentrations of conjugates of pokeweed antiviral protein and monoclonal antibodies, which do not inhibit proliferation of normal CD4+ T cells or CD4-dependent responses. These conjugates inhibit HIV-1 protein synthesis and also strongly inhibit HIV-1 production in activated CD4+ T cells from infected patients.

  14. Inhibition of Pneumococcal Adherence to Human Nasopharyngeal Epithelial Cells by Anti-PsaA Antibodies

    PubMed Central

    Romero-Steiner, Sandra; Pilishvili, Tamar; Sampson, Jacquelyn S.; Johnson, Scott E.; Stinson, Annie; Carlone, George M.; Ades, Edwin W.

    2003-01-01

    The role of pneumococcal (Pnc) surface adhesin A (PsaA) in the adherence of Streptococcus pneumoniae (pneumococcus) to host cells is not well defined. We examined the effect of anti-PsaA antibodies in an inhibition of adherence assay using Detroit 562 nasopharyngeal human epithelial cells. Rabbit polyclonal (Pab) anti-recombinant PsaA (rPsaA) sera, a purified mouse monoclonal antibody (MAb) (MAb 6F62G8E12), and 22 healthy adult sera with known anti-PsaA IgG levels (obtained by enzyme-linked immunosorbent assay) were evaluated for their abilities to inhibit Pnc adherence to confluent monolayers (measured as percent reduction in CFU counts compared to those of uninhibited controls). Pnc adherence was dependent on capsular phenotype (no or low adherence for opaque strains). With an inoculum of 104 to 105 bacteria/well, the mean ± standard deviation count in controls was 163 ± 32 CFU/well for transparent strains. Low adherence was observed for a PsaA-minus mutant even at higher inoculum doses. Mean percent inhibitions of adherence with Pab and MAb were 54 and 50%, respectively. Adult sera showed inhibition in a dose-response fashion with a range of 98 to 8%, depending on the serum anti-PsaA antibody concentration. Absorption of Pab with rPsaA restored Pnc adherence to control levels. Absorption of sera with a PsaA-minus mutant did not result in a significant decrease (P >0.05) of inhibition of adherence activity. Additionally, nearly 100% of Pnc adherence was inhibited by lipidated rPsaA at 2.5 μg/ml. Our data support the argument that PsaA is an adhesin that mediates Pnc adherence to human nasopharyngeal cells. This functional assay may be useful in evaluating antibodies elicited in response to PsaA vaccination. PMID:12626450

  15. Antibodies toward high-density lipoprotein components inhibit paraoxonase activity in patients with systemic lupus erythematosus.

    PubMed

    Batuca, J R; Ames, P R J; Isenberg, D A; Alves, J Delgado

    2007-06-01

    Patients with systemic lupus erythematosus (SLE) have an increased incidence of vascular disease, and oxidative stress is recognized as an important feature in this condition, despite the underlying mechanisms not being fully understood. In these patients, an interaction between lipoproteins and the immune system has been suggested, but most studies have only looked at antibodies against oxidized low-density lipoproteins. This study was undertaken to determine the presence of antibodies directed against high-density lipoproteins (HDL) and to identify a possible association between these antibodies and paraoxonase (PON), an antioxidant enzyme present in HDL. Plasma from 55 patients with SLE was collected and IgG aHDL and antiapolipoprotein A-I (aApo A-I) antibodies were assessed by enzyme-linked immunosorbent assay. Standardization of the method was performed in a control population of 150 healthy subjects. Plasma levels above 5 standard deviations of the mean of the control population were considered positive. PON activity was assessed by quantification of p-nitrophenol formation (micromol/mL/min). Patients with SLE had higher titers of aHDL (P < 0.0001) and aApo A-I (P < 0.0001) antibodies, and lower PON activity (P < 0.0001) than healthy controls. There was also a direct correlation between the titers of aHDL and aApo A-I antibodies (r = 0.61; P < 0.0001). PON activity was inversely correlated with aApo A-I (P = 0.0129) antibody levels. Anti-HDL and aApo A-I antibodies from patients with high titers were isolated and subsequently incubated with human HDL. These antibodies reduced PON activity up to a maximum of 70.2% and 78.4%, respectively. This study showed the presence of aHDL and aApo A-I antibodies in patients with SLE. These antibodies were associated with reduced PON activity in plasma, and the in vitro inhibition assay confirmed a direct inhibition of the enzyme activity.

  16. Characterization of a monoclonal antibody that specifically inhibits triosephosphate isomerase activity of Taenia solium.

    PubMed

    Víctor, Sanabria-Ayala; Yolanda, Medina-Flores; Araceli, Zavala-Carballo; Lucía, Jiménez; Abraham, Landa

    2013-08-01

    In the present study, we obtained and characterized partially a monoclonal antibody (4H11D10B11 mAb) against triosephosphate isomerase from Taenia solium (TTPI). This antibody recognized the enzyme by both ELISA and western blot and was able to inhibit its enzymatic activity in 74%. Moreover, the antigen-binding fragments (Fabs), products of digestion of the monoclonal antibody with papain, retained almost the same inhibitory effect. We determined the binding site by ELISA; synthetic peptides containing sequences from different non-conserved regions of the TTPI were confronted to the 4H11D10B11 mAb. The epitope recognized by the monoclonal antibody was located on peptide TTPI-56 (ATPAQAQEVHKVVRDWIRKHVDAGIADKARI), and an analysis of mimotopes, obtained with the 4H11D10B11 mAb, suggests that the epitope spans the sequence WIRKHVDAGIAD, residues 193-204 of the enzyme. This epitope is located within helix 6, next to loop 6, an essential active loop during catalysis. The antibody did not recognize triosephosphate isomerase from man and pig, definitive and intermediary hosts of T. solium, respectively. Furthermore, it did not bind to the catalytic site, since kinetic analysis demonstrated that inhibition had a non-competitive profile.

  17. A novel monoclonal antibody targeting coxsackie virus and adenovirus receptor inhibits tumor growth in vivo

    PubMed Central

    Kawada, Manabu; Inoue, Hiroyuki; Kajikawa, Masunori; Sugiura, Masahito; Sakamoto, Shuichi; Urano, Sakiko; Karasawa, Chigusa; Usami, Ihomi; Futakuchi, Mitsuru; Masuda, Tohru

    2017-01-01

    To create a new anti-tumor antibody, we conducted signal sequence trap by retrovirus-meditated expression method and identified coxsackie virus and adenovirus receptor (CXADR) as an appropriate target. We developed monoclonal antibodies against human CXADR and found that one antibody (6G10A) significantly inhibited the growth of subcutaneous as well as orthotopic xenografts of human prostate cancer cells in vivo. Furthermore, 6G10A also inhibited other cancer xenografts expressing CXADR, such as pancreatic and colorectal cancer cells. Knockdown and overexpression of CXADR confirmed the dependence of its anti-tumor activity on CXADR expression. Our studies of its action demonstrated that 6G10A exerted its anti-tumor activity primarily through both antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Moreover, 6G10A reacted with human tumor tissues, such as prostate, lung, and brain, each of which express CXADR. Although we need further evaluation of its reactivity and safety in human tissues, our results show that a novel anti-CXADR antibody may be a feasible candidate for cancer immunotherapy. PMID:28074864

  18. Epigallocatechin-3-gallate potently inhibits the in vitro activity of hydroxy-3-methyl-glutaryl-CoA reductase[S

    PubMed Central

    Cuccioloni, Massimiliano; Mozzicafreddo, Matteo; Spina, Michele; Tran, Chi Nhan; Falconi, Maurizio; Eleuteri, Anna Maria; Angeletti, Mauro

    2011-01-01

    Hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) is the rate-controlling enzyme of cholesterol synthesis, and owing to its biological and pharmacological relevance, researchers have investigated several compounds capable of modulating its activity with the hope of developing new hypocholesterolemic drugs. In particular, polyphenol-rich extracts were extensively tested for their cholesterol-lowering effect as alternatives, or adjuvants, to the conventional statin therapies, but a full understanding of the mechanism of their action has yet to be reached. Our work reports on a detailed kinetic and equilibrium study on the modulation of HMGR by the most-abundant catechin in green tea, epigallocatechin-3-gallate (EGCG). Using a concerted approach involving spectrophotometric, optical biosensor, and chromatographic analyses, molecular docking, and site-directed mutagenesis on the cofactor site of HMGR, we have demonstrated that EGCG potently inhibits the in vitro activity of HMGR (Ki in the nanomolar range) by competitively binding to the cofactor site of the reductase. Finally, we evaluated the effect of combined EGCG-statin administration. PMID:21357570

  19. The G-quadruplex-forming aptamer AS1411 potently inhibits HIV-1 attachment to the host cell

    PubMed Central

    Perrone, Rosalba; Butovskaya, Elena; Lago, Sara; Garzino-Demo, Alfredo; Pannecouque, Christophe; Palù, Giorgio; Richter, Sara N.

    2016-01-01

    AS1411 is a G-rich aptamer that forms a stable G-quadruplex structure and displays antineoplastic properties both in vitro and in vivo. This oligonucleotide has undergone phase 2 clinical trials. The major molecular target of AS1411 is nucleolin (NCL), a multifunctional nucleolar protein also present in the cell membrane where it selectively mediates the binding and uptake of AS1411. Cell-surface NCL has been recognised as a low-affinity co-receptor for human immunodeficiency virus type 1 (HIV-1) anchorage on target cells. Here we assessed the anti-HIV-1 properties and underlying mechanism of action of AS1411. The antiviral activity of AS1411 was determined towards different HIV-1 strains, host cells and at various times post-infection. Acutely, persistently and latently infected cells were tested, including HIV-1-infected peripheral blood mononuclear cells from a healthy donor. Mechanistic studies to exclude modes of action other than virus binding via NCL were performed. AS1411 efficiently inhibited HIV-1 attachment/entry into the host cell. The aptamer displayed antiviral activity in the absence of cytotoxicity at the tested doses, therefore displaying a wide therapeutic window and favourable selectivity indexes. These findings, besides validating cell-surface-expressed NCL as an antiviral target, open the way for the possible use of AS1411 as a new potent and promisingly safe anti-HIV-1 agent. PMID:27032748

  20. Simultaneous knockdown of BRAF and expression of INK4A in melanoma cells leads to potent growth inhibition and apoptosis

    SciTech Connect

    Zhao Yanhua; Zhang Yan; Yang Zhen; Li, Albert; Dong Jianli

    2008-06-06

    Abnormal BRAF and p16INK4A co-exist in 60% of melanomas. BRAF mutation also occurs in 80% of benign nevi where it turns-on p16INK4A resulting in proliferative senescence; loss of p16INK4A removes the inhibitory block leading to melanoma development. Since only melanomas with wild-type BRAF have amplified CDK4 and cyclin D1 genes, p16INK4A-CDK4/6-cyclin D pathway is viewed as linearly downstream of BRAF. Thus, co-occurrence of aberrant BRAF and INK4A may be remnant of changes during melanoma formation without functional significance. To explore this notion, we simultaneously knocked down BRAF (via siRNA) and expressed INK4A cDNA in melanoma cells and observed enhanced growth inhibition. Notably, although each alone had no statistically significant effect on apoptosis, co-expression of BRAF siRNA and INK4A cDNA caused potent apoptosis, which was associated with up-regulation of BIM and down-regulation of BCL2. Our results suggest that aberrant BRAF and INK4A cooperate to promote proliferation and survival of melanoma cells.

  1. Chemically modified, non-anticoagulant heparin derivatives are potent galectin-3 binding inhibitors and inhibit circulating galectin-3-promoted metastasis.

    PubMed

    Duckworth, Carrie A; Guimond, Scott E; Sindrewicz, Paulina; Hughes, Ashley J; French, Neil S; Lian, Lu-Yun; Yates, Edwin A; Pritchard, D Mark; Rhodes, Jonathan M; Turnbull, Jeremy E; Yu, Lu-Gang

    2015-09-15

    Concentrations of circulating galectin-3, a metastasis promoter, are greatly increased in cancer patients. Here we show that 2- or 6-de-O-sulfated, N-acetylated heparin derivatives are galectin-3 binding inhibitors. These chemically modified heparin derivatives inhibited galectin-3-ligand binding and abolished galectin-3-mediated cancer cell-endothelial adhesion and angiogenesis. Unlike standard heparin, these modified heparin derivatives and their ultra-low molecular weight sub-fractions had neither anticoagulant activity nor effects on E-, L- or P-selectin binding to their ligands nor detectable cytotoxicity. Intravenous injection of such heparin derivatives (with cancer cells pre-treated with galectin-3 followed by 3 subcutaneous injections of the derivatives) abolished the circulating galectin-3-mediated increase in lung metastasis of human melanoma and colon cancer cells in nude mice. Structural analysis using nuclear magnetic resonance and synchrotron radiation circular dichroism spectroscopies showed that the modified heparin derivatives bind to the galectin-3 carbohydrate-recognition domain. Thus, these chemically modified, non-anticoagulant, low-sulfated heparin derivatives are potent galectin-3 binding inhibitors with substantial potential as anti-metastasis/cancer drugs.

  2. Human Memory B Cells Producing Potent Cross-Neutralizing Antibodies against Human Parechovirus: Implications for Prevalence, Treatment, and Diagnosis

    PubMed Central

    Benschop, K. S. M.; Koen, G.; Claassen, Y. B.; Wagner, K.; Bakker, A. Q.; Wolthers, K. C.

    2015-01-01

    ABSTRACT The family Picornaviridae is a large and diverse group of positive-sense RNA viruses, including human enteroviruses (EVs) and human parechoviruses (HPeVs). The human immune response against EVs and HPeVs is thought to be mainly humoral, and an insufficient neutralizing antibody (Ab) response during infection is a risk factor and can ultimately be life threatening. The accessibility of different antigenic sites and observed cross-reactivity make HPeVs a good target for development of therapeutic human monoclonal antibodies (MAbs). In this study, we generated two different human MAbs specific for HPeV by screening culture supernatants of Ab-producing human B cell cultures for direct neutralization of HPeV1. Both MAbs showed HPeV1-specific neutralization as well as neutralization of HPeV2. One antibody, AM18, cross-neutralized HPeV4, -5, and -6 and coxsackievirus A9 (CV-A9). VP1 capsid protein-specific assays confirmed that AM18 bound VP1 of HPeV1, -2, and -4 with high affinity (11.5 pM). In contrast, the HPeV1-specific MAb AM28, which neutralized HPeV1 even more efficiently than did AM18, showed no cross-reactivity with HPeV3 to -6 or other EVs and did not bind any of the capsid proteins, suggesting that AM28 is specific for a conformation-dependent, nonlinear epitope on the virus. The discovery of MAbs that are cross-reactive between HPeVs may help development of HPeV treatment options with antibodies and vaccine design based on epitopes recognized by these antibodies. IMPORTANCE HPeV infections are widespread among young children and adults, causing a broad range of disease. Infections can be severe and life threatening, while no antiviral treatment is available. Given that the absence of neutralizing Abs is a risk factor for severe disease in infants, treatment of picornavirus infections with MAbs would be a therapeutic option. To study antibody neutralization of HPeV in more detail, we generated two different HPeV1-specific human MAbs. Both MAbs show HPe

  3. Plasmodium vivax Invasion of Human Erythrocytes Inhibited by Antibodies Directed against the Duffy Binding Protein

    PubMed Central

    Grimberg, Brian T; Udomsangpetch, Rachanee; Xainli, Jia; McHenry, Amy; Panichakul, Tasanee; Sattabongkot, Jetsumon; Cui, Liwang; Bockarie, Moses; Chitnis, Chetan; Adams, John; Zimmerman, Peter A; King, Christopher L

    2007-01-01

    Background Plasmodium vivax invasion requires interaction between the human Duffy antigen on the surface of erythrocytes and the P. vivax Duffy binding protein (PvDBP) expressed by the parasite. Given that Duffy-negative individuals are resistant and that Duffy-negative heterozygotes show reduced susceptibility to blood-stage infection, we hypothesized that antibodies directed against region two of P. vivax Duffy binding protein (PvDBPII) would inhibit P. vivax invasion of human erythrocytes. Methods and Findings Using a recombinant region two of the P. vivax Duffy binding protein (rPvDBPII), polyclonal antibodies were generated from immunized rabbits and affinity purified from the pooled sera of 14 P. vivax–exposed Papua New Guineans. It was determined by ELISA and by flow cytometry, respectively, that both rabbit and human antibodies inhibited binding of rPvDBPII to the Duffy antigen N-terminal region and to Duffy-positive human erythrocytes. Additionally, using immunofluorescent microscopy, the antibodies were shown to attach to native PvDBP on the apical end of the P. vivax merozoite. In vitro invasion assays, using blood isolates from individuals in the Mae Sot district of Thailand, showed that addition of rabbit anti-PvDBPII Ab or serum (antibodies against, or serum containing antibodies against, region two of the Plasmodium vivax Duffy binding protein) (1:100) reduced the number of parasite invasions by up to 64%, while pooled PvDBPII antisera from P. vivax–exposed people reduced P. vivax invasion by up to 54%. Conclusions These results show, for what we believe to be the first time, that both rabbit and human antibodies directed against PvDBPII reduce invasion efficiency of wild P. vivax isolated from infected patients, and suggest that a PvDBP-based vaccine may reduce human blood-stage P. vivax infection. PMID:18092885

  4. Anti-syntaxin antibodies inhibit calcium-dependent catecholamine secretion from permeabilized chromaffin cells.

    PubMed

    Gutierrez, L M; Quintanar, J L; Viniegra, S; Salinas, E; Moya, F; Reig, J A

    1995-01-05

    Adrenomedullary chromaffin cells release catecholamines in response to the intracellular calcium rise upon stimulation by different secretagogues. The presence of syntaxin 1, a protein presumably involved in docking of synaptic vesicles to presynaptic membranes, has been investigated in chromaffin cells. The study using two different monoclonal antibodies shows that syntaxin 1 is present in the chromaffin cell membrane fraction. Functional experiments demonstrate that anti-syntaxin antibodies inhibit calcium-dependent secretion in permeabilized cells. These results suggest that syntaxin 1 is an important component of the secretory machinery in chromaffin cells.

  5. Ebolavirus Nucleoprotein C-Termini Potently Attract Single Domain Antibodies Enabling Monoclonal Affinity Reagent Sandwich Assay (MARSA) Formulation

    PubMed Central

    Sherwood, Laura J.; Hayhurst, Andrew

    2013-01-01

    Background Antigen detection assays can play an important part in environmental surveillance and diagnostics for emerging threats. We are interested in accelerating assay formulation; targeting the agents themselves to bypass requirements for a priori genome information or surrogates. Previously, using in vitro affinity reagent selection on Marburg virus we rapidly established monoclonal affinity reagent sandwich assay (MARSA) where one recombinant antibody clone was both captor and tracer for polyvalent nucleoprotein (NP). Hypothesizing that the closely related Ebolavirus genus may share the same Achilles' heel, we redirected the scheme to see whether similar assays could be delivered and began to explore their mechanism. Methods and Findings In parallel we selected panels of llama single domain antibodies (sdAb) from a semi-synthetic library against Zaire, Sudan, Ivory Coast, and Reston Ebola viruses. Each could perform as both captor and tracer in the same antigen sandwich capture assay thereby forming MARSAs. All sdAb were specific for NP and those tested required the C-terminal domain for recognition. Several clones were cross-reactive, indicating epitope conservation across the Ebolavirus genus. Analysis of two immune shark sdAb revealed they also targeted the C-terminal domain, and could be similarly employed, yet were less sensitive than a comparable llama sdAb despite stemming from immune selections. Conclusions The C-terminal domain of Ebolavirus NP is a strong attractant for antibodies and enables sensitive sandwich immunoassays to be rapidly generated using a single antibody clone. The polyvalent nature of nucleocapsid borne NP and display of the C-terminal region likely serves as a bountiful affinity sink during selections, and a highly avid target for subsequent immunoassay capture. Combined with the high degree of amino acid conservation through 37 years and across wide geographies, this domain makes an ideal handle for monoclonal affinity reagent

  6. Mining the human autoantibody repertoire: isolation of potent IL17A-neutralizing monoclonal antibodies from a patient with thymoma.

    PubMed

    Beerli, Roger R; Bauer, Monika; Fritzer, Andrea; Rosen, Lindsey B; Buser, Regula B; Hanner, Markus; Maudrich, Melanie; Nebenfuehr, Mario; Toepfer, Jorge Alejandro Sepulveda; Mangold, Susanne; Bauer, Anton; Holland, Steven M; Browne, Sarah K; Meinke, Andreas

    2014-01-01

    Anti-cytokine autoantibodies have been widely reported to be present in human plasma, both in healthy subjects and in patients with underlying autoimmune conditions, such as autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) or thymic epithelial neoplasms. While often asymptomatic, they can cause or facilitate a wide range of diseases including opportunistic infections. The potential therapeutic value of specific neutralizing anti-cytokine autoantibodies has not been thoroughly investigated. Here we used mammalian cell display to isolate IL17A-specific antibodies from a thymoma patient with proven high-titer autoantibodies against the same. We identified 3 distinct clonotypes that efficiently neutralized IL17A in a cell-based in vitro assay. Their potencies were comparable to those of known neutralizing antibodies, including 2, AIN457 (secukinumab) and ixekizumab that are currently in clinical development for the treatment of various inflammatory disorders. These data clearly demonstrate that the human autoantibody repertoire can be mined for antibodies with high therapeutic potential for clinical development.

  7. Mining the human autoantibody repertoire: Isolation of potent IL17A-neutralizing monoclonal antibodies from a patient with thymoma

    PubMed Central

    Beerli, Roger R; Bauer, Monika; Fritzer, Andrea; Rosen, Lindsey B; Buser, Regula B; Hanner, Markus; Maudrich, Melanie; Nebenfuehr, Mario; Toepfer, Jorge Alejandro Sepulveda; Mangold, Susanne; Bauer, Anton; Holland, Steven M; Browne, Sarah K; Meinke, Andreas

    2014-01-01

    Anti-cytokine autoantibodies have been widely reported to be present in human plasma, both in healthy subjects and in patients with underlying autoimmune conditions, such as autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) or thymic epithelial neoplasms. While often asymptomatic, they can cause or facilitate a wide range of diseases including opportunistic infections. The potential therapeutic value of specific neutralizing anti-cytokine autoantibodies has not been thoroughly investigated. Here we used mammalian cell display to isolate IL17A-specific antibodies from a thymoma patient with proven high-titer autoantibodies against the same. We identified 3 distinct clonotypes that efficiently neutralized IL17A in a cell-based in vitro assay. Their potencies were comparable to those of known neutralizing antibodies, including 2, AIN457 (secukinumab) and ixekizumab that are currently in clinical development for the treatment of various inflammatory disorders. These data clearly demonstrate that the human autoantibody repertoire can be mined for antibodies with high therapeutic potential for clinical development. PMID:25484038

  8. Characterization of the structural determinants required for potent mechanism-based inhibition of human cytochrome P450 1A1 by cannabidiol.

    PubMed

    Yamaori, Satoshi; Okushima, Yoshimi; Yamamoto, Ikuo; Watanabe, Kazuhito

    2014-05-25

    We previously demonstrated that cannabidiol (CBD) was a potent mechanism-based inhibitor of human cytochrome P450 1A1 (CYP1A1). However, the moiety of CBD that contributes to the potent mechanism-based inhibition of human CYP1A1 remains unknown. Thus, the effects of compounds structurally related to CBD on CYP1A1 activity were examined with recombinant human CYP1A1 in order to characterize the structural requirements for potent inactivation by CBD. When preincubated in the presence of NADPH for 20min, olivetol, which corresponds to the pentylresorcinol moiety of CBD, enhanced the inhibition of the 7-ethoxyresorufin O-deethylase activity of CYP1A1. In contrast, d-limonene, which corresponds to the terpene moiety of CBD, failed to inhibit CYP1A1 activity in a metabolism-dependent manner. Pentylbenzene, which lacks two free phenolic hydroxyl groups, also did not enhance CYP1A1 inhibition. On the other hand, preincubation of the CBD-2'-monomethyl ether (CBDM) and CBD-2',6'-dimethyl ether (CBDD) enhanced the inhibition of CYP1A1 activity. Inhibition by cannabidivarin (CBDV), which possessed a propyl side chain, was strongly potentiated by its preincubation. Orcinol, which has a methyl group, augmented CYP1A1 inhibition, whereas its derivative without an alkyl side chain, resorcinol, did not exhibit any metabolism-dependent inhibition. The preincubation of CBD-hydroxyquinone did not markedly enhance CYP1A1 inhibition. We further confirmed that olivetol, CBDM, CBDD, CBDV, and orcinol, as well as CBD (kinact=0.215min(-1)), inactivated CYP1A1 activity; their kinact values were 0.154, 0.0638, 0.0643, 0.226, and 0.0353min(-1), respectively. These results suggest that the methylresorcinol structure in CBD may have structurally important roles in the inactivation of CYP1A1.

  9. Inhibition of HIV-1 entry by antibodies: potential viral and cellular targets

    PubMed Central

    Phogat, S.; Wyatt, R. T.; Hedestam, G. B. Karlsson

    2008-01-01

    Phogat S, Wyatt RT, Karlsson Hedestam GB (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA; Department of Microbiology Tumor and Cell Biology, Karolinska Institutet, Stockholm; and the Swedish Institute for Infectious Disease Control, Solna, Sweden). Inhibition of HIV-1 entry by antibodies: potential viral and cellular targets (Review). Vaccine-induced antibodies that interfere with viral entry are the protective correlate of most existing prophylactic vaccines. However, for highly variable viruses such as HIV-1, the ability to elicit broadly neutralizing antibody responses through vaccination has proven to be extremely difficult. The major targets for HIV-1 neutralizing antibodies are the viral envelope glycoprotein trimers on the surface of the virus that mediate receptor binding and entry. HIV-1 has evolved many mechanisms on the surface of envelope glyco-proteins to evade antibody-mediated neutralization, including the masking of conserved regions by glycan, quaternary protein interactions and the presence of immunodominant variable elements. The primary challenge in the development of an HIV-1 vaccine that elicits broadly neutralizing antibodies therefore lies in the design of suitable envelope glycoprotein immunogens that circumvent these barriers. Here, we describe neutralizing determinants on the viral envelope glyco-proteins that are defined by their function in receptor binding or by rare neutralizing antibodies isolated from HIV-infected individuals. We also describe the nonvariable cellular receptors involved in the HIV-1 entry process, or other cellular proteins, and ongoing studies to determine if antibodies against these proteins have efficacy as therapeutic reagents or, in some cases, as vaccine targets to interfere with HIV-1 entry. PMID:17598813

  10. A humanized anti-DLL4 antibody promotes dysfunctional angiogenesis and inhibits breast tumor growth

    PubMed Central

    Jia, Xuelian; Wang, Wenyi; Xu, Zhuobin; Wang, Shijing; Wang, Tong; Wang, Min; Wu, Min

    2016-01-01

    Blockage of Delta-like 4 (DLL4)-directed Notch signaling induces excessive tip cell formation and endothelial proliferation resulting in dysfunctional angiogenesis in tumors. MMGZ01, as a murine anti-human DLL4 monoclonal antibody, specifically binds to human DLL4 and blocks Notch pathway. Here, the structure of MMGZ01 variable fragment (Fv) was established and framework region (FR) residues which supported complementarily determining region (CDR) loop conformation were identified. Important residues interactions were also identified through docking MMGZ01 Fv with antigen epitope in DLL4. To humanize the murine antibody, we modified MMGZ01 Fv through CDR grafting and the reconstructed antibody (H3L2) maintained similar structure and binding affinity to parental MMGZ01 after back mutation of 12 canonical murine residues in the FRs. Meanwhile, H3L2 promoted human umbilical vein endothelial cell (HUVEC) proliferation through inhibiting DLL4-directed Notch pathway. Moreover, in MDA-MB-231-bearing nude mice, H3L2 induced dysfunctional angiogenesis and tumor cell apoptosis and showed superior anti-tumor activity. In conclusion, H3L2 is an ideal humanized antibody that inhibits tumor growth through targeting DLL4-Notch pathway and has attracting potentials for clinical applications. PMID:27301650

  11. Crystal Structure of the Hendra Virus Attachment G Glycoprotein Bound to a Potent Cross-Reactive Neutralizing Human Monoclonal Antibody

    PubMed Central

    Xu, Kai; Rockx, Barry; Xie, Yihu; DeBuysscher, Blair L.; Fusco, Deborah L.; Zhu, Zhongyu; Chan, Yee-Peng; Xu, Yan; Luu, Truong; Cer, Regina Z.; Feldmann, Heinz; Mokashi, Vishwesh; Dimitrov, Dimiter S.; Bishop-Lilly, Kimberly A.; Broder, Christopher C.; Nikolov, Dimitar B.

    2013-01-01

    The henipaviruses, represented by Hendra (HeV) and Nipah (NiV) viruses are highly pathogenic zoonotic paramyxoviruses with uniquely broad host tropisms responsible for repeated outbreaks in Australia, Southeast Asia, India and Bangladesh. The high morbidity and mortality rates associated with infection and lack of licensed antiviral therapies make the henipaviruses a potential biological threat to humans and livestock. Henipavirus entry is initiated by the attachment of the G envelope glycoprotein to host cell membrane receptors. Previously, henipavirus-neutralizing human monoclonal antibodies (hmAb) have been isolated using the HeV-G glycoprotein and a human naïve antibody library. One cross-reactive and receptor-blocking hmAb (m102.4) was recently demonstrated to be an effective post-exposure therapy in two animal models of NiV and HeV infection, has been used in several people on a compassionate use basis, and is currently in development for use in humans. Here, we report the crystal structure of the complex of HeV-G with m102.3, an m102.4 derivative, and describe NiV and HeV escape mutants. This structure provides detailed insight into the mechanism of HeV and NiV neutralization by m102.4, and serves as a blueprint for further optimization of m102.4 as a therapeutic agent and for the development of entry inhibitors and vaccines. PMID:24130486

  12. Inactivated poliovirus type 2 vaccine delivered to rat skin via high density microprojection array elicits potent neutralising antibody responses

    PubMed Central

    Muller, David A.; Pearson, Frances E.; Fernando, Germain J.P.; Agyei-Yeboah, Christiana; Owens, Nick S.; Corrie, Simon R.; Crichton, Michael L.; Wei, Jonathan C.J.; Weldon, William C.; Oberste, M. Steven; Young, Paul R.; Kendall, Mark A. F.

    2016-01-01

    Polio eradication is progressing rapidly, and the live attenuated Sabin strains in the oral poliovirus vaccine (OPV) are being removed sequentially, starting with type 2 in April 2016. For risk mitigation, countries are introducing inactivated poliovirus vaccine (IPV) into routine vaccination programs. After April 2016, monovalent type 2 OPV will be available for type 2 outbreak control. Because the current IPV is not suitable for house-to-house vaccination campaigns (the intramuscular injections require health professionals), we developed a high-density microprojection array, the Nanopatch, delivered monovalent type 2 IPV (IPV2) vaccine to the skin. To assess the immunogenicity of the Nanopatch, we performed a dose-matched study in rats, comparing the immunogenicity of IPV2 delivered by intramuscular injection or Nanopatch immunisation. A single dose of 0.2 D-antigen units of IPV2 elicited protective levels of poliovirus antibodies in 100% of animals. However, animals receiving IPV2 by IM required at least 3 immunisations to reach the same neutralising antibody titres. This level of dose reduction (1/40th of a full dose) is unprecedented for poliovirus vaccine delivery. The ease of administration coupled with the dose reduction observed in this study points to the Nanopatch as a potential tool for facilitating inexpensive IPV for mass vaccination campaigns. PMID:26911254

  13. Inactivated poliovirus type 2 vaccine delivered to rat skin via high density microprojection array elicits potent neutralising antibody responses.

    PubMed

    Muller, David A; Pearson, Frances E; Fernando, Germain J P; Agyei-Yeboah, Christiana; Owens, Nick S; Corrie, Simon R; Crichton, Michael L; Wei, Jonathan C J; Weldon, William C; Oberste, M Steven; Young, Paul R; Kendall, Mark A F

    2016-02-25

    Polio eradication is progressing rapidly, and the live attenuated Sabin strains in the oral poliovirus vaccine (OPV) are being removed sequentially, starting with type 2 in April 2016. For risk mitigation, countries are introducing inactivated poliovirus vaccine (IPV) into routine vaccination programs. After April 2016, monovalent type 2 OPV will be available for type 2 outbreak control. Because the current IPV is not suitable for house-to-house vaccination campaigns (the intramuscular injections require health professionals), we developed a high-density microprojection array, the Nanopatch, delivered monovalent type 2 IPV (IPV2) vaccine to the skin. To assess the immunogenicity of the Nanopatch, we performed a dose-matched study in rats, comparing the immunogenicity of IPV2 delivered by intramuscular injection or Nanopatch immunisation. A single dose of 0.2 D-antigen units of IPV2 elicited protective levels of poliovirus antibodies in 100% of animals. However, animals receiving IPV2 by IM required at least 3 immunisations to reach the same neutralising antibody titres. This level of dose reduction (1/40th of a full dose) is unprecedented for poliovirus vaccine delivery. The ease of administration coupled with the dose reduction observed in this study points to the Nanopatch as a potential tool for facilitating inexpensive IPV for mass vaccination campaigns.

  14. Inhibition of human factor VIIIa by anti-A2 subunit antibodies.

    PubMed Central

    Lollar, P; Parker, E T; Curtis, J E; Helgerson, S L; Hoyer, L W; Scott, M E; Scandella, D

    1994-01-01

    Human inhibitory alloantibodies and autoantibodies to Factor VIII (FVIII) are usually directed toward the A2 and/or C2 domains of the FVIII molecule. Anti-C2 antibodies block the binding of FVIII to phospholipid, but the mechanism of action of anti-A2 antibodies is not known. We investigated the properties of a patient autoantibody, RC, and a monoclonal antibody, 413, that bind to the region which contains the epitopes of all anti-A2 alloantibodies or autoantibodies studied to date. mAb 413 and RC were noncompetitive inhibitors of a model intrinsic Factor X activation complex (intrinsic FXase) consisting of Factor IXa, activated FVIII (FVIIIa), and synthetic phospholipid vesicles, since they decreased the Vmax of intrinsic FXase by > 95% at saturating concentrations without altering the Km. This indicates that RC and mAb 413 either block the binding of FVIIIa to FIXa or phospholipid or interfere with the catalytic function of fully assembled intrinsic FXase, but they do not inhibit the binding of the substrate Factor X. mAb 413 did not inhibit the increase in fluorescence anisotropy that results from the binding of Factor VIIIa to fluorescein-5-maleimidyl-D-phenylalanyl-prolyl-arginyl-FIXa (Fl-M-FPR-FIXa) on phospholipid vesicles in the absence of Factor X, indicating it does not inhibit assembly of intrinsic FXase. Addition of Factor X to Fl-M-FPR-FIXa, FVIIIa, and phospholipid vesicles produced a further increase in fluorescence anisotropy and a decrease in fluorescence intensity. This effect was blocked completely by mAb 413. We conclude that anti-A2 antibodies inhibit FVIIIa function by blocking the conversion of intrinsic FXase/FX complex to the transition state, rather than by interfering with formation of the ground state Michaelis complex. PMID:8200986

  15. Inhibition of Giardia lamblia excystation by antibodies against cyst walls and by wheat germ agglutinin.

    PubMed Central

    Meng, T C; Hetsko, M L; Gillin, F D

    1996-01-01

    Although excystation is crucial to the initiation of infection by Giardia lamblia, little is known about the regulation of this important process. We have been able to reliably induce excystation in vitro by mimicking cyst passage through the stomach and upper small intestine by the exposure of in vitro-derived cysts to an acidic, reducing environment (stage I) followed by protease treatment at a slightly alkaline pH (stage II). Preexposure of cysts to polyclonal rabbit antiserum against purified cyst walls (PCWs) or to wheat germ agglutinin (WGA) inhibited excystation by > 90%. Adsorption of either ligand with PCWs eliminated inhibition, demonstrating specificity for cyst wall epitopes. Inhibition by WGA was reversed by either chitotriose or sialic acid, while inhibition by polyclonal antibodies against PCWs (anti-PCW) was reversed only by sialic acid, which also inhibited binding of both ligands to intact cysts and to cyst wall antigens in immunoblots. Binding of anti-PCW did not affect acidification of cyst cytoplasm during stage I. Exposure of cysts to anti-PCW and WGA prior to, but not after, stage II was sufficient to inhibit excystation, and inhibition could be partially reversed by increasing the protease concentration during stage II. A 7- to 10-fold higher proportion of WGA- and anti-PCW-treated cysts than control cysts remained intact after stage II. Our results suggest that these ligands, which bind cyst wall epitopes, inhibit excystation, most likely by interfering with proteolysis of cyst wall glycoproteins during stage II. PMID:8675320

  16. Recombinant receptor-binding domain of SARS-CoV spike protein expressed in mammalian, insect and E. coli cells elicits potent neutralizing antibody and protective immunity.

    PubMed

    Du, Lanying; Zhao, Guangyu; Chan, Chris C S; Sun, Shihui; Chen, Min; Liu, Zhonghua; Guo, Hongxiang; He, Yuxian; Zhou, Yusen; Zheng, Bo-Jian; Jiang, Shibo

    2009-10-10

    Severe acute respiratory syndrome (SARS) is a newly emerging infectious disease. The potential recurrence of the disease from animal reservoirs highlights the significance of development of safe and efficient vaccines to prevent a future SARS epidemic. In this study, we expressed the recombinant receptor-binding domain (rRBD) in mammalian (293T) cells, insect (Sf9) cells, and E. coli, respectively, and compared their immunogenicity and protection against SARS-CoV infection in an established mouse model. Our results show that all rRBD proteins expressed in the above systems maintained intact conformation, being able to induce highly potent neutralizing antibody responses and complete protective immunity against SARS-CoV challenge in mice, albeit the rRBD expressed in 293T cells elicited stronger humoral immune responses with significantly higher neutralizing activity (P<0.05) than those expressed in Sf9 and E. coli cells. These results suggest that all three rRBDs are effective in eliciting immune responses and protection against SARS-CoV and any of the above expression systems can be used for production of rRBD-based SARS subunit vaccines. Preference will be given to rRBD expressed in mammalian cells for future evaluation of the vaccine efficacy in a non-human primate model of SARS because of its ability to refold into a native conformation more readily and to induce higher level of neutralizing antibody responses than those expressed in E. coli and insect cells.

  17. Inhibition of the HIV-1 and HIV-2 proteases by a monoclonal antibody.

    PubMed Central

    Lescar, J.; Brynda, J.; Rezacova, P.; Stouracova, R.; Riottot, M. M.; Chitarra, V.; Fabry, M.; Horejsi, M.; Sedlacek, J.; Bentley, G. A.

    1999-01-01

    The monoclonal antibody 1696, directed against the HIV-1 protease, displays strong inhibitory effects toward the catalytic activity of the enzyme of both the HIV-1 and HIV-2 isolates. This antibody cross-reacts with peptides that include the N-terminus of the enzyme, a region that is well conserved in sequence among different viral strains and which, furthermore, is crucial for homodimerization to the active enzymatic form. This observation, as well as antigen-binding studies in the presence of an active site inhibitor, suggest that 1696 inhibits the HIV protease by destabilizing its active homodimeric form. To characterize further how the antibody 1696 inhibits the HIV-1 and HIV-2 proteases, we have solved the crystal structure of its Fab fragment by molecular replacement and refined it at 3.0 A resolution. The antigen binding site has a deep cavity at its center, which is lined mainly by acidic and hydrophobic residues, and is large enough to accommodate several antigen residues. The structure of the Fab 1696 could form a starting basis for the design of alternative HIV protease-inhibiting molecules of broad specificity. PMID:10631984

  18. Diagnosis of typhoid fever: detection of Salmonella typhi porins-specific antibodies by inhibition ELISA.

    PubMed Central

    Nandakumar, K S; Palanivel, V; Muthukkaruppan, V

    1993-01-01

    Porins are highly immunogenic outer membrane proteins of Salmonella. Sera from typhoid patients contained a high level of IgG antibodies directed to porins of Salm. typhi. Since porins are highly conserved proteins, anti-porins antibodies both from typhoid patients and healthy normals reacted with porins from several Gram-negative bacteria. Therefore, in order to improve the specificity of detecting Salm. typhi porins-specific antibodies, an inhibition ELISA was developed using enzyme-conjugated MoAbs (MP1 and MPN4) specific to Salm. typhi porins. Sera from typhoid patients with positive haemoculture (16 out of 17) inhibited the binding of MP1 to porins, thus showing a positive test for typhoid, whereas sera from patients with other Gram-negative bacterial infections (n = 7) and from healthy volunteers (66 out of 67) were found to be negative. The sensitivity, specificity, accuracy, positive predictive value and negative predictive value of this assay were 94.1, 98.7, 97.8, 94.1 and 98.7% respectively. The validity of our inhibition ELISA for typhoid was higher than that of the Widal test. The diagnosis of typhoid fever as early as 3 days after the onset of fever, using a single specimen is possible. PMID:8222322

  19. Complement inhibition as potential new therapy for antibody-mediated rejection.

    PubMed

    Eskandary, Farsad; Wahrmann, Markus; Mühlbacher, Jakob; Böhmig, Georg A

    2016-04-01

    Antibody-mediated rejection (ABMR) is a leading cause of kidney allograft failure. While the exact mechanisms contributing to donor-specific antibody (DSA)-triggered tissue injury are still incompletely understood, complement activation via the classical pathway is believed to be one of the key players. There is now growing interest in complement blockade as an antirejection treatment. One attractive strategy may be inhibition of terminal complex formation using anti-C5 antibody eculizumab. Anecdotal reports, case series, and a unique cohort of flow crossmatch-positive live donor kidney transplant recipients subjected to eculizumab-based desensitization have demonstrated successful prevention and reversal of acute clinical ABMR. Nevertheless, maybe due to complement activation steps proximal of C5 or even complement-independent mechanisms, subclinical rejection processes that might culminate in chronic injury were found to escape inhibition. Larger studies designed to clarify the actual clinical value of terminal complement inhibition as an antirejection treatment are currently underway. In addition, alternative concepts, such as therapies that target key component C1, are currently under development, and we will see in the near future whether new strategies in the pipeline will have the potential to beneficially impact clinical practice.

  20. VNAR single-domain antibodies specific for BAFF inhibit B cell development by molecular mimicry.

    PubMed

    Häsler, Julien; Flajnik, Martin F; Williams, Gareth; Walsh, Frank S; Rutkowski, J Lynn

    2016-07-01

    B cell-activating factor (BAFF) plays a dominant role in the B cell homeostasis. However, excessive BAFF promotes the development of autoreactive B-cells and several antibodies have been developed to block its activity. Bispecific antibodies with added functionality represent the next wave of biologics that may be more effective in the treatment of complex autoimmune disease. The single variable domain from the immunoglobulin new antigen receptor (VNAR) is one of the smallest antibody recognition units that could be combined with monospecific antibodies to develop bispecific agents. We isolated a panel of BAFF-binding VNARs with low nM potency from a semi-synthetic phage display library and examined their functional activity. The anti-BAFF VNARs blocked the binding of BAFF to all three of its receptors (BR3, TACI and BCMA) and the presence of the conserved DXL receptor motif found in the CDR3 regions suggests molecular mimicry as the mechanism of antagonism. One clone was formatted as an Fc fusion for functional testing and it was found to inhibit both mouse and human BAFF with equal potency ex vivo in a splenocyte proliferation assay. In mice, subchronic administration reduced the number of immature and transitional intermediates B cells and mature B cell subsets. These results indicate that VNAR single domain antibodies function as selective B-cell inhibitors and offer an alternative molecular format for targeting B-cell disorders.

  1. Prevalence of hemagglutination-inhibition and neutralizing antibodies to arboviruses in horses of java.

    PubMed

    Widjaja, S; Soekotjo, W; Hartati, S; Jennings, G B; Corwin, A L

    1995-03-01

    A study was conducted to measure the prevalence of hemagglutination-inhibition (HI) and neutralizing antibodies against two arboviruses (Chikungunya and Japanese encephalitis virus) in horses of Java, Indonesia. Blood specimens were collected from a sample of 112 horses at two stables: Pulo Mas, a racing track-horse complex, located in a residential area in North Jakarta, and Pamulang, a riding school, located in a rural environment of West Jaya. Sera were tested by the HI assay and plaque reduction neutralization test. JEV antibodies were detected by HI in 58 (52%) of the horses, while only 11 (10%) had Chikungunya antibodies by HI. The proportion of Pamulang horses infected with JEV (66%) was significantly higher than found among Pulo Mas horses (40%) screened (p < 0.01). Of the 58 horses with JEV antibodies by HI, 52 (90%) were found to have specific neutralization antibodies to JEV. HI and neutralization tests on horse sera indicated that the risk to alpha virus infections was minimal in horses surveyed from Java. However, there was a high risk of JEV infection among the same population.

  2. Antibody

    MedlinePlus

    An antibody is a protein produced by the body's immune system when it detects harmful substances, called antigens. Examples ... microorganisms (bacteria, fungi, parasites, and viruses) and chemicals. Antibodies may be produced when the immune system mistakenly ...

  3. Inhibiting angiogenesis with human single-chain variable fragment antibody targeting VEGF.

    PubMed

    Hosseini, Hossien; Rajabibazl, Masoumeh; Ebrahimizadeh, Walead; Dehbidi, Gholamreza Rafiei

    2015-01-01

    Vascular endothelial growth factor (VEGF) is a highly specific angiogenesis factor which has crucial roles in the angiogenesis of tumors. Anti-angiogenesis agents can inhibit growth and metastasis of tumor cells. Single-chain variable fragments (scFv) have the same affinity as whole antibodies and smaller size, thus result in more tissue permeability and higher production yield. In this research we aim to isolate a human scFv antibody against VEGF that inhibits angiogenesis. For that, we have used human scFv phage library to isolate a specific scFv antibody against binding site of VEGF. The human scFv phage library was amplified according to the manufacture protocol and panned against recombinant VEGF. ScFv antibody was isolated after five rounds of panning. Phage ELISA was used for detection of the highest affinity binder (HR6). Soluble HR6 scFv was expressed in non-suppressor strain of Escherichia coli HB2151 and purified using Ni-NTA chromatography. In vivo and in vitro function of the HR6 scFv was analyzed by chorioallantoic membrane assay and endothelial cell proliferation assay on VEGF stimulated HUVECs. Result of the cross reactivity showed that HR6 scFv specifically bounds to VEGF. The affinity was calculated to be 1.8×10(-7)M. HR6 could stop HUVEC proliferation in a dose dependent manner and anti-angiogenesis activity was observed using 10μg of HR6 in chorioallantoic membrane assay. In this work, we demonstrate that a HR6 scFv selected from human library phage display specifically blocks VEGF signaling, furthermore, this scFv has an anti-angiogenesis effect and because of its small size has more tissue diffusion. The HR6 antibody was isolated form a human library thus, it is not immunogenic for humans and could serve as a potential therapeutic agent in cancer.

  4. Invasion of erythrocytes in vitro by Plasmodium falciparum can be inhibited by monoclonal antibody directed against an S antigen.

    PubMed

    Saul, A; Cooper, J; Ingram, L; Anders, R F; Brown, G V

    1985-11-01

    A monoclonal antibody has been produced which binds to the heat stable S antigen present in the FCQ-27/PNG isolate of Plasmodium falciparum. This monoclonal antibody also inhibits the invasion in vitro of erythrocytes by malarial merozoites thus demonstrating that the S antigens of Plasmodium falciparum may be a target of protective immune responses.

  5. Neuraminidase inhibiting antibody responses in pigs differ between influenza A virus N2 lineages and by vaccine type

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The neuraminidase (NA) protein of influenza A viruses (IAV) has important functional roles in the viral replication cycle. Antibodies specific to NA can reduce viral replication and limit disease severity, but are not routinely measured. We analyzed NA inhibiting (NI) antibody titers in serum and re...

  6. Ouabain-specific antibodies: immunochemical properties and reversal of Na+, K+-activated adenosine triphosphatase inhibition

    PubMed Central

    Smith, Thomas W.

    1972-01-01

    Antibodies with high affinity and specificity for the cardiac glycoside ouabain were raised in rabbits. The antigen used was a conjugate of ouabain linked through its rhamnose moiety to terminal α-amino groups of poly D,L alanyl-human serum albumin. Ouabain-specific antibodies were present as early as 3 wk, and rose steadily in titer over the initial 20-33 wk of immunization. Levels as high as 6.5 mg specific immunoglobulin per ml antiserum were reached in one rabbit at the end of 45 wk. The average intrinsic association constants for ouabain were 1.3 × 109 M-1 and 1.6 × 109 M-1 in antisera studied in detail, and there was evidence of restricted heterogeneity of binding site affinities. A high degree of specificity was demonstrated. Significant cross-reactivity occurred only with other cardioactive steroid compounds such as acetyl strophanthidin, digoxin, and digitoxin, while endogenous steroids did not cross-react even when present in 1000-fold excess. A rapid and convenient radioimmunoassay procedure for plasma or urine ouabain concentrations was developed using these antibodies. Competition between ouabain-3H tracer and unlabeled ouabain for specific antibody binding sites allowed the measurement of ouabain concentrations as low as 0.1 ng/ml or less without need for extraction procedures. The high association constants observed in these studies permit antibody reversal of established myocardial effects of ouabain. Both blockade and reversal of ouabain inhibition of canine myocardial microsomal Na+, K+-activated ATPase by antibody were documented, suggesting a possible mechanism for reversal of cellular effects. PMID:4260123

  7. Human Antibodies against a Purified Glucosylceramide from Cryptococcus neoformans Inhibit Cell Budding and Fungal Growth

    PubMed Central

    Rodrigues, Marcio L.; Travassos, Luiz R.; Miranda, Kildare R.; Franzen, Anderson J.; Rozental, Sonia; de Souza, Wanderley; Alviano, Celuta S.; Barreto-Bergter, Eliana

    2000-01-01

    A major ceramide monohexoside (CMH) was purified from lipidic extracts of Cryptococcus neoformans. This molecule was analyzed by high-performance thin-layer chromatography (HPTLC), gas chromatography coupled with mass spectrometry, and fast atom bombardment-mass spectrometry. The cryptococcal CMH is a β-glucosylceramide, with the carbohydrate residue attached to 9-methyl-4,8-sphingadienine in amidic linkage to 2-hydroxyoctadecanoic acid. Sera from patients with cryptococcosis and a few other mycoses reacted with the cryptococcal CMH. Specific antibodies were purified from patients' sera by immunoadsorption on the purified glycolipid followed by protein G affinity chromatography. The purified antibodies to CMH (mainly immunoglobulin G1) bound to different strains and serological types of C. neoformans, as shown by flow cytofluorimetry and immunofluorescence labeling. Transmission electron microscopy of yeasts labeled with immunogold-antibodies to CMH and immunostaining of isolated cell wall lipid extracts separated by HPTLC showed that the cryptococcal CMH predominantly localizes to the fungal cell wall. Confocal microscopy revealed that the β-glucosylceramide accumulates mostly at the budding sites of dividing cells with a more disperse distribution at the cell surface of nondividing cells. The increased density of sphingolipid molecules seems to correlate with thickening of the cell wall, hence with its biosynthesis. The addition of human antibodies to CMH to cryptococcal cultures of both acapsular and encapsulated strains of C. neoformans inhibited cell budding and cell growth. This process was complement-independent and reversible upon removal of the antibodies. The present data suggest that the cryptococcal β-glucosylceramide is a fungal antigen that plays a role on the cell wall synthesis and yeast budding and that antibodies raised against this component are inhibitory in vitro. PMID:11083830

  8. Inhibition of kinesin-driven microtubule motility by monoclonal antibodies to kinesin heavy chains

    PubMed Central

    1988-01-01

    We have prepared and characterized seven mouse monoclonal antibodies (SUK 1-7) to the 130-kD heavy chain of sea urchin egg kinesin. On immunoblots, SUK 3 and SUK 4 cross-reacted with Drosophila embryo 116- kD heavy chains, and SUK 4, SUK 5, SUK 6, and SUK 7 bound to the 120-kD heavy chains of bovine brain kinesin. Three out of seven monoclonal antikinesins (SUK 4, SUK 6, and SUK 7) caused a dose-dependent inhibition of sea urchin egg kinesin-induced microtubule translocation, whereas the other four monoclonal antibodies had no detectable effect on this motility. The inhibitory monoclonal antibodies (SUK 4, SUK 6, and SUK 7) appear to bind to spatially related sites on an ATP- sensitive microtubule binding 45-kD chymotryptic fragment of the 130-kD heavy chain, whereas SUK 2 binds to a spatially distinct site. None of the monoclonal antikinesins inhibited the microtubule activated MgATPase activity of kinesin, suggesting that SUK 4, SUK 6, and SUK 7 uncouple this MgATPase activity from motility. PMID:2974459

  9. Human single-chain variable fragment antibody inhibits macrophage migration inhibitory factor tautomerase activity.

    PubMed

    Tarasuk, Mayuri; Poungpair, Ornnuthchar; Ungsupravate, Duangporn; Bangphoomi, Kunan; Chaicumpa, Wanpen; Yenchitsomanus, Pa-Thai

    2014-03-01

    Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine, secreted from a variety of immune cells, that regulates innate and adaptive immune responses. Elevation of MIF levels in plasma correlates with the severity of inflammatory diseases in humans. Inhibition of MIF or its tautomerase activity ameliorates disease severity by reducing inflammatory responses. In this study, the human single-chain variable fragment (HuScFv) antibody specific to MIF was selected from the human antibody phage display library by using purified recombinant full-length human MIF (rMIF) as the target antigen. Monoclonal HuScFv was produced from phage-transformed bacteria and tested for their binding activities to rMIF by indirect enzyme-linked immunosorbent assay as well as to native MIF by western blot analysis and immunofluorescence assay. The HuScFv with highest binding signal to rMIF also inhibited the tautomerase activities of both rMIF and native MIF in human monoblastic leukemia (U937) cells in a dose-dependent manner. Mimotope searching and molecular docking concordantly demonstrated that the HuScFv interacted with Lys32 and Ile64 in the MIF tautomerase active site. To the best of our knowledge, this is the first study to focus on MIF-specific fully-human antibody fragment with a tautomerase-inhibitory effect that has potential to be developed as anti-inflammatory biomolecules for human use.

  10. Synthetic nanoparticle vaccines produced by layer-by-layer assembly of artificial biofilms induce potent protective T-cell and antibody responses in vivo.

    PubMed

    Powell, Thomas J; Palath, Naveen; DeRome, Mary E; Tang, Jie; Jacobs, Andrea; Boyd, James G

    2011-01-10

    Nanoparticle vaccines induce potent immune responses in the absence of conventional adjuvant due to the recognition by immune cells of the particle structures, which mimic natural pathogens such as viruses and bacteria. Nanoparticle vaccines were fabricated by constructing artificial biofilms using layer-by-layer (LbL) deposition of oppositely charged polypeptides and target designed peptides on CaCO(3) cores. LbL nanoparticles were efficiently internalized by dendritic cells in vitro by a mechanism that was at least partially phagocytic, and induced DC maturation without triggering secretion of inflammatory cytokines. LbL nanoparticle delivery of designed peptides to DC resulted in potent cross-presentation to CD8+ T-cells and more efficient presentation to CD4+ T-cells compared to presentation of soluble peptide. A single immunization of mice with LbL nanoparticles containing designed peptide induced vigorous T-cell responses characterized by a balanced effector (IFNγ) and Th2 (IL-4) ELISPOT profile and in vivo CTL activity. Mice immunized with LbL nanoparticles bearing ovalbumin-derived designed peptides were protected from challenge with Listeria monocytogenes ectopically expressing ovalbumin, confirming the relevance of the CTL/effector T-cell responses. LbL nanoparticles also elicited antibody responses to the target epitope but not to the matrix components of the nanoparticle, avoiding the vector or carrier affect that hampers utility of other vaccine platforms. The potency and efficacy of LbL nanoparticles administered in aqueous suspension without adjuvant or other formulation additive, and the absence of immune responses to the matrix components, suggest that this strategy may be useful in producing novel vaccines against multiple diseases.

  11. Potent and Broad Inhibition of HIV-1 by a Peptide from the gp41 Heptad Repeat-2 Domain Conjugated to the CXCR4 Amino Terminus

    PubMed Central

    Haggarty, Beth S.; Duong, Jennifer; Jordon, Andrea P. O.; Romano, Josephine; DeClercq, Joshua J.; Gregory, Philip D.; Riley, James L.; Holmes, Michael C.

    2016-01-01

    HIV-1 entry can be inhibited by soluble peptides from the gp41 heptad repeat-2 (HR2) domain that interfere with formation of the 6-helix bundle during fusion. Inhibition has also been seen when these peptides are conjugated to anchoring molecules and over-expressed on the cell surface. We hypothesized that potent anti-HIV activity could be achieved if a 34 amino acid peptide from HR2 (C34) were brought to the site of virus-cell interactions by conjugation to the amino termini of HIV-1 coreceptors CCR5 or CXCR4. C34-conjugated coreceptors were expressed on the surface of T cell lines and primary CD4 T cells, retained the ability to mediate chemotaxis in response to cognate chemokines, and were highly resistant to HIV-1 utilization for entry. Notably, C34-conjugated CCR5 and CXCR4 each exhibited potent and broad inhibition of HIV-1 isolates from diverse clades irrespective of tropism (i.e., each could inhibit R5, X4 and dual-tropic isolates). This inhibition was highly specific and dependent on positioning of the peptide, as HIV-1 infection was poorly inhibited when C34 was conjugated to the amino terminus of CD4. C34-conjugated coreceptors could also inhibit HIV-1 isolates that were resistant to the soluble HR2 peptide inhibitor, enfuvirtide. When introduced into primary cells, CD4 T cells expressing C34-conjugated coreceptors exhibited physiologic responses to T cell activation while inhibiting diverse HIV-1 isolates, and cells containing C34-conjugated CXCR4 expanded during HIV-1 infection in vitro and in a humanized mouse model. Notably, the C34-conjugated peptide exerted greater HIV-1 inhibition when conjugated to CXCR4 than to CCR5. Thus, antiviral effects of HR2 peptides can be specifically directed to the site of viral entry where they provide potent and broad inhibition of HIV-1. This approach to engineer HIV-1 resistance in functional CD4 T cells may provide a novel cell-based therapeutic for controlling HIV infection in humans. PMID:27855210

  12. A peroxovanadium compound induces Xenopus oocyte maturation: inhibition by a neutralizing anti-insulin receptor antibody.

    PubMed

    Cummings, C; Zhu, L; Sorisky, A; Liu, X J

    1996-05-01

    Synthetic peroxovanadium compounds are a new class of potent inhibitors of protein phosphotyrosine phosphatases. These compounds exhibit insulin-like activity both in vitro and in experimental animals. However, the molecular mechanism by which these compounds exert their biological effect is not well defined. We demonstrate here that several of these compounds induce Xenopus oocyte maturation in vitro, as indicated by germinal vesicle breakdown. Using one of these compounds for further studies, we show that the induction is dose-dependent and is accompanied by activation of maturation promoting factor as well as activation of Xenopus MAP kinase. Like insulin, bpV(pic) causes an acute accumulation of PI(3,4,5)P3 (phosphotidylinositol-3,4,5-trisphosphate), a product of PI 3-kinase. More importantly, bpV(pic)-induced oocyte maturation was abolished by microinjection of a neutralizing monoclonal anti-insulin receptor antibody (17A3) into oocytes or preincubation of oocytes with a PI 3-kinase inhibitor (wortmannin). These results suggest that bpV(pic) acts upstream of the Xenopus IGF-1 receptor in the induction of meiotic maturation, presumably by neutralizing an inhibitory protein tyrosine phosphatase(s) that may regulate the receptor. Finally, using an oocyte-follicle cell complex that responded to human chorionic gonadotropin (hCG) to undergo GVBD, we showed that injection of 17A3 anti-insulin receptor antibody into oocytes did not affect hCG-induced oocyte maturation.

  13. Antibody-Mediated Inhibition of TNFR1 Attenuates Disease in a Mouse Model of Multiple Sclerosis

    PubMed Central

    Williams, Sarah K.; Maier, Olaf; Fischer, Roman; Fairless, Richard; Hochmeister, Sonja; Stojic, Aleksandar; Pick, Lara; Haar, Doreen; Musiol, Sylvia; Storch, Maria K.; Pfizenmaier, Klaus; Diem, Ricarda

    2014-01-01

    Tumour necrosis factor (TNF) is a proinflammatory cytokine that is known to regulate inflammation in a number of autoimmune diseases, including multiple sclerosis (MS). Although targeting of TNF in models of MS has been successful, the pathological role of TNF in MS remains unclear due to clinical trials where the non-selective inhibition of TNF resulted in exacerbated disease. Subsequent experiments have indicated that this may have resulted from the divergent effects of the two TNF receptors, TNFR1 and TNFR2. Here we show that the selective targeting of TNFR1 with an antagonistic antibody ameliorates symptoms of the most common animal model of MS, experimental autoimmune encephalomyelitis (EAE), when given following both a prophylactic and therapeutic treatment regime. Our results demonstrate that antagonistic TNFR1-specific antibodies may represent a therapeutic approach for the treatment of MS in the future. PMID:24587232

  14. Inhibition of antibody formation during continual stimulation with a strong immunogen

    PubMed Central

    Gras, J.; Roca, Mercedes; Ayats, Rosa; Castro, Rosa; Duran, F.

    1974-01-01

    Long persisting antigenic stimulation at immunogenic levels leads to a profound inhibition of antibody formation. With Brucellus abortus, there is first a brief and high IgM response. IgG antibody titres remain at a low level for some days, and then begin a slow and progressive increase, leading to a rather persistent maximum, and finally after about 300 days, to the state of inhibition. When the same total dose is given with monthly intervals, the effect is quite different, with similar IgM and IgG peaks being observed after each dose. The inhibited animals respond moderately to a ten-fold higher antigen dose, and only with IgG. Six months after interruption of the persistent antigenic stimulus, a strong response can be obtained after a new antigenic stimulation, with a substantial proportion of IgM. It is concluded that persistent antigenic stimulation plays a major role in the change from IgM to IgG synthesis. PMID:4212089

  15. Antibodies against Thrombospondin-Related Anonymous Protein Do Not Inhibit Plasmodium Sporozoite Infectivity In Vivo

    PubMed Central

    Gantt, Soren; Persson, Cathrine; Rose, Keith; Birkett, Ashley J.; Abagyan, Ruben; Nussenzweig, Victor

    2000-01-01

    Thrombospondin-related anonymous protein (TRAP), a candidate malaria vaccine antigen, is required for Plasmodium sporozoite gliding motility and cell invasion. For the first time, the ability of antibodies against TRAP to inhibit sporozoite infectivity in vivo is evaluated in detail. TRAP contains an A-domain, a well-characterized adhesive motif found in integrins. We modeled here a three-dimensional structure of the TRAP A-domain of Plasmodium yoelii and located regions surrounding the MIDAS (metal ion-dependent adhesion site), the presumed business end of the domain. Mice were immunized with constructs containing these A-domain regions but were not protected from sporozoite challenge. Furthermore, monoclonal and rabbit polyclonal antibodies against the A-domain, the conserved N terminus, and the repeat region of TRAP had no effect on the gliding motility or sporozoite infectivity to mice. TRAP is located in micronemes, secretory organelles of apicomplexan parasites. Accordingly, the antibodies tested here stained cytoplasmic TRAP brightly by immunofluorescence. However, very little TRAP could be detected on the surface of sporozoites. In contrast, a dramatic relocalization of TRAP onto the parasite surface occurred when sporozoites were treated with calcium ionophore. This likely mimics the release of TRAP from micronemes when a sporozoite contacts its target cell in vivo. Contact with hepatoma cells in culture also appeared to induce the release of TRAP onto the surface of sporozoites. If large amounts of TRAP are released in close proximity to its cellular receptor(s), effective competitive inhibition by antibodies may be difficult to achieve. PMID:10816526

  16. Antibodies against thrombospondin-related anonymous protein do not inhibit Plasmodium sporozoite infectivity in vivo.

    PubMed

    Gantt, S; Persson, C; Rose, K; Birkett, A J; Abagyan, R; Nussenzweig, V

    2000-06-01

    Thrombospondin-related anonymous protein (TRAP), a candidate malaria vaccine antigen, is required for Plasmodium sporozoite gliding motility and cell invasion. For the first time, the ability of antibodies against TRAP to inhibit sporozoite infectivity in vivo is evaluated in detail. TRAP contains an A-domain, a well-characterized adhesive motif found in integrins. We modeled here a three-dimensional structure of the TRAP A-domain of Plasmodium yoelii and located regions surrounding the MIDAS (metal ion-dependent adhesion site), the presumed business end of the domain. Mice were immunized with constructs containing these A-domain regions but were not protected from sporozoite challenge. Furthermore, monoclonal and rabbit polyclonal antibodies against the A-domain, the conserved N terminus, and the repeat region of TRAP had no effect on the gliding motility or sporozoite infectivity to mice. TRAP is located in micronemes, secretory organelles of apicomplexan parasites. Accordingly, the antibodies tested here stained cytoplasmic TRAP brightly by immunofluorescence. However, very little TRAP could be detected on the surface of sporozoites. In contrast, a dramatic relocalization of TRAP onto the parasite surface occurred when sporozoites were treated with calcium ionophore. This likely mimics the release of TRAP from micronemes when a sporozoite contacts its target cell in vivo. Contact with hepatoma cells in culture also appeared to induce the release of TRAP onto the surface of sporozoites. If large amounts of TRAP are released in close proximity to its cellular receptor(s), effective competitive inhibition by antibodies may be difficult to achieve.

  17. Muscle palmitate uptake and binding are saturable and inhibited by antibodies to FABP(PM).

    PubMed

    Turcotte, L P; Swenberger, J R; Tucker, M Z; Yee, A J; Trump, G; Luiken, J J; Bonen, A

    2000-07-01

    Studies show that uptake of long-chain fatty acids (LCFA) across the plasma membranes (PM) may occur partly via a carrier-mediated process and that the plasma membrane fatty acid-binding protein (FABP(PM)) may be a component of this system. To test the hypothesis that FABP(PM) is involved in transsarcolemmal transport of LCFA in muscle, we measured palmitate uptake in giant sarcolemmal vesicles and palmitate binding to PM proteins in rat muscles, (1) in the presence of increasing amounts of unbound palmitate and (2) in the absence or presence of antibody to FABP(PM). Both palmitate uptake and binding were found to be saturable functions of the unbound palmitate concentration with calculated Vmax values of 10.5 +/- 1.2 pmol/mg protein/15 sec and 45.6 +/- 2.9 nmol/mg protein/15 min and Km values of 12.8 +/- 3.8 and 18.4 +/- 1.8 nmol/L, respectively. The Vmax values for both palmitate uptake and binding were significantly decreased by 75-79% in the presence of a polyclonal antibody to the rat hepatic FABP(PM). Antibody inhibition was found to be dose-dependent and specific to LCFA. Glucose uptake was not affected by the presence of the antibody to FABP(PM). Palmitate uptake and binding were also inhibited in the presence of trypsin and phloretin. These results support the hypothesis that transsarcolemmal LCFA transport occurs in part by a carrier-mediated process and that FABP(PM) is a component of this process in muscle.

  18. Induction of Potent and Long-Lived Antibody and Cellular Immune Responses in the Genitorectal Mucosa Could be the Critical Determinant of HIV Vaccine Efficacy

    PubMed Central

    Chanzu, Nadia; Ondondo, Beatrice

    2014-01-01

    The field of HIV prevention has indeed progressed in leaps and bounds, but with major limitations of the current prevention and treatment options, the world remains desperate for an HIV vaccine. Sadly, this continues to be elusive, because more than 30 years since its discovery there is no licensed HIV vaccine. Research aiming to define immunological biomarkers to accurately predict vaccine efficacy have focused mainly on systemic immune responses, and as such, studies defining correlates of protection in the genitorectal mucosa, the primary target site for HIV entry and seeding are sparse. Clearly, difficulties in sampling and analysis of mucosal specimens, as well as their limited size have been a major deterrent in characterizing the type (mucosal antibodies, cytokines, chemokines, or CTL), threshold (magnitude, depth, and breadth) and viral inhibitory capacity of HIV-1-specific immune responses in the genitorectal mucosa, where they are needed to immediately block HIV acquisition and arrest subsequent virus dissemination. Nevertheless, a few studies document the existence of HIV-specific immune responses in the genitorectal mucosa of HIV-infected aviremic and viremic controllers, as well as in highly exposed persistently seronegative (HEPS) individuals with natural resistance to HIV-1. Some of these responses strongly correlate with protection from HIV acquisition and/or disease progression, thus providing significant clues of the ideal components of an efficacious HIV vaccine. In this study, we provide an overview of the key features of protective immune responses found in HEPS, elite and viremic controllers, and discuss how these can be achieved through mucosal immunization. Inevitably, HIV vaccine development research will have to consider strategies that elicit potent antibody and cellular immune responses within the genitorectal mucosa or induction of systemic immune cells with an inherent potential to home and persist at mucosal sites of HIV entry. PMID

  19. Breakthrough of SIV strain smE660 challenge in SIV strain mac239-vaccinated rhesus macaques despite potent autologous neutralizing antibody responses.

    PubMed

    Burton, Samantha L; Kilgore, Katie M; Smith, S Abigail; Reddy, Sharmila; Hunter, Eric; Robinson, Harriet L; Silvestri, Guido; Amara, Rama R; Derdeyn, Cynthia A

    2015-08-25

    Although the correlates of immunological protection from human immunodeficiency virus or simian immunodeficiency virus infection remain incompletely understood, it is generally believed that medium to high titers of serum neutralizing antibodies (nAbs) against the challenge virus will prevent infection. This paradigm is based on a series of studies in which passive transfer of HIV-specific nAbs protected rhesus macaques (RMs) from subsequent mucosal challenge with a chimeric human/simian immunodeficiency virus. However, it is unknown whether nAb titers define protection in the setting of active immunization. Here we determined serum nAb titers against breakthrough transmitted/founder (T/F) SIVsmE660-derived envelope glycoprotein (Env) variants from 14 RMs immunized with SIVmac239-based DNA-prime/modified vaccinia virus Ankara-boost vaccine regimens that included GM-CSF or CD40L adjuvants and conferred significant but incomplete protection against repeated low-dose intrarectal challenge. A single Env variant established infection in all RMs except one, with no identifiable genetic signature associated with vaccination breakthrough compared with T/F Envs from four unvaccinated monkeys. Breakthrough T/F Env pseudoviruses were potently neutralized in vitro by heterologous pooled serum from chronically SIVsmE660-infected monkeys at IC50 titers exceeding 1:1,000,000. Remarkably, the T/F Env pseudoviruses from 13 of 14 monkeys were also susceptible to neutralization by autologous prechallenge serum at in vitro IC50 titers ranging from 1:742-1:10,832. These titers were similar to those observed in vaccinated RMs that remained uninfected. These data suggest that the relationship between serum nAb titers and protection from mucosal SIV challenge in the setting of active immunization is more complex than previously recognized, warranting further studies into the balance between immune activation, target cell availability, and protective antibody responses.

  20. Characterization of electrogenic bromosulfophthalein transport in carnation petal microsomes and its inhibition by antibodies against bilitranslocase.

    PubMed

    Passamonti, Sabina; Cocolo, Alessandra; Braidot, Enrico; Petrussa, Elisa; Peresson, Carlo; Medic, Nevenka; Macri, Francesco; Vianello, Angelo

    2005-07-01

    Bilitranslocase is a rat liver plasma membrane carrier, displaying a high-affinity binding site for bilirubin. It is competitively inhibited by grape anthocyanins, including aglycones and their mono- and di-glycosylated derivatives. In plant cells, anthocyanins are synthesized in the cytoplasm and then translocated into the central vacuole, by mechanisms yet to be fully characterized. The aim of this work was to determine whether a homologue of rat liver bilitranslocase is expressed in carnation petals, where it might play a role in the membrane transport of anthocyanins. The bromosulfophthalein-based assay of rat liver bilitranslocase transport activity was implemented in subcellular membrane fractions, leading to the identification of a bromosulfophthalein carrier (K(M) = 5.3 microm), which is competitively inhibited by cyanidine 3-glucoside (Ki = 51.6 microm) and mainly noncompetitively by cyanidin (Ki = 88.3 microm). Two antisequence antibodies against bilitranslocase inhibited this carrier. In analogy to liver bilitranslocase, one antibody identified a bilirubin-binding site (Kd = 1.7 nm) in the carnation carrier. The other antibody identified a high-affinity binding site for cyanidine 3-glucoside (Kd = 1.7 microm) on the carnation carrier only, and a high-affinity bilirubin-binding site (Kd = 0.33 nm) on the liver carrier only. Immunoblots showed a putative homologue of rat liver bilitranslocase in both plasma membrane and tonoplast fractions, isolated from carnation petals. Furthermore, only epidermal cells were immunolabeled in petal sections examined by microscopy. In conclusion, carnation petals express a homologue of rat liver bilitranslocase, with a putative function in the membrane transport of secondary metabolites.

  1. Catechins and procyanidins of Ginkgo biloba show potent activities towards the inhibition of β-amyloid peptide aggregation and destabilization of preformed fibrils.

    PubMed

    Xie, Haiyan; Wang, Jing-Rong; Yau, Lee-Fong; Liu, Yong; Liu, Liang; Han, Quan-Bin; Zhao, Zhongzhen; Jiang, Zhi-Hong

    2014-04-22

    Catechins and procyanidins, together with flavonoid glycosides and terpene trilactones, are three important categories of components in the standard extract of Ginkgo biloba leaves (EGb761). In this research, catechins and proanthocyanidins were found to exist in both the extract of Ginkgo leaves and Ginkgo products. By comparing with reference compounds, six of them were identified as (+)-catechin, (-)-epicatechin, (-)-gallocatechin, (-)-epigallocatechin and procyanidins B1 and B3. The activities of these polyphenols in the inhibition of Aβ42 aggregation and the destabilization of preformed fibrils were evaluated using biochemical assays, which showed that all six of the polyphenols, as well as a fraction of the extract of Ginkgo biloba leaves (EGb) containing catechins and procyanidins, exerted potent inhibitory activities towards Aβ42 aggregation and could also destabilize the performed fibrils. Catechins and procyanidins can therefore be regarded as the potent active constituents of EGb761 in terms of their inhibition of Aβ42 aggregation and destabilization of the fibrils. Although quantitative mass spectroscopic analysis revealed that the catechins and procyanidins are only present in low concentrations in EGb761, these components should be studied in greater detail because of their potent inhibitory effects towards Aβ42 aggregation and their ability to destabilize preformed fibrils, especially during the quality control of Ginkgo leaves and the manufacture of Ginkgo products.

  2. Inhibition of autologous mixed lymphocyte reaction by monoclonal antibodies specific for the beta chain of HLA-DR antigens.

    PubMed

    Kasahara, M; Ikeda, H; Ogasawara, K; Ishikawa, N; Okuyama, T; Fukasawa, Y; Kojima, H; Kunikane, H; Hawkin, S; Ohhashi, T

    1984-09-01

    Recent studies using rabbit antisera to the separated HLA-DR alpha and beta subunits have suggested that alpha chain-specific, but not beta chain-specific, antisera inhibit T cell proliferative responses in primary and secondary human autologous mixed lymphocyte reaction (AMLR). In the present study, with the aid of sequential co-precipitation assays and Western blotting methods, a monoclonal rat alloantibody 1E4, specific for the beta chain of rat class II molecules carrying an Ia determinant Ba-2.7, was characterized to recognize a monomorphic determinant located on the beta chain of DR antigens. This antibody and a murine monoclonal antibody HU-4, also specific for the beta chain of DR antigens, strongly inhibited both primary and secondary AMLR through a mechanism distinct from an antibody-dependent cell-mediated cytotoxicity reaction. These results indicate that the inhibition of AMLR is not a unique feature of DR alpha-specific antibodies.

  3. Targeting Attenuated Interferon-α to Myeloma Cells with a CD38 Antibody Induces Potent Tumor Regression with Reduced Off-Target Activity

    PubMed Central

    Pogue, Sarah L.; Taura, Tetsuya; Bi, Mingying; Yun, Yong; Sho, Angela; Mikesell, Glen; Behrens, Collette; Sokolovsky, Maya; Hallak, Hussein; Rosenstock, Moti; Sanchez, Eric; Chen, Haiming; Berenson, James; Doyle, Anthony; Nock, Steffen; Wilson, David S.

    2016-01-01

    Interferon-α (IFNα) has been prescribed to effectively treat multiple myeloma (MM) and other malignancies for decades. Its use has waned in recent years, however, due to significant toxicity and a narrow therapeutic index (TI). We sought to improve IFNα’s TI by, first, attaching it to an anti-CD38 antibody, thereby directly targeting it to MM cells, and, second, by introducing an attenuating mutation into the IFNα portion of the fusion protein rendering it relatively inactive on normal, CD38 negative cells. This anti-CD38-IFNα(attenuated) immunocytokine, or CD38-Attenukine™, exhibits 10,000-fold increased specificity for CD38 positive cells in vitro compared to native IFNα and, significantly, is ~6,000-fold less toxic to normal bone marrow cells in vitro than native IFNα. Moreover, the attenuating mutation significantly decreases IFNα biomarker activity in cynomolgus macaques indicating that this approach may yield a better safety profile in humans than native IFNα or a non-attenuated IFNα immunocytokine. In human xenograft MM tumor models, anti-CD38-IFNα(attenuated) exerts potent anti-tumor activity in mice, inducing complete tumor regression in most cases. Furthermore, anti-CD38-IFNα(attenuated) is more efficacious than standard MM treatments (lenalidomide, bortezomib, dexamethasone) and exhibits strong synergy with lenalidomide and with bortezomib in xenograft models. Our findings suggest that tumor-targeted attenuated cytokines such as IFNα can promote robust tumor killing while minimizing systemic toxicity. PMID:27611189

  4. Structural definition of a potent macrophage activating factor derived from vitamin D3-binding protein with adjuvant activity for antibody production.

    PubMed

    Yamamoto, N

    1996-10-01

    Incubation of human vitamin D3-binding protein (Gc protein), with a mixture of immobilized beta-galactosidase and sialidase, efficiently generated a potent macrophage activating factor, a protein with N-acetylgalactosamine as the remaining sugar. Stepwise incubation of Gc protein with immobilized beta-galactosidase and sialidase, and isolation of the intermediates with immobilized lectins, revealed that either sequence of hydrolysis of Gc glycoprotein by these glycosidases yields the macrophage-activating factor, implying that Gc protein carries a trisaccharide composed of N-acetylgalactosamine and dibranched galactose and sialic acid termini. A 3 hr incubation of mouse peritoneal macrophages with picomolar amounts of the enzymatically generated macrophage-activating factor (GcMAF) resulted in a greatly enhanced phagocytic activity. Administration of a minute amount (10-50 pg/mouse) of GcMAF resulted in a seven- to nine-fold enhanced phagocytic activity of macrophages. Injection of sheep red blood cells (SRBC) along with GcMAF into mice produced a large number of anti-SRBC antibody secreting splenic cells in 2-4 days.

  5. The mechanism of potent GTP cyclohydrolase I inhibition by 2,4-diamino-6-hydroxypyrimidine: requirement of the GTP cyclohydrolase I feedback regulatory protein.

    PubMed

    Kolinsky, Monica A; Gross, Steven S

    2004-09-24

    Inhibition of GTP cyclohydrolase I (GTPCH) has been used as a selective tool to assess the role of de novo synthesis of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) in a biological system. Toward this end, 2,4-diamino-6-hydroxypyrimidine (DAHP) has been used as the prototypical GTPCH inhibitor. Using a novel real-time kinetic microplate assay for GTPCH activity and purified prokaryote-expressed recombinant proteins, we show that potent inhibition by DAHP is not the result of a direct interaction with GTPCH. Rather, inhibition by DAHP in phosphate buffer occurs via an indirect mechanism that requires the presence of GTPCH feedback regulatory protein (GFRP). Notably, GFRP was previously discovered as the essential factor that reconstitutes inhibition of pure recombinant GTPCH by the pathway end product BH4. Thus, DAHP inhibits GTPCH by engaging the endogenous feedback inhibitory system. We further demonstrate that L-Phe fully reverses the inhibition of GTPCH by DAHP/GFRP, which is also a feature in common with inhibition by BH4/GFRP. These findings suggest that DAHP is not an indiscriminate inhibitor of GTPCH in biological systems; instead, it is predicted to preferentially attenuate GTPCH activity in cells that most abundantly express GFRP and/or contain the lowest levels of L-Phe.

  6. Inhibition of viral reverse transcriptase and human sperm DNA polymerase by anti-sperm antibodies.

    PubMed Central

    Witkin, S S; Higgins, P J; Bendich, A

    1978-01-01

    The IgG fraction of serum from a rabbit immunized with detergent-prepared human sperm nuclei inhibited the DNA polymerase activities in human sperm and seminal fluid as well as the partially purified reverse transcriptase of the baboon endogenous type-C retrovirus (BEV). The analogous enzymes from lysates of oncogenic type-C viruses was unaffected. IgG from the serum of individual partners from infertile marriages similarly inhibited both purified BEV reverse transcriptase and human sperm DNA polymerase, but not a DNA polymerase isolated from human prostatic fluid. The data suggest that BEV reverse transcriptase and the human sperm DNA polymerase are antigenically related. Furthermore, the sperm appears to be auto-antigenic and the antibodies thus formed may be capable of interfering with reproductive success. PMID:82498

  7. Xanthatin, a novel potent inhibitor of VEGFR2 signaling, inhibits angiogenesis and tumor growth in breast cancer cells.

    PubMed

    Yu, Yao; Yu, Jing; Pei, Chong Gang; Li, Yun Yan; Tu, Ping; Gao, Gui Ping; Shao, Yi

    2015-01-01

    Anti-angiogenesis targeting vascular endothelial growth factor receptor 2 (VEGFR2) has emerged as an important tool for cancer treatment. In this study, we described a novel VEGFR2 inhibitor, xanthatin, which inhibits tumor angiogenesis and growth. The biochemical profiles of xanthatin were investigated using kinase assay, migration assay, tube formation, Matrigel plug assay, western blot, immunofluorescence and human tumor xenograft model. Xanthatin significantly inhibited growth, migration and tube formation of human umbilical vascular endothelial cell as well as inhibited vascular endothelial growth factor (VEGF)-stimulated angiogenesis. In addition, it inhibited VEGF-induced phosphorylation of VEGFR2 and its downstream signaling regulator. Moreover, xanthatin directly inhibit proliferation of breast cancer cells MDA-MB-231. Oral administration of xanthatin could markedly inhibit human tumor xenograft growth and decreased microvessel densities (MVD) in tumor sections. Taken together, these preclinical evaluations suggest that xanthatin inhibits angiogenesis and may be a promising anticancer drug candidate.

  8. Function-blocking ERBB3 antibody inhibits the adaptive response to RAF inhibitor

    PubMed Central

    Kugel, Curtis H.; Hartsough, Edward J.; Davies, Michael A.; Setiady, Yulius Y.; Aplin, Andrew E.

    2014-01-01

    ERBB3/HER3 expression and signaling is upregulated in mutant BRAF melanoma as an adaptive, pro-survival response to FDA-approved RAF inhibitors. Since compensatory ERBB3 signaling counteracts the effects of RAF inhibitors, co-targeting ERBB3 may increase the efficacy of RAF inhibitors in mutant BRAF models of melanoma. Here we corroborate this concept by showing that the ERBB3 function-blocking monoclonal antibody huHER3-8 can inhibit neuregulin-1 (NRG1) activation of ERBB3 and downstream signaling in RAF-inhibited melanoma cells. Targeting mutant BRAF in combination with huHER3-8 decreased cell proliferation and increased cell death in vitro, and decreased tumor burden in vivo, compared to targeting either mutant BRAF or ERBB3 alone. Further, the likelihood of a durable tumor response in vivo was increased when huHER3-8 was combined with RAF inhibitor PLX4720. Together, these results offer a preclinical proof of concept for the application of ERBB3 neutralizing antibodies to enhance the efficacy of RAF inhibitors in melanoma to delay or prevent tumor re-growth. Insofar as ERBB3 is often upregulated in response to other kinase-targeted therapeutics, findings may have implications for other cancers as well. PMID:25035390

  9. Inhibition of fibroblast growth factor receptor 3-dependent lung adenocarcinoma with a human monoclonal antibody

    PubMed Central

    Yin, Yongjun; Ren, Xiaodi; Smith, Craig; Guo, Qianxu; Malabunga, Maria; Guernah, Ilhem; Zhang, Yiwei; Shen, Juqun; Sun, Haijun; Chehab, Nabil; Loizos, Nick; Ludwig, Dale L.; Ornitz, David M.

    2016-01-01

    ABSTRACT Activating mutations in fibroblast growth factor receptor 3 (FGFR3) have been identified in multiple types of human cancer and in congenital birth defects. In human lung cancer, fibroblast growth factor 9 (FGF9), a high-affinity ligand for FGFR3, is overexpressed in 10% of primary resected non-small cell lung cancer (NSCLC) specimens. Furthermore, in a mouse model where FGF9 can be induced in lung epithelial cells, epithelial proliferation and ensuing tumorigenesis is dependent on FGFR3. To develop new customized therapies for cancers that are dependent on FGFR3 activation, we have used this mouse model to evaluate a human monoclonal antibody (D11) with specificity for the extracellular ligand-binding domain of FGFR3, that recognizes both human and mouse forms of the receptor. Here, we show that D11 effectively inhibits signaling through FGFR3 in vitro, inhibits the growth of FGFR3-dependent FGF9-induced lung adenocarcinoma in mice, and reduces tumor-associated morbidity. Given the potency of FGF9 in this mouse model and the absolute requirement for signaling through FGFR3, this study validates the D11 antibody as a potentially useful and effective reagent for treating human cancers or other pathologies that are dependent on activation of FGFR3. PMID:27056048

  10. Inhibition of tumorigenesis driven by different Wnt proteins requires blockade of distinct ligand-binding regions by LRP6 antibodies

    PubMed Central

    Ettenberg, Seth A.; Charlat, Olga; Daley, Michael P.; Liu, Shanming; Vincent, Karen J.; Stuart, Darrin D.; Schuller, Alwin G.; Yuan, Jing; Ospina, Beatriz; Green, John; Yu, Qunyan; Walsh, Renee; Schmitz, Rita; Heine, Holger; Bilic, Sanela; Ostrom, Lance; Mosher, Rebecca; Hartlepp, K. Felix; Zhu, Zhenping; Fawell, Stephen; Yao, Yung-Mae; Stover, David; Finan, Peter M.; Porter, Jeffery A.; Sellers, William R.; Klagge, Ingo M.; Cong, Feng

    2010-01-01

    Disregulated Wnt/β-catenin signaling has been linked to various human diseases, including cancers. Inhibitors of oncogenic Wnt signaling are likely to have a therapeutic effect in cancers. LRP5 and LRP6 are closely related membrane coreceptors for Wnt proteins. Using a phage-display library, we identified anti-LRP6 antibodies that either inhibit or enhance Wnt signaling. Two classes of LRP6 antagonistic antibodies were discovered: one class specifically inhibits Wnt proteins represented by Wnt1, whereas the second class specifically inhibits Wnt proteins represented by Wnt3a. Epitope-mapping experiments indicated that Wnt1 class-specific antibodies bind to the first propeller and Wnt3a class-specific antibodies bind to the third propeller of LRP6, suggesting that Wnt1- and Wnt3a-class proteins interact with distinct LRP6 propeller domains. This conclusion is further supported by the structural functional analysis of LRP5/6 and the finding that the Wnt antagonist Sclerostin interacts with the first propeller of LRP5/6 and preferentially inhibits the Wnt1-class proteins. We also show that Wnt1 or Wnt3a class-specific anti-LRP6 antibodies specifically block growth of MMTV-Wnt1 or MMTV-Wnt3 xenografts in vivo. Therapeutic application of these antibodies could be limited without knowing the type of Wnt proteins expressed in cancers. This is further complicated by our finding that bivalent LRP6 antibodies sensitize cells to the nonblocked class of Wnt proteins. The generation of a biparatopic LRP6 antibody blocks both Wnt1- and Wnt3a-mediated signaling without showing agonistic activity. Our studies provide insights into Wnt-induced LRP5/6 activation and show the potential utility of LRP6 antibodies in Wnt-driven cancer. PMID:20713706

  11. 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

  12. Laurenditerpenol, a New Diterpene from the Tropical Marine Alga Laurencia intricata Potently Inhibits HIF-1 Mediated Hypoxic Signaling in Breast Tumor Cells

    PubMed Central

    Mohammed, Kaleem A.; Hossain, Chowdhury Faiz; Zhang, Lei; Bruick, Richard K.; Zhou, Yu-Dong; Nagle, Dale G.

    2010-01-01

    The degree of tumor hypoxia correlates with advanced disease stages and treatment resistance. The transcription factor hypoxia-inducible factor-1 (HIF-1) promotes tumor cell adaptation and survival under hypoxic conditions. Therefore, specific HIF-1 inhibitors represent an important new class of potential tumor-selective therapeutic agents. A T47D human breast tumor cell-based reporter assay was used to examine extracts of plants and marine organisms for inhibitors of HIF-1 activation. Bioassay-guided fractionation of the lipid extract of the red alga Laurencia intricata yielded a structurally novel diterpene laurenditerpenol (1). The structure of 1 was determined spectroscopically. The relative configurations of the substituents of each ring system were assigned based on NOESY correlations. The absolute configurations of positions C-1 was determined by the modified Mosher ester procedure (directly in NMR tubes). Compound 1 potently inhibited hypoxia-activated HIF-1 (IC50: 0.4 μM) and hypoxia-induced VEGF (a potent angiogenic factor) in T47D cells. Compound 1 selectively inhibits HIF-1 activation by hypoxia but not iron chelator induced activation. Further, 1 suppresses tumor cell survival under hypoxic conditions without affecting normoxic cell growth. Compound 1 inhibits HIF-1 by blocking the induction of the oxygen-regulated HIF-1α protein. Mitochondrial respiration studies revealed that 1 suppresses oxygen consumption. PMID:15620241

  13. A small molecule antagonist of chemokine receptors CCR1 and CCR3. Potent inhibition of eosinophil function and CCR3-mediated HIV-1 entry.

    PubMed

    Sabroe, I; Peck, M J; Van Keulen, B J; Jorritsma, A; Simmons, G; Clapham, P R; Williams, T J; Pease, J E

    2000-08-25

    We describe a small molecule chemokine receptor antagonist, UCB35625 (the trans-isomer J113863 published by Banyu Pharmaceutical Co., patent WO98/04554), which is a potent, selective inhibitor of CCR1 and CCR3. Nanomolar concentrations of UCB35625 were sufficient to inhibit eosinophil shape change responses to MIP-1alpha, MCP-4, and eotaxin, while greater concentrations could inhibit the chemokine-induced internalization of both CCR1 and CCR3. UCB35625 also inhibited the CCR3-mediated entry of the human immunodeficiency virus-1 primary isolate 89.6 into the glial cell line, NP-2 (IC(50) = 57 nm). Chemotaxis of transfected cells expressing either CCR1 or CCR3 was inhibited by nanomolar concentrations of the compound (IC(50) values of CCR1-MIP-1alpha = 9.6 nm, CCR3-eotaxin = 93.7 nm). However, competitive ligand binding assays on the same transfectants revealed that considerably larger concentrations of UCB35625 were needed for effective ligand displacement than were needed for the inhibition of receptor function. Thus, it appears that the compound may interact with a region present in both receptors that inhibits the conformational change necessary to initiate intracellular signaling. By virtue of its potency at the two major eosinophil chemokine receptors, UCB35625 is a prototypic therapy for the treatment of eosinophil-mediated inflammatory disorders, such as asthma and as an inhibitor of CCR3-mediated human immunodeficiency virus-1 entry.

  14. Antibodies against a Plasmodium falciparum antigen PfMSPDBL1 inhibit merozoite invasion into human erythrocytes.

    PubMed

    Sakamoto, Hirokazu; Takeo, Satoru; Maier, Alexander G; Sattabongkot, Jetsumon; Cowman, Alan F; Tsuboi, Takafumi

    2012-03-02

    One approach to develop a malaria blood-stage vaccine is to target proteins that play critical roles in the erythrocyte invasion of merozoites. The merozoite surface proteins (MSPs) and the erythrocyte-binding antigens (EBAs) are considered promising vaccine candidates, for they are known to play important roles in erythrocyte invasion and are exposed to host immune system. Here we focused on a Plasmodium falciparum antigen, PfMSPDBL1 (encoded by PF10_0348 gene) that is a member of the MSP3 family and has both Duffy binding-like (DBL) domain and secreted polymorphic antigen associated with merozoites (SPAM) domain. Therefore, we aimed to characterize PfMSPDBL1 as a vaccine candidate. Recombinant full-length protein (rFL) of PfMSPDBL1 was synthesized by a wheat germ cell-free system, and rabbit antiserum was raised against rFL. We show that rabbit anti-PfMSPDBL1 antibodies inhibited erythrocyte invasion of wild type parasites in vitro in a dose dependent manner, and the specificity of inhibitory activity was confirmed using PfMSPDBL1 knockout parasites. Pre-incubation of the anti-PfMSPDBL1 antibodies with the recombinant SPAM domain had no effect on the inhibitory activity suggesting that antibodies to this region were not involved. In addition, antibodies to rFL were elicited by P. falciparum infection in malaria endemic area, suggesting the PfMSLDBL1 is immunogenic to humans. Our results suggest that PfMSPDBL1 is a novel blood-stage malaria vaccine candidate.

  15. Dual control mechanism for heme oxygenase: tin(IV)-protoporphyrin potently inhibits enzyme activity while markedly increasing content of enzyme protein in liver.

    PubMed Central

    Sardana, M K; Kappas, A

    1987-01-01

    Tin(IV)-protoporphyrin (Sn-protoporphyrin) potently inhibits heme degradation to bile pigments in vitro and in vivo, a property that confers upon this synthetic compound the ability to suppress a variety of experimentally induced and naturally occurring forms of jaundice in animals and humans. Utilizing rat liver heme oxygenase purified to homogeneity together with appropriate immunoquantitation techniques, we have demonstrated that Sn-protoporphyrin possesses the additional property of potently inducing the synthesis of heme oxygenase protein in liver cells while, concurrently, completely inhibiting the activity of the newly formed enzyme. Substitution of tin for the central iron atom of heme thus leads to the formation of a synthetic heme analogue that regulates heme oxygenase by a dual mechanism, which involves competitive inhibition of the enzyme for the natural substrate heme and simultaneous enhancement of new enzyme synthesis. Cobaltic(III)-protoporphyrin (Co-protoporphyrin) also inhibits heme oxygenase activity in vitro, but unlike Sn-protoporphyrin it greatly enhances the activity of the enzyme in the whole animal. Co-protoporphyrin also acts as an in vivo inhibitor of heme oxygenase; however, its inducing effect on heme oxygenase synthesis is so pronounced as to prevail in vivo over its inhibitory effect on the enzyme. These studies show that certain synthetic heme analogues possess the ability to simultaneously inhibit as well as induce the enzyme heme oxygenase in liver. The net balance between these two actions, as reflected in the rate of heme oxidation activity in the whole animal, appears to be influenced by the nature of the central metal atom of the synthetic metalloporphyrin. Images PMID:3470805

  16. TGF-β3 Inhibits Antibody Production by Human B Cells

    PubMed Central

    Tsuchida, Yumi; Sumitomo, Shuji; Ishigaki, Kazuyoshi; Suzuki, Akari; Kochi, Yuta; Tsuchiya, Haruka; Ota, Mineto; Komai, Toshihiko; Inoue, Mariko; Morita, Kaoru; Okamura, Tomohisa; Yamamoto, Kazuhiko; Fujio, Keishi

    2017-01-01

    TGF-β is a pleotropic cytokine involved in various biological processes. Of the three isoforms of TGF-β, TGF-β1 has long been recognized as an important inhibitory cytokine in the immune system and has been reported to inhibit B cell function in both mice and humans. Recently, it has been suggested that TGF-β3 may play an important role in the regulation of immune system in mice. Murine CD4+CD25-LAG3+ regulatory T cells suppress B cell function through the production of TGF-β3, and it has been reported that TGF-β3 is therapeutic in a mouse model of systemic lupus erythematosus. The effect of TGF-β3 on human B cells has not been reported, and we herein examined the effect of TGF-β3 on human B cells. TGF-β3 suppressed B cell survival, proliferation, differentiation into plasmablasts, and antibody secretion. Although the suppression of human B cells by TGF-β1 has long been recognized, the precise mechanism for the suppression of B cell function by TGF-β1 remains elusive; therefore, we examined the effect of TGF-β1 and β3 on pathways important in B cell activation and differentiation. TGF-β1 and TGF-β3 inhibited some of the key molecules of the cell cycle, as well as transcription factors important in B cell differentiation into antibody secreting cells such as IRF4, Blimp-1, and XBP1. TGF-β1 and β3 also inhibited B cell receptor signaling. Our results suggest that TGF-β3 modifying therapy might be therapeutic in autoimmune diseases with B cell dysregulation in humans. PMID:28052118

  17. Complement Inhibition for Prevention and Treatment of Antibody-Mediated Rejection in Renal Allograft Recipients.

    PubMed

    Jordan, S C; Choi, J; Kahwaji, J; Vo, A

    2016-04-01

    Therapeutic interventions aimed at the human complement system are recognized as potentially important strategies for the treatment of inflammatory and autoimmune diseases because there is often evidence of complement-mediated injury according to pathologic assessments. In addition, there are a large number of potential targets, both soluble and cell bound, that might offer potential for new drug development, but progress in this area has met with significant challenges. Currently, 2 drugs are approved aimed at inhibition of complement activation. The first option is eculizumab (anti-C5), which is approved for the treatment of paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. Eculizumab has also been studied in human transplantation for the treatment and prevention of antibody-mediated rejection (ABMR). Initial data from uncontrolled studies suggested a significant benefit of eculizumab for the prevention of ABMR in highly HLA-sensitized patients, but a subsequent randomized, placebo-controlled trial failed to meet its primary endpoint. Anecdotal data, primarily from case studies, showed benefits in treating complement-mediated ABMR. A second approved complement-inhibiting therapy is C1 esterase inhibitor (C1-INH), which is approved for use in patients with hereditary angioedema, a condition caused by mutations in the gene that codes for C1-INH. A recent placebo-controlled trial of C1-INH for prevention of ABMR in HLA-sensitized patients found that the drug was safe, with evidence for inhibition of systemic complement activation and complement-activating donor-specific antibodies. Other drugs are now under development.

  18. Inhibition of enzyme activity of Rhipicephalus (Boophilus) microplus triosephosphate isomerase and BME26 cell growth by monoclonal antibodies.

    PubMed

    Saramago, Luiz; Franceschi, Mariana; Logullo, Carlos; Masuda, Aoi; Vaz, Itabajara da Silva; Farias, Sandra Estrazulas; Moraes, Jorge

    2012-10-12

    In the present work, we produced two monoclonal antibodies (BrBm37 and BrBm38) and tested their action against the triosephosphate isomerase of Rhipicephalus (Boophilus) microplus (RmTIM). These antibodies recognize epitopes on both the native and recombinant forms of the protein. rRmTIM inhibition  by BrBm37 was up to 85% whereas that of BrBrm38 was 98%, depending on the antibody-enzyme ratio. RmTIM activity was lower in ovarian, gut, and fat body tissue extracts treated with BrBm37 or BrBm38 mAbs. The proliferation of the embryonic tick cell line (BME26) was inhibited by BrBm37 and BrBm38 mAbs. In summary, the results reveal that it is possible to interfere with the RmTIM function using antibodies, even in intact cells.

  19. Anti-phospholipid human monoclonal antibodies inhibit CCR5-tropic HIV-1 and induce β-chemokines

    PubMed Central

    Liao, Hua-Xin; Alam, S. Munir; Scearce, Richard M.; Plonk, M. Kelly; Kozink, Daniel M.; Drinker, Mark S.; Zhang, Ruijun; Xia, Shi-Mao; Sutherland, Laura L.; Tomaras, Georgia D.; Giles, Ian P.; Kappes, John C.; Ochsenbauer-Jambor, Christina; Edmonds, Tara G.; Soares, Melina; Barbero, Gustavo; Forthal, Donald N.; Landucci, Gary; Chang, Connie; King, Steven W.; Kavlie, Anita; Denny, Thomas N.; Hwang, Kwan-Ki; Chen, Pojen P.; Thorpe, Philip E.; Montefiori, David C.

    2010-01-01

    Traditional antibody-mediated neutralization of HIV-1 infection is thought to result from the binding of antibodies to virions, thus preventing virus entry. However, antibodies that broadly neutralize HIV-1 are rare and are not induced by current vaccines. We report that four human anti-phospholipid monoclonal antibodies (mAbs) (PGN632, P1, IS4, and CL1) inhibit HIV-1 CCR5-tropic (R5) primary isolate infection of peripheral blood mononuclear cells (PBMCs) with 80% inhibitory concentrations of <0.02 to ∼10 µg/ml. Anti-phospholipid mAbs inhibited PBMC HIV-1 infection in vitro by mechanisms involving binding to monocytes and triggering the release of MIP-1α and MIP-1β. The release of these β-chemokines explains both the specificity for R5 HIV-1 and the activity of these mAbs in PBMC cultures containing both primary lymphocytes and monocytes. PMID:20368576

  20. Inhibition of Enzyme Activity of Rhipicephalus (Boophilus) microplus Triosephosphate Isomerase and BME26 Cell Growth by Monoclonal Antibodies

    PubMed Central

    Saramago, Luiz; Franceschi, Mariana; Logullo, Carlos; Masuda, Aoi; Vaz, Itabajara da Silva; Farias, Sandra Estrazulas; Moraes, Jorge

    2012-01-01

    In the present work, we produced two monoclonal antibodies (BrBm37 and BrBm38) and tested their action against the triosephosphate isomerase of Rhipicephalus (Boophilus) microplus (RmTIM). These antibodies recognize epitopes on both the native and recombinant forms of the protein. rRmTIM inhibition by BrBm37 was up to 85% whereas that of BrBrm38 was 98%, depending on the antibody-enzyme ratio. RmTIM activity was lower in ovarian, gut, and fat body tissue extracts treated with BrBm37 or BrBm38 mAbs. The proliferation of the embryonic tick cell line (BME26) was inhibited by BrBm37 and BrBm38 mAbs. In summary, the results reveal that it is possible to interfere with the RmTIM function using antibodies, even in intact cells. PMID:23202941

  1. Significant decrease of ADP release rate underlies the potent activity of dimethylenastron to inhibit mitotic kinesin Eg5 and cancer cell proliferation

    SciTech Connect

    Sun, Linlin; Sun, Xiaodong; Xie, Songbo; Yu, Haiyang; Zhong, Diansheng

    2014-05-09

    Highlights: • DIMEN displays higher anti-proliferative activity than enastron. • DIMEN induced mitotic arrest and apoptosis more significantly than enastron. • DIMEN blocked the conformational change of ADP-binding pocket more effectively. • DIMEN hindered ADP release more potently than enastron. - Abstract: Eg5 is a mitotic kinesin that plays a crucial role in the formation of bipolar mitotic spindles, by hydrolyzing ATP to push apart anti-parallel microtubules. Dimethylenastron is potent specific small molecule inhibitor of Eg5. The mechanism by which dimethylenastron inhibits Eg5 function remains unclear. By comparing with enastron, here we report that dimethylenastron prevents the growth of pancreatic and lung cancer cells more effectively, by halting mitotic progression and triggering apoptosis. We analyze their interactions with ADP-bound Eg5 crystal structure, and find that dimethylenastron binds Eg5 motor domain with higher affinity. In addition, dimethylenastron allosterically blocks the conformational change of the “sandwich”-like ADP-binding pocket more effectively. We subsequently use biochemical approach to reveal that dimethylenastron slows ADP release more significantly than enastron. These data thus provide biological, structural and mechanistic insights into the potent inhibitory activity of dimethylenastron.

  2. Inhibition of insulin degradation by hepatoma cells after microinjection of monoclonal antibodies to a specific cytosolic protease.

    PubMed Central

    Shii, K; Roth, R A

    1986-01-01

    Four monoclonal antibodies were identified by their ability to bind to 125I-labeled insulin covalently linked to a cytosolic insulin-degrading enzyme from human erythrocytes. All four antibodies were also found to remove more than 90% of the insulin-degrading activity from erythrocyte extracts. These antibodies were shown to be directed to different sites on the enzyme by mapping studies and by their various properties. Two antibodies recognized the insulin-degrading enzyme from rat liver; one inhibited the erythrocyte enzyme directly; and two recognized the enzyme after gel electrophoresis and transfer to nitrocellulose filters. By this latter procedure and immunoprecipitation from metabolically labeled cells, the enzyme from a variety of tissues was shown to be composed of a single polypeptide chain of apparent Mr 110,000. Finally, these monoclonal antibodies were microinjected into the cytoplasm of a human hepatoma cell line to assess the contribution of this enzyme to insulin degradation in the intact cell. In five separate experiments, preloading of cells with these monoclonal antibodies resulted in an inhibition of insulin degradation of 18-54% (average 39%) and increased the amount of 125I-labeled insulin associated with the cells. In contrast, microinjection of control antibody or an extraneous monoclonal antibody had no effect on insulin degradation or on the amount of insulin associated with the cells. Moreover, the monoclonal antibodies to the insulin-degrading enzyme caused no significant inhibition of degradation of another molecule, low density lipoprotein. Thus, these results support a role for this enzyme in insulin degradation in the intact cell. Images PMID:2424018

  3. Inhibition of HIV-1 Env-Mediated Cell-Cell Fusion by Lectins, Peptide T-20, and Neutralizing Antibodies

    PubMed Central

    Yee, Michael; Konopka, Krystyna; Balzarini, Jan; Düzgüneş, Nejat

    2011-01-01

    Background: Broadly cross-reactive, neutralizing human monoclonal antibodies, including 2F5, 2G12, 4E10 and IgG1 b12, can inhibit HIV-1 infection in vitro at very low concentrations. We examined the ability of these antibodies to inhibit cell-cell fusion between Clone69TRevEnv cells induced to express the viral envelope proteins, gp120/gp41 (Env), and highly CD4-positive SupT1 cells. The cells were loaded with green and red-orange cytoplasmic fluorophores, and fusion was monitored by fluorescence microscopy. Results: Cell-cell fusion was inhibited completely by the carbohydrate binding proteins (CBPs), Hippeastrum hybrid (Amaryllis) agglutinin (HHA), and Galanthus nivalis (Snowdrop) agglutinin (GNA), and by the peptide, T-20, at relatively low concentrations. Anti-gp120 and anti-gp41 antibodies, at concentrations much higher than those required for neutralization, were not particularly effective in inhibiting fusion. Monoclonal antibodies b12, m14 IgG and 2G12 had moderate inhibitory activity; the IC50 of 2G12 was about 80 µg/ml. Antibodies 4E10 and 2F5 had no inhibitory activity at the concentrations tested. Conclusions: These observations raise concerns about the ability of neutralizing antibodies to inhibit the spread of viral genetic material from infected cells to uninfected cells via cell-cell fusion. The interaction of gp120/gp41 with cell membrane CD4 may be different in cell-cell and virus-cell membrane fusion reactions, and may explain the differential effects of antibodies in these two systems. The fluorescence assay described here may be useful in high throughput screening of potential HIV fusion inhibitors. PMID:21660189

  4. Oblongifolin M, an active compound isolated from a Chinese medical herb Garcinia oblongifolia, potently inhibits enterovirus 71 reproduction through downregulation of ERp57.

    PubMed

    Wang, Mengjie; Dong, Qi; Wang, Hua; He, Yaqing; Chen, Ying; Zhang, Hong; Wu, Rong; Chen, Xinchun; Zhou, Boping; He, Jason; Kung, Hsiang-Fu; Huang, Canhua; Wei, Yuquan; Huang, Jian-dong; Xu, Hongxi; He, Ming-Liang

    2016-02-23

    There is no effective drug to treat EV71 infection yet. Traditional Chinese herbs are great resources for novel antiviral compounds. Here we showed that Oblongifolin M (OM), an active compound isolated from Garcinia oblongifolia, potently inhibited EV71 infection in a dose dependent manner. To identify its potential effectors in the host cells, we successfully identified 18 proteins from 52 differentially expressed spots by comparative proteomics studies. Further studies showed that knockdown of ERp57 inhibited viral replication through downregulating viral IRES (internal ribosome entry site) activities, whereas ectopic expression of ERp57 increased IRES activity and partly rescued the inhibitory effects of OM on viral replication. We demonstrated that OM is an effective antiviral agent; and that ERp57 is one of its cellular effectors against EV71 infection.

  5. Oblongifolin M, an active compound isolated from a Chinese medical herb Garcinia oblongifolia, potently inhibits enterovirus 71 reproduction through downregulation of ERp57

    PubMed Central

    Wang, Hua; He, Yaqing; Chen, Ying; Zhang, Hong; Wu, Rong; Chen, Xinchun; Zhou, Boping; He, Jason; Kung, Hsiang-Fu; Huang, Canhua; Wei, Yuquan; Huang, Jian-dong; Xu, Hongxi; He, Ming-Liang

    2016-01-01

    There is no effective drug to treat EV71 infection yet. Traditional Chinese herbs are great resources for novel antiviral compounds. Here we showed that Oblongifolin M (OM), an active compound isolated from Garcinia oblongifolia, potently inhibited EV71 infection in a dose dependent manner. To identify its potential effectors in the host cells, we successfully identified 18 proteins from 52 differentially expressed spots by comparative proteomics studies. Further studies showed that knockdown of ERp57 inhibited viral replication through downregulating viral IRES (internal ribosome entry site) activities, whereas ectopic expression of ERp57 increased IRES activity and partly rescued the inhibitory effects of OM on viral replication. We demonstrated that OM is an effective antiviral agent; and that ERp57 is one of its cellular effectors against EV71 infection. PMID:26848777

  6. AP24534, a Pan-BCR-ABL Inhibitor for Chronic Myeloid Leukemia, Potently Inhibits the T315I Mutant and Overcomes Mutation-Based Resistance

    SciTech Connect

    O’Hare, Thomas; Shakespeare, William C.; Zhu, Xiaotian; Eide, Christopher A.; Rivera, Victor M.; Wang, Frank; Adrian, Lauren T.; Zhou, Tianjun; Huang, Wei-Sheng; Xu, Qihong; Metcalf, III, Chester A.; Tyner, Jeffrey W.; Loriaux, Marc M.; Corbin, Amie S.; Wardwell, Scott; Ning, Yaoyu; Keats, Jeffrey A.; Wang, Yihan; Sundaramoorthi, Raji; Thomas, Mathew; Zhou, Dong; Snodgrass, Joseph; Commodore, Lois; Sawyer, Tomi K.; Dalgarno, David C.; Deininger, Michael W.N.; Druker, Brian J.; Clackson, Tim

    2010-09-07

    Inhibition of BCR-ABL by imatinib induces durable responses in many patients with chronic myeloid leukemia (CML), but resistance attributable to kinase domain mutations can lead to relapse and a switch to second-line therapy with nilotinib or dasatinib. Despite three approved therapeutic options, the cross-resistant BCR-ABL{sup T315I} mutation and compound mutants selected on sequential inhibitor therapy remain major clinical challenges. We report design and preclinical evaluation of AP24534, a potent, orally available multitargeted kinase inhibitor active against T315I and other BCR-ABL mutants. AP24534 inhibited all tested BCR-ABL mutants in cellular and biochemical assays, suppressed BCR-ABL{sup T315I}-driven tumor growth in mice, and completely abrogated resistance in cell-based mutagenesis screens. Our work supports clinical evaluation of AP24534 as a pan-BCR-ABL inhibitor for treatment of CML.

  7. AP24534, a Pan-BCR-ABL Inhibitor for Chronic Myeloid Leukemia, Potently Inhibits the T315I Mutant and Overcomes Mutation-Based Resistance

    PubMed Central

    O’Hare, Thomas; Shakespeare, William C.; Zhu, Xiaotian; Eide, Christopher A.; Rivera, Victor M.; Wang, Frank; Adrian, Lauren T.; Zhou, Tianjun; Huang, Wei-Sheng; Xu, Qihong; Metcalf, Chester A.; Tyner, Jeffrey W.; Loriaux, Marc M.; Corbin, Amie S.; Wardwell, Scott; Ning, Yaoyu; Keats, Jeffrey A.; Wang, Yihan; Sundaramoorthi, Raji; Thomas, Mathew; Zhou, Dong; Snodgrass, Joseph; Commodore, Lois; Sawyer, Tomi K.; Dalgarno, David C.; Deininger, Michael W.N.; Druker, Brian J.; Clackson, Tim

    2009-01-01

    SUMMARY Inhibition of BCR-ABL by imatinib induces durable responses in many patients with chronic myeloid leukemia (CML), but resistance attributable to kinase domain mutations can lead to relapse and a switch to second-line therapy with nilotinib or dasatinib. Despite three approved therapeutic options, the cross-resistant BCR-ABLT315I mutation and compound mutants selected on sequential inhibitor therapy remain major clinical challenges. We report design and pre-clinical evaluation of AP24534, a potent, orally available multi-targeted kinase inhibitor active against T315I and other BCR-ABL mutants. AP24534 inhibited all tested BCR-ABL mutants in cellular and biochemical assays, suppressed BCR-ABLT315I-driven tumor growth in mice, and completely abrogated resistance in cell-based mutagenesis screens. Our work supports clinical evaluation of AP24534 as a pan-BCR-ABL inhibitor for treatment of CML. PMID:19878872

  8. Triosephosphate isomerase of Taenia solium (TTPI): phage display and antibodies as tools for finding target regions to inhibit catalytic activity.

    PubMed

    Sanabria-Ayala, Víctor; Belmont, Iaraset; Abraham, Landa

    2015-01-01

    Previous studies demonstrated that antibodies against triosephosphate isomerase of Taenia solium (TTPI) can alter its enzymatic catalysis. In the present study, we used antibodies produced against the NH2-terminal region of TTPI (1/3NH2TTPI) and the phage display technology to find target regions to inhibit TTPI activity. As a first step, we obtained polyclonal antibodies against non-conserved regions from the 1/3NH2TTPI, which had an inhibitory effect of about 74 % on catalytic activity. Afterward, they were used to screen a library of phage-displayed dodecapeptides; as a result, 41 phage mimotope clones were isolated and grouped according to their amino acid sequence, finding the consensus A1 (VPTXPI), A2 (VPTXXI), B (LTPGQ), and D (DPLPR). Antibodies against selected phage mimotope clones were obtained by rabbit's immunization; these ones clearly recognized TTPI by both Western blot and ELISA. However, only the mimotope PDTS16 (DSVTPTSVMAVA) clone, which belongs to the VPTXXI consensus, raised antibodies capable of inhibiting the TTPI catalytic activity in 45 %. Anti-PDTS16 antibodies were confronted to several synthetic peptides that encompass the 1/3NH2TTPI, and they only recognized three, which share the motif FDTLQK belonging to the helix-α1 in TTPI. This suggests that this motif is the main part of the epitope recognized by anti-PDTS16 antibodies and revealed its importance for TTPI catalysis.

  9. Electrochemical detection of anti-tau antibodies binding to tau protein and inhibition of GSK-3β-catalyzed phosphorylation.

    PubMed

    Esteves-Villanueva, Jose O; Martic-Milne, Sanela

    2016-03-01

    Tau protein hyperphosphorylation triggers tau aggregation and its toxicity, leading to neuronal death and cell-to-cell toxicity. Hence, inhibition of protein kinases is a viable tool toward reduction of tau toxicity. By targeting various epitopes of Tau441 protein immobilized on Au surface, the protein kinase inhibition by anti-tau antibodies was measured by surface electrochemistry. The electrochemical impedance spectroscopy was used to measure the charge transfer resistance (Rct) of nonphosphorylated tau-Au film (nTau-Au) and compared with the phosphorylated tau-Au film (pTau-Au). The pTau-Au films were characterized by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS), which indicated high phosphorus content. The Rct factor was used as the measure of inhibition efficacies by anti-tau antibodies (D8, A10, P262, and Tau46) in addition to antibody formulation intravenous immunoglobulin (IVIG). The Rct factor for pTau-Au in the absence of antibodies was 0.25 ± 0.08, indicating a dramatic decrease in Rct on phosphorylation. The Rct factors for Tau46 and A10 were 0.57 ± 0.22 and 0.65 ± 0.26, respectively, indicating phosphorylation inhibition. All antibodies exhibited similar binding to nTau-Au. The proposed electrochemical assay may be used for detection of other posttranslational modifications.

  10. Potent inhibition of endopeptidase 24.16 and endopeptidase 24.15 by the phosphonamide peptide N-(phenylethylphosphonyl)-Gly-L-Pro-L-aminohexanoic acid.

    PubMed

    Barelli, H; Dive, V; Yiotakis, A; Vincent, J P; Checler, F

    1992-10-15

    A phosphonamide peptide, N-(phenylethylphosphonyl)-Gly-L-Pro-L-aminohexanoic acid, previously shown to block Clostridium histolyticum collagenases, was examined as a putative inhibitor of endopeptidase 24.16 and endopeptidase 24.15. Hydrolysis of two endopeptidase 24.16 substrates, i.e. 3-carboxy-7-methoxycoumarin (Mcc)-Pro-Leu-Gly-Pro-D-Lys-dinitrophenyl (Dnp) and neurotensin, were completely and dose-dependently inhibited by the phosphonamide inhibitor with KI values of 0.3 and 0.9 nM respectively. In addition, the phosphonamide peptide inhibited the hydrolysis of benzoyl (Bz)-Gly-Ala-Ala-Phe-(pAB) p-aminobenzoate and neurotensin by endopeptidase 24.15 with about a 10-fold lower potency (KI values of 5 and 7.5 nM respectively). The selectivity of this inhibitor towards several exo- and endo-peptidases belonging to the zinc-containing metallopeptidase family established that a 1 microM concentration of this inhibitor was unable to affect leucine aminopeptidase, carboxypeptidase A, angiotensin-converting enzyme and endopeptidase 24.11. The present paper therefore reports on the first hydrophilic highly potent endopeptidase 24.16 inhibitor and describes the most potent inhibitory agent directed towards endopeptidase 24.15 developed to date. These tools should allow one to assess the contribution of endopeptidase 24.16 and endopeptidase 24.15 to the physiological inactivation of neurotensin as well as other neuropeptides.

  11. More potent inhibition of human CYP2A6 than mouse CYP2A5 enzyme activities by derivatives of phenylethylamine and benzaldehyde.

    PubMed

    Rahnasto, M; Raunio, H; Poso, A; Juvonen, R O

    2003-05-01

    1. A rapid 96-well plate assay method was developed and validated to measure liver microsomal coumarin 7-hydroxylation in vitro. 2. The method was used to test inhibition of human and mouse CYP2A enzymes by three phenylethylamine derivatives 2-(p-tolyl)-ethylamine, amphetamine, 2-phenylethylamine and benzaldehyde, and two of its derivatives, 4-methylbenzaldehyde and 4-methoxybenzaldehyde. 3. The benzaldehyde derivatives were more potent inhibitors of CYP2A5 than the phenylethylamines. The K(ic) value of 4-methylbenzaldehyde was 3.4 micro M and for 4-methoxybenzaldehyde it was 0.86 micro M for CYP2A5. 4. Amphetamine is a weak inhibitor of CYP2A6, whereas benzaldehyde is a suicide inhibitor with K(inact) = 0.16 min(-1) and K(I) = 18 micro M. The K(ic) values of 2-phenylethylamine, 2-(p-tolyl)-ethylamine, 4-methylbenzaldehyde and 4-methoxybenzaldehyde were 1.13, 0.23, 0.36 and 0.73 micro M for CYP2A6, respectively. 5. Novel potent inhibitors were found for CYP2A6 and, except for 4-methoxybenzaldehyde, all the compounds inhibited CYP2A5 and CYP2A6 enzymes differentially. These data add to the refinement of CYP2A enzyme active sites and provide chemical leads for developing novel chemical inhibitors of the CYP2A6 enzyme.

  12. Water extracts of cinnamon and clove exhibits potent inhibition of protein glycation and anti-atherosclerotic activity in vitro and in vivo hypolipidemic activity in zebrafish.

    PubMed

    Jin, Seori; Cho, Kyung-Hyun

    2011-07-01

    Advanced glycation end products contribute to the pathogenesis of diabetic complications and atherosclerosis. Aqueous extracts of ground pepper, cinnamon, rosemary, ginger, and clove were analyzed and tested for anti-atherosclerotic activity in vitro and in vivo using hypercholesterolemic zebrafish. Cinnamon and clove extracts (at final 10 μg/mL) had the strongest anti-glycation and antioxidant activity in this study. Cinnamon and clove had the strongest inhibition of activity against copper-mediated low-density lipoprotein (LDL) oxidation and LDL phagocytosis by macrophages. Cinnamon or clove extracts had potent cholesteryl ester transfer protein (CETP) inhibitory activity in a concentration-dependent manner. They exhibited hypolipidemic activity in a hypercholesterolemic zebrafish model; the clove extract-treated group had a 68% and 80% decrease in serum cholesterol and TG levels, respectively. The clove extract-fed group had the smallest increase in body weight and height and the strongest antioxidant activity following a 5-week high cholesterol diet. Hydrophilic ingredients of cinnamon and clove showed potent activities to suppress the incidence of atherosclerosis and diabetes via strong antioxidant potential, prevention of apoA-I glycation and LDL-phagocytosis, inhibition of CETP, and hypolipidemic activity. These results suggest the potential to develop a new functional dietary agent to treat chronic metabolic diseases, such as hyperlipidemia and diabetes.

  13. Potent and selective inhibition of varicella-zoster virus (VZV) by nucleoside analogues with an unusual bicyclic base.

    PubMed

    McGuigan, C; Yarnold, C J; Jones, G; Velázquez, S; Barucki, H; Brancale, A; Andrei, G; Snoeck, R; De Clercq, E; Balzarini, J

    1999-11-04

    We herein report the discovery of an entirely new category of potent antiviral agents based on novel deoxynucleoside analogues with unusual bicyclic base moieties. Target structures, previously known as byproducts in Pd-catalyzed coupling of terminal alkynes with 5-iodo-nucleosides, are recognized herein for the first time to be potent and selective inhibitors of varicella-zoster virus (VZV) in vitro. As an unusual structure-activity relationship we noted the absolute requirement of a long alkyl side chain, with an optimum length of C(8)-C(10), for antiviral activity. We thus report the synthesis and characterization of a series of chain-modified analogues and their extensive in vitro evaluation. The lead compounds have a ca. 300-fold enhancement in anti-VZV activity over the reference compound acyclovir, with no detectable in vitro cytotoxicity. The novel structure of these compounds, coupled with their ease of synthesis, excellent antiviral profile, and promising physical properties, makes them of great interest for possible antiviral drug development.

  14. CSL311, a novel, potent, therapeutic monoclonal antibody for the treatment of diseases mediated by the common β chain of the IL-3, GM-CSF and IL-5 receptors.

    PubMed

    Panousis, Con; Dhagat, Urmi; Edwards, Kirsten M; Rayzman, Veronika; Hardy, Matthew P; Braley, Hal; Gauvreau, Gail M; Hercus, Timothy R; Smith, Steven; Sehmi, Roma; McMillan, Laura; Dottore, Mara; McClure, Barbara J; Fabri, Louis J; Vairo, Gino; Lopez, Angel F; Parker, Michael W; Nash, Andrew D; Wilson, Nicholas J; Wilson, Michael J; Owczarek, Catherine M

    2016-01-01

    The β common-signaling cytokines interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-5 stimulate pro-inflammatory activities of haematopoietic cells via a receptor complex incorporating cytokine-specific α and shared β common (βc, CD131) receptor. Evidence from animal models and recent clinical trials demonstrate that these cytokines are critical mediators of the pathogenesis of inflammatory airway disease such as asthma. However, no therapeutic agents, other than steroids, that specifically and effectively target inflammation mediated by all 3 of these cytokines exist. We employed phage display technology to identify and optimize a novel, human monoclonal antibody (CSL311) that binds to a unique epitope that is specific to the cytokine-binding site of the human βc receptor. The binding epitope of CSL311 on the βc receptor was defined by X-ray crystallography and site-directed mutagenesis. CSL311 has picomolar binding affinity for the human βc receptor, and at therapeutic concentrations is a highly potent antagonist of the combined activities of IL-3, GM-CSF and IL-5 on primary eosinophil survival in vitro. Importantly, CSL311 inhibited the survival of inflammatory cells present in induced sputum from human allergic asthmatic subjects undergoing allergen bronchoprovocation. Due to its high potency and ability to simultaneously suppress the activity of all 3 β common cytokines, CSL311 may provide a new strategy for the treatment of chronic inflammatory diseases where the human βc receptor is central to pathogenesis. The coordinates for the βc/CSL311 Fab complex structure have been deposited with the RCSB Protein Data Bank (PDB 5DWU).

  15. Env-2dCD4 S60C complexes act as super immunogens and elicit potent, broadly neutralizing antibodies against clinically relevant human immunodeficiency virus type 1 (HIV-1).

    PubMed

    Killick, Mark A; Grant, Michelle L; Cerutti, Nichole M; Capovilla, Alexio; Papathanasopoulos, Maria A

    2015-11-17

    The ability to induce a broadly neutralizing antibody (bNAb) response following vaccination is regarded as a crucial aspect in developing an effective vaccine against human immunodeficiency virus type 1 (HIV-1). The bNAbs target the HIV-1 envelope glycoprotein (Env) which is exposed on the virus surface, thereby preventing cell entry. To date, conventional vaccine approaches such as the use of Env-based immunogens have been unsuccessful. We expressed, purified, characterized and evaluated the immunogenicity of several unique HIV-1 subtype C Env immunogens in small animals. Here we report that vaccine immunogens based on Env liganded to a two domain CD4 variant, 2dCD4(S60C) are capable of consistently eliciting potent, broadly neutralizing antibody responses in New Zealand white rabbits against a panel of clinically relevant HIV-1 pseudoviruses. This was irrespective of the Env protein subtype and context. Importantly, depletion of the anti-CD4 antibodies appeared to abrogate the neutralization activity in the rabbit sera. Taken together, this data suggests that the Env-2dCD4(S60C) complexes described here are "super" immunogens, and potentially immunofocus antibody responses to a unique epitope spanning the 2dCD4(60C). Recent data from the two available anti-CD4 monoclonal antibodies, Ibalizumab and CD4-Ig (and bispecific variants thereof) have highlighted that the use of these broad and potent entry inhibitors could circumvent the need for a conventional vaccine targeting HIV-1. Overall, the ability of the unique Env-2dCD4(S60C) complexes to elicit potent bNAb responses has not been described previously, reinforcing that further investigation for their utility in preventing and controlling HIV-1/SIV infection is warranted.

  16. Epitope mapping for the monoclonal antibody that inhibits intramolecular electron transfer in flavocytochrome b2.

    PubMed Central

    Lê, K H Diêp; Mayer, Martine; Lederer, Florence

    2003-01-01

    Flavocytochrome b(2) (yeast L-lactate dehydrogenase) carries one FMN and one protohaem IX on each of its four subunits. The prosthetic groups are bound to separate domains, the haem domain (residues 1-99) and the flavin domain (residues 100-485), which interact for electron transfer between lactate-reduced FMN and haem b(2); in vivo, the latter reduces cytochrome c. In the crystal structure, one haem domain out of two is mobile. Previously we have described a monoclonal antibody, raised against the tetramer, that only recognizes the native haem domain and prevents electron transfer between flavin and haem, while having no effect on flavin reduction by the substrate [Miles, Lederer and Lê (1998) Biochemistry 37, 3440-3448]. In order to understand the structural basis of the uncoupling between the domains, we proceeded to site-directed mutagenesis, so as to map the epitope on the surface of the haem domain. We analysed the effects of 14 mutations at 12 different positions, located mostly in the domain interface or at its edge; we also analysed the effect of replacing protohaem IX with its dimethyl ester. We used as criteria the antibody-mediated inhibition of cytochrome c reduction by flavocytochrome b(2), competitive ELISA tests and surface plasmon resonance. We have thus defined a minimal epitope surface on the haem domain; it encompasses positions 63, 64, 65, 67, 69 and 70 and one or both haem propionates. When the haem and flavin domains are docked for electron transfer, the 65, 67 and 70 side chains, as well as the haem propionates, are excluded from solvent. The present results thus indicate that, when bound, the antibody acts as a wedge between the domains and constitutes a physical barrier to electron transfer. PMID:12646042

  17. Therapeutic antibody targeting of indoleamine-2,3-dioxygenase (IDO2) inhibits autoimmune arthritis.

    PubMed

    Merlo, Lauren M F; Grabler, Samantha; DuHadaway, James B; Pigott, Elizabeth; Manley, Kaylend; Prendergast, George C; Laury-Kleintop, Lisa D; Mandik-Nayak, Laura

    2017-02-20

    Rheumatoid arthritis (RA) is a debilitating inflammatory autoimmune disease with no known cure. Recently, we identified the immunomodulatory enzyme indoleamine-2,3-dioxygenase 2 (IDO2) as an essential mediator of autoreactive B and T cell responses driving RA. However, therapeutically targeting IDO2 has been challenging given the lack of small molecules that specifically inhibit IDO2 without also affecting the closely related IDO1. In this study, we develop a novel monoclonal antibody (mAb)-based approach to therapeutically target IDO2. Treatment with IDO2-specific mAb alleviated arthritis in two independent preclinical arthritis models, reducing autoreactive T and B cell activation and recapitulating the strong anti-arthritic effect of genetic IDO2 deficiency. Mechanistic investigations identified FcγRIIb as necessary for mAb internalization, allowing targeting of an intracellular antigen traditionally considered inaccessible to mAb therapy. Taken together, our results offer preclinical proof of concept for antibody-mediated targeting of IDO2 as a new therapeutic strategy to treat RA and other autoantibody-mediated diseases.

  18. Antibody-Based Detection and Inhibition of Vaginolysin, the Gardnerella vaginalis Cytolysin

    PubMed Central

    Randis, Tara M.; Kulkarni, Ritwij; Aguilar, Jorge L.; Ratner, Adam J.

    2009-01-01

    Bacterial vaginosis (BV) is the most common vaginal infection worldwide and is associated with significant adverse sequelae. We have recently characterized vaginolysin (VLY), the human-specific cytotoxin produced by Gardnerella vaginalis and believed to play a critical role in the pathogenesis of BV and its associated morbidities. We hypothesize that novel antibody-based strategies may be useful for detection of VLY and for inhibition of its toxic effects on human cells. Using purified toxin as an immunogen, we generated polyclonal rabbit immune serum (IS) against VLY. A western blot of G. vaginalis lysate was probed with IS and a single band (57 kD) identified. Immunofluorescence techniques using IS detected VLY production by G. vaginalis. In addition, we have developed a sandwich ELISA assay capable of VLY quantification at ng/ml concentrations in the supernatant of growing G. vaginalis. To investigate the potential inhibitory role of IS on VLY-mediated cell lysis, we exposed human erythrocytes to VLY or VLY pretreated with IS and determined the percent hemolysis. Pretreatment with IS resulted in a significant reduction in VLY-mediated lysis. Similarly, both human cervical carcinoma cells and vaginal epithelial cells exhibited reduced cytolysis following exposure to VLY with IS compared to VLY alone. These results confirm that antibody-based techniques are an effective means of VLY detection. Furthermore, VLY antiserum functions as an inhibitor of VLY–CD59 interaction, mitigating cell lysis. These strategies may have a potential role in the diagnosis and treatment of BV. PMID:19370149

  19. Structural basis for inhibition of erythrocyte invasion by antibodies to Plasmodium falciparum protein CyRPA

    PubMed Central

    Chen, Lin; Xu, Yibin; Wong, Wilson; Thompson, Jennifer K; Healer, Julie; Goddard-Borger, Ethan D; Lawrence, Michael C; Cowman, Alan F

    2017-01-01

    Plasmodium falciparum causes malaria in humans with over 450,000 deaths annually. The asexual blood stage involves invasion of erythrocytes by merozoites, in which they grow and divide to release daughter merozoites, which in turn invade new erythrocytes perpetuating the cycle responsible for malaria. A key step in merozoite invasion is the essential binding of PfRh5/CyRPA/PfRipr complex to basigin, a step linked to the formation of a pore between merozoites and erythrocytes. We show CyRPA interacts directly with PfRh5. An invasion inhibitory monoclonal antibody to CyRPA blocks binding of CyRPA to PfRh5 and complex formation thus illuminating the molecular mechanism for inhibition of parasite growth. We determined the crystal structures of CyRPA alone and in complex with an antibody Fab fragment. CyRPA has a six-bladed β-propeller fold, and we identify the region that interacts with PfRh5. This functionally conserved epitope is a potential target for vaccines against P. falciparum. DOI: http://dx.doi.org/10.7554/eLife.21347.001 PMID:28195530

  20. Inhibition of human tumor xenograft growth in nude mice by a conjugate of monoclonal antibody LA22 to epidermal growth factor receptor with anti-tumor antibiotics mitomycin C

    SciTech Connect

    Shao Wei; Zhao Shan; Liu Zhaofei; Zhang Jianzhong; Ma Shujun; Sato, J. Denry; Zhang Peng; Tong Mei; Han Jiping; Wang Yan; Bai Dongmei; Wang Fan . E-mail: wangfan@bjmu.edu.cn; Sun Le . E-mail: lsun@welsonpharma.com

    2006-10-20

    Anti-EGFR monoclonal antibodies LA22 and Erbitux bind to different epitopes of EGFR. The chemimmunoconjugates of MMC with LA22 or Erbitux were prepared, and in vitro cytotoxicity assays with A549 cells showed that LA22-MMC was much more potent than Erbitux or Erbitux-MMC. Viabilities of A549 cells treated with LA22-MMC, Erbitux or Erbitux-MMC were 35%, 94%, and 81%, respectively. Immunoscintigraphy of xenografts of human A431 and A549 cells in nude mice both showed that {sup 125}I-labeled-LA22-MMC enriched in tumor sites prominently. Most importantly, in vivo assays showed LA22-MMC was significantly more effective than free drug MMC in the treatment of subcutaneous xenografts of human A431 cells in nude mice (83% inhibition for LA22-MMC and 30% for MMC). We concluded that LA22-MMC could be a very potent drug for treatment of solid tumors.

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

    SciTech Connect

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

    1986-02-01

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

  2. A unique anti-CD115 monoclonal antibody which inhibits osteolysis and skews human monocyte differentiation from M2-polarized macrophages toward dendritic cells.

    PubMed

    Haegel, Hélène; Thioudellet, Christine; Hallet, Rémy; Geist, Michel; Menguy, Thierry; Le Pogam, Fabrice; Marchand, Jean-Baptiste; Toh, Myew-Ling; Duong, Vanessa; Calcei, Alexandre; Settelen, Nathalie; Preville, Xavier; Hennequi, Marie; Grellier, Benoit; Ancian, Philippe; Rissanen, Jukka; Clayette, Pascal; Guillen, Christine; Rooke, Ronald; Bonnefoy, Jean-Yves

    2013-01-01

    Cancer progression has been associated with the presence of tumor-associated M2-macrophages (M2-TAMs) able to inhibit anti-tumor immune responses. It is also often associated with metastasis-induced bone destruction mediated by osteoclasts. Both cell types are controlled by the CD115 (CSF-1R)/colony-stimulating factor-1 (CSF-1, M-CSF) pathway, making CD115 a promising target for cancer therapy. Anti-human CD115 monoclonal antibodies (mAbs) that inhibit the receptor function have been generated in a number of laboratories. These mAbs compete with CSF-1 binding to CD115, dramatically affecting monocyte survival and preventing osteoclast and macrophage differentiation, but they also block CD115/CSF-1 internalization and degradation, which could lead to potent rebound CSF-1 effects in patients after mAb treatment has ended. We thus generated and selected a non-ligand competitive anti-CD115 mAb that exerts only partial inhibitory effects on CD115 signaling without blocking the internalization or the degradation of the CD115/CSF-1 complex. This mAb, H27K15, affects monocyte survival only minimally, but downregulates osteoclast differentiation and activity. Importantly, it inhibits monocyte differentiation to CD163(+)CD64(+) M2-polarized suppressor macrophages, skewing their differentiation toward CD14(-)CD1a(+) dendritic cells (DCs). In line with this observation, H27K15 also drastically inhibits monocyte chemotactic protein-1 secretion and reduces interleukin-6 production; these two molecules are known to be involved in M2-macrophage recruitment. Thus, the non-depleting mAb H27K15 is a promising anti-tumor candidate, able to inhibit osteoclast differentiation, likely decreasing metastasis-induced osteolysis, and able to prevent M2 polarization of TAMs while inducing DCs, hence contributing to the creation of more efficient anti-tumor immune responses.

  3. Small molecule kinase inhibitor LRRK2-IN-1 demonstrates potent activity against colorectal and pancreatic cancer through inhibition of doublecortin-like kinase 1

    PubMed Central

    2014-01-01

    Background Doublecortin-like kinase 1 (DCLK1) is emerging as a tumor specific stem cell marker in colorectal and pancreatic cancer. Previous in vitro and in vivo studies have demonstrated the therapeutic effects of inhibiting DCLK1 with small interfering RNA (siRNA) as well as genetically targeting the DCLK1+ cell for deletion. However, the effects of inhibiting DCLK1 kinase activity have not been studied directly. Therefore, we assessed the effects of inhibiting DCLK1 kinase activity using the novel small molecule kinase inhibitor, LRRK2-IN-1, which demonstrates significant affinity for DCLK1. Results Here we report that LRRK2-IN-1 demonstrates potent anti-cancer activity including inhibition of cancer cell proliferation, migration, and invasion as well as induction of apoptosis and cell cycle arrest. Additionally we found that it regulates stemness, epithelial-mesenchymal transition, and oncogenic targets on the molecular level. Moreover, we show that LRRK2-IN-1 suppresses DCLK1 kinase activity and downstream DCLK1 effector c-MYC, and demonstrate that DCLK1 kinase activity is a significant factor in resistance to LRRK2-IN-1. Conclusions Given DCLK1’s tumor stem cell marker status, a strong understanding of its biological role and interactions in gastrointestinal tumors may lead to discoveries that improve patient outcomes. The results of this study suggest that small molecule inhibitors of DCLK1 kinase should be further investigated as they may hold promise as anti-tumor stem cell drugs. PMID:24885928

  4. Selective inhibition of EZH2 by ZLD1039 blocks H3K27methylation and leads to potent anti-tumor activity in breast cancer

    PubMed Central

    Song, Xuejiao; Gao, Tiantao; Wang, Ningyu; Feng, Qiang; You, Xinyu; Ye, Tinghong; Lei, Qian; Zhu, Yongxia; Xiong, Menghua; Xia, Yong; Yang, Fangfang; Shi, Yaojie; Wei, Yuquan; Zhang, Lidan; Yu, Luoting

    2016-01-01

    Enhancer of zeste homolog 2 (EZH2) is a candidate oncogenic driver due to its prevalent overexpression and aberrant repression of tumor suppressor genes in diverse cancers. Therefore, blocking EZH2 enzyme activity may present a valid therapeutic strategy for the treatment of cancers with EZH2 overexpression including breast cancers. Here, we described ZLD1039 a potent, highly selective, and orally bioavailable small molecule inhibitor of EZH2, which inhibited breast tumor growth and metastasis. ZLD1039 considerably inhibited EZH2 methyltransferase activity with nanomolar potency, decreased global histone-3 lysine-27 (H3K27) methylation, and reactivated silenced tumor suppressors connected to increased survival of patients with breast cancer. Comparable to conditional silencing of EZH2, its inhibition by ZLD1039 decreased cell proliferation, cell cycle arrest, and induced apoptosis. Comparably, treatment of xenograft-bearing mice with ZLD1039 led to tumor growth regression and metastasis inhibition. These data confirmed the dependency of breast cancer progression on EZH2 activity and the usefulness of ZLD1039 as a promising treatment for breast cancer. PMID:26868841

  5. Potent and synergistic neutralization of human immunodeficiency virus (HIV) type 1 primary isolates by hyperimmune anti-HIV immunoglobulin combined with monoclonal antibodies 2F5 and 2G12.

    PubMed

    Mascola, J R; Louder, M K; VanCott, T C; Sapan, C V; Lambert, J S; Muenz, L R; Bunow, B; Birx, D L; Robb, M L

    1997-10-01

    Three antibody reagents that neutralize primary human immunodeficiency virus type 1 (HIV-1) isolates were tested for magnitude and breadth of neutralization when used alone or in double or triple combinations. Hyperimmune anti-HIV immunoglobulin (HIVIG) is derived from the plasma of HIV-1-infected donors, and monoclonal antibodies (MAbs) 2F5 and 2G12 bind to distinct regions of the HIV-1 envelope glycoprotein. The antibodies were initially tested against a panel of 15 clade B HIV-1 isolates, using a single concentration that is achievable in vivo (HIVIG, 2,500 microg/ml; MAbs, 25 microg/ml). Individual antibody reagents neutralized many of the viruses tested, but antibody potency varied substantially among the viruses. The virus neutralization produced by double combinations of HIVIG plus 2F5 or 2G12, the two MAbs together, or the triple combination of HIVIG, 2F5, and 2G12 was generally equal to or greater than that predicted by the effect of individual antibodies. Overall, the triple combination displayed the greatest magnitude and breadth of neutralization. Synergistic neutralization was evaluated by analyzing data from dose-response curves of each individual antibody reagent compared to the triple combination and was demonstrated against each of four viruses tested. Therefore, combinations of polyclonal and monoclonal anti-HIV antibodies can produce additive or synergistic neutralization of primary HIV-1 isolates. Passive immunotherapy for treatment or prophylaxis of HIV-1 should consider mixtures of potent neutralizing antibody reagents to expand the magnitude and breadth of virus neutralization.

  6. Potent and synergistic neutralization of human immunodeficiency virus (HIV) type 1 primary isolates by hyperimmune anti-HIV immunoglobulin combined with monoclonal antibodies 2F5 and 2G12.

    PubMed Central

    Mascola, J R; Louder, M K; VanCott, T C; Sapan, C V; Lambert, J S; Muenz, L R; Bunow, B; Birx, D L; Robb, M L

    1997-01-01

    Three antibody reagents that neutralize primary human immunodeficiency virus type 1 (HIV-1) isolates were tested for magnitude and breadth of neutralization when used alone or in double or triple combinations. Hyperimmune anti-HIV immunoglobulin (HIVIG) is derived from the plasma of HIV-1-infected donors, and monoclonal antibodies (MAbs) 2F5 and 2G12 bind to distinct regions of the HIV-1 envelope glycoprotein. The antibodies were initially tested against a panel of 15 clade B HIV-1 isolates, using a single concentration that is achievable in vivo (HIVIG, 2,500 microg/ml; MAbs, 25 microg/ml). Individual antibody reagents neutralized many of the viruses tested, but antibody potency varied substantially among the viruses. The virus neutralization produced by double combinations of HIVIG plus 2F5 or 2G12, the two MAbs together, or the triple combination of HIVIG, 2F5, and 2G12 was generally equal to or greater than that predicted by the effect of individual antibodies. Overall, the triple combination displayed the greatest magnitude and breadth of neutralization. Synergistic neutralization was evaluated by analyzing data from dose-response curves of each individual antibody reagent compared to the triple combination and was demonstrated against each of four viruses tested. Therefore, combinations of polyclonal and monoclonal anti-HIV antibodies can produce additive or synergistic neutralization of primary HIV-1 isolates. Passive immunotherapy for treatment or prophylaxis of HIV-1 should consider mixtures of potent neutralizing antibody reagents to expand the magnitude and breadth of virus neutralization. PMID:9311792

  7. Anti-myeloma activity of a multi targeted kinase inhibitor, AT9283, via potent Aurora Kinase and STAT3 inhibition either alone or in combination with lenalidomide

    PubMed Central

    Santo, Loredana; Hideshima, Teru; Cirstea, Diana; Bandi, Madhavi; Nelson, Erik A.; Gorgun, Gullu; Rodig, Scott; Vallet, Sonia; Pozzi, Samantha; Patel, Kishan; Unitt, Christine; Squires, Matt; Hu, Yiguo; Chauhan, Dharminder; Mahindra, Anuj; Munshi, Nikhil C.; Anderson, Kenneth C.; Raje, Noopur

    2014-01-01

    Purpose Aurora Kinases, whose expression is linked to genetic instability and cellular proliferation, are under investigation as novel therapeutic targets in multiple myeloma (MM). Here, we investigated the preclinical activity of a small molecule–multi-targeted kinase inhibitor, AT9283, with potent activity against Aurora kinase A (AURKA), Aurora kinase B (AURKB) and Janus Kinase 2/3. Experimental design We evaluated the in vitro anti myeloma activity of AT9283 alone and in combination with lenalidomide and the in vivo efficacy by using a Xenograft mouse model of human MM. Results Our data demonstrated AT9283 induced cell growth inhibition and apoptosis in MM. Studying the apoptosis mechanism of AT9283 in MM, we observed features consistent with both AURKA and AURKB inhibition, e.g increase of cells with polyploid DNA content, decrease in phospho-Histone H3, and decrease of phospho-Aurora A. Importantly, AT9283 also inhibited STAT3 tyrosine phosphorylation in MM cells. Genetic depletion of STAT3, AURKA or AURKB showed growth inhibition of MM cells, suggesting a role of AT9283-induced inhibition of these molecules in the underlying mechanism of MM cell death. In vivo studies demonstrated decreased MM cell growth and prolonged survival in AT9283-treated mice compared to controls. Importantly, combination studies of AT9283 with lenalidomide showed significant synergistic cytotoxicity in MM cells, even in the presence of bone marrow stromal cells (BMSCs). Enhanced cytotoxicity was associated with increased inhibition of pSTAT3 and pERK. Conclusions Demonstration of in vitro and in vivo anti-MM activity of AT9283 provides the rationale for the clinical evaluation of AT9283 as monotherapy and in combination in patients with MM. PMID:21430070

  8. A novel sulindac derivative that potently suppresses colon tumor cell growth by inhibiting cGMP phosphodiesterase and β-catenin transcriptional activity.

    PubMed

    Whitt, Jason D; Li, Nan; Tinsley, Heather N; Chen, Xi; Zhang, Wei; Li, Yonghe; Gary, Bernard D; Keeton, Adam B; Xi, Yaguang; Abadi, Ashraf H; Grizzle, William E; Piazza, Gary A

    2012-06-01

    Nonsteroidal anti-inflammatory drugs (NSAIDs) have been widely reported to inhibit tumor growth by a COX-independent mechanism, although alternative targets have not been well defined or used to develop improved drugs for cancer chemoprevention. Here, we characterize a novel sulindac derivative referred to as sulindac benzylamine (SBA) that does not inhibit COX-1 or COX-2, yet potently inhibits the growth and induces the apoptosis of human colon tumor cells. The basis for this activity appears to involve cyclic guanosine 3',5',-monophosphate phosphodiesterase (cGMP PDE) inhibition as evident by its ability to inhibit cGMP hydrolysis in colon tumor cell lysates and purified cGMP-specific PDE5, increase intracellular cGMP levels, and activate cGMP-dependent protein kinase G at concentrations that suppress tumor cell growth. PDE5 was found to be essential for colon tumor cell growth as determined by siRNA knockdown studies, elevated in colon tumor cells as compared with normal colonocytes, and associated with the tumor selectivity of SBA. SBA activation of PKG may suppress the oncogenic activity of β-catenin as evident by its ability to reduce β-catenin nuclear levels, Tcf (T-cell factor) transcriptional activity, and survivin levels. These events preceded apoptosis induction and appear to result from a rapid elevation of intracellular cGMP levels following cGMP PDE inhibition. We conclude that PDE5 and possibly other cGMP degrading isozymes can be targeted to develop safer and more efficacious NSAID derivatives for colorectal cancer chemoprevention.

  9. Aggregation of macrophages and fibroblasts is inhibited by a monoclonal antibody to the hyaluronate receptor

    SciTech Connect

    Green, S.J.; Underhill, C.B. ); Tarone, G. )

    1988-10-01

    To examine the role of the hyaluronate receptor in cell to cell adhesion, the authors have employed the K-3 monoclonal antibody (MAb) which specifically binds to the hyaluronate receptor and blocks its ability to interact with hyaluronate. In the first set of experiments, they investigated the spontaneous aggregation of SV-3T3 cells, which involves two distinct mechanisms, one of which is dependent upon the presence of divalent cation and the other is independent. The divalent cation-independent aggregation was found to be completely inhibited by both intact and Fab fragments of the K-3 MAb. In contrast, the K-3 MAb had no effect on the divalent cation-dependent aggregation of cells. In a second set of experiments, we examined alveolar macrophages. The presence of hyaluronate receptors on alveolar macrophages was demonstrated by the fact that detergent extracts of these cells could bind ({sup 3})hyaluronate, and this binding was blocked by the K-3 MAb. Immunoblot analysis of alveolar macrophages showed that the hyaluronate receptor had a M{sub r} of 99,500, which is considerably larger than the 85,000 M{sub r} for that on BHK cells. When hyaluronate was added to suspensions of alveolar macrophages, the cells were induced to aggregate. This effect was inhibited by the K-3 MAb, suggesting that the hyaluronate-induced aggregation was mediated by the receptor.

  10. 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

  11. INHIBITION OF IN VITRO FERTILIZATION IN THE HAMSTER BY ANTIBODIES RAISED AGAINST THE RAT SPERM PROTEIN SP22

    EPA Science Inventory

    INHIBITION OF IN VITRO FERTILIZATION IN THE HAMSTER BY ANTIBODIES RAISED AGAINST THE RAT SPERM PROTEIN SP22. SC Jeffay*, SD Perreault, KL Bobseine*, JE Welch*, GR Klinefelter, US EPA, Research Triangle Park, NC.
    SP22, a rat sperm membrane protein that is highly-correlated w...

  12. Inhibiting CD146 by its Monoclonal Antibody AA98 Improves Radiosensitivity of Cervical Cancer Cells

    PubMed Central

    Cheng, Huawen

    2016-01-01

    Background Cervical cancer is one of the major causes of cancer death of females worldwide. Radiotherapy is considered effective for cervical cancer treatment, but the low radiosensitivity found in some cases severely affects therapeutic outcomes. This study aimed to reveal the role of CD146, an important adhesion molecule facilitating tumor angiogenesis, in regulating radiosensitivity of cervical cancer cells. Material/Methods CD146 protein expression was compared in normal cells, cervical cancer cells with lower radiosensitivity, and cervical cancer cells with higher sensitivity from cervical squamous cell carcinoma patients. Anti-CD146 monoclonal antibody AA98 was used to inhibit CD146 in human cervical cancer SiHa cells with relatively low radiosensitivity, and then the cell survival and apoptosis changes after radiation were detected by colony formation assay and flow cytometry. Results CD146 protein was significantly up-regulated in cervical cancer cells (P<0.001), especially in cancer cells with lower radiosensitivity. The SiHa cells treated with AA98 showed more obvious inhibition in cell survival (P<0.05) and promotion in cell apoptosis (P<0.01) after radiation, compared to the untreated cells. More dramatic changes in apoptotic factors Caspase 3 and Bcl-XL were also detected in AA98-treated cells. Conclusions These results indicate that inhibiting CD146 improves the effect of radiation in suppressing SiHa cells. This study shows the potential of CD146 as a target for increasing radiosensitivity of cervical cancer cells, which might allow improvement in treatment outcome in cervical cancer. Further studies are necessary for understanding the detailed mechanism of CD146 in regulating radiosensitivity. PMID:27647179

  13. Immune inhibition of virus release from human and nonhuman cells by antibody to viral and host cell determinants.

    PubMed

    Shariff, D M; Davies, J; Desperbasques, M; Billstrom, M; Geerligs, H J; Welling, G W; Welling-Wester, S; Buchan, A; Skinner, G R

    1991-01-01

    Immune inhibition of release of the DNA viruses, herpes simplex virus types 1 and 2 and pseudorabies virus by anti-viral and anti-host cell sera occurred while two RNA viruses, influenza and encephalomyocarditis, were inhibited only by anti-viral sera (not anti-host cell sera). Simian virus 40 and surprisingly two herpes viruses, bovine mamillitis and equine abortion, were not inhibited by either anti-viral or anti-host sera. Using the herpes simplex virus model, inhibition of virus release was detected in different cells of human and nonhuman origin with cross-inhibition between cell lines of different origin; thus, this form of immunotherapy may not require antibody to be tissue or organ specific. Evidence of inhibition of virus release from neoplastic and leukemic cell lines suggests possible application of this approach to control of virus-mediated leukoproliferative pathology (e.g. Burkitt's lymphoma or adult T cell leukemia).

  14. Potent and selective chemical probe of hypoxic signalling downstream of HIF-α hydroxylation via VHL inhibition

    NASA Astrophysics Data System (ADS)

    Frost, Julianty; Galdeano, Carles; Soares, Pedro; Gadd, Morgan S.; Grzes, Katarzyna M.; Ellis, Lucy; Epemolu, Ola; Shimamura, Satoko; Bantscheff, Marcus; Grandi, Paola; Read, Kevin D.; Cantrell, Doreen A.; Rocha, Sonia; Ciulli, Alessio

    2016-11-01

    Chemical strategies to using small molecules to stimulate hypoxia inducible factors (HIFs) activity and trigger a hypoxic response under normoxic conditions, such as iron chelators and inhibitors of prolyl hydroxylase domain (PHD) enzymes, have broad-spectrum activities and off-target effects. Here we disclose VH298, a potent VHL inhibitor that stabilizes HIF-α and elicits a hypoxic response via a different mechanism, that is the blockade of the VHL:HIF-α protein-protein interaction downstream of HIF-α hydroxylation by PHD enzymes. We show that VH298 engages with high affinity and specificity with VHL as its only major cellular target, leading to selective on-target accumulation of hydroxylated HIF-α in a concentration- and time-dependent fashion in different cell lines, with subsequent upregulation of HIF-target genes at both mRNA and protein levels. VH298 represents a high-quality chemical probe of the HIF signalling cascade and an attractive starting point to the development of potential new therapeutics targeting hypoxia signalling.

  15. Potent and selective chemical probe of hypoxic signalling downstream of HIF-α hydroxylation via VHL inhibition

    PubMed Central

    Frost, Julianty; Galdeano, Carles; Soares, Pedro; Gadd, Morgan S.; Grzes, Katarzyna M.; Ellis, Lucy; Epemolu, Ola; Shimamura, Satoko; Bantscheff, Marcus; Grandi, Paola; Read, Kevin D.; Cantrell, Doreen A.; Rocha, Sonia; Ciulli, Alessio

    2016-01-01

    Chemical strategies to using small molecules to stimulate hypoxia inducible factors (HIFs) activity and trigger a hypoxic response under normoxic conditions, such as iron chelators and inhibitors of prolyl hydroxylase domain (PHD) enzymes, have broad-spectrum activities and off-target effects. Here we disclose VH298, a potent VHL inhibitor that stabilizes HIF-α and elicits a hypoxic response via a different mechanism, that is the blockade of the VHL:HIF-α protein–protein interaction downstream of HIF-α hydroxylation by PHD enzymes. We show that VH298 engages with high affinity and specificity with VHL as its only major cellular target, leading to selective on-target accumulation of hydroxylated HIF-α in a concentration- and time-dependent fashion in different cell lines, with subsequent upregulation of HIF-target genes at both mRNA and protein levels. VH298 represents a high-quality chemical probe of the HIF signalling cascade and an attractive starting point to the development of potential new therapeutics targeting hypoxia signalling. PMID:27811928

  16. Potent and selective chemical probe of hypoxic signalling downstream of HIF-α hydroxylation via VHL inhibition.

    PubMed

    Frost, Julianty; Galdeano, Carles; Soares, Pedro; Gadd, Morgan S; Grzes, Katarzyna M; Ellis, Lucy; Epemolu, Ola; Shimamura, Satoko; Bantscheff, Marcus; Grandi, Paola; Read, Kevin D; Cantrell, Doreen A; Rocha, Sonia; Ciulli, Alessio

    2016-11-04

    Chemical strategies to using small molecules to stimulate hypoxia inducible factors (HIFs) activity and trigger a hypoxic response under normoxic conditions, such as iron chelators and inhibitors of prolyl hydroxylase domain (PHD) enzymes, have broad-spectrum activities and off-target effects. Here we disclose VH298, a potent VHL inhibitor that stabilizes HIF-α and elicits a hypoxic response via a different mechanism, that is the blockade of the VHL:HIF-α protein-protein interaction downstream of HIF-α hydroxylation by PHD enzymes. We show that VH298 engages with high affinity and specificity with VHL as its only major cellular target, leading to selective on-target accumulation of hydroxylated HIF-α in a concentration- and time-dependent fashion in different cell lines, with subsequent upregulation of HIF-target genes at both mRNA and protein levels. VH298 represents a high-quality chemical probe of the HIF signalling cascade and an attractive starting point to the development of potential new therapeutics targeting hypoxia signalling.

  17. Potent inhibition of DNA unwinding and ATPase activities of pea DNA helicase 45 by DNA-binding agents.

    PubMed

    Pham, Xuan Hoi; Tuteja, Narendra

    2002-06-07

    Pea DNA helicase 45 (PDH45) is an ATP-dependent DNA unwinding enzyme, with intrinsic DNA-dependent ATPase activity [Plant J. 24 (2000) 219]. We have determined the effect of various DNA-binding agents, such as daunorubicin, ethidium bromide, ellipticine, cisplatin, nogalamycin, actinomycin C1, and camptothecin on the DNA unwinding and ATPase activities of the plant nuclear DNA helicase PDH45. The results show that all the agents except actinomycin C1, and camptothecin inhibited the helicase (apparent K(i) values ranging from 1.5 to 7.0 microM) and ATPase (apparent K(i) values ranging from 2.5 to 11.9 microM) activities. This is the first study to show the effect of various DNA-binding agents on the plant nuclear helicase and also first to demonstrate inhibition of any helicase by cisplatin. Another striking finding that the actinomycin C1 and ellipticine act differentially on PDH45 as compared to pea chloroplast helicase suggests that the mechanism of DNA unwinding could be different in nucleus and chloroplast. These results suggest that the intercalation of the inhibitors into duplex DNA generates a complex that impedes translocation of PDH45, resulting in both the inhibitions of unwinding activity and ATP hydrolysis. This study would be useful to obtain a better understanding of the mechanism of plant nuclear DNA helicase unwinding and the mechanism by which these agents can disturb genome integrity.

  18. Scaffold-hopping of bioactive flavonoids: Discovery of aryl-pyridopyrimidinones as potent anticancer agents that inhibit catalytic role of topoisomerase IIα.

    PubMed

    Priyadarshani, Garima; Amrutkar, Suyog; Nayak, Anmada; Banerjee, Uttam C; Kundu, Chanakya N; Guchhait, Sankar K

    2016-10-21

    A strategy of scaffold-hopping of bioactive natural products, flavones and isoflavones, leading to target-based discovery of potent anticancer agents has been reported for the first time. Scaffold-hopped flavones, 2-aryl-4H-pyrido[1,2-a]pyrimidin-4-ones and the scaffold-hopped isoflavones, 3-aryl-pyrido[1,2-a]pyrimidin-4-ones were synthesized via Pd-catalyzed activation-arylation methods. Most of the compounds were found to exhibit pronounced human topoisomerase IIα (hTopoIIα) inhibitory activities and several compounds were found to be more potent than etoposide (a hTopoIIα-inhibiting anticancer drug). These classes of compounds were found to be hTopoIIα-selective catalytic inhibitors while not interfering with topoisomerase I and interacted with DNA plausibly in groove domain. Cytotoxicities against various cancer cells, low toxicity in normal cells, and apoptotic effects were observed. Interestingly, compared to parent flavones/isoflavones, their scaffold-hopped analogs bearing alike functionalities showed significant/enhanced hTopoIIα-inhibitory and cytotoxic properties, indicating the importance of a natural product-based scaffold-hopping strategy in the drug discovery.

  19. Potent and highly selective human immunodeficiency virus type 1 (HIV-1) inhibition by a series of alpha-anilinophenylacetamide derivatives targeted at HIV-1 reverse transcriptase.

    PubMed Central

    Pauwels, R; Andries, K; Debyser, Z; Van Daele, P; Schols, D; Stoffels, P; De Vreese, K; Woestenborghs, R; Vandamme, A M; Janssen, C G

    1993-01-01

    In vitro evaluation of a large chemical library of pharmacologically acceptable prototype compounds in a high-capacity, cellular-based screening system has led to the discovery of another family of human immunodeficiency virus type 1 (HIV-1) inhibitors. Through optimization of a lead compound, several alpha-anilinophenylacetamide (alpha-APA) derivatives have been identified that inhibit the replication of several HIV-1 strains (IIIB/LAI, RF, NDK, MN, HE) in a variety of host cell types at concentrations that are 10,000- to 100,000-fold lower than their cytotoxic concentrations. The IC50 of the alpha-APA derivative R 89439 for HIV-1 cytopathicity in MT-4 cells was 13 nM. The median 90% inhibitory concentration (IC90) in a variety of host cells was 50-100 nM. Although these alpha-APA derivatives are active against a tetrahydroimidazo [4,5,1-jk][1,4]benzodiazepin-2(1H)-thione-(TIBO)-resistant HIV-1 strain, they do not inhibit replication of HIV-2 (strains ROD and EHO) or simian immunodeficiency virus (strains Mac251, mndGB1, and agm3). An HIV-1 strain containing the Tyr181-->Cys mutation in the reverse transcriptase region displayed reduced sensitivity. alpha-APA derivative R 89439 inhibited virion and recombinant reverse transcriptase of HIV-1 but did not inhibit that of HIV-2. Reverse transcriptase inhibition depended upon the template/primer used. The relatively uncomplicated synthesis of R 89439, its potent anti-HIV-1 activity, and its favorable pharmacokinetic profile make R 89439 a good candidate for clinical studies. PMID:7680476

  20. Transforming growth factor beta is a potent inhibitor of interleukin 1 (IL-1) receptor expression: proposed mechanism of inhibition of IL-1 action

    PubMed Central

    1990-01-01

    Transforming growth factor beta (TGF-beta) acts as a potent inhibitor of the growth and functions of lymphoid and hemopoietic progenitor cells. Cell proliferation depends not only on the presence of growth factors, but also on the development of specific receptor-signal transducing complexes. We therefore investigated whether the inhibitory actions of TGF-beta could be mediated by inhibition of growth factor receptors. TGF-beta inhibited the constitutive level of interleukin 1 receptor (IL-1R) expression on several murine lymphoid and myeloid progenitor cell lines, as well as IL-1R expression induced by interleukin 3 (IL-3) on normal murine and human bone marrow cells. Furthermore, treatment of bone marrow progenitor cells with TGF-beta concomitantly inhibited the ability of IL-1 to promote high proliferative potential (HPP) colony formation as well as blocked IL-1- induced IL-2 production by EL-4 6.1 cells. These findings provide the first evidence that the inhibitory action of TGF-beta on the growth and functional activities of hematopoietic and T cells is associated with a reduction in the cell surface receptor expression for IL-1. PMID:2143773

  1. A novel and selective inhibitor of PKC ζ potently inhibits human breast cancer metastasis in vitro and in mice.

    PubMed

    Wu, Jing; Liu, Shuye; Fan, Zhijuan; Zhang, Lei; Tian, Yaqiong; Yang, Rui

    2016-06-01

    Cell motility and chemotaxis play pivotal roles in the process of tumor development and metastasis. Protein kinase C ζ (PKC ζ) mediates epidermal growth factor (EGF)-stimulated chemotactic signaling pathway through regulating cytoskeleton rearrangement and cell adhesion. The purpose of this study was to develop anti-PKC ζ therapeutics for breast cancer metastasis. In this study, a novel and high-efficient PKC ζ inhibitor named PKCZI195.17 was screened out through a substrate-specific strategy. MTT assay was used to determine the cell viability of human breast cancer MDA-MB-231, MDA-MB-435, and MCF-7 cells while under PKCZI195.17 treatment. Wound-healing, chemotaxis, and Matrigel invasion assays were performed to detect the effects of PKCZI195.17 on breast cancer cells migration and invasion. Adhesion, actin polymerization, and Western blotting were performed to detect the effects of PKCZI195.17 on cells adhesion and actin polymerization, and explore the downsteam signaling mechanisms involved in PKC ζ inhibition. MDA-MB-231 xenograft was used to measure the in vivo anti-metastasis efficacy of PKCZI195.17. The compound PKCZI195.17 selectively inhibited PKC ζ kinase activity since it failed to inhibit PKC α, PKC β, PKC δ, PKC η, AKT2, as well as FGFR2 activity. PKCZI195.17 significantly impaired spontaneous migration, chemotaxis, and invasion of human breast cancer MDA-MB-231, MDA-MB-435, and MCF-7 cells, while PKCZI195.17 did not obviously inhibited cells viability. PKCZI195.17 also inhibited cells adhesion and actin polymerization through attenuating the phosphorylations of integrin β1, LIMK, and cofilin, which might be the downstream effectors of PKC ζ-mediated chemotaxis in MDA-MB-231 cells. Furthermore, PKCZI195.17 suppressed the breast cancer metastasis and increased the survival time of breast tumor-bearing mice. In summary, PKCZI195.17 was a PKC ζ-specific inhibitor which dampened cancer cell migration and metastasis and may serve as a novel

  2. Inhibiting the Aurora B Kinase Potently Suppresses Repopulation During Fractionated Irradiation of Human Lung Cancer Cell Lines

    SciTech Connect

    Sak, Ali; Stuschke, Martin; Groneberg, Michael; Kuebler, Dennis; Poettgen, Christoph; Eberhardt, Wilfried E.E.

    2012-10-01

    Purpose: The use of molecular-targeted agents during radiotherapy of non-small-cell lung cancer (NSCLC) is a promising strategy to inhibit repopulation, thereby improving therapeutic outcome. We assessed the combined effectiveness of inhibiting Aurora B kinase and irradiation on human NSCLC cell lines in vitro. Methods and Materials: NSCLC cell lines were exposed to concentrations of AZD1152-hydroxyquinazoline pyrazol anilide (AZD1152-HQPA) inhibiting colony formation by 50% (IC50{sub clone}) in combination with single dose irradiation or different fractionation schedules using multiple 2-Gy fractions per day up to total doses of 4-40 Gy. The total irradiation dose required to control growth of 50% of the plaque monolayers (TCD50) was determined. Apoptosis, G2/M progression, and polyploidization were also analyzed. Results: TCD50 values after single dose irradiation were similar for the H460 and H661 cell lines with 11.4 {+-} 0.2 Gy and 10.7 {+-} 0.3 Gy, respectively. Fractionated irradiation using 3 Multiplication-Sign 2 Gy/day, 2 Multiplication-Sign 2 Gy/day, and 1 Multiplication-Sign 2 Gy/day schedules significantly increased TCD50 values for both cell lines grown as plaque monolayers with increasing radiation treatment time. This could be explained by a repopulation effect per day that counteracts 75 {+-} 8% and 27 {+-} 6% of the effect of a 2-Gy fraction in H460 and H661 cells, respectively. AZD1152-HQPA treatment concomitant to radiotherapy significantly decreased the daily repopulation effect (H460: 28 {+-} 5%, H661: 10 {+-} 4% of a 2-Gy fraction per day). Treatment with IC50{sub clone} AZD1152-HPQA did not induce apoptosis, prolong radiation-induced G2 arrest, or delay cell cycle progression before the spindle check point. However, polyploidization was detected, especially in cell lines without functional p53. Conclusions: Inhibition of Aurora B kinase with low AZD1152-HQPA concentrations during irradiation of NSCLC cell lines affects repopulation during

  3. Inhibition of in vitro limb cartilage differentiation by syndecan-3 antibodies.

    PubMed

    Seghatoleslami, M R; Kosher, R A

    1996-09-01

    The transmembrane heparan sulfate proteoglycan syndecan-3 is transiently expressed in high amounts during the cellular condensation process that characterizes the onset of limb cartilage differentiation. During condensation, limb mesenchymal cells become closely juxtaposed and undergo cell-cell and cell-matrix interactions that are necessary to trigger cartilage differentiation and cartilage-specific gene expression. To test directly the possible involvement of syndecan-3 in regulating the onset of limb chondrogenesis, we examined the effect of polyclonal antibodies against a syndecan-3 fusion protein on the chondrogenic differentiation of chick limb mesenchymal cells in micromass culture. Syndecan-3 antiserum elicits a dose-dependent inhibition of the accumulation of Alcian blue-stainable cartilage matrix by high density limb mesenchymal cell micromass cultures (2 x 10(5) cells/10 microliters) and a corresponding reduction in steady-state levels of mRNAs for cartilage-characteristic type II collagen and the core protein of the cartilage proteoglycan aggrecan. In preimmune serum-treated control cultures proliferating cells are limited to the periphery of areas of cartilage matrix deposition, whereas large numbers of proliferating cells are uniformly distributed throughout the undifferentiated cultures supplemented with syndecan-3 antiserum. Limb mesenchymal cells cultured at lower densities (1 x 10(5) cells/10 microliters) in the presence of preimmune serum form extensive precartilage condensations characterized by the close juxtaposition of rounded cells by day 2 of culture. In contrast, in the presence of syndecan-3 antiserum, the cells fail to aggregate but rather remain flattened and spatially separated from one another, suggeting that syndecan-3 antibodies impair the formation of precartilage condensations. These results indicate that syndecan-3 plays an important role in regulating the onset of limb chondrogenesis, perhaps by mediating the cell-cell and cell

  4. mTOR kinase inhibitors promote antibody class switching via mTORC2 inhibition.

    PubMed

    Limon, Jose J; So, Lomon; Jellbauer, Stefan; Chiu, Honyin; Corado, Juana; Sykes, Stephen M; Raffatellu, Manuela; Fruman, David A

    2014-11-25

    The mammalian target of rapamycin (mTOR) is a kinase that functions in two distinct complexes, mTORC1 and mTORC2. In peripheral B cells, complete deletion of mTOR suppresses germinal center B-cell responses, including class switching and somatic hypermutation. The allosteric mTORC1 inhibitor rapamycin blocks proliferation and differentiation, but lower doses can promote protective IgM responses. To elucidate the complexity of mTOR signaling in B cells further, we used ATP-competitive mTOR kinase inhibitors (TOR-KIs), which inhibit both mTORC1 and mTORC2. Although TOR-KIs are in clinical development for cancer, their effects on mature lymphocytes are largely unknown. We show that high concentrations of TOR-KIs suppress B-cell proliferation and differentiation, yet lower concentrations that preserve proliferation increase the fraction of B cells undergoing class switching in vitro. Transient treatment of mice with the TOR-KI compound AZD8055 increased titers of class-switched high-affinity antibodies to a hapten-protein conjugate. Mechanistic investigation identified opposing roles for mTORC1 and mTORC2 in B-cell differentiation and showed that TOR-KIs enhance class switching in a manner dependent on forkhead box, subgroup O (FoxO) transcription factors. These observations emphasize the distinct actions of TOR-KIs compared with rapamycin and suggest that TOR-KIs might be useful to enhance production of class-switched antibodies following vaccination.

  5. In vitro and in vivo anti-angiogenesis effect of shallot (Allium ascalonicum): a heat-stable and flavonoid-rich fraction of shallot extract potently inhibits angiogenesis.

    PubMed

    Seyfi, Parivash; Mostafaie, Ali; Mansouri, Kamran; Arshadi, Delnia; Mohammadi-Motlagh, Hamid-Reza; Kiani, Amir

    2010-09-01

    This study has been undertaken to elucidate the anti-angiogenic properties of shallot extract in vitro and in vivo and also to define the responsible fraction and its stability. After preparation of the extract of shallot bulbs with 50% ethanol, the extract was successively fractionated into n-hexane, ethyl acetate, n-butanol and aqueous fractions. The ethyl acetate fraction was further fractionated to three subfractions using thin layer chromatography. Anti-angiogenic activity of fractions and subfractions were examined on human umbilical vein endothelial cells (HUVECs) in collagen matrix and chicken chorioallantoic membrane (CAM) models. Among the fractions, ethyl acetate fraction and one of its subfractions potently inhibited angiogenesis in vitro and in vivo. Furthermore, ethyl acetate fraction sustained its inhibitory effect significantly even after treatment in high thermal and low pH conditions. These findings provided a useful basis for further investigations on shallot as a useful herb with therapeutic or preventive activity against angiogenesis related disorders.

  6. Inhibition of the development of morphine tolerance by a potent dual mu-delta-opioid antagonist, H-Dmt-Tic-Lys-NH-CH2-Ph.

    PubMed

    Jinsmaa, Yunden; Marczak, Ewa D; Balboni, Gianfranco; Salvadori, Severo; Lazarus, Lawrence H

    2008-10-01

    Three analogues of the dual mu-/delta-antagonist, H-Dmt-Tic-R-NH-CH2-Ph (R = 1, Lys-Z; 2, Lys-Ac; 3, Lys) were examined in vivo: 1 and 2 exhibited weak bioactivity, while 3 injected intracerebroventricularly was a potent dual antagonist for morphine- and deltorphin C-induced antinociception comparable to naltrindole (delta-antagonist), but 93% as effective as naloxone (nonspecific opioid receptor antagonist) and 4% as active as CTOP, a mu antagonist. Subcutaneous or oral administration of 3 antagonized morphine-induced antinociception indicating passage across epithelial and blood-brain barriers. Mice pretreated with 3 before morphine did not develop morphine tolerance indicative of a potential clinical role to inhibit development of drug tolerance.

  7. Potent inhibition of angiotensin AT1 receptor signaling by RGS8: importance of the C-terminal third exon part of its RGS domain.

    PubMed

    Song, Dan; Nishiyama, Mariko; Kimura, Sadao

    2016-10-01

    R4/B subfamily RGS (regulator of G protein signaling) proteins play roles in regulation of many GPCR-mediated responses. Multiple RGS proteins are usually expressed in a cell, and it is difficult to point out which RGS protein species are functionally important in the cell. To evaluate intrinsic potency of these RGS proteins, we compared inhibitory effects of RGS1, RGS2, RGS3, RGS4, RGS5, RGS8 and RGS16 on AT1 receptor signaling. Intracellular Ca(2+) responses to angiotensin II were markedly attenuated by transiently expressed RGS2, RGS3 and RGS8, compared to weak inhibition by RGS1, RGS4, RGS5 and RGS16. N-terminally deleted RGS2 (RGS2 domain) lost this potent inhibitory effect, whereas RGS domains of RGS3 and RGS8 showed strong inhibition similar to those of the full-length proteins. To investigate key determinants that specify the differences in potency, we constructed chimeric domains by replacing one or two of three exon parts of RGS8 domain with the corresponding part of RGS5. The chimeric RGS8 domains containing the first or the second exon part of RGS5 showed strong inhibitory effects similar to that of wild type RGS8, but the chimeric domain with the third exon part of RGS5 lost its activity. On the contrary, replacement of the third exon part of RGS5 with the corresponding residues of RGS8 increased the inhibitory effect. The role of the third exon part of RGS8 domain was further confirmed with the chimeric RGS8/RGS4 domains. These results indicate the potent inhibitory activity of RGS8 among R4/B subfamily proteins and importance of the third exon.

  8. Inhibition of antibody response to Pseudomonas exotoxin and an immunotoxin containing Pseudomonas exotoxin by 15-deoxyspergualin in mice.

    PubMed

    Pai, L H; FitzGerald, D J; Tepper, M; Schacter, B; Spitalny, G; Pastan, I

    1990-12-15

    Immunotoxins are potent cell-killing agents that may be useful in the treatment of cancer. The early production of neutralizing antibodies to immunotoxins is one of the major limiting factors for their use in humans. 15-Deoxyspergualin (DSG), a derivative of spergualin, which is a metabolite of Bacillus laterosporus, has been found to have immunosuppressive activity in rodents, dogs, and primates. We examined the suppressive activity of DSG on the antibody response to Pseudomonas exotoxin in mice by enzyme-linked immunosorbent assay. Male BDF1 mice were immunized with a single dose of a nontoxic mutant of Pseudomonas exotoxin (40 micrograms) and then treated with i.p. injections of DSF at a dose of 10 mg/kg for 3 days. Although antibodies to Pseudomonas exotoxin were observed within 7 days in the control group, there was complete suppression of antibody production in the DSG-treated group. Immunosuppression has also been observed in animals immunized with multiple doses (10 mg x 7 d) of Pseudomonas exotoxin and treated with DSG at a dose of 5 mg/kg for 21 days. Similar immunosuppression was observed in mice given multiple doses of the immunotoxin, anti-Tac-LysPE40. We conclude that the immunosuppressive activity of DSG may be useful in increasing the duration of immunotoxin treatment.

  9. D-Amino acid oxidase-induced oxidative stress, 3-bromopyruvate and citrate inhibit angiogenesis, exhibiting potent anticancer effects.

    PubMed

    El Sayed, S M; El-Magd, R M Abou; Shishido, Y; Yorita, K; Chung, S P; Tran, D H; Sakai, T; Watanabe, H; Kagami, S; Fukui, K

    2012-10-01

    Angiogenesis is critical for cancer growth and metastasis. Steps of angiogenesis are energy consuming, while vascular endothelial cells are highly glycolytic. Glioblastoma multiforme (GBM) is a highly vascular tumor and this enhances its aggressiveness. D-amino acid oxidase (DAO) is a promising therapeutic protein that induces oxidative stress upon acting on its substrates. Oxidative stress-energy depletion (OSED) therapy was recently reported (El Sayed et al., Cancer Gene Ther, 19, 1-18, 2012). OSED combines DAO-induced oxidative stress with energy depletion caused by glycolytic inhibitors such as 3-bromopyruvate (3BP), a hexokinase II inhibitor that depleted ATP in cancer cells and induced production of hydrogen peroxide. 3BP disturbs the Warburg effect and antagonizes effects of lactate and pyruvate (El Sayed et al., J Bioenerg Biomembr, 44, 61-79, 2012). Citrate is a natural organic acid capable of inhibiting glycolysis by targeting phosphofructokinase. Here, we report that DAO, 3BP and citrate significantly inhibited angiogenesis, decreased the number of vascular branching points and shortened the length of vascular tubules. OSED delayed the growth of C6/DAO glioma cells. 3BP combined with citrate delayed the growth of C6 glioma cells and decreased significantly the number and size of C6 glioma colonies in soft agar. Human GBM cells (U373MG) were resistant to chemotherapy e.g. cisplatin and cytosine arabinoside, while 3BP was effective in decreasing the viability and disturbing the morphology of U373MG cells.

  10. Inhibition of autologous mixed lymphocyte reaction by monoclonal antibodies specific for the β chain of HLA-DR antigens

    PubMed Central

    Kasahara, M.; Ikeda, H.; Ogasawara, K.; Ishikawa, N.; Okuyama, T.; Fukasawa, Y.; Kojima, H.; Kunikane, H.; Hawkin, S.; Ohhashi, T.; Natori, T.; Wakisaka, A.; Kikuchi, Y.; Aizawa, M.

    1984-01-01

    Recent studies using rabbit antisera to the separated HLA-DR α and β subunits have suggested that α chain-specific, but not β chain-specific, antisera inhibit T cell proliferative responses in primary and secondary human autologous mixed lymphocyte reaction (AMLR). In the present study, with the aid of sequential co-precipitation assays and Western blotting methods, a monoclonal rat alloantibody 1E4, specific for the β chain of rat class II molecules carrying an Ia determinant Ba-2.7, was characterized to recognize a monomorphic determinant located on the β chain of DR antigens. This antibody and a murine monoclonal antibody HU-4, also specific for the β chain of DR antigens, strongly inhibited both primary and secondary AMLR through a mechanism distinct from an antibody-dependent cell-mediated cytotoxicity reaction. These results indicate that the inhibition of AMLR is not a unique feature of DR α-specific antibodies. ImagesFigure 2 PMID:6205985

  11. Microtubule-dependent control of cell shape and pseudopodial activity is inhibited by the antibody to kinesin motor domain

    PubMed Central

    1993-01-01

    One of the major functions of cytoplasmic microtubules is their involvement in maintenance of asymmetric cell shape. Microtubules were considered to perform this function working as rigid structural elements. At the same time, microtubules play a critical role in intracellular organelle transport, and this fact raises the possibility that the involvement of microtubules in maintenance of cell shape may be mediated by directed transport of certain cellular components to a limited area of the cell surface (e.g., to the leading edge) rather than by their functioning as a mechanical support. To test this hypothesis we microinjected cultured human fibroblasts with the antibody (called HD antibody) raised against kinesin motor domain highly conserved among the different members of kinesin superfamily. As was shown before this antibody inhibits kinesin-dependent microtubule gliding in vitro and interferes with a number of microtubule-dependent transport processes in living cells. Preimmune IgG fraction was used for control experiments. Injections of fibroblasts with HD antibody but not with preimmune IgG significantly reduced their asymmetry, resulting in loss of long processes and elongated cell shape. In addition, antibody injection suppressed pseudopodial activity at the leading edge of fibroblasts moving into an experimentally made wound. Analysis of membrane organelle distribution showed that kinesin antibody induced clustering of mitochondria in perinuclear region and their withdrawal from peripheral parts of the cytoplasm. HD antibody does not affect either density or distribution of cytoplasmic microtubules. The results of our experiments show that many changes of phenotype induced in cells by microtubule-depolymerizing agents can be mimicked by the inhibition of motor proteins, and therefore microtubule functions in maintaining of the cell shape and polarity are mediated by motor proteins rather than by being provided by rigidity of tubulin polymer itself. PMID

  12. YL529, a novel, orally available multikinase inhibitor, potently inhibits angiogenesis and tumour growth in preclinical models

    PubMed Central

    Xu, Youzhi; Lin, Hongjun; Meng, Nana; Lu, Wenjie; Li, Guobo; Han, Yuanyuan; Dai, Xiaoyun; Xia, Yong; Song, Xiangrong; Yang, Shengyong; Wei, Yuquan; Yu, Luoting; Zhao, Yinglan

    2013-01-01

    Background and Purpose Targeted chemotherapy using small-molecule inhibitors of angiogenesis and proliferation is a promising strategy for cancer therapy. Experimental Approach YL529 was developed via computer-aided drug design, de novo synthesis and high-throughput screening. The biochemical, pharmacodynamic and toxicological profiles of YL529 were investigated using kinase and cell viability assays, a mouse tumour cell-containing alginate bead model, a zebrafish angiogenesis model and several human tumour xenograft models in athymic mice. Key Results In vitro, YL529 selectively inhibited the activities of VEGFR2/VEGFR3 and serine/threonine kinase RAF kinase. YL529 inhibited VEGF165-induced phosphorylation of VEGFR2, as well as the proliferation, migration, invasion and tube formation of human umbilical vascular endothelial cells. It also significantly blocked vascular formation and angiogenesis in the zebrafish model. Moreover, YL529 strongly attenuated the proliferation of A549 cells by disrupting the RAF/mitogen-activated protein (MAP) or extracellular signal-regulated kinase (Erk) kinase (MEK) kinase kinase/MAPK pathway. Oral administration of YL529 (37.5–150 mg−1·kg−1·day−1) to nude mice bearing established tumour xenografts significantly prevented the growth (60–80%) of A549, SPC-A1, A375, OS-RC-2 and HCT116 tumours without detectable toxicity. YL529 markedly reduced microvessel density and increased tumour cell apoptosis in the tumours formed in mice inoculated with the lung cancer cells, SPC-A1 and A549, and the colon carcinoma cells, HCT116. Conclusions and Implications YL529, an orally active multikinase inhibitor, shows therapeutic potential for solid tumours, and warrants further investigation as a possible anticancer agent. PMID:23594209

  13. Broadly Neutralizing Hemagglutinin Stalk-Specific Antibodies Induce Potent Phagocytosis of Immune Complexes by Neutrophils in an Fc-Dependent Manner.

    PubMed

    Mullarkey, Caitlin E; Bailey, Mark J; Golubeva, Diana A; Tan, Gene S; Nachbagauer, Raffael; He, Wenqian; Novakowski, Kyle E; Bowdish, Dawn M; Miller, Matthew S; Palese, Peter

    2016-10-04

    Broadly neutralizing antibodies that recognize the conserved hemagglutinin (HA) stalk have emerged as exciting new biotherapeutic tools to combat seasonal and pandemic influenza viruses. Our general understanding of the mechanisms by which stalk-specific antibodies achieve protection is rapidly evolving. It has recently been demonstrated that broadly neutralizing HA stalk-specific IgG antibodies require Fc-Fcγ receptor (FcγR) interactions for optimal protection in vivo Here we examine the neutrophil effector functions induced by stalk-specific antibodies. As the most abundant subset of blood leukocytes, neutrophils represent a critical innate effector cell population and serve an instrumental role in orchestrating downstream adaptive responses to influenza virus infection. Yet, the interplay of HA stalk-specific IgG, Fc-FcγR engagement, and neutrophils has remained largely uncharacterized. Using an in vitro assay to detect the production of reactive oxygen species (ROS), we show that human and mouse monoclonal HA stalk-specific IgG antibodies are able to induce the production of ROS by neutrophils, while HA head-specific antibodies do not. Furthermore, our results indicate that the production of ROS is dependent on Fc receptor (FcR) engagement and phagocytosis. We went on to assess the ability of monoclonal HA stalk-specific IgA antibodies to induce ROS. Consistent with our findings for monoclonal IgGs, only HA stalk-specific IgA antibodies elicited ROS production by neutrophils. This induction is dependent on the engagement of FcαR1. Taken together, our findings describe a novel FcR-dependent effector function induced by HA stalk-specific IgG and IgA antibodies, and importantly, our studies shed light on the mechanisms by which HA stalk-specific antibodies achieve protection.

  14. Broadly Neutralizing Hemagglutinin Stalk-Specific Antibodies Induce Potent Phagocytosis of Immune Complexes by Neutrophils in an Fc-Dependent Manner

    PubMed Central

    Mullarkey, Caitlin E.; Bailey, Mark J.; Golubeva, Diana A.; Tan, Gene S.; Nachbagauer, Raffael; He, Wenqian; Novakowski, Kyle E.; Bowdish, Dawn M.; Miller, Matthew S.

    2016-01-01

    ABSTRACT Broadly neutralizing antibodies that recognize the conserved hemagglutinin (HA) stalk have emerged as exciting new biotherapeutic tools to combat seasonal and pandemic influenza viruses. Our general understanding of the mechanisms by which stalk-specific antibodies achieve protection is rapidly evolving. It has recently been demonstrated that broadly neutralizing HA stalk-specific IgG antibodies require Fc-Fcγ receptor (FcγR) interactions for optimal protection in vivo. Here we examine the neutrophil effector functions induced by stalk-specific antibodies. As the most abundant subset of blood leukocytes, neutrophils represent a critical innate effector cell population and serve an instrumental role in orchestrating downstream adaptive responses to influenza virus infection. Yet, the interplay of HA stalk-specific IgG, Fc-FcγR engagement, and neutrophils has remained largely uncharacterized. Using an in vitro assay to detect the production of reactive oxygen species (ROS), we show that human and mouse monoclonal HA stalk-specific IgG antibodies are able to induce the production of ROS by neutrophils, while HA head-specific antibodies do not. Furthermore, our results indicate that the production of ROS is dependent on Fc receptor (FcR) engagement and phagocytosis. We went on to assess the ability of monoclonal HA stalk-specific IgA antibodies to induce ROS. Consistent with our findings for monoclonal IgGs, only HA stalk-specific IgA antibodies elicited ROS production by neutrophils. This induction is dependent on the engagement of FcαR1. Taken together, our findings describe a novel FcR-dependent effector function induced by HA stalk-specific IgG and IgA antibodies, and importantly, our studies shed light on the mechanisms by which HA stalk-specific antibodies achieve protection. PMID:27703076

  15. Rapid Discovery and Structure–Activity Relationships of Pyrazolopyrimidines That Potently Suppress Breast Cancer Cell Growth via SRC Kinase Inhibition with Exceptional Selectivity over ABL Kinase

    PubMed Central

    2016-01-01

    Novel pyrazolopyrimidines displaying high potency and selectivity toward SRC family kinases have been developed by combining ligand-based design and phenotypic screening in an iterative manner. Compounds were derived from the promiscuous kinase inhibitor PP1 to search for analogs that could potentially target a broad spectrum of kinases involved in cancer. Phenotypic screening against MCF7 mammary adenocarcinoma cells generated target-agnostic structure–activity relationships that biased subsequent designs toward breast cancer treatment rather than to a particular target. This strategy led to the discovery of two potent antiproliferative leads with phenotypically distinct anticancer mode of actions. Kinase profiling and further optimization resulted in eCF506, the first small molecule with subnanomolar IC50 for SRC that requires 3 orders of magnitude greater concentration to inhibit ABL. eCF506 exhibits excellent water solubility, an optimal DMPK profile and oral bioavailability, halts SRC-associated neuromast migration in zebrafish embryos without inducing life-threatening heart defects, and inhibits SRC phosphorylation in tumor xenografts in mice. PMID:27115835

  16. Discovery of Potent Cysteine-Containing Dipeptide Inhibitors against Tyrosinase: A Comprehensive Investigation of 20 × 20 Dipeptides in Inhibiting Dopachrome Formation.

    PubMed

    Tseng, Tien-Sheng; Tsai, Keng-Chang; Chen, Wang-Chuan; Wang, Yeng-Tseng; Lee, Yu-Ching; Lu, Chung-Kuang; Don, Ming-Jaw; Chang, Chang-Yu; Lee, Ching-Hsiao; Lin, Hui-Hsiung; Hsu, Hung-Ju; Hsiao, Nai-Wan

    2015-07-15

    Tyrosinase is an essential copper-containing enzyme required for melanin synthesis. The overproduction and abnormal accumulation of melanin cause hyperpigmentation and neurodegenerative diseases. Thus, tyrosinase is promising for use in medicine and cosmetics. Our previous study identified a natural product, A5, resembling the structure of the dipeptide WY and apparently inhibiting tyrosinase. Here, we comprehensively estimated the inhibitory capability of 20 × 20 dipeptides against mushroom tyrosinase. We found that cysteine-containing dipeptides, directly blocking the active site of tyrosinase, are highly potent in inhibition; in particular, N-terminal cysteine-containing dipeptides markedly outperform the C-terminal-containing ones. The cysteine-containing dipeptides, CE, CS, CY, and CW, show comparative bioactivities, and tyrosine-containing dipeptides are substrate-like inhibitors. The dipeptide PD attenuates 16.5% melanin content without any significant cytotoxicity. This study reveals the functional role of cysteine residue positional preference and the selectivity of specific amino acids in cysteine-containing dipeptides against tyrosinase, aiding in developing skin-whitening products.

  17. Clotrimazole as a Potent Agent for Treating the Oomycete Fish Pathogen Saprolegnia parasitica through Inhibition of Sterol 14α-Demethylase (CYP51)

    PubMed Central

    Warrilow, Andrew G. S.; Hull, Claire M.; Rolley, Nicola J.; Parker, Josie E.; Nes, W. David; Smith, Stephen N.

    2014-01-01

    A candidate CYP51 gene encoding sterol 14α-demethylase from the fish oomycete pathogen Saprolegnia parasitica (SpCYP51) was identified based on conserved CYP51 residues among CYPs in the genome. It was heterologously expressed in Escherichia coli, purified, and characterized. Lanosterol, eburicol, and obtusifoliol bound to purified SpCYP51 with similar binding affinities (Ks, 3 to 5 μM). Eight pharmaceutical and six agricultural azole antifungal agents bound tightly to SpCYP51, with posaconazole displaying the highest apparent affinity (Kd, ≤3 nM) and prothioconazole-desthio the lowest (Kd, ∼51 nM). The efficaciousness of azole antifungals as SpCYP51 inhibitors was confirmed by 50% inhibitory concentrations (IC50s) of 0.17 to 2.27 μM using CYP51 reconstitution assays. However, most azole antifungal agents were less effective at inhibiting S. parasitica, Saprolegnia diclina, and Saprolegnia ferax growth. Epoxiconazole, fluconazole, itraconazole, and posaconazole failed to inhibit Saprolegnia growth (MIC100, >256 μg ml−1). The remaining azoles inhibited Saprolegnia growth only at elevated concentrations (MIC100 [the lowest antifungal concentration at which growth remained completely inhibited after 72 h at 20°C], 16 to 64 μg ml−1) with the exception of clotrimazole, which was as potent as malachite green (MIC100, ∼1 μg ml−1). Sterol profiles of azole-treated Saprolegnia species confirmed that endogenous CYP51 enzymes were being inhibited with the accumulation of lanosterol in the sterol fraction. The effectiveness of clotrimazole against SpCYP51 activity (IC50, ∼1 μM) and the concentration inhibiting the growth of Saprolegnia species in vitro (MIC100, ∼1 to 2 μg ml−1) suggest that clotrimazole could be used against Saprolegnia infections, including as a preventative measure by pretreatment of fish eggs, and for freshwater-farmed fish as well as in leisure activities. PMID:25085484

  18. Antiproliferative and proapoptotic activity of GUT-70 mediated through potent inhibition of Hsp90 in mantle cell lymphoma

    PubMed Central

    Jin, L; Tabe, Y; Kimura, S; Zhou, Y; Kuroda, J; Asou, H; Inaba, T; Konopleva, M; Andreeff, M; Miida, T

    2011-01-01

    Background: Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma with poor prognosis, requiring novel anticancer strategies. Methods: Mantle cell lymphoma cell lines with known p53 status were treated with GUT-70, a tricyclic coumarin derived from Calophyllum brasiliense, and the biological and biochemical consequences of GUT-70 were studied. Results: GUT-70 markedly reduced cell proliferation/viability through G1 cell cycle arrest and increased apoptosis, with greater sensitivity in mutant (mt)-p53-expressing MCL cells than in wild-type (wt)-p53-bearing cells. Mechanistically, GUT-70 showed binding affinity to heat-shock protein 90 (Hsp90) and ubiquitin-dependent proteasomal degradation of Hsp90 client proteins, including cyclin D1, Raf-1, Akt, and mt-p53. Depletion of constitutively overexpressed cyclin D1 by GUT-70 was accompanied by p27 accumulation and decreased Rb phosphorylation. GUT-70 induced mitochondrial apoptosis with Noxa upregulation and Mcl-1 downregulation in mt-p53 cells, but Mcl-1 accumulation in wt-p53 cells. Noxa and Mcl-1 were coimmunoprecipitated, and activated BAK. Treatment with a combination of GUT-70 and bortezomib or doxorubicin had synergistic antiproliferative effects in MCL cells that were independent of p53 status. Conclusion: GUT-70 has pronounced antiproliferative effects in MCL with mt-p53, a known negative prognostic factor for MCL, through Hsp90 inhibition. These findings suggest that GUT-70 has potential utility for the treatment of MCL. PMID:21139584

  19. 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.

  20. Potent anti-tumour activity of a novel conditionally replicating adenovirus for melanoma via inhibition of migration and invasion

    PubMed Central

    Jiang, G; Yang, C-S; Xu, D; Sun, C; Zheng, J-N; Lei, T-C; Liu, Y-Q

    2014-01-01

    Background: Conditionally replicating adenoviruses (CRAds) represent a novel class of oncological therapeutic agents. One strategy to ensure tumour targeting is to place the essential viral genes under the control of tumour-specific promoters. Ki67 has been selected as a cancer gene therapy target, as it is expressed in most malignant cells but is barely detectable in most normal cells. This study aimed to investigate the effects of a Ki67 promoter-controlled CRAd (Ki67-ZD55-IL-24) on the proliferation and apoptosis of melanoma cells. Methods: Melanoma cells were independently treated with Ki67-ZD55-IL-24, ZD55-IL-24, Ki67-ZD55, and ZD55-EGFP. The cytotoxic potential of each treatment was assessed using cell viability measurements. Cell migration and invasion were assayed using cell migration and invasion assays. Apoptosis was assayed using the annexin V-FITC assay, western blotting, reverse transcriptase PCR (RT–PCR), haematoxylin and eosin (H&E) staining, and the TUNEL assay. Results: Our results showed that Ki67-ZD55-IL-24 had significantly enhanced anti-tumour activity as it more effectively induced apoptosis in melanoma cells than the other agents. Ki67-ZD55-IL-24 also caused the most significant inhibition of cell migration and invasion of melanoma cells. Furthermore, apoptosis was induced more effectively in melanoma xenografts in nude mice. Conclusions: This strategy holds promising potential for the further development of an effective approach to treat malignant melanoma. PMID:24714752

  1. Anti-apical-membrane-antigen-1 antibody is more effective than anti-42-kilodalton-merozoite-surface-protein-1 antibody in inhibiting plasmodium falciparum growth, as determined by the in vitro growth inhibition assay.

    PubMed

    Miura, Kazutoyo; Zhou, Hong; Diouf, Ababacar; Moretz, Samuel E; Fay, Michael P; Miller, Louis H; Martin, Laura B; Pierce, Mark A; Ellis, Ruth D; Mullen, Gregory E D; Long, Carole A

    2009-07-01

    Apical membrane antigen 1 (AMA1) and the 42-kDa merozoite surface protein 1 (MSP1(42)) are leading malaria vaccine candidates. Several preclinical and clinical trials have been conducted, and an in vitro parasite growth inhibition assay has been used to evaluate the biological activities of the resulting antibodies. In a U.S. phase 1 trial with AMA1-C1/Alhydrogel plus CPG 7909, the vaccination elicited anti-AMA1 immunoglobulin G (IgG) which showed up to 96% inhibition. However, antibodies induced by MSP1(42)-C1/Alhydrogel plus CPG 7909 vaccine showed less than 32% inhibition in vitro. To determine whether anti-MSP1(42) IgG had less growth-inhibitory activity than anti-AMA1 IgG in vitro, the amounts of IgG that produced 50% inhibition of parasite growth (Ab(50)) were compared for rabbit and human antibodies. The Ab(50)s of rabbit and human anti-MSP1(42) IgGs were significantly higher (0.21 and 0.62 mg/ml, respectively) than those of anti-AMA1 IgGs (0.07 and 0.10 mg/ml, respectively) against 3D7 parasites. Ab(50) data against FVO parasites also demonstrated significant differences. We further investigated the Ab(50)s of mouse and monkey anti-AMA1 IgGs and showed that there were significant differences between the species (mouse, 0.28 mg/ml, and monkey, 0.14 mg/ml, against 3D7 parasites). Although it is unknown whether growth-inhibitory activity in vitro reflects protective immunity in vivo, this study showed that the Ab(50) varies with both antigen and species. Our data provide a benchmark for antibody levels for future AMA1- or MSP1(42)-based vaccine development efforts in preclinical and clinical trials.

  2. Sclerostin antibody inhibits skeletal deterioration in mice exposed to partial weight-bearing.

    PubMed

    Spatz, J M; Ellman, R; Cloutier, A M; Louis, L; van Vliet, M; Dwyer, D; Stolina, M; Ke, H Z; Bouxsein, M L

    2017-02-01

    Whereas much is known regarding the musculoskeletal responses to full unloading, little is known about the physiological effects and response to pharmacological agents in partial unloading (e.g. Moon and Mars) environments. To address this, we used a previously developed ground-based model of partial weight-bearing (PWB) that allows chronic exposure to reduced weight-bearing in mice to determine the effects of murine sclerostin antibody (SclAbII) on bone microstructure and strength across different levels of mechanical unloading. We hypothesize that treatment with SclAbII would improve bone mass, microarchitecture and strength in all loading conditions, but that there would be a greater skeletal response in the normally loaded mice than in partially unloaded mice suggesting the importance of combined countermeasures for exploration-class long duration spaceflight missions. Eleven-week-old female mice were assigned to one of four loading groups: normal weight-bearing controls (CON) or weight-bearing at 20% (PWB20), 40% (PWB40) or 70% (PWB70) of normal. Mice in each group received either SclAbII (25mg/kg) or vehicle (VEH) via twice weekly subcutaneous injection for 3 weeks. In partially-unloaded VEH-treated groups, leg BMD decreased -5 to -10% in a load-dependent manner. SclAbII treatment completely inhibited bone deterioration due to PWB, with bone properties in SclAbII-treated groups being equal to or greater than those of CON, VEH-treated mice. SclAbII treatment increased leg BMD from +14 to +18% in the PWB groups and 30 ± 3% in CON (p< 0.0001 for all). Trabecular bone volume, assessed by μCT at the distal femur, was lower in all partially unloaded VEH-treated groups vs. CON-VEH (p< 0.05), and was 2-3 fold higher in SclAbII-treated groups (p< 0.001). Midshaft femoral strength was also significantly higher in SclAbII vs. VEH-groups in all-loading conditions. These results suggest that greater weight bearing leads to greater benefits of SclAbII on bone

  3. Sclerostin antibody inhibits skeletal deterioration in mice exposed to partial weight-bearing

    NASA Astrophysics Data System (ADS)

    Spatz, J. M.; Ellman, R.; Cloutier, A. M.; Louis, L.; van Vliet, M.; Dwyer, D.; Stolina, M.; Ke, H. Z.; Bouxsein, M. L.

    2017-02-01

    Whereas much is known regarding the musculoskeletal responses to full unloading, little is known about the physiological effects and response to pharmacological agents in partial unloading (e.g. Moon and Mars) environments. To address this, we used a previously developed ground-based model of partial weight-bearing (PWB) that allows chronic exposure to reduced weight-bearing in mice to determine the effects of murine sclerostin antibody (SclAbII) on bone microstructure and strength across different levels of mechanical unloading. We hypothesize that treatment with SclAbII would improve bone mass, microarchitecture and strength in all loading conditions, but that there would be a greater skeletal response in the normally loaded mice than in partially unloaded mice suggesting the importance of combined countermeasures for exploration-class long duration spaceflight missions. Eleven-week-old female mice were assigned to one of four loading groups: normal weight-bearing controls (CON) or weight-bearing at 20% (PWB20), 40% (PWB40) or 70% (PWB70) of normal. Mice in each group received either SclAbII (25 mg/kg) or vehicle (VEH) via twice weekly subcutaneous injection for 3 weeks. In partially-unloaded VEH-treated groups, leg BMD decreased -5 to -10% in a load-dependent manner. SclAbII treatment completely inhibited bone deterioration due to PWB, with bone properties in SclAbII-treated groups being equal to or greater than those of CON, VEH-treated mice. SclAbII treatment increased leg BMD from +14 to +18% in the PWB groups and 30 ± 3% in CON (p < 0.0001 for all). Trabecular bone volume, assessed by μCT at the distal femur, was lower in all partially unloaded VEH-treated groups vs. CON-VEH (p < 0.05), and was 2-3 fold higher in SclAbII-treated groups (p < 0.001). Midshaft femoral strength was also significantly higher in SclAbII vs. VEH-groups in all-loading conditions. These results suggest that greater weight bearing leads to greater benefits of SclAbII on bone mass

  4. Comparison of Serum Hemagglutinin and Neuraminidase Inhibition Antibodies After 2010–2011 Trivalent Inactivated Influenza Vaccination in Healthcare Personnel

    PubMed Central

    Laguio-Vila, Maryrose R.; Thompson, Mark G.; Reynolds, Sue; Spencer, Sarah M.; Gaglani, Manjusha; Naleway, Allison; Ball, Sarah; Bozeman, Sam; Baker, Steven; Martínez-Sobrido, Luis; Levine, Min; Katz, Jackie; Fry, Alicia M.; Treanor, John J.

    2015-01-01

    Background. Most inactivated influenza vaccines contain purified and standardized hemagglutinin (HA) and residual neuraminidase (NA) antigens. Vaccine-associated HA antibody responses (hemagglutination inhibition [HAI]) are well described, but less is known about the immune response to the NA. Methods. Serum of 1349 healthcare personnel (HCP) electing or declining the 2010–2011 trivalent-inactivated influenza vaccine ([IIV3], containing A/California/7/2009 p(H1N1), A/Perth/16/2009 [H3N2], B/Brisbane/60/2008 strains) were tested for NA-inhibiting (NAI) antibody by a modified lectin-based assay using pseudotyped N1 and N2 influenza A viruses with an irrelevant (H5) HA. Neuraminidase-inhibiting and HAI antibody titers were evaluated approximately 30 days after vaccination and end-of-season for those with polymerase chain reaction (PCR)-confirmed influenza infection. Results. In 916 HCP (68%) receiving IIV3, a 2-fold increase in N1 and N2 NAI antibody occurred in 63.7% and 47.3%, respectively. Smaller responses occurred in HCP age >50 years and those without prior 2009–2010 IIV3 nor monovalent A(H1N1)pdm09 influenza vaccinations. Forty-four PCR-confirmed influenza infections were observed, primarily affecting those with lower pre-exposure HAI and NAI antibodies. Higher pre-NAI titers correlated with shorter duration of illness for A(H1N1)pdm09 virus infections. Conclusions. Trivalent-inactivated influenza vaccine is modestly immunogenic for N1 and N2 antigens in HCP. Vaccines eliciting robust NA immune responses may improve efficacy and reduce influenza-associated morbidity. PMID:25884004

  5. Antibodies against 70-kD heat shock cognate protein inhibit mediated nuclear import of karyophilic proteins

    PubMed Central

    1992-01-01

    Previously, we found that anti-DDDED antibodies strongly inhibited in vivo nuclear transport of nuclear proteins and that these antibodies recognized a protein of 69 kD (p69) from rat liver nuclear envelopes that showed specific binding activities to the nuclear location sequences (NLSs) of nucleoplasmin and SV-40 large T-antigen. Here we identified this protein as the 70-kD heat shock cognate protein (hsc70) based on its mass, isoelectric point, cellular localization, and partial amino acid sequences. Competition studies indicated that the recombinant hsc70 expressed in Escherichia coli binds to transport competent SV-40 T-antigen NLS more strongly than to the point mutated transport incompetent mutant NLS. To investigate the possible involvement of hsc70 in nuclear transport, we examined the effect of anti-hsc70 rabbit antibodies on the nuclear accumulation of karyophilic proteins. When injected into the cytoplasm of tissue culture cells, anti-hsc70 strongly inhibited the nuclear import of nucleoplasmin, SV- 40 T-antigen NLS bearing BSA and histone H1. In contrast, anti-hsc70 IgG did not prevent the diffusion of lysozyme or 17.4-kD FITC-dextran into the nuclei. After injection of these antibodies, cells continued RNA synthesis and were viable. These results indicate that hsc70 interacts with NLS-containing proteins in the cytoplasm before their nuclear import. PMID:1332978

  6. Alpha-lipoic acid potently inhibits peroxynitrite-mediated DNA strand breakage and hydroxyl radical formation: implications for the neuroprotective effects of alpha-lipoic acid.

    PubMed

    Jia, Zhenquan; Zhu, Hong; Vitto, Michael J; Misra, Bhaba R; Li, Yunbo; Misra, Hara P

    2009-03-01

    Alpha-lipoic acid (LA) has recently been reported to afford protection against neurodegenerative disorders in humans and experimental animals. However, the mechanisms underlying LA-mediated neuroprotection remain an enigma. Because peroxynitrite has been extensively implicated in the pathogenesis of various forms of neurodegenerative disorders, this study was undertaken to investigate the effects of LA in peroxynitrite-induced DNA strand breaks, a critical event leading to peroxynitrite-elicited cytotoxicity. Incubation of phi X-174 plasmid DNA with the 3-morpholinosydnonimine (SIN-1), a peroxynitrite generator, led to the formation of both single- and double-stranded DNA breaks in a concentration- and time-dependent fashion. The presence of LA at 100-1,600 microM was found to significantly inhibit SIN-1-induced DNA strand breaks in a concentration-dependent manner. The consumption of oxygen induced by 250 microM SIN-1 was found to be decreased in the presence of high concentrations of LA (400-1,600 microM), indicating that LA at these concentrations may affect the generation of peroxynitrite from auto-oxidation of SIN-1. It is observed that incubation of the plasmid DNA with authentic peroxynitrite resulted in a significant formation of DNA strand breaks, which could also be dramatically inhibited by the presence of LA (100-1,600 microM). EPR spectroscopy in combination with spin-trapping experiments, using 5,5-dimethylpyrroline-N-oxide (DMPO) as spin trap, resulted in the formation of DMPO-hydroxyl radical adduct (DMPO-OH) from authentic peroxynitrite and LA at 50-1,600 microM inhibited the adduct signal. Taken together, these studies demonstrate for the first time that LA can potently inhibit peroxynitrite-mediated DNA strand breakage and hydroxyl radical formation. In view of the critical involvement of peroxynitrite in the pathogenesis of various neurodegenerative diseases, the inhibition of peroxynitrite-mediated DNA damage by LA may be responsible, at least

  7. A novel highly potent trivalent TGF-β receptor trap inhibits early-stage tumorigenesis and tumor cell invasion in murine Pten-deficient prostate glands.

    PubMed

    Qin, Tai; Barron, Lindsey; Xia, Lu; Huang, Haojie; Villarreal, Maria M; Zwaagstra, John; Collins, Cathy; Yang, Junhua; Zwieb, Christian; Kodali, Ravindra; Hinck, Cynthia S; Kim, Sun Kyung; Reddick, Robert L; Shu, Chang; O'Connor-McCourt, Maureen D; Hinck, Andrew P; Sun, Lu-Zhe

    2016-12-27

    The effects of transforming growth factor beta (TGF-β) signaling on prostate tumorigenesis has been shown to be strongly dependent on the stage of development, with TGF-β functioning as a tumor suppressor in early stages of disease and as a promoter in later stages. To study in further detail the paradoxical tumor-suppressive and tumor-promoting roles of the TGF-β pathway, we investigated the effect of systemic treatment with a TGF-β inhibitor on early stages of prostate tumorigenesis. To ensure effective inhibition, we developed and employed a novel trivalent TGF-β receptor trap, RER, comprised of domains derived from the TGF-β type II and type III receptors. This trap was shown to completely block TβRII binding, to antagonize TGF-β1 and TGF-β3 signaling in cultured epithelial cells at low picomolar concentrations, and it showed equal or better anti-TGF-β activities than a pan TGF-β neutralizing antibody and a TGF-β receptor I kinase inhibitor in various prostate cancer cell lines. Systemic administration of RER inhibited prostate tumor cell proliferation as indicated by reduced Ki67 positive cells and invasion potential of tumor cells in high grade prostatic intraepithelial neoplasia (PIN) lesions in the prostate glands of Pten conditional null mice. These results provide evidence that TGF-β acts as a promoter rather than a suppressor in the relatively early stages of this spontaneous prostate tumorigenesis model. Thus, inhibition of TGF-β signaling in early stages of prostate cancer may be a novel therapeutic strategy to inhibit the progression as well as the metastatic potential in patients with prostate cancer.

  8. A novel highly potent trivalent TGF-β receptor trap inhibits early-stage tumorigenesis and tumor cell invasion in murine Pten-deficient prostate glands

    PubMed Central

    Qin, Tai; Barron, Lindsey; Xia, Lu; Huang, Haojie; Villarreal, Maria M.; Zwaagstra, John; Collins, Cathy; Yang, Junhua; Zwieb, Christian; Kodali, Ravindra; Hinck, Cynthia S.; Kim, Sun Kyung; Reddick, Robert L.; Shu, Chang; O’Connor-McCourt, Maureen D.; Hinck, Andrew P.; Sun, Lu-Zhe

    2016-01-01

    The effects of transforming growth factor beta (TGF-β) signaling on prostate tumorigenesis has been shown to be strongly dependent on the stage of development, with TGF-β functioning as a tumor suppressor in early stages of disease and as a promoter in later stages. To study in further detail the paradoxical tumor-suppressive and tumor-promoting roles of the TGF-β pathway, we investigated the effect of systemic treatment with a TGF-β inhibitor on early stages of prostate tumorigenesis. To ensure effective inhibition, we developed and employed a novel trivalent TGF-β receptor trap, RER, comprised of domains derived from the TGF-β type II and type III receptors. This trap was shown to completely block TβRII binding, to antagonize TGF-β1 and TGF-β3 signaling in cultured epithelial cells at low picomolar concentrations, and it showed equal or better anti-TGF-β activities than a pan TGF-β neutralizing antibody and a TGF-β receptor I kinase inhibitor in various prostate cancer cell lines. Systemic administration of RER inhibited prostate tumor cell proliferation as indicated by reduced Ki67 positive cells and invasion potential of tumor cells in high grade prostatic intraepithelial neoplasia (PIN) lesions in the prostate glands of Pten conditional null mice. These results provide evidence that TGF-β acts as a promoter rather than a suppressor in the relatively early stages of this spontaneous prostate tumorigenesis model. Thus, inhibition of TGF-β signaling in early stages of prostate cancer may be a novel therapeutic strategy to inhibit the progression as well as the metastatic potential in patients with prostate cancer. PMID:27863384

  9. 125I-labeled anti-bFGF monoclonal antibody inhibits growth of hepatocellular carcinoma

    PubMed Central

    Hu, Peng-Hui; Pan, Lan-Hong; Wong, Patrick Ting-Yat; Chen, Wen-Hui; Yang, Yan-Qing; Wang, Hong; Xiang, Jun-Jian; Xu, Meng

    2016-01-01

    AIM: To investigate the inhibitory efficacy of 125I-labeled anti-basic fibroblast growth factor (bFGF) monoclonal antibody (mAb) in hepatocellular carcinoma (HCC). METHODS: bFGF mAb was prepared by using the 1G9B9 hybridoma cell line with hybridization technology and extracted from ascites fluid through a Protein G Sepharose affinity column. After labeling with 125I through the chloramine-T method, bFGF mAb was further purified by a Sephadex G-25 column. Gamma radiation counter GC-1200 detected radioactivity of 125I-bFGF mAb. The murine H22 HCC xenograft model was established and randomized to interventions with control (phosphate-buffered saline), 125I-bFGF mAb, 125I plus bFGF mAb, bFGF mAb, or 125I. The ratios of tumor inhibition were then calculated. Expression of bFGF, fibroblast growth factor receptor (FGFR), platelet-derived growth factor, and vascular endothelial growth factor (VEGF) mRNA was determined by quantitative reverse transcriptase real-time polymerase chain reaction. RESULTS: The purified bFGF mAb solution was 8.145 mg/mL with a titer of 1:2560000 and was stored at -20 °C. After coupling, 125I-bFGF mAb was used at a 1: 1280000 dilution, stored at 4 °C, and its specific radioactivity was 37 MBq/mg. The corresponding tumor weight in the control, 125I, bFGF mAb, 125I plus bFGF mAb, and 125I-bFGF mAb groups was 1.88 ± 0.25, 1.625 ± 0.21, 1.5 ± 0.18, 1.41 ± 0.16, and 0.98 ± 0.11 g, respectively. The tumor inhibition ratio in the 125I, bFGF mAb, 125I plus bFGF mAb, and 125I-bFGF mAb groups was 13.6%, 20.2%, 25.1%, and 47.9%, respectively. Growth of HCC xenografts was inhibited significantly more in the 125I-bFGF mAb group than in the other groups (P < 0.05). Expression of bFGF and FGFR mRNA in the 125I-bFGF mAb group was significantly decreased in comparison with other groups (P < 0.05). Groups under interventions revealed increased expression of VEGF mRNA (except for 125I group) compared with the control group. CONCLUSION: 125I-bFGF m

  10. [Inhibition of expontaneous cytotoxicity and antibody dependency by rheumatoid synovial fluid].

    PubMed

    Noguera Hernando, E; Kreisler, M; Durantez, A; Larrea Gayarre, A; de Landazuri, M O; Cruz Martínez, J

    1978-01-01

    A number of authors have pointed out a diminution of ADCC (Antibody dependent cellular cytotoxicity) in lymphocytes from peripheral blood of patients with rheumatoid arthritis (RA). It has also been found that the addition of rheumatoid serum inhibits ADCC and also spontaneous cellular cytotoxicity (SCC). This effect could be the result of blocking of effector cell receptors for the Fc fragment of IgG by anti-immunoglobulins and/or immune complexes, present in great quantities in rheumatoid serum. We investigated the effect of synovial fluid on the ADCC and SCC shown by purified suspensions of lymphocytes from healthy donors and RA patients towards chicken erythrocytes tagged with 51 Cr. The samples of synovial fluid from patients with RA or arthrosis did not influence per se the spontaneous release of 51 Cr, once their complement had been removed. Seven-eight of the rheumatoid synovial fluid (RSF) produced a significant decline (p less than 0.01) of SCC. Lymphocytes from the peripheral blood of RA patients showed a greater decline in SCC after the addition of RSF than those from healthy subjects (p less than 0.02). In 14/16 RSF and 5/7 samples of arthrosis synovial fluid (ASF) the ability to diminish ADCC significantly (P less than 0.01) was shown. RSF maintained this inhibitory effect in 1:40 and 1:80 dilutions, whereas in these conditions ASF had no effect on ADCC. RSF and ASF, before their complement was removed, showed an opposite effect, provoking an increase in cytotoxic activity, both SCC and ADCC, though in different proportions. These experiments show that RSF, like rheumatoid serum, inhibits ADCC and SCC, possibly by the same mechanism which blocks the Fc receptors by means of immune complexes, and coincides in its general lines with the recent findings of Díaz Jouanen et al. The pathogenetic implications of this phenomenon are difficult to clarify at present. Its occurrence in vivo would represent the establishment of a local block of cytotoxic

  11. Hemagglutination inhibiting antibody persistence 1 year after influenza vaccination in Korean children and adolescents.

    PubMed

    Kang, Eun Kyeong; Eun, Byung Wook; Kim, Nam Hee; Kim, Yun Kyung; Lim, Jung Sub; Kim, Dong Ho

    2016-12-01

    This study aimed to assess the 1-y immunogenicity of influenza vaccines and the association between immunogenicity at 1 m and further influenza infections in children aged 6 m to 18 y. Serum hemagglutination inhibition (HI) antibody titers and GMTs were determined for the recommended influenza strains 0, 1, 6, and 12 m post-vaccination. The serological evidence of influenza infections were defined as the increase of HI titer (HI ≥1:40 and 4-fold rise). The seroprotection rates for strains A(H1N1), A(H3N2), and B were 91.2%, 87.6%, and 87.6%, respectively, at 1 month (n = 174). These rates were 76.5%, 64.7%, and 54.6%, respectively, at 12 m. The seroprotection rates and GMTs for influenza A(H1N1) and A(H3N2) were higher at 12 m than at 0 m (p < 0.05) but not for B. There were 39 subjects (42 cases) of serological influenza infections. Subjects with seroprotection at 1 m post-vaccination had showed fewer serologic A(H1N1) (10.1 vs 54.5%) and A(H3N2) (7.2 vs 38.1%) infections than the ones with HI titer <1:40 during follow-up (P < 0.01). In conclusion, influenza vaccines used during the 2008-09 season induced adequate 1-y immunogenicity for A(H1N1) and A(H3N2). The immunogenicity at one month after vaccination influenced further serological influenza infections.

  12. Post-Streptococcal Auto-Antibodies Inhibit Protein Disulfide Isomerase and Are Associated with Insulin Resistance

    PubMed Central

    Aran, Adi; Weiner, Karin; Lin, Ling; Finn, Laurel Ann; Greco, Mary Ann; Peppard, Paul; Young, Terry; Ofran, Yanay; Mignot, Emmanuel

    2010-01-01

    Post-streptococcal autoimmunity affects millions worldwide, targeting multiple organs including the heart, brain, and kidneys. To explore the post-streptococcal autoimmunity spectrum, we used western blot analyses, to screen 310 sera from healthy subjects with (33%) and without (67%) markers of recent streptococcal infections [anti-Streptolysin O (ASLO) or anti-DNAse B (ADB)]. A 58 KDa protein, reacting strongly with post-streptococcal sera, was identified as Protein Disulfide Isomerase (PDI), an abundant protein with pleiotropic metabolic, immunologic, and thrombotic effects. Anti-PDI autoantibodies, purified from human sera, targeted similar epitopes in Streptolysin O (SLO, P51-61) and PDI (P328-338). The correlation between post-streptococcal status and anti-human PDI auto-immunity was further confirmed in a total of 2987 samples (13.6% in 530 ASLO positive versus 5.6% in 2457 ASLO negative samples, p<0.0001). Finally, anti-PDI auto-antibodies inhibited PDI-mediated insulin degradation in vitro (n = 90, p<0.001), and correlated with higher serum insulin (14.1 iu/ml vs. 12.2 iu/ml, n = 1215, p = 0.039) and insulin resistance (Homeostatic Model Assessment (HOMA) 4.1 vs. 3.1, n = 1215, p = 0.004), in a population-based cohort. These results identify PDI as a major target of post-streptococcal autoimmunity, and establish a new link between infection, autoimmunity, and metabolic disturbances. PMID:20886095

  13. Monoclonal Antibodies Targeting the Alpha-Exosite of Botulinum Neurotoxin Serotype/A Inhibit Catalytic Activity

    PubMed Central

    Fan, Yongfeng; Geren, Isin N.; Dong, Jianbo; Lou, Jianlong; Wen, Weihua; Conrad, Fraser; Smith, Theresa J.; Smith, Leonard A.; Ho, Mengfei; Pires-Alves, Melissa; Wilson, Brenda A.; Marks, James D.

    2015-01-01

    The paralytic disease botulism is caused by botulinum neurotoxins (BoNT), multi-domain proteins containing a zinc endopeptidase that cleaves the cognate SNARE protein, thereby blocking acetylcholine neurotransmitter release. Antitoxins currently used to treat botulism neutralize circulating BoNT but cannot enter, bind to or neutralize BoNT that has already entered the neuron. The light chain endopeptidase domain (LC) of BoNT serotype A (BoNT/A) was targeted for generation of monoclonal antibodies (mAbs) that could reverse paralysis resulting from intoxication by BoNT/A. Single-chain variable fragment (scFv) libraries from immunized humans and mice were displayed on the surface of yeast, and 19 BoNT/A LC-specific mAbs were isolated by using fluorescence-activated cell sorting (FACS). Affinities of the mAbs for BoNT/A LC ranged from a KD value of 9.0×10−11 M to 3.53×10−8 M (mean KD 5.38×10−9 M and median KD 1.53×10−9 M), as determined by flow cytometry analysis. Eleven mAbs inhibited BoNT/A LC catalytic activity with IC50 values ranging from 8.3 ~73×10−9 M. The fine epitopes of selected mAbs were also mapped by alanine-scanning mutagenesis, revealing that the inhibitory mAbs bound the α-exosite region remote from the BoNT/A LC catalytic center. The results provide mAbs that could prove useful for intracellular reversal of paralysis post-intoxication and further define epitopes that could be targeted by small molecule inhibitors. PMID:26275214

  14. Radiometric cytolysis inhibition assay, a new rapid test for neutralizing antibodies to intact and trypsin-cleaved poliovirus

    SciTech Connect

    Hovi, T.; Roivainen, M.

    1989-04-01

    We have developed a new rapid test, the radiometric cytolysis inhibition assay (RACINA), for the determination of neutralizing poliovirus antibodies. HeLa cells prelabeled with /sup 51/Cr, (/sup 3/H)leucine, or, preferentially, with (/sup 3/H)uridine are used as sensitive quantitative indicators of residual infectious virus. Both suspensions and monolayer cultures of the indicator cells can be used. Neutralization of a fraction of a high-titer virus preparation can be scored after the first replication cycle at 8 to 10 h. By lowering the incubation temperature to 30/degree/C, the completion of the cytolysis due to the first replication cycle of poliovirus was delayed beyond 21 h. This makes it possible to use the RACINA, unlike the standard microneutralization assay, for measuring antibodies to trypsin-cleaved polioviruses. The RACINA was found to be as sensitive as and more reproducible than the standard microneutralization assay in the measurement of neutralizing poliovirus antibodies. The RACINA is a rapid and reliable test for neutralizing antibodies and in principle it may be applicable for quantitation of neutralizing antibodies to other cytolytic agents as well.

  15. Antibodies against synthetic epitopes inhibit the enzymatic activity of mutalysin II, a metalloproteinase from bushmaster snake venom.

    PubMed

    Ferreira, R N; Machado de Avila, R A; Sanchez, E F; Maria, W S; Molina, F; Granier, C; Chávez-Olórtegui, C

    2006-12-15

    Mutalysin II (mut-II), a 22.5kDa zinc endopeptidase isolated from bushmaster (Lachesis muta muta) snake venom, is a direct acting fibrin(ogen)olytic proteinase. It induces monoclonal and polyclonal antibodies which efficiently neutralize the hemorrhagic effect of L. muta and several Bothrops whole venoms. To characterize epitopes of protective antibodies we have used the Spot method of multiple peptide synthesis to prepare 64 overlapping dodecapeptides frameshifted by three residues, covering the complete amino acid sequence of mut-II. The rabbit anti-mut-II antibodies binding pattern to peptides revealed several continuous antigenic regions: one in the N-terminal part, two in the central region and the other in the C-terminal of mut-II. By using homology modelling, a three-dimensional model of mut-II was built which showed that epitopes are surface exposed. Anti-peptide antibodies were raised against three peptides (one representative of each epitope region) covalently coupled as a mixture to keyhole limpet hemocyanin. Purified IgG from the resulting anti- peptide antibodies cross-reacted with mut-II and induced a dose-dependent inhibition of the mut-II catalyzed proteolysis of fibrinogen.

  16. Hemagglutination by psittacine beak and feather disease virus and use of hemagglutination inhibition for detection of antibodies against the virus.

    PubMed

    Ritchie, B W; Niagro, F D; Latimer, K S; Steffens, W L; Pesti, D; Lukert, P D

    1991-11-01

    Conditions for psittacine beak and feather disease (PBFD) virus hemagglutination and hemagglutination-inhibition (HI) test reactions are defined. The PBFD virus was found to hemagglutinate cockatoo and some guinea pig erythrocytes. The HI test was used to assay serum antibody titer in birds with active PBFD virus infections and in others that had been exposed to diseased birds. On the basis of HI antibody titers in psittacine birds that had been exposed to PBFD virus, but remained clinically normal, we suggest that some birds exposed to the virus are able to mount an effective immune response. Birds with active PBFD virus infections had lower antibody values than did birds that had been exposed to the virus, but remained clinically normal. On the basis of these findings, the ability to develop a suitable HI antibody response may be crucial in determining the disease status of susceptible birds exposed to the PBFD virus. If HI antibodies are found to have neutralizing activity, then the fact that a high HI titer was induced in birds inoculated with purified PBFD virus might suggest that an immunization program would be effective in preventing PBFD virus infections.

  17. Inhibition of preS1-hepatocyte interaction by an array of recombinant human antibodies from naturally recovered individuals

    PubMed Central

    Sankhyan, Anurag; Sharma, Chandresh; Dutta, Durgashree; Sharma, Tarang; Chosdol, Kunzang; Wakita, Takaji; Watashi, Koichi; Awasthi, Amit; Acharya, Subrat K.; Khanna, Navin; Tiwari, Ashutosh; Sinha, Subrata

    2016-01-01

    Neutralizing monoclonal antibodies are being found to be increasingly useful in viral infections. In hepatitis B infection, antibodies are proven to be useful for passive prophylaxis. The preS1 region (21–47a.a.) of HBV contains the viral hepatocyte-binding domain crucial for its attachment and infection of hepatocytes. Antibodies against this region are neutralizing and are best suited for immune-based neutralization of HBV, especially in view of their not recognizing decoy particles. Anti-preS1 (21–47a.a.) antibodies are present in serum of spontaneously recovered individuals. We generated a phage-displayed scFv library using circulating lymphocytes from these individuals and selected four preS1-peptide specific scFvs with markedly distinct sequences from this library. All the antibodies recognized the blood-derived and recombinant preS1 containing antigens. Each scFv showed a discrete binding signature, interacting with different amino acids within the preS1-peptide region. Ability to prevent binding of the preS1 protein (N-terminus 60a.a.) to HepG2 cells stably expressing hNTCP (HepG2-hNTCP-C4 cells), the HBV receptor on human hepatocytes was taken as a surrogate marker for neutralizing capacity. These antibodies inhibited preS1-hepatocyte interaction individually and even better in combination. Such a combination of potentially neutralizing recombinant antibodies with defined specificities could be used for preventing/managing HBV infections, including those by possible escape mutants. PMID:26888694

  18. Inhibition of preS1-hepatocyte interaction by an array of recombinant human antibodies from naturally recovered individuals.

    PubMed

    Sankhyan, Anurag; Sharma, Chandresh; Dutta, Durgashree; Sharma, Tarang; Chosdol, Kunzang; Wakita, Takaji; Watashi, Koichi; Awasthi, Amit; Acharya, Subrat K; Khanna, Navin; Tiwari, Ashutosh; Sinha, Subrata

    2016-02-18

    Neutralizing monoclonal antibodies are being found to be increasingly useful in viral infections. In hepatitis B infection, antibodies are proven to be useful for passive prophylaxis. The preS1 region (21-47a.a.) of HBV contains the viral hepatocyte-binding domain crucial for its attachment and infection of hepatocytes. Antibodies against this region are neutralizing and are best suited for immune-based neutralization of HBV, especially in view of their not recognizing decoy particles. Anti-preS1 (21-47a.a.) antibodies are present in serum of spontaneously recovered individuals. We generated a phage-displayed scFv library using circulating lymphocytes from these individuals and selected four preS1-peptide specific scFvs with markedly distinct sequences from this library. All the antibodies recognized the blood-derived and recombinant preS1 containing antigens. Each scFv showed a discrete binding signature, interacting with different amino acids within the preS1-peptide region. Ability to prevent binding of the preS1 protein (N-terminus 60a.a.) to HepG2 cells stably expressing hNTCP (HepG2-hNTCP-C4 cells), the HBV receptor on human hepatocytes was taken as a surrogate marker for neutralizing capacity. These antibodies inhibited preS1-hepatocyte interaction individually and even better in combination. Such a combination of potentially neutralizing recombinant antibodies with defined specificities could be used for preventing/managing HBV infections, including those by possible escape mutants.

  19. Potent and Selective Inhibitors of Trypanosoma cruzi Triosephosphate Isomerase with Concomitant Inhibition of Cruzipain: Inhibition of Parasite Growth through Multitarget Activity.

    PubMed

    Aguilera, Elena; Varela, Javier; Birriel, Estefanía; Serna, Elva; Torres, Susana; Yaluff, Gloria; de Bilbao, Ninfa Vera; Aguirre-López, Beatriz; Cabrera, Nallely; Díaz Mazariegos, Selma; de Gómez-Puyou, Marieta Tuena; Gómez-Puyou, Armando; Pérez-Montfort, Ruy; Minini, Lucia; Merlino, Alicia; Cerecetto, Hugo; González, Mercedes; Alvarez, Guzmán

    2016-06-20

    Triosephosphate isomerase (TIM) is an essential Trypanosoma cruzi enzyme and one of the few validated drug targets for Chagas disease. The known inhibitors of this enzyme behave poorly or have low activity in the parasite. In this work, we used symmetrical diarylideneketones derived from structures with trypanosomicidal activity. We obtained an enzymatic inhibitor with an IC50 value of 86 nm without inhibition effects on the mammalian enzyme. These molecules also affected cruzipain, another essential proteolytic enzyme of the parasite. This dual activity is important to avoid resistance problems. The compounds were studied in vitro against the epimastigote form of the parasite, and nonspecific toxicity to mammalian cells was also evaluated. As a proof of concept, three of the best derivatives were also assayed in vivo. Some of these derivatives showed higher in vitro trypanosomicidal activity than the reference drugs and were effective in protecting infected mice. In addition, these molecules could be obtained by a simple and economic green synthetic route, which is an important feature in the research and development of future drugs for neglected diseases.

  20. Compounds isolated from the aerial part of Crataegus azarolus inhibit growth of B16F10 melanoma cells and exert a potent inhibition of the melanin synthesis.

    PubMed

    Mustapha, Nadia; Bzéouich, Imèn Mokdad; Ghedira, Kamel; Hennebelle, Thierry; Chekir-Ghedira, Leila

    2015-02-01

    Poor therapeutic results have been reported for treatment of malignant melanoma; therefore in this study, we have investigated inhibitory capacity of vitexin-2''-O-rhamnoside as well as the extract from which it was isolated, i.e. the ethyl acetate extract obtained from the leaves of Crataegus azarolus, on mouse melanoma (B16F10) proliferation. Cell viability was determined using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In addition, amounts of melanin and tyrosinase were measured spectrophotometrically at 475nm. Ethyl acetate extract and vitexin-2''-O-rhamnoside exhibited significant anti-proliferative activity against B16F10 melanoma cells after incubation for 48hours with IC50s of 50μg/mL and 20μM, respectively. Furthermore, these two compounds have the ability to reduce the melanin content by inhibiting the tyrosinase activity of B16F10 cells. Thus, further investigations are merited to ascertain their potential application in treating hyperpigmentation disorders.

  1. C-5-Modified Tetrahydropyrano-Tetrahydofuran-Derived Protease Inhibitors (PIs) Exert Potent Inhibition of the Replication of HIV-1 Variants Highly Resistant to Various PIs, including Darunavir

    PubMed Central

    Aoki, Manabu; Hayashi, Hironori; Yedidi, Ravikiran S.; Martyr, Cuthbert D.; Takamatsu, Yuki; Aoki-Ogata, Hiromi; Nakamura, Teruya; Nakata, Hirotomo; Das, Debananda; Yamagata, Yuriko; Ghosh, Arun K.

    2015-01-01

    ABSTRACT We identified three nonpeptidic HIV-1 protease inhibitors (PIs), GRL-015, -085, and -097, containing tetrahydropyrano-tetrahydrofuran (Tp-THF) with a C-5 hydroxyl. The three compounds were potent against a wild-type laboratory HIV-1 strain (HIV-1WT), with 50% effective concentrations (EC50s) of 3.0 to 49 nM, and exhibited minimal cytotoxicity, with 50% cytotoxic concentrations (CC50) for GRL-015, -085, and -097 of 80, >100, and >100 μM, respectively. All the three compounds potently inhibited the replication of highly PI-resistant HIV-1 variants selected with each of the currently available PIs and recombinant clinical HIV-1 isolates obtained from patients harboring multidrug-resistant HIV-1 variants (HIVMDR). Importantly, darunavir (DRV) was >1,000 times less active against a highly DRV-resistant HIV-1 variant (HIV-1DRVRP51); the three compounds remained active against HIV-1DRVRP51 with only a 6.8- to 68-fold reduction. Moreover, the emergence of HIV-1 variants resistant to the three compounds was considerably delayed compared to the case of DRV. In particular, HIV-1 variants resistant to GRL-085 and -097 did not emerge even when two different highly DRV-resistant HIV-1 variants were used as a starting population. In the structural analyses, Tp-THF of GRL-015, -085, and -097 showed strong hydrogen bond interactions with the backbone atoms of active-site amino acid residues (Asp29 and Asp30) of HIV-1 protease. A strong hydrogen bonding formation between the hydroxyl moiety of Tp-THF and a carbonyl oxygen atom of Gly48 was newly identified. The present findings indicate that the three compounds warrant further study as possible therapeutic agents for treating individuals harboring wild-type HIV and/or HIVMDR. IMPORTANCE Darunavir (DRV) inhibits the replication of most existing multidrug-resistant HIV-1 strains and has a high genetic barrier. However, the emergence of highly DRV-resistant HIV-1 strains (HIVDRVR) has recently been observed in vivo and in

  2. Bedaquiline, an FDA-approved antibiotic, inhibits mitochondrial function and potently blocks the proliferative expansion of stem-like cancer cells (CSCs).

    PubMed

    Fiorillo, Marco; Lamb, Rebecca; Tanowitz, Herbert B; Cappello, Anna Rita; Martinez-Outschoorn, Ubaldo E; Sotgia, Federica; Lisanti, Michael P

    2016-08-01

    Bedaquiline (a.k.a., Sirturo) is an anti-microbial agent, which is approved by the FDA for the treatment of multi-drug resistant pulmonary tuberculosis (TB). Bedaquiline is a first-in-class diaryl-quinoline compound, that mechanistically inhibits the bacterial ATP-synthase, and shows potent activity against both drug-sensitive and drug-resistant TB. Interestingly, eukaryotic mitochondria originally evolved from engulfed aerobic bacteria. Thus, we hypothesized that, in mammalian cells, bedaquiline might also target the mitochondrial ATP-synthase, leading to mitochondrial dysfunction and ATP depletion. Here, we show that bedaquiline has anti-cancer activity, directed against Cancer Stem-like Cells (CSCs). More specifically, we demonstrate that bedaquiline treatment of MCF7 breast cancer cells inhibits mitochondrial oxygen-consumption, as well as glycolysis, but induces oxidative stress. Importantly, bedaquiline significantly blocks the propagation and expansion of MCF7-derived CSCs, with an IC-50 of approx. 1-μM, as determined using the mammosphere assay. Similarly, bedaquiline also reduces both the CD44+/CD24low/- CSC and ALDH+ CSC populations, under anchorage-independent growth conditions. In striking contrast, bedaquiline significantly increases oxygen consumption in normal human fibroblasts, consistent with the fact that it is well-tolerated in patients treated for TB infections. As such, future pre-clinical studies and human clinical trials in cancer patients may be warranted. Interestingly, we also highlight that bedaquiline shares certain structural similarities with trans-piceatannol and trans-resveratrol, which are known natural flavonoid inhibitors of the mitochondrial ATP-synthase (complex V) and show anti-aging properties.

  3. Bedaquiline, an FDA-approved antibiotic, inhibits mitochondrial function and potently blocks the proliferative expansion of stem-like cancer cells (CSCs)

    PubMed Central

    Fiorillo, Marco; Lamb, Rebecca; Tanowitz, Herbert B.; Cappello, Anna Rita; Martinez-Outschoorn, Ubaldo E.; Sotgia, Federica; Lisanti, Michael P.

    2016-01-01

    Bedaquiline (a.k.a., Sirturo) is an anti-microbial agent, which is approved by the FDA for the treatment of multi-drug resistant pulmonary tuberculosis (TB). Bedaquiline is a first-in-class diaryl-quinoline compound, that mechanistically inhibits the bacterial ATP-synthase, and shows potent activity against both drug-sensitive and drug-resistant TB. Interestingly, eukaryotic mitochondria originally evolved from engulfed aerobic bacteria. Thus, we hypothesized that, in mammalian cells, bedaquiline might also target the mitochondrial ATP-synthase, leading to mitochondrial dysfunction and ATP depletion. Here, we show that bedaquiline has anti-cancer activity, directed against Cancer Stem-like Cells (CSCs). More specifically, we demonstrate that bedaquiline treatment of MCF7 breast cancer cells inhibits mitochondrial oxygen-consumption, as well as glycolysis, but induces oxidative stress. Importantly, bedaquiline significantly blocks the propagation and expansion of MCF7-derived CSCs, with an IC-50 of approx. 1-μM, as determined using the mammosphere assay. Similarly, bedaquiline also reduces both the CD44+/CD24low/− CSC and ALDH+ CSC populations, under anchorage-independent growth conditions. In striking contrast, bedaquiline significantly increases oxygen consumption in normal human fibroblasts, consistent with the fact that it is well-tolerated in patients treated for TB infections. As such, future pre-clinical studies and human clinical trials in cancer patients may be warranted. Interestingly, we also highlight that bedaquiline shares certain structural similarities with trans-piceatannol and trans-resveratrol, which are known natural flavonoid inhibitors of the mitochondrial ATP-synthase (complex V) and show anti-aging properties. PMID:27344270

  4. CARMIL is a potent capping protein antagonist: identification of a conserved CARMIL domain that inhibits the activity of capping protein and uncaps capped actin filaments.

    PubMed

    Uruno, Takehito; Remmert, Kirsten; Hammer, John A

    2006-04-14

    Acanthamoeba CARMIL was previously shown to co-purify with capping protein (CP) and to bind pure CP. Here we show that this interaction inhibits the barbed end-capping activity of CP. Even more strikingly, this interaction drives the uncapping of actin filaments previously capped with CP. These activities are CP-specific; CARMIL does not inhibit the capping activities of either gelsolin or CapG and does not uncap gelsolin-capped filaments. Although full-length (FL) CARMIL (residues 1-1121) possesses both anti-CP activities, C-terminal fragments like glutathione S-transferase (GST)-P (940-1121) that contain the CARMIL CP binding site are at least 10 times more active. We localized the full activities of GST-P to its C-terminal 51 residues (1071-1121). This sequence contains a stretch of 25 residues that is highly conserved in CARMIL proteins from protozoa, flies, worms, and vertebrates (CARMIL Homology domain 3; CAH3). Point mutations showed that the majority of the most highly conserved residues within CAH3 are critical for the anti-CP activity of GST-AP (862-1121). Finally, we found that GST-AP binds CP approximately 20-fold more tightly than does FL-CARMIL. This observation together with the elevated activities of C-terminal fragments relative to FL-CARMIL suggests that FL-CARMIL might exist primarily in an autoinhibited state. Consistent with this idea, proteolytic cleavage of FL-CARMIL with thrombin generated an approximately 14-kDa C-terminal fragment that expresses full anti-CP activities. We propose that, after some type of physiological activation event, FL-CARMIL could function in vivo as a potent CP antagonist. Given the pivotal role that CP plays in determining the global actin phenotype of cells, our results suggest that CARMIL may play an important role in the physiological regulation of actin assembly.

  5. The metalloendopeptidase nardilysin (NRDc) is potently inhibited by heparin-binding epidermal growth factor-like growth factor (HB-EGF).

    PubMed Central

    Hospital, Véronique; Nishi, Eiichiro; Klagsbrun, Michael; Cohen, Paul; Seidah, Nabil G; Prat, Annik

    2002-01-01

    Nardilysin (N-arginine dibasic convertase, or NRDc) is a cytosolic and cell-surface metalloendopeptidase that, in vitro, cleaves substrates upstream of Arg or Lys in basic pairs. NRDc differs from most of the other members of the M16 family of metalloendopeptidases by a 90 amino acid acidic domain (DAC) inserted close to its active site. At the cell surface, NRDc binds heparin-binding epidermal growth factor-like growth factor (HB-EGF) and enhances HB-EGF-induced cell migration. An active-site mutant of NRDc fulfills this function as well as wild-type NRDc, indicating that the enzyme activity is not required for this process. We now demonstrate that NRDc starts at Met(49). Furthermore, we show that HB-EGF not only binds to NRDc but also potently inhibits its enzymic activity. NRDc-HB-EGF interaction involves the 21 amino acid heparin-binding domain (P21) of the growth factor, the DAC of NRDc and most probably its active site. Only disulphide-bonded P21 dimers are inhibitory. We also show that Ca(2+), via the DAC, regulates both NRDc activity and HB-EGF binding. We conclude that the DAC is thus a key regulatory element for the two distinct functions that NRDc fulfills, i.e. as an HB-EGF modulator and a peptidase. PMID:12095415

  6. Applying a dual process model of self-regulation: The association between executive working memory capacity, negative urgency, and negative mood induction on pre-potent response inhibition.

    PubMed

    Gunn, Rachel L; Finn, Peter R

    2015-03-01

    This study tested a dual-process model of self-control where the combination of high impulsivity (negative urgency - NU), weak reflective / control processes (low executive working memory capacity - E-WMC), and a cognitive load is associated with increased failures to inhibit pre-potent responses on a cued go/no-go task. Using a within-subjects design, a cognitive load with and without negative emotional load was implemented to consider situational factors. Results suggested that: (1) high NU was associated with low E-WMC; (2) low E-WMC significantly predicted more inhibitory control failures across tasks; and (3) there was a significant interaction of E-WMC and NU, revealing those with low E-WMC and high NU had the highest rates of inhibitory control failures on all conditions of the task. In conclusion, results suggest that while E-WMC is a strong independent predictor of inhibitory control, NU provides additional information for vulnerability to problems associated with self-regulation.

  7. The metalloendopeptidase nardilysin (NRDc) is potently inhibited by heparin-binding epidermal growth factor-like growth factor (HB-EGF).

    PubMed

    Hospital, Véronique; Nishi, Eiichiro; Klagsbrun, Michael; Cohen, Paul; Seidah, Nabil G; Prat, Annik

    2002-10-01

    Nardilysin (N-arginine dibasic convertase, or NRDc) is a cytosolic and cell-surface metalloendopeptidase that, in vitro, cleaves substrates upstream of Arg or Lys in basic pairs. NRDc differs from most of the other members of the M16 family of metalloendopeptidases by a 90 amino acid acidic domain (DAC) inserted close to its active site. At the cell surface, NRDc binds heparin-binding epidermal growth factor-like growth factor (HB-EGF) and enhances HB-EGF-induced cell migration. An active-site mutant of NRDc fulfills this function as well as wild-type NRDc, indicating that the enzyme activity is not required for this process. We now demonstrate that NRDc starts at Met(49). Furthermore, we show that HB-EGF not only binds to NRDc but also potently inhibits its enzymic activity. NRDc-HB-EGF interaction involves the 21 amino acid heparin-binding domain (P21) of the growth factor, the DAC of NRDc and most probably its active site. Only disulphide-bonded P21 dimers are inhibitory. We also show that Ca(2+), via the DAC, regulates both NRDc activity and HB-EGF binding. We conclude that the DAC is thus a key regulatory element for the two distinct functions that NRDc fulfills, i.e. as an HB-EGF modulator and a peptidase.

  8. Applying a dual process model of self-regulation: The association between executive working memory capacity, negative urgency, and negative mood induction on pre-potent response inhibition

    PubMed Central

    Gunn, Rachel L.; Finn, Peter R.

    2014-01-01

    This study tested a dual-process model of self-control where the combination of high impulsivity (negative urgency – NU), weak reflective / control processes (low executive working memory capacity - E-WMC), and a cognitive load is associated with increased failures to inhibit pre-potent responses on a cued go/no-go task. Using a within-subjects design, a cognitive load with and without negative emotional load was implemented to consider situational factors. Results suggested that: (1) high NU was associated with low E-WMC; (2) low E-WMC significantly predicted more inhibitory control failures across tasks; and (3) there was a significant interaction of E-WMC and NU, revealing those with low E-WMC and high NU had the highest rates of inhibitory control failures on all conditions of the task. In conclusion, results suggest that while E-WMC is a strong independent predictor of inhibitory control, NU provides additional information for vulnerability to problems associated with self-regulation. PMID:25530648

  9. Complement inhibition enables tumor delivery of LCMV glycoprotein pseudotyped viruses in the presence of antiviral antibodies

    PubMed Central

    Evgin, Laura; Ilkow, Carolina S; Bourgeois-Daigneault, Marie-Claude; de Souza, Christiano Tanese; Stubbert, Lawton; Huh, Michael S; Jennings, Victoria A; Marguerie, Monique; Acuna, Sergio A; Keller, Brian A; Lefebvre, Charles; Falls, Theresa; Le Boeuf, Fabrice; Auer, Rebecca A; Lambris, John D; McCart, J Andrea; Stojdl, David F; Bell, John C

    2016-01-01

    The systemic delivery of therapeutic viruses, such as oncolytic viruses or vaccines, is limited by the generation of neutralizing antibodies. While pseudotyping of rhabdoviruses with the lymphocytic choriomeningitis virus glycoprotein has previously allowed for multiple rounds of delivery in mice, this strategy has not translated to other animal models. For the first time, we provide experimental evidence that antibodies generated against the lymphocytic choriomeningitis virus glycoprotein mediate robust complement-dependent viral neutralization via activation of the classical pathway. We show that this phenotype can be capitalized upon to deliver maraba virus pseudotyped with the lymphocytic choriomeningitis virus glycoprotein in a Fischer rat model in the face of neutralizing antibody through the use of complement modulators. This finding changes the understanding of the humoral immune response to arenaviruses, and also describes methodology to deliver viral vectors to their therapeutic sites of action without the interference of neutralizing antibody. PMID:27909702

  10. Proteolytic processing of poliovirus polypeptides: antibodies to polypeptide P3-7c inhibit cleavage at glutamine-glycine pairs

    SciTech Connect

    Hanecak, R.; Semler, B.L.; Anderson, C.W.; Wimmer, E.

    1982-07-01

    Proteolytic processing of poliovirus polypeptides was examined by the addition of antibodies directed against the viral proteins P3-7c and P2-X to a cell-free translation extract prepared from infected HeLa cells. Antisera to P3-7c specifically inhibited in vitro processing at Gln-Gly pairs. Partial amino acid sequence analysis revealed a second Tyr-Gly pair that is utilized in protein processing. Neither Tyr-Gly cleavage is affected by antibody to P3-7C. Anti-P3-7c antibodies react not only with P3-7c but also with P3-6a and P3-2, two viral polypeptides NH/sub 2/-coterminal with P3-7c. Preimmune and anti-P2-X antibodies had no effect on the processing of poliovirus proteins in vitro. The authors conclude that the activity responsible for processing poliovirus polypeptides at Gln-Gly pairs resides in the primary structure of P3-7c and not in P2-X.

  11. A short treatment with an antibody to sclerostin can inhibit bone loss in an ongoing model of colitis.

    PubMed

    Eddleston, Alison; Marenzana, Massimo; Moore, Adrian R; Stephens, Paul; Muzylak, Mariusz; Marshall, Diane; Robinson, Martyn K

    2009-10-01

    Chronic inflammation leads to bone loss, and increased fracture rates have been reported in a number of human chronic inflammatory conditions. The study reported here investigates the skeletal effects of dosing a neutralizing antibody to the bone regulatory protein sclerostin in a mouse model of chronic colitis. When dosed prophylactically, an antibody to sclerostin (Scl-AbI) did not reduce the weight loss or histological changes associated with colitis but did prevent inflammation-induced bone loss. At the end of the experiment, Scl-AbI-treated animals had a significantly higher femoral BMD (+27%, p < 0.05) than control antibody (Cntrl-Ab)-treated animals. In a second experiment, treatment with Scl-AbI was delayed until colitis had developed, by which time the mechanical properties of femurs in colitic animals were significantly worse than those of healthy age-matched control mice (maximum load, -26%, p < 0.05; energy, -37%, p < 0.05; ultimate strength, -33%, p < 0.05; elastic modulus, -17%, p < 0.05). A short treatment with Scl-AbI halted bone loss and reversed the decline of both intrinsic and extrinsic mechanical properties of the femur such that, after 19 days of treatment, the bone mechanical properties in the Scl-AbI-treated animals were not significantly different from those of noncolitic age-matched controls. Serum markers of bone formation and resorption suggested that the antibody to sclerostin stimulated osteoblast activity and inhibited osteoclast-mediated bone resorption.

  12. Growth inhibition in a brain metastasis model by antibody delivery using focused ultrasound-mediated blood-brain barrier disruption.

    PubMed

    Kobus, Thiele; Zervantonakis, Ioannis K; Zhang, Yongzhi; McDannold, Nathan J

    2016-09-28

    these brain metastases. Interestingly, only some of the rats responded to the treatment. We did not observe a difference in tumor volume at the start of the treatment, nor in HER2 expression or in contrast-enhancement on MRI between the responders and non-responders to explain this. Better understanding of why certain animals respond is needed and will help in translating this technique to the clinic. In conclusion, we demonstrate that BBB disruption using focused ultrasound in combination with antibody therapy can inhibit growth of breast cancer brain metastasis.

  13. Haemagglutination-inhibiting antibodies against arboviruses of the families Togaviridae and Bunyaviridae in birds caught in southern Moravia, Czechoslovakia.

    PubMed

    Juricová, Z; Hubálek, Z; Halouzka, J; Pellantová, J; Chytil, J

    1987-01-01

    A total of 295 birds belonging to 19 species of 7 families of wild Passeriformes were examined by haemagglutination-inhibition test. The birds were caught for an international research program "Balt" at the time of autumn migration (August-September 1984). Their blood sera were examined for antibodies against 6 arbovirus antigens of the genera Alphavirus (Sindbis-SIN) and Flavivirus (tick-borne encephalitis-TBE, West Nile-WN) and family Bunyaviridae (Tahyna-TAH, Calovo-CVO and Bhanja-BHA). Antibodies against all studied viruses were detected at different frequencies: SIN 6.4%, TBE 7.1%, WN 9.7%, TAH 16.3%, CVO 12.1%, and BHA 1.0%.

  14. Immunoreactions involving platelets. III. Quantitative aspects of platelet agglutination, inhibition of clot retraction, and other reactions caused by the antibody of quinidine purpura.

    PubMed

    SHULMAN, N R

    1958-05-01

    Quantitative aspects of platelet agglutination and inhibition of clot retraction by the antibody of quinidine purpura were described. The reactions appeared to depend on formation of types of antibody-quinidine-platelet complexes which could fix complement but complement was not necessary for these reactions. Complement fixation was at least 10 times more sensitive than platelet agglutination or inhibition of clot retraction for measurement and detection of antibody activity. Although it has been considered that antibodies of drug purpura act as platelet lysins in the presence of complement and that direct lysis of platelets accounts for development of thrombocytopenia in drug purpura, the present study suggests that attachment of antibody produces a change in platelets which is manifested in vitro only by increased susceptibility to non-specific factors which can alter the stability of platelets in the absence of antibody. The attachment of antibody to platelets in vivo may only indirectly affect platelet survival. In contrast to human platelets, dog, rabbit, and guinea pig platelets, and normal or trypsin-treated human red cells did not agglutinate, fix complement, or adsorb antibody; and intact human endothelial cells did not fix complement or adsorb antibody. Rhesus monkey platelets were not agglutinated by the antibody but did adsorb antibody and fix complement although their activity in these reactions differed quantitatively from that of human platelets. Cinchonine could be substituted for quinidine in agglutination and inhibition of clot retraction reactions but quinine and cinchonidine could not. Attempts to cause passive anaphylaxis in guinea pigs with the antibody of quinidine purpura were not successful.

  15. Influenza nucleoprotein DNA vaccination by a skin targeted, dry coated, densely packed microprojection array (Nanopatch) induces potent antibody and CD8(+) T cell responses.

    PubMed

    Fernando, Germain J P; Zhang, Jin; Ng, Hwee-Ing; Haigh, Oscar L; Yukiko, Sally R; Kendall, Mark A F

    2016-09-10

    DNA vaccines have many advantages such as thermostability and the ease and rapidity of manufacture; for example, in an influenza pandemic situation where rapid production of vaccine is essential. However, immunogenicity of DNA vaccines was shown to be poor in humans unless large doses of DNA are used. If a highly efficacious DNA vaccine delivery system could be identified, then DNA vaccines have the potential to displace protein vaccines. In this study, we show in a C57BL/6 mouse model, that the Nanopatch, a microprojection array of high density (>21,000 projections/cm(2)), could be used to deliver influenza nucleoprotein DNA vaccine to skin, to generate enhanced antigen specific antibody and CD8(+) T cell responses compared to the conventional intramuscular (IM) delivery by the needle and syringe. Antigen specific antibody was measured using ELISA assays of mice vaccinated with a DNA plasmid containing the nucleoprotein gene of influenza type A/WSN/33 (H1N1). Antigen specific CD8(+) T cell responses were measured ex-vivo in splenocytes of mice using IFN-γ ELISPOT assays. These results and our previous antibody and CD4(+) T cell results using the Nanopatch delivered HSV DNA vaccine indicate that the Nanopatch is an effective delivery system of general utility that could potentially be used in humans to increase the potency of the DNA vaccines.

  16. High mannose-specific lectin (KAA-2) from the red alga Kappaphycus alvarezii potently inhibits influenza virus infection in a strain-independent manner.

    PubMed

    Sato, Yuichiro; Morimoto, Kinjiro; Hirayama, Makoto; Hori, Kanji

    2011-02-11

    The carbohydrate binding profile of the red algal lectin KAA-2 from Kappaphycus alvarezii was evaluated by a centrifugal ultrafiltration-HPLC method using pyridylaminated oligosaccharides. KAA-2 bound exclusively to high mannose type N-glycans, but not to other glycans such as complex type, hybrid type, or the pentasaccharide core of N-glycans. This lectin exhibited a preference for an exposed α1-3 Man on a D2 arm in a similar manner to Eucheuma serra agglutinin (ESA-2), which shows various biological activities, such as anti-HIV and anti-carcinogenic activity. We tested the anti-influenza virus activity of KAA-2 against various strains including the recent pandemic H1N1-2009 influenza virus. KAA-2 inhibited infection of various influenza strains with EC50s of low nanomolar levels. Immunofluorescence microscopy using an anti-influenza antibody demonstrated that the antiviral activity of KAA-2 was exerted by interference with virus entry into host cells. This mechanism was further confirmed by the evidence of direct binding of KAA-2 to a viral envelope protein, hemagglutinin (HA), using an ELISA assay. These results indicate that this lectin would be useful as a novel antiviral reagent for the prevention of infection.

  17. Dual targeting of mTORC1 and mTORC2 by INK-128 potently inhibits human prostate cancer cell growth in vitro and in vivo.

    PubMed

    Jiang, Shang-Jun; Wang, Shuo

    2015-09-01

    Both mammalian target of rapamycin (mTOR) complexes 1 and 2 (mTORC1/2) are often over-activated in prostate cancer cells and are associated with cancer progression. In the current study, we evaluated the potential anti-prostate cancer activity of INK-128, an ATP-competitive mTORC1/2 dual inhibitor, both in vitro and in vivo. Our results showed that INK-128 exerted potent anti-proliferative activity in established (PC-3 and LNCaP lines) and primary (patient-derived) human prostate cancer cells by inducing cell apoptosis. The latter was evidenced by increase of annexin V percentage, formation of cytoplasmic histone-associated DNA fragments, and cleavage of caspase-3. INK-128-induced prostate cancer cell apoptosis and cytotoxicity were alleviated upon pretreatment of cells with the pan-caspase inhibitor z-VAD-FMK or the specific caspase-3 inhibitor z-DVED-FMK. At the molecular level, INK-18 blocked mTORC1/2 activation in PC-3 cells and LNCaP cells and downregulated mTOR-regulated genes including cyclin D1, hypoxia-inducible factor 1α (HIF-1α), and HIF-2α. ERK-MAPK activation and androgen receptor expression were, however, not affected by INK-128 treatment. In vivo, oral administration of INK-128 significantly inhibited growth of PC-3 xenografts in nude mice. The preclinical results of this study suggest that INK-128 could be further investigated as a promising anti-prostate cancer agent.

  18. tert-Butylphenylacetylene Is a Potent Mechanism-Based Inactivator of Cytochrome P450 2B4: Inhibition of Cytochrome P450 Catalysis by Steric Hindrance

    PubMed Central

    Zhang, Haoming; Lin, Hsia-lien; Walker, Vyvyca J.; Hamdane, Djemel

    2009-01-01

    We have demonstrated that 4-(tert-butyl)-phenylacetylene (tBPA) is a potent mechanism-based inactivator for cytochrome P450 2B4 (P450 2B4) in the reconstituted system. It inactivates P450 2B4 in a NADPH- and time-dependent manner with a KI of 0.44 μM and kinact of 0.12 min−1. The partition ratio was approximately zero, indicating that inactivation occurs without the reactive intermediate leaving the active site. Liquid chromatography-mass spectrometry analyses revealed that tBPA forms a protein adduct with a 1:1 stoichiometry. Peptide mapping of the tBPA-modified protein provides evidence that tBPA is covalently bound to Thr302. This is consistent with results of molecular modeling that show the terminal carbon of the acetylenic group is only 3.65 Å away from Thr302. To characterize the effect of covalent modification of Thr302, tBPA-modified P450 2B4 was purified to homogeneity from the reconstituted system. The Soret band of tBPA-modified protein is red-shifted by 5 to 422 nm compared with unmodified protein. Benzphetamine binding to the modified P450 2B4 causes no spin shift, indicating that substrate binding and/or the heme environment has been altered by covalently bound tBPA. Cytochrome P450 reductase reduces the unmodified and tBPA-modified P450s at approximately the same rate. However, addition of benzphetamine stimulates the rate of reduction of unmodified P450 2B4 by ∼20-fold but only marginally stimulates reduction of the tBPA-modified protein. This large discrepancy in the stimulation of the first electron transfer by benzphetamine strongly suggests that the impairment of P450 catalysis is due to inhibition of benzphetamine binding to the tBPA-modified P450 2B4. PMID:19720728

  19. Inhibition by yeast killer toxin-like antibodies of oral Streptococci adhesion to tooth surfaces in an ex vivo model.

    PubMed Central

    Conti, Stefania; Magliani, Walter; Arseni, Simona; Frazzi, Raffaele; Salati, Antonella; Ravanetti, Lara; Polonelli, Luciano

    2002-01-01

    BACKGROUND: Monoclonal (KTmAb) and recombinant (KTscFv) anti-idiotypic antibodies, representing the internal image of a yeast killer toxin, proved to be microbicidal in vitro against important eukaryotic and prokaryotic pathogens such as Candida albicans, Pneumocystis carinii, Mycobacterium tuberculosis, Staphylococcus aureus, S. haemolyticus, Enterococcus faecalis, E. faecium, and Streptococcus pneumoniae, including multidrug-resistant strains. KTmAb and KTscFv exerted a strong therapeutic effect in well-established animal models of candidiasis and pneumocystosis. Streptococcus mutans is the most important etiologic agent of dental caries that might result from the metabolic end products of dental plaque. Effective strategies to reduce the disease potential of dental plaque have considered the possibility of using antibiotics or antibodies against oral streptococci in general and S. mutans in particular. In this study, the activity of KTmAb and KTscFv against S. mutans and the inhibition and reduction by KTmAb of dental colonization by S. mutans and other oral streptococci in an ex vivo model of human teeth were investigated. MATERIALS AND METHODS: KTscFv and KTmAb were used in a conventional colony forming unit (CFU) assay against a serotype C strain of S. mutans, and other oral streptococci (S. intermedius, S. mitis, S. oralis, S. salivarius). An ex vivo model of human teeth submerged in saliva was used to establish KTmAb potential of inhibiting or reducing the adhesion to dental surfaces by S. mutans and other oral streptococci. RESULTS: KTmAb and KTscFv kill in vitro S. mutans and other oral streptococci. KTmAb inhibit colonization of dental surfaces by S. mutans and oral streptococci in the ex vivo model. CONCLUSIONS: Killer antibodies with antibiotic activity or their engineered derivatives may have a potential in the prevention of dental caries in vivo. PMID:12428062

  20. Structural insight in the inhibition of adherence of F4 fimbriae producing enterotoxigenic Escherichia coli by llama single domain antibodies.

    PubMed

    Moonens, Kristof; Van den Broeck, Imke; Okello, Emmanuel; Pardon, Els; De Kerpel, Maia; Remaut, Han; De Greve, Henri

    2015-02-24

    Enterotoxigenic Escherichia coli that cause neonatal and post-weaning diarrhea in piglets express F4 fimbriae to mediate attachment towards host receptors. Recently we described how llama single domain antibodies (VHHs) fused to IgA, produced in Arabidopsis thaliana seeds and fed to piglets resulted in a progressive decline in shedding of F4 positive ETEC bacteria. Here we present the structures of these inhibiting VHHs in complex with the major adhesive subunit FaeG. A conserved surface, distant from the lactose binding pocket, is targeted by these VHHs, highlighting the possibility of targeting epitopes on single-domain adhesins that are non-involved in receptor binding.

  1. Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Single Domain Antibodies Are Potent Inhibitors of Low Density Lipoprotein Receptor Degradation.

    PubMed

    Weider, Elodie; Susan-Resiga, Delia; Essalmani, Rachid; Hamelin, Josée; Asselin, Marie-Claude; Nimesh, Surendra; Ashraf, Yahya; Wycoff, Keith L; Zhang, Jianbing; Prat, Annik; Seidah, Nabil G

    2016-08-05

    Single domain antibodies (sdAbs) correspond to the antigen-binding domains of camelid antibodies. They have the same antigen-binding properties and specificity as monoclonal antibodies (mAbs) but are easier and cheaper to produce. We report here the development of sdAbs targeting human PCSK9 (proprotein convertase subtilisin/kexin type 9) as an alternative to anti-PCSK9 mAbs. After immunizing a llama with human PCSK9, we selected four sdAbs that bind PCSK9 with a high affinity and produced them as fusion proteins with a mouse Fc. All four sdAb-Fcs recognize the C-terminal Cys-His-rich domain of PCSK9. We performed multiple cellular assays and demonstrated that the selected sdAbs efficiently blocked PCSK9-mediated low density lipoprotein receptor (LDLR) degradation in cell lines, in human hepatocytes, and in mouse primary hepatocytes. We further showed that the sdAb-Fcs do not affect binding of PCSK9 to the LDLR but rather block its induced cellular LDLR degradation. Pcsk9 knock-out mice expressing a human bacterial artificial chromosome (BAC) transgene were generated, resulting in plasma levels of ∼300 ng/ml human PCSK9. Mice were singly or doubly injected with the best sdAb-Fc and analyzed at day 4 or 11, respectively. After 4 days, mice exhibited a 32 and 44% decrease in the levels of total cholesterol and apolipoprotein B and ∼1.8-fold higher liver LDLR protein levels. At 11 days, the equivalent values were 24 and 46% and ∼2.3-fold higher LDLR proteins. These data constitute a proof-of-principle for the future usage of sdAbs as PCSK9-targeting drugs that can efficiently reduce LDL-cholesterol, and as tools to study the Cys-His-rich domain-dependent sorting the PCSK9-LDLR complex to lysosomes.

  2. Psychological Factors Capable of Preventing the Inhibition of Antibody Responses in Separated Infant Monkeys.

    ERIC Educational Resources Information Center

    Coe, Christopher L.; And Others

    1987-01-01

    Capacity of infant monkeys to mount an antibody response to viral challenge was evaluated after monkeys' removal from their mothers in several social and physical environments. Results indicated that trauma of separation was reduced when infants were familiar with the separation environment or familiar social companions were available. (PCB)

  3. Antibody-mediated inhibition of Nogo-A signaling promotes neurite growth in PC-12 cells

    PubMed Central

    Yazdi, Iman K; Taghipour, Nima; Hmaidan, Sarah; Palomba, Roberto; Scaria, Shilpa; Munoz, Alvaro; Boone, Timothy B; Tasciotti, Ennio

    2016-01-01

    The use of a monoclonal antibody to block the neurite outgrowth inhibitor Nogo-A has been of great interest for promoting axonal recovery as a treatment for spinal cord injury. While several cellular and non-cellular assays have been developed to quantify the bioactive effects of Nogo-A signaling, demand still exists for the development of a reliable approach to characterize the effectiveness of the anti-Nogo-A antibody. In this study, we developed and validated a novel cell-based approach to facilitate the biological quantification of a Nogo-A antibody using PC-12 cells as an in vitro neuronal cell model. Changes in the mRNA levels of the neuronal differentiation markers, growth-associated protein 43 and neurofilament light-polypeptide, suggest that activation of the Nogo-A pathway suppresses axonal growth and dendrite formation in the tested cell line. We found that application of anti-Nogo-A monoclonal antibody can significantly enhance the neuronal maturity of PC-12 cells by blocking the Nogo-A inhibitory effects, providing enhanced effects on neural maturity at the molecular level. No adverse effects were observed on cell viability. PMID:27027860

  4. VEGFR2 targeted antibody fused with MICA stimulates NKG2D mediated immunosurveillance and exhibits potent anti-tumor activity against breast cancer

    PubMed Central

    Wang, Youfu; Ren, Xueyan; Wang, Tong; Chen, Zhiguo; Tang, Mingying; Sun, Fumou; Li, Zhaoting; Wang, Min; Zhang, Juan

    2016-01-01

    Binding of MHC class I-related chain molecules A and B (MICA/B) to the natural killer (NK) cell receptor NK group 2, member D (NKG2D) is thought critical for activating NK-mediated immunosurveillance. Angiogenesis is important for tumor growth and interfering with angiogenesis using the fully human IgG1 anti-VEGFR2 (vascular endothelial growth factor receptor 2) antibody (mAb04) can be effective in treating malignancy. In an effort to make mAb04 more effective we have generated a novel antibody fusion protein (mAb04-MICA) consisting of mAb04 and MICA. We found that mAb04-MICA maintained the anti-angiogenic and antineoplastic activities of mAb04, and also enhanced immunosurveillance activated by the NKG2D pathway. Moreover, in human breast tumor-bearing nude mice, mAb04-MICA demonstrated superior anti-tumor efficacy compared to combination therapy of mAb04 + Docetaxel or Avastin + Docetaxel, highlighting the immunostimulatory effect of MICA. In conclusion, mAb04-MICA provided new inspiration for anti-tumor treatment and had prospects for clinical application. PMID:26909862

  5. Plasmodium falciparum 19-kilodalton merozoite surface protein 1 (MSP1)-specific antibodies that interfere with parasite growth in vitro can inhibit MSP1 processing, merozoite invasion, and intracellular parasite development.

    PubMed

    Moss, David K; Remarque, Edmond J; Faber, Bart W; Cavanagh, David R; Arnot, David E; Thomas, Alan W; Holder, Anthony A

    2012-03-01

    Merozoite surface protein 1 (MSP1) is a target for malaria vaccine development. Antibodies to the 19-kDa carboxy-terminal region referred to as MSP1(19) inhibit erythrocyte invasion and parasite growth, with some MSP1-specific antibodies shown to inhibit the proteolytic processing of MSP1 that occurs at invasion. We investigated a series of antibodies purified from rabbits immunized with MSP1(19) and AMA1 recombinant proteins for their ability to inhibit parasite growth, initially looking at MSP1 processing. Although significant inhibition of processing was mediated by several of the antibody samples, there was no clear relationship with overall growth inhibition by the same antibodies. However, no antibody samples inhibited processing but not invasion, suggesting that inhibition of MSP1 processing contributes to but is not the only mechanism of antibody-mediated inhibition of invasion and growth. Examining other mechanisms by which MSP1-specific antibodies inhibit parasite growth, we show that MSP1(19)-specific antibodies are taken up into invaded erythrocytes, where they persist for significant periods and result in delayed intracellular parasite development. This delay may result from antibody interference with coalescence of MSP1(19)-containing vesicles with the food vacuole. Antibodies raised against a modified recombinant MSP1(19) sequence were more efficient at delaying intracellular growth than those to the wild-type protein. We propose that antibodies specific for MSP1(19) can mediate inhibition of parasite growth by at least three mechanisms: inhibition of MSP1 processing, direct inhibition of invasion, and inhibition of parasite development following invasion. The balance between mechanisms may be modulated by modifying the immunogen used to induce the antibodies.

  6. Investigations on the presence of antibodies to papova viruses in patients with different forms of cancer and in other categories of patients or apparently healthy subjects. Note II. Hemagglutination-inhibiting serum antibodies to BK virus.

    PubMed

    Stoian, M; Nastac, E; Pucă, D; Hozoc, M; Bolocan, J; Serban, A

    1981-01-01

    Hemagglutination-inhibiting serum antibodies to BK virus (BKV were detected in patients with different forms of cancer and in blood donors (positivity percentages: 67.04 and 57.78, respectively). No such antibodies were found in a group of children 1 to 14 years of age. The data point to the presence of a latent human infection with BKV in the population of Romania.

  7. Chimeric, divalent and tetravalent anti-CD19 monoclonal antibodies with potent in vitro and in vivo antitumor activity against human B-cell lymphoma and pre-B acute lymphoblastic leukemia cell lines.

    PubMed

    Liu, Xiao-Yun; Pop, Laurentiu M; Tsai, Lydia; Pop, Iliodora V; Vitetta, Ellen S

    2011-07-15

    CD19 is an attractive therapeutic target for treating human B-cell tumors. In our study, chimeric (c) divalent (cHD37) and tetravalent (cHD37-DcVV) anti-CD19 monoclonal antibodies (MAbs) were constructed, expressed and evaluated for their binding to human 19-positive (CD19(+)) tumor cell lines. They were also tested for proapoptotic activity and the ability to mediate effector functions. The antitumor activity of these MAbs was further tested in mice xenografted with the CD19(+) Burkitt's lymphoma cell line, Daudi or the pre-B acute lymphoblastic leukemia (ALL) cell line, NALM-6. The cHD37 and cHD37-DcVV MAbs exhibited specific binding and comparable proapoptotic activity on CD19(+) tumor cell lines in vitro. In addition, the cHD37 and cHD37-DcVV MAbs were similar in their ability to mediate antibody-dependent cell-mediated phagocytosis (ADCP). However, the tetravalent cHD37-DcVV MAb bound more avidly, had a slower dissociation rate, and did not internalize as well. It also had enhanced antibody-dependent cellular cytotoxicity (ADCC) with human but not murine effector cells. The cHD37 and cHD37-DcVV MAbs exhibited comparable affinity for the human neonatal Fc receptor (FcRn) and similar pharmacokinetics (PKs) in mice. Moreover, all the HD37 constructs were similar in extending the survival of mice xenografted with Daudi or NALM-6 tumor cells. Therefore, the cHD37 and cHD37-DcVV MAbs have potent antitumor activity and should be further developed for use in humans. Although not evident in mice, due to its increased ability to mediate ADCC with human but not mouse effector cells, the cHD37-DcVV MAb should have superior therapeutic efficacy in humans.

  8. A novel mechanism for antibody-based anthrax toxin neutralization: inhibition of prepore-to-pore conversion.

    PubMed

    Mechaly, Adva; Levy, Haim; Epstein, Eyal; Rosenfeld, Ronit; Marcus, Hadar; Ben-Arie, Einat; Shafferman, Avigdor; Ordentlich, Arie; Mazor, Ohad

    2012-09-21

    Protective antigen (PA), a key component of anthrax toxin, mediates the entry of lethal factor (LF) or edema factor (EF) through a membranal pore into target cells. We have previously reported the isolation and chimerization of cAb29, an anti-PA monoclonal antibody that effectively neutralizes anthrax toxin in an unknown mechanism. The aim of this study was to elucidate the neutralizing mechanism of this antibody in vitro and to test its ability to confer post-exposure protection against anthrax in vivo. By systematic evaluation of the steps taking place during the PA-based intoxication process, we found that cAb29 did not interfere with the initial steps of intoxication, namely its ability to bind to the anthrax receptor, the consecutive proteolytic cleavage to PA(63), oligomerization, prepore formation, or LF binding. However, the binding of cAb29 to the prepore prevented its pH-triggered transition to the transmembranal pore, thus preventing the last step of intoxication, i.e. the translocation of LF/EF into the cell. Epitope mapping, using a phage display peptide library, revealed that cAb29 binds the 2α(1) loop in domain 2 of PA, a loop that undergoes major conformational changes during pore formation. In vivo, we found that 100% of anthrax-infected rabbits survived when treated with cAb29 12 h after exposure. In conclusion, these experiments demonstrate that cAb29 exerts its potent neutralizing activity in a unique manner by blocking the prepore-to-pore conversion process.

  9. Growth inhibition of human lung adenocarcinoma cells by antibodies against epidermal growth factor receptor and by ganglioside GM3: involvement of receptor-directed protein tyrosine phosphatase(s).

    PubMed

    Suarez Pestana, E; Greiser, U; Sánchez, B; Fernández, L E; Lage, A; Perez, R; Böhmer, F D

    1997-01-01

    Growth of the EGF receptor-expressing non-small-cell lung carcinoma cell line H125 seems to be at least partially driven by autocrine activation of the resident EGF receptors. Thus, the possibility of an EGF receptor-directed antiproliferative treatment was investigated in vitro using a monoclonal antibody (alpha EGFR ior egf/r3) against the human EGF receptor and gangliosides which are known to possess antiproliferative and anti-tyrosine kinase activity. The moderate growth-inhibitory effect of alpha EGFR ior egf/r3 was strongly potentiated by the addition of monosialoganglioside GM3. Likewise, the combination of alpha EGFR ior egf/r3 and GM3 inhibited EGF receptor autophosphorylation activity in H125 cells more strongly than either agent alone. A synergistic inhibition of EGF receptor autophosphorylation by alpha EGFR ior egf/r3 and GM3 was also observed in the human epidermoid carcinoma cell line A431. In both cell lines, the inhibition of EGF receptor autophosphorylation by GM3 was prevented by pretreatment of the cells with pervanadate, a potent inhibitor of protein tyrosine phosphatases (PTPases). Also, GM3 accelerated EGF receptor dephosphorylation in isolated A431 cell membranes. These findings indicate that GM3 has the capacity to activate EGF receptor-directed PTPase activity and suggest a novel possible mechanism for the regulation of cellular PTPases.

  10. Growth inhibition of human lung adenocarcinoma cells by antibodies against epidermal growth factor receptor and by ganglioside GM3: involvement of receptor-directed protein tyrosine phosphatase(s).

    PubMed Central

    Suarez Pestana, E.; Greiser, U.; Sánchez, B.; Fernández, L. E.; Lage, A.; Perez, R.; Böhmer, F. D.

    1997-01-01

    Growth of the EGF receptor-expressing non-small-cell lung carcinoma cell line H125 seems to be at least partially driven by autocrine activation of the resident EGF receptors. Thus, the possibility of an EGF receptor-directed antiproliferative treatment was investigated in vitro using a monoclonal antibody (alpha EGFR ior egf/r3) against the human EGF receptor and gangliosides which are known to possess antiproliferative and anti-tyrosine kinase activity. The moderate growth-inhibitory effect of alpha EGFR ior egf/r3 was strongly potentiated by the addition of monosialoganglioside GM3. Likewise, the combination of alpha EGFR ior egf/r3 and GM3 inhibited EGF receptor autophosphorylation activity in H125 cells more strongly than either agent alone. A synergistic inhibition of EGF receptor autophosphorylation by alpha EGFR ior egf/r3 and GM3 was also observed in the human epidermoid carcinoma cell line A431. In both cell lines, the inhibition of EGF receptor autophosphorylation by GM3 was prevented by pretreatment of the cells with pervanadate, a potent inhibitor of protein tyrosine phosphatases (PTPases). Also, GM3 accelerated EGF receptor dephosphorylation in isolated A431 cell membranes. These findings indicate that GM3 has the capacity to activate EGF receptor-directed PTPase activity and suggest a novel possible mechanism for the regulation of cellular PTPases. Images Figure 5 Figure 6 PMID:9010029

  11. [Possible relation between viruses and oromaxillofacial tumors. V. Demonstration of hemagglutination-inhibiting anti-BK virus antibodies in patients with tumors of the parotid gland].

    PubMed

    Stoian, M; Zaharia, O; Suru, M; Constantinescu, E; Goldstein, I; Nastac, E

    1987-01-01

    Anti-BK-virus hemagglutination inhibiting antibodies were revealed in 81.8% of the patients with parotid gland tumors. Results of the investigations conducted on oromaxillofacial tumors including the parotid gland ones are discussed from the point of view of the presence of viral antigens (herpes-, SV40 and BK-viruses) and of specific antibodies. Possible implication of the papova viruses in the etiopathogenesis of the parotid gland tumors in humans are also discussed.

  12. Synthetic Antibodies Inhibit Bcl-2-associated X Protein (BAX) through Blockade of the N-terminal Activation Site*

    PubMed Central

    Uchime, Onyinyechukwu; Dai, Zhou; Biris, Nikolaos; Lee, David; Sidhu, Sachdev S.; Li, Sheng; Lai, Jonathan R.; Gavathiotis, Evripidis

    2016-01-01

    The BCL-2 protein family plays a critical role in regulating cellular commitment to mitochondrial apoptosis. Pro-apoptotic Bcl-2-associated X protein (BAX) is an executioner protein of the BCL-2 family that represents the gateway to mitochondrial apoptosis. Following cellular stresses that induce apoptosis, cytosolic BAX is activated and translocates to the mitochondria, where it inserts into the mitochondrial outer membrane to form a toxic pore. How the BAX activation pathway proceeds and how this may be inhibited is not yet completely understood. Here we describe synthetic antibody fragments (Fabs) as structural and biochemical probes to investigate the potential mechanisms of BAX regulation. These synthetic Fabs bind with high affinity to BAX and inhibit its activation by the BH3-only protein tBID (truncated Bcl2 interacting protein) in assays using liposomal membranes. Inhibition of BAX by a representative Fab, 3G11, prevented mitochondrial translocation of BAX and BAX-mediated cytochrome c release. Using NMR and hydrogen-deuterium exchange mass spectrometry, we showed that 3G11 forms a stoichiometric and stable complex without inducing a significant conformational change on monomeric and inactive BAX. We identified that the Fab-binding site on BAX involves residues of helices α1/α6 and the α1-α2 loop. Therefore, the inhibitory binding surface of 3G11 overlaps with the N-terminal activation site of BAX, suggesting a novel mechanism of BAX inhibition through direct binding to the BAX N-terminal activation site. The synthetic Fabs reported here reveal, as probes, novel mechanistic insights into BAX inhibition and provide a blueprint for developing inhibitors of BAX activation. PMID:26565029

  13. Antibody-Mediated Rejection of Arterialised Venous Allografts Is Inhibited by Immunosuppression in Rats

    PubMed Central

    Varga, Martin; Oliverius, Martin; Kuhn, Stephanie; Feldbrügge, Linda; Krenzien, Felix; Hau, Hans-Michael; Wiltberger, Georg; Schmelzle, Moritz; Jonas, Sven

    2014-01-01

    Objectives and Design We determined in a rat model (1) the presence and dynamics of alloantibodies recognizing MHC complexes on quiescent Brown-Norway (BN) splenic cells in the sera of Lewis (LEW) recipients of Brown-Norway iliolumbar vein grafts under tacrolimus immunosuppression; and (2) the presence of immunoglobulins in the wall of acute rejected vein allografts. Materials and Methods Flow cytometry was used for the analysis of day 0, 14 and 30 sera obtained from Lewis recipients of isogeneic iliolumbar vein grafts (group A) or Brown-Norway grafts (group B, C) for the presence of donor specific anti-MHC class I and II antibodies. Tacrolimus 0.2 mg/kg daily was administered from day 1 to day 30 (group C). Histology was performed on day 30. Results Sera obtained preoperatively and on day 30 were compared in all groups. The statistically significant decrease of anti MHC class I and II antibody binding was observed only in allogenic non-immunosuppressed group B (splenocytes: MHC class I - day 0 (93%±7% ) vs day 30 (66%±7%), p = 0.02, MHC class II - day 0 (105%±3% ) vs day 30 (83%±5%), p = 0.003; B-cells: MHC class I - day 0 (83%±5%) vs day 30 (55%±6%), p = 0.003, MHC class II - day 0 (101%±1%) vs day 30 (79%±6%), p = 0.006; T-cells: MHC class I - day 0 (71%±7%) vs day 30 (49%±5%), p = 0.04). No free clusters of immunoglobulin G deposition were detected in any experimental group. Conclusion Arterialized venous allografts induce strong donor-specific anti-MHC class I and anti-MHC class II antibody production with subsequent immune-mediated destruction of these allografts with no evidence of immunoglobulin G deposition. Low-dose tacrolimus suppress the donor-specific antibody production. PMID:24618652

  14. Dengue Type Four Viruses with E-Glu345Lys Adaptive Mutation from MRC-5 Cells Induce Low Viremia but Elicit Potent Neutralizing Antibodies in Rhesus Monkeys

    PubMed Central

    Li, Xiao-Feng; Tsai, Meng-Ju; Hsiao, Hung-Ju; Peng, Jia-Guan; Sue, Shih-Che; Qin, Cheng-Feng; Wu, Suh-Chin

    2014-01-01

    Knowledge of virulence and immunogenicity is important for development of live-attenuated dengue vaccines. We previously reported that an infectious clone-derived dengue type 4 virus (DENV-4) passaged in MRC-5 cells acquired a Glu345Lys (E-E345K) substitution in the E protein domain III (E-DIII). The same cloned DENV-4 was found to yield a single E-Glu327Gly (E-E327G) mutation after passage in FRhL cells and cause the loss of immunogenicity in rhesus monkeys. Here, we used site-directed mutagenesis to generate the E-E345K and E-E327G mutants from DENV-4 and DENV-4Δ30 infectious clones and propagated in Vero or MRC-5 cells. The E-E345K mutations were consistently presented in viruses recovered from MRC-5 cells, but not Vero cells. Recombinant E-DIII proteins of E345K and E327G increased heparin binding correlated with the reduced infectivity by heparin treatment in cell cultures. Different from the E-E327G mutant viruses to lose the immunogencity in rhesus monkeys, the E-E345K mutant viruses were able to induce neutralizing antibodies in rhesus monkeys with an almost a 10-fold lower level of viremia as compared to the wild type virus. Monkeys immunized with the E-E345K mutant virus were completely protected with no detectable viremia after live virus challenges with the wild type DENV-4. These results suggest that the E-E345K mutant virus propagated in MRC-5 cells may have potential for the use in live-attenuated DENV vaccine development. PMID:24959738

  15. Dengue type four viruses with E-Glu345Lys adaptive mutation from MRC-5 cells induce low viremia but elicit potent neutralizing antibodies in rhesus monkeys.

    PubMed

    Lin, Hsiao-Han; Lee, Hsiang-Chi; Li, Xiao-Feng; Tsai, Meng-Ju; Hsiao, Hung-Ju; Peng, Jia-Guan; Sue, Shih-Che; Qin, Cheng-Feng; Wu, Suh-Chin

    2014-01-01

    Knowledge of virulence and immunogenicity is important for development of live-attenuated dengue vaccines. We previously reported that an infectious clone-derived dengue type 4 virus (DENV-4) passaged in MRC-5 cells acquired a Glu345Lys (E-E345K) substitution in the E protein domain III (E-DIII). The same cloned DENV-4 was found to yield a single E-Glu327Gly (E-E327G) mutation after passage in FRhL cells and cause the loss of immunogenicity in rhesus monkeys. Here, we used site-directed mutagenesis to generate the E-E345K and E-E327G mutants from DENV-4 and DENV-4Δ30 infectious clones and propagated in Vero or MRC-5 cells. The E-E345K mutations were consistently presented in viruses recovered from MRC-5 cells, but not Vero cells. Recombinant E-DIII proteins of E345K and E327G increased heparin binding correlated with the reduced infectivity by heparin treatment in cell cultures. Different from the E-E327G mutant viruses to lose the immunogencity in rhesus monkeys, the E-E345K mutant viruses were able to induce neutralizing antibodies in rhesus monkeys with an almost a 10-fold lower level of viremia as compared to the wild type virus. Monkeys immunized with the E-E345K mutant virus were completely protected with no detectable viremia after live virus challenges with the wild type DENV-4. These results suggest that the E-E345K mutant virus propagated in MRC-5 cells may have potential for the use in live-attenuated DENV vaccine development.

  16. Allo-immunization elicits CCR5 antibodies, SDF-1 chemokines, and CD8-suppressor factors that inhibit transmission of R5 and X4 HIV-1 in women.

    PubMed

    Wang, Y; Underwood, J; Vaughan, R; Harmer, A; Doyle, C; Lehner, T

    2002-09-01

    Studies in humans suggest that allo-immunization induces CC-chemokines, CD8-suppressor factors (SF) and anti-HIV immunity. Here we report that allo-immunization with unmatched leucocytes from partners of women with recurrent spontaneous abortion elicits specific antibodies to the CCR5 receptor. Such antibodies inhibit replication of M-tropic HIV-1 (R5) and MIP-1beta-mediated chemotaxis. These CCR5 antibodies were also found in the sera of multiparous women that were naturally immunized by semi-allogeneic fetal antigens. The specificity of these antibodies was demonstrated by adsorption with CCR5 transfected HEK-293 cells, a baculovirus CCR5 preparation and a peptide of the 2nd extra-cellular loop of CCR5. Allo-immunization also stimulated increased concentrations of the CXC chemokine, SDF-1alpha and CD8-SF that inhibit T-tropic HIV-1 (X4) replication. We suggest that allo- immunization may elicit (a) CC chemokines, CCR5 antibodies and CD8-SF that inhibit M-tropic HIV-1 infection and (b) the CXC chemokine SDF-1alpha and CD8-SF that inhibit T-tropic HIV-1 infection.

  17. In vitro inhibitory effects of non-steroidal anti-inflammatory drugs on 4-methylumbelliferone glucuronidation in recombinant human UDP-glucuronosyltransferase 1A9--potent inhibition by niflumic acid.

    PubMed

    Mano, Yuji; Usui, Takashi; Kamimura, Hidetaka

    2006-01-01

    The inhibitory potencies of non-steroidal anti-inflammatory drugs (NSAIDs) on UDP-glucuronosyltransferase (UGT) 1A9 activity were investigated in recombinant human UGT1A9 using 4-methylumbelliferone (4-MU) as a substrate for glucuronidation. 4-MU glucuronidation (4-MUG) showed Michaelis-Menten kinetics with a Km value of 6.7 microM. The inhibitory effects of the following seven NSAIDs were investigated: acetaminophen, diclofenac, diflunisal, indomethacin, ketoprofen, naproxen and niflumic acid. Niflumic acid had the most potent inhibitory effect on 4-MUG with an IC50 value of 0.0341 microM. The IC50 values of diflunisal, diclofenac and indomethacin were 1.31, 24.2, and 34.1 microM, respectively, while acetaminophen, ketoprofen and naproxen showed less potent inhibition. Niflumic acid, diflunisal, diclofenac and indomethacin inhibited 4-MUG competitively with Ki values of 0.0275, 0.710, 53.3 and 69.9 microM, respectively, being similar to each IC50 value. In conclusion, of the seven NSAIDs investigated, niflumic acid was the most potent inhibitor of recombinant UGT1A9 via 4-MUG in a competitive manner.

  18. Broadly neutralizing alphavirus antibodies bind an epitope on E2 and inhibit entry and egress

    PubMed Central

    Fox, Julie M.; Long, Feng; Edeling, Melissa A.; Lin, Hueylie; van Duijl-Richter, Mareike K.S.; Fong, Rachel H.; Kahle, Kristen M.; Smit, Jolanda M.; Jin, Jing; Simmons, Graham; Doranz, Benjamin J.; Crowe, James E.; Fremont, Daved H.; Rossmann, Michael G.; Diamond, Michael S.

    2015-01-01

    SUMMARY We screened a panel of mouse and human monoclonal antibodies (MAbs) against chikungunya virus and identified several with inhibitory activity against multiple alphaviruses. Passive transfer of broadly neutralizing MAbs protected mice against infection by chikungunya, Mayaro, and O’nyong’nyong alphaviruses. Using alanine-scanning mutagenesis, loss-of-function recombinant proteins and viruses, and multiple functional assays, we determined that broadly neutralizing MAbs block multiple steps in the viral lifecycle including entry and egress, and bind to a conserved epitope on the B domain of the E2 glycoprotein. A 16 Å resolution cryo-electron microscopy structure of a Fab fragment bound to CHIKV E2 B domain provided an explanation for its neutralizing activity. Binding to the B domain was associated with repositioning of the A domain of E2 that enabled cross-linking of neighboring spikes. Our results suggest that B domain antigenic determinants could be targeted for vaccine or antibody therapeutic development against multiple alphaviruses of global concern. PMID:26553503

  19. Broadly Neutralizing Alphavirus Antibodies Bind an Epitope on E2 and Inhibit Entry and Egress.

    PubMed

    Fox, Julie M; Long, Feng; Edeling, Melissa A; Lin, Hueylie; van Duijl-Richter, Mareike K S; Fong, Rachel H; Kahle, Kristen M; Smit, Jolanda M; Jin, Jing; Simmons, Graham; Doranz, Benjamin J; Crowe, James E; Fremont, Daved H; Rossmann, Michael G; Diamond, Michael S

    2015-11-19

    We screened a panel of mouse and human monoclonal antibodies (MAbs) against chikungunya virus and identified several with inhibitory activity against multiple alphaviruses. Passive transfer of broadly neutralizing MAbs protected mice against infection by chikungunya, Mayaro, and O'nyong'nyong alphaviruses. Using alanine-scanning mutagenesis, loss-of-function recombinant proteins and viruses, and multiple functional assays, we determined that broadly neutralizing MAbs block multiple steps in the viral lifecycle, including entry and egress, and bind to a conserved epitope on the B domain of the E2 glycoprotein. A 16 Å resolution cryo-electron microscopy structure of a Fab fragment bound to CHIKV E2 B domain provided an explanation for its neutralizing activity. Binding to the B domain was associated with repositioning of the A domain of E2 that enabled cross-linking of neighboring spikes. Our results suggest that B domain antigenic determinants could be targeted for vaccine or antibody therapeutic development against multiple alphaviruses of global concern.

  20. Inhibition of cell surface mediated plasminogen activation by a monoclonal antibody against alpha-Enolase.

    PubMed

    López-Alemany, Roser; Longstaff, Colin; Hawley, Stephen; Mirshahi, Massoud; Fábregas, Pere; Jardí, Merce; Merton, Elizabeth; Miles, Lindsey A; Félez, Jordi

    2003-04-01

    Localization of plasmin activity on leukocyte surfaces plays a critical role in fibrinolysis as well as in pathological and physiological processes in which cells must degrade the extracellular matrix in order to migrate. The binding of plasminogen to leukocytic cell lines induces a 30- to 80-fold increase in the rate of plasminogen activation by tissue-type (tPA) and urokinase-type (uPA) plasminogen activators. In the present study we have examined the role of alpha-enolase in plasminogen activation on the cell surface. We produced and characterized a monoclonal antibody (MAb) 11G1 against purified alpha-enolase, which abrogated about 90% of cell-dependent plasminogen activation by either uPA or tPA on leukocytoid cell lines of different lineages: B-lymphocytic, T-lymphocytic, granulocytic, and monocytic cells. In addition, MAb 11G1 also blocked enhancement of plasmin formation by peripheral blood neutrophils and monocytes. In contrast, MAb 11G1 did not affect plasmin generation in the presence of fibrin, indicating that this antibody did not interact with fibrinolytic components in the absence of cells. These data suggest that, although leukocytic cells display several molecules that bind plasminogen, alpha-enolase is responsible for the majority of the promotion of plasminogen activation on the surfaces of leukocytic cells.

  1. CCR5 antibodies HGS004 and HGS101 preferentially inhibit drug-bound CCR5 infection and restore drug sensitivity of Maraviroc-resistant HIV-1 in primary cells

    SciTech Connect

    Latinovic, Olga; Reitz, Marvin; Le, Nhut M.; Foulke, James S.; Faetkenheuer, Gerd; Lehmann, Clara; Redfield, Robert R.; Heredia, Alonso

    2011-03-01

    R5 HIV-1 strains resistant to the CCR5 antagonist Maraviroc (MVC) can use drug-bound CCR5. We demonstrate that MVC-resistant HIV-1 exhibits delayed kinetics of coreceptor engagement and fusion during drug-bound versus free CCR5 infection of cell lines. Antibodies directed against the second extracellular loop (ECL2) of CCR5 had greater antiviral activity against MVC-bound compared to MVC-free CCR5 infection. However, in PBMCs, only ECL2 CCR5 antibodies HGS004 and HGS101, but not 2D7, inhibited infection by MVC resistant HIV-1 more potently with MVC-bound than with free CCR5. In addition, HGS004 and HGS101, but not 2D7, restored the antiviral activity of MVC against resistant virus in PBMCs. In flow cytometric studies, CCR5 binding by the HGS mAbs, but not by 2D7, was increased when PBMCs were treated with MVC, suggesting MVC increases exposure of the relevant epitope. Thus, HGS004 and HGS101 have antiviral mechanisms distinct from 2D7 and could help overcome MVC resistance.

  2. Antibody-mediated FOXP3 protein therapy induces apoptosis in cancer cells in vitro and inhibits metastasis in vivo.

    PubMed

    Heinze, Emil; Baldwin, Scott; Chan, Grace; Hansen, James; Song, Jason; Clements, Douglas; Aragon, Robert; Nishimura, Robert; Reeves, Mark; Weisbart, Richard

    2009-07-01

    In addition to its immune suppressive function in T-regulatory cells, the nuclear transcription factor, FOXP3, has been identified as a tumor suppressor. To evaluate the clinical efficacy of monoclonal antibody (mAb) 3E10 Fv antibody-mediated FOXP3 protein therapy of cancer, the Fv-FOXP3 fusion protein produced in Pichia pastoris was tested on breast, ovarian, and colon cancer cells in vitro, and with colon cancer cells in vivo in a mouse model of colon cancer metastasis to liver. Treatment with Fv-FOXP3 resulted in dose-dependent cell death of cancer cells in vitro. Apoptosis was established as a mechanism of cell death by demonstrating increased production of the p17 activated fragment of caspase-3 by cancer cells in response to Fv-FOXP3 and inhibition of cell killing by the caspase inhibitor, Z-VAD-FMK. Fv-FOXP3 treatment resulted in clinically significant reduction in tumor burden in a syngeneic model of colon cancer metastasis to liver in Balb/c mice. These results represent the first demonstration of effective full-length FOXP3 protein therapy and emphasize the clinical potential of mAb 3E10 as an intracellular and intranuclear delivery vehicle of FOXP3 for prevention and treatment of cancer metastasis.

  3. Antithyroglobulin antibody

    MedlinePlus

    Thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Hypothyroidism - thyroglobulin antibody; Thyroiditis - thyroglobulin antibody; Graves disease - thyroglobulin antibody; Underactive thyroid - thyroglobulin antibody

  4. The antigenic determinants on HIV p24 for CD4+ T cell inhibiting antibodies as determined by limited proteolysis, chemical modification, and mass spectrometry.

    PubMed

    Williams, Jason G; Tomer, Kenneth B; Hioe, Catarina E; Zolla-Pazner, Susan; Norris, Philip J

    2006-11-01

    In the last decade, mass spectrometry has been employed by more and more researchers for identifying the proteins in a macromolecular complex as well as for defining the surfaces of their binding interfaces. This characterization of protein-protein interfaces usually involves at least one of several different methodologies in addition to the actual mass spectrometry. For example, limited proteolysis is often used as a first step in defining regions of a protein that are protected from proteolysis when the protein of interest is part of a macromolecular complex. Other techniques used in conjunction with mass spectrometry for determining regions of a protein involved in protein-protein interactions include chemical modification, such as covalent cross-linking, acetylation of lysines, hydrogen-deuterium exchange, or other forms of modification. In this report, both limited proteolysis and chemical modification were combined with several mass spectrometric techniques in efforts to define the protein surface on the HIV core protein, p24, recognized by two different monoclonal human antibodies that were isolated from HIV+ patients. One of these antibodies, 1571, strongly inhibits the CD4+ T cell proliferative response to a known epitope (PEVIPMFSALSEGATP), while the other antibody, 241-D, does not inhibit as strongly. The epitopes for both of these antibodies were determined to be discontinuous and localized to the N-terminus of p24. Interestingly, the epitope recognized by the strongly inhibiting antibody, 1571, completely overlaps the T cell epitope PEVIPMFSALSEGATP, while the antibody 241-D binds to a region adjacent to the region of p24 recognized by the antibody 1571. These results suggest that, possibly due to epitope competition, antibodies produced during HIV infection can negatively affect CD4+ T cell-mediated immunity against the virus.

  5. Infectivity-Neutralizing and Hemagglutinin-Inhibiting Antibody Responses to Respiratory Coronavirus Infections of Cattle in Pathogenesis of Shipping Fever Pneumonia

    PubMed Central

    Lin, Xiaoqing; O'Reilly, Kathy L.; Burrell, Mamie L.; Storz, Johannes

    2001-01-01

    Respiratory bovine coronaviruses (RBCV) emerged as an infectious agent most frequently isolated from respiratory tract samples of cattle with acute respiratory tract diseases. Infectivity-neutralizing (IN) and hemagglutinin-inhibiting (HAI) antibodies induced by RBCV infections were monitored in sequential serum samples collected from cattle during a naturally evolving and experimentally monitored epizootic of shipping fever pneumonia (SFP). Cattle nasally shedding RBCV at the beginning of the epizootic started with low levels of serum IN and HAI antibodies. An increase in serum IN antibody after day 7 led to reduction of virus shedding in nasal secretions by the majority of the cattle between days 7 and 14. A substantial rise in the serum HAI antibody was observed during the initial phase among the sick but not the clinically normal cattle which were infected with RBCV. The RBCV isolation-positive cattle that developed fatal SFP had minimal serum IN and HAI antibodies during the course of disease development. Cattle that remained negative in RBCV isolation tests entered this epizootic with high levels of serum IN and HAI antibodies, which dramatically increased during the next two weeks. Protection against SFP was apparently associated with significantly higher levels of serum IN antibodies at the beginning of the epizootic. The RBCV-neutralizing activity is associated with serum immunoglobulin G (IgG), particularly the IgG2 subclass, while RBCV-specific HAI antibody is related to both serum IgG and IgM fractions. PMID:11238222

  6. Analysis of Anti-Influenza Virus Neuraminidase Antibodies in Children, Adults, and the Elderly by ELISA and Enzyme Inhibition: Evidence for Original Antigenic Sin

    PubMed Central

    Rajendran, Madhusudan; Ermler, Megan E.; Bunduc, Paul; Amanat, Fatima; Izikson, Ruvim; Cox, Manon; Palese, Peter; Eichelberger, Maryna

    2017-01-01

    ABSTRACT Antibody responses to influenza virus hemagglutinin provide protection against infection and are well studied. Less is known about the human antibody responses to the second surface glycoprotein, neuraminidase. Here, we assessed human antibody reactivity to a panel of N1, N2, and influenza B virus neuraminidases in different age groups, including children, adults, and the elderly. Using enzyme-linked immunosorbent assays (ELISA), we determined the breadth, magnitude, and isotype distribution of neuraminidase antibody responses to historic, current, and avian strains, as well as to recent isolates to which these individuals have not been exposed. It appears that antibody levels against N1 neuraminidases were lower than those against N2 or B neuraminidases. The anti-neuraminidase antibody levels increased with age and were, in general, highest against strains that circulated during the childhood of the tested individuals, providing evidence for “original antigenic sin.” Titers measured by ELISA correlated well with titers measured by the neuraminidase inhibition assays. However, in the case of the 2009 pandemic H1N1 virus, we found evidence of interference from antibodies binding to the conserved stalk domain of the hemagglutinin. In conclusion, we found that antibodies against the neuraminidase differ in magnitude and breadth between subtypes and age groups in the human population. (This study has been registered at ClinicalTrials.gov under registration no. NCT00336453, NCT00539981, and NCT00395174.) PMID:28325769

  7. Generation and characterization of small single domain antibodies inhibiting human tumor necrosis factor receptor 1.

    PubMed

    Steeland, Sophie; Puimège, Leen; Vandenbroucke, Roosmarijn E; Van Hauwermeiren, Filip; Haustraete, Jurgen; Devoogdt, Nick; Hulpiau, Paco; Leroux-Roels, Geert; Laukens, Debby; Meuleman, Philip; De Vos, Martine; Libert, Claude

    2015-02-13

    The cytokine TNF is a well known drug target for several inflammatory diseases such as Crohn disease. Despite the great success of TNF blockers, therapy could be improved because of high costs and side effects. Selective inhibition of TNF receptor (TNFR) 1 signaling holds the potential to greatly reduce the pro-inflammatory activity of TNF, thereby preserving the advantageous immunomodulatory signals mediated by TNFR2. We generated a selective human TNFR1 inhibitor based on Nanobody (Nb) technology. Two anti-human TNFR1 Nbs were linked with an anti-albumin Nb to generate Nb Alb-70-96 named "TNF Receptor-One Silencer" (TROS). TROS selectively binds and inhibits TNF/TNFR1 and lymphotoxin-α/TNFR1 signaling with good affinity and IC50 values, both of which are in the nanomolar range. Surface plasmon resonance analysis reveals that TROS competes with TNF for binding to human TNFR1. In HEK293T cells, TROS strongly reduces TNF-induced gene expression, like IL8 and TNF, in a dose-dependent manner; and in ex vivo cultured colon biopsies of CD patients, TROS inhibits inflammation. Finally, in liver chimeric humanized mice, TROS antagonizes inflammation in a model of acute TNF-induced liver inflammation, reflected in reduced human IL8 expression in liver and reduced IL6 levels in serum. These results demonstrate the considerable potential of TROS and justify the evaluation of TROS in relevant disease animal models of both acute and chronic inflammation and eventually in patients.

  8. Effective Inhibition of Bone Morphogenetic Protein Function by Highly Specific Llama-Derived Antibodies.

    PubMed

    Calpe, Silvia; Wagner, Koen; El Khattabi, Mohamed; Rutten, Lucy; Zimberlin, Cheryl; Dolk, Edward; Verrips, C Theo; Medema, Jan Paul; Spits, Hergen; Krishnadath, Kausilia K

    2015-11-01

    Bone morphogenetic proteins (BMP) have important but distinct roles in tissue homeostasis and disease, including carcinogenesis and tumor progression. A large number of BMP inhibitors are available to study BMP function; however, as most of these antagonists are promiscuous, evaluating specific effects of individual BMPs is not feasible. Because the oncogenic role of the different BMPs varies for each neoplasm, highly selective BMP inhibitors are required. Here, we describe the generation of three types of llama-derived heavy chain variable domains (VHH) that selectively bind to either BMP4, to BMP2 and 4, or to BMP2, 4, 5, and 6. These generated VHHs have high affinity to their targets and are able to inhibit BMP signaling. Epitope binning and docking modeling have shed light into the basis for their BMP specificity. As opposed to the wide structural reach of natural inhibitors, these small molecules target the grooves and pockets of BMPs involved in receptor binding. In organoid experiments, specific inhibition of BMP4 does not affect the activation of normal stem cells. Furthermore, in vitro inhibition of cancer-derived BMP4 noncanonical signals results in an increase of chemosensitivity in a colorectal cancer cell line. Therefore, because of their high specificity and low off-target effects, these VHHs could represent a therapeutic alternative for BMP4(+) malignancies.

  9. A Potent HER3 Monoclonal Antibody That Blocks Both Ligand-Dependent and -Independent Activities: Differential Impacts of PTEN Status on Tumor Response.

    PubMed

    Xiao, Zhan; Carrasco, Rosa A; Schifferli, Kevin; Kinneer, Krista; Tammali, Ravinder; Chen, Hong; Rothstein, Ray; Wetzel, Leslie; Yang, Chunning; Chowdhury, Partha; Tsui, Ping; Steiner, Philipp; Jallal, Bahija; Herbst, Ronald; Hollingsworth, Robert E; Tice, David A

    2016-04-01

    HER3/ERBB3 is a kinase-deficient member of the EGFR family receptor tyrosine kinases (RTK) that is broadly expressed and activated in human cancers. HER3 is a compelling cancer target due to its important role in activation of the oncogenic PI3K/AKT pathway. It has also been demonstrated to confer tumor resistance to a variety of cancer therapies, especially targeted drugs against EGFR and HER2. HER3 can be activated by its ligand (heregulin/HRG), which induces HER3 heterodimerization with EGFR, HER2, or other RTKs. Alternatively, HER3 can be activated in a ligand-independent manner through heterodimerization with HER2 in HER2-amplified cells. We developed a fully human mAb against HER3 (KTN3379) that efficiently suppressed HER3 activity in both ligand-dependent and independent settings. Correspondingly, KTN3379 inhibited tumor growth in divergent tumor models driven by either ligand-dependent or independent mechanisms in vitro and in vivo Most intriguingly, while investigating the mechanistic underpinnings of tumor response to KTN3379, we discovered an interesting dichotomy in that PTEN loss, a frequently occurring oncogenic lesion in a broad range of cancer types, substantially blunted the tumor response in HER2-amplified cancer, but not in the ligand-driven cancer. To our knowledge, this represents the first study ascertaining the impact of PTEN loss on the antitumor efficacy of a HER3 mAb. KTN3379 is currently undergoing a phase Ib clinical trial in patients with advanced solid tumors. Our current study may help us optimize patient selection schemes for KTN3379 to maximize its clinical benefits. Mol Cancer Ther; 15(4); 689-701. ©2016 AACR.

  10. Biocompatible chitosan nanoparticles as an efficient delivery vehicle for Mycobacterium tuberculosis lipids to induce potent cytokines and antibody response through activation of γδ T cells in mice

    NASA Astrophysics Data System (ADS)

    Das, Ishani; Padhi, Avinash; Mukherjee, Sitabja; Dash, Debi P.; Kar, Santosh; Sonawane, Avinash

    2017-04-01

    The activation of cell-mediated and humoral immune responses to Mycobacterium tuberculosis (Mtb) is critical for protection against the pathogen and nanoparticle-mediated delivery of antigens is a more potent way to induce different immune responses. Herein, we show that mice immunized with Mtb lipid-bound chitosan nanoparticles (NPs) induce secretion of prominent type-1 T-helper (Th-1) and type-2 T-helper (Th-2) cytokines in lymph node and spleen cells, and also induces significantly higher levels of IgG, IgG1, IgG2 and IgM in comparison to control mice. Furthermore, significantly enhanced γδ-T-cell activation was observed in lymph node cells isolated from mice immunized with Mtb lipid-coated chitosan NPs as compared to mice immunized with chitosan NPs alone or Mtb lipid liposomes. In comparison to CD8+ cells, significantly higher numbers of CD4+ cells were present in both the lymph node and spleen cells isolated from mice immunized with Mtb lipid-coated chitosan NPs. In conclusion, this study represents a promising new strategy for the efficient delivery of Mtb lipids using chitosan NPs to trigger an enhanced cell-mediated and antibody response against Mtb lipids.

  11. Biocompatible chitosan nanoparticles as an efficient delivery vehicle for Mycobacterium tuberculosis lipids to induce potent cytokines and antibody response through activation of γδ T-cells in mice.

    PubMed

    Das, Ishani; Padhi, Avinash; Mukherjee, Sitabja; Dash, Debi; Kar, Santosh; Sonawane, Avinash

    2017-02-16

    Activation of cell mediated and humoral immune responses to Mycobacterium tuberculosis (Mtb) are critical for protection and that nanoparticles mediated delivery of antigens are more potent in inducing different immune responses. Herein, we show that mice immunized with Mtb lipid bound chitosan nanoparticles(NPs) induce secretion of prominent type 1 T helper (Th1) and type 2 T helper (Th2) cytokines in lymph node and spleen cells, and also induced significantly higher levels of IgG, IgG1, IgG2 and IgM in comparison to control mice. Furthermore, significantly enhanced γδ-T cell activation was observed in lymph node cells isolated from mice immunized with Mtb lipid coated chitosan-NPs as compared to mice immunized with chitosan-NPs alone or Mtb lipid liposomes. In comparison to CD8+ cells, significantly higher CD4+ cells were present in both the lymph node and spleen cells isolated from mice immunized with Mtb lipid coated chitosan NP. In conclusion, this study represents a promising new strategy for efficient delivery of Mtb lipids using chitosan NPs to trigger enhanced cell mediated and antibody response against Mtb lipids.

  12. A novel human Fab antibody for Trop2 inhibits breast cancer growth in vitro and in vivo.

    PubMed

    Lin, Hong; Zhang, Huiling; Wang, Jun; Lu, Meiping; Zheng, Feng; Wang, Changjun; Tang, Xiaojun; Xu, Ning; Chen, Renjie; Zhang, Dawei; Zhao, Ping; Zhu, Jin; Mao, Yuan; Feng, Zhenqing

    2014-03-01

    Human trophoblastic cell surface antigen 2 (Trop2) has been suggested as an oncogene, which is associated with the different types of tumors. In this study, a human Fab antibody against Trop2 extracellular domain was isolated from phage library by phage display technology, and characterized by ELISA, FACS, fluorescence staining and Western blotting analysis. MTT, apoptosis assay and wound healing assay were employed to evaluate the inhibitory effects of Trop2 Fab on breast cancer cell growth in vitro, while tumor-xenograft model was employed to evaluate the inhibitory effects on breast cancer growth in vivo. The results showed that Trop2 Fab inhibited the proliferation, induced the apoptosis and suspended the migration of MDA-MB-231 cells in a dose dependent manner. The expression caspase-3 was activated, and the expression of Bcl-2 was reduced while that of Bax was elevated in MDA-MB-231 cells by treating with Trop2 Fab. In addition, Trop2 Fab inhibited the growth of breast cancer xenografts and the expression of Bcl-2 was reduced while that of Bax was elevated in xenografts. Trop2 Fab, which was isolated successfully in this research, is a promising therapeutic agent for the treatment of Trop2 expressing breast cancer.

  13. An inhibition enzyme immuno assay exploring recombinant invariant surface glycoprotein and monoclonal antibodies for surveillance of surra in animals.

    PubMed

    Rudramurthy, G R; Sengupta, P P; Ligi, M; Rahman, H

    2017-02-20

    The present study is aimed at the development of inhibition ELISA (I-ELISA) exploring monoclonal antibodies (MAbs) and recombinant invariant surface glycoprotein. The extracellular domain (ED) of invariant surface glycoprotein (ISG-75) from Trypanosoma evasni has been heterologously expressed in Pichia pastoris (X-33). The recombinant ISG-75 (rISG-75ED) was characterized by immunoblot and ELISA, followed by the production of MAbs against rISG-75ED. The MAbs were characterized by immunoblot and then explored in the development of I-ELISA for the detection of surra. The diagnostic potential of the developed test has been evaluated using 1192 field sera sample including cattle, buffalo, donkey, horse and camel. The statistical analysis of the data showed optimum combination of diagnostic sensitivity and specificity at 98.8% and 99.2% respectively, with cut-off percentage inhibition (PI) value of >45. The Cohen's kappa coefficient of agreement was found to be 0.98. Hence, the diagnostic test developed in the present study can be exploited as a potential and reliable tool in the serodiagnosis and surveillance of surra in animals.

  14. A Dual-Modality Herpes Simplex Virus 2 Vaccine for Preventing Genital Herpes by Using Glycoprotein C and D Subunit Antigens To Induce Potent Antibody Responses and Adenovirus Vectors Containing Capsid and Tegument Proteins as T Cell Immunogens

    PubMed Central

    Mahairas, Gregory G.; Shaw, Carolyn E.; Huang, Meei-Li; Koelle, David M.; Posavad, Christine; Corey, Lawrence; Friedman, Harvey M.

    2015-01-01

    ABSTRACT We evaluated a genital herpes prophylactic vaccine containing herpes simplex virus 2 (HSV-2) glycoproteins C (gC2) and D (gD2) to stimulate humoral immunity and UL19 (capsid protein VP5) and UL47 (tegument protein VP13/14) as T cell immunogens. The HSV-2 gC2 and gD2 proteins were expressed in baculovirus, while the UL19 and UL47 genes were expressed from replication-defective adenovirus vectors. Adenovirus vectors containing UL19 and UL47 stimulated human and murine CD4+ and CD8+ T cell responses. Guinea pigs were either (i) mock immunized; (ii) immunized with gC2/gD2, with CpG and alum as adjuvants; (iii) immunized with the UL19/UL47 adenovirus vectors; or (iv) immunized with the combination of gC2/gD2-CpG/alum and the UL19/UL47 adenovirus vectors. Immunization with gC2/gD2 produced potent neutralizing antibodies, while UL19 and UL47 also stimulated antibody responses. After intravaginal HSV-2 challenge, the mock and UL19/UL47 adenovirus groups developed severe acute disease, while 2/8 animals in the gC2/gD2-only group and none in the combined group developed acute disease. No animals in the gC2/gD2 or combined group developed recurrent disease; however, 5/8 animals in each group had subclinical shedding of HSV-2 DNA, on 15/168 days for the gC2/gD2 group and 13/168 days for the combined group. Lumbosacral dorsal root ganglia were positive for HSV-2 DNA and latency-associated transcripts for 5/8 animals in the gC2/gD2 group and 2/8 animals in the combined group. None of the differences comparing the gC2/gD2-only group and the combined group were statistically significant. Therefore, adding the T cell immunogens UL19 and UL47 to the gC2/gD2 vaccine did not significantly reduce genital disease and vaginal HSV-2 DNA shedding compared with the excellent protection provided by gC2/gD2 in the guinea pig model. IMPORTANCE HSV-2 infection is a common cause of genital ulcer disease and a significant public health concern. Genital herpes increases the risk of

  15. Anti-human HB-EGF monoclonal antibodies inhibiting ectodomain shedding of HB-EGF and diphtheria toxin binding.

    PubMed

    Hamaoka, Miki; Chinen, Ichino; Murata, Takuya; Takashima, Seiji; Iwamoto, Ryo; Mekada, Eisuke

    2010-07-01

    HB-EGF is a member of the EGF family of growth factors that bind and activate the EGF receptor. HB-EGF is synthesized as a membrane-anchored protein (proHB-EGF), and then proteolytically cleaved, resulting in the mitogenically active soluble form. ProHB-EGF functions as the receptor for the diphtheria toxin (DT). HB-EGF plays pivotal roles in pathophysiological processes, including cancer. Monoclonal antibodies (mAbs) specific for HB-EGF could be an important tool in HB-EGF research. However, few such mAbs have been established to date. In this study, we newly generated seven clones of hybridoma-derived mAbs by immunizing HB-EGF null mice with recombinant human HB-EGF protein. All mAbs specifically bound to human HB-EGF but not to mouse HB-EGF. Epitope mapping analysis showed that most of the mAbs recognized the EGF-like domain. Although none of the newly isolated mAbs directly inhibited the mitogenic activity of HB-EGF for EGFR-expressing cells, some strongly inhibited DT-binding. Interestingly, some of the mAbs efficiently inhibited ectodomain shedding of proHB-EGF, and consequently prevented the cell growth of the EGFR-expressing cells in a co-culture system with proHB-EGF-expressing cells. Hence, these new anti-HB-EGF mAbs may advance clinical as well as basic research on HB-EGF.

  16. Antibodies associated with heparin-induced thrombocytopenia (HIT) inhibit activated protein C generation: new insights into the prothrombotic nature of HIT

    PubMed Central

    Krishnaswamy, Sriram; Rauova, Lubica; Zhai, Li; Hayes, Vincent; Amirikian, Karine; Esko, Jeffrey D.; Bougie, Daniel W.; Aster, Richard H.; Cines, Douglas B.; Poncz, Mortimer

    2011-01-01

    Heparin-induced thrombocytopenia (HIT) is caused by antibodies that recognize complexes between platelet factor 4 (PF4) and heparin or glycosaminoglycan side chains. These antibodies can lead to a limb- and life-threatening prothrombotic state. We now show that HIT antibodies are able to inhibit generation of activated protein C (aPC) by thrombin/thrombomodulin (IIa/TM) in the presence of PF4. Tetrameric PF4 potentiates aPC generation by formation of complexes with chondroitin sulfate (CS) on TM. Formation of these complexes occurs at a specific molar ratio of PF4 to glycosaminoglycan. This observation and the finding that the effect of heparin on aPC generation depends on the concentration of PF4 suggest similarity between PF4/CS complexes and those that bind HIT antibodies. HIT antibodies reduced the ability of PF4 to augment aPC formation. Cationic protamine sulfate, which forms similar complexes with heparin, also enhanced aPC generation, but its activity was not blocked by HIT antibodies. Our studies provide evidence that complexes formed between PF4 and TM's CS may play a physiologic role in potentiating aPC generation. Recognition of these complexes by HIT antibodies reverses the PF4-dependent enhancement in aPC generation and may contribute to the prothrombotic nature of HIT. PMID:21772054

  17. The trimeric serine protease HtrA1 forms a cage-like inhibition complex with an anti-HtrA1 antibody.

    PubMed

    Ciferri, Claudio; Lipari, Michael T; Liang, Wei-Ching; Estevez, Alberto; Hang, Julie; Stawicki, Scott; Wu, Yan; Moran, Paul; Elliott, Mike; Eigenbrot, Charles; Katschke, Kenneth J; van Lookeren Campagne, Menno; Kirchhofer, Daniel

    2015-12-01

    High temperature requirement A1 (HtrA1) is a trypsin-fold serine protease implicated in the progression of age-related macular degeneration (AMD). Our interest in an antibody therapy to neutralize HtrA1 faces the complication that the target adopts a trimeric arrangement, with three active sites in close proximity. In the present study, we describe antibody 94, obtained from a human antibody phage display library, which forms a distinct macromolecular complex with HtrA1 and inhibits the enzymatic activity of recombinant and native HtrA1 forms. Using biochemical methods and negative-staining EM we were able to elucidate the molecular composition of the IgG94 and Fab94 complexes and the associated inhibition mechanism. The 246-kDa complex between the HtrA1 catalytic domain trimer (HtrA1_Cat) and Fab94 had a propeller-like organization with one Fab bound peripherally to each protomer. Low-resolution EM structures and epitope mapping indicated that the antibody binds to the surface-exposed loops B and C of the catalytic domain, suggesting an allosteric inhibition mechanism. The HtrA1_Cat-IgG94 complex (636 kDa) is a cage-like structure with three centrally located IgG94 molecules co-ordinating two HtrA1_Cat trimers and the six active sites pointing into the cavity of the cage. In both complexes, all antigen-recognition regions (paratopes) are found to bind one HtrA1 protomer and all protomers are bound by a paratope, consistent with the complete inhibition of enzyme activity. Therefore, in addition to its potential therapeutic usefulness, antibody 94 establishes a new paradigm of multimeric serine protease inhibition.

  18. Negative regulation of HLA-DR expression on endothelial cells by anti-blood group A/B antibody ligation and mTOR inhibition.

    PubMed

    Iwasaki, Kenta; Miwa, Yuko; Uchida, Kazuharu; Kodera, Yasuhiro; Kobayashi, Takaaki

    2017-02-01

    Donor-specific antibody (DSA), particularly against HLA class II, is a major cause of chronic antibody-mediated rejection (CAMR) after transplantation, although ABO-incompatible kidney transplantation has recently demonstrated favorable graft outcomes. The condition of no injury even in the presence of anti-donor antibody has been referred to as "accommodation", which would be one of the key factors for successful long-term graft survival. The purpose of this study was to analyze the beneficial effect of anti-blood group A/B antibody ligation on endothelial cells against HLA-DR antibody-mediated, complement-dependent cytotoxicity (CDC). Blood group A/B-expressing endothelial cells EA.hy926 or Human Umbilical Vein Endothelia Cells (HUVEC) were incubated with IFNγ in the presence or absence of anti-blood group A/B antibody or mTOR inhibitor (mTOR-i) for 48h. The effects on signaling pathway, HLA expression, complement regulatory factors, and CDC were investigated. Expression of HLA-DR on EA.hy926 or HUVEC were successfully elicited by IFNγ treatment, although little or no expression was observed in quiescent cells. Pre-incubation with anti-blood group A/B antibody had resistance to HLA-DR antibody-mediated CDC against IFNγ-treated cells in a concentration-dependent manner. This finding was ascribed to decreased expression of HLA-DR by post-translational regulation and increased expression of CD55/59, which was related to ERK and mTOR pathway inhibition. mTOR-i also inhibited HLA-DR expression by itself. Furthermore, the combination of mTOR-I and anti-blood group A/B ligation had an additive effect in preventing HLA-DR antibody-mediated CDC. Anti-blood group A/B antibody might play a preventive role in CAMR. Inhibition of the ERK and mTOR pathways may contribute to the development of a novel treatment in the maintenance period after transplantation.

  19. Voltage-induced inhibition of antigen-antibody binding at conducting optical waveguides.

    PubMed

    Liron, Zvi; Tender, Leonard M; Golden, Joel P; Ligler, Frances S

    2002-06-01

    Optical waveguides coated with electrically conducting indium-tin oxide (ITO) are demonstrated here as a new class of substrate for fluorescent immunosensors. These waveguides combine electrochemical control with evanescent excitation and image-based detection. Presented here are preliminary results utilizing these waveguides that demonstrate influence of waveguide voltage on antigen binding. Specifically, waveguide surfaces were bisected into electrically addressable halves, anti-ovalbumin immobilized in patterns on their surfaces, and a 1.3 V bias applied between waveguide halves in the presence of Cy5-labeled ovalbumin in 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl and 0.05% Tween-20. Fluorescence imaging indicated that binding of the antigen to positively biased waveguide halves was inhibited nearly 10-fold compared with negatively biased waveguide halves and unbiased controls. Furthermore, it is shown that ovalbumin binding to positively biased waveguide regions is regenerated after removal of applied voltage. These results suggest that electrochemical control of immunosensor substrates can be used as a possible strategy toward minimizing cross-reactive binding and/or nonspecific adsorption, immunosensor regeneration, and controlled binding.

  20. Effect of Low Molecular Weight Heparins (LMWHs) on antiphospholipid Antibodies (aPL) – Mediated Inhibition of Endometrial Angiogenesis

    PubMed Central

    Di Nicuolo, Fiorella; Castellani, Roberta; Veglia, Manuela; Stinson, John; Scambia, Giovanni; Di Simone, Nicoletta

    2012-01-01

    Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by vascular thrombosis and/or pregnancy morbidity in the presence of circulating antiphospholipid antibodies (aPL). Different pathogenic mechanisms for aPL-mediated pregnancy failure have been proposed. In particular a direct effect of aPL on both maternal and fetal side of the placental tissue has been reported, since their reactivity with β2-glycoprotein I (β2GPI) makes them adhere to trophoblast and human endometrial endothelial cell (HEEC) membranes. β2GPI can be recognized by aPL that, once bound, interfere with both trophoblast functions and with the HEEC differentiation. APS patients can be successfully treated with Low Molecular Weight Heparin (LMWH). Recent reports suggest that LMWH acts through mechanisms alternative to its well known anticoagulant effect, because of its ability to bind β2GPI. In our previous studies, we showed that LMWH is able to reduce the aPL binding to trophoblasts and restore cell invasiveness and differentiation. So far, however, no study has described its effects on endometrial angiogenesis. The aim of our research was to evaluate whether two LMWHs, tinzaparin and enoxaparin, have an effect on the aPL-inhibited endometrial angiogenesis. This prompted us to investigate: (i) in vitro HEEC angiogenesis through a Matrigel assay; (ii) VEGF secretion by ELISA; (iii) matrix metalloproteinase-2 (MMP-2) activity by gelatin zymography; (iv) Nuclear Factor-κB (NF-κB) DNA binding activity by colorimetric assay; (v) STAT-3 activation by a sandwich-ELISA kit. Furthermore, using an in vivo murine model we investigated the LMWHs effects on angiogenesis. We demonstrated that the addition of LMWHs prevents aPL-inhibited HEEC angiogenesis, both in vitro and in vivo, and is able to restore the aPL inhibited NF-κB and/or STAT-3 activity, the VEGF secretion and the MMPs activity. The demonstration of a beneficial role for LMWHs on the aPL-inhibited HEEC angiogenesis might

  1. Fcγ Receptor-induced Soluble Vascular Endothelial Growth Factor Receptor-1 (VEGFR-1) Production Inhibits Angiogenesis and Enhances Efficacy of Anti-tumor Antibodies*

    PubMed Central

    Justiniano, Steven E.; Elavazhagan, Saranya; Fatehchand, Kavin; Shah, Prexy; Mehta, Payal; Roda, Julie M.; Mo, Xiaokui; Cheney, Carolyn; Hertlein, Erin; Eubank, Timothy D.; Marsh, Clay; Muthusamy, Natarajan; Butchar, Jonathan P.; Byrd, John C.; Tridandapani, Susheela

    2013-01-01

    Monocytes/macrophages are potent mediators of antitumor antibody therapy, where they engage target cells via Fcγ receptors (FcγR). Binding of these cells to opsonized tumor targets elicits cytokine production, phagocytosis, and antibody-mediated cellular cytotoxicity. Here we show for the first time that activation of monocyte FcγR results in the secretion of soluble vascular endothelial growth factor receptor-1 (VEGFR-1/sFlt-1), which serves to antagonize VEGF-mediated angiogenesis and tumor growth. Consistent with this, using a murine solid tumor model of antibody therapy, we show that sFlt-1 is involved in restricting tumor growth. Analyzing the mechanism of induction of sFlt-1, we found that the Erk and PI3K pathways were required for transcription, and NF-κB was required for translation. Upon closer examination of the role of NF-κB, we found that a microRNA, miR181a, negatively regulates FcγR-mediated sFlt-1 production and that NF-κB serves to antagonize this microRNA. Taken together, these results demonstrate a novel and biologically important function of monocytes and macrophages during antibody therapy. PMID:23902770

  2. Fcγ receptor-induced soluble vascular endothelial growth factor receptor-1 (VEGFR-1) production inhibits angiogenesis and enhances efficacy of anti-tumor antibodies.

    PubMed

    Justiniano, Steven E; Elavazhagan, Saranya; Fatehchand, Kavin; Shah, Prexy; Mehta, Payal; Roda, Julie M; Mo, Xiaokui; Cheney, Carolyn; Hertlein, Erin; Eubank, Timothy D; Marsh, Clay; Muthusamy, Natarajan; Butchar, Jonathan P; Byrd, John C; Tridandapani, Susheela

    2013-09-13

    Monocytes/macrophages are potent mediators of antitumor antibody therapy, where they engage target cells via Fcγ receptors (FcγR). Binding of these cells to opsonized tumor targets elicits cytokine production, phagocytosis, and antibody-mediated cellular cytotoxicity. Here we show for the first time that activation of monocyte FcγR results in the secretion of soluble vascular endothelial growth factor receptor-1 (VEGFR-1/sFlt-1), which serves to antagonize VEGF-mediated angiogenesis and tumor growth. Consistent with this, using a murine solid tumor model of antibody therapy, we show that sFlt-1 is involved in restricting tumor growth. Analyzing the mechanism of induction of sFlt-1, we found that the Erk and PI3K pathways were required for transcription, and NF-κB was required for translation. Upon closer examination of the role of NF-κB, we found that a microRNA, miR181a, negatively regulates FcγR-mediated sFlt-1 production and that NF-κB serves to antagonize this microRNA. Taken together, these results demonstrate a novel and biologically important function of monocytes and macrophages during antibody therapy.

  3. Inhibition of pancreatic carcinoma by homo- and heterocombinations of antibodies against EGF-receptor and its kin HER2/ErbB-2

    PubMed Central

    Maron, Ruth; Schechter, Bilha; Mancini, Maicol; Mahlknecht, Georg; Yarden, Yosef; Sela, Michael

    2013-01-01

    Due to intrinsic aggressiveness and lack of effective therapies, prognosis of pancreatic cancer remains dismal. Because the only molecular targeted drug approved for pancreatic ductal adenocarcinoma is a kinase inhibitor specific to the epidermal growth factor receptor (EGFR), and this receptor collaborates with another kinase, called HER2 (human EGF-receptor 2), we assumed that agents targeting EGFR and/or HER2 would effectively retard pancreatic ductal adenocarcinoma. Accordingly, two immunological strategies were tested in animal models: (i) two antibodies able to engage distinct epitopes of either EGFR or HER2 were separately combined, and (ii) pairs of one antibody to EGFR and another to HER2. Unlike the respective single monoclonal antibodies, which induced weak effects, both types of antibody combinations synergized in animals in terms of tumor inhibition. Immunological cooperation may not depend on receptor density, antigenic sites, or the presence of a mutant RAS protein. Nevertheless, both types of antibody combinations enhanced receptor degradation. Future efforts will examine the feasibility of each strategy and the potential of combining them to achieve sustained tumor inhibition. PMID:24003140

  4. Design and synthesis of 2-(3-benzo[b]thienyl)-6,7-methylenedioxyquinolin-4-one analogues as potent antitumor agents that inhibit tubulin assembly.

    PubMed

    Chang, Yu-Hsun; Hsu, Mei-Hua; Wang, Sheng-Hung; Huang, Li-Jiau; Qian, Keduo; Morris-Natschke, Susan L; Hamel, Ernest; Kuo, Sheng-Chu; Lee, Kuo-Hsiung

    2009-08-13

    As part of our continuing investigation of azo-flavonoid derivatives as potential anticancer drug candidates, a series of 2-aryl-6,7-methylenedioxyquinolin-4-one analogues was designed and synthesized. The design combined structural features of 2-(2-fluorophenyl)-6,7-methylenedioxyquinolin-4-one (CHM-1), a previously discovered compound with potent in vivo antitumor activity, and 2-arylquinolin-4-ones, identified by CoMFA models. The newly synthesized analogues were evaluated for cytotoxicity against seven human cancer cell lines, and structure-activity relationship (SAR) correlations were established. Analogues 1, 37, and 39 showed potent cytotoxicity against different cancer cell lines. Compound 1 demonstrated selective cytotoxicity against Hep 3B (hepatoma) cells. Compound 37 was cytotoxic against HL-60 (leukemia), HCT-116 (colon cancer), Hep 3B (hepatoma), and SK-MEL-5 (melanoma) cells. Compound 39 exhibited broad cytotoxicity against all seven cancer cell lines, with IC50 values between 0.07 and 0.19 microM. Results from mechanism of action studies revealed that these new quinolone derivatives function as antitubulin agents.

  5. Antibodies against MAEBL Ligand Domains M1 and M2 Inhibit Sporozoite Development In Vitro

    PubMed Central

    Preiser, Peter; Rénia, Laurent; Singh, Naresh; Balu, Bharath; Jarra, William; Voza, Tatiana; Kaneko, Osamu; Blair, Peter; Torii, Motomi; Landau, Irène; Adams, John H.

    2004-01-01

    MAEBL is a type 1 membrane protein that is implicated in the merozoite invasion of erythrocytes and sporozoite invasion of mosquito salivary glands. This apical organelle protein is structurally similar to the ebl erythrocyte binding proteins, such as EBA-175, except that the tandem ligand domains of MAEBL are similar to part of the extracellular domain of apical membrane antigen 1 and not the Duffy binding-like domain. Although midgut and salivary gland sporozoites are morphologically similar, salivary gland sporozoites undergo a period of new gene expression after infecting the salivary glands, display distinct phenotypic differences, and are more infectious for the mammalian host. The objectives of this project were to determine the molecular form of MAEBL in the infectious salivary gland sporozoites and whether the ligand has a role in the sporozoite development to exoerythrocytic stages in hepatocytes. We determined that MAEBL is newly expressed in salivary gland sporozoites and in a form distinct from what is present in the midgut sporozoites or present in erythrocytic stages. Both ligand domains (M1 and M2) were expressed as part of a full-length membrane form of MAEBL in the salivary gland sporozoites in contrast to the other stages that retain only the M2 ligand domain as part of the membrane form of the protein. Antisera developed against the cysteine-rich regions of the extracellular portion of MAEBL inhibited sporozoite development to exoerythrocytic forms in vitro. Together these data indicate that MAEBL has a role in this third developmental stage in the life cycle of the malaria parasite. Thus, MAEBL is another target for pre-erythrocytic-stage vaccine development against malaria parasites. PMID:15155670

  6. A novel rabbit anti-hepatocyte growth factor monoclonal neutralizing antibody inhibits tumor growth in prostate cancer cells and mouse xenografts

    SciTech Connect

    Yu, Yanlan; Chen, Yicheng; Ding, Guoqing; Wang, Mingchao; Wu, Haiyang; Xu, Liwei; Rui, Xuefang; Zhang, Zhigen

    2015-08-14

    The hepatocyte growth factor and its receptor c-Met are correlated with castration-resistance in prostate cancer. Although HGF has been considered as an attractive target for therapeutic antibodies, the lack of cross-reactivity of monoclonal antibodies with human/mouse HGFs is a major obstacle in preclinical developments. We generated a panel of anti-HGF RabMAbs either blocking HGF/c-Met interaction or inhibiting c-Met phosphorylation. We selected one RabMAb with mouse cross-reactivity and demonstrated that it blocked HGF-stimulated downstream activation in PC-3 and DU145 cells. Anti-HGF RabMAb inhibited not only the growth of PC-3 cells but also HGF-dependent proliferation in HUVECs. We further demonstrated the efficacy and potency of the anti-HGF RabMAb in tumor xenograft mice models. Through these in vitro and in vivo experiments, we explored a novel therapeutic antibody for advanced prostate cancer. - Highlights: • HGF is an attractive target for castration-refractory prostate cancer. • We generated and characterized a panel of anti-HGF rabbit monoclonal antibodies. • More than half of these anti-HGF RabMAbs was cross-reactive with mouse HGF. • Anti-HGF RabMAb blocks HGF-stimulated phosphorylation and cell growth in vitro. • Anti-HGF RabMAb inhibits tumor growth and angiogenesis in xenograft mice.

  7. Soluble, but not immobilized, anti-IgM antibody inhibits post-activation events leading to T-cell-dependent B-cell differentiation.

    PubMed Central

    Zamorano, J; Rivas, D; Gayo, A; Mozo, L; Gutiérrez, C

    1995-01-01

    The potential for surface immunoglobulin-binding ligands to modify B-cell differentiation responses induced by activated T cells has been investigated. Activated T cells in human splenic mononuclear cells cultured on anti-CD3-coated plates induced B cells to produce large amounts of IgM and IgG. In this experimental system, cross-linking of B-cell antigen receptors by soluble, bivalent monoclonal or polyclonal anti-IgM antibodies completely inhibited IgM production, and greatly diminished IgG production, in a dose-dependent manner. Similar results were obtained using a F(ab')2 fragment of a goat anti-IgM antibody. Inhibition of B-cell differentiation by bivalent cross-linking reagents did not require the presence of antigen-presenting cells (APC), as comparable results were obtained in co-cultures of purified T and B cells. In contrast, enhanced immunoglobulin secretion was seen when surface IgM was cross-linked using anti-IgM antibody immobilized on the culture plate. Interestingly, activated T cells induced similar levels of expression on B cells of the activation antigens CD23, CD25 and CD71, and of class II molecules, irrespective of any treatment with soluble or immobilized anti-IgM antibody. This indicates that soluble anti-IgM specifically inhibits B-cell differentiation without altering initial events of T-cell-dependent B-cell activation. Images Figure 3 Figure 5 PMID:7642212

  8. An antibody against the surfactant protein A (SP-A)-binding domain of the SP-A receptor inhibits T cell-mediated immune responses to Mycobacterium tuberculosis.

    PubMed

    Samten, Buka; Townsend, James C; Sever-Chroneos, Zvjezdana; Pasquinelli, Virginia; Barnes, Peter F; Chroneos, Zissis C

    2008-07-01

    Surfactant protein A (SP-A) suppresses lymphocyte proliferation and IL-2 secretion, in part, by binding to its receptor, SP-R210. However, the mechanisms underlying this effect are not well understood. Here, we studied the effect of antibodies against the SP-A-binding (neck) domain (alpha-SP-R210n) or nonbinding C-terminal domain (alpha-SP-R210ct) of SP-R210 on human peripheral blood T cell immune responses against Mycobacterium tuberculosis. We demonstrated that both antibodies bind to more than 90% of monocytes and 5-10% of CD3+ T cells in freshly isolated PBMC. Stimulation of PBMC from healthy tuberculin reactors [purified protein derivative-positive (PPD+)] with heat-killed M. tuberculosis induced increased antibody binding to CD3+ cells. Increased antibody binding suggested enhanced expression of SP-R210, and this was confirmed by Western blotting. The antibodies (alpha-SP-R210n) cross-linking the SP-R210 through the SP-A-binding domain markedly inhibited cell proliferation and IFN-gamma secretion by PBMC from PPD+ donors in response to heat-killed M. tuberculosis, whereas preimmune IgG and antibodies (alpha-SP-R210ct) cross-linking SP-R210 through the non-SP-A-binding, C-terminal domain had no effect. Anti-SP-R210n also decreased M. tuberculosis-induced production of TNF-alpha but increased production of IL-10. Inhibition of IFN-gamma production by alpha-SP-R210n was abrogated by the combination of neutralizing antibodies to IL-10 and TGF-beta1. Together, these findings support the hypothesis that SP-A, via SP-R210, suppresses cell-mediated immunity against M. tuberculosis via a mechanism that up-regulates secretion of IL-10 and TGF-beta1.

  9. Investigations on the presence of antibodies to papova viruses in patients with different forms of cancer and in other categories of patients or apparently healthy subjects. Note III. Hemagglutination-inhibiting serum antibodies to polyoma virus.

    PubMed

    Stoian, M; Hozoc, M; Pucă, D; Serban, A; Bolocan, J; Nastac, E

    1981-01-01

    Hemagglutination-inhibiting antibodies to polyoma virus could be detected in sera from apparently healthy subjects, patients with nonmalignant respiratory diseases and patients with different forms of cancer. The positivity percentages (12.97, 12.92 and 16.36, respectively) and the titers recorded were lower than in the case of BK virus. The results obtained suggest a slight antigenic relationship between polyoma and BK virus.

  10. The Cell Wall as the Antigenic Site for Antibodies Stimulating Ingestion (MSF) and Inhibition (BIF) of Brucella in Macrophages from Normal and Immune Animals

    PubMed Central

    Ralston, Doris J.; Elberg, S. S.

    1971-01-01

    Following extraction with hot trichloracetic acid and digestion with trypsin, deoxyribonuclease and ribonuclease, cell wall residues of Brucella melitensis strain Rev I contained an antigenic moiety capable of removing antibodies responsible for stimulation of ingestion, inhibition of Brucella growth by macrophages, and agglutination of Brucella. The preparation was dermonecrotic for both normal and immune rabbits and, when injected in oil, stimulated antibody production. The peptidoglycan-containing structure was degraded by egg-white lysozyme and enzymes from normal and immune macrophages with an accompanying loss of dermonecrotic and serumabsorbing capacity. PMID:5582072

  11. The role of antibody in the inhibition of the growth of Meth.A tumour in syngeneic experiments in vivo and in vitro.

    PubMed

    Farram, E; Festenstein, H; de Giorgi, L

    1978-09-01

    An in vitro technique for detecting anti-tumour responses was studied and shown to involve non- T cells. Further examination of the effector mechanism revealed that tumour inhibition was antibody mediated, probably through complement dependent lysis; ADCC was considered unlikely. The amount of antibody involved was small, as shown by indirect immunofluorescence labelling of tumour cells, but was nevertheless effective in vitro and in causing regression of tumours in vivo. These findings may have important implications for the manipulation of the host responses to tumours.

  12. Monoclonal antibody affinity purification of a Leishmania membrane glycoprotein and its inhibition of leishmania-macrophage binding.

    PubMed Central

    Chang, C S; Chang, K P

    1986-01-01

    Specific monoclonal antibody coupled to Affi-Gel 10 was used to purify a major membrane glycoprotein of Leishmania mexicana amazonensis, one of a group of parasitic protozoa that specifically infect mammalian macrophages. Immobilized antigen was eluted at a 34% efficiency with buffers at either pH 2.5 or 11 or with MgCl2, but only the antigen eluted under basic conditions could be readsorbed to the immunobeads. Sephacryl S-300 gel filtration of the purified antigen gave a single peak of protein estimated to have a molecular mass of 400 kDa. However, NaDodSO4/polyacrylamide gel electrophoresis showed a single band of this protein with an apparent molecular mass of 63 kDa. The antigen is an N-linked glycoprotein, as indicated by its increase in electrophoretic mobility after treatment with endoglycosidase H and by its binding to lentil lectin-Sepharose, elutable with methyl alpha-D-mannoside and methyl alpha-D-glucoside. Purified antigen inhibits the binding of leishmania cells to macrophages by 50%, suggesting that it may play a role in the process of infection. Images PMID:3079902

  13. Development of Potent and Selective Indomethacin Analogues for the Inhibition of AKR1C3 (Type 5 17β-Hydroxysteroid Dehydrogenase/Prostaglandin F Synthase) in Castrate-Resistant Prostate Cancer

    PubMed Central

    2013-01-01

    Castrate-resistant prostate cancer (CRPC) is a fatal, metastatic form of prostate cancer. CRPC is characterized by reactivation of the androgen axis due to changes in androgen receptor signaling and/or adaptive intratumoral androgen biosynthesis. AKR1C3 is upregulated in CRPC where it catalyzes the formation of potent androgens. This makes AKR1C3 a target for the treatment of CRPC. AKR1C3 inhibitors should not inhibit AKR1C1/AKR1C2, which inactivate 5α-dihydrotestosterone. Indomethacin, used to inhibit cyclooxygenase, also inhibits AKR1C3 and displays selectivity over AKR1C1/AKR1C2. Parallel synthetic strategies were used to generate libraries of indomethacin analogues, which exhibit reduced cyclooxygenase inhibitory activity but retain AKR1C3 inhibitory potency and selectivity. The lead compounds inhibited AKR1C3 with nanomolar potency, displayed >100-fold selectivity over AKR1C1/AKR1C2, and blocked testosterone formation in LNCaP-AKR1C3 cells. The AKR1C3·NADP+·2′-des-methyl-indomethacin crystal structure was determined, and it revealed a unique inhibitor binding mode. The compounds reported are promising agents for the development of therapeutics for CRPC. PMID:23432095

  14. Visible-light-induced CO release from a therapeutically viable tryptophan-derived manganese(I) carbonyl (TryptoCORM) exhibiting potent inhibition against E. coli.

    PubMed

    Ward, Jonathan S; Lynam, Jason M; Moir, James; Fairlamb, Ian J S

    2014-11-10

    The first visible-light-activated carbon-monoxide-releasing molecule (CO-RM) to exhibit a potent effect against Escherichia coli is described. The easily prepared tryptophan-derived manganese-containing complex (TryptoCORM) released 1.4 moles of CO at 465 nm, and 2 moles at 400 nm. A comprehensive synthetic, mechanistic and microbiological study into the behaviour of TryptoCORM is reported. The complex is thermally stable (i.e., does not release CO in solution in the absence of light), shows low toxicity against mammalian cells and releases tryptophan on photoinduced degradation, all of which point to TryptoCORM being therapeutically viable.

  15. Differential inhibition of recombinant bovine GH (rbGH) activity in vitro by in vivo enhancing monoclonal antibodies.

    PubMed

    Beattie, J; Phillips, K; Borromeo, V

    2001-04-01

    We have previously described the effects of complexing recombinant bovine growth hormone (rbGH) with the in vivo enhancing monoclonal antibodies (Mabs) OA11 and OA15 and the non-enhancing Mab OA14 on the subsequent activity of GH in different tissue culture models. We reported that all of these Mabs caused the inhibition of GH-stimulated Jak-2 tyrosine kinase phosphorylation in the GH responsive pre-adipocyte cell line 3T3-F442A. However, using the mouse myeloid cell line FDC-P1 transfected with the full length ovine GH receptor (GHR), we subsequently found that OA11 and OA14 remained inhibitory with respect to the end point measurement of GH stimulated mitogenesis but that OA15 had no inhibitory effect on GH stimulated mitogenesis in this cell line. In order to correlate longer term mitogenic effects of Mab-GH complexes with signalling events in this transfected cell line model, we now report on the effects of complexing with Mab on the subsequent GH stimulated phosphorylation of Stat5b (signal transducer and activator of transcription). In agreement with our data for the mitogenic activity of GH-Mab complexes, we found that OA11 and OA14 inhibit GH activation of Stat5b but that OA15 is not inhibitory. Further to this, the dose-response effect of both OA11 and OA14 on the GH stimulation of Stat5b in the FDC-P1-oGHR transfected cells correlates with the previously described dose-response effects for both Mabs in the context of GH stimulation of mitogenic effects. We conclude that in this oGHR transfected cell line model, Mab effects on short and long term GH signalling events are tightly correlated. The observation that neither of the in vivo enhancing Mabs--OA11 or OA15--amplifies the response to GH in our transfected cell line model, coupled with the differential nature of Mab effects on GH activity (OA11--inhibition; OA15--no effect) may argue for an in vivo mechanism for enhancement of GH activity.

  16. Functional and molecular effects of mercury compounds on the human OCTN1 cation transporter: C50 and C136 are the targets for potent inhibition.

    PubMed

    Galluccio, Michele; Pochini, Lorena; Peta, Valentina; Iannì, Maria; Scalise, Mariafrancesca; Indiveri, Cesare

    2015-03-01

    The effect of mercury compounds has been tested on the organic cation transporter, hOCTN1. MeHg(+), Hg(2+), or Cd(2+) caused strong inhibition of transport. 1,4-Dithioerythritol (DTE), cysteine (Cys), and N-acetyl-l-cysteine reversed (NAC) the inhibition at different extents. 2-Aminoethyl methanethiosulfonate hydrobromide (MTSEA), a prototype SH reagent, exerted inhibition of transport similar to that observed for the mercurial agents. To investigate the mechanism of action of mercurials, mutants of hOCTN1 in which each of the Cys residues was substituted by Ala have been constructed, over-expressed in Escherichia coli, and purified. Tetraethylammonium chloride (TEA) uptake mediated by each mutant in proteoliposomes was comparable to that of wild type (WT). IC50 values of the WT and mutants for the mercury compounds were derived from dose-response analyses. The mutants C50A and C136A showed significant increase of IC50 indicating that the 2 Cys residues were involved in the interaction with the mercury compounds and inhibition of the transporter. The double mutant C50A/C136A was constructed; the lack of inhibition confirmed that the 2 Cys residues are the targets of mercury compounds. MTSEA showed similar behavior with respect to the mercurial reagents with the difference that increased IC50 was observed also in the C81A mutant. Similar results were obtained when transport was measured as acetylcholine uptake. Ethyl mercury (Thimerosal) inhibited hOCTN1 as well. C50A, C50A/C136A and, at very lower extent, C136A showed increased IC50 indicating that C50 was the major target of this mercury compound. The homology model of hOCTN1 was built using as template PiPT and validated by the experimental data on mutant proteins.

  17. Hemolytic disease of the fetus and newborn due to anti-Ge3: combined antibody-dependent hemolysis and erythroid precursor cell growth inhibition.

    PubMed

    Blackall, Douglas P; Pesek, Gina D; Montgomery, Matthew M; Oza, Krishna K; Arndt, Patricia A; Garratty, George; Shahcheraghi, Ali; Denomme, Gregory A

    2008-10-01

    The Gerbich (Ge) antigens are a collection of high-incidence antigens carried on the red blood cell membrane glycoproteins, glycophorins C and D. Antibodies against these antigens are uncommon, and there have been only rare case reports of hemolytic disease of the fetus and newborn due to anti-Ge. In this case report, we present a neonate with severe anemia and hyperbilirubinemia due to anti-Ge3. Routine and special laboratory studies undertaken in this case suggested two mechanisms for the patient's hemolysis and persistent anemia. Antibody-dependent hemolysis was associated with early-onset hyperbilirubinemia, anemia, and a mild reticulocytosis, and inhibition of erythroid progenitor cell growth was associated with late anemia and normal bilirubin and reticulocyte values. Though rare, anti-Ge3 can be a dangerous antibody in pregnancy. Affected neonates may require intensive initial therapy and close follow-up for at least several weeks after delivery.

  18. The Investigational Drug VT-1129 Is a Highly Potent Inhibitor of Cryptococcus Species CYP51 but Only Weakly Inhibits the Human Enzyme

    PubMed Central

    Warrilow, Andrew G. S.; Parker, Josie E.; Price, Claire L.; Nes, W. David; Garvey, Edward P.; Hoekstra, William J.; Schotzinger, Robert J.; Kelly, Diane E.

    2016-01-01

    Cryptococcosis is a life-threatening disease often associated with HIV infection. Three Cryptococcus species CYP51 enzymes were purified and catalyzed the 14α-demethylation of lanosterol, eburicol, and obtusifoliol. The investigational agent VT-1129 bound tightly to all three CYP51 proteins (dissociation constant [Kd] range, 14 to 25 nM) with affinities similar to those of fluconazole, voriconazole, itraconazole, clotrimazole, and ketoconazole (Kd range, 4 to 52 nM), whereas VT-1129 bound weakly to human CYP51 (Kd, 4.53 μM). VT-1129 was as effective as conventional triazole antifungal drugs at inhibiting cryptococcal CYP51 activity (50% inhibitory concentration [IC50] range, 0.14 to 0.20 μM), while it only weakly inhibited human CYP51 activity (IC50, ∼600 μM). Furthermore, VT-1129 weakly inhibited human CYP2C9, CYP2C19, and CYP3A4, suggesting a low drug-drug interaction potential. Finally, the cellular mode of action for VT-1129 was confirmed to be CYP51 inhibition, resulting in the depletion of ergosterol and ergosta-7-enol and the accumulation of eburicol, obtusifolione, and lanosterol/obtusifoliol in the cell membranes. PMID:27161631

  19. First-in-Human Evaluation of the Safety and Immunogenicity of an Intranasally Administered Replication-Competent Sendai Virus–Vectored HIV Type 1 Gag Vaccine: Induction of Potent T-Cell or Antibody Responses in Prime-Boost Regimens

    PubMed Central

    Nyombayire, Julien; Anzala, Omu; Gazzard, Brian; Karita, Etienne; Bergin, Philip; Hayes, Peter; Kopycinski, Jakub; Omosa-Manyonyi, Gloria; Jackson, Akil; Bizimana, Jean; Farah, Bashir; Sayeed, Eddy; Parks, Christopher L.; Inoue, Makoto; Hironaka, Takashi; Hara, Hiroto; Shu, Tsugumine; Matano, Tetsuro; Dally, Len; Barin, Burc; Park, Harriet; Gilmour, Jill; Lombardo, Angela; Excler, Jean-Louis; Fast, Patricia; Laufer, Dagna S.; Cox, Josephine H.

    2017-01-01

    Background. We report the first-in-human safety and immunogenicity assessment of a prototype intranasally administered, replication-competent Sendai virus (SeV)–vectored, human immunodeficiency virus type 1 (HIV-1) vaccine. Methods. Sixty-five HIV-1–uninfected adults in Kenya, Rwanda, and the United Kingdom were assigned to receive 1 of 4 prime-boost regimens (administered at 0 and 4 months, respectively; ratio of vaccine to placebo recipients, 12:4): priming with a lower-dose SeV-Gag given intranasally, followed by boosting with an adenovirus 35–vectored vaccine encoding HIV-1 Gag, reverse transcriptase, integrase, and Nef (Ad35-GRIN) given intramuscularly (SLA); priming with a higher-dose SeV-Gag given intranasally, followed by boosting with Ad35-GRIN given intramuscularly (SHA); priming with Ad35-GRIN given intramuscularly, followed by boosting with a higher-dose SeV-Gag given intranasally (ASH); and priming and boosting with a higher-dose SeV-Gag given intranasally (SHSH). Results. All vaccine regimens were well tolerated. Gag-specific IFN-γ enzyme-linked immunospot–determined response rates and geometric mean responses were higher (96% and 248 spot-forming units, respectively) in groups primed with SeV-Gag and boosted with Ad35-GRIN (SLA and SHA) than those after a single dose of Ad35-GRIN (56% and 54 spot-forming units, respectively) or SeV-Gag (55% and 59 spot-forming units, respectively); responses persisted for ≥8 months after completion of the prime-boost regimen. Functional CD8+ T-cell responses with greater breadth, magnitude, and frequency in a viral inhibition assay were also seen in the SLA and SHA groups after Ad35-GRIN boost, compared with those who received either vaccine alone. SeV-Gag did not boost T-cell counts in the ASH group. In contrast, the highest Gag-specific antibody titers were seen in the ASH group. Mucosal antibody responses were sporadic. Conclusions. SeV-Gag primed functional, durable HIV-specific T

  20. Inhibition of an Allergen–Antibody Reaction Related to Japanese Cedar Pollinosis Using DNA Aptamers Against the Cry j 2 Allergen

    PubMed Central

    Ogihara, Kazumasa; Savory, Nasa; Abe, Koichi; Yoshida, Wataru; Arakawa, Mitsuru; Asahi, Masahiko; Kamohara, Seika

    2015-01-01

    Japanese cedar pollinosis is one of the most prevalent allergies in Japan. Reducing the allergen content of pollen plays a major role in the alleviation of allergy symptoms. Aptamers, oligonucleotides with an affinity for specific molecules, have great potential for reducing allergic activity. In this study, we report that the anti-Cry j 2 aptamers, CJ2-04 and CJ2-08, inhibited allergen–antibody reactions between Cry j 2, one of the major allergens in Japanese cedar pollen, and immunoglobulin E in serum collected from a patient with Japanese cedar pollinosis. In addition, the suppression of Ca2+ mobilization in basophils, which is related to degranulation, was observed in samples preincubated with either of these DNA aptamers. This study indicates that anti-Cry j 2 aptamers may inhibit allergen–antibody reactions and suppress the induction of Japanese cedar pollinosis, possibly leading to a novel external defense against this and other types of allergens. PMID:26484654

  1. A dodecylamine derivative of cyanocobalamin potently inhibits the activities of cobalamin-dependent methylmalonyl-CoA mutase and methionine synthase of Caenorhabditis elegans.

    PubMed

    Bito, Tomohiro; Yabuta, Yukinori; Ichiyanagi, Tsuyoshi; Kawano, Tsuyoshi; Watanabe, Fumio

    2014-01-01

    In this study, we showed that cyanocobalamin dodecylamine, a ribose 5'-carbamate derivative of cyanocobalamin, was absorbed and accumulated to significant levels by Caenorhabditis elegans and was not further metabolized. The levels of methylmalonic acid and homocysteine, which serve as indicators of cobalamin deficiency, were significantly increased in C. elegans treated with the dodecylamine derivative, indicating severe cobalamin deficiency. Kinetic studies show that the affinity of the cyanocobalamin dodecylamine derivative was greater for two cobalamin-dependent enzymes, methylmalonyl-CoA mutase and methionine synthase, compared with their respective coenzymes, suggesting that the dodecylamine derivative inactivated these enzymes. The dodecylamine derivative did not affect the levels of mRNAs encoding these enzymes or those of other proteins involved in intercellular cobalamin metabolism, including methylmalonyl-CoA mutase (mmcm-1), methylmalonic acidemia cobalamin A complementation group (mmaa-1), methylmalonic aciduria cblC type (cblc-1), and methionine synthase reductase (mtrr-1). In contrast, the level of the mRNAs encoding cob(I)alamin adenosyltransferase (mmab-1) was increased significantly and identical to that of cobalamin-deficient C. elegans. These results indicate that the cyanocobalamin-dodecylamine derivative acts as a potent inhibitor of cobalamin-dependent enzymes and induces severe cobalamin deficiency in C. elegans.

  2. Molecular basis of vitamin E action. Tocotrienol potently inhibits glutamate-induced pp60(c-Src) kinase activation and death of HT4 neuronal cells.

    PubMed

    Sen, C K; Khanna, S; Roy, S; Packer, L

    2000-04-28

    HT4 hippocampal neuronal cells were studied to compare the efficacy of tocopherols and tocotrienol to protect against glutamate-induced death. Tocotrienols were more effective than alpha-tocopherol in preventing glutamate-induced death. Uptake of tocotrienols from the culture medium was more efficient compared with that of alpha-tocopherol. Vitamin E molecules have potent antioxidant properties. Results show that at low concentrations, tocotrienols may have protected cells by an antioxidant-independent mechanism. Examination of signal transduction pathways revealed that protein tyrosine phosphorylation processes played a central role in the execution of death. Activation of pp60(c-Src) kinase and phosphorylation of ERK were observed in response to glutamate treatment. Nanomolar amounts of alpha-tocotrienol, but not alpha-tocopherol, blocked glutamate-induced death by suppressing glutamate-induced early activation of c-Src kinase. Overexpression of kinase-active c-Src sensitized cells to glutamate-induced death. Tocotrienol treatment prevented death of Src-overexpressing cells treated with glutamate. alpha-Tocotrienol did not influence activity of recombinant c-Src kinase suggesting that its mechanism of action may include regulation of SH domains. This study provides first evidence describing the molecular basis of tocotrienol action. At a concentration 4-10-fold lower than levels detected in plasma of supplemented humans, tocotrienol regulated unique signal transduction processes that were not sensitive to comparable concentrations of tocopherol.

  3. Inhibition of p85, the non-catalytic subunit of phosphatidylinositol 3-kinase, exerts potent antitumor activity in human breast cancer cells

    PubMed Central

    Folgiero, V; Di Carlo, S E; Bon, G; Spugnini, E P; Di Benedetto, A; Germoni, S; Pia Gentileschi, M; Accardo, A; Milella, M; Morelli, G; Bossi, G; Mottolese, M; Falcioni, R

    2012-01-01

    The phosphoinositide 3-kinases (PI3Ks) are heterodimers consisting of the catalytic subunit p110 and the regulatory subunit p85. The PI3K/Akt pathway is strongly deregulated in breast cancer (BC) representing one of the mechanisms of resistance to therapies. Therefore, the identification of inhibitors of PI3K components represents one of the main goals to produce therapeutic agents. Here, we evaluated the efficacy of a phosphopeptide 1257 (P-1257) that targeting p85 strongly inhibits PI3K activity. We tested the effects of P-1257 administration in vitro and in vivo using BC cells expressing different levels of ErbB-2 and resistant or responsive to Trastuzumab. We demonstrated that inhibition of p85 activity by P-1257 induces cell death and sensitizes JIMT-1 and KPL-4 ErbB-2-overexpressing BC cells to Trastuzumab treatment. It is noteworthy that P-1257 delivery in vivo by electroporation or liposomes significantly inhibits the proliferation of tumor cells engrafted at subcutaneous and visceral sites. Overall, our data indicate that the p85 subunit is a valid target for therapeutic approaches and suggest that the structure of the peptide used in our study could be utilized for the development of novel drugs to apply in combination with therapies that fail to cure BCs with high PI3K activity. PMID:23222510

  4. Soluble human CD4 elicits an antibody response in rhesus monkeys that inhibits simian immunodeficiency virus replication

    SciTech Connect

    Watanabe, Mamoru; Chen, Zheng W.; Tsubota, Hiroshi; Lord, C.I.; Levine, C.G.; Letvin, N.L. )

    1991-01-01

    Rhesus monkeys infected with the simian immunodeficiency virus of macaques (SIV{sub mac}) demonstrate significant virologic and clinical improvement as a result of treatment with human recombinant soluble CD4 (rsCD4). The authors show that human rsCD4 does not efficiently inhibit SIV{sub mac} replication in bone marrow macrophages of rhesus monkeys and does not significantly augment bone marrow hematopoietic colony formation in vitro. However, plasma of human rsCD4-treated rhesus monkeys does exhibit significant anti-SIV{sub mac} activity in vitro. Plasma of these animals efficiently blocks SIV{sub mac} replicaton in peripheral blood lymphocytes and bone marrow macrophages. It also increases granulocyte/macrophage colony formation in vitro by bone marrow cells of SIV{sub mac}-infected monkeys. This plasma and the IgG fraction of plasma from a rhesus monkey immunized with human rsCD4 in adjuvant demonstrate reactivity with a soluble form of the rhesus monkey CD4 molecule, exhibit binding to CD4{sup +} but not CD8{sup +} concanavalin A-activated rhesus monkey peripheral blood lymphocytes, and precipitate the CD4 molecule from surface-labeled activated rhesus monkey peripheral blood lymphocytes. Moreover, anti-viral activity is demonstrable in the IgG fraction of plasma from a human rsCD4-immunized monkey. These studies raise the possibility that a modified human CD4 molecule serving as an immunogen might elicit an antibody response that could potentially induce a beneficial therapeutic response in human immunodeficiency virus-infected individuals.

  5. CWF-145, a novel synthetic quinolone derivative exerts potent antimitotic activity against human prostate cancer: Rapamycin enhances antimitotic drug-induced apoptosis through the inhibition of Akt/mTOR pathway.

    PubMed

    Hung, Chao-Ming; Lin, Ying-Chao; Liu, Liang-Chih; Kuo, Sheng-Chu; Ho, Chi-Tang; Way, Tzong-Der

    2016-12-25

    CWF-145, a synthetic 2-phenyl-4-quinolone derivative exerted potent cytotoxicity against prostate cancer. CWF-145 inhibited prostate cancer cell lines PC-3, DU-145 and LNCap. It had a very low IC50 about 200 nM against castrate-resistant prostate cancer (CRPC) PC-3. We found that CWF-145 had a similar effect to clinical trial antimitotic agents in cancer cells and normal cells. CWF-145 arrested cell cycle at G2/M phase by binding to the β-tubulin at the colchicine-binding site then disrupted microtubule polymerization. Furthermore, the damaged microtubule affected the Akt/mammalian target of rapamycin (mTOR) signaling pathway. Our data showed that CWF-145 activated Akt and mTOR expression to increase emi1 accumulation and inhibit APC. The increased cyclin B1 and securin arrested cell cycle at G2/M phase. Moreover, we showed that Akt activation markedly increased resistance to microtubule-directed agents, including CWF-145, colchicine, and paclitaxel. Interestingly, rapamycin inhibited Akt-mediated therapeutic resistance, indicating that these effects were dependent on mTOR. Taken together, these observations suggest that activation of the Akt/mTOR signaling pathway can promote resistance to chemotherapeutic agents that do not directly target metabolic regulation. These data may provide insight into potentially synergistic combinations of anticancer therapies.

  6. Mononuclear copper(II) complexes with 3,5-substituted-4-salicylidene-amino-3,5-dimethyl-1,2,4-triazole: synthesis, structure and potent inhibition of protein tyrosine phosphatases.

    PubMed

    Ma, Ling; Lu, Liping; Zhu, Miaoli; Wang, Qingming; Li, Ying; Xing, Shu; Fu, Xueqi; Gao, Zengqiang; Dong, Yuhui

    2011-06-28

    Six copper complexes of Schiff base ligands containing 3,5-substituted-4-salicylideneamino-3,5-dimethyl-1,2,4-triazole have been synthesized and well characterized. The structures of complexes 1 and 2 were determined by X-ray crystal analysis. Fluorescence and potentiometric study indicated that in the physiological pH range, one ligand was dissociated from the complexes to form 1:1 mononucleus copper complexes. The complexes potently inhibit protein tyrosine phosphatase 1B (PTP1B), T-cell protein tyrosine phosphatase (TCPTP), megakaryocyte protein tyrosine phosphatase 2 (PTP-MEG2) and Src homology phosphatase 1 (SHP-1) with 3-4 fold selectivity against PTP1B over TCPTP and PTP-MEG2, and 3-9 fold over SHP-1, but display almost no inhibition against Src homology phosphatase 2 (SHP-2). Complex 1 inhibits PTP1B with a competitive model with K(i) of 30 nM. Substitution with small groups at the phenyl of the ligand does not obviously influence the inhibitory ability of the complexes.

  7. A large-tumor-antigen-specific monoclonal antibody inhibits DNA replication of simian virus 40 minichromosomes in an in vitro elongation system.

    PubMed Central

    Stahl, H; Dröge, P; Zentgraf, H; Knippers, R

    1985-01-01

    In productively infected cells, a fraction of large-tumor antigen (T antigen) is tightly bound to replicating simian virus 40 (SV40) minichromosomes and does not dissociate at salt concentrations of greater than 1 M NaCl. We present electronmicrograms demonstrating the presence of T antigen on the replicated sections of replicating SV40 minichromosomes. We also show that the fraction of tightly bound T antigen is recognized by antibodies from mouse tumor serum and, more specifically, by a particular T-antigen-specific monoclonal antibody, PAb 1630. A second T-antigen-specific monoclonal antibody, PAb 101, does not react with the T-antigen fraction remaining on replicating SV40 chromatin at high salt concentrations. We used an in vitro replication system which allows, via semiconservative DNA replication, the completion of in vivo-initiated replicative intermediate DNA molecules. We show that monoclonal antibody PAb 1630, but not monoclonal antibody PAb 101, inhibits viral DNA replication. We discuss the possibility that SV40 T antigen may play a role in chain elongation during SV40 chromatin replication. Images PMID:2985809

  8. A comparison of a blocking ELISA and a haemagglutination inhibition assay for the detection of antibodies to Avibacterium (Haemophilus) paragallinarum in sera from artificially infected chickens.

    PubMed

    Sun, H; Miao, D; Zhang, P; Gong, Y; Blackall, P J

    2007-10-01

    The ability of blocking ELISAs and haemagglutination-inhibition (HI) tests to detect antibodies in sera from chickens challenged with either Avibacterium (Haemophilus) paragallinarum isolate Hp8 (serovar A) or H668 (serovar C) was compared. Serum samples were examined weekly over the 9 weeks following infection. The results showed that the positive rate of serovar A specific antibody in the B-ELISA remained at 100% from the second week to the ninth week. In chickens given the serovar C challenge, the highest positive rate of serovar C specific antibody in the B-ELISA appeared at the seventh week (60% positive) and was then followed by a rapid decrease. The B-ELISA gave significantly more positives at weeks 2, 3, 7, 8 and 9 post-infection for serovar A and at week 7 post-infection for serovar C. In qualitative terms, for both serovar A and serovar C infections, the HI tests gave a lower percentage of positive sera at all time points except at 9 weeks post-infection with serovar C. The highest positive rate for serovar A HI antibodies was 70% of sera at the fourth and fifth weeks post-infection. The highest rate of serovar C HI antibodies was 20% at the fifth and sixth weeks post-infection. The results have provided further evidence of the suitability of the serovar A and C B-ELISAs for the diagnosis of infectious coryza.

  9. Novel mode of action of the calcium antagonist mibefradil (Ro 40-5967): potent immunosuppression by inhibition of T-cell infiltration through allogeneic endothelium.

    PubMed Central

    Blaheta, R A; Hailer, N P; Brude, N; Wittig, B; Oppermann, E; Leckel, K; Harder, S; Scholz, M; Weber, S; Encke, A; Markus, B H

    1998-01-01

    Cyclosporin A reduces the mitotic activity of allosensitized lymphocytes, but fails to limit emigration of these cells into the donor organ. However, the modulation of both lymphocyte proliferation and infiltration are desirable characteristics of immunosuppressive therapy. The calcium-channel blocker, verapamil, has recently been shown to effectively prevent the transmigration of CD4+ and CD8+ T cells through allogeneic endothelium. Mibefradil (Ro 40-5967) represents a new generation of calcium antagonists with high potency and long-term activity. To evaluate the immunosuppressive potential of this drug, the influence of mibefradil on lymphocyte adhesion to, horizontal locomotion along, and penetration through allogeneic endothelium (HUVEC) was performed. When lymphocytes were prestimulated for 24 hr with mibefradil, adhesion and penetration were dose-dependently reduced. The adhesion ID50 values were 3.4 microM (CD4+ T cells) versus 9.2 microM (CD8+ T cells) and 2.1 microM (CD4+ T cells) versus 3.9 microM (CD8+ T cells) with regard to penetration. Mibefradil also effectively blocked horizontal locomotion. Specific down-regulation of T-cell binding to the P-selection receptor (ID50: CD4+ T cells, 0.8 microM: CD8+ T cells, 1.2 microM) and to the intracellular adhesion molecule-1 (ICAM-1) receptor (ID50: CD4+ T cells, 1.9 microM; CD8+ T cells, 1.5 microM) by mibefradil seems to be responsible for the decreased adhesion and penetration rates. Reduction of intracellular F-actin in T lymphocytes could diminish cell locomotion. In conclusion, the potent suppressive properties of mibefradil support its use as a co-medication in cyclosporin A-based immunosuppressive therapy. PMID:9741343

  10. Potent Inhibition of Pseudogymnoascus destructans, the Causative Agent of White-Nose Syndrome in Bats, by Cold-Pressed, Terpeneless, Valencia Orange Oil

    PubMed Central

    Boire, Nicholas; Zhang, Sean; Khuvis, Joshua; Lee, Rick; Rivers, Jennifer; Crandall, Philip; Keel, M. Kevin; Parrish, Nicole

    2016-01-01

    The causative agent of White-nose Syndrome (WNS), Pseudogymnoascus destructans, has been shown to be fatal to several species of bats in North America. To date, no compounds or chemical control measures have been developed which eliminates the growth of the fungus in the environment or in affected animals. In the current study, we evaluated the activity of cold-pressed, terpeneless orange oil (CPT) against multiple isolates of P. destructans in vitro. For all assays, a modified Kirby-Bauer disk diffusion assay was used. Standardized spore suspensions were prepared, adjusted to a specific optical density, and used to plate fungal lawns. Plates were incubated at either 15°C or 4°C for up to 6 months and checked at regular intervals for growth. Once controls had grown, zones of inhibition were measured (mm) on test plates and compared to those obtained using current antifungal drugs. All P. destructans isolates were completely inhibited by 100% CPT (10 μL) at 1 month of incubation regardless of temperature (4°C and 15°C). Complete inhibition persisted up to 6 months following a single exposure at this concentration. Of the standard antifungals, only amphotericin B demonstrated any activity, resulting in zone diameters ranging from 58 mm to 74 mm. CPT, at the highest concentration tested (100%), had no significant effect against a variety of other environmental organisms including various filamentous fungi, bacteria and aerobic actinomycetes. Given that CPT is relatively non-toxic, the possibility exists that the all-natural, mixture could be used as an environmental pre-treatment to eradicate P. destructans from bat habitats. Additional studies are needed to assess any undesirable effects of CPT on bat behavior and health and overall impacts on other members of the interconnected ecosystem(s). PMID:26849057

  11. A novel 7-bromoindirubin with potent anticancer activity suppresses survival of human melanoma cells associated with inhibition of STAT3 and Akt signaling.

    PubMed

    Liu, Lucy; Kritsanida, Marina; Magiatis, Prokopios; Gaboriaud, Nicolas; Wang, Yan; Wu, Jun; Buettner, Ralf; Yang, Fan; Nam, Sangkil; Skaltsounis, Leandros; Jove, Richard

    2012-11-01

    STAT3 and Akt signaling have been validated as potential molecular targets for treatment of cancers including melanoma. These small molecule inhibitors of STAT3 or Akt signaling are promising for developing anti-melanoma therapeutic agents. MLS-2438, a novel 7-bromoindirubin, a derivative of the natural product indirubin, was synthesized with a bromo-group at the 7-position on one indole ring and a hydrophilic group at the 3'-position on the other indole ring. We tested the anticancer activity of MLS-2438 and investigated its mechanism of action in human melanoma cell lines. Here, we show that MLS-2438 inhibits viability and induces apoptosis of human melanoma cells associated with inhibition of STAT3 and Akt signaling. Several pro-apoptotic Bcl-2 family proteins are involved in the MLS-2438 mediated apoptosis. MLS-2438 inhibits Src kinase activity in vitro and phosphorylation of JAK2, Src, STAT3 and Akt in cultured cancer cells. In contrast to the decreased phosphorylation levels of JAK2, Src, STAT3 and Akt, phosphorylation levels of the MAPK (Erk1/2) signaling protein were not reduced in cells treated with MLS-2438. These results demonstrate that MLS-2438, a novel natural product derivative, is a Src inhibitor and potentially regulates kinase activity of JAK2 and Akt in cancer cells. Importantly, MLS-2438 suppressed tumor growth with low toxicity in a mouse xenograft model of human melanoma. Our findings support further development of MLS-2438 as a potential small-molecule therapeutic agent that targets both STAT3 and Akt signaling in human melanoma cells.

  12. Potent Inhibition of Pseudogymnoascus destructans, the Causative Agent of White-Nose Syndrome in Bats, by Cold-Pressed, Terpeneless, Valencia Orange Oil.

    PubMed

    Boire, Nicholas; Zhang, Sean; Khuvis, Joshua; Lee, Rick; Rivers, Jennifer; Crandall, Philip; Keel, M Kevin; Parrish, Nicole

    2016-01-01

    The causative agent of White-nose Syndrome (WNS), Pseudogymnoascus destructans, has been shown to be fatal to several species of bats in North America. To date, no compounds or chemical control measures have been developed whi